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PubMed2002

The ontogeny of human drug-metabolizing enzymes: phase II conjugation enzymes and regulatory mechanisms.

Changes in phase II drug-metabolizing enzyme expression during development, as well as the balance between phase I and phase II enzymes, can significantly alter the pharmacokinetics for a given drug or toxicant. Although our knowledge is incomplete, many of the phase II enzymes are expressed early in development. There is evidence for glutathione S-transferase A1/A2 (GSTA1/A2), GSTM, and GSTP1 in fetal liver, lung and kidney, although tissue-specific patterns and changes with time are observed. N-Acetyltransferase 1 (NAT1) activity also has been reported throughout gestation in fetal liver, adrenal glands, lung, kidney, and intestine. Only postnatal changes in NAT1 expression were apparent. Nothing is known about human NAT2 developmental expression. Some UDP-glucuronosyltransferase and sulfotransferase isoforms also are detectable in fetal liver and other tissues by the first or second trimester, and substantial changes in isoform expression patterns, as well as overall expression levels, are observed with increasing maturity. Finally, expression of both epoxide hydrolases 1 and 2 (EPHX1 and EPHX2) is observed in fetal liver, and for the former, increased expression with time has been documented. Less is known about ontogenic molecular control mechanisms. Limited data suggest that the hepatocyte nuclear factor and CCAAT/enhancer binding protein families are critical for fetal liver drug-metabolizing enzyme expression whereas D element binding protein and related factors may regulate postnatal hepatic expression. There is a paucity of data regarding mechanisms for the onset of extrahepatic fetal expression or specific mechanisms determining temporal switches, such as those observed within the CYP3A and flavin-containing monooxygenase families.

Acetyltransferases↗
PubMed1993

Elevation of serum phase II enzymes by anticarcinogenic enzyme inducers: markers for a chemoprotected state?

Inducers of Phase II enzymes, already consumed by humans as food additives, medicines or as constituents of vegetables, can prevent experimental carcinogenesis. Since protection is neither carcinogen- nor organ-specific, clinical trials are already underway to establish the efficacy of 'anticarcinogenic enzyme inducers' (i.e. oltipraz). However, efficient and cost-effective assays to establish the dose wherein a putative anticarcinogen can raise Phase II enzyme levels are lacking. We tested the proposal that serum Phase II enzyme activities would be dependent on relative tissue levels by measuring quinone reductase and glutathione S-transferase activities in sera of mice treated with dietary 2(3)-tert-butyl-4-hydroxyanisole (BHA) or dimethyl fumarate. Serum activities were significantly elevated in animals with increased tissue specific activities of these Phase II enzymes. Increasing concentrations of BHA in the diet from 0.05-0.5% increased hepatic specific activities of both QR and GST from two to six-fold, and increases in serum activities were well correlated to increases observed in the liver (r2 > or = 0.95). There was no evidence for an elevation of serum alanine aminotransferase levels. Thus, in the absence of serological evidence for hepatocellular damage, increased serum Phase II enzyme activities can be correlated to tissue levels. Our results suggest that similar assays tailored to human sera will not only be useful in the execution of chemoprevention trials, but also to assess the role that Phase II enzyme induction plays in the prevention of cancer by fruits and vegetables.

Adult↗
PubMed1992

Effects of cooked brussels sprouts on cytochrome P-450 profile and phase II enzymes in liver and small intestinal mucosa of the rat.

Male Wistar rats were given semi-synthetic diets supplemented with 0, 2.5, 5 and 20% cooked Brussels sprouts for 2, 7, 14 or 28 days. The effects on several cytochrome P-450 enzymes and phase II enzymes (glutathione S-transferase (GST), glucuronyl transferases 1 and 2 (GT1 and GT2) and DT-diaphorase (DTD)) in the liver and small intestinal mucosa were investigated. From 2 days of exposure onwards Brussels sprouts induced P4501A2 and--to a lesser extent--P4501A1 apoprotein levels in the liver, whereas in the small intestine markedly enhanced P4502B apoprotein levels could be detected. No enhanced P4503A apoprotein levels were observed. The 5 and 20% sprouts diets increased the intestinal pentoxyresorufin depentylation (PROD, 4.5-9-fold), and the hydroxylation of testosterone at the 16 alpha- and 16 beta-site (2.6-4.2-fold) after 2 days of exposure. In addition, the 20% sprouts died also enhanced the intestinal ethoxyresorufin deethylation (EROD) activity (c. 5-fold), the hepatic EROD and PROD activities (c. 2-fold) and the formation of 6 beta-hydroxytestosterone (c. 1.6-fold); the formation of 2 alpha-hydroxytestosterone in the liver was decreased (to c. 70% of the control value). GST activity was induced both in the liver (5 and 20% diet) and intestine (20% diet only) throughout the experiment. The 20% sprouts diet enhanced the hepatic DTD and GT1 activities, whereas the GT2 activity was decreased. The induction of DTD in the small intestine after 2 days (2.5-3.2-fold with 5 and 20% sprouts diets, respectively) diminished during the experiment. These results indicate that dietary exposure to cooked Brussels sprouts for only 2 days can change the metabolic activities of several phase II enzymes and cytochrome P-450 enzymes, of which P4502B is the predominant form induced in the small intestine.

