Midori Sakamoto

Response of murine epidermal keratinocyte cultures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD): Comparison of haired and hairless genotypes☆

Based on the observation that congenic haired and hairless mice differ in the hyperkeratinizing/hyperproliferative epidermal response following topical exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo, it has been proposed that this response in mice segregates with the Ah locus (which controls for the Ah receptors) and the hr locus (for hairlessness) (Knutson and Poland, 1982). In the present studies this hypothesis was tested by comparing the response of epidermal keratinocytes derived from genotypically segregated newborn haired and hairless HRS/J mice to TCDD exposure in in vitro cultures. the parameters monitored were stimulation of cell proliferation (as measured by protein content and rate of tritiated thymidine incorporation in cultures), epidermal transglutaminase activity, cornified cell envelope formation, and keratin staining with Rhodanile blue. Results suggested that the sensitivity of HRS/J haired and hairless mouse epidermal keratinocyte cultures to TCDD exposure in vitro was very similar. In both cell cultures all parameters monitored were stimulated by TCDD exposure in a similar dose-dependent manner. This suggests that physiologic factors beyond the epidermal cells may be involved in expression of the different responses seen in the skin of mice to TCDD in vivo.

Vitamin A deficiency and the induction of cutaneous toxicity in murine skin by TCDD.

The mechanisms involved in the induction of toxicity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a prototype for a group of toxic polyhalogenated aromatic hydrocarbons, are largely unknown. To test the hypothesis that TCDD-induced toxicity involves the reduction of vitamin A levels, we investigated the role of vitamin A deficiency in modulating the cutaneous response of congenic haired (+/+) and hairless (hr/hr) mice to TCDD. Hairless mice are recognized as sensitive models for expression of TCDD-induced cutaneous toxicity. Haired mice normally do not develop a cutaneous response to TCDD. Mice raised on a vitamin A-deficient diet, and age- and sex-matched controls raised on standard chow, were treated topically with TCDD and their cutaneous responses monitored histologically. Body weights and thymus gland weights were monitored as additional parameters of toxicity. Liver and skin vitamin A levels were determined by HPLC. Vitamin A depletion by itself had no effect on the normal cutaneous histology of the haired phenotype, nor were any changes in cutaneous morphology attributable to TCDD toxicity observed in haired, TCDD-treated animals even when they were severely vitamin A depleted. On the other hand, in hairless mice, vitamin A deficiency caused a distinct increase in keratinization of dermal epithelial cysts, and an increase in the sensitivity of these cysts to TCDD-induced hyperkeratinization. TCDD-induced body weight loss and atrophy of the thymus gland were not affected by the vitamin A status of either the haired or hairless animals. Analysis of vitamin A levels in skin and liver, following topical treatment of mice with TCDD, indicated that TCDD exposure did not affect cutaneous levels, but did significantly lower liver levels of vitamin A. These experiments suggest that although systemic vitamin A deficiency may potentiate the expression of TCDD-induced toxicity in skin of hairless mice, expression of TCDD-induced toxicity probably involves more complex mechanisms than a reduction in vitamin A levels.

Effect of TCDD on the density of Langerhans cells in murine skin

Tetrachlorodibenzo-p-dioxin (TCDD) is a prototype for a group of toxic polyhalogenated aromatic hydrocarbons. We have studied the effect of TCDD on skin, specifically the difference in cutaneous response of congenic haired (hr/+) and hairless (hr/hr) mice. Topical application of 0.6 microgram of TCDD induces epidermal hyperplasia/hyperkeratinization in the skin of hr/hr mice, but does not affect the epidermis of congenic hr/+ littermates. Suppression of various parameters of the immune response has been found to be another effect of TCDD exposure in experimental animals. In the present study, we investigated the effect of topical treatment with TCDD on the density of epidermal immune cells, the Langerhans cells (LC), in the skin of hr/hr and hr/+ mice. Results showed that TCDD-induced epidermal hyperplasia/hyperkeratinization in skin of hr/hr mice is accompanied by an increase in the density of LC. In the skin of hr/+ mice, in which TCDD exposure does not induce hyperplastic changes, LC densities are not affected. The increase in LC densities in TCDD-treated hr/hr mouse skin did not result in increased sensitivity of the skin to contact hypersensitization with dinitrofluorobenzene, as measured by changes in ear thickness. When hr/hr murine skin was grafted into skin of hr/+ mice and the entire dorsal skin (including the graft) treated with TCDD, LC were increased in the grafted skin, but not in the surrounding hr/+ skin. Conversly, when hr/+ murine skin was grafted into hr/hr mice and both treated with TCDD, there was no increase in the density of LC in the grafted hr/+ skin. Concomitant treatment of hairless mice with TCDD and with indomethacin did not affect the increase in the density of LC induced by TCDD treatment alone. These findings suggest that TCDD-induced epidermal changes in hr/hr murine skin involve production of factors which mediate the increase in epidermal LC.

The hr locus and the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in newborn mice.

In mice, the recessive mutation hairless (hr) controls the cutaneous response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but its influence on TCDDs systemic toxicity is unclear. To clarify this, we compared the effects of lactational TCDD exposure on standardized litters of newborn HRS/J mice homozygous for either hr or +that were fostered by haired dams exposed to 0, 6, 8 or 12 μg TCDD/kg body weight on postnatal day 0. At 12 μg/kg, TCDD was lethal to both haired and hairless pups. At the lower doses (6 and 8 μg/kg) the survival of hr/hr pups was significantly lower than +/+ pups. Affected pups succumbed following a 1 to 2-day period of cachexia and wasting. As has been reported for other mouse strains, TCDD exposure impacted on their neonatal development and lessened the time to eye opening for both haired and hairless pups. However, the hairless animals were affected at lower doses than were the haired. The results of this study document that the hr/hr genotype does influence the systemic toxicity of TCDD in mice.