Polymorphisms in the carcinogen detoxification genes CYB5A and CYB5R3 and breast cancer risk in African American women.

PURPOSE: Cytochrome b 5 (encoded by CYB5A) and NADH cytochrome b 5 reductase (encoded by CYB5R3) detoxify aromatic and heterocyclic amine mammary carcinogens found in cigarette smoke. We hypothesized that CYB5A and CYB5R3 polymorphisms would be associated with breast cancer risk in women. METHODS: We characterized the prevalence of 18 CYB5A and CYB5R3 variants in genomic DNA from African American (AfrAm) and Caucasian (Cauc) women from the Carolina Breast Cancer Study population (1,946 cases and 1,747 controls) and determined their associations with breast cancer risk, with effect modification by smoking. RESULTS: A CYB5R3 variant, I1M+6T (rs8190370), was significantly more common in breast cancer cases (MAF 0.0238) compared with controls (0.0169, p = 0.039); this was attributable to a higher MAF in AfrAm cases (0.0611) compared with AfrAm controls (0.0441, p = 0.046; adjusted OR 1.41, CI 0.98-2.04; p = 0.062). When smoking was considered, I1M+6T was more strongly associated with breast cancer risk in AfrAm smokers (adjusted OR 2.10, 1.08-4.07; p = 0.028) compared with never smokers (OR = 1.21; 0.77-1.88; p for interaction = 0.176). I1M+6T and three additional CYB5R3 variants, -251T, I8-1676C, and *392C, as well as two CYB5A variants, 13G and I2-992T, were significantly more common in AfrAms compared with Caucs. CONCLUSIONS: CYB5R3 I1M+6C>T should be considered in future molecular epidemiologic studies of breast cancer risk in AfrAms. Further, variants in CYB5A and CYB5R3 should be considered in the evaluation of other tumors in AfrAms that are associated with aromatic and heterocyclic amine exposures, to include prostate, bladder, and colon cancers.

Resequencing of the TMF-1 (TATA Element Modulatory Factor) regulated protein (TRNP1) gene in domestic and wild canids.

BACKGROUND: Cortical folding is related to the functional organization of the brain. The TMF-1 regulated protein (TRNP1) regulates the expansion and folding of the mammalian cerebral cortex, a process that may have been accelerated by the domestication of dogs. The objectives of this study were to sequence the TRNP1 gene in dogs and related canid species, provide evidence of its expression in dog brain and compare the genetic variation within dogs and across the Canidae. The gene was located in silico to dog chromosome 2. The sequence was experimentally confirmed by amplifying and sequencing the TRNP1 exonic and promoter regions in 72 canids (36 purebred dogs, 20 Gy wolves and wolf-dog hybrids, 10 coyotes, 5 red foxes and 1 Gy fox). RESULTS: A partial TRNP1 transcript was isolated from several regions in the dog brain. Thirty genetic polymorphisms were found in the Canis sp. with 17 common to both dogs and wolves, and only one unique to dogs. Seven polymorphisms were observed only in coyotes. An additional 9 variants were seen in red foxes. Dogs were the least genetically diverse. Several polymorphisms in the promoter and 3'untranslated region were predicted to alter TRNP1 function by interfering with the binding of transcriptional repressors and miRNAs expressed in neural precursors. A c.259_264 deletion variant that encodes a polyalanine expansion was polymorphic in all species studied except for dogs. A stretch of 15 nucleotides that is found in other mammalian sequences (corresponding to 5 amino acids located between Pro58 and Ala59 in the putative dog protein) was absent from the TRNP1 sequences of all 5 canid species sequenced. Both of these aforementioned coding sequence variations were predicted to affect the formation of alpha helices in the disordered region of the TRNP1 protein. CONCLUSIONS: Potentially functionally important polymorphisms in the TRNP1 gene are found within and across various Canis species as well as the red fox, and unique differences in protein structure have evolved and been conserved in the Canidae compared to all other mammalian species.

