Ann. occup. Hyg., Vol. 48, No. 1, pp. 65–73, 2004
© 2004 British Occupational Hygiene Society
Published by Oxford University Press
DOI: 10.1093/annhyg/meg081
Determination of Keratin Protein in a Tape-stripped
Skin Sample from Jet Fuel Exposed Skin
YI-CHUN E. CHAO and LEENA A. NYLANDER-FRENCH*
Department of Environmental Sciences and Engineering, School of Public Health, The University of
North Carolina at Chapel Hill, Chapel Hill, NC 27599-7400, USA
Received 28 March 2003; in final form 20 May 2003
Chemical contaminants or their metabolites may bind to and react with keratin proteins in the
stratum corneum of the skin. Here, we present a tape-stripping method for the removal and
quantification of keratin from the stratum corneum for normalization of extracted concentrations of naphthalene (as a marker for jet fuel exposure) from 12 human volunteers before and
after exposure to jet fuel (JP-8). Due to the potential for removal of variable amounts of squamous tissue from each tape-strip sample, keratin was extracted and quantified using a modified
Bradford method. Confirmation of the extraction of keratin was verified by western blotting
using a monoclonal mouse anti-human cytokeratin antibody. Naphthalene was quantified in the
sequential tape strips collected from the skin between 10 and 25 min after a single dose of JP-8
was initially applied. The penetration of jet fuel into the stratum corneum was demonstrated by
the fact that the average mass of naphthalene recovered by a tape strip decreased with
increased exposure time and subsequent tape strips and that the evaporation of naphthalene
was observed to be negligible. There were no significant differences in the amount of keratin or
naphthalene removed by tape strips between males and females, between age groups, races or
degrees of skin pigmentation. We conclude that (i) the amount of keratin removed with tape
strips was not affected by up to a 25 min exposure to JP-8 and (ii) there was a substantial
decrease in the amount of keratin removed with consecutive tape strips from the same site, thus,
adjusting the amount of naphthalene by the amount of keratin measured in a tape-strip sample
should improve the interpretation of the amount of this analyte using this sampling approach.
Although we found that normalization of the naphthalene to the amount of keratin in the tapestrip samples did not affect the ability of this method to quantify the dermal exposure to JP-8
under these laboratory conditions, the actual concentration of naphthalene (as a marker for JP8 exposure) per unit of keratin in a tape-strip sample can be determined using this method and
may prove to be required when measuring occupational exposures under field conditions.
Keywords: colorimetric protein assay; dermal exposure; jet fuel; JP-8; keratin; naphthalene; skin; stratum corneum;
tape-stripping
ical factors that influence skin penetration. Methods
to assess the significance of dermal exposure are
limited in both number and scope. Because the skin is
a large complex organ with xenobiotic metabolism
and dynamic immune response systems (Mukhtar,
1992; Marzulli and Maibach, 1996), the technology
used to assess dermal exposure to hazardous chemicals must be able to assay these complex interactions.
Methods developed to measure the quantity of
chemical contaminants deposited directly on the skin
under occupational or experimental conditions
include the use of passive exposure patches, clothing,
skin swabs, liquid rinses and tracers. While these
methods provide information on the mass of a chem-
INTRODUCTION
Many environmental chemicals can partially or fully
breach the skin of exposed individuals where they
may be metabolized and interact with dermal macromolecules or the skin immune system and/or be
systemically absorbed and distributed to other potential target sites. Studies of skin toxicity have mainly
focused on methods for evaluating skin irritation and
allergic reactions or investigating the physicochem-
*Author to whom correspondence should be addressed.
Tel: +1-919-966-3826; Fax: +1-919-966-4711; e-mail:
[email protected]
65
66
Y.-C. E. Chao and L. A. Nylander-French
ical contaminant that may have been deposited on
unprotected skin, they fail to relate the amount of
contamination on the skin to the amount actually
absorbed into and through the skin and consequently
made available for systemic uptake (total body dose).
None of these approaches discerns the difference
between chemicals absorbed into the non-viable
keratinized layers of the skin, and that ultimately
diffuse through the skin resulting in systemic
exposure, and those that react at the site of contact
with the non-viable and/or viable components of the
skin. Most importantly, these methods are difficult to
standardize for routine use in occupational field
settings. Biological monitoring has been used to
indirectly measure dermal exposure. However, the
use of biological monitoring methods to determine
transdermal exposure and absorption requires several
assumptions about the absorption of contaminants in
the lung, tidal volumes, respiratory rates and the movement of materials onto and through the epidermis.
None of the currently used methods can be used to
measure the actual dose resulting from reactions with
the skin in situ or to indicate the potential for detrimental effects to the skin per se. Therefore, development of new methods that are capable of measuring
contamination concentration in the skin is required in
order to improved quantification of dermal exposure
and risk assessment.
The tape-stripping technique has been used for the
determination of chemical penetration at different
depths in the skin by using adhesive tape to remove
target cells (Rougier et al., 1983, 1985, 1986; Dupuis
et al., 1984, 1986; Weigmann et al., 1999; Hostynek
et al., 2001a,b). We have successfully modified the
technique to measure dermal deposition of UV radiation-curable acrylate coatings (Nylander-French,
2000) and jet fuel exposure in occupational settings
(Mattorano et al., 2003). Here, we report development of a colorimetric method for quantification of
keratin from tape-stripped samples and the effect of
topically applied jet fuel on the amount of keratin that
can be removed by the adhesive tape strips. This
method allows normalization and correction for variable amounts of tissue removed per strip and determination of the actual concentration of naphthalene
in each tape-strip sample.
