Chemical Toxicology

Chemistry Group

Recent advances in genomic, metabonomic and proteomic research, coupled with the availability of novel tools and methods to analyze the products of altered gene expression, have provided new insights into mechanisms of toxicity evoked by xenobiotics. While these advances promise to revolutionize our ability to characterize hazard, the challenge in the near future is to establish a body of available knowledge to serve as a foundation for applying the data generated by these new methods to risk assessment. The scope of toxicology experimentation performed with these new technologies is still relatively limited. Few of these data are publicly available and broad consensus on the application and interpretation of these data has not been reached in international regulatory and scientific arenas. As such, there is significant need for a coordinated evaluation.

Work in genomics will result in a greater understanding of the mechanisms of chemical toxicity. This understanding will come through the determination of relationships between chemical exposure and changes in genome-wide gene, and protein expression patterns or changes in patterns of metabolites. An understanding of the consequences of pattern changes is critically important in developing a complete understanding of toxicological processes because gene expression is altered either directly or indirectly as a result of toxicant exposure in almost all cases. The spectrum of the altered genes or proteins then determines the type and outcome of the toxic response. Viewed in this manner, patterns of gene and protein expression, or alterations in endogenous metabolism, can be used as markers of exposure and as methods for identifying mechanisms of toxicity. The simultaneous analysis of thousands of end points will allow toxicologists to take a new look at toxicological issues that cannot be completely unraveled using nongenomic techniques. Genomics will likely be used in the immediate future to better address a number of key toxicological issues, including mode of action, dose-response relationships, chemical interactions and hazard identification in chemical mixtures, and human exposure assessment.

The primary goal of toxicogenomics is to understand biological responses to environmental stressors and identify agents that are a significant risk to human health. Toxicogenomics is a powerful tool for improving human risk assessment because it will measure specific changes in gene expression in humans and other species that are exposed to drugs or other agents and to profile the process of disease progression. Careful data analysis could identify similar patterns in different species, leading to a "signature" for a given pathway of toxicity or disease state in humans. Once signatures are identified using large scale, global microarray analysis, it will then be possible to develop smaller, multi-chemical and multi-pathway arrays that can be used to assess the potential toxicity of chemicals in a rapid, prospective manner. This would result in better interspecies extrapolation, greater confidence in animal models, reduction in the number of animals needed for testing, faster testing, and most importantly, insights into pathways of toxicity and disease processes and their mechanisms heretofore unattainable using less developed technologies.

The mission of the Tox/Path Workgroup is to provide fundamental toxicology and pathology expertise in the design and conduct of NCT sponsored studies in-house and extramurally in order to assure genomic and proteomic data can be interpreted in the correct phenotypic context. The Tox/Path Workgroup  efforts within the NCT consists of four components; 1) an intramural program; 2) liaison activities with the National Toxicology Program; 3) collaboration with the International Life Sciences Institute; and 4) liaison with the extramural Toxicogenomic Research Consortium. The close association of the Tox/Path Workgroup with the Microarray Center, NTP, ILSI and the TRC provides an opportunity to integrate fundamental toxicology with the new genomic, proteomic, and metabonomic technologies.

Intramural Toxicology/Pathology Program: Conduct of a typical study in-house comprises collaboration with other staff members at NIEHS and Toxicogenomics Research Consortium during the design phase of NCT studies; performance of a comprehensive literature review; selection of the disease model to study (hepatic necrosis, renal tubular necrosis, apoptosis, etc.); selection of agent(s) known to induce the disease process and structural analogs that do not elicit the disease; design the study (dose ranges to produce toxic and nontoxic responses or pharmacological responses, duration of exposure/recovery); protocol preparation (both animal and chemical protocols); procurement of chemical and animals; acquisition of in-life data; dose formulation; administration of chemical; monitor study animals to assess toxicity; collection of tissues (blood, liver, kidney, urine) and data at termination; processing of tissues in an appropriate manner for analysis (blood for hematology, serum for clinical chemistry proteomics, tissues into liquid nitrogen, urine at ‚80†C, tissues into neutral buffered formalin and/or fixative for ultrastructural studies); monitor histopathology and ultrastructure preparation and clinical chemistry and hematology analyses; provide pathology evaluation and review; provide oversight of entry of clinical chemistry and pathology data into TDMS; archive data from clinical chemistry measurements; and analysis of all data (clinical chemistry, histopathology, ultrastructure) for complete toxicological characterization prior to genomic, metabonomic and/or proteomic analyses. Support for the pathology aspects of the studies are also provided by the Cellular and Molecular Pathology Branch.

Adverse reactions to drugs may account for 2-5% of the hospitalized cases of jaundice and for more than 40% of the cases of "hepatitis" among patients over 50. Acetaminophen is an intrinsic hepatotoxicant that appears to cause direct cytotoxicity. It is the most common cause of liver failure in the UK following ingestion with suicidal intent. The hepatic injury is produced by a toxic metabolite. Administration of acetaminophen in doses that exceed the hepatic detoxifying capacity, results in its oxidation by the cytochrome P450 system to N-acetyl-p-benzoquinone. Acetaminophen is an attractive chemical for the National Center for Toxicogenomics because of its direct cytotoxicity for hepatocytes, because therapeutic misadventure provides a human subpopulation for comparative studies and because it has been widely studied. The metabolism and toxicity have been well described. The Tox/Path Workgroup is finishing a series of studies to document the gene expression changes associated with Acetaminophen. The first study addressed the importance of the dosing regimen for subsequent NCT toxicology studies. Male Fisher 344 rats were dosed once by oral gavage (the human route of exposure) to four doses of acetaminophen a) a therapeutic dose; b) a high therapeutic dose; c) a toxic but reversible dose; and d) a potentially fatal dose. Phenotypic analyses were performed by histopathology, hematology, ultrastructure, and urinary metabonomic evaluations. Liver, kidney and blood were or will be analyzed for gene expression changes and proteomic analyses. Significant toxicity induced by the top two doses was identified by serum ALT and ASP, hemolysis, histopathology and ultrastructure evaluation. There were over 300 genes up or down regulated at the top dose but far fewer at the next lower dose, indicative of the sharp dose-response curve for this chemical. While there were genes alterations in the two therapeutic doses, they were fewer and of less magnitude, possibly indicative of the pharmacology actions of the drug, although there was an indication that some toxic genes were expressed at the lower doses, but of less magnitude that at the higher doses.

Differential gene expression from the individual rats at various time points and dose levels are being analyzed for correlation with individual histologic and clinical chemistry changes. The liver undergoes dramatic metabolic changes with food ingestion; changes that result in marked changes in gene expression and susceptibility to toxicity. This is factor often ignored in toxicology studies. Feeding and drinking water studies result in exposure during the night while gavage, dermal and inhalation studies result in exposure during the resting phase. Day and nighttime acetaminophen studies are also underway in collaboration with the National Toxicology Program (NTP). These studies will provide insight on the time of day and the metabolic status of the liver on hepatic toxicity induced by acetaminophen. Our hypothesis is that metabolic status of the liver during exposure will profoundly affect toxicity.

Collaboration with the National Toxicology Program: Tox/Path Workgroup also is the liaison between the NCT and the National Toxicology Program. We interact with both groups to maximize the communication between these two programs in order to optimize the utilization of resources. Our goal is the incorporation of the latest technologies into NTP toxicity assessments by bringing genomics experience of the NCT to the NTP. We plan to provide animals on test by the NTP to the NCT in order to utilize the animals in their toxicity testing and reduce repetitive testing. Our current efforts on this behalf include participation in the design of the toxicogenomic portion of two NTP studies- the effect of circadian rhythm on gene expression and the toxicity of the algal toxin microcystin). Dr. Cunningham also serves as Chemical Selection Coordinator for the National Toxicology Program Interagency Committee for Chemical Evaluation and Coordination (ICCEC). The ICCEC is composed of representatives of all agencies of the Federal government involved in issues of chemical toxicity testing and research and functions to provide oversight and guidance to maintain high standards of research and testing in chemical toxicology. He is Project Officer for the contract 'Chemical Disposition in Mammals' at the Stanford Research International, Menlo Park, CA.; Project Leader for the Peroxisome Proliferation Initiative which is evaluating WY-14,643, gemfibrozil, 2,4-dichlorophenoxyacetic acid, and dibutyl phthalate; and Project Leader for the class study for toxicological evaluation of alpha, beta-unsaturated ketones (methyl vinyl ketone, ethyl vinyl ketone, cyclohexene-1-one and methyl styryl ketone), and Study Director for the toxicological assessment of various chemicals. In 1999, Dr. Cunningham was detailed to the newly formed National Center for Toxicogenomics, headquartered at NIEHS. He will coordinate the activities of the Toxicology/Pathology Group with responsibility for overseeing comprehensive toxicological evaluation of in-life studies conducted in support of the Microarray, Proteomics, and Gene Expression Groups.

Collaboration with the International Life Sciences Institute: The Tox/Path Workgroup is the NCT leader for collaboration with the International Life Sciences Institute Initiative for Genomics and Proteomics Nephrotoxicity Workgroup. We interact with scientists from many private and governmental institutes to design, conduct and interpret toxicity studies on 3 nephrotoxicants: cisplatin, gentamycin and puromycin. Primary responsibility has focused on the conduct of a subchronic puromycin study in rats. We also serve as the repository for samples and data for that study. We also collaborate in the data analysis from all three studies and provide data and samples to others from the puromycin study. This interaction includes monthly conference calls and bimonthly meetings as well as laboratory analyses.

Collaboration with the Toxicogenomics Research Consortium: The Tox/Path Workgroup is also providing the universal control RNA samples to be used by all members of the Toxicogenomics Research Consortium (TRC) Steering Committee and Cooperative Research Project (CRP) members. We were chosen to plan and conduct the in-life portion of the collaboration to provide RNA that will be used by all Consortium members as control RNA for future experiments in C57 black male mice. The RNA pool is composed of RNA from liver, kidney, lung, brain and spleen and Arabodopsis mRNA.

Selected Publications

1. Fostel, J.M., Choi, D., Zwickl, C., Morrison,N., Bao,W., Richard, A., Yang, C., Bruno, M.E., Heinloth, A.N., Madenspacher,J.H., Merrick,B.A., Paules, R.S., Tomer,K.B., Wetmore, B.A., Tennant, R., Cunningham, M.L., Boorman, G.A., Irwin, R., Garcia,A., Papoian, T., Brown, R., Stevens, J. and Waters, M.D.: Chemical Effects in Biological Systems – Data Dictionary (CEBS-DD) for capture and integration of biological study design description, conventional phenotypes and ‘omics data, Toxicological Sciences, 88, 585-601, 2005.
2. Sanders, J.M., Burka, L.T., Smith, C.S., Black, W., James, R. and Cunningham, M.L.: Differential expression of CYP1A, 2B and 3A genes in the F344 rat following exposure to a polybrominated diphenyl ether mixture or individual components, Toxicological Sciences, 88, 127-133, 2005.
3. Dunnick, J., Blackshear, P., Kissling, G., Cunningham, M.L., Parker, J., Nyska, A.: Critical pathways in heart function: Bis(2-chloroethoxy)methane-induced heart gene change in F344 rats. Toxicologic Pathology, 34, 348-356, 2006.
4. Rusyn, I., Peters, J.M. and Cunningham, M.L.: Modes of action and species-specific effects of di-(2-ethylhexyl)phthalate in the liver. Critical Reviews in Toxicology, 36, 459-479, 2006.
5. Powell, C.L., Kosyk, O., Ross, P.K., Schoonhoven, R., Boysen, G., Swenberg, J.A., Heinloth, A.N., Boorman, G.A., Cunningham, M.L., Paules, R.S. and Rusyn, I. (2006) Phenotypic achoring of acetaminophen-induced oxidative stress with gene expression profiles in rat liver. Toxicological Sciences, 93, 213-222, 2006.
6. Coecke, S., Ahr, H., Blaauboer, B.J., Bremer, S., Casati, S., Castell, J., Combes, R., Corvi, R., Crespi, C.L., Cunningham, M.L., Elaut, G., Eletti, B., Freidig, A., Gennari, A., Ghersi-Egea, J.-F., Guillouzo, A., Hartung, T., Hoet, P., Ingelman-Sundberg, M., Munn, S., Janssens, W., Ladstetter, B., Leahy, D., Long, A., Meneguz, A., Monshouwer, M., Morath, S., Nagelkerke, F., Pelkonen, O., Ponti, J. Prieto, P., Richert, L., Sabbioni, E., Schaack, B., Steiling, W., Testai, E., Vericat, J.-A. and Worth, A. Metabolism: a Bottleneck in In Vitro Toxicological Test Development: Alternatives to Laboratory Animals, 34, 49-54, 2006.
7. Cunningham, M.L.: Putting the fun into functional toxicogenomics. Toxicological Sciences 92, 347-348, 2006.
8. Wyde, M.E., Cunningham, M.L. et al.: NTP technical report on the toxicology and carcinogenesis studies of alpha-methylstyrene (CAS No. 98-83-9) in F344/N rats and B6C3F1 mice (inhalation studies), NTP Toxicity Report Series Number 543, 2006.
9. Alsarra, I.A., Brockman, W.G., Cunningham, M.L. and Badr, M.Z.: Hepatocellular proliferation in response to agonists of peroxisome proliferators-activated receptor alpha: a role for kupffer cells? Journal of Carcinogenesis 5, 26-31, 2006.
10. Woods, C.G., Burns, A.M., Maki, A., Tak, W., Bradford, B.U., Cunningham, M.L., Conner, H.D., Kadiiska, M.B., Mason, R.P., Peters, J.M. and Rusyn, I.: Continuous exposure to peroxisome proliferators leads to a sustained increase in oxidant production in mouse liver that is dependent upon PPARalpha but not NADPH oxidase. Free Radicals in Biology and Medicine, in press, 2007.
11. Cunningham, M.L.: NTP toxicity report on toxicity studies of Wyeth-14,643 administered in feed to Harlan Sprague-Dawley rats, B6C3F1 mice, and Syrian hamsters, NTP Toxicity Report Series # 62, in press, 2007.
12. Woods, C.G., Kosyk, O., Bradford, B.U., Ross, P.K., Burns, A.M., Cunningham, M.L., Quo, P., Ibrahim, J.G. , Rusyn I.: Time course investigation of PPARalpha- and Kupffer cell-dependent effects of WY-14,643 in mouse liver using microarray gene expression. Toxicology and applied pharmacology, 225, 267-277, 2007.

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