Differential Relationship between the Carbon Chain Length of Jet Fuel Aliphatic Hydrocarbons and Their Ability to Induce Cytotoxicity vs. Interleukin-8 Release in Human Epidermal Keratinocytes

Chi-Chung Chou, Jim E. Riviere and Nancy A. Monteiro-Riviere1

Center for Chemical Toxicology Research and Pharmacokinetics (CCTRP), North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606

Received February 26, 2002; accepted April 29, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Jet fuels are complex mixtures of hydrocarbons known to cause dermal toxicity and to increase the release of proinflammatory cytokines by human epidermal keratinocytes (HEK). However, the dermatotoxic effects of individual hydrocarbons remain unclear. Since aliphatic hydrocarbons make up more than 80% of the hydrocarbons formulated in jet fuels, the objective of this study was to assess acute cytotoxicity and IL-8 release induced by individual aliphatic hydrocarbons without a vehicle. Ten aliphatic hydrocarbons with carbon (C) chain lengths ranging from 6 to 16 were dosed neat on HEK grown on 96-well plates. Acute exposure (1, 5, and 15 min) to aliphatic hydrocarbons significantly increased HEK mortality such that the increase in cytotoxicity corresponded with the decrease in carbon chain length. Extended exposure time did not increase cytotoxicity significantly until 15 min of exposure by short-chain hydrocarbons (C <= 11). There were differences between the aliphatic hydrocarbons with respect to their effects on IL-8 release. IL-8 concentration was increased significantly by 3- to 10-fold, with the highest increase found after exposure to hydrocarbons in the C9–C13 range. These studies indicated that individual aliphatic hydrocarbons are toxic to HEK cells and are capable of inducing proinflammatory cytokines. Higher cytotoxicity by shorter-chain aliphatic hydrocarbons did not correlate to increased ability to stimulate IL-8 release, which peaked at mid-chain lengths, suggesting a different structure-activity relationship for these two toxicological endpoints in keratinocyte cell cultures.

Key Words: keratinocytes; aliphatic hydrocarbons; cytotoxicity; interleukin-8; jet fuels.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Millions of civilians and military personnel are regularly exposed to hydrocarbon fuels, including gasoline and kerosene-based jet fuels. Exposure to hydrocarbon fuels has been consistently shown to cause histological changes (Kabbur et al., 2001Go; Monteiro-Riviere et al., 2001Go; Rhyne et al., 2002Go) and induce inflammatory responses (Broddle et al., 1996Go; Freeman et al., 1990Go, 1993Go) in the skin. While there is increasing concern in the potential for dermatotoxicity of hydrocarbon fuels, little is known about the toxicities of individual jet fuel components nor their contribution to the cutaneous toxicity seen with the final fuel mixture. Jet fuels consist of more than 228 heterogeneous aliphatic and aromatic hydrocarbons (Committee on Toxicology, 1996Go). Each of the major hydrocarbon components could exert one or more toxicological effects in the skin. In order to dissect the dermatotoxicity of such a complex mixture, the toxic potential of the major hydrocarbon components must be individually assessed.

Aliphatic hydrocarbons are the primary hydrocarbon components (81%) of jet fuels, and exhibit a broad range of carbon chain length (9% C8–C9, 65% C10–C14, and 7% C15–C17). Some studies suggest that they are also more likely to be sequestered in the epidermis than aromatic hydrocarbons (Baynes et al., 2000Go; McDougal et al., 2000Go; Riviere et al., 1999Go). Previous studies in our laboratory have suggested that the hydrocarbons common to Jet A, JP-8, and JP8-100 fuels may be responsible for the proinflammatory responses seen in the skin (Allen et al., 2000Go; Monteiro-Riviere et al., 2001Go).

