David R. Dixon

Effects of cadmium on nuclear integrity and DNA repair efficiency in the gill cells of Mytilus edulis L

Although the effects of heavy metals on marine invertebrate species are well studied in term of their toxicity and bioaccumulation, less is known about their genotoxicity. The aim of this investigation was to assess the DNA damaging potential of cadmium (Cd) in an important pollution sentinel organism, the mussel Mytilus edulis. Cadmium is one of the most toxic and widespread heavy metals found in the marine environment, and is a recognised carcinogen in mammals. Based on the results of the comet assay (alkaline single cell gel electrophoresis), Cd was found not to be genotoxic in mussel gill cells under acute and chronic exposure conditions, whereas pre-exposure to low concentrations of Cd was found to enhance the genotoxicity of another mutagen, hydrogen peroxide (H2O2). The effects of H2O2 were normally reversible when cells were transferred to clean saline buffer. However, in cells that had been pre-treated with Cd, in vivo or in vitro, we observed a decrease in this post-treatment DNA repair. The effects of Cd were reversed by zinc which suggests that the inhibitory effect of Cd on DNA repair was due to the displacement of zinc ions from active sites on proteins involved in the repair process (a property already described for mammals). Moreover, since Cd inhibits or delays the onset of apoptosis (programmed cell death), this removes one of the main defence mechanisms responsible for protecting the organism against neoplasia. There appears to be a close similarity between the effects of Cd on marine molluscs and mammals.

Evaluation of the comet assay as a method for the detection of DNA damage in the cells of a marine invertebrate, Mytilus edulis L. (Mollusca: Pelecypoda)

The potential application of the comet assay for monitoring the effect of DNA damaging agents on the marine mussel, Mytilus edulis (an important pollution indicator organism), was explored. A detailed investigation of the baseline levels of single-strand breaks in isolated gill cells, and how they were affected by age/size of animal, time since collection, feeding regime, in vivo vs. in vitro exposure conditions, and by antioxidant supplementation was undertaken. The level of cometing in untreated controls was found to be highly variable over time (fluctuations between low and very high DNA damage occurred over just 14 days post collection). No difference was observed between age/size and feeding regime of the animals. On exposure to 0, 100, 500 and 1000 microM H2O2, it was observed that the in vitro exposure produced a markedly more homogeneous dose response compared to the in vivo studies (where gill cells were exposed as a tissue). An important finding of our research was the effect of prior supplementation of the animals diet with 1 mg/ml alpha-tocopherol acetate (vitamin E compound), which resulted in a marked reduction in the levels of DNA damage expressed by the negative controls, without influencing the actual response to H2O2 (0, 5, 25, and 100 microM) and N-nitrosodimethylamine, NDMA (0, 5, 25, and 100 mM). The effect of vitamin E supplementation was to increase the sensitivity of the comet assay at the lower end of the dose range. This study demonstrated the potential application of the comet assay to the gill cells of the mussel, M. edulis. Although preliminary findings suggest that antioxidant supplementation can improve the sensitivity of the assay by lowering the baseline damage in untreated animals, our conclusion is that the assay has more potential for use in an in vitro context for the screening of agents destined for release or disposal into the marine environment.

Intron-targeted PCR: a new approach to survey neutral DNA polymorphism in bivalve populations

PCR (polymerase chain reaction) amplification of non-coding introns in phylogenetically widespread genes, using DNA primers based on the conserved exon sequences, provides a widely applicable strategy for finding DNA polymorphisms in eukaryotic genomes. Polymorphisms in introns provide a new source of potentially neurtral genetic markers for use in population biology. Here we use this approach to design PCR primers for an intron of calmodulin genes. We show that there are at least two calmodulin genes in mussels of theMytilus edulis species complex, and using gene- and species-specific primers we resolve two alleles at a calmodulin intron locus. Population surveys using PcR of adult mussel DNA reveal that genotype frequencies at most sites surveyed in England, Scotland and Italy, conform to Hardy-Weinberg equilibrium, suggesting that this is a novel neutral genetic marker. The data also provide preliminary evidence for restricted gene flow between mussel populations on the west and northeast coasts of Britain, and for local effects around the Thames estuary.

