Ameliorating effect of β-carotene on ethylmethane sulphonate-induced genotoxicity in the fish Oreochromis mossambicus

Mutation Research 542 (2003) 1–13

Ameliorating effect of ␤-carotene on ethylmethane sulphonate-induced genotoxicity in the fish Oreochromis mossambicus Bibhas Guha, Anisur Rahman Khuda-Bukhsh∗ Department of Zoology, University of Kalyani, Kalyani 741235, West Bengal, India Received 10 January 2003; received in revised form 9 June 2003; accepted 30 July 2003

Abstract Genotoxic effects have been assessed in the fish Oreochromis mossambicus treated separately and conjointly with 0.2% ethylmethane sulphonate (EMS) and 0.05% ␤-carotene (BC) during five different time periods (6, 24, 48, 72 and 96 h) by analysis of endpoints such as chromosome aberrations, abnormal red blood cell nuclei, abnormal sperm morphology and protein contents (both qualitative and quantitative) of selected tissues, viz. muscle, heart, eye, brain, gill, liver, spleen and kidney. In addition, the relative efficacy of three doses of BC 0.02, 0.05 and 0.1%, in ameliorating genotoxic effects of 0.2% EMS was also tested after a treatment period of 48 h. EMS caused chromosomal aberrations, nuclear anomalies in red blood cells, abnormal sperm morphology and an apparent alteration of protein synthesis in various tissues. Some of these genotoxic effects of EMS appeared to be ameliorated by all three doses of BC, of which the 0.02% dose showed a marginally better efficacy. © 2003 Elsevier B.V. All rights reserved. Keywords: Genotoxicity; Antimutagen; ␤-Carotene; Tilapia; Chromosome aberrations; Nuclear and protein anomalies

1. Introduction In recent years, water pollution caused by various toxic substances has become a matter of great human concern and warrants their testing for potential genotoxic effects on aquatic organisms. Fish, particularly those living in rivers or confined waters, also run the risk of direct or indirect exposure to various chemical mutagens present in the run-offs along with various toxicants, pesticides, industrial wastes, etc. Therefore, it has also become necessary to search for possible protective agents or anti-mutagens.

∗ Corresponding author. Tel.: +91-033-25828750x315(O)/25828768(R). E-mail address: [email protected] (A.R. Khuda-Bukhsh).

Ethyl methanesulphonate (EMS), an alkylating agent, has been extensively used in genetic research for its ability to induce artificial mutagenesis. It is known to alter a base in the DNA, causing mutations in a wide variety of organisms [1]. EMS has also been reported to cause extensive chromosomal damage in Notobranchius rochowi [2], nuclear damage (micronuclei) in peripheral blood in Umbra pygmaea [3], and both chromosomal damage and nuclear anomalies in tilapia, Oreochromis mossambicus and the Indian climbing perch, Anabas testudineus [4,5]. In fact, because of lack of a suitable karyotype in most of the fish, testing of the genotoxic potential of an agent on fish has to be extrapolated mainly from cytogenetical assays carried out only on some particular fish species having a suitable karyotype. Therefore, it could be of added benefit to find toxicological

1383-5718/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2003.07.012

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B. Guha, A.R. Khuda-Bukhsh / Mutation Research 542 (2003) 1–13

endpoints other than the conventional ones that could help determine genotoxicity, particularly in those fish that have unsuitable karyotypes for genotoxicity assessment. With this in mind, evaluation of additional parameters such as a qualitative assay of protein profiles was taken up recently in two fish models, O. mossambicus and A. testudineus [4,5], along with the more conventional cytogenetic protocols. The toxicity endpoints used in these studies include (i) cytogenetic assay through the study of chromosomal aberrations, micronuclei and abnormal red blood cell nuclei, abnormal sperm morphology, and (ii) protein assay by analysis of protein contents and gel profiles in nine selected tissues, viz. dorsal muscle, ventral muscle, heart, eye, brain, gill, liver, spleen and kidney, in treated and control fish. The results not only showed that EMS had considerable damaging effects on somatic chromosomes and nuclei of peripheral blood cells in these two fish species, but also demonstrated visible alterations in their protein profiles in all tissues studied, some of which were quite different from those in controls. Vitamin C, an antioxidant, was shown to significantly reduce the EMS-induced cytogenetic and protein damage in O. mossambicus [4]. On the other hand, BC, the pro-vitamin A, another antioxidant, had not been tested earlier in any fish for its possible ability to protect against chemical mutagens, although anti-clastogenic effects of BC had earlier been reported in bone marrow cells of mice [6] against some cyclophosphamides. BC has also been shown to have anti-carcinogenic and anti-mutagenic effects in mammalian test systems [7,8]. Therefore, the primary objectives of the present investigation were (i) to test if BC could have a similar ameliorating effect on EMS-induced genotoxicity in fish, and if it did, (ii) to find out whether a minimum effective dose could be suggested to counteract EMS-induced genotoxicity, and (iii) to see if protein endpoints can be of some added advantage in indicating the extent of genotoxicity, or of recovery processes.