Administration, Oral↗
PubMed1998

Polymorphism of phase II enzymes: identification of new enzymes and polymorphic variants by database analysis.

The Phase II enzymes of xenobiotic metabolism are characterized by a high level of substrate diversity and genetic polymorphism. Genetic polymorphism of the Phase II enzymes can be of substantial clinical significance as some variants have differences in substrate specificity, stability and levels of expression. Variation in these factors can give rise to abnormal drug metabolism and susceptibility to carcinogens and toxins. A new approach to the discovery of additional members of Phase II enzyme families and the identification of polymorphic variants using searches of the EST databases has been investigated. The examples provided demonstrate that relatively simple search strategies can be highly productive.

Amino Acid Sequence↗
PubMed2006

Phase I and Phase II enzyme polymorphisms and childhood cancer.

Childhood cancers continue to be challenging clinical entities whose etiology, demographic characteristics, clinical progression, treatment efficacy, and outcomes remain incompletely understood. Research suggests that multiple environmental and genetic factors may play crucial roles in the pathophysiology of many of these malignancies. Recent attention has been directed to the role of carcinogen metabolizing enzymes in the etiology and progression of cancer in both adults and children due to their multitude of polymorphic variants and their intimate interaction with environmental factors. In particular, xenobiotic metabolizing enzymes (XME), which are intimately involved in the activation and deactivation of many environmental carcinogens, have become an area of significant interest. Traditionally, these enzymes have been classified into either phase I or phase II enzymes depending on their substrates, activity, and occasionally based on their sequence in the metabolic pathways, and have been demonstrated to have numerous polymorphic variants. Phase I enzymes predominantly consist of cytochrome enzymes responsible for mixed function oxidase activity, whereas phase II enzymes are frequently conjugation reactions necessary for drug metabolism or the further metabolism of phase I enzyme products. Current research has discovered numerous interactions between polymorphisms in these enzymes and changes in cancer susceptibility, treatment efficacy, and clinical outcomes in childhood cancer. Furthermore, studies of polymorphisms in these enzymes have demonstrated to have synergistic/antagonistic interactions with other XME polymorphisms and demonstrate variable influences on disease pathophysiology depending on the patient's ethnic background and environmental milieu. Continuing research on the role of polymorphisms in phase I and phase II enzymes will likely further elucidate the intimate role of these polymorphisms with environmental factors in the etiology of childhood cancer.

Adolescent↗
PubMed1995

Phase II enzymes and bioactivation.

