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Purpose: Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy., Experimental Design: PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3ε transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti-PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque., Results: We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ε bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell-mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF -mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti-PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile., Conclusions: These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies., (©2020 American Association for Cancer Research.)

Bisexual people face unique stressors related to their sexual orientation, but identity invalidation (experiences in which others deny, negate, or refuse to accept an individual's identity) has received limited empirical attention. As such, the goals of the current study were to examine manifestations of bisexual identity invalidation and their consequences for wellbeing, identity, and relationships. Fifty-two bisexual and other non-monosexual (e.g., pansexual, queer) participants completed a qualitative survey that asked them to describe their most stressful experience of identity invalidation. Three coders analyzed the data using a consensual qualitative research approach, and the coding scheme was confirmed by two auditors. Most participants (85%) had experienced identity invalidation. They described five perceived reasons: (1) others did not understand or accept bisexuality; (2) the gender of their partner did not fit with others' beliefs about bisexuality; (3) others believed they were confused; (4) others believed they were "faking" it; and (5) others rejected bisexuality for religious reasons. Participants described consequences of identity invalidation including negative emotions, identity-related challenges, and relational difficulties. These findings suggest that identity invalidation is a common experience among bisexual people and it may be an important intervention target.

Covalent attachment of poly(ethylene glycol) (PEG) to therapeutic proteins has been used to prolong in vivo exposure of therapeutic proteins. We have examined pharmacokinetic, biodistribution, and biophysical profiles of three different tumor necrosis factor alpha (TNF) Nanobody-40 kDa PEG conjugates: linear 1 × 40 KDa, branched 2 × 20 kDa, and 4 × 10 kDa conjugates. In accord with earlier reports, the superior PK profile was observed for the branched versus linear PEG conjugates, while all three conjugates had similar potency in a cell-based assay. Our results also indicate that (i) a superior PK profile of branched versus linear PEGs is likely to hold across species, (ii) for a given PEG size, the extent of PEG branching affects the PK profile, and (iii) tissue penetration may differ between linear and branched PEG conjugates in a tissue-specific manner. Biophysical analysis (R(g)/R(h) ratio) demonstrated that among the three protein-PEG conjugates the linear PEG conjugate had the most extended time-average conformation and the most exposed surface charges. We hypothesized that these biophysical characteristics of the linear PEG conjugate accounts for relatively less optimal masking of sites involved in elimination of the PEGylated Nanobodies (e.g., intracellular uptake and proteolysis), leading to lower in vivo exposure compared to the branched PEG conjugates. However, additional studies are needed to test this hypothesis.

Current risk assessments of 1,3-butadiene (BD*) are complicated by limited evidence of its carcinogenicity in humans. Hence, there is a critical need to identify early events and factors that account for the heightened sensitivity of mice to BD-induced carcinogenesis and to deter-mine which animal model, mouse or rat, is the more useful surrogate of potency for predicting health effects in BD-exposed humans. HEI sponsored an earlier investigation of mutagenic responses in mice and rats exposed to BD, or to the racemic mixture of 1,2-epoxy-3-butene (BDO) or of 1,2,3,4-diepoxybutane (BDO2; Walker and Meng 2000). In that study, our research team demonstrated (1) that the frequency of mutations in the hypoxanthine-guanine phosphoribosyl transferase (Hprt) gene of splenic T cells from BD-exposed mice and rats could be correlated with the species-related differences in cancer susceptibility; (2) that mutagenic-potency and mutagenic-specificity data from mice and rats exposed to BD or its individual epoxy intermediates could provide useful information about the BD metabolites responsible for mutations in each species; and (3) that our novel approach to measuring the mutagenic potency of a given chemical exposure as the change in Hprt mutant frequencies (Mfs) over time was valuable for estimating species-specific differences in mutagenic responses to BD exposure and for predicting the effect of BD metabolites in each species. To gain additional mode-of-action information that can be used to inform studies of human responses to BD exposure, experiments in the current investigation tested a new set of five hypotheses about species-specific patterns in the mutagenic effects in rodents of exposure to BD and BD metabolites: 1. Repeated BD exposures at low levels that approach the occupational exposure limit for BD workers (set by the U.S. Occupational Safety and Health Administration) are mutagenic in female mice. 2. The differences in mutagenic responses of the Hprt gene to BD in similarly exposed rodents of a given species (reported in various earlier studies) are primarily associated with age-related thymus activity and trafficking of T cells and with sex-related differences in BD metabolism. 3. The mutagenic potency of the stereochemical forms of BD's epoxy intermediates plays a significant role in the species-related mutagenicity of BD. 4. The hydrolysis-detoxification pathway of BD through 1,2-dihydroxy-3-butene (BD-diol) is a major contributor to mutagenicity at high-level BD exposures in mice and rats. 5. Significant and informative species-specific differences in mutation spectra can be identified by examining both large- and small-scale genetic alterations in the Hprt gene of BD-exposed mice and rats. The first four hypotheses were tested by exposing mice and rats to BD, meso-BDO2, or BD-diol and measuring Hprt Mfs as the primary biomarker. For this, we used the T-cell-cloning assay of lymphocytes isolated from the spleens of exposed and control (sham-exposed) mice and rats. The first hypothesis was tested by exposing female B6C3F1 mice (4 to 5 weeks of age) by inhalation for 2 weeks (6 hours/day, 5 days/week) to 0 or 3 ppm BD. Hprt Mfs were measured at the time of peak mutagenic response after exposure for this age of mice. We then compared the resulting data to those from mutagenicity studies with mice of the same age that had been exposed in a similar protocol to higher levels of BD (Walker and Meng 2000). In mice exposed to 3 ppm BD (n = 27), there was a significant 1.6-fold increase over the mean background Hprt Mf in control animals (n = 24, P = 0.004). Calculating the efficiency of Hprt mutant induction, by dividing induced Hprt Mfs by the respective BD exposure levels, demonstrated that the mutagenic potency of 3 ppm BD was twice that of 20 ppm BD and almost 20 times that of 625 or 1250 ppm BD in exposed female mice. Sample-size calculations based on the Hprt Mf data from this experiment demonstrated the feasibility of conducting a future experiment to find out whether induced Mfs at even lower exposure levels (between 0.1 and 1.0 ppm BD) fit the supralinear exposure-response curve found with exposures between 3.0 and 62.5 ppm BD, or whether they deviate from the curve as Mf values approach the background levels found in control animals. The second hypothesis was tested by estimating mutagenic potency for female mice exposed by inhalation for 2 weeks to 0 or 1250 ppm BD at 8 weeks of age and comparing this estimate to that reported for female mice exposed to BD in a similar protocol at 4 to 5 weeks of age (Walker and Meng 2000). For these two age groups, the shapes of the mutant splenic T-cell manifestation curves were different, but the mutagenic burden was statistically the same. These results support our contention that the disparity in responses reported in earlier Hprt-mutation studies of BD-exposed rodents is related more to age-related T-cell kinetics than to age-specific differences in the metabolism of BD. The third hypothesis was tested by estimating mutagenic potency for female mice and rats (4 to 5 weeks of age) exposed by inhalation to 2 or 4 ppm meso-BDO2 and comparing these estimates to those previously obtained for female mice and rats of the same age and exposed in a similar protocol to (+/-)-BDO2 (Meng et al. 1999b; Walker and Meng 2000). These exposures to stereospecific forms of BDO2 caused equivalent mutagenic effects in each species. This suggests that the small differences in the mutagenic potency of the individual stereoisomers of BDO2 appear to be of less consequence in characterizing the sources of BD-induced mutagenicity than the much larger differences between the mutagenic potencies of BDO2 and the other two BD epoxides (BDO and 1,2-dihydroxy-3,4-epoxybutane [BDO-diol]). The fourth hypothesis was tested in several experiments. First, female and male mice and rats (4 to 5 weeks of age) were exposed by nose only for 6 hours to 0, 62.5, 200, 625, or 1250 ppm BD or to 0, 6, 18, 24, or 36 ppm BD-diol primarily to establish BD and BD-diol exposure levels that would yield similar plasma concentrations of BD-diol. Second, animals were exposed in inhalation chambers for 4 weeks to 0, 6, 18, or 36 ppm BD-diol to determine the mutagenic potency estimates for these exposure levels and to compare these estimates with those reported for BD-exposed female mice and rats (Walker and Meng 2000) in which similar blood levels of BD-diol had been achieved. Measurements of plasma concentrations of BD-diol (via a gas chromatography and mass spectrometry [GC/MS] method developed for this purpose) showed these results: First, BD-diol accumulated in a sublinear manner during a single 6-hour exposure to more than 200 ppm BD. Second, BD-diol accumulated in a linear manner during single (6-hour) or repeated (4-week) exposure to 6 or 18 ppm BD and in a sublinear manner with increasing levels of BD-diol exposure. Third, exposure of female mice and rats to 18 ppm BD-diol produced plasma concentrations equivalent to those produced by exposure to 200 ppm BD (exposure to 36 ppm BD-diol produced plasma concentrations of about 25% of those produced by exposure to 625 ppm BD). In general, 4-week exposure to 18 or 36 ppm BD-diol was significantly mutagenic in female and male mice and rats. The differences in mutagenic responses between the species and sexes were not remarkable, except that the mutagenic effects were greatest in female mice. The substantial differences in the exposure-related accumulation of BD-diol in plasma after rodents were exposed to more than 200 ppm BD compared with the relatively small differences in the mutagenic responses to direct exposures to 6, 18, or 36 ppm BD-diol in female mice provided evidence that the contribution of BD-diol-derived metabolites to the overall mutagenicity of BD has a narrow range of effect that is confined to relatively high-level BD exposures in mice and rats. This conclusion was supported by the results of parallel analyses of adducts in mice and rats concurrently exposed to BD-diol (Powley et al. 2005b), which showed that the exposure-response curves for the formation of N-(2,3,4-trihydroxybutyl)valine (THB-Val) in hemoglobin, formation of N7-(2,3,4-trihydroxybutyl)guanine (THB-Gua) in DNA, and induction of Hprt mutations in exposed rodents were remarkably similar in shape (i.e., supralinear). Combined, these data suggest that trihydroxybutyl (THB) adducts are good quantitative indicators of BD-induced mutagenicity and that BD-diol-derived BDO-diol (the major source of the adducts) might be largely responsible for mutagenicity in rodents exposed to BD-diol or to hight levels of BD. The mutagenic-potency studies of meso-BDO2 and BD-diol reported here, combined with our earlier studies of BD, (+/-) BDO, and(+/-)-BDO2 (Walker and Meng 2000), revealed important trends in species-specific mutagenic responses that distinguish the relative degree to which the epoxy intermediates contribute to mutation induction in rodents at selected levels of BD exposures. These data as a whole suggest that , in mice, BDO2 largely causes mutations at exposures less than 62.5 ppm BD and that BD-diol-derived metabolites add to these mutagenic effects at higher BD exposures. In rats, it appears that the BD-diol pathway might account for nearly all the mutagenicity at the hight-level BD exposures where significant increases in Hprt Mfs are found and cancers are induced. Additional exposure-response studies of hemoglobin and DNA adducts specifics to BDO2, BDO-diol, and other reactive intermediates are needed to determine more definitively the relative contribution of each metabolite to the DNA alkylation and mutation patterns induced by BD exposure in mice and rats. For the fifth hypothesis, a multiplex polymerase chain reaction (PCR) procedure for the analysis of genomic DNA mutations in the Hprt gene of mice was developed. (ABSTRACT TRUNCATED)

