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Efficacy of vitamin-C (l -ascorbic acid) in reducing genotoxicity in fish (Oreochromis mossambicus) induced by ethyl methane sulphonate
Chemosphere 47 (2002) 49–56 www.elsevier.com/locate/chemosphere
Efficacy of vitamin-C (L -ascorbic acid) in reducing genotoxicity in fish (Oreochromis mossambicus) induced by ethyl methane sulphonate B. Guha, A.R. Khuda-Bukhsh
*
Department of Zoology, Kalyani University, Kalyani-741235, India Received 23 March 2001; accepted 1 November 2001
Abstract The genotoxic effects of ethyl methane sulphonate (EMS) have been assessed in a fish, Oreochromis mossambicus with endpoints including chromosome aberrations, abnormal red blood cell nuclei, abnormal sperm morphology, and protein content (both qualitative and quantitative) of selected tissues, namely, muscle, heart, eye, brain, gill, liver, spleen and kidney. EMS caused chromosomal aberrations, nuclear anomalies in red blood cells, abnormal sperm morphology, and alteration of protein synthesis in various tissues. Some of the EMS toxicity appeared to be modulated and ameliorated in this fish by vitamin-C treatment. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Genotoxicity; Antimutagen; Tilapia; Chromosome aberrations; Micronuclei; Sperm head anomaly
1. Introduction Ethyl methane sulphonate (EMS) is an alkylating agent which has been extensively used in genetic research for its ability to alter a base that is already incorporated into a DNA molecule and thereby is of prime importance in the induction of artificial mutations. The clastogenic, mutagenic, teratogenic and carcinogenic effects of EMS have been extensively studied in various mammals (see Kihlman, 1966). Fish, particularly those living in rivers or confined waters, also run the risk of direct or indirect exposure to various chemical mutagens collected along with the run-offs containing various toxicants, pesticides, industrial wastes, etc. Therefore, it is of interest to study the effects of such mutagens on the genome of fish. Similarly, vitamin-C (L -ascorbic acid; VC) is believed to have anti-oxidant nature (Machlin
*
Corresponding author. Tel.: +91-33-5828768/5828750x315. E-mail address: [email protected] (A.R. Khuda-Bukhsh).
and Bendich, 1987; Hayatsu et al., 1988; Sato et al., 1990) and has been quite extensively tested in various mammalian models for its ability to modulate cytogenetic toxicity of certain common pesticides (Hoda and Sinha, 1991, 1993; Khan and Sinha, 1992, 1993). VC has also been reported to have anticarcinogenic (Cameron, 1979; Pauling et al., 1985), anticlastogenic (Gebhart et al., 1985; Krishna et al., 1986; Hoda et al., 1991; Hoda and Sinha, 1993; Bose and Sinha, 1994) and even antimutagenic (Guttenplan, 1977; Shamberger, 1984; Raina and Gurtoo, 1985) roles in a variety of test systems, from microorganisms to cell-free systems or cells in culture in mammalian models, but again its role in modulating cytogenetic damage has not been ascertained in any fish. Therefore, in the present investigation the effectiveness of using a fish model (Oreochromis mossambicus) for the study of the genotoxic effects of EMS and modulation of EMS toxicity by VC was addressed. The toxicity endpoints that this study looked at were chromosomal aberrations, abnormal red blood cell nuclei, abnormal sperm morphology and protein content and gel profiles of nine selected tissues from vital organs, namely, muscle, heart, eye, brain, gill, liver,
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B. Guha, A.R. Khuda-Bukhsh / Chemosphere 47 (2002) 49–56
spleen and kidney. Abnormalities induced by toxicant in any of these tissues may have implications in fish health and yield. While chromosomal, nuclear and sperm head anomalies would point to genomic changes caused by the treatment of EMS, the protein parameters would bear further support to functional changes due to genomic toxicity and/or damage. To our knowledge, such a two-step assessment has not been previously conducted with fish.
2. Materials and methods Live specimens of O. mossambicus weighing between 18 and 25 g were collected from a local fresh water pond and were acclimated for 5–6 d in a concrete vat containing fresh tap water (stored from deep tubewell) and fed with artificial diet (rice bran þ wheat þ oil cake). Separate vats were maintained under the same conditions in which 40–42 specimens were kept for the experiments. Six live specimens each were injected with optimally low doses that produced fairly quantifiable changes (obtained through range-finding trials): (1) 0.2% EMS, (2) 0.02% ascorbic acid, (3) 0.2% EMS and 0.02% ascorbic acid and (4) double distilled water @ 1 ml/100 g of body weight for each of the six fixation intervals (viz., 6, 12, 24, 48, 72 and 96 h; since the peak damaging effect induced by EMS seemed to be obtained at 48 h and there was a declining trend till 96 h, fixation interval beyond 96 h was not considered). Injected specimens were removed into smaller vats containing tap water and were fed with artificial diet till sacrificed. Specimens injected with double distilled water (being the ‘‘vehicle’’ for EMS and VC) served as controls. For chromosome analysis, experimental and control fish specimens were injected intramuscularly with 0.05% colchicine @ 1 ml/100 g bw about 3 h prior to sacrifice and their somatic chromosomes were prepared as per the citrate-flame drying technique (see Khuda-Bukhsh, 1979). 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). Small nonrefractile circular or ovoid chromatin bodies resembling nucleus in staining property and either attached to or detached from the nucleus were considered as micronuclei (MN). The frequency of anomalous nuclei (AN, i.e. nuclear boundaries with various unorthodox shapes) was recorded as per the method of Carrasco et al. (1990) and nuclear and cytoplasmic volumes were measured (Porichha et al., 1998) to calculate nucleocytoplasmic ratio (Cyt/Nu ratio ¼ Vc =Vn where Vc is the volume of cytoplasm and Vn is the volume of nucleus;
nuclei with geometrically bizarre shapes were excluded from the purview of the study) in the normal erythrocytes (Khuda-Bukhsh et al., 2000) to examine if the ratio could give any indication of the genotoxic status of the cell. For the study of sperm head abnormality, epididymes of testis of the males were dissected and inner contents were taken out in fish––Ringer and smears were prepared on slides and stained with Giemsa (Manna and Biswas, 1988). For biochemical study nine different tissues, namely, dorsal muscle, ventral muscle, heart, eye, brain, gill, liver, spleen and kidney were removed after 48 h of treatment from each of the treated and control specimens separately and were subjected to the SDS-PAGE technique (Laemmli, 1970) using 7.5% separating gel in Tris–glycine buffer for visualizing different sub-fractions of protein bands; for the quantitative assay the technique of Lowry et al. (1951) was followed. For the analysis of data, ANOVA was first conducted to ascertain significant gross differences, if any, between different series (viz., EMS-treated, EMS plus VC-treated or DWtreated series) and among different fixation intervals. To further pinpoint the difference between control and treated series, Student’s t-test was conducted and the level of significance determined by using the Fisher and Yates statistical tables (Fisher and Yates, 1963). The similarity index was calculated on the basis of molecular weight of individual bands arranged into size classes of 10 by adopting the following formula (Shaw, 1970): SI ðsimilarity indexÞ ¼ n=0:5 ðN1 þ N2 Þ
ð1Þ
where n is the frequency of identical protein fractions of both individuals to be compared and N1 and N2 represented the number of all fractions of the electropherograms of both the individuals under test. The percentage of protection was calculated by subtracting the total percentage of abnormality (e.g. chromosome aberrations, or MN) encountered in the EMS plus VC-treated series from that of the percentage of respective abnormality encountered in the only EMStreated series.
