title: Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences.
abstract: 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.
authors.affiliation: University of Konstanz, Molecular Toxicology Group, Department of Biology, 78457 Konstanz, Germany.
authors.forename: Aswin
authors.initials: A
authors.lastname: Mangerich
authors.affiliation: University of Konstanz, Molecular Toxicology Group, Department of Biology, 78457 Konstanz, Germany.
authors.forename: Malgorzata
authors.initials: M
authors.lastname: Debiak
authors.affiliation: University of Konstanz, Molecular Toxicology Group, Department of Biology, 78457 Konstanz, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Birtel
authors.affiliation: University of Konstanz, Molecular Toxicology Group, Department of Biology, 78457 Konstanz, Germany.
authors.forename: Viviane
authors.initials: V
authors.lastname: Ponath
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Balszuweit
authors.affiliation: University of Konstanz, Molecular Toxicology Group, Department of Biology, 78457 Konstanz, Germany.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Lex
authors.affiliation: University of Konstanz, Molecular Toxicology Group, Department of Biology, 78457 Konstanz, Germany.
authors.forename: Rita
authors.initials: R
authors.lastname: Martello
authors.affiliation: University of Konstanz, Molecular Toxicology Group, Department of Biology, 78457 Konstanz, Germany.
authors.forename: Waltraud
authors.initials: W
authors.lastname: Burckhardt-Boer
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Romano
authors.initials: R
authors.lastname: Strobelt
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Siegert
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Horst
authors.initials: H
authors.lastname: Thiermann
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany; Walther-Straub-Institute of Pharmacology and Toxicology, 80336 Munich, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Steinritz
authors.affiliation: Bundeswehr Institute of Pharmacology and Toxicology, 80937 Munich, Germany.
authors.forename: Annette
authors.initials: A
authors.lastname: Schmidt
authors.affiliation: University of Konstanz, Molecular Toxicology Group, Department of Biology, 78457 Konstanz, Germany. Electronic address: alexander.buerkle@uni-konstanz.de.
authors.forename: Alexander
authors.initials: A
authors.lastname: Bürkle
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000931:Antidotes / Q000633:toxicity; D018906:Antineoplastic Agents, Alkylating / Q000633:toxicity*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002619:Chemical Warfare Agents / Q000633:toxicity*; D002699:Chlorambucil / Q000633:toxicity; D018736:DNA Adducts / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D042822:Genomic Instability / Q000187:drug effects; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D008466:Mechlorethamine / Q000633:toxicity; D048629:Micronuclei, Chromosome-Defective / Q000139:chemically induced; D009151:Mustard Gas / Q000031:analogs & derivatives / Q000633:toxicity*; D009588:Nitrogen Mustard Compounds / Q000633:toxicity*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors / Q000633:toxicity; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors
chemical_list: D000931:Antidotes; D018906:Antineoplastic Agents, Alkylating; D002619:Chemical Warfare Agents; D018736:DNA Adducts; D009588:Nitrogen Mustard Compounds; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D002699:Chlorambucil; D000246:Adenosine Diphosphate Ribose; D008466:Mechlorethamine; C031278:2-chloroethyl ethyl sulfide; D011065:Poly(ADP-ribose) Polymerases; D009151:Mustard Gas
keywords: Genotoxicity; HaCaT; Mass spectrometry; Micronucleus; Nitrogen mustard; PARP inhibitors; Poly(ADP-ribose); Sulfur mustard