Naphthalene cytotoxicity in microsomal epoxide hydrolase deficient mice.

Naphthalene (NA) is a ubiquitous pollutant to which humans are widely exposed. 1,2-Dihydro-1,2-dihydroxynaphthalene (NA-dihydrodiol) is a major metabolite of NA generated by microsomal epoxide hydrolase (mEH). To investigate the role of the NA-dihydrodiol and subsequent metabolites (i.e. 1,2-naphthoquinone) in cytotoxicity, we exposed both male and female wild type (WT) and mEH null mice (KO) to NA by inhalation (5, 10, 20 ppm for 4h). NA-dihydrodiol was ablated in the KO mice. High-resolution histopathology was used to study site-specific cytotoxicity, and formation of naphthalene metabolites was measured by HPLC in microdissected airways. Swollen and vacuolated airway epithelial cells were observed in the intra- and extrapulmonary airways of all mice at and below the current OSHA standard (10 ppm). Female mice may be more susceptible to this acute cytotoxicity. In the extrapulmonary airways, WT mice were more susceptible to damage than KO mice, indicating that the metabolites associated with mEH-mediated metabolism could be partially responsible for cytotoxicity at this site. The level of cytotoxicity in the mEH KO mice at all airway levels suggests that non-mEH metabolites are contributing to NA cellular damage in the lung. Our results indicate that the apparent contribution of mEH-dependent metabolites to toxicity differs by location in the lung. These studies suggest that metabolites generated through the mEH pathway may be of minor importance in distal airway toxicity and subsequent carcinogenesis from NA exposure.

Lung effects of inhaled corticosteroids in a rhesus monkey model of childhood asthma.

BACKGROUND: The risks for infants and young children receiving inhaled corticosteroid (ICS) therapy are largely unknown. Recent clinical studies indicate that ICS therapy in pre-school children with symptoms of asthma result in decreased symptoms without influencing the clinical disease course, but potentially affect postnatal growth and development. The current study employs a primate experimental model to identify the risks posed by ICS therapy. OBJECTIVE: To (1) establish whether ICS therapy in developing primate lungs reverses pulmonary pathobiology associated with allergic airway disease (AAD) and (2) define the impact of ICS on postnatal lung growth and development in primates. METHODS: Infant rhesus monkeys were exposed, from 1 through 6 months, to filtered air (FA) with house dust mite allergen and ozone using a protocol that produces AAD (AAD monkeys), or to FA alone (Control monkeys). From three through 6 months, the monkeys were treated daily with ICS (budesonide) or saline. RESULTS: Several AAD manifestations (airflow restrictions, lavage eosinophilia, basement membrane zone thickening, epithelial mucin composition) were reduced with ICS treatment, without adverse effects on body growth or adrenal function; however, airway branching abnormalities and intraepithelial innervation were not reduced. In addition, several indicators of postnatal lung growth and differentiation: vital capacity, inspiratory capacity, compliance, non-parenchymal lung volume and alveolarization, were increased in both AAD and Control monkeys that received ICS treatment. CONCLUSIONS AND CLINICAL RELEVANCE: Incomplete prevention of pathobiological changes in the airways and disruption of postnatal growth and differentiation of airways and lung parenchyma in response to ICS pose risks for developing primate lungs. These responses also represent two mechanisms that could compromise ICS therapy's ability to alter clinical disease course in young children.

Equine cytochrome P450 2C92: cDNA cloning, expression and initial characterization.

Substantial gaps exist in our knowledge of the metabolic clearance of therapeutic agents in horses. Accordingly, a cytochrome P450 monooxygenase in the 2C family was cloned from an equine liver, sequenced and expressed in a baculovirus expression system. Catalytic activities of the recombinant protein were measured with a number of substrates. The protein, assigned CYP2C92, displayed optimal catalytic activity with diclofenac using molar ratios of CYP2C92 to NADPH CYP450 reductase of 1:18. Addition of cytochrome b(5) to diclofenac incubations had no significant effect on metabolic turnover. CYP2C92 catalyzed diclofenac metabolism was 20-fold slower than the human counterpart, CYP2C9. CYP2C92 demonstrated comparable tolbutamide and (S)-warfarin hydroxylase activity compared to CYP2C9, upon addition of b(5) to the reactions. The results of this study demonstrate substantial interspecies differences in metabolism of substrates by CYP2C orthologues in the horse and human and support the need to fully characterize this enzyme system in equids.