MATERIALS AND METHODS
Chemicals
Jet fuel (JP-8) was provided by the US Air Force
Research Laboratory (Wright-Patterson Air Force
Base, OH). Since JP-8 is a performance specification
fuel and the composition can vary, the US Air Force
uses a ‘generic’ JP-8 for toxicological research
purposes so that data from individual laboratories
will be comparable. JP-8 is a kerosene-based distillate of crude oil consisting primarily of the C9–C16
hydrocarbons, including naphthalene. Because of the
chemical complexity of JP-8, naphthalene (C10H8,
CAS 91-20-3) was chosen as a marker of exposure to
JP-8 since (i) it is one of the 13 most prevalent chemicals which comprise 29% of the base fuel in JP-8
(naphthalene represents 1.1% v/v of JP-8) (Potter and
Simmons, 1998; Riviere et al., 1999), (ii) it is easily
identified by gas chromatography/mass spectrometry
(GC/MS) at low concentrations, (iii) it was not present
in the tape adhesive, (iv) it was not found in control
(blank) skin tape-strip samples and (v) naphthalene
(or its metabolites) is commonly used as a marker for
exposure in other sampling media, e.g. ambient air,
exhaled breath, urine and blood, by other investigators (Riviere et al., 1999; McDougal et al., 2000;
Baynes et al., 2001; Kanikkannan et al., 2001a,b).
Solid naphthalene (99%+ scintillation grade; Aldrich,
Milwaukee, WI) and solid deuterated naphthalene
(naphthalene-d8, 98%; Aldrich) were used as a
standard and as an internal standard, respectively.
Acetone (nanograde; Mallinckrodt Baker, Paris, KY)
was used as an extraction solvent to remove JP-8
components from the tape-strip samples and as a
dissolving solution for solid naphthalene and naphthalene-d8.
Experimental procedure
The study population consisted of 12 subjects
(seven females and five males) with an average age
of 34.3 ± 7.51 yr (range 23–46). Racially, the population was divided into Caucasians (n = 4), Asians (n =
6) and African-Americans (n = 2). Eight subjects had
light skin, which tans with little or no burning (Skin
type I); two of the subjects had light/fair skin, which
burns easily in the sun (Skin type II); two subjects
had naturally pigmented brown skin (Skin type III).
The study was approved by the Institutional Review
Board on Research Involving Human Subjects
(School of Public Health, The University of North
Carolina at Chapel Hill, Chapel Hill, NC).
Exposure to jet fuel was conducted in an acrylic
exposure chamber (20.3 cm width × 20.3 cm length ×
18.7 cm height, total volume 7706 cm3) (Fig. 1).
After the subject’s volar arm was placed into the
exposure chamber, an attached two-well aluminum
application chamber (2.5 cm width × 4.0 cm length ×
1.3 cm height with a total area of 10 cm2/well) was
adjusted against the arm using a spring-loaded rod
that applied constant pressure to define the exposure
site and to prevent jet fuel from spreading outside the
exposure site during the experiment. The two wells
were 4 cm apart and an aluminum tab (2.2 cm long
and 0.2 cm thick) was placed in the center of each
well and secured to extend 0.08 cm below the
chamber–skin surface. The tab was designed to keep
the skin flat and prevent the exposure site from
‘doming’ inside the application chamber and cause
the fuel to pool around the interior walls when pres-
Tape-strip skin sampling
67
time (Nylander-French, 2000). The adhesive tape
strip was removed slowly with constant force at an
∼45° angle. Four successive tape strips (five in total)
were carefully applied to the same site immediately
after the previous tape strip was removed and the new
strip was also retained on the skin for 2 min. In the
first experiment (keratin analysis), the tape strip was
rolled with the adhesive side facing out and placed
into a 2 ml cryovial for keratin extraction and quantification by colorimetric assay. In the second experiment (naphthalene analysis), the tape strip was folded
and placed in a 20 ml scintillation vial containing 5 ml
of nanograde acetone and 20 µl of 25 µg/ml naphthalene-d8. The vials were placed on a rotation shaker
for 30 min at 250 r.p.m. and stored at 4°C until
analyzed by GC/MS. At the end of each exposure, the
aluminum application chamber was rinsed with 5 ml
of acetone to determine the amount of residual JP-8
left in the chamber. The rinsing solution was transferred to a 20 ml scintillation vial, concentrated to
0.5 ml using compressed nitrogen and analyzed by
GC/MS chromatography in the same manner as the
naphthalene tape-strip samples.
Fig. 1. Exposure chamber.
sure was applied to seal the application chamber
against the skin. The exposure chamber was also
equipped with an opening for placement of Tenax
sampling tubes for the measurement of naphthalene
evaporation within the exposure chamber during the
experiment.
The study was divided into two experiments for
each subject. In the first experiment, the amount of
keratin collected by each tape strip was determined.
In the second experiment, the amount of naphthalene
removed by the tape strips after JP-8 exposure was
determined. In both experiments, two sites on the
ventral surface of each lower arm were exposed to
JP-8. Each site received a single application of 25 µl
of JP-8, which was retained on the non-occluded skin
for 10, 15, 20 or 25 min. For all subjects, 10 and 15
min exposure sites were on the right arm and the 20
and 25 min sites were on the left arm. Tape-strip
samples were also collected from one unexposed site
on the subject’s right arm. These experiments were
conducted 1 month apart, thereby allowing sufficient
time for the skin to recover from the first experiment.
Application of JP-8 was conducted through a
septum on the top of the exposure chamber using a 50
µl syringe with a blunt needle (Hamilton, Reno, NV).
