Characterisation of biotransformation enzyme activities and DNA integrity in isolated cells of the digestive gland of the common mussel, Mytilus edulis L

Characterisation of biotransformation and antioxidant enzyme activities and DNA integrity was carried out on isolated cells of the major biotransformation organ (digestive gland) of Mytilus edulis as a first step in developing a cell culture system for use in toxicology. Digestive gland cells were isolated by trypsin or non-protease tissue dissociation procedures, followed by filtration and differential centrifugation. Both dissociation methods produced a mixture of smaller cell types and large digestive cells at a viability of over 90% (EOSIN Y exclusion). The specific activities (per milligramme of protein) of 10 000× g supernatants from freshly isolated digestive gland mixed-cell populations produced by either dissociation method were similar to those from whole digestive gland for the antioxidant enzymes superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6), but from 3- to 10-fold lower for the biotransformation enzyme activities benzo[a]pyrene hydroxylase, glutathione- S-transferase (EC 2.5.1.18; substrate: 1-chloro-2,4-dinitrobenzene) and NADPH-dependent DT-diaphorase (EC 1.6.99.2). Cells produced by trypsin dissociation showed significant increased DNA strand breakage as measured by the single cell electrophoretic ‘comet’ assay, viz.% comet tail DNA increased from 10.3±2.3 (control) to 19.8±4.1 (0.01% w/v trypsin) to 23.7±3.2 (0.1%) (mean±S.D.). Cell yields from digestive gland were only slightly lower for non-enzymic compared to trypsin dissociation for the same time period of dissociation, indicating the former as a preferred method of cell culture preparation.