Prevention of naphthalene-induced pulmonary toxicity by glutathione prodrugs: roles for glutathione depletion in adduct formation and cell injury.

Naphthalene is metabolized in the lung and liver to reactive intermediates by cytochrome P450 enzymes. These reactive species deplete glutathione, covalently bind to proteins, and cause necrosis in Clara cells of the lung. The importance of glutathione loss in naphthalene toxicity was investigated by using the glutathione prodrugs (glutathione monoethylester or cysteine-glutathione mixed disulfide) to maintain glutathione pools during naphthalene exposure. Mice given a single intraperitoneal injection of naphthalene (1.5 mmol/kg) were treated with either prodrug (2.5 mmol/kg) 30 min later. Both compounds effectively maintained glutathione levels and decreased naphthalene-protein adducts in the lung and liver. However, cysteine-glutathione mixed disulfide was more effective at preventing Clara cell injury. To study the prodrugs in Clara cells without the influence of hepatic naphthalene metabolism and circulating glutathione, dose-response and time-course studies were conducted with intrapulmonary airway explant cultures. Only the ester of glutathione raised GSH in vitro; however, both compounds limited protein adducts and cell necrosis. In vitro protection was not associated with decreased naphthalene metabolism. We conclude that (1) glutathione prodrugs can prevent naphthalene toxicity in Clara cells, (2) the prodrugs effectively prevent glutathione loss in vivo, and (3) cysteine-glutathione mixed disulfide prevents naphthalene injury in vitro without raising glutathione levels.

Glutathione depletion is a major determinant of inhaled naphthalene respiratory toxicity and naphthalene metabolism in mice.

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.

Naphthalene-induced respiratory tract toxicity: metabolic mechanisms of toxicity.

The lung, which is in intimate contact with the external environment, is exposed to a number of toxicants both by virtue of its large surface area and because it receives 100% of the cardiac output. Lung diseases are a major disease entity in the U.S. population ranking third in terms of morbidity and mortality. Despite the importance of these diseases, key issues remain to be resolved regarding the interactions of chemicals with lung tissue and the factors that are critical determinants of chemical-induced lung injury. The importance of cytochrome P450 monooxygenase dependent metabolism in chemical-induced lung injury in animal models was established over 25 years ago with the furan, 4-ipomeanol. Since then, the significance of biotransformation and the reasons for the high degree of pulmonary selectivity for a myriad of different chemicals has been well documented, mainly in rodent models. However, with many of these chemicals there are substantial differences in the susceptibility of rats vs. mice. Even within the same species, varied levels of the respiratory tract respond differently. Thus, key pieces of data are still missing when evaluating the applicability of data generated in rodents to primates, and as a result of this, there are substantial uncertainties within the regulatory community with regards to assessing the risks to humans for exposure to some of these chemicals. For example, all of the available data suggest that the levels of cytochrome P450 monooxygenases in rodent lungs are 10-100 times greater than those measured in the lungs of nonhuman primates or in man. At first glance, this suggests that a significant margin of safety exists when evaluating the applicability of rodent studies in the human, but the issues are more complex. The intent of this review is to outline some of the work conducted on the site and species selective toxicity and metabolism of the volatile lung toxic aromatic hydrocarbon, naphthalene. We argue that a complete understanding of the cellular and biochemical mechanisms by which this and other lung toxic compounds generate their effects in rodent models with subsequent measurement of these cellular and biochemical events in primate and human tissues in vitro will provide a far better basis for judging whether the results of studies done in rodent models are applicable to humans.

Inhaled naphthalene causes dose dependent Clara cell cytotoxicity in mice but not in rats.

Current OSHA standards for naphthalene exposure are set at 10 ppm (time-weighted average) with a standard threshold exposure concentration of 15 ppm. While several studies have thoroughly delineated the time course and dose response of injury by naphthalene administered ip, the pattern and severity of injury by inhalation exposure are unknown. These studies compare the regiospecific and dose-dependent cytotoxicity of naphthalene after inhalation exposure. Mice and rats were exposed for 4 h to naphthalene vapor at concentrations of 0-110 ppm. In rats, no injury was observed in the lung epithelium at exposure concentrations up to 100 ppm. Exposures as low as 2 ppm produced proximal airway injury in mice, with increased severity in a concentration-dependent fashion up to 75 ppm. Terminal airways of exposed mice exhibited little or no injury at low concentrations (1-3 ppm). Exposures of 8.5 ppm or higher were required to produce injury to Clara cells in the terminal airways. In contrast, administration of naphthalene (300 mg/kg) extended the injury pattern toward the lobar bronchus. We conclude (1) the pattern of injury to naphthalene is highly dependent on the route of exposure, (2) lung injury to inhaled naphthalene is species dependent, and (3) Clara cells of mouse airways are exquisitely sensitive to inhaled naphthalene at concentrations well below the current OSHA standard for human exposure.

