Effect Of Aroclor1254 On B(a)P-induced DNA Damage And Metabolic Enzyme Mechanism

Polychlorinated biphenyls (PCBs), a family of industrial synthetics composed of chlorinated biphenyls congeners, are a class of persistent organic pollutants, being concerned internationally today. They were widely used in a variety of industrial applications due to their insulating and fire retardant properties. Because of the improper treatment after use, PCBs persist extensively in the environment around world. Since PCBs were found in the environment in the 1960s, their pollution has spread all over the world including air, water, soil, food and all kinds of biological samples. So they attract a worldwide attention on their possible adverse effects on the ecology and the human beings.Aroclor1254 is a kind of commercial PCBs mixture, being widely used as insulating material, plasticizer, liquid for transmission etc, and has been detected in many kinds of biological samples. In Ames test and genetic toxicity tests in mammalian cells in vitro, Aroclor1254 didn't show inherent toxicity. Enzymology researches indicate that Arolcor1254 can induce cytochrome P450 and appear to be a good metabolic enzyme inducer. Cytochrome P450 1A1 (CYP1A1), participates in the metabolic process of many precarcinogens and mutagenic agents, playing an important role in activition of precarcinogens such as benzo(a)pyrene (B(a)P) to the ultimate (proximate). As a result, Aroclor1254 may enhance genotoxicity of B(a)P in organism. To date most researchers paid attention on genotoxicity of Aroclor1254 or B(a)P alone, while few documents have been reported involving interaction between Aroclor1254 and B(a)P in organism.Consequently in the present study, we detected CYP1A1 activity, DNA strand breaks and formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in HepG2 cells exposed to B(a)P after pretreatment with Aroclor1254 using EROD assay, single cell gel electrophoresis (SCGE) and high-performance liquid chromatography- electrochemical detection (HPLC-EC) assay, respectively, to evaluate the effects of Aroclor1254 on B(a)P-induced genotoxicity and metabolic enzyme mechanism.The paper is consisted of two parts:The first part:The effect of Aroclor1254 on B(a)P-induced CYP1A1 activity in HepG2 cellsHepG2 cells were pretreated with low, medium and high concentrations of Aroclor1254 (11.5, 23.0 and 46.0μmol/L) for 24h and then exposed to B(a)P (50.0μmol/L). DMSO (10 ml/L) was used as solvent control. UV spectrophotometry and fluorescence spectrophotometry were used to detected activity of the metabolic enzyme CYP1A1 (Ethoxyresorufin-O-deethylase, EROD) in HepG2 cells in vitro. The results showed as follow: (1) In HepG2 cells treated with B(a)P (50.0μmol/L) and medium and high concentrations of Aroclor 1254 (23.0 and 46.0μmol/L), EROD activities were 0.164±0.011, 0.173±0.018, 0.217±0.034 pmol/min/mg protein, respectively, which were significantly higher than that in solvent control (0.116±0.010 pmol/min/mg protein); (2) After pretreatment of HepG2 cells with Aroclor1254 (23.0, 46.0μmol/L), B(a)P-induced EROD activities were 0.216±0.027, 0.254±0.017 pmol/min/mg protein, which increased by 32% and 55% as compared with B(a)P alone. Our study indicated that both Aroclor1254 and B(a)P might induce activity of CYP1A1 in HepG2 cells at certain concentrations and Aroclor1254 enhanced B(a)P-induced CYP1A1 activity in HepG2 cells.The second part:The effect of Aroclor1254 on B(a)P-induced DNA damage in HepG2 cells HepG2 cells were pretreated with low, medium and high concentrations of Aroclor1254 (11.5, 23.0 and 46.0μmol/L) for 24 hours and then exposed to B(a)P (50μmol/L). DMSO (10 ml/L) was used as solvent control. SCGE and HPLC-EC assays were applied to detect DNA single-strand breaks and 8-OHdG in HepG2 cells, respectively. The results showed as follow: (1) In HepG2 cells treated with B(a)P alone, average Oliver tail moment (OTM) and 8-OHdG level (8-OHdG/106dG) were 1.66±0.21 and 23.31±6.02, respectively, which were significantly higher that in solvent control (0.79±0.15 and 12.31±3.24); (2) In Aroclor 1254 treated group (11.5, 23.0, 46.0μmol/L), average OTM were 0.88±0.20, 1.01±0.15 and 1.10±0.16, and 8-OHdG levels (8-OHdG/106dG) were 19.57±7.57, 22.80±9.16 and 31.74±9.25, respectively. No significant increase of OTM and 8-OHdG level were shown in HepG2 cells treated with Aroclor1254 alone in comparision with the solvent control, only except high concentration of Aroclor1254 (46μmol/L); (3) After pretreatment of HepG2 cells with different concentrations of Aroclor1254 (11.5, 23.0 and 46.0μmol/L), B(a)P-induced DNA strand breaks (OTM: 2.14±0.22, 2.43±0.32 and 2.71±0.31) increased by 29%, 46% and 63%, and 8-OHdG levels (8-OHdG/106dG: 32.50±3.81, 49.23±16.66 and 60.36±18.04) increased by 39%, 111% and 159% as compared with B(a)P alone. There was a significant positive correlation between OTM and EROD activity in HepG2 cells (r=0.958, P<0.05), as well as between 8-OHdG levels and EROD activity (r=0.992, P<0.05).Our study suggested that Aroclor1254 might enhance B(a)P-induced DNA damage in HepG2 cells, which should imply a synergistic effect of Aroclor1254 on the genotoxicity of B(a)P. Aroclor1254 enzyme induction might have contributed to enhanced DNA damage caused by B(a)P, based on increased avtivity of metabolivc enzymy after pretreatment of Aroclor1254.