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An introduction about genotoxicology methods as tools for monitoring aquatic ecosystem: present status and future perspectives.

Introduction

The demand for a clean and immune supply of water for drinking, agriculture and recreation has accrued since the last few decades. The global production of chemicals has increased from 1 million ton in 1930 to more than 400 smillion tons during the year 2005 and about 100,000 different substances have been registered in the market, of which 30,000 to 70,000 are in daily use [1]. Most of the chemicals used on land enter the aquatic environment via., wastewater from agricultural, industrial and domestic sources including municipal sewage treatment plants [2]. In some instances, these chemicals are capable of permanently altering the genetic material of exposed organisms, i.e., they are mutagens. In recent years, reports on the occurrences of malignancies and other biological conditions in aquatic organisms following exposure to suspected genotoxins have increased. Such exposure of aquatic fauna not only poses a high risk for non-target organisms including man via food chain in the ecological context, but also may lead to heritable mutations and loss in the genetic diversity with significant implications of the long term survival of exposed population. The studies related to the impact of genotoxic exposure in an organism, therefore, not only cover an attempt to elucidate the mechanism(s) involving direct changes to the genetic material, but also the genotoxic effects and changes at the level of gene pool and population [3].

The uncontrolled presence of genotoxins in aquatic environment poses significant toxicological risks to a myriad of non-target organisms and finds its way to the food chain, threatening the ecological balance and the biodiversity of the nature [4]. This is an unwanted situation and justifies the monitoring of the aquatic environment for the presence of genotoxins. Although it is clear that progress in any field of research depends primarily on the development of its methodology. In this context, recent advances in the development of techniques and diagnostic tools have certainly enhanced the understanding of how an organism interacts with its environment at individual, cellular and molecular level, and substantial progress has been made to assess the genotoxin induced genetic damage in the aquatic biota [3]. The biomonitoring of aquatic genotoxicity is cardinal for many folds. Initially, the detection of genotoxic effects in aquatic environment is needed to assess the health of aquatic organisms. Second, from the ecological perspective, the protection of genetic diversity in natural populations is important for population survival [5]. The exposure to genotoxins can damage the DNA of living cells and if these DNA lesions are not repaired, they can commence a cascade of biological consequences at the cellular, individual, community and finally at the population level [6]. The relevance of detecting the genotoxic risk assessment in contaminated aquatic environment(s) was firstly perceived in the late 1970's. Since then several tests have been flourished for evaluating genotoxicity in aquatic organisms. The possibility of using changes in DNA integrity to the genetic material as markers of exposure and effect of genotoxicants has been previously investigated [7]. The presence of DNA adducts has been taken as evidence of exposure to specific genotoxins [8]. These tests rely on the premise that any changes to DNA may have chronic and profound consequences [9]. Although some studies have highlighted the state of the art of methodologies from genotoxicological perspective [10-12], this review addresses this question again and to place it in somewhat a broader context. This review addresses the genotoxicity tests developed to date besides delving the latest knowledge and thinking on these cardinal approaches for the assessment of aquatic health, management and conservation.

Genotoxicology- A Cursory Look

Genotoxicology is the systematic investigation of the effect(s) that a physical or chemical agent can exert on the genetic system of an organism and when present in our environment, interaction of such chemicals may give rise to cumulative effect(s), with genetic consequences for the future of the affected species. The genotoxicity is often confused with mutagenicity; but while all mutagens are genotoxic, not all genotoxic substances are mutagenic. The alteration can have direct or indirect effects on the DNA: the induction of mutations, mistimed event activation, and direct DNA damage leading to mutations. The primary objective of the genetic toxicology, however, is to detect and analyze the potential hazard of those agents that are highly specific for interaction with nucleic acids and produce alterations in genetic material at subtoxic concentration. Thus, its first aim is to describe the outcome produced by toxic substances in various test species, but only from the genetic point of view and to draw conclusions that can be extrapolated to man. The next aim is to investigate the mechanism(s) of action of the toxic substances, and on the basis of this knowledge to assess the environmental risks [13]. The beginning of the genotoxicology was marked by H.J. Muller's discovery of sex linked recessive lethal mutation in Drosophila induced by the x-rays. The impetus in evolving the field of genotoxicity came from the study of Alexander-Hollaender, who led the foundation of the Environmental Mutagen Society (EMS), in USA [14]. The EMS and, in particular, its applied role in genotoxicology, has had a profound positive impact on many disciplines in toxicology and environmental risk assessment [15]. In 1973, after the establishment of International Association of Environmental Mutagen Societies (IAEMS), the beginning of active research in the field of genotoxicology was marked. National and international agencies became increasingly interested in mutagenesis, recognizing its importance not only for genetic diseases but also for carcinogenesis [13]. In the United States, agencies like National Institute of Health (NIH), National Toxicology Programme (NTP), Environmental Protection Agency (EPA), Food and Drug Administration (FDA) have supported the basic research in mutagenesis.

Agents that induce alterations in genetic material at sub-toxic exposure level are classified as genotoxic. These agents usually have common chemical or physical properties facilitating their interaction with genetic material. Most genotoxic agents damage nucleic acids at high exposure concentrations evoking acute and nonspecific cytotoxic effects in wide range of cellular processes. Thus, genetic toxicology has evolved to play a dual role in safety evaluation programmes. One role is the implementation of mutagenicity testing and risk assessment methods to evaluate the impact of genotoxic agents found in the environment, more specifically of those whose presence may alter the integrity of gene pool. The second role is the application of genetic test methods to the detection and mechanistic understanding of carcinogenic chemicals [16]. However, the ecological impacts of many genotoxins still remain poorly understood. The history of genotoxicology and electronic data collections may be reviewed online as the internet offers a wide range of digital resources for the field of genotoxicology. The popular Web based resources are TOXNET, GENE-TOX, TOXLINE, MEDLINE, PUBMED, Gateway, Entrez, Hazardous Substances Data Bank, CHEMIDPLUS at NLM, etc., As the molecular biology and computational tools improve, new areas such as structural activity relationship analysis, mutational spectra databases and toxicogenomics, which are the emerging areas within genotoxicology, have online resources as well [17].

Role of Piscine Model in Genotoxicology

Research on the genotoxic effects of different pollutants to fish has been primarily directed at determining the potential for chemical mutagenesis in these organisms. However, clastogenic activity may be a risk factor for genetic diseases in fish population, in addition to carcinogenesis. There is also continuing interest in developing in vivo genotoxicity assays with piscine model systems for assessing health risk to higher vertebrates [12]. Aquaculture is one of the most rapidly expanding food industries since fish have always been an important source of food for humans and, for large proportion of the population they represent a major source of protein [18]. On the other hand, the aquatic environment is a sink for many chemicals, as illustrated by the occasionally high pollution levels and frequencies of chemical spills, and fish have been the target of overt chemical pollution [1]. Fish have received particular attention as monitor system in the surveillance of aquatic ecosystem and indirectly the human health. For example, marine fish and shell fish, which are a major source of protein in many countries, are often contaminated with high concentrations of methyl mercury. Induction of chromosomal damage has been found in the lymphocytes of persons exposed to methyl mercury through consumption of contaminated fish [19].

In conventional eco-toxicity testing strategies, fish has been regarded as an indispensable component of integrated toxicity testing strategies for the aquatic organisms. The current guidelines at the OECD level are a good example of this with fish-targeted guidelines covering acute toxicity [20], early life stage toxicity [21] and juvenile growth test [22]. In particular, fish acute toxicity tests play an important role in environmental risk assessment and hazard classification, because they allow for first estimates of the relative toxicity of various chemicals in various species [23]. They have become widely required for the aquatic hazard assessment of new and existing synthetic substances as well as for use in the evaluation of complex effluents. Thus, in the "base set" of data requirements for all substances which are produced or imported in quantities of one ton or more per year, the acute toxicity for freshwater fish [20] is a mandatory component. Furthermore, no single species is universally sensitive to all chemicals. The single species LC50 data are questionable with respect to accuracy and, in more general terms, to toxicological relevance. Thus, it seems largely accepted that there is no most susceptible species that could be used in a possible conservative testing approach [24]. The retreat of the toxicity tests with the single most sensitive species is to ignore any knowledge of the complexity of ecosystems. Therefore, the susceptibility and tolerance of most fish populations may be hard to predict from single standard acute toxicity test. Multiple tests on various species are necessary to get an appropriate estimate of the range of susceptibilities to chemical substances.

The current spurt in the fish cytogenetical studies had its origin in the standardization of the newer techniques and in the realization of an immense applied value of the cytogenetic data of fish. The fish genotoxicity indeed stands on the threshold of a golden era with great prospects of its being used in applied research, such as monitoring of genotoxic agents [25]. Since most of the chemicals and pesticides used terrestrially end up finally in the aquatic system, it is the fish that are affected and should be monitored for these subtle but genetically damaging changes [26]. Induction of cytogenetic damage in fish could serve to monitor not only selected genotoxic agents in the laboratory, but also the presence of genotoxicants in surface waters and different aqueous ecosystems [27]. Several testing protocols have been developed earlier which permit evaluation of the mutagenic properties of various chemicals and physical agents. Short term tests include acute toxicity, sister chromatid analysis, gross chromosomal changes, formation of micronuclei and effects on DNA. Long term studies can be carried out on mutagenicity and general toxicity. The modes of administration may be oral, intraperitoneal or in aqueous environment [28]. Exposure of fish to genotoxic agents may occur either by the digestive route or by the respiratory route following absorption of water borne chemical through the gills [12]. The fish is able to convert xenobiotics to active metabolites. It has also been successfully used to demonstrate the presence of genotoxic agents in the aquatic environment [12,27].

