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Appearance of interleukin lα relates dna interstrand cross‐links and cytotoxicity in cultured human keratinocytes exposed to bis‐(2‐chloroethyl)sulfide
Author(s) -
Pu Y.,
Lin P.,
Vaughan F. L.,
Bernstein I. A.
Publication year - 1995
Publication title -
journal of applied toxicology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.784
H-Index - 87
eISSN - 1099-1263
pISSN - 0260-437X
DOI - 10.1002/jat.2550150609
Subject(s) - cytotoxicity , microbiology and biotechnology , ethidium bromide , trypan blue , dna , chemistry , dna damage , cell culture , biochemistry , biology , cell , in vitro , genetics
The utility of an increase in the level of interleukin lα (IL‐1α) 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 (μM BCES at 37°C for 30 min. The amounts of IL‐1α 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‐1α. 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‐1α 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‐1α 72 h after exposure. These data suggest that the appearance of IL‐1α can be used to quantify the cytotoxicity resulting from BCES‐mediated damage to cellular DNA and that the 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.