3. Results and discussion 3.1. Chromosome aberration study Normal metaphase complements (Fig. 1, PM 1) were carefully examined for any possible clastogenic change due to various treatments. Representative photomicrographs of various types of chromosomal aberrations like, chromatid break (PM 2), ring (PM 3), acentric fragment (PM 4), pulverization (PM 5), C-mitotic effect (PM 6) etc. have been provided mainly from EMStreated fish. However, a few aberrations of minor nature
B. Guha, A.R. Khuda-Bukhsh / Chemosphere 47 (2002) 49–56
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Fig. 1. PM 1–6: photomicrographs of metaphase complements: normal (PM 1) and aberrated, chromatid break, CB (PM 2), ring, R (PM 3), acentric fragment, AF (PM 4), pulverization (PM 5) and C-mitotic effect (PM 6); PM 7–12: showing erythrocytes with micronuclei, MN (PM 7–8) and anomalous nuclei, AN (PM 9–12); PM 13–16: showing sperm with normal (PM 13) and abnormal head shapes, SHA (PM 14–16); PM 17–20: showing gel electropherograms of 9 different tissues of control (PM 17) and treated (PM 18–20) fish; PM 18 ¼ ascorbic acid treated, PM 19 ¼ EMS treated and PM 20 ¼ EMS þ ascorbic acid conjointly treated. DM: Dorsal muscle, VM: Ventral muscle, H: Heart, E: Eye, Br: Brain, G: Gill, Li: Liver, Sp: Spleen, K: Kidney.
were also recorded in fish injected with distilled water (DW) and also in VC-injected fish. The summarized data on the frequencies and types of aberrations ob-
served in various treated and control fishes have been provided in Table 1. It would be revealed from the data that the frequencies of aberrations in the DW-injected
52 Table 1 Showing frequency distribution of chromosome aberrations, 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% ascorbic acid (T2 ) separately and 0.2% EMS þ 0:02% ascorbic acid conjointly (T3 ) and their respective double distilled water controls (C) at different fixation intervals Series
6
C T1 T2 T3
2:58 0:24 10:21 0:83 1:58 0:53 8:25 1:23
C T1 T2 T3
3:19 0:38 10:96 1:51 2:76 0:21 8:48 0:89
C T1 T2 T3
4:49 1:14 14:57 1:63 2:45 0:46 10:03 1:03
C T1 T2 T3
4:11 1:39 19:46 0:67 2:62 0:94 14:37 1:20
C T1 T2 T3
5:08 0:90 13:88 0:70 3:71 0:66 11:42 0:64
C T1 T2 T3
4:82 1:19 11:63 1:09 4:01 0:73 9:41 1:65
12
24
48
72
96
Chromosome aberration
Nuclear anomalies
% of CA SE
% of MNE SE
% of protection
% of AN SE
% of protection
% of Cyt/ Nu SE
– – – –
– – – –
– – – –
– – – –
– – – –
1:63 0:07 6:18 0:85 1:28 0:08 2:37 0:55
– – – –
– – – –
– – – –
– – – –
– – – –
2:30 0:52 4:43 0:84 2:16 0:64 2:57 0:17
12:52 0:83 11:97 0:72 12:15 0:73 13:14 1:06
2:14 0:12 10:29 0:54 1:83 0:27 2:80 0:16
% of protection
1.96
2.48
4.54a
5.09b
2.46a
2.22
0:05 0:01 0:21 0:03 0:05 0:02 0:07 0:02 0:06 0:02 0:23 0:04 0:05 0:02 0:12 0:02 0:06 0:02 0:17 0:03 0:04 0:02 0:11 0:03 0:04 0:01 0:13 0:02 0:03 0:02 0:11 0:03
0.14b
0.11a
0.06
0.02
Sperm head anomaly
0:83 0:08 1:26 0:09 0:57 0:05 0:71 0:08 0:87 0:05 1:15 0:04 0:58 0:03 0:81 0:07 0:96 0:12 1:21 0:10 0:51 0:03 0:60 0:06 0:88 0:16 1:13 0:14 0:52 0:02 0:76 0:11
0.55c
0.34b
0.61c
0.37
10:32 1:13 13:56 1:06 13:33 0:78 13:35 1:23 9:79 0:37 11:85 0:29 12:48 0:45 13:80 0:50 11:10 1:08 12:85 1:17 12:76 0:60 13:30 0:65
Difference T1 vs T3
1.17
0.21
1.95b
0.45
% of SHA SE
1:82 0:65 8:12 0:99 2:17 0:63 3:19 0:35 1:62 0:54 5:56 1:37 1:78 0:43 3:04 0:33 1:63 0:25 4:82 0:08 1:63 0:25 2:32 0:22
% of protection
3.81a
1.86
7.49c
4.93b
2.52
2.50c
Chromosome aberrations (CA) include gap, break, centric fusion, translocation, fragment, pulverisation, ring, terminal association, polyploidy, aneuploidy, stickiness, C-mitotic effect, precocious centromeric separation, constriction, etc. No. of individuals examined in each series/fixation intervals ¼ 6; cells scored per individual ¼ 500 for CA, 20 000 for MNE, AN etc. 6000 for SHA. a p < 0:05. b p < 0:01. c p < 0:001.
B. Guha, A.R. Khuda-Bukhsh / Chemosphere 47 (2002) 49–56
Fixation time intervals (h)
B. Guha, A.R. Khuda-Bukhsh / Chemosphere 47 (2002) 49–56
fishes and the VC-injected fishes were relatively low as compared to EMS-treated fish, particularly at early intervals, and were slightly increased at longer intervals. The aberration frequencies in the EMS-injected fishes at different intervals were generally strikingly higher than in the DW-injected controls or in the VC-injected fish (p < 0:05 and p < 0:01), particularly up to 48 h, after which the aberration frequencies gradually declined. However, the injection of ascorbic acid in distilled water medium to EMS-treated fish considerably reduced the aberration frequencies as compared to only EMS-treated fish at all the fixation intervals (see Table 1), of which the differences at 24, 48 and 72 h were statistically significant (p < 0:05 and p < 0:01).
