Biomedical subjects

Toshiharu Horie

Publications and source records attributed to Toshiharu Horie.

At least 19 recordsLinked to original sources

PubMed2006

Functional analysis of mouse and monkey multidrug resistance-associated protein 2 (Mrp2).

We investigated the intrinsic transport activity of mouse and monkey Mrp2 and compared it with that of rat and dog Mrp2 reported previously. Mrp2 cDNAs were isolated from BALB/c and Macaca fascicularis liver, respectively, and vesicle transport studies were performed using recombinant Mrp2s expressed in insect Sf9 cells. ATP-dependent transport of [3H]leukotriene C4 (LTC4), [3H]17beta-estradiol 17-(beta-D-glucuronide) (E217betaG), [3H]bromosulfophthalein (BSP), and [3H]cholecystokinin octapeptide (CCK-8) were readily detected for all Mrp2s. A species difference in the intrinsic transport activity was apparent for LTC4 (monkey > mouse, dog > rat) and BSP (rat, dog, monkey > mouse). In addition to the difference in the transport activity, complex kinetic profiles were also evident in CCK-8, where a cooperative transport site was observed. Moreover, the transport of [3H]E217betaG by mouse and monkey Mrp2 was quite different from that of rat and dog Mrp2 in that 1) there was practically only nonsaturable uptake for [3H]E217betaG and 2) 4-methylumbelliferon glucuronide (Mrp2 modulator) showed a concentration-dependent stimulatory effect on the transport of [3H]E217betaG in mouse and monkey Mrp2, whereas rat and dog transport activity was inhibited by the modulator. In conclusion, although the substrate specificity is similar, the intrinsic transport activity differs from one species to another. This is due not only to the difference in the Km and Vmax values, but also the qualitatively different mode of substrate and modulator recognition exhibited by different species.

Adenosine Triphosphate↗

PubMed2006

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.

Animals↗

PubMed2006

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.

ATP-Binding Cassette Transporters↗

PubMed2006

Transporters as a determinant of drug clearance and tissue distribution.

Transporters play an important role in the processes of drug absorption, distribution and excretion. In this review, we have focused on the involvement of transporters in drug excretion in the liver and kidney. The rate of transporter-mediated uptake and efflux determines the rate of renal and hepatobiliary elimination. Transporters are thus important as a determinant of the clearance in the body. Even when drugs ultimately undergo metabolism, their elimination rate is sometimes determined by the uptake rate mediated by transporters. Transporters regulate the pharmacological and/or toxicological effect of drugs because they limit their distribution to tissues responsible for their effect and/or toxicity. For example, the liver-specific distribution of some statins via organic anion transporters helps them to produce their high pharmacological effect. On the other hand, as in the case of metformin taken up by organic cation transporter 1, drug distribution to the tissue(s) may enhance its toxicity. As transporter-mediated uptake is a determinant of the drug elimination rate, drug-drug interactions involving the process of transporter-mediated uptake can occur. In this review, we have introduced some examples and described their mechanisms. More recently, some methods to analyze such transporter-mediated transport have been reported. The estimation of the contributions of transporters to the net clearance of a drug makes it possible to predict the net clearance from data involving drug transport in transporter-expressing cells. Double transfected cells, where both uptake and efflux transporters are expressed on the same polarized cells, are also helpful for the analysis of the rate of transporter-mediated transcellular transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Down-regulation of hepatic cytochrome P450 enzymes associated with cisplatin-induced acute renal failure in male rats.

