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Sister-chromatid exchange in developing eggs of Mytilus galloprovincialis Lmk. (bivalvia)
Mutation Research, 174 (1986) 207-211
207
Elsevier MRLett. 0872
Sister-chromatid exchange in developing eggs of Lmk. (Bivalvia)
Mytilus galloprovincial&
R. Brunetti, I. Gola and F. Majone Dipartimento di Biologia, Universitb di Padova, Via Loredan 10, 35100 Padova (Italy)
(Accepted 24 February 1986)
Summary A simple and rapid experimental technique for detecting SCE in developing eggs of M y t i l u s galloprovincialis Lmk. (Bivalvia) was set up. Eggs, fertilized in the laboratory, were treated with nitrilotriacetic acid (NTA), HgCI2 and HgCI2 plus NTA (1 : 1). Results confirm the high genotoxicity of mercury, indicating an absence of activity by NTA and a lack of interaction between NTA and the metal. Eggs from mussels collected in two stations characterized by different levels of sea-waste and hydrocarbon pollution presented a significant difference in their SCE frequencies. This indicates that a genetic effect can also be produced in gametes. This system may be used for laboratory studies as well as for monitoring the environmental genotoxicity of marine pollutants.
Sister-chromatid exchange (SCE), that is the reciprocal interchange of DNA between sister chromatids, is easily visualized in metaphase chromosomes (Korenberg and Freedlender, 1974) and has been applied to study chromosome damage both in vitro and in vivo (Latt et al., 1981). Since SCE can be induced by subtoxic doses of carcinogens and mutagens (Gebhart, 1981), its analysis offers the possibility of a rapid, sensitive and quantitative assay of genetic damage. In particular the study of SCE in vivo has become increasingly relevant for the screening of environmental mutagens and carcinogens. Both non-mammalian and mammalian in vivo systems are available for SCE studies and a variety of somatic and germ tissues have been analyzed for
SCE induction by chemical mutagens (Latt et al., 1981). Even though it is known that genetic toxicants are present in polluted marine environments and may represent a real threat to marine organisms, only a few cytogenetic studies have been performed on marine systems because of certain difficulties encountered, namely the low yield of second replication metaphases (Kligerman, 1979; Alink et al., 1980; Dixon and Clarke, 1982; Van der Gaag and Van de Kerkhoff, 1985; Van de Kerkhoff and Van der Gaag, 1985). Therefore, we decided to undertake a study of SCE in developing eggs of M y t i l u s galloprovincialis Lmk. on the assumption that such material presents the advantage of a high yield of cells with a short cell cycle.
0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (BiomedicalDivision)
208
Materials and methods
Specimens of Mytilus galloprovincialis Link. were collected from areas of the Chioggia basin (Venice lagoon). M. galloprovincialis is a dioecious bivalve widely spread in the Mediterranean sea and it is intensively bred for commercial purposes. Its karyotype consists of 28 chromosomes (ThiriotQuievreux and Ayraud, 1982). Reproduction takes place from October to May (Da Ros et al., 1985) in the lagoon, the temperature of the sea water being less than 15°C. In the laboratory, spawning was induced by transferring mussels into seawater preheated to 24°C. Eggs from several (5-6) females were pooled together and fertilized with sperm from a single male. When the first cleavage appeared (after about 1 h), fertilized eggs were harvested with a plankton net and distributed into glass beakers with 300 ml of filtered seawater (0.45 # pore size). Then bromodeoxyuridine (BUdR, Sigma) and HgC12, nitrilotriacetic acid (NTA, B.H. Schilling) and HgC12 plus NTA (molar ratio: l / l ) , were directly added to the cultures at the concentrations
m
Fig. 1. Metaphase from developing eggs of M. galloprovincialis with sister-chromatid differentiation ( x 1200).
