Elevated cardiac 3-deoxyglucosone, a highly reactive intermediate in glycation reaction, in doxorubicin-induced cardiotoxicity in rats.

3-Deoxyglucosone (3-DG) is a highly reactive carbonyl intermediate in glycation reaction (also known as Maillard reaction) and plays an important role in diabetic complications. We investigated the potential involvement of 3-DG in doxorubicin (DXR)-induced cardiotoxicity. Male Crl:CD(SD) rats received intravenous injections of DXR at 2mg/kg, once weekly, for 6 weeks, with/without daily intraperitoneal treatment with 3-DG scavenging agents, i.e., aminoguanidine (AG, 25mg/kg/day) and pyridoxamine (PM, 60mg/kg/day). Cardiac levels of 3-DG, thiobarbituric acid reactive substances (TBARS), fructosamine, and pentosidine, plasma glucose levels and cardiac troponin I (cTnI), echocardiography, and histopathology were assessed at 4 and 6 weeks after treatment. Cardiac 3-DG levels were significantly increased by DXR treatment at 4 and 6 weeks. Cardiac fructosamine levels and plasma glucose were not altered by DXR; however, TBARS levels in the heart were significantly increased at 4 and 6 weeks, suggesting that the enhanced generation of 3-DG is not attributed to any abnormal glycemic status, but may be related to oxidative stress by DXR. An advanced glycation end-product, pentosidine, was significantly increased by DXR treatment at 6 weeks. Intervention by AG and PM ameliorated the DXR-induced echocardiographic abnormalities, increased cTnI in plasma, and histopathological lesion as well as normalizing the elevation of 3-DG and pentosidine levels. These results suggest that 3-DG is generated by DXR and involved, at least in part, in the pathogenesis of DXR-cardiotoxicity through glycation reaction.

Metabolic activation of hepatotoxic drug (benzbromarone) induced mitochondrial membrane permeability transition.

The risk of drug-induced liver injury (DILI) is of great concern to the pharmaceutical industry. It is well-known that metabolic activation of drugs to form toxic metabolites (TMs) is strongly associated with DILI onset. Drug-induced mitochondrial dysfunction is also strongly associated with increased risk of DILI. However, it is difficult to determine the target of TMs associated with exacerbation of DILI because of difficulties in identifying and purifying TMs. In this study, we propose a sequential in vitro assay system to assess TM formation and their ability to induce mitochondrial permeability transition (MPT) in a one-pot process. In this assay system, freshly-isolated rat liver mitochondria were incubated with reaction solutions of 44 test drugs preincubated with liver microsomes in the presence or absence of NADPH; then, NADPH-dependent MPT pore opening was assessed as mitochondrial swelling. In this assay system, several hepatotoxic drugs, including benzbromarone (BBR), significantly induced MPT in a NADPH-dependent manner. We investigated the rationality of using BBR as a model drug, since it showed the most prominent MPT in our assay system. Both the production of a candidate toxic metabolite of BBR (1',6-(OH)2 BBR) and NADPH-dependent MPT were inhibited by several cytochrome P450 (CYP) inhibitors (clotrimazole and SKF-525A, 100µM). In summary, this assay system can be used to evaluate comprehensive metabolite-dependent MPT without identification or purification of metabolites.

Mitochondrial iron accumulation exacerbates hepatic toxicity caused by hepatitis C virus core protein.

Patients with long-lasting hepatitis C virus (HCV) infection are at major risk of hepatocellular carcinoma (HCC). Iron accumulation in the livers of these patients is thought to exacerbate conditions of oxidative stress. Transgenic mice that express the HCV core protein develop HCC after the steatosis stage and produce an excess of hepatic reactive oxygen species (ROS). The overproduction of ROS in the liver is the net result of HCV core protein-induced dysfunction of the mitochondrial respiratory chain. This study examined the impact of ferric nitrilacetic acid (Fe-NTA)-mediated iron overload on mitochondrial damage and ROS production in HCV core protein-expressing HepG2 (human HCC) cells (Hep39b cells). A decrease in mitochondrial membrane potential and ROS production were observed following Fe-NTA treatment. After continuous exposure to Fe-NTA for six days, cell toxicity was observed in Hep39b cells, but not in mock (vector-transfected) HepG2 cells. Moreover, mitochondrial iron ((59)Fe) uptake was increased in the livers of HCV core protein-expressing transgenic mice. This increase in mitochondrial iron uptake was inhibited by Ru360, a mitochondrial Ca(2+) uniporter inhibitor. Furthermore, the Fe-NTA-induced augmentation of mitochondrial dysfunction, ROS production, and cell toxicity were also inhibited by Ru360 in Hep39b cells. Taken together, these results indicate that Ca(2+) uniporter-mediated mitochondrial accumulation of iron exacerbates hepatocyte toxicity caused by the HCV core protein.

Sustained intrahepatic glutathione depletion causes proteasomal degradation of multidrug resistance-associated protein 2 in rat liver.

Multidrug resistance-associated protein 2 (MRP2) is a member of a family of efflux transporters that are involved in biliary excretion of organic anions from hepatocytes. Disrupted canalicular localization and decreased protein expression of MRP2 have been observed in patients with chronic cholestatic disorder and hepatic failure without a change in its mRNA expression. We have previously demonstrated that post-transcriptional regulation of the rapid retrieval of rat MRP2 from the canalicular membrane to the intracelluar compartment occurs under conditions of acute (~30min) oxidative stress. However, it is unclear whether MRP2 expression is decreased during its sustained internalization during chronic oxidative stress. The present study employed buthionine sulfoximine (BSO) to induce chronic oxidative stress in the livers of Sprague-Dawley rats and then examined the protein expression and localization of MRP2. Canalicular MRP2 localization was altered by BSO treatment for 2h without changing the hepatic protein expression of MRP2. While the 8h after exposure to BSO, hepatic MRP2 protein expression was decreased, and the canalicular localization of MRP2 was disrupted without changing the mRNA expression of MRP2. The BSO-induced reduction in MRP2 protein expression was suppressed by pretreatment with N-benzyloxycarbonyl (Cbz)-Leu-Leu-leucinal ( MG-132), a proteasomal inhibitor. Furthermore, the modification of MRP2 by small ubiquitin-relatedmodifier 1 (SUMO-1) was impaired in BSO-treated rat liver,while that by ubiquitin (Ub) and MRP2 was enhanced. Taken together, the results of this study suggest the sustained periods of low GSH content coupled with altered modification of MRP2 by Ub/SUMO-1 were accompanied by proteasomal degradation of MRP2.

Ezrin regulates the expression of Mrp2/Abcc2 and Mdr1/Abcb1 along the rat small intestinal tract.

Multidrug resistance-associated protein 2 (MRP2)/ATP-binding cassette protein C2 (ABCC2) and multidrug resistance protein 1 (MDR1)/ABCB1 are well-known efflux transporters located on the brush border membrane of the small intestinal epithelia, where they limit the absorption of a broad range of substrates. The expression patterns of MRP2/ABCC2 and MDR1/ABCB1 along the small intestinal tract are tightly regulated. Several reports have demonstrated the participation of ERM (ezrin/radixin/moesin) proteins in the posttranslational modulation of MRP2/ABCC2 and MDR1/ABCB1, especially with regard to their membrane localization. The present study focused on the in vivo expression profiles of MRP2/ABCC2, MDR1/ABCB1, ezrin, and phosphorylated ezrin to further elucidate the relationship between the efflux transporters and the ERM proteins. The current results showed good correlation between the phosphorylation status of ezrin and Mrp2/Abcc2 expression along the gastrointestinal tract of rats and between the expression profiles of both ezrin and Mdr1/Abcb1 in the small intestine. We also demonstrated the involvement of conventional protein kinase C isoforms in the regulation of ezrin phosphorylation. Furthermore, experiments conducted with wild-type (WT) ezrin and a T567A (Ala substituted Thr) dephosphorylated mutant showed a decrease in membrane surface-localized and total expressed MRP2/ABCC2 in T567A-expressing vs. WT ezrin-expressing Caco-2 cells. In contrast, T567A- and WT-expressing cells both showed an increase in membrane surface-localized and total expressed MDR1/ABCB1. These findings suggest that the phosphorylation status and the expression profile of ezrin differentially direct MRP2/ABCC2 and MDR1/ABCB1 expression, respectively, along the small intestinal tract.