Unique gene expression patterns in liver and kidney associated with exposure to chemical toxicants.

DNA arrays containing 260 unique genes involved in phase I and II metabolism, heat shock, DNA repair, inflammation, transcription, and housekeeping were used to examine gene expression patterns in liver and kidney in response to five classes of chemicals (polyaromatic hydrocarbons: benzo(a)pyrene, 3-methylcholanthrene; DNA alkylators: dimethylnitrosamine, ethylnitrosourea; peroxisome proliferators: diethylhexylphthalate, clofibrate; heavy metals: CdCl(2), HgCl(2); and oxidative stressors: CCl(4), bromobenzene). Time course experiments in mice were carried out in both tissues for each chemical and dose-response studies were used to further evaluate several of these chemicals. Each pair of chemicals yielded a similar pattern of gene expression distinct from the other four classes of chemicals. Both peroxisome proliferators up-regulated Cyp4a10, acyl-CoA thioesterase, and insulin-like growth factor binding protein-1, whereas the DNA alkylators altered the expression of monokine induced by gamma-interferon, the metallothioneins, p21, and several acute phase proteins. For each of the five classes of chemicals tested, several genes that were induced or repressed were common in each chemical exposure, whereas other genes were unique for that specific class of compound. Both time and dose are important factors in differentiating between chemical classes. Likewise, comparison of changes in messenger RNA expression between the kidney and liver of treated animals indicates that gene arrays may be useful in determining the comparative toxicity of chemicals in various tissues but that exposure to uncharacterized chemicals will have to be monitored in several tissues.

Early events in naphthalene-induced acute Clara cell toxicity. II. Comparison of glutathione depletion and histopathology by airway location.

One of the presumed roles of intracellular glutathione (GSH) is the protection of cells from injury by reactive intermediates produced by the metabolism of xenobiotics. To establish whether GSH depletion is a critical step in the initiation of events that lead to cytotoxicity by P450-activated cytotoxicants, naphthalene, a well-defined Clara cell cytotoxicant, was administered to mice (200 mg/kg) by intraperitoneal injection. Shortly after injection (1, 2, and 3 h), intracellular GSH content was assessed by high performance liquid chromatography or quantitative epifluorescent imaging microscopy and compared with the degree of cytotoxicity as assessed by high resolution histopathology. In highly susceptible airways (distal bronchioles), GSH decreased by 50% in 1 h. Cytoplasmic vacuolization was not visible until 2 h, when GSH had decreased by an additional 50%. By 3 h, cytoplasmic blebbing was extensive. In minimally susceptible airways (lobar and proximal bronchi), GSH depletion varied widely within the population; a small proportion of the cells lost greater than 50% of their GSH by 2 h and a significant percentage of the cells retained most of their GSH throughout the entire 3 h. Cytoplasmic vacuolization was apparent in some of the cells at 2 h but not visible in any cells at 3 h. We conclude that (1) loss of intracellular GSH is an early event that precedes initial signs of cellular damage in Clara cell cytotoxicity; (2) this pattern of loss in relation to early injury is found both in highly susceptible and minimally susceptible airway sites; (3) there is wide cell-to-cell heterogeneity in the response; (4) the heterogeneity in the response profile varies between populations in highly susceptible and minimally susceptible sites; and (5) once the intracellular GSH concentration within the entire cell population drops below a certain threshold, the initial phase of injury becomes irreversible.

Factors modulating the epithelial response to toxicants in tracheobronchial airways.

As one of the principal interfaces between the organism and the environment, the respiratory system is a target for a wide variety of toxicants and carcinogens. The cellular and architectural complexity of the respiratory system appears to play a major role in defining the focal nature of the pulmonary response to environmental stressors. This review will address the biological factors that modulate the response of one of the major target compartments within the respiratory system, the tracheobronchial airway tree. Individual airway segments respond uniquely to toxic stress and this response involves not only the target cell population, e.g. epithelium, but also other components of the airway wall suggesting a trophic interaction within all components of the airway wall in maintaining steady state and responding to injury. A number of biological factors modulate the nature of the response, including: (1) metabolic potential at specific sites for activation and detoxification; (2) the nature of the local inflammatory response; (3) age of the organism at the time of exposure; (4) gender of the exposed organism; (5) history of previous exposure; and (6) species and strain of the organism exposed.

