Benzene Oxide Research Articles

Diiron N-bridged species bearing phthalocyanine ligand catalyzes oxidation of methane, propane and benzene under mild conditions

Transformation of methane, the most abundant and the least reactive compound of natural gas to valuable products is one of the most difficult chemical problems of great practical importance. In Nature, methane monooxygenase enzymes transform methane to methanol in water under physiological conditions. However, chemical analogs for such a transformation are unknown. Here, we show the mild and efficient aqueous oxidation of methane by hydrogen peroxide, an ecologically and biologically relevant oxidant catalyzed by supported μ-nitrido diiron phthalocyanine dimer, (FePctBu4)2N . This bio-inspired complex containing a stable Fe – N – Fe motif catalyzes the oxidation of methane to methanol which is further transformed to formaldehyde and formic acid as is demonstrated using13CH4and18O labelling. (FePctBu4)2N - H2O2system shows a high activity in the oxidation of benzene to phenol which occurs via formation of benzene oxide and exhibits NIH shift typically accociated with biological oxidation. Mechanistic features of oxidation of methane and benzene as well as detected intermediate hydroperoxo- and high valent oxo diiron complexes support an O-atom transfer reaction mechanism relevant to bio-oxidation.

N‐Bridged Diiron Phthalocyanine Catalyzes Oxidation of Benzene with H2O2 via Benzene Oxide with NIH Shift Evidenced by Using 1,3,5‐[D3]Benzene as a Probe

Kinetic isotope effect: Incorporation of 18O from H218O2 in phenol in the presence of H216O and 16O2 and detection of benzene oxide as well as NIH shift in the course of the oxidation of PhH indicates that μ-nitrido diiron phthalocyanine operates by a biooxidation-like mechanism (see figure).

Hemoglobin Adducts of Benzene Oxide in Neonatal and Adult Dried Blood Spots

Adducts of reactive chemicals with hemoglobin (Hb) or human serum albumin can be used as biomarkers of internal doses of carcinogens. Because dried blood spots are easier to collect and store than conventional venous blood samples, they encourage applications of biomarkers of exposure in large epidemiologic studies. In addition, neonatal dried blood spot can be used to investigate chemical exposures in utero. Here, we report a simple method to isolate Hb from dried blood spot with high recovery and purity using the addition of ethanol to aqueous dried blood spot extracts. To prove the concept that dried blood spot-derived proteins can be used to assay for adducts, we measured Hb adducts of benzene oxide, a reactive metabolite of the ubiquitous air pollutant benzene in nine neonatal and nine adult dried blood spots (from volunteer subjects), using a gas chromatography-mass spectrometry method that we had previously developed. For comparison, benzene oxide-Hb adducts were measured in the same nine adult subjects using Hb that had been isolated and purified using our conventional method for venous blood. The geometric mean of benzene oxide-Hb levels in all dried blood spot samples ranged from 27.7 to 33.1 pmol/g globin. Neither of the comparisons of mean (logged) benzene oxide-Hb levels between sources (adult conventional versus adult dried blood spot and adult dried blood spot versus newborn dried blood spot) showed a significant difference. Based upon the estimated variance of the benzene oxide-Hb levels, we had 80% power to detect a 1.7-fold difference in geometric mean levels of benzene oxide-Hb in our sample of nine subjects.

Evidence from Mechanistic Probes for Distinct Hydroperoxide Rearrangement Mechanisms in the Intradiol and Extradiol Catechol Dioxygenases

Three mechanistic probes were used to investigate whether the Criegee rearrangement step of catechol 1,2-dioxygenase (CatA) from Acinetobacter sp. proceeds via a direct 1,2-acyl migration, via homolytic O-O cleavage, or via a benzene oxide-oxepin rearrangement. Incubation of CatA with 3-chloroperoxybenzoic acid led to the formation of a 9:1 mixture of 2-chlorophenol and 3-chlorophenol, via a mechanism involving O-O homolytic cleavage. Incubation of CatA with 2-hydroperoxy-2-methylcyclohexanone led to formation of 5,6-diketoheptan-1-ol, also consistent with an O-O homolytic cleavage mechanism, and not consistent with a direct 1,2-acyl migration. No reaction product was isolated from incubation of CatA with 6-hydroxymethyl-6-methylcyclohexa-2,4-dienone, an analogue that is able to undergo the benzene oxide-oxepin rearrangement, but not able to undergo O-O homolytic cleavage. In contrast, incubation of extradiol dioxygenase MhpB from Escherichia coli with 6-hydroxymethyl-6-methylcyclohexa-2,4-dienone led to the formation of a 2-tropolone ring expansion product, consistent with a direct 1,2-alkenyl migration for extradiol cleavage. Taken together, the results imply different mechanisms for the Criegee rearrangement steps of intradiol and extradiol catechol dioxygenases: a direct 1,2-alkenyl migration for extradiol cleavage and an O-O homolytic cleavage mechanism for intradiol cleavage.

