Biomedical subjects

V M Lakshmi

Publications and source records attributed to V M Lakshmi.

At least 19 recordsLinked to original sources

PubMed1997

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.

Animals↗

PubMed1996

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.

Acetylation↗

PubMed1996

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.

Aminobiphenyl Compounds↗

PubMed1996

NADPH-dependent oxidation of benzidine by rat liver.

This study used liver microsomes from control an naphthoflavone-treated rats to evaluate NADPH-dependent oxidation of benzidine. With microsomes from beta-naphthoflavone-treated rats, the rates of formation of aqueous soluble metabolite (HPLC analysis) and protein and DNA binding were 835 +/- 81, 14.5 +/- 1.8 and 0.71 +/- 0.14 pmol/mg/min respectively. beta-Naphthoflavone treatment elicited 12.3-, 1.8- and 14.2-fold increases in benzidine metabolism compared with controls as judged by HPLC and protein and DNA binding respectively. For microsomes from treated animals, Km and Vmax values were 47 +/- 6 micromol and 1.13 +/- 0.16 nmol/mg protein/min respectively. All of the metabolic parameters were inhibited to varying degrees by glutathione (1 or 10 mM), N-acetylmethionine (10 mM) and ascorbic acid (10 mM). Following glutathione addition, at least two new metabolite peaks were observed, representing -6% of the total radioactivity recovered by HPLC. Neither metabolite was 3-(glutathion-S-yl)benzidine. Cytochrome P450 inhibitors (10 micro) specific for different members of cytochrome gene families 1-3 indicated that benzidine was metabolized by cytochrome P450 1A1/1A2. Ellipticine and alpha-naphthoflavone, specific 1A1/1A2 inhibitors, elicited 50% inhibition at -0.2 and 0.5 micro respectively. Electron impact and negative ion chemical ionization mass spectro- metry identified the aqueous soluble metabolite as 3-hydroxybenzidine. The lability of 3-hydroxybenzidine observed at pH > 7.0 was prevented by ascorbic acid. Thus, cytochrome P450 1A1/1A2 NADPH-dependent metabolism of benzidine to 3-hydroxybenzidine was demonstrated.

Animals↗

PubMed1996

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.

Benzidines↗

PubMed1995

Prostaglandin H synthetase-mediated metabolism of dopamine: implication for Parkinson's disease.

Differences in prostaglandin H synthetase (PHS) activity in the substantia nigra of age- and postmortem interval-matched parkinsonian, Alzheimer's, and normal control brain tissue were assessed. Prostaglandin E2 (PGE2, an index of PHS activity) was higher in substantia nigra of parkinsonian brain tissue than Alzheimer's or control tissue. Incubation of substantia nigra slices with arachidonic acid (AA) increased PGE2 synthesis. Dopamine stimulated PHS synthesis of PGE2. [3H]Dopamine was activated by PHS to electrophilic intermediate(s) that covalently bound to DNA, microtubulin protein, bovine serum albumin, and sulfhydryl reagents. When AA was replaced by hydrogen peroxide, PHS/H2O2-supported binding proceeded at rates similar to those observed with PHS/AA. Indomethacin and aspirin inhibited AA-mediated cooxidation of dopamine but not H2O2-mediated metabolism. PHS-mediated metabolism of dopamine was not affected by monoamine oxidase inhibitors. Substrate requirements and effects of specific inhibitors suggest cooxidation of dopamine is mediated by the hydroperoxidase activity of PHS. 32P-postlabeling was used to detect dopamine-DNA adducts. PHS/AA activation of dopamine in the presence of DNA resulted in the formation of five dopamine-DNA adducts, i.e., 23, 43, 114, 70, and 270 amol/micrograms DNA. DNA adduct formation was PHS, AA, and dopamine dependent. PHS catalyzed cooxidation of dopamine in dopaminergic neuronal degeneration is discussed.

Absorption↗

PubMed1995

Glucuronidation of N-hydroxy metabolites of N-acetylbenzidine.

