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Susceptibility to genetic damage and cell types in Mediterranean mussels
Marine Environmental Research 54 (2002) 487–491 www.elsevier.com/locate/marenvrev
Susceptibility to genetic damage and cell types in Mediterranean mussels L. Dolcetti, P. Venier* University of Padova, Department of Biology, Via Bassi 58/B, 35131 Padova, Italy
Abstract Micronucleus (MN) frequency is generally accepted as a marker of chromosomal damage and has been studied in a variety of cells and species. In previous work, we detected significant dose-related MN increases in the epithelial-like gill cells and agranular haemocytes of Mytilus galloprovincialis treated with benzo[a]pyrene, a well-known mutagenic pollutant. In addition, we have studied micronuclei and other nuclear abnormalities in mussels collected from the Venice lagoon (Italy). Frequency changes, possibly related to genotoxic/toxic stress, in both granular and micronucleated cells from gills and haemolymph, were detected. Environmental data suggest the effect of genotoxic pollutants and the importance of cell replication in the interpretation of micronucleus frequencies. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mytilus; Genotoxic pollutants; Micronuclei; Nuclear abnormalities; Cell replication
Micronuclei are small cytoplasmatic chromatin bodies surrounded by membrane. Their formation depends on cell replication since they become evident when chromosome fragments or whole chromosomes are excluded from the main nucleus during cell division. A number of molecular mechanisms can lead to micronucleus formation (e.g. cellular processing of DNA strand breaks, mitotic loss of acentric fragments, altered segregation of whole chromosomes) and micronucleus-like structures also appear during apoptosis (Heddle et al., 1991; Stopper & Muller, 1997). In 1976 Countryman and Heddle proposed the micronucleus (MN) as an index of chromosomal damage and it has subsequently been studied in cells from humans, rodents, plants, fishes and invertebrates. MN and other cytogenetic abnormalities have also been investigated in various coastal bivalve species (Bolognesi, Landini, Roggieri, Fabbri, & Viarengo, 1999; Bresler et al., 1999; Venier, Maron, & Canava, 1997). Recently, the MN test on Mytilus sp was described among other stress * Corresponding author. Tel.: +39-049-8276284; fax: +39-049-8276280. E-mail address: [email protected] (P. Venier). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00142-3
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L. Dolcetti, P. Venier / Marine Environmental Research 54 (2002) 487–491
indexes and suggested for large scale monitoring in the frame of the MED POL Programme initiatives (Viarengo, Burlando, Giordana, Bolognesi, & Gabrielides, 2000). Microscopic preparations from mussel tissues usually require haemocyte withdrawal (e.g. by injecting the posterior adductor muscle) or enzymatic dissociation of gill cells. Fixation of cell suspensions occurs before or after their transfer to slides (by spreading or cytocentrifugation) and standard Giemsa staining is commonly applied. MN frequencies are determined on cells with preserved cytoplasm, following well-known criteria for MN classification (Bolognesi et al., 1999; Venier et al., 1997). Previously, we treated Mediterranean mussels with increasing doses of benzo[a]pyrene, a genotoxic chemical capable of inducing DNA adducts, strand breaks and other toxic effects. Significant MN formation (up to 11.71 vs. 3.97%) was evident in the subpopulation of epithelial-like gill cells, distinguishable from other cells smaller in size with more compact nuclear chromatin and higher nucleus:cytoplasm ratio. In the same tissue, granular cells, probably granular haemocytes, were found (14.58 and 2.46–19.32% in control and treated mussels, respectively). Parallel and significant MN increases (up to 16.27 vs. 6.91%) were detected in the haemolymph hyalinocytes (granular haemocytes were 57.92 and 24.94–65.24% in control and treated mussels, respectively) (Venier et al., 1997). Following essentially the same