Journal Article

Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (−)benzo[<i>a</i>]pyrene-7R-<i>trans</i>-7,8-dihydrodiol

Jennifer S. Mammen, Gary S. Pittman, Ying Li, Fadi Abou-Zahr, Bassem A. Bejjani, Douglas A. Bell, Paul T. Strickland and Thomas R. Sutter

in Carcinogenesis

Volume 24, issue 7, pages 1247-1255
Published in print July 2003 | ISSN: 0143-3334
Published online July 2003 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/bgg088
Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (−)benzo[a]pyrene-7R-trans-7,8-dihydrodiol

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Genetic differences that underlie inter-individual variation in the metabolism of common carcinogens are important potential sources of cancer susceptibility. Cytochrome P450 1B1 (CYP1B1), a central enzyme in the activation of the ubiquitous environmental carcinogen benzo[a]pyrene (B[a]P), has several genetic variants. This study investigated six rare mutations and four common polymorphisms for their effects on B[a]P metabolism. Five missense mutations associated with congenital glaucoma (Gly61Glu, Gly365Trp, Asp374Asn, Pro437Leu and Arg469Tryp) dramatically decreased the capacity of CYP1B1 to convert (−)benzo[a]pyrene-7R-trans-7,8-dihyrodiol (B[a]P-7,8-diol) to (±)benzo[a]pyrene-r-7,t-8-dihydrodiol-9,10-epoxides. These five mutations resulted in enzymes with 3–12% of normal activity when assayed in vitro using an Saccharomyces cerevisiae microsomal expression system. A 10 bp deletion mutation produced no detectable protein or activity. In contrast, proteins containing all possible combinations of four common single nucleotide polymorphisms (Arg48Gly, Ala199Ser, Val432Leu, Asn453Ser) had modest effects on B[a]P-7,8-diol metabolism. Michaelis–Menten analysis suggested that two alleles, Arg48, Ala119, Val432, Ser453 (RAVS) and Arg48, Ala119, Leu432, Ser453 (RALS), have KM values 2-fold lower than Arg48, Ala119, Val432, Ser453 (RAVN): 1.4 ± 0.3 and 1.3 ± 0.4 µM, respectively, compared with 2.8 ± 0.8 µM (P < 0.05). However, these differences could not be confirmed with direct measurements of rate at low substrate concentration. There were no significant differences for either of two other kinetic parameters, kcat or kcat/KM. Allele frequency analysis in three populations reveals the Ser453 variant is rare among those of Asian (<1%) and African ancestry (<4%), and more common in individuals of European ancestry (16%). Haplotypes containing the Ser453 variant were uncommon; only RALS was detectable in our small populations. The RALS allele occurred between 0.5% in Asians and 15% in Europeans. Our study demonstrates that rare, disease-associated mutations in CYP1B1 significantly decrease the enzyme's metabolism of B[a]P-7,8-diol; however, our results do not identify any major differences in this metabolism due to four common single amino acid polymorphisms.

Keywords: B[a]P, benzo[a]pyrene; B[a]P-7,8-diol, (−)benzo[a]pyrene-7R-trans-7,8-dihydrodiol; CYP, cytochrome P450; DE1, (±)B[a]P-r-7,t-8-dihydrodiol-c-9,10-epoxide; DE2, (±)B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide; EH, Estimated Haplotypes; mEH, microsomal epoxide hydrolase; RTTC, (±)benzo[a]pyrene-r-7,t-8,t-9,c-10-tetrahydrotetrol

Journal Article.  6704 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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