Carbon-monoxide poisoning in young drug addicts due to indoor use of a gasoline-powered generator.

We report six fatal cases of unintentional carbon-monoxide poisoning which occurred in a house occupied by young people. The source of carbon monoxide was a gasoline-powered generator. For all victims, an external body examination was carried out and blood and urine samples collected. Blood carboxyhaemoglobin (COHb) was performed using an automated visible spectrophotometric analysis. Blood-alcohol level quantification was performed using gas chromatography and drug screening in urine was performed by a one-step manual qualitative immunochromatography (Syva Rapid test, Behring Diagnostics Inc.) for benzoylecgonine (the main metabolite of cocaine in urine), morphine, 11-nor-Delta(9)-THC-9-COOH (cannabinoids) and d-methamphetamine. In all victims the COHb value was as high or higher than 65%. No alcohol was found in blood samples, but urine samples were positive for methamphetamine, cocaine and cannabis in five cases and for opiates in one case. In four victims, the urine sample was positive for at least three drugs. The availability and accuracy of rapid toxicological screening is an important tool for the medical examiner at the immediate scene of a clinical forensic examination.

Suicide by electrocution with low-voltage current.

Three cases of suicide by electrocution with low-voltage current were observed in five years (1994-1998) by medical clinical forensic examiners of an Emergency Forensic Unit of the Paris suburb among 2,000 external death examinations. The cases involved one woman, aged 72 and two men, aged 38 and 41. In the last two cases, electric burns were retrieved under bared electric wires, placed on the arms or fingers in order to realize a hand-to-hand electric circuit involving the heart muscle. In the other case, the electric circuit between mouth and foot also involved the heart muscle. Household low-voltage current delivered (220 V in France) had a sufficient strength to induce local muscular paralysis and heart fibrillation. In the three cases, blood samples taken have retrieved very high levels of muscular enzymes (CPK, LDH) correlated to the mechanism of electric death. The rareness of suicide by electrocution and its forensic characteristics are detailed in order to help the clinical forensic examiners, prosecutors, and police officers concerned by such death examinations.

Influence of endothelial cells on structure, biochemistry and functionality of epidermis reconstructed on synthetic porous membrane.

The model of keratinocytes cultured on a synthetic porous membrane at the air-liquid interface leads to the formation of a pluristratified and cornified epidermis with histological and biochemical characteristics near those observed in vivo. In the present study, we evaluated the effect of proliferative endothelial cells on epidermalization. Keratinocytes were grown in three culture conditions: in defined medium (DM; control), in medium previously conditioned by proliferative endothelial cells (CM) and in medium with proliferative endothelial cells (pEC). The structures of reconstructed epidermis were analyzed by electron microscopy, their biochemistry by DNA, protein and cytokine analyses and finally their functionality was evaluated by estradiol and water absorption testing. Ultrastructural analysis showed a well-developed and cornified epidermis for each culture condition. In addition, living epidermis was thinner in the presence of endothelial cells, revealing faster epidermal differentiation. DNA and protein analyses were in accordance with these results. Secreted soluble factors varied according to culture conditions. At 37 degreesC, the permeability of reconstructed epidermis in DM, in CM or with pEC was 5- to 10-fold higher than that of native human epidermis with both tracers. Laminin coating of the inserts led to similar absorption results except for the DM where the barrier function to estradiol was decreased 2-fold. At 32 degreesC, reconstructed and native epidermis were, respectively, 1.5- and 2-fold less permeable to estradiol compared to 37 degreesC. In conclusion, this model is adequate for fundamental and pharmacological studies since it allows the study of interactions between two cell types without their direct contact as well as percutaneous absorption tests directly performed in the modified culture chamber.

Protective effect of curcuminoids on epidermal skin cells under free oxygen radical stress.

Curcuminoids from Curcuma longa L. (Zingiberaceae) protected normal human keratinocytes from hypoxanthine/ xanthine oxidase injury. Since curcuminoids synergistically inhibited nitroblue tetrazolium reduction, a decrease in superoxide radical formation leading to lower levels of cytotoxic hydrogen peroxide was proposed as an explanation for this protective effect.

Importance of sebaceous glands in cutaneous penetration of an antiandrogen: target effect of liposomes.

The significance of the sebaceous gland pathway in the cutaneous permeation of an antiandrogen, 4-[3-(4-hydroxybutyl)-4,4-dimethyl -2,5-dioxo-1-imidazolidinyl]-2-(trifluoromethyl)benzonitrile (RU 58841), was studied with normal hairless rat skin and an induced scar hairless rat skin without sebaceous glands. RU 58841 was dissolved in an alcoholic solution and encapsulated in liposomes for comparison. After 24 h, the cumulative percentage of RU 58841 absorbed in vitro was 3-4-fold higher in the normal skin than in the scar skin; in the case of liposomes, the accