Modulation of Rat Liver Regeneration after Partial Hepatectomy by Dietary Cholesterol.

INTRODUCTION: The aim of study was to evaluate impact of long-term dietary cholesterol overload on the cholesterol homeostasis and liver regeneration. MATERIAL AND METHODS: Serum lipid parameters, 14C-cholesterol incorporation, liver DNA synthesis and protein expression was determined in partially hepatectomized (PH) rats fed with a standard (SLD) or hypercholesterolemic (CHOL) diet. RESULTS: 29-day intake of CHOL diet before PH produced increase in serum total cholesterol, LDL lipoprotein, and triglyceride concentration. PH provoked decrease in serum total cholesterol and triglyceride concentration in both groups. PH was associated with increase in serum ALT activity more pronounced in CHOL animals. Hepatic DNA synthesis was increased after PH in both groups, but lower in CHOL. Hypercholesterolemic diet reduced the absorption of radiolabelled cholesterol in intestine and then activity in blood and liver. The 14C-cholesterol hepatic activities tend to increase after PH in both groups. CHOL diet produced up-regulation of Acyl-CoA:cholesterol acyltransferase-2 protein expression. PH was associated with increase of LDL receptor and Acyl-CoA:cholesterol acyltransferase-2 protein expression in both dietary groups. DISCUSSION: Liver regeneration after PH is negatively influenced by CHOL diet. The increased uptake of cholesterol in the liver after PH associated with up-regulation of LDL receptor protein expression suggests preferential use of extrahepatic cholesterol by the liver.

Iron depletion induces hepatic secretion of biliary lipids and glutathione in rats.

Iron depletion (ID) has been shown to induce the liver expression of Cyp7a1, the rate-limiting enzyme initiating conversion of cholesterol to bile acids (BA), although the effect on bile acids metabolism and bile production is unknown. Therefore, we investigated changes in bile secretion and BA synthesis during diet-induced iron depletion (ID) in rats. ID increased bile flow along with augmented biliary excretion of bile acids, glutathione, cholesterol and phospholipids. Accordingly, we found transcriptional upregulation of the Cyp7a1, Cyp8b1, and Cyp27a1 BA synthetic enzymes, as well as induction of the Abcg5/8 cholesterol transporters in ID rat livers. In contrast, intravenous infusion of 3H-taurocholate failed to elicit any difference in biliary secretion of this compound in the ID rats. This corresponded with unchanged expression of canalicular rate-limiting transporters for BA as well as glutathione. We also observed that ID substantially changed the spectrum of BA in bile and decreased plasma concentrations of BA and cholesterol. Experiments with differentiated human hepatic HepaRG cells confirmed human CYP7A1 orthologue upregulation resulting from reduced iron concentrations. Results employing a luciferase reporter gene assay suggest that the transcriptional activation of the CYP7A1 promoter under ID conditions works independent of farnesoid X (FXR), pregnane X (PXR) and liver X (LXRα) receptors activation. It can be concluded that this study characterizes the molecular mechanisms of modified bile production as well as cholesterol as along with BA homeostasis during ID. We propose complex upregulation of BA synthesis, and biliary cholesterol secretion as the key factors affected by ID.

LC-MS/MS method for the determination of haemanthamine in rat plasma, bile and urine and its application to a pilot pharmacokinetic study.

Evidence gathered in various studies points to the fact that haemanthamine, an isoquinoline alkaloid, has multiple medicinally interesting characteristics, including antitumor, antileukemic, antioxidant, antiviral, anticonvulsant and antimalarial activity. This work presents, for the first time, a universal LC-MS/MS method for analysis of haemanthamine in plasma, bile and urine which has been verified in a pilot pharmacokinetic experiment on rats. Chromatographic separation was performed on a pentafluorophenyl core-shell column in gradient elution mode with a mobile phase consisting of acetonitrile-methanol-ammonium formate buffer. A sample preparation based on liquid-liquid extraction with methyl tert-butyl ether was employed with ambelline used as an internal standard. Quantification was performed using LC-MS-ESI(+) in Selected Reaction Monitoring mode. The method was validated according to the European Medicines Agency guideline in a concentration range of 0.1-10 µmol/L in plasma, bile and urine. The concentration-time profiles of haemanthamine in plasma, bile and urine after a single i.v. bolus of 10 mg/kg have been described for the first time. The presented study addresses the lack of information on haemanthamine pharmacokinetics and also introduces a new universal method of haemanthamine analysis in complex biological matrices. Copyright © 2015 John Wiley & Sons, Ltd.

