Stability of activating systems for in vitro mutagenesis assays: enzyme activity and activating ability following long-term storage at - 85 degrees C

Abstract

Activating systems for in vitro mutagenesis assays are commonly prepared and stored at low temperature until required. The objective of the studies reported here was to determine the long-term stability of activating systems stored at - 85 degrees C. A broad range of microsomal enzymes in the postmitochondrial supernatant (PMS) and the microsomal fraction of livers from Aroclor 1254 treated rats were studied in conjunction with the ability of these fractions to catalyse the conversion of dimethylnitrosamine (DMN) and benzo(a)pyrene (B(a)P) to products mutagenic to Chinese hamster ovary (CHO) cells and Salmonella typhimurium TM677. Biphenyl-2- and biphenyl-4-hydroxylase showed a rapid decline in activity on storage, epoxide hydratase activity increased with storage and other enzyme activities studied were relatively stable for up to 32 weeks. No consistent trends in the ability of either the microsomes or the PMS to catalyze DMN or B(a)P induced mutation were observed for up to 12 weeks with CHO cells and 24 weeks with bacteria. It is concluded that low temperature storage of activating systems is an acceptable procedure. However, the results also indicate that certain enzyme activities change during storage, suggesting that aberrant results may be obtained when stored activating systems are used in in vitro tests to screen for mutagens.