Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites.

The formation of reactive metabolites through biotransformation is the suspected cause of many adverse drug reactions. Testing for the propensity of a drug to form reactive metabolites has increasingly become an integral part of lead-optimization strategy in drug discovery. DNA reactivity is one undesirable facet of a drug or its metabolites and can lead to increased risk of cancer and reproductive toxicity.

Comparison of the 17 alpha-hydroxylase/C17,20-lyase activities of porcine, guinea pig and bovine P450c17 using purified recombinant fusion proteins containing P450c17 linked to NADPH-P450 reductase.

The cDNAs for cytochrome P450c17 (P450c17) of three species, pig, guinea pig, and cow, representing three families of mammals (suidae, procaviidae, and bovidae, respectively) were each engineered into an expression plasmid (pCWori+). The P450c17 domain of the coding sequence was connected to a truncated form of rat NADPH-P450 reductase by a linker sequence encoding two amino acids (SerThr). These fusion proteins were expressed in E.

The effect of reciprocal active site mutations in human cytochromes P450 1A1 and 1A2 on alkoxyresorufin metabolism.

Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 --> Ala, were constructed, expressed in Escherichia coli, and purified by Ni-NTA affinity chromatography. In nearly every case, the residue replacement led to loss of 7-methoxy- and 7-ethoxyresorufin O-dealkylase activity compared to the wild-type enzymes, except for the P450 1A1 S122T mutation which increased both activities. Mutations at position 382 in both P450 1A1 and 1A2 shifted substrate specificity from one enzyme to another, confirming the importance of this residue.

Characterization of substrate binding to cytochrome P450 1A1 using molecular modeling and kinetic analyses: case of residue 382.

Key residue Val-382 in P450 1A1 has been predicted to interact with the alkoxy chain of resorufin derivatives. Therefore, we undertook a detailed analysis of substrate mobility in the active site of the P450 1A1 homology model and assessed the effect of mutations at position 382. Dynamic trajectories of 7-methoxy-, 7-ethoxy-, and 7-pentoxyresorufin indicated that 7-ethoxyresorufin would be oxidized most efficiently by the wild-type enzyme.

Microsomal cytochrome P450 levels and activities of isolated rat livers perfused with albumin.

Purpose: We recently showed that the perfusion of isolated rat livers with perfusates containing bovine serum albumin (BSA) would significantly stimulate the release of tumor necrosis factor (TNF)-alpha. Here, we hypothesize that BSA-induced increase in the release of TNF-alpha, and possibly other cytokines, would affect cytochrome P450 (CYP)-mediated drug metabolism.

Methods: Rat livers were perfused ex vivo for 1, 2, or 3 h with a physiologic buffer containing or lacking 1% BSA (n = 4-5/group).