Toxicity mechanisms of chlorpyrifos and trifloxystrobin pesticides in human keratinocytes : 클로르피리포스와 트리플록시스트로빈 농약의 피부세포 내 독성 기전 연구

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수의과대학 수의학과
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서울대학교 대학원
PesticidesPesticide exposureToxicity mechanismReactive oxygen speciesHuman skin keratinocyte
학위논문 (박사)-- 서울대학교 대학원 : 수의학과, 2017. 2. 채찬희.
Pesticides have provided significant benefits including plant disease control and increased crop yields since they were developed and utilized. However, pesticide causes many adverse effects on non-target organisms including human, which necessitate precise toxicity tests and risk assessment. Integumentary system is a main exposure site of pesticide exposure. Therefore, it is important to investigate toxicity mechanism of pesticides especially in skin. Chlorpyrifos, and trifloxystrobin are widely-used pesticides for both agricultural and residential application. Although various studies have reported toxicity and health-related effects by their exposure, the toxicity mechanism in skin cells has not been well-characterized. The present study determined the potential mechanism involved in skin toxicity of chlorpyrifos and trifloxystrobin using HaCaT human skin keratinocyte cell line.
First, after treating chlorpyrifos to HaCaT cells, we examined changes in the cells. Chlorpyrifos triggered reactive oxygen species (ROS) generation and mitochondrial oxidative stress. We focused on NLRP3 inflammasome, known to induce innate immune response. We used mitochondrial ROS (mROS) scavenger mitoTEMPO to demonstrate a role for mROS in NLRP3 inflammasome and programmed cell death induced by chlorpyrifos. These results showed that chlorpyrifos provoked NLRP3 inflammasome and pyroptosis/apoptosis via an increase of mROS in HaCaT cells, and could propose that chlorpyrifos induces innate immune response and skin inflammation through activating the NLRP3 inflammasome in skin epithelial cells.
Second, skin toxicity mechanism of trifloxystrobin was explored using HaCaT cells. Following treatment of trifloxystrobin, cell viability, and subsequent Annexin V-FITC/propidium iodide assay, TUNEL assay and Western blotting were performed to investigate the cell death mechanism of trifloxystrobin. Exposure to trifloxystrobin resulted in diminished viability of HaCaT cells in both a time- and concentration-dependent manner. The cell death was derived through apoptotic pathways in the HaCaT cells. Furthermore, we explored the effect of trifloxystrobin on TRAIL-mediated extrinsic apoptosis using siRNA transfection. Knockdown of death receptor 5 suppressed trifloxystrobin-provoked apoptosis. These results indicate that trifloxystrobin induces TRAIL-mediated apoptosis and has an inhibitory effect in keratinocytes that can interfere with the barrier function and integrity of the skin.
Third, we investigated the impact of trifloxystrobin on exposed skin at the cellular organelle level in skin keratinocytes. HaCaT cells were treated with trifloxystrobin for 48 h and trifloxystrobin showed detrimental effects on mitochondria evidenced by altered mitochondrial membrane potential and morphology. To identify autophagic degradation of the damaged mitochondria, confocal imaging and Western blotting were performed. Trifloxystrobin induced autophagy and mitophagy-related proteins in HaCaT cells. The mitoTEMPO was applied to further explore the mechanism of trifloxystrobin-mediated mitophagy, and it alleviated the effects on mitophagy induction. Our findings indicated that mitochondrial damage and mitophagy may play a role in trifloxystrobin-induced toxicity in human keratinocytes and this could be suggested as a mechanism of cutaneous diseases developed by exposure.
Taken together, these findings could be suggested as mechanisms of cutaneous diseases developed by the pesticide exposure. Furthermore, our studies can contribute to the assessment of pesticide-induced diseases and be considered in the management of pesticide use and its adverse effects.
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College of Veterinary Medicine (수의과대학)Dept. of Veterinary Medicine (수의학과)Theses (Ph.D. / Sc.D._수의학과)
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