Age dependence of hepatic transport in control and phenobarbital-pretreated rats.

Eosine is excreted in rat bile unchanged, which makes it suitable for the study of age dependent changes in hepatic uptake and excretion. Bile flow was approximately 40 μl/kg/min in 20-day-old rats and twice as high in 30-day-old animals. In 60- and 120-day-old rats the bile volume was decreased, moreover in 220-day-old ones it fell to the level of 20-day-old rats. The biliary excretion of eosine (150 μmol/kg i.v.) was highest in 60-day-old rats, however, the biliary flow reached its peak in 30-day-old rats. After phenobarbital (PB) pretreatment (75 mg/kg i.p. daily for five days) each age group showed enhancement in liver weight and bile volume. On the other hand, the hepatic concentration of eosine did not change after PB pretreatment caused an increase in the biliary excretion of eosine in 30-, 60-, 120- and 220-day-old rats but no significant change in 20-day-old animals. Our results indicate that the hepatic transport in young rats was immature and was not induced by PB. However, PB can increase the low excretion rate in old rats.     

The influence of cage bedding on the metabolism of sulphobromophthalein sodium by an hepatic cytosol-located enzyme system.

The use of cage bedding prepared from pinewood shavings has been shown to be associated with an increase in the activity of sulphobromophthalein sodium (BSP) S-aryltransferase in the hepatic cytosol in rats houses on this substance. This increase was associated with enhanced secretion rates of dye into the bile due to an elevation in the biliary excretion rate of conjugated BSP. Analysis of the hepatic dye content at the time of maximal excretion of BSP into the bile indicated that this phenomenon was due to increased intrahepatic conjugation of BSP. This observation emphasizes the importance of considering environmental factors that may influence results when designing experiments on hepatic metabolism.     

Effects of two-thirds hepatectomy on sulfobromophthalein handling by the rat liver

The modifications in the hepatic transport of sulfobromophthalein (BSP) were studied after partial hepatectomy (p.h.) in Wistar rats. The biliary excretion of BSP, injected i.v. at 150 mumol/kg, decreased in the early periods after p.h., with a disappearance of the choleretic effect induced by the dye in sham-operated animals. The impairment in the biliary BSP excretion corresponded to the conjugated fraction and was accompanied by a lowered glutathione S-transferase activity in the liver.     

Effects of colchicine on the hepatocellular transport of indocyanine green in the rat

The effects of colchicine on plasma elimination and biliary excretion of indocyanine green (ICG) and sulfobromophthalein (BSP) in rats were examined. Elimination of two different doses of ICG (6 mg and 20 mg/kg body weight) from plasma was significantly delayed when rats were treated with colchicine (3 mg/kg body weight) 3 h prior to the administration of the dye. On the other hand, disappearance of BSP (100 mg/kg) from plasma was not influenced by colchicine. The fact that the difference in the ICG elimination from plasma between colchicine-treated and saline-treated rats was minimal in the early period (i.e., 2 min after administration of the dye), but evident after its half-life (i.e., 10 min, when 6 mg/kg body weight of ICG was given), suggested that colchicine mainly affected the hepatocellular transport of ICG rather than the uptake of the dye by hepatocytes. Colchicine also significantly reduced the excretion of ICG (6 mg and 20 mg/kg) into bile but did not alter that of BSP (100 mg and 200 mg/kg). On the other hand, the same amount of lumicolchicine (3 mg/kg) did not have any effect on the biliary excretion of ICG. These results suggested that ICG is transported through hepatocytes into bile with the aid of the cytoplasmic microtubular system, whereas BSP is handled by hepatocytes in a different way.     

The influence of cadmium on the biliary excretion of eosine and bromsulphthalein and on microsomal monooxygenase activities in the rat

Cadmium sulfate x 8/3 H2O (0.3, 0.6, 1.5 and 2.0 mg/kg) administered simultaneously with eosine and bromsulphthalein (120 mumol/kg i.v.) did not significantly change the biliary excretion of the dyes. After a 3 days pretreatment with 2.0 mg/kg CdSO4 i.p. a body weight loss and an increase in the relative liver weight when calculated on body weight were observed together with an enhanced bile flow, but the excretion of the dyes was not markedly influenced. Ethylmorphine N-demethylation and ethoxycoumarin O-deethylation activities as well as microsomal cytochrome P-450 concentration were diminished by about 50%. It can be concluded that in male rats the hepatic microsomal monooxygenase system is more sensitive towards cadmium than the hepatic transport system for organic anions.     

