| Summary: | 碩士 === 國立陽明大學 === 口腔生物研究所 === 101 === Department of Health in Taiwan has documented that oral cancer is the sixth leading causes of cancer death, and the fourth for man. 90% of oral cancer are oral squamous cell carcinoma. About 88% of oral cancer patients have the habit of betel nut chewing, indicating the occurrence of oral cancer and exposure to external carcinogens are highly correlated. Piper betle inflorescence which contains high concentration of safrole, is a unique ingredient of betel quid ( BQ ) in Taiwan. The saliva concentration of safrole in a betel-quid chewer could be as high as 420 μM. Metabolic activation of safrole is initiated through cytochrome P450 and the following generation of an active metabolite can form DNA adduct(s) and initiate carcinogenesis. Previous results showed that safrole increased lipid peroxide generation and hydrogen peroxide. Safrole activated cytosolic arylhydrocarbon receptor ( AhR ) , and then induce CYP1A expression, whereas the AhR protein level in total lysate was not affected. In addition, safrole enhanced oxidative stress and increased Nrf2 protein level in total lysate.
My study aims to know effects of safrole on cytochrome P4501A and its regulatory mechanisms in human oral epidermal cancer cells: the enhancement of benzo(a)pyrene ( B[a]P ) mutagenecity. Comet assay show that pre-exposure safrole and then treatment of B[a]P would enhanced the DNA fragmentation. We used the 4x XRE reporter assay found out that safrole activated cytosolic arylhydrocarbon receptor ( AhR ), and induce CYP1A expression at 30h, whereas the AhR protein level in total lysate was not affected. On the other hand, safrole also induce oxidative stress pathway. Nrf2 protein was increased. The AhR antagonist ( α-napthoflavone and resveratrol ) reduced the safrole-induced XRE activity. Safrole induced CYP1A RNA and protein were decreased by the AhR antagonist and Si-RNA. Safrole induced CYP1A1/2 expression were highly correlated with AhR pathway. In addition to using the CYP1A2 mechanism based inhibitors furafylline, makes safrole induced CYP1A1/2 mRNA expression decreased on behalf of the CYP1A2 involved in the metabolism of safrole. Safrole also enhanced oxidative stress and increased Nrf2 protein level in total lysate. Pretreatment the antioxidant N-acetylcystein and catalase didn’t effect the oxidative stress after treatment 420 μM safrole at 6 hours. On the other hand, we found methylenedioxybenzenes ( isosafrole、hydroxychavicol、myristicin and eugenol ) increased XRE activity and CYP1A expression on OECM-1 cells. Current results demonstrated that safrole induced CYP1A partly through AhR. Safrole may be a factor of the enhancement of the toxicity of AhR ligands, such as B[a]P and increase the risk of oral carcinogenesity. In conclusion, these results demonstrate that safrole can activate 4x XRE promoter sequence and make Nrf2 translocate to nucleus, induce downstream gene CYP1A and NQO1 through AhR and Nrf2. Numerous carcinogens could be bioactived by CYP1A, Safrole may be a possible factor to enhance toxic compound, such as B[a]P to enhance the risk of oral carcinogenesity.
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