Glutathione conjugation of bromosulfophthalein in relation to hepatic glutathione content in the rat in vivo and in the perfused rat liver

Glutathione Conjugation of Bromosulfophthalein in Relation to Hepatic Glutathione Content in the Rat In Vivo and in the Perfused Rat Liver C. A . WILCO SNEL, 1 K . SANDY PANG, 2 AND GERARD J . M U L D E R 1

The r e l a t i o n b e t w e e n t h e rate o f g l u t a t h i o n e (GSH) c o n j u g a t i o n a n d h e p a t i c GSH c o n t e n t w a s studied in t h e rat i n v i v o and t h e i n s i t u s i n g l e - p a s s - p e r f u s e d rat liver p r e p a r a t i o n w i t h b r o m o s u l f o p h t h a l e i n (BSP) as t h e m o d e l substrate. The biliary e x c r e t i o n of t h e BSP-GSH c o n j u g a t e a n d t h e h e p a t i c GSH c o n t e n t w e r e m o n i t o r e d simultaneously during intravenous infusions with BSP in t h e rat i n vivo, a n d during liver p e r f u s i o n s w i t h BSPc o n t a i n i n g p e r f u s i o n m e d i u m . Rats w e r e p r e t r e a t e d w i t h single or m u l t i p l e d o s e s o f b u t h i o n i n e sulfoximine, an inhibitor o f the de n o v o s y n t h e s i s o f GSH. Surprisingly, t h e e x c r e t i o n o f t h e BSP-GSH c o n j u g a t e w a s sust a i n e d at a high rate, despite a virtually c o m p l e t e depletion o f h e p a t i c GSH, b o t h in the rat i n v i v o as well as in t h e p e r f u s e d rat liver. The results indicate that GSH w a s still available for c o n j u g a t i o n w i t h B S P after a p p a r e n t d e p l e t i o n o f t h e h e p a t i c GSH pool, p r e s u m a b l y b e c a u s e of a residual de n o v o s y n t h e s i s of GSH in t h e liver. Despite t h e multiple p r e t r e a t m e n t w i t h b u t h i o n i n e sulfoximine, t h e de n o v o GSH s y n t h e s i s w a s sufficient to s u s t a i n a h i g h rate o f GSH c o n j u g a t i o n o f BSP. The cosubstrateKm for GSH c o n j u g a t i o n o f B S P in the liver w a s estim a t e d to be very small (approximately 0.3/~mol/g): the e x c r e t i o n rate o f the BSP-GSH c o n j u g a t e w a s o n l y imo paired at m i n i m a l h e p a t i c GSH levels. (HEPATOLOGY 1995;21:1387-1394.)

Glutathione (GSH) conjugation is an important biotransformation reaction of xenobiotics and endogenous compounds containing electrophilic groups. The reaction is catalyzed by the glutathione-S-transferases (Enzyme Committee 2.5.1.18) and requires the endogenous tripeptide GSH. We have studied the dependence of GSH conjugation on hepatic GSH in the rat in vivo

Abbreviations: GSH, glutathione; BSP, bromosulfophthalein; BSO, D,Lbuthionine-[S,R]-sulfoximine; DEM, diethyl maleate; TLC, thin layer chromatography; HPLC, high-pressure liquid chromatography; v/v/v, volume to volume to volume; w/v, weight to volume. From the 1Division of Toxicology, Leiden/Amsterdam Center for Drug Research, Leiden University, Leiden, The Netherlands; and the ~Faculty of Pharmacy, University of Toronto, Toronto, Canada. Received June 30, 1994; accepted December 1, 1994. Address reprint requests to: Gerard J. Mulder, PhD, Leiden/Amsterdam Center for Drug Research, Leiden University, PO Box 9503, 2300 RA Leiden, The Netherlands. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2105-002353.00/0

with racemic 2-bromoisovalerylurea as the model substrate.1 However, 2-bromoisovalerylurea is not ideal for this purpose, because the derived GSH conjugates are further metabolized and not only excreted from the liver into bile, but also into blood. 2 Consequently, measurement of biliary excretion rate of the GSH conjugates alone leads to an underestimation of the hepatic GSH conjugation rate. Moreover, the urinary excretion of the 2-bromoisovalerylurea mercaptures does not necessarily reflect the efflux of GSH conjugates from the liver into blood, because extrahepatic GSH conjugation of 2-bromoisovalerylurea may occur (unpublished data). In this study, we used the liver diagnostic dye bromosulfophthalein (BSP) as a substrate to examine the role of hepatic GSH in GSH conjugation. BSP is exclusively metabolized via GSH conjugation to give the BSP-GSH conjugate. 3-6Degradation of BSP-GSH to cysteinylglycine and cysteine conjugates, as well as formation of di-GSH conjugates and derived metabolites have been reported, 7 but these pathways are quantitatively negligible. BSP elimination from blood involves carrier-mediated uptake of BSP by the liver, s-l° enzymatic conversion to BSP-GSH, and excretion of BSP-GSH (and unchanged BSP) in bile by an adenosine triphosphatedependent organic anion tranporter. 1~BSP-GSH efflux from the liver into blood has been shown to be absent or negligible. 12-14BSP distributes into all tissues of the body, '5'16 but its metabolism seems to be confined to the liver; extrahepatic GSH conjugation of BSP has not been reported thus far. The rate-limiting step for the excretion of BSP-GSH into bile has been extensively studied: induction of GSH conjugation with phenobarbital, butylated hydroxy-anisol, or trans-stilbene oxide results in increased excretion rates of BSP-GSH in bile, 17'~s whereas inhibition of GSH conjugation with clofibric acid, TM tienilic acid, 2° or ethacrynic acid, 2~and depletion of GSH with diethyl maleate 22'2s or methyliodide24 lead to a reduced rate of BSP-GSH excretion. Moreover, the half-life of biliary excretion of BSP-GSH is shorter after administration of BSP-GSH than after administration of BSP. 25 A multiple indicator dilution study with BSP has shown that the influx-rate constant is higher than the sequestration-rate constant. 26 The above results collectively suggest that the GSH conjugation in the

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liver d e t e r m i n e s the r a t e of biliary excretion of BSPGSH. Therefore, B S P s e e m e d ideally suitable to s t u d y G S H conjugation in relation to h e p a t i c G S H content. MATERIALS AND METHODS Chemicals. Bromosulfophthalein (BSP), taurocholic acid and 5-sulfosalicylic acid were purchased from Janssen Chimica, Geel, Belgium. D,L-Buthionine-[S,R]-sulfoximine (BSO) was purchased from Sigma Chemical Co., St. Louis, MO. Reduced GSH and diethyl maleate (DEM) were from E. Merck, Darmstadt, Germany. 5,5'-Dithio-bis-(2-nitrobenzoic acid) was from BDH Chemicals Ltd., Poole, England. [3~S]-LCysteine was from Amersham Intl. Plc, Amersham, England. The bromosulfophthalein-glutathione conjugate (BSP-GSH) was prepared according to the method of Whelan et al 6 with minor modifications. 1~All other chemicals and reagents were of analytical or HPLC grade. Animals. Male outbred WU rats (a Wistar-derived strain; specific pathogen-free quality) were obtained from the breeding unit of the Sylvius Laboratories, University of Leiden. Rats were housed in macrolon cages on standard hard-wood bedding in a temperature-controlled (22 ° to 24°C) and humidity-controlled (50%-60%) room with alternating 12 hour-on and 12 hour-off illumination cycles. They were allowed free access to SRMA 1210 lab chow pellets (Hope Farms, Woerden, The Netherlands) and tap water till the day of the experiment. Intravenous BSP Infusions in the R a t In Vivo. Rats (215 to 235 g body weight) were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg). Throughout the experiment, the body temperature was kept at 38°C with an electrical heating pad. The bile duct and the external jugular vein were cannulated as described previouslyS After surgery BSO (2 mmol/kg; in saline) was administered intravenously via the cannula in the jugular vein over a period of 2 minutes. One hour later, an infusion with BSP was started at an infusion rate of 1.5 #mol/min/kg. Taurocholic acid was coinfused at an infusion rate of 6 #mol/min/kg to maintain the bile flow at a constant level. A solution of BSP and taurocholic acid in saline was administered via the jugular vein at a constant flow rate of 1.9 mL/hr; concentrations of BSP and taurocholic acid were modified according to the body weights of individual rats. Throughout the 6 hours of infusion, bile was collected in toto in 15-minute fractions. The volume of the bile samples was determined gravimetrically, assuming a density of 1 g/mL. Bile samples were stored at -20°C until analysis. Liver biopsy specimens were taken at 0, 52, 112, 172, 232, 292, and 352 minutes (_+1 minutes) after the start of the infusion; 20-50-mg samples were snipped from the edge of the left lateral lobe. Immediately hereafter, biopsy samples were frozen in liquid nitrogen and stored at -80°C. They were analyzed within 2 days after the experiment. To minimize bleeding from the liver, a small piece of a gelatin-based hemostatic sponge (Willospon, Willpharma, Zwanenburg, The Netherlands) was applied at the site of excision. In two separate experiments, rats were pretreated and infused as described above; in addition, arterial blood (100 #L) was sampled via a cannula in the carotid artery at 52, 112, 172, 232, 292, and 352 minutes, and immediately diluted with 100 #L of a 12.9 #mol/L sodium citrate solution in saline. After sampling, 150 #L of heparin solution (20 IU/mL; in saline) was injected into the carotid artery in order to prevent the formation of blood clots in the cannula. Sampling of liver biopsy specimens was omitted in these experiments because considerable blood loss from the liver by repeated hepariniza-

