**2.1 Metals and monoamines levels**

316 Herbicides – Properties, Synthesis and Control of Weeds

systems in animal (mostly rat) and human models (Rosso et al., 2000; Barnekow et al., 2001; Charles et al., 2001). Doses of 50 mg/kg bw/day of 2,4-D have been reported to increase ventral prostate weight in rats. Treatment of human prostate cancer cell cultures with 10 nM 2,4-D enhanced the androgenic activity of dihydroxytestosterone (DHT) on cell proliferation and transactivation (Kim et al., 2005). In cultured chinese-hamster ovary cells, 2.0 to 10.0 µg/ml 2,4-D were reported to produce DNA damage and sister chromatid exchange (Gonzalez et al., 2005). Importantly, although the 2,4-D toxicity in low doses is controversial, the U.S. Environmental Protection Agency (U.S. EPA, 2006) established a LD50 of 639

There could be particular situations in which the susceptibility of a population exposed to environmental pollutants can be dangerously enhanced. This may be the case for many rural populations subjected to some specific nutritional deficiencies, as often observed in developing countries. Such situation may be worthy of attention during the development

It has been recently found that 2,4-D administered to lactating rats can pass to suckling pups, an can also inhibit the suckling-induced hormone release in the mother. Thus, gestational and lactational periods –including the neonatal and prepubertal stages– seem to be particularly favorable for the induction of 2,4-D effects in rodents (Stürtz et al., 2000;

In human studies, prenatal exposure to 2,4-D was associated with mental retardation of the children (Casey, 1984). Comparable animal experiments in chicken and rats showed that prenatal exposure altered some behavioral patterns of the offspring (Sanders & Rogers,

In the rat, one critical period for normal maturation during growth seems to be that corresponding to the perinatal development of the brain—''the brain growth spurt'' spanning the first 3 or 4 weeks of life (Diaz & Samson, 1980). Therefore, exposure of rats to pesticides during the first weeks of life would have adverse effects on growth and behavior, as well as on the locomotor activity, as affected by anatomical changes. Noteworthy, the age

This selective susceptibility of the developing nervous system may be due to several toxicokinetic factors and a partial lack of a blood–brain barrier (BBB) in the fetus. In humans,

Gupta et al. (1999) have shown that different classes of pesticides are able to change the permeability characteristics of the BBB in rats when administered during some susceptible periods of the BBB development, and that this effect may persist after exposure for variable periods. An altered BBB may render the nervous system more vulnerable to other toxics that

Therefore, although the developing nervous system has some capacity to adapt to or compensate for early perturbations, many chemical agents have been shown more toxic on

the BBB is not fully developed until the middle of the first year of life (Rodier, 1995).

stage, especially concerning the endocrine and nervous systems.

**2. Adverse effects on developing nervous system** 

at exposure is an important factor (Kolb & Wishaw, 1989).

the developing than on the adult nervous system (Tilson, 1998).

would not be able to pass the BBB otherwise.

mg/kg based on rat studies.

1981; Sjoden & Soderberg, 1972).

2006).

Studies in well-fed or undernourished rat offsprings showed that the mechanisms for the induction of the above effects would include some changes in brain monoaminergic system (Ferri et al., 2000) and in iron (Fe), copper (Cu) and zinc (Zn) brain levels (Ferri et al., 2003).

Importantly, the combination of neonatal undernourishment plus mothers' exposure at 2,4- D low dose (70 mg/kg bw) induced a higher modification of the measured parameters than those induced by undernourishment or 2,4-D exposure alone. The data showed a different pup's brain areas susceptibility to the 2,4-D effects and an increased vulnerability to the herbicide, including an increased mortality at a higher dose (100 mg/kg bw), a feature which was not observed in well-nourished animals.

In addition, the results suggest that malnutrition or exposure to 2,4-D exert their effects independently (Tables 1 & 2) (Ferri et al., 2003) and the fact that the alterations observed are very different according to the area involved, reinforces the idea of a selective susceptibility for each brain region.

