Effect of chemopreventive compounds from Brassica vegetables on NAD(P)H:quinone reductase and induction of DNA strand breaks in murine hepa1c1c7 cells.

We have compared the effects of aqueous extracts of cooked Brussels sprouts, isolated glucosinolates and their breakdown products on the activity of quinone reductase [NADPH:quinone-reductase] (QR) and on DNA strand breaks induced by hydrogen peroxide in murine hepa1c1c7 cells. QR activity was not significantly altered after incubation of the cells with Brussels sprouts extracts. However, some of the glucosinolates and in particular their myrosinase-catalysed hydrolysis products and the degradation product of indole-glucosinolates, indole-3-carbinole (I3C), di(indol-3-yl)-methane (DIM) and 2,3-bis(indol-3-ylmethyl)indole (TRI) effectively induced QR activity. Isolated isothiocyanates did not influence the QR activity. The extracts of cooked and autolysed Brussels sprouts and some glucosinolates inhibited the DNA strand breaks induced by 100 microM hydrogen peroxide. Maximum inhibition was by 20-38% after 24 h of preincubation. Hydrolysis of the glucosinolates by myrosinase decreased the inhibitory effects, whereas I3C, DIM or TRI had no effect on DNA damage. Accordingly, the protective effect of Brussels sprouts constituents against induction of oxidative DNA damage appears to be unrelated to enzyme inducing properties via the antioxidant responsive element. Both of these effects could be part of the suggested cancer preventive effect of cruciferous vegetables.     

Evaluation of urinary N-acetyl cysteinyl allyl isothiocyanate as a biomarker for intake and bioactivity of Brussels sprouts.

Isothiocyanates (ITC), glucosinolate hydrolysis products from Brussels sprouts (BS) and other cruciferous vegetables, are considered to protect the body from cancer by induction of detoxification enzymes such as quinone reductase (QR). Urinary N-acetyl-cysteine (NAC) conjugates of ITC have been proposed as biomarkers of crucifer intake. Here we asked if dietary intake and induction of detoxification enzymes are dose-related to urinary NAC conjugate appearance. Male F344 rats (4/group) received an AIN 76B-40 diet containing 0, 10 or 20% freeze-dried BS for 6 days. A human subject ingested 500 g BS. Urinary AITC-NAC was identified in human and rat urine. Ten and 20% BS diets caused a 1.4- and 2.3-fold induction of QR in the pancreas, a 1.5- and 2.5-fold induction in liver and a 3.1- and 3.6-fold induction in colonic epithelium, respectively. Liver and pancreatic QR induction was dose-related, whereas induction of QR in colon was less different between the two doses. Excretion of the conjugate was dose-related only on day 1, and unrelated to dose after day 2. These results suggest that urinary NAC–AITC is a qualitative biomarker for ingestion and bioactivity of BS, but that it may not be dose-related when rats are fed continuously for 2 or more days.     

4-Methylsulfanyl-3-butenyl isothiocyanate derived from glucoraphasatin is a potent inducer of rat hepatic phase II enzymes and a potential chemopreventive agent

The objective of this study was to establish whether the phytochemical glucoraphasatin, a glucosinolate present in cruciferous vegetables, and its corresponding isothiocyanate, 4-methylsulfanyl-3-butenyl isothiocyanate, up-regulate enzymes involved in the detoxification of carcinogens and are thus potential chemopreventive agents. Glucoraphasatin and myrosinase were isolated and purified from Daikon sprouts and Sinapis alba L., respectively. Glucoraphasatin (0–10 μM) was incubated for 24 h with precision-cut rat liver slices in the presence and absence of myrosinase, the enzyme that converts the glucosinolate to the isothiocyanate. The intact glucosinolate failed to influence the O-dealkylations of methoxy- and ethoxyresorufin or the apoprotein expression of CYP1 enzymes. Supplementation with myrosinase led to an increase in the dealkylation of methoxyresorufin, but only at the highest concentration of the glucosinolate, and CYP1A2 expression. In the absence of myrosinase, glucoraphasatin caused a marked increase in epoxide hydrolase activity at concentrations as low as 1 μM paralleled by a rise in the enzyme protein expression; at the highest concentration only, a rise was also observed in glucuronosyl transferase activity, but other phase II enzyme systems were unaffected. Addition of myrosinase to the glucoraphasatin incubation maintained the rise in epoxide hydrolase and glucuronosyl transferase activities, further elevated quinone reductase and glutathione S-transferase activities, and increased total glutathione concentrations. It is concluded that at low concentrations, glucoraphasatin, either intact and/or through the formation of 4-methylsulfanyl-3-butenyl isothiocyanate, is a potent inducer of hepatic enzymes involved in the detoxification of chemical carcinogens and merits further investigation for chemopreventive activity.     

