Pseudoenzymatic reduction of N-hydroxy-2-acetylaminofluorene to 2-acetylaminofluorene mediated by cytochrome P450.
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N-hydroxy-2-acetylaminofluorene (N-OH-AAF) was reduced to 2-acetylaminofluorene by rat liver microsomes in the presence of both NAD(P)H and FAD under anaerobic conditions. The microsomal reduction proceeds as if it were an enzymatic reaction. However, when the microsomes were boiled, the activity was not abolished, but was enhanced. The activity was also observed with cytochrome P450 2B1 alone, without NADPH-cytochrome P450 reductase, in the presence of these cofactors. Hematin also exhibited a significant reducing activity in the presence of both a reduced pyridine nucleotide and FAD. The activities of microsomes, cytochrome P450 2B1 and hematin were also observed upon the addition of photochemically reduced FAD instead of both NAD(P)H and FAD. The microsomal reduction of N-OH-AAF appears to be a non-enzymatic reaction by the reduced flavin, catalyzed by the heme group of cytochrome P450. (+info)
Purification and characterization of N-hydroxy-2-acetylaminofluorene sulfotransferase from rat liver.
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N-Hydroxy-2-acetylaminofluorene (N-OH-2-AAF) sulfotransferase is an enzyme that catalyzes the sulfate transfer from the active sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), to N-OH-2-AAF to form a highly reactive product acetylaminofluorene N-sulfate. It has been purified about 2000-fold with a yield of over 12% from adult Sprague-Dawley male rat livers by an eight-step procedure. The final preparation was homogeneous on analytrical disc gel electrophoresis. The purified enzyme had activity toward p-nitrophenol with an approximately 1600-fold increase in specific activity over the crude homogenate, but it had almost no detectable activity toward steroids such as estrone, beta-estradiol, testosterone, dehydroisoandrosterone, and corticosterone. There was also very little sulfation activity toward serotonin and L-tyrosine methyl ester. The optimal pH for the enzyme activity is approximately 6.3 when measured in sodium phosphate buffer. Mg2+ at 6 to 9 mM could increase the enzyme activity up to 30%. Mn2+ activated the enzyme only slightly at very low concentrations. Zn2+, Co2+, Cu2+, and Ni2+ were all strongly inhibitory, but Ca2+ had very little effect. Thiol compounds were found to have a stabilizing effect and thiol-blocking reagents were potent inhibitors for this enzyme. The pure enzyme was very unstable especially in diluet solutions. The isoelectric point (pl) of the enzyme is 5.66 +/- 0.07. The molecular weight of the native enzyme was 68,000 +/- 500 as estimated by Sephadex G-100 and G-200 gel filtrations. A single component with molecular weight of 38,250 +/- 1,350 was observed on sodium dodecyl sulfate gel electrophoresis in the absence and presence of 2-mercaptoethanol. Comparison of the enzyme activity in mail and female rat livers at each stage of purification revealed that there was only a trace amount of N-OH-2-AAF sulfotransferase present in the female rat liver. (+info)
The contribution of UDP-glucuronosyltransferase 1A9 on CYP1A2-mediated genotoxicity by aromatic and heterocyclic amines.
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The importance of environmental and dietary arylamines, and heterocyclic amines in the etiology of human cancer is of growing interest. These pre-carcinogens are known to undergo bioactivation by cytochrome P450 (CYP)-directed oxidation, which then become substrates for the UDP-glucuronosyltransferases (UGTs). Thus, glucuronidation may contribute to the elimination of CYP-mediated reactive intermediate metabolites, preventing a toxic event. In this study, human UGTs were analyzed for their ability to modulate the mutagenic actions of N-hydroxy-arylamines formed by CYP1A2. Studies with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15 expressed in heterologous cell culture confirmed that UGT1A9 glucuronidated the mutagenic arylamines N-hydroxy-2-acetylaminofluorene (N-hydroxy-2AAF) and 2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-hydroxy-PhIP). To examine the mutagenic potential of these agents, a genotoxicity assay was employed using Salmonella typhimurium NM2009, a bacterial strain expressing the umuC SOS response gene fused to a beta-galactosidase reporter lacZ gene. DNA modification results in the induction of the umuC gene and subsequent enhancement of beta-galactosidase activity. Both N-hydroxy-2AAF and N-hydroxy-PhIP stimulated a dose-dependent increase in bacterial beta-galactosidase activity. In addition, the procarcinogens 2AAF and PhIP were efficiently bioactivated to bacterial mutagens when incubated with Escherichia coli membranes expressing CYP1A2 and NADPH reductase. CYP1A2 generated 2AAF- and PhIP-mediated DNA damage, but only the action of N-hydroxy-2AAF was blocked by expressed UGT1A9. These results indicate that UGT1A9 can control the outcome of a genotoxic response. The results also indicate that while a potential toxicant such as N-hydroxy-PhIP can serve as substrate for glucuronidation, its biological actions can exceed the capacity of the detoxification pathway to prevent the mutagenic episode. (+info)
Irradiation-induced adduct formation of RNA with carcinogenic arylamine derivatives.
