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Dermal toxicity and microscopic alterations by JP-8 jet fuel components in vivo in rabbit
Environmental Toxicology and Pharmacology 16 (2004) 153–161
Dermal toxicity and microscopic alterations by JP-8 jet fuel components in vivo in rabbit Somnath Singh1 , Jagdish Singh∗ Department of Pharmaceutical Sciences, College of Pharmacy, North Dakota State University, Fargo, ND 58105, USA Accepted 8 December 2003
Abstract In this study, we investigated the skin irritation, macroscopic and microscopic barrier alteration in vivo in rabbits from aliphatic and aromatic components of jet propellant-8 (JP-8) jet fuel. Macroscopic barrier properties were evaluated by measuring transepidermal water loss (TEWL), skin capacitance, and skin temperature; microscopic changes were observed by light microscopy. Draize visual scoring system was used to measure skin irritation. We found significant (P < 0.05) increase in temperature at the site of all chemically saturated patches immediately after patch removal in comparison to the control site. Tridecane (TRI) produced a greater increase in temperature and capacitance at all time points than all the other components of JP-8. Both the aliphatic and aromatic components increased the TEWL at all time points. Tridecane produced greater increase in TEWL followed by naphthalene (NAP), 1-methylnaphthalene (1-MN), 2-metylnaphthalene (2-MN), tetradecane (TET), and dodecane (DOD). All of the above components of JP-8 caused moderate to severe erythema and edema, which were not resolved to the baseline even after 24 h of patch removal. Light microscopy revealed an increase in epidermal thickness (ET), and decrease in length and thickness of collagen fibers’ bundle by the above components of JP-8. These results suggest potential dermatotoxicity from the JP-8 components. © 2004 Elsevier B.V. All rights reserved. Keywords: JP-8 components; TEWL; Skin irritation; Skin capacitance; Light microscopy
1. Introduction Jet propellant-8 (JP-8) jet fuel is kerosene based multicomponent mixture of aromatic and aliphatic hydrocarbons, without the lower molecular weight fraction (Mattie et al., 1991). Hence, it is less volatile and has a higher flash point than earlier used jet fuel (JP-4). JP-8 has been associated with toxicity in animal models and humans. Significant reduction in fetal body weight was found in pregnant rat fed JP-8 orally (Cooper and Mattie, 1996). JP-8 induces apoptosis in rat lung epithelial cells, primary mouse T lymphocytes, Jorkat T lymphoma cells, and monocytic cells (Stoica et al., 2001). Induction of necrosis in fibrob-
∗ Corresponding author. Tel.: +1-701-231-7943; fax: +1-701-231-8333. E-mail address: [email protected] (J. Singh). 1 Present address: Department of Pharmacy Sciences, School of Pharmacy and Health Professions, Creighton University Medical Center, Omaha, NE 68178, USA.
1382-6689/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2003.12.001
lasts and keratinocytes including cytotoxicity and altered histology were found in human skin grafts treated with JP-8 (Rosenthal et al., 2001). Four different cell lines exposed to JP-8 showed cytotoxicity and electrophysiological toxicity (Geraldine et al., 2000). Exposure to aromatic components of JP-8 resulted in concentration-dependent decrease in pain sensitivity in rats and respiratory rate in mice (Korsak et al., 1998). Studies in mice have shown that short-term, low concentration JP-8 exposure had significant effects on the immune system, e.g., decreased viable immune cell numbers, decreased immune organ weights, and loss of immune function that persisted for extended periods of time (Harris et al., 1997). There is direct risk of carcinogenesis of the skin itself, since petroleum middle distillates such as kerosene have been shown to act as strong carcinogens in a mouse skin assay (Clark et al., 1988). Aliphatic and aromatic components in JP-8 can cause occupational dermatitis (Baynes et al., 2001) and skin irritation after dermal exposure (Kabbur et al., 2001).
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There is a great potential for human exposure to JP-8. Splashes during refueling, handling of engine part coated with JP-8, contact with sides of fuel tanks during fuel maintenance operations, contact with fuel leaks on the undersides of aircraft or on ramp are potentials sources of dermal exposure (McDougal et al., 2000). Skin irritation is the non-immunologic evocation of normal or exaggerated reaction in a tissue by application of a stimulus (Singh and Maibach, 1998). There is not yet an adequately validated in vitro model available to predict the skin irritation of chemicals. A prevalidation study on in vitro tests for acute skin irritation was carried out to evaluate EpiDermTM , EPISKINTM , PREDISKINTM , the non-perfused pig ear method, and the mouse skin integrity function test. It was concluded that none of these tests was ready for inclusion in a formal validation study on in vitro tests for acute skin irritation (Fentem et al., 2001; Zuang et al., 2002). The standard way to forecast skin irritation is by so called predictive tests on man or animals (Draize et al., 1944). TEWL is an well-accepted method for quantifying material innocuousness (Idson, 1978) and provides robust method for assessing damage to the stratum corneum (SC). Irritation tends to reduce the efficiency of stratum corneum barrier function and results in an increase in TEWL. This is sometimes associated with a decrease in skin water content (Wilhelm et al., 1989) and increase in skin temperature (Grice et al., 1971). Hence, measurement of skin capacitance or skin hydration and skin temperature may also be used to assess irritation (Thiele and Malten, 1973; Serban et al., 1981). Light microscopy examines the structure of the cells in skin, and provides a direct visual evidence of any anatomical changes (Monteiro-Riviere, 1991). Skin is anatomically divided into two principal components, the outer epidermis and underlying dermis. Within these two primary divisions, numerous cell types and specialized adnexial structures can be identified. By light microscopic study, structure to function relationship can be overviewed (Roberts and Walters, 1998). JP-8 is a complex mixture composed of both aliphatic and aromatic hydrocarbons, the latter of which are associated with DNA damage and carcinogenesis. The most damaging components are the most permeable (Rosenthal et al., 2001). Due to the chemical complexity of JP-8, one cannot assess the dermal toxicity of entire fuel components. Therefore, we selected representative three aromatic and three aliphatic components, which widely differed in their lipophilicity, binding to stratum corneum, and permeability through the skin (Singh et al., 2002). In this study, we determined microscopic alteration, skin barrier perturbation, and skin irritation in vivo in rabbits from model aromatic (naphthalene (NAP), 1-methylnaphthalene (1-MN), and 2-metylnaphthalene (2-MN)) and aliphatic (dodecane (DOD), tridecane (TRI), and tetradecane (TET)) components of JP-8.
2. Materials and methods 2.1. Materials JP-8 jet fuel was obtained from Wright–Patterson Air Force Base, Ohio. DOD was purchased from Mallinckrodt Chemical Works, St. Louis, MO; TRI and 1-MN from Aldrich Chemical Company, Milwaukee, WI; TET from EM Science, Gibbstown, NJ; 2-MN from Acros Organic, NJ; and NAP from Sigma Chemical, St. Louis, MO. Ten percent neutrally buffered formalin (Accustain® ), AccumateTM tissue embedding/infiltrating medium, alcoholic solution of eosin Y with phloxine, hematoxylin solution Gill No. 3, glycerolgelatin mounting medium were purchased from Sigma Diagnostics, St. Louis, MO, USA. Chrysoidine G was procured from Fluka Chemie AG, Switzerland. Haye’s chambers were purchased from Acaderm, Inc., Menlo park, CA, USA. Rabbits (New Zealand, white, male, 10 weeks old, and approximately 1.5 kg body weight) were purchased from Harlan, Indianapolis, IN, USA. All other chemicals used were of analytical grade. 2.2. Methods 2.2.1. Skin integrity and irritation in rabbits Skin barrier function and primary irritation were measured in vivo in rabbits. The dorsal region of each rabbit was shaven carefully avoiding skin damages with the help of Oster Golden A5® single speed clipper using size 40 blade. Rabbit was acclimatized for 24 h before taking any measurements. Environmental conditions were monitored: room temperature 20–26 ◦ C, relative humidity 46–58%. TEWL was measured quantitatively with a TewameterTM (Courage & Khazaka, Cologne, FRG) using Fick’s law of diffusion. The probe was held on the skin until a stable TEWL value established (1 min). The electrical capacitance (device internal unit (i.u.)) of the skin surface was measured using CorneometerTM SM 820 (Courage & Khazaka, Cologne, FRG). CorneometerTM SM 820 measures the content of skin moisture by exploiting completely different dielectric constant of water (81) and other substances (mostly <7). The measuring capacitor shows changes of capacitance according to the moisture content of the skin. A glass lamina separates the metallic tracks (gold) in the probe head from the skin in order to prevent current conduction in the sample. An electric scatter field penetrates the skin during the measurement and the dielectricity is determined. One track builds up a surplus of electrons (minus charge) the other lacks of electrons (plus charge). An electric field between the tracks with alternating attraction develops. During the measurement the scatterfield penetrates the very first layer of the skin and determines the dielectricity (Courage & Khazaka, 1998). Skin temperature was measured using an infrared thermometer (Omega Engineering Inc., Stamford, CT).
