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We previously reported the in vitro and in vivo induction of thioredoxin reductase (TR) by sulforaphane (SF) purified from broccoli. The present study was designed to determine whether this induction is mediated by putative antioxidant response elements (ARE) found in the promoter. Luciferase reporter constructs were built using the TR promoter sequence. Sulforaphane, tert-butylhydroquinone and beta-napthoflavone, as well as the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), increased luciferase activity in HepG2 cells transfected with the reporter construct (P < 0.0001). Quinone reductase (QR) is an enzyme with a well-characterized ARE, and QR reporter constructs built as positive controls showed similar patterns of induction. Mutation of the core sequence of a putative ARE in the TR promoter drastically decreased inducibility by SF, but mutations in nonconsensus areas of the ARE and outside of the ARE did not affect inducibility. Results from electrophoretic mobility shift assay analysis corroborated mutated reporter gene findings. Induction by TPA was not affected by mutation of the putative ARE. Se plus SF, and SF alone were equally effective for induction of TR reporter luciferase activity (P < 0.0001); Se alone had no effect. Se and SF independently increased TR activity (P < 0.0001) and when combined, increased TR activity synergistically (P = 0.036). These data suggest that TR is transcriptionally regulated by electrophilic compounds via an ARE in the 5' region of the gene, and that this mechanism is unrelated to the established Se-dependent induction of selenoproteins.

The discovery of bioactive components in foods is exciting, suggesting the possibility of improved public health through diet. Yet the content of bioactive components in plant food varies, making quality control and intake recommendations problematic. Variation in content of bioactive components in fruits and vegetables depends upon both genetics and environment, including growing conditions, harvest and storage, processing and meal preparation. Cruciferous vegetables, which contain both anticarcinogenic and antioxidant properties, are excellent examples to illustrate the problem in assessing health benefits of foods that vary in content of bioactive components. In broccoli, the content of both glucosinolates and their bioactive hydrolysis products varies with genotype, environment and processing. Antioxidant vitamins and flavonoid content varies also. Here we review the influences of genetics, environment and post-harvest processing on content of bioactive components in broccoli, an area that is presently only partly understood. Reporting a range for bioactive component content can help the public to make informed choices about diet. For the future, research into the mechanisms behind this variation can lead to an understanding of genetic regulation of these variations, resulting in the generation of a consistent supply of nutritionally enhanced plant foods on the market.

Flavor and glucosinolate contents of multiple broccoli cultivars (Brassica oleracea var. italica) were compared to determine whether a relationship existed between sensory and chemical attributes. Glucosinolates have been associated with the anticarcinogenic activity and flavor of cruciferous vegetables. A trained sensory descriptive panel developed 19 descriptive terms for aroma and flavor attributes and evaluated 19 broccoli cultivars. All 19 genotypes were assayed for individual and total glucosinolate content by HPLC (high performance liquid chromatography). Broccoli varieties differed significantly in their sensory and glucosinolate profiles. Using correlation and principal component analysis, we found little evidence that the glucosinolates were responsible for the distinctive flavor notes of broccoli.

Broccoli can positively impact human health in a number of ways including acting as a vehicle for the delivery of promising antioxidants. Studies in non-cellular assay systems indicate that broccoli extracts have a high capacity for scavenging free radicals or interrupting free radical reactions. Here we report the effect of broccoli extracts on oxidative stress in HepG2 cells using the dichlorofluorescein-diacetate (DCFH-DA) assay. Our results confirm the association between broccoli extracts and enhanced antioxidant activity while providing additional evidence for protection against reactive oxygen species (ROS) at the cellular level. In HepG2 cells, the level of protection against AAPH-induced ROS differed among broccoli genotypes tested. We found that the HepG2 cell assay provided more information about the antioxidative lipid soluble fraction than the commonly reported ORAC assay.

Sulforaphane (SF), a glucosinolate-derived isothiocyanate found in cruciferous vegetables, is considered an anticarcinogenic component in broccoli. Sulforaphane induces a battery of detoxification enzymes, including quinone reductase (QR). Induction is thought to be mediated through a common regulatory region termed the antioxidant response element (ARE). To test the hypothesis that the antioxidant selenoprotein thioredoxin reductase (TR) may be induced as part of this coordinated host-defense response to dietary anticarcinogenic compounds, TR activity was measured in livers of rats pair-fed diets containing SF and/or broccoli (n = 6/group). At the doses used, neither SF nor broccoli alone significantly elevated TR activity, whereas treatments containing both broccoli and SF caused a significant increase in TR activity. Glutathione peroxidase (GSH-Px), a second selenium-dependant enzyme with antioxidant activity, was downregulated in rats fed both SF and broccoli, compared to the control diet.A second experiment, using mouse hepatoma Hepa1c1c7 cells, tested whether an interaction exists between selenium (Se) and SF in TR inducibility, since Se is known to induce TR activity. Selenium (2.5mgr;M) plus SF (2.0mgr;M) caused significantly greater TR activity than either treatment alone. All treatments with added Se or SF caused significantly greater TR activities than no Se or SF treatment. Glutathione peroxidase activity was elevated by Se, but not by SF. These data suggest that TR, known to be regulated by Se, is also upregulated as part of a host response to the dietary anticarcinogen SF, a trait not shared by another Se-dependent enzyme, GSH-Px.