After the desired exposure time (10, 15, 20 or 25
min), the arm was removed from the exposure
chamber and an adhesive tape strip (Cover-Roll™
tape; Beiersdorf AG, Germany), pre-cut to 2.5 cm ×
4.0 cm (10 cm2), was applied to the exposed site and
removed using clean forceps after a 2 min adhesion
Keratin analysis
Keratin extraction from the tape strip was
performed by modification of the protocol as outlined
by Dreher et al. (1998). An aliquot of 1 ml of 1 M
NaOH was added to a 2 ml cryovial with the rolled
tape strip and the tube was vortexed at various intervals over a 2 h period. After 2 h, the sample was
stored at 4°C overnight. The following morning, 1 ml
of 1 M HCl was added and the sample was vortexed
again.
In the preliminary study, the Bio-Rad DC protein
assay kit, which uses a modified Lowry assay (BioRad Laboratories, Hercules, CA), was used to
attempt to quantify the extracted keratin. However,
the Cover-Roll™ tape with its woven polyester
backing and polyacrylate adhesive gave a false positive result in the assay. Thus, another colorimetric
method based on a modified Bradford assay (Amresco,
Solon, OH) was attempted and no positive result was
found from the tape alone. A standard curve was
prepared using commercially available human
keratin (Sigma, St Louis, MO). A 100 µl aliquot of
unknown sample was added to a 1.5 ml microcentrifuge tube or, for the keratin standard, a known
concentration of keratin (Sigma) was aliquoted and
the volume brought to 100 µl with 1 M NaCl. One
milliliter of Bradford reagent was added and the
sample was vortexed. The sample was allowed to
stand at room temperature for 2 min before absorbance was determined at 595 nm using a UV spectrophotometer (Beckman DU640; Beckman Instruments,
Palo Alto, CA). A standard curve was generated by
plotting absorbance at 595 nm versus protein concentration.
68
Y.-C. E. Chao and L. A. Nylander-French
Electrophoresis and molecular weight comparison
Since the prominent keratin proteins in stratum
corneum (non-viable epidermis) are keratin 1 and
keratin 10, we used a NuPAGE™ (Novex, San Diego,
CA) vertical gel electrophoresis system to determine
the approximate molecular weight of the proteins that
were extracted by the tape-stripping method. The
NuPAGE™ protocol was followed for sample preparation for both the extracted samples and the protein
standards (Mark12™; Novex). A 4–12% Bis–Tris gel
with a 4–12% MES running buffer was run at 200 V.
A recirculating water bath was used to maintain the
buffer temperature at 9°C for the duration of the 45 min
run time. Staining of the gel was performed using the
Novex™ Colloidal Blue Kit protocol for NuPAGE™
Bis–Tris gels. The protein bands present in the
extracted samples displayed on the gels after staining
had a molecular weight of ∼66.3 kDa and ≥55.4 kDa
as determined by comparison with the Mark12™
wide range protein standard.
Western analysis
In order to confirm that the proteins removed from
the tape were keratin proteins, based on molecular
weight of electrophoretically separated proteins, we
performed western blotting. Ten micrograms of
extracted stratum corneum protein was run on a 4–
15% Tris–HCl vertical minigel blotting system (BioRad). The gel was run at 100 V with a 1× SDS
running buffer. The gel was western blotted onto a
PVDF membrane at 100 V for 1 h at 4°C using the
Trans-Blot Cell assembly (Bio-Rad). The filter was
blocked with 5% milk in phosphate-buffered saline
and 0.1% Tween (PBST). The primary antibody, a
monoclonal mouse anti-human cytokeratin antibody
(Dako Corp., Carpinteria, CA) was diluted 1:2000
and incubated with the filter overnight. The filter was
rinsed with PBST and an immunoPure™ goat antimouse IgG, peroxidase-conjugated secondary antibody obtained from Pierce (Rockford, IL) was added
in 1% milk at a dilution of 1:5000 for 45 min. The
filter was subsequently rinsed in PBST and detection
of the antibody complex was performed by using the
SuperSignal™ West Pico Chemiluminescent Substrata
kit (Pierce). Autoradiographic hyperfilm was used to
detect the signal after film exposures lasting from 1 to
15 s.
The two dominant protein bands identified by
western autoradiography had a molecular weight of
<75 kDa and >50 kDa as determined by comparison
with the RPN 800 protein ladder (Amersham, Piscataway, NJ). The molecular weights correspond to the
vertical gel electrophoresis analysis and to the known
molecular weight of the human stratum corneum
keratin 1 and keratin 10, which were 68 and 56.5
kDa, respectively.
Naphthalene analysis
Tape-strip samples. Prior to GC/MS analysis,
adhesive tape was removed from each vial using
clean forceps and any remaining solution in the tape
was squeezed back into the vial. Samples were concentrated from 5 to 0.5 ml using compressed nitrogen.
The remaining solution was transferred to 2 ml amber
autoinjector vials for GC/MS analysis.
A Thermoquest Trace gas chromatograph equipped
with an AS 2000 autoinjector and a Finnigan Polaris
Q quadrupole ion trap mass spectrometry detector
(ThermoQuest, Austin, TX), operated in electron
ionization mode, was used for chemical analysis. The
GC column was an RTX-5MS (30 m, 0.25 mm inside
diameter, 0.25 µm film thickness) (Restek Corp.,
Bellefonte, PA). Helium was used as the carrier gas
and the column flow was controlled via an electronic
pressure control system and maintained in constant
flow mode at 1.5 ml/min, with vacuum compensation
enabled. Injector and detector temperature were both
225°C. The oven temperature was initially held at
45°C for 2 min and then increased at 2°C/min to 72°C
and held for 23 min. After naphthalene and naphthalene-d8 eluted (∼34 min), the oven temperature was
increased at 50°C per min to 280°C and held for 20
min to remove later eluting compounds present in jet
fuel. Sample injections (1 µl) were made in the
split/splitless mode.