Evaluation of urinary N-acetyl cysteinyl allyl isothiocyanate as a biomarker for intake and bioactivity of Brussels sprouts

Isothiocyanates (ITC), glucosinolate hydrolysis products from Brussels sprouts (BS) and other cruciferous vegetables, are considered to protect the body from cancer by induction of detoxification enzymes such as quinone reductase (QR). Urinary N-acetyl-cysteine (NAC) conjugates of ITC have been proposed as biomarkers of crucifer intake. Here we asked if dietary intake and induction of detoxification enzymes are dose-related to urinary NAC conjugate appearance. Male F344 rats (4/group) received an AIN 76B-40 diet containing 0, 10 or 20% freeze-dried BS for 6 days. A human subject ingested 500 g BS. Urinary AITC-NAC was identified in human and rat urine. Ten and 20% BS diets caused a 1.4- and 2.3-fold induction of QR in the pancreas, a 1.5- and 2.5-fold induction in liver and a 3.1- and 3.6-fold induction in colonic epithelium, respectively. Liver and pancreatic QR induction was dose-related, whereas induction of QR in colon was less different between the two doses. Excretion of the conjugate was dose-related only on day 1, and unrelated to dose after day 2. These results suggest that urinary NAC–AITC is a qualitative biomarker for ingestion and bioactivity of BS, but that it may not be dose-related when rats are fed continuously for 2 or more days.     

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.     

Altered CYP expression and function in response to dietary factors: potential roles in disease pathogenesis

Increasing evidence implicates dietary factors in the progression of diseases, including certain cancers, diabetes and obesity. Diet also regulates the expression and function of CYP genes, which impacts on drug elimination and may also significantly affect disease pathogenesis. Upregulation of CYPs 2E1 and 4A occurs after feeding of experimental diets that are high in fats or carbohydrates; these diets also promote hepatic lipid infiltration, which is a component of the metabolic syndrome that characterises obesity. Increased availability of lipid substrates for CYPs can enhance free radical production and exacerbate tissue injury. Similar processes may also occur in other models of experimental disease states that exhibit a component of altered nutrient utilization. Food-derived chemicals, including constituents of cruciferous vegetables and fruits, modulate CYP expression and the expression of genes that encode cytoprotective phase II enzymes. Certain dietary indoles and flavonoids activate CYP1A expression either by direct ligand interaction with the aryl hydrocarbon receptor (AhR) or by augmenting the interaction of the AhR with xenobiotic response elements in CYP1A1 and other target genes. Other dietary chemicals, including methylenedioxyphenyl (MDP) compounds and isothiocyanates also modulate CYP gene expression. Apart from altered CYP regulation, a number of dietary agents also inhibit CYP enzyme activity, leading to pharmacokinetic interactions with coadministered drugs. A well described example is that of grapefruit juice, which contains psoralens and possibly other chemicals, that inactivate intestinal CYP3A4. Decreased presystemic oxidation by this CYP increases the systemic bioavailability of drug substrates and the likelihood of drug toxicity. Dietary interactions may complicate drug therapy but inhibition of certain CYP reactions may also protect the individual against toxic metabolites and free radicals generated by CYPs. Chemicals in teas and cruciferous vegetables may also inhibit human CYP enzymes that have been implicated in the bioactivation of chemical carcinogens. Thus, food constituents modulate CYP expression and function by a range of mechanisms, with the potential for both deleterious and beneficial outcomes.     

Effects of a water-soluble extract of rosemary and its purified component rosmarinic acid on xenobiotic-metabolizing enzymes in rat liver.

The effects of a water-soluble extract (WSE) of rosemary and its purified antioxidant rosmarinic acid (RA) on xenobiotic metabolizing enzymes (XME) were studied in rat liver after dietary administration. The modulation of phase I enzymes such as cytochrome P450 (CYP) 1A, 2B, 2E1, 3A, and phase II enzymes such as glutathione S-transferase (GST), quinone reductase (QR) and UDP-glucuronosyltransferase (UGT) was evaluated by measuring enzyme activities with specific substrates. Protein levels of CYPs and rGST A1/A2, A3/A5, M1, M2 and P1 were measured using antibodies in Western blots. Caffeic acid was also studied because it results from RA biotransformation in rat after oral administration. Male SPF Wistar rats received the different compounds at 0.5% (w/w) incorporated into their diet for 2 weeks. WSE, containing RA, flavones and monoterpenes enhanced CYP 1A1, 2B1/2, 2E1 and GST (especially rGST A3/A5, M1 and M2), QR and UGT. On the contrary, no modification of XME was observed in response to RA or CA (except for a slight increase of UGT activity after CA treatment). The induction of XME by WSE could be attributed to flavones, monoterpenes or an additive effect of all components.     

