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Ameliorative effects of vitamin supplementation on ethyl methane sulphonate-induced genotoxicity in a fish, Anabas testudineus
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Ecotoxicology and Environmental Safety 68 (2007) 63–70 www.elsevier.com/locate/ecoenv
Ameliorative effects of vitamin supplementation on ethyl methane sulphonate-induced genotoxicity in a fish, Anabas testudineus Bibhas Guha1, Jayanta Kumar Das, Anisur Rahman Khuda-Bukhsh Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India Received 11 October 2005; received in revised form 27 April 2006; accepted 5 June 2006 Available online 14 August 2006
Abstract The efficacy of 0.02% vitamin C (VC; L-ascorbic acid) and 0.05% b-carotene (BC) at the rate of 1 ml/100 g of body weight in amelioration of ethyl methane sulphonate (EMS)-induced genotoxicity has been studied in an Indian endemic fish, Anabas testudineus by using several cytogenetical endpoints like chromosome aberrations, micronuclei (MN) and abnormal nuclei (AN), and sperm head anomaly at 6, 24, 48, 72 and 96 h after treatment, as compared to suitable controls (distilled water (DW)-treated control for EMS and VC-treated fish, and 1% alcohol-treated control for BC-treated fish). Both VC and BC reduced EMS-induced genotoxicity at all the fixation intervals as compared to their respective controls. Additionally, effects of two more doses of VC (0.01% and 0.05%) and BC (0.02% and 0.1%) were analyzed at 72 h after treatment (at the peak period of EMS genotoxicity) for testing their relative efficacy in amelioration of EMS-induced cytogenetical damage in this fish. All the three doses of both VC and BC appeared to reduce the EMSinduced genotoxicity in this fish to a variable extent, of which the higher dose of VC appeared to give marginally better protection while the dose–response relationship was inconclusive for BC. The results of this study can lead to future research for exploring if low doses of these vitamins may be useful in protecting fish from genotoxic damage on exposure to mutagenic agents in small confined/stagnant waters. r 2006 Elsevier Inc. All rights reserved. Keywords: Anabas testudineus; Antimutagen; Ascorbic acid; b-carotene; Genotoxicity
1. Introduction Ethylmethane sulphonate (EMS), an alkylating agent is an extensively used chemical in genetic research for its clastogenic, mutagenic, teratogenic, and carcinogenic effects not only in various mammalian models but also for its similar actions on a few species of fish (Hooftman, 1981; Hooftman and Vink, 1981; Hooftman and de Raat, 1982; Guha and Khuda-Bukhsh, 2002a) including an Indian endemic species Anabas testudineus (Guha and Khuda-Bukhsh, 2002b). In recent years, however, the emphasis has been shifted to the search of agents that can either reverse or ameliorate the clastogenic/mutagenic potentials of genotoxic agents. On this count several Corresponding author.
E-mail addresses: [email protected], khudabukhsh_48@ rediffmail.com (A.R. Khuda-Bukhsh). 1 Presently at: Department of Zoology, School of Science, Netaji Subhas Open University, 1, Woodburn Park, Kolkata-700 020, India. 0147-6513/$ - see front matter r 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2006.06.001
vitamins, mainly in view of their non-toxic nature, have been extensively investigated for their possible protective role against artificial mutagenesis in some suitable mammalian models; e.g., vitamin C (VC) (Shamberger, 1984; Hayatsu et al., 1988; Hoda and Sinha, 1991,1993; Khan and Sinha, 1992, 1993, 1996), b-carotene (BC) (Burton and Ingold, 1984; Hayatsu et al., 1988), vitamin A (Dharmshila and Sinha, 1990; Sinha and Dharmshila, 1994), vitamin E (Hayatsu et al., 1988), etc. Positive protective ability of VC and BC against EMS-induced genotoxicity had earlier been reported in a cichlid fish Oreochromis mossambicus (Guha and Khuda-Bukhsh, 2002a, 2003). But the role of either VC or BC in modulating EMS-induced clastogenicity/ mutagenicity had not been tested before in the Indian climbing perch, A. testudineus, which has the ability to survive considerable periods of time outside water with the aid of its specialized accessory respiratory organ, more efficient in aerial mode of respiration. Therefore, the present investigation was undertaken to test the hypothesis
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that VC or BC can also ameliorate EMS-induced genotoxicity in another unrelated fish taxon, A. testudineus. The present study included several widely accepted cytogenetical end points, like study of chromosomal aberrations (CA), induction of micronuclei (MN), abnormal nuclei (AN), and sperm head abnormality (SHA) in the experimental and control specimens. Further, the relative efficacy of three different doses each of VC and BC have also been tested to ascertain if there is any dose–response relationship in the process of amelioration of EMS-induced genotoxicity in this fish species. 2. Materials and methods Healthy specimens of A. testudineus (Bloch) (family: Anabantidae) weighing between 18 and 25 g and measuring between 8 and 10 cm were collected from a local freshwater pond and were acclimated for 10 days at ambient temperature (28–32 1C) in a concrete vat (circular vat with a diameter and a height each of 1 m, half-filled with freshwater, stored from deep tube well) and fed with artificial diet (rice bran+wheat+oil cake) before they were used for the experiments. Separate sets of six specimens each were injected intramuscularly (bilaterally) with optimally low doses (that produced fairly quantifiable cytogenetical changes as revealed from range-finding trials) of: (1) 0.2% EMS, (2) 0.02% VC, (3) 0.2% EMS plus 0.02% VC, (4) 0.05% BC (dissolved in 1% ethyl alcohol; Alc), (5) 1% Alc (being the ‘‘vehicle’’ of BC), (6) 0.2% EMS plus 1% Alc, (7) 0.2% EMS plus 0.05% BC, and (8) distilled water (DW), each at the rate of 1 ml/100 g of body weight and were sacrificed at five different specific time periods after injection (subsequently mentioned as ‘‘fixation intervals’’), namely, 6, 24, 48, 72, and 96 h (since the peak damaging effect induced by EMS seemed to be obtained at 72 h and there was a declining trend until 96 h, fixation intervals beyond 96 h were not considered). Additionally, 5–6 specimens each were also injected with 0.05% and 0.1% VC and also 0.02% and 0.1% BC separately and conjointly with 0.2% EMS each at the rate of 1 ml/100 g bw to evaluate the relative efficacy of the different doses of VC or BC, if any. Injected specimens were transferred into smaller vats with a diameter and height of 1 ft, half-filled with freshwater at ambient temperature, and were fed with artificial diet until sacrificed. Specimens injected intramuscularly with double DW (being the ‘‘vehicle’’ of EMS) and with 1% Alc (being the ‘‘vehicle’’ of BC) at 1 ml/100 g bw served as controls. The nonnatural route was favored because the genotoxic effects or their ameliorations could be correlated for the exact dose of test chemicals injected into the body rather than leaving the fish to ingest these either with food or water resulting in differential intake/assimilation or removal through feces/excretion for different specimens. Besides, the possibility of differential absorption through surface and the management problem related with periodical changes of water medium with the test chemicals could also be avoided by the injection route. For chromosome analysis, experimental and control fish specimens of both sexes were injected intramuscularly with 0.05% colchicine at 1 ml/ 100 g bw about 3 h prior to sacrifice and their kidney chromosomes were prepared as per the citrate-flame drying technique (Khuda-Bukhsh, 1979). Clastogenic changes, both on individual chromosomes (e.g., breaks, constrictions, centric fusions, translocations, fragments, etc.) or on the whole chromosome set (e.g., polyploidy, pulverisation, stickiness, etc.) were recorded in 500 metaphase spreads from each series. In view of the small size of the chromosomes or owing to unfavorable disposition, sometimes some confusion regarding the determination of the exact nature of aberration, e.g., erosion or constriction, made placement of aberrations to a particular type difficult, for which reason the more important and clear aberrations were put under ‘‘major’’ types and the relatively less significant ones including the gaps and the other ‘‘difficult-to-place’’ ones were pulled together under the ‘‘other’’ types. The total aberration frequencies, i.e., the sum total of ‘‘major’’ and ‘‘other’’ type aberrations,
obtained from six specimens of each series were taken into account in the statistical analysis. For micronucleus testing, blood was collected from living fish specimens at different fixation intervals by puncturing their caudal peduncle and blood films were drawn on clean grease-free slides. Semidried slides were dipped in 90% ethyl alcohol briefly and allowed to air-dry. Air-dried slides were stained in May-Grunwald stain as per the procedure of Schmid (1976). The frequency of anomalous nuclei was recorded as per the method of Carrasco et al. (1990). For the study of SHA, epididymes of testis of the males were dissected and inner contents taken out in Fish-Ringer’s solution. Thin smears of the content were prepared on slides and stained with Giemsa. Sperm showing normal and abnormal head morphologies were recorded following the methods adopted by Manna and Biswas (1988). For the determination of % of suppression, calculation was made by following the formula: % suppression ¼ 100{(100/% number of abnormality observed in EMS treated series) multiplied by % number of abnormality observed in vitamin plus EMS treated series}; e.g., % Suppr. of CA between T1 vs. T3 series ¼ 100{(100/% of CA in T1) % of CA in T3} where T1 represents % number 0.2% EMS and T3 represents % data of 0.2% EMS plus 0.02% VC. The data were subjected to Andersion–Dearling Normality test and found to be within normal range. For further analysis of data, student’s t-test was conducted between data of treated and control fishes for the same fixation interval to test the level of significance, if any. Additionally, one-way ANOVA using SPSS 10.0 software program was performed for treated and control fishes to test the level of significance, if any, among different variables at different fixation intervals. During observation, the observer was ‘‘blinded’’ as to the origin of the slides (i.e., whether it was of the ‘‘control’’ or ‘‘treated’’ fish), which were only decoded after the completion of observation of the total lot of slides.
