National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan.
Mutat Res. 2011 Jul 14;723(1):58-64. doi: 10.1016/j.mrgentox.2011.04.002. Epub 2011 Apr 29.
The fungal toxin, Ochratoxin A (OTA), is a common contaminant in human food and animal feed. The present study evaluated micronucleus (MN) induction by OTA in comparison with its ability to induce cytotoxicity and DNA damage in two mammalian cell lines, CHO-K1-BH(4) Chinese hamster ovary cells and TK6 human lymphoblastoid cells. Micronuclei were evaluated by flow cytometry, cytotoxicity was estimated by relative population doubling (RPD), while direct DNA damage and oxidative DNA damage were measured with the Comet assay, performed without and with digestion by formamidopyrimidine-DNA glycosylase (fpg). For the MN and cytotoxicity measurements, the cell lines were treated for 24h (CHO cells) or 27h (TK6 cells) with 5-25μM OTA in the absence of exogenous metabolic activation. The OTA treatments resulted in concentration-responsive increases in cytotoxicity, with higher concentrations of the agent being more cytotoxic in CHO cells than TK6 cells. 15μM OTA produced positive responses for MN induction and hypodiploid events (a measure of aneugenicity) in both cell lines; this concentration of OTA also produced cytotoxicity near to the recommended limit for the assay (45±5% RPD). A time course assay with TK6 cells indicated that at least 4h of OTA treatment were required to produce a positive MN response. For the Comet assay DNA damage assessments, the cell lines were treated with 5-50μM OTA for 4h. Direct DNA damage was detected in TK6 cells, but not CHO cells, while concentration-related increases in fpg-sensitive sites were detected for both cell lines. The consistent association of oxidative DNA damage with OTA exposure suggests its involvement in producing OTA-induced clastogenicity and aneugenicity; however, based on its detection in TK6 cells direct DNA damage could be involved in any human risk posed by OTA exposure.
真菌毒素赭曲霉毒素 A（OTA）是人类食品和动物饲料中常见的污染物。本研究评估了 OTA 诱导微核（MN）的能力，并将其与在两种哺乳动物细胞系，CHO-K1-BH(4)中国仓鼠卵巢细胞和 TK6 人淋巴母细胞中的细胞毒性和 DNA 损伤的诱导能力进行比较。微核通过流式细胞术进行评估，细胞毒性通过相对群体倍增（RPD）来估计，而直接 DNA 损伤和氧化 DNA 损伤则通过 Comet 测定法进行测量，该测定法在没有和存在解旋酶（fpg）消化的情况下进行。对于 MN 和细胞毒性测量，在不存在外源性代谢激活的情况下，用 5-25μM OTA 处理细胞系 24h（CHO 细胞）或 27h（TK6 细胞）。OTA 处理导致细胞毒性呈浓度依赖性增加，该试剂的较高浓度在 CHO 细胞中比 TK6 细胞的细胞毒性更高。15μM OTA 在两种细胞系中均引起 MN 诱导和亚二倍体事件（一种着丝粒外遗传效应的测量）的阳性反应；该浓度的 OTA 也在接近测定法建议的限（45±5%RPD）的情况下产生细胞毒性。用 TK6 细胞进行的时间过程测定表明，至少需要 4h 的 OTA 处理才能产生阳性 MN 反应。对于 Comet 测定法的 DNA 损伤评估，用 5-50μM OTA 处理细胞系 4h。直接 DNA 损伤在 TK6 细胞中检测到，但在 CHO 细胞中未检测到，而在两种细胞系中均检测到与浓度相关的 fpg 敏感位点增加。氧化 DNA 损伤与 OTA 暴露的一致关联表明其参与产生 OTA 诱导的断裂剂和着丝粒外遗传效应；然而，基于其在 TK6 细胞中的检测，直接 DNA 损伤可能涉及 OTA 暴露对人类造成的任何风险。