IL-1 receptor blockade alleviates endotoxin-mediated impairment of renal drug excretory functions in rats.

The aim of our study was to investigate whether two potent anti-inflammatory agents, dexamethasone and anakinra, an IL-1 receptor antagonist, may influence acute kidney injury (AKI) and associated drug excretory functions during endotoxemia (LPS) in rats. Ten hours after LPS administration, untreated endotoxemic rats developed typical symptoms of AKI, with reduced GFR, impaired tubular excretion of urea and sodium, and decreased urinary excretion of azithromycin, an anionic substrate for multidrug resistance-transporting proteins. Administration of both immunosuppressants attenuated the inflammatory response, liver damage, AKI, and increased renal clearance of azithromycin mainly by restoration of GFR, without significant influence on its tubular secretion. The lack of such an effect was related to the differential effect of both agents on the renal expression of individual drug transporters. Only dexamethasone increased the urinary clearance of bile acids, in accordance with the reduction of the apical transporter (Asbt) for their tubular reabsorption. In summary, our data demonstrated the potency of both agents used for the prevention of AKI, imposed by endotoxins, and for the restoration of renal drug elimination, mainly by the improvement of GFR. The influence of both drugs on altered tubular functions and the expression of drug transporters was differential, emphasizing the necessity of knowledge of transporting pathways for individual drugs applied during sepsis. The effect of anakinra suggests a significant contribution of IL-1 signaling to the pathogenesis of LPS-induced AKI.

Boldine enhances bile production in rats via osmotic and farnesoid X receptor dependent mechanisms.

Boldine, the major alkaloid from the Chilean Boldo tree, is used in traditional medicine to support bile production, but evidence to support this function is controversial. We analyzed the choleretic potential of boldine, including its molecular background. The acute- and long-term effects of boldine were evaluated in rats either during intravenous infusion or after 28-day oral treatment. Infusion of boldine instantly increased the bile flow 1.4-fold in healthy rats as well as in animals with Mrp2 deficiency or ethinylestradiol induced cholestasis. This effect was not associated with a corresponding increase in bile acid or glutathione biliary excretion, indicating that the effect is not related to stimulation of either bile acid dependent or independent mechanisms of bile formation and points to the osmotic activity of boldine itself. We subsequently analyzed bile production under conditions of changing biliary excretion of boldine after bolus intravenous administration and found strong correlations between both parameters. HPLC analysis showed that bile concentrations of boldine above 10 µM were required for induction of choleresis. Importantly, long-term pretreatment, when the bile collection study was performed 24-h after the last administration of boldine, also accelerated bile formation despite undetectable levels of the compound in bile. The effect paralleled upregulation of the Bsep transporter and increased biliary clearance of its substrates, bile acids. We consequently confirmed the ability of boldine to stimulate the Bsep transcriptional regulator, FXR receptor. In conclusion, our study clarified the mechanisms and circumstances surrounding the choleretic activity of boldine.

Multidrug Resistance-Associated Protein 2 Deficiency Aggravates Estrogen-Induced Impairment of Bile Acid Metabolomics in Rats.