Applications of Genotoxicological Methods in Monitoring Aquatic Environment

The genotoxicity of various chemical agents in fish and other aquatic organisms have been assessed by several assays. These assays differ in their end points and specificities to explore the genotoxic effects induced by various toxicants at different levels in biological systems, but most of them are having their own limitations.

Chromosomal Aberration Test

Chromosomal aberrations (CA) are one of the important biological consequences of exposure of the target organism to genotoxic agents. Chromosomal studies have received considerable attention in recent years, in part from a growing interest in the evaluation of genotoxicity of environmental toxicants and carcinogens. Chromosomal changes leading to mutations were first described in Oenothera [29]. During 1930-1940 the CA along with their mechanism, behaviour and fate, following irradiation was studied in some insects like Drosophila and in plant systems, i.e., Allium and Tradescantia. The protocols gradually developed following the discovery of colchicine which was able to arrest cells at metaphase. However, studies of chemical effects of chromosomes were mainly limited to academic exercises till 1970's, when concern over the deteriorating environmental situation led to an increasing interest for effective short term assays to screen mutagens and carcinogens. The cytogenetic tests emerged as a very important component of these systems [30].

CA may be defined as a missing, extra, or irregular portion of chromosome. A karyotype refers to a full set of chromosomes from an organism that can be compared to a normal karyotype for the species via genetic testing. CA usually occurs when there is an error in cell division following meiosis or mitosis. Cytogenic effects can be studied either in whole animals (in vivo) or in cells grown in culture (in vitro). In both cases, the animal or cell is exposed to the test substance and then afterwards treated with a metaphase-arresting Colchicine or Colcimide. Following suitable staining the metaphase cells are analysed microscopically for the presence of aberration. There are many types of CA and can be organized into two basic groups, structural and numerical aberrations.

The pioneering studies of Howard and Pelc [31] demonstrated that between two mitoses, the intervening interphase has three phases designated as G1 (pre-DNA synthetic), S (DNA synthesis) and G2 (postsynthetic). Ionising radiation induced chromosome type of aberrations in G1, chromatid type of aberrations in G2 and most of the S phase. True radiomimetic chemicals, such as bleomycin and neocarcinostatin induce similar pattern of aberrations as ionising radiation. However, chemical mutagens, which do not induce directly DNA strand breaks but cause other lesions, were shown to induce only chromatid type aberrations irrespective of the DNA synthetic stage treated. An intervening S phase is necessary to visualize the aberrations. Short wave UV induces aberrations in a similar way as chemical mutagens. On this basis, chromosome breaking agents (clastogens) can be classified as S dependent and independent agents [32].

The morphological and numerical alteration of chromosome carrying DNA (genes) results in a quantitative alteration or rearrangement of genes and has been substantially studied in various research areas. The mammalian in vivo chromosomal aberration test has been successfully used for the detection of structural aberrations induced by the test substance in the bone marrow cells of animals, usually rodents [33,34]. In fact the chromosomal aberration test using cultured mammalian cells is one of the sensitive methods to predict environmental mutagens or carcinogens, and considered as complementary test to the Salmonella microsome assay (Ames test) by some workers [35].

Studies using Chromosomal Aberration Test in Fish

Of the estimated 20,000 fish species, a mere 500 have been studied for complete karyotype. In Indian context, the figure is far too less; the fish cytologists have determined the karyotypes of only about 125 species to a satisfactory extent [25]. Genotoxic studies in different fish species using cytogenetic analysis have been reported by a number of workers [27]. Exposure of fish to various toxicants for a prolonged period, even at low levels, leads to chromosomal aberrations including gene alterations [25,36,37]. An advantage of chromosomal studies is that they reveal a measure of sub-lethal effects of xenobiotics in vivo.

Most fish typically have high chromosome numbers, greater than 48 with small acrocentric chromosomes [38]. Given to this nature, different techniques have been used to perform cytogenetic studies; direct in vivo and indirect in vitro. Direct techniques are based on the use of colchicine to block quickly proliferating cell population of kidney, spleen and gill epithelia at the metaphase stage of the cell cycle. Just after the fish is sacrificed, cell samples are taken and treated for slide preparations [39]. The indirect test technique involve tissue cultures [40], that allow for the application of perfect doses of colchicine and the use of mitotic stimulants such as phytohaemeagglutinin (PHA). Each of this procedure has been optimized to obtain large number of well spread metaphases and used regularly for karyotypic analysis, and to assess the impact of genotoxic agents.

Fish as an in vivo model can be quite promising for screening the mutagenic properties of chemicals and environmental pollutants specially the agricultural runoff that routinely pollute our water bodies [12]. Kligerman is credited to develop a cytogenetic fish model (Umbra limi) in which chromosome damage was later accurately detected using physical and chemical agents [39]. The toxicity of different pesticides has been associated with changes in replication of DNA and DNA aberration that leads to mutation and hyperproliferation of cells due to local irritation [41]. In fish DNA repairing takes place at a much lower speed than in mammals [42], which render fish as a sentinel organism as far as bio-monitoring studies are concerned [4,12]. There is rich documented literature witnessing research on molecular level of different fish species showing ill effects of pesticides on genes and DNA levels [43-45].

The genotoxicity of contaminated water in the field and laboratory, for various in vivo and in vitro monitoring systems have successfully employed the fish cells. CA has been observed after exposure of fish species to different chemicals. Exposure of Channa punctatus to Dichlorvos (0.01 ppm) caused chromatid gaps, centromeric gaps, attenuation, chromatid breaks, extra fragments, and stubbed arm in kidney cells [25]; while exposure of the same species to fenvalerate caused chromatid separation, chromatid break, deletion, fragments, gaps, and ring type chromosomes [46]. Interestingly longer exposure was associated with lower frequency of DNA aberrations. Cypermethrin caused changes in nucleic acids (RNA and DNA) in gonadal tissue of Colisa fasciatus. This test was equally effective in cultured cells and embryos in some parallel studies of fish and as well as on cell lines of Umbra limi [2,43,47].

There are several studies demonstrating the suitability of conventional cytogenetic techniques like chromosomal aberration test for the assessment of genotoxic effects of various toxicants using fish as bioindicator. Table 1 presents a list of some important studies, using fish as a model, to evaluate genotoxicity by chromosomal aberration test.

Micronucleus Assay (MNT)

The MNT is recognized as one of the most simple, reliable and successful assays for the in vivo evaluation of genotoxicity of various toxicants. Micronuclei (MN) were first described in the cytoplasm of erythrocytes more than a century ago and were called "fragment of nuclear material" by Howell or "intraglobularies corpuscules" in the terminology of Jolly in the late 18th century and early 1900. These structures are known by the haematologists as "Howell-Jolly bodies" [72]. It is now well-established that MN mainly originate from acentric chromosome fragments, acentric chromatid fragments or whole chromosomes that fail to be included in the daughter nuclei at the completion of telophase during mitosis because they did not attach properly with the spindle during the segregation process in anaphase [73]. These displaced chromosomes or chromosome fragments are eventually enclosed by a nuclear membrane and, except for their smaller size, are morphologically similar to nuclei after conventional nuclear staining. Scoring MN in interphase nuclei is technically easier and faster than scoring of chromosomal aberration in metaphase nuclei [27].

Salvadori highlighted that, whatever the type of DNA damage, MN is originated during cell division [74]. The DNA damage due to exposure to mutagenic agents is expressed in micronucleus form just after one cycle of cell division and is dependent on the proportion of cells under division. The increased interest in environmental genotoxicity studies went ahead with the development of a great number of tests to evaluate genotoxic effects in aquatic environments. Considering this, MNT, one of the most popular and promising tests on ecotoxicology, represents a cytogenetic indicator of DNA damage for over 30 years [73].

The MNT is a simple and sensitive assay for "in vivo and in vitro" evaluation of genotoxicity in aquatic environment as a part of the biomonitoring programs for several days followed by microscopic analysis of erythrocytes, gill, liver, kidney and fin cells [27]. Fish serves as useful genetic model for the assessment of pollution in aquatic ecosystems [75]. Fish respond to toxic agents similar to higher vertebrates and can allow the assessment of substances that are potentially hazardous to humans. Fish can, in fact, be the sentinel organisms that can indicate the risk of human exposure to drinking water contaminated with genotoxicants [72].

