Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “BENZIDINE”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 37 records · Page 2Linked to original sources
PubMed1988

Mechanism of formation and structural characterization of DNA adducts derived from peroxidative activation of benzidine.

Although the peroxidative metabolism of benzidine and other carcinogenic arylamines has been regarded as a significant pathway in extrahepatic tissues, the mechanism of peroxidase-mediated covalent binding to DNA has remained unclear. In this study, we have compared the metabolic activation of benzidine by prostaglandin H synthase, horseradish peroxidase, chloroperoxidase, and lactoperoxidase. All four peroxidases mediated the binding of benzidine to DNA and equimolar amounts of hydrogen peroxide (or arachidonic acid) and benzidine were required for the maximal binding in the system with either horseradish peroxidase or prostaglandin H synthase. In reactions containing both synthetic [3H]benzidine diimine and [14C]benzidine, rapid equilibration of both compounds was evident through the formation of its charge-transfer complex and the 14C-associated binding to DNA. However, the total binding (3H and 14C) correlated with the concentration of benzidine diimine rather than that of the charge-transfer complex. Two major and one minor deoxyguanosine adducts (P-I, P-III and P-IV, respectively) were isolated after the enzymatic hydrolysis of the benzidine-modified DNA. P-I was identified as N-(deoxyguanosin-8-yl)-benzidine, which was the major adduct formed by reaction of benzidine diimine with DNA. P-IV, which was also formed on reaction of benzidine diimine with DNA, was consistent with an N-(deoxyguanosin-N2-yl)-benzidine structure. P-III, which was formed only in the peroxidase incubations with DNA, was characterized as a novel N,3-(deoxyguanosin-N7,C8-yl)-benzidine derivative. Furthermore, this DNA adduct was shown to arise by the action of the peroxidase on DNA that had been previously modified by benzidine diimine. These results indicate that the two-electron oxidation product of benzidine, benzidine diimine, is the predominant reactive intermediate for the DNA binding mediated by peroxidases.

Arachidonic Acid↗
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↗
PubMed2004

Comparative metabolic activation of benzidine and N-acetylbenzidine by prostaglandin H synthase.

Benzidine and N-acetylbenzidine are activated to genotoxic metabolite(s) within the urothelial target tissue, with phase-I and phase-II enzymes being relevant. In principle, both benzidine and N-acetylbenzidine are activated by prostaglandin H synthase (PHS) to reactive intermediates. However, the relative impacts of benzidine and N-acetylbenzidine in this process remain unclear. Two experimental in vitro systems were used in the present comparative investigation: ram seminal vesicle microsomes rich in PHS and porcine urinary bladder epithelial cells (PUBEC) as a model system mimicking the general metabolic situation within the human urothelium. Benzidine, N-acetylbenzidine and N,N'-diacetylbenzidine were incubated with ram seminal vesicle microsomes and arachidonic acid and control incubations were performed with heat-inactivated microsomes. The metabolic disappearance of benzidine, N-acetylbenzidine or N,N'-diacetylbenzidine indicated a rapid turnover by PHS of benzidine and a slower turnover of N-acetylbenzidine. There was almost no PHS-associated metabolism of N,N'-diacetylbenzidine, suggesting that diacetylation of benzidine could represent a pathway of biological inactivation. Under similar conditions, incubations were performed with ram seminal vesicles and benzidine or N-acetylbenzidine upon addition of calf thymus DNA. After re-isolation of the DNA and 32P-postlabeling, with benzidine 2 distinct adducts were found of unknown nature, and with N-acetylbenzidine a single adduct appeared with co-migrated with the N'-(3'-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine. PUBEC cells were also incubated with benzidine or N-acetylbenzidine. No DNA adduct was found with benzidine, but a total of five adducts was produced from N-acetylbenzidine. The major adduct again co-migrated with N'-(3'-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine. When benzidine was incubated with PUBEC cells N-acetylbenzidine and, with some delay, N,N'-diacetylbenzidine were formed. Application of Lineweaver-Burk plots for the formation of N-acetylbenzidine from benzidine revealed a K(m) of 56.4 microM and a Vmax of 7.05 nmol/h per 10(6) PUBEC cells. The investigations generally support a key role of N-acetylbenzidine at the target site of the urothelium.

Animals↗
PubMedDate not supplied

The role of acetylation in benzidine metabolism and DNA adduct formation in dog and rat liver.