indicated in Results. 2 h later, colchicine (Sigma; final concentration: 0.05 mg/ml) was added. 4 h after fertilization, egss were harvested and exposed to seawater diluted with 0.6°70 KC1 (10 min for each of 3 seawater:KC1 solutions in the ratios 2: l, 1:1, 1:2). During the last 5 min, eggs in the 3rd solution were also exposed to trypsin (Difco; final concentration: 2.5 mg/ml). Then eggs were fixed in ethanol: acetic acid (3 : 1). SCE were detected on chromosome preparations (Fig. 1) obtained from second-generation metaphase cells by staining with Giemsa (Korenberg and Freedlender, 1974), following the already described procedures (Majone and Levis, 1979). Since our data did not satisfy the assumptions for an analysis of variance, statistical comparisons were done using the nonparametric techniques of Wilcoxon (2 samples) and Kruskal-Wallis (more than 2 samples) (Sokal and Rohlf, 1981). Results
In Table l, the mean number of SCE/metaphase in eggs treated with NTA (5 mg/1), HgC12 (0.03 mg/1) and HgCI2 (0.03 mg/l) plus NTA (0.027 mg/l; molar ratio: 1/1) are given. Treatments were performed in the presence of BUdR (10-3 M or 2×10 -3 M). It can be observed that the mean number of SCE/metaphase is significantly higher with respect to the control, after treatment with HgCl2. On the contrary, no difference can be noticed between NTA treatment and control, or between treatment with HgC12 and HgCI2 plus NTA. To verify the effect of the BUdR concentration we compared controls and treatments for different BUdR doses. No statistically significant difference was noticed, and this suggests the absence of any interaction between BUdR and the other chemicals, at least for the presently tested BUdR concentrations. In Table 2 we compare the mean numbers of SCE/metaphase in developing eggs from animals collected in two stations which were quite near each other - - P and NP -- characterized by very different degrees of eutrophication (Brunetti et al.,
209
TABLE 1 INDUCTION OF SCE BY NTA AND HgCI2 IN DEVELOPING EGGS OF Mytilus galloprovincialis Lmk., AFTER EXPOSURE TO DIFFERENT DOSES OF BUdR Treatment
NTA (5 mg/l) HgC12 (0.03 mg/1) HgCI2 (0.03 mg/l) + NTA (ratio 1 : 1) NTA (5 mg/ml) HgCl2 (0.03 mg/l) HgC12 (0.03 mg/l) + NTA (ratio 1 : 1)
BUdR (M)
Number Mean Variance a of meta- number of phases SCE/ metaphase
Discussion
10- 3
20
2.40
0.88
10- 3
20
4.65
1.61 -~
10- 3
20
4.05
0.47
3 3
30 30
2.23 2.70
0.81 / 1.11 ) ns
2× 10 -3
30
4.93
2× l0 -3
30
4.77
1.17 ~) _~ n 2.19
2×10 2×10
ns
)
a n s , not significantly different (P_>0.05); s, significantly different (P<0.001). The statistical comparison between data obtained with the two doses of BUdR point out that the differences are not significant (P_>0.05) for all series of data (controis; NTA; HgCI2; HgCI2 plus NTA).
1983) and hydrocarbon pollution (Fossato and Dolci, 1977). In 1975, the latter authors detected very different amounts of aliphatic hydrocarbons in soft parts of mussels from station P (No. 24 in their paper) and from station NP (No. 27), with mean annual values of 14.5 and 0.6 mg/100 g of
TABLE 2 SCE DETECTED IN DEVELOPING EGGS OF MUSSELS FROM TWO DIFFERENT STATIONS Station
Number of metaphases
Mean number of SCE/ metaphase
Variance
NP P
40 44
2.15 4.74
l.ll ) 3.12 ~
s, significantly different (P<0.001).
wet flesh, respectively. As can be seen in Table 2, the mean number of SCE/metaphase in eggs from mussels collected in station P is more than double that from mussels collected in station NP.
