Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous Self-Pollinated Sunflower Lines

*Victoria Mykhailenko, Viktor Kyrychenko, Alexander Bragin and Dmitry Chuiko*

## **Abstract**

A majority of sunflower lines and hybrids were based on starting material obtained by traditional methods; so the issues of developing new trends in extending the genetic diversity of this crop require constant attention of scientists. At present, induced mutagenesis along with hybridization has become a leading method for generating new forms of crops. Their success depends largely on availability and assortment of starting material. Induction of mutations is a way to create it. The main value of induced mutagenesis for breeding is determined by opportunities to solve problems that are impossible or difficult to solve by traditional methods. The choice of an effective concentration (dose) of a mutagen is very important, since the frequency and range of mutations depend not only on the mutagen itself but also on its dose and exposure. In addition, it is relevant to search for new mutagens with reduced harmful effects at the same level of mutability. Cytological analysis of chromosomal aberrations is an important method of evaluation and identification of mutagenic effects. In this section, studies into chemical and physical mutagenesis in breeding, exemplified by new modern homozygous self-pollinated sunflower lines, are summarized; methodical recommendations on the use of induced mutagenesis in sunflower breeding are presented; and methods of generation, investigation, and further use of mutations are rationalized.

**Keywords:** gamma rays, dimethyl sulfate, mutagenesis, meiosis, mutation, self-pollinated line, breeding, sunflower

## **1. Introduction**

In comparison with other oil crops, sunflower produces the highest oil yield per unit area (on average 750 kg/ha in Ukraine), which makes this crop a major oil crop. The oil content in seeds of released hybrids is 50–52%, and in breeding hybrids—up to 60%.

The nutritional value of sunflower oil is determined by high content of polyunsaturated fatty linoleic acid (55–60%), which has a significant biological activity and accelerates the metabolism of cholesterol esters in the body, which has a positive effect on health. Sunflower oil also contains ingredients that are very valuable to the human body, such as phosphatides, sterols, and vitamins (A, D, E, K). The nutritional value of sunflower seeds per 100 g is as follows: energy, 2445 kJ; proteins, 20.8 g; fats, 51.5 g (of which saturated fats account for 4.5 g; monounsaturated ones, 18.5 g; and polyunsaturated ones, 23.1 g); and carbohydrates, 20 g. This makes sunflower a valuable food product.

Induced mutagenesis allows developing new starting material with various morphological and physiological features and biochemical parameters, increasing the frequency and expanding the assortment of original mutations within a short time.

Mutations are a source of expansion of the genetic diversity of sunflower, which in its turn is a starting material for the breeding of this crop. Radiation and chemical mutagens are used to produce artificial mutations in sunflower [1].

The strongest chemical mutagens (supermutagens), which cause a several hundred-fold increase in the frequency of mutations, include ethyleneamine, diethyl sulfate, dimethyl sulfate, nitrosoethylurea, nitrosomethylurea, hydrogen peroxide, etc. [2].

Since Wetterer's first attempts to gamma-irradiate sunflower seeds in 1911, Shull and Mitchell's experiments in 1933 [3], Soldatov's achievements [4], reports of contemporary scientists Kalaydzhan [5], Lacombe [6], Soroka [7], Cvejic [8, 9], Lyakh [10], Vasin [11, 12], Kyrychenko [13], Škorić [14], and many others, a considerable progress have been achieved in enriching the sunflower gene pool by induced mutagenesis. However, despite considerable advances, due to continuous refreshment of starting material, induced mutagenesis has been and remains an important method for developing new and improving existent starting material in breeding.

Our purpose was to obtain self-pollinated sunflower lines with genetic mutations induced by chemical and physical mutagens that can be used to improve features of the sunflower crop and to develop methodological approaches for studying mutant generations.

## **2. Means and mechanisms of experimental mutation induction**

When researchers obtain and control new hereditary changes in plants during their experiments, some completely new possibilities to create breeding initial material appear.

Since Watson and Crick decoded the structure of DNA, characterized the mechanism of its replication and discovered the system of recording genetic information, highlighting the genetic nature of mutations, it became evident that the primary cause of any mutation is the primary disorders in the DNA structure, which are in the process of cell metabolism can be realized in true mutations or repaired and restored to their original state.

Primary abnormalities induced in hereditary structures of an organism under the influence of natural or artificial factors can cause the appearance of two types of mutations—point ones, caused by disorders in the original structures of the DNA molecule, and chromosomal ones, caused by qualitative or quantitative changes in the chromosomal systems of cells.

Primary disorders in DNA structures are not repaired to their original state; they initiate the processes of gene (point) mutation formation. Such disorders include replacement of nitrogen base pairs (transversions); the inclusion of additional complementary pairs of nucleotides (duplication); loss of nucleotide pairs in the structure of a DNA molecule (deletions); 1800 rotation of nucleotide pairs (inversions), etc.

**45**

molecules.

originated.

possible recombinations.

as study objects.

treatment.

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous…*

Initial DNA integrity disorders may result in chromosome rupture. In this case open sections of chromosomal filaments can be combined reaching their original state or form new combinations. Thus, there are chromosomal mutations that are

Mutations can attach a molecule of sugar (deoxyribose), phosphate, or a nitrogenous base to the nucleotides. For example, despite the fact that deoxyribose is the only sugar group in DNA, it is not desirable to exclude the possibility of accidental incorporation of individual ribose molecules into DNA. In such cases, the phosphate and nitrogenous bases of the ribonucleotide may be like those of the dezoxyribonucleotides. Phosphoric acid, as a component of DNA and RNA, may contain

Disorders induced by irradiation in DNA molecules can affect phosphodiester, sugar-phosphate, glycosyl, and other chemical bonds. As a result, single or doublethread breaks occur, as well as the destruction of nitrogenous bases. For example, when dry seeds are irradiated, the nitrogenous bases included in the DNA structure may be converted in to thymine, guanine radicals, etc., which are sufficiently stable

The alkylating compounds including DMS are a source to introduce the methyl radicals (CH3), ethyl (C2H5), etc. into the molecules, thereby providing an alkylation reaction. They are characterized by a wide range of mutagenic effects, inducing simple and complex substitutions as well as breaks in DNA molecules. All nitrogenous bases, phosphoric acid residue, and even deoxyribose residue are alkylated. As a result of alkylation reactions, the purine bases are most likely to fall out DNA strand, causing the formation of voids at the corresponding points in the molecule. It is obvious that the mechanism of mutagenesis in the alkylation of DNA bases is associated with a violation in the accuracy of the auto-reproduction of DNA

All these events eventually result in changes within DNA molecules that manifest themselves as mutations, most of which are lethal. However, many mutations are viable. They are involved in the process of gene recombination, and as a result they are integrated in their functions with other genes of the genotype where they

The plant genotype has a significant effect on the specificity and level of mutations. Therefore, selection of starting material plays a significant role in obtaining valuable mutants. Generally, the best area-specific forms are recommended to use as sources, which need refining in terms of individual characteristics and features. Constant self-pollinated forms are the best for mutational breeding, as their mutations can be easily and reliably identified. Therefore, working with mutagenesis, one should apply different methods of isolation of nurseries and mutant plants in order to prevent biological contamination and occurrence, along with mutations, of

Twelve new homozygous, self-pollinated sunflower lines from a genetic collection of the Plant Production Institute named after V.Ya. Yuriev, which are of breeding value and differ in several morphological and biochemical features, were taken

Chemical supermutagen dimethyl sulfate (DMS) and gamma rays were used to induce genetic variability. Two hundred fifty seeds were used in each variant of

**3. Research methodology: selection of starting material**

*DOI: http://dx.doi.org/10.5772/intechopen.89563*

radioactive P32 atom instead of a normal phosphorus one.

in the dry state and sufficiently reactive when wetted.

characterized by a wide diversity.

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous… DOI: http://dx.doi.org/10.5772/intechopen.89563*

Initial DNA integrity disorders may result in chromosome rupture. In this case open sections of chromosomal filaments can be combined reaching their original state or form new combinations. Thus, there are chromosomal mutations that are characterized by a wide diversity.

Mutations can attach a molecule of sugar (deoxyribose), phosphate, or a nitrogenous base to the nucleotides. For example, despite the fact that deoxyribose is the only sugar group in DNA, it is not desirable to exclude the possibility of accidental incorporation of individual ribose molecules into DNA. In such cases, the phosphate and nitrogenous bases of the ribonucleotide may be like those of the dezoxyribonucleotides. Phosphoric acid, as a component of DNA and RNA, may contain radioactive P32 atom instead of a normal phosphorus one.

Disorders induced by irradiation in DNA molecules can affect phosphodiester, sugar-phosphate, glycosyl, and other chemical bonds. As a result, single or doublethread breaks occur, as well as the destruction of nitrogenous bases. For example, when dry seeds are irradiated, the nitrogenous bases included in the DNA structure may be converted in to thymine, guanine radicals, etc., which are sufficiently stable in the dry state and sufficiently reactive when wetted.

The alkylating compounds including DMS are a source to introduce the methyl radicals (CH3), ethyl (C2H5), etc. into the molecules, thereby providing an alkylation reaction. They are characterized by a wide range of mutagenic effects, inducing simple and complex substitutions as well as breaks in DNA molecules. All nitrogenous bases, phosphoric acid residue, and even deoxyribose residue are alkylated. As a result of alkylation reactions, the purine bases are most likely to fall out DNA strand, causing the formation of voids at the corresponding points in the molecule. It is obvious that the mechanism of mutagenesis in the alkylation of DNA bases is associated with a violation in the accuracy of the auto-reproduction of DNA molecules.

All these events eventually result in changes within DNA molecules that manifest themselves as mutations, most of which are lethal. However, many mutations are viable. They are involved in the process of gene recombination, and as a result they are integrated in their functions with other genes of the genotype where they originated.

## **3. Research methodology: selection of starting material**

The plant genotype has a significant effect on the specificity and level of mutations. Therefore, selection of starting material plays a significant role in obtaining valuable mutants. Generally, the best area-specific forms are recommended to use as sources, which need refining in terms of individual characteristics and features. Constant self-pollinated forms are the best for mutational breeding, as their mutations can be easily and reliably identified. Therefore, working with mutagenesis, one should apply different methods of isolation of nurseries and mutant plants in order to prevent biological contamination and occurrence, along with mutations, of possible recombinations.

Twelve new homozygous, self-pollinated sunflower lines from a genetic collection of the Plant Production Institute named after V.Ya. Yuriev, which are of breeding value and differ in several morphological and biochemical features, were taken as study objects.

Chemical supermutagen dimethyl sulfate (DMS) and gamma rays were used to induce genetic variability. Two hundred fifty seeds were used in each variant of treatment.

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

makes sunflower a valuable food product.

peroxide, etc. [2].

generations.

material appear.

repaired and restored to their original state.

the chromosomal systems of cells.

activity and accelerates the metabolism of cholesterol esters in the body, which has a positive effect on health. Sunflower oil also contains ingredients that are very valuable to the human body, such as phosphatides, sterols, and vitamins (A, D, E, K). The nutritional value of sunflower seeds per 100 g is as follows: energy, 2445 kJ; proteins, 20.8 g; fats, 51.5 g (of which saturated fats account for 4.5 g; monounsaturated ones, 18.5 g; and polyunsaturated ones, 23.1 g); and carbohydrates, 20 g. This

Induced mutagenesis allows developing new starting material with various morphological and physiological features and biochemical parameters, increasing the frequency and expanding the assortment of original mutations within a short time. Mutations are a source of expansion of the genetic diversity of sunflower, which in its turn is a starting material for the breeding of this crop. Radiation and chemi-

cal mutagens are used to produce artificial mutations in sunflower [1].

for developing new and improving existent starting material in breeding.

**2. Means and mechanisms of experimental mutation induction**

The strongest chemical mutagens (supermutagens), which cause a several hundred-fold increase in the frequency of mutations, include ethyleneamine, diethyl sulfate, dimethyl sulfate, nitrosoethylurea, nitrosomethylurea, hydrogen

Since Wetterer's first attempts to gamma-irradiate sunflower seeds in 1911, Shull and Mitchell's experiments in 1933 [3], Soldatov's achievements [4], reports of contemporary scientists Kalaydzhan [5], Lacombe [6], Soroka [7], Cvejic [8, 9], Lyakh [10], Vasin [11, 12], Kyrychenko [13], Škorić [14], and many others, a considerable progress have been achieved in enriching the sunflower gene pool by induced mutagenesis. However, despite considerable advances, due to continuous refreshment of starting material, induced mutagenesis has been and remains an important method

Our purpose was to obtain self-pollinated sunflower lines with genetic mutations induced by chemical and physical mutagens that can be used to improve features of the sunflower crop and to develop methodological approaches for studying mutant

When researchers obtain and control new hereditary changes in plants during their experiments, some completely new possibilities to create breeding initial

Primary abnormalities induced in hereditary structures of an organism under the influence of natural or artificial factors can cause the appearance of two types of mutations—point ones, caused by disorders in the original structures of the DNA molecule, and chromosomal ones, caused by qualitative or quantitative changes in

Primary disorders in DNA structures are not repaired to their original state; they initiate the processes of gene (point) mutation formation. Such disorders include replacement of nitrogen base pairs (transversions); the inclusion of additional complementary pairs of nucleotides (duplication); loss of nucleotide pairs in the structure of a DNA molecule (deletions); 1800 rotation of nucleotide pairs (inversions), etc.

Since Watson and Crick decoded the structure of DNA, characterized the mechanism of its replication and discovered the system of recording genetic information, highlighting the genetic nature of mutations, it became evident that the primary cause of any mutation is the primary disorders in the DNA structure, which are in the process of cell metabolism can be realized in true mutations or

**44**

As to chemical mutagenesis, seeds in capron partly loosened sacs were soaked in 0.01 and 0.05% DMS solutions (prepared on distilled water, as some mutagens tend to degrade rapidly in tap water). We prepared these solutions under a hood, wearing rubber gloves: crushed an ampoule with mutagen in water. Depending on the object, the treatment time ranges 2–24 h. With sunflower seeds, the exposure was 18 hours, with periodic stirring. The mutagen/treated seeds ratio (volume/weight) was 10:1. In addition, to accelerate the process of solution penetration through the seed coat, it is recommended to ultrasound seeds for a short time (1–7 min) [15]. After treatment of seeds, in order to reduce the damaging effect, we washed them out for 1 h in running tap water and then sowed in soil on the same day.

Dimethyl sulfate (DMS) is a chemical supermutagen, an alkylating compound, which breaks chromosomes, leading to a large number of chromosomal inversions.

Studying physical mutagenesis on new sunflower lines, we used gamma rays from the radioactive isotope Co60, which has a relatively high irradiation uniformity. Dry seeds were once irradiated on a "Theratron Elit 80" Ionizing Radiation Source Cobalt 60 at Kharkiv Regional Oncology Hospital.

In a research on induced mutagenesis, a great attention is paid to concentrations (doses) of mutagens, which affect the number and quality of mutations; therefore, we used the most effective for agricultural crops DMS concentrations (0.01 and 0.05%) and doses of gamma rays (120 and 150 Gy).

Seeds of corresponding sunflower lines soaked in distilled water were used as controls.

When working with mutagens, which are poisonous and sometimes volatile, one should strictly follow the safety regulations and have appropriate equipment and rooms [16, 17].

## **4. Generation, evaluation, and further use of mutations: M1 generation**

Mutagen-treated seeds were sown in mutant nurseries: M1 nursery (area = 20 m2 ; single-row plots comprising 250 plants each), M2 nursery (area = 40 m2 ; single-row plots comprising 25 plants each), and M3 nursery (area = 50 m2 ; single-row plots comprising 25 plants each). The sowing scheme was 70 × 25 cm. Seeds were sown with manual planters within the optimal timeframe (2nd–3rd 10 days of May). Winter wheat was the forecrop. Mutant plants in the experimental plots were harvested by cutting and manually threshed.

We observed the expected decrease in the field germinability in the M1 generation, and the higher concentration or dose of the mutagenic factor was, the more drastically the germinability is reduced. Our data indicate that the phenotypic effect of gamma rays is stronger than the DMS effect.

The highest frequency of phenotypic changes was noticed with 150 Gy gamma irradiation (42.9%); the frequency of phenotypic changes after DMS treatment was only 27–28%. The plant development was delayed and was followed by death. Among the DMS-treated plants, there were no such phenomena; therefore the used concentrations of this chemical are not lethal (**Table 1**).

Mutagenic factors affect biochemical processes in seeds, impairing metabolism and causing unnatural changes, which in its turn influences vital processes in seeds and plants emerging from them. Therefore, studies of microspore formation (meiosis) are a reliable way to investigate the genetic variability of organisms at the cellular level and to the evaluate effects of mutagenic factors on chromosomes of pollen mother cells (PMC) of sunflower lines.

**47**

anthers.

**Table 1.**

Gamma rays

meiosis were as follows:

acid 3:1) for 24 h.

disappears.

acetocarmine.

alcohol burner until boiling.

glass and examined under a microscope.

anthers. Green star phase.

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous…*

**Number of plants from treated seeds**

Control No treatment 250 220 220 0 0.0 DMS 0.01 250 218 157 61 28.0

**Number of phenotypically unchanged plants**

0.05 250 208 152 56 27.0

120 250 38 22 16 42.1 150 250 28 16 12 42.9

**Number of phenotypically changed plants**

**Phenotypic effect (%)**

Microspores in anthers of flowering plants are the final result of meiosis, which

The genotypes of the new self-pollinated lines—sterility fixers (Kh1002B and Kh1008B) and lines— pollen fertility restorers (Kh06134V and Kh201V) pre-treated with chemical mutagen DMS at concentrations of 0.01 and 0.05% or gamma-

The steps of microslide preparation to investigate chromosomes in sunflower

1.Collection of specimens in the field—calathidium segments (d = 2–3 cm) with

2.Fixation of the specimens in Clark's solution (absolute alcohol/glacial acetic

5.Staining the specimens in 2% aceto-orcein for 12–24 h. Aceto-orcein solution was prepared as follows: dissolve 1 g of dye in 45 ml of glacial acetic acid and 55 ml of distilled water. Dissolution is carried out in a reflux flask in a water bath for 30–60 min. After cooling, the solution of aceto-orcein is filtered and placed in a glass stoppered bottle. As a part of the study, we demonstrated that aceto-orcein was more effective for staining sunflower chromosomes than

6.The stained specimen is placed on a mount in a drop of 45% acetic acid or in a drop of 0.5% aceto-orcein, covered with a cover slip and heated above an

7.The slide is carefully crushed with a match to get a cell monolayer under the

Meiosis was examined under a Micromed XS-5520 microscope at magnification of 40× and 100×. Oil immersion (special immersion oil, cedar oil, or glycerol)

3.Washing out the specimens in 70% ethanol until the odor of acetic acid

can be traced on temporary and permanent microslides made from immature

irradiated at doses of 120 and 150 Gy were studied.

*Phenotype effect in the M1 generation of sunflower (average across lines).*

4.Storage of the specimens in 70% ethanol.

