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Benzidine undergoes N-acetylation and following CYP1A2-catalyzed N-hydroxylation undergoes O-acetylation catalyzed by N-acetyltransferase 1 (NAT1). Benzidine exposure is associated with urinary bladder cancer but the effect of NAT1 genetic polymorphism on individual risk remains unclear. We used Chinese hamster ovary (CHO) cells transfected with human CYP1A2 and NAT1*4 allele (reference) or NAT1*14B (variant) to investigate the effects of dose and NAT1 polymorphism on benzidine metabolism and genotoxicity. Rates of benzidine N-acetylation in vitro were higher in CHO cells transfected with NAT1*4 compared to NAT1*14B. CHO cells transfected with NAT1*14B exhibited greater N-acetylation rates in situ than cells transfected with NAT1*4 at low doses of benzidine expected with environmental exposures but not at higher doses. NAT1*14B exhibited over tenfold lower apparent KM which resulted in higher intrinsic clearance for benzidine N-acetylation compared to CHO cells transfected with NAT1*4. Benzidine-induced hypoxanthine phosphoribosyl transferase (HPRT) mutations were higher in CHO cells transfected with NAT1*14B than with NAT1*4 (p < 0.001). Benzidine caused concentration-dependent increase in γ-H2AX signal (indicative of DNA double-strand breaks) in CHO cells transfected with NAT1*4 or NAT1*14B. CHO cells transfected with NAT1*14B exhibited significantly higher level of DNA damage than with NAT1*4 (p < 0.0001). Benzidine-induced ROS did not differ significantly (p > 0.05) between CHO cells transfected with NAT1*4 or NAT1*14B except at 50 μM. Levels of benzidine-induced DNA damage and reactive oxygen species (ROS) showed strong dose-dependent correlation. Our findings support human studies associating NAT1*14B with increased incidence or severity of urinary bladder cancer in workers exposed to benzidine. [ABSTRACT FROM AUTHOR]

The rabbit was the initial animal model to investigate the acetylation polymorphism expressed in humans. Use of the rabbit model is compromised by lack of a rapid non-invasive method for determining acetylator phenotype. Slow acetylator phenotype in the rabbit results from deletion of the N-acetyltransferase 2 ( NAT2) gene. A relatively quick and non-invasive method for identifying the gene deletion was developed and acetylator phenotypes confirmed by measurement of N- and O-acetyltransferase activities in hepatic cytosols. Rabbit liver cytosols catalyzed the N-acetylation of sulfamethazine ( p = 0.0014), benzidine ( p = 0.0257), 4-aminobiphenyl ( p = 0.0012), and the O-acetylation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP; p = 0.002) at rates significantly higher in rabbits possessing NAT2 gene than rabbits with NAT2 gene deleted. In contrast, hepatic cytosols catalyzed the N-acetylation of p-aminobenzoic acid (an N-acetyltransferase 1 selective substrate) at rates that did not differ significantly ( p > 0.05) between rabbits positive and negative for NAT2. The new NAT2 genotyping method facilitates use of the rabbit model to investigate the role of acetylator polymorphism in the metabolism of aromatic and heterocyclic amine drugs and carcinogens. [ABSTRACT FROM AUTHOR]

Recently, rs11892031[A] has been identified in a genome-wide association study (GWAS) to confer increased risk of urinary bladder cancer (UBC). To confirm this association and additionally study a possible relevance of exposure to urinary bladder carcinogens, we investigated the IfADo UBC study group, consisting of eight case-control series from different regions including 1,805 cases and 2,141 controls. This analysis was supplemented by a meta-analysis of all published data, including 13,395 cases and 54,876 controls. Rs11892031 A/A was significantly associated with UBC risk in the IfADo case-control series adjusted to cigarette smoking, gender, age and ethnicity (OR = 1.18; 95% CI = 1.02-1.37; P = 0.026). In the meta-analysis, a convincing association with UBC risk was obtained (OR = 1.19; 95% Cl = 1.12-1.26; P < 0.0001). Interestingly, the highest odds ratios were obtained for individual case-control series with a high degree of occupational exposure to polycyclic aromatic hydrocarbons and aromatic amines: cases with suspected occupational UBC (OR = 1.41) and cases from the highly industrialized Ruhr area (OR = 1.98) compared with Ruhr area controls (all combined OR = 1.46). Odds ratios were lower for study groups with no or a lower degree of occupational exposure to bladder carcinogens, such as the Hungary (OR = 1.02) or the ongoing West German case-control series (OR = 1.06). However, the possible association of rs11892031[A] with exposure to bladder carcinogens still should be interpreted with caution, because in contrast to the differences between the individual study groups, interview-based data on occupational exposure were not significantly associated with rs11892031. In conclusion, the association of rs11892031[A] with UBC risk could be confirmed in independent study groups. [ABSTRACT FROM AUTHOR]