The folded shape of mammalian brains allows the cerebral cortex and other structures to attain a large surface area to fit into the skull. Over the past several thousand years, the domestication of dogs has led to increased folding as well as a great variety of skull size and shape amongst various breeds. Cortical folding, also known as gyrification, is regulated by numerous genes, including one that encodes the TMF-1 regulated protein (TRNP1). The TRNP1 protein in the brain affects the development of specialized cells in the brain that are involved in gyrification. It is not known whether a functionally distinct TRNP1 protein in dogs is responsible for the increased folding. Variations in the DNA sequence of the gene that encodes TRNP1 may be responsible for these dramatic changes in brain structure in dogs. This study sought to discover the differences in the TRNP1 DNA sequence in seventy-two canids, represented by thirty-six dogs of various breeds, twenty gray wolves and wolf-dog hybrids, ten coyotes, five red foxes and one gray fox.After finding evidence of the expression of this gene in dog brain, we located thirty genetic changes or variants in the canids, with seventeen common to both dogs and wolves, and only one unique to dogs. Another seven of these genetic variants were observed only in coyotes. An additional nine variants were seen in red foxes. Dogs were the least genetically diverse species, an expected result of the inbreeding that characterizes domestication. Several of these changes may affect the function of the TRNP1 gene by affecting the binding of other biomolecules to regions in the DNA which regulate this gene. This study also found two other changes, one only found in dogs, and the other one only found in canids (compared to all other mammalian TRNP1 proteins) may change the length and three-dimensional structure and hence the function of the TRNP1 protein. This study concluded that numerous, potentially functionally significant dissimilarities in the TRNP1 gene exist between dogs and their wild relatives, as well as between canid and all other mammalian species.

Evaluation of polymorphisms in the sulfonamide detoxification genes NAT2, CYB5A, and CYB5R3 in patients with sulfonamide hypersensitivity.

OBJECTIVE: To determine whether polymorphisms in the sulfonamide detoxification genes, CYB5A (encoding cytochrome b(5)), CYB5R3 (encoding cytochrome b(5) reductase), or NAT2 (encoding N-acetyltransferase 2) were over-represented in patients with delayed sulfonamide drug hypersensitivity, compared with control patients who tolerated a therapeutic course of trimethoprim-sulfamethoxazole without adverse event. METHODS: DNA from 99 nonimmunocompromised patients with sulfonamide hypersensitivity who were identified from the Personalized Medicine Research Project at the Marshfield Clinic, and from 99 age-matched, race-matched, and sex-matched drug-tolerant controls, were genotyped for four CYB5A and five CYB5R3 polymorphisms, and for all coding NAT2 SNPs. RESULTS: CYB5A and CYB5R3 SNPs were found at low allele frequencies (<3-4%), which did not differ between hypersensitive and tolerant patients. NAT2 allele and haplotype frequencies, as well as inferred NAT2 phenotypes, also did not differ between groups (60 vs. 59% slow acetylators). Finally, no difference in NAT2 status was found in a subset of patients with more severe hypersensitivity signs (drug reaction with eosinophilia and systemic symptoms) compared with tolerant patients. CONCLUSION: We found no evidence of a substantial involvement of these nine CYB5A or CYB5R3 polymorphisms in sulfonamide hypersensitivity risk, although minor effects cannot be completely ruled out. Despite careful medical record review and full resequencing of the NAT2 coding region, we found no association of NAT2 coding alleles with sulfonamide hypersensitivity (predominantly cutaneous eruptions) in this adult Caucasian population.

Evaluation of sulfonamide detoxification pathways in haematologic malignancy patients prior to intermittent trimethoprim-sulfamethoxazole prophylaxis.

