Arsine gas is a potent hemolytic agent. Early work suggested glutathione depletion preceded, and oxygen required for hemolysis to occur. This study developed an in vitro model of arsine hemolysis, using the solubility of arsine gas in aqueous solutions. A total of 75% of the arsine was taken up into the cells within 5 minutes. Hemolysis occurred after 1-2 hours and reached 40-50%. Glutathione depletion occurred, but only after hemolysis reached its maximum. Increasing intracellular glutathione did not prevent hemolysis. The use of an intracellular chelator, monomethyldimercaptosuccinic acid did not prevent hemolysis. Hemolysis occurred in an oxygen excluding atmosphere but carboxyhemoglobin prevented hemolysis. Glutathione depletion is not a critical first step in arsine induced hemolysis. The interaction of arsine with the heme site of hemoglobin is critical to hemolysis. It is likely that a free radical intermediate of oxygen or arsine is the ultimate hemolytic agent.

Acetaldehyde, the first metabolite of ethanol, has been shown to bind to proteins to form protein-acetaldehyde adducts in vivo and in vitro. The effects of acetaldehyde have been implicated in diseases associated with alcoholism. In the present study, we have extended the observations by studying three different protein-acetaldehyde adducts in vitro, and hair keratin-acetaldehyde adduct in alcohol-fed mice in vivo. Our studies reported here suggest that our enzyme-linked immunosorbent assay (ELISA) is able to detect the stable protein-acetaldehyde adducts. In our preliminary application of the indirect ELISA assay in the chronically alcohol-fed mice, we found that there were significantly increased levels of hair keratin-acetaldehyde adducts in the 5-week alcohol group, 8-week alcohol group, including different alcohol diet groups, compared to the normal control group. We suggest that our indirect ELISA assay has a potential as a biochemical parameter for alcoholism in the clinical settings, although the further study should be performed. Fluorescent techniques, including fluorescent HPLC and fluorescent spectrophotometry were also discussed.

The identification of chlorinated organic compounds in drinking supplies has resulted in an interest in the metabolism and toxicity of chlorinated aldehydes. Another possible factor in the metabolism of chloroaldehydes is the endogenaous tripeptide glutathione. The formation of glutathioneconjugates with chloroaldehydes may increase or decrease their rate of reaction with the aldehyde dehydrogenase enzyme. This study found that the rat liver aldehyde dehydrogenase isozymes lost activity with time regardless of storage conditions. In vitro assays of enzyme activity confirmed substrate specificity for the chloroaldehydes and the isozymes. The reaction rate of glutathione depletion by monochloroacetaldehyde was the highest of the aldehydes. The addition of glutathione to the activity assays resulted in a decrease in Vmax but few significant changes in the Km values. Glutathione concentration was depleted by aldehydes in solution. Glutathione effected the metabolism of aldehydes by aldehyde dehydrogenase isozymes.

No description available.

4-Vinylcyclohexene (VCH), an industrial chemical, causes destruction of small pre-antral follicles (F1) in mice. Previous studies suggested that VCH is hepatically bioactivated to its ovotoxic metabolite, vinylcyclohexene diepoxide (VCD), by the cytochrome P450 enzymes, likely Cyp 2E1, Cyp 2A, and Cyp 2B. Additionally, microsomal epoxide hydrolase (mEH) likely participates in detoxifying the epoxide metabolites. The role of ovarian metabolism (bioactivation/detoxification) of VCH and its metabolites is not known. The hypothesis of this dissertation research was that ovarian metabolism contributes to VCH-induced ovotoxicity. These studies investigated whether the mouse ovary expresses (a) mRNA, (b) total protein, and (c) functional protein for several metabolic enzymes (Cyp 2E1, Cyp 2A, Cyp 2B, mEH), as well as, (d) the effects of VCH/VCD dosing on these parameters. Female B6C3F1 mice were dosed (i.p.) daily (15 d) with VCH (7.4 mmol/kg) or VCD (0.57 mmol/kg). Ovaries were removed and enzymatically digested and sorted into specific ovarian fractions (F1, small preantral; F2, large preantral; F3, antral; Int, interstitial cells) for mRNA analysis by realtime PCR, fixed for immunohistochemistry by confocal microscopy, or homogenized for functional assays. Basal expression was detected for mRNA encoding Cyp 2E1, Cyp 2A, Cyp 2B, and mEH in all ovarian fractions. In vivo dosing with VCH/VCD differentially altered expression, as expression increased for all enzymes in targeted F1 follicles. All enzymes were also distributed throughout the ovary, with high immunostaining intensity in the Int. In vivo dosing with VCH/VCD also affected protein distribution. Utilizing model substrates, catalytic activity was evaluated in ovarian fractions or whole ovaries. Basal activity was detected for Cyp 2E1, Cyp 2B, and mEH, while VCH dosing only induced activities for Cyp 2E1 and mEH. Taken together, the ovary has the metabolic capacity to be involved in metabolic reactions. Bioactivation is likely via Cyp 2E1 in the Int. cells. Although the relative contribution of ovarian metabolism in VCH-induced ovotoxicity is not known, the ovary likely plays a greater role in detoxification, due to greater levels of mEH activity. Thus, the ovary may provide a metabolic contribution to mediating the effects of ovotoxicants.

