Dibromosulfophthalein Research Articles

Effects of P-glycoprotein and Mrp2 inhibitors on the hepatobiliary disposition of Rhodamine 123 and its glucuronidated metabolite in isolated perfused rat livers

The hepatobiliary disposition of rhodamine 123 (RH-123) and its glucuronidated (RH-Glu) and deacylated (RH-110) metabolites were studied in an isolated perfused rat liver (IPRL) model in the presence and absence of P-glycoprotein (P-gp) and Mrp2 inhibitors. A single dose (180 µg) of RH-123 was added to a recirculating perfusate in the absence (Control) or presence of cyclosporine A (CyA) or dibromosulfophthalein (DBSP) in the perfusate. Serial (0–90 min) perfusate and bile and terminal liver samples were collected for analysis by HPLC. In the Control livers, 25.4 ± 2.2% (mean ± SD) of the dose was recovered as RH-123 (11.7 ± 2.0%) and RH-Glu (13.2 ± 0.9%) in the bile. Whereas CyA substantially (90%) reduced (p < 0.001) the biliary excretion of RH-123 without affecting the excretion of RH-Glu, DBSP reduced the biliary excretion of RH-Glu by >80% (p < 0.001) with no effect on the biliary excretion of RH-123. Mass balance studies showed that DBSP, in addition to reducing the biliary clearance of RH-Glu, also strongly inhibited the glucuronidation of RH-123, an effect that was confirmed in vitro using the glucuronidation marker umbelliferone. It is concluded that the use of RH-123 in an IPRL model may serve as a dual marker for the determination of the altered functions of P-gp and/or Mrp2. © 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:455–466, 2010

Activation of plasma membrane reduced glutathione transport in death receptor apoptosis of HepG2 cells

Cells undergoing apoptosis release reduced glutathione (GSH) into the extracellular space; however, the physiological significance and the mechanism behind the GSH export remain unclear. The present study demonstrates that GSH is released by HepG2 cells undergoing Fas, tumor necrosis factor α (TNFα), or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-stimulated cell death. GSH release was observed at times when extracellular lactate dehydrogenase (LDH) activity and propidium iodide (PI) incorporation were low, suggesting that the GSH release does not occur because of nonspecific cell damage, but is occurring through a specific transport system. Caspase 3-like proteases were activated before GSH was released, indicating that protease may be involved in signaling GSH release. To investigate the mechanism of GSH release, studies were performed in the presence of GSH transport inhibitors, as well as 25 mM GSH in the media. Two organic anion transporter inhibitors, probenecid and dibromosulfophthalein (DBSP), were effective in inhibiting Fas-stimulated GSH release. The addition of 25 mM GSH to the extracellular media also prevented the loss of intracellular GSH and delayed cell death. These findings suggest that an organic anion transporter is involved in GSH release during apoptosis, and that maintenance of intracellular GSH levels during apoptosis provides protection for the cell.

Role of Rat Multidrug Resistance Protein 2 in Plasma and Biliary Disposition of Dibromosulfophthalein after Microsomal Enzyme Induction

We have previously demonstrated that microsomal enzyme inducers phenobarbital (PB) and pregnenolone-16α-carbonitrile (PCN), but not 3-methylcholanthrene (3-MC) and benzo(a)pyrene (BaP), increase expression and function of rat Multidrug Resistance Protein 2 (Mrp2), a canalicular organic anion transporter. Thus, the purpose of this study was to determine whether Mrp2 protein induction alters the biliary and plasma dispositions of dibromosulfophthalein (DBSP). After four daily ip injections of PB, PCN, 3-MC, BaP, or vehicle, DBSP (100 mg/kg) was injected iv and was measured in blood and bile over a 40-min period. PB and PCN significantly enhanced plasma disappearance and biliary excretion of DBSP, whereas 3-MC and BaP did not. To determine whether the enhanced plasma disappearance and biliary excretion was entirely due an increase in Mrp2, PCN was also administered ip daily for 4 days to Mrp2-null Eisai hyperbilirubinemic (EHBR) rats and then injected iv with DBSP. PCN significantly increased plasma DBSP disappearance in EHBR rats during early time intervals (2–20 min), but not at later time intervals (25–40 min). PCN did not increase DBSP biliary excretion in EHBR rats, but actually decreased it at later time intervals. In summary, the increase in Mrp2 protein after microsomal enzyme induction is responsible for increased biliary DBSP excretion. Furthermore, the increase in Mrp2 protein after microsomal enzyme induction is not responsible for the enhanced plasma DBSP disappearance at early time points, yet may influence plasma DBSP disappearance at later time points. This study also demonstrates the importance of compensatory hepatic transporters in eliminating DBSP by alternative pathways other than Mrp2.