Enzyme-catalysed synthesis and reactions of benzene oxide/oxepine derivatives of methyl benzoates

A series of twelve benzoate esters was metabolised, by species of the Phellinus genus of wood-rotting fungi, to yield the corresponding benzyl alcohol derivatives and eight salicylates. The isolation of a stable oxepine metabolite, from methyl benzoate, allied to evidence of the migration and retention of a carbomethoxy group (the NIH Shift), during enzyme-catalysed ortho-hydroxylation of alkyl benzoates to form salicylates, is consistent with a mechanism involving an initial arene epoxidation step. This mechanism was confirmed by the isolation of a remarkably stable, optically active, substituted benzene oxide metabolite of methyl 2-(trifluoromethyl)benzoate, which slowly converted into the racemic form. The arene oxide was found to undergo a cycloaddition reaction with 4-phenyl-1,2,4-triazoline-3,5-dione to yield a crystalline cycloadduct whose structure and racemic nature was established by X-ray crystallography. The metabolite was also found to undergo some novel benzene oxide reactions, including epoxidation to give an anti-diepoxide, base-catalysed hydrolysis to form a trans-dihydrodiol and acid-catalysed aromatisation to yield a salicylate derivative via the NIH Shift of a carbomethoxy group.

愛媛県における大気エアロゾル中多環芳香族炭化水素類（ＰＡＨｓ）の濃度レベル，変動の特徴とその起源

Concentrations of particulate polycyclic aromatic hydrocarbons(PAHs) in ambient air were investigated every month during the period Apr. 2004 to Mar. 2006 at two sampling sites in Ehime Prefecture; Site-N in Niihama city industrial area and Site-U in Uwajima city nonindustrial area. The sampling was carried out by high volume air sampler on quarts filters, extracted with dichloromethane and analyzed by HPLC with programmable fluorescence detection. Heavy metals and water soluble ios in particulate samples and benzene in air samples were also analyzed in this study. Annual average of total PAHs concentrations ranged from 4.5 to 4.7 ng/m3 at Site-N, higher than in Site-U (2.0∼3.4 ng/m3). Estimated risks of each PAHs based on unit risk were in the order of 10-7∼10-5, but overall risk was higher than objective criteria suggested by Central Environmental Council, Japan. B[a]P contributed above 50 % followed by DB[a,h]A. To have a complete knowledge on PAHs in atmosphere, their levels in the gaseous phase should also be monitored. Major source of PAHs would be vehicles especially diesel cars because of their diagnostic ratios of B[ghi]P/IP, IP/(IP+B[ghi]P) and (B[b]F+B[k]F)/B[ghi]P ranged within values reported for diesel exhausts so far. This view can be substantiated by the observed high correlation of PAHs with benzene and carbon oxide. Indexes of sources were selected by hierarchical cluster analysis on datasets of PAHs, metals and ions at Site-U which has no significant sources from industries. Then temporal variations of them and back trajectories at points in time were analyzed. It appears that high concentrations of anthropogenic metals and NH4SO4 are observed when the air mass was originated and passed through low-altitudes on industrial regions of China. Although PAHs showed significant correlation with these metals, they would be increased mainly by the effect of atmospheric stability.

