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Title: Synergistic effects of UVA and benzo[a]pyrene on cytotoxicity, oxidative stress and apoptosis of KB cells
Other Titles: Zi wai xian A yu ben bing pi dui KB xi bao yu xi bao du xing, yang hua ying ji ji xi bao diao wang zhu fang mian zhi xie tong xiao ying
紫外線 A 與苯並芘對 KB 細胞于細胞毒性, 氧化應激及細胞凋亡諸方面之協同效應
Authors: Zhang, Xiaowei (張效偉)
Department: Dept. of Biology and Chemistry
Degree: Master of Philosophy
Issue Date: 2003
Publisher: City University of Hong Kong
Subjects: Apoptosis
Cell-mediated cytotoxicity
Killer cells
Polycyclic aromatic hydrocarbons
Ultraviolet radiation
Notes: CityU Call Number: QR185.8.K54 Z43 2003
Includes bibliographical references (leaves 66-76)
Thesis (M.Phil.)--City University of Hong Kong, 2003
xi, 76 leaves : ill. (some col.) ; 30 cm.
Type: Thesis
Abstract: Recent studies have indicated that the toxicity of polycyclic aromatic hydrocarbons (PAHs) may be enhanced after exposure to solar ultraviolet (UV) light. Two possible mechanisms may be involved in toxicity enhancement. UV may modify the structure of PAHs, and result in products that are more toxic than the parental compounds (photomodification). Alternatively, UV may enhance PAH molecules to produce reactive oxygen species (ROS) by photosenitization. Using benzo[a]pyrene (BaP) as a model PAH, and human oral epidermoid carcinoma cells (KB) as a cell model, this thesis aims to study the mechanisms involved in the toxicity enhancement of PAHs by UV. Experiments were designed to: 1) evaluate the cytotoxicity of photomodified products produced after BaP was irradiated by solar UV light; 2) investigate the relationship between the production of intracellular ROS and formation of 8-Hydroxy-2'deoxyguanosine (8-OHdG), a specific marker for DNA oxidation, in cells co-exposed to BaP and UVA; and 3) examine the role of intracellular ROS production and the integrity of mitochondria in programmed cell death (apoptosis) induced by co-exposure to UVA and BaP. In solution, 28% of BaP is degraded upon irradiation of solar UV (72.5 +- 5 mW /cm2 for 24 hour). Results of MTT (3-(4, 5-dimethylthiazol-2-y1)-2, 5- diphenyl tetrazolium bromide) assays however, revealed no significant difference in cytotoxicity between BaP and its photomodified products after UV irradiation. In contrast, a significant decrease in cell viability (60%) was observed when cells pretreated with BaP (3.3 uM) were irradiated by UVA (0.50 mW /cm2 of for 20 min.) Our results indicated that the observed enhancement in cytotoxicity after coexposure to UVA and BaP is caused by photosensitization, not photomodification. To investigate the relationship between production of ROS and formation of 8-OHdG, KB cells were exposed to BaP for 24 h, followed by exposure to UVA (365 nm) or UVB (3 12 nm). The levels of intracellular ROS were measured directly using flow cytometry coupled with the use of a fluorescent probe, dihydrorhodamine 123 (DHR-123). Levels of 8-OHdG were measured using high performance liquid chromatography coupled with electrochemical detection (HPLC-ECD). Under the same dose of UV energy (0.10 mW /cm2, 20 min), a substantial increase in ROS was found in the UVA plus BaP (13.3 uM) treatment (2.9 fold, as compared with UVA control), while a lower level of ROS production (1.3 fold, as compared with UVB control) was found in the treatment of UVB plus BaP (13.3 uM). These results indicated that BaP acts primarily as a photosensitizer of UVA, but not UVB. Furthermore, the increased intracellular ROS production was dependent upon both concentration of BaP and dose of UVA. A linear relationship between levels of ROS and 8-OHdG formation in cells co-exposed to BaP and UVA was clearly evident. To study the role of ROS in apoptosis induced by BaP and UVA, cells were pre-incubated with BaP (750 nM) for 24 h, followed by UVA irradiation (0.30 or 0.60 J/cm2) before further culture. DNA content, mitochondrial integrity and micronucleus induction in KB cells were investigated. Fluorescence microscopy studies showed intracellular localization of BaP was closely associated with a burst of ROS within mitochondria of KB cells co-exposed to BaP and UVA, indicating that mitochondria are the most susceptible organelles. Cell cycle analysis indicated that significant accumulation of sub-GI cells were induced by BaP treatment in darkness. However, under UVA irradiation, BaP induced S-phase arrest was released after 20 h further culture. Swelling of the mitochondrial membrane was found in cells coexposed to UVA and BaP after a further 6 h culture, indicating that the increase in intracellular ROS production could lead to mitochondrial dysfunction, and consequently the release of cytochrome c into the cytosol. Furthermore, the frequency of occurrence of micronuclei increased in cells treated with BaP and UVA after being cultured for a further 24 h, showing that increased ROS production could enhance genotoxicity. Overall, results suggested that the synergistic effects induced by solar UV and BaP are mediated through the enhancement of intracellular ROS production resulting from photosensitization. The intracellular ROS burst could increase genotoxicity by impairing DNA and its repairing systems, as well as mitochondria dysfunctions and consequently apoptosis.
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