Fish micronucleus assay to assess genotoxic potential of arsenic at its guideline exposure in aquatic environment Amod Kumar, Vibudh

P.Kesari & Parimal K.Khan

BioMetals An International Journal on the Role of Metal Ions in Biology, Biochemistry and Medicine ISSN 0966-0844 Volume 26 Number 2 Biometals (2013) 26:337-346 DOI 10.1007/s10534-013-9620-8

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Biometals (2013) 26:337346 DOI 10.1007/s10534-013-9620-8

Fish micronucleus assay to assess genotoxic potential of arsenic at its guideline exposure in aquatic environment
Amod Kumar Vibudh P. Kesari Parimal K. Khan

Received: 12 November 2012 / Accepted: 7 March 2013 / Published online: 14 March 2013 Springer Science+Business Media New York 2013

Abstract The exposure to arsenic, a potential genotoxic carcinogen in humans, via drinking water is a serious worldwide health hazard. The arsenic content of 10 lg L-1 in drinking water, however, has been established as its guideline standard (maximum contaminant limit) that has been estimated to pose minimum risk to cancer. Since micronucleus induction in the erythrocytes of sh is a sensitive indicator of genotoxic agents in water, the piscine micronucleus assay was used in the present experiment to assess the genotoxic potential of arsenic at its various exposure levels including the guideline value for drinking water. The experiments were conducted in two different species of shes, the pond murrel (Channa punctatus) and the goldsh (Carassius auratus). Signicant increases in the frequency of micronucleated erythrocytes were documented in a dose-dependent manner in both Channa and Carassius. The shes, however, exhibited variations in inter-specic sensitivity to micronucleus induction following arsenic exposure. The exposure level of arsenic at its guideline value for

drinking water, therefore, exhibited marked genotoxicity in shes. Keywords Arsenic Genotoxicity Fish Guideline value Micronucleus

Introduction An increasing public health hazard of global concern is related to arsenic in drinking water (Ng et al. 2003; Nordstrom 2002; NRC 2001). Arsenic is a toxic metalloid (ATSDR 2007) and an established human carcinogen (IARC 2004). It is ubiquitously present in our living environment owing to its release mainly from geochemical sources (as occurs in more than 245 minerals) as well as from a variety of anthropogenic activities (Bissen and Frimmel 2003; Florea et al. 2004). The average level of arsenic in groundwater is typically 12 lg L-1, but its level well in excess of 1,000 lg L-1 has been measured at many places in several countries including India (Chakraborti et al. 2009; Kar et al. 2010; Saha 2009; Smith and Smith 2004; van Halem et al. 2009). Consequently, drinking water with elevated concentration of arsenic is the predominant source of arsenic exposure in humans, affecting about 130 million people worldwide (Lubin et al. 2007; WHO 2008). Arsenic accounts for multisystem adverse health effects in humans, leading to pleiotropic injury including cancers of skin, lung, liver, kidney and

A. Kumar V. P. Kesari P. K. Khan (&) Toxicogenetics Laboratory, Department of Zoology, Patna University, Patna 800 005, India e-mail: parimal_khan@yahoo.co.in Present Address: A. Kumar Department of Zoology, K. M. College, University of Delhi, New Delhi, India

