Charatda Punvittayagul

Effect of pinocembrin isolated from Boesenbergia pandurata on xenobiotic-metabolizing enzymes in rat liver.

Pinocembrin, 5, 7-dihydroxyflavanone, is one of the flavanones found in the rhizomes of Boesenbergia pandurata. Previous study demonstrated that pinocembrin was neither toxic nor mutagenic to male rats. This study evaluated the effects of pinocembrin on phase I and II xenobiotic-metabolizing enzymes in rat liver. It was found that heme oxygenase activity significantly increased in 10 and 100 mg/kg bw of pinocembrin treated groups (p<0.05). However, pinocembrin did not affect the activities of NADPH: cytochrome P450 reductase, NADPH: quinone reductase, UDP-glucuronosyltransferase and glutathione-S-transferase. It also did not affect the expression of phase I metabolizing enzymes, including CYP1A1, CYP2B1, CYP2C11, CYP2E1, CYP3A2, and NADPH: cytochrome P450 reductase. In conclusion, short-term treatment of pinocembrin in Wistar rats increased the activity of heme oxygenase but did not affect on the activities of other phase II xenobiotic-metabolizing enzymes or the expression of cytochrome P450 enzymes.

Mutagenicity and antimutagenicity of hydrophilic and lipophilic extracts of Thai northern purple rice

Purple rice (Oryza sativa L. var. indica) cv. Kum Doisaket is cultivated in northern Thailand. This study evaluated the mutagenic and antimutagenic properties of hydrophilic and lipophilic components of purple rice using the Ames test. The seed and hull of purple rice were extracted with hexane, methanol, ethanol, and water. The methanol extracts had the highest amounts of phenolic acids and flavonoids, while the hexane extracts contained large amount of tocols and γ-oryzanol. None of the extracts were mutagenic in Salmonella typhimurium strains TA98 and TA100. The hexane extract of rice hull and the methanol extract of rice seed were strongly effective against aflatoxin B1- and 2-amino-3, 4 dimethylimidazo (4, 5-f) quinoline-induced mutagenesis, while aqueous extracts showed weakly antimutagenic properties. All extracts with the exception of aqueous extracts enhanced the number of revertant colonies from benzo (a) pyrene induced-mutagenesis. None of the extracts inhibited mutagenesis induced by the direct mutagens 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide and sodium azide. The hull extracts showed more potent antimutagenicity than the seed extracts. Based on a chemical analysis, γ-oryzanol and γ-tocotrienol in the hull and cyanidin-3-glucoside and peonidin-3-glucoside in the seed are candidate antimutagens in purple rice. The antimutagenic mechanisms of purple rice might be related to either modulation of mutagen metabolizing enzymes or direct attack on electrophiles. These findings supported the use of Thai purple rice as a cancer chemopreventive agent.

Purple Rice Bran Extract Attenuates the Aflatoxin B1-Induced Initiation Stage of Hepatocarcinogenesis by Alteration of Xenobiotic Metabolizing Enzymes

Pigmented rice bran has been suggested to be a valuable source of beneficial phytochemicals. We investigated genotoxic and anti-genotoxic effects of purple rice bran extract (PRBE) in rats using a liver micronucleus assay. Purple rice bran was extracted with methanol, obtaining large amounts of phenolic compounds, including anthocyanins and small amounts of gamma-oryzanol. The experimental protocols were divided into two sets. Male rats were divided into three groups. Group 1 was a negative control, while Groups 2 and 3 were fed with 100 and 500 mg/kg bw of PRBE, respectively, for 28 days. PRBE had no effect on micronucleus formation or xenobiotic metabolizing enzymes in rat liver. Experiments concerning the effect of PRBE on AFB1 showed that PRBE significantly lessened the amount of micronucleated hepatocytes in AFB1 treated rats. Furthermore, it modulated metabolic activation of AFB1 metabolism in the liver by suppressing activity and protein expression of CYP1A2, CYP3A and CYP 450 reductase, and enhancing phase II enzymes including GST and UGT. Overall, purple rice bran extract was not genotoxic in rats. It exhibited anti-genotoxicity by modulation some xenobiotic enzymes active in AFB1 metabolism.

