研究雙特松對HepG2細胞之DNA修復的影響;The impact on DNA repair in HepG2 cells treated with Dicrotophos

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Title:	研究雙特松對HepG2細胞之DNA修復的影響;The impact on DNA repair in HepG2 cells treated with Dicrotophos
Authors:	林儀臻;Lin, I-Chen
Contributors:	生命科學系
Keywords:	雙特松;DNA損傷;DNA修復;BER;NER;MMR;HR;NHEJ;HepG2;Dicrotophos;DNA damage;DNA repair;BER;NER;MMR;HR;NHEJ;HepG2
Date:	2023-08-16
Issue Date:	2024-09-19 15:21:25 (UTC+8)
Publisher:	國立中央大學
Abstract:	有機磷類 (Organophosphate, OP) 是一種用於防治害蟲的殺蟲劑。由於其實用性高且對環境毒性低，它們被認為是使用最廣泛的殺蟲劑。雙特松 (Dicrotophos, Dic) 是一種含有有機磷的殺蟲劑，由於其目標害蟲之種類十分廣泛，故被大量地使用於各國。儘管其有效性已被證實，但已有推測認為Dic可能與人類的基因毒性作用、基因永久突變與疾病有關。然而，關於此基因毒性之損傷的機制卻尚未有詳細的闡述。先前，透過鹼性彗星試驗，我們揭露了Dic可能引起DNA單股斷裂的能力。為了進一步找出此單股斷裂可能造成的影響，我們從先前處理過Dic的細胞中獲取無細胞提取物 (cell-free extract)，再利用彗星試驗之體外DNA 修復檢測來評估其鹼基切除修復 (Base excision repair, BER) 和核苷酸切除修復 (Nucleotide excision repair, NER) 的活性。在有Dic處理過的組別中，其所產生的DNA尾巴之長度較未處理過之對照組短，說明BER和NER修復蛋白之切割能力皆受到了Dic的抑制。因此，我們可以得出BER和NER在Dic處理後均被抑制之結論。有鑑於未修復之單股斷裂有可能發展成DNA之雙股斷裂，我們進一步使用中性彗星試驗和H2AX之磷酸化 (γH2AX) 以檢測Dic處理後，細胞所產生之雙股斷裂的情形。兩種試驗皆顯示在Dic處理後，細胞雙股斷裂之情形有顯著升高的現象。為了進一步再探討細胞針對此雙股斷裂之情形的反應機制，我們分別利用了同源重組修復 (Homologous recombination, HR) 試劑與西方墨點法來評估HR與非同源性末端黏合之修復 (Non-homologous end joining, NHEJ) 的活性或修復蛋白之基因表現量。結果顯示，在HR活性試驗中，與未處理過之對照組相比，Dic顯著地抑制了HR之修復活性。此外，NHEJ之重要相關基因Ku80蛋白表現量，同樣在經Dic處理後也顯著降低。考慮到錯誤配對之DNA修復 (Mismatch repair, MMR) 是防止DNA複製與修復之錯誤的重要機制，我們再度進一步利用西方墨點法測定了在Dic處理後，MMR之重要相關基因msh2和mlh1的基因表現情形。結果顯示，在Dic處理過之細胞中，其MSH2和MLH1之蛋白表現量皆有顯著下調的趨勢，表示MMR修復之效能有可能在Dic處理過後發生改變。總結來說，我們揭示了在Dic處理後所引起的基因毒性可以對多個DNA修復途徑之活性產生負面的影響。本研究針對雙特松之潛在負面影響提供了更詳細的分子機制，可有助於調節人體健康的發展。;Organophosphates (OP) are a group of insecticides used for pest control. Due to their high level of utility, yet relatively low environmental toxicity, they are considered the most widely used insecticides. Dicrotophos (Dic), an OP insecticide, is extensively used due to its broad spectrum of target pests. Despite its effectiveness, Dic has been speculated to be asso-ciated with genotoxic effects, gene mutagenicity, and diseases in humans. However, the mechanism in terms of genotoxic insults has not been fully explicated. Previously, by using the alkaline comet assay, we revealed Dic could induce DNA sin-gle-strand breaks (SSBs). To further determine the response of SSBs, we assessed the activity of Base excision repair (BER) and Nucleotide excision repair (NER) via the comet-based in vitro DNA repair assay, utilizing a cell-free extract derived from cells that were formerly subjected to Dic. In the Dic-treated groups, the length of the DNA tail was shorter compared to the non-exposed groups, indicating the incision ability of BER/NER repair enzymes was decreased by Dic. Thus, we concluded that both BER and NER were suppressed after Dic exposure. Given the fact that unrepaired SSBs could potentially develop into DNA double-strand breaks (DSBs), we further evaluate the extent of DSBs following Dic exposure by implementing the neutral comet assay, along with the assessment of the expression level of the phosphorylated form of H2AX (γH2AX). Both assays revealed a notable increase in the level of DSBs after Dic ex-posure. To determine the response of DSBs, homologous recombination (HR) activity and non-homologous end joining (NHEJ) were evaluated via the HR assay kit and western blot, respectively. Our results showed Dic significantly attenuated the HR repair activity com-pared to the non-exposed control. Moreover, the expression level of the NHEJ-associated gene, Ku80 was also significantly reduced after Dic exposure. Considering that Mismatch repair (MMR) is a vital mechanism for preventing errors in DNA replication and repair, we were prompted to investigate the expression of MMR-associated genes, msh2 and mlh1, fol-lowing Dic exposure. Our results showed that both MSH2 and MLH1 protein expression were notably downregulated in Dic-treated cells, suggesting the alteration of the MMR effi-cacy might occur after Dic exposure. Collectively, we highlighted that genotoxicity caused by Dic exposure negatively modulates the activity of multiple DNA repair pathways. This present study provides a more detailed mechanism into the potential adverse effects of Di-crotophos, which may further help in modulating health complications.
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