metallothionein and 2-tert-butylhydroquinone

Xenobiotic metabolism has been proposed to play a role in modulating the rate of aging. Xenobiotic metabolizing enzymes (XME) are expressed at higher levels in calorically restricted mice (CR) and in GH/IGF-I-deficient, long-lived mutant mice. In this study, we show that many phase I XME genes are similarly upregulated in additional long-lived mouse models, including "crowded litter" (CL) mice, whose lifespan has been increased by food restriction limited to the first 3 wk of life, and in mice treated with rapamycin. Induction in the CL mice lasts at least through 22 mo of age, but induction by rapamycin is transient for many of the mRNAs. Cytochrome P-450s, flavin monooxygenases, hydroxyacid oxidase, and metallothioneins were found to be significantly elevated in similar proportions in each of the models of delayed aging tested, whether these were based on mutation, diet, drug treatment, or transient early intervention. The same pattern of mRNA elevation could be induced by 2 wk of treatment with tert-butylhydroquinone, an oxidative toxin known to activate Nrf2-dependent target genes. These results suggest that elevation of phase I XMEs is a hallmark of long-lived mice and may facilitate screens for agents worth testing in intervention-based lifespan studies.

Topics: Animals; Antioxidants; Caloric Restriction; Cytochrome P-450 Enzyme System; Female; Flavins; Gene Expression Regulation; Hydroquinones; Liver; Longevity; Male; Metallothionein; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Mixed Function Oxygenases; Oxidoreductases; Sirolimus; Up-Regulation; Xenobiotics

Phenolic antioxidants, such as tBHQ [2,5-di-(t-butyl)-1,4-hydroquinone], induce Mt1 (metallothionein 1) gene expression and accumulation of MT protein. Induction of Mt1 mRNA does not depend on protein synthesis, and correlates with oxidation-reduction functions of the antioxidants. In the present study, we analysed the biochemical pathway of the induction. Induction depends on the presence of MTF-1 (metal-activated transcription factor 1), a transcription factor that is required for metal-induced transcription of Mt1, but does not require nuclear factor erythroid 2-related factor 2, a tBHQ-activated CNC bZip (cap 'n' collar basic leucine zipper) protein, that is responsible for regulating genes encoding phase II drug-metabolizing enzymes. Moreover, tBHQ induces the expression of MRE-beta Geo, a reporter gene driven by five metal response elements that constitute an optimal MTF-1 binding site. Reconstitution of Mtf1 -null cells with MTF-1 restores induction by both zinc and tBHQ. Unlike activation of phase II genes by tBHQ, induction of Mt1 expression does not occur in the presence of EDTA, when cells are cultured in zinc-depleted medium, or in cells with reduced intracellular 'free' zinc due to overexpression of ZnT1, a zinc-efflux transporter, indicating that induction requires zinc. In addition, fluorescence imaging reveals that tBHQ increases cytoplasmic free zinc concentration by mobilizing intracellular zinc pools. These findings establish that phenolic antioxidants activate Mt1 transcription by a zinc-dependent mechanism, which involves MTF-1 binding to metal regulator elements in the Mt1 gene promoter.

Topics: Animals; Antioxidants; Carcinoma, Hepatocellular; Cell Line; Cell Line, Tumor; Cricetinae; DNA-Binding Proteins; Embryo, Mammalian; Fibroblasts; Hydroquinones; Kidney; Liver Neoplasms, Experimental; Metallothionein; Mice; NF-E2-Related Factor 2; Oxidation-Reduction; RNA, Messenger; Trans-Activators; Transcription Factor MTF-1; Transcription Factors; Transcription, Genetic; Zinc

To investigate a possible role of phosphorylation in the signal transduction pathways responsible for transcriptional regulation of drug-metabolizing enzymes, we tested seven specific tyrosine kinase inhibitors (tyrphostins) for their effects on NAD(P)H:quinone oxidoreductase-1 (NQO1) mRNA levels in mouse hepatoma Hepa-1c1c7 (Hepa-1) cells and chose to study AG879 further. The potent electrophile tert-butylhydroquinone (tBHQ) is known to activate NQO1 gene transcription via the electrophile response element (EPRE). Among the tyrphostins tested, tyrphostin AG879 was unique in preventing the accumulation of tBHQ-induced NQO1 mRNA; this effect was dependent on the AG879 dose and was also sensitive to the time when AG879 was added relative to the beginning of tBHQ treatment. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin; TCDD) is known to activate Cyp1a1 gene transcription by way of aromatic hydrocarbon response elements (AHREs). We found that AG879 also prevents, to a lesser extent, the AHRE-mediated induction of CYP1A1 and NQO1 mRNA by dioxin. Zinc or cadmium is known to activate metallothionein (Mt1) gene transcription via the metal response element (MRE). AG879 induced MT1 mRNA, and AG879 did not block zinc- or cadmium-induced MT1 mRNA, indicating that the effects of AG879 on NQO1 or CYP1A1 mRNA levels cannot be generalized to all transcripts. Using transient transfection of EPRE-, AHRE-, or MRE-driven luciferase reporter gene constructs in Hepa-1 cells, we showed that the inhibitory effects of AG879 occurred at the level of EPRE- and AHRE-mediated transcription, but that AG879 did not affect the MRE-driven transcriptional response. These data suggest that AG879 might inhibit an unknown tyrosine kinase(s) whose activity is essential for EPRE- and AHRE-mediated trans-activation of certain mammalian genes. These results also indicate that some sharing of common signal transduction pathways might exist in the regulation of genes involved in drug metabolism that also respond to oxidative stress.

Topics: Animals; Cytochrome P-450 CYP1A1; Dioxins; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Induction; Enzyme Inhibitors; Hydroquinones; Metallothionein; Mice; Polychlorinated Dibenzodioxins; Protein-Tyrosine Kinases; Quinone Reductases; RNA, Messenger; Time Factors; Transcriptional Activation; Tumor Cells, Cultured; Tyrphostins

Oxidative stress (tert-butylhydroquinone) rapidly induced metallothionein-I gene expression in mouse Hepa cells, and this effect was mediated predominantly through metal response promoter elements in transient transfection assays. In vivo genomic footprinting of the mouse metallothionein-I promoter after treatment of Hepa cells with hydrogen peroxide, tert-butylhydroquinone, or zinc suggested a rapid increase in occupancy of the metal response elements. More subtle changes also occurred in the constitutive genomic footprint at the composite major late transcription factor/antioxidant response element. This element may, in part, mediate induction by hydrogen peroxide. Electrophoretic mobility shift assays demonstrated a rapid (30 min) increase in the DNA binding activity of metal-responsive transcription factor-1 in Hepa cells treated with any of these inducers. In control cells, upstream stimulatory factor binding with the major late transcription factor site, and a nuclear protein complex distinct from AP-1, but specific for the antioxidant response element, were detected. The amounts of these complexes were not altered after these treatments. These studies indicate that metal-responsive transcription factor-1 plays a role in activating mouse metallothionein-I gene transcription in response to reactive oxygen species.

Topics: Animals; Antioxidants; Base Sequence; Binding Sites; Cell Line; DNA Footprinting; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation; Hydrogen Peroxide; Hydroquinones; Metallothionein; Mice; Molecular Sequence Data; Oxidation-Reduction; Oxidative Stress; Promoter Regions, Genetic; RNA, Messenger; Transcription Factor AP-1; Transcription Factor MTF-1; Transcription Factors; Transcription, Genetic; Transfection; Zinc