AIMS: Patients with haematologic malignancies have a reportedly high incidence of sulfamethoxazole (SMX) hypersensitivity. The objective of this study was to determine whether deficiencies in sulfonamide detoxification pathways, to include glutathione (GSH) and ascorbate (AA), and cytochrome b(5) (b5) and cytochrome b(5) reductase (b5R), were prevalent in these patients. A secondary pilot objective was to determine whether the incidence of drug hypersensitivity following intermittent trimethoprim-SMX (TMP-SMX) prophylaxis approached that reported for high dose daily regimens. METHODS: Forty adult patients with haematologic malignancies (HM) and 35 healthy adults were studied; an additional 13 HM patients taking ascorbate supplements (HM-AA) were also evaluated. Twenty-two of 40 HM patients were prescribed and were compliant with TMP-SMX 960 mg three to four times weekly. RESULTS: There were no significant differences between HM and healthy groups in plasma AA (median 37.2 µm vs. 33.9 µm) or red blood cell GSH (1.9 mmvs. 1.8 mm). However, plasma AA was correlated significantly with leucocyte b5/b5R reduction (r= 0.39, P= 0.002). Deficient b5/b5R activities were not found in HM patients. In fact, patients with chronic lymphocytic leukaemia or myeloma had significantly higher median activities (80.7 µmol mg(-1) min(-1)) than controls (18.9 µmol mg(-1) min(-1), P= 0.008). After 3-4 weeks of treatment, no patients developed SMX-specific T cells and only one patient developed rash. CONCLUSIONS: Deficiencies of blood antioxidants and b5/b5R reduction were not found in this population with haematologic malignancies, and the development of skin rash and drug-specific T cells appeared to be uncommon with intermittent TMP-SMX prophylaxis.

Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction.

OBJECTIVES: NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA. METHODS: Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semiquantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced. RESULTS: Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06-1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (P<0.0001, r=0.42; P=0.01, r=0.23, respectively), and expression of both proteins correlated with one another (P<0.0001; r=0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency. CONCLUSION: These studies indicate that although novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low-frequency cSNPs seem to only minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.

Combined ascorbate and glutathione deficiency leads to decreased cytochrome b5 expression and impaired reduction of sulfamethoxazole hydroxylamine.

Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T-cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b (5) (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity toward SMX-NO and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO-related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue-drug adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b (5), as well as significant decreases in SMX-HA reduction in SMX-NO-treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in patients with AIDS with antioxidant depletion.

Glucuronidation of polychlorinated biphenylols and UDP-glucuronic acid concentrations in channel catfish liver and intestine.

Polychlorinated biphenylols (OH-PCBs) are potentially toxic polychlorinated biphenyl metabolites that can be eliminated by glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs). OH-PCBs with a 3,5-dichloro-4-hydroxy substitution pattern have been detected in blood from humans and wildlife, suggesting slow elimination. In this study we assessed the glucuronidation of 4-OH-PCBs with zero, one, or two chlorine atoms flanking the 4-hydroxyl group and zero to four chlorine atoms in the aphenolic ring in microsomes from channel catfish liver and proximal intestine. Product formation was quantitated with [(14)C]UDP-glucuronic acid (UDPGA). Physiological concentrations of UDPGA were measured in preparations of liver and intestine. When the OH-PCB concentrations were varied in the presence of saturating UDPGA concentrations, glucuronidation V(max) values were higher in hepatic than in intestinal microsomes (0.40-3.4 and 0.12-0.78 nmol/min/mg of protein, respectively), whereas the K(m) values were generally lower for intestine (0.042-0.47 mM) than for liver (0.11-1.64 mM). In both tissues V(max) values with 3,5-dichloro-4-OH-PCBs were lower than with the corresponding 3-chloro-4-OH-PCBs. Varying the UDPGA concentrations in the presence of saturating concentrations of OH-PCB showed that the K(m) for UDPGA was lower in intestine (27 microM) than in liver (690 microM). The measured concentration of UDPGA in catfish liver (246-377 nmol/g) was lower than the K(m) for UDPGA, suggesting that in vivo rates of glucuronidation may be suboptimal, whereas in intestine the measured UDPGA concentration (71-258 nmol/g) was higher than the K(m) for UDPGA. Although liver has a greater glucuronidation capacity than proximal intestine, the properties of intestinal UGTs in channel catfish enable them to efficiently glucuronidate low concentrations of OH-PCBs.

