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Enzymes from the pentose phosphate pathway (PPP) are potential drug targets for the development of new drugs against Trypanosoma brucei, the causative agent of African sleeping disease: for instance, the 6-phosphogluconate dehydrogenase is currently studied actively for such purposes. Structural and functional studies are necessary to better characterize the associated enzymes and compare them to their human homologues, in order to undertake structure-based drug design studies on such targets. In this context, the crystal structure of 6-phosphogluconolactonase (6PGL) from T. brucei, the second enzyme from PPP, was determined at 2.1 Angstroms resolution. Comparison of its sequence and structure to other related proteins in the 6PGL family with a known structure (Thermotoga maritima Tm6GPL 1PBT and Vibrio cholerae Vc6PGL (1Y89), which have not been discussed in print), or in the glucosamine-6-phosphate-deaminase family (hexameric Escherichia coli 1DEA and monomeric Bacillus subtilis 2BKV), allowed the identification of the 6PGL active site. In addition to the analysis of the crystal structure, 3D NMR interaction studies and docking experiments are reported here. Key residues involved in substrate binding or in catalysis were identified.

Background: Mechanisms underlying prevention by beta-naphthoflavone (beta-NF) of mammary carcinogenesis initiated with 7,12-dimethylbenz[a]anthracene (DMBA) in the rat were elucidated., Methods and Results: Treatment of female Sprague-Dawley rats with beta-NF at 40 mg/kg b.wt. for 4 days by oral gavage in corn oil before a single oral dose of DMBA (112 mg/kg b.wt.) suppressed mammary gland carcinogenesis as shown by an increase in the median latent period from 10 to 24 weeks and a 60% decrease in the multiplicity of mammary adenocarcinomas. In contrast, a 20-day treatment with beta-NF starting 3 weeks after DMBA had no significant effects on mammary tumorigenesis. The activities of phase I and phase II enzymes were examined in the liver and mammary gland 24 h after treatment of rats with beta-NF, DMBA, or beta-NF followed by DMBA as in the first bioassay. Treatment with either beta-NF or DMBA increased the hepatic activities of cytochrome P450 (CYP)1A1, 1A2, and 2B1/2, and glutathione S-transferase, and the mammary activity of CYP1A1. The activity of mammary CYP2B1/2 induced by DMBA was decreased by beta-NF. In the liver, the increase of UDP-glucuronosyl transferase (GT) activity in rats treated with beta-NF and DMBA was 2.3-fold greater than in rats treated with DMBA alone. Thus, treatment with beta-NF likely increased the rate of glucuronidation of DMBA dihydrodiols leading to carcinogen detoxification. The levels of the DMBA adducts determined by 32P-postlabeling of the mammary gland DNA were decreased in the beta-NF-pretreated rats., Conclusion: The beta-NF-induced increase in the hepatic UDP-GT activity and decrease in the mammary DNA-DMBA adducts occurred under the same treatment regimen that led to suppression of DMBA-induced mammary carcinogenesis.

Indole-3-carbinol (I3C), present in cruciferous vegetables, and its major in vivo product 3,3'-diindolylmethane (DIM), have been reported to suppress estrogen-responsive cancers. This effect may be mediated through the modification of cytochrome P450 (CYP) complement and activities leading to estrogen detoxification. We examined the effects of a 4-day treatment of female Sprague-Dawley rats with DIM at 8.4 and 42 mg/kg body weight (bwt), on the hepatic CYP protein level, CYP1A1, 1A2, 2B1/2 and 3A1/2 probe activities and CYP-dependent metabolism of 17beta-estradiol (E2) and estrone (E1). At 42 mg/kg bwt, DIM effected a small increase (2.8-fold) in CYP1A1 activity, and at both dose levels it reduced CYP3A1/2 activity by approximately 40%. At the higher dose level, DIM decreased the rates of oxidation of E2 to 4-OH-E2, 4-OH-E1, 6alpha-OH-E2 and 6(alpha+beta)-OH-E1 by 39, 44, 71 and 60%, respectively, and E1 to 6(alpha+beta)-OH-E1 by 39%. These effects were considerably different from those of I3C reported by us previously. We also examined the effects of DIM and I3C on the hepatic microsomal metabolism of tamoxifen (TAM). Whereas metabolism of TAM was unaffected by DIM, formation of N-desmethyl-TAM (and its presumed derivative) was increased approximately 3-fold by I3C at 250 mg/kg bwt. Since N-desmethyl-TAM is transformed to a genotoxic metabolite, dietary exposure to I3C may enhance hepatic carcinogenicity of TAM in the rat. The differences between I3C and DIM in CYP-mediated activities and metabolism indicate that DIM is not a proximate intermediate in the mechanism of action of I3C.

