A Japanese Patient with Gastric Cancer and Dihydropyrimidine Dehydrogenase Deficiency Presenting with DPYD Variants.

A 63-year-old Japanese male with stomach adenocarcinoma received oral 5-fluorouracil derivative, cisplatin and trastuzumab chemotherapy. On day 8, severe diarrhea and mucositis developed; chemotherapy was stopped. On day 14, the patient developed renal dysfunction and febrile neutropenia. He also suffered from pneumonia due to Candida albicans. Systemic symptoms improved after intensive conservative treatment. Best supportive care was continued until the patient died from gastric cancer. The dihydropyrimidine dehydroge-nase protein level was low at 3.18 U/mg protein. The result of DPYD genotyping revealed three variants at posi-tions 1615 (G > A), 1627 (A > G), and 1896 (T > C) in exons 13, 13, and 14, respectively.

Magnolol protects PC12 cells from hydrogen peroxide or 6-hydroxydopamine induced cytotoxicity.

Magnoliae Cortex contains a range of bioactive components including terpenes (e.g. α-, ß- and γ-eudesmol), phenylpropanoids (e.g. honokiol and magnolol) and alkaloids (e.g. magnocurarine). We recently reported that pretreatment of PC12 cells with Magnoliae Cortex extract significantly suppresses cytotoxicity induced by H2O2 or 6-hydroxydopamine (6-OHDA) through the induction of drug-metabolizing and antioxidant enzymes. In this study, we investigated whether honokiol and magnolol, which are known to be active components of Magnoliae Cortex, induce drug-metabolizing enzymes and antioxidant enzymes in PC12 cells. We also examined the cytoprotective effect of honokiol and magnolol against H2O2 or 6-OHDA induced cell death in PC12 cells. Our results revealed that honokiol and magnolol induced both NAD(P)H:quinone oxidoreductase 1 (NQO1) and catalase enzyme activities in a concentration-dependent manner. Pretreatment of PC12 cells with magnolol suppressed toxicity induced by H2O2 or 6-OHDA. However, pretreatment of PC12 cells with honokiol showed only a suppressive effect on toxicity induced by H2O2. Our results suggest that the cytoprotective effect of Magnoliae Cortex extract on PC12 cells is mainly attributable to magnolol and only partially to honokiol.

4-(Hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone glucuronide has the potential to form 2'-deoxyguanosine and N-acetylcysteine adducts.

Cigarette smoking is a risk factor for the development of various cancers, such as lung, nasal, liver and bladder cancers. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, is implicated in human lung cancer. NNK-induced DNA adducts are found in target tissues for NNK carcinogenesis. NNK is activated by cytochrome P450 dependent α-hydroxylation at either the methylene carbon or methyl carbon adjacent to the N-nitroso group. The former leads to the formation of the methylating agent, and the latter produce the pyridyloxobutylating agent. NNK and some of its metabolites are further metabolized by UDP-glucuronosyltransferases (UGTs). Glucuronides generally are much less active than the parent aglycon therefore the glucuronides of NNK-related metabolites are thought to be inactive. However, 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone glucuronide (HO-methyl NNK glucuronide) can be transported to the target organs of NNK carcinogenesis where subsequent hydrolysis causes the release of the reactive intermediate. Regeneration of HO-methyl NNK could play an important role in the tissue-specific carcinogenicity of NNK. In the present study, we investigated the reactivity of HO-methyl NNK glucuronide toward 2'-deoxyguanosine (dGuo) and N-acetylcysteine (NAC; used as a models for thiol groups on proteins). The reaction mixtures of HO-methyl NNK glucuronide and dGuo or NAC were analyzed by LCMS-IT-TOF-MS. We also employed 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone, a pyridyloxobutylating agent, to confirm the formation of pyridyloxobutylated adducts. Thus, we determined the production of pyridyloxobutylated dGuo and NAC adducts. Our results suggest HO-methyl NNK glucuronide could generate a reactive intermediate in the tissues and then form adducts with proteins and DNA.

Procyanidins from Cinnamomi Cortex promote proteasome-independent degradation of nuclear Nrf2 through phosphorylation of insulin-like growth factor-1 receptor in A549 cells.