Effects of Food Natural Products on the Biotransformation of PCBs.

Many food products, particularly fruits and vegetables, contain natural products that affect biotransformation enzymes. These may be expected to affect the rate of biotransformation of PCBs that are metabolized by the affected enzymes. The first step in PCB metabolism is cytochrome P450-dependent monooxygenation. Natural products present in cruciferous vegetables have been shown to selectively up-regulate CYP1A1 and CYP1A2 isozymes on chronic ingestion, and may lead to increased metabolism of those PCB congeners that are substrates for the induced P450s. On the other hand, several natural products selectively inhibit monooxygenation, especially in the intestine, and may lead to increased bioavailability and reduced metabolism of dietary PCBs. Food natural products are known to affect phase II pathways important in the detoxication of hydroxylated PCBs, namely UDP-glucuronosyltransferase and PAPS-sulfotransferase. Continual dietary exposure to chrysin and quercetin, found in fruits and vegetables, induces UGT1A1 and may reduce exposure to hydroxylated PCBs through increased glucuronidation. These and other natural products are also inhibitors of glucuronidation and sulfonation, potentially leading to transient decreases in the elimination of hydroxylated PCBs. In summary, the expected effects of food natural products on PCB biotransformation are complex and may be biphasic, with initial inhibition followed by enhanced biotransformation through monooxygenation and conjugation pathways.

Glucuronidation in the polar bear (Ursus maritimus).

Polar bears bioaccumulate lipophilic pollutants, including polychlorinated biphenyls (PCBs), into their bodies from their exclusive diet of marine organisms. Hydroxylated PCB metabolites (OH-PCBs) have been found in plasma, presumably due to CYP-dependent biotransformation of PCBs in liver. Little is known about the phase 2 metabolism of hydroxylated xenobiotics in polar bears. The objective of this study was to examine UDP-glucuronosyltransferase (UGT) activity with OH-PCBs and a hydroxylated polycyclic aromatic hydrocarbon, 3-hydroxy-benzo(a)pyrene (3-OH-BaP), in polar bear liver. Samples of frozen polar bear liver were used to prepare microsomes. UGT activity with 3-OH-BaP in Brij-treated microsomes, measured by a fluorescence assay, was readily measurable with protein concentrations in assay tubes of up to 10 g/ml, but dropped off very sharply at higher protein concentrations. The apparent Km for 3-OH-BaP was 1.71 +/- 0.04 microM, and Vmax 1.26 +/- 0.16 nmol/min/mg protein (mean +/- SD, n=3). UGT activities with a model tetrachloro-OH-PCB (4'-OH-CB72) and a model hexachloro-OH-PCB (4'-OH-CB159) were assayed with [14-C]-UDPGA and separation of the [14-C]-glucuronide by ion-pair extraction and thin-layer chromatography. [14-C]-glucuronide conjugates were readily formed by polar bear liver microsomes in the absence of added substrate, apparently from contaminants present in liver. This phenomenon was not observed using hepatic microsomes from laboratory-held catfish. Glucuronidation efficiency was much higher with 4'-OH-CB72 (Km 7.3 microM; Vmax 1.55 nmol/min/mg) than 4'-OH-CB159 (Km 16.1 microM; Vmax 0.46 nmol/min/mg). The identities of the aglycones present in polar bear liver are not known, but could include OH-PCBs or hydroxylated metabolites of other persistent organic pollutants. This study demonstrates that UGT with high activity for 3-OH-BaP and other substrates is present in polar bear liver.

Individual variability in the detoxification of carcinogenic arylhydroxylamines in human breast.