Glucuronidation of N-hydroxy arylamines is thought to be a necessary step in their initiation of bladder cancer. This was evaluated for the N-hydroxy metabolites of N-acetylbenzidine (ABZ). N'-Hydroxy-N-acetylbenzidine (N'-HA), N-hydroxy-N-acetylbenzidine (N-HA) and N-hydroxy- N,N'-diacetylbenzidine (N-HDA) were synthesized. Except for N'-HA, these compounds were quite stable. Ascorbic acid and/or acidic pH increased the stability of N'-HA. When each N-hydroxy compound was added to reaction mixtures containing [14C]UDP-glucuronic acid, 3 mM ascorbic acid and human liver microsomes a new product was detected by HPLC. Emulgen 911 was a better detergent than Triton X-100 for expressing microsomal activity, with maximal glucuronidation observed with 0.3% Emulgen 911. At 0.125 mM amine the rate of glucuronidation was N-HDA >> N'-HA = benzidine > ABZ > N-HA. In contrast, at 0.5 mM amine the rate of glucuronidation of N-HA was only exceeded by N-HDA. At pH 5.5 and 37 degrees C the t1/2 for the enzymatically prepared glucuronide conjugates of ABZ, N'-HA and N-HA were 7.5 min and 3.5 and 1.8 h respectively. For N-HDA > 90% of this glucuronide remained after 24 h. At pH 7.4 and 37 degrees C the t1/2 for the glucuronide conjugates of ABZ and N-HA were 2.3 and 2 h respectively, with the amounts remaining after 24 h for N'-HA and N-HDA being 75 and 90% respectively. At pH 6.5 the t1/2 for N'-HA was 14 h. Thus only glucuronides of ABZ and N'-HA exhibit pH-dependent changes in t1/2. Compared with ABZ, glucuronides the N-hydroxy metabolites are more stable at acidic pH. Acidic urine would be more likely to hydrolyze the glucuronide conjugate of ABZ than those of its N-hydroxy metabolites. Because these results are different from that hypothesized for arylmonoamines, a new model was developed to explain the role of N-oxidation, N-glucuronidation and N-acetylation in the carcinogenesis of benzidine, an aryldiamine.

Acetylation↗

PubMed1995

N-acetylbenzidine and N,N'-diacetylbenzidine formation by rat and human liver slices exposed to benzidine.

The extent to which N-acetylbenzidine and N,N'-diacetylbenzidine are formed may influence benzidine-induced carcinogenesis. This study compared the formation of these metabolites by rat and human liver slices. The relationship between the NAT2 genotype and the formation of these acetylated products was also evaluated in humans. In rat liver slices incubated with 0.05 mM [3H]benzidine for 1 h (n = 3), N-acetylbenzidine and N,N'-diacetylbenzidine represented 8.8 +/- 3.6 and 73 +/- 2.5% respectively of the total radioactivity recovered by HPLC. No unmetabolized benzidine was observed. This suggests that an equilibrium exists between benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine in rat liver slice incubations which favors N,N'-diacetylbenzidine formation. In the presence of 0.1 mM paraoxon, a deacetylase inhibitor, N-acetylbenzidine and N,N'-diacetylbenzidine increased to 13 +/- 0.6 and 79 +/- 0.3% respectively. Within 2 h after incubating human liver slices with 0.014 mM [3H]benzidine (n = 8), benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine represented 19 +/- 5, 34 +/- 4 and 1.6 +/- 0.5%, respectively, of the total radioactivity recovered by HPLC. Thus in the human, conditions in liver slices favor N-acetylbenzidine rather than N,N'-diacetylbenzidine formation. With paraoxon, benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine represented 2 +/- 0.4, 24 +/- 4 and 51 +/- 3%, respectively. This resulted in a 32-fold increase in N,N'-diacetylbenzidine formation. Individuals with rapid NAT2 genotypes formed 1.4-fold more N-acetylbenzidine than slow acetylators. However, this increase was not significant. There was no apparent correlation of N,N'-diacetylbenzidine formation with NAT2 genotype. Similar results were observed when human slices were incubated with 0.09 mM [3H]benzidine. Deacetylase, perhaps more than N-acetyltransferase, influences hepatic metabolism and subsequent carcinogenesis of benzidine in man. These results help explain the species and organ specificity of benzidine carcinogenesis.

Aged↗

PubMed1994

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.

Benzidines↗

PubMed1994

Human liver glucuronidation of benzidine.