s
Treatment with HgC12 caused a doubling of the mean number of SCE/cell in developing eggs of Mytilus galloprovincialis Lmk. (Bivalvia) just as observed, at the same doses, in mammalian cells cultured in vitro (Montaldi et al., 1985), thus confirming the high genotoxicity of this metal (L6onard et al., 1983). On the contrary, treatments with NTA and with NTA plus HgCI2 indicated an absence of genotoxicity of NTA in the SCE assay on Mytilus eggs and a lack of interaction between NTA and the metal, as also observed in other systems (Montaldi et al., 1985; Ved Brat and Williams, 1984). The mean number of SCE/cell in eggs of mussels from the polluted area was significantly higher than the value found in the unpolluted area (Table 2). This indicates that a genetic effect is also produced on gametes, and furthermore suggests a possible use of the SCE technique on this biological system not only for laboratory studies but also for monitoring the environmental genotoxicity of marine pollutants. It should be noticed that the mean number of SCE/cell in eggs from the two stations is lower than that reported for larval or adult stages of marine Polychaeta (Pesch and Pesch, 1980) and Mytilus edulis (Dixon and Clarke, 1982; Harrison and Jones, 1982); in particular, it is lower than that found in the gills of adult mussels from the same stations (Majone et al., in preparation). We are presently analyzing SCE in adult mussel tissues and it seems as though it might be possible to examine these genetic effects simultaneously in embryonic and in parental cells. Until now, the study on the induction of SCE by chemical mutagens in marine organisms was hampered by the lack of a suitable test organism. A variety of animals were tested, namely larval a n d / o r adult stages of the mussel M. edulis (Dixon
210
and Clarke, 1982; Harrison and Jones, 1982), polychaete Neanthes arenaceodentata (Pesch and Pesch, 1980) and fishes (Kligerman, 1979; Van der Gaag and Van de Kerkhoff, 1985; Van de Kerkhoff and Van der Gaag, 1985). Although in vivo SCE assays with these organisms produced promising results, many problems concerning the reliability of this technique were pointed out. The main difficulties were: (i) the unreliability of the SCE staining procedure (Van der Gaag and Van de Kerkhoff, 1985; Van de Kerkhoff and Van der Gaag, 1985); (ii) the time required for the BUdR exposure (Dixon and Clarke, 1982); (iii) the low yield of second-division cells, resulting in a long time being required for scoring (Dixon and Clarke, 1982; Van der Gaag and Van de Kerkhoff, 1985; Van de Kerkhoff and Van der Gaag, 1985); (iv) the difficulty in obtaining complete metaphases for SCE detection (Harrison and Jones, 1982). In the present work, some of these difficulties have been overcome. Namely, the time required by the experimental procedure and particularly by the BUdR incorporation has been radically shortened (to 2-3 h) as compared with the time (few days) required with the adult mussel or with other systems (Kligerman, 1979; Alink et al., 1980; Pesch and Pesch, 1980). The shortness of the adopted experimental procedure makes the analysis of thirdgeneration metaphases also possible. These are better suited than the second-generation metaphases for investigations of the SCE formation mechanism (Schwartzman et al., 1979). In conclusion, this system closely resembles in vivo the environmental conditions, it allows a simple experimental procedure, and finally it offers the advantage of making possible the study of cytogenetic responses from marine organisms in their first developmental stages.