Frequent p53 and H-ras Mutations in Benzene- and Ethylene Oxide-Induced Mammary Gland Carcinomas from B6C3F1 Mice

Benzene and ethylene oxide are multisite carcinogens in rodents and classified as human carcinogens by the National Toxicology Program. In 2-year mouse studies, both chemicals induced mammary carcinomas. We examined spontaneous, benzene-, and ethylene oxide-induced mouse mammary carcinomas for p53 protein expression, using immunohistochemistry, and p53 (exons 5-8) and H-ras (codon 61) mutations using cycle sequencing techniques. p53 protein expression was detected in 42% (8/19) of spontaneous, 43% (6/14) of benzene-, and 67% (8/12) of ethylene oxide-induced carcinomas. However, semiquantitative evaluation of p53 protein expression revealed that benzene- and ethylene oxide-induced carcinomas exhibited expression levels five- to six-fold higher than spontaneous carcinomas. p53 mutations were found in 58% (7/12) of spontaneous, 57% (8/14) of benzene-, and 67% (8/12) of ethylene oxide-induced carcinomas. H-ras mutations were identified in 26% (5/19) of spontaneous, 50% (7/14) of benzene-, and 33% (4/12) of ethylene oxide-induced carcinomas. When H-ras mutations were present, concurrent p53 mutations were identified in 40% (2/5) of spontaneous, 71% (5/7) of benzene-, and 75% (3/4) of ethylene oxide-induced carcinomas. Our results demonstrate that p53 and H-ras mutations are relatively common in control and chemically induced mouse mammary carcinomas although both chemicals can alter the mutational spectra and more commonly induce concurrent mutations.

Albumin Adducts of Electrophilic Benzene Metabolites in Benzene-Exposed and Control Workers

BackgroundMetabolism of benzene produces reactive electrophiles, including benzene oxide (BO), 1,4-benzoquinone (1,4-BQ), and 1,2-benzoquinone (1,2-BQ), that are capable of reacting with blood proteins to produce adducts.ObjectivesThe main purpose of this study was to characterize relationships between levels of albumin adducts of these electrophiles in blood and the corresponding benzene exposures in benzene-exposed and control workers, after adjusting for important covariates. Because second blood samples were obtained from a subset of exposed workers, we also desired to estimate within-person and between-person variance components for the three adducts.MethodsWe measured albumin adducts and benzene exposures in 250 benzene-exposed workers (exposure range, 0.26–54.5 ppm) and 140 control workers (exposure range < 0.01–0.53 ppm) from Tianjin, China. Separate multiple linear regression models were fitted to the logged adduct levels for workers exposed to benzene < 1 ppm and ≥ 1 ppm. Mixed-effects models were used to estimate within-person and between-person variance components of adduct levels.ResultsWe observed nonlinear (hockey-stick shaped) exposure–adduct relationships in log-scale, with inflection points between about 0.5 and 5 ppm. These inflection points represent air concentrations at which benzene contributed marginally to background adducts derived from smoking and from dietary and endogenous sources. Adduct levels were significantly affected by the blood-collection medium (serum or plasma containing either heparin or EDTA), smoking, age, and body mass index. When model predictions of adduct levels were plotted versus benzene exposure ≥ 1 ppm, we observed marked downward concavity, particularly for adducts of the benzoquinones. The between-person variance component of adduct levels increased in the order 1,2-BQ < 1,4-BQ < BO, whereas the within-person variance components of the three adducts followed the reverse order.ConclusionsAlthough albumin adducts of BO and the benzoquinones reflect exposures to benzene ≥ 1 ppm, they would not be useful biomarkers of exposure at ambient levels of benzene, which tend to be < 0.01 ppm, or in those working populations where exposures are consistently < 1 ppm. The concavity of exposure–adduct relationships is consistent with saturable metabolism of benzene at air concentrations > 1 ppm. The surprisingly large effect of the blood-collection medium on adduct levels, particularly those of the benzoquinones, should be further investigated.

The Loss of Carbon Dioxide from Activated Perbenzoate Anions in the Gas Phase: Unimolecular Rearrangement via Epoxidation of the Benzene Ring

