Estimation of proinflammatory biomarkers of skin irritation by dermal microdialysis following exposure with irritant chemicals.

The aim of the present study was to quantify the release of proinflammatory biomarkers by dermal microdialysis after topical exposure with irritant chemicals, Jet fuel (JP-8) and xylene in rat skin. Occlusive dermal exposure (2h) was carried out with 230microl of JP-8 or xylene using Hill top chambers((R)). Linear microdialysis probes (10mm) were inserted in the dermis under urethane anesthesia. The dialysis fluid was pumped at a flow rate of 2microl/min and the dialysate was collected for 7h following probe insertion. The expression of substance P (SP), calcitonin-gene related peptide (CGRP) and prostaglandin E(2) (PGE(2)) in the dialysate following microdialysis was measured by enzyme immunoassay (EIA). The effect of pretreatment with an SP antagonist (SR-140333) and a PGE(2) inhibitor (celecoxib), 6 and 18h before the application of JP-8 was also assessed to further establish the sensitivity of the microdialysis set up. On similar lines, untreated and capsaicin treated control experiments were performed to compare with the SP release following JP-8 treatment. Further, we also investigated the SP release following topical application of xylene. The mean concentrations of SP after the application of JP-8 (90.01+/-3.31) and 3h after its removal (58.66+/-9.36) indicated that JP-8 induced significantly higher release of SP as compared to the baseline value (P<0.05). The release of SP following JP-8 treatment (58.66+/-9.36pg/ml) was comparable to capsaicin (58.18+/-11.29pg/ml). JP-8 exposure resulted in a significant increase (P<0.001) in PGE(2) levels over the baseline control at the end of 1 and 2h of exposure. JP-8 treatment also produced significant increase (P<0.001) in PGE(2) levels as compared to the untreated control during occlusion and 1h following its removal. There was a significant drop (P<0.05) in the PGE(2) levels by the end of 3h following exposure. Pretreatment with SR-140333 and celecoxib significantly reduced (P<0.05) SP and PGE(2) release induced by JP-8. The mean concentrations of SP following xylene exposure (25.50+/-8.80pg/ml) and 3h after its removal (34.37+/-5.61pg/ml) indicated its skin irritation potential. Unlike JP-8, xylene produced a significant increase in SP release only after the removal of occlusion. Pretreatment with SR-140333 significantly blocked the xylene induced SP release. CGRP was not detected in any of the samples. This study demonstrates that dermal microdialysis can be used to quantify skin irritation potential of JP-8 and related irritant chemicals.

Effect of methyl substitution of benzene on the percutaneous absorption and skin irritation in hairless rats.

The permeation rate and skin retention of benzene and methylbenzenes were assessed in vitro using hairless rat skin. The effects of unocclusive dermal exposures of these chemicals (15 microl every 2h for 8h a day for 4 days) on the transepidermal water loss (TEWL), erythema and skin histopathology were measured in CD hairless rats. The expression of IL-1 alpha and TNF-alpha in the skin and blood were measured at the end of dermal exposures. The flux of benzene was about 1.5-, 2.5- and 80-fold higher than toluene, xylene and tetramethyl benzene isomers (TMB), respectively, and the values were inversely correlated with molecular weight (r(2)=0.7455) and logoctanol-water partition coefficient (r(2)=0.7831). The retention of chemicals in stratum corneum (SC) was in the order of TMB>xylene>toluene approximately benzene. The TEWL and erythema data demonstrated that the irritation was in the following order: TMB>xylene>benzene. The histo-pathological examination showed that xylene and TMB induced granulocyte infiltration, swelling of the epidermis, and extensive disruption and damage of stratum corneum. Likewise, the expression of IL-1 alpha in the blood and TNF-alpha in the skin after dermal exposures was higher for TMB followed by xylene and benzene compared to control. In conclusion, the aromatic hydrocarbon chemicals induced cumulative irritation upon low-level repeat exposures for a 4-day period and the irritation increased with the number of methyl groups of benzene. The affinity of the chemical to SC and their gradual accumulation in the skin in the present study is the reason for the differences in the skin irritation profiles of different aromatic chemicals. Our ultimate goal is to develop a biologically based model that connects skin retention of chemical to the skin irritation response. The findings of the present study will be helpful in understanding the role of these chemicals in the jet fuel and various petroleum based fuels in inducing skin irritation response.

