Chem.-BioL Interactions, 68 (1988)85--98 85 Elsevier ScientificPublishers Ireland Ltd. IDENTIFICATION OF S-(2,5-DIHYDROXYPHENYL)-CYSTEINE AND S42,5-DIHYDROXYPHENYL)-N-ACETYL-CYSTEINE AS URINARY METABOLITES OF ACETAMINOPHEN IN THE MOUSE. EVIDENCE FOR/~BENZOQUINONE AS A REACTIVE INTERME- DIATE IN ACETAMINOPHEN METABOLISM GARY A. PASCOE, CARL JOHAN CALLEMAN and THOMAS A. BAILLE* Department of Medicinal Chemistry, School of Pharmacy, BG-20, University of Washingto~ Seattle, WA 98195 (U.S.A./ (Received September 22rid, 1987) (Revision received April 4th, 1988) (Accepted April 4th, 1988) SUMMARY S-(2,5-Dihydroxyphenyl)-cysteine and S-(2,5-dihydroxyphenyl~N-acetyl- cysteine [the cysteine- and N-acetyl-cysteine adducts, respectively, of hydro- quinone (HQ)] were identified and quantified in the urine of mice administered [ring-U-14C]acetaminophen [14C]APAP, 200 mg kg -1, i.p.). Urine was collected for 24 h and fractionated by HPLC to isolate the above adducts. These conjugates were then converted to a common derivative, viz. O,O',S-tris-acetyl-3-thiwhydroquinone, which was characterized by GC/MS. Neither of the HQ adducts was detected in the urine of control mice which had not received APAP. Quantification of urinary HQ-cysteine and HQ-N-acetyl- cysteine was performed by HPLC techniques, which indicated that these conjugates accounted for approx. 1.5°/o of the administered dose of APAP after 24 h, a figure which is equivalent to 6.3o/o of the corresponding APAP- thiol conjugates in the urine. These findings provide strong indirect evidence that p-benzoquinone is formed as a reactive, but apparently non-hepatotoxic, metabolite of APAP in vivo. Key words: Acetaminophen metabolism - Thiol adducts -- Benzoquinone *To whomcorrespondenceshould be addressed. Abbreviations: APAP, acetaminophen, 4-hydroxyacetanilide; BZQ, p-benzoquinone; GSH, gluta- thione; HQ, hydroquinone; HQ-cysteine, S-(2,5-dihydroxyphenyl)-cysteine; HQ-N-acetyl-cysteine, S-(2,5-dihydroxyphenyl)-N-acetyl-cysteine; MU, methylene units; NAPQI, N-acetyl-p-benzoquinone imine. 0009-2797/88/$03.50 © 1988 Elsevier ScientificPublishers Ireland Ltd. Printed and Published in Ireland