Influence of toluene co-exposure on the metabolism and genotoxicity of benzene in mice using continuous and intermittent exposures.

Benzene exposure in occupational settings often occurs with concurrent exposure to toluene, the methyl-substituted derivative of benzene. Toluene is also readily metabolized by CYP450 isozymes although oxidation primarily occurs in the methyl group. While earlier mouse studies addressing co-exposure to benzene and toluene at high concentrations demonstrated a reduction in benzene-induced genotoxicity, we have previously found, using an intermittent exposure regimen with lower concentrations of benzene (50 ppm) and toluene (100 ppm), that toluene enhances benzene-induced clastogenic or aneugenic bone marrow injury in male CD-1 mice with significantly increased CYP2E1, and depleted GSH and GSSG levels. The follow-up study reported here also used the same daily and total co-exposures but over consecutive days and compared the effects of co-exposure on genotoxicity and metabolism in CD-1 mice both with and without buthionine sulfoximine (BSO) treatment to deplete GSH. In this study the toluene co-exposure doubled the genotoxic response (as determined by the erythrocyte micronucleus test) to benzene alone. Further, GSH depletion caused a reduction in this genotoxicity in both benzene exposed and benzene/toluene co-exposed mice. The results are discussed in terms of the analyses of urinary metabolites from this consecutive day study and the intermittent exposure study as well as levels of CYP2E1, epoxide hydrolase, quinone reductase, alcohol dehydrogenase, and aldehyde dehydrogenase activities. The results suggest that the presence of glutathione is necessary for benzene genotoxicity either as a metabolite conjugate or through an indirect mechanism such as TNF-induced apoptosis.

Homology modelling of human CYP2E1 based on the CYP2C5 crystal structure: investigation of enzyme-substrate and enzyme-inhibitor interactions.

The construction of a homology model of human cytochrome P450 2E1 (CYP2E1) is reported, based on the CYP2C5 crystallographic template. A relatively high degree of primary sequence homology (identity=59%), as expected for proteins of the same CYP family, ensured a straightforward generation of the 3-dimensional model due to relatively few deletions and insertions of amino acid residues with respect to the CYP2C5 crystal structure. Probing the CYP2E1 model with typical substrates of the enzyme showed a good agreement with experimental information in the form of positions of metabolism for substrates, and with site-directed mutagenesis data on certain residues. Furthermore, quantitative relationships between substrate binding affinity and various structural parameters associated with the substrate molecules facilitated the formulation of a procedure for estimating relative binding energy and, consequently, K(m) or K(D) values towards the CYP2E1 enzyme. This method has been based on a consideration of the active site interactions between substrates and key amino acid residues lining the haem pocket, together with compound lipophilicity data from partition coefficients.

Human carcinogens: an evaluation study via the COMPACT and HazardExpert procedures.

The results of computer-optimized molecular parametric analysis of chemical toxicity (COMPACT) and HazardExpert evaluations on 14 established human carcinogens are reported. The concordances between COMPACT and carcinogenicity (71%) and between HazardExpert and carcinogenicity (57%) are significantly improved when taken in combination, where all 14 carcinogens are correctly identified by the two systems used in conjunction. However, if a negative energy of the highest occupied molecular orbital (E(HOMO)) value is regarded as evidence of electrophilic reactivity likely to give rise to mutagenicity and carcinogenicity, then 13/14 (93%) of the carcinogens are correctly identified by combination with the COMPACT procedure alone. It is possible, therefore, to establish likely carcinogenicity arising from either P450 mediation (CYP1 and CYP2E) or compound electrophilicity via the employment of a straightforward approach to molecular and electronic structure calculation, a process that can be performed in a relatively short time frame (i.e., less than 1 hour per chemical) and at a low cost.

Application of process chemistry and SAR modelling to the evaluation of health findings of lower olefins.

