The objective of this proposal is to develop an Ames mutagenicity test that uses cloned human cytochromes P450 to activate promutagens to their reactive forms. By incorporating the activating enzymes into the test organism (Salmonella typhimurium) using a recombinant DNA approach, human enzymes can be used making the test more relevant to human exposure. In addition, the undefined nature of the tissue homogenate would be replaced with cloned enzymes of known structure and activity, and the activation can take place inside the bacterial cell rather than outside, thereby facilitating the interaction of potentially short-lived mutagens with the target DNA. If successful, this recombinant mutagenicity test will enhance sensitivity for many mutagens and will provide information concerning the human enzymes involved in mutagen activation.
The specific aims of this proposal are: 1) Increasing P450 2E1 expression through modification of the N-terminal region, identification and modification of protein determinants that impede translation or promote enzyme degradation, and increasing P450 reductase levels (which appear to be limiting in these cells) by placing the P450 reductase cDNA ahead of the p450 cDNA; 2) Changing the antibiotic resistance of the P450/P450 reductase coexpression plasmid so that it is compatible with pKM101, an ampicillin-resistant R-factor plasmid carrying the mucAB genes which increases error-prone DNA repair and thus increases sensitivity to mutagens; and 3) Coexpressing additional enzymes that may be involved in P4502E1-catalyzed nitrosamine activation, including cytochrome b5.
|Bhuyan, K C; Bhuyan, D K; Chiu, W et al. (1991) Desferal-Mn(III) in the therapy of diquat-induced cataract in rabbit. Arch Biochem Biophys 288:525-32|