2. Materials and Methods Living healthy specimens of O. mossambicus (Peters) (family: Cichlidae) weighing between 18 and 25 g were collected from a local fresh water pond and were acclimated for a few days in a concrete vat con-

taining fresh tap water (stored from a deep tube well) and fed with artificial diet (rice bran+wheat+oil cake) for about 7 days before they were used for the present study. Six live specimens each were injected bilaterally with optimally low doses that produced well quantifiable changes (as revealed from range-finding trials), viz. (1) 0.2% EMS, (2) 0.05% BC, (3) 1% alcohol (being the ‘vehicle’ of BC), (4) 0.2% EMS plus 1% alcohol, (5) 0.2% EMS plus 0.05% BC and (6) double-distilled water at 1 ml/100 g of body weight for each of five fixation intervals, viz. 6, 24, 48, 72 and 96 h (since the peak damaging effect induced by EMS seemed to appear at 48 h, with a declining trend up until 96 h, a fixation time beyond 96 h was not considered). Additionally, five to six specimens were each injected either with 0.02 or 0.1% BC alone or in combination with 0.2% EMS (through bilateral injections) to evaluate the comparative efficacy of the different doses of BC at 48 h. Injected specimens were transferred to smaller vats containing tap water and fed artificial diet until they were sacrificed. Specimens injected with double-distilled water (being the ‘vehicle’ of EMS) served as controls. The non-natural injection route (i.e. no exposure to medium containing the test chemicals) was favored because the genotoxic effects or their amelioration could be known 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 and assimilation or removal via the faces for different specimens. Besides, the possibility of differential absorption through the skin could be avoided by choosing the injection route, apart from the higher cost and management problems related with periodical change of water medium contaminated with test chemicals. For chromosome analysis, experimental and control fish specimens were injected intramuscularly with 0.05% colchicine at 1 ml/100 g bw about 3 h prior to sacrifice and their somatic chromosomes were prepared as per the citrate-flame drying technique [9]. Clastogenic changes, both on individual chromosomes, e.g. breaks, constrictions, gaps, centric fusion, translocation, fragments, etc. or on the whole chromosome set, e.g. polyploidy, pulverization, stickiness, etc. were recorded in 500 metaphase spreads from each series. In view of the small size of the chromosomes, or owing to an unfavorable disposition, confusion

B. Guha, A.R. Khuda-Bukhsh / Mutation Research 542 (2003) 1–13

regarding the determination of the exact nature of the aberration, e.g. gaps, erosion, or constriction made it difficult to classify the aberrations in a particular category. Therefore, 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’ aberrations were pooled together under the ‘other types’. The total aberration frequencies, i.e. the sum total of ‘major’ and ‘other’ type of 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 intervals after dosing by puncturing their caudal peduncle, and blood films were drawn on clean grease-free slides. Semi-dried slides were briefly dipped in 90% ethyl alcohol and allowed to air-dry. Air-dried slides were stained in May–Grunwald stain as per the procedure of Schmid [10], to determine the occurrence of micronuclei, if any. Micronuclei are single - rarely multiple -, supernumerary nuclear structures, often observed as a result of xenobiotic treatment within the cytoplasm of an erythrocyte. They have the following characteristics: a distinct outline to the structure, a round, almond or ovoid shape, a diameter that is 1/5th to 1/20th of the erythrocyte, staining characteristics consistent with the chromatin in the main nucleus, and dissociated from or connected to the main nucleus by a very thin basophilic strand. The frequency of anomalous nuclei was recorded as per the method of Carrasco et al. [11] and nuclear and cytoplasmic volumes were measured according to the method suggested by Samuels [12]. The nucleo-cytoplasmic ratio in normal erythrocytes was then calculated accordingly [4,13]. For the study of sperm head abnormality, epididymes of the testis were dissected, and their inner contents taken out in Fish–Ringer medium; smears were prepared on slides with the Fish–Ringer and stained with Giemsa [14]. For biochemical study, nine different tissues, namely, dorsal muscle (DM), ventral muscle (VM), heart (H), eye (E), brain (Br), gill (G), liver (Li), spleen (Sp) and kidney (K) were removed after 48 h of treatment from each of the treated and control specimens separately and subjected to the SDS-PAGE technique [15] using a 7.5% separating gel in Tris–glycine buffer for visualizing different sub-fractions of protein bands. The data of individual and consistently

