Anti-inflammatory and Quinone Reductase-Inducing Compounds from Beilschmiedia mannii.

Previous studies on the therapeutic potential of plant species found in the diet of chimpanzees living in Taï National Park have shown that they could be potential candidates for the search of new molecules useful for humans. Based on the screening of some of these plants, the fruits of Beilschmiedia mannii, whose dichloromethane extract showed cancer chemopreventive properties, were selected. Bioactivity-guided fractionation of the extract resulted in the isolation and identification of two γ-pyrones, including desmethoxydihydromethysticin (1: ), found in a natural source for the first time, and a new congener, beilschmiediapyrone (2: ), as well as five known alkamides (3:  - 7: ). Their structures were established by using nuclear magnetic resonance spectroscopy and mass spectrometry methods. The isolated compounds were evaluated for their cancer chemopreventive potential by using quinone reductase induction and nuclear factor-kappa B inhibition tests in Hepa 1c1c7 and HEK-293/NF-κB-Luc cells, respectively. Among them, compounds 1: and 2: were the most active. The concentrations to double the quinone reductase activity were 7.5 µM for compound 1: and 6.1 µM for compound 2: . Compounds 1: and 2: inhibited nuclear factor-kappa B with IC50 values of 2.1 and 3.4 µM, respectively. These results are promising with regard to cancer chemoprevention, especially because this plant is also used for cooking by the local population around the Taï forest.

Phenolics from Barleria cristata var. Alba as carcinogenesis blockers against menadione cytotoxicity through induction and protection of quinone reductase.

BACKGROUND: There are increasing interests in natural compounds for cancer chemoprevention. Blocking agents represent an important class of chemopreventive compounds. They prevent carcinogens from undergoing metabolic activation and thereby suppressing their interaction with cellular macromolecular targets. METHODS: The effect of phenolic compounds isolated from Barleria cristata var. alba as chemopreventive agent was evaluated. The ethyl acetate fraction of B. cristata was subjected to different chromatographic techniques for isolation of its major phenolic compounds. The isolated compounds were evaluated for their potential to induce the cancer chemopreventive enzyme marker NAD(P)H quinonereductase 1 (NQO1) in murine Hepa-1c1c7 cell model. RESULTS: The ethyl acetate fraction of B. cristata var. alba yielded five known compounds identified as verbascoside (1), isoverbascoside (2), dimethoxyverbascoside (3), p-hydroxy benzoic acid (4), and apigenin-7-O-glucoside (5). Among the tested compounds, isoverbascoside (2) was shown to potently induce the activity of the enzyme in a dose -dependent manner. As a functional assay for detoxification, compound 2 was the strongest to protect Hepa-1c1c7 against the toxicity of menadione, a quinone substrate for NQO1. CONCLUSION: This effect seemed to be attributed to the compound's potential to induce both the catalytic activity and protein expression of NQO1 as revealed by enzyme assay and Western blotting, respectively.

Expressions of some antioxidant genes in SH-SY5Y cells treated with ß-lapachone, morphine and electromagnetic field.

ß-Lapachone (ß-Lap), morphine (Mor), and electromagnetic field (EMF) generate reactive oxygen species. The goal of the present study was to examine the effects of Mor and EMF, in combination with ß-Lap on the cell growth inhibition and expression of several antioxidant genes. The 0.50 mT intensity of 50 Hz EMF and two exposure conditions ("15 min field-on/15 min field-off" and "30 min field-on continuously") on SH-SY5Y cells were used. The effects of Mor and EMF, in combination with ß-Lap on cell growth inhibition and the expression levels of several antioxidant genes (NQO1, NQO2, SOD1, SOD2, CAT, GSTO1, GSTM2, GSTM3, GSTP1, MGST1, MGST3) in SH-SY5Y cells were measured. The relative mRNA levels were calculated according to the [Formula: see text]. Whereas NQO1 mRNA level decreased in the "15 min field-on/15 min field-off" condition, the expression level of NQO2 was increased. Both NQO1 and NQO2 expressions increased in Mor treated cells. IC50 values of ß-Lap in combination with Mor, EMF, and "Mor + EMF" were higher than cells treated only with ß-Lap. The NQO1 expression level in the cells treated with ß-Lap was higher than the other treatments, indicating that ß-Lap induces the expression of NQO1. Moreover, multiple linear regression analysis indicated that NQO1 mRNA levels were associated positively with ß-Lap and negatively with EMF. At least in part, the mRNA levels of NQO1 were associated with IC50 values of ß-Lap in designed treatments. There is a negative association between mRNA levels of NQO1 and IC50 values of ß-Lap but not NQO2.

