Alan R. Buckpitt

Characterisation of the toxic metabolite(s) of naphthalene

The toxicity of naphthalene and its metabolites has been investigated in vitro. Both naphthalene and its metabolite 1-naphthol were bioactivated by human hepatic microsomes to metabolite(s) which were toxic to mononuclear leucocytes (MNL). However 1-naphthol was more cytotoxic than naphthalene (49.8 +/- 13.9% vs. 19.0 +/- 10.0% cell death; P < 0.01), indicating that the toxicity of naphthalene is dependent on the bioactivation of 1-naphthol. CYP2E1-induced rat liver microsomes increased metabolism of naphthalene by 13% compared to control microsomes with a concomitant increase in both 1-naphthol and dihydrodiol formation. The cytotoxicity of naphthalene but not of 1-naphthol was increased by CYP2E1 induction, indicating that separate enzymes are involved in the bioactivation of 1-naphthol. The metabolites of 1-naphthol, 1,2-naphthoquinone (51.4 +/- 6.6% cell death) and 1,4-naphthoquinone (49.1 +/- 3.4% cell death) were directly toxic to MNL and depleted glutathione to 1.0% of the control levels. Both quinones were also genotoxic to human lymphocytes. In contrast, the primary metabolite of naphthalene, the 1,2-epoxide (0-100 microM) was neither cytotoxic nor genotoxic, and did not deplete glutathione. In conclusion, our data suggests that the cytotoxicity and genotoxicity of naphthalene is associated with the formation of quinones from 1-naphthol rather than naphthalene-1,2-epoxide.

Allergic asthma induced in rhesus monkeys by house dust mite (Dermatophagoides farinae)

To establish whether allergic asthma could be induced experimentally in a nonhuman primate using a common human allergen, three female rhesus monkeys (Macaca mulatta) were sensitized with house dust mite (Dermatophagoides farinae) allergen (HDMA) by subcutaneous injection, followed by four intranasal sensitizations, and exposure to allergen aerosol 3 hours per day, 3 days per week for up to 13 weeks. Before aerosol challenge, all three monkeys skin-tested positive for HDMA. During aerosol challenge with HDMA, sensitized monkeys exhibited cough and rapid shallow breathing and increased airway resistance, which was reversed by albuterol aerosol treatment. Compared to nonsensitized monkeys, there was a fourfold reduction in the dose of histamine aerosol necessary to produce a 150% increase in airway resistance in sensitized monkeys. After aerosol challenge, serum levels of histamine were elevated in sensitized monkeys. Sensitized monkeys exhibited increased levels of HDMA-specific IgE in serum, numbers of eosinophils and exfoliated cells within lavage, and elevated CD25 expression on circulating CD4(+) lymphocytes. Intrapulmonary bronchi of sensitized monkeys had focal mucus cell hyperplasia, interstitial infiltrates of eosinophils, and thickening of the basement membrane zone. We conclude that a model of allergic asthma can be induced in rhesus monkeys using a protocol consisting of subcutaneous injection, intranasal instillation, and aerosol challenge with HDMA.

Varying effects of sulfhydryl nucleophiles on acetaminophen oxidation and sulfhydryl adduct formation

Abstract The effects of glutathione, cysteine, N-acetylcysteine, cysteamine, α-mercaptopropionylglycine and methionine on the NADPH-dependent metabolism and covalent binding of acetaminophen have been examined in mouse liver microsomal incubations. With the exception of methionine, all of the nucleophiles decreased covalent binding by forming adducts with the electrophilic metabolite of acetaminophen. The adducts were measured quantitatively by high pressure liquid chromatography. In contrast to glutathione, N-acetylcysteine and α-mercaptopropionylglycine, both cysteamine and cysteine in high concentrations also decreased covalent binding of acetaminophen through another mechanism, inhibition of the formation of the reactive acetaminophen metabolite. These results indicate that both inhibition of metabolite formation and detoxification of metabolite by sulfhydryl adduct formation are mechanisms that can be important in reducing acetaminophen toxicity in overdosed patients treated with these nucleophiles.

Tolerance to multiple doses of the pulmonary toxicant, naphthalene.

