Metabolic, behavioural, and psychosocial risk factors and cardiovascular disease in women compared with men in 21 high-income, middle-income, and low-income countries: an analysis of the PURE study.

BACKGROUND: There is a paucity of data on the prevalence of risk factors and their associations with incident cardiovascular disease in women compared with men, especially from low-income and middle-income countries. METHODS: In the Prospective Urban Rural Epidemiological (PURE) study, we enrolled participants from the general population from 21 high-income, middle-income, and low-income countries and followed them up for approximately 10 years. We recorded information on participants' metabolic, behavioural, and psychosocial risk factors. For this analysis, we included participants aged 35-70 years at baseline without a history of cardiovascular disease, with at least one follow-up visit. The primary outcome was a composite of major cardiovascular events (cardiovascular disease deaths, myocardial infarction, stroke, and heart failure). We report the prevalence of each risk factor in women and men, their hazard ratios (HRs), and population-attributable fractions (PAFs) associated with major cardiovascular disease. The PURE study is registered with ClinicalTrials.gov, NCT03225586. FINDINGS: In this analysis, we included 155 724 participants enrolled and followed-up between Jan 5, 2005, and Sept 13, 2021, (90 934 [58·4%] women and 64 790 [41·6%] men), with a median follow-up of 10·1 years (IQR 8·5-12·0). At study entry, the mean age of women was 49·8 years (SD 9·7) compared with 50·8 years (9·8) in men. As of data cutoff (Sept 13, 2021), 4280 major cardiovascular disease events had occurred in women (age-standardised incidence rate of 5·0 events [95% CI 4·9-5·2] per 1000 person-years) and 4911 in men (8·2 [8·0-8·4] per 1000 person-years). Compared with men, women presented with a more favourable cardiovascular risk profile, especially at younger ages. The HRs for metabolic risk factors were similar in women and men, except for non-HDL cholesterol, for which high non-HDL cholesterol was associated with an HR for major cardiovascular disease of 1·11 (95% CI 1·01-1·21) in women and 1·28 (1·19-1·39) in men, with a consistent pattern for higher risk among men than among women with other lipid markers. Symptoms of depression had a HR of 1·09 (0·98-1·21) in women and 1·42 (1·25-1·60) in men. By contrast, consumption of a diet with a PURE score of 4 or lower (score ranges from 0 to 8), was more strongly associated with major cardiovascular disease in women (1·17 [1·08-1·26]) than in men (1·07 [0·99-1·15]). The total PAFs associated with behavioural and psychosocial risk factors were greater in men (15·7%) than in women (8·4%) predominantly due to the larger contribution of smoking to PAFs in men (ie, 1·3% [95% CI 0·5-2·1] in women vs 10·7% [8·8-12·6] in men). INTERPRETATION: Lipid markers and depression are more strongly associated with the risk of cardiovascular disease in men than in women, whereas diet is more strongly associated with the risk of cardiovascular disease in women than in men. The similar associations of other risk factors with cardiovascular disease in women and men emphasise the importance of a similar strategy for the prevention of cardiovascular disease in men and women. FUNDING: Funding sources are listed at the end of the Article.

Metabolism and disposition of bladder carcinogens in rat and guinea pig: possible mechanism of guinea pig resistance to bladder cancer.

The metabolism and disposition of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) were studied in rat and guinea pig. Rat is susceptible whereas guinea pig is resistant to FANFT-induced bladder cancer. Rats and guinea pigs were p.o. administered either 2-[14C]ANFT or 2-[14C]FANFT (100 mg/kg), and 18-h urine and feces were collected. Tissue distribution of radiolabel was determined. In both species, the highest concentrations of radioactivity expressed as nmol/g tissue were observed in the urine and intestines. Urinary metabolites were separated by high-performance liquid chromatography and radioactivity determined by radioanalytical detection. FANFT was not detected in urine from either species under any experimental condition. More ANFT was observed in urine following FANFT than ANFT administration. This deformylation-dependent excretion of FANFT was demonstrated in both species and has been previously described as renal metabolic/excretory coupling. Less ANFT, the carcinogen more proximate than FANFT, is excreted in guinea pigs compared with rats. A unique ANFT metabolite was identified in guinea pig but not rat urine. This metabolite represented 80 and 18% of radioactivity recovered in guinea pig urine following ANFT and FANFT administration, respectively. A metabolite produced by guinea pig liver and kidney microsomes in the presence of uridine-5'-diphosphoglucuronic acid coeluted with this unique metabolite. The urinary metabolite was characterized using hydrolytic enzymes, acid hydrolysis, and mass spectrometry and identified as an ANFT-N-glucuronide. A unique UDP-glucuronosyl-transferase appears to be responsible, at least in part, for the reduced amount of free ANFT excreted by guinea pigs compared with rats. Reduced levels of urinary ANFT observed in guinea pigs may partially explain the resistance of this species to FANFT-induced bladder cancer.