3. Results 3.1. Chromosome aberration study Normal metaphase complements of A. testudineus (PM 1) were carefully examined for any possible clastogenic/ mutagenic change due to treatments with the test chemicals or distilled water. Representative photomicrographs of various types of chromosomal aberrations like, chromatid break (PM 2), ring (PM 3), pulverization (PM 4), acentric fragment (PM 5), terminal association and stretching (PM 6), etc. have been provided mainly from different treated fishes (see Fig. 1). An analysis of data revealed that the frequencies of aberrations in the EMS-treated series were significantly higher than in the DW-treated series showing the clastogenic effect of EMS (Table 1) in this fish. On the other hand, the treatment of 0.02% VC also produced a small amount of CA as compared to DW-treated controls. The percentages of CA in the conjointly treated EMS+0.02% VC series were, however, found to be reduced as compared to only EMS-treated series at all the fixation intervals and the percentages of suppression values were significant (Po0.05–0.001) except at 24 h (see Table 1). Similar protective effects (Po0.05–0.01) were also found to be rendered by 0.05% BC to EMS-treated fish at all fixation intervals (see Table 1) except at 96 h, when the data were compared against values of the EMS+1% Alc-treated fish. On the other hand, in fish conjointly treated with both 1% alcohol and EMS, CA were considerably enhanced as compared to only EMS-
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Like CA, the frequency of MN and AN were also elevated in fish treated with 1% Alc along with 0.2% EMS. However, only BC (dissolved in 1% alcohol) also produced quite appreciable amount of MN and AN at all fixation intervals. ANOVA revealed the protective efficacy of 0.02% VC (T3) against 0.2% EMS (T1)-treated series at 24 and 96 h (a ¼ Po0.05), and of 0.05% BC (a ¼ Po0.05) (T7) against 0.2% EMS plus 1% Alc (T6) treated at 48 and 96 h with regard to micronucleated erythrocytes (MNE) (Histogram 1b in Appendix A). Further, the analysis also revealed the protective efficacy (a ¼ Po0.05, b ¼ Po0.01) of 0.05% BC (T7) against T6 at 48 and 96 h in respect of anomalous nuclei (AN) (Histogram 1c in Appendix A). 3.3. Sperm head abnormality study
Fig. 1. Photomicrographs of normal (PM 1) and aberrated (PM 2–6) metaphase complements of A. testudineus; chromatid break (PM 2), ring (PM 3), pulverization (PM 4), acentric fragment (PM 5), terminal association and stretching (PM 6). Normal erythrocytes (PM 7), micronucleated erythrocytes (PM 8–10), anomalous nuclei (PM 11–12); sperm with normal (PM 13) and abnormal head shapes (PM 14–16). CB ¼ chromatid break, R ¼ ring, F ¼ fragment, STR ¼ stretching, TA ¼ terminal association, MN ¼ micronucleated erythrocytes, ANanomalous nuclei, SHA ¼ sperm head anomaly and Bar ¼ 10 mm.
treated fish, showing some additive effects of alcohol to EMS-induced clastogenicity. Analysis of variance (ANOVA) revealed the protective efficacy (a ¼ Po0.05) of 0.02% VC (T3) at 96 h with regard to chromosome aberrations (CA) against 0.2% EMS (T1)-treated series (Histogram 1a in Appendix A). 3.2. Micronucleus study Micronucleated erythrocytes (MN) (PM 8–10) and erythrocytes with abnormal nuclei (AN) (PM 11–12) were observed in much greater number in fish treated with EMS alone and in fish treated with EMS+1% alcohol as compared to the numbers of MN and AN encountered in the only DW-treated or 1% alcohol-treated fish (Table 1). Further, EMS-treated fish counter injected with VC showed fewer micronuclei and anomalous nuclei as compared to only EMS-injected fish at all the fixation intervals examined, of which the percentages of suppression were statistically significant at 24, 48, 72 and 96 h (Po0.01) for micronuclei and at 72 h (Po0.01) for abnormal nuclei. Similar results were obtained in case of EMS+BC-treated fish, where the percentages of suppression, as compared to EMS+Alc-treated fish, were significant at all fixation intervals (Po0.05) except at 6 h (see Table 1) for MN and for AN, at 48, 72 and 96 h (Po0.05).
Sperm with normal (PM 13) and abnormal (PM 14–16) head shapes were encountered from different treated series. The frequency of sperm with abnormal head was less in EMS+VC (0.02%)-treated fish as compared to only EMStreated fish at all the fixation intervals, of which the percentages of suppression observed at 24 and 96 h were statistically significant (Po0.05) (see Table 1). In case of BC (0.05%) and EMS-treated series the percentages of abnormal sperm head were less than in the EMS+Alctreated series and the differences were statistically significant at all the fixation intervals (Po0.05–0.01; see Table 1). Fish treated with only 1% alcohol induced more genotoxic effects at all the fixation intervals than in the DW-treated controls. Alcohol also appeared to show an additive effect when treated conjointly with EMS. ANOVA revealed the protective efficacy (a ¼ Po0.05; b ¼ Po0.01) of 0.05% BC (T7) against 0.2% EMS plus 1% Alc (T6) at 6, 24 and 96 h in regard to sperm head anomaly (SHA) (Histogram 1d in Appendix A). 3.4. Dose–response study The combined treatment of each of the three doses of VC (i.e., 0.02%, 0.05%, and 0.1%) with 0.2% EMS showed a reduced frequency of chromosome aberrations at 72 h (Po0.001) as compared to those treated only with 0.2% EMS. However, the higher dose (0.1% VC) apparently reduced the cytogenetical effect appreciably (see Table 2). On the other hand, there was no clear indication of BC having a direct dose–response relationship as the medium dose showed apparently the maximum protective effect (Po0.05–0.01). In case of MN, the higher dose (0.1%) and in case of AN the lower dose (0.02%) of VC appeared to provide greater protection (Po0.05–0.01; see Table 2). The reverse was true in case of the conjoint treatment of BC and EMS, as the lower dose (0.02%) reduced the MN frequency to the maximum (Po0.05), as compared to that of EMS+Alc treated fish. However, the medium dose (0.05%) of BC reduced the frequency of AN (Po0.05; see Table 2) to a greater extent.
