**Table 1.** *concentrationtoxicmetalsin*

*The of different potentially (mg/L) drinking, ground, and surface water and in sediments and vegetables (mg/kg).*

#### *Health Risks of Potentially Toxic Metals Contaminated Water DOI: http://dx.doi.org/10.5772/intechopen.92141*

**Sample**

**68**

Ground water Maru Town, Nigeria

Groundwater

Groundwater

Ground water

Sediments

Sediments

Sediments Sediments

Sediments

Sediments

Sediments

Sediments

Vegetables

Vegetables

Vegetables

Supermarket

 of the

0.002–0.040

0.0006–0.028

0.009–0.126

 0.22–2.65

 1.12–3.88

0.0005–0.07

 0.010–

0.0012–0.016

 0.0012–

0.0065

(Co)

0.096

 0.012–0.223

 0.13–2.47

 1.31–3.95

0.0019–0.065

 0.012–

0.002–0.020

 0.0005–

[44]

0.033 (Co)

0.291

Florida

 Bangladesh

 0.03–2.4

 0.03–22.6

 0.4–52.3

 0.33–95.5

 0.0.02–61.5

 0.03–1.02

 0.63–1.33

 0.02–32

[41–43]

(Mn)

 Bay of Bengal

 0.03–0.06

 0.61–0.79

 0.38–0.66

 River Ganga

1.7

69.9

29.8

67.8

26.7 0.01–1.42

 0.01–0.23

26.7

 River Ghaghara

 0.21–0.28

 61.3–84.7

 2.8–11.7

 13.3–17.6

 10.7–14.3

 15.3–25.6

 River Gomati

 1.9–8.4

 River of Philippines

Kabul

4.4–7.1

 75.5–92.5 32.8–131.8

 29.4–217.1

9.0–95.4

35.8–90.9

3.7–15.0

[37]

11.4–18.4

[38]

(Co)

[39]

[40]

 76.8–263.3

 10.9–15.3

 8.1–88.3

 32.8–54.6

 69.1–85.1 12.1–98.1

4.1–25.3

[36]

(Co)

 Mashavera Basin

Georgia

 River Raohe, Chia

 0.00–1.60

1.5–1.7

 26.4–28

 347.8–410.7

 423.3–458.9

 29.6–37.2

 22.3–22.5

 13.5–97.1

 15.6–793.5

 11.2–52.9

 16.0–222.2

 18.4–66.4

 12.9–318.0

 Singhbhum,

 India

 0.01–0.08

 0.04–0.28

 Iran

 Albania

0.0–0.0006

0.006

0.0075–0.078

0.001–0.0058

0.09 0.08–0.42

 0.03–0.14

0.008–0.009

[31]

*Heavy Metal Toxicity in Public Health*

[32]

0.07–4.45

[33]

(Fe)

[13]

0.00–0.02

[34]

(Hg)

[35]

**Source** **Cd**

**Cr** 0.0–0.99

 0.0–0.33

0.012–0.087

**Cu**

**Zn**

**Pb**

**Ni** 0.00–

0.0056

**As**

 **Others** [30]

**Concentration**

 **of metal (mg/L) or (mg/kg)**


**Fish species**

**71**

*Oreochromis*

*Finfish* *Shrimp*

*Oyster* *Tilapia zillii*

*Malapterurus*

*Clarias gariepinus* *Clarias batrachus*

*Barbuss harpeyi* *Barbus xanthopterus*

Bodo Creek, Niger Delta, Nigeria

Tigris River in Baghdad

Local fish ponds of Ludhiana city and Sutlej River

Niger River, Nigeria

 *electricus*

Niger River, Nigeria

 *niloticus*

Burullus Lake, Egypt

Lower Gangetic Delta, India

Lower Gangetic Delta, India

Lower Gangetic Delta, India

Niger River, Nigeria

**Source**

**Tissue** **Cd**

Muscles

Whole Body Whole Body Whole Body

Gill

Muscle

Intestine

Gill

Muscle

Intestine

Gill

Muscle

Intestine

 Liver Muscle

Kidney

Gills

Liver

Muscle

Gills

Liver

Muscle

 0.8

 0.5–1.6

 0.7–0.8

 0.7–0.8

 1.1

 2.7–2.8

 2.2–2.6

 0.7–0.8

 0.7–0.9

 1.8–2.1

 2.2–2.5

 2.1–2.5

 1.2–1.3

 1.1–1.2

 1.3–1.6

 0.97–1.2

 1.6–1.6

 0.6–0.7

 0.80

 1.05

 1.3–2.9

 2.5–2.7

 0.5–0.8

 0.9

 2.05

 2.3–2.4

 2.2–2.5

 1.1–1.2

 1.05–1.1

 1.5–1.6

[65]

 4.45

 2.14

 4.65

 56.83

 8.04

 3.09

 0.24

 2.10

 3.79

 29.42

 4.48

 0.68

 3.74

 3.69

 3.48

 67.78

 11.12

 3.13

[58]

 0.033

 0.04

 0.055

 0.049

 0.032

 0.056

 0.119

 0.049

 0.094

 BDL

 0–1.5

 0–1.32

 0.45

 0.85

 0.39 1.3–53.1

6.2–109.2

8.7–69.1

 21.4–202.8

8.92

5.42

12.20

5.77

3.74

5.74

5.55

3.38

6.6

 ND

ND

 ND

ND

 ND

ND

 ND

ND

 ND

ND

 ND

ND

 ND

ND

 ND

ND

*Health Risks of Potentially Toxic Metals Contaminated Water*

 ND

ND

 [64]

*DOI: http://dx.doi.org/10.5772/intechopen.92141*

 0–8

 11.7–213.7

 0–10

 2.0–111.5

 0–3.05

 4.70

 0.46

[62]

[63]

 **Cr**

 **Cu**

 **Zn**

 **Pb**

 **As Hg**

**Concentration**

 **of metal (mg/kg)**


#### *Health Risks of Potentially Toxic Metals Contaminated Water DOI: http://dx.doi.org/10.5772/intechopen.92141*

**Fish species**

**70**

*Cyprinus carpio*

*Labeo rohita* *Cyprinus carpio*

*Wallago attu*

*Labeo rohita* *Channa striatus*

Kolleru Lake, India

Kolleru Lake, India

Indus River Mianwali, Pakistan

Indus River Mianwali, Pakistan

Sardaryab, tributary of River Kabul

Sardaryab, tributary of River Kabul

**Source**

**Tissue** **Cd**

Gills Liver Muscles

Gills Liver Muscles

Gills Liver Kidney Muscles

Gills Liver Kidney Muscles

Gills

Liver

Kidney

Muscles

Gills

Liver

Kidney

Muscles

 0.45

 0.85

 0.53

 0.46

 1.30

 0.16

 0.47

 1.03

 1.21

 1.33

0.25

0.14

 0.48

 0.33

 0.99

 1.01

 1.35

 0.32

 0.59

 0.03

 1.14

 1.30

 0.32

 0.02

 0.09

 0.38

 0.22

 0.25

 0.72

 0.23

 0.94

 0.55

 0.62

 0.69

 1.06

 1.04

 1.48

0.27

0.07

0.04

0.12

0.20

 0.38

 0.19

 0.09

 0.19

 0.3

0.05

 [61]

 **Cr** 0.154

0.188

0.024

0.133

0.165

0.019

2.9–9.4

3–5.4

1.55–3.5

1.03–2.63

4.5–9.5

12.5–20

2.0–6.5

6.5–15.2

 2.9–5.5

—

0.83–2.0

 1.4–3.5

—

0.0–0.5

 11.5–21

—

0.9–1.0

 3–5.9

—

0.45–1.92

 1.1–1.67

—

0.46–1.9

 1.4–2.5

—

0.48–0.9

 2.5–3.4

—

0.45–1.1

 0.77–1.4

—

0.5–1.87

[60]

 0.01

 0.02

 0.000

 0.071

 0.088

 0.161

 0.018

 0.058

 0.024

 0.016

 0.018

 0.000

*Heavy Metal Toxicity in Public Health*

 0.089

 0.07

 0.142

 0.024

 0.074

 0.041

[59]

 **Cu**

 **Zn**

 **Pb**

 **As Hg**

**Concentration**

 **of metal (mg/kg)**


**Fish species**

**73**

*Oncorhynchus*

**Table 2.**

*The* 

*concentration*

 *of different potentially*

 *toxic metals in different parts of freshwater*

 *fish.*

 *mykiss*

Hamadan Province, Iran

Whole Body 0.17–13.74

Muscle

Liver

 0.17–13.74

 0.17–11.88

**Source**

**Tissue** **Cd**

 **Cr**

 **Cu**

 **Zn**

 **Pb** 0.34–70.17 0.34–35.19

1.29–70.17

*Health Risks of Potentially Toxic Metals Contaminated Water*

*DOI: http://dx.doi.org/10.5772/intechopen.92141*

[73]

 **As Hg**

**Concentration**

 **of metal (mg/kg)**

**Reference**

*Heavy Metal Toxicity in Public Health*


*The concentration of different potentially toxic metals in different parts of freshwater fish.*

#### *Health Risks of Potentially Toxic Metals Contaminated Water DOI: http://dx.doi.org/10.5772/intechopen.92141*

**Fish species**

**72**

*Callinectes amnicola*

*Chrysichthys*

*Alosa immaculata*

*Cyprinus carpio* *Cyprinus carpio*

*Silurus glanis* *Silurus glanis* *Sander lucioperca* *Tilapia mossambica*

*Oreochromis*

*Lutjanus griseus*

*Lutjanus stellatus*

*Thunnus albacares*

Pearl Delta river China Pearl Delta river China

Egypt

 *niloticus*

Lakes of Coimbatore,

 India

*nigrodigitatus*

Densu River, Ghana

Danube River Danube River Danube River, Belgrade, Siberia

Danube River, Belgrade, Siberia

Danube River Danube River

Water bodies of

Aurangabad,

 India

Gill Skin Liver Muscle Muscle

Gill

Liver

Whole Body Whole Body Whole Body

 0.06

 0.07

 0.03

 1.76–2.22

 1.47–1.85

 1.26–1.59

3.66–4.58

1.80–2.74

1.56–2.16

2.98–4.23

 0.26–0.32 8.49–9.69

9.9–11.3

11.74–13.42

 25.08–35.4

 8.07–13.24

0.03

0.04

0.32

[72]

 1.53

 0.39

[71]

 21.05–29.71

 6.89–11.30

 18.38–25.94

 5.91–9.69

[70]

 0.29–0.36

 0.19–0.26

 0.32–0.46

Bodo Creek, Niger Delta, Nigeria

**Source**

**Tissue** **Cd**

Leg

Gill

Muscle

carapace

Muscles Whole Body Whole Body

 Whole Body

 Whole Body Whole Body Whole Body

 0.04

 0.09

 0.08

 0.014

 0.084

 0.09

0.59

5.34 5.10 0.207 0.235 0.07 0.11

0.65 0.58

0.036 0.014

0.17

0.23

 0.3 [69]

 0.33

[68]

[67]

 2.34

0.19 0.37

 [11] [66]

 2.4–5.43

 0.00–1.13

 0.38–2.02

 0.43–3.78

 **Cr**

 **Cu**

 **Zn**

 **Pb** 0.3–1.13 0.01–0.42

0.01–0.62

*Heavy Metal Toxicity in Public Health*

0.00–0.47

[27]

 **As Hg**

**Concentration**

 **of metal (mg/kg)**


**Fish species**

**75**

*Liocarcinus*

*Rapana venosa*

*Mytilus*  *Otolithes ruber* *Lutjanus johnii*

*Lagocephalus*

*Hirundichthys*

*Cypselurus spilopterus*

*Sardina* *Xiphias gladius* *Brachydeuterus*

*Pennahia anea* *Arius maculatus* *Decapterus maraudsi*

*Megalaspis cordyla*

*Bramidae*

Terengganu

Terengganu

Terengganu

 Coastal Area, Malaysia

 Coastal Area, Malaysia

 Coastal Area, Malaysia

 Soft tissue

 Soft tissue

 Soft tissue

 1.53

 0.31

 0.2

 *auritus*

*coromandelensis*

Southeast coast of India

Southeast coast of India Algerian coasts, Algeria Algerian coast, Algeria Fishing Habour Ghana,

Coastal Waters, Malaysia

 *sceleratus*

North-eastern

Mediterranean

 part of Turkey

 Muscle

Skin

Soft tissue Soft tissue Soft tissue Soft tissue

Muscle Muscle

Liver

Gill

Muscle

Liver

Gill

 0.02–0.19

 0.23–0.98

 0.04–0.09

 0.02–0.21

 0.44–0.69

 0.03–0.21

 0.57

 0.55

 0.02

 0.02

 0.113–0.217

 0.10–0.15

 0.168–0.209

0.26–0.28

0.26–0.33

 2.15–3.30

2.13

3.90

0.42

0.94–4.38

6.7–19.8

1.08–6.52

0.83–3.68

17.94–55

1.65–6.95

0.64

1.16

0.98

 15.14

 0.09

 10.42

 0.02

 7.97

 0.17

[84]

 282–528

 0.24–0.5

 356–558

 0.1–1.16

 23–48.6

 0.15–0.36

 48.2–115.7

 0.45–1.96

 7–114.4

 0.96–1.26

 17.7–26.3

 0.14–0.41

 2.28

0.2 0.31

 [11] [83]

 0.56

 0.62

[82]

 017–0.19

 3–3.28

 0.20–0.24

[81]

 3.34–6.45

 0.34–0.58

 0.045–0.139

 0.20–0.36

 0.276–0.518

 51.4–86.63

 1.46–2.56

[80]

*Health Risks of Potentially Toxic Metals Contaminated Water*

*galloprovincialis*

Persian Gulf, Iran

Persian Gulf, Iran

 *depurator*

Samsun coasts of the Black Sea Turkey

 Soft tissue Soft tissue Soft tissue Soft tissue Soft tissue

 0.17–0.38

 0.21–0.47

 0.08

 0.085

 0.07

**Source**

**Tissue** **Cd**

 **Cr**

 **Cu** 7.7 4.3 9.2

14

 0.50 1.98–2.98 2.53–3.12

[79]

*DOI: http://dx.doi.org/10.5772/intechopen.92141*

9

 0.12

19

 0.48

[78]

 **Zn**

 **Pb**

 **As Hg**

**Concentration**

 **of metal (mg/kg)**


#### *Health Risks of Potentially Toxic Metals Contaminated Water DOI: http://dx.doi.org/10.5772/intechopen.92141*

**Fish species**

**74**

*Mullet*

Southeast Coast of Indian Ocean

**Source**

**Tissue** **Cd**

Gills

Gonad

Muscle

Skin

Whole Body

 Muscle Gonad

Whole Body

Skin

Whole Body Whole Body Whole Body Whole Body Whole Body Whole Body Whole Body

 Whole Body Whole Body Whole Body Whole Body

Soft tissue

 0.08–0.45

 12.7–38.0

 59.2–133.5

 87–191

 2.36–17.5

[77]

 0.007

 0.04

 0.004

 0.003

 0.004

 0.007

 0.001

 0.001

 0.004

 0.060

 0.027

*Crab* *Shrimp*

*Euthynnus affinis* *Pampus argenteus* *Decapterus macrosoma*

*Leiognathus*

 *daura*

*Fenneropenaeus*

*Lates calcarifer* *Johnius belangerii*

*Chirocentrus*

*Arius maculatus*

*Parastromateus*

*Oyster*

Gulf of Chabahar

 *niger*

 *dorab*

Red Sea, Jeddah Coast, Saudi Arabia

 *indicus*

Tok Bali Port, Malaysia Tok Bali Port, Malaysia Tok Bali Port, Malaysia Tok Bali Port, Malaysia Tok Bali Port, Malaysia

Southeast Coast of Indian Ocean

Southeast Coast of Indian Ocean

 0.005

 0.010

 0.013

 0.001

 0.013

 **Cr**

 **Cu** 0.092

0.192

0.034

0.016

0.085

0.243

0.330

0.061

0.082

0.2

0.70

0.038

0.064

0.018

0.34

0.49

1.35

1.45

1.15

2.55

 22.29

 1.0

 25.95

 0.85

 22.91

 1.0

 23.33

 1.45

 6.5

 0.20

[75]

[76]

 14.4

 0.008

 5.45

 0.003

 5.29

 0.001

 4.83

 0.024

 62.4

 0.3

[57]

 0.088

 0.007

 0.233

 0.268

 0.259

 0.013

 0.244

 0.150

 0.228

 0.237

 0.176

 0.026

 0.018

 0.000

 0.074

 0.009

*Heavy Metal Toxicity in Public Health*

 0.284

 0.302

 0.087

 0.043

[74]

 **Zn**

 **Pb**

 **As Hg**

**Concentration**

 **of metal (mg/kg)**


**Fish species**

**77**

*Pseudotolithus*

 *typus*

**Source**

**Tissue** **Cd**

Gill

Bone

Muscle

Head

Eye

Gill

Bone

Muscle

 0.0–0.140

0.0–0.02

*Cyprinus carpio L,*

*Cyprinus carpio L,*

**Table 3.**

*The* 

*concentration*

 *of different potentially*

 *toxic metals in different tissues of marine organisms.*

Ala gul wetland (Iran)

Alma gul wetland (Iran)

 0.32

 0.37

 0.59 1.23–39.4

1.23–4.4

 19.15–117.4

 2.1–8.7

 1.15–47.7

 0–21.86

[86]

 0.38

 0.11

 0.32

 0.37

 0.62

 0.42

 0.12

*Health Risks of Potentially Toxic Metals Contaminated Water*

*DOI: http://dx.doi.org/10.5772/intechopen.92141*

 0.30

 0.37

 0.0

 0.39

 0.01

 0.28

 0.23

 0.0

 0.38

 0.16

 0.31

 0.36

 0.0

 0.36

 0.07

 0.31

 0.38

 0.39

 0.39

 0.12

 0.81

 0.86

 0.94

 0.94

 0.27

 **Cr**

 **Cu**

 **Zn**

 **Pb**

 **As Hg**

**Concentration**

 **of metal (mg/kg)**

**Reference**

*Heavy Metal Toxicity in Public Health*


 **3.** *The concentration of different potentially toxic metals in different tissues of marine organisms.*

**Table**

#### *Health Risks of Potentially Toxic Metals Contaminated Water DOI: http://dx.doi.org/10.5772/intechopen.92141*

**Fish species**

**76**

*Selaroides leptolepis*

*Epinephelus*

*Rastrellige* *Nibea soldado* *Pristipomoides*

*Priacanthus*

*Siganus*  *Thunnus obesus* *Trichiurus lepturus*

*canaliculatus*

 *tayenus*

 *filamentosus*

 *lanceolatus*

Terengganu

Terengganu

Terengganu

Terengganu

Terengganu

Terengganu

Terengganu

Western and Central pacific ocean

Coastal Waters of Ondo State, Nigeria

 Coastal Area, Malaysia

 Coastal Area, Malaysia

 Coastal Area, Malaysia

 Coastal Area, Malaysia

 Coastal Area, Malaysia

 Coastal Area, Malaysia

 Coastal Area, Malaysia

 Soft tissue

 Soft tissue

 Soft tissue

 Soft tissue

 Soft tissue

 Soft tissue

 Soft tissue

 Soft tissue

 Muscle

Head

Eye

Gill

Bone

Muscle

Head

Eye

Gill

Bone

Muscle

Head

Eye

 0.29

 0.32

 0.39

 0.39

 0.04

 0.27

 0.26

 0.38

 0.38

 0.71

 0.0

 0.0

 0.46

 0.36

 0.10

*Pseudotolithus*

 *senegalensis*

 0.32

 0.38

 0.59

 0.39

 0.11

 1.14

 1.28

 1.88

 1.29

 0.40

 0.49

 0.62

 0.0

 0.65

 0.14

 0.26

 0.37

 0.0

 0.36

 0.34

 0.0

 0.0

 0.58

 0.35

 0.1

*Pentanmius*

 *guigarius*

 0.32

 0.35

 0.59

 0.10

 0.12

 0.48

 0.49

 0.87

 0.51

 0.15

 0.37

 0.35

 0.0

 0.38

 0.20

 0.21

 0.32

 0.0

 0.38

 0.20

 0.0

 0.0

 0.0

 0.34

 0.09

[85]

 0.10

 0.08

 0.08

 0.12

 0.25

 0.64

 0.66

**Source**

**Tissue** **Cd**

 **Cr**

 **Cu** 0.68

0.83

0.5

0.29

0.25

0.42

0.68

 11.60

 0.15 0.929

 6.63

 0.13

 4.88

 ND

 5.91

 ND

 9.39

 0.73

*Heavy Metal Toxicity in Public Health*

 12.51

 0.11

 11.28

 0.14

 **Zn**

 **Pb**

 **As Hg**

**Concentration**

 **of metal (mg/kg)**

#### **7. Potentially toxic metals-resistance**

The potentially toxic metals (Cd, Cu, Pb, Zn, Cr (III), Cr (VI), and Hg) aren't only toxic to human health but also enrich antibiotic resistant microbes particularly bacteria. Co-selection of an antibiotic and metal resistance in bacteria is extremely important because it promotes antibiotic resistance in bacteria even in the absence of antibiotics. Co-selection occurs by two mechanisms:

**Toxic metal**

Cu Produces hyperoxide radicals by interaction with cell membrane. Enzymatic activities are inhibited and cellular functions are disrupted.

Hg Hg inactivates the

**79**

enzymatic activities, interferes in the protein synthesis and DNA function, and disrupts cell membrane. Destroys the biological membranes as the mercuric ions are lipid soluble so easily passed through biological membranes.

**Mechanism of action Mechanism of**

*Health Risks of Potentially Toxic Metals Contaminated Water*

*DOI: http://dx.doi.org/10.5772/intechopen.92141*

**resistance**

Resistant genes are located on plasmids and transposons and transfers in between bacterial species.

Hg resistant genes are located on plasmids and transposons. The resistance mechanism involves the reduction of Hg2+ ions to Hg in the cytoplasm of the bacteria by the enzyme mercuric reductase which is encoded with the merA gene.

**Antibiotic categories / generic names**

Vancomycin, Amphenicol

Quinolone; Sulphonamide

Penicillin; Cephalosporin; Nitrofuran

Sulphonamide, Chloramphenicol, Ampicillin, Streptomycetin, Augmentin

Fluoroquinolone, Quinolone

Sulphonamide; Cephalosporin; Macrolide

Tetracycline *Klebsiella* spp.

Carbapenem *Pseudomonas*

Erythromycin soil bacteria of

Fluoroquinolone *E. coli* [92];

Cephalosporin *Fecal Enterococci* [91]

Macrolide *E. faecium* [98]

[100]

[101]

[103] Tetracycline *E. coli*; *Citrobacter*

[105]

Penicillin *Salmonella* spp.

Teicoplanin *Salmonella* spp. [102]

Cephalosporin *E. coli* [107];

**Pathogen(s)**

[89, 90]; *Fecal Enterococci* [91]; *E. coli* [92]; *E. faecium* [93]

*aeruginosa* [94]

from ship [96]

Scotland [90]; *Enterococcus faecium*

*E. faecium* [93]

*Enterobacter* spp.; *P. aeruginosa* [97]

*Salmonella enterica* [99]; Fecal Gramnegative bacteria

*E. coli*, *Citrobacter* spp., Klebsiella spp.

[102]; *Serratia* spp.

spp.; *Klebsiella* spp. [104]; *E. faecium*

*E. coli*; *Citrobacter* spp.; *Enterobacter* spp.; *Klebsiella* spp.; *Proteus* spp. [106]

*Citrobacter* spp.; *Enterobacter* spp.; *Klebsiella* spp.; *Proteus* spp. [106]

*E. coli* [92]

[93, 95] Chloramphenicol *Bacillus* spp. isolated

[95]

*Enterococcus faecium*


Besides these two mechanisms, co-selection is additionally promoted by coregulatory mechanism which occurs when different resistant genes is controlled by one regulator gene [88]. The impact of the potentially toxic metals on the antibiotic resistant bacterial strain is a given in **Table 4**.

#### **8. Toxicity of potentially toxic metals**

World Health Organization has reported that globally in the year 2015 approximately 8.8 million deaths were due to cancer, presence of potentially toxic metals beyond permissible limits within the environment is one among the main factors of the death because the endocrine system is disrupted by these metals. When the food or drinking water containing potentially toxic metals beyond their maximum tolerance concentration is ingested, the metabolism of living cells in the body is negatively affected [122]. The immune and hematopoietic systems in human and animals also are adversely affected on exposure to the mixtures of those metals [122]. Li et al. [123] reported that the main cause for human bone diseases is the presence of the potentially toxic metals beyond their permissible limit within the aquatic environment. Potentially toxic metals Pb, Hg, Cd, As, and Cr in living cells causes cytotoxicity [124] and oxidative stress [124], leading to the damages of antioxidants, enzyme inhibition, apoptosis (programmed cell death), loss of DNA repair mechanism, protein dysfunction, and damage to lipid peroxidase and of the membrane.

#### **8.1 Cadmium**

Cadmium in human causes Itai-Itai disease, liver/kidney lesions, hepato-colic effects [125], carcinoma, prostatic adenocarcinoma, osteoporosis, hypertension, disorder, and kidney lesions including enlargement, nuclear, and mitochondrial damages, and histological changes; decreased antioxidant power of kidney also


**7. Potentially toxic metals-resistance**

*Heavy Metal Toxicity in Public Health*

those metals [87].

resistant bacterial strain is a given in **Table 4**.

**8. Toxicity of potentially toxic metals**

membrane.

**8.1 Cadmium**

**78**

of antibiotics. Co-selection occurs by two mechanisms:

The potentially toxic metals (Cd, Cu, Pb, Zn, Cr (III), Cr (VI), and Hg) aren't only toxic to human health but also enrich antibiotic resistant microbes particularly bacteria. Co-selection of an antibiotic and metal resistance in bacteria is extremely important because it promotes antibiotic resistance in bacteria even in the absence

i. Co-resistance: when antibiotics and these metals co-exist within the same environment, these metals influence some antibiotic resistant bacteria to survive in more polluted environment. Co-resistance occurs when two or more different resistant genes are present on the same genetic elements (plasmid, transposon, Integron) or are present within the same bacterial strain which provides resistant to different compounds. The rise of antibiotic resistant genes is directly correlated with the concentration of

ii. Cross-resistance: cross resistance occurs when antibiotics and potentially toxic metals target the same microbes leading to generic detoxification of genes by reducing intracellular concentration of antibiotics and metals, and

Besides these two mechanisms, co-selection is additionally promoted by coregulatory mechanism which occurs when different resistant genes is controlled by one regulator gene [88]. The impact of the potentially toxic metals on the antibiotic

World Health Organization has reported that globally in the year 2015 approximately 8.8 million deaths were due to cancer, presence of potentially toxic metals beyond permissible limits within the environment is one among the main factors of the death because the endocrine system is disrupted by these metals. When the food or drinking water containing potentially toxic metals beyond their maximum tolerance concentration is ingested, the metabolism of living cells in the body is negatively affected [122]. The immune and hematopoietic systems in human and animals also are adversely affected on exposure to the mixtures of those metals [122]. Li et al. [123] reported that the main cause for human bone diseases is the presence of the potentially toxic metals beyond their permissible limit within the aquatic environment. Potentially toxic metals Pb, Hg, Cd, As, and Cr in living cells causes cytotoxicity [124] and oxidative stress [124], leading to the damages of antioxidants, enzyme inhibition, apoptosis (programmed cell death), loss of DNA repair mechanism, protein dysfunction, and damage to lipid peroxidase and of the

Cadmium in human causes Itai-Itai disease, liver/kidney lesions, hepato-colic effects [125], carcinoma, prostatic adenocarcinoma, osteoporosis, hypertension, disorder, and kidney lesions including enlargement, nuclear, and mitochondrial damages, and histological changes; decreased antioxidant power of kidney also

enhanced efflux occurs during cross-resistance [87].


**Toxic metal**

Pb Lead induces

mutagenicity, inhibits enzyme activities and transcription. Pb in the bacteria destroys the nucleic acid.

*DOI: http://dx.doi.org/10.5772/intechopen.92141*

Ni Ni replaces the essential metals from metalloprotein. The activity of the enzymes is retarded as Ni binds the catalytic site of the enzyme. Ni causes oxidative stress which results in the enhanced DNA damage, protein impairment, and lipid peroxidation.

As Disrupts enzymatic

utilization.

Co Produces non B12 cobalt protein.

**Table 4.**

**81**

functions in the cell and interferes in the phosphate uptake and

**Mechanism of action Mechanism of**

*Health Risks of Potentially Toxic Metals Contaminated Water*

**resistance**

Resistance mechanism is due to adsorption of lead by extracellular polysaccharides, cell exclusion and ion efflux to the cell exterior.

The resistance mechanism is due to energy-dependent Ni efflux pump induced by cnr which is promoted by a chemo-osmotic proton-antiporter system.

Activation of efflux pumps due to cross resistance between arsenic and antibiotics is the main mechanism

The gene Czc, affects the inner and outer membranes and removes the cobalt from the cytoplasm

*Impact of potentially toxic metals on some bacterial strain resistant to antibiotics.*

**Antibiotic categories / generic names**

Macrolide, Quinolone

Aminoglycoside *Citrobacter* spp. [104]

Penicillin *Enterobacter* spp. [107]

Teicoplanin *E. faecium* [105] Quinolone *Klebsiella* spp. [106] Sulphonamide *Proteus* spp. [106**]** Vancomycin *Enterobacter* spp.;

Amphenicol *Salmonella* Spp. [114] Fluoroquinolone *P. aeruginosa* [115] Tetracycline *Shigella* spp. [116]

Quinolone *E. coli* [111] Tetracycline Soil bacteria of

Penicillin *Salmonella* spp. [102]

Amphenicol *E. faecium* [105] Sulfonamide *Citrobacter* spp. [112]

Penicillin *E. coli* [107] Sulfonamide *Salmonella* spp. [117]

Tetracycline *E. coli* [118] Amphenicol *Salmonella* spp. [119]

Aminoglycoside *Citrobacter* spp. [106]

Macrolide *Enterobacter* spp. [120]

Penicillin *Salmonella* spp. [102]

*Klebsiella* spp. [106]

Quinolone *E. coli* [111] Sulfamethoxazole *P. aeruginosa* [121**]** Vancomycin *Enterobacter* spp.;

**Pathogen(s)**

*Klebsiella* spp. [106]

Scotland [90]

*E. coli* [111]


#### *Health Risks of Potentially Toxic Metals Contaminated Water DOI: http://dx.doi.org/10.5772/intechopen.92141*

**Toxic metal**

Zn Due to affinity of Zn to thiol group, the Zn metal is toxic to bacteria, retards glycolysis,

*Heavy Metal Toxicity in Public Health*

inhibition.

Cr Due to strong oxidizing potential the metal Cr damages the cells of the microbes,inhibits the oxygen uptake, growth and elongation of lag

Cd Cd in bacteria denatures the protein, interacts with calcium metabolism, damages the cell membrane, hinders cell division and transcription, and also affects the nucleic acid.

**80**

phase.

transmembrane proton translocation and acid tolerance in the bacterial cell. Also decreases the biomass causing growth

**Mechanism of action Mechanism of**

**resistance**

Resistance to Zn is found in Gram negative and gram positive bacteria and resistance is mainly via *czr*C gene

Cr in microbes affects basal energy metabolism, protein oxidative stress protection, DNA repair, detoxification of enzymes, efflux pumps,

homeoostasis

Resistance to Cd in Gram negative bacteria affects Czc and Ncc genes and encoding dsbA gene needed for disulphite formation while in Gram positive bacteria resistance to Cd is with the CdA pump

**Antibiotic categories / generic names**

Norfloxacin, Augmentin, Gentamicin, Ampicillin

Penicillin, Teicoplanin

Fluoroquinolone, quinolone

Tetracycline, Carbapenem

Cephalosporin, Tetracycline

Nitrofuran, Teicoplanin

Quinolone, Vancomycin

Augmentin, Ampicillin

Fluoroquinolone, Quinolone

Penicillin, Tetracycline

Amphenicol, Cephalosporin Methicillin, Sulfonamide, Aminoglycoside

Aminoglycoside *E. coli*; *Citrobacter*

Vancomycin *E. faecium* [100]

Carbapenem *Pseudomonas*

Methicillin *Staphylococcus*

Penicillin *Enterobacter* spp. [97]

Sulfonamide *Klebsiella* spp. [112]

[96**]**

[101]

[112]

Macrolide *E. faecium* [98]

Ampicillin Bacteria

**Pathogen(s)**

spp.; *Enterobacter* spp.; *Klebsiella* spp.; *Salmonella* spp.; *P. aeruginosa* [108]

Bacteria isolated from the soil of Kenya [109]

*aeruginosa* [94]

*Salmonella* spp. [102]

*E. coli*; *Citrobacter* spp. [101]

*aureus* [110]

Soil bacteria of Scotland [90**]**

*P. aeruginosa* [97]

*Salmonella* spp. [102]

Bacteria isolated from the soil of Kenya [109]

isolated from ship

*E. coli*; *Citrobacter* spp.; *Klebsiella* spp.

*P. aeruginosa* [113]

*E. coli*; *Citrobacter* spp.; *Klebsiella* spp.

