Ann. occup. Hyg., Vol. 48, No. 1, pp. 65-73, 2004
© 2004 British Occupational Hygiene Society
Published by Oxford University Press
Determination of Keratin Protein in a Tape-stripped Skin Sample from Jet Fuel Exposed Skin
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
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 tape-strip 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 JP-8 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
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 physicochemical 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 chemical 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 x 20.3 cm length x 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 x 4.0 cm length x 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 pressure 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.
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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-RollTM tape; Beiersdorf AG, Germany), pre-cut to 2.5 cm x 4.0 cm (10 cm2), was applied to the exposed site and removed using clean forceps after a 2 min adhesion 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.
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 (Bio-Rad Laboratories, Hercules, CA), was used to attempt to quantify the extracted keratin. However, the Cover-RollTM 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.
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 NuPAGETM (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 NuPAGETM protocol was followed for sample preparation for both the extracted samples and the protein standards (Mark12TM; 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 NovexTM Colloidal Blue Kit protocol for NuPAGETM 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 Mark12TM 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 (Bio-Rad). The gel was run at 100 V with a 1x 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 immunoPureTM goat anti-mouse 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 SuperSignalTM 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 x 6.3 mm o.d. x 5.0 mm i.d., fabricated in-house) with an open diffusion channel of 1.5 cm x 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 Tenax-packed, cryogen-free focusing cold trap maintained 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 x 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) 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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).
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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.
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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).
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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 strips compared with Caucasians and African-Americans (P = 0.0002 and 0.0103, respectively) (Table 4). However, no such differences were observed at other exposure times.
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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 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.
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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 (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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have developed a tape-stripping technique to measure the concentration of naphthalene in JP-8-exposed 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 Nylander-French, 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 JP-8. 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 tape-strip sample, this method will be a powerful tool to measure concentration of a compound in the skin and to determine dermal exposure.
We conclude that the actual concentration of naphthalene (as a marker for jet fuel exposure) per unit of keratin can be determined using this tape-stripping 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).
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FOOTNOTES |
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* Author to whom correspondence should be addressed. Tel: +1-919-966-3826; Fax: +1-919-966-4711; e-mail: leena_french{at}unc.edu
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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