Skin irritation usually has an inflammatory component. Different chemical irritants can trigger different inflammatory processes initiated by different proinflammatory cytokines (Nickoloff, 1991Go). Among them, IL-8 has been shown to increase significantly in response to a variety of chemical irritants (Effendy et al., 2000Go) and osmotic and oxidative stressors (Terunuma et al., 2001Go) and UV irradiation (Clydesdale et al., 2001Go), following the activation of primary proinflammatory cytokines interleukin-1 (IL-1a, IL-1b) and tumor necrosis factor-{alpha} (TNF-{alpha}); (Corsini and Galli, 2000Go). The nonspecific nature of IL-8 as a general mediator, inducible by various external stimuli, makes it an ideal biomarker to assess chemical irritants that possess an inflammatory component. We have previously demonstrated IL-8 and TNF-{alpha} responses by low levels (0.01%) of jet fuels (Allen et al., 2000Go, 2001bGo) and jet fuel components (Allen et al., 2001aGo) in an in vitro model. In these studies, IL-8 was more consistently released than TNF-{alpha} by human epidermal keratinocytes (HEK; Allen et al., 2001bGo). The release of other secondary cytokines (IL-4, IL-6, and granulocyte-macrophage colony-stimulating factor, GM-CSF) in response to jet fuel exposure have not been well studied. IL-8 was selected as the biomarker of choice, over the other secondary cytokines, for the assessment of early inflammatory responses in HEK cells exposed directly to aliphatic hydrocarbons.

Although the primary concerns regarding the skin toxicity of jet fuels is for extended and repeated exposures, such exposures occur typically at concentrations well below the permissible exposure limits. It is not well documented whether high concentrations and acute exposure would result in similar or unexpected skin toxicity (Harris et al., 2000Go). In addition, in vitro dermatological studies usually use vehicles such as DMSO (Dudley et al., 2001Go), ethanol (Allen et al., 2001bGo; Grant et al., 2000Go; Rosenthal et al., 2001Go), and cyclodextrin (Allen et al., 2001aGo) in order to solubilize the lipophilic hydrocarbons into the media or dosing solution. The irritant nature of these vehicles often confounds the interpretation of direct cellular toxicity in the skin.

The objective of this study was to examine the effects of neat aliphatic hydrocarbon exposure on HEK cytotoxicity and IL-8 production as a biomarker of cutaneous irritation. The elimination of the vehicle allows for the intrinsic toxicity of these aliphatic hydrocarbons to be evaluated independently. In order to further elucidate which specific jet fuel hydrocarbon(s) are responsible for the dermatotoxicity, and whether carbon chain length is a contributing factor to which the type of toxicity and/or the degree of toxicity are exhibited, 10 aliphatic hydrocarbons in the carbon range of 6 to 16 (Table 1Go), representing the major aliphatic hydrocarbon clusters found in jet fuels (Basak et al., 2000Go), were investigated for their proinflammatory and cytotoxic effects to HEK.


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TABLE 1 Structure, Formula, and Average Percentage Presence of Major Aliphatic Hydrocarbons in Jet Fuels Used in This Study
 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Test compounds.
Ten aliphatic hydrocarbons, with carbon chain lengths ranging from 6 to 16, were selected for this study (Table 1Go). Cyclohexane (C6), n-octane (C8), n-nonane (C9), n-decane (C10), n-undecane (C11), n-dodecane (C12), n-tridecane (C13), n-tetradecane (C14), n-pentadecane (C15), and n-hexadecane (C16), all with greater than 98% purity, were purchased from Sigma Chemical Co. (St. Louis, MO). Jet A, a base jet fuel devoid of performance additives and JP-8, a fuel consisting of Jet A plus a performance additive package, were kindly supplied by Major T. Miller from Wright Patterson Air Force Base. All chemicals were dosed under sterile conditions to prevent contamination.