Taurine supplementation induces multidrug resistance protein 2 and bile salt export pump expression in rats and prevents endotoxin-induced cholestasis

The effect of oral taurine supplementation on endotoxin-induced cholestasis was investigated in rat liver. At 12h following lipopolysaccharide (LPS) injection (4mg/kg body weight i.p.) bile flow and bromosulfophthalein (BSP) and taurocholate (TC) excretion were determined in the perfused liver and the expression of the canalicular transporters multidrug resistance protein 2 (Mrp2) and bile salt export pump (Bsep) was analyzed. Injection of LPS induced a significant decrease of bile flow ( 2.2+/-0.2 microl/g liver wet weight/min vs 3.3+/-0.1 microl/g liver wet weight in controls), biliary BSP excretion (10.8+/-2.2 nmol/g/min vs 21.0+/-3.8 nmol/g/min), and biliary TC excretion (114+/-23 nmol/g/min vs 228+/-8 nmol/g/min). These effects were due to transporter retrieval from the canalicular membrane and downregulation of Mrp2 and Bsep expression. In taurine-supplemented rats bile flow was 30% higher than that in untreated rats and the expression of Mrp2 and Bsep protein was increased two- to threefold. In taurine-supplemented rats there was no significant reduction of bile flow or of BSP and TC excretion at 12h following LPS injection. This protective effect of taurine was due to higher Mrp2 and Bsep protein levels compared to nonsupplemented LPS-treated rats, whereas relative Mrp2 retrieval from the canalicular membrane induced by LPS was not significantly different. LPS-induced tumor necrosis factor alpha and interleukin-1beta release were lower in taurine-fed rats; however, downregulation of Mrp2 and Bsep expression by LPS was delayed but not prevented. The data show that oral supplementation of taurine induces Mrp2 and Bsep expression and may prevent LPS-induced cholestasis.     

The effect of chlordiazepoxide hydrochloride on the isolated perfused rat liver.

The effects of chlordiazepoxide-hydrochloride (CDZ) on the isolated perfused rat liver were examined. CDZ administration decreased bile flow, biliary excretion of sulfobromophthalein (BSP) and hepatic uptake of BSP. The addition of CDZ to the perfusate of livers obtained from phenobarbital (Pb) pretreated rats led to 50% greater reductions in bile flow, concentration of BSP in bile and hepatic uptake of BSP. The adverse effects of CDZ on BSP excretion per g liver, however, did not appear to be enhanced by Pb pretreatment. The complex nature of the interrelationship of the effects of Pb and of CDZ on the control liver prevented differentiation of the role of CDZ from that of a metabolite on the adverse effect on liver function.     

Induction of ornithine decarboxylase and S-adenosylmethionine decarboxylase by sulfobromophthalein in rats

The administration of sulfobromophthalein (BSP, 0.5 mmol/kg, ip.) increased ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAMDC) activities to 30-fold and 5-fold, respectively, of the controls at 12 hr in the liver of rats. Parallel to the increase in ODC, there was an increase in hepatic putrescine content. However, spermine content tended to decrease. BSP increased ODC and SAMDC activities and putrescine content, but decreased spermine content, in a dose-dependent manner. Pretreatment of rats with actinomycin D and cycloheximide almost completely blocked the BSP-mediated increase of ODC and SAMDC activities. Pretreatment with glutathione (GSH) failed to inhibit BSP-mediated increase of ODC and SAMDC activities. In addition, the administration of BSP-GSH conjugate (0.5 mmol/kg, iv.) did not produce the increase of ODC and SAMDC activities. Pretreatment with phenobarbital and 3-methylcholanthrene did not inhibit BSP-mediated increase of ODC and SAMDC. The results indicate that BSP could cause changes in hepatic polyamine content due to the induction of ODC and SAMDC.     

