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 μ m 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 μ m 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 μ m 5SG-TBHQ induced a cytotoxic effect. In these experiments, LDH leakage was 33.5 ± 0.6%. With these cells, inhibition of apical γ-glutamyltranspeptidase (γGT) activity by acivicin, which was not toxic by itself, decreased 5SG-TBHQ-induced LDH leakage to 19.3 ± 1.2%, whereas 6SG-TBHQ (also 250μ m)-induced LDH leakage was increased to 55.3 ± 1.0%. Co-incubation of RPT cells with SG-TBHQs in the presence of 1.5 m m 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 γGT-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.
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