The mechanism of the down-regulation of hepatic transporters in rats with indomethacin-induced intestinal injury.

BACKGROUND: Previously, we reported that hepatic transporters were down-regulated consistent with intestinal injury in indomethacin (IDM)-treated rats. AIM: The purpose of this study was to characterize this mechanism of the down-regulation of hepatic transporters in IDM-treated rats. METHODS: Hepatic nuclear receptor expressions, oxidative stress condition and the expression of hepatic transporters were evaluated in rats with IDM-induced intestinal injury with or without the administration of mucosal protectant ornoprostil, a prostaglandin E1 analogue, or aminoguanidine (AG), an iNOS inhibitor. RESULTS: All the nuclear receptors examined in the present study, which regulates hepatic transporters, were decreased by the administration of IDM. Hepatic glutathione, an indicator of oxidative stress, was significantly reduced compared with control. We then determined the expression of hepatic transporters by semi-quantitative real-time RT-PCR and Western blot analysis in IDM-treated rats with or without the administration of ornoprostil or AG. Ornoprostil recovered the gene expression of Oatp1a1, Oatp1b2 and Mrp2 and protein expression of Mrp2 while it had no effect on Oatp1a1 and Oatp1b2 proteins. These results indicated that the gene expression of hepatic transporters was down-regulated in association with the intestinal injury. On the other hand, there is no effect of AG on the reduced gene expression of hepatic Oatp1a1, Oatp1b2 and Mrp2. In protein expression, AG slightly recovered Mrp2 expression accompanied by a partial decrease in portal NO levels. CONCLUSIONS: We suggest that the transcriptional process influenced by a dysfunction of hepatic nuclear receptors as well as the effect of NO on the post-transcriptional process due to intestinal injury are partially involved in the down-regulation of hepatic transporters.

Clinical significance of organic anion transporting polypeptides (OATPs) in drug disposition: their roles in hepatic clearance and intestinal absorption.

Organic anion transporting polypeptide (OATP) family transporters accept a number of drugs and are increasingly being recognized as important factors in governing drug and metabolite pharmacokinetics. OATP1B1 and OATP1B3 play an important role in hepatic drug uptake while OATP2B1 and OATP1A2 might be key players in intestinal absorption and transport across blood-brain barrier of drugs, respectively. To understand the importance of OATPs in the hepatic clearance of drugs, the rate-determining process for elimination should be considered; for some drugs, hepatic uptake clearance rather than metabolic intrinsic clearance is the more important determinant of hepatic clearances. The importance of the unbound concentration ratio (liver/blood), K(p,uu) , of drugs, which is partly governed by OATPs, is exemplified in interpreting the difference in the IC(50) of statins between the hepatocyte and microsome systems for the inhibition of HMG-CoA reductase activity. The intrinsic activity and/or expression level of OATPs are affected by genetic polymorphisms and drug-drug interactions. Their effects on the elimination rate or intestinal absorption rate of drugs may sometimes depend on the substrate drug. This is partly because of the different contribution of OATP isoforms to clearance or intestinal absorption. When the contribution of the OATP-mediated pathway is substantial, the pharmacokinetics of substrate drugs should be greatly affected. This review describes the estimation of the contribution of OATP1B1 to the total hepatic uptake of drugs from the data of fold-increases in the plasma concentration of substrate drugs by the genetic polymorphism of this transporter. To understand the importance of the OATP family transporters, modeling and simulation with a physiologically based pharmacokinetic model are helpful.

Interaction of Mrp2 with radixin causes reversible canalicular Mrp2 localization induced by intracellular redox status.

