Nrf2-ARE-Dependent Alterations in Zinc Transporter mRNA Expression in HepG2 Cells

<div><p>Zinc transporters are solute carrier family members. To date, 10 zinc transporters (ZnTs) and 14 Zrt-, Irt-like proteins (ZIPs) have been identified. ZnTs control intracellular zinc levels by effluxing zinc from the cytoplasm into the extracellular fluid, intracellular vesicles, and organelles; ZIPs also contribute to control intracellular zinc levels with influxing zinc into the cytoplasm. Recently, changes in zinc transporter expression have been observed in some stress-induced diseases, such as Alzheimer’s disease and diabetes mellitus. However, little is known regarding the mechanisms that regulate zinc transporter expression. To address this, we have investigated the effect of a well-established stress response pathway, the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway, on zinc transporter mRNA levels. Exposure to 10⁻⁴ M <i>tert</i>-butylhydroquinone (<i>t</i>-BHQ), which activates Nrf2-ARE signaling, for 6 h significantly increases ZnT-1, ZnT-3, and ZnT-6 mRNAs levels, and significantly decreases ZnT-10 and ZIP-3 mRNA levels. These changes are not observed with 10⁻⁶ M <i>t</i>-BHQ, which does not activate Nrf2-ARE signaling. Furthermore, <i>t</i>-BHQ exposure does not affect metal responsive element transcription, a <i>cis</i> element that is activated in response to intracellular free zinc accumulation. From these results, we believe that the transcription of ZnT-1, ZnT-3, ZnT-6, ZnT-10, and ZIP-3 is influenced by the Nrf2-ARE signal transduction pathway.</p></div

The effect of <i>t</i>-BHQ treatment on metal transcriptional factor-1 (MTF-1) mRNA expression.

<p>HepG2 cells were treated with the denoted concentration of <i>t</i>-BHQ or medium including 0.1% (v/v) DMSO as a control for 6 h. The mRNA level was determined using qRT-PCR with SYBR^® Green. Data are normalized to ß-actin expression. Bars represent the mean ± SD of 4 samples.</p

Antioxidant responsive element (ARE) luciferase reporter activity following <i>t</i>-BHQ treatment.

<p>HepG2 cells were cotransfected with pGL4.37, a firefly luciferase vector encoding the ARE, and the <i>Renilla</i> luciferase vector pGL4.74 prior to treatment with the denoted concentration of <i>t</i>-BHQ for 6 h. Firefly and <i>Renilla</i> luciferase activities were subsequently determined and firefly luciferase activity was normalized to the <i>Renilla</i> luciferase activity to control for transfection efficiency. Data are expressed as relative activity values compared with vehicle control (0.1% DMSO = 100%). Bars indicate the mean ± SD of 3 (10⁻⁶ M <i>t</i>-BHQ) or 4 (Control and 10⁻⁴ M <i>t</i>-BHQ) samples. ***p < 0.001 compared with control.</p

The effect of <i>tert</i>-butylhydroquinone (<i>t</i>-BHQ) treatment on zinc transporter mRNA levels.

<p>HepG2 cells were treated with the denoted concentration of <i>t</i>-BHQ for 6 h. The expression of each mRNA was determined by qRT-PCR with SYBR^® Green. Data are normalized to ß-actin mRNA expression. Values represent the mean ± standard deviation (SD) of 4 samples. *p < 0.05, ***p < 0.001 compared with vehicle treated control (0.1% dimethyl sulfoxide; DMSO) cells.</p

Metal responsive element (MRE)-luciferase reporter activity following <i>t</i>-BHQ treatment.

<p>HepG2 cells were cotransfected with the firefly luciferase vector encoding the MRE, pGL4.40, and the pGL4.74 <i>Renilla</i> luciferase vector. Transfected cells were treated with control (0.1% (v/v) DMSO), <i>t</i>-BHQ, or 0.1 mM ZnSO₄ (as a positive control) for 6 h and luciferase activities were determined. Firefly luciferase activity was normalized to <i>Renilla</i> luciferase activity to control for transfection efficiency. Data are expressed as relative activity values compared with control (0.1% DMSO = 100%). Bars indicate the mean ± SD of 4 samples. ***p < 0.001 compared with control.</p

Quantitative real-time polymerase chain reaction (qRT-PCR) primer sequences and product sizes.

