Abstract

In this study, we have studied the cytotoxicity and genotoxic potency of 3 pro-oxidants; H₂O₂, menadione and KBrO₃ in different dosing scenarios, namely acute (1-day dosing) and chronic (5-days). For this purpose, relative population doubling (RPD%) and mononucleated micronucleus (MN) test were used. TK6 cells and NH32 were employed in in vitro experiments. In the study, the total acute dose was divided into 5 days for each prooxidant chemicals by dose fractionation (1/5th per day) method. Acute dosing was compared to chronic dosing. The oxidative stress caused by the exposure of cells with pro-oxidant chemicals to the cells was determined by an optimized 2′,7′-dichlorofluorescein diacetate (DCFHDA) test method. The antioxidant levels of the cell lines were altered with buthionine sulfoxide (BSO) and N-acetyl cysteine (NAC), and the effect of antioxidant capacity on the MN formation in the cells was observed with this method. In the case of H₂O₂ and menadione, fractional dosing has been observed to result in lower toxicity and lower genotoxicity. But in the case of KBrO₃, unlike the other 2 pro-oxidants, higher MN induction was observed with fractionated doses. DCFHDA test clearly demonstrated ROS induction with H₂O₂ and menadione but not with KBrO₃. Unexpectedly, DCFHDA test demonstrated that KBrO₃ did not cause an increase ROS levels in both acute and chronic dosing, suggesting an alternative ROS induction mechanism. It was also observed that, treatment with BSO and NAC, caused increasing and decreasing of MN fold change respectively, allowing further ROS specific mechanisms to be explored. Hence, dose fractionation expectedly caused less MN, cytotoxicity and ROS formation with H₂O₂ and menadione exposure, but not with KBrO₃. This implies a unique mechanism of action for KBrO₃ induced genotoxicity. Chronic dosing in vitro may be a valuable approach allowing better understanding of how chemicals damage DNA and pose human hazards.

中文翻译：

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长期给药和 p53 状态对体外促氧化化学物质遗传毒性的影响

摘要

在这项研究中，我们研究了 3 种促氧化剂的细胞毒性和基因毒性效力；H ₂ O ₂、甲萘醌和KBrO ₃不同给药方案，即急性（1天给药）和慢性（5天给药）。为此，使用相对群体倍增（RPD%）和单核微核（MN）测试。TK6细胞和NH32用于体外实验。在该研究中，通过剂量分割（每天 1/5）方法将每种促氧化化学物质的总急性剂量分为 5 天。将急性给药与慢性给药进行比较。通过优化的2',7'-二氯荧光素二乙酸酯（DCFHDA）测试方法测定细胞暴露于促氧化化学物质引起的氧化应激。用丁硫氨酸亚砜（BSO）和N-乙酰半胱氨酸（NAC）改变细胞系的抗氧化水平，并观察抗氧化能力对细胞内MN形成的影响。对于H ₂ O ₂和甲萘醌，已观察到分次给药可导致较低的毒性和较低的遗传毒性。_{但就 KBrO 3}而言，与其他 2 种促氧化剂不同，分次剂量观察到更高的 MN 诱导作用。DCFHDA 测试清楚地证明了 H ₂ O _{2和甲萘醌的 ROS 诱导作用，但 KBrO 3}则不然。出乎意料的是，DCFHDA 测试表明 KBrO ₃在急性和慢性给药中均不会导致 ROS 水平增加，这表明存在另一种 ROS 诱导机制。还观察到，BSO 和 NAC 处理分别引起 MN 倍数变化的增加和减少，从而允许进一步探索 ROS 特异性机制。因此，剂量分割预计会导致 H ₂ O _{2和甲萘醌暴露的 MN、细胞毒性和 ROS 形成减少，但 KBrO 3}则不会。_{这意味着 KBrO 3}诱导的遗传毒性具有独特的作用机制。体外长期给药可能是一种有价值的方法，可以更好地了解化学物质如何损害 DNA 并对人类造成危害。