Cancer Chemopreventive Effect of Spirogyra Neglecta (Hassall) Kützing on Diethylnitrosamine-Induced Hepatocarcinogenesis in Rats

Spirogyra neglecta, a freshwater green alga, is a local food in the northern and northeastern parts of Thailand. This investigation explored the anticarcinogenicity of S neglecta and its possible cancer chemopreventive mechanisms in rats divided into 14 groups. Groups 1 and 10 served as positive and negative control groups, respectively. Groups 1-9 were intraperitoneally injected with diethylnitrosamine (DEN) once a week for 3 weeks. Groups 10-14 received normal saline instead. One week after the last DEN injection, groups 2-5 were administered for 9 consecutive weeks various doses of S neglecta extract (SNE) and dried S neglecta (SND), mixed with basal diet. Groups 6-9 and 11-14 similarly were administered various doses of SNE and SND starting from the first week of the experiment. Administration of SNE and SND was not associated with formation of glutathione-S- transferase placental form (GST-P) positive foci in rat liver. SNE and SND during initiation phase significantly reduced the number of GST-P positive foci in rats injected with DEN. The number of GST-P also diminished in groups treated with SNE and SND after injection with DEN, except for the low dose extract group. SNE showed stronger anticarcinogenic potency than SND. Furthermore, SNE also decreased the number of Ki-67 positive cells. However, the numbers of TUNEL-positive cells in the liver of the SNE-treated groups were not statistically different from the controls. The GST activity in 50 mg/kg bw of SNE and 1% of SND groups was significantly increased as compared to the positive control. In conclusion, Spirogyra neglecta (Hassall) Kutzing showed cancer chemopreventive properties at the early stages of diethylnitrosamine-induced hepatocarcinogenesis in rats. Possible inhibitory mechanisms include enhancement of the activities of some detoxifying enzymes and/or suppression of precancerous cells.

Effects of pinocembrin on the initiation and promotion stages of rat hepatocarcinogenesis

Pinocembrin (5, 7-dihydroxyflavanone) is a flavanone extracted from the rhizome of Boesenbergia pandurata. Our previous studies demonstrated that pinocembrin had no toxicity or mutagenicity in rats. We here evaluated its effects on the initiation and promotion stages in diethylnitrosamine-induced rat hepatocarcinogenesis, using short- and medium-term carcinogenicity tests. Micronucleated hepatocytes and liver glutathione-S-transferase placental form foci were used as end point markers. Pinocembrin was neither mutagenic nor carcinogenic in rat liver, and neither inhibited nor prevented micronucleus formation as well as GST-P positive foci formation induced by diethylnitrosamine. Interestingly, pinocembrin slightly increased the number of GST-P positive foci when given prior to diethylnitrosamine injection.

Cleistocalyx nervosum Extract Ameliorates Chemical-Induced Oxidative Stress in Early Stages of Rat Hepatocarcinogenesis

PURPOSE To study the effect of Cleistocalyx nervosum extract (CE) on diethylnitrosamine (DEN) and phenobarbital (PB) induced oxidative stress in early stages of rat hepatocarcinogenesis. MATERIALS AND METHODS Male Wistar rats were divided into 4 groups, with Group 1 as a negative control and Group 2 was a positive control receiving DEN injections once a week and PB in drinking water for 6 weeks. Two weeks before DEN initiation and PB treatment, Groups 3 and 4, were fed with 500 and 1000 mg/kg of CEs, respectively, for 8 weeks. RESULTS A number of GST-P-positive foci, preneoplastic lesions, in the liver were markedly increased in carcinogen administered rats, but was comparatively decreased in rats treated with 1000 mg/kg of CE. The CE reduced malondialdehyde in serum and in the livers of rats treated with DEN and PB. Moreover, CE significantly increased the activities of glutathione peroxidase and catalase in rat liver. CONCLUSIONS CE appeared to exert its chemopreventive effects by modulating antioxidant status during DEN and PB induced early stages of hepatocarcinogenesis in rats.

Antimutagenicity and anticlastogenicity of glutinous purple rice hull using in vitro and in vivo testing systems

Glutinous purple rice (Oryza sativa L.var. indica) contains high amounts of nutrients and phytochemicals. Rice hull is a low-value waste product, yet contains many bioactive ingredients. The genotoxicity and antigenotoxicity of the dicholoromethane extract of glutinous purple rice hull were investigated. The amounts of phenolic compounds, flavonoids and γ-oryzanol of hull extract were 174.0±55.0, 220.0± 7.5, and 23.6±0.1 mg/g extract, respectively. Lower amounts of tocopherols and tocotrienols were detected. The mutagenicity and antimutagenicity of the extract were determined using a Salmonella mutation assay. Rice hull extract was not mutagenic in either strain TA98 or TA100 in the presence and absence metabolic activation. The extract showed antimutagenicity against AFB1 and MeIQ, but did not against AF-2 and NaN3. It enhanced the mutagenic activity of benzo(a)pyrene in TA100 with metabolic activation. The toxicity and genotoxicity of dichloromethane extract of glutinous purple rice hull were further studied in an animal model. The extract was non-toxic to rats at a single dose of 2000 mg/kg bw of extract following the OECD Guideline 425 protocol. The clastogenicity and anticlastogenicity of the extract were studied using a rat liver micronucleus assay. The oral administration of 500 mg/kg bw of glutinous purple rice hull extract for 28 days did not affect the number of micronucleated hepatocytes or mitotic index in rats, but it significantly decreased the number of micronucleated hepatocytes in diethylnitrosamine-initiated rats. Furthermore, the dichloromethane rice hull extract significantly enhanced glutathione-S-transferase activity in the livers of diethylnitrosamine-initiated rats. In conclusion, dichloromethane extract of glutinous purple rice hull presented antimutagenic and anticlastogenic potential. The inhibitory mechanism of this rice hull extract might be partly due to either induction of detoxifying enzymes or inhibition of metabolizing enzymes.