Acknowledgements We are grateful to Prof. A.G. Levis for his critical reading of the manuscript. This work was supported by grants from the National Research Council of Italy (C.N.R., P.F. 'Medicina Preventiva e Riabilitativa'), the Venetia Region ('Centro
di Alta Specializzazione in Cancerogenesi Ambientale'), and the Italian Ministry of Education (M.P.I.). References Alink, G.M., E.M.H. Frederix-Wolters, M.A. van der Gaag, J.F.J. van de Kerkhoff and C.L.M. Poels (1980) Induction of sister-chromatid exchanges in fish exposed to Rhine water, Mutation Res., 78, 369-374. Brunetti, R., M.G. Marin, L. Beghi and M. Bressan (1983) Study of the pollution in the Venetian lagoon's lower basin during the period 1974-1981, Riv. Idrobiol., 22(1), 1-58. Da Ros, L., M. Bressan and M.G. Marin (1985) Reproductive cycle of the mussel (Mytilus galloprovincialis Lmk.) in the Venice lagoon, Boll. Zool., 52, 223-229. Dixon, D.R., and K.R. Clarke (1982) Sister chromatid exchange: a sensitive method for detecting damage caused by exposure to environmental mutagens in the chromosome of adult Mytilus edulis, Mar. Biol. Lett., 3, 163-172. Fossato, V.U., and F. Dolci (1977) lnquinamento da idrocarburi nel bacino centrale e meridionale della Laguna Veneta, Arch. Oceanogr. Limnol., 19, 47-54. Gebhart, E. (1981) Sister chromatid exchange (SCE) and structural chromosome aberration in mutagenicity testing, Hum. Genet., 58, 235-254. Harrison, F.L., and I.M. Jones (1982) An in vivo sisterchromatid exchange assay in the larvae of the mussel Mytilus edulis: response to 3 mutagens, Mutation Res., 105,235-242. Kligerman, A.D. (1979) Induction of sister chromatid exchanges in the central mudminnow following in vivo exposure to mutagenic agents, Mutation Res., 64, 205-217. Korenberg, J.R., and B.H. Freedlender (1974) Giemsa technique for the detection of sister chromatid exchanges, Chromosoma, 48, 355-360. Latt, S.A., J. Allen, S.E. Bloom, A. Carrano, E. Falke, E. Schneider, R. Schreck, R. Tice, B. Whitfield and S. Wolff (1981) Sister-chromatid exchanges: a report of the Gene-Tox Program, Mutation Res., 87, 17-62. L6onard; A., P. Jacquet and R.R. Lauwerys (1983) Mutagenicity and teratogenicity of mercury compounds, Mutation Res., 114, 1-18. Majone, F., and A.G. Levis (1979) Chromosome aberrations and sister chromatid exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds, Mutation Res., 67, 231-238. Montaldi, A., L. Zentilin, P. Venier, 1. Gola, V. Bianchi, S. Paglialunga and A.G. Levis (1985) Interaction of nitrilotriacetic acid with heavy metals in the induction of sister chromatid exchanges in cultured mammalian cells, Environ. Mutagen., 7, 381-390. Perry, R., P.W.W. Kirk, T. Stephenson and J.N. Lester (1984) Environmental aspects of the use of NTA as a detergent builder, Water Res., 18, 225-276.
211 Pesch, G.G., and C.E. Pesh (1980) Neanthes arenaceodentata (Polychaeta: Anellida), a proposed cytogenetic model for marine genetic toxicology, Can. J. Fish. Aquat. Sci., 37, 1225-1228. Schwartzman, J.B., F. Cortes, A. Gonzales-Fernandez, C. Gutierrez and J.F. Lopez-Saez (1979) On the nature of sister chromatid exchanges in 5-bromodeoxyuridine-substituted chromosomes, Genetics, 92, 1251-1264. Sokal, R.R., and F.J. Rohlf (1981) Biometry, 2nd edn., Freeman, San Francisco. Thiriot-Quievreux, C., and N. Ayraud (1982) Les caryotypes de quelques esp~ces de bivalves et de gasteropodes marins, Mar. Biol., 70, 165-172.