Ten broccoli (Brassica oleracea L. (Botrytis Group)) accessions were grown in several environments to estimate glucosinolate (GS) variability associated with genotype, environment, and genotype × environment interaction and to identify differences in the stability of GSs in broccoli florets. Significant differences in genetic variability were identified for aliphatic GSs but not for indolyl GSs. The percentage of GS variability attributable to genotype for individual aliphatic compounds ranged from 54.2% for glucoraphanin to 71.0% for progoitrin. For total indolyl GSs, the percentage of variability attributable to genotype was only 12%. Both qualitative and quantitative differences in GSs were detected among the genotypes. Ten-fold differences in progoitrin, glucoraphanin, and total aliphatic GS levels were observed between the highest and lowest genotypes. Only two lines, Eu8-1 and VI-158, produced aliphatic GSs other than glucoraphanin in appreciable amounts. Differences in stability of these compounds among the cultivars were also observed between fall and spring plantings. Results suggest that genetic factors necessary for altering the qualitative and quantitative aliphatic GS profiles are present within existing broccoli germplasm, which makes breeding for enhanced cancer chemoprotectant activity feasible. (Wattenberg, 1985). Bioactive breakdown products from indi- vidual GSs differ in their potency as inducers of these enzymes

Antioxidant capacity of hydrophilic and lipophilic extracts from eight broccoli genotypes was compared using the oxygen radical absorbance capacity (ORAC) assay. Each genotype was analyzed for carotenoid, tocopherol, ascorbic acid, and flavonoid content. Results indicate that the antioxidant capacity of hydrophilic extracts ranged from 65.8 to 121.6 micromol trolox equivalents (TE)/g of tissue, and the capacity of lipophilic extracts ranged from 3.9 to 17.5 micromol TE/g. Ascorbic acid and flavonoid content of the hydrophilic extracts did not explain the total variation in antioxidant capacity of those extracts, suggesting either the presence of other antioxidant components that have yet to be identified or that the known antioxidants are producing synergistic effects. The carotenoids did correlate with antioxidant capacity of the lipophilic extracts and accounted for the majority of the variability in that fraction. The variability in hydrophilic and lipophilic antioxidant capacity found among these genotypes suggests that potential efficacy from antioxidants will vary considerably from genotype to genotype.

The anticarcinogenic properties of broccoli are believed to be due to modification of detoxification enzymes by a group of isothiocyanates, hydrolysis products of glucosinolates, particularly sulforaphane. We previously showed that the nitrile crambene (1-cyano-2-hydroxy-3-butene), present in most Brassica vegetables, induces hepatic quinone reductase activity when administered to rats. In this study, we compared the effects of seven daily oral doses of crambene (50 mg/kg rat/day) and sulforaphane (50 mg/kg rat/day) on induction of hepatic quinone reductase activity in Fischer 344 rats. The two treatments produced similar effects, with crambene and sulforaphane producing 1.5- and 1.7-fold induction in hepatic quinone reductase activity, respectively. Additionally, we evaluated the effect of crambene on quinone reductase activity in Hepa 1c1c7 cells, because this system had been shown to possess high sensitivity to sulforaphane and is commonly used for screening anticarcinogenic compounds. Crambene (5 mM) induced quinone reductase activity and caused cell cycle arrest in the G2/M phase in mouse Hepa 1c1c7 cells, rat H4IIEC3 cells, and human Hep G2 cells (95% viability). Doses of crambene needed for induction of quinone reductase in cell culture were approximately 100-fold greater than effective doses of sulforaphane. These findings indicate that hepatoma cell lines may not accurately reflect relative potency of anticarcinogens in Fischer 344 rats.

Methyl jasmonate (MeJA) spray treatments were applied to the kale varieties 'Dwarf Blue Curled Vates' and 'Red Winter' in replicated field plantings in 2010 and 2011 to investigate alteration of glucosinolate (GS) composition in harvested leaf tissue. Aqueous solutions of 250 µM MeJA were sprayed to saturation on aerial plant tissues four days prior to harvest at commercial maturity. The MeJA treatment significantly increased gluconasturtiin (56%), glucobrassicin (98%), and neoglucobrassicin (150%) concentrations in the apical leaf tissue of these genotypes over two seasons. Induction of quinone reductase (QR) activity, a biomarker for anti-carcinogenesis, was significantly increased by the extracts from the leaf tissue of these two cultivars. Extracts of apical leaf tissues had greater MeJA mediated increases in phenolics, glucosinolate concentrations, GS hydrolysis products, and QR activity than extracts from basal leaf tissue samples. The concentration of the hydrolysis product of glucoraphanin, sulforphane was significantly increased in apical leaf tissue of the cultivar 'Red Winter' in both 2010 and 2011. There was interaction between exogenous MeJA treatment and environmental conditions to induce endogenous JA. Correlation analysis revealed that indole-3-carbanol (I3C) generated from the hydrolysis of glucobrassicin significantly correlated with QR activity (r = 0.800, P

Camelina sativa L. Crantz is under development as a novel oilseed crop, yet bioefficacy of camelina phytochemicals is unknown. Defatted camelina seed meal contains two major aliphatic glucosinolates (GSLs), glucoarabin (9-(methylsulfinyl)nonylglucosinolate; GSL 9) and glucocamelinin (10-(methylsulfinyl)decylglucosinolate; GSL 10), with traces of a third, 11(methylsulfinyl)undecylglucosinolate and several flavonoids, mostly quercetin glycosides. In Hepa1c1c7 cells, hydrolyzed GSLs (hGSLs) 9 and 10 upregulated the phase II detoxification enzyme quinone reductase (NQO1), with no effect on cytochrome P450 (CYP) 1A1 activity. Isobologram graphs revealed synergy of NQO1 induction for a combination of hGSL 9 and quercetin. These findings suggest that defatted camelina seed meal should be evaluated for anticancer activity, similar to broccoli and other Brassicaceae family members. Interestingly, synergy of NQO1 induction was also seen for physiologically relevant doses of sulforaphane (SF) and quercetin, two key bioactives present in broccoli. This suggests that SF within broccoli may be more potent than purified SF.