#### **2.2 Oxidative stress**

Different studies suggest some functional relationships between the oxidative status of the Central Nervous System (CNS) and the protecting level of catecholamines (Kumiko et al., 2001) and metals, like Fe and Cu, the major generators of reactive oxygen species –ROS- in Alzheimer's disease (Huang et al., 1999), related with a decreased glutathione (GSH) content (Dringer, 2000) and also involved in Fenton's and Haber Weiss' redox reactions . (Halliwell & Gutteridge, 1998; Milton, 2004). Other data have shown that 2,4-D affects the redox chain, thus altering cell energetic metabolism and redox balance (Palmeira et al., 1994; Sulik et al., 1998; Bukowska et al., 2003; Duchnowicz et al., 2002).

In rat pups, exposure to 2,4-D through breast milk induced a number of changes in different brain areas, such as disparate changes in the activity of some protective enzymes, an increase in reactive oxygen species (ROS) levels, and a depletion of reduced glutathione (GSH) content (Tables 3, 4 & 5, respectively) (Ferri et al., 2007).

Therefore, as long as a high oxygen consumption by the CNS increases its sensitivity to oxidative stress (Emerit et al., 2004), the observed changes in the levels of metal ions and neurotransmitters, particularly catecholamines, as well as the oxidative status imbalance, would point out oxidative stress as one possible mechanism of adverse 2,4-D effects on the CNS.

318 Herbicides – Properties, Synthesis and Control of Weeds

Oxidative Stress as a Possible Mechanism

**Cu,Zn-SOD** 

**Mn-SOD** 

**Se-GPx** 

**noSe-GPx** 

abbreviations as in the text

exposed to 2,4-D.

**2,4-D** 38.01.4\*

the text.

as in the text.

( 15.7%)

**2,4-D** 1.250.38 1.290.20 0.820.18\*

20.60.5\* ( 15.7%)

of Toxicity of the Herbicide 2,4-Dichlorophenoxyacetic Acid (2,4-D) 319

**DMSO** 2330 119 1950 200 2460 150 2730 330 2330 200 1950 120 2320 160 **2,4-D** 2400 93 2450 310\* 2540 220 2610 240 2980 320\* 2140 90 2400 120

**DMSO** 250 110 250 80 310 120 320 130 270 90 240 120 370 140 **2,4-D** 284 135 150 70 280 100 280 90 390 60 290 110 350 110

**2,4-D** 1978 133\* 2200 200\* 2250 150\* 2530 170 2690 210 1850 120\* 2810 210

**DMSO** 31.52 1.24 30.00 1.43 28.19 2.10 25.62 9.10 29.05 1.14 31.93 1.06 28.89 1.85 **2,4-D** 26.76 1.14\* 24.19 1.90\* 22.29 2.86\* 29.52 2.10\* 32.29 1.00\* 27.57 1.03\* 27.97 1.56

**DMSO** 17.71 0.69 20.80 1.08 18.55 0.62 18.18 0.98 20.94 0.69 22.65 0.77 15.81 0.69 **2,4-D** 20.32 0.94\* 18.99 0.99 15.65 0.46\* 17.95 1.05 19.90 0.87 19.35 0.82\* 17.01 0.71

**Brain PFc Str Cereb Hipp MB Hyp** 

**Brain PFc Str Cereb Hipp MB Hyp** 

( 37.4%) 0.800.22 0.940.24 0.700.15\*

( 11.1%) 23.71.1 18.11.1 23.80.7\*

( 17.2%) 21.11.3

( 34.0%) 1.080.30

**Enzime Treatment Brain PFc Str Cereb Hipp MB Hyp** 

**CAT DMSO** 2556 150 2950 250 2740 200 2300 130 2580 160 2360 200 2740 <sup>150</sup>

( 22.5%) ( 25.4%) ( 17.9%) ( 21.6%)