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appearing bands in the six specimens of each series were pooled and a computer-simulated gel profile was obtained. For the quantitative assay, the technique of Lowry et al. [16] was followed. For determining statistical significance, if any, Student’s t-test was conducted between data of treated and control fish [17]. During observation, the observer was ‘blinded’ as to the origin of cytogenetic slides or materials for biochemical assays, which were only decoded after the experiment. 3. Results 3.1. Chromosome aberration study Normal metaphase complements (Fig. 1) were carefully examined for any possible clastogenic change due to treatments with the test chemicals or distilled water. Of the various types of chromosomal aberrations recorded, representative photomicrographs of a few types like chromatid break (Fig. 2), acentric fragment (Fig. 3), centric fusion (Fig. 4), pulverization (Fig. 5), and precocious centromeric separation (Figs. 6 and 7) are shown mainly from EMS-treated fishes. Analysis of the data revealed that the frequency of aberrations in the EMS-treated series was significantly higher than in the distilled water (DW)-treated controls, showing the clastogenic effect of EMS (Table 1). The treatment with only 0.05% BC produced slightly more CA than in the DW-treated control. When EMS-treated fish was injected with 1% alcohol (the vehicle of BC), the chromosome aberrations were considerably enhanced, apparently showing that the genotoxic effect of EMS was aggravated by alcohol. On the other hand, the percentage of chromosome aberrations in the EMS + BC (0.05%)-treated series were found to be reduced as compared to that of EMS + 1% alcohol-treated fish at all time points, but it was significant only at 6, 48 and 96 h (P < 0.01–0.001) (see Table 1). Each of the three doses of BC (i.e. 0.02, 0.05 and 0.1%) when applied in combination with 0.2% EMS reduced chromosome aberrations at 48 h to a considerable extent (P < 0.001) compared with those in fishes treated with 0.2% EMS+1% alcohol. However, 0.02% BC appeared to provide the maximum antagonistic effect (see Table 2).

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Figs. 1-16. Photomicrographs of normal (1) and aberrant (2–6) metaphase complements of Oreochromis mossambicus; chromatid break – CB (2), fragment – F (3), centric fusion – CF (4), pulverisation (5), precocious centromeric separation – PCS (6); normal erythrocytes (7), micronucleated erythrocytes (8–10) and anomalous nuclei (11 and 12); sperm with normal (13) and abnormal head shapes (14–16).

3.2. Micronucleus study Micronucleated erythrocytes (MN) (Figs. 8–10) and erythrocytes with abnormal nuclei (AN) (Figs. 11 and 12) were observed in much greater number in fish

treated with EMS alone and in fish treated with EMS+ 1% alcohol. However, the numbers of MN and AN observed in the DW- or BC-treated fish (Table 1) were much lower. EMS-treated fish counter-injected with BC showed fewer micronuclei and anomalous nuclei

Table 1 Showing frequency distribution of chromosome aberrations (CA), micronucleated erythrocytes (MNE), anomalous nuclei (AN), nucleo-cytoplasmic ratio (Cyt/Nu ratio) and sperm head abnormality (SHA) of O. mossambicus treated with 0.2% EMS (T1 ), 0.05% BC (T2 ), 1% alcohol (T3 ), separately and 0.2% EMS + 1% alcohol (T4 ), 0.2% EMS + 0.05% BC conjointly (T5 ) and their respective double distilled water controls (C) at different fixation intervals Fixation time intervals (h)

6

48

72

96

Chromosome aberrations in kidney cells % major types CA

% other types CA

Total % of CA ± S.E.

C T1 T2 T3 T4 T5

0.23 3.57 0.75 1.05 4.57 2.05

2.06 6.64 4.78 3.66 8.24 5.13

2.29 10.21 5.53 4.71 12.81 7.18

± ± ± ± ± ±

0.27 0.83 0.94 0.84 1.33 0.93

C T1 T2 T3 T4 T5

0.79 4.92 1.18 0.74 5.36 2.61

2.58 9.65 6.15 4.47 11.19 9.74

3.37 14.57 7.33 5.21 16.55 12.35

± ± ± ± ± ±

1.23 1.63 1.12 1.04 1.79 0.98

C T1 T2 T3 T4 T5

0.70 7.40 0.63 0.69 6.33 2.74

3.25 12.06 4.39 6.50 16.06 8.65

3.94 19.46 5.02 7.19 22.38 11.39

± ± ± ± ± ±

1.36 0.67 0.78 1.13 2.01 1.56

C T1 T2 T3 T4 T5

0.60 2.84 0.99 0.47 4.48 2.78

2.99 11.05 7.52 6.07 10.95 10.74

3.59 13.88 8.50 6.54 15.42 13.52

± ± ± ± ± ±

0.92 0.70 0.63 1.12 3.21 2.08

C T1 T2 T3 T4 T5

0.47 4.28 0.48 0.46 4.72 1.21

3.74 7.35 6.67 5.06 13.93 6.04

4.21 11.63 7.14 5.51 18.65 7.25

± ± ± ± ± ±

0.97 1.09 1.83 0.74 2.28 1.78

Micronucleus in peripheral erythrocytes % suppr.

