Article pubs.acs.org/est
PI3K/Akt Pathway Mediates Nrf2/ARE Activation in Human L02 Hepatocytes Exposed to Low-Concentration HBCDs Wen Zou,† Cen Chen,† Yufang Zhong,† Jing An,*,† Xinyu Zhang,† Yingxin Yu,† Zhiqiang Yu,*,‡ and Jiamo Fu‡ †
Institute of Environmental Pollution and Health, School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, P. R. China ‡ State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, P. R. China S Supporting Information *
ABSTRACT: We investigated the effects of hexabromocyclododecanes (HBCDs) at environmentally relevant concentrations on human L02 hepatocytes and explored possible underlying molecular mechanism(s), focusing on functional interactions between the phosphatidylinositol 3-kinase/protein kinase B (PI3K/ Akt) and nuclear factor-erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) pathways. The results showed that low concentrations of HBCDs could stimulate cell proliferation in a “DNA-dependent protein kinase catalytic subunit” (DNA-PKcs)-dependent manner, increase protein levels and nuclear translocation of transcription factor Nrf2, and upregulate expression of its target gene heme oxygenase-1 (HO-1). Electrophoretic mobility-shift assays (EMSAs) showed that ARE was a prominent element for HO-1 induction after lowconcentration HBCDs exposure. The relationship between PI3K/Akt pathway and Nrf2/HO-1 axis was demonstrated by the finding that pretreatment with PI3K inhibitors (wortmannin, LY294002) attenuated the upregulation of Nrf2 expression induced by HBCDs exposure. Furthermore, knock-down of DNA-PKcs through small interfering RNA blocked Nrf2/HO-1 axis activation in L02 cells exposed to low-concentration HBCDs. Moreover, DNA-PKcs and phosphorylated Akt at Ser473 proved to be crucial in regulating the Nrf2-ARE pathway. Thus, the PI3K/Akt pathway is essential in regulating Nrf2-ARE pathway activation in L02 cells induced by low-concentration HBCDs.
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reported in the human adipose tissue, milk, and blood,12−14 with concentrations up to 190 μg/kg lipid. In fact, many researchers have recently turned their attention to the “environmentally relevant dose”, the dose based on the internal concentration of the toxicant, rather than the dose administered.15 To improve understanding the adverse effects and potential health risk of HBCDs, it is necessary to obtain adequate toxicological data at environmentally relevant doses, as well as to investigate the underlying molecular mechanism. DNA-dependent protein kinase catalytic subunit (DNAPKcs) belongs to the phosphatidylinositol 3-kinase (PI3K) superfamily. It has been identified as one of the phosphorylating enzymes for protein kinase B (Akt) at Ser473.16,17 Previous studies indicated that DNA-PKcs and the PI3K/Akt pathway could respond to oxidative stress, modulating various intracellular downstream signaling events such as cell growth, apoptosis, and differentiation.18,19 Nuclear factor-erythroid 2related factor 2 (Nrf2) is also one of the most versatile
INTRODUCTION Hexabromocyclododecanes (HBCDs) are widely used as brominated flame retardants (BFRs) additives in polystyrene foams, thermal insulation, building materials, upholstery textiles, and electronic products.1 To date, HBCDs have become the third most frequently used BFRs in the world, following tetrabromobisphenol A (TBBPA) and polybrominated diphenyl ethers (PBDE).2 Due to their persistence in environment, long-range transport, bioaccumulation, and various toxicity,3 HBCDs are currently under assessment for inclusion in the Stockholm Convention on persistent organic pollutants (POPs).4 Previous studies indicated that HBCDs exposure might induce hepatotoxicity, endocrine disruption, neurotoxicity, and reproductive/developmental toxicity.5,6 Animal experiments also demonstrated that the liver is a major target organ of HBCDs exposure for mammals7,8 and fish.9 Relatively high concentration HBCDs exposure (30 mg/kg per day, 28 d) could increase liver size and weight.6,10 Long-term HBCDs exposure (130 mg/kg per day) caused a series of pathological changes such as hepatic nodules, fatty infiltration, liver necrosis, and even liver tumors.11 However, most of these studies were performed at significantly higher concentrations than those © XXXX American Chemical Society
Received: April 24, 2013 Revised: September 30, 2013 Accepted: October 4, 2013
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dx.doi.org/10.1021/es401791s | Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Environmental Science & Technology
Article
performed with Hoechst 33342. The fluorescence was observed under a fluorescence microscope (Olympus BX-51, Japan). Electrophoretic Mobility-Shift Assay (EMSA). EMSAs were performed as described elsewhere with some modifications.37 Cytoplasmic and nuclear extracts of cells were prepared with a nuclear and cytoplasmic extraction reagent kit (NEPER). DNA−protein binding reactions were conducted according to the manufacturer’s protocol for the EMSA/GelShift Kit. The reaction mixtures were loaded on 4% nondenaturing polyacrylamide gels, which were then blotted onto nylon membranes and subjected to chemiluminescence detection. Western Blotting. Western blotting was performed according to our previous study.33 Briefly, cells were lysed using NE-PER to obtain cytoplasmic and nuclear extracts or using mammalian protein extraction reagent (M-PER) to collect total protein. Equal amounts of protein were subjected to 8% or 10% sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane, which was then incubated with primary antibodies and corresponding secondary antibodies. The blots were visualized using chemiluminescence, and optical densities of individual bands were quantified using the Chemi-Imager digital imaging system (Alpha Innotech, San Leandro, CA, USA). Small Interfering RNA-Mediated Knock-down of DNAPKcs in L02 Cells. L02 cells were transfected with control or DNA-PKcs siRNA using Lipofectamine 2000 as described previously.36 Specific siRNA targeting DNA-PKcs and a nontargeting control siRNA were provided by Prof. Ping-Kun Zhou (Beijing Institute of Radiation Medicine, Beijing, China). L02-C and L02-N1 cells were stably transfected with a control construct and a specific siRNA targeting the DNA-PKcs catalytic motif (nucleotides 11637−11655), respectively. Statistical Analyses. All experiments were performed in triplicate, and data are expressed as means ± SEM. All data were subjected to one-way ANOVA analysis and Tukey’s post hoc test for comparisons. A p value
