Plasma membrane damage detected by nucleic acid leakage.

Molecular toxicology Pub Date : 1989-01-01
E Fortunati, V Bianchi
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Abstract

Among the indicators of membrane damage, the leakage of intracellular components into the medium is the most directly related to the perturbations of the membrane molecular organization. The extent of the damage can be evaluated from the size of the released components. We have designed a protocol for the detection of membrane leakage based on the preincubation of cells with tritiated adenine for 24 h, followed by a 24-h chase in nonradioactive medium. The treatment takes place when the distribution of the precursor among its end products has reached the plateau, and thus the differences of radioactivity in the fractions obtained from the control and treated cultures (medium, nucleotide pool, RNA, DNA) correspond to actual quantitative variations induced by the test chemical. Aliquots of the medium are processed to determine which percentage of the released material is macromolecular, in order to distinguish between mild and severe membrane damage. The origin of the extracellular radioactivity can be recognized from the variations of RNA counts in the treated cells. DNA radioactivity is used to evaluate the number of cells that remain attached to the plates in the different conditions of treatment. By this means, generalized permeabilization of membranes to macromolecules is distinguished from complete solubilization of only a subpopulation of cells. We present some examples of application of the protocol with detergents (LAS, SDS, Triton X-100) and with Cr(VI), which damages cell membranes by a different mechanism of action.

核酸渗漏检测质膜损伤。
在膜损伤的指标中,胞内组分向介质的渗漏与膜分子组织的扰动最直接相关。损坏的程度可以从释放部件的大小来评估。我们设计了一种检测膜渗漏的方案,该方案基于将细胞与氚化腺嘌呤预孵育24小时,然后在非放射性介质中进行24小时的追踪。当前体在其最终产物中的分布达到平台时,就进行处理,因此从对照和处理的培养物(培养基、核苷酸池、RNA、DNA)中获得的组分的放射性差异与测试化学品引起的实际定量变化相对应。处理等分的介质,以确定释放的物质中大分子物质的百分比,以区分轻度和严重的膜损伤。细胞外放射性的来源可以从处理细胞中RNA计数的变化中识别出来。DNA放射性用于评估在不同处理条件下仍附着在培养皿上的细胞数量。通过这种方法,将膜对大分子的普遍渗透与仅对一小部分细胞的完全溶解区分开来。我们提出了一些应用该方案的例子,洗涤剂(LAS, SDS, Triton X-100)和Cr(VI),它们通过不同的作用机制破坏细胞膜。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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