Induction of multidrug resistance-associated protein 2 in liver, intestine and kidney of streptozotocin-induced diabetic rats.

Many studies have demonstrated that Mrp2 is highly regulated in some physiopathological situations. The aim of this study was to investigate effects of diabetes mellitus on function and expression of multidrug resistance-associated protein 2 (Mrp2) in rat liver, kidney and intestine. Diabetic rats were induced by an intraperitoneal administration of streptozotocin (65 mg/kg) and randomly divided into diabetic (DM) rats and insulin-treated diabetic rats. Sulfobromophthalein (BSP), a substrate of Mrp2, was used to evaluate Mrp2 function in vivo. Data from excretion experiments demonstrated that compared with normal rats, diabetes markedly enhanced BSP excretion via bile, urine and intestinal perfusate, which contributed to the elevated plasma clearance of BSP after intravenous administration of 45 µmol/kg BSP. Western blot results showed higher levels of hepatic, renal and intestinal Mrp2 protein in DM rats, although no difference was observed in renal Mrp2. Insulin treatment partly reversed these alterations. Induction of Mrp2 by diabetes was in parallel with the increase in bile flow, levels of biliary and plasma total bile acid (TBA), and plasma conjugated bilirubin in DM rats. Diabetes may enhance Mrp2 function and expression in liver, kidney and intestine, which might be due to insulin deficiency, increased TBA and conjugated bilirubin.

Pharmacokinetics, tissue distribution and excretion of a new photodynamic drug deuxemether.

Deuxemether was a new photodynamic drug effective for many kinds of solid tumor therapy, which was mainly composed of 3-(or 8-)-(1-methoxyethyl)-8-(or 3-)-(1-hydroxyethyl)-deutero-porphyrin IX (MHD) and 3,8-di(1-methoxyethyl)-deuteroporphyrin IX (DMD). The aims of this study were to elucidate its pharmacokinetic characteristics, tissue distribution, plasma protein binding and excretion properties and underlying mechanisms of deuxemether in rats based on the simultaneous determination of MHD and DMD. The pharmacokinetic profiles of both MHD and DMD in rats after intravenous doses were linear and best fitted to a two compartment model, characterized with a rapid distribution phase (MHD: t(1/2)alpha, 0.09-0.14 h; DMD: t 1/2 alpha, 0.07-0.11h) and a relatively slow elimination phase (MHD: t 1/2 beta, 2.03-3.20 h; DMD: t 1/2 beta, 2.51-3.20 h). The tissue distributions of MHD and DMD in rats were rather limited as evidenced from their low distribution volume (0.75-1.70 L/kg) and the results of tissue distribution study. Protein binding of MHD and DMD were moderate (65.36-89.99% for MHD; 45.43-76.23% for DMD), independent of drug concentrations and similar between human and rat plasma over a concentration range of 0.50-50.0 microg/mL. Both MHD and DMD were predominantly (>74.1%) eliminated from rats as the parent drugs through the hepatobiliary systems and finally excreted into the feces. The multidrug resistance-associated proteins 2 (MRP2) inhibitors, bromosulfophthalein and probenecid, substantially inhibited the hepatobiliary elimination of MHD and DMD while the P-gp inhibitor digoxin had little effect, suggesting that MRP2 may contribute to the rapid and extensive hepatobiliary excretion of deuxemether. There were no significant differences between MHD and DMD for all pharmacokinetic characteristics studied. In conclusion, this study provided firstly the full pharmacokinetic characteristics and mechanisms of deuxemether, which would be helpful for its clinical regiment design.

Biliary excretion of glutathione and glutathione disulfide in the rat. Regulation and response to oxidative stress.