Hepatic drug metabolism is impaired in experimental animals and humans with renal diseases. An anticancer drug, cisplatin induces acute renal failure (ARF) in rats. Under the same experimental conditions, cisplatin causes down-regulation of hepatic cytochrome P450 (P450) enzymes in an isozyme selective manner. The present study examined the pathological role of ARF in the down-regulation of hepatic P450 enzymes in the cisplatin-treated rats. Male rats with single dose of intraperitoneally cisplatin (5 mg/kg) caused marked changes in renal parameters, BUN and serum creatinine but not hepatic parameters, serum alanine aminotransferase or aspartate aminotransferase. The rats also suffered from down-regulation of hepatic microsomal CYP2C11 and CYP3A2, male specific P450 isozymes, but not CYP1A2, CYP2E1, or CYP2D2. The decrease in serum testosterone level was also observed in injured rats, which was consistent with the selective effects on male specific P450 enzymes. Protection of rats against cisplatin-induced ARF by dimethylthiourea, a hydroxyl radical scavenger, also protected rats against the decrease in serum testosterone levels and the down-regulation of CYP2C11 and CYP3A2. Carboplatin, an analogue to cisplatin but no ARF inducer, did not cause decrease in serum testosterone levels and down-regulation of hepatic male specific P450 enzymes. These results suggest that down-regulation of hepatic P450 enzymes in male rats given cisplatin is closely related to the cisplatin-induced ARF and the resultant impairment of testis function.

Acute Disease↗

PubMed2006

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.

Animals↗

PubMed2006

Aged garlic extract protects against methotrexate-induced apoptotic cell injury of IEC-6 cells.

Gastrointestinal toxicity is one of the most serious side effects of methotrexate (MTX) treatment. The side effects often disrupt the cancer chemotherapy. We previously reported that aged garlic extract (AGE) protects the small intestine of rats from MTX-induced damage. In this study, the protection of AGE against MTX-induced damage of IEC-6 cells originating from the rat jejunum crypt was investigated. MTX decreased the viability of IEC-6 cells, but this effect was prevented by AGE (0.5%). The MTX-induced apoptosis of IEC-6 cells was depressed by AGE. These results indicated that AGE protects IEC-6 cells from the MTX-induced damage. AGE may be useful in cancer chemotherapy with MTX because it reduces MTX-induced intestinal damage.

Animals↗

PubMed2005

Apical/basolateral surface expression of drug transporters and its role in vectorial drug transport.

It is well known that transporter proteins play a key role in governing drug absorption, distribution, and elimination in the body, and, accordingly, they are now considered as causes of drug-drug interactions and interindividual differences in pharmacokinetic profiles. Polarized tissues directly involved in drug disposition (intestine, kidney, and liver) and restricted distribution to naive sanctuaries (blood-tissue barriers) asymmetrically express a variety of drug transporters on the apical and basolateral sides, resulting in vectorial drug transport. For example, the organic anion transporting polypeptide (OATP) family on the sinusoidal (basolateral) membrane and multidrug resistance-associated protein 2 (MRP2/ABCC2) on the apical bile canalicular membrane of hepatocytes take up and excrete organic anionic compounds from blood to bile. Such vectorial transcellular transport is fundamentally attributable to the asymmetrical distribution of transporter molecules in polarized cells. Besides the apical/basolateral sorting direction, distribution of the transporter protein between the membrane surface (active site) and the intracellular fraction (inactive site) is of practical importance for the quantitative evaluation of drug transport processes. The most characterized drug transporter associated with this issue is MRP2 on the hepatocyte canalicular (apical) membrane, and it is linked to a genetic disease. Dubin-Johnson syndrome is sometimes caused by impaired canalicular surface expression of MRP2 by a single amino acid substitution. Moreover, single nucleotide polymorphisms in OATP-C/SLC21A6 (SLCO1B1) also affect membrane surface expression, and actually lead to the altered pharmacokinetic profile of pravastatin in healthy subjects. In this review article, the asymmetrical transporter distribution and altered surface expression in polarized tissues are discussed.

Animals↗

PubMed2005

Enhanced glucose absorption in the rat small intestine following repeated doses of 5-fluorouracil.