Human N-acetylation of benzidine: role of NAT1 and NAT2.

These studies were designed to assess metabolism of benzidine and N-acetylbenzidine by N-acetyltransferase (NAT) NAT1 and NAT2. Metabolism was assessed using human recombinant NAT1 and NAT2 and human liver slices. For benzidine and N-acetylbenzidine, Km and Vmax values were higher for NAT1 than for NAT2. The clearance ratios (NAT1/NAT2) for benzidine and N-acetylbenzidine were 54 and 535, respectively, suggesting that N-acetylbenzidine is a preferred substrate for NAT1. The much higher NAT1 and NAT2 Km values for N-acetylbenzidine (1380 +/- 90 and 471 +/- 23 microM, respectively) compared to benzidine (254 +/- 38 and 33.3 +/- 1.5 microM, respectively) appear to favor benzidine metabolism over N-acetylbenzidine for low exposures. Determination of these kinetic parameters over a 20-fold range of acetyl-CoA concentrations demonstrated that NAT1 and NAT2 catalyzed N-acetylation of benzidine by a binary ping-pong mechanism. In vitro enzymatic data were correlated to intact liver tissue metabolism using human liver slices. Samples incubated with either [3H]benzidine or [3H]N-acetylbenzidine had a similar ratio of N-acetylated benzidines (N-acetylbenzidine + N',N'-diacetylbenzidine/ benzidine) and produced amounts of N-acetylbenzidine > benzidine > N,N'-diacetylbenzidine. With [3H]benzidine, p-aminobenzoic acid, a NAT1-specific substrate, increased the amount of benzidine and decreased the amount of N-acetylbenzidine produced, resulting in a decreased ratio of acetylated products. This is consistent with benzidine being a NAT1 substrate. N-Acetylation of benzidine or N-acetylbenzidine by human liver slices did not correlate with the NAT2 genotype. However, a higher average acetylation ratio was observed in human liver slices possessing the NAT1*10 compared to the NAT1*4 allele. Thus, a combination of human recombinant NAT and liver slice experiments has demonstrated that benzidine and N-acetylbenzidine are both preferred substrates for NAT1. These results also suggest that NAT1 may exhibit a polymorphic expression in human liver.

Acidic urine pH is associated with elevated levels of free urinary benzidine and N-acetylbenzidine and urothelial cell DNA adducts in exposed workers.

We evaluated the influence of urine pH on the proportion of urinary benzidine (BZ) and N-acetylbenzidine present in the free, unconjugated state and on exfoliated urothelial cell DNA adduct levels in 32 workers exposed to BZ in India. Postworkshift urine pH was inversely correlated with the proportions of BZ (r = -0.78; P < 0.0001) and N-acetylbenzidine (r = -0.67; P < 0.0001) present as free compounds. Furthermore, the average of each subject's pre- and postworkshift urine pH was negatively associated with the predominant urothelial DNA adduct (P = 0.0037, adjusted for urinary BZ and metabolites), which has been shown to cochromatograph with a N-(3'-phosphodeoxyguanosin-8-yl)-N'-acetylbenzidine adduct standard. Controlling for internal dose, individuals with urine pH < 6 had 10-fold higher DNA adduct levels compared to subjects with urine pH > or = 7. As reported previously, polymorphisms in NAT1, NAT2, and GSTM1 had no impact on DNA adduct levels. This is the first study to demonstrate that urine pH has a strong influence on the presence of free urinary aromatic amine compounds and on urothelial cell DNA adduct levels in exposed humans. Because there is evidence that acidic urine has a similar influence on aromatic amines derived from cigarette smoke, urine pH, which is influenced by diet, may be an important susceptibility factor for bladder cancer caused by tobacco in the general population.

The glutathione S-transferase M1 (GSTM1) null genotype and benzidine-associated bladder cancer, urine mutagenicity, and exfoliated urothelial cell DNA adducts.

Multiple studies in the general population have suggested that subjects with the glutathione S-transferase M1 (GSTM1)-null genotype, who lack functional GSTM1, are at higher risk for bladder cancer. To evaluate the impact of the GSTM1-null genotype on bladder cancer caused by occupational exposure to benzidine and to determine its influence on benzidine metabolism, we carried out three complementary investigations: a case-control study of bladder cancer among workers previously exposed to benzidine in China, a cross-sectional study of urothelial cell DNA adducts and urinary mutagenicity in workers currently exposed to benzidine in India, and a laboratory study of the ability of human GSTM1 to conjugate benzidine and its known metabolites in vitro. There was no overall increase in bladder cancer risk for the GSTM1-null genotype among 38 bladder cancer cases and 43 controls (odds ratio, 1.0; 95% confidence interval, 0.4-2.7), although there was some indication that highly exposed workers with the GSTM1-null genotype were at greater risk of bladder cancer compared to similarly exposed workers without this allele. However, the GSTM1 genotype had no impact on urothelial cell DNA adduct and urinary mutagenicity levels in workers currently exposed to benzidine. Furthermore, human GSTM1 did not conjugate benzidine or its metabolites. These results led us to conclude that the GSTM1-null genotype does not have an impact on bladder cancer caused by benzidine, providing a contrast to its association with elevated bladder cancer risk in the general population.