3.2. Micronucleus study Micronucleated erythrocytes (MN) (PM 7, 8) and erythrocytes with abnormal nuclei (AN) (PM 9–12) were observed in much greater number in fish treated with EMS. However, a few were also encountered in the DWand VC-treated fish. The frequencies of MN and AN observed in various treated and control fishes have been summarized in Table 1. A critical analysis of the data would also reveal that EMS-treated fish counter-injected with VC showed fewer micronuclei and anomalous nuclei as compared to only EMS-injected fish at the four fixation intervals examined (see Table 1), of which the differences were statistically significant at 24 and 48 h (p < 0:05 and p < 0:01). The Cyt/Nu ratio appeared to be generally higher in the EMS- and VC-treated fish as compared to DW-treated controls. The differences in the ratio were, however, statistically significant only at 72 h (p < 0:05). But when compared between fishes treated with only EMS and EMS þ VC, the Cyt/Nu ratios were found to be greater in the EMS plus VC-treated fish except at 48 h. Thus, the ratios did not necessarily denote toxicity status of the cells and can not really be useful as a toxicity parameter. However, the ratio might have some relationship with differential hydration status of the cells treated with different agents. Further, when a correlation between the ratio and the occurrence of MN or AN was considered, the Cyt/Nu ratio appeared to increase slightly in the fish showing greater number of micronuclei at some fixation intervals like 24, 48, and 72 h, but no such apparent relationship could be substantiated with the frequency of AN and Cyt/Nu.
3.3. Sperm head abnormality study Sperm with normal (PM 13) and abnormal (PM 14– 16) head shapes were encountered in both control and treated fish. The frequencies of sperm with abnormal
53
head shapes have been summarized in Table 1. The data reveal that the frequency of sperm with abnormal head was less in EMS þ VC-treated fish as compared to only EMS-treated fish at all the fixation intervals (see Table 1), of which the differences observed at 6, 24, 48 and 96 h were statistically significant at various levels (p < 0:05, 0.01 and 0.001).
3.4. Qualitative analysis The number and summarized data of gel–electrophoretic band profiles in different tissues of fish injected with distilled water, ascorbic acid, and EMS separately and EMS þ ascorbic acid conjointly have been presented in Table 2 and photographs of a few representative electropherograms provided (PM 17–20). From the data it would be revealed that in all the tissues the number of bands was decreased in the EMS-treated fish as compared to DW-treated controls except for liver and spleen tissues in which the number of bands was greater in EMS-treated fish. In general, the less number of bands would signify the loss of certain species of proteins while the gain in number of bands would speak for the synthesis of new species of protein as a result of specific treatment. However, sometimes the number of bands remaining same, the type of proteins may be different if the individual band in electropherograms of different treatment or control series does not have the same molecular weight or the nature and intensity of staining of bands differ between the bands in question. Therefore, a further critical study was made by assigning the band characteristics of the different treatment and control series (Table 2). In the ascorbic acid-treated fish the number of bands was greater than in the DW-treated fish for some tissues (i.e. eye, gill, liver and spleen) whereas it was less in number in the other tissues (i.e., dorsal muscle, heart, brain and kidney). This would mean that ascorbic acid might have acted differentially on different organs, either owing to their more favorable absorption potential and/or occurrence of greater number of working domains (for scavenging/anti-oxidant activity) in certain tissues, or else because of their differential metabolic degradation/denaturation or loss of function in certain tissues. The number of bands in the EMS þ VC-treated fish was generally greater than in the only EMS-treated fish, which would also imply that VC possibly restored damaged protein by the synthesis of the lost ones. Further, in order to have a quick view on the extent of change in band profiles in the different tissues of treated and control fishes, a similarity index has been calculated on the basis of the molecular weight and provided in Table 3. This would also give an idea as to the extent to which individual tissue showed modulations in their protein profiles as a result of EMS treatment followed by VC treatment.
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Table 2
Showing number of bands, range of relative mobility (Rm) and range of molecular weight of SDS-PAGE total protein profiles and amount of total protein (mg/gm) in different tissues of O. mossambicus treated with 0.2% EMS (T1 ), 0.02% ascorbic acid (T2 ) separately and 0.2% EMS þ 0:02% ascorbic acid conjointly (T3 ) and their respective double distilled water controls (C) at 48 h fixation interval Series
Tissues DM
VM
H
E
Br
G
Li
Sp
K
23 23 23 23
22 14 18 25
23 18 25 29
29 14 23 30
21 14 29 26
21 22 26 22
13 15 17 18
27 22 26 27
0.138–0.976 0.053–0.947 0.31–0.980 0.014–0.960
0.034–0.936 0.37–0.946 0.317–0.906 0.034–0.973
0.021–0.969 0.01–0.978 0.086–0.978 0.043–0.982
0.031–0.969 0.032–0.961 0.022–0.964 0.037–0.948
0.028–0.969 0.068–0.977 0.022–0.978 0.036–0.953
0.024–0.976 0.083–0.947 0.022–0.986 0.036–0.953
0.031–0.976 0.068–0.902 0.018–0.982 0.037–0.963
0.041–0.976 0.071–0.976 0.022–0.978 0.049–0.962
29.9–235.1 32.1–290.0 29.6–154.1 31.1–318.7
32.8–303.3 32.2–127.2 35.5–151.5 30.1–303.6
30.4–313.7 29.7–324.3 29.7–267.0 29.5–296.9
32.6–305.9 31.0–304.9 30.8–313.1 32.0–301.4
30.4–308.5 29.8–279.5 29.7–313.1 31.6–302.2
29.9–311.1 32.1–269.3 29.2–313.1 31.6–302.2
29.9–305.9 35.9–279.5 29.5–315.8 30.9–301.4
29.9–296.2 29.9–277.3 29.7–313.1 30.9–292.7
13.2 11.8 17.9 16.6
15.7 11.4 14.7 25.0
16.6 14.4 21.1 26.2
19.5 15.4 24.7 26.2
24.5 15.9 33.6 30.0
34.6 15.7 35.0 39.5
50.0 40.9 26.5 55.9
62.2 31.2 33.1 34.8
Number of bands C T1 T2 T3
26 20 25 29
Range of Rm C T1 T2 T3
0.038–0.974 0.226–0.947 0.054–0.946 0.022–0.968
Range of molecular weight C T1 T2 T3
30.0–300.7 32.1–189.5 32.2–288.8 30.5–313.1
Amount of protein C T1 T2 T3
14.8 13.0 18.3 20.2
DM: Dorsal muscle, VM: Ventral muscle, H: Heart, E: Eye, Br: Brain, G: Gill, Li: Liver, Sp: Spleen, K: Kidney.
Table 3 Showing similarity indices on molecular weights of SDS-PAGE total protein profiles in different tissues of O. mossambicus treated with 0.2% EMS (T1 ), 0.02% ascorbic acid (T2 ) separately and 0.2% EMS þ 0:02% ascorbic acid conjointly (T3 ) and their respective double distilled water controls (C) at 48 h fixation interval Series C vs T1 C vs T2 T1 vs T3
Tissues DM
VM
H
E
Br
G
Li
Sp
K
0.74 0.54 0.64
0.69 0.74 0.65
0.67 0.60 0.65
0.57 0.63 0.54
0.50 0.73 0.41
0.50 0.64 0.60
0.77 0.75 0.77
0.64 0.73 0.59
0.68 0.69 0.63
DM: Dorsal muscle, VM: Ventral muscle, H: Heart, E: Eye, Br: Brain, G: Gill, Li: Liver, Sp: Spleen, K: Kidney.
