Objectives-NADH cytochrome b 5 reductase (b5R) and cytochrome b 5 (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 semi-quantified 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 (V max /K m ) 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 while novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low frequency cSNPs only appear to 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.

ABSTRACT: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 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.

Glutathione-S-transferase enzymes (GSTs) play an important role in the detoxification of environmental carcinogens. Defective GST genotypes are over-represented in human cancers; in particular, low activity GSTT1 genotypes are risk factors for non-Hodgkin lymphoma. We hypothesized that defective GSTT1 genotypes would be associated with lymphoma risk in dogs. To address this, we resequenced the exons, splice junctions, and 3'-UTR of canine GSTT1 in dogs with lymphoma (n = 93) and age-matched unaffected dogs (n = 86). Of 27 canine GSTT1 variants identified, the I2+28 G>A was significantly associated with lymphoma [odds ratio (OR) 6.26, 95% confidence interval (CI), 1.77-22.2], with the AA genotype found in 18.3% of affected dogs but only 3.5% of controls (P = 0.002). This intronic variant was predicted to perturb GSTT1 mRNA splicing, and may increase lymphoma risk by impairing detoxification of environmental chemicals. Confirmation of this finding in a larger population of dogs may support the inclusion of GSTT1 genotyping in epidemiologic studies of canine lymphoma risk.

Background Delayed hypersensitivity (HS) reactions to potentiated sulfonamide antimicrobials occur in both dogs and humans, and involve an intermediate hydroxylamine metabolite that is detoxified by cytochrome b5 and NADH cytochrome b5 reductase. Hypothesis/Objectives We hypothesized that polymorphisms in the genes (CYB5A and CYB5R3) encoding these 2 enzymes would be associated with risk of sulfonamide HS in dogs. Animals A total of 18 dogs with delayed HS to potentiated sulfonamide antimicrobials and 16 dogs that tolerated (TOL) a therapeutic course of these drugs without adverse effect. Methods CYB5A and CYB5R3 were sequenced from canine liver, and the promoter, exons, and 3′ untranslated regions of both genes were resequenced from genomic DNA obtained from all dogs. Results Multiple polymorphisms were found in both genes. When controlled for multiple comparisons, the 729GG variant in CYB5R3 was significantly overrepresented in dogs with sulfonamide HS (78% of dogs), compared to TOL dogs (31%; P = .003). Conclusions and Clinical Importance The CYB5R3 729GG variant may contribute to the risk of sulfonamide HS in dogs. Functional characterization of this polymorphism, as well as genotyping in a larger number of HS and TOL dogs, is warranted.

Objective To determine whether polymorphisms in the sulfonamide detoxification genes, CYB5A (encoding cytochrome b5), CYB5R3 (encoding cytochrome b5 reductase), or NAT2 (encoding N-acetyltransferase 2) were over-represented in patients with delayed sulfonamide drug hypersensitivity, compared to control patients that tolerated a therapeutic course of trimethoprim-sulfamethoxazole without adverse event. Methods DNA from 99 non-immunocompromised patients with sulfonamide hypersensitivity that were identified from the Personalized Medicine Research Project at the Marshfield Clinic, and from 99 age-, race-, and gender-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 (less than 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; DRESS) compared to tolerant patients. Conclusions We found no evidence for a substantial involvement of these 9 CYB5A or CYB5R3 polymorphisms in sulfonamide HS risk, although minor effects cannot be completely ruled out. Despite careful medical record review and full re-sequencing of the NAT2 coding region, we found no association of NAT2 coding alleles with sulfonamide hypersensitivity (predominantly cutaneous eruptions) in this adult Caucasian population.

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.

ObjectivesGlutathione‐S‐transferases (GSTs) detoxify reactive xenobiotics, and defective GST gene polymorphisms increase cancer risk in humans. A low activity GST‐theta variant was previously found in research beagles. The purpose of our study was to determine the molecular basis for this phenotype and its allele frequency in pet dogs.MethodsBanked livers from 45 dogs of various breeds were screened for low GST‐theta activity by the substrate 1,2‐dichloro‐4‐nitrobenzene (DCNB), and were genotyped for variants in a novel canine GST gene, GSTT5. Whole‐genome sequences from 266 dogs were genotyped at one discovered variant GSTT5 locus.ResultsCanine livers ranged 190‐fold in GST‐theta activities, and a GSTT5 exon coding variant 385_390delGACCAG (Asp129_Gln130del) was significantly associated with low activity (P < 0.0001) and a marked decrease in hepatic protein expression (P = 0.0026). Recombinant expression of variant GSTT5 led to a 92% decrease in V max for DCNB (P = 0.0095). The minor allele frequency (MAF) for 385_390delGACCAG was 0.144 in 45 dog livers, but was significantly higher in beagles (0.444) versus nonbeagles (0.007; P = 0.0004). The homozygous genotype was significantly over‐represented in Pembroke Welsh corgis (P < 0.0001) based on available whole‐genome sequence data.ConclusionsAn Asp129_Gln130del variant in canine GSTT5 is responsible for marked loss of GST‐theta enzyme activity. This variant is significantly over‐represented in purpose‐bred laboratory beagles and in Pembroke Welsh corgis. Additional work will determine the prevalence of this variant among other purebred dogs, and will establish the substrate range of this polymorphic canine enzyme with respect to common environmental carcinogens.

Purpose Cytochrome b5 (encoded by CYB5A) and NADH cytochrome b5 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 (1946 cases and 1747 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 to controls (0.0169, P =0.039); this was attributable to a higher MAF in AfrAm cases (0.0611) compared to 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 to 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 to Caucs. Conclusions CYB5R3 I1M+6 C>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.