RESPIRATORY TOXICOLOGY RESEARCH CORE
DESCRIPTION:
As a primary interface between the organism and its environment, the respiratory system is the target for a wide range of toxicants including reactive gases, agricultural chemicals, and airborne pathogens and particles. Even agricultural chemicals that have the lungs as a target but are not incorporated into dusts or sprays, can still cause toxicity by ingestion of contaminated food and drinking water or absorption through the skin. In the Central Valley of California, the encroachment of significant urban populations into prime agricultural land
has produced impaction of atmospheric pollution on agricultural hazards and vice versa. The goals of this core are to: 1) promote interdisciplinary research elucidating the cellular, metabolic and molecular mechanisms defining the response of the respiratory system to environmental toxicants and 2) provide information relative to the harmful effects of photochemical air pollution and other inhaled toxicants which facilitate understanding the relative susceptibility of humans to harm from exposure to agricultural contaminants.
Core investigators have six objectives:
1. Define the cellular basis of pulmonary response to toxicant exposure
2. Identify the metabolic mechanisms for toxicity of xenobiotics which target the lungs
3. Define the mechanisms regulating the development of tolerance resulting from repeated, long-term exposure
4. Identify the factors responsible for the varying susceptibility among human subpopulations
5. Establish the nature of the synergism of exposure to multiple toxicants on acute lung toxicity
6. Validate biomarkers of respiratory tract exposure and toxicity
MEMBERS:
Charles Plopper, Ph.D., Core Leader, Professor, Department of Anatomy, Physiology and Cell Biology
Alan Buckpitt, Ph.D., Professor, Department of Molecular Biosciences
Bruce Hammock, Ph.D., Professor, Department of Entomology
Jerold Last, Ph.D., Professor, Department of Internal Medicine, Division of Pulmonary/Critical Care Medicine
Kent Pinkerton, Ph.D., Professor in Residence; Department of Anatomy, Physiology and Cell Biology
Hanspeter Witschi, Ph.D., Professor, Department of Molecular Biosciences
Reen Wu, Ph.D., Professor, Center for Comparative Respiratory Biology
KEY WORDS:
* Lung
* PM
* P450
* Ozone
* Airway
* Fibrosis
* Epoxide Hydrolase
* Development
PROGRESS REPORT:
This highly interactive group of investigators is pursuing the core's research objectives through seventeen (17) different projects which fall under the following general groupings: 1) metabolic mechanisms of bioactivated lung toxicity in adults and newborns; 2) cellular mechanisms of lung injury by inhaled particles and oxidant gases; 3) impact of co-exposure to lung toxicants. There is considerable overlap between headings with some projects actually being part of more than one group.
Bioactivated toxicants: Center investigators have extended the use of fluorescent dyes which are live cell reagents for identifying in situ the distribution of cells killed by the oxidant gas ozone throughout the air passages to primates, neonates, and rats co-exposed to ozone and bioactivated toxicants and particles (PM). Core members are combining this with strategies for defining the glutathione content of individual epithelial cells, also in whole-mount lung preparations. Specific inhibitors have been identified for one of the critical pulmonary P450 isozymes (CYP2F2). The sensitivity of in situ hybridization techniques for measuring cellular biosynthetic activity have been extended to co-exposure studies. One of the critical macromolecules for lung cell function which forms adducts with metabolites of bioactivated toxicants has been identified as actin and the core is now defining the impact of exposure on target cell cytoskeleton. These new findings are moving the core more closely to the point of being able to define the mechanisms of cellular injury from lung-targeted toxicants at the cellular level in the three-dimensional context of the local environment within the lung. Because many metabolites of bioactivated xenobiotics (such as limonen and cinnamaldehyde) are metabolized by and can deplete glutathione-based pathways, a focus under this group is to define the role of glutathione in protection from acute injury and development of tolerance. These studies have demonstrated a substantial heterogeneity of glutathione within populations of murine Clara cells both in vivo and in situ and have suggested that this heterogeneity may be an important factor in determining the susceptibility of lung cells to cytotoxicants requiring glutathione for alteration in cytotoxicity. The most current exciting new findings are the ability to map the alterations in the distribution of plemorized and nonplemorized actin in cells undergoing toxic stress from reactive oxidant metabolites of polyaromatic hydrocarbons. Using the whole mount approach, the distribution of both F and G actin alter during this process. A new nucleation site is established at the apex of the target cell which proceeds to partition off cytoplasm with injured organelles from the rest of the cell. This occurs approximately 3 hours post glutathione depletion. When glutathione is depleted from cells by inhibitors of glutathione synthesis, the tolerance developed in cells by repeated exposure to lung toxicants rapidly disappears. Preliminary data in isolated cells indicates that one of the key factors in development of tolerance is an active GSH-X pump which removes bound toxicants from the cell. Inhibitor studies indicate that this removal also depletes the GSH pool and puts these cells at further risk to injury. New rules from the U.S. E.P.A. allow registration of "natural metabolites" as pest control agents with limited toxicological data. These materials include natural products such as limonene and cinnamaldehyde.
