Narumi Sugihara

Anti- and pro-oxidative effects of flavonoids on metal-induced lipid hydroperoxide-dependent lipid peroxidation in cultured hepatocytes loaded with α-linolenic acid

Lipid hydroperoxide (LOOH)-dependent lipid peroxidation was induced in alpha-linolenic acid (LNA)-loaded hepatocytes by adding Fe, Cu, V, or Cd ions at concentrations from 20 to 500 microM. The effects of structurally related flavonoids at concentrations from 10 to 500 microM on the lipid peroxidation were examined. The results with regard to each flavonoid subclass are as follows: (i) Flavonols such as myricetin, quercetin, fisetin, and kaempferol, but not morin, showed dose-dependent antioxidative activity against metal-induced lipid peroxidation at all metal concentrations. Myricetin, quercetin, and fisetin were the most effective antioxidants, although their efficacies depended on the metal ion. Kaempferol and morin had antioxidative activity equal to the other flavonols in the presence of Cu ions, but were much less effective for the other three metal ions. (ii) Flavones, luteolin, apigenin, and chrysin were antioxidative at low Fe concentrations, but were pro-oxidative at high Fe concentrations. Luteolin exhibited antioxidative activity similar to that of catechol-containing flavonols in the presence of the other three metal ions. Apigenin and chrysin also acted as pro-oxidants with V or with all metal ions, respectively. (iii) Taxifolin, a flavanone, also showed both anti- and prooxidative activity, depending on Fe concentrations, but with other metal showed only antioxidative activity ions. (iv) Epigallocatechin, a flavanol, was antioxidative with all metal ions, and its activity was similar to that of catechol-containing flavonols. The various effects of flavonoids on metal-induced lipid peroxidation in LNA-loaded hepatocytes is discussed with regard to the change in redox potential of flavonoid-metal complexes.

Presence or absence of a gallate moiety on catechins affects their cellular transport

The accumulation of (—)‐epicatechin (EC), a non‐gallate catechin, was significantly lower than that of (—)‐epicatechin gallate (ECG), a gallate catechin, in Caco‐2 cells. Using Caco‐2 cell monolayers cultured in transwells, the transport of catechins in the basolateral‐to‐apical direction was much higher than that in the apical‐to‐basolateral direction, suggesting the involvement of an efflux transporter. Moreover, the results suggest that involvement of a transporter in EC efflux is greater than that for ECG. Treatment with transporter inhibitors MK571, quinidine or mitoxantrone, which inhibit MRP2, P‐glycoprotein (P‐gp) and BCRP, respectively, led to an increase in the accumulation of EC into Caco‐2 cells and a decrease in the Papp ratio (Papp B→A/Papp A→B) for EC. These transporters seemed to be involved in EC efflux. BCRP was not an efflux transporter for ECG, and the influences of MRP2 and P‐gp on ECG efflux were lower than for EC. Thus, efflux transporters appear to be responsible for the difference in cellular accumulation of EC versus ECG, suggesting that the presence or absence of a gallate moiety in the catechin structure influences the transporters.

Blood ammonia levels and hepatic encephalopathy induced by CCl4 in rats

An investigation of the mechanism of development of hepatic encephalopathy induced by CCl4 was performed in rats. CCl4 (1.0 ml/kg three times per week for over 10 weeks) caused hepatic encephalopathy in 80% of the treated rats. Accompanying the hepatic encephalopathy were hematemesis, abdominal dropsy, and hyperammonemia, conditions observed in hepatic coma patients. The blood ammonia levels were tremendously increased in only those rats with hepatic encephalopathy. Hepatic activities of carbamylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), important enzymes of the urea cycle, were significantly inhibited by CCl4. However, the causality between the inhibition of CPS or ASS activity and the increase in blood ammonia levels was not observed. On the other hand, the content of ATP, which is a substrate of CPS and ASS, was decreased by 60% in liver of rats with hepatic encephalopathy. The activity of Mg2+-ATPase which can decompose hepatic ATP was increased by 60 and 300% in mitochondria and microsomes, respectively, of livers of rats with CCl4-induced encephalopathy. There was a good correlation between the decreased hepatic ATP content and the increased mitochondrial Mg2+-ATPase activity. Furthermore, there was also a good correlation between the increase in blood ammonia levels and the increase in Mg2+-ATPase activity in microsomes. These findings suggest that hyperammonemia, which was produced by the decrease in hepatic content and by the inhibition of CPS and ASS, may play an important role in induction of hepatic encephalopathy.