A colloquium entitled Phase II enzymes and bioactivation was held during the 10th International Symposium on Microsomes and Drug Oxidations in Toronto, Ont., on July 20, 1994. This colloquium was a tribute in recognition of the contributions by Dr. James R. Gillette in advancing our understanding of drug metabolism and chemical toxicity. A major focus of the colloquium was formation of conjugates such as those with glutathione (GSH) that may not lead to detoxification but to bioactivation. The GSH conjugates may be further metabolized to reactive species that cause toxicity. The nephrotoxicity of hydroquinone and bromobenzene is mediated via quinone - glutathione conjugates, and is manifested in cellular changes, including induction of the gadd-153 and hsp-70 mRNA. The formation of GSH conjugates is also involved in the bioactivation of the vicinal dihalopropane 1,2-dibromo-3-chloropropane; cytotoxic lesions are observed in the kidney and testes The evidence indicates that conjugation is mediated by the GSH S-transferases. The symposium also covered aspects of the importance of conjugation in the pharmacokinetics of certain drugs. Conjugation reactions including sulfation are markedly influenced by the manner in which the liver processes the drug. Characteristics such as erythrocyte binding, as in the case of acetaminophen, become limiting factors in the conjugation reactions. Conjugation reactions can lead to a different outcome, such as acquired drug resistance. Conjugation of metallothioneins with the alkylating mustard drugs melphalan and chlorambucil can lead to the formation of protein adducts. Conjugation of reactive intermediates with these small molecular weight proteins may be considered as a phase II reaction and a mechanism of detoxification. A different pathway for the metabolism of xenobiotics is catalyzed by the carboxylesterases, a family of enzymes that is involved in hydrolysis of chemical compounds, generally leading to detoxification. Three rat esterases have been purified, cloned, and characterized. Two forms, hydrolase A and hydrolase B, are present in liver microsomes in a number of species, including the human. These are also detected in extrahepatic tissues. A third esterase, hydrolase S, is found in rat liver microsomes and rat serum, and may be a serum carboxylesterase secreted from the liver. A better knowledge of esterases will advance our understanding of pharmacokinetics and mechanisms of the effects of chemicals such as phenacetin and acetaminophen, two drugs that Dr. Gillette has worked with extensively. The data presented herein reflect the new and innovative approaches that have been adopted to investigate various aspects of chemical toxicity and drug metabolism. These data also indicate that significant insights are likely to come from integrated approaches utilizing established toxicological techniques together with those from other disciplines, including molecular biology and analytical chemistry.

Animals↗
PubMed1997

Soy feeding induces phase II enzymes in rat tissues.

The ability of soy to induce phase II detoxification enzymes was evaluated in male Sprague-Dawley rats. Soybeans contain biologically active compounds that are known inducers of phase II enzyme activity. Rats were fed soy flour (SF) or soy protein isolate (SPI) to provide 75% of total protein as soy. Rats were given free access to food for one- and two-week periods before enzyme activity was compared with that of casein control groups (AIN-93G). Hepatic glutathione S-transferase (GST) activity was significantly greater in rats fed SF for one and two weeks and in rats fed SPI for two weeks than in controls. Quinone reductase activity was significantly greater (12- to 14-fold) in the colon of rats fed SF and SPI for two weeks and in serum (1.8- to 2-fold) in the SF group at one and two weeks. Liver, kidney, and small intestine uridine 5'-diphosphate-glucuronosyl transferase activity was significantly increased in the SPI and SF groups at two weeks. A time dependence in induction of phase II enzymes was observed in several tissues. There was no significant difference in total liver glutathione in either diet group compared with controls. The data indicate that dietary soy enhances phase II enzyme activity, especially quinone reductase and uridine 5'-diphosphate-glucuronosyl transferase, which could lead to protection from potentially harmful xenobiotics.

Animals↗
PubMed2001

Longterm stability of phase I and phase II enzymes of porcine liver cells in flat membrane bioreactors.

Recently, researchers have focused on the use of bioartificial liver devices to support patients with fulminant hepatic failure. Our team developed a cell-based flat membrane bioreactor (FMB). In this, porcine liver cells were maintained in 3D-coculture between two gel layers in a sandwich configuration for 3 weeks to study the influence of this bioreactor technique on the preservation of basic, not induced activities of phase I and phase II enzymes. First, the time and substrate dependencies of the following enzymes were measured: ethoxyresorufin-O-deethylase (EROD, CYP 1A1/1A2) and ethoxycoumarin-O-deethylase (ECOD, CYP 2B6) as phase I enzymes, and glutathione-S-transferase (GST), UDP-glucuronosyltransferase (UGT) and sulfotransferase (ST) as phase II enzymes. To find optimal test conditions Michaelis-Menten kinetics were calculated. Next, different potential inducers were tested to find out the most effective compounds. Based on these results, the basic, not induced levels of the different enzymes were determined in the flat membrane bioreactor. Furthermore, the response of these enzyme activities to the chosen inducers was investigated to examine whether the cells keep their ability for drug-drug interactions. Basic, not induced activities of both phase I enzymes and the phase II enzymes GST and UGT were maintained at nearly the initial levels during the complete period of study. In addition, it was possible to induce these enzymes twice or three times in a weekly interval. In contrast, the basic, not induced activity of ST increased during the first 10 days of culture. It stabilized then and was maintained steady. As in short-term investigations, no reaction of the ST-activity towards any inducer could be obtained. These results prove that porcine liver cells preserve their phase I and phase II activities and respond to inducing drugs over 3 weeks in culture. Therefore, the flat membrane bioreactor is not only suitable for investigating drug metabolism, drug-drug interactions, and enzyme induction but also for supporting liver functions.