HEPATOLOGYMay 1995 tion was anticipated. Blood samples were stored at -20°C until analysis. Effect of Multiple BSO Administration and DEM. The effect of multiple BSO administration was evaluated, using rats that had been pretreated with BSO (4 mmol/kg; in saline) administered via intraperitoneal injection, 24 hours and 6 hours before the infusion. All subsequent procedures were identical to those described under the previous caption, including the intravenous administration of BSO (2 mmol/kg) 1 hour before the start of the infusion. The effect of DEM on the biliary excretion of BSP-GSH was studied using the methodology described under the previous caption. At t = 180 minutes, the infusion was briefly interrupted for the intravenous administration of DEM (1 mmo]/ kg); at this point, the hepatic GSH pool was expected to be nearly completely depleted. To assess a direct effect of DEM on the biliary BSP-GSH excretion, control experiments were performed in which synthetic BSP-GSH, instead of BSP, was infused. The BSP-GSH infusion rate (1.1 #mol/min/kg) corresponded to the BSP-GSH excretion rate observed during a BSP infusion of 1.5 #mol/min/kg (see the Results section). Taurocholic acid was coinfused at an infusion rate of 6 #mol/ min/kg. DEM (1 mmol/kg) was administered at t = 180 minutes. In three rats that had been treated with multiple BSO injections as outlined above the possible incorporation of [35S]-L-cysteine into the BSP-GSH conjugate was investigated. After 180 minutes on the above BSP infusion a bolus dose of [35S]-cysteine (47 #Ci in 0.25 mL 1 mmol/L L-cysteine in saline) was administered intravenously. Incorporation of radiolabel into BSP-GSH was determined by thin layer chromatography (TLC) of bile samples; in some cases bile samples were analyzed by high-pressure liquid chromatography (HPLC) to confirm the TLC localization of radioactivity in BSP-GSH. For the HPLC method bile was diluted 10-fold with 10 mmol/L sodium phosphate buffer, pH 6.0; the separation system is described below. One-minute fractions of eluent were collected and counted for radioactivity after addition of 4 mL of liquid scintillation cocktail (Emulsifier Safe, Packard Instruments, Meriden, CT). For the TLC method, 10 #L bile was applied to Silicagel 60 F254 plates, and the plates were developed with n-propanol/water/glacial acetic acid = 10/5/1 volume to volume to volume (v/v/v). The BSPGSH spots were identified by phosphorous imaging of radioactivity and quantitated by liquid scintillation counting. Single Pass In Situ Rat Liver Perfusions. Rats (body weight, 280 to 340 g) were used as liver donors. The surgical procedure was conducted under pentobarbital anesthesia (60 mg/kg) as described previously. 2s The perfusion medium consisted of 20% volume to volume (v/v) washed bovine red blood cells (courtesy of Slachthuis Leiden B.V., Leiden, The Netherlands), 1% weight to volume (w/v) bovine serum albumin (Sigma Chemical Co, St. Louis, MO), 0.3% w/v glucose, and 50 #mol/L BSP in Krebs-Henseleit bicarbonate solution, buffered to pH 7.4. Perfusate was oxygenated with 95% oxygen: 5% carbon dioxide and kept at 37°C in a 500 mL reservoir. Perfusions were performed for 65 minutes at a constant flow rate of 10 mL/min in a single-pass fashion as described previously. 1~ Liver viability during the perfusion was assessed on the basis of gross appearance, bile flow, and oxygen consumption. Bile was collected in toto in 5-minute fractions into preweighed vials. Volumes of the bile samples were determined gravimetrically, assuming a density for bile of 1 g/mL. Venous outflow was sampled at the Tmidof the bile collection intervals. Three samples were taken from the reservoir, and the mean value was used to denote the BSP input concentra-

HEPATOLOGYVol. 21, No. 5, 1995 tion. On the basis of BSP input (Cin) and BSP output (Gout) concentrations, hepatic extraction ratios were calculated as: (Cin - Cout)/Cin. Liver biopsies were sampled as described above at 7.5, 17.5, 27.5, 37.5, 47.5, and 57.5 minutes (_+1 minute). No measures were taken to prevent loss of perfusate via the cutting edges; a minimal loss of perfusate (<2%) was observed after removal of six biopsy specimens. Bile and perfusate samples were stored at -20°C until analysis. Liver biopsy specimens were stored at -80°C and analyzed within 2 days after the experiment. Rats that were used as liver donors had undergone one of the following pretreatments: (1) intraperitoneal injections of BSO (4 mmol/kg; in saline) administered 24 and 6 hours before the perfusion, and intravenous injection of BSO (2 mmol/ kg; in saline) administered 1 hour before the perfusion via a cannula in the jugular vein; (2) same as (1), but with injection of saline instead of the BSO solution; (3) same as (1), but in addition, administration of BSP (100 #mol/kg; in saline) administered 1 hour before the perfusion via a cannula in the jugular vein; and (4) intraperitoneal injection of DEM (4 mmol/kg) administered 1 hour before the perfusion. Quantitation of BSP and BSP-GSH in Bile, Blood, and Perfusate. BSP and BSP-GSH in bile and blood were quantitated by previously reported HPLC methods, 12but with some modifications. The separation of BSP and BSP-GSH was achieved on a stainless steel column (150 × 3 m m i.d.), packed with 5 #m-particles of Spherisorb ODS2 reversed-phase material. The system comprised an S1000-solvent delivery system, a $2110 low-pressure gradient mixer, a $2000 HPLC controller (Sykam, Gauting, Germany), and a Promis II autosampler (Separations Analytical Instruments, Emmen, The Netherlands). Ultraviolet detection was achieved with a spectrophotometer (Waters Associates, Milford, MA) at a wavelength of 254 nm. Elution solvent A was a 10 mmol/L sodium phosphate buffer, pH 6.0; solvent B was acetonitrile. The following elution profile was used at a constant flow rate of 0.55 mL/ min: isocratic conditions (0% B) were maintained for 2.5 minutes followed by a linear gradient to 30% B in 15 minutes. Hereafter, the system was reverted to the initial conditions in 2.5 minutes and allowed an equilibration interval of 5 minutes for the next injection. Bile and blood samples were processed for HPLC analysis as described previously. 12 BSP in perfusate was quantitated colorimetrically as described previously. 12 Quantitation of GSH in Liver Biopsies. GSH in liver biopsy specimens was determined by the colorimetric method of Ellman 29'3° for the quantitation of nonprotein thiols. Briefly, the liver biopsy specimen was weighed and subsequently homogenized by ultrasonication in 500 #L of ice-cold 5% w/v 5sulfosalicylic acid. These manipulations were performed rapidly and on ice. The samples were centrifuged (10 minutes at 13,000 rpm) and 500 #L of the supernatant was added to 1.0 mL 0.5 mol/L Tris-HC1 buffer, pH 9.0, containing 20 mmol/L edetic acid. The absorbance of this mixture was read at 412 nm before and after addition of 50 #L 10 mmol/L 5,5'dithio-bis-[2-nitrobenzoic acid], dissolved in 0.1 mol/L sodium phosphate buffer, pH 7.0, containing 20 mmol/L edetic acid. A series of GSH solutions in the concentration range of 10 to 250 #mol/L, dissolved in 5% (w/v) 5-sulfosalicylic acid, furnished the calibration curve. Because this colorimetric method does not differentiate between GSH and other hepatic nonprotein thiols, a number of liver biopsies (GSH content, 0.3 to 5 #mol/g) were analyzed on the same day by the colorimetric method and by a GSHspecific HPLC method. This HPLC method, which was modified from that of ()zcimder et al, 31 involves separation of GSH

