Concentration- and time-dependent genomic changes in the mouse urinary bladder following exposure to arsenate in drinking water for up to 12 weeks.

Inorganic arsenic (As(i)) is a known human bladder carcinogen. The objective of this study was to examine the concentration dependence of the genomic response to As(i) in the urinary bladders of mice. C57BL/6J mice were exposed for 1 or 12 weeks to arsenate in drinking water at concentrations of 0.5, 2, 10, and 50 mg As/l. Urinary bladders were analyzed using gene expression microarrays. A consistent reversal was observed in the direction of gene expression change: from predominantly decreased expression at 1 week to predominantly increased expression at 12 weeks. These results are consistent with evidence from in vitro studies of an acute adaptive response that is suppressed on longer exposure due to downregulation of Fos. Pathways with the highest enrichment in gene expression changes were associated with epithelial-to-mesenchymal transition, inflammation, and proliferation. Benchmark dose (BMD) analysis determined that the lowest median BMD values for pathways were above 5 mg As/l, despite the fact that pathway enrichment was observed at the 0.5 mg As/l exposure concentration. This disparity may result from the nonmonotonic nature of the concentration-responses for the expression changes of a number of genes, as evidenced by the much fewer gene expression changes at 2 mg As/l compared with lower or higher concentrations. Pathway categories with concentration-related gene expression changes included cellular morphogenesis, inflammation, apoptosis/survival, cell cycle control, and DNA damage response. The results of this study provide evidence of a concentration-dependent transition in the mode of action for the subchronic effects of As(i) in mouse bladder cells in the vicinity of 2 mg As(i)/l.

Extrapolating the acute behavioral effects of toluene from 1- to 24-h exposures in rats: roles of dose metric and metabolic and behavioral tolerance.

Recent research on the acute effects of volatile organic compounds suggests that extrapolation from short (∼1 h) to long durations (up to 4 h) may be improved by using estimates of brain toluene concentration (Br[Tol]) instead of cumulative inhaled dose (C × t) as a metric of dose. This study compared predictions of these two dose metrics on the acute behavioral effects of inhaled toluene in rats during exposures up to 24 h in duration. We first evaluated estimates of Br[Tol] with a physiologically based toxicokinetic (PBTK) model for rats intermittently performing an operant task while inhaling toluene for up to 24 h. Exposure longer than 6 h induced P450-mediated metabolism of toluene. Adjusting the corresponding parameters of the PBTK model improved agreement between estimated and observed values of Br[Tol] in the 24-h exposure scenario. Rats were trained to perform a visual signal detection task and were then tested while inhaling toluene (0, 1125, and 1450 ppm for 24 h and 1660 ppm for 21 h). Tests occurred at times yielding equivalent C × t products but different estimates of Br[Tol], and also at 1 and 6 h afterexposure. Effects of toluene were better predicted by Br[Tol] than by C × t. However, even using Br[Tol] as the dose metric (after accounting for metabolic induction), acute dose-effect functions during 24-h exposures were shifted to the right relative to 1-h exposures, indicating that a dynamic behavioral tolerance also developed during prolonged exposure to toluene.

Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in C57BL6 mice following subchronic exposure to arsenate in drinking water.

