title: TNF-alpha expression patterns as potential molecular biomarker for human skin cells exposed to vesicant chemical warfare agents: sulfur mustard (HD) and Lewisite (L).
abstract: Studies were conducted to examine the effect of two vesicant chemical warfare agents (VCWA), one of them an arsenical, on cytokine gene expression in normal human epidermal keratinocyte (NHEK) cells. We tested 2,2'-dichlorethylsulfide (sulfur mustard, military designation HD) and 2,chlorovinyldichloroarsine (Lewisite, military designation L), which have significant differences in their chemical, physical, and toxicological properties. Human tumor necrosis factor-alpha (hTNF-alpha) cytokine was detected by using the enzyme-linked immunosorbent assay, a protein multiplex immunoassay, Luminex100, and reverse transcription-polymerase chain reaction (RT-PCR). The messenger RNA expression of hTNF-alpha was determined to provide a semi-quantitative analysis. HD-stimulated NHEK induced secretion of hTNF-alpha in a dose-dependent manner. Dose response effect of Lewisite decreased hTNF-alpha levels. Time-response data indicated that the maximum response for HD occurred at 24 h with an associated cytotoxic concentration of 10(-4) mol/L. NHEK cells stimulated with 10(-4) mol/L HD for 24 h at 37 degrees C increased detectable levels of hTNF-alpha from 5 to 28 ng/ml at an index of cell viability between 85 to 93% as detected by Luminex100. Our results indicated that the increased levels of hTNF-alpha by HD are dependent on the primary cultures, cell densities, and chemical properties of the stimulation. Lewisite under the same conditions as HD caused a reduction of hTNF-alpha from control levels of 1.5 ng/ml to 0.3 ng/ml after stimulation (10(-4) mol/L), with an index of cell viability of reverse similar 34%. We analyzed the transcriptional of hTNF-alpha gene and found that HD (10(-6) to 10(-4) mol/L) activates hTNF-alpha gene in cultured NHEK and that L at 10(-6) to 10(-4) mol/L markedly reduces hTNF-alpha gene. We conclude that the pro-inflammatory mediator, hTNF-alpha, could be a potential biomarker for differentiating between exposure of HD or L.
authors.affiliation: Drug Assessment Division, US Army Medical Research Institute of Chemical Defense, Edgewood Area, Aberdeen Proving Ground, MD, USA. carmen.arroyo@amedd.army.mil
authors.forename: C M
authors.initials: CM
authors.lastname: Arroyo
authors.forename: D L
authors.initials: DL
authors.lastname: Burman
authors.forename: D W
authors.initials: DW
authors.lastname: Kahler
authors.forename: M R
authors.initials: MR
authors.lastname: Nelson
authors.forename: C M
authors.initials: CM
authors.lastname: Corun
authors.forename: J J
authors.initials: JJ
authors.lastname: Guzman
authors.forename: M A
authors.initials: MA
authors.lastname: Smith
authors.forename: E D
authors.initials: ED
authors.lastname: Purcell
authors.forename: B E
authors.initials: BE
authors.lastname: Hackley
authors.forename: S-D
authors.initials: SD
authors.lastname: Soni
authors.forename: C A
authors.initials: CA
authors.lastname: Broomfield
mesh_terms: D000328:Adult; D001152:Arsenicals / Q000494:pharmacology*; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002619:Chemical Warfare Agents / Q000494:pharmacology*; D016207:Cytokines / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007509:Irritants / Q000494:pharmacology*; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009151:Mustard Gas / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
chemical_list: D001152:Arsenicals; D015415:Biomarkers; D002619:Chemical Warfare Agents; D016207:Cytokines; D007509:Irritants; D014409:Tumor Necrosis Factor-alpha; C035965:lewisite; D009151:Mustard Gas