Methodological issues in biomonitoring of low level exposure to benzene.
Data from a pilot study on unmetabolized benzene and trans,trans muconic acid (t,t-MA) excretion in filling station attendants and unexposed controls were used to afford methodological issues in the biomonitoring of low benzene exposures (around 0.1 ppm). Urinary concentrations of benzene and t,t-MA were measured by dynamic head-space capillary GC/FID and HPLC, respectively. The accuracy of the HPLC determination of t,t-MA was assessed in terms of inter- and intra-method reliability. The adequacy of urinary t,t-MA and benzene as biological markers of low benzene exposure was evaluated by analysing the relationship between personal exposure to benzene and biomarker excretion. Filling station attendants excreted significantly higher amounts of benzene, but not of t,t-MA, than controls. Adjusting for occupational benzene exposure, smokers excreted significantly higher amounts of t,t-MA, but not of unmetabolized benzene, than nonsmokers. A comparative analysis of the present and previously published biomonitoring surveys showed a good inter-study agreement regarding the amount of t,t-MA and unmetabolized benzene excreted (about 0.1-0.2 mg/l and 1-2 micrograms/l, respectively) per unit of exposure (0.1 ppm). For each biomarker, based on the distribution of parameters observed in the pilot study, we calculated the minimum sample size required to estimate the population mean with given confidence and precision. (+info)
The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases and catalyzes aromatic monohydroxylation of benzene, toluene, and phenol.
The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase alpha-subunit (XamoA); aamB, encoding the 88-residue oxygenase gamma-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase beta-subunit (XamoE); and aamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of sigma54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase alpha-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase from Rhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacter strain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture of o-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studies Xanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of the aam gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol. (+info)
Benzene-induced uncoupling of naphthalene dioxygenase activity and enzyme inactivation by production of hydrogen peroxide.
Naphthalene dioxygenase (NDO) is a multicomponent enzyme system that oxidizes naphthalene to (+)-cis-(1R,2S)-1,2-dihydroxy-1, 2-dihydronaphthalene with consumption of O2 and two electrons from NAD(P)H. In the presence of benzene, NADH oxidation and O2 utilization were partially uncoupled from substrate oxidation. Approximately 40 to 50% of the consumed O2 was detected as hydrogen peroxide. The rate of benzene-dependent O2 consumption decreased with time, but it was partially increased by the addition of catalase in the course of the O2 consumption by NDO. Detailed experiments showed that the total amount of O2 consumed and the rate of benzene-induced O2 consumption increased in the presence of hydrogen peroxide-scavenging agents, and further addition of the terminal oxygenase component (ISPNAP) of NDO. Kinetic studies showed that ISPNAP was irreversibly inactivated in the reaction that contained benzene, but the inactivation was relieved to a high degree in the presence of catalase and partially relieved in the presence of 0.1 mM ferrous ion. Benzene- and naphthalene-reacted ISPNAP gave almost identical visible absorption spectra. In addition, hydrogen peroxide added at a range of 0.1 to 0.6 mM to the reaction mixtures inactivated the reduced ISPNAP containing mononuclear iron. These results show that hydrogen peroxide released during the uncoupling reaction acts both as an inhibitor of benzene-dependent O2 consumption and as an inactivator of ISPNAP. It is proposed that the irreversible inactivation of ISPNAP occurs by a Fenton-type reaction which forms a strong oxidizing agent, hydroxyl radicals (. OH), from the reaction of hydrogen peroxide with ferrous mononuclear iron at the active site. Furthermore, when [14C]benzene was used as the substrate, cis-benzene 1,2-dihydrodiol formed by NDO was detected. This result shows that NDO also couples a trace amount of benzene to both O2 consumption and NADH oxidation. (+info)
Adenosylcobalamin-mediated methyl transfer by toluate cis-dihydrodiol dehydrogenase of the TOL plasmid pWW0.