Effect of some indole derivatives on xenobiotic metabolism and xenobiotic-induced toxicity in cultured rat liver slices.

In this study the effect of some indole derivatives on xenobiotic metabolizing enzymes and xenobiotic-induced toxicity has been examined in cultured precision-cut liver slices from male Sprague-Dawley rats. While treatment of rat liver slices for 72 hours with 2-200 microM of either indole-3-carbinol (I3C) or indole-3-acetonitrile (3-ICN) had little effect on cytochrome P-450 (CYP)-dependent enzyme activities, enzyme induction was observed after in vivo administration of I3C. The treatment of rat liver slices with 50 microM 3,3'-diindolylmethane (DIM; a dimer derived from I3C under acidic conditions) for 72 hours resulted in a marked induction of CYP-dependent enzyme activities. DIM appears to be a mixed inducer of CYP in rat liver slices having effects on CYP1A, CYP2B and CYP3A subfamily isoforms. Small increases in liver slice reduced glutathione levels and glutathione S-transferase activity were also observed after DIM treatment. While aflatoxin B1 and monocrotaline produced a concentration-dependent inhibition of protein synthesis in 72-hour-cultured rat liver slices, cytotoxicity was markedly reduced in liver slices cultured with 50 microM DIM. These results demonstrate that cultured rat liver slices may be employed to evaluate the effects of chemicals derived from cruciferous and other vegetables on CYP isoforms. In addition, liver slices can also be utilized to examine the ability of such chemicals to modulate xenobiotic-induced toxicity.     

Effect of dietary indole-3-carbinol on intestinal and hepatic monooxygenase, glutathione S-transferase and epoxide hydrolase activities in the rat

Male Sprague-Dawley rats were fed on purified diets supplemented with 50–500 ppm indole-3-carbinol (I3C), a compound present in cruciferous vegetables, or with 25% Brussels sprouts (Brassica oleracea) for 10 days after a 1-wk equilibration on a purified diet. Cytosolic and microsomal fractions were prepared from liver and intestinal mucosae. Intestinal aryl hydrocarbon hydroxylase (AHH) activity was increased significantly (P < 0.05) over basal levels by I3C at 50 ppm (a 6.1-fold increase), at 125 ppm (11.8-fold), at 250 ppm (14.1-fold) and at 500 ppm (20.2-fold) and by 25% Brussels sprouts (3.6-fold). Intestinal ethoxycoumarin O-deethylase (ECD) activity was also significantly increased by I3C, the increases being 4.6-, 8.7-, 9.3- and 11.2-fold with 50, 125, 250 and 500 ppm I3C, respectively, and 3.2-fold with the sprouts diet. Hepatic AHH and ECD were not increased significantly by any of these dietary treatments. Hepatic and intestinal glutathione S-transferase (GST) activities were increased (P < 0.05) 1.9- and 1.6-fold, respectively, by the sprouts diet but were not significantly affected even by 500 ppm I3C. Microsomal epoxide hydrolase (EH) activity of the small intestine was increased 2.0-fold by the sprouts diet but was unaffected by I3C. Hepatic cytochrome P-450 was increased 1.3-fold by the sprouts diet although I3C at 500 ppm only produced a 1.1-fold increase. A no-effect-level for I3C on intestinal monooxygenase induction was estimated to be between 16 and 25 ppm, supporting the contention that I3C can account for much of the monooxygenase induction observed when experimental animals are fed diets high in cruciferous vegetables. The results also indicate that Brassica oleracea contains other compounds which are responsible for the induction of GST and EH activities.     