Many studies demonstrated that 5-fluorouracil (5-FU) treatment of rodents caused the damage of small intestine, resulting in the malabsorption, while we recently found that repeated administration of 5-FU to rats increased Na(+)-dependent glucose absorption in the small intestine. This study investigated the cause of enhanced glucose absorption. 3-O-methyl-d-glucose (3-OMG) absorption was examined using the everted intestine technique. d-Glucose uptake, phlorizin binding, Western blot analysis and membrane fluidity were examined using small intestinal brush-border membrane vesicles (BBMV). Repeated oral administration of 5-FU to rats increased Na(+)-dependent 3-OMG absorption in the small intestine, while alkaline phosphatase activity in the small intestine decreased. Na(+)/K(+)-ATPase activity of 5-FU-treated rats was about three-fold higher than that of control rats. Although the amount of Na(+)-dependent glucose co-transporter (SGLT1) in 5-FU-treated rats decreased, the overshoot magnitude of d-glucose uptake in BBMV was not altered. Maximum binding of phlorizin in 5-FU-treated rats was 1.5-fold larger than that of control rats, but not altered the maximal rate of d-glucose absorption, Michaelis constant of d-glucose and dissociation constant of phlorizin. The membrane fluidity of 5-FU-treated rats increased. The enhanced d-glucose absorption in 5-FU-treated rats seems to occur secondarily due to the activation of Na(+)/K(+)-ATPase activity in basolateral membranes (BLM). Because the amounts of SGLT1 in 5-FU-treated rats decreased, the increase of turnover rate of SGLT1 and/or an expression of unknown Na(+)-dependent glucose co-transporter with high affinity for d-glucose and phlorizin sensitivity would contribute to the enhancement of d-glucose transport in 5-FU-treated rats.

3-O-Methylglucose↗

PubMed2005

Acyl glucuronidation of fluoroquinolone antibiotics by the UDP-glucuronosyltransferase 1A subfamily in human liver microsomes.

Acyl glucuronidation is an important metabolic pathway for fluoroquinolone antibiotics. However, it is unclear which human UDP-glucuronosyltransferase (UGT) enzymes are involved in the glucuronidation of the fluoroquinolones. The in vitro formation of levofloxacin (LVFX), grepafloxacin (GPFX), moxifloxacin (MFLX), and sitafloxacin (STFX) glucuronides was investigated in human liver microsomes and cDNA-expressed recombinant human UGT enzymes. The apparent Km values for human liver microsomes ranged from 1.9 to 10.0 mM, and the intrinsic clearance values (calculated as Vmax/Km) had a rank order of MFLX > GPFX > STFX > > LVFX. In a bank of human liver microsomes (n = 14), the glucuronidation activities of LVFX, MFLX, and STFX correlated highly with UGT1A1-selective beta-estradiol 3-glucuronidation activity, whereas the glucuronidation activity of GPFX correlated highly with UGT1A9-selective propofol glucuronidation activity. Among 12 recombinant UGT enzymes, UGT1A1, 1A3, 1A7, and 1A9 catalyzed the glucuronidation of these fluoroquinolones. Results of enzyme kinetics studies using the recombinant UGT enzymes indicated that UGT1A1 most efficiently glucuronidates MFLX, and UGT1A9 most efficiently glucuronidates GPFX. In addition, the glucuronidation activities of MFLX and STFX in human liver microsomes were potently inhibited by bilirubin with IC50 values of 4.9 microM and 4.7 microM, respectively; in contrast, the glucuronidation activity of GPFX was inhibited by mefenamic acid with an IC50 value of 9.8 microM. These results demonstrate that UGT1A1, 1A3, and 1A9 enzymes are involved in the glucuronidation of LVFX, GPFX, MFLX, and STFX in human liver microsomes, and that MFLX and STFX are predominantly glucuronidated by UGT1A1, whereas GPFX is mainly glucuronidated by UGT1A9.

Animals↗

PubMed2005

Mrp2/Abcc2 transport activity is stimulated by protein kinase Calpha in a baculo virus co-expression system.