Van de Kerkhoff, J.F.J., and M.A. van der Gaag (1985) Some factors affecting optimal differential staining of sister chromatids in vivo in the fish Nothobranchius rachowi, Mutation Res., 143, 39-43. Van der Gaag, M.A., and F.J.F. van de Kerkhoff (1985) The development of an in vivo SCE-assay in the fish Nothobranchius rachowi, 4th Int. Conf. Environ. Mut., Stockholm, June 1985, Abstract p. 40. Ved Brat, S., and G.N. Williams (1984) Nitrilotriacetic acid does not induce sister-chromatid exchanges in hamster or human cells, Fd. Chem. Toxicol., 3, 211-215. Communicated by A. Abbondandolo
207
Elsevier MRLett. 0872
Sister-chromatid exchange in developing eggs of Lmk. (Bivalvia)
Mytilus galloprovincial&
R. Brunetti, I. Gola and F. Majone Dipartimento di Biologia, Universitb di Padova, Via Loredan 10, 35100 Padova (Italy)
(Accepted 24 February 1986)
Summary A simple and rapid experimental technique for detecting SCE in developing eggs of M y t i l u s galloprovincialis Lmk. (Bivalvia) was set up. Eggs, fertilized in the laboratory, were treated with nitrilotriacetic acid (NTA), HgCI2 and HgCI2 plus NTA (1 : 1). Results confirm the high genotoxicity of mercury, indicating an absence of activity by NTA and a lack of interaction between NTA and the metal. Eggs from mussels collected in two stations characterized by different levels of sea-waste and hydrocarbon pollution presented a significant difference in their SCE frequencies. This indicates that a genetic effect can also be produced in gametes. This system may be used for laboratory studies as well as for monitoring the environmental genotoxicity of marine pollutants.
Sister-chromatid exchange (SCE), that is the reciprocal interchange of DNA between sister chromatids, is easily visualized in metaphase chromosomes (Korenberg and Freedlender, 1974) and has been applied to study chromosome damage both in vitro and in vivo (Latt et al., 1981). Since SCE can be induced by subtoxic doses of carcinogens and mutagens (Gebhart, 1981), its analysis offers the possibility of a rapid, sensitive and quantitative assay of genetic damage. In particular the study of SCE in vivo has become increasingly relevant for the screening of environmental mutagens and carcinogens. Both non-mammalian and mammalian in vivo systems are available for SCE studies and a variety of somatic and germ tissues have been analyzed for
SCE induction by chemical mutagens (Latt et al., 1981). Even though it is known that genetic toxicants are present in polluted marine environments and may represent a real threat to marine organisms, only a few cytogenetic studies have been performed on marine systems because of certain difficulties encountered, namely the low yield of second replication metaphases (Kligerman, 1979; Alink et al., 1980; Dixon and Clarke, 1982; Van der Gaag and Van de Kerkhoff, 1985; Van de Kerkhoff and Van der Gaag, 1985). Therefore, we decided to undertake a study of SCE in developing eggs of M y t i l u s galloprovincialis Lmk. on the assumption that such material presents the advantage of a high yield of cells with a short cell cycle.
0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (BiomedicalDivision)
208
Materials and methods
Specimens of Mytilus galloprovincialis Link. were collected from areas of the Chioggia basin (Venice lagoon). M. galloprovincialis is a dioecious bivalve widely spread in the Mediterranean sea and it is intensively bred for commercial purposes. Its karyotype consists of 28 chromosomes (ThiriotQuievreux and Ayraud, 1982). Reproduction takes place from October to May (Da Ros et al., 1985) in the lagoon, the temperature of the sea water being less than 15°C. In the laboratory, spawning was induced by transferring mussels into seawater preheated to 24°C. Eggs from several (5-6) females were pooled together and fertilized with sperm from a single male. When the first cleavage appeared (after about 1 h), fertilized eggs were harvested with a plankton net and distributed into glass beakers with 300 ml of filtered seawater (0.45 # pore size). Then bromodeoxyuridine (BUdR, Sigma) and HgC12, nitrilotriacetic acid (NTA, B.H. Schilling) and HgC12 plus NTA (molar ratio: l / l ) , were directly added to the cultures at the concentrations
m
Fig. 1. Metaphase from developing eggs of M. galloprovincialis with sister-chromatid differentiation ( x 1200).