3.5. Quantitative analysis The quantitative data on total protein contents of different tissues in different control and treated series have been presented in Table 2. It would be revealed from the data that as compared to the DW-treated series the total protein contents in the EMS-treated series were less in all the tissues, whereas in the ascorbic acid-treated series the content was greater or similar in all tissues except spleen and kidney which showed lesser amount of total protein. On the other hand in the combined EMS þ VC-treated series the total protein contents were greater than in either distilled water or EMS- or ascorbic acid-treated series. In other words apparently the inhibitory effects of EMS on protein synthesis was not only removed by ascorbic acid treatment but the con-
joint treatment of EMS þ VC 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. A close scrutiny of the data presented in Table 2 would also reveal that the response in different tissues was slightly different, which would also corroborate the hypothesis that ascorbic acid may have a somewhat preferential action in certain tissues depending on the necessity and its ability to help synthesize certain proteins (e.g., collagen) damaged/lost due to EMS treatment. In the present study, EMS was found to induce various types of chromosome aberrations, nuclear anomalies including micronuclei formation, abnormal sperm head shapes, change in gel–electrophoretic band profiles and total protein contents of nine different tissues
B. Guha, A.R. Khuda-Bukhsh / Chemosphere 47 (2002) 49–56
examined, which would amply corroborate the genotoxic and mutagenic potentials of EMS in O. mossambicus. Earlier Hooftman (1981) reported enhanced occurrence of chromosome aberrations in Notobranchius rochowi as a result of EMS and benzo[a]pyrene treatments. 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. 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 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 (Cohn, 1979). Therefore, the change of gel–electrophoretic band profiles and total protein contents observed in the EMS-treated fish was an additional indication of what might actually represent faulty/conformationally altered proteins, as a consequence of damage in DNA that emanated altered signals for protein synthesis. Alternatively, it is also possible that some of the altered protein might owe their origin to the direct/indirect action of reactive oxygen species or reactive nitrogen species produced due to cellular toxicity. Further, it would be revealed from the results of the present study that EMS produced genotoxic effects which appeared to increase up to 48 h after treatment but the effects were sustained through longer intervals to a considerable extent. Interestingly the injection of ascorbic acid (0.02%) appeared to modulate the genotoxic effects at all fixation intervals and ameliorated the EMS toxicity to a statistically significant extent at several of these fixation intervals, as indicated by both the cytogenetical and biochemical protocols used. The relatively short span of damaging activity of EMS might partly be due to the inherent ability of fish to excrete the toxic substance at the earliest and partly to its ability to detoxify the chemical by metabolic degradation. The exact mechanism by which VC modulates or minimizes the cytogenetical toxicity of EMS is not known. However, it is known that ascorbic acid is an anti-oxidant which might inhibit the oxidative metabolism of EMS and thus could prevent the production of mutagenic electrophilic metabolites (Goncharova and Kuzhir, 1989). Further, as ascorbic acid has marked nucleophilic properties it might intercept reactive electrophilic metabolites produced by EMS, thereby preventing their attack on nucleophilic sites on DNA and hence blocking adduct formation (Liehr et al., 1989). Also as part of a redox buffer system VC can scavenge harmful free radical metabolites/reactive oxygen species (Sato et al., 1990). Thus, the general protective effect of VC observed against EMS-induced genotoxicity by a twostep study in this fish could actually be accomplished
55
through one or many of these inhibition mechanisms as suggested by others for some other models (De Flora and Ramel, 1988; Khan and Sinha, 1993, 1996; Hoda and Sinha, 1993). Alternatively, since VC has a positive role to play in collagen (a component of protein) formation, it might have acted as the external reductant that is required in the conversion of proline to hydroxyproline in the synthesis of collagen protein in certain tissues that might have been damaged by EMS treatment.
Acknowledgements The authors are grateful to ICAR, Government of India, New Delhi, for providing financial support of the work. References Bose, S., Sinha, S.P., 1994. Modulation of ochratoxin-produced genotoxity in mice by vitamin-C. Fd. Chem. Toxic 32, 533–537. Cameron, E., 1979. In: Hanck, A., Ritzel, G. (Eds.), Vitamin C––Recent Advances and Aspects in Virus Diseases, Cancer and in Lipid Metabolism. Huber, Bern, pp. 9–24. Carrasco, K.R., Tilbury, K.L., Myers, M.S., 1990. Assessment of the piscine micronucleus test as in situ biological indicator of chemical contaminant effects. Can. J. Fish. Acu. Sci. 47, 2123–2136. Cohn, N.S., 1979. Elements of Cytology, second ed. Freeman Book Company, Delhi. De Flora, S., Ramel, C., 1988. Mechanisms of inhibitors of mutagenesis: classification and overview. Mutat. Res. 202, 285–306. Fisher, R.A., Yates, F., 1963. Statistical Tables for Biological, Agricultural and Medical Research, sixth ed. Oliver and Boyd, Edinburgh. Gebhart, E., Wagner, H., Grziwok, K., Behnsen, H., 1985. The action of anticlastogens in human lymphocyte cultures and their modification by rat liver S9 II. Studies with vitamin C and E. Mutat. Res. 149, 83–94. Goncharova, R.I., Kuzhir, T.D., 1989. A comparative study of the antimutagenic effects of antioxidants on chemical mutagenesis in Drosophila melanogaster. Mutat. Res. 214, 257–265. Guttenplan, J.B., 1977. Inhibition by L -ascorbate of bacterial mutagenesis induced by two N-nitroso compounds. Nature 268, 368–370. Hayatsu, H., Arimoto, S., Negishi, T., 1988. Dietary inhibitors of mutagenesis and carcinogenesis. Mutat. Res. 202, 429–446. Hoda, Q., Sinha, S.P., 1991. Minimization of cytogenetic toxicity of malathion by Vitamin C. J. Nutri. Sci. Vitaminol. 34, 329–339. Hoda, Q., Bose, S., Sinha, S.P., 1991. Vitamin C mediated minimisation of malathion and rogor-induced mitoinhibition and clastogeny. Cytologia 56, 389–397. Hoda, Q., Sinha, S.P., 1993. Vitamin C-mediated minimisation of rogor-induced genotoxicity. Mutat. Res. 299, 29–36.