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Radiolysis of N2O-saturated solutions of transfer RNA (tRNA) and the arylacethydroxamic acids, N-hydroxy-N-2-acetylaminofluorene and N-hydroxy-N-4-acetylaminobiphenyl; their corresponding acetamides, 2-acetylaminofluorene and 4-acetylaminofluorene; or the O-glucuronide of N-hydroxy-N-2-acetylaminofluorene resulted in adduct formation of the nucleic acid with these carcinogenic arylamine derivatives. The yield of adducts on irradiation of the arylacethdroxamic acids with tRNA was greater than that for their corresponding acetamides or the O-glucuronide. The fluorenylacethydroxamic acid and acetamide were also more reactive than the biphenyl analogs. Adduct formation resulting from radiolysis of tRNA and the arylacethydroxamic acids or the O-glucuronide proceeded with retention of both the aromatic nucleus and the N-acetyl group. The yields of adducts were much greater for irradiated mixtures than for irradiation of either component alone followed by mixing. Evaluation of the data shows that initial modification of the tRNA or the carcinogen can lead to adduct formation. In the case of primary radical attack of the nucleic acid, it has been shown that short-lived reactive RNA intermediates are responsible for a major fraction of the observed yield of adducts in the irradiated mixtures. Comparative studies showed that irradiation under conditions that favor reaction of oxidizing radicals enhanced formation of the adducts. Oxygen was shown to protect RNA from irradiation-induced binding of the arylacethydroxamine acids due to competition of O2 with the carcinogen for the reactive RNA intermediates. (+info)
N-hydroxy-2-acetylaminofluorene inhibition of rat live RNA polymerases.
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Administration of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) to rats inhibits liver nuclear RNA synthesis. This effect is reflected in an in vitro inhibition of RNA synthesis by isolated whole nuclei. The decreased RNA synthesis can be accounted for entirely by an inhibition of the RNA polymerase activities quantitatively solubilized and partially purified from these nuclei. Both nucleolar and nucleoplasmic polymerases are affected. A similar inhibition of the polymerases was demonstrated in intact nuclei by inactivating the endogenous template with actinomycin D and assaying the polymerases with an added exogenous template, poly(deoxyadenylate-deoxythymidylate). Chromatin was prepared from similar nuclear preparations by two methods, differing in the extent to which they remove endogenous polymerase activity. Each chromatin preparation was transcribed with added Escherichia coli or partially purified rat liver nucleoplasmic RNA polymerase respectively. With either polymerase and either chromatin preparation, no inhibition of the template activity of chromatin isolated from N-OH-AAF-treated animals could be detected. It is concluded that N-OH-AAF is a potent inhibitor of rat liver nuclear RNA synthesis and that the mechanism of this inhibition is inactivation of the RNA polymerases. At the same time, N-OH-AAF leaves the chromatin template, at least quantitatively, intact for the synthesis of RNA. The implications of such an effect of N-OH-AAF on RNA synthesis are discussed. (+info)
Biochemical and morphological changes in hepatic nuclear membranes produced by N-hydroxy-2-acetylaminofluorene.
