Shirou Itagaki

In vitro and in vivo antioxidant properties of chlorogenic acid and caffeic acid.

Dietary polyphenols are thought to be beneficial for human health as antioxidants. Coffee beans contain a common polyphenol, chlorogenic acid. Chlorogenic acid is the ester of caffeic acid and quinic acid. Although these polyphenols have received much attention, there is little evidence indicating a relationship between the effect and the rate of absorption. In this study, we focused on the beneficial effects of chlorogenic acid and caffeic acid, a major metabolite of chlorogenic acid. We carried out in vitro and in vivo experiments. In the in vitro study, caffeic acid had stronger antioxidant activity than that of chlorogenic acid. The uptake of chlorogenic acid by Caco-2 cells was much less than that of caffeic acid. The physiological importance of an orally administered compound depends on its availability for intestinal absorption and subsequent interaction with target tissues. We then used an intestinal ischemia-reperfusion model to evaluate antioxidant activities in vivo. We found that both chlorogenic acid and caffeic acid had effects on intestinal ischemia-reperfusion injury. Since caffeic acid has a stronger antioxidant activity than that of chlorogenic acid and chlorogenic acid is hydrolyzed into caffeic acid in the intestine, it is possible that caffeic acid plays a major role in the protective effect of chlorogenic acid against ischemia-reperfusion injury.

Association between risk of myopathy and cholesterol-lowering effect: A comparison of all statins

In the present study, we examined the mechanisms underlying the cytotoxicity of pitavastatin, a new statin, and we compared the in vitro potencies of muscle cytotoxicity using a prototypic embryonal rhabdomyosarcoma cell line (RD cells), a typical side effect of statins and compared the cholesterol-lowering effects of statins using Hep G2 hepatoma cells. Pitavastatin reduced the number of viable cells and caused caspase-9 and -3/7 activation in a time- and concentration-dependent manner. The comparison of cytotoxities of statins showed that statins significantly reduced cell viability and markedly enhanced activity of caspase-3/7 in concentration-dependent manner. On the other hand, the effects of hydrophilic statins, pravastatin, rosuvastatin were very weak. The rank order of cytotoxicity was cerivastatin > simvastatin acid> fluvastatin > atorvastatin > lovastatin acid > pitavastatin >> rosuvastatin, pravastatin. Statin-induced cytotoxicity is associated with these partition coefficients. On the other hand, the cholesterol-lowering effect of statins did not correlate with these partition coefficients and cytotoxicity. Thus, it is necessary to consider the association between risk of myopathy and cholesterol-lowering effect of a statin for precise use of statins.

Expression Level of ABCG2 in the Placenta Decreases from the Mid Stage to the End of Gestation

The aim of this study was to elucidate the expression pattern of ABCG2 in the placenta from the mid stage to the end of gestation. rABCG2 expression was investigated in rats on the 14th gestation day (gd) and the 20th gd. Expression of the rABCG2 gene and expression of rABCG2 protein in the placenta were detected on gd 14 by RT-PCR and Western blot analysis respectively. The expression level of rABCG2 on gd 20 was less than that on gd 14. We investigated whether progesterone, secreted from the placenta, regulates the expression of ABCG2 in BeWo cells. Expression levels of the ABCG2 gene and protein in BeWo cells were decreased by progesterone treatment. We conclude that progesterone plays a role in reduction in the expression level of ABCG2 in the placenta with the advance of gestation from the mid stage to the end of gestation.

Placental Folate Transport during Pregnancy

The aim of this study was to elucidate the mechanism of folate transport in the placenta over the course of pregnancy. We found that folate receptor α (FRα) and reduced folate carrier (RFC) localized on the apical side of human placental villi. Since folate binding to placental brush-border membrane vesicles (BBMVs) was strongly inhibited by phosphatidylinositol-specific phospholipase C (PI-PLC) treatment, it is possible that FRα, a glycosyl phosphatidylinositol linked glycoprotein, is a candidate for folate uptake from maternal blood to the placenta. Moreover, additional inhibitory effects of thiamine pyrophosphate (TPP) and hemin on folate uptake after PI-PLC treatment suggested that not only FRα but also RFC and heme carrier protein 1 (HCP1) are involved in the folate transport mechanism in the human placenta. It was also found that accumulation of folate after intravenous injection increased with the progress of gestation in the rat placenta and the fetus. Furthermore, increases in the expression levels of mRNA of rFRα, rRFC, and rHCP1 in the rat placenta during pregnancy were observed. These findings suggest that FRα, RFC, and HCP1 are important carriers of folate in the placenta during pregnancy. The results of this study suggest that increases in the expression levels of FRα, RFC, and HCP1 in the placenta play an important role in the response to increased need for folate for the placenta and fetus during development with the progress of gestation.