44.0b

25.4

49.1c

12.3

61.1b

% of MNE ± S.E. 0.00 0.12 0.07 0.04 0.14 0.08

± ± ± ± ± ±

0.00 0.04 0.03 0.02 0.06 0.02

0.05 0.21 0.11 0.10 0.27 0.13

± ± ± ± ± ±

0.01 0.03 0.02 0.02 0.04 0.01

0.06 0.23 0.09 0.07 0.25 0.14

± ± ± ± ± ±

0.02 0.02 0.01 0.01 0.03 0.03

0.06 0.17 0.08 0.06 0.31 0.09

± ± ± ± ± ±

0.02 0.02 0.02 0.01 0.05 0.01

0.04 0.13 0.05 0.04 0.18 0.07

± ± ± ± ± ±

0.01 0.02 0.01 0.01 0.03 0.01

% suppr.

42.86

51.85b

44.00a

71.00b

61.11b

% of AN ± S.E. 0.52 1.54 1.80 0.98 2.02 1.28

± ± ± ± ± ±

0.08 0.19 0.24 0.22 0.89 0.29

0.43 1.26 0.68 0.81 1.77 1.21

± ± ± ± ± ±

0.08 0.09 0.14 0.09 0.43 0.09

0.47 1.15 0.79 1.51 1.85 0.85

± ± ± ± ± ±

0.05 0.04 0.21 0.24 0.31 0.12

0.46 1.21 0.85 1.88 1.67 0.91

± ± ± ± ± ±

0.12 0.10 0.31 0.31 0.38 0.17

0.48 1.13 0.73 1.13 1.95 0.72

± ± ± ± ± ±

0.16 0.14 0.19 0.12 0.29 0.21

% suppr.

36.6

31.6

54.1a

45.5

63.1b

Sperm Head Anomaly

% of Cyt/Nu ± S.E. 10.52 11.15 9.34 10.84 13.08 12.10

± ± ± ± ± ±

1.14 0.48 0.63 0.82 0.86 0.55

10.52 11.97 12.96 12.05 12.35 12.41

± ± ± ± ± ±

0.83 0.72 0.70 0.92 0.34 0.72

10.32 13.56 13.16 11.59 13.76 13.42

± ± ± ± ± ±

1.13 1.06 0.63 1.88 0.56 0.61

9.79 11.85 12.70 11.15 12.47 14.93

± ± ± ± ± ±

0.37 0.29 1.13 1.26 0.84 1.15

11.10 12.85 12.49 9.77 13.05 15.17

± ± ± ± ± ±

1.08 1.17 1.48 0.31 1.07 0.76

Difference T4 vs T5

0.98

−0.06

0.34

−2.46

−2.12

% of SHA ± S.E. 1.63 6.18 2.48 1.77 8.81 5.92

± ± ± ± ± ±

0.07 0.85 0.72 0.31 1.21 0.72

2.14 10.29 2.83 2.36 10.70 5.78

± ± ± ± ± ±

0.12 0.54 0.61 0.34 1.34 0.64

1.82 8.12 2.66 2.19 10.35 5.87

± ± ± ± ± ±

0.65 0.39 0.32 0.32 0.98 0.28

1.62 8.56 3.72 1.70 8.34 3.79

± ± ± ± ± ±

0.54 1.37 0.64 0.32 1.64 0.97

1.63 4.82 3.00 1.81 6.59 3.65

± ± ± ± ± ±

0.25 0.08 0.51 0.24 0.88 0.68

% suppr.

32.8

46.0b

43.3b

54.6a

B. Guha, A.R. Khuda-Bukhsh / Mutation Research 542 (2003) 1–13

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Series

44.6a

Major type CA include break, centric fusion, translocation, fragment, pulverization, ring, constriction, polyploidy, aneuploidy, Other type CA include gap, stickiness, precocious centromeric separation, terminal association, erosion, condensation and other unclassified aberrations, No. of individuals examined in each series/fixation intervals = 6; cells scored per individual = 500 for CA, 20000 for MNE and AN, 130 for Cyt/Nu ratio, 6000 for SHA. a P < 0.05. b P < 0.01. c P < 0.001.

