BiochemicalPharmacolo~, Vol. 43, No. 3, pp. 417-482, 1992. Printed in Great Britain.

am-2952/E ss.00 + 0.00 @ 1992. Pergmon Press plc

INHIBITION OF BILIARY EXCRETION OF INDOCYANINE GREEN BY THE THIOL-OXIDIZING AGENT, DIAZENEDICARBOXYLIC ACID BIS[N,N’-DIMETHYLAMIDE]
HARUHIKO YOSHIDA,* MAKOTO IIJIMA, YUKIO KURONUMA and TAKASHI HARADA Second Department of Internal Medicine, Dokkyo University School of Medicine, Mibu-Kitakobayashi, Tochigi 321-02, Japan

(Receioed 30 July 1991; accepted 17 October 1991) Abstract-Biliary excretion of Indocyanine green (ICG) in Sprague-Dawley rats during constant intravenous infusion of the dye in uiuo was inhibited by intraneritoneally administered diaxenedicarboxvlic acid bis[N,N’-dimethylamide] (diamide, 0.5 mmol/kg body wt), a ghrtathione-specific thiol-oxidi&ng agent. Sienificant inhibition of ICG excretion was observed also when ICG was iniected ranidlv 90 min ager d&ride administration. Disappearance of ICG from the plasma was not affected by diamide. Oxidized glutathione in bile increased transiently following diamide administration but returned to the basal level within 30 min. Hepatic concentrations of reduced and oxidized glutathione were not different from those of controls when determined 90 min after diamide administration. The inhibition of ICG excretion was completely prevented by subsequent administration of dithiothreitol (0.5 mmol/kg) 30min after that of diamide. The results, therefore, suggest that the biiary excretion of ICG was inhibited by secondary changes in the redox status of thiols in hepatocytes caused by a transient increase in oxidized glutathione. _

Indocyamnine green (ICGt) is carried in the plasma bound to lipoproteins [l, 21 and is extracted exclusively by the liver, to be excreted into bile without metabolic modifications [3]. Several proteins on the sinusoidal membranes of hepatocytes have been indicated to be transporters of several organic anions [4-61, and they may also transport ICG across the membrane. However, little is known about the mechanism of biliary excretion of the dye. We have found that a thiol-oxidizing agent, diamide [7-91, markedly inhibits the biliary excretion of ICG in rats when administered i.p., and in this study the mechanism of this inhibition was investigated.
MATERIALS AND

distilled water (18 mg/mL) and diluted with 4 vol. of physiological saline. The solution was infused via PE-50 tubing carmulated into the jugular vein at a constant rate of O.O5mL/min/kg body wt (i.e. 180 pg ICG/min/kg body wt). The bile was collected at lo-min intervals. After 6Omin of infusion, the biliary excretion rate of ICG approached a plateau and diamide (0.5 mmol/kg dissolved in mL/kg physiological saline) was administered i.p. Changes in bile flow and KG excretion were observed for the next 90 mm while ICG was infused at the same rate.
Effect of diamide on glutathione in the bile and liuer. Since the reduced form of glutathione (GSH)

METHODS

Diamide and acivicin were purchased from the Sigma Chemical Co. (St Louis, MO, U.S.A.). ICG was purchased from the Daiichi Seiyaku Co. (Tokyo, Japan). Other chemicals used were of analytical grade. Sprague-Dawley rats (weighing 300-350 g), fed ad lib. on conventional rat food, were used in all experiments. The common bile duct was cannulated with PE10 polyethylene tubing while the rat was anesthetized with pentobarbital (50mg/kg body wt). The bile volume was measured gravimetrically using 1 as the specific gravity. KG concentration was determined spectrophotometrically by absorbance at 805 nm. Constant infusion of KG. ICG was dissolved in * Corresponding (81) 282-86-6481. 7 Abbreviations: diaxenedicarboxylic reduced glutathione;
author. Tel. (81) 282-87-2147; FAX ICG, Indocyanine green; diamide, acid bis[N,N’-dimethylamide]; GSH, GSSG, oxidized glutathione. 477

is rapidly auto-oxidized to its oxidized form (GSSG) in untreated bile [lo], the effects of diamide on GSH and GSSG in the bile were examined in another series of experiments. An irreversible y-glutamyltranspe tidase inhibitor, acivicin (20 pmol/kg in 0.4 mL Pkg distilled water), was administered retrogradely into the bile duct to inhibit intraductular hydrolysis of glutathione [ll, 121. Bile was collected in tubes, each containing 0.2mL of 5% metaphosphoric acid, to inhibit oxidation of GSH [lo]. Diamide (0.5 mmol/kg i.p.) was administered and the bile was collected for the next 90min. At the end of each experiment, the liver was excised and homogenized with phosphate-buffered 0.25 mol/L sucrose ( pH 7.4). The homogenate (25% w/v) was mixed with a volume of 10% trichloroacetic acid and the deproteinized supernatant was obtained by centrifugation [ 131. Glutathione concentration in bile and liver was determined by the method of Tietze [13] as total glutathione (GSH + GSSG). GSSG concentration was determined independently (as GS equivalent)

