Effects Of PCB153 On DNA Damage And DNA Repair-related Enzyme Expression Induced By PBDE-47 In Human Neuroblastoma SH-SY5Y Cells In Vitro

Polybrominated diphenyl ethers (PBDEs) is a class of newly found Persistent Organic Pollutants (POPs), which is extensively used in electronic products,textiles, rubber,plastics, and building materials. Becase of structure similarity with PCBs, PBDEs have characters of high liposolubility, chemical stability, low degradation, and low evaporability which contribute to environmental persistence and bioamplification by the way of physical leakage and chemical binding. The results of environmental epidemiology study demonstrated that PBDEs were extensively existed in environmental media (air, water, soil and sludge), animal body (fish, whale and birds), and human breast milk, blood and adipose tissues. PBDEs present a potential risk to the environment and human health.Some studies indicated that PBDEs possessed neurotoxicology, hepatotoxicity, immunotoxicity, reproduction toxicology, endocrine disrupting, and carcinogenicity both in vivo and in vitro. Other studies have shown that PBDEs can cause persistent dysfunction in adult animals, which was manifested as spontaneous deranged behavior, learning and memory defects, and dysfunctions in the cholinergic system that worsen with age. However its correlate mechanisms remains unclear. It has been reported that oxidative stress due to administration of PBDEs induced lipid peroxidation and oxidative DNA damage. 2,2', 4,4'-tetrabromodiphenyl ether (PBDE-47) is one of the highest congener present in biological samples as well as in human body.PCBs is a class of traditonal POPs, which have been identified as"human possible carcinogenesis material"and"animal clear carcinogenesis material". Although there is a decrease tendency in environment, PCBs still occupy a relative high level in enviroment, so they still belong to an important class of persistant organic pollutants. Of the various congeners, PCB153 is much higher present in human body than other PCBs. Some studies indicated PCBs can induce oxidative stress, micronuclei formation and other DNA damage effects. Furthermore, epidemiology studies demonstrated that PBDEs and PCBs were extensively co-present in environment compartants, More important is PBDEs have been detected from human breast milk, blood and adiposes. So it is imperative to study the mode of action of these two materials when they are co-present in certain material. In addition, PBDEs and PCBs have similar toxicological endpoint, which can induce oxidative stress and DNA damage effects. So we wonder that whether or not they will have synergistic actions on DNA damage if they are simultaneously exposed to human. Whether or not oxidative stress is involved in the process of DNA damage induced by PBDE-47. In order to answer these questions, we performed a set of molecular biological analyses such as ROS, DNA strand breakage, and 8-OHdG in SH-SY5Y cells to assess the effects of PBDE-47 and PCB153 on oxidative stress and DNA damage.DNA repair system plays a very important role in maintaining the integrity and instability of DNA frame, cells survival as well as normal physiological functions. After DNA damage, a specific set of DNA repair enzymes that catalyze cellular response to DNA damage according to the degree and category of DNA damage. So we could evaluate the DNA damage degree indirectly by studying changes of DNA repair genes. XRCC1 (X-ray repair cross-complementing gene 1) is the first mammal gene that affects cell sensitivity to ionizing radiation. XRCC1 gene involved in rejoining DNA strand breaks and can repair DNA damage caused by endogenous and exogenous factors, including oxidative damage by associating with other proteins such as PARP (ADP-ribose polymerase), DNA ligase III and DNA polymeraseβdirectly. XRCC3 (X-ray repair cross-complementing gene 3) mainly repair DNA Double Strand Breakage (DSB) by Nucleotide Excision Repair way (NER). So the present study we used XRCC1 and XRCC3 to judge the degree of DNA damage according to their different expressions. Providing the further mechanisms underlying of PBDE-47 and the mode of action induced by PBDE-47 and PCB153 in SH-SY5Y cell in vitro.This paper is consisted of two parts:Part I Effects of PBDE-47 and/or PCB153 on oxidative stress and DNA damage in SH-SY5Y cells in vitro.Objective To explore the effects of PBDE-47 and/or PCB153 on oxidative stress and DNA damage in SH-SY5Y cell in vitro. Methods SH-SY5Y cells were incubated with different concentrations of 1,5,10μM PBDE-47 or/and 5μM PCB153 and antioxidant n-acetyl cysteine (NAC 100μM) for 24 h in vitro. ROS level, percentage of DNA in the tail, olive tail moment (OTM) and 8-OHdG were measured respectively. Results PBDE-47 increased percentage of DNA in the tail and OTM significantly at doses of 5μM and above in a concentration-dependent manner compared to the control (P<0.05). The percentage of DNA in the tail were significantly higher in cells treated by 5μM PBDE-47+5μM PCB153 and 10μM PBDE-47+5μM PCB153 groups than that in their corresponding doses of PBDE-47 groups or PCB153 group, OTM was significantly higher in cells treated by 5μM PBDE-47+5μM PCB153 group than that in their corresponding doses of PBDE-47 groups or PCB153 group (P<0.05). The percentage of DNA in the tail and OTM of cells treated by the groups added NAC were obviously lower than that of their corresponding groups not added it (P< 0.05). The interactive action was observed between PBDE-47 and PCB153 in DNA olive tail moment (F=23.74, P<0.01). In addition, ROS formation and 8-OHdG level were significantly increased in 5 and 10μM combined groups compared to their corresponding concentrations of PBDE-47 groups or PCB153 group (P<0.05). The groups added with NAC dramatically decreased ROS level, DNA damage and 8-OHdG level compared to their corresponding groups not added it (P<0.05).Conclusions PBDE-47 can induce DNA damage, PBDE-47 combined with PCB153 may increase the effects on DNA damage in SH-SY5Y cells in vitro, oxidative stress may play an important role in DNA damage induced by PBDE-47.Part II Effects of PBDE-47 and/or PCB153 on DNA repair-related enzyme expression in SH-SY5Y cells in vitro.Objective To explore the effects of PBDE-47 and/or PCB153 on DNA repair-related enzyme in SH-SY5Y cells in vitro. Methods SH-SY5Y cells were incubated with different concentrations of PBDE-47 and/or PCB153 as described in the part I, DNA repair-related enzyme XRCC1 and XRCC3 mRNA expressions were measured by using RT-PCR. Results Significant decreased XRCC1 mRNA expression were observed at 10μM PBDE-47, 5μM PBDE-47+5μM PCB153, and 10μM PBDE-47+5μM PCB153 groups while significant increased XRCC3 mRNA expression were observed at 10μM PBDE-47 and 10μM PBDE-47+5μM PCB153 compared to the control group (P<0.05). XRCC1 mRNA expression in cells treated with 100μM NAC+10μM PBDE-47+5μM PCB153 group was significantly higher than that in 10μM PBDE-47+5μM PCB153 group (P<0.05). XRCC3 mRNA expression in cells treated with PBDE-47 anti-oxidant groups were significantly lower than those of their corresponding non-antioxidant groups (P<0.05). Correlation analysis showed that a negative relationship was observed between ROS level and XRCC1 mRNA (r=0.83) and no obvious relationship was observed between ROS level and XRCC3 mRNA (r=0.42, P﹥0.05). Furthermore, there was a negative relationship between DNA OTM and XRCC1 mRNA expression while a positive relationship between DNA OTM and XRCC3 mRNA expression (r1=0.73, r3=0.77, P<0.05). Conclusions PBDE-47 can effect the expression of DNA repair-related enzyme. ROS inhibited the mRNA expression of XRCC1. The inhibited DNA repair-related enzyme may be partly the reason for DNA damage induced by PBDE-47 in vitro.