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The visual scoring system was used to evaluate primary skin irritation manifested as erythema: very slight erythema barely perceptible # 1; well defined erythema # 2; moderate to severe erythema # 3; severe erythema, beet redness to slight eschar formation, injuries in depth # 4; and edema: very slight edema, barely perceptible # 1; slight edema, edges of area well defined by definite raising # 2; moderate edema, area raised approximately 1 mm # 3; severe edema, raised more than 1 mm, and extending beyond area of exposure # 4 (Draize et al., 1944). However, skin irritation is scored 0 through 3 according to Organization of Economic Cooperation and Development (OECD) guidelines: grade 0 no reaction; grade 1 weakly positive reaction (usually characterized by mild erythema and/or dryness across most of the treatment site); grade 2 moderately positive reaction (usually distinct erythema or dryness, possibly spreading beyond the treatment site); grade 3 strongly positive reaction (strong and often spreading erythema with edema and/or scabbing) (OECD Draft Document, 1997). Dorsal skin of each rabbit was marked into one cm2 area at three places on the left side and three places on the right side. Each rabbit had three test sites and three control sites. Baseline measurements were taken corresponding to test and control sites. Haye’s occlusive test chamber saturated with test chemical (50 l) was placed at the test sites and chemical untreated chamber on the control sites; secured in place by pressing each chamber. After 24 h, patches were removed from both control and test sites. Skin measurements (e.g., transepidermal water loss (TEWL), skin capacitance and visual Draize scores) were taken at 0, 1, 2, 4, and 24 h after removal of the patches. The primary planned comparisons involved the assessment of skin reactions to test chemicals in terms of measured responses. Response variables for the analysis were formed by subtracting the measurements recorded at the control site from that at the corresponding test site. This removed any occlusion effects, which may have been common to the test and control sites. There was no need to anesthetize rabbits during experimentation because all procedures and measurements (TEWL, capacitance, and Draize visual scores) were non-invasive and painless. Rabbits were euthanized immediately after completion of the experiment by injecting pentobarbital sodium intravenously via ear vein at the dose of 100 mg/kg of body weight. All the experiments were performed in replicates of six. 2.2.2. Light microscopy of JP-8 exposed skin Skin samples from different groups of rabbits were taken by punch biopsy immediately after patch removal and after 24 h of patch removal from the chemically treated and control sites. In each case, rabbits were euthanized before punch biopsy. We also took skin sample from the region where no patch was applied. These samples were fixed in 10% neutrally buffered formalin solution (Accustain® ). For light microscopy study, these samples were washed with water to remove excess fixative. Then, the samples
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were dehydrated by transferring successively to increasing strengths of alcohol, and cleared by passing through toluene. Finally, the samples were infiltrated and embedded in molten paraffin (AccumateTM ), cooled at room temperature and paraffin blocks were prepared. Sections of 10 m were cut by rotory microtome, mounted on a glass slide with the help of glycerogelatin, stained with chrysoidine, hematoxylin and counter stained with eosin. At last, they were dehydrated, cleared, cover slipped, and examined under microscope (Humasons, 1972; Luna, 1968). Microscopic structures observed were photographed by Polaroid® Microcam camera on 339 Polaroid Auto Films. epidermal thickness (ET), length and thickness of collagen fibers’ bundle were measured quantitatively under a 100× lens (Meiji Microscope, Osaka, Japan) with the help of a Cole-Parmer Video Caliper (Model 49910-20, Cole-Parmer, Chicago, IL, USA).
3. Results Fig. 1 shows the changes in temperature at different time points (0, 1, 2, 4, and 24 h) after removal of patches in comparison to temperature at the same site before application of patches. We found significant (P < 0.05) increase in temperature at chemically treated sites immediately (0 h) after removal of patches in comparison to temperature before application of patches. Greater increase (P < 0.05) in temperature was observed at TRI treated site at all time points followed by NAP, 1-MN, and 2-MN. However, we did not observe an increase in the temperature (P > 0.05) at 4 h or 24 h of patch removal than before application of the patches in case of NAP, 1-MN, and 2-MN. At control site, temperatures at different time points after removal of patch were not significantly (P < 0.05) different. Fig. 2 shows the change in capacitance at different time points after removal of patches in comparison to capacitance at the same site before application of the patches. TRI caused greater increase in capacitance than other chemicals, however, the increase was not significant (P > 0.05) except at 0 h, in comparison to the control at the same time point. There was an increase in the skin capacitance immediately after removal of the patches in all cases followed by gradual decrease at later time points. Fig. 3 shows changes in the TEWL values at different time points (0, 1, 2, 4, and 24 h) after removal of patches in comparison to TEWL at the same site before application of patches. There was no significant (P > 0.05) increase in TEWL at control sites at all time points. All the chemicals caused an increase in TEWL at all time points after removal of the patches in comparison to TEWL before application of the patches. TRI caused greater increase in TEWL followed by NAP, 1-MN, 2-MN, TET, and DOD. Increase in TEWL by TRI was significant (P < 0.05) at all time points in comparison to increase in TEWL at control sites. Tables 1 and 2 show Draize scores for erythema and edema, respectively, at different time points after removal
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Fig. 1. Changes in the temperature at 0, 1, 2, 4, and 24 h after patch removal in comparison to temperature at the same site before application of patch. All data are mean ± S.D., n = 6.
Fig. 2. Changes in the capacitance at 0, 1, 2, 4, and 24 h after patch removal in comparison to temperature at the same site before application of patch. All data are mean ± S.D., n = 6. Table 1 In vivo in rabbit erythema scores at different time points after removal of patches Chemicals
Draize score for erythema (mean ± S.D., n = 6) Before
Control JP-8 DOD TRI TET NAP 1-MN 2-MN
0 0 0 0 0 0 0 0
± ± ± ± ± ± ± ±
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0h 0 1 1 2 1 2 2 2
± ± ± ± ± ± ± ±
1h 0.0 0.4 0.5 0.8 0.5 0.5 0.4 0.4
0 1 1 2 1 2 2 2
± ± ± ± ± ± ± ±
2h 0.0 0.4 0.5 0.5 0.8 0.5 0.4 0.4
0 1 1 2 1 2 2 2
± ± ± ± ± ± ± ±
4h 0.0 0.4 0.4 0.6 0.9 0.8 0.4 0.4
0 1 1 2 1 1 1 1
± ± ± ± ± ± ± ±
24 h 0.0 0.5 0.0 0.6 0.9 0.8 0.5 0.5
0 2 1 2 1 2 1 2
± ± ± ± ± ± ± ±
0.0 0.8 0.4 0.8 0.8 0.8 0.5 0.9
Control: patch without chemical; DOD: dodecane; TRI: tridecane; TET: tetradecane; NAP: naphthalene; 1-MN: 1-methylnaphthalene; 2-MN: 2-methylnaphthalene.