The mass spectrometry was operated in the
selected ion monitoring (SIM) mode. Ions at m/z 128
and 102 were monitored for naphthalene and ions at
m/z 136 and 108 for naphthalene-d8. These ions were
selected based on fragmentation patterns of the
compounds observed while analyzing fuel samples
with the GC/MS operated in the SCAN mode. The
limit of quantification was 0.3 ng/cm3.
Air samples. The amount of evaporated naphthalene from the skin was measured using two
aluminum tubes (90 mm × 6.3 mm o.d. × 5.0 mm i.d.,
fabricated in-house) with an open diffusion channel
of 1.5 cm × 5.0 mm i.d. and containing 0.1 g of 20/35
mesh Tenax TA (SKC Iin., Eighty Four, PA) in series
and a sampling pump operating at 1.4 l/min flow rate.
The analysis of naphthalene was conducted
according to a published procedure (Egeghy et al.,
2000). Briefly, before sample collection, tubes were
initially conditioned at 250°C for 30 min followed by
3 min at 225°C with a continuous flow of ultra-high
purity helium gas at a rate of 45 ml/min using an
automatic thermal desorption system (Model ATD
400; Perkin-Elmer Corp., Norwalk, CT) to remove
traces of naphthalene. Tubes were shielded from light
and stored at room temperature before analysis.
Desorption of collected samples was carried out for 2
min at 225°C to transfer analytes onto a Tenaxpacked, cryogen-free focusing cold trap maintained
Tape-strip skin sampling
at –30°C using a Perkin-Elmer ATD 400 automatic
thermal desorption system. In order to transfer the
contents to the analytical column via a fused silica
transfer line (maintained at 200°C), the cold trap was
rapidly heated to 225°C and maintained at this
temperature for 0.1 min. Naphthalene content was
analyzed with a Hewlett Packard 6890 Series II gas
chromatograph (Hewlett Packard Corp., Palo Alto,
CA) equipped with a HNU PI-52-02A photoionization detector (PID) with a 9.5 eV lamp (HNU Systems
Inc., Newton, MA). Separation was achieved with a
megabore DB-1 column (60 m × 0.53 mm i.d.
dimethylpoolysiloxane, 1.5 µm film thickness; J&W
Scientific, Folsom, CA). Ultra-high purity helium
was used as the carrier gas (flow rate 8 ml/min). The
oven temperature was held at 40°C for 5 min, then
increased at 10°C/min to 75°C, then increased at
5.5°C/min to 175°C and, finally, increased at
50°C/min to a final temperature of 260°C and held
for 6 min. Chromatograms were manually integrated
using Hewlett Packard gas chromatography ChemStation software. Naphthalene was identified by the
retention time of 21.95 min.
Samples were quantified against external naphthalene standards prepared by injecting 2 µl of known
concentration of naphthalene into the Tenax tubes.
Naphthalene standards (100, 50, 25, 12.5, 6.25, 2.5,
1, 0.5, 0.25, 0.125, 0.05, 0.025 and 0.0125 mg/ml)
were prepared by serial dilution from a stock solution
of 200 mg naphthalene dissolved in 2 ml of hexane
(100 mg/ml). A calibration curve was determined by
linear least squares regression. The limit of quantification was 1.0 µg/m3.
Statistical analysis
All statistical analyses employed the SAS System
Software (SAS Institute, Cary, NC). Normality of the
data was investigated using histograms and the
Shapiro–Wilks test. For all exposure times, the measured mass of keratin and naphthalene in tape-strip
samples were normally distributed while naphthalene
concentration (normalized for the mass of keratin)
69
was log normally distributed. A one-way analysis of
variance was used to investigate differences in average
mass of keratin and naphthalene between different
consecutive tapes (1st to 5th) and/or different
exposure times (10, 15, 20 and 25 min). Covariate
analyses were performed by Proc NPAR1WAY to
investigate the effect of covariates on the removed
mass of keratin and naphthalene. Recovery of naphthalene was calculated based on the best estimate of the
amount of naphthalene (42 500 ng) in 25 µl of
applied JP-8.
RESULTS
Keratin in tape strips
The average mass of keratin removed with sequential tape strips from unexposed and JP-8-exposed
sites at different exposure times are presented in
Table 1. The average mass of keratin removed by a
tape strip decreased with sequential tape strips both at
unexposed and JP-8-exposed sites. For unexposed
sites, the average mass of keratin in individual tape
strips varied from 154 ± 75.3 µg/cm2 for the first tape
strips to 52.7 ± 17.3 µg/cm2 for the fifth tape strips.
For JP-8-exposed sites, the average mass of keratin in
individual tape strips varied from 122 ± 59.8 µg/cm2
for the first tape strips to 59.9 ± 22.5 µg/cm2 for the
fifth tape strips. There was no significant difference
between the average mass of keratin removed from
unexposed sites and JP-8-exposed sites when
compared with either the total mass of keratin on five
sequential tape strips (P = 0.798) or on each tape strip
at different exposure times (all P > 0.158).
The effects of covariates including gender, age,
race and skin type upon total mass of keratin
(µg/cm2) removed by five sequential tape strips at
different exposure times were not observed to be
significant (Table 2). The observed minor differences
for age group, ethnicity and skin type between 15 and
20 min exposure sites may be attributed to the small
number of individuals in this study.