Glucuronide conjugates of 4-aminobiphenyl and its N-hydroxy metabolites. pH stability and synthesis by human and dog liver.

Glucuronide conjugates of arylamines are thought to be important in the carcinogenic process. This study investigated the pH stability and synthesis of glucuronide conjugates of 4-aminobiphenyl and its N-hydroxy metabolites by human and dog liver. Both dog and human liver slices incubated with 0.06 mM [3H]-4-aminobiphenyl produced the N-glucuronide of 4-aminobiphenyl as the major product. After 2 hr of incubation, the N-glucuronide of 4-aminobiphenyl represented 52 and 27% of the total radioactivity recovered by HPLC in dog and human, respectively. When 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, or N-hydroxy-N-acetyl-4-aminobiphenyl was added to human microsomes containing [14C]UDP-glucuronic acid, a new product peak was detected by HPLC. At 0.5 mM, the rate of glucuronidation was N-hydroxy-N-acetyl-4-aminobiphenyl > N-hydroxy-4-aminobiphenyl > 4-aminobiphenyl. The rate of formation of the N-glucuronide of 4-aminobiphenyl was similar to that observed with benzidine and N-acetylbenzidine. The glucuronides of 4-aminobiphenyl and N-hydroxy-4-aminobiphenyl were both acid labile with T1/2 values of 10.5 and 32 min, respectively, at pH 5.5. The glucuronide of N-hydroxy-N-acetyl-4-aminobiphenyl was not acid labile with T1/2 values at pH 5.5 and 7.4 of 55 and 68 min, respectively. The glucuronide of 4-aminobiphenyl was the most acid labile conjugate examined. Thus, the glucuronide of 4-aminobiphenyl is a major product of dog and human liver slice metabolism and likely to play an important role in the carcinogenic process.

Hypochlorous acid-mediated activation of N-acetylbenzidine to form N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine.

Hypochlorous acid (HOCl), a chemically reactive oxidant, is an important component of the inflammatory response and may contribute to carcinogenesis. This study assessed the possible activation of N-acetylbenzidine (ABZ) by HOCI to form a specific DNA adduct, N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine. HOCl was incubated with 0.06 mM 3H-ABZ, and transformation assessed by HPLC. Similar results were observed at pH 5.5 or 7.4. A linear increase in transformation was observed from 0.025 to 0.1 mM HOCl with up to 80% of ABZ changed. Approximately, 2 nmoles of HOCI oxidized 1 nmole of ABZ. N-oxidation products of ABZ metabolism, such as N'-hydroxy-N-acetylbenzidine, were not detected. Oxidation of ABZ was prevented by taurine, DMPO, glutathione, and ascorbic acid, whereas mannitol was without effect. Results are consistent with a radical mechanism. In the presence of 2'-deoxyguanosine 3'-monophosphate (dGp), a new product (dGp-ABZ) was observed. The same adduct was observed with DNA. dGp-ABZ was found to be quite stable (>80% remaining) at 70 degrees C in pH 5.5 (60 min) and 7.4 (240 min). Electrospray mass spectrometry indicated that dGp-ABZ was N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine, and this was confirmed by NMR. 32P-postlabeling in combination with TLC and HPLC determined that the adduct made by either HOCl or prostaglandin H synthase oxidation of ABZ in the presence of dGp or DNA was dGp-ABZ. Thus, HOCI activates ABZ to form dGp-ABZ and may be responsible for the presence of this adduct in peripheral white blood cells from workers exposed to benzidine. Reaction of ABZ with HOCl provides an easy, convenient method for preparing dGp-ABZ.

Benzidine-DNA adduct levels in human peripheral white blood cells significantly correlate with levels in exfoliated urothelial cells.

