Induction Of Detoxifying Enzymes Research Articles

The role of detoxification enzymes in the susceptibility of Brevipalpus californicus exposed to acaricide and insecticide mixtures

The intense spraying of pesticides to control different arthropod pests has resulted in negative side effects for the management of pests. It was previously discovered that exposure to non-acaricidal insecticides alone or in a mixture, results in lower efficiency of the acaricide spirodiclofen used for Brevipalpus spp. control. We investigate here whether the induced expression of detoxification enzymes by non-lethal insecticides may antagonize spirodiclofen toxicity. Brevipalpus californicus mites exposed to the insecticide phosmet alone or in combination with spirodiclofen showed increased activity of P450 monooxygenases (P450s). No antagonistic effects in mite mortality were observed by the combination of phosmet and spirodiclofen. On the other hand, mites exposed to the insecticide imidacloprid alone or in combination with spirodiclofen showed an increase in the activity of P450s, carboxylcholinesterases (CCE), and glutathione-S-transferases (GST). An antagonistic effect on mite mortality was observed when mites were exposed to the LC25 of spirodiclofen combined with the field rate treatment of imidacloprid. The addition of PBO (a P450 monooxygenase inhibitor) to the mixture of spirodiclofen and imidacloprid resulted in a synergistic effect over mite mortality but the addition of DEM (a GST inhibitor) resulted in an antagonist effect. Taken together, this study showed that the combination of imidacloprid with spirodiclofen is antagonistic for the control of B. californicus, and this results from the induction of detoxification enzymes, such as P450s, CCE, and GST. The use of inhibitors highlights the role of these enzymes in the antagonism of the mixture.

Anticancer property of allyl sulfides derived from garlic (Allium sativum L.)

Several lines of evidences imply that garlic (Allium sativum L.) is one of the potent vegetable for the cancer prevention. Fresh garlic extract, garlic powder, garlic oil, and organosulfur compounds derived from garlic have been reported to exhibit the anticancer effect through their anticarcinogenic, antimutagenic, and antitumor properties. Evidences from experimental studies, those which have been performed primarily at molecular basis with garlic are introduced in this review. Especially, we focused on allyl sulfides as promising compounds derived from garlic for cancer prevention, and discussed the anticancer properties of allyl sulfides in terms of anticarcinogenic, antimutagenic and antigenotoxic activity, induction of detoxification enzymes, cell cycle inhibition as well as induction of apoptosis.

Formation and Biological Targets of Quinones: Cytotoxic versus Cytoprotective Effects.

Quinones represent a class of toxicological intermediates, which can create a variety of hazardous effects in vivo including, acute cytotoxicity, immunotoxicity, and carcinogenesis. In contrast, quinones can induce cytoprotection through the induction of detoxification enzymes, anti-inflammatory activities, and modification of redox status. The mechanisms by which quinones cause these effects can be quite complex. The various biological targets of quinones depend on their rate and site of formation and their reactivity. Quinones are formed through a variety of mechanisms from simple oxidation of catechols/hydroquinones catalyzed by a variety of oxidative enzymes and metal ions to more complex mechanisms involving initial P450-catalyzed hydroxylation reactions followed by two-electron oxidation. Quinones are Michael acceptors, and modification of cellular processes could occur through alkylation of crucial cellular proteins and/or DNA. Alternatively, quinones are highly redox active molecules which can redox cycle with their semiquinone radical anions leading to the formation of reactive oxygen species (ROS) including superoxide, hydrogen peroxide, and ultimately the hydroxyl radical. Production of ROS can alter redox balance within cells through the formation of oxidized cellular macromolecules including lipids, proteins, and DNA. This perspective explores the varied biological targets of quinones including GSH, NADPH, protein sulfhydryls [heat shock proteins, P450s, cyclooxygenase-2 (COX-2), glutathione S-transferase (GST), NAD(P)H:quinone oxidoreductase 1, (NQO1), kelch-like ECH-associated protein 1 (Keap1), IκB kinase (IKK), and arylhydrocarbon receptor (AhR)], and DNA. The evidence strongly suggests that the numerous mechanisms of quinone modulations (i.e., alkylation versus oxidative stress) can be correlated with the known pathology/cytoprotection of the parent compound(s) that is best described by an inverse U-shaped dose–response curve.