Table 1. The average mass of stratum corneum keratin (µg/cm2) removed by five successive tape strips at different JP-8 exposure
times
Tape strip
Unexposed
(µg/cm2 ± SD)
10 min (µg/cm2 ± 15 min
SD)
(µg/cm2 ± SD)
20 min
(µg/cm2 ± SD)
25 min
(µg/cm2 ± SD)
P value
1st tape
154 ± 75.3a
135 ± 60.7a
90.8 ± 56.9b
138 ± 43.7a
123 ± 70.1c
0.158
2nd tape
107 ±
140 ±
56.0a
98.0 ± 53.9
108 ±
104 ± 60.3d
0.306
3rd tape
82.0 ± 41.7d
82.2 ± 24.5d
81.1 ± 40.7
88.2 ± 35.8d
87.9 ± 39.5
0.981
4th tape
69.8 ± 27.4
74.9 ± 22.7d
72.7 ± 35.8
78.9 ± 36.8
69.5 ± 36.7
0.951
5th tape
52.8 ± 17.3
56.7 ± 18.3
65.4 ± 24.3
56.3 ± 17.7
61.1 ± 29.5
0.672
All tapes
465 ± 163
489 ± 139
408 ± 190
469 ± 124
445 ± 199
0.798
39.6c
45.8d
different from tape strips 3–5; all P values ≤ 0.011.
different from the first tape strip at unexposed and 20 min sites; P = 0.030 and P = 0.035, respectively.
cSignificantly different from tape strips 4 and 5; all P values ≤ 0.028.
dSignificantly different from tape strip 5; all P values ≤ 0.043.
aSignificantly
bSignificantly
70
Y.-C. E. Chao and L. A. Nylander-French
Table 2. The average total mass of stratum corneum keratin (µg/cm2) removed by five consecutive tape strips at different JP-8
exposure times by gender, age group, race and skin type
n
Unexposed
(µg/cm2 ± SD)
10 min
(µg/cm2 ± SD)
15 min
(µg/cm2 ± SD)
20 min
(µg/cm2 ± SD)
25 min
(µg/cm2 ± SD)
Females
7
493 ± 160
506 ± 136
363 ± 167
460 ± 141
397 ± 170
Males
5
427 ± 177
465 ± 155
471 ± 221
480 ± 108
512 ± 237
<35 yr
6
538 ± 173
545 ± 107
530 ± 177a
554 ± 72.8a
536 ± 212
>35 yr
6
393 ± 125
433 ± 153
286 ± 110
384 ± 104
354 ± 149
Caucasian
4
403 ± 122
483 ± 172
331 ± 108
510 ± 49.6b
394 ± 110
Asian
6
538 ± 185
529 ± 132
530 ±
505 ± 121c
545 ± 211
African-American
2
372 ± 99.8
380 ± 28.0
197 ± 105
279 ± 14.4
247 ± 168
Skin type Id
8
524 ± 162
549 ± 118
501 ± 156e
507 ± 103e
530 ± 181
IIf
2
325 ± 118
360 ± 164
246 ± 69.7
505 ± 85.3
306 ± 48.4
2
372 ± 99.8
380 ± 28.0
197 ± 105
279 ± 14.4
247 ± 168
Skin type
Skin type IIIg
172c
aSignificantly
different from age >35 years; P = 0.017 at 15 min site and P = 0.008 at 20 min site.
different from African-American; P = 0.004.
cSignificantly different from African-American; P = 0.046 at 15 min site and P = 0.047 at 20 min site.
dTans with little or no burning.
eSignificantly different from Skin type III; P = 0.033 at 15 min site and P = 0.017 at 20 min site.
fBurns in the sun.
gNaturally pigmented, brown.
bSignificantly
Table 3. The average mass of naphthalene (ng/cm2) removed by five successive tape strips at different JP-8 exposure times
Tape strip
10 min (ng/cm2 ± SD)
15 min (ng/cm2 ± SD)
20 min (ng/cm2 ± SD)
25 min (ng/cm2 ± SD)
P value
1st tape
2510 ±
2410 ± 875
1870 ± 678
1570 ± 1120
0.042
674a
2nd tape
30.9 ± 13.2
31.6 ± 15.7
33.9 ± 11.5
29.9 ± 15.2
0.923
3rd tape
8.45 ± 2.61
10.1 ± 3.76
9.84 ± 2.72
9.78 ± 3.45
0.623
4th tape
5.66 ± 1.13
6.38 ± 2.72
6.48 ± 1.21
6.12 ± 1.59
0.737
5th tape
4.59 ± 1.05
5.36 ± 2.03
5.42 ± 1.21
5.47 ± 1.32
0.491
All tapes
2560 ± 681b
1620 ± 1130
0.046
aSignificantly
bSignificantly
2470 ± 888
1920 ± 685
different from the first tape strip at 20 and 25 min sites; P = 0.037 and P = 0.028, respectively.
different from the all tape strips at 20 and 25 min sites; P = 0.040 and P = 0.029, respectively.
Naphthalene
In this study, each subject was exposed to a single
dose of 25 µl of JP-8 at four different exposure times.
During the exposure, naphthalene could either penetrate into the skin, remain in the walls of the application chamber or evaporate. Therefore, the amount of
naphthalene was measured (i) in the tape strips, (ii) in
the application chamber at the end of each exposure
and (iii) in the air during exposure.
Naphthalene in the tape strips
The average mass of naphthalene (ng/cm2) removed
by sequential tape strips from JP-8-exposed skin sites
at different exposure times (10, 15, 20 and 25 min) is
presented in Table 3. For each individual exposure
time, the average mass of naphthalene decreased
significantly from the first tape strips to the fifth tape
strips (all P < 0.0001). However, no differences were
observed between the 4th and 5th tape strips at 15, 20
and 25 min sites (P = 0.352, 0.066 and 0.329, respectively). The highest average mass of naphthalene
(2510 ± 674 ng/cm2) was removed with the first tape
strips at the 10 min site, which was significantly
different from the naphthalene removed with the first
tape strips at the 20 min site (P = 0.037) and at the
25 min site (P = 0.028). No significant differences
were observed between other sequential tape strips
(2nd to 5th) at different exposure times (all P >
0.491). The average total mass of naphthalene
removed with all five sequential tape strips at 10 min
sites was significantly different from the 20 and 25
min sites (P = 0.040 and 0.029, respectively).