*E. coli* [111]

**Table 4.**

*Impact of potentially toxic metals on some bacterial strain resistant to antibiotics.*

disrupts mineral balance within the body, causing dysfunctions of sexual glands and skeltel diseases. A psychomotor function of the brain is bogged down in the presence of Cd. The toxicity of cadmium to cell is because Cd can displace vitamin C and E from their metabolically active sites, decrease in absorption of calcium by intestine and enhanced dissolution of bone calcium causing disorder in the normal bone metabolism processes. Cd an endocrine disrupter causes neuro-developmental toxicity. Toxicity of Cd in fishes includes immune suppression and immune dysfunction.

bladder cancer also is caused by As. The prolonged exposure to arsenic also affects

Mercury is the third top hazardous substance. Aquatic organisms convert inorganic mercury to methyl mercury which inactivates Na+/K+ ATPase. Hg with the production of reactive oxygen causes neurotoxic effects in human including death of neuronal cells, cognitive dysfunction, and Alzheimer's disease. As Hg is an endocrine disrupter, during pregnancy exposure to metal causes long-term damages to new born as mercury disrupts the influences the maternal-fetal balance. Minamata disease, renal toxicity, skin, nose irritation, damage to central systema nervosum,

hearing speech, and visual disorders are another health risks to human.

Copper an integral part of several enzymes in small amount (0.9 mg daily uptake) is an essential metal for animals and plants. Deficiency of copper in human causes anemia, a low number of leucocytes, defects in animal tissue, and osteoporosis in infants. The copper within the body beyond its permissible limit causes hematemesis, jaundice, melena, damage to central nervous system, liver, and kidney problems. Wilson's disease a genetic disease is additionally caused by copper.

Nickel, a natural occurring metal, exists in a number of mineral forms and is an ingredient of chocolate, steel, and other metal products, pigments, valves and of batteries. Excess uptake of nickel by human causes asthma, pneumonia, allergies, heart disorder, skin rashes, and miscarriage. Chances of development of carcinoma, nose cancer, larynx cancer, and prostatic adenocarcinoma also are enhanced.

Cobalt is an essential metal for the life as it is the integral part of vitamin B12 (cobalamin). Human when exposed to the higher concentration of cobalt causes decreased pulmonary function, asthma, interstitial lung disease, wheezing, and dyspnoea and reduces pulmonary function. Respiratory tract hyperplasia, pulmonary fibrosis, increase in number of red blood cells, emphysema, paralysis of the systema nervosum, seizures, growth retardation, and thyroid deficiency are diseases occurs in human at a really high concentration of the metal.

As zinc plays a crucial role in number of metallo enzymes viz., dehydrogenase, alkaline phosphatase, carbonic anhydrase, leucine amino peptidase, superoxide dismutase, and deoxyribosenucleic acid (DNA) and ribosenucleic acid (RNA) polymerase is an essential metal in humans and animals. Over exposures to zinc in human causes dry or pharyngitis, chest tightness, headache, increased indices of pulmonary inflammation, nausea, decrease in the activity of copper metallo enzyme, decreased HDL-cholesterol level, immuno toxicity, and gastrointestinal

central systema nervosum.

*Health Risks of Potentially Toxic Metals Contaminated Water*

*DOI: http://dx.doi.org/10.5772/intechopen.92141*

**8.5 Mercury**

**8.6 Copper**

**8.7 Nickel**

**8.8 Cobalt**

**8.9 Zinc**

effects.

**83**

#### **8.2 Lead**

Out of about four million tons of lead used per annum globally about three million tons is discharged within the environment. In the physical body, 90–95% of the intruding lead is accumulated within the bones which combine with bone minerals and organic matter causes rise in the blood lead level. Accumulation of lead within the bones affects the acid-base equilibrium causing calcium deficiency. Pb within the body lowers the active vitamin D3 level and parathyroid level within the plasma affecting somatic cell function viz., decrease in the secretion of γcarboxyglutamic acid containing protein. Clinical studies have shown that if the extent of Pb in the drinking water is 50 μg/L, the blood lead level within the human is going to be about 30 μg/L; if the extent increased further, the lead level within the breast fed babies are enhanced causing hindrance within the bone development. If the blood lead level in children exceeds 75 μg/L, it causes coma, convulsions, and eventually death. Pb also affects central systema nervosum, renal, cardiovascular, neurological, and musculoskeletal systems. Pb influences the heme synthesizing enzymes by replacing Zn within the heme synthesis. Lead disrupts biosynthesis of hemoglobin, metabolism of Fe, Zn, and Cu, and of vitamin D within the body, and also causes cognitive impairment. Pb in physical body also acts as nephrotoxicants. Lead in fish's body also affects immune system [122]. Long-term exposure to the low concentration of a mix of Cd, As, and Pb in human and other animal cause hepatotoxic (damage to the liver) effects [123].

#### **8.3 Chromium**

The annual output of Cr globally is approximately 7.5 million tons. The secretion of Collagen-Type I which helps in the bone fracture healing is suppressed in the presence of chromium ion. Chromium in human causes nose ulcers, asthma, DNA damage, hemolysis, damage to liver, kidney, and carcinoma.

#### **8.4 Arsenic**

Smith et al. [126] after their research studies reported that if the person get 50 μg/L of arsenic (daily) then 13 out of 1000 individuals will suffer with lung, liver, kidney or bladder cancer. Skin lesions are by the uptake of 0.0012 mg/kg/day of arsenic. Bhattacharya et al. [125] found that low concentration of arsenic for long period damages liver in human and other animals. Enlargement of kidney, nuclear, and mitochondrial damages, histological changes, and decreased antioxidant power of kidney is additionally caused by arsenic in human. Arsenic also causes neurotoxic effects in human with the assembly of the reactive oxygen species which incorporates death of neuronal cells, cognitive dysfunction, and Alzheimer's disease. Cognitive impairment, deafness, hypertension, anemia dementia, hematemesis, and

bladder cancer also is caused by As. The prolonged exposure to arsenic also affects central systema nervosum.

#### **8.5 Mercury**

disrupts mineral balance within the body, causing dysfunctions of sexual glands and skeltel diseases. A psychomotor function of the brain is bogged down in the presence of Cd. The toxicity of cadmium to cell is because Cd can displace vitamin C and E from their metabolically active sites, decrease in absorption of calcium by intestine and enhanced dissolution of bone calcium causing disorder in the normal bone metabolism processes. Cd an endocrine disrupter causes neuro-developmental

toxicity. Toxicity of Cd in fishes includes immune suppression and immune

Out of about four million tons of lead used per annum globally about three million tons is discharged within the environment. In the physical body, 90–95% of the intruding lead is accumulated within the bones which combine with bone minerals and organic matter causes rise in the blood lead level. Accumulation of lead within the bones affects the acid-base equilibrium causing calcium deficiency. Pb within the body lowers the active vitamin D3 level and parathyroid level within the plasma affecting somatic cell function viz., decrease in the secretion of γcarboxyglutamic acid containing protein. Clinical studies have shown that if the extent of Pb in the drinking water is 50 μg/L, the blood lead level within the human is going to be about 30 μg/L; if the extent increased further, the lead level within the breast fed babies are enhanced causing hindrance within the bone development. If the blood lead level in children exceeds 75 μg/L, it causes coma, convulsions, and eventually death. Pb also affects central systema nervosum, renal, cardiovascular, neurological, and musculoskeletal systems. Pb influences the heme synthesizing enzymes by replacing Zn within the heme synthesis. Lead disrupts biosynthesis of hemoglobin, metabolism of Fe, Zn, and Cu, and of vitamin D within the body, and also causes cognitive impairment. Pb in physical body also acts as nephrotoxicants. Lead in fish's body also affects immune system [122]. Long-term exposure to the low concentration of a mix of Cd, As, and Pb in human and other animal cause

The annual output of Cr globally is approximately 7.5 million tons. The secretion of Collagen-Type I which helps in the bone fracture healing is suppressed in the presence of chromium ion. Chromium in human causes nose ulcers, asthma, DNA

Smith et al. [126] after their research studies reported that if the person get 50 μg/L of arsenic (daily) then 13 out of 1000 individuals will suffer with lung, liver, kidney or bladder cancer. Skin lesions are by the uptake of 0.0012 mg/kg/day of arsenic. Bhattacharya et al. [125] found that low concentration of arsenic for long period damages liver in human and other animals. Enlargement of kidney, nuclear, and mitochondrial damages, histological changes, and decreased antioxidant power of kidney is additionally caused by arsenic in human. Arsenic also causes neurotoxic effects in human with the assembly of the reactive oxygen species which incorporates death of neuronal cells, cognitive dysfunction, and Alzheimer's disease. Cognitive impairment, deafness, hypertension, anemia dementia, hematemesis, and

hepatotoxic (damage to the liver) effects [123].

damage, hemolysis, damage to liver, kidney, and carcinoma.

dysfunction.

*Heavy Metal Toxicity in Public Health*

**8.3 Chromium**

**8.4 Arsenic**

**82**

**8.2 Lead**

Mercury is the third top hazardous substance. Aquatic organisms convert inorganic mercury to methyl mercury which inactivates Na+/K+ ATPase. Hg with the production of reactive oxygen causes neurotoxic effects in human including death of neuronal cells, cognitive dysfunction, and Alzheimer's disease. As Hg is an endocrine disrupter, during pregnancy exposure to metal causes long-term damages to new born as mercury disrupts the influences the maternal-fetal balance. Minamata disease, renal toxicity, skin, nose irritation, damage to central systema nervosum, hearing speech, and visual disorders are another health risks to human.

#### **8.6 Copper**

Copper an integral part of several enzymes in small amount (0.9 mg daily uptake) is an essential metal for animals and plants. Deficiency of copper in human causes anemia, a low number of leucocytes, defects in animal tissue, and osteoporosis in infants. The copper within the body beyond its permissible limit causes hematemesis, jaundice, melena, damage to central nervous system, liver, and kidney problems. Wilson's disease a genetic disease is additionally caused by copper.

#### **8.7 Nickel**

Nickel, a natural occurring metal, exists in a number of mineral forms and is an ingredient of chocolate, steel, and other metal products, pigments, valves and of batteries. Excess uptake of nickel by human causes asthma, pneumonia, allergies, heart disorder, skin rashes, and miscarriage. Chances of development of carcinoma, nose cancer, larynx cancer, and prostatic adenocarcinoma also are enhanced.

#### **8.8 Cobalt**

Cobalt is an essential metal for the life as it is the integral part of vitamin B12 (cobalamin). Human when exposed to the higher concentration of cobalt causes decreased pulmonary function, asthma, interstitial lung disease, wheezing, and dyspnoea and reduces pulmonary function. Respiratory tract hyperplasia, pulmonary fibrosis, increase in number of red blood cells, emphysema, paralysis of the systema nervosum, seizures, growth retardation, and thyroid deficiency are diseases occurs in human at a really high concentration of the metal.

#### **8.9 Zinc**

As zinc plays a crucial role in number of metallo enzymes viz., dehydrogenase, alkaline phosphatase, carbonic anhydrase, leucine amino peptidase, superoxide dismutase, and deoxyribosenucleic acid (DNA) and ribosenucleic acid (RNA) polymerase is an essential metal in humans and animals. Over exposures to zinc in human causes dry or pharyngitis, chest tightness, headache, increased indices of pulmonary inflammation, nausea, decrease in the activity of copper metallo enzyme, decreased HDL-cholesterol level, immuno toxicity, and gastrointestinal effects.

### **9. Conclusions**

• Contamination of ground and surface water by potentially toxic metals is a worldwide problem.

**References**

[1] Li L, Yang X. The essential element manganese, oxidative stress, and metabolic diseases: Links and

*DOI: http://dx.doi.org/10.5772/intechopen.92141*

*Health Risks of Potentially Toxic Metals Contaminated Water*

on soil and plant grown. Waste Management. 2017;**64**:117-132. DOI: 10.1016/j.wasman.2017.03.002

[9] Galitskaya IV, Rama Mohan K, Keshav Krishna A, Batral GI, Eremina ON, Putilina VS, et al. Assessment of soil and groundwater contamination by heavy metals and metalloids in Russian and Indian megacities. Procedia Earth and Planetary Science. 2017;**17**:674-677

[10] Rezania S, Taib SM, Md Din MF,

phytotechnology: Heavy metals removal by diverse aquatic plants species from wastewater. Journal of Hazardous Materials. 2016;**318**:587-599

[11] Gbogbo F, Arthur-Yartel A, Bondzie JA, Dorleku WP, Dadzie S, Kwansa-Bentum B, et al. Risk of heavy metal ingestion from the consumption of two commercially valuable species of fish from the fresh and coastal waters of Ghana. PLoS One. 2018;**13**(3):e0194682. DOI: 10.1371/journal.pone.0194682

[12] Ahmed MK, Parvin E, Islam MM, Akter MS, Khan S, Al-Mamun MH. Lead- and cadmium-induced

histopathological changes in gill, kidney and liver tissue of freshwater climbing perch *Anabas testudineus* (Bloch, 1792). Chemistry and Ecology. 2014;**30**:

Concentration and pollution assessment

[14] Kulkarni HV, Mladenov N, Datta S, Chatterjee D. Influence of monsoonal recharge on arsenic and dissolved organic matter in the Holocene and Pleistocene aquifers of the Bengal Basin.

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interactions. Oxidative Medicine and Cellular Longevity. 2018;**2018**:7580707.

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[3] Ali H, Khan E, Ilahi I. Environmental

hazardous heavy metals: Environmental

bioaccumulation. Journal of Chemistry. 2019;**2019**:6730305. DOI: 10.1155/2019/

[5] Nizami G, Rehman S. Assessment of heavy metals and their effects on quality of water of rivers of Uttar Pradesh, India: A review. Environmental

Toxicology and Chemistry. 2018;**2**:65-71

[6] Paul D. Research on heavy metal pollution of river ganga: A review. Annals of Agrarian Science. 2017;**15**:

[7] Toth G, Hermann T, Da Silva MR, Montanerella L. Heavy metals in agricultural soils of the European Union with implications for food safety. Environment International. 2016;**88**:

[8] Sharma B, Sarkar A, Singh P, Singh RP. Agricultural utilization of biosolids: A review on potential effects

chemistry and ecotoxicology of

[4] Jiao Z, Li H, Song M, Wang L. Ecological risk assessment of heavy metals in water and sediment of the Pearl River estuary, China. Materials Science and Engineering. 2018;**394**: 052055. DOI: 10.1088/1757-899X/394/5/

persistence, toxicity, and

6730305

052055

278-286

299-309

**85**

DOI: 10.1155/2018/.580707


### **Declaration**

No original data is utilized in this review; all information is accessed from published work.

#### **Author details**

Om Prakash Bansal Chemistry Department, D.S. College, Aligarh, India

\*Address all correspondence to: drop1955@gmail.com

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Health Risks of Potentially Toxic Metals Contaminated Water DOI: http://dx.doi.org/10.5772/intechopen.92141*

#### **References**

**9. Conclusions**

aquifers.

citizenry.

**Declaration**

published work.

**Author details**

**84**

Om Prakash Bansal

Chemistry Department, D.S. College, Aligarh, India

\*Address all correspondence to: drop1955@gmail.com

provided the original work is properly cited.

worldwide problem.

*Heavy Metal Toxicity in Public Health*

abnormalities in the children.

• Contamination of ground and surface water by potentially toxic metals is a

• The major route of the groundwater and aquatic contamination by potentially toxic metals are the leaching from toxic industrial waste dumps, municipal landfills, and leaching of agricultural chemicals from soils into the upper

• Potentially toxic metals contaminated vegetables and fruits; fishes, seafood, and drinking water are the most sources of the ingestion of those metals by the

• A number of biological and biochemical processes are disrupted in the physical body by accumulation of those metals. These metals also cause developmental

• These potentially toxic metals promote the spread of antibiotic resistant genes

No original data is utilized in this review; all information is accessed from

© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

which causes the ineffectiveness of broad- spectrum antibiotics.

[1] Li L, Yang X. The essential element manganese, oxidative stress, and metabolic diseases: Links and interactions. Oxidative Medicine and Cellular Longevity. 2018;**2018**:7580707. DOI: 10.1155/2018/.580707

[2] Zwolak A, Sarzyńska M, Szpyrka E, Stawarczyk K. Sources of soil pollution by heavy metals and their accumulation in vegetables: A review. Water, Air, and Soil Pollution. 2019;**230**:164. DOI: 10.1007/s11270-019-4221-y

[3] Ali H, Khan E, Ilahi I. Environmental chemistry and ecotoxicology of hazardous heavy metals: Environmental persistence, toxicity, and bioaccumulation. Journal of Chemistry. 2019;**2019**:6730305. DOI: 10.1155/2019/ 6730305

[4] Jiao Z, Li H, Song M, Wang L. Ecological risk assessment of heavy metals in water and sediment of the Pearl River estuary, China. Materials Science and Engineering. 2018;**394**: 052055. DOI: 10.1088/1757-899X/394/5/ 052055

[5] Nizami G, Rehman S. Assessment of heavy metals and their effects on quality of water of rivers of Uttar Pradesh, India: A review. Environmental Toxicology and Chemistry. 2018;**2**:65-71

[6] Paul D. Research on heavy metal pollution of river ganga: A review. Annals of Agrarian Science. 2017;**15**: 278-286

[7] Toth G, Hermann T, Da Silva MR, Montanerella L. Heavy metals in agricultural soils of the European Union with implications for food safety. Environment International. 2016;**88**: 299-309

[8] Sharma B, Sarkar A, Singh P, Singh RP. Agricultural utilization of biosolids: A review on potential effects on soil and plant grown. Waste Management. 2017;**64**:117-132. DOI: 10.1016/j.wasman.2017.03.002

[9] Galitskaya IV, Rama Mohan K, Keshav Krishna A, Batral GI, Eremina ON, Putilina VS, et al. Assessment of soil and groundwater contamination by heavy metals and metalloids in Russian and Indian megacities. Procedia Earth and Planetary Science. 2017;**17**:674-677

[10] Rezania S, Taib SM, Md Din MF, Dahalan FA, Kamyab H. Comprehensive review on phytotechnology: Heavy metals removal by diverse aquatic plants species from wastewater. Journal of Hazardous Materials. 2016;**318**:587-599

[11] Gbogbo F, Arthur-Yartel A, Bondzie JA, Dorleku WP, Dadzie S, Kwansa-Bentum B, et al. Risk of heavy metal ingestion from the consumption of two commercially valuable species of fish from the fresh and coastal waters of Ghana. PLoS One. 2018;**13**(3):e0194682. DOI: 10.1371/journal.pone.0194682

[12] Ahmed MK, Parvin E, Islam MM, Akter MS, Khan S, Al-Mamun MH. Lead- and cadmium-induced histopathological changes in gill, kidney and liver tissue of freshwater climbing perch *Anabas testudineus* (Bloch, 1792). Chemistry and Ecology. 2014;**30**: 532-540

[13] Wei J, Duan M, Li Y, et al. Concentration and pollution assessment of heavy metals within surface sediments of the Raohe Basin, China. The Scientific Reporters. 2019;**9**:13100. DOI: 10.1038/s 415 98-019-49724-7

[14] Kulkarni HV, Mladenov N, Datta S, Chatterjee D. Influence of monsoonal recharge on arsenic and dissolved organic matter in the Holocene and Pleistocene aquifers of the Bengal Basin. Science of the Total Environment. 2018; **637–638**:588-599. DOI: 10.1016/j. scitonev.2018.05.009

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[16] Jiang Z, Li P, Tu J, Wei D, Zhang R, Wang Y, et al. Arsenic in geothermal systems of Tengchong, China: Potential contamination on freshwater resources. International Biodeterioration & Biodegradation. 2018;**128**:28-35. DOI: 10.1016/j.ibiod.2016.05.013

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[82] Mehouel F, Bouayad L,

Hammoudi AH, Ayadi Q, Regad F. Evaluation of the heavy metals (mercury, lead, and cadmium)

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Coimbatore district, Tamilnadu, India. International Research Journal of Pharmacy. 2017;**8**(1):41-45. DOI: 10.7897/2230-8407.08018

[71] Leung HM, Leung AO, Wang HS,

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[73] Reyahi-Khoram M, Setayesh-Shiri F, Cheraghi M. Study of the heavy metals (Cd and Pb) content in the tissues of rainbow trouts from Hamedan

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[74] Sulieman HMA, Suliman EAM. Appraisal of heavy metal levels in some marine organisms gathered from the Vellar and Uppanar estuaries southeast coast of Indian Ocean. Journal of Taibah University for Science. 2019;**13**:

[75] Nasyitah SN, Ahmad ZA,

Khairul NM, Ley JL, Kyoung-Woong K. Bioaccumulation of heavy metals in Maricultured fish, *Lates calcarifer* (Barramudi), *Lutjanus campechanus* (red snapper) and *Lutjanus griseus* (Grey snapper). Chemosphere. 2018;**197**:

338-343

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*niloticus* collected from lakes of

2015;**4**:511-520

[83] Bashir FA, Alhemmali EM. Analysis of some heavy metal in marine fish in muscle, liver and gill tissue in two marine fish spices from Kapar coastal waters, Malaysia. In: The Second Symposium on Theories and Applications of Basic and Biosciences. Misrata, Libya; 2015

[84] Rosli MNR, Samat SB, Yasir MS, Yusof MFM. Analysis of heavy metal accumulation in fish at Terengganu coastal area, Malaysia. Sains Malaysiana. 2018;**47**(6):1277-1283. DOI: 10.17576/ jsm-2018-4706-24

[85] Olusola JO, Festus AA. Assessment of heavy metals in some marine fish species relevant to their concentration in water and sediment from coastal waters of Ondo state, Nigeria. Journal of Marine Science: Research & Development. 2015;**5**:163. DOI: 10.4172/ 2155-9910.1000163

[86] Bandpei AM, Bay A, Zafarzadeh A, Hassanzadeh V. Bioaccumulation of heavy metals muscle of common carp fish (Cyprinus carpio L, 1758) from Ala gul and Alma gul wetlands of Golestan and consumption risk assessment. International Journal of Medical Research & Health Science. 2016;**5**:267-273

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[88] Pal C, Asiani K, Arya S, Rensing C, Stek DJ, DGJ L, et al. Metal resistance and its association with antibiotic resistance. Advances in Microbial Physiology. 2017;**70**:261-301. DOI: 10.1016/bs.ampbs.2017.02.001

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[102] Ali NM, Mazhar SA, Mazhar B, Imtiaz A, Andleeb S. Antibacterial activity of different plant extracts and antibiotics on pathogenic bacterial isolates from wheat field water. Pakistan Journal of Pharmaceutical Sciences. 2017;**30**(4):1321-1325

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[103] Mirzaei N, Rastegari H, Kargar M. Antibiotic resistance pattern among gram negative mercury resistant bacteria isolated from contaminated environments. Jundishapur Journal of Microbiology. 2013;**6**(10):634-639

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Health. 2017;**15**(4):566-579

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2017;**30**(4):1321-1325

1474.1482

939-946

Characterization of heavy metal and antibiotic resistant bacteria isolated from Aliaga ship dismantling zone, eastern Aegean Sea, Turkey.

International Journal of Environmental

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[101] Anssour L, Messai Y, Estepa V, Torres C, Bakour R. Characteristics of

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Research. 2013;**7**(4):895-902

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[114] Mourão J, Marçal S, Ramos P, Campos J, Machado J, Peixe L, et al. Tolerance to multiple metal stressors in emerging non-typhoidal MDR *Salmonella* serotypes: A relevant role for copper in anaerobic conditions. The Journal of Antimicrobial Chemotherapy. 2016;**71**(8):2147-2157

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[116] Manegabe BJ, Marie-Médiatrice NK, Barr Dewar J, Christian SB. Antibiotic resistance and tolerance to heavy metals demonstrated by environmental pathogenic bacteria isolated from the Kahwa River, Bukavu town, Democratic Republic of the Congo. International Journal of Environmental Studies. 2017; **74**(2):290-302

[117] Sandegren L, Linkevicius M, Lytsy B, Melhus A, Andersson DI. Transfer of an *Escherichia coli* ST131 multiresistance cassette has created a *Klebsiella pneumoniae*-specific plasmid associated with a major nosocomial outbreak. Journal of Antimicrobial Chemotherapy. 2012;**67**:74-83. DOI: 10.1093/jac/dkr40.5

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[121] Oyetibo GO, Ilori MO, Adebusoye SA, Obayori OS, Amund OO. Bacteria with dual resistance to elevated concentrations of heavy metals and antibiotics in Nigerian contaminated systems. Environmental Monitoring and Assessment. 2010;**168** (1–4):305-314

[122] Ojedokun AT, Bello OS. Sequestering heavy metals from wastewater using cow dung. Water Resources and Industry. 2016;**13**:7-13

[123] Li JJ, Li-Na P, Shan W, Meng-Da Z. Advances in the effect of heavy metals in aquatic environment on the health risks for bone. Earth and Environmental Science. 2018;**186**:012057. DOI: 10.1088/1755-1315/186/3/012057

[124] Hernández-García A, Romero D, Gómez-Ramírez P, María-Mojica P, Martínez-López E, García-Fernández AJ. In vitro evaluation of cell death induced by cadmium, lead and their binary mixtures on erythrocytes of common buzzard (*Buteo buteo*). Toxicology In Vitro. 2014;**28**:300-306. DOI: 10.1016/j.tiv.2013.11.005

[125] Bhattacharya PT, Misra SR, Mohsina Hussain M. Nutritional aspects of essential trace elements in oral health and disease: An extensive review. Scientifica. 2016;**2016**:5464373. DOI: 10.1155/2016/5464373

[126] Smith AH, Lingas EO, Rahman M. Contamination of drinking-water by arsenic in Bangladesh: A public health emergency. Bulletin of the World Health Organization. 2000;**78**(9): 1093-1103

**95**

**1. Introduction**

**Chapter 6**

**Abstract**

Toxicologic Characteristics of

Physical-Chemical Properties,

Biological Accumulation, and

Morphological-Functional

*and Marina Alexandrovna Zemlyanova*

*Nina Vladimirovna Zaitseva*

to 10.3–5.15 mg/kg via gastric tube.

exposure, long-term effects, inhalation and oral route

Nanodisperse Manganese Oxide:

Properties at Various Exposure Types

Nanosized manganese oxide has excellent prospects. Some data imply that its particles can be toxic when introduced in various ways, and it requires further examination of this nanomaterial. The authors conducted research of nanodisperse MnO2 water suspension at intragastric, inhalation, and skin-resorptive introduction into small rodents and obtained profound characteristics of its toxic effects, determined target organs and revealed dose-dependent effects. The substance was characterized with acute toxicity, and its bioaccumulation under long-term exposure caused morphofunctional disorders in brain, lipid peroxidation activation, and lower antioxidant system activity. The authors detected vessel hyperemia, subarachnoid hemorrhages, brain edema with perivascular and pericellular spaces dilatation, nerve fiber demyelinization, and focal dystrophic changes in vessels endothelium. After a long-term introduction in doses from 0.25 to 2.5 mg/kg, oxidizing-antioxidant imbalance occurred, neurotransmitters and electrolytes balance was violated, and there was also brush border epithelium insufficiency. Nanodisperse MnO2 water suspension in doses equal to 2.5 and 0.25 mg/kg at intragastric introduction into Wistar rats did not have embryotoxic or teratogenic effects. It did not have any mutagenic effects in doses equal to 10.3 and 5.15 mg/kg or gonadotoxic effects either when introduced into Wistar male rats in doses equal

**Keywords:** nanodisperse magnesium oxide, toxicity, acute exposure, chronic

Nowadays, one can see rapid growth in worldwide development and commercialization of nanoindustries and nanotechnological products in overall

#### **Chapter 6**

Amund OO. Bacteria with dual

*Heavy Metal Toxicity in Public Health*

[122] Ojedokun AT, Bello OS. Sequestering heavy metals from wastewater using cow dung. Water Resources and Industry. 2016;**13**:7-13

Science. 2018;**186**:012057. DOI: 10.1088/1755-1315/186/3/012057

Martínez-López E, García-

common buzzard (*Buteo buteo*). Toxicology In Vitro. 2014;**28**:300-306.

DOI: 10.1016/j.tiv.2013.11.005

[125] Bhattacharya PT, Misra SR,

10.1155/2016/5464373

1093-1103

**94**

Mohsina Hussain M. Nutritional aspects of essential trace elements in oral health and disease: An extensive review. Scientifica. 2016;**2016**:5464373. DOI:

[126] Smith AH, Lingas EO, Rahman M. Contamination of drinking-water by arsenic in Bangladesh: A public health emergency. Bulletin of the World Health Organization. 2000;**78**(9):

[124] Hernández-García A, Romero D, Gómez-Ramírez P, María-Mojica P,

Fernández AJ. In vitro evaluation of cell death induced by cadmium, lead and their binary mixtures on erythrocytes of

(1–4):305-314

resistance to elevated concentrations of heavy metals and antibiotics in Nigerian contaminated systems. Environmental Monitoring and Assessment. 2010;**168**

[123] Li JJ, Li-Na P, Shan W, Meng-Da Z. Advances in the effect of heavy metals in aquatic environment on the health risks for bone. Earth and Environmental

## Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties, Biological Accumulation, and Morphological-Functional Properties at Various Exposure Types

*Nina Vladimirovna Zaitseva and Marina Alexandrovna Zemlyanova*

#### **Abstract**

Nanosized manganese oxide has excellent prospects. Some data imply that its particles can be toxic when introduced in various ways, and it requires further examination of this nanomaterial. The authors conducted research of nanodisperse MnO2 water suspension at intragastric, inhalation, and skin-resorptive introduction into small rodents and obtained profound characteristics of its toxic effects, determined target organs and revealed dose-dependent effects. The substance was characterized with acute toxicity, and its bioaccumulation under long-term exposure caused morphofunctional disorders in brain, lipid peroxidation activation, and lower antioxidant system activity. The authors detected vessel hyperemia, subarachnoid hemorrhages, brain edema with perivascular and pericellular spaces dilatation, nerve fiber demyelinization, and focal dystrophic changes in vessels endothelium. After a long-term introduction in doses from 0.25 to 2.5 mg/kg, oxidizing-antioxidant imbalance occurred, neurotransmitters and electrolytes balance was violated, and there was also brush border epithelium insufficiency. Nanodisperse MnO2 water suspension in doses equal to 2.5 and 0.25 mg/kg at intragastric introduction into Wistar rats did not have embryotoxic or teratogenic effects. It did not have any mutagenic effects in doses equal to 10.3 and 5.15 mg/kg or gonadotoxic effects either when introduced into Wistar male rats in doses equal to 10.3–5.15 mg/kg via gastric tube.

**Keywords:** nanodisperse magnesium oxide, toxicity, acute exposure, chronic exposure, long-term effects, inhalation and oral route

#### **1. Introduction**

Nowadays, one can see rapid growth in worldwide development and commercialization of nanoindustries and nanotechnological products in overall

production chain; such products and technologies are considered to belong to a market segment of new technologies (the sixth technological structure) [1]. As per US Congressional Research Service experts assessment, world market of finished products and goods containing nanocomponents and nanomaterials now amounts to more than 1 trillion US dollars; it comprises more than 800 consumer goods produced with the use of nanotechnologies; by 2020, more than 15% of overall goods output in the world will be produced with the use of nanodevelopments, and the volume of this market in various sectors will be equal to more than 3 trillion US dollars. Aggregate volume of investment into scientific research and start-ups related to nanotechnologies received from various sources worldwide is estimated to amount to almost 20 billion US dollars. Annual nanoindustries market growth is expected to reach 20–30% [2]. In experts' opinion, nanoindustries and nanotechnologies (together with other technologies) are already facilitating transfer to new technological structure based on renewable energy sources, intellectual power engineering technologies, construction of completely new energy-efficient buildings, hydrogen technologies application, electrical and hybrid vehicle creation, 3D printer design and implementation, etc.

Nowadays, nanoclusters evolve quite intensely all over the world (more than 1700 cluster organizations in 260 European regions) and in Russia (more than 330 participating organizations). World market segments of nanomaterials develop as their commercial use in such key spheres as aerospace, health care, biotechnologies, power engineering, electronics and IT, processing industries, and consumer goods sector, grows rapidly [1, 3]. All this becomes apparent in the attitudes the European Union (EU) has toward the matter, declaring that nanotechnology is one of the Key Emerging Technologies 2020 Strategy. Its enormous potential for innovation has fostered large investments in developing new consumer products and industrial applications. The outlooks for a rapid growth in the sector have raised not only hopes and high expectations, but also societal concerns about the adequacy of nanotechnology regulation. Indeed, despite their clear benefits, engineered nanomaterials pose environmental and health risks [4].

All these processes prove the necessity to systematically examine potential dangers and threats for human activity, which are related to large-scale spread of nanotechnologies and nanobiotechnologies. In spite of all their undeniable innovative properties, nanomaterials including those containing metal nanoparticles may cause certain health risk at all stages of production and product consumption due to their specific physical-chemical properties. They may also be dangerous for human environment objects and lead to grave social and economic consequences.

The challenges that researchers encountered provoked the need to develop reliable methods for characterization of nanoparticles released from various product matrices into complex biological, environmental and food media, and for the assessment of their human and environmental exposure, hazard, and risk [4]. Special attention is paid to detecting correlation between physical properties (i.e., size, shape, surface structure, and aggregation degree) of nanomaterials and toxic response induction in biological structures. This research direction has been actively developed in Federal Scientific Center for Medical and Preventive Health Risk Management Technologies (Perm) for many years. Physical properties of a number of widely spread metal-oxide nanoparticles as well as peculiarities of their biological and toxic effects exerted at various exposure types have not been studied sufficiently; so the research goal is to systemize the knowledge on the subject and to make it more precise.

**97**

МnO2 aerosol.