Oxidative stress is a feature of cholestatic syndrome and induces multidrug resistance-associated protein 2 (Mrp2) internalization from the canalicular membrane surface. We have previously shown that the activation of a novel protein kinase C (nPKC) by oxidative stress regulates Mrp2 internalization. The internalized Mrp2 was recycled to the canalicular surface in a protein kinase A (PKA)-dependent manner after intracellular glutathione (GSH) levels were replenished. However, the putative phosphorylation targets of these protein kinases involved in reversible Mrp2 trafficking remain unclear. In this study, we investigated the effect of changing the intrahepatic redox status on the C-terminal phosphorylation status of radixin (p-radixin), which links Mrp2 to F-actin, and the interaction of p-radixin with Mrp2 in rat hepatocytes. We detected a significant decrease in the amount of p-radixin that co-immunoprecipitated with Mrp2 after tertiary-butylhydroperoxide (t-BHP) treatment. After treatment with GSH-ethylester (GSH-EE), the phosphorylation level became the same as that of the control. A PKC and protein phosphatase (PP)-1/2A inhibitor, but not a PP-2A selective inhibitor, prevented the t-BHP-induced decrease of p-radixin and subsequent canalicular Mrp2 localization. In contrast, a PKA inhibitor affected the recovery process facilitated by GSH-EE treatment. In conclusion, the interaction of p-radixin with Mrp2 was decreased by the activation of PKC and PP-1 under oxidative stress conditions which subsequently led to Mrp2 internalization, whereas the interaction of p-radixin and Mrp2 was increased by the activation of PKA during recovery from oxidative stress.

Mechanistic differences in permeation behavior of supersaturated and solubilized solutions of carbamazepine revealed by nuclear magnetic resonance measurements.

A solid dispersion (SPD) of carbamazepine (CBZ) with hydroxypropyl methylcellulose acetate succinate (HPMC-AS) was prepared by the spray drying method. The apparent solubility (37 °C, pH 7.4) of CBZ observed with the SPD was over 3 times higher than the solubility of unprocessed CBZ. The supersaturated solution was stable for 7 days. A higher concentration of CBZ in aqueous medium was also achieved by mixing with Poloxamer 407 (P407), a solubilizing agent. From permeation studies of CBZ using Caco-2 monolayers and dialysis membranes, we observed improved CBZ permeation across the membrane in the supersaturated solution of CBZ/HPMC-AS SPD. On the contrary, the CBZ-solubilized P407 solution exhibited poor permeation by CBZ. The chemical shifts of CBZ on the (1)H NMR spectrum from CBZ/HPMC-AS SPD solution were not altered significantly by coexistence with HPMC-AS. In contrast, an upfield shift of CBZ was observed in the CBZ/P407 solution. The spin-lattice relaxation time (T(1)) over spin-spin relaxation time (T(2)) indicated that the mobility of CBZ in the HPMC-AS solution was much lower than that in water. Meanwhile, the mobility of CBZ in P407 solution was significantly higher than that in water. NMR data indicate that CBZ does not strongly interact with HPMC-AS. CBZ mobility was suppressed due to self-association and microviscosity around CBZ, which do not affect permeation behavior. Most of the CBZ molecules in the CBZ/P407 solution were solubilized in the hydrophobic core of P407, and a few were free to permeate the membrane. The molecular state of CBZ, as evaluated by NMR measurements, directly correlated with permeation behavior.

Bile salt export pump inhibitors are associated with bile acid-dependent drug-induced toxicity in sandwich-cultured hepatocytes.

Drug-induced liver injury (DILI) is a major reason for the dropout of candidate compounds from drug testing and the withdrawal of pharmaceuticals from clinical use. Among the various mechanisms of liver injury, the accumulation of bile acids (BAs) within hepatocytes is thought to be a primary mechanism for the development of DILI. Although bile salt export pump (BSEP) dysfunction is considered a susceptibility factor for DILI, little is known about the relationship between drug-induced BSEP dysfunction and BA-dependent hepatotoxicity. Furthermore, few methods are at hand for the systematic and quantitative evaluation of BA-dependent DILI. This study aimed to construct a model of DILI by employing sandwich-cultured hepatocytes (SCHs). SCHs can be used to assess functions of canalicular transporters such as BSEP and the activity of metabolic enzymes. Here, the impact of 26 test compounds (ritonavir, troglitazone, etc.) was investigated on BA-dependent cytotoxicity in SCHs. SCHs were exposed to each compound for 24h with or without BAs (glycochenodeoxycholic acid, deoxycholic acid, etc.). As a result, BA-dependent toxicity was observed for 11 test compounds in SCHs treated in the presence of BAs, while no signs of toxicity were observed for SCHs treated in the absence of BAs. Of the 11 compounds, nine were known BSEP inhibitors. Moreover, for some compounds, an increase in the severity of BA-dependent toxicity was observed in SCHs that were co-treated with 1-aminobenzotriazole, a non-selective inhibitor of cytochrome P450 (CYP450)-mediated drug metabolism. These results indicate that the SCH-based model is likely to prove useful for the evaluation of BA-dependent DILI, including the effects of drug metabolism and BSEP inhibition on liver injury.

Population pharmacokinetic analysis of letrozole in Japanese postmenopausal women.

PURPOSE: Letrozole is an orally active aromatase inhibitor for the treatment of breast cancer. The objectives of this study were to examine the pharmacokinetic profile of letrozole in Japanese subjects and to identify factors that influence variability in the pharmacokinetics of letrozole using population pharmacokinetic (PPK) analysis. METHODS: Twenty-five healthy postmenopausal Japanese women were enrolled in the study and received 2.5 mg letrozole once daily for 14 or 28 days. A PPK model was developed using NONMEM software. Age, body weight (WT), AST, ALT, total bilirubin, serum creatinine (CRE), and genotype of CYP2A6 were studied as covariates. Estrone, estrone sulfate, and estradiol in plasma were measured as pharmacodynamic markers. RESULTS: CYP2A6 genotype, CRE, and AST were significant covariates for apparent systemic clearance (CL/F), and WT was a significant covariate for apparent distribution volume (Vd/F). Population mean estimates of CL/F and Vd/F in subjects without CYP2A6 mutation were 1.03 × (CRE/0.70)(-1.27) × (AST/17.5)(-0.793) L/h and 94.2 × (WT/51.1)(1.12) L respectively. CL/F in subjects possessing 1 and 2 CYP2A6 mutation alleles were 84.3% and 44.8% of the value in the subjects without mutation respectively. Estrogen levels fell to below detection limits in most subjects after letrozole administration. Three mild and transient adverse events (upper respiratory tract inflammation, arthralgia, and vomiting) were reported in the study. CONCLUSIONS: CYP2A6 genotype largely influences CL/F of letrozole. Genetic polymorphism of CYP2A6 and body weight will be causes of ethnic difference in PK. However, dose adjustment is not necessary, because of the wide therapeutic range.

Oxidative stress is a triggering factor for LPS-induced Mrp2 internalization in the cryopreserved rat and human liver slices.

Cholestasis develops during inflammation and is characterized as occurring under oxidative stress. We have described the internalization of multidrug resistance-associated protein 2 (Mrp2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid or lipopolysaccharide (LPS)-induced acute oxidative stress in rat liver. However, it remains unclear whether canalicular Mrp2 internalization is observed in human liver under conditions of acute oxidative stress. In this study, we examined the effect of dimerumic acid (DMA), an antioxidant and found in traditional Chinese medicine, on endotoxin-induced Mrp2 internalization in rat and human liver slices. At 1.5h following LPS treatment (100microg/mL), canalicular Mrp2 localization was disrupted without changing the expression of Mrp2 protein or the integrity of filamentous actin in the rat and human liver slices. Pretreatment with DMA (10microM) counteracted LPS-induced subcellular distribution of Mrp2. Our data clearly indicated that LPS-induced short-term rapid retrieval of Mrp2 from the canalicular surface resulted from LPS-induced oxidative stress in rat and human liver slices.

Multiple mechanisms underlying troglitazone-induced mitochondrial permeability transition.