Host suitability and diet mixing influence activities of detoxification enzymes in adult Japanese beetles

Induction of cytochrome P450, glutathione S transferase (GST), and carboxylesterase (CoE) activity was measured in guts of the scarab Popillia japonica Newman, after consumption of single or mixed plant diets of previously ranked preferred (rose, Virginia creeper, crape myrtle and sassafras) or non-preferred hosts (boxelder, riverbirch and red oak). The goal of this study was to quantify activities of P450, GST and CoE enzymes in the midgut of adult P. japonica using multiple substrates in response to host plant suitability (preferred host vs non-preferred hosts), and single and mixed diets. Non-preferred hosts were only sparingly fed upon, and as a group induced higher activities of P450, GST and CoE than did preferred hosts. However, enzyme activities for some individual plant species were similar across categories of host suitability. Similarly, beetles tended to have greater enzyme activities after feeding on a mixture of plants compared to a single plant type, but mixing per se does not seem as important as the species represented in the mix. Induction of detoxification enzymes on non-preferred hosts, or when switching between hosts, may explain, in part, the perceived feeding preferences of this polyphagous insect. The potential consequences of induced enzyme activities on the ecology of adult Japanese beetles are discussed.

Broccoli sprout extract induces detoxification-related gene expression and attenuates acute liver injury.

To investigate the effects of broccoli sprout extract (BSEx) on liver gene expression and acute liver injury in the rat. First, the effects of BSEx on liver gene expression were examined. Male rats were divided into two groups. The Control group was fed the AIN-76 diet, and the BSEx group was fed the AIN-76 diet containing BSEx. After a 10-d feeding period, rats were sacrificed and their livers were used for DNA microarray and real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses. Next, the effects of BSEx on acute liver injury were examined. In experiments using acute liver injury models, 1000 mg/kg acetaminophen (APAP) or 350 mg/kg D-galactosamine (D-GalN) was used to induce injury. These male rats were divided into four groups: Control, BSEx, Inducer (APAP or D-GalN), and Inducer+BSEx. The feeding regimens were identical for the two analyses. Twenty-four hours following APAP administration via p.o. or D-GalN administration via i.p., rats were sacrificed to determine serum aspartate transaminase (AST) and alanine transaminase (ALT) levels, hepatic glutathione (GSH) and thiobarbituric acid-reactive substances accumulation and glutathione-S-transferase (GST) activity. Microarray and real-time RT-PCR analyses revealed that BSEx upregulated the expression of genes related to detoxification and glutathione synthesis in normal rat liver. The levels of AST (70.91 ± 15.74 IU/mL vs 5614.41 ± 1997.83 IU/mL, P < 0.05) and ALT (11.78 ± 2.08 IU/mL vs 1297.71 ± 447.33 IU/mL, P < 0.05) were significantly suppressed in the APAP + BSEx group compared with the APAP group. The level of GSH (2.61 ± 0.75 nmol/g tissue vs 1.66 ± 0.59 nmol/g tissue, P < 0.05) and liver GST activity (93.19 ± 16.55 U/g tissue vs 51.90 ± 16.85 U/g tissue, P < 0.05) were significantly increased in the APAP + BSEx group compared with the APAP group. AST (4820.05 ± 3094.93 IU/mL vs 12465.63 ± 3223.97 IU/mL, P < 0.05) and ALT (1808.95 ± 1014.04 IU/mL vs 3936.46 ± 777.52 IU/mL, P < 0.05) levels were significantly suppressed in the D-GalN + BSEx group compared with the D-GalN group, but the levels of AST and ALT in the D-GalN + BSEx group were higher than those in the APAP + BSEx group. The level of GST activity was significantly increased in the D-GalN + BSEx group compared with the D-GalN group (98.04 ± 15.75 U/g tissue vs 53.15 ± 8.14 U/g tissue, P < 0.05). We demonstrated that BSEx protected the liver from various types of xenobiotic substances through induction of detoxification enzymes and glutathione synthesis.