Isothiocyanates, generated from the hydrolysis of glucosinolates in plants of the Brassicaceae family, promote health, including anticancer bioactivity. Hydrolysis requires the plant enzyme myrosinase, giving myrosinase a key role in health promotion by brassica vegetables. Myrosinase measurement typically involves isolating crude protein, potentially underestimating activity in whole foods. Myrosinase activity was estimated using unextracted fresh tissues of five broccoli and three kale cultivars, measuring the formation of allyl isothiocyanate (AITC) and/or glucose from exogenous sinigrin. A correlation between AITC and glucose formation was found, although activity was substantially lower measured as glucose release. Using exogenous sinigrin or endogenous glucoraphanin, concentrations of the hydrolysis products AITC and sulforaphane correlated (r = 0.859; p = 0.006), suggesting that broccoli shows no myrosinase selectivity among sinigrin and glucoraphanin. Measurement of AITC formation provides a novel, reliable estimation of myrosinase-dependent isothiocyanate formation suitable for use with whole vegetable food samples.

Broccoli is rich in bioactive components, such as sulforaphane and indole-3-carbinol, which may impact cancer risk. The glucosinolate profile of broccoli can be manipulated through treatment with the plant stress hormone methyl jasmonate (MeJA). Our objective was to produce broccoli with enhanced levels of indole glucosinolates and determine its impact on prostate carcinogenesis. Brassica oleracea var. Green Magic was treated with a 250 μM MeJA solution 4 days prior to harvest. MeJA-treated broccoli had significantly increased levels of glucobrassicin, neoglucobrassicin, and gluconasturtiin (P < .05). Male transgenic adenocarcinoma of mouse prostate (TRAMP) mice (n = 99) were randomized into three diet groups at 5-7 weeks of age: AIN-93G control, 10% standard broccoli powder, or 10% MeJA broccoli powder. Diets were fed throughout the study until termination at 20 weeks of age. Hepatic CYP1A was induced with MeJA broccoli powder feeding, indicating biological activity of the indole glucosinolates. Following ∼ 15 weeks on diets, neither of the broccoli treatments significantly altered genitourinary tract weight, pathologic score, or metastasis incidence, indicating that broccoli powder at 10% of the diet was ineffective at reducing prostate carcinogenesis in the TRAMP model. Whereas broccoli powder feeding had no effect in this model of prostate cancer, our work demonstrates the feasibility of employing plant stress hormones exogenously to stimulate changes in phytochemical profiles, an approach that may be useful for optimizing bioactive component patterns in foods for chronic-disease-prevention studies.

Camelina sativa L. Crantz is under development as a novel oil seed crop yet there are few studies about the bioefficacy of camelina. Camelina seed meal contains several potentially bioactive compou...

Cruciferous vegetables contain secondary metabolites termed glucosinolates that break down to products that upregulate hepatic detoxification enzymes. We have previously shown that a mixture of four major glucosinolate breakdown products from Brussels sprouts interact to produce synergistic induction of phase II detoxification enzymes. Here we tested the hypothesis that this synergism is at the level of transcription and is due to the interaction between the oral bifunctional inducer, indole-3-carbinol (I3C), and monofunctional inducer, crambene (1-cyano 2-hydroxy 3-butene). Adult male rats were treated by gavage with either corn oil (vehicle); crambene (50 mg/kg), I3C (56 mg/kg), or a mix of crambene and I3C at the doses shown. Given orally, I3C alone and crambene with I3C caused significant induction of CYP1A activity and CYP1A1 mRNA levels, whereas crambene alone had no significant effect on CYP1A activity or mRNA levels. Crambene and I3C individually caused induction of glutathione S-transferase (GST) and quinone reductase (QR) activity. The mixture of crambene and I3C caused induction of GST and QR that was significantly greater than the sum of the induction by individual treatments. Upregulation of total GST activity was not as great as that of QR, possibly because some subunits did not show this effect. GST Ya2 mRNA showed a synergistic upregulation by crambene and I3C, while Yc1 and Yc2 showed only an additive response. We speculate that this different regulation is partly due to differences in gene sequences within the antioxidant response element and xenobiotic response element in the regulatory region of GST Ya2 compared to those within the regulatory region of the Yc1/Yc2 subunits.

We investigated the effects of estrogen on global myocardial ischemia-reperfusion injury in rats that were ovariectomized (Ovx), sham-operated, or ovariectomized and then given 17β-estradiol (E2β) supplementation (Ovx+E2β). Hearts were excised, cannulated, perfused with and then immersed in chilled (4°C) cardioplegia solution for 30 min, and then retrogradely perfused with warm (37°C), oxygenated Krebs-Henseleit bicarbonate buffer for 120 min. The coronary flow rate, first derivative of left ventricular pressure, and nitrite production were all significantly lower in Ovx than in sham-operated or Ovx+E2β hearts. However, coronary flow rates or nitrate production were not consistently different throughout the entire reperfusion period. Ca2+accumulated more in Ovx rat hearts than in sham-operated or Ovx+E2β hearts, and mitochondrial respiratory function was lower in Ovx hearts than in hearts from the other two groups. Marked interstitial edema and contraction bands were seen in hematoxylin-eosin-stained sections of Ovx rat hearts but not in hearts from either of the other groups. Hematoxylin-basic fuchsin-picric acid-stained sections revealed fewer viable myocytes in hearts from the Ovx group than from the sham or Ovx+E2β group. Transmission electron microscopy demonstrated more severely damaged mitochondria and ultrastructural damage to myocytes in Ovx rat hearts. Our results indicate that estrogen plays a cardioprotective role in global myocardial ischemia-reperfusion injury in female rats.

