Abstract
Primary cell cultures of the major bio transformation organ in two species of aquatic invertebrate from different phyla, i. e. principally the digestive gland of the marine mussel Mytilus edulis (Mollusca) and also the hepatopancreas of the red swamp crayfish Procambarus clarkii (Arthropoda), as well as M. edulis gill, were developed and investigated for potential use in toxicological studies. Suspension cultures of M. edulis digestive gland cells contained a mixture of smaller cell types (basophilic cells, duct epithelial cells, crypt or undifferentiated cells, blood cells and lipid-rich cells) and large lysosomal digestive cells. Smaller cell types survived at a viability of over 50% after 2 weeks in culture whereas the more fragile digestive cells deteriorated rapidly (viability below 50% after 2 days). No cell proliferation was observed. Analysis of DNA integrity in freshly isolated M. edulis digestive gland cells using the single cell electrophoresis ('comet') assay revealed that trypsin caused significant DNA strand breakage, indicating that the use of trypsin in tissue dissociation procedures should be avoided. Freshly isolated M. edulis digestive gland cells had similar or slightly lower biotransformation enzyme (benzo[a]pyrene hydroxylase[BPH], glutathione S-transferase [GST] and NADH-DT diaphorase [DTD]) and antioxidant enzyme (superoxide dismutase [SOD], catalase) activities than comparable subcellular fractions of whole tissue. A time-course study in M. edulis digestive gland cells cultured for 4 days revealed changes in enzyme activities possibly indicative of differential cell survival, i. e. biotransformation enzyme (GST, DTD and cytochrome P450-dependent BPH) activities decreased more rapidly over time than antioxidant enzyme (SOD and catalase) activities. This may be due to the capacity for xenobiotic metabolism being primarily located in the large digestive cells compared to antioxidant functions being found in all cell types. Exposure of M. edulis digestive gland cells to benzo[a]pyrene resulted in elevated protein levels in Western blot analysis using anti-fish CYP1A and hsp70 antibodies, but identification of the former was not possible due to the low specificity of the CYP1A antibodies employed. In an attempt to apply the primary cell culture in a novel approach to biomonitoring, a comparison of two field populations of M. edulis using radiolabelled digestive gland cells was undertaken using 2-D gel electrophoresis. Digestive gland cells from a polluted site showed significant elevations of at least 10 newly synthesized proteins of 30-57 kDa molecular weight. Finally, in order to compare tissue- and species-specific differences, cell dissociation techniques for M. edulis digestive gland cells were applied to M. edulis gill cells and P. clarkii hepatopancreas cells and the toxic action of the organophosphate pesticide fenitrothion via metabolism to fenitroxon was investigated. Enzyme activities studied were acetylcholinesterase (AChE), carboxyl esterase (CbE), BPH and GST in M. edulis cells, and AChE, 7-ethoxyresorufin O-deethylase (EROD) and GST in P. clarkii cells. In all three primary cell cultures, the same pattern of enzyme modulation became apparent with esterase activities (AChE, CbE) being inhibited, and GST activity slightly elevated upon exposure to fenitrothion. M. edulis and P. clarkii cells metabolized fenitrothion to its toxic metabolite, fentrooxon, and a second metabolite, 3-methyl-4-nitrophenol was detected in P. clarkii cells but not in M. edulis cells. The use of CYP-inhibitors indicated that CYP plays a major role in the metabolism of fenitrothion but that different CYP isoforms may be involved in M. edulis compared to P. clarkii. . Overall, it was demonstrated that primary cell cultures from M. edulis digestive gland and P. clarkii hepatopancreas contain cell types, enzyme and metabolic characteristics similar to intact tissues, and that short-term studies using these cell cultures can make a significant contribution to understanding xenobiotic metabolism and cellular toxicity in sentinel species. This may aid the interpretation of ecotoxicological and biomonitoring studies.