The relationship of exposure and tissue concentration of parent chemical and metabolites over prolonged exposure is a critical issue for chronic toxicities mediated by metabolite(s) rather than parent chemical alone. This is an issue for AsV because its trivalent metabolites have unique toxicities and relatively greater potency compared to their pentavalent counterparts for many endpoints. In this study, dose-dependency in tissue distribution and urinary excretion for inorganic arsenic and its methylated metabolites was assessed in female C57Bl/6 mice exposed to 0, 0.5, 2, 10 or 50 ppm arsenic (as arsenate, AsV) in their drinking water for 12 weeks. No adverse effects were observed and body weight gain did not differ significantly among groups. Urinary excretion of arsenite monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), dimethylarsinic acid (DMAV), and trimethylarsine oxide (TMAO) increased linearly with dose, whereas AsV and monomethylarsonic acid (MMAV) excretion was non-linear with respect to dose. Total tissue arsenic accumulation was greatest in kidney > lung > urinary bladder >>> skin > blood > liver. Monomethyl arsenic (MMA, i.e. MMA(III)+MMAV) was the predominant metabolite in kidney, whereas dimethylarsenic (DMA, i.e., DMA(III)+DMAV) was the predominant metabolite in lung. Urinary bladder tissue had roughly equivalent levels of inorganic arsenic and dimethylarsenic, as did skin. These data indicate that pharmacokinetic models for arsenic metabolism and disposition need to include mechanisms for organ-specific accumulation of some arsenicals and that urinary metabolite profiles are not necessarily reflective of target tissue dosimetry.

Tissue distribution and urinary excretion of inorganic arsenic and its methylated metabolites in mice following acute oral administration of arsenate.

The relationship of exposure dose and tissue concentration of parent chemical and metabolites is a critical issue in cases where toxicity may be mediated by a metabolite or by parent chemical and metabolite acting together. This has emerged as an issue for inorganic arsenic (iAs), because both its trivalent and pentavalent methylated metabolites have unique toxicities; the methylated trivalent metabolites also exhibit greater potency than trivalent inorganic arsenic (arsenite, As(III)) for some endpoints. In this study, the time-course tissue distributions for iAs and its methylated metabolites were determined in blood, liver, lung, and kidney of female B6C3F1 mice given a single oral dose of 0, 10, or 100 micromol As/kg (sodium arsenate, As(V)). Compared to other organs, blood concentrations of iAs, mono- (MMA), and dimethylated arsenic (DMA) were uniformly lower across both dose levels and time points. Liver and kidney concentrations of iAs were similar at both dose levels and peaked at 1 h post dosing. Inorganic As was the predominant arsenical in liver and kidney up to 1 and 2 h post dosing, with 10 and 100 micromol As/kg, respectively. At later times, DMA was the predominant metabolite in liver and kidney. By 1 h post dosing, concentrations of MMA in kidney were 3- to 4-fold higher compared to other tissues. Peak concentrations of DMA in kidney were achieved at 2 h post dosing for both dose levels. Notably, DMA was the predominant metabolite in lung at all time points following dosing with 10 micromol As/kg. DMA concentration in lung equaled or exceeded that of other tissues from 4 h post dosing onward for both dose levels. These data demonstrate distinct organ-specific differences in the distribution and methylation of iAs and its methylated metabolites after exposure to As(V) that should be considered when investigating mechanisms of arsenic-induced toxicity and carcinogenicity.

A concise review of the toxicity and carcinogenicity of dimethylarsinic acid.

Dimethylarsinic acid (DMA) has been used as a herbicide (cacodylic acid) and is the major metabolite formed after exposure to tri- (arsenite) or pentavalent (arsenate) inorganic arsenic (iAs) via ingestion or inhalation in both humans and rodents. Once viewed simply as a detoxification product of iAs, evidence has accumulated in recent years indicating that DMA itself has unique toxic properties. DMA induces an organ-specific lesion--single strand breaks in DNA--in the lungs of both mice and rats and in human lung cells in vitro. Mechanistic studies have suggested that this damage is due mainly to the peroxyl radical of DMA and production of active oxygen species by pulmonary tissues. Multi-organ initiation-promotion studies have demonstrated that DMA acts as a promotor of urinary bladder, kidney, liver and thyroid gland cancers in rats and as a promotor of lung tumors in mice. Lifetime exposure to DMA in diet or drinking water also causes a dose-dependent increase in urinary bladder tumors in rats, indicating that DMA is a complete carcinogen. These data collectively suggest that DMA plays a role in the carcinogenesis of inorganic arsenic.