5

6

Fixation time intervals (h)

Series

48

C T1 T2 T3 T4 T5 T6 T7 T8 T9

Chromosome aberrations in kidney cells % major types CA

% other types CA

Total % of CA ± S.E.

0.70 7.40 0.47 0.63 0.89 0.69 6.33 2.78 2.74 3.22

3.25 12.06 4.42 4.39 4.67 6.50 16.06 6.82 8.65 7.60

3.94 19.46 4.88 5.02 5.56 7.19 22.38 9.60 11.39 10.82

± ± ± ± ± ± ± ± ± ±

1.36 0.67 0.62 0.78 0.33 1.13 2.01 0.96 1.56 0.72

Micronucleus in peripheral erythrocytes % suppr.

57.1c 49.1c 51.7c

% of MNE ± S.E. 0.06 0.23 0.07 0.09 0.08 0.07 0.25 0.17 0.14 0.13

± ± ± ± ± ± ± ± ± ±

0.02 0.02 0.01 0.01 0.02 0.01 0.03 0.03 0.03 0.03

% suppr.

32.00 44.00a 48.00a

% of AN ± S.E. 0.47 1.15 0.57 0.79 0.55 1.51 1.85 0.75 0.85 0.97

± ± ± ± ± ± ± ± ± ±

0.05 0.04 0.12 0.21 0.07 0.24 0.31 0.12 0.12 0.42

% suppr.

59.5b 54.1a 47.6

Sperm Head Anomaly

% of Cyt/Nu ± S.E. 10.32 13.56 12.43 13.16 13.42 11.59 13.76 13.21 13.42 14.93

± ± ± ± ± ± ± ± ± ±

1.13 1.06 0.96 0.63 0.61 1.88 0.56 0.86 0.81 3.15

Difference T6 vs T7 , T6 vs T8 , T6 vs T9 0.55 0.34 −1.17

% of SHA ± S.E. 1.82 8.12 1.92 2.66 2.76 2.19 10.35 3.56 5.87 4.56

± ± ± ± ± ± ± ± ± ±

0.65 0.39 0.18 0.32 0.78 0.32 0.98 0.48 0.28 1.21

% suppr.

65.6c 43.3b 55.9b

Major type CA include break, centric fusion, translocation, fragment, pulverization, ring, constriction, polyploidy, 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 = 6; cells scored per individual = 500 for CA, 20000 for MNE and AN, 130 for Cyt/Nu ratio, 6000 for SHA. a P < 0.05. b P < 0.01. c P < 0.001.

B. Guha, A.R. Khuda-Bukhsh / Mutation Research 542 (2003) 1–13

Table 2 Showing frequency distribution of chromosome aberrations (CA), micronucleated erythrocytes (MNE), anomalous nuclei (AN), nucleo-cytoplasmic ratio (Cyt/Nu ratio) and sperm head abnormality (SHA) of O. mossambicus treated with 0.2 % EMS (T1 ), 0.02 % BC (T2 ), 0.05% BC (T3 ), 0.1% BC (T4 ), 1% alcohol (T5 ) separately and 0.2% EMS + 1% alcohol (T6 ), 0.2% EMS + 0.02% BC (T7 ), 0.2% EMS + 0.05% BC (T8 ), 0.2% EMS + 0.1% BC (T9 ) and conjointly and their respective double distilled water controls (C) at 48 h fixation intervals

B. Guha, A.R. Khuda-Bukhsh / Mutation Research 542 (2003) 1–13

compared with fish that received EMS+1% alcohol at all time points examined, the differences being statistically significant at 24, 48, 72 and 96 h (P < 0.05–0.01) for micronuclei and at 48 and 96 h (P < 0.05–0.01) for abnormal nuclei. Like chromosome aberrations, the data showed the enhancing effect of alcohol, as the percentage in the EMS + 1% alcohol-treated series were higher than in the series that received only EMS. However, at all time points BC-treated fish showed slightly more MN than those treated only with 1% alcohol, while this was true for AN at 6 h only. Further, the Cyt/Nu ratio appeared to increase in the combined EMS/BC-treated series than in the EMS + 1% alcohol-treated series, except at 6 and 48 h. The combined treatment of BC and EMS also showed smaller numbers of MN and AN than in the EMS + 1% alcohol-treated fish for all the three doses of BC. In the case of MN, 0.05% BC and 0.1% BC appeared to provide greater protection (P < 0.05) than 0.02% BC. However, in case of AN, 0.02% BC appeared to have a stronger protective effect (P < 0.01) than the higher doses (0.05 and 0.1% BC) at 48 h (see Table 2). However, the data of nucleo-cytoplasmic ratios did not provide any specific correlation between the different treatment series except to note that apparently the lowest ratio was prevalent in the DW-treated control (see Table 2). 3.3. Sperm head abnormality study Sperm with normal (Fig. 13) and abnormal (Figs. 14–16) head shapes were observed in both control and treated fish (Table 1). The frequency of sperm with abnormal heads was less in EMS + BC (0.05%)-treated fish compared with the EMS + 1% alcohol group, the differences being statistically significant at all time points and in all cases except for the 6 h samples (P < 0.05–0.01) (see Table 1). Alcohol apparently had its damaging effect in all the fixation intervals as compared to the DW-treated controls. Alcohol also produced a higher percentage of sperm head abnormality when applied together with EMS. When EMS-treated fish were counter-injected with either of the three doses of BC, the extent of sperm head abnormality was less than in the EMS + 1% alcohol-treated fish for all doses. However, 0.02% BC