CYP2E1 has been reported to have an essential role in alcohol-mediated increases in hepatic steatosis and acetaminophen hepatotoxicity. We found that pretreatment of Cyp2e1(-/-) mice with ethanol plus isopentanol, the predominant alcohols in alcoholic beverages, for 7 days resulted in micro- and macrovesicular steatosis in the livers of all mice, as well as a dramatic increase in acetaminophen hepatotoxicity. In Cyp2e1(-/-) mice administered up to 600 mg acetaminophen/kg alone and euthanized 7 h later, there was no increase in serum levels of ALT. In Cyp2e1(-/-) mice pretreated with ethanol and isopentanol, subsequent exposure to 400 or 600 mg acetaminophen/kg resulted in centrilobular necrosis in all mice with maximal elevation in serum levels of ALT. Acetaminophen-mediated liver damage was similar in males and females. Hepatic microsomal levels of APAP activation in untreated females were similar to those in males treated with the alcohols. However, the females, like the males, required pretreatment with the alcohols in order to increase APAP hepatotoxicity. These findings suggest that, in the Cyp2e1(-/-) mice, the alcohol-mediated increase in acetaminophen hepatotoxicity involves the contribution of other factors, in addition to induction of CYP(s) that activate acetaminophen. Alternatively, CYP-mediated activation of acetaminophen measured in vitro may not reflect the actual activity in vivo. Our findings that a 7-day treatment with ethanol and isopentanol causes extensive hepatic steatosis and increases acetaminophen hepatotoxicity in Cyp2e(-/-) mice indicate that CYP2E1 is not essential for either response.

Ethanol and isopentanol are the predominant alcohols in alcoholic beverages. We have reported previously that pretreatment of rats with a liquid diet containing 6.3% ethanol plus 0.5% isopentanol for 7 days results in a synergistic increase in acetaminophen hepatotoxicity, compared with rats treated with either alcohol alone. Here, we investigated the role of CYP3A in acetaminophen hepatotoxicity associated with the combined alcohol treatment. Triacetyloleandomycin, a specific inhibitor of CYP3A, protected rats pretreated with ethanol along with isopentanol from acetaminophen hepatotoxicity. At both 0.25 and 0.5 g acetaminophen/kg, triacetyloleandomycin partially prevented elevations in serum levels of alanine aminotransferase. At 0.25 g acetaminophen/kg, triacetyloleandomycin completely protected 6 of 8 rats from histologically observed liver damage, and partially protected the remaining 2 rats. At 0.5 g acetaminophen/kg, triacetyloleandomycin decreased histologically observed liver damage in 7 of 15 rats. In rats pretreated with ethanol plus isopentanol, CYP3A, measured immunohistochemically, was decreased by acetaminophen treatment. This effect was prevented by triacetyloleandomycin. These results suggest that CYP3A has a major role in acetaminophen hepatotoxicity in animals administered the combined alcohol treatment. We also found that exposure to ethanol along with 0.1% isopentanol for only 3 days resulted in maximal increases in acetaminophen hepatotoxicity by the combined alcohol treatment, suggesting that short-term consumption of alcoholic beverages rich in isopentanol may be a risk for developing liver damage from acetaminophen.

The cytosolic glutathione S-transferases (GSTs) are a family of phase II detoxifying isoenzymes that catalyze the interaction of the tripeptide thiol glutathione (GSH) with a wide variety of reactive and often toxic or carcinogenic electrophilic substrates. Pancreatic GSTs, however, have only been partially characterized. In this study, pancreatic cytosolic GSTs from male Fisher 344 rats were semipurified by affinity chromatography and then analyzed for isoenzyme content by chromatofocusing (fast protein liquid chromatography) and for subunit content by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography. In addition, polyclonal rabbit antisera were produced against homodimeric isoenzymes purified from rat liver and kidney, including the alpha class isoenzymes 1-1 and 2-2, the mu class isoenzyme 4-4, and the pi class isoenzyme 7-7. These antisera were used in immunohistochemical (IHC) studies of the distribution of the pancreatic GSTs. A range of 0.5-1.6% of the total protein in rat pancreatic cytosol was found to be GST protein. The most abundant subunits present were the pi subunit 7 and mu subunits 3 and 4. Using modified methodology, smaller amounts of the alpha subunit 2 and the mu subunit 6 were detected, whereas very small amounts of the alpha subunits 1 and 8 were present. The IHC demonstrated that the GSTs were in large part limited to the duct system of the exocrine pancreas, with positive staining of endothelial cells and stroma observed for the alpha and mu subunits. Isoenzymes containing the alpha subunit 2 were preferentially expressed in centroacinar cells and small ductules, whereas those containing the mu subunit 4 and the pi subunit 7 were more prevalent within larger ductules and ducts. The lumens of the largest ducts also contained the two subunits 4 and 7. It is concluded that the acinar cells of the exocrine pancreas may lack the protection against electrophilic toxic and carcinogenic agents provided by the ductular system by GSTs.

This commentary focuses on the roles of CYP3A and CYP2E in alcohol-mediated increases in acetaminophen hepatotoxicity. CYP2E has been considered to be the main form of P450 responsible for such toxicity in animals and humans. However, CYP3A, which is also induced by alcohol, has been shown to have a greater affinity for acetaminophen than CYP2E. Previous experiments implicating CYP2E in alcohol-mediated increases in acetaminophen hepatotoxicity have used inhibitors of this form of P450 that are now proving to be non-specific. Triacetyloleandomycin (TAO) is a potent inhibitor of CYP3A that maintains specificity in vitro over a large concentration range. In rats treated with ethanol or the combination of ethanol and isopentanol, the major higher chain alcohol in alcoholic beverages, TAO protects animals from increases in acetaminophen hepatotoxicity, suggesting a major role of CYP3A. CYP2E may not have a major role due to the rapid loss of induced levels in the absence of continued exposure to ethanol. Knockout mice, which are being used to define the role of particular proteins in biological responses, have been developed for CYP2E1 and CYP1A2 but not CYP3A. Cyp2e1(-/-) and Cyp1a2(-/-) mice are more resistant to acetaminophen hepatotoxicity than wild-type strains, even though the amounts of the other forms of P450s are unaltered in the liver. These findings suggest that the relative amounts of P450s and not just kinetic characteristics determine their role in acetaminophen hepatotoxicity. The clinical implications of the findings that CYP3A can have a major role in acetaminophen-mediated hepatotoxicity are discussed.