Application of modelling techniques to the planning of in vitro arsenic kinetic studies.

A kinetic model describing the hepatic methylation of arsenite [As(III)] was developed on the basis of limited data from in vitro mechanistic studies. The model structure is as follows: sequential enzymic methylation of arsenite to its monomethylated (MMA) and dimethylated (DMA) products by first-order and Michaelis-Menten kinetics, respectively; uncompetitive inhibition of the formation of DMA by As(III); and first-order reversible binding of As(III), MMA and DMA to cytosolic proteins. Numerical sensitivity analysis was used to evaluate systematically the impact of changes in input parameters on model responses. Sensitivity analysis was used to investigate the possibility of designing experiments for robust testing of the uncompetitive inhibition hypothesis, and for further refining the model. Based on the sensitivity analysis, the MMA concentration is the most important response on which to focus. The parameters V(max) and k(i) can be reliably estimated by using the same concentration time-course data at intermediate initial arsenite concentrations of 1--5microM at 30 +/- 5 minutes. K(m) must be estimated independently of V(max), since the two parameters are highly correlated at all times, and the optimal experimental conditions would include lower initial concentrations of arsenite (0.1--0.5microM) and earlier time-points (about 8--18 minutes). The use of initial arsenite concentrations much above 5microM would not yield additional useful information, because the sensitivity coefficients for MMA, protein-bound MMA, DMA and protein-bound DMA tend to become extremely small or exhibit erratic trends. Overall trends in the sensitivity analysis indicated the desirability of performing measurements at times shorter than 60 minutes. This work demonstrates that physiological modelling and sensitivity analysis can be efficient tools for experimental planning and hypothesis testing when applied in the earliest phases of kinetic model development, thus allowing more-efficient and more-directed experimentation, and minimising the use of laboratory animals.

Dose-dependent effects on tissue distribution and metabolism of dimethylarsinic acid in the mouse after intravenous administration.

Most mammals methylate inorganic arsenic to dimethylarsinic acid (DMA). This organic arsenical causes organ-specific toxicity and is a multi-organ tumor promoter. The objective of this study was to examine whether dose could affect the distribution and metabolism of DMA. Female B6C3F1 mice (3-4/time point) were administered 1.11 or 111 mg/kg of DMA (1 microCi of [14C] or unlabeled) intravenously and killed serially (5-480 min). Blood was separated into plasma and red blood cell fractions and liver, kidney and lung were removed, weighed and homogenized. Tissue samples were oxidized and analyzed for DMA-derived radioactivity. Blood and several organs of the non-radioactive DMA-treated animals were digested in acid and analyzed by hydride generation atomic absorption spectrophotometry for DMA and metabolites. Concentration-time profiles showed a biexponential decrease of DMA-derived radioactivity in all tissues examined. Kidney had the highest concentration (1-20% dose/gm) of radioactivity of all tissues up to 60 min post-administration. Concentration of radioactivity was greater in plasma than red blood cells at 5 and 15 min and then was similar for the remaining time points. A dose-dependent effect on the concentration of radioactivity was observed in the lung. The retention of radioactivity in the lung was altered compared with liver and kidney, with a much longer t1/2beta and a disproportionate increase in area under the curve with increased dose. No methylated or demethylated products of DMA were detected in blood or any organ up to 8 h post-exposure. The dose-dependent distribution of DMA in the lung may have a role in the toxic effects DMA elicits in this organ.

An integrated pharmacokinetic and pharmacodynamic study of arsenite action. 1. Heme oxygenase induction in rats.