<p>Quantitative real-time polymerase chain reaction (qRT-PCR) primer sequences and product sizes.</p

Dose-dependent alteration in heme oxygenase-1 mRNA expression following <i>t</i>-BHQ treatment.

<p>HepG2 cells were treated with the denoted concentration of <i>t</i>-BHQ or vehicle (0.1% (v/v) DMSO) for 6 h prior to quantification of mRNA expression by qRT-PCR with SYBR^® Green. Data are normalized to ß-actin expression. Bars indicate the mean ± SD of 4 samples. ***p < 0.001 compared with control.</p

<i>t</i>-BHQ dose-dependent changes in HepG2 cell cytotoxicity.

<p>Cells were treated with the denoted concentration of <i>t</i>-BHQ or vehicle (0.1% (v/v) DMSO) for 6 h and cell viability was assessed using the WST-8 assay. Values represent the mean ± SD of 5 samples. ***p < 0.001 compared with control (100 ± 4.8%).</p

Restoration of Dioxin-Induced Damage to Fetal Steroidogenesis and Gonadotropin Formation by Maternal Co-Treatment with α-Lipoic Acid

<div><p>2,3,7,8-Tetrachlorodibenzo-<em>p</em>-dioxin (TCDD), an endocrine disruptor, causes reproductive and developmental toxic effects in pups following maternal exposure in a number of animal models. Our previous studies have demonstrated that TCDD imprints sexual immaturity by suppressing the expression of fetal pituitary gonadotropins, the regulators of gonadal steroidogenesis. In the present study, we discovered that all TCDD-produced damage to fetal production of pituitary gonadotropins as well as testicular steroidogenesis can be repaired by co-treating pregnant rats with α-lipoic acid (LA), an obligate co-factor for intermediary metabolism including energy production. While LA also acts as an anti-oxidant, other anti-oxidants; <em>i.e.</em>, ascorbic acid, butylated hydroxyanisole and edaravone, failed to exhibit any beneficial effects. Neither wasting syndrome nor CYP1A1 induction in the fetal brain caused through the activation of aryl hydrocarbon receptor (AhR) could be attenuated by LA. These lines of evidence suggest that oxidative stress makes only a minor contribution to the TCDD-induced disorder of fetal steroidogenesis, and LA has a restorative effect by targeting on mechanism(s) other than AhR activation. Following a metabolomic analysis, it was found that TCDD caused a more marked change in the hypothalamus, a pituitary regulator, than in the pituitary itself. Although the components of the tricarboxylic acid cycle and the ATP content of the fetal hypothalamus were significantly changed by TCDD, all these changes were again rectified by exogenous LA. We also provided evidence that the fetal hypothalamic content of endogenous LA is significantly reduced following maternal exposure to TCDD. Thus, the data obtained strongly suggest that TCDD reduces the expression of fetal pituitary gonadotropins to imprint sexual immaturity or disturb development by suppressing the level of LA, one of the key players serving energy production.</p> </div

The LA-specific recovery from a TCDD-induced reduction in the fetal expression of gonadotropins: A–C, pituitary levels of LHβ, FSHβ and LH/FSH α-subunit mRNAs, respectively; and D, serum content of LH.

<p>Gonadotropin mRNAs in the fetuses (GD20), the parents of which were treated with TCDD (GD15) and anti-oxidants (GD15-20), were determined by RT-PCR. The level of gonadotropin mRNA was normalized by β-actin mRNA. Serum LH (GD20 fetuses) was determined by ELISA. In the white bar control, pregnant rats were treated with DMSO alone or anti-oxidant dissolved in DMSO. In the shaded bar control, dams were given aqueous NaCl alone or anti-oxidant dissolved in this solution. The pituitaries and sera of all male fetuses in one dam were pooled to become one analytical unit. Each bar represents the mean value relative to the control ± SEM of 5 (panels A-C) or 7 (panel D) dams. *p<0.05 and **p<0.01, from the respective controls (A-C) or between a pair indicated (<i>D</i>).</p