Diesel exhaust particle-induced alterations of pulmonary phase I and phase II enzymes of rats

Although diesel exhaust particles (DEP) are known to produce pulmonary disorders, the xenobiotic metabolic pathways associated with DEP detoxification and bioactivation remain unclear. In this study, the effect of acute exposure of DEP on phase I and phase II enzymes of rat lung was investigated. Intratracheal administration of DEP produced an induction of cytochrome P-450 (CYP) 1A1 enzyme protein and activity at 1 d postexposure, with the enzyme level returning to control at 5 d postexposure. On the other hand, carbon black (CB), a particle control, did not show any induction of CYP1A1 protein or enzyme activity. However, both DEP and CB significantly decreased CYP2B1 protein and enzyme activity at 1 d postexposure. The decrease in CYP2B1 enzyme protein and activity by DEP or CB treatment was observed up to 7 d postexposure. DEP and CB treatments also significantly attenuated glutathione S-transferase (GST)-pi protein at 1 d postexposure. Both DEP and CB at 35 mg/kg significantly decreased the activities of GST and catalase at 1 and 7 d postexposure. DEP, but not CB, significantly induced quinone reductase (QR) activity at 7 d postexposure. This study suggests that DEP may induce CYP1A1 and QR enzymes via a chemical effect, while the carbonaceous core may be involved in the attenuation of CYP2B1, GST, and catalase proteins and enzyme activities.     

Regulation of CYP1A1 gene expression by the antioxidant tert-butylhydroquinone.

CYP1A1, a major phase I enzyme, plays an important role in the metabolism of polycyclic aromatic hydrocarbons and in the chemical activation of xenobiotics to carcinogenic derivatives. The phenolic antioxidant tert-butylhydroquinone (tBHQ), often used as a food preservative, is generally considered to act only as a mono-functional inducer of phase II enzymes, thereby exerting chemo-protection. However, we recently observed that tBHQ elevated the activity of an aryl hydrocarbon receptor (AhR) response element (DRE)-driven luciferase reporter in human colon carcinoma cells (Caco-2). Therefore, we studied the effects of tBHQ on the activity of a DRE-driven reporter, CYP1A1 mRNA expression, and CYP1A enzyme activity in Caco-2 cells and human HepG2 hepatoma cells. We found tBHQ caused induction of reporter activity and CYP1A1 expression and activity in Caco-2 and HepG2 cells. Moreover, tBHQ combined with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased reporter activity and mRNA expression in Caco-2 cells in an additive manner. By contrast, tBHQ decreased TCDD-mediated induction of reporter activity and CYP1A1 mRNA expression in HepG2 cells. Resveratrol, an AhR antagonist, repressed the induction of CYP1A1 by tBHQ. Cotransfection of HepG2 cells with a dominant negative AhR nuclear translocator mutant abolished the tBHQ-induced CYP1A1 reporter activity. These findings indicate that CYP1A1 may be induced by the antioxidant tBHQ via an AhR-dependent mechanism.     

Effect of ergot alkaloids associated with fescue toxicosis on hepatic cytochrome P450 and antioxidant proteins

Intake of ergot alkaloids found in endophyte-infected tall fescue grass is associated with decreased feed intake and reduction in body weight gain. The liver is one of the target organs of fescue toxicosis with upregulation of genes involved in xenobiotic metabolism and downregulation of genes associated with antioxidant pathways. It was hypothesized that short-term exposure of rats to ergot alkaloids would change hepatic cytochrome P450 (CYP) and antioxidant expression, as well as reduce antioxidant enzyme activity and hepatocellular proliferation rates. Hepatic gene expression of various CYPs, selected nuclear receptors associated with the CYP induction, and antioxidant enzymes were measured using real-time PCR. Hepatic expression of CYP, antioxidant and proliferating cell nuclear antigen (PCNA) proteins were measured using Western blots. The CYP3A1 protein expression was evaluated using primary rat hepatocellular cultures treated with ergovaline, one of the major ergot alkaloids produced by fescue endophyte, in order to assess the direct role of ergot alkaloids in CYP induction. The enzyme activities of selected antioxidants were assayed spectrophotometrically. While hepatic CYP and nuclear receptor expression were increased in ergot alkaloid-exposed rats, the expression and activity of antioxidant enzymes were reduced. This could potentially lead to increased oxidative stress, which might be responsible for the decrease in hepatocellular proliferation after ergot alkaloid exposure. This study demonstrated that even short-term exposure to ergot alkaloids can potentially induce hepatic oxidative stress which can contribute to the pathogenesis of fescue toxicosis.     

