| Summary: | 碩士 === 中山醫學院 === 毒理學研究所 === 86 ===
This thesis was to study the toxicity of environmental carcinogenbenzo[a]pyrene (B[a]P) and cooking oil fume (COF) in lung cell lines (CL-3, MRC-5 and A427). First we examined effects of 24 hrs treatment with 1 or 10 μM B[a]P on cell growth. Viable CL-3 cell number was reduced to only 42% or 22% of control at 96 hr after removal of B[a]P. However, viable A427 cell number was similar between control and B[a]P treated cells. Second, we utilized the Comet assay to detect DNA single strand break. After 10 μM B[a]P treatment for 24 hrs, the tail moment of CL-3 cell increased from 10.15 mm to 38.81 mm, indicating the presence of DNA single strand breaks. However, no DNA strand break was observed in 10 μM B[a]P-treated A427 cell. To understand the mechanism of difference in B[a]P sensitivity between CL-3 and A427 cell, the following experiments were performed. B[a]P is metabolically activated by cytochrome P450 1A1 (CYP1A1) and, on the other hand, also induces CYP1A1 enzyme activity through activation of aryl hydrocarbon receptor (AhR). We found that CYP1A1 enzyme actiivty, as measured by aryl hydrocarbon hydroxylase (AHH) assay, was induced by 10 μM B[a]P in CL-3 cell. However, B[a]P treatment failed to induce CYP1A1 activity in A427 cells. Pretreatment with α-naphthoflavone decreased effects of B[a]P on CYP1A1 induction and cell growth in CL-3 cell. AhR was only activated by B[a]P in CL-3 cell instead of A427 cell, as measured by gel retardation assay. Glutathione s-transferase (GST) plays a role in detoxification pathway of B[a]P . We found that GST enzyme activity in A427 cell (21.16 nmol/min/mg) was higher than in CL-3 cell (8.22 pmol/min/mg). In addition, CL-3 cell lacked GST M1 gene. In summary, B[a]p failed to activate AhR and induced CYP1A1 enzyme activity in A427 cell and A427 cell has high GST enzyme activity. These may explain the resistance to B[a]p in A427cell.
COF inhibited gromth of CL-3 and MRC-5 cells. Treatment with 200*g/ml COF for 24 hrs reduced viable cell number to 39% and 46% of control 96 hrlater. COF also induced DNA damage as detected by the Comet assay. After treatment with 200 *g/ml COF for 24 hrs, tail moment in CL-3 and MRC-5 cells were respectively increased from 10.15 mm tp 42.16 mm and 1.92mm to 42.16 mm. CYP IAI enzyme activity in CL-3 cell was only slightly increased by 10 *M B[a]P treatment. Oxidative stress was induced by treatmenr with 200 *g/ml COF in CL-3 cell, as indicated by formation of 8-OH deoxy-guanosine (8-OH dG).P retreatment with n-acetylcysteine (NAC) or dicoumarol reduced effect of B[a]P on cell growth in CL-3 and MRC-5 cells. From these data we concluded that COF inhibited cell growth and caused DNA damage in MRC-5 and CL-3 cells. COF toxicity may related to oxidative stress and require metabolic activation by NAD(P)H quinone oxidoreductase (NQO1) in CL-3 and MRC-5 cells.
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