Mutation Research, 191 (1987) 157-161 Elsevier
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Persistence of micronuclei in the marine mussel, Mytilus after treatment with mitomycin C Franca Majone,
galloprovincialis,
Riccardo Brunetti, Isabella Gola and Angelo Gino Levis
Department of Biology, University of Padua, Via Loredan 10, 35131 Padua (Italy)
(Accepted 19 March 1987) Ke.vwords: Mytilus galloprovincialis; Micronuclei, persistence; Mitomycin C.
Summary The frequency of micronuclei induced by mitomycin C (MMC) in cells of the gill tissue of the marine mussel, M y t i l u s galloprovincialis Lmk., was determined over a long period (up to 40-52 days) following treatment. Two doses of MMC ( 0 . 5 × 1 0 -v and 10 - 7 M) were tested at 13°C and 23°C, temperatures representative of the winter and summer thermic conditions of the Mediterranean Sea. In all cases, the frequency of micronuclei was significantly increased by MMC and declined after treatment until it reached a plateau level, significantly higher than the control value. This persisted for a very long time. The frequency of micronuclei induced by a second treatment with MMC performed on the 28th day, did not differ significantly from that produced by the first treatment at the same dose. Temperature did not influence the pattern of the described phenomena to a significant extent. The reason for the persistence of an increased frequency of micronuclei is discussed, and a system is proposed for evaluating the genotoxicity of water pollutants present long before sampling.
Only a few studies on the induction of cytogenetic effects (SCE and chromosomal aberrations) of chemical mutagens on marine animals have been performed because of difficulties encountered in these systems, such as the low yield of metaphases (Kligerman, 1979; Dixon and Clarke, 1982; Van der Gaag, 1985). However, we have recently shown (Brunetti et al., 1986) that developing eggs of the mussel, M y t i l u s galloprovincialis, can easily be studied for SCE induction, and represent a useful system for laboratory investigations as well as for monitoring the environmental genotoxicity of marine pollutants. On the other Correspondence: Dr. Franca Majone, Department of Biology, University of Padua, Via Loredan 10, 35131 Padua (Italy).
hand, as the mussel is a filter-feeding animal, the main environmental impact should be at the level of the gill tissue. For this reason, we studied the induction of micronuclei in this tissue. It should be easier to observe micronuclei than other types of cytogenetic effect (chromosomal aberrations and SCE) because they are detected in interphase, even if cellular proliferation is required. This paper describes the induction of micronuclei by mitomycin C and the persistence of micronuclei after the end of treatment in the gill cells of M. galloprovincialis. In other papers we report the application of the present assay to screening the genotoxic effects of defined chemical pollutants, such as heavy metals and nitrilotriacetic acid, after experimental treatment (Gola
0165-7992/87/$ 03.50 (~) 1987 Elsevier Science Publishers B.V. (Biomedical Division)
158 et al., 1986), or of unknown water contaminants after environmental exposure (Brunetti et al., 1987 a,b,c).
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Material and m e t h o d s
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Adult specimens of the mussel, Mytilus galloprovincialis Lmk. (major axis about 5 cm), collected from only one station in the southern basin of the Lagoon of Venice, were acclimatised for a week in a laboratory in running sea water of salinity 34%o to 36%o and at a controlled temperature of 13 +_ I°C. The experimental population was sampled at regular intervals and the frequency of micronuclei in the gill tissue was determined. The entire population was divided into two samples which were treated for 48 h with mitomycin C (MMC) at concentrations of 10-7 M and 0.5 x 10 7 M, respectively. The two samples were tested at regular intervals until the frequencies of micronuclei, increased by MMC, had again dropped to stable levels. A second experiment with mussels collected from the same station was performed using the same modalities, but at a temperature of 23 _+ I°C. The temperatures of 13 and 23°C were chosen as being representative of the mean winter and summer thermic conditions of the Mediterranean Sea. To determine whether the response to the chemical mutagen was enhanced by previous treatment, 28 days after the first treatment with MMC, two samples of 10 mussels were exposed to the same doses of MMC at the two temperatures. To determine the frequency of micronuclei, the gills were removed, lacerated and filtered to obtain a cellular suspension; then the cells were fixed in ethanol:acetic acid (3:1) and centrifuged at 1000 r.p.m, for 5 min. The resuspended cells were spread on slides, air-dried and stained with 5% Giemsa. Each sample consisted of 8 mussels, and 1000 cells per mussel were scored to determine the frequency both of mitoses (mitotic index) and of micronuclei under an oil-immersion objective ( x 100). According to the suggestion of Tates et al. (1980), micronuclei (Fig. 1) were only scored when (i) their chromatin structure and colour intensity
e
f
Fig. 1. Mitoses and micronuclei in cells of the gill of untreated specimens of M. galloprovincialis: (a) prophase; (b) metaphase; (c), (d), (e), (f), micronuclei in interphase cells. In (f) two micronuclei of different diameter are present.