Epidemiology studies show increased leukemia mortality among styrene butadiene rubber (SBR) workers but not among butadiene monomer production employees. A detailed review of the SBR manufacturing process indicates that sodium dimethyldithiocarbamate (DMDTC) introduced into the SBR manufacturing process for a period in the 1950s coincides with increased leukemia mortality. Using the Computer-Optimized Molecular Parametric Analysis of Chemical Toxicity (COMPACT), we assessed the enzyme (cytochrome P450) substrate specificity of an olefin series including 1,3-butadiene (BD) and also modeled its interaction with DMDTC. These analyses showed correlation of a structural/electronic parameter--the COMPACT radius--with the presence or absence of cytogenetic activity and also found that DMDTC would inhibit the oxidative metabolism of BD at least at high concentrations. Both DMDTC and its diethyl analog (DEDTC) bind with CYP 2E1 and CYP 2A6. Both of these isoforms are important in the initial oxidative metabolism of butadiene and other olefins. In co-exposure studies in mice of DMDTC with BD or with epoxybutene (EB), we found that there was a reduced increase in genotoxic activity based on micronuclei induction compared with BD or EB exposure alone. Treatment with DMDTC significantly increased the protein carbonyl contents of hepatic microsomes compared with that of controls, a finding that may be related to DMDTC's activity as a prooxidant. Co-exposure with DMDTC and EB increased hepatic microsomal carbonyls to levels significantly greater than those of DMDTC-treated mice, while EB administration in the absence of DMDTC did not change protein carbonyls relative to those of controls. The increase in hepatic microsomal protein carbonyls suggests that DMDTC may modulate EB metabolism towards the formation of reactive intermediates that react with proteins. The present molecular modeling and mechanistic studies suggest that co-exposure of BD and DMDTC is a plausible biological hypothesis regarding increased leukemia risk among SBR workers.

Molecular modelling of human CYP2E1 by homology with the CYP102 haemoprotein domain: investigation of the interactions of substrates and inhibitors within the putative active site of the human CYP2E1 isoform.

1. The construction of a three-dimensional model of human CYP2E1 is reported. It is based on homology with the haemoprotein domain of the unusual bacterial P450, CYP102, which is of known crystal structure. 2. Interactive docking of a number of human CYP2E1 substrates is consistent with their known positions of CYP2E1-mediated metabolism, where specific interactions with key active site amino acid side-chains appear to rationalize the binding and orientation of substrate molecules. 3. Amino acid residues within the putative active site of human CYP2E1, including those associated with the binding of substrates and inhibitors, are shown to correspond with those identified by site-directed mutagenesis experiments conducted on CYP2 family isoforms, and they are known to affect substrate metabolism regioselectivity. 4. Consequently, it was found that the CYP2E1 active site exhibits complementarity with the structural characteristics of known substrates and inhibitors of this enzyme, including their relatively low molecular weights and disposition of hydrogen bond-forming groups.

Potential biomarkers of benzene exposure.

Biological markers or biomarkers of exposure are indicators for the evaluation of the internal dose of a xenobiotic. Biomarkers integrate exposure from all routes and sources. This review presents a short overview of potential biomarkers of benzene exposure currently under investigation, the methodology used for their determination, and experimental findings and their usefulness and specificity in assessing exposure to benzene. Potential biomarkers of benzene exposure are benzene, benzene metabolites, and adducts formed by reactive benzene metabolites with cellular constituents. The potential biomarkers of benzene exposure described in this review are: (1) benzene, the parent hydrocarbon; (2) ring-hydroxylated urinary metabolites, phenol, catechol, hydroquinone, and 1,2,4-trihydroxybenzene; (3) trans,trans-muconic acid, a urinary ring-opened metabolite; (4) N-acetyl-S-(2,5-dihydroxyphenyl)-L-cysteine, a urinary metabolite of benzene, phenol, and hydroquinone; (5) S-phenylmercapturic acid, a glutathione-derived adduct; (6) N7-phenylguanine, a DNA adduct; and (7) S-phenylcysteine and N-phenyl-valine, hemoglobin/protein-derived adducts.

Oncogenicity studies of inhaled methyl tertiary-butyl ether (MTBE) in CD-1 mice and F-344 rats.

