Analysis of interleukin-8 release from normal human epidermal keratinocytes exposed to aliphatic hydrocarbons: delivery of hydrocarbons to cell cultures via complexation with α-cyclodextrin
Introduction
Jet A, JP-8 and JP-8+100 are the three predominant jet fuels used today. Jet A is the commercial fuel while JP-8 and JP-8+100 are military fuels used by both US and NATO forces. Most jet fuel toxicology studies have focused on their capacity for pulmonary toxicity. However, recent emphasis on the dermal toxicity of jet fuels has uncovered that they have the capacity for traversing the stratum corneum barrier, and that they can incite a biological response (Riviere, Brooks, Monteiro-Riviere, Budsaba, & Smith, 1999, Ullrich, 1999, Monteiro-Riviere, Inman, Rhyne, & Riviere, 2000). Furthermore, the individual components of the fuels may traverse the epidermis at varying rates, or become sequestered within the epidermis to increase their exposure time to the viable keratinocytes (Riviere, Brooks, Monteiro-Riviere, Budsaba, & Smith, 1999, McDougal, Pollard, Weisman, Garrett, & Miller, 2000, Baynes, Brookes, & Riviere, 2001). Much of the toxicological concern for jet fuels has focused on the recent addition of performance additives. Jet A is a kerosene-cut jet fuel base composed of a complex mixture of 228 aliphatic and aromatic hydrocarbon compounds. JP-8 is a mixture of Jet A plus three additives, an icing inhibitor (diethylene glycol monomethyl ether), an antistatic compound (Stadis 450) and a corrosion inhibitor (DC1-4A/8Q21). JP-8+100 contains JP-8 and an additional additive package (an antioxidant, chelator, detergent and dispersant). Recent studies in our laboratory have revealed that jet fuels induce the production and release of interleukin-8 by normal human epidermal keratinocytes (NHEK) (Allen et al., 2000). However, these studies demonstrated that there were no statistical differences among the responses of NHEK to the individual jet fuels. This has led to the current hypothesis that the hydrocarbon components, common to all of the jet fuels, may lead to the proinflammatory cytokine generation.
McDougal et al. (2000) provided a gas chromatographic analysis of the most prevalent hydrocarbon components in a single lot of JP-8. Because jet fuels are synthesized based on performance characteristics rather than chemical composition, their lot-to-lot component concentrations are variable. However, there are broad compositional limits for the concentrations of aromatic (22%) and aliphatic (81%) hydrocarbons. Most of the aliphatic hydrocarbons (65%) are between 10 and 14 carbons in length (McDougal et al., 2000). These data can be use to assume general trends in component composition for a particular lot of jet fuel. Because there are so many different components in jet fuels, it would take endless hours to study the toxicity of each one. Therefore, clustering them into groups allows for a broader survey for future predictions of similar components (Basak et al., 2000). To test the hypothesis that common cluster components have similar biological effects, we have chosen three aliphatic components in the 10–14 carbon range (undecane, dodecane, tridecane) and one aliphatic component outside this class (hexadecane). An additional rationale for selecting these specific components is the fact that they were among the most prevalent in the analysis of McDougal et al. (2000).
One significant problem with tissue culture systems is the necessity for solubilizing the compound of choice into aqueous media. Because jet fuels are complex mixtures that may contain both hydrophobic and hydrophilic components they can be adequately solubilized by mixing with absolute ethanol (Grant et al., 2000; R. Young and M. Witten, personal communication). However, the individual hydrocarbons by themselves are completely insoluble in water. Previously, we have attempted to solubilize individual hydrocarbons using multiple organic solvents as well as multiple surfactants with limited success (unpublished observation).
Cyclodextrins have been used in the pharmaceutical arena for over 30 years. They are cyclic oligosaccharides that have six to eight dextrose molecules joined together. This arrangement creates a lipophilic “open cavity” region and a hydrophilic exterior (Stella and Rajewski, 1997). This allows for a hydrophobic molecule to associate with the lipophilic region and form an inclusion complex. The number of dextrose molecules determines the size of the lipophilic central cavity and thus the size of hydrophobic molecule it can accommodate. Aliphatic chains generally fit best into an α-cyclodextrin (six dextroses) while aromatic groups fit best into a β-cyclodextrin (seven dextroses) (Yalkowsky, 1999). The encapsulation of branched alkanes into cyclodextrin inclusion complexes has been used successfully to deliver them to mammalian cells (Janz and Shacter, 1991). The lipophilic cavity is exposed, and therefore the encapsulated hydrocarbon can come in contact with the very lipophilic cell membrane and dissociate from the complex to interact with the cell.
These studies represent novel data demonstrating the successful solubilization of four aliphatic hydrocarbons by complexation with α-cyclodextrin. Furthermore, these data also demonstrate that the aliphatic hydrocarbon components in jet fuels are capable alone to cause keratinocyte proinflammatory cytokine induction.
Section snippets
Inclusion complex formation
Inclusion complexes were formed according to the method described in Janz and Shacter (1991). In order to determine the concentration of hydrocarbon in the inclusion complexes, initial experiments were performed using 14C-labeled dodecane as a marker component for aliphatic hydrocarbons. Briefly, hydrocarbons (2 mm) were added to a 4 mm solution of α-cyclodextrin and allowed to stir for 24 h at room temperature. The resulting inclusion complexes were pelleted by centrifugation at 3500 g and
Inclusion complex formation
The mean percent recovery in the α-cyclodextrin inclusion complex pellet for dodecane was 91%. This value was used to determine the approximate concentration of each hydrocarbon in their respective inclusion complexes using the specific activities of a known concentration of each isotope. Owing to their structural similarities, undecane, tridecane and hexadecane were assumed to be complexed with α-cyclodextrin similarly to dodecane.
Cytotoxicity of hydrocarbons
Cells treated with only α-cyclodextrin showed no significant
Discussion
Recent studies have shown that repeated and prolonged topical exposures to jet fuels result in skin irritation (Kinkhead, Sallins, & Wolfe, 1992, Baker, Dodd, McDougal, & Miller, 1999). However, to date the causative fuel components have not been elucidated. Previous studies in our lab (Allen et al., 2000) have demonstrated that Jet A, JP-8 and JP-8+100 all cause NHEK to produce and release pro-inflammatory cytokines such as IL-8. However, these studies also revealed that no significant
Acknowledgements
This work represents partial fulfillment of the requirement for the degree of Doctor of Philosophy (D. Allen) and was partially supported by the US Air Force Office of Scientific Research FQ8671-98-000-462.
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