Cytotoxicity of food preservatives in cultured rat hepatocytes loaded with linolenic acid

We investigated the ability of eight food preservatives to induce lipid peroxidation in normal and alpha-linolenic acid (LNA)-loaded cultured rat hepatocytes. On the addition of sodium dehydroacetate (DHA-Na), potassium sorbate (SA-K) or thiabendazole (TBZ) to the cell culture, lipid peroxidation, assessed in terms of the production of malondialdehyde (MDA), was induced in LNA-loaded cells, but not in normal cells. At the low concentrations, induction of lipid peroxidation in LNA-loaded cells was highest with TBZ, whereas at high concentrations DHA-Na greatly induced lipid peroxidation. The occurrence of lipid peroxidation in LNA-loaded cells was accompanied by a decrease in cellular GSH levels with the three preservatives and by a decrease in cellular protein-SH levels with DHA-Na and TBZ. Furthermore, cell injury, measured by the release of LDH, was produced in LNA-loaded cells exposed to DHA-Na and SA-K. The addition of TBZ caused substantial cell injury in normal cells, and even greater injury in LNA-loaded cells. The prevention of lipid peroxidation in LNA-loaded hepatocytes by addition of an antioxidant, N,N-diphenyl-p-phenylenediamine (DPPD) almost completely prevented DHA-Na- and SA-K-induced cell injury, and reduced TBZ-induced cell injury. The addition of diphenyl (DP), o-phenylphenol (OPP) or butyl p-hydroxybenzoate (BHB) caused severe cell injury, in association with a marked decrease in cellular levels of both of GSH and protein-SH in both groups of cells. However, lipid peroxidation was not detectable in either group of cells exposed to these preservatives. Sodium propionate (PA-Na) and sodium benzoate (BA-Na) had little effect on any cytotoxic parameter in either group of cells.

Influence of gallate and pyrogallol moieties on the intestinal absorption of (-)-epicatechin and (-)-epicatechin gallate.

UNLABELLEDnThe cellular accumulation of individual catechins was measured as an index of intestinal absorption to clarify the interactions among catechins. The cellular accumulation of (-)-epicatechin (EC) increased in the presence of other catechins. The ability of gallate catechin such as (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) to increase the cellular accumulation of EC was greater than that of nongallate catechins. Gallic acid octyl ester (GAO) also increased the cellular accumulation of EC by 426% as compared with that in untreated cells. Conversely, the cellular accumulation of ECG was not influenced by other catechins, but it increased by 54% in the presence of GAO. Experiments using GAO derivatives indicated that the gallate moiety required the presence of a catechol group and a neighboring carbonyl group, whereas the pyrogallol moiety, without a neighboring carbonyl group, required 3 hydroxyl groups to increase the cellular accumulation of EC. Furthermore, gallate esters required long carbon chains to increase the same. The experiment using EGCG, GAO, or their derivatives indicated that the ability of gallate or pyrogallol moiety to increase the cellular accumulation of EC was restricted by their hydrophobicity. These results suggest that the co-administration of foods containing functional materials such as gallate or pyrogallol moieties, increases the intestinal absorption of catechin.nnnPRACTICAL APPLICATIONnThe cellular accumulation of (-)-epicatechin increased by the gallate or pyrogallol moiety in catechin structure. The interaction among catechins appeared to affect intestinal absorption of catechin. The bioavailability of catechin may be improved by co-administration of functional foods.

Inhibitory Effect of Flavonoids on the Efflux of N-Acetyl 5-Aminosalicylic Acid Intracellularly Formed in Caco-2 Cells

