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Acrylonitrile (AN) is an industrial vinyl monomer that is acutely toxic. When administered to rats, AN covalently binds to tissue proteins in a dose-dependent but nonlinear manner [Benz, F. W., Nerland, D. E., Li, J., and Corbett, D. (1997) Fundam. Appl. Toxicol. 36, 149-156]. The nonlinearity in covalent binding stems from the fact that AN rapidly depletes liver glutathione after which the covalent binding to tissue proteins increases disproportionately. The identity of the tissue proteins to which AN covalently binds is unknown. The experiments described here were conducted to begin to answer this question. Male Sprague-Dawley rats were injected subcutaneously with 115 mg/kg (2.2 mmol/kg) [2,3-(14)C]AN. Two hours later, the livers were removed, homogenized, and fractionated into subcellular components, and the radioactively labeled proteins were separated on SDS-PAGE. One set of labeled proteins was found to be glutathione S-transferase (GST). Specific labeling of the mu over the alpha class was observed. Separation of the GST subunits by HPLC followed by scintillation counting showed that AN was selective for subunit rGSTM1. Mass spectral analysis of tryptic digests of the GST subunits indicated that the site of labeling was cysteine 86. The reason for the high reactivity of cysteine 86 in rGSTM1 was hypothesized to be due to its potential interaction with histidine 84, which is unique in this subunit.

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Three markers of acute acrylonitrile (AN) intoxication, namely, tissue glutathione (GSH), tissue cyanide (CN), and covalent binding to tissue protein, were studied as a function of dose and time. Doses administered and responses expected were 20 mg/kg (LD0), 50 mg/kg (LD10), 80 mg/kg (LD50), and 115 mg/kg (LD90). Liver GSH was the most sensitive marker of AN exposure. At 80 mg/kg AN, virtually complete depletion of liver GSH was observed within 30 min with no recovery through 120 min. Kidney GSH showed a similar, but less intense depletion; while blood and brain GSH were more refractory to AN. Whole blood and brain CN rose progressively during the first 60 min in a dose-dependent fashion. At the lowest dose, CN levels decreased thereafter, whereas, at the three higher doses, CN levels were maintained or continued to increase through 120 min. At the highest dose, blood and brain CN remained at acutely toxic levels through 240 min. Covalent binding increased rapidly in all tissues during the first 30 min at all doses. At the lowest dose, little additional covalent binding was observed beyond 30 min, while at the three higher doses, covalent binding increased, although at a slower rate. The data indicate that these three biologic markers of acute AN intoxication respond dramatically in a time-dependent manner in the toxic dosage range. Furthermore, the data provide evidence that AN toxicity is gated by GSH depletion in liver with the resultant termination of AN detoxification.

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The dose dependence of acrylonitrile (AN) covalent binding to tissue protein, following a single acute exposure over a 100-fold range in dose, was measured. Covalent binding was a linear function of AN dose in the lower dose range (0.02-0.95 mmol AN/kg). The slopes of the dose-response curves indicated that tissues varied by nearly 10-fold in their reactivity with AN. The relative order of covalent binding was as follows: blood > > kidney = liver > forestomach = brain > glandular stomach > > muscle. Similar dose-response behavior was observed for globin total covalent binding and for globin N-(2-cyanoethyl) valine (CEValine) adduct formation. The latter adduct was found to represent only 0.2% of the total AN adduction to globin. Regression of tissue protein binding versus globin total covalent binding or globin CEValine adduct indicated that both globin biomarkers could be used as surrogates to estimate the amount of AN bound to tissue protein. At higher AN doses, above approximately 1 mmol/kg, a sharp break in the covalent binding dose-response curve was observed. This knot value is explained by the nearly complete depletion of liver glutathione and the resultant termination of AN detoxification. The toxicity of AN is known to increase sharply above this dose. The data suggest that a comparison of specific tissue proteins labeled by AN above and below this threshold dose may provide some insight into the mechanism of AN-induced toxicity.