In India, studies on MNT have been initiated by Manna who reported presence of micronuclei in erythrocytes and chromosomal aberrations in Oreochromis mossambicus treated with aldrin, cadmium chloride and x-rays [76]. The percentage of micronuclei was low but chromosomal aberrations were higher in x-rays treated specimens. Das and Nanda noted an increase in micronuclei frequency in the erythrocytes of Heteropneustes fossilis exposed to mitomycin C and paper mill effluent [77]. Barat employed the MNT to screen the genotoxic effect of the pesticide Malathion on Channa punctatus at concentration of 0.033 mL/L for periods of 5,11 and 16 days and reported significant increase in the frequency of MN than that of the control [78]. MNT have been reported to be sensitive for detecting the genotoxic potential of the low levels of malathion pesticide in two fish species namely Mystus vittatus and C. punctatus in aqueous medium and there was also dose dependent increase in MN formation [79]. The study of Ateeq showed a positive dose-response relationship in fish for genotoxic and cytotoxic endpoints when exposed to two herbicides namely 2,4-dichlorophenoxyacetic acid (2,4-D) and butachlor [80]. The genotoxic endpoints in the study were MNT and chromosomal aberration test using C. punctatus as an experimental model. Ahmad studied the effect of pentachlorophenol (PCP) on fish genome using haematoxylin-eosin technique and observed that the frequency of micronucleated erythrocytes increased with the increase in exposure time [81]. Recently, Farah reported the strong genotoxic effects of PCP and 2,4-D in fresh water fish C. punctatus, using MNT and chromosomal aberration test [82]. The ameliorating effect of ethanolic neem leaf extract was also carried out in the study.

MNT has become a very popular tool for assessment of genotoxic potential of various chemical agents by using fish as a model. Table 2 shows some studies on fish, for the evaluation of genotoxicity of various xenobiotic agents, using MNT.

Comet Assay

Since Singh launched the alkaline Single Cell Gel Electrophoresis (SCGE) protocol or comet assay to the scientific community, its uses and applications has been accreting [105]. The thematic areas of its current employment in evaluating the genotoxicology are vast, either in vitro or in vivo, both in the laboratory and in the field, terrestrial or aquatic. The comet assay developed by Singh and his coworkers was successfully acclimatized to fish erythrocytes with few modifications. The authors concluded that the assay as "extremely sensitive and should be useful in detecting DNA damage caused by environmental pollutants." Since the past two decades, this premonitory statement has been recurrent and increasingly reinforced by an array of scientific publications, exploring a wide diversity of approaches, i.e., in vitro [106], in vivo [107], and as well as surveying wild native specimens [108]. Furthermore, in recent years we have witnessed a greater profusion of publications. For example in the last year (2014), 72 articles were published (according to a literature search on PubMed) evaluating DNA damage by comet assay in fish exposed to various genotoxins (Figure 1). This vast utilization of fish should also be regarded as a sign of primary concern of genetic ecotoxicologists over the health status of aquatic ecosystems.

[FIGURE 1 OMITTED]

The popularity of this test is due to its sensitivity, simplicity, cost effectiveness and time efficiency due to automatic scoring of the comets by using the image-analysis software. Furthermore, the German Federal Environmental Agency proposes the comet assay as a useful test for a reliable detection of genotoxins in surface waters. Alkaline comet assay is capable of detecting a wide variety of DNA damages such as DNA single-strand breaks, double-strand breaks, oxidatively induced base damages and alkali-labile DNA length: width ratio of the DNA mass in a variety of fish cells (erythrocytes, liver, gill and kidney) exposed to contaminated water samples, and the amount of damage increased with the duration of exposure. It is well-established that comet assay is applicable, virtually, to all species. A clear demonstration of this polyvalence is the finding that, since last two decades, this assay was successfully adapted to more than 90 fish species. This wide range of species includes mostly bony fish (Class Osteichthyes), both rayfinned fish (Subclass Actinopterygii), the overwhelming majority of cases, and lobe-finned fish (Subclass Sarcopterygii) like Arapaima gigas [109]. The jawless fish (Class Agnatha) are represented with an interesting study with sea lamprey (Petromyzon marinus) describing the relationship between sperm DNA damage and fertilizing ability [110], while cartilaginous fish (Class Chondrichthyes) are completely unexplored. Some recent landmark studies on comet assay using piscine model are summarized in Table 3. In what concerns to the type of agent/contaminant tested, the application of comet assay in the field of aquatic genotoxicology has accompanied the evolution of other subareas of environmental toxicology involving piscine models.

Recent Developments and Correlations with other Biomarkers

For evaluating the environmental samples in the field of genotoxicology, recent advancement has been achieved [131]. A transgenic zebrafish has been established for the detection of genotoxins. It carries plasmids that are having the [rp.sup.SL] gene of Escherichia coli as a mutational target gene [132]. Another transgenic fish, that bikes multiple copies of a bacteriophage lambda vector harboring the cII gene as a mutational target, has been established by a technique originally developed for lambda transgenic rodents [133]. The [p.sup.53] tumor suppressor gene, which is known to be implicated in cancer development, has been investigated as a possible biomarker for genotoxin in fish cells [134].

The combination of genotoxicity tests with other biomarkers to evaluate the genotoxin(s) based genetic damage has been assessed in many studies (Figure 2). Some of them show a positive correlation between the results given by the genotoxicity tests and other biomarkers. For instance, in one of our previous study [12], a positive correlation between oxidative stress and genotoxicity was observed on endosulfan (persistent organochlorine pesticide) exposure. Our data suggested that chronic exposure to endosulfan resulted in an increased oxidative stress, which was reflected by increase in LPO in erythrocytes, in a time and concentration dependent manner; antioxidative enzymes like glutathione, superoxide dismutase and catalase also fluctuated significantly in the fish blood of all the treatment groups. This type of positive correlation was also supported by many recent studies [37,135]. It is also interesting to note that the different genotoxicity tests also showed positive correlation between them. For instance, the studies performed by Mouchet showed a positive correlation between DNA strands breaks detection and micronuclei induction at most of the times [136,137]. This result was expected since the comet assay reveals primary DNA damages and the micronucleus test measures unrepaired lesions that result from the non-repaired primary DNA damages, which are likely to be inherited by subsequent generations of cells.

[FIGURE 2 OMITTED]

In recent years fish also acted as sentinel organism for screening the natural products to evaluate their pharmacological activities [138140]. Fish, living in different aquatic environments also carry the risk of direct or indirect exposure to various xenobiotics, including pesticide residues [141]. In fish, pharmacological studies are still in preliminary stage and only few reports are available. One such report concerns about the antimutagenic and anticarcinogenic activity of chlorophyllin towards aflatoxin in rainbow trout [142]. The ameliorating effect of vitamin C, [beta]-carotene and azadirachtin (principle compound of neem) against genotoxicity of ethyl methanesulfonate and cadmium chloride has been demonstrated in a fish, Oreochromis mossambicus [58]. Recently the antimutagenic effect of neem leaves extract in freshwater fish, Channa punctatus has been evaluated by cytogenetic tests [143].

In the recent years, as a sign of maturity, intensive attention has been devoted to the interference of non-contamination related factors, i.e., biotic and abiotic, with the genotoxicity expression. This is an important knowledge to permit an accurate assessment of the contribution genotoxins to the measured DNA damage. In this direction, hypoxia and hyperoxia, considered as critical stressors in the aquatic environment, were tested in Cyprinus carpio, revealing that both conditions increase oxidative DNA damage by 25% when compared to normoxic conditions [144]. Another study depicted that acute extreme exercise results in oxidative DNA damage in Leuciscus cephalus, suggesting that fish living in fast flowing contaminated waters are at intense risk [145]. The evaluation of the consequences of germ cell DNA damage on progeny outcomes has been regarded as a strategy to reflect potential chronic impacts of aquatic genotoxins in fish, since genetic damage in such cells, if unchecked, can be carried on to next generations. Therefore, it was concluded that a strong positive correlation exists between the DNA damage in sperm from parental fish (Salmo trutta and Salvelinus alpinus), exposed to the alkylating agent methyl methanesulfonate, and the incidence of skeletal abnormalities in the offspring; clearly indicating that DNA damage had been inherited [146]. The risk assessment of DNA-damaged germ cells in the reproduction is strictly acceptable in fish because of external fertilization or embryo development [125], since both gametes and embryos can be directly exposed to waterborne genotoxicants. This approach can represent an additional contribution to predict the impact of DNA damage on recruitment rate, progeny fitness, and thereby, on the population dynamics. Furthermore, it has been also evaluated that due to the transfer of damaged DNA to the future generation, the offsprings were subjected to elevated teratogenicity and mortality, depicting a strong relationship between genotoxicity and the declining wild populations [147].

Conclusion

The studies reviewed here clearly demonstrate that environmental genotoxicology holds the key to early detection and monitoring of genotoxins in aquatic environments, particularly when piscine model in taken into consideration. Fish serves as useful genetic model and the significance of assessment of genotoxicity in fish lies in the fact that higher vertebrates, including humans feeding on fish, are easily exposed to the genotoxic agents that are trapped in fish body. Ecotoxicological characteristics of fish such as its wide distribution and availability throughout the year, easy maintenance in the aquaria, cost effectiveness and commercial importance make fish an excellent model for genotoxicity studies. Furthermore, fish cells retain important traits like poikilothermic behaviour, unique xenobiotic metabolism, and low rate of repair mechanism [148], thus, making fish as sentinel organism for biomonitoring studies.