*DOI: http://dx.doi.org/10.5772/intechopen.89563*

**Number of treated seeds**

**(%)/dose (Gy)**

**Mutagen Concentration** 

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous… DOI: http://dx.doi.org/10.5772/intechopen.89563*


## **Table 1.**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

As to chemical mutagenesis, seeds in capron partly loosened sacs were soaked in 0.01 and 0.05% DMS solutions (prepared on distilled water, as some mutagens tend to degrade rapidly in tap water). We prepared these solutions under a hood, wearing rubber gloves: crushed an ampoule with mutagen in water. Depending on the object, the treatment time ranges 2–24 h. With sunflower seeds, the exposure was 18 hours, with periodic stirring. The mutagen/treated seeds ratio (volume/weight) was 10:1. In addition, to accelerate the process of solution penetration through the seed coat, it is recommended to ultrasound seeds for a short time (1–7 min) [15]. After treatment of seeds, in order to reduce the damaging effect, we washed them

out for 1 h in running tap water and then sowed in soil on the same day.

Source Cobalt 60 at Kharkiv Regional Oncology Hospital.

0.05%) and doses of gamma rays (120 and 150 Gy).

Dimethyl sulfate (DMS) is a chemical supermutagen, an alkylating compound, which breaks chromosomes, leading to a large number of chromosomal

Studying physical mutagenesis on new sunflower lines, we used gamma rays from the radioactive isotope Co60, which has a relatively high irradiation uniformity. Dry seeds were once irradiated on a "Theratron Elit 80" Ionizing Radiation

In a research on induced mutagenesis, a great attention is paid to concentrations (doses) of mutagens, which affect the number and quality of mutations; therefore, we used the most effective for agricultural crops DMS concentrations (0.01 and

Seeds of corresponding sunflower lines soaked in distilled water were used as

**4. Generation, evaluation, and further use of mutations: M1 generation**

comprising 25 plants each). The sowing scheme was 70 × 25 cm. Seeds were sown with manual planters within the optimal timeframe (2nd–3rd 10 days of May). Winter wheat was the forecrop. Mutant plants in the experimental plots were

single-row plots comprising 250 plants each), M2 nursery (area = 40 m2

plots comprising 25 plants each), and M3 nursery (area = 50 m2

harvested by cutting and manually threshed.

of gamma rays is stronger than the DMS effect.

pollen mother cells (PMC) of sunflower lines.

concentrations of this chemical are not lethal (**Table 1**).

Mutagen-treated seeds were sown in mutant nurseries: M1 nursery (area = 20 m2

We observed the expected decrease in the field germinability in the M1 generation, and the higher concentration or dose of the mutagenic factor was, the more drastically the germinability is reduced. Our data indicate that the phenotypic effect

The highest frequency of phenotypic changes was noticed with 150 Gy gamma irradiation (42.9%); the frequency of phenotypic changes after DMS treatment was only 27–28%. The plant development was delayed and was followed by death. Among the DMS-treated plants, there were no such phenomena; therefore the used

Mutagenic factors affect biochemical processes in seeds, impairing metabolism

and causing unnatural changes, which in its turn influences vital processes in seeds and plants emerging from them. Therefore, studies of microspore formation (meiosis) are a reliable way to investigate the genetic variability of organisms at the cellular level and to the evaluate effects of mutagenic factors on chromosomes of

;

; single-row

; single-row plots

When working with mutagens, which are poisonous and sometimes volatile, one should strictly follow the safety regulations and have appropriate equipment and

**46**

inversions.

controls.

rooms [16, 17].

*Phenotype effect in the M1 generation of sunflower (average across lines).*

Microspores in anthers of flowering plants are the final result of meiosis, which can be traced on temporary and permanent microslides made from immature anthers.

The genotypes of the new self-pollinated lines—sterility fixers (Kh1002B and Kh1008B) and lines— pollen fertility restorers (Kh06134V and Kh201V) pre-treated with chemical mutagen DMS at concentrations of 0.01 and 0.05% or gammairradiated at doses of 120 and 150 Gy were studied.

The steps of microslide preparation to investigate chromosomes in sunflower meiosis were as follows:


Meiosis was examined under a Micromed XS-5520 microscope at magnification of 40× and 100×. Oil immersion (special immersion oil, cedar oil, or glycerol) was used to study slides at magnification of 100×. To document and illustrate the results, microphotographs were taken with a Nikon D 3200 kit VR camera equipped with a special Asian Microscope Adapter.

Cells with meiosis disorders were counted by metaphase-anaphase method: the percentage of cells with abnormalities was calculated related to the total number of cells under examination.

Analysis of meiosis in archisporial cells showed considerable effects of DMS and gamma rays on chromosomes in the M1, which manifested themselves as occurrence of significant chromosomal aberrations compared to the control (P < 0.99). The effect level depended on the mutagen exposure.

For example, after DMS treatment, the percentage of cells with abnormalities ranged within 7–14% (0.01%) and 12–20% (0.05%), significantly exceeding the control. After gamma irradiation, the percentage of cells with abnormalities ranged within 16–19% (120 Gy) and 20–25% (150 Gy), significantly exceeding the control.

Comparison of the results showed that the effect of gamma rays on meiosis of the lines under investigation significantly differed (P < 0.99) from that of DMS. Gamma rays resulted in the occurrence of more abnormal tetrads in the M1 compared to DMS treatment. After irradiation, the percentage of abnormal tetrads ranged from 16.00% in line Kh1008B (120 Gy) to 27.10% in line Kh201V (150 Gy), whereas in DMS - treated lines, the percentage of abnormal tetrads ranged from 1.55% in line Kh201V (0.01%) to 21.65% in line Kh1008B (0.05%).

We observed normalization of meiosis and elimination of cells with abnormalities in subsequent mutant generations of sunflower compared to the M1.

In line Kh06134V, the percentage of cells with abnormalities in different phases of meiosis in the M2 varied within 8.09–8.69% (0.01 and 0.05% DMS) and within 5.96–8.16% (120 and 150 Gy gamma irradiation). In the M3, the percentage of aberrations varied within 3.36–4.09% after 0.01 and 0.05% DMS treatment and within 4.29–5.34% after 120 and 150 Gy gamma irradiation.

In line Kh201V, the percentage of cells with abnormalities in the M2 varied within 4.53–8.45% after DMS treatment and within 7.79–9.48% after gamma irradiation. In the M3, these values were 2.54–4.96 and 2.15–3.48%, respectively.

In line Kh1002B, the percentage of cells with abnormalities in the M2 varied within 6.06–4.89% after DMS treatment and within 6.91–7.44% after gamma irradiation. In the M3, these values were 3.35–4.66 and 3.60–4.83%, respectively.

In line Kh1008B, we noted 4.92–6.95% of cells with abnormalities in the M2 after DMS treatment and 6.42–10.77% after gamma irradiation. In the M3, these values were 2.15–3.57 and 3.09–5.26%, respectively (**Figure 1**).

The identified meiotic abnormalities in mutants were manifested as a chromosome lag during the formation of metaphase plate, impaired chromosome distribution in metaphase II, distorted metaphase plates, a chromosome lag in anaphase, asynchronous division during the second stage of meiosis, formation of pentads, triads, dyads, etc. (**Figure 2**).

Note. 1, outsider chromosomes in anaphase I; 2, asynchronous division during the second stage of meiosis; 3, chromosomes outside the metaphase plate in metaphase I; 4, abnormal tetrads.

All the specimens had phenotypic alterations (bent stem, dwarfism, absence of generative organs, chlorophyll deficit, deformation of generative organs, etc.) during subsequent development (**Figure 3**).

To prevent cross-pollination between different sunflower lines, individual inflorescences had been isolated the day before semiflorets opened, the offspring of which were to be examined the next year as the M2 families. Concurrently, controls, non-treated with mutagens lines, were isolated.

**49**

**Figure 1.**

*M1–M3 sunflower (%).*

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous…*

During the vegetation period, phenological observations of the growth and development of mutant plants were conducted; the field germinability was determined; cytological analysis was performed; and biometric measurements were

*Normalization of meiosis and gradual attenuation of the mutagenic effects of DMS and gamma rays in the* 

*DOI: http://dx.doi.org/10.5772/intechopen.89563*

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous… DOI: http://dx.doi.org/10.5772/intechopen.89563*

#### **Figure 1.**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

with a special Asian Microscope Adapter.

effect level depended on the mutagen exposure.

cells under examination.

control.

was used to study slides at magnification of 100×. To document and illustrate the results, microphotographs were taken with a Nikon D 3200 kit VR camera equipped

Cells with meiosis disorders were counted by metaphase-anaphase method: the percentage of cells with abnormalities was calculated related to the total number of

Analysis of meiosis in archisporial cells showed considerable effects of DMS and gamma rays on chromosomes in the M1, which manifested themselves as occurrence of significant chromosomal aberrations compared to the control (P < 0.99). The

For example, after DMS treatment, the percentage of cells with abnormalities ranged within 7–14% (0.01%) and 12–20% (0.05%), significantly exceeding the control. After gamma irradiation, the percentage of cells with abnormalities ranged within 16–19% (120 Gy) and 20–25% (150 Gy), significantly exceeding the

Comparison of the results showed that the effect of gamma rays on meiosis of the lines under investigation significantly differed (P < 0.99) from that of DMS. Gamma rays resulted in the occurrence of more abnormal tetrads in the M1 compared to DMS treatment. After irradiation, the percentage of abnormal tetrads ranged from 16.00% in line Kh1008B (120 Gy) to 27.10% in line Kh201V (150 Gy), whereas in DMS - treated lines, the percentage of abnormal tetrads ranged from

We observed normalization of meiosis and elimination of cells with abnormali-

In line Kh06134V, the percentage of cells with abnormalities in different phases of meiosis in the M2 varied within 8.09–8.69% (0.01 and 0.05% DMS) and within 5.96–8.16% (120 and 150 Gy gamma irradiation). In the M3, the percentage of aberrations varied within 3.36–4.09% after 0.01 and 0.05% DMS treatment and within

In line Kh201V, the percentage of cells with abnormalities in the M2 varied within 4.53–8.45% after DMS treatment and within 7.79–9.48% after gamma irradiation. In the M3, these values were 2.54–4.96 and 2.15–3.48%, respectively. In line Kh1002B, the percentage of cells with abnormalities in the M2 varied within 6.06–4.89% after DMS treatment and within 6.91–7.44% after gamma irradiation. In the M3, these values were 3.35–4.66 and 3.60–4.83%, respectively.

In line Kh1008B, we noted 4.92–6.95% of cells with abnormalities in the M2 after DMS treatment and 6.42–10.77% after gamma irradiation. In the M3, these values

The identified meiotic abnormalities in mutants were manifested as a chromosome lag during the formation of metaphase plate, impaired chromosome distribution in metaphase II, distorted metaphase plates, a chromosome lag in anaphase, asynchronous division during the second stage of meiosis, formation of pentads,

Note. 1, outsider chromosomes in anaphase I; 2, asynchronous division during the second stage of meiosis; 3, chromosomes outside the metaphase plate in meta-

All the specimens had phenotypic alterations (bent stem, dwarfism, absence of generative organs, chlorophyll deficit, deformation of generative organs, etc.)

To prevent cross-pollination between different sunflower lines, individual inflorescences had been isolated the day before semiflorets opened, the offspring of which were to be examined the next year as the M2 families. Concurrently, controls,

1.55% in line Kh201V (0.01%) to 21.65% in line Kh1008B (0.05%).

4.29–5.34% after 120 and 150 Gy gamma irradiation.

were 2.15–3.57 and 3.09–5.26%, respectively (**Figure 1**).

triads, dyads, etc. (**Figure 2**).

phase I; 4, abnormal tetrads.

during subsequent development (**Figure 3**).

non-treated with mutagens lines, were isolated.

ties in subsequent mutant generations of sunflower compared to the M1.

**48**

*Normalization of meiosis and gradual attenuation of the mutagenic effects of DMS and gamma rays in the M1–M3 sunflower (%).*

During the vegetation period, phenological observations of the growth and development of mutant plants were conducted; the field germinability was determined; cytological analysis was performed; and biometric measurements were

#### **Figure 2.**

*Microphotographs of meiotic abnormalities in mutant generations of self-pollinated sunflower lines.*

#### **Figure 3.**

*Phenotypic effects of chemical and physical mutagens in the M1 sunflower. 1, chlorophyll-deficient shoots from gamma-irradiated seeds (150 Gy); 2, 4, 6, morphoses induced by DMS (0.05%) in the early stages of plant development; 3, 5, 7, chlorophyll morphoses induced by DMS (0.05%); 10, deformation of generative organs induced by DMS (0.01%); 8, stem fasciation induced by DMS (0.05%); 9, absence of generative organs induced by gamma irradiation (120 Gy).*

#### **Figure 4.**

*Morphological changes observed in the M1 of self-pollinated line Kh06134V: 1, xantha chlorophyll mutation 'golden tip' (0.05% DMS), and 2, "purple tint of leaves" mutation (0.01% DMS).*

made (plant height measured 20 days after anthesis, calathidium diameter, and number of leaves per plant). Mutant plants were evaluated for the following parameters: oil content (%), 1000-seed weight, and fatty acid composition of oil.

In the M1, there were a lot of plants with different phenotypic developmental defects compared to the controls. However, one should keep in mind that most of them were so-called morphoses and consequences of phenotypic variability; such changes are not inherited and disappear in M2.

It is impossible to detect recessive mutations in M1 plants, since of 2 alleles of a gene, as a rule, one allele only mutates, and the altered recessive allele is always

**51**

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous…*

paired with the unchanged dominant allele (AA–Aa); therefore, selection of muta-

Only dominant mutations found by some researchers in some crops (usually in wheat) after exposure to chemical supermutagens can be detected in M1. Thus, examining the M1 of self-pollinated line Kh06134V, we distinguished some morphological changes: a chlorophyll-deficient mutation (xantha) called "golden top" (0.01% DMS) and a mutation of a purple tint of leaves (0.05% DMS), which is

M2 is sown by families and single plants or by continuous sowing according to

Plants were selected in the second mutant generation by visible morphological and physiological alterations to obtain macromutants as well as well-developed plants without visible alterations to find biochemical mutations and micromutations of quantitative traits. In addition, seeds from families without changes in the

Analysis of the mutant frequency in the M2 showed that gamma rays (120 and 150 Gy) produced more plants with alterations than DMS (0.01 and 0.05%). The percentage of plants with alterations after gamma irradiation ranged 36.0–36.4%, while the percentage of plants with alterations after DMS treatment was within 9.6–9.8%. We noted the individual genotypic responses of the lines to the increase in the concentration and dose of mutagens. The rise in the number of plants with alterations in the M2 depended on the increase in the concentration of DMS and the

In particular, in line Kh1002B, the total frequency of alterations was 3.2% with 0.01% DMS and 3.5% with 0.05% DMS, whereas plants with alterations were much

In line Kh1334V, the total frequency of plants with alterations was 3.4 and 3.3%

0.01 and 0.05% DMS produced 8.9 and 13.1% of plants with alterations, respectively, in line Kh201V. However, the effect of gamma rays was more conspicuous, and the total frequency of plants with alterations was 32.1 and 37.5% after 120 and

Most of the alterations observed in the M2 of the gamma-irradiated lines were nonheritable modifications found in early stages of the plant development, which disappeared during growth, whereas most of the DMS-induced alterations detected in different stages of the plant development were stable. The mutation nature of the

We studied inheritance of mutant traits in the M3 and subsequent generations. We also assessed the new mutant lines for breeding value and tested them for economically valuable traits, intending to involve constant valuable forms in hybridiza-

Having evaluated the alterations, we identified mutants noticeable for oil content in seeds, fatty acid composition of oil, 1000-seed weight, and resistance to

changes in the M2 was finally established by inheritance in the M3 families.

tion and heterosis breeding in order to obtain new sunflower hybrids.

more numerous with gamma rays (120 Gy–22.6%, 150 Gy–27.8%) (**Table 2**). 0.01% DMS-treated line Kh06134V gave the total frequency of plants with alterations of 14.7%, and the total frequency of plants with alterations after 0.05% DMS treatment was 10.0%. Gamma irradiation produced significantly more plants with alterations: 120 Gy produced 36.6% of plants with alterations, and

stably expressed in subsequent mutant generations (**Figure 4**).

**5. M2 and M3 generations, investigation, and use of mutations**

variants of mutagenic treatment, with optimal convenient density.

*DOI: http://dx.doi.org/10.5772/intechopen.89563*

M2 were sampled to reveal them in the M3.

dose of gamma rays (**Table 2**).

150 Gy–47.5% (**Table 2**).

with 0.01 and 0.05% DMS, respectively (**Table 2**).

150 Gy exposure, respectively (**Table 2**).

the pathogen of sunflower downy mildew.

tions is started with M2.

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous… DOI: http://dx.doi.org/10.5772/intechopen.89563*

paired with the unchanged dominant allele (AA–Aa); therefore, selection of mutations is started with M2.

Only dominant mutations found by some researchers in some crops (usually in wheat) after exposure to chemical supermutagens can be detected in M1. Thus, examining the M1 of self-pollinated line Kh06134V, we distinguished some morphological changes: a chlorophyll-deficient mutation (xantha) called "golden top" (0.01% DMS) and a mutation of a purple tint of leaves (0.05% DMS), which is stably expressed in subsequent mutant generations (**Figure 4**).

## **5. M2 and M3 generations, investigation, and use of mutations**

M2 is sown by families and single plants or by continuous sowing according to variants of mutagenic treatment, with optimal convenient density.

Plants were selected in the second mutant generation by visible morphological and physiological alterations to obtain macromutants as well as well-developed plants without visible alterations to find biochemical mutations and micromutations of quantitative traits. In addition, seeds from families without changes in the M2 were sampled to reveal them in the M3.

Analysis of the mutant frequency in the M2 showed that gamma rays (120 and 150 Gy) produced more plants with alterations than DMS (0.01 and 0.05%). The percentage of plants with alterations after gamma irradiation ranged 36.0–36.4%, while the percentage of plants with alterations after DMS treatment was within 9.6–9.8%. We noted the individual genotypic responses of the lines to the increase in the concentration and dose of mutagens. The rise in the number of plants with alterations in the M2 depended on the increase in the concentration of DMS and the dose of gamma rays (**Table 2**).

In particular, in line Kh1002B, the total frequency of alterations was 3.2% with 0.01% DMS and 3.5% with 0.05% DMS, whereas plants with alterations were much more numerous with gamma rays (120 Gy–22.6%, 150 Gy–27.8%) (**Table 2**).

0.01% DMS-treated line Kh06134V gave the total frequency of plants with alterations of 14.7%, and the total frequency of plants with alterations after 0.05% DMS treatment was 10.0%. Gamma irradiation produced significantly more plants with alterations: 120 Gy produced 36.6% of plants with alterations, and 150 Gy–47.5% (**Table 2**).

In line Kh1334V, the total frequency of plants with alterations was 3.4 and 3.3% with 0.01 and 0.05% DMS, respectively (**Table 2**).

0.01 and 0.05% DMS produced 8.9 and 13.1% of plants with alterations, respectively, in line Kh201V. However, the effect of gamma rays was more conspicuous, and the total frequency of plants with alterations was 32.1 and 37.5% after 120 and 150 Gy exposure, respectively (**Table 2**).

Most of the alterations observed in the M2 of the gamma-irradiated lines were nonheritable modifications found in early stages of the plant development, which disappeared during growth, whereas most of the DMS-induced alterations detected in different stages of the plant development were stable. The mutation nature of the changes in the M2 was finally established by inheritance in the M3 families.