were similar to or weaker than those of the main nucleus, (ii) their borders were distinctly recognizable, suggesting the presence of a nuclear membrane, (iii) they were 'roundish', and (iv) they were included within the same cell cytoplasm. Because the variances of the frequencies of micronuclei were found to be homogeneous, data were statistically processed by means of the analysis of variance (anova) (Sokal and Rohlf, 1981). Results
Mitoses and micronuclei (Fig. 1) are easily detected in cells of the gill of M. galloprovincialis. The mitotic index in untreated mussels was 6.25_+ 1.06 (mean _+ S.E. as determined in cell preparations from 4 mussels). The baseline frequencies of micronuclei in the pooled controls of the two experimental populations did not differ significantly and ranged around the value of 2.2/1000 cells (Fig. 2). The effects of MMC on the frequency of micronuclei at the end of the treatments were not proportional to the doses used, but were statistically different (P<0.001). After treatment with MMC, the frequency of micronuclei declined as a function of time, until a plateau level about twice that of untreated mussels was reached (Fig. 2). As early as the 8th day after
159
some days later with the higher dose of MMC and the lower temperature, as indicated by the reduction in F values (not shown). A difference between the frequency of micronuclei in the series treated with different doses of MMC persisted even after 40-52 days. The temperature did not produce statistically significant differences in the pattern of the phenomena described, with the exception of the attainment of the plateau phase. Fig. 2 also reports the effect of a second treatment with MMC performed 28 days after the first treatment with the same doses: the frequencies of micronuclei induced by the 2nd treatment did not differ from those produced by the 1st treatment.
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Fig. 2. Induction of micronuclei by mitomycin C (MMC) in cells of the gill of M. galloprovincialis. Means (8 animals per sample) and 95% confidence limits (only half intervals are drawn) of micronuclei after treatment with 0.5 × 10 7 M ( • ) and 10 V M ( ° ) M M C , a t 2 3 o C ( A ) and 13°C (B). Samples were collected before ( ~ ) and at various times after the end of a 48 h MMC treatment ( ¥ ) . A second treatment with the same MMC doses ( ¥ ¥ ) was performed 28 days after the first one. At both temperatures, the frequencies of micronuclei in the series treated with the 2 different doses of MMC show a statistically significant difference (P<0.001 at 13°C and P < 0.05 at 23°C), until the plateau phase is reached. The mean values in pooled samples from the plateau phase differ significantly (P<0.001) from those obtained in pooled controis. The frequencies of micronuclei induced by the second treatment (~A),~ ) do not differ significantly from those scored immediately after the first treatment at the same MMC doses.
