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In Vitro Study of Trichloroethylene-induced Keratinocytes Damage and Protective Role of Vitamin E and Ginkgo Biloba Leaf Extracts

Author: ZhuQiXing
Tutor: ZhangXueJun；YangSen
School: Anhui Medical University,
Course: Dermatology and Venereology
Keywords: Trichloroethylene keratinocytes oxidative stress nitric oxide apoptosis Vitamin E Ginkgo biloba leave extracts
CLC: R751
Type: PhD thesis
Year: 2005
Downloads: 183
Quote: 1
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Abstract

Trichloroethylene (TCE) is an organic solvent, commonly used in a variety of industrial processes, including removing grease from metal parts or lenses and dry cleaning. Epidemiological investigations have shown that TCE - induced skin lesions through direct contact are becoming increasingly prevalent in developing countries including China, which received considerable attention in occupational health fields. However, the mechanisms underlying TCE derma-toxicity and effective protection approaches are not yet available, which limited treatment and prevention of TCE-induced skin lesion. Keratinocytes (KCs) are the structural backbone of the epidermis and the outermost layer of the skin that forms an environmental barrier, and there is increasing evidence that KCs play an active role in the pathogenesis of skin diseases. So investigating TCE-induced damage on KCs and searching for potentially useful preventive approaches would provide a rationale for further exploring the mechanism of TCE-induced skin lesions and developing more effective measures in prevention and treatment of the TCE-associated skin lesions.In the present study, we used normal human epidermal keratinocytes as an in vitro model to test whether TCE would exert a potent cytotoxic effect on the KCs, including cell viability, membrane integrity, oxidative stress, nitric oxide (NO) and nitric oxide synthase (NOS) activity and mRNA expression, morphologic changes of cellular substructure and apoptosis. To explore the mechanism of TCE-induced irritative contact dermatitis and assess the protective effects of Ginkgo biloba leave extracts (EGb) and Vitamin E against the TCE-induced KCs damage.Objective To investigate the damage of TCE on normal human epidermal KCs in vitro culture, explore the mechanism by which TCE induced KCs damage, and assess the protective effects of antioxidant Vitamin E and EGb on TCE-induced human KCs damage.Methods KCs were obtained from normal skin digested with 0.25% trypsin by two steps, and cultured in K-SFM medium in vitro. After mixing the KCs derived from 3 or more different donors together, the following assays were carried out with or without antioxidant Vitamin E and EGb. Neutral red uptake (NRU) assay was used to measure the NR50 of TCE on KCs and determine the esposure dose of TCE. Cell membrane integrity was assessed by lactate dehydrogenase (LDH) release test. Malondialdehyde (MDA) contents, superoxide dismutase (SOD) activity, reactive oxygen radical (ROS) levels, NO and NOS activity were measuree by kits. NOS mRNA expression were assessed by RT-PCR. DNA gel electrophoresis, transmission electron microscopy and flow cytometry were used to observe DNA fragment, morphologic changes, DNA contents, and cell apoptosis rate respectively.Results (1) NRU assay showed that TCE caused a dose-dependent decrease in cell viability. The NR50 value of TCE on cultured human KCs was found to be 4.5252 mmol/L with a 95% confidence interval of (3.9221, 5.1284); NR50 of the positive control’s SLS on cultured human KCs was 0.2773 mmol/L within the 95% confidence interval of (0.1930, 0.3615) provided by SOP. A time- and concentration-dependent release of LDH was observed at 1, 2, 3, 4 h after the cells were exposed to different doses of TCE. TCE also caused an increase in MDA content and ROS level, whilst an inhibition of SOD activity, in a concentration-dependent manner. NO and NOS activity was elevated in time- and concentration-mode in KCs exposed to TCE. iNOS mRNA expression was up-regulated time- and concentration- dependently, while cNOS mRNA expression was up-regulated concentration- dependently only. In addition, TCE-induced KCs apoptosis was observed in vitro. TCE caused not only Ladder-DNA fragment, but also the typical morphologic changes of apoptosis under electron microscope and proportional increase of apoptotic cells dose-dependently by FCM assay.(2) After pre-treatment of the cells for 2 h with vitamin E at the concentrations of 10-200uunol/L, the cell viability impaired by TCE 5.0 mmol/L was restored to the normal level with the dose-dependence. This suggests that Vitamin E exerted a significantly protective effect on TCE-impaired cell viability. Consisting with this. Vitamin E significantly reduced the LDH release induced by TCE (2.0 mmol/L) at the same doses-dependence. Meanwhile, the elevated level of either MDA, ROS, NO, or NOS and the lowered activity of SOD were inhibited significantly by vitamin E at the different concentrations in a dose-dependent manner. The up-regulation of iNOS mRNA and cNOS mRNA expression in KCs exposured to TCE were also inhibited by Vitamine E at 72 h. After treatment with vitamin E (150*ttmol/L)for 2 h, the apoptosis could not be detected by using of DNA gel electrophoresis , morphologic observation under electronic microscope, and counting the number of apoptotic cells.(3) The cell viability lowered by TCE (5.0 mmol/L) was restored to normal level after treatment for 2 h with EGb (10-200mg/l). Additionally, EGb significantly inhibited the increase of LDH release caused by TCE (2.0 mmol/L) in a dose-dependent manner. TCE at the concentration of 2.0 mmol/L increased the level of MDA, ROS, and NO, activated NOS, and inhibited the activity of SOD, which were blocked by EGb with concentration-dependence. EGb also inhibited the up-regulation expression of iNOS mRNA and cNOS mRNA in KCs caused by TCE (2.0 mmol/L) at 72 h, which consisted with the change of NOS activity. After pre-treatment of the cells with 150mg/L, the ladder was not detected, the cells presented no morphologic difference from the control and apoptosis rate was substantially decreased.

In Vitro Study of Trichloroethylene-induced Keratinocytes Damage and Protective Role of Vitamin E and Ginkgo Biloba Leaf Extracts

Abstract

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