The lung of adult mammals is the target for a wide range of environmental toxicants. Many of these toxicants, including furans, and chlorinated and aromatic hydrocarbons, require metabolism by the cytochrome P450 monooxygenase system to produce toxicity. The nonciliated bronchiolar epithelial (or Clara) cell is the primary target cell for a large number of these compounds. Clara cells differentiate postnatally in most species. Differentiation includes both the acquisition of cellular features characteristic of mature Clara cells and a 10-fold increase in pulmonary cytochrome P450 monooxygenase activity during the postnatal period. Investigators have found that differences in both cellular characteristics and P450 monooxygenase expression alter Clara cell susceptibility and probably affect lung toxicity in neonates and postnatal animals in two species, rabbits and mice. In mice, post-natal differentiation of the phase II enzyme systems indicate that there is a substantial mismatch in some of these pathways in comparison to the level of bioactivation potential in neonatal animals. This apparently accounts for a substantial portion of the elevated susceptibility in neonates compared to adults. A second striking feature of bioactivated toxicity in neonates is that targeted injury to Clara cells in early phases of postnatal development results in a failure of the local epithelium to go through the repair phases observed in the adult. It appears that at least three stages of the repair process are down-regulated in neonates: proliferation of progenitor cells, migration of daughter cells into the wound area, and differentiation of cells migrating into the wound.
The pulmonary cytotoxicity of nitronaphthalene, a Clara cell cytotoxicant, depends upon cytochrome P-450 monooxygenase-mediated metabolism. Nitroaromatics such as nitronaphthalene are major components of diesel exhaust emissions. Humans living in urban areas are exposed simultaneously to both nitroaromatics (and other P-450 mediated cytotoxicants) and to high levels of oxidant air pollutants. Previous studies indicate that exposure to ozone produces marked alterations in P-450 monooxygenase activity. Short-term ozone exposure results in focal injury to the centriacinus, a target site shared by nitronaphthalene. Long-term exposure to ozone produces a reorganized cell population tolerant to further oxidant injury. It now appears that the potential effects of exposure to ozone alter the response of the lung to nitronaphthalene. The core has now identified a wide range of metabolites from P-450 mediated activation of 1-nitronaphthalene which indicate primarily P-450 based metabolism, but could suggest some reductive metabolism as well. This heterogeneity in the metabolic characteristics may be the mechanism for the dichotomous toxicity experienced in the lung with varying doses. Different airway levels respond to different levels of dose by varying the cell population targeted for injury.
The core is developing markers of exposure, effect and susceptibility for chemicals which produce lung toxicity in animals. These studies are based on the premise that the development of markers capable of signaling that an exposure has been at a level sufficient to result in cytotoxicity is dependent upon a complete understanding of essential biochemical and metabolic steps involved in toxicity in animals. Accordingly, this work defines the importance of specific protein and nonprotein targets of electrophilic intermediates to cytotoxic injury for three chemicals that produce focal injury to the respiratory epithelium by virtue of cytochrome P450-dependent metabolism, naphthalene, nitronaphthalene and trichloroethylene.
Particles and gases: A critical need in this area is sensitive assays for defining response to exposure.
The core investigators have evaluated a variety of methods for their sensitivity in detecting injury from particulate exposure. It appears to be critical that assays which evaluate injury effects can be applied to site-selected responses. To date, permeability studies have not identified clear patterns of irreversible cellular injury to carbon and nitrate particles, even in association with environmental levels of ozone. However, cell proliferation assays have identified focal sites where cellular responses involve activation of mitosis. These sites correspond relatively closely to aerodynamic models of particle deposition. They are at airway branch points and in terminal bronchiole-alveolar duct junctions.