Effects of Benzyl Isothiocyanate and Its N-Acetylcysteine Conjugate on Induction of Detoxification Enzymes in Hepa1c1c7 Mouse Hepatoma Cells

The induction of detoxification enzymes by benzyl isothiocyanate (BITC) and its synthetic N-acetyl-L-cysteine (NAC) conjugate (NAC-BITC) was examined in Hepa1c1c7 murine hepatoma cells. BITC and NAC-BITC inhibited Hepa1c1c7 cell growth in a dose-dependent manner. Cell growth was 4.5~57.2% lower in Hepa1c1c7 cells treated with 0.1~10 μM BITC than in control-treated Hepa1c1c7 cells. The NAC-BITC treatment had a similar inhibitory pattern on Hepa1c1c7 cell growth; 0.5 μM and 10 μM NAC-BITC decreased cell growth by 13.6% and 47.4%, respectively. Treatment of Hepa1c1c7 cells with 0.1~2.0 μM BITC also elicited a dose-response effect on the induction of quinone reductase quinone reductase (QR) activity and QR mRNA expression. Treatment with 1 μM and 2 μM BITC caused 1.8- and 2.8-fold inductions of QR mRNA, respectively. By comparison, treatment with 1 μM and 2 μM NAC-BITC caused 1.6- and 1.9-fold inductions of QR mRNA, respectively. Cytochrome P450 (CYP) 1A1 and CYP2E1 induction were lower in 0.1~2 μM BITC-treated cells than in control-treated cells. CYP2E1 activity was 1.2-fold greater in 0.1 μM NAC-BITC-treated cells than in control-treated cells. However, the CYP2E1 activity of cells treated with higher concentrations (i.e., 1~2 μM) of NAC-BITC was similar to the activity of control-treated cells. Considering the potential of isothiocyanatesto prevent cancer, these results provide support for the use of BITC and NAC-BITC conjugates as chemopreventive agents.

Bifunctional Chemopreventive Effects ofAdenocaulon himalaicumThrough Induction of Detoxification Enzymes and Apoptosis

Phase II detoxification enzymes are known to play essential roles in the detoxification and elimination of activated carcinogens during tumor initiation, while apoptosis is one of the most important chemopreventive targets for inhibiting tumor promotion in cancer. In this study, we investigated the cancer chemopreventive activity of two plant extracts, the ethanolic extract of Adenocaulon himalaicum (AHE) and the butanolic fraction of AHE (AHB). Both, the AHE and AHB induced NQO1 activity and had relatively high chemoprevention indices (CI=12.4). The AHE and AHB were associated with increased NQO1 and HO-1 mRNA levels via Nrf2-ARE pathway activation. In addition, the AHB increased CYP1A1 activity through AhR-XRE pathway activation. We also found that the AHE and AHB induced apoptosis, as evidenced by phosphatidylserine externalization, an increase in the sub-G0/G1 content, chromatin condensation, poly(ADP-ribose) polymerase cleavage, and p53 induction. These data suggest that AHE and AHB act as bifunctional inducers and that their chemopreventive effects result from the biphasic induction of phase II detoxification enzymes and apoptosis. Therefore, these results suggest that A. himalaicum plant extracts have potential for use as chemopreventive agents for the prevention and/or treatment of human cancers.

Acaricide, Fungicide and Drug Interactions in Honey Bees (Apis mellifera)