Troglitazone, a thiazolidinedione class antidiabetic drug, was withdrawn from the market because of its severe idiosyncratic hepatotoxicity. It causes a mitochondrial permeability transition (MPT), which may in part contribute to its hepatotoxicity. In the present study, the mechanism of troglitazone mitochondrial toxicity was investigated in isolated rat liver mitochondria. Mitochondrial swelling induced by 10 µM troglitazone was attenuated by bromoenol lactone (BEL), an inhibitor of Ca²+-independent phospholipase A2 (iPLA2). In contrast, that induced by 50 µM troglitazone was exacerbated by BEL. This exacerbation was diminished by addition of 2mM glutathione, an antioxidant. Oxygen consumption by state 3 respiration in isolated mitochondria was also decreased by troglitazone, but it was not affected by BEL. Mitochondrial swelling induced by 10 µM troglitazone was completely attenuated in the absence of Ca²+ while that induced by 50 µM troglitazone was not affected. Addition of 1 µM cyclosporin A (CsA), an inhibitor of MPT pores, completely attenuated swelling induced by 10 µM troglitazone while it only partly diminished that induced by 50 µM troglitazone. Thus, the MPT induced by 10 and 50 µM troglitazone are regulated by different mechanism; the MPT induced by 10 µM troglitazone is regulated by the activation of iPLA2 and caused by the opening of CsA-regulating MPT pores followed by accumulation of Ca²+ in mitochondria, while that induced by 50 µM troglitazone is partly regulated by reactive oxygen species and mainly caused by the opening of CsA-insensitive MPT pores.

Correlation between apical localization of Abcc2/Mrp2 and phosphorylation status of ezrin in rat intestine.

The multidrug resistance-associated protein 2/ATP-binding cassette transporter family C2 (Mrp2/Abcc2) is an ATP-dependent export pump that mediates the transport of a variety of organic anions. Abcc2 is mainly expressed on the canalicular membrane of hepatocytes and also the brush-border membrane of intestinal epithelial cells. We have previously reported that Abcc2 is rapidly internalized from the canalicular membrane during acute oxidative stress, which induces protein kinase C (PKC) activation in rat liver. However, it has not been elucidated whether PKC is involved in the regulation of Abcc2 localization in other tissues. In this study, we investigated this issue in rat intestinal epithelia. Exposure to thymeleatoxin, a conventional PKC (cPKC) activator, for 20 min reduced the cumulative glutathione S-bimane efflux for 40 min via Abcc2 from 30.3 +/- 2.1 nmol/cm to 18.1 +/- 1.6 nmol/cm. Likewise, the Abcc2 expression in the brush-border membrane of the small intestine was reduced to half that of the control without changing the total amount of Abcc2 present in the homogenate. Immunoprecipitation analysis suggested an interaction between Abcc2 and ezrin, a scaffolding protein that is dominantly expressed in the intestine. Thymeleatoxin treatment decreased the amount of the active form (C-terminally phosphorylated form) of ezrin and the amount of Abcc2 that coimmunoprecipitated with ezrin. These results indicate that cPKC activation diminishes the protein-protein interaction between ezrin and Abcc2. In conclusion, the phosphorylation status of ezrin correlates with the cell surface expression of Abcc2 in the rat small intestine, which may be regulated by cPKC.

Long-lasting inhibition of the transporter-mediated hepatic uptake of sulfobromophthalein by cyclosporin a in rats.