Competition of pravastatin and dibromosulfophthalein on the electroneutral and electrogenic tetrabromosulfophthalein uptake in rat liver

The effect of pravastatin (PVST) and dibromosulfophthalein (DBSP) on the hepatic uptake of bromosulfophthalein (BSP) was investigated in liver plasma membrane vesicles enriched on basolateral fraction. BSP uptake was measured either in the absence (electroneutral component) or in the presence of a membrane potential (electrogenic component). PVST and DBSP inhibited both components of BSP uptake. The Dixon and Cornish-Bowden plot of the data revealed a competitive type of inhibition for PVST and DBSP on the electroneutral portion of BSP uptake. The inhibition constants ( K i, mM) were 0.650±0.050 for PVST and 0.020±0.005 for DBSP. These data indicate that PVST, DBSP and BSP share a common electroneutral transport system at basolateral liver plasma membrane. On the contrary, PVST exerted a non-competitive type of inhibition on the electrogenic component of BSP uptake ( K i=0.210±0.040 mM) and DBSP produced a mixed type of inhibition ( K i=0.018±0.002 mM). These results indicate that PVST does not compete with the electrogenic BSP carrier system at the level of the substrate binding site but modulates its functionality and that DBSP shares the electrogenic component of BSP transport and also modulates the performance of this carrier system.

Molecular structure influence in the recognition of phthaleins by the electrogenic organic anion carrier at the sinusoidal plasma membrane level in the liver.

The hepatocytic uptake of cholephilic organic anions occurs by carrier-mediated mechanisms. Electrogenic and electroneutral transport systems have been described. The aim of this study was to determine the dissociation constant (Kd) of the Electrogenic Carrier System(s) (ECS) for tetrabromosulfophthalein (BSP), dibromosulfophthalein (DBSP), tetrabromophthalein (TBP), tetrabromosulfonephthalein (TBS) and thymol blue (TB). Kd (uM) values for ECS-organic anion complexes were: ECS-BSP = 3.61 +/- 0.18; ECS-DBSP = 11.61 +/- 1.32; ECS-TBP = 0.51 +/- 0.08; ECS-TBS = 1.31 +/- 0.25; ECS-TB = 9.44 +/- 1.80. From these data, it is possible to conclude that molecular characteristics of the organic anions are important factors in determining the dissociation constant for the electrogenic hepatic carrier(s). In this sense, the addition of two sulphonic groups on the phenolic ring; the presence of a sulphonic on the benzenic ring and the absence of two or four bromines on the molecule confers a lower affinity for ECS.

Biliary excretion of estradiol-17 beta-glucuronide in the rat

Estradiol-17beta-glucuronide (E217G) is a cholestatic agent and is considered to be related to the pathogenesis of intrahepatic cholestasis of pregnancy. In the current study, we examined the mechanism of the biliary excretion of E217G and estradiol metabolites in rats. Biliary excretion of tracer doses of [3H]estradiol-17beta-glucuronide and [14C]estradiol or [3H]taurocholate and ]14C]vinblastine, a P-glycoprotein (P-GP) substrate, intravenously administered as a bolus to bile-drained control rats or EHBR was studied. Biliary excretion of E217G and estradiol metabolites EHBR was markedly delayed. Analyses of biliary metabolites after estradiol injection showed less polar conjugates in EHBR. In contrast, the excretion of taurocholate and vinblastine (VLB) was only slightly delayed in EHBR. Although phenothiazine treatment to induce the expression of P-GP increased biliary vinblastine excretion, it did not affect biliary excretion of a tracer dose of [3H]estradiol-17beta-glucuronide. However, phenothiazine treatment inhibited the cholestasis induced by E217G infused at the rate of 0.075 micromol/min/100g for 20 minutes and increased biliary E217G excretion. Sulfobromophthalein infusion (0.2 micromol/min/100 g body 0 weight) markedly inhibited the biliary excretion of E217G and estradiol metabolites, whereas dibromosulfophthalein (DBSP) at the same infusion rate had no effect. These findings indicate that EG17G is excreted into bile by a canalicular organic anion carrier for sulfobromopthalein (BSP), not for DBSP, under physiological conditions, and that P-GP influences E217G excretion only at a high dose. under physiological conditions, and that P-GP influence s E217G excretion only at a high dose. (Hepatology 1996 Mar;23(3):607-13)

