title: Cytotoxicity of S-(1,2,3,4,4-pentachlorobutadienyl)-L cysteine after apical and basolateral exposure of LLC-PK monolayers. Involvement of an amino acid transport system.
abstract: Glutathione conjugation and subsequent formation of cysteine conjugates are key steps in the nephrotoxicity of halogenated alkenes. In this metabolic activation several organs are involved. However little is known about the transporters responsible for the uptake of cysteine conjugates. Recent evidence suggest that amino acid transporters play a role in this uptake. Monolayers of LLC-PK1 cells, a kidney cell line, were exposed to S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine (PCBD-CYS). Cytotoxicity was used as a parameter for PCBD-CYS uptake. Basolateral exposure (1 h: 400 microM and 16 h: 25 microM) to PCBD-CYS resulted in a much higher aminooxyacetic acid inhibitable cytotoxicity than apical exposure, suggesting a preferential basolateral uptake of PCBD-CYS. Exposure to PCBD-CYS in the absence of sodium did not result in a decrease of the cytotoxicity, suggesting a sodium independency of the PCBD-CYS uptake. Amino acids and amino acid analogues were used as diagnostic compounds in the further identification of the PCBD-CYS transporter. In cis-inhibition experiments monolayers were co-incubated with PCBD-CYS and these diagnostic compounds during one hour. System L substrates such as 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) and cycloleucine did not inhibit cytotoxicity. D-Tryptophan, a model inhibitor of System T, caused a strong inhibition. System L has, in contrast to System T, a high sensitivity to trans-stimulation. Pre-loading the monolayers with the diagnostic compounds should cause an increase in cytotoxicity when System L is involved. Neither System L substrates such as BCH and cycloleucine nor D-tryptophan increased cytotoxicity. These results suggest a preferential basolateral uptake of PCBD-CYS in LLC-PK1 monolayers and involvement of an amino acid transporter with characteristics of System T.
authors.affiliation: Dept. of Toxicology, Agricultural University Wageningen, The Netherlands.
authors.forename: J J
authors.initials: JJ
authors.lastname: Mertens
authors.forename: E A
authors.initials: EA
authors.lastname: Keukens
authors.forename: M
authors.initials: M
authors.lastname: Appel
authors.forename: B
authors.initials: B
authors.lastname: Spenkelink
authors.forename: J H
authors.initials: JH
authors.lastname: Temmink
authors.forename: P J
authors.initials: PJ
authors.lastname: van Bladeren
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002070:Butadienes / Q000493:pharmacokinetics / Q000633:toxicity*; D002352:Carrier Proteins / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives* / Q000493:pharmacokinetics / Q000633:toxicity; D007674:Kidney Diseases / Q000139:chemically induced* / Q000378:metabolism; D012964:Sodium / Q000494:pharmacology; D013552:Swine
chemical_list: D000596:Amino Acids; D002070:Butadienes; D002352:Carrier Proteins; C039330:S-pentachlorobuta-1,3-dien-yl-cysteine; D012964:Sodium; D003545:Cysteine