Effect of lupeol on antioxidants and xenobiotic enzymes in N-Butyl-N-(4-hydroxybutyl) nitrosamine induced bladder carcinogenesis in experimental rats.

OBJECTIVE: Urothelial carcinoma of the bladder is a common malignancy ranked 9th with an estimated 356,600 new cases diagnosed annually worldwide. The study showed the protective effects of Lupeol in N-Butyl-N-(4-hydroxybutyl) nitrosamine induced bladder carcinogenesis in in vivo experimental model. Forty male healthy wistar rats were selected randomly divided into four groups. Group I rats served as healthy control. Group II rats were treated with BBN (150 mg/gavage/twice a week) for 8 weeks. Group III rats were treated with BBN + Lupeol [ Lupeol (50 mg/kg bw/day) treatment was started 1 week prior to the BBN treatment, and it was orally administered for 8 weeks]. Group IV rats were treated with Lupeol alone (50 mg/kg bw/day) for 8 weeks. All the experimental rats were maintained and euthanized at 32nd week. Serum and bladder tissues were collected and examined for biochemical parameters, serum markers and histopathological evaluation. Preventive (BBN + Lupeol) group modulates the activity of antioxidant enzymes such as Superoxide dismutase, Catalase, Reduced glutathione, Glutathione Peroxidase, Thiobarbituric acid reactive substances (TBARS) and drug metabolizing enzymes such as Cytochrome P450, Cytochrome b5, NADPH Cytochrome c reductase, NADPH- Quinone Oxidoreductase 1 and Glutathione-S-transferase when compared to BBN treated rats. Serological markers such as Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) were significantly (P<0.05) decreased in preventive lupeol treated groups. Lupeol supplementation protects BBN induced bladder carcinogenesis in experimental rats by its antioxidant, anti-inflammatory and antiproliferative properties.

Peanut sprout extract attenuates cisplatin-induced ototoxicity by induction of the Akt/Nrf2-mediated redox pathway.

OBJECTIVE: Cisplatin is commonly used to treat solid tumors. However, permanent hearing loss is a major side effect of cisplatin chemotherapy and often results in dose reduction of the cisplatin chemotherapy. Peanut sprouts show cytoprotective properties owing to their antioxidant activities. This study was designed to investigate the effect of peanut sprout extract (PSE) on cisplatin-induced ototoxicity in an auditory cell line, HEI-OC1 cells. METHODS: Cells were exposed to cisplatin for 24 h, with or without pre-treatment with PSE, cell viability was examined using the MTT assay. Apoptotic cells were identified by double staining with Hoechst 33258 and propidium iodide. Western blot analysis was performed to examine apoptotic proteins including C-PARP and C-caspase, anti-apoptotic protein Bcl-2, and Nrf2 redox system activation. Mitochondrial reactive oxygen species (ROS) were investigated to examine whether PSE could scavenge cisplatin-induced ROS. Real-time PCR analyses were performed to investigate the mRNA levels of antioxidant enzymes including NQO1, HO-1, GPx2, Gclc, and catalase. RESULTS: The cisplatin-treated group showed reduced cell viability, increased apoptotic properties and markers, and increased ROS levels. PSE pre-treatment before cisplatin exposure significantly increased cell viability and reduced apoptotic properties and ROS production. These effects resulted from the up-regulation of antioxidant genes, including NQO1, HO-1, GPx2, Gclc, and catalase through Akt phosphorylation and Nrf2 activation. CONCLUSION: Our results demonstrate that PSE protects from cisplatin-induced cytotoxicity by activating the antioxidant effects via the Akt/Nrf-2 pathway in this auditory cell line, and indicate that PSE may provide novel treatment to prevent cisplatin-induced ototoxicity.