Diamide (0. ICG was infused at a rate of lgOpg/kg/ min from t = 0 to t = 150. Vertical lines indicate 1 SD.p. Bile flow rates also decreased following diamide administration. Bolus ICG injection studies were performed to determine the duration and reversibility of the effect of diamide. In group B. N = 4) and controls (broken line.5 mmol/kg.) was administered at t = 60. Rats in group A demonstrated a marked decrease in bile flow rate following diamide administration. biliary excretion rates of ICG began to decrease 5 min after the administration of diamide and remained lower than the maximum level throughout the observation period (Fig. Plasma disappearance of KG. 3). ICG was dissolved in distilled water (5 mg/kg in 1 mL/kg) and injected rapidly into the jugular vein. blood (0. Bile was collected at lO-min intervals. 4). ICG concentration in the bile was lower in group A (but not in group B) than .5 mmol/kg i. YOSHIDA et al. physiological saline instead of diamide. 1). rats were pretreated with diamide (0. GSH concentration was calculated as the difference between the two values. i. Changes in the excretion rate of GSH were not significant.3 mL each) was drawn from the femoral vein 5. while that in group D was 73% (Fig. Biliary excretion rates of GSSG increased during the first 20 min after diamide administration but returned subsequently to basal level (Fig. and the exponential regression was calculated using the least-squares method. Cumulative ICG excretion after 90 min was 44% of the administered dose in rats in group A. 5). Bile was collected for 90 min after ICG injection. Rats were divided into four groups. those in group D. Top: bile flow rates of diamide-treated rats (solid line. The biliary excretion rates of ICG after bolus injection were lower in group A than in group D (controls) throughout the observation period (Fig. Bottom: biliary excretion rates of KG of diamide-treated rats (solid line) and controls (broken line).418 H. 10.5 mmol/kg in mL/kg physiological saline. GSH and GSSG concentrations in the liver determined 90 min after diamide administration did not differ from control levels (Table 1).p.p. The serum was separated by centrifugation and the concentration of ICG was measured. ICG excretion rates did not decrease in group B. The time-course of the disappearance of ICG from the plasma was revealed to be monoexponential. Bolus injection of ZCG.) was also administered 60min prior to the ICG injection. with the maximum decrease between 15 and 20min after administration (Fig. 1. Effect of diamide on bile flow rate and ICG excretion. i. by the method of Griffith [14] using 4-vinylpyridine to mask GSH at the final concentration of 0. N = 4). However. and 15 min after the injection of ICG.5% (v/v). 2). In groups A and B. Rats in group C received dithiothreitol only.) 90 min prior to the ICG injection. In several experiments with bolus ICG injection. dithiothreitol (0. diamide iv) 0 20 40 80 80 100 120 140 time (min) Fig. RESULTS In the constant ICG infusion studies. ICG (180pglkglmin.

6). a non-selective thiol-reducing agent. therefore.98 f 0. Diamide (0. i. Biliary excretion rates of GSH (open bars) and GSSG (hatched bars) are shown. This indicates that the oxidixing effect of diamide on GSH was almost completely eliminated 90min after the administration of diamide. I 479 ip) f time (mtn) Fig. Values are means of four experiments with diamide-treats rats (bars with solid line) and controls (bars with broken line). The disappearance DISCU~IO~ The time-course of the disappearance of ICG from the plasma was not altered by diamide treatment. On the other hand. However.08 * 0. Bile was collected at IO-min intervals.p. Table 1.5mmoVkg. in group D between 5 and 30 min atier ICG injection of ICG from the plasma in group A was not different to that in group D (Table 2). ICG excretion was still significantly lower in diamide-treated rats than in controls when injected at that time.62 f 0. values are means and SD). and the hepatic levels of GSH and GSSG determined 90 min after diamide treatment were not different from those of controls. The effect of diamide on ICG excretion was completely antagonized by the subsequent administration of dithiothreitol. GSSG produced in the cell is avidly reduced to GSH by the cellular GSSG reduct~~NADPH system and excessive GSSG. Thus. GSSG in the bile had returned to its normal level 30min after diamide administration.29 GSSG (pmol/g liver) 0. 30min after diamide. Effect of diamide on biliary excretion of glutathione.Smmoi/kg. the kinetic studies by Kosower et al. (Fig. Hepatic concentration of GSH and GSSG GSH &mol/g liver) Diamide-treated rats Controls 4.51 4. suggesting that diamide affected not only the redox status of cellular ~uta~one but also that of other thiols.Values are means + SD. Diamide oxidizes GSH to GSSG chemically (uide infra). [9] demonstrated that diamide reacts with GSH much more rapidly than it does with other substances. when GSSG in the bile had already returned to its basal level. Nevertheless. cellular GSSG increased by diamide is unlikely to have directly affected ICG excretion. broken line. diamide-treated.06 0.Bihary excretion of ICG diamide (0. indicating that diamide inhibited the biliary excretion of ICG from the liver without affecting the hepatic extraction of the dye. controls. protein thiol-disulfide status in the cell. N = 4. 90 min prior to the preparationof liver homogenate.08 Hepatic concentration of GSH and GSSG.5 mmol/kg) was administered i. We.) was admi~te~d at t = 0. known to be an important determinant of enzyme function and cell homeostasis. indicating that the conversion of GSH to GSSG is the most important immediate consequence of diamide treatment. Diamide oxidizes not only GSH but also several other natural electron donors in uifro f8].11 + 0. conclude that ICG excretion was inhibited by secondary changes in the redox status of cellular . if any. is actively excreted into the bile [15-171. 2. Diamide (O. Changes in bile flow fate are shown (solid line. may be disturbed by changes in the redox status of glutathione through thiol-disuhide exchanges catalysed by thiol-transferase [18]. One standard deviation was less than 10% of corresponding mean values.p.