In a cross-sectional study of 33 workers exposed to benzidine and benzidine dyes and 15 non-exposed controls, we previously reported that exposure status and internal dose of benzidine metabolites were strongly correlated with the levels of specific benzidine-DNA adducts in exfoliated urothelial cells. We also evaluated DNA adduct levels in peripheral white blood cells (WBC) of a subset of 18 exposed workers and 7 controls selected to represent a wide range of adducts in exfoliated urothelial cells. Samples were coded and then DNA was analyzed using 32P-postlabeling, along with n-butanol extraction. One adduct, which co-chromatographed with a synthetic N-(3'-phospho-deoxyguanosin-8-yl)-N'-acetylbenzidine standard, predominated in those samples with adducts present. The median level (range) of this adduct in WBC DNA was 194.4 (3.2-975) RAL x 10(9) in exposed workers and 1.4 (0.1-6.4) in the control subjects (p = 0.0002, Wilcoxon Rank Sum Test). There was a striking correlation between WBC and exfoliated urothelial cell adduct levels (Pearson r = 0.84, p < 0.001) among exposed subjects. In addition, the sum of urinary benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine correlated with the levels of this adduct in both tissues. This is the first study in humans to show a relationship for a specific carcinogen adduct in a surrogate tissue and in urothelial cells, the target for urinary bladder cancer.

Metabolism of aromatic and heterocyclic amine bladder carcinogens: bioanalytical considerations.

Aromatic and heterocyclic amines are environmental chemicals which can cause bladder cancer in man. Because these chemicals cause carcinomas at a site distal to their portals of entry, metabolic processes are involved in initiation of their carcinogenic effects. N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and its deformylated analogue, ANFT, were used as model compounds to assess metabolism. Electrochemical properties of ANFT made liquid chromatography with electrochemical detection a specific and sensitive method for analysis. Peroxidatic metabolism of ANFT by prostaglandin H synthase (PHS) in the presence of N-acetylcysteine resulted in the formation of 2-amino-4-(5-nitro-2-furyl)-5-(N-acetylcystein-S-yl)thiazole (ANFT-MA). This thioether product has an oxidation potential significantly lower than ANFT. Rat urinary excretion of ANFT-MA was significantly decreased with peroxidase inhibitors, 6-n-propyl-2-thiouracil and methimazole. Inhibitors did not alter excretion of ANFT or prostaglandin E2, a PHS product of arachidonic acid metabolism. 1H and 13C-NMR were selected to explore potential structural differences between ANFT and FANFT which might explain preferential PHS metabolism of ANFT. Evidence for a "zwitterion" configuration for ANFT but not FANFT was observed. ANFT in the "zwitterion" configuration would be a better reducing co-substrate. Chemical synthesis and GC-MS fragmentation patterns identified 3-(2,3-dihydro-1-methyl-2-pyrrolyl)pyridine as a peroxidatic product of nicotine metabolism. This peroxidatic product was found in urine from a cigarette smoker in an amount approximately 6% that observed for continine. Thus, a potential rôle for peroxidative metabolism was demonstrated in man.

Mechanism of 3-(glutathion-S-yl)-benzidine formation.

The formation of thioether conjugates is an important mechanism for inactivation of carcinogens. 3-(Glutathion-S-yl)-benzidine (BZ-SG) formation prevents benzidinediimine and peroxidase-mediated benzidine binding to DNA. Benzidinediimine is the two-electron oxidized product of benzidine thought to be the reactive intermediate involved in peroxidase-mediated binding of benzidine to DNA. Diimine interacts with benzidine to form a dimeric complex known as the charge-transfer complex. The latter is in equilibrium with the cation radical. This study evaluated the mechanism by which BZ-SG forms. Benzidinediimine was synthesized and used to study the formation of BZ-SG. With 0.05 mM benzidinediimine, BZ-SG formation was optimum at pH 4.5 and with glutathione at 0.05 to 0.1 mM. By monitoring specific absorption spectra, the reduction of benzidinediimine at pH 4.5 was evaluated. The t1/2 for diimine decay (425 nm) and maximum absorbance of the charge-transfer complex (600 nm) were each at approximately 5 min. Within 10 min, the maximum amount of benzidine had formed from diimine. BZ-SG formation followed the decay of diimine. The relationship between benzidinediimine and benzidine, with respect to BZ-SG formation, was assessed at a fixed concentration of glutathione (0.05 mM) and a fixed total concentration of amine and diimine (0.05 mM). In three separate experiments, each of these three components was radiolabeled independent of the other two components. Experiments with [3H]glutathione indicated that conjugate formation was dependent upon diimine, and not benzidine. With [3H]benzidinediimine or [3H]benzidine, two different calculations were necessary to assess conjugate formation. For [3H]benzidinediimine, the calculation considered that only the radiolabeled diimine formed conjugate, while with [3H]benzidine, a specific activity calculation was necessary to demonstrate that conjugate formation was dependent upon diimine. With 0.05 mM [3H]benzidine, horseradish peroxidase-catalyzed formation of BZ-SG was optimum between 0.05 and 0.0625 mM H2O2. The latter is consistent with conversion of benzidine to diimine before formation of BZ-SG. Specific inhibitors and the absence of oxygen uptake indicated the lack of involvement of cation, thiyl, and carbon-centered radicals. The results are consistent with the existence of the charge-transfer complex and with benzidinediimine reacting with glutathione to form BZ-SG.

