Chromium (VI)-induced transformation is enhanced by Zn deficiency in BALB/c 3T3 cells.

Hexavalent chromium [Cr(VI)] is a carcinogenic heavy metal that is reduced to intermediate oxidation states, such as Cr(V) and Cr(IV), in the process of forming stable Cr(III) forms; it is these intermediate forms that are thought to be responsible for much of the DNA damage and mutations that are induced by Cr(VI). Metallothionein (MT), a heavy metal-binding protein, is induced by zinc and other heavy metals and protects cells from the toxic effects of these metals by sequestering them. MT cannot bind Cr, but by scavenging reactive oxygen species through its cysteine residues, it may act as a protective factor against Cr(VI)-induced DNA lesions by reducing Cr(VI) directly to Cr(III), thereby avoiding the creation of the toxic intermediates. Here, we showed that Zn deficiency decreased MT expression in BALB/3T3 clone A31-1-1 cells and caused them to become highly susceptible to Cr(VI)-induced transformation. To obtain Zn-deficient cultures, cells were cultured in medium supplemented with 10% Chelex(®)-100 chelating resin-treated FBS. The increase in susceptibility to transformation was abolished by culturing the cells with supplemental Zn (50 µM). Previously, we reported that Cr(VI) inhibits MT transcription by preventing the zinc-dependent formation of a complex of metal response element-binding transcription factor-1 (MTF-1) and the co-activator p300. Our results suggest that the carcinogenicity of Cr(VI) is enhanced by MTF-1 dysfunction.

Chromium (VI) inhibits mouse metallothionein-I gene transcription by modifying the transcription potential of the co-activator p300.

The production of the heavy metal-binding proteins, the metallothioneins (MTs), is induced by heavy metals such as Zn, Cd, and Hg. MTs maintain Zn homeostasis and attenuate heavy metal-induced cytotoxicity by sequestering these metals and lowering their intracellular concentrations. Previously, we had reported that Zn induced the formation of a co-activator complex containing metal response element-binding transcription factor-1 (MTF-1) and the histone acetyltransferase (HAT), p300, which plays an essential role in the activation of MT-1 transcription. In addition, we had shown that Cr(VI) inhibits Zn-induced MT-1 transcription by preventing the Zn-dependent formation of the MTF-1-p300 complex. In the current study, we have shown that the inhibition by Cr(VI) was partially overcome by the overexpression of p300 or MTF-1 in an MT-I promoter-driven luciferase reporter assay system and have used real-time RT-PCR to determine MT-I mRNA levels. It has been reported that Cr(VI) inhibits CYP1A1 transcription by crosslinking histone deacetylase (HDAC) to the promoter. The crosslink inhibits the recruitment of p300 to the MT-1 promoter and blocks HAT-dependent transactivation by p300. However, our results demonstrate that trichostatin A, an HDAC inhibitor, could not block the inhibitory effects of Cr(VI) on MT-1 transcription and that there were no significant differences in the in vitro inhibitory effects of Cr(VI), Cr(III), and Zn on p300 HAT activity. This suggests that the inhibitory effects of Cr(VI) on MT-I transcription may be due to its effects on the HAT-independent transactivation ability rather than the HAT-dependent, HDAC release-related transactivation ability of p300.

The zinc-sensing transcription factor MTF-1 mediates zinc-induced epigenetic changes in chromatin of the mouse metallothionein-I promoter.

Metallothionein (MT) is a small, cysteine-rich protein active in zinc homeostasis, cadmium detoxification, and protection against reactive oxygen species. Mouse MT-I gene transcription is regulated by metal response element-binding transcription factor-1 (MTF-1), which is recruited to the promoter by zinc. We examined alterations in the chromatin structure of the MT-I promoter associated with enhanced transcriptional activation. MTF-1 proved essential for zinc-induced epigenetic changes in the MT-I promoter. Chromatin immunoprecipitation assays demonstrated that zinc treatment rapidly decreased Lys4-trimethylated and Lys9-acetylated histone H3 in the promoter and decreased total histone H3 but not histone H3.3. Micrococcal nuclease sensitivity of the MT-I promoter was increased by zinc. Thus, the chromatin structure in the promoter may be locally disrupted by zinc-induced nucleosome removal. Without MTF-1 these changes were not observed, and an MTF-1 deletion mutant recruited to the MT-I promoter by zinc that did not recruit the coactivator p300 or activate MT-I transcription did not affect histone H3 in the MT-I promoter in response to zinc. Interleukin-6, which induces MT-I transcription independently of MTF-1, did not reduce histone H3 levels in the promoter. Rapid disruption of nucleosome structure at the MT-I promoter is mediated by zinc-responsive recruitment of an active MTF-1-coactivator complex.

Ethanol-induced expression of glutamate-cysteine ligase catalytic subunit gene is mediated by NF-kappaB.

Glutamate-cysteine ligase is a rate-limiting enzyme in the de novo synthesis of glutathione, a known scavenger of electrophiles and reactive oxygen species. Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by nuclear factor erythroid 2-related factor 2 (Nrf2). It has been reported that ethanol induces human GCLC production via Nrf2-mediated transactivation of the antioxidant-responsive element (ARE). Here, the luciferase reporter assay revealed the presence of an ethanol-responsive element in the human GCLC promoter; it spanned bases -1432 to -832 in hepatocytes and HepG2 cells transfected with cytochrome P450 2E1 (CYP2E1). The region lacked an ARE but had a putative nuclear factor-kappaB (NF-kappaB) element. NF-kappaB DNA-binding activity was activated in response to ethanol treatment. CYP2E1 expression was required for GCLC promoter-driven gene expression and the activation of NF-kappaB. Thus ethanol-induced GCLC transcription is mediated by not only Nrf2 but also NF-kappaB.

Chromium(VI) inhibits mouse metallothionein-I gene transcription by preventing the zinc-dependent formation of an MTF-1-p300 complex.

Mouse MT-I (metallothionein-I) transcription is regulated by MTF-1 (metal-response-element-binding transcription factor-1) which is recruited to the promoter in response to zinc. Cr(VI) [chromium(VI)] pretreatment blocks zinc-activation of the endogenous MT-I gene and attenuates zinc-activation of MT-I-promoter-driven luciferase reporter genes in transient transfection assays. Chromatin immunoprecipitation assays revealed that Cr(VI) only modestly reduces recruitment of MTF-1 to the MT-I promoter in response to zinc, but drastically reduces the recruitment of RNA polymerase II. These results suggest that Cr(VI) inhibits the ability of MTF-1 to transactivate this gene in response to zinc. Zinc has recently been shown to induce the formation of a co-activator complex containing MTF-1 and the histone acetyltransferase p300 which plays an essential role in the activation of MT-I transcription. In the present study, co-immunoprecipitation assays demonstrated that Cr(VI) pretreatment blocks the zinc-induced formation of this co-activator complex. Thus Cr(VI) inhibits mouse MT-I gene expression in response to zinc by interfering with the ability of MTF-1 to form a co-activator complex containing p300 and recruiting RNA polymerase II to the promoter.