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

Abstract

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.