Our objective was to determine whether steam blanching, storage and preparation affected concentrations of sulforaphane (SF), sulforaphane nitrile (SFN), cyanohydroxybutene (CHB), iberin (I) or iberin nitrile (IN) in fresh and frozen broccoli. Broccoli (var. “Arcadia”) was grown in St. Charles, IL over three seasons. Samples were steam blanched (2 min at 93 ± 5°C) within 24h of harvest, frozen and stored at −20°C up to 90 days, and fresh broccoli was stored at 4°C up to 21 days. Samples were analyzed uncooked or microwave cooked. SF, SFN, I, IN and CHB were determined by GC in dichloromethane extracts from lyophilized samples. Rates of loss for CHB and SF were similar during storage of fresh broccoli. Blanching, storage, and microwave cooking decreased (p < 0.01) concentrations of each compound in fresh and frozen broccoli.

CYP2E is considered the only form of cytochrome P450 responsible for ethanol-mediated increases in acetaminophen hepatotoxicity. However, in experimental systems used for investigating ethanol-mediated increases in acetaminophen hepatotoxicity, animals are withdrawn from ethanol for 16 to 24 hr before the administration of acetaminophen to ensure the clearance of ethanol from the circulation. In rats, CYP2E has been shown to decrease to control levels after this time period of withdrawal from ethanol. We have previously shown in cultured human and rat hepatocytes, and in intact rats, that ethanol induces CYP3A in addition to CYP2E. To determine if there might be a role for CYP3A in ethanol-mediated APAP hepatotoxicity in addition to the recognized role for CYP2E, we investigated the effect of triacetyloleandomycin (TAO) on acetaminophen hepatotoxicity in ethanol-pretreated rats, as well as the effect of 11 hr withdrawal from ethanol on hepatic levels of CYP3A and CYP2E. TAO was dissolved in saline instead of dimethylsulfoxide, the solvent most usually employed, since dimethylsulfoxide inhibits CYP2E. Rats were administered 6.3% ethanol as part of the Lieber-DeCarli diet for 7 days, followed by replacement of the liquid diet with water for 11 hr. This 11-hr withdrawal from ethanol resulted in a decrease in hepatic levels of ethanol-induced CYP2E; however, considerable induction was still evident. There was no significant decrease in CYP3A. TAO completely prevented the histologically observed liver damage from acetaminophen in ethanol-pretreated rats, but did not prevent the increase in serum levels of AST. In ethanol-pretreated rats, exposure to APAP in the absence of TAO was associated with a 75% decrease in CYP3A, compared to animals exposed to APAP in the presence of TAO. These results suggest that CYP3A may have been suicidally inactivated by acetaminophen in the absence of TAO. Our findings suggest that CYP3A has a major role in ethanol-mediated increases in acetaminophen hepatotoxicity.

Spray treatment of methyl jasmonate (MeJA) has been shown to increase glucosinolate (GS) concentrations and health-promoting activity in Brassica vegetables. Since there is no reported standardized protocol, several MeJA treatment studies have been conducted to maximize human health bioactivity using the F1 broccoli cultivar 'Green Magic'.Foliar MeJA application 4 days prior to harvest of broccoli at commercial maturity resulted in enhanced total GS concentrations. Although a single application of 250 µmol L(-1) MeJA maximized GS concentrations in broccoli florets, two days of consecutive treatments (4 and 3 days prior to harvest) of 250 µmol L(-1) MeJA further enhanced neoglucobrassicin concentrations and floret extract quinone reductase (QR)-inducing activity. With increasing concentrations of MeJA in spray applications to broccoli florets, concentrations of the glucosinolates glucoraphanin, gluconasturtiin and neoglucobrassicin and the isothiocyanate sulforaphane as well as anticancer and anti-inflammatory bioactivities as measured by QR induction and inhibition of nitric oxide (NO) production respectively were significantly increased. Concentrations of these phytochemicals showed strong positive correlations with QR-inducing and NO-inhibitory activities.These application protocols were found to maximize GS and GS hydrolysis product concentrations and putatively enhance the health-promoting properties of broccoli heads for consumers.

The in vivo bioavailability of the flavone-C-glycosides has been little studied compared to their O-glycoside analogues, which are both more common in nature and considered more easily hydrolyzed than C-glycosides, by both enterocytes and gut microbiota. In this study, we used vitexin-2-O-xyloside (VOX), an apigenin-8-C-glucoside-2-O-xyloside, purified from seeds of Swiss chard (Beta vulgaris cicla), to investigate VOX absorption into portal blood compared to its aglycone, apigenin. We used a rat model in which we ligated the ileo- and colo-caecal junctions, then administered apigenin or VOX directly into the caecum. Blood samples were drawn from the portal vein at timed intervals over 40 min. The kinetic profile of appearance in portal blood of the compounds and their metabolites was evaluated by HPLC-ESI-MS. Apigenin was found in portal blood both as the aglycone and as an apigenin-glucuronide derivative. The VOX was found unchanged and as a reduced monoglycoside, which underwent glucuronidation. By collecting the bile, we confirmed that the liver received unchanged VOX, which was returned to the gut by enterohepatic recirculation for reabsorption from the ileum. The amount of apigenin and VOX remaining in the caecum accounted for ∼15% and ∼26%, respectively. These data show for the first time that the C-glycoside VOX is absorbed unchanged and undergoes enterohepatic recirculation in addition to hydrolysis to the monoglycoside, reduction and conjugation to form a bioavailable glucuronide.

