Grantee Research Project Results
2015 Progress Report: Toxicogenetics of tetrachloroethylene metabolism and toxicity: Using Collaborative Cross mouse population approach to address remaining gaps in human health assessments
EPA Grant Number: R835612Title: Toxicogenetics of tetrachloroethylene metabolism and toxicity: Using Collaborative Cross mouse population approach to address remaining gaps in human health assessments
Investigators: Rusyn, Ivan , Chiu, Weihsueh A , Wright, Fred A.
Current Investigators: Rusyn, Ivan , Wright, Fred A.
Institution: Texas A & M University , North Carolina State University at Raleigh
EPA Project Officer: Aja, Hayley
Project Period: September 1, 2014 through March 30, 2017
Project Period Covered by this Report: September 1, 2014 through August 31,2015
Project Amount: $800,000
RFA: Susceptibility and Variability in Human Response to Chemical Exposure (2013) RFA Text | Recipients Lists
Research Category: Chemical Safety for Sustainability
Objective:
Tetrachloroethylene (perchloroethylene, PERC) is a high-production chemical of great concern to both risk assessors and public health officials worldwide. It has long been assumed that PERC metabolism and toxicity closely mimic those of trichloroethylene (TCE), a structurally similar chlorinated solvent. However, recent human health assessments of these chemicals determined that major differences in toxicokinetics and toxicodynamics exist between TCE and PERC. Furthermore, it was concluded that critical gaps remain in understanding of the human health hazard of PERC, including toxicokinetics, toxicodynamics and population variability. Thus, the long-term objective of this research proposal is to uncover the mechanistic linkages between the genome (e.g., variation in DNA sequence among individuals), metabolism (e.g., formation of organ-specific toxic intermediates), and adverse molecular events (e.g., transcriptional changes associated with toxicity) in response to PERC.
The central hypotheses of this research are that: (i) genetic variability-associated differences in PERC metabolism affect organ-specific toxicity of PERC; and (ii) a population-based experimental design utilizing Collaborative Cross (CC) can be used to exploit the variability in toxicity responses to better characterize uncertainties in human health assessments. The proposal includes three specific objectives:
Specific Objective 1: To characterize variability in the toxicokinetics of PERC by using the Collaborative Cross mouse model of the human population.
Specific Objective 2: To characterize variability in the toxicodynamics of PERC by evaluating inter-strain differences in dose-dependent effects on the liver and kidney in a sub-acute study.
Specific Objective 3: To evaluate the effects of inter-strain variability in PERC metabolism on liver and kidney toxicity in a sub-chronic study.
Progress Summary:
Most of the work in the past year has focused on the activities under Specific Objective 1. To date, we have accomplished the following: 45 strains of CC mice (adult male mice ~ 260 total) have been exposed to a single dose of PERC (1,000 mg/kg, i.g.) and have been euthanized from 1-24 hr (n=1/strain/time point) post dose to evaluate PERC toxicokinetics. We also dosed 1 mouse/strain with vehicle (5% alkamuls-el620 in saline) to assess the toxicodynamics of PERC at 24 hrs.
With respect to PERC toxicokinetics profiling we: 1) analyzed the most abundant PERC oxidative metabolite trichloroacetic acid (TCA) in liver, kidney, and serum across every time point; and 2) characterized TCA levels in brain, gonadal fat pad, and lung in nine strains of CC mice (based on liver, kidney, and serum TCA data, these nine strains were selected as representative strains of the population for further toxicokinetic profiling).
With respect to PERC toxicodynamics, we accomplished the following by conducting experiments in liver and kidney of treated CC mice: 1) evaluated peroxisome proliferator activated receptor-alpha (PPARα) activation by PERC via qRT-PCR using Acox1 and Cyp4a10 as biomarkers; 2) assessed CYP2E1 protein levels; 3) measured organ to body weight ratios; and 4) examined liver and kidney histopathology (H&E).
We have observed the following results so far: 1) TCA area under the curve (AUC) varies by ~10-fold in liver, ~6-fold in kidney, ~25-fold in serum, and ~5-fold in brain across the mouse CC population (max/min); 2) variability is observed not only in TCA AUC, but also in Tmax, Cmax, and half-life; 3) mice treated with a single 1,000 mg/kg dose of PERC lose significant body weight (~6% on average compared to vehicle); 4) no consistent differences in organ/body weight ratios were observed; 5) minimal to mild micro/macrovesiscular steatosis is observed in some strains after PERC treatment, while no kidney injury was observed; 6) there is large variability of basal hepatic CYP2E1 levels in liver and kidney tissues across the population; 7) at a population level, no induction of CYP2E1 was observed following PERC treatment; 8) at a population level, signification induction of PPARα-responsive genes is observed in the liver; 9) the degree of PPARα induction varies by ~2 orders of magnitude across the population; and 10) no correlations are observed between liver TCA levels and basal hepatic CYP2E1 protein levels, hepatic induction of PPARα-responsive genes, body weight loss, or degree of steatosis.
These observations are significant because they support both of our original hypotheses, specifically that CC mouse population is a valuable research tool for exploring the extent and the molecular mechanisms of inter-individual variability in toxicokinetics and toxicodynamics of PERC toxicity.
Future Activities:
1) First, we will analyze PERC levels and conjugative metabolites in the same tissues in which TCA was measured. Then, we will use these TCA/PERC toxicokinetic data to refine the PERC PBPK model to incorporate population-level variability using a Bayesian approach. We anticipate data acquisition to take approximately 3 additional months.
2) While toxicity testing is ongoing, we will work on the pipeline for genome-wide analysis of genetic susceptibility loci that may explain the variability in responses to PERC among CC strains. This then will be applied to the PERC toxicokinetic data to understand genetic determinants of PERC metabolism, and to the future data from 4- and 13-week toxicity data to understand genetic determinants of PERC-associated target organ toxicity.
3) We will select 10 CC strains for the 4-week toxicity studies based on variability in levels of TCA and/or conjugative metabolites. We also will base our strain selection on the availability of these CC lines (e.g., if one strain will not breed well enough to supply us with enough similarly aged animals to accomplish robust statistical power [n=10/dose group], then another strain may be selected). We will conduct the 4 week exposure study in year 2 of funding and will analyze tissues in a way similar to that detailed in the results for the acute study.
Journal Articles:
No journal articles submitted with this report: View all 14 publications for this projectSupplemental Keywords:
genome-wide association, toxicity pathway, kidney, liver, mouse, population, tetrachloroethylene, PERC, perchloroethyleneProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.