BackgroundChemical analysis shows that honey bees (Apis mellifera) and hive products contain many pesticides derived from various sources. The most abundant pesticides are acaricides applied by beekeepers to control Varroa destructor. Beekeepers also apply antimicrobial drugs to control bacterial and microsporidial diseases. Fungicides may enter the hive when applied to nearby flowering crops. Acaricides, antimicrobial drugs and fungicides are not highly toxic to bees alone, but in combination there is potential for heightened toxicity due to interactive effects.Methodology/Principal FindingsLaboratory bioassays based on mortality rates in adult worker bees demonstrated interactive effects among acaricides, as well as between acaricides and antimicrobial drugs and between acaricides and fungicides. Toxicity of the acaricide tau-fluvalinate increased in combination with other acaricides and most other compounds tested (15 of 17) while amitraz toxicity was mostly unchanged (1 of 15). The sterol biosynthesis inhibiting (SBI) fungicide prochloraz elevated the toxicity of the acaricides tau-fluvalinate, coumaphos and fenpyroximate, likely through inhibition of detoxicative cytochrome P450 monooxygenase activity. Four other SBI fungicides increased the toxicity of tau-fluvalinate in a dose-dependent manner, although possible evidence of P450 induction was observed at the lowest fungicide doses. Non-transitive interactions between some acaricides were observed. Sublethal amitraz pre-treatment increased the toxicity of the three P450-detoxified acaricides, but amitraz toxicity was not changed by sublethal treatment with the same three acaricides. A two-fold change in the toxicity of tau-fluvalinate was observed between years, suggesting a possible change in the genetic composition of the bees tested.Conclusions/SignificanceInteractions with acaricides in honey bees are similar to drug interactions in other animals in that P450-mediated detoxication appears to play an important role. Evidence of non-transivity, year-to-year variation and induction of detoxication enzymes indicates that pesticide interactions in bees may be as complex as drug interactions in mammals.

The Influence of Curcumin, Quercetin, and Eicosapentaenoic Acid on the Expression of Phase II Detoxification Enzymes in the Intestinal Cell Lines HT-29, Caco-2, HuTu 80, and LT97

Curcumin, quercetin, and eicosapentaenoic acid (EPA) are 3 natural compounds with the capacity to reduce adenoma burden in patients with familial adenomatous polyposis (FAP). The mechanistic basis of this anticarcinogenic capacity is largely unknown, but it was suggested that induction of detoxification enzymes is involved. Therefore, the effects of low-dose curcumin, quercetin, and EPA on phase II detoxification enzymes UDP-glucuronosyltransferase (UGT), glutathione S-transferase (GST), as well as on glutathione (GSH) content were analyzed in 4 cell line models of intestinal carcinogenesis. HT-29, HuTu 80, and Caco-2 intestinal cancer cells and LT97 colon adenoma cells from a patient with FAP were treated with low-dose noncytotoxic concentrations of curcumin, quercetin, and EPA. GST enzyme activity was measured by spectrophotometry, and expression of GSTA1, GSTM1, GSTP1, GSTT1, and UGT1 by Western blotting. Cytosolic GSH levels were determined by high performance liquid chromatography. An inducing effect of curcumin and quercetin on GST or UGT was seen in Caco-2, LT97, and HuTu 80 cells. GSH levels were reduced by quercetin and EPA in HT-29 cells and induced by curcumin in Caco-2 cells. In LT97 cells, GST activity and expression was reduced, but UGT1 expression was induced by curcumin and quercetin; whereas EPA only decreased GST or UGT levels. In summary, enhancement of the detoxification capacity by low dose of the potential anticarcinogens curcumin, quercetin, or EPA seems only a minor factor in explaining their anticarcinogenic properties.

Induction of Detoxification Enzymes by Quercetin in the Silkworm

Quercetin is one of the most abundant flavonoids and the defense secondary metabolites in plants. In this study, the effect of quercetin on the growth of the silkworm larvae was investigated. Cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), and carboxylesterases (COE) were assayed after exposure to different concentrations of quercetin for 3 d (short-term) and 7 d (long-term), respectively. The results showed that the weight gain of the silkworm larvae significantly decreased after the larvae were treated by different concentrations of quercetin except for the treatment with 0.5% quercetin. Activities of P450, GST, and COE were induced by 0.5 or 1% concentration of quercetin. In the midgut, the induction activity of P450s was reached to the highest level (2.3-fold) by 1% quercetin for 7 d, the highest induction activities of GSTs toward CHP and CDNB were 4.1-fold and 2.6-fold of controls by 1% quercetin after 7 d exposure, respectively. For COEs, the highest activity (2.3-fold) was induced by 0.5% quercetin for 7 d. However, P450s in whole body were higher inducible activities in short-term treatment than those in long-term treatment. The responses of eight cytochrome P450 (CYP) genes belonged to CYP6 and CYP9 families and seven GST genes were detected with real-time polymerase chain reaction. In addition, the genes induced by quercetin significantly were confirmed by qRT-PCR. CYP6AB5, CYP6B29, and GSTe8 were identified as inducible genes, of which the highest induction levels were 10.9-fold (0.5% quercetin for 7 d), 6.2-fold (1% quercetin for 7 d), and 7.1-fold (1% quercetin for 7 d), respectively.