Multidrug resistance-associated protein 2 (Mrp2) mediates biliary secretion of anionic endobiotics and xenobiotics. Genetic alteration of Mrp2 leads to conjugated hyperbilirubinemia and predisposes to the development of intrahepatic cholestasis of pregnancy (ICP), characterized by increased plasma bile acids (BAs) due to mechanisms that are incompletely understood. Therefore, this study aimed to characterize BA metabolomics during experimental Mrp2 deficiency and ICP. ICP was modeled by ethinylestradiol (EE) administration to Mrp2-deficient (TR) rats and their wild-type (WT) controls. Spectra of BAs were analyzed in plasma, bile, and stool using an advanced liquid chromatography-mass spectrometry (LC-MS) method. Changes in BA-related genes and proteins were analyzed in the liver and intestine. Vehicle-administered TR rats demonstrated higher plasma BA concentrations consistent with reduced BA biliary secretion and increased BA efflux from hepatocytes to blood via upregulated multidrug resistance-associated protein 3 (Mrp3) and multidrug resistance-associated protein 4 (Mrp4) transporters. TR rats also showed a decrease in intestinal BA reabsorption due to reduced ileal sodium/bile acid cotransporter (Asbt) expression. Analysis of regulatory mechanisms indicated that activation of the hepatic constitutive androstane receptor (CAR)-Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway by accumulating bilirubin may be responsible for changes in BA metabolomics in TR rats. Ethinylestradiol administration to TR rats further increased plasma BA concentrations as a result of reduced BA uptake and increased efflux via reduced Slco1a1 and upregulated Mrp4 transporters. These results demonstrate that Mrp2-deficient organism is more sensitive to estrogen-induced cholestasis. Inherited deficiency in Mrp2 is associated with activation of Mrp3 and Mrp4 proteins, which is further accentuated by increased estrogen. Bile acid monitoring is therefore highly desirable in pregnant women with conjugated hyperbilirubinemia for early detection of intrahepatic cholestasis.

Pravastatin modulates liver bile acid and cholesterol homeostasis in rats with chronic cholestasis.

BACKGROUND AND AIM: The administration of pravastatin to patients with cholestatic liver disease has suggested the potential of the drug with regard to reducing raised plasma cholesterol and bile acid levels. Information about the mechanisms associated with this effect is lacking. Thus, the aim of the present study is to evaluate pravastatin effects on the liver bile acid and cholesterol homeostasis in healthy and cholestatic rats. METHODS: Control sham-operated and reversibly bile duct-obstructed (BDO) rats were treated with pravastatin (1 or 5 mg/kg) or the vehicle alone for 7 days after surgery. RESULTS: Lower doses of pravastatin reduced bile acid plasma concentrations in cholestatic animals. The effect was associated with reduced liver mRNA expression of Cyp7a1, Cyp8b1, Mrp2, Ugt1a1 and the increased expression of Bsep. In addition, BDO-induced increase in the liver content of cholesterol was normalized by pravastatin. The change was accompanied by the reduced liver expression of Hmg-CoA reductase, LDL receptor, and Acat2, and induced the expression of Abca1 and Mdr2. These changes corresponded with the upregulation of nuclear receptors LXRα and PPARα, and the downregulation of FXR, CAR, SREBP-2 and HNF1α. High doses of pravastatin lacked any positive effects on bile acids and cholesterol homeostasis, and blocked bile formation through the reduction of the biliary excretion of bile acids. CONCLUSIONS: Pravastatin rendered a positive reduction in BDO-induced increases in plasma bile acid concentrations and cholesterol liver content, mainly through the transcriptionally-mediated downregulation of genes involved in the synthesis of these compounds in the liver.

Metformin impairs bile acid homeostasis in ethinylestradiol-induced cholestasis in mice.