The unimolecular reactivities of a range of perbenzoate anions (X-C6H5CO3-), including the perbenzoate anion itself (X = H), nitroperbenzoates (X = para-, meta-, ortho-NO2), and methoxyperbenzoates (X = para-, meta-OCH3) were investigated in the gas phase by electrospray ionization tandem mass spectrometry. The collision-induced dissociation mass spectra of these compounds reveal product ions consistent with a major loss of carbon dioxide requiring unimolecular rearrangement of the perbenzoate anion prior to fragmentation. Isotopic labeling of the perbenzoate anion supports rearrangement via an initial nucleophilic aromatic substitution at the ortho carbon of the benzene ring, while data from substituted perbenzoates indicate that nucleophilic attack at the ipso carbon can be induced in the presence of electron-withdrawing moieties at the ortho and para positions. Electronic structure calculations carried out at the B3LYP/6-311++G(d,p) level of theory reveal two competing reaction pathways for decarboxylation of perbenzoate anions via initial nucleophilic substitution at the ortho and ipso positions, respectively. Somewhat surprisingly, however, the computational data indicate that the reaction proceeds in both instances via epoxidation of the benzene ring with decarboxylation resulting--at least initially--in the formation of oxepin or benzene oxide anions rather than the energetically favored phenoxide anion. As such, this novel rearrangement of perbenzoate anions provides an intriguing new pathway for epoxidation of the usually inert benzene ring.

Progress of Epidemiological and Molecular Epidemiological Studies on Benzene in China

Benzene is an organic solvent that has been used in industry for about 100 years throughout the world. Since 1973, a series of toxicological and molecular epidemiological studies on benzene were conducted by researchers at the Chinese Academy of Preventive Medicine (CAPM) (1973-1986) and subsequently by a collaboration between the CAPM and the National Cancer Institute (NCI) in the United States that began in 1986, which was joined by investigators from the University of California at Berkeley, the University of North Carolina at Chapel Hill, and New York University. The findings demonstrated that the risk of leukemia and lymphoma among benzene-exposed workers was significantly increased, with elevated risks for leukemia present not only at higher exposure but also among workers exposed to under 10 ppm. Therefore, the benzene permissible level was decreased to 1.8 ppm (6 mg/m(3)) and benzene-induced leukemia is treated as an occupational cancer in China. The benzene permissible level is 1.0 in the United States and in several other developed countries and it has been suggested to be decreased to 0.5 ppm (ACGIH). A number of potential biomarkers are related to benzene exposure and poisoning. Some of these are benzene oxide-protein adducts, chromosome aberration of lymphocytes, and GPA mutations in erythrocytes, a decrease in B cell and CD4(-)T cell counts in peripheral blood, and altered expression of CXCL16, ZNF331, JUN, and PF4 in lymphocytes. Variation in multiple benzene metabolizing genes may be associated with risk of benzene hematotoxicity, including CYP2E1, MPO, NQO1, and GSTT1.

Physiologically‐Based Pharmacokinetic Modeling of Benzene in Humans: A Bayesian Approach

Benzene is myelotoxic and leukemogenic in humans exposed at high doses (>1 ppm, more definitely above 10 ppm) for extended periods. However, leukemia risks at lower exposures are uncertain. Benzene occurs widely in the work environment and also indoor air, but mostly below 1 ppm, so assessing the leukemia risks at these low concentrations is important. Here, we describe a human physiologically-based pharmacokinetic (PBPK) model that quantifies tissue doses of benzene and its key metabolites, benzene oxide, phenol, and hydroquinone after inhalation and oral exposures. The model was integrated into a statistical framework that acknowledges sources of variation due to inherent intra- and interindividual variation, measurement error, and other data collection issues. A primary contribution of this work is the estimation of population distributions of key PBPK model parameters. We hypothesized that observed interindividual variability in the dosimetry of benzene and its metabolites resulted primarily from known or estimated variability in key metabolic parameters and that a statistical PBPK model that explicitly included variability in only those metabolic parameters would sufficiently describe the observed variability. We then identified parameter distributions for the PBPK model to characterize observed variability through the use of Markov chain Monte Carlo analysis applied to two data sets. The identified parameter distributions described most of the observed variability, but variability in physiological parameters such as organ weights may also be helpful to faithfully predict the observed human-population variability in benzene dosimetry.

Experimental Detection of One Case of Benzene Epoxidation by a Peroxy Radical and Computational Prediction of Another

Reaction of the beta-styryl radical with O2 in benzene results in a low yield of benzene oxide, which is shown by isotopic labeling to arise from the solvent. Ab initio and DFT calculations elucidate the mechanism of this reaction, and identify the properties of other radicals that should be more effective promoters of the reaction. The CN radical is found to be one candidate.

