Limitations of hairless mouse skin as a model for in vitro permeation studies through human skin: hydration damage

Hairless mouse skin currently provides a popular model membrane for studies in human percutaneous absorption. Although some similarities between the two skin types have been demonstrated, the effects of prolonged hydration on hairless mouse skin have not previously been rigorously examined. We have measured in vitro the effects of hydration at 31 degrees C on the permeabilities of hairless mouse skin and human abdominal and scalp skin to a model polar compound (water) and a lipid material (hexanol). The permeability of hairless mouse skin rose dramatically, especially to water (fiftyfold increase), whereas the human skin was more stable. We also compared the effects of stripping the stratum corneum with the effects of 8-d hydration for hairless mouse and human abdominal skin. Hydration of hairless mouse skin was as effective as tape-stripping in eliminating the stratum corneum barrier, whereas stripping human skin was far more damaging than hydration, suggesting that prolonged hydration mechanically disrupted mouse skin but not human skin. Histological examination of fresh and hydrated tissues confirmed this suggestion. We therefore recommend that hairless mouse skin is not used as a model for human tissue during in vitro permeation studies under conditions of long-term hydration, i.e., greater than three days.     

Permeation of chromium salts through human skin in vitro

Chromium permeation studies were performed on full thickness human skin in diffusion cells. All samples were analysed for the total chromium content by graphite furnace Zeeman-corrected atomic absorption spectrometry. Some samples were analysed by an ion chromatographic method permitting the simultaneous determination of Cr(VI) and Cr(III) as well. The amounts of chromium found in all skin layers were significantly higher when potassium dichromate was applied to the skin compared with chromium chloride or chromium nitrate. Chromium could only be detected in the recipient phase after application of the dichromate solution. Chromium skin levels increased with increasing concentrations of applied chromium salts up to 0.034 M Cr. The amount of chromium in recipient phase and skin layers increased with increasing pH when the applied solution contained potassium dichromate. This was ascribed to a decreased skin barrier function of the skin. The amount of chromium found in all skin layers after application of chromium chloride decreased with increasing pH due to lower solubility of the salt. The % of chromium found in the recipient phase as chromium(VI) increased with increasing total chromium concentration indicating a limited reduction ability of the skin in vitro.     

Permeation of nickel salts through human skin in vitro

The impact of occlusion and the role of the counter ion on the permeation rate of nickel ions ill rough skin have been examined using excised human skin. A highly sensitive electrochemical method was used to quantify the amount of nickel permeating the skin. The investigations show that nickel ions are capable of permeating the skin barrier when applied under occlusion. The process is slow, having a lag time of approximately 50 h. The permeation rate, and thus the physical amount being bioavailable is considerably increased when aqueous nickel chloride is used in comparison with aqueous nickel sulphate With respect to nickel permeation of the skin, we conclude that the choice of salt is an important consideration in patch testing.     

In vitro percutaneous penetration: evaluation of the utility of hairless mouse skin

The permeability barrier of hairless mouse skin has been determined in vitro after exposure of the epidermal surface to volumes of acetone typically used in human in vivo skin penetration studies. It has been shown that the transport of tritiated water (when applied for limited 5-h periods) across hairless mouse skin is not affected by acetone treatments of approximately 15 microliters/cm2. Submersion of the membranes between aqueous donor and receptor phases for periods greater than 24 h, however, leads to significant and catastrophic barrier impairment. The acetone dose in the experiments reported is greater than that employed in vivo when the solvent is used to deposit a penetrant on human skin. We suggest, therefore, that acetone-mediated facilitation of percutaneous absorption in humans is unlikely. A further conclusion of this work is that in vitro solvent-deposition penetration experiments using hairless mouse skin should provide reliable transport information for at least 48 h postadministration. Although hairless mouse skin is more permeable than its human counterpart, in vitro measurements using the murine barrier should, therefore, provide useful and relevant guidelines for risk assessment calculations and bioavailability determinations.     

