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.
Showing the most recent 10 out of 34 publications