We identified and characterized a methyl transfer activity of the toluate cis-dihydrodiol (4-methyl-3,5-cyclohexadiene-cis-1, 2-diol-1-carboxylic acid) dehydrogenase of the TOL plasmid pWW0 towards toluene cis-dihydrodiol (3-methyl-4,5-cyclohexadiene-cis-1, 2-diol). When the purified enzyme from the recombinant Escherichia coli containing the xylL gene was incubated with toluene cis-dihydrodiol in the presence of NAD+, the end products differed depending on the presence of adenosylcobalamin (coenzyme B12). The enzyme yielded catechol in the presence of adenosylcobalamin, while it gave 3-methylcatechol in the absence of the cofactor. Adenosylcobalamin was transformed to methylcobalamin as a result of the enzyme reaction, which indicates that the methyl group of the substrate was transferred to adenosylcobalamin. Other derivatives of the cobalamin such as aquo (hydroxy)- and cyanocobalamin did not mediate the methyl transfer reaction. The dehydrogenation and methyl transfer reactions were assumed to occur concomitantly, and the methyl transfer reaction seemed to depend on the dehydrogenation. To our knowledge, the enzyme is the first dehydrogenase that shows a methyl transfer activity as well. (+info)
Determination of the urinary benzene metabolites S-phenylmercapturic acid and trans,trans-muconic acid by liquid chromatography-tandem mass spectrometry.
To investigate how various levels of exposure affect the metabolic activation pathways of benzene in humans and to examine the relationship between urinary metabolites and other biological markers, we have developed a sensitive and specific liquid chromatographic-tandem mass spectrometric assay for simultaneous quantitation of urinary S-phenylmercapturic acid (S-PMA) and trans,trans-muconic acid (t,t-MA). The assay involves spiking urine samples with [13C6]S-PMA and [13C6]t,t-MA as internal standards and clean up of samples by solid-phase extraction with subsequent analysis by liquid chromatography coupled with electrospray-tandem mass spectrometry-selected reaction monitoring (LC-ES-MS/MS-SRM) in the negative ionization mode. The efficacy of this assay was evaluated in human urine specimens from smokers and non-smokers as the benzene-exposed and non-exposed groups. The coefficient of variation of runs on different days (n = 8) for S-PMA was 7% for the sample containing 9.4 microg S-PMA/l urine, that for t,t-MA was 10% for samples containing 0.07 mg t,t-MA/l urine. The mean levels of urinary S-PMA and t,t-MA in smokers were 1.9-fold (P = 0.02) and 2.1-fold (P = 0.03) higher than those in non-smokers. The mean urinary concentration (+/-SE) was 9.1 +/- 1.7 microg S-PMA/g creatinine [median 5.8 microg/g, ranging from not detectable (1 out of 28) to 33.4 microg/g] among smokers. In non-smokers' urine the mean concentration was 4.8 +/- 1.1 microg S-PMA/g creatinine (median 3.6 microg/g, ranging from 1.0 to 19.6 microg/g). For t,t-MA in smokers' urine the mean (+/-SE) was 0.15 +/- 0.03 mg/g creatinine (median 0.11 mg/ g, ranging from 0.005 to 0.34 mg/g); the corresponding mean value for t,t-MA concentration in non-smokers' urine was 0.07 +/- 0.02 mg/g creatinine [median 0.03 mg/g, ranging from undetectable (1 out of 18) to 0.48 mg/g]. There was a correlation between S-PMA and t,t-MA after logarithmic transformation (r = 0.41, P = 0.005, n = 46). (+info)
Metabolism of [14C]phenol in the isolated perfused mouse liver.
A previous report from this laboratory focused on the metabolism of [14C]benzene (BZ) in the isolated, perfused, mouse liver (C. C. Hedli, et al., 1997, Toxicol. Appl. Pharmacol. 146, 60-68). Whereas administration of BZ to mice results in bone marrow depression (R. Snyder et al., 1993, Res. Commun. Chem. Pathol. Pharmacol. 20, 191-194), administration of phenol (P), the major metabolite of BZ, does not. It was, therefore, of interest to determine whether the metabolic fate of P produced during BZ metabolism differed from that of P metabolized in the absence of BZ. Mouse livers were perfused with a solution of [14C]P in both the orthograde (portal vein to central vein) and retrograde (central vein to portal vein) direction to investigate the metabolic zonation of enzymes involved in P hydroxylation and conjugation. Perfusate samples were collected, separated by HPLC, and tested for radioactivity. Unconjugated metabolites were identified by comparing their retention times with nonradiolabeled standards, which were detected by UV absorption. Conjugated metabolites were identified and collected on the basis of radiochromatogram results, hydrolyzed enzymatically, and identified by co-chromatography with unlabeled BZ metabolites. The objective was to compare and quantify the metabolites formed during the perfusion of P in the orthograde and retrograde directions and to compare the orthograde P-perfusion results with the orthograde BZ results reported previously. Regardless of the direction of P perfusion, the major compounds released from the liver were P. phenylgucuronide, phenylsulfate, hydroquinone (HQ), and HQ glucuronide. A comparison of the results of perfusing P in the orthograde versus the retrograde direction showed that more P was recovered unchanged and more HQ was formed during retrograde perfusion. The results suggest that enzymes involved in P hydroxylation are generally closer to the central vein than those involved in conjugation, and that during retrograde perfusion, P metabolism may be limited by the sub-optimal conditions of perfusion. Comparison of the orthograde perfusion studies of P and BZ revealed that a larger percentage of the radioactivity released from the liver was identified as unconjugated HQ after BZ perfusion than after P perfusion. In addition, the amount of radioactivity covalently bound to liver macromolecules was measured after each perfusion and determined to be proportional to the amount of HQ and HQG detected in the perfusate samples. (+info)
Environmental exposure to volatile organic compounds among workers in Mexico City as assessed by personal monitors and blood concentrations.