protocol (haemolymph withdrawal and dispasedigestion of gills, cytocentrifugation, fixation in methanol and Giemsa staining) we determined the individual proportion of granulocytes (%) and micronucleated cells (%) in gills and haemolymph of mussels collected from the Venice lagoon area (2– 4000 cells/mussel, five mussels/site), then calculated mean and standard deviations per mussel group, and statistically significant differences (G test). Representative images of gill cells and haemocytes are shown in Fig. 1. Epitheliallike gill cells appear with or without cilia, some cells with cytoplasmatic inclusions but still discernible from typical granulocytes (Fig. 1a). Aggregated and broken cells, cells with ‘‘golden’’ inclusions or abnormal cytoplasm were excluded from scoring. Granular and agranular haemocytes were easily categorized (Fig. 1b) while mitotic figs. were rare or difficult to recognize (Fig. 1c). The percentage of granular cells was analyzed in gills and haemolymph of mussels from different sites and time periods (Spring–Summer of 1997, 1999, 2000) (Venier, 2000; Venier, Pampanin, & Libertini, 1998). This percentage was considerably smaller in gills than in haemolymph (mean and standard deviation for 12 mussel groups were 8.43 4.48 and 47.91 12.46, respectively). In all sampling campaigns, the coupled comparison between each industrial site and the related reference site revealed significant increases in granular cells in both tissues (P40.001). Overall, the granular cell fraction was substantially lower in the reference (N=3) vs. industrial (N=6) sites (3.16 0.37 vs. 10.31 2.35 in gills and 34.57 13.85 vs. 53.82 5.52 in haemolymph). In 1997 and 1999, significant MN increases were also found in native mussels from industrial sites compared with the related reference site (e.g. 8.85 vs. 6.37% and 4.59 vs. 1.42% in 1997; 3.74 vs. 0.73% and 3.98 vs. 2.04%, in 1999; mean values in gills
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Fig. 1. Representative gill cells (a) and haemocytes (b, c) from Mytilus galloprovincialis (Shandon Cytospin 3, Giemsa staining). Main gill cell with cilia, cytoplasmatic inclusions and micronucleus (a, arrow). Granular and agranular haemocytes with apparent micronucleus (b, arrow) and mitotic figure (c, arrow).
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L. Dolcetti, P. Venier / Marine Environmental Research 54 (2002) 487–491
and haemolymph, respectively; P40.05). The most recent data (Fig. 2) did not follow the same trend, indicating unaltered MN frequency in the main gill cells (Fig. 2a) and significantly lower values (P40.001) in agranular haemocytes (Fig. 2b) for the industrial sites. The parallel substantial increase of nuclear haemocyte abnormalities (including incomplete MN) suggests inhibition of cell replication possibly caused by toxic/genotoxic agents and influencing MN formation. The infrequent mitotic figures ( < 1%) did not allow proper estimation of cell replication (nuclear abnormalities and mitotic cells are seldom evaluated in studies on aquatic organisms) (Barsiene & Lovejoy, 2000). Compared with human-based studies (see the HUman MicroNucleus Project; Bonassi et al., 2001), the assessment of MN frequencies in mussels needs to be developed further in order to understand the influence of different variables on MN
Fig. 2. Mean frequencies of MN, other nuclear abnormalities and mitotic figures in gills (a) and haemolymph (b) of mussels collected from offshore to the inner lagoon (sites 1, 2 and 3, the latter in the industrial district) in Spring–Summer 2000. Significant frequency differences of micronucleated haemocytes (sites 1 vs. 3, P40.001) and nuclear haemocyte abnormalities (sites 1 vs. 2 and 1 vs. 3, P40.001) were found.
L. Dolcetti, P. Venier / Marine Environmental Research 54 (2002) 487–491
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frequency (laboratory protocol, scoring criteria, and host factors) and to integrate proper cytogenetic data in synthetic indexes of mussel health status. Grant sponsors: Fondazione per la Ricerca sul Cancro-Ancona, MURST1999 (9905218993_004).