Regulation of the biliary excretion of reduced glutathione (GSH) and glutathione disulfide (GSSG) and responses to selected model toxins were examined in male Sprague-Dawley rats. In control and phenobarbital-pretreated rats in which the intrahepatic concentration of GSH was modulated by the administration of diethyl maleate or acetaminophen, the biliary concentration of GSH was consistently lower than, but directly proportional to, the intrahepatic concentration of GSH. Furthermore, increments in bile flow produced by the infusion of sulfobromophthalein (BSP)-glutathione were associated with proportional increases in the biliary excretion of GSH, suggesting that GSH passes into bile passively along a concentration gradient. In contrast, GSSG appears to be secreted into bile against a steep concentration gradient. An increased hepatic production and biliary excretion of GSSG resulted from the administration of t-butyl hydroperoxide. Measurement of biliary GSSG and BSP during a constant infusion of the GSH adduct of BSP indicated that GSSG shares a common excretory mechanism with GSH adducts. Diquat, nitrofurantoin, and paraquat also markedly stimulated the biliary excretion of GSSG. On a molar basis, these compounds generated much more GSSG than a direct substrate for glutathione peroxidase such as t-butyl hydroperoxide, indicating that the compounds undergo redox-cycling with concomitant production of hydrogen peroxide. Aminopyrine (0.8 mmol/kg) also significantly increased biliary GSSG. This increase, however, was associated with a proportional increase in bile flow and in the biliary excretion of GSH such that the GSSG/GSH ratio in bile did not change. Acetaminophen and chloroform, two compounds generating electrophilic metabolites that deplete intrahepatic GSH, led to a progressive decrease in the biliary excretion of GSH and GSSG. Furosemide and dimethylnitrosamine, the electrophilic metabolites of which do not deplete hepatic GSH, minimally altered biliary GSH and GSSG. Similarly, carbon tetrachloride and iproniazid, which yield organic radical metabolites that can peroxidize membrane lipids, did not increase the biliary excretion of GSSG. This finding indicates that membrane-bound lipid hydroperoxides may not be good substrates for glutathione peroxidases. The measurement of the biliary excretion of GSSG and of the GSSG/GSH ratio in bile is a sensitive index of oxidative stress in vivo and thus complements other in vivo parameters for the study of reactive intermediates of xenobiotics such as the determination of covalent binding, the formation of lipid hydroxy acids, and the depletion of intracellular GSH.

[Pharmacokinetic study of cancer chemotherapy].

We reported that the rate of conversion of lactone to carboxylate forms of irinotecan (CPT-11) and its metabolites plays a major role in the biliary excretion of these compounds. Sulfobromophthalein partially inhibited the secretion of SN-38-glucronide into the gastrointestinal lumen, whereas little change was seen in that of active metabolite SN-38. Co-administration of sulphobromophthalein with CPT-11 might lower the late-onset gastrointestinal toxicity observed during treatment with CPT-11 without lowering anticancer activity. In the ileum, the level of transport in the direction form the serosal layer to mucosal layer was significantly greater than that in the direction form the mucosal layer to serosal layer, whereas a significant difference was not observed in the jejunum. This secretory transport required metabolic energy was diminished by sulfobromophthalein. A specific transport system plays a major role in the secretion of SN-38 and that this secretory transport system predominantly exists in the ileum. Uptake of SN-38 was significantly reduced at 4 degrees C. Baicalin inhibited the uptake of SN-38. A specific transport system mediates the uptake of SN-38 across the apical membrane in Caco-2 cells. Inhibition of this transporter would be a useful means for reducing late-onset diarrhea.

Does paracellular permeability play a role in cholephilic dye-induced cholestasis?

Changes in hepatic paracellular permeability were investigated during the development of cholephilic dye-induced cholestasis in rats. For this purpose, four dyes with different cholestatic potency (phenol red, sulfobromophthalein, bromcresol green and rose bengal) were infused at a high, potentially damaging dose (280 nmol/min per 100 g body wt., i.v.), and changes in paracellular permeability were continuously monitored by measuring the access into bile of the permeability probe -14C-sucrose. The cholestatic potency of the different dyes was: rose bengal > bromcresol green > sulfobromophthalein > phenol red. All dyes increased [14C]sucrose bile-to-plasma ratio, producing a displacement towards curves of higher permeability. The capability of the dyes to increase biliary permeability followed the same order as their respective cholestatic potencies. The possible implications of the present results for cholephilic dye-induced cholestasis are discussed.