Toxic vents and DNA damage: first evidence from a naturally contaminated deep-sea environment

Levels of DNA strand breakage were measured, using the comet assay, in cells from vent mussels, Bathymodiolus azoricus, from three contrasting vent fields on the mid Atlantic Ridge. Different levels of DNA damage were recorded in untreated mussels, shortly after collection, and it was animals from the shallowest, and less active, Menez Gwen vent field (840-m depth), which showed the greatest amount of damage. In contrast to animals from two deeper and putatively more toxic sites, Menez Gwen animals went on to repair this damage and were able to survive under laboratory conditions at 1 bar pressure for several months. Animals from the two deeper sites showed both higher levels of initial mortality and a much reduced capacity for survival at 1 bar. The differences in DNA damage levels at the time of collection were interpreted as an expression of differences in cell viability/enzyme activity rather than a reflection of any differences in their natural environmental conditions. Small B. azoricus showed a capacity to repair DNA damage, whereas this ability appeared to be lacking in large individuals. By reproducing at a relatively early age, the deep-sea vent fauna may be able to resist the toxic effects of its environment by exploiting this natural, stage specific capacity to repair damaged DNA.

Molecular evolution and diversification of the vestimentiferan tube worms

The Vestimentifera, or deep-sea tube worms, comprise an ecologically and anatomically unusual group of marine invertebrates, with poorly understood biogeography, ecology, phylogenetic affinities and evolutionary radiation. To gain insight into evolutionary diversification within the group, we have used a molecular biological approach. We report the cloning of a region of 28S ribosomal DNA from representatives of five vestimentiferan genera plus, for comparison, a polychaete and a perviate pogonophore. Phylogenetic analyses using these DNA sequences confirm that Ridgeia and Tevnia are closely related genera. The analyses also lead us to propose the hypothesis that the earliest vestimentiferan lineage to diverge gave rise to the genus Lamellibrachia only. In addition, our comparative DNA sequence data now provide a means to use molecular methods for identification of deep-sea tube worms; we employed this approach to demonstrate that the first vestimentiferan specimen from the eastern Atlantic Ocean belongs to the genus Lamellibrachia . DNA-based identification should have wide applications in the study of vestimentiferan biogeography and ecology.

Development of an in vivo genotoxicity assay using the marine worm Platynereis dumerilii (Polychaeta: Nereidae)

An in vivo genotoxicity test system has been developed using the embryo-larval stages of the marine annelid, Platynereis dumerilii (Polychaeta: Nereidae). This species is representative of an ecologically important group of marine invertebrates, it is amenable to laboratory culture and has a well defined and stable karyotype (2n=28) which is suitable for the analysis of a range of cytogenetic endpoints, including chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs). An evaluation of the cell cycle kinetics using the embryo-larval stages allowed selection of exposure times for cytogenetic work. Subsequently, 12-h-old embryos were exposed to reference mutagens, dissolved in sea water, in the presence of 5-bromodeoxyuridine (BrdU) for 12 h (SCE analysis) or 8 h (CA analysis) at 15 +/- 1 degree C, by which time they had reached the first larval stage (20-24h). Dose response-relationships for cytotoxicity, SCEs and CAs were observed for both direct acting mutagens (methyl methanesulfonate, mitomycin C) and mutagens which require metabolic activation (cyclophosphamide, benzo[a]pyrene). The sensitivity of the embryo-larval stages of P. dumerilii to both direct and indirect acting mutagens, their suitability for laboratory culture, together with the presence of a good karyotype and chromosome morphology for cytogenetic analyses, makes this species a potentially valuable in vivo model for marine genotoxicity testing.

Assessment of developmental effects, cytotoxicity and genotoxicity in the marine polychaete (Platynereis dumerilii) exposed to disinfected municipal sewage effluent.

While sodium hypochlorite is widely used as a disinfectant for municipal sewage effluents and power station cooling waters discharged into coastal environments, there is limited information on the potential in vivo genotoxicity of such disinfection procedures to marine organisms. Using a recently developed test system based on the marine polychaete Platynereis dumerilii, we have evaluated impacts based on embryo-larval development, cytotoxicity and genotoxicity following exposure to disinfected settled (primary) effluent from a municipal sewage treatment works (STW). Sewage samples were collected from Newton Abbot STW, Devon, UK and then disinfected with sodium hypochlorite based on standard operational procedures. Exposure of polychaetes to dilutions of disinfected sewage in seawater (20 +/- 1 degree C) led to a marked reduction in normal embryo-larval development (7 h EC50 from 0.57-1.88% (v/v), n = 4), with a simultaneous increase in cytotoxicity. Following the calculation of the Maximum Tolerated Dose (MTD), based on developmental and cytotoxic effects, the organisms were also analysed for the induction of chromosomal aberrations. This investigation demonstrated the absence of genotoxicity in polychaetes exposed in vivo to sewage disinfected with sodium hypochlorite. These observations extend our previously published studies in which polychaetes exposed to non-disinfected sewage, while showing developmental toxicity and cytotoxicity, did not exhibit any evidence of cytogenetic damage.