References Barsiene, J., & Lovejoy, D. B. (2000). International Review Hydrobiology, 85(5–6), 663–672. Bolognesi, C., Landini, E., Roggieri, P., Fabbri, R., & Viarengo, A. (1999). Environ. Molec. Mutagenesis, 33(4), 287–292. Bonassi, S., Fenech, M., Lando, C., Lin, Y. P., Ceppi, M., Chang, W. P., Holland, H., Kirsch-Volders, M., Zeiger, E., Ban, S. Y., Barale, R., Bigatti, M. P., Bolognesi, C., Jia, C., Di Giorgio, M., Ferguson, L. R., Fucic, A., Lima, O. G., Hrelia, P., Krishnaja, A. P., Lee, T. K., Migliore, L., Mikhalevich, L., Mirkova, E., Mosesso, P., Muller, W. U., Odagiri, Y., Scarfi, M. R., Szabova, E., Vorobtsova, I., Vral, A., & Zijno, A. (2001). Environmental and Molecular Mutagenesis, 37(1), 31–45. Bresler, V., Bissinger, V., Abelson, A., Dizer, H., Sturm, A., Kratke, R., Fishelson, L., & Hansen, P. D. (1999). Helgoland Marine Research, 53(3–4), 219–243. Heddle, J. A., Cimino, M. C., Hayashi, M., Romagna, F., Shelby, M. D., Tucker, J. D., Vanparys, P., & MacGregor, J. T. (1991). Environ. Molec. Mutagenesis, 18, 277–291. Stopper, H., & Muller, S. O. (1997). Toxicology in Vitro, 11, 661–667. Venier, P., Pampanin, D., & Libertini, A. (1998). 28th Annual Meeting EEMS (p. 240). Salzburg. Venier, P., Maron, S., & Canova, S. (1997). Mutation Research, 390(1/2), 33–44. Venier, P. (2000). Project 2023/3E-B, Final Report. Viarengo, A., Burlando, B., Giordana, A., Bolognesi, C., & Gabrielides, G. P. (2000). Marine Environmental Research, 49(5), 483–486.
Susceptibility to genetic damage and cell types in Mediterranean mussels L. Dolcetti, P. Venier* University of Padova, Department of Biology, Via Bassi 58/B, 35131 Padova, Italy
Abstract Micronucleus (MN) frequency is generally accepted as a marker of chromosomal damage and has been studied in a variety of cells and species. In previous work, we detected significant dose-related MN increases in the epithelial-like gill cells and agranular haemocytes of Mytilus galloprovincialis treated with benzo[a]pyrene, a well-known mutagenic pollutant. In addition, we have studied micronuclei and other nuclear abnormalities in mussels collected from the Venice lagoon (Italy). Frequency changes, possibly related to genotoxic/toxic stress, in both granular and micronucleated cells from gills and haemolymph, were detected. Environmental data suggest the effect of genotoxic pollutants and the importance of cell replication in the interpretation of micronucleus frequencies. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mytilus; Genotoxic pollutants; Micronuclei; Nuclear abnormalities; Cell replication
Micronuclei are small cytoplasmatic chromatin bodies surrounded by membrane. Their formation depends on cell replication since they become evident when chromosome fragments or whole chromosomes are excluded from the main nucleus during cell division. A number of molecular mechanisms can lead to micronucleus formation (e.g. cellular processing of DNA strand breaks, mitotic loss of acentric fragments, altered segregation of whole chromosomes) and micronucleus-like structures also appear during apoptosis (Heddle et al., 1991; Stopper & Muller, 1997). In 1976 Countryman and Heddle proposed the micronucleus (MN) as an index of chromosomal damage and it has subsequently been studied in cells from humans, rodents, plants, fishes and invertebrates. MN and other cytogenetic abnormalities have also been investigated in various coastal bivalve species (Bolognesi, Landini, Roggieri, Fabbri, & Viarengo, 1999; Bresler et al., 1999; Venier, Maron, & Canava, 1997). Recently, the MN test on Mytilus sp was described among other stress * Corresponding author. Tel.: +39-049-8276284; fax: +39-049-8276280. E-mail address: [email protected] (P. Venier). 0141-1136/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(02)00142-3
488