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Fig. 3. Changes in the TEWL at 0, 1, 2, 4, and 24 h after patch removal in comparison to temperature at the same site before application of patch. All data are mean ± S.D., n = 6.
of TET. TRI decreased CL greater (P < 0.05) than others did. Likewise, all the chemicals decreased CT significantly (P < 0.05) in comparison to the control in both 0 and 24 h skin samples except 24 h TET skin sample. We did not find significant (P < 0.05) difference in ET, CL, and CT for control sample taken at 0 and 24 h after patch removal (Table 3, Fig. 4a and b). Fig. 5a and b shows light micrographs of TRI treated samples taken at 24 and 0 h after patch removal, respectively. We found that increase in ET in 0 h sample was higher than that in 24 h sample. SC was found detached at some places 0 h TRI treated sample. However, CL and CT were not different. Fig. 6a–d shows light micrographs of control, TRI treated after 0 h, TRI treated after 24 h, and TET treated sample after 24 h of patch removal, respectively. We found decrease in CL and CT for TRI treated 0 and 24 h samples in comparison to control (Fig. 6a–c). In TET treated 24 h sample (Fig. 6d) decrease in CL was found lower than 0 h sample as well as other chemicals (Table 3) but increase in ET was more than other chemicals.
of chemical containing patches. All of the chemicals and JP-8 itself caused moderate to severe erythema and edema, which were not resolved to the baseline level even after 24 h of patch removal. Table 3 shows epidermal thickness, collagen fibers’ bundle length (CL), and collagen fibers’ bundle thickness (CT) at 0 and 24 h of patch removal. We did not find any significant (P < 0.05) difference in ET, CL, and CT between the skin samples from the control sites (patch without chemical) and from the sites without patch as well as test chemicals at both time points. All chemically treated sites showed significant (P < 0.05) increase in ET in comparison to the control sites at 0 h after patch removal. However, this increase in ET was not significant (P < 0.05) in samples taken at 24 h of patch removal except for TET treated samples (Fig. 6d). We found significant (P < 0.05) decrease in CL in skin samples taken from sites immediately (0 h) after removal of chemically treated patches in comparison to the control, which were not found to regain the original length in samples taken 24 h after chemically treated patch removal except in case Table 2 In vivo in rabbit edema scores at different time points after removal of patches Chemicals
Draize score for erythema (mean ± S.D., n = 6) Before
Control JP-8 DOD TRI TET NAP 1-MN 2-MN
0 0 0 0 0 0 0 0
± ± ± ± ± ± ± ±
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0h 0 2 2 2 2 2 2 2
± ± ± ± ± ± ± ±
1h 0.0 0.5 0.9 0.8 0.6 0.8 0.5 0.9
0 2 2 2 2 3 2 2
± ± ± ± ± ± ± ±
2h 0.0 0.1 0.9 0.8 0.7 0.0 0.8 0.9
0 1 1 2 1 2 2 2
± ± ± ± ± ± ± ±
4h 0.0 0.5 0.5 0.8 0.4 0.9 0.5 0.9
0 2 2 2 1 3 1 2
± ± ± ± ± ± ± ±
24 h 0.0 0.4 0.8 0.9 0.5 0.0 0.5 0.9
0 3 2 3 1 3 1 3
± ± ± ± ± ± ± ±
0.0 0.0 0.1 0.0 0.4 0.0 0.5 0.0
Control: patch without chemical; DOD: dodecane; TRI: tridecane; TET: tetradecane; NAP: naphthalene; 1-MN: 1-methylnaphthalene; 2-MN: 2-methylnaphthalene.
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Table 3 Quantitative microscopic alterations by JP-8 jet fuel components in vivo in rabbit JP-8 components
ET
WPWC Control JP-8 DOD TRI TET NAP 1-MN 2-MN
23.0 20.3 32.3 43.2 46.4 50.7 43.8 41.5 42.3
0h
24 h ± ± ± ± ± ± ± ± ±
5.9 4.1 10.1 9.9 9.9 13.8 12.4 12.5 11.7
24.2 24.5 24.9 25.2 26.6 35.1 28.2 27.6 26.9
± ± ± ± ± ± ± ± ±
5.3 6.8 4.3 4.6 7.1 5.1 7.8 8.2 4.8
CL (m, mean ± S.D., n = 25)
CT
0h
0h
56.99 54.03 44.80 42.18 34.78 43.16 38.85 40.05 39.64
24 h ± ± ± ± ± ± ± ± ±
16.9 13.5 11.6 4.3 8.5 9.3 9.8 11.2 8.8
58.6 62.3 44.2 42.8 36.5 55.3 43.7 42.2 42.3
± ± ± ± ± ± ± ± ±
17.0 10.3 9.9 12.7 9.9 12.6 10.5 7.1 7.3
12.5 11.2 9.0 9.0 8.1 8.5 8.3 8.3 8.4
24 h ± ± ± ± ± ± ± ± ±
4.2 1.5 1.9 1.1 1.4 1.7 1.2 0.2 1.4
12.1 11.6 8.7 8.4 7.2 10.9 7.4 7.4 7.4
± ± ± ± ± ± ± ± ±
3.1 1.8 1.5 1.3 1.3 1.6 1.7 1.1 1.9
ET: epidermal thickness; CL: collagen fibers’ bundle length; CT: collagen fibers’ bundle thickness; WPWC: site without patch and chemical; control: patch without chemical; DOD: dodecane; TRI: tridecane; TET: tetradecane; NAP: naphthalene; 1-MN: 1-methylnaphthalene; 2-MN: 2-methylnaphthalene.
Fig. 4. Light micrographs of control (patch without JP-8 component) skin samples (a) control at 24 h and (b) control at 0 h (E, epidermis; CFB, collagen fibers’ bundle) (40×).
4. Discussion Skin regulates the body temperature. Any barrier perturbation may lead to increase in temperature (Thiele and van Senden, 1966). JP-8 has been implicated in skin barrier per-
turbation due to extraction of lipids and proteins from SC (Singh and Singh, 2001). Hence, the test chemicals increased the temperature. TRI caused greater increase in temperature because of its greater ability of extracting lipid and protein from SC (Singh and Singh, 2003).
Fig. 5. (a) Light micrograph of skin sample after 24 h of patch removal containing tridecane and (b) light micrograph of skin sample after 0 h of patch removal containing tridecane (key: E, epidermis; CFB, collagen fibers’ bundle; FB, fibroblast) (40×).
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Fig. 6. Light micrographs of skin sample showing changes in collagen fibers’ bundles: (a) control at 0 h, (b) 0 h after removing tridecane containing patch, (c) 24 h after removing tridecane containing patch and (d) 24 h after removing tetradecane containing patch (key: E, epidermis; CFB, collagen fibers’ bundle; FB, fibroblast) (40×).
