Relationship Of PCBs/B(a)P Combination Induced Genotoxicity And Metabolic Enzyme

Polychlorinated biphenyls (PCBs) are a class of chlorinated hydrocarbons which belong to the family of persistent organic pollutants (POPs) that have been widely used in a variety of industrial application. Because of the improper handling after industrial production, PCBs have been released into the environment. In 2001, 127 countries signed the"Stockholm Convention on Persistent Organic Pollutants"which aimed at prohibiting or limiting the utilization of POPs. PCB has been listed as one of the 12 most persistent organic pollutants on the planet. As the representative of polycyclic aromatic hydrocarbons (PAHs), benzo(a)pyrene(B(a)P) is the first discovered environmental carcinogen, which exhibit strong carcinogenic properties. Because of usually co-existing of PCBs and B(a)P in many environmental medium and human samples[1][2], the investigation about the combined adverse effect of these pollutants and related mechanism have the strong practical meaning.In the present study, we chosen PCB153 and PCB126 as the research objects because they are not only the members of PCBs family which belong to POPs, but also have their own characteristics. PCB153 is one of the most detectable PCBs in the human blood and milk and PCB126 is one of the most toxic PCBs. From the view of enzyme induced mode, PCB153 is the phenobarbital-type(PB-type) inducer while PCB126 is 3-methylcholanthrene-type(3-MC type) inducer. From the view of molecular constitution, PCB153 is the non-coplanar congeners but PCB126 is coplanar congeners. Evidence indicated that PCB153 and PCB126 can catalyze the activation of P450 enzyme[3-5], which plays the key role in metabolism activation of many PAH mixture including B(a)P from pre-carcinogenic chemicals to ultimate carcinogenic. Therefore, PCB153 and PCB126 probably enhance the B(a)P-induced inherent toxicity through metabolic enzyme induction. To test if altered enzyme activities induced by PCBs would influence the toxicity of B(a)P, HepG2 cells were combined treated with PCB153 or PCB126 and B(a)P to investigate the interaction between PCBs and B(a)P, further exploring the possible mechanisms.The metabolically competent human hepatoma cell line HepG2 was treated with different concentrations of PCB153, PCB126 and B(a)P alone, or treated with PCB153 or PCB126 and B(a)P together. For combined treatment groups, HepG2 cells were pre-treated with different concentrations of PCB153 or PCB126 for 48h, then co-treated with B(a)P and PCB153 or PCB126 together for another 24h. Enzyme activities of CYP1A1, CYP1A2, CYP2B and GST, formation of micronuclei (MN) and mRNA expression of Ah receptor were analyzed through fluorescence spectrophotometry, colorimetric method, cytokinesis -block micro micronucleus method (CBMN) and reverse transcriptase PCR (RT-PCR) respectively to investigate the combined effect of PCB126 or PCB 153 and B(a)P on genotoxicity in HepG2 cells and related metabolic enzyme mechanism.α-Naphthoflavone (ANF) as inhibitor of P450 enzyme was used in the test to comfirm the key effect of phaseⅠe nzyme on genetoxic changes in combined groups of PCBs and B(a)P .The whole study is composed of the following three parts:PartⅠCombined effect of PCB153 and B(a)P on genotoxicity and metabolic enzyme changes in HepG2 cellsHepG2 cells were treated with different concentrations of PCB153 (0.1, 1, 10, and 100μmol/L) and B(a)P (50μmol/L) for 72h alone, or pretreated with PCB153 for 48h then co-treated with B(a)P (50μmol/L) and PCB153 together for another 24h. DMSO (10ml/L) was used as solvent control. Ethoxyresorufin-O-deethylase (EROD), mthoxyresorufin -O-deethylase (MROD) and pentoxyresorufin-O-deethylase (PROD) were detected as marker enzyme of CYP1A1, CYP1A2 and CYP2B respectively. Meanwhile activities of glutathione S-transferase (GST), formation of MN and nucleus division index (NDI) were also analyzed. The experiments were carried out again after adding the phase I enzyme inhibitor ANF. The results were as follows:1. EROD activities in HepG2 cells treated with B(a)P (50μmol/L) and PCB153 (0.1, 1, 10 and 100μmol/L) alone were significantly increased (1.05±0.05, 0.81±0.02, 