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urinary bladder (Abernathy et al. 2003; Ng 2005; Rahman et al. 2009). A serious concern of arsenic exposure is its marked genotoxic effect in humans as well as in animals (Basu et al. 2001; Gebel 2001). Exposure to arsenicals causes increased frequency of micronucleus-induction (Basu et al. 2002, 2004; Chakraborty et al. 2006; Ghosh et al. 2006; Khan et al. 2013; Lewinska et al. 2007; Tian et al. 2001; Vuyyuri et al. 2006; Yadav and Trivedi 2009a), chromosomal aberrations and DNA strand breaks (Biswas et al. 2006; Dopp et al. 2004; Kesari et al. 2012; Lee et al. 2007; Mahata et al. 2003; Palus et al. 2005; Yadav and Trivedi 2009b) in various in vivo and in vitro test systems. The World Health Organization (Sobsey and Bartram 2003; WHO 2008) has formulated guidelines to ensure the quality of drinking water, and established a provisional guideline standard of arsenic (10 lg L-1) for drinking water quality (GDWQ) that has been estimated to pose a minimum risk to cancer based on human cancer data and cost benet balance. The United States Environmental Protection Agency (USEPA 2001) has also recommended a maximum contaminant limit (MCL) of 10 lg L-1 of arsenic for US drinking water. In contrast, Walker and Fosbury (2009) have shown recently that there is an increased risk of toxicity even at the low exposure level of the guideline value of arsenic (10 lg L-1) in drinking water. The potential of arsenic at its guideline value, however, has not been evaluated to ascertain any discernible genotoxic effect. Fishes can act as sentinel organisms for indicating the potential for exposure of human populations to genotoxic chemicals in drinking water (Al-Sabti and Metcalfe 1995). They have been shown to respond in a manner similar to mammals in genotoxicity assessment (Jha 2004), and hence, are frequently used to assess the potential risk associated with contaminants in aquatic environment (Lakra and Nagpure 2009). Micronucleus-induction among the multiple genotoxicity biomarkers, is a reliable indicator of chromosomal breakage and damage (Fenech 2002; Heddle et al. 1991). Accordingly, micronucleus formation in piscine erythrocytes is one of the most sensitive indicators of genotoxic agents in water (Bolognesi et al. 2006; Ergene et al. 2007; Hayashi et al. 1998; Udroiu 2006), and is perhaps the best biomarker to assess arsenic exposure (Marchiset-Ferlay et al. 2012). Freshwater shes including Channa living in arsenic-

contaminated waters have shown to accumulate inorganic arsenic in their tissues (Jankong et al. 2007; Kar et al. 2011). Furthermore, shes exhibit elevated incidence of micronucleus induction upon exposure to high levels of arsenic (Ahmed et al. 2011; Ramirez and Garcia 2005; Yadav and Trivedi 2009a). Since the genotoxicity of arsenic has been documented particularly at its high exposure levels, the present work investigates the genotoxic potential of arsenic at its lower exposure levels, especially at the guideline exposure through water, compared to the magnitude of response at higher exposure levels using micronucleus induction in shes as a favoured cytogenetic biomarker.

Materials and methods Test organisms The present study was carried out on the two different species of shes, the red cap Oranda goldsh (Carassius auratus, Cypriniformes) and the common pond murrel (Channa punctatus, Ophiocephaliformes). The goldsh was chosen because of its proven sensitivity to genotoxic chemicals (Cavas 2011; Deguchi et al. 2007; Lourenco et al. 2010; Masuda et al. 2004). Channa is also an excellent test organism because of its several ecotoxicological characteristics including its sensitivity and resistance to arsenic toxicity (Allen and Rana 2004; Yadav and Trivedi 2009a, b). Healthy and active specimens of Channa punctatus and Carassius auratus, with an average body weight of 30 2 and 20 5 gm respectively, were procured from local sources (Patna, India). Fish were then acclimated to laboratory conditions for 2 weeks in glass aquaria containing water collected from a local lentic source with no detectable arsenic content. They were fed once a day with food pellets (with no detectable amount of arsenic) during the period of study. Test substance and exposure levels Arsenic trioxide (As2O3; CAS#: 1327-53-3 UN#: 1561; sd NE cHEM, Mumbai, India) was used as a source of arsenic exposure in the present experiment because of being the most widely used commercial compound of arsenic (ATSDR 2008). Since it is an

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amphoteric oxide which is moderately soluble in water and forms a weak acid but it can be dissolved readily in an alkaline solution where it gives arsenite oxyanions, the major form of the arsenic in water. The stock solution of arsenic trioxide (As2O3) was, therefore, prepared in 1 M NaOH. It was then added to aquaria water in estimated volumes to achieve four different levels of arsenic in water (Table 1) including its guideline value for drinking water (10 lg L-1). Specimens of Channa and Carassius were segregated into two different sets of various experimental groups including a negative control and a positive control (Tables 1, 2). The groups were exposed to different levels of arsenic in water for a period of 15 consecutive days to simulate the condition of a subchronic exposure. Since arsenic is a well-established genotoxic agent, the group exposed to highest arsenic level (1,000 lg L-1) was selected for positive control while the group exposed to aquaria water without arsenic addition was used as negative control, following the scheme of Ramirez and Garcia (2005). Each experimental group with six specimens, either of Channa or of Carassius, was maintained in a separate glass aquarium at a water temperature of 26 2 C with permanent aeration. Water of aquaria, under static renewal system, was changed on alternate day, and the respective concentration of arsenic, maintained by adding fresh stock solution, was analyzed by Atomic Absorption Spectrophotometer (Perkin Elmer, Analyst 200) following the standard method based on hydride generation (APHA 1998).
Table 1 Frequency prole of micronucleus (MN) formation in erythrocytes of sh, Channa punctatus, upon exposure to arsenic Experimental groups Exposure levels (lg L ) C T1 T2 T3 T4
a -1