Similar to keratin, none of the covariates (genders,
age groups, races and skin types) significantly
affected the average mass of naphthalene removed by
all five sequential tape strips at different exposure
times, except at 25 min where Asians (n = 6) showed
a lower average mass of naphthalene removed by
Tape-strip skin sampling
71
Table 4. The average total mass of naphthalene (ng/cm2) removed by five consecutive tape strips at different JP-8 exposure times
by gender, age group, race and skin type
n
10 min (µg/cm2 ± SD)
15 min (µg/cm2 ± SD)
20 min (µg/cm2 ± SD)
25 min (µg/cm2 ± SD)
Females
6
2600 ± 635
2370 ± 1040
1990 ± 541
1840 ± 1160
Males
5
2510 ± 807
2580 ± 759
1830 ± 889
1360 ± 1170
<35 r
5
2600 ± 806
2710 ± 669
1820 ± 824
1480 ± 1320
>35 yr
6
2520 ± 636
2260 ± 1050
2000 ± 615
1740 ± 1070
Caucasian
4
3140 ± 561
3160 ± 390
2300 ± 764
2740 ± 673
Asian
5
2230 ± 542
2150 ± 660
1640 ± 589
African-American
2
2200 ± 602
1870 ± 1600
1860 ± 775
1960 ± 903
Skin type Ib
7
2630 ± 810
2360 ± 656
1980 ± 756
1350 ± 1310
Skin type IIc
2
2660 ± 159
3440 ± 224
1780 ± 777
2230 ± 541
Skin type IIId
2
2200 ± 602
1870 ± 1600
1860 ± 775
1960 ± 903
587 ± 85.8a
aSignificantly
different from Caucasians (P = 0.0002) and African-Americans (P = 0.0103).
with little or no burning.
cBurns in the sun.
dNaturally pigmented, brown.
bTans
Table 5. The results of the best estimates for the total amount of naphthalene recovered at different JP-8 exposure times, the
amount of naphthalene removed from the skin by five successive tape strips, evaporation and chamber residue losses
Tape strips (ng ± SD)
Recovery efficiency (% ± SD)
Total chamber loss (ng ± SD)
Recovery efficiency (% ± SD)
Evaporation (ng ± SD)
Recovery efficiency (% ± SD)
Chamber residue (ng ± SD)
Recovery efficiency (% ± SD)
Total naphthalene (ng ± SD)
Total recovery efficiency (% ± SD)
10 min
15 min
20 min
25 min
25 600 ± 6810
24700 ± 8880
19 200 ± 6850
16200 ± 11300
60.2 ± 16.0
58.0 ± 20.9
45.2 ± 16.1
38.1 ± 26.7
7020 ± 3100
6990 ± 2460
7080 ± 2520
5790 ± 2500
16.5 ± 7.30
16.4 ± 5.80
16.7 ± 5.92
13.6 ± 5.88
616 ± 207
1240 ± 436
2160 ± 786
3070 ± 1580
1.45 ± 0.49
2.93 ± 1.02
5.08 ± 1.85
7.21 ± 3.72
6400 ± 3090
5750 ± 2510
4930 ± 2360
2720 ± 2130
15.1 ± 7.30
32600 ± 8030
76.7 ± 18.9
tape strips compared with Caucasians and AfricanAmericans (P = 0.0002 and 0.0103, respectively)
(Table 4). However, no such differences were
observed at other exposure times.
Naphthalene recovery
The best estimates for the amounts of naphthalene
removed from the skin as well as evaporation and
chamber residues at different exposure times are
presented in Table 5. The amount of naphthalene
removed with five sequential tape strips and total
chamber loss decreased with increasing exposure
time in a linear manner (R2 = 0.942). The measured
amounts of naphthalene residue in the application
chamber, based on the best estimate of the amount of
naphthalene applied in JP-8, were 15.1, 13.5, 11.6
and 6.41% for the 10, 15, 20 and 25 min exposure
times, respectively. The decrease in the average mass
of naphthalene residue in the application chamber
with longer exposure time was expected due to
13.5 ± 5.91
11.6 ± 5.56
6.41 ± 5.02
31600 ± 10 400
29300 ± 8160
22000 ± 12500
74.4 ± 24.5
61.8 ± 19.2
51.8 ± 29.5
potential evaporation and skin absorption. The evaporation of naphthalene from the skin during exposure
was 1.45, 2.93, 5.08 and 7.21% of the best estimate of
the applied amount of naphthalene during the 10, 15,
20 and 25 min exposures, respectively. The average
mass of evaporated naphthalene increased with
increasing exposure time and showed a linear relationship for the 12 subjects measured over these four
exposure time points (R2 = 0.993). The total naphthalene recovery varied from 77% for 10 min exposure
to 52% for 25 min exposure, indicating a rapid penetration of naphthalene into the skin.
Normalization of naphthalene against keratin
In order to obtain a concentration profile of naphthalene in the stratum corneum (ng/µg keratin) after
exposure to JP-8, the mass of naphthalene (ng/cm2)
removed by each tape strip was normalized by
dividing by the mass of keratin (µg/cm2) removed by
the matching tape strip. Naphthalene concentrations
72
Y.-C. E. Chao and L. A. Nylander-French
Table 6. The average logtransformed naphthalene concentration [ln(ng/µg keratin)] in five successive tape strips at different JP-8
exposure times
Tape strip
15 min
ln(ng/µg) ± SD
1st tape
2.95 ± 0.543
3.36 ± 0.704a
2.64 ± 0.560
2.46 ± 1.27
0.073
2nd tape
–1.47 ± 0.908
–1.04 ± 1.14
–1.14 ± 0.525
–1.15 ± 1.18
0.747
3rd tape
–2.30 ± 0.480
–1.83 ± 1.10
–2.13 ± 0.701
–2.21 ± 0.665
0.525
4th tape
–2.51 ± 0.487
–2.33 ± 0.958
–2.44 ± 0.568
–2.33 ± 0.829
0.926
5th tape
–2.50 ± 0.477
–2.48 ± 0.739
–2.31 ± 0.484
–2.27 ± 0.659
0.759
aSignificantly
20 min
ln(ng/µg) ± SD
25 min
ln(ng/µg) ± SD
P value
10 min
ln(ng/µg) ± SD
different from the first tape strip at 20 min site; P = 0.016.