Administration of glutathione and lipid peroxidation induced during fasting

It is well known that lipid peroxidation may be initiated or exaggerated by conditions leading to hepatic GSH depletion or altered GSH/GSSG ratio. In our study we evaluated the effects of GSH administration on hepatic, bile and plasma GSH, GSSG and MDA in rats depleted of the tripeptide by a prolonged. fasting. An exteriorized biliary-duodenal fistula was established and GSH or saline solution was administered i.p. for a period of 6h. Rats treated with GSH exhibited an increased GSH and decreased GSSG biliary excretion. Whereas in control rats an opposite pattern was observed, namely enhanced GSSG and decreased GSH biliary excretion. While hepatic GSH and GSSG concentrations were comparable in the two groups, a significant increase in liver and plasma MDA production was found in controls compared to GSH treated rats. Our data suggest a protective role of GSH against the production of lipoperoxidation as evidenced by the decrease of hepatic, biliary and plasma MDA levels and by a decreased percentage of biliary GSSG. In addition, the significant increase of biliary GSH excretion, observed in rats treated with GSH compared to controls, may be due to an increased supply of the tripeptide which is known to be preferentially excreted into bile in the reduced form.     

Multiplicity of Hepatic Excretory Mechanisms for Organic Anions

Previous studies based upon competition between different organic anions for biliary excretion in vivo have suggested that all organic anions share a common hepatic secretory mechanism. Corriedale sheep with an inherited defect in organic anion excretion by the liver were used to study this problem directly without the need for competition studies, the results of which are difficult to analyze. Maximal biliary excretion of sulfobromphthalein (BSP) in mutant Corriedale sheep was less than 7% of that observed in normal sheep whereas maximal biliary excretion of taurocholate, the major organic anion in sheep bile, was not different in mutant and normal sheep. Taurocholate infusion enhanced maximal hepatic excretion of BSP in normal but not in mutant sheep. These studies of an inheritable disorder which appears to be identical to the Dubin-Johnson syndrome in man, demonstrate that taurocholate excretion requires at least one step in biliary excretion which is not required by other organic anions such as bile pigment, porphyrins, drugs, and dyes.     

Effect of the glutathione S-transferase inhibitor, tienilic acid, on biliary excretion of sulphobromophthalein

Tienilic acid, a phenoxyacetic acid diuretic, reduces the amount of total sulphobromophthalein (BSP) excretion in the isolated perfused rat liver (IPRL). This reduction was primarily by reduction in excretion of conjugated BSP, with excretion of unchanged BSP being relatively unaffected. TA also reduces the amount of conjugated BSP formed in vitro, indicating that its effect in the IPRL may be by means of inhibiting the glutathione S-transferase enzymes involved in the formation of the conjugate. It would appear that a reduction in the biliary excretion of BSP cannot be taken to be an indication of reduced liver function in a general sense.     

Hepatic organic anion transport kinetics and bile flow during short-term total parenteral nutrition in the rabbit

Plasma disappearance of sulfobromophthalein (BSP) after an intravenous bolus (5 mg/kg) was determined in six lab chow-fed (LCF) rabbits and in six rabbits maintained on total parenteral nutrition (TPN) for 5 days. A common bile duct cannula enabled measurements of bile flow and biliary BSP excretion. Compartmental analysis of the biexponential plasma disappearance curve yielded three fractional transfer rates, plasma to liver (hepatic uptake), liver to plasma (reflux), and liver to bile (canalicular excretion). The transfer rates for hepatic uptake were 0.253 +/- 0.061/min for LCF and 0.147 +/- 0.040/min for TPN (P less than 0.01) and for the canalicular excretion of BSP were 0.038 +/- 0.019/min for LCF and 0.019 +/- 0.002/min for TPN (P less than 0.05). Model-computed rates for BSP excretion in bile over 60 min were lower with TPN (61%) than with LCF (80%); the measured excretory rates were 53% for TPN rabbits and 75% of injected dose for LCF animals. Basal biliary flow was reduced by 50% in the TPN group. With a two-compartmental model, assuming two pools and three transfer rates, we have demonstrated for the first time significant decreases in hepatic uptake and canalicular excretion of the organic anion BSP during TPN. A decrease in hepatic blood flow due to the enteral fast of TPN could have contributed in part to the decreased hepatic uptake. But, because the second exponent of the biexponential curve is independent of hepatic blood flow, the decrease in liver to bile transfer rate is a true approximation of a diminished canalicular excretory capacity during TPN. It is concluded that the movement of organic anions along the hepatic BSP/bilirubin transport system is impaired early during TPN.     