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This paper describes a technique for quantitative analysis of 2-oxoglutarate (alpha-ketoglutarate) in biological samples using liquid chromatography with electrochemical detection (LC-EC). This method utilizes a simplified, efficient sample preparation designed to select for 2-oxoglutarate and similar compounds by derivatization with phenylhydrazine. The response was linear over the range from 62.5 to 1000 ng/ml. The least quantifiable concentration was 62.5 ng/ml and the least detectable concentration was 25 ng/ml. To test the ability of the assay to measure 2-oxoglutarate in biological samples, this method was used to quantitate the 2-oxoglutarate content in chick osteoblast cultures and to determine the ability of the assay to accurately measure a standard addition of 500 ng/ml 2-oxoglutarate when added to a sample of the forementioned groups. The 2-oxoglutarate content of these cells was 6.67 +/- 1.20 ng/micrograms DNA or 105 +/- 18 ng/cell layer (mean +/- 95% confidence interval) and the assay accurately measured the standard addition. This method was also used to quantitate 2-oxoglutarate content in whole embryonic chick calvariae containing 6.40 +/- 0.95 ng/mg dry bone weight or 37.5 +/- 5.5 ng/bone. This assay provides significantly lower detection limits than the currently available procedures and is suitable for determination of 2-oxoglutarate in biological samples where very low amounts of 2-oxoglutarate are found. This method is the first application of LC-EC for quantitating 2-oxoglutarate.

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Benzene is metabolized to one or more hematotoxic species. Saturation of benzene metabolism could limit the production of toxic species. Saturation of phase II enzymes involved in the conjugation of the phenolic metabolites of benzene also could affect the hematotoxicity of benzene. To investigate the latter possibility, we exposed male Swiss mice, via the inhalation route, to various concentrations of benzene for 6 h per day for 5 days. Following termination of the final exposure the mice were killed and the levels of phenylsulfate and phenylglucuronide in the blood determined. Spleen weights were recorded and the number of white blood cells counted. At low benzene exposure concentrations phenylsulfate is the major conjugated form of phenol in the blood. At high exposure concentrations, phenylglucuronide is the predominant species. The reductions in spleen weight and white blood cell numbers correlated with the concentration of phenylsulfate in the blood, but are most probably not causally related.

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The metabolite 2-(S-glutathionyl)hydroquinone is formed when a microsomal incubation mixture containing either benzene or phenol is supplemented with glutathione. This metabolite is derived from the conjugation of benzoquinone, an oxidation product of hydroquinone. However, neither the glutathione conjugate or its mercapturate, N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine, have been identified as metabolites resulting from in vivo metabolism of benzene, phenol, or hydroquinone. To determine if a hydroxylated mercapturate is produced in vivo, we treated male Sprague-Dawley rats with either benzene (600 mg/kg), phenol (75 mg/kg), or hydroquinone (75 mg/kg) and collected the urine for 24 hr. HPLC coupled with electrochemical detection confirmed the presence of a metabolite that was chromatographically and electrochemically identical to N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine. The metabolite was isolated from the urine samples and treated with diazomethane to form the N-acetyl-S-(2,5-dimethoxyphenyl)-L-cysteine methyl ester derivative. The mass spectra obtained from these samples were identical to that of an authentic sample of the derivative. The results of these experiments indicate that benzene, phenol, and hydroquinone are metabolized in vivo to benzoquinone and excreted as the mercapturate, N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine.

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Thiol-containing antidotes for acute acrylonitrile (AN) toxicity may exert their action by chemically reacting with AN, by replacing critical sulfhydryl groups cyanoethylated by AN, and by detoxifying cyanide produced from AN metabolism. We have evaluated the ability of the optical isomers of cysteine and N-acetylcysteine to act as antidotes against AN toxicity in order to assess the relative importance of each of these three antidotal mechanisms. The toxicity of AN was determined in male Sprague-Dawley rats and compared to the toxicity determined after treatment with 2 mmol/kg of thiol antidote by computing a protective index (median lethal dose with antidote/median lethal dose without antidote). The protective indices of L-cysteine, D-cysteine, N-acetyl-L-cysteine, and N-acetyl-D-cysteine were 2.03, 1.97, 1.76, and 1.25, respectively. Measurements of urinary mercapturates, derived from the non-oxidative pathway of AN metabolism, indicated that none of the antidotes was able to significantly increase the excretion of these metabolites. Blood cyanide generated from the oxidative metabolism of AN and butyronitrile was also determined. All of the antidotes, except N-acetyl-D-cysteine, lowered blood cyanide levels. A comparison of these results with the predicted relative abilities of the enantiomers to participate in each of the three antidotal mechanisms leads to the conclusion that, under these experimental conditions, the best correlation exists with the cyanide detoxification mechanism.

