Cytotoxic effects on the plasma membrane of human diploid fibroblasts--a comparative study of leakage tests.

Confluent monolayers of human diploid lung fibroblasts were treated with cytolytic agents. The induced membrane damage was investigated by different test systems. Changes of membrane permeability were compared with morphological alterations. Four tests employed leakage of cytoplasmic markers of different sizes as criteria of membrane damage. Radioactive markers of the following decreasing size order were used: [3H]RNA (MW greater than 200,000) greater than 51Cr greater than [3H]nucleotides greater than [1-14C]alpha-amino-isobutyric acid (AIB, MW 103). Uptake of trypan blue was employed as a fifth criterior of changed membrane permeability. A comparison between the tests indicated the following order of sensitivity for detection of membrane damage: leakage of AIB-label greater than leakage of nucleotide label greater than leakage of 51Cr-label=uptake of trypan blue= morphological changes greater than leakage of RNA-label. Two distinct types of leakage patterns were evident: 1. Upon incubation with Triton X-100 all four release curves coincided. This was considered as representing large functional "holes". 2. With the polyene amphotericin B the smallest marker (AIB) was released to a strikingly greater extent than other markers. This was regarded as representing very small functional "holes". Melittin from bee venom and theta-toxin from Clostridium perfringens induced leakage patterns of two intermediate types. The results indicate that a combination of several different size markers may be useful for characterizing induced membrane lesions.