( 15.10%) ( 19.4%) ( 20.9%) ( 15.2%) ( 11.1%) ( 13.6%)

( 14.7%) ( 15.6%) ( 14.6%)

Enzyme activities are expresed as miliUnits per miligram of protein. Values indicate means ± SEM. Values between brackets are % of increase () or decrease (), respectively, with respect to each DMSO control value. \*p < 0.05, n= 6/group. 100 mg 2,4-D/kg cw of mother. PFc (Pre frontal cortex, Str (Striatum), Hipp (Hippocampus), Hyp (Hypothalamus), MB (Midbarin), Cereb (Cerebellum), Cu,Zn-SOD (Copper,Zinc superoxide dismutase), Mn-SOS (Manganese superoxide dismutase), CAT (catalase), Se-GPx (selenium-glutathione peroxidase), noSe-GPx (non selenium-glutathione peroxidase),and other

Table 3. Protective Enzymes Activities in brain areas of 25-old-day pups lactationally

25.10.7\*

n= 6/group. 100 mg 2,4-D/kg cw of mother. PFc (Pre frontal cortex, Str (Striatum), Hipp

Table 4. ROS levels in brain areas of 25-old-day pups lactationally exposed to 2,4-D.

**DMSO** 45.12.5 17.80.7 22.60.8 24.00.9 18.01.0 20.3 1.0 22.61.2

ROS levels are expresed as IF per mg of protein. Values indicate means ± SEM. Values between brackets are % of increase () or decrease (), respectively, with respect to each DMSO control value.; \*p < 0.05,

(Hippocampus), Hyp (Hypothalamus), MB (Midbarin), Cereb (Cerebellum), other abbreviations as in

**DMSO** 1.220.40 1.230.29 1.310.24 0.790,19 0.880.19 1.060.12 1.070.41

GSH levels are expresed as microgram per miligram of protein. Values indicate means ± SEM. Values between brackets are % of increase () or decrease (), respectively, with respect to each DMSO control value. \*p < 0.05, n= 6/group. 100 mg 2,4-D/kg bw of mother. PFc (Pre frontal cortex, Str (Striatum), Hipp (Hippocampus), Hyp (Hypothalamus), MB (Midbarin), Cereb (Cerebellum), other abbreviations

Table 5. GSH levels in brain areas of 25-old-day pups lactationally exposed to 2,4-D.

( 25.6%) ( 27.9%)


Monoamine content is expressed as pMol/mg of tissue. Values indicate means ± SEM. Values between brackets are % of increase () or decrease (), respectively, with respect to each DMSO control value.\*p < 0.05; \*\*p < 0.01; n= 6/group; 100 mg 2,4-D/kg cw of mother. PFc (Pre frontal cortex), Str (Striatum), Hipp (Hippocampus), Hyp (Hypothalamus), MB (Midbarin), Cereb (Cerebellum), NE (Norepinephrine), DA (Dopamine), DOPAC (3,4-Dihydroxyphenylacetic acid), HVA (Homovanillic Acid), TRP (Tryptophan), 5-HT (Serotonin) and 5-5-HIAA (Hydroxyindoleacetic acid); other abbreviations as indicated in the text.

Table 1. Monoamine levels in different brain areas of 25-day-old, 2,4-D-expossed pups.


Metal contents are expressed as micrograms per gram of wet tissue. Values indicate means ± SEM. Values between brackets are % of increase () or decrease (), respectively, with respect to each DMSO control value. \*p < 0.05 with reference to DMSO control values. \*\*p <0 .01 with reference to DMSO control values; n= 6/group. 100 mg 2,4-D/kg cw of mother. PFc (Pre frontal cortex, Str (Striatum), Cereb (Cerebellum), Hipp (Hippocampus), MB (Midbarin), Hyp (Hypothalamus), and other abbreviations as in the text.