0.87±0.03, 1.10±0.09 and 1.38±0.14 pmol/min/mg protein) compared with solvent control (0.72±0.03pmol/min/mg protein) (P<0.01); Same concentrations of B(a)P and PCB153 enhanced MROD activities (1.01±0.05, 2.44±0.07, 2.81±0.09, 3.34±0.14 and 3.80±0.30pmol/min/mg protein) compared with solvent control (0.83±0.03pmol/min/mg protein) (P<0.01); PROD activities in HepG2 cells treated with the same concentrations of B(a)P and PCB153 alone were also significantly increased (1.48±0.09, 1.65±0.06, 2.51±0.20, 2.77±0.31 and 3.67±0.31pmol/min/mg protein) compared with solvent control (1.08±0.04pmol/min/mg protein) (P<0.01); GST activities were increased in HepG2 cells treated with B(a)P (2.14±0.06) and decreased in all treated groups of PCB153 (0.85±0.18, 0.70±0.05, 0.65±0.10 and 0.29±0.14U/mgprot) compared with solvent control (1.09±0.13U/mgprot) (P<0.05).After pre-treatment with PCB153 (0.1,1,10 and 100μmol/L) for 48h, the HepG2 cells were co-treated with PCB153 and B(a)P(50μmol/L) together for another 24h. The results were as follows: (1) Enhanced EROD activities were found in combined treatment groups (1.27±0.01, 1.39±0.06, 1.47±0.08 and 1.85±0.06pmol/min/mg protein) compared with group treated with B(a)P alone (P<0.01) and they increased 22%, 34%, 42% and 80% respectively. The factorial design multivariable ANOVA showed synergistic effect of PCB153 and B(a)P on EROD induction. (2) Enhanced MROD activities were found in combined treatment groups (3.10±0.08, 3.33±0.06, 3.44±0.01 and 3.58±0.06pmol/min/mg protein) compared with group treated with B(a)P alone (P<0.001) and they increased 209%, 232%, 243% and 357% respectively. The factorial design multivariable ANOVA showed synergistic effect of PCB153 and B(a)P on MROD induction at low concentrations of PCB153 and antagonistic effect at high concentrations of PCB153. (3) Enhanced PROD activities were found in combined treatment groups (1.64±0.24, 2.45±0.07, 2.61±0.27 and 3.66±0.32 pmol/min/mg protein) compared with group treated with B(a)P alone (P<0.01) and they increased 16%, 80%, 113% and 218% respectively. The factorial design multivariable ANOVA showed the interaction between PCB153 and B(a)P hasn't any meaning. (4) GST activities were lower in combined treatment groups (2.12±0.19, 1.73±0.06, 1.66±0.09 and 0.56±0.06 U/mgprot) then group treated with B(a)P alone (P<0.01) and they decreased 2%, 41%, 48% and 158% respectively, The factorial design multivariable ANOVA showed antagonistic effect between PCB153 and B(a)P. ANF showed significant inhibition effect on EROD, MROD and PROD activities and induced effect on GST activities in group treated with PCB153 alone and combined treatment groups of PCB153 and B(a)P.2. The MN rates in HepG2 cells treated with B(a)P (50μmol/L) and PCB153 (0.1, 1, 10 and 100μmol/L) alone were 52±6.00‰, 27±1.15‰, 26±2.00‰, 29±4.16‰and 49±4.04‰. Compared with solvent control (23±3.05‰), only B(a)P and 100μmol/Lof PCB153 enhanced MN formation. Another concentrations of PCB153 had no effect on MN formation and NDI.; In combined treatment groups (except group of 0.1μmol/L of PCB153 and B(a)P) enhanced MN formation was found (71±6.11‰, 76±4.73‰and 83±8.00‰) compared with group treated with B(a)P alone (P<0.05) and they increased 19%, 24% and 31% respectively.. The factorial design multivariable ANOVA showed synergistic effect of PCB153 and B(a)P on MN formation. After adding of ANF, the MN formations in combined groups were inhibited markedly except combined group of 100μmol/L of PCB153 and B(a)P.In summary for this part, PCB153 and B(a)P induced the phase I enzyme CYP1A1, CYP1A2 and CYP2B activities at certain concentration. There was a synergistic effect of PCB153 and B(a)P combination on phase I enzyme induction. PCB153 inhabited phase II enzyme GST activities. PCB153 and B(a)P combination showed antagonistic effect on GST activities. PCB153 had no effect in MN formation except the highest concentration. PCB153 and B(a)P combination showed synergistic effect on MN formation. ANF inhibited phase I enzyme and enhanced phase II enzyme which contributed to MN formation. These confirmed that PCB153 had significant effect on the metabolic enzymes which play the key role in enhancement of genotoxicity of