Oncogenicity studies of methyl tertiary-butyl ether (MTBE) vapor were conducted in CD-1 mice and Fischer 344 rats. Fifty animals of each sex per species per group were exposed for 6 h a day, 5 days per week to 0 (control), 400, 3000 and 8000 ppm MTBE vapor in air for 18 months (mice) and 24 months (rats). Both species showed reversible central nervous system depression at 8000 ppm for the first week of exposure, which continued for mice for the study duration. For the 8000 ppm mice, reduced body weight gain and early mortality prior to terminal euthanasia were exposure related. In the males, these deaths appear to be due to exacerbation of uropathy or dysuria, which occurs spontaneously in this strain. Increases in absolute and relative liver (both sexes) and kidney weight (males only) were seen at 3000 and 8000 ppm and decreases in brain and spleen weights were also noted (the latter decreases were without microscopic lesions and occurred at 8000 ppm only). An increase in hepatocellular hypertrophy occurred in both sexes at the two highest concentrations. The only neoplastic lesion found in this study in mice was an increased incidence of hepatocellular adenomas in females at the 8000 ppm exposure. In a follow-up study, a statistically significant elevation of cell proliferation in female mouse liver has been shown to occur following 5 days, but not 28 days, of exposure to 8000 ppm MTBE, suggesting that MTBE induces mitogenesis. For male rats, early euthanasia was required at week 82 and week 97 for the 8000 and 3000 ppm groups, respectively, due to excessive mortality from a severe progressive nephrosis. The end stage of this process appeared earlier in the male rats of all MTBE exposure groups; the incidence of this lesion and mortality for exposed females was comparable to control females. No exposure-related changes in hematological parameters were observed for any group at any time point, but a decrease in corticosterone levels was seen for male rats from the 8000 ppm group. Absolute and relative kidney and liver weight increases occurred in 3000 and 8000 ppm exposure groups, but the liver weight change was not accompanied by histopathological change. At study termination, increases in the incidence and severity of a chronic nephropathy in males from all exposure groups and in females exposed to 3000 and 8000 ppm was associated with secondary lesions of hyperplasia of the parathyroid and mineralization of tissues. Renal tubular cell tumors were increased in male rats exposed to 3000 and 8000 ppm. This may be associated with an accumulation of protein (stainable by Mallory's Heidenhain) in kidney tubular epithelial cells after 4 weeks of exposure. An increased incidence of interstitial cell adenomas of the testes was seen in males exposed to 3000 and 8000 ppm but was believed to be an artefact of an unusually low control incidence and not considered to be exposure related. Based on the above effects, the no-observed-effect level (NOEL) for chronic toxicity is 400 ppm, and the NOEL for carcinogenic effects is 3000 ppm (mice) and 400 ppm (rats).

Molecular modelling of CYP2E1 enzymes from rat, mouse and man: an explanation for species differences in butadiene metabolism and potential carcinogenicity, and rationalization of CYP2E substrate specificity.

Molecular modelling of substrates of cytochrome P4502E1 (CYP2E1) within the putative active site region of CYP2E1 constructed from the CYP102 crystal structure is reported. Structural characteristics of CYP2E1 substrates, such as molecular size, energy levels and polarity, calculated via molecular orbital procedures provide correlations with toxicity and carcinogenicity; and species differences in CYP2E1-mediated metabolism are rationalized in terms of interactions with putative active site amino acid residues, including Thr-437 and Phe-181. In particular, the activation of buta-1,3-diene can be explained by active site modelling with CYP2E1 enzymes sequenced from rat, mouse and man, where there is a non-conservative change T437H between rodent and human isozymes, together with a conservative change I438V between mouse and rat CYP2E1.

Chronic toxicity and carcinogenic evaluation of diisononyl phthalate in rats.

