Chemical-induced Skin Irritation

Irritation of the skin by chemicals can be a major problem in the workplace and the home.  Contact dermatitis is a very common occupational disease that may account for up to 15% of all occupational illness with annual costs (due to lost time and medical expenses) of up to $1 billion every year.  Because our skin is the interface with our immediate environment, there is potential for contact with chemicals directly (splash, immersion or aerosol) or from contact with contaminated surfaces. Once chronically irritated, our skin may never completely recover and become a lifelong problem.  When chronically irritated, the skin may become red, scaly or chapped, itchy or sensitive to touch. Chemicals may irritate the skin by causing an immune response, disrupting the epidermal barrier, being cytotoxic to any of the 30 types of skin cells, causing oxidative stress at a cellular level or any combination of these effects.  The specific biological mechanisms by which various chemicals cause irritation are poorly understood, but some of the biological responses are well recognized.  Cytokine release (IL-1a, TNFa, IL-6 and IL-8 etc.) initiates the inflammatory cascade and growth factor release (PDGF, TGFa, TGFβ, EGF, and GM-CSF etc.) causes increased epidermal turnover.  It is a goal of our laboratory to understand the “triggers” and the process of chemical-induced skin irritation so that therapeutic and/or prophylactic treatments can be developed.

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Measures of Irritation

In our dermal toxicology laboratory, we focus on chemical-induced irritation in the laboratory animal as a model for human responses.   Rats are a particularly good model because they are large enough to allow a carefully controlled exposure and provide large enough pieces of skin for genomic and proteomic analysis.  They are used as the experimental species for many toxicology and pharmacology studies. In addition, the rat genome has been mapped, which tremendously assists in understanding the biological response of the skin to volatile organic chemicals.
 
We compared the early histological responses of F-344 rats and Hartley guinea pigs to chloropentafluorobenzene (CPFB) and 1,2-dichlorobenzene (DCB) every half hour for up to 4 hours (McDougal et al. 1997).  Light and transmission electron microscopy showed that the guinea pig response was more rapid and more severe than the rat response. The guinea pig response was more variable than the rat and as a result we focused subsequent studies on the rat.  After four hours, CPFB caused more severe PMN margination and inflammation than DCB in both species, although the compounds are closely related. 

In a repeated dose study, we evaluated the irritation of three jet fuels (JP-4, JP-8 and JP-8+100) following repeated application to the skin daily for up to 28 days and subsequent recovery from irritation 7, 14 and 21 days after the end of the exposure (Baker et al. 1999).  All three fuels were very irritating and no differences could be discerned between them; however, all three treatment groups recovered during the post treatment period and they were no different from control after 21 days of non-treatment.

Xylene is an organic solvent and component of many products including gasoline and jet fuel.  In an attempt to try to understand the early response after a brief (1 hour) cutaneous exposure, we look at histological changes and changes in interleukin-1 alpha (IL-1a) and inducible nitric oxide synthase (iNOS) at 1, 2, 4 & 6 hours after the beginning of the exposure (Gunasekar et al. 2003).  We use a Hill Top Chamber® held in place with a rodent harness for the occluded chemical exposures.

R Jacket (Lomir Biomedical) with Velcro on the side not shown	Gauze-filled exposure chamber with velcro on back

Western blots showed that IL-1a increased by about 80% right after the exposure and iNOS increased by 60% four hours after the exposure. We also found epidermal-dermal separation and granulocyte infiltration by 6 hours after the beginning of the exposures.  We concluded that IL-1a and iNOS might serve as early indicators of skin irritation.

The results of a similar (1 hour) exposure using JP-8 in rats showed significant changes in both IL-1a and iNOS as measured by enzyme linked immunosorbent assay (ELISA) and western blot (Kabbur et al. 2001).  These changes were localized primarily to the epidermis by immunohistochemical localization.  Histologically, we found granulocyte infiltration by 2 hours.  We concluded that the response to a brief JP-8 exposure was rapid and that we could see changes in biochemical parameters before they were apparent visually.

In an attempt to understand more about these chemical-induced irritant responses we looked at changes in oxidative species (a measure of oxidative stress) and low molecular weight DNA (a measure of apoptotic cell death) with both m-xylene (Rogers et al. 2001b) and JP-8 (Rogers et al. 2001a).  We found that both oxidative species and low molecular weight DNA were increased by 2 hours with xylene.  With JP-8 exposure, oxidative species were found at the end of the exposure, but low molecular weight DNA was not significantly different until 4 hours.  We suggested that these could potentially be predictive biomarkers of irritant effects.

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Gene Expression

When gene expression studies became more widely available with microarrays, we investigated the effects of brief exposures on gene expression in the skin with some different skin irritants that may have had mechanisms of action that were distinct.  We investigated effects of a surfactant (10% sodium lauryl sulfate in water, SLS), an undiluted organic solvent (m-xylene) and an undiluted “green” solvent (d-limonene) after 1 hour exposures using the Affymetrix Rat Toxicology chip with approximately 850 transcripts (Rogers et al. 2003).  We looked at untreated skin to identify basal gene expression in the skin.  The majority of the active genes in normal skin were related to metabolism, oxidative/cellular stress and signal transduction.  We measured changes in gene expression at the end of the exposure and 4 hours after the beginning of the exposure and this table shows that the gene responses (combined for both time points) to the surfactant, SLS, was less than the gene changes with the solvents. 

Also, there were 43 of the same genes increased with xylene and limonene at one hour, but the SLS response was unique.  This suggests that surfactants and solvents may cause skin irritation by different mechanisms.