N-acetyl 5-aminosalicylic acid (5-AcASA) that was intracellularly formed from 5-aminosalicylic acid (5-ASA) at 200 μM was discharged 5.3, 7.1, and 8.1-fold higher into the apical site than into the basolateral site during 1, 2, and 4-hour incubations, respectively, in Caco-2 cells grown in Transwells. The addition of flavonols (100 μM) such as fisetin and quercetin with 5-ASA remarkably decreased the apically directed efflux of 5-AcASA. When 5-ASA (200 μM) was added to Caco-2 cells grown in tissue culture dishes, the formation of 5-AcASA decreased, and, in addition, the formed 5-AcASA was found to be accumulated within the cells in the presence of such flavonols. Thus, the decrease in 5-AcASA efflux by such flavonols was attributed not only to the inhibition of N-acetyl-conjugation of 5-ASA but to the predominant cellular accumulation of 5-AcASA. Various flavonoids also had both of the effects with potencies that depend on their specific structures. The essential structure of flavonoids was an absence of a hydroxyl substitution at the C5 position on the A-ring of flavone structure for the inhibitory effect on the N-acetyl-conjugation of 5-ASA, and a presence of hydroxyl substitutions at the C3′ or C4′ position on the B-ring of flavone structure for the promoting effect on the cellular accumulation of 5-AcASA. Both the decrease in 5-AcASA apical efflux and the increase in 5-AcASA cellular accumulation were also caused by MK571 and indomethacin, inhibitors of MRPs, but not by quinidine, cyclosporin A, P-glycoprotein inhibitors, and mitoxantrone, a BCRP substrate. These results suggest that certain flavonoids suppress the apical efflux of 5-AcASA possibly by inhibiting MRPs pumps located on apical membranes in Caco-2 cells.N-acetyl 5-aminosalicylic acid (5-AcASA) that was intracellularly formed from 5-aminosalicylic acid (5-ASA) at 200 microM was discharged 5.3, 7.1, and 8.1-fold higher into the apical site than into the basolateral site during 1, 2, and 4-hour incubations, respectively, in Caco-2 cells grown in Transwells. The addition of flavonols (100 microM) such as fisetin and quercetin with 5-ASA remarkably decreased the apically directed efflux of 5-AcASA. When 5-ASA (200 microM) was added to Caco-2 cells grown in tissue culture dishes, the formation of 5-AcASA decreased, and, in addition, the formed 5-AcASA was found to be accumulated within the cells in the presence of such flavonols. Thus, the decrease in 5-AcASA efflux by such flavonols was attributed not only to the inhibition of N-acetyl-conjugation of 5-ASA but to the predominant cellular accumulation of 5-AcASA. Various flavonoids also had both of the effects with potencies that depend on their specific structures. The essential structure of flavonoids was an absence of a hydroxyl substitution at the C5 position on the A-ring of flavone structure for the inhibitory effect on the N-acetyl-conjugation of 5-ASA, and a presence of hydroxyl substitutions at the C3 or C4 position on the B-ring of flavone structure for the promoting effect on the cellular accumulation of 5-AcASA. Both the decrease in 5-AcASA apical efflux and the increase in 5-AcASA cellular accumulation were also caused by MK571 and indomethacin, inhibitors of MRPs, but not by quinidine, cyclosporin A, P-glycoprotein inhibitors, and mitoxantrone, a BCRP substrate. These results suggest that certain flavonoids suppress the apical efflux of 5-AcASA possibly by inhibiting MRPs pumps located on apical membranes in Caco-2 cells.

Determination of malondialdehyde by high-performance liquid chromatography using 4-(2-phthalimidyl)benzohydrazide as a pre-column fluorescent labelling reagent

A high-performance liquid chromatographic method was developed for the determination of malondialdehyde after conversion into 1-[4-(2-phthalimidyl)-benzoyl]pyrazole with the fluorescent labelling reagent 4-(2-phthalimidyl)benzohydrazide. The labelling reaction was carried out at 50 °C for 60 min in the presence of phosphoric acid. The extent of conversion of malondialdehyde into the fluorescent derivative was approximately 100%. The fluorescent derivative was separated on an ODP-50 column with elution using 40% aqueous acetonitrile and detected by fluorescence at 320 nm (excitation) and 385 nm (emission). The detection limit (signal-to-noise ratio = 3) was 8 fmol per injection (20 µl). The relative standard deviations for within-day and day-to-day precision were 3.18 and 3.53%(0.5 µmol l–1, n= 8), respectively. The method was applied to the determination of malondialdehyde generated from isolated rat hepatocytes stimulated with tert-butyl hydroperoxide.