Inheritance of a nuclear DNA polymorphism assayed in single bivalve larvae

We examined the inheritance of theMytilus edulis CaM-1 Intron 3 locus, a non-coding DNA locus with two potentially neutral length-variants. The polymerase chain reaction (PCR) was used to determine theCaM-1 genotype for 799 larvae obtained from 11 laboratory crosses. Larvae were typed singly only 8 to 24 h after fertilization. We find evidence that each allele can be inherited from either sex, that there are no barriers to fertilization between gametes of different genotypes, and that most larvae have genotypes compatible with Mendelian inheritance of the locus. Deviations from expected genotype frequencies were found in some crosses; we suggest that a contributing factor is aneuploidy in early larvae, but a major cause is more likely to be selection at a locus linked toCaM-1, occurring under the artificial laboratory culture conditions. This study demonstrates the feasibility of applying molecular genetic techniques to early larval stages of marine bivalves, and presents a new non-destructive biopsy method for DNA analysis from living adult mussels.

Ocean-ridge segmentation and vent tubeworms (Vestimentifera) in the NE Pacific

Abstract Vestimentiferan tube worms are important components of the hydrothermal vent and cold seep communities of the Pacific Ocean. The distribution and geographic intraspecies variation of Ridgeia piscesae and Lamellibrachia barhami were examined in the region of the Explorer (1 site), Juan de Fuca (33 sites) and Gorda (2 sites) Ridges and the nearby Cascadia Subduction zone. Isozyme electrophoresis, DNA restriction fragment length polymorphism (RFLP) and DNA sequencing techniques have been used. Ridgeia piscesae is widespread at hot vent sites along the ridges. The transform offset between Explorer and Juan de Fuca does not appear to impede gene flow (isozyme and RFLP data). Hydrographic conditions close to the Juan de Fuca Ridge favour bidirectional along-axis transport of planktonic larvae and some cross-axis transport. Rapid colonization by R. piscesae has been observed at a new vent site on CoAxial Segment of the Juan de Fuca Ridge, supporting the idea of a pool of larvae in the water overlying the ridge. The 360 km offset between southern Juan de Fuca and Gorda Ridges is associated with a significant level of genetic differentiation (shown by RFLP) which indicates some interruption to larval dispersal at this scale. The occurrence of the cold-seep species Lamellibrachia barhami is confirmed at one hydrothermal site, the sedimented Middle Valley on Juan de Fuca Ridge. Vestimentiferans appear to have a tremendous dispersal, location and adaptation capability. It is only at the thousand kilometre scale, that complete barriers to dispersal occur.

Evaluation of the genotoxicity of municipal sewage effluent using the marine worm Platynereis dumerilii (Polychaeta: Nereidae).

Samples of settled (primary) effluent were collected from a municipal sewage treatment works at Newton Abbot, Devon, UK, a site which discharges primary effluent via long sea pipeline into the English Channel (minimum of 200-fold initial dilution). Sewage samples were collected during the period February-April 1995 and were analysed for standard physico-chemical parameters (ammonia, chemical oxygen demand, conductivity, non-purgeable organic carbon and settled solids). Samples were also tested for cytotoxicity, genotoxicity, and for developmental effects in the embryo-larval stages of the marine worm, Platynereis dumerilii. Exposure to sewage concentrations of > or = 10% (v/v) in seawater at 20 +/- 1 degrees C led to a marked reduction in normal embryo-larval development (7 h EC50 values from 10% to 18% v/v, n = 5). There was also evidence of a simultaneous delay in the cell cycle progression (as determined by sister chromatid differential staining) following embryo-larval exposures to sewage concentrations of > or = 10% (v/v). Following the calculation of the Maximum Tolerated Dose (MTD), based on cytotoxic and developmental effects, cells from the same embryo-larvae were analysed for chromosomal aberrations (CAs). Results were consistent for all samples tested, demonstrating the absence of cytogenetic damage following the in vivo exposure of polychaete embryo-larvae to settled sewage.