As Center investigators become able to understand the mechanisms underlying responses of lung cells to dust exposure, they will be better able to define the fibrogenic potential of a given dust and to define strategies to protect cells from the damaging effects of such dusts and, hopefully, to protect lungs from environmentally-induced diseases. The size of particles thought to be involved in adverse health effects are in the size range below 10 (so called PM10 fraction). However, smaller, finer particles below 2.5 may actually present the greatest health risk due to their ability to penetrate deeply into the lungs. A number of epidemiological studies have shown a strong association between exposure to particulate air pollutants and increased morbidity and mortality. Deaths are usually attributed to a vague
entity called "cardiopulmonary failure. However, no mechanistic basis is available to explain the phenomenon. The core is evaluating the biological effects of inhaled fine particles composed of carbon and nitrate and is now beginning to assess the impact of these particles on lung toxicity in neonates, as well as very old animals.
Co-exposure: The group is highly concerned about the impact of exposure to a second pollutant on the effects of the first. In addition to ozone co-exposure with bioactivated cytotoxicants, the core has also investigated mixture of two oxidant gases (O3 and NO2) and tobacco smoke.
Male Sprague-Dawley rats exposed for 6 hours a night to 0.8 ppm of ozone for 90 days and then exposed to nitronaphthalene showed a wide range of responses which varied significantly from filtered air-exposed animals treated with the same doses of nitronaphthalene. Ozone-exposed animals administered high doses of nitronaphthalene had significantly greater mortality than filtered air-exposed animals. At lower doses, terminal bronchioles in ozone-exposed animals exhibited greater injury than those in filtered air-exposed animals administered the same dose. At high doses, minor daughter epithelium was extensively exfoliated in filtered air-exposed animals, but less severely injury in ozone-exposed animals suggesting that chronic ozone exposure increases the overall susceptibility of acute toxicity of cytochrome P-450 activated compounds and alters the epithelial response on a site-specific basis.
Center investigators want to characterize cigarette sidestream smoke (SS) concentration dependency of IUGR (intrauterine growth retardation) and to establish whether IUGR exposure is time-dependent; particularly, investigators will test the hypothesis that there are critical intervals during gestation where intrauterine development might be influenced by exposure to SS. There is evidence that cigarette SS produces IUGR in rats. Pregnant Sprague-Dawley rats were exposed to side stream cigarette smoke for 6 hours a day, at a concentration of 1 mg/m3 of TSP, on days 3, 6-10, and 13-17 of pregnancy. Not only did these exposures reduce mean pup weight without altering fetus number or maternal body weight, but they also altered the distribution and density of neuroepithelial bodies within the airways. It appears that they may have also had a negative impact on local proliferation and responses and that SS may negatively impact lung repair. Controls were kept in an identical chamber without smoke exposure. The animals were killed on day 20 of gestation. No differences were found in maternal body weight gain or average daily food consumption between the smoke-exposed and control groups. The number of fetuses and of implantation sites per litter were comparable among the groups. However, there was a small but significant reduction in mean pup weight.
PUBLICATIONS:
1. Canada, A.T., S.R. Schulman, D.W. Winsett, K.E. Pinkerton and D. Costa. 1998. Hyperoxia-induced airway hyperreactivity in neonatal guinea pigs is not inflammation dependent. Inhalation Toxicology 10:15-25.
2. Chang, M. M-J., R. Wu, C.G. Plopper and D.M. Hyde. 1998. IL-8 is one of the major chemokines produced by monkey airway epithelium after ozone-induced injury. American Journal of Physiology 275, Lung Cellular and Molecular Physiology 19:L524-532.
3. Debernard, S., C. Morisseau, T.F. Severson, L. Feng, H. Wojtasek, G.D. Prestwich and B.D. Hammock. 1998. Expression and characterization of the recombinant juvenile hormone epoxide hydrolase (JHEH) from Manduca sexta. Insect Biochem. Mol. Biol. 28:409-419.
4. Farman, C.A., Watkins, K., van Hoozen, B., Last, J.A., Witschi, H. and Pinkerton, K.E. Centriacinar remodeling and sustained procollagen gene expression following exposure to ozone and nitrogen dioxide. American Journal of Respiratory Cell and Molecular Biology, in press (1998).