We studied inheritance of mutant traits in the M3 and subsequent generations. We also assessed the new mutant lines for breeding value and tested them for economically valuable traits, intending to involve constant valuable forms in hybridization and heterosis breeding in order to obtain new sunflower hybrids.

Having evaluated the alterations, we identified mutants noticeable for oil content in seeds, fatty acid composition of oil, 1000-seed weight, and resistance to the pathogen of sunflower downy mildew.

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

*Microphotographs of meiotic abnormalities in mutant generations of self-pollinated sunflower lines.*

made (plant height measured 20 days after anthesis, calathidium diameter, and number of leaves per plant). Mutant plants were evaluated for the following param-

*Morphological changes observed in the M1 of self-pollinated line Kh06134V: 1, xantha chlorophyll mutation* 

*Phenotypic effects of chemical and physical mutagens in the M1 sunflower. 1, chlorophyll-deficient shoots from gamma-irradiated seeds (150 Gy); 2, 4, 6, morphoses induced by DMS (0.05%) in the early stages of plant development; 3, 5, 7, chlorophyll morphoses induced by DMS (0.05%); 10, deformation of generative organs induced by DMS (0.01%); 8, stem fasciation induced by DMS (0.05%); 9, absence of generative organs induced* 

In the M1, there were a lot of plants with different phenotypic developmental defects compared to the controls. However, one should keep in mind that most of them were so-called morphoses and consequences of phenotypic variability; such

It is impossible to detect recessive mutations in M1 plants, since of 2 alleles of a gene, as a rule, one allele only mutates, and the altered recessive allele is always

eters: oil content (%), 1000-seed weight, and fatty acid composition of oil.

*'golden tip' (0.05% DMS), and 2, "purple tint of leaves" mutation (0.01% DMS).*

changes are not inherited and disappear in M2.

**50**

**Figure 4.**

**Figure 2.**

**Figure 3.**

*by gamma irradiation (120 Gy).*


**Table 2.**

*Relative frequencies of major mutations induced by DMS and gamma rays in the M2 sunflower, % (exemplified by 4 lines).*

The traits of 1000-seed weight and oil content in seeds followed different patterns, depending on the genotypes of the self-pollinated lines. Thus, in the M3 there were genotypes, in which DMS treatment and gamma irradiation increased the content of oil in seeds (Od973B, Kh1002B, Mkh845B, X0816B, Kh06135V, Kh1334V, and Kh201V) and 1000-seed weight (Kh808B, Kh1002B, Mkh845B, Kh0816V, Kh785V, Kh1334V, and Kh201V). On the whole, 1000-seed weight insignificantly varied in the M3 (2–10%).

The mutants with increased content of oil in seeds are listed below: Kh1002B No 224 (0.05% DMS), 50%, and No 876 (150 Gy gamma rays), 48% (46% in the control); Kh0816V No. 422 (0.01% DMS), 50% (53% in the control); Kh1334V No. 609 (0.01% DMS), 48%, and No. 658 (0.05% DMS), 46% (43% in the control); and Kh201V No 685 (0.01% DMS), 54%, and No 1143 (150 Gy gamma rays), 52% (48% in the control) (**Figure 5**).

The mutants with increased 1000-seed weight are listed below: Mkh845B No. 385 (0.05% DMS), 64 g; No. 996 (150 Gy gamma rays), 67 g (48 g in the control);

**53**

control) (**Figure 6**).

*1000-seed weight in the mutant families (g).*

**Figure 6.**

**Figure 5.**

**6. Conclusions**

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous…*

Kh06134V No. 1029 (120 Gy gamma rays), 40 g (32 g in the control); Kh785V No. 596 (0.05% DMS), 51 g (34 g in the control); Kh1334V No. 645 (0.05% DMS), 75 g (53 g in the control); and Kh201V No 1146 (150 Gy gamma rays), 63 g (47.1 g in the

Biochemical analysis of oil from mutant sunflower seeds highlighted plants with increased content of linoleic acid of up to 70% (63% in the control) from line Kh201V. Among the mutants obtained from line Kh1334V, there were DMS-induced variants with increased contents of oleic and behenic acids (0.85% vs. 0.64% in the

As exemplified by the M1–M3 mutant generations of sunflower, an important scientific challenge of determining peculiarities of the variability of quantitative and qualitative traits under the influence of DMS (0.01 and 0.05%) and gamma rays (120 and 150 Gy) was theoretically described, and a new solution to it was suggested. The frequency and range of mutational variability in the M2 were summarized, and the inheritance of the mutant traits in subsequent generations was established. Chromosomal abnormalities in meiosis were characterized, and the

control), and such a combination is valuable for breeding (**Table 3**).

*DOI: http://dx.doi.org/10.5772/intechopen.89563*

*Oil content in seeds of the mutant families (%).*

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous… DOI: http://dx.doi.org/10.5772/intechopen.89563*

#### **Figure 5.**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

**The total frequency of mutations (%)**

Kh1002B 0.01% DMS 3.2 0.6 1.5 1.13

Kh06134V 0.01% DMS 10.0 1.9 3.3 4.7

Kh1334V 0.01% DMS 3.4 0.6 1.5 1.3

Kh201V 0.01% DMS 8.9 2.0 4.1 2.9

DMS 0.01% 9.6 3.1 3.7 2.8

γ-rays 120 Gy 36.0 11.3 16.3 8.3

**Frequency of chlorophyll mutations (%)**

0.05% DMS 3.5 0.9 2.1 0.6 120 Gy γ-rays 22.6 10.7\* 7.1\* 4.8 150 Gy γ-rays 27.8\* 2.8 13.9 11.1\* LSD 05 1.5 0.8 1.0 0.8

0.05% DMS 14.7\* 3.5 5.9 5.3 120 Gy γ-rays 36.6 3.3 20.0 13.3\* 150 Gy γ-rays 47.5\* 16.4\* 21.3 9.8 LSD 05 4.6 2.6 3.3 3.0

0.05% DMS 3.3 1.7\* 0.7 0.9 LSD 05 1.4 0.8 0.8 0.8

0.05% DMS 13.1\* 4.4\* 5.0 3.7 120 Gy γ-rays 32.1 5.2 18.7 8.2 150 Gy γ-rays 37.5\* 8.6\* 18.4 10.5\* LSD 05 4.1 2.2 3.1 1.6

0.05% 9.8 3.2 3.8 2.8

150 Gy 36.4 8.2 18.9 9.3

**Frequency of morphological mutations (%)**

**Frequency of economically valuable mutations (%)**

**Mutagen concentration/ dose**

**Original line**

The traits of 1000-seed weight and oil content in seeds followed different patterns, depending on the genotypes of the self-pollinated lines. Thus, in the M3 there were genotypes, in which DMS treatment and gamma irradiation increased the content of oil in seeds (Od973B, Kh1002B, Mkh845B, X0816B, Kh06135V, Kh1334V, and Kh201V) and 1000-seed weight (Kh808B, Kh1002B, Mkh845B, Kh0816V, Kh785V, Kh1334V, and Kh201V). On the whole, 1000-seed weight insignificantly

*Relative frequencies of major mutations induced by DMS and gamma rays in the M2 sunflower, % (exemplified* 

The mutants with increased content of oil in seeds are listed below: Kh1002B No 224 (0.05% DMS), 50%, and No 876 (150 Gy gamma rays), 48% (46% in the control); Kh0816V No. 422 (0.01% DMS), 50% (53% in the control); Kh1334V No. 609 (0.01% DMS), 48%, and No. 658 (0.05% DMS), 46% (43% in the control); and Kh201V No 685 (0.01% DMS), 54%, and No 1143 (150 Gy gamma rays), 52% (48%

The mutants with increased 1000-seed weight are listed below: Mkh845B No. 385 (0.05% DMS), 64 g; No. 996 (150 Gy gamma rays), 67 g (48 g in the control);

**52**

varied in the M3 (2–10%).

Average across 12 lines

*\* Difference significant at LSD 05.*

**Table 2.**

*by 4 lines).*

in the control) (**Figure 5**).

*Oil content in seeds of the mutant families (%).*

#### **Figure 6.**

*1000-seed weight in the mutant families (g).*

Kh06134V No. 1029 (120 Gy gamma rays), 40 g (32 g in the control); Kh785V No. 596 (0.05% DMS), 51 g (34 g in the control); Kh1334V No. 645 (0.05% DMS), 75 g (53 g in the control); and Kh201V No 1146 (150 Gy gamma rays), 63 g (47.1 g in the control) (**Figure 6**).

Biochemical analysis of oil from mutant sunflower seeds highlighted plants with increased content of linoleic acid of up to 70% (63% in the control) from line Kh201V. Among the mutants obtained from line Kh1334V, there were DMS-induced variants with increased contents of oleic and behenic acids (0.85% vs. 0.64% in the control), and such a combination is valuable for breeding (**Table 3**).

## **6. Conclusions**

As exemplified by the M1–M3 mutant generations of sunflower, an important scientific challenge of determining peculiarities of the variability of quantitative and qualitative traits under the influence of DMS (0.01 and 0.05%) and gamma rays (120 and 150 Gy) was theoretically described, and a new solution to it was suggested. The frequency and range of mutational variability in the M2 were summarized, and the inheritance of the mutant traits in subsequent generations was established. Chromosomal abnormalities in meiosis were characterized, and the



**55**

**Author details**

Ukraine

Victoria Mykhailenko1

and Dmitry Chuiko1

Kharkiv, Ukraine

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous…*

breeding-genetic values of induced mutations as well as possibilities of their use in breeding were evaluated. Methodological peculiarities of the mutational breeding of sunflower as a cross-pollinated crop were defined, and new mutants with changed features were detected. In addition, this study allowed us to conclude that DMS was more effective than gamma rays for the induction of valuable for breeding

Thus, induced mutagenesis is a major component of the complex breeding process of creation of new parental lines and hybrids of sunflower with economi-

mutations in new homozygous self-pollinated sunflower lines.

\*, Viktor Kyrychenko2

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

provided the original work is properly cited.

1 Department of Genetics, Selection and Seed Production, Faculty of Agronomy, Kharkiv National Agrarian University Named after V.V. Dokuchayev, Kharkiv,

© 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,

2 Plant Production Institute Named after V.Ya. Yuryev of NAAS of Ukraine,

, Alexander Bragin1

*DOI: http://dx.doi.org/10.5772/intechopen.89563*

cally valuable characteristics.

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous… DOI: http://dx.doi.org/10.5772/intechopen.89563*

breeding-genetic values of induced mutations as well as possibilities of their use in breeding were evaluated. Methodological peculiarities of the mutational breeding of sunflower as a cross-pollinated crop were defined, and new mutants with changed features were detected. In addition, this study allowed us to conclude that DMS was more effective than gamma rays for the induction of valuable for breeding mutations in new homozygous self-pollinated sunflower lines.

Thus, induced mutagenesis is a major component of the complex breeding process of creation of new parental lines and hybrids of sunflower with economically valuable characteristics.

## **Author details**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

**54**

**Original line**

Kh201V

Control

> №742

№694 №1133

> Kh1334V

DMS, 0.05% DMS, 0.01% γ-rays, 120 Gy

Control

> №659

№642 №628 №609

> **Table 3.**

*Fatty acid composition of oil from the M3 sunflower seeds.*

DMS, 0.05% DMS, 0.05% DMS, 0.01% DMS, 0.01%

**Mutant**

**Mutagen, (concentration/dose)**

**Palmitic**

**C16:0**

6.67 6.40 6.71 7.25 3.43 3.29 3.71 3.83 3.54

0.15

3.40

86.90

3.92

0.35

0.83

0.17

3.85

87.25

2.75

0.47

0.84

0.12

3.52

88.48

2.15

0.30

0.85

0.11

3.54

89.10

2.00

0.30

0.85

0.11

3.78

87.28

3.51

0.35

0.64

0.80

3.47

17.55

70.54

0.12

0.11

0.56

4.00

25.34

62.85

0.13

0.21

0.41

4.95

16.72

70.79

0.15

0.37

0.47

3.87

25.34

62.75

0.28

0.24

**C16:1**

**C18:0**

**C18:1**

**C18:2**

**C18:3**

**C22:0**

**Palmitoleic**

**Stearic**

**Oleic**

**Linoleic**

**Linolenic**

**Behenic**

Victoria Mykhailenko1 \*, Viktor Kyrychenko2 , Alexander Bragin1 and Dmitry Chuiko1

1 Department of Genetics, Selection and Seed Production, Faculty of Agronomy, Kharkiv National Agrarian University Named after V.V. Dokuchayev, Kharkiv, Ukraine

2 Plant Production Institute Named after V.Ya. Yuryev of NAAS of Ukraine, Kharkiv, Ukraine

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

© 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.

## **References**

[1] Olsen O, Wang X, Von Wettstein D. Sodium azide mutagenesis: Preferential generation of A.T--> G.C transitions in the barley antl8 gene. Proceedings of the National Academy of Sciences of the United States of America. 1993;**90**(17):8043-8047

[2] Maliuta SS. Mutagenesis. In: Ecological Encyclopedia: 3 volumes. К.: TOV "Tsentr Ekolohichnoi Osvity ta Informatsii"; 2007. p. 321. (in Ukrainian)

[3] Berezina NM. Pre-sowing irradiation of seeds of agricultural plants. In: Corresponding Member of AS USSR A.M. Kuzin. Moscow: Agropromizdat; 1964. pp. 188-189. (in Russian)

[4] Soldatov KI. Chemical mutagenesis in sunflower breeding. In: Proceeding 7th Internat. Sunflower Conf. 1976. pp. 352-357

[5] Kalaydzhan AA. Chemical mutagenesis in sunflower breeding: Author's abstract of the thesis for a Candidate Degree in Agricultural Sciences: Specialty 06.01.05 "Breeding and Seed Production"; Krasnodar. 1998. 48 p. (in Russian)

[6] Lacombe S. A dominant mutation for high oleic acid content in sunflower (*Helianthus annuus* L.) seed is genetically linked to a single oleatedesaturase RFLP locus. Molecular Breeding. 2001;**8**(2):129-137. DOI: 10.1023/A:1013358711651

[7] Soroka AI. Mutational variability in sunflower after exposure of immature corcules to mutagen. Nauk.-Tekhn. Biul. Instytutu Oliinykh Kultur. 2013;**18**:19- 24. (in Russian)

[8] Cvejic S. Radio sensitivity of sunflower restorer lines to different mutagenic treatments. In: Proceed. 5th Confer. of Young Scientists

and Specialists; Krasnodar. 2009. pp. 255-259

[9] Cvejić S. Mutation breeding for changed quality in sunflower. In: Cvejić S, Miladinović D, Jocić S, editors. Mutagenesis: Exploring Genetic Diversity of Crops. Wageningen, Netherlands: Wageningen Academic Publishers; 2014. pp. 3379-3388

[10] Lyakh V. Influence of mature and immature sunflower seed treatment with ethylmethanesulphonate on mutation spectrum and frequency. Helia. 2005;**28**(43):87-98

[11] Vasin VA. Genetic variability in sunflower after exposure of mature and immature seeds to ethylmethane sulfonate: Author's abstract of the thesis for a Candidate Degree in Agricultural Sciences: Specialty: 03.00.15; Kyiv. 2008. 48 p. (in Ukrainian)

[12] Vasin VA. Effect of ethylmethane sulfonate exposure of mature and immature seeds of sunflower on the frequency and assortment of mutations in the M2. Fiziologiya i Biokhimiya Kulturnykh Rasteniy. 2006;**38**(1):34-44. (in Russian)

[13] Kyrychenko VV. Chemical mutagens and improvement of sunflower lines. Selekthiia i Nasinnytstvo.1988;**80**:19-22. (in Ukrainian)

[14] Škorić D et al. Sunflower genetics and breeding: International monography. Novi Sad: Serbian Academy of Sciences and Arts, Branch. 2012;XV:520s

[15] Lysikov VN. Results of using chemical mutagens for breeding and genetic studies on maize in Moldaviya. In: Mutational Breeding. M.: Nauka; 1968. pp. 58-62. (in Russian)

[16] Zoz NN. Methods of using chemical mutagens in agricultural crop breeding.

**57**

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous…*

*DOI: http://dx.doi.org/10.5772/intechopen.89563*

In: Mutational Breeding. M.: Nauka;

[17] Artemchuk IP. Effects of mutagen exposure on the mutation frequency in winter wheat. Fiziologiya i Biokhimiya Kulturnykh Rasteniy. 2003;**3**:222-228.

1968. pp. 23-27. (in Russian)

(in Ukrainian)

*Generation, Evaluation, and Prospects of Further Use of Mutations Based on New Homozygous… DOI: http://dx.doi.org/10.5772/intechopen.89563*

In: Mutational Breeding. M.: Nauka; 1968. pp. 23-27. (in Russian)

[17] Artemchuk IP. Effects of mutagen exposure on the mutation frequency in winter wheat. Fiziologiya i Biokhimiya Kulturnykh Rasteniy. 2003;**3**:222-228. (in Ukrainian)

**56**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

and Specialists; Krasnodar. 2009.

[9] Cvejić S. Mutation breeding for changed quality in sunflower. In: Cvejić S, Miladinović D, Jocić S, editors.

Mutagenesis: Exploring Genetic Diversity of Crops. Wageningen, Netherlands: Wageningen Academic Publishers; 2014. pp. 3379-3388

[10] Lyakh V. Influence of mature and immature sunflower seed treatment with ethylmethanesulphonate on mutation spectrum and frequency.

[11] Vasin VA. Genetic variability in sunflower after exposure of mature and immature seeds to ethylmethane sulfonate: Author's abstract of the thesis for a Candidate Degree in Agricultural Sciences: Specialty: 03.00.15; Kyiv. 2008.

[12] Vasin VA. Effect of ethylmethane sulfonate exposure of mature and immature seeds of sunflower on the frequency and assortment of mutations in the M2. Fiziologiya i Biokhimiya Kulturnykh Rasteniy. 2006;**38**(1):34-44.

[13] Kyrychenko VV. Chemical mutagens and improvement of sunflower lines. Selekthiia i Nasinnytstvo.1988;**80**:19-22.

[14] Škorić D et al. Sunflower genetics and breeding: International monography. Novi Sad: Serbian Academy of Sciences

and Arts, Branch. 2012;XV:520s

[15] Lysikov VN. Results of using chemical mutagens for breeding and genetic studies on maize in Moldaviya. In: Mutational Breeding. M.: Nauka;

1968. pp. 58-62. (in Russian)

[16] Zoz NN. Methods of using chemical mutagens in agricultural crop breeding.

Helia. 2005;**28**(43):87-98

48 p. (in Ukrainian)

(in Russian)

(in Ukrainian)

pp. 255-259

[1] Olsen O, Wang X, Von

**References**

[2] Maliuta SS. Mutagenesis. In:

1964. pp. 188-189. (in Russian)

[5] Kalaydzhan AA. Chemical mutagenesis in sunflower breeding: Author's abstract of the thesis for a Candidate Degree in Agricultural Sciences: Specialty 06.01.05 "Breeding and Seed Production"; Krasnodar. 1998.

Informatsii"; 2007. p. 321.