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appeared to show statistically significant protection that was stronger (P < 0.001) than that shown by 0.05 and 0.1% BC (P < 0.05; see Table 2) against EMS + 1% alcohol-induced sperm head abnormality. 3.4. Qualitative analysis Data on gel-electrophoretic band profiles in nine different tissues of fish injected with distilled water, one of three doses of BC, alcohol and EMS separately and in combination (i.e. EMS + one of three doses of BC, and EMS + 1% alcohol) have been critically analyzed. Computer-generated electropherograms of two representative tissues (dorsal muscle and heart muscle) for each treatment series are given in Figs. 17 and 18, to allow effective band-to-band comparison (Figs. 17 and 18). Among the tissues examined, the number of bands was lower in the EMS-treated fish than in the DW-treated controls except for liver tissue in which the number of bands was higher in EMS-treated fish. Single treatment with BC and alcohol produced a higher number of bands than EMS treatment, except for liver tissues, whereas in BC-treated fish the number of bands went down. The combined treatment with EMS and BC (0.05%) showed a higher number of bands than treatment with EMS + 1% alcohol except for eye, liver and spleen tissues, where the band number was lower. Among the three doses of BC (i.e. 0.02, 0.05 and 0.1%) applied in combination with EMS, 0.05% BC appeared to increase the band numbers more strongly than that produced by EMS+1% alcohol, in all tissues except spleen and liver. However, in the series treated with BC only (all three doses), the 0.05% BC treatment did not always yield a maximum number of bands in most of the tissues, although in some tissues it did (e.g. DM, H and K), whereas the maximum increase in band number was apparently caused by the lower dose (0.02%) in most tissues. The treatment with 1% alcohol changed the number and other characteristics of bands as compared to DW-treated fish but there was no clear-cut trend, because the number of bands in the alcohol-treated fish was increased in some tissues (e.g. H, Li and S) while it was decreased in others (i.e. DM, E, G and K). In the EMS + alcohol-treated fish the number of bands was increased as compared to fish treated with EMS only, in all tissues examined.

Mol. Wt. Marker

DW

0.2% EMS + 1% Alc

0.2% EMS + 0.1% BC

0.2% EMS +0.05% BC

0.2% EMS+0.02% BC

1% Alc

0.1% BC

0.05% BC

0.02% BC

B. Guha, A.R. Khuda-Bukhsh / Mutation Research 542 (2003) 1–13

0.2% EMS

8

-205.5

-97.4 -68.0

+

-43.0 -29.0

Fig. 17. Gel electropherograms of dorsal muscle of fishes of each treatment series.

-205.0

-97.4 -68.0 -43.0 -29.0 Fig. 18. Gel electropherograms of heart muscle of fishes of each treatment series.

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3.5. Quantitative analysis The quantitative data on total protein contents of nine different tissues in different control and treated series have been critically analyzed. As compared to the DW-treated series the total protein content in the EMS-treated series was less in all tissues, whereas in the BC-treated group the content was higher in all tissues except spleen. On the other hand, in the combined EMS + BC (0.05%)-treated series the total protein content was greater than in either distilled water or EMS-treated fish except for brain, liver and spleen tissue. However, in the EMS + 1% alcohol-treated fish, there was an increase of total protein content in all the tissues as compared with the EMS + 0.05% BC-treated group. In the fish treated with alcohol only, the amount of protein was higher in all tissues than in the BC-treated fish except for brain tissue. Apparently, the inhibitory effects of EMS on protein synthesis were not only abolished by the BC treatment, but the combined treatment of EMS+BC might have actually helped synthesis of more proteins in some unknown way, and some of these could be new ones as revealed from their gel electrophoretic characteristics. The treatment of EMS with either one of the three doses of BC considerably increased the total protein content, as compared to the EMS+1% alcohol-treated fish, in most tissues. Of the three doses of BC, 0.02% BC appeared to have greater increasing effect in certain tissues (e.g. DM, Br and Li). Interestingly EMS + alcohol treatment also raised the total protein contents in all the tissues as compared to treatment with either EMS or control treatment.

