AMES MUTAGENICITY TESTING WITH RECOMBINANT HUMAN P450S

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The Ames test for mutagenicity is a widely used protocol to estimate the genotoxicity and potential carcinogenicity of drugs and chemicals. This test measures the frequency of reversion of histidine auxotrophs of Salmonella typhimurium following their incubation in the presence of the test chemical and a metabolic activating system. This activating system is most often the 9000 x g supernatant (S9 fraction) of a tissue homogenate, most commonly derived from rodent liver. The presence of activating enzymes, notably the cytochromes P450, in this tissue homogenate promotes the conversion of promutagens and procarcinogens to their active metabolites, which are then capable of binding to cellular macromolecules, including DNA. The present proposal would eliminate the use of laboratory animals as a source of the activating enzyme preparation by directly expressing these enzymes in the test bacterium (S. typhimurium). Moreover, several shortcomings of the Ames test as presently used would be eliminated: 1) the undefined nature of the tissue homogenate would be replaced with enzymes of known structure and activity; and 2) it would be possible to use cloned human, rather than rodent, enzymes, and thereby more accurately estimate the risk to man of exposure to the test chemical; and 3) the activation can take place inside the bacterial cell, rather than outside, facilitating the interaction of potentially short-lived mutagens with the target DNA and thereby enhancing the sensitivity of the assay. The specific aims of this proposal are: 1) To coexpress a human cytochrome P450 with cytochrome P450 reductase in S. typhimurium and determine the intracellular levels of these enzymes by functional and immunochemical assays; if necessary, to modify the expression vector (a plasmid containing one or more copies of the P450 and reductase cDNAs, and one or more promoters) so as to optimize the ratio of the two expressed enzymes for maximum activity; 2) To characterize the enzymatic parameters of the optimized system with appropriate substrate(s), and, importantly, to demonstrate that the specific P450-catalyzed monooxygenase activity is present in live bacterial cultures; 3) To establish the utility of the transformed S. typhimurium cells in an Ames test by adding a test substance known to cause genetic mutations and scoring for histidine revertants, with comparison to bacterial cells not containing a P450 expression system, but incubated in the presence or absence of a human liver S9 fraction; and 4) To expand the "recombinant P450 Ames test" by establishing a panel of S. typhimurium recombinants containing a series of human cytochromes P450, focussing on those most often associated with the activation of carcinogenic and mutagenic chemicals, and verifying the utility of this system.