Metabolism and excretion of nitrofurothiazole bladder carcinogens.

Nitrofurothiazoles such as 2-amino-4-(5-nitro-2-furyl) thiazole (ANFT) and N-formylated ANFT (FANFT) are model compounds used in the study of chemical carcinogenesis. FANFT is a more potent uroepithelial carcinogen than ANFT but previous studies have shown extensive deformylation of FANFT to ANFT in vivo and ANFT to be the putative proximate carcinogen. To investigate this paradox, disposition of radiolabeled FANFT and ANFT was determined in rats prepared for clearance experiments. After 2 hr, 27.3 +/- 2.1% of recovered ANFT was excreted in urine compared to 44.2 +/- 1.8% of FANFT (P less than .001). In addition, approximately 20% of both FANFT and ANFT were excreted in bile after 2 hr. The disposition of nonradiolabeled FANFT and ANFT was also determined. The urinary excretion rate for ANFT with i.v. ANFT administration was 0.9 +/- 0.1 nmol/min. Following i.v. FANFT administration, the urinary excretion rate for ANFT was 49.7 +/- 8.6 nmol/min (P less than .001). The elimination half-lives were 23 +/- 3 and less than 5 min, for ANFT and FANFT, respectively. The differences in renal handling of ANFT and FANFT could not be accounted for by differences in protein binding. Large differences were found in urinary metabolite excretion between FANFT and ANFT administration. These results demonstrate deformylation dependent excretion (renal metabolic/excretory coupling) exists for FANFT resulting in much higher concentrations of ANFT reaching the urinary tract than when ANFT only is administered. Biliary excretion accounts for significant early clearance of both ANFT and FANFT. Renal metabolic/excretory coupling may explain the difference in uroepithelial carcinogenicity between FANFT and ANFT.

Rat liver cytochrome P450 metabolism of N-acetylbenzidine and N,N'-diacetylbenzidine.

To provide the information necessary for assessing risk and preventing tumorigenesis, the metabolism of N-acetylbenzidine and N,N'-diacetylbenzidine was assessed with rat liver microsomes from control and beta-naphthoflavone-treated rats. The oxidation of [3H]N-acetylbenzidine to [3H]N'-hydroxy-N-acetylbenzidine (N'HA), [3H]N-hydroxy-N-acetylbenzidine (NHA), and 3H-ring oxidation products was assessed. For [3H]N,N'-diacetylbenzidine, the formation of [3H]N-hydroxy-N,N'-diacetylbenzidine (NHDA) and the 3H-ring oxidation product was assessed. With beta-naphthoflavone-treated microsomes, the rate of NHA formation was 8-fold more than observed with control. Although significant formation of ring-oxidation products was demonstrated, the formation of N'HA was at the limit of detection. With control microsomes, N'HA was a major metabolite with more N'HA (49 +/- 6 pmol/mg protein/min) produced than NHA (38 +/- 5). Whereas the oxidation of N,N'-diacetylbenzidine was not observed with control microsomes, significant formation of NHDA (421 +/- 49 pmol/mg protein/min) and ring-oxidation (182 +/- 28) product was observed with beta-naphthoflavone-treated microsomes. Metabolism of [3H]N-acetylbenzidine and [3H]N,N'-diacetylbenzidine by beta-naphthoflavone-treated microsomes was completely inhibited by the specific cytochrome P4501A1/1A2 inhibitors alpha-naphthoflavone and ellipticine at 10 microM. Except for the < 30% inhibition observed with the cytochrome P4502E1 inhibitor (disulfiram), inhibitors of cytochrome P4503A1/3A2 (troleandomycin) and P4502C6 (sulfinpyrazone) were not effective at 10 microM. N'HA formation by control microsomes was not prevented by any of these inhibitors. Conditions that inhibit flavin-dependent monooxygenase metabolism, methimazole (1 mM), and heat treatment (37 degrees C for 60 min) were also ineffective in preventing N'HA formation. The nonspecific cytochrome P450 inhibitor SKF-525A (10 microM) exhibited a partial dose-response inhibition (maximum 41% of complete reaction mixture) of N'HA formation, but did not alter NHA formation. In contrast, the nonspecific cytochrome P450 inhibitor, 2,4-dichloro-6-phenylphenoxyethylamine prevented formation of both N'HA and NHA. beta-Naphthoflavone treatment increased [3H]N-acetylbenzidine binding to DNA, but not [3H]N,N'-diacetylbenzidine. Binding of both compounds to DNA was inhibited by ellipticine. N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine was detected by 32P-postlabeling in microsomal incubations with N-acetylbenzidine, but not N,N'-diacetylbenzidine. More adduct was detected with control than beta-naphthoflavone-treated microsomes. Results are consistent with cytochrome P4501A1/1A2 playing the major role in N-acetylbenzidine and N,N'-diacetylbenzidine metabolism by liver microsomes from control and beta-naphthoflavone-treated rats. The formation of N'HA by control, but not by beta-naphthoflavone-treated, rats and its insensitivity to inhibition by cytochrome P4501A1/1A2 inhibitors were unexpected.