Cyclosporin A (CsA) is a well known inhibitor of the organic anion-transporting polypeptide (OATP/Oatp) family transporters, causing a large number of transporter-mediated drug-drug interactions in clinical situations. In the present study, we examined the inhibitory effect of CsA on the hepatic uptake of sulfobromophthalein (BSP) in rats, focusing on a long-lasting inhibition. Twenty-one hours after the subcutaneous administration of CsA, the hepatic clearance of BSP was decreased. The liver uptake index study revealed that hepatic uptake of BSP was reduced in CsA-treated rats for at least 3 days. Comparison of uptake studies using isolated hepatocytes prepared from control and CsA-treated rats showed that hepatic uptake in CsA-treated rats was decreased. In primary cultured hepatocytes, after preincubation with CsA, the uptake of [(3)H]BSP was reduced even after removal of CsA from the incubation buffer although a preincubation time dependence was not observed. However, the expression of Oatp1a1 and Oatp1b2, which are involved in the hepatic uptake of BSP, and the amount of intrahepatic glutathione, a driving force of Oatp1a1, did not change in CsA-treated rats. Thus, we can conclude that CsA modulates the transporter function sustainably. It can cause a potent in vivo drug-drug interaction. The modulation of transporters is not caused by reduced expression or driving force of transporters. It may be affected by CsA accumulated in the liver or its metabolites. The inhibitory effect of CsA on the transporter-mediated uptake of BSP cannot be explained by a simple competitive mechanism and a novel mechanism should be considered.

Down-regulation of hepatic cytochrome P450 enzymes in rats with trinitrobenzene sulfonic acid-induced colitis.

Hepatic cytochrome P450 (P450) enzymes are down-regulated during inflammation. In this study, an animal model of inflammatory bowel disease was subjected to characterization of hepatic P450 expression under inflammatory conditions. Rats were treated intracolonically with 100 mg/kg trinitrobenzene sulfonic acid (TNBS) dissolved in 30% ethanol, and homogenates of colonic mucosa and hepatic microsomes of the rats were prepared. The colitis was accompanied by appearance of higher levels of portal endotoxin, interleukin-6, and nitric oxide metabolites and decreases in contents and activities for hepatic CYP3A2, CYP2C11, and, to a lesser extent, CYP1A2 and CYP2E1. Nimesulide, a preferential COX-2 inhibitor, protected rats with TNBS-induced colitis (TNBS-colitis) against the down-regulation of hepatic CYP3A2. Polymyxin B, which neutralizes endotoxin, curcumin, which has anti-inflammatory properties, and gadolinium chloride, which inactivates macrophages, attenuated the down-regulation of CYP3A2. Similar effects were observed in other P450s such as CYP2C11, but the agents were less effective in attenuating the down-regulation. Our data suggest that endogenous substances leaked from damaged colon in the rats with TNBS-colitis activate Kupffer cells, leading to down-regulation of hepatic P450s with differential susceptibility to the inflammatory stimuli. The colitis model, instead of exogenous administration of lipopolysaccharide or cytokines, could be applied to the study on mechanisms for altered hepatic P450 expression and other liver functions under mild inflammatory conditions.

Hepatitis C virus core protein triggers abnormal porphyrin metabolism in human hepatocellular carcinoma cells.

Porphyria cutanea tarda (PCT), the most common of the human porphyrias, arises from a deficiency of uroporphyrinogen decarboxylase. Studies have shown a high prevalence of hepatitis C virus (HCV) infection in patients with PCT. While these observations implicate HCV infection as a risk factor for PCT pathogenesis, the mechanism of interaction between the virus and porphyrin metabolism is unknown. This study aimed to assess the effect of HCV core protein on intracellular porphyrin metabolism to elucidate the link between HCV infection and PCT. The accumulation and excretion of porphyrins after treatment with 5-aminolevulinic acid, a porphyrin precursor, were compared between cells stably expressing HCV core protein and controls. Cells expressing HCV core protein had lower amounts of intracellular protoporphyrin IX and heme and had higher amounts of excreted coproporphyrin III, the oxidized form of coproporphyrinogen III, compared with controls. These observations suggest that HCV core protein affects porphyrin metabolism and facilitates the export of excess coproporphyrinogen III and/or coproporphyrin III, possibly via porphyrin transporters. Real-time PCR analysis revealed that the presence of HCV core protein increased the mRNA expression of porphyrin exporters ABCG2 and FLVCR1. Western blot analysis showed a higher expression level of FLVCR1, but not ABCG2, as well as a higher expression level of mature ALAS1, which is the rate-limiting enzyme in the heme synthesis pathway, in HCV core protein-expressing cells compared with controls. The data indicate that HCV core protein induced abnormal intracellular porphyrin metabolism, with an over-excretion of coproporphyrin III. These findings may partially account for the susceptibility of HCV-infected individuals to PCT development.