Inhalation of subclinical doses of sarin suppresses the antibody-forming cell (AFC) response, T-cell mitogenesis, and serum corticosterone (CORT) levels, and high doses of sarin cause lung inflammation. However, the duration of these changes is not known. In these studies, rats were exposed to a subclinical dose of sarin (0.4 mg/m3/h/day) for 1 or 5 days, and immune and inflammatory parameters were assayed up to 8 weeks before sarin exposure. Our results showed that the effects of a 5-day sarin exposure on the AFC response and T-cell receptor (TCR)-mediated Ca2+ response disappeared within 2-4 weeks after sarin exposure, whereas the CORT and adrenocorticotropin hormone (ACTH) levels remained significantly decreased. Pretreatment of rats with chlorisondamine attenuated the effects of sarin on the AFC and the TCR-mediated Ca2+ response, implicating the autonomic nervous system (ANS) in the sarin-induced changes in T-cell function. Moreover, exposure to a single or five repeated subclinical doses of sarin upregulated the mRNA expression of proinflammatory cytokines in the lung, which is associated with the activation of NFkappaB in bronchoalveolar lavage cells. These effects were lost within 2 weeks of sarin inhalation. Our results suggest that while sarin-induced changes in T cells and cytokine gene expression were short lived, suppression of CORT and ACTH levels were relatively long lived and might represent biomarkers of sarin exposure. Moreover, while the effects of sarin on T-cell function were regulated by the ANS, the decreased CORT levels by sarin might result from its effects on the hypothalamus-pituitary-adrenal axis.

The disposition of [14C]methyltetrahydrofuran (14C-MTHF) in rats and mice was determined by following changes in the radioactivity in tissue and excreta with time after dosing. MTHF administered orally (1, 10, or 100 mg/kg) or intravenously (1 mg/kg) to either rats or mice was rapidly metabolized and excreted with <8% (mice) or 8-22% (rats) of the dose remaining in the body after 24 h (1 and 10 mg/kg doses) or 72 h (100 mg/kg dose). Based on recovery of radioactivity in excreta (other than feces) and tissues (other than the gastrointestinal [GI] tract), absorption of orally administered MTHF was essentially complete (93-100%). There were no overt signs of toxicity observed at any dose studied. The major route of excretion in mice was in urine followed by exhaled CO2. In rats the major route of excretion was exhaled CO2 followed by urinary excretion. The excretion of exhaled volatile organic compounds (VOC) was dose-dependent in both species; at lower doses exhaled VOC represented 1-5% of dose, but at the highest dose (100 mg/kg) this proportion rose to 14% (mice) and 27% (rats). Analysis of the VOCs exhaled at the high dose indicated that the increase was due to exhalation of the parent compound, 14C-MTHF. Analysis of urine showed three highly polar peaks in the mouse urine and two polar peaks in the rat urine. Because the 14C label in MTHF was in the methyl group, the polar metabolites were considered likely due to the one-carbon unit getting into the metabolic pool and labeling intermediate dietary metabolites.

Studies were performed to determine if the detoxification pathway of 1,3-butadiene (BD) through 3-butene-1,2-diol (BD-diol) is a major contributor to mutagenicity in BD-exposed mice and rats. First, female and male mice and rats (4-5 weeks old) were exposed by nose-only for 6h to 0, 62.5, 200, 625, or 1250 ppm BD or to 0, 6, 18, 24, or 36 ppm BD-diol primarily to establish BD and BD-diol exposure concentrations that yielded similar plasma levels of BD-diol, and then animals were exposed in inhalation chambers for 4 weeks to BD-diol to determine the mutagenic potency estimates for the same exposure levels and to compare these estimates to those reported for BD-exposed female mice and rats where comparable blood levels of BD-diol were achieved. Measurements of plasma levels of BD-diol (via GC/MS methodology) showed that (i) BD-diol accumulated in a sub-linear fashion during single 6-h exposures to >200 ppm BD; (ii) BD-diol accumulated in a linear fashion during single or repeated exposures to 6-18 ppm BD and then in a sub-linear fashion with increasing levels of BD-diol exposure; and (iii) exposures of mice and rats to 18 ppm BD-diol were equivalent to those produced by 200 ppm BD exposures (with exposures to 36 ppm BD-diol yielding plasma levels approximately 25% of those produced by 625 ppm BD exposures). Measurements of Hprt mutant frequencies (via the T cell cloning assay) showed that repeated exposures to 18 and 36 ppm BD-diol were significantly mutagenic in mice and rats. The resulting data indicated that BD-diol derived metabolites (especially, 1,2-dihydroxy-3,4-epoxybutane) have a narrow range of mutagenic effects confined to high-level BD (>or=200 ppm) exposures, and are responsible for nearly all of the mutagenic response in the rat and for a substantial portion of the mutagenic response in the mouse following high-level BD exposures.

Experiments were performed: (i) to investigate potential age- and gender-dependent differences in mutagenic responses in T cells following exposures of B6C3F1 mice and F344 rats by inhalation for 2 weeks to 0 or 1250 ppm butadiene (BD), and (ii) to determine if exposures for 2 weeks to 62.5 ppm BD produce a mutagenic effect in female rats. To evaluate the effect of age on mutagenic response, mutant manifestation curves for splenic T cells of female mice exposed at 8-9 weeks of age were defined by measuring Hprt mutant frequencies (MFs) at multiple time points after BD exposure using a T cell cloning assay and comparing the resulting mutagenic potency estimate (calculated as the difference of areas under the mutant manifestation curves of treated versus control animals) to that reported for female mice exposed to BD in the same fashion beginning at 4-5 weeks of age. The shapes of the mutant T cell manifestation curves for spleens were different [e.g., the maximum BD-induced MFs in older mice (8.0+/-1.0 [S.D.]x10(-6)) and younger mice (17.8+/-6.1 x 10(-6)) were observed at 8 and 5 weeks post-exposure, respectively], but the mutagenic burden was the same for both age groups. To assess the effect of gender on mutagenic response, female and male rodents were exposed to BD at 4-5 weeks of age and Hprt MFs were measured when maximum MFs are expected to occur post-exposure. The resulting data demonstrated that the pattern for mutagenic susceptibility from high-level BD exposure is female mice>male mice>female rats>male rats. Exposures of female rats to 62.5 ppm BD caused a minor but significant mutagenic response compared with controls (n=16/group; P=0.03). These results help explain part of the differing outcomes/interpretations of data in earlier Hprt mutation studies in BD-exposed rodents.