Many lines of evidence demonstrate that transcription factor nuclear factor-E2-related factor 2 (Nrf2) plays essential roles in cancer cell proliferation and resistance to chemotherapy, thereby indicating that suppression of abnormal Nrf2 activation is needed for a new therapeutic approach. Our previous studies reported that procyanidins prepared from Cinnamomi Cortex extract (CCE) have an ability to suppress cytoprotective enzymes and cell proliferation in human cancer cells with activated Nrf2. In the present study, we investigated the mechanism of CCE procyanidin-mediated antagonization of Nrf2. CCE procyanidin treatment rapidly reduced nuclear Nrf2 expression and phosphorylated insulin-like growth factor-1 receptor (IGF-1R) in A549 cells. Nrf2 protein expression in A549 cells with reduced IGF-1R expression and function was not affected by treatment with CCE procyanidins, which suggested that CCE procyanidins decreased Nrf2 through IGF-1R. Nrf2 suppression by CCE procyanidins was mitigated in the presence of protease inhibitors, not proteasome inhibitors. In addition, CCE procyanidin treatment led to enhancement of nuclear cysteine protease activity in A549 cells. Our findings suggest a novel mechanism by which CCE procyanidins can promote proteasome-independent degradation of nuclear Nrf2 through IGF-1R phosphorylation and cysteine protease activation.

Selective antagonization of activated Nrf2 and inhibition of cancer cell proliferation by procyanidins from Cinnamomi Cortex extract.

Nuclear factor-E2-related factor 2 (Nrf2) is an important transcription factor and plays a central role in inducible expression of many cytoprotective genes. Recent studies have reported that various cancer cells having unrestrained Nrf2 due to its overexpression exhibit increased proliferation and resistance to chemotherapy. Suppression of abnormal Nrf2 activation is needed for a new therapeutic approach against these cancers. Our previous study found that procyanidins prepared from Cinnamomi Cortex extract (CCE) have an ability to suppress Nrf2-regulated enzyme activity and Nrf2 expression in human lung cancer A549 cells. In the present study, we investigated the effect of CCE procyanidins on Nrf2 activity and cell proliferation in several cancer cells, which have normal or constitutively active Nrf2. Interestingly, CCE procyanidin treatment selectively reduced Nrf2 expression and inhibited cell proliferation in cancer cells that overexpress Nrf2, but these phenomena were not seen in cells with low Nrf2 expression. Moreover, transfection assay demonstrated that CCE procyanidins had selective inhibition of activated Nrf2. These results suggest that CCE procyanidins might be an effective cancer therapeutic agent to selectively suppress abnormal Nrf2 activation responsible for enhanced proliferation.

5-Fluorouracil Chemotherapy for Dihydropyrimidine Dehydrogenase-deficient Patients: Potential of the Dose-escalation Method.

BACKGROUND: Dihydropyrimidine dehydrogenase (DPD) degrades approximately 85% of administered 5-fluorouracil (5-FU). With a reported high mortality rate, chemotherapy is generally contraindicated for patients with DPD deficiency. PATIENTS AND METHODS: Chemotherapy was initiated for a 73-year-old man with DPD deficiency. Capecitabine was administered in incrementally increasing doses, beginning with a single pill while monitoring plasma 5-FU concentration, and neutrophil and platelet counts. RESULTS: DPD protein level was 2.35 U/mg. After increasing the capecitabine dose to 1,800 mg, oxaliplatin and bevacizumab were added. Subsequent DPD protein measurement showed that the level had increased to approximately 12-fold the one before chemotherapy. Sequencing of all 23 exons of DPYD gene revealed a mutation of guanine to thymine in exon 11 (1156 G>T). CONCLUSION: This is the first report to indicate that DPD activity can be induced. These findings may provide early indications of a new method for chemotherapy for DPD-deficient patients.