Although glucuronidation is considered an important pathway in aromatic amine-induced bladder cancer, benzidine glucuronidation has not been assessed in humans. Glucuronidation of benzidine was assessed with human liver microsomes and slices. Emulgen 911-treated microsomes exhibited a Km for benzidine of 0.8 +/- 0.06 mM and a Vmax of 4.2 +/- 0.7 nmol/mg protein/min. A variety of agents were tested for their ability to inhibit benzidine N-glucuronide formation. At 0.25 mM, estriol, 17-epiestriol, bilirubin, hyodeoxycholic acid and cyproheptadine were good inhibitors (< 50% of control). Dose-dependent inhibition studies with estriol, testosterone and 4-aminobiphenyl demonstrated that each agent reached a plateau as its concentration was increased. When these agents were combined at maximal inhibitory concentrations, additive inhibition was observed. These results suggest that more than one UDP-glucuronosyltransferase metabolizes benzidine. The cDNA clones pUDPGTh-1 and -2 encode transferases which metabolize hyodeoxycholic acid and estrogen derivatives, but neither transferase catalyzed benzidine glucuronidation. Slices were used to assess metabolism by intact tissue and converted [3H]benzidine (0.09 mM) to N-acetyl-benzidine. N-Glucuronides of both benzidine and N-acetylbenzidine were observed and represented 14-37% of the total recovered radioactivity. The amount of N-acetylbenzidine N'-glucuronide observed was proportional to the amount of N-acetylbenzidine produced. Thus, N-glucuronidation appears to represent a major pathway for metabolism of benzidine in humans. The extent of N-acetylation affects the proportion of benzidine and N-acetylbenzidine glucuronidated by human liver slices.

Aged↗

PubMed1993

N-acetylbenzidine-N'-glucuronidation by human, dog and rat liver.

While N-glucuronidation is an important pathway for metabolism of aromatic amines, it has not been demonstrated for N-acetylbenzidine. A glucuronide of N-acetylbenzidine was synthesized and identified by mass spectrometry as N-acetylbenzidine-N'-glucuronide. This N'-glucuronide is acid labile with a t1/2 of 4 min at pH 5.3. A similar acid lability was also observed with benzidine-N-glucuronide. The formation of N-acetylbenzidine-N'-glucuronide was assessed with liver slices and microsomes prepared from human, dog and rat. When 0.014 mM [3H]N-acetylbenzidine was incubated with human liver slices a significant amount of N-acetylbendizine-N'-glucuronide was produced (8-26% of the total radioactivity recovered). With higher concentrations of [3H]N-acetylbenzidine (1 mM) rat slices also produced N-acetylbenzidine-N'-glucuronide. However, N'-glucuronide formation was not detected with dog liver slices incubated with either 0.014 or 1 mM [3H]N-acetylbenzidine. N-Acetylbenzidine-N'-glucuronide formation was observed with microsomes prepared from human, dog and rat. To assess maximum activity four detergents were used at two concentrations. With or without detergent activation the relative amount of glucuronidation was human > > dog > rat. The rate of benzidine N-glucuronide formation was 4.3- and 1.6-fold greater than N-acetylbenzidine-N'-glucuronide in dog and rat respectively, while in human both rates were similar (1.1-fold). With or without detergent activation the relative amount of benzidine-N-glucuronide formation was human > dog > > rat. N-Glucuronidation of [3H]N,N'-diacetylbenzidine was not observed. Thus N-actylbenzidine-N'-glucuronide formation appears to be an important pathway for metabolism of N-acetylbenzidine, especially in humans. Due to their acid lability, formation of the N-glucuronides of N-acetylbenzidine and benzidine provides a mechanism for hepatic detoxification and accumulation of these carcinogens in the bladder. A new model is described illustrating the effect of N-glucuronidation and the influence of N-acetylation on arylmono- and aryldiamine-induced bladder carcinogenesis.

Animals↗

PubMed1993

Liver NADPH-dependent oxidation of the 5-nitrofurans, FANFT and ANFT, by guinea pig and rat.

1. Oxidative metabolism of the bladder carcinogens FANFT/ANFT was examined in vitro in guinea pig (resistant species) relative to rat (susceptible species). 2. The total rate of ANFT hepatic metabolism by guinea pig (soluble metabolites plus protein bound, 354 pmol/min per mg protein) was approx. 4 times that in rat. 3. The total rate of FANFT metabolism was similar in both species and approx. one-quarter that for ANFT in guinea pig. In rat, the rate of total metabolism of FANFT and ANFT was similar. 4. Cytochrome P450 inhibitors, 2,4-dichloro-6-phenylphenoxyethylamine, 7,8-benzoflavone, and n-octylamine largely inhibited metabolism in guinea pig, but had little effect in rat. 5. H.p.l.c. analysis of ANFT metabolites indicated distinctly different products in guinea pig compared to rat. 7,8-Benzoflavone decreased metabolite formation by 80% in guinea pig, but only 30% in rat. 6. Flavin-dependent monooxygenases may participate in metabolism of these carcinogens in rat, but not guinea pig. 7. Because ANFT is thought to be a more proximate carcinogen than FANFT, the increased rate of ANFT metabolism and the formation of different products in guinea pig compared to rat may partially explain the resistance of guinea pig to FANFT-induced bladder cancer.