L. Dolcetti, P. Venier / Marine Environmental Research 54 (2002) 487–491
indexes and suggested for large scale monitoring in the frame of the MED POL Programme initiatives (Viarengo, Burlando, Giordana, Bolognesi, & Gabrielides, 2000). Microscopic preparations from mussel tissues usually require haemocyte withdrawal (e.g. by injecting the posterior adductor muscle) or enzymatic dissociation of gill cells. Fixation of cell suspensions occurs before or after their transfer to slides (by spreading or cytocentrifugation) and standard Giemsa staining is commonly applied. MN frequencies are determined on cells with preserved cytoplasm, following well-known criteria for MN classification (Bolognesi et al., 1999; Venier et al., 1997). Previously, we treated Mediterranean mussels with increasing doses of benzo[a]pyrene, a genotoxic chemical capable of inducing DNA adducts, strand breaks and other toxic effects. Significant MN formation (up to 11.71 vs. 3.97%) was evident in the subpopulation of epithelial-like gill cells, distinguishable from other cells smaller in size with more compact nuclear chromatin and higher nucleus:cytoplasm ratio. In the same tissue, granular cells, probably granular haemocytes, were found (14.58 and 2.46–19.32% in control and treated mussels, respectively). Parallel and significant MN increases (up to 16.27 vs. 6.91%) were detected in the haemolymph hyalinocytes (granular haemocytes were 57.92 and 24.94–65.24% in control and treated mussels, respectively) (Venier et al., 1997). Following essentially the same protocol (haemolymph withdrawal and dispasedigestion of gills, cytocentrifugation, fixation in methanol and Giemsa staining) we determined the individual proportion of granulocytes (%) and micronucleated cells (%) in gills and haemolymph of mussels collected from the Venice lagoon area (2– 4000 cells/mussel, five mussels/site), then calculated mean and standard deviations per mussel group, and statistically significant differences (G test). Representative images of gill cells and haemocytes are shown in Fig. 1. Epitheliallike gill cells appear with or without cilia, some cells with cytoplasmatic inclusions but still discernible from typical granulocytes (Fig. 1a). Aggregated and broken cells, cells with ‘‘golden’’ inclusions or abnormal cytoplasm were excluded from scoring. Granular and agranular haemocytes were easily categorized (Fig. 1b) while mitotic figs. were rare or difficult to recognize (Fig. 1c). The percentage of granular cells was analyzed in gills and haemolymph of mussels from different sites and time periods (Spring–Summer of 1997, 1999, 2000) (Venier, 2000; Venier, Pampanin, & Libertini, 1998). This percentage was considerably smaller in gills than in haemolymph (mean and standard deviation for 12 mussel groups were 8.43 4.48 and 47.91 12.46, respectively). In all sampling campaigns, the coupled comparison between each industrial site and the related reference site revealed significant increases in granular cells in both tissues (P40.001). Overall, the granular cell fraction was substantially lower in the reference (N=3) vs. industrial (N=6) sites (3.16 0.37 vs. 10.31 2.35 in gills and 34.57 13.85 vs. 53.82 5.52 in haemolymph). In 1997 and 1999, significant MN increases were also found in native mussels from industrial sites compared with the related reference site (e.g. 8.85 vs. 6.37% and 4.59 vs. 1.42% in 1997; 3.74 vs. 0.73% and 3.98 vs. 2.04%, in 1999; mean values in gills
L. Dolcetti, P. Venier / Marine Environmental Research 54 (2002) 487–491
489
Fig. 1. Representative gill cells (a) and haemocytes (b, c) from Mytilus galloprovincialis (Shandon Cytospin 3, Giemsa staining). Main gill cell with cilia, cytoplasmatic inclusions and micronucleus (a, arrow). Granular and agranular haemocytes with apparent micronucleus (b, arrow) and mitotic figure (c, arrow).
