Activation of poly [ADP-ribose] polymerase in endothelial cells and keratinocytes: Role in an in vitro model of sulfur mustard-mediated vesication

Although endothelial cells and keratinocytes appear to be the primary cellu lar targets of sulfur mustard (SM), the role of the nuclear enzyme poly (AD P-ribose) polymerase (PARP) in SM-induced vesication has not been clearly d efined. PARP is thought to play a crucial role in DNA repair mechanisms fol lowing exposure to alkylating agents like SM. Using a combination of fluore scence microscopy and biochemical assays, we tested the hypothesis that SM causes activation of PARP in endothelial cells and keratinocytes with subse quent loss of nicotinamide adenine dinucleotide (NAD) and depletion of aden osine triphosphate (ATP) levels. To determine if PARP activation accounts f or SM-induced vesication, keratinocyte adherence and permeability of endoth elial monolayers were measured as in vitro correlates of vesication. As ear ly as 2 to 3 h after exposure to SM concentrations as low as 250 mu M, dram atic changes were induced in keratinocyte morphology and microfilament arch itecture. Exposure to 500 mu M SM induced a fourfold increase in PARP activ ity in endothelial cells, and a two- to threefold increase in keratinocytes . SM induced a dose-related loss of NAD(+) in both endothelial cells and ke ratinocytes, ATP levels fell to similar to 50% of control levels in respons e to SM concentrations greater than or equal to 500 mu M. SM concentrations greater than or equal to 250 mu M significantly reduced keratinocyte adher ence as early as 3 h after exposure. Endothelial monolayer permeability inc reased substantially with concentrations of SM >250 mu M. These observation s support the hypothesis that the pathogenic events necessary for SM-induce d vesication (i.e,, capillary leak and loss of keratinocyte adherence) at h igher vesicating doses of SM (greater than or equal to 500 mu M) may depend on NAD loss with PARP activation and subsequent ATP-dependent effects on m icrofilament architecture. Vesication developing as a result of exposure to lower concentrations of SM presumably occurs by mechanisms that do not dep end on loss of cellular ATP (e.g., apoptosis and direct SM-mediated damage to integrins and the basement membrane). (C) 1999 Academic Press.