diethyl-maleate and naphthalene

Naphthalene (NA) is a semivolatile aromatic hydrocarbon to which humans are exposed from a variety of sources. NA results in acute cytotoxicity to respiratory epithelium in rodents. Cytochrome P450-dependent metabolic activation to form reactive intermediates and loss of soluble cellular thiols (glutathione) are critical steps in NA toxicity, but the precise mechanisms by which this chemical results in cellular injury remain unclear. Protein thiols are likely targets of reactive NA metabolites. Loss of these, through adduction or thiol oxidation mechanisms, may be important underlying mechanisms for NA toxicity. To address the hypothesis that loss of thiols on specific cellular proteins is critical to NA-induced cytotoxicity, we compared reduced to oxidized thiol ratios in airway epithelial cell proteins isolated from lungs of mice treated with NA or the nontoxic glutathione depletor, diethyl maleate (DEM). At 300 mg/kg doses, NA administration resulted in a greater than 85% loss of glutathione levels in the airway epithelium, which is similar to the loss observed after DEM treatment. Using differential fluorescent maleimide labeling followed by 2DE separation of proteins, we identified more than 35 unique proteins that have treatment-specific differential sulfhydryl oxidation. At doses of NA and DEM that produce similar levels of glutathione depletion, Cy3/Cy5 labeling ratios were statistically different for 16 nonredundant proteins in airway epithelium. Proteins identified include a zinc finger protein, several aldehyde dehydrogenase variants, beta-actin, and several other structural proteins. These studies show distinct patterns of protein thiol alterations with the noncytotoxic DEM and the cytotoxic NA.

Topics: Animals; Chromatography, High Pressure Liquid; Electrophoresis, Gel, Two-Dimensional; Epithelial Cells; Glutathione; Male; Maleates; Mice; Naphthalenes; Oxidation-Reduction; Respiratory Mucosa; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Sulfhydryl Compounds; Tandem Mass Spectrometry

Naphthalene (NA) is metabolized to highly reactive intermediates that are primarily detoxified by conjugation to glutathione (GSH). Intraperitoneal administration of naphthalene causes substantial loss of both hepatic and respiratory GSH, yet only respiratory tissues are injured in mice. The liver supplies GSH to other organs via the circulation, making it unclear whether respiratory GSH losses reflect in situ respiratory depletion or decreased hepatic supply. To address this concern, mice were exposed to naphthalene by inhalation (1.5-15 ppm; 2-4 h), thereby bypassing first-pass hepatic involvement. GSH levels and histopathology were monitored during the first 24 h after exposure. Half of the mice were given the GSH depletor diethylmaleate (DEM) 1 hour before naphthalene exposure. Lung and nasal GSH levels rapidly decreased (50-90%) in mice exposed to 15 ppm naphthalene, with cell necrosis throughout the respiratory tract becoming evident several hours later. Conversely, 1.5 ppm naphthalene caused moderate GSH loss and only injured the nasal olfactory epithelium. Neither naphthalene concentration depleted hepatic GSH. Animals pretreated with DEM showed significant GSH loss and injury in nasal and intrapulmonary airway epithelium at both naphthalene concentrations. DEM treatment, perhaps by causing significant GSH loss, decreased water-soluble naphthalene metabolite formation by 48% yet increased NA-protein adducts 193%. We conclude that (1) GSH depletion occurs in airways independent of hepatic function; (2) sufficient GSH is not supplied by the liver to maintain respiratory GSH pools, or to prevent injury from inhaled naphthalene; and (3) GSH loss precedes injury and increases protein adduct formation.

Topics: Administration, Inhalation; Animals; Animals, Outbred Strains; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Drug Therapy, Combination; Glutathione; Inactivation, Metabolic; Liver; Lung; Male; Maleates; Mice; Naphthalenes; Olfactory Mucosa; Respiratory Tract Diseases

