Biochemical Pharmacology, Vol. 37, No. 7, pp. 1287-1291, 1988. 0006-2952/88 $3.00 + 0.00 Printed in Great Britain. t~ 1988. Pergamon Press plc IMPAIRMENT OF BROMOSULFOPHTHALEIN HEPATIC TRANSPORT AND CHOLESTASIS INDUCED BY DIETHYL MALEATE IN THE RABBIT RAFAEL JIMI~NEZ, OLGA LARRUBIA, MARIA J. MONTE and ALEJANDRO ESTELLER Department of Physiology and Pharmacology, University of Salamanca, 37007, Salamanca, Spain (Received 1 June 1987; accepted 1 October 1987) Abstract--The present study was designed to investigate the effect of hepatic glutathione depletion induced by intraperitoneal administration of diethyl maleate (DEM) on the maximum biliary transport (Tm) and on the biliary excretion of bromosulfophthalein (BSP) in anaesthetized rabbits when the dye was perfused endovenously at doses exceeding Tm. The Tm of total BSP (BSPt) and that of conjugated BSP (BSPc) were significantly reduced after DEM administration whereas that of unconjugated BSP (BSPu) was markedly increased. A reduction in the biliary excretion of BSPt and BSPc, in the percentage of BSPc, in the cumulative excretion of BSPt and in the percent-dose recovery were also observed. However, no change in hepatic glutathione S- transferase activity was noted after DEM. The cholestasis observed following DEM administration coursed with falls in the biliary secretion of sodium, chloride and bicarbonate. Cellular glutathione (GSH)* depletion induced by different methods has proved to be a useful tool in detoxification and drug metabolism since distur- bances in conjugation may impair the biliary excretion of different xenobiotics such as BSP [1-3]. The most common chemical compound employed to induce cellular GSH depletion is diethyl maleate, an unsaturated a~,fl-carbonyl compound which reacts with GSH to yield derivatives that are excreted in the bile and which produce a choleretic effect in species such as the rat, the dog and the rabbit [4-7]. In the latter species, unlike what happens in the former, the choleretic response to DEM is less intense; it does not last as long and is followed by intense cholestasis, metabolic acidosis and a pro- nounced reduction in the biliary excretion of HCO~- [7], whose secretion--specially high in this species---plays an important role in bile formation [3, 8]. Moreover, it has been reported that BSP, a xenobiotic with a low Tm in rabbits [9, 10], induces choleresis [9], cholestasis [10] or no effect at all [11]. Additionally, in view of the fact that BSP and DEM conjugation is catalyzed by GSH-S-transferases, whose activity is very low in the rabbit [12], the aim of the present work was to assess the effect of hepatic GSH depletion by DEM administration on biliary BSP excretion and on other biliary, hepatic and blood parameters. MATERIALS AND METHODS Animals and experimental procedures. Male New Zealand rabbits weighing between 1.5 and 2.0 kg were used. The animals were housed in environ- * Abbreviations used: BSPt, BSPc, BSPu, total, con- jugated and unconjugated bromosulfophthalein; DEM, diethyl maleate; GSH, glutathione; Tin, maximum biliary transport. mental conditions and fed a standard diet. Food but not water was withheld for 18 hr prior to surgery. After pentobarbital anaesthesia (30 mg/kg i.v.) tra- cheotomy was performed followed by a median ven- tral laparotomy. The cystic duct and the pylorus were ligated. The choledocus and the left femoral artery and vein were cannulated as described previously [7]. A cannula was also introduced into the first part of the duodenum. The body temperature of the rabbits, measured with a rectal probe, was kept between 38.5-39.5 ° with an electric heating pad. After an equilibrium period of 30 min to allow bile flow to stabilize, bile was collected in previously weighed tubes over four hours at intervals of 10 or 15 min. Part of the samples taken (< 10%) was frozen at -40 ° until later assay. An aliquot of bile equivalent to that taken every 10-15 min was later infused into the duodenum. After collecting four baseline 15 min samples con- tinuous BSP infusion was started at a dose of 0.88 Ixmol/min/kg and maintained over a period of 60-180 min. In one group of rabbits DEM was administered at 120 rain at a dose of 3.2 mmol/kg mixed 1/1 (v/v) with corn oil; another group received corn oil alone and was used as the control group. Sixty microlitres of blood samples were obtained from the femoral artery at 0, 60, 120 and 180 min for the determination of pOE, pCO2, pH and HCO~- concentrations. At minutes 5, 10, 15, 20, 40, 60, 90, 120, 150 and 180 after the start of BSP infusion, blood samples were obtained for the analysis of plasma BSP levels. The animals were killed by exsanguination. Livers were flushed with an ice-cold NaC1 solution (0.9%, w/v) injected via the hepatic vein. Following this the livers and bladders were removed. Liver samples were freeze-clamped in liquid nitrogen for later analysis of the amounts BSP and GSH and GSH-S- transferase activity. 1287