Talarolutins A-D: Meroterpenoids from an endophytic fungal isolate of Talaromyces minioluteus.

Four meroterpenoids [talarolutins A-D] and one known compound [purpurquinone A] were characterized from an endophytic fungal isolate of Talaromyces minioluteus (G413), which was obtained from the leaves of the medicinal plant milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)]. The structures of talarolutins A-D were determined by the analysis of various NMR and MS techniques. The relative and absolute configuration of talarolutin A was determined by X-ray diffraction analysis. A combination of NOESY data and comparisons of ECD spectra were employed to assign the relative and absolute configuration of the other analogs. Talarolutins B-D were tested for cytotoxicity against human prostate carcinoma (PC-3) cell line, antimicrobial activity, and induction of quinone reductase; no notable bioactivity was observed in any assay.

Chlorella ethanol extract induced phase II enzyme through NFE2L2 (nuclear factor [erythroid-derived] 2-like 2, NRF2) activation and protected ethanol-induced hepatoxicity.

In this study, we investigated the hepatoprotective effects of ethanol extracts from Chlorella vulgaris (CH) on animals. We measured its effect on the quinone reductase (QR) activity in Hepa1c1c7 cells, finding that CH induced a significantly higher QR activity in these cells. We isolated the active fraction (CH F4-2) from CH using chromatography methods. CH F4-2 may activate cellular antioxidant enzymes through upregulation of the Nrf2 pathway in hepatocarcinoma cells with CH F4-2 (25.0-200 µg/mL) for 48 h. Furthermore, CH F4-2 increased the expression of NQO1 [ NAD(P)H: quinone oxidoreductase, also known as QR], heme oxygenase-1, and glutathione-S-transferase P. Moreover, we found that ethanol-induced hepatic pathological changes-elevations in glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, γ-glutamyltransferase, and lactate dehydrogenase-were significantly decreased. The inhibitory effect of CH on alcohol-induced liver injury was associated with the suppression of alcohol-induced increases in intestinal permeability. The ethanol extract from CH was found to induce QR activation, making it a potentially good candidate for a hepatoprotection agent.

Natural potential therapeutic agents of neurodegenerative diseases from the traditional herbal medicine Chinese dragon's blood.

ETHNOPHARMACOLOGICAL RELEVANCE: Dragon's blood has been used as a famous traditional medicine since ancient times by many cultures. It is a deep red resin, obtained from more than 20 different species of four distinct genera. Red resin of Dracaena cochinchinensis S.C. Chen, known as Chinese dragon's blood or Yunnan dragon's blood, has been shown to promote blood circulation, alleviate inflammation, and to treat stomach ulcers, diarrhea, diabetes, and bleeding. This study investigated an effective approach to identify natural therapeutic agents for neurodegeneration from herbal medicine. The dichloride extract and isolated effective constituents of Chinese dragon's blood showed quinone oxidoreductase 1 (NQO1) inducing activity and anti-inflammatory effect significantly, which are therapy targets of various neurodegenerative diseases. MATERIALS AND METHODS: Multiple chromatography and spectra analysis were utilized to afford effective constituents. Then Hepa 1c1c7 and BV-2 cells were employed to assay their NQO1 inducing and anti-inflammatory activities, respectively. RESULTS: Bioactivities guided isolation afforded 21 effective constituents, including two new polymers cochinchinenene E (1), cochinchinenene F (2) and a new steroid dracaenol C (16). The main constituent 3 (weight percent 0.2%), 5 (weight percent 0.017%), 4 (weight percent 0.009%), 9 (weight percent 0.094%), 10 (weight percent 0.017%) and 8 (weight percent 0.006%) are responsible for the anti-inflammatory activities of Chinese dragon's blood. While, new compounds 1, 2 and known compounds 5, 11 showed good NQO1 inducing activities. The brief feature of the activities and structures was discussed accordingly. CONCLUSION: Overviewing the bioactivities and phytochemical study result, 4'-hydroxy-2,4-dimethoxydihydrochalcone (3) and pterostilbene (5) as effective constituents of Chinese dragon's blood, were found to be potential candidate therapeutic agents for neurodegenerative diseases.