Cell culture and dosing regimen.
Cryopreserved adult normal human epidermal keratinocytes (approximately 260K cells/vial) were purchased from Clonetics Corp. (San Diego, CA) and passage on to three 75-cm2 culture flasks, each containing 15 ml of serum-free keratinocyte growth medium (KGM-2; from keratinocytes basal media supplemented with 0.1 ng/ml human epidermal growth factor, 5 µg/ml insulin, 0.4% bovine pituitary extract, 0.1% hydrocortisone, 0.1% transferrin, 0.1% epinephrine, and 50 µg/ml gentamicin/50 ng/ml amphotericin-B). The culture flasks were maintained in a humidified incubator at 37°C with a 95% O2/5% CO2 atmosphere. After reaching 70–80% confluency, the keratinocytes were harvested and plated immediately in 96-well tissue culture-treated plastic ware (Corning, Acton, MA) at a density of 6–8 K cells/well. Once HEK were grown to 80% confluency in the plates, the media was aspirated from each well and the cells were dosed (in triplicate) with 40 µl/well of designated test compound for various lengths of predetermined exposure times (1, 5, or 15 min). The three exposure times were chosen based on a pilot study to evaluate the cell mortality as a function of exposure times. For the purpose of the study, the exposure times were determined (1) to simulate acute exposure where an accidental spill occurs (1 min), (2) to find the longest exposure time without significantly causing cell death (15 min), and (3) to find an optimal exposure time where the cells would maintain approximately 50% viability after exposure to most aliphatic hydrocarbons, so that cytokine production could be reliably evaluated (5 min). The exposure was terminated by aspirating the test compounds with a vacuum. This was immediately followed with a rinse of 100 µl/well KGM-2 medium. After replacing the rinse medium with 200 µl of fresh KGM-2, the plates were returned to the incubator for subsequent sampling. At scheduled time points (see below), the cell media were collected and frozen immediately at –80°C for later IL-8 determination. After sample collection, the cells in each well were evaluated for mortality. In order to evaluate whether the cytotoxicity by hydrocarbons was an acute event or a continuous process, HEK mortality was determined at 4 (n = 7) and 24 (n = 3) h after dosing treatments. For cytokine release, IL-8 concentrations were measured at 0, 1, 4, 8, and 24 h after a 1-min (n = 3) or 5-min (n = 3) exposure of HEK to the test hydrocarbons. IL-8 concentration after a 15-min exposure was not evaluated in light of high cell mortality under this condition.

Cytotoxicity assays.
Cytotoxicity was assessed by determining the percentage mortality of HEK cells after aliphatic hydrocarbon exposure. Cell mortality was determined by the neutral red (NR) uptake method, as described by Borenfreund and Puerner (1985). Briefly, 50 µg/ml NR in KGM-2 (NR medium) was preincubated at 37°C overnight and 200 µl was added to the treated wells after sample collection. Following a 3-h incubation at 37°C in a 95% O2/5% CO2 environment, the medium was replaced by 200 µl of wash/fix solution (1% CaCl2 in 0.5% formaldehyde) for 2 min and NR was extracted with 100 µl of 1% acetic acid/49% H2O/50% ethanol. Color formation after 20 min was evaluated by measuring absorption at 550 nm in an ELISA reader (Multiskan RC, Labsystem, Helsinki, Finland). Absorbance values were then normalized against KGM-2 control wells and expressed as percentage mortality relative to control wells (0% mortality), which is proportional to the cytotoxicity.

