Michele A. Medinsky

Reduction of benzene metabolism and toxicity in mice that lack CYP2E1 expression

Transgenic CYP2E1 knockout mice (cyp2e1-/-) were used to investigate the involvement of CYP2E1 in the in vivo metabolism of benzene and in the development of benzene-induced toxicity. After benzene exposure, absence of CYP2E1 protein was confirmed by Western blot analysis of mouse liver samples. For the metabolism studies, male cyp2e1-/- and wild-type control mice were exposed to 200 ppm benzene, along with a radiolabeled tracer dose of [14C]benzene (1.0 Ci/mol) by nose-only inhalation for 6 hr. Total urinary radioactivity and all radiolabeled individual metabolites were reduced in urine of cyp2e1-/- mice compared to wild-type controls during the 48-hr period after benzene exposure. In addition, a significantly greater percentage of total urinary radioactivity could be accounted for as phenylsulfate conjugates in cyp2e1-/- mice compared to wild-type mice, indicating the importance of CYP2E1 in oxidation of phenol following benzene exposure in normal mice. For the toxicity studies, male cyp2e1-/-, wild-type, and B6C3F1 mice were exposed by whole-body inhalation to 0 ppm (control) or 200 ppm benzene, 6 hr/day for 5 days. On Day 5, blood, bone marrow, thymus, and spleen were removed for evaluation of micronuclei frequencies and tissue cellularities. No benzene-induced cytotoxicity or genotoxicity was observed in cyp2e1-/- mice. In contrast, benzene exposure resulted in severe genotoxicity and cytotoxicity in both wild-type and B6C3F1 mice. These studies conclusively demonstrate that CYP2E1 is the major determinant of in vivo benzene metabolism and benzene-induced myelotoxicity in mice.

Recent developments in methanol toxicity

The disposition of methanol and its putative toxic metabolite formate has been studied in humans, non-human primates, and rodents after exposure to high, neurotoxic doses. The rate at which rodents detoxify formate is more rapid than that of primates. Formate, an endogenous biological substrate, is detoxified by metabolism to CO2 via a tetrahydrofolate-(THF) dependent pathway. Species with high hepatic THF levels, such as rodents, are less sensitive to the neurotoxic effects of large methanol doses compared with species with low THF levels, such as primates. Data on the capacity of primates to detoxify formate derived from inhalation of low levels of methanol are critical for assessing human risk from methanol fuels. Female cynomolgus monkeys exposed to low concentrations of [14C]methanol (10-200 ppm) for 2 h have blood levels of methanol-derived formate that are 100- to 1000-fold lower than endogenous levels of formate. Healthy human volunteers exposed at rest or during exercise to 200 ppm methanol for 6 h or exposed to 20 mg/kg orally have elevated blood levels of methanol, but blood formate concentrations are not significantly increased above endogenous concentrations. Deficiencies in THF may prolong blood levels of formate and increase the likelihood of toxic effects. Limited studies in non-human primates with low THF levels exposed to 900 ppm methanol for 2 h have shown that concentrations of methanol-derived formate in blood remain below endogenous levels. Thus human populations may not be at added risk of neurotoxic effects resulting from exposure to low levels of methanol.

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.

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.

Development of hepatic lesions in male Fischer-344 rats fed AIN-76A purified diet.

n Abstractn n The suitability of the AIN-76A diet for Fischer-344 rats was investigated. This diet, proposed by the American Institute of Nutrition for use when a purified diet composed of refined ingredients and added vitamins and minerals is required, was tested in Sprague-Dawley rats. Male weanling Fischer-344 rats were fed three different lots of the AIN-76A diet from two suppliers. Increase in body weights and food consumption were compared to animals fed a cereal-based control diet. Animals were sacrificed at various intervals and tissues were taken for histopathological observation. By 8 weeks moderate to marked periportal lipidosis developed in livers of all rats fed the AIN-76A diet. Liver-body weight ratios over the 8-week period were significantly higher in rats fed AIN-76A diets compared to rats fed the control diet. However, growth rates of rats fed the AIN-76A diet were similar to growth rates of controls. Some rats fed the AIN-76A diet developed severe hemorrhagic lesions. The AIN-76A diet in its present form is not suitable for use with male Fischer-344 rats.n n

A simulation model describing the metabolism of inhaled and ingested selenium compounds

Abstract Organ distribution and retention of the trace element selenium were studied in rats after inhalation of selenious acid and elemental selenium aerosols. A simulation model of selenium metabolism was developed from data obtained. Although the rate of absorption of these two compounds into blood was different, once absorbed both chemical forms behaved identically. This indicates that both compounds joined the same selenium metabolic pool. The simulation model was extended to estimate the levels of selenium in people inhaling selenium compounds normally found in urban atmospheres and in individuals exposed to selenium compounds in the workplace. Predictions of the model for uptake and organ retention of selenium from the environment were compared to measured organ burdens in humans. Inhalation of selenium compounds at levels normally present in urban atmospheres does not contribute significantly to human body burdens of selenium. The most probable source of selenium in human tissues is due to ingestion of selenium in food. However, selenium present in the workplace, in concentrations at the timeweighted threshold limit value, may pose an inhalation hazard to at least some of the workers.