(normalized for the mass of keratin) were observed to
be log normally distributed. The average log-transformed naphthalene concentrations for each tape strip
at different exposure times are presented in Table 6.
The average log-transformed naphthalene concentration for the first tape strips was 2.85 ± 0.868 and
decreased to –2.39 ± 0.586 for the fifth tape strips. No
significant differences were observed between the
naphthalene concentrations for matching tape strips
at different exposure sites (all P > 0.073), except for
the first tape strips between the 15 and 20 min
exposure sites (P = 0.0156). Thus, the data indicates
that normalization of the removed naphthalene with
the removed amount of keratin in each tape-strip
sample did not affect the ability of this tape-strip
method to quantify the dermal exposure to JP-8 when
naphthalene was used as a marker for exposure to jet
fuel.
DISCUSSION
We have developed a tape-stripping technique to
measure the concentration of naphthalene in JP-8exposed skin by normalizing the amount of naphthalene to the amount of keratin in each tape strip
collected from individuals with normal skin (no
atopics were included in the study). For this purpose,
we developed a keratin protein extraction method and
modified a colorimetric method based on the Bradford method for protein quantification to determine
the amount of keratin removed from the stratum
corneum with each sequential tape strip after
exposure to jet fuel. Previously, a GC/MS analytical
method was developed to detect naphthalene in the
tape-strip samples (Mattorano et al., 2003).
We observed that the sequential tape strips
removed a decreasing but consistent amount of
keratin from both unexposed and JP-8-exposed skin,
indicating that the average mass of keratin removed
with tape strips was not affected by exposure to jet
fuel nor by gender, age, ethnicity or degree of skin
pigmentation. Thus, the colorimetric assay for quantification of keratin provides a useful and reliable
method for determination of keratin in tape-strip
samples and has major advantages compared with
weighing, which is time consuming, subject to errors
due to humidity and cumbersome to perform in the
field (Marttin et al., 1996). Also, optical spectroscopy, which is useful for the investigation of the
depth profile of stratum corneum, is not reliable for
quantification of keratin in the tape strips due to the
influence of light scatter (Marttin et al., 1996;
Weigmann et al., 1999; Boeniger and NylanderFrench, 2002).
The results also show that we were able to quantify
naphthalene in the sequential tape strips collected
from the skin even 25 min after a single dose of JP-8
was initially applied. Penetration of the jet fuel into
the stratum corneum was demonstrated by the fact
that the average mass of naphthalene recovered with
tape strips decreased with increasing exposure time
to a finite source of jet fuel and subsequent tape strips
and that evaporation of naphthalene was observed to
be negligible. Furthermore, similarly to keratin, the
average mass of naphthalene recovered by tape-stripping from unexposed and JP-8-exposed sites was not
affected by gender, age, ethnicity or degree of skin
pigmentation.
With the tape-stripping method we were able to
obtain a concentration profile for naphthalene in the
stratum corneum (ng/µg keratin) after exposure to JP8. The naphthalene concentrations in the stratum
coneum were observed to be log normally distributed. In this study, keratin and naphthalene were quantified using different tape-strip samples collected
1 month apart at approximately the same sites on the
arm in order to allow the skin to fully recover from
the previous tape stripping. Despite this deficiency,
the fact that consistent amounts of both keratin and
naphthalene were removed by sequential tape strips
independent of potential confounders, i.e. gender,
age, race and degree of skin pigmentation, speaks for
the reliability and usefulness of this method. Currently,
we are modifying this method to allow simultaneous
quantification of both keratin and naphthalene on the
same tape strip, thus, allowing measurement of the
exact concentration of a compound in the skin. By
incorporating both measurements on a single tapestrip sample, this method will be a powerful tool to
measure concentration of a compound in the skin and
to determine dermal exposure.
Tape-strip skin sampling
We conclude that the actual concentration of
naphthalene (as a marker for jet fuel exposure) per
unit of keratin can be determined using this tapestripping method. Under laboratory conditions
normalization of the naphthalene to the amount of
keratin removed in the tape-strip samples did not
affect the ability of this method to quantify dermal
exposure to JP-8. However, normalization may be
required when measuring occupational exposures
under field settings due to variable exposures and
potential changes in skin conditions. The tape-stripping technique as described or with some modifications is generally applicable to assessing dermal
exposure to other compounds (Cullander et al., 2000;
Kristiansen et al., 2000). However, investigation of
variations in skin condition (dry versus moist skin,
skin defects, etc.) to determine the potential impact of
these on the sampling method is warranted. Furthermore, the influence of compounds that readily react
and/or are metabolized in the stratum corneum and
workplace conditions (e.g. occlusion, temperature)
need to be determined. Although no significant differences were observed by amount of keratin proteins
removed with samples evaluated thus far, larger
population studies are warranted to investigate
factors that might influence the amount of stratum
corneum removed by tape-stripping.
