http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, Early Online: 1–7 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2014.900066
RESEARCH ARTICLE
Assessment of genotoxic and mutagenic potential of hexavalent chromium in the freshwater fish Labeo rohita (Hamilton, 1822) Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Newcastle on 08/22/14 For personal use only.
Naresh Sahebrao Nagpure, Rashmi Srivastava, Ravindra Kumar, Basdeo Kushwaha, Satish Kumar Srivastava, Pavan Kumar, and Anurag Dabas Molecular Biology and Biotechnology Division, National Bureau of Fish Genetic Resources, Lucknow, U. P., India
Abstract
Keywords
The present study was undertaken to investigate the genotoxicity and mutagenicity of sublethal concentrations of hexavalent chromium (potassium dichromate) in the Indian major carp, Labeo rohita. The 96 h LC50 value of potassium dichromate estimated was 118 mg L1 by probit analysis using SPSS (version 16.0) software. Based on 96 h LC50 value, three sublethal test concentrations of potassium dichromate (29.5, 59.0 and 88.5 mg L1) were selected and specimens were exposed in vivo to these test concentrations for 96 h. The mutagenic and genotoxic effects of potassium dichromate were evaluated in gill and blood cells using micronucleus (MN) test and comet assay. In general, significant (p50.05) effects due to the concentrations and the exposure durations were observed in exposed specimens. The MN induction was highest at 96 h at all the test concentrations in the peripheral blood. A similar trend was observed for the DNA damage, measured in terms of percentage of tail DNA, in erythrocyte and gill cells. The study indicated hazardous effect of the hexavalent chromium to fish and other aquatic organisms and indirectly to human beings.
Acute toxicity, genotoxicity, hexavalent chromium, Labeo rohita, mutagenicity
Introduction Pollution of water resources is a relentless and emergent problem. Despite the existence of relevant legislation, the contamination of the aquatic environment by toxic chemical pollutants continues to occur through domestic and industrial effluents (Claxton et al., 1998; Kushwaha et al., 2012; White & Rasmussen, 1998). Heavy metals, like chromium, cadmium, lead, mercury, are aquatic pollutants of an indispensible group having bio-accumulative and nonbiodegradable properties. Their excessive contamination to aquatic ecosystem has augmented the environmental and health concerns worldwide (Kasherwani et al., 2009; Kumar et al., 2013; Suruchi & Khanna, 2011; Velma et al., 2009; Yilmaz et al., 2010). Chromium is the 6th most abundant heavy metal in the earth crust and its compounds are known to have toxic, genotoxic, mutagenic and carcinogenic effects to man and animals (Mount & Hockett, 2000; Patlolla et al., 2008; Stohs & Bagchi, 1995; Wise et al., 2006; Von Burg & Liu, 1993). Both trivalent chromium (Cr III) and hexavalent chromium
Address for correspondence: Dr. Ravindra Kumar, Molecular Biology and Biotechnology Division, National Bureau of Fish Genetic Resources (Indian Council of Agricultural Research), Canal Ring Road, P.O. Dilkusha, Lucknow - 226 002, India. Tel: (0522) 2442440, 2442441. Fax: (0522) 2442403. E-mail: [email protected]
History Received 19 January 2013 Revised 26 October 2013 Accepted 26 December 2013 Published online 20 March 2014
(Cr VI) are biologically active, but differ in their ability to cross biological membranes (Leonard & Lauwerys, 1980; Singh et al., 1998). According to Sugiyama (1992), Cr(VI) is a strong oxidizing agent able to form chromate and dichromate ions in the environment and its compounds have been declared as a potent occupational carcinogen among the workers of chrome plating, stainless steel and pigment industries. The hexavalent chromate enters into cells via the surface transport system and inside the cell it is reduced to trivalent chromium which induces genotoxic effects in the cell (Sugiyama, 1992). Micronucleus test, chromosomal aberrations and DNA damage assays have been used for assessing genotoxicity of various chemicals in different animals (Cavas & ErgeneGozukara, 2005; De Lemos et al., 2001; Farag et al., 2006, Kushwaha et al., 2000; Kumar et al., 2010; Kushwaha et al., 2012). However, comet assay (or single cell gel electrophoresis assay) is more popular because it can be performed quickly and provides increased sensitivity for detecting low levels of DNA damage (Ali et al., 2008; Kumar et al., 2010; Kushwaha et al., 2000; Nagpure et al., 2007; Nwani et al., 2010; Pandey et al., 2006; Talapatra et al., 2006). As a result, the comet assay is being employed to assess the genotoxicity of various chemical compounds in several test organisms (Ali et al., 2008; Kumar et al., 2010; Kushwaha et al., 2012; Nwani et al., 2010; Talapatra et al., 2006). Micronucleus assay has also been extensively used in situ for detection of clastogenic and aneugenic effect of chemicals
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because it is formed by chromosome fragment or whole chromosome that lag at cell division due to the lack of centromere or damage. Some studies have shown that chromium compounds induced DNA damage in different manners, viz. DNA single and double-strand breaks generating chromosomal aberrations, micronucleus formation, sister chromatid exchanges, formation of DNA adducts, and alteration in DNA replication and transcription (Ali et al., 2008; Kumar et al., 2010; Kushwaha et al., 2000; Kushwaha et al., 2003; Matsumoto et al., 2006; Nwani et al., 2010; O’Brien et al., 2001; Zhitkovich et al., 1996). Labeo rohita (Hamilton, 1822), commonly known as ‘‘rohu’’, is the most widely cultured species in the country among the Indian major carps. Out of 3.9 million metric tons of total freshwater aquaculture production, contribution of carps alone is about 90%, with at least 50% of which is contributed by the rohu. L. rohita can serve as an excellent indicator of water quality and environmental pollution in aquatic systems (Vutukuru et al., 2007). The gills are the main target of direct contact with contaminants and play an important role in metal uptake, storage, and transfer to the internal compartments via blood transport. Hence, an attempt has been made for the evaluation of tissue specific alterations in genetic content due to metal exposure. In the present study, acute toxicity of potassium dichromate was estimated to evaluate the Cr(VI) induced mutagenicity and genotoxicity in erythrocyte and gill tissues of L. rohita. The acquired information of the present study will help in determination of safe levels of chromium for aquatic survival and in implementation of strategies for bioremediation of chromium.