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Hooftman, R.N., 1981. The induction of chromosome aberrations in Notobranchius rochowi (Pisces: Cyprinodontidae) after treatment with ethyl methanesulphonate or benzo[a]pyrene. Mutat. Res. 91, 347–352. Hooftman, R.N., de Raat, W.K., 1982. Induction of nuclear anomalies (micronuclei) in peripheral blood erythrocytes of eastern mudminnow Umbra pygmaea by ethyl methane sulphonate. Mutat. Res. 104, 147–152. Khan, P.K., Sinha, S.P., 1992. Dose-dependent minimisation of cytogenetic toxicity of endosulphan by vitamin C. Cytologia 57, 217–221. Khan, P.K., Sinha, S.P., 1993. Antimutagenic efficacy of higher doses of vitamin C. Mutat. Res. 298, 157–161. Khan, P.K., Sinha, S.P., 1996. Ameliorating effect of Vitamin C on murine sperm toxicity induced by three pesticides (endosulfan, phosphamidon and mancozeb). Mutagenesis 11, 33–36. Kihlman, B.A., 1966. Action of Chemicals on Dividing Cells. Prentice-Hall, Englewood cliffs, NJ. Krishna, G., Nath, J., Ong, T., 1986. Inhibition of cyclophosphamide and mytomycin C-induced sister chromatid exchanges in mice by vitamin C. Cancer Res. 46, 2670– 2674. Khuda-Bukhsh, A.R., 1979. Chromosomes in three species of fishes, Aplocheilus panchax (Cyprinodontidae), Lates calcarifer (Percidae) and Gadusia chapra (Clupeidae). Caryologia 32, 161–169. Khuda-Bukhsh, A.R., Chakrabarti, J., Mallick, P., Mohanty, K.C., 2000. Genotoxic studies in natural populations of fish as an indicator of aquatic pollution in the Hooghly–Matlah estuary system. In: Jana, B.B., Banerjee, R.D., Guterstam, B., Heeb, J. (Eds.), Waste Recycling and Resource Management in the Developing World, Proc. Int. Conf. Ecol. Eng., Science City, Calcutta, 23–27, November 1998. pp. 377–385. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4 . Nature 227, 680–685. Liehr, J.G., Roy, D., Gladek, A., 1989. Mechanism of inhibition of estrogen-induced renal carcinogenesis in male Syrian hamster by Vitamin C. Carcinogenesis 10, 1983– 1988. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265–275.
Machlin, L., Bendich, A., 1987. Free radical tissue damage: protective role of antioxidant nutrients. FASEB J 1, 1983– 1998. Manna, G.K., Biswas, S.K., 1988. Induction of abnormal sperm head in tilapia fish as means of testing genotoxic potentiality of the bacterium, Pseudomonas aeruginosa. La Kromosomo II 51–52, 1659–1664. Pauling, L., Nixon, J.C., Stiff, F., Marcuson, R., Dunham, W.B., Barth, R., Bensch, K., Herman, Z.S., Blaisdell, B.E., Tsao, C., Preder, M., Andrews, V., Willoughby, R., Zuckerland, E., 1985. Effect of dietary ascorbic acid on the incidence of mammary tumors in RIII mice. Proc. Natl. Acad. Sci. USA 82, 5185–5189. Porichha, S.K., Sarangi, P.K., Prasad, R., 1998. Cytogenetic effect of chlorpyriphos in Channa punctatus. In: Manna, G.K., Roy, S.C. (Eds.). Proc. IXth All India Cong. Cyto. Genet., Chandigarh, 22–25 November, 1996. Pers. Cytol. Genet. 9 631–638. Raina, V., Gurtoo, H.L., 1985. Effect of vitamin A, C and E on aflatoxin B1 induced mutagenesis in Salmonella typhimurium TA-98 and TA-100. Teratogen. Carcinogen. Mutagen. 5, 29–40. Sato, K., Niki, E., Shimasaki, H., 1990. Free radical-mediated chain oxidation of low density lipoprotein and its synergistic inhibition by Vitamin E and Vitamin C. Arch. Biochem. Biophy. 279, 402–405. Schmid, W., 1976. In: Hollaender, A. (Ed.), Chemical Mutagens: Principles and Methods of their Detection, vol. 4. Plenum Press, New York, pp. 31–53. Shamberger, R.J., 1984. Genetic toxicology of ascorbic acid. Mutat. Res. 133, 135–159. Shaw, C.R., 1970. How many genes evolved? Biochem. Genet. 4, 275–283. Mr. Bibhas Guha, M.Sc., b. 13th Dec., 1973, Formerly Senior Research Fellow of ICAR, New Delhi, working on mutagenesis and antimutagenesis of fish. Dr. A.R. Khuda-Bukhsh, M.Sc., Ph.D.: b. 26th September, 1948, Reader in Zoology, University of Kalyani, published over 100 research papers, completed several research projects granted by UGC, ICAR, CSIR, DOE, Government of India, some 20 students awarded Ph.D. degree under his supervision, field of research: fish cytogenetics, mutagenesis and antimutagenesis; aphid cytogenetics, mammalian mutagenesis and antimutagenesis.
Efficacy of vitamin-C (L -ascorbic acid) in reducing genotoxicity in fish (Oreochromis mossambicus) induced by ethyl methane sulphonate B. Guha, A.R. Khuda-Bukhsh
*
Department of Zoology, Kalyani University, Kalyani-741235, India Received 23 March 2001; accepted 1 November 2001
Abstract The genotoxic effects of ethyl methane sulphonate (EMS) have been assessed in a fish, Oreochromis mossambicus with endpoints including chromosome aberrations, abnormal red blood cell nuclei, abnormal sperm morphology, and protein content (both qualitative and quantitative) of selected tissues, namely, muscle, heart, eye, brain, gill, liver, spleen and kidney. EMS caused chromosomal aberrations, nuclear anomalies in red blood cells, abnormal sperm morphology, and alteration of protein synthesis in various tissues. Some of the EMS toxicity appeared to be modulated and ameliorated in this fish by vitamin-C treatment. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Genotoxicity; Antimutagen; Tilapia; Chromosome aberrations; Micronuclei; Sperm head anomaly
1. Introduction Ethyl methane sulphonate (EMS) is an alkylating agent which has been extensively used in genetic research for its ability to alter a base that is already incorporated into a DNA molecule and thereby is of prime importance in the induction of artificial mutations. The clastogenic, mutagenic, teratogenic and carcinogenic effects of EMS have been extensively studied in various mammals (see Kihlman, 1966). Fish, particularly those living in rivers or confined waters, also run the risk of direct or indirect exposure to various chemical mutagens collected along with the run-offs containing various toxicants, pesticides, industrial wastes, etc. Therefore, it is of interest to study the effects of such mutagens on the genome of fish. Similarly, vitamin-C (L -ascorbic acid; VC) is believed to have anti-oxidant nature (Machlin
*
Corresponding author. Tel.: +91-33-5828768/5828750x315. E-mail address: [email protected] (A.R. Khuda-Bukhsh).
and Bendich, 1987; Hayatsu et al., 1988; Sato et al., 1990) and has been quite extensively tested in various mammalian models for its ability to modulate cytogenetic toxicity of certain common pesticides (Hoda and Sinha, 1991, 1993; Khan and Sinha, 1992, 1993). VC has also been reported to have anticarcinogenic (Cameron, 1979; Pauling et al., 1985), anticlastogenic (Gebhart et al., 1985; Krishna et al., 1986; Hoda et al., 1991; Hoda and Sinha, 1993; Bose and Sinha, 1994) and even antimutagenic (Guttenplan, 1977; Shamberger, 1984; Raina and Gurtoo, 1985) roles in a variety of test systems, from microorganisms to cell-free systems or cells in culture in mammalian models, but again its role in modulating cytogenetic damage has not been ascertained in any fish. Therefore, in the present investigation the effectiveness of using a fish model (Oreochromis mossambicus) for the study of the genotoxic effects of EMS and modulation of EMS toxicity by VC was addressed. The toxicity endpoints that this study looked at were chromosomal aberrations, abnormal red blood cell nuclei, abnormal sperm morphology and protein content and gel profiles of nine selected tissues from vital organs, namely, muscle, heart, eye, brain, gill, liver,
0045-6535/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 1 ) 0 0 3 0 8 - 3
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spleen and kidney. Abnormalities induced by toxicant in any of these tissues may have implications in fish health and yield. While chromosomal, nuclear and sperm head anomalies would point to genomic changes caused by the treatment of EMS, the protein parameters would bear further support to functional changes due to genomic toxicity and/or damage. To our knowledge, such a two-step assessment has not been previously conducted with fish.