Biliary excretion of estradiol-17β-glucuronide in the rat

Estradiol-17β-glucuronide (E217G) is a cholestatic agent and is considered to be related to the pathogenesis of intrahepatic cholestasis of pregnancy. In the current study, we examined the mechanism of the biliary excretion of E217G and estradiol metabolites in rats. Biliary excretion of tracer doses of [3H]estradiol-17β-glucuronide and [14C]estradiol or [3H]taurocholate and ]14C]vinblastine, a P-glycoprotein (P-GP) substrate, intravenously administered as a bolus to bile-drained control rats or EHBR was studied. Biliary excretion of E217G and estradiol metabolites EHBR was markedly delayed. Analyses of biliary metabolites after estradiol injection showed less polar conjugates in EHBR. In contrast, the excretion of taurocholate and vinblastine (VLB) was only slightly delayed in EHBR. Although phenothiazine treatment to induce the expression of P-GP increased biliary vinblastine excretion, it did not affect biliary excretion of a tracer dose of [3H]estradiol-17β-glucuronide. However, phenothiazine treatment inhibited the cholestasis induced by E217G infused at the rate of 0.075 μmol/min/100g for 20 minutes and increased biliary E217G excretion. Sulfobromophthalein infusion (0.2 μmol/min/100 g body 0 weight) markedly inhibited the biliary excretion of E217G and estradiol metabolites, whereas dibromosulfophthalein (DBSP) at the same infusion rate had no effect. These findings indicate that EG17G is excreted into bile by a canalicular organic anion carrier for sulfobromopthalein (BSP), not for DBSP, under physiological conditions, and that P-GP influences E217G excretion only at a high dose. under physiological conditions, and that P-GP influence s E217G excretion only at a high dose.

Biliary excretion of glycyrrhizin in rats: kinetic basis for multiplicity in bile canalicular transport of organic anions.

To examine the presence of multiplicity for the biliary excretion of xenobiotic conjugates, we studied the disposition of glycyrrhizin (GR), which has glucuronide within its molecular structure and has the ability to inhibit the biliary excretion of liquiritigenin (LG) glucuronides. GR was administered intravenously as a bolus to Sprague-Dawley (SD) rats which received an i.v. infusion of inhibitors (dibromosulfophthalein (DBSP) and indocyanine green (ICG)) at their transport maximum rates. Biliary excretion of GR was also examined in Eisai hyperbilirubinemic rats (EHBR), which have a hereditary defect in the canalicular transport system of several organic anions. Infusion of ICG did not affect the biliary excretion of GR, whereas infusion of DBSP reduced it significantly. The plasma concentration of GR was increased by DBSP but not by ICG. In EHBR, the biliary excretion of GR was severely impaired, resulting in an increase in the plasma concentration of GR. These findings suggest (1) that the biliary excretion of GR is mediated by the system which is shared by DBSP and LG glucuronides but not by ICG and (2) that this system is hereditarily defective in EHBR. Together with our previous findings, the multiplicity for the biliary excretion of organic anions is shown.

Pharmacokinetic modeling of the sinusoidal efflux of anionic ligands from the isolated perfused rat liver: the influence of albumin.