Piscine model have been used in several eukaryotic genotoxicity and mutagenicity tests, which include its use in CA test, MNT and above all comet assay. Based on the studies reviewed, MNT has been widely used to screen the genotoxic effects of the pesticides. It has several advantages over CA test such as additional detection of aneugens, technically less demanding, ease of scoring and possibility of automation and micronuclei arise from two important types of genetic damage, i.e., clastogenesis and spindle disruption. Furthermore, MNT is often more sensitive cytogenetic test with the promising results. Comet assay has broad applicability when applied to fish, providing a sensitive, reliable, rapid and versatile system for the study of environmental genotoxicity. However, one of the virtues of this assay is unquestionably its cost-effectiveness, compared to other genotoxicity techniques. Many disciplines in the aquatic genotoxicology are integrated in the task of identifying and comprehending each toxicant mode of activity in organisms, which is responsible for clinical signs of intoxication and death. The combined use of genotoxicity biomarkers, especially the comet assay with other biomarkers as well as standardization and interlaboratory calibration are recommended to further strengthen its use in environmental assessment studies.

The elucidation of the type of DNA damage that is generated and the critical monitoring of DNA repair through lesion-specific enzymes during the comet assay, will add value to this assay in future genotoxicological studies for monitoring and risk assessment of aquatic organisms. Furthermore, it could also assist to evaluate the potential causes of their declining populations in specific environments. Despite the evidence here highlighted toward a functional association between genotoxicity measured at individual level and a negative impact at population level, so far, DNA damage measured by comet assay in fish has not succeeded to garner enough recognition to be incorporated into national and international risk assessment protocols, even though the comparison between this and other potential biomarkers as already showed higher efficiency in the distinction between impacted and reference sites. The unequivocal and convincing demonstration of its ecological relevance is probably the greatest challenge to comet assay on the next decade.

Overall, these few examples clearly illustrate that the application of the genotoxicity tests, particularly, the comet assay in genotoxicity assessment remains as purposeful as challenging. The swift integration of new methodological improvements to the comet assay protocol like DNA repair enzyme modifications shows that genotoxicologists are constantly improving approaches and protocols. Furthermore, it must be noticed, as showed by the reviewed studies, that ecotoxicology is probably one of the most diversified and complex field of research where genotoxicity assessment is surveyed as routine. As such, one may expect another decade of successful, although constantly improving, application of the genotoxicity.

In spite the fact that the new technologies are emerging, there is still no single ultimate solution for monitoring water quality. Consequently, it is important for the researchers to determine the most suitable genotoxicity tests that suits the needs and the facilities of their research institute, and then to select the available test model, as in research we have to always start with what we have.

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References

[1.] Schwarzenbach RP, Escher BI, Fenner K, Hofstetter TB, Johnson CA (2006) The challenge of micropollutants in aquatic systems. Science 313: 1072-1077.

[2.] Ohe T, Watanabe T, Wakabayashi K (2004) Mutagens in surface waters: a review. Mutat Res 567: 109-149.

[3.] Jha AN (1998) Use of aquatic invertebrates in genotoxicological studies. Mutat Res 153: 1-10.

[4.] Dar SA, Yousuf AR, Balkhi MH, Ganai FA, Ali MN (2014) Oxidative stress in the freshwater cyprinid crucian carp (Carassius carassius L.) upon chronic exposure to endosulfan. Toxicological & Environmental Chemistry 96: 906-916.

[5.] Jha AN, Cheung VV, Foulkes ME, Hill SJ, Depledge MH (2000) Detection of Genotoxins in the Marine Environment: Adoption and Evaluation of an Integrated Approach Using the Embryo-Larval Stages of the Marine Mussel, Mytilusedulis. Mutat Res 464: 213-228.

[6.] Lee R, Steinert S (2003) Use of the Single Cell Gel Electrophoresis/Comet Assay for Detecting DNA Damage in Aquatic (Marine and Freshwater) Animals. Mutat Res/ Reviews in Mutation Research 544: 43-64.

[7.] McCarthy JF, Shugart LR (1990) Biological Markers of Environmental Contamination. In: McCarthy, J.F. and Shugart, L.R., Eds., Biomarkers of Environmental Contamination, Lewis Publishers, Boca Raton pp: 3-14.

[8.] Shugart LR (1999) Structural Damage to DNA in Response to Toxicant Exposure. In: Forbes, V.E., Ed., Genetics and Ecotoxicology, Taylor & Francis, London pp: 151-167.

[9.] Lam PK, Gray JS (2003) The Use of Biomarkers in Environmental Monitoring Programmes. Marine Pollution Bulletin 46: 182-186.

[10.] Obiakor MO, Okonkwo JC, Nnabude PC, Ezeonyejiaku CD (2012) EcoGenotoxicology: Micronucleus Assay in Fish Erythrocytes as in Situ Aquatic Pollution Biomarker: A Review. Journal of Animal Science Advances 2: 123 133.

[11.] Osman AGM (2014) Genotoxicity Tests and Their Contributions in Aquatic Environmental Research. Journal of Environmental Protection 5: 1391-1399.

[12.] Dar SA, Yousuf AR, Balkhi MH, Ganai FA, Bhat FA (2015) Assessment of endosulfan induced genotoxicity and mutagenicity manifested by oxidative stress pathways in freshwater cyprinid fish crucian carp (Carassius carassius L.). Chemosphere 120: 273-283.

[13.] Moutschen J (1985) Introduction to genetic toxicology, John Wiley and Sons Press, New-York.

[14.] Hoffman GR (1996) Genetic toxicology. In Casarett and Doull's Toxicology: The basic science of poisions, 5th (ed), McGraw Hill.

[15.] Brusick D, Auletta A (1985) Developmental status of bioassays in genetic toxicology. Mutat Res 153: 1-10.

[16.] Depledge M, Fossi M (1994) The role of biomarkers in environmental assessment. Invert Ecotoxicol 3: 161-172.

[17.] Young RR (2002) Genetic toxicology: web resources. Toxicology 173:103-121.

[18.] Nagel R, Isberner K (1998) Testing of chemicals with fish- a critical evaluation of tests with special regard to zebrafish. In: Fish Ecotoxicology, Braunbeck T, Hinton DE, Streit B (eds.), Birkhauser pp: 337-352.

[19.] WHO (1990) International program on chemical safety. Environmental Health Criteria 101: Methylmercury. Geneva: World Health Organization pp: 144.

[20.] OECD (1992) OECD Guidelines for the Testing of Chemicals. Section 2: Effects on on Biotic Systems Test No. 203: Acute Toxicity for Fish, Paris, France.

[21.] OECD (1992b) Guidelines for the Testing of Chemicals. Section 2: Effects on Biotic Systems Test No. 210: Fish, Early-Life Stage Toxicity Test, Paris, France.

[22.] OECD (2000) Guidelines for the Testing of Chemicals. Section 2: Effect on Biotic Systems Test No.215: Fish, Juvenile Growth Test, Paris, France.

[23.] Wedekind, C, von Siebenthal B, Gingold R (2007) The weaker points of fish acute toxicity tests and how tests on embryos can solve some issues. Environ Pollution 148: 385-390.

[24.] Fent K (2003) Okotoxikologie. Georg Thieme Verlag pp: 332.

[25.] Rishi KK, Grewal S (1995) Chromosome aberration test for the insecticide, dichlorvos, of fish chromosomes. Mutat Res 344: 1-4.

[26.] Ondarza PM, Gonzalez M, Fillmann G, Miglioranza KSB (2014) PBDEs, PCBs and organochlorine pesticides distribution in edible fish from Negro River basin, Argentinean Patagonia. Chemosphere 94: 135-142.

[27.] Al-Sabti K, Metcalfe CD (1995) Fish micronuclei for assessing genotoxicity in water. Mutat Res 343: 121-135.

[28.] Kligerman AD (1982). In: Hsu TC (Ed), Cytogenetic asssays of environmental mutagens. Allenheld, Osmum pp: 161.

[29.] de Vries H (1918) Mass mutations and twin hybrids in Oenothera grandiflora Ait, Bot Gaz 65: 377-422.

[30.] Sharma AK, Sharma A (1980) Chromosome techniques-theory and practice, 3rd (ed) Butterworths, London pp: 711.

[31.] Howard A, Pelc SR (1951) Synthesis of nucleoprotein in bean root cells, Nature 167: 599-600.

[32.] Kihlman BA (1977) Caffeine and Chromosomes, Elsevier, Amsterdam pp: 504.

[33.] Amer SM, Fahmy MA, Aly FA, Farghaly AA (2002) Cytogenetic studies on the effect of feeding mice with stored wheat grains treated with malathion. Mutat Res 513: 1-10.

[34.] Patlolla AK, Tchounwou P (2005) Cytogenetic evaluation of arsenic trioxide toxicity in Sprague- Dawley rats. Mutat Res 587: 126-133.

[35.] Suzuki H, Ikeda T, Yamagishi T, Nakaike S, Nakane S, et al. (1995) Efficient induction of chromosome-type aberrations by topoisomerase II inhibitors closely associated with stabilization of the cleavable complex in cultured fibroblastic cells. Mutat Res 328: 151-161.

[36.] Yadav KK, Trivedi SP (2009) Chromosomal aberrations in a fish, Channa punctata after in vivo exposure to three heavy metals. Mutat Res/Genet Toxicol Environ Mutagen 678: 7-12.

[37.] Ansari RA, Rahman S, Kaur M, Anjum S, Raisuddin S (2011) In vivo cytogenetic and oxidative stress-inducing effects of cypermethrin in freshwater fish, Channa punctata Bloch. Ecotoxicol Environ Saf 74: 150-156.