2. Materials and methods Live specimens of O. mossambicus weighing between 18 and 25 g were collected from a local fresh water pond and were acclimated for 5–6 d in a concrete vat containing fresh tap water (stored from deep tubewell) and fed with artificial diet (rice bran þ wheat þ oil cake). Separate vats were maintained under the same conditions in which 40–42 specimens were kept for the experiments. Six live specimens each were injected with optimally low doses that produced fairly quantifiable changes (obtained through range-finding trials): (1) 0.2% EMS, (2) 0.02% ascorbic acid, (3) 0.2% EMS and 0.02% ascorbic acid and (4) double distilled water @ 1 ml/100 g of body weight for each of the six fixation intervals (viz., 6, 12, 24, 48, 72 and 96 h; since the peak damaging effect induced by EMS seemed to be obtained at 48 h and there was a declining trend till 96 h, fixation interval beyond 96 h was not considered). Injected specimens were removed into smaller vats containing tap water and were fed with artificial diet till sacrificed. Specimens injected with double distilled water (being the ‘‘vehicle’’ for EMS and VC) served as controls. For chromosome analysis, experimental and control fish specimens were injected intramuscularly with 0.05% colchicine @ 1 ml/100 g bw about 3 h prior to sacrifice and their somatic chromosomes were prepared as per the citrate-flame drying technique (see Khuda-Bukhsh, 1979). 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). Small nonrefractile circular or ovoid chromatin bodies resembling nucleus in staining property and either attached to or detached from the nucleus were considered as micronuclei (MN). The frequency of anomalous nuclei (AN, i.e. nuclear boundaries with various unorthodox shapes) was recorded as per the method of Carrasco et al. (1990) and nuclear and cytoplasmic volumes were measured (Porichha et al., 1998) to calculate nucleocytoplasmic ratio (Cyt/Nu ratio ¼ Vc =Vn where Vc is the volume of cytoplasm and Vn is the volume of nucleus;
nuclei with geometrically bizarre shapes were excluded from the purview of the study) in the normal erythrocytes (Khuda-Bukhsh et al., 2000) to examine if the ratio could give any indication of the genotoxic status of the cell. For the study of sperm head abnormality, epididymes of testis of the males were dissected and inner contents were taken out in fish––Ringer and smears were prepared on slides and stained with Giemsa (Manna and Biswas, 1988). For biochemical study nine different tissues, namely, dorsal muscle, ventral muscle, heart, eye, brain, gill, liver, spleen and kidney were removed after 48 h of treatment from each of the treated and control specimens separately and were subjected to the SDS-PAGE technique (Laemmli, 1970) using 7.5% separating gel in Tris–glycine buffer for visualizing different sub-fractions of protein bands; for the quantitative assay the technique of Lowry et al. (1951) was followed. For the analysis of data, ANOVA was first conducted to ascertain significant gross differences, if any, between different series (viz., EMS-treated, EMS plus VC-treated or DWtreated series) and among different fixation intervals. To further pinpoint the difference between control and treated series, Student’s t-test was conducted and the level of significance determined by using the Fisher and Yates statistical tables (Fisher and Yates, 1963). The similarity index was calculated on the basis of molecular weight of individual bands arranged into size classes of 10 by adopting the following formula (Shaw, 1970): SI ðsimilarity indexÞ ¼ n=0:5 ðN1 þ N2 Þ
ð1Þ
where n is the frequency of identical protein fractions of both individuals to be compared and N1 and N2 represented the number of all fractions of the electropherograms of both the individuals under test. The percentage of protection was calculated by subtracting the total percentage of abnormality (e.g. chromosome aberrations, or MN) encountered in the EMS plus VC-treated series from that of the percentage of respective abnormality encountered in the only EMStreated series.
3. Results and discussion 3.1. Chromosome aberration study Normal metaphase complements (Fig. 1, PM 1) were carefully examined for any possible clastogenic change due to various treatments. Representative photomicrographs of various types of chromosomal aberrations like, chromatid break (PM 2), ring (PM 3), acentric fragment (PM 4), pulverization (PM 5), C-mitotic effect (PM 6) etc. have been provided mainly from EMStreated fish. However, a few aberrations of minor nature
B. Guha, A.R. Khuda-Bukhsh / Chemosphere 47 (2002) 49–56
51
Fig. 1. PM 1–6: photomicrographs of metaphase complements: normal (PM 1) and aberrated, chromatid break, CB (PM 2), ring, R (PM 3), acentric fragment, AF (PM 4), pulverization (PM 5) and C-mitotic effect (PM 6); PM 7–12: showing erythrocytes with micronuclei, MN (PM 7–8) and anomalous nuclei, AN (PM 9–12); PM 13–16: showing sperm with normal (PM 13) and abnormal head shapes, SHA (PM 14–16); PM 17–20: showing gel electropherograms of 9 different tissues of control (PM 17) and treated (PM 18–20) fish; PM 18 ¼ ascorbic acid treated, PM 19 ¼ EMS treated and PM 20 ¼ EMS þ ascorbic acid conjointly treated. DM: Dorsal muscle, VM: Ventral muscle, H: Heart, E: Eye, Br: Brain, G: Gill, Li: Liver, Sp: Spleen, K: Kidney.