Environmental arsenic exposure is associated with human cancers of the skin, lung, kidney, and bladder. Mechanisms of arsenic toxicity and carcinogenicity, however, remain poorly studied and merit further investigation. The present studies address the effects of arsenic metabolism on toxicity, under the hypothesis that arsenic metabolism results in both detoxication and bioactivation. Inorganic arsenic and methylated metabolites were tested for toxicity in cultured cells, demonstrating that both detoxication and bioactivation occurs with metabolism, dependent upon methylation and valence state. Monomethylated MMAIII , the most toxic metabolite in cultured cells was also a potent inhibitor of pyruvate dehydrogenase and was more lethal than arsenite in hamsters, illustrating its role as an arsenic bioactivation product. The lung is an established target of arsenic exposure. Arsenic also crosses placenta during pregnancy, reaching the developing fetus. Given this evidence, the present studies investigate the ability of arsenic to target the developing lung following in utero exposure to low doses of arsenic during fetal development. Fetal rats were exposed to 500 mug/L arsenic via maternal drinking water, from conception to embryonic day eighteen. In order to assess toxicogenetic alterations in the developing lung, subtractive hybridization was used to create a cDNA library of arsenic-induced differential gene expression. This library consisted of 326 clones that were subsequently spotted on a cDNA microarray, including those involved in lung development and in formation of the extracellular matrix. In order to model effects of arsenic on gene expression in the developing lung, microarrays were conducted utilizing cultured lung cells dosed with four sub-cytotoxic doses of arsenic for up to fourteen days. These arrays showed that arsenic modulates a decreasing number of genes over the time course and that genes are primarily upregulated following short exposures. Selected array and subtracted library gene expression was also evaluated by quantitative real time PCR and western immunoblotting. Additional microarrays were conducted with 500 ppb arsenic treated fetal lung tissue using a commercial cDNA microarray, revealing perturbations in cellular proliferation and angiogenesis genes in vivo. Collectively, these studies indicate that lung development can be perturbed by gestational arsenic exposure.

Isoniazid (INH) is an antimicrobial used around the world in the treatment and chemoprophylaxis of tuberculosis. Hepatotoxicity is a well-recognized adverse effect of INH therapy. Metabolites of INH, namely hydrazine (HD) and acetylhydrazine (AcHD), are believed to be responsible for this hepatotoxicity. Studies were initiated to test the hypothesis that HD and not AcHD administration results in alterations in hepatic lipid homeostasis. In adult male C57B1/6J mice doses up to 300 mg AcHD/kg, p.o. did not produce liver damage. In contrast, exposure to HD resulted in time- and dose-dependent decreases in plasma cholesterol as well as lipid accumulation leading to liver damage. Hepatic gene expression profiles were determined after administration of HD or AcHD (100 mg/kg, p.o.). The expression of genes involved in lipid synthesis, transport and metabolism, as well as genes associated with necrosis were altered by HD In contrast, AcHD produced fewer changes and did not result in the differential expression of genes involved in lipid accumulation or necrosis. Several of the genes changed by HD exposure are regulated by PPARalpha. The involvement of PPARalpha in HD-mediated steatosis was investigated in PPARalpha-deficient mice. Administration of HD to these mice induced greater hepatic lipid accumulation and macrovesicular degeneration than did its administration to wild-type mice. This is consistent with the role of PPARalpha in removing lipids from liver. The pathology and the microarray data suggest that the PPARalpha-deficient mice are less capable of meeting the demands of HD-mediated increases in hepatic lipid presentation. In the wild-type animals, PPARalpha is activated in response to HD-induced increases in fatty acids. Consequently, these mice are better able to compensate for the lipid accumulation. This is the first demonstration of a critical role for PPARalpha in response to HD-induced steatosis.

