Alan Buckpitt - US grants

Extrapolation of toxicologic data derived in animals to the human is a complex task that is especially difficult with those agents where there are marked species differences in the response and where there is a lack of epidemiologic data. There are a number of volatile industrial and environmental chemicals including naphthalene, dichloroethylene and trichloroethylene that result in dose-dependent necrosis of the pulmonary Clara cells of the mouse but in no injury to rat or hamster lung. Naphthalene-induced lung injury depends upon cytochrome P450 mediated metabolism and correlates with the glutathione (GSH) depletion and covalent binding by reactive metabolites to pulmonary macromolecules. However, covalent binding in nontarget tissues was higher than in the lung suggesting that then nature of reactive, potentially cytotoxic metabolites differed. Subsequent examination of the microsomal metabolism of naphthalene has demonstrated an excellent correlation between the formation of a particular reactive metabolite (trapped as a GSH adduct) and the species/organo-selectivity in naphthalene-induced cytotoxicity. These studies propose to determine whether urinary mercapturic acids derived from these GSH adducts can be used as a probe to examine the activity and metabolic selectivity of the pulmonary monooxygenase system. The importance stems from the fact that in vitro studies of human lung P450 xenobiotic metabolism have consistently failed to demonstrate significant activity possible because of artifacts introduced during tissue preparation. The goal of these studies is to provide a means to examine interindividual variations in activity and selectivity of the human lung monooxygenases. The proposed studies will develop methods for the extraction and HPLC separation and quantitation of urinary naphthalene mercapturates. Each of the isomeric GSH adducts will be administered to animals to determine their metabolic fate. The excretion of each of the isomers will be monitored in both rats and mice exposed to naphthalene by inhalation and by ip injection to determine whether the rates of excretion of the isomers are dependent upon the first pass organ (liver or lung) and the species exposed.

environmental toxicology; cytotoxicity; respiratory toxin; toxicant interaction; lung disorder; naphthalenes; glutathione transferase; pollution related respiratory disorder; detoxification; protein biosynthesis; respiratory epithelium; necrosis; enzyme mechanism; cytochrome P450; biotransformation; glutathione; toxin metabolism; tissue /cell culture; ultraviolet spectrometry; Macaca mulatta; hamsters; laboratory mouse; nuclear magnetic resonance spectroscopy; radiotracer; dialysis; electron microscopy; high performance liquid chromatography; density gradient ultracentrifugation; autoradiography; scintillation counter;

environmental contamination; environmental toxicology; diagnosis design /evaluation; waste disposal;

Approaches for detecting an exposure to a potentially toxic substance vary with the chemical under consideration and with the types of exposure (duration, level of exposure, route of exposure). Previously used biomarkers have varied in complexity from detection of the parent compound or a primary metabolite in the urine to detecting an adduct (mainly to hemoglobin or albumin) in blood. Urinary metabolites tend to be relatively easily measured but suffer from the standpoint that samples must be collected relatively soon after exposure. Hemoglobin adducts have the advantage of being long-lived and thus adducts tend to accumulate after continuous low level exposure. In both cases however, it is difficult to link the presence of a metabolite in the urine or an adduct in the blood stream with a specific toxicity. The work proposed in this application is to develop a biomarker(s) for a group of compounds that undergo metabolic activation to produce cell selective injury to pulmonary bronchiolar Clara cells. These compounds include naphthalene, nitronaphthalene, and, if time permits, several chlorinated ethylenes. These compounds, as well as close structural analogs, have been identified frequently in hazardous waste sites. The proposed work is based on the finding that naphthalene is metabolized to reactive intermediates that selectively arylate two proteins (15-16 kDa) in target cell populations and on preliminary data suggesting that arylation of these proteins may play a critical role in the cell injury that ensues after naphthalene administration. Development of a biomarker(s) that is tightly correlated with the intracellular targets for the toxicant, should allow discrimination of exposures that are above and below the threshold for toxicity. A variety of biochemical and morphologic approaches will be applied to this problem. A system for the isolation and short term culture of dissected pulmonary airways will be used to determine the formation of adducted macromolecules in target cell populations as well as the fate of the adducts. In all cases, quantitative morphometry will be used to assess toxicity and this will be related to adduct formation to carefully define the relationship between interaction at specific cellular targets and cell injury. Adducts will be identified by determining N-terminal sequences of the adducted protein and tryptic digests as well as from cDNA. The chemical nature of the adducts will be determined by tandem mass spectrometry and by in vitro incubations of the suspected proximate metabolites with protein target(s). The disposition kinetics of the adducted proteins will be assessed to determine appropriate sampling site (can adducts or primary decomposition products be detected in urine?, blood?) and to determine the half life of these products. Multiple route, multiple dose exposures in animals will be used to establish appropriate conditions for use of the biomarkers developed in the project.