We have investigated the time-course of gene expression changes in the epidermis for up to 8 hours after a 1-hour exposure to JP-8 (McDougal et al. 2006). After exposure,we separated the epidermis from the rest of the skin with a cryotome and isolated total mRNA. Gene expression was studied with microarray techniques (Affymetrix RGU-34-A).  Changes from sham-treatments were analyzed and characterized.  We found consistent two-fold increases in gene expression of 27 transcripts at one, four and eight hours after the beginning of the one-hour exposure that were related primarily to structural proteins, cell signaling, inflammatory mediators, growth factors and enzymes.  Analysis of pathways changed showed that several signaling pathways were increased at one hour and that the most significant changes at eight hours were in metabolic pathways, many of which were down regulated.  Based on the one-hour changes in gene expression, we hypothesize that the “trigger” of the JP-8-induced, epidermal, stress response is a “physical” disruption of osmotic, oxidative and membrane stability which releases preformed and biologically active IL-1a which activates gene expression in the signaling pathways and results in the inflammatory, apoptotic and growth responses.

Schematic of the irritant effects of JP-8

We are currently exploring pharmacological manipulation of the irritant cascade in the epidermis by epidermal injections of antagonists to some of the signaling molecules.  We will preadminister the antagonists before the irritant chemical exposure and look at changes in mRNA and protein levels when compared to the non pretreated skin.  We expect that we will be able to use this as a tool to better understand the very early events in the irritant cascade.

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Systems biology models

Another tool that will help in the understanding of the process of chemical-induced irritation is a systems biology model for the specific IL-1/ IL-6 inflammatory pathway (McDougal et al. 2005). This project is in collaboration with the USEPA’s National Center for Computational Toxicology.

We have developed a preliminary kinetic model of the interleukin 1 (IL-1)-stimulated intracellular signaling pathway in epidermal keratinocytes as an initial effort toward the pharmacodynamic modeling of the skin.  On exposure to external stimuli, such as chemical irritants, the skin secretes various cytokines and chemokines and evokes a cascade of events in the subcutaneous tissue.  Therefore, the pharmacodynamic process of the skin primarily involves the responses of the skin cells to these endogenous proteins.  Among them, one of central importance is IL-1, a proinflammatory cytokine that mediates the host defense activities of the skin.  The model captures the series of biochemical events initiated from IL-1a binding to IL-1 receptor (type I) on the cell surface that activates the transcriptional factor nuclear factor (NFK-B) and leads to production of a responsive protein, IL-6.
The future for us in this area is to integrate the pharmacological manipulation studies with the systems biology models to provide useful tool for understanding and predicting the molecular biology of chemical-induced irritation in the epidermis.

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Reference List

Baker, W., Dodd, D., McDougal, J. N., and Miller, T. E. Repeated Dose Skin Irritation Study on Jet Fuels - A Histopathological Study. AFRL-HE-WP-TR-1999-0022. 1999. Wright Patterson AFB, Air Force Research Laboratory. (link to Baker tr 1999)

Gunasekar, P. G., Rogers, J. V., Kabbur, M. B., Garrett, C. M., Brinkley, W. W., and McDougal, J. N. (2003). Molecular and histological responses in rat skin exposed to m-xylene. J. Biochem. Mol. Toxicol. 17(2), 92-94.PM:12717741

Kabbur, M. B., Rogers, J. V., Gunasekar, P. G., Garrett, C. M., Geiss, K. T., Brinkley, W. W., and McDougal, J. N. (2001). Effect of JP-8 jet fuel on molecular and histological parameters related to acute skin irritation. Toxicol. Appl. Pharmacol. 175(1), 83-88.PM:11509030

McDougal, J. N., Garrett, C. M., Amato, C. M., and Berberich, S. J. (2006). Effects of brief cutaneous JP-8 fuel exposures on time-course of gene expression in the epidermis. Toxicological Sciences doi: 10.1093/toxsci/kfl154.http://toxsci.oxfordjournals.org/

McDougal, J. N., Grabau, J. H., Dong, L., Mattie, D. R., and Jepson, G. W. (1997). Inflammatory damage to skin by prolonged contact with 1,2-dichlorobenzene and chloropentafluorobenzene. Microsc. Res. Tech. 37(3), 214-220.PM:9144633

McDougal, J. N., Zheng, Y., Zhang, Q., and Conolly, R. (2005). Biologically Based Pharmacokinetic and Pharmacodynamic Models of the Skin. In Dermal Absorption Models in Toxicology and Pharmacology (J.E.Riviere, Ed.), pp. 89-112. Taylor and Francis, London UK.

Rogers, J. V., Garrett, C. M., and McDougal, J. N. (2003). Gene expression in rat skin induced by irritating chemicals. J. Biochem. Mol. Toxicol. 17(3), 123-137.PM:12815608

Rogers, J. V., Gunasekar, P. G., Garrett, C. M., Kabbur, M. B., and McDougal, J. N. (2001a). Detection of oxidative species and low-molecular-weight DNA in skin following dermal exposure with JP-8 jet fuel. J. Appl. Toxicol. 21(6), 521-525.PM:11746201

Rogers, J. V., Gunasekar, P. G., Garrett, C. M., and McDougal, J. N. (2001b). Dermal exposure to m-xylene leads to increasing oxidative species and low molecular weight DNA levels in rat skin. J. Biochem. Mol. Toxicol. 15(4), 228-230.PM:11673852

James N. McDougal
Office and Lab: 216 Health Sciences
Phone: (937) 775-3697
Fax: (937) 775-7221
E-Mail: james.mcdougal@wright.edu