3,3',4,4'-tetrachloroazoxybenzene (TCAOB), and 3,3',4,4'-tetrachloroazobenzene (TCAOB), which are structurally similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are formed as byproducts during the synthesis of industrial products. They exhibit Ah receptor binding characteristics and activities similar to those defined for TCDD in vitro at equal moral doses. However, they do not express toxicities in vivo at equal molar doses. The reduced in vivo toxicity can possibly be attributed to differences in the absorption and dispositional kinetics of TCAOB and TCAB as compared to TCDD. Thus, in this study, the absorption and dispositional kinetics of TCAOB and TCAB were examined in male F-344 rats. To address this, the animals received ¹⁴C-TCAB or ¹⁴C-TCAOB and the excretion of [¹⁴C] was monitored over 96 hr. For TCAB and TCAOB, the majority of the dose was eliminated within 48 hr regardless of the route of administration. The primary route of elimination was via the feces, and significant quantities of [¹⁴C] were eliminated in the urine. Pharmacokinetic parameters indicate that the compounds are readily cleared from the blood (TCAB: t₁/₂=4 hr, CLs=12 ml/kg(min); TCAOB t1/2=7 hr, CLs=12 ml/kg(min)). By contrast, TCDD has a half-life of 16-31 days and a elimination rate of 1-2% of the [¹⁴C] -dose per day in the bile/feces with no urinary elimination. Thus, TCAOB and TCAB are eliminated faster than TCDD. Urinary metabolite analysis following administration of TCAOB or TCAB revealed a variety of dichlorolaniline conjugates, which indicates the role of azo reduction in their formation. Several metabolites were present in the bile including glucuronide conjugates of dichloroaniline (DCA) and a putative glucuronide of TCAB (formed from TCAOB as well as TCAB). Clearly, the azo bond is responsible for the enhanced elimination of TCAOB and TCAB as compared to TCDD. The removal of the intestinal flora by the antibiotic pretreatment reduced the total reductive activity but did not eliminate it completely. This indicated that the rapid metabolism and elimination of these two compounds was a result of the combination of azo-reduction by both gut flora and mammalian tissues. The production of dichloroaniline by the in vitro liver homogenate strengthens this conclusion. Since dichloroaniline was produced over time by the liver enzymes following administration of either TCAOB or TCAB, the liver thus contributes to the reduction of the azo bond.

In lung preparations, As(V) was reduced to As(III) [first order rate constant of 0.0104/min]; As(III) was oxidized to As(V) [first order rate constant of 0.005/min], methylated to MMA [K(m) = 5.383μM, V(max) = 0.00031 μmol/liter/min/mg], and complexed with GSH; MMA was converted to DMA [K(m) = 63.4 μM, V(max) = 0.0000384 μmol/liter/min/mg]; and arsine was oxidized to As(III) and As(V) and methylated. Toxicity of As(III), As(V), MMA, DMA, and arsine was assessed by measuring effects on cell and slice viability hsp32 induction, and production of DNA single strand breaks. Because all species of arsenic did not produce effects, it was possible to deduce an "active" form of arsenic from these studies. Pulmonary arsenic metabolism was modeled using SIMUSOLV. This model indicated that arsine disposition cannot be explained solely by oxidation to As(III) before methylation or further oxidation occurs. The concentration of arsenic species present in toxicity studies were predicted with this model and correlated to observed effects. There was good correlation between reduction of As(V) to As(III) with toxicity and hsp32 induction. However, the effects observed for arsine did not correlate with oxidation to arsenite.

Studies were initiated to characterize the acute and sub-chronic pulmonary responses to inhaled Jet-Propulsion Fuel 8 (JP-8) aerosol. At 24 to 30 hours following JP-8 exposure, physiological, biochemical, cellular, and morphological techniques were used to assay for lung injury. In addition, C57BL/6 and B6.A.D.(Ahyd mice were utilized in the acute study to determine if responsiveness to aryl hydrocarbon hydroxylase (AHH) induction modulates toxicity. JP-8 contains aromatic compounds that are known substrates for AHH. Acute lung injury was evident by increased respiratory permeability that was accompanied by alveolar macrophage infiltration and activation, following exposure to occupational permissible levels of JP-8. Morphological alterations were characterized by terminal bronchiolar lesions of vacuolization and necrosis. AHH responsiveness did not appear to influence the severity of JP-8 induced lung injury. Lung injury following sub-chronic inhalation was found to be progressive in nature. Repeated exposure induced alveolar hemorrhage and alveolar macrophage cytotoxicity. Morphological changes progressed to include epithelial denudation of bronchiolar airways and vacuolization of alveolar type II epithelial cells and adjacent endothelia. The pulmonary clearance of JP-8 following inhalation exposures was determined indirectly by analysis for tetradecane content within lung homogenates from exposed mice. Clearance of JP-8 following acute exposure was determined to have a half-life of 43 minutes and increased by 14 minutes following a toxic sub-chronic exposure. The relatively rapid pulmonary clearance of JP-8 following either acute or sub-chronic exposure implies that JP-8 induced lung injury was independent of pulmonary retention. Studies were also performed to determine if non-cytotoxic concentrations of JP-8 or tetradecane could decrease bronchial epithelial barrier function. Studies showed that one hour of exposure to JP-8 or tetradecane could significantly enhance paracellular permeability to mannitol in the BEAS-2B human bronchial epithelial cell line, at two hours after exposure. Bronchial epithelial permeability appeared to be more sensitive to tetradecane than JP-8. Subsequent recovery studies determined that JP-8 and tetradecane-induced decreases in barrier function reach a maximum at 12 hours and barrier function returns to control by 48 hours post-exposure. These results indicate that JP-8 induced lung injury may be initiated by changes in airway barrier function.