Down regulation of aryl sulfotransferase IV (AST IV) in promotion/progression of liver carcinogenesis by N-2-fluorenylacetamide (2-FAA) has been established. This study examined whether the C-9 oxidized metabolites of 2-FAA, which have recently been shown to promote diethylnitrosamine (DEN)-initiated liver carcinogenesis in male Sprague-Dawley rats, effect the above change. Hence, in DEN-initiated rats, the effects of promoting regimens of 9-OH-2-FAA or 9-oxo-2-FAA, 15 oral doses at 50 and 100 mumol/kg of body weight, were compared to those of 2-FAA at 50 mumol/kg of body weight and of the vehicle on the activity of N-hydroxy(OH)-2-FAA sulfotransferase (ST), an isozyme of AST IV and AST IV expression and distribution. Relative to the vehicle, treatment with the fluorenyl compounds led to decreased levels in hepatic N-OH-2-FAA ST activity and development of hepatic nodules and tumors which had still lower levels of the ST activity than the respective remnant livers. At approximately 8 months after treatment with the C-9-oxidized compounds at doses twice that of 2-FAA, the extents of decreases in the hepatic N-OH-2-FAA ST activity and cytosolic AST IV protein in tumors were comparable to those with 2-FAA. Immunocytochemical analysis showed close association of AST IV deficiency with neoplastic liver lesions. In comparison to N-OH-2-FAA, 9-OH-2-FAA had only low and 9-oxo-2-FAA lacked sulfate acceptor activity in the presence of male rat liver cytosol or AST IV. At 3.3-fold greater concentration than N-OH-2-FAA, 9-oxo-2-FAA inhibited (27%) the sulfate acceptor activity of N-OH-2-FAA in the presence of AST IV, which suggested interference by 9-oxo-2-FAA at the active site. Although the C-9-oxidized compounds do not appear to be substrates for N-OH-2-FAA ST, their ability to cause a decrease in N-OH-2-FAA ST activity and protein similar to that of 2-FAA supports their role in hepatocarcinogenesis. Whereas 9-OH-2-FAA had a 3.9-fold greater sulfate acceptor activity in the presence of female than male rat liver cytosol and inhibited dehydroepiandrosterone ST activity of female rat liver, N-OH-2-FAA and 9-oxo-2-FAA inhibited estrone ST activity of male rat liver, suggesting that the C-9-oxidized compounds as well as N-OH-2-FAA are substrates for STs other than AST IV.

The possibility that the interaction of C-nitroso aromatics with polyunsaturated fatty acids (PUFA) causes lipid peroxidation was investigated through determination of conjugated diene and malodialdehyde (MDA) formation after anaerobic/aerobic vs. aerobic incubations of nitrosobenzene (NOB) or 2-nitrosofluorene (2-NOF) with linoleic, linolenic or arachidonic acid or methyl linolenate. Anaerobic incubation of NOB or 2-NOF with linolenic acid at the molar ratio of 1:1 for 24 h yielded approximately 5.5-13% of the PUFA as conjugated diene which appeared stable upon exposure to air. Interaction of PUFA and 2-NOF or NOB yielded MDA, the amounts of which were significantly greater when 24-h anaerobic preceded 1-6-h aerobic incubation. Furthermore, the differences in the amounts of MDA resulting from 24- and 0-h anaerobic incubations were significantly greater when the molar ratio of 2-NOF (or NOB) to PUFA was increased (2.0 greater than 1.0 greater than 0.5). Superoxide dismutase or catalase had no effect on the yields of MDA following either anaerobic/aerobic or aerobic incubations of PUFA and 2-NOF. EDTA (1 or 10 microM) had no effect on the yields of MDA from aerobic incubations, but it decreased the amounts of MDA (by approximately 30 or 60%, respectively) from anaerobic/aerobic incubations. The data suggested that inhibition by EDTA was due to chelation of trace iron, which following anaerobic interaction of PUFA and 2-NOF might have been reduced to Fe2+ and contributed to the enhanced lipid peroxidation. Thus, adduction of C-nitroso aromatics to PUFA yields radical species which directly and/or via reaction with trace iron lead to lipid peroxidation. The lipophilicity of C-nitroso aromatics suggests that this process may be of consequence in their mutagenesis/carcinogenesis.

