| Summary: | 碩士 === 國立中興大學 === 環境工程學系所 === 98 === Benzo[a]pyrene (BaP) is classified as a carcinogen in humans and in laboratory animals. It is generated through incomplete combustion of hydrocarbons. Animal studies and epidemiological surveys have demonstrated that BaP induces adverse biological effects, including induction of oxdative stress, DNA damage, and cancer. The objective of this research is to examine the effects of one of the polyaromatic hydrocarbons (PAHs), i.e. BaP, in modulating the estrogen-induced induction of cytotoxic response, oxidative stress, altered gene expression, and DNA damage in human MCF-7 and MDA-MB-231 breast cancer cells.
The first part of this research was to investigate whether the BaP induces oxdative stress and DNA damage in human breast cancer cells. Results indicated that BaP induced concertration- and time-dependent increases in cytotoxic response in human breast cancer cells and that the degree of cytotoxic response induced by BaP was greater in MCF-7 cells than in MDA-MB-231cells. Further, BaP induced ROS formation in both MCF-7 and MDA-MB-231cells after exposure for up to 72 h. Additionally, the extent of DNA strand breaks were determined by the Comet assay. After 72h of exposure, significant increases in DNA strand breaks were detected in MCF-7 and MDA-MB-231 cells exposed to BaP at concertrations 1 and 10 μM. This finding suggests that BaP induced concentration- and time-dependent increases in the extent of DNA strand breaks in both MCF-7 and MDA-MB-231 cells.
To determine the effects of AhR and ERα status on the induction of DNA strand breaks by BaP, we incubated MCF-7 cells with BaP for 72h in the presence of an AhR or an ERα inhibitor. Results clearly showed that in the presence of a AhR inhibitor significant decreases in DNA strand breaks were detected in MCF-7 cells treated with BaP for 72h. Co-treatment of BaP with a ERα inhibitor further enhanced the extent of DNA strand breaks in MCF-7 cells. Furthermore, the addition of Catalase and Superoxide dismutases to the incubate reduced the BaP-mediated induction of DNA strand breaks in MCF-7 cells. We noticed that treatment of cells with BaP resulted in significant increase in the expression of CYP1A1 protein level in MCF-7 whereas slight increase CYP1A1 protein level was observed in BaP-treated MDA-MB-231 cells.
The second part of this research was to uncover the effect of BaP pretreatment and ERα status on the induction of oxdative stress and DNA damage by 17 beta -estradiol (E2) in human MCF-7 and MDA-MB-231 breast cancer cells. Pretreatment of BaP did not modulate the extent of the cytotoxic response and oxidative stress induced by estrogen in MCF-7 and MDA-MB-231 cells. In contrast, we observed that E2 (10 nM) alone induced DNA strand breaks in MDA-MB-231 cells as measured by the Comet assay. Further investigation indicated that the DNA-damaging and repairing effects induced by E2 in MDA-MB-231 cells were completely blocked by pretreatment of BaP (10 μM for 72h ). On the contrary, with BaP pretreatment, significant increases in DNA strand breaks and CYP1A1 expression were detected in MCF-7 cells exposed to E2. Overall, this evidence suggests that similar to dioxins, BaP is capable of inducing imbalances in the expression of enzymes responsible for the bioactivation of estrogen leading to the subsequent accumulation of DNA damage in MCF-7 and MDA-MB-231 cells. Furthermore we confirmed that ERα plays a protective role in modulating the induction of DNA damage by E2 in human breast cancer cells.
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