Intracerebroventricular 4-methylcatechol (4-MC) ameliorates chronic pain associated with depression-like behavior via induction of brain-derived neurotrophic factor (BDNF)

Neuropathic pain concurrent with mood disorder from peripheral nerve injury is a serious clinical problem that significantly affects quality of life. Recent studies have suggested that a lack of brain-derived neurotrophic factor (BDNF) in the limbic system may cause this pain-emotion. BDNF is induced in cultured neurons by 4-methylcatechol (4-MC), but the role of 4-MC-induced BDNF in pain-emotion is poorly understood. Thus, we assessed the possible involvement of BDNF in brain in depression-like behavior during chronic pain following peripheral nerve injury. In addition, we examined whether intracerebroventricular (i.c.v.) 4-MC prevents chronic pain in rats and produces an antidepressant effect. Sprague-Dawley rats implanted intracerebroventricularly with a PE-10 tube were subjected to chronic constriction injury (CCI). Pain was assessed by a reduction in paw withdrawal latency (PWL) to heat stimuli after CCI. We also used a forced swimming testing (FST; time of immobility, in seconds) from day 14 to day 21 after CCI. Modulation of pain and emotional behavior was performed by injection of PD0325901 (a MEK1/2 inhibitor). 4-MC (100 nM) was continuously administered i.c.v. for 3 days during the period from day 14 to day 21 after CCI. To block analgesic and antidepressant effects, anti-BDNF antibody or K252a (a TrkB receptor inhibitor) was injected in combination with 4-MC. Naloxone was also coadministered to confirm the analgesic effect of 4-MC. During the chronic stage after CCI, the rats showed a sustained decrease in PWL (thermal hyperalgesia) associated with extension of the time of immobility (depression-like behavior). PD0325901 significantly reduced the decrease in PWL and the increased time of immobility after CCI. The decreased PWL and increased time of immobility were also reduced by 4-MC and by treatment with an ERK1/2 inhibitor. These effects of 4-MC i.c.v. were reversed by anti-BDNF and K252a. The analgesic effect of 4-MC i.c.v. was also antagonized by naloxone. Based on these results, we suggest that a lack of BDNF and activation of ERK1/2 in the pain-emotion network in the CNS may be involved in depression-like behavior during chronic pain. 4-MC i.c.v. ameliorates chronic pain and depression-like behavior by producing of BDNF and normalization of ERK1/2 activation. Therefore, enhancement of BDNF may be a new treatment strategy for chronic pain associated with depression.     

The biliary excretion of sulfbromophthalein in the pig

The characteristics of the hepatic metabolism of Sulfbromophthalein (BSP) have not been described previously for the pig. This is an important deficiency, since the pig is particularly suitable for studies of hepatic physiology and pharmacology which might apply to man. The aim of these experiments was to establish the pattern of serum clearance and biliary excretion of BSP and to determine that dose which would produce a maximal concentration in bile. A dose response and pattern of biliary excretion of BSP was studied at three dose levels administered either as a single bolus of a continuous infusion. All experiments were performed in conscious, conditioned pigs. The patterns of serum clearance and biliary excretion were found to be similar to other laboratory animals and to man. Maximary biliary concentration of BSP was achieved by a single bolus of 5-9 mumol/kg or a constant infusion of 0-59 mumol/kg/min. At these dose levels no significant alteration in bile flow was demonstrated nor was there any correlation between bile flow and BSP excretion. Supra-maximal doses produced a significant increase in bile flow and with these doses there was a significant positive correlation between bile flow and BSP excretion.     