The oxidation of carcinogenic hydroxamic acids, N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) and N-hydroxy-N-3-fluorenylacetamide (N-OH-3-FAA) catalyzed by horseradish peroxidase (HRP) or cytochrome c in the presence of H2O2 was investigated. HRP/H2O2 was a more efficient system in oxidation of both hydroxamic acids and the standard substrate, guaiacol, then cytochrome c/H2O2. Peroxidative activity of cytochrome c was shown after incubation with Triton X-100 and H2O2 for 20 min at room temperature in 0.05 M phosphate buffer (pH 7.5) or in 0.1 M sodium acetate (pH 6.0) without Triton X-100. Both hydroxamic acids were oxidized to nitroxyl free radicals as shown by electron spin resonance (ESR) spectroscopy. These radicals dismutated to equimolar amounts of 2- or 3-nitrosofluorene and acetate esters of the corresponding hydroxamic acids as shown by thin layer chromatography and spectrophotometric analysis of the products. In addition, large amounts of the N-fluorenylamides were generated in the reactions with cytochrome c/H2O2 system. Of the products, only 2- or 3-nitrosofluorene per se or when generated from the oxidation of the hydroxamic acids, interacted with lecithin (1 mg/ml) to yield ESR signals of the immobilized nitroxyl free radicals. In contrast to HRP/H2O2 system, in which the initial velocity of the radical formation was too fast to measure and the maximal concentrations of the nitroxyl free radicals of both hydroxamic acids were similar, in the cytochrome c/H2O2 system the nitroxyl free radical of N-OH-2-FAA formed at a 6-fold faster rate and accumulated at a 2-fold higher concentration than the radical of N-OH-3-FAA. In both enzyme systems, the persistence of the signal and the length of time before it had decreased to one half its maximum were several-fold longer for the nitroxyl free radical of N-OH-3-FAA than for that of N-OH-2-FAA. These data showed that these nitroxyl free radicals differed in their kinetic properties. One electron oxidation of N-OH-3-FAA by HRP/H2O2 system and of both isomeric hydroxamic acids by cytochrome c/H2O2 system are reported for the first time in this work and may be considered an activation reaction in carcinogenesis by these compounds.

A method for the differentiation of chlorinated and brominated products from peroxidative oxidation of mixtures of the halides is presented. Chlorination or bromination of monochlorodimedone (MCD) by fungal chloroperoxidase (CPO) was measured by loss of MCD absorbance. Although the Vmax was similar for both halides [approximately 0.08 mM (2 min)-1], the apparent Km for chlorination was 10 times greater than that for bromination (5.88 vs 0.67 mM). Chlorination was also quantitated as I3- produced from N-chlorotaurine and I-. The Vmax [0.076 mM (2 min)-1] and apparent Km (6.31 mM) determined by this method agreed with those determined with MCD. Selective reduction by H2O2 of the I-oxidizing potential of N-bromotaurine allowed determination of the brominated product from the difference between the amounts of halogenated MCD and N-chlorotaurine. The brominated product predominated at saturating and at physiologic halide levels. Hence, it is suggested that Br- plays a significant role in halogenation even though in vivo levels of Cl- are equal to or greater than 1000 times those Br-.

A method for the identification and quantitation of carcinogeniic O-acetates of fluorenylhydroxamic acids by high-pressure liquid chromatography is described. The adsorbent is Corasil II and a mixture of ethyl acetate-n-hexane (1:1) is used as the solvent. N-Acetoxy-2-fluorenylacetamide or N-acetoxy-3-fluorenylacetamide are separable as single peaks from N-acetoxy-4-fluorenylacetamide and from N-acetoxy-2-fluorenylbenzamide. The peak height traced by the recorder is linearly proportional to the amount of compound in the effluent. The method can be utilized for the detection of 0.1-1.0 mug of compound. The method has been used to identify the products of the decomposition of N-acetoxy-2-fluorenylacetamide in aqueous media.

A method for the assay of glucuronidation of C- and N-hydroxylated metabolites of the carcinogen N-2-fluorenylacetamide is described. The method employs UDP-[U-14C ))glucuronic acid and Baker C18 extraction columns for separation of the glucuronides from their aglycones and from the glucuronic acid. The 14C-labeled glucuronides, generated by rat liver microsomes, are eluted from the columns with 30% (v/v) methanol after prewashing the columns and elution of the radioactivity of 14C-glucuronic acid with 1 mM ammonium acetate, pH 6.9. The radioactivity of the eluates is measured by scintillation counting. The method is modified for assays of glucuronidation of alpha-naphthol and p-nitrophenol in that 1 mM phosphoric acid is used instead of 1 mM ammonium acetate, and the method is potentially adaptable to other aglycones. By monitoring radioactivity or uv absorbance of the column eluates, it is shown that all aglycones, except p-nitrophenol, are retained on the columns during elution of their glucuronides with 30% (v/v) methanol and are eluted only when absolute methanol is used. The identity of the glucuronides is shown by their response to hydrolysis by beta-glucuronidase in the presence and absence of D-saccharic acid-1,4-lactone and, in some instances, by chromatographic and spectral analyses of the released aglycones.

N-Hydroxy-N-2-fluorenylacetamide, a proximate carcinogenic metabolite of N-2-fluorenylacetamide, is oxidized largely to 2-nitrosofluorene by lactoperoxidase or extract of peroxidative activity of rat uterus in an H2O2- and Br- -dependent reaction. Evidence is presented that the oxidizing species includes OBr- (HOBr). This novel oxidation may be involved in carcinogenesis by N-arylhydroxamic acids.