Epilobium hirsutum alters xenobiotic metabolizing CYP1A1, CYP2E1, NQO1 and GPx activities, mRNA and protein levels in rats.

CONTEXT: Natural products have attracted increasing interests due to their use in flavoring, nutrition, cosmetics, pharmacy and medicine. Epilobium hirsutum L. (Onagraceae) is known for its analgesic, antimicrobial, and antiproliferative activity. CYP1A1 and CYP2E1, xenobiotic metabolizing enzymes, serve as a metabolic activation route yielding reactive metabolites that are eliminated by the action of NQO1 and glutathione peroxidase (GPx) enzymes. OBJECTIVE: This study investigated in vivo effects of Epilobium hirsutum (EH) on CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA expressions in liver. MATERIALS AND METHODS: Male Wistar Albino rats were injected with EH at a dose of 37.5 mg/kg i.p. daily for 9 d. CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA levels were determined by enzyme assays, Western blotting and qPCR, respectively. RESULTS: CYP1A1 associated ethoxyresorufin-O-deethylase activity of control and EH-treated animals were found as 6.54 ± 1.21 and 4.48 ± 1.67 nmol/min/mg, respectively. CYP2E1 associated aniline 4-hydroxylase of control and EH group were 0.537 ± 0.011 and 0.109 ± 0.01 nmol/min/mg, respectively. However, EH treatment increased the GPx and NQO1 activities from 0.069 ± 0.015 to 0.107 ± 0.026 nmol/min/mg and from 163.34 ± 92 to 588.3 ± 14 nmol/min/mg, respectively. Furthermore, protein and mRNA expression analysis revealed that CYP1A1 and CYP2E1 levels were decreased while those of NQO1 and GPx increased after EH treatment. DISCUSSION AND CONCLUSION: Our current data suggest that the metabolism of xenobiotics, including drugs, may be altered due to changes in the expression and activity of these proteins by EH.

Carnosic acid induces the NAD(P)H: quinone oxidoreductase 1 expression in rat clone 9 cells through the p38/nuclear factor erythroid-2 related factor 2 pathway.

The anticarcinogenic effect of rosemary has been partly attributed to the modulation of the activity and expression of phase II detoxification enzymes. Here we compared the effects of phenolic diterpenes from rosemary on the expression of NAD(P)H: quinone oxidoreductase 1 (NQO1) in rat Clone 9 liver cells. Cells were treated with 1-20 µmol/L of carnosic acid (CA) or carnosol (CS) for 24 h. Both CA and CS dose dependently increased NQO1 enzyme activity and protein expression, and the induction potency of CA was stronger than that of CS. The increase in NQO1 enzyme activity in cells treated with 10 µmol/L CA and CS was 4.1- and 1.9-fold, respectively (P < 0.05). RT-PCR showed that CA and CS induced NQO1 mRNA in a dose-dependent manner. Furthermore, CA dose dependently induced transcription of nuclear factor erythroid-2 related factor 2 (Nrf2) and antioxidant response element (ARE)-luciferase reporter activity. Silencing of Nrf2 expression alleviated NQO1 protein expression and ARE-luciferase activity by CA. Moreover, the phosphorylation of p38 was mainly stimulated in the presence of CA. Pretreatment with SB203580 or silencing of p38 expression inhibited Nrf2 activation and NQO1 induction. These results suggest that the increased NQO1 expression by CA is likely related to the p38-Nrf2 pathway and help to clarify the possible molecular mechanism of action of rosemary phenolic compounds in drug metabolism and cancer prevention.