Formation and stability of 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone glucuronide, a stable form of reactive intermediate produced from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, in mice.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific nitrosamine, induced lung tumors in rodents and is likely involved in human lung cancer. 4-(Hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (HO-methyl NNK) glucuronide, a glucuronide of the reactive intermediate of NNK, has been identified in rats. The aim of this study is to estimate the role of HO-methyl NNK glucuronide in the tumorigenic effects of NNK. We investigated the urinary excretion and tissue distribution of HO-methyl NNK glucuronide in A/J mice, which are susceptible to NNK carcinogenesis, and C57BL/6J mice, which are resistant to NNK carcinogenesis. The cumulative urinary excretion of the HO-methyl NNK glucuronide in the C57BL/6J mice was more than 20 times higher than in the A/J mouse urine. Tissue concentrations of HO-methyl NNK glucuronide were also higher in the C57BL/6J mice than in the A/J mice. Assessment of the stability of HO-methyl NNK glucuronide in liver homogenates at physiological pH conditions showed that more than 60% of the glucuronide remained until 2 hr of incubation. These results suggested that HO-methyl NNK glucuronide is likely to be a detoxified metabolite and could be one reason for differences in the susceptibility to NNK tumorigenesis between the two strains. Once HO-methyl NNK is formed in tissues, C57BL/6J mice have a high ability to form HO-methyl NNK glucuronide so that HO-methyl NNK, the reactive intermediate formed from NNK, is readily excreted in urine as a stable form.

Tissue distributions of 4-(hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone glucuronide, a stable form of reactive intermediate produced from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, in the rat.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), induces lung tumors in rodents and has been suggested as a causative factor in human lung cancer. NNK is activated by α-hydroxylation at either the methyl or methylene carbon adjacent to the N-nitroso group to yield unstable intermediates that spontaneously decompose to produce alkylating agents. 4-(Hydroxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (HO-methyl NNK) glucuronide, a glucuronide of the reactive intermediate of NNK has been identified. However, there are no available data concerning HO-methyl NNK glucuronide. In the present study, we investigated the tissue distribution of HO-methyl NNK glucuronide in control and phenobarbital (PB)-treated rats after intraperitoneal administration of NNK. In PB-treated rats, HO-methyl NNK glucuronide was detected in plasma, kidney, liver, lung, and pancreas. On the contrary, in the control rats, HO-methyl NNK glucuronide was detected only in plasma, kidney and liver at low concentrations compared with PB-treated rats. The results of cumulative urinary excretion of HO-methyl NNK glucuronide in Wistar and Gunn rats suggested that PB-inducible UDP-glucuronosyltransferase 2B isoforms mainly contribute to the formation of HO-methyl NNK glucuronide.

Enhanced sensitivity of A549 cells to the cytotoxic action of anticancer drugs via suppression of Nrf2 by procyanidins from Cinnamomi Cortex extract.

Nuclear factor-E2-related factor 2 (Nrf2) is an important cytoprotective transcription factor because Nrf2-regulated enzymes play a key role in antioxidant and detoxification processes. Recent studies have reported that lung cancer cells overexpressing Nrf2 exhibit increased resistance to chemotherapy. Suppression of overexpressed Nrf2 is needed for a new therapeutic approach against lung cancers. In the present study, we found that Cinnamomi Cortex extract (CCE) has an ability to suppress Nrf2-regulated enzyme activity and Nrf2 expression in human lung cancer A549 cells with high Nrf2 activity. Moreover, we demonstrated that CCE significantly enhances sensitivity of A549 cells to the cytotoxic action of doxorubicin and etoposide as well as increasing the intracellular accumulation of both drugs. These results suggest that CCE might be an effective concomitant agent to reduce anticancer drug resistance derived from Nrf2 overexpression. Bioactivity-guided fractionation revealed that procyanidin tetramers and pentamers contained in CCE were active components in suppressing Nrf2.

Involvement of different human glutathione transferase isoforms in the glutathione conjugation of reactive metabolites of troglitazone.