Materials and methods Experimental animals and chemical Freshwater fish L. rohita were procured from the fish farm of Aquaculture Research Training Unit of NBFGR at Chinhat, Lucknow for the experimentation purpose. The adult fish specimens were taken with an average age of approximately eight months. After transportation to laboratory, the specimens were given prophylactic treatment by bathing them twice in 0.05% KMnO4 solution for two min to avoid any dermal infections. They had an average (±S.D) wet weight and length of 21.0 ± 2.5 g and 13.0 ± 1.2 cm, respectively. The fish were then acclimatized for one month under laboratory condition before start of the experiment as suggested by Bennett & Dooley (1982). Potassium dichromate (K2Cr2O7; 99.9% Extra pure AR, Maximum limits of impurities, Chloride 0.001%, Sulphate 0.005%, Iron 0.002%, Copper 0.001% and Sodium 0.002%), manufactured by Sisco Research Laboratories (SRL) Pvt. Ltd., Mumbai, India, was procured and used for the present study. Acute toxicity bioassay A definite test was performed in static system to determine the 96 h LC50 of potassium dichromate in L. rohita, following the standard methods (OECD, 1992). The range finding test was carried out prior to the definitive test to determine the concentration range of the test solution. Ten specimens were randomly placed in each aquarium, filled with 50 litres of
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water, with loading density of 4.2 g L1. Fish were exposed to nominal potassium dichromate concentrations of 110, 120, 130, 140, 150 and 160 mg L1. The exposure at each concentration was replicated twice along with a control in normal water. The physicochemical characteristics of test water, namely temperature, pH, conductivity, dissolved oxygen, and total hardness, were analyzed by standard methods (American Public Health Association, American Water Works Association, Water Pollution Control Federation, 2005) during the experimentation period. Fish mortality in each aquarium was recorded at the intervals of 24, 48, 72 and 96 h. The criteria for death were no gill movement and no reaction to gentle prodding. Dead fish were immediately removed from the experiment. Determination of sublethal concentrations The 96 h LC50 values (with 95% confidence limits) of potassium dichromate for freshwater carp L. rohita was estimated as 118.71 mg L1. Based on the 96 h LC50 value, the three test concentrations of potassium dichromate, viz. sublethal concentration I (SL-I; 1/4th of LC50 ¼ 29.5 mg L1), sublethal concentration II (SL-II; 1/2nd of LC50 ¼ 59 mg L1) and sublethal concentration III (SL-III; 3/4th of LC50 ¼ 88.5 mg L1) were estimated for in vivo exposure experiment. In vivo exposure experiment The fish specimens were exposed to the three aforementioned test concentrations of potassium dichromate in a static system. Healthy fish specimens of approximately same size in average (±S.D.) weight (21 ± 2.5 g) and length (13 ± 1.2 cm) were randomly selected and transferred to aquarium containing 50 litres of water containing different concentration of potassium dichromate along with one control. The exposure was continued up to 96 h and the tissue sampling was done at the intervals of 24, 48, 72 and 96 h at the rate of five specimens per sampling duration. For each test concentration and control, the experiment was replicated twice. This study was conducted following the OECD guideline in the static test conditions (OECD, 1992). Isolation of blood and gill tissues On each sampling, peripheral blood samples were collected from the caudal vein of both the control and treated specimens (5–6 fishes from each group on the consecutive days of exposure) using heparinized syringes for comet assay and micronuclei test. Further, the specimens were sacrificed, after anaesthesization with ethylene glycol monophenyl ether, by decapitation followed by dissection and the removal of gills tissues. The tissue samples were placed on ice for its viability and immediately processed for genotoxicity assessment. The physicochemical properties of test water, namely temperature, pH, conductivity, dissolved oxygen, chloride, total hardness and total alkalinity were analyzed by standard methods (American Public Health Association, American Water Works Association, Water Pollution Control Federation, 2005).
Genotoxicity assessment of chromium in Labeo rohita
DOI: 10.3109/01480545.2014.900066
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Comet assay (CA) The comet assay or alkaline single-cell gel electrophoresis was performed as a three layer procedure (Singh et al., 1988) with slight modifications (McKelvey-Martin et al., 1993). The blood cells were suspended in chilled phosphate buffered saline (PBS, pH 7) and the gill tissue (75 mg) was homogenized in PBS (pH 7) followed by centrifugation at 4000 rpm at 4 C for 5 min. Cell viability was evaluated using trypan blue exclusion test (Anderson et al., 1994) and the cell suspension showing 485% cell viability were further processed for CA. In brief, 20 mL of cell suspension was mixed with 80 mL of 0.5% low melting point agarose (LMPA) and layered on one end frosted glass slide, which was already coated with a layer of 200 mL of 1% normal agarose. The slide was again coated with third layer of 100 mL LMPA and covered with coverslip. After solidification, the coverslip was removed and the slide was immersed in cold lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10, and 10% DMSO & 1% TritonX-100 added fresh) and kept at 4 C overnight. After lysis, the slides were placed in a horizontal electrophoresis tank side by side. The tank was filled with fresh solution (1 mM Na2EDTA, 300 mM NaOH, 0.2% DMSO, pH 13.5) to a level of approximately 0.25 cm above the slides. The slides were left in the solution for 20 min to allow unwinding, and the electrophoresis was performed for 20 min at 16 V, 300 mA. The slides were then neutralized gently with 0.4 M Tris buffer at pH 7.5 and stained with 75 mL ethidium bromide (20 mg/mL). Two slides per specimen prepared and 25 cells per slide (200 cells per concentration) were scored randomly under florescent microscope (Leica-DMLS, Leica Microsystems GmbH, Wetzlar, Germany) equipped with appropriate filters and the cells analyzed using image analysis computer software (Komet–5.5 Kinetic Imaging, Merseyside, UK). The parameter selected for quantification of DNA damage was %Tail DNA (¼100 – %Head DNA), as determined by the software. Micronucleus test (MNT) Peripheral blood was smeared on pre-cleaned slides. After fixation in pure ethanol for 20 min, the smeared slide was allowed to air-dry, stained with 6% Giemsa for 30 min., washed in tap water, dried on hot plate and mounted in DPX (Di-N-butyle Phthalate in Xylene). Two slides were prepared from each specimen, and a total of 2000 erythrocyte cells were examined from each slide under 100X magnification. Small, non refractive, circular or ovoid chromatin bodies displaying same staining and focusing patterns, as the main nucleus, were scored as micronuclei (Al-Sabti & Metcalfe, 1995). Statistical analyses One way analysis of variance (ANOVA) was applied to compare the means obtained for various parameters among different concentrations and durations. The % MN frequencies were compared between concentrations within duration using Mann–Whitney test. A p value less than 0.05 were considered statistically significant. Lethal concentration values of potassium dichromate were calculated using SPSS (version 16.0,
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Chicago, IL) computer program with 95% confidence limits. The values for the biomarkers are expressed as mean ± SE (n ¼ 5).
Results Physicochemical properties of the test solution The temperature varied from 22.4 to 23.3 C, whereas the pH ranged from 7.4 to 7.6. The dissolved oxygen concentration was 6.6 to 7.2 mg L1. During the experimentation, the chloride, total hardness and total alkalinity of the test water ranged 42–44, 170–176 and 270–280 mg L1 as CaCO3, respectively. 96 h LC50 of potassium dichromate Potassium dichromate caused acute toxicity in a concentration dependent manner. Experimental conditions produced no mortality in the control. When administered at 160 and 180 mg L1 doses, potassium dichromate induced 100% cumulative mortality in the two replicate groups within 96 h of exposure. In the present study, the 96 h lethal concentrations of potassium dichromate to L. rohita have been presented in Table 1. The 96 h LC50 value was estimated to be 118 mg L1. Induction of DNA damage The single strand DNA breaks representing in the form of % tail DNA increased significantly (p50.05) with increasing concentrations and durations in the erythrocyte and gill tissues of potassium dichromate exposed fish specimens when compared to control. The highest DNA damage in both the tissues was found at 88.5 mg L1 concentration and 96 h post exposure. The DNA damage measured in terms of %tail DNA was found to be increased significantly (p50.05) from 9.16 to 20.08% in erythrocytes of exposed specimens, while in gill cells it increased from 12.60 to 23.01% with the progression of experiment (Figures 1 and 2 and Table 2). Induction of micronucleus The results of MN induction in erythrocytes of L. rohita after exposure to different test concentrations and sampling times of potassium dichromate are presented in Table 3. There was a significant increase in MN frequencies at all the exposed concentrations along with exposure durations when compared to control (Figure 3). The lowest concentration of potassium
Table 1. Various 96 h lethal concentrations of chromium in L. rohita. 95% Confidence limits LC values LC10 LC20 LC30 LC40 LC50 LC60 LC70 LC80 LC90
1
Concentrations (mg L )
Lower
Upper
100.618 106.493 110.941 114.888 118.705 122.648 127.012 132.317 140.042
1.855 4.062 7.141 11.553 18.087 28.248 45.246 76.621 122.953
118.150 121.993 124.946 127.649 130.408 133.557 137.823 146.542 206.434
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Figure 1. Fluorescence microscopy visualization of DNA damage induced by hexavalent chromium during acute exposure at 29.5 mg L1, 59.0 mg L1 and 88.5 mg L1 test concentrations using the Comet Assay in red blood cells of Labeo rohita. Red blood cells isolated from (a) control and (b) exposed fish. Ethidium bromide stained nuclei following electrophoresis shows ‘‘comet tail’’ in exposed cells. (Bar represents 10 m).