DESCRIPTION (provided by applicant): The respiratory tract is an important target for environmental agents, which require P450 dependent metabolism to ellicit toxicity. Naphthalene (NA) and close structural congeners produce injury to airway Clara cells which is species selective. Human exposure to naphthalenes occurs from a variety of sources including cigarette smoke and polluted air. Nitronaphthalenes (NN)(and structural congeners) are generated in the atmosphere from NA and are a direct byproduct of diesel exhaust but do not show species selective toxicity. The overarching goal of work outlined in this application is to identify biochemical and metabolic mechanisms for the toxicity of these agents in animals with the aim of determining the relevance of these mechanisms in humans. The following hypotheses will be tested: 1) epoxides generated from the parent hydrocarbons are proximate intermediates in both toxicity and protein adduct formation, 2) these electrophiles interact irreversibly with a number of proteins involved in antioxidant protection and protein folding (protein disulfide isomerase, peroxiredoxin, heat shock proteins and glutathione transferase) 3) by depleting glutathione these electrophiles enhance the redox sensitivity of these same proteins, 4) glutathione transferase pi plays a key role in the inactivation of toxic metabolites generated from both compounds and 5) the liver plays a role in generating metabolites which are released to the circulation and enhance the susceptibility of the lung to metabolites generated in situ. These studies will use a number of mouse models in which key enzymes involved in the detoxication of the epoxides (epoxide hydrolase, glutathione transferase pi), a key enzyme in quinone redox cycling (NADPH quinone oxidoreductase) and the redox partner for CYP450 in the liver have been genetically disrupted. Adducts with, and loss of sulfhydryls on individual proteins will be measured using 2D electrophoresis. These studies are expected to identify appropriate biomarker targets and metabolic proteins where polymorphisms could be important in altering individual susceptibility.

animal tissue; respiratory system; biological products; Primates;

toxin metabolism; environmental toxicology; biomedical facility; chromatography; nuclear magnetic resonance spectroscopy; mass spectrometry;

animal tissue; environmental toxicology; respiratory system; biological products; Primates;

DESCRIPTION (Adapted from the Investigator's Abstract): Nononcogenic pulmonary diseases are the third leading cause of death in the US and are a major factor in morbidity and disability. Pulmonary cancer is the leading cause of cancer-related deaths. Although cigarette smoking is a major etiologic factor in these diseases, exposure to chemicals in the workplace and in the environment may be important as well. Numerous studies in laboratory animals have demonstrated the importance of the lung as a target for both inhaled and ingested chemicals. Because the studies in rodents have shown high variability in species and regional sensitivity within the lung to these chemicals, the applicability of studies in rodents for estimating the risks of exposure to humans is questionable. There is no understanding of how the factors thought to be critical for determining lung toxicity in rodents applies to the anatomically, cellularly, and metabolically distinct human lung. Among the factors likely to influence the susceptibility of the lung to toxic chemicals are the cellular distribution and catalytic activities of the key metabolic enzymes (cytochrome P450 monooxygenases) which bioactivate inert chemicals to reactive, lung toxic metabolites. These studies test the postulate that some rodent lung toxicants metabolically activated by P450 also produce focal injury in human lung. Three hypotheses will be tested: 1) the Rhesus macaque, which is anatomically and cellularly similar to humans, is an appropriate surrogate for humans in evaluating chemical-induced lung toxicity, 2) the cellular distribution of monooxygenases in human and primate lungs is highly focal making those cells particularly susceptible to cytotoxicants, and 3) the catalytic activities of pulmonary P450's in monkeys are similar to the human. The toxicity of three rodent lung toxicants, naphthalene (NA), 1-nitronaphthalene (NN) and 4-ipomeanol (IPO) will be evaluated in vitro in lungs of Rhesus monkeys, humans, and in sensitive rodent models (positive controls) and in vivo in monkeys. The cellular distribution of P450 isozymes responsible for the bioactivation of lung toxicants in rodents (CYP2B, CYP2E, CYP2F, CYP4B) will be defined in monkey and human lung. The catalytic activity of recombinant P450s (rodent, monkey and human) will be assessed with NA, NN and IPO. This work should provide a better understanding of the similarities and differences in the biologic response of lungs of rodents, monkeys, and humans to chemicals that undergo metabolic activation by the cytochrome P450 monooxygenase system.