Cancer chemopreventive activity and metabolism of isoliquiritigenin, a compound found in licorice.

Isoliquiritigenin (2',4',4-trihydroxychalcone; ILG), a chalcone found in licorice root and many other plants, has shown potential chemopreventive activity through induction of phase II enzymes such as quinone reductase-1 in murine hepatoma cells. In this study, the in vivo metabolism of ILG was investigated in rats. In addition, ILG glucuronides and ILG-glutathione adducts were observed in human hepatocytes and in livers from rats treated with ILG. ILG glucuronides were detected in both plasma and rat liver tissues. In addition, in a full-term cancer chemoprevention study conducted with 7,12-dimethylbenz(a)anthracene-treated female Sprague-Dawley rats, dietary administration of ILG slightly increased tumor latency but had a negative effect on the incidence of mammary tumors starting at approximately 65 days after 7,12-dimethylbenz(a)anthracene administration. Further, no significant induction of phase II enzymes was found in mammary glands, which is consistent with the low level of ILG observed in these tissues. However, ILG significantly induced quinone reductase-1 activity in the colon, and glutathione as well as glutathione S-transferase in the liver. Analysis of mRNA expression in tissues of rats treated with ILG supported these findings. These results suggest that ILG should be tested for chemopreventive efficacy in nonmammary models of cancer.

Chemical synthesis and biological evaluation of a NAD(P)H:quinone oxidoreductase-1 targeted tripartite quinone drug delivery system.

NAD(P)H:quinone oxidoreductase-1 (NQO1) is a potential target for therapeutic intervention but attempts to exploit NQO1 using quinone-based bioreductive prodrugs have been largely compromised by toxicity to organs that inherently express high levels of NQO1. In an attempt to circumvent this problem, this study describes the development of a tripartite quinone-based drug delivery system, the ultimate objective of which is to release a targeted therapeutic agent following the reduction of a quinone "trigger" by NQO1. Molecular modeling of drug/NQO1 interactions were conducted prior to the synthesis of N-{4-[bis-(2-chloroethyl)-amino]-phenyl}-beta,beta,2,4,5-pentamethyl-3,6-dioxo-1,4-cyclohexadiene-1-propanamide (prodrug 1). Prodrug 1 is a good substrate for purified NQO1 (V(max) and K(m) values of 11.86 +/- 3.09 micromol/min/mg and 2.70 +/- 1.14 micromol/L, respectively) and liquid chromatography-mass spectrometry analysis of the metabolites generated showed that lactone 3 and aniline mustard 4 were generated in a time- and NQO1-dependent manner. Chemosensitivity studies showed that prodrug 1 is selectively toxic to cells that overexpress NQO1 under aerobic conditions, and comet assay analysis confirmed the presence of elevated interstrand cross-links in NQO1-rich compared with NQO1-deficient cells. Hypoxic sensitization (hypoxic cytotoxicity ratio = 15.8) was observed in T47D cells that overexpress cytochrome P450 reductase. In conclusion, the results of this study provide mechanistic proof of principle that a tripartite benzoquinone drug delivery system is enzymatically reduced to release an active therapeutic agent. Further development of this concept to fine-tune substrate specificity for specific reductases and/or the inclusion of alternative therapeutic agents is warranted.

Betalains, phase II enzyme-inducing components from red beetroot (Beta vulgaris L.) extracts.