The first epithelial surface encountered by inhaled materials is the epithelium of the respiratory tract. The epithelium is lined by a fluid (ELF) that can be sampled by a saline wash (lavage) of the area of interest. This technique, known as bronchoalveolar lavage (BAL), provides a means of sampling a body fluid that can provide valuable information on the reaction of the lung to inhaled materials. The most common responses measured are indicators of an inflammatory response, the most sensitive of which is an influx of neutrophils. In the extracellular fluid, levels of beta-glucuronidase activity indicate activation of macrophages, and lactate dehydrogenase activity indicates cytotoxicity. Other pro- and anti-inflammatory soluble factors that can be measured in BAL fluid include secretory products of macrophages and epithelial cells, such as tumor necrosis factor alpha, fibronectin, interleukin-1, various chemotactic factors (including IL-8, MIP-2), growth factors, proteases, and antiproteases. Oxidative stress can be measured by the levels of reduced glutathione in ELF, and increased levels of alkaline phosphatase indicate increased Type II cell secretions. Allergic responses are indicated by increased eosinophils and factors such as histamine and arachidonate metabolites in BAL fluid. BAL analysis can be used as a complementary technique with more traditional measures of lung injury, such as histopathology or radiology. The advantage of BAL analysis is that one can pick up early indicators of biochemical changes leading to later morphological changes in a disease process. A second advantage is that the BAL fluid analyses are quantitative, and dose-response measures can be obtained. In large animals, one can do repeated lavages to follow a disease process; in small animals, one can use serial sacrifices in similarly exposed rodents to achieve the same goal. Research related to the use of BAL fluid analyses to detect lung damage has been conducted at the Lovelace Respiratory Research Institute with funding from various sources including the US Department of Energy and the US Environmental Protection Agency.

Malpractice law is frequently justified by the claim that it improves health care services but this belief remains untested. Using a multiple case study in 16 remote rural areas in New Zealand, this study examined the effects of formal quasi-judicial investigations on the quality of health care services. The study found that the fragile local health systems were damaged by the quasi-judicial investigations of the medical disciplinary body and became less efficient and less user-friendly. A few doctors left rural practice and were difficult to replace. The remaining health workers responded to the investigations in a negative manner, losing confidence, enthusiasm and motivation for work; they performed in a less efficient manner, working more slowly, setting up barriers to access, ordering more tests and referring more to secondary care. Complainants also appeared to have been disadvantaged as a consequence of having complained.

1,3-Butadiene (BD) is a monomer produced in petrochemical production facilities and from several combustion sources. The United States Environmental Protection Agency has defined BD as a probable human carcinogen. Methods for assessing exposure and internal dose are therefore of critical interest, and one technique is the measurement of urinary metabolites. Here we describe methods for measuring two urinary metabolites, N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (referred to as MI) and an isomeric mixture of the regio- and stereoisomers (R)/(S)-N-acetyl-S-(1-(hydroxymethyl)-2-propen-yl)-L-cysteine and (R)/(S)-N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine (referred to as MII). The method is based on isolation of the metabolites by solid-phase extraction and measurement using liquid chromatography and triple quadrupole mass spectrometry (LC-MS(3)). The LC-MS(3) allowed good selectivity with minimal sample preparation. Assay accuracy was within 10% or better, with substantial improvement in accuracy accompanying the commercial availability of deuterated internal standards for both compounds. Assay precision and linearity passed rigorous validation criteria, and precision-based limits of quantitation values were 12 and 1 ng/mL for MI and MII, respectively. Data are shown from analysis of human urine from occupationally exposed individuals and rat urine from BD exposures conducted to investigate rodent metabolic profiles. Both of these data sets clearly show that this assay can discern previously described relationships between BD exposure and the production of MI/MII.

This unit describes how bronchoalveolar lavage can be used in laboratory animals to sample the epithelial lining fluid of the lung for information on the degree of pulmonary inflammation induced by exposure to an airborne toxicant. The technique allows quantitative assessment of inflammatory responses and is valuable for providing dose-response information in exposed animals. Lavage fluid samples may also be used for proteomic analyses, and the protein expression profiles may be used to address specific mechanistic questions.

A multiinstitutional, transitional epidemiologic study was conducted with a worker population in the Czech Republic to evaluate the utility of a continuum of non-disease biological responses as biomarkers of exposure to 1,3-butadiene (BD)* in an industrial setting. The study site included two BD facilities in the Czech Republic. Institutions that collaborated in the study were the University of Vermont (Burlington, Vermont, USA); the Laboratory of Genetic Ecotoxicology (Prague, the Czech Republic); Shell International Chemicals, BV (Amsterdam, The Netherlands); the University of North Carolina at Chapel Hill (Chapel Hill, North Carolina, USA); University of Texas Medical Branch at Galveston (Galveston, Texas, USA); Leiden University (Leiden, The Netherlands); and the Health and Safety Laboratory (Sheffield, United Kingdom). Male volunteer workers (83) participated in the study: 24 were engaged in BD monomer production, 34 in polymerization activities, and 25 plant administrative workers served as unexposed control subjects. The BD concentrations experienced by each exposed worker were measured by personal monitor on approximately ten separate occasions for 8-hour workshifts over a 60-day exposure assessment period before biological samples were collected. Coexposures to styrene, benzene, and toluene were also measured. The administrative control workers were considered to be a homogeneous, unexposed group for whom a series of 28 random BD measurements were taken during the exposure assessment period. Questionnaires were administered in Czech to all participants. At the end of the exposure assessment period, blood and urine samples were collected at the plant; samples were. fractionated, cryopreserved, and kept frozen in Prague until they were shipped to the appropriate laboratories for specific biomarker analysis. The following biomarkers were analyzed: * polymorphisms in genes involved in BD metabolism (Prague and Burlington); * urinary concentrations of 1-hydroxy-2-(N-acetylcysteinyl)-3-butene and 2-hydroxy-1-(N-acetylcysteinyl)-3-butene (M2 [refers to an isomeric mixture of both forms]) (Amsterdam); * urinary concentrations of 1,2-dihydroxy-4-(N-acetylcysteinyl)-butane (M1) (Amsterdam); * concentrations of the hemoglobin (Hb) adducts N-(1-[hydroxymethyl]-2-propenyl)valine and N-(2-hydroxy-3-butenyl)valine (HBVal [refers to an isomeric mixture of both forms]) (Amsterdam); * concentrations of the Hb adduct N-(2,3,4-trihydroxybutyl)valine (THBVal) (Chapel Hill); * T cell mutations in the hypoxanthine phosphoribosyltransferase (HPRT) gene (autoradiographic assay in Galveston with slide review in Burlington; cloning assay in Leiden with mutational spectra determined in Burlington); and * chromosomal aberrations by the conventional method and by fluorescence in situ hybridization [FISH]), and cytogenetic changes (sister chromatid exchanges [SCEs] (Prague). All assay analysts were blinded to worker and sample identity and remained so until all work in that laboratory had been completed and reported. Assay results were sent to the Biometry Facility in Burlington for statistical analyses. Analysis of questionnaire data revealed that the three exposure groups were balanced with respect to age and years of residence in the district, but the control group had significantly more education than the other two groups and included fewer smokers. Group average BD exposures were 0.023 mg/m3 (0.010 ppm) for the control group, 0.642 mg/m3 (0.290 ppm) for the monomer group, and 1.794 mg/m3 (0.812 ppm) for the polymer group; exposure levels showed considerable variability between and within individuals. Styrene exposures were significantly higher in the polymer group than in the other two groups. We found no statistically significant differences in the distributions of metabolic genotypes over the three exposure groups; genotype frequencies were consistent with those previously reported for this ethnic and national population. Although some specific genotypes were associated with quantitative differences in urinary metabolite concentrations or Hb adduct dose-response characteristics, none indicated a heightened susceptibility to BD. Concentrations of both the M2 and M1 urinary metabolites and both the HBVal and THBVal Hb adducts were significantly correlated with group and individual mean BD exposure levels; the Hb adducts were more strongly correlated than the urinary metabolites. By contrast, no significant relations were observed between BD exposures and HPRT gene mutations (whether determined by the auto-radiographic or the cloning method) or any of the cytogenetic biomarkers (whether determined by the conventional method or FISH analysis). Neither the mutational nor the cytogenetic responses showed any association with genotypes. The molecular spectrum of HPRT mutations in BD-exposed workers showed a high frequency of deletions; but the same result was found in the unexposed control subjects, which suggests that these were not due to BD exposure. This lack of association between BD exposures and genetic effects persisted even when control subjects were excluded from the analyses or when we conducted regression analyses of individual workers exposed to different levels of BD.

Coexposure to subclinical levels of nerve gas and to heat stress may have induced some of the clinical symptoms of the Gulf War Syndrome. We tested the hypothesis that single or repeated subclinical exposure to sarin, particularly under conditions of heat stress, would impair regulation of body temperature and locomotor activity. Male F344 rats were housed at 25 degrees C or under mild heat stress at 32 degrees C and were exposed 1 h/day for 1, 5, or 10 days to 0, 0.2, or 0.4 mg/m(3) of sarin in a nose-only exposure system. Body temperature and activity were monitored continuously by telemetry during exposure and 1 month postexposure. Exposed rats showed no clinical symptoms of toxicity such as tremors, despite evidence of reduced red blood cell cholinesterase activity. Heat stress consistently elevated body temperature in unexposed animals, particularly during the dark period when animals are most active. Inhalation of sarin gas at the two subclinical levels did not affect body temperature acutely in a biologically meaningful manner after the first exposure nor after 5 or 10 repeated exposures, either at thermoneutral ambient temperature or during chronic heat stress. There were no consistent effects of sarin or housing temperature on activity. The data suggest that subclinical levels of sarin have minimal effects on temperature regulation and locomotor activity under these observation conditions.