Figure 2. Fluorescence microscopy visualization of DNA damage induced by acute exposure to hexavalent chromium at 29.5 mg L1, 59.0 mg L1 and 88.5 mg L1 test concentrations using Comet Assay in gill cells of L. rohita. Gill cells isolated from (a) control and (b) exposed fish showing comet tail. (Bar represents 10 m).
Table 2. % Tail DNA in erythrocytes and gill cells of L. rohita exposed to different test concentrations of potassium dichromate. Exposure duration (h) Tissue
Test concentrations (mg L1)
Erythrocyte
24 A
Control 29.5 59.0 88.5 Control 29.5 59.0 88.5
Gill
48
72 A
4.12 ± 0.28 9.29 ± 0.04cA1 10.14 ± 0.32bA1 11.25 ± 0.31aB1 4.82 ± 0.31A 10.93 ± 0.08cB2 11.74 ± 0.09bA2 13.57 ± 0.17aB2
4.12 ± 0.28 9.16 ± 0.3bA1 9.83 ± 0.29abA1 10.48 ± 0.07aA1 4.82 ± 0.31A 10.5 ± 0.16bA2 11.23 ± 0.27abA2 11.48 ± 0.11aA2
96 A
4.12 ± 0.28 9.46 ± 0.95cA1 12.61 ± 0.16bB2 15.12 ± 0.04aC2 4.82 ± 0.31A 11.98 ± 0.16cC2 14.86 ± 0.17bcB1 16.78 ± 0.26aC1
4.12 ± 0.28A 11.66 ± 0.58cB1 13.82 ± 0.26bC2 20.08 ± 0.21aD2 4.82 ± 0.31A 12.06 ± 0.15cC1 17.04 ± 0.57bC1 23.01 ± 0.42aD1
Mean percentage of tail DNA (±SE) in the erythrocyte and gill cells (n ¼ 200 cells/concentration/duration) of L. rohita exposed to different sub-lethal concentration of potassium dichromate at different exposure durations. Values with different alphabet (lowercase) superscripts differ significantly (p50.05) between test concentrations within tissue and exposure duration. Values with different alphabets (uppercase) differ significantly (p50.05) between exposure durations within test concentration and tissue. Values with different numeric superscripts differ significantly (p50.05) between tissues within test concentration and exposure duration. Table 3. Micronuclei frequencies in erythrocytes of L. rohita exposed to potassium dichromate at different test concentrations and exposure durations. Exposure duration (h) 1
Test concentrations (mg L ) Control 29.5 59.0 88.5
24
48 a1
0.027 ± 0.023 0.072 ± 0.031a2 0.182 ± 0.035a3 0.296 ± 0.028a4
72 a1
0.028 ± 0.021 0.117 ± 0.026ab2 0.218 ± 0.27b3 0.268 ± 0.041a4
96 a1
0.028 ± 0.022 0.152 ± 0.02b3 0.225 ± 0.11b4 0.432 ± 0.03b5
0.029 ± 0.021a1 0.163 ± 0.029b3 0.273 ± 0.041b4 0.504 ± 0.029b5
Mean percentage of micronucleus frequencies (±SE) in erythrocytes of L. rohita (n ¼ 2000) exposed to different sub-lethal concentrations of potassium dichromate at different exposure durations. Values with numeric superscript differ significantly (p50.05) between exposure durations within test concentration. Values with alphabet superscript differ significantly (p50.05) between test concentrations within exposure duration.
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DOI: 10.3109/01480545.2014.900066
Genotoxicity assessment of chromium in Labeo rohita
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Figure 3. Cytological visualization of micronuclei induced by exposure to hexavalent chromium at 29.5 mg L1, 59.0 mg L1 and 88.5 mg L1 test concentrations using Micronuclei Test in red blood cells of L. rohita. Red blood cell isolated from (a) control showing no micronuclei and (b) exposed fish showing micronuclei as marked by an arrow. (Bar represents 10 m).
dichromate treatment in fish specimen induced MN frequency of 0.072% in blood erythrocyte in 24 h which was significantly increased to 0.163% with 96 h exposure. Similar trend was observed for SL-II in which the MN frequency of 0.182% in 24 h increased to 0.273% after 96 h. At the highest concentration, the MN frequency significantly increased from 0.296% at 24 h exposure to 0.504% after 96 h duration. The MN frequency increased with increasing concentrations and exposure durations and highest induction in MN frequencies was observed at SL-III concentration after 96 h post exposure.