To determine whether benzidine is acetylated in dog, like rat, the metabolism of benzidine was assessed with dog and rat liver slices. Slices were incubated with 0.05 mM [3M]benzidine for 4 h. Media and cellular DNA were analyzed for acetylated benzidine metabolites and adducts. In rat, benzidine was rapidly converted to acetylated metabolites. At 1 h, benzidine, N-acetylbenzidine, and N,N'-diacetylbenzidine represented 5%, 23%, and 54%, respectively, of the total radioactivity in media. Within 2 h, 75% of the radioactivity was N,N'-diacetylbenzidine. In dog, 45% of the radioactivity was present in metabolites more polar than benzidine by 4 h. No N-acetylated metabolites were observed in dog liver slice media. To identify acetylated benzidine DNA adducts, N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine was prepared and identified by FAB MS. This nucleoside adduct was used to synthesize N-(deoxyguanosin-8-yl)-N-acetylbenzidine and N'-(deoxyguanosin-8-yl)-N-acetylbenzidine. Nucleoside adducts from slices incubated with [3H]benzidine were analyzed by HPLC. With this method of analysis, the 3H-material did not correlate with the synthetic adduct standards. To improve sensitivity and identify liver adducts, a 32P-postlabeling method was developed. 2'-Deoxyguanosine 3'-monophosphate adduct standards of acetylated benzidine were prepared. 32P-Postlabeling analysis demonstrated that rat liver contained only N'-(3'-monophosphodeoxyguanosine-8-yl)-N-acetylbenzidine after a 1- or 4-h exposure to benzidine. In contrast, no acetylated adducts were detected in dog. Results indicate that dog is a nonacetylator with respect to benzidine. The availability of acetylated benzidine nucleotide standards allowed unambiguous identification of N'-(3'-monophosphodeoxyguanosin-8-yl)-N-acetylbenzidine as the adduct present in rat liver slices. These nucleotide adduct standards will be useful in subsequent studies in animals and human.

Acetylation↗
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↗
PubMed1996

In vitro percutaneous absorption of benzidine in complex mechanistically defined chemical mixtures.

Little work has been done on the topical absorption of the bladder carcinogen benzidine. Since humans are more likely to be exposed to chemical mixtures than to a single chemical, a program was developed in these laboratories to examine the cumulative effect of complex mixtures on percutaneous absorption of important toxicants such as benzidine. In this investigation, a mixture is defined as a physical combination consisting of a marker chemical and several other chemicals, each of which can have independent and/or synergistic effects on dermal penetration and absorption of the marker chemical. Ten mixtures, consisting of a marker chemical (benzidine, B), a solvent (acetone, A or DMSO, D), a surfactant (0 or 10% sodium lauryl sulfate, SL), a vasodilator (0 or 180 microg methyl nicotinate, M), and a reducing agent (0 or 2% SnCl2, s) were employed in this study. Isolated perfused porcine skin flaps (IPPSFs), which have proven to be a suitable in vitro model for assessing dermal absorption and toxicity, and flow-through diffusion cell systems were utilized. The extent of benzidine absorption in skin sections dosed with either B + A (0.94% dose) or B + D (1.01% dose) was similar to that when IPPSFs were dosed with either B + A (0.54% dose) or B + D (1.31% dose). However, flux vs time profiles were different when the two in vitro methods were compared. For mixtures containing (1) DMSO only or acetone only or (2) solvents containing SL + M, benzidine absorption was enhanced when compared with other mixtures. Compared to acetone, DMSO appears to enhance dermal penetration of benzidine in most of the mixtures. Compared to other mixtures evaluated, SnCl2 inhibited benzidine absorption irrespective of solvent present. SnCl2 also appears to inhibit benzidine penetration in DMSO mixtures containing SL only, but not in acetone mixtures. It is proposed that chemical-chemical interactions between benzidine and SnCl2 may be inhibiting benzidine absorption and chemical-biological interactions between M + SL and skin may be enhancing benzidine absorption. Across all mixtures, maximum observed benzidine absorption was almost 3% of the topical dose over 8 hr, but maximum penetration was 22% over the same time period which would suggest a potential for greater systemic exposure over longer time frames. This work underscores the need to study potentially toxic chemicals in mixture exposure scenarios since the interactions observed would confound risk assessment based on single chemical data.

Animals↗
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↗