Contribution of organic anion transporting polypeptide OATP2B1 to amiodarone accumulation in lung epithelial cells

The accumulation mechanisms of amiodarone (AMD) involving transporters in lung alveolar epithelial type II cells were studied. The uptake of AMD was examined using human alveolar epithelial-derived cell line A549 as a model. AMD was transported by the carrier-mediated system, and the apparent K(m) and V(max) values were 66.8+/-30.3 muM and 49.7+/-9.7 nmol/mg protein/5 min, respectively. The uptake of AMD by A549 cells was Na(+)-independent and was inhibited by substrates of human organic anion transporting polypeptide (OATP). The inhibition profiles were similar to the inhibitory effects of several compounds on OATP2B1-mediated E-3-S transport, and RT-PCR analysis showed mRNA expression of OATP2B1 and 1B3 in A549 cells. SiRNAs targeted to the OATP2B1 gene decreased the OATP2B1 mRNA expression level in A549 cells up to about 50% and reduced the uptake of AMD up to about 40%. These results indicate that AMD uptake mediated by carriers, including OATP2B1, might lead to accumulation of AMD in the lung and AMD-induced pulmonary toxicity (AIPT).

Liver X receptor regulates expression of MRP2 but not that of MDR1 and BCRP in the liver.

Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily. Multidrug resistance-associated protein 2 (MRP2), multidrug resistance 1 (MDR1) and breast cancer resistance protein (BCRP) play an important role in the efflux of a broad range of endogenous and xenobiotic compounds from hepatocytes. Since the effects of LXR activation on there transporters have been obscure, we investigated the effects of LXR agonists, TO901317 and 25-hydroxycholesterol, on MRP2, MDR1, BCRP expression in HepG2 cells and the rat liver. In an in vitro study, TO901317 increased ABCA1, an LXR target gene, and MRP2 mRNA and protein levels. On the other hand, TO901317 had little effect on MDR1 and BCRP mRNA levels. In an in vivo study, Abca1 and Mrp2 mRNA and protein levels were increased by TO901317, but TO901317 had no effect on Mdr1a and Bcrp mRNA levels in the rat liver. Moreover, TO901317-induced MRP2 mRNA expression was blocked by LXRalpha knockdown. In this study, we demonstrated that LXR activation induced expression of MRP2 but not that of MDR1 and BCRP in hepatocytes. The results suggest that agonists for LXR activate transcription of the MRP2 gene in order to promote excretion of endogenous and xenobiotic compounds from hepatocytes into bile.

Uptake of irinotecan metabolite SN-38 by the human intestinal cell line Caco-2

PurposeThe aim of this study was to investigate the transport mechanisms of transporters that contribute to the intestinal uptake of 7-ethyl-10-hydroxycamptothecin (SN-38).MethodsHuman intestinal epithelial Caco-2 cells were used to investigate the mechanistic basis of transepithelial uptake of SN-38. We investigated the characteristics of SN-38 uptake into Caco-2 cells. The effects of baicalin and sulfobromophthalein (BSP) on the uptake of SN-38 by Caco-2 cells were examined.ResultsUptake of SN-38 was significantly reduced at 4°C. Baicalin inhibited the uptake of SN-38 in a concentration-dependent manner. BSP significantly reduced the uptake of SN-38. However, probenecid, pravastatin and grepafloxacin did not affect the uptake of SN-38.ConclusionsThe results suggest that a specific transport system mediates the uptake of SN-38 across the apical membrane in Caco-2 cells.

Regulation mechanism of ABCA1 expression by statins in hepatocytes.