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Murine L-929 cells were treated with benzene or a series of benzene metabolites, washed and then interferon-alpha/beta was induced with polyriboinosinic-polyribocytidylic acid. Exposure of the cells to benzene or phenol, a monocyclic metabolite of benzene, did not affect interferon-alpha/beta induction. However, exposure of the cells to p-benzoquinone, hydroquinone or catechol, dihydroxy- and diketo-metabolites of benzene, resulted in a severe inhibition of interferon-alpha/beta production. There was no significant loss of viability of the cell cultures. Additional studies with p-benzoquinone indicated that inhibition of interferon-alpha/beta was reversible and could be abrogated by addition of reduced glutathione to the cell cultures.

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Phenol and related compounds are toxic agents to which humans are exposed. Analysis of phenolic compounds may be used as an index of exposure. In this report a method for analysis of phenol, phenylglucuronide, phenylsulfate, and related mono- and dihydroxybenzenes as their propanoate esters by gas chromatography/mass spectrometry is presented. The use of this derivatization technique increases the sensitivity of the analysis by improving chromatographic performance and introducing a traceable "reporter" group into the analyte. This method has also been adapted, by using selected ion monitoring (SIM), to the quantitative analysis of phenol.

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We exposed spleen cells from female Swiss/Webster mice to benzene and benzene metabolites to determine the effects of such exposure on interferon gamma induction by concanavalin A. Exposing the cells to benzene or phenol did not affect interferon gamma production, but exposing them to p-benzoquinone, catechol, or hydroquinone significantly inhibited interferon gamma production. Cell viability, as determined by trypan blue viability staining, was not influenced by the chemical treatment. When interferon gamma production was inhibited, the inhibition was dose dependent. The time of optimum production of interferon gamma after exposure to concanavalin A was not affected by treatment of the cells with p-benzoquinone. These data indicate the importance of dihydroxy and diketo metabolites as immunotoxic derivatives of benzene.

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In vivo treatment of randombred Swiss Webster mice with polyriboinosinic-polyribocytidylic acid (poly l X poly C) inhibited the induction of cytochrome P-450's by both 3-methylcholanthrene [(MCA) CAS: 56-49-5] and phenobarbitol [(PB) CAS: 50-06-6]. Concomitant treatments with poly l X poly C and a single dose of MCA inhibited the induction of P-450's for 24 hours and delayed the obtainment of the MCA-induced P-450 levels for approximately 48-72 hours. When cytochrome P-450 levels were induced by four successive daily treatments of MCA or PB and when poly l X poly C was given on only the 1st day, induction of P-450's was completely suppressed for 24 hours and obtainment of the maximal P-450 level was delayed by 72-96 hours. Treatment with poly l X poly C of animals preinduced for P-450's by four successive daily treatments with either PB or MCA decreased the P-450 content to the noninduced basal level within 24 hours. The effect was temporary in the MCA-treated mice since P-450 content recovered to the MCA-preinduced levels within 72 hours. PB-dependent P-450 induction was short lived, and no recovery occurred after poly l X poly C treatment of PB-preinduced mice. Reduced hepatic cytochrome P-450 contents correlated with decreased abilities of liver homogenates to metabolically activate benzo [a]pyrene (CAS: 50-32-8) and N-acetyl-2-aminofluorene (CAS: 53-96-3), as scored in an Ames Salmonella typhimurium revertant assay.

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In vivo induction of gamma interferon (IFN-gamma) by sensitization of mice with Mycobacterium bovis strain BCG and subsequent challenge with tuberculin depressed the ability of liver homogenates from treated animals to metabolically activate promutagens. The Ames Salmonella typhimurium revertant assay was used for analyses of metabolic conversion of promutagens by liver homogenates. Relative to the mutant frequencies determined with control liver homogenates, induction of IFN-gamma depressed the abilities of homogenates from treated animals to activate N-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), and benzo[a]pyrene (BP) by 55%, 44% and 95%, respectively. Within 18-24 h of Aroclor 1254 treatment, liver P-450 content had increased 43%, and the relative mutant yields per unit protein for all three promutagens had approximately doubled. In vivo induction of IFN-gamma suppressed the Aroclor 1254-dependent increases in mutagenesis by AAF (63%), AFB1 (90%), and BP (reduced to a level 23% below non-Aroclor 1254 treatment). In all cases, the levels of depression of promutagen activation qualitatively correlated with cytochrome P-450 content and the induction of IFN-gamma.