As long as horny layer (SC) is intact, capacitance should not be influenced by the increase in the amount of tissue fluid contained beneath the SC. Even a small scratched wound in the test area, which exposed the underlying living tissue, results in a marked increase in capacitance (Tagami et al., 1980). The penetration depth of the electrical scatter field was very small which resulted in measurement of moisture only on skin surface. The very short measuring time (1 s with single measurement) prevented occlusion effects. The sophisticated probe electronics provided temperature stability to the probe constant but low pressure of the probe head (approximately 1.6 N) provided exact, reproducible measurement (Courage & Khazaka, 1998). Hence, all the test chemicals increased capacitance because water from viable epidermis might have rushed up due to perturbed barrier. This is supported by higher increase in capacitance with TRI. In certain cases, the TEWL tend to increase with increasing capacitance. A possible reason for this could be an increase in skin moisture, induced by occlusion, which is also known to reduce diffusional resistance of the SC (Marie et al., 1992). A lack of correlation between TEWL and the hydration status of the SC has also been reported (Triebskorn et al., 1983). We found increase in TEWL for all of the
chemicals due to rupture of skin barrier and increase in temperature. Water loss by skin is thought to be a temperature dependent diffusion phenomenon through a water barrier located in horny layer (Thiele and Malten, 1973). There was no significant (P < 0.05) increase in TEWL for control at different time points. Moreover, we subtracted the TEWL of control sites from the corresponding test sites. Hence, increase in TEWL cannot be attributed to occlusion effect. Irritation tends to reduce the efficiency of the SC barrier function. We found perturbation in macroscopic barrier as evidenced by significant (P < 0.05) increase in temperature, capacitance, and TEWL at test sites, in comparison to control, after 0 h of chemically saturated patch removal. Diffusibility of a chemical substance through the SC is an important factor affecting irritation which depends on a complex interaction of variables; temperature, degree of hydration, mainly physical properties of the penetrating substance, and damage to the structural or chemical integrity of the SC membrane itself (Mathias and Maibach, 1978). Diffusibility through a membrane increases as the temperature of the membrane increases (Rothenberg et al., 1977). Increasing the water content of the SC (membrane hydration) as indicated by higher capacitance values generally
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enhances percutaneous absorption. JP-8 components have been found to extract SC lipids and proteins (Singh et al., 2002) resulting in damage to chemical integrity of the SC. Therefore, all of the JP-8 components caused moderate to severe erythema and edema. Light microscopy provides visual evidences of the microscopic changes in the skin. We did not find any significant (P < 0.05) differences with respect to ET, CL, and CT in skin samples taken from sites having patch without any chemical after 0 and 24 h of patch removal (Table 3, Fig. 4a and b). Hence, any microscopic alteration observed in chemically treated sites could be attributed to the presence of chemical rather than patch itself. We observed significant increase in epidermal thickness (Table 3) and detachment of SC at some places, which indicates perturbation of SC barrier properties (Fig. 5b). Increase in ET may be due to rushing of water/liquid from the underlying viable region assisted by SC and other barrier perturbation. The dermis, a critical organ of the body, not only provides the nutritive, immune, and other support systems for the epidermis through a thin papillary layer adjacent to the epidermis but also plays a role in temperature, pressure, and pain regulation. The dermis consists of collagenous fibers (70%), and elastic connective tissue, in a semigel matrix of mucopolysaccharides (Roberts and Walters, 1998). In the TRI treated skin samples (Fig. 6b and c) collagen fibers’ bundles were found shorter than control (Fig. 6a). TRI decreased CL more than (Table 3) others because it is more permeable to skin. Due to very high lipophilicity TET binds with SC and virtually forms a depot, therefore, it would reach to the dermis in comparatively less amount than other chemicals (Singh and Singh, 2003). That’s why, we found greater increase (recovery) in CL after 24 h of patch removal from CL of 0 h TET sample in comparison to other chemicals (Table 3 and Fig. 6d) than other chemicals. In control, the collagen fibers were bundled together and hence, appeared coarse and aggregated (Fig. 6a). In treated skin, collagen fibers’ bundles were found shortened less coarse and rarefied (Fig. 6b and c). In cross-section, collagen fibers appear wrapped over each other in the form of bundle. We put double adjustable crosshairs with the help of video caliper around the main collagen fibers’ bundle excluding branched collagen fibers’ bundle. These crosshairs formed a rectangle which diagonal we took as an approximation of length of collagen fibers’ bundle. Although this is not exact length of collagen fibers’ bundle, but it indicates the effect of JP-8/JP-8 components on collagen fibers’ bundle. Collagen fibers are fundamental to animal development because they provide a mechanical basis for molecular and cell attachment and stabilize the shape and form of growing tissues (Kadler et al., 2000). These changes in collagen fibers’ bundle due to dermal absorption of JP-8 components might impair the growth of different epidermal skin layers, which may lead to dermatotoxicity. These changes in collagen fibers’ bundle due to dermal absorption of JP-8 components may lead to dermatotoxicity.
The main cells present in the dermis are fibroblasts, which produce the connective tissue components of collagen, laminin, fibronectin and vitronectin; mast cells, which are involved in the immune and inflammatory responses; and melanocytes, involved in the production of the pigment melanin (Roberts and Walters, 1998). Fig. 4 shows fewer fibroblasts in control skin. However, Fig. 6b shows a greater number of fibroblasts in TRI treated skin than the control, which suggest some kinds of immune reaction and inflammatory processes caused by it (Ulrich, 1999; Ulrich and Lyons, 2001; Singh and Singh, 2001). In conclusion, we observed increase in temperature, capacitance, TEWL and severe to moderate erythema and edema with all test chemicals. However, TRI brought greater changes in the above parameters. The quantitative microscopic measurement results showed increase in epidermal thickness, and decrease in collagen fibers’ bundle length and thickness. This constitutes potential dermatotoxicity from these chemicals.
Acknowledgements We acknowledge the financial support from the US Department of Defense, Air Force Office of Scientific Research Grant # F 49620-99-1-0223. We also thank Sumeet Rastogi for his help during in vivo irritation experiments. References Baynes, R.E., Brooks, J.D., Budsaba, K., Smith, C.E., Riviere, J.E., 2001. Mixture effects of JP-8 additives on the dermal disposition of jet fuel components. Toxicol. Appl. Pharmacol. 175, 269. Clark, C.R., Walter, M.K., Farguson, P.W., Katchen, M., 1988. Comparative dermal carcinogenesis of shale and petroleum-derived distillates. Toxicol. Ind. Health 4, 11. Cooper, J.R., Mattie, D.R., 1996. Developmental toxicity of JP-8 Jet fuel in the rat. J. Appl. Toxicol. 16, 197. Courage & Khazaka, 1998. Information and Operating Instructions for the Corneometer CM 820® . p. 13. Draize, J.H., Woodard, G., Calvery, H.O., 1944. Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther. 82, 377. Fentem, J.H., Briggs, D., Chesne, C., Elliott, G.R., Harbell, J.W., Heylings, J.R., Portes, P., Roguet, R., van de Sandt, J.J., Botham, P.A., 2001. A prevalidation study on in vitro tests for acute skin irritation, results and evaluation by the Management Team. Toxicol. In Vitro 15, 57. Geraldine, M.G., Shaffer, K.M., Winfred, Y.K., David, A.S., Joseph, J.P., 2000. Investigation of in vitro toxicity of jet fuels JP-8 and jet A. Drug Chem. Toxicol. 23, 279. Grice, K., Sattar, H., Sharratt, M., Baker, H., 1971. Skin temperature and transepidermal water loss. J. Invest. Dermatol. 57, 108. Harris, D.T., Sakiesrewa, D., Robledo, R.F., Witten, M., 1997. Protection from JP-8 jet fuel induced immunotoxicity by administration of aerosolized substance P. Toxicol. Ind. Health 13, 571. Humasons, G.L., 1972. Animal Tissue Techniques. W.H. Freeman and Company, San Francisco, pp. 7, 45–47, 156–158. Idson, D.R., 1978. In vivo measurement of transepidermal water loss. J. Soc. Cosmet. Chem. 29, 573.
S. Singh, J. Singh / Environmental Toxicology and Pharmacology 16 (2004) 153–161 Kabbur, M.B., Rogers, J.V., Gunasekar, P.G., Garrett, C.M., Geiss, K.T., Brinkley, W.W., McDougal, J.N., 2001. Effect of JP-8 jet fuel on molecular and histological parameters related to acute skin irritation. Toxicol. Appl. Pharmacol. 175, 83. Kadler, K.E., David, F.H., Helen, G., Tobias, S., 2000. Tip-mediated fusion involving unipolar collagen fibrils accounts for rapid fibril elongation, the occurrence of fibrillar branched networks in skin and the paucity of collagen fibril ends in vertebrates. Matrix Biol. 19, 359. Korsak, Z., Wanda, M., Rydzynski, K., 1998. Toxic effects of acute inhalation exposure to 1-methylnaphthalene and 2-methylnaphthalene in experimental animals. Int. J. Occup. Environ. Health 11, 335. Luna, L.G., 1968. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. McGraw-Hill, New York, p. 30. Marie, L., Hakan, O., Tony, A., Ylva, W.L., 1992. Friction, capacitance and transepidermal water loss (TEWL) in dry atopic and normal skin. Br. J. Dermatol. 126, 137. Mathias, C.G., Maibach, H.I., 1978. Dermatotoxicology monographs. I. Cutaneous irritation: factors influencing the response to irritants. Clin. Toxicol. 13, 333. Mattie, D.R., Alden, C.L., Newell, T.K., Gaworski, C.L., Flemming, C.D., 1991. A 90-day continuous vapor inhalation toxicity study of JP-8 jet fuel followed by 20 or 21 months of recovery in Fischer 344 rats and C56BL/6 mice. Toxicol. Pathol. 19, 77. McDougal, J.N., Pollard, D.L., Weisman, W., Garrett, C.M., Miller, T.E., 2000. Assessment of skin absorption and penetration of JP-8 jet fuel and its components. Toxicol. Sci. 55, 247. Monteiro-Riviere, N.A., 1991. Comparative anatomy, physiology, and biochemistry of mammalian skin. In: Hobson, D.W. (Ed.), Dermal and Ocular Toxicology: Fundamental and Methods. CRC Press, Boston, p. 6. OECD Draft Document, 1997. OECD Guideline for the Testing of Chemicals. http://www.oecd.org/ehs/test/flags.htm. Roberts, M.S., Walters, K.A., 1998. The relationship between structure and barrier function of skin. In: Roberts, M.S., Walters, K.A. (Eds.), Dermal Absorption and Toxicity Assessment, Marcel Dekker, New York, p. 1. Rosenthal, D.S., Cynthia, M.G., Liu, W.F., Stoica, B.A., Simulson, M.E., 2001. Mechanism of JP-8 jet fuel cell toxicity. II. Induction of necrosis in skin fibroblasts and keratinocytes and modulation of levels of Bcl-2 family members. Toxicol. Appl. Pharmacol. 171, 107. Rothenberg, H.W., Menne, T., Sjolin, K.E., 1977. Temperature dependent primary irritant dermatitis from lemon perfume. Contact Dermatitis 3, 37. Serban, G.P., Henry, S.M., Cotty, V.F., Marcus, A.D., 1981. In vivo evaluation of skin lotions by electrical capacitance and conductance. J. Soc. Cosmet. Chem. 32, 421.
