Trichloroethylene (Tri) is a major environmental contaminant and is an established animal carcinogen. Human health risk assessment for Tri is difficult because of marked sex- and species-dependent differences in metabolism, toxicity, and target organ specificity. Toxic and carcinogenic effects of Tri in the kidneys are due to its metabolism by glutathione conjugation, subsequent metabolism to the cysteine conjugation, S-(1,2 dichlorovinyl)-L-cysteine (DCVC), and metabolism of DCVC by the cysteine conjugate b-lyase to form reactive compounds. Rats are the most susceptible species to Tri-induce kidney toxicity but there is much disagreement about the kidney as a target organ for Tri in humans. This proposal will use confluent primary cultures of human proximal tubular (hPT) cells as a model to determine factors that may contribute to the relatively low susceptibility of humans to Tri-induced kidney toxicity. Cells will be obtained from fresh human kidney tissue by collagenase digestion and will be cultured under serum-free, hormonally-defined conditions. These cells maintain expression of several proximal tubular functions during culture. The proposal will address three hypotheses: 1) Lower rates of metabolism and/or transport account for the relatively low susceptibility of hPT cells to DCVC; 2) DCVC produces renal tubular cell death in hPT cells by both necrosis and apoptosis; and 3) DCVC-induced alteration in mitochondrial function are causally associated with apoptosis in hPT cells. Previous work defined rates of glutathione conjugation of Tri in human liver and kidney tissue and showed that this initial step of the metabolic pathway cannot account for differences in susceptibility of human kidney tissue to Tri-induced toxicity. Furthermore, DCVC is known to be the penultimate toxic metabolite of Tri by this pathway. Accordingly, these studies will use DCVC as the primary test agent. The first hypothesis will be addressed by measurement of DCVC metabolism by the b-lyase and the N-acetyltransferase, which forms the mercapturate, N-acetyl-S-(1,2 or 2,2-dichlorovinyl)-L-cysteine (NAcDCVC). Rates of NAcDCVC deacetylation will also be quantitated. Transport of DCVC and NAcDCVC across by basolateral and brush-border membranes will be quantitated and characterized in hPT cells grown on filter inserts. The second hypothesis will be addressed by defining precise exposure conditions (time, concentration) whereby DCVC produces either necrosis or apoptosis. Necrosis will be measured by release of a cytosolic enzyme whereas apoptosis will be assessed by several assays, including cell cycle analysis, DNA fragmentation, and annexin staining by flow cytometry, cytochrome c release, and bcl-2 expression. The third hypothesis will be tested by correlating DCVC-induced changes in various measurers of mitochondrial function with the onset and severity of apoptosis. These studies will enhance our understanding of how DCVC produces renal cell injury in the human kidney and should serve as a model for analysis of species differences in responses to other nephrotoxic chemicals and should enhance our ability to evaluate human susceptibility to chemically induced renal injury.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
1R01ES008828-01A2
Application #
2908529
Study Section
Special Emphasis Panel (ZRG1-ALTX-4 (01))
Program Officer
Mullins, Christopher V
Project Start
1999-09-15
Project End
2002-08-31
Budget Start
1999-09-15
Budget End
2000-08-31
Support Year
1
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Wayne State University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
City
Detroit
State
MI
Country
United States
Zip Code
48202
Lash, Lawrence H; Putt, David A; Benipal, Bavneet (2014) Multigenerational study of chemically induced cytotoxicity and proliferation in cultures of human proximal tubular cells. Int J Mol Sci 15:21348-65
Xu, Feng; Papanayotou, Irene; Putt, David A et al. (2008) Role of mitochondrial dysfunction in cellular responses to S-(1,2-dichlorovinyl)-L-cysteine in primary cultures of human proximal tubular cells. Biochem Pharmacol 76:552-67
Lash, Lawrence H; Putt, David A; Cai, Hongliang (2008) Drug metabolism enzyme expression and activity in primary cultures of human proximal tubular cells. Toxicology 244:56-65
Lash, Lawrence H; Putt, David A; Huang, Paul et al. (2007) Modulation of hepatic and renal metabolism and toxicity of trichloroethylene and perchloroethylene by alterations in status of cytochrome P450 and glutathione. Toxicology 235:11-26
Lash, Lawrence H; Putt, David A; Hueni, Sarah E et al. (2007) Interactive toxicity of inorganic mercury and trichloroethylene in rat and human proximal tubules: effects on apoptosis, necrosis, and glutathione status. Toxicol Appl Pharmacol 221:349-62
Lash, Lawrence H; Putt, David A; Cai, Hongliang (2006) Membrane transport function in primary cultures of human proximal tubular cells. Toxicology 228:200-18
Lash, Lawrence H; Putt, David A; Parker, Jean C (2006) Metabolism and tissue distribution of orally administered trichloroethylene in male and female rats: identification of glutathione- and cytochrome P-450-derived metabolites in liver, kidney, blood, and urine. J Toxicol Environ Health A 69:1285-309
Lash, Lawrence H; Putt, David A; Hueni, Sarah E et al. (2005) Molecular markers of trichloroethylene-induced toxicity in human kidney cells. Toxicol Appl Pharmacol 206:157-68
Krause, Renee J; Lash, Lawrence H; Elfarra, Adnan A (2003) Human kidney flavin-containing monooxygenases and their potential roles in cysteine s-conjugate metabolism and nephrotoxicity. J Pharmacol Exp Ther 304:185-91
Lash, Lawrence H; Putt, David A; Hueni, Sarah E et al. (2003) Roles of necrosis, Apoptosis, and mitochondrial dysfunction in S-(1,2-dichlorovinyl)-L-cysteine sulfoxide-induced cytotoxicity in primary cultures of human renal proximal tubular cells. J Pharmacol Exp Ther 305:1163-72

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