were also recorded in fish injected with distilled water (DW) and also in VC-injected fish. The summarized data on the frequencies and types of aberrations ob-
served in various treated and control fishes have been provided in Table 1. It would be revealed from the data that the frequencies of aberrations in the DW-injected
52 Table 1 Showing frequency distribution of chromosome aberrations, 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% ascorbic acid (T2 ) separately and 0.2% EMS þ 0:02% ascorbic acid conjointly (T3 ) and their respective double distilled water controls (C) at different fixation intervals Series
6
C T1 T2 T3
2:58 0:24 10:21 0:83 1:58 0:53 8:25 1:23
C T1 T2 T3
3:19 0:38 10:96 1:51 2:76 0:21 8:48 0:89
C T1 T2 T3
4:49 1:14 14:57 1:63 2:45 0:46 10:03 1:03
C T1 T2 T3
4:11 1:39 19:46 0:67 2:62 0:94 14:37 1:20
C T1 T2 T3
5:08 0:90 13:88 0:70 3:71 0:66 11:42 0:64
C T1 T2 T3
4:82 1:19 11:63 1:09 4:01 0:73 9:41 1:65
12
24
48
72
96
Chromosome aberration
Nuclear anomalies
% of CA SE
% of MNE SE
% of protection
% of AN SE
% of protection
% of Cyt/ Nu SE
– – – –
– – – –
– – – –
– – – –
– – – –
1:63 0:07 6:18 0:85 1:28 0:08 2:37 0:55
– – – –
– – – –
– – – –
– – – –
– – – –
2:30 0:52 4:43 0:84 2:16 0:64 2:57 0:17
12:52 0:83 11:97 0:72 12:15 0:73 13:14 1:06
2:14 0:12 10:29 0:54 1:83 0:27 2:80 0:16
% of protection
1.96
2.48
4.54a
5.09b
2.46a
2.22
0:05 0:01 0:21 0:03 0:05 0:02 0:07 0:02 0:06 0:02 0:23 0:04 0:05 0:02 0:12 0:02 0:06 0:02 0:17 0:03 0:04 0:02 0:11 0:03 0:04 0:01 0:13 0:02 0:03 0:02 0:11 0:03
0.14b
0.11a
0.06
0.02
Sperm head anomaly
0:83 0:08 1:26 0:09 0:57 0:05 0:71 0:08 0:87 0:05 1:15 0:04 0:58 0:03 0:81 0:07 0:96 0:12 1:21 0:10 0:51 0:03 0:60 0:06 0:88 0:16 1:13 0:14 0:52 0:02 0:76 0:11
0.55c
0.34b
0.61c
0.37
10:32 1:13 13:56 1:06 13:33 0:78 13:35 1:23 9:79 0:37 11:85 0:29 12:48 0:45 13:80 0:50 11:10 1:08 12:85 1:17 12:76 0:60 13:30 0:65
Difference T1 vs T3
1.17
0.21
1.95b
0.45
% of SHA SE
1:82 0:65 8:12 0:99 2:17 0:63 3:19 0:35 1:62 0:54 5:56 1:37 1:78 0:43 3:04 0:33 1:63 0:25 4:82 0:08 1:63 0:25 2:32 0:22
% of protection
3.81a
1.86
7.49c
4.93b
2.52
2.50c
Chromosome aberrations (CA) include gap, break, centric fusion, translocation, fragment, pulverisation, ring, terminal association, polyploidy, aneuploidy, stickiness, C-mitotic effect, precocious centromeric separation, constriction, etc. No. of individuals examined in each series/fixation intervals ¼ 6; cells scored per individual ¼ 500 for CA, 20 000 for MNE, AN etc. 6000 for SHA. a p < 0:05. b p < 0:01. c p < 0:001.
B. Guha, A.R. Khuda-Bukhsh / Chemosphere 47 (2002) 49–56
Fixation time intervals (h)
B. Guha, A.R. Khuda-Bukhsh / Chemosphere 47 (2002) 49–56
fishes and the VC-injected fishes were relatively low as compared to EMS-treated fish, particularly at early intervals, and were slightly increased at longer intervals. The aberration frequencies in the EMS-injected fishes at different intervals were generally strikingly higher than in the DW-injected controls or in the VC-injected fish (p < 0:05 and p < 0:01), particularly up to 48 h, after which the aberration frequencies gradually declined. However, the injection of ascorbic acid in distilled water medium to EMS-treated fish considerably reduced the aberration frequencies as compared to only EMS-treated fish at all the fixation intervals (see Table 1), of which the differences at 24, 48 and 72 h were statistically significant (p < 0:05 and p < 0:01).
3.2. Micronucleus study Micronucleated erythrocytes (MN) (PM 7, 8) and erythrocytes with abnormal nuclei (AN) (PM 9–12) were observed in much greater number in fish treated with EMS. However, a few were also encountered in the DWand VC-treated fish. The frequencies of MN and AN observed in various treated and control fishes have been summarized in Table 1. A critical analysis of the data would also reveal that EMS-treated fish counter-injected with VC showed fewer micronuclei and anomalous nuclei as compared to only EMS-injected fish at the four fixation intervals examined (see Table 1), of which the differences were statistically significant at 24 and 48 h (p < 0:05 and p < 0:01). The Cyt/Nu ratio appeared to be generally higher in the EMS- and VC-treated fish as compared to DW-treated controls. The differences in the ratio were, however, statistically significant only at 72 h (p < 0:05). But when compared between fishes treated with only EMS and EMS þ VC, the Cyt/Nu ratios were found to be greater in the EMS plus VC-treated fish except at 48 h. Thus, the ratios did not necessarily denote toxicity status of the cells and can not really be useful as a toxicity parameter. However, the ratio might have some relationship with differential hydration status of the cells treated with different agents. Further, when a correlation between the ratio and the occurrence of MN or AN was considered, the Cyt/Nu ratio appeared to increase slightly in the fish showing greater number of micronuclei at some fixation intervals like 24, 48, and 72 h, but no such apparent relationship could be substantiated with the frequency of AN and Cyt/Nu.
3.3. Sperm head abnormality study Sperm with normal (PM 13) and abnormal (PM 14– 16) head shapes were encountered in both control and treated fish. The frequencies of sperm with abnormal
53
head shapes have been summarized in Table 1. The data reveal that the frequency of sperm with abnormal head was less in EMS þ VC-treated fish as compared to only EMS-treated fish at all the fixation intervals (see Table 1), of which the differences observed at 6, 24, 48 and 96 h were statistically significant at various levels (p < 0:05, 0.01 and 0.001).
3.4. Qualitative analysis The number and summarized data of gel–electrophoretic band profiles in different tissues of fish injected with distilled water, ascorbic acid, and EMS separately and EMS þ ascorbic acid conjointly have been presented in Table 2 and photographs of a few representative electropherograms provided (PM 17–20). From the data it would be revealed that in all the tissues the number of bands was decreased in the EMS-treated fish as compared to DW-treated controls except for liver and spleen tissues in which the number of bands was greater in EMS-treated fish. In general, the less number of bands would signify the loss of certain species of proteins while the gain in number of bands would speak for the synthesis of new species of protein as a result of specific treatment. However, sometimes the number of bands remaining same, the type of proteins may be different if the individual band in electropherograms of different treatment or control series does not have the same molecular weight or the nature and intensity of staining of bands differ between the bands in question. Therefore, a further critical study was made by assigning the band characteristics of the different treatment and control series (Table 2). In the ascorbic acid-treated fish the number of bands was greater than in the DW-treated fish for some tissues (i.e. eye, gill, liver and spleen) whereas it was less in number in the other tissues (i.e., dorsal muscle, heart, brain and kidney). This would mean that ascorbic acid might have acted differentially on different organs, either owing to their more favorable absorption potential and/or occurrence of greater number of working domains (for scavenging/anti-oxidant activity) in certain tissues, or else because of their differential metabolic degradation/denaturation or loss of function in certain tissues. The number of bands in the EMS þ VC-treated fish was generally greater than in the only EMS-treated fish, which would also imply that VC possibly restored damaged protein by the synthesis of the lost ones. Further, in order to have a quick view on the extent of change in band profiles in the different tissues of treated and control fishes, a similarity index has been calculated on the basis of the molecular weight and provided in Table 3. This would also give an idea as to the extent to which individual tissue showed modulations in their protein profiles as a result of EMS treatment followed by VC treatment.
