Pramod Aryal

Use of genetically engineered Salmonella typhimurium OY1002/1A2 strain coexpressing human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase and bacterial O-acetyltransferase in SOS/umu assay.

The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)–catalyzed N‐hydroxylation and subsequent esterification by O‐acetyltransferase (O‐AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH‐P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113–120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW″/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O‐AT gene in the pOA101‐expressing umuC″lacZ gene) in S. typhimurium TA1535. In addition, as an O‐AT–deficient strain, we developed the OY1003/1A2 strain by introducing pCW″/1A2:hNPR and pOA101 into O‐AT–deficient S. typhimurium TA1535/1,8‐DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and ≧0 nmol isoniazid/min/mg protein as the O‐AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2‐amino‐1,4‐dimethylimidazo[4,5‐f]quinoline (MeIQ), 2‐amino‐3‐methylimidazo[4,5‐f]quinoline (IQ), 2‐amino‐3,8‐dimethylimidazo[4,5‐f]quinoxaline (MeIQx), 2‐aminoanthracene, 2‐amino‐6‐methyldipyrido[1,2‐a::3,2′‐d]imidazole, 3‐amino‐1,4‐dimethyl‐5H‐pyrido[4,3‐b]indole, and 3‐amino‐1‐methyl‐5H‐pyrido[4,3‐a]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O‐acetyltransferase. Environ. Mol. Mutagen. 36:121–126, 2000.

Use of heterologously-expressed cytochrome P450 and glutathione transferase enzymes in toxicity assays

Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1B1, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity.