Project Number: 9278234 Project Title: Use of a Hepatocyte Model for Identifying Biomarkers Division: DART Project Officer (PO): Cynthia Striley PO Degree: Ph.D. PO Area of Expertise (scientific discipline): Analytical Chemistry Project Start Date: 10/98 Project End Date: 09/01 Total Budget over the Project Period: $147,387 Goal/Sub-Goal: Chronic Effects of Pesticide Exposure/Exposure Assessment Research Challenge/Issue: Biological monitoring techniques which detect exposures to workplace chemicals within a person’s body (usually by testing urine or blood) are essential for the conduct of many occupational health studies. A model system using human liver fractions from 200+ donor livers to study individual variability in the metabolism of foreign chemicals, particularly agricultural chemicals, was developed. This model will be used to develop specific biological monitoring methods that can be used to assess workers' exposure to occupational chemicals. Pesticide exposure constitutes a potentially serious health risk to agricultural workers. Urinary biological monitoring is an effective tool for assessing exposure to these pesticides especially when dermal exposure occurs as is the case with pesticide applicators. In general, when a person is exposed to foreign chemical, it is metabolically altered, primarily in the liver, and excreted in one or more metabolically altered forms - i.e., as metabolites - rather than in its original form. Knowledge of the nature of excreted urinary metabolites, therefore, is key to the development of effective urinary biomonitoring methods so the most appropriate metabolites (biomarkers) can be measured. A lack of knowledge of the individual variability of chemical metabolism limits biomarker development. Since some individuals are more susceptible to adverse health effects, this study will address this issue of inter-individual variability by developing an in vitro model system with which metabolism of xenobiotic chemicals can be studied. The ability to identify the most appropriate metabolites to use for biomonitoring will allow for more accurate exposure assessment and hence improved intervention measures. Further, because more complete knowledge of the variability in how individuals metabolize chemicals can enhance risk assessment research, the USEPA has agreed to fund part of this research. Activities: A model system using subcellular fractions of human hepatocytes, containing Phase I and Phase II metabolic enzymes was used to simulate metabolism in humans in vivo. The hepatocyte preparations from approximately 150 human livers were provided by the International Institute for the Advancement of Medicine (IIAM). The concentrations of various metabolizing enzymes in the fractions were determined immunochemically. Then, occupationally relevant agrochemicals were incubated with the preparations and the resulting metabolites extracted and analyzed using conventional chromatographic techniques. Immunochemical assays (IAs) were developed for the appropriate metabolites of these agrochemicals. These methods were then used to measure individual metabolites in samples of urine from field studies.
A hepatocyte library was procured (156 hepatic samples; age distribution 2 days to 60 years). Gender, race, age and lifestyle information is available on all of the samples. The entire library was analyzed for UDP-glucuronosyltransferase activity, and CYP450 forms important in human metabolism towards agrochemicals were immunologically determined in a pilot group of 40 hepatic samples. Using the protein assay from the pilot study the entire library was analyzed for glucuronosyltransferase and glutathione transferase activity. In addition characterization of the following cytochrome P450 (CYP450) forms were performed: CYP1A2; CYP2E1; CYP3A; CYP2B6; CYP2D6; CYP2A6; CYP2C9; CYP2C19; CYP4A and CYP1B. Polymerase chain reaction (PCR) genotyping was completed by Dr. Dan Nebert at the University of Cincinnati's Medical Center in FY00-01. The hepatocyte library grew in FY00 to more than 200 samples. In addition, we procured extra-hepatic tissues (lung, jejunum and kidney). These samples aided in the estimation of total body metabolism as well as cases where there is specific target organ toxicity