Supplementary data for: Induction of Detoxification Enzymes by Quercetin in the Silkworm

Supplementary data for: Induction of Detoxification Enzymes by Quercetin in the Silkworm

Induction of Detoxification Enzymes by Feeding Unblanched Brussels Sprouts Containing Active Myrosinase to Mice for 2 Wk

In cruciferous vegetables, myrosinase metabolizes the relatively inactive glucosinolates into isothiocyanates and other products that have the ability to increase detoxification enzyme expression. Thus, maintaining myrosinase activity during food preparation may be critical to receiving the maximum benefit of consumption of Brussels sprouts or other cruciferous vegetables. To test the importance of maintaining myrosinase activity for maximizing bioactivity, experimental diets containing 20% unblanched (active myrosinase) or 20% blanched (inactivated myrosinase) freeze-dried Brussels sprouts and a nutrient-matched control diet were evaluated in vitro and in vivo for their ability to induce detoxification enzymes. Treatment of immortalized HepG2 human hepatoma cells with the unblanched Brussels sprout diet caused a greater increase quinone activity compared to the blanched Brussels sprout diet. C3H/HeJ mice fed the unblanched Brussels sprout diets for 2 wk had significantly higher plasma sulforaphane concentrations. Liver expression of CYP1A1 and epoxide hydrolase, measured using real-time PCR, was correlated with the plasma concentration of sulforaphane. In the lung, expression of epoxide hydrolase, thioredoxin reductase, UDP glucuronosyltransferase, quinone reductase, heme oxygenase, CYP1A1, CYP1A2, and CYP1B1 were also correlated with the plasma concentration of sulforaphane. Together these data demonstrate that, as predicted by the in vitro experiment, in vivo exposure to Brussels sprouts with active myrosinase resulted in greater induction of both phase I and phase II detoxification enzymes in the liver and the lungs that correlated with plasma sulforaphane concentrations.

Response of the CutwormSpodoptera liturato Sesame Leaves or Crude Extracts in Diet

The effects of extracts of sesame, Sesamum indicum L. (Liamiales: Pedaliaceae), and whole leaves of some selected cultivars of sesame were tested using a natural host Spodoptera litura (F.) (Lepidoptera: Noctuidae). Indices taken using the immature stages include; diet utilization, growth and development and induction of detoxification enzymes. The results indicate that S. litura generally selects its food amongst cultivars within 6 hours after food presentation. Growth and development of the insect is controlled also by plant acceptability and quality. Although all the cultivars tested significantly limit insect growth and development the variety 56S-radiatum did not allow a complete life cycle as pupation from first instar stage was 0%. Generally the crucial period for immature S. litura was the larval period, especially the first two instars where the weight of an insect fed on an experimental diet was three times lower than that of a control diet. The larval developmental period was greater than 40 days as compared to 17 days for insects fed a control diet. S. litura also had lowered efficiency in utilizing ingested food, from a low of 13% in a sesame cultivar to 45% in the control diet. The key detoxification enzyme was a glutathione s-transferase that was confirmed by a 6-fold increase between S. litura fed a plant cultivar vs. a control diet towards the substrate 1,2-dichloro-4-nitrobenzene. First and second instars of S. litura have a relatively reduced detoxification of enzymes in response to plant cultivar diets leading to low survival. A 3% v/w crude extract of the cultivars increased enzyme induction towards all the tested substrates.