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

**2. Utilization prospects for nanosized particles of manganese oxide and** 

Nanosized particles of manganese oxide (МnO2) represent a prospective nanomaterial, which can be used for creating high-technology components applicable in nanoelectronics, nano-optics, and nanochemistry [5]. Nowadays, nanodisperse МnO2 is used as an active component in portable power sources, solar batteries, electrical appliances, accelerators, and sorbents [6, 7]. A possibility to use threadlike nanosized particles of МnO2 for sensor electrodes creation is of particular interest for researchers [7]. Planned production of matrices based on nanosized МnO2 for nanomagnetic materials and sorbents, nanoaccelerators, and semiconductor thermistors, can reach up to 1000 tons per year and is considered to be "mass production" [8]. Here, direct exposure of workers involved in the production process, as well as population living in areas influenced by such production, becomes quite possible. A possibility that the substance is introduced into the atmosphere in the form of aerosol is determined by technological processes as nanoparticles of МnO2 are emitted in their course. Such processes include vacuum-ultrasound laser ablation applied in producing matrices of nanomagnetic materials and sorbents [9]. Laser ablation in suspension of nanodisperse МnO2 includes local impulse-continuous heating of the substance, sublimation, crystallization, and hydrodynamic processes, which cause formation of nanodisperse

Formation of nanodisperse МnO2 aerosol occurs in production of sensory electrodes, biosensors, [6], cathode accelerators, and semiconductor thermistors [7], when electrochemical deposition is applied; this process means covering graphite rods with stable hydrosol of disperse МnO2 nanoparticles in electroplating baths. Sewage formed in the processes contains МnO2, and they can get into surface wells. Today, aerosol of nanodisperse МnO2 can be found in civil engineering and chemical production facilities. Working area air at a production facility manufacturing potash fertilizers was examined; the research results proved that МnO2 particles were present in the air; thus, there were about 4435–7330 million/m3 particles sized 45–95 nm at a carrier driver workplace in a milling workshop (МnO2

operator workplace in a granulating workshop. Manganese particles were identified among those nanoparticles via mass-spectrometry technique with inductive-bound plasma; Agilent 7500cx mass-spectrometer with octopole reaction collision cell was applied (Agilent Technologies Inc., USA) [10]. There are some data implying that МnO2 particles can be toxic when introduced in various ways, and it requires

Detailed research on nanosized particles toxicity involves a wide range of tasks according to the recommendations set forth by Good laboratory practice (GLP): toxicology evaluation, single dose, repeated dose, toxicokinetics, genotoxicity, reproductive and developmental toxicology, and local toxicity. According to these recommendations, the authors have assessed toxicity of nanosized МnO2. All the animals before the experiment underwent 14-day quarantine and were placed in standard cages made of polypropylene, two animals in each. Cages were in a ventilated room. Air temperature in the room was constant and equal to 23.0 ± 2.0°

air humidity was 60.0 ± 5.0%. The animals received semisynthetic nutrition with food and biological value, which completely satisfied all physiological needs. They also had free access to food and water. All procedures and examinations on animals were performed in full conformity with guide for the care and use of laboratory

). Nanoparticles sized 5–25 nm

C, and

were registered at a machine

content in working area air was equal to 0.3 mg/m3

in concentrations equal to 4588–11,423 million/m3

further examination of this nanomaterial.

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

**sources of their introduction into environment**

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

#### **2. Utilization prospects for nanosized particles of manganese oxide and sources of their introduction into environment**

Nanosized particles of manganese oxide (МnO2) represent a prospective nanomaterial, which can be used for creating high-technology components applicable in nanoelectronics, nano-optics, and nanochemistry [5]. Nowadays, nanodisperse МnO2 is used as an active component in portable power sources, solar batteries, electrical appliances, accelerators, and sorbents [6, 7]. A possibility to use threadlike nanosized particles of МnO2 for sensor electrodes creation is of particular interest for researchers [7]. Planned production of matrices based on nanosized МnO2 for nanomagnetic materials and sorbents, nanoaccelerators, and semiconductor thermistors, can reach up to 1000 tons per year and is considered to be "mass production" [8]. Here, direct exposure of workers involved in the production process, as well as population living in areas influenced by such production, becomes quite possible. A possibility that the substance is introduced into the atmosphere in the form of aerosol is determined by technological processes as nanoparticles of МnO2 are emitted in their course. Such processes include vacuum-ultrasound laser ablation applied in producing matrices of nanomagnetic materials and sorbents [9]. Laser ablation in suspension of nanodisperse МnO2 includes local impulse-continuous heating of the substance, sublimation, crystallization, and hydrodynamic processes, which cause formation of nanodisperse МnO2 aerosol.

Formation of nanodisperse МnO2 aerosol occurs in production of sensory electrodes, biosensors, [6], cathode accelerators, and semiconductor thermistors [7], when electrochemical deposition is applied; this process means covering graphite rods with stable hydrosol of disperse МnO2 nanoparticles in electroplating baths. Sewage formed in the processes contains МnO2, and they can get into surface wells. Today, aerosol of nanodisperse МnO2 can be found in civil engineering and chemical production facilities. Working area air at a production facility manufacturing potash fertilizers was examined; the research results proved that МnO2 particles were present in the air; thus, there were about 4435–7330 million/m3 particles sized 45–95 nm at a carrier driver workplace in a milling workshop (МnO2 content in working area air was equal to 0.3 mg/m3 ). Nanoparticles sized 5–25 nm in concentrations equal to 4588–11,423 million/m3 were registered at a machine operator workplace in a granulating workshop. Manganese particles were identified among those nanoparticles via mass-spectrometry technique with inductive-bound plasma; Agilent 7500cx mass-spectrometer with octopole reaction collision cell was applied (Agilent Technologies Inc., USA) [10]. There are some data implying that МnO2 particles can be toxic when introduced in various ways, and it requires further examination of this nanomaterial.

Detailed research on nanosized particles toxicity involves a wide range of tasks according to the recommendations set forth by Good laboratory practice (GLP): toxicology evaluation, single dose, repeated dose, toxicokinetics, genotoxicity, reproductive and developmental toxicology, and local toxicity. According to these recommendations, the authors have assessed toxicity of nanosized МnO2. All the animals before the experiment underwent 14-day quarantine and were placed in standard cages made of polypropylene, two animals in each. Cages were in a ventilated room. Air temperature in the room was constant and equal to 23.0 ± 2.0° C, and air humidity was 60.0 ± 5.0%. The animals received semisynthetic nutrition with food and biological value, which completely satisfied all physiological needs. They also had free access to food and water. All procedures and examinations on animals were performed in full conformity with guide for the care and use of laboratory

*Heavy Metal Toxicity in Public Health*

implementation, etc.

consequences.

make it more precise.

materials pose environmental and health risks [4].

production chain; such products and technologies are considered to belong to a market segment of new technologies (the sixth technological structure) [1]. As per US Congressional Research Service experts assessment, world market of finished products and goods containing nanocomponents and nanomaterials now amounts to more than 1 trillion US dollars; it comprises more than 800 consumer goods produced with the use of nanotechnologies; by 2020, more than 15% of overall goods output in the world will be produced with the use of nanodevelopments, and the volume of this market in various sectors will be equal to more than 3 trillion US dollars. Aggregate volume of investment into scientific research and start-ups related to nanotechnologies received from various sources worldwide is estimated to amount to almost 20 billion US dollars. Annual nanoindustries market growth is expected to reach 20–30% [2]. In experts' opinion, nanoindustries and nanotechnologies (together with other technologies) are already facilitating transfer to new technological structure based on renewable energy sources, intellectual power engineering technologies, construction of completely new energy-efficient buildings, hydrogen technologies application, electrical and hybrid vehicle creation, 3D printer design and

Nowadays, nanoclusters evolve quite intensely all over the world (more than 1700 cluster organizations in 260 European regions) and in Russia (more than 330 participating organizations). World market segments of nanomaterials develop as their commercial use in such key spheres as aerospace, health care, biotechnologies, power engineering, electronics and IT, processing industries, and consumer goods sector, grows rapidly [1, 3]. All this becomes apparent in the attitudes the European Union (EU) has toward the matter, declaring that nanotechnology is one of the Key Emerging Technologies 2020 Strategy. Its enormous potential for innovation has fostered large investments in developing new consumer products and industrial applications. The outlooks for a rapid growth in the sector have raised not only hopes and high expectations, but also societal concerns about the adequacy of nanotechnology regulation. Indeed, despite their clear benefits, engineered nano-

All these processes prove the necessity to systematically examine potential dangers and threats for human activity, which are related to large-scale spread of nanotechnologies and nanobiotechnologies. In spite of all their undeniable innovative properties, nanomaterials including those containing metal nanoparticles may cause certain health risk at all stages of production and product

consumption due to their specific physical-chemical properties. They may also be dangerous for human environment objects and lead to grave social and economic

The challenges that researchers encountered provoked the need to develop reliable methods for characterization of nanoparticles released from various product matrices into complex biological, environmental and food media, and for the assessment of their human and environmental exposure, hazard, and risk [4]. Special attention is paid to detecting correlation between physical properties (i.e., size, shape, surface structure, and aggregation degree) of nanomaterials and toxic response induction in biological structures. This research direction has been actively developed in Federal Scientific Center for Medical and Preventive Health Risk Management Technologies (Perm) for many years. Physical properties of a number of widely spread metal-oxide nanoparticles as well as peculiarities of their biological and toxic effects exerted at various exposure types have not been studied sufficiently; so the research goal is to systemize the knowledge on the subject and to

**96**

animals (ILAR, DELS) [11] and requirements set forth by Ethics Committee of Federal Scientific Center for Medical and Preventive Health Risk Management Technologies.

#### **3. Results and discussion**

#### **3.1 Physical and chemical properties of nanodisperse magnesium oxide**

The following substances were tested during the experiment: water suspension of nanodisperse magnesium oxide (III, IV) (manganese (III, IV) oxide, CAS Registry Number 1313-13-09). IUPAC name: manganese oxide and manganese (III, IV) oxide. Synonyms: manganese dioxide, manganese binoxide, and manganese peroxide [12].

According to the results of studying the particle size and shape with independent methods, it is found that the МnO2 sample tested was a nanomaterial. This is evidenced by the study results of the water suspension test sample (МnO2 concentration is 36.0 ± 2.3 mg/cm3 ) with a residual CTAB in the suspension below the detection threshold (0.00001 mg/cm3 ). The particle size distribution (crosssectional dimension determined with dynamic laser light scattering) is represented in the bar chart as follows: 13 nm (1.2% of the total number of particles), 15–29 nm (94.4% of the total number of particles), and 33–100 nm (4.1% of the total number of particles). The maximum peak value of the particle size made 19 ± 4 nm (41.2% of the total number of particles) (**Table 1**).

Scanning electron microscopy revealed that the particles being visualized exceeded 20 nm in size. The difference with the previous method may be due to a failed focus of the scanning microscope on the nanoparticles smaller than 20 nm, despite sensitivity of 3–10 nm, as stated in its data sheet. As seen in particles are of filamentary shape (97.8% of the total number of particles). The determined sizes and the shape of particles are confirmed by atomic force microscopy (**Figures 1** and **2**).

The textural characteristics of resulting material studied showed that the adsorption-desorption isotherm of nitrogen corresponds to the type IV (isotherm with a distinct capillary condensation), and the shape of the hysteresis loop belongs to H3 type with the distinct area of mesopores filling within the range of relative pressures (*p/p0*) 0.7–1. In other words, mesopores filling at higher relative pressures verifies the presence of large diameter mesopores (**Figure 3**).

The maximum pore size distribution occurs in the range of ~10 nm. The specific surface area (SBET) of the nanosized particles, calculated by Brunauer et al. [13], amounted to 150.2 ± 2.6 m<sup>2</sup> /g. The total pore volume was equal to 0.676 cm3 /g (the total pores volume (Vtot) was calculated from the amount of nitrogen adsorbed at a relative pressure p/p0 ≈ 0.99. Pore size distribution was determined by the desorption isotherms by Barrett et al. [14]).

The water suspension of nanodisperse magnesium oxide had the following physicochemical characteristics: сhemical formula: MnO2, smiles: О═Mn═O, molar mass: 6.9368 g/mol, chemical composition of the nanosized phase: metal, presence of solvent: matrix-bidistilled water, particle charge: neutral at рН = 7.4, resistance to aggregation: particles are prone to aggregation, hydrophobicity: hydrophilic substance, Bp at 76 mm Hg: 3127°C, Bm: 1080°C; vapor tension (mm Hg) and volatility (mg/m3 20°C): undetermined, specific weight: 4.8 g/cm3 , water solubility: insoluble, oil/water ratio: undetermined, and aggregation state: in water at 20 and 35°C—dark brown solid substance, in air at 20 and 35°C—dark brown powder, of high strength and hardness.

**99**

**Figure 1.**

**Table 1.**

**introduction into a body**

*dispersive X-ray attachment for microanalysis (Bruker, Germany) [15].*

*analyzer Horiba LB-550 (Horiba, Japan) [14].*

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

**Size of particles in a suspension, micron Proportion of particles, %**

0.0131 1.2 0.0150 9.6 0.0171 16.4 0.0196 20.0 0.0225 21.2 0.0257 13.9 0.0295 13.2 0.0338 0.44 0.0387 0.38 0.0443 0.64 0.0507 0.90 0.0581 0.73 0.0666 0.42 0.0762 0.39 0.0873 0.15

**3.2 Acute toxicity study of nanodisperse manganese oxide at various types of** 

*Distribution of MnO2 nanoparticles in a water suspension versus particle size using dynamic light scattering* 

Over the last decade, a lot of researchers have dedicated their work to practical application of a priority nanomaterial, namely, nanodisperse МnO2 [16]. They are particularly interested in examining possibilities to use threadlike nanosized МnO2 particles for sensory electrodes creation [6, 7] or cathode accelerator creation [7] at

**3.2.1 Acute toxicity study of MnO2 nanoparticles under inhalation exposure**

*Scanning electron microscopy image of MnO2 nanoparticles, electron microscopy with a high-resolution scanning microscope (3–10 nm, max magnification of 300,000X) S-3400 N (Hitachi, Japan) with energy* 

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*


#### **Table 1.**

*Heavy Metal Toxicity in Public Health*

**3. Results and discussion**

concentration is 36.0 ± 2.3 mg/cm3

the detection threshold (0.00001 mg/cm3

of the total number of particles) (**Table 1**).

Technologies.

peroxide [12].

(**Figures 1** and **2**).

amounted to 150.2 ± 2.6 m<sup>2</sup>

volatility (mg/m3

high strength and hardness.

tion isotherms by Barrett et al. [14]).

animals (ILAR, DELS) [11] and requirements set forth by Ethics Committee of Federal Scientific Center for Medical and Preventive Health Risk Management

**3.1 Physical and chemical properties of nanodisperse magnesium oxide**

The following substances were tested during the experiment: water suspension of nanodisperse magnesium oxide (III, IV) (manganese (III, IV) oxide, CAS Registry Number 1313-13-09). IUPAC name: manganese oxide and manganese (III, IV) oxide. Synonyms: manganese dioxide, manganese binoxide, and manganese

According to the results of studying the particle size and shape with independent methods, it is found that the МnO2 sample tested was a nanomaterial. This is evidenced by the study results of the water suspension test sample (МnO2

sectional dimension determined with dynamic laser light scattering) is represented in the bar chart as follows: 13 nm (1.2% of the total number of particles), 15–29 nm (94.4% of the total number of particles), and 33–100 nm (4.1% of the total number of particles). The maximum peak value of the particle size made 19 ± 4 nm (41.2%

Scanning electron microscopy revealed that the particles being visualized exceeded 20 nm in size. The difference with the previous method may be due to a failed focus of the scanning microscope on the nanoparticles smaller than 20 nm, despite sensitivity of 3–10 nm, as stated in its data sheet. As seen in particles are of filamentary shape (97.8% of the total number of particles). The determined sizes and the shape of particles are confirmed by atomic force microscopy

The textural characteristics of resulting material studied showed that the adsorption-desorption isotherm of nitrogen corresponds to the type IV (isotherm with a distinct capillary condensation), and the shape of the hysteresis loop belongs to H3 type with the distinct area of mesopores filling within the range of relative pressures (*p/p0*) 0.7–1. In other words, mesopores filling at higher relative pressures

The maximum pore size distribution occurs in the range of ~10 nm. The specific surface area (SBET) of the nanosized particles, calculated by Brunauer et al. [13],

total pores volume (Vtot) was calculated from the amount of nitrogen adsorbed at a relative pressure p/p0 ≈ 0.99. Pore size distribution was determined by the desorp-

The water suspension of nanodisperse magnesium oxide had the following physicochemical characteristics: сhemical formula: MnO2, smiles: О═Mn═O, molar mass: 6.9368 g/mol, chemical composition of the nanosized phase: metal, presence of solvent: matrix-bidistilled water, particle charge: neutral at рН = 7.4, resistance to aggregation: particles are prone to aggregation, hydrophobicity: hydrophilic substance, Bp at 76 mm Hg: 3127°C, Bm: 1080°C; vapor tension (mm Hg) and

20°C): undetermined, specific weight: 4.8 g/cm3

insoluble, oil/water ratio: undetermined, and aggregation state: in water at 20 and 35°C—dark brown solid substance, in air at 20 and 35°C—dark brown powder, of

/g. The total pore volume was equal to 0.676 cm3

/g (the

, water solubility:

verifies the presence of large diameter mesopores (**Figure 3**).

) with a residual CTAB in the suspension below

). The particle size distribution (cross-

**98**

*Distribution of MnO2 nanoparticles in a water suspension versus particle size using dynamic light scattering analyzer Horiba LB-550 (Horiba, Japan) [14].*

#### **Figure 1.**

*Scanning electron microscopy image of MnO2 nanoparticles, electron microscopy with a high-resolution scanning microscope (3–10 nm, max magnification of 300,000X) S-3400 N (Hitachi, Japan) with energy dispersive X-ray attachment for microanalysis (Bruker, Germany) [15].*

#### **3.2 Acute toxicity study of nanodisperse manganese oxide at various types of introduction into a body**

#### **3.2.1 Acute toxicity study of MnO2 nanoparticles under inhalation exposure**

Over the last decade, a lot of researchers have dedicated their work to practical application of a priority nanomaterial, namely, nanodisperse МnO2 [16]. They are particularly interested in examining possibilities to use threadlike nanosized МnO2 particles for sensory electrodes creation [6, 7] or cathode accelerator creation [7] at

#### **Figure 2.**

*The 3D-pattern of the MnO2 nanodisperse particles, atomic force microscopy with the use of solver-PRO microscope (NT-MDT, Russian) [10].*

#### **Figure 3.**

*Nanodisperse MnO2: (a) nitrogen isotherm adsorption-desorption and (b) pore size distribution d (nm) [15].*

covering graphite rods via electrochemical deposition, up-to-date sorbents with the use of vacuum-ultrasound laser ablation [17, 18]. Inhalation exposure of workers to nanosized МnO2 particles is quite possible during such manufacturing processes as these particles are emitted into working area air. In relation to that, wider utilization of nanosized МnO2 in industrial production as well as providing workers' safety in the process requires more profound studies on toxicity of nanodisperse МnO2 when it enters a body being inhaled as an aerosol.

The authors used water suspension of nanodisperse MnO2 in concentration equal to 36.0 ± 2.3 mg/cm3 as an examined substance. To make comparison, microdisperse MnO2 with concentration in manganese water suspension equal to 40.31 ± 1.6 mg/cm3 was used. The particles size amounted to 5.5–37.0 μm (particles' share 67.0%). The size of microdisperse MnO2 particles is 194–1300 times greater than the nanodisperse MnO2 particles size. Specific surface area of MnO2 nanoparticles (Brunauer-Emmett-Teller technique [13]) was equal to 150.23 m<sup>2</sup> /g, which was 1.2 times higher than microparticle-specific surface area (130 m<sup>2</sup> /g). This property can cause high MnO2 nanoparticles reactivity *in vitro* and *in vivo* [19].

To determine acute toxicity parameters, the authors completed an experiment on pubescent Wistar rats (male and female) with body weight equal to 190 ± 10 g. They examined and assessed acute toxicity of nanodisperse MnO2 water suspension at inhalation introduction as an aerosol in accordance with Interstate Standard "Testing techniques on determining chemical products effects exerted on a human body: acute inhalation toxicity technique determining acute toxicity class

**101**

1.8 mg/m3

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

(ATC technique)" (OECD, Test No 436:2008, IDT) (2012). Inhalation introduction into the experimental animals' bodies was modeled in inhalation system with integrated software; and a chamber for a whole body was used in the process (TSE Systems GmbH, Germany). The experimental animals were divided into three groups (experimental groups were 1 and 2, and group 3 was a control one, n = 18 animals). Experimental group 1 was exposed to the examined substance inhalation at substance nominal concentration in the chamber equal to 0.05 mg/l; concentration for experimental group 2 was equal to 0.5 mg/l. The exposure lasted for 4 hours; the animals received no nutrition during the process. After inhalation exposure, the animals were observed during next 96 hours to detect possible delayed substance toxicity. Control group was exposed to inhalation of distilled water in the form of an aerosol under analogous conditions. The water conformed to TU 6-09-2502-77. After observation period for the animals from experimental groups 1 and 2 was over, they were taken out of the experiment via sparing euthanasia. Brains were extracted with a special instrument and fixed in 5% solution of buffered neutral formalin. Fixed tissue pieces were dehydrated in Excelsior ES automatic histological processor (Thermo Scientific, Germany). The finished specimens were examined in Axio Lab A1 lightoptical microscope, micropictures were taken with the use of Mikroskopkamera

AxioCam ERc 5 s (Carl Zeiss, Germany) at magnification equal to ×400.

MnO2 aerosol at actual concentration equal to 0.029 ± 0.001 mg/dm3

; for mice during 7 hours, 49 mg/m3

ized with evident neurotoxic effects, which started to occur in male and female rats from experimental group 1 after 3 hours of exposure. Animals had movement coordination disorders, took unusual postures, and showed weaker reaction to sound stimulus; all these effects remained in survivor animals during 48 hours after exposure. When actual concentration was equal to 0.472 ± 0.005 mg/dm3

started to suffer from apparent respiratory failure after 30 minutes of exposure. Most rats in experimental group 2 (three male and two female) died within 150–190 minutes after the experiment started; their death was caused by acute respiratory failure. One female rat died 2 hours after the experiment was over. The animals were sluggish before their death, they took lateral position, and demonstrated no reaction to sound stimulus or motion activity. Death of the experimental animals was not established. CL50 of the examined nanodisperse MnO2 amounted to 0.12 mg/l. This concentration is within 0.05–0.5 mg/l range, which allows to define the tested substance as having the second hazard class "ATC technique" (OECD, Test No. 436:2008, IDT). It is known that minimal toxic concentration (TCL0) of nanosized МnO2 for rats at inhalation introduction during 24 hours amounts to

dose (LDL0) for rats at intratracheal introduction amount to 45 mg/kg. Safety specification does not contain any additional information regarding errors, confidence limits, or animals sex. Morphologic changes in brain tissues of the rats from

The results of all research performed with independent techniques application helped to detect that the examined MnO2 sample was a nanomaterial. The examination of the tested substance suspension in concentration equal to 36.0 ± 2.3 mg/cm3 proves it. Particle size distribution on the bar graph is as follows (particles crosssection size is presented): 13 nm (1.2% of the total particles number), 15–29 nm (94.4%), and 33–100 nm (4.1%). Maximum peak of particles size corresponded to 19 ± 4 nm (41.2%). Scanning electron microscopy technique enabled detecting that visualized particles size exceeded 20 nm. Assessment of nanoparticles quantity in the inhalation chamber area showed that when nanodisperse fraction is fed into the chamber and transfers into aerosol, it does not agglomerate to micrometer range (**Figures 4** and **5**). The size of most particles (99% from the total quantity) does not exceed 100 nm at the examined actual concentrations after 2–4 hours of exposure. Clinical picture of acute intoxication at inhalation exposure to nanodisperse

was character-

(data taken from Minimal lethal

, rats

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

#### *Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

(ATC technique)" (OECD, Test No 436:2008, IDT) (2012). Inhalation introduction into the experimental animals' bodies was modeled in inhalation system with integrated software; and a chamber for a whole body was used in the process (TSE Systems GmbH, Germany). The experimental animals were divided into three groups (experimental groups were 1 and 2, and group 3 was a control one, n = 18 animals). Experimental group 1 was exposed to the examined substance inhalation at substance nominal concentration in the chamber equal to 0.05 mg/l; concentration for experimental group 2 was equal to 0.5 mg/l. The exposure lasted for 4 hours; the animals received no nutrition during the process. After inhalation exposure, the animals were observed during next 96 hours to detect possible delayed substance toxicity. Control group was exposed to inhalation of distilled water in the form of an aerosol under analogous conditions. The water conformed to TU 6-09-2502-77. After observation period for the animals from experimental groups 1 and 2 was over, they were taken out of the experiment via sparing euthanasia. Brains were extracted with a special instrument and fixed in 5% solution of buffered neutral formalin. Fixed tissue pieces were dehydrated in Excelsior ES automatic histological processor (Thermo Scientific, Germany). The finished specimens were examined in Axio Lab A1 lightoptical microscope, micropictures were taken with the use of Mikroskopkamera AxioCam ERc 5 s (Carl Zeiss, Germany) at magnification equal to ×400.

The results of all research performed with independent techniques application helped to detect that the examined MnO2 sample was a nanomaterial. The examination of the tested substance suspension in concentration equal to 36.0 ± 2.3 mg/cm3 proves it. Particle size distribution on the bar graph is as follows (particles crosssection size is presented): 13 nm (1.2% of the total particles number), 15–29 nm (94.4%), and 33–100 nm (4.1%). Maximum peak of particles size corresponded to 19 ± 4 nm (41.2%). Scanning electron microscopy technique enabled detecting that visualized particles size exceeded 20 nm. Assessment of nanoparticles quantity in the inhalation chamber area showed that when nanodisperse fraction is fed into the chamber and transfers into aerosol, it does not agglomerate to micrometer range (**Figures 4** and **5**). The size of most particles (99% from the total quantity) does not exceed 100 nm at the examined actual concentrations after 2–4 hours of exposure.

Clinical picture of acute intoxication at inhalation exposure to nanodisperse MnO2 aerosol at actual concentration equal to 0.029 ± 0.001 mg/dm3 was characterized with evident neurotoxic effects, which started to occur in male and female rats from experimental group 1 after 3 hours of exposure. Animals had movement coordination disorders, took unusual postures, and showed weaker reaction to sound stimulus; all these effects remained in survivor animals during 48 hours after exposure. When actual concentration was equal to 0.472 ± 0.005 mg/dm3 , rats started to suffer from apparent respiratory failure after 30 minutes of exposure.

Most rats in experimental group 2 (three male and two female) died within 150–190 minutes after the experiment started; their death was caused by acute respiratory failure. One female rat died 2 hours after the experiment was over. The animals were sluggish before their death, they took lateral position, and demonstrated no reaction to sound stimulus or motion activity. Death of the experimental animals was not established. CL50 of the examined nanodisperse MnO2 amounted to 0.12 mg/l. This concentration is within 0.05–0.5 mg/l range, which allows to define the tested substance as having the second hazard class "ATC technique" (OECD, Test No. 436:2008, IDT). It is known that minimal toxic concentration (TCL0) of nanosized МnO2 for rats at inhalation introduction during 24 hours amounts to 1.8 mg/m3 ; for mice during 7 hours, 49 mg/m3 (data taken from Minimal lethal dose (LDL0) for rats at intratracheal introduction amount to 45 mg/kg. Safety specification does not contain any additional information regarding errors, confidence limits, or animals sex. Morphologic changes in brain tissues of the rats from

*Heavy Metal Toxicity in Public Health*

*microscope (NT-MDT, Russian) [10].*

covering graphite rods via electrochemical deposition, up-to-date sorbents with the use of vacuum-ultrasound laser ablation [17, 18]. Inhalation exposure of workers to nanosized МnO2 particles is quite possible during such manufacturing processes as these particles are emitted into working area air. In relation to that, wider utilization of nanosized МnO2 in industrial production as well as providing workers' safety in the process requires more profound studies on toxicity of nanodisperse МnO2 when

*Nanodisperse MnO2: (a) nitrogen isotherm adsorption-desorption and (b) pore size distribution d (nm) [15].*

*The 3D-pattern of the MnO2 nanodisperse particles, atomic force microscopy with the use of solver-PRO* 

The authors used water suspension of nanodisperse MnO2 in concentration

microdisperse MnO2 with concentration in manganese water suspension equal to

share 67.0%). The size of microdisperse MnO2 particles is 194–1300 times greater than the nanodisperse MnO2 particles size. Specific surface area of MnO2 nanopar-

To determine acute toxicity parameters, the authors completed an experiment on pubescent Wistar rats (male and female) with body weight equal to 190 ± 10 g. They examined and assessed acute toxicity of nanodisperse MnO2 water suspension at inhalation introduction as an aerosol in accordance with Interstate Standard

ticles (Brunauer-Emmett-Teller technique [13]) was equal to 150.23 m<sup>2</sup>

can cause high MnO2 nanoparticles reactivity *in vitro* and *in vivo* [19].

"Testing techniques on determining chemical products effects exerted on a human body: acute inhalation toxicity technique determining acute toxicity class

1.2 times higher than microparticle-specific surface area (130 m<sup>2</sup>

as an examined substance. To make comparison,

was used. The particles size amounted to 5.5–37.0 μm (particles'

/g, which was

/g). This property

it enters a body being inhaled as an aerosol.

equal to 36.0 ± 2.3 mg/cm3

40.31 ± 1.6 mg/cm3

**Figure 3.**

**Figure 2.**

**100**

#### **Figure 4.**

*Nanoparticle concentration in the inhalation chamber air at actual MnO2 concentration equal to 0.029 ± 0.001 mg/dm3 [15].*

**Figure 5.**

*Nanoparticle concentration in the inhalation chamber air at actual MnO2 concentration equal to 0.472 ± 0.005 mg/dm3 [15].*

experimental group 1 in comparison with control group were characterized with the following pathologic disorders: brain substance vessels were filled with blood insignificantly or moderately and had focal endothelium swelling and perivascular spaces dilatation. The most apparent changes in brain tissue of the rats from experimental group 1 occurred in cerebellum. Neurons and neuroglia cells were characterized with grave ischemic damages in the form of wrinkling and pyknosis. Nerve fibers of brain tissue looked spongy and were unevenly colored, had fizzy contours, and focal prolapse of glial elements (**Figure 6**).

**103**

**Figure 6.**

*space dilatation [15].*

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

The results obtained in the course of the research indicate that the examined nanosized МnO2 particles can cause neurotoxic effect and respiratory failure; the combination of these two factors could lead to the animals' death. The obtained results are confirmed by research performed by a number of authors [20, 21], which prove that МnO2 nanoparticles exert toxic effects at inhalation exposure. Thus, after 24-hour exposure, catalytic generation of active oxygen forms (AOF) in human alveolar epithelial cells [20] increased; level of extracellular and intracellular oxidized glutathione form (GSSG) also grew by 30 and 80% correspondingly [20, 21]. Manganese oxide (IV) nanoparticles sized up to 30 nm are able to penetrate into neuron-like PC-12 cells of a brain at inhalation exposure via olfactory nerve

Here, slight mitochondrial activity inhibition occurs; dose-dependent decrease in dopamine and its metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid) takes place. This process is accompanied with a multiple AOF growth [20, 24] and becomes apparent in experimental animals through neurodegenerating disorders as early as after 2 or 3 weeks of exposure [20, 23, 24]. It is proved that МnO2 nanoparticles (III, IV) can accumulate in brain cells [25, 26]. In particular, astrocytes are able to accumulate МnO2 nanoparticles and produce AOF [25, 27, 28]. This process is accompanied with protein cleavage activation mediated by caspase-3 and protein kinase Сδ (these are enzymes that participate in apoptosis, necrosis, and inflammatory processes), as well as phosphorylation cycle activation [25, 26]. As particles concentration increases, level of p38 mutagen-active protein kinase grows linearly; this protein kinase activates apoptotic mechanism of untimely cell death [24, 29–31]. Tumor necrosis factor-α doubles in olfactory bulb, frontal cortex, midbrain, and striate body [27]. If inhalation exposure to МnO2 nanoparticles (III, IV) is long-term, time-depending activation of transferrin in dopaminergic nervous cells is detected, as well as structural changes in Beclin 1 and LC3 proteins, which, in its turn, can be an evidence of potential autophagia process activation [20]. As per data taken from the annotated scientific literature, it is proved that toxic effects exerted on nervous system cells can be caused both by nanoparticles [25, 29] and by microdisperse analog at a low-dose exposure [32]. Disorders in neurons membranes functions can underlie the neurotoxic action mechanism; such disorders result from membranes lipid peroxidation which in its turn is caused by direct cytotoxic effect of nanoparticles determined for dopaminergic

*Cerebellum of a Wistar rat after acute inhalation exposure to nanodisperse MnO2 aerosol at actual MnO2*

*×400): А is cerebellum tissue without changes, green; B is ischemia focus (grave ischemic changes), edema (damage zone is outlined); С is motor neuron of cerebellum subcortex; and D is glia cells with pericellular* 

 *(painted with hematoxylin-eosin,* 

*concentration in the inhalation chamber area equal to 0.029 ± 0.001 mg/dm3*

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

[22] and accumulate in astrocytes [17, 23].

#### *Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

The results obtained in the course of the research indicate that the examined nanosized МnO2 particles can cause neurotoxic effect and respiratory failure; the combination of these two factors could lead to the animals' death. The obtained results are confirmed by research performed by a number of authors [20, 21], which prove that МnO2 nanoparticles exert toxic effects at inhalation exposure. Thus, after 24-hour exposure, catalytic generation of active oxygen forms (AOF) in human alveolar epithelial cells [20] increased; level of extracellular and intracellular oxidized glutathione form (GSSG) also grew by 30 and 80% correspondingly [20, 21]. Manganese oxide (IV) nanoparticles sized up to 30 nm are able to penetrate into neuron-like PC-12 cells of a brain at inhalation exposure via olfactory nerve [22] and accumulate in astrocytes [17, 23].