7-Alkoxycoumarin O-Dealkylase↗
PubMed2000

7-Methylsulfinylheptyl and 8-methylsulfinyloctyl isothiocyanates from watercress are potent inducers of phase II enzymes.

Watercress is an exceptionally rich dietary source of beta-phenylethyl isothiocyanate (PEITC). This compound inhibits phase I enzymes, which are responsible for the activation of many carcinogens in animals, and induces phase II enzymes, which are associated with enhanced excretion of carcinogens. In this study, we show that watercress extracts are potent inducers of quinone reductase (QR) in murine hepatoma Hepa 1c1c7 cells, a widely adopted assay for measuring phase II enzyme induction. However, contrary to expectations, this induction was not associated with PEITC (which is rapidly lost to the atmosphere upon tissue disruption due to its volatility) or a naturally occurring PEITC-glutathione conjugate, but with 7-methylsulfinyheptyl and 8-methylsulfinyloctyl isothiocyanates (ITCs). While it was confirmed that PEITC does induce QR (5 microM required for a two-fold induction in QR), 7-methylsulfinyheptyl and 8-methylsulfinyloctyl ITCs were more potent inducers (0.2 microM and 0.5 microM, respectively, required for a two-fold induction in QR). Thus, while watercress contains three times more phenylethyl glucosinolate than methylsulfinylalkyl glucosinolates, ITCs derived from methylsulfinylalkyl glucosinolates may be more important phase II enzyme inducers than PEITC, having 10 - to 25-fold greater potency. Analysis of urine by liquid chromatography-mass spectroscopy (LC-MS) following consumption of watercress demonstrated the presence of N:-acetylcysteine conjugates of 7-methylsulfinylheptyl, 8-methylsulfinyloctyl ITCs and PEITC, indicating that these ITCs are taken up by the gut and metabolized in the body. Watercress may have exceptionally good anticarcinogenic potential, as it combines a potent inhibitor of phase I enzymes (PEITC) with at least three inducers of phase II enzymes (PEITC, 7-methylsulfinylheptyl ITC and 8-methylsulfinyloctyl ITC). The study also demonstrates the application of LC-MS for the detection of complex glucosinolate-derived metabolites in plant extracts and urine.

Acetylcysteine↗
PubMed2001

Antimutagenic activity of organosulfur compounds from Allium is associated with phase II enzyme induction.

In a previous study, we showed that naturally occurring organosulfur compounds (OSCs) from garlic and onion modulated the activation of carcinogen via the alteration of cytochromes P450. The present study was undertaken to determine the incidence of the in vivo induction of phase II enzymes by individual OSCs on the genotoxicity of several carcinogens. Diallyl sulfide (DAS), diallyl disulfide (DADS), dipropyl sulfide (DPS) and dipropyl disulfide (DPDS), were administered by gavage (1mmol/kg) to male SPF Wistar rats for 4 consecutive days. The effects of treatments on phase II enzymes and on the genotoxicity of carcinogens were evaluated with hepatic cytosols and microsomes from OSCs-treated rats. DADS strongly increased all the phase II enzymes activities examined, i.e. total glutathione S-transferase (GST) activity, mu GST activity, quinone reductase (QR) activity and epoxide hydrolase (EH) activity. In addition, DADS strongly increased the protein level of rGSTP1. QR activity, total and mu GST activities were also increased by DAS and DPDS whereas DPS increased only mu GST activity and QR activity. To assess the repercussions of these inductions on the genotoxicity of carcinogens, the effects of cytosols or microsomes from OSCs-treated rats on the mutagenicity of (+)-anti-7beta,8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), styrene oxide (SO) and 4-nitroquinoline 1-oxide (4-NQO) were measured in the Ames test. DADS showed a very effective antimutagenic activity against BPDE, SO and 4-NQO. DAS reduced the mutagenicity of BPDE and SO. In contrast, DPS and DPDS showed little efficient antimutagenic activity since they only reduced the mutagenicity of BPDE and 4-NQO, respectively. Interestingly, DADS appeared to be as effective as ethoxyquin, a model inducer of phase II enzymes, in both inducing phase II enzymes and inhibiting the mutagenicity of carcinogens. This study demonstrated that the antimutagenic activities of OSCs against several ultimate carcinogens were closely related to their ability to induce phase II enzymes.

