Activation of procarcinogens by human cytochrome P450 enzymes expressed in Escherichia coli. Simplified bacterial systems for genotoxicity assays.
Bacterial assays were used to examine the activation of 14 known procarcinogens by cytochrome P450 (P450) enzymes. Human P450s 1A1, 1A2 and 3A4 were expressed in Escherichia coli 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 procarcinogens by P450s. The patterns of activation of the 14 individual chemicals were consistent with the literature developed using human liver microsomes, 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 arylamines and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Recombinant P450 3A4 was found to be more active than P450 1A2 in the activation of aflatoxin B1 at all substrate concentrations examined.
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232.
T
T
Shimada
E M
EM
Gillam
P
P
Sandhu
Z
Z
Guo
R H
RH
Tukey
F P
FP
Guengerich
D000595:Amino Acid Sequence; D001571:Benzoflavones / Q000494:pharmacology; D001711:Biotransformation; D002273:Carcinogens / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests / Q000379:methods*; D011994:Recombinant Proteins / Q000494:pharmacology
D001571:Benzoflavones; D002273:Carcinogens; D011994:Recombinant Proteins; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System