Here, slight mitochondrial activity inhibition occurs; dose-dependent decrease in dopamine and its metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid) takes place. This process is accompanied with a multiple AOF growth [20, 24] and becomes apparent in experimental animals through neurodegenerating disorders as early as after 2 or 3 weeks of exposure [20, 23, 24]. It is proved that МnO2 nanoparticles (III, IV) can accumulate in brain cells [25, 26]. In particular, astrocytes are able to accumulate МnO2 nanoparticles and produce AOF [25, 27, 28]. This process is accompanied with protein cleavage activation mediated by caspase-3 and protein kinase Сδ (these are enzymes that participate in apoptosis, necrosis, and inflammatory processes), as well as phosphorylation cycle activation [25, 26]. As particles concentration increases, level of p38 mutagen-active protein kinase grows linearly; this protein kinase activates apoptotic mechanism of untimely cell death [24, 29–31]. Tumor necrosis factor-α doubles in olfactory bulb, frontal cortex, midbrain, and striate body [27]. If inhalation exposure to МnO2 nanoparticles (III, IV) is long-term, time-depending activation of transferrin in dopaminergic nervous cells is detected, as well as structural changes in Beclin 1 and LC3 proteins, which, in its turn, can be an evidence of potential autophagia process activation [20].

As per data taken from the annotated scientific literature, it is proved that toxic effects exerted on nervous system cells can be caused both by nanoparticles [25, 29] and by microdisperse analog at a low-dose exposure [32]. Disorders in neurons membranes functions can underlie the neurotoxic action mechanism; such disorders result from membranes lipid peroxidation which in its turn is caused by direct cytotoxic effect of nanoparticles determined for dopaminergic

#### **Figure 6.**

*Heavy Metal Toxicity in Public Health*

**102**

**Figure 5.**

*0.472 ± 0.005 mg/dm3*

 *[15].*

**Figure 4.**

*0.029 ± 0.001 mg/dm3*

 *[15].*

experimental group 1 in comparison with control group were characterized with the following pathologic disorders: brain substance vessels were filled with blood insignificantly or moderately and had focal endothelium swelling and perivascular spaces dilatation. The most apparent changes in brain tissue of the rats from experimental group 1 occurred in cerebellum. Neurons and neuroglia cells were characterized with grave ischemic damages in the form of wrinkling and pyknosis. Nerve fibers of brain tissue looked spongy and were unevenly colored, had fizzy

*Nanoparticle concentration in the inhalation chamber air at actual MnO2 concentration equal to* 

*Nanoparticle concentration in the inhalation chamber air at actual MnO2 concentration equal to* 

contours, and focal prolapse of glial elements (**Figure 6**).

*Cerebellum of a Wistar rat after acute inhalation exposure to nanodisperse MnO2 aerosol at actual MnO2 concentration in the inhalation chamber area equal to 0.029 ± 0.001 mg/dm3 (painted with hematoxylin-eosin, ×400): А is cerebellum tissue without changes, green; B is ischemia focus (grave ischemic changes), edema (damage zone is outlined); С is motor neuron of cerebellum subcortex; and D is glia cells with pericellular space dilatation [15].*

neurons [25, 29, 33]. This effect can be more apparent for nanodisperse particles in comparison with microdisperse analog effects due to the fact that nanoparticles have a greater specific surface area.

Mechanism of nanodisperse MnO2 neurotoxicity is related to the ability to generate free radicals and to interact with proteins. Manganese oxide nanoparticles are able to actively generate free radicals when they interact with bilipid layer of cell membranes [25, 30]. When interacting with cell membranes, they stimulate excessive creation of active oxygen forms (AOF), which is accompanied with high catalytic activity and cell apoptosis [25, 26, 30]. Oxidized (GSSG) and reduced glutathione form (GSH) content is one of the parameters showing oxidative stress level. It is proved that if alveolar epithelial cells are exposed to МnO2 particles for 24 hours, the level of extracellular and intracellular GSSG increases by 30 and 80% correspondingly; at the same time, caspase-3 activity grows and this enzyme is known to induce apoptosis processes. GSH concentration increases after 24-hour exposure to the examined substance, and it can be caused by activation of γ-glutamylcysteine synthetase synthesis and more active feed system of cystine and glutamate amino acids, which are substrates for synthesis of reduced glutathione form [25]. A significant increase in GSSG in a cell induced by МnO2 nanoparticles can be caused by manganese particles entering reduction reaction with superoxide formation, which, under superoxide dismutase effects, is converted into oxygen and hydrogen peroxide. Then, hydrogen peroxide decomposes with the help of reduced glutathione form, and it leads to GSSG increase. Another possible way of hydrogen peroxide transformation in a cell is hydroxyl radicals creation in the presence of manganese ions; these radicals are also able to oxidize GSH with creation of GSSG [26].

Clinical picture of acute intoxication, detailed in the previous works by the authors [34], confirms the mechanism of toxic effects exerted by nanodisperse MnO2 particles which a number of authors describe in above-mentioned scientific works [27, 28]. Respiratory failure evolvement can also be related to potential ability of the examined nanoparticles to cause inflammatory changes with consequent apoptosis of alveolar epithelial cells. At the same time, МnO2 nanoparticles have greater resistance to mucociliary clearance; therefore, they are in a longer contact with respiratory tract cells in comparison with microdisperse analog [28].

#### **3.2.2 Examination of acute toxicity which nanodisperse MnO2 has at oral introduction**

Intensive development of the major promising nanotechnological spheres such as nano-optics, nanoelectronics, pharmacology, chemistry, and metallurgy has direct influence on growth of nanodisperse MnO2 production volumes. Nanosized MnO2 particles are widely used in production of matrixes for nanomagnetic materials and sorbents [35], nanoaccelerators, semiconductor thermistors [36]. Wastes of such productions can get into sewage and then into water reservoirs serving as sources for drinking water supply. MnO2 nanoparticles are known to have nonspherical form, which makes for lower speed of their excretion out of a body with the help of immune system's phagocytes through lymphatic ducts and causes their longer contacts with body tissues [19]. In relation to that it is reasonable to think that profound examination of nanodisperse МnO2 aerosol toxicity at oral introduction with water into a body is of great importance for securing production workers safety and safety of population living in areas influenced by such productions.

The authors examined nanodisperse MnO2 water suspension with particles sized 15–29 nm. Particles were thread-like and had surface area equal to 150.2 ± 2.6 m2 /g. MnO2 concentration in nanodisperse solution was equal to 36.0 ± 2.3 mg/cm3 .

**105**

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

share 67.0%) (dynamic light scattering technique with the use of Microtrac S3500 laser analyzer (Microtrac, the USA)). The size of microdisperse MnO2 particles is 194–1300 times greater than nanodisperse MnO2 particle size. Specific surface area of MnO2 nanoparticles (Brunauer–Emmett–Teller technique [13]) was equal to

/g, which was 1.2 times higher than microparticles specific surface area

results of comprehensive acute experiments performed in accordance with methodical guidelines [10] on nonlinear male white mice with body weight equal to 27 ± 2 г (M ± m) (n = 70); the animals belonged to a conventional category. Nanodisperse MnO2 was introduced into mice's bodies a single time via gastric tube in various doses: group 1 received 2000 mg/kg of body weight, group 2–3500 mg/kg, and group 3–5000 mg/kg. The tested sample was introduced in a form of water suspension in volume equal to 1–2% of the animals' body weight. Microdisperse MnO2 was introduced into mice from experimental groups 4, 5, and 6 in the same doses as in groups 1, 2, and 3. The tested samples were introduced in the same way. Control group 7 received water a single time via gastric tube in the same volume. The observation term after the tested substances was introduced amounted to 14 days. Mice were

The experiment revealed that clinical picture of acute intoxication at introducing nano- and microdisperse MnO2 solutions is uniform and nonspecific. Animals from experimental groups 1–6 suffered from hyperexcitability and convulsions during the first 20 minutes of the experiment; then inhibition state occurred, animals' reaction to sound and pain became weak, they had hypopnoe. Animals in experimental groups 2 and 3 died mostly during the first 24 hours; in group 1, during 48 hours; and in comparison groups 5 and 6 in the period from 48 to 72 hours. No mice from group 4 or 7 died in the course of the experiment. It was detected that if nanodisperse MnO2 solution was once introduced into a body via gastric tube, LD50 amounted to 2340 ± 602.6 mg/kg of body weight (third hazard class); in case of microdisperse MnO2 solution introduction, the dose was equal to 6000 ± 485.6 mg/kg (fourth hazard class). Average death time (TL50) for mice after intragastric introduction of nanodisperse MnO2 solution amounted to 35.2 hours. When microdisperse MnO2 solution was introduced, TL50 amounted to 32 hours. Cumulation index amounted to 0.79 for nanodisperse MnO2 particles. If cumulation index is >5, one can assume that nanoparticles are hypercumulative. Cumulation index is equal to 0 for microdisperse MnO2 particles. If cumulation index is equal to

0, one can assume that particles are moderately cumulative [39].

Analysis of changes, which occurred in venous blood of the experimental animals, revealed that nanoparticles of disperse MnO2 solution in a dose equal to 3500 and 5000 mg/kg exert more apparent toxic effects on erythrocytes and thrombocytes in comparison with a microdisperse analog. It is confirmed by polychromatocytes occurrence as their share amounted to 20 and 35% of the total erythrocytes number in peripheral blood of mice from experimental groups 2 and 3 correspondingly. When microdisperse MnO2 was introduced in the same doses polychromatocytes share in mice's blood amounted to 10 and 15% correspondingly. 10–20% of erythrocytes in blood of mice from those groups contained pathologic Jolly bodies and it was two times higher than the same parameter in blood of mice from experimental groups 5 and 6 correspondingly. Nanodisperse MnO2 solution caused massive thrombocytes aggregation in blood of mice from experimental groups 2 and 3. Microdisperse MnO2 solution when introduced into mice in a dose

/g). Acute toxicity parameters of nanodisperse MnO2 were assessed as per the

was used. The particle size amounted to 5.5–37.0 μm (particles'

The detailed description of synthesis and physical and chemical properties of nanodisperse MnO2 is given in the precious works [37, 38]. To make comparison, microdisperse MnO2 with concentration in manganese water suspension equal to

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

kept in cages: five animals in each.

40.31 ± 1.6 mg/cm3

150.23 m2

(130 m2

#### *Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

The detailed description of synthesis and physical and chemical properties of nanodisperse MnO2 is given in the precious works [37, 38]. To make comparison, microdisperse MnO2 with concentration in manganese water suspension equal to 40.31 ± 1.6 mg/cm3 was used. The particle size amounted to 5.5–37.0 μm (particles' share 67.0%) (dynamic light scattering technique with the use of Microtrac S3500 laser analyzer (Microtrac, the USA)). The size of microdisperse MnO2 particles is 194–1300 times greater than nanodisperse MnO2 particle size. Specific surface area of MnO2 nanoparticles (Brunauer–Emmett–Teller technique [13]) was equal to 150.23 m2 /g, which was 1.2 times higher than microparticles specific surface area (130 m2 /g). Acute toxicity parameters of nanodisperse MnO2 were assessed as per the results of comprehensive acute experiments performed in accordance with methodical guidelines [10] on nonlinear male white mice with body weight equal to 27 ± 2 г (M ± m) (n = 70); the animals belonged to a conventional category. Nanodisperse MnO2 was introduced into mice's bodies a single time via gastric tube in various doses: group 1 received 2000 mg/kg of body weight, group 2–3500 mg/kg, and group 3–5000 mg/kg. The tested sample was introduced in a form of water suspension in volume equal to 1–2% of the animals' body weight. Microdisperse MnO2 was introduced into mice from experimental groups 4, 5, and 6 in the same doses as in groups 1, 2, and 3. The tested samples were introduced in the same way. Control group 7 received water a single time via gastric tube in the same volume. The observation term after the tested substances was introduced amounted to 14 days. Mice were kept in cages: five animals in each.

The experiment revealed that clinical picture of acute intoxication at introducing nano- and microdisperse MnO2 solutions is uniform and nonspecific. Animals from experimental groups 1–6 suffered from hyperexcitability and convulsions during the first 20 minutes of the experiment; then inhibition state occurred, animals' reaction to sound and pain became weak, they had hypopnoe. Animals in experimental groups 2 and 3 died mostly during the first 24 hours; in group 1, during 48 hours; and in comparison groups 5 and 6 in the period from 48 to 72 hours. No mice from group 4 or 7 died in the course of the experiment. It was detected that if nanodisperse MnO2 solution was once introduced into a body via gastric tube, LD50 amounted to 2340 ± 602.6 mg/kg of body weight (third hazard class); in case of microdisperse MnO2 solution introduction, the dose was equal to 6000 ± 485.6 mg/kg (fourth hazard class). Average death time (TL50) for mice after intragastric introduction of nanodisperse MnO2 solution amounted to 35.2 hours. When microdisperse MnO2 solution was introduced, TL50 amounted to 32 hours. Cumulation index amounted to 0.79 for nanodisperse MnO2 particles. If cumulation index is >5, one can assume that nanoparticles are hypercumulative. Cumulation index is equal to 0 for microdisperse MnO2 particles. If cumulation index is equal to 0, one can assume that particles are moderately cumulative [39].

Analysis of changes, which occurred in venous blood of the experimental animals, revealed that nanoparticles of disperse MnO2 solution in a dose equal to 3500 and 5000 mg/kg exert more apparent toxic effects on erythrocytes and thrombocytes in comparison with a microdisperse analog. It is confirmed by polychromatocytes occurrence as their share amounted to 20 and 35% of the total erythrocytes number in peripheral blood of mice from experimental groups 2 and 3 correspondingly. When microdisperse MnO2 was introduced in the same doses polychromatocytes share in mice's blood amounted to 10 and 15% correspondingly. 10–20% of erythrocytes in blood of mice from those groups contained pathologic Jolly bodies and it was two times higher than the same parameter in blood of mice from experimental groups 5 and 6 correspondingly. Nanodisperse MnO2 solution caused massive thrombocytes aggregation in blood of mice from experimental groups 2 and 3. Microdisperse MnO2 solution when introduced into mice in a dose

*Heavy Metal Toxicity in Public Health*

GSH with creation of GSSG [26].

**introduction**

ticles have a greater specific surface area.

neurons [25, 29, 33]. This effect can be more apparent for nanodisperse particles in comparison with microdisperse analog effects due to the fact that nanopar-

Mechanism of nanodisperse MnO2 neurotoxicity is related to the ability to generate free radicals and to interact with proteins. Manganese oxide nanoparticles are able to actively generate free radicals when they interact with bilipid layer of cell membranes [25, 30]. When interacting with cell membranes, they stimulate excessive creation of active oxygen forms (AOF), which is accompanied with high catalytic activity and cell apoptosis [25, 26, 30]. Oxidized (GSSG) and reduced glutathione form (GSH) content is one of the parameters showing oxidative stress level. It is proved that if alveolar epithelial cells are exposed to МnO2 particles for 24 hours, the level of extracellular and intracellular GSSG increases by 30 and 80% correspondingly; at the same time, caspase-3 activity grows and this enzyme is known to induce apoptosis processes. GSH concentration increases after 24-hour exposure to the examined substance, and it can be caused by activation of γ-glutamylcysteine synthetase synthesis and more active feed system of cystine and glutamate amino acids, which are substrates for synthesis of reduced glutathione form [25]. A significant increase in GSSG in a cell induced by МnO2 nanoparticles can be caused by manganese particles entering reduction reaction with superoxide formation, which, under superoxide dismutase effects, is converted into oxygen and hydrogen peroxide. Then, hydrogen peroxide decomposes with the help of reduced glutathione form, and it leads to GSSG increase. Another possible way of hydrogen peroxide transformation in a cell is hydroxyl radicals creation in the presence of manganese ions; these radicals are also able to oxidize

Clinical picture of acute intoxication, detailed in the previous works by the authors [34], confirms the mechanism of toxic effects exerted by nanodisperse MnO2 particles which a number of authors describe in above-mentioned scientific works [27, 28]. Respiratory failure evolvement can also be related to potential ability of the examined nanoparticles to cause inflammatory changes with consequent apoptosis of alveolar epithelial cells. At the same time, МnO2 nanoparticles have greater resistance to mucociliary clearance; therefore, they are in a longer contact

with respiratory tract cells in comparison with microdisperse analog [28].

**3.2.2 Examination of acute toxicity which nanodisperse MnO2 has at oral** 

Intensive development of the major promising nanotechnological spheres such as nano-optics, nanoelectronics, pharmacology, chemistry, and metallurgy has direct influence on growth of nanodisperse MnO2 production volumes. Nanosized MnO2 particles are widely used in production of matrixes for nanomagnetic materials and sorbents [35], nanoaccelerators, semiconductor thermistors [36]. Wastes of such productions can get into sewage and then into water reservoirs serving as sources for drinking water supply. MnO2 nanoparticles are known to have nonspherical form, which makes for lower speed of their excretion out of a body with the help of immune system's phagocytes through lymphatic ducts and causes their longer contacts with body tissues [19]. In relation to that it is reasonable to think that profound examination of nanodisperse МnO2 aerosol toxicity at oral introduction with water into a body is of great importance for securing production workers safety and safety of population living in areas influenced by such productions.

The authors examined nanodisperse MnO2 water suspension with particles sized 15–29 nm. Particles were thread-like and had surface area equal to 150.2 ± 2.6 m2

MnO2 concentration in nanodisperse solution was equal to 36.0 ± 2.3 mg/cm3

/g.

.

**104**

equal to 5000 mg/kg (experimental group 6) led to only sporadic occurrence of thrombocytes aggregation. There were no pathologic changes in blood slides of mice from experimental group 7.

Morphological examinations after introducing nanodisperse MnO2 solution in a dose equal to 3500 mg/kg revealed changes in all examined organs taken out of mice. There was significant dilatation and hyperemia of veins in liver and kidneys, considerable blood overflow in veins of cardiac muscle and epicardium, as well as in heart chambers. There were hemorrhages detected in medullar substance of kidneys. Proliferative processes occurred in macrophage and lymphoid systems of experimental animals. An increased number of macrophages and their activation was detected in liver, in both kidneys and, in particular, in lungs (**Figure 7**). Kupffer cells in liver are enlarged and bulged into sinusoid capillaries lumen; mesangial cells in renal bodies are hypertrophic, and numerous alveolar macrophages contain phagocyte material. Lymphoid nodules in spleen white pulp are enlarged, tend to fuse, and there is no typical division into zones in them. Red pulp prevails in the organ together with diffuse and focal lymphatization occurrence. Proliferation of cells from lymphocytes and macrophages range leads to leukocytic infiltration of parenchymatous organs. Numerous periportal and intralobular lymphoidhistiocytic infiltrates occur in liver; perivascular and intertubular ones are observed in kidneys. Organ infiltration with lymphocytes is so active that it can penetrate through blood-brain barrier; lymphoid infiltrates can be detected even in white substance of cerebral hemispheres (**Figures 8**–**10**).

When a microdisperse MnO2 solution in a dose equal to 3500 mg/kg was introduced, it led to some dilatation of sinusoid capillaries in liver and vessels of renal microcirculatory bed. Capsule lumen in some renal corpuscles was dilated. Occasional lymph-histiocytic infiltration occurred in parenchymatous organs. Infiltrates were located only in connective tissue and were rather small. As opposed to group 2, animals from group 5 had no infiltrates in brain hemispheres cortex. There was only slight perivascular and moderate pericellular edema located mostly in granular layer.

Therefore, mice from group 2 after a single introduction of nanodisperse MnO2 solution in a dose equal to 3500 mg/kg via gastric tube had more apparent morphological changes in internal organs than mice from group 5 (microdisperse analog). These changes were detected in circulatory system and characterized with dilatation and hyperemia of veins in all the studied organs. Blood overflow in veins of cardiac

#### **Figure 7.**

*Macrophage reaction in a lung of a mouse from experimental group 2, which died during the first day after nanodisperse MnO2 in a dose equal to 3500 mg/kg, was introduced. The specimen was painted with hematoxylin and eosin, and the magnification was equal to ×1000 [38].*

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**Figure 10.**

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

*A nonlinear mouse from experimental group 2, which died during the first day after nanodisperse MnO2 in a dose equal to 3500 mg/kg, was introduced. Lymph-histiocytic intralobular infiltrates in a liver. The specimen is* 

*A nonlinear mouse from experimental group 2, which died during the first day after nanodisperse MnO2 in a dose equal to 3500 mg/kg, was introduced. Lymph-histiocytic perivascular infiltrates in a kidney. The specimen* 

*A nonlinear mouse from experimental group 2, which died during the first day after nanodisperse MnO2 in a dose equal to 3500 mg/kg, was introduced. The specimen is painted with hematoxylin and eosin, and magnification is equal to ×400. Lymphocytic infiltration in white substance of cerebral hemispheres [38].*

*painted with hematoxylin and eosin, and magnification is equal to ×200 [38].*

*is painted with hematoxylin and eosin, and magnification is equal to ×200 [38].*

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

**Figure 8.**

**Figure 9.**

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

#### **Figure 8.**

*Heavy Metal Toxicity in Public Health*

from experimental group 7.

in granular layer.

substance of cerebral hemispheres (**Figures 8**–**10**).

equal to 5000 mg/kg (experimental group 6) led to only sporadic occurrence of thrombocytes aggregation. There were no pathologic changes in blood slides of mice

When a microdisperse MnO2 solution in a dose equal to 3500 mg/kg was introduced, it led to some dilatation of sinusoid capillaries in liver and vessels of renal microcirculatory bed. Capsule lumen in some renal corpuscles was dilated. Occasional lymph-histiocytic infiltration occurred in parenchymatous organs. Infiltrates were located only in connective tissue and were rather small. As opposed to group 2, animals from group 5 had no infiltrates in brain hemispheres cortex. There was only slight perivascular and moderate pericellular edema located mostly

Therefore, mice from group 2 after a single introduction of nanodisperse MnO2 solution in a dose equal to 3500 mg/kg via gastric tube had more apparent morphological changes in internal organs than mice from group 5 (microdisperse analog). These changes were detected in circulatory system and characterized with dilatation and hyperemia of veins in all the studied organs. Blood overflow in veins of cardiac

*Macrophage reaction in a lung of a mouse from experimental group 2, which died during the first day after nanodisperse MnO2 in a dose equal to 3500 mg/kg, was introduced. The specimen was painted with* 

*hematoxylin and eosin, and the magnification was equal to ×1000 [38].*

Morphological examinations after introducing nanodisperse MnO2 solution in a dose equal to 3500 mg/kg revealed changes in all examined organs taken out of mice. There was significant dilatation and hyperemia of veins in liver and kidneys, considerable blood overflow in veins of cardiac muscle and epicardium, as well as in heart chambers. There were hemorrhages detected in medullar substance of kidneys. Proliferative processes occurred in macrophage and lymphoid systems of experimental animals. An increased number of macrophages and their activation was detected in liver, in both kidneys and, in particular, in lungs (**Figure 7**). Kupffer cells in liver are enlarged and bulged into sinusoid capillaries lumen; mesangial cells in renal bodies are hypertrophic, and numerous alveolar macrophages contain phagocyte material. Lymphoid nodules in spleen white pulp are enlarged, tend to fuse, and there is no typical division into zones in them. Red pulp prevails in the organ together with diffuse and focal lymphatization occurrence. Proliferation of cells from lymphocytes and macrophages range leads to leukocytic infiltration of parenchymatous organs. Numerous periportal and intralobular lymphoidhistiocytic infiltrates occur in liver; perivascular and intertubular ones are observed in kidneys. Organ infiltration with lymphocytes is so active that it can penetrate through blood-brain barrier; lymphoid infiltrates can be detected even in white

**106**

**Figure 7.**

*A nonlinear mouse from experimental group 2, which died during the first day after nanodisperse MnO2 in a dose equal to 3500 mg/kg, was introduced. Lymph-histiocytic intralobular infiltrates in a liver. The specimen is painted with hematoxylin and eosin, and magnification is equal to ×200 [38].*

#### **Figure 9.**

*A nonlinear mouse from experimental group 2, which died during the first day after nanodisperse MnO2 in a dose equal to 3500 mg/kg, was introduced. Lymph-histiocytic perivascular infiltrates in a kidney. The specimen is painted with hematoxylin and eosin, and magnification is equal to ×200 [38].*

#### **Figure 10.**

*A nonlinear mouse from experimental group 2, which died during the first day after nanodisperse MnO2 in a dose equal to 3500 mg/kg, was introduced. The specimen is painted with hematoxylin and eosin, and magnification is equal to ×400. Lymphocytic infiltration in white substance of cerebral hemispheres [38].*

muscle, epicardium and all heart chambers was particularly evident. Mice from group 5 had no peculiar morphological changes in circulatory system organs. Mice from group 2 had proliferative processes in lymphoid and macrophage systems. These processes were morphologically apparent through hypertrophy of thymus lobules cortex, lymphoid follicles of spleen white pulp and lymphatization of spleen red pulp, and macrophage activation in liver, kidneys and lungs. Such morphological changes did not occur in mice from group 5. Proliferation of lymphoid cells and macrophages leads to histioleukocytic infiltration of parenchymatous organs. And here, processes caused by nanodisperse MnO2 solution impact are more apparent than those caused by microdisperse MnO2 solution. Morphological changes are determined by vascular disorders in venous bed, which result in hemorrhages in parenchymatous organs, especially in kidneys. There were none of such morphological changes in mice when microdisperse MnO2 solution was introduced into them. The analysis of the obtained results allows to make a conclusion that nanodisperse MnO2 solution at a single introduction via gastric tube has LD50 equal to 2340 ± 602.6 mg/kg (third hazard class), which is 2.6 times higher than LD50 equal to 6000 ± 485.6 mg/kg at a single introduction of microdisperse MnO2 solution (fourth hazard class). Nanodisperse MnO2 solution at single introduction into mice via gastric tube in a dose equal to 3500 mg/kg exerts hemotoxic effects, which reveal themselves in a form of pathologic inclusions in erythrocytes and increased thrombocytes aggregation. There were also certain circulation disorders observed such as vein dilatation and hyperemia, as well as filling of their lumen with erythrocytes, which leads to hemorrhages in all the studied organs. Proliferative processes in lymphoid and macrophage systems were detected; they result in hypertrophy of thymus lobule cortex and lymphoid follicles of spleen white pulp, lymphatization of spleen red pulp, and macrophages activation in liver, kidneys and lungs.

#### **3.3 Studying subchronic and chronic toxicity of nanodisperse manganese oxide water suspension at intragastric introduction via gastric tube**

**Studying subchronic toxicity of nanodisperse МnO2 water suspension at intragastric introduction via gastric tube**: nowadays, there is a growing interest in using МnO2 nanoparticles as a sorbent and catalyst for complex purification of liquid radioactive wastes, which are dangerous for human health. But МnO2 nanoparticles can get into sewage in the process and later they can be found in surface water reservoirs used for drinking water supply to population. In relation to that, study of toxic effects exerted by МnO2 nanoparticles at oral introduction with drinking water matters a lot if one wants to assess their safety.

Experimental research dedicated to nanodisperse MnO2 were performed with intragastric introduction via gastric tube during 90 days. During this particular research, nanodisperse MnO2 water suspension was introduced into Wistar rats (males and females) with body weight equal to 200 ± 10 g (n = 100) via gastric tube daily for 90 days. Animals were divided into four experimental groups and 1 control group, 20 animals in each. Nanodisperse MnO2 water suspension was introduced in the following doses: group 1—257.7 mg/kg (1/10 LD50), group 2—51.54 mg/kg (1/50 LD50), group 3—10.3 mg/kg (1/250 LD50 3 ), group 4—5.15 mg/kg (1/500 LD50), and group 5 (control one)—distilled water in volume equal. Nanodisperse MnO2 water suspension dispersion was accomplished directly before carrying out the research and later on a weekly basis during 90 days, while the experiment was lasting in order to achieve even particles distribution in a volume of liquid; the procedure was done with the use of ultrasound at room temperature under continuous pulsation at 65%-power for 2 minutes. The following parameters were assessed: body weight dynamics, as well as changes in biochemical parameters of neurons functions and

**109**

concentrations [27].

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

oxidation-antioxidant system balance. Morphological changes in brain tissues were

Body weight dynamics analysis performed on rats from group 1 revealed authentic decrease in this parameter by 7.7% by the 30th day of the experiment in comparison with the baseline. Body weight was recovered by the 90th day. Decrease in body weight of rats from group 1 authentically differed from this parameter in control group during the whole experiment. In all other groups, authentic increase in body weight of experimental animals was detected. Examination and assessment of biochemical parameters characterizing neurotransmitters balance and oxidationantioxidant system at long-term introduction of nanodisperse MnO2 water suspension proves negative effects occurrence; these effects are dose depending. There was an authentic increase in lipid hydroperoxide level in blood serum of rats on the 30th day of the experiment when doses were 10.0–260 mg/kg of body weight; this parameter was 1.4–1.9 times higher than the baseline in rats from the control group (р < 0.001). Malonic dialdehyde level in blood serum of rats was 1.6–2.0 times higher than the baseline of this parameter in control group rats, the doses being equal (р < 0.001). On the 90th day, high levels of lipids hydroperoxides and malonic dialdehyde in blood serum remained. The order of discrepancy between the baseline and parameters in the control group was 1.3–1.9 times (р < 0.001). When a dose was equal to 5.0 mg/kg, there were not any authentic discrepancies between these parameters and the baseline, or between them and control group parameters during the whole experiment. When doses were 10.0–260 mg/kg, an authentic decrease in Cu/Zn-SOD and OAS in blood serum of rats was registered; this decrease was dose dependent. During the whole experiment, Cu/Zn-SOD level was on average from 1.4 to 4.6 times lower than the baseline and the control group parameter (р < 0.001–0.002). OAS level was 1.6–5.4 times lower (р < 0.001). When a dose was equal to 5.0 mg/kg, there were no authentic discrepancies in OAS level

studied via histological specimens microscopy (magnification ×400).

between experimental and control group during the whole experiment.

Assessment of basic neurotransmitters content in blood serum of rats on the 90th day of the experiment revealed authentic increase in glutamate level and decrease in GABA level in comparison with the baseline and control group. Changes in these parameters were dose dependent. Glutamate level in blood serum grew from 2 to 3.8 times depending on a dose (р < 0.001). GABA level in blood serum decreased 2.3–2.7 times (р < 0.001). When a dose was equal to 5.0 mg/kg, there was no authentic decrease in the analyzed parameters in blood serum against control group parameters. The obtained results show that, given their small size and high penetrability, МnO2 nanoparticles can penetrate through blood-brain barrier and cause morphological-functional disorders in various sections of central nervous system when introduced into a body in different ways even in relatively small

The detected activation of cell membranes lipid peroxidation and imbalance in CNS neuromediators at intragastric introduction of nanodispersed MnO2, in Wistar rats, are confirmed by the results obtained by a number of authors at other ways of introduction into a body (for example, at intranasal, intratracheal, and inhalation introduction). As a number of authors state, when МnO2 particle dose equals to 2.63 mg/kg and intranasal introduction of this dose lasts for 6 weeks, neurotoxicity can be observed as relative refractory period of a tail nerve grows [27]. Intratracheal introduction of МnO2 nanoparticles in a dose equal to 2.63 mg/kg during 6 weeks leads to significant decrease in body weight, longer absolute refractory period of a tail nerve, and lower animals movability [27]. Neurotoxicity of МnO2 nanoparticles at intratracheal introduction in doses equal to 2.63 and 5.26 mg/kg can be seen through increase in latent period of cortical potential occurrence (total response of large cortex neurons populations to synchronous impulses flow coming to them

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

#### *Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

oxidation-antioxidant system balance. Morphological changes in brain tissues were studied via histological specimens microscopy (magnification ×400).

Body weight dynamics analysis performed on rats from group 1 revealed authentic decrease in this parameter by 7.7% by the 30th day of the experiment in comparison with the baseline. Body weight was recovered by the 90th day. Decrease in body weight of rats from group 1 authentically differed from this parameter in control group during the whole experiment. In all other groups, authentic increase in body weight of experimental animals was detected. Examination and assessment of biochemical parameters characterizing neurotransmitters balance and oxidationantioxidant system at long-term introduction of nanodisperse MnO2 water suspension proves negative effects occurrence; these effects are dose depending. There was an authentic increase in lipid hydroperoxide level in blood serum of rats on the 30th day of the experiment when doses were 10.0–260 mg/kg of body weight; this parameter was 1.4–1.9 times higher than the baseline in rats from the control group (р < 0.001). Malonic dialdehyde level in blood serum of rats was 1.6–2.0 times higher than the baseline of this parameter in control group rats, the doses being equal (р < 0.001). On the 90th day, high levels of lipids hydroperoxides and malonic dialdehyde in blood serum remained. The order of discrepancy between the baseline and parameters in the control group was 1.3–1.9 times (р < 0.001). When a dose was equal to 5.0 mg/kg, there were not any authentic discrepancies between these parameters and the baseline, or between them and control group parameters during the whole experiment. When doses were 10.0–260 mg/kg, an authentic decrease in Cu/Zn-SOD and OAS in blood serum of rats was registered; this decrease was dose dependent. During the whole experiment, Cu/Zn-SOD level was on average from 1.4 to 4.6 times lower than the baseline and the control group parameter (р < 0.001–0.002). OAS level was 1.6–5.4 times lower (р < 0.001). When a dose was equal to 5.0 mg/kg, there were no authentic discrepancies in OAS level between experimental and control group during the whole experiment.

Assessment of basic neurotransmitters content in blood serum of rats on the 90th day of the experiment revealed authentic increase in glutamate level and decrease in GABA level in comparison with the baseline and control group. Changes in these parameters were dose dependent. Glutamate level in blood serum grew from 2 to 3.8 times depending on a dose (р < 0.001). GABA level in blood serum decreased 2.3–2.7 times (р < 0.001). When a dose was equal to 5.0 mg/kg, there was no authentic decrease in the analyzed parameters in blood serum against control group parameters. The obtained results show that, given their small size and high penetrability, МnO2 nanoparticles can penetrate through blood-brain barrier and cause morphological-functional disorders in various sections of central nervous system when introduced into a body in different ways even in relatively small concentrations [27].