The distribution of the polymorphic alleles of the genes coding for glutathione S-transferases (GSTs) M1 and T1 was compared with the results of cytological grading of exfoliated urothelial cells (Pap test) in a non-diseased high-risk group of workers formerly exposed to benzidine in the Shanghai dyestuff industry (n=317). All subjects were genotyped for GSTT1 and M1 gene polymorphism by allele-specific PCR. Individuals were stratified according to their job and duration of exposure. A subgroup of 78 individuals with cytological gradings of grade III or higher in the Pap test showed a significant under-representation of the combination of GSTT1 0/0 and M1 0/0 genotypes compared with 238 subjects with a cytological classification lower than grade III (OR 0.55, 95% CI 0.31–0.98, P=0.04). These results suggest that neither the GSTM1 0/0 or GSTT1 0/0 genotype alone nor their combination had a clear association with cytopathological changes in exfoliated urothelial cells from individuals previously exposed to benzidine in Shanghai. This contradicts the results of studies indicating that the GSTM1 0/0 genotype is associated with an increased risk for bladder cancer in the general population, mostly outside China. [ABSTRACT FROM AUTHOR]

Covalent binding of benzidine and some congeners to hemoglobin was studied in female Wistar rats after oral administration. Hemoglobin adducts were hydrolyzed under alkaline conditions, and the arylamines extracted and analysed by HPLC with electrochemical detector. With benzidine, three cleavage products were observed, the major component being monoacetylbenzidine. This indicates that 4-nitroso-4′-N-acetylaminobiphenyl is the major reactive metabolite in erythrocytes. In addition benzidine and 4-aminobiphenyl were identified. The latter indicates a hitherto unknown metabolic pathway of benzidine. With 3,3′-dichlorobenzidine-dihydrochloride, 3,3′-dimethoxybenzidine and 3,3′-dimethylbenzidine two cleavage products were observed, the parent diamines being present in excess to or in amounts comparable to the monoacetyl derivative. With 3,3′,5,5′-tetramethylbenzidine a hemoglobin adduct could not be found. When the azo dye direct red 28 was administered to the animals, the three cleavage products typical for benzidine were found, indicating that benzidine became bioavailable after reductive cleavage of the azo compound. In this case the fraction of 4-aminobiphenyl was greater than after benzidine. It is proposed to use the analysis of hemoglobin adducts in human blood to control the exposure of individuals to these carcinogenic chemicals in the course of biochemical effect monitoring. [ABSTRACT FROM AUTHOR]

The effects of ethanol-feeding to rats, over a 6-week period, on the activation of genotoxic compounds of different chemical classes, requiring metabolic conversion to exert their mutagenic activity, were studied in isolated rat hepatocytes. The influence of such treatment on cytochrome P-450 content and N-acetylation in isolated hepatocytes was also investigated. Benzidine (BZ), dimethylnitrosamine (DMN), diethylnitrosamine (DEN), isoniazid (INH) and cyclophosphamide (CP) were more effectively activated to products mutagenic towards Salmonella typhimurium by hepatocytes from ethanol-pretreated rats than by hepatocytes from controls. The mutagenic potency of 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) was not influenced by ethanol pretreatment. Ethanol consumption was found to be associated with increased cytochrome P-450 content and enhanced N-acetylation in the isolated hepatocytes. Our results support the hypothesis that an alteration of the hepatic drug-metabolizing system may be responsible for the ethanol-induced increase in susceptibility to certain genotoxic compounds. [ABSTRACT FROM AUTHOR]

Environmental dyes and their derivatives, some of which are genotoxic, must be transported within the body to the tissues which they affect. One mechanism for this can be observed directly by crossed immunoelectrophoresis (X-IEP). Binding of these chemicals to certain serum proteins changes electrophoretic and immunoprecipitation morphology in X-IEP patterns. This is demonstrated here for four azo dyes derived from benzidine, 3,3′-dimethylbenzidine, and 3,3′-dimethoxybenzidine, and their parent aromatic amines. Direct Red 2 (a 3,3′-dimethylbenzidine-based dye), Direct Blue 15 (a 3,3′-dimethoxybenzidine-based dye), Direct Black 38 (a benzidinebased dye), and Evans Blue (a 3,3′-dimethylbenzidine-based dye) all bound to albumin, α-lipoprotein, β-lipoprotein, and hemopexin. Direct Red 2 only slightly affected the mobilities of these proteins. Direct Blue 15 bound also to prealbumin and α-antichymotrypsin, and degraded C globulin. Direct Black 38 and Evans Blue bound to numerous additional proteins. Evans Blue bound variably to proteins of sera from different individuals, suggesting that there are individual differences in serum protein binding capabilities for these chemicals. Of the three derivatives of the benzidine dyes, only 3,3′-dimethylbenzidine caused changes in X-IEP patterns, indicating its binding to the serum proteins. This chemical differentially affected sub-populations of α-lipoprotein, either by altering its electrophoretic mobility or inhibiting its recognition by antibodies. Autoradiographic analyses demonstrated the binding of benzidine and 3,3′-dimethylbenzidine to both α- and β-lipoproteins. [ABSTRACT FROM AUTHOR]