Groups of 110 Fischer 344 rats/sex were fed diisononyl phthalate (DINP) at dietary levels of 0, 0.03, 0.3, and 0.6 wt% for periods up to 2 years. Interim sacrifices of 10 predesignated rats/sex/dose were at 6, 12, and 18 months with surviving animals sacrificed at 24 months. At study termination, survival was in excess of 60% for every group. At the mid or high dose, the following biological effects were noted: slight decreases in food consumption and body weight; slight increase in mortality; a dose-related increase in relative organ weights of liver and kidney; and some slight effects on urinalysis, hematologic, and clinical chemistry parameters. No peroxisome induction was observed in livers of treated rats compared with controls. No clear treatment-related nonneoplastic or neoplastic lesions were found. However, mononuclear cell leukemia (MNCL) and changes known to be associated with an increased incidence of MNCL were seen in the mid-dose and high-dose groups. A literature review suggests that MNCL is a common finding in aging F344 rats and that this increased incidence in rats treated with DINP is not relevant to man. A clear no-observed-effect level was demonstrated for all biological end points at a dietary level of 0. 03 wt% or approximately 17 mg/kg/day of DINP.

Determination of leukemogenic benzene exposure concentrations: refined analyses of the Pliofilm cohort.

Biologic data on benzene metabolite doses, cytotoxicity, and genotoxicity often show that these effects do not vary directly with cumulative benzene exposure (i.e., concentration times time, or c x t). To examine the effect of an alternate exposure metric, we analyzed cell-type specific leukemia mortality in Pliofilm workers. The work history of each Pliofilm worker was used to define each worker's maximally exposed job/department combination over time and the associated long-term average concentration associated with the maximally exposed job (LTA-MEJ). Using this measure, in conjunction with four job exposure estimates, we calculated SMRs for groups of workers with increasing LTA-MEJs. The analyses suggest that a critical concentration of benzene exposure must be reached in order for the risk of leukemia or, more specifically, AMML to be expressed. The minimum concentration is between 20 and 60 ppm depending on the exposure estimate and endpoint (all leukemias or AMMLs only). We believe these analyses are a useful adjunct to previous analyses of the Pliofilm data. They suggests that (a) AMML risk is shown only above a critical concentration of benzene exposure, measured as a long-term average and experienced for years, (b) the critical concentration is between 50 and 60 ppm when using a median exposure estimate derived from three previous exposure assessments, and is between 20 and 25 ppm using the lowest exposure estimates, and (c) risks for total leukemia are driven by risks for AMML, suggesting that AMML is the cell type related to benzene exposure.

Isoprene cancer risk and the time pattern of dose administration.

Most statistical risk assessment models assume that equal, measured on scale such as mg/kg/day, create equal tumor risks. This equivalent dose metric (EDM) hypothesis allows risks to be extrapolated from high concentrations to low-concentrations and from and species, sex, and strain to another, since it implies that all administered dose histories corresponding to the same total dose create the same risk. This paper tests the EDM hypothesis using data on tumor rates in B6C3F mice administered isoprene via inhalation. Its major conclusion is that the EDM hypothesis does not hold for isoprene. For example, it appears that exposure concentration has a greater impact on tumor rates than weeks of exposure. To predict tumor probabilities, the time pattern of dose administration must be considered. The asymmetric effects of concentration, hours-per-day, and number of days of exposure on tumor risks imply that complex dynamic risk models may be needed to accurately describe dose-time-response relations. The traditional concept of a dose-response relation as a static curve relating a numerical summary of dose to a numerical summary of response probability is probably not predictively useful for chemicals such as isoprene, and extrapolations of risk based on the EDM hypothesis could be misleading for such chemicals.

Genotoxicity and twenty-eight-day subchronic toxicity studies on tertiary amyl methyl ether.