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Table 1 Frequency distribution of chromosome aberrations (CA), micronucleated erythrocytes (MNE), anomalous nuclei (AN), and sperm head abnormality (SHA) of Anabas testudineus treated with 0.2% EMS (T1), 0.02% VC (T2), 0.2% EMS+0.02% VC (T3), 0.05% BC (T4), 1% alcohol (T5), 0.2% EMS+1% alcohol (T6), 0.2% EMS+0.05% BC (T7) and their respective DW-treated controls (C) at different fixation intervals Fixation time intervals
6h
24 h
48 h
72 h
96 h
Series
Chromosome aberrations in kidney cells
Micronuclei and anomalous nuclei in peripheral erythrocytes
% major types CA
% other %7SE types CA
%7SE
C T1 T2 T3
0.00 2.40 0.00 1.49
1.40 5.90 1.05 5.46
1.4070.28 8.3070.32 1.0570.12 6.9570.48
T4 T5 T6 T7
0.71 0.51 3.94 1.87
3.57 3.08 5.17 3.50
4.2970.82 3.5970.78 9.1171.08 5.3770.32
C T1 T2 T3
0.44 4.69 0.51 3.32
1.80 3.67 2.02 3.71
2.2170.58 8.3770.60 2.5370.23 7.0370.82
T4 T5 T6 T7
0.74 0.49 4.31 2.09
3.19 2.46 5.02 3.95
3.9270.63 2.9670.18 10.0570.92 6.0570.60
C T1 T2 T3
0.24 3.49 0.24 3.01
2.62 6.28 1.70 3.66
2.8670.82 9.7770.67 1.9470.18 6.6770.62
T4 T5 T6 T7
0.43 0.71 4.71 1.99
3.66 2.62 5.46 3.19
4.0971.08 3.3370.72 10.1771.12 5.1870.62
C T1 T2 T3
0.00 4.73 0.00 3.33
1.65 8.18 1.44 4.79
1.6570.23 12.9170.67 1.4470.12 8.1370.32
T4 T5 T6 T7
0.73 0.25 5.20 2.86
2.43 2.01 8.00 5.71
3.1670.52 2.2670.13 13.2070.92 8.5770.48
C T1 T2 T3
0.00 2.73 0.50 2.25
0.93 7.35 1.76 4.49
0.9270.18 10.0870.42 2.2670.21 6.7470.60
T4 T5 T6 T7
0.41 0.26 3.63 2.14
2.86 3.16 6.05 5.48
3.4770.54 3.4270.58 9.6870.82 7.6270.58
% suppr.
T1 vs. T3 16.27a
T1 vs. T7 41.06a
T1 vs. T3 16.01
T1 vs. T7 39.80b
T1 vs. T3 31.73b
T1 vs. T7 49.07b
T1 vs. T3 37.03c
T1 vs. T7 35.08b
T1 vs. T3 32.06b
T1 vs. T7 21.28
0.0070.00 0.0870.03 0.0070.00 0.0470.02 0.0370.01 0.0270.01 0.1170.04 0.0470.02 0.0470.01 0.1870.02 0.0770.02 0.1070.01 0.0770.01 0.0370.01 0.2270.04 0.1170.02 0.0270.01 0.1770.02 0.0770.01 0.0970.01 0.0970.02 0.0270.01 0.1870.02 0.1270.01 0.0270.01 0.2170.02 0.0670.01 0.1270.01 0.0770.01 0.0370.01 0.2370.03 0.1470.02 0.0270.01 0.1470.01 0.0670.01 0.0970.01 0.0670.01 0.0370.01 0.1770.02 0.1070.02
% suppr. %7SE
T1 vs. T3 50.00
T1 vs. T7 63.64
T1 vs. T3 44.44b
T1 vs. T7 50.00a
T1 vs. T3 47.06b
T1 vs. T7 33.33a
T1 vs. T3 42.86b
T1 vs. T7 39.13a
T1 vs. T3 35.71b
T1 vs. T7 41.18a
0.9870.12 7.9371.12 2.1370.52 5.8570.32 4.9870.54 3.6271.08 8.5471.41 6.2371.08 1.0270.21 10.4170.72 3.2370.60 8.3771.73 6.1271.23 3.3670.60 8.9170.82 8.1670.64 1.0970.23 9.4370.45 3.4370.82 8.7971.23 5.3170.82 5.0170.92 10.1471.12 6.7970.73 1.2370.17 8.1870.79 3.5170.38 4.7070.52 5.5970.98 2.8270.58 8.4870.98 5.8570.58 0.8670.12 7.4770.52 3.9170.78 6.8671.62 6.6871.12 4.5270.82 11.2371.38 6.7171.32
Sperm head anomaly
% suppr. %7SE
T1 vs. T3 26.23
T1 vs. T7 27.05
T1 vs. T3 19.60
T1 vs. T7 8.42
T1 vs. T3 6.79
T1 vs. T7 33.04a
T1 vs. T3 42.55b
T1 vs. T7 31.01a
T1 vs. T3 8.17
T1 vs. T7 40.25a
0.8770.11 4.2870.53 1.0870.13 3.0970.60 2.9870.64 1.9470.32 6.5170.78 4.1070.42 0.8170.13 7.7670.73 1.0270.12 3.9770.62 2.1570.78 1.9870.76 6.7570.34 3.8470.78 0.5170.21 6.2570.82 1.0570.09 4.5670.89 2.5270.32 1.7370.28 10.0071.20 5.1770.82 0.5270.09 5.2570.98 1.6770.18 4.6070.60 2.9370.36 2.5670.49 10.4971.09 4.3470.62 0.7170.10 5.7370.73 1.0970.32 2.8870.38 3.1070.68 1.7570.42 7.5270.48 4.4670.41
% suppr.
T1 vs. T3 27.80
T1 vs. T7 37.02a
T1 vs. T3 48.84a
T1 vs. T7 43.11b
T1 vs. T3 27.04
T1 vs. T7 48.30b
T1 vs. T3 12.38
T1 vs. T7 58.63b
T1 vs. T3 49.74a
T1 vs. T7 40.69b
Major type CA include break, centric fusion, translocation, fragment, pulverization, ring, constriction, polyploidy, and aneuploidy. Other types of CA include stickiness, precocious centromeric separation, terminal association, erosion, condensation and other unclassified aberrations. No. of individuals examined in each series/fixation intervals ¼ 6; cells scored per individual ¼ 500 for CA, 20,000 for MNE and AN, 130 for Cyt/Nu ratio, 6000 for SHA. a ¼ po0.05, b ¼ po0.01, c ¼ po0.001.
Table 2 Frequency distribution of chromosome aberrations, micronucleated erythrocytes (MNE), anomalous nuclei (AN) and sperm head abnormality (SHA) of Anabas testudineus treated with 0.2 % EMS (T1), 0.02 % VC (T2), 0.05% VC (T3), 0.1% VC (T4), 0.2% EMS+0.02% VC (T5), 0.2% EMS+0.05% VC (T6), 0.2% EMS+0.1% VC (T7), 0.02 % BC (T8), 0.05% BC (T9), 0.1% BC (T10), 1% Alc (T11), 0.2% EMS+1% Alc (T12), 0.2% EMS+0.02% BC (T13), 0.2% EMS+0.05% BC (T14), 0.2% EMS+0.1% BC (T15) and their respective DW-water treated controls (C) at 72 h fixation intervals
72 h
Series
Chromosome aberrations in kidney cells
% otherr types CA
% of CA7SE
C T1 T2 T3 T4 T5
0.00 4.73 0.00 0.00 0.00 3.33
1.65 8.18 1.44 1.11 1.28 4.79
1.6570.23 12.9170.67 1.4470.12 1.1170.21 1.2870.08 8.1370.32
T6
2.93
4.53
7.4770.42
T7
2.50
4.06
6.5670.23
T8 T9 T10 T11 T12 T13
0.48 0.73 1.03 0.25 4.22 2.79
2.85 2.43 4.36 2.01 8.98 6.74
3.8170.72 3.1670.52 5.3871.02 2.2670.13 13.2070.92 9.5370.83
T14
2.88
5.78
8.5770.48
T15
2.88
7.74
10.6270.52
% of suppr.
T1 vs. T5 37.03c T1 vs. T6 42.14c T1 vs. T7 49.19c
T12 vs. T13 27.10a T12 vs. T14 35.08b T12 vs. T15 19.55a
Sperm head anomaly
% of MNE7SE % of suppr.
% of AN7SE
% of SHA7SE
0.0270.01 0.2170.02 0.0670.01 0.0270.01 0.0470.01 0.1270.01
1.2370.17 8.1870.79 3.5170.38 3.3570.98 2.8970.321 4.7070.52
0.1270.02 0.1170.03 0.0770.01 0.0770.01 0.0970.02 0.0370.01 0.2370.03 0.1270.03 0.1470.02 0.1570.02
T1 vs. T5 42.86a T1 vs. T6 42.86a T1 vs. T7 47.62b
T12 vs. T13 47.83a T12 vs. T14 39.13a T12 vs. T15 34.79
6.5670.81 5.7970.72 3.0470.71 5.5970.98 3.5270.83 2.2870.58 8.4870.98 6.8271.03 5.8570.58 5.8870.59
% of suppr.
T1 vs. T5 42.55b T1 vs. T6 19.80 T1 vs. T7 29.22
T12 vs. T13 19.58 T12 vs. T14 31.01a T12 vs. T15 30.66a
0.5270.09 5.2570.98 1.6770.18 0.9070.21 1.0570.32 4.6070.60 2.9570.38 2.5870.28 2.4570.70 2.9370.36 3.0870.82 2.5670.49 10.4971.09 4.8070.33 4.3470.62 4.1870.62
% of suppr.
