Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences

Abstract

Letters ; 244 (2016). -S. 56-71 https://dx.doi.org/10.1016/j.toxlet.2015In particular, we recorded substance specific as well as dose and time dependent PARylation responses using independent bioanalytical methods based on single cell immuno fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence based protocol for the detection of N7 ETE guanine DNA adducts, the excision rate of CEES induced DNA adducts was not affected by PARP inhibition. Furthermore, while CEES induced moderate changes in cellular NAD + levels, annexin V/PI flow cytometry analysis revealed that these changes did not affect CEES induced short term cytotoxicity 24 h after treatment. In contrast, PARP inhibition impaired cell proliferation and clonogenic survival, and potentiated micronuclei formation of HaCaT cells upon CEES treatment. Similarly, PARP inhibition affected clonogenic survival of cells treated with bi functional mustards such as SM and HN2.In conclusion, we demonstrate that PARylation plays a functional role in mustard induced cellular stress response with substance specific differences. Since PARP inhibitors exhibit therapeutic potential to treat SM related pathologies and to sensitize cancer cells for mustard based chemotherapy, potential long term effects of PARP inhibition on genomic stability and carcinogenesis should be carefully considered when pursuing such a strategy.

Sulfur mustard resistant keratinocytes obtained elevated glutathione levels and other changes in the antioxidative defense mechanism

“…Moreover, the resistant cells showed significantly higher levels of NAD + , NADH and therefore total NAD than the sensitive cells. Supporting our data, the NAD + depletion often described in the literature could also not be validated by even treatment with up to 3 mM CEES and 24 h (Mangerich et al, 2016). The NAD + decrease seems to be concentration dependent and might be achieved by increasing the SM doses adequately.…”

“…The accumulation of glucose-6-phosphate leads to induction and secretion of proteases resulting in nuclear and cytoplasmic damage (Graham et al, 2005). The NAD + depletion often mentioned in the literature could not be validated by other studies (Mangerich et al, 2016). Otherwise, niacin, the vitamin from which NAD + and NADP + are synthesized, showed protective effects against DNA-damaging agents (Jacobson et al, 2007).…”

Death of Monocytes through Oxidative Burst of Macrophages and Neutrophils: Killing in Trans

“…Cells were treated with 1 mM H 2 O 2 for 5 min before they were fixed in ice cold methanol for 7 min at -20°C [15]. Cover slips were washed 3 x 5 min in PBS and samples were blocked in 5% dry milk in PBS-T for 1 h at RT.…”

Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions

“…However, the moderate PARP activation induced by CEES presumably leads only to minor changes in intracellular NAD + concentrations, which speaks against the hypothesis that SM causes massive PARP activation resulting in ATP depletion, necrotic cell death, and subsequent tissue inflammation. For a detailed cell culture study characterizing the PARylation response induced by bifunctional mustards as well as a discussion of the therapeutic potential of PARP inhibitors in SM related pathologies the reader is referred to an accompanying paper (Mangerich et al, 2016) It is interesting to note that PAR formation after H 2 O 2 peaks already at 5 min post treatment (Martello et al, 2013), whereas maximum PARylation was observed 10 min post CEES treatment. One possibility for the different PARylation dynamics are substance specific toxicokinetics.…”

“…This study provides the basis for a broader characterization of sulfur and nitrogen mustard induced PARylation response and cellular consequences thereof, which is described in an accompa nying article (Mangerich et al, 2016).…”