Intraperitoneal administration of single doses of the volatile aromatic hydrocarbon, naphthalene, resulted in dose-dependent bronchiolar epithelial cell necrosis in mice. Twenty-four hours after a dose of 50 mg/kg, swelling of Clara cells with some exfoliation of epithelial cells was evident in half of the treated animals. At doses of 100 mg/kg small numbers of necrotic and swollen cells with pyknotic nuclei were observed. At 200 mg/kg there were substantial numbers of bronchiolar epithelial cells sloughed into the airway lumen, apical projections were virtually absent, and there were large numbers of cells with pyknotic nuclei. In contrast, bronchiolar airways from mice treated with naphthalene daily for 7 days at doses of 50, 100, or 200 mg/kg/day differed only slightly from controls. Significant protection to bronchiolar epithelial cell necrosis produced by 300 mg/kg naphthalene was afforded by seven daily injections of 200 but not 50 or 100 mg/kg naphthalene. A gradual recovery in sensitivity to the 300 mg/kg challenge dose of naphthalene was observed as the time between the last 200 mg/kg naphthalene dose increased from 24 to 144 hr. Daily administration of 200 mg/kg but not 50 or 100 mg/kg naphthalene for 7 days resulted in a selective decrease in the rate of formation of 1R,2S-naphthalene oxide by mouse lung but not liver microsomal enzymes. This selective decrease in pulmonary microsomal formation of 1R,2S-oxide continued in animals killed 48, 96, and 144 hr after the last administration of 200 mg/kg. Alterations in the rate of formation of reactive, covalently bound naphthalene metabolites in lung microsomes were not observed, nor were there any differences in the levels of covalently bound reactive metabolites in vivo between tolerant and control animals. These studies are consistent with other work showing that the lung loses susceptibility to the acute injury arising from repeated exposure to pneumotoxicants. In contrast to other studies with naphthalene where alterations in the levels of covalently bound reactive metabolites in the lung closely paralleled the extent and severity of bronchiolar injury, these studies clearly separate necrosis from covalent binding. Although the correlation was not absolute, it appears that formation of 1R,2S-oxide by microsomal enzymes in vitro is a better overall marker of decreased sensitivity to naphthalene-induced bronchiolar necrosis than is reactive metabolite binding either in vivo or in vitro.

Liver cytosol catalyzed conjugation of reduced glutathione with a reactive metabolite of acetaminophen

Abstract The effect of liver cytosol enzymes on the rate of formation of a glutathione (GSH) conjugate and on the rate of covalent binding of acetaminophen to microsomal proteins was studied in vitro. Mouse liver microsomes were incubated with [ 14 C ] acetaminophen , GSH, a NADPH-generating system, and Sephadex G-25-treated mouse liver supernatant. The addition of liver cytosol to the microsomal incubation increased the rate of conjugate production, as measured by high pressure liquid chromatography and decreased the rate of covalent binding of the reactive intermediate of acetaminophen to microsomal proteins at all concentrations of GSH and acetaminophen studied. The effect of the liver supernatant enzymes was most pronounced at low GSH concentrations. Although cysteine and N-acetyl- l -cysteine form conjugates with acetaminophen, the cytosol preparations did not facilitate conjugate formation with these nucleophiles. Cytosol enzymes may be important in the detoxification of acetaminophen particularly when the liver concentration of GSH is low and may contribute to the marked “threshold” effect seen with acetaminophen toxicity.

Asthma/Allergic Airways Disease : Does Postnatal Exposure to Environmental Toxicants Promote Airway Pathobiology?

The recent, dramatic increase in the incidence of childhood asthma suggests a role for environmental contaminants in the promotion of interactions between allergens and the respiratory system of young children. To establish whether exposure to an environmental stressor, ozone (O3), and an allergen, house dust mite (HDMA), during early childhood promotes remodeling of the epithelial-mesenchymal trophic unit (EMTU) of the tracheobronchial airway wall by altering postnatal development, infant rhesus monkeys were exposed to cyclic episodes of filtered air (FA), HDMA, O3, or HDMA plus O3. The following alterations in the EMTU were found after exposure to HDMA, O3, or HDMA plus O3: (1) reduced airway number; (2) hyperplasia of bronchial epithelium; (3) increased mucous cells; (4) shifts in distal airway smooth muscle bundle orientation and abundance to favor hyperreactivity; (5) interrupted postnatal basement membrane zone differentiation; (6) modified epithelial nerve fiber distribution; and (7) reorganization of the airway vascular and immune system. Conclusions: cyclic challenge of infants to toxic stress during postnatal lung development modifies the EMTU. This exacerbates the allergen response to favor development of intermittent airway obstruction associated with wheeze. And, exposure of infants during early postnatal lung development initiates compromises in airway growth and development that persist or worsen as growth continues, even with cessation of exposure.

Bioactivation of the pulmonary toxicants naphthalene and 1-nitronaphthalene by rat CYP2F4.