An ultrastructural study of ultraviolet-induced elastic fiber damage in hairless mouse skin

Ultrastructural visualization of mouse elastic fibers has been hampered by the small size and sparseness of the fibers. Furthermore, with conventional uranyl acetate and lead citrate stains, the elastin matrix remains electron-lucent so that even severe elastosis is elusive. The addition of tannic acid to the staining procedure renders the elastin matrix extremely electron dense and easily located even at low scanning power. Microfibrils, as with conventional stains, are electron-dense and clearly visible. This study describes, with the aid of tannic acid, normal hairless mouse elastic fibers and their progressive degeneration with increasing exposure to ultraviolet radiation. Results with the two staining methods are contrasted. In addition, photodamaged mouse and human elastic fibers are compared for differences and similarities.     

Percutaneous penetration of squaric acid and its esters in hairless mouse and human skin in vitro

Summary Squaric acid diethylester and squaric acid dibutylester have been used in contact sensitization therapy of alopecia areata. This study investigated the application of these esters or squaric acid alone to hairless mouse and human skin in vitro to determine squaric acid flux from the various preparations. Measurable amounts of squaric acid were delivered through skin by squaric acid itself, but flux was lower than for that delivered by the two esters. These results support the proposal by Noster that the esters combine with a protein to form an antigen while squaric acid can not and that this explains why the esters are active in contact sensitization and the acid is not. We suggest that the results of previous studies showing that the diethyl ester of squaric acid was a less effective sensitizer than the dibutyl ester may have been due to decomposition of the ethyl ester to squaric acid.     

UVA- and UVB-induced changes in hairless mouse skin collagen

UVA- and UVB-induced alterations in dermal collagen were investigated in a murine animal model. Groups of hairless mice were exposed to UVA and UVB for 28 weeks at a dose of 60 J/cm² three times weekly and 0.06 J/cm² three times weekly, respectively. Untreated animals were used as controls. Every 4 weeks dorsal skin was examined for quantitative and qualitative changes in dermal collagen. Neither UVA nor UVB caused a significant alteration in total skin collagen content. However, after UVA treatment the ability of skin collagen to be digested by pepsin decreased dramatically (up to 65% of skin collagen remained insoluble after 4 months), whereas exposure to UVB had no significant effect. Furthermore a shift in the ratio of 1(I,III) chains to 2(I) chains was detected after UVA exposure. The amount of type V collagen in mouse skin, as determined by a sensitive ELISA method, was markedly decreased after UVA treatment, but not after UVB treatment.This work was presented in part at the Jahrestagung der Arbeitsgemeinschaft Dermatologische Forschung 1988     

Changes in arachidonic acid metabolism in UV-irradiated hairless mouse skin

This study was conducted to investigate the metabolism of arachidonic acid in the skin of hairless mice exposed to UVA, PUVA, UVB, and UVC irradiation. The main products of arachidonic acid in the epidermis were hydroxyeicosatetraenoic acid (HETE), PGE2, and PGD2. Dermis displayed a lower lipoxygenase activity (expressed as HETE production) than the epidermis and showed no detectable cyclooxygenase activity, i.e., no prostaglandin production. The main changes observed in UV-induced inflammatory reactions were as follows. 1. A 5-fold increase in dermal HETE production in PUVA-treated animals and a 29% reduction in epidermal HETE formation after UVC treatment. 2. A marked decrease of PGD2 and a marked increase of PGE2 formation due to alterations of PGH2 metabolism in the UVB-treated group; however, cyclooxygenase activity was unchanged. These changes in arachidonic acid metabolism in the skin may be of pathophysiologic importance in UV-induced inflammatory reaction.     

Metabolism of prednisolone 21-acetate in hairless mouse skin.

We investigated the hydrolytic activity of prednisolone 21-acetate (PNA) to prednisolone (PN) in an enzyme solution composed of esterase and skin homogenates from hairless mice. The values of the Michaelis-Menten constant obtained from hairless mouse skin and esterase solution were 14.2 and 10.2 microM, respectively; conversely, the value of the maximum rate from hairless mouse and esterase solution were 0.67 and 1,886 nmol/min/mg protein, respectively. To examine the effect of enzymatic inhibitors on hydrolytic activity, five enzymatic inhibitors, 3,4-dichloroisocoumarine (DCIC), N-tosyl-L-phenylalanine chloromethyl ketone, iodoacetamide, p-hydroxymercuribenzoic acid (HMBA) and sodium dodecylsulfate, were added to the enzyme solution. Sixty-eight percent of hydrolytic activity in skin homogenates were not deactivated by DCIC which completely inhibited the enzymatic activity in esterase solution. We also studied the localization of hydrolytic enzyme with a subcellular faction: 66 and 11% of specific activity existed in microsome (Ms) and cytosol (Cp) fractions, indicating that the hydrolytic activity of PNA was included mainly in the Ms fraction. Hydrolytic activity in Ms and Cp fractions was different from sensitivity to enzymatic inhibitor; DCIC inhibited activity in the Ms fraction and, on the other hand, HMBA inhibited it in the Cp fraction. Therefore, Ms and Cp fractions in skin homogenates include a different esterase isoform and the metabolism of PNA to PN in hairless mouse skin is mediated by these isoforms.     