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The effect of N-hydroxy-2-acetylaminofluorene on the ultrastructure and synthesis of hepatic neclear membranes was evaluated in partially hepatectomized rats. The incorporation of L-[4,5-3H]leucine into two nuclear membrane fractions increased within 2 hr after hepatic resection and reached a peak at 20 hr. After partial hepatectomy, the decay of radioactivity in nuclear membrane proteins labeled with L-[4,5-3H]leucine revealed similar half-lives for the two membrane fractions when compared to those obtained from sham-operated animals. The protein concentration of the nuclear membrane fraction of higher density decreased sharply within 2 hr after partial hepatectomy and remained low throughout a 20-hr postoperative period. Polyacrylamide gel electrophoresis of both nuclear membrane fractions showed a similar composition. Nine proteins were resolved, varying from 21,000 to 190,000 daltons. The two major protein bands were in the range of 50,000 and 70,000 daltons, respectively. Treatment of partially hepatectomized animals with N-hydroxy-2-acetylaminofluorene showed marked dilation of the nuclear envelope and rough endoplasmic reticulum in situ upon electron microscopic examination. Vacuolization and evagination of the perinuclear membranes were also noticeable in isolated nuclei obtained from carcinogen-treated rats. Inhibition by N-hydroxy-2-acetylaminofluorene of the incorporation of L-[4,5-3H]leucine into the nuclear membranes was dose-dependent and remained depressed throughout a 60-min labeling period. These results suggest that the inhibitory effects on RNA and protein synthesis previously shown to be produced by this arylhydroxylamine hepatocarcinogen may lead to disruption of the morphology and synthesis of the nuclear envelope. (+info)
Suppressive role of indole on 2-acetylaminofluorene hepatotoxicity.
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Indole is known to suppress the hepatotoxicity and carcinogenicity of 2-acetylaminofluorene (AAF) in rats and hamsters. For elucidation of the mechanism of its protective role, 2 experiments were conducted using young male rats. In the 1st experiment, the 24-hr biliary excretion of N-hydroxy-2-acetylaminofluorene (N-OH-AAF)-glucuronide was measured after 2 and 4 weeks of dietary administration of 0.03% AAF with or without 1.6% indole. The amount of [9-14C]N-OH-AAF that was excreted as the glucuronide following a single i.p. injection of [9-14C]AAF was lower after 2 weeks in animals fed AAF and indole, as compared to those fed AAF alone [1.5 +/- 1.2% versus 19.6 +/- 3.6% S.E. (p less than 0.001)]. After 4 weeks of AAF administration without indole, the biliary excretion fell to 4.8 +/- 2.1%. This was also significantly higher than that of the animals fed both AAF and indole [1.8 +/- 1.2% (p less than 0.025)]. The suppressive role of indole on the conjugate excretion was also reflected in a decreased biliary excretion of all [9-14C]AAF metabolites in animals treated with indole alone. In the 2nd experiment, the protective action of indole was assessed by survival following daily i.p. injections of N-OH-AAF and Na2SO4 solution. Na2SO4 increased the hepatotoxicity of N-OH-AAF. Indole suppressed the toxicity of N-OH-AAF even in the presence of Na2SO4. This protective role of indole was partially overcome only when excess sulfate was coadministered. These results indicate that indole suppresses the biliary excretion of the O-glucuronide of N-OHAAF during the initial exposure of the animal to the carcinogen, possibly reflecting decreased N-OH-AAF formation. Indole also modifies the metabolism of AAF FOLLOWING N-hydroxylation, perhaps activating N-OH-AAF, depending upon the concentration of sulfate available. (+info)
Horseradish peroxidase/hydrogen peroxide-catalyzed oxidation of the carcinogen N-hydroxy-N-acetyl-2-aminofluorene as effected by cyanide and ascorbate.
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Horseradish peroxidase and H2O2 mediate N-hydroxy-N-acetyl-2-aminofluorene (N-OH-AAF) conversion into two more potent carcinogens, 2-nitrosofluorene and N-acetoxy-N-acetyl-2-aminofluorene. Optical studies of this system indicate that horseradish peroxidase is operating as a peroxidase with N-OH-AAF as the electron donor. Our studies confirm the earlier finding that 2-nitrosofluorene and N-acetoxy-N-acetyl-2-aminofluorene are the products of the type II enzyme-mediated oxidation of N-OH-AAF, but surprisingly, the results with the type VI enzyme indicate that more 2-nitrosofluorene was formed and, in addition, another product absorbing at 245 nm was formed. If ascorbate is present in the N-OH-AAF/horseradish peroxidase/H2O2 system, ascorbate is oxidized preferentially. Cyanide, a known inhibitor of the peroxidase, does not inhibit when N-OH-AAF is the electron donor. The reaction products are the same in the presence or absence of cyanide. (+info)