Effects of commonly consumed vegetables on hepatic xenobiotic-metabolizing enzymes in the mouse

To investigate the effects of consuming a variety of common vegetables on the activities of xenobiotic-metabolizing enzymes in the mouse liver, male C57BL/6 mice were fed purified diets supplemented isocalorically with 20% freeze-dried powdered kidney bean, soya bean, alfalfa, cauliflower, mustard greens, carrot, kale, Brussels sprouts, beet, egg plant or onion or with 40% sweet potato. Hepatic ethoxycoumarin O-deethylase (ECD) activity was increased significantly (P less than or equal to 0.05) over the controls in the mice fed diets containing kidney bean (1.5-fold), alfalfa (1.6-fold), cauliflower (2.2-fold), mustard greens (1.2-fold), carrots (1.2-fold) and kale (1.3-fold). No significant increases in ECD activity were seen in the other groups. Aryl hydrocarbon hydroxylase (AHH) activity was assayed in the cauliflower and Brussels sprouts groups and was found to be unaffected by these diets. Glutathione S-transferase (GST) and epoxide hydratase (EH) activities were increased (P less than or equal to 0.05) by soya bean (1.2- and 1.6-fold respectively), Brussels sprouts (2.0- and 1.6-fold), cauliflower (1.2- and 1.6-fold), alfalfa (1.3- and 2.0-fold) and onion (1.8- and 2.3-fold). No significant increases in GST or EH activities were seen in the other groups. Of the twelve vegetables investigated, only three (sweet potato, beet and egg plant) had no statistically significant effects on any of the xenobiotic-metabolizing parameters tested. However, the beet diet caused an apparent inhibition of ECD activity (74% of control) and the sweet potato diet caused an apparent increase (1.3-fold) in GST activity, although statistical significance could not be established at P less than or equal to 0.05.     

Dietary influences on rat hepatic and intestinal DT-diaphorase activity

DT-diaphorase (DTD) is a flavoprotein that catalyses the two-electron reduction of various redox dyes and quinones such as menadione and phylloquinone. It has been proposed that this enzyme may have a protective effect against cancer, as the two-electron reduction prevents the formation of toxic oxygen metabolites that may be generated as a result of the one-electron reduction catalysed by enzymes such as NADPH-cytochrome P-450 reductase. The effects of a purified diet supplemented with either 25% Brussels sprouts, phylloquinone (2.5 or 25 ppm) or 250 ppm indole-3-carbinol on hepatic and intestinal DTD activity in adult male Sprague-Dawley rats have been determined. One group was fed on the purified diet and dosed with 3-methylcholanthrene (20 mg/kg), 24 hr before being killed. Hepatic DTD activity was increased 3-fold in the indole-3-carbinol group, 4.4-fold in the sprouts-fed animals and 8.2-fold in the 3-methylcholanthrene-treated animals. Neither level of phylloquinone affected hepatic DTD activity. Intestinal DTD activity was increased 2.1-fold in the indole-3-carbinol group, 3.7-fold in the sprouts-fed animals and 4.3-fold in the 3-methylcholanthrene group. In animals given 25 ppm phylloquinone, intestinal enzyme activity was 60% of the control level, while no effect was noted in those given 2.5 ppm phylloquinone. Although increases in the activities of intestinal xenobiotic-metabolizing enzymes resulting from dietary influences are well documented, the increase in hepatic DTD activity seen in response to vegetable consumption has not been reported. The significance of these results in relation to the possible protective effects of dietary cruciferous vegetables against cancer is discussed.     

Induction of hepatic phase II drug-metabolizing enzymes by 1,7-phenanthroline in rats is accompanied by induction of MRP3.

The purpose of the present study was to evaluate the effect of 1,7-phenanthroline (PH), which has been proposed to be a selective phase II enzyme inducer, on the gene expression of xenobiotic transporters, as well as hepatic and renal drug-metabolizing enzymes. After oral administration of PH for 3 days to male Sprague-Dawley rats, mRNA levels in liver (75 and 150 mg/kg doses) and kidney (75 mg/kg dose only) were determined using real-time quantitative polymerase chain reaction. At 150 mg/kg/day, PH treatment resulted in significant increases in hepatic mRNA levels of Mrp3 (36-fold), UGT1A6 (20-fold), UGT2B1 (4-fold), and quinone reductase (QR, 5-fold), compared with the vehicle-treated group. Similar increases in Mrp3 (99-fold), UGT1A6 (17-fold), UGT2B1 (3-fold), and QR (11-fold) mRNA levels were observed in the liver after PH treatment of rats at 75 mg/kg/day. In contrast, the expression levels of CYP2C11 and Oatp2 were decreased by approximately 80 and 50%, respectively. In addition, PH (75 mg/kg/day) elicited statistically significant changes in renal gene expression of CYP3A1, UGT1A6, QR, and Mrp3, but the magnitude of renal Mrp3 induction was less than 2-fold over control. Although PH is known to modulate hepatic glucuronidation in vivo, these data indicated that PH induced mRNA levels of the efflux transporter, Mrp3, which may also affect the disposition of xenobiotics.