Workers’ exposures and potential health risks to air toxics in a petrochemical complex assessed by improved methodology

This study was designed to comprehensively evaluate workers' potential health risks of exposure to 39 air toxics in the Ta-sher Petrochemical Complex. Open-Path Fourier Transform Infrared Spectroscopy (OP-FTIR) was used to measure concentrations of air toxics. We used the measured worksite concentrations between 1997 and 1999 at 11 companies in the petrochemical complex, employing 3,100 on-site workers. The 39 measured air toxics included 10 chemicals with acute reference exposure levels (RELa), 19 chemicals with chronic reference exposure levels (RELc), and 3 chemicals classified as Class 1 or 2A human carcinogens by the International Agency for Research on Cancer (IARC). We then used RELa to calculate the hazard index of acute health effects (HI ( A )) for workers in individual plants. We also calculated the hazard index of chronic health effects (HIc) and cancer risks for all workers in the entire petrochemical complex. Workers in five companies had HI ( A ) greater than 1 because of toluene, benzene, methyl ethyl ketone, chloroform and isopropanol exposures. Workers in this petrochemical complex had HIc greater than 1 because of acrylonitrile, 1,3-butadiene, hydrogen cyanide, and n,n-dimethylformamide exposures. Risk of hematopoietic system cancer because of benzene and ethylene oxide exposure, and respiratory system cancer because of 1,3-butadiene exposure was estimated to be 3.1-6.1 x 10(-4) and 5.2-7.1 x 10(-4), respectively. Our findings indicated that workers in the petrochemical complex might have excess cancer and noncancer risks due to acute or chronic exposures to air toxics from multiple emission sources.

Development of an efficient route to hyperbranched poly(arylene ether sulfone)s

AbstractA two‐step route to an AB2 monomer that underwent polymerization via nucleophilic aromatic substitution to afford hyperbranched poly(arylene ether sulfone)s (HB PAES) was developed. The synthesis of 3,5‐difluoro‐4′‐hydroxydiphenyl sulfone (4) was accomplished by the reaction of 3,5‐difluorophenylmagnesium bromide with 4‐methoxyphenylsulfonyl chloride, followed by deprotection of the phenol group with HBr in acetic acid. The polymerization of 4 in the presence of 3,4,5‐trifluorophenylsulfonyl benzene or tris(3,4,5‐trifluorophenyl)phosphine oxide as a core molecule afforded HB PAES with number‐average molecular weights ranging from 3400 to 8400 Da and polydispersity index values ranging from 1.5 to 4.8. The presence of cyclic oligomeric species, formed by an intramolecular cyclization process, was a contributing factor to the relatively low molecular weights. The degree of branching (DB) of the HB PAES samples was estimated by a comparison of the 19F NMR spectra of the polymer samples with those of a series of model compounds, and DB values ranging from 0.51 to 0.70 were determined. The glass‐transition temperatures for the HB PAES samples were in the range of 205–222 °C, as determined by differential scanning calorimetry. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43:3178–3187, 2005

Protein adducts as biomarkers of human benzene metabolism

We used cysteinyl adducts of serum albumin (Alb) to investigate the production of two reactive benzene metabolites, namely, benzene oxide (BO) and 1,4-benzoquinone (1,4-BQ) in workers exposed to benzene. Adducts were measured in 160 benzene-exposed workers who did not use respiratory protection (based upon individual geometric mean benzene exposure levels: median = 5.27 ppm, interquartile range = 2.14–13.4 ppm, range = 0.074–328 ppm) and 101 local controls, from populations in Shanghai and Tianjin, China. After isolation of Alb, these adducts (designated as BO-Alb and 1,4-BQ-Alb) were cleaved from the protein with methanesulfonic acid and trifluoroacetic anhydride and measured by gas chromatography-mass spectrometry. Although BO-Alb and 1,4-BQ-Alb were measured in all subjects, levels of both adducts were 2.4-fold greater (median value) in exposed subjects than in controls (interquartile-fold range = 1.63–4.05 for BO-Alb and 1.64–3.69 for 1,4-BQ-Alb). Log–log plots of the individual adduct levels versus exposure were quasi-linear with straight-line slopes of about 0.3 for both BO-Alb and 1,4-BQ-Alb. Since these log-space slopes were significantly less than one, we infer that adduct production was nonlinear, i.e., less-than proportional to benzene exposure, over the indicated range. This behavior points to saturation of CYP2E1 as a critical metabolic consequence of high exposure to benzene in humans. Thus, the biologically effective dose of BO and 1,4-BQ should be proportionally greater in persons exposed to low rather than high levels of benzene.