Sampling of blood Fish were anaesthetized with benzocaine (0.1 g L-1) at the time of blood sampling and the samples of blood (*0.2 mL/sh) were drawn from caudal vein with the help of 1 mL tuberculin syringe, pre-rinsed with EDTA. Preparation of slides Thin smears of blood were made on clean slides by slide drawn method, air-dried for overnight followed by their xation in methanol. Slides were then stained by MayGruenwaldGiemsa staining technique and nally rinsed in running tap water to remove stain particles. Screening of slides A uniform criteria as suggested by Fenech et al. (2003) was adopted for scoring the micronuclei. Non-refractile structures, that were in the same focal plane as the main nucleus, resembled nuclei in colour and staining intensity and had diameter less than one-third of the main nucleus, were scored as micronuclei. Slides were coded and scored blind, and were analyzed by a light microscope using its oil immersion lens (1,0009). About 12,000 erythrocytes were screened in each experimental group of Channa punctatus for the detection of micronuclei. The number of erythrocytes examined in each experimental group of Carassius auratus, however, varied from 2,100 to 4,200. The micronucleated cells were scored for each sh, and the

Total cells scored

Total cells with MN No. % SE 0.86 0.08 1.41 0.10* 1.73 0.11* 2.87 0.15* 3.82 0.17*

Table 2 Frequency prole of micronucleus (MN) formation in the erythrocytes of goldsh, Carassius auratus, upon exposure to arsenic Experimental groups Exposure levels (lg L-1) C T1 T2 T3
a

Total cells scored

Total cells with MN No. % SE 2.37 0.23 5.37 0.42* 6.46 0.51* 8.52 0.60*

00 10a 50b 500c 1,000d

11,617 12,338 12,375 12,051 12,113

100 175 215 346 463

00 10a 50b 1,000c

4,209 2,811 2,291 2,100

100 151 148 179

* Signicant difference with control at p \ 0.05 (Z test) Guideline value (maximum contaminant limit) of arsenic for drinking water National standard of arsenic for drinking water in India High levels of arsenic content in groundwater

* Signicant difference with control at p \ 0.05 (Z test) Guideline value (maximum contaminant limit) of arsenic for drinking water National standard of arsenic for drinking water in India High level of arsenic content in groundwater

b c

c, d

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values so obtained were further pooled together in their respective experimental groups. The individual and pooled means of micronucleus frequency (MN%), to be used separately for statistical analysis, were calculated as follows: MN% Number of micronucleated cells 100 Total number of cells screened

Statistical evaluation The data of micronucleus frequency so obtained were evaluated statistically with the help of data analysis and graphing software developed by Origin Lab (Northampton, MA) after applying one-way analysis of variance (ANOVA) followed by an equality of proportion test (Z test), and the differences were deemed signicant at p \ 0.05. The difference in the response of two species of shes to arsenic (i.e. inter-specic difference in micronucleus induction), if any, was also analyzed by comparing the slopes of their regression lines.

micronucleatd cells in Carassius increased to 5.37 % (151 among 2,811 erythrocytes) in T1 group, 6.46 % (148 among 2,291 erythrocytes) in T2 group and 8.52 % (179 among 2,100 erythrocytes) in T3 group. A comparison of the slopes of regression lines (Fig. 1) exhibited a signicant difference in the response of two species of shes to arsenic. The cells of Carassius, compared to Channa, responded well to increasing concentrations of arsenic.