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from other thiols by ion-pair reversed-phase HPLC and amperometric detection of on-line generated bromine, which oxidizes thiols and related compounds. The sample pretreatment was identical to that described above for the colorimetric method. Good correlation was shown to exist between the colorimetric method and the HPLC method (r 2 = .98). From the small intercept (y = .12 #mol/g), it was concluded that the hepatic nonprotein thiol content consists nearly exclusively of GSH. Therefore, the colorimetric assay essentially provides a good estimation of the hepatic GSH content. RESULTS

I n t r a v e n o u s B S P Infusions in The R a t In Vivo. To c o r r e l a t e t h e r a t e of G S H c o n j u g a t i o n of B S P w i t h t h e h e p a t i c G S H c o n t e n t in t h e r a t in vivo, B S P w a s inf u s e d in r a t s t h a t h a d b e e n p r e t r e a t e d w i t h BSO. Prel i m i n a r y e x p e r i m e n t s h a d s h o w n t h a t inhibition of G S H s y n t h e s i s b y BSO p r e t r e a t m e n t w a s n e c e s s a r y in o r d e r to achieve a sufficiently h i g h e x t e n t of G S H depletion. Therefore, we could not p e r f o r m t h e s e experi m e n t s in r a t s w i t h o u t BSO t r e a t m e n t . Coinfusion of taurocholic acid w a s e s s e n t i a l to k e e p t h e bile flow m o r e or less c o n s t a n t for t h e d u r a t i o n of t h e B S P infusion. Biliary excretion r a t e s of B S P - G S H w e r e m e a s u r e d to estimate the hepatic GSH conjugation rate. B i l i a r y excretion r a t e s of b o t h B S P - G S H a n d B S P w e r e r e l a t i v e l y c o n s t a n t w i t h i n 30 m i n u t e s a f t e r t h e s t a r t of t h e infusion a n d a m o u n t e d to a b o u t 1.1 a n d 0.4 # m o l / m i n / k g , r e s p e c t i v e l y (Fig. 1A a n d 1B); t h u s , the infused B S P w a s c o m p l e t e l y r e c o v e r e d in bile. D u r ing this initial period, c o n c e n t r a t i o n s of B S P in blood g r a d u a l l y i n c r e a s e d f r o m 70 to 120 # m o l / L w i t h o u t r e a c h i n g s t e a d y - s t a t e a n d t h e a p p a r e n t blood c l e a r a n c e of B S P (calculated as B S P infusion r a t e / B S P blood conc e n t r a t i o n ) d e c r e a s e d f r o m 4.8 to 2.8 m L / m i n . T h e hepatic G S H c o n t e n t declined f r o m 4.0 #mol/g to a minim u m level of 0.3 #mol/g in t h e course of 4 h o u r s (Fig. 1C). A d d i t i o n a l a n a l y s i s w i t h a m o r e selective H P L C s y s t e m (see M a t e r i a l s a n d Methods) confirmed this G S H level in t h e liver; it w a s not possible to f u r t h e r d e c r e a s e t h e G S H c o n t e n t (see below). I n a n u m b e r of liver biopsy s p e c i m e n s , we d e t e r m i n e d t h e G S H conc e n t r a t i o n in cytosol, a f t e r c e n t r i f u g a t i n g t h e liver hom o g e n a t e (in 0.15 mol/L KC1, p r e p a r e d w i t h a P o t t e r E l v e h j e m h o m o g e n i z e r ) for 15 m i n u t e s a t 9,000g. T h e r e s i d u a l G S H w a s p r e s e n t in t h e p o s t m i t o c h o n d r i a l fraction a n d did not d e c r e a s e f u r t h e r d u r i n g t h e various e x p e r i m e n t s (see below). W h e n G S H w a s d e p l e t e d to this m i n i m u m level, t h e B S P - G S H excretion r a t e declined f r o m 1,100 n m o l / m i n / k g to a n e w p l a t e a u of 800 n m o l / m i n / k g . T h e excretion r a t e did not d e c r e a s e below this level, in spite of the a p p a r e n t depletion of h e p a t i c G S H . Effect of Multiple BSO Administration a n d DEM. The effect of a m o r e c o m p l e t e inhibition of t h e de novo synt h e s i s of G S H w a s a s s e s s e d b y a d m i n i s t r a t i o n of t h r e e BSO doses o v e r a 24-hour period before the B S P infusion. T h e initial h e p a t i c G S H c o n t e n t in t h e s e experim e n t s (2.1 #mol/g) w a s c o n s i d e r a b l y lower t h a n in exp e r i m e n t s following a single BSO p r e t r e a t m e n t (4.0