Discussion Chromium induced genotoxic and mutagenic effect in L. rohita at various exposed concentrations along with exposure time duration. The micronuclei induction and DNA damage in erythrocytes and gill tissues was highest at the highest test concentration of 88.5 mg/L. Thus, the present study revealed that chromium is responsible for inducing micronuclei formations and DNA damage in erythrocyte and gills tissue of the fish. The combined approaches using these assays will help in providing broad perspective in aquatic toxicology, as fish gill cells are constantly being exposed to environmental pollutants while blood cells are simple to be handled. Fish are often used as sentinel organism for ecotoxicological studies because they play a number of roles in the trophic web, accumulate toxic substances and respond to low concentration of mutagens (Cavas & Ergene-Gozukara, 2005). Therefore, the use of fish biomarkers as indices of the effects of pollution, are of increasing importance and can permit early detection of aquatic environmental problems (Lopez-Barea, 1996; Van Der Oost et al., 2003). The test result of the LC50 of the present study at 96 h was 118.71 mg L1 which indicated that potassium dichromate is very toxic to fish. Our estimate is slightly lower than the 96 h LC50 values of hexavalent chromium (142 mg/L) in L. rohita (Jaffri et al., 2003) and comparatively higher than the 96 h LC50 value 43.7 mg L1 of potassium dichromate for tilapia, Oreochromis spp., as reported by Abbas & Ali (2007) and 91.5 mg L1 for Oreochromis areus as estimated by
Yilmaz et al., (2010). Interestingly, the 96 h LC50 111.45 mg/ L of the same toxicant in L. rohita (Vutukuru, 2005) was quite similar to our present study. This variation may be due to the disparity and hardiness of the experimental species, age, body size of the individuals and water quality parameters (Al-Akel, 1996). Genotoxicity can occur due to many physico-chemical agents that result in a wide variety of potential damages to the genetic material, ranging from various DNA adducts to single- and double-strand breakages, DNA-DNA and DNAprotein cross-links or even chromosomal breakage (Cavalcanti et al., 2010; Wang et al., 2009). The comet assay has been considered as sensitive, rapid and reliable method of quantitatively measuring DNA damage in eukaryotic and prokaryotic cells (Bajpayee et al., 2005; Cotelle & Ferard, 1999). It is increasingly being used in testing of substances such as industrial chemicals, biocides, agrochemicals, food additives and pharmaceuticals for genotoxicity testing (Brendler-Schwaab et al., 2005). The assay is favoured among other cytogenetic methods used for the detection of DNA damage (Buschini et al., 2003) as it is capable of detecting wide variety of DNA damage such as DNA singlestrand breaks (Sharbel, 2004). Micronucleus assay detects injuries that survive at least one mitotic cycle and allows for measurement of chromosome breakage or chromosome loss (Kirsch-Volders et al., 2003). The combined application of both the assays were suggested for the testing of genotoxins to understand the mechanisms underlying mutagenicity (Van Goethem et al., 1997). Therefore, comet assay and micronucleus assay were undertaken in the present study for evaluation of acute toxicity and genotoxicity of hexavalent chromium as also supported by Ahmed et al. (2013). The present study revealed that the Cr(VI) is genotoxic to the Indian major carp L. rohita. Our results are corroborated with the previous reports (Ahmad et al., 2006). In another study, medaka fin cell lines, exposed to Cr(VI) to examine the genotoxic potentials, have demonstrated double strand DNA breaks and chromosome damage in a concentration-dependent manner (Goodale et al., 2008). Abbas & Ali (2007) studied the genotoxic potential of Cr(VI) in Orechromis spp. using
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RAPD-PCR after exposure to sub lethal concentrations (43.7 mg L1) for 24 and 96 h. The results showed polymorphic bands in long time exposed fish, whereas no polymorphic bands found in short time exposed fish. The polymorphic bands appear due to fragmented DNA strands. Furthermore, studies have reported the genotoxic potential of Cr(VI) in Pimephales promelas (De Lemos et al., 2001) and in Channa punctatus (Kumar et al., 2012) based on the induction of micronucleus in erythrocytes. The DNA damage at higher test concentrations in the erythocytes and gills could be due to the elevated levels of hydroperoxides in both the tissues compared to their controls (Velma & Tchounwou, 2010). Further, the Cr (VI) is known as one of the core toxicants for degeneration of secondary gill lamellae (Sesha Srinivas & Rao, 1998). We applied the alkaline comet assay to evaluate total DNA strand breaks in the erythrocyte and gill cells of L. rohita exposed in vivo to different test concentrations of potassium dichromate. The data obtained showed that the frequencies of % tail DNA damage for all concentrations of potassium dichromate, tested in both tissues, were significantly higher (p50.01) than the control. For the tissues, higher the concentration and duration of potassium dichromate exposure, the higher the % tail DNA damage. While, the % tail DNA damage in gill tissues was slightly higher than the erythrocytes and the DNA damage was both dose- and time dependent. Hence, Cr(VI) specifically altered the gills at genetic level and further, the subtle effects seen at low concentrations suggest that gills are more sensitive to Cr (VI) toxicity than erythrocytes.
Conclusion The present study revealed that chromium induced DNA damage in erythrocyte and gill tissues of L. rohita as measured by the comet assay and micronuclei test. The acute toxicity estimates will eventually help to determine the safe dose of the chemical for a species and to establish water quality criteria for planning remedial measures to reduce the chromium pollution. The combined approaches using these assays will help in providing broad perspective in aquatic toxicology, as fish gill cells are constantly being exposed to environmental pollutants, while blood cells are simple to be handled. There is a great potential to make use of these assays for assessing detailed information on cellspecific genotoxic and mutagenic effects, inter-individual variability, adaptability, and further contributing in formulation of strategies and measures for the conservation of fish diversity.
Acknowledgements The authors are grateful to the Director, National Bureau of Fish Genetics Resources, Lucknow for providing necessary facilities and to Uttar Pradesh Council of Science & Technology (UP-CST), Lucknow for financial assistance.
Declaration of interest There are no conflicts of statement among the authors and with funding agency.
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Kumar P, Kumar R, Nagpure NS, et al. (2013). Genotoxicity and antioxidant enzyme activity induced by hexavalent chromium in Cyprinus carpio after in vivo exposure. Drug Chem Toxicol 36:451–460. Kumar R, Nagpure NS, Kushwaha B, et al. (2010). Investigation of the genotoxicity of Malathion to freshwater teleost fish Channa punctatus (Bloch) using micronucleus test and comet assay. Arch Environ Contam Toxicol 58:123–130. Kushwaha B, Srivastava SK, Singh B, et al. (2000). Evaluation of comet assay and micronuclei test as genotoxic assays in Channa punctatus. Nat Acad Sci Lett 23:177–179. Kushwaha B, Nagpure NS, Srivastava SK, et al. (2003). Variation of micronuclei in peripheral blood cells of Channa punctatus. The Indian J Anim Sci 73:1192–1193. Kushwaha B, Pandey S, Sharma S, et al. (2012). In situ assessment of genotoxic and mutagenic potential of polluted river water in Channa punctatus and Mystus vittatus. Inter Aquat Res 4:16. doi:10.1186/ 2008-6970-4-16. Leonard A, Lauwerys RR. (1980). Carcinogenicity and mutagenicity of chromium. Mutat Res 76:227–239. Lopez-Barea J. (1996). Biomarkers to detect environmental pollution. Toxicol Lett 88:77–79. Matsumoto ST, Mantovani MS, Malaguttii MIA, et al. (2006). Genotoxicity and mutagenicity of water contaminated with tannery effluents, as evaluated by the micronucleus test and comet assay using the fish Oreochromis niloticus and chromosome aberrations in onion root-tips. Genet Mol Biol 29:148–158. McKelvey-Martin VJ, Green MH, Schmezer P, et al. (1993). The single cell gel electrophoresis assay (comet assay): a European review. Mutat Res 288:47–63. Mount DR, Hockett JR. (2000). Use of toxicity identification evaluation methods to characterize, identify and confirm hexavalent chromium toxicity in an industrial effluent. Water Res 34:1379–1385. Nagpure NS, Kumar R, Kushwaha B, et al. (2007). Genotoxicity assessment in fishes, A practical approach. Lucknow, India: NBFGR, 126 p. Nwani CD, Lakra WS, Nagpure NS, et al. (2010). Mutagenic and genotoxic effects of carbosulfan in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis. Food Chem Toxicol 48:202–208. O’Brien T, Xu J, Patierno SR. (2001). Effects of glutathione on chromium-induced DNA cross linking and DNA polymerase arrest. Mol Cell Biochem 222:173–182. OECD (Organization for Economic Cooperation and Development). (1992). Guideline for the testing of chemicals: fish, acute toxicity test, Document 203 p. Pandey S, Nagpure NS, Kumar R, et al. (2006). Genotoxicity evaluation of acute doses of endosulfan to fresh water teleost Channa punctatus (Bloch) by alkaline single cell gel electrophoresis. Ecotoxicol Environ Saf 65:56–56. Patlolla AK, Barnes C, Yedjou C, et al. (2008). Oxidative stress, DNA damage, and antioxidant enzyme activity induced by hexavalent chromium in Sprague Dawley rats. Environ Toxicol 24:66–73. Sharbel W. (2004). Monitoring DNA damage following radiation exposure using cytokinesis–block micronucleus method and alkaline single-cell gel electrophoresis. Clinica Chimica Acta 347:15–24. Sesha Srinivas V, Rao BM. (1998). Chromium induced alterations in the gill of the freshwater teleost fish, Labeo rohita. Ind J Compar Anim Physiol 17:31–33.