The detected activation of cell membranes lipid peroxidation and imbalance in CNS neuromediators at intragastric introduction of nanodispersed MnO2, in Wistar rats, are confirmed by the results obtained by a number of authors at other ways of introduction into a body (for example, at intranasal, intratracheal, and inhalation introduction). As a number of authors state, when МnO2 particle dose equals to 2.63 mg/kg and intranasal introduction of this dose lasts for 6 weeks, neurotoxicity can be observed as relative refractory period of a tail nerve grows [27]. Intratracheal introduction of МnO2 nanoparticles in a dose equal to 2.63 mg/kg during 6 weeks leads to significant decrease in body weight, longer absolute refractory period of a tail nerve, and lower animals movability [27]. Neurotoxicity of МnO2 nanoparticles at intratracheal introduction in doses equal to 2.63 and 5.26 mg/kg can be seen through increase in latent period of cortical potential occurrence (total response of large cortex neurons populations to synchronous impulses flow coming to them

*Heavy Metal Toxicity in Public Health*

muscle, epicardium and all heart chambers was particularly evident. Mice from group 5 had no peculiar morphological changes in circulatory system organs. Mice from group 2 had proliferative processes in lymphoid and macrophage systems. These processes were morphologically apparent through hypertrophy of thymus lobules cortex, lymphoid follicles of spleen white pulp and lymphatization of spleen red pulp, and macrophage activation in liver, kidneys and lungs. Such morphological changes did not occur in mice from group 5. Proliferation of lymphoid cells and macrophages leads to histioleukocytic infiltration of parenchymatous organs. And here, processes caused by nanodisperse MnO2 solution impact are more apparent than those caused by microdisperse MnO2 solution. Morphological changes are determined by vascular disorders in venous bed, which result in hemorrhages in parenchymatous organs, especially in kidneys. There were none of such morphological changes in mice when microdisperse MnO2 solution was introduced into them. The analysis of the obtained results allows to make a conclusion that nanodisperse MnO2 solution at a single introduction via gastric tube has LD50 equal to 2340 ± 602.6 mg/kg (third hazard class), which is 2.6 times higher than LD50 equal to 6000 ± 485.6 mg/kg at a single introduction of microdisperse MnO2 solution (fourth hazard class). Nanodisperse MnO2 solution at single introduction into mice via gastric tube in a dose equal to 3500 mg/kg exerts hemotoxic effects, which reveal themselves in a form of pathologic inclusions in erythrocytes and increased thrombocytes aggregation. There were also certain circulation disorders observed such as vein dilatation and hyperemia, as well as filling of their lumen with erythrocytes, which leads to hemorrhages in all the studied organs. Proliferative processes in lymphoid and macrophage systems were detected; they result in hypertrophy of thymus lobule cortex and lymphoid follicles of spleen white pulp, lymphatization of

spleen red pulp, and macrophages activation in liver, kidneys and lungs.

**water suspension at intragastric introduction via gastric tube**

drinking water matters a lot if one wants to assess their safety.

LD50), group 3—10.3 mg/kg (1/250 LD50

**3.3 Studying subchronic and chronic toxicity of nanodisperse manganese oxide** 

**Studying subchronic toxicity of nanodisperse МnO2 water suspension at intragastric introduction via gastric tube**: nowadays, there is a growing interest in using МnO2 nanoparticles as a sorbent and catalyst for complex purification of liquid radioactive wastes, which are dangerous for human health. But МnO2 nanoparticles can get into sewage in the process and later they can be found in surface water reservoirs used for drinking water supply to population. In relation to that, study of toxic effects exerted by МnO2 nanoparticles at oral introduction with

Experimental research dedicated to nanodisperse MnO2 were performed with intragastric introduction via gastric tube during 90 days. During this particular research, nanodisperse MnO2 water suspension was introduced into Wistar rats (males and females) with body weight equal to 200 ± 10 g (n = 100) via gastric tube daily for 90 days. Animals were divided into four experimental groups and 1 control group, 20 animals in each. Nanodisperse MnO2 water suspension was introduced in the following doses: group 1—257.7 mg/kg (1/10 LD50), group 2—51.54 mg/kg (1/50

3

group 5 (control one)—distilled water in volume equal. Nanodisperse MnO2 water suspension dispersion was accomplished directly before carrying out the research and later on a weekly basis during 90 days, while the experiment was lasting in order to achieve even particles distribution in a volume of liquid; the procedure was done with the use of ultrasound at room temperature under continuous pulsation at 65%-power for 2 minutes. The following parameters were assessed: body weight dynamics, as well as changes in biochemical parameters of neurons functions and

), group 4—5.15 mg/kg (1/500 LD50), and

**108**

and caused by afferent irritator) in visual, auditory, and the first somatosensory area. This effect can be determined by disorders in neuron membranes functions as a result of membrane lipid peroxidation accompanied with calcium homeostasis disorders [23]. Nanoparticles and microparticles of МnO2 in concentration equal to 100 μg/kg in saline were injected into rats once a 2 weeks during 14 weeks, and it allowed to determine that both substances caused authentic increase in dextrose and cholesterol level. МnO2 nanoparticles lowered the level of high-density lipoproteins at the sixth, twelfth, and fourteenth week, while microdisperse analog caused such decrease at the twelfth week only. Researchers detected no authentic changes in triglyceride concentration. Changes in biochemical profile might be caused by oxidizing possibilities, which МnO2 nanoparticles have [40]. An injection of МnO2 nanoparticles into a rat's brain (in substantia nigra and tegmentum ventral area) in concentration equal to 87 μg/μl in 1 μl causes changes in locomotor abilities and spatial memory, which are related to dopaminergic neuron disorders and inflammation caused by nanoparticles. МnO2 nanoparticles lead to early symptoms of extrapyramidal disorders with selective loss of dopaminergic neurons [27].

Histological specimens made of experimental animals' brains were assessed; the assessment results allowed to detect morphologic changes in tissue structure depending on a dose of nanodisperse MnO2 water suspension. After a dose equal to 260 mg/kg of body weight a day had been introduced, the authors detected substantial vessels hyperemia in brain hemispheres cortex and cerebellum together with erythrocytes diapedesis and formation of subarachnoid hemorrhages focuses. There was also brain edema with perivascular and pericellular spaces dilatation (**Figure 11**). Focuses of nerve fibers demyelinization were detected as white substance fibers were unevenly painted and lighter areas with fizzy contours occurred.

After a 50 mg/kg dose focal perivascular and pericellular spaces dilatations form in brain cortex layers, but cortex layers still remain differentiated (**Figure 12**). Vessels in brain cortex tissues substance have thin walls, are moderately filled with blood, the endothelium is flattened, and there are small focal subarachnoid hemorrhages and focal neurons dystrophy zones. A slight perivascular spaces dilatation occurred after a 10 mg/kg of body weight a day (**Figure 13**). But when a dose was equal to 5 mg/kg of body weight a day, morphologic picture of brain and cerebellum tissues corresponded to that of control group; structure patterns were preserved in all the sections (**Figure 14**).

#### **Figure 11.**

*Brain hemispheres cortex of a rat at intragastric introduction of nanodisperse MnO2 water suspension in a dose equal to 260 mg/kg of body weight a day, 90th day of the experiment. There are dilated perivascular and pericellular spaces in brain cortex layers. Painted with hematoxylin and eosin, magnification ×400 [41].*

**111**

**Figure 14.**

*magnification ×100 [41].*

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

*Brain hemispheres cortex of a rat at intragastric introduction of nanodisperse MnO2 water suspension in a dose equal to 50 mg/kg of body weight a day, 90th day of the experiment. There are dilated perivascular and pericellular spaces in brain cortex layers. Painted with hematoxylin and eosin, magnification ×400 [41].*

*Brain hemispheres cortex of a rat at intragastric introduction of nanodisperse MnO2 water suspension in a dose equal to 10 mg/kg of body weight a day, 90th day of the experiment. There is slight perivascular spaces dilatation in brain hemispheres cortex. painted with hematoxylin and eosin, magnification ×100 [41].*

*Cerebellum of a rat at intragastric introduction of nanodisperse MnO2 water suspension in a dose equal to 5 mg/kg of body weight a day, 90th day of the experiment. Tissue structure pattern in all cerebellum sections is preserved and corresponds to that of control group. Painted with hematoxylin and eosin,* 

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

**Figure 12.**

**Figure 13.**

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

#### **Figure 12.**

*Heavy Metal Toxicity in Public Health*

all the sections (**Figure 14**).

and caused by afferent irritator) in visual, auditory, and the first somatosensory area. This effect can be determined by disorders in neuron membranes functions as a result of membrane lipid peroxidation accompanied with calcium homeostasis disorders [23]. Nanoparticles and microparticles of МnO2 in concentration equal to 100 μg/kg in saline were injected into rats once a 2 weeks during 14 weeks, and it allowed to determine that both substances caused authentic increase in dextrose and cholesterol level. МnO2 nanoparticles lowered the level of high-density lipoproteins at the sixth, twelfth, and fourteenth week, while microdisperse analog caused such decrease at the twelfth week only. Researchers detected no authentic changes in triglyceride concentration. Changes in biochemical profile might be caused by oxidizing possibilities, which МnO2 nanoparticles have [40]. An injection of МnO2 nanoparticles into a rat's brain (in substantia nigra and tegmentum ventral area) in concentration equal to 87 μg/μl in 1 μl causes changes in locomotor abilities and spatial memory, which are related to dopaminergic neuron disorders and inflammation caused by nanoparticles. МnO2 nanoparticles lead to early symptoms of extrapyramidal disorders with selective loss of dopaminergic neurons [27].

Histological specimens made of experimental animals' brains were assessed; the assessment results allowed to detect morphologic changes in tissue structure depending on a dose of nanodisperse MnO2 water suspension. After a dose equal to 260 mg/kg of body weight a day had been introduced, the authors detected substantial vessels hyperemia in brain hemispheres cortex and cerebellum together with erythrocytes diapedesis and formation of subarachnoid hemorrhages focuses. There was also brain edema with perivascular and pericellular spaces dilatation (**Figure 11**). Focuses of nerve fibers demyelinization were detected as white substance fibers were unevenly painted and lighter areas with fizzy contours occurred. After a 50 mg/kg dose focal perivascular and pericellular spaces dilatations form

in brain cortex layers, but cortex layers still remain differentiated (**Figure 12**). Vessels in brain cortex tissues substance have thin walls, are moderately filled with blood, the endothelium is flattened, and there are small focal subarachnoid hemorrhages and focal neurons dystrophy zones. A slight perivascular spaces dilatation occurred after a 10 mg/kg of body weight a day (**Figure 13**). But when a dose was equal to 5 mg/kg of body weight a day, morphologic picture of brain and cerebellum tissues corresponded to that of control group; structure patterns were preserved in

*Brain hemispheres cortex of a rat at intragastric introduction of nanodisperse MnO2 water suspension in a dose equal to 260 mg/kg of body weight a day, 90th day of the experiment. There are dilated perivascular and pericellular spaces in brain cortex layers. Painted with hematoxylin and eosin, magnification ×400 [41].*

**110**

**Figure 11.**

*Brain hemispheres cortex of a rat at intragastric introduction of nanodisperse MnO2 water suspension in a dose equal to 50 mg/kg of body weight a day, 90th day of the experiment. There are dilated perivascular and pericellular spaces in brain cortex layers. Painted with hematoxylin and eosin, magnification ×400 [41].*

#### **Figure 13.**

*Brain hemispheres cortex of a rat at intragastric introduction of nanodisperse MnO2 water suspension in a dose equal to 10 mg/kg of body weight a day, 90th day of the experiment. There is slight perivascular spaces dilatation in brain hemispheres cortex. painted with hematoxylin and eosin, magnification ×100 [41].*

#### **Figure 14.**

*Cerebellum of a rat at intragastric introduction of nanodisperse MnO2 water suspension in a dose equal to 5 mg/kg of body weight a day, 90th day of the experiment. Tissue structure pattern in all cerebellum sections is preserved and corresponds to that of control group. Painted with hematoxylin and eosin, magnification ×100 [41].*

Brain cortex tissues layers are differentiated quite well. External granular layer is formed out of solid bunch consisting of numerous small neurons; pyramidal layer of brain cortex is wide and consists of polymorphous neurons. Internal granular layer of brain cortex and cerebellum is thin, noncontinuous, made of small pyramidal and stellate cells; ganglionic layer cells in brain cortex tissues are large, polymorphous with dark nucleuses, diffusely located; there is a great number of diverse neurons (they differ in form and size) in polymorphous cells layer. Ganglionic layer in cerebellum tissue has one raw of Purkinje cells with well-developed eosinophilic granular cytoplasm and rounded dark nucleuses. Molecular layer in cerebellum tissue is spongy and contains a small number of minor cells. White substance in brain and cerebellum tissue is made of evenly painted nerve fibers bunches and rounded glia cells. Brain vessels have thin walls and are feebly or moderately filled with blood.

Comparison of pathomorphologic effects occurring at intragastric introduction of nanodisperse MnO2 water suspension and microdisperse MnO2 water suspension in a dose equal to 10 mg/kg during 90 days showed that pathomorphologic changes in brain and cerebellum tissues were much more apparent and wide-spread when nanodisperse MnO2 was introduced. After introduction of nanodisperse MnO2 water suspension more apparent changes occurred in circulatory system in a form of hemodynamic disorders with focal feeble and moderate vessels hyperemia in brain, liver, lungs, kidneys, and heart; there were also subarachnoid hemorrhages in brain. Changes in lymphatic system became evident through feeble and moderate perivascular lymph-macrophage infiltrates in lungs tissue penetrating into adjacent alveoli. Pathologic changes in macrophage system were apparent through alveolar macrophages activation with formation of small bunches in alveoli lumen. After introduction of microdisperse MnO2 water suspension changes are represented by focal vessels hyperemia, subarachnoid hemorrhages in brain, small focal lymphmicrophage infiltrates in lungs and gastrointestinal tract. Besides, after introduction of nanodisperse MnO2 water suspension there was perivascular and pericellular spaces dilatation in brains, feebly apparent perivascular thin fibrous cardiosclerosis and feebly apparent focal protein dystrophy in hepatocytes; no such effects were detected after microdisperse MnO2 introduction.

All the obtained materials were summarized; it allowed to make an assumption on a possible mechanism of toxic impact exerted by nanodisperse MnO2 particles at oral introduction. Lipid peroxidation activation caused by direct damaging impact exerted by nanodisperse MnO2 particles on bilipid layer of cytoplasmatic membrane can underlie the whole process [42]. High levels of lipid hydroperoxides and malonic dialdehyde in blood serum are the evidence of this effect. Lower levels of Cu/Zn-SOD and OAS in blood serum prove antioxidation processes insufficiency. The results obtained in the research showed that astrocytes and neurons membranes are the first targets influenced by nanodisperse MnO2 water suspension at oral introduction, just as at inhalation one [21, 26]. In case of oral introduction it can be determined by apparent ability of nanodisperse MnO2 particles to penetrate into blood from gastrointestinal tract. MnO2 nanoparticles reach brain tissues coming from bloodstream through capillary endothelial cells of blood brain barrier and accumulate in astrocytes [26]. Damaged astrocytes due to enhanced peroxidation of cells membranes lipids and active oxygen forms occurrence can lose their ability to capture and neutralize excessive quantities of "exciting" amino acid, namely glutamate; thus, excitotoxic effect evolves [43]. It becomes apparent as glutamate concentration in blood serum grows and GABA content in it decreases.

Morphological changes in brain tissues prove pathogenetic impact exerted by MnO2 nanoparticles which was detected in the process of biochemical parameters assessment. Degenerative changes evolvement can be caused by direct oxidative

**113**

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

Some authors present data showing that chronic effects exerted by МnO2 nanoparticles lead to its accumulation in liver tissues with their consequent damage [45]. Histopathologic examinations revealed that МnO2 nanoparticles had toxic influence on liver and kidneys [46]. There is an assumption that manganese is transported to organs with significant mitochondria content (to liver, pancreas and

Thus, the research revealed that nanodisperse MnO2 water suspension, when being introduced daily into Wistar rats via gastric tube in doses equal to 260, 50, 10 mg/kg of body weight/a day during 90 days, causes lipid peroxidation activation (higher levels of lipid hydroperoxides and malonic dialdehyde in blood serum), and decrease in antioxidation system activity (lower OAS and Cu/Zn-SOD concentrations in blood serum). MnO2 nanoparticles damage neurons and astrocytes membranes and lead to improper neurotransmitters ratio (higher glutamate

concentration and lower GABA in blood serum). Pathomorphologic brain disorders occurred, such as vessels hyperemia, subarachnoid hemorrhages, brain edema with perivascular and pericellular spaces dilatation, focuses of nerve fibers demyeliniza-

When a dose of nanodisperse MnO2 water suspension was equal to 5 mg/kg of

To examine chronic toxicity of nanodisperse MnO2 water suspension, a comprehensive experiment was performed on white male Wistar rats with body weight equal to 90 ± 5 g (n = 360). specific and integral parameters of negative effects in target organs were studied at daily intragastric introduction of nanodisperse MnO2 water suspension via gastric tube. The experiment lasted for 180 days. The tested substance was daily introduced intragastrically via gastric tube, one time a day, the interval between introductions amounted to 24 hours. Mn concentration in water suspension in terms of

suspension was introduced in the following doses: group 1–2.5 mg/kg (1/1000 LD50);

(control one)—distilled water in volume equal. The substance concentration in the suspension was controlled weekly. There were several assessments of animals' overall health state, their survival rate, and body weight dynamics; these assessments took place just before the experiment beginning, then on the 30th day, 60th day, 90th day, 120th day, 150th day, and 180th day of the experiment. Blood samples were taken from animals of all groups out of a caudal vein; biochemical and hematologic parameters were detected before the experiment beginning, on the 30th day and on the 180th day.

group 2–0.25 mg/kg (1/10,000 LD50); group 3–0.05 mg/kg (1/50,000 LD50

. Nanodisperse MnO2 water

3

); group 4

**Studying chronic toxicity of nanodisperse MnO2 water suspension at intragastric introduction via gastric tube**: toxicity of nanodisperse MnO2 water suspension was examined at intragastric introduction via gastric tube under chronic experiment conditions; the examination was accomplished in full conformity with Methodical guidelines on toxicological-hygienic assessment of nanomaterial safety and Methodical guidelines on assessment order when assessing toxic effects exerted

hypophysis in particular) where it is accumulated very fast [47].

tion, focal dystrophic changes in vessels endothelium.

by nanomaterials on laboratory animals [48, 49].

MnO2 was determined it amounted to 41.37 ± 2.5 mg/cm3

body weight/a day the substance did not exert any toxic effects.

influence which nanoparticles have on neurons, glia cells, and vessels endothelium. The detected effects can only result from direct contact between nanoparticles and brain tissues which is the evidence of probable penetration through blood-brain barrier. The results of examining morphologic changes in brain tissues of Wistar rats, which were obtained at sub-acute intragastric introduction of nanodispersed MnO2, enrich the existing data at sub-chronic intragastric introduction of МnO2 nanoparticles, obtained by other authors. When МnO2 nanoparticles are once a day introduced into rats' bodies orally during 28 days they are absorbed, accumulated in tissues after exposure and reveal their toxicity in doses lower than in case of micro-

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

disperse analog [44].

#### *Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

influence which nanoparticles have on neurons, glia cells, and vessels endothelium. The detected effects can only result from direct contact between nanoparticles and brain tissues which is the evidence of probable penetration through blood-brain barrier. The results of examining morphologic changes in brain tissues of Wistar rats, which were obtained at sub-acute intragastric introduction of nanodispersed MnO2, enrich the existing data at sub-chronic intragastric introduction of МnO2 nanoparticles, obtained by other authors. When МnO2 nanoparticles are once a day introduced into rats' bodies orally during 28 days they are absorbed, accumulated in tissues after exposure and reveal their toxicity in doses lower than in case of microdisperse analog [44].

Some authors present data showing that chronic effects exerted by МnO2 nanoparticles lead to its accumulation in liver tissues with their consequent damage [45]. Histopathologic examinations revealed that МnO2 nanoparticles had toxic influence on liver and kidneys [46]. There is an assumption that manganese is transported to organs with significant mitochondria content (to liver, pancreas and hypophysis in particular) where it is accumulated very fast [47].

Thus, the research revealed that nanodisperse MnO2 water suspension, when being introduced daily into Wistar rats via gastric tube in doses equal to 260, 50, 10 mg/kg of body weight/a day during 90 days, causes lipid peroxidation activation (higher levels of lipid hydroperoxides and malonic dialdehyde in blood serum), and decrease in antioxidation system activity (lower OAS and Cu/Zn-SOD concentrations in blood serum). MnO2 nanoparticles damage neurons and astrocytes membranes and lead to improper neurotransmitters ratio (higher glutamate concentration and lower GABA in blood serum). Pathomorphologic brain disorders occurred, such as vessels hyperemia, subarachnoid hemorrhages, brain edema with perivascular and pericellular spaces dilatation, focuses of nerve fibers demyelinization, focal dystrophic changes in vessels endothelium.

When a dose of nanodisperse MnO2 water suspension was equal to 5 mg/kg of body weight/a day the substance did not exert any toxic effects.

**Studying chronic toxicity of nanodisperse MnO2 water suspension at intragastric introduction via gastric tube**: toxicity of nanodisperse MnO2 water suspension was examined at intragastric introduction via gastric tube under chronic experiment conditions; the examination was accomplished in full conformity with Methodical guidelines on toxicological-hygienic assessment of nanomaterial safety and Methodical guidelines on assessment order when assessing toxic effects exerted by nanomaterials on laboratory animals [48, 49].

To examine chronic toxicity of nanodisperse MnO2 water suspension, a comprehensive experiment was performed on white male Wistar rats with body weight equal to 90 ± 5 g (n = 360). specific and integral parameters of negative effects in target organs were studied at daily intragastric introduction of nanodisperse MnO2 water suspension via gastric tube. The experiment lasted for 180 days. The tested substance was daily introduced intragastrically via gastric tube, one time a day, the interval between introductions amounted to 24 hours. Mn concentration in water suspension in terms of MnO2 was determined it amounted to 41.37 ± 2.5 mg/cm3 . Nanodisperse MnO2 water suspension was introduced in the following doses: group 1–2.5 mg/kg (1/1000 LD50); group 2–0.25 mg/kg (1/10,000 LD50); group 3–0.05 mg/kg (1/50,000 LD50 3 ); group 4 (control one)—distilled water in volume equal. The substance concentration in the suspension was controlled weekly. There were several assessments of animals' overall health state, their survival rate, and body weight dynamics; these assessments took place just before the experiment beginning, then on the 30th day, 60th day, 90th day, 120th day, 150th day, and 180th day of the experiment. Blood samples were taken from animals of all groups out of a caudal vein; biochemical and hematologic parameters were detected before the experiment beginning, on the 30th day and on the 180th day.

*Heavy Metal Toxicity in Public Health*

detected after microdisperse MnO2 introduction.

with blood.

Brain cortex tissues layers are differentiated quite well. External granular layer is formed out of solid bunch consisting of numerous small neurons; pyramidal layer of brain cortex is wide and consists of polymorphous neurons. Internal granular layer of brain cortex and cerebellum is thin, noncontinuous, made of small pyramidal and stellate cells; ganglionic layer cells in brain cortex tissues are large, polymorphous with dark nucleuses, diffusely located; there is a great number of diverse neurons (they differ in form and size) in polymorphous cells layer. Ganglionic layer in cerebellum tissue has one raw of Purkinje cells with well-developed eosinophilic granular cytoplasm and rounded dark nucleuses. Molecular layer in cerebellum tissue is spongy and contains a small number of minor cells. White substance in brain and cerebellum tissue is made of evenly painted nerve fibers bunches and rounded glia cells. Brain vessels have thin walls and are feebly or moderately filled

Comparison of pathomorphologic effects occurring at intragastric introduction of nanodisperse MnO2 water suspension and microdisperse MnO2 water suspension in a dose equal to 10 mg/kg during 90 days showed that pathomorphologic changes in brain and cerebellum tissues were much more apparent and wide-spread when nanodisperse MnO2 was introduced. After introduction of nanodisperse MnO2 water suspension more apparent changes occurred in circulatory system in a form of hemodynamic disorders with focal feeble and moderate vessels hyperemia in brain, liver, lungs, kidneys, and heart; there were also subarachnoid hemorrhages in brain. Changes in lymphatic system became evident through feeble and moderate perivascular lymph-macrophage infiltrates in lungs tissue penetrating into adjacent alveoli. Pathologic changes in macrophage system were apparent through alveolar macrophages activation with formation of small bunches in alveoli lumen. After introduction of microdisperse MnO2 water suspension changes are represented by focal vessels hyperemia, subarachnoid hemorrhages in brain, small focal lymphmicrophage infiltrates in lungs and gastrointestinal tract. Besides, after introduction of nanodisperse MnO2 water suspension there was perivascular and pericellular spaces dilatation in brains, feebly apparent perivascular thin fibrous cardiosclerosis and feebly apparent focal protein dystrophy in hepatocytes; no such effects were

All the obtained materials were summarized; it allowed to make an assumption on a possible mechanism of toxic impact exerted by nanodisperse MnO2 particles at oral introduction. Lipid peroxidation activation caused by direct damaging impact exerted by nanodisperse MnO2 particles on bilipid layer of cytoplasmatic membrane can underlie the whole process [42]. High levels of lipid hydroperoxides and malonic dialdehyde in blood serum are the evidence of this effect. Lower levels of Cu/Zn-SOD and OAS in blood serum prove antioxidation processes insufficiency. The results obtained in the research showed that astrocytes and neurons membranes are the first targets influenced by nanodisperse MnO2 water suspension at oral introduction, just as at inhalation one [21, 26]. In case of oral introduction it can be determined by apparent ability of nanodisperse MnO2 particles to penetrate into blood from gastrointestinal tract. MnO2 nanoparticles reach brain tissues coming from bloodstream through capillary endothelial cells of blood brain barrier and accumulate in astrocytes [26]. Damaged astrocytes due to enhanced peroxidation of cells membranes lipids and active oxygen forms occurrence can lose their ability to capture and neutralize excessive quantities of "exciting" amino acid, namely glutamate; thus, excitotoxic effect evolves [43]. It becomes apparent as glutamate

concentration in blood serum grows and GABA content in it decreases.

Morphological changes in brain tissues prove pathogenetic impact exerted by MnO2 nanoparticles which was detected in the process of biochemical parameters assessment. Degenerative changes evolvement can be caused by direct oxidative

**112**

The authors selected parameters for assessing animals body responses at chronic introduction of the tested substance in accordance with Methodical guidelines on toxicological-hygienic assessment of nanomaterial safety [48]. Animals were taken out of the experiment on the 180th day via sparing euthanasia with carbon dioxide. After euthanasia they were autopsied, and microscopic assessment of morphological changes in internal organs followed. Nanodisperse MnO2 particles were identified in blood of experimental animals at intragastric introduction via gastric tube on the 180th day of the experiment.

The obtained results revealed that hair state, motion activity, food consumption, and body weight of experimental animals in experimental groups did not have any discrepancies with the same parameters in the control group during the whole experiment. Body weight dynamics of animals from experimental groups did not differ authentically from body weight dynamics of animals from control group. There were no deaths of experimental animals in experiment and control groups at introduction of nanodisperse MnO2water suspension during the whole observation period.

Basing on the analysis of changes in hematologic and biochemical blood parameters of Wistar rats from groups 1 and 2 against group 4, the following negative effects, which characterize toxicity of nanosized MnO2 water suspension, were highlighted:


**115**

agglomeration.

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

in blood serum of animals from group 1 on the 20th day of the experiment was 1.3 times lower; and in group 2, 1.5 times lower than in group 4; this parameter in groups 1 and 2 was 2.2 times lower than in group 4 on the 180th day of the experiment (р = 0.0011). There were not any authentic changes in these

3.*Inflammatory effect:* inflammatory effect was detected in groups 1 and 2 as per 1.6 times increase in CRP level on the 30th day of the experiment; on the 180th day, it was 1.9 times higher than in control group (р = 0.001–0.002). Animals from group 1 had higher TNF on the 30th day of the experiment (5.5 times higher), and animals from group 2, 6.8 times higher in comparison with the same parameter in group 4 (р = 0.001). On the 180th day of the experiment,

4.*Insufficiency of bowels brush border epithelium:* lower activity of β-galactosidase in blood serum may indicate that insufficiency of brush border epithelium in bowels evolves. This parameter was 3.9 times lower in group 1 and 2.9 times lower in group 2 in comparison with group 4 on the 180th day of the experiment (р = 0.001). There were not any changes in this parameter in group 3

5.*Electrolyte balance violation* electrolyte balance violation was detected in group 1 as there was an authentic decrease in K level in blood serum on the 180th day. The level was 1.3 times lower than the same parameter level in

6.*Sensitization:* sensitization occurrence was detected as per EO increase in blood on the 30th day of the experiment. It grew 1.8 times in group 1 and 1.9 times in group 2 in comparison with this parameter level in group 4 (р = 0.013–0.026). EO-LY level increased 1.3–1.7 times and 1.3 times against the control parameter (р = 0.001–0.036). MO level decreased 1.6 times in group 1 on the 30th day; later this parameter grew 1.5 times on the 180th day (р = 0.006–0.023). Sensitization in animals from groups 1 and 2 remained on the 180th day as MO quantity increased 1.4–1.45 times, NE quantity decreased 1.5–1.7 times, and EO quantity decreased 3.7–5.1 times against group 4 (р = 0.001–0.016). There were not any authentic changes in the examined parameters in group 3 in comparison with control group during the whole experiment. As the remaining biochemical parameters were assessed, no authentic discrepancies were revealed between experimental

No authentic changes in the examined parameters were detected against the control group when nanodisperse MnO2 water suspension was introduced in a dose equal to 0.05 mg/kg during 180 days. Electronic microscopy of animals' whole blood did not reveal any nanodisperse particles in blood of animals from group 4. X-ray spectrometry analysis did not reveal any Mn particles either (**Figure 15**). When nanodisperse MnO2 water suspension was introduced intragastrically via gastric tube into Wistar rates in a dose equal to 0.25 mg/kg (1/10,000 LD50) during 180 days there were bunches of particles which were ellipsoid-shaped and needle-shaped on electronic images. There were not any such particles in blood of rats from the control group (**Figure 16**). The size of these particles lies within 90–130 nm range, and it can be determined by their

TNF increased 4.4 times in group 1 and 5.4 times in group 2.

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

parameters in animals from group 3.

against the control level.

group 4 (р = 0.011).

groups and control group.

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

in blood serum of animals from group 1 on the 20th day of the experiment was 1.3 times lower; and in group 2, 1.5 times lower than in group 4; this parameter in groups 1 and 2 was 2.2 times lower than in group 4 on the 180th day of the experiment (р = 0.0011). There were not any authentic changes in these parameters in animals from group 3.


No authentic changes in the examined parameters were detected against the control group when nanodisperse MnO2 water suspension was introduced in a dose equal to 0.05 mg/kg during 180 days. Electronic microscopy of animals' whole blood did not reveal any nanodisperse particles in blood of animals from group 4. X-ray spectrometry analysis did not reveal any Mn particles either (**Figure 15**). When nanodisperse MnO2 water suspension was introduced intragastrically via gastric tube into Wistar rates in a dose equal to 0.25 mg/kg (1/10,000 LD50) during 180 days there were bunches of particles which were ellipsoid-shaped and needle-shaped on electronic images. There were not any such particles in blood of rats from the control group (**Figure 16**). The size of these particles lies within 90–130 nm range, and it can be determined by their agglomeration.

*Heavy Metal Toxicity in Public Health*

day of the experiment.

period.

highlighted:

The authors selected parameters for assessing animals body responses at chronic introduction of the tested substance in accordance with Methodical guidelines on toxicological-hygienic assessment of nanomaterial safety [48]. Animals were taken out of the experiment on the 180th day via sparing euthanasia with carbon dioxide. After euthanasia they were autopsied, and microscopic assessment of morphological changes in internal organs followed. Nanodisperse MnO2 particles were identified in blood of experimental animals at intragastric introduction via gastric tube on the 180th

The obtained results revealed that hair state, motion activity, food consumption, and body weight of experimental animals in experimental groups did not have any discrepancies with the same parameters in the control group during the whole experiment. Body weight dynamics of animals from experimental groups did not differ authentically from body weight dynamics of animals from control group. There were no deaths of experimental animals in experiment and control groups at introduction of nanodisperse MnO2water suspension during the whole observation

Basing on the analysis of changes in hematologic and biochemical blood parameters of Wistar rats from groups 1 and 2 against group 4, the following negative effects, which characterize toxicity of nanosized MnO2 water suspension, were

1.*Oxidizing-antioxidant balance:* lipid peroxidation activation was detected as per lipid hydroperoxide growth in blood serum and MDA growth in blood plasma. Lipid hydroperoxide concentration in blood serum of experimental animals grew on the 30th day of the experiment; it grew 2.5 times in group 1 and 1.6 times in group 2 in comparison with the same parameter in group 4. On the 180th day of the experiment, these parameters were 2.0 and 1.7 times higher in comparison with the group 4 (р = 0.001–0.003). Increased MDA content was detected in experimental animals from group 1 and 2 on the 30th day of the experiment, and it was 1.9 times higher than in group 4 (р = 0.001–0.002). This parameter was at the same level as in group 4 on the 180th day of the experiment. There was a decrease in antioxidation system activity. On the 30th day of the experiment, animals from group 1 had authentic 1.4 times lower activity of Cu/Zn-SOD than the same parameter in group 4. This parameter in animals from group 2 was 1.3 times lower than in group 4. On the 180th day of the experiment, Cu/ Zn-SOD in animals from group 1 was 1.5 times lower; and in animals from group 2, 1.4 times lower (р = 0.001–0.005). Level of AOS in animals from group 1 was 4.7 times lower than in animals from group 4 on the 30th day of the experiment; the same parameter in group 2 was 5.9 times lower. On the 180th day, AOS in group 1 was 3.5 times lower; and in group 2, 4.1 times lower (р = 0.001–0.002). AOS and Cu/ZN-SOD parameters in group 3 corresponded to the same parameters in group 4 during the whole experiment.