Analysis of IL-8.
Cell medium was thawed and assayed in triplicate for IL-8 concentration, using an enzyme-linked immunosorbent assay (ELISA) cytoset kit (Biosource International, Camarillo, CA). Briefly, human anti-IL-8 monoclonal antibody at 1 µg/ml in phosphate-buffered solution (PBS) (pH 7.4) was coated overnight onto 96-well immunoassay plates (Greiner, Germany) at 4°C. Nonspecific binding was blocked with 0.5% bovine serum albumin in PBS for 2 h at room temperature. After washing 3 times with normal saline, 100 µl of samples or standards were added to each well, followed immediately by the addition of a biotinylated mouse antihuman monoclonal anti-IL-8 antibody (0.5 µg/ml) and incubated for 2 h at room temperature. Following another wash, a streptavidin/horseradish peroxidase conjugate (1:2500) was then added to each well and the plates incubated for 30 min at room temperature. In the final step, plates were washed and developed in the dark for 30 min with o-phenylenediamine dihydrochloride plus urea hydrogen peroxide (Sigma Fast, Sigma Chemical Co., St. Louis, MO) at room temperature. Sample IL-8 concentrations were first determined by comparing the absorbance value at 450 nm to a standard curve on a Multiskan RC plate reader. The final IL-8 concentration (pg/ml) for each treatment and time was calculated using Genesis Lite, Ver. 3 for Windows software (Labsystems, Helsinki, Finland) with data normalized by viability in corresponding wells.

Statistics.
The HEK percent cell mortality and total IL-8 concentration from each treatment and time were statistically compared with its own media controls, using ANOVA (SAS 6.12 for Windows; SAS Institute, Cary, NC). Multiple comparisons among different hydrocarbons and jet fuels were also conducted within each exposure length and sampling time by Duncan's multiple comparison procedure. Statistical differences were set at the p < 0.05 level.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxicity of Hydrocarbons
Cytotoxicity was expressed as HEK % mortality compared to the average death of media controls (0%). All treatments with the aliphatic hydrocarbons caused significant increases (p < 0.001) in HEK % mortality except at 4 h after a 1-min exposure to hexadecane. There were no statistically significant (p > 0.05) differences in cytotoxicity with regard to exposure time for aliphatic hydrocarbons with greater than 11 carbons (C > 11). Hydrocarbons with fewer than 12 carbons (C < 12) exhibited an increase in cytotoxicity that was evident with longer exposure time; a significant increase in % mortality was found after 15 min of exposure compared to 1 min of exposure. The difference in cytotoxicity was significant between the 5- and 15-min exposures (Fig. 1Go) with even shorter carbon chain lengths (C9, C8, and C6). HEK mortality also progressed with time after 1- and 5-min exposure. Cell mortality at 24 h was significantly (p < 0.05) greater than at 4 h; indicating that cells continued to die even after the removal of hydrocarbons, rather than recovering from acute exposure. Exposure of HEK to Jet A, the base jet fuel with mixed lengths of hydrocarbons, showed moderate (similar to C11 and C12 at 4 h) to low (similar to C14 to C16 at 24 h) cytotoxicity compared to the individual hydrocarbons. HEK mortality after JP-8 exposure was similar to that of Jet A (see below, Fig. 3Go).



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FIG. 1. HEK mortality after exposure to aliphatic hydrocarbons and Jet A. HEK mortality was evaluated at either 4 or 24 h after exposure to aliphatic hydrocarbons without vehicle for 1, 5, or 15 min. Data represent % mortal cells (mean ± SEM) compared to controls; (a) significantly different from 1-min exposure/evaluated at 4 h; (b) significantly different from 5-min exposure/evaluated at 4 h.

 


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FIG. 3. (A) Cytotoxicity (mean ± SEM, n = 14) after exposure to Jet A, JP-8, and 10 different aliphatic hydrocarbons, grouped by carbon chain length. (B) IL-8 release (mean ± SEM, n = 6) after exposure to Jet A, JP-8, and 10 different aliphatic hydrocarbons grouped by carbon chain length. Hydrocarbons with the same letter and case were not statistically different (p > 0.05).

 
Hydrocarbon Induction of IL-8 Release
With the exception of cyclohexane, acute exposures (1- and 5-min) to aliphatic hydrocarbons resulted in a significant increase in IL-8 release over control cells at 4, 8, and 24 h (Fig. 2Go). There were significant differences among aliphatic hydrocarbons with respect to their effects on IL-8 release. The highest release of IL-8 occurred with hydrocarbons with C9 and C13 chain lengths (Fig. 2Go). Five minutes of exposure to the different hydrocarbons resulted in significantly higher levels of IL-8 release compared to the 1-min exposure.