Much of the work in toxicology has focused on delineating the effects of a single chemical entity often at high doses and over short time periods. However, humans are more often exposed to multiple chemicals over long time periods and at lower doses than generally used experimentally. Thus, there is a need to understand potential interactions of exposure to multiple chemical entities at both the cellular and whole organism level. The current request proposes to take advantage of recent developments in analysis of gene expression with high density microarrays to explore the use of this technology to identify alterations associated with exposure to multiple chemicals. This work will build on recent findings showing that the cytotoxicity of the metabolically activated, systemic pulmonary injurant, 1- nitronaphthalene, is considerably enhanced by preexposure to ozone. Two approaches will be utilized. Arrays of genes coding for both Phase I and Phase II metabolizing enzymes, enzymes involved in the synthesis and degradation of glutathione, several heat shock proteins and housekeeping genes will be prepared. mRNA isolated from control and treated (nitronaphthalene, ozone and nitronaphthalene plus ozone) rat lung will be used as a template for synthesis of cDNA labeled with fluorescent tags (CY-3 (control) and CY-5 (treated)) and these will be hybridized to the arrayed targets to determine whether treatments cause up or down regulation of genes likely to control the metabolic activation or detoxication of nitronaphthalene. Parallel quantitative histopathology studies will be done to confirm the severity of the pulmonary lesion in all treatment groups. In the second approach, clones from a control rat lung library will be arrayed on glass slides and screened against labeled mRNA from control (CY-3) and treated (CY-5) animals. Clones showing up or down regulation will be sequenced for identification. These studies will test the validity of using DNA arrays to rapidly screen changes in gene expression in response to mixtures of lung toxicants. The combination of dose and time course response studies which include detailed examination of tissues by histopathology will define cellular/molecular events that occur in response to chemical exposure and are expected to explore the validity of using DNA arrays to screen potential chemical interactions. By examining library clones, these studies may identify new genes whose regulation is altered by chemical exposure.

DESCRIPTION: (Investigator?s Abstract): This application requests funds to purchase a spotter, reader and associated software for establishing a DNA microarray center at UC Davis. The center will provide access for 18 NIH funded investigators to the technology and equipment necessary for generating custom DNA chips. In addition, three centers (Superfund, the NIEHS Center for Agricultural Chemicals and the California Primate Research Center) and students in a number of NIH supported training programs (training grants in Environmental Toxicology, Environmental Pathology, Pulmonary Medicine) will benefit from access to this equipment. As EST sets are established and accessibility of the expertise and equipment needed become greater, we anticipate that there will be more users than listed in the current application. The recent introduction of high throughput techniques for genome wide expression analysis has opened a number of opportunities to understand the variety and complexity of cellular responses to various manipulations. In addition, these approaches have allowed a rapid and more detailed view of alterations in gene expression patterns in response to diseases, which in turn has greatly accelerated the identification of potential drug targets for the treatment of these diseases. While a number of research questions can be answered by the use of DNA arrays, access to the equipment and the cost of commercial arrays has hampered full application of these approaches by many investigators. Much of the work outlined by the major users in this application focuses on changes in target tissues/cells including cells of the pulmonary, reproductive, cardiovascular, neurologic and dermatologic systems in response to a diverse set of agents. In all of these studies the intent is to identify changes, which occur in gene regulation in response to the toxicant, and as a lead toward understanding the critical events leading to toxicity. A second group of investigators will apply these approaches to study of alterations in response to diseases including schizophrenia as well as those associated with susceptibility to viral infections. In all cases the ready availability of flexible technology for generation of sufficient numbers of chips to allow detailed, thorough analysis of changes occurring in transcriptional regulation will both broaden the questions that can be posed by this group of investigators and will increase the speed with which they can be answered.

Lung diseases are a significant cause of morbidity and mortality in the US population. Exposure to chemicals in air, water and food contributes to these diseases. A number of chemicals undergo bioactivation to produce selective lung injury in rodents. This project focuses on naphthalene (NA) and 1-nitronaphthalene (NN), which are present in superfund waste sites and which represent a class of environmentally important compounds. Both chemicals undergo metabolism to electrophilic intermediates which become bound covalently to proteins in target cells. Protein binding is a key component of cytotoxicity. We have demonstrated that the proteins targeted are similar in rodents and primate nasal epithelium. This underscores the need to develop biomarkers which are tightly tied to toxicity. The central hypothesis is: /the formation of key protein adducts with reactive metabolites of NA/NN in target respiratory tissue is causally related to cytotoxicity; measurement of adducted peptides/proteins in urine and nasal brushings provides a highly sensitive molecular signature of exposure and effect./ The proposed work builds on recent findings showing 1) high specific activity adducts in urine of NA-treated mice, and 2) numerous identified protein adducts in lungs and nasal cavity of mice, rats and monkeys. The proposed studies will utilize antibodies from project 3 to trap and concentrate adducted peptides from the urine and to evaluate adduct levels in animal model systems. These approaches will be validated for their ability to assess adduct levels following exposures to small amounts of 14C-NA by accelerator mass spectrometry (Core A), will utilize the mass spectrometry facilities of cores A and B for analysis of adducted peptides and will depend heavily on the proteomics services offered by Core B. Overall, these studies are expected to: 1) yield fundamental information on how adducted proteins, formed in respiratory tissues, are handled in the whole animal, 2) explore concentration-response relationships at environmentally relevant concentrations, 3) provide methods which can be applied to exposed human populations, which will be useful-with modification-for assessing risk of other metabolically activated chemicals and 4) provide methods which can clarify the importance of genetic polymorphisms in genes responsible for the biodisposition of chemicals like NA and NN.