indicated in Results. 2 h later, colchicine (Sigma; final concentration: 0.05 mg/ml) was added. 4 h after fertilization, egss were harvested and exposed to seawater diluted with 0.6°70 KC1 (10 min for each of 3 seawater:KC1 solutions in the ratios 2: l, 1:1, 1:2). During the last 5 min, eggs in the 3rd solution were also exposed to trypsin (Difco; final concentration: 2.5 mg/ml). Then eggs were fixed in ethanol: acetic acid (3 : 1). SCE were detected on chromosome preparations (Fig. 1) obtained from second-generation metaphase cells by staining with Giemsa (Korenberg and Freedlender, 1974), following the already described procedures (Majone and Levis, 1979). Since our data did not satisfy the assumptions for an analysis of variance, statistical comparisons were done using the nonparametric techniques of Wilcoxon (2 samples) and Kruskal-Wallis (more than 2 samples) (Sokal and Rohlf, 1981). Results
In Table l, the mean number of SCE/metaphase in eggs treated with NTA (5 mg/1), HgC12 (0.03 mg/1) and HgCI2 (0.03 mg/l) plus NTA (0.027 mg/l; molar ratio: 1/1) are given. Treatments were performed in the presence of BUdR (10-3 M or 2×10 -3 M). It can be observed that the mean number of SCE/metaphase is significantly higher with respect to the control, after treatment with HgCl2. On the contrary, no difference can be noticed between NTA treatment and control, or between treatment with HgC12 and HgCI2 plus NTA. To verify the effect of the BUdR concentration we compared controls and treatments for different BUdR doses. No statistically significant difference was noticed, and this suggests the absence of any interaction between BUdR and the other chemicals, at least for the presently tested BUdR concentrations. In Table 2 we compare the mean numbers of SCE/metaphase in developing eggs from animals collected in two stations which were quite near each other - - P and NP -- characterized by very different degrees of eutrophication (Brunetti et al.,
209
TABLE 1 INDUCTION OF SCE BY NTA AND HgCI2 IN DEVELOPING EGGS OF Mytilus galloprovincialis Lmk., AFTER EXPOSURE TO DIFFERENT DOSES OF BUdR Treatment
NTA (5 mg/l) HgC12 (0.03 mg/1) HgCI2 (0.03 mg/l) + NTA (ratio 1 : 1) NTA (5 mg/ml) HgCl2 (0.03 mg/l) HgC12 (0.03 mg/l) + NTA (ratio 1 : 1)
BUdR (M)
Number Mean Variance a of meta- number of phases SCE/ metaphase
Discussion
10- 3
20
2.40
0.88
10- 3
20
4.65
1.61 -~
10- 3
20
4.05
0.47
3 3
30 30
2.23 2.70
0.81 / 1.11 ) ns
2× 10 -3
30
4.93
2× l0 -3
30
4.77
1.17 ~) _~ n 2.19
2×10 2×10
ns
)
a n s , not significantly different (P_>0.05); s, significantly different (P<0.001). The statistical comparison between data obtained with the two doses of BUdR point out that the differences are not significant (P_>0.05) for all series of data (controis; NTA; HgCI2; HgCI2 plus NTA).
1983) and hydrocarbon pollution (Fossato and Dolci, 1977). In 1975, the latter authors detected very different amounts of aliphatic hydrocarbons in soft parts of mussels from station P (No. 24 in their paper) and from station NP (No. 27), with mean annual values of 14.5 and 0.6 mg/100 g of
TABLE 2 SCE DETECTED IN DEVELOPING EGGS OF MUSSELS FROM TWO DIFFERENT STATIONS Station
Number of metaphases
Mean number of SCE/ metaphase
Variance
NP P
40 44
2.15 4.74
l.ll ) 3.12 ~
s, significantly different (P<0.001).
wet flesh, respectively. As can be seen in Table 2, the mean number of SCE/metaphase in eggs from mussels collected in station P is more than double that from mussels collected in station NP.