Cholestatic and choleretic effect are well known for protein kinase C activator and inhibitor, respectively. However, post-translational regulation, especially the effect of phosphorylation status of the biliary transporters on their intrinsic transport activity has not been fully understood. In this study, effect of phosphorylation on the transport activity of Mrp2, a biliary organic anion transporter, was examined in membrane vesicles isolated from Sf9 cells co-expressing excess amount of protein kinase Calpha (PKCalpha). Mrp2-mediated transport activity was enhanced to three-fold by co-expressing PKCalpha. At the same time, phosphorylation of Mrp2 was also detected. The Km and Vmax values for the transport of [3H]estradiol-17beta-D-glucuronide exhibited a 1.5-fold decrease and a 1.9-fold increase, respectively. Probenecid (100 microM) and benzylpenicillin (1 mM), both are activator of Mrp2, did not stimulated the transport activity of phosphorylated Mrp2. On the other hand, transport activity was further stimulated by Estron-3-sulfate and taurocholic acid. Similar mechanism that occurred in the presence of probenecid and benzylpenicillin, but different from that occurred in the presence of Estron-3-sulfate and taurocholic acid seems to be involved in the stimulation. Considering the discrepancy between the previous in vivo inhibitory effect of PKC activators and our in vitro stimulatory effect of PKCalpha on Mrp2 transport activity, direct modulation of Mrp2-transport activity may be minor if any under in vivo condition.

ATP-Binding Cassette Transporters↗

PubMed2005

Enhanced absorption of 3-O-methyl glucose following gastrointestinal injury induced by repeated oral administration of 5-FU in mice.

The absorption of nutrients is mainly mediated by specific carriers and generally retarded following gastrointestinal injury. The aim of this study was to assess the effect of repeated oral administration of 5-fluorouracil (5-FU) on the intestinal absorption of glucose by using 3-O-methyl-D-glucose (3-OMG), a glucose analogue that is not metabolized, as a probe. Repeated administration of 5-FU (60 mg/kg/day for 3 days) readily induced intestinal mucosal injury assessed by visual observation and loss of intestinal wet weight. At the same time, the carrier-dependent absorption clearance of 3-OMG was increased 1.8-fold, while the carrier-independent absorption assessed by L-glucose transport was not affected. Phloretin, a glucose transporter 2 (GLUT2) inhibitor, completely abolished the absorption of 3-OMG in both control and 5-FU-treated mice, indicating the specific effect on the carrier-dependent process. Protein and mRNA expressions of GLUT2 were significantly higher in 5-FU-treated mice compared to the control mice. Sodium (Na(+)) glucose co-transporter 1 (SGLT1) expressions were also moderately elevated in 5-FU-treated mice. Concomitantly, the uptake of D-glucose into both isolated brush border and basolateral membrane vesicles was significantly increased. These results indicate that repeated oral administration of 5-FU did not hamper, but unexpectedly induced, SGLT1 and GLUT2 expression to enhance glucose absorption.

3-O-Methylglucose↗

PubMed2005

Involvement of mitochondrial permeability transition in acetaminophen-induced liver injury in mice.

BACKGROUND/AIMS: Although mitochondria have been demonstrated as primary targets in acetaminophen hepatotoxicity, the mechanism for mitochondria-mediated toxicity has not been defined. We examined the role of mitochondrial permeability transition (MPT) in the acetaminophen-induced liver injury. METHODS: Male CD-1 mice were given intraperitoneally acetaminophen (350 mg/kg) without or with cyclosporin A (50 mg/kg), a specific inhibitor of MPT. Serum alanine aminotransferase (ALT), a marker of liver injury, and other biochemical parameters were determined. RESULTS: Acetaminophen-induced ALT leakage was attenuated by co-administration of cyclosporin A. Cyclosporin A did not affect acetaminophen-induced early decrease in hepatic reduced glutathione (GSH) contents, indicating lack of the effect on the metabolic activation. Acetaminophen-induced decrease in mitochondrial GSH and ATP contents, and cytosolic leakage of cytochrome c were attenuated by cyclosporin A, suggesting that mitochondrial oxidative stress and ATP depletion resulting from MPT are principle mechanisms involved in acetaminophen-induced liver injury. Mitochondrial swelling by calcium was exacerbated in the mitochondria isolated from the acetaminophen-treated mice. In vitro exposure of intact mitochondria to N-acetyl-p-benzoquinone imine (NAPQI) with calcium caused mitochondrial swelling. CONCLUSIONS: The present data indicate that the MPT is the principal mechanism in the acetaminophen-induced liver injury and NAPQI is a candidate to open the transition pore.

Acetaminophen↗

PubMed2004

Ethacrynic-acid-induced glutathione depletion and oxidative stress in normal and Mrp2-deficient rat liver.

