Single amino acid mutations, but not common polymorphisms, decrease the activity of CYP1B1 against (-)benzo[a]pyrene-7R-trans-7,8-dihydrodiol.
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.
Mammen
Jennifer S
JS
Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe St, Baltimore, MD 21205, USA.
Pittman
Gary S
GS
Li
Ying
Y
Abou-Zahr
Fadi
F
Bejjani
Bassem A
BA
Bell
Douglas A
DA
Strickland
Paul T
PT
Sutter
Thomas R
TR
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D044383:Black People / Q000235:genetics; D065633:Cytochrome P-450 CYP1B1; D017931:DNA Primers / Q000737:chemistry; D004101:Dihydroxydihydrobenzopyrenes / Q000378:metabolism*; D005787:Gene Frequency; D005838:Genotype; D005901:Glaucoma / Q000201:enzymology* / Q000235:genetics*; D006239:Haplotypes; D006801:Humans; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009245:NADH Dehydrogenase / Q000378:metabolism; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism
D017931:DNA Primers; D004101:Dihydroxydihydrobenzopyrenes; C017228:benzo(a)pyrene 7,8-dihydrodiol; D001189:Aryl Hydrocarbon Hydroxylases; C585496:CYP1B1 protein, human; D065633:Cytochrome P-450 CYP1B1; D009245:NADH Dehydrogenase