Administration, Oral↗
PubMed2006

Sulforaphane-stimulated phase II enzyme induction inhibits cytokine production by airway epithelial cells stimulated with diesel extract.

Airborne particulate pollutants, such as diesel exhaust particles, are thought to exacerbate lung and cardiovascular diseases through induction of oxidative stress. Sulforaphane, derived from cruciferous vegetables, is the most potent known inducer of phase II enzymes involved in the detoxification of xenobiotics. We postulated that sulforaphane may be able to ameliorate the adverse effects of pollutants by upregulating expression of endogenous antioxidant enzymes. Stimulation of bronchial epithelial cells with the chemical constituents of diesel particles result in the production of proinflammatory cytokines. We first demonstrated a role for phase II enzymes in regulating diesel effects by transfecting the airway epithelial cell line (BEAS-2B) with the sentinel phase II enzyme NAD(P)H: quinine oxidoreductase 1 (NQO1). IL-8 production in response to diesel extract was significantly reduced in these compared with untransfected cells. We then examined whether sulforaphane would stimulate phase II induction and whether this would thereby ablate the effect of diesel extracts on cytokine production. We verified that sulforaphane significantly augmented expression of the phase II enzyme genes GSTM1 and NQO1 and confirmed that sulforaphane treatment increased glutathione S-transferase activity in epithelial cells without inducing cell death or apoptosis. Sulforaphane pretreatment inhibited IL-8 production by BEAS-2B cells upon stimulation with diesel extract. Similarly, whereas diesel extract stimulated production of IL-8, granulocyte-macrophage colony-stimulating factor, and IL-1beta from primary human bronchial epithelial cells, sulforaphane pretreatment inhibited diesel-induced production of all of these cytokines. Our studies show that sulforaphane can mitigate the effect of diesel in respiratory epithelial cells and demonstrate the chemopreventative potential of phase II enzyme enhancement.

Base Sequence↗
PubMed2004

Phase II enzyme inducer, sulforaphane, inhibits UVB-induced AP-1 activation in human keratinocytes by a novel mechanism.

Ultraviolet (UV) light-induced activation of activator protein-1 (AP-1), resulting at least in part from oxidative stress, promotes skin carcinogenesis. It has not yet been determined whether elevating cellular phase II enzymes and glutathione (GSH) levels inhibits the AP-1 activation. We have, therefore, examined the effects of two well-known inducers of phase II enzymes, sulforaphane (SF) and tert-butylhydroquinone (tBHQ), on UVB-induced AP-1 activation, with an AP-1-luciferase reporter plasmid that was stably transfected into human HaCaT keratinocytes (HCL14 cells). Exposure of HCL14 cells to SF or tBHQ led to the induction of quinone reductase-1 (QR-1), a marker of global cellular phase II enzymes, as well as elevation of cellular GSH levels. Incubation of the cells with 1-10 microM SF or 11-45 microM tBHQ for 24 h resulted in up to 1.4-fold and 1.7-fold increase of QR-1 activity, respectively, and up to 1.5-fold and 1.6-fold increases in cellular GSH levels, respectively. AP-1 activation was dramatically enhanced by irradiating HCL14 cells with 250 J/m(2) of UVB. While the above SF treatment dose-dependently reduced the UVB-induced AP-1 activation in HCL14 cells, the tBHQ treatment did not, suggesting that elevating cellular phase II enzymes and GSH levels may not lead to inhibition of UVB-induced AP-1 activation. Indeed, depleting cellular GSH by 80% did not affect UVB-induced AP-1 activation either. Subsequent electrophoretic mobility shift assays (EMSA) showed that SF added directly to the EMSAs inhibited AP-1 DNA binding activity, whereas tBHQ was ineffective. Taken together, our results indicated that elevating phase II enzymes and GSH levels in human keratinocytes does not lead to significant inhibition of UVB-induced AP-1 activation. The inhibitory effect of SF on UVB-induced AP-1 activation appears to be at least partly due to the direct inhibition of AP-1 DNA binding activity. This direct effect of SF on AP-1 DNA binding is a novel mechanism for the action of a drug inhibitor of AP-1 activation.