Discussion Catton (1951) extensively studied the blood cell formation in certain teleost shes. The nucleated erythrocytes of the circulating blood of shes arise from lymphoid haematoblasts in their haematopoitic organs, chiey the intertubular tissue of the kidney. The small lymphoid haematoblasts initially develop into proerythroblasts which then proliferate to form erythroblasts that frequently undergo mitosis and nally differentiate into reticulocytes (immature or polychromatic erythrocytes). Cells at the stage of reticulocytes are released into the peripheral circulation, where they subsequently mature into normochromatic erythrocytes. Although the duration of cell cycle and maturation time of erythrocytes has been well documented in mammalian system, little information pertinent to teleosts is available, partly because the cell cycle varies with temperature in these poikilotherms

Results Exposure to arsenic in sh was found to induce micronuclei in their erythrocytes. Signicant increases in micronucleated cells were observed in all the exposure levels including exposure level at the guideline value of arsenic for drinking water. Most of the affected erythrocytes were found to contain only one micronucleus in them, but some with two or more micronuclei were also encountered. The frequency of micronucleus induction increased with corresponding increase in the exposure level, suggesting a dosedependent increase in genotoxic indices. The control group of Channa (Table 1) exhibited only 100 micronucleated cells among 11,617 erythrocytes (0.86 %). Exposure to arsenic in Channa increased the frequency of micronucleated cells to 1.41 % (175 among 12,338 erythrocytes) in T1 group, 1.73 % (215 among 12,375 erythrocytes) in T2 group, 2.87 % (346 among 12,051 erythrocytes) in T3 group and 3.82 % (463 among 12,133 erythrocytes) in T4 group. In the control group of Carassius (Table 2), a total of 100 micronucleated cells were also scored which, however, account for 2.37 % of 4,209 erythrocytes screened. Upon exposure to arsenic, the frequency of

Fig. 1 Regression analysis of arsenic exposure and micronucleus induction in two species of shes, Channa and Carassius

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(Fange 1986; Nikinmaa 1990). In goldsh, maturation of erythrocytes occur in 1620 days (Murad and Houston 1992) and requires almost similar duration in many other shes (Dinnen et al. 1987; Fischer et al. 1998). The micronucleus assay requires that a substantial fraction of the cell population treated with a genotoxic agent must undergo mitosis so that the chromosomal anomalies induced during the rst cell cycle are visible as micronuclei in the cytoplasm during the second or subsequent cell cycles (Tates et al. 1980). Micronuclei are formed by the condensation of acentric chromosomal fragments or entire lagging chromosomes that are not incorporated into the daughter nuclei following anaphase in the dividing cells (erythroblasts). They appear as separate, small nuclei, in addition to main nucleus, in the cytoplasm of daughter cells (maturing erythrocytes). Since micronuclei cannot be observed until after the rst cell cycle, the frequencies of micronucleus within a cell population are highly dependent on the kinetics of cell proliferation. In shes, a peak in micronucleated erythrocytes occurs 15 days after exposure as per the available literature (Udroiu 2006). The design of present study with an exposure period of 15 consecutive days at an almost constant temperature followed by harvesting of cells on the next day, therefore, appears to be in accordance with the blood cell kinetics of experimental shes. The results of present study exhibited signicant genotoxic potential of arsenic in a dose-dependent manner at all the levels of exposure. The arsenic level of 10 lg L-1 in water (its current provisional guideline value for drinking water quality) was, therefore, found to produce marked genetic damage, inducing signicant genotoxicity in shes. Based on regression analysis of the degree of responses in the two species of shes upon exposure to arsenic, Carassius was found to be more sensitive than Channa. The variability in inter-specic sensitivity in shes to micronucleus induction has also been shown earlier (Grisolia and Cordeiro 2000). Contrary to this, both the species of sh exhibited almost a similar pattern of dose-dependent increase in the overall frequency of micronucleated cells which has also been documented in other shes (Hooftman and de Raat 1982). In shes, micronucleus formation, therefore, appears to be a sensitive indicator of arsenic genotoxicity which might have been induced by the arsenic mediated