2.*Neurotransmitter balance violation:* animals in group 1 had 1.4 times lower

dopamine level in blood serum on the 30th day of the experiment; and animals in group 2, 1.7 times lower level than animals in group 4 (р = 0.002). On the 180th day of the experiment, dopamine decreased 1.7 times in group 1 and 1.9 times in group 2. By the 30th day of the experiment, there was an increase in glutamate level in blood serum in groups 1 and 2 in comparison with group 4; this parameter was 2.5 and 3.3 times higher correspondingly (р = 0.0011). By the 180th day of the experiment, glutamate level in group 1 was 1.9 times higher than in group 4 and 3.7 times higher in group 2 (р = 0.001). GABA level

**114**

In spite of the fact that X-ray spectrometry analysis did not reveal any Mn content in blood of experimental animals, which received a dose equal to 0.25 mg/kg (and it can be due to spatial resolution inaccuracy of this technique), visualized particles were considered to be Mn nanosized particles as they are identical with the pure suspension sample of the tested substance and are not detected in blood of animals from the control group.

**Figure 15.** *X-ray spectrometry analysis of whole blood taken from a male Wistar rat form the control group [50].*

#### **Figure 16.**

*X-ray spectrometry analysis of whole blood taken from an experimental animal, which received nanodisperse MnO2 in a dose equal to 0.25 mg/kg [50].*

**117**

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

Embryotoxicity means capability of a chemical to exert negative influence on offspring during the initial period of pregnancy, i.e., the period between conception and embryo formation. Teratogenicity means capability of a chemical to cause malformations and deviations in offspring postnatal development when a female

Embryotoxic and teratogenic effects exerted by nanodisperse MnO2 were examined and assessed at oral introduction with water in accordance with methodical guidelines on studying embryotoxic effects of chemicals at hygienic validation of their maximum permissible concentration in water of water objects [52]. The experiment was performed on white senior male and female Wistar rats with body weight equal to 200 ± 10 g (n = 90). All the animals before the experiment underwent 14-day quarantine and were placed in standard cages made of polypropylene, two animals in each. Rats were divided into three groups, 15 animals in each. Groups 1 and 2 were experimental ones, and group 3 was a control one. To get female rats pregnant, intact male rats and intact female rats were made to mate under control conditions during two estrous cycles. The day of sperm detection in vaginal smear of a female rat via microscopy was thought to be the first day of pregnancy. Nanodisperse MnO2 water suspension was introduced into pregnant female rats daily one time a day via gastric tube from the first to the twenty-first pregnancy day in two doses: 2.50 mg/kg (1/1000 LD50) and 0.25 mg/kg (1/10,000 LD50). Control group of rats received distilled water. Changes in general condition and behavior of experimental animals were registered during the whole experiment. Rat's body weight was measured on the first, eighth, fourteenth and twenty-first day of pregnancy. Rats were taken out of the experiment via sparing euthanasia with carbon dioxide on the twenty-first day of pregnancy. Pregnant female rats were dissected just after euthanasia. Assessment was performed in two stages. On the first stage, overall disorders in fetus development (embryotoxicity) were assessed. On the second stage, the focus was on occurrence of congenital malformations in internal

The experiment results showed that pregnant female rats from groups 1 and 2 had ordinary motion activity during the observation period. Innate reflexes and reactions to external irritants were normal and rats ate their forage willingly. Their fur was clean, shiny, and smooth. Visible mucous tunics were physiologically colored without any discharge. Appearance, behavior, and body weight dynamics of rats from experimental groups did not have any authentic discrepancies from the same parameters of rats from control group during the whole observation period. There were not any signs of animals intoxication or death in both experimental groups during the whole observation period. The examination results showed that all the embryotoxicity parameters (number of implantation points, number of viable fetuses, and number of resorptions) of pregnant female rats receiving nanodisperse MnO2 water suspension via gastric tube formed during first to twenty-first day of pregnancy in doses equal to 2.5 and 0.25 mg/kg had no authentic discrepancies with the same parameters of rats from control group (р > 0.05). Fetuses from each litter did not have any visible external congenital malformations when being examined externally. There were not any authentic discrepancies between body weight or cranio-caudal body dimensions of fetuses from experimental and control groups. There were no morphologic changes in internal organs or skeletal system of fetuses from groups 1 and 2 at intragastric introduction of nanodisperse MnO2

**4. Examining and assessing potential reproductive and mutagenic toxicity of nanodisperse manganese oxide water suspension at oral** 

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

body is exposed to this chemical during pregnancy [51].

organs and skeletal system of fetuses (teratogenicity).

**introduction with water**

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

#### **4. Examining and assessing potential reproductive and mutagenic toxicity of nanodisperse manganese oxide water suspension at oral introduction with water**

Embryotoxicity means capability of a chemical to exert negative influence on offspring during the initial period of pregnancy, i.e., the period between conception and embryo formation. Teratogenicity means capability of a chemical to cause malformations and deviations in offspring postnatal development when a female body is exposed to this chemical during pregnancy [51].

Embryotoxic and teratogenic effects exerted by nanodisperse MnO2 were examined and assessed at oral introduction with water in accordance with methodical guidelines on studying embryotoxic effects of chemicals at hygienic validation of their maximum permissible concentration in water of water objects [52]. The experiment was performed on white senior male and female Wistar rats with body weight equal to 200 ± 10 g (n = 90). All the animals before the experiment underwent 14-day quarantine and were placed in standard cages made of polypropylene, two animals in each. Rats were divided into three groups, 15 animals in each. Groups 1 and 2 were experimental ones, and group 3 was a control one. To get female rats pregnant, intact male rats and intact female rats were made to mate under control conditions during two estrous cycles. The day of sperm detection in vaginal smear of a female rat via microscopy was thought to be the first day of pregnancy. Nanodisperse MnO2 water suspension was introduced into pregnant female rats daily one time a day via gastric tube from the first to the twenty-first pregnancy day in two doses: 2.50 mg/kg (1/1000 LD50) and 0.25 mg/kg (1/10,000 LD50). Control group of rats received distilled water. Changes in general condition and behavior of experimental animals were registered during the whole experiment. Rat's body weight was measured on the first, eighth, fourteenth and twenty-first day of pregnancy. Rats were taken out of the experiment via sparing euthanasia with carbon dioxide on the twenty-first day of pregnancy. Pregnant female rats were dissected just after euthanasia. Assessment was performed in two stages. On the first stage, overall disorders in fetus development (embryotoxicity) were assessed. On the second stage, the focus was on occurrence of congenital malformations in internal organs and skeletal system of fetuses (teratogenicity).

The experiment results showed that pregnant female rats from groups 1 and 2 had ordinary motion activity during the observation period. Innate reflexes and reactions to external irritants were normal and rats ate their forage willingly. Their fur was clean, shiny, and smooth. Visible mucous tunics were physiologically colored without any discharge. Appearance, behavior, and body weight dynamics of rats from experimental groups did not have any authentic discrepancies from the same parameters of rats from control group during the whole observation period. There were not any signs of animals intoxication or death in both experimental groups during the whole observation period. The examination results showed that all the embryotoxicity parameters (number of implantation points, number of viable fetuses, and number of resorptions) of pregnant female rats receiving nanodisperse MnO2 water suspension via gastric tube formed during first to twenty-first day of pregnancy in doses equal to 2.5 and 0.25 mg/kg had no authentic discrepancies with the same parameters of rats from control group (р > 0.05). Fetuses from each litter did not have any visible external congenital malformations when being examined externally. There were not any authentic discrepancies between body weight or cranio-caudal body dimensions of fetuses from experimental and control groups.

There were no morphologic changes in internal organs or skeletal system of fetuses from groups 1 and 2 at intragastric introduction of nanodisperse MnO2

*Heavy Metal Toxicity in Public Health*

from the control group.

In spite of the fact that X-ray spectrometry analysis did not reveal any Mn content in blood of experimental animals, which received a dose equal to 0.25 mg/kg (and it can be due to spatial resolution inaccuracy of this technique), visualized particles were considered to be Mn nanosized particles as they are identical with the pure suspension sample of the tested substance and are not detected in blood of animals

*X-ray spectrometry analysis of whole blood taken from an experimental animal, which received nanodisperse* 

*X-ray spectrometry analysis of whole blood taken from a male Wistar rat form the control group [50].*

**116**

**Figure 16.**

**Figure 15.**

*MnO2 in a dose equal to 0.25 mg/kg [50].*

water suspension into Wistar rats from first to twenty-first day of pregnancy; there were no discrepancies between morphologic characteristics of fetuses internal organs and fetuses skeletal system between experimental and control groups. Fetuses from experimental groups and control group had multilayer flat epidermis with increased number of layers up to eight cells; derma cells were spindle-shaped; fibers were tightly located and had numerous hair follicles. Subcutaneous fatty tissue was in the form of sporadic small lipocyte bunches (**Figure 17**). Small intestine wall is fully formed; limbic epithelium has characteristic appearance; Paneth cells are large and located not only in crypts bottom area, but also in villi epithelium. Beaker cells are sporadic (**Figure 18**).

Cartilaginous elements are mature; eosinophilic intracellular substance prevails in most cartilages (**Figures 19** and **20**).

There were numerous focuses of indirect and direct osteogenesis. Skeletal muscles are with small diameter of fibers and banding that is not clearly visible. Brain hemispheres cortex is cellular, layers are poorly differentiated and prevail over medulla. Cardiac histiocytes are with small section and large nucleuses. Fibers are spongy. Connective tissue content in cardiac muscle is minimal. Banding is not detected. Marrow is cellular; its bulk is represented by cells of erythropoetic type; there are no lipocytes.

**Examining and assessing mutagenic activity of nanodisperse MnO2 water suspension at oral introduction with water**: micronucleus test is a widespread technique applied for assessing mutagenic activity of new unknown chemicals; the test was independently worked out and implemented by Heddle and Schmid in early 1970s [53]. Micronucleuses are small DNA-containing formations consisting of acentric chromosome fragments. During telophase, these fragments can become a part of daughter cells nucleuses or form singular or numerous micronucleuses in cytoplasm. The test is based on microscopic detection of cells with micronucleuses. Spontaneous frequency of cells with micronucleuses amounts to 0.1–0.2% [54].

Data on mutagenic activity, which nanosized MnO2 particles, may be contradictory. The authors have not been able to find any research works proving apparent mutagenic properties of МnO2 nanoparticles. At the same time, some experts state that МnO2 nanoparticles are undoubtedly genotoxic "in vivo" [55]. There are data given by a number of researchers that manganese nanoparticles (52.1 ± 23.8 nm) at 24-hour exposure on PC-12 cells in concentration equal to 10 mg/cm3 are able to inhibit PARK2 gene and tyrosine hydroxylase gene expression (The latter is an enzyme that catalyzes the first limiting stage of catecholamines synthesis, including dopamine.) It is detected that manganese nanoparticles enhance SNCA gene

#### **Figure 17.**

*Epidermis of Wistar rat fetus on the twenty-first day of pregnancy at intragastric introduction of nanodisperse MnO2 water suspension via gastric tube. Painted with hematoxylin-eosin, magnification ×200 [10].*

**119**

**Figure 20.**

*magnification ×200 [10].*

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

*Small intestine of a Wistar rat fetus on the twenty-first day of pregnancy at intragastric introduction of nanodisperse MnO2 water suspension via gastric tube. Painted with hematoxylin-eosin, magnification ×200 [10].*

*Brainpan and brain of a Wistar rat fetus on the twenty-first day of pregnancy at intragastric introduction of nanodisperse MnO2 water suspension via gastric tube. Painted with hematoxylin-eosin, magnification ×200 [10].*

expression, which makes α-synucleins double in cells participating in various neurodegenerating disorders evolvement. After nanodisperse, МnO2 was orally introduced into Wistar rats in doses equal to 300 and 1000 mg/kg during 28 days; there

*Ribs and intercostal muscles of a Wistar rat fetus on the twenty-first day of pregnancy at intragastric introduction of nanodisperse MnO2 water suspension via gastric tube. Painted with hematoxylin-eosin,* 

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

**Figure 18.**

**Figure 19.**

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

#### **Figure 18.**

*Heavy Metal Toxicity in Public Health*

Beaker cells are sporadic (**Figure 18**).

in most cartilages (**Figures 19** and **20**).

there are no lipocytes.

water suspension into Wistar rats from first to twenty-first day of pregnancy; there were no discrepancies between morphologic characteristics of fetuses internal organs and fetuses skeletal system between experimental and control groups. Fetuses from experimental groups and control group had multilayer flat epidermis with increased number of layers up to eight cells; derma cells were spindle-shaped; fibers were tightly located and had numerous hair follicles. Subcutaneous fatty tissue was in the form of sporadic small lipocyte bunches (**Figure 17**). Small intestine wall is fully formed; limbic epithelium has characteristic appearance; Paneth cells are large and located not only in crypts bottom area, but also in villi epithelium.

Cartilaginous elements are mature; eosinophilic intracellular substance prevails

There were numerous focuses of indirect and direct osteogenesis. Skeletal muscles are with small diameter of fibers and banding that is not clearly visible. Brain hemispheres cortex is cellular, layers are poorly differentiated and prevail over medulla. Cardiac histiocytes are with small section and large nucleuses. Fibers are spongy. Connective tissue content in cardiac muscle is minimal. Banding is not detected. Marrow is cellular; its bulk is represented by cells of erythropoetic type;

**Examining and assessing mutagenic activity of nanodisperse MnO2 water suspension at oral introduction with water**: micronucleus test is a widespread technique applied for assessing mutagenic activity of new unknown chemicals; the test was independently worked out and implemented by Heddle and Schmid in early 1970s [53]. Micronucleuses are small DNA-containing formations consisting of acentric chromosome fragments. During telophase, these fragments can become a part of daughter cells nucleuses or form singular or numerous micronucleuses in cytoplasm. The test is based on microscopic detection of cells with micronucleuses. Spontaneous frequency of cells with micronucleuses amounts to 0.1–0.2% [54]. Data on mutagenic activity, which nanosized MnO2 particles, may be contradictory. The authors have not been able to find any research works proving apparent mutagenic properties of МnO2 nanoparticles. At the same time, some experts state that МnO2 nanoparticles are undoubtedly genotoxic "in vivo" [55]. There are data given by a number of researchers that manganese nanoparticles (52.1 ± 23.8 nm)

at 24-hour exposure on PC-12 cells in concentration equal to 10 mg/cm3

to inhibit PARK2 gene and tyrosine hydroxylase gene expression (The latter is an enzyme that catalyzes the first limiting stage of catecholamines synthesis, including dopamine.) It is detected that manganese nanoparticles enhance SNCA gene

*Epidermis of Wistar rat fetus on the twenty-first day of pregnancy at intragastric introduction of nanodisperse* 

*MnO2 water suspension via gastric tube. Painted with hematoxylin-eosin, magnification ×200 [10].*

are able

**118**

**Figure 17.**

*Small intestine of a Wistar rat fetus on the twenty-first day of pregnancy at intragastric introduction of nanodisperse MnO2 water suspension via gastric tube. Painted with hematoxylin-eosin, magnification ×200 [10].*

#### **Figure 19.**

*Brainpan and brain of a Wistar rat fetus on the twenty-first day of pregnancy at intragastric introduction of nanodisperse MnO2 water suspension via gastric tube. Painted with hematoxylin-eosin, magnification ×200 [10].*

#### **Figure 20.**

*Ribs and intercostal muscles of a Wistar rat fetus on the twenty-first day of pregnancy at intragastric introduction of nanodisperse MnO2 water suspension via gastric tube. Painted with hematoxylin-eosin, magnification ×200 [10].*

expression, which makes α-synucleins double in cells participating in various neurodegenerating disorders evolvement. After nanodisperse, МnO2 was orally introduced into Wistar rats in doses equal to 300 and 1000 mg/kg during 28 days; there was an increased number of DNA damages in leucocytes, and also an increased number of micronucleuses and chromosome aberrations in marrow cells.

Potential mutagenic activity of nanodisperse МnO2 water suspension in polychromatocytes (reticulocytes) of mammals marrow was assessed via micronucleus test [53]. The experiment was carried out on С57В1/6 white male mice with body weight equal to 20.0 ± 1.0 g (n = 24). The experiment lasted for 2 days. Experimental animals were divided into four groups (six animals in each); groups 1 and 2 were experimental ones, group 3 was negative control, and group 4 was positive control. Nanodisperse МnO2 water suspension was once introduced via gastric tube in two doses: group 1 received 10.3 mg/kg (1/250 LD50), group 2–5.15 mg/kg dose (1/500 LD50), and group 3 (negative control)—distilled water in a volume equal to 0.2 cm3 group 4 (positive control)—cyclophosphamide water suspension was once introduced intraperitoneally into mice from in a dose equal to 20 mg/kg in a volume equal to 0.2 cm3 . Cyclophosphamide is known to be cytogenetically active [13].

Thus, nanodisperse MnO2 water suspension at a single intragastric introduction via gastric tube into С57В1/6 male mice in doses equal to 10.3 and 5.15 mg/kg does not cause increased micronucleuses formation in vivo and, consequently, does not have any mutagenic effects. As per other authors' data, there is no information on possible penetration of МnO2 nanoparticles into cells nucleuses. It reduces the risk of direct contact between examined particles and cellular DNA [23]. DNA damage can occur through activation of lipid peroxidation and excessive AOF production from damaged membranes, which leads to cytokines induction (TNF-a tumor necrosis factor) and DNA damage. It can result in transcription factors activation, NF-KB in particular, which is responsible for polygenic expression. As a result, apoptotic mechanism is activated, or programmed cells death is inhibited, which can cause tumor activity [56, 57]. Some authors state that manganese nanoparticles (52.1 ± 23.8 nm) when exerting a 24-hour effect on PC-12 cells in concentration equal to 10 mg/cm3 are able to inhibit PARK2 gene expression and gene of tyrosine hydroxylase (an enzyme that catalyzes the first limiting stage of catecholamine synthesis, dopamine included). It is proved that manganese nanoparticles enhance SNCA gene expression which leads to double increase in α-synucleins in cells taking part in evolvement of various neurodegenerating disorders. When nanodisperse МnO2 was introduced into Wistar rats orally in doses equal to 300 and 1000 mg/kg during 28 days DNA damages in leucocytes increased, a number of micronucleuses and chromosome aberrations in marrow cells grew. These changes were accompanied with inhibition of various ATPases activity; here changes in ALAT, ASAT, and LDG activity in liver, kidneys, and blood serum were dose depending [44]. We could not find any research proving apparent mutagenic properties of МnO2 particles. At the same time, a number of authors showed that МnO2 nanoparticles had evident genotoxicity "in vivo" [43].

**Examining and assessing gonadotoxic activity (screening) of МnO2 oxide water suspension at oral introduction with water**: gonadotoxicity of nanodisperse MnO2 water suspension was examined on laboratory animals under subchronic experiment conditions in accordance with guidelines 2492-81 "On studying chemicals gonadotoxicity at hygienic standardization in water of water reservoirs" and international recommendations [58, 59]. The authors examined gonads of white male Wistar rats with body weight equal to 190 ± 20 g (n = 40). Experimental animals were divided into five groups, eight animals in each. Water suspension containing nanodisperse MnO2 was introduced in a concentration equal to 36.0 ± 2.3 mg/cm3 , into animals from experimental groups via gastric tube once a day every day in following doses: group 1 received 257.7 mg/kg (1/10 LD50), group 2–51.54 mg/kg (1/50 LD50), group 3–10.3 mg/kg (1/250 LD50),

**121**

**Figure 22.**

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

group 4–5.15 mg/kg (1/500 LD50), and group 5 (control)—distilled water in a volume equal. The experiment lasted for 90 days. Animals euthanasia was accomplished with carbon dioxide. Then, a special instrument was applied to take out epididymis and make a longitudinal cut along it. Extraction of the epididymis in

copy was performed with the use of МС 100Х microscope (Micros, Austria). The task was to assess such functional and morphometric parameters as sperm mass, total sperm quantity, number of alive sperm, sperm mobility duration, osmotic and

*A spermatozoon neck pathology in a male Wistar rat at oral introduction of nanodisperse МnO2 in a dose* 

*A spermatozoon neck and head pathology in a male Wistar rat at oral introduction of nanodisperse МnO2 in a* 

acidic resistance of sperm, relative quantity of sperm pathologies.

of 0.9% NaCl solution was performed during 2 minutes at room temperature

С. Sperm quantity was calculated in Goryaev chamber. Specimens micros-

The obtained results revealed authentic 2.18 times decrease in sperm quantity in male rats from group 1 against the control group (р < 0.05). Male rats from group 2 had authentically 1.7 times lower quantity of sperm than rats from the control group. There were no authentic discrepancies in sperm number between male rats from groups 3, 4 and 5 and the control group. There was an authentic 1.2–1.3 times decrease in osmotic and acidic resistance of sperm in rats from groups 1 and 2 against the control group parameter (р < 0.05). There were no discrepancies in this parameter in rats from groups 3, 4 and 5 and the control group. Such morphological changes as sperm head, tail, and neck pathology occurred in groups 1 and 2 1.7–10.3 times more frequently than in the control group (**Figures 21** and **22**). This parameter in groups 3, 4, and 5 did not have any discrepancies with the control

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

10 cm3

being 22°

group parameter.

**Figure 21.**

*equal to 51.54 mg/kg, magnification ×100 [55].*

*dose equal to 257.7 mg/kg, magnification ×200 [55].*

#### *Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*

group 4–5.15 mg/kg (1/500 LD50), and group 5 (control)—distilled water in a volume equal. The experiment lasted for 90 days. Animals euthanasia was accomplished with carbon dioxide. Then, a special instrument was applied to take out epididymis and make a longitudinal cut along it. Extraction of the epididymis in 10 cm3 of 0.9% NaCl solution was performed during 2 minutes at room temperature being 22° С. Sperm quantity was calculated in Goryaev chamber. Specimens microscopy was performed with the use of МС 100Х microscope (Micros, Austria). The task was to assess such functional and morphometric parameters as sperm mass, total sperm quantity, number of alive sperm, sperm mobility duration, osmotic and acidic resistance of sperm, relative quantity of sperm pathologies.

The obtained results revealed authentic 2.18 times decrease in sperm quantity in male rats from group 1 against the control group (р < 0.05). Male rats from group 2 had authentically 1.7 times lower quantity of sperm than rats from the control group. There were no authentic discrepancies in sperm number between male rats from groups 3, 4 and 5 and the control group. There was an authentic 1.2–1.3 times decrease in osmotic and acidic resistance of sperm in rats from groups 1 and 2 against the control group parameter (р < 0.05). There were no discrepancies in this parameter in rats from groups 3, 4 and 5 and the control group. Such morphological changes as sperm head, tail, and neck pathology occurred in groups 1 and 2 1.7–10.3 times more frequently than in the control group (**Figures 21** and **22**). This parameter in groups 3, 4, and 5 did not have any discrepancies with the control group parameter.

#### **Figure 21.**

*Heavy Metal Toxicity in Public Health*

water in a volume equal to 0.2 cm3

cytogenetically active [13].

equal to 10 mg/cm3

had evident genotoxicity "in vivo" [43].

equal to 36.0 ± 2.3 mg/cm3

equal to 20 mg/kg in a volume equal to 0.2 cm3

was an increased number of DNA damages in leucocytes, and also an increased number of micronucleuses and chromosome aberrations in marrow cells. Potential mutagenic activity of nanodisperse МnO2 water suspension in polychromatocytes (reticulocytes) of mammals marrow was assessed via micronucleus test [53]. The experiment was carried out on С57В1/6 white male mice with body weight equal to 20.0 ± 1.0 g (n = 24). The experiment lasted for 2 days. Experimental animals were divided into four groups (six animals in each); groups 1 and 2 were experimental ones, group 3 was negative control, and group 4 was positive control. Nanodisperse МnO2 water suspension was once introduced via gastric tube in two doses: group 1 received 10.3 mg/kg (1/250 LD50), group 2–5.15 mg/kg dose (1/500 LD50), and group 3 (negative control)—distilled

water suspension was once introduced intraperitoneally into mice from in a dose

hydroxylase (an enzyme that catalyzes the first limiting stage of catecholamine synthesis, dopamine included). It is proved that manganese nanoparticles enhance SNCA gene expression which leads to double increase in α-synucleins in cells taking part in evolvement of various neurodegenerating disorders. When nanodisperse МnO2 was introduced into Wistar rats orally in doses equal to 300 and 1000 mg/kg during 28 days DNA damages in leucocytes increased, a number of micronucleuses and chromosome aberrations in marrow cells grew. These changes were accompanied with inhibition of various ATPases activity; here changes in ALAT, ASAT, and LDG activity in liver, kidneys, and blood serum were dose depending [44]. We could not find any research proving apparent mutagenic properties of МnO2 particles. At the same time, a number of authors showed that МnO2 nanoparticles

**Examining and assessing gonadotoxic activity (screening) of МnO2 oxide** 

**water suspension at oral introduction with water**: gonadotoxicity of nanodisperse MnO2 water suspension was examined on laboratory animals under subchronic experiment conditions in accordance with guidelines 2492-81 "On studying chemicals gonadotoxicity at hygienic standardization in water of water reservoirs" and international recommendations [58, 59]. The authors examined gonads of white male Wistar rats with body weight equal to 190 ± 20 g (n = 40). Experimental animals were divided into five groups, eight animals in each. Water suspension containing nanodisperse MnO2 was introduced in a concentration

tube once a day every day in following doses: group 1 received 257.7 mg/kg (1/10 LD50), group 2–51.54 mg/kg (1/50 LD50), group 3–10.3 mg/kg (1/250 LD50),

Thus, nanodisperse MnO2 water suspension at a single intragastric introduction via gastric tube into С57В1/6 male mice in doses equal to 10.3 and 5.15 mg/kg does not cause increased micronucleuses formation in vivo and, consequently, does not have any mutagenic effects. As per other authors' data, there is no information on possible penetration of МnO2 nanoparticles into cells nucleuses. It reduces the risk of direct contact between examined particles and cellular DNA [23]. DNA damage can occur through activation of lipid peroxidation and excessive AOF production from damaged membranes, which leads to cytokines induction (TNF-a tumor necrosis factor) and DNA damage. It can result in transcription factors activation, NF-KB in particular, which is responsible for polygenic expression. As a result, apoptotic mechanism is activated, or programmed cells death is inhibited, which can cause tumor activity [56, 57]. Some authors state that manganese nanoparticles (52.1 ± 23.8 nm) when exerting a 24-hour effect on PC-12 cells in concentration

are able to inhibit PARK2 gene expression and gene of tyrosine

, into animals from experimental groups via gastric

group 4 (positive control)—cyclophosphamide

. Cyclophosphamide is known to be

**120**

*A spermatozoon neck pathology in a male Wistar rat at oral introduction of nanodisperse МnO2 in a dose equal to 51.54 mg/kg, magnification ×100 [55].*

#### **Figure 22.**

*A spermatozoon neck and head pathology in a male Wistar rat at oral introduction of nanodisperse МnO2 in a dose equal to 257.7 mg/kg, magnification ×200 [55].*

Therefore, nanodisperse МnO2 water suspension does not exert any gonadotoxic effects on male Wistar rats when it is introduced into them via gastric tube during 90 days in doses equal to 10.3–5.15 mg/kg.

#### **5. Conclusion**

Contemporary research in nanotoxicology calls for studying and systemizing miscellaneous toxic effects exerted by new nanomaterials at various introduction ways and exposure period. Special attention is paid to determining target organs and negative effects caused by nanoparticles impacts, which do not occur after exposure to analog microparticles. To obtain comprehensive characteristics, one should study remote toxic effects such as embryotoxicity, gonadotoxicity, mutagenic activity occurrence, etc.

The authors conducted experimental research of nanodisperse МnO2 water suspension at intragastric, inhalation, and skin-resorptive introduction into small rodents (mice and Wistar rats) with various exposure periods. It allowed to obtain a sufficiently profound and detailed characteristics of the toxic effects exerted by this substance, to determine the target organs and to reveal dose-dependent effects.

The obtained knowledge provides better understanding of toxic impact exerted by nanosized metal oxides, which have great potential of application in human activities. It is vital for working out efficient measures aimed at providing safety in production processes and in nanomaterials application.

#### **Acknowledgements**

The authors are grateful to specialists working at the multiphase disperse system laboratory of Technical Chemistry Institute (the Russian Academy of Sciences, Urals Branch), specialists working at Chemical Technologies Department of Perm National Research Polytechnic University, specialists working at Human Ecology and Life Activity Safety Department of Perm State National Research University, as they all contributed into providing data and materials for this edition.

#### **Notation and abbreviations**


**123**

**Author details**

provided the original work is properly cited.

Management Technologies", Perm, Russian

\*Address all correspondence to: zem@fcrisk.ru

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Nina Vladimirovna Zaitseva and Marina Alexandrovna Zemlyanova\* FBSI "Federal Scientific Center for Medical and Preventive Health Risk

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties…*

LD50 dose causing death of animals in quantity 50% TL50 time causing death of animals in quantity 50%

*DOI: http://dx.doi.org/10.5772/intechopen.83499*

GABA γ-aminobutyric acid LP lipid peroxidation MDA malonic dialdehyde

Cu/Zn-SOD Cu/Zn-superoxide dismutase OAS overall antioxidant state DNA deoxyribonucleic acid

*Toxicologic Characteristics of Nanodisperse Manganese Oxide: Physical-Chemical Properties… DOI: http://dx.doi.org/10.5772/intechopen.83499*


### **Author details**

*Heavy Metal Toxicity in Public Health*

genic activity occurrence, etc.

**Acknowledgements**

**Notation and abbreviations**

XRD X-ray diffraction

AOF active oxygen forms GSSG oxidized glutathione form GSH reduced glutathione form

LDL0 minimal lethal dose

IT information technology EU European Union

SUN sustainable nanotechnologies GLP good laboratory practice

CAS chemical abstracts service

CTAB cetyltrimethylammonium bromide ILAR, Institute for Laboratory Animal Research

DELS division on earth and life studies

TCL0 minimal toxic concentration

**5. Conclusion**

90 days in doses equal to 10.3–5.15 mg/kg.

Therefore, nanodisperse МnO2 water suspension does not exert any gonadotoxic effects on male Wistar rats when it is introduced into them via gastric tube during

Contemporary research in nanotoxicology calls for studying and systemizing miscellaneous toxic effects exerted by new nanomaterials at various introduction ways and exposure period. Special attention is paid to determining target organs and negative effects caused by nanoparticles impacts, which do not occur after exposure to analog microparticles. To obtain comprehensive characteristics, one should study remote toxic effects such as embryotoxicity, gonadotoxicity, muta-

The authors conducted experimental research of nanodisperse МnO2 water suspension at intragastric, inhalation, and skin-resorptive introduction into small rodents (mice and Wistar rats) with various exposure periods. It allowed to obtain a sufficiently profound and detailed characteristics of the toxic effects exerted by this substance, to determine the target organs and to reveal dose-dependent effects. The obtained knowledge provides better understanding of toxic impact exerted

by nanosized metal oxides, which have great potential of application in human activities. It is vital for working out efficient measures aimed at providing safety in

laboratory of Technical Chemistry Institute (the Russian Academy of Sciences, Urals Branch), specialists working at Chemical Technologies Department of Perm National Research Polytechnic University, specialists working at Human Ecology and Life Activity Safety Department of Perm State National Research University, as

they all contributed into providing data and materials for this edition.

IUPAC International Union of Pure and Applied Chemistry

CL50 concentration causing death of animals in quantity 50%

The authors are grateful to specialists working at the multiphase disperse system

production processes and in nanomaterials application.

**122**

Nina Vladimirovna Zaitseva and Marina Alexandrovna Zemlyanova\* FBSI "Federal Scientific Center for Medical and Preventive Health Risk Management Technologies", Perm, Russian

\*Address all correspondence to: zem@fcrisk.ru

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Toxicology. 2004;**16**:437-445. DOI: 10.1080/08958370490439597

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manganese oxide safety (III, IV). Nutrition Issues. 2012;**81**(5):13-19

assessment of cumulation. Hygiene and

[41] Zaitseva N, Zemlyanova М, Zvezdin V, Akafyeva T, Mazunina D, Dovbyish А. Effects of subchronic exposure manganese oxide nanoparticles on the central nervous system, lipid

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[42] Sakon S, Xue X, Takekawa M, Sasazuki T, Okazaki T, Kojima Y, et al. NF-kB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death. The EMBO Journal. 2003;**22**(15):3898-3909.

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ijms12096267

H. Nanoparticles and neurotoxicity. Journal of Molecular Science. 2011;**12**:6267-6280. DOI: 10.3390/

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nanoparticles in Wistar rats after 28 days of repeated oral exposure. Journal of Applied Toxicology. 2013;**33**(10):1165-

[40] Mousavi Z, Hassanpourezatti M, Najafizadeh P, Rezagholian S, Safi Rhamanifar M, Nosrati N. Effects of subcutaneous injection MnO2 micro- and nanoparticles on blood glucose level and lipid profile in rat. The Iranian Journal of Medical Sciences. 2016;**41**(6):518-524

[39] Shtabskiy B. Quantitative

Sanitary. 1973;**8**:24-28

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[33] Li T, Shi T, Li X, Zeng S, Yin L, Pu Y. Effects of nano-MnO2 on dopaminergic neurons and the spatial learning capability of rats. International Journal of Environmental Research and Public Health. 2014;**11**(8):7918-7930. DOI: 10.3390/ijerph110807918

[34] Zemlyanova M, Zvezdin V, Akafieva T. Inhalation toxicity of nanodispersed manganese oxide aerosol. Labor Medicine and Industrial Ecology.

