Assessment of the role of DNA damage and repair in the survival of primary cultures of rat cutaneous keratinocytes exposed to bis(2-chloroethyl)sulfide

Abstract

Toxicity manifests itself as vesication in human skin exposed topically to bis(2-chloroethyl)sulfide (BCES). The destruction of the proliferating population of epidermal cells is a major component of the pathogenic process. Available data strongly suggest that damage to cellular DNA is a critical factor in the loss of these cells. However, the influence of DNA repair on this toxic response has not been adequately studied. Therefore, a study was undertaken to ascertain the influence of DNA repair on the survival of primary monolayer cultures of rat cutaneous keratinocytes exposed to BCES. The sensitive nucleoid sedimentation assay was employed for the determination of DNA damage in cultures exposed to very low levels of BCES. Initial experiments demonstrated that within 1 hr of exposure to as little as 0.1 microM BCES the structural integrity of cellular DNA was compromised, presumably resulting from the appearance of single-strand breaks in the nucleic acid. This same effect was demonstrated in basal cells derived from a stratified, cornified culture grown at the air-liquid interface and exposed topically to the vesicant. Further studies with the monolayer culture demonstrated that the gross structural integrity of the DNA in cells exposed to as much as 5 microM BCES was completely restored within the first 22 hr following the exposure. However, this repair process appeared to be inefficient since a depression of thymidine incorporation into DNA and a significant loss of DNA were exhibited in exposed cultures as long as 72 hr after the initial exposure.

The use of doxycycline as a protectant against sulphur mustard in HaCaT cells

“…The increased DNA fragmentation observed following HD exposure is a well known feature of this agent (Ribeiro et al, 1991;Mol et al, 1993). However, although the combined co-and post-treatment of HaCaT cells with doxycycline partially attenuated this response in HD exposed cultures in the present study, it did not enhance cell culture viability.…”

DNA damage, signalling and repair after exposure of cells to the sulphur mustard analogue 2-chloroethyl ethyl sulphide

“…The toxicity of SM is thought to be mediated by the alkylation of nucleic acids and proteins, although the exact mechanisms are not clear (Papirmeister et al, 1985;Ribeiro et al, 1991). Several studies have demonstrated that SM is able to modify DNA via the formation of DNA mono-adducts and crosslinks.…”

Cross-Linking Interferes with Assessing Sulfur Mustard-Induced DNA Damage in Human Peripheral Blood Lymphocytes Using the Comet Assay

“…This is in agreement with the findings of others who evaluated by electrophoresis total genomic DNA extracted from SMexposed PBL (Meier and Millard 1998); DNA fragmentation was detected as early as 2 h after exposure to 300 µM SM. In another study, using the nucleoid sedimentation assay, it was demonstrated that within 1 h of exposure to as little as 0.1 µM SM, the structural integrity of DNA from rat cutaneous keratinocytes was compromised, assumed to be the result of DNA single-strand breakage (Ribeiro et al 1991). …”

“…Since there is an overall increase in the amount of DNA damage detected over time, cross-link repair appears to take place sooner and/or at a higher rate than strand break repair. Previous studies with rat cutaneous keratinocytes suggest that at low SM concentrations, cells are capable of completely repairing SM-induced DNA damage within 22 h following exposure, but at high concentrations the damage is so extensive that the cell loses its ability to completely repair damaged DNA (Ribeiro et al 1991). Up to at least 6 h following SM exposure, the rate of DNA single strand break formation may be higher than the rate of DNA single strand break repair, which would also contribute to the overall effect.…”