5. Gilman, S.D., S.J. Gee, B.D. Hammock, J.S.Vogel, K. Haack, B.A. Buchholz, S.P. Freeman, R.C. Wester, X. Hui and H.I. Maibach. 1998. Analytical performance of accelerator mass spectrometry and liquid scintillation counting for detection of 14C-labeled atrazine metabolites in human urine. Anal. Chem. 70(16):3463-3469.
6. Goldkorn, T., Balaban, N., Matsukuma, K., Chea, V., Gould, R. Last, J.A., Chan, C., and Chavez, C. EGF Receptor Phosphorylation and Signaling is Targeted by H2O2 Redox Stress. American Journal of Respiratory Cell and Molecular Biology, 19:786-798, 1998.
7. Hammock, B.D. 1998. Status of recombinant baculoviruses in insect pest control. Review in Toxicology. In: Pesticides and the Future: Minimizing Chronic Exposure of Humans and the Environment, (Kuhr, R.J., and N. Motoyama, eds), pp: 205-221. IOS Press, Amsterdam, The Netherlands.
8. Hammock, B.D. 1998. Toxic Ooze. Chemical & Engineering News. 76:28.
9. Harris, A.S., I. Wengatz, M. Wortberg, S.B. Kreissig, S.J. Gee, and B.D. Hammock. 1998. Development and application of immunoassays for biological and environmental monitoring. In: Multiple Stresses in Ecosystems, (Cech Jr., J.J., B.W. Wilson, D.G. Crosby, eds.), pp. 135-153. Lewis Publishers, Boca Raton, Florida.
10. Hoover, K., S.A. Alaniz, J.L. Yee, D.M. Rocke, B.D. Hammock and S.S. Duffey. 1998. Dietary protein and chlorogenic acid effect on baculoviral disease of noctuid (Lepidoptera: Noctuidae) larvae. Environ. Entomol. 27(5):1264-1272.
11. Hoover, K., M.J. Stout, S.A. Alaniz, B.D. Hammock, and S.S. Duffey. 1998. Influence of induced plant defenses in cotton and tomato on the efficacy of baculoviruses on noctuid larvae. J. Chem. Ecol. 24(2):253-271.
12. Hoover, K., J.L. Yee, C.M. Schultz, D.M. Rocke, B.D. Hammock, and S.S. Duffey. 1998. Effects of plant identity and chemical constituents on the efficacy of a baculovirus against Heliothis virescens. J. Chem. Ecol. 24(2):221-252.
13. Jaeger L.L., A.D. Jones, and B.D. Hammock. 1998. Development of an enzyme-linked immunosorbent assay for atrazine mercapturic acid in human urine. Chem. Res. Toxicol. 11(4):342-352.
14. Ji, C.M., F.H. Royce, U. Truong, C.G. Plopper, G. Singh and K.E. Pinkerton. 1998. Maternal exposure to environmental tobacco smoke alters Clara cell secretory protein expression in fetal rat lung. American Journal of Physiology: Lung Cellular and Molecular Physiology, 870-876.
15. Last, J.A., Gould, R., Grimmer, K., Duff, Z. and VanHoozen, B. Lung Collagen Types are Altered in Rats Chronically Exposed to Ozone. Inhalation Toxicology. 10:101-111, 1998.
16. Lee, C., K.C. Watt, A-M Chang, C.G. Plopper, A.R. Buckpitt and K.E. Pinkerton. 1998. Site-selective differences in cytochrome P450 isoform activities: Comparison of expression in rat and rhesus monkey lung and induction in rats. Drug Metabolism and Disposition 26(5):396-400.
17. Madl, A.K., D.W. Wilson, H.J. Segall and K.E. Pinkerton. 1998. Alteration in lung particle translocation, macrophage function, and microfilament arrangement in monocrotaline-treated rats. Toxicology and Applied Pharmacology, 153:28-38.
18. Mitchell, A.E., J. Zheng, B.D. Hammock, M. Lo Bello, and A.D. Jones. 1998. Structural and functional consequences of haloenol lactone inactivation of murine and human glutathione S-transferase. Biochemistry 37(19):6752-6759.