treatment, the frequencies of micronuclei in the subsequent samples did not differ statistically from each other, but the plateau phase was attained
MMC is a well known genotoxic agent, whose mutagenic and clastogenic effects have been reported both in mammalian systems (gatt, 1974; Perry and Evans, 1975; Perry, 1980; Kliesch et al., 1981) and in marine organisms (Dixon and Clarke, 1982; Harrison and Jones, 1982; Krishnaja and Rege, 1982; Das and Nanda, 1986). In the latter, the genotoxic effect of MMC was detected as increased SCE frequencies in larvae or gill tissues of the mussel, M. edulis (Harrison and Jones, 1982; Dixon and Clarke, 1982), as induction of chromosomal aberrations in different tissues of the fish, Boleophthalrnus dussumieri (Krishnaja and Rege, 1982), and as micronuclei in the erythrocytes of the fish, Heteropneustesfossilis (Das and Nanda, 1986). In the present study, we demonstrated that MMC is capable of increasing the frequency of micronuclei in gill cells of the mussel, Mytilus galloprovincialis. MMC also slows down the cell cycle in mammalian cells cultured in vitro (Perry and Evans, 1975), and the same effect was observed in the gill of M. galloprovincialis (Majone, unpublished observations). Micronuclei can only be detected in cells undergoing mitosis, and so low frequencies of micronuclei in treated cells may be due to the inhibition of mitotic activity rather than to a reduced level of genotoxic damage (Tates et al., 1983; Das and Nanda, 1986). In our conditions, in spite of a
160
reduced mitotic activity, the frequency of micronuclei significantly increased after M M C treatment, suggesting that a high number of damaged cells undergoing mitosis was able to produce micronuclei. Moreover, as the micronucleated cells can accumulate with time (Tares et al., 1983), the high frequency of micronuclei observed in our experiments at the end of MMC treatment was probably due to both the increased rate of micronuclei formation and the accumulation process. Up until the 8th day after the end of treatment with MMC, the frequency of micronuclei decayed as a consequence of mitotic activity of nonmicronucleated cells. After the 8th day, the frequency of micronuclei became stable around a value about twice the control level. This should indicate that in the treated gill the number of cells producing micronuclei was higher than the background number of micronuclei-producing cells present in the controls. The constant frequency of micronuclei 8 days after treatment represented the balance between the production of new micronucleated cells and the progressive elimination of micronuctei through the normal mitotic activity. High frequencies of micronuclei, even some time after treatment with chemical mutagens, were observed in cells of rat liver (Tates et al., 1980, 1983) - - a cell system with almost no mitotic activity - - where the frequency of micronuclei was measured after induction of D N A synthesis. A persistent 'preclastogenic' damage, rather than the stability of micronuclei formed just after treatment, was held responsible for the occurrence of micronucleated cells after considerable intervals of time (Tates et al., 1983). The low but significant number of mitoses also observed in mussel species other than M. galloprovincialis (Dixon and Clarke, 1982) might be due to cells that gradually replace the preexisting ones, as histological investigations seem to indicate (Sunila, 1986). The persistence of a high frequency of micronuclei for a long time after treatment might be due to such a gradual replacement. Anyway, the effects observed by us should be the result of damage produced by the treatments in
cells that undergo delayed mitosis. In conclusion, our data indicate the suitability of this system for detecting the induction of micronuclei in experimental conditions and for monitoring environmental genotoxicants (Gola et al., 1986; Brunetti et al., 1987a,b,c). Moreover, the pattern of production of micronuclei some considerable time after treatment suggests that the frequency of micronuclei in the gills of mussels from natural populations could be used as a parameter for evaluating the genotoxicity of water pollutants acting long before sampling. Since the micronuclei presently examined resulted from treatment with MMC, a clastogenic substance producing mainly chromosome breakage, it would be interesting to study the kinetics of induction of the micronuclei by other substances acting as spindle poisons and producing micronuclei without breaking the chromosomes.
Acknowledgements This study was supported by grants from the National Research Council of Italy (C.N.R., P.F. 'Oncologia'), the Venetian Region, and the Italian Ministry of Education (M.P.I.).
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161 adult Mytilus edulis, Mar. Biol. Lett., 3, 163-172. Gola, 1., R. Brunetti, F. Majone and A.G. Levis (1986) Applications of the micronucleus test to a marine organism treated with N T A and insoluble heavy metals, Atti Ass. Genet. It., 32, 95-96. Harrison, F.L., and I.M. Jones (1982) An in vivo sister chromatid exchange assay in the larvae of the mussel Mytilus edulis: response to 3 mutagens, Mutation Res., 105,235-242. Kliesch, D., N. Danford and I.D. Adler (1981) Micronucleus test and bone marrow chromosome analysis: A comparison of two methods in vivo for evaluating chemically induced chromosomal alterations, Mutation Res., 80, 321-332. Kligerman, A.D. (1979) Induction of sister chromatid exchanges in the central m u d m i n n o w following in vivo exposure to mutagenic agents, Mutation Res., 64, 205-217. Krishnaja, A . P . , and M.S. Rege (1982) Induction of chromosomal aberrations in fish Boleophthalmus dussumieri after exposure in vivo to mitomycin C and heavy metals mercury, selenium and chromium, Mutation Res., 102, 71-82. Latt, S.A. (1974) Sister chromatid exchanges, indices of h u m a n chromosome damage and repair: detection by fluorescence and induction by mitomycin C, Proc. Natl. Acad. Sci. (U.S.A.), 71, 3162-3166. Perry, P. (1980) Chemical mutagens and sister chromatid exchange, in: F.J. de Serres and A. Hollaender (Eds.),
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