Rat heme oxygenase (HO) activity was used as a specific (among forms of arsenic) and sensitive biomarker of effect for orally administered sodium arsenite in rats. Time course studies showed that HO was induced in rat liver from 2 to 48 h in both rat liver and kidney. Hepatic and renal inorganic arsenic (iAs) concentrations were high at times preceding a high degree of HO induction. At times following pronounced HO induction, tissue dimethylarsinic acid concentrations were high. Dose-response studies of arsenite showed substantial HO induction in liver at doses of 30 micromol/kg and higher and in the kidney at doses of 100 micromol/kg and higher. Doses of 10 (in liver) and of 30 micromol/kg (in kidney) sodium arsenite given by gavage did not significantly induce rat HO activity. Speciation of tissue total arsenic into iAs, methylarsonic acid (MMA), and dimethylarsinic acid (DMA) permits us to link tissue iAs and HO enzyme induction. There was a linear relationship between tissue inorganic arsenic (iAs) concentration and tissue HO in individual rats (r(2) = 0.780 in liver and r(2) = 0.797 in kidney). Nonlinear relationships were observed between administered arsenite dose and either liver or kidney iAs concentration. Overall, there was a sublinear relationship between administered arsenite and biological effect in rats. Teratogenesis Carcinog. Mutagen. 19:385-402, 1999. Published 1999 Wiley-Liss, Inc.

Strain-dependent disposition of inorganic arsenic in the mouse.

Recent studies have suggested that polymorphisms in the methylation of inorganic arsenic (iAs) exist in animals and humans. Methylation of iAs is an important step in the elimination of arsenic. The objective of this study was to examine whether there are differences in iAs disposition, and hence methylation, between three strains of mice. Ninety-day-old female mice (strains: C3H/HeNCrlBR, C57BL/6NCrlBR, and B6C3F1/CrlBR) were administered [73As]arsenate or [73As]arsenite orally at dose levels of 0.5 or 5.0 mg As/kg. Another group of mice were administered [73As]arsenate (5.0 mg As/kg) intraperitoneally (i.p.). Disposition of [73As] was assessed by whole-body counting, and analysis of urine, feces and tissues for radioactivity. Urine was analyzed by chromatography for arsenic metabolites. Several strain- and dose-related effects in the disposition of [73As] were observed with both arsenicals. After oral administration, the clearance of [73As]arsenate, measured by whole-body counting, was dependent on the strain. However, because there was no strain dependence on clearance of [73As]arsenate administered i.p., the effect after oral administration may be due to a difference in absorption of arsenate between the strains. With increased oral dose of arsenate and arsenite, the clearance of [73As] was slower and there was higher tissue retention of [73As]. The percentage of metabolites excreted in urine also was affected by the administered dose. With increased dose, the percentage of arsenite and monomethylarsonic acid were significantly increased, and dimethylarsinic acid decreased. However, our results suggest there is no overall difference between these strains of mice with respect to disposition of iAs. A better understanding of the role of phenotype in the disposition and toxicity of iAs would reduce the uncertainty in arsenic risk assessment.

Influence of gender and acetone pretreatment on benzene metabolism in mice exposed by nose-only inhalation.

Benzene (BZ) requires oxidative metabolism catalyzed by cytochrome P-450 2E1 (CYP 2E1) to exert its hematotoxic and genotoxic effects. We previously reported that male mice have a two-fold higher maximum rate of BZ oxidation compared with female mice; this correlates with the greater sensitivity of males to the genotoxic effects of BZ as measured by micronuclei induction and sister chromatid exchanges. The aim of this study was to quantitate levels of BZ metabolites in urine and tissues, and to determine whether the higher maximum rate of BZ oxidation in male mice would be reflected in higher levels of hydroxylated BZ metabolites in tissues and water-soluble metabolites in urine. Male and female B6C3F, mice were exposed to 100 or 600 ppm 14C-BZ by nose-only inhalation for 6 h. An additional group of male mice was pretreated with 1% acetone in drinking water for 8 d prior to exposure to 600 ppm BZ; this group was used to evaluate the effect of induction of CYP 2E1 on urine and tissue levels of BZ and its hydroxylated metabolites. BZ, phenol (PHE), and hydroquinone (HQ) were quantified in blood, liver, and bone marrow during exposure and postexposure, and water-soluble metabolites were analyzed in urine in the 48 h after exposure. Male mice exhibited a higher flux of BZ metabolism through the HQ pathway compared with females after exposure to either 100 ppm BZ (32.0 2.03 vs. 19.8 2.7%) or 600 ppm BZ (14.7 1.42 vs. 7.94 + 0.76%). Acetone pretreatment to induce CYP 2E1 resulted in a significant increase in both the percent and mass of urinary HQ glucuronide and muconic acid in male mice exposed to 600 ppm BZ. This increase was paralleled by three- to fourfold higher steady-state concentrations of PHE and HQ in blood and bone marrow of acetone-pretreated mice compared with untreated mice. These results indicate that the higher maximum rate of BZ metabolism in male mice is paralleled by a greater proportion of the total flux of BZ through the pathway for HQ formation, suggesting that the metabolites formed along this pathway may be responsible for the genotoxicity observed following BZ exposure.