4. Discussion In view of ever increasing levels of pollution caused by a wide variety of toxic substances in various water bodies, testing for potential genotoxic effects on non-target aquatic organisms has assumed considerable significance. As fish may act as ‘sentinel’ organisms for indicating aquatic pollution, several species have been successfully used as test materials for detecting genotoxic activity in the aquatic environment [2,18–22]. Analysis of metaphase chromosomes in fish for the occurrence of chromosome aberrations and sister-chromatid exchange (SCE) in order to detect as

9

well as quantify the extent of genotoxicity or point mutation induced by an agent has proven to be useful only in a few fish models [23–25]. The majority of fish species are not suitable for the chromosomal assay because of their small size and large diploid number of chromosomes without any marked variation in size. Therefore, assay of the peripheral erythrocytes in fish for the occurrence of micronuclei (MN) and different types of nuclear abnormalities has been adopted as a good substitute for the chromosomal assay, and based on information on the frequency of occurrence of micronuclei or nuclei with abnormal shape, a monitoring system for potential genotoxicity of an agent has been proposed [3–5,26–30]. Another acceptable marker of genotoxic stress since the work of Wyrobek et al. [31] has been the assay of abnormality of sperm head shape. This protocol has only seldom been utilized before in testing genotoxicity in fish [4,5,14,29,30] due to difficulties in obtaining male meiotic activity round the year (i.e. for periodicity of breeding). In the present study, a critical record of nucleocytoplasmic ratios in different series has been added to see if there could be any indication of a correlation between the extent of genotoxicity and a shift of the ratio. But although there was an apparent increase at 48 h of the ratio when all the three doses of BC were applied along with EMS, as compared with treatment with only BC, the ratio was rather erratic in other treatment series. Thus, it is not very clear whether the differences were in fact a result of differential hydration status of the cells rather than being of any meaningful measure of genotoxicity levels. Incidentally, a critical analysis of gel electrophoretic band profiles of total protein was perhaps not considered by earlier workers to be of practical value in genotoxicity testing, as they either preferred only suitable fish models for their studies, or else depended solely on induction of micronuclei and nuclear anomalies as valid indicators of genotoxicity where chromosomal studies proved to be difficult. It is in the latter cases that the protein assay could be of some additional benefit. The results of the present investigation demonstrated that there were distinct alterations in the band numbers and profiles along with visible effects on cytogenetical parameters in the treated fish as compared to controls, and that various low doses of BC showed some protection/recovery in terms of the

10

B. Guha, A.R. Khuda-Bukhsh / Mutation Research 542 (2003) 1–13

protocols used. Therefore, the assay of protein as an additional parameter along with the study of micronuclei and anomalous nuclei could provide more convincing evidence of the genotoxic potential of mutagens/toxicants/pollutants to which many economically important species of fish (e.g. carps) with unsuitable karyotypes can be exposed. In addition, even in fish with which cytogenetic studies can be done without much difficulty, changes in protein profiles as an endpoint of genotoxicity testing can also be recommended. Effects of a mutagen on protein structure/synthesis can also give important clues, which may have direct or indirect implications for their health and yield in response to different agents [4,5,32,33]. Although, strictly speaking, the appearance or disappearance of protein bands may not always be related to cytogenetical changes in general, these changes may indeed reflect to a certain extent, or may have some correlations with the ‘stress-proteins’ or ‘heat-shock proteins’ that unfailingly appear in response to chemical or physical insult [34]. Further, the present observations on protein profiles can also represent a first step in the process of determining whether or not the chemical-induced altered DNA is expressed as a specifically altered protein product(s). This speculation is in accordance with the evidence provided by Dang et al. [35] that the expression of certain classes of proteins (e.g. metallothionein, stress proteins, etc.) are affected in very specific ways by exposure to cadmium and other chemicals that produce genotoxic stress. EMS has been extensively tested in various mammalian models and this mono-functional alkylating agent has been reported to cause major chromosomal aberrations including chromosomal breakage in mammalian cells, by binding to DNA regions rich in G–C base pairs, causing those regions to become unstable or by disrupting the DNA backbone, perhaps in regions where protein is bound [1,36]. Therefore, the change of gel-electrophoretic band profiles and total protein contents observed in the EMS-treated fish might actually reflect damage in DNA or the protein-synthesizing system. However, since protein synthesis is controlled at various steps, the marked variation in protein bands in O. mossambicus by the treatment with EMS would imply that either the mutagen had affected the DNA so as to produce altered mRNA which led to synthesis of altered proteins manifested as bands with altered gel electrophoretic properties, or else the effect could