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Singh, J., Maibach, H.I., 1998. Irritancy of topical chemicals and transdermal delivery system. In: Roberts, M.S., Walters, K.A. (Eds.), Dermal Absorption and Toxicity Assessment. Marcel Dekker, New York, pp. 281–296. Singh, S., Singh, J., 2001. Dermal toxicity: effect of jet propellant-8 fuel exposure on the biophysical, macroscopic and microscopic properties of porcine skin. Environ. Toxicol. Pharmacol. 10, 123. Singh, S., Zhao, K., Singh, J., 2002. In vitro permeability and binding of hydrocarbons in pig ear and human abdominal skin. Drug Chem. Toxicol. 25, 83. Singh, S., Singh, J., 2003. Percutaneous absorption, biophysical, and macroscopic barrier properties of porcine skin exposed to major components of JP-8 jet fuel. Environ. Toxicol. Pharmacol. 14, 77. Stoica, B.A., Boulares, A.H., Rosenthal, D.S., Iyer, S., Hamilton, I.D.G., Simulson, M.E., 2001. Mechanism of JP-8 jet fuel toxicity. I. Induction of apoptosis in rat lung epithelial Cells. Toxicol. Appl. Pharmacol. 171, 94. Tagami, H., Ohi, H., Iwatsuki, K., Kanamaru, Y., Yamada, M., Ichijo, B., 1980. Evaluation of the skin surface hydration in vivo by electrical measurement. J. Invest. Dermatol. 75, 500. Thiele, F.A.J., Malten, K.E., 1973. Evaluation of the skin damage I. Br. J. Dermatol. 89, 373. Thiele, F.A.J., van Senden, K.G., 1966. Relationship between skin temperature and the insensible perspiration of the human skin. J. Invest. Dermatol. 47, 307. Triebskorn, A., Gloor, M., Greiner, F., 1983. Comparative investigations on the water content of the stratum corneum using different methods of measurement. Dermatologica 167, 64. Ulrich, S.E., 1999. Dermal application of JP-8 jet fuel induces immune suppression. Toxicol. Sci. 52, 61. Ulrich, S.E., Lyons, H.J., 2001. Mechanisms involved in the immunotoxicity induced by dermal application of JP-8 jet fuel. Toxicol. Sci. 58, 290. Wilhelm, K.P., Surber, C., Maibach, H.I., 1989. Quantification of sodium lauryl sulfate irritant dermatitis in man: comparison of four techniques: skin color reflectance, transepidermal water loss, laser Doppler flow measurement and visual scores. Arch. Dermatol. Res. 281, 291. Zuang, V., Balls, M., Botham, P.A., Coquette, A., Corsini, E., Curren, R.D., Elliott, G.R., Fentem, J.H., Heylings, J.R., Liebsch, M., Medina, J., Roguet, R., van de Sandt, J.J., Wiemann, C., Worth, A.P., 2002. Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. The European Center for the Validation of Alternative Methods Skin Irritation Task Force Report 2. Altern. Lab. Anim. 30, 109.
Dermal toxicity and microscopic alterations by JP-8 jet fuel components in vivo in rabbit Somnath Singh1 , Jagdish Singh∗ Department of Pharmaceutical Sciences, College of Pharmacy, North Dakota State University, Fargo, ND 58105, USA Accepted 8 December 2003
Abstract In this study, we investigated the skin irritation, macroscopic and microscopic barrier alteration in vivo in rabbits from aliphatic and aromatic components of jet propellant-8 (JP-8) jet fuel. Macroscopic barrier properties were evaluated by measuring transepidermal water loss (TEWL), skin capacitance, and skin temperature; microscopic changes were observed by light microscopy. Draize visual scoring system was used to measure skin irritation. We found significant (P < 0.05) increase in temperature at the site of all chemically saturated patches immediately after patch removal in comparison to the control site. Tridecane (TRI) produced a greater increase in temperature and capacitance at all time points than all the other components of JP-8. Both the aliphatic and aromatic components increased the TEWL at all time points. Tridecane produced greater increase in TEWL followed by naphthalene (NAP), 1-methylnaphthalene (1-MN), 2-metylnaphthalene (2-MN), tetradecane (TET), and dodecane (DOD). All of the above components of JP-8 caused moderate to severe erythema and edema, which were not resolved to the baseline even after 24 h of patch removal. Light microscopy revealed an increase in epidermal thickness (ET), and decrease in length and thickness of collagen fibers’ bundle by the above components of JP-8. These results suggest potential dermatotoxicity from the JP-8 components. © 2004 Elsevier B.V. All rights reserved. Keywords: JP-8 components; TEWL; Skin irritation; Skin capacitance; Light microscopy
1. Introduction Jet propellant-8 (JP-8) jet fuel is kerosene based multicomponent mixture of aromatic and aliphatic hydrocarbons, without the lower molecular weight fraction (Mattie et al., 1991). Hence, it is less volatile and has a higher flash point than earlier used jet fuel (JP-4). JP-8 has been associated with toxicity in animal models and humans. Significant reduction in fetal body weight was found in pregnant rat fed JP-8 orally (Cooper and Mattie, 1996). JP-8 induces apoptosis in rat lung epithelial cells, primary mouse T lymphocytes, Jorkat T lymphoma cells, and monocytic cells (Stoica et al., 2001). Induction of necrosis in fibrob-
∗ Corresponding author. Tel.: +1-701-231-7943; fax: +1-701-231-8333. E-mail address: [email protected] (J. Singh). 1 Present address: Department of Pharmacy Sciences, School of Pharmacy and Health Professions, Creighton University Medical Center, Omaha, NE 68178, USA.
1382-6689/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2003.12.001
lasts and keratinocytes including cytotoxicity and altered histology were found in human skin grafts treated with JP-8 (Rosenthal et al., 2001). Four different cell lines exposed to JP-8 showed cytotoxicity and electrophysiological toxicity (Geraldine et al., 2000). Exposure to aromatic components of JP-8 resulted in concentration-dependent decrease in pain sensitivity in rats and respiratory rate in mice (Korsak et al., 1998). Studies in mice have shown that short-term, low concentration JP-8 exposure had significant effects on the immune system, e.g., decreased viable immune cell numbers, decreased immune organ weights, and loss of immune function that persisted for extended periods of time (Harris et al., 1997). There is direct risk of carcinogenesis of the skin itself, since petroleum middle distillates such as kerosene have been shown to act as strong carcinogens in a mouse skin assay (Clark et al., 1988). Aliphatic and aromatic components in JP-8 can cause occupational dermatitis (Baynes et al., 2001) and skin irritation after dermal exposure (Kabbur et al., 2001).