[35] Amatucci GG, Pereira N. Fluoride based electrode materials for advanced energy storage devices. Journal of Fluorine Chemistry. 2007;**128**:243-262. DOI: 10.1016/j.jfluchem.2006.11.016

[36] Hua M, Zhang S, Pan B, Zhang W, Lv L, Zhang Q. Heavy metal removal from water/wastewater by nanosized metal oxides: A review. Journal of Hazardous Materials. 2012;**211-212**:317- 331. DOI: 10.1016/j.jhazmat.2011.10.016

[37] Zaitseva N, Zemlyanova M, Zvezdin V, Akafyeva T. Biological effects of manganese oxide nanoparticles after peroral intake. Journal of Pharmacy and Nutrition Sciences. 2013;**3**(4):231-237

[38] Zaitseva N, Zemlyanova М, Zvezdin V, Saenko Е, Tarantin А, Makhmudov R, et al. Toxicological-hygienic assessment of nanodisperse and microdisperse

2015;**12**:13-17

**126**

[47] Deng Q, Liu J, Li Q, Ket C, Liu Z, Shen Y, et al. Interaction of occupational manganese exposure and alcohol drinking aggravates the increase of liver enzyme concentrations from a cross-sectional study in China. Environmental Health. 2013;**12**:30. DOI: 10.1186/1476-069X-12-30

[48] Onishhenko GG, Tutel'jan VA, Hotimchenko SA, Gmoshinskij IV, et al. Toxicologic-Hygienic Assessment of Nanomaterial Safety: MU 1.2.2520-09. Moscow: Federal Center for Hygiene and Epidemiology of Rospotrebnadzor; 2009. 43 p

[49] Onishhenko GG, Bragina IV, Aksenova OI, Zavistjaeva TJu, Assessment et al. Order when Assessing Toxic Effects Exerted by Nanomaterials on Laboratory Animals. MU1.2.2869-11. Moscow: Federal center for Hygiene and Epidemiology of Rospotrebnadzor; 2011. 23 p

[50] Zaitseva N, Zemlyanova M, Zvezdin V, Sayenko Y. Toxicological and hygienic safety assessment of the aqueous suspension of nano-dispersed silicon dioxide, synthesized using liquidcrystal templating. Health Risk Analysis. 2013;**1**:65-72

[51] Sanockij I, Sidorov K, editors. Russian Glossary of Selected Terms from Preventive Toxicology. Moscow: Centr mezhdunar. proektov GKNT; 1982. 68 p

[52] Methodical Guidelines on Studying Embryotoxic Effects of Chemicals at

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[53] Ledebur MV, Schmid W. The micronucleus test methodological aspects. Mutation Research/ Fundamental and Molecular Mechanisms of Mutagenesis. 1973;**19**(1):109-117. DOI: 10.1016/0027-5107(73)90118-8

[54] Manual on experimental (preclinical) study of new pharmacological substances. Edited by a corresponding member of the RAMS, Prof. R.N. Habriev. Moscow: Methodical Guidelines; 2005. 832 p. Available from: https://elibrary.ru/item. asp?id=19116235&

[55] Minigalieva IA, Katsnelson BA, Privalova LI, Sutunkova MP, Gurvich VB, Shur VY, et al. Attenuation of combined nickel (II) oxide and manganese (II, III) oxide nanoparticles' adverse effects with a complex of bioprotectors. International Journal of Molecular Sciences. 2015;**16**(9):22555-22583. DOI: 10.3390/ ijms160922555

[56] Pahl H. Activators and target genes of Rel/NF–kB transcription factors. Oncogene. 1999;**18**:6853-6866. DOI: 10.1038/sj.onc.1203239

[57] Takizawa H, Ohtoshi T, Kawasaki S, Kohyama T, Desaki M, Kasama T, et al. Diesel exhaust particles induce NF-kB activation in human bronchial epithelial cells in vitro: Importance in cytokine transcription. Journal of Immunology. 1999;**162**:4705-4711. DOI: 0022-1767/99/\$02.00

[58] Guidelines for the Study Gonadotoxic Action of Chemical Substances in Hygienic Rationing of Water Reservoirs. MU 2492-81 [Internet]. 1981. Available from: http://docs.cntd.ru/document/ 675400365 [Accessed: 11 December 2018]

[59] ICH Harmonised Tripartite Guideline [Internet]. 1993. Available from: https://www.ich.org/fileadmin/ Public\_Web\_Site/ICH\_Products/ Guidelines/Efficacy/E7/Step4/E7\_ Guideline.pdf [Accessed: 11 December 2018]

**129**

**Chapter 7**

**Abstract**

designed to treat specific cancers.

**1. Introduction**

**Keywords:** cell cycle, iron chelation, redox, ROS, ferroptosis

Intracellular Iron Concentration

and Distribution Have Multiple

Iron is essential for numerous cellular reactions that require oxygen transfer. Iron deficiency is a common problem in humans and is the most common nutritional disease worldwide. However, excess cellular iron can be toxic. Maintenance of iron hemostasis utilizes specialized pathways responsible for iron transport, iron uptake by cells, and appropriate cellular distribution of iron for utilization or storage. This chapter reviews how iron depletion is associated with inhibition of cellular proliferation and cell cycle arrest at different parts of the cell cycle. These effects are based on the effective chelation of iron, and more importantly on differences in various tissue responses to both iron depletion and iron toxicity. These differences may explain why in some tissues, particularly rapidly growing cancer cells, iron depletion causes cell cycle arrest and apoptosis, a form of programed cell death. Other neoplastic tissues are more prone to the toxic effects of iron, which can induce autophagic cell death (termed ferroptosis) via reactive oxygen species resulting in lysosomal degradation of cellular constituents. An appreciation of these differences can be utilized by novel pharmaceutical agents discussed below

Iron is mainly used in oxygen transfer reactions necessary for moving oxygen to tissues by heme moieties including hemoglobin and myoglobin, enzymes necessary for oxidative phosphorylation, and oxygen transfer reactions by enzymes containing iron sulfur compounds [1, 2]. Compared to other essential micronutrients, iron is found in relatively high (micromolar) concentrations in tissues and serum. Although essential for these oxygen transfer reactions, excess iron is toxic [1–4]. Therefore complex pathways have evolved to maintain proper iron hemostasis utilizing specialized proteins responsible for iron transport, iron uptake by cells, and appropriate cellular distribution of iron for utilization or storage [1, 2]. Based on iron needs, promoters control transcription of proteins involved in iron homeostasis. Additionally, specialized elements regulate mRNA translation called iron regulatory elements act in a coordinated manner to rapidly regulate concentrations of transferrin receptor (necessary for transferrin bound iron cellular uptake) when more iron is needed, and ferritin, (the iron storage protein) when the cellular iron

Effects on Cell Cycle Events

*Paul Seligman and Gamini Siriwardana*

#### **Chapter 7**

*Heavy Metal Toxicity in Public Health*

of Water Reservoirs. MU 2492-81 [Internet]. 1981. Available from: http://docs.cntd.ru/document/ 675400365 [Accessed: 11 December

[59] ICH Harmonised Tripartite Guideline [Internet]. 1993. Available from: https://www.ich.org/fileadmin/ Public\_Web\_Site/ICH\_Products/ Guidelines/Efficacy/E7/Step4/E7\_ Guideline.pdf [Accessed: 11 December

2018]

2018]

**128**

## Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events

*Paul Seligman and Gamini Siriwardana*

#### **Abstract**

Iron is essential for numerous cellular reactions that require oxygen transfer. Iron deficiency is a common problem in humans and is the most common nutritional disease worldwide. However, excess cellular iron can be toxic. Maintenance of iron hemostasis utilizes specialized pathways responsible for iron transport, iron uptake by cells, and appropriate cellular distribution of iron for utilization or storage. This chapter reviews how iron depletion is associated with inhibition of cellular proliferation and cell cycle arrest at different parts of the cell cycle. These effects are based on the effective chelation of iron, and more importantly on differences in various tissue responses to both iron depletion and iron toxicity. These differences may explain why in some tissues, particularly rapidly growing cancer cells, iron depletion causes cell cycle arrest and apoptosis, a form of programed cell death. Other neoplastic tissues are more prone to the toxic effects of iron, which can induce autophagic cell death (termed ferroptosis) via reactive oxygen species resulting in lysosomal degradation of cellular constituents. An appreciation of these differences can be utilized by novel pharmaceutical agents discussed below designed to treat specific cancers.

**Keywords:** cell cycle, iron chelation, redox, ROS, ferroptosis

#### **1. Introduction**

Iron is mainly used in oxygen transfer reactions necessary for moving oxygen to tissues by heme moieties including hemoglobin and myoglobin, enzymes necessary for oxidative phosphorylation, and oxygen transfer reactions by enzymes containing iron sulfur compounds [1, 2]. Compared to other essential micronutrients, iron is found in relatively high (micromolar) concentrations in tissues and serum. Although essential for these oxygen transfer reactions, excess iron is toxic [1–4]. Therefore complex pathways have evolved to maintain proper iron hemostasis utilizing specialized proteins responsible for iron transport, iron uptake by cells, and appropriate cellular distribution of iron for utilization or storage [1, 2]. Based on iron needs, promoters control transcription of proteins involved in iron homeostasis. Additionally, specialized elements regulate mRNA translation called iron regulatory elements act in a coordinated manner to rapidly regulate concentrations of transferrin receptor (necessary for transferrin bound iron cellular uptake) when more iron is needed, and ferritin, (the iron storage protein) when the cellular iron

concentration is high [1, 4]. Ferritin concentrations both intracellular, and small amounts of secreted ferritin found in serum, are increased when there is excess iron, as well as in inflammatory conditions such as infections or cancer, presumably to inhibit iron utilization as well as protect against cellular iron toxicity. Transferrin receptor density is much higher in cells that require more iron, such as cells that synthesize hemoglobin and cells that are actively proliferating [5].

Agents that interfere with iron uptake or iron utilization have been used to treat cancer. One such agent used in limited clinical studies is gallium, a relatively inert metal that binds to transferrin and inhibits cellular iron uptake and utilization [6]. Another is the iron chelator deferioxamine (DFO) used in the treatment of neuroblastoma [7]. DFO has been considered the "gold standard" as a treatment for iron overload [1, 4]. However, newer iron chelators are not only more practical (oral gastrointestinal absorption) but have improved iron chelation efficacy and a more rapid onset of action. They are also lipophilic, however, and hence can potentially confound other biologic processes [8], including lipid peroxidation and autophagy if used for iron depletion only. Lipophilicity may have more potential for cancer treatment, particularly in combination with carefully chosen chemotherapeutic agents [8, 9], since utilizing the iron chelation effect alone combined with agents that specifically inhibit DNA synthesis, for example, may result in less combined efficacy [10].

Iron, therefore, is a requirement for cellular proliferation, particularly rapidly growing cells (including cancer cells). Clinical measurements of iron status in epidemiologic studies have shown a lower incidence of cancer in iron depleted individuals [11, 12], better survival in patients whose tumors retain less iron, and a higher incidence in those with or at risk for iron overload [13, 14].

Cellular iron depletion caused by the use of iron chelators *in vitro* is associated with inhibition of cellular proliferation attributable to cell cycle arrest [15–17]. Initially the deficit was ascribed only to inhibition of ribonucleotide reductase (RNR) an iron dependent enzyme necessary for DNA synthesis. The iron facilitates formation of a tyrosyl free radical at the active site of the M2 protein subunit of RNR [18, 19]. Hydroxyurea (HU), a cancer chemotherapy agent well absorbed after oral administration, is converted to a free radical nitroxide *in vivo* and quenches the tyrosyl free radical of RNR. Inhibition of RNR is associated with an early S phase block (sometimes described as a G1/S block). Some studies have indicated that the S phase block associated with ribonucleotide reductase inhibition might be distinguished from a G1 block, which may also occur with iron depletion [20, 21].

#### **2. Iron depletion causes inhibition of cellular proliferation and at least two blocks in the cell cycle**

More recently, an important advantage for studying cell cycle events has been the development of newer reagents that better pinpoints these events. In particular, antibodies that recognize cell cycle specific phosphorylation events, such as kinase activation status, have proven quite useful. Our more recent studies have shown that the two blocks caused by HU vs. iron chelation can be distinguished by different cell cycle events. In these studies we utilized neuroblastoma cell lines that are relatively sensitive to growth inhibition by iron depletion [22]. Although several other cell lines have shown both G1 and S phase inhibition by iron chelation, we chose the SKNSH neuroblastoma line because of reproducible predictable growth rates as well as consistently diploid chromosomal makeup and consistent contact inhibition with greater than 90% of cells in (G0) G1. SKNSH cells uniformly respond to various stimuli including those that promote cell proliferation. Examples of these promoters include simply subculture

**131**

*duplicate.*

**Figure 1.**

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events*

into serum containing media. These conditions make the cell line particularly useful for the study of very early cell cycle events after the stimulus for proliferation.

Recent studies have indicated at least one reason for the G1 block is an iron requirement for psrc activation. Src is inactive in the absence of iron as demonstrated by both lack of phosphorylation of Y416 (active site) and persistent phosphorylation at the inactivating Y527. Progression of mammalian cells into S phase from late G1 also requires the activation of cdk2 by cyclin E. Cdk2 bound to P27 is in an inactive state. In our studies, although the rapid activation of src with iron repletion was short-lived, it was rapidly followed by P27 degradation allowing

*This figure and legend were taken from Siriwardana and Seligman [23]. Cyclin E levels decrease after 1 h of release from the DFO block. Confluent neuroblastoma cells were serum starved for 24 h then split and plated into media, one set with DFO (lanes 3–6, 9–12) and one with no DFO (lanes 1–2, 7–8) and were incubated for 24 h. After 24 h, one set of cells that was in DFO was replaced with CM (release). Medium in the others were replaced with CM (continuously in medium without DFO). The medium in all plates was aspirated 1 h later and the cells were lysed using 200 μL of hot SDS loading buffer. Forty microliter samples of the lysate were separated by 10% SDS-PAGE and the proteins were transferred to PVDF membranes. This was first probed for cyclin E. Then the membrane was stripped and was probed for β-actin. All treatments were conducted in* 

In these studies, timed experiments used sequential blocking by specific agents including aphidicolin (aph) (DNA polymerase inhibitor causing G1/S block), HU (RNR inhibition), specific kinase inhibitors (i.e., psrc or pcdk2 inhibition), and DFO. Cells were > 90 in G1 phase due to contact inhibition and stimulated to proliferate by subculture in "fresh" medium in 10% fetal calf serum (FCS) Timed studies were performed at the time of the proliferation stimulus, the addition of these specific agents, and "release" (wash out) of these agents. "Release medium" containing heat inactivated FCS was added after the attached cells were extensively washed with phosphate buffered saline. In previous studies we found that medium such as RPMI (supposedly with no added iron salts or FCS) actually contains 1–2 μmol/L of contaminating iron salts and about 0.6 μmol iron/L presumably bound to the transferrin in fetal calf serum. The initial DFO effect was associated with G1 arrest, persistence of cyclin E protein but inhibition of cyclin E/cdk2 complex activity, and no measurable cyclin A protein. DFO washout and media replacement resulted in the rapid disappearance of cyclin E (**Figure 1**). The block by aph was associated with a slight widening of G1 phase suggesting arrest at G1/S, but more importantly cyclin A protein was evident with no discernible cyclin E present. In contrast HU treatment exhibited a block in early to mid-S phase. DFO added slightly before release from aph showed cell cycle changes similar to HU suggesting that this block was due to RNR inhibition (**Figure 2**). These results were confirmed by studies of a unique cell line with a much higher rate of proliferation that consistently was resistant to the G1 effects of iron depletion. It also exhibited persistent cyclin A protein,

*DOI: http://dx.doi.org/10.5772/intechopen.86399*

perhaps due to less contact inhibition.

#### *Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events DOI: http://dx.doi.org/10.5772/intechopen.86399*

into serum containing media. These conditions make the cell line particularly useful for the study of very early cell cycle events after the stimulus for proliferation.

In these studies, timed experiments used sequential blocking by specific agents including aphidicolin (aph) (DNA polymerase inhibitor causing G1/S block), HU (RNR inhibition), specific kinase inhibitors (i.e., psrc or pcdk2 inhibition), and DFO. Cells were > 90 in G1 phase due to contact inhibition and stimulated to proliferate by subculture in "fresh" medium in 10% fetal calf serum (FCS) Timed studies were performed at the time of the proliferation stimulus, the addition of these specific agents, and "release" (wash out) of these agents. "Release medium" containing heat inactivated FCS was added after the attached cells were extensively washed with phosphate buffered saline. In previous studies we found that medium such as RPMI (supposedly with no added iron salts or FCS) actually contains 1–2 μmol/L of contaminating iron salts and about 0.6 μmol iron/L presumably bound to the transferrin in fetal calf serum. The initial DFO effect was associated with G1 arrest, persistence of cyclin E protein but inhibition of cyclin E/cdk2 complex activity, and no measurable cyclin A protein. DFO washout and media replacement resulted in the rapid disappearance of cyclin E (**Figure 1**). The block by aph was associated with a slight widening of G1 phase suggesting arrest at G1/S, but more importantly cyclin A protein was evident with no discernible cyclin E present. In contrast HU treatment exhibited a block in early to mid-S phase. DFO added slightly before release from aph showed cell cycle changes similar to HU suggesting that this block was due to RNR inhibition (**Figure 2**). These results were confirmed by studies of a unique cell line with a much higher rate of proliferation that consistently was resistant to the G1 effects of iron depletion. It also exhibited persistent cyclin A protein, perhaps due to less contact inhibition.

Recent studies have indicated at least one reason for the G1 block is an iron requirement for psrc activation. Src is inactive in the absence of iron as demonstrated by both lack of phosphorylation of Y416 (active site) and persistent phosphorylation at the inactivating Y527. Progression of mammalian cells into S phase from late G1 also requires the activation of cdk2 by cyclin E. Cdk2 bound to P27 is in an inactive state. In our studies, although the rapid activation of src with iron repletion was short-lived, it was rapidly followed by P27 degradation allowing

#### **Figure 1.**

*Heavy Metal Toxicity in Public Health*

efficacy [10].

**two blocks in the cell cycle**

concentration is high [1, 4]. Ferritin concentrations both intracellular, and small amounts of secreted ferritin found in serum, are increased when there is excess iron, as well as in inflammatory conditions such as infections or cancer, presumably to inhibit iron utilization as well as protect against cellular iron toxicity. Transferrin receptor density is much higher in cells that require more iron, such as cells that

Agents that interfere with iron uptake or iron utilization have been used to treat cancer. One such agent used in limited clinical studies is gallium, a relatively inert metal that binds to transferrin and inhibits cellular iron uptake and utilization [6]. Another is the iron chelator deferioxamine (DFO) used in the treatment of neuroblastoma [7]. DFO has been considered the "gold standard" as a treatment for iron overload [1, 4]. However, newer iron chelators are not only more practical (oral gastrointestinal absorption) but have improved iron chelation efficacy and a more rapid onset of action. They are also lipophilic, however, and hence can potentially confound other biologic processes [8], including lipid peroxidation and autophagy if used for iron depletion only. Lipophilicity may have more potential for cancer treatment, particularly in combination with carefully chosen chemotherapeutic agents [8, 9], since utilizing the iron chelation effect alone combined with agents that specifically inhibit DNA synthesis, for example, may result in less combined

Iron, therefore, is a requirement for cellular proliferation, particularly rapidly growing cells (including cancer cells). Clinical measurements of iron status in epidemiologic studies have shown a lower incidence of cancer in iron depleted individuals [11, 12], better survival in patients whose tumors retain less iron, and a

Cellular iron depletion caused by the use of iron chelators *in vitro* is associated with inhibition of cellular proliferation attributable to cell cycle arrest [15–17]. Initially the deficit was ascribed only to inhibition of ribonucleotide reductase (RNR) an iron dependent enzyme necessary for DNA synthesis. The iron facilitates formation of a tyrosyl free radical at the active site of the M2 protein subunit of RNR [18, 19]. Hydroxyurea (HU), a cancer chemotherapy agent well absorbed after oral administration, is converted to a free radical nitroxide *in vivo* and quenches the tyrosyl free radical of RNR. Inhibition of RNR is associated with an early S phase block (sometimes described as a G1/S block). Some studies have indicated that the S phase block associated with ribonucleotide reductase inhibition might be distinguished from a G1 block, which may also occur with iron depletion [20, 21].

**2. Iron depletion causes inhibition of cellular proliferation and at least** 

More recently, an important advantage for studying cell cycle events has been the development of newer reagents that better pinpoints these events. In particular, antibodies that recognize cell cycle specific phosphorylation events, such as kinase activation status, have proven quite useful. Our more recent studies have shown that the two blocks caused by HU vs. iron chelation can be distinguished by different cell cycle events. In these studies we utilized neuroblastoma cell lines that are relatively sensitive to growth inhibition by iron depletion [22]. Although several other cell lines have shown both G1 and S phase inhibition by iron chelation, we chose the SKNSH neuroblastoma line because of reproducible predictable growth rates as well as consistently diploid chromosomal makeup and consistent contact inhibition with greater than 90% of cells in (G0) G1. SKNSH cells uniformly respond to various stimuli including those that promote cell proliferation. Examples of these promoters include simply subculture

synthesize hemoglobin and cells that are actively proliferating [5].

higher incidence in those with or at risk for iron overload [13, 14].

**130**

*This figure and legend were taken from Siriwardana and Seligman [23]. Cyclin E levels decrease after 1 h of release from the DFO block. Confluent neuroblastoma cells were serum starved for 24 h then split and plated into media, one set with DFO (lanes 3–6, 9–12) and one with no DFO (lanes 1–2, 7–8) and were incubated for 24 h. After 24 h, one set of cells that was in DFO was replaced with CM (release). Medium in the others were replaced with CM (continuously in medium without DFO). The medium in all plates was aspirated 1 h later and the cells were lysed using 200 μL of hot SDS loading buffer. Forty microliter samples of the lysate were separated by 10% SDS-PAGE and the proteins were transferred to PVDF membranes. This was first probed for cyclin E. Then the membrane was stripped and was probed for β-actin. All treatments were conducted in duplicate.*

for activation of cdk2 by its phosphorylation at THR 160 presumably resulting in activation of the cdk2/cyclin E complex (**Figures 3–5**).

These events in turn were followed by rapid disappearance of cyclin E protein, presumably due to cdk2/cyclin E complex activity, and appearance of cyclin A protein allowing cells to proceed into and through S phase. Inhibition of src kinase activity may account for decreased downstream events ascribed to iron depletion in other cell lines including inhibition of cyclin D synthesis [25], an event not seen in SKNSH due to constitutive expression of cyclin D [23]. For example, we have found that the S phase kinase associated protein (Skp2), which is responsible for promoting p27 degradation, was not upregulated in the presence of DFO [26]. In contrast, p27 was not degraded after release from the iron block in the presence of the proteasome inhibitor MG132 (unpublished data). Other studies have asserted lack of p27 degradation does not allow for activation of cdk2, but more recently iron chelation was found to cause specific inactivation of cdk2 by persistence of the p21 inhibitor [27].

#### **Figure 2.**

*This figure and legend was adapted from Siriwardana and Seligman [23]. Cyclin A is absent in DFO-treated neuroblastoma cells but occurs in cells arrested with aphidicolin or hydroxyurea. Serum starved neuroblastoma cells for 24 h were subcultured in CM (FCS), or RPMI/10% FCS with hydroxyurea, aphidicolin or DFO. The dishes were incubated for 24 h and the cells were harvested in 0.5 mL of cold PBS, a 50 μL portion of cells was used for FACS and the rest were centrifuged, supernatant removed and added with 200 μL of hot SDS loading buffer. Forty microliter samples of the lysate were separated by 10% SDS-PAGE and the proteins were transferred to PVDF membranes. This was probed for cyclin A. Then, the membrane was stripped and was probed for β-actin. All treatments were conducted in duplicate.*

#### **Figure 3.**

*This figure and legend were adapted from Siriwardana and Seligman [24].Confluent SKNSH cells were sub-cultured into RPMI/10% heat-inactivated FCS with 100 μm DFO and incubated. After 20 h the medium was replaced with new medium containing no DFO and 10% heat deactivated FCS. The cells were harvested at regular intervals beginning 15 min after aspirating the medium and adding hot SDS loading buffer. Westerns were performed as described and Src p416 levels were determined. Thereafter, the blot was stripped and probed for B Actin. Each treatment was conducted in duplicate. Based on densitometry, pSrc/B-Actin (average of duplicates) for RM vs. RM with DFO, respectively. 0 time 0.35 vs. 0.29; 15 min 0.78 vs. 0.28; 30 min 0.55 vs. 0.21; 60 min 0.31 vs. 0.26.*

**133**

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events*

*This figure and legend were adapted from Siriwardana and Seligman [24]. Total p27 levels decrease rapidly after release from the DFO block. Confluent SKNSH cells were sub-cultured into RPMI/10% FCS with 100 μm DFO and incubated. After 20 h the medium was replaced with new medium as RM. The cells were harvested at regular intervals beginning 15 min after aspirating the medium and adding hot SDS loading buffer. Westerns were performed as described and (total) p27 levels were determined. Thereafter, the blot was stripped and* 

In recent unpublished studies we have found evidence that cells blocked at G1/S by aphidicolin, a DNA polymerase inhibitor, do not proceed into S phase if the cells are depleted of iron. The cells remain in G1/S, a block distinguished from both the block in G1 associated with iron depletion alone, as well as the block associated with RNR inhibition by iron depletion. Progression of cells into S phase after release from aphidicolin is prevented with the use of DFO and not with the src inhibitor, AZD, suggesting iron depletion has an additional cell cycle arrest mechanism independent of src inhibition by iron depletion causing G1 arrest as well as the later arrest due to

*This figure and figure legend were adapted from Siriwardana and Seligman [24]. Confluent SKNSH cells were sub-cultured into RPMI/10% FCS (CM) with 100 um DFO and incubated. After 20 h the medium was replaced with new medium containing no DFO and 10% heat deactivated FCS (RM). The cells were harvested at regular intervals beginning at 15 min after RM added, the medium was aspirated and hot SDS loading buffer was added. Westerns were performed as described and pcdk2 levels were determined. Thereafter, the blot was stripped and probed for total cdk2 and then B Actin. Each treatment was conducted in duplicate.*

inhibition of RNR caused by iron depletion (similar to the HU arrest),

used in order to measure DNA content utilizing propidium iodide.

Cells were incubated in aph or HU in RPMI with 10% F CS for 24 h. Then the cells received either no addition, DFO, or AZD for 16 h ahead of release from aph or HU. This 16 h time point was chosen because our previous studies have shown that DFO required at least several hours to effectively remove the "chelatable" cellular iron, a reference to iron that is readily bioavailable, as opposed to, for example, iron stored in ferritin. At 16 h (a total of about 40 h) these time periods were chosen because of extensive studies detailing the rate of proliferation of SKNSH cells with or without added agents, to ensure <10% of the cells are dead (by trypan blue exclusion), and allow time for the vast majority of cells to exhibit the block desired, including allowing a minority of the cells to "recycle" after division to the area of the block. The cells were released from aph or HU to medium, RPMI (10%FCS) with no addition, with DFO or with AZD. The cells were harvested 6 h later and a florescent activated cell sorter was

*DOI: http://dx.doi.org/10.5772/intechopen.86399*

*probed for B Actin. Each treatment was conducted in duplicate.*

**Figure 4.**

**Figure 5.**

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events DOI: http://dx.doi.org/10.5772/intechopen.86399*

#### **Figure 4.**

*Heavy Metal Toxicity in Public Health*

activation of the cdk2/cyclin E complex (**Figures 3–5**).

*probed for β-actin. All treatments were conducted in duplicate.*

for activation of cdk2 by its phosphorylation at THR 160 presumably resulting in

These events in turn were followed by rapid disappearance of cyclin E protein, presumably due to cdk2/cyclin E complex activity, and appearance of cyclin A protein allowing cells to proceed into and through S phase. Inhibition of src kinase activity may account for decreased downstream events ascribed to iron depletion in other cell lines including inhibition of cyclin D synthesis [25], an event not seen in SKNSH due to constitutive expression of cyclin D [23]. For example, we have found that the S phase kinase associated protein (Skp2), which is responsible for promoting p27 degradation, was not upregulated in the presence of DFO [26]. In contrast, p27 was not degraded after release from the iron block in the presence of the proteasome inhibitor MG132 (unpublished data). Other studies have asserted lack of p27 degradation does not allow for activation of cdk2, but more recently iron chelation was found to cause specific inactivation of cdk2 by persistence of the p21 inhibitor [27].

*This figure and legend was adapted from Siriwardana and Seligman [23]. Cyclin A is absent in DFO-treated neuroblastoma cells but occurs in cells arrested with aphidicolin or hydroxyurea. Serum starved neuroblastoma cells for 24 h were subcultured in CM (FCS), or RPMI/10% FCS with hydroxyurea, aphidicolin or DFO. The dishes were incubated for 24 h and the cells were harvested in 0.5 mL of cold PBS, a 50 μL portion of cells was used for FACS and the rest were centrifuged, supernatant removed and added with 200 μL of hot SDS loading buffer. Forty microliter samples of the lysate were separated by 10% SDS-PAGE and the proteins were transferred to PVDF membranes. This was probed for cyclin A. Then, the membrane was stripped and was* 

*This figure and legend were adapted from Siriwardana and Seligman [24].Confluent SKNSH cells were sub-cultured into RPMI/10% heat-inactivated FCS with 100 μm DFO and incubated. After 20 h the medium was replaced with new medium containing no DFO and 10% heat deactivated FCS. The cells were harvested at regular intervals beginning 15 min after aspirating the medium and adding hot SDS loading buffer. Westerns were performed as described and Src p416 levels were determined. Thereafter, the blot was stripped and probed for B Actin. Each treatment was conducted in duplicate. Based on densitometry, pSrc/B-Actin (average of duplicates) for RM vs. RM with DFO, respectively. 0 time 0.35 vs. 0.29; 15 min 0.78 vs. 0.28; 30 min 0.55 vs. 0.21;* 

**132**

**Figure 3.**

*60 min 0.31 vs. 0.26.*

**Figure 2.**

*This figure and legend were adapted from Siriwardana and Seligman [24]. Total p27 levels decrease rapidly after release from the DFO block. Confluent SKNSH cells were sub-cultured into RPMI/10% FCS with 100 μm DFO and incubated. After 20 h the medium was replaced with new medium as RM. The cells were harvested at regular intervals beginning 15 min after aspirating the medium and adding hot SDS loading buffer. Westerns were performed as described and (total) p27 levels were determined. Thereafter, the blot was stripped and probed for B Actin. Each treatment was conducted in duplicate.*

#### **Figure 5.**

*This figure and figure legend were adapted from Siriwardana and Seligman [24]. Confluent SKNSH cells were sub-cultured into RPMI/10% FCS (CM) with 100 um DFO and incubated. After 20 h the medium was replaced with new medium containing no DFO and 10% heat deactivated FCS (RM). The cells were harvested at regular intervals beginning at 15 min after RM added, the medium was aspirated and hot SDS loading buffer was added. Westerns were performed as described and pcdk2 levels were determined. Thereafter, the blot was stripped and probed for total cdk2 and then B Actin. Each treatment was conducted in duplicate.*

In recent unpublished studies we have found evidence that cells blocked at G1/S by aphidicolin, a DNA polymerase inhibitor, do not proceed into S phase if the cells are depleted of iron. The cells remain in G1/S, a block distinguished from both the block in G1 associated with iron depletion alone, as well as the block associated with RNR inhibition by iron depletion. Progression of cells into S phase after release from aphidicolin is prevented with the use of DFO and not with the src inhibitor, AZD, suggesting iron depletion has an additional cell cycle arrest mechanism independent of src inhibition by iron depletion causing G1 arrest as well as the later arrest due to inhibition of RNR caused by iron depletion (similar to the HU arrest),

Cells were incubated in aph or HU in RPMI with 10% F CS for 24 h. Then the cells received either no addition, DFO, or AZD for 16 h ahead of release from aph or HU. This 16 h time point was chosen because our previous studies have shown that DFO required at least several hours to effectively remove the "chelatable" cellular iron, a reference to iron that is readily bioavailable, as opposed to, for example, iron stored in ferritin. At 16 h (a total of about 40 h) these time periods were chosen because of extensive studies detailing the rate of proliferation of SKNSH cells with or without added agents, to ensure <10% of the cells are dead (by trypan blue exclusion), and allow time for the vast majority of cells to exhibit the block desired, including allowing a minority of the cells to "recycle" after division to the area of the block. The cells were released from aph or HU to medium, RPMI (10%FCS) with no addition, with DFO or with AZD. The cells were harvested 6 h later and a florescent activated cell sorter was used in order to measure DNA content utilizing propidium iodide.

**Figures 6–10** shows DNA profiles of cells treated as indicated above. Our previous studies documented that effective chelation of intracellular iron by DFO *in vitro* took at least several hours [20]. Therefore cells incubated in aph for 2 days exhibited the expected G1/S block and after adding "release" medium with no addition for 6 h cells were almost exclusively in early and mid-S phase (**Figures 6** and **7**). Aph treatment with DFO added only one half hour before "wash out" [so then is the DFO also washed out?], even with the DFO added to the release medium for 6 h, showed an almost identical profile as cells without DFO added (**Figure 8**). However, when DFO was added 16 h before release, and the cells were released in medium containing DFO for 6 h the profile showed cells still arrested in G1/S (**Figures 9** and **10**).