s
Treatment with HgC12 caused a doubling of the mean number of SCE/cell in developing eggs of Mytilus galloprovincialis Lmk. (Bivalvia) just as observed, at the same doses, in mammalian cells cultured in vitro (Montaldi et al., 1985), thus confirming the high genotoxicity of this metal (L6onard et al., 1983). On the contrary, treatments with NTA and with NTA plus HgCI2 indicated an absence of genotoxicity of NTA in the SCE assay on Mytilus eggs and a lack of interaction between NTA and the metal, as also observed in other systems (Montaldi et al., 1985; Ved Brat and Williams, 1984). The mean number of SCE/cell in eggs of mussels from the polluted area was significantly higher than the value found in the unpolluted area (Table 2). This indicates that a genetic effect is also produced on gametes, and furthermore suggests a possible use of the SCE technique on this biological system not only for laboratory studies but also for monitoring the environmental genotoxicity of marine pollutants. It should be noticed that the mean number of SCE/cell in eggs from the two stations is lower than that reported for larval or adult stages of marine Polychaeta (Pesch and Pesch, 1980) and Mytilus edulis (Dixon and Clarke, 1982; Harrison and Jones, 1982); in particular, it is lower than that found in the gills of adult mussels from the same stations (Majone et al., in preparation). We are presently analyzing SCE in adult mussel tissues and it seems as though it might be possible to examine these genetic effects simultaneously in embryonic and in parental cells. Until now, the study on the induction of SCE by chemical mutagens in marine organisms was hampered by the lack of a suitable test organism. A variety of animals were tested, namely larval a n d / o r adult stages of the mussel M. edulis (Dixon
210
and Clarke, 1982; Harrison and Jones, 1982), polychaete Neanthes arenaceodentata (Pesch and Pesch, 1980) and fishes (Kligerman, 1979; Van der Gaag and Van de Kerkhoff, 1985; Van de Kerkhoff and Van der Gaag, 1985). Although in vivo SCE assays with these organisms produced promising results, many problems concerning the reliability of this technique were pointed out. The main difficulties were: (i) the unreliability of the SCE staining procedure (Van der Gaag and Van de Kerkhoff, 1985; Van de Kerkhoff and Van der Gaag, 1985); (ii) the time required for the BUdR exposure (Dixon and Clarke, 1982); (iii) the low yield of second-division cells, resulting in a long time being required for scoring (Dixon and Clarke, 1982; Van der Gaag and Van de Kerkhoff, 1985; Van de Kerkhoff and Van der Gaag, 1985); (iv) the difficulty in obtaining complete metaphases for SCE detection (Harrison and Jones, 1982). In the present work, some of these difficulties have been overcome. Namely, the time required by the experimental procedure and particularly by the BUdR incorporation has been radically shortened (to 2-3 h) as compared with the time (few days) required with the adult mussel or with other systems (Kligerman, 1979; Alink et al., 1980; Pesch and Pesch, 1980). The shortness of the adopted experimental procedure makes the analysis of thirdgeneration metaphases also possible. These are better suited than the second-generation metaphases for investigations of the SCE formation mechanism (Schwartzman et al., 1979). In conclusion, this system closely resembles in vivo the environmental conditions, it allows a simple experimental procedure, and finally it offers the advantage of making possible the study of cytogenetic responses from marine organisms in their first developmental stages.