Applications of gene arrays in environmental toxicology: fingerprints of gene regulation associated with cadmium chloride, benzo(a)pyrene, and trichloroethylene.

Toxicity testing of unknown chemicals currently uses a number of short-term bioassays. These tests are costly and time consuming, require large numbers of animals, and generally focus on a single end point. The recent development of DNA arrays provides a potential mechanism for increasing the efficiency of standard toxicity testing through genome-wide assessments of gene regulation. In this study, we used DNA arrays containing 148 genes for xenobiotic metabolizing enzymes, DNA repair enzymes, heat shock proteins, cytokines, and housekeeping genes to examine gene expression patterns in the liver in response to cadmium chloride, benzo(a)pyrene (BaP), and trichloroethylene (TCE). Dose-response studies were carried out in mice for each chemical; each produced a unique pattern of gene induction. As expected, CdCl2 markedly up-regulated metallothionine I and II (5- to 10,000-fold at the highest doses) and several of the heat shock/stress response proteins and early response genes. In contrast, administration of BaP up-regulated only Cyp1a1 and Cyp1a2 genes and produced no significant increases in any of the stress response genes or any of the DNA repair genes present on the array. Likewise, TCE-induced gene induction was highly selective; only Hsp 25 and 86 and Cyp2a were up-regulated at the highest dose tested. Microarray analysis with a highly focused set of genes is capable of discriminating between different classes of toxicants and has potential for differentiating highly noxious versus more subtle toxic agents. These data suggest that use of microarrays to evaluate the potential hazards of unknown chemicals or chemical mixtures must include multiple doses and time points to provide effective assessments of potential toxicity of these substances.

Metabolic capabilities of CYP2F2 with various pulmonary toxicants and its relative abundance in mouse lung subcompartments.

The tissue- and species-selective toxicity of a number of pulmonary toxicants has been attributed to the presence and distribution of activating enzymes with high k(cat) in target airways of susceptible species. The mouse is especially sensitive to a variety of metabolically activated lung toxicants. Recombinant CYP2F2 (mouse) was recently shown to effectively metabolize the species-selective pulmonary toxicant naphthalene. Here we show that the pulmonary toxicants 1-nitronaphthalene and 2-methylnaphthalene are metabolized readily with high k(cat) values (17.1 and 67.6 min(-1), respectively) to potentially cytotoxic intermediates at biologically relevant K(m) values (21.5 and 3.7 microM, respectively). Additionally, anthracene and benzo[a]pyrene are both metabolized by CYP2F2 (0.14 +/- 0.04 and 0.04 +/- 0.00 nmol/nmol/min, respectively), albeit at much lower rates. The levels of total CYP in mouse airways are considerably higher than those in parenchyma and trachea, and this is consistent with much higher rates of naphthalene metabolism in microsomal preparations from airways compared with the other subcompartments. The data suggest that CYP2F2 is a prominent cytochrome P450 in mouse lung that metabolizes a number of pulmonary toxicants. The presence of CYP2F2 may be important in the susceptibility of the mouse to metabolically activated pulmonary toxicants.

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.

Long-term exposure to ozone increases acute pulmonary centriacinar injury by 1-nitronaphthalene: I. Region-specific enzyme activity.

To test whether exposure to ozone alters pulmonary cytochrome P450 monooxygenase-mediated metabolism of xenobiotics, rates of 1-nitronaphthalene (1-NN) metabolism were measured in microsomes prepared from trachea, intrapulmonary airways, and distal lung of rats exposed to filtered air (FA) or ozone (O(3)) (0.8 ppm 8 h/day for 90 days). Regioisomeric glutathione conjugates derived from intermediate epoxides were measured by HPLC. Compared with FA, rates of glutathione conjugate formation in distal lung (including the central acinus) were elevated 2-fold in O(3)-exposed rats. Activity for cytochrome P450 2B, the isozyme thought to be responsible for the metabolic activation of 1-NN, was increased 3-fold in the distal lung of O(3)- compared with FA-exposed rats. There was a 2 +/- 0. 5-fold increase in immunodetectable CYP 2B protein in microsomes from the same lung subcompartment (P <.05). Immunodetectable protein was expressed in nonciliated epithelial (or "Clara") cells and not associated with ciliated epithelial cells. No differences between O(3)- and FA-exposed rats were noted in 1-NN metabolism or CYP 2B activity in trachea or intrapulmonary airways. This study emphasizes that cellular and biochemical alterations associated with long-term O(3) exposure vary considerably by location within the lung. Long-term exposure to O(3) elevates both CYP 2B activity and 1-NN metabolism in an airway-specific manner.