[38.] Cucchi C, Baruffaldi A (1990) A new method for karyological studies in teleost fish. J Fish Biol 37: 71-75.

[39.] Kligerman AD, Bloom SE, Howell WM (1975) Umbra limi: a model for the study of the chromosomal aberrations in fishes. Mutat Res 31: 225-33.

[40.] Ribas G, Surrales J, Carbonell E, Xamena N, Creus A, et al. (1996) Genotoxicity of the herbicides alachlor and maleic hydrazide in cultured human lymphocytes. Mutagenesis 11: 221-227.

[41.] Benford DJ, Price SC, Lawrence JN, Grasso P, Bremmer JN (1994). Investigations of the genotoxicity and cell proliferative activity of dichlorvos in mouse forestomach. Toxicol 92: 203-215.

[42.] Espina NG, Wesis P (1995) DNA repair in fish from polluted estuaries. Mar Environ Res 39: 309-312.

[43.] Hose JE, Brown ED (1998) Field applications of the piscine anaphase aberration test: lessons from the Exxon Valdez oil spill. Mutat Res 399: 167-178.

[44.] Gartiser S, Stiene G, Hartmann A, Zipperle J (2001) Einsatz von Desinfektionsmitteln im Krankenhausbereich Ursache fur okotoxische and gentoxische Effekte im Krankakenhausabwasser? (German) Vom Wasser 96: 71-88.

[45.] Cavas T, Konen S (2007) Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay. Mutagenesis 22: 263-268.

[46.] Saxena KK, Chaudhari R (2010) Study of chromosomal abnormalities in C. punctatus expose to fenvalerate. J App Nat Sci 2: 70-73.

[47.] Singh SK, Singh SK, Yadav RP (2010) Toxicological and Biochemical Alterations of Cypermethrin (Synthetic Pyrethroids) Against Freshwater Teleost Fish Colisa fasciatus at Different Season. World J Zool 5: 25-32.

[48.] Hooftman RN (1981) The induction of chromosomal aberrations in Notobranchus rechowi (Pises: Cyprinodontidae) after treatment with ethylmethane sulphonate or benzo (a) pyrene. Mutat Res 91: 347-352.

[49.] Krishnaja AP, Rege MS (1982) Induction of chromosomal aberrations in fish Boleophthalmus dussumieri after exposure in vivo to Mitomycin-C and heavy metals mercury, selenium and chromium. Mutat Res 102: 71-82.

[50.] Manna GK, Mukherjee PK (1986) Effect of organophosphate insecticide melathion on chromosome, cell division and total muscle proteins of the cichlid fish Tilapia. In Pers Cytol and Genet 5: 225-235.

[51.] Gadhia PK, Dholakia, AH, Gadhia M (1990) Cadmium nitrate induced chromosomal aberrations in a common carp Cyrprinus carpio. Aquacultura Hungarica 6: 19-23.

[52.] Odeigah C, Osaneyinpeju O (1995) Genotoxic effects of two industrial effluents and ethylmethane sulfonate in Clarius lazera. Food Chem Toxicol 33: 501-505.

[53.] Ali N, Ahmad W (1998) Effect of Pentachlorophenol on chromosomes of a catfish Heteropneustus fossilis. Indian J Expt Biol 36: 304-307.

[54.] Arockia R, Selvanayagam M (1998) Genotoxic effects of fenvalerate on the chromosomes of fish Oreochromis mossambicus (peters). Poll Res 17: 119 122.

[55.] Mathew ANP, Jahageerdar S (1999) Effect of heavy metal on the karyotype of Channa punctatus. Indian J Fish 46: 167-172.

[56.] Das P, John G (1999) Induction of sister chromatid exchanges and chromosome aberrations invivo in Etroplus suratensis following exposure to organophosphorus pesticides. Toxicol Lett 104: 111-116.

[57.] Mahrous K, Abdou H (2001) Cytogenetical studies on some Nile fish. J Egypt Ger Soc Zool Histology Histochemistry & Genetics 36: 133-144.

[58.] Guha B, Khuda-Bukhsh AR (2002) Efficacy of vitamin-C (l-ascorbic acid) in reducing genotoxicity in fish (Oreochromis mossambicus) induced by ethyl methane sulphonate. Chemosphere 47: 49-56.

[59.] Saxena KK, Rana R (2005) Genotoxic effect of cypermethrin in a fresh water fish Channa punctatus. Proceedings of International Transfer of Technology Initiative Workshop on Parasitology and Genotoxicity in Sustainable Aquaculture, Allahabad pp: 26.

[60.] Velmurugan B, Ambrose T, Selvanayagam M (2006) Genotoxic evaluation of lambda-cyhalothrin in Mystus gulio. Journal of Environmental Biology 27: 247-250.

[61.] Abumourad IM, El- Sherbeny KM, Farghaly AA (2006) Detection of DNA damage of Clarias gariepinus exposed to 2,4 -D using chromosomal aberrations and RAPD assays. Bulletin of Agriculture, Cairo University 57: 658-700.

[62.] Gadhiai PK, Gadhia M, Georje S, Vinod KR, Pithawala M (2008) Induction of chromosomal aberrations in mitotic chromosomes of fish Boleophthalmus dussumieri after exposure in vivo to antineoplastics Bleomycin, Mitomycin-C and Doxorubicin. Indian Journal of Science and Technology 1: 1-6.

[63.] Rita AJJ, Milton JMC (2008) Karyomorphological analysis of the fresh water cichlid Oreochromis mossambicus (Peters) exposed to carbamate pesticide methomyl (Lannate). J Adv Zool 29: 57-61.

[64.] Mohamed MM, El-fiky SA, Soheir YM, Abeer AI (2008) Cytogenetic studies on the effect of copper sulfate and lead acetate pollution on Oreochromis niloticus fish. Journal of cell Biology 3: 51-60.

[65.] Malik MK, Parmesh K, Reenu S, Rishi S (2009) Genotoxic effect of paper mill effluent on chromosomes of fish Channa punctatus. Current World Environment 4: 353-357.

[66.] Tripathi N, Bajpai S, Tripathi M (2009) Genotoxic alterations induced by fluoride in asian catfish, clarias batrachus (linn.) Research report Fluoride 42: 292-296.

[67.] Mahmoud A, Mohamed Z, Ghada Y, Khaled S (2010) Cytogenetical studies on some River Nile species from polluted and nonpolluted Aquatic habitats. Egypt Acad J biolog Sci 2: 1-8.

[68.] Yadav AS, Bhatnagar A, Kaur M, (2010) Assessment of Genotoxic Effects of Butachlor in Fresh Water Fish, Cirrhinus mrigala (Hamilton). Research Journal of Environmental Toxicology 4: 223-230.

[69.] Yadav A, Bhatnagar A, Kaur M (2013) Aberrations in the Chromosomes of Cirrhinus mrigala (Hamilton) upon Exposure to Butachlor. Iranian Journal of Toxicology 7: 858-865.

[70.] Kaur H, Kalotra R, Walia GK, Handa D (2013) Genotoxic effects of dyeing industry effluent on a freshwater fish, Cirrhinus mrigala by chromosomal aberration test. International Journal of Pharmacy and Biological Sciences 3: 423-431.

[71.] Dar SA, Yousuf AR, Balkhi MH, Ganai FA, Bhat FA (2014b) Investigation of the genotoxicity of endosulfan to freshwater cyprinid fish crucian carp (Carassius carassius L.) using the micronucleus and chromosomal aberration as biomarkers. Nucleus 57: 87-98.

[72.] Kirsch-Volders M, Sofuni T, Aardema M, Albertini S, Fenech M, et al. (2003) Report from the in vitro micronucleus assay working group. Mutat Res 540: 153-163.

[73.] Fenech M (2010) The lymphocyte cytokinesis-block micronucleus cytome assay and its application in radiation biodosimetry. Health Phys 98: 234-243.

[74.] Salvadori D, Ribeiro L, Fenech M (2003) Micronuclei test in human cells in vitro, In: Environmental Mutagenesis (Ribeiro L, Salvadori D, Marques E., Eds). Canoas: ULBRA pp: 201-223.

[75.] Park E, Lee J, Etoh H (1993) Fish Cell line (ULF-23HU). Derived from the Fin of the Central Mudminnow (Umbra limi): Suitable Characteristics for Clastogenicity Assay. In Vitro Cell Dev Biol 25: 987-994.

[76.] Manna GK, Banerje G, Gupta SM (1985) Micronuclei test in the peripheral erythrocytes of the exotic fish Oreochromis mossambicus. The Nucleus 28: 176-179.

[77.] Das RK, Nanda NK (1986) Induction of micronuclei in peripheral erythrocytes of fish H. fossilis by mitomycin C and paper mill effluent. Mutation Research 175: 67-76.

[78.] Barat A, Sahoo PK, Nagpure NS, Ponniah AG (1997) Micronuclei test in peripheral blood in air breathing fish Channa punctatus treated with Malathion. In; Proc. National Symposium on Fish Genetics and Biodiversity Conservation for sustainable production (eds. Ponniah AG, Das P, Verma SR) NATCON Pub 5: 415-417.

[79.] Kushwaha B, Srivastava SK, Singh B, Nagpure NS, Ponniah AG (2000) Evaluation of comet assay and micronuclei test as genotoxicity assays in Channa punctatus. Nat Acad Sci Letters 23: 177-182.