Methyl jasmonate (MeJA) treatment can significantly increase glucosinolate (GS) concentrations in Brassica vegetables and potentially enhance anticancer bioactivity. Although MeJA treatment may promote ethylene biosynthesis, which can be detrimental to postharvest quality, there are no previous reports of its effect on cauliflower postharvest quality. To address this, cauliflower curds in field plots were sprayed with either 0.1 % Triton X-100 (control) or 500 μM MeJA solutions four days prior to harvest, then stored at 4 °C. Tissue subsamples were collected after 0, 10, 20, and 30 days of postharvest storage and assayed for visual color change, ethylene production, GS concentrations, and extract quinone reductase inductive activity. MeJA treatment increased curd GS concentrations of glucoraphanin, glucobrassicin, and neoglucobrassicin by 1.5, 2.4, and 4.6-fold over controls, respectively. MeJA treated cauliflower showed significantly higher quinone reductase activity, a biomarker for anticancer bioactivity, without reducing visual color and postharvest quality for 10 days at 4 °C storage.

Although the full mechanisms are not yet elucidated, research into the mechanism of toxicity of aluminum (Al) on bone formation and remodeling and on hematopoietic tissue is ongoing. In contrast little information exists on the interactive effects of systemic Al and the kidney. In bone, both clinically and experimentally, high doses of Al inhibit remodeling, slowing both osteoblast and osteoclast activities and producing osteomalacia and adynamic bone disease. In contrast, while very low levels of Al are mitogenic in bones of experimental animals, the effect of low levels of Al in humans is unknown. Aluminum has been shown to have its mitogenic action at the osteoblast, but whether the effect on resorption is viz osteoblast-directed changes in osteoclast activity has not yet been determined. Parathyroid hormone (PTH) levels are disrupted by Al in humans and animals. Whether altered PTH levels play a major or even a minor role in Al-dependent osteotoxicity requires clarification. In hematopoietic tissue, Al causes a microcytic anemia, not reversible by iron. Friend leukemia cells treated with Al have been reported to accumulate excess iron, without incorporating it into ferritin or heme. It is not yet known which steps in iron metabolism are disrupted by Al, if they involve a single mechanism of action, or even if this disruption in iron metabolism accounts for the anemia seen in Al toxicosis. In kidney, research is needed to evaluate Al nephrotoxicity; there are almost no studies in this area. Furthermore, research is needed to evaluate mechanisms of renal Al excretion, presently shown by one study to occur at the distal tubule. Such studies might well throw light on whether Al plays a role in aggravating renal insufficiency, or whether the role of the kidney in Al toxicosis is limited to the causative effect of renal compromise on Al accumulation. In summary, while a number of mechanisms have been proposed for the toxic action of Al, no single mechanism emerges to explain these diverse effects of systemic Al. Recommendations for future research are presented and summarized in Table 1.

Effect of pre-harvest methyl jasmonate (MeJA) and post-harvest 1-methylcyclopropene (1-MCP) treatments on broccoli floret glucosinolate (GS) concentrations and quinone reductase (QR, an in vitro anti-cancer biomarker) inducing activity were evaluated two days prior to harvest, at harvest and at 10, 20, and 30 days of post-harvest storage at 4 °C. MeJA treatments four days prior to harvest of broccoli heads was observed to significantly increase floret ethylene biosynthesis resulting in chlorophyll catabolism during post-harvest storage and reduced product quality. Post-harvest treatment with 1-methylcyclopropene (1-MCP), which competitively binds to protein ethylene receptors, maintained post-harvest floret chlorophyll concentrations and product visual quality in both control and MeJA-treated broccoli. Transcript abundance of BoPPH, a gene which is responsible for the synthesis of pheophytinase, the primary enzyme associated with chlorophyll catabolism in broccoli, was reduced by 1-MCP treatment and showed a significant, negative correlation with floret chlorophyll concentrations. The GS, glucobrassicin, neoglucobrassicin, and gluconasturtiin were significantly increased by MeJA treatments. The products of some of the GS from endogenous myrosinase hydrolysis [sulforaphane (SF), neoascorbigen (NeoASG), N-methoxyindole-3-carbinol (NI3C), and phenethyl isothiocyanate (PEITC)] were also quantified and found to be significantly correlated with QR. Sulforaphane, the isothiocyanate hydrolysis product of the GS glucoraphanin, was found to be the most potent QR induction agent. Increased sulforaphane formation from the hydrolysis of glucoraphanin was associated with up-regulated gene expression of myrosinase (BoMyo) and the myrosinase enzyme co-factor gene, epithiospecifier modifier1 (BoESM1). This study demonstrates the combined treatment of MeJA and 1-MCP increased QR activity without post-harvest quality loss.

Acetaminophen (APAP) hepatotoxicity was investigated in rats fed ethanol and isopentanol alone or in combination in a liquid diet for 7 days. Serum levels of aspartate aminotransferase (AST) and histological examination of liver slices were used to assess hepatotoxicity. At 7 hr after intragastric administration of 0.5 or 1.0 g APAP/kg, there was no significant increase in serum levels of AST in rats treated with APAP alone, or in rats pretreated with ethanol or isopentanol alone followed by APAP. There was mild central lobular congestion in the livers of rats pretreated with ethanol alone followed by APAP. In contrast, in rats pretreated with the combination of ethanol and isopentanol, administration of APAP caused a dramatic increase in serum levels of AST, along with marked central lobular necrosis, including steatosis and ischemic changes. Hepatic glutathione levels were decreased to 40-50% of control values in APAP-treated rats that had been pretreated with ethanol either alone or in combination with isopentanol. The serum concentrations of APAP were significantly lower in rats pretreated with the combination of ethanol and isopentanol followed by 1 g APAP/kg than in rats treated with APAP alone, suggesting a greater rate of APAP metabolism. We had reported previously that combined treatment of rats with ethanol and isopentanol resulted in additive to synergistic increases in CYP3A, with no further increases in CYP2E than that caused by ethanol alone. CYP3A may, therefore, be responsible for the increased APAP hepatotoxicity caused by the combined alcohol treatment.