(in Ukrainian)

pp. 352-357

48 p. (in Russian)

Ecological Encyclopedia: 3 volumes. К.: TOV "Tsentr Ekolohichnoi Osvity ta

[3] Berezina NM. Pre-sowing irradiation of seeds of agricultural plants. In: Corresponding Member of AS USSR A.M. Kuzin. Moscow: Agropromizdat;

[4] Soldatov KI. Chemical mutagenesis in sunflower breeding. In: Proceeding 7th Internat. Sunflower Conf. 1976.

[6] Lacombe S. A dominant mutation for high oleic acid content in sunflower

[7] Soroka AI. Mutational variability in sunflower after exposure of immature corcules to mutagen. Nauk.-Tekhn. Biul. Instytutu Oliinykh Kultur. 2013;**18**:19-

[8] Cvejic S. Radio sensitivity of sunflower restorer lines to different mutagenic treatments. In: Proceed. 5th Confer. of Young Scientists

(*Helianthus annuus* L.) seed is genetically linked to a single oleatedesaturase RFLP locus. Molecular Breeding. 2001;**8**(2):129-137. DOI:

10.1023/A:1013358711651

24. (in Russian)

Wettstein D. Sodium azide mutagenesis: Preferential generation of A.T--> G.C transitions in the barley antl8 gene. Proceedings of the National Academy of Sciences of the United States of America. 1993;**90**(17):8043-8047

**59**

**Chapter 4**

**Abstract**

Ecotoxicology

*and Roumiana Metcheva*

little-known information.

**1. Introduction**

Nefarious, but in a Different

*Peter Vladislavov Ostoich, Michaela Beltcheva* 

Way: Comparing the Ecotoxicity,

Gene Toxicity and Mutagenicity

of Lead (Pb) and Cadmium (Cd)

in the Context of Small Mammal

Lead and cadmium are long established toxic and carcinogenic metals. Still, the mechanisms of their interaction with eukaryotic DNA are not unequivocally understood. New data provide evidence on the influence of both metals on DNA repair, particularly non-homologous end joining (NHEJ) and mismatch repair (MMR). This may help explain the weak direct mutagenicity of both Pb2+ and Cd2+ ions in the Ames test, as opposed to the proven carcinogenicity of both metals; it has long been proposed that lead and cadmium may induce an imbalance in mammalian systems of DNA damage repair and promote genomic instability. While new evidence for mechanistic interactions of metals with DNA repair emerges, some of the old questions involving dose distribution, pathways of exposure and bioaccumulation/ detoxification kinetics still remain valid. To help place the current state of the art in the genetic toxicology of lead and cadmium within the context of ecotoxicology, the current authors propose an integrative approach and offer a review of other authors' work as well as some of their own data on systemic and organ-specific toxicities in laboratory mice. The current chapter is a comparative analysis of the state of the art in the specific toxicity and genotoxicity of Pb and Cd, presenting some new and

**Keywords:** lead (Pb), cadmium (Cd), genotoxicity, ecotoxicology, physiological

The last several decades have seen an increase in scientific and public interest in the problem of environmental contamination as a consequence of human activities. A wide variety of chemicals is released into the environment from different sources, either intentionally or as a result of accidents, prompting

reactions, DNA damage and repair, cell signaling, laboratory mice

## **Chapter 4**

Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity of Lead (Pb) and Cadmium (Cd) in the Context of Small Mammal Ecotoxicology

*Peter Vladislavov Ostoich, Michaela Beltcheva and Roumiana Metcheva*

## **Abstract**

Lead and cadmium are long established toxic and carcinogenic metals. Still, the mechanisms of their interaction with eukaryotic DNA are not unequivocally understood. New data provide evidence on the influence of both metals on DNA repair, particularly non-homologous end joining (NHEJ) and mismatch repair (MMR). This may help explain the weak direct mutagenicity of both Pb2+ and Cd2+ ions in the Ames test, as opposed to the proven carcinogenicity of both metals; it has long been proposed that lead and cadmium may induce an imbalance in mammalian systems of DNA damage repair and promote genomic instability. While new evidence for mechanistic interactions of metals with DNA repair emerges, some of the old questions involving dose distribution, pathways of exposure and bioaccumulation/ detoxification kinetics still remain valid. To help place the current state of the art in the genetic toxicology of lead and cadmium within the context of ecotoxicology, the current authors propose an integrative approach and offer a review of other authors' work as well as some of their own data on systemic and organ-specific toxicities in laboratory mice. The current chapter is a comparative analysis of the state of the art in the specific toxicity and genotoxicity of Pb and Cd, presenting some new and little-known information.

**Keywords:** lead (Pb), cadmium (Cd), genotoxicity, ecotoxicology, physiological reactions, DNA damage and repair, cell signaling, laboratory mice

## **1. Introduction**

The last several decades have seen an increase in scientific and public interest in the problem of environmental contamination as a consequence of human activities. A wide variety of chemicals is released into the environment from different sources, either intentionally or as a result of accidents, prompting

widespread concern about the effects of anthropogenic contamination on the biota. While many organic pollutants such as pesticides and petroleum refining products are subject to environmental degradation by physical, chemical, or biological pathways, heavy metals and their compounds typically retain their toxicity over long periods of time. Recently, important advances have been made in the understanding of the gene toxicity and mutagenicity of heavy metals in the environment [1–6]. For instance, it has been established that the gene toxicity of lead (Pb2+) and cadmium (Cd2+) ions is not due to direct DNA-metal interactions [2, 3]. It has been demonstrated that Cd2+ affects DNA repair pathways, particularly the non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs) at very low concentrations (<30 μmol) in several *in vitro* test systems [4, 5]. Nevertheless, some questions regarding the gene toxicity and mutagenicity of lead and cadmium remain open. For instance, several authors have noted that *in vivo* test systems are much more sensitive than *in vitro* systems (i.e., cell cultures) with respect to lead-induced endpoints for genotoxicity assessment (chromosomal aberrations, micronuclei, sister chromatid exchanges, comet assay endpoints) [7, 8]. In practice this means that animal models, especially rodents, are much more suitable for analysis of the genotoxicity of Pb2+ than cell cultures. When considering cadmium, useful mechanistic data on mutagenicity and comutagenicity has been obtained with *in vitro* test systems [4, 5]. Still, the question of the importance of Cd2+ as genotoxic agent in living mammalian organisms remains open. One study has reported lead-induced genomic instability in the progeny of mice exposed to Pb2+ *in utero* [9]. It is still unclear if this phenomenon has been observed by other authors and how common heavy metal-induced genomic instability is. If parental exposure to toxic metals can influence the stability of the genome in subsequent generations, this is potentially very alarming and could influence the current standards and permissible limits for occupational and environmental exposure. Last but not least, toxic metals seldom occur alone in contaminated sites. For instance, non-ferrous metal smelters typically emit a cocktail of toxic chemical elements in the atmosphere. This means that an accurate environmental risk assessment should be performed on a case-by-case basis, and that both ecotoxicological biomonitoring, and more general attempts to resolve the problem of heavy metal genotoxicity and mutagenicity, should be concerned not with a single toxicant but rather a plurality of different toxic agents present in a given locality. A number of studies have been performed with wild rodents exposed environmentally to complex contamination including Pb2+ and Cd2+ [10–23]. While these studies include endpoints for scoring genetic damage (chromosomal aberrations, micronuclei, comet tail length and tail moment) relatively little is understood about the molecular mechanisms underpinning the genotoxicity of complex mixtures of toxic metals.

In summary, from the perspective of ecotoxicology, it is well-established that Cd2+ and Pb2+ are genotoxic metal ions, especially in complex organisms. At the same time, knowledge about the mechanisms for heavy metal genotoxicity is scarce, with anecdotal evidence for interactions with DNA repair systems in complex vertebrate organisms, and relatively little knowledge of how the gene toxicity of Pb2+ and Cd2+ fits into the bigger picture of the specific physiological reactions of terrestrial vertebrates to toxic metals. For the purposes of the current study, the main questions regarding lead and cadmium gene toxicity are the following:

1.What are the specific molecular mechanisms, responsible for the gene toxicity of Pb2+ and Cd2+? How does intoxication with heavy metals lead to detectable chromosomal damage and mutagenesis? What are the similarities and differences when considering the gene toxicity of lead and cadmium?

**61**

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity…*

2.Can we draw conclusions about the comprehensive biological effects of heavy metals? For instance, it has long been established that terrestrial vertebrates respond to Pb2+ and Cd2+ by increased expression of detoxifying proteins (metallothioneins) and increased biosynthesis of glutathione. While there is evidence for adaptive responses, how does this apply to genetic damage

3.What are the effects of complex environmental pollution? How do complex

4.Last but not least, what are the prospects, challenges, and potential answers from future studies dealing with the gene toxicity of Pb2+ and Cd2+?

In order to provide, in part, answers to these four questions, the current study aims to analyze the state-of-the-art in what is known about the genotoxicity of lead and cadmium within the context of ecotoxicology. The current authors have employed a wide scope of sources in order to synthesize what is currently known and understood about the gene toxicity of Pb2+ and Cd2+, and conduct a comparative analysis of the two metals. In addition, insight and information is provided from a personal set of sources and experience, which are not widely publicized. Finally, the current article discusses several potential directions for future studies in the gene toxicity of heavy metals and proposes an integrated, trans-disciplinary approach to solving the problems, associated with the ecotoxicity and gene toxicity

**2.1 Ecotoxicity, bioaccumulation patterns, and specific organ toxicities**

Lead (Pb) is present in the Earth's crust at comparatively low concentrations (0.121 ppb) and has four stable isotopes (204Pb, 206Pb, 107Pb, and 208Pb) [24]. Although a comparatively rare metal, it has been historically one of the first industrially mined chemical elements. Contemporary sources estimate annual primary production of lead to be 4.6 million metric tons [25]. While Pb has been released in the atmosphere during manufacturing processes and combustion of fossil fuels, leading to global trace contamination, the main concern has been strong local contamination in the vicinity of mining, refining and smelting processes, as well as localized accidental releases. The toxicity of lead has been suspected since ancient times, with authors arguing mass poisoning from the metal in Ancient Rome due to its use for water pipes, glassmaking, and in winemaking processes [26]. Contemporary ecotoxicological research is concerned mainly with local contamination with Pb, with several important impact sites identified in Europe: Bukowno in Poland, Nitra, Slovakia, Asenovgrad, Bulgaria, and the Coto Doñana area in Spain [12, 15, 16, 18, 19, 22, 27–29]. The studies in these areas have dealt mainly with biomonitor species of wild rodents, and have investigated bioaccumulation of lead and other toxic metals, as well as endpoints for the determination of gene toxicity. Regardless of the zoomonitor used (typically, the wood mouse, *Apodemus sylvaticus*, yellow-necked mouse, *Apodemus flavicollis*, bank vole, *Myodes glareolus*, common vole, *Microtus arvalis*, Algerian mouse, *Mus spretus*), similar tendencies for bioaccumulation of Pb in the organisms of small mammals have been detected, and often correlated with the induction of genetic damage (chromosome aberrations, micronuclei). These studies have demonstrated significant effects of heavy metal

*DOI: http://dx.doi.org/10.5772/intechopen.89850*

induced by heavy metals?

of Pb2+ and Cd2+.

**2. Lead (Pb)**

mixtures of metallic toxicants affect organisms?

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity… DOI: http://dx.doi.org/10.5772/intechopen.89850*


In order to provide, in part, answers to these four questions, the current study aims to analyze the state-of-the-art in what is known about the genotoxicity of lead and cadmium within the context of ecotoxicology. The current authors have employed a wide scope of sources in order to synthesize what is currently known and understood about the gene toxicity of Pb2+ and Cd2+, and conduct a comparative analysis of the two metals. In addition, insight and information is provided from a personal set of sources and experience, which are not widely publicized. Finally, the current article discusses several potential directions for future studies in the gene toxicity of heavy metals and proposes an integrated, trans-disciplinary approach to solving the problems, associated with the ecotoxicity and gene toxicity of Pb2+ and Cd2+.

## **2. Lead (Pb)**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

genotoxicity of complex mixtures of toxic metals.

In summary, from the perspective of ecotoxicology, it is well-established that Cd2+ and Pb2+ are genotoxic metal ions, especially in complex organisms. At the same time, knowledge about the mechanisms for heavy metal genotoxicity is scarce, with anecdotal evidence for interactions with DNA repair systems in complex vertebrate organisms, and relatively little knowledge of how the gene toxicity of Pb2+ and Cd2+ fits into the bigger picture of the specific physiological reactions of terrestrial vertebrates to toxic metals. For the purposes of the current study, the main questions regarding lead and cadmium gene toxicity are the following:

1.What are the specific molecular mechanisms, responsible for the gene toxicity of Pb2+ and Cd2+? How does intoxication with heavy metals lead to detectable chromosomal damage and mutagenesis? What are the similarities and differ-

ences when considering the gene toxicity of lead and cadmium?

widespread concern about the effects of anthropogenic contamination on the biota. While many organic pollutants such as pesticides and petroleum refining products are subject to environmental degradation by physical, chemical, or biological pathways, heavy metals and their compounds typically retain their toxicity over long periods of time. Recently, important advances have been made in the understanding of the gene toxicity and mutagenicity of heavy metals in the environment [1–6]. For instance, it has been established that the gene toxicity of lead (Pb2+) and cadmium (Cd2+) ions is not due to direct DNA-metal interactions [2, 3]. It has been demonstrated that Cd2+ affects DNA repair pathways, particularly the non-homologous end joining (NHEJ) of DNA double-strand breaks (DSBs) at very low concentrations (<30 μmol) in several *in vitro* test systems [4, 5]. Nevertheless, some questions regarding the gene toxicity and mutagenicity of lead and cadmium remain open. For instance, several authors have noted that *in vivo* test systems are much more sensitive than *in vitro* systems (i.e., cell cultures) with respect to lead-induced endpoints for genotoxicity assessment (chromosomal aberrations, micronuclei, sister chromatid exchanges, comet assay endpoints) [7, 8]. In practice this means that animal models, especially rodents, are much more suitable for analysis of the genotoxicity of Pb2+ than cell cultures. When considering cadmium, useful mechanistic data on mutagenicity and comutagenicity has been obtained with *in vitro* test systems [4, 5]. Still, the question of the importance of Cd2+ as genotoxic agent in living mammalian organisms remains open. One study has reported lead-induced genomic instability in the progeny of mice exposed to Pb2+ *in utero* [9]. It is still unclear if this phenomenon has been observed by other authors and how common heavy metal-induced genomic instability is. If parental exposure to toxic metals can influence the stability of the genome in subsequent generations, this is potentially very alarming and could influence the current standards and permissible limits for occupational and environmental exposure. Last but not least, toxic metals seldom occur alone in contaminated sites. For instance, non-ferrous metal smelters typically emit a cocktail of toxic chemical elements in the atmosphere. This means that an accurate environmental risk assessment should be performed on a case-by-case basis, and that both ecotoxicological biomonitoring, and more general attempts to resolve the problem of heavy metal genotoxicity and mutagenicity, should be concerned not with a single toxicant but rather a plurality of different toxic agents present in a given locality. A number of studies have been performed with wild rodents exposed environmentally to complex contamination including Pb2+ and Cd2+ [10–23]. While these studies include endpoints for scoring genetic damage (chromosomal aberrations, micronuclei, comet tail length and tail moment) relatively little is understood about the molecular mechanisms underpinning the

**60**

#### **2.1 Ecotoxicity, bioaccumulation patterns, and specific organ toxicities**

Lead (Pb) is present in the Earth's crust at comparatively low concentrations (0.121 ppb) and has four stable isotopes (204Pb, 206Pb, 107Pb, and 208Pb) [24]. Although a comparatively rare metal, it has been historically one of the first industrially mined chemical elements. Contemporary sources estimate annual primary production of lead to be 4.6 million metric tons [25]. While Pb has been released in the atmosphere during manufacturing processes and combustion of fossil fuels, leading to global trace contamination, the main concern has been strong local contamination in the vicinity of mining, refining and smelting processes, as well as localized accidental releases. The toxicity of lead has been suspected since ancient times, with authors arguing mass poisoning from the metal in Ancient Rome due to its use for water pipes, glassmaking, and in winemaking processes [26]. Contemporary ecotoxicological research is concerned mainly with local contamination with Pb, with several important impact sites identified in Europe: Bukowno in Poland, Nitra, Slovakia, Asenovgrad, Bulgaria, and the Coto Doñana area in Spain [12, 15, 16, 18, 19, 22, 27–29]. The studies in these areas have dealt mainly with biomonitor species of wild rodents, and have investigated bioaccumulation of lead and other toxic metals, as well as endpoints for the determination of gene toxicity. Regardless of the zoomonitor used (typically, the wood mouse, *Apodemus sylvaticus*, yellow-necked mouse, *Apodemus flavicollis*, bank vole, *Myodes glareolus*, common vole, *Microtus arvalis*, Algerian mouse, *Mus spretus*), similar tendencies for bioaccumulation of Pb in the organisms of small mammals have been detected, and often correlated with the induction of genetic damage (chromosome aberrations, micronuclei). These studies have demonstrated significant effects of heavy metal

contamination on the biota, and have proven the importance of continuing monitoring studies in contaminated ecosystems.

The biokinetics and specific organ and tissue toxicities of Pb have been actively investigated in animal models since the late 1950s, initially employing radioactive tracer isotopes such as 203Pb and 210Pb [30, 31]. This has led to the development of several biokinetic models for the metal in mammalian organisms [31–33]. The Harley-Kneip six-compartent model, developed with the use of primates, is considered to be one of the first informative biokinetic models for lead absorption, distribution and elimination (**Figure 1**).

As evident from the model, a significant percentage of ingested lead (~80%) is excreted without being absorbed by the gut. At the same time, the coefficient for absorption from the bloodstream into bone λ12 = 0.34–0.11 is significantly higher than the coefficient for release of Pb from the bones into the bloodstream (λ21 = 1.73 x 10<sup>−</sup><sup>3</sup> ). In practice, this means that once a significant amount of lead is absorbed into the bones, it is practically impossible to eliminate it. The Harley-Kneip model also emphasizes the differences between juvenile and adult organisms, with juvenile animals much more susceptible to lead bioaccumulation [32]. To a varying level, Pb is also absorbed in the liver, kidneys, and the nervous system. It has been established that, in mammalian organisms, if the metal reaches sustained blood levels above 80 μg/dL, practically every organ and system is affected [24].

The primary targets for lead intoxication are the hematopoietic system, the nervous system and the liver. At sustained blood levels above 50 μg/dL, Pb inhibits

**63**

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity…*

the enzymes delta-aminolevulinic acid dehydratase (ALAD) and ferrochelatase, leading to impaired erythrocyte biogenesis, disturbances in erythrocyte kinetics, and anemia [34]. Several authors report an inhibition of the immune system at blood levels above 50 μg/dL, as well as histopathological lesions in the bone marrow at levels exceeding 100 μg/dL [23, 35, 36]. Death from anemia occurs at blood levels above 150 μg/dL. The nervous system is particularly sensitive in young individuals, and it has been established that Pb levels in blood exceeding 18 μg/dL lead to cognitive disturbances; it has been reported that very low doses cause neuronal apoptosis in rats [37]. In cases of chronic and sub-chronic lead intoxication, there is significant liver damage. Macroscopically, the liver increases in size; steatosis, hyperplasia and disruption of the liver microvasculature, as well as focal necrosis, have been observed at doses above 40 μg/dL, with marked changes in the activity of alanine

and aspartate transaminase (ALT, and AST) and kidney damage [8].

