TOXICOLOGICAL SCIENCES 69, 226 –233 (2002)
Copyright © 2002 by the Society of Toxicology
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-Riviere 1
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
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 3to 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 midchain 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.
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., 2001; Monteiro-Riviere et al., 2001; Rhyne et al., 2002)
and induce inflammatory responses (Broddle et al., 1996; Freeman et al., 1990, 1993) 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
1
To whom correspondence should be addressed. Fax: (919) 513-6358.
E-mail: [email protected].
226
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, 1996). 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., 2000; McDougal et al., 2000; Riviere et al.,
1999). 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., 2000; Monteiro-Riviere et al., 2001).
Skin irritation usually has an inflammatory component. Different chemical irritants can trigger different inflammatory
processes initiated by different proinflammatory cytokines
(Nickoloff, 1991). Among them, IL-8 has been shown to
increase significantly in response to a variety of chemical
irritants (Effendy et al., 2000) and osmotic and oxidative
stressors (Terunuma et al., 2001) and UV irradiation (Clydesdale et al., 2001), following the activation of primary proinflammatory cytokines interleukin-1 (IL-1a, IL-1b) and tumor
necrosis factor-␣ (TNF-␣); (Corsini and Galli, 2000). 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-␣ responses by
low levels (0.01%) of jet fuels (Allen et al., 2000, 2001b) and
jet fuel components (Allen et al., 2001a) in an in vitro model.
In these studies, IL-8 was more consistently released than
TNF-␣ by human epidermal keratinocytes (HEK; Allen et al.,
2001b). The release of other secondary cytokines (IL-4, IL-6,
and granulocyte-macrophage colony-stimulating factor, GMCSF) in response to jet fuel exposure have not been well
227
TOXICITY OF ALIPHATIC HYDROCARBONS
TABLE 1
Structure, Formula, and Average Percentage Presence of Major Aliphatic Hydrocarbons in Jet Fuels Used in This Study
Name
Formula
Structure
MW
% (w/w)
Cyclohexane
C 6H 12
84.2
1.0
n-Octane
C 8H 18
114.2
0.8
n-Nonane
C 9H 20
128.3
1.1
n-Decane
C 10H 22
142.3
3.8
n-Undecane
C 11H 24
156.3
7.3
n-Dodecane
C 12H 26
170.3
4.7
n-Tridecane
C 13H 28
184.4
4.4
n-Tetradecane
C 14H 30
198.4
3.0
n-Pentadecane
C 15H 32
212.4
1.6
n-Hexadecane
C 16H 34
226.5
0.4
Note. MW, molecular weight; % (w/w), weight/weight.
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., 2000). In addition, in
vitro dermatological studies usually use vehicles such as
DMSO (Dudley et al., 2001), ethanol (Allen et al., 2001b;
Grant et al., 2000; Rosenthal et al., 2001), and cyclodextrin
(Allen et al., 2001a) 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 1), representing
the major aliphatic hydrocarbon clusters found in jet fuels
(Basak et al., 2000), were investigated for their proinflammatory and cytotoxic effects to HEK.
MATERIALS AND METHODS
Test compounds. Ten aliphatic hydrocarbons, with carbon chain lengths
ranging from 6 to 16, were selected for this study (Table 1). 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 nhexadecane (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-cm 2 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 ␮g/ml insulin, 0.4% bovine pituitary extract, 0.1% hydrocortisone, 0.1% transferrin, 0.1% epinephrine, and 50 ␮g/ml gentamicin/50 ng/ml
amphotericin-B). The culture flasks were maintained in a humidified incubator
at 37°C with a 95% O 2/5% CO 2 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
␮l/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
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CHOU, RIVIERE, AND MONTEIRO-RIVIERE
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 ␮l/well KGM-2 medium. After replacing the rinse
medium with 200 ␮l 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 ␮g/ml NR in KGM-2 (NR
medium) was preincubated at 37°C overnight and 200 ␮l was added to the
treated wells after sample collection. Following a 3-h incubation at 37°C in a
95% O 2/5% CO 2 environment, the medium was replaced by 200 ␮l of wash/fix
solution (1% CaCl 2 in 0.5% formaldehyde) for 2 min and NR was extracted
with 100 ␮l of 1% acetic acid/49% H 2O/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 ␮g/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 ␮l 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 ␮g/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
Cytotoxicity of Hydrocarbons
Cytotoxicity was expressed as HEK % mortality compared
to the average death of media controls (0%). All treatments
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.
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 5and 15-min exposures (Fig. 1) 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. 3).
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.
2). 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
TOXICITY OF ALIPHATIC HYDROCARBONS
229
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).
and C13 chain lengths (Fig. 2). Five minutes of exposure to the
different hydrocarbons resulted in significantly higher levels of
IL-8 release compared to the 1-min exposure.
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
230
CHOU, RIVIERE, AND MONTEIRO-RIVIERE
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. 2B). 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
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).
shown in Figure 3A, 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. 3B). The smallest fold increase in IL-8 release
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., 2000, 2001a) using both IL-8 and TNF-␣ 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, 2001). 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., 2001) 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, 2001; Feghali and Wright, 1997;
Takashima and Bergstresser, 1996), to our knowledge IL-10
was the only other secondary proinflammatory cytokine that
has been investigated in regard to jet fuel toxicity (Ullrich,
1999; Ullrich and Lyons, 2000). 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.
TOXICITY OF ALIPHATIC HYDROCARBONS
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. 3), 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., 2001; McDougal et al., 2000; Riviere et al., 1999). Cytotoxicity of this nature could be related to the ability of shortchain 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, 1995; Kemeny et al., 1994), mimicking an
increased dosing time. This was supported by several dermal
absorption studies (Baynes et al., 2000; McDougal et al., 2000;
Riviere et al., 1999) 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 middlelength carbon chain (C12–C14) have been reported earlier. The
n-alkanes have been shown to exhibit maximal skin tumorpromotion activity at chain lengths of C12–C14 (Baxter and
Miller, 1987; Sice, 1966). Earlier investigators had also demonstrated that skin irritation peaked at C14 (Brown and Box,
1970). 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., 1999), have a specific affinity for
aliphatic hydrocarbons through their long-chain aliphatic tails.
Whichever mechanism(s) are involved, the results have impli-
231
cations 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, 1984; Flynn, 1990; Scheuplein and Blank, 1971).
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., 1987). In this study, ringshaped 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., 2001; Oberholzer et
al., 2001; Reich et al., 2001), nuclear factor kappa B (NF-␬B)
(Cruz et al., 2001; Takizawa et al., 2000; Valacchi et al., 2001)
and p38 (Arbabi and Maier, 2002) 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., 1996; Senftleben and Karin, 2002). IL-1b and
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CHOU, RIVIERE, AND MONTEIRO-RIVIERE
TNF-␣ will likely parallel IL-8 response based on earlier
findings (Allen et al., 2000, 2001a,b).
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.
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