(1/124) Peroxygenase metabolism of N-acetylbenzidine by prostaglandin H synthase. Formation of an N-hydroxylamine.
Synthesis of prostaglandin H2 by prostaglandin H synthase (PHS) results in a two-electron oxidation of the enzyme. An active reduced enzyme is regenerated by reducing cofactors, which become oxidized. This report examines the mechanism by which PHS from ram seminal vesicle microsomes catalyzes the oxidation of the reducing cofactor N-acetylbenzidine (ABZ). During the conversion of 0.06 mM ABZ to its final end product, 4'-nitro-4-acetylaminobiphenyl, a new metabolite was observed when 1 mM ascorbic acid was present. Similar results were observed whether 0.2 mM arachidonic acid or 0.5 mM H2O2 was used as the substrate. This metabolite co-eluted with synthetic N'-hydroxy-N-acetylbenzidine (N'HA), but not with N-hydroxy-N-acetylbenzidine. The new metabolite was identified as N'HA by electrospray ionization/MS/MS. N'HA represented as much as 10% of the total radioactivity recovered by high pressure liquid chromatography. When N'HA was substituted for ABZ, PHS metabolized N'HA to 4'-nitro-4-acetylaminobiphenyl. Inhibitor studies demonstrated that metabolism was due to PHS, not cytochrome P-450. The lack of effect of 5,5-dimethyl-1-pyrroline N-oxide, mannitol, and superoxide dismutase suggests the lack of involvement of one-electron transfer reactions and suggests that hydroxyl radicals and superoxide are not sources of oxygen or oxidants. Oxygen uptake studies did not demonstrate a requirement for molecular oxygen. When [18O]H2O2 was used as the substrate, 18O enrichment was observed for 4'-nitro-4-acetylaminobiphenyl, but not for N'HA. A 97% enrichment was observed for one atom of 18O, and a 17 +/- 7% enrichment was observed for two 18O atoms. The rapid exchange of 18O-N'HA with water was suggested to explain the lack of enrichment of N'HA and the low enrichment of two 18O atoms into 4'-nitro-4-acetylaminobiphenyl. Results demonstrate a peroxygenase oxidation of ABZ and N'HA by PHS and suggest a stepwise oxidation of ABZ to N'-hydroxy, 4'-nitroso, and 4'-nitro products. (+info)
(2/124) 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. (+info)
(3/124) Ultrastructural localization of light-induced lipid peroxides in the rat retina.
PURPOSE: Localization of light-induced lipid peroxides in the rat retina at an ultrastructural level as benzidine-reactive substances. METHODS: Long-Evans rats with nondilated pupils were exposed to intense light of 6000 lux for 12 or 24 hours. Control animals were kept under physiological light conditions. Rats with dilated pupils were exposed to a light intensity of 50 lux or 150,000 lux for 1 hour. For ultrastructural localization the enucleated eyes were fixed in a 0.1-M cacodylate buffer (pH 7.4) containing 2% glutaraldehyde for 2 hours. Pieces of the superior part of the central eyecup were incubated overnight with tetramethylbenzidine (TMB; pH 3.0) at 4 degrees C, postfixed with 1.5% OSO4, and embedded for electron microscopy. RESULTS: In animals exposed to intense light, electron-dense structures appeared exclusively throughout the rod outer segments after an irradiation of 6000 lux for 24 hours or 150,000 lux for 1 hour and were absent in animals with nondilated pupils kept at physiological light conditions. Dilation of the pupils leads to the appearance of electron-dense structures after just 1 hour of 50 lux, whereas rats with nondilated pupils withstand even a 12-hour irradiation with 6000 lux. No electron-dense structures were found when no TMB was used in incubation. CONCLUSIONS: The appearance of electron-dense structures in the rod outer segments depends on the incubation with TMB and intensive light exposure of the rat. Dilation of the pupils lowers the threshold for the emergence of electron-dense structures significantly. This strongly supports the view that light-induced lipid peroxides in the rat retina are localized at an ultrastructural level as benzidine-reactive substances. This protocol presents a tool for the generation and ultrastructural localization of lipid peroxides in rat retinas. (+info)
(4/124) Glucuronidation of benzidine and its metabolites by cDNA-expressed human UDP-glucuronosyltransferases and pH stability of glucuronides.