Due to low direct mutagenicity levels in the Ames test, lead (Pb) was initially thought not to be directly mutagenic [38]. Nevertheless, evidence soon accumulated that the metal was responsible for producing chromosomal aberrations in occupationally exposed workers and environmentally exposed human populations [31, 35]. Since the 1970s different *in vitro* and *in vivo* studies have been conducted regarding the potential of lead compounds to damage genomic DNA in mammals. The table below presents several informative studies conducted on the gene toxicity of lead using different *in vitro* test systems and endpoints, arranged chronologically

The studies cited provide evidence that lead is mutagenic and clastogenic under certain circumstances. While older studies show relatively weak clastogenicity of Pb when considering chromosomal aberrations [39, 40], newer publications report genotoxicity by using more sensitive endpoints, such as the induction of sister chromatid exchanges (SCE), tail length in the comet assay, and induction of γH2AX foci, indicating DNA double-strand breaks [6, 43, 44]. It should be noted that the study indicating the highest toxicity of Pb, uses lead chromate (PbCrO4), which means its effects could be due to the inherent gene toxicity of hexavalent chromium [44]. Several authors have noted the greater sensitivity of *in vivo* test systems when studying the gene toxicity of lead [7, 9]. For the purposes of the current study, several sources dealing with *in vivo* models have been selected (**Table 2**).

It should be noted that, in contrast to *in vitro* test systems not almost all tests with Pb administration to living animals show evidence for genotoxic effects. Not only that, some authors have noted a very close dose dependence of effects on Pb concentrations in living organisms, as well as trans-generational accumulation of chromosomal aberrations after exposure of mice *in utero* [9]. From the viewpoint of ecotoxicology, this means that the risks from environmental exposure to lead compounds are often underestimated when using *in vitro* test systems and only *in vivo* models can provide an accurate assessment of genetic risk to the biota. Much discussion has taken place concerning the molecular mechanisms of Pb-induced genetic damage. For instance, in the last two decades it has been accepted that lead interferes with the mechanisms for DNA repair, which is evident with studies analyzing Pb as a co-mutagen with other agents such as UV light, X-rays and methylnitronitrosoguanidine (MNNG) [50]. While it is accepter that the metal can inhibit DNA repair, the mechanisms of DNA damage induction *per se* are not well understood. For instance, it has been conclusively demonstrated that Pb and Cd do not interact with DNA directly under physiological conditions [3]. On the other hand, other authors have noted that Pb and other toxic metals can

*DOI: http://dx.doi.org/10.5772/intechopen.89850*

**2.2 Gene toxicity and mutagenicity**

(**Table 1**).

#### **Figure 1.** *Biokinetic model for the metabolism of lead in mammalian organisms [32].*

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity… DOI: http://dx.doi.org/10.5772/intechopen.89850*

the enzymes delta-aminolevulinic acid dehydratase (ALAD) and ferrochelatase, leading to impaired erythrocyte biogenesis, disturbances in erythrocyte kinetics, and anemia [34]. Several authors report an inhibition of the immune system at blood levels above 50 μg/dL, as well as histopathological lesions in the bone marrow at levels exceeding 100 μg/dL [23, 35, 36]. Death from anemia occurs at blood levels above 150 μg/dL. The nervous system is particularly sensitive in young individuals, and it has been established that Pb levels in blood exceeding 18 μg/dL lead to cognitive disturbances; it has been reported that very low doses cause neuronal apoptosis in rats [37]. In cases of chronic and sub-chronic lead intoxication, there is significant liver damage. Macroscopically, the liver increases in size; steatosis, hyperplasia and disruption of the liver microvasculature, as well as focal necrosis, have been observed at doses above 40 μg/dL, with marked changes in the activity of alanine and aspartate transaminase (ALT, and AST) and kidney damage [8].

### **2.2 Gene toxicity and mutagenicity**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

toring studies in contaminated ecosystems.

distribution and elimination (**Figure 1**).

x 10<sup>−</sup><sup>3</sup>

contamination on the biota, and have proven the importance of continuing moni-

The biokinetics and specific organ and tissue toxicities of Pb have been actively investigated in animal models since the late 1950s, initially employing radioactive tracer isotopes such as 203Pb and 210Pb [30, 31]. This has led to the development of several biokinetic models for the metal in mammalian organisms [31–33]. The Harley-Kneip six-compartent model, developed with the use of primates, is considered to be one of the first informative biokinetic models for lead absorption,

As evident from the model, a significant percentage of ingested lead (~80%) is excreted without being absorbed by the gut. At the same time, the coefficient for absorption from the bloodstream into bone λ12 = 0.34–0.11 is significantly higher than the coefficient for release of Pb from the bones into the bloodstream (λ21 = 1.73

). In practice, this means that once a significant amount of lead is absorbed into the bones, it is practically impossible to eliminate it. The Harley-Kneip model also emphasizes the differences between juvenile and adult organisms, with juvenile animals much more susceptible to lead bioaccumulation [32]. To a varying level, Pb is also absorbed in the liver, kidneys, and the nervous system. It has been established that, in mammalian organisms, if the metal reaches sustained blood levels

The primary targets for lead intoxication are the hematopoietic system, the nervous system and the liver. At sustained blood levels above 50 μg/dL, Pb inhibits

above 80 μg/dL, practically every organ and system is affected [24].

*Biokinetic model for the metabolism of lead in mammalian organisms [32].*

**62**

**Figure 1.**

Due to low direct mutagenicity levels in the Ames test, lead (Pb) was initially thought not to be directly mutagenic [38]. Nevertheless, evidence soon accumulated that the metal was responsible for producing chromosomal aberrations in occupationally exposed workers and environmentally exposed human populations [31, 35]. Since the 1970s different *in vitro* and *in vivo* studies have been conducted regarding the potential of lead compounds to damage genomic DNA in mammals. The table below presents several informative studies conducted on the gene toxicity of lead using different *in vitro* test systems and endpoints, arranged chronologically (**Table 1**).

The studies cited provide evidence that lead is mutagenic and clastogenic under certain circumstances. While older studies show relatively weak clastogenicity of Pb when considering chromosomal aberrations [39, 40], newer publications report genotoxicity by using more sensitive endpoints, such as the induction of sister chromatid exchanges (SCE), tail length in the comet assay, and induction of γH2AX foci, indicating DNA double-strand breaks [6, 43, 44]. It should be noted that the study indicating the highest toxicity of Pb, uses lead chromate (PbCrO4), which means its effects could be due to the inherent gene toxicity of hexavalent chromium [44].

Several authors have noted the greater sensitivity of *in vivo* test systems when studying the gene toxicity of lead [7, 9]. For the purposes of the current study, several sources dealing with *in vivo* models have been selected (**Table 2**).

It should be noted that, in contrast to *in vitro* test systems not almost all tests with Pb administration to living animals show evidence for genotoxic effects. Not only that, some authors have noted a very close dose dependence of effects on Pb concentrations in living organisms, as well as trans-generational accumulation of chromosomal aberrations after exposure of mice *in utero* [9]. From the viewpoint of ecotoxicology, this means that the risks from environmental exposure to lead compounds are often underestimated when using *in vitro* test systems and only *in vivo* models can provide an accurate assessment of genetic risk to the biota.

Much discussion has taken place concerning the molecular mechanisms of Pb-induced genetic damage. For instance, in the last two decades it has been accepted that lead interferes with the mechanisms for DNA repair, which is evident with studies analyzing Pb as a co-mutagen with other agents such as UV light, X-rays and methylnitronitrosoguanidine (MNNG) [50]. While it is accepter that the metal can inhibit DNA repair, the mechanisms of DNA damage induction *per se* are not well understood. For instance, it has been conclusively demonstrated that Pb and Cd do not interact with DNA directly under physiological conditions [3]. On the other hand, other authors have noted that Pb and other toxic metals can



#### **Table 1.**

*Exemplary studies on the genotoxicity of lead compounds* in vitro*.*

induce a pro-oxidative state in living organisms at comparatively low concentrations (<30–50 μmol) [50, 51]. In summary, it can be said that the genotoxicity of lead works at the following levels:


While the basics of lead genotoxicity have been confirmed, and the metal has been confirmed as reproductively toxic and carcinogenic in mammalian species,

**65**

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity…*

1–15 mg/kg Pb in food

kg PB intraperitoneally

> 117 mg/kg in food

CD-1 mice 0.01–1 μM Inhalation,

1 mg/l in drinking water

21.5 mg/kg Pb in food

ICR mice 50–200 mg/

**Dose Exposure Endpoint Effect**

1% Pb in food 2 weeks CA Increase in

3 days

90 days Comet

3–16 months CA Increase in

Injection SCE Increase in

3 days MN test Increase

Comet assay

assay

17 days MN test Increase

CA

CA

SCE

in MN frequency

Increase in tail length

Increase in tail length

in MN frequency

SCE

SCE Increase in

much remains to be investigated regarding the molecular mechanisms of the

**3.1 Ecotoxicity, bioaccumulation patterns, and specific organ toxicities**

metric tons per year [25]. Similarly to lead, the main concern regarding

Cd-associated contamination is local pollution of terrestrial and riverine ecosystems. The toxicity of cadmium was discovered after the start of its extraction from polymetallic ores, with one example being the "itai-itai" disease in the Toyama prefecture of Japan, attributed after 1950 to Cd poisoning [52]. In Europe sites, severely polluted with cadmium are comparatively rare. One exception is the area of Bukowno in Poland, where there is significant local contamination [16, 53, 54]. Several studies deal with the ecotoxicity of Cd with the use of zoomonitors (mainly yellow-necked mice, *Ap. flavicollis* and bank voles, *M. glareolus,* but also the common magpie, *Pica pica*) [53, 54]. While in Europe the element is mostly present as a trace contaminant in cases of polymetallic pollution, the main concern for cadmium contamination are the countries where most of it is mined and produced, namely China, South Korea, Japan, Mexico, Canada and Kazakhstan.

Cadmium (Cd) is a malleable, silvery-white metal present in the Earth's crust in concentrations of 01–0.5 ppm, having five stable isotopes (108Cd, 110Cd, 111Cd, 112Cd, and 114Cd) [24]. Discovered as a separate element within zinc ores in 1817, it is a toxicant, associated primarily with the late industrial age. Mined at a large scale since the 1920s, the metal is currently produced at a level of 23,000–24,000

interactions of Pb2+ with mammalian DNA repair systems.

*Exemplary studies on the genotoxicity of lead compounds* in vivo*.*

**3. Cadmium (Cd)**

*DOI: http://dx.doi.org/10.5772/intechopen.89850*

**Test system**

> a/SW mice

*Macaca fascicularis*

Sprague-Dawley rats

Kunming mice

> *Mus spretus*

**tested**

Lead acetate

Lead acetate

Lead acetate

Lead acetate

Lead acetate

Lead acetate

Lead acetate

**Authors Substance** 

Muro and Goyer [45]

Deknudt et al. [46]

Sharma et al. [47]

Robbiano et al. [48]

Valverde et al. [49]

Yuan and Tang [9]

Tapisso et al. [21]

**Table 2.**

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity… DOI: http://dx.doi.org/10.5772/intechopen.89850*


**Table 2.**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

CHO cells 10<sup>−</sup><sup>6</sup>

lymphocytes 10<sup>−</sup><sup>3</sup>

CH V79 cells 0.5–10

lymphocytes 1–100

CHO AA8 cells

lung fibroblasts

EJ30 carcinoma

 to 10<sup>−</sup><sup>3</sup> M

 to 10<sup>−</sup><sup>5</sup> M

μM

10<sup>−</sup><sup>6</sup> to 10<sup>−</sup><sup>8</sup> M

μM

0.1–5 μM

30–1000 μM

**Test system Dose Exposure Endpoint Effect**

16 h CA No effect, except

3 h CA No effect

mutation

48–60 h CA No effect

assay

Comet assay

γH2AX foci

foci

Telomere score

24–48 h CA Increase of %

44 h HPRT

1 h Comet

24 h γH2AX

for increase of gaps

Co-mutagenicity with UV light

Increase in tail length and % tail DNA

metaphases with damage

Increased % tail DNA

Dose-dependent increase of γH2AX foci

Dose-dependent increase of γH2AX foci

> Telomere instability

SCE Increase in SCE

SCE Increase in SCE

**Authors Substance** 

Bauchinger and Schmid [39]

Gasiorek and Bauchinger [40]

Hartwig et al. [41]

Cai and Arenaz [42]

Wozniak and Blasiak [43]

Xie et al. [44]

Pottier et al. [6]

**Table 1.**

**tested**

Lead acetate

Lead acetate

Lead acetate

Lead nitrate

Lead acetate

Lead chromate

> Lead nitrate

*Exemplary studies on the genotoxicity of lead compounds* in vitro*.*

induce a pro-oxidative state in living organisms at comparatively low concentrations (<30–50 μmol) [50, 51]. In summary, it can be said that the genotoxicity of lead

inhibition of key enzymes like glutathione-S-transferase (GST); disruption

2.Induction of genomic DNA damage; inhibition of key DNA repair systems such as base excision repair (BER) and disruption of telomere maintenance [6].

3.Mutagenesis, clastogenesis, tumor initiation and promotion, increase in the levels of apoptosis in some tissues, reproductive toxicity, organ and system

While the basics of lead genotoxicity have been confirmed, and the metal has been confirmed as reproductively toxic and carcinogenic in mammalian species,

1.Induction of reactive oxygen species (ROS) by Fenton-like reactions;

of lysosomal membranes and induction of apoptosis [51].

**64**

works at the following levels:

toxicities [37].

*Exemplary studies on the genotoxicity of lead compounds* in vivo*.*

much remains to be investigated regarding the molecular mechanisms of the interactions of Pb2+ with mammalian DNA repair systems.

## **3. Cadmium (Cd)**

## **3.1 Ecotoxicity, bioaccumulation patterns, and specific organ toxicities**

Cadmium (Cd) is a malleable, silvery-white metal present in the Earth's crust in concentrations of 01–0.5 ppm, having five stable isotopes (108Cd, 110Cd, 111Cd, 112Cd, and 114Cd) [24]. Discovered as a separate element within zinc ores in 1817, it is a toxicant, associated primarily with the late industrial age. Mined at a large scale since the 1920s, the metal is currently produced at a level of 23,000–24,000 metric tons per year [25]. Similarly to lead, the main concern regarding Cd-associated contamination is local pollution of terrestrial and riverine ecosystems. The toxicity of cadmium was discovered after the start of its extraction from polymetallic ores, with one example being the "itai-itai" disease in the Toyama prefecture of Japan, attributed after 1950 to Cd poisoning [52]. In Europe sites, severely polluted with cadmium are comparatively rare. One exception is the area of Bukowno in Poland, where there is significant local contamination [16, 53, 54]. Several studies deal with the ecotoxicity of Cd with the use of zoomonitors (mainly yellow-necked mice, *Ap. flavicollis* and bank voles, *M. glareolus,* but also the common magpie, *Pica pica*) [53, 54]. While in Europe the element is mostly present as a trace contaminant in cases of polymetallic pollution, the main concern for cadmium contamination are the countries where most of it is mined and produced, namely China, South Korea, Japan, Mexico, Canada and Kazakhstan.

The toxicity of cadmium was discovered after animal studies in the period 1955– 1970 [52, 55, 56]. In mammalian organisms, the metal affects primarily the kidneys, liver, pancreas, and, at higher levels, the nervous system [55]. As an established IARC Group 1 carcinogen, Cd increases the risk of lung cancer at low doses, and causes pneumonitis and lung edema at higher doses [52]. Nevertheless, the main target organ for chronic Cd intoxication are the kidneys, where the metal is accumulated, causing proteinuria, hypophosphatemia, histopathological changes in the kidney tissue, and loss of kidney function [57]. High chronic and sub-chronic dose burdens cause histopathological changes in the liver [58, 59]. Due to its antagonistic and antimetabolic activity against necessary elements such as Zn, Cu, and Ca, as well as its interference with a variety of DNA-binding enzymes, cadmium is considered toxic at high levels to all organs and systems [24, 57]. Unlike Pb, which has a strong tendency for bioaccumulation in the animal organism, Cd has higher rates of clearance from mammalian organisms due to the action of metallothionein (MT) proteins—low molecularweight, highly conserved molecules, which bind non-specifically to dietary elements such as Zn, Se, Cu, as well as toxic elements like Cd, Hg, Ag, As, and, to a much lesser extent, Pb [54, 60]. Metallothioneins bind Cd2+ ions in mammals, form Cd-MT complexes, which are excreted through the kidneys, thereby detoxifying, to some extent, low levels of cadmium. Nevertheless, although this system is inducible and upregulated by the presence of toxic metals in the body, it gets saturated at high doses, being unable to compensate high dose burdens of toxic metals [54]. Due to the inefficiency of existing biological detoxication systems, as well as the tendency of the metal for bioaccumulation in plants and animals, Cd is considered very dangerous even at low doses where no physical symptoms are present. It is, therefore, not surprising that a variety of biomonitoring studies for Cd have been conducted [22, 61].

## **3.2 Gene toxicity and mutagenicity**

The debate regarding the genotoxicity of cadmium continued for decades until recently [52]. This was due primarily to the fact that initially, using the Ames test, Cd was demonstrated to have very low mutagenicity. This, on the other hand, contradicted data demonstrating that the metal was a powerful carcinogen in mammals [24, 62]. At the same time, cadmium-induced inhibition of DNA repair systems and, consequently, co-genotoxicity, has been reported consistently since the late 1980s [56, 63]. Due to these relatively early observations on DNA repair inhibition, most *in vitro* studies have focused on the role of Cd as a co-genotoxin when combined with other genotoxic agents, for instance, ionizing and UV radiation, DNA intercalators and DNA alkylating agents [5, 63]. Data on cadmiuminduced genotoxicity from several investigations with *in vitro* test models are presented in **Table 3**.

All the studies cited typically provide evidence for co-mutagenicity of Cd with known mutagens such as UV light, DNA alkylating agents such as methylnitronitrosoguanidine (MNNG), and ionizing radiation. Comparably to *in vitro* studies with Pb, older experimental work with cadmium provides evidence for comutagenicity (although not direct mutagenicity) of the metal, while newer work, utilizing more sensitive endpoints, provides evidence for specific mechanisms such as DNA repair inhibition [4, 5].

While *in vitro* studies highlight Cd as a powerful co-mutagen due to DNA repair inhibition, several *in vivo* studies have shown that cadmium can be genotoxic (particularly clastogenic) at low doses. The results of several such investigations are presented in **Table 4**.

The *in vivo* studies above demonstrate cadmium genotoxicity at acute sublethal doses. It should be noted that in these studies, no separate co-mutagen is required,

**67**

**Table 3.**

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity…*

unlike in the *in vitro* models. Even though they prove conclusively that cadmium is genotoxic to mammals, they have a major shortcoming from an ecotoxicological point of view. Namely, the dose administration is either by injection or by oral gavage, which means that the observed effects of cadmium are due to acute exposure, as opposed to chronic and sub-chronic intoxication, which can be achieved by dosing the animal with food, water, or by inhalation means. One of the studies deals with minisatellite DNA instability, demonstrating that Cd intoxication can lead to instability in the non-coding segments of mammalian genomic DNA [69].