Characterization of the Mechanisms Involved in the Increased Renal Elimination of Bromosulfophthalein During Cholestasis: Involvement of Oatp1

The kidneys and liver are the major routes for organic anion elimination. We have recently shown that acute obstructive jaundice is associated with increased systemic and renal elimination of two organic anions, p-aminohippurate and furosemide, principally excreted through urine. This study examined probable adaptive mechanisms involved in renal elimination of bromosulfophthalein (BSP), a prototypical organic anion principally excreted in bile, in rats with acute obstructive jaundice. Male Wistar rats underwent bile duct ligation (BDL rats). Pair-fed sham-operated rats served as controls. BSP renal clearance was performed by conventional techniques. Renal organic anion-transporting polypeptide 1 (Oatp1) expression was evaluated by immunoblotting and IHC. Excreted, filtered, and secreted loads of BSP were all higher in BDL rats compared with Sham rats. The higher BSP filtered load resulted from the increase in plasma BSP concentration in BDL rats, because glomerular filtration rate showed no difference with the Sham group. The increase in the secreted load might be explained by the higher expression of Oatp1 observed in apical membranes from kidneys of BDL animals. This likely adaptation to hepatic injury, specifically in biliary components elimination, might explain, at least in part, the huge increase in BSP renal excretion observed in this experimental model. (J Histochem Cytochem 57:449–456, 2009)     

Sex-specific extraction of organic anions by the rat liver

The capacity for hepatic elimination of some compounds is different in males and females and differential expression of a number of sinusoidal and canalicular transporters exists. However, the specific events underlying the functional differences are not understood. To determine how sex influences sinusoidal and canalicular organic anion transport, bile duct-cannulated livers from mature Sprague-Dawley rats of both sexes were single-pass perfused with saline containing the model organic anions bromosulphophthalein (BSP), carboxyfluorescein (CF), carboxyfluorescein diacetate (CFDA) or 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Assay of effluent perfusate anion concentration showed that BSP, but not DIDS, extraction was significantly higher in male versus female rats. At 20 min perfusion with 50 microM BSP the mean effluent concentration was 5.6 and 20.1 microM in, respectively, male and female rats. HPLC confirmed that the effluent perfusate concentration of BSP was higher in female as compared with male rats and was not contributed to by its glutathione conjugate. With 25 microM DIDS, the effluent concentration reached 7.3 (male) and 8.2 microM (female), indicating high extraction efficiency. In contrast to BSP and DIDS, CF extraction was very low (<20%) so that differences between male and females could not be assessed. Biliary BSP and CF excretion were, respectively, 3.5- and 4-fold higher in male rats. Neither sinusoidal efflux nor biliary excretion of CF was sex-dependent with a higher cytoplasmic load of CF (during CFDA perfusion). Our results suggest that differences in sinusoidal uptake are responsible for the sex-specific hepatic excretion of some organic anions.     

The biliary excretion of {3H}-25-hydroxyvitamin D3 following chronic ethanol administration in the rat

The biliary excretion of {³H}-25-hydroxyvitamin D₃ was studied in rats fed a diet containing 36% of total calories as ethanol and in pair-fed controls. {³H}-25-hydroxyvitamin D₃ was injected i.v. and the biliary appearance of {³H}-compounds studied over a period of 80 minutes. Choleresis was not affected by chronic ethanol feeding averaging 98.7±10.3 μl min^?1 kg^?1 in Controls and 101.8±8.7 μl min^?1 kg^?1 in ethanol-fed animals. The total biliary excretion of {³H} was found to be 29% higher in ethanol-treated than control rats and represented 6.3±0.4% and 4.9±0.4% (p<0.05) of the injected dose at the end of the experiment. The biliary excretion rate as well as the biliary concentration of {³H}-25-hydroxyvitamin D³ congeners were significantly increased by the ethanol treatment from the 25th to the 80th minute after the i.v. injection of {³H}-25-hydroxyvitamin D₃. The plasma and liver {³H} concentrations were not significantly affected by the ethanol treatment. These results suggest that, in the rat, chronic ethanol ingestion promotes the biliary loss of {³H}-25-hydroxyvitamin D₃. The higher concentration of {³H}-compounds into the bile of ethanol treated rats also suggests the presence of excretory pathway(s) which could be, at least in part, independent of bile flow. This increased loss of {³H}-25-hydroxyvitamin D³ into the bile may be a contributing factor in the impaired vitamin D status in alcoholics.     