Dose-dependent effects on the disposition of monomethylarsonic acid and dimethylarsinic acid in the mouse after intravenous administration.

The organic arsenicals monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) are the primary metabolites of inorganic arsenic, a known human carcinogen. The objective of this study was to examine if dose would affect the excretion and terminal tissue disposition of MMA and DMA in the mouse. 14C-MMA (4.84 and 484 mumol/kg) and -DMA (8.04 and 804 mumol/kg) were administered to female mice via the tail vein. The mice were placed in metabolism cages for collection of urine (1, 2, 4, 8, 12, and 24 h) and feces (24 h). The animals were then sacrificed at 24 h and tissues were removed and analyzed for radioactivity. The urine was also analyzed for parent compound and metabolites. Urinary excretion of MMA- and DMA-derived radioactivity predominated over fecal excretion. Dose did not affect the overall urinary excretion of both compounds. However, fecal excretion was significantly lower in the low-dose MMA-treated animals as opposed to in the high-dose group, whereas in the high-dose DMA-treated group excretion was lower than in the low-dose DMA group. The retention of radioactivity was low (< 2% of dose) and the distribution pattern similar for both compounds, with carcass > liver > kidney > lung. The concentration of radioactivity (% dose/g tissue) was greater in kidney than in liver, lung, and blood for both compounds. The distribution and concentration of MMA-derived radioactivity was significantly greater in the liver and lung of the high-dose group. The MMA-treated animals excreted predominantly MMA in urine and lower amounts of DMA (< 10% of the dose). The percentage excreted as DMA was significantly higher in the low-dose MMA group. In the urine of DMA-treated animals, an unstable metabolite and the parent compound were detected. Overall, it appears the dose of organic arsenical administered has a minimal effect on its excretion and terminal tissue disposition in the mouse. The rapid elimination and low retention of MMA and DMA explain in part their low acute toxicity.

Influence of dietary selenium on the disposition of arsenate in the female B6C3F1 mouse.