B(a)P. PartⅡ: Combined effect of PCB126 and B(a)P on genotoxicity and metabolic enzyme changes in HepG2 cellsHepG2 cells were treated with different concentrations of PCB126 (0.01, 0.1, 1, 10nmol/L) and B(a)P (50μmol/L) alone, or pretreated with different concentrations of PCB126 for 48h then co-treated with B(a)P and PCB126 together for another 24h. Activities of EROD, MROD, PROD and GST, formation of MN and NDI were analyzed respectively. The experiments were carried out again after adding the phase I enzyme inhibitor ANF. The results were as follows:1. EROD activities in HepG2 cells treated with B(a)P (50μmol/L) and PCB126 (0.01, 0.1,1 and 10nmol/L) alone were significantly increased (1.05±0.05, 1.14±0.20, 1.50±0.28, 1.63±0.25 and 2.10±0.14 pmol/min/mg protein), compared with solvent control (0.72±0.03pmol/min/mg protein) (P<0.01); same concentrations of B(a)P and PCB126 enhanced MROD activities (1.01±0.05, 2.66±0.35, 2.84±0.12, 3.12±0.07 and 3.17±0.04pmol/min/mg protein), compared with solvent control (0.83±0.03pmol/min/mg protein) (P<0.05), PROD activities in HepG2 cells treated with same concentrations of B(a)P and PCB126 alone were also significantly increased(1.48±0.09, 1.01±0.35, 1.59±0.22, 1.66±0.17 and 1.95±0.11pmol/min/mg protein), compared with solvent control (1.08±0.04pmol/min/mg protein) (P<0.05) except the 0.01μmol/L PCB126; GST activities were increased in HepG2 cells treated with B(a)P (2.14±0.06) and decreased in all treated groups of PCB126 (1.11±0.15, 0.29±0.04, 0.26±0.02, 0.25±0.02 and 0.24±0.12U/mgprot), compared with solvent control (0.83±0.13 U/mgprot) (P<0.05).After pre-treatment with PCB126 (0.01, 0.1,1 and 10nmol/L) for 48h, the HepG2 cells were co-treated with PCB126 and B(a)P (50μmol/L) together for another 24h. The results were as followes: (1) Enhanced EROD activities were found in combined treatment groups (1.89±0.01, 1.90±0.09, 1.96±0.07 and 1.99±0.18pmol/min/mg protein), compared with group treated with B(a)P alone (P<0.001) and they increased 117%, 189%, 195% and 198% respectively. The factorial design multivariable ANOVA showed synergistic effect of PCB126 and B(a)P on EROD induction at low concentrations of PCB126 and antagonistic effect at the highest concentration of PCB126. (2) Enhanced MROD were found in combined treatment groups (3.15±0.32, 3.43±0.24, 3.46±0.05 and 3.84±0.03pmol/min /mg protein), compared with group treated with B(a)P alone(P<0.001), and they increased 214%, 342%, 345% and 383% respectively. The factorial design multivariable ANOVA showed synergistic effect of PCB126 and B(a)P on MROD induction. (3) PROD activities were found in combined treatment groups (0.45±0.04, 1.12±0.10, 2.19±0.18 and 2.25±0.07 pmol/min/mg protein), compared with group treated with B(a)P alone(P<0.01), and they decreased at combined groups of 0.01,0.1nmol/L PCB126 and B(a)P, but increased 71%and 117% separately at combined groups of 1,10nmol/L PCB126 and B(a)P.The factorial design, multivariable ANOVA showed antagonistic effect of PCB126 and B(a)P on PROD induction except the combined group of 1nmol/L PCB126 and B(a)P. (4) GST activities were lower in combined treatment groups (1.11±0.17, 0.99±0.11, 0.85±0.08 and 0.71±0.11 U/mgprot) then group treated with B(a)P alone(P<0.05), and they decreased 1%, 13%, 26% and 40% respectively. The factorial design multivariable ANOVA show synergistic effect between PCB126 and B(a)P. ANF showed significant inhibition effect on EROD, MROD, and PROD activities and induced effect on GST activities in group treated with PCB126 alone and combined treatment groups of PCB126 and B(a)P.2 The MN rates in HepG2 cells treated with B(a)P (50μmol/L) and PCB126 (0.01, 0.1,1 and 10nmol/L) alone were 52±6.00‰, 22±8.14‰, 30±10.5‰, 35±7.76‰and 36±6.50‰, compared with solvent control (23±3.05‰), only B(a)P enhanced MN formation. The PCB126 had no effect on MN formation and NDI; In combined treatment groups enhanced MN formation was found (52±4.51‰, 73±5.51‰, 79±4.24‰and 86±2.00‰, compared with group treated with B(a)P alone(P<0.05) except the 0.01nmol/L PCB126 combined with B(a)P group. And they increased 21%,, 27%, and 34% at last three concentrations of PCB126 respectively. The factorial design multivariable ANOVA