Table 2. Effects of 2,4-D on iron, zinc and copper levels in different brain areas of wellnourished pups.


**AREA Treatment NE DA DOPAC HVA TRP 5-HT 5-HIAA PFc DMSO** 0.93 0.04 3.20 0.44 0.97 0.09 0.28 0.03 20.71 0.61 1.07 0.13 1.08 0.07  **2,4-D** 1.10 0.06\* 2.01 0.30\* 0.90 0.17 0.25 0.03 11.26 0.51\*\* 1.48 0.09\* 1.03 0.05

**Str DMSO** 4.24 0.42 20.79 1.61 9.37 0.48 3.19 0.12 27.46 1.61 2.98 0.31 2.84 0.29  **2,4-D** 2.36 0.44\* 17.03 3.31 6.96 0.69\*\* 2.05 0.32\*\* 28.06 1.76 1.85 0.25\* 2.81 0.42

**Hipp DMSO** 0.91 0.12 0.70 0.09 0.44 0.07 0.30 0.04 5.55 0.35 0.74 0.09 1.63 0.11  **2,4-D** 1.67 0.24\* 0.92 0.09\* 0.58 0.06\* 0.50 0.05\* 3.68 0.20\*\* 1.10 0.13\* 1.75 0.10

**Hyp DMSO** 9.05 1.19 1.58 0.35 1.46 0.21 1.08 0.20 3.81 0.26 1.52 0.13 3.09 0.59  **2,4-D** 13.57 1.44\* 1.80 0.33 1.03 0.17 1.14 0.18 2.96 0.35 2.08 0.31 2.68 0.26

**MB DMSO** 3.16 0.57 1.54 0.31 0.65 0.14 0.36 0.06 31.88 1.21 2.79 0.21 3.54 0.40  **2,4-D** 3.96 0.17 1.96 0.17 0.78 0.12 0.24 0.02 23.34 0.97\*\* 4.14 0.19\*\* 4.78 0.37\* (27%) (48%) (35%) **Cereb DMSO** 1.58 0.12 0.17 0.06 0.31 0.01 0.09 0.01 7.39 0.55 0.46 0.03 0.48 0.03  **2,4-D** 2.44 0.08\*\* 0.21 0.04 0.29 0.01 0.13 0.01 4.86 0.21\*\* 0.43 0.04 0.43 0.02

Monoamine content is expressed as pMol/mg of tissue. Values indicate means ± SEM. Values between brackets are % of increase () or decrease (), respectively, with respect to each DMSO control value.\*p < 0.05; \*\*p < 0.01; n= 6/group; 100 mg 2,4-D/kg cw of mother. PFc (Pre frontal cortex), Str (Striatum),

(Norepinephrine), DA (Dopamine), DOPAC (3,4-Dihydroxyphenylacetic acid), HVA (Homovanillic Acid), TRP (Tryptophan), 5-HT (Serotonin) and 5-5-HIAA (Hydroxyindoleacetic acid); other

Table 1. Monoamine levels in different brain areas of 25-day-old, 2,4-D-expossed pups.

**Metal Treatment PFc Str Cereb Hipp MB Hyp Fe DMSO** 19.58 ± 2.36 17.07 ± 0.90 16.43 ± 1.27 14.23 ± 0.73 18.79 ± 2.03 19.48 ± 2.25

(16.04 %)

13.10 ± 2.00\*\* (61.58%)

15.75 ± 2.65 20.26 ± 0.68\*

**Zn DMSO** 12.51 ± 1.20 34.10 ± 2.40 24.40 ± 1.90 25.40 ± 2.10 32.65 ± 1.10 29.80 ± 3.40

**Cu DMSO** 1.85 ± 0.08 2.25 ± 0.11 1.88 ± 0.07 1.84 ± 0.02 2.21 ± 0.19 1.95 ± 0.08

2.38 ± 0.21 2.20 ± 0.08\*\*

Metal contents are expressed as micrograms per gram of wet tissue. Values indicate means ± SEM. Values between brackets are % of increase () or decrease (), respectively, with respect to each DMSO control value. \*p < 0.05 with reference to DMSO control values. \*\*p <0 .01 with reference to DMSO control values; n= 6/group. 100 mg 2,4-D/kg cw of mother. PFc (Pre frontal cortex, Str (Striatum), Cereb (Cerebellum), Hipp (Hippocampus), MB (Midbarin), Hyp (Hypothalamus), and other abbreviations as

