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

Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In 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 24h 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.

Mangerich A ，Debiak M ，Birtel M ，Ponath V ，Balszuweit F ，Lex K ，Martello R ，Burckhardt-Boer W ，Strobelt R ，Siegert M ，Thiermann H ，Steinritz D ，Schmidt A ，Bürkle A ... -  《-》

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.

Sulfur mustard (SM) is a bifunctional alkylating agent with a long history of use as a chemical weapon. Although its last military use is dated for the eighties of the last century, a potential use in terroristic attacks against civilians remains a significant threat. Thus, improving medical therapy of mustard exposed individuals is still of particular interest. PARP inhibitors were recently brought into the focus as a potential countermeasure for mustard-induced pathologies, supported by the availability of efficient compounds successfully tested in cancer therapy. PARP activation after SM treatment was reported in several cell types and tissues under various conditions; however, a detailed characterization of this phenomenon is still missing. This study provides the basis for such studies by developing and optimizing experimental conditions to investigate poly(ADP-ribosyl)ation (PARylation) in HaCaT keratinocytes upon treatment with the monofunctional alkylating agent 2-chloroethyl ethyl sulfide ("half mustard", CEES). By using an immunofluorescence-based approach, we show that optimization of experimental conditions with regards to the type of solvent, dilution factors and treatment procedure is essential to obtain a homogenous PAR staining in HaCaT cell cultures. Furthermore, we demonstrate that different CEES treatment protocols significantly influence the cytotoxicity profiles of treated cells. Using an optimized treatment protocol, our data reveals that CEES induces a dose- and time-dependent dynamic PARylation response in HaCaT cells that could be completely blocked by treating cells with the clinically relevant pharmacological PARP inhibitor ABT888 (also known as veliparib). Finally, siRNA experiments show that CEES-induced PAR formation is predominantly due to the activation of PARP1. In conclusion, this study provides a detailed analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to study the molecular mechanism of PARP1 activation and its functional consequences after mustard treatment in general. Such a study is presented in an accompanying article (Mangerich et al., 2016).

Debiak M ，Lex K ，Ponath V ，Burckhardt-Boer W ，Thiermann H ，Steinritz D ，Schmidt A ，Mangerich A ，Bürkle A ... -  《-》

Inhibition of poly(ADP-ribose) polymerase (PARP) influences the mode of sulfur mustard (SM)-induced cell death in HaCaT cells.

Kehe K ，Raithel K ，Kreppel H ，Jochum M ，Worek F ，Thiermann H ... -  《ARCHIVES OF TOXICOLOGY》

Modified immunoslotblot assay to detect hemi and sulfur mustard DNA adducts.

Sulfur mustard (SM) is an old chemical warfare agent causing blisters (vesicant). Skin toxicity is thought to be partly caused by SM induced DNA damage. SM and the hemi mustard 2-chloroethyl ethyl sulfide (CEES) are bi- and monofunctional DNA alkylating agents, respectively. Both chemicals react especially with N7 guanine. The most abundant adducts are 7-hydroxyethylthioethylguanine for SM (61%) and 7-ethyl thioethylguanine for CEES. Thus, DNA alkylation should serve as a biomarker of SM exposure. A specific monoclonal antibody (2F8) was previously developed to detect SM and CEES adducts at N7 position by means of immunoslotblot (ISB) technique (van der Schans et al. (2004) [16]). Nitrogen mustards (HN-1, HN-2, HN-3) are alkylating agents with structural similarities, which can form DNA adducts with N7 guanine. The aim of the presented work was to modify the van der Schans protocol for use in a field laboratory and to test the cross reactivity of the 2F8 antibody against nitrogen mustards. Briefly, human keratinocytes were exposed to SM and CEES (0-300μM, 60min) or HN-1, HN-2, HN-3 (120min). After exposure, cells were scraped and DNA was isolated and normalized. 1μg DNA was transferred to a nitrocellulose membrane using a slotblot technique. After incubation with 2F8 antibody, the DNA adducts were visualized with chromogen staining (3,3'-diaminobenzidine (DAB), SeramunGrün). Blots were photographed and signal intensity was quantified. In general, DAB was superior to SeramunGrün stain. A staining was seen from 30nM to 300μM of SM or CEES, respectively. However, statistically significant DNA adducts were detected after CEES and SM exposure above 30μM which is below the vesicant threshold. No signal was observed after HN-1, HN-2, HN-3 exposure. The total hands-on time to complete the assay was about 36h. Further studies are necessary to validate SM or CEES exposure in blister roofs of exposed patients.

Kehe K ，Schrettl V ，Thiermann H ，Steinritz D ... -  《-》

Poly (ADP-ribose) polymerase (PARP) is essential for sulfur mustard-induced DNA damage repair, but has no role in DNA ligase activation.

Concurrent activation of poly (ADP-ribose) polymerase (PARP) and DNA ligase was observed in cultured human epidermal keratinocytes (HEK) exposed to the DNA alkylating compound sulfur mustard (SM), suggesting that DNA ligase activation could be due to its modification by PARP. Using HEK, intracellular 3H-labeled NAD+ (3H-adenine) was metabolically generated and then these cells were exposed to SM (1 mM). DNA ligase I isolated from these cells was not 3H-labeled, indicating that DNA ligase I is not a substrate for (ADP-ribosyl)ation by PARP. In HEK, when PARP was inhibited by 3-amino benzamide (3-AB, 2 mM), SM-activated DNA ligase had a half-life that was four-fold higher than that observed in the absence of 3-AB. These results suggest that DNA repair requires PARP, and that DNA ligase remains activated until DNA damage repair is complete. The results show that in SM-exposed HEK, DNA ligase I is activated by phosphorylation catalysed by DNA-dependent protein kinase (DNA-PK). Therefore, the role of PARP in DNA repair is other than that of DNA ligase I activation. By using the DNA ligase I phosphorylation assay and decreasing PARP chemically as well as by PARP anti-sense mRNA expression in the cells, it was confirmed that PARP does not modify DNA ligase I. In conclusion, it is proposed that PARP is essential for efficient DNA repair; however, PARP participates in DNA repair by altering the chromosomal structure to make the DNA damage site(s) accessible to the repair enzymes.

Bhat KR ，Benton BJ ，Ray R 《-》