Cadmium Exposure Sensitizes NIH3T3 Cells To Bisphenol A-Induced DNA Oxidative Damage

Objective:Cadmium (Cd) or Bisphenol A (BPA), with its widespread distribution and ubiquitous presence in human daily life, is easy for human to contact with through the common route such as oral or breath exposure. Previous studies have shown the possible genotoxicity of Cd or BPA in vitro or vivo and also found that Cd at non-cytotoxic concentrations may enhance genotoxicity of other DNA damaging agents. However, there is no research about the toxicity of combined exposure to Cd and BPA Therefore, our objective is to investigate if Cd exposure at non-cytotoxic dose enhance genotoxicity of BPA. In this study, the NIH3T3 cells were pretreated without or with 5 or 10 μ M Cd for 24h and then treated with 10μM BPA, then cell viability, LDH release, DNA damage, cell cycle distribution and apoptosis were subsequently detected. The results show that Cd or BPA treatment alone failed to cause any significant change in all indexes compared with control, however, interestingly, the treatment of combined Cd and BPA significantly increases cytotoxicity, DNA damage, G2 phase arrest, the percentage of TUNEL positive cells and cleaved-PARP expression level. These findings infer that Cd exposure at non-totoxic dose increase genotoxicity of BPA and offer theoretical foundation for further best protection of harm effects induced by Cd and BPA exposure. However, the mechanisms of Cd at non-cytotoxic dose sensitizing cells to BPA exposure need for further research.Objective:Cadmium chloride (CdCl2) or Bisphenol A (BPA), with its widespread distribution and ubiquitous presence in human daily life, is easy for human to contact with through the common route such as oral or breath exposure. Previous studies have shown that Cd at non-cytotoxic concentrations may enhance genotoxicity of other DNA damaging agents such as γ-rays or benzo[a]pyrene (B[a]P). However, there is no research about the toxicity of combined exposure to Cd and BPA. Therefore, our objective is to investigate if Cd exposure at non-toxic dose enhance genotoxicity of BPA and research the possible mechnism.Method:The NIH3T3 cells In the logarithmic phase were plated, after adherence, cells were pretreated without or with 5 or 10 μ M CdCl2 for 24h and then administrated without or with 2..10 or 50 μ M BPA for another 24h. In this study, there exists four groups:(1) control; (2) only CdCl2 treated group:cells were just treated with 5 or 10 μM CdCl2 for 24h; (3) only BPA treated group:cells were only treated with 2n 10 or 50 μM BPA for 24h; (4) CdCl2 +BPA treated group:after adherence, cells firstly were pretreated with 5 or 10 u M CdCl2 for 24h and then conducted with 2、10 or 50 μ M BPA for another 24h. Following by BPA treatment, the Cell counting Kit-8(CCK-8) was used to detect cell viability, LDH release levels were determined by Cytotoxicity LDH detection kit; Reactive oxygen(ROS) detection kit was conducted to detect the levels of ROS; DNA damage was determined by alkaline comet assay and Y H2AX expression levels were measured by immunofluorescence assay and Western blot; Flow cytometry was employed to examine cell cycle distribution; TUNEL staining and the expression levels of cleaved-PARP detected by Western blot were performed to assess cells apoptosis.Results:① Compared to control, in only CdCl2 treated group,5 or 10 μ M CdCl2 treatment failed to cause any significant change (P>0.05) in all indexes such as cell viability、LDH release levels、ROS levels、comet parameters (Tail DNA%、TL、TM、OTM)、γH2AX expression levels、 cell cycle distribution、 the percentage of TUNEL positive cells and cleaved-PARP expression levels.② In only BPA treated group,2 u M or 10 u M BPA treatment is not enough to induce any dramatic change on cell viability、LDH release levels、ROS levels、comet parameters (Tail DNA%、TL、TM、OTM、 γH2AX expression levels、cell cycle distribution、the percentage of TUNEL positive cells and cleaved-PARP expression levels when compared with control group(P>0.05); however,50 u M BPA treatment in comparison with control decreased cell viability, increased LDH release levels、ROS generation、comet parameters (Tail DNA%、TL、TM、OTM), as well as Y H2AX expression levels, induced G2 phase arrest and the increase of the percentage of TUNEL positive cells and cleaved-PARP expression levels(P<0.05).③CdCl2 exposure at non-cytotoxic dose increase genotoxicity of BPA. CdCl2+BPA treated group compared with only BPA treated group:5 or 10 μM CdCl2 pretreatment sensitized NIH3T3 cells to 10 μM or 50 μM BPA-induced cell viability inhibition, the increase of LDH release、comet parameters (Tail DNA%、TL、TM、OTM)、γH2AX expression levels、 G2 phase arrest as well as the increase of the percentage of TUNEL positive cells and cleaved-PARP expression levels(P<0.05); Noteworthily, compared with only 2μM BPA treatment,5 u M CdCl2 pre-exposure have not been enough to sensitize NIH3T3 cells to 2 u M BPA-induced DNA damage, with no significant change on above indexes (P>0.05), nevertheless,10 μ M CdCl2 pre-exposure can change the indexes above induced by only 2 μ M BPA treatment(P<0.05).Conclusion:CdCl2 pre-exposure at non-cytotoxic dose of 5 or 10 μ M increased genotoxicity of BPA to NIH3T3cells, even sensitized cells to BPA at non-cytotoxic dose of 2 or 10 μ M induced DNA damage. One of the mechanisms that CdCl2 accelerated cells to BPA-induced DNA damage was associated with increased intracellular ROS generation. This study inferred that exposure to non-toxic dose CdCl2 may sensitize organism to toxicants such as BPA- induced injure, except that, these data not only appealed to us to cause more attention on the hazard of cadmium and BPA exposure, but also offered theoretical foundation for related government search further best protection of harm effects induced by Cd and BPA exposure. However, the mechanisms of Cd at non-cytotoxic dose sensitizing cells to BPA-induced genotoxicity need for further research.