Effects of hydrophilic compounds in purple rice husk on AFB 1 -induced mutagenesis

Rice husk has been shown to possess cancer chemopreventive activity. This study focused on mutagenicity and antimutagenicity of the crude acidified methanolic extract of purple rice husk and its solvent- partitioned fractions using a Salmonella mutation assay and rat liver micronucleus test. The purple rice husk extracts were not mutagenic in S. typhimurium strain TA98, but were mutagenic in TA100 (crude extract and its hydrophilic fractions). There was no significant mutagenicity of purple rice husk extracts in rat liver. The rice husks possessed antimutagenicity against AFB1-induced mutagenesis in both bacterial and animal models. The dichloromethane-partitioned fraction (DPF) showed the highest antimutagenicity. Vanillic acid, a major phenolic compound found in DPF, exhibited antimutagenicity against AFB1 in both bacterial and rat models. The inhibitory mechanism was associated with the induction of detoxifying enzymes in AFB1 metabolism of rat liver. These findings suggested that vanillic acid may be a principal antimutagenic compound in purple rice husk.

Anticlastogenicity and Anticarcinogenicity of Purple Rice Extract in Rats

ABSTRACT Oryza sativa L. var. indica cv. Kum Doi Saket is a pigmented rice variety grown in northern Thailand. Our previous study found that the methanol extract of purple rice seed had the highest level of antimutagenicity in a Salmonella mutation assay. The present study was designed to evaluate its in vivo anticlastogenic and anticarcinogenic potentials. The purple rice extract had no acute toxicity on rats. The oral administration of 1,000 mg/kg body weight (bw) of the extract for 28 days did not increase the number of micronucleated hepatocytes. Interestingly, it significantly reduced the amount of micronucleus formation in the liver of diethylnitrosamine (DEN)-treated rats. The inhibitory mechanism involved the induction of hepatic glutathione S-transferase (GST) activity. In addition, oral administration of 500 mg/kg bw extract for 10 weeks significantly decreased the number of hepatic GST placental form positive foci, but did not modulate the number of colonic aberrant crypt foci in DEN- and dimethylhydrazine-initiated rats. In conclusion, the methanol extract of purple rice seed showed no toxicity, clastogenicity, or carcinogenicity in laboratory rats. It did display chemopreventive activity against the early stages of rat hepatocarcinogenesis.

Augmentation of diethylnitrosamine–induced early stages of rat hepatocarcinogenesis by 1,2-dimethylhydrazine

Abstract Diethylnitrosamine (DEN) and 1,2-dimethylhydrazine (DMH) are classical carcinogens used in experimental rodent carcinogenesis. However, the interaction effects of these carcinogens on biochemical and molecular changes during carcinogenesis have not been investigated. Therefore, the effect of DEN and DMH co-administration on preneoplastic lesion formation and its molecular mechanism in rats were determined. Triple intraperitoneal administrations of DEN were made before, during or after double subcutaneous injections of DMH. At week 8 of the experiment, the preneoplastic hepatic glutathione-S-transferase placental form (GST-P) positive foci and colonic aberrant crypt foci (ACF) were analyzed. The combined treatment of these carcinogens increased toxicity to rats. Administration of DMH alone did not induce hepatic GST-P positive foci, while co-treatment with DMH enhanced hepatic GST-P positive foci formation. However, DEN did not influence the size or number of colonic ACF. The treatment with DMH alone induced CYP2E1 and P450 reductase, demonstrating that DMH enhanced DEN metabolism in DEN- and DMH-treated rats. These findings were related to increases in hepatic O6-methylguanine DNA adducts and hepatotoxicity, which are associated with the induction of cell proliferation and liver cancer development. DEN-induced early stages of rat hepatocarcinogenesis were synergistically promoted by DMH via metabolic enzyme induction leading to enhanced DNA mutation and hepatocarcinogenicity.