[80.] Ateeq B, Farah MA, Ali MN, Ahmad W (2002) Induction of micronuclei and erythrocyte alterations in the catfish Clarias batrachus by 2,4-dichlorophenoxyacetic acid and butachlor. Mutat Res 518: 135-144.

[81.] Ahmad W, Ali MN, Farah MA, Ateeq B (2002) Computerized automated morphometric assay including frequencyestimation of pentachlorophenol induced nuclear anomalies (micronucleus) in catfish Heteropneustes fossilis. Chromosoma 110: 570-574.

[82.] Farah MA, Ateeq B, Ali MN, Ahmad W (2002) Evaluation of genotoxicity of PCP and 2,4-D by micronucleus test in freshwater fish Channa punctatus. Ecotoxicol Environ Saf 54: 25-29.

[83.] Al-Sabti K (1986) Comparative micronucleated erythrocyte cell induction in three cyprinids by five carcinogenic-mutagenic chemicals. Cytobios 47: 147-154.

[84.] Al-Sabti K (1992) Micronuclei induction in pike (Esox lucius) in Swedish lakes contaminated with radiocesium. Cytobios 70: 27-32.

[85.] De Flora S, Vigano L, D'Agostini F, Camoirano A, Bagnasco M, et al. (1993) Multiple genotoxicity biomarkers in fish exposed in situ to polluted river water. Mutat Res 319: 167-177.

[86.] Al-Sabti K (1994) Micronuclei induced by selenium, mercury, methylmercury and their mixtures in binucleated blocked fish erythrocyte cell. Mutat Res 320: 157-163.

[87.] Belpaeme K, Delbeke K, Zhu L, Kirsch-Volders M (1996) Cytogenetic Studies of PCB77 on Brown Trout (Salmo trutta fario) Using the Micronucleus Test and the Alkaline Comet Assay. Mutagenesis 11: 485-492.

[88.] Marlasca MJ, Sanpera C, Riva MC, Sala R, Crespo S (1998) Hepatic alterations and induction of micronuclei in rainbow trout (Oncorhynchus mykiss) exposed to a textile industry effluent. Histol Histopathol 13: 703-712.

[89.] Campana MA, Panzeri AM, Moreno VJ, Dulout FN (1999) Genotoxic evaluation of the pyrethroid lambda-cyhalothrin using the micronucleus test in erythrocytes of the fish Cheirodon interruptus interruptus. Mutation Research 438: 155-161.

[90.] Matsumoto FE, Colus IMS (2000) Micronucleus frequencies in Astyanax bimaculatus (Characidae) treated with cyclophosphamide or vinblastine sulfate. Micronucleu Genetics and Molecular Biology 23: 489-492.

[91.] Ayllon F, Garcia-Vaszquez E (2000) Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test. Mutat Res 467: 177-186.

[92.] Rodriguez-Cea A, Ayllon F, Garcia-Vazquez E (2003) Micronucleus test in freshwater fish species: An evaluation of its sensitivity for application in field surveys. Ecotoxicol Environ Safety 56: 442-448.

[93.] Buschini A, Martino A, Gustavino B, Monfrinotti M, Poli P, et al. (2004) Comet assay and micronuclei test in circulating erythrocytes of Cyprinus carpio specimens exposed in situ to lake waters treated with disinfectants of potabilization. Mutat Res 557: 119-129.

[94.] Cavas T, Garanko NN, Arkhipchuk VV (2005) Induction of micronuclei and binuclei in blood, gill and liver cells of fishes subchronically exposed to cadmium chloride and copper sulphate. Food and Chemical Toxicology 43: 569-574.

[95.] Grisolia C, Oliveira A, Bonfim H, Klautau-Guimaraes M (2005) Genotoxicity evaluation of domestic sewage in a municipal wastewater treatment plant. Genet Mol Biol 28: 334-338.

[96.] Bolognesi C, Perrone E, Roggieri B, Pampanin DM, Sciutto A (2006) Assessment of micronuclei induction in peripheral erythrocytes of fish exposed to xenobiotics under controlled conditions. Aquat Toxicol 78: 93-98.

[97.] Andreikenaite L, Barsiene J, Vosyliene M (2007) Studies of micronuclei and other nuclear abnormalities in blood of rainbow trout (Oncorhynchus mykiss) treated with heavy metal mixture and road maintenance salts. Acta Zoolog Lituanica 17: 213-219.

[98.] Ali F, El-Shehawi A, Seehy M (2008) Micronucleus test in fish genome: A sensitive monitor for aquatic pollution. Afr J Biotechnol 7: 606-612

[99.] Ali D, Nagpure NS, Kumar S, Kumar R, Kushwaha B, et al. (2009) Assessment of genotoxic and mutagenic effects of chlorpyrifos in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis. Food and Chemical Toxicology 47: 650-656.

[100.] Kumar R, Nagpure NS, Kushwaha B, Srivastava SK, Lakra WS (2010) Investigation of the Genotoxicity of Malathion to Freshwater Teleost Fish Channa punctatus (Bloch) Using the Micronucleus Test and Comet Assay. Arch Environ Contam Toxicol 58: 123-130.

[101.] Vera CJ, Soloneski S, Larramendy ML (2010) Genotoxic and cytotoxic effects of the formulated insecticide Aficida[R] on Cnesterodon decemmaculatus (Jenyns, 1842) (Pisces: Poeciliidae). Mutation Research 703: 180-186.

[102.] Polard T, Jean S, Gauthier L, Laplanche C, Merlina G, et al. (2011) Mutagenic impact on fish of runoff events in agricultural areas in south-west France. Aquatic Toxicology 101: 126-134.

[103.] Bucker A, Carvalho MS, Conceicao MB, Alves-gomes JA (2012) Micronucleus test and comet assay in erythrocytes of the Amazonian electric fish Apteronotus bonapartii exposed to benzene. J Braz Soc Ecotoxicol 7: 65-73.

[104.] Gadhave PD, Brar RS, Banga HS, Dhawan A (2014) A-cyhalothrin induced genotoxicity in freshwater fish, Labeo rohita. Veterinary World 7: 412-415.

[105.] Singh NP, McCoy MT, Tice RR, Schneider EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 75: 184-191.

[106.] Kienzler A, Tronchere X, Devaux A, Bony S (2012) Assessment of RTG-W1, RTL-W1, and PLHC-1 fish cell lines for genotoxicity testing of environmental pollutants by means of a Fpg-modified Comet Assay. Toxicol In Vitro 26: 500-510.

[107.] Palanikumar L, Kumaraguru AK, Ramakritinan CM (2013) Biochemical and genotoxic response of naphthalene to fingerlings of milkfish Chanos chanos. Ecotoxicology 22: 1111-1122.

[108.] Laroche J, Gauthier O, Quiniou L, Devaux A, Bony S, et al. (2013) Variation patterns in individual fish responses to chemical stress among estuaries, seasons and genders: the case of the European flounder (Platichthys flesus) in the Bay of Biscay. Environ Sci Pollut Res Int 20: 738-748.

[109.] Groff AA, daSilva J, Nunes EA, Ianistcki M, Guecheva TN, et al. (2010) UVA/ UVB-induced genotoxicity and lesion repair in Colossoma macropomum and Arapaima gigas Amazonian fish. J Photoche Photobiol Biol 99: 93-99.

[110.] Ciereszko A, Wolfe TD, Dabrowski K (2005) Analysis of DNA damage in sea lamprey (Petromyzon marinus) spermatozoa by UV, hydrogen peroxide, and the toxicant bisazir. Aquat Toxicol 73: 128-138.

[111.] Regoli F, Winston GW, Gorbi S, Frenzilli G, Nigro M, et al. (2003) Integrating enzymatic responses to organic chemical exposure with total oxyradical absorbing capacity and DNA damage in the European eel Anguilla anguilla: toward development of a more holistic biomarker assessment, Environ Toxicol Chem 22: 2120-2129.

[112.] Masuda S, Deguchi Y, Masuda Y, Watanabe T, Nukaya H, et al. (2004) Genotoxicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and 4-amino-3,30-dichloro-5,40dinitro-biphenyl (ADDB) in goldfish (Carassius auratus) using the micronucleus test and the comet assay. Mutat Res 560: 33-40.

[113.] Diekmann M, Waldmann P, Schnurstein A, Grummt T, Braunbeck T, et al. (2004) On the relevance of genotoxicity for fish populations II: genotoxic effects in zebrafish (Danio rerio) exposed to 4-nitroquinoline-1-oxide in a complete life-cycle test. Aquat Toxicol 68: 27-37.

[114.] Ateeq A, Farah MA, Ahmad W (2005) Detection of DNA damage by alkaline single cell gel electrophoresis in 2,4-dichlorophenoxyacetic-acid- and butachlor exposed erythrocytes of Ciarias batrachus. Ecotoxicol Environ Saf 62: 348-354.

[115.] Pandey S, Nagpure NS, Kumar R, Sharma S, Srivastava SK, et al. (2006) Genotoxicity evaluation of acute doses of endosulfan to freshwater teleost Channa punctatus (Bloch) by alkaline single-cell gel electrophoresis. Ecotoxicol Environ Saf 65: 56-61.