Null mutation of glutathione transferase (GST) M1 and GSTT1 was reported to correlate statistically with an abnormal increase in the plasma levels of alanine aminotransferase or aspartate aminotransferase caused by troglitazone in diabetic patients (Clin Pharmacol Ther, 73:435-455, 2003). This clinical evidence leads to the hypothesis that GSH conjugation catalyzed by GSTT1 and GSTM1 has a role in the elimination of reactive metabolites of troglitazone. However, the contribution of GST isoforms expressed in human liver to the detoxification of reactive metabolites of troglitazone has not yet been clarified. We investigated the involvement of human GST isoforms in the GSH conjugation of reactive metabolites of troglitazone using recombinant GST enzymes. Five reported GSH conjugates of reactive metabolites were produced from troglitazone after incubation with liver microsomes, NADPH, and GSH in a GSH concentration-dependent manner. Addition of human recombinant GSTA1, GSTA2, GSTM1, or GSTP1 protein to the incubation mixture further increased the GSH conjugates. However, the addition of GSTT1 did not show any catalytic effect. It is of interest that one of the reactive metabolites with a quinone structure was predominantly conjugated with GSH by GSTM1. Thus, we demonstrated that the GST isoforms contributed differently to the GSH conjugation of individual reactive metabolites of troglitazone, and GSTM1 is the most important GST isoform in the GSH conjugation of a specific reactive metabolite produced from the cytotoxic, quinone-form metabolite of troglitazone.

Dietary diacetylene falcarindiol induces phase 2 drug-metabolizing enzymes and blocks carbon tetrachloride-induced hepatotoxicity in mice through suppression of lipid peroxidation.

Falcarindiol is a diacetylenic natural product containing unique carbon-carbon triple bonds. Mice were orally administrated falcarindiol (100 mg/kg), and drug-metabolizing and antioxidant enzymes were monitored in several tissues of mice. Treatment with falcarindiol was found to increase glutathione S-transferase (GST) and NAD(P)H: quinone oxidoreductase 1 activities in liver, small intestine, kidney, and lung. No changes were observed in cytochrome P450 (CYP) 1A known to activate procarcinogens. Western blot analysis revealed that various GST subunits including GSTA4, which plays an important role in the detoxification of alkenals produced from lipid peroxides, were induced in liver, small intestine, and kidney of falcarindiol-treated mice. Additionally, we investigated the protective effects of falcarindiol against hepatotoxicity induced by carbon tetrachloride (CCl(4)) and the mechanism of its hepatoprotective effect. Pretreatment with falcarindiol prior to the administration of CCl(4) significantly suppressed both an increase in serum alanine transaminase/aspartate transaminase (ALT/AST) activity and an increase in hepatic thiobarbituric acid reactive substance levels without affecting CCl(4)-mediated degradation of CYP2E1. Formation of hexanoyl-lysine and 4-hydroxy-2(E)-nonenal-histidine adducts, lipid peroxidation biomarkers, in homogenates from the liver of CCl(4)-treated mice was decreased in the group of mice pretreated with falcarindiol. These results suggest that the protective effects of falcarindiol against CCl(4) toxicity might, in part, be explained by anti-lipid peroxidation activity associated with the induction of the GSTs including GSTA4.

Cooperation of NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferases reduces menadione cytotoxicity in HEK293 cells.

Previous studies have shown that NAD(P)H:quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of menadione (2-methyl-1,4-naphthoquinone, also known as vitamin K3). However, menadiol (2-methyl-1,4-naphthalenediol) formed from menadione by NQO1-mediated reduction continues to be an unstable substance, which undergoes the reformation of menadione with concomitant formation of reactive oxygen species (ROS). Hence, we focused on the roles of phase II enzymes, with particular attention to UDP-glucuronosyltransferases (UGTs), in the detoxification process of menadione. In this study, we established an HEK293 cell line stably expressing NQO1 (HEK293/NQO1) and HEK293/NQO1 cell lines with doxycycline (DOX)-regulated expression of UGT1A6 (HEK293/NQO1/UGT1A6) and UGT1A10 (HEK293/NQO1/UGT1A10), and evaluated the role of NQO1 and UGTs against menadione-induced cytotoxicity. Our results differed from those of previous studies. HEK293/NQO1 was the most sensitive cell line to menadione cytotoxicity among cell lines established in this study. These phenomena were also observed in HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells in which the expression of UGT was suppressed by DOX treatment. On the contrary, HEK293/NQO1/UGT1A6 and HEK293/NQO1/UGT1A10 cells without DOX treatment were resistant to menadione-induced cytotoxicity. These results demonstrated that NQO1 is not a detoxification enzyme for menadione and that UGT-mediated glucuronidation of menadiol is the most important detoxification process.

Activation of the Nrf2/ARE pathway via S-alkylation of cysteine 151 in the chemopreventive agent-sensor Keap1 protein by falcarindiol, a conjugated diacetylene compound.