Acknowledgements We are grateful to Prof. A.G. Levis for his critical reading of the manuscript. This work was supported by grants from the National Research Council of Italy (C.N.R., P.F. 'Medicina Preventiva e Riabilitativa'), the Venetia Region ('Centro
di Alta Specializzazione in Cancerogenesi Ambientale'), and the Italian Ministry of Education (M.P.I.). References Alink, G.M., E.M.H. Frederix-Wolters, M.A. van der Gaag, J.F.J. van de Kerkhoff and C.L.M. Poels (1980) Induction of sister-chromatid exchanges in fish exposed to Rhine water, Mutation Res., 78, 369-374. Brunetti, R., M.G. Marin, L. Beghi and M. Bressan (1983) Study of the pollution in the Venetian lagoon's lower basin during the period 1974-1981, Riv. Idrobiol., 22(1), 1-58. Da Ros, L., M. Bressan and M.G. Marin (1985) Reproductive cycle of the mussel (Mytilus galloprovincialis Lmk.) in the Venice lagoon, Boll. Zool., 52, 223-229. Dixon, D.R., and K.R. Clarke (1982) Sister chromatid exchange: a sensitive method for detecting damage caused by exposure to environmental mutagens in the chromosome of adult Mytilus edulis, Mar. Biol. Lett., 3, 163-172. Fossato, V.U., and F. Dolci (1977) lnquinamento da idrocarburi nel bacino centrale e meridionale della Laguna Veneta, Arch. Oceanogr. Limnol., 19, 47-54. Gebhart, E. (1981) Sister chromatid exchange (SCE) and structural chromosome aberration in mutagenicity testing, Hum. Genet., 58, 235-254. Harrison, F.L., and I.M. Jones (1982) An in vivo sisterchromatid exchange assay in the larvae of the mussel Mytilus edulis: response to 3 mutagens, Mutation Res., 105,235-242. Kligerman, A.D. (1979) Induction of sister chromatid exchanges in the central mudminnow following in vivo exposure to mutagenic agents, Mutation Res., 64, 205-217. Korenberg, J.R., and B.H. Freedlender (1974) Giemsa technique for the detection of sister chromatid exchanges, Chromosoma, 48, 355-360. Latt, S.A., J. Allen, S.E. Bloom, A. Carrano, E. Falke, E. Schneider, R. Schreck, R. Tice, B. Whitfield and S. Wolff (1981) Sister-chromatid exchanges: a report of the Gene-Tox Program, Mutation Res., 87, 17-62. L6onard; A., P. Jacquet and R.R. Lauwerys (1983) Mutagenicity and teratogenicity of mercury compounds, Mutation Res., 114, 1-18. Majone, F., and A.G. Levis (1979) Chromosome aberrations and sister chromatid exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds, Mutation Res., 67, 231-238. Montaldi, A., L. Zentilin, P. Venier, 1. Gola, V. Bianchi, S. Paglialunga and A.G. Levis (1985) Interaction of nitrilotriacetic acid with heavy metals in the induction of sister chromatid exchanges in cultured mammalian cells, Environ. Mutagen., 7, 381-390. Perry, R., P.W.W. Kirk, T. Stephenson and J.N. Lester (1984) Environmental aspects of the use of NTA as a detergent builder, Water Res., 18, 225-276.
211 Pesch, G.G., and C.E. Pesh (1980) Neanthes arenaceodentata (Polychaeta: Anellida), a proposed cytogenetic model for marine genetic toxicology, Can. J. Fish. Aquat. Sci., 37, 1225-1228. Schwartzman, J.B., F. Cortes, A. Gonzales-Fernandez, C. Gutierrez and J.F. Lopez-Saez (1979) On the nature of sister chromatid exchanges in 5-bromodeoxyuridine-substituted chromosomes, Genetics, 92, 1251-1264. Sokal, R.R., and F.J. Rohlf (1981) Biometry, 2nd edn., Freeman, San Francisco. Thiriot-Quievreux, C., and N. Ayraud (1982) Les caryotypes de quelques esp~ces de bivalves et de gasteropodes marins, Mar. Biol., 70, 165-172.
Van de Kerkhoff, J.F.J., and M.A. van der Gaag (1985) Some factors affecting optimal differential staining of sister chromatids in vivo in the fish Nothobranchius rachowi, Mutation Res., 143, 39-43. Van der Gaag, M.A., and F.J.F. van de Kerkhoff (1985) The development of an in vivo SCE-assay in the fish Nothobranchius rachowi, 4th Int. Conf. Environ. Mut., Stockholm, June 1985, Abstract p. 40. Ved Brat, S., and G.N. Williams (1984) Nitrilotriacetic acid does not induce sister-chromatid exchanges in hamster or human cells, Fd. Chem. Toxicol., 3, 211-215. Communicated by A. Abbondandolo