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There is a great potential for human exposure to JP-8. Splashes during refueling, handling of engine part coated with JP-8, contact with sides of fuel tanks during fuel maintenance operations, contact with fuel leaks on the undersides of aircraft or on ramp are potentials sources of dermal exposure (McDougal et al., 2000). Skin irritation is the non-immunologic evocation of normal or exaggerated reaction in a tissue by application of a stimulus (Singh and Maibach, 1998). There is not yet an adequately validated in vitro model available to predict the skin irritation of chemicals. A prevalidation study on in vitro tests for acute skin irritation was carried out to evaluate EpiDermTM , EPISKINTM , PREDISKINTM , the non-perfused pig ear method, and the mouse skin integrity function test. It was concluded that none of these tests was ready for inclusion in a formal validation study on in vitro tests for acute skin irritation (Fentem et al., 2001; Zuang et al., 2002). The standard way to forecast skin irritation is by so called predictive tests on man or animals (Draize et al., 1944). TEWL is an well-accepted method for quantifying material innocuousness (Idson, 1978) and provides robust method for assessing damage to the stratum corneum (SC). Irritation tends to reduce the efficiency of stratum corneum barrier function and results in an increase in TEWL. This is sometimes associated with a decrease in skin water content (Wilhelm et al., 1989) and increase in skin temperature (Grice et al., 1971). Hence, measurement of skin capacitance or skin hydration and skin temperature may also be used to assess irritation (Thiele and Malten, 1973; Serban et al., 1981). Light microscopy examines the structure of the cells in skin, and provides a direct visual evidence of any anatomical changes (Monteiro-Riviere, 1991). Skin is anatomically divided into two principal components, the outer epidermis and underlying dermis. Within these two primary divisions, numerous cell types and specialized adnexial structures can be identified. By light microscopic study, structure to function relationship can be overviewed (Roberts and Walters, 1998). JP-8 is a complex mixture composed of both aliphatic and aromatic hydrocarbons, the latter of which are associated with DNA damage and carcinogenesis. The most damaging components are the most permeable (Rosenthal et al., 2001). Due to the chemical complexity of JP-8, one cannot assess the dermal toxicity of entire fuel components. Therefore, we selected representative three aromatic and three aliphatic components, which widely differed in their lipophilicity, binding to stratum corneum, and permeability through the skin (Singh et al., 2002). In this study, we determined microscopic alteration, skin barrier perturbation, and skin irritation in vivo in rabbits from model aromatic (naphthalene (NAP), 1-methylnaphthalene (1-MN), and 2-metylnaphthalene (2-MN)) and aliphatic (dodecane (DOD), tridecane (TRI), and tetradecane (TET)) components of JP-8.
2. Materials and methods 2.1. Materials JP-8 jet fuel was obtained from Wright–Patterson Air Force Base, Ohio. DOD was purchased from Mallinckrodt Chemical Works, St. Louis, MO; TRI and 1-MN from Aldrich Chemical Company, Milwaukee, WI; TET from EM Science, Gibbstown, NJ; 2-MN from Acros Organic, NJ; and NAP from Sigma Chemical, St. Louis, MO. Ten percent neutrally buffered formalin (Accustain® ), AccumateTM tissue embedding/infiltrating medium, alcoholic solution of eosin Y with phloxine, hematoxylin solution Gill No. 3, glycerolgelatin mounting medium were purchased from Sigma Diagnostics, St. Louis, MO, USA. Chrysoidine G was procured from Fluka Chemie AG, Switzerland. Haye’s chambers were purchased from Acaderm, Inc., Menlo park, CA, USA. Rabbits (New Zealand, white, male, 10 weeks old, and approximately 1.5 kg body weight) were purchased from Harlan, Indianapolis, IN, USA. All other chemicals used were of analytical grade. 2.2. Methods 2.2.1. Skin integrity and irritation in rabbits Skin barrier function and primary irritation were measured in vivo in rabbits. The dorsal region of each rabbit was shaven carefully avoiding skin damages with the help of Oster Golden A5® single speed clipper using size 40 blade. Rabbit was acclimatized for 24 h before taking any measurements. Environmental conditions were monitored: room temperature 20–26 ◦ C, relative humidity 46–58%. TEWL was measured quantitatively with a TewameterTM (Courage & Khazaka, Cologne, FRG) using Fick’s law of diffusion. The probe was held on the skin until a stable TEWL value established (1 min). The electrical capacitance (device internal unit (i.u.)) of the skin surface was measured using CorneometerTM SM 820 (Courage & Khazaka, Cologne, FRG). CorneometerTM SM 820 measures the content of skin moisture by exploiting completely different dielectric constant of water (81) and other substances (mostly <7). The measuring capacitor shows changes of capacitance according to the moisture content of the skin. A glass lamina separates the metallic tracks (gold) in the probe head from the skin in order to prevent current conduction in the sample. An electric scatter field penetrates the skin during the measurement and the dielectricity is determined. One track builds up a surplus of electrons (minus charge) the other lacks of electrons (plus charge). An electric field between the tracks with alternating attraction develops. During the measurement the scatterfield penetrates the very first layer of the skin and determines the dielectricity (Courage & Khazaka, 1998). Skin temperature was measured using an infrared thermometer (Omega Engineering Inc., Stamford, CT).
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The visual scoring system was used to evaluate primary skin irritation manifested as erythema: very slight erythema barely perceptible # 1; well defined erythema # 2; moderate to severe erythema # 3; severe erythema, beet redness to slight eschar formation, injuries in depth # 4; and edema: very slight edema, barely perceptible # 1; slight edema, edges of area well defined by definite raising # 2; moderate edema, area raised approximately 1 mm # 3; severe edema, raised more than 1 mm, and extending beyond area of exposure # 4 (Draize et al., 1944). However, skin irritation is scored 0 through 3 according to Organization of Economic Cooperation and Development (OECD) guidelines: grade 0 no reaction; grade 1 weakly positive reaction (usually characterized by mild erythema and/or dryness across most of the treatment site); grade 2 moderately positive reaction (usually distinct erythema or dryness, possibly spreading beyond the treatment site); grade 3 strongly positive reaction (strong and often spreading erythema with edema and/or scabbing) (OECD Draft Document, 1997). Dorsal skin of each rabbit was marked into one cm2 area at three places on the left side and three places on the right side. Each rabbit had three test sites and three control sites. Baseline measurements were taken corresponding to test and control sites. Haye’s occlusive test chamber saturated with test chemical (50 l) was placed at the test sites and chemical untreated chamber on the control sites; secured in place by pressing each chamber. After 24 h, patches were removed from both control and test sites. Skin measurements (e.g., transepidermal water loss (TEWL), skin capacitance and visual Draize scores) were taken at 0, 1, 2, 4, and 24 h after removal of the patches. The primary planned comparisons involved the assessment of skin reactions to test chemicals in terms of measured responses. Response variables for the analysis were formed by subtracting the measurements recorded at the control site from that at the corresponding test site. This removed any occlusion effects, which may have been common to the test and control sites. There was no need to anesthetize rabbits during experimentation because all procedures and measurements (TEWL, capacitance, and Draize visual scores) were non-invasive and painless. Rabbits were euthanized immediately after completion of the experiment by injecting pentobarbital sodium intravenously via ear vein at the dose of 100 mg/kg of body weight. All the experiments were performed in replicates of six. 2.2.2. Light microscopy of JP-8 exposed skin Skin samples from different groups of rabbits were taken by punch biopsy immediately after patch removal and after 24 h of patch removal from the chemically treated and control sites. In each case, rabbits were euthanized before punch biopsy. We also took skin sample from the region where no patch was applied. These samples were fixed in 10% neutrally buffered formalin solution (Accustain® ). For light microscopy study, these samples were washed with water to remove excess fixative. Then, the samples
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were dehydrated by transferring successively to increasing strengths of alcohol, and cleared by passing through toluene. Finally, the samples were infiltrated and embedded in molten paraffin (AccumateTM ), cooled at room temperature and paraffin blocks were prepared. Sections of 10 m were cut by rotory microtome, mounted on a glass slide with the help of glycerogelatin, stained with chrysoidine, hematoxylin and counter stained with eosin. At last, they were dehydrated, cleared, cover slipped, and examined under microscope (Humasons, 1972; Luna, 1968). Microscopic structures observed were photographed by Polaroid® Microcam camera on 339 Polaroid Auto Films. epidermal thickness (ET), length and thickness of collagen fibers’ bundle were measured quantitatively under a 100× lens (Meiji Microscope, Osaka, Japan) with the help of a Cole-Parmer Video Caliper (Model 49910-20, Cole-Parmer, Chicago, IL, USA).