Concurrent bromosulphophthalein and antipyrine administration to assess liver blood flow and hepatic enzymes in rats.

This study investigated the feasibility of using concurrent iv administration of antipyrine (15 mg/kg body weight) and bromosulphophthalein (BSP; 25 mg/kg) in the rat. Antipyrine is used as an index of hepatic drug metabolism and BSP is used to assess hepatic blood flow. Plasma concentrations of BSP were described using biexponential phases, while antipyrine plasma concentrations were monoexponential. No significant difference was observed between antipyrine pharmacokinetic parameters in concurrent BSP rats when compared with controls. There was also no significant difference between BSP pharmacokinetic parameters in concurrent antipyrine rats when compared with controls, except in the alpha value (P less than 0.05). This indicates that BSP distribution may be affected by concurrent antipyrine administration. Therefore, simultaneous administration of both substrates is not acceptable to study hepatic blood flow. Another iv combination dose (25 mg BSP/kg body weight, followed by 15 mg antipyrine/kg 0.5 hr later) and a dose of 15 mg antipyrine/kg body weight only was administered to rats pretreated with phenobarbital (90 mg/kg body weight) for 6 days. Pharmacokinetic parameters of BSP, beta, k21, k23 and plasma clearance, in the pretreated rats were significantly different from non-pretreated rats. No significant difference was observed in the pharmacokinetic parameters of antipyrine between the combination dose and antipyrine dose in the phenobarbital-pretreated rats. The half-lives of antipyrine in both pretreated groups decreased approximately by 70%, while the clearance increased four times compared with controls. The volume of distribution in these animals did not change as a result of phenobarbital pretreatment. This suggests that a 25 mg BSP/kg body weight dose followed by 15 mg antipyrine/kg 0.5 hr later may be a feasible approach to study liver blood flow, as well as hepatic efficiency in rats.

Absorption of organic anions as model drugs following application to rat liver surface in-vivo.

Absorption of organic anions (phenol red, bromphenol blue and bromosulphonphthalein) has been studied after their application to rat liver surface in-vivo, employing a cylindrical glass cell (i.d. 9 mm, area 0.64 cm2). Each drug appeared gradually in the blood with the peak level at about 1 h, after which its concentration declined slowly. Absorbed model drug was efficiently excreted into the bile. These observations appear to indicate the possibility of drug absorption from liver surface membrane. Absorption of model drugs was estimated to be more than 59% in 6 h. The biliary recovery and metabolism of phenol red did not change as compared with that after intravenous administration.

Comparison of bromosulphthalein and indocyanine green clearance in sheep.

The kinetics of indocyanine green removal from the blood after a single intravenous injection were measured in sheep and compared with those of a single dose of bromosulphthalein given to the same animals 48 hours later. A single dose of 1.5 mg kg-1 of indocyanine green (ICG) was cleared in a double exponential manner. The hepatic uptake of ICG was not significantly different from that of bromosulphthalein (BSP) but its rate of excretion in bile was significantly lower (P < 0.05) and its rate of plasma reflux significantly higher (P < 0.05). The plasma distribution volumes of the two compounds were similar but the mean (SD) plasma clearance of ICG (435 [228.6] ml min-1) was significantly lower than that of BSP (626 [291.9] ml min-1) (P < 0.05). The retention of ICG 30 minutes after the injection was significantly higher than that of BSP (P < 0.01).

Effect of age on liver function.

Serum concentrations of bilirubin, alkaline phosphatase, and GOT as well as the retention of BSP and 131I-rose bengal were determined in 43 normal subjects over 50 years old for the purpose of evaluating age-dependent variations. The normalcy of the liver in all subjects was confirmed by biopsy. The values of serum bilirubin, alkaline phosphatase, and GOT were unaltered with increasing age. Age also had no effect on the retention of BSP and 131I-rose bengal, in contrast to some previous studies in which the normalcy of the liver was not satisfactorily established. No variations between the sexes were seen in our study. The median of the 45 minute BSP retention test was 4.2 percent, with a ninth decile of 7.5 percent. The median of the retention of 131I-rose bengal was 32 percent, with a ninth decile of 39.4 percent.