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Singh NP, McCoy MT, Tice RR, Schneider EL. (1988). A simple technique for quantization of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191. Singh J, Bridgewater LC, Patierno SR. (1998). Differential sensitivity of chromium-mediated DNA interstrand crosslinks and DNA-protein crosslinks to disruption by alkali and EDTA. Toxicol Sci 45:72–76. Sugiyama M. (1992). Role of physiological antioxidants in chromium (VI)-induced cellular injury. Free Radic Biol Med 12:397–407. Suruchi, Khanna P. (2011). Assessment of heavy metal contamination in different vegetables grown in and around urban areas. Res J Environ Toxicol 5:162–179. Stohs SJ, Bagchi D. (1995). Oxidative mechanisms in the toxicity of metal ions. Free Rad Biol Med 18:321–336. Talapatra SN, Ganguly P, Mukhopadhyyay A, Banerjee S. (2006). Assessment of genetic biomarkers with special reference to micronucleated and binucleated erythrocytes in two fish species grown at Industrial vicinity of thermal power plants, Kolkata, India. Asian J Water Environ Poll 4:139–144. Van Der Oost R, Beyer J, Vermeulen NPE. (2003). Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ Toxicol Pharm 13:57–149. Van Goethem F, Lison D, Kirsch-Volders M. (1997). Comparative evaluation of the in vitro micronucleus test and the alkaline single cell gel electrophoresis assay for the detection of DNA damaging agents: genotoxic effects of cobalt powder, tungsten carbide and cobalt– tungsten carbide. Mutat Res 392:31–43. Velma V, Vutukuru SS, Tchounwou PB. (2009). Ecotoxicology of hexavalent chromium in freshwater fish: a critical review. Rev Environ Health 24:129–145. Velma V, Tchounwou PB. (2010). Chromium induced biochemical, genotoxic and histopathologic effects in liver and kidney of Goldfish, Carassius auratus. Mutat Res 30 698:43–51. Von Burg R, Liu D. (1993). Chromium and hexavalent chromium. J Appl Toxicol 13:225–230. Vutukuru SS. (2005). Acute effects of hexavalent chromium on survival, oxygen consumption, hematological parameters and some biochemical profiles of the Indian Major Carp, Labeo rohita. Int J Environ Res Public Health 2:456–462. Vutukuru SS, Prabhath NA, Raghavender M, Yerramilli A. (2007). Effect of arsenic and chromium on the serum amino-transferases activity in Indian major carp, Labeo rohita. Int J Environ Res Public Health 4:224–227. Wang J, Sawyer JR, Chen L, et al. (2009). The mouse lymphoma assay detects recombination, deletion, and aneuploidy. Toxicol Sci 109: 96–105. White P, Rasmussen JB. (1998). The genotoxic hazard of domestic wastes in surface waters. Mutat Res 410:223–236. Wise SS, Holmes AL, Xie H, et al. (2006). Chronic exposure to particulate chromate induces spindle assembly checkpoint bypass in human lung cells. Chem Res Toxicol 19:1492–1498. Yilmaz AB, Turan C, Toker T. (2010). Uptake and distribution of hexavalent chromium in tissues (gill, skin and muscle) of a freshwater fish, Tilapia, Oreochromis aureus. J Environ Chem Ecotoxicol 2: 28–33. Zhitkovich A, Voitkun V, Costa M. (1996). Formation of the amino acidDNA complexes by hexavalent and trivalent chromium in vitro: importance of trivalent chromium and the phosphate group. Biochemistry 35:7275–7282.
RESEARCH ARTICLE
Assessment of genotoxic and mutagenic potential of hexavalent chromium in the freshwater fish Labeo rohita (Hamilton, 1822) Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Newcastle on 08/22/14 For personal use only.
Naresh Sahebrao Nagpure, Rashmi Srivastava, Ravindra Kumar, Basdeo Kushwaha, Satish Kumar Srivastava, Pavan Kumar, and Anurag Dabas Molecular Biology and Biotechnology Division, National Bureau of Fish Genetic Resources, Lucknow, U. P., India
Abstract
Keywords
The present study was undertaken to investigate the genotoxicity and mutagenicity of sublethal concentrations of hexavalent chromium (potassium dichromate) in the Indian major carp, Labeo rohita. The 96 h LC50 value of potassium dichromate estimated was 118 mg L1 by probit analysis using SPSS (version 16.0) software. Based on 96 h LC50 value, three sublethal test concentrations of potassium dichromate (29.5, 59.0 and 88.5 mg L1) were selected and specimens were exposed in vivo to these test concentrations for 96 h. The mutagenic and genotoxic effects of potassium dichromate were evaluated in gill and blood cells using micronucleus (MN) test and comet assay. In general, significant (p50.05) effects due to the concentrations and the exposure durations were observed in exposed specimens. The MN induction was highest at 96 h at all the test concentrations in the peripheral blood. A similar trend was observed for the DNA damage, measured in terms of percentage of tail DNA, in erythrocyte and gill cells. The study indicated hazardous effect of the hexavalent chromium to fish and other aquatic organisms and indirectly to human beings.
Acute toxicity, genotoxicity, hexavalent chromium, Labeo rohita, mutagenicity
Introduction Pollution of water resources is a relentless and emergent problem. Despite the existence of relevant legislation, the contamination of the aquatic environment by toxic chemical pollutants continues to occur through domestic and industrial effluents (Claxton et al., 1998; Kushwaha et al., 2012; White & Rasmussen, 1998). Heavy metals, like chromium, cadmium, lead, mercury, are aquatic pollutants of an indispensible group having bio-accumulative and nonbiodegradable properties. Their excessive contamination to aquatic ecosystem has augmented the environmental and health concerns worldwide (Kasherwani et al., 2009; Kumar et al., 2013; Suruchi & Khanna, 2011; Velma et al., 2009; Yilmaz et al., 2010). Chromium is the 6th most abundant heavy metal in the earth crust and its compounds are known to have toxic, genotoxic, mutagenic and carcinogenic effects to man and animals (Mount & Hockett, 2000; Patlolla et al., 2008; Stohs & Bagchi, 1995; Wise et al., 2006; Von Burg & Liu, 1993). Both trivalent chromium (Cr III) and hexavalent chromium
Address for correspondence: Dr. Ravindra Kumar, Molecular Biology and Biotechnology Division, National Bureau of Fish Genetic Resources (Indian Council of Agricultural Research), Canal Ring Road, P.O. Dilkusha, Lucknow - 226 002, India. Tel: (0522) 2442440, 2442441. Fax: (0522) 2442403. E-mail: [email protected]
History Received 19 January 2013 Revised 26 October 2013 Accepted 26 December 2013 Published online 20 March 2014