To some extent, the molecular mechanisms of DNA damage induction by Cd2+ ions are better understood than those of Pb2+-induced gene toxicity. It has been demonstrated that, at doses above 30 μM, cadmium down-regulates a key system for DNA DSB repair, namely non-homologous end-joining [4, 5]. Evidence suggests that the kinetics and formation of γH2AX foci are impaired at doses greater than 30 μM, with DNA-PKcs catalytic activity falling off at cadmium concentrations at doses of 200 μM [4, 5]. It has been established, as well, that at these doses the metal initially over-activates the system of homologous recombination repair, which may promote genomic instability [4]. Nevertheless, the induction of DNA damage in *in vivo* models by cadmium alone does not show a clear dose-response curve [52]. El-Ghor et al. have demonstrated a significant increase in microsatellite instability in rats exposed to cadmium [69]. Nevertheless, this methodology is controversial, both due to the unknown relationship of microsatellite DNA stability to the overall

**Dose Exposure Endpoint Effect**

4 h Mutagenicity β-Gal gene

DNA synthesis

X-ray damage

activity

1–100 μM 24 h γH2AX foci Disruption of

24 h DNA repair Inhibition of DNA

24 h UV damage Co-mutagenicity

HR activity Upregulation

Micronuclei Dose-dependent

DNA repair Inhibition at doses

inactivation

DSB repair

Inhibition of DNA synthesis

with UV light

Inhibition of DNA DSB repair

Inhibition of DNA DSB repair by NHEJ

of homologous recombination

> γH2AX foci kinetics

increase of micronuclei

above 30 μM

*DOI: http://dx.doi.org/10.5772/intechopen.89850*

**Authors Substance** 

Takahashi et al. [64]

Nocentini [56]

Snyder et al. [65]

Viau et al. [4]

Pereira et al. [5] **tested**

Cadmium chloride

Cadmium chloride

Cadmium chloride

Cadmium chloride, cadmium acetate

Cadmium chloride

Nevertheless, this methodology is still very controversial.

**Test system**

*E. coli* CHS26

Human fibroblasts

HMEC-1 endothelial cells

ZF-4 zebrafish cells

*Exemplary studies on the genotoxicity of cadmium compounds* in vitro*.*

HeLa cells 10<sup>−</sup><sup>8</sup>

10<sup>−</sup><sup>8</sup> to 10<sup>−</sup><sup>4</sup> M

10<sup>−</sup><sup>7</sup> to 10<sup>−</sup><sup>2</sup> M

 to 10<sup>−</sup><sup>3</sup> M

1–100 μM 24 h NHEJ

## *Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity… DOI: http://dx.doi.org/10.5772/intechopen.89850*

unlike in the *in vitro* models. Even though they prove conclusively that cadmium is genotoxic to mammals, they have a major shortcoming from an ecotoxicological point of view. Namely, the dose administration is either by injection or by oral gavage, which means that the observed effects of cadmium are due to acute exposure, as opposed to chronic and sub-chronic intoxication, which can be achieved by dosing the animal with food, water, or by inhalation means. One of the studies deals with minisatellite DNA instability, demonstrating that Cd intoxication can lead to instability in the non-coding segments of mammalian genomic DNA [69]. Nevertheless, this methodology is still very controversial.

To some extent, the molecular mechanisms of DNA damage induction by Cd2+ ions are better understood than those of Pb2+-induced gene toxicity. It has been demonstrated that, at doses above 30 μM, cadmium down-regulates a key system for DNA DSB repair, namely non-homologous end-joining [4, 5]. Evidence suggests that the kinetics and formation of γH2AX foci are impaired at doses greater than 30 μM, with DNA-PKcs catalytic activity falling off at cadmium concentrations at doses of 200 μM [4, 5]. It has been established, as well, that at these doses the metal initially over-activates the system of homologous recombination repair, which may promote genomic instability [4]. Nevertheless, the induction of DNA damage in *in vivo* models by cadmium alone does not show a clear dose-response curve [52]. El-Ghor et al. have demonstrated a significant increase in microsatellite instability in rats exposed to cadmium [69]. Nevertheless, this methodology is controversial, both due to the unknown relationship of microsatellite DNA stability to the overall


#### **Table 3.**

*Exemplary studies on the genotoxicity of cadmium compounds* in vitro*.*

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

The toxicity of cadmium was discovered after animal studies in the period 1955– 1970 [52, 55, 56]. In mammalian organisms, the metal affects primarily the kidneys, liver, pancreas, and, at higher levels, the nervous system [55]. As an established IARC Group 1 carcinogen, Cd increases the risk of lung cancer at low doses, and causes pneumonitis and lung edema at higher doses [52]. Nevertheless, the main target organ for chronic Cd intoxication are the kidneys, where the metal is accumulated, causing proteinuria, hypophosphatemia, histopathological changes in the kidney tissue, and loss of kidney function [57]. High chronic and sub-chronic dose burdens cause histopathological changes in the liver [58, 59]. Due to its antagonistic and antimetabolic activity against necessary elements such as Zn, Cu, and Ca, as well as its interference with a variety of DNA-binding enzymes, cadmium is considered toxic at high levels to all organs and systems [24, 57]. Unlike Pb, which has a strong tendency for bioaccumulation in the animal organism, Cd has higher rates of clearance from mammalian organisms due to the action of metallothionein (MT) proteins—low molecularweight, highly conserved molecules, which bind non-specifically to dietary elements such as Zn, Se, Cu, as well as toxic elements like Cd, Hg, Ag, As, and, to a much lesser extent, Pb [54, 60]. Metallothioneins bind Cd2+ ions in mammals, form Cd-MT complexes, which are excreted through the kidneys, thereby detoxifying, to some extent, low levels of cadmium. Nevertheless, although this system is inducible and upregulated by the presence of toxic metals in the body, it gets saturated at high doses, being unable to compensate high dose burdens of toxic metals [54]. Due to the inefficiency of existing biological detoxication systems, as well as the tendency of the metal for bioaccumulation in plants and animals, Cd is considered very dangerous even at low doses where no physical symptoms are present. It is, therefore, not surprising that a

variety of biomonitoring studies for Cd have been conducted [22, 61].

The debate regarding the genotoxicity of cadmium continued for decades until recently [52]. This was due primarily to the fact that initially, using the Ames test, Cd was demonstrated to have very low mutagenicity. This, on the other hand, contradicted data demonstrating that the metal was a powerful carcinogen in mammals [24, 62]. At the same time, cadmium-induced inhibition of DNA repair systems and, consequently, co-genotoxicity, has been reported consistently since the late 1980s [56, 63]. Due to these relatively early observations on DNA repair inhibition, most *in vitro* studies have focused on the role of Cd as a co-genotoxin when combined with other genotoxic agents, for instance, ionizing and UV radiation, DNA intercalators and DNA alkylating agents [5, 63]. Data on cadmiuminduced genotoxicity from several investigations with *in vitro* test models are

All the studies cited typically provide evidence for co-mutagenicity of Cd with known mutagens such as UV light, DNA alkylating agents such as methylnitronitrosoguanidine (MNNG), and ionizing radiation. Comparably to *in vitro* studies with Pb, older experimental work with cadmium provides evidence for comutagenicity (although not direct mutagenicity) of the metal, while newer work, utilizing more sensitive endpoints, provides evidence for specific mechanisms such

While *in vitro* studies highlight Cd as a powerful co-mutagen due to DNA repair

The *in vivo* studies above demonstrate cadmium genotoxicity at acute sublethal doses. It should be noted that in these studies, no separate co-mutagen is required,

inhibition, several *in vivo* studies have shown that cadmium can be genotoxic (particularly clastogenic) at low doses. The results of several such investigations are

**3.2 Gene toxicity and mutagenicity**

presented in **Table 3**.

presented in **Table 4**.

as DNA repair inhibition [4, 5].

**66**


#### **Table 4.**

*Exemplary studies on the genotoxicity of cadmium compounds* in vivo*.*

stability of coding genomic DNA, and the method of Cd intoxication used (oral gavage versus the more common method of administering via food or water). The available literature leads the current authors to believe that cadmium acts as a *tumor promoter*, with initiating events being diverse other factors (ionizing radiation background, metabolic reactive oxygen species, or other genotoxic factors). With respect to reproductive toxicity and cadmium-induced genomic instability, there is reason to believe that cadmium is reproductively toxic at high doses and can cause transmissible genetic damage in the progeny of exposed individuals. Still, much more research (both mechanistic studies and eco-toxicological experimentation) is needed to demonstrate conclusively the potential of the metal to change the genetic structure of exposed populations.

## **4. Comparing lead and cadmium as genotoxic agents**

#### **4.1 Induction of DNA damage**

It has been demonstrated that both Pb and Cd do not bind DNA directly, nor induce DNA damage due to DNA-metal interactions [3, 41]. At the same time, it is well-established that the metals promote the generation of reactive oxygen species and interact with redox signaling, disrupting cell homeostasis in organs and tissues

**69**

strand breaks.

area for future research.

polluted by ecological accidents [15, 19].

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity…*

and promoting a pro-oxidative state [41, 71]. In addition, specific target enzymes for Cd2+ have been identified—these include specifically several zinc-finger proteins like p53, XPA, PARP-1 and NF-ĸB. This would indicate increased potential of cadmium ions to act as tumor promoters even at low concentrations [41, 71].

On the other hand, it has been observed that Cd alone, at physiological concentrations, is a more significant causal agent of chromosomal aberrations in *in vivo* models, thereby acting more strongly as a mutagen and clastogen [3]. This is probably due to stronger induction of ROS and disruption of cellular redox signaling [72].

Little is understood about the interactions of lead with DNA repair systems. While several studies show disruption of γH2AX foci kinetics and, therefore, disruption of DNA DSB repair, and one study highlights a disruption of telomere maintenance, no mechanistic data exists to suggest how exactly Pb2+ ions interfere

Much more is known about the influence of Cd2+ ions with DNA repair. For instance, the tendency of this metal ion to displace zinc from zinc-finger DNAbinding enzymes leads to a disruption in the nucleotide-excision repair system (NER), which can explain the co-mutagenicity of cadmium with agents such as UV light and DNA alkylating chemicals [56, 63]. There have been a few studies analyzing the effects of cadmium on key DNA DSB repair systems [4, 5]. What these authors have established that, in selected *in vitro* models, even at concentrations lower than 30 μM, cadmium chloride inhibits non-homologous end-joining (NHEJ), over-activates the MRE-11-dependent homologous recombination (HR) and telomere maintenance, and leads to a general disturbance in γH2AX foci kinetics (a very sensitive indicator for DNA damage and repair), as well as a sharp decrease in DNA-PKcs catalytic activity, indicating inability to repair double-

While cadmium has undoubtedly been better studied as a genotoxic and cogenotoxic agent, lead (Pb) is also a significant genotoxin, albeit at significantly higher concentrations (>10-fold or more). Pointing out the exact mechanisms of the interaction of Pb with mammalian DNA repair system remains a valid topical

**5. Gene toxicity of lead and cadmium in the context of ecotoxicology**

Mechanistic studies, both *in vivo* and *in vitro*, are informative when trying to understand the basic principles of heavy metal genotoxicity. Nevertheless, what is the significance of environmental exposure to Pb and Cd? Typically environmental exposure occurs chronically or sub-chronically through food, drinking water and inhalation, and happens at comparatively low doses. In addition, exposure patterns are complex. For instance, pollution is often polymetallic, with an added variety of other organic and inorganic chemicals. Studies have been conducted in localities where pollution from lead-zinc smelters and mines is present, such as Asenovgrad in Bulgaria and Bukowno in Poland [10, 12, 14, 16, 18, 22, 27] as well as in areas,

The answers that these studies give us is that each studied locality has its own pollution pattern, leading to its own "fingerprint" of systemic toxicity and gene toxicity. For instance, it has been demonstrated that for BALB/c laboratory mice, exposed to 1% polymetallic industrial dust through food, the contents of the heavy

*DOI: http://dx.doi.org/10.5772/intechopen.89850*

**4.2 Interactions with DNA repair systems**

with DNA repair and the DNA damage response [6, 44].

#### *Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity… DOI: http://dx.doi.org/10.5772/intechopen.89850*

and promoting a pro-oxidative state [41, 71]. In addition, specific target enzymes for Cd2+ have been identified—these include specifically several zinc-finger proteins like p53, XPA, PARP-1 and NF-ĸB. This would indicate increased potential of cadmium ions to act as tumor promoters even at low concentrations [41, 71].

On the other hand, it has been observed that Cd alone, at physiological concentrations, is a more significant causal agent of chromosomal aberrations in *in vivo* models, thereby acting more strongly as a mutagen and clastogen [3]. This is probably due to stronger induction of ROS and disruption of cellular redox signaling [72].

#### **4.2 Interactions with DNA repair systems**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

**Test system**

Swiss albino mice

ICR mice 1 mg/

Swiss albino mice

Wistar rats

Sprague-Dawley rats

*Exemplary studies on the genotoxicity of cadmium compounds* in vivo*.*

0.4– 6.75 mg/ kg body weight

kg body weight

1–7.6 mg/ kg body weight

2.93 mg/ kg body weight

40–80 mg/ kg body weight

Oral gavage

Oral gavage

**Dose Exposure Endpoint Effect**

Injection SCE Increase in

Injection MN test Increase

Injection SCE Increase in

Minisatellite DNA

SCE

CA

in MN frequency

in MN frequency

CA

SCE

CA

Minisatellite instability

tail length

CA Increase in

CA Increase in

CA Increase in

Comet assay Increase in

MN test Increase

**Authors Substance** 

Mukherjee et al. [66]

Privezentsev et al. [67]

Fahmy and Aly [68]

El-Ghor et al. [69]

Wada et al. [70]

**Table 4.**

**tested**

Cadmium chloride

Cadmium chloride

Cadmium chloride

Cadmium chloride

Cadmium chloride

stability of coding genomic DNA, and the method of Cd intoxication used (oral gavage versus the more common method of administering via food or water). The available literature leads the current authors to believe that cadmium acts as a *tumor promoter*, with initiating events being diverse other factors (ionizing radiation background, metabolic reactive oxygen species, or other genotoxic factors). With respect to reproductive toxicity and cadmium-induced genomic instability, there is reason to believe that cadmium is reproductively toxic at high doses and can cause transmissible genetic damage in the progeny of exposed individuals. Still, much more research (both mechanistic studies and eco-toxicological experimentation) is needed to demonstrate conclusively the potential of the metal to change the genetic

It has been demonstrated that both Pb and Cd do not bind DNA directly, nor induce DNA damage due to DNA-metal interactions [3, 41]. At the same time, it is well-established that the metals promote the generation of reactive oxygen species and interact with redox signaling, disrupting cell homeostasis in organs and tissues

**68**

structure of exposed populations.

**4.1 Induction of DNA damage**

**4. Comparing lead and cadmium as genotoxic agents**

Little is understood about the interactions of lead with DNA repair systems. While several studies show disruption of γH2AX foci kinetics and, therefore, disruption of DNA DSB repair, and one study highlights a disruption of telomere maintenance, no mechanistic data exists to suggest how exactly Pb2+ ions interfere with DNA repair and the DNA damage response [6, 44].

Much more is known about the influence of Cd2+ ions with DNA repair. For instance, the tendency of this metal ion to displace zinc from zinc-finger DNAbinding enzymes leads to a disruption in the nucleotide-excision repair system (NER), which can explain the co-mutagenicity of cadmium with agents such as UV light and DNA alkylating chemicals [56, 63]. There have been a few studies analyzing the effects of cadmium on key DNA DSB repair systems [4, 5]. What these authors have established that, in selected *in vitro* models, even at concentrations lower than 30 μM, cadmium chloride inhibits non-homologous end-joining (NHEJ), over-activates the MRE-11-dependent homologous recombination (HR) and telomere maintenance, and leads to a general disturbance in γH2AX foci kinetics (a very sensitive indicator for DNA damage and repair), as well as a sharp decrease in DNA-PKcs catalytic activity, indicating inability to repair doublestrand breaks.

While cadmium has undoubtedly been better studied as a genotoxic and cogenotoxic agent, lead (Pb) is also a significant genotoxin, albeit at significantly higher concentrations (>10-fold or more). Pointing out the exact mechanisms of the interaction of Pb with mammalian DNA repair system remains a valid topical area for future research.

## **5. Gene toxicity of lead and cadmium in the context of ecotoxicology**

Mechanistic studies, both *in vivo* and *in vitro*, are informative when trying to understand the basic principles of heavy metal genotoxicity. Nevertheless, what is the significance of environmental exposure to Pb and Cd? Typically environmental exposure occurs chronically or sub-chronically through food, drinking water and inhalation, and happens at comparatively low doses. In addition, exposure patterns are complex. For instance, pollution is often polymetallic, with an added variety of other organic and inorganic chemicals. Studies have been conducted in localities where pollution from lead-zinc smelters and mines is present, such as Asenovgrad in Bulgaria and Bukowno in Poland [10, 12, 14, 16, 18, 22, 27] as well as in areas, polluted by ecological accidents [15, 19].

The answers that these studies give us is that each studied locality has its own pollution pattern, leading to its own "fingerprint" of systemic toxicity and gene toxicity. For instance, it has been demonstrated that for BALB/c laboratory mice, exposed to 1% polymetallic industrial dust through food, the contents of the heavy metals Pb and Cd increase steadily in a 90-day experiment, while at the same time the incidence of chromosome aberrations peaks at the 45-day midpoint, indicating the possibility of an adaptive response [18]. Similar results have been obtained with wild rodents from the same locality in different time frames [20]. Another area of research, which is currently active and productive, is heavy metal detoxification, particularly with the use of zeolite sorbents [29]. From the viewpoint of ecotoxicology, it is already known how chronic and sub-chronic doses of Pb and Cd affect the organism separately, but more research (including mechanistic studies) is needed in order to understand the effects of complex pollution patterns on living organisms.

The available data on the gene toxicity and eco-toxicity of Pb and Cd leads the current authors to believe that more significant research needs to be done in two main areas:


Finally, connections should be made to existing occupational safety and environmental legislation regarding the use of Pb and Cd worldwide. Some of the safety concerns regarding the two elements stem from the fact that heavy metals and their compounds are highly persistent in the environment. Additionally, gene toxicity, especially in the case of cadmium, have caused EU authorities to propose banning the use, mining and refining of Cd within the EU entirely. Since effects of Pb and Cd on genomic instability in the progeny of mammalian species have been observed [9, 69], but are not well understood, it is advisable that safety approaches to Cd and Pb have a "conservative approach," meaning that exposure tolerance limits and environmental releases should be as low as possible in order to mitigate risk to humans and the biosphere.

## **6. Conclusion**

The current work has analyzed the state-of-the art in what is known about the gene toxicity of lead and cadmium in an ecotoxicological context. Cd has been demonstrated as a powerful co-mutagen in *in vitro* test systems and as a direct mutagen *in vivo*. While Pb is generally a less potent inductor of chromosome aberrations, it has still been demonstrated to be genotoxic, particularly *in vivo*. While many studies have been conducted on the environmental exposure to Pb and Cd and their compounds, the interactions of the two metals as genotoxic agents are not yet fully understood. Two main challenges remain for future research in ecotoxicology and toxicogenetics: the combination of mechanistic *in vivo* and *in vitro* studies with ecotoxicological research, in order to understand better the specific pathways of heavy metal-induced gene toxicity, and future research on the detoxication of Pb and Cd and the mitigation of their gene toxicity.