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FIG. 1. Biliary excretion ofBSP-GSH (A) and unconjugated BSP (B), and the hepatic GSH content (C) during a 6-hour intravenous (IV) infusion in the rat in vivo under the following conditions: ( - - [] - - ) rats received an IV injection of BSO (2 mmol/kg) 1 hour before the start o f a BSP infusion (1.5 #mol/min/kg); ( - - • - - ) same as the first group, but in addition, rats received an IV injection of DEM (1 mmol/kg) at t = 180 minutes; ( - - A - - ) same as the first group, but in addition, rats received intraperitoneal injections of BSO (4 mmol/kg) 24 and 6 hours before the experiment. Data shown are mean _+ SD; n = 5 for the first group; n = 4 for the second and third groups.

#mol/g), and GSH depletion was attained within the first 2 hours of the infusion (Fig. 1). The biliary excretion rate of BSP-GSH in these rats failed to reach constancy and decreased to 300 to 400 nmol/min/kg within the first 2 hours of the infusion (Fig. 1A). An increased biliary excretion of unconjugated BSP partially compensated for the decreased excretion of BSP-GSH (Fig. 1B). The effect of DEM on the BSP-GSH excretion after depletion of hepatic GSH was studied by intravenous injection of DEM, 180 minutes after the start of the BSP infusion to rats pretreated with a single BSO dose. Immediately upon DEM administration, the biliary excretion rate of BSP-GSH decreased abruptly from 1,100 to 200 nmol/min/kg (Fig. 1A). Concurrently, the biliary excretion of unconjugated BSP increased from 400 to 700 nmol/min/kg (Fig. 1B). DEM administration failed to reduce the GSH content below the minimum level of 0.3 #mol/g (Fig. 1C). An eventual direct effect of DEM on the biliary BSP-GSH excretion was evaluated by administration of DEM to rats that received a BSPGSH infusion (instead o f a BSP infusion) at an infusion rate of 1.1 #mol/min/kg, under otherwise identical conditions. DEM, which was administered after 180 minutes, had no effect on the biliary excretion rate of BSPGSH (Fig. 2). To test whether GSH biosynthesis still could occur in the BSO-pretreated rats, [3~S]-L-cysteine was injected intravenously 3 hours after start of the BSP infusion in rats that had received the multiple BSO-pretreatment. Incorporation of radioactivity into BSP-GSH excreted in bile was measured by HPLC and TLC of the bile samples collected after the [3~S]-cysteine injection. The radioactivity excretion was maximal in the second 15minute bile fraction after injection. Subsequently, it decreased rapidly to a much lower level. Some 2% of

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the radioactivity was excreted in bile over the 3-hour period following the injection. Most of this radioactivity, in particular in the first few fractions, was present in the BSP-GSH conjugate (Fig. 3), as was confirmed by HPLC: some 90% of radioactivity in the first two fractions, decreasing to 50% to 60% in later fractions. The excretion of the BSP-GSH conjugate was more-orless constant during the 3 hours after [35S]-cysteine injection, but the incorporation of the radioactivity showed a sharp peak immediately following the injection, and subsequently decreased to a much lower steady level (Fig. 3). In Situ Rat Liver Perfusions. To assess the role of the liver in the continued biliary excretion of BSP-GSH after depletion of hepatic GSH, BSP conjugation was studied in relation to hepatic GSH content in the in situ single-pass perfused rat liver preparation. The BSP concentration in influent perfusate was 50 pmol/ L, a concentration that was devoid of toxic effects to the liver. 13 The GSH content in livers of nonpretreated rats declined from 4.7 to 3.3 #mol/g during the course of the perfusion. The hepatic GSH content in the multiple BSO pretreated group was much lower and decreased from 1.8 to 0.9 #mol/g (Fig. 4C). Yet, the steady-state biliary excretion rates of BSP-GSH (approximately 160 nmol/min) and unconjugated BSP (approximately 5 nmol/min) were not different between these two experimental groups (Fig. 4A and 4B). The hepatic extraction ratio of BSP from perfusate was the same for these two experimental groups: the value (approximately 0.3) remained contant during the course of the perfusion (Fig. 4D). Mass balance was attained at steady-state: the uptake rate of BSP from the perfusate equalled the total biliary excretion rates of BSP-GSH and BSP. The decrease in hepatic GSH content during the perfusion

