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.
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.
Tewari-Singh
Neera
N
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.
Gu
Mallikarjuna
M
Agarwal
Chapla
C
White
Carl W
CW
Agarwal
Rajesh
R
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*
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
J Biol Chem. 2000 May 26;275(21):16202-12
10821866
J Burns Wounds. 2007 Oct 30;7:e7
18091984
Biogerontology. 2010 Aug;11(4):421-36
20084458
Mol Biol Cell. 2002 Mar;13(3):978-88
11907276
Nat Rev Cancer. 2003 Mar;3(3):155-68
12612651
Toxicol Appl Pharmacol. 2009 Jan 1;234(1):128-34
18977373
Oncogene. 2009 Jun 18;28(24):2314-23
19421147
J Biol Chem. 2003 Mar 7;278(10):8531-40
12482751
Arch Toxicol. 2008 Jul;82(7):461-70
18046540
Eplasty. 2007 Nov 27;8:e2
18213398
J Biochem Mol Toxicol. 2004;18(6):300-12
15674844
J Burns Wounds. 2007 Jul 10;7:e2
17846661
J Appl Toxicol. 2000 Dec;20 Suppl 1:S81-6
11428649
Free Radic Biol Med. 2010 May 1;48(9):1188-96
20138141
Toxicology. 2009 Sep 1;263(1):20-5
18602966
Cell Prolif. 2007 Feb;40(1):1-13
17227291
Toxicol Appl Pharmacol. 2008 Jul 1;230(1):97-108
18342354
Eplasty. 2008 Jun 10;8:e32
18615149
EMBO J. 2004 Aug 18;23(16):3386-96
15272308
J Biochem Mol Toxicol. 2000;14(6):291-302
11083082
J Biol Chem. 2005 May 27;280(21):20375-83
15792956
Carcinogenesis. 2001 Apr;22(4):661-4
11285203
Oncogene. 2006 Feb 16;25(7):1053-69
16205633
Oncogene. 2002 Feb 7;21(7):1108-16
11850828
J Invest Dermatol. 1997 Dec;109(6):722-7
9406811
J Biol Chem. 2008 Sep 12;283(37):25455-25467
18606816
Arch Toxicol. 2006 Oct;80(10):662-70
16552503
Br J Pharmacol. 2004 Mar;141(5):795-802
14769780
Mol Cancer Ther. 2007 Jun;6(6):1718-27
17575103
Environ Mol Mutagen. 2004;44(2):151-5
15278918
J Appl Toxicol. 2000 Dec;20 Suppl 1:S87-91
11428650
J Biol Chem. 2004 Dec 17;279(51):53272-81
15489221
Mol Cancer Ther. 2006 Dec;5(12):3294-302
17172433
Life Sci. 2007 May 30;80(24-25):2199-201
17229443
J Biochem Mol Toxicol. 2004;18(6):289-99
15674843
Eplasty. 2008 Mar 26;8:e16
18438446
J Pharmacol Exp Ther. 2009 Mar;328(3):732-9
19064720
Free Radic Biol Med. 2009 Dec 1;47(11):1640-51
19761830
Toxicology. 2009 Sep 1;263(1):12-9
19651324
Nature. 2000 Nov 23;408(6811):433-9
11100718
Toxicol Appl Pharmacol. 2007 Jun 15;221(3):363-71
17482225
J Appl Toxicol. 2000 Dec;20 Suppl 1:S43-9
11428642
Toxicol In Vitro. 2009 Oct;23(7):1396-405
19619636
World J Gastroenterol. 2004 Jan 15;10(2):155-60
14716813
Toxicology. 2005 Oct 30;214(3):198-209
16084004
J Burns Wounds. 2005 Jan 05;4:e1
16921406
J Appl Toxicol. 2005 Mar-Apr;25(2):115-28
15747377
Toxicol Appl Pharmacol. 2008 Jun 15;229(3):342-50
18400241
Toxicol Appl Pharmacol. 2002 Oct 15;184(2):116-26
12408956
Toxicology. 2009 Apr 5;258(1):39-46
19167455
Toxicology. 2009 Mar 29;257(3):105-12
19111594
Mol Cell Biochem. 2002 May-Jun;234-235(1-2):185-93
12162432
Prog Cell Cycle Res. 2003;5:393-411
14593734
Cancer Sci. 2006 Oct;97(10):984-9
16925578
Biochemistry. 2000 Jun 27;39(25):7559-69
10858306
Cytometry A. 2005 Nov;68(1):1-9
16184611
Genes Dev. 2001 Sep 1;15(17):2177-96
11544175
Toxicol Sci. 2009 Mar;108(1):194-206
19075041
Clin Exp Dermatol. 2006 Jan;31(1):1-5
16309468
J Biol Chem. 2003 Apr 25;278(17):14806-11
12588868
Br J Pharmacol. 2002 Sep;137(2):245-52
12208782