Cytochrome b(5) (b5) and NADH cytochrome b(5) reductase (b5R) detoxify reactive hydroxylamine (NHOH) metabolites of known arylamine and heterocyclic amine mammary carcinogens. The aim of this study was to determine whether NHOH reduction for the prototypic arylamine 4-aminobiphenyl (4-ABP) was present in human breast and to determine whether variability in activity was associated with single nucleotide polymorphisms (SNPs) in the coding, promoter, and 3'untranslated region (UTR) regions of the genes encoding b5 (CYB5A) and b5R (CYB5R3). 4-ABP-NHOH reduction was readily detected in pooled human breast microsomes, with a K(m) (280µM) similar to that found with recombinant b5 and b5R, and a V(max) of 1.12 ± 0.19 nmol/min/mg protein 4-ABP-NHOH reduction varied 75-fold across 70 individual breast samples and correlated significantly with both b5 (80-fold variability) and b5R (14-fold) immunoreactive protein. In addition, wide variability in b5 protein expression was significantly associated with variability in CYB5A transcript levels, with a trend toward the same association between b5R and CYB5R3. Although a sample with a novel coding SNP in CYB5A, His22Arg, was found with low reduction and b5 expression, no other SNPs in either gene were associated with outlier activity or protein expression. We conclude that b5 and b5R catalyze the reduction of 4-ABP-NHOH in breast tissue, with very low activity, protein, and messenger RNA expression in some samples, which cannot be attributed to promoter, coding, or 3'UTR SNPs. Further studies are underway to characterize the transcriptional regulation of CYB5A and CYB5R3 and begin to understand the mechanisms of individual variability in this detoxification pathway.

Sulfonation of environmental chemicals and their metabolites in the polar bear (Ursus maritimus).

Although its habitat comprises mostly remote regions of the Arctic, the polar bear is subject to bioaccumulation of persistent environmental pollutants. Along with their phase I metabolites, they are potential substrates for detoxification via sulfonation and glucuronidation. The capability of polar bear liver to sulfonate a structurally diverse group of environmental chemicals, that is, 3-hydroxybenzo[a]pyrene (3-OH-B[a]P), triclosan, 4'-hydroxy-3,3',4,5'-tetrachlorobiphenyl (4'OH-PCB79), 4'-hydroxy-2,3,3',4,5,5'-hexachlorobiphenyl (4'-OH-PCB159), 4'-hydroxy-2,3,3',5,5',6-hexachlorobiphenyl (4'-OH-PCB165), the methoxychlor metabolite 2-(4-methoxyphenyl)-2-(4-hydroxyphenyl)-1,1,1-trichloroethane (OHMXC), tris(4-chlorophenyl)-methanol (TCPM), and pentachlorophenol (PCP) was investigated. The glucuronidation of 3-OH-B[a]P was also studied. Enzyme activity was assayed by incubation of liver cytosol or microsomes derived from three adult male polar bears with 3'-phosphoadenosine-5'-phosphosulfate or uridine 5'-diphosphoglucuronic acid and substrate, followed by fluorometric or radiochemical thin-layer chromatographic analysis. The efficiency of sulfonation decreased in the order 3-OH-B[a]P >>> triclosan >> 4'-OH-PCB79 > OHMXC > 4'-OH-PCB165 > TCPM > 4'-OH-PCB159 > PCP, all of which produced detectable sulfate conjugates. The 3-OH-B[a]P substrate was readily sulfonated and glucuronidated (apparent K(m) 0.41, 1.4 microM, and apparent V(max) 0.50, 3.00 nmol/min/mg, respectively). UDP-glucuronic acid kinetics suggested the presence of multiple enzymes glucuronidating 3-OH-B[a]P. Substrate inhibition was observed for the sulfonation of 3-OH-B[a]P and 4'OH-PCB79 (K(i) 1.0 and 217 microM, respectively). Triclosan was the most rapidly sulfated (apparent V(max) 1008 pmol/min/mg) of the substrates tested. Since sulfonation of an acyclic tertiary alcoholic group, as in TCPM, has not previously been reported, we also examined TCPM conjugation in humans and catfish, both of which formed TCPM-sulfate. The hexachlorinated polychlorinated biphenylols, TCPM, and PCP were poor substrates for sulfonation, suggesting that this may be one reason why these substances and structurally similar xenobiotics persist in polar bears.