Diseased skin models in the hairless guinea pig: in vivo percutaneous absorption

We observed hydrocortisone and benzoic acid absorption in relation to experimentally induced in vivo damaged skin models in the hairless guinea pig. Radioactivity of the drugs in urine was calculated as absorbed dose. Both drugs have different patterns of excretion in urine. Damaging the skin abolished some barrier function and increased the absorption of both model drugs. With cellophane-tape-stripped skin, the absorption was increased 3 times and 2 times for each drug, respectively. Irritation with 2% sodium lauryl sulfate increased absorption by a ratio of 2-4 times. Defatting with chloroform/methanol (2:1) mixture increased absorption to the greatest extent (5- and 2.7-fold). The possible mechanism of a delipidization effect was considered in view of a visible skin lesion and inflammatory reaction. Precautions are proposed for those with damaged/diseased skin in dealing with topical medications or handling solvents.     

Specific induction of metallothionein in hairless mouse skin by zinc and dexamethasone

The stimulation of metallothionein (MT) synthesis in mouse skin after i.p. treatments with various doses of dexamethasone or zinc was demonstrated. Specific MT mRNA induction was determined by Northern analysis. Zinc was a more efficient inducer than dexamethasone. The maximal MT accumulation occurs after i.p. injections of 50-100 mg dexamethasone/kg body weight. The possible role of MT in the skin is briefly discussed.     

UVB-induced collagen changes in the skin of the hairless albino mouse

Biochemical techniques have been used to measure ultraviolet-B (UVB)-induced changes in dermal collagen composition. Hairless albino mice were irradiated dorsally with a daily dose of 62 mJ/cm2 UVB for 12, 24, 30, and 36 weeks. Nonirradiated controls were housed under identical conditions. Additional groups were irradiated for similar periods and kept for a further 6-24 weeks without irradiation. Skin samples were taken from dorsal and ventral (nonirradiated) surfaces and types I and III collagen were quantified densitometrically after cyanogen bromide digestion and polyacrylamide gel electrophoresis. Type III collagen was expressed as a percentage of the total types I and III collagen and the ratio of dorsal/ventral type III (D/V III) was determined for each mouse. The ratio increased significantly in irradiated animals whereas it decreased in the corresponding period in control animals. In irradiated mice withdrawn from UV exposure the ratio D/V III tended to revert to control levels. These data are in agreement with those of our previous human studies, which showed an increase in type III collagen in sun-exposed skin when compared with covered sites.     

Topical estrogens: their effects on connective tissue synthesis in hairless mouse skin

Skin is an important target organ for estrogens. The major reported effects of estrogens are as regulators of connective tissue molecules, namely collagen and hyaluronic acid. We investigated the regulation of connective tissue synthesis by topical estrogens in a hairless mouse model of photodamaged skin, which has been previously shown to respond to topical retinoids. The naturally occurring estrogen, 17beta-estradiol (17beta-E) and a close stereoisomer, 17alpha-estradiol (17alpha-E), were found to be as effective as all- trans-retinoic acid in stimulating the development of new connective repair zones in photodamaged skin. Furthermore, 17beta-E and 17alpha-E caused a skin thickening response in normal hairless mouse skin after three daily treatments. Skin thickening is due to water accumulation as a result of estrogen-induced hyaluronan synthesis. Our results show that topical estrogens are important regulators of connective tissue synthesis in photodamaged skin as well as normal skin. These findings are consistent with reports from human studies in which estrogen has been found to stimulate collagen production. We also demonstrated that 17alpha-E, previously thought to be a weak or inactive estrogen, is less potent than 17beta-E, but nonetheless topically effective in stimulating connective tissue synthesis.     