Animals↗

PubMed1993

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)

Biotransformation↗

PubMed1992

Mechanism of formation of the thioether conjugate of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT).

The formation of thioether conjugates is an important pathway for inactivation of certain carcinogens. This study assessed the mechanism by which the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT) forms a glutathione conjugate (ANFT-SG). Peroxidatic metabolism of ANFT, in the presence of glutathione, results in ANFT-SG formation. Both prostaglandin H synthase and horseradish peroxidase can catalyze this reaction. Metabolism of the reducing co-substrates ANFT, phenol, and aminopyrine elicit increases in oxidized glutathione (GSSG). ANFT-SG formation is potentiated by phenol and aminopyrine. tert-Nitrosobutane (tNB), a thiyl radical trap, prevented increases in both GSSG and ANFT-SG. Increasing concentrations of ANFT elicited corresponding increases in both GSSG and ANFT-SG. Peroxidatic metabolism of ANFT in the presence of glutathione, but not in the absence of glutathione, resulted in oxygen uptake. The formation of GSSG and oxygen uptake are consistent with the presence of thiyl radicals during ANFT metabolism. 5,5-Dimethyl-1-pyrroline N-oxide, a thiyl radical trap, was not as effective as tNB in inhibiting the formation of ANFT-SG and GSSG. Ascorbic acid, a reducing cosubstrate and antioxidant, was very effective in preventing ANFT-SG and GSSG formation, while the strong nucleophile methionine was ineffective. To clarify effects of different test agents, their effects on aminopyrine cation radical formation were assessed. Results are consistent with ANFT reacting with thiyl radicals to form ANFT-SG. ANFT appears to be a thiyl radical trap. Peroxidatic metabolism of ANFT probably results in the formation of a cation radical rather than a carbon-centered radical.

Carcinogens↗

PubMed1992

Role of N-glucuronidation in benzidine-induced bladder cancer in dog.

The mechanism by which benzidine induces bladder cancer in dog was evaluated by assessing metabolism of [3H]benzidine by dog liver slices and microsomes. Slices incubated with 0.05 mM [3H]benzidine exhibited a 32.5 min incubated with 0.05 mM [3H]benzidine exhibited a 32.5 min peak, which was also produced when microsomal incubations were supplemented with UDP-glucuronic acid. In contrast to microsomes, very little of the 32.5 min peak was produced with the 100,000 g supernatant fraction. Microsomal metabolism was increased 5-fold by pretreatment with Triton X-100. Very little activity was observed with rat microsomes in either the presence or absence of Triton X-100. This metabolite was also generated by incubating benzidine with glucuronic acid at 4 degrees C for 3 days. Thermospray MS identified this metabolite as benzidine N-glucuronide. At 37 degrees C, the t1/2 stability of purified N-glucuronide was 99, 25 and 3 min in dog urine adjusted to pH 7.3, 6.3 and 5.3 respectively. The N-glucuronide was quite stable at pH 9.3, in dog plasma, and in aprotic solvents for 4 h at 37 degrees C. Relative to benzidine, its N-glucuronide is weakly bound to plasma proteins but not more reactive with DNA. Thus, detoxification by liver provides a mechanism for accumulation of benzidine in acidic urine, uptake of benzidine into bladder epithelium, and activation of benzidine in bladder. The liver and N-glucuronidation play a potentially important role in the species specificity of benzidine carcinogenesis.

Animals↗

PubMed1991

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.

Animals↗

PubMed1990

Thermospray high performance liquid chromatography/mass spectrometric identification of a bladder carcinogen metabolite isolated from guinea pig urine.

An in vivo urinary metabolite of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl) thiazole was isolated from guinea pig urine and was identified by direct analysis using thermospray mass spectrometry/high-performance liquid chromatography as 1-(2-amino-4-(5-nitro-2-furyl)-2-thiazolyl)-1-deoxy-beta-D-glucopyran uronic acid. The structure of this metabolite was also established by chemical synthesis. Both positive and negative ion thermospray mass spectrometry of the conjugate showed fragment ions resulting from cleavage across the pyran ring of the glucuronic acid comprising of aglycone moiety. These characteristic fragment ions may be diagnostic for identification of N-glucuronides from O-glucuronides.

Animals↗

PubMed1990

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.

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