[116.] Kim IY, Hyun CK (2006) Comparative evaluation of the alkaline comet assay with the micronucleus test for genotoxicity monitoring using aquatic organisms. Ecotoxicol Environ Saf 64: 288-297.

[117.] Pellacani C, Buschini A, Furlini M, Poli P, Rossi C (2006) A battery of in vivo and in vitro tests useful for genotoxic pollutant detection in surface waters. Aquat Toxicol 77: 1-10.

[118.] Del Barga, Frenzilli G, Scarcelli V, Nigro M, Malmvarn A, et al. (2006) DNA strand breaks induced by algal extracts (Poiysiphonia fucoides) in rainbow trout. Mar Environ Res 2: 283-286.

[119.] Inzunza B, Orrego R, Penalosa M, Gavilan JF, Barra R (2006) Analysis of CYP4501A1, PAHs metabolites in bile, and genotoxic damage in Oncorhynchus mykiss exposed to Biobio River sediments, Central Chile. Ecotoxicol Environ Saf 65: 242-251.

[120.] Deguchi Y, Toyoizumi T, Masuda S, Yasuhara A, Mohri S, et al. (2007) Evaluation of mutagenic activities of leachates in landfill sites by micronucleus test and comet assay using goldfish. Mutat Res 627: 178-185.

[121.] Cavas T, Konen S (2007) Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay. Mutagenesis 22: 263-268.

[122.] Vanzella TP, Martinez CBR, Colus IMS (2007) Genotoxic and mutagenic effects of diesel oil water soluble fraction on a neotropical fish species. Mutat Res 631: 36-43.

[123.] Kosmehl T, Hallare AV, Braunbeck T, Hollert H (2008) DNA damage induced by genotoxicants in zebrafish (Danio rerio) embryos after contact exposure to freeze-dried sediment and sediment extracts from Laguna Lake (The Philippines) as measured by the comet assay. Mutat Res 650: 1-14.

[124.] Grisolia CK, Rivero CG, Starling F, Silva IC, Barbosa AC, et al. (2009) Profile of micronucleus frequencies and DNA damage in different species of fish in a eutrophic tropical lake. Genet Mol Biol 32: 138-143.

[125.] Perez-Cerezales S, Martinez-Paramo S, Beirao J, Herraez MP (2010) Fertilization capacity with rainbow trout DNA-damaged sperm and embryo developmental success. Reproduction 139: 989-997.

[126.] Mohanty G, Mohanty J, Nayak AK, Mohanty S, Dutta SK (2011) Application of comet assay in the study of DNA damage and recovery in rohu (Labeo rohita) fingerlings after an exposure to phorate, an organophosphate pesticide. Ecotoxicology 20: 283-292.

[127.] Pandey AK, Nagpure NS, Trivedi SP, Kumar R, Kushwaha B (2011) Profenofos induced DNA damage in freshwater fish, Channa punctatus (Bloch) using alkaline single cell gel electrophoresis. Mutation Research 726: 209- 214.

[128.] Guilherme S, Gaivao I, Santos MA, Pacheco M (2012) DNA damage in fish (Anguilla anguilla) exposed to a glyphosate-based herbicide--elucidation on the organ-specificity and the role of oxidative stress. Mutat Res 743: 1-9.

[129.] Ternjej I, Gaurina Srcek V, Mihaljevic Z, Kopjar N (2013) Cytotoxic and genotoxic effects of water and sediment samples from gypsum mining area in channel catfish ovary (CCO) cells. Ecotoxicol Environ Saf 98: 119-127.

[130.] Taju G, Abdul Majeed S, Nambi KS, Sahul Hameed AS (2014) In vitro assay for the toxicity of silver nanoparticles using heart and gill cell lines of Catla catla and gill cell line of Labeo rohita. Comp Biochem Physiol CToxicol Pharmacol 161: 41-52.

[131.] OSPAR Commission (2002) Survey on genotoxicity test methds for the evaluation of waste water within whole effluent assessment. London.

[132.] Amanuma K, Takeda H, Amanuma H, Aoki Y (2000) Transgenic zebra fish for detecting mutations caused by compounds in aquatic environments. Natl Biotechnol 18: 62-65.

[133.] Winn RN, Norris MB, Brayer KJ, Torres C, Muller SL (2000) Detection of mutations in transgenic fish carrying a bacteriophage lambda CII transgene. Proc Natl Acad Sci 97: 12655-12660.

[134.] Bhaskaran A, May D, Rand-Weaver M, Tyler CR (2000) Molecular characterization of the first non- mammalian p73 cDNA. Comp Biochem Physiol B Biochem Mol Biol 126: 49-57.

[135.] Ahmad I, Maria VL, Oliveira M, Pacheco M, Santos MA (2006) Oxidative stress and genotoxic effects in gill and kidney of Anguilla anguilla L. exposed to chromium with or without pre-exposure to b-naphthoflavone. Mutat Res 608: 16-28.

[136.] Mouchet F, Gauthier L, Mailhes C, Ferrier V, Devaux A (2005) Comparative study of the Comet Assay and the micronucleus test in amphibian larvae (Xenopus laevis) using benzo(a)pyrene, ethyl methanesulfonate, and methyl methanesulfonate: establishment of a positive control in the amphibian Comet Assay. Environ Toxicol 20: 74-84.

[137.] Mouchet F, Gauthier L, Mailhes C, Ferrier V, Devaux A (2006) Comparative evaluation of genotoxicity of captan in amphibian larvae (Xenopus laevis and Pleurodeles waltl) using the Comet Assay and the micronucleus test. Environ Toxicol 21: 264-277.

[138.] Abdel-Daim MM, Abdelkhalek NK, Hassan AM (2015) Antagonistic activity of dietary allicin against deltamethrin-induced oxidative damage in freshwater Nile tilapia; Oreochromis niloticus. Ecotoxicol Environ Saf 111: 146-152.

[139.] Abdelkhalek NK, Ghazy EW, Abdel-Daim MM (2015) Pharmacodynamic interaction of Spirulina platensis and deltamethrin in freshwater fish Nile tilapia, Oreochromis niloticus: impact on lipid peroxidation and oxidative stress. Environ Sci Pollut Res Int 22: 3023-3031.

[140.] Dar SA (2015) Mutagenic Effects of Endosulfan in Crucian Carp (Carassius carassius L.) and the Role of Podophyllum hexandrum Royle as an Antimutagen. PhD thesis, University of Kashmir, Srinagar, India.

[141.] Dar SA, Yousuf AR, Balkhi MH, Ali MN, Bhat FA (2014) In-vivo cytogenetic damage in freshwater cyprinid crucian carp (Carassius carassius L.) upon endosulfan exposure. Journal of Research and Development 14: 47-56.

[142.] Dashwood R, Negishi T, Hayatsu H, Breinholt V, Hendricks J, et al. (1998) Chemopreventive properties of chlorophylls towards aflatoxin B1: a review of the antimutagenicity and anticarcinogenicity data in rainbow trout. Mutat Res 399: 245-253.

[143.] Farah MA, Ateeq B, Ahmad W (2006) Antimutagenic effect of neem leaves extract in freshwater fish, Channa punctatus evaluated by cytogenetic tests. The Science of the Total Environment 364: 200-214.

[144.] Mustafa SA, Al-Subiai SN, Davies SJ, Jha AN (2011) Hypoxia-induced oxidative DNA damage links with higher level biological effects including specific growth rate in common carp, Cyprinus carpio L. Ecotoxicology 20: 1455-1466.

[145.] Aniagu SO, Day N, Chipman JK, Taylor EW, Butler PJ, et al. (2006) Does exhaustive exercise result in oxidative stress and associated DNA damage in the chub (Leuciscus cephalus)? Environ Mol Mutagen 47: 616-623.

[146.] Devaux A, Fiat L, Gillet C, Bony S (2011) Reproduction impairment following paternal genotoxin exposure in brown trout (Salmo trutta) and Arctic charr (Salvelinus alpinus). Aquat Toxicol 101: 405-411.

[147.] Fabender C, Braunbeck T (2013) Reproductive and genotoxic effects in zebrafish after chronic exposure to methyl methanesulfonate in a multigeneration study. Ecotoxicology 22: 825-837.

[148.] Kumar R, Pandey S, Sharma S (2005) Study of sister chromatid exchanges for assessment of genotoxicity. In: Kapour D, Nagpour NS (eds), Training on genotoxic assays in fishes. National Bureau of Genetic Resources, Dilkusha, Telibagh, India pp: 68.

Citation: Dar SA, Yousuf AR, Balkhi MH (2016) An Introduction about Genotoxicology Methods as Tools for Monitoring Aquatic Ecosystem: Present Status and Future Perspectives. Fish Aquac J 7: 158. doi:10.4172/21503508.1000158

Sabzar Ahmad Dar (1) *, Abdul Rehman Yousuf (1) and Masood-ul-Hassan Balkhi (2)

(1) Limnology and Fisheries Laboratory, Centre of Research for Development (CORD), University of Kashmir, Srinagar, Jammu and Kashmir, India

(2) Division of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), Jammu and Kashmir, India

* Corresponding author: Sabzar Ahmad Dar, Limnology and Fisheries Laboratory, Centre of Research for Development (CORD), University of Kashmir, Srinagar, Jammu and Kashmir, India, Tel: 91-09797970976; E-mail: sabzar.cord@gmail.com

Received December 30, 2015; Accepted February 10, 2016; Published February 17, 2016
Table 1: Summary of some landmark genotoxicity studies in fish
using chromosomal aberration test: a review.

