Long-term exposure to trichloroethylene (TE) causes renal cancer in rats. Dichloroacetylene (DA), a breakdown product of TE, is both nephrotoxic and neurotoxic. TE and DA are metabolized to the glutathione (GSH S-conjugate and cysteine S-conjugate (= S-(1,2-dichlorovinyl)-L-cysteine, DCVC) which are toxic. DCVC causes selective damage to the S3 nephron segment; mitochondria are vulnerable. Cysteine S-conjugate beta-lyases are pyridoxal 5'-phosphate-dependent enzymes that convert DCVC and similar compounds to pyruvate, ammonia and a toxic fragment. The fragment kills renal cells by reacting with macromolecules, depleting cellular thiols and stimulating peroxidative damage. Kynureninase and cytosolic glutamine transaminase K (cytGTK) have cysteine S-conjugate beta-lyase activity. However, kynureninase is inactivated by DCVC and the lyase activity of cytGTK is supported by alpha-keto acids present in vivo only at low levels. A high molecular weight (Mr) multi-subunit lyase (recently discovered by the investigator) is present in the S3 segment and in kidney cytosol and mitochondria, and is inactivated by physiological levels of alpha-ketoglutarate. Therefore, it may be the principal enzyme responsible for selective renal damage.
The aim of the current proposal is to purify the high Mr lyase from rat kidney cytosol (and mitochondria) and clone and sequence the subunits. Antibodies will be generated and used to determine the cellular/subcellular location of the enzyme/subunits and contribution of the enzyme to total beta-lyase activity of the kidney. The Xenopus oocyte expression system will be used to determine the relative importance of each subunit to overall structure/catalytic function in the holoenzyme. LLC-PK1 cells lacking mitochondria and heterologous expression in a mammalian cell line should provide useful tools to study the role of the high Mr enzyme in the toxicity of GSH-/cysteine S-conjugates. The proposed work will provide insights into the relationship of the high Mr lyase to other PLP enzymes. If the high Mr lyase is shown to be the principal agent for bioactivation of nephrotoxic conjugates the results will have medical importance. TE is a common pollutant. Risks to humans heavily exposed to TE (or similar compounds) may be minimized by regimens that inhibit the high Mr lyase.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES008421-03
Application #
2749706
Study Section
Special Emphasis Panel (ZRG4-ALTX-1 (01))
Project Start
1997-08-01
Project End
2001-07-31
Budget Start
1998-08-01
Budget End
1999-07-31
Support Year
3
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Winifred Masterson Burke Med Research Institute
Department
Type
DUNS #
780676131
City
White Plains
State
NY
Country
United States
Zip Code
10605
Pinto, John T; Krasnikov, Boris F; Alcutt, Steven et al. (2014) Kynurenine aminotransferase III and glutamine transaminase L are identical enzymes that have cysteine S-conjugate ?-lyase activity and can transaminate L-selenomethionine. J Biol Chem 289:30950-61
Jeitner, Thomas M; Cooper, Arthur J L (2014) Inhibition of human glutamine synthetase by L-methionine-S,R-sulfoximine-relevance to the treatment of neurological diseases. Metab Brain Dis 29:983-9
Vauzour, David; Pinto, John T; Cooper, Arthur J L et al. (2014) The neurotoxicity of 5-S-cysteinyldopamine is mediated by the early activation of ERK1/2 followed by the subsequent activation of ASK1/JNK1/2 pro-apoptotic signalling. Biochem J 463:41-52
Hallen, André; Jamie, Joanne F; Cooper, Arthur J L (2013) Lysine metabolism in mammalian brain: an update on the importance of recent discoveries. Amino Acids 45:1249-72
Tsikas, Dimitrios; Evans, Christopher E; Denton, Travis T et al. (2012) Stable isotope gas chromatography-tandem mass spectrometry determination of aminoethylcysteine ketimine decarboxylated dimer in biological samples. Anal Biochem 430:4-15
Halamkova, Lenka; Mailloux, Shay; Halamek, Jan et al. (2012) Enzymatic analysis of ýý-ketoglutaramate--a biomarker for hyperammonemia. Talanta 100:7-11
Cooper, Arthur J L; Krasnikov, Boris F; Pinto, John T et al. (2012) Comparative enzymology of (2S,4R)4-fluoroglutamine and (2S,4R)4-fluoroglutamate. Comp Biochem Physiol B Biochem Mol Biol 163:108-20
Bridges, Christy C; Krasnikov, Boris F; Joshee, Lucy et al. (2012) New insights into the metabolism of organomercury compounds: mercury-containing cysteine S-conjugates are substrates of human glutamine transaminase K and potent inactivators of cystathionine ?-lyase. Arch Biochem Biophys 517:20-9
Cooper, Arthur Jl; Pinto, John T; Callery, Patrick S (2011) Reversible and irreversible protein glutathionylation: biological and clinical aspects. Expert Opin Drug Metab Toxicol 7:891-910
Pinto, John T; Lee, Jeong-In; Sinha, Raghu et al. (2011) Chemopreventive mechanisms of ýý-keto acid metabolites of naturally occurring organoselenium compounds. Amino Acids 41:29-41

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