Clara-cell populations show a high degree of variation in susceptibility to injury by bioactivated cytotoxicants. Because glutathione (GSH) is critical for detoxification of electrophilic metabolites, heterogeneity in Clara cell GSH levels may lead to a wide range of cytotoxic responses. This study was designed to define the distinct GSH pools within Clara cells, characterize heterogeneity within the population, and examine whether heterogeneity contributes to susceptibility. Using fluorescent imaging combined with high-performance liquid chromatography analysis, semiquantitative measurements were obtained by evaluation of GSH using monochlorobimane and monobromobimane. In steady-state conditions, the GSH measured in isolated cells was in the femtomole range, but varied 4-fold between individual cells. Clara cells analyzed in situ and in vitro confirmed this heterogeneity. The response of these cells to compounds that modulate GSH was also variable. Diethylmaleate depleted GSH, whereas GSH monoethylester augmented it. However, both acted nonuniformly in isolated Clara cells. The depletion of intracellular GSH caused a striking decrease in cell viability upon incubation with naphthalene (NA). The sulfhydryl-binding fluorochrome BODIPY, which colocalized with tetramethylrosamine, a mitochondrial dye, demonstrated by confocal microscopy that cellular sulfhydryls are highest in the mitochondria, next-highest in cytoplasm, and lowest in the nucleus. These pools responded differently to modulators of GSH. We concluded that the steady-state intracellular GSH of Clara cells exists in distinct pools and is highly heterogeneous within the population, and that the heterogeneity of GSH levels corresponds closely to the response of Clara cells to injury by NA.

Topics: Acetylcysteine; Animals; Boron Compounds; Bridged Bicyclo Compounds, Heterocyclic; Cell Nucleus; Cell Survival; Cells, Cultured; Chromatography, High Pressure Liquid; Cytoplasm; Cytotoxins; Epithelial Cells; Fluorescent Dyes; Glutathione; Lung; Maleates; Microscopy, Fluorescence; Mitochondria; Naphthalenes

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)

Topics: Animals; Buthionine Sulfoximine; Electrophoresis, Polyacrylamide Gel; Glutathione; Hemoglobins; Male; Maleates; Methionine Sulfoximine; Mice; Naphthalenes; Protein Binding; Serum Albumin; Sulfhydryl Compounds

Previous studies have suggested a significant role for reproductive tract glutathione in protecting against chemical-induced germ-cell mutations. Therefore, a number of compounds were tested for their ability to perturb glutathione levels in the testes and epididymides as well as liver following single acute dosages to rats. Phorone (250 mg/kg), isophorone (500 mg/kg), and diethyl maleate (500 mg/kg) significantly reduced glutathione in the liver and in both reproductive organs examined. Methyl iodide (100 mg/kg), trimethyl phosphate (600 mg/kg), naphthalene (500 mg/kg), acetaminophen (1500 mg/kg), and pentachlorophenol (25 mg/kg) affected hepatic and epididymal glutathione, but had little or no effect on testicular levels. The ability of isophorone to enhance the covalent binding of tritiated ethyl methanesulfonate (3H-EMS) to spermatocytes was assessed. Perturbation of reproductive tract glutathione by isophorone treatment significantly enhanced the extent of 3H-EMS-induced binding to sperm heads. The temporal pattern of ethylations in sperm heads was consistent with the stage of sperm development known to be susceptible to ethylations by EMS. Therefore, chemical-induced lowering of glutathione in the male reproductive tract may be a mechanism for potentiation of chemical-induced germ-cell mutations.

Topics: Animals; Cyclohexanes; Cyclohexanones; Epididymis; Glutathione; Hydrocarbons, Iodinated; Ketones; Male; Maleates; Naphthalenes; Organophosphates; Rats; Rats, Inbred Strains; Testis

As a measure of glutathione (GSH) conjugation, urinary, fecal and biliary excretion of thioethers and hepatic GSH content were measured in rhesus monkeys following administration of single doses of naphthalene and diethylmaleate (DEM). Naphthalene had little or no effect on hepatic GSH content and the excretion of thioethers into urine, feces or bile of rhesus monkeys which is similar to that observed in chimpanzees and humans and is in contrast to results obtained from rats. Apparently, conjugation of naphthalene and/or its metabolites with GSH does not play a major role in the metabolism of naphthalene in primates, whereas it is one of the major pathways in rodents. Rhesus monkeys, like chimpanzees, excreted about 13% of the various doses of DEM (30, 75 and 200 mg/kg) as thioethers into urine which is half of that excreted by rats. Six hrs after administration of 200 mg/kg DEM, the hepatic GSH content was decreased by 90% in the rhesus monkey. During the first day after this dose (200 mg/kg), the increase in the excretion of thioethers into bile corresponded to about 15% of the dose of DEM administered. Since fecal excretion of thioethers corresponded to only 1% of the dose and urinary excretion represented 12% of the dose, it appears that biliary thioethers of DEM are reabsorbed from the intestine and then excreted into urine. It appears that the rhesus monkey as well as the chimpanzee is, whereas the rat is not, a good animal model to study GSH-related conjugation reactions with predictive value for man.

Topics: Animals; Bile; Biotransformation; Feces; Female; Glutathione; Liver; Macaca mulatta; Male; Maleates; Naphthalenes; Sulfides; Time Factors