(Cr VI) are biologically active, but differ in their ability to cross biological membranes (Leonard & Lauwerys, 1980; Singh et al., 1998). According to Sugiyama (1992), Cr(VI) is a strong oxidizing agent able to form chromate and dichromate ions in the environment and its compounds have been declared as a potent occupational carcinogen among the workers of chrome plating, stainless steel and pigment industries. The hexavalent chromate enters into cells via the surface transport system and inside the cell it is reduced to trivalent chromium which induces genotoxic effects in the cell (Sugiyama, 1992). Micronucleus test, chromosomal aberrations and DNA damage assays have been used for assessing genotoxicity of various chemicals in different animals (Cavas & ErgeneGozukara, 2005; De Lemos et al., 2001; Farag et al., 2006, Kushwaha et al., 2000; Kumar et al., 2010; Kushwaha et al., 2012). However, comet assay (or single cell gel electrophoresis assay) is more popular because it can be performed quickly and provides increased sensitivity for detecting low levels of DNA damage (Ali et al., 2008; Kumar et al., 2010; Kushwaha et al., 2000; Nagpure et al., 2007; Nwani et al., 2010; Pandey et al., 2006; Talapatra et al., 2006). As a result, the comet assay is being employed to assess the genotoxicity of various chemical compounds in several test organisms (Ali et al., 2008; Kumar et al., 2010; Kushwaha et al., 2012; Nwani et al., 2010; Talapatra et al., 2006). Micronucleus assay has also been extensively used in situ for detection of clastogenic and aneugenic effect of chemicals
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because it is formed by chromosome fragment or whole chromosome that lag at cell division due to the lack of centromere or damage. Some studies have shown that chromium compounds induced DNA damage in different manners, viz. DNA single and double-strand breaks generating chromosomal aberrations, micronucleus formation, sister chromatid exchanges, formation of DNA adducts, and alteration in DNA replication and transcription (Ali et al., 2008; Kumar et al., 2010; Kushwaha et al., 2000; Kushwaha et al., 2003; Matsumoto et al., 2006; Nwani et al., 2010; O’Brien et al., 2001; Zhitkovich et al., 1996). Labeo rohita (Hamilton, 1822), commonly known as ‘‘rohu’’, is the most widely cultured species in the country among the Indian major carps. Out of 3.9 million metric tons of total freshwater aquaculture production, contribution of carps alone is about 90%, with at least 50% of which is contributed by the rohu. L. rohita can serve as an excellent indicator of water quality and environmental pollution in aquatic systems (Vutukuru et al., 2007). The gills are the main target of direct contact with contaminants and play an important role in metal uptake, storage, and transfer to the internal compartments via blood transport. Hence, an attempt has been made for the evaluation of tissue specific alterations in genetic content due to metal exposure. In the present study, acute toxicity of potassium dichromate was estimated to evaluate the Cr(VI) induced mutagenicity and genotoxicity in erythrocyte and gill tissues of L. rohita. The acquired information of the present study will help in determination of safe levels of chromium for aquatic survival and in implementation of strategies for bioremediation of chromium.
Materials and methods Experimental animals and chemical Freshwater fish L. rohita were procured from the fish farm of Aquaculture Research Training Unit of NBFGR at Chinhat, Lucknow for the experimentation purpose. The adult fish specimens were taken with an average age of approximately eight months. After transportation to laboratory, the specimens were given prophylactic treatment by bathing them twice in 0.05% KMnO4 solution for two min to avoid any dermal infections. They had an average (±S.D) wet weight and length of 21.0 ± 2.5 g and 13.0 ± 1.2 cm, respectively. The fish were then acclimatized for one month under laboratory condition before start of the experiment as suggested by Bennett & Dooley (1982). Potassium dichromate (K2Cr2O7; 99.9% Extra pure AR, Maximum limits of impurities, Chloride 0.001%, Sulphate 0.005%, Iron 0.002%, Copper 0.001% and Sodium 0.002%), manufactured by Sisco Research Laboratories (SRL) Pvt. Ltd., Mumbai, India, was procured and used for the present study. Acute toxicity bioassay A definite test was performed in static system to determine the 96 h LC50 of potassium dichromate in L. rohita, following the standard methods (OECD, 1992). The range finding test was carried out prior to the definitive test to determine the concentration range of the test solution. Ten specimens were randomly placed in each aquarium, filled with 50 litres of
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water, with loading density of 4.2 g L1. Fish were exposed to nominal potassium dichromate concentrations of 110, 120, 130, 140, 150 and 160 mg L1. The exposure at each concentration was replicated twice along with a control in normal water. The physicochemical characteristics of test water, namely temperature, pH, conductivity, dissolved oxygen, and total hardness, were analyzed by standard methods (American Public Health Association, American Water Works Association, Water Pollution Control Federation, 2005) during the experimentation period. Fish mortality in each aquarium was recorded at the intervals of 24, 48, 72 and 96 h. The criteria for death were no gill movement and no reaction to gentle prodding. Dead fish were immediately removed from the experiment. Determination of sublethal concentrations The 96 h LC50 values (with 95% confidence limits) of potassium dichromate for freshwater carp L. rohita was estimated as 118.71 mg L1. Based on the 96 h LC50 value, the three test concentrations of potassium dichromate, viz. sublethal concentration I (SL-I; 1/4th of LC50 ¼ 29.5 mg L1), sublethal concentration II (SL-II; 1/2nd of LC50 ¼ 59 mg L1) and sublethal concentration III (SL-III; 3/4th of LC50 ¼ 88.5 mg L1) were estimated for in vivo exposure experiment. In vivo exposure experiment The fish specimens were exposed to the three aforementioned test concentrations of potassium dichromate in a static system. Healthy fish specimens of approximately same size in average (±S.D.) weight (21 ± 2.5 g) and length (13 ± 1.2 cm) were randomly selected and transferred to aquarium containing 50 litres of water containing different concentration of potassium dichromate along with one control. The exposure was continued up to 96 h and the tissue sampling was done at the intervals of 24, 48, 72 and 96 h at the rate of five specimens per sampling duration. For each test concentration and control, the experiment was replicated twice. This study was conducted following the OECD guideline in the static test conditions (OECD, 1992). Isolation of blood and gill tissues On each sampling, peripheral blood samples were collected from the caudal vein of both the control and treated specimens (5–6 fishes from each group on the consecutive days of exposure) using heparinized syringes for comet assay and micronuclei test. Further, the specimens were sacrificed, after anaesthesization with ethylene glycol monophenyl ether, by decapitation followed by dissection and the removal of gills tissues. The tissue samples were placed on ice for its viability and immediately processed for genotoxicity assessment. The physicochemical properties of test water, namely temperature, pH, conductivity, dissolved oxygen, chloride, total hardness and total alkalinity were analyzed by standard methods (American Public Health Association, American Water Works Association, Water Pollution Control Federation, 2005).