4. 3. However. N = 4): received physiological saline.p. (A) (open circles. Values are the means of cumulative biliary excretion as percentages of injected dose. For experimental protocol and symbols. it is unlikely that ICG crosses plasma membranes by simple diffusion. Akerboom et al. as suggested by the selective uptake by hepatocytes. Values are means and SD. ‘g 30. the inhibition of ICG excretion by diamide was not caused by changes in the putative canalicular transporter for multiple organic anions. It is possible that the transporter has affinity also for ICG.24]. ICG (5 mg/kg) was rapidly injected via the jugular vein at t = 0. Thus. 5 2 20.480 H. 0”’ 0 10 ( 20 30 40 50 60 70 90 90 time (mid Fig. thiols other than glutathione due to the transient increase in GSSG. the effect of diamide on the biliary excretion of the sulfobromophthalein-glutathione conjugate or bilirubin is transient and disappears when GSSG in the bile returns to the normal level (unpublished observations). and is involved in the biliary excretion of the dye. Cumulative ICG excretion after bolus injection. at t = -60). another organic anion. [19] reported the inhibition of biliary excretion of taurocholate by diamide and other agents as occurring by similar mechanisms. (B) (open triangles. Bile was collected at 5-min intervals. 3. whether diamide was administered during KG transport . :. B p 40.5 mmol/kg i. Those thiols in the hepatocytes appeared to be essential for the biliary excretion of ICG.p. An ATP-dependent anion transporter on the canalicular membrane has been indicated as being responsible for the canalicular of these substances and is defective in the mutants [23. Biliary excretion of several organic anions such as bilirubin glucuronides. YOSHIDAet al. 60 2 . N = 6): pretreated with diamide (0. N = 6): pretreated with both dlamide (same as in A) and dithiothreitol (OS mmol/kg i. and (D) (closed circles. :: 10. time (mid Fig. Biliary excretion rate of ICG after bolus injection. Although ICG is relatively lipophilic. N = 4): pretreated with dithiothreitol only. sulfobromophthalein and GSSG is impaired in two mutant rat strains [2&22]. (C) (closed triangles. The inhibition of ICG excretion by diamide was associated with marked decreases in bile flow rate.$ 60. see legend to Fig. at t = -90).

However. Diamide (0. As shown in Fig.5 mmol/kg) was administered i-p. and 15 min after bolus injection (5 mg/kg) assuming C = C. the initial decrease of bile flow following ICG injection was partially reversed by 25min when the ICG concentration in the hepatocytes was presumably lower than it was initially but was still increasing in the bile as shown in Fig. therefore. ICG concentration in bile after bolus injection. 90 min prior to the KG injection.Biliary excretion of KG ICG (5mg/kg. 6. Changes in bile flow rate after bolus injection of ICG. where C is the ICG concentration at time t and Co is the apparent initial concentration. ICG accumulated in hepatocytes will decrease bile formation. 6. composition in segments 90min prior to the bolus injection of it is possible that diamide altered the of bile. The partitioning to biliary lipid particles is different between ICG and more Table 2.247 (0. Unfortunately no technique is currently available to . rather unlikely that the precipitation of ICG in the biliary tree played a major role in decreasing ICG excretion. Ranges in parentheses arc those of mean6 f 1 SD.36) (“Yz27g) Monoexponential regression was calculated from the serum concentration of ICG 5..exp(-r In 2/T*). see legend to Fig. ICG at high hepatic concentrations is itself anti-choleretic [26-281 without affecting bile salt excretion [29] and toxic effects of ICG on mitochondrial functions in vitro have been reported [30]. 3. 3. Disappearance of ICG from plasma Co (mg/mL) Diamide-treated rats Controls 0. see legend to Fig. Thus. iv) J 481 0’ -10 ’ 0 10 20 30 40 50 60 70 60 time (mid Fig. 20 30 40 50 60 70 80 90 time (mid Fig.89 (4*1:zo1) (4. diamide may selectively inhibit ICG excretion by causing cholestasis in the biliary tree if the lipid composition of bile is altered by diamide so that the solubility of ICG only is affected. 5.09-5. Values are mean and SD (not for triangles). 10. predisposing ICG to precipitate of the biliary tree and causing cholestasis outside the hepatocytes.2iiao9) TW Wn) 4. For experimental protocol and symbols. 5. hydrophilic organic anions [25]. Values are means and SD. For experimental protocol and symbols. Thus. It is. Thus. N = 4. infusion or ICG.

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