Under basal conditions, the interaction of the cytosolic protein Kelch-like ECH-associated protein 1 (Keap1) with the transcription factor nuclear factor-E2-related factor 2 (Nrf2) results in a low level of expression of cytoprotective genes whose promoter region contains the antioxidant response element (ARE). In response to oxidants and electrophiles, Nrf2 is stabilized and accumulates in the nucleus. The mechanism for this effect has been proposed to involve thiol-dependent modulation of Keap1, leading to loss of its ability to negatively regulate Nrf2. We previously reported that falcarindiol (heptadeca-1,9(Z)-diene-4,6-diyne-3,8-diol), which occurs in Apiaceae and the closely related Araliaceae plants, causes nuclear accumulation of Nrf2 and induces ARE-regulated enzymes. Here, we report the mechanism of Nrf2 induction by falcarindiol. NMR analysis revealed that the conjugated diacetylene carbons of falcarindiol acted as electrophilic moieties to form adducts with a cysteine (Cys) thiol. In addition, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and circular dichroism spectroscopy, it was demonstrated that falcarindiol alkylated Cys residues in Keap1 and altered the Keap1 secondary structure. Transfection studies using the purified Keap1 protein, a luciferase reporter construct, and an Nrf2-expressing plasmid indicated that the intact Keap1 protein suppressed Nrf2-mediated ARE-luciferase activity. On the other hand, the falcarindiol-alkylated Keap1 protein did not suppress such activity. Treatment of HEK293 cells overexpressing Keap1 with falcarindiol generated a high molecular weight (HMW) form of Keap1. Furthermore, the Cys151 residue in Keap1 was found to be uniquely required for not only the formation of HMW Keap1 but also an increase in ARE-luciferase activity by falcarindiol. Our results demonstrate that falcarindiol having conjugated diacetylene carbons covalently modifies the Cys151 residue in Keap1 and that the inactivation of Keap1 by falcarindiol leads to activation of the Nrf2/ARE pathway.

Induction of antioxidant and phase 2 drug-metabolizing enzymes by falcarindiol isolated from Notopterygium incisum extract, which activates the Nrf2/ARE pathway, leads to cytoprotection against oxidative and electrophilic stress.

In the present study, we isolated falcarindiol from Notopterygium incisum and investigated the effect of falcarindiol on the expression of antioxidant enzymes (AOEs), such as catalase, and phase 2 drug-metabolizing enzymes (DMEs), such as glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1, in a cultured cell line from normal rat liver, Clone 9 cells. Exposure of Clone 9 cells to falcarindiol resulted in the significant induction of AOEs and phase 2 DMEs. Western blot analysis and transfection studies using a luciferase reporter construct demonstrated that the induction of AOEs and phase 2 DMEs by falcarindiol was caused through the Nrf2/ARE (nuclear factor-E2-related factor 2/antioxidant response element) pathway. Pretreatment of cells with falcarindiol accelerated the detoxification of a potentially toxic quinone (menadione) and mitigated menadione-induced cytotoxicity. We found that falcarindiol was a novel inducer of AOEs and phase 2 DMEs and falcarindiol might exhibit chemopreventive activity.

Inhibitory effects of herbal extracts on the activity of human sulfotransferase isoform sulfotransferase 1A3 (SULT1A3).

Sulfotransferase 1A3 (SULT1A3) is a phase II detoxifying enzyme of xenobiotics predominantly expressed in the intestinal epithelium. Recent increase in the use of herbal extracts as dietary supplements may lead to an increase in the possibility of dietary supplement-drug interactions. The purpose of the present study was to investigate the effects of 18 herbal extracts on SULT1A3 activity and the possibility of interaction between medicinal drugs and herbal extracts. We examined the inhibitory potencies of 18 herbal extracts on the sulfation of dopamine, a typical substrate of SULT1A3, and ritodrine, a beta(2) stimulant, by human recombinant SULT1A3. The sulfation of dopamine was inhibited by extracts of banaba, green tea, Rafuma, grape seed, peanut seed coat, gingko biloba leaf, St. John's wort, gymnema and milkthistle. The IC(50) values of these herbal extracts were lower than the putative gastrointestinal concentration when the recommended dose was ingested. On the other hand, chlorella extract and rutin showed no inhibitory effects and wheat, mulberry and siberian ginseng had IC(50) values exceedingly higher than the putative gastrointestinal concentration. The inhibitory profiles of herbal extracts for the sulfation of ritodrine were comparable to those for the sulfation of dopamine. In conclusion, the extracts of herbs such as banaba and green tea potently inhibited SULT1A3 activity. These extracts may increase the bioavailability of drugs whose bioavailabilities were limited by the function of SULT1A3 on the intestinal epithelium.