The use of S-phenylmercapturic acid as a biomarker in molecular epidemiology studies of benzene

S-Phenylmercapturic acid ( S-PMA), is a urinary metabolite of benzene, thought to be derived from the condensation product of benzene oxide with glutathione. S-PMA may be determined by GC, HPLC (UV or fluorescence detection), GC–MS, LC–MS/MS or immunoassays. The limit of sensitivities of most of these techniques is 1 μg/l urine or below. It has been suggested that S-PMA may have value as a biomarker for low level human exposure to benzene, in view of the facts that urinary excretion of S-PMA has been found to be related to airborne benzene in occupationally exposed workers, and that only low background levels of S-PMA have been found in control subjects. We have evaluated the use of S-PMA as a biomarker, using a commercially available analytical service, in a multicentre European study of populations exposed to varying levels of benzene, in Italy (Milan, Genoa) and in Bulgaria (Sofia). These were filling station attendants, urban policemen, bus drivers, petrochemical workers and referents (a total of 623 subjects). S-PMA was measured at the end of the work shift by an immunoassay procedure. Urinary benzene (in Milan only) and the benzene metabolite trans,trans-muconic acid ( t,t-MA) were measured before and after the work shift. Air-borne benzene was measured as a monitor of exposure. Urinary benzene was the most discriminatory biomarker and showed a relationship with airborne benzene at all levels of exposure studied (including groups exposed to <0.1 ppm benzene), whereas t,t-MA and S-PMA, as determined by immunoassay, were suitable only in the highest exposed workers (petrochemical industry, geometric mean 1765 μg/m 3 (0.55 ppm) benzene). All three biomarkers were positively correlated with smoking as measured by urinary cotinine).

Investigation of cysteinyl protein adducts of benzene diolepoxide

Metabolism of benzene is fairly complex yielding several reactive metabolites that could form adducts with macromolecules such as DNA and proteins. Systemic availability of these reactive species can be estimated by measuring the corresponding adducts of these species with serum albumin or hemoglobin. We have shown previously that serum albumin adducts of benzene oxide and 1,4-benzoquinone increased in a dose-dependent manner in rodents and humans exposed to benzene. However, formation of macromolecular adducts of other reactive metabolites, such as benzene diolepoxide and E, E-muconaldehyde, has not received much attention despite the demonstrated mutagenicity and/or carcinogenicity of these metabolites. We studied the reaction of isomers of benzene diolepoxide, namely syn-((±)BDE1) and anti-BDE ((±)BDE2), with sulfhydryl groups in vitro. The half-life of BDE2 in 0.1 M ammonium acetate buffer (pH 7.6) was (5.46 h) longer that that in serum albumin in the same buffer (3.83 h) indicating the reaction of BDE with nucleophilic sites on albumin. Reaction products between these and l-cysteine, and GSH were identified and characterized by mass spectrometry in an attempt to develop an assay to quantify these adducts in serum albumin.

Use of genetically modified mouse models to assess pathways of benzene-induced bone marrow cytotoxicity and genotoxicity

Benzene induces bone marrow cytotoxicity and chromosomal breaks as a primary mode of action for the induction of bone marrow toxicity. Our research group has used genetically modified mouse models to examine metabolic and genomic response pathways involved in benzene induced cytotoxicity and genotoxicity in bone marrow and in hematopoietic stem cells (HSC). We review our studies using NQO1−/− mice and mEH−/− mice to examine the roles of these enzymes, NAD(P)H:quinone oxidoreductase-1 ( NQO1) and microsomal epoxide hydrolase ( mEH) in mediating benzene-induced toxicity. NQO1 catalyzes the detoxication of benzene quinone metabolites and mEH catalyzes the hydrolysis of benzene oxide. Our studies using gene expression profiling of bone marrow and enriched HSC populations isolated from the bone marrow of benzene-exposed mice demonstrate differential gene expression responses of key genes induced by inhaled benzene. These studies show that benzene toxicity is regulated by a number of genetic pathways that affect the production of reactive metabolites and DNA damage response pathways in a target tissue.