Crude aqueous and ethanolic extracts of root tissue of red (Rd) and high-pigment (HP) beet (Beta vulgaris L.) strains exhibited antioxidant and phase II enzyme-inducing activities, and these extracts were fractionated using Sephadex LH-20 chromatography. These bioactivities tended to become co-enriched in early and late eluting fractions, comprising 5-25% of the material recovered from the column. Liquid chromatography-mass spectrometry (MS) was used to resolve and identify multiple betalain components in the most potent quinone reductase (QR)-inducing fractions. Active fractions were found to contain vulgaxanthins I and II, and (iso)betanin, but other components remained unidentified. Two of the isolated active fractions were incorporated into rodent diets at 10-150 ppm over a 2-mo period to assess bioavailability and in vivo efficacy for phase II enzyme induction in various organs. No statistically significant effect of diet was obtained, and wide ranges of tissue enzyme levels among individual animals were observed. This lack of effect and diversity in response to diet may be related to the wide range in absorptive capacity of and/or insufficient level or enrichment of the active agents or to difficulties in assessing such activity in vivo. Subsequent to the animal studies, betanin was isolated in pure form, identified by MS analysis, and confirmed to be QR inducers in the bioassay.

Induction of quinone reductase by sulforaphane and sulforaphane N-acetylcysteine conjugate in murine hepatoma cells.

Broccoli belongs to a group of vegetables termed cruciferous vegetables and characterized by their glucosinolate content. These glucosinolates are secondary metabolites that, upon hydrolysis, release bioactive isothiocyanates (ITCs). Bioactive ITCs are considered to protect the body from cancer by inducing detoxification enzymes such as quinone reductase (QR). This has the potential to make dietary choice a powerful strategy for achieving protection against carcinogenesis, mutagenesis, and other forms of toxicity from xenobiotic electrophiles and reactive forms of oxygen. The bioactive ITC sulforaphane (SF) is the hydrolysis product of glucoraphanin, the predominant aliphatic glucosinolate in broccoli. Because SF appears more potent than many other ITCs in induction of detoxification enzymes, it may have potential as a dietary cancer-preventative agent. One potential concern is that SF is highly reactive and has a very short half-life in the body, forming a glutathione conjugate that is further metabolized to the N-acetyl-L-cysteine conjugate (SF-NAC), the major excretory product found in the urine. However, the conjugate is a reversible complex, able to release free SF. The objective of this study was to compare QR-inducing activity by SF and its major metabolite SF-NAC, in murine hepatoma cells. Both SF and SF-NAC caused dose-related cell growth inhibition and QR induction. SF, 1 and 2 microM, resulted in a 3.0- and 3.5-fold induction of QR, respectively, and the same concentrations of SF-NAC caused a similar, although somewhat greater, induction of QR, 3.8- and 4.5-fold, respectively. These results strengthen the basis for considering that an effective therapeutic form of SF may be the ITC conjugate, formed in situ or given in place of purified ITC as prophylactic treatment to individuals at high risk for cancer.

Olive oil consumption during pregnancy and lactation in rats influences mammary cancer development in female offspring.

This study examined the effects of variety and quantity of dietary fat consumed by rats during pregnancy and lactation on female offspring's response to chemically induced mammary cancer. Groups of six female rats were fed diets containing 7% corn oil (7-CO), 15% CO (15-CO), 7% olive oil (7-OO), or 15% OO (15-OO) for 5 wk prior to, and during, pregnancy and lactation. Female offspring (n = 15 per group) were fed a 7-CO diet, and mammary cancer was induced with 7,12-dimethylbenz[a]anthracene (DMBA). Three months following cancer induction tumor incidence and size were recorded, and markers of apoptosis, serum estrogen concentrations, and hepatic phase II enzymes were measured. Tumor incidence was 47% in offspring born to mothers fed the 7-OO diet, rose to 67% in 7-CO and 15-OO offspring, and reached 86% in 15-CO. A trend toward smaller tumors was observed in the 7-OO group, and offspring of mothers fed high-fat diets had significantly more tumors. Estradiol levels at the end of lactation were significantly lower in mothers fed 7-OO but were similar in all groups of offspring. In tumor tissue, Bcl-2 expression was highest in the 15-CO offspring, and Bak expression was significantly higher in rats exposed to OO. A distinct trend toward increased caspase-3 expression (20 kDa) was observed in the 7-OO offspring, and both low-fat diets significantly elevated caspase activity. In healthy mammary tissue, rats exposed to low-fat diets had significantly higher caspase-3 (32-kDa) levels, and caspase-3 activity was significantly higher in the healthy tissue from both OO groups. Hepatic quinone reductase activity was significantly lower in offspring of mothers fed the low-fat diets. These results indicate that perinatal exposure to OO may have a protective effect against future development of mammary cancer in female offspring, whereas high-fat diets fed to pregnant and lactating rats, in particular CO, may be deleterious.