ATP-binding cassette transporter A1 (ABCA1) is predicted to be involved in the control of apolipoprotein AI-mediated cholesterol efflux: biosynthesis of high-density lipoprotein (HDL). However, the effects of HMG-CoA reductase inhibitors (statins) on ABCA1 in the liver and the precise mechanisms of their actions have been obscure. The aims of this study were to determine whether statins (atorvastatin (Ato) and pitavastatin (Pit)) affect hepatic ABCA1 expression and to clarify the mechanisms of their actions using HepG2 cells and the rat liver. We examined alterations in mRNA and protein levels of ABCA1 and peroxisome proliferator-activated receptors (PPARs) by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. In vitro and in vivo studies suggested that Pit increases ABCA1 mRNA level, but not Ato. Pit greatly increased Abca1 mRNA level and also increased the amount of plasma HDL and the mRNA level of PPARα. Clofibrate (PPARα agonist) increased ABCA1 expression in HepG2 cells and rat primary hepatocytes more than did PPAR β/δ and γ agonists. Pit-induced ABCA1 expression alteration was blocked by GW6471 (PPARα antagonist) and by PPARα knockdown. In this study, we demonstrated that Pit affect ABCA1 expression via PPARα in hepatocytes. The strategy to target a PPARα agonist in the liver can lead to increases in ABCA1 expression and HDL level.

Involvement of Cholesterol Membrane Transporter Niemann-Pick C1-Like 1 in the Intestinal Absorption of Lutein

PURPOSE Lutein is a carotenoid mainly found in green leafy vegetables and is located in the macula lutea in the human eye. Since humans cannot synthesize lutein de novo, it must be digested as food. The physiological importance of an orally administered compound depends on its interaction with target tissues. It is therefore important to clarify the absorption mechanism in the intestine. Cholesterol membrane transporters Niemann-Pick C1 Like 1 (NPC1L1) and scavenger receptor class B type 1 (SR-B1) are involved in the intestinal absorption of highly lipophilic compounds including cholesterol. Ezetimibe, a selective inhibitor of intestinal NPC1L1, is the widespread lipid-lowering agent. It is important to investigate the possibility of food-drug interactions in order to prevent undesirable and harmful clinical consequences. The aim of this work was to determine whether NPC1L1, SR-B1 and other transporters are involved in absorption of lutein. METHODS Caco-2 cells were used for accumulation and permeability study of lutein. Lutein concentration was determined by an HPLC system. The cDNA of transporters was isolated from total RNA of Caco-2 cells, and the expression of these transporters was confirmed by RT-PCR (reverse transcription-polymerase chain reaction). RESULTS Ezetimibe inhibited up to 40% of lutein accumulation by Caco-2 cell monolayers. Block lipid transport 1 (BLT-1), a selective chemical inhibitor of SR-B1, also inhibited lutein accumulation by Caco-2 cells. On the other hand, ATP-depletion reagents (sodium fluoride and sodium azide or carbonyl cyanide m-chlorophenylhydrazone) did not influence the accumulation or permeation of lutein significantly. CONCLUSIONS The results show that lutein absorption is, at least in part, mediated by influx transporters NPC1L1 and SR-B1 rather than mediated by efflux transporters such as ABC (ATP-binding cassette) transporters.

Bicarbonate supplementation as a preventive way in statins-induced muscle damage

PURPOSE The aim of this study was to evaluate the bicarbonate-induced improvement of statins, cerivastatin, simvastatin acid and lovastatin acid -induced apoptosis using rat myoblast cell line (L6) as a model of in vitro skeletal muscle and of cerivastatin-induced muscle damage in vivo study. METHODS Statin-induced reduction of cell viability and apoptosis was measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay and caspase assay. In vivo, we evaluated plasma creatine phosphokinase (CPK) level in cerivastatin-treated rat. RESULTS Bicarbonate prevented cerivastatin-, simvastatin- acid and lovastatin acid -induced reduction of cell viability, morphological change and caspase activation in L6 cells. Moreover, in the in vivo study, bicarbonate prevented cerivastatin-induced increase in CPK concentrations. CONCLUSIONS These results from in vitro and in vivo studies support that bicarbonate supplementation prevented statin-induced muscle damage.