oxidative stress (Kitchin and Ahmad 2003; Kitchin and Conolly 2010) following the production of reactive oxygen species (ROS). The resulting oxidative stress seems to produce damage in DNA and chromosomes and may even lead to carcinogenesis (Aposhian and Aposhian 2006; Dopp et al. 2010a; Hei and Filipic 2004; Nesnow et al. 2002; Schwerdtle et al. 2003; Soto-Reyes et al. 2005; Yamanaka et al. 2004).The arsenic induced oxidative damage in DNA (Jomova et al. 2011; Kessel et al. 2002; Liu and Jan 2000), and its involvement in micronucleus induction has been demonstrated in mammalian cells (Ho et al. 2000). Recently, arsenic induced oxidative stress has also been documented in shes (Bagnyukova et al. 2007; Bhattacharya and Bhattacharya 2007; Ventura-Lima et al. 2009a, b), showing comparable similarity with mammals in this regard (Ventura-Lima et al. 2011). The detailed mechanisms of arsenic mediated ROS formation remain elucidative. Evidences suggest that oxidative methylation of arsenic during its biotransformation, found to be associated with oxidative damage to DNA (Kojima et al. 2009; Ruiz-Ramos et al. 2009), is one of the proposed mechanisms of ROS formation (Aposhian and Aposhian 2006). The methylated (biotransformed) trivalent species of arsenic, reported to be biologically more active than its pentavalent form (Dopp et al. 2010b), have been considered as the major source of arsenic genotoxicity (Mass et al. 2001; Kligerman et al. 2003). The methylated arsenic species can release redox-active iron from ferritin, which then promotes the conversion of superoxide radical and hydrogen peroxide into highly reactive hydroxyl radicals (Ahmad et al. 2000). The arsenic mediated ROS generation induces oxidative DNA damage through the oxidation of DNA bases (mainly producing oxidized guanines), leading to the formation of DNA adducts and DNA protein crosslinks that ultimately causes DNA strand breaks (Bau et al. 2002; Marnett 2000; Wang et al. 2001). Substantial increase in arsenic induced DNA strand breaks was observed when the cells were subjected to digestion by the enzyme formamidopyrimidineDNA glycosilase, known to catalyze the excision of oxidized bases to remove DNA adducts (Cadet et al. 2000), and proteinase K, effective in releasing DNA-strand breaks from DNAprotein cross-links (Mouron et al. 2006; Wang et al. 2002). It, therefore, seems as if these DNA strand breaks had already existed but were bound by proteins.

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The genotoxic effect of arsenic, due to its high afnity for the sulfhydryl groups of proteins and enzymes (Castro et al. 2009; Samuel et al. 2005; Vahter and Concha 2001), may also be mediated, at least in part, by the oxidative damage induced by arsenic in them. Interaction of arsenic with SH-groups induces structural modication in proteins (Wang et al. 2004), leading to the inactivation of many enzymes (Akter et al. 2005). Arsenic mediated oxidative damage in enzymes is also reported to interfere with the DNA repair mechanisms, such as nucleotide excision repair (Andrew et al. 2003, 2006) and base excision repair (Sykora and Snow 2008), by either inhibiting ligation or down regulating the gene expression of DNA repair enzymes, particularly the DNA polymerase b (De Vizcaya-Ruiz et al. 2009; Ding et al. 2009; Piatek et al. 2008). Recently, Lai et al. (2011) have observed an important role of DNA polymerase b in repairing arsenic induced DNA damage since its deciency exacerbates arsenic induced genotoxic effects and its overexpression effectively protects the cell from arsenic induced oxidative stresses. Arsenic exposure has also been shown to deplete glutathione, a thiol based non-protein antioxidant, due to the involvement of later in arsenic metabolism or due to the thiol preference of arsenic for direct binding (Maiti and Chatterjee 2001). Therefore, the arsenic mediated inhibition of the enzymes of DNA repair and the decrease in glutathione level drastically affect the cellular protective mechanisms against ROS which further contributed to increased level of oxidative damage in cells, exacerbating arsenic genotoxicity (Frechet et al. 2001). Furthermore, arsenic binding to tubulin is also known to cause perturbation of spindle apparatus and induction of chromosome endo-reduplication leading to aneuploidy, polyploidy and even mitotic arrest (Kitchin and Wallace 2008; Kligerman et al. 2005; McCabe et al. 2000; Valko et al. 2005). The two vertebrate species, human and sh, exhibit perceptible genetic damage upon exposure to arsenic. The result obtained in shes, however, cannot be directly extrapolated in humans due to difference in pharmacokinetics and pharmacodynamics between them. Since no threshold of arsenic for its genotoxic effect in humans has been established, and almost all studies have been done at higher arsenic levels, further epidemiological studies, using micronucleus induction as a biomarker of arsenic (carcinogenic) exposure,

in populations exposed to low levels of arsenic (including its guideline value) are highly pertinent for better understanding of the pathogenesis of arsenic.

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