The nerve gas sarin is a potent cholinergic agent, and exposure to high doses may cause neurotoxicity and death. Subclinical exposures to sarin have been postulated to contribute to the Gulf War syndrome; however, the biological effects of subclinical exposure are largely unknown. In this communication, evidence shows that subclinical doses (0.2 and 0.4 mg/m(3)) of sarin administered by inhalation to F344 rats for 1 h/day for 5 or 10 days inhibited the anti-sheep red blood cell antibody-forming cell response of spleen cells without affecting the distribution of lymphocyte subpopulations in the spleen. Moreover, sarin suppressed T cell responses, including the concanavalin A (Con A) and the anti-alphabeta-T cell receptor (TCR) antibody-induced T cell proliferation and the rise in the intracellular calcium following TCR ligation. These concentrations of sarin altered regional but not total brain acetylcholinesterase activity. Interestingly, serum corticosterone levels of the sarin-treated animals were dramatically lower than the control animals, indicating that sarin-induced immunosuppression did not result from the activation of the hypothalamus-pituitary-adrenal (HPA) axis. Pretreatment of animals with the ganglionic blocker chlorisondamine abrogated the inhibitory effects of sarin on spleen cell proliferation in response to Con A and anti-TCR antibodies. These results suggest that the effects of sarin on T cell responsiveness are mediated via the autonomic nervous system and are independent of the HPA axis.

The purpose of this study was to determine whether exposure to levels of sarin causing no overt clinical signs would cause more subtle, adverse health effects that persisted after the exposure ended. Inhalation exposures of male Fischer 344 rats to 0, 0.2, or 0.4 mg/m(3) of sarin for 1 h/day for 1, 5, or 10 days under normal (25 degrees C) and heat-stressed (32 degrees C) conditions were completed and observations were made at 1 day and 1 month after the exposures. The sarin exposures had no observed effects on body weight, respiration rate, and minute volume during exposure nor in body temperature and activity during the 30-day recovery period. There was no evidence of cellular changes in brain determined by routine histopathology nor of any increase in apoptosis. Brain mRNA for interleukin (IL)-1beta, tumor necrosis factor-alpha, and IL-6 was increased in a dose-dependent manner. Autoradiographic studies demonstrated that M1 cholinergic receptor site densities were unchanged at 1 day after repeated exposures with or without heat stress. At 30 days, there was a decrease in M1 receptors in the olfactory tubercle (with and without heat), and, with heat stress, M1 sites also decreased in a dose-dependent manner in the frontal cortex, anterior olfactory nucleus, and hippocampus. M3 receptor sites were not affected by sarin exposure alone. In the presence of heat stress, there was an upregulation in binding site densities in the frontal cortex, olfactory tubercle, anterior nucleus, and striatum immediately after exposure, and these effects persisted at 30 days. Although red blood cell acetylcholinesterase (AChE) was not greatly inhibited by the 1-day exposure, there were 30 and 60% inhibitions after repeated exposures at the low and high doses, respectively. Histochemical staining for AChE demonstrated that sarin exposure alone reduced AChE in the cerebral cortex, striatum, and olfactory bulb. Sarin exposure under heat stress reduced AChE staining in the hippocampus, an area important for memory function. Thus, repeated exposures under heat-stress conditions, to levels of sarin that would not be noticed clinically, resulted in delayed development of brain alterations in cholinergic receptor subtypes that may be associated with memory loss and cognitive dysfunction.

Background: Surfactant abnormalities have been described in bacterial pneumonia., Objective: To determine the safety and effect of exogenous surfactant replacement in patients with ventilator-associated pneumonia (VAP)., Methods: Patients with VAP were randomized in a double-blind study to receive either an artificial surfactant (Exosurf) consisting mostly of disaturated phospholipids (DSPL) or saline via a continuous nebulizer system for 5 days. Patients underwent bronchoscopy and bronchoalveolar lavage (BAL) prior to and after 4 days of therapy., Results: Twenty-two patients were randomized, with 8 receiving Exosurf. There was no detected difference in outcome between the saline- and Exosurf-treated patients in terms of days on ventilator, 30-day or hospital mortality. At the follow-up lavage, the patients treated with Exosurf had a significant rise in the level of DSPL (p < 0.05), while the saline group did not, suggesting delivery of drug. Also at the follow-up lavage, the percentage of neutrophils in the BAL fell in the Exosurf patients (p < 0.01), but not in the saline group., Conclusion: Exogenous surfactant replacement given to patients with VAP increased the amount of DSPL retrieved by BAL. This treatment was associated with a fall in the neutrophil response to pneumonia., (Copyright 2002 S. Karger AG, Basel)

1,3-Butadiene (BD), which is used to manufacture synthetic rubber, is a mutagen and carcinogen. Because past occupational exposures have been associated with an increased risk of leukemia, there has been a dramatic reduction in workplace exposure standards. The health benefits of these reduced levels of occupational exposure to BD will be difficult to evaluate using relatively insensitive traditional epidemiological studies; however, biomarkers can be used to determine whether there are genotoxic effects associated with recent exposures to BD. In past studies of BD-exposed workers in Southeast Texas, we observed an increase in the frequency of lymphocytes with mutations in a reporter gene, hprt. Frequencies of hprt mutant cells correlated with air levels of BD and with the concentration of a BD metabolite in urine. Average exposures to 1-3 parts per million (p.p.m.) of BD were associated with a threefold increase in hprt variant (mutant) frequencies (Vfs). We now report results from a follow-up study of workers in a synthetic rubber plant in Southeast Texas. Thirty-seven workers were evaluated on three occasions over a 2-week period for exposure to BD by the use of personal organic vapor monitors and by determining the concentration of a BD metabolite in urine. The frequency of hprt mutants was determined, by autoradiography, with lymphocyte samples collected 2 weeks after the final exposure measurement. Based on their work locations, the study participants were assigned to high-exposure (N=22) or low-exposure (N=15) groups. The BD exposure, +/-standard error, of the workers in the high-exposure group (1.65+/-0.52 p.p.m.) was significantly greater than the low-exposure group (0.07+/-0.03 p.p.m.; P<0.01). The frequency of hprt mutant lymphocytes was also significantly different in the two groups (high, 10.67+/-1.5 x 10(-6); low, 3.54+/-0.6 x 10(-6); P<0.001). The concentration of the urine metabolite was greater in the high-exposure group, but the difference was not significant. The correlation coefficient between hprt Vf and BD exposure levels was r=0.44 (CI(95), 0.11-0.69; P=0.011). This study reproduced the findings from a previous study at this plant. Although studies of butadiene-exposed workers in other countries have not detected an effect of exposure on frequencies of hprt mutant lymphocytes, we have repeatedly observed this result in our studies in Texas.

Olefinic compounds are commercially valuable because they form useful polymeric substances. The same chemical property (presence of double bonds) that makes the olefins useful may also cause them to be toxic in the body. The double bonds of olefins can be oxidized by cytochrome P450 enzymes to epoxides, which are electrophiles that can react with DNA and may cause alterations in the genetic information carried by that macromolecule. Epoxides can be rendered inactive toward DNA by binding to proteins, by hydrolysis to diols through epoxide hydrolase enzymes (EHs), or by forming conjugates with glutathione via glutathione S-transferase (GST) activities. The balance between the oxidizing enzymatic activities and the hydrolyzing or conjugating enzymatic activities in the livers of different species can influence the potential toxicity of the olefins. The location of the enzymes and the potential for concerted reactions in which epoxides are inactivated immediately after formation will also influence the potential toxicity of the olefins. Cytochrome P450 enzymes and EHs are in microsomes located in the rough endoplasmic reticulum surrounding the nucleus where the DNA is located. GST is in the cytoplasm of the cell. In the case of 1,3-butadiene (BD), such enzymatic differences may strongly influence the toxicity in different species. The mouse, in which BD is a potent multi-site carcinogen, has the lowest microsomal EH activity of any species. This allows the monoepoxides formed in the microsomes by cytochrome P450 enzymes to be further oxidized to the highly genotoxic diepoxide (DEB), and both epoxides can either be released into the blood for distribution throughout the body or can react with DNA in the nucleus. The rat, in which BD is a weak carcinogen, has much higher levels of microsomal EH, and only trace amounts of DEB enter the bloodstream. Major BD metabolites in primates suggest that the hydrolysis pathway is even more prominent in primates than in rats. Data suggest that BD will be much less toxic in primates than in mice. Considering these quantitative differences in metabolism may help to reduce the uncertainties in extrapolating animal data on olefin toxicity to health risk assessments for humans exposed to the compounds.

A study was conducted to test the hypothesis that repeated low level exposures to 1,3-butadiene (BD), approaching the OSHA occupational threshold for this chemical, produce a significant mutagenic response in mice. Female B6C3F1 mice (4-5 weeks of age) were exposed by inhalation for 2 weeks (6 h/day, 5 days/week) to 0 or 3 ppm BD, and then necropsied at 4 weeks after the cessation of exposures to measure the frequency of mutations (MF) at the Hprt locus using the T-lymphocyte clonal assay. At necropsy, T cells were isolated from spleen and cultured in the presence of mitogen, growth factors, and a selection agent. Cells were scored for growth on days 8-9 after plating to determine cloning efficiencies (CEs) and Hprt MFs. There was a marginal but significant reduction in the growth of splenic T cells from mice exposed to 3 ppm (n=27) compared with control mice (n=24) (P=0.004), suggesting the occurrence of BD-induced cytotoxicity at this low exposure concentration. In addition, the average Hprt MF in mice exposed to 3 ppm BD [1.54+/-0.82 (S.D.)x10(-6)] was significantly increased by 1.6-fold over the average control value of 0.96+/-0.51 (S.D.)x10(-6) (P=0.004). Comparisons of these data to earlier Hprt mutagenicity studies of mice exposed to high concentrations of BD (where significant mutagenic but not cytotoxic effects were observed) indicate that the ability to detect the cytotoxic and mutagenic responses of T cells to low levels of BD was enhanced by using a much larger sample size than usual for both the control and treatment groups. Additional analyses of the quantitative relationships between CE and MF demonstrated that CE had no significant effect upon MF values in sham-exposed control mice or mice exposed to low-level BD. Furthermore, the approaches for assessing the impact of CE and clonality on Hprt MFs in these control and BD-exposed mice were applied with the same rigor as in in vivo Hprt mutagenicity studies in human children. The overall study results support the conclusion that short-term low-level BD exposure is mutagenic in the mouse.

