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 a s metabolizing system in the Salmonella mutagenicity assay. The liver cells were isolated with EDTA perfusion without the addition of collag enase and had a viability of 96% as judged by trypan blue exclusion. W hen freshly isolated liver parenchymal were cryopreserved with a compu ter controlled freezing protocol and stored at -196 degrees C they had a mean viability of 89% after thawing. Furthermore, freshly isolated cells were stored at 0 degrees C in University of Wisconsin organ tran splantation solution. After 1 day of hypothermic storage they had a vi ability of 95%. Four different indirect mutagens, 2-aminoanthracene, b enzo[a]pyrene, 7,12-dimetylbenz[a]anthracene and cyclophosphamide, wer e used with the liver cells as metabolizing system in the preincubatio n assay with Salmonella typhimurium TA100. After cryopreservation, liv er parenchymal cells were able to activate all tested indirect mutagen s to ultimate mutagens. However, the induction of revertants was lower with three of the four tested compounds. Only 2-aminoanthracene was a ctivated to the same extent by freshly isolated and cryopreserved live r cells. 7-Hydroxymethyl-12-methylbenz[a]anthracene, which is activate d to its ultimate mutagen by sulfotransferase, also induced a reduced mutagenic effect with cryopreserved liver cells in comparison to fresh ly isolated liver parenchymal cells. This indicates that phase I and p hase II enzyme activities are effected by cryopreservation. However, i dentical mutation frequencies were obtained when freshly isolated live r parenchymal cells or 1 day hypothermically preserved liver parenchym al cells were used in the cell-mediated Salmonella mutagenicity test. The use of hypothermic short-time storage of liver parenchymal cells c ould help to make the liver cell-mediated genotoxicity test simpler an d thereby more attractive.