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FIG. 2. (A) IL-8 concentration (mean ± SEM, pg/ml) of HEK after 1-min exposure to 10 different aliphatic hydrocarbons (n = 3). (B) IL-8 concentration (mean ± SEM, pg/ml) of HEK after a 5-min exposure to 10 different aliphatic hydrocarbons (n = 3). Hydrocarbons with the same letter and bar were not statistically different (p > 0.05).

 
Carbon Chain Length and Toxicity
Multiple comparisons of mortality and IL-8 concentration between individual hydrocarbons exhibited a general similarity among groups of hydrocarbons with similar carbon chain length. Aside from cyclohexane, which had a minimal release of IL-8, the similarities can best be categorized by sorting the test hydrocarbons into 3 clusters of consecutive carbon chain length: C8–C10 (octane, nonane, and decane), C11–C13 (undecane, dodecane, tridecane), and C14–C16 (tetradecane, pentadecane, and hexadecane). Statistically, each group showed a significant difference between groups but there were very few significant differences within each carbon chain group. As shown in Figure 3AGo, cytotoxicity inversely correlated with carbon chain length. The average % cell mortality after exposure to C8–C10 was significantly greater than exposure to C11–C13, which was significantly greater than C14–C16. The increase in mortality with time was more evident in short-chain hydrocarbons. In contrast, there is no linear relationship between carbon chain length and the ability to stimulate IL-8 release (Fig. 3BGo). The smallest fold increase in IL-8 release was found at C8–C10 (4 h), which was similar to Jet A and JP-8 (4 h) but was significantly different from C11–C13 at 4 h. At 24 h, there were no significant differences between C14–C16 and the two fuel types in IL-8 release, but C14–C16 and the two jet fuels were significantly different from C8–C10 and C11–C13. Dodecane was the lowest of C11–C13 range that resemble C14–C16 in expression of IL-8 (Fig. 2BGo). These data suggest that hydrocarbon chain length, in the range studied for C6 to C16, demonstrated a different pattern of toxicity for the endpoints of mortality versus IL-8 release. In contrast, the comparison of Jet A and JP-8 indicated that despite their differences in additive compositions, there was no significant difference between the two jet fuels on these two endpoints, suggesting that the performance additives, when present in the jet fuel, exhibited no additional cellular toxicity to HEK cells based on these two endpoints.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Assessment of jet fuel dermal toxicity is complex in that jet fuels are a mixture of hundreds of hydrocarbons/performance additives, each having different chemical compositions and absorption characteristics through the skin barrier. In order to delineate the mechanisms of toxicity on such a complex mixture, the toxicological effects of each component chemical should provide the most insight into understanding the overall mechanisms of dermatotoxicity. The similarity of Jet A to JP-8 responses in HEK for both cytotoxicity and IL-8 release suggests that additives in JP-8 are not primarily responsible for dermal toxicity. This is consistent with our previous studies (Allen et al., 2000Go, 2001aGo) using both IL-8 and TNF-{alpha} as endpoints. One of the principal JP-8 additives, diethylene glycol monomethyl ether has also been reported to have little cellular toxicity (Geiss and Frazier, 2001Go). Thus, in HEK cultures additives do not modulate cellular toxicity. In contrast, such additives could differentially modulate the absorption of different hydrocarbon fuel components through intact skin (Baynes et al., 2001Go) and produce different patterns of jet fuel toxicity as a function of hydrocarbon penetration.