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Several 5-halopyrimidinones have been shown to have many different biological activities. These include interferon induction, antitumor effects, modulation of immune responses, and polyclonal B-cell activation. The present study was carried out to determine the effects of treatment of mice with two 5-halopyrimidinones, 2-amino-5-bromo-6-phenyl-4(3H)-pyrimidinone (ABPP) and 2-amino-5-iodo-6-phenyl-4(3H)-pyrimidinone (AIPP), on the murine cytochrome P-450 system. Administration of ABPP or AIPP to mice by a dosage regimen similar to that resulting in interferon induction by these chemicals resulted in a significant depression in liver cytochrome P-450 levels. These results suggest that 5-halopyrimidinones can depress cytochrome P-450 levels and that this depression may affect the metabolism of other drugs by cytochrome P-450.

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The induction of hepatic glutathione S-transferase by ethanol was investigated in male Swiss-Webster mice using a liquid diet. After a 7-day feeding period, mice that received 18, 27 or 36% of their calories as ethanol exhibited significant increases in the specific activity of glutathione S-transferase when 1,2-dichloro-4-nitrobenzene (DCNB), p-nitrobenzylchloride (NBC) and 1,2-epoxy-3-(p-nitrophenoxy)propane (ENP) were used as substrates. The observed increases in activity appeared to be related to the concentration of ethanol in the diet. Thus, mice fed a diet with 36% of the calories as ethanol exhibited the greatest increase in specific activity (DCNB, 75%; NBC, 60%, ENP, 34%). Pair-fed mice demonstrated similar changes in enzymatic activity. A time-course study indicated a 4-day feeding period was not sufficient to elicit significant induction, but a significant increase was apparent by day 7. This increase was maintained or increased through day 14. By comparison, 0.5 mg of phenobarbital/ml of diet produced a greater increase in enzymatic activity (DCNB, 449%; NBC, 227%; ENP, 219%). These results suggest that ethanol does induce glutathione S-transferase, but it is a relatively poor inducer of this enzyme.

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Mice were injected sc with benzene or one of its metabolites, phenol, catechol, or hydroquinone. The ability of these compound to inhibit erythropoiesis was quantified by measuring the incorporation of 59Fe into developing erythrocytes. Benzene decreased 59Fe incorporation into developing erythrocytes in a dose-dependent manner. Maximum inhibition was observed when benzene was administered 48 hr prior to initiation of the 59Fe uptake test. The three metabolites of benzene also significantly inhibited 59Fe incorporation when they were administered 48 hr prior to initiation of 59Fe uptake assay. The degree of inhibition observed with the metabolites was not as great as that observed with benzene. Coadministration of the microsomal mixed-function oxidase inhibitor, 3-amino-1,2,4-triazole, abolished the erythropoietic toxicity of benzene and phenol but had no effect on the catechol- or hydroquinone-induced toxicity.

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Multiple forms of cytosolic benzene dihydrodiol dehydrogenase (designated DD1, DD2, DD3, and DD4 according to order of elution from DEAE-cellulose column) were purified to homogeneity from liver of male Swiss-Webster mice, primarily by DEAE-cellulose, affinity, gel filtration, and hydroxylapatite chromatography. The purified enzymes were shown to have specific activities of 3.7, 0.94, 0.98, and 0.76 units/mg of protein, respectively, when assayed with 1.8 mM benzene dihydrodiol and 2.3 mM NADP+. DD1 and DD2 were monomers with molecular weights of 30,000 and 34,000, respectively, while DD3 and DD4 were dimers in the native state with molecular weights of 64,000 and 65,000. The isoelectric points were 8.1, 6.2, 5.5, and 5.4, respectively. The different forms of dihydrodiol dehydrogenase were also studied with respect to their Michaelis constants and substrate specificity.

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Gamma interferon (IFN-gamma), induced by sensitization of mice with Mycobacterium bovis strain BCG followed by challenge with tuberculin, was partially purified by adsorption on silicic acid. Administration of the partially purified IFN-gamma to mice significantly depressed levels of liver microsomal cytochrome P-450 to an equivalent extent as crude IFN-gamma.

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Induction of gamma (Type II) interferon results in depression of the cytochrome P-450 system of mice. The gamma interferon is induced by sensitization of mice with Mycobacterium bovis strain BCG followed by challenge with tuberculin. The degree of depression of the cytochrome P-450 system correlates with the levels of interferon induced. passive transfer of exogenous gamma interferon also results in depression of the murine cytochrome P-450 system. In the present study the metabolism of diphenylhydantoin, a drug metabolized by the cytochrome P-450 system, was examined in mice in which gamma interferon was induced. The metabolism of diphenylhydantoin was severely inhibited in mice in which interferon was induced, and the level of inhibition correlated with the liter of gamma interferon induced. Passive transfer of gamma interferon also depressed the metabolism of diphenylhydantoin by the murine cytochrome P-450 system.

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