Oxidative stress in the liver is sometimes accompanied by cholestasis. We investigated the localization and role of multidrug-resistance-associated protein (Mrp) 2, a biliary transporter involved in bile-salt-independent bile flow, under ethacrynic acid (EA)-induced acute oxidative stress. Normal Sprague-Dawley rat (SDR) and Mrp2-deficient Eisai hyperbilirubinemic rat (EHBR) livers were perfused with 500 microM EA. The release of glutamic pyruvic transaminase (GPT) and thiobarbituric-acid-reactive substances (TBARS) from EHBR liver was markedly delayed compared with that from SDR liver. This is mainly due to the higher basal level of glutathione (GSH) in EHBR liver (59.1 +/- 0.3 nmol/mg protein) compared with SDR liver (39.7 +/- 1.5 nmol/mg protein). EA similarly induced a rapid reduction in GSH followed by mitochondrial permeability transition in the isolated mitochondria from both SDR and EHBR. Internalization of Mrp2 was detected before nonspecific disruption of the canalicular membrane and GPT release in SDR liver perfused with 100 microM EA. SDR liver preperfused with hyperosmolar buffer (405 mosmol/L) for 30 min induced internalization of Mrp2 without changing the basal GSH level, while elimination of hepatic GSH by 300 microM EA perfusion was significantly delayed thereafter. Concomitantly, hepatotoxicity assessed by the release of GPT and TBARS was also significantly attenuated under hyperosmolar conditions. In conclusion, preserved cytosolic and intramitochondrial GSH is the key factor involved in the acute hepatotoxicity induced by EA and its susceptibility could be altered by the presence of Mrp2.

ATP-Binding Cassette Transporters↗

PubMed2004

Functional analysis of dog multidrug resistance-associated protein 2 (Mrp2) in comparison with rat Mrp2.

We investigated whether the species difference in the biliary excretion activity of some Mrp2 substrates was attributable to the intrinsic transport potential or the expression level of Mrp2, especially in rat and dog. Dog Mrp2 cDNA was isolated from beagle dog liver, and a vesicle transport study was performed using recombinant rat and dog Mrp2 expressed in insect Sf9 cells. The ATP-dependent transport of 17beta-estradiol 17-(beta-D-glucuronide) ([3H]E(2)17betaG) and leukotriene C4 ([3H]LTC4), normalized by the absolute protein expression level, was similar in both Mrp2s. The Mrp2 protein expression in dog liver was only 10% of that in rat liver and was comparable with the reported difference in the biliary excretion clearance of temocaprilat as Mrp2 substrate. In contrast to LTC4, unique transport kinetics for E(2)17betaG were evident in dog Mrp2. In addition to the high-affinity site with a K(m) value of 3.25 +/- 0.10 microM, which is similar to that in rat Mrp2 (4.81 +/- 1.21 microM), dog Mrp2 has an additional low-affinity site (>>75 microM), which makes a major contribution to the transport of E(2)17betaG (65% of the total transport capacity at tracer concentration). In summary, the difference in the biliary excretion activity of Mrp2 substrates between rat and dog depends on the Mrp2 protein expression level rather than the intrinsic transport activity of the transporter molecules. The unique transport properties of glucuronide conjugates by dog Mrp2 may lead to the species difference involving the drug-drug interaction or drug-induced hyperbilirubinemia on the bile canalicular membrane.

ATP-Binding Cassette Transporters↗

PubMed2004

Protective effect of fosfomycin on gentamicin-induced lipid peroxidation of rat renal tissue.

Fosfomycin is clinically recognized to reduce the aminoglycoside antibiotics-induced nephrotoxicity. However, little has been clarified why fosfomycin protects the kidney from the aminoglycosides-induced nephrotoxicity. Gentamicin, a typical aminoglycoside, is reported to cause lipid peroxidation. We focused on lipid peroxidation induced by gentamicin as a mechanism for the aminoglycosides-induced nephrotoxicity. The aim of this study is to investigate the effect of fosfomycin on the gentamicin-induced lipid peroxidation. In rat renal cortex mitochondria, fosfomycin was shown to depress the gentamicin-induced lipid peroxidation, which was evaluated by formation of thiobarbituric acid reactive substances (TBARS). Interestingly, this effect was observed in rat renal cortex mitochondria, but not in rat liver microsomes. However, fosfomycin did not affect lipid peroxidation of arachidonic acid caused by gentamicin with iron. Fosfomycin inhibited the gentamicin-induced iron release from rat renal cortex mitochondria. These results indicated that fosfomycin inhibited the gentamicin-induced lipid peroxidation by depressing the iron release from mitochondria. This may possibly be one mechanism for the protection of fosfomycin against the gentamicin-induced nephrotoxicity.