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(8 to 14 pmol per liver) equalled or exceeded the total amount of BSP-GSH excreted in bile (8 to 9 #mol), both in the nonpretreated group and the multiple BSOpretreated group. Because multiple BSO pretreatment alone failed to deplete the hepatic GSH content completely, an additional series of experiments were conducted in which hepatic GSH was further depleted with an intravenous bolus dose of BSP (100 #mol/kg), administered 1 hour before the start of the perfusion. The use of BSP as a depleting agent did not interfere with its use as a substrate during the perfusion, because the bolus dose was almost completely eliminated at the start of the perfusion (data not shown). The combination of multiple BSO-pretreatment plus BSP-administration effectively decreased the hepatic GSH content to its minireal level (Fig. 4C). Yet, the biliary excretion rate of BSP-GSH reached an average plateau level of as much as 70 nmol/min (Fig. 4A). However, large interindividual differences were found to exist at this level of depletion. The biliary excretion of unconjugated BSP amounted to approximately 8 nmol/min (Fig. 4B). The hepatic extraction ratio declined from 0.3 to 0.2 in the course of the perfusion (Fig. 4D). The decrease in hepatic GSH during the perfusion (2 #mol/g) was less than the total amount of BSP-GSH excreted in bile (3 to 4 #mol/g). Depletion of hepatic GSH without inhibition of GSH biosynthesis was achieved by pretreatment of rats with an intraperitoneal injection of DEM; this pretreatment also depleted the hepatic GSH content to 0.3 #mol/g (Fig. 4C). The biliary excretion rate of BSP-GSH slowly increased to approximately 70 nmol/min (Fig. 4A). The hepatic extraction of BSP from perfusate declined from 0.3 to 0.2 in the course of the perfusion (Fig. 4D). The biliary excretion ofunconjugated BSP (8 nmol/min) was slightly increased, compared with the nonpretreated and multiple BSO-pretreated group (Fig. 4B). The decrease in hepatic GSH (0.4 pmol per liver) in the course of the perfusion was less than the total amount of BSPGSH excreted in bile (3 to 4 pmol). DISCUSSION

In order to study the relation between GSH conjugation rate of BSP and hepatic GSH content, the biliary excretion of BSP-GSH and the hepatic GSH content were monitored simultaneously during intravenous infusions with BSP in the rat in vivo, and during rat liver perfusions with BSP-containing perfusate. The rats used in these experiments were extensively pretreated with BSO, an inhibitor of the de novo synthesis of GSH. The hepatic GSH content was typically depleted to a minimum of approximately 0.3 #mol/g. This remainder could not be attributed to mitochondrial GSH, as the same amount was recovered in the postmitochondrial fraction prepared from liver biopsies at this level of GSH depletion. However, it is unlikely that this residual amount of GSH was available for conjugation with BSP, because both BSP and DEM failed to deplete it.

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SNEL, PANG, AND MULDER

HEPATOLOGY May 1995

It is possible that this represents GSH of nonparenchymal cells that cannot be used for BSP conjugation. Surprisingly, in the rat in vivo, the biliary excretion of BSP-GSH continued at a high rate (800 nmol/min/ kg) after virtually complete depletion of hepatic GSH. One possible explanation would be that the conjugation rate of BSP exceeded the T~x for biliary excretion of BSP-GSH, so that BSP-GSH accumulated in the liver; BSP-GSH might subsequently have been released into bile when conjugation itself was impaired by GSH depletion. However, intrahepatic accumulation of BSPGSH is inconsistent with the rapid achievement of steady-state in BSP-GSH excretion and with the rapid decline of BSP-GSH excretion rates, observed on termination of the BSP infusion after 6 hours (data not shown). Moreover, the Tm~xfor biliary excretion of BSPGSH was not attained in these infusions: higher BSPGSH excretion rates were observed when the BSP infusion rate was further increased (data not shown). In addition, the coinfusion of taurocholic acid reduces the

intrahepatic storage capacity of BSP-GSH and raises the Tma x for biliary excretion of BSP-GSH. 32'33 The high rate of residual GSH conjugation after depletion of hepatic GSH suggested that GSH was still available for conjugation of BSP, presumably as a result of de novo synthesis of GSH in the liver (or possibly in extrahepatic tissues). Such an explanation implies that the de novo synthesis of GSH was not fully inhibited by a single BSO pretreatment. Therefore, the experiments were repeated in rats that had received 3 BSO doses over a 24-hour period. BSP-GSH excretion rates after depletion of hepatic GSH were lower in the multiple BSO-pretreated rats than in the rats that had received a single BSO dose but still amounted to 0.3 to 0.4 #mol/min/kg. This suggested that multiple BSO pretreatment still allowed for some residual GSH synthesis. This was confirmed by the finding that PS]cysteine is still readily incorporated into BSP-GSH excreted in bile in these rats. Intravenously injected [3~S]-cysteine is very rapidly eliminated from plasma

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HEPATOLOGY Vol. 21, No. 5, 1995