Development of phase II xenobiotic metabolizing enzymes in differentiating murine clara cells.

Glutathione S-transferases (GSTs) and epoxide hydrolases (EHs) protect cells from exogenous insult by detoxifying electrophilic compounds. Little is known about these enzyme systems during postnatal lung development. This study was designed to help establish whether the heightened neonatal susceptibility of the lung to bioactivated cytotoxicants is the result of inadequate ability to detoxify reactive intermediates. We compared the distribution of immunoreactive protein and enzymatic activity of GSTs and EHs in isolated distal airways during pre- and postnatal development in lungs of mice from 16 days gestation to 9 weeks postnatal age (adult). GST alpha, mu, and pi class protein expression in fetal and postnatal lung varied by isozyme and age. Isozymes alpha and mu are expressed at low levels before birth, high levels on postnatal day 7, low levels between postnatal days 14 and 21, high levels at postnatal day 28, and slightly lower levels in adults. Immunoreactive protein of isozyme pi has a peak expression on gestational day 18 and again on postnatal day 4, is undetectable at postnatal day 21, and is at peak levels in the adult mouse lung. GST activity in distal airways increased with age. Microsomal EH protein expression increased in intensity with age, while activity was similar in airways from all ages. We conclude that in the mouse lung (1) cellular expression of glutathione S-transferase varies by age and isozyme and does not increase with increasing age, (2) airway glutathione S-transferase activity increases with increasing age and does not correlate with immunoreactive protein expression, and (3) airway microsomal epoxide hydrolase activity does not increase, even though immunoreactive protein expression does increase with age.

Elevated airway GSH resynthesis confers protection to Clara cells from naphthalene injury in mice made tolerant by repeated exposures.

Repeated exposures to Clara cell cytotoxicants, such as naphthalene (NA), render target cell populations resistant to further acute injury. Previous studies suggest that alterations in bioactivation enzymes in target sites (bronchioles) of tolerant mice are insufficient to account for the marked reduction in susceptibility. Mice were made tolerant by seven daily injections of NA. GSH in the terminal airways was 2.7-fold greater in tolerant mice than in vehicle controls and a NA (300 mg/kg) challenge dose did not produce injury. Tolerant mice, allowed to recuperate for 96 h after the seventh NA injection, were again susceptible to NA injury, and terminal airway GSH levels had declined to control levels. To determine whether alterations in GSH resynthesis account for tolerance, the activity of gamma-glutamylcysteine synthetase (gamma-GCS) was measured or mice were treated with a combination of buthionine sulfoximine (BSO), a gamma-GCS inhibitor, and NA. gamma-GCS activity was elevated in resistant airways of tolerant mice. Tolerant mice treated with both BSO and NA appeared as susceptible to injury as NA-challenged controls. We conclude that GSH is critical for Clara cell resistance to NA injury in tolerant mice because: 1) GSH levels in target airways from NA-tolerant animals are elevated; 2) after a 96-h recuperation period, tolerant mice had lower GSH levels and are again susceptible to NA injury; 3) alterations in the activity of gamma-GCS correspond with changes in susceptibility to NA injury; and 4) inhibition of gamma-GCS with BSO increases susceptibility to NA injury in tolerant mice.

Heterogeneity of clara cell glutathione. A possible basis for differences in cellular responses to pulmonary cytotoxicants.

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.

Species differences in the regio- and stereoselectivity of 1-nitronaphthalene metabolism.