Oxidative stress and Mrp2 internalization.

Oxidative stress in the liver is sometimes accompanied by cholestasis. We have described the internalization of multidrug resistance-associated protein 2/ATP-binding cassette transporter family 2 (Mrp2/Abcc2), a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress in rat liver. However, the signaling pathway and regulatory molecules have not been investigated. In the present study, we investigated the mechanism of EA-induced Mrp2 internalization using isolated rat hepatocyte couplets (IRCHs). The Mrp2 index, defined as the ratio of Mrp2-positive canalicular membrane staining in IRCHs per number of cell nuclei, was significantly reduced by treatment with EA. This reduction was abolished by a nonspecific protein kinase C (PKC) inhibitor Gö6850, a Ca(2+) chelator, EGTA, but not by a protein kinase A (PKA)-selective inhibitor, a Ca(2+)-dependent conventional PKC (cPKC) inhibitor Gö6976, or a protein kinase G (PKG) inhibitor (1 microM). Moreover, an increase in the intracellular Ca(2+) level and NO release into medium were observed shortly after the EA treatment. Both of these increases, as well as Mrp2 internalization, were completely blocked by EGTA. In conclusion, EA produced a reduction in GSH, Ca(2+) elevation, NO production, and nPKC activation in a sequential manner, finally leading to Mrp2 internalization.

Mitochondrial permeability transition as a potential determinant of hepatotoxicity of antidiabetic thiazolidinediones.

Troglitazone, a thiazolidinedione class of antidiabetic agent, causes serious idiosyncratic hepatotoxicity. Troglitazone is metabolized to a reactive metabolite that covalently binds to cellular macromolecules, but the role of the covalent adduct in the hepatotoxicity is controversial. Because troglitazone has been found to cause cytotoxicity to hepatocytes along with mitochondrial dysfunction, we investigated the effects of troglitazone and other thiazolidinediones on mitochondrial function by using liver mitochondria fraction isolated from male CD-1 mice. Incubation of energized mitochondria with succinate in the presence of Ca2+ and troglitazone induced mitochondrial swelling, and the swelling was partially inhibited by cyclosporin A. Troglitazone also induced decreases in mitochondrial membrane potential and mitochondrial Ca2+ accumulation. These results demonstrate that troglitazone induces mitochondrial permeability transition (MPT). Similar results were obtained for ciglitazone, whereas rosiglitazone and pioglitazone, which are less hepatotoxic than troglitazone, had little effect on these mitochondria functions. It is therefore possible that the troglitazone-induced opening of MPT pore, which is not induced by rosiglitazone or pioglitazone, may contribute to the hepatotoxicity induced specifically by troglitazone.

Naproxen-induced oxidative stress in the isolated perfused rat liver.

We previously showed that naproxen induced the oxidative stress in the liver microsomes and the isolated hepatocytes of rats. In this study, the in situ effect of naproxen on the rat liver tissue was investigated, using the isolated perfused liver from the view-point of the naproxen-induced hepatotoxicity. The leakage of glutamic-oxaloacetic transaminase (GOT) from the perfused liver and appearance of thiobarbituric acid reactive substances (TBARS) in the perfusate increased with the progress of perfusion after a lag time of about 1h. The naproxen-perfusion of the liver decreased the biliary excretion of glutathione (GSH) and oxidized glutathione, glutathione disulfide (GSSG) prior to TBARS production and GOT leakage. GSSG content in the naproxen-perfused liver was significantly higher than in the control. TBARS appeared in the perfusate of the naproxen-perfused liver for 30 min, but not in the control. The biliary excretion clearance (CL(bile)) of indocyanine green (ICG), a reagent for testing the liver function, in the liver perfused with naproxen decreased to a half of that in the liver perfused without naproxen. Thus, the naproxen-induced oxidative stress in the liver was shown to affect the physiological function of liver through the impairment of biliary excretion, which is recognized as a detoxification system.