Effects of Brief Cutaneous JP-8 Jet Fuel Exposures on TimeCourse of Gene Expression in the EpidermisJames N. McDougal,*,1Carol M. Garrett,* Carol M. Amato,* and Steven J. Berberich†*Department of Pharmacology and Toxicology and †Department of Biochemistry and Molecular Biology,Wright State University, Dayton, Ohio 45435Received September 21, 2006; accepted October 27, 2006The jet fuel jet propulsion fuel 8 (JP-8) has been shown to causean inflammatory response in the skin, which is characterizedhistologically by erythema, edema, and hyperplasia. Studies inlaboratory animal skin and cultured keratinocytes have identifieda variety of changes in protein levels related to inflammation,oxidative damage, apoptosis, and cellular growth. Most of thesestudies have focused on prolonged exposures and subsequenteffects. In an attempt to understand the earliest responses of theskin to JP-8, we have investigated changes in gene expression inthe epidermis for up to 8 h after a 1-h cutaneous exposure in rats.After exposure, we separated the epidermis from the rest of theskin with a cryotome and isolated total mRNA. Gene expressionwas studied with microarray techniques, and changes from shamtreatments were analyzed and characterized. We found consistenttwofold increases in gene expression of 27 transcripts at 1, 4, and8 h after the beginning of the 1-h exposure that were relatedprimarily to structural proteins, cell signaling, inflammatorymediators, growth factors, and enzymes. Analysis of pathwayschanged showed that several signaling pathways were increased at1 h and that the most significant changes at 8 h were in metabolicpathways, many of which were downregulated. These resultsconfirm and expand many of the previous molecular studies withJP-8. Based on the 1-h changes in gene expression, we hypothesizethat the trigger of the JP-8–induced, epidermal stress response isa physical disruption of osmotic, oxidative, and membranestability which activates gene expression in the signaling pathwaysand results in the inflammatory, apoptotic, and growth responsesthat have been previously identified.Key Words: cutaneous; jet fuel; signaling pathway; inflamma-tory; apoptotic; response; JP-8.Jet propulsion fuel 8 (JP-8) is a multipurpose hydrocarbonfuel that is related to kerosene and used by the U.S. and NorthAtlantic Treaty Organization (NATO) military organizations.Starting in the 1980’s, a 20-year conversion process has led toJP-8 being the primary fuel for aircraft, ground vehicles,cooking stoves, and personnel heaters for the U.S. and NATOforces (National Research Council, 2003). Compared to thefuel predecessor, JP-4, the desirable characteristics of JP-8 area higher flash point and lower proportions of the cancer-causing, low–molecular weight hydrocarbons such as benzene,toluene, and xylene (Zeiger and Smith, 1998). Greater potentialfor cutaneous exposures is a consequence of the reducedvolatility and increased use. There have been anecdotal reportsof increased skin irritation in jet mechanics with JP-8 comparedto JP-4, and JP-8 has been shown to be more irritating than JP-4in rat studies (Baker et al., 1999). JP-8 exposure to the skin oflaboratory animals causes erythema and edema, as indicatedvisually and histologically, at periods of 1 h to 4 weeks inhairless rats, rats, and pigs (Kabbur et al., 2001; Kanikkannanet al. , 2001, 2002; Monteiro-Riviere et al., 2001). Local andsystemic toxicity of JP-8 from cutaneous exposures has beenrecently reviewed (McDougal and Rogers, 2004).The physical signs of JP-8 skin irritation are well character-ized in laboratory animals, but the molecular mechanisms arenot well understood. JP-8–induced irritation of the epidermishas been characterized histologically by erythema, edema, andhyperplasia, which increased in severity with exposure time(Baker et al., 1999; Monteiro-Riviere et al., 2001, 2004).Reversible epidermal barrier disruption is seen histologically(Monteiro-Riviere et al., 2001) and functionally with measure-ments of transepidermal water loss (Kanikkannan et al., 2001,2002; Monteiro-Riviere et al., 2001). Swollen mitochondriawith disrupted cristae in the epidermis can occur after 5 h of JP-8 exposure in the pig (Monteiro-Riviere et al., 2004). Theseearly-occurring mitochondrial changes are consistent with JP-8–induced oxidative stress (Rogers et al., 2001) and reversal ofchanges in the delayed hypersensitivity response with antiox-idants (Ramos et al., 2004). Nucleolar margination andsegregation found after 4 days of treatment in pigs suggestthat JP-8 induces abnormalities in DNA signal transduction(Monteiro-Riviere et al., 2004). Seven-day cutaneous treatmentwith JP-8 in rats showed increased neutrophilic infiltration andincreases in transcripts of some of the inflammatory chemo-kines (CXCL1, CXCL2, CCL2, CCL3, and CCL11) andinterleukin-6 (IL-6) in the skin (Gallucci et al., 2004). In this1To whom correspondence should be addressed at Department of Pharma-cology and Toxicology, Wright State University, Health Sciences Building 216,Boonshoft School of Medicine, 3640 Colonel Glenn Highway, Dayton, OH45435. Fax: (937) 775-2001. E-mail: [email protected].Ó The Author 2006. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.For Permissions, please email: [email protected] SCIENCES 95(2), 495–510 (2007)doi:10.1093/toxsci/kfl154Advance Access publication November 3, 2006same study, a cytokine (CCL2), tumor necrosis factor (TNF-a),and IL-1b protein levels were increased. JP-8–exposed whole-skin levels of IL-1a and inducible nitric oxide synthase proteinhave been shown to change as early as 1 h, before any histolo-gical or visual changes occur (Kabbur et al., 2001). The im-munoregulatory cytokine IL-10 has been detected in the serumof mice beginning 48 h after JP-8 cutaneous exposure (Ullrich,1999). JP-8 suppresses cell-mediated immunity in mice afterinhalation (Harris et al., 2000) and cutaneous (Ramos et al.,2002) exposures. These studies suggest that the inflammatoryresponses (oxidative damage, necrosis, apoptosis, lipid synthe-sis, and components of an immune response) of the skin to JP-8involve a variety of pathways. It appears that some of thesemolecular changes precede the visible or microscopic changesthat are identified after exposure, but the trigger and sequence ofthe pathway responses is unclear. It has been suggested thatpreformed and
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