Mitigation of nitrofurantoin-induced toxicity in the perfused rat liver.

1. Nitrofurantoin is an antimicrobial agent which produces hepatotoxicity caused by the redox cycling of the nitro group and its radical anion. This futile cycling triggers a complex series of events known collectively as oxidative stress. 2. Our goal was to determine treatment strategies which could mitigate nitrofurantoin-induced toxicity in the isolated perfused rat liver. We co-infused various agents which blocked early or late events in the progression to toxicity. Tissue levels of glutathione and protein thiols were measured as indicators of the progression to toxicity and lactate dehydrogenase leakage into the perfusate was used as a marker of irreversible cell death. 3. Five treatments significantly (P < 0.05) decreased LDH leakage (reported as thousands of units accumulated in perfusate at 300 min, mean+/-standard error, n = 3-4) when compared to nitrofurantoin alone (274 +/- 37). These treatments were adenosine-2'-monophosphate (120 +/- 53), penicillamine (90 +/- 29), N-(2-mercaptopropionyl)-glycine (120 +/- 49) and bromosulfophthalein with (80 +/- 29) or without 5,5'-difluro-1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraace tic acid (101 +/- 46). Two other treatments, N-acetylcysteine (183 +/- 7) and dithiothreitol (166 +/- 59) delayed the onset of toxicity. Finally, calpeptin (319 +/- 34) which blocks activation of nonlysosomal proteases was ineffective. 4. We concluded that early intervention on the pathway to toxicity was most effective. The strategies detailed here may prove beneficial in treating hepatotoxicity seen following nitrofurantoin therapy.

Comparison of sulfobromophthalein and indocyanine green clearances in the dog.

Sulfobromophthalein (BSP) and indocyanine green (ICG) were given in single-dose IV injections to dogs and plasma disappearances were measured. The BSP was given at a dose of 5 mg/kg of body weight, and ICG was given at doses of 1.0 and 1.5 mg/kg. The half-life for BSP was 4.2 +/- 0.8 minutes, and for ICG at the 1.0 mg/kg dose, 9.0 +/- 2.0 minutes, and at the 1.5 mg/kg dose, 8.4 +/- 1.9 minutes. The disappearance rate (percentage per minute) for BSP was 17.0 +/- 3.2, and for ICG at the small dose level, 8.1 +/- 1.7, and at the large dose level, 8.7 +/- 2.2. The clearances (ml/min/kg) for BSP were 15.4 +/- 6.2 and for ICG at the small dose level, 3.7 +/- 0.7, and at the large dose level, 3.9 +/- 0.7. The 30-minute percentage retentions for BSP were 1.9 +/- 1.1 and for ICG at the small dose level, 14.7 +/- 5.0, and for ICG at the large dose level, 11.4 +/- 3.0. All of the differences between BSP and ICG means for these values measured were statistically significant (P less than 0.01). There were no significant differences between sexes or between the ICG doses for any of the values (P greater than 0.05).

Comparison of sulfobromophthalein and indocyanine green clearances in the cat.

Plasma disappearances of sulfobromophthalein (BSP) and indocyanine green (ICG) given in single-dose IV injections to cats were measured. The BSP was given at a dose of 5 mg/kg of body weight; ICG was given at a dose of 1.5 mg/kg of body weight. The half-life, disappearance rate (percent per minute) and clearance (ml/min/kg) were determined during the initial phase of plasma dye elimination. The half-life for BSP was 2.2 +/- 0.7 minutes, and for ICG, 3.8 +/- 0.9 minutes. The disappearance rate for BSP was 34.7 +/- 10.1, and for ICG, 19.2 +/- 4.5. The clearance for BSP was 26.3 +/- 23.1 and for ICG, 8.6 +/- 4.1. The 30-minute percentage retention for BSP was 0.6 +/- 0.8% and for ICG, 7.3 +/- 2.9%. In the cat, ICG was cleared from the plasma less rapidly than BSP. There were no significant differences between males and females for these values. For both BSP and ICG, a significantly shorter half-life, faster disappearance rate, and lower 30-minute percentage retention were observed in the cat, compared with values observed in the dog given similar dye dosages. Clearance curves for BSP and ICG in the cat were similar to those in the dog, indicating that the 2 species may have similar volumes of dye distribution and excretory mechanisms. The results of the present study indicate that the 30-minute plasma level following a 5 mg/kg dose of BSP in normal cats is often too low for measurement, whereas the 30-minute plasma concentration of ICG following a 1.5 mg/kg dose is adequate. For the 30-minute dye retention test in the cat, ICG may be a preferred diagnostic agent.