Interactions between arsenic (As) and selenium (Se) at the metabolic level are multifaceted and complex. These interactions are of practical significance because populations in various parts of the world are simultaneously exposed to inorganic As in drinking water and Se mainly in the diet at varying levels. The primary goal of this study was to investigate whether differing dietary Se status would alter the profile of urinary metabolites or their time course for elimination after exposure to arsenate [As(V)]. Weanling female B6C3F1 mice were maintained for 28 d on either a control diet of powdered rodent meal sufficient in Se (A, 0.2 ppm) or Torula yeast-based (TYB) diets deficient (B, 0.02 ppm Se), sufficient (C, 0.2 ppm Se), or excessive (D, 2.0 ppm Se) in Se; mice then received by oral gavage 5 mg (As)/kg as sodium [73As] arsenate. The time course for elimination of total arsenic and metabolites in urine was measured over a 48-h period, and total arsenic was determined in feces and tissues at 48 h. Mice on the Se excess diet excreted a significantly higher percentage of urinary As as inorganic As, with a significantly decreased ratio of organic to inorganic As compared to Se-sufficient mice, suggesting that As methylation was decreased. Mice on the Se-deficient diet appeared to eliminate As(V), arsenite, and dimethylarsinic acid (DMA) in urine more slowly than Se-sufficient mice; however, further studies are required to confirm this finding. Mice on the Se-sufficient meal diet (A) excreted significantly less (by percent) arsenate-derived radioactivity in urine and more in feces compared to mice on the Se-sufficient TYB diet (C), with total elimination being similar for both groups. This indicates that mice on the meal diet absorbed significantly less As(V) than mice on the TYB diet, and this may be due to more fiber or "bulk" in the meal diet. This finding emphasizes the importance of considering dietary composition when interpreting and comparing As disposition studies. Overall this study provides suggestive evidence that dietary Se status alters As metabolism and disposition. This indicates that dietary Se status may be an issue that should be considered in the design and interpretation of epidemiologic studies.

Mechanistic considerations in benzene physiological model development.

Benzene, an important industrial solvent, is also present in unleaded gasoline and cigarette smoke. The hematotoxic effects of benzene in humans are well documented and include aplastic anemia, pancytopenia, and acute myelogenous leukemia. However, the risks of leukemia at low exposure concentrations have not been established. A combination of metabolites (hydroquinone and phenol, for example) may be necessary to duplicate the hematotoxic effect of benzene, perhaps due in part to the synergistic effect of phenol on myeloperoxidase-mediated oxidation of hydroquinone to the reactive metabolite benzoquinone. Because benzene and its hydroxylated metabolites (phenol, hydroquinone, and catechol) are substrates for the same cytochrome P450 enzymes, competitive interactions among the metabolites are possible. In vivo data on metabolite formation by mice exposed to various benzene concentrations are consistent with competitive inhibition of phenol oxidation by benzene. In vitro studies of the metabolic oxidation of benzene, phenol, and hydroquinone are consistent with the mechanism of competitive interaction among the metabolites. The dosimetry of benzene and its metabolites in the target tissue, bone marrow, depends on the balance of activation processes such as enzymatic oxidation and deactivation processes such as conjugation and excretion. Phenol, the primary benzene metabolite, can undergo both oxidation and conjugation. Thus the potential exists for competition among various enzymes for phenol. Zonal localization of phase I and phase II enzymes in various regions of the liver acinus also impacts this competition. Biologically based dosimetry models that incorporate the important determinants of benzene flux, including interactions with other chemicals, will enable prediction of target tissue doses of benzene and metabolites at low exposure concentrations relevant for humans.

Exposure in a household using gasoline-contaminated water.

Contamination of drinking water with petroleum products is an increasingly common problem. Physicians are often asked to advise patients about such exposures. This study assessed household exposure from gasoline-contaminated drinking water in a New England household. A sampling strategy was designed to estimate inhalation and ingestion exposure to benzene and three other aromatic hydrocarbons typically found in gasoline-contaminated water. The estimated inhaled doses of all agents were similar to the estimated ingested dose. Over half the inhaled dose of all four agents was associated with shower activities as was over half the estimated total dose by all routes of exposure. Under these conditions, discontinuing ingestion of water contaminated with these agents may decrease the dose of benzene by less than one third, whereas discontinuing both ingestion and showering may decrease the dose of benzene by over three quarters. This limited study suggests that routes of exposure other than ingestion are important and should receive attention in the regulatory and risk-assessment process.

Differences in rates of benzene metabolism correlate with observed genotoxicity.