3. Results Fig. 1 shows the changes in temperature at different time points (0, 1, 2, 4, and 24 h) after removal of patches in comparison to temperature at the same site before application of patches. We found significant (P < 0.05) increase in temperature at chemically treated sites immediately (0 h) after removal of patches in comparison to temperature before application of patches. Greater increase (P < 0.05) in temperature was observed at TRI treated site at all time points followed by NAP, 1-MN, and 2-MN. However, we did not observe an increase in the temperature (P > 0.05) at 4 h or 24 h of patch removal than before application of the patches in case of NAP, 1-MN, and 2-MN. At control site, temperatures at different time points after removal of patch were not significantly (P < 0.05) different. Fig. 2 shows the change in capacitance at different time points after removal of patches in comparison to capacitance at the same site before application of the patches. TRI caused greater increase in capacitance than other chemicals, however, the increase was not significant (P > 0.05) except at 0 h, in comparison to the control at the same time point. There was an increase in the skin capacitance immediately after removal of the patches in all cases followed by gradual decrease at later time points. Fig. 3 shows changes in the TEWL values at different time points (0, 1, 2, 4, and 24 h) after removal of patches in comparison to TEWL at the same site before application of patches. There was no significant (P > 0.05) increase in TEWL at control sites at all time points. All the chemicals caused an increase in TEWL at all time points after removal of the patches in comparison to TEWL before application of the patches. TRI caused greater increase in TEWL followed by NAP, 1-MN, 2-MN, TET, and DOD. Increase in TEWL by TRI was significant (P < 0.05) at all time points in comparison to increase in TEWL at control sites. Tables 1 and 2 show Draize scores for erythema and edema, respectively, at different time points after removal
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Fig. 1. Changes in the temperature at 0, 1, 2, 4, and 24 h after patch removal in comparison to temperature at the same site before application of patch. All data are mean ± S.D., n = 6.
Fig. 2. Changes in the capacitance at 0, 1, 2, 4, and 24 h after patch removal in comparison to temperature at the same site before application of patch. All data are mean ± S.D., n = 6. Table 1 In vivo in rabbit erythema scores at different time points after removal of patches Chemicals
Draize score for erythema (mean ± S.D., n = 6) Before
Control JP-8 DOD TRI TET NAP 1-MN 2-MN
0 0 0 0 0 0 0 0
± ± ± ± ± ± ± ±
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0h 0 1 1 2 1 2 2 2
± ± ± ± ± ± ± ±
1h 0.0 0.4 0.5 0.8 0.5 0.5 0.4 0.4
0 1 1 2 1 2 2 2
± ± ± ± ± ± ± ±
2h 0.0 0.4 0.5 0.5 0.8 0.5 0.4 0.4
0 1 1 2 1 2 2 2
± ± ± ± ± ± ± ±
4h 0.0 0.4 0.4 0.6 0.9 0.8 0.4 0.4
0 1 1 2 1 1 1 1
± ± ± ± ± ± ± ±
24 h 0.0 0.5 0.0 0.6 0.9 0.8 0.5 0.5
0 2 1 2 1 2 1 2
± ± ± ± ± ± ± ±
0.0 0.8 0.4 0.8 0.8 0.8 0.5 0.9
Control: patch without chemical; DOD: dodecane; TRI: tridecane; TET: tetradecane; NAP: naphthalene; 1-MN: 1-methylnaphthalene; 2-MN: 2-methylnaphthalene.
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Fig. 3. Changes in the TEWL at 0, 1, 2, 4, and 24 h after patch removal in comparison to temperature at the same site before application of patch. All data are mean ± S.D., n = 6.
of TET. TRI decreased CL greater (P < 0.05) than others did. Likewise, all the chemicals decreased CT significantly (P < 0.05) in comparison to the control in both 0 and 24 h skin samples except 24 h TET skin sample. We did not find significant (P < 0.05) difference in ET, CL, and CT for control sample taken at 0 and 24 h after patch removal (Table 3, Fig. 4a and b). Fig. 5a and b shows light micrographs of TRI treated samples taken at 24 and 0 h after patch removal, respectively. We found that increase in ET in 0 h sample was higher than that in 24 h sample. SC was found detached at some places 0 h TRI treated sample. However, CL and CT were not different. Fig. 6a–d shows light micrographs of control, TRI treated after 0 h, TRI treated after 24 h, and TET treated sample after 24 h of patch removal, respectively. We found decrease in CL and CT for TRI treated 0 and 24 h samples in comparison to control (Fig. 6a–c). In TET treated 24 h sample (Fig. 6d) decrease in CL was found lower than 0 h sample as well as other chemicals (Table 3) but increase in ET was more than other chemicals.
of chemical containing patches. All of the chemicals and JP-8 itself caused moderate to severe erythema and edema, which were not resolved to the baseline level even after 24 h of patch removal. Table 3 shows epidermal thickness, collagen fibers’ bundle length (CL), and collagen fibers’ bundle thickness (CT) at 0 and 24 h of patch removal. We did not find any significant (P < 0.05) difference in ET, CL, and CT between the skin samples from the control sites (patch without chemical) and from the sites without patch as well as test chemicals at both time points. All chemically treated sites showed significant (P < 0.05) increase in ET in comparison to the control sites at 0 h after patch removal. However, this increase in ET was not significant (P < 0.05) in samples taken at 24 h of patch removal except for TET treated samples (Fig. 6d). We found significant (P < 0.05) decrease in CL in skin samples taken from sites immediately (0 h) after removal of chemically treated patches in comparison to the control, which were not found to regain the original length in samples taken 24 h after chemically treated patch removal except in case Table 2 In vivo in rabbit edema scores at different time points after removal of patches Chemicals
Draize score for erythema (mean ± S.D., n = 6) Before
Control JP-8 DOD TRI TET NAP 1-MN 2-MN
0 0 0 0 0 0 0 0
± ± ± ± ± ± ± ±
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
0h 0 2 2 2 2 2 2 2
± ± ± ± ± ± ± ±
1h 0.0 0.5 0.9 0.8 0.6 0.8 0.5 0.9
0 2 2 2 2 3 2 2
± ± ± ± ± ± ± ±
2h 0.0 0.1 0.9 0.8 0.7 0.0 0.8 0.9
0 1 1 2 1 2 2 2
± ± ± ± ± ± ± ±
4h 0.0 0.5 0.5 0.8 0.4 0.9 0.5 0.9
0 2 2 2 1 3 1 2
± ± ± ± ± ± ± ±
24 h 0.0 0.4 0.8 0.9 0.5 0.0 0.5 0.9
0 3 2 3 1 3 1 3
± ± ± ± ± ± ± ±
0.0 0.0 0.1 0.0 0.4 0.0 0.5 0.0
Control: patch without chemical; DOD: dodecane; TRI: tridecane; TET: tetradecane; NAP: naphthalene; 1-MN: 1-methylnaphthalene; 2-MN: 2-methylnaphthalene.