in our rats34: after 5 minutes only some 15% of the original level of radioactivity is found in plasma. The peak of [35S]-cysteine incorporation in the second bile fraction collected after injection seems to reflect this pattern. The fact that the incorporation peaks very early after [35S]-cysteine injection suggests that the biosynthesis of GSH under our conditions of a high GSH requirement for BSP conjugation is still very rapid, in spite of the BSO treatment. The finding that the specific radioactivity of BSP-GSH peaks briefly and decreases subsequently again (Fig. 3) can be explained if we assume that GSH biosynthesis continues during the BSP infusion in our BSO-treated rats, using the available cysteine pool. Immediately on injection of [35S]cysteine, there will be rapid mixing of the radioactive cysteine with a rapidly exchanging cysteine pool, that will be labeled at a high specific radioactivity. Subsequently, the [35S]-cysteine will further equilibrate with the (total) body pool of free cysteine, leading to a much lower specific radioactivity of the cysteine pool that is used later for GSH biosynthesis. In summary, these results confirm that in spite of extensive BSO treatment, at least in some compartments GSH biosynthesis is still possible. The effect of DEM on BSP-GSH excretion after depletion of hepatic GSH was evaluated by administration of an intravenous dose of DEM at t = 180 minutes. Immediately after DEM administration, the BSP-GSH excretion rate decreased drastically, most likely as a result of competition between BSP and DEM for the immediate consumption of newly synthesized GSH. Competition between BSP-GSH and DEM-GSH conjugates for hepatobiliary transport carriers must be excluded, because the BSP-GSH excretion was not depressed by DEM during an infusion with synthetic BSP-GSH. This possibility was assessed because intraperitoneal administration of a higher dose of DEM (4.3 mmol/kg) has been shown to reduce the biliary excretion of infused BSP-GSH. 3~ Although the liver is the predominant organ involved in uptake and GSH conjugation of BSP, 1~'16 it cannot be excluded that GSH in extrahepatic tissues is also used for conjugation of BSP. To assess the role of the liver exclusively, we also studied the rate o f G S H conjugation of BSP in relation to hepatic GSH content in the in situ perfused rat liver preparation. Maximal rates of GSH conjugation of BSP were attained in nondepleted (4 to 5 #mol/g) and partially depleted (1 to 2 #tool/g) livers. Under these conditions, the GSH conjugation of BSP was not rate-limited by GSH availability. Although there were erythrocytes in this perfusion system, we have shown that incubation for several hours of BSP with erythrocytes did not result in a measurable degree of conjugation (Snel et al ~2 and unpublished results); therefore, all conjugation must occur in the liver in this system. The biliary excretion of BSP-GSH was sustained at about half maximal rates when the hepatic GSH content was depleted to the minimum level of 0.3 pmol/g by pretreatment of the rats with DEM or with the combination of multiple BSO administration

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and a BSP bolus dose. Evidently, the cosubstrate-Km for GSH conjugation of BSP in the liver is very low. The correlation between the steady-state biliary excretion rate of BSP-GSH and the hepatic GSH content at the Tmld of the corresponding bile collection interval shows a cosubstrate-Km value of 0.2-0.3 #mol/g (Fig. 5). This is lower than the cosubstrate-Km for GSH conjugation of intravenously infused 2-bromoisovalerylurea, as determined in the rat in vivo. 1 In liver perfusion experiments with rats that were pretreated with the combination of BSO and BSP, the amount of BSP-GSH excreted in bile during the perfusion (4 #mol) exceeded the decrease in liver GSH content (2 #tool). The discrepancy was much larger in BSP infusion experiments in the rat in vivo, following multiple BSO pretreatment: the amount of BSP-GSH excreted in bile between t = 180 and t = 360 min (14 pmol) greatly exceeded the decrease in hepatic GSH content (< 1 pmol). This indicates that de novo synthesis of GSH in the liver (and possibly also in extrahepatic tisues) continued, in spite of the BSO pretreatment. This lack of sensitivity to BSO may be restricted to a GSH pool that is used for conjugation of BSP: even a single BSO pretreatment effectively inhibited the conjugation of BSO, as observed in infusion experiments with 2 BSO, conducted under identical conditions as described above for BSP. 1 In this article, we have shown that the cosubstrateKm for GSH conjugation of BSP in the liver is very low (0.2 to 0.3 pmol/g). In combination with the incomplete inhibition of the GSH synthesis by BSO, the conjugation of BSP is not much impaired, in spite of severe GSH depletion. The significance may become apparent if other, toxic substrates are enzymatically conjugated

1394

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with the same efficiency as BSP; in that case, severe GSH depletion in the liver need not necessarily potentiate their toxicity, because their conjugation may still be little impaired. REFERENCES

1. Polhuijs M, Lankhaar G, Mulder GJ. The relationship between glutathione content in the liver and glutathione conjugation rate in the rat in vivo. Biochem J 1992;285:401-404. 2. te Koppele JM, Dogterom P, Vermeulen NPE, Meijer DKF, Van der Gen A, Mulder GJ. Pharmacokinetics and stereoselectivity of metabolism and excretion of ~-bromoisovalerylurea in vivo and in the perfused liver. J Pharmacol Exp Ther 1986;239:905914. 3. Krebs JS, Brauer RW. Metabolism of sulfobromophthalein sodium (BSP) in the rat. Am J Physiol 1958; 194:37-43. 4. Grodsky GM, Carbone JV, Fanska R. Identification of metabolites of sulfobromophthalein. J Clin Invest 1959;38:1981-1988. 5. Combes B, Stakelum GS. Conjugation of sulfobromophthalein sodium with glutathione in thioether linkage by the rat. J Clin Invest 1960;39:1214-1222. 6. Whelan G, Hoch J, Combes B. A direct assessment of the importance of conjugation for biliary transport of sulfobromophthalein sodium. J Lab Clin Med. 1970;75:542-557. 7. Sano K, Kinosita I, Mihara R, Ikegami Y, Uesugi T. High-performance liquid chromatographic determination of sulfobromophthalein and its conjugates. J Chromatogr Biomed Appl 1992;578:63-70. 8. Scharschmidt BF, Waggoner JG, Berk PD. Hepatic organic anion uptake in the rat. J Clin Invest 1975;56:1280-1292. 9. Min AD, Johansen KL, Campbell CG, WolkoffAW. Role of chloride and intracellular pH on the activity of the rat hepatocyte organic anion transporter. J Clin Invest 1991;87:1496-1502. 10. Potter BJ, Blades BF, Shepard MD, Thung SM, Berk PD. The kinetics of sulfobromophthalein uptake by rat liver sinusoidal vesicles. Biochim Biophys Acta 1987;898:159-171. 11. Jansen PLM, Groothuis GMM, Peters WH, Meijer DKF. Selective hepatobiliary transport defect for organic anions and neutral steroids in mutant rats with hereditary conjugated hyperbilirubinemia. HEPATOLOGY1987; 7:71-76. 12. Snel CAW, Zhao Y, Mulder GJ, Pang KS. Methods for the quantitation of bromosulfophthalein and its glutathione conjugate in biological fluids. Anal Biochem 1993;212:28-34. 13. Zhao Y, Snel CAW, Mulder GJ, Pang KS. Localization of glutathione conjugation activities towards bromosulfophthalein in perfused rat liver: studies with the multiple indicator dilution technique. Drug Metab Dispos 1993;21:1070-1078. 14. Chen EH, Gumucio JJ, Ho NH, Gumucio DL. Hepatocytes of zones 1 and 3 conjugate sulfobromophthalein with glutathione. HEPATOLOGY1984;4:467-476. 15. Klaassen CD. Plasma disappearance and biliary excretion ofsulfobromopthalein and phenol-3,6-dibromphthalein disulfonate after microsomal enzyme induction. Biochem Pharmacol 1970; 19:1241-1249. 16. Klaassen CD. Extrahepatic distribution of sulfobromophthalein. Can J Physiol Pharmacol 1975;53:120-123. 17. Yam J, Reeves M, Roberts RJ. Comparison of sulfobromophthalein (BSP) and sulfobromophthalein-glutathione(BSP-GSH) disposition under conditions of altered liver function and in the isolated perfused rat liver. J Lab Clin Med 1976;87:373-383.