19. Morisseau, C., G. Du, J.W. Newman and B.D. Hammock. 1998. Mechanism of mammalian soluble epoxide hydrolase inhibition by chalcone oxide derivatives. Arch. Biochem. Biophys. 356(2):214-228.
20. Moskowitz, H., R. Herrmann, A.D. Jones, and B D. Hammock. 1998. A depressant insect-selective toxin analog from the venom of the scorpion Leiurus quinquestriatus hebraeus, purification and structure/function characterization. Eur. J. Biochem. 254:44-49.
21. Nakagawa, Y., M. Sadilek, E. Lehmberg, R. Herrmann, R. Herrmann, H. Moskowitz, Y.M. Lee, B.A. Thomas, R. Shimizu, M. Kuroda, A.D. Jones, and B.D. Hammock. 1998. Rapid purification and molecular modeling of AaIT peptides from venom of Androctonus australis. Arch. Insect Biochem. Physiol. 38:53-65.
22. Pinkerton, KE; Weller, BL; Ménache, MG; Plopper, CG. 1998. Consequences of prolonged inhalation of ozone on F344/N rats: collaborative studies. Part XIII. A comparison of changes in the tracheobronchial epithelium and pulmonary acinus in male rats at 3 and 20 months. Research Report/Health Effects Institute, 65:1-32.
23. Pinot, F., E.D. Caldas, C. Schmidt, D.G. Gilchrist, A.D. Jones, C.K. Winter and B.D. Hammock. 1997. Characterization of epoxide hydrolase activity in Alternaria alternata f. sp. lycopersici. Possible involvement in toxin production. Mycopathologia 140:51-58.
24. Plopper, C.G., G.E. Hatch, V. Wong, X. Duan, A.J. Weir, B.K. Tarkington, R.B. Devlin, S. Becker and A.R. Buckpitt. 1998. Relationship of inhaled ozone concentration to acute tracheobronchial epithelial injury, site-specific ozone dose, and glutathione depletion in rhesus monkeys. American Journal of Respiratory Cell and Molecular Biology 19:387-399.
25. Reimer, G.J., S.J. Gee, and B.D. Hammock. 1998. Comparison of a time-resolved fluorescence immunoassay and an enzyme-linked immunosorbent assay for the analysis of atrazine in water. J. Agric. Food Chem. 46(8):3353-3358.
26. Sanborn, J.R., S. J. Gee, S.D. Gilman, Y. Sugawara, A.D. Jones, J. Rogers, F. Szurdoki, L.H. Stanker, D.W. Stoutamire, and B.D. Hammock. 1998. Hapten synthesis and antibody polychlorinated dibenzo-p-dioxin immunoassays. J. Agric. Food Chem. 46(6):2407-2416.
27. Smiley-Jewell, S.M., S.J. Nishio, A.J. Weir and C.G. Plopper. 1998. Neonatal Clara cell toxicity by 4-ipomeanol alters bronchiolar organization in adult rabbits. American Journal of Physiology 274, Lung Cellular and Molecular Physiology 18:L485-498.
28. Sugawara, Y, S.J. Gee, J.R. Sanborn, S.D. Gilman, and B.D. Hammock. 1998. Development of a highly sensitive enzyme-linked immunosorbent assay based on polyclonal antibodies for the detection of polychlorinated dibenzo-p-dioxins. Anal. Chem. 70:1092-1099.
29. Watt, K.C., C.G. Plopper, A.J. Weir, B. Tarkington and A.R. Buckpitt. 1998. Cytochrome P450 2E1 in rat tracheobronchial airways: response to ozone exposure. Toxicology and Applied Pharmacology 149:195-202.
30. Wengatz, I., D.W. Stoutamire, S.J. Gee, and B.D. Hammock. 1998. Development of an enzyme-linked immunosorbent assay for the detection of the pyrethroid insecticide fenpropathrin. J. Agric. Food Chem. 46(6):2211-2221.
31. Witschi HP, Espiritu I, Yu M and Willits NH (1998) The effects of phenethyl isothio-cyanate (PEITC), N-acetylcysteine (NAC) and green tea on tobacco smoke-induced lung tumors in strain A/J mice. Carcinogenesis 19: 1789-1794.
32. Witschi HP (1998) Tobacco smoke as a mouse lung carcinogen. Experimental Lung Res. 24: 385-394.