Cryopreserved and hypothermically stored rat liver parenchymal cells as metabolizing system in the Salmonella mutagenicity assay

Freshly isolated and preserved rat liver parenchymal cells were used as metabolizing system in the Salmonella mutagenicity assay. The liver cells were isolated with EDTA perfusion without the addition of collagenase and had a viability of 96% as judged by trypan blue exclusion. When freshly isolated liver parenchymal were cryopreserved with a computer controlled freezing protocol and stored at −196°C they had a mean viability of 89% after thawing. Furthermore, freshly isolated cells were stored at 0°C in University of Wisconsin organ transplantation solution. After 1 day of hypothermic storage they had a viability of 95%. Four different indirect mutagens, 2-aminoanthracene, benzo[ a]pyrene, 7,12-dimethylbenz[ a]anthracene and cyclophosphamide, were used with the liver cells as metabolizing system in the preincubation assay with Salmonella typhimurium TA100. After cryopreservation, liver parenchymal cells were able to activate all tested indirect mutagens to ultimate mutagens. However, the induction of revertants was lower with three of the four tested compounds. Only 2-aminoanthracene was activated to the same extent by freshly isolated and cryopreserved liver cells. 7-Hydroxymethyl-12-methylbenz[ a]anthracene, which is activated to its ultimate mutagen by sulfotransferase, also induced a reduced mutagenic effect with cryopreserved liver cells in comparison to freshly isolated liver parenchymal cells. This indicates that phase I and phase II enzyme activities are effected by cryopreservation. However, identical mutation frequencies were obtained when freshly isolated liver parenchymal cells or 1 day hypothermically preserved liver parenchymal cells were used in the cell-mediated Salmonella mutagenicity test. The use of hypothermic short-time storage of liver parenchymal cells could help to make the liver cell-mediated genotoxicity test simpler and thereby more attractive.