The overall objective of this study was to evaluate a continuum of biomarkers in blood and urine for their sensitivities as indicators of low level occupational exposures to 1,3 butadiene (BD). The study design was largely cross-sectional, with biological samples collected within a short timeframe. Personal 8-h BD exposure measures were made on several occasions over a 60-day period for each potentially exposed worker in order provide maximum accuracy for this independent variable and to accommodate the different expression intervals of the several biomarkers. Co-exposures to styrene, toluene and benzene were also measured. The study included 24 BD monomer production workers (mean BD exposure=0.642 mg/m(3)), 34 polymerization workers (mean BD exposure=1.794 mg/m(3)) and 25 controls (mean BD exposure=0.023 mg/m(3)). The several biomarkers were measured by a consortium of investigators at different locations in the US and Europe. These biomarkers included: (1) metabolic genotypes (CYP2E1, EH, GST M1, GST T1, ADH2, ADH3), determined in Prague and Burlington, VT; (2) urinary M1 and M2 metabolites (1,2-dihydroxy-4-[N-acetylcysteinyl]-butane and 1-hydroxy-2-[N-acetylcysteinyl]-3-butene, respectively), determined in Albuquerque, NM and Leiden; (3) hemoglobin adducts (N-[2-dihydroxy-3-butenyl]valine=HBVal and N-[2,3,4-trihydroxybutyl]valine=THBVal), determined in Amsterdam and Chapel Hill, NC, respectively; (4) HPRT mutations determined by autoradiographic assay in Galveston, TX, with slides re-read in Burlington, VT; (6) hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutations determined by cloning assay in Leiden with mutational spectra characterized in Burlington, VT; (7) sister chromatid exchanges and chromosome aberrations determined by standard methods and FISH analysis in Prague. Urinary M1 and M2 metabolites and HBVal and THBVal hemoglobin adducts were all significantly correlated with BD exposure levels, with adducts being the most highly associated. No significant relationships were observed between BD exposures and HPRT mutations or any of the cytogenetic endpoints, regardless of method of assay.

Subclinical, repeated exposures of F344 rats to sarin resulted in brain alterations in densities of chlonergic receptor subtypes that may be associated with memory loss and cognitive dysfunction. The exposures also depressed the immune system. The rat appears to be a good model for studying the effects of subclinical exposure to a nerve gas.

The carcinogenicity of 1,3-butadiene (BD) varies greatly in the rodent species in which 2-year bioassay studies were completed. This raises the question of whether the risk of BD exposure in humans is more like that of the sensitive species, the mouse, or more like that of the resistant species, the rat. Numerous studies have indicated that one reason for the species differences in response to BD is that the blood and tissues of BD-exposed mice contain high levels of both the mono- and the diepoxide metabolite, while the tissue and blood of exposed rats contain very little of the diepoxide. The diepoxide is far more mutagenic than the monoepoxide, and so it is reasonable that the diepoxide plays a major role in tumor induction in the mouse. If the diepoxide is the primary carcinogen, the presence of the diepoxide in the blood of exposed individuals should be an indicator of risk from BD exposure. In this study, we report that the diepoxide is sufficiently stable to be excreted into the urine of exposed rodents and that the urinary levels of the diepoxide reflect the relative levels of the compound in the blood of the two species. The conclusion is that urinary diepoxide should be investigated as a potential biomarker of the formation of the diepoxide in humans exposed to BD.

1,3-Butadiene (BD) is carcinogenic and mutagenic in B6C3F1 mice. BD inhalation induces an increased frequency of specific base substitution mutations in the bone marrow and spleen of B6C3F1 lacI transgenic mice. BD is bioactivated to at least three mutagenic metabolites: 1,2-epoxybutene (EB), 1,2-epoxy-3,4-butanediol (EBD), and 1,2,3,4-diepoxybutane (DEB), however, the contribution of these individual metabolites to the in vivo mutational spectrum of BD is uncertain. In the present study, lacI transgenic mice were exposed by inhalation (6h per day, 5 days per week for 2 weeks) to 0 or 29.9ppm of the BD metabolite, EB to assess its contribution to the in vivo mutational spectrum of BD. No increase in lacI mutant frequency was observed in the bone marrow or spleen of EB-exposed mice. The lack of mutagenicity in the bone marrow or spleen likely relate to insufficient levels of EB reaching these tissues. The lacI mutant frequency was increased 2.7-fold in the lungs of EB-exposed mice (mean+/-S.D., 9.9+/-3.0x10(-5)) compared to air control mice (3.6+/-0.7x10(-5)). DNA sequence analysis of 65 and 66 mutants from the lungs of air control and EB-exposed mice, respectively, revealed an increase in the frequency of two categories of base substitution mutation and deletions. Like mice exposed to BD, EB-exposed mice had an increased frequency of A:T-->T:A transversions. However, in contrast to the BD mutational spectra, G:C-->A:T transitions at 5'-CpG-3' sequences, occurred with increased frequency in the EB-exposed mice. The increased frequency of deletions as well as the induction of two tandem mutations and a tandem deletion in the lungs of EB-exposed mice are also inconsistent with previous mutational spectra from BD-exposed mice or EB-exposed cells in culture. We hypothesize that the direct in vivo mutagenicity and further in situ metabolism of EB in the lungs of EB-exposed mice played a prominent role in the generation of the current mutational spectrum.

The chronic (2-yr) inhalation toxicity of 1,3-butadiene (BD), a chemical used in large quantities to make rubber and plastics, differs greatly between mice and rats. Mice develop lung tumors after exposures to concentrations as low as 6.25 ppm, whereas rats develop mammary tumors only after exposures to 1000-8000 ppm BD. Extensive research has been carried out to determine where humans fit into this susceptibility range. Species differences in rates of metabolism of BD have been noted, but inconsistencies in metabolism data from different laboratories and some problems in the fit of physiologically based pharmacokinetic (PBPK) models with experimental data have left uncertainties. The experiments reported here are intended to clarify the issue of human metabolism of BD and to determine if metabolism of BD in cynomolgus monkeys is similar enough to metabolism in humans to use in vivo data from monkeys for PBPK modeling. The results indicate that for the reactions studied (oxidation of BD to the mono- and diepoxide), BD is metabolized substantially the same in monkey and human hepatic microsomes. The human metabolism data agreed with that reported earlier when the in vitro metabolism of BD was studied at low BD concentrations. Finally, BD at high concentrations was found to inhibit the further oxidation of its metabolite, the monoepoxide. Incorporation of this information on the competition between BD and its first oxidation product for CYP2E1 should improve the fit of PBPK models.

Reports in the literature suggest that one reason for the greater sensitivity of mice to the carcinogenicity of 1,3-butadiene (BD) is that exposed mice metabolize much more of the BD to 1,2,3,4-diepoxybutane (BDO2) than do exposed rats. The purpose of this study was to determine the tumorigenicity of BDO2 in rats and in mice exposed to the same concentration of the agent. Female B6C3F1 mice and Sprague-Dawley rats, 10 to 11 weeks old, 56 per group, were exposed by inhalation to 0, 2.5, or 5.0 ppm BDO2, 6 hours/day, 5 days/week for 6 weeks. Preliminary dosimetry studies in rodents exposed for 6 hours to 12 ppm BDO2 indicated that blood levels would be expected to be approximately 100 and 200 pmol/g at the two exposure concentrations in the rat and twice those levels in the mouse. During the 6-week exposure, the mice at the high exposure level showed signs of labored breathing during the last week, and four mice died. In the others, however, the respiratory symptoms disappeared after exposure ended. Rats showed no clinical signs of toxicity during exposure but developed labored breathing after the end of the exposure leading to the death of 13 rats within 3 months. At the end of the exposure, some animals (8 per group) were evaluated for the acute toxicity resulting from the BDO2 exposure. The remaining exposed rats and mice were held for 18 months for observation of tumor development. At the end of the exposure, rats had no biologically significant alteration in standard hematological parameters, but mice had a dose-dependent increase in neutrophils and decrease in lymphocytes. In both species the significant histopathologic lesions were in the nose, concentrated around the main airflow pathway. Necrosis, inflammation, and squamous metaplasia of the nasal mucosa, as well as atrophy of the turbinates, were all present at the end of exposure to 5.0 ppm. Within 6 months, necrosis and inflammation subsided, but squamous metaplasia remained in the mice. In rats that died after exposure, squamous metaplasia was seen in areas of earlier inflammation and, in other rats, extended beyond those areas with time. The metaplasia was severe enough to restrict and occlude the nasopharyngeal duct. Later, keratinizing squamous cell carcinomas developed from the metaplastic foci in rats but not mice. At the end of 18 months, the only significant increase in neoplasia in the exposed rats was a dose-dependent increase in neoplasms of the nasal mucosa (0/47, 12/48, and 21/48 for the control, 2.5 ppm, and 5.0 ppm exposures, respectively). Neoplasia of the nasal mucosa did not increase significantly in the mice; neoplastic lesions in the mice were observed in reproductive organs, lymph nodes, bone, liver, Harderian gland, pancreas, and lung. The only significant increase in neoplasms in a single organ in the mice was in the Harderian gland (0/40, 2/42, and 5/36 for the control, 2.5 ppm, and 5.0 ppm exposures, respectively). This tumor accounts for the apparent trend toward an increase in total neoplastic lesions in mice as a function of dose (10/40, 7/42, and 16/36 for control, 2.5 ppm, and 5.0 ppm exposures, respectively). These findings indicate that the metabolite of BD, BDO2, is carcinogenic in the respiratory tract of rats. An increase in respiratory tract tumors was not observed in similarly exposed mice despite the fact that preliminary studies indicated mice should have received twice the dose to tissue compared with the rats. High cytosolic activity of detoxication enzymes in the mouse may account, in part, for the differences in response.

