© 1994 Oxford University Press
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Activation of procarcinogens by human cytochrome P450 enzymes expressed in Escherichia coli. Simplified bacterial systems for genotoxicity assays
Department of Biochemistry and Center in Molecular Toxicology Vanderbilt University School of Medicine Nashville, TN 37232
1Departments of Pharmacology and Medicine, University of California at San Diego, La Jolla, CA 92093, USA
2Osaka Prefectural Institute of Public Health Nakamichi, Higashinari-ku, Osaka 537, Japan
3Department of Physiology and Pharmacology, University of Queensland St Lucia, Queensland 4072, Australia
4Smith Kline & Beecham. King of Prussia, PA 19406, USA
5To whom correspondence should be addressed
Bacterial assays were used to examine the activation of 14 known procarcinogens by cytochrome P450 (P450) enzymest
. Human P450s 1A1, 1A2 and 3A4 were expressed in Escherichia coil with slight modification of their N-terminal sequences. Genotoxicity was measured by the induction of the SOS response in Salmonella typhimurium NM2009 (TA1535/pSK1002/pNM12), which contains a umuC regulatory sequence attached to the lacZ reporter gene. Conditions for analysis were examined using E.coli membranes and purified enzymes. Membrane fractions, fortified with NADPH-P450 reductase, were found to be useful preparations for measuring activation of the procarcinogens. Conditions of linearity were established for these assays and the systems were applied to several particular problems related to bioactivation of procarcino gens by P450s. The patterns of activation of the 14 indi vidual chemicals were consistent with the literature developed using human liver niicrosomes, purified liver P450s and other approaches. The P450s expressed in bacterial membranes could be inhibited by antibodies. 7,8-Benzoflavone inhibited P450s 1A1 and 1A2 and stimulated P450 3A4 in the membranes. The contributions of P450s 1A1 and 1A2 were distinguished with some of the arylam ines and 7,8-dihydroxy-7,8-dihydrobenzo[a] Recom binant P450 3A4 was found to be more active than P450 1A2 in the activation of allatoxin B1 at all substrate concentrations examined.
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