Skin toxicity of jet fuels: ultrastructural studies and the effects of substance P

Abstract

Topical exposure to jet fuel is a significant occupational hazard. Recent studies have focused on dermal absorption of fuel and its components, or alternatively, on the biochemical or immunotoxicological sequelae to exposure. Surprisingly, morphological and ultrastructural analyses have not been systematically conducted. Similarly, few studies have compared responses in skin to that of the primary target organ, the lung. The focus of the present investigation was 2-fold: first, to characterize the ultrastructural changes seen after topical exposure to moderate doses (335 or 67 μl/cm²) of jet fuels [Jet A, Jet Propellant (JP)-8, JP-8+100] for up to 4 days in pigs, and secondly, to determine if co-administration of substance P (SP) with JP-8 jet fuel in human epidermal keratinocyte cell cultures modulates toxicity as it does to pulmonary toxicity in laboratory animal studies. The primary change seen after exposure to all fuels was low-level inflammation accompanied by formation of lipid droplets in various skin layers, mitochondrial and nucleolar changes, cleft formation in the intercellular lipid lamellar bilayers, as well as disorganization in the stratum granulosum–stratum corneum interface. An increased number of Langerhans cells were also noted in jet fuel-treated skin. These changes suggest that the primary effect of jet fuel exposure is damage to the stratum corneum barrier. SP administration decreased the release of interleukin (IL)-8 normally seen in keratinocytes after JP-8 exposure, a response similar to that reported for SP's effect on JP-8 pulmonary toxicity. These studies provide a base upon which biochemical and immunological data collected in other model systems can be compared.

Introduction

Approximately 5 billion gallons of JP-8 are used each year by U.S. and N.A.T.O. military services. Jet fuels used in commercial and military aircraft are a complex multicomponent mixture consisting of hundreds of aromatic and aliphatic hydrocarbons. Military fuels are designated as members of the Jet Propellant (JP) series with JP-4 and JP-7 being the primary fuels used in earlier years. In the 1980s, the Department of Defense chose JP-8 as its primary fuel for aircraft, ground vehicles, and other equipment such as tent heaters and generators. The primary military jet fuel, JP-8, is a kerosene-cut commercial jet fuel base, Jet A, to which additives are added to make up <2% of the formulation [a corrosion inhibitor DC1-4A, an antistatic compound Stadis 450, and an icing inhibitor diethylene glycol monomethyl ether (DIEGME)]. JP-8+100 is JP-8 with an additional additive package consisting of the antioxidant BHT, a chelator, a detergent, and dispersant components. In the base fuel, 13 components, present at greater than 1% v/v concentrations, comprise the bulk of the formulation (dodecane, tridecane, undecane, 2,6-dimethylundecane, pentadecane, 2-methylundecane, tetradecane, heptylcyclohexane, 1-methylnaphthalene, 2-methylnaphthalene, 2,6-dimethylnaphthalene, naphthalene, and 1,2,3,4-tetramethylbenzene).

Topical exposure to jet fuels has been shown to induce a broad spectrum of toxicological effects in the skin ATSDR, 1995, ATSDR, 1998, Grant et al., 2000, Henz, 1998, Kabbur et al., 2001, Monteiro-Riviere et al., 2001a, National Research Council, 1996, National Research Council, 2003, Rhyne et al., 2002, Ullrich, 1999, Zeiger and Smith, 1998. There have been assorted studies on acute dermal exposure to jet fuel, although most have been observational and lack mechanistic data. Very high dose dermal exposure in rodents (e.g. 2–8 g/kg/day × 5 days/week × 13 weeks in mice; 0.5 g/kg/day × 5 days/week × 103 weeks in female mice) may be lethal. Lower-level exposures have been implicated in causing nonspecific irritation ATSDR, 1998, Schultz et al., 1981. Many studies quoted in general jet fuel toxicology reviews lack precise dosimetry that makes interpretation difficult. Studies of dermal jet fuel exposure to humans are limited. In one early report, there was a dose-dependent increase in dermatitis from acute exposures to 55–85% solutions of kerosene (1.5 ml of a solution applied to “midback” with an occlusive tape for 24 h), with no effect from a more dilute 40% solution (Tagami and Ogino, 1973). Again, controls were not performed in this study making interpretation difficult. In a pathology study by Lupulescu and Birmingham, 1975, Lupulescu and Birmingham, 1976, topical application of 1.0 ml of kerosene impaired epidermal protein synthesis, but not DNA replication nor collagen synthesis. Hyperemia, cellular damage of the epidermis, and mild edema also occurred following 90 min of this acute exposure. Histological changes included disorganization of the cells, cytolysis, and enlarged intercellular spaces in the stratum corneum and stratum spinosum cells of the epidermis. Chronic dermal exposure to longer-chain aliphatics (C14–C25) may result in skin tumors (reviewed in Ritchie et al., 2001), as does exposure to middle distillate steams that contain components similar to jet fuels Freeman et al., 1993, Nessel et al., 1999. Skin sensitization assays have demonstrated some level of irritation after exposure to the older fuels that may lead to chemical burns. Repeated patch testing does not consistently produce evidence of sensitization ATSDR, 1995, 1996). In general, acute exposure to moderate concentrations of jet fuel or kerosene results in skin irritation and hyperplasia Nessel et al., 1999, Walborg et al., 1998. Application of liquid jet fuels under occluded dosing dramatically increased erythema and reduced hairless rat skin barrier function as assessed by transepidermal water loss (TEWL) Kanikkannan et al., 2001, Kanikkannan et al., 2002, which is consistent with lipid and protein extraction from porcine skin seen after jet fuel exposure (Singh and Singh, 2001). Our group has found that lower-level exposure, which more closely mimics occupational scenarios, produces less adverse effects Monteiro-Riviere et al., 2001a, Rhyne et al., 2002.

