title: Biological and molecular mechanisms of sulfur mustard analogue-induced toxicity in JB6 and HaCaT cells: possible role of ataxia telangiectasia-mutated/ataxia telangiectasia-Rad3-related cell cycle checkpoint pathway.
abstract: Effective medical treatment and preventive measures for chemical warfare agent sulfur mustard (HD)-caused incapacitating skin toxicity are lacking, because of limited knowledge of its mechanism of action. The proliferating basal epidermal cells are primary major sites of attack during HD-caused skin injury. Therefore, employing mouse JB6 and human HaCaT epidermal cells, here, we investigated the molecular mechanism of HD analogue 2-chloroethyl ethyl sulfide (CEES)-induced skin cytotoxicity. As compared to the control, up to 1 mM CEES treatment of these cells for 2, 4, and 24 h caused dose-dependent decreases in cell viability and proliferation as measured by DNA synthesis, together with S and G2-M phase arrest in cell cycle progression. Mechanistic studies showed phosphorylation of DNA damage sensors and checkpoint kinases, ataxia telangiectasia-mutated (ATM) at ser1981 and ataxia telangiectasia-Rad3-related (ATR) at ser428 within 30 min of CEES exposure, and modulation of S and G2-M phase-associated cell cycle regulatory proteins, which are downstream targets of ATM and ATR kinases. Hoechst-propidium iodide staining demonstrated that CEES-induced cell death was both necrotic and apoptotic in nature, and the latter was induced at 4 and 24 h of CEES treatment in HaCaT and JB6 cells, respectively. An increase in caspase-3 activity and both caspase-3 and poly(ADP-ribose)polymerase (PARP) cleavage coinciding with CEES-caused apoptosis in both cell lines suggested the involvement of the caspase pathway. Together, our findings suggest a DNA-damaging effect of CEES that activates ATM/ATR cell cycle checkpoint signaling as well as caspase-PARP pathways, leading to cell cycle arrest and apoptosis/necrosis in both JB6 and HaCaT cells. The identified molecular targets, quantitative biomarkers, and epidermal cell models in this study have the potential and usefulness in rapid development of effective prophylactic and therapeutic interventions against HD-induced skin toxicity.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, 12700 East 19th Avenue, Box C238 P-15, Research 2, Aurora, Colorado 80045, USA.
authors.forename: Neera
authors.initials: N
authors.lastname: Tewari-Singh
authors.forename: Mallikarjuna
authors.initials: M
authors.lastname: Gu
authors.forename: Chapla
authors.initials: C
authors.lastname: Agarwal
authors.forename: Carl W
authors.initials: CW
authors.lastname: White
authors.forename: Rajesh
authors.initials: R
authors.lastname: Agarwal
mesh_terms: D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000078404:Epidermal Cells; D006801:Humans; D051379:Mice; D009151:Mustard Gas / Q000009:adverse effects / Q000031:analogs & derivatives*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012867:Skin / Q000187:drug effects*; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C031278:2-chloroethyl ethyl sulfide; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; C576897:Atr protein, mouse; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D053148:Caspase 3; D009151:Mustard Gas
references.citation: J Biol Chem. 2000 May 26;275(21):16202-12
references.pmid: 10821866
references.citation: J Burns Wounds. 2007 Oct 30;7:e7
references.pmid: 18091984
references.citation: Biogerontology. 2010 Aug;11(4):421-36
references.pmid: 20084458
references.citation: Mol Biol Cell. 2002 Mar;13(3):978-88
references.pmid: 11907276
references.citation: Nat Rev Cancer. 2003 Mar;3(3):155-68
references.pmid: 12612651
references.citation: Toxicol Appl Pharmacol. 2009 Jan 1;234(1):128-34
references.pmid: 18977373
references.citation: Oncogene. 2009 Jun 18;28(24):2314-23
references.pmid: 19421147
references.citation: J Biol Chem. 2003 Mar 7;278(10):8531-40
references.pmid: 12482751
references.citation: Arch Toxicol. 2008 Jul;82(7):461-70
references.pmid: 18046540
references.citation: Eplasty. 2007 Nov 27;8:e2
references.pmid: 18213398
references.citation: J Biochem Mol Toxicol. 2004;18(6):300-12
references.pmid: 15674844
references.citation: J Burns Wounds. 2007 Jul 10;7:e2