Acknowledgements—The authors appreciate the contribution of
all the volunteers who participated in this study. We also thank
Gregory Lacks for keratin sample analysis and Drs Peter
Egeghy and Stephen M. Rappaport for their assistance in
naphthalene evaporation analysis. This work was supported in
part by the US Air Force through a subcontract with Texas
Tech University (1331/0489-01), NIOSH Pilot Project
Research Training Grant (T42/CCT410423-09) and National
Institute of Environmental Health Sciences (P42ES05948).
REFERENCES
Baynes RE, Brooks JD, Budsaba K, Smith CE, Riviere JE.
(2001) Mixture effects of JP-8 additives on the dermal disposition of jet fuel components. Toxicol Appl Pharmacol; 175:
269–81.
Boeniger MF, Nylander-French LA. (2002) Comparison of
three methods for determining removal of stratum corneum
using adhesive tape strips. International Conference on
Occupational & Environmental Exposures of Skin to Chemicals: Science & Policy, Hilton Crystal City, 8–11 September, 2002.
Cullander C, Jeske S, Imbert D, Grant PG, Bench G. (2000) A
quantitative minimally invasive assay for the detection of
metals in the stratum corneum. J Pharm Biomed Anal; 22:
265–79.
Dreher F, Arens A, Hostynek JJ, Mudumba S, Ademola J,
Maibach HI. (1998) Colorimetric method for quantifying
human stratum corneum removed by adhesive-tape stripping. Acta Derm Venereol; 78: 186–9.
Dupuis D, Rougier A, Roguet R, Lotte C, Kalopissis G. (1984)
In vivo relationship between horny layer reservoir effect and
percutaneous absorption in human and rat. J Invest Dermatol; 82: 353–6.
73
Dupuis D, Rougier A, Roguet R, Lotte C. (1986) The measurement of the stratum corneum reservoir: a simple method to
predict the influence of vehicles on in vivo percutaneous
absorption. Br J Dermatol; 115: 233–8.
Egeghy PP, Tornero-Velez R, Rappaport SM. (2000) Environmental and biological monitoring of benzene during selfservice automobile refueling. Environ Health Perspect; 108:
1195–1202.
Hostynek JJ, Dreher F, Nakada T, Schwindt D, Anigbogu A,
Maibach HI. (2001a) Human stratum corneum adsorption of
nickel salts. Investigation of depth profiles by tape stripping
in vivo. Acta Derm Venereol Suppl (Stockh); 212: 11–8.
Hostynek JJ, Dreher F, Pelosi A, Anigbogu A, Maibach HI.
(2001b) Human stratum corneum penetration by nickel. In
vivo study of depth distribution after occlusive application of
the metal as powder. Acta Derm Venereol Suppl (Stockh);
212: 5–10.
Kanikkannan N, Burton S, Patel R, Jackson T, Shaik MS, Singh
M. (2001a) Percutaneous permeation and skin irritation of
JP-8+100 jet fuel in a porcine model. Toxicol Lett; 119:
133–42.
Kanikkannan N, Patel R, Jackson T, Shaik MS, Singh M.
(2001b) Percutaneous absorption and skin irritation of JP-8
(jet fuel). Toxicology; 161: 1–11.
Kristiansen J, Christensen JM, Hendriksen T, Henrik-Nielsen
N, Menné T. (2000) Determination of nickel in fingernails
and forearm skin (stratum corneum). Anal Chem Acta; 403:
265–272.
Marttin E, Neelissen-Subnel MT, De Haan FH, Bodde HE.
(1996) A critical comparison of methods to quantify stratum
corneum removed by tape stripping. Skin Pharmacol; 9: 69–
77.
Marzulli FN, Maibach HI. (1996) Dermatotoxicology. New
York: Taylor & Francis.
Mattorano DA, Kupper LL, Nylander-French LA. (2003)
Estimating dermal exposure to jet fuel (naphthalene) from
adhesive tape-strip samples. Ann Occup Hyg; submitted for
publication.
McDougal JN, Pollard DL, Weisman W, Garrett CM, Miller
TE. (2000) Assessment of skin absorption and penetration of
JP-8 jet fuel and its components. Toxicol Sci; 55: 247–55.
Mukhtar H. (1992) Pharmacology of the skin. Boca Raton, FL:
CRC Press.
Nylander-French LA. (2000) A tape-stripping method for
measuring dermal exposure to multifunctional acrylates.
Ann Occup Hyg; 44: 645–51.
Potter TL, Simmons KE. (1998) Total petroleum hydrocarbon
criteria working group series. In Composition of petroleum
mixtures, Vol. 2. Amherst, MA: Amherst Scientific Publishers.
Riviere JE, Brooks JD, Monteiro-Riviere NA, Budsaba K,
Smith CE. (1999) Dermal absorption and distribution of topically dosed jet fuels jet-A, JP-8, and JP-8(100). Toxicol
Appl Pharmacol; 160: 60–75.
Rougier A, Dupuis D, Lotte C, Roguet R, Schaefer H. (1983) In
vivo correlation between stratum corneum reservoir function
and percutaneous absorption. J Invest Dermatol; 81: 275–8.
Rougier A, Dupuis D, Lotte C, Roguet R. (1985) The measurement of the stratum corneum reservoir. A predictive method
for in vivo percutaneous absorption studies: influence of
application time. J Invest Dermatol; 84: 66–8.
Rougier A, Dupuis D, Lotte C, Roguet R, Wester RC, Maibach
HI. (1986) Regional variation in percutaneous absorption in
man: measurement by the stripping method. Arch Dermatol
Res; 278: 465–9.
Weigmann H, Lademann J, Meffert H, Schaefer H, Sterry W.
(1999) Determination of the horny layer profile by tape stripping in combination with optical spectroscopy in the visible
range as a prerequisite to quantify percutaneous absorption.
Skin Pharmacol Appl Skin Physiol; 12: 34–45.