1-Nitronaphthalene (1-NN) is a mutagenic nitroaromatic that has been detected in emissions from both heavy- and light-duty diesel engines, as well as in urban airborne particles. 1-NN is a cytochrome P450-bioactivated, nonciliated bronchiolar epithelial (Clara) cell cytotoxicant. Our recent studies demonstrated that 1-NN was metabolized by rat lung and liver microsomal enzymes to six 1-NN GSH conjugates via intermediate C(5),C(6)- and C(7),C(8)-epoxides. These studies examined the metabolism of 1-NN in mouse, and compared the differences in rates of 1-NN GSH conjugate formation between the two species. HPLC radioactivity profiles demonstrated that seven different conjugates were generated in mouse lung and liver microsomal incubations. Six of the seven conjugates corresponded with those observed in incubations with rat microsomes. Mass spectrometry of the new conjugate yielded a m/z 497 (M+H) and identical daughter ions as in the other six conjugates when analyzed by mass spectrometry in electrospray positive ion mode. The major conjugate generated in mouse and rat lung microsomal incubations was conjugate 4 (1-nitro-7-glutathionyl-8-hydroxy-7, 8-dihydronaphthalene). In comparison, the formation of conjugate 6 (1-nitro-5-hydroxy-6-glutathionyl-5,6-dihydronaphthalene) predominated in mouse liver, whereas in rat liver, conjugate 5, a diastereomer of conjugate 6, was generated at the highest rate. We concluded that the rates of formation of regio- and stereoisomeric epoxides from 1-NN differed substantially in target and nontarget tissues, but there was no clear pattern of correlation of tissue susceptibility to the rate or metabolite produced.

Development of a toxicological gene array and quantitative assessment of this technology.

High-density arrays of DNA bound to solid substrates offer a powerful approach to identifying changes in gene expression in response to toxicants. While DNA arrays have been used to explore qualitative changes in gene regulation, less attention has focused on the quantitative aspects of this technology. Arrays containing expressed sequence tags for xenobiotic metabolizing enzymes, proteins associated with glutathione regulation, DNA repair enzymes, heat shock proteins, and housekeeping genes were used to examine gene expression in response to beta-naphthoflavone (beta-NF). Upregulation of cytochrome P4501a1 (Cyp1a1) and 1a2 in mouse liver was maximal 8 h after beta-NF administration. Significant upregulation of Cyp1a2 was noted at beta-NF doses as low as 0.62 and 1.2 mg/kg when gene expression was measured by microarray or Northern blotting, respectively. Maximal Cyp1a2 induction is 5-fold by Northern analysis and 10-fold by microarray. Induction of Cyp1a1 was 15- and 20-fold by Northern and microarray analysis, respectively. The coefficient of variation for spot to spot and slide to slide comparisons was <15%; this variability was smaller than interanimal variability (18-60%). Comparison of mRNA expression in control animals indicated that there are differences in labeling/detection associated with Cy3/Cy5 dyes; accordingly, experiments must include methods for establishing baseline signals for all genes. We conclude that the dynamic range and sensitivity of DNA microarrays on glass slides is comparable to Northern blotting analysis and that variability of the data introduced during spotting and hybridization is less than the interanimal variability.

Hepatic and pulmonary enzyme activities in horses.

OBJECTIVE: To determine hepatic and pulmonary phase-I and phase-II enzyme activities in horses. SAMPLE POPULATION: Pulmonary and hepatic tissues from 22 horses that were 4 months to 32 years old. PROCEDURE: Pulmonary and hepatic tissues from horses were used to prepare cytosolic (glutathione S-transferase and soluble epoxide hydrolase) and microsomal (cytochrome P450 monooxygenases) enzymes. Rates of microsomal metabolism of ethoxyresorufin, pentoxyresorufin, and naphthalene were determined by high-performance liquid chromatography. Activities of glutathione S-transferase and soluble epoxide hydrolase were determined spectrophotometrically. Cytochrome P450 content was determined by carbon monoxide bound-difference spectrum of dithionite-reduced microsomes. Activity was expressed relative to total protein concentration. RESULTS: Microsomal protein and cytochromeP450 contents were detectable in all horses and did not vary with age. Hepatic ethoxyresorufin metabolism was detected in all horses; by comparison, pulmonary metabolism of ethoxyresorufin and hepatic and pulmonary metabolism of pentoxyresorufin were detected at lower rates. Rate of hepatic naphthalene metabolism remained constant with increasing age, whereas rate of pulmonary naphthalene metabolism was significantly lower in weanlings (ie, horses 4 to 6 months old), compared with adult horses. Hepatic glutathione S-transferase activity (cytosol) increased with age; however, these changes were not significant. Pulmonary glutathione S-transferase activity (cytosol) was significantly lower in weanlings than adult horses. Hepatic and pulmonary soluble epoxide hydrolase did not vary with age of horses. CONCLUSIONS AND CLINICAL RELEVANCE: Activity of cytochrome P450 isoforms that metabolize naphthalene and glutathione S-transferases in lungs are significantly lower in weanlings than adult horses, which suggests reduced ability of young horses to metabolize xenobiotics by this organ.