be a result of protein degradation due to cytotoxicity [4,5,33]. From the trend in the effects seen at various time points, it would emerge that EMS apparently produced genotoxic effects that increased up to 48 h after treatment, but the effects were sustained through longer intervals to a considerable extent. Interestingly, the injection of BC (0.05%) appeared to modulate the genotoxic effects at all time points and ameliorated the EMS-induced toxicity to a statistically significant extent at several of these time points, as indicated by both the cytogenetic and biochemical assays. Although the modulating effect of BC on EMSinduced genotoxicity has been documented here in a fish model for the first time, it was reported earlier that BC has a protective effect against experimentally induced tumors [9]. Alam et al. [37] reported inhibitory activity of BC against carcinogenesis in the salivary glands of rats. Mathews-Roth [38] reported a similar activity in the skin of hairless mice. A BC-rich diet has also been claimed to protect animals from UV light-induced carcinogenesis and from UV + psoralen-induced carcinogenesis [39]. Further, BC has been shown to inhibit both the initiation and promotion of carcinogenesis [40] in hamsters. Similarly, a carotene-rich diet has been correlated with a low risk of cancer [41]. Most of these anti-cancer activities have basically been explained in terms of the radical-quenching properties of the agents involved. BC absorbs oxygen from the air and becomes colorless. As an antioxidant, this compound is expected to quench active oxygen species and to trap free radicals [8]. Therefore, it may very well be possible that the favorable modulations of the EMS-induced genotoxicity are achieved by the radical-scavenging property of BC [6,7,42]. However, under certain conditions anti-oxidants have been reported to have potential to act as pro-oxidants [43]. For example, BC is known to have antioxidant activity by scavenging DNA-damaging reactive oxygen species, thereby giving protection against cancer. Some recent studies suggest that ascorbate sometimes increases DNA damage in humans. Similarly BC may also behave as a pro-oxidant in the lung of smokers [44,45]. Interestingly enough, BC by itself, when applied in this fish model, was found to show some chromosome damaging effects, but much of this could be due to the alcohol in which it was dissolved.

B. Guha, A.R. Khuda-Bukhsh / Mutation Research 542 (2003) 1–13

(a) 14

(b)

11

0.25

12 0.2

% of MN

% of CA

10 8 6

0.15 0.1

4 0.05 2 0

0 0.02%

0.05%

0.02%

0.10%

BC

(c)

BC

EMS+BC

1.6

(d) 20

1.4

18

0.10%

EMS+BC

16

Cyt/Nu ratio

1.2

% of AN

0.05%

Doses of BC

Doses of BC

1 0.8 0.6 0.4

14 12 10 8 6 4

0.2

2 0

0 0.02%

0.05%

0.02%

0.10%

BC

EMS+BC

(e)

0.05%

0.10%

Doses of BC

Doses of BC BC

EMS+BC

7 6

% of SHA

5 4 3 2 1 0 0.02%

0.05%

0.10%

Doses of BC BC

EMS+BC

Fig. 19. Graphs showing dose-response of 0.02, 0.05 and 0.1% BC treatment in respect of CA (a), MN (b), AN (c), Cyt/Nu ratio (d) and SHA (e) produced by 0.2% EMS in Orecochromis mossambicus. CB, chromatid break; F, fragment; CF, centric fusion; PCS, precocious centromeric separation; MN, micronucleated erythrocytes; AN, anomalous nuclei; SHA, sperm head anomaly; DM, dorsal muscle; VM, ventral muscle; H, heart; E, eye; Br, brain; G, gill; Li, liver; S, spleen and K, kidney. Bar, 10 ␮m.

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On the other hand, the protective ability of BC against the effects of EMS could be based on its antioxidant activity. Conversely, the reported pro-oxidant activity of this vitamin may also be responsible for the insignificant amount of chromosome damage observed when this vitamin was given alone. So far as the dose-response relationship in the EMS-treated fish is concerned, it was not very clear and conclusive. However, there was an apparent increase in antagonistic/protective effect with the increase of dose of the vitamin up to a certain level (Fig. 19), after which an additional increase in dose did not help any further. In any case, all three doses were quite low, the lowest dose (0.02%) showing marginally better protection in case of most parameters.

Acknowledgements The authors are grateful to ICAR, Government of India, New Delhi, for providing financial support of the work.

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