Amino acid positions 69-132 of UGT1A9 are involved in the C-glucuronidation of phenylbutazone.

Phenylbutazone (PB) is known to be biotransformed to its O- and C-glucuronide. Recently, we reported that PB C-glucuronide formation is catalyzed by UGT1A9. Interestingly, despite UGT1A8 sharing high amino acid sequence identity with UGT1A9, UGT1A8 had no PB C-glucuronidating activity. In the present study, we constructed eight UGT1A9/UGT1A8 chimeras and evaluated which region is important for PB C-glucuronide formation. All of the chimeras and UGT1A8 and UGT1A9 had 7-hydroxy-(4-trifluoromethyl)coumarin (HFC) O-glucuronidating activity. The K(m) values for HFC glucuronidation of UGT1A8, UGT1A9 and their chimeras were divided into two types, UGT1A8 type (high K(m)) and UGT1A9 type (low K(m)), and these types were determined according to whether their amino acids at positions 69-132 were those of UGT1A8 or UGT1A9. Likewise, PB O-glucuronidating activity was also detected by all of the chimeras, and their K(m) values were divided into two types. On the contrary, PB C-glucuronidating activity was detected by UGT1A9((1-132))/1A8((133-286)), UGT1A9((1-212))/1A8((213-286)), UGT1A8((1-68))/1A9((69-286)), and UGT1A8((1-68))/1A9((69-132))/1A8((133-286)) chimeras. The region 1A9((69-132)) was common among chimeras having PB C-glucuronidating activity. Of interest is that UGT1A9((1-68))/1A8((69-132))/1A9((133-286)) had lost PB C-glucuronidation activity, but retained activities of PB and HFC O-glucuronidation. These results strongly suggested that amino acid positions 69-132 of UGT1A9 are responsible for chemoselectivity for PB and affinity to substrates such as PB and HFC.

Plasma profiling of intact isoflavone metabolites by high-performance liquid chromatography and mass spectrometric identification of flavone glycosides daidzin and genistin in human plasma after administration of kinako.

The roles of isoflavones in the prevention of several hormone-dependent cancers and osteoporosis are of great interest. Despite many pharmacokinetics studies of the isoflavones, the actual types of conjugates circulating in the body and the position(s) of conjugation sites on the flavone skeleton are still uncertain because, in general, conjugated compounds in biological fluids have been evaluated by measuring the free aglycones obtained after selective enzymatic hydrolysis. Using an high-performance (HPLC)-UV-diode-array detector (DAD) method combined with solid-phase extraction, we have obtained HPLC profiles of isoflavone glycosides [daidzin (Din) and genistin (Gin)] and of intact isoflavone metabolites in human plasma: daidzein, genistein, daizein-7-glucuronide, daidzein-4'-glucuronide, genistein-7-glucuronide, genistein-4'-glucuronide, daidzein-7-sulfate, daidzein-4'-sulfate, genistein-7-sulfate, and genistein-4'-sulfate. We investigated the plasma profile of intact isoflavone metabolites in plasma obtained 1 to-7 h after orally administration of 50 g of kinako (baked soybean powder) to two healthy volunteers. The results of DAD analysis indicated that the main isoflavone metabolite peaks were identified on the HPLC chromatogram. Furthermore, the intact glycosides Din and Gin were detected in 1-h plasma samples by their positive electrospray ionization mass spectra, demonstrating that the glycosides Din and Gin can be absorbed from the gut.

UDP-glucuronosyltransferases 1A6 and 1A10 catalyze reduced menadione glucuronidation.