Metformin, an oral antidiabetic drug, recently demonstrated a reducing effect on bile acids (BA) plasma concentrations in one patient with intrahepatic cholestasis of pregnancy (ICP) by unknown mechanism. Therefore, the aim of the present study was to examine the effect of metformin on BA homeostasis and related molecular pathways in the liver and intestine using a mouse model of ICP. The cholestasis was induced in female C57BL/6 mice by repeated administration of ethinylestradiol (10 mg/kg BW s.c.) and/or metformin (150 mg/kg BW orally) over 5 consecutive days with subsequent bile collection and molecular analysis of samples. We demonstrated that metformin significantly increased the rate of bile secretion in control mice. This increase was BA dependent and was produced both by increased liver BA synthesis via induced cholesterol 7α-hydroxylase (Cyp7a1) and by increased BA reabsorption in the ileum via induction of the apical sodium-dependent BA transporter (Asbt). In contrast, metformin further worsened ethinylestradiol-induced impairment of bile secretion. This reduction was also BA dependent and corresponded with significant downregulation of Bsep, and Ntcp, major excretory and uptake transporters for BA in hepatocytes, respectively. The plasma concentrations of BA were consequently significantly increased in the metformin-treated mice. Altogether, our data indicate positive stimulation of bile secretion by metformin in the intact liver, but this drug also induces serious impairment of BA biliary secretion, with a marked increase in plasma concentrations in estrogen-induced cholestasis. Our results imply that metformin should be used with caution in situations with hormone-dependent cholestasis, such as ICP.

Up-regulation of renal Mdr1 and Mrp2 transporters during amiodarone pretreatment in rats.

Although amiodarone (AMD) is known to produce drug-drug interactions through inhibition of transporter-mediated excretion of drugs, its impact on these mechanisms during chronic treatment has not been described yet. Therefore, the aim of this study was to investigate the influence of AMD pretreatment on the main multidrug transporting proteins, Mdr1 and Mrp2, in the liver and kidney. The expression of the transporters and pharmacokinetics of their substrates, rhodamine-123 (Rho123) and endogenous conjugated bilirubin (CB), were evaluated in rats after either AMD oral pretreatments (4-14 days) or single intravenous bolus. AMD pretreatment of all durations up-regulated renal Mdr1 and Mrp2 protein expression to 155-190% and 152-223% of the control values, respectively. In agreement, we observed a corresponding increase in renal clearance of both substrates. Hepatic expression was increased only for Mdr1 to 234-270% of controls, which was associated with increased biliary elimination of amiodarone without change in Rho123 biliary clearance. Interestingly, hepatic expression of another Mdr transporter, Mdr2, was progressively decreased by amiodarone administration. Acute administration of AMD reduced Rho123 biliary clearance by 64%. Our results indicate that repeated administration of AMD to rats is associated with significant increase in hepatic and renal expression of Mdr1 and Mrp2 transporters, which may contribute to variability in pharmacokinetics of AMD and simultaneously applied drugs.

Methotrexate released in vitro from bone cement inhibits human stem cell proliferation in S/G2 phase.

Methotrexate (MTX) released from bone cement showed a useful local effect in animal models of bone tumours. However, local toxic reactions such as impaired wound healing were observed in areas surrounding the MTX-loaded implant. Therefore, we hypothesised that MTX released from bone cement would have harmful effects on human mesenchymal stem cells (MSC)-one of the basic components of bone marrow and tissue reparatory processes. Moreover, elution of MTX was calculated from implants prepared either with liquid or powdered MTX. During the 28-day incubation, the cement compounded with liquid MTX showed the highest elution rate of the drug. MTX released from pellets produced a significant decrease in proliferation of MSC as a consequence of a blockade of their cell cycle in the S/G2 phase. These findings indicate impairment of stem cell function in marginal areas surrounding the MTX-loaded cement and may help to explain problems with regeneration of tissues in these locations.

Evaluation of Neutrophil Gelatinase-Associated Lipocalin as a Predictor of Glomerular Filtration Rate and Amikacin Clearance During Early Rat Endotoxemia: Comparison with Traditional Endogenous and Exogenous Biomarkers.