Naphthalene, a ubiquitous environmental contaminant, produces cytotoxicity in nonciliated bronchiolar epithelial (Clara) cells in mice; rats are refractory to lung cytotoxicity from naphthalene. In contrast, 1-nitronaphthalene is a potent toxicant in both species. Naphthalene is metabolized by CYP2F to a 1,2-epoxide, the first and obligate step in events leading to cytotoxicity. 1-Nitronaphthalene is metabolized to both the 5,6- and the 7,8-epoxides with the 7,8-epoxide predominating in lung. Previous studies have demonstrated recombinant CYP2F2 (mouse) to efficiently metabolize both naphthalene and 1-nitronaphthalene. To better understand the mechanism for the unique toxicity profiles for both compounds, a CYP2F ortholog (CYP2F4) was isolated from rat lung and expressed using a baculovirus system. Recombinant CYP2F4 efficiently generates 1R,2S-naphthalene oxide (Km = 3 μM, Vmax = 107 min-1) and the 5,6- and 7,8-epoxides of 1-nitronaphthalene (Km = 18 μM, Vmax = 25 min-1 based on total generated glutathione conjugates). Kinetics and regio/stereoselectivity of rat CYP2F4 were indistinguishable from mouse CYP2F2. These results, combined with our recent immunomapping studies demonstrating minimal pulmonary CYP2F expression in rats, indicate that CYP2F expression is the factor most clearly associated with susceptibility to naphthalene-induced pneumotoxicity. CYP2F4 failed to display an enhanced ability to bioactivate 1-nitronaphthalene, an ability that could have potentially compensated for the lower CYP2F pulmonary expression levels in the rat, yet equal species susceptibilities. These results suggest the importance of other P450 enzymes in the epoxidation/bioactivation of 1-nitronaphthalene. Expression of recombinant CYP2F1 (human) yielded an immunoreactive protein with no detectable CO-difference spectrum suggesting inadequate heme incorporation.

Mechanisms of lung injury by systemically administered chemicals

In this paper we will attempt to provide some explanations for the organ selectivity of four different pneumotoxicants (monocrotaline, naphtalene, 3-methylindole, and 4-ipomeanol)

Reactive naphthalene metabolite binding to hemoglobin and albumin

Earlier work has shown that the murine Clara cell cytotoxicant, naphthalene, is metabolized to reactive metabolites which deplete glutathione or, in the absence of sufficient glutathione, become bound covalently to tissue macromolecules. Correlations between bound metabolite levels in the lung with injury suggests an association between reactive metabolite binding and toxicity. In this study we examine the formation of covalent naphthalene adducts with hemoglobin and albumin in mice to determine whether these serve as useful indices of exposure and metabolism for a chemical which shows a glutathione threshold. Covalent binding of radioactivity from [3H]naphthalene to both albumin and hemoglobin was dose dependent and a glutathione threshold was observed. At early times after naphthalene administration, the formation of albumin adducts was 10- to 30-fold higher than that of hemoglobin adducts. Hemoglobin and albumin adduct levels decreased by apparent first-order processes with half-lives of 11.5 and 1.8 days, respectively. These half-lives are consistent with the turnover of these blood proteins in the mouse. Pretreatment with buthionine sulfoximine resulted in higher levels of albumin adduct but in no alteration of hemoglobin adduct levels in comparison with control. In contrast, diethylmaleate pretreatment increased the level of hemoglobin adduct but not albumin adduct. The antibody to naphthalene mercapturates recognized the hemoglobin adduct(s) but not the albumin adduct(s). Comparison of the data from ELISA (standardized using hydroxymercaptodihydronaphthalene) and radiochemical analysis yielded curves with identical slopes; the absolute levels of adduct found by ELISA were approximately half those measured with radiochemical techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of Metabolically Stable Inhibitors of Mammalian Microsomal Epoxide Hydrolase

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to a number of diseases, such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. We recently demonstrated that fatty amides, such as elaidamide, represent a new class of potent inhibitors of mEH. While these compounds are very active on recombinant mEH in vitro, they are quickly inactivated in liver extracts reducing their value in vivo. We investigated the effect of structural changes on mEH inhibition potency and microsomal stability. Results obtained indicate that the presence of a small alkyl group alpha to the terminal amide function and a thio-ether beta to this function increased mEH inhibition by an order of magnitude while significantly reducing microsomal inactivation. The addition of a hydroxyl group 9-10 carbons from the terminal amide function resulted in better inhibition potency without improving microsomal stability. The best compound obtained, 2-nonylsulfanyl-propionamide, is a competitive inhibitor of mEH with a K I of 72 nM. Furthermore, this new inhibitor significantly reduces mEH diol production in ex vivo lungs exposed to naphthalene, underlying the usefulness of the inhibitors described herein. These novel inhibitors could be valuable tools to investigate the physiological and biological roles of mEH.