references.pmid: 17846661
references.citation: J Appl Toxicol. 2000 Dec;20 Suppl 1:S81-6
references.pmid: 11428649
references.citation: Free Radic Biol Med. 2010 May 1;48(9):1188-96
references.pmid: 20138141
references.citation: Toxicology. 2009 Sep 1;263(1):20-5
references.pmid: 18602966
references.citation: Cell Prolif. 2007 Feb;40(1):1-13
references.pmid: 17227291
references.citation: Toxicol Appl Pharmacol. 2008 Jul 1;230(1):97-108
references.pmid: 18342354
references.citation: Eplasty. 2008 Jun 10;8:e32
references.pmid: 18615149
references.citation: EMBO J. 2004 Aug 18;23(16):3386-96
references.pmid: 15272308
references.citation: J Biochem Mol Toxicol. 2000;14(6):291-302
references.pmid: 11083082
references.citation: J Biol Chem. 2005 May 27;280(21):20375-83
references.pmid: 15792956
references.citation: Carcinogenesis. 2001 Apr;22(4):661-4
references.pmid: 11285203
references.citation: Oncogene. 2006 Feb 16;25(7):1053-69
references.pmid: 16205633
references.citation: Oncogene. 2002 Feb 7;21(7):1108-16
references.pmid: 11850828
references.citation: J Invest Dermatol. 1997 Dec;109(6):722-7
references.pmid: 9406811
references.citation: J Biol Chem. 2008 Sep 12;283(37):25455-25467
references.pmid: 18606816
references.citation: Arch Toxicol. 2006 Oct;80(10):662-70
references.pmid: 16552503
references.citation: Br J Pharmacol. 2004 Mar;141(5):795-802
references.pmid: 14769780
references.citation: Mol Cancer Ther. 2007 Jun;6(6):1718-27
references.pmid: 17575103
references.citation: Environ Mol Mutagen. 2004;44(2):151-5
references.pmid: 15278918
references.citation: J Appl Toxicol. 2000 Dec;20 Suppl 1:S87-91
references.pmid: 11428650
references.citation: J Biol Chem. 2004 Dec 17;279(51):53272-81
references.pmid: 15489221
references.citation: Mol Cancer Ther. 2006 Dec;5(12):3294-302
references.pmid: 17172433
references.citation: Life Sci. 2007 May 30;80(24-25):2199-201
references.pmid: 17229443
references.citation: J Biochem Mol Toxicol. 2004;18(6):289-99
references.pmid: 15674843
references.citation: Eplasty. 2008 Mar 26;8:e16
references.pmid: 18438446
references.citation: J Pharmacol Exp Ther. 2009 Mar;328(3):732-9
references.pmid: 19064720
references.citation: Free Radic Biol Med. 2009 Dec 1;47(11):1640-51
references.pmid: 19761830
references.citation: Toxicology. 2009 Sep 1;263(1):12-9
references.pmid: 19651324
references.citation: Nature. 2000 Nov 23;408(6811):433-9
references.pmid: 11100718
references.citation: Toxicol Appl Pharmacol. 2007 Jun 15;221(3):363-71
references.pmid: 17482225
references.citation: J Appl Toxicol. 2000 Dec;20 Suppl 1:S43-9
references.pmid: 11428642
references.citation: Toxicol In Vitro. 2009 Oct;23(7):1396-405
references.pmid: 19619636
references.citation: World J Gastroenterol. 2004 Jan 15;10(2):155-60
references.pmid: 14716813
references.citation: Toxicology. 2005 Oct 30;214(3):198-209
references.pmid: 16084004
references.citation: J Burns Wounds. 2005 Jan 05;4:e1
references.pmid: 16921406
references.citation: J Appl Toxicol. 2005 Mar-Apr;25(2):115-28
references.pmid: 15747377
references.citation: Toxicol Appl Pharmacol. 2008 Jun 15;229(3):342-50
references.pmid: 18400241
references.citation: Toxicol Appl Pharmacol. 2002 Oct 15;184(2):116-26
references.pmid: 12408956
references.citation: Toxicology. 2009 Apr 5;258(1):39-46
references.pmid: 19167455
references.citation: Toxicology. 2009 Mar 29;257(3):105-12
references.pmid: 19111594
references.citation: Mol Cell Biochem. 2002 May-Jun;234-235(1-2):185-93
references.pmid: 12162432
references.citation: Prog Cell Cycle Res. 2003;5:393-411
references.pmid: 14593734
references.citation: Cancer Sci. 2006 Oct;97(10):984-9
references.pmid: 16925578
references.citation: Biochemistry. 2000 Jun 27;39(25):7559-69
references.pmid: 10858306
references.citation: Cytometry A. 2005 Nov;68(1):1-9
references.pmid: 16184611
references.citation: Genes Dev. 2001 Sep 1;15(17):2177-96
references.pmid: 11544175
references.citation: Toxicol Sci. 2009 Mar;108(1):194-206
references.pmid: 19075041
references.citation: Clin Exp Dermatol. 2006 Jan;31(1):1-5
references.pmid: 16309468
references.citation: J Biol Chem. 2003 Apr 25;278(17):14806-11
references.pmid: 12588868
references.citation: Br J Pharmacol. 2002 Sep;137(2):245-52
references.pmid: 12208782