Anticarcinogenic Agents↗
PubMed2005

Role of PI3K/Akt and MEK/ERK signaling pathways in sulforaphane- and erucin-induced phase II enzymes and MRP2 transcription, G2/M arrest and cell death in Caco-2 cells.

Isothiocyanate sulforaphane is an extensively studied cancer chemopreventive agent in human diet. In this study, the effects of sulforaphane (SFN) and its sulfide analog, erucin (ERN), have been examined on the induction of the phase II enzymes, quinine oxidoreductase (NQO1) and UDP-glucuronosyl transferase (UGT1A1), multidrug transporter (MRP2), cell cycle arrest and cell death in human colon adenocarcinoma Caco-2 cells. Additionally, the roles of PI3K/Akt and MEK/ERK signaling pathways have been assessed in these sulforaphane- and erucin-induced events. Although erucin and sulforaphane have similar IC50 values (21 and 23 microM after 72 h treatment), erucin was more effective in the induction of G2/M accumulation, depletion of mitochondrial potential, induction of cell death and mRNA induction of phase II enzymes and MRP2. Erucin (20 microM) induced the mRNAs of NQO1, UGT1A1 and MRP2 by 11.1-, 11.6- and 6.7-fold, whereas sulforaphane (20 microM) induced 3.3-, 5.3- and 2.2-fold, respectively. Both erucin and sulforaphane induced activation (phosphorylation) of ERK1/2 and Akt kinases but had no effect on JNK and p38 activation. Erucin-induced phase II enzyme transcriptions were decreased by PI3K and MEK1 inhibitors (LY294002 and PD98059), but the decreases in sulforaphane-induced transcription were less marked. Erucin induced a large increase in G2/M cell number than sulforaphane. The ability of kinase inhibitors to overcome G2/M block was low with the exception of PD98059 in sulforaphane-treated cells. Both, sulforaphane and erucin at high concentrations induced accumulation of sub-G1 cells, cell death and dissipation of mitochondrial membrane potential. Taken together, these results demonstrate that PI3K/Akt and MEK/ERK signals are important intracellular mediators in erucin- and sulforaphane-mediated phase II enzyme transcription and cell cycle arrest in Caco-2 cells.

Caco-2 Cells↗
PubMed1996

Phase II enzyme expression in rat liver in response to the antiestrogen tamoxifen.

The genotoxicity and carcinogenicity of tamoxifen have been attributed to metabolic activation of tamoxifen to an electrophile. Phase II enzymes are known to be involved in the metabolism of the drug and possibly in the formation or elimination of the active metabolite. To determine the effects of tamoxifen on phase II enzyme expression, the drug was administered to F344 rats, and hepatic glutathione S-transferase (GST), UDP-glucuronosyltransferase (UGT), and sulfotransferase (ST) expression was evaluated. Some of the tamoxifen-induced effects, including dramatic suppression of selected GST enzymes and activity, were observed at a dose in rats that is directly equivalent, on a mg/kg b.w. basis, to the doses used for breast cancer treatment. Most of the observed responses are not consistent with the previously described phenobarbital-like properties of tamoxifen and could be the result of the partial agonist activity of tamoxifen at the estrogen receptor. Northern blot analysis was performed with isozyme-specific oligonucleotide probes for rat GST, ST, and UGT. In addition, GST subunit protein levels were assayed by high-performance liquid chromatography. In females, tamoxifen treatment resulted in a 60% suppression of GST Ya1 mRNA and protein levels and a 40% suppression of GST Ya2 levels. In males, tamoxifen treatment suppressed GST Ya1 expression approximately 60%, and GST Ya2 expression was suppressed at low doses but induced above control at high doses. Male GST Yc1 was induced approximately 80% over control. The expression of all other major forms of rat hepatic GST subunit protein, including GST Yb1, Yb2, Yb3, Yp, and Yl, was unaffected by tamoxifen treatment. GST conjugation activity toward delta 5-androstene-3,17-dione, a GST Ya1- and Ya2-specific substrate, was suppressed approximately 40% in both sexes, consistent with our protein and mRNA data. Total GST activity, as measured by the rate of chlorodinitrobenzene conjugation, was not changed. Tamoxifen also produced a dose-dependent increase in UGT2B1 mRNA, a phenobarbital-inducible enzyme; mRNA levels reached 210 and 420% of control in females and males, respectively. In addition, mRNA levels for ST2A2, a female-specific ST gene, were suppressed 50% in females and induced 120% over control in males. mRNA expression for all other forms of rat liver UGT and ST isozymes that were tested was not significantly affected by tamoxifen treatment. Overall, these results demonstrate that tamoxifen has significant effects on hepatic phase II enzyme expression that may have implications for the carcinogenicity and/or therapeutic activity of the drug.