Reactions of Benzene Oxide with Thiols Including Glutathione

S-Phenylmercapturic acid is a minor metabolite of benzene used as a biomarker for human benzene exposures. The reaction of intracellular glutathione with benzene oxide-oxepin, the initial metabolite of benzene, is presumed to give 1-(S-glutathionyl)-cyclohexa-3,5-dien-2-ol, which undergoes dehydration to S-phenylglutathione, the precursor of S-phenylmercapturic acid. To validate the proposed route to S-phenylglutathione, reactions of benzene oxide-oxepin with glutathione and other sulfur nucleophiles have been studied. The reaction of benzene oxide with an excess of aqueous sodium sulfide, followed by acetylation, gave bis-(6-trans-5-acetoxycyclohexa-1,3-dienyl)sulfide, the structure of which was proved by X-ray crystallography. Reactions of benzene oxide-oxepin in a 95:5 (v/v) mixture of phosphate buffer in D2O with (CD3)2SO were monitored by 1H NMR spectroscopy. In the absence of glutathione, the half-life of benzene oxide-oxepin was ca. 34 min at 25 degrees C and pD 7.0. The half-life was not affected in the range of 2-15 mM glutathione in the presence and absence of a commercial sample of human glutathione S-transferase (at pH 7.0, 8.0, 8.5, or 10.0). The adduct 1-(S-glutathionyl)-cyclohexa-3,5-diene-2-ol was identified in these reaction mixtures, especially at higher pH, by mass spectrometry and by its acid-catalyzed decomposition to S-phenylglutathione. Incubation of benzene oxide with N-acetyl-L-cysteine at 37 degrees C and pH 10.0 and subsequent mass spectrometric analysis of the mixture showed formation of pre-S-phenylmercapturic acid and the dehydration product, S-phenylmercapturic acid. The data validate the premise that benzene oxide-oxepin can be captured by glutathione to give (1R,2R)- and/or (1S,2S)-1-(S-glutathionyl)-cyclohexa-3,5-dien-2-ol, which dehydrate to S-phenylglutathione. The capture is a relatively inefficient process at pH 7 that is accelerated at higher pH. These studies account for the observation that the metabolism of benzene is dominated by the formation of phenol. The pathway leading to S-phenylmercapturic acid is necessarily minor on account of the low efficiency of benzene oxide capture by glutathione at pH 7 vs spontaneous rearrangement to phenol.

Substituent effects on tautomerization of oxepine to benzene oxide: a Hammett study via ab initio

In order to find the susceptibility of the oxepine to benzene oxide tautomerization to the substituent effects, equilibrium constants are found for conversion of 3-(X)-oxepines to their corresponding 2-(X)-benzene oxides (Series 1), as well as 4-(X)-oxepines to analogous 3-(X)- benzene oxides (Series 2) where X=H, NH 2, OCH 3, CH 3, CF 3, F, Cl, Br, NO 2 and CN. For the equilibria related to Series 1, the K eq are: 31.29 ( H), 0.01 (NH 2), 498.58 (OCH 3), 2.71 (CH 3), 4.19 (CF 3), 0.31 (F), 1.03 (Cl), 1.72 (Br), 0.31 (NO 2) and 1.89 (CN). For Series 2, the K eq are: 31.29 ( H), 47.73 (NH 2), 7.97 (OCH 3), 16.48 (CH 3), 44.61 (CF 3), 52.81 (F), 52.81(Cl), 64.67 (Br), 374.21 (NO 2) and 107.31(CN). Experimental K eq has only been reported for X=H, which appears to consistent with that calculated in this report. Using B3LYP/6-31G* Hammett ρ value of 0.8 and 0.6 are obtained for Series 1 and 2, respectively; suggesting that electron withdrawing groups slightly shift the equilibrium in favor of benzene oxide. All rate constants ( k r), activation electronic energies (Δ E #), enthalpies (Δ H #) and Gibbs free energies (Δ G #) for 1 ⇌ 2 tautomerizations, are calculated at HF, DFT (B3LYP) and MP2 levels of theory, using 6-31G* standard basis set.