54
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Table 2
Showing number of bands, range of relative mobility (Rm) and range of molecular weight of SDS-PAGE total protein profiles and amount of total protein (mg/gm) in different tissues of O. mossambicus treated with 0.2% EMS (T1 ), 0.02% ascorbic acid (T2 ) separately and 0.2% EMS þ 0:02% ascorbic acid conjointly (T3 ) and their respective double distilled water controls (C) at 48 h fixation interval Series
Tissues DM
VM
H
E
Br
G
Li
Sp
K
23 23 23 23
22 14 18 25
23 18 25 29
29 14 23 30
21 14 29 26
21 22 26 22
13 15 17 18
27 22 26 27
0.138–0.976 0.053–0.947 0.31–0.980 0.014–0.960
0.034–0.936 0.37–0.946 0.317–0.906 0.034–0.973
0.021–0.969 0.01–0.978 0.086–0.978 0.043–0.982
0.031–0.969 0.032–0.961 0.022–0.964 0.037–0.948
0.028–0.969 0.068–0.977 0.022–0.978 0.036–0.953
0.024–0.976 0.083–0.947 0.022–0.986 0.036–0.953
0.031–0.976 0.068–0.902 0.018–0.982 0.037–0.963
0.041–0.976 0.071–0.976 0.022–0.978 0.049–0.962
29.9–235.1 32.1–290.0 29.6–154.1 31.1–318.7
32.8–303.3 32.2–127.2 35.5–151.5 30.1–303.6
30.4–313.7 29.7–324.3 29.7–267.0 29.5–296.9
32.6–305.9 31.0–304.9 30.8–313.1 32.0–301.4
30.4–308.5 29.8–279.5 29.7–313.1 31.6–302.2
29.9–311.1 32.1–269.3 29.2–313.1 31.6–302.2
29.9–305.9 35.9–279.5 29.5–315.8 30.9–301.4
29.9–296.2 29.9–277.3 29.7–313.1 30.9–292.7
13.2 11.8 17.9 16.6
15.7 11.4 14.7 25.0
16.6 14.4 21.1 26.2
19.5 15.4 24.7 26.2
24.5 15.9 33.6 30.0
34.6 15.7 35.0 39.5
50.0 40.9 26.5 55.9
62.2 31.2 33.1 34.8
Number of bands C T1 T2 T3
26 20 25 29
Range of Rm C T1 T2 T3
0.038–0.974 0.226–0.947 0.054–0.946 0.022–0.968
Range of molecular weight C T1 T2 T3
30.0–300.7 32.1–189.5 32.2–288.8 30.5–313.1
Amount of protein C T1 T2 T3
14.8 13.0 18.3 20.2
DM: Dorsal muscle, VM: Ventral muscle, H: Heart, E: Eye, Br: Brain, G: Gill, Li: Liver, Sp: Spleen, K: Kidney.
Table 3 Showing similarity indices on molecular weights of SDS-PAGE total protein profiles in different tissues of O. mossambicus treated with 0.2% EMS (T1 ), 0.02% ascorbic acid (T2 ) separately and 0.2% EMS þ 0:02% ascorbic acid conjointly (T3 ) and their respective double distilled water controls (C) at 48 h fixation interval Series C vs T1 C vs T2 T1 vs T3
Tissues DM
VM
H
E
Br
G
Li
Sp
K
0.74 0.54 0.64
0.69 0.74 0.65
0.67 0.60 0.65
0.57 0.63 0.54
0.50 0.73 0.41
0.50 0.64 0.60
0.77 0.75 0.77
0.64 0.73 0.59
0.68 0.69 0.63
DM: Dorsal muscle, VM: Ventral muscle, H: Heart, E: Eye, Br: Brain, G: Gill, Li: Liver, Sp: Spleen, K: Kidney.
3.5. Quantitative analysis The quantitative data on total protein contents of different tissues in different control and treated series have been presented in Table 2. It would be revealed from the data that as compared to the DW-treated series the total protein contents in the EMS-treated series were less in all the tissues, whereas in the ascorbic acid-treated series the content was greater or similar in all tissues except spleen and kidney which showed lesser amount of total protein. On the other hand in the combined EMS þ VC-treated series the total protein contents were greater than in either distilled water or EMS- or ascorbic acid-treated series. In other words apparently the inhibitory effects of EMS on protein synthesis was not only removed by ascorbic acid treatment but the con-
joint treatment of EMS þ VC 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. A close scrutiny of the data presented in Table 2 would also reveal that the response in different tissues was slightly different, which would also corroborate the hypothesis that ascorbic acid may have a somewhat preferential action in certain tissues depending on the necessity and its ability to help synthesize certain proteins (e.g., collagen) damaged/lost due to EMS treatment. In the present study, EMS was found to induce various types of chromosome aberrations, nuclear anomalies including micronuclei formation, abnormal sperm head shapes, change in gel–electrophoretic band profiles and total protein contents of nine different tissues
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examined, which would amply corroborate the genotoxic and mutagenic potentials of EMS in O. mossambicus. Earlier Hooftman (1981) reported enhanced occurrence of chromosome aberrations in Notobranchius rochowi as a result of EMS and benzo[a]pyrene treatments. 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. 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 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 (Cohn, 1979). Therefore, the change of gel–electrophoretic band profiles and total protein contents observed in the EMS-treated fish was an additional indication of what might actually represent faulty/conformationally altered proteins, as a consequence of damage in DNA that emanated altered signals for protein synthesis. Alternatively, it is also possible that some of the altered protein might owe their origin to the direct/indirect action of reactive oxygen species or reactive nitrogen species produced due to cellular toxicity. Further, it would be revealed from the results of the present study that EMS produced genotoxic effects which appeared to increase up to 48 h after treatment but the effects were sustained through longer intervals to a considerable extent. Interestingly the injection of ascorbic acid (0.02%) appeared to modulate the genotoxic effects at all fixation intervals and ameliorated the EMS toxicity to a statistically significant extent at several of these fixation intervals, as indicated by both the cytogenetical and biochemical protocols used. The relatively short span of damaging activity of EMS might partly be due to the inherent ability of fish to excrete the toxic substance at the earliest and partly to its ability to detoxify the chemical by metabolic degradation. The exact mechanism by which VC modulates or minimizes the cytogenetical toxicity of EMS is not known. However, it is known that ascorbic acid is an anti-oxidant which might inhibit the oxidative metabolism of EMS and thus could prevent the production of mutagenic electrophilic metabolites (Goncharova and Kuzhir, 1989). Further, as ascorbic acid has marked nucleophilic properties it might intercept reactive electrophilic metabolites produced by EMS, thereby preventing their attack on nucleophilic sites on DNA and hence blocking adduct formation (Liehr et al., 1989). Also as part of a redox buffer system VC can scavenge harmful free radical metabolites/reactive oxygen species (Sato et al., 1990). Thus, the general protective effect of VC observed against EMS-induced genotoxicity by a twostep study in this fish could actually be accomplished
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through one or many of these inhibition mechanisms as suggested by others for some other models (De Flora and Ramel, 1988; Khan and Sinha, 1993, 1996; Hoda and Sinha, 1993). Alternatively, since VC has a positive role to play in collagen (a component of protein) formation, it might have acted as the external reductant that is required in the conversion of proline to hydroxyproline in the synthesis of collagen protein in certain tissues that might have been damaged by EMS treatment.
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