Animals↗

PubMed2004

Multidrug resistance-associated protein 2 (MRP2) enhances 4-hydroxynonenal-induced toxicity in Madin-Darby canine kidney II cells.

4-Hydroxy-trans-2,3-nonenal (HNE) is a toxic end product of lipid peroxidation. This multifunctional aldehyde reacts with proteins, phospholipids, and nucleic acids, consequently activating/inactivating enzymes, affecting signal transduction and gene expression. HNE is mainly detoxified by glutathione (GSH) conjugation. In our previous report, we showed that GSH conjugates of 4-hydroxynonenal (HNE-SG) are substrates of multidrug resistance-associated protein 2 (MRP2). MRP2 has been shown to export HNE-SG conjugates into the extracellular space. In the present study, the role of MRP2 in the detoxification of HNE was studied using Madin-Darby canine kidney II (MDCK II) cells expressing human MRP2. MRP2 reduced the intracellular accumulation of HNE-SG conjugate but unexpectedly increased the susceptibility of cells to HNE. The viability of cells was reduced to approximately 70% in the presence of 62.5 microM HNE in MDCK II cells expressing MRP2, whereas MDCK II cells remained unaffected. MRP2 accelerated the elimination of intracellular GSH via a conjugation reaction with HNE (half-life of GSH was 30.1 and 12.2 min for MDCK II cells and MDCK II cells expressing MRP2, respectively). Moreover, the consumption of GSH was unlimited in MDCK II cells expressing MRP2, finally resulting in necrosis. These results indicate that MRP2 has an adverse effect during the detoxification of HNE in MDCK II cells and suggest that expression of MRP2 may enhance the damage caused by oxidative stress.

Adenosine Triphosphate↗

PubMed2004

Endotoxin-mediated disturbance of hepatic cytochrome P450 function and development of endotoxin tolerance in the rat model of dextran sulfate sodium-induced experimental colitis.

Hepatobiliary abnormalities have been described in patients with chronic inflammatory bowel diseases. Hepatic cytochrome P450 (P450)-dependent drug-metabolizing enzyme activities and susceptibility to a hepatotoxin, d-galactosamine, were determined in rats with dextran sulfate sodium (DSS)-induced colitis to assess whether liver function is affected in the model of inflammatory bowel disease. Colitis was induced by treatment of rats with 3% DSS in drinking water for 7 days. Liver microsomes for enzyme activities and serum for biological analysis were prepared from the rats with colitis, along with untreated and lipopolysaccharide (LPS)-treated rats. Other rats received intraperitoneal injection of d-galactosamine to assess their susceptibility to the toxin-induced liver injury. Treatment of rats with DSS resulted in not only colitis but also decreases in hepatic P450-dependent drug-metabolizing enzyme activities. Elevated endotoxin was found in portal blood, which was not associated with liver injury. The potency and the isoform selectivity in the suppression of the P450 enzymes by DSS treatment were similar to those of LPS-treated rats. Coadministration of antibiotics, polymyxin B or metronidazole, with DSS protected rats from decreases in some but not all P450 enzyme activities, indicating partial involvement of bacterial endotoxin in the P450 decreases. The rats with colitis were less susceptible than untreated rats to d-galactosamine-induced liver injury and TNF-alpha production, suggesting development of endotoxin tolerance in DSS-colitis. In conclusion, these results suggest that the DSS-colitis leads to endotoxin-mediated down-regulation of hepatic P450 enzymes and protection against d-galactosamine-induced liver injury, probably due to endotoxin tolerance.

Administration, Oral↗