T1 vs. T5 10.38 T1 vs. T6 43.81 T1 vs. T7 50.86
T12 vs. T13 54.24b T12 vs. T14 58.63b T12 vs. T15 60.15b
Major type CA include break, centric fusion, translocation, fragment, pulverization, ring, constriction, polyploidy and aneuploidy. Other type CA include stickiness, precocious centromeric separation, terminal association, erosion, condensation, and other unclassified aberrations. No. of individuals examined in each series/fixation intervals ¼ 6; cells scored per individual ¼ 500 for CA, 20,000 for MNE and AN, 130 for Cyt/Nu ratio, 6000 for SHA. a ¼ po0.05, b ¼ po0.01, c ¼ po0.001.
ARTICLE IN PRESS
% major types CA
Micronuclei and anomalous nuclei in peripheral erythrocytes
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Fixation time intervals
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The combined treatment of each of the three doses of VC and EMS reduced the frequency of sperm head abnormality as compared to those treated only with EMS. The higher dose (0.1%) of VC appeared to show more suppression of damage to sperm head than the other two doses (see Table 2). Similarly, there was an indication of the higher dose of BC (0.1%) being slightly more effective (Po0.01; see Table 2) in rendering protection to sperm head than the other two doses. ANOVA revealed the protective efficacy of 0.02% VC (T5) against 0.2% EMS (T1) and 0.02% BC (T13), 0.05% BC (T14) and 0.1% BC (T15) against 0.2% EMS plus 1% Alc (T12) in regard to CA (b ¼ Po0.01), and in regard to AN (b ¼ Po0.01) at 72 h. Similarly, there was protective ability (a ¼ Po0.05) of 0.02% BC (T13), 0.05% BC (T14) and 0.1% BC (T15) against 0.2% EMS plus 1% Alc (T12) in regard to SHA (Histogram 2 in Appendix A).
4. Discussion EMS has been extensively tested in a wide variety of animals and this mono-functional alkylating agent has been reported to cause major chromosomal aberrations including chromosomal breakage in mammalian models, by binding to DNA regions rich in G–C base pairs, causing those region to become unstable or by disrupting the main chain of DNA, perhaps in regions where protein is bound (see Malacinski and Freifelder, 1988). In the present study, EMS was found to induce various types of chromosome aberrations, nuclear anomalies including micronuclei formation and abnormal sperm head shapes. Earlier, Hooftman (1981) reported the induction of chromosome aberrations in killifish, Notobranchius rachowi after the treatment with ethylmethane sulphonate or benzo(a)pyrene. Subsequently, Hooftman and de Raat (1982) reported the induction of nuclear anomalies including micronuclei formation in the erythrocytes of another fish, Umbra pygmeae, treated with EMS. Micronuclei are one or rarely multiple supernumerary nuclear structures often observed as a result of xenobiotic treatment within the cytoplasm of an erythrocyte having the following characteristics: a distinct outline to the structure, a round, almond or ovoid 1 shape, a diameter 15 20 th of the erythrocyte, staining characteristics consistent with chromatin of the main nucleus, either dissociated from or connected to the main nucleus by a very thin basophilic strand. Micronuclei are believed to derive from chromosomal fragments or whole chromosomes that are not incorporated into daughter nuclei at the time of cell division. Therefore, micronucleus testing has been used extensively in fish in vivo and recommended as a potent parameter to designate clastogenic efficiency of a chemical (Heddle et al., 1983; Metcalf, 1988; Matsumoto and Co’lus, 2000; Grisolia and Cordeiro, 2000; Porto et al., 2005) although an in vitro binucleated blocked hepatic cell technique has also been found to be useful for genotoxicity testing in fish (Al-Sabti, 1995).
In the present study, the genotoxic effects of EMS gradually increased up to 72 h after treatment but the genotoxic effects appeared to decline thereafter, except for sperm head abnormality. Interestingly, the genotoxic effects of EMS were considerably reduced when different doses of VC or BC were also injected alongside. Such protective effects of some vitamins have also been reported against various mutagens and toxicants and linked mainly to their anti-oxidant nature (Machlin and Bendich, 1987; Hayatsu et al., 1988; Sato et al., 1990) and also for their nucleophilic character in some cases (Krishna et al., 1986). Alternatively, the antimutagenic effect of VC has also been suggested to be mediated by various detoxication enzymes (Goncharova, 1984). VC has also been suggested to prevent the production of electrophilic metabolites (Goncharova and Kuzhir, 1989). As VC has marked nucleophilic properties it might intercept reactive electrophilic metabolites, thereby preventing their attack on nucleophilic sites on DNA, and hence blocking the adduct formation (Bhattacharya et al., 1987; Liehr et al., 1989). However, VC has also been claimed to exert its protective effect through multiple inhibition mechanisms (Flora and Ramel, 1988). Interestingly enough, the treatment of ascorbic acid alone to the fish produced some amount of chromosome damage, nuclear anomalies and sperm head anomaly. Stich et al. (1979) demonstrated the chromosome breaking ability of VC in mammalian cells earlier. This might be possible as sometimes antioxidants under certain conditions have been reported to have potential to act as prooxidants (Wang and Russel, 1999; Devsagayam and Kamat, 2000). VC apparently has anti-spermatotoxic activity as its administration also protected sperm from EMS-induced damage. Various mutagens particularly pesticides have earlier been reported to induce abnormality in sperm head and thereby adversely affect male reproductive potentiality by causing decrease in sperm count (Whorton et al., 1977). Further, various types of structural alterations in chromosomes have been reported that led to pairing impairments among homologues and division-disruptive changes in spermatocytes of mice (Bhunya and Behera, 1987; Pandey et al., 1990). Mutagens affecting germ cells generally induce sperm head abnormalities in mice (Topham, 1980) due to alterations in testicular DNA and sperm chromatin structure (Evenson et al., 1986). Many enzymatic functions of vitamin C are essential for the normal integrity and function of testes, i.e., the synthesis, development and maintenance of normal sperm (Dawson et al., 1990). 5. Conclusions Although in the present study, there was no conclusive indication of dose–response relationship particularly with regard to the protection rendered by BC against EMS in fish, VC had some positive dose–response in rendering
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protection. Khan and Sinha (1993) also reported a differential dose–response of vitamin C in mice. They reported that higher dose of VC (10, 20 and 40 mg/kg bw/ day) exhibited a greater combating ability against clastogenecity induced by several pesticides. However, the lower doses of VC also reduced toxin-induced cytogenetic damage to a considerable extent but they could not bring the frequency of damages down to the control level (Hoda and Sinha, 1991; Hoda et al., 1992; Sinha and Bose, 1992). It was argued that higher dose of VC could possibly compensate for the urinary loss of VC (Yokogoshi et al., 1983) and/or might be required for maintaining its threshold level for antioxidant activities in tissues (Wayner et al., 1986). Although EMS has been reported to cause extensive chromosomal damage in a variety of fish species, e.g., N.s rochowi (Hooftman, 1981), Umbra pygmaea (Hooftman and de Raat, 1982), A. testudineus (Guha and KhudaBukhsh, 2002b), modulating effect of BC on EMS-induced genotoxicity has only been documented so far in one species of fish, Oreochromis mossambicus (Guha and Khuda-Bukhsh, 2002a, 2003). However, similar protective effect of BC on genotoxicity induced by some other mutagen, cyclophosphamide (CPA) has also been reported in mice (Mukherjee et al., 1991; Salvadori et al., 1992) and on human hepatoma cells (Salvadori et al, 1993). As an antioxidant, BC is expected to quench active oxygen species and to trap free radicals (Burton and Ingold, 1984; Hayatsu et al., 1988; Sato et al., 1990; Mukherjee et al., 1991) in the same manner as VC. Therefore, the favorable modulations of the EMS-induced genotoxicity by both VC and BC might have been achieved by the common radical scavenging property of both. Interestingly, in all the treatment series of EMS plus Alc, there appeared to be some increased damage when individual data were compared to only EMS-treated lots, thereby showing some additive damaging effect of alcohol when conjointly treated with EMS. Thus, the genotoxic potentials of EMS have been confirmed in this fish, which proved to act as a good cytogenetical model. It may be pointed out that relatively low doses of both the vitamins injected intramuscularly could quite effectively ameliorate the genotoxic effects induced by EMS in this fish, as revealed from the different cytogenetical endpoints studied. Therefore, for this experiment using injection route to be eventually useful in the context of fish farming, it remains to be demonstrated whether these vitamins placed in water or food would also be protective, particularly in closed and stagnant water bodies contaminated with such mutagenic agents.
Acknowledgments The authors are grateful to ICAR, Government of India, New Delhi, for providing financial support of the work.