Characterization of chemopreventive agents from the dichloromethane extract of Eurycorymbus cavaleriei by liquid chromatography–ion trap mass spectrometry

In the present study, we examined the potential chemopreventive activity of dichloromethane extract of Eurycorymbus cavaleriei by investigating the change of constitutions after incubation with glutathione (GSH). The major constitutions in the dichloromethane extract of E. cavaleriei were cumarin compounds and their cleavage pattern was examined by LC–MS-MS and the characteristic product ions at m/ z 206 and 207 were helpful to determine the substitutions of coumarinolignoid compounds. The mechanism of conjugations of 5′-demethylaquillochin and its isomer with GSH was discussed and validated through analysis of the conjugations of reference compound 6-hydroxy-7-methoxycoumarin with GSH by LC–MS-MS and NMR spectrum. The relative ability to induce the detoxification enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1) of nine coumarin compounds was tested which also showed 5′-demethylaquillochin exhibited the most potential chemopreventive ability. These observations suggest that 5′-demethylaquillochin and its isomer from the dichloromethane extract of E. cavaleriei have potential as chemopreventive agents through induction of detoxification enzymes.

Raphasatin Degradation Product Induces Detoxification Enzyme Activity

Epidemiologic studies suggest that cruciferous vegetable consumption lowers cancer risk, likely through the induction of detoxification enzymes. Glucosinolates are defensive secondary metabolites found in cruciferous vegetables which yield several myrosinase metabolites upon tissue disruption including isothiocyanates (ITC). ITCs have been shown to induce phase I and phase II detoxification enzymes both in vivo and in vitro. Spanish black radish (SBR) (Raphanus sativus L. var. niger) is a member of the Cruciferae family that has a high concentration of a glucosinolate unique to the radish family, glucoraphasatin (GRH). The predominant myrosinase‐metabolite of GRH hydrolysis is 4‐methylthio‐3‐butenyl isothiocyanate (raphasatin). Raphasatin was undetectable in 24‐hour hydrolysis of whole radish preparations. Raphasatin levels peaked 30 minutes after the initiation of myrosinase‐catalyzed hydrolysis of purified GRH, but then rapidly decreased and were undetectable after 24 hours using GC‐MS. This 24‐hour myrosinase hydrolysis of GRH induced quinone reductase activity dose‐dependently in the human hepatoma HepG2 cell line. These data suggest that a raphasatin degradation product is able to induce phase II detoxification enzymes in a manner similar to raphasatin.

Abstract A43: Hops (humulus lupulus) induce detoxification enzymes in vivo

Abstract A43 Extracts of hop strobiles, the female parts of Humulus lupulus L., are popular botanical dietary supplements as alternative treatment for menopausal symptoms (1). Cancer preventive properties of hops, which are in part mediated through the induction of detoxification enzymes, have frequently been reported (2). This effect has been mainly attributed to one of its major constituents, xanthohumol (XN) (3). However, the chemopreventive effects have so far only been demonstrated in vitro and have not been confirmed in vivo. Therefore, we conducted an in vivo study in adult female rats and analyzed the effect of oral administration of a standardized hop extract (7.5 g extract/kg BW per day, containing 2% XN) or s.c. injection of XN (20 mg/day) on detoxification enzymes, such as NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione-S-transferase (GST). Analysis of various tissues revealed that while pure XN and oral hops significantly induced NQO1 activity in liver and colon tissue, only oral hops and not pure xanthohumol induced NQO1 activity in the uterus. Interestingly, hops but not xanthohumol significantly induced GST activity in the liver and mammary gland suggesting that other hop constituents and/or an additional mechanism might be responsible for these effects. Transcription of many detoxification enzymes is regulated through the antioxidant response element (ARE) and its transcription factor Nrf2, which is repressed under basal conditions by Keap1 (4). It has been shown that XN alkylates Keap1 and therefore enhances the concentration of free Nrf2 in the nucleus which results in ARE activation (5). Another pathway leading to ARE activation is mediated by protein kinase C (PKC) that phosphorylates Nrf2 leading to accumulation of Nrf2 in the nucleus (6). To investigate whether this pathway is involved in the observations described above, additional experiments with HepG2-ARE-luciferase C8 cells were performed. The cells were treated with hop extract or XN with or without the selective PKC inhbitor, Ro-32-0432, and the ARE-luciferase induction was determined. The results revealed that hops- but not XN-regulated ARE induction is in part mediated by PKC. Possible active compounds are under investigation. These results suggest that hop dietary supplements have the potential as preventive agents against cancer through induction of detoxifying enzymes. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A43.