BACKGROUND AND OBJECTIVES: Renal elimination of amikacin and other aminoglycosides is slowed down in sepsis-induced acute kidney injury increasing the risk of adverse effects. Since neutrophil gelatinase-associated lipocalin (NGAL) and aminoglycosides share the mechanisms for renal excretion, the predictive power of NGAL was examined towards the changes in amikacin pharmacokinetics during early endotoxemia in anesthetized Wistar rats. METHODS: Endogenous biomarkers of inflammation and acute kidney injury were assessed including NGAL in saline-injected controls and two groups of rats challenged with an intravenous injection of bacterial lipopolysaccharide (5 mg/kg)-a fluid-resuscitated group (LPS) and a fluid-resuscitated group infused intravenously with 8 µg/kg/h terlipressin (LPS-T). Sinistrin and amikacin were infused to measure glomerular filtration rate (GFR) and amikacin clearance (CLam). The investigations included blood gas analysis, chemistry and hematology tests and assessment of urine output, creatinine clearance (CLcr) and sinistrin clearance (CLsini). RESULTS: Within 3 h of injection, systemic and renal inflammatory responses were induced by lipopolysaccharide. Gene and protein expression of NGAL was increased in the kidneys and the concentrations of NGAL in the plasma (pNGAL) and urine rose 4- to 38-fold (P < 0.01). The decreases in CLam and the GFR markers (CLcr, CLsini) were proportional, reflecting the extent to which endotoxemia impaired the major elimination mechanism for the drug. Terlipressin attenuated lipopolysaccharide-induced renal dysfunction (urine output, CLcr, CLsini) and accelerated CLam. The pNGAL showed a strong association with the CLsini (rs = - 0.77, P < 0.0005). Concerning prediction of CLam, pNGAL was comparable to CLcr (mean error - 24%) and inferior to CLsini (mean error - 6.4%), while the measurement of NGAL in urine gave unsatisfactory results. CONCLUSIONS: During early endotoxemia in the rat, pNGAL has a moderate predictive ability towards CLam. Clinical studies should verify whether pNGAL can support individualized dosing of aminoglycosides to septic patients.

Alteration of methotrexate biliary and renal elimination during extrahepatic and intrahepatic cholestasis in rats.

Methotrexate (MTX), an important anticancer and immunosuppressive agent, has been suggested for the treatment of primary biliary cirrhosis. However, the drug's pharmacodynamics and toxicity is dependent on its concentrations in plasma which in turn are directly related to MTX's elimination in the liver and kidney. Therefore, the aim of this study was to evaluate changes in MTX biliary and renal excretion during either intrahepatic or obstructive cholestasis in rats. The steady state pharmacokinetic parameters of MTX were evaluated in rats one (BDO1) or seven (BDO7) days after bile duct obstruction (BDO) or 18 h after administration of lipopolysaccharide (LPS). In comparison to the respective control groups, biliary and total clearances of MTX were decreased to 12% and 49% in the BDO1 group, to 5% and 56% in the BDO7 animals, and to 42% and 43% in the LPS group, respectively. Renal clearance of MTX was unchanged in BDO groups, but decreased to 23% of controls in the LPS animals. The serum biochemistry and expression of main hepatic MTX transporters (Mrp2, Mrp3, Mrp4, Bcrp, Oatp1a1, Oatp1a4 and Oatp1b2) confirmed the pathological cholestatic changes in the liver and partly elucidated the cause of changes in MTX pharmacokinetic parameters. In conclusion, this study is the first describing marked alteration of MTX hepatic and renal elimination induced by cholestasis in rats. Moreover, the reported changes in MTX pharmacokinetics and respective transporter expression suggest important mechanistic differences between the two widely used cholestatic models.

Iron overload reduces synthesis and elimination of bile acids in rat liver.

Excessive iron accumulation in the liver, which accompanies certain genetic or metabolic diseases, impairs bile acids (BA) synthesis, but the influence of iron on the complex process of BA homeostasis is unknown. Thus, we evaluated the effect of iron overload (IO) on BA turnover in rats. Compared with control rats, IO (8 intraperitoneal doses of 100 mg/kg every other day) significantly decreased bile flow as a consequence of decreased biliary BA secretion. This decrease was associated with reduced expression of Cyp7a1, the rate limiting enzyme in the conversion of cholesterol to BA, and decreased expression of Bsep, the transporter responsible for BA efflux into bile. However, IO did not change net BA content in faeces in response to increased intestinal conversion of BA into hyodeoxycholic acid. In addition, IO increased plasma cholesterol concentrations, which corresponded with reduced Cyp7a1 expression and increased expression of Hmgcr, the rate-limiting enzyme in de novo cholesterol synthesis. In summary, this study describes the mechanisms impairing synthesis, biliary secretion and intestinal processing of BA during IO. Altered elimination pathways for BA and cholesterol may interfere with the pathophysiology of liver damage accompanying liver diseases with excessive iron deposition.