Studies of jet fuel exposure also have been conducted in swine, an accepted animal model for human skin. Absorption of hydrocarbon markers from jet fuels has been documented in porcine skin (Riviere et al., 1999). Histologically, in swine across all fuel types (Jet A, JP-8, JP-8+100) exposure to fabric-occluded low doses (67 μl/cm²) of fuel resulted in increased epidermal thickness, intraepidermal and subcorneal microabscesses, and altered enzyme histochemistry patterns when dosed for 4 days in vivo Monteiro-Riviere et al., 2001a, Rhyne et al., 2002. JP-8+100 exposure tended to have more severe effects. This supports anecdotal data that dermal reactions among workers were more pronounced in JP-8+100 compared to other fuels.

Recent toxicological investigations have started shedding light on some potential mechanisms of action. Ullrich et al. have reported in a series of studies Ramos et al., 2002, Ullrich, 1999, Ullrich and Lyons, 2000 that low-dose (50 μl/day) repeated (5 days) JP-8 exposure to C3H/HeN mice suppressed contact and delayed hypersensitivity responses, including depressing the protective effect of prior vaccinations. They hypothesized that IL-10 and PGE₂ are produced by keratinocytes that distribute systemically and down-regulated the cell-mediated immune response. Blocking PGE₂ release by a selective cyclooxygenase-2 inhibitor significantly reversed this immunosuppression. Gavage studies suggest that the immunotoxicity seen is regulated by an aryl hydrocarbon receptor-independent mechanism (Dudley et al., 2001). Our laboratory Allen et al., 2000, Allen et al., 2001a has shown that JP-8 increases pro-inflammatory cytokine production of TNFα and IL-8 in human keratinocytes within 4 h of JP-8 exposure. There is no appreciable difference in reaction after exposure to Jet A, JP-8, or JP-8+100. This is the same time frame seen in rats, where IL-1α and inducible nitrous oxide synthetase (iNOS) expression was increased after JP-8 exposure (Kabbur et al., 2001). Finally, Rosenthal et al. (2001) have shown that JP-8 induces cell necrosis in keratinocyte cultures at levels that induced apoptosis in lung epithelial cells. Higher levels of fuel exposure resulted in differential modulation of pro- and anti-survival members of the Bcl-2 gene family. Inhalational exposure to JP-8 is known to produce significant adverse effects Harris et al., 1997, Harris et al., 2000a, Hays et al., 1995, Pfaff et al., 1995, Robledo and Witten, 1998, Robledo and Witten, 1999, Stoica et al., 2001. Lung toxicity is characterized by increased pulmonary resistance, interstitial edema, endothelial damage, and apoptotic cell death. Suppression of cell-mediated immune response has been observed after inhalational exposure, as discussed above an effect also seen topically in mice. Administration of the tachykinin substance P (SP) ameliorates this pulmonary toxicity, although administration of a SP antagonist worsened the toxicity. This JP-8 response was similar to that seen with tetradecane exposure.

Most of these studies have been conducted with the complete jet fuels, and thus there is no firm grasp on which specific hydrocarbon, or class, are responsible for the toxicity seen. Studies have been conducted to assess if all hydrocarbon components of fuel are equi-toxic relative to their ability to induce cytokine release (IL-8), and furthermore, if there was a relationship between direct cytotoxicity and cytokine release Allen et al., 2000, Allen et al., 2001b, Chou et al., 2002, Chou et al., 2003. For the aliphatics (C6–C16) tested, increased cytotoxicity was associated with decreased chain length. In contrast, maximum IL-8 release peaked at chain lengths of C10–C13. Responses of keratinocytes to aromatic component exposure were significantly more complex. At cytotoxic doses, IL-8 release paralleled rank order of toxicity. In contrast, many of these compounds significantly decreased IL-8 release at non-cytotoxic (<5% lethality) doses. Methylnaphthalene, dimethylnaphthalene, and naphthalene increased IL-8 release. Both the aliphatic and aromatic studies strongly suggest that cytotoxicity, and ability to cause IL-8 release, are independent toxicological endpoints; and multiple hydrocarbon components are capable of producing toxicological sequelae that mimic complete jet fuel exposure.

The focus of the manuscript is to ultrastructurally characterize early changes in vivo after topical application of jet fuels to porcine skin. A number of mechanisms have been proposed for the low-level jet fuel adverse effects seen, however, little in vivo data exist to see if these biochemical changes (e.g. cytokine release, altered barrier function) have morphological manifestations. Special fixation and staining techniques that preserve lipid architecture were employed to assess ultrastructural damage to lipid lamellae that constitute the stratum corneum barrier. The relationship of dermal to inhalational toxicity deserves attention because the cellular response (necrosis versus apoptosis) appears different, although both result in local epithelial immunosuppression. To investigate this linkage, keratinocytes will be exposed to the same substance P that ameliorated the pulmonary effects in mice. Combined, these studies should shed light both on the effects at a cellular level of low-level jet fuel exposure as well as start to provide additional evidence on the pathways involved in modulating the mechanism of jet fuel toxicity.