Table 2. Effects of 2,4-D on iron, zinc and copper levels in different brain areas of well-

**2,4-D 70 mg/kg** 1.97 ± 0.18 2.17 ± 0.13 2.00 ± 0.10 2.01 ± 0.20 2.16 ± 0.21 1.91 ± 0.16

(17.02%)

(23.31%)

21.50 ± 0.85 27.89 ± 1.60\*

24.40 ± 0.70 34.30 ± 3.50\*

(9.80%)

(35.04%)

2.23 ± 0.17\* (21.19%)

16.65 ± 2.65 17.53 ± 0.86 12.86 ± 1.20 15.54 ± 0.55 20.95 ± 1.76

13.98 ± 1.80 14.95 ± 0.35 21.31 ± 2.68

24.35 ± 1.54\*\* (25.42%)

29.55 ± 1.74 27.11 ± 2.56

2.14 ± 0.20 1.91 ± 0.15

26.37 ± 3.20

(20%) (37%) (46%) (38%)

(44%) (26%) (36%) (38%)

(83%) (31%%) (31%) (66%) (34%) (49%)

(54%) (34%)

Hipp (Hippocampus), Hyp (Hypothalamus), MB (Midbarin), Cereb (Cerebellum), NE

(50%)

abbreviations as indicated in the text.

 **2,4-D 70 mg/kg** 24.48 ± 1.83\*

 **2,4-D 100 mg/kg** 29.48 ± 2.19\*

 **2,4-D 100 mg/kg** 17.93 ± 2.00\*

in the text.

nourished pups.

**2,4-D 100 mg/kg** 2.31 ± 0.21\*

(25.05 %)

(50.56 %)

(43.32%)

( 24.86%)

 **2,4-D 70 mg/kg** 14.64 ± 2.20 28.63 ± 2.40\*


Enzyme activities are expresed as miliUnits per miligram of protein. Values indicate means ± SEM. Values between brackets are % of increase () or decrease (), respectively, with respect to each DMSO control value. \*p < 0.05, n= 6/group. 100 mg 2,4-D/kg cw of mother. PFc (Pre frontal cortex, Str (Striatum), Hipp (Hippocampus), Hyp (Hypothalamus), MB (Midbarin), Cereb (Cerebellum), Cu,Zn-SOD (Copper,Zinc superoxide dismutase), Mn-SOS (Manganese superoxide dismutase), CAT (catalase), Se-GPx (selenium-glutathione peroxidase), noSe-GPx (non selenium-glutathione peroxidase),and other abbreviations as in the text

Table 3. Protective Enzymes Activities in brain areas of 25-old-day pups lactationally exposed to 2,4-D.


ROS levels are expresed as IF per mg of protein. Values indicate means ± SEM. Values between brackets are % of increase () or decrease (), respectively, with respect to each DMSO control value.; \*p < 0.05, n= 6/group. 100 mg 2,4-D/kg cw of mother. PFc (Pre frontal cortex, Str (Striatum), Hipp (Hippocampus), Hyp (Hypothalamus), MB (Midbarin), Cereb (Cerebellum), other abbreviations as in the text.

Table 4. ROS levels in brain areas of 25-old-day pups lactationally exposed to 2,4-D.