Animals↗
PubMed1999

Soy induces phase II enzymes but does not inhibit dimethylbenz[a]anthracene-induced carcinogenesis in female rats.

Isoflavones in soy may play a role in the prevention of cancer through their capacity to affect antioxidant or protective phase II enzyme activities. This study evaluated the effects of dietary isoflavone levels on the induction of antioxidant and phase II enzyme activities and inhibition of breast carcinogenesis. Female Sprague-Dawley rats (36 d) were fed one of four purified diets with casein, or with soy containing three levels of isoflavonoids (0.03, 0.4 or 0.81 mg/g diet; low, middle and high level of isoflavones, respectively). After 2 wk, enzyme activity was determined of rats (n = 6-7) from each diet group. Liver glutathione peroxidase and glutathione reductase activities, blood glutathione levels, kidney glutathione S-transferase and colon quinone reductase (QR) activities were greater in rats consuming the high isoflavone diet compared to rats consuming the casein diet. Kidney QR and liver, kidney, small intestine, and colon UDP-glucuronosyltransferase activities were greater in rats fed the high isoflavone diet compared to rats fed the casein and low-isoflavone diets. Liver and blood oxidized glutathione were lower in rats fed the high-isoflavone diet compared to those fed the low-isoflavone diet. A subset of rats (n = 86) was fed the purified diets for 2 wk and intubated with dimethylbenz[a]anthracene or peanut oil and palpated weekly for tumors. At 13 wk, there was an inverse relationship (R(2) = 0.911, P < 0.09) between tumor incidence and increasing isoflavone intake. These data support the mechanism of soy and soy isoflavones as antioxidant and phase II enzyme inducers, but not as tumor inhibitors.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

The effects of dietary ellagic acid on rat hepatic and esophageal mucosal cytochromes P450 and phase II enzymes.

Ellagic acid (EA), a naturally occurring plant polyphenol possesses broad chemoprotective properties. Dietary EA has been shown to reduce the incidence of N-2-fluorenylacetamide-induced hepatocarcinogenesis in rats and N-nitrosomethylbenzylamine (NMBA)-induced rat esophageal tumors. In this study changes in the expression and activities of specific rat hepatic and esophageal mucosal cytochromes P450 (P450) and phase II enzymes following dietary EA treatment were investigated. Liver and esophageal mucosal microsomes and cytosol were prepared from three groups of Fisher 344 rats which were fed an AIN-76 diet containing no EA or 0.4 or 4.0 g/kg EA for 23 days. In the liver total P450 content decreased by up to 25% and P450 2E1-catalyzed p-nitrophenol hydroxylation decreased by 15%. No changes were observed in P450 1A1, 2B1 or 3A1/2 expression or activities or cytochrome b5 activity. P450 reductase activity decreased by up to 28%. Microsomal epoxide hydrolase (mEH) expression decreased by up to 85% after EA treatment, but mEH activities did not change. The hepatic phase II enzymes glutathione S-transferase (GST), NAD(P)H:quinone reductase [NAD-(P)H:QR] and UDP glucuronosyltransferase (UDPGT) activities increased by up to 26, 17 and 75% respectively. Assays for specific forms of GST indicated marked increases in the activities of isozymes 2-2 (190%), 4-4 (150%) and 5-5 (82%). In the rat esophageal mucosa only P450 1A1 could be detected by Western blot analysis and androstendione was the only P450 metabolite of testosterone detectable. However, there were no differences in the expression of P450 1A1, the formation of androstendione or NAD(P)H:QR activities between control and EA-fed rats in the esophagus. Although there was no significant decrease in overall GST activity, as measured with 1-chloro-2,4-dinitrobenzene (CDNB), there was a significant decrease in the activity of the 2-2 isozyme (66% of control). In vitro incubations showed that EA at a concentration of 100 microM inhibited P450 2E1, 1A1 and 2B1 activities by 87, 55 and 18% respectively, but did not affect 3A1/2 activity. Using standard steady-state kinetic analyses, EA was shown to be a potent non-competitive inhibitor of both liver microsomal ethoxyresorufin O-deethylase and p-nitrophenol hydroxylase activities, with apparent Ki values of approximately 55 and 14 microM respectively. In conclusion, these results demonstrate that EA causes a decrease in total hepatic P450 with a significant effect on hepatic P450 2E1, increases some hepatic phase II enzyme activities [GST, NAD-(P)H:QR and UDPGT] and decreases hepatic mEH expression. It also inhibits the catalytic activity of some P450 isozymes in vitro. Thus the chemoprotective effect of EA against various chemically induced cancers may involve decreases in the rates of metabolism of these carcinogens by phase I enzymes, due to both direct inhibition of catalytic activity and modulation of gene expression, in addition to effects on the expression of phase II enzymes, thereby enhancing the ability of the target tissues to detoxify the reactive intermediates.