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Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv. 2006.06.001.
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Ecotoxicology and Environmental Safety 68 (2007) 63–70 www.elsevier.com/locate/ecoenv
Ameliorative effects of vitamin supplementation on ethyl methane sulphonate-induced genotoxicity in a fish, Anabas testudineus Bibhas Guha1, Jayanta Kumar Das, Anisur Rahman Khuda-Bukhsh Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani-741235, West Bengal, India Received 11 October 2005; received in revised form 27 April 2006; accepted 5 June 2006 Available online 14 August 2006
Abstract The efficacy of 0.02% vitamin C (VC; L-ascorbic acid) and 0.05% b-carotene (BC) at the rate of 1 ml/100 g of body weight in amelioration of ethyl methane sulphonate (EMS)-induced genotoxicity has been studied in an Indian endemic fish, Anabas testudineus by using several cytogenetical endpoints like chromosome aberrations, micronuclei (MN) and abnormal nuclei (AN), and sperm head anomaly at 6, 24, 48, 72 and 96 h after treatment, as compared to suitable controls (distilled water (DW)-treated control for EMS and VC-treated fish, and 1% alcohol-treated control for BC-treated fish). Both VC and BC reduced EMS-induced genotoxicity at all the fixation intervals as compared to their respective controls. Additionally, effects of two more doses of VC (0.01% and 0.05%) and BC (0.02% and 0.1%) were analyzed at 72 h after treatment (at the peak period of EMS genotoxicity) for testing their relative efficacy in amelioration of EMS-induced cytogenetical damage in this fish. All the three doses of both VC and BC appeared to reduce the EMSinduced genotoxicity in this fish to a variable extent, of which the higher dose of VC appeared to give marginally better protection while the dose–response relationship was inconclusive for BC. The results of this study can lead to future research for exploring if low doses of these vitamins may be useful in protecting fish from genotoxic damage on exposure to mutagenic agents in small confined/stagnant waters. r 2006 Elsevier Inc. All rights reserved. Keywords: Anabas testudineus; Antimutagen; Ascorbic acid; b-carotene; Genotoxicity
1. Introduction Ethylmethane sulphonate (EMS), an alkylating agent is an extensively used chemical in genetic research for its clastogenic, mutagenic, teratogenic, and carcinogenic effects not only in various mammalian models but also for its similar actions on a few species of fish (Hooftman, 1981; Hooftman and Vink, 1981; Hooftman and de Raat, 1982; Guha and Khuda-Bukhsh, 2002a) including an Indian endemic species Anabas testudineus (Guha and Khuda-Bukhsh, 2002b). In recent years, however, the emphasis has been shifted to the search of agents that can either reverse or ameliorate the clastogenic/mutagenic potentials of genotoxic agents. On this count several Corresponding author.
E-mail addresses: [email protected], khudabukhsh_48@ rediffmail.com (A.R. Khuda-Bukhsh). 1 Presently at: Department of Zoology, School of Science, Netaji Subhas Open University, 1, Woodburn Park, Kolkata-700 020, India. 0147-6513/$ - see front matter r 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ecoenv.2006.06.001
vitamins, mainly in view of their non-toxic nature, have been extensively investigated for their possible protective role against artificial mutagenesis in some suitable mammalian models; e.g., vitamin C (VC) (Shamberger, 1984; Hayatsu et al., 1988; Hoda and Sinha, 1991,1993; Khan and Sinha, 1992, 1993, 1996), b-carotene (BC) (Burton and Ingold, 1984; Hayatsu et al., 1988), vitamin A (Dharmshila and Sinha, 1990; Sinha and Dharmshila, 1994), vitamin E (Hayatsu et al., 1988), etc. Positive protective ability of VC and BC against EMS-induced genotoxicity had earlier been reported in a cichlid fish Oreochromis mossambicus (Guha and Khuda-Bukhsh, 2002a, 2003). But the role of either VC or BC in modulating EMS-induced clastogenicity/ mutagenicity had not been tested before in the Indian climbing perch, A. testudineus, which has the ability to survive considerable periods of time outside water with the aid of its specialized accessory respiratory organ, more efficient in aerial mode of respiration. Therefore, the present investigation was undertaken to test the hypothesis
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that VC or BC can also ameliorate EMS-induced genotoxicity in another unrelated fish taxon, A. testudineus. The present study included several widely accepted cytogenetical end points, like study of chromosomal aberrations (CA), induction of micronuclei (MN), abnormal nuclei (AN), and sperm head abnormality (SHA) in the experimental and control specimens. Further, the relative efficacy of three different doses each of VC and BC have also been tested to ascertain if there is any dose–response relationship in the process of amelioration of EMS-induced genotoxicity in this fish species. 2. Materials and methods Healthy specimens of A. testudineus (Bloch) (family: Anabantidae) weighing between 18 and 25 g and measuring between 8 and 10 cm were collected from a local freshwater pond and were acclimated for 10 days at ambient temperature (28–32 1C) in a concrete vat (circular vat with a diameter and a height each of 1 m, half-filled with freshwater, stored from deep tube well) and fed with artificial diet (rice bran+wheat+oil cake) before they were used for the experiments. Separate sets of six specimens each were injected intramuscularly (bilaterally) with optimally low doses (that produced fairly quantifiable cytogenetical changes as revealed from range-finding trials) of: (1) 0.2% EMS, (2) 0.02% VC, (3) 0.2% EMS plus 0.02% VC, (4) 0.05% BC (dissolved in 1% ethyl alcohol; Alc), (5) 1% Alc (being the ‘‘vehicle’’ of BC), (6) 0.2% EMS plus 1% Alc, (7) 0.2% EMS plus 0.05% BC, and (8) distilled water (DW), each at the rate of 1 ml/100 g of body weight and were sacrificed at five different specific time periods after injection (subsequently mentioned as ‘‘fixation intervals’’), namely, 6, 24, 48, 72, and 96 h (since the peak damaging effect induced by EMS seemed to be obtained at 72 h and there was a declining trend until 96 h, fixation intervals beyond 96 h were not considered). Additionally, 5–6 specimens each were also injected with 0.05% and 0.1% VC and also 0.02% and 0.1% BC separately and conjointly with 0.2% EMS each at the rate of 1 ml/100 g bw to evaluate the relative efficacy of the different doses of VC or BC, if any. Injected specimens were transferred into smaller vats with a diameter and height of 1 ft, half-filled with freshwater at ambient temperature, and were fed with artificial diet until sacrificed. Specimens injected intramuscularly with double DW (being the ‘‘vehicle’’ of EMS) and with 1% Alc (being the ‘‘vehicle’’ of BC) at 1 ml/100 g bw served as controls. The nonnatural route was favored because the genotoxic effects or their ameliorations could be correlated for the exact dose of test chemicals injected into the body rather than leaving the fish to ingest these either with food or water resulting in differential intake/assimilation or removal through feces/excretion for different specimens. Besides, the possibility of differential absorption through surface and the management problem related with periodical changes of water medium with the test chemicals could also be avoided by the injection route. For chromosome analysis, experimental and control fish specimens of both sexes were injected intramuscularly with 0.05% colchicine at 1 ml/ 100 g bw about 3 h prior to sacrifice and their kidney chromosomes were prepared as per the citrate-flame drying technique (Khuda-Bukhsh, 1979). Clastogenic changes, both on individual chromosomes (e.g., breaks, constrictions, centric fusions, translocations, fragments, etc.) or on the whole chromosome set (e.g., polyploidy, pulverisation, stickiness, etc.) were recorded in 500 metaphase spreads from each series. In view of the small size of the chromosomes or owing to unfavorable disposition, sometimes some confusion regarding the determination of the exact nature of aberration, e.g., erosion or constriction, made placement of aberrations to a particular type difficult, for which reason the more important and clear aberrations were put under ‘‘major’’ types and the relatively less significant ones including the gaps and the other ‘‘difficult-to-place’’ ones were pulled together under the ‘‘other’’ types. The total aberration frequencies, i.e., the sum total of ‘‘major’’ and ‘‘other’’ type aberrations,
obtained from six specimens of each series were taken into account in the statistical analysis. For micronucleus testing, blood was collected from living fish specimens at different fixation intervals by puncturing their caudal peduncle and blood films were drawn on clean grease-free slides. Semidried slides were dipped in 90% ethyl alcohol briefly and allowed to air-dry. Air-dried slides were stained in May-Grunwald stain as per the procedure of Schmid (1976). The frequency of anomalous nuclei was recorded as per the method of Carrasco et al. (1990). For the study of SHA, epididymes of testis of the males were dissected and inner contents taken out in Fish-Ringer’s solution. Thin smears of the content were prepared on slides and stained with Giemsa. Sperm showing normal and abnormal head morphologies were recorded following the methods adopted by Manna and Biswas (1988). For the determination of % of suppression, calculation was made by following the formula: % suppression ¼ 100{(100/% number of abnormality observed in EMS treated series) multiplied by % number of abnormality observed in vitamin plus EMS treated series}; e.g., % Suppr. of CA between T1 vs. T3 series ¼ 100{(100/% of CA in T1) % of CA in T3} where T1 represents % number 0.2% EMS and T3 represents % data of 0.2% EMS plus 0.02% VC. The data were subjected to Andersion–Dearling Normality test and found to be within normal range. For further analysis of data, student’s t-test was conducted between data of treated and control fishes for the same fixation interval to test the level of significance, if any. Additionally, one-way ANOVA using SPSS 10.0 software program was performed for treated and control fishes to test the level of significance, if any, among different variables at different fixation intervals. During observation, the observer was ‘‘blinded’’ as to the origin of the slides (i.e., whether it was of the ‘‘control’’ or ‘‘treated’’ fish), which were only decoded after the completion of observation of the total lot of slides.