Dihydroisotanshinone I protects against menadione-induced toxicity in a primary culture of rat hepatocytes.

Dihydroisotanshinone I is a phenanthrenequinone derivative isolated from the roots of Salvia trijuga Diels. The present study demonstrated the hepatoprotective effect of dihydroisotanshinone I against menadione-induced cytotoxicity in a primary culture of rat hepatocytes. Pretreating the cells with dihydroisotanshinone I at concentrations ranging from 2.5 microM to 20 microM for 24 hours caused dose-dependent protection against hepatotoxicity induced by menadione. Intracellular glutathione level and activity of DT-diaphorase have been suggested to play important roles in menadione-induced cytotoxicity. However, treating the hepatocytes with 20 microM dihydroisotanshinone I for 24 hours did not cause a significant change in glutathione level and DT-diaphorase activity. On the contrary, adding dihydroisotanshinone I to freshly isolated hepatocytes at concentrations between 50 nM to 200 nM inhibited NADH-induced superoxide production dose-dependently as indicated by the decrease of lucigenin-amplified chemiluminescence. In addition, dihydroisotanshinone I at concentrations ranging from 5 microM to 20 microM inhibited tert-butyl hydroperoxide-induced lipid peroxidation dose-dependently in isolated hepatocytes as indicated by the level of malondialdehyde. These results suggest that the protective action of dihydroisotanshinone I against menadione-induced hepatotoxicity is attributed to its antioxidant properties including the free radical scavenging activity and inhibition of lipid peroxidation. Abbreviations. DTD:DT-diaphorase GSH:glutathione LDH:lactate dehydrogenase MDA:malondialdehyde MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide TBHP: tert-butyl hydroperoxide

Selective induction of phase II enzymes in the urinary bladder of rats by allyl isothiocyanate, a compound derived from Brassica vegetables.

Allyl isothiocyanate (AITC) is formed from sinigrin, a glucosinolate that is present in many Brassica vegetables. In the present study, the effect of various dose levels of AITC on the activities of the phase II detoxification enzymes quinone reductase (QR) and glutathione S-transferase (GST) in rat tissues has been examined. High dose levels of AITC, given daily for 5 days, increased the activity of QR and/or GST in the liver, kidneys, lungs, spleen, urinary bladder, glandular and nonglandular stomach, duodenum, jejunum, ileum, cecum, and colon plus rectum of the animals. At low doses, however, increases in enzyme activity were observed only in the urinary bladder of the rats, with a significant effect being recorded in this tissue at a dose of AITC of only 10 micromol/kg/day. The effect of duration of exposure to AITC on enzyme levels in the urinary bladder was also investigated, with rats receiving the isothiocyanate each day for 1-21 days. In the case of QR, a plateau of activity was reached after 15 daily doses of AITC, but GST activity continued to increase with continued exposure, and no plateau was reached after 21 doses. The dose level of AITC found to be effective in rats approaches the level that could be achieved through human consumption of Brassica vegetables, suggesting that induction of phase II enzymes by food-derived isothiocyanates could contribute to the lower incidence of bladder cancer observed in individuals who regularly consume such vegetables.

Inhibition of azoxymethane-induced aberrant crypt foci formation in rat colorectum by whole leaf Aloe arborescens Miller var. natalensis Berger.