N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine formation by peroxidative metabolism.

N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine (dGp-ABZ) is thought to play an important role in initiation of benzidine-induced bladder cancer in humans. This report assesses the possible formation of this adduct by peroxidatic activation of N-acetylbenzidine (ABZ). Adduct formation was measured by 32P-post-labeling. Ram seminal vesicle microsomes were used as a source of prostaglandin H synthase (PHS). The peroxidatic activity of PHS was compared with that for horseradish peroxidase. Both peroxidases converted ABZ to dGp-ABZ whether DNA or 2'-deoxyguanosine 3'-monophosphate (dGp) was present. Following 32P-post-labeling, the enzymatic and synthetic adduct were extracted from PEI-cellulose plates and were shown to have the same HPLC elution profiles for the bisphosphate adduct (32P-dpGp-ABZ). Treatment of the enzymatic and synthetic bisphosphate adduct with nuclease P1 yielded a product that eluted at the same time from the HPLC (32P-dpG-ABZ). Additional experiments demonstrated that the PHS-derived 5'-monophosphate (dpG-ABZ) and 3'-monophosphate (dGp-ABZ) adducts were also identical to their corresponding synthetic standard. With comparable amounts of total ABZ metabolism, PHS produced approximately 40-fold more dGp-ABZ than horseradish peroxidase (1943 +/- 339 versus 49 +/- 7.8 fmol/mg dGp). Adduct formation was dependent upon the presence of peroxidase and the specific substrate, i.e. arachidonic acid or H2O2. Adduct formation by PHS was inhibited by indomethacin (0.1 mM), ascorbic acid (1 mM) and glutathione (10 mM), but not by 5,5-dimethyl-1-pyrroline N-oxide (DMPO) (100 mM), a radical scavenger. Horseradish peroxidase adduct formation was also inhibited by ascorbic acid and glutathione. In addition, DMPO elicited greater than a 96% inhibition. Results demonstrate peroxidatic metabolism of ABZ to form dGp-ABZ. The mechanism of dGp-ABZ formation by PHS and horseradish peroxidase may be different.

N-glucuronidation of benzidine and its metabolites. Role in bladder cancer.

Workers exposed to high levels of benzidine have a 100-fold increased incidence of bladder cancer. This review evaluates the overall metabolism of benzidine to determine pathways important to initiation of bladder cancer. Upon incubation of benzidine with liver slices from rats, dogs, and humans, different proportions of this diamine were N-acetylated and N-glucuronidated. With dogs, a non-acetylator species, N-glucuronidation was the major pathway. In contrast, little glucuronidation was observed in rats with N, N'-diacetylbenzidine, the major metabolite of benzidine. Human liver slices demonstrated both extensive N-acetylation and N-glucuronidation. Differences between rats and humans were attributed to rapid deacetylation by human liver with N-acetylbenzidine rather than an accumulation of N, N'-diacetylbenzidine. N-Acetylbenzidine oxidative metabolism was also observed. The acid lability of glucuronide products of benzidine, N-acetylbenzidine, and oxidation products of N-acetylbenzidine metabolism was assessed. N-Glucuronides of benzidine, N-acetylbenzidine, and N'-hydroxy-N-acetylbenzidine were acid-labile, with the latter having a much longer half-time than the former two glucuronides. Because bladder epithelium contains relatively high levels of prostaglandin H synthase and not cytochrome P450, the peroxidative metabolism of N-acetylbenzidine was assessed. N'-(3'-Monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine was the only DNA adduct detected. This adduct is also the major adduct detected in bladder cells from workers exposed to benzidine. In urine from these workers, an inverse relationship between urine pH and levels of free (unconjugated) benzidine and N-acetylbenzidine was observed. A similar inverse relationship was observed for urine pH and levels of bladder cell N'-(3'-monophospho-deoxyguanosin-8-yl)-N-acetylbenzidine. These results suggest multiple pathways (acetylation, glucuronidation, peroxidation) in multiple organs (liver, blood, kidney, bladder) are important in benzidine-induced bladder cancer.

Human and Escherichia coli beta-glucuronidase hydrolysis of glucuronide conjugates of benzidine and 4-aminobiphenyl, and their hydroxy metabolites.