Benzene, an important component in gasoline, is a widely distributed environmental contaminant that has been linked to known health effects in animals and humans, including leukemia. In Mexico City, environmental benzene levels, which may be elevated because of the heavy traffic and the poor emission control devices of older vehicles, may pose a health risk to the population. To assess the potential risk, portable passive monitors and blood concentrations were used to survey three different occupational groups in Mexico City. Passive monitors measured the personal exposure of 45 workers to benzene, ethylbenzene, toluene, o-xylene and m-/p-xylene during a work shift. Blood concentrations of the above volatile organic compounds (VOCs), methyl tert-butyl ether, and styrene were measured at the beginning and the end of a work shift. Passive monitors showed significantly higher (p > 0.0001) benzene exposure levels among service station attendants (median = 330 microg/m3; range 130-770) as compared to street vendors (median = 62 microg/m3; range 49-180) and office workers (median = 44 microg/m3, range 32-67). Baseline blood benzene levels (BBLs) for these groups were higher than those reported for similar populations from Western countries (median = 0.63 microg/L, n = 24 for service station attendants; median = 0.30 microg/L, n = 6 for street vendors; and median = 0.17 microgr;g/L, n = 7 for office workers). Nonsmoking office workers who were nonoccupationally exposed to VOCs had BBLs that were more than five times higher than those observed in a nonsmoking U.S. population. BBLs of participants did not increase during the work shift, suggesting that because the participants were chronically exposed to benzene, complex pharmacokinetic mechanisms were involved. Our results highlight the need for more complete studies to assess the potential benefits of setting environmental standards for benzene and other VOCs in Mexico. (+info)
Microbial communities associated with anaerobic benzene degradation in a petroleum-contaminated aquifer.
Microbial community composition associated with benzene oxidation under in situ Fe(III)-reducing conditions in a petroleum-contaminated aquifer located in Bemidji, Minn., was investigated. Community structure associated with benzene degradation was compared to sediment communities that did not anaerobically oxidize benzene which were obtained from two adjacent Fe(III)-reducing sites and from methanogenic and uncontaminated zones. Denaturing gradient gel electrophoresis of 16S rDNA sequences amplified with bacterial or Geobacteraceae-specific primers indicated significant differences in the composition of the microbial communities at the different sites. Most notable was a selective enrichment of microorganisms in the Geobacter cluster seen in the benzene-degrading sediments. This finding was in accordance with phospholipid fatty acid analysis and most-probable-number-PCR enumeration, which indicated that members of the family Geobacteraceae were more numerous in these sediments. A benzene-oxidizing Fe(III)-reducing enrichment culture was established from benzene-degrading sediments and contained an organism closely related to the uncultivated Geobacter spp. This genus contains the only known organisms that can oxidize aromatic compounds with the reduction of Fe(III). Sequences closely related to the Fe(III) reducer Geothrix fermentans and the aerobe Variovorax paradoxus were also amplified from the benzene-degrading enrichment and were present in the benzene-degrading sediments. However, neither G. fermentans nor V. paradoxus is known to oxidize aromatic compounds with the reduction of Fe(III), and there was no apparent enrichment of these organisms in the benzene-degrading sediments. These results suggest that Geobacter spp. play an important role in the anaerobic oxidation of benzene in the Bemidji aquifer and that molecular community analysis may be a powerful tool for predicting a site's capacity for anaerobic benzene degradation. (+info)