title: Cytotoxicity of 2-tert-butyl hydroquinone glutathione conjugates after apical and basolateral exposure of rat renal proximal tubular cell monolayers.
abstract: Confluent monolayers of renal proximal tubular (RPT) cells cultured on porous supports were used to investigate the cytotoxicity induced by glutathione conjugates of 2-tert-butyl-(1,4)-hydroquinone (SG-TBHQ) after apical and basolateral exposure. As judged from lactate dehydrogenase (LDH) leakage, cytotoxicity was observed after basolateral exposure of monolayers to 250 and 500 mum 2-tert-butyl-5-(glutathion-S-yl)hydroquinone (5SG-TBHQ). In these experiments, LDH leakage was 22.3 +/- 1.9 and 32.2 +/- 1.9%, respectively. Basolateral exposure of monolayers to 250 and 500 mum 6SG-TBHQ resulted in LDH leakage of 22.2 +/- 2.5 and 30.0 +/- 2.7%, respectively. The double conjugate, 2-tert-butyl-3,6-(diglutathione-S-yl)hydroquinone (3,6SG-TBHQ), was not toxic and LDH leakage was about control level (15.0%). Basolaterally located probenecid-sensitive organic anion transporters did not seem to play a part in the cytotoxic effect. However, when RPT cell monolayers were cultured in 24-well tissue culture plates, apical challenge with 250 mum 5SG-TBHQ induced a cytotoxic effect. In these experiments, LDH leakage was 33.5 +/- 0.6%. With these cells, inhibition of apical gamma-glutamyltranspeptidase (gammaGT) activity by acivicin, which was not toxic by itself, decreased 5SG-TBHQ-induced LDH leakage to 19.3 +/- 1.2%, whereas 6SG-TBHQ (also 250mum)-induced LDH leakage was increased to 55.3 +/- 1.0%. Co-incubation of RPT cells with SG-TBHQs in the presence of 1.5 mm ascorbic acid (AA) pointed to a pro-oxidant rather than an antioxidant effect of AA. Superoxide dismutase and catalase completely abolished SG-TBHQ-induced cytotoxicity. Since cultured RPT cells lack N-acetylation of cysteine conjugates, the N-deacetylation/N-acetylation ratio cannot have a vital role in renal toxicity of quinone thioethers in this in vitro system. It seems, therefore, that the cytotoxicity observed is mainly the result of extracellular redox cycling of SG-TBHQs. The lack of toxicity of 6SG-TBHQ after apical exposure could be due to detoxification by gammaGT-mediated cysteinylglycine- or cysteine-conjugate formation followed by cyclization, as shown for other related quinone glutathione conjugates. The relative importance of the observed effects for the in vivo situation is discussed.
authors.affiliation: Department of Toxicology, Agricultural University, Tuinlaan 5, PO Box 8000,6700 EA Wageningen, The Netherlands.
authors.forename: H E
authors.initials: HE
authors.lastname: Haenen
authors.forename: E
authors.initials: E
authors.lastname: Bleijlevens
authors.forename: H
authors.initials: H
authors.lastname: Elzerman
authors.forename: J H
authors.initials: JH
authors.lastname: Temmink
authors.forename: J H
authors.initials: JH
authors.lastname: Koeman
authors.forename: P J
authors.initials: PJ
authors.lastname: Van Bladeren