Although glucuronidation is considered a necessary step in aromatic amine-induced bladder cancer, the specific enzymes involved are not known. This study assessed the capacity of five different human recombinant UDP-glucuronosyltransferases expressed in COS-1 cells to glucuronidate benzidine, its metabolites and 4-aminobiphenyl. [(14)C]UDP-glucuronic acid was used as co-substrate. UGT1A1, UGT1A4 and UGT1A9 each metabolized all of the aromatic amines. UGT1A9 exhibited the highest relative rates of metabolism with preference for the two hydroxamic acids, N-hydroxy-N-acetylbenzidine and N-hydroxy-N,N'-diacetylbenzidine. UGT1A9 metabolized 4-aminobiphenyl approximately 50% faster than benzidine or N-acetylbenzidine. UGT1A4 N-glucuronidated N'-hydroxy- N-acetylbenzidine at the highest relative rate compared with the other transferases. UGT1A6 was effective in metabolizing only four of the eight aromatic amines tested. UGT1A1 demonstrated more extensive metabolism of the hydroxamic acid, N-hydroxy-N,N'-diacetylbenzidine, and the ring oxidation product, 3-OH-N,N'-diacetylbenzidine, than it did for the other six amines. UGT2B7 was the only product of the UGT2 gene family examined and it metabolized all the aromatic amines at similar low relative levels compared with a preferred substrate, 4-OH-estrone. The K(m) values for N-acetylbenzidine metabolism by UGT1A1 and UGT1A4 were 0.37 +/- 0.14 and 1.8 +/- 0.4 mM, respectively. The O-glucuronide of 3-OH-N,N'-diacetylbenzidine was not hydrolyzed during a 24 h 37 degrees C incubation at either pH 5. 5 or 7.4. Likewise, the O-glucuronide of 3-OH-benzidine was stable at pH 7.4, with 52% remaining at pH 5.5 after 24 h. These results suggest the following relative ranking of transferase metabolism: UGT1A9 > UGT1A4 > > UGT2B7 > UGT1A6 approximately UGT1A1. The relative pH stability of O-glucuronides is consistent with a role in detoxification and excretion of aromatic amines, while the acid lability of N-glucuronides is consistent with delivery of these amines to the bladder epithelium for activation, resulting in DNA adducts which may lead to mutations. (+info)
(5/124) H(2)O(2) detection from intact mitochondria as a measure for one-electron reduction of dioxygen requires a non-invasive assay system.
Evaluation of the existence of superoxide radicals (O*-(2)), the site of generation and conditions required for one-e(-) transfer to oxygen from biological redox systems is a prerequisite for the understanding of the deregulation of O(2) homeostasis leading to oxidative stress. Mitochondria are increasingly considered the major O*-(2) source in a great variety of diseases and the aging process. Contradictory reports on mitochondrial O*-(2) release prompted us to critically investigate frequently used O*-(2) detection methods for their suitability. Due to the impermeability of the external mitochondrial membrane for most constituents of O*-(2) detection systems we decided to follow the stable dismutation product H(2)O(2). This metabolite was earlier shown to readily permeate into the cytosol. With the exception of tetramethylbenzidine none of the chemical reactants indicating the presence of H(2)O(2) by horseradish peroxidase-catalyzed absorbance change were suited due to solubility problems or low extinction coefficients. Tetramethylbenzidine-dependent H(2)O(2) detection was counteracted by rereduction of the dye through e(-) carriers of the respiratory chain. Although the fluorescent dyes scopoletin and homovanillic acid were found to be suited for the detection of mitochondrial H(2)O(2) release, fluorescence change was strongly affected by mitochondrial protein constituents. The present study has resolved this problem by separating the detection system from H(2)O(2)-producing mitochondria. (+info)
(6/124) Examination of low-incidence brain tumor responses in F344 rats following chemical exposures in National Toxicology Program carcinogenicity studies.
Neoplasms in the brain are uncommon in control Fischer 344 (F344) rats; they occur at a rate of less than 1% in 2-yr toxicity/carcinogenicity studies. Furthermore, only 10 of nearly 500 studies conducted by the National Toxicology Program (NTP) showed any evidence of chemically related neoplastic effects in the brain. Generally, the brain tumor responses were considered equivocal, because the characteristics of potential neurocarcinogenic agents (such as statistically significant increased incidences, decreased latency and/or survival, and demonstration of dose-response relationships) were not observed. A thorough examination, including comparisons with a well-established historical database, is often critical in evaluating rare brain tumors. Chemicals that gave equivocal evidence of brain tumor responses were generally associated with carcinogenicity at other sites, and many chemicals were mutagenic when incubated with metabolic activating enzymes. Other factors that were supportive of the theory that marginal increases in brain tumor incidence were related to chemical exposure were that (a) some of the tumors were malignant, (b) no brain neoplasms were observed in concurrent controls from some studies, and/or (c) brain tumors were also seen following exposure to structurally related chemicals. In 2-yr studies in F344 rats (studies conducted by the NTP), equivocal evidence of carcinogenicity was observed for the following 9 chemicals: isoprene, bromoethane, chloroethane, 3,3'-dimethylbenzidine dihydrochloride, 3,3'-dimethoxybenzidine dihydrochloride, furosemide, C.I. direct blue 15, diphenhydramine hydrochloride, and 1-H-benzotriazole. Glycidol was the only chemical evaluated by the NTP with which there was clear evidence of brain tumor induction in F344 rats. Clarification of the potential neurocarcinogenic risks of chemicals that produce equivocal evidence of a brain tumor response in conventional 2-yr rodent studies may be aided by the use of transgenic mouse models that exhibit genetic alterations that reflect those present in human brain tumors as well as by the use of in utero exposures. (+info)
(7/124) A new group of potent inducers of differentiation in murine erythroleukemia cells.
This report identifies a group of compounds, polymethylene bisacetamides (acetylated diamines), which are potent inducers of erythroid differentiation in murine erythroleukemia cells. A known inducing agent, N-methylacetamide, was dimerized through varying numbers of methylenes in an attempt to increase the local effective concentration at adjacent target sites. The simple dimer was no more effective than N-methylacetamide alone; introduction of five to eight methylenes between acetamide groups substantially increased the effectiveness of these compounds. The hexamethylene bisacetamide was active between 0.5 mM and 5 mM; the percentage of cells induced and the rate at which they were recruited to differentiation was dependent upon the concentration of inducer within this range. At 5 mM hexamethylene bisacetamide essentially the entire population (greater than 99%) was induced to a pathway of erythroid differentiation which was greater differentiation of the cultured cells than with any inducer yet tested. (+info)
(8/124) 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. (+info)