GSH levels are expresed as microgram per miligram of protein. Values indicate means ± SEM. Values between brackets are % of increase () or decrease (), respectively, with respect to each DMSO control value. \*p < 0.05, n= 6/group. 100 mg 2,4-D/kg bw of mother. PFc (Pre frontal cortex, Str (Striatum), Hipp (Hippocampus), Hyp (Hypothalamus), MB (Midbarin), Cereb (Cerebellum), other abbreviations as in the text.

Table 5. GSH levels in brain areas of 25-old-day pups lactationally exposed to 2,4-D.

Oxidative Stress as a Possible Mechanism

**Hydroxyl radical** 

**Carbonyl groups** 

**Total Thiols** 

**MDA** 

**GST** 

**CAT** 

**Se-GPx** 

**3.2 Ovary** 

(Agarwal et al., 2006).

Abbreviations as in the text.

Table 6. Oxidative parameters in ventral prostate.

species stimulates antioxidant activity (Celik & Tuluce, 2007).

**GR** 

of Toxicity of the Herbicide 2,4-Dichlorophenoxyacetic Acid (2,4-D) 321

**Control** 3.25±0.34 2.97±0.39 1.09±0.13 **2,4-D** 8.75±0.61\* (169%) 6.53±0.09\* (119%) 2.03±0.18\* (85%)

**Control** 3.54±0.12 10.66±1.07 7.02±0.88 **2,4-D** 4.84±0.11\* (37%) 15.01±1.32\* (47%) 12.42±1.11\* (77%)

**Control** 491±12 642±86 341±24 **2,4-D** 520±14 748±14 333±8

**Control** 29.09±0.32 27.74±3.74 38.27± 2.14 **2,4-D** 41.91±3.05\* (44%) 42.69±3.13\* (54%) 47.48 ± 2.54\* (24%)

**Control** 8.93±0.67 10.53±2.53 13.37±2.09 **2,4-D** 19.07±2,45\* (113%) 15.95±1.04\* (45) 18.14±0.26\* (36)

**Control** 10.93±1,20 5.44±0.21 5.99±0.21 **2,4-D** 14.74±1.26\* (35%) 11.44±0.34\* (110%) 7.77±0.39\* (28%)

**Control** 312±18 518±57 562±32 **2,4-D** 436±33\* (36) 880±41\* (70%) 530±13

**Control** 10.05±0.86 33.01±2.52 31.09±4.36 **2,4-D** 10.47±1.78 37.75±3.48 10.57±0.05\* (72%)

Hydroxy radical are expresed as 2,3 dihydroxybenzoic acid/salicilc acid rario; carbonyl groups and total thiols are expresed as micromol per miligram of protein; MDA is expresed as nanomol per microgram of protein. GST, CAT and GR activities are expresed as Units per miligram of protein; and Se-GPx is expresed as miliUnits per miligram of protein. Each value is the mean ± SEM. Values between brackets are % of increase () or decrease (); \*p < 0.05, n= 6/group. 70 mg 2,4-D/kg cw of mother.

Therefore, the 2,4-D-induced increase in all ROS level, lipid peroxidation and protein oxidation may have caused some critical oxidative stress in ventral prostate. Nevertheless, the increased activity of some antioxidant enzymes in the prostate could have not been strong enough as to counteract the oxidative stress produced by the herbicide at different stages of rat development. Moreover, it is not a general rule that increase in oxidative

The complex ovarian structure varies widely during differentiation. Free radicals play important regulating roles during the ovarian follicular cycle, possibly through inhibition of steroid production (Behrman et al., 2001). There is also a delicate balance between ROS and antioxidant enzymes in the ovarian tissues (Agarwal et al., 2005). Non-physiological effects of free radicals include premature ovarian follicular atresia via cell apoptosis. Many pesticides— e.g. the xenoestrogen pesticide methoxychlor — can induce oxidative stress and apoptosis in the ovary (Gupta et al., 2006). Moreover, clinical studies have reported increased levels of reactive oxygen species associated to a decreased female fertility

**PND 45 PND 60 PND 90** 
