Potential pitfalls associated with testing of enzyme preparations in the Salmonella/microsome assay

The effect of a sample of food enzyme preparations on S9 activity was evaluated in bacterial mutation assays with the Salmonella typhimurium strains TA98 and TA100 using benzo(a)pyrene, 2-aminoanthracene and 2-aminofluorene as model compounds. Under the experimental conditions applied, Aspergillus oryzae protease and porcine pancreas trypsin, applied at low non-toxic doses, proved to effectively inhibit the metabolic activation of benzo(a)pyrene by Aroclor induced rat liver 9, while the activation of 2-aminoanthracene and 2-aminofluorene was only marginally affected. The tolerance of metabolic activation of 2-aminoanthracene to the presence of proteolytic enzymes, compared to the strong inhibition elicited on the metabolic activation of benzo(a)pyrene, points to the involvement of different components of liver S9 in their biotransformation. Overall, data indicate that the use of 2-aminoanthracene as positive control in the Ames test can give a misleading indication of S9 proficiency, and thus it should be used with caution or in conjunction with other chemicals, especially in the testing of crude enzyme preparations in which proteases may be present as minor components.