Benzene (BZ) requires oxidative metabolism via cytochrome P450 2E1 (CYP 2E1) to exert its hematotoxic and genotoxic effects. Male mice are two- to threefold more sensitive to the genotoxic effects of BZ as measured by micronuclei induction and sister chromatid exchanges. The purpose of our study was to investigate sex-related differences in the metabolism of BZ, phenol (PHE) and hydroquinone (HQ) in order to understand the metabolic basis for sex-dependent differences in BZ genotoxic susceptibility in mice. Rates of BZ oxidation were quantitated using closed chamber gas uptake studies with male and female B6C3F1 mice exposed to initial low (400-500 ppm), intermediate (1200-1300 ppm), and high (2600-2800 ppm) BZ concentrations. Acetone-pretreated and diethyldithiocarbamate-pretreated male mice were also studied to determine the extent to which induction and inhibition of CYP 2E1, respectively, would alter in vivo BZ oxidation rates. Elimination of PHE and HQ from blood was also compared in male and female mice to complement previously reported data on sex-related differences in urinary excretion of conjugated metabolites following iv administration of PHE. Based on PBPK model analysis, the optimized rate of metabolism (Vmax) of BZ was almost twofold higher in male mice (14.0 mumol/hr-kg) than in female mice (7.9 mumol/hr-kg); both male and female mice gas-uptake data were well fit with a KM of 3.0 microM. Pretreatment of male mice with 1% acetone in drinking water for 8 days to specifically induce CYP 2E1 enhanced the rate of BZ oxidation by approximately fivefold (Vmax = 75 mumol/hr-kg), while diethyldithiocarbamate pretreatment (320 mg/kg ip 30 min prior to uptake study) completely inhibited BZ oxidation (Vmax = 0 mumol/hr-kg). Thus, both pretreatment regimens are potentially useful investigative tools to study the metabolic basis for benzene toxicity. Elimination of PHE from blood was significantly faster in male mice, while elimination of HQ did not differ between male and female mice. Previous data indicated male mice produce more of the oxidized and conjugated metabolite, HQ glucuronide, after PHE administration, suggesting that HQ production from PHE is greater in male mice. Taken together, these data support the hypothesis that the greater sensitivity of male mice to the genotoxic effects of BZ compared to females is a function of greater oxidative metabolism toward both BZ and PHE in male mice. These data also suggest that differences in hepatic human CYP 2E1 activity may be an important factor to consider when evaluating human risk for benzene-induced hematotoxic and genotoxic effects.

Benzene: a case study in parent chemical and metabolite interactions.

Benzene, an important industrial solvent, is also present in unleaded gasoline and cigarette smoke. The hematotoxic effects of benzene in humans are well documented and include aplastic anemia and pancytopenia, and acute myelogenous leukemia. A combination of metabolites (hydroquinone and phenol for example) is apparently necessary to duplicate the hematotoxic effect of benzene, perhaps due in part to the synergistic effect of phenol on myeloperoxidase-mediated oxidation of hydroquinone to the reactive metabolite benzoquinone. Since benzene and its hydroxylated metabolites (phenol, hydroquinone and catechol) are substrates for the same cytochrome P450 enzymes, competitive interactions among the metabolites are possible. In vivo data on metabolite formation by mice exposed to various benzene concentrations are consistent with competitive inhibition of phenol oxidation by benzene. In vitro studies of the metabolic oxidation of benzene, phenol and hydroquinone are consistent with the mechanism of competitive interaction among the metabolites. The dosimetry of benzene and its metabolites in the target tissue, bone marrow, depends on the balance of activation processes such as enzymatic oxidation and deactivation processes such as conjugation and excretion. Phenol, the primary benzene metabolite, can undergo both oxidation and conjugation. Thus, the potential exists for competition among various enzymes for phenol. However, zonal localization of Phase I and Phase II enzymes in various regions of the liver acinus regulates this competition. Biologically-based dosimetry models that incorporate the important determinants of benzene flux, including interactions with other chemicals, will enable prediction of target tissue doses of benzene and metabolites at low exposure concentrations relevant for humans.