Tertiary amyl methyl ether (TAME) is an oxygenate with a potential role as a component in reformulated gasolines. The genotoxic potential of TAME was assessed in an Ames assay and a mouse micronucleus assay. The Ames assay was carried out using five standard salmonella strains and doses ranging from 100 to 10,000 micrograms per plate. Tertiary amyl methyl ether was not mutagenic in any of the strains, either with or without metabolic activation. In the micronucleus assay, mice were given a single intraperitoneal injection of TAME at doses of 0.15, 0.375 or 0.75 g kg-1. Bone marrow samples were collected and evaluated for micronucleus formation at 24, 48 and 72 h after dosing. No elevation in micronucleus frequency was observed at any dose or at any of the collection times. Thus, TAME was not clastogenic to mouse bone marrow under the conditions of this study. Preliminary test data indicated that the acute oral LD50 for TAME in Sprague-Dawley rats was ca. 2.1 g kg-1. In the 28-day subchronic study, Sprague-Dawley rats of both sexes were dosed orally with vehicle, 0.125, 0.5 or 1.0 g kg-1 day-1 TAME in corn oil at a dose volume of 2 ml/kg-1. Dosing continued 7 days a week for a period of 28 days. Deaths of two out of 10 animals in the high-dose group (1 g kg-1 day-1) appeared to be compound related. Food consumption and body weights were reduced in the high-dose male group relative to controls; otherwise, clinical observations were minimal.(ABSTRACT TRUNCATED AT 250 WORDS)

Subchronic feeding study of four white mineral oils in dogs and rats.

Subchronic 90-day feeding studies were conducted on four highly refined white mineral oils to determine any potential for toxicity in Long-Evans rats (20 per sex per dose level) and beagle dogs (4 per sex per dose level). Each oil was fed at dietary dose levels of 300 ppm and 1500 ppm (w/w). No treatment-related effects of toxicological importance were detected in daily observations of general health or in periodic assessments of food consumption and body weight, hematology, serum clinical chemistry, and urinalysis. Observations in dogs suggested that the white oils produced mild laxative effects. Gross and histopathologic examinations, as well as measurements of organ weights, did not reveal any macroscopic or microscopic changes which could be due to treatment. In addition, special staining by Oil Red O of liver, mesenteric lymph nodes, spleen, gastrointestinal tract, stomach, and kidneys indicated no evidence of oil or lipid deposition. A special re-examination of tissues from female and male rats, in response to more recent conflicting data from the Fischer 344 strain, found no histopathologic signs of macrophage accumulation and/or microgranuloma formation in liver, spleen, or mesenteric lymph nodes. These data indicate that repeated exposure to relatively high levels of white mineral oils in the diets does not produce significant subchronic toxicity in Long-Evans rats or beagle dogs.

An industrial hygiene methodology developed in support of a retrospective morbidity case-control study.

An industrial hygiene methodology that was developed in support of an epidemiologic case-control study is described. This study was conducted to investigate a potentially increased incidence of colorectal cancer among employees who worked at a unit that manufactured polypropylene by a heavy diluent process. Retrospective epidemiologic case-control studies typically have exposure assessment problems because industrial hygiene monitoring data are often not available. Misleading job titles are another problem that can provide a poor framework for estimating exposures. In addition, a job title-based exposure assessment assumes individual work patterns are not important in assessing worker exposures, although this is contradictory to industrial hygiene experience. The design of the case-control study provided an innovative industrial hygiene approach to circumvent these typical exposure estimating problems. The industrial hygiene methodology of the case-control study included assessing historical exposures, developing an exposure estimating matrix, selecting candidate etiologic agents based on a joint toxicologic and industrial hygiene review, administering a work activity questionnaire, calculating exposure scores, and conducting a statistical analysis. The study design also provided an additional exposure measurement independent of the toxicologic and industrial hygiene review. This provided an opportunity to compare the likelihood of exposure misclassification and, as expected, showed that a more detailed exposure estimate resulted in less misclassification.

An adenomatous polyp case-control study to assess occupational risk factors following a workplace colorectal cancer cluster.