Genotoxicity assessment of chromium in Labeo rohita
DOI: 10.3109/01480545.2014.900066
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Comet assay (CA) The comet assay or alkaline single-cell gel electrophoresis was performed as a three layer procedure (Singh et al., 1988) with slight modifications (McKelvey-Martin et al., 1993). The blood cells were suspended in chilled phosphate buffered saline (PBS, pH 7) and the gill tissue (75 mg) was homogenized in PBS (pH 7) followed by centrifugation at 4000 rpm at 4 C for 5 min. Cell viability was evaluated using trypan blue exclusion test (Anderson et al., 1994) and the cell suspension showing 485% cell viability were further processed for CA. In brief, 20 mL of cell suspension was mixed with 80 mL of 0.5% low melting point agarose (LMPA) and layered on one end frosted glass slide, which was already coated with a layer of 200 mL of 1% normal agarose. The slide was again coated with third layer of 100 mL LMPA and covered with coverslip. After solidification, the coverslip was removed and the slide was immersed in cold lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH 10, and 10% DMSO & 1% TritonX-100 added fresh) and kept at 4 C overnight. After lysis, the slides were placed in a horizontal electrophoresis tank side by side. The tank was filled with fresh solution (1 mM Na2EDTA, 300 mM NaOH, 0.2% DMSO, pH 13.5) to a level of approximately 0.25 cm above the slides. The slides were left in the solution for 20 min to allow unwinding, and the electrophoresis was performed for 20 min at 16 V, 300 mA. The slides were then neutralized gently with 0.4 M Tris buffer at pH 7.5 and stained with 75 mL ethidium bromide (20 mg/mL). Two slides per specimen prepared and 25 cells per slide (200 cells per concentration) were scored randomly under florescent microscope (Leica-DMLS, Leica Microsystems GmbH, Wetzlar, Germany) equipped with appropriate filters and the cells analyzed using image analysis computer software (Komet–5.5 Kinetic Imaging, Merseyside, UK). The parameter selected for quantification of DNA damage was %Tail DNA (¼100 – %Head DNA), as determined by the software. Micronucleus test (MNT) Peripheral blood was smeared on pre-cleaned slides. After fixation in pure ethanol for 20 min, the smeared slide was allowed to air-dry, stained with 6% Giemsa for 30 min., washed in tap water, dried on hot plate and mounted in DPX (Di-N-butyle Phthalate in Xylene). Two slides were prepared from each specimen, and a total of 2000 erythrocyte cells were examined from each slide under 100X magnification. Small, non refractive, circular or ovoid chromatin bodies displaying same staining and focusing patterns, as the main nucleus, were scored as micronuclei (Al-Sabti & Metcalfe, 1995). Statistical analyses One way analysis of variance (ANOVA) was applied to compare the means obtained for various parameters among different concentrations and durations. The % MN frequencies were compared between concentrations within duration using Mann–Whitney test. A p value less than 0.05 were considered statistically significant. Lethal concentration values of potassium dichromate were calculated using SPSS (version 16.0,
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Chicago, IL) computer program with 95% confidence limits. The values for the biomarkers are expressed as mean ± SE (n ¼ 5).
Results Physicochemical properties of the test solution The temperature varied from 22.4 to 23.3 C, whereas the pH ranged from 7.4 to 7.6. The dissolved oxygen concentration was 6.6 to 7.2 mg L1. During the experimentation, the chloride, total hardness and total alkalinity of the test water ranged 42–44, 170–176 and 270–280 mg L1 as CaCO3, respectively. 96 h LC50 of potassium dichromate Potassium dichromate caused acute toxicity in a concentration dependent manner. Experimental conditions produced no mortality in the control. When administered at 160 and 180 mg L1 doses, potassium dichromate induced 100% cumulative mortality in the two replicate groups within 96 h of exposure. In the present study, the 96 h lethal concentrations of potassium dichromate to L. rohita have been presented in Table 1. The 96 h LC50 value was estimated to be 118 mg L1. Induction of DNA damage The single strand DNA breaks representing in the form of % tail DNA increased significantly (p50.05) with increasing concentrations and durations in the erythrocyte and gill tissues of potassium dichromate exposed fish specimens when compared to control. The highest DNA damage in both the tissues was found at 88.5 mg L1 concentration and 96 h post exposure. The DNA damage measured in terms of %tail DNA was found to be increased significantly (p50.05) from 9.16 to 20.08% in erythrocytes of exposed specimens, while in gill cells it increased from 12.60 to 23.01% with the progression of experiment (Figures 1 and 2 and Table 2). Induction of micronucleus The results of MN induction in erythrocytes of L. rohita after exposure to different test concentrations and sampling times of potassium dichromate are presented in Table 3. There was a significant increase in MN frequencies at all the exposed concentrations along with exposure durations when compared to control (Figure 3). The lowest concentration of potassium
Table 1. Various 96 h lethal concentrations of chromium in L. rohita. 95% Confidence limits LC values LC10 LC20 LC30 LC40 LC50 LC60 LC70 LC80 LC90
1
Concentrations (mg L )
Lower
Upper
100.618 106.493 110.941 114.888 118.705 122.648 127.012 132.317 140.042
1.855 4.062 7.141 11.553 18.087 28.248 45.246 76.621 122.953
118.150 121.993 124.946 127.649 130.408 133.557 137.823 146.542 206.434
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Figure 1. Fluorescence microscopy visualization of DNA damage induced by hexavalent chromium during acute exposure at 29.5 mg L1, 59.0 mg L1 and 88.5 mg L1 test concentrations using the Comet Assay in red blood cells of Labeo rohita. Red blood cells isolated from (a) control and (b) exposed fish. Ethidium bromide stained nuclei following electrophoresis shows ‘‘comet tail’’ in exposed cells. (Bar represents 10 m).
Figure 2. Fluorescence microscopy visualization of DNA damage induced by acute exposure to hexavalent chromium at 29.5 mg L1, 59.0 mg L1 and 88.5 mg L1 test concentrations using Comet Assay in gill cells of L. rohita. Gill cells isolated from (a) control and (b) exposed fish showing comet tail. (Bar represents 10 m).
Table 2. % Tail DNA in erythrocytes and gill cells of L. rohita exposed to different test concentrations of potassium dichromate. Exposure duration (h) Tissue
Test concentrations (mg L1)
Erythrocyte
24 A
Control 29.5 59.0 88.5 Control 29.5 59.0 88.5
Gill
48
72 A
4.12 ± 0.28 9.29 ± 0.04cA1 10.14 ± 0.32bA1 11.25 ± 0.31aB1 4.82 ± 0.31A 10.93 ± 0.08cB2 11.74 ± 0.09bA2 13.57 ± 0.17aB2
4.12 ± 0.28 9.16 ± 0.3bA1 9.83 ± 0.29abA1 10.48 ± 0.07aA1 4.82 ± 0.31A 10.5 ± 0.16bA2 11.23 ± 0.27abA2 11.48 ± 0.11aA2
96 A
4.12 ± 0.28 9.46 ± 0.95cA1 12.61 ± 0.16bB2 15.12 ± 0.04aC2 4.82 ± 0.31A 11.98 ± 0.16cC2 14.86 ± 0.17bcB1 16.78 ± 0.26aC1
4.12 ± 0.28A 11.66 ± 0.58cB1 13.82 ± 0.26bC2 20.08 ± 0.21aD2 4.82 ± 0.31A 12.06 ± 0.15cC1 17.04 ± 0.57bC1 23.01 ± 0.42aD1
Mean percentage of tail DNA (±SE) in the erythrocyte and gill cells (n ¼ 200 cells/concentration/duration) of L. rohita exposed to different sub-lethal concentration of potassium dichromate at different exposure durations. Values with different alphabet (lowercase) superscripts differ significantly (p50.05) between test concentrations within tissue and exposure duration. Values with different alphabets (uppercase) differ significantly (p50.05) between exposure durations within test concentration and tissue. Values with different numeric superscripts differ significantly (p50.05) between tissues within test concentration and exposure duration. Table 3. Micronuclei frequencies in erythrocytes of L. rohita exposed to potassium dichromate at different test concentrations and exposure durations. Exposure duration (h) 1
Test concentrations (mg L ) Control 29.5 59.0 88.5
24
48 a1
0.027 ± 0.023 0.072 ± 0.031a2 0.182 ± 0.035a3 0.296 ± 0.028a4
72 a1
0.028 ± 0.021 0.117 ± 0.026ab2 0.218 ± 0.27b3 0.268 ± 0.041a4
96 a1
0.028 ± 0.022 0.152 ± 0.02b3 0.225 ± 0.11b4 0.432 ± 0.03b5
0.029 ± 0.021a1 0.163 ± 0.029b3 0.273 ± 0.041b4 0.504 ± 0.029b5
Mean percentage of micronucleus frequencies (±SE) in erythrocytes of L. rohita (n ¼ 2000) exposed to different sub-lethal concentrations of potassium dichromate at different exposure durations. Values with numeric superscript differ significantly (p50.05) between exposure durations within test concentration. Values with alphabet superscript differ significantly (p50.05) between test concentrations within exposure duration.