Individuals exposed to carcinogenic aromatic amines excrete arylamine N- and O-glucuronide metabolites. This study assessed the susceptibility of selected glucuronides to hydrolysis by human and Escherichia coli beta-glucuronidase. N- or O-glucuronides were prepared with the following aglycones: benzidine, N-acetylbenzidine, N'-hydroxy-N-acetylbenzidine, N-hydroxy-N-acetylbenzidine, N-hydroxy-N,N'-diacetylbenzidine, 3-hydroxy-N,N'-diacetylbenzidine, 3-hydroxy-benzidine, 4-aminobiphenyl, N-hydroxy-4-aminobiphenyl, and N-hydroxy-N-acetyl-4-aminobiphenyl. The (3)H- and (14)C-labeled glucuronides were prepared with human or rat liver microsomes using UDP-glucuronic acid as cosubstrate. Each of the 10 glucuronides (6-12 microM) was incubated at pH 5.5 or 7.0 with either human recombinant (pure) or E. coli (commercial preparation) beta-glucuronidase for 30 min at 37 degrees C. Hydrolysis was measured by HPLC. Reaction conditions were optimized, using the O-glucuronide of N-hydroxy-N,N'-diacetylbenzidine. Both enzymes preferentially hydrolyzed O-glucuronides over N-glucuronides and distinguished between structural isomers. With E. coli beta-glucuronidase at pH 7.0, selectivity was demonstrated by the complete hydrolysis of N-hydroxy-N-acetyl-4-aminobiphenyl O-glucuronide in the presence of N-acetylbenzidine N-glucuronide, which was not hydrolyzed. Metabolism by both enzymes was completely inhibited by the specific beta-glucuronidase inhibitor saccharic acid-1,4-lactone (0.5 mM). The concentration of human beta-glucuronidase necessary to achieve significant hydrolysis of glucuronides was substantially more than the amount of enzyme reported previously to be present in urine under either normal or pathological conditions. The bacterial enzyme may hydrolyze O-glucuronides, but not N-glucuronides, in urine at neutral pH. Thus, the nonenzymatic hydrolysis of N-glucuronides by acidic urine is likely a more important source of free amine than enzymatic hydrolysis.

'Revised' mass spectral identification of 2-amino-4-(5-nitro-2-furyl)thiazole metabolites.

The electron ionization mass spectra of 2-amino-4-(5-nitro-2-furyl)thiazole metabolites obtained from microsomal incubations and chemical syntheses were studied. The identities of the metabolites were established by chemical ionization, high resolution, and metastable measurements. The compounds studied showed multiple modes of cleavage, skeletal rearrangements and hydrogen back-transfer.

Phenylbutazone peroxidatic metabolism and conjugation.

Phenylbutazone, a nonsteroidal anti-inflammatory drug, elicits therapeutic as well as toxic effects by unknown pathways. Phenylbutazone was shown to form a conjugate with the heterocyclic amine bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT). To understand further the reactivity of these compounds, this study was conducted to identify the conjugate formed and determine the mechanism of conjugate formation. Both prostaglandin H synthase and horseradish peroxidase catalyzed conjugate formation. This conjugate was identified by 1H-NMR to be 4-[2-amino-4-(5-nitro-2-furyl)-5-thiazolyl]-4-butyl-1,2-diphenyl-3,5- pyrazolidinedione. Phenylbutazone-mediated oxygen uptake was inhibited by ANFT (0.1 mM) and the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (200 mM) and tert-nitrosobutane (4 mM). By contrast, phenol (0.005 to 0.25 mM) and aminopyrine (0.4 mM) stimulated oxygen uptake. None of these agents mediated oxygen uptake in the absence of phenylbutazone. Conjugate formation was significantly increased by phenol (0.005-0.25 mM) and aminopyrine (0.4 mM), as well as in the absence of oxygen. Conjugate formation was inhibited by 5,5-dimethyl-1-pyrroline-N-oxide (200 mM), tert-nitrosobutane (4 mM), ascorbic acid (2 mM), and 95% oxygen. Horseradish peroxidase initiated conjugate formation at much lower concentrations than it metabolized ANFT. The stoichiometric relationship between phenylbutazone and ANFT, with respect to conjugate formation, was complex. With the concentration of ANFT fixed at 0.05 mM, phenylbutazone exhibited saturation kinetics with a Km of 0.2 mM. In contrast, saturation kinetics were not observed with ANFT.Km values for ANFT varied with the concentration of phenylbutazone used.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of peroxidative activation of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT): comparison with benzidine.

