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

Carcinogenesis. 2003 Jul;24(7):1247-55. doi: 10.1093/carcin/bgg088. Epub 2003 Jun 5.

Abstract

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 microM, respectively, compared with 2.8+/-0.8 microM (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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Aryl Hydrocarbon Hydroxylases / genetics
  • Aryl Hydrocarbon Hydroxylases / metabolism*
  • Blacks / genetics
  • Cytochrome P-450 CYP1B1
  • DNA Primers / chemistry
  • Dihydroxydihydrobenzopyrenes / metabolism*
  • Gene Frequency
  • Genotype
  • Glaucoma / enzymology*
  • Glaucoma / genetics*
  • Haplotypes
  • Humans
  • Microsomes / enzymology
  • Microsomes / metabolism*
  • Mutagenesis, Site-Directed
  • Mutation, Missense*
  • NADH Dehydrogenase / metabolism
  • Oxidation-Reduction
  • Polymerase Chain Reaction
  • Polymorphism, Restriction Fragment Length
  • Polymorphism, Single Nucleotide*
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism

Substances

  • DNA Primers
  • Dihydroxydihydrobenzopyrenes
  • benzo(a)pyrene 7,8-dihydrodiol
  • Aryl Hydrocarbon Hydroxylases
  • CYP1B1 protein, human
  • Cytochrome P-450 CYP1B1
  • NADH Dehydrogenase