Dosing-time-dependent variation in biliary excretion of flomoxef in rats

We previously reported that the biliary excretion of flomoxef, an oxacephem antibiotic, was greater after dosing at 21:00 than at 09:00h in diurnally active human subjects. The present study was undertaken to examine whether the biliary excretion of flomoxef is also dependent on its dosing time in rats. Adult male Wistar rats were housed under light on at 07:00h and off at 19:00h. Bile fluid was completely drained through a polyethylene catheter from conscious animals. Flomoxef (20 mg/kg) was injected into the tail vein at 09:00 or 21:00h by a cross-over design, and drained bile fluid was collected for 8h after each dosing. The maximum concentration of biliary flomoxef was significantly greater and its total excretion tended to be greater after dosing at 09:00 than 21:00h. These results suggest the biliary excretion of flomoxef is enhanced after dosing at the beginning of the rest period in rats, as it is in humans.     

Mrp2 is involved in benzylpenicillin-induced choleresis

Benzylpenicillin (PCG; 180 micromol/kg), a classic beta-lactam antibiotic, was intravenously given to Sprague-Dawley (SD) rats and multidrug resistance-associated protein 2 (Mrp2)-deficient Eisai hyperbilirubinemic rats (EHBR). A percentage of the [(3)H]PCG was excreted into the bile of the rats within 60 min (SD rats: 31.7% and EHBR: 4.3%). Remarkably, a transient increase in the bile flow ( approximately 2-fold) and a slight increase in the total biliary bilirubin excretion were observed in SD rats but not in the EHBR after PCG administration. This suggests that the biliary excretion of PCG and its choleretic effect are Mrp2-dependent. Positive correlations were observed between the biliary excretion rate of PCG and bile flow (r(2) = 0.768) and more remarkably between the biliary excretion rate of GSH and bile flow (r(2) = 0.968). No ATP-dependent uptake of [(3)H]PCG was observed in Mrp2-expressing Sf9 membrane vesicles, whereas other forms of Mrp2-substrate transport were stimulated in the presence of PCG. GSH efflux mediated by human MRP2 expressed in Madin-Darby canine kidney II cells was enhanced in the presence of PCG in a concentration-dependent manner. In conclusion, the choleretic effect of PCG is caused by the stimulation of biliary GSH efflux as well as the concentrative biliary excretion of PCG itself, both of which were Mrp2 dependent.     

Short- and long-term effects of biliary drainage on hepatic cholesterol metabolism in the rat.

The present study concerns short- and long-term effects of interruption of the enterohepatic circulation (EHC) on hepatic cholesterol metabolism and biliary secretion in rats. For this purpose, we employed a technique that allows reversible interruption of the EHC, during normal feeding conditions, and excludes effects of anaesthesia and surgical trauma. [3H]Cholesteryl oleate-labelled human low-density lipoprotein (LDL) was injected intravenously in rats with (1) chronically (8 days) interrupted EHC, (2) interrupted EHC at the time of LDL injection and (3) intact EHC. During the first 3 h after interruption of the EHC, bile flow decreased to 50% and biliary bile acid, phospholipid and cholesterol secretion to 5%, 11% and 19% of their initial values respectively. After 8 days of bile diversion, biliary cholesterol output and bile flow were at that same level, but bile acid output was increased 2-3-fold and phospholipid output was about 2 times lower. The total amount of cholesterol in the liver decreased after interruption of the EHC, which was mainly due to a decrease in the amount of cholesteryl ester. Plasma disappearance of LDL was not affected by interruption of the EHC. Biliary secretion of LDL-derived radioactivity occurred 2-4 times faster in chronically interrupted rats as compared with the excretion immediately after interruption of the EHC. Radioactivity was mainly in the form of bile acids under both conditions. This study demonstrates the very rapid changes that occur in cholesterol metabolism and biliary lipid composition after interruption of the EHC. These changes must be taken into account in studies concerning hepatic metabolism of lipoprotein cholesterol and subsequent secretion into bile.