Menadione (2-methyl-1,4-naphthoquine), also known as vitamin K3, has been widely used as a model compound in the field of oxidative stress-related research. The metabolism of menadione has been studied, and it is known that menadione undergoes a two-electron reduction by NAD(P)H:Quinone oxidoreductase 1 (NQO1) after which the reduced form of menadione (2-methyl-1,4-naphthalenediol, menadiol) is glucuronidated and excreted in urine. To investigate which human UDP-glucuronosyltransferase (UGT) isoforms participate in the glucuronidation of menadiol reduced by NQO1 from menadione, we first constructed heterologously expressed NQO1 in Sf9 cells and tested the menadiol glucuronidating activity of 16 human recombinant UGT isoforms. Of the 16 UGT isoforms, UGTs 1A6, 1A7, 1A8, 1A9, and 1A10 catalyzed menadiol glucuronidation, and, of these, UGTs 1A6 and 1A10 catalyzed menadiol glucuronidation at much higher rates than the other UGTs. Menadiol was regioselectively glucuronidated in the manner of 4-position>1-position by UGTs 1A7, 1A8, 1A9, and 1A10. In contrast to these UGTs, only UGT1A6 exhibited 1-menadiol-preferential glucuronidating activity. The results suggest possible detoxification pathways for quinones via NQO1 reduction followed by UGT glucuronidation.

Redox cycling of 9,10-phenanthraquinone to cause oxidative stress is terminated through its monoglucuronide conjugation in human pulmonary epithelial A549 cells.

9,10-Phenanthraquinone (PQ), a component of airborne particulate matter, causes marked cellular protein oxidation and cytotoxicity through a two-electron reduction to 9,10-dihydroxyphenanthrene (PQH2), which is associated with the propagation of reactive oxygen species (K. Taguchi et al., Free Radic. Biol. Med. 43:789-799, 2007). In the present study, we explored a biotransformation pathway for the detoxification of PQ. Exposure of human pulmonary epithelial A549 cells to PQ resulted in a time-dependent appearance of an unknown metabolite in the medium that was identified as the monoglucuronide of PQH2 (PQHG). Whereas a variety of isozymes of uridine 5'-diphosphate glucuronosyltransferase (UGTs) are responsible for PQHG formation, UGT1A10 and UGT1A6 were particularly effective catalysts for glucuronide conjugation. In cell-free systems, PQ exhibited a rapid thiol oxidation and subsequent oxygen consumption in the presence of dithiothreitol, whereas PQHG did not. Unlike the parent compound, PQHG completely lost the ability to oxidize cellular proteins and cause cell death in A549 cells. In addition, deletion of the transcription factor Nrf2 decreased PQHG formation and increased PQ-mediated toxicity of mouse primary hepatocytes. Thus, we conclude that PQHG is a metabolite of PQ, generated through PQH2, that terminates its redox cycling and transports it to extracellular space.

Inhibitory effects of various beverages on human recombinant sulfotransferase isoforms SULT1A1 and SULT1A3.

Sulfotransferase (SULT) 1A1 and SULT1A3 play important roles in the presystemic inactivation of beta(2) agonists in the liver and intestine, respectively. The study aimed to investigate the inhibitory effects of grapefruit juice, orange juice, green tea, black tea and oolong tea and their constituents on the activities of SULT1A1 and SULT1A3. The activities of both SULT1A1 and SULT1A3 were significantly inhibited by all the beverages investigated at a concentration of 10%. The beverage constituents were tested in concentration ranges considered to be physiologically relevant. The grapefruit constituent, quercetin, completely inhibited SULT1A1, while quercetin and naringin both partially inhibited SULT1A3. The orange constituents, tangeretin and nobiletin, also completely inhibited SULT1A1. The tea constituents, (-)-epicatechin gallate and (-)-epigallocatechin gallate, both almost completely inhibited SULT1A1 and SULT1A3. Moreover, the theaflavin and thearubigin fractions of black tea both completely inhibited SULT1A1 and strongly inhibited SULT1A3. The inhibitory action of green tea on SULT1A3 was competitive, while that of black tea and oolong tea was mixed competitive/non-competitive. Mechanism-based inhibition was not observed with any beverage. In conclusion, various beverages, especially teas, inhibit the function of SULT1A3, and therefore may have the potential to increase the bioavailability of orally administered substrates of SULT1A3, such as beta(2) agonists.