This effect of iron chelation by DFO causing a block at G1/S is distinct from inhibition of src kinase by DFO treatment in G1 phase. Treatment with the specific src kinase inhibitor AZD 0530 (AZD) showed almost identical DNA profile results as DFO treatment during G1 phase [24]. When AZD was added to cells 16 h before release from Aph and continued in release medium (no addition), 6 h later the profile showed results similar to no AZD treatment with the vast majority of cells in S phase (**Figures 11** and **12**).

Cells in HU for 48 h at the time of release into fresh medium are in early S phase, indicating inhibition of DNA synthesis caused by depletion of deoxyribonucleotides due to inhibition of RNR. These cells will proceed through S phase 6 h later after wash out (**Figures 13** and **14**). However, when DFO is added to the HU containing media 16 h before release most of the cells appear to be in G1/S phase, at the time of release. Moreover, when the same cells are released into medium containing DFO, the vast majority have still have not entered S phase 6 h later, although there are still a few cells in early S.

Studies using inhibitors of cdk2 activity were performed to determine its requirement for cell cycle progression. We hypothesized that iron depletion caused by DFO at G1 and S phase was not an effect of RNR inhibition, but a different process necessary to exit G1/S phase. We therefore assessed the effects of two cdk2 inhibitors JNJ7706621 (JNJ) and BMS 387032 (BMS). Both inhibitors affect the activity of other cyclin dependent kinases but have some specificity for cdk2. These agents have been used in humans as investigational study drugs in cancer protocols (supported by Johnson and Johnson and Bristol Myers Squibb respectively).

**135**

**Figure 8.**

**Figure 7.**

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events*

As expected adding both inhibitors to cells treated with aph overnight, and maintaining their presence upon aph release (6 h) showed persistent G1/S phase arrest (**Figures 15** and **16**). Moreover, when BMS [28] was added to HU treated cells overnight and maintained after release, about 85% of cells remained in G1/S. The percentage of cells in S and G2/M phase when JNJ was added under these conditions showed about 20–25% of cells in S and G2/M phase, perhaps showing a different

In conclusion the studies shown in **Figures 6–16** indicate there is a third cell cycle block caused by iron depletion distinct from the G1 block associated with psrc inhibition and the early to mid—S phase block caused by inhibition of RNR. The third putative block similar to the G1/S block seen with DNA polymerase inhibition

effect of BMS under these conditions (data not shown).

*Aph 6 h after release with DFO in release medium but no DFO added before.*

(Aph) and with the less specific cdk2 inhibitors (**Figure 17**).

*DOI: http://dx.doi.org/10.5772/intechopen.86399*

*Six hours after release into medium (no addition).*

**Figure 6.** *Aph at time of release.*

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events DOI: http://dx.doi.org/10.5772/intechopen.86399*

**Figure 7.** *Six hours after release into medium (no addition).*

*Heavy Metal Toxicity in Public Health*

S phase (**Figures 11** and **12**).

are still a few cells in early S.

**Figures 6–10** shows DNA profiles of cells treated as indicated above. Our previous studies documented that effective chelation of intracellular iron by DFO *in vitro* took at least several hours [20]. Therefore cells incubated in aph for 2 days exhibited the expected G1/S block and after adding "release" medium with no addition for 6 h cells were almost exclusively in early and mid-S phase (**Figures 6** and **7**). Aph treatment with DFO added only one half hour before "wash out" [so then is the DFO also washed out?], even with the DFO added to the release medium for 6 h, showed an almost identical profile as cells without DFO added (**Figure 8**). However, when DFO was added 16 h before release, and the cells were released in medium containing DFO for 6 h the profile showed cells still arrested in G1/S (**Figures 9** and **10**). This effect of iron chelation by DFO causing a block at G1/S is distinct from inhibition of src kinase by DFO treatment in G1 phase. Treatment with the specific src kinase inhibitor AZD 0530 (AZD) showed almost identical DNA profile results as DFO treatment during G1 phase [24]. When AZD was added to cells 16 h before release from Aph and continued in release medium (no addition), 6 h later the profile showed results similar to no AZD treatment with the vast majority of cells in

Cells in HU for 48 h at the time of release into fresh medium are in early S phase, indicating inhibition of DNA synthesis caused by depletion of deoxyribonucleotides due to inhibition of RNR. These cells will proceed through S phase 6 h later after wash out (**Figures 13** and **14**). However, when DFO is added to the HU containing media 16 h before release most of the cells appear to be in G1/S phase, at the time of release. Moreover, when the same cells are released into medium containing DFO, the vast majority have still have not entered S phase 6 h later, although there

Studies using inhibitors of cdk2 activity were performed to determine its requirement for cell cycle progression. We hypothesized that iron depletion caused by DFO at G1 and S phase was not an effect of RNR inhibition, but a different process necessary to exit G1/S phase. We therefore assessed the effects of two cdk2 inhibitors JNJ7706621 (JNJ) and BMS 387032 (BMS). Both inhibitors affect the activity of other cyclin dependent kinases but have some specificity for cdk2. These agents have been used in humans as investigational study drugs in cancer protocols (supported by Johnson and Johnson and Bristol Myers Squibb respectively).

**134**

**Figure 6.**

*Aph at time of release.*

**Figure 8.** *Aph 6 h after release with DFO in release medium but no DFO added before.*

As expected adding both inhibitors to cells treated with aph overnight, and maintaining their presence upon aph release (6 h) showed persistent G1/S phase arrest (**Figures 15** and **16**). Moreover, when BMS [28] was added to HU treated cells overnight and maintained after release, about 85% of cells remained in G1/S. The percentage of cells in S and G2/M phase when JNJ was added under these conditions showed about 20–25% of cells in S and G2/M phase, perhaps showing a different effect of BMS under these conditions (data not shown).

In conclusion the studies shown in **Figures 6–16** indicate there is a third cell cycle block caused by iron depletion distinct from the G1 block associated with psrc inhibition and the early to mid—S phase block caused by inhibition of RNR. The third putative block similar to the G1/S block seen with DNA polymerase inhibition (Aph) and with the less specific cdk2 inhibitors (**Figure 17**).

**Figure 9.** *Aph at time of release with DFO added for 16 h before rebase.*

#### **Figure 10.**

*Six hours after release from aph and DFO with DFO in release medium. [G1-88%, S-5%, G2/M-5% "dead" cells in this case are shown mostly to the left of the G1 peak and are discounted].*

In initial Western blot experiments we found that DFO reduces the active cdk2 and cyclin A, whereas src inhibition does not. Cells were incubated in aph for 24 h, followed by addition of DFO for 16 h. Cells treated with aph had measurable src kinase, cdk2 kinase and cyclin A protein as shown in our prior studies. As expected, cells treated with AZD showed decreased src kinase activity in both incubation times but adequate levels of cyclin A and cdk2 kinase. Taken together these results strongly suggest that another block caused by iron depletion is associated with an event that occurs after src kinase activity, but before the initiation of DNA synthesis.

However, further studies need to be performed to assess the significance and longer-term effects of the decreased cyclin A and pcdk2 with the longer DFO

**137**

etc., see below).

**Figure 12.**

**Figure 11.**

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events*

incubation. These further studies may provide evidence as to why the longer iron depletion affects synthesis of cell cycle related proteins. These studies should initially put an emphasis on inhibition of promoters (i.e., E2F isoforms, or NFR2,

Until 10 years ago programed cell death with a specific DNA "ladder" measured on gels, particularly in cancer cells, was ascribed to a process called apoptosis [29, 30]. Evidence for apoptosis was often used to validate anti-neoplastic agents studied *in vitro*. Further studies later showed that apoptosis associated with cells treated with cancer chemotherapeutic agents, or a normal process such as depletion of

**2.1 Selected iron associated cell death and the oxidative state**

*DOI: http://dx.doi.org/10.5772/intechopen.86399*

*Aph time of release with AZD added 16 h before release.*

*Aph 6 h after release with AZD in release medium.*

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events DOI: http://dx.doi.org/10.5772/intechopen.86399*

**Figure 11.** *Aph time of release with AZD added 16 h before release.*

*Heavy Metal Toxicity in Public Health*

*Aph at time of release with DFO added for 16 h before rebase.*

**136**

synthesis.

**Figure 10.**

**Figure 9.**

In initial Western blot experiments we found that DFO reduces the active cdk2 and cyclin A, whereas src inhibition does not. Cells were incubated in aph for 24 h, followed by addition of DFO for 16 h. Cells treated with aph had measurable src kinase, cdk2 kinase and cyclin A protein as shown in our prior studies. As expected, cells treated with AZD showed decreased src kinase activity in both incubation times but adequate levels of cyclin A and cdk2 kinase. Taken together these results strongly suggest that another block caused by iron depletion is associated with an event that occurs after src kinase activity, but before the initiation of DNA

*Six hours after release from aph and DFO with DFO in release medium. [G1-88%, S-5%, G2/M-5% "dead"* 

*cells in this case are shown mostly to the left of the G1 peak and are discounted].*

However, further studies need to be performed to assess the significance and longer-term effects of the decreased cyclin A and pcdk2 with the longer DFO

**Figure 12.** *Aph 6 h after release with AZD in release medium.*

incubation. These further studies may provide evidence as to why the longer iron depletion affects synthesis of cell cycle related proteins. These studies should initially put an emphasis on inhibition of promoters (i.e., E2F isoforms, or NFR2, etc., see below).

#### **2.1 Selected iron associated cell death and the oxidative state**

Until 10 years ago programed cell death with a specific DNA "ladder" measured on gels, particularly in cancer cells, was ascribed to a process called apoptosis [29, 30]. Evidence for apoptosis was often used to validate anti-neoplastic agents studied *in vitro*. Further studies later showed that apoptosis associated with cells treated with cancer chemotherapeutic agents, or a normal process such as depletion of

**Figure 14.** *Six hours after release in medium with no addition.*

specific antibody producing B cells, apoptosis was mainly described as a caspasedependent process [31]. Recently, several non-apoptotic regulated processes that result in cell death have been discovered [32]. One of these processes has been termed ferroptosis [32]. Ferroptosis is best described as autophagy that results in cell death, or autophagic cell death [32]. Overall this process is a combination of the cell's response to toxicity including but not limited to cancer chemotherapy. When first described, autophagy was deemed a cell's response to toxicity including any toxin, chemotherapeutic insult or even hypoxia [33, 34]. The process was associated with lysosomal degradation of cellular constituents including some organelles [33, 34]. The initiation of autophagy is not thought to be cell cycle specific. Until recently, it was thought to be primarily a protective mechanism by which cells

**139**

**Figure 15.**

**Figure 16.**

be in remission [35–37].

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events*

entered senescence, (arrested in G0/G1 phase), presumably before "start" [35]. This autophagic process was seldom thought to be a cause of cell death. Some have hypothesized that when cancer cells enter senescence a subset become cancer initiating cells, or cancer stem cells resulting in re-emergence of a cancer thought to

Studies have also indicated that senescence associated reprogramming promotes cancer "stemness" that is enriched in relapse tumors [38], resulting in highly aggressive growth potential after escape from G0/G1 cell cycle blockade [38]. Ferroptosis was first described in cancer cells with activation of a common oncogene called RAS [39]. Inappropriate Ras activity results in autonomous cell proliferation. An inhibitor of cell proliferation activated by unregulated RAS was a small molecule called erastin [32, 40]. Erastin was found to increase processes associated with autophagy. By some

*DOI: http://dx.doi.org/10.5772/intechopen.86399*

*Aph at time of release with JNJ. JNJ added before release.*

*Six hours after release continued in release medium (no aph).*

**Figure 13.**

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events DOI: http://dx.doi.org/10.5772/intechopen.86399*

**Figure 15.** *Aph at time of release with JNJ. JNJ added before release.*

**Figure 16.** *Six hours after release continued in release medium (no aph).*

entered senescence, (arrested in G0/G1 phase), presumably before "start" [35]. This autophagic process was seldom thought to be a cause of cell death. Some have hypothesized that when cancer cells enter senescence a subset become cancer initiating cells, or cancer stem cells resulting in re-emergence of a cancer thought to be in remission [35–37].

Studies have also indicated that senescence associated reprogramming promotes cancer "stemness" that is enriched in relapse tumors [38], resulting in highly aggressive growth potential after escape from G0/G1 cell cycle blockade [38]. Ferroptosis was first described in cancer cells with activation of a common oncogene called RAS [39]. Inappropriate Ras activity results in autonomous cell proliferation. An inhibitor of cell proliferation activated by unregulated RAS was a small molecule called erastin [32, 40]. Erastin was found to increase processes associated with autophagy. By some

*Heavy Metal Toxicity in Public Health*

**138**

**Figure 14.**

*Six hours after release in medium with no addition.*

**Figure 13.**

*HU at time of release.*

specific antibody producing B cells, apoptosis was mainly described as a caspasedependent process [31]. Recently, several non-apoptotic regulated processes that result in cell death have been discovered [32]. One of these processes has been termed ferroptosis [32]. Ferroptosis is best described as autophagy that results in cell death, or autophagic cell death [32]. Overall this process is a combination of the cell's response to toxicity including but not limited to cancer chemotherapy. When first described, autophagy was deemed a cell's response to toxicity including any toxin, chemotherapeutic insult or even hypoxia [33, 34]. The process was associated with lysosomal degradation of cellular constituents including some organelles [33, 34]. The initiation of autophagy is not thought to be cell cycle specific. Until recently, it was thought to be primarily a protective mechanism by which cells

#### **Figure 17.**

*Simplified cell cycle figure to illustrate steps that require iron. Progression to the next phase requires activation of specific cyclin dependent kinases that depend on binding to each phase specific cyclin(s). X-mid G1 block at least partially caused by iron requirement for psrc activation, Y-putative G1/S block that occurs before DNA synthesis, Z-S phase block at least partly caused by inhibition of RNR. Based on implicating different promoters or changes in the redox state it is quite possible, an iron requirement is necessary in allowing completion of the cell cycle.*

accounts [41] increased cellular iron with associated increased reactive oxygen species (ROS) contributed to lipid peroxidation resulting in cell death, thus fulfilling the diagnosis of ferroptosis. In certain instances initiation of this process can influence some neoplastic cells to become more sensitive to chemotherapy [42, 43]. The process may also be accelerated by a decrease in ferroportin [44], the only protein that promotes cellular iron efflux, resulting in a net increase in cellular bioavailable iron [45]. Therefore, the use of passive iron chelation (such as DFO) may actually protect certain proliferating cells from autophagy during cell cycle arrest. This process may be of benefit for normal cells during proliferation [46, 47], but disadvantageous for certain cancer cells during treatment.

Although the precise definition of ferroptosis requires evidence for autophagic cell death, in some instances apoptosis may also be part of the process [48]. Some studies suggest lysosomal leak degrades ferritin, releasing ferritin iron that stimulates lipid peroxidation and is associated with changes of autophagy that can lead to cell death [49]. More recently, p53 has been identified as a ferroptosis inducer by inhibiting cystine uptake, decreasing the cells ability to counteract oxidative processes possibly via decreased GSH, resulting in increased cellular ROS and sensitivity to ferroptosis. Less reducing potential, such as erastin's mechanism of action, was thought to be the main cause of differences in the oxidative state among cell types, but iron concentration and/or iron distribution also plays a part. The p53 effect is thought to be metabolic in nature and not directly related to p53 effects on cell cycle, but it has stimulated interest in novel anticancer agents [50, 51]. It is quite possible that more efficient lipid soluble iron chelators may be more effective in

**141**

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events*

promoting cell death from autophagy due to processes associated with ferroptosis

For example, a recent study has shown that polyamines via several pathways are involved with cancer cell proliferation, and these polyamine pathways are stimulated by iron. Thus, specific iron chelation not only inhibits cell growth by mechanisms detailed above, but also inhibits the cellular proliferation caused by the stimulating effect of activated polyamines [52]. Another study has shown that increasing levels of the CDK inhibitor p21 are associated with iron chelation [27], which can have variable effects on inhibition of cell growth depending on the type of chelator as well as the cell type [53]. The study that emphasizes some of these concepts, from the laboratory of DR Richardson, involves "targeting" oncogenic nuclear factor kappa B signaling [54] with redox-active agents. Under normal conditions nuclear factor kappa B signaling occurs under different immunologic conditions [55]. However, aberrant activation of this pathway results in tumorigenesis and unregulated cancer cell proliferation. A hypothesis advanced is the use of lipophilic thiosemicarbazone chelators, first studied extensively in 2006 as having potent antitumor activity, that could potentially overcome resistance to chemotherapeutics [56]. The new studies [54, 57] detail that thiosemicarbazones "form redox-active metal complexes that generate high ROS levels." It is explained that nuclear factor kappa B signaling is actually activated when ROS is in sub-lethal amounts. However, higher ROS generation will inhibit this signaling as one adjunct toward cancer cell death, as opposed to lower ROS generation leading to autophagy, leading to cellular senescence with resistance to anticancer therapy and possible tumor progression. Cell death (ferroptosis) as opposed to autophagic senescence can depend on the tissue type, the redox state, the dose of the lipophilic chelator

In recent studies now in press we found that in genomic studies of acute myelogenous leukemia (AML) patients that had low ferroportin message had significantly improved survival compared to those in the higher message group. Using AML cell lines and AML patient derived cells we confirmed that low ferroportin expression compared with high expression resulted in greater iron uptake, faster rates of proliferation, and more sensitivity to chemotherapy. At least in AML where survival is dependent on chemotherapy response higher rates of cellular iron incorporation

Iron deficiency caused by chelation has been shown to inhibit cellular proliferation, particularly in rapidly growing cancer cells *in vitro* since the 1970s. Since then, iron depletion has been shown to block specific cell cycle processes associated with events in G1, S and probably the G1/S phase transition. Clinical studies of iron depletion have supported many of these findings, and a few select investigational clinical agents that interfere with iron or deplete cells of iron have been utilized in cancer chemotherapy studies. Data are presented regarding specific cell cycle events associated with iron depletion, and some differences in these events among different cell types are described. Passive iron chelation may better pinpoint a step in the metabolic or cell proliferation pathways that require iron. Moreover, the concept of ferroptosis associated with excess iron causing autophagic cell death has resulted in a plethora of studies. These investigations have generated renewed interest in lipophilic iron chelators that result in differential iron binding to sub cellular areas based on the malignant cell type. The resulting generation of ROS, inhibition of oncogenic tumor promoters, and associated autophagic cell death (with a certain

*DOI: http://dx.doi.org/10.5772/intechopen.86399*

used and the amount of bioavailable iron.

may improve survival [58].

**3. Conclusion**

such as lipid peroxidation.

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events DOI: http://dx.doi.org/10.5772/intechopen.86399*

promoting cell death from autophagy due to processes associated with ferroptosis such as lipid peroxidation.

For example, a recent study has shown that polyamines via several pathways are involved with cancer cell proliferation, and these polyamine pathways are stimulated by iron. Thus, specific iron chelation not only inhibits cell growth by mechanisms detailed above, but also inhibits the cellular proliferation caused by the stimulating effect of activated polyamines [52]. Another study has shown that increasing levels of the CDK inhibitor p21 are associated with iron chelation [27], which can have variable effects on inhibition of cell growth depending on the type of chelator as well as the cell type [53]. The study that emphasizes some of these concepts, from the laboratory of DR Richardson, involves "targeting" oncogenic nuclear factor kappa B signaling [54] with redox-active agents. Under normal conditions nuclear factor kappa B signaling occurs under different immunologic conditions [55]. However, aberrant activation of this pathway results in tumorigenesis and unregulated cancer cell proliferation. A hypothesis advanced is the use of lipophilic thiosemicarbazone chelators, first studied extensively in 2006 as having potent antitumor activity, that could potentially overcome resistance to chemotherapeutics [56]. The new studies [54, 57] detail that thiosemicarbazones "form redox-active metal complexes that generate high ROS levels." It is explained that nuclear factor kappa B signaling is actually activated when ROS is in sub-lethal amounts. However, higher ROS generation will inhibit this signaling as one adjunct toward cancer cell death, as opposed to lower ROS generation leading to autophagy, leading to cellular senescence with resistance to anticancer therapy and possible tumor progression. Cell death (ferroptosis) as opposed to autophagic senescence can depend on the tissue type, the redox state, the dose of the lipophilic chelator used and the amount of bioavailable iron.

In recent studies now in press we found that in genomic studies of acute myelogenous leukemia (AML) patients that had low ferroportin message had significantly improved survival compared to those in the higher message group. Using AML cell lines and AML patient derived cells we confirmed that low ferroportin expression compared with high expression resulted in greater iron uptake, faster rates of proliferation, and more sensitivity to chemotherapy. At least in AML where survival is dependent on chemotherapy response higher rates of cellular iron incorporation may improve survival [58].

#### **3. Conclusion**

*Heavy Metal Toxicity in Public Health*

accounts [41] increased cellular iron with associated increased reactive oxygen species (ROS) contributed to lipid peroxidation resulting in cell death, thus fulfilling the diagnosis of ferroptosis. In certain instances initiation of this process can influence some neoplastic cells to become more sensitive to chemotherapy [42, 43]. The process may also be accelerated by a decrease in ferroportin [44], the only protein that promotes cellular iron efflux, resulting in a net increase in cellular bioavailable iron [45]. Therefore, the use of passive iron chelation (such as DFO) may actually protect certain proliferating cells from autophagy during cell cycle arrest. This process may be of benefit for normal cells during proliferation [46, 47], but disadvantageous for

*Simplified cell cycle figure to illustrate steps that require iron. Progression to the next phase requires activation of specific cyclin dependent kinases that depend on binding to each phase specific cyclin(s). X-mid G1 block at least partially caused by iron requirement for psrc activation, Y-putative G1/S block that occurs before DNA synthesis, Z-S phase block at least partly caused by inhibition of RNR. Based on implicating different promoters or changes in the redox state it is quite possible, an iron requirement is necessary in allowing completion of the* 

Although the precise definition of ferroptosis requires evidence for autophagic cell death, in some instances apoptosis may also be part of the process [48]. Some studies suggest lysosomal leak degrades ferritin, releasing ferritin iron that stimulates lipid peroxidation and is associated with changes of autophagy that can lead to cell death [49]. More recently, p53 has been identified as a ferroptosis inducer by inhibiting cystine uptake, decreasing the cells ability to counteract oxidative processes possibly via decreased GSH, resulting in increased cellular ROS and sensitivity to ferroptosis. Less reducing potential, such as erastin's mechanism of action, was thought to be the main cause of differences in the oxidative state among cell types, but iron concentration and/or iron distribution also plays a part. The p53 effect is thought to be metabolic in nature and not directly related to p53 effects on cell cycle, but it has stimulated interest in novel anticancer agents [50, 51]. It is quite possible that more efficient lipid soluble iron chelators may be more effective in

**140**

**Figure 17.**

*cell cycle.*

certain cancer cells during treatment.

Iron deficiency caused by chelation has been shown to inhibit cellular proliferation, particularly in rapidly growing cancer cells *in vitro* since the 1970s. Since then, iron depletion has been shown to block specific cell cycle processes associated with events in G1, S and probably the G1/S phase transition. Clinical studies of iron depletion have supported many of these findings, and a few select investigational clinical agents that interfere with iron or deplete cells of iron have been utilized in cancer chemotherapy studies. Data are presented regarding specific cell cycle events associated with iron depletion, and some differences in these events among different cell types are described. Passive iron chelation may better pinpoint a step in the metabolic or cell proliferation pathways that require iron. Moreover, the concept of ferroptosis associated with excess iron causing autophagic cell death has resulted in a plethora of studies. These investigations have generated renewed interest in lipophilic iron chelators that result in differential iron binding to sub cellular areas based on the malignant cell type. The resulting generation of ROS, inhibition of oncogenic tumor promoters, and associated autophagic cell death (with a certain

extent of apoptosis) suggest these compounds might be useful chemotherapeutic agents. In the future, based on differences in neoplastic tissues, a balance between inhibiting cell proliferation by iron depletion and cell death associated with excess iron will require further study to maximize both events using investigational agents. For example, one transcription factor, NRF2, is known for modulating cellular iron homeostasis and is thought to decrease ferroportin in macrophages, presumably as a way to decrease bioavailable and potentially toxic iron in other cells. However, under pathologic conditions, this transcription factor may increase iron retention by malignant cells [59]. Hepcidin, a hormone made in the liver, under normal conditions is stimulated by inflammation and results in degradation of ferroportin in macrophages. This presumably allows for increased storage of iron and lower iron in serum [45]. However, more needs to be known about how hepcidin affects tumor cells under pathologic conditions [45]. In another example it was found that in estrogen receptor positive breast cancer cells, ferroportin message was significantly reduced with estrogen treatment. Of further interest, a functional estrogen response element was identified within a ferroportin promoter that would repress ferroportin expression [60]. Unfortunately, pharmaceutical companies have a protective interest in new agents, especially those that have potential in the lucrative cancer treatment market. Therefore they are hesitant to publish studies showing biologic effects of an agent, particularly if the effect might be construed as leading to a potentially toxic event.

### **Conflict of interest**

There are no conflicts of interest to declare.

### **Author details**

Paul Seligman\* and Gamini Siriwardana School of Medicine, University of Colorado, Aurora, Colorado, USA

\*Address all correspondence to: paul.seligman@ucdenver.edu

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**143**

1992;**15**:319-322

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events*

Oncotarget. 2015;**6**(22):18748-18779. DOI: 10.18632/oncotarget.4349

[9] Potuckova E, Jansova H, Machacek M, Vavrova A, Haskova P, Tichotova L. Quantitative analysis of the antiproliferative activity of combinations of selected iron-chelating agents and clinically used anti-neoplastic drugs.

[10] Chang YC, Lo WJ, Huang YT, Lin CL, Feng CC, Lin HT. Deferasirox has strong anti-leukemia activity but may antagonize the anti-leukemia effect of doxorubicin. Leukemia and Lymphoma. 2017;**58**(9):2176-2184. DOI:

10.1080/10428194.2017.1280604

[11] Pinnix Z, Miller L, Wang R, D'Agostino T, Kute M. Ferroportin and iron regulation in breast cancer progression and prognosis. Science Translational Medicine. 2010;**2**(43):56

[12] Zacharski L, Chow B, Hoes P, Shamayeva G, Baron J, Dalman R. Decreased cancer risk after iron reduction in patients with peripheral arterial disease: results from a randomized trial. Journal of the National Cancer Institute.

[13] Lenarduzzi M, Hui AB, Yue S, Ito E, Shi W, Williams J. Hemochromatosis

[14] Liu X, Lv C, Luan X, Lv M. C282Y polymorphism in the HFE gene is associated with risk of breast cancer. Tumour Biology. 2013;**34**:2759-2764

[15] Hoffbrand A, Ganeshaguru K, Hooton J, Tattersall M. Effect of iron deficiency and deferoxamine on DNA synthesis in human cells. British Journal

of Hematology. 1986;**33**:517

enhances tumor progression via upregulation of intracellular iron in head and neck cancer. PLoS One.

2008;**100**:996-1002

2013;**8**

PLoS One. 2014;**9**

*DOI: http://dx.doi.org/10.5772/intechopen.86399*

[1] Seligman P, Klausner R, Huebers A. Molecular mechanisms of iron metabolism. In: Molecular Basis of Blood Disease. Neinhaus A, Majerus P, Stamatoyanopoulous G, Leder P editors.

Phila: Saunders; 1987:219-244

[2] Bogdan A, Miyazawa M, Hashimoto K, Tsuji Y. Regulators of iron homeostasis: New players in metabolism, cell death and disease. Trends in Biochemical Sciences. 2016;**41**(3):274-286. DOI: 10.1016/j.

[3] MacKenzie E. Intracellular iron transport and storage from molecular mechanisms to health implications. Antioxidants & Redox Signaling.

[4] Hentze M, Muckenthaler M, Andrews N. Balancing acts: molecular control of mammalian iron metabolism.

[5] Chitambur C, Massey E, Seligman P. Regulation of transferrin receptor expression on human leukemia cells during proliferation and induction of differentiation. The Journal of Clinical Investigation. 1983;**72**:1314-1325

[6] Seligman P, Crawford E. Treatment of advanced transitional cell carcinoma of the bladder with continuous-infusion gallium nitrate. Journal of the National Cancer Institute. 1991;**83**:1582-1584

American Journal of Clinical Oncology.

[8] Lui GY, Kovacevic Z, Richardson V, Merlot AM, Kalinowski DS, Richardson DR. Targeting cancer by binding iron: Dissecting cellular signaling pathways.

[7] Caniglia L. Deferoxamine, cyclophosphamide, etoposide, carboplatin, and thiotepa (D-CeCat): A new cytoreductive chelationchemotherapy regimen in patients with advanced neuroblastoma.

tibs.2015.11.9012

2008;**10**:997-1030

Cell. 2004;**117**:285-297

*Intracellular Iron Concentration and Distribution Have Multiple Effects on Cell Cycle Events DOI: http://dx.doi.org/10.5772/intechopen.86399*

#### **References**

*Heavy Metal Toxicity in Public Health*

to a potentially toxic event.

There are no conflicts of interest to declare.

**Conflict of interest**

**Author details**

extent of apoptosis) suggest these compounds might be useful chemotherapeutic agents. In the future, based on differences in neoplastic tissues, a balance between inhibiting cell proliferation by iron depletion and cell death associated with excess iron will require further study to maximize both events using investigational agents. For example, one transcription factor, NRF2, is known for modulating cellular iron homeostasis and is thought to decrease ferroportin in macrophages, presumably as a way to decrease bioavailable and potentially toxic iron in other cells. However, under pathologic conditions, this transcription factor may increase iron retention by malignant cells [59]. Hepcidin, a hormone made in the liver, under normal conditions is stimulated by inflammation and results in degradation of ferroportin in macrophages. This presumably allows for increased storage of iron and lower iron in serum [45]. However, more needs to be known about how hepcidin affects tumor cells under pathologic conditions [45]. In another example it was found that in estrogen receptor positive breast cancer cells, ferroportin message was significantly reduced with estrogen treatment. Of further interest, a functional estrogen response element was identified within a ferroportin promoter that would repress ferroportin expression [60]. Unfortunately, pharmaceutical companies have a protective interest in new agents, especially those that have potential in the lucrative cancer treatment market. Therefore they are hesitant to publish studies showing biologic effects of an agent, particularly if the effect might be construed as leading

**142**

provided the original work is properly cited.

Paul Seligman\* and Gamini Siriwardana

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

School of Medicine, University of Colorado, Aurora, Colorado, USA

\*Address all correspondence to: paul.seligman@ucdenver.edu

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[2] Bogdan A, Miyazawa M, Hashimoto K, Tsuji Y. Regulators of iron homeostasis: New players in metabolism, cell death and disease. Trends in Biochemical Sciences. 2016;**41**(3):274-286. DOI: 10.1016/j. tibs.2015.11.9012

[3] MacKenzie E. Intracellular iron transport and storage from molecular mechanisms to health implications. Antioxidants & Redox Signaling. 2008;**10**:997-1030

[4] Hentze M, Muckenthaler M, Andrews N. Balancing acts: molecular control of mammalian iron metabolism. Cell. 2004;**117**:285-297

[5] Chitambur C, Massey E, Seligman P. Regulation of transferrin receptor expression on human leukemia cells during proliferation and induction of differentiation. The Journal of Clinical Investigation. 1983;**72**:1314-1325

[6] Seligman P, Crawford E. Treatment of advanced transitional cell carcinoma of the bladder with continuous-infusion gallium nitrate. Journal of the National Cancer Institute. 1991;**83**:1582-1584

[7] Caniglia L. Deferoxamine, cyclophosphamide, etoposide, carboplatin, and thiotepa (D-CeCat): A new cytoreductive chelationchemotherapy regimen in patients with advanced neuroblastoma. American Journal of Clinical Oncology. 1992;**15**:319-322

[8] Lui GY, Kovacevic Z, Richardson V, Merlot AM, Kalinowski DS, Richardson DR. Targeting cancer by binding iron: Dissecting cellular signaling pathways.

Oncotarget. 2015;**6**(22):18748-18779. DOI: 10.18632/oncotarget.4349

[9] Potuckova E, Jansova H, Machacek M, Vavrova A, Haskova P, Tichotova L. Quantitative analysis of the antiproliferative activity of combinations of selected iron-chelating agents and clinically used anti-neoplastic drugs. PLoS One. 2014;**9**

[10] Chang YC, Lo WJ, Huang YT, Lin CL, Feng CC, Lin HT. Deferasirox has strong anti-leukemia activity but may antagonize the anti-leukemia effect of doxorubicin. Leukemia and Lymphoma. 2017;**58**(9):2176-2184. DOI: 10.1080/10428194.2017.1280604

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### *Edited by John Kanayochukwu Nduka and Mohamed Nageeb Rashed*

It is often said that the "dosage" of any substance determines its remedy or poison effect. Heavy metal sources encompass sewage, pesticides, fertilizers, environmental contamination, occupational exposure/contact through inhalation, ingestion, and skin. Before the advent of technology/the industrial revolution, communicable diseases ravaged the human race but this seems to have given way to non-communicable diseases such as cancers, renal failure, hormonal distortion enzymes, inhibition of fetal growth, and DNA damage causing negative health issues due to heavy metals. This book brings to the fore probably the most recent experimental research/review on heavy metal contamination, remediating techniques, cellular tissue damage, and toxicological and antioxidant effects of heavy metals. It is hoped that its contents will make interesting reading for all.

Published in London, UK © 2020 IntechOpen © Roungchai / iStock

Heavy Metal Toxicity in Public Health

Heavy Metal Toxicity

in Public Health

*Edited by John Kanayochukwu Nduka* 

*and Mohamed Nageeb Rashed*