3. Results 3.1. Chromosome aberration study Normal metaphase complements of A. testudineus (PM 1) were carefully examined for any possible clastogenic/ mutagenic change due to treatments with the test chemicals or distilled water. Representative photomicrographs of various types of chromosomal aberrations like, chromatid break (PM 2), ring (PM 3), pulverization (PM 4), acentric fragment (PM 5), terminal association and stretching (PM 6), etc. have been provided mainly from different treated fishes (see Fig. 1). An analysis of data revealed that the frequencies of aberrations in the EMS-treated series were significantly higher than in the DW-treated series showing the clastogenic effect of EMS (Table 1) in this fish. On the other hand, the treatment of 0.02% VC also produced a small amount of CA as compared to DW-treated controls. The percentages of CA in the conjointly treated EMS+0.02% VC series were, however, found to be reduced as compared to only EMS-treated series at all the fixation intervals and the percentages of suppression values were significant (Po0.05–0.001) except at 24 h (see Table 1). Similar protective effects (Po0.05–0.01) were also found to be rendered by 0.05% BC to EMS-treated fish at all fixation intervals (see Table 1) except at 96 h, when the data were compared against values of the EMS+1% Alc-treated fish. On the other hand, in fish conjointly treated with both 1% alcohol and EMS, CA were considerably enhanced as compared to only EMS-
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Like CA, the frequency of MN and AN were also elevated in fish treated with 1% Alc along with 0.2% EMS. However, only BC (dissolved in 1% alcohol) also produced quite appreciable amount of MN and AN at all fixation intervals. ANOVA revealed the protective efficacy of 0.02% VC (T3) against 0.2% EMS (T1)-treated series at 24 and 96 h (a ¼ Po0.05), and of 0.05% BC (a ¼ Po0.05) (T7) against 0.2% EMS plus 1% Alc (T6) treated at 48 and 96 h with regard to micronucleated erythrocytes (MNE) (Histogram 1b in Appendix A). Further, the analysis also revealed the protective efficacy (a ¼ Po0.05, b ¼ Po0.01) of 0.05% BC (T7) against T6 at 48 and 96 h in respect of anomalous nuclei (AN) (Histogram 1c in Appendix A). 3.3. Sperm head abnormality study
Fig. 1. Photomicrographs of normal (PM 1) and aberrated (PM 2–6) metaphase complements of A. testudineus; chromatid break (PM 2), ring (PM 3), pulverization (PM 4), acentric fragment (PM 5), terminal association and stretching (PM 6). Normal erythrocytes (PM 7), micronucleated erythrocytes (PM 8–10), anomalous nuclei (PM 11–12); sperm with normal (PM 13) and abnormal head shapes (PM 14–16). CB ¼ chromatid break, R ¼ ring, F ¼ fragment, STR ¼ stretching, TA ¼ terminal association, MN ¼ micronucleated erythrocytes, ANanomalous nuclei, SHA ¼ sperm head anomaly and Bar ¼ 10 mm.
treated fish, showing some additive effects of alcohol to EMS-induced clastogenicity. Analysis of variance (ANOVA) revealed the protective efficacy (a ¼ Po0.05) of 0.02% VC (T3) at 96 h with regard to chromosome aberrations (CA) against 0.2% EMS (T1)-treated series (Histogram 1a in Appendix A). 3.2. Micronucleus study Micronucleated erythrocytes (MN) (PM 8–10) and erythrocytes with abnormal nuclei (AN) (PM 11–12) were observed in much greater number in fish treated with EMS alone and in fish treated with EMS+1% alcohol as compared to the numbers of MN and AN encountered in the only DW-treated or 1% alcohol-treated fish (Table 1). Further, EMS-treated fish counter injected with VC showed fewer micronuclei and anomalous nuclei as compared to only EMS-injected fish at all the fixation intervals examined, of which the percentages of suppression were statistically significant at 24, 48, 72 and 96 h (Po0.01) for micronuclei and at 72 h (Po0.01) for abnormal nuclei. Similar results were obtained in case of EMS+BC-treated fish, where the percentages of suppression, as compared to EMS+Alc-treated fish, were significant at all fixation intervals (Po0.05) except at 6 h (see Table 1) for MN and for AN, at 48, 72 and 96 h (Po0.05).
Sperm with normal (PM 13) and abnormal (PM 14–16) head shapes were encountered from different treated series. The frequency of sperm with abnormal head was less in EMS+VC (0.02%)-treated fish as compared to only EMStreated fish at all the fixation intervals, of which the percentages of suppression observed at 24 and 96 h were statistically significant (Po0.05) (see Table 1). In case of BC (0.05%) and EMS-treated series the percentages of abnormal sperm head were less than in the EMS+Alctreated series and the differences were statistically significant at all the fixation intervals (Po0.05–0.01; see Table 1). Fish treated with only 1% alcohol induced more genotoxic effects at all the fixation intervals than in the DW-treated controls. Alcohol also appeared to show an additive effect when treated conjointly with EMS. ANOVA revealed the protective efficacy (a ¼ Po0.05; b ¼ Po0.01) of 0.05% BC (T7) against 0.2% EMS plus 1% Alc (T6) at 6, 24 and 96 h in regard to sperm head anomaly (SHA) (Histogram 1d in Appendix A). 3.4. Dose–response study The combined treatment of each of the three doses of VC (i.e., 0.02%, 0.05%, and 0.1%) with 0.2% EMS showed a reduced frequency of chromosome aberrations at 72 h (Po0.001) as compared to those treated only with 0.2% EMS. However, the higher dose (0.1% VC) apparently reduced the cytogenetical effect appreciably (see Table 2). On the other hand, there was no clear indication of BC having a direct dose–response relationship as the medium dose showed apparently the maximum protective effect (Po0.05–0.01). In case of MN, the higher dose (0.1%) and in case of AN the lower dose (0.02%) of VC appeared to provide greater protection (Po0.05–0.01; see Table 2). The reverse was true in case of the conjoint treatment of BC and EMS, as the lower dose (0.02%) reduced the MN frequency to the maximum (Po0.05), as compared to that of EMS+Alc treated fish. However, the medium dose (0.05%) of BC reduced the frequency of AN (Po0.05; see Table 2) to a greater extent.
