The Cytological Effects Of HBCDs On Human Hepatocyte And The Potential Molecular Mechanism

Hexabromocyclododecanes (HBCDs) are widely used as additive brominatedflame retardants (BFRs) in polystyrene foams, thermal insulation building materials,upholstery textiles and electronic products. Recent studies indicate that HBCDs havebecome ubiquitous organic contaminants and present some characteristics ofpersistent organic pollutants (POPs) such as bioaccumulation, long-rangetransportation and toxicity, which will threaten the environment and human health.For example, HBCDs have been detected in practically all environmental media (suchas air, sediment and soil), animal tissues and even human tissues.Recent data have shown that HBCDs have little acute toxicity at concentrationsless than their water solubility. Although extensive studies of HBCDs toxicity havebeen conducted at high doses (μM level) which are much higher than human actualexposure doses (≤nM level), the biological effects of HBCDs at environmentallyrelevant concentrations cannot been authenticly evaluated. Recent toxicologicaladvances have revealed that long-term exposure to HBCDs resulted in hepatotoxicity,endocrine disruption, neurotoxicity and reproductive effects. Liver has been identifiedas a major target organ for HBCDs in animals. The objective of this study is toinvestigate the effects of HBCDs at environmentally relevant low concentrations inhuman normal hepatocyte cell line L02, to explore the possible underlying molecularmechanism, and to further explain the relationship of HBCDs with hepatomadevelopment.We investigated biological effects of HBCDs (10-14M-100μM) in L02cells suchas cell growth, apoptosis, intracellular ROS levels, DNA damage, mitochondrialdamage and cytosolic Ca2+to initially identify the toxic effect dose range.Additionally, proliferation-related genes PCNA and apoptosis-related gene Apaf-1protein expression levels were measured by western blot in order to further clarify theeffects of the low dose HBCDs on proliferation and apoptosis. Based on cell growthand oxidative stress, we detected the expressions and phosphorylation levels of keysignaling molecules around PI3K/Akt and Nrf2-ARE pathways by western blot, EMSA and immunofluorescence with the assists of RNA interference and PI3Kinhibitors to explore the underlying molecular regulation mechanism of low doseHBCDs-induced cell proliferation and oxidative stress.The results showed that high concentration of HBCDs had toxic effects on L02cells, as shown by inhibition of cell survival, increase of apoptosis and DNA damage.The ROS overload and elevation of cytosolic Ca2+were attributed to Apaf-1mediatedmitochondrial apoptosis pathway induced by high concentration HBCDs exposure.However, low concentration of HBCDs could stimulate cell proliferation of L02cells,which might be associated with enhancement of PCNA expression.The environmentally relevant low concentrations of HBCDs could activatePI3K/Akt pathway by the phosphorylation of Akt at the position of Ser473, whichmediated Nrf2-ARE pathway activation, as evidenced by an increased protein leveland nuclear translocation of Nrf2and up-regulating its target gene HO-1expression.Additionally, we found that low concentration HBCDs pretreatment couldsignificantly attenuated the cell death, ROS formation and DNA damage induced bysubsequent high concentration HBCDs. Thus, low concentration HBCDs were likelyto generate an antioxidative protctive effect by activating PI3K/Akt and Nrf2-AREpathways, which consequently maintain intracellular redox homeostasis to ensure theproliferation of L02. However, on the other hand, it has been reported thattumorigenesis can be associated with the sustained activation of PI3K/Akt andNrf2-ARE pathways. Therefore, chronic exposure to low concentration HBCDs mightresult in a sustained, weak upregulation of PI3K/Akt and Nrf2-ARE pathways, whichmight induce mutations of normal hepatocyte cells and potentially cause/promotetumor formation and development.