The effect of occlusive and unocclusive exposure to xylene and benzene on skin irritation and molecular responses in hairless rats.

Aromatic hydrocarbons readily penetrate the skin on dermal exposure, leading to irritation, inflammation and cytotoxicity. The effects of short-term occlusive and long-term unocclusive dermal exposure to benzene and xylene on the skin irritation response (transepidermal water loss (TEWL), skin moisture content and erythema) and cytokine/chemokine expression (interleukin-1alpha (IL-1alpha), tumor necrosis factor-alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1)) were investigated in hairless rats. Occlusive dermal exposure was carried out with 230 microL of the chemicals for 1 h using Hill top chambers. In unocclusive dermal exposure, 15 microL of the chemicals were applied to the skin every 2 h, for 8 h a day, for 4 days. The occlusive dermal exposure revealed a clear difference in the TEWL and erythema response of these chemicals (xylene>benzene) whereas unocclusive exposure revealed similar TEWL and erythema scores for both benzene and xylene. The expression of IL-1alpha was elevated 2.5- and 3.8-fold in response to occlusive and unocclusive exposure, respectively, vs control (P<0.01) for both the chemicals (benzene and xylene). Similarly, TNF-alpha levels were elevated about 2.4- and 6.0-fold as a result of occlusive and unocclusive exposure, respectively, vs control (P<0.01). These results show that unocclusive exposure induced significantly higher TNF-alpha expression than occlusive exposure (P<0.05). The MCP-1 expression in blood was slightly elevated compared with the control group, but this increase was not statistically significant (P>0.05). Similarly, MCP levels in skin were increased approximately 1.7- and 1.8-fold by occlusive and unocclusive exposure, respectively, compared with the control group (P<0.05). Our study demonstrates that the skin irritation profiles of benzene and xylene are similar and unocclusive long-term exposure to small amounts of these chemicals can induce more skin irritation and cytokine response than occlusive exposure.

Percutaneous absorption and skin irritation upon low-level prolonged dermal exposure to nonane, dodecane and tetradecane in hairless rats.

Even though the dermal toxicity of hydrocarbon fuels has been well established in the literature, there is little information available on the dermal penetration kinetics and irritation potential of the individual hydrocarbons. The penetration and skin retention of nonane, dodecane and tetradecane was assessed in vitro using hairless rats' skin. The effects of unocclusive dermal exposures of these chemicals (15 microL every 2 h for 8 h a day for four days) on the transepidermal water loss (TEWL) and erythema were measured in CD hairless rats. The expression of interleukin 1alpha (IL- 1alpha) and TNF-alpha in the skin and blood were measured at the end of dermal exposures. The flux of dodecane was 3- and 77-fold higher than nonane and tetradecane. The retention of chemicals in stratum corneum (SC) was in the order of tetradecane > dodecane > nonane, and directly correlated to the log Kp (r2 = 0.9900) and molecular weight of the chemicals (r2 = 0.8782). The TEWL and erythema data indicate that irritation was in the following order: tetradecane > dodecane > nonane. Likewise, the expression of IL-lalpha in the blood and TNF-alpha in the skin after dermal exposures was higher for tetradecane followed by dodecane and nonane compared to control. In conclusion, the aliphatic hydrocarbon chemicals of the present study induced cumulative irritation upon low-level repeat exposures for a four-day period. The affinity of the chemicals to SC and their gradual accumulation in the skin in the present study is the probable cause for the differences in the skin irritation profiles of different aliphatic chemicals. The findings of the present study will be helpful in understanding the skin irritation response of the chemicals in humans; indeed the reality check arises from dermal exposures in humans and human experience in occupational handling of these chemicals.