Gymnaster koraiensis (GK) is a Korean herb used in folk medicine and recently found to positively impact liver health. Because GK contains polyacetylenes and because other polyacetylenes have been found to exhibit anti-inflammatory and anti-cancer activities, we hypothesized that the polyacetylene gymnasterkoreayne B (GKB), known to increase hepatic detoxification enzymes, may also exert anti-inflammatory and anti-cancer activities. We fed male C57BL/6 mice purified AIN93G diets containing no additions, GKB or the GKB-rich fraction of an ethanol extract (GE) from GK to determine if these diets would slow or prevent either inflammation or inflammation-enhanced colon cancer, using the dextran sulfate sodium/azoxymethane mouse model. The GKB (500 μmol/kg diet daily) showed some anti-inflammatory activity, but GE, containing an equal dose of GKB, protected strongly against both inflammation and colon cancer, decreasing adenocarcinomas by 90%. These data support further research into GK as a promising potential anti-cancer agent.

Glucosinolates and their breakdown products (nitriles) have long been implicated as toxic factors when feeding rapeseed (Brassica napus) meals and crambe (Crambe abyssinica) meals to poultry. Accordingly, various methods have been developed to remove these compounds from the meals to enhance their value as feed supplements. Glucosinolates and nitriles were extracted from commercially processed, defatted crambe meal by washing with water or various solvent-water mixtures: 50% isopropanol, 50% acetone, or 50% ethanol. In addition, crambe seed was extruded and extracted in the laboratory with isopropanol or hexane. Water washing of commercially defatted meal proved to be the most effective method of extraction, removing 95% of the glucosinolates and nitriles. Meals were fed to 7-d-old broiler chicks at 10% of the diet for 14 d. Weight gain decreased (P < .05) in most groups; however a greater decrease (P < .01) was observed in birds fed meals with high glucosinolate content. Feed intake also decreased (P < .05) in most groups; consequently, feed efficiencies were similar for all groups. No changes in serum chemistries, triiodothyronine, thyroxine, or tissue lesions were associated with glucosinolate or nitrile intake. A relationship (P < .05, r = .74) was found between weight gain and glucosinolate intake. No correlation was found between feed intake and meal glucosinolate or nitrile concentrations.

The time- and dose-dependent accumulation of aluminum (Al) in liver and its effect on several hepatic systems were investigated in female Swiss-Webster mice given intraperitoneal (ip) Al lactate. A single dose of 50, 75, or 100 mg Al/kg produced significant depletion of hepatic glutathione (GSH) and cytochrome P450 (P450) and an increase in heme oxygenase (HO) activity when measured at 6 hr. However, no increase in hepatic malondialdehyde (MDA) was seen at this time. Using a dose of 100 mg Al/kg, Al rapidly accumulated in liver (0.29 +/- 0.03 vs 0.12 +/- 0.04 micrograms Al/mg protein at 6 hr). Hepatic GSH was significantly depleted at 2, 4, and 6 hr postdosing, HO activity was significantly increased at 6, 12, 24, 48, and 96 hr postdosing, hepatic cytochrome P450 was decreased at 12, 18, 24, and 48 hr postdosing, while hepatic MDA was not significantly elevated until 24 hr postdosing. Twenty-four hours following three daily doses of 50 mg Al/kg, HO activity was increased, P450 levels were depleted, and hepatic Al content was significantly increased, although hepatic MDA was unaffected. We conclude that parenteral Al produces a course of hepatic damage that begins with GSH depletion and proceeds through a sustained increase in HO activity, with cytochrome P450 loss being an early event and production of hepatic MDA being a late event.

The bacterium Salmonella typhimurium TA98 (TA98) is commonly used in the Ames test to detect frameshift mutations. Published Ames test results for fluoride mutagenicity are inconsistent. We considered that this might be due to an unnoticed variable in experimental conditions. Al and F, both present in finished drinking water, form a stable complex. The effect of Al and F uptake by TA98 cells was evaluated and any effect of Al on Ames test results for F was also determined. A known number of TA98 cells in 0.1 M potassium phosphate buffer (PPB, pH 7.4) was incubated with buffer only, and various concentrations of Al as AlCl3, F as NaF, or aluminum and fluoride (Al: F, 1: 62.5 w/w) for 1 h at 37 degrees C in a shaking incubator. Neither F (19 and 190 ppm), Al (0.3 and 3 ppm), nor Al:F (7.6 ppb-3 ppm: 0.45-190 ppm) was found to be mutagenic as assessed by a modification of the Ames standard plate incorporation assay. Intracellular Al accumulated in a concentration-dependent manner from 0.5 to 4.5 ppm, then decreased as Al was increased to 9.5 ppm. Intracellular F was below the limit of detection (0.2 ppm) even when the medium contained 589 ppm F. However, F was taken up from media containing greater than 200 ppm F, providing that aluminum was also present. Experiments carried out using N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer (100 mM, pH 7.4) gave similar results for F uptake, suggesting the Al requirement for F uptake is not an artifact caused by buffer chelation. However, Al uptake was not biphasic from HEPES buffer, but increased with dose over the range studied (1.5-9.0 ppm). While F did not appear to cause mutations in the presence or absence of Al, it was concluded that the mutagenicity test, using TA98, is not suitable for the evaluation of F mutagenicity.

Antioxidant foods and ingredients are an important component of the food industry. In the past, antioxidants were used primarily to control oxidation and retard spoilage, but today many are used because of putative health benefits. However, the traditional message that oxidative stress, which involves the production of reactive oxygen species (ROS), is the basis for chronic diseases and aging is being reexamined. Accumulating evidence suggests that ROS exert essential metabolic functions and that removal of too many ROS can upset cell signaling pathways and actually increase the risk of chronic disease. It is imperative that the food industry be aware of progress in this field to present the science relative to foods in a forthright and clear manner. This may mean reexamining the health implications of adding large amounts of antioxidants to foods.