In view of the large numbers of hydrocarbons that are components of jet fuels, only one cytokine was evaluated as a biomarker of the irritation response to complement the cytotoxicity assay (manifested by cell mortality). The choice of IL-8 was based on the assumption that it provided broad recognition of irritation and has high reproducibility in response to jet fuel exposure in HEK cells, as discussed earlier. Although the inflammatory cytokine network has been extensively studied (Cavaillon, 2001Go; Feghali and Wright, 1997Go; Takashima and Bergstresser, 1996Go), to our knowledge IL-10 was the only other secondary proinflammatory cytokine that has been investigated in regard to jet fuel toxicity (Ullrich, 1999Go; Ullrich and Lyons, 2000Go). The specific mechanisms by which the IL-8 response is triggered cannot be discerned by monitoring IL-8 alone; however, changes in IL-8 release serve as a robust biomarker for cutaneous irritation.

The most important findings of this study were that there were significant differences among aliphatic hydrocarbon chain lengths with respect to their effects on HEK mortality versus IL-8 release. Rather than a general increase corresponding to decreased carbon chain length, as was seen in the HEK mortality, the increase in IL-8 production showed a peak in response around C9–C13. The greater effect on cytotoxicity did not necessarily correlate to a higher potential to increase IL-8 release (Fig. 3Go), suggesting that the mechanisms by which each hydrocarbon causes dermal toxicity are different. The increase in short chain cytotoxicity could be the result of the enhanced hydrocarbon penetration in the cell as seen in the isolated perfusion skin flap and diffusion cell model (Baynes et al., 2001Go; McDougal et al., 2000Go; Riviere et al., 1999Go). Cytotoxicity of this nature could be related to the ability of short-chain hydrocarbons to disrupt membrane function.

The higher cytokine release with intermediate carbon chain lengths might be associated with an enhanced membrane residence time, which enable them to interact with IL-8 receptors, (Aggarwal and Puri, 1995Go; Kemeny et al., 1994Go), mimicking an increased dosing time. This was supported by several dermal absorption studies (Baynes et al., 2000Go; McDougal et al., 2000Go; Riviere et al., 1999Go) assessing the dermal disposition of hydrocarbons, and it was found that dodecane (C12) had a significantly greater residual surface concentration than octane (C8). However, in a monolayer the differences in the absorption rate and residence time due to membrane barriers would be less important than for intact skin models. It is therefore feasible that the intrinsic toxicity of each hydrocarbon contributes to the differences in expression of cytotoxicity of complete fuels. It is unfortunate that intrinsic potency and the delivery of the chemical will always be confounded when using a cell culture model. The ability of detecting intracellular concentration of individual hydrocarbons would be imperative to the differentiation of these two factors.

The maximal biological effects peaked with the middle-length carbon chain (C12–C14) have been reported earlier. The n-alkanes have been shown to exhibit maximal skin tumor-promotion activity at chain lengths of C12–C14 (Baxter and Miller, 1987Go; Sice, 1966Go). Earlier investigators had also demonstrated that skin irritation peaked at C14 (Brown and Box, 1970Go). The underlying mechanism was not clear. The maximal IL-8 release peaking at C9 to C13 in this study might be the result of a balance between membrane and/or intracellular availability of the hydrocarbons characterized by a wide range of partition coefficients (5.18 for C8 and 8.46 for C16). In addition to IL-8 receptors, certain membrane components could also selectively facilitate the retention and/or transportation of hydrocarbons through the membrane. For instance, the CD1 family (CD1a, CD1b, CD1c and CD1d) proteins, which are responsible for the T-cell recognition of lipophilic antigens (Moody et al., 1999Go), have a specific affinity for aliphatic hydrocarbons through their long-chain aliphatic tails. Whichever mechanism(s) are involved, the results have implications for safety measures since the middle-range aliphatic hydrocarbons (C10–14) are the most abundant hydrocarbons, which constitute 65% of the total base jet fuel. One should also realize that the artificial conditions of cell culture preclude a direct correlation of chemical contact time in culture to an accidental occupational exposure. The results indicated that a noncytotoxic increase in exposure time (1 to 5 min) could result in a significant increase in the release of the proinflammatory cytokine IL-8. Therefore, reducing total hydrocarbon exposure is very important.