Synergistic effects of flavonoids and ascorbate on enhancement in DNA degradation induced by a bleomycin-Fe complex

Flavonoids were examined for synergistic effects with ascorbate on enhancement of DNA degradation induced by a bleomycin(BLM)–Fe complex. The synergistic effects of flavonoids and ascorbate on DNA degradation induced by the BLM–Fe complex were observed to be greater with flavonoids such as isorhamnetin, kaempferol and morin, which accelerated oxidation more markedly in the presence, than in the absence of BLM. Conversely, myricetin and fisetin, which showed oxidation barely accelerated by the addition of BLM, inhibited DNA degradation promoted by ascorbate. Consequently, there was a good correlation between oxidation of flavonoids accelerated by BLM and the extent of DNA degradation promoted synergistically with ascorbate. Our previous studies indicated that oxidation of flavonoids accelerated by BLM and DNA degradation promoted by flavonoids were not correlated with Fe(III)-reducing activity of flavonoids. Those results suggest that Fe(III)-reducing activity of flavonoids is not the only factor determining DNA degradation-promoting activity induced by the BLM–Fe complex. On the other hand, in a Fenton reaction, degradation of 2-deoxy-d-ribose promoted by flavonoids was correlated to the Fe(III)-reducing activity of flavonoids. However, there was not a synergistic interaction between flavonoids and ascorbate in the degradation of 2-deoxy-d-ribose. Therefore, it is suggested that the synergistic DNA degradation caused by flavonoids and ascorbate in the BLM–Fe redox cycle arose from the difference in the reductive processes in which flavonoids and ascorbate mainly act.

Hepatic ATP content and hyperammonemia induced by CCl4 in rats

An investigation of the mechanism of development of hyperammonemia observed in CCl4-induced hepatic encephalopathy was performed in rats. CCl4 (1.0 ml/kg 3 times per week for over 10 weeks) caused a severe hyperammonemia and depletion of hepatic ATP contents in only those rats with hepatic encephalopathy. However, CCl4 (1.0 ml/kg 3 times per week for 7 weeks) did not cause hepatic encephalopathy and did not change in blood ammonia levels. Administration of 2,4-dinitrophenol (2,4-DNP) in these CCl4-treated rats caused hepatic encephalopathy within 30 min after injection and then the increase of 140 micrograms/dl in blood ammonia levels and the decrease of 80% in hepatic ATP contents were observed. However, the administration of 2,4-DNP in CCl4-untreated rats did not cause hepatic encephalopathy within 30 min after injection although the increase of 70 micrograms/dl in blood ammonia levels and the decrease of 80% in hepatic ATP contents were observed. Hepatic activities of carbamylphosphate synthetase (CPS) and argininosuccinate synthetase (ASS), important enzymes of the urea cycle, were significantly inhibited by 85% and 60% respectively, in rats treated with CCl4 plus 2,4-DNP. However, in rats treated with 2,4-DNP and without CCl4, the hepatic activities of CPS and ASS were inhibited only 25% and 0%, respectively. These findings suggest that the severe hyperammonemia, which may be produced by the decrease of hepatic ATP content and the inhibition of CPS and ASS, may play an important role in induction of hepatic encephalopathy.

N-terminal domain of the cholesterol transporter Niemann–Pick C1-like 1 (NPC1L1) is essential for α-tocopherol transport

Both cholesterol and α-tocopherol are essential lipophilic nutrients for humans and animals. Although cholesterol in excess causes severe problems such as coronary heart disease, it is a necessary component of cell membranes and is the precursor for the biosynthesis of steroid hormones and bile acids. Niemann-Pick C1-like 1 (NPC1L1) is a cholesterol transporter that is highly expressed in the small intestine and liver in humans and plays an important role in cholesterol homeostasis. Cholesterol promotes NPC1L1 endocytosis, which is an early step in cholesterol uptake. Furthermore, α-tocopherol is the most active form of vitamin E, and sufficient amounts of vitamin E are critical for health. It has been reported that NPC1L1 mediates α-tocopherol absorption; however, the mechanisms underlying this process are unknown. In this study, we found that treatment of cells that stably express NPC1L1-GFP with α-tocopherol promotes NPC1L1 endocytosis, and the NPC1L1 inhibitor, ezetimibe, efficiently prevents the α-tocopherol-induced endocytosis of NPC1L1. Cholesterol binding to the N-terminal domain (NTD) of NPC1L1 (NPC1L1-NTD) is essential for NPC1L1-mediated cholesterol absorption. We found that α-tocopherol competitively binds NPC1L1-NTD with cholesterol. Furthermore, when cells stably expressed NPC1L1ΔNTD-GFP, α-tocopherol could not induce the endocytosis of NPC1L1ΔNTD. Taken together, these results demonstrate that NPC1L1 recognizes α-tocopherol via its NTD and mediates α-tocopherol uptake through the same mechanism as cholesterol absorption.