DNA Damage Initiated by the Sulfur Mustard Analog Mechlorethamine (methylbis(2‐chloroethyl)amine) in Mouse Keratinocytes is Associated with an Oxidative Stress Response

Abstract

Sulfur mustard and nitrogen mustard (HN2, mechlorethamine) are bifunctional alkylating agent that cause dermal inflammation, edema and blistering. Skin keratinocytes are known to be important targets for mustards, which form both monofunctional DNA adducts and inter- and intrastrand DNA cross-links in the cells leading to cytotoxicity. In these studies, we used PAM212 mouse keratinocytes to analyze mechanisms underlying HN2 toxicity. Treatment of PAM212 cells with HN2 (3-30 μM) caused cytostasis; this was associated with a cell cycle block in late S phase and G2M phase. HN2 also caused DNA double strand breaks as shown by increased expression of phospho-H2A.X, a H2A histone variant that binds to double stranded DNA breaks creating a repair recognition domain. HN2-induced phosphorylation of H2A.X was both time- and concentration-dependent. Maximal increases were evident in cells at 24 h post HN2. HN2 induced double strand breaks were correlated with increases in the cellular antioxidants heme oxygenase-1 (HO-1) and heat shock protein27 (hsp27), which peaked 3 h and 6 h post HN2-treatment. These findings are consistent with the idea that HN2 initiates a DNA damage response in mouse keratinocytes that leads to induction of an adaptive stress response. Interfering with DNA strand breaks may be an effective strategy to mitigate mustard-induced damage in keratinocytes. Support: AR055073 and ES005022