Effect of Haemonchus contortus infection on the clearance of antipyrine, sulfobromophthalein, chloramphenicol, and sulfathiazole in lambs.

A study was made to determine the effect of Haemonchus contortus parasitic infection in lambs on the clearance of several IV administered drugs. Clearance of sulfobromophthalein or sulfathiazole from the plasma of lambs was unaffected by infection with H contortus. Clearance of antipyrine was enhanced by the infection, and thiabendazole treatment did not alter this effect. Clearance of chloramphenicol (CAP), administered as the succinate ester (CAPS), was not changed by the infection, but it was increased after treatment with thiabendazole. Changes in the mean body residence time and initial plasma concentration of CAPS and CAP after treatment with thiabendazole indicate that hydrolysis of CAPS to CAP was reduced. High concentrations of CAPS apparently enhanced its own elimination directly rather than via the expected sequence involving hydrolysis, glucuronidation, and excretion of CAP-glucuronide. Enhanced clearance of antipyrine following infection of lambs with H contortus can be explained in at least 2 ways. First, it is possible that the lambs did not have mature amounts of hepatic drug metabolizing enzyme activity as reported by other investigators, which may be explained by breed differences or animal husbandry practices. Second, infection of lambs by H contortus may have triggered an inductive response in hepatic cytochrome P-450-mediated activities, which might result via a generalized enhancement in hepatic protein synthesis associated with the physiologic response to replace plasma proteins and other blood components lost through gastrointestinal hemorrhage caused by the active feeding of adult worms. Other phase-II reactions such as acetylation, glucuronidation, and glutathione-S-transferase apparently were not affected.

Down-regulation of hepatic transporters for BSP in rats with indomethacin-induced intestinal injury.

Previous reports have demonstrated that an intestinal injury causes hypofunctions of the liver associated with down-regulations of cytochrome P450, but an influence on hepatic transporters remains unclear. Here, we tested hepatic transporter functions in a rat model of bowel injury using indomethacin (IDM). After administration of IDM (8.5 mg/kg, i.p., 3 d), the rats suffered the intestinal impairment indicated by a reduction of alkaline phosphatase activity in mucosa. In vivo pharmacokinetic experiments of bromosulfophthalein (BSP) showed that there was a reduction in its plasma elimination rate and cumulative biliary excretion in IDM-treated rats and systemic and biliary clearances reduced to nearly 50% of the control group. Protein expressions in plasma membrane and mRNA levels of organic anion transporting polypeptide 1b2 (Oatp1b2) and multidrug resistance-associated protein 2 (Mrp2), which play hepatic BSP uptake and biliary excretion, respectively, in the liver were significantly reduced following the IDM treatment. In portal plasma, the levels of proinflammatory cytokines were unchanged, while the level of nitric oxide metabolites (NO2- + NO3-) increased to 6.5-fold that of the control. The time-course on IDM treatment indicated that, firstly, intestinal injury was induced, the NO level increased, and the hepatic Oatp1b2 and Mrp2 expression began to fall followed by an increase in plasma ALT. In conclusion, IDM-induced injury to the small intestine causes the hypofunction of hepatic Oatp1b2 and Mrp2 independently on the hepatic impairment, and NO arising from bowel injury may be one of key factors for it through the remote effect.