The mechanism of activation of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) was investigated by comparison with benzidine. In comparison with benzidine, ANFT has a higher electrochemical potential (approximately 700 mV) and is less effective as a reducing co-substrate for either prostaglandin H synthase (PHS) or horseradish peroxidase. Activation was monitored by measuring binding to protein (BSA) and DNA. ANFT binding to protein was reduced by indomethacin, a fatty acid cyclooxygenase inhibitor; phenol and aminopyrine, competitive reducing co-substrates; ascorbic acid, an antioxidant; and glutathione, thioether conjugate formation. These results are consistent with those previously reported for benzidine and demonstrate a peroxide co-substrate requirement, interaction of peroxidase with amine, formation of reactive intermediates and inactivation of reactive intermediates. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO), a radical trap, also reduced ANFT binding to protein. Similar results were observed whether activation by PHS or horseradish peroxidase was investigated. Peroxidative activation of ANFT and benzidine to bind DNA was inhibited by these test agents in a manner similar to that observed with protein except that DMPO did not reduce binding. In addition, 2-methyl-2-nitrosopropane and methyl viologen, which are radical traps, and methionine and p-nitrobenzyl-pyridine, which are strong nucleophiles, did not reduce ANFT or benzidine binding to DNA. These agents also did not prevent binding of benzidinediimine, the two-electron product of benzidine oxidation, to polydeoxyguanosine. Glutathione inhibited diimine binding by forming a conjugate. Results demonstrate that activation of ANFT to bind protein and DNA is similar to benzidine. Peroxidative activation of benzidine occurs by both one- and two-electron oxidation. A similar mechanism would explain ANFT binding to protein (one electron) and DNA (two electron).

Glucuronidation of N-acetylbenzidine by human liver.

N-Glucuronidation is an important pathway in aromatic amine metabolism. This study assessed N-glucuronidation of N-acetylbenzidine by human liver slices and microsomes. With slices, considerable metabolism of [3H]N-acetylbenzidine (0.2 mM) was observed during a 2-hr incubation. N-Acetylbenzidine N'-glucuronide represented significant metabolism in four different human liver samples (6-33% of the total recovered radioactivity following HPLC). Benzidine (11-43%), benzidine N-glucuronide (8-11%), and N,N'-diacetylbenzidine (0-2%) were also formed. The kinetics of N-acetylbenzidine N'-glucuronide formation were investigated using Triton X-100-pretreated microsomes. Data were best described by a two-component Michaelis-Menten model composed of both high-affinity (low KM) and low-affinity (high KM) UDP-glucuronsyltranosferases. The high- and low-affinity KMs were 0.36 +/- 0.02 and 1.07 +/- 0.12 mM, respectively. To help identify the UDP-glucuronosyltransferases metabolizing N-acetylbenzidine, 23 transferase substrates were tested for their ability to inhibit glucuronidation. At 0.25 mM, bilirubin, estriol, and 17-epiestriol were good inhibitors (< 50% of control). Dose-response inhibition studies with estriol and 4-aminobiphenyl demonstrated that each agent reached a plateau as its concentration was increased. IC50 for estriol and 4-aminobiphenyl was 0.15 +/- 0.03 and 0.57 +/- 0.06 mM, respectively. Complimentary inhibition was observed when these agents were combined at maximal inhibitory concentrations. These results suggest that more than one UDP-glucuronosyltransferase metabolizes N-acetylbenzidine. N-Glucuronidation represents a major pathway for N-acetylbenzidine metabolism in humans.

Reactive nitrogen oxygen species metabolize N-acetylbenzidine.

A close association has been reported for certain types of cancers influenced by aromatic amines and infection/inflammation. Reactive nitric oxygen species (RNOS), components of the inflammatory response, are bactericidal and tumoricidal, and contribute to the deleterious effects attributed to inflammation on normal tissues. This study assessed the possible transformation of the aromatic amine N-acetylbenzidine (ABZ) by RNOS. RNOS were generated by various conditions to react with ABZ, and samples were evaluated by HPLC. Conditions which generate nitrogen dioxide radical (NO(2)(-) + myeloperoxidase + H(2)O(2), ONOO(-), and NO(2)(-) + HOCl) produced primarily a single new product termed 3'-nitro-ABZ. The myeloperoxidase-catalyzed reaction with 0.3 mM NO(2)(-) was completely inhibited by 1 mM cyanide, and not effected by 100 mM chloride with or without 1 mM taurine. In contrast, conditions which generate N(2)O(3), such as spermine NONOate, did not produce 3'-nitro-ABZ, but rather two compounds termed 4'-OH-AABP and AABP. (1)H NMR and mass spectrometry identified 3'-nitro-ABZ as 3'-nitro-N-acetylbenzidine, 4'-OH-AABP as 4'-OH-4-acetylaminobiphenyl, and AABP as 4-acetylaminobiphenyl. Human polymorphonuclear neutrophils incubated with [(3)H]ABZ and stimulated with beta-phorbol 12-myristate 13-acetate produced 3'-nitro-ABZ in the presence of NO(2)(-) (0.1-1 mM). Neutrophil 3'-nitro-ABZ formation was verified by mass spectrometry and was consistent with myeloperoxidase oxidation of NO(2)(-). The results demonstrate that ABZ forms unique products in the presence of nitrosating and nitrating RNOS, which could influence the carcinogenic process and serve as biomarkers for these reactive species.