Appearance of interleukin lα relates dna interstrand cross‐links and cytotoxicity in cultured human keratinocytes exposed to bis‐(2‐chloroethyl)sulfide

Abstract

The utility of an increase in the level of interleukin 1 alpha (IL-1 alpha) as an indicator of cytotoxicity from exposure to bis-(2-chloroethyl) sulfide (BCES) was evaluated in submerged monolayer cultures of human cutaneous keratinocytes. Four-day-old cultures were exposed to 1-100 microM BCES at 37 degrees for 30 min. The amounts of IL-1 alpha in the medium at and in cells 72 h after exposure were measured immunologically with an enzyme-linked immunosorbent assay using monoclonal antibody to human IL-1 alpha. The antibody was conjugated with peroxidase for visualization. Cell viability was measured concomitantly using the trypan blue exclusion technique. The degree of interstrand cross-linking as a measure of damage in the cellular DNA was determined by measuring the fluorescence resulting from the intercalation of ethidium bromide into double-stranded molecules that remained in heat-denatured DNA isolated from cells that had been exposed to BCES. A high correlation was observed between the dose-responsive increase in the level of IL-1 alpha in the medium and in the cells, and the dose-responsive decrease that took place in the fraction of viable cells in exposed cultures. The dose-responsive increase in the interstrand cross-linking found in the DNA of cells immediately after exposure to BCES also correlated with the increase in IL-1 alpha 72 h after exposure. These data suggest that the appearance of IL-1 alpha can be used to quantify the cytotoxicity resulting from BCES-medicated damage to cellular DNA and that degree of cross-linking in the DNA immediately after exposure to BCES is predictive of the level of cytotoxicity in an exposed culture 3 days later.