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Table 1 Frequency distribution of chromosome aberrations (CA), micronucleated erythrocytes (MNE), anomalous nuclei (AN), and sperm head abnormality (SHA) of Anabas testudineus treated with 0.2% EMS (T1), 0.02% VC (T2), 0.2% EMS+0.02% VC (T3), 0.05% BC (T4), 1% alcohol (T5), 0.2% EMS+1% alcohol (T6), 0.2% EMS+0.05% BC (T7) and their respective DW-treated controls (C) at different fixation intervals Fixation time intervals
6h
24 h
48 h
72 h
96 h
Series
Chromosome aberrations in kidney cells
Micronuclei and anomalous nuclei in peripheral erythrocytes
% major types CA
% other %7SE types CA
%7SE
C T1 T2 T3
0.00 2.40 0.00 1.49
1.40 5.90 1.05 5.46
1.4070.28 8.3070.32 1.0570.12 6.9570.48
T4 T5 T6 T7
0.71 0.51 3.94 1.87
3.57 3.08 5.17 3.50
4.2970.82 3.5970.78 9.1171.08 5.3770.32
C T1 T2 T3
0.44 4.69 0.51 3.32
1.80 3.67 2.02 3.71
2.2170.58 8.3770.60 2.5370.23 7.0370.82
T4 T5 T6 T7
0.74 0.49 4.31 2.09
3.19 2.46 5.02 3.95
3.9270.63 2.9670.18 10.0570.92 6.0570.60
C T1 T2 T3
0.24 3.49 0.24 3.01
2.62 6.28 1.70 3.66
2.8670.82 9.7770.67 1.9470.18 6.6770.62
T4 T5 T6 T7
0.43 0.71 4.71 1.99
3.66 2.62 5.46 3.19
4.0971.08 3.3370.72 10.1771.12 5.1870.62
C T1 T2 T3
0.00 4.73 0.00 3.33
1.65 8.18 1.44 4.79
1.6570.23 12.9170.67 1.4470.12 8.1370.32
T4 T5 T6 T7
0.73 0.25 5.20 2.86
2.43 2.01 8.00 5.71
3.1670.52 2.2670.13 13.2070.92 8.5770.48
C T1 T2 T3
0.00 2.73 0.50 2.25
0.93 7.35 1.76 4.49
0.9270.18 10.0870.42 2.2670.21 6.7470.60
T4 T5 T6 T7
0.41 0.26 3.63 2.14
2.86 3.16 6.05 5.48
3.4770.54 3.4270.58 9.6870.82 7.6270.58
% suppr.
T1 vs. T3 16.27a
T1 vs. T7 41.06a
T1 vs. T3 16.01
T1 vs. T7 39.80b
T1 vs. T3 31.73b
T1 vs. T7 49.07b
T1 vs. T3 37.03c
T1 vs. T7 35.08b
T1 vs. T3 32.06b
T1 vs. T7 21.28
0.0070.00 0.0870.03 0.0070.00 0.0470.02 0.0370.01 0.0270.01 0.1170.04 0.0470.02 0.0470.01 0.1870.02 0.0770.02 0.1070.01 0.0770.01 0.0370.01 0.2270.04 0.1170.02 0.0270.01 0.1770.02 0.0770.01 0.0970.01 0.0970.02 0.0270.01 0.1870.02 0.1270.01 0.0270.01 0.2170.02 0.0670.01 0.1270.01 0.0770.01 0.0370.01 0.2370.03 0.1470.02 0.0270.01 0.1470.01 0.0670.01 0.0970.01 0.0670.01 0.0370.01 0.1770.02 0.1070.02
% suppr. %7SE
T1 vs. T3 50.00
T1 vs. T7 63.64
T1 vs. T3 44.44b
T1 vs. T7 50.00a
T1 vs. T3 47.06b
T1 vs. T7 33.33a
T1 vs. T3 42.86b
T1 vs. T7 39.13a
T1 vs. T3 35.71b
T1 vs. T7 41.18a
0.9870.12 7.9371.12 2.1370.52 5.8570.32 4.9870.54 3.6271.08 8.5471.41 6.2371.08 1.0270.21 10.4170.72 3.2370.60 8.3771.73 6.1271.23 3.3670.60 8.9170.82 8.1670.64 1.0970.23 9.4370.45 3.4370.82 8.7971.23 5.3170.82 5.0170.92 10.1471.12 6.7970.73 1.2370.17 8.1870.79 3.5170.38 4.7070.52 5.5970.98 2.8270.58 8.4870.98 5.8570.58 0.8670.12 7.4770.52 3.9170.78 6.8671.62 6.6871.12 4.5270.82 11.2371.38 6.7171.32
Sperm head anomaly
% suppr. %7SE
T1 vs. T3 26.23
T1 vs. T7 27.05
T1 vs. T3 19.60
T1 vs. T7 8.42
T1 vs. T3 6.79
T1 vs. T7 33.04a
T1 vs. T3 42.55b
T1 vs. T7 31.01a
T1 vs. T3 8.17
T1 vs. T7 40.25a
0.8770.11 4.2870.53 1.0870.13 3.0970.60 2.9870.64 1.9470.32 6.5170.78 4.1070.42 0.8170.13 7.7670.73 1.0270.12 3.9770.62 2.1570.78 1.9870.76 6.7570.34 3.8470.78 0.5170.21 6.2570.82 1.0570.09 4.5670.89 2.5270.32 1.7370.28 10.0071.20 5.1770.82 0.5270.09 5.2570.98 1.6770.18 4.6070.60 2.9370.36 2.5670.49 10.4971.09 4.3470.62 0.7170.10 5.7370.73 1.0970.32 2.8870.38 3.1070.68 1.7570.42 7.5270.48 4.4670.41
% suppr.
T1 vs. T3 27.80
T1 vs. T7 37.02a
T1 vs. T3 48.84a
T1 vs. T7 43.11b
T1 vs. T3 27.04
T1 vs. T7 48.30b
T1 vs. T3 12.38
T1 vs. T7 58.63b
T1 vs. T3 49.74a
T1 vs. T7 40.69b
Major type CA include break, centric fusion, translocation, fragment, pulverization, ring, constriction, polyploidy, and aneuploidy. Other types of CA include stickiness, precocious centromeric separation, terminal association, erosion, condensation and other unclassified aberrations. No. of individuals examined in each series/fixation intervals ¼ 6; cells scored per individual ¼ 500 for CA, 20,000 for MNE and AN, 130 for Cyt/Nu ratio, 6000 for SHA. a ¼ po0.05, b ¼ po0.01, c ¼ po0.001.
Table 2 Frequency distribution of chromosome aberrations, micronucleated erythrocytes (MNE), anomalous nuclei (AN) and sperm head abnormality (SHA) of Anabas testudineus treated with 0.2 % EMS (T1), 0.02 % VC (T2), 0.05% VC (T3), 0.1% VC (T4), 0.2% EMS+0.02% VC (T5), 0.2% EMS+0.05% VC (T6), 0.2% EMS+0.1% VC (T7), 0.02 % BC (T8), 0.05% BC (T9), 0.1% BC (T10), 1% Alc (T11), 0.2% EMS+1% Alc (T12), 0.2% EMS+0.02% BC (T13), 0.2% EMS+0.05% BC (T14), 0.2% EMS+0.1% BC (T15) and their respective DW-water treated controls (C) at 72 h fixation intervals
72 h
Series
Chromosome aberrations in kidney cells
% otherr types CA
% of CA7SE
C T1 T2 T3 T4 T5
0.00 4.73 0.00 0.00 0.00 3.33
1.65 8.18 1.44 1.11 1.28 4.79
1.6570.23 12.9170.67 1.4470.12 1.1170.21 1.2870.08 8.1370.32
T6
2.93
4.53
7.4770.42
T7
2.50
4.06
6.5670.23
T8 T9 T10 T11 T12 T13
0.48 0.73 1.03 0.25 4.22 2.79
2.85 2.43 4.36 2.01 8.98 6.74
3.8170.72 3.1670.52 5.3871.02 2.2670.13 13.2070.92 9.5370.83
T14
2.88
5.78
8.5770.48
T15
2.88
7.74
10.6270.52
% of suppr.
T1 vs. T5 37.03c T1 vs. T6 42.14c T1 vs. T7 49.19c
T12 vs. T13 27.10a T12 vs. T14 35.08b T12 vs. T15 19.55a
Sperm head anomaly
% of MNE7SE % of suppr.
% of AN7SE
% of SHA7SE
0.0270.01 0.2170.02 0.0670.01 0.0270.01 0.0470.01 0.1270.01
1.2370.17 8.1870.79 3.5170.38 3.3570.98 2.8970.321 4.7070.52
0.1270.02 0.1170.03 0.0770.01 0.0770.01 0.0970.02 0.0370.01 0.2370.03 0.1270.03 0.1470.02 0.1570.02
T1 vs. T5 42.86a T1 vs. T6 42.86a T1 vs. T7 47.62b
T12 vs. T13 47.83a T12 vs. T14 39.13a T12 vs. T15 34.79
6.5670.81 5.7970.72 3.0470.71 5.5970.98 3.5270.83 2.2870.58 8.4870.98 6.8271.03 5.8570.58 5.8870.59
% of suppr.
T1 vs. T5 42.55b T1 vs. T6 19.80 T1 vs. T7 29.22
T12 vs. T13 19.58 T12 vs. T14 31.01a T12 vs. T15 30.66a
0.5270.09 5.2570.98 1.6770.18 0.9070.21 1.0570.32 4.6070.60 2.9570.38 2.5870.28 2.4570.70 2.9370.36 3.0870.82 2.5670.49 10.4971.09 4.8070.33 4.3470.62 4.1870.62
% of suppr.