noted. Assays to determine biomarkers of exposure have been developed. In an internal collaboration with DBBS, PAEB an in vitro metabolism assay for toluene was developed and applied to an interspecies study to validate the predictability of animal models. Elucidation of metabolic pathway and biomarkers of exposure to Azinphos-methyl, Pipron, chloraceranilides (metolalchlor, alachlor) and triazines (atrazine, cyanazine) were investigated. A newly acquired HPLC system facilitated rapid analysis of metabolites and putative biomarkers. Outputs: The following peer reviewed papers were published as a result of this project. Lipscomb, J. C., Barton, H. A., Tornero-Velez, R., Evans, M.V., Alcasey, S, Snawder J.E. and Laskey, J. “The metabolic rate constants and specific activity of human and rat hepatic cytochrome P-450 2E1 toward toluene and chloroform.” J Toxicol Environ Health A, 67(7):537-553 (2004) NN: 20024735 Lipscomb, J. C., Teuschler, L. K., Swartout, J. C., Striley, C.A.F. and Snawder J. E. “Variance of Microsomal Protein and Cytochrome P450 2E1 and 3A Forms in Adult Human Liver.” Toxicol Mech Method 13, 45 (2003). [Cited 6 times] Davis, R. R., Murphy, W. J., Snawder, J. E., Striley, C.A.F., Henderson, D., Khan, A., Krieg, E. F. “Susceptibility to the ototoxic properties of toluene is species specific” Hearing Research 166, 24 (2002). [Cited 2 times] NN: 20022397 Lipscomb, J.C., Striley C.A.F. and Snawder, J.E. "Human interindividual variability in the expression of cytochrome P450 forms critical to xenobiotic metabolism." Toxicologist 2000 Mar; 54(1):181. This paper was awarded the “Outstanding Presentation in Risk Assessment” from the Society of Toxicology. NN: 20020983 Snawder, J.E. and Lipscomb, J.C., “Interindividual variance of cytochrome P450 forms in human hepatic microsomes: correlation of individual forms with xenobiotic metabolism and
implications in risk assessment.” Regul Toxicol Pharmacol 32(2):200-209 (2000). Winner of the Alice Hamilton award in the Biological Sciences Category in 2001, NN: 20020814 The following presentations were given as a result of this project. Striley, C.A.F. Snawder, J.E., and Biagini, R.E. "Use of a Human Microsomal Library for Metabolite Elucidation, Assay Development and Biological Monitoring of Occupationally Relevant Compounds." Immunochemistry Summit IX /221st American Chemical Society National Meeting, San Diego, CA, April (2001). Striley, C.A.F. and Snawder, J.E. "Clinical Biological Monitoring Strategies: Use of a Hepatic Microsomal Library for Metabolite Elucidation of Occupationally Relevant Compounds". NIOSH Salt Fork Interdivisional Conference, Mineral Wells, WV, November (2000). Forms in Human Hepatic Microsomes: Correlation of Individual Forms with Xenobiotic Metabolism Federation of American Societies for Experimental Biology Conference, Washington. (1999). Intermediate Outcomes: Finalization of the total body model of xenobiotic metabolism with USEPA researchers that take into account the age, race and gender. This model is being developed with a special emphasis on its applicability to juvenile risk assessment. USEPA has used results from research developed in cooperation with NIOSH to re-evaluate uncertainty factors used in risk assessments and have applied them to new risk assessments and dose-response models. End Outcomes: Research findings pertaining to inter-individual variation in metabolism was determined for various human populations (age, race, gender, BMI) by analyzing a library of hepatic (liver) tissue for various enzymes (CYP450). Differences were used when calculating total internal dose and risk of adverse outcome due to occupational exposure to chemicals. EPA has used the data from this work to re-evaluate uncertainty factors used in risk assessments and have applied them to new risk assessments. External Factors: Collaboration with the USEPA and International Institute for the Advancement of Medicine was key to the success of this project and the resultant peer-reviewed publications. Future Directions: Hepatic, jejunal and renal tissue will continue to be used to simulate human metabolism determine how inter-individual variation impacts chemical exposures. Stored tissues will be shared with researchers/partners in the future.