This study contains a pharmacokinetic analysis on the efflux of organic anions from the liver into the bloodstream (sinusoidal efflux) with specific reference to the influence of albumin. The net sinusoidal efflux rate of dibromosulfophthalein (DBSP) from preloaded livers, being the resultant of sinusoidal efflux and reuptake of ligand by hepatocytes downstream the sinusoid, can be strongly increased by the presence of bovine serum albumin (BSA), a protein having multiple binding sites for DBSP. We previously attributed this effect to a reduction of reuptake through extracellular binding of the organic anion to the protein, rather than to an intrinsic stimulatory effect on the actual membrane transport process from the cells. In the present study we tested this hypothesis using a pharmacokinetic multicompartment liver model. This model resembles the parallel tube model in that the liver is described by several compartments placed in series instead of a single well-stirred compartment and it takes into account rates of dissociation and association in binding to proteins in the sinusoidal space. The model parameters were fitted from the sinusoidal efflux and biliary excretion data from efflux experiments measuring the stimulatory effect of various concentrations of BSA. Equilibrium binding of DBSP to albumin as well as the dissociation rate constant (koff) were determined in vitro with rapid filtration techniques. The experimental data could not be fitted satisfactorily when using the experimentally obtained values of the protein association and dissociation rate constants (kon and koff). However, they could be simulated accurately assuming 16 times higher values for the association and dissociation rate constant compared to those determined in vitro. Time constants of the perfusate flow, liver (re)uptake, and protein association and dissociation indicate that binding equilibrium does not exist within the sinusoids and that, in particular at low protein concentrations, the net sinusoidal efflux rate is association rate-limited: A large fraction of the ligand effluxed from the cell into the median is taken up by the hepatocyte before binding to the proteins occurs. Higher kon and koff values predicted by the model might indicate altered DBSP-albumin binding characteristics upon passage through the liver but alternatively can be explained by an intrinsic effect of albumin on the carrier-mediated efflux process. Efflux experiments showed a marked stimulatory effect of the protein on sinusoidal efflux but only a moderate effect on biliary excretion, despite a strong decrease in liver content. These patterns indicate that sinusoidal efflux and biliary excretion occur from two different intracellular compartments that equilibrate slowly.

Mechanistic aspects of uptake and sinusoidal efflux of dibromosulfophthalein in the isolated perfused rat liver

Using the isolated perfused rat liver technique we examined whether the accumulation and sinusoidal efflux processes of the organic anion dibromosulfophthalein (DBSP) are dependent on the intracellular ATP content, chloride concentration in the perfusion medium as well as temperature of the medium and whether they are mediated by the same or by separate carrier mechanisms. The net sinusoidal efflux rate, being the resultant of sinusoidal efflux and re-uptake, was decreased more than 50% after lowering the medium temperature from 37 to 26° indicating that the efflux process is carrier-mediated. The uptake rate was decreased only 18% after lowering the medium temperature to 26°. Lowering of the hepatic ATP content for more than 80% clearly decreased the DBSP uptake rate but not the sinusoidal efflux rate. These observations indicate that these opposing transport steps probably are mediated by two separate carriers. Additional evidence for this hypothesis originated from the observation that sinusoidal efflux of DBSP was decreased about 30% whereas hepatic uptake of the substrate remained unaltered after replacing chloride in the perfusion medium with gluconate. In summary, we conclude that uptake and sinusoidal efflux of DBSP are mediated by two separate carrier systems that are influenced differently by ATP depletion, temperature lowering and presence of Cl − gradients.

Secretion of the organic anion harmol sulfate from liver into blood: Evidence for a carrier-mediated mechanism