We examined the modifying effect of whole-leaf Aloe arborescens Miller var. natalensis Berger (designated as 'ALOE') on azoxymethane (AOM)-induced aberrant crypt foci (ACF), putative preneoplastic lesions, in the rat colorectum. Male F344 rats (4 weeks old) were fed the basal diet, or experimental diets containing 1% or 5% ALOE for 5 weeks. One week later, all rats except those in the vehicle-treated groups were injected s.c. with AOM (15 mg/kg, once weekly for 3 weeks). At 9 weeks of age, all the rats were killed, and the colorectum and liver were evaluated for ACF and cytosolic quinone reductase (QR; a phase 2 enzyme), respectively. In rats given AOM and ALOE (1% or 5% in diet) the numbers of ACF/colorectum, aberrant crypts/colorectum, aberrant crypts/focus and large ACF/colorectum were significantly decreased compared with those of rats given AOM alone (all p < 0.01). No ACF were found in rats treated without AOM. In addition, ALOE significantly increased cytosolic QR activity in the liver (p < 0.01). These results indicated that ALOE inhibited the development of AOM-induced ACF in the rat colorectum, with increased QR activity in the liver, and therefore suggested that ALOE might have a chemopreventive effect against colon carcinogenesis at least in the initiation stage.

Potent induction of phase 2 enzymes in human prostate cells by sulforaphane.

Two population-based, case-control studies have documented reduced risk of prostate cancer in men who consume cruciferous vegetables. Cruciferae contain high levels of the isothiocyanate sulforaphane. Sulforaphane is known to bolster the defenses of cells against carcinogens through up-regulation of enzymes of carcinogen defense (phase 2 enzymes). Prostate cancer is characterized by an early and near universal loss of expression of the phase 2 enzyme glutathione S-transferase (GST)-pi. We tested whether sulforaphane may act in prostatic cells by increasing phase 2 enzyme expression. The human prostate cancer cell lines LNCaP, MDA PCa 2a, MDA PCa 2b, PC-3, and TSU-Pr1 were treated with 0.1-15 microM sulforaphane in vitro. LNCaP was also treated with an aqueous extract of broccoli sprouts. Quinone reductase enzymatic activity, a surrogate of global phase 2 enzyme activity, was assayed by the menadione-coupled reduction of tetrazolium dye. Expression of NQO-1, GST-alpha, gamma-glutamylcysteine synthetase-heavy and -light chains, and microsomal GST was assessed by Northern blot analysis. Sulforaphane and broccoli sprout extract potently induce quinone reductase activity in cultured prostate cells, and this induction appears to be mediated by increased transcription of the NQO-1 gene. Sulforaphane also induces expression of gamma-glutamylcysteine synthetase light subunit but not the heavy subunit, and this induction is associated with moderate increases in intracellular glutathione levels. Microsomal and alpha-class glutathione transferases were also induced transcriptionally. Sulforaphane induces phase 2 enzyme expression and activity significantly in human prostatic cells. This induction is accompanied by, but not because of, increased intracellular glutathione synthesis. Our findings may help explain the observed inverse correlation between consumption of cruciferae and prostate cancer risk.

Low doses of diallyl disulfide, a compound derived from garlic, increase tissue activities of quinone reductase and glutathione transferase in the gastrointestinal tract of the rat.

Diallyl disulfide (DADS), a substance that is formed from the organosulfur compounds present in garlic, is known to increase tissue activities of the phase II detoxification enzymes quinone reductase (QR) and glutathione transferase (GT) in animals. In previous experiments, however, high doses of DADS were employed and only a limited range of tissues were examined. In the present studies, increased activities of QR and GT were recorded in the forestomach, glandular stomach, duodenum, jejunum, ileum, cecum, colon, liver, kidneys, spleen, heart, lungs, and urinary bladder of rats given DADS over a wide range of dose levels. Large variations in response were recorded among the different organs, with forestomach, duodenum, and jejunum being the most sensitive to enzyme induction by DADS. In these organs, significant increases in QR activity were observed at a dose of only 0.3 mg/kg/day. Such a dose level is close to that which may be achieved through human consumption of garlic, suggesting that induction of phase II enzymes may contribute to the protection that is afforded by this vegetable against cancer of the gastrointestinal tract in humans.