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Figure 3. Cytological visualization of micronuclei induced by exposure to hexavalent chromium at 29.5 mg L1, 59.0 mg L1 and 88.5 mg L1 test concentrations using Micronuclei Test in red blood cells of L. rohita. Red blood cell isolated from (a) control showing no micronuclei and (b) exposed fish showing micronuclei as marked by an arrow. (Bar represents 10 m).
dichromate treatment in fish specimen induced MN frequency of 0.072% in blood erythrocyte in 24 h which was significantly increased to 0.163% with 96 h exposure. Similar trend was observed for SL-II in which the MN frequency of 0.182% in 24 h increased to 0.273% after 96 h. At the highest concentration, the MN frequency significantly increased from 0.296% at 24 h exposure to 0.504% after 96 h duration. The MN frequency increased with increasing concentrations and exposure durations and highest induction in MN frequencies was observed at SL-III concentration after 96 h post exposure.
Discussion Chromium induced genotoxic and mutagenic effect in L. rohita at various exposed concentrations along with exposure time duration. The micronuclei induction and DNA damage in erythrocytes and gill tissues was highest at the highest test concentration of 88.5 mg/L. Thus, the present study revealed that chromium is responsible for inducing micronuclei formations and DNA damage in erythrocyte and gills tissue of the fish. The combined approaches using these assays will help in providing broad perspective in aquatic toxicology, as fish gill cells are constantly being exposed to environmental pollutants while blood cells are simple to be handled. Fish are often used as sentinel organism for ecotoxicological studies because they play a number of roles in the trophic web, accumulate toxic substances and respond to low concentration of mutagens (Cavas & Ergene-Gozukara, 2005). Therefore, the use of fish biomarkers as indices of the effects of pollution, are of increasing importance and can permit early detection of aquatic environmental problems (Lopez-Barea, 1996; Van Der Oost et al., 2003). The test result of the LC50 of the present study at 96 h was 118.71 mg L1 which indicated that potassium dichromate is very toxic to fish. Our estimate is slightly lower than the 96 h LC50 values of hexavalent chromium (142 mg/L) in L. rohita (Jaffri et al., 2003) and comparatively higher than the 96 h LC50 value 43.7 mg L1 of potassium dichromate for tilapia, Oreochromis spp., as reported by Abbas & Ali (2007) and 91.5 mg L1 for Oreochromis areus as estimated by
Yilmaz et al., (2010). Interestingly, the 96 h LC50 111.45 mg/ L of the same toxicant in L. rohita (Vutukuru, 2005) was quite similar to our present study. This variation may be due to the disparity and hardiness of the experimental species, age, body size of the individuals and water quality parameters (Al-Akel, 1996). Genotoxicity can occur due to many physico-chemical agents that result in a wide variety of potential damages to the genetic material, ranging from various DNA adducts to single- and double-strand breakages, DNA-DNA and DNAprotein cross-links or even chromosomal breakage (Cavalcanti et al., 2010; Wang et al., 2009). The comet assay has been considered as sensitive, rapid and reliable method of quantitatively measuring DNA damage in eukaryotic and prokaryotic cells (Bajpayee et al., 2005; Cotelle & Ferard, 1999). It is increasingly being used in testing of substances such as industrial chemicals, biocides, agrochemicals, food additives and pharmaceuticals for genotoxicity testing (Brendler-Schwaab et al., 2005). The assay is favoured among other cytogenetic methods used for the detection of DNA damage (Buschini et al., 2003) as it is capable of detecting wide variety of DNA damage such as DNA singlestrand breaks (Sharbel, 2004). Micronucleus assay detects injuries that survive at least one mitotic cycle and allows for measurement of chromosome breakage or chromosome loss (Kirsch-Volders et al., 2003). The combined application of both the assays were suggested for the testing of genotoxins to understand the mechanisms underlying mutagenicity (Van Goethem et al., 1997). Therefore, comet assay and micronucleus assay were undertaken in the present study for evaluation of acute toxicity and genotoxicity of hexavalent chromium as also supported by Ahmed et al. (2013). The present study revealed that the Cr(VI) is genotoxic to the Indian major carp L. rohita. Our results are corroborated with the previous reports (Ahmad et al., 2006). In another study, medaka fin cell lines, exposed to Cr(VI) to examine the genotoxic potentials, have demonstrated double strand DNA breaks and chromosome damage in a concentration-dependent manner (Goodale et al., 2008). Abbas & Ali (2007) studied the genotoxic potential of Cr(VI) in Orechromis spp. using
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RAPD-PCR after exposure to sub lethal concentrations (43.7 mg L1) for 24 and 96 h. The results showed polymorphic bands in long time exposed fish, whereas no polymorphic bands found in short time exposed fish. The polymorphic bands appear due to fragmented DNA strands. Furthermore, studies have reported the genotoxic potential of Cr(VI) in Pimephales promelas (De Lemos et al., 2001) and in Channa punctatus (Kumar et al., 2012) based on the induction of micronucleus in erythrocytes. The DNA damage at higher test concentrations in the erythocytes and gills could be due to the elevated levels of hydroperoxides in both the tissues compared to their controls (Velma & Tchounwou, 2010). Further, the Cr (VI) is known as one of the core toxicants for degeneration of secondary gill lamellae (Sesha Srinivas & Rao, 1998). We applied the alkaline comet assay to evaluate total DNA strand breaks in the erythrocyte and gill cells of L. rohita exposed in vivo to different test concentrations of potassium dichromate. The data obtained showed that the frequencies of % tail DNA damage for all concentrations of potassium dichromate, tested in both tissues, were significantly higher (p50.01) than the control. For the tissues, higher the concentration and duration of potassium dichromate exposure, the higher the % tail DNA damage. While, the % tail DNA damage in gill tissues was slightly higher than the erythrocytes and the DNA damage was both dose- and time dependent. Hence, Cr(VI) specifically altered the gills at genetic level and further, the subtle effects seen at low concentrations suggest that gills are more sensitive to Cr (VI) toxicity than erythrocytes.
Conclusion The present study revealed that chromium induced DNA damage in erythrocyte and gill tissues of L. rohita as measured by the comet assay and micronuclei test. The acute toxicity estimates will eventually help to determine the safe dose of the chemical for a species and to establish water quality criteria for planning remedial measures to reduce the chromium pollution. The combined approaches using these assays will help in providing broad perspective in aquatic toxicology, as fish gill cells are constantly being exposed to environmental pollutants, while blood cells are simple to be handled. There is a great potential to make use of these assays for assessing detailed information on cellspecific genotoxic and mutagenic effects, inter-individual variability, adaptability, and further contributing in formulation of strategies and measures for the conservation of fish diversity.
Acknowledgements The authors are grateful to the Director, National Bureau of Fish Genetics Resources, Lucknow for providing necessary facilities and to Uttar Pradesh Council of Science & Technology (UP-CST), Lucknow for financial assistance.
Declaration of interest There are no conflicts of statement among the authors and with funding agency.
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DOI: 10.3109/01480545.2014.900066
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