In the liver, drugs with phenolic groups can be converted to sulfate or glucuronide conjugates and are then transported into bile or back into the bloodstream. The mechanism for transport of drugs and drug conjugates from the hepatocytes into the blood at the sinusoidal side of the cell are not well defined. In the case of carrier-mediated transport of these strongly polar conjugates, saturability of transport and mutual competition between structurally related compounds would be anticipated. This competitive aspect was investigated by using two organic anions, dibromosulfophthalein (DBSP) and harmol sulfate. The latter compound was generated by the hepatocytes from harmol, which was continuously infused into the rat in vivo and in isolated perfused rat livers. In addition we loaded the perfused rat livers with preformed harmol sulfate and studied its efflux rate to the perfusate as influenced by DBSP. In steady state, about 80% of harmol was sulfated and 20% was glucuronidated. Harmol sulfate was mainly excreted in the urine, the glucuronide was equally excreted in urine and bile. DBSP lowered the urinary excretion of harmol sulfate by about 30% which was due to a decrease in plasma concentration. However, renal clearance of harmol sulfate (3.2 ± 0.2 ml/min) was unchanged. At the same time DBSP doubled the biliary clearance of harmol sulfate (1.0 ± 0.1 and 2.2 ± 0.2 ml/min in controls and DBSP studies respectively). DBSP decreased glucuronide excretion both in urine and bile. The increase in biliary output and decrease in urinary excretion of harmol sulfate is explained by competitive interaction between the organic anion DBSP and harmol sulfate at the sinusoidal level. Efflux experiments in single pass perfused isolated livers showed a clear decrease of harmol sulfate transport from liver into plasma by DBSP and provided evidence for such an inhibitory phenomenon ( t 1 2 of efflux was 3.58 ± 0.21 compared with 2.46 ± 0.07 min for controls). These results indicate that organic anion transport from the hepatocyte into the blood stream is very likely carrier-mediated. A decrease in renal output of drug conjugates therefore may not only be due to a decrease in the conjugation process but also to a lower liver to blood transport rate which at the same time may produce a higher biliary output.

Pharmacokinetics of the hepatic transport of organic anions: influence of extra- and intracellular binding on hepatic storage of dibromosulfophthalein and interactions with indocyanine green.

The influence of intracellular and extracellular protein binding on the hepatic storage and biliary elimination of dibromosulfophthalein (DBSP) was studied in isolated perfused rat liver. Under first order kinetic conditions the amount of DBSP in the liver at a given plasma concentration (hepatic storage) was determined by extracellular binding to albumin and intracellular binding to the cytosolic Y and Z proteins as well as concentrative membrane transport from plasma into the liver. At higher doses, extensive binding of DBSP to intracellular organelles also occurred while liver cytosol/plasma concentration gradients of unbound DBSP were much lower. Hepatic storage increased with decreasing albumin concentration in the perfusate of isolated perfused rat livers. However, it was shown that this parameter is dose-dependent, and errors can be introduced in its calculation if nonlinearity of sinusoidal and canalicular transport processes as well as nonlinear protein binding are not taken into account. The influence of another organic anion, indocyanine green (ICG) on the hepatic storage, subcellular distribution, and elimination of DBSP was subsequently studied. At equimolar amounts the presence of ICG resulted in a 50% decrease in hepatic clearance and hepatic distribution volume of DBSP. It was inferred that these changes are due to an inhibition of carrier-mediated transport across the sinusoidal and canalicular membrane and preferential displacement from intracellular binding sites. In contrast DBSP in equimolar amount enhanced the initial disappearance rate and biliary excretion of ICG, probably due to increasing its free fraction in plasma. It is concluded that the level and mechanism of interaction of two drugs within the eliminating organ can be characterized by combining clearance studies with data on subcellular and extracellular binding.

Acinar heterogeneity in hepatic transport of dibromosulfophthalein and ouabain studied by autoradiography, normal and retrograde perfusions and computer simulation

