Plasma membrane damage detected by nucleic acid leakage.
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.
Fortunati
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Department of Biology, University of Padua, Padova, Italy.
Bianchi
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V
D000225:Adenine; D000476:Alkanesulfonates / Q000494:pharmacology; D017738:Alkanesulfonic Acids; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects*; D004247:DNA / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology*; D005020:Ethyl Methanesulfonate / Q000494:pharmacology; D009696:Nucleic Acids / Q000378:metabolism*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011192:Potassium Dichromate / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013501:Surface-Active Agents / Q000494:pharmacology*; D014316:Tritium; D014343:Trypan Blue
D000476:Alkanesulfonates; D017738:Alkanesulfonic Acids; D003902:Detergents; D009696:Nucleic Acids; D013501:Surface-Active Agents; C030641:alkylbenzyl sulfonic acid; D014316:Tritium; D012967:Sodium Dodecyl Sulfate; D011092:Polyethylene Glycols; D012313:RNA; D017830:Octoxynol; D004247:DNA; D005020:Ethyl Methanesulfonate; D014343:Trypan Blue; D000225:Adenine; D011192:Potassium Dichromate