490
L. Dolcetti, P. Venier / Marine Environmental Research 54 (2002) 487–491
and haemolymph, respectively; P40.05). The most recent data (Fig. 2) did not follow the same trend, indicating unaltered MN frequency in the main gill cells (Fig. 2a) and significantly lower values (P40.001) in agranular haemocytes (Fig. 2b) for the industrial sites. The parallel substantial increase of nuclear haemocyte abnormalities (including incomplete MN) suggests inhibition of cell replication possibly caused by toxic/genotoxic agents and influencing MN formation. The infrequent mitotic figures ( < 1%) did not allow proper estimation of cell replication (nuclear abnormalities and mitotic cells are seldom evaluated in studies on aquatic organisms) (Barsiene & Lovejoy, 2000). Compared with human-based studies (see the HUman MicroNucleus Project; Bonassi et al., 2001), the assessment of MN frequencies in mussels needs to be developed further in order to understand the influence of different variables on MN
Fig. 2. Mean frequencies of MN, other nuclear abnormalities and mitotic figures in gills (a) and haemolymph (b) of mussels collected from offshore to the inner lagoon (sites 1, 2 and 3, the latter in the industrial district) in Spring–Summer 2000. Significant frequency differences of micronucleated haemocytes (sites 1 vs. 3, P40.001) and nuclear haemocyte abnormalities (sites 1 vs. 2 and 1 vs. 3, P40.001) were found.
L. Dolcetti, P. Venier / Marine Environmental Research 54 (2002) 487–491
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frequency (laboratory protocol, scoring criteria, and host factors) and to integrate proper cytogenetic data in synthetic indexes of mussel health status. Grant sponsors: Fondazione per la Ricerca sul Cancro-Ancona, MURST1999 (9905218993_004).
References Barsiene, J., & Lovejoy, D. B. (2000). International Review Hydrobiology, 85(5–6), 663–672. Bolognesi, C., Landini, E., Roggieri, P., Fabbri, R., & Viarengo, A. (1999). Environ. Molec. Mutagenesis, 33(4), 287–292. Bonassi, S., Fenech, M., Lando, C., Lin, Y. P., Ceppi, M., Chang, W. P., Holland, H., Kirsch-Volders, M., Zeiger, E., Ban, S. Y., Barale, R., Bigatti, M. P., Bolognesi, C., Jia, C., Di Giorgio, M., Ferguson, L. R., Fucic, A., Lima, O. G., Hrelia, P., Krishnaja, A. P., Lee, T. K., Migliore, L., Mikhalevich, L., Mirkova, E., Mosesso, P., Muller, W. U., Odagiri, Y., Scarfi, M. R., Szabova, E., Vorobtsova, I., Vral, A., & Zijno, A. (2001). Environmental and Molecular Mutagenesis, 37(1), 31–45. Bresler, V., Bissinger, V., Abelson, A., Dizer, H., Sturm, A., Kratke, R., Fishelson, L., & Hansen, P. D. (1999). Helgoland Marine Research, 53(3–4), 219–243. Heddle, J. A., Cimino, M. C., Hayashi, M., Romagna, F., Shelby, M. D., Tucker, J. D., Vanparys, P., & MacGregor, J. T. (1991). Environ. Molec. Mutagenesis, 18, 277–291. Stopper, H., & Muller, S. O. (1997). Toxicology in Vitro, 11, 661–667. Venier, P., Pampanin, D., & Libertini, A. (1998). 28th Annual Meeting EEMS (p. 240). Salzburg. Venier, P., Maron, S., & Canova, S. (1997). Mutation Research, 390(1/2), 33–44. Venier, P. (2000). Project 2023/3E-B, Final Report. Viarengo, A., Burlando, B., Giordana, A., Bolognesi, C., & Gabrielides, G. P. (2000). Marine Environmental Research, 49(5), 483–486.