Background: The aim of this study was to investigate whether BGD activity is of additional value in the assessment of pulmonary inflammation caused by coal dust exposure., Design and Methods: Ex-coalminers were included in this study. Forty-eight healthy male subjects, without a relevant medical history, were used as controls., Results: In ex-coalminers serum BGD activity was higher compared to the control group. Moreover, ex-coalminers with a normal chest radiograph and normal serum LDH demonstrated elevated serum BGD compared to the control group. However, no relation was found in the total group of ex-coalminers between serum BGD activity and pulmonary function parameters., Conclusions: Our study adds in vivo human evidence to the already existing animal data that BGD is a potential biomarker useful in monitoring pulmonary inflammation caused by coal dust exposure.

Background: The objective of the study was to evaluate the additional value of beta-glucuronidase (BGD), a lysosomal enzyme in the analysis of transudative and exsudative pleural effusions, especially between malignant and non-malignant effusions., Design and Methods: Pleural fluid samples obtained from four respective diagnostic groups: transudates parapneumonic effusions, malignant effusions or pleuritis carcinomatosa, and empyema were evaluated., Results: Beta-glucuronidase was significantly different between transudative and exsudative effusions (p<0.001) as well as between parapneumonic and malignant effusions (p<0.03), parapneumonic effusions and empyema (p<0.002), and malignant and empyema (p<0.002), respectively. Logistic regression analysis yielded a weak discrimination between the parapneumonic and malignant groups., Conclusions: Beta-glucuronidase activity differed between pleural effusions of various origin. However, including BGD in the biochemical work-up of pleural effusions did not reveal discriminatory value in the assessment of the classification of these effusions.

Previous studies suggest that the greater sensitivity of mice, compared to rats, to the carcinogenicity of 1,3-butadiene (BD) is linked to higher rates of BD metabolism to butadiene diepoxide (BDO2) by mice than rats. The purpose of this study was to determine the tumorigenicity of BDO2 in mice and rats exposed by inhalation to the same concentrations of the agent. Female B6C3F1 mice and Sprague-Dawley rats, 10-11 weeks old, 56/group, were exposed to 0, 2.5, or 5.0 ppm BDO2, 6 h/day, 5 days/week for 6 weeks. At the end of the BDO2 exposure, 8 animals/group were evaluated for toxicity. The remainder of the exposed rats and mice were held for up to 18 months for observation of tumor development. At the end of the exposure, rats had no biologically significant alteration in standard hematological parameters, but mice had a dose-dependent increase in neutrophils and decrease in lymphocytes. Most of the significant lesions in both species were in the nose, concentrated around the main airflow pathway. Necrosis, inflammation, and squamous metaplasia of the nasal mucosa, as well as atrophy of the turbinates, were all present in animals exposed to 5.0 ppm. In mice, necrosis and inflammation subsided within 6 months, but squamous metaplasia remained. In rats that died after exposure, squamous metaplasia was seen in areas of earlier inflammation and extended beyond those areas with time. The metaplasia was severe enough to restrict and occlude the nasopharyngeal duct. Later, keratinizing squamous-cell carcinomas developed from metaplastic foci in rats, but these were not seen in mice. At the end of 18 months, the only significant increase in neoplasia in the exposed rats was a dose-dependent increase in neoplasms of the nasal mucosa (0/47, 12/48, and 21/48 for the control, 2.5 ppm, and 5.0 ppm exposures, respectively). Neoplasia of the nasal mucosa did not increase significantly in the mice. Neoplastic lesions in the mice were observed in reproductive organs, lymph nodes, bone, liver, Harderian gland, pancreas, and lung, but the only significant increase in neoplasms in a single organ in the mice was in the Harderian gland (0/40, 2/42, and 5/36 for the control, 2.5 ppm, and 5.0 ppm exposures, respectively). This tumor accounts for the apparent trend toward an increase in total neoplastic lesions in mice as a function of dose (10/40, 7/42, and 16/36 for control, 2.5 ppm, and 5.0 ppm, respectively). These findings indicate that the metabolite of BD, BDO2, is carcinogenic in the upper respiratory tract of rats. An increase in upper respiratory tract tumors was not observed in similarly exposed mice, despite the fact that preliminary studies indicated mice should have received twice the dose to tissue than did the rats. Higher cytosolic activity of detoxication enzymes has been reported in the liver and lung cells of the mouse compared to the rat, and this may account, in part, for the differences in response. The transport of externally administered BDO2, into the cell and through the cytoplasm, might allow detoxication of the molecule before it reaches critical sites on the DNA. The results indicate that the site of formation of the BDO2 is important for tumor induction.

Butadiene diepoxide (BDO2), a metabolite of 1,3-butadiene (BD) and potent mutagen, is suspected to be a proximate carcinogen in the multisite tumorigenesis in B6C3F1 mice exposed to BD. Rats, in contrast to mice, do not form much BDO2 when exposed to BD, and they do not form cancers after exposure to the low levels of BD at which mice develop lung and heart tumors. Tests were planned to determine the direct carcinogenic potential of BDO2 in similarly exposed rats and mice, to see if they would develop tumors of the lung (the most sensitive target organ in BD-exposed mice) or other target tissues. The objective of the current series of studies was to assess the acute toxicity and dosimetry to blood and lung of BDO2 administered by various routes to B6C3F1 mice and Sprague-Dawley rats. The studies were needed to aid in the design of the carcinogenesis study. Initial studies using intraperitoneal injection of BDO2 were designed to determine the rate at which each of the species cleared the compound from the body; the clearance was equally fast in both species. A second study was designed to determine if the highly reactive BDO2, when deposited in the lung, would enter the bloodstream from the lung; intratracheally instilled BDO2 did enter the bloodstream, indicating that exposure via the lungs would result in BDO2 reaching other organs of the body. In a third study, rats and mice were exposed by inhalation for 6 h to 12 ppm BDO2 to determine blood and lung levels of the compound. Concentrations of BDO2 in the lung immediately after the exposure were 2 to 3 times higher than in the blood in both species (approximately 500 and 1000 pmol/g blood in the rat and mouse, respectively). As expected, mice received a higher dose/g tissue than did rats, consistent with the higher minute volume/kg body weight of the mice. The inhalation dosimetry study was followed by a histopathology study to determine the acute toxicity to rodents following a single, 6-h exposure to 18 ppm BDO2. No clinical signs of toxicity were observed; lesions were confined to the olfactory epithelium where areas of necrosis were observed. Analysis of bronchoalveolar lavage fluid did not indicate pulmonary inflammation. Based on these findings, an attempt was made to expose rats and mice repeatedly (for 7 days) to 10 and 20 ppm BDO2, but these exposure concentrations proved too toxic, due to inflammation of the nasal mucosa and occlusion of the nasal airway, a lesion that cannot be tolerated by obligate nose breathers. Finally, the toxicity of rats and mice exposed 6 h/day for 5 days to 0, 2.5, or 5.0 ppm BDO2 was determined. The repeated exposures caused no clinical signs of toxicity, nor were any lesions observed in the respiratory tract or other major organs. Therefore, the final design selected for the carcinogenesis study comprised exposing the rats and mice for 6 h/day, 5 days/week for 6 weeks to 0, 2.5, or 5.0 ppm BDO2.