High soluble endoglin levels regulate cholesterol homeostasis and bile acids turnover in the liver of transgenic mice.

AIMS: Increased plasma soluble endoglin concentrations (sEng) are frequently detected in metabolic disorders accompanied with hypercholesterolemia in serum, but effect of sEng on the cholesterol biochemistry is unknown. Cholesterol and bile acids (BA) are important products of liver metabolism with numerous functions within the organism. Turnover of these substances requires precise regulation due to potential toxicities during their cumulation. In this study, we hypothesized that high sEng levels affect cholesterol homeostasis and BA turnover in mice liver. MAIN METHODS: Nine-month-old transgenic male mice overexpressing human sEng and wild-type mice underwent plasma, bile, stool, and organ samples analysis by analytical, qRT-PCT and Western blot methods. KEY FINDINGS: sEng mice demonstrated decreased plasma total and LDL cholesterol concentrations due to upregulation of hepatic Sr-b1 and Ldlr receptors, increased liver cholesterol content, and increased Abcg8-mediated cholesterol efflux into bile. sEng also increased conversion of cholesterol into bile acids (BA) via upregulation of Cyp7a1 and increased Mdr1 expression. Plasma concentrations of BA were increased in sEng mice due to their enhanced reabsorption via ileum. Increased hepatic disposition of BA led to their increased biliary excretion coupled with choleretic activity. SIGNIFICANCE: For the first time, we have shown that high sEng plasma levels affect cholesterol and BA homeostasis on the basis of complex liver and intestinal effects. The significance of these findings for pathophysiology of diseases associated with increased sEng concentrations remains to be elucidated in prospective studies.

P-glycoprotein function and expression during obstructive cholestasis in rats.

OBJECTIVES: The present study was aimed at evaluation of in vivo biliary and renal excretion of rhodamine 123 (Rho123), a P-glycoprotein (P-gp) substrate, in rats during either acute or chronic cholestasis induced by bile duct obstruction (BDO). METHODS: The Rho123 clearance study was performed either one (BDO1) or seven (BDO7) days after BDO. Bile flow was reconstituted, and bile and urine were collected after steady-state plasma concentration of Rho123 was attained. Tissue expression of P-gp was evaluated by quantitative immunohistochemistry, and immunoblotting. RESULTS: Significant up-regulation of the liver P-gp protein was observed in acute and chronic cholestasis. Primary periportal location of P-gp was enlarged also to pericentral areas. In the kidneys, immunohistochemistry showed pancellular increase in P-gp after 1 day of BDO, which subsided after 7 days of BDO. Nevertheless, biliary and renal clearances (CL(Bile) and CL(R)) of Rho123 did not reflect the induction of P-gp expression. While CL(Bile) was reduced one day after cholestasis and restored on the seventh day, the CL(R) was preserved in BDO1 group and reduced in BDO7 group without change in glomerular filtration rate. In parallel, biliary and renal clearances of conjugated bilirubin were significantly reduced in both cholestatic groups compared with controls. CONCLUSION: These findings suggest that extrahepatic cholestasis causes time-dependent changes in elimination of Rho123 which do not exactly reflect alteration of P-gp expression in the rat liver and kidney. These data may help to explain impaired elimination of P-gp substrates after short-term cholestasis that may commonly occur in clinical practice.

Zonation of multidrug resistance-associated protein 2 in rat liver after induction with dexamethasone.