HEPATOLOGYMay 1995 18. Gregus Z, Klaassen CD. Role ofligandin as a binding-proteinand as an enzyme in the biliary excretion of sulfobromophthalein. J Pharmacol Exp Ther 1982;221:242-246. 19. Foliot A, Touchard D, Celier C. Impairment of hepatic glutathione-S-transferase activity as a cause of reduced biliary sulfobromophthalein excretion in clofibrate treated rats. Biochem Pharmacol 1984;33:2929-2834. 20. Fehring SI, Ahokas JT. Effect of the glutathione-S-transferase inhibitor, tienilic acid, on biliary excretion of sulfobromophthalein. Chem Biol Interactions 1989;69:23-32. 21. James SI, Ahokas JT. Modulation of sulfobromophthalein excretion by ethacrynic acid. Xenobiotica 1992;22:1433-1439. 22. Aza MJ, Gonzalez J, Esteller A. Effects of diethyl maleate pretreatment on biliary excretion and choleretic action of sulfobromophthalein in rats. Arch Int Pharmacodyn 1986;281:321-331. 23. Varga F, Fischer E, Szily TS. Biliary excretion ofbromsulphthalein and glutathione conjugation of bromsulphthalein in rats pretreated with diethyl maleate. Biochem Pharmacol 1974;23:26172623. 24. Priestly BG, Plaa GL. Sulfobromophthalein metabolism and excretion in rats with iodomethane-induced depletion of hepatic glutathione. J Pharmacol Exp Ther 1970; 174:221-231. 25. Klaassen CD. Hepatic excretory function in the newborn rat. J Pharmacol Exp Ther 1973;184:721-728. 26. Theilmann L, Stollman YR, Arias IM, WolkoffAW. Does Z-protein have a role in transport of bilirubin and bromosulfophthalein by isolated perfused rat liver? HEPATOLOGY1984;4:923-926. 27. Mulder GJ, Scholtens E, Meijer DKF. Collection of urine and bile from the rat. Methods Enzymol 1981;77:21-30. 28. Pang KS, Cherry WF, Accaputo J, Schwab AJ, Goresky CA. Combined hepatic arterial-portal venous and hepatic arterial-hepatic venous perfusions to probe the abundance of drug metabolizing activities: Perihepatic O-deethylation activity for phenacetin and periportal sulfation activity for acetominophen in the once through rat liver preparation. J Pharmacol Exp Ther 1988; 247:690-700. 29. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-77. 30. Sedlak J, Lindsay RH. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 1968;25:192-205. 31. (~zcimder M, Louter AHJ, Lingeman H, Voogt WH, Frei RW, Bloemendal M. Determination of oxidized, reduced and proteinbound glutathione in eye lenses by high-performance liquid chromatography and electrochemical detection. J Chromatogr 1991;570:19-28. 32. Gregus Z, Fischer E. Effect of sodium taurocholate on hepatic uptake and biliary excretion of organic anions in rats. Arch Int Pharmacodyn Ther 1979;240:180-192. 33. Cadranel JF, Dumont M, Mesa VA, Degott C, Touchard D, Erlinger S. Effect of chronic administration of cyclosporin A on hepatic uptake and biliary secretion in bromosulfophthalein in rats. Dig Dis Sci 1991;36:221-224. 34. Mulder GJ. Dietary determinants of drug response: cysteine and methionine in the diet and cofactor availability for conjugation of xenobiotics with sulfate, glutathione and taurine. In: Plaa GL, du Souich P, and Erill S, eds. Interactions between drugs and chemicals in industrialized societies. Amsterdam: Elsevier, 1987:71-84, 35. Combes B, Backof B. Effect of diethyl maleate on the biliary excretion rate of infused sulfobromophthalein-glutathione. Biochem Pharmacol 1982;31:2669-2674.