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

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.

Novel CYP1B1 and Known PAX6 Mutations in Anterior Segment Dysgenesis (ASD)

“…However, the common CYP1B1 SNP haplotypes themselves have no strong influence on enzyme activity. [29][30][31] The most likely explanation for these patients is that the second mutation has not yet been identified.…”

Correlations of Genotype with Phenotype in Indian Patients with Primary Congenital Glaucoma

“…1,8 It has been shown that this mutation significantly reduces the enzyme's metabolism. 9 P193 and E229 amino acid (aa) residues are also conserved among various members of the cytochrome P450 superfamily. 10 Mutational analyses of CYP1B1 coding exons revealed homozygous mutations in 30 of 43 Indian patients described in this study.…”

“…Those mutations at the N-terminus hinge region or C terminus CCSs are expected to interfere with fundamental properties of the cytochrome P450 molecule, such as proper folding, heme binding, and formation of stable hemoprotein complex, substrate accommodation, and interaction with the redox partner, and to decrease significantly the enzyme's metabolism. 1,8,9 Frameshift mutations causing premature stop codons in the open reading frames would result in functional null alleles. 1,10 Several CYP1B1 mutations would cause conformational changes in the DNA which in turn affect the structure function relationship of CYP1B1.…”

Antenatal care and perinatal outcomes in Kwale district, Kenya

“…"The antenatal period clearly presents opportunities for reaching pregnant women with a number of interventions that may be vital to their health and well-being and that of their infants" [1]. The putative benefits of antenatal care (ANC) to babies include increased growth, reduced risk of infection and increased survival [2].…”

“…Some elements of the ANC package (tetanus toxoid, screening for pre-eclampsia, screening and treatment of asymptomatic bacteriuria and syphilis) have been shown to be cost-effective in a Sub-Saharan African context [3]. Although it cannot be claimed that ANC is the solution to high maternal and perinatal mortality in the developing world (since "few life-threatening complications can be prevented antenatally" [1]) ensuring the provision of ANC may help progress to the Millennium Development Goals for maternal and child mortality [4]. …”