Fish                Chemical(s)/pollutants               Reference

Notobranchus        Ethylmethane-sulphonate and            [48]
rechowi             benzo(a)pyrene

Boleophthalmus      Mitomycin-C, mercury, selenium         [49]
dussumieri          and chromium

Oreochromis         Malathion                              [50]
mossambicus

Cyprinus carpio     Cadmium nitrate                        [51]

Clarius lazera      Ethylmethane sulfonate                 [52]

Channa punctatus    Dichlorvos                             [25]

Heteropneustus      Pentachlorophenol                      [53]
fossilis

Oreochrochromis     Fenvalerate                            [54]
mossambicus

Channa              Heavy metals                           [55]
punctatus

Etroplus            Methyl parathion and Phosphamidon      [56]
suratensis

Oreochromis         Agricultural and industrial            [57]
niloticus and       waste water
Clarias lazera

Oreochromis         Ethylmethane sulfonate                 [58]
mossambicus

Channa              Cypermethrin                           [59]
punctatus

Mystus gulio        Lambda-cyhalothrin                     [60]

Clarias             2,4-dichlorophenoxyacetic acid         [61]
gariepinus

Boleophthalmus      Bleomycin, Mitomycin-C and             [62]
dussumieri          Doxorubicin

Oreochromis         Carbamate pesticide methomyl           [63]
mossambicus

Oreochromis         Copper sulphate and lead acetate       [64]
niloticus

Channa              Paper mill effluent                    [65]
punctatus

Clarias             Fluoride                               [66]
batrachus

Oreochromis         Sewage discharge                       [67]
niloticus and
Tilapia zillii

Channa              Fenvalerate                            [46]
punctatus

Cirrhinus           Butachlor                              [68]
mrigala

Channa              Cypermethrin                           [37]
punctatus

Cirrhinus           Butachlor                              [69]
mrigala

Cirrhinus           Dyeing industry effluent               [70]
mrigala

Carassius           Endosulfan                            [12,71]
carassius

Table 2: Assessment of genetic damage by micronucleus assay in fish
after in vivo exposure to genotoxicants.

Fish                    Chemical(s)/pollutants       Reference

Oreochromis             Aldrin, cadmium chloride       [76]
mossambicus             and x-rays

Cyprinus carpio and     Aflatoxin B1, arochlor         [83]
Tinca tinca             1254, benzidene,
                        benzo(a)pyrene and 20-
                        methylechloanthrene

Heteropneustes          Mitocycin C and paper          [77]
fossilis                mill effluent:
                        allylformate

Esox lucius             Radiocesium                    [84]

Oncorhynchus mykiss     In situ to heavily             [85]
                        polluted tributary of
                        the River Po (Northern
                        Italy)

Carassius auratus       Selenium, mercury and          [86]
gibelio                 methyl-mercury

Salmo truttafario       PCB77                          [87]

Channa punctatus        Malathion                      [78]

Oncorhynchus mykiss     A textile industry             [88]
                        effluent

Cheirodon interruptus   Pyrethroid A-                  [89]
interruptus             cyhalothrin

Astyanax bimaculatus    Cyclophosphamide,              [90]
                        vinblastine sulfate

Channa punctatus        Malathion                      [79]

Clarias batrachus       2,4-dichlorophenoxyacetic      [80]
                        acid and butachlor

Heteropneustes          Pentachlorophenol              [81]
fossilis

Channa punctatus        Pentachlorophenol and          [82]
                        2,4-dichlorophenoxyacetic
                        acid

Anguilla Anguilla,      Metals, hydrocarbons,          [92]
Phoxinus phoxinus and   pesticides
Salmo trutta

Cyprinus carpio         Disinfectants (sodium          [93]
                        hypochlorite, peracetic
                        acid and chloride dioxide)

Cyprinus carpio,        Cadmium chloride and           [94]
Carassius gibelio,      copper sulphate
Corydoras paleatus

Oreochromis niloticus   Domestic sewage                [95]
and Tilapia rendalli

Scophthalmus maximus    Dialkyl phthalate,             [96]
                        bisphenol-A,
                        tetrabromodiphenyl ether

Oncorhynchus mykiss     Mixture of heavy metals        [97]

Clarias gariepinus,     Heavy metals                   [98]
Oreochromis niloticus
and Oreochromis
aureus

Channa punctatus        Chlorpyrifos                   [99]

Channa punctatus        Malathion                      [100]

Cnesterodon             Aficida[R] (insecticide)       [101]
decemmaculatus

Carassius carassius     Agricultural runoff            [102]

Apteronotus             Benzene                        [103]
bonapartii

Labeo rohita            A-cyhalothrin                  [104]

Carassius               Endosulfan                    [12,71]
carassius

Table 3: Genotoxicity studies in fish using the comet assay
published during the last twelve years.

Fish                  Cell type            Agent           Exposure

Anguilla            Erythrocytes           B(a)P
Anguilla                                    b-BF
                                       Arochlor 1254
                                            TCDD

Carassius           Erythorocytes       PBTA-6 (dye)         3-9 h
auratus

Danio              Hepatocytes and   4-nitroquinoline-
rerio                gill cells           1-oxide

Clarias             Erythrocytes        Herbicides:         48-96 h
batrachus                                  2,4-D
                                         Butachlor

Channa                Gill and           Endosulfan         24-96 h
punctatus          kidney tissues       (pesticide)

Cyprinus           Gill and liver           MNNG            7 days
carpio                  cells

C. carpio           Erythrocytes     Lake water (Italy)     20 days

Oncorhynchus        Erythrocytes       Algal extracts       7 days
mykiss                                 (P. fucoides)

O. mykissO          Erythrocytes       Sediments from       21 days
                                        Biobio River
                                          (Chile)

C. auratus          Erythrocytes       Leachate from         96 h
                                       landfill sites
                                          (Japan)

C. auratus          Erythrocytes         Glyphosate        2-6 days

Plotosus            Erythrocytes        Diesel water        6-96 h;
lineatus                              soluble fraction      15 days
                                           (DWSF)

D. rerio               Freshly         Sediments fiom         3 h
                   fertilized eggs    Laguna lake (the
                                        Philippines)

Geophagus           Erythrocytes       Eutrophication     Field study
brasiliensis,
Cichla
temensis,
Hoplias
malabaricus,
C. carpio and
Steindachnerina
insculpita

O. mykiss            Spermatozoa      Cryopreservation

Labeo rohita        Erythrocytes,         Phorate           24-96 h
                   liver and gill
                        cells

C. punctatus         Gill cells          Profenofos         24-96 h

A. anguilla        Blood and liver    Glyphosate-based      24-72 h
                                         herbicide

Ictalurus          Ovary cell line   Water and sediment   Field study
punctatus                            from gypsum mining
                                            area

C. catla, L.       Heart and gill          Silver            96 h
rohita               cell lines        nanoparticles

Fish                  Concentration      Response     Reference

Anguilla              0.1-50 mg/kg           +          [111]
Anguilla              0.1-50 mg/kg
                      0.1-50 mg/kg
                      0.01-2 mg/kg

Carassius              1-100 mg/kg           +          [112]
auratus

Danio               0, 0.1,0.3, 1.1,         +          [113]
rerio              and 2.9 [micro]g/l

Clarias                 25-75 ppm           C-R         [114]
batrachus               1-2.5 ppm

Channa                  4-10 ppb            C-R         [115]
punctatus

Cyprinus            0.01-1 ppm (mg/l)        +          [116]
carpio

C. carpio          Seasonal difference       +          [117]

Oncorhynchus              0.5%               +          [118]
mykiss

O. mykissO                PAHs               +          [119]
                       (2000-7000
                       ng/g d.w.)

C. auratus                                   +          [120]

C. auratus              5-15 ppm            D-R         [121]

Plotosus           Diesel oil added to
lineatus             4 parts water.
                   Water phase diluted                  [122]
                     to 50% WSF with
                   dechlorinated water

                    430 mg/ml (100%)
D. rerio            (1:1); 215 mg/ml         +          [123]
                   (50%); 107.5 mg/ml
                          (25%)

Geophagus                                    +          [124]
brasiliensis,
Cichla
temensis,
Hoplias
malabaricus,
C. carpio and
Steindachnerina
insculpita

O. mykiss                                    +          [125]

Labeo rohita         0.001,0.002 and        C-R         [126]
                        0.01 ppm

C. punctatus         0.58, 1.16 and         C-R         [127]
                        1.74 ppb,

A. anguilla           58 and 116 g           +          [128]
                       [l.sup.-1]

Ictalurus                                    +          [129]
punctatus

C. catla, L.          Environmental          +          [130]
rohita                  realistic
                      concentration

(+) = DNA damage; (-) = No DNA damage;
(C-R) = Concentration dependent response
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Title Annotation:Research Article: Open Access
Author:Dar, Sabzar Ahmad; Yousuf, Abdul Rehman; Balkhi, Masood-ul-Hassan
Publication:Fisheries and Aquaculture Journal
Article Type:Report
Date:Jan 1, 2016
Words:10871
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