The species specific response to 1,3-butadiene (BD), an important industrial chemical, was investigated by determining the influence of exposure duration and exposure concentration on the mutagenicity of BD in mice and rats and by defining the spectra of mutations in the Hprt gene T-cell mutants from control and BD-exposed mice. Female B6C3F1 mice and F344 rats (4-5 weeks old) were exposed by inhalation to 0, 20, 62.5, or 625 ppm of BD for up to 4 weeks (6 h/day, 5 days/week). Groups of control and exposed animals (n=4-12/group) were necropsied at multiple time points after exposure and the T-cell cloning assay was used to measure Hprt mutant frequencies in lymphocytes isolated from spleen. Mutant clones collected from control and BD-exposed mice were propagated and analyzed by RT-PCR to produce Hprt cDNA for sequencing. In animals necropsied 4 weeks after 2 or 4 weeks of BD exposure (0 or 625 ppm), the rate of accumulation of mutations was greater in mice than in rats. Supra-linear dose-response curves were observed in BD-exposed mice, indicating a higher efficiency of mutant induction at lower concentrations of BD. The mutagenic potency estimates (represented by the differences in the areas under the mutant T-cell 'manifestation' curves of treated vs. control animals) in mice were 11 and 61 following 4 weeks of exposures to 62.5 and 625 ppm of BD, respectively, while mutant frequencies (Mfs) in rats were significantly increased only at 625 ppm BD (mutagenic potency of 7). Molecular analysis of Hprt cDNA from expanded T-cell clones from control and BD-exposed mice demonstrated an increased frequency of mutants in exposed animals that likely contain large deletions in the Hprt gene (P=0.016). These data indicate that both exposure duration and exposure concentration are important in determining the magnitude of mutagenic response to BD, and that mutagenic and carcinogenic properties of BD in mice may be related more to the ability of its metabolites to cause chromosomal deletions than to produce point mutations., (Copyright 1999 Elsevier Science B.V.)

The purpose of this study was to determine if Hprt mutant frequency (Mf) data from rodents exposed directly to individual epoxy metabolites of 1,3-butadiene (BD) can be used to identify the relative significance of each intermediate in the mutagenicity of BD in mice vs. rats. To this end, the relative contributions of the racemic mixtures of BD monoepoxide (BDO) and BD diepoxide (BDO(2)) to BD-induced mutagenicity was investigated by exposing mice and rats to selected concentrations of BDO and BDO(2) (i.e., 2.5 and 4.0 ppm, respectively) and comparing the mutagenic potency of each intermediate to that of BD (at 62.5 ppm) when comparable blood levels of metabolites are achieved (in the mouse). Female B6C3F1 mice and F344 rats (4-5 weeks old) were exposed to rac-BDO (0, 2.5, or 25 ppm) or (+/-)-BDO(2) (0, 2, 4 ppm) by inhalation for 4 weeks (6 h/day, 5 days/week), and then groups of control and exposed animals (n=3-12/group) were necropsied at multiple time points post-exposure for measuring Hprt Mfs in splenic lymphocytes (via the T-cell cloning assay) and estimating mutagenic potencies (represented by the difference in the areas under the mutant T-cell 'manifestation' curves of treated vs. control animals). The resulting Mf data, along with the extant metabolism data, suggest that at lower BD exposures (

1,3-Butadiene (BD) is a high-volume chemical used in the production of rubber and plastic. BD is a potent carcinogen in mice and a much weaker carcinogen in rats, and has been classified as a probable human carcinogen. Upon metabolic activation in vivo, it forms DNA-reactive metabolites, 1,2-epoxy-3-butene (EB), 1,2:3, 4-diepoxybutane (DEB), and 3,4-epoxy-1,2-butanediol (EBD). The molecular dosimetry of N-7 guanine adduct formation by these metabolites of BD in liver, lung, and kidney of B6C3F1 mice and F344 rats exposed to 0, 20, 62.5, or 625 ppm BD was studied. The adducts, racemic and meso forms of N-7-(2,3,4-trihydroxybut-1-yl)guanine (THB-Gua), N-7-(2-hydroxy-3-buten-1-yl)guanine (EB-Gua I), and N-7-(1-hydroxy-3-buten-2-yl)guanine (EB-Gua II), were isolated from DNA by neutral thermal hydrolysis, desalted on solid-phase extraction cartridges, and quantitated by LC/ESI(+)/MS/MS. The number of adducts per 10(6) normal guanine bases for a given adduct was higher in mice than rats exposed to 625 ppm BD, but generally similar at lower levels of exposure. The THB-Gua adducts were the most abundant (6-27 times higher than EB-Gua) and exhibited a nonlinear exposure-response relationship. In rats, the exposure-response curves for the formation of THB-Gua adducts reached a plateau after 62.5 ppm, suggesting saturation of metabolic activation. The number of THB-Gua adducts continued to increase in mice between 62.5 and 625 ppm BD. In contrast, the less common EB-Gua adducts had a linear exposure-response relationship in both species. Combining the information from this study with previous data on BD metabolism, we were able to estimate the number of THB-Gua that resulted from DEB and EBD, and conclude that most of the THB-Gua is formed from EBD. We hypothesize that most of the EBD arises from the immediate conversion of DEB to EBD within the endoplasmic reticulum. This study highlights the need for measurements of the levels of EBD in tissues of rats and mice and for the development of a unique biomarker for DEB that is available for binding to DNA.

It has been suggested that alterations in bronchoalveolar lavage fluid (BALF) reflect pathologic changes in the lung. Cytoplasmatic enzymes such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and LDH isoenzymes are recognized indicators of cell damage or death. The aim of this study was to determine whether there is a relation between the enzyme activity and the cell types present in BALF. Therefore, BALF samples obtained from patients with various pulmonary disorders were studied. Out of these samples a group with mainly polymorphonuclear neutrophils (PMNs; n = 15; Group I) and another with mainly alveolar macrophages (AMs; n = 10; Group II) were selected. Additionally, the value of analysis of lysed cells in BALF for assessment of LDH-isoenzyme patterns was examined. The cell-free fraction of BALF of Group II showed lower LDH and ALP activity compared to Group I. The LDH-isoenzyme pattern also differed, with the LDH3/LDH5 ratios being lower in all BALF samples with predominantly PMNs than in BALF samples with predominantly AMs. Lysis of the cells present in the BALF samples by sonication prior to LDH-isoenzyme analysis provided no additional information beyond that found by analysis of the cell-free BALF. In conclusion, determination of enzyme activity appears to be useful in monitoring pulmonary inflammation.

1,3-Butadiene (BD), a compound used extensively in the rubber industry, is weakly carcinogenic in Sprague-Dawley rats after chronic exposures to concentrations of 1000 and 8000 ppm. Conversely, in B6C3F1 mice, tumors occur after chronic exposures to concentrations as low as 6.25 ppm. Previously, we have shown that tissue concentrations of the mutagenic BD metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), are present in greater concentrations in mice than in rats following acute exposures to low levels (100 ppm or less). This disparity was particularly significant for the diepoxide. We hypothesized that if these epoxides are involved in the carcinogenic response of BD, then they will also be present in rat tissues at relatively high concentrations following exposures to 8000 ppm BD. In the present study, concentrations of the BD epoxides, BDO and BDO2, were determined in blood of female Sprague-Dawley rats following a single 6-h exposure and 10 repeated exposures to a target concentration of 8000 ppm BD. Concentrations of these epoxides were also determined in a number of other tissues, including the primary rat target organ-mammary gland-following 10 repeated exposures. Blood concentrations of BDO were 4030 pmol/g +/- 191 following a 6-h exposure and were 18% lower following the 10-day exposure. Blood concentrations of BDO2, following the 8000 ppm exposures, were very similar to those previously observed after exposures to 62.5 ppm BD (11 +/- 1 and 17 +/- 1 pmol/g following exposures of 6h and 6h/day for 10 days, respectively.) Concentrations of BDO ranged from 740 +/- 110 (femur) to 8990 +/- 1150 (fat) pmol/g tissue. Concentrations of BDO2 were similar among eight tissues analyzed, ranging from 5 +/- 1 (femur) to 17 +/- 3 (heart) pmol/g tissue. Tissue concentrations of butadiene monoepoxide were increased by 17- to 50-fold in tissues from rats exposed by inhalation to 8000 ppm BD as compared to tissues from rats exposed to 62.5 ppm BD. Based on earlier studies at our institute the internal dose of BD increases approximately 14-fold in the 8000 ppm-exposed rats compared to rats exposed to 62.5 ppm BD. Concentrations of butadiene diepoxide in rat tissues following an exposure to 8000 ppm BD were similar to those observed in rat tissues following exposures to 62.5 ppm BD. This study shows that pathways responsible for the accumulation of BDO2 in rats are saturated following low-level BD exposures. This suggests that the primary determinant of BD tumorigenicity in rats is not butadiene diepoxide. The high levels of BDO observed in rat mammary tissue suggest that this metabolite may be a more important determinant of BD carcinogenesis in the rat., (Copyright 1998 Academic Press.)

1,3-Butadiene (BD), a compound used extensively in the rubber industry, is a potent carcinogen in mice and a weak carcinogen in rats in chronic carcinogenicity bioassays. While many chemicals are known to alter their own metabolism after repeated exposures, the effect of exposure prior to BD on its in vivo metabolism has not been reported. The purpose of the present research was to examine the effect of repeated exposure to BD on tissue concentrations of two mutagenic BD metabolites, butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2). Concentrations of BD epoxides were compared in several tissues of rats and mice following a single exposure or ten repeated exposures to a target concentration of 62.5 ppm BD. Female Sprague-Dawley rats and female B6C3F1 mice were exposed to BD for 6 h or 6 h x 10 days. BDO and BDO2 were quantified in blood and several other tissues following preparation by cryogenic vacuum distillation and analysis by multidimensional gas chromatography-mass spectrometry. Blood and lung BDO concentrations did not differ significantly (P < or = 0.05) between the two exposure regimens in either species. Following multiple exposures to BD, BDO levels were 5- and 1.6-fold higher (P < or = 0.05) in mammary tissue and 2- and 1.4-fold higher in fat tissue of rats and mice, respectively, as compared with single exposures. BDO2 levels also increased in rat fat tissue following multiple exposures to BD. However, in mice, levels of this metabolite decreased by 15% in fat, by 28% in mammary tissue and by 34% in lung tissue following repeated exposures to BD. The finding that the mutagenic epoxide BDO, which is the precursor to the highly mutagenic BDO2, accumulates in rodent fat may be important in assessing the potential risk to humans from inhalation of BD.