Metabolism of butadiene by mice, rats, and humans: a comparison of physiologically based toxicokinetic model predictions and experimental data

1,3-Butadiene is a carcinogen in rats and mice, with mice being substantially more sensitive than rats. Our recent research is directed toward obtaining a better understanding of the cancer risk of butadiene in humans by evaluating species-dependent differences in the formation of the toxic metabolites epoxybutene and diepoxybutane. The recent data include in vitro studies on butadiene metabolism using tissues from humans, rats, and mice as well as experimental data and physiological model predictions for butadiene in blood and butadiene epoxides in blood, lung, and liver after exposure of rats and mice to inhaled butadiene. The findings suggest that humans would be more like rats and less like mice regarding the formation of butadiene epoxides. These research findings permit a reassessment of some default options that are used in carcinogen risk assessments. The research approach employed can be a useful strategy for developing mechanistic and toxicokinetic data to supplant default assumptions used in carcinogen risk assessments.

Systemic absorption of selenious acid and elemental selenium aerosols in rats

Absorption of Se from the nasal passages, lungs, gastrointestinal tract, and skin was studied in Fischer-344 rats. Radiolabeled selenious acid and elemental Se particles were administered by inhalation, gavage, nasal instillation, and iv injection. Selenious acid was always absorbed into the general circulation more rapidly and to a greater extent than elemental Se. By 4 h after inhalation of selenious acid and elemental Se aerosols, 94% of the selenious acid and 57% of the elemental Se deposited in lungs was absorbed into blood. Of the selenious acid instilled into nasal passages, 18% was absorbed into blood; 16% of the elemental Se was absorbed. Gastrointestinal absorption was 87% for selenious acid and 50% for elemental Se. Selenious acid solutions were also painted onto the pelts of rats. From 10 to 30% of the selenious acid was absorbed through the skin. Following inhalation or injection of either Se compound, most of the Se was excreted in the urine. Significantly more Se appeared in feces of animals receiving elemental Se by gavage than animals receiving selenious acid. Results indicate that if people were to absorb inhaled Se from the upper respiratory tract in a manner similar to that of rats, one-third more selenious acid would be absorbed into the general circulation than elemental Se. All Se deposited in the lungs would be absorbed into blood. However, selenious acid would be absorbed more rapidly than elemental Se.

Toxicity of selenium compounds to alveolar macrophages

Selenium compounds released into urban atmospheres as a result of fossil fuel combustion may pose an inhalation hazard to people. Two chemical forms of selenium produced during coal combustion and present in combustion effluent are selenious acid. H2SeO3, and elemental selenium, Se. In an attempt to determine the toxicity of selenium compounds relative to other trace elements, the cytotoxicity of H2SeO3 and Se to rabbit alveolar macrophages in vitro was investigated. Macrophages were obtained by lung lavage and exposed in tissue culture after 20 h. Neither selenious acid nor elemental selenium caused cell lysis at concentrations which decreased total cell viability. Selenious acid was an order of magnitude more toxic then elemental selenium. Elemental selenium was similar in toxicity to environmental contaminants such as CdCl2 and V2O5. These in vitro cytotoxicity data can be used to predict the risk posed to people inhaling selenium compounds at levels found in urban atmospheres.

The application of physiologically based pharmacokinetic/ pharmacodynamic (PBPK/PD) modeling to understanding the mechanism of action of hazardous stances

Much of toxicology research is focused on elucidating the nature of the mechanisms through which various xenobiotics exert their toxic effects. The central issue in extrapolating laboratory experiments to the human situation is whether mechanisms which are operative in laboratory animals are similar to mechanisms operating in humans. The underlying assumption is that understanding mechanisms permits rational extrapolation between species, across routes of exposure, or from high to low doses. There are two general classes of mechanisms of action. First, there are the mechanisms that result in the translation of an exposure concentration to the effective dose at the target site. The mechanisms that are operative at a pharmacokinetic level include those that are physiologically driven and those that are metabolically based. Second are mechanisms through which the dose at the target site elicits the ultimate adverse response. These are pharmacodynamic in nature and refer to the action of the effective dose at the target site. Altered gene regulation, cytotoxicity, and cell proliferation are examples of processes involving potential adverse effects at the target site. A quantitative understanding of the mechanisms involved in going from exposure to dose and dose to response can aid in answering the question of whether or not these mechanisms in animals and humans are similar or different.