Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay
Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay
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DOI：
10.1002/cbf.3016
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发表时间：
2014-04-01
影响因子：
3.6
通讯作者：
Collins, Andrew R.
中科院分区：
文献类型：
--
作者：
Akor-Dewu, Maryam B.;El Yamani, Naouale;Collins, Andrew R.
用彗星试验保存用于DNA损伤分析的人血细胞通常包括通过离心法分离单个核细胞，将其悬浮在冰冻介质中，然后缓慢冷冻至-80℃的繁琐过程。最近的一份出版物(Al-Salmani et al.Free Rad Biol Med 2011；51：719-725)描述了一种简单的方法，将少量全血冷冻到-20或-80摄氏度；在随后的解冻时，进行彗星分析，没有迹象表明快速冷冻导致DNA链断裂。然而，全血中的白细胞(无论是新鲜的还是冷冻的)对过氧化氢的损伤具有异常的抵抗力，因此不能使用常见的抗氧化状态测试(对过氧化氢断裂的抵抗力)。我们通过从解冻的血液中分离白细胞来改进这种方法；我们发现，经过三次洗涤后，细胞对过氧化氢的反应正常。此外，我们还使用甲酰胺基嘧啶DNA糖基化酶测定了分离的白细胞中特定的内源性碱基损伤(氧化嘌呤)。在一项对10名受试者的血液样本的研究中，过氧化氢敏感性和内源性损伤都反映了细胞的抗氧化状态，两者显著相关。这种改进的样本收集和储存方法在可用血量有限的情况下特别适用，并在生物监测和生态毒理学研究中具有很大潜力，其中样本是在现场或远离检测实验室的地点获得的。版权所有(C)2013 John Wiley&Sons，Ltd.
Preservation of human blood cells for DNA damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to -80 degrees Ca laborious process. A recent publication (Al-Salmani et al. Free Rad Biol Med 2011; 51: 719-725) describes a simple method in which small volumes of whole blood are frozen to -20 or -80 degrees C; on subsequent thawing, the comet assay is performed, with no indication of elevated DNA strand breakage resulting from the rapid freezing. However, leucocytes in whole blood (whether fresh or frozen) are abnormally resistant to damage by H2O2, and so a common test of antioxidant status (resistance to strand breakage by H2O2) cannot be used. We have refined this method by separating the leucocytes from the thawed blood; we find that, after three washes, the cells respond normally to H2O2. In addition, we have measured specific endogenous base damage (oxidized purines) in the isolated leucocytes, using the enzyme formamidopyrimidine DNA glycosylase. In a study of blood samples from 10 subjects, H2O2 sensitivity and endogenous damageboth reflecting the antioxidant status of the cellscorrelated significantly. This modified approach to sample collection and storage is particularly applicable when the available volume of blood is limited and has great potential in biomonitoring and ecogenotoxicology studies where samples are obtained in the field or at sites remote from the testing laboratory. Copyright (c) 2013 John Wiley & Sons, Ltd.