showed synergistic effect of PCB126 and B(a)P on MN formation. After adding of ANF, the MN formations in combined groups were inhibited obviously.In summary for this part, PCB126 and B(a)P induced the phase I enzyme CYP1A1, CYP1A2 and CYP2B activities at certain concentrations. There was a synergistic effect of PCB126 and B(a)P combination on CYP1A1, CYP1A2 and GST activities and antagonistic effect on CYP2B activities. PCB126 had no effect on MN formation. PCB126 and B(a)P combination showed synergistic effect on MN formation. ANF inhibited phase I enzyme and enhanced phase II enzyme which contributed to MN formation. These confirmed that PCB126 had significant effect on the metabolic enzymes which play the key role in enhancement of genotoxicity of B(a)P.As an Ah receptor ligand, PCB126 can enhance the enzyme activities by influence the Ah receptor. So we investigated the effect of PCB126 and B(a)P combination on mRNA expression of Ah receptor in the third part.PartⅢ: Combined effect of PCB126 and B(a)P on Ah receptor gene expression in HepG2 cellsHepG2 cells were treated with different concentrations of PCB126 (0.01, 0.1, 1, 10nmol/L) and B(a)P (50μmol/L) alone, or pretreated with different concentrations of PCB126 for 48h then treated with B(a)P and PCB126 together for another 24h. DMSO (10ml/L) was used as solvent control. The mRNA expression of Ah receptor was analyzed through RT-PCR kit. The experiments were carried out again after adding the phase I enzyme inhibitor ANF. The results showed that the expression of Ah receptor mRNA in HepG2 cells treated with B(a)P and PCB126 alone were significantly increased (0.111±0.018, 0.258±0.036, 0.416±0.068, 0.717±0.041 and 0.929±0.060) compared with solvent control (0.015±0.004) (P<0.001). Enhanced expression levels of Ah receptor mRNA were found in combined treatment groups (0.424±0.011, 0.616±0.033, 0.823±0.040 and 0.886±0.007) compared with group treated with B(a)P alone(P<0.001) and they increased 31%, 51%, 72% and 78% respectively. Ah receptor mRNA expressions were inhibited obviously after ANF added. Pearson correlation analyses show that the CYP1A1 and CYP1A2 enzyme activities were significantly positive correlated with the mRNA expression of Ah receptor (r=0.838, P<0.01; r=0.900, P<0.01), and mRNA expressions of Ah receptor were significantly positive correlated with MN rates (r=0.899, P<0.01). This findings indicated that Ah receptor-dependent CYP1A induction contributed to enhancement of MN formation and Ah receptor play the key role in synergistic effect of PCB126 in combination with B(a)P.In summary for this part, PCB126 and B(a)P induced Ah receptor mRNA expression at certain concentrations. PCB126 and B(a)P combination showed synergistic effect. As 3-MC enzyme inducer type, PCB126 induced enzyme activities mainly via Ah receptor pathway. As high affinity ligand of Ah receptor, PCB126 can enhance B(a)P-induced genetic toxicity through inducing the Ah receptor to increase the phase I enzyme activities.The main conclusion and the new ideas:The main conclusion:(1) PCBs can increase the phase I enzyme and inhibit the phase II enzyme, therefore enhance the genetic toxicity of B(a)P.(2) As non-coplanar congeners of PCB, PCB153 obviously enhanced the genetic toxicity of B(a)P via effect on metabolic enzymes although related mechanism is still unclear.(3) As coplanar congeners of PCB, PCB126 obviously enhanced the genetic toxicity of B(a)P via Ah receptor pathway by which phase I enzyme activities were increased. It proved that the phase I enzyme play the key role in B(a)P-induced genetic toxicity by combination of PCBs and B(a)P.The new ideas:(1) Establishing the combined-effects model of PCBs-PAH in mammalian cell line in vitro at the first time. Providing effect of changing the phase I and II enzyme activities on enhancement of B(a)P-induced genetic toxicity. Providing the theoretical foundation for further research combined effect of PCBs and B(a)P on human health.(2) Discovering the inhibited effect of PCB153 and PCB126 on GST activities at certain range of concentrations for the first time. Providing the theoretical foundation for the further research of the phase II enzyme activities affected by PCBs.