To assess potential etiologic factors among a population of Texas polypropylene workers previously found to have an excess of colorectal cancer (concentrated among mechanical and process workers), the authors conducted an adenomatous polyp case-control study. Cases (n = 24) were workers found to have adenomatous polyps during a company-sponsored colorectal cancer screening program, while controls (n = 72) were workers found to be free of polyps. Exposure assessment utilized a thorough industrial hygiene and toxicologic review of operations in conjunction with work activity interviews of cases and controls to develop chemical-specific exposure scores weighted by time, exposure level, and frequency of exposure. Stratified analyses and logistic regression found that cases tended to have higher exposure to pre-extrusion polymer plus additives (odds ratio (OR) = 2.6, 90% confidence interval (CI) 1.1-6.3) and higher exposure to certain finishing additives (OR = 4.8, 90% CI 1.5-15.3). Analyses by job category or area of the plant did not indicate additional risk factors. The exposures identified in this study are discussed in light of the available toxicologic data on these and related compounds. The need for confirmatory experimental and epidemiologic studies is noted.

Future directions--toxicology studies of 1,3-butadiene and isoprene.

Examination of a profile of data already existing on 1,3-butadiene shows adequate knowledge in many areas of toxicology that are conventionally required in hazard identification. However, while much progress has been made in areas of metabolism and pharmacokinetics, further studies would be worthwhile to improve mechanistic understanding such as the examination of alternate metabolic pathways, the generation of interspecies scaling factors, and an assessment of the relevance of various tumor sites. In this respect, data pertaining to repeated and pulse exposures of rodents and primates would be helpful. Another important aspect is the need to understand any human health implications of the observed 1,3-butadiene-induction of the murine leukemia virus. In this respect, studies have been pursued that include the comparison of leukemogenesis in congenetic strains, the leukemogenicity of viral isolates in rodent carcinogenicity and human cell culture studies, and the mechanisms of activation of ecotropic proviral sequences. Molecular epidemiological and toxicological research is ongoing in rodents and primates to evaluate hemoglobin adduct formation as an index of 1,3-butadiene exposure. Challenges of specificity, sensitivity, and simplification of current procedures need to be overcome. Recent mutagenicity and metabolism data suggest that structurally-related isoprene may have carcinogenic potential. The use of interstrain comparative studies and data in a second species is discussed, as well as proposed metabolism studies.

Inhalation toxicity of high flash aromatic naphtha.

A petroleum distillate--a high aromatic naphtha--consisting of a 50/50 blended mixture of equivalent products. SHELLSOL A* and SOLVESSO 100**, containing C9 isomers (75 percent) particularly trimethyl benzenes, was examined for systemic toxicity in rats by inhalation exposure. A preliminary 13-week inhalation study with SHELLSOL A had resulted in liver and kidney weight increases in female rats at the high (7400 mg/m3) and medium (3700 mg/m3) exposure levels, and a low grade anaemia in females at all exposure levels (7400, 3700 and 1800 mg/m3). The follow-up 12-month inhalation study in rats described here used atmosphere generated from the SHELLSOL A/SOLVESSO 100 blend of 1800, 900 and 450 mg/m3. Initial reduction in body weight gain occurred in both male and female rats at the higher exposures. Various statistically significant haematological changes were transiently seen in males up to six months, but were not considered biologically significant. High exposure male liver and kidney weights were increased at 6 and 12 months but, in the absence of histopathological changes, were considered to be physiological adaptive responses. No treatment-related histopathological abnormalities were found. It is concluded that chronic exposure to this high aromatic naphtha is without systemic toxicity in rats under the conditions of these studies.

An association of upper respiratory cancer with exposure to diethyl sulfate.

A morbidity and mortality study of workers at an alcohol manufacturing plant which included several weak acid isopropyl alcohol units and a strong acid ethanol unit is described. An excess mortality of upper respiratory cancer was found and associated with work on the strong acid ethanol unit. The strong acid ethanol process used resulted in high concentrations of diethyl sulfate, which has been shown to be carcinogenic in animals, and the unit, which closed on 1975, had significant opportunities for worker exposure to diethyl sulfate. These facts, plus previous reports of excess upper respiratory cancer on strong acid isopropyl alcohol units with similarly high concentrations of the animal carcinogen diisopropyl sulfate, lead to the tentative conclusion that diethyl sulfate was primarily responsible for the ethanol unit cancer cases. In the modern weak acid isopropyl alcohol plants, where only trace amounts of diisopropyl sulfate are present and exposures are much lower, the problems found on the old strong acid units do not exist.