The glucosinolate hydrolysis product 1-isothiocyanato-3-(methylsulfinyl)-propane (IMSP), also known as iberin, is consumed in the average human (US) diet at approximately 1 mumol/kg/day. The chemoprotective effects observed with the consumption of cruciferous vegetables may be due to the presence of specific glucosinolate hydrolysis products either within the crucifers, or formed after ingestion of the crucifers. The mechanism of chemoprotection may be through selective induction of components of Phase II xenobiotic metabolizing enzymes. The influence of repeated administration of low concentrations of IMSP by gavage on components of Phase I and Phase II xenobiotic metabolizing systems was examined in the liver and small intestine of male Fischer 344 rats. Doses of 1, 10 and 100 mumol IMSP/kg, administered by gavage for 7 days, did not alter weight gain, or hepatic and renal weights, relative to body weight, and did not cause any histological lesions. Intestinal glutathione S-transferase (GST) activity and NAD(P)H:quinone reductase (QR) activities were significantly elevated to 3.1 and 8.1 times control values, respectively, at the 100 mumol/kg dose only. The administration of IMSP at 1, 10 or 100 mumol/kg had no significant effect on hepatic Phase I enzymes activities (cytochrome P-450 concentrations, ethoxycoumarin O-deethylase [ECD] and aminopyrine N-demethylase [AND] activities) or Phase II enzyme activities (GST, QR and UDP-glucuronosyltransferase [UDP-GT] activities towards 1-naphthol or 4-hydroxybiphenyl), at any of the doses tested and no effect on intestinal enzyme activities at doses below 100 mumol IMSP/kg. It is concluded that IMSP does not have a significant influence on induction of the Phase I or Phase II xenobiotic metabolizing enzymes in rats when tested at doses approximating those found in the human diet.

The combination of caffeine with acetaminophen (APAP) is used widely in the treatment of headache. The effects of caffeine on APAP-induced hepatotoxicity and APAP bioactivation by liver microsomes from uninduced mice and from mice pretreated with various agents that induce cytochrome P450 were studied. When 1 mM caffeine was included, the rate of glutathione-APAP conjugate (GS-APAP) formation was increased significantly by 33 and 39% in microsomes from phenobarbital (PB)- and dexamethasone (DEX)-treated mice, respectively, whereas this parameter was decreased 39 and 12% by caffeine in microsomes from beta-naphthoflavone (beta NF)- and acetone-treated mice, respectively. A 5 mM concentration of caffeine increased GS-APAP formation by 47, 107 and 117% in microsomes from control, PB-, and DEX-treated mice, respectively, and decreased it 39 and 25% in microsomes from beta NF- and acetone-treated mice, respectively. Caffeine was a competitive inhibitor of APAP bioactivation in microsomes from beta NF- and acetone-treated mice. While caffeine increased APAP bioactivation in microsomes from uninduced, PB-, and DEX-treated mice, the apparent Km values for APAP were increased by caffeine, indicating that this enhancement was not due to a direct effect of caffeine on APAP binding to cytochrome P450 but may be due to an effect of caffeine on the substrate-enzyme complex. The variable effect of caffeine on APAP hepatotoxicity correlated with the effect of caffeine on APAP bioactivation by liver microsomes, regardless of pretreatment. Lack of correlation of aminopyrine N-demethylase, but good correlation of erythromycin N-demethylase activity with the extent of caffeine enhancement of APAP bioactivation following PB or DEX treatment suggests that a murine P450 subfamily similar to the rat P450 3A subfamily may be the candidate in mediating the stimulatory effect of caffeine on APAP bioactivation and APAP-induced hepatotoxicity.

Aging is associated with oxidative stress and heightened inflammatory response to infection. Dietary interventions to reduce these changes are therefore desirable. Broccoli contains glucoraphanin, which is converted to sulforaphane (SFN) during digestion. SFN increases antioxidants including NAD (P) H quinone oxidoreductase (NQO1) and inhibits inflammatory cytokines. We hypothesized that dietary broccoli would support an antioxidant response in brain and periphery and inhibit LPS-induced inflammation and sickness. Adult and aged mice were fed control or 10% broccoli diet for 4 wk prior to an LPS injection. Social interactions were assessed 2, 4, 8, and 24 h following LPS, and mRNA quantified in liver and brain at 24 h. Dietary broccoli did not prevent LPS-induced decrease in social interactions in young or aged mice. Interleukin (IL)-1β expression was unaffected by broccoli consumption and was induced by LPS in brain and liver of adult and aged mice (p

Cyanohydroxybutene (CHB) is reported to be hepatotoxic in male Fischer 344 rats at an oral dose of 300 mg/kg and, while no longer hepatotoxic, pancreatotoxic at 200 mg/kg. In addition, the 200 mg/kg dose causes a persistent elevation in hepatic and pancreatic glutathione (GSH). This study was conducted to determine if smaller doses of CHB could cause GSH elevation in the absence of toxicity. A single oral dose of 100 mg/kg or multiple lower doses (50 mg/kg daily for 3 days or 30 mg/kg for 6 days) caused a significant and persistent increase in pancreatic GSH, although hepatic levels were unchanged. Ten milligrams per kilogram, even daily for 24 days, was without effect on hepatic or pancreatic GSH. Neither a single oral dose of 100 mg/kg nor multiple lower doses were associated with toxicity. However, when either 100 or 50 mg/kg were administered intravenously, pancreatic apoptosis was observed. In animals dosed with 100 mg/kg iv, mixed histiocytic and suppurative inflammation and frank pancreatic necrosis also developed and were associated with elevated plasma lipase and amylase. The animals receiving CHB intravenously also exhibited elevated GSH levels in both pancreas and liver. This study shows that oral doses between 30 and 100 mg CHB/kg can be used to elevate GSH levels without any pancreatotoxicity. However, a single 50 mg CHB/kg dose given intravenously causes apoptosis, while 100 mg/kg causes severe pancreatotoxicity with necrosis.