In addition to carbon chain length, the structural or electron configuration of the chain could also affect the chemical properties and hence the toxicological effects it can elicit (Dugard and Scott, 1984Go; Flynn, 1990Go; Scheuplein and Blank, 1971Go). Earlier studies using structure-activity relationships on approximately 800 ring and nonring chemicals did not clearly elucidate whether one type of functional group is more likely to cause skin irritation (Enslein et al., 1987Go). In this study, ring-shaped cyclohexane, while clearly was the most cytotoxic to HEK, induced only a minimal IL-8 response, smaller than the control level. The preliminary investigation on the dermatotoxicity of ring-configured aromatic hydrocarbons to HEK cells noted that some, but not all, tested aromatic hydrocarbons cause a significant decrease in IL-8 (unpublished data), suggesting the opposing effect in IL-8 production involved more than the difference in ring-versus-chain configurations. Furthermore, despite the higher toxicities of decane, undecane, and tridecane (C10–C13 group), dodecane (C12) tends to stimulate less IL-8 release compared to the other hydrocarbons in the same group, once more implicating a higher level structural element correlate to toxicity than simple chain length. Therefore, the two responses seen after jet fuel exposure (acute cytotoxicity versus irritation) may be due to different components exerting different mechanisms, where shorter chain aliphatics (C6–C9) cause cytotoxicity and others (C10–C13) modulate immune response. To complicate this scenario further, topical hydrocarbons may also extract intercellular skin lipids, which potentially could increase dermal absorption, and thus toxicity, in an intact animal.

Finding the specific mechanisms by which hydrocarbons cause dermatotoxicity warrants further study. The current results suggest that pathways involved in the regulation of IL-8 expression might be related to the underlying mechanisms for hydrocarbon toxicity. Bear in mind that the regulation of inflammatory cytokines is an interactive network rather than separated pathways. IL-10 (Farkas et al., 2001Go; Oberholzer et al., 2001; Reich et al., 2001Go), nuclear factor kappa B (NF-{kappa}B) (Cruz et al., 2001Go; Takizawa et al., 2000Go; Valacchi et al., 2001Go) and p38 (Arbabi and Maier, 2002Go) pathways are the most feasible targets worth further investigations in view of their direct regulatory relationship to IL-8 and connections to other proinflammatory mediators such as nitric oxide (Bruch-Gerharz et al., 1996Go; Senftleben and Karin, 2002Go). IL-1b and TNF-{alpha} will likely parallel IL-8 response based on earlier findings (Allen et al., 2000Go, 2001aGo,bGo).

Finally, it is important to note that the toxicological effects of individual hydrocarbons carried different weight in the overall mixture toxicities, because aliphatic hydrocarbons are not all present in equal fractions in jet fuels. This issue is even more complicated by possible synergistic effects among coexisting hydrocarbon components. Therefore, it is equally as important to interpret the toxicities of hydrocarbons by clusters with respect to their compositional presence in the fuels or by different clusters with which different toxicological endpoints are indicated. The statistical analysis in this study has led us to a meaningful reclustering of hydrocarbons by their carbon chain length, which is similar to their fractional presence in the base fuel. In conclusion, we have characterized 10 aliphatic hydrocarbons in their ability to induce proinflammatory cytokine IL-8 release and cytotoxicity to HEK cells. Significant differences in individual dermatotoxicity, with regard to the carbon chain length, were detected. Specifically, the relationship of aliphatic hydrocarbon chain length to cell mortality is different than to the induction of IL-8 release in human epidermal keratinocytes.


    ACKNOWLEDGMENTS
 
This work was supported by the U.S. Air force office of Scientific Research F49620–01–1–0080. Portions of this work were presented at the 41st Annual Meeting of the Society of Toxicology, Nashville, TN. We thank Mr. Alfred O. Inman for his assistance.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (919) 513-6358. E-mail: nancy_monteiro{at}ncsu.edu. Back


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