Angelica sinensis and Its Alkylphthalides Induce the Detoxification Enzyme NAD(P)H: Quinone Oxidoreductase 1 by Alkylating Keap1

The roots of Angelica sinensis (Oliv.) Diels (Dang Gui; Apiaceae) have a long history in traditional Chinese medicine as a remedy for women's disorders and are often called "lady's ginseng". Currently, extracts of A. sinensis are commonly included in numerous dietary supplements used for women's health and as antiaging products. In the present study, we examined the potential chemopreventive activity of A. sinensis extracts by measuring the relative ability to induce the detoxification enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1). The lipophilic partitions showed strong NQO1 induction with concentrations to double the enzyme activity (CD) of 5.5 +/- 0.7 (petroleum ether) and 3.9 +/- 0.5 microg/mL (chloroform). Fractionation led to the isolation of phenolic esters and alkylphthalides, especially Z-ligustilide, the main lipophilic compound, which showed strong NQO1 inducing properties (CD = 6.9 +/- 1.9 microM). Transcription of many detoxifying enzymes is regulated through the antioxidant response element (ARE) and its transcription factor Nrf2, which is repressed under basal conditions by Keap1. However, exposure to electrophilic inducers that alkylate Keap1 results in higher concentrations of free Nrf2 and ARE activation. The ARE reporter activity was therefore analyzed in HepG2-ARE-C8 cells after incubation with lipophilic extracts of A. sinensis or ligustilide for 24 h. Under these conditions, both the extract and the ligustilide increased ARE-luciferase reporter activity in a dose-dependent manner. Incubation of ligustilide with GSH and subsequent LC-MS-MS analysis revealed that ligustilide as well as oxidized ligustilide species covalently modified GSH. In addition, using MALDI-TOF mass spectrometry and LC-MS-MS, it was demonstrated that the lipophilic extracts, ligustilide, and monooxygenated ligustilide alkylated important cysteine residues in human Keap1 protein, thus activating Nrf2 and transcription of ARE regulated genes. These observations suggest that A. sinensis dietary supplements standardized to ligustilide have potential as chemopreventive agents through induction of detoxification enzymes.

Expression profile of human cells in culture exposed to glycidamide, a reactive metabolite of the heat-induced food carcinogen acrylamide

Recent findings of acrylamide in many common foods have sparked renewed interest in assessing human health hazards and the long-term risk associated with exposure to vinyl compounds. Acrylamide is tumorigenic at high doses in rodents and has been classified as a probable human carcinogen. However, cancer risk projections in the population remain problematic because the molecular pathogenesis of acrylamide at the low level of dietary uptake is not understood. In particular, the question of whether specific transcriptional responses may amplify or mitigate the known genotoxicity of acrylamide has never been examined. Here, we used high-density DNA microarrays and PCR validations to assess genome-wide messenger profiles induced by glycidamide, the more reactive metabolite of acrylamide. The expression changes resulting from glycidamide treatment of human epithelial cells are characterized by the induction of detoxification enzymes, several members of the glutathione system and antioxidant factors. Low-dose experiments indicate that the up-regulation of epoxide hydrolase 1 represents the most sensitive transcriptional biomarker of glycidamide exposure. At higher concentrations, glycidamide induces typical markers of tumor progression such as steroid hormone activators, positive regulators of nuclear factor-κB, growth stimulators and apoptosis inhibitors. Concomitantly, growth suppressors and cell adhesion molecules are down-regulated. The main implication of these findings for risk assessment is that low concentrations of glycidamide elicit cytoprotective reactions whereas transcriptional signatures associated with tumor progression may be expected only at doses that exceed the range of ordinary dietary exposures.