**71**

**Author details**

Sofia, Bulgaria

Peter Vladislavov Ostoich\*, Michaela Beltcheva and Roumiana Metcheva

\*Address all correspondence to: p.ostoich@gmail.com

provided the original work is properly cited.

Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences,

© 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,

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity…*

This work is supported by the National Science Fund of the Republic of Bulgaria, Project DN 04/1, 13.12.2016: "Study of the combined effect of the natural radioactivity background, the UV radiation, the climate changes and the cosmic rays on model groups of plant and animal organisms in mountain ecosystems".

*DOI: http://dx.doi.org/10.5772/intechopen.89850*

**Acknowledgements**

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity… DOI: http://dx.doi.org/10.5772/intechopen.89850*

## **Acknowledgements**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

on living organisms.

have been conducted.

humans and the biosphere.

and Cd and the mitigation of their gene toxicity.

**6. Conclusion**

main areas:

metals Pb and Cd increase steadily in a 90-day experiment, while at the same time the incidence of chromosome aberrations peaks at the 45-day midpoint, indicating the possibility of an adaptive response [18]. Similar results have been obtained with wild rodents from the same locality in different time frames [20]. Another area of research, which is currently active and productive, is heavy metal detoxification, particularly with the use of zeolite sorbents [29]. From the viewpoint of ecotoxicology, it is already known how chronic and sub-chronic doses of Pb and Cd affect the organism separately, but more research (including mechanistic studies) is needed in order to understand the effects of complex pollution patterns

The available data on the gene toxicity and eco-toxicity of Pb and Cd leads the current authors to believe that more significant research needs to be done in two

1.Mechanistic studies dealing with the specific effects of the two metals on DNA repair systems. This is especially true for Pb, since lead-induced chromosomal aberrations in mammalian cells at low doses are a well-established fact, but no concrete mechanistic studies on the effects of Pb on DNA repair systems

2.Ecotoxicological studies highlighting the effects of different cocktails of pollutants in a given locality on a standardized test system. Suitable *in vitro* systems, which have been proposed include metabolically competent human and rat hepatoma cell lines, which have been used for the study of metabolically

Finally, connections should be made to existing occupational safety and environmental legislation regarding the use of Pb and Cd worldwide. Some of the safety concerns regarding the two elements stem from the fact that heavy metals and their compounds are highly persistent in the environment. Additionally, gene toxicity, especially in the case of cadmium, have caused EU authorities to propose banning the use, mining and refining of Cd within the EU entirely. Since effects of Pb and Cd on genomic instability in the progeny of mammalian species have been observed [9, 69], but are not well understood, it is advisable that safety approaches to Cd and Pb have a "conservative approach," meaning that exposure tolerance limits and environmental releases should be as low as possible in order to mitigate risk to

The current work has analyzed the state-of-the art in what is known about the gene toxicity of lead and cadmium in an ecotoxicological context. Cd has been demonstrated as a powerful co-mutagen in *in vitro* test systems and as a direct mutagen *in vivo*. While Pb is generally a less potent inductor of chromosome aberrations, it has still been demonstrated to be genotoxic, particularly *in vivo*. While many studies have been conducted on the environmental exposure to Pb and Cd and their compounds, the interactions of the two metals as genotoxic agents are not yet fully understood. Two main challenges remain for future research in ecotoxicology and toxicogenetics: the combination of mechanistic *in vivo* and *in vitro* studies with ecotoxicological research, in order to understand better the specific pathways of heavy metal-induced gene toxicity, and future research on the detoxication of Pb

activated genotoxins for over two decades [73].

**70**

This work is supported by the National Science Fund of the Republic of Bulgaria, Project DN 04/1, 13.12.2016: "Study of the combined effect of the natural radioactivity background, the UV radiation, the climate changes and the cosmic rays on model groups of plant and animal organisms in mountain ecosystems".

## **Author details**

Peter Vladislavov Ostoich\*, Michaela Beltcheva and Roumiana Metcheva Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences, Sofia, Bulgaria

\*Address all correspondence to: p.ostoich@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.

## **References**

[1] Ferraro M, Fenocchio A, Mantovani M, Ribeiro C, Cestari M. Mutagenic effects of tributyltin and inorganic lead (Pb II) on the fish *H. Malabaricus* as evaluated using the comet assay and the piscine micronucleus and chromosome aberration tests. Genetics and Molecular Biology. 2004;**27**(1):27-33

[2] Valverde M, Fortoul T, Diaz-Barriga F, Mejia J, Rojas E. Induction of genotoxicity by cadmium chloride inhalation in several organs of CD-1 mice. Mutagenesis. 2000;**15**:109-114

[3] Valverde M, Trejo C, Rojas E. Is the capacity of lead acetate and cadmium chloride to induce genotoxic damage due to direct DNA–metal interaction? Mutagenesis. 2001;**16**(3):265-270

[4] Viau M, Gastaldo J, Bencokova Z, Joubert A, Foray N. Cadmium inhibits non-homologous end-joining and over-activates the MRE-11-dependent repair pathway. Mutation Research. 2008;**654**:13-21

[5] Pereira S, Cavalie I, Camilleri V, Gilbin R, Adam-Guillermin C. Comparative genotoxicity of aluminium and cadmium in embryonic zebrafish cells. Mutation Research. 2013;**750**:19-26

[6] Pottier G, Viau M, Ricoul M, Shim G, Bellamy M, Cuceu C, et al. Lead exposure induces telomere instability in human cells. PLoS One. 2013;**8**(6):1-8

[7] Garcia-Leston J, Mendez J, Pasaro E, Laffon B. Genotoxic effects of lead: An updated review. Environment International. 2010;**36**(1):623-636

[8] Wang L, Li J, Li J, Liu Z. Effects of lead and/or cadmium on the oxidative damage of rat kidney cortex mitochondria. Biological Trace Element Research. 2010;**137**:69-78

[9] Yuan X, Tang C. The accumulation effect of lead on DNA damage in mice blood cells of three generations and the protection of selenium. Journal of Environmental Science and Health. 2001;**36**(1):501-508

[10] Wlostowski T. Heavy metals in the liver of *Clethrionomys glareolus* (Schreber, 1780) and *Apodemus sylvaticus* (Pallas, 1771) from forests contaminated with coal-industry fumes. Ekologia Polska. 1987;**35**:115-129

[11] Ma W, Denneman W, Faber J. Hazardous exposure of ground-living small mammals to cadmium and lead in contaminated terrestrial ecosystems. Archives of Environmental Contamination and Toxicology. 1991;**20**:266-270

[12] Topashka-Ancheva M, Metcheva R. Bioaccumulation of heavy metals and chromosome aberrations in small mammals from industrially polluted region in Bulgaria. In: Contributions to the Zoogeography and Ecology of the Eastern Mediterranean Region. Vol. 1. 1999. pp. 69-74

[13] Gdula-Argasinska J, Sawicka-Kapusta K. Effect of heavy metals pollution on rodents from six forest sites of Malopolska district. In: 11th Annual Meeting of SETAC Europe, Madrid, Spain. 2001. pp. 1-5

[14] Ieradi L, Zima J, Allegra F, Kotlanova E, Campanella L, Grossi R, et al. Evaluation of genotoxic damage in wild rodents from a polluted area in the Czech Republic. Folia Zoologica. 2003;**52**(1):57-66

[15] Tanzarella C, Degrassi F, Cristaldi M, Moreno S, Lascialfari A, Chiuchiarelli G, et al. Genotoxic damage in free-living Algerian mouse (*Mus spretus*) after the Coto Doñana ecological

**73**

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity…*

[22] Martiniakova M, Omelka R, Stawarz R, Formicki G. Accumulation of lead, cadmium, nickel, iron, copper and zinc in bones of small mammals from polluted areas in Slovakia. Polish Journal of Environmental Studies.

[23] Tete N, Durfort M, Reiffel D, Scheifler A, Sanchez-Chardi M.

[24] Luckey T, Venugopal B. Metal Toxicity in Mammals: Physiologic and Chemical Basis for Metal Toxicity. New York: Plenum Press; 1977. 238p

[25] Graedel T. Metal stocks in society: Scientific synthesis. International Resources Workshop Proceedings.

[26] Nriagu J. Saturnine gout among Roman aristocrats: Did lead poisoning contribute to the fall of the empire? New England Journal of Medicine.

Histopathology related to cadmium and lead bioaccumulation in chronically exposed wood mice, *Apodemus sylvaticus*, around a former smelter. Science of the Total Environment.

2012;**21**(1):153-158

2014;**481**(1):167-177

2010;**1**(1):59-90

1983;**308**(1):660-663

2003;**91**:54-61

2015;**7**(1):12-29

[27] Metcheva R, Teodorova S,

Topashka-Ancheva M. A comparative analysis of the heavy metals and toxic elements loading indicated by small mammals in different Bulgarian regions. Acta Zoologica Bulgarica. 2001;**53**:61-80

[28] Festa F, Cristaldi M, Ieradi L, Moreno S, Cozzi R. The comet assay for the detection of DNA damage in *Mus spretus* from Doñana National Park. Environmental Research.

[29] Beltcheva M, Metcheva R, Topashka-Ancheva M, Popov N, Teodorova S, Heredia-Rojas J, et al. Zeolites versus lead toxicity. Journal of Bioequivalence & Bioavailability.

*DOI: http://dx.doi.org/10.5772/intechopen.89850*

disaster. Environmental Pollution.

[16] Damek-Poprawa M, Sawicka-Kapusta K. Damage to the liver, kidney and testis with reference to burden of heavy metals in yellow-necked mice from areas around steelworks and zinc smelters in Poland. Toxicology.

[17] Sánchez-Chardi A, Penarroja-Matutano C, Oliveira Ribeiro CA, Nadal J. Bioaccumulation of metals and effects of a landfill in small mammals. Part II. The wood mouse, *Apodemus sylvaticus*. Chemosphere.

[18] Metcheva R, Topashka-Ancheva M,

Teodorova S. Influence of lead and cadmium on some genetic and physiological parameters of laboratory mice. In: Cato M, editor. Environmental Research Trends. 2007.

[19] Udroiu I, Ieradi L, Tanzarella C, Moreno S. Biomonitoring of Doñana National Park using the Algerian mouse (*Mus spretus*) as a sentinel species. Fresenius Environmental Bulletin.

[20] Topashka-Ancheva M, Metcheva R, Teodorova S. A comparative analysis of the heavy metals loading of small mammals in different Bulgarian regions. II. Chromosomal aberrations and blood pathology. Ecotoxicology

2001;**115**(1):43-48

2003;**186**:1-10

2007;**70**:101-110

pp. 205-230

2008;**17**(9):1519-1525

and Environmental Safety.

[21] Tapisso J, Marques C, da Luz Mathias M, Ramalhinho M. Induction of micronuclei and sister chromatid exchange in bone-marrow cells and abnormalities in sperm of Algerian mice (*Mus spretus*) exposed to cadmium, lead and zinc. Mutation Research/Fundamental and Molecular

Mechanisms of Mutagenesis.

2009;**678**(1):59-64

2003;**54**(2):188-193

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity… DOI: http://dx.doi.org/10.5772/intechopen.89850*

disaster. Environmental Pollution. 2001;**115**(1):43-48

[16] Damek-Poprawa M, Sawicka-Kapusta K. Damage to the liver, kidney and testis with reference to burden of heavy metals in yellow-necked mice from areas around steelworks and zinc smelters in Poland. Toxicology. 2003;**186**:1-10

[17] Sánchez-Chardi A, Penarroja-Matutano C, Oliveira Ribeiro CA, Nadal J. Bioaccumulation of metals and effects of a landfill in small mammals. Part II. The wood mouse, *Apodemus sylvaticus*. Chemosphere. 2007;**70**:101-110

[18] Metcheva R, Topashka-Ancheva M, Teodorova S. Influence of lead and cadmium on some genetic and physiological parameters of laboratory mice. In: Cato M, editor. Environmental Research Trends. 2007. pp. 205-230

[19] Udroiu I, Ieradi L, Tanzarella C, Moreno S. Biomonitoring of Doñana National Park using the Algerian mouse (*Mus spretus*) as a sentinel species. Fresenius Environmental Bulletin. 2008;**17**(9):1519-1525

[20] Topashka-Ancheva M, Metcheva R, Teodorova S. A comparative analysis of the heavy metals loading of small mammals in different Bulgarian regions. II. Chromosomal aberrations and blood pathology. Ecotoxicology and Environmental Safety. 2003;**54**(2):188-193

[21] Tapisso J, Marques C, da Luz Mathias M, Ramalhinho M. Induction of micronuclei and sister chromatid exchange in bone-marrow cells and abnormalities in sperm of Algerian mice (*Mus spretus*) exposed to cadmium, lead and zinc. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis. 2009;**678**(1):59-64

[22] Martiniakova M, Omelka R, Stawarz R, Formicki G. Accumulation of lead, cadmium, nickel, iron, copper and zinc in bones of small mammals from polluted areas in Slovakia. Polish Journal of Environmental Studies. 2012;**21**(1):153-158

[23] Tete N, Durfort M, Reiffel D, Scheifler A, Sanchez-Chardi M. Histopathology related to cadmium and lead bioaccumulation in chronically exposed wood mice, *Apodemus sylvaticus*, around a former smelter. Science of the Total Environment. 2014;**481**(1):167-177

[24] Luckey T, Venugopal B. Metal Toxicity in Mammals: Physiologic and Chemical Basis for Metal Toxicity. New York: Plenum Press; 1977. 238p

[25] Graedel T. Metal stocks in society: Scientific synthesis. International Resources Workshop Proceedings. 2010;**1**(1):59-90

[26] Nriagu J. Saturnine gout among Roman aristocrats: Did lead poisoning contribute to the fall of the empire? New England Journal of Medicine. 1983;**308**(1):660-663

[27] Metcheva R, Teodorova S, Topashka-Ancheva M. A comparative analysis of the heavy metals and toxic elements loading indicated by small mammals in different Bulgarian regions. Acta Zoologica Bulgarica. 2001;**53**:61-80

[28] Festa F, Cristaldi M, Ieradi L, Moreno S, Cozzi R. The comet assay for the detection of DNA damage in *Mus spretus* from Doñana National Park. Environmental Research. 2003;**91**:54-61

[29] Beltcheva M, Metcheva R, Topashka-Ancheva M, Popov N, Teodorova S, Heredia-Rojas J, et al. Zeolites versus lead toxicity. Journal of Bioequivalence & Bioavailability. 2015;**7**(1):12-29

**72**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

[9] Yuan X, Tang C. The accumulation effect of lead on DNA damage in mice blood cells of three generations and the protection of selenium. Journal of Environmental Science and Health.

[10] Wlostowski T. Heavy metals in the liver of *Clethrionomys glareolus* (Schreber, 1780) and *Apodemus sylvaticus* (Pallas, 1771) from forests contaminated with coal-industry fumes.

Ekologia Polska. 1987;**35**:115-129

[11] Ma W, Denneman W, Faber J. Hazardous exposure of ground-living small mammals to cadmium and lead in contaminated terrestrial

Contamination and Toxicology.

1991;**20**:266-270

1. 1999. pp. 69-74

Spain. 2001. pp. 1-5

2003;**52**(1):57-66

ecosystems. Archives of Environmental

[12] Topashka-Ancheva M, Metcheva R. Bioaccumulation of heavy metals and chromosome aberrations in small mammals from industrially polluted region in Bulgaria. In: Contributions to the Zoogeography and Ecology of the Eastern Mediterranean Region. Vol.

[13] Gdula-Argasinska J, Sawicka-Kapusta K. Effect of heavy metals pollution on rodents from six forest sites of Malopolska district. In: 11th Annual Meeting of SETAC Europe, Madrid,

[14] Ieradi L, Zima J, Allegra F,

[15] Tanzarella C, Degrassi F, Cristaldi M, Moreno S,

Lascialfari A, Chiuchiarelli G, et al. Genotoxic damage in free-living Algerian mouse (*Mus spretus*) after the Coto Doñana ecological

Kotlanova E, Campanella L, Grossi R, et al. Evaluation of genotoxic damage in wild rodents from a polluted area in the Czech Republic. Folia Zoologica.

2001;**36**(1):501-508

[1] Ferraro M, Fenocchio A,

**References**

Biology. 2004;**27**(1):27-33

[2] Valverde M, Fortoul T, Diaz-Barriga F, Mejia J, Rojas E. Induction of genotoxicity by cadmium

2000;**15**:109-114

2008;**654**:13-21

chloride inhalation in several organs of CD-1 mice. Mutagenesis.

[3] Valverde M, Trejo C, Rojas E. Is the capacity of lead acetate and cadmium chloride to induce genotoxic damage due to direct DNA–metal interaction? Mutagenesis. 2001;**16**(3):265-270

[4] Viau M, Gastaldo J, Bencokova Z, Joubert A, Foray N. Cadmium inhibits non-homologous end-joining and over-activates the MRE-11-dependent repair pathway. Mutation Research.

[5] Pereira S, Cavalie I, Camilleri V, Gilbin R, Adam-Guillermin C.

