Induction of human NAD(P)H:quinone oxidoreductase (NQO1) gene expression by the flavonol quercetin.

Flavonoids are plant polyphenolic compounds ubiquitous in fruits, vegetables and herbs. The flavonol quercetin is one of the most abundant dietary flavonoids. It has diverse biological properties in cultured cells, including cytoprotection, and exhibits antitumorigenic effects in animal models. The mechanism(s) for the protective properties of flavonoids are currently unknown but may involve modulation of phase II detoxifying enzymes. We have investigated the effect of quercetin on expression and enzymatic activity of one of the major phase II detoxification systems, NAD(P)H:quinone oxidoreductase (NQO1) in the MCF-7 human breast carcinoma cell line. We show that treatment of MCF-7 cells for 24 h with 15 microM quercetin results in a twofold increase in NQO1 protein levels and enzyme activity, and a three- to fourfold increase in NQO1 mRNA expression. We found that when these cells were transiently transfected with a luciferase (Luc) reporter plasmid containing two copies of the antioxidant response element (ARE) of the human NQO1 gene linked to a minimal viral promoter, quercetin caused an approximately twofold increase in Luc activity. Quercetin failed to increase Luc activity in cells transfected with a reporter vector containing a mutated ARE. The increase in NQO1 transcription in response to quercetin suggests that dietary plant polyphenols can stimulate transcription of phase II detoxifying systems, potentially through an ARE-dependent mechanism. Induction of the human NQO1 gene by dietary polyphenolics could afford protection against carcinogenic chemicals in molecular pathways utilizing the ARE.

Characterization of hepatic iron overload following dietary administration of dicyclopentadienyl iron (Ferrocene) to mice: cellular, biochemical, and molecular aspects.

A unique organic form of iron (dicyclopentadienyl iron; ferrocene) has been used to further elucidate specific hepatic histopathologic, biochemical, and molecular parameters associated with dietary iron overload. Male C57BL/6Ibg mice fed a diet containing 0.04-0.2% w/w ferrocene for 115 days displayed severe hepatic siderosis of hepatocytes accompanied by a 15-fold induction of nonheme iron content compared to control mice receiving a diet with normal amounts of iron. The ferrocene treatment led to significant increases in hepatocellular necrosis as measured by plasma alanine aminotransferase activity. Histological assessment of hepatic fibrosis revealed mild increases in collagen deposition localized with accumulations of hemosiderin primarily in centrilobular hepatocytes. Hepatic fibrosis was confirmed by measurement of hepatic hydroxyproline content that was increased 4-fold in ferrocene-fed animals compared to control animals not ingesting ferrocene. Hepatic siderosis was accompanied by significant increases in hepatic malondialdehyde content suggesting the ferrocene-induced iron burden initiated lipid peroxidation in vivo. Expression of the heavy-chain isoform of ferritin mRNA and protein measured in liver after ferrocene feeding was increased approximately 8- and 2-fold, respectively, compared to the appropriate controls. These results, using an organic form of iron fed to genetically well-characterized inbred mice, provide new additional insight into the specific molecular and biochemical events that occur in association with histopathologic changes initiated by iron-induced liver injury. These data support the hypothesis that peroxidation of cellular membrane lipids is an important mechanism involved in the toxicity of excess hepatic iron and possibly the initiation of liver fibrogenesis. The results presented here also provide novel in vivo evidence documenting the cellular modulation of ferritin in response to the toxic effects of hepatic iron overloading and iron-mediated oxidative stress.

Formation of liver microsomal MDA-protein adducts in mice with chronic dietary iron overload.

Lipid peroxidation has been proposed to be a major mechanism involved in the pathophysiology of hepatic iron overload. Hepatic microsomal lipid peroxidation has been demonstrated in animals with dietary iron overload, and major products of lipid peroxidation with known cytotoxicity, such as malondialdehyde (MDA), may be involved in iron-induced hepatocellular injury by covalent binding to microsomal proteins. This investigation examined whether DBA/2Ibg mice fed a diet enriched with ferrocene-iron for 16 weeks, results in hepatic lipid peroxidation, and if liver microsomes contain proteins adducted by MDA. Chronic iron feeding to mice resulted in a severe hepatic iron overload with hepatic stores of iron 12-fold greater than those measured in control mice and a three-fold increase in hepatic concentrations of MDA, indicating the occurrence of iron-induced lipid peroxidation in vivo. Hepatic collagen content was increased by over three-fold (p < 0.05) in iron-fed mice as compared to control animals, suggesting increased fibrogenesis. Using rabbit antiserum specific for MDA amine protein adducts and immunoprecipitation-Western blotting, we documented formation of 10 liver microsomal proteins adducted by MDA in iron overload mice (approximate molecular weights; 214, 140, 129, 121, 103, 83, 62, 60, 48, and 43-kD). Control mice did not exhibit positive immunostaining for these protein adducts. The incubation of synthetic MDA with liver microsomes isolated from untreated mice demonstrated formation of MDA-adducted proteins with molecular weights comparable to those detected following in vivo iron overload. The data from this animal study are the first to demonstrate that lipid-derived aldehydes produced from hepatic iron overload in vivo, covalently bind and hence, chemically modify numerous proteins in microsomes. These data suggest that MDA modified proteins in microsomes may play a role in a sequence of events that lead to cell injury during metal-induced liver damage.

Association of glutathione S-transferase isozyme-specific induction and lipid peroxidation in two inbred strains of mice subjected to chronic dietary iron overload.