Modulation of Human Glutathione S-Transferases by Polyphenon E Intervention

Green tea consumption has been associated with decreased risk of certain types of cancers in humans. Induction of detoxification enzymes has been suggested as one of the biochemical mechanisms responsible for the cancer-preventive effect of green tea. We conducted this clinical study to determine the effect of repeated green tea polyphenol administration on a major group of detoxification enzymes, glutathione S-transferases (GST). A total of 42 healthy volunteers underwent a 4-week washout period by refraining from tea or tea-related products. At the end of the washout period, a fasting blood sample was collected, and plasma and lymphocytes were isolated for assessment of GST activity and level. Following the baseline evaluation, study participants underwent 4 weeks of green tea polyphenol intervention in the form of a standardized Polyphenon E preparation at a dose that contains 800 mg epigallocatechin gallate (EGCG) once a day. Polyphenon E was taken on an empty stomach to optimize the oral bioavailability of EGCG. Upon completion of the intervention, samples were collected for postintervention GST assessment. Four weeks of Polyphenon E intervention enhanced the GST activity in blood lymphocytes from 30.7 +/- 12.2 to 35.1 +/- 14.3 nmol/min/mg protein, P = 0.058. Analysis based on baseline activity showed that a statistically significant increase (80%, P = 0.004) in GST activity was observed in individuals with baseline activity in the lowest tertile, whereas a statistically significant decrease (20%, P = 0.02) in GST activity was observed in the highest tertile. In addition, Polyphenon E intervention significantly increased the GST-pi level in blood lymphocytes from 2,252.9 +/- 734.2 to 2,634.4 +/- 1,138.3 ng/mg protein, P = 0.035. Analysis based on baseline level showed that this increase was only significant (P = 0.003) in individuals with baseline level in the lowest tertile, with a mean increase of 80%. Repeated Polyphenon E administration had minimal effects on lymphocyte GST-mu and plasma GST-alpha levels. There was a small but statistically significant decrease (8%, P = 0.003) in plasma GST-alpha levels in the highest tertile. We conclude that 4 weeks of Polyphenon E administration resulted in differential effects on GST activity and level based on baseline enzyme activity/level, with GST activity and GST-pi level increased significantly in individuals with low baseline enzyme activity/level. This suggests that green tea polyphenol intervention may enhance the detoxification of carcinogens in individuals with low baseline detoxification capacity.

The environmental estrogen, nonylphenol, activates the constitutive androstane receptor.

Nonylphenol (NP) and its parent compounds, the nonylphenol ethoxylates are some of the most prevalent chemicals found in U.S. waterways. NP is also resistant to biodegradation and is a known environmental estrogen, which makes NP a chemical of concern. Our data show that NP also activates the constitutive androstane receptor (CAR), an orphan nuclear receptor important in the induction of detoxification enzymes, including the P450s. Transactivation assays demonstrate that NP increases murine CAR (mCAR) transcriptional activity, and NP treatment can overcome the inhibitory effects of the inverse agonist, androstanol, on mCAR activation. Treatment of wild-type (CAR +/+) mice with NP at 50 or 75 mg/kg/day increases Cyp2b protein expression in a dose-dependent manner as demonstrated by Western blotting, and was confirmed by quantitative reverse transcription-PCR of Cyp2b10 transcript levels. CAR-null (CAR -/-) mice show no increased expression of Cyp2b following NP treatment, indicating that CAR is required for NP-mediated Cyp2b induction. In addition, NP increases the translocation of CAR into the nucleus, which is the key step in the commencement of CAR's transcriptional activity. NP also induced CYP2B6 in primary human hepatocytes, and increased Cyp2b10 messenger RNA and protein expression in humanized CAR mice, indicating that NP is an activator of human CAR as well. In conclusion, NP is a CAR activator, and this was demonstrated in vitro with transactivation assays and in vivo with transgenic CAR mouse models.