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Table 3 Quantitative microscopic alterations by JP-8 jet fuel components in vivo in rabbit JP-8 components
ET
WPWC Control JP-8 DOD TRI TET NAP 1-MN 2-MN
23.0 20.3 32.3 43.2 46.4 50.7 43.8 41.5 42.3
0h
24 h ± ± ± ± ± ± ± ± ±
5.9 4.1 10.1 9.9 9.9 13.8 12.4 12.5 11.7
24.2 24.5 24.9 25.2 26.6 35.1 28.2 27.6 26.9
± ± ± ± ± ± ± ± ±
5.3 6.8 4.3 4.6 7.1 5.1 7.8 8.2 4.8
CL (m, mean ± S.D., n = 25)
CT
0h
0h
56.99 54.03 44.80 42.18 34.78 43.16 38.85 40.05 39.64
24 h ± ± ± ± ± ± ± ± ±
16.9 13.5 11.6 4.3 8.5 9.3 9.8 11.2 8.8
58.6 62.3 44.2 42.8 36.5 55.3 43.7 42.2 42.3
± ± ± ± ± ± ± ± ±
17.0 10.3 9.9 12.7 9.9 12.6 10.5 7.1 7.3
12.5 11.2 9.0 9.0 8.1 8.5 8.3 8.3 8.4
24 h ± ± ± ± ± ± ± ± ±
4.2 1.5 1.9 1.1 1.4 1.7 1.2 0.2 1.4
12.1 11.6 8.7 8.4 7.2 10.9 7.4 7.4 7.4
± ± ± ± ± ± ± ± ±
3.1 1.8 1.5 1.3 1.3 1.6 1.7 1.1 1.9
ET: epidermal thickness; CL: collagen fibers’ bundle length; CT: collagen fibers’ bundle thickness; WPWC: site without patch and chemical; control: patch without chemical; DOD: dodecane; TRI: tridecane; TET: tetradecane; NAP: naphthalene; 1-MN: 1-methylnaphthalene; 2-MN: 2-methylnaphthalene.
Fig. 4. Light micrographs of control (patch without JP-8 component) skin samples (a) control at 24 h and (b) control at 0 h (E, epidermis; CFB, collagen fibers’ bundle) (40×).
4. Discussion Skin regulates the body temperature. Any barrier perturbation may lead to increase in temperature (Thiele and van Senden, 1966). JP-8 has been implicated in skin barrier per-
turbation due to extraction of lipids and proteins from SC (Singh and Singh, 2001). Hence, the test chemicals increased the temperature. TRI caused greater increase in temperature because of its greater ability of extracting lipid and protein from SC (Singh and Singh, 2003).
Fig. 5. (a) Light micrograph of skin sample after 24 h of patch removal containing tridecane and (b) light micrograph of skin sample after 0 h of patch removal containing tridecane (key: E, epidermis; CFB, collagen fibers’ bundle; FB, fibroblast) (40×).
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Fig. 6. Light micrographs of skin sample showing changes in collagen fibers’ bundles: (a) control at 0 h, (b) 0 h after removing tridecane containing patch, (c) 24 h after removing tridecane containing patch and (d) 24 h after removing tetradecane containing patch (key: E, epidermis; CFB, collagen fibers’ bundle; FB, fibroblast) (40×).
As long as horny layer (SC) is intact, capacitance should not be influenced by the increase in the amount of tissue fluid contained beneath the SC. Even a small scratched wound in the test area, which exposed the underlying living tissue, results in a marked increase in capacitance (Tagami et al., 1980). The penetration depth of the electrical scatter field was very small which resulted in measurement of moisture only on skin surface. The very short measuring time (1 s with single measurement) prevented occlusion effects. The sophisticated probe electronics provided temperature stability to the probe constant but low pressure of the probe head (approximately 1.6 N) provided exact, reproducible measurement (Courage & Khazaka, 1998). Hence, all the test chemicals increased capacitance because water from viable epidermis might have rushed up due to perturbed barrier. This is supported by higher increase in capacitance with TRI. In certain cases, the TEWL tend to increase with increasing capacitance. A possible reason for this could be an increase in skin moisture, induced by occlusion, which is also known to reduce diffusional resistance of the SC (Marie et al., 1992). A lack of correlation between TEWL and the hydration status of the SC has also been reported (Triebskorn et al., 1983). We found increase in TEWL for all of the
chemicals due to rupture of skin barrier and increase in temperature. Water loss by skin is thought to be a temperature dependent diffusion phenomenon through a water barrier located in horny layer (Thiele and Malten, 1973). There was no significant (P < 0.05) increase in TEWL for control at different time points. Moreover, we subtracted the TEWL of control sites from the corresponding test sites. Hence, increase in TEWL cannot be attributed to occlusion effect. Irritation tends to reduce the efficiency of the SC barrier function. We found perturbation in macroscopic barrier as evidenced by significant (P < 0.05) increase in temperature, capacitance, and TEWL at test sites, in comparison to control, after 0 h of chemically saturated patch removal. Diffusibility of a chemical substance through the SC is an important factor affecting irritation which depends on a complex interaction of variables; temperature, degree of hydration, mainly physical properties of the penetrating substance, and damage to the structural or chemical integrity of the SC membrane itself (Mathias and Maibach, 1978). Diffusibility through a membrane increases as the temperature of the membrane increases (Rothenberg et al., 1977). Increasing the water content of the SC (membrane hydration) as indicated by higher capacitance values generally
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enhances percutaneous absorption. JP-8 components have been found to extract SC lipids and proteins (Singh et al., 2002) resulting in damage to chemical integrity of the SC. Therefore, all of the JP-8 components caused moderate to severe erythema and edema. Light microscopy provides visual evidences of the microscopic changes in the skin. We did not find any significant (P < 0.05) differences with respect to ET, CL, and CT in skin samples taken from sites having patch without any chemical after 0 and 24 h of patch removal (Table 3, Fig. 4a and b). Hence, any microscopic alteration observed in chemically treated sites could be attributed to the presence of chemical rather than patch itself. We observed significant increase in epidermal thickness (Table 3) and detachment of SC at some places, which indicates perturbation of SC barrier properties (Fig. 5b). Increase in ET may be due to rushing of water/liquid from the underlying viable region assisted by SC and other barrier perturbation. The dermis, a critical organ of the body, not only provides the nutritive, immune, and other support systems for the epidermis through a thin papillary layer adjacent to the epidermis but also plays a role in temperature, pressure, and pain regulation. The dermis consists of collagenous fibers (70%), and elastic connective tissue, in a semigel matrix of mucopolysaccharides (Roberts and Walters, 1998). In the TRI treated skin samples (Fig. 6b and c) collagen fibers’ bundles were found shorter than control (Fig. 6a). TRI decreased CL more than (Table 3) others because it is more permeable to skin. Due to very high lipophilicity TET binds with SC and virtually forms a depot, therefore, it would reach to the dermis in comparatively less amount than other chemicals (Singh and Singh, 2003). That’s why, we found greater increase (recovery) in CL after 24 h of patch removal from CL of 0 h TET sample in comparison to other chemicals (Table 3 and Fig. 6d) than other chemicals. In control, the collagen fibers were bundled together and hence, appeared coarse and aggregated (Fig. 6a). In treated skin, collagen fibers’ bundles were found shortened less coarse and rarefied (Fig. 6b and c). In cross-section, collagen fibers appear wrapped over each other in the form of bundle. We put double adjustable crosshairs with the help of video caliper around the main collagen fibers’ bundle excluding branched collagen fibers’ bundle. These crosshairs formed a rectangle which diagonal we took as an approximation of length of collagen fibers’ bundle. Although this is not exact length of collagen fibers’ bundle, but it indicates the effect of JP-8/JP-8 components on collagen fibers’ bundle. Collagen fibers are fundamental to animal development because they provide a mechanical basis for molecular and cell attachment and stabilize the shape and form of growing tissues (Kadler et al., 2000). These changes in collagen fibers’ bundle due to dermal absorption of JP-8 components might impair the growth of different epidermal skin layers, which may lead to dermatotoxicity. These changes in collagen fibers’ bundle due to dermal absorption of JP-8 components may lead to dermatotoxicity.
The main cells present in the dermis are fibroblasts, which produce the connective tissue components of collagen, laminin, fibronectin and vitronectin; mast cells, which are involved in the immune and inflammatory responses; and melanocytes, involved in the production of the pigment melanin (Roberts and Walters, 1998). Fig. 4 shows fewer fibroblasts in control skin. However, Fig. 6b shows a greater number of fibroblasts in TRI treated skin than the control, which suggest some kinds of immune reaction and inflammatory processes caused by it (Ulrich, 1999; Ulrich and Lyons, 2001; Singh and Singh, 2001). In conclusion, we observed increase in temperature, capacitance, TEWL and severe to moderate erythema and edema with all test chemicals. However, TRI brought greater changes in the above parameters. The quantitative microscopic measurement results showed increase in epidermal thickness, and decrease in collagen fibers’ bundle length and thickness. This constitutes potential dermatotoxicity from these chemicals.
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