Many simple organic molecules including solvents, agrichemicals, drugs and drug candidates become cytotoxic upon biotransformation to reactive electrophilic metabolites. The liver is a major site of foreign compound metabolism and is often a major target for drug and chemical toxicity. Hepatotoxicity remains the single most important factor in the failure of drug candidates late in development (when such failure is extremely expensive in terms of lost investment of resources), and of approved drugs early in open clinical usage (when the cost of failure can also include severe or even fatal injury to patients). Understanding the mechanism(s) by which chemicals cause cytotoxic liver injury remains a major challenge of enormous scientific and practical importance. A major focus of our laboratory has been the identification and characterization of chemically reactive metabolites, the protein adducts they form in living cells, and the specific proteins that become adducted. We have hypothesized that there exists a common subset of cellular proteins whose covalent modification by reactive metabolites initiates cytotoxic events through intracellular signaling mechanisms. Our recent work has identified 28 such candidate proteins. Bioinformatic analysis reveals that 21 of them interact with 165 partner proteins, many of which are members of the MAP kinase signaling pathway. Encouraged by these observations, we propose to test our original hypothesis more thoroughly by (1) expanding significantly the list of known target proteins for chemically-reactive metabolites of several paradigm hepatotoxicants to look for commonality (i.e. functional proteomics);(2) applying bioinformatic methods to analyze target proteins and their first-neighbor interacting partners to uncover common signaling pathways that lead to cytotoxic outcomes;(3) testing the hypothesis that hepatocytes are able to recognize and respond to adducted proteins per se by delivering pre-adducted proteins directly into hepatocytes using protein transduction domains and observing the cells for cytotoxic responses and/or changes in intracellular signaling pathways associated with hepatotoxicity.

Public Health Relevance

Many drugs and chemicals give rise to chemically-reactive metabolites that modify cellular proteins leading to cytotoxicity. We have identified certain proteins whose modification may play a general role in this process, and we now propose to search more broadly for other such proteins and their interacting cellular partners that may form part of a general process underlying reactive metabolite cytotoxicity.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM021784-34
Application #
8240112
Study Section
Xenobiotic and Nutrient Disposition and Action Study Section (XNDA)
Program Officer
Okita, Richard T
Project Start
1978-08-01
Project End
2014-03-31
Budget Start
2012-04-01
Budget End
2014-03-31
Support Year
34
Fiscal Year
2012
Total Cost
$296,658
Indirect Cost
$94,850
Name
University of Kansas Lawrence
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
076248616
City
Lawrence
State
KS
Country
United States
Zip Code
66045
Hanzlik, Robert P; Koen, Yakov M; Fang, Jianwen (2013) Bioinformatic analysis of 302 reactive metabolite target proteins. Which ones are important for cell death? Toxicol Sci 135:390-401
Koen, Yakov M; Sarma, Diganta; Hajovsky, Heather et al. (2013) Protein targets of thioacetamide metabolites in rat hepatocytes. Chem Res Toxicol 26:564-74
Koen, Yakov M; Sarma, Diganta; Williams, Todd D et al. (2012) Identification of protein targets of reactive metabolites of tienilic acid in human hepatocytes. Chem Res Toxicol 25:1145-54
Hanzlik, Robert P; Fang, Jianwen; Koen, Yakov M (2009) Filling and mining the reactive metabolite target protein database. Chem Biol Interact 179:38-44
Ikehata, Keisuke; Duzhak, Tatyana G; Galeva, Nadezhda A et al. (2008) Protein targets of reactive metabolites of thiobenzamide in rat liver in vivo. Chem Res Toxicol 21:1432-42
Ji, Tao; Ikehata, Keisuke; Koen, Yakov M et al. (2007) Covalent modification of microsomal lipids by thiobenzamide metabolites in vivo. Chem Res Toxicol 20:701-8
Koen, Yakov M; Gogichaeva, Natalia V; Alterman, Michail A et al. (2007) A proteomic analysis of bromobenzene reactive metabolite targets in rat liver cytosol in vivo. Chem Res Toxicol 20:511-9
Hanzlik, Robert P; Koen, Yakov M; Theertham, Bhargav et al. (2007) The reactive metabolite target protein database (TPDB)--a web-accessible resource. BMC Bioinformatics 8:95
Koen, Yakov M; Yue, Weimin; Galeva, Nadezhda A et al. (2006) Site-specific arylation of rat glutathione s-transferase A1 and A2 by bromobenzene metabolites in vivo. Chem Res Toxicol 19:1426-34
Yue, Weimin; Koen, Yakov M; Williams, Todd D et al. (2005) Use of isotopic signatures for mass spectral detection of protein adduction by chemically reactive metabolites of bromobenzene: studies with model proteins. Chem Res Toxicol 18:1748-54

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