BACKGROUND AND AIM: The present study was aimed to evaluate the hepatic zonation of multidrug resistance-associated protein 2 (mrp2), an important drug transporter, and its potential changes during the induction of its expression by known inducer, dexamethasone (DEX). METHODS: The hepatic expression of mrp2 was studied by immunohistochemistry with consequent quantification by measurement of integral optical densities of mrp2 staining in the periportal and perivenous areas of the liver acinus in control and DEX-pretreated rats (1 mg/kg daily per os for 4 days). Overall changes in mrp2 expression and function produced by DEX were monitored using Western blotting and an in vivo clearance study of endogenous-conjugated bilirubin, a mrp2 substrate. RESULTS: In the control animals, a quantitative image analysis revealed the primary periportal localization of mrp2 within the liver acinus with the expression of mrp2 being 16.7-fold of that in the perivenous area. After DEX pretreatment, the expression of mrp2 increased, especially in the perivenous hepatocytes. The overall expression of mrp2 increased 3.2-fold in comparison with the control group. This observation was confirmed by Western blotting, which showed a 1.3-fold increase in the mrp2 protein after DEX pretreatment. The functional consequences of the induced mrp2 protein in the livers of the DEX-pretreated rats were demonstrated by the increased biliary excretion of conjugated bilirubin. CONCLUSION: In conclusion, these results indicate the zonation of mrp2 protein expression primarily to periportal hepatocytes. The induction by DEX produced spatially disproportional changes with an increase in the mrp2 protein being most prominent in the perivenous hepatocytes.

Increased melibiose/rhamnose ratio in bile of rats with acute cholestasis.

BACKGROUND AND AIM: Melibiose/rhamnose permeability test is used for noninvasive intestinal mucosa barrier testing. However, the possible escape route of the absorbed saccharides through either intact or impaired blood-biliary barriers has not so far been explored. The objective of the present study was therefore two-fold: First, to describe in detail the biliary pharmacokinetics of melibiose and rhamnose in rats; second, to evaluate the changes of both sugars' pharmacokinetics upon impairment of the blood-biliary barrier by acute extrahepatic cholestasis in rats. METHODS: Bile duct obstructed (BDO), sham-operated and intact (unoperated) male Wistar rats were administered, 24 h after the appropriate intervention, with a single intravenous dose of melibiose and rhamnose, and a 4-h pharmacokinetic study was performed. RESULTS: In intact animals, the biliary excretion of melibiose and rhamnose was only 0.06% and 0.4% of the administered dose, respectively, while the urinary excretion accounted for 70.6% and 61.7%, respectively. In BDO animals, the biliary excretion rate of both saccharides, especially that of melibiose, was increased with a consequent 4.4-fold rise of the biliary melibiose/rhamnose ratio, the accepted paracellular permeability indicator. Both, the renal clearance of melibiose and the urinary melibiose/rhamnose ratio remained uninfluenced by cholestasis. CONCLUSION: The present study is the first to describe in detail pharmacokinetic parameters and the biliary excretion of melibiose and rhamnose in healthy and cholestatic rats. The altered melibiose/rhamnose biliary excretion ratio in BDO rats indicates that the test is able to detect the impairment of the blood-biliary barrier in acute extrahepatic cholestasis.

Resveratrol modifies biliary secretion of cholephilic compounds in sham-operated and cholestatic rats.

AIM: To investigate the effect of resveratrol on biliary secretion of cholephilic compounds in healthy and bile duct-obstructed rats. METHODS: Resveratrol (RSV) or saline were administered to rats by daily oral gavage for 28 d after sham operation or reversible bile duct obstruction (BDO). Bile was collected 24 h after the last gavage during an intravenous bolus dose of the Mdr1/Mrp2 substrate azithromycin. Bile acids, glutathione and azithromycin were measured in bile to quantify their level of biliary secretion. Liver expression of enzymes and transporters relevant for bile production and biliary secretion of major bile constituents and drugs were analyzed at the mRNA and protein levels using qRT-PCR and Western blot analysis, respectively. The TR-FRET PXR Competitive Binding Assay kit was used to determine the agonism of RSV at the pregnane X receptor. RESULTS: RSV increased bile flow in sham-operated rats due to increased biliary secretion of bile acids (BA) and glutathione. This effect was accompanied by the induction of the hepatic rate-limiting transporters for bile acids and glutathione, Bsep and Mrp2, respectively. RSV also induced Cyp7a1, an enzyme that is crucial for bile acid synthesis; Mrp4, a transporter important for BA secretion from hepatocytes to blood; and Mdr1, the major apical transporter for xenobiotics. The findings were supported by increased biliary secretion of azithromycin. The TR-FRET PXR competitive binding assay confirmed RSV as a weak agonist of the human nuclear receptor PXR, which is a transcriptional regulator of Mdr1/Mrp2. RSV demonstrated significant hepatoprotective properties against BDO-induced cirrhosis. RSV also reduced bile flow in BDO rats without any corresponding change in the levels of the transporters and enzymes involved in RSV-mediated hepatoprotection. CONCLUSION: Resveratrol administration for 28 d has a distinct effect on bile flow and biliary secretion of cholephilic compounds in healthy and bile duct-obstructed rats.