The alpha-class glutathione S-transferases are proposed to play a prominent role in catalyzing the conjugation of glutathione with electrophilic aldehydic products of lipid peroxidation. The effect of iron-induced lipid peroxidation on induction of glutathione S-transferase (GST) isozymes A1 and A4 in the livers of male C57/BL6Ibg and DBA/J2Ibg mice was studied. C57 and DBA mice were fed for 4 months on a diet supplemented with iron as ferrocene and then were assessed for liver injury, hepatic iron loading, indices of lipid peroxidation, GST activity, and induction of GST isozymes A1 and A4. Iron-treated animals displayed a loss in body weight from pair-fed controls and had large increases in hepatic non-heme iron with concomitant liver injury, as measured by serum alanine aminotransferase. Hepatic lipid hydroperoxides, a direct measure of oxidized membrane lipids, were significantly increased only in C57 mice, but hepatic concentrations of reduced glutathione (GSH) were significantly increased in both inbred strains. Total GST activity toward 1-chloro-2,4-dinitrobenzene was significantly increased in C57 mice but not in DBA. Western blot studies using polyclonal antibodies specific for GST A1 and A4 revealed significant increases of 1.5-2.0-fold in these GST isoforms in both inbred strains. These results in a unique murine model for hepatic iron overload further support recent in vivo studies (Khan et al., Toxicol. Appl. Pharmacol., 131, 63-72, 1995) that have associated induction of GST A4 with protection against oxidative stress-induced lipid peroxidation. The observed increases in lipid hydroperoxides, hepatic GSH, GST activity, and GST A1 and A4 protein strongly support the hypothesis that induction of GST A1 and A4 represents an important protective event in the detoxification of electrophilic products of lipid peroxidation.

Alcohol mediates increases in hepatic and serum nonheme iron stores in a rat model for alcohol-induced liver injury.

The notion that prolonged ethanol consumption promotes hepatocellular damage through interactions with iron was evaluated in rats fed ethanol with or without supplemental dietary carbonyl iron. The individual and combined pro-oxidant potential of these agents was evaluated in terms of their ability to perturb iron homeostasis and initiate hepatocellular injury. Sprague-Dawely rats received a high fat liquid diet for 8 weeks supplemented with: 35% ethanol-derived calories (Alcohol group), 0.02 to 0.04% (w/v) carbonyl iron (Iron group), ethanol plus carbonyl iron (Alcohol + Iron group), or a diet containing carbohydrate-derived isocaloric calories (Control group). Hepatic and serum nonheme iron stores were significantly elevated (p < 0.05) in all treatment groups, compared with the Controls. Catalytically active low-molecular weight iron was detected in rats consuming alcohol and was markedly elevated (p < 0.05) in rats ingesting iron alone or iron in combination with alcohol. Elevations in serum ALT indicated significant hepatocellular injury in rats ingesting only alcohol, but was most prominent in the rats consuming ethanol in combination with iron (p < 0.05). Significant hepatic fatty infiltration, increased hydroxyproline content, and perturbations in reduced glutathione were also observed in the Alcohol and Iron treatment groups. Histochemical assessment of hepatic iron sequestration revealed that alcohol feeding resulted in deposition of ferric iron in the centrilobular area of the liver lobule. This unique alcohol-mediated iron deposition was histologically graded above Control group and was observed in both hepatocytes and Kupffer cells. Data presented herein suggest that alcohol alone or in combination with iron results in rather specific lobular patterns of hepatic iron deposition relevant to iron overload observed in human alcoholics. Furthermore, data suggest that alcohol- and iron-initiated prefibrotic events occur before extensive hepatocellular necrosis.

The enzymatic formation and chemical reactivity of quinone methides correlate with alkylphenol-induced toxicity in rat hepatocytes.

The effects of o-alkyl substituents on both the cytochrome P450-catalyzed oxidation of phenols to p-quinone methides (QM's; 4-methylene-2,5-cyclohexadien-1-ones), and on the rates of nucleophilic additions to the 4-methylene carbon of QM's were investigated. The derivatives of 4-methylphenol studied were BHT (2,6-di-tert-butyl), BHTOH [6-tert-butyl-2-(hydroxy-tert-butyl)], BDMP (2-tert-butyl-6-methyl), BMP (2-tert-butyl), TMP (2,6-dimethyl), and DMP (2-methyl). QM formation was estimated to be in the range 0.17-0.70 nmol/(nmol of P450.min) in rat liver microsomes and 16-62 pmol/(10(6) cells.min) in isolated rat hepatocytes. QM's derived from BHT (BHT-QM), BHTOH (BHTOH-QM), BDMP (BDMP-QM), and TMP (TMP-QM) were synthesized and their rates of reaction with water and reduced glutathione (GSH) determined. BDMP-QM and TMP-QM were the most reactive, BHT-QM was consumed relatively slowly, and BHTOH-QM displayed intermediate reactivity. These variations in rate were rationalized by differences in hydrogen bonding with the carbonyl oxygen, which affects positive charge density at the site of nucleophilic attack. The loss of hepatocyte viability during incubations with BMP, BDMP, and BHTOH was preceded by GSH depletion. Pretreatment of hepatocytes with diethyl maleate exacerbated alkylphenol toxicity, and metyrapone protected the cells. These data, together with information on the formation and reactivity of QM's, strongly support the proposal that QM's mediate the toxicity of alkylated 4-methylphenols in rat hepatocytes.