This study is aimed to investigate the relative involvement of periportal (zone 1) and perivenous (zone 3) hepatocytes in the uptake and biliary excretion of the organic anion dibromosulfophthalein (DBSP) and the uncharged cardiac-glycoside ouabain. The localization in the acinus of [ 35S]BSP (sulfobromophthalein, the tetra-bromo-analogue of DBSP) and [ 3H]ouabain administered to livers perfused with normal and retrograde flow, was detected by autoradiography. The plasma disappearance and biliary excretion rates of DBSP and [ 3H]ouabain were determined in normal and retrograde perfusions. In addition, computer simulations were performed to predict the effect of reversal of the perfusate flow on the plasma disappearance and biliary excretion rate curves and on the concentration of label in zones 1 and 3. Autoradiography showed that 2 and 10 min after injection of [ 35S]BSP to normally and retrogradely perfused livers, the label was uniformly distributed in the liver acinus. The same results were found 30 sec and 10 min after injection of [ 3H]ouabain to normally and retrogradely perfused livers. The plasma disappearance and biliary excretion rate of DBSP were slightly faster in retrograde perfusions compared to normal perfusions both with and without a basal bile salt infusion of 15 μmole/hr. This could not be explained by an acinar heterogeneity with respect to any of the DBSP transport steps (plasma to liver, liver to plasma, liver to bile) as was shown by computer simulations. The plasma disappearance and biliary excretion rate of ouabain were similar in normal and retrograde perfusions. It is concluded that periportal and perivenous hepatocytes are equally involved in the uptake of (D)BSP and ouabain from the medium. However, due to the particular distribution patterns no conclusions can be drawn from normal and retrograde perfusions about the relative involvement of these cells in biliary excretion, as was shown by computer simulation. The unaffected kinetic behaviour of the retrogradely perfused livers indicated that no liver damage occurs during retrograde perfusion with respect to transport function.

Formation of a metabolite of dibromosulfophthalein (DBSP) in man.

In bile specimens from postoperative patients with biliary drainage following cholecystectomy, in addition to unchanged dibromosulfophthalein (DBSP), a single polar metabolite of DBSP was found after i.v. injection of 5 mg/kg of the diagnostic dye. This metabolite, which has not previously been detected, was resistant to beta-glucuronidase and arylsulfatase and was remarkably stable in strongly acid and alkaline solutions. It exhibited the same spectrum and colour change interval as unchanged DBSP. Further studies of its identity revealed that it gave a ninhydrin-positive reaction and that its Rf-value on TLC could be restored by Raney-nickel reduction. Amino-acid analysis after reduction and acid hydrolysis showed an increase in glutamic acid and alanine that can be considered as splitting products of conjugated glutathione following these procedures. Estimation of the quantity of this possible glutathione conjugate indicates that it is formed less rapidly than the glutathione derivative of the tetrabromoanalogue BSP, and that it represents up to 25% of the total dye excreted in bile. The observed metabolism of DBSP in man may complicate its use in the study of hepatic transport function, and negates the previous assumption that, as in certain other animal species, the dye is excreted unchanged.

Pharmacokinetics of biliary excretion in man V. Dibromosulfophthalein.

The pharmacokinetics of dibromosulfophthalein (DBSP), the 3,6-dibromo analogue of BSP, was studied in 7 patients with a biliary fistula, 52 h after cholecystectomy, and in 6 gynaecological patients with an indwelling urethral catheter, following extirpation of the uterus i.e. with an intact enterohepatic circulation. Plasma protein binding determined by ultrafiltration was 98–99% up to a concentration of 700 µg/ml. After an intravenous bolus injection of DBSP 5 mg/kg, a biexponential plasma decay was found in both groups, with a rapid initial t1/2 of 2–6 min and a slow secondary phase of 33–109 min (mean 66 min) in the cholecystectomy patients, and 10–30 min (mean 19 min) in the gynaecological patients. The biliary excretion rate varied considerably between the patients and was highly correlated with bile flow. Biliary output amounted to a maximum of 86% of the dose in 24 h. The excretion rate curves showed ascending and descending phases, the mean terminal t1/2 being 65 min. Urinary excretion was 3–11% of the dose in 8 h in the gynaecological patients (mean 6%) and 6–31% in the cholecystectomy group (mean 16%). Renal clearance of unbound DBSP was about ten-times greater than the glomerular filtration rate, which indicates tubular secretion. A two compartment model with elimination from the peripheral and central compartments was selected because of these data. Analysis of the plasma-disappearance curves indicated an initial plasma clearance of 500–600 ml/min, which suggests that hepatic uptake will be very dependent on flow. Steady state (biliary) clearance was about 400 ml/min in the gynaecological group and approximately half that in the cholecystectomy patients; V1 tended to be higher and V2 to be lower in the latter group. It is concluded that biliary excretion rate of DBSP in patients with a biliary fistula is probably depressed by the postoperative bile drainage and the lack of enterohepatic cycling of bile salts.