The pathways of metabolism of benzene appear to be qualitatively similar in all species studied thus far. However, there are quantitative differences in the fraction of benzene metabolized by the different pathways. These species differences become important for risk assessments based on animal data. Mice have a greater overall capacity to metabolize benzene than rats or primates, based on mass balance studies conducted in vivo using radiolabled benzene. Mice and monkeys metabolize more of the benzene to hydroquinone metabolites than do rats or chimpanzees, especially at low doses. Nonhuman primates metabolize less of the benzene to muconic acid than do rodents or humans. In all species studied, a greater proportion of benzene is converted to hydroquinone and ring-breakage metabolites at low doses than at high doses. This finding should be considered in attempting to extrapolate the toxicity of benzene observed at high doses to predicted toxicity at low doses. Because ring-breakage metabolites and hydroquinone have both been implicated in the toxicity of benzene, the higher formation of those metabolites in the mouse may partially explain why mice are more sensitive to benzene than are rats. Metabolism of benzene in humans, the species of interest, does not exactly mimic that of any animal species studied. More information on the urinary and blood metabolites of occupationally exposed people is required to determine the fractional conversion of benzene to putative toxic metabolites and the degree of variability present in human subjects.

Levels of butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2) were compared in tissues of male Sprague-Dawley rats and male B6C3F1 mice and in tissues of male and female Sprague-Dawley rats following inhalation exposures to 62.5 ppm 1,3-butadiene (BD). In male rats, BDO2 levels were highest in blood and were present at a concentration of only 5 +/- 1 pmol/g. Following a 6-h exposure, the concentration of BDO2 in the blood, femurs, lung and fat of female rats was 3 to 7-fold that of male rats. Levels of BDO were similar in tissues of female and male rats. Generally, levels of BDO were approximately 3 to 8-fold greater in mouse tissues as compared with rat tissues following 4-h exposures to BD. In blood, 204 +/- 15 pmol/g BDO2 was detected in male mice, while in rats, blood BDO2 levels were 5 +/- 1 pmol/g. This study shows marked species differences in tissue levels of BD epoxides, particularly BDO2, in rats and mice, and is the first to show gender differences in BD metabolism.

Species differences in the metabolism of 1,3-butadiene (BD) have been studied in an effort to explain the major differences observed in the responses of mice, the sensitive species, and rats, the resistant species, to the toxicity of inhaled BD. BD is metabolized by the same metabolic pathways in all species studied, but there are major species differences in the quantitative aspects of those pathways. Of the species studied, mice are the most efficient at metabolizing BD to the initial metabolite, the monoepoxide (BDO). Mice either convert most of the BDO to the diepoxide (BDO2), the most mutagenic of the BD metabolites, or form conjugates of the BDO with glutathione (GSH). Rats, on the other hand, are less active at forming BDO, oxidize very little of the BDO to BDO2, and form GSH conjugates with either the BDO or its hydrolysis product, butenediol. Primates convert even less of inhaled BD to BDO and hydrolyze most of the BDO to the butenediol. The extent to which primates form BDO2 is unknown. Because of the association of high levels of the highly mutagenic BDO2 with the sensitive rodent strain, it is important to determine the production of this metabolite in primates, particularly humans.

Differences among laboratory animal species in the pulmonary carcinogenicity of chronic inhalation exposure to diesel exhaust have raised several important interpretive issues. Under similar heavy exposure conditions, it is clear that diesel exhaust is a pulmonary carcinogen in rats, but not in Syrian hamsters. Previous reports give conflicting views of the response of mice, which is presently considered equivocal. This report describes carcinogenicity results from a bioassay of CD-1 mice conducted in parallel with a previously reported bioassay of F344 rats (Mauderly et al. (1987) Fundam. Appl. Toxicol. 9, 208-221). Exposure to whole diesel exhaust 7 hr/day, 5 days/week for 24 months at soot concentrations of 0.35, 3.5, or 7.1 mg/m3 caused accumulations of soot in mouse lungs similar to those in lungs of rats and, like the results from rats, did not significantly affect survival or body weight. In contrast to the dose-related neoplastic response of rats, however, the exposures of mice did not increase the incidence of lung neoplasms. This finding is consistent with other data showing that mice, as well as Syrian hamsters, differ from rats in their lung neoplastic and nonneoplastic responses to heavy, chronic inhalation exposure to diesel exhaust soot and several other particles. Although rodents serve as useful indicators of potential human carcinogenic hazards, it is not yet clear which, if any, rodent species have lung neoplastic responses that are useful for quantitative predictions of human lung cancer risk from chronic inhalation of poorly soluble, respirable particles.

A major public health concern is the degree to which environmental or occupational exposures to exogenous chemicals result in adverse health effects. Biological markers have the potential for helping to answer this important question by providing links between markers of exposures and markers of early stages of the development of disease. However, that potential requires in-depth, mechanistic research to be fully realized. Biological markers of exposure have been extensively investigated, and mathematical models of the toxicokinetics of agents have been developed to relate exposures to internal doses. The field of clinical medicine has long used clinical signs and symptoms to detect disease. However, the critical area of research needed to improve the application of biomarkers to environmental health research is mechanistic research to link dose to critical tissues to the development of early, pre-clinical signs of developing disease. Only if the mechanism of disease induction is known can one determine the 'biologically effective' dose and the earliest biological changes leading to disease.

Repeated inhalation of nickel subsulfide (Ni3S2) by F344/N rats for 3 months results in chronic active inflammation in the lung and atrophy of the olfactory epithelium. The primary purpose of this study was to determine early responses of the respiratory tract to inhaled Ni3S2 in rats and to track the course of development of such lesions in rats exposed for up to 22 days. A secondary purpose was to obtain an improved estimate of the half-time for clearance of Ni from Ni3S2-exposed lungs. Groups of F344/N rats were exposed to 0, 0.6 or 2.5 mg Ni3S2/m3, 6 h/day for 1-22 days. Histopathological changes in nose and lung, as well as biochemical and cytological changes in lung, as measured in bronchoalveolar lavage fluid (BALF) and lung tissue, alveolar macrophage (AM) viability and Ni concentration in lung were evaluated. Inflammatory lung lesions in rats exposed to 2.5 mg Ni3S2/m3 peaked in intensity after 4 days of exposure. Minimal degeneration of the olfactory epithelium was noted in the 2.5 mg Ni3S2/m3-exposed rats after day 4 of exposure, with atrophy of the olfactory epithelium occurring in rats killed at 22 days. Lactate dehydrogenase, beta-glucuronidase and total protein in BALF were significantly elevated within 7 days of exposure while alkaline phosphatase activity was significantly depressed. AM viability was significantly reduced after 2 days of exposure. Concentrations of Ni in lung increased rapidly during the first 7 days of exposure, but more slowly thereafter. Lung burden data from this and a previous study suggest a clearance half-time for Ni of 3.5-8 days. Results indicate that Ni3S2 is relatively soluble in lung and inhalation of concentrations near the current Threshold Limit Value of 1 mg Ni/m3 can produce detrimental changes in the respiratory tract of rats after only a few days of exposure.

1,3-Butadiene (BD), a compound used extensively in the rubber industry, is carcinogenic in the male and female Sprague-Dawley rats after chronic exposures to 1000 and 8000 p.p.m. In terms of incidence of tumors the majority were in mammary tissue, thus the incidence of tumors in female rats exceeded that in males in chronic carcinogenicity studies. In the present study the production and disposition of butadiene monoepoxide (BDO) and butadiene diepoxide (BDO2), mutagenic BD metabolites, were examined in male and female Sprague-Dawley rats following a low level inhalation exposure to BD. The rats were exposed to a target concentration of 62.5 p.p.m. BD by nasal inhalation for 6 h. Immediately after exposure blood, bone marrow, lung and fat samples were removed from all the animals and mammary tissue was removed from females. The samples were prepared by cryogenic-vacuum line distillation and analyzed for the epoxides using multidimensional gas chromatography-mass spectrometry. Levels of BDO in the blood were 25.9 +/- 2.9 and 29.4 +/- 2 pmol/g in male and females respectively. The levels of this metabolite were also similar in males and females in the other tissues examined. The greatest amounts of BDO were in fat (175 +/- 21 and 203 +/- 13 pmol/g in males and females respectively). Levels of BDO2 were approximately 5-fold greater in the blood of female rats compared with male rats. In the other tissues examined BDO2 was also consistently greater in tissues from females. In fat BDO2 was present at a concentration of 7.7 +/- 1.3 and 1.1 +/- 0.1 pmol/g tissue in females and males respectively. Mammary tissue from female rats contained 10.5 +/- 2.4 pmol/g BDO2, a level slightly lower than that observed in blood. The ratios of the two epoxides differed markedly between males and females in all tissues examined. Differences were most pronounced in lung and fat tissues, where BDO/BDO2 ratios were 9 and 0.6 (lung) and 159 and 26 (fat) for males and females respectively. This study is the first to describe a gender difference in the metabolism of BD. The greater production of the highly mutagenic BDO2 in females may play a role in the increased incidence of mammary tumors after chronic exposure to BD.