Animals↗
PubMed2005

Expression of human phase II enzymes in chimeric mice with humanized liver.

We clarified that major human cytochrome P450 (P450) enzymes were expressed in a chimeric mouse line established recently in Japan, in which the liver could be replaced by more than 80% with human hepatocytes. In this study, we investigated major human phase II enzymes such as UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), N-acetyltransferase (NAT), and glutathione S-transferase (GST) in the livers of chimeric mice by mRNA, protein, and enzyme activity using reverse transcription-polymerase chain reaction, Western blot analysis, and high-performance liquid chromatography, respectively. Human UGT, SULT, NAT, and GST mRNA were expressed in the liver of the chimeric mice, and UGT2B7, SULT1E1, SULT2A1, and GSTA1 proteins could be detected. The expression of mRNA and protein was correlated with the human albumin (hAlb) concentration in mouse blood, the replacement of which by human hepatocytes could be estimated by the hAlb concentration in the blood of the chimeric mice, because the chimeric mice produce human albumin. The enzyme activities, such as morphine 6-glucuronosyltransferase activity and estrone 3-sulfotransferase activity, activities that are specific to humans but not to mice, were increased in a hAlb concentration-dependent manner. The chimeric mice with humanized liver with nearly 90% replacement by human hepatocytes demonstrated almost the same protein contents of human phase II enzymes and enzyme activities as those of the donor. In conclusion, the chimeric mice exhibited an efficient capacity of drug conjugation similar to that in humans. These chimeric mice expressed human phase II enzymes as well as P450s, suggesting that they could be a useful animal model in drug development.

Animals↗
PubMed2004

Modulation of 3-methylcholanthrene-induced rat hepatic and renal cytochrome P450 and phase II enzymes by plant phenols: protocatechuic and tannic acids.

Naturally occurring phenolics, protocatechuic and tannic acids have been reported to be inhibitors of chemical mutagenesis and carcinogenesis in experimental models. Here, we have studied the effect of pretreatment with these compounds on MC-induced cytochrome P450 and phase II enzymes in rats. The male Wistar rats were treated intraperitoneally with protocatechuic acid and tannic acid in the dose of 50mg/kg every 3 days for 2 weeks. MC was administered at the 12th day of phenolics treatment. The activities of EROD (CYP1A1), MROD (CYP1A2), PROD (CYP2B), PNPH (CYP2E1), GST, UDPGT, NQO1 were measured in the liver and kidney. Protocatechuic acid treatment minimally reduced the MC-induced EROD and MROD, but the observed differences were statistically significant. This compound was also a weak inhibitor of hepatic PNPH. Moreover, Western blot analysis with CYP1A1/1A2- and CYP2E1-specific antibodies showed the same effect in the levels of hepatic CYP1A1/1A2 and CYP2E1. Minimal decrease of renal constitutive (by 23%) and more significant reduction of induced form (by 66%) of PNPH was found as result of treatment with protocatechuic acid. Tannic acid alone had no effect on cytochrome P450 enzymes while in combination with MC this polyphenol minimally enhanced the MC induction of MROD and in greater extent PNPH in liver. The treatment with protocatechuic acid alone enhanced slightly the activities of all three phase II enzymes in liver. The pretreatment with this phenolic of the MC-induced rats however significantly increased the activities of hepatic GST and NQO1 in comparison with MC-treated group. In kidney MC-induced activity of NQO1 was reduced (about 43%) to the control level by tannic acid pretreatment. The results of our present study indicate that in rat the prolonged treatment with protocatechuic acid affects differently the activities of CYP and phase II enzyme when compared to tannic acid. Moreover, the effect of this polyphenols significantly depends on the method of treatment.

Animals↗