Dose-, route-, and sex-dependent urinary excretion of phenol metabolites in B6C3F1 mice.

Phenol is the major oxidized metabolite of benzene, a known human leukemogen and ubiquitous environmental pollutant. Unlike benzene, phenol does not induce tumors in mice following oral exposure; benzene also exhibits sex-related differences in genotoxicity to bone marrow cells that are not observed following phenol administration. We studied the urinary excretion of phenol metabolites in mice as a means to further investigate the metabolic basis for differences in benzene- and phenol-induced toxicity. Male and female B6C3F1 mice (n = 3/group) were exposed to 15, 40, 100, or 225 mumol [14C]phenol/kg by i.v. tail vein injection (6 microCi/mouse). First-pass intestinal metabolism of phenol was evaluated by comparison of urinary excretion of phenol metabolites following i.v. administration with additional groups of male mice that received the same dose levels by oral gavage. Mice were placed in glass metabolism cages, and urine was collected over dry ice for 48 h. Urinary metabolites were separated by high-pressure liquid chromatography (HPLC) and quantified by liquid scintillation spectrometry. Urinary excretion of conjugated metabolites of phenol was dose-dependent in both male and female mice administered phenol by i.v. injection or gavage. The major urinary metabolites of phenol were phenol sulfate (PS), phenol glucuronide (PG), and hydroquinone glucuronide (HQG). Sulfation was the dominant pathway at all dose levels, but decreased as a percent of the excreted dose with a concomitant increase in glucuronidation as the dose level increased. Male mice consistently excreted a higher proportion of phenol as the oxidized conjugated metabolite, HQG, compared to female mice, suggesting that male mice oxidize phenol to hydroquinone more rapidly than female mice. Increased oxidation of phenol to hydroquinone by male mice compared to female mice is consistent with both the greater sensitivity of male mice to the genotoxic effects of benzene and the greater potency of hydroquinone compared to phenol as a genotoxicant. Intestinal conjugation of phenol prior to absorption was significant only at low doses and thus alone does not provide an explanation for the lack of carcinogenicity of phenol in bioassays conducted at much higher dose levels.

Extent and implications of interspecies differences in the intestinal hydrolysis of certain glucuronide conjugates.

1. Interspecies differences in the intestinal hydrolysis of glucuronide conjugates (phenolphthalein glucuronide, 4-methylumbelliferone glucuronide, morphine-3-glucuronide) were evaluated in mouse, rat and rabbit small intestine and caecum. 2. beta-Glucuronidase activity in the caecum was 50-200-fold higher than in the small intestine for all species and substrates studied. 3. There was evidence of a similarity between species in the capacity of the gut contents cultures from the proximal and distal small intestine to hydrolyse phenolphthalein glucuronide and 4-methylumbelliferone glucuronide. 4. Morphine was not liberated from morphine-3-glucuronide in detectable amounts in the proximal and distal small intestine. 5. Species- and substrate-specific differences were identified in the capacity of the caecal microbiota to hydrolyse the glucuronide conjugates studied. 6. The capacity of the rabbit caecal microbiota to hydrolyse all three glucuronides was significantly lower than those of both rat and mouse. 7. Morphine was metabolized to codeine in low, but detectable levels in all three species.

Inhibition of beta-glucuronidase in human urine by ascorbic acid.

The effects of oral supplementation of ascorbic acid (1500 mg d-1) on urinary beta-glucuronidase (beta-G) activity was assessed in a double-blind crossover study design over a 3-week period. The subjects on ascorbic acid treatment displayed a statistically significant (P less than 0.05) decrease in beta-G with an approximate decrease of 25%.

Isolation of pathogenic Aspergillus species from commercially-prepared potting media.

Twelve commercially-prepared potting soils were screened for the presence of pathogenic Aspergillus species. Pathogenic Aspergillus species were isolated from 67% of the soils. A fumigatus was isolated from 42% and A. flavus and A. niger from 33%.