Skin barrier disruption by acetone: observations in a hairless mouse skin model

To disrupt the barrier function of the skin, different in vivo methods have been established, e.g., by acetone wiping or tape-stripping. In this study, the acetone-induced barrier disruption of hairless mice was investigated in order to establish a reliable model to study beneficial, long-term effects on barrier recovery after topical application. For both treatments (i.e., acetone treatment and tape-stripping) the transepidermal water loss directly after disruption and the subsequent barrier recovery profile were similar. Histological assessment showed significant lower number of corneocyte layers in acetone-treated and tape-stripped skin compared to untreated skin, while there was no statistical difference between the two treatments. Lipid analysis of acetone-treated skin revealed that only small fraction of lipids were extracted consisting of predominantly nonpolar lipids. Importantly, the ratio of the barrier lipids, i.e., cholesterol, free fatty acids and ceramides, remained similar between control and acetone-treated skin. This reflects the undisrupted lipid organization, as determined by small-angle X-ray diffraction measurements: the long-periodicity lamellar phase was still present after acetone treatment. Our results contradict earlier studies which reported no mechanical stratum corneum removal, a substantial extraction of lipids and disruption in lipid organization. In conclusion, our studies demonstrate that barrier disruption due to acetone treatment is mainly due to removal of corneocytes.     

Determination and correlation of in vitro viability for hairless mouse and human neonatal whole skin and stratum corneum/epidermis

Background and design: Viable tissue is essential to assess the rate and extent of biotransformation during percutaneous absorption in vitro. We assessed the viability of hairless mouse whole skin (WS) and stratum corneum/epidermis (SCE) and human neonatal SCE following separation from the dermis by EDTA phosphate-buffered saline (EDTA-PBS) incubation or by heat treatment by measuring the conversion of dextrose to lactate. Lactate concentrations in receptor fluid samples were determined using a Sigma diagnostic lactate determination kit. A standard curve was prepared and samples assayed spectrophotometrically at 340 nm using a lambda 2β spectrophotometer. Standard curves were prepared for each experiment and correlation coefficient values ( r ) were calculated. Results: Our results showed that heirless mouse SCE was associated with glucose conversion to lactic acid at an increased rate if incubated in EDTA-PBS for 4 h and used immediately. Lactate production was greater with the dermis present (EDTA-PBS WS). The rate of glucose to lactate conversion in hairless mouse SCE was 20–25% of that found in WS. Compared with Dulbecco’s modified PBS (DMPBS)-treated WS controls, the rate of lactate production in EDTA-PBS-treated WS was nearly a 50% less. Heat treatment in water at 60° C to separate SCE from hairless mouse WS appeared to eliminate viability. Viability of hairless mouse SCE, as measured by glucose conversion to lactate, was comparable to human neonatal SCE. Conclusions: These results suggest that the dermis is a significant contributor to glucose metabolism and that incubation in EDTA-PBS is a contributing factor to the overall decrease in metabolic capacity of the tissue. As a result of these findings, hairless mouse SCE appears to be useful as a model for human neonatal SCE in percutaneous absorption studies. Received: 1 September 1995     

Development of an in vivo animal model for skin penetration in hairless rats assessed by mass balance

The aim of the study was to develop an in vivo animal model for studies of the penetration of topically applied drugs into the skin of hairless rats. Protective appliances were designed for non-occluded and finite-dose application of topical formulations. The design allowed 2 test sites for each rat and free mobility throughout the test period. By consecutive tape stripping, monitored by measurements of transepidermal water loss and confirmed by histological examination of skin biopsies, 10 tape strippings were found to remove the stratum corneum completely. For assessment of the model, (14)C-salicylic acid and (14)C-butyl salicylate were topically applied. Rapid and differentiated percutaneous absorption of both compounds were shown by urinary excretion data. For (14)C-salicylic acid the amount on the skin surface, in the stratum corneum and in the viable skin was determined. Total mass balance on the applied radioactivity was performed and a recovery of 90 +/- 2% was achieved. The radioactivity found in the protective appliances (<10%) was explained by lateral skin diffusion of the model compounds into the dressings.