T1 vs. T5 10.38 T1 vs. T6 43.81 T1 vs. T7 50.86
T12 vs. T13 54.24b T12 vs. T14 58.63b T12 vs. T15 60.15b
Major type CA include break, centric fusion, translocation, fragment, pulverization, ring, constriction, polyploidy and aneuploidy. Other type CA include stickiness, precocious centromeric separation, terminal association, erosion, condensation, and other unclassified aberrations. No. of individuals examined in each series/fixation intervals ¼ 6; cells scored per individual ¼ 500 for CA, 20,000 for MNE and AN, 130 for Cyt/Nu ratio, 6000 for SHA. a ¼ po0.05, b ¼ po0.01, c ¼ po0.001.
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% major types CA
Micronuclei and anomalous nuclei in peripheral erythrocytes
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Fixation time intervals
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The combined treatment of each of the three doses of VC and EMS reduced the frequency of sperm head abnormality as compared to those treated only with EMS. The higher dose (0.1%) of VC appeared to show more suppression of damage to sperm head than the other two doses (see Table 2). Similarly, there was an indication of the higher dose of BC (0.1%) being slightly more effective (Po0.01; see Table 2) in rendering protection to sperm head than the other two doses. ANOVA revealed the protective efficacy of 0.02% VC (T5) against 0.2% EMS (T1) and 0.02% BC (T13), 0.05% BC (T14) and 0.1% BC (T15) against 0.2% EMS plus 1% Alc (T12) in regard to CA (b ¼ Po0.01), and in regard to AN (b ¼ Po0.01) at 72 h. Similarly, there was protective ability (a ¼ Po0.05) of 0.02% BC (T13), 0.05% BC (T14) and 0.1% BC (T15) against 0.2% EMS plus 1% Alc (T12) in regard to SHA (Histogram 2 in Appendix A).
4. Discussion EMS has been extensively tested in a wide variety of animals and this mono-functional alkylating agent has been reported to cause major chromosomal aberrations including chromosomal breakage in mammalian models, by binding to DNA regions rich in G–C base pairs, causing those region to become unstable or by disrupting the main chain of DNA, perhaps in regions where protein is bound (see Malacinski and Freifelder, 1988). In the present study, EMS was found to induce various types of chromosome aberrations, nuclear anomalies including micronuclei formation and abnormal sperm head shapes. Earlier, Hooftman (1981) reported the induction of chromosome aberrations in killifish, Notobranchius rachowi after the treatment with ethylmethane sulphonate or benzo(a)pyrene. Subsequently, Hooftman and de Raat (1982) reported the induction of nuclear anomalies including micronuclei formation in the erythrocytes of another fish, Umbra pygmeae, treated with EMS. Micronuclei are one or rarely multiple supernumerary nuclear structures often observed as a result of xenobiotic treatment within the cytoplasm of an erythrocyte having the following characteristics: a distinct outline to the structure, a round, almond or ovoid 1 shape, a diameter 15 20 th of the erythrocyte, staining characteristics consistent with chromatin of the main nucleus, either dissociated from or connected to the main nucleus by a very thin basophilic strand. Micronuclei are believed to derive from chromosomal fragments or whole chromosomes that are not incorporated into daughter nuclei at the time of cell division. Therefore, micronucleus testing has been used extensively in fish in vivo and recommended as a potent parameter to designate clastogenic efficiency of a chemical (Heddle et al., 1983; Metcalf, 1988; Matsumoto and Co’lus, 2000; Grisolia and Cordeiro, 2000; Porto et al., 2005) although an in vitro binucleated blocked hepatic cell technique has also been found to be useful for genotoxicity testing in fish (Al-Sabti, 1995).
In the present study, the genotoxic effects of EMS gradually increased up to 72 h after treatment but the genotoxic effects appeared to decline thereafter, except for sperm head abnormality. Interestingly, the genotoxic effects of EMS were considerably reduced when different doses of VC or BC were also injected alongside. Such protective effects of some vitamins have also been reported against various mutagens and toxicants and linked mainly to their anti-oxidant nature (Machlin and Bendich, 1987; Hayatsu et al., 1988; Sato et al., 1990) and also for their nucleophilic character in some cases (Krishna et al., 1986). Alternatively, the antimutagenic effect of VC has also been suggested to be mediated by various detoxication enzymes (Goncharova, 1984). VC has also been suggested to prevent the production of electrophilic metabolites (Goncharova and Kuzhir, 1989). As VC has marked nucleophilic properties it might intercept reactive electrophilic metabolites, thereby preventing their attack on nucleophilic sites on DNA, and hence blocking the adduct formation (Bhattacharya et al., 1987; Liehr et al., 1989). However, VC has also been claimed to exert its protective effect through multiple inhibition mechanisms (Flora and Ramel, 1988). Interestingly enough, the treatment of ascorbic acid alone to the fish produced some amount of chromosome damage, nuclear anomalies and sperm head anomaly. Stich et al. (1979) demonstrated the chromosome breaking ability of VC in mammalian cells earlier. This might be possible as sometimes antioxidants under certain conditions have been reported to have potential to act as prooxidants (Wang and Russel, 1999; Devsagayam and Kamat, 2000). VC apparently has anti-spermatotoxic activity as its administration also protected sperm from EMS-induced damage. Various mutagens particularly pesticides have earlier been reported to induce abnormality in sperm head and thereby adversely affect male reproductive potentiality by causing decrease in sperm count (Whorton et al., 1977). Further, various types of structural alterations in chromosomes have been reported that led to pairing impairments among homologues and division-disruptive changes in spermatocytes of mice (Bhunya and Behera, 1987; Pandey et al., 1990). Mutagens affecting germ cells generally induce sperm head abnormalities in mice (Topham, 1980) due to alterations in testicular DNA and sperm chromatin structure (Evenson et al., 1986). Many enzymatic functions of vitamin C are essential for the normal integrity and function of testes, i.e., the synthesis, development and maintenance of normal sperm (Dawson et al., 1990). 5. Conclusions Although in the present study, there was no conclusive indication of dose–response relationship particularly with regard to the protection rendered by BC against EMS in fish, VC had some positive dose–response in rendering
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protection. Khan and Sinha (1993) also reported a differential dose–response of vitamin C in mice. They reported that higher dose of VC (10, 20 and 40 mg/kg bw/ day) exhibited a greater combating ability against clastogenecity induced by several pesticides. However, the lower doses of VC also reduced toxin-induced cytogenetic damage to a considerable extent but they could not bring the frequency of damages down to the control level (Hoda and Sinha, 1991; Hoda et al., 1992; Sinha and Bose, 1992). It was argued that higher dose of VC could possibly compensate for the urinary loss of VC (Yokogoshi et al., 1983) and/or might be required for maintaining its threshold level for antioxidant activities in tissues (Wayner et al., 1986). Although EMS has been reported to cause extensive chromosomal damage in a variety of fish species, e.g., N.s rochowi (Hooftman, 1981), Umbra pygmaea (Hooftman and de Raat, 1982), A. testudineus (Guha and KhudaBukhsh, 2002b), modulating effect of BC on EMS-induced genotoxicity has only been documented so far in one species of fish, Oreochromis mossambicus (Guha and Khuda-Bukhsh, 2002a, 2003). However, similar protective effect of BC on genotoxicity induced by some other mutagen, cyclophosphamide (CPA) has also been reported in mice (Mukherjee et al., 1991; Salvadori et al., 1992) and on human hepatoma cells (Salvadori et al, 1993). As an antioxidant, BC is expected to quench active oxygen species and to trap free radicals (Burton and Ingold, 1984; Hayatsu et al., 1988; Sato et al., 1990; Mukherjee et al., 1991) in the same manner as VC. Therefore, the favorable modulations of the EMS-induced genotoxicity by both VC and BC might have been achieved by the common radical scavenging property of both. Interestingly, in all the treatment series of EMS plus Alc, there appeared to be some increased damage when individual data were compared to only EMS-treated lots, thereby showing some additive damaging effect of alcohol when conjointly treated with EMS. Thus, the genotoxic potentials of EMS have been confirmed in this fish, which proved to act as a good cytogenetical model. It may be pointed out that relatively low doses of both the vitamins injected intramuscularly could quite effectively ameliorate the genotoxic effects induced by EMS in this fish, as revealed from the different cytogenetical endpoints studied. Therefore, for this experiment using injection route to be eventually useful in the context of fish farming, it remains to be demonstrated whether these vitamins placed in water or food would also be protective, particularly in closed and stagnant water bodies contaminated with such mutagenic agents.
Acknowledgments The authors are grateful to ICAR, Government of India, New Delhi, for providing financial support of the work.
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Appendix A. Supplementary materials Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv. 2006.06.001.
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