Novel and potent anti-tumor and anti-metastatic di-2-pyridylketone thiosemicarbazones demonstrate marked differences in pharmacology between the first and second generation lead agents.

Di(2-pyridyl)ketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di(2-pyridyl)ketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) are novel, highly potent and selective anti-tumor and anti-metastatic drugs. Despite their structural similarity, these agents differ in their efficacy and toxicity in-vivo. Considering this, a comparison of their pharmacokinetic and pharmaco/toxico-dynamic properties was conducted to reveal if these factors are involved in their differential activity. Both compounds were administered to Wistar rats intravenously (2 mg/kg) and their metabolism and disposition were studied using UHPLC-MS/MS. The cytotoxicity of both thiosemicarbazones and their metabolites was also examined using MCF-7, HL-60 and HCT116 tumor cells and 3T3 fibroblasts and H9c2 cardiac myoblasts. Their intracellular iron-binding ability was characterized by the Calcein-AM assay and their iron mobilization efficacy was evaluated. In contrast to DpC, Dp44mT undergoes rapid demethylation in-vivo, which may be related to its markedly faster elimination (T1/2 = 1.7 h for Dp44mT vs. 10.7 h for DpC) and lower exposure. Incubation of these compounds with cancer cells or cardiac myoblasts did not result in any significant metabolism in-vitro. The metabolism of Dp44mT in-vivo resulted in decreased anti-cancer activity and toxicity. In conclusion, marked differences in the pharmacology of Dp44mT and DpC were observed and highlight the favorable pharmacokinetics of DpC for cancer treatment.

Deferoxamine but not dexrazoxane alleviates liver injury induced by endotoxemia in rats.

The purpose of the present study was to compare the activity of two different clinically available iron chelators on the development of acute liver injury after administration of the bacterial endotoxin (lipopolysaccharide [LPS]) in rats. Lipopolysaccharide was administered either alone or after pretreatment with dexrazoxane (DEX) or deferoxamine (DFO). Control groups received only saline or its combination with either chelator. After 8 h, untreated LPS rats developed liver injury, with signs of inflammation and oxidative stress. Lipopolysaccharide reduced plasma iron concentrations in association with increased production of hepcidin and the reduced liver expression of ferroportin. Administration of chelating agents to LPS animals showed distinct effects. Although both drugs were able to reduce liver iron content, together with corresponding changes in hepcidin and ferroportin expressions, only DFO showed a protective effect against liver injury despite relatively small liver concentrations. In sharp contrast, DEX failed to improve any hallmark of liver injury and even worsened the GSH/GSSG ratio, the indicator of oxidative stress in the tissue. High-performance liquid chromatography-mass spectrometry analysis showed marked liver accumulation of iron-chelating metabolite of DEX (ADR-925), whereas the parent compound was undetectable. Further downregulation of transporters involved in bile formation was observed after DFO in the LPS group as well as in healthy animals. Neither chelator imposed significant liver injury in healthy animals. In conclusion, we demonstrated marked differences in the modulation of endotoxemic liver impairment between two iron chelators, implicating that particular qualities of chelating agents may be of crucial importance.