[6] Pottier G, Viau M, Ricoul M,

Comparative genotoxicity of aluminium and cadmium in embryonic zebrafish cells. Mutation Research. 2013;**750**:19-26

Shim G, Bellamy M, Cuceu C, et al. Lead exposure induces telomere instability in human cells. PLoS One. 2013;**8**(6):1-8

[7] Garcia-Leston J, Mendez J, Pasaro E, Laffon B. Genotoxic effects of lead: An updated review. Environment International. 2010;**36**(1):623-636

[8] Wang L, Li J, Li J, Liu Z. Effects of lead and/or cadmium on the oxidative damage of rat kidney cortex mitochondria. Biological Trace Element

Research. 2010;**137**:69-78

Mantovani M, Ribeiro C, Cestari M. Mutagenic effects of tributyltin and inorganic lead (Pb II) on the fish *H. Malabaricus* as evaluated using the comet assay and the piscine micronucleus and chromosome

aberration tests. Genetics and Molecular

[30] Catellino N, Aloj S. Determination of the elimination constants of Pb-210 from various rat tissues. Folia Medica. 1964;**47**(1):238-248

[31] Rabinowitz M. Historical perspective on lead biokinetic models. Environmental Health Perspectives. 1998;**106**(6):1461-1465

[32] Kneip T, Mallon R, Harley N. Biokinetic modeling for mammalian metabolism. Neurotoxicology. 1983;**4**:189-192

[33] O'Flaherty E. Physiologically based models for bone seeking elements. V: Lead absorption and disposition in childhood. Toxicology and Applied Pharmacology. 1995;**131**:297-308

[34] Goyer R, Rhine B. Pathological effects of lead. International Review of Experimental Pathology. 1973;**12**(1):23-37

[35] Zelikoff J, Thomas P. Immunotoxicology of Environmental and Occupational Metals. London: Taylor and Francis; 2005. 382p

[36] Tham C, Chakravarthy S, Haleagrahara N, De Alvis R. Morphological study of bone marrow to assess the effects of lead acetate on haemopoiesis and aplasia and the ameliorating role of *Carica papaya* extract. Experimental and Therapeutic Medicine. 2013;**5**(2):648-654

[37] Ahmed M, Ahmed M, Meki A, Abd-Raboh N. Neurotoxic effects of lead on rats: Relationship to apoptosis. International Journal of Health Sciences. 2013;**7**(2):192-199

[38] Winder C, Bonin T. The genotoxicity of lead. Mutation Research. 1993;**285**(1):117-124

[39] Bauchinger M, Schmid E. Chromosome analysis of cultures of Chinese hamster cells after treatment with lead acetate. Mutation Research. 1972;**14**:95-100

[40] Gasiorek K, Bauchinger M. Chromosome changes in human lymphocytes after separate and combined treatment with divalent salts of lead, cadmium, and zinc. Environmental Mutagenesis. 1981;**3**:513-518

[41] Hartwig A, Schlepegrell R, Beyersmann D. Indirect mechanism of lead-induced genotoxicity in cultured mammalian cells. Mutation Research. 1990;**241**:75-82

[42] Cai M, Arenaz P. Antimutagenic effect of crown ethers on heavy metalinduced sister chromatid exchanges. Mutagenesis. 1998;**13**(1):27-32

[43] Wozniak K, Blasiak J. In vitro genotoxicity of lead acetate: Induction of single and double DNA strand breaks and DNA-protein cross-links. Mutation Research. 2003;**535**:127-139

[44] Xie H, Wise S, Holmes A, Xu B, Wakeman T, Pelsue S. Carcinogenic lead chromate induces DNA double-strand breaks in human lung cells. Mutation Research. 2005;**586**:160-172

[45] Muro L, Goyer R. Chromosome damage in experimental lead poisoning. Archives of Pathology. 1969;**87**:660-663

[46] Deknudt G, Colle A, Gerber G. Chromosomal abnormalities in lymphocytes from monkeys poisoned with lead. Mutation Research. 1977;**45**:77-83

[47] Sharma R, Jacobson-Kram D, Lemmon M, Bakke J, Galperin I, Blazak W. Sister chromatid exchange and cell replication kinetics in fetal and maternal cells after treatment with chemical teratogens. Mutation Research. 1985;**158**:217-231

[48] Robbiano L, Carrozzino R, Porta Puglia C, Corbu C, Brambilla G.

**75**

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity…*

[56] Nocentini S. Inhibition of DNA replication and repair by cadmium in mammalian cells. Protective interaction

[57] Nordberg G. Historical perspectives on cadmium toxicology. Toxicology

[58] Rikans L, Yamano T. Mechanisms

hepatotoxicity. Journal of Biochemical

histopathological changes in the kidneys and liver of bank voles *Myodes glareolus* kept in different group densities. Ecotoxicology. 2012;**21**(8):2235-2243

[60] Bremner I. Mammalian absorption, transport and excretion of cadmium. In: Webb M, editor. The Chemistry, Biochemistry and Biology of Cadmium. Amsterdam: Elsevier Holland; 1979.

of zinc. Nucleic Acids Research.

and Applied Pharmacology.

of cadmium-mediated acute

and Molecular Toxicology.

[59] Salinska A, Wlostowski T, Zambrzyczka E. Effect of dietary cadmium and/or lead on

1987;**15**:4211-4225

2009;**238**:192-200

1999;**14**(2):110-117

pp. 175-193

[61] Shore R, Douben P. The ecotoxicological significance of cadmium intake and residues in terrestrial small mammals. Ecotoxicology and Environmental

Safety. 1994;**29**:101-112

1992;**118**:367-375

2002;**110**(5):797-799

[62] Rossmann T, Roy N, Lin W. Is cadmium genotoxic? IARC Publications.

[63] Hartwig A, Asmuss A, Ehleben I, Herzer U, Kostelac D, Pelzer A, et al. Interference of toxic metal ions with DNA repair processes and cell cycle control: Molecular mechanisms. Environmental Health Perspectives.

[64] Takahashi K, Imaeda T, Kawazoe Y. Effect of metal ions on the adaptive

*DOI: http://dx.doi.org/10.5772/intechopen.89850*

Correlation between induction of DNA

Diaz-Barriga F, Mejia J, del Castillo E. Genotoxicity induced in CD-1 mice by inhaled lead: Differential organ response. Mutagenesis. 2002;**17**:55-61

[50] Hartwig A. Role of DNA repair inhibition of lead and cadmiuminduced genotoxicity: A review. Environmental Health Perspectives.

[51] Van den Bussche J, Soarez E. Lead induces oxidative stress and phenotypic markers of apoptosis in *Saccharomyces cerevisae*. Applied Microbiology and Biotechnology. 2011;**90**(2):679-687

fragmentation and micronuclei formation in kidney cells from rats and humans, and tissue-specific carcinogenic activity. Toxicology and Applied Pharmacology.

1999;**161**:153-159

1994;**102**(3):45-50

[52] Waalkes M. Cadmium carcinogenesis in review. Journal of Inorganic Biochemistry.

[53] Wlostowski T, Krasowska A, Bonda E. Photoperiod affects hepatic and renal cadmium accumulation, metallothionein induction, and cadmium toxicity in the wild bank vole (*Clethrionomys glareolus*). Ecotoxicology and Environmental

[54] Wlostowski T, Dmowski K, Bonda-Ostaszewska E. Cadmium accumulation, metallothionein and glutathione levels, and histopathological changes in the kidneys and liver of magpie (*Pica pica*) from a zinc smelter area. Ecotoxicology.

2000;**79**:241-244

Safety. 2004;**58**:29-36

2010;**19**:1066-1073

[55] Friberg L, Piscator M,

Nordberg G, Kjellstrom T. Cadmium in the Environment. 2nd ed. Cleveland, Ohio: Chemical Rubber Co.; 1974. 176p

[49] Valverde M, Fortoul T,

*Nefarious, but in a Different Way: Comparing the Ecotoxicity, Gene Toxicity and Mutagenicity… DOI: http://dx.doi.org/10.5772/intechopen.89850*

Correlation between induction of DNA fragmentation and micronuclei formation in kidney cells from rats and humans, and tissue-specific carcinogenic activity. Toxicology and Applied Pharmacology. 1999;**161**:153-159

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

with lead acetate. Mutation Research.

[40] Gasiorek K, Bauchinger M. Chromosome changes in human lymphocytes after separate and combined treatment with divalent salts of lead, cadmium, and zinc. Environmental Mutagenesis.

[41] Hartwig A, Schlepegrell R,

Beyersmann D. Indirect mechanism of lead-induced genotoxicity in cultured mammalian cells. Mutation Research.

[42] Cai M, Arenaz P. Antimutagenic effect of crown ethers on heavy metalinduced sister chromatid exchanges. Mutagenesis. 1998;**13**(1):27-32

[43] Wozniak K, Blasiak J. In vitro genotoxicity of lead acetate: Induction of single and double DNA strand breaks and DNA-protein cross-links. Mutation

[44] Xie H, Wise S, Holmes A, Xu B, Wakeman T, Pelsue S. Carcinogenic lead chromate induces DNA double-strand breaks in human lung cells. Mutation

[45] Muro L, Goyer R. Chromosome damage in experimental lead poisoning. Archives of Pathology. 1969;**87**:660-663

[46] Deknudt G, Colle A, Gerber G. Chromosomal abnormalities in lymphocytes

from monkeys poisoned with lead. Mutation Research. 1977;**45**:77-83

[47] Sharma R, Jacobson-Kram D, Lemmon M, Bakke J, Galperin I, Blazak W. Sister chromatid exchange and cell replication kinetics in fetal and maternal cells after treatment with chemical teratogens. Mutation Research.

[48] Robbiano L, Carrozzino R, Porta Puglia C, Corbu C, Brambilla G.

1985;**158**:217-231

Research. 2003;**535**:127-139

Research. 2005;**586**:160-172

1972;**14**:95-100

1981;**3**:513-518

1990;**241**:75-82

[30] Catellino N, Aloj S. Determination of the elimination constants of Pb-210 from various rat tissues. Folia Medica.

perspective on lead biokinetic models. Environmental Health Perspectives.

[33] O'Flaherty E. Physiologically based models for bone seeking elements. V: Lead absorption and disposition in childhood. Toxicology and Applied Pharmacology. 1995;**131**:297-308

[34] Goyer R, Rhine B. Pathological effects of lead. International Review of Experimental Pathology.

[36] Tham C, Chakravarthy S, Haleagrahara N, De Alvis R.

Medicine. 2013;**5**(2):648-654

[38] Winder C, Bonin T. The

[39] Bauchinger M, Schmid E. Chromosome analysis of cultures of Chinese hamster cells after treatment

2013;**7**(2):192-199

1993;**285**(1):117-124

[37] Ahmed M, Ahmed M, Meki A, Abd-Raboh N. Neurotoxic effects of lead on rats: Relationship to apoptosis. International Journal of Health Sciences.

genotoxicity of lead. Mutation Research.

Morphological study of bone marrow to assess the effects of lead acetate on haemopoiesis and aplasia and the ameliorating role of *Carica papaya* extract. Experimental and Therapeutic

[35] Zelikoff J, Thomas P. Immunotoxicology of Environmental and Occupational Metals. London: Taylor and Francis;

1964;**47**(1):238-248

1998;**106**(6):1461-1465

1983;**4**:189-192

1973;**12**(1):23-37

2005. 382p

[31] Rabinowitz M. Historical

[32] Kneip T, Mallon R, Harley N. Biokinetic modeling for mammalian metabolism. Neurotoxicology.

**74**

[49] Valverde M, Fortoul T, Diaz-Barriga F, Mejia J, del Castillo E. Genotoxicity induced in CD-1 mice by inhaled lead: Differential organ response. Mutagenesis. 2002;**17**:55-61

[50] Hartwig A. Role of DNA repair inhibition of lead and cadmiuminduced genotoxicity: A review. Environmental Health Perspectives. 1994;**102**(3):45-50

[51] Van den Bussche J, Soarez E. Lead induces oxidative stress and phenotypic markers of apoptosis in *Saccharomyces cerevisae*. Applied Microbiology and Biotechnology. 2011;**90**(2):679-687

[52] Waalkes M. Cadmium carcinogenesis in review. Journal of Inorganic Biochemistry. 2000;**79**:241-244

[53] Wlostowski T, Krasowska A, Bonda E. Photoperiod affects hepatic and renal cadmium accumulation, metallothionein induction, and cadmium toxicity in the wild bank vole (*Clethrionomys glareolus*). Ecotoxicology and Environmental Safety. 2004;**58**:29-36

[54] Wlostowski T, Dmowski K, Bonda-Ostaszewska E. Cadmium accumulation, metallothionein and glutathione levels, and histopathological changes in the kidneys and liver of magpie (*Pica pica*) from a zinc smelter area. Ecotoxicology. 2010;**19**:1066-1073

[55] Friberg L, Piscator M, Nordberg G, Kjellstrom T. Cadmium in the Environment. 2nd ed. Cleveland, Ohio: Chemical Rubber Co.; 1974. 176p

[56] Nocentini S. Inhibition of DNA replication and repair by cadmium in mammalian cells. Protective interaction of zinc. Nucleic Acids Research. 1987;**15**:4211-4225

[57] Nordberg G. Historical perspectives on cadmium toxicology. Toxicology and Applied Pharmacology. 2009;**238**:192-200

[58] Rikans L, Yamano T. Mechanisms of cadmium-mediated acute hepatotoxicity. Journal of Biochemical and Molecular Toxicology. 1999;**14**(2):110-117

[59] Salinska A, Wlostowski T, Zambrzyczka E. Effect of dietary cadmium and/or lead on histopathological changes in the kidneys and liver of bank voles *Myodes glareolus* kept in different group densities. Ecotoxicology. 2012;**21**(8):2235-2243

[60] Bremner I. Mammalian absorption, transport and excretion of cadmium. In: Webb M, editor. The Chemistry, Biochemistry and Biology of Cadmium. Amsterdam: Elsevier Holland; 1979. pp. 175-193

[61] Shore R, Douben P. The ecotoxicological significance of cadmium intake and residues in terrestrial small mammals. Ecotoxicology and Environmental Safety. 1994;**29**:101-112

[62] Rossmann T, Roy N, Lin W. Is cadmium genotoxic? IARC Publications. 1992;**118**:367-375

[63] Hartwig A, Asmuss A, Ehleben I, Herzer U, Kostelac D, Pelzer A, et al. Interference of toxic metal ions with DNA repair processes and cell cycle control: Molecular mechanisms. Environmental Health Perspectives. 2002;**110**(5):797-799

[64] Takahashi K, Imaeda T, Kawazoe Y. Effect of metal ions on the adaptive

response induced by N-methyl-N-nitrosourea in *Escherichia coli*. Biochemical and Biophysical Research Communications. 1988;**157**:1124-1130

[65] Snyder RD, Davis GF, Lachmann PJ. Inhibition by metals of X-ray and ultraviolet-induced DNA repair in human cells. Biological Trace Element Research. 1989;**21**:389-398

[66] Mukherjee A, Giri A, Sharma A, Talukder G. Relative efficacy of shortterm tests in detecting genotoxic effects of cadmium chloride in mice *in vivo*. Mutation Research. 1988;**206**(2):285-295

[67] Privezentsev K, Sirota N, Gaziev A. The genotoxic effects of cadmium studied *in vivo*. Tsitologiya i genetika. 1996;**30**(3):45-51

[68] Fahmy A, Aly F. *In vivo* and *in vitro* studies on the genotoxicity of cadmium chloride in mice. Journal of Applied Toxicology. 2000;**20**:231-238

[69] El-Ghor A, Noshy M, El Ashmaoui H, Eid J, Hassanane M. Microsatellite instability at three microsatellite loci (D6mit3, D9mit2 and D15Mgh1) located in different common fragile sites of rats exposed to cadmium. Mutation Research. 2010;**696**(2):160-166

[70] Wada K, Fukuyama T, Nakashima N, Matsumoto K. Assessment of the *in vivo* genotoxicity of cadmium chloride, chloroform, and D,L-menthol as coded test chemicals using the alkaline comet assay. Mutation Research. 2015;**786**:114-119

[71] Xu J, Wise J, Wang L, Schumann K, Zhang Z, Shi X. Dual roles of oxidative stress in metal carcinogenesis. Journal of Environmental Pathology, Toxicology and Oncology. 2017;**36**(4):345-376

[72] Hartwig A. Metal interaction with redox regulation: An integrating concept in metal carcinogenesis?

Free Radical Biology and Medicine. 2013;**55**:63-72

[73] Knasmüller S, Parzefall W, Sanyal R, Ecker S, Schwab C, Uhl M, et al. Use of metabolically competent human hepatoma cells for the detection of mutagens and antimutagens. Mutation Research. 1998;**402**:185-202

**77**

**Chapter 5**

**Abstract**

**1. Introduction**

Response

DCLK1 and DNA Damage

*Michael Bronze and Parthasarathy Chandrakesan*

**Keywords:** DNA damage, DDR, ATM, ATR, DCLK1

*Janani Panneerselvam, Dongfeng Qu, Courtney Houchen,* 

Genome integrity is constantly monitored by sophisticated cellular networks, collectively termed as the DNA damage response (DDR). The DDR is a signaling network that includes cell cycle checkpoints and DNA repair and damage tolerance pathways. Failure of the DDR or associated events causes various diseases, including cancer. DDR is primarily mediated by phosphatidylinositol-3-kinase-like protein kinase (PIKKs) family members ataxia-telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related protein (ATR). However, one of the many unanswered questions regarding these signal-transduction pathways is: how does the cell turn the DDR signals on? There was no conclusive demonstration of the involvement of a specific sensory kinase in DDR signals until our recent research on the DCLK1 role in regulating ATM after genotoxic injury. Currently, various studies are demonstrating the importance of DCLK1 in DNA damage response. Here, we discuss the novel insights into the role of DCLK1 in DNA damage response.

DNA damage exists in all cellular organisms, and DNA, the genetic material in each living cell is the fundamental unit of life and its integrity and stability are essential to life [1]. However, DNA is not passive; rather, it is a chemical unit subject to be attacked from a range of endogenous and environmental damaging agents. The endogenous damages are the damage caused by reactive oxygen species or metabolic byproducts, and DNA metabolization; exogenous damages are caused by external agents, like radiations, toxins, chemicals, and microorganisms [2]. In response to the DNA damage, cells rapidly recruit a sophisticated network which is called DNA damage-response (DDR) systems. DDR systems include DNA repair mechanisms, damage tolerance processes, and cell-cycle checkpoint pathways [3]. Failure of DDR causes genomic instability which results in various diseases including immune deficiency, neurological degeneration, premature aging, and severe cancer susceptibility [2, 4]. Indeed, great progress has been made towards understanding the mechanisms of DDR in homeostasis, carcinogenesis and cancer advancement but much remains to delineate how the DDR network systems are regulated. Furthermore, how the DDR network is formed and how it is fine-tuned by upstream and downstream mediators or signaling pathways that support the homeostasis or disease progression required to understand. While the rapid activation of DDR against the

## **Chapter 5**

*Genotoxicity and Mutagenicity - Mechanisms and Test Methods*

Free Radical Biology and Medicine.

[73] Knasmüller S, Parzefall W, Sanyal R, Ecker S, Schwab C, Uhl M, et al. Use of metabolically competent human hepatoma cells for the detection

of mutagens and antimutagens. Mutation Research. 1998;**402**:185-202

2013;**55**:63-72

response induced by N-methyl-N-nitrosourea in *Escherichia coli*. Biochemical and Biophysical Research Communications. 1988;**157**:1124-1130

[65] Snyder RD, Davis GF,

1988;**206**(2):285-295

[67] Privezentsev K, Sirota N, Gaziev A. The genotoxic effects of cadmium studied *in vivo*. Tsitologiya i

genetika. 1996;**30**(3):45-51

Toxicology. 2000;**20**:231-238

[68] Fahmy A, Aly F. *In vivo* and *in vitro* studies on the genotoxicity of cadmium chloride in mice. Journal of Applied

[69] El-Ghor A, Noshy M, El Ashmaoui H, Eid J, Hassanane M. Microsatellite instability at three microsatellite loci (D6mit3, D9mit2 and D15Mgh1) located in different common fragile sites of rats exposed to cadmium. Mutation Research. 2010;**696**(2):160-166

[70] Wada K, Fukuyama T, Nakashima N, Matsumoto K. Assessment of the *in vivo* genotoxicity of cadmium chloride, chloroform, and D,L-menthol as coded test chemicals using the alkaline comet assay. Mutation Research.

[71] Xu J, Wise J, Wang L, Schumann K, Zhang Z, Shi X. Dual roles of oxidative stress in metal carcinogenesis. Journal of Environmental Pathology, Toxicology and Oncology. 2017;**36**(4):345-376

[72] Hartwig A. Metal interaction with redox regulation: An integrating concept in metal carcinogenesis?

Lachmann PJ. Inhibition by metals of X-ray and ultraviolet-induced DNA repair in human cells. Biological Trace Element Research. 1989;**21**:389-398

[66] Mukherjee A, Giri A, Sharma A, Talukder G. Relative efficacy of shortterm tests in detecting genotoxic effects of cadmium chloride in mice *in vivo*. Mutation Research.

**76**

2015;**786**:114-119
