A rapid, simple in vitro screening test, using [(3)H]glutathione and l-[(35)S]cysteine as trapping agents, to detect reactive intermediates of xenobiotics.
Reactive intermediates, formed by biotransformation of xenobiotics, play an important role in drug toxicity. We have developed a rapid in vitro screening test for the detection of such intermediates. The unlabelled xenobiotic substrate is incubated with a post-mitochondrial supernatant, or a microsomal fraction of rat liver in the presence of NADPH and [(3)H]glutathione (GSH). Reactive intermediates formed are trapped with GSH, forming a radiolabelled GSH adduct. Analysis of the incubation medium by HPLC shows the formation of a new radioactive peak, corresponding to this adduct. To make the test more specific, similar incubations are done with l-[(35)S]cysteine: reactive intermediates are likely to be trapped by this agent, while substrates for GSH-transferase give a negative response with l-[(35)S]cysteine. The test has been used with 44 substrates; of the 18 expected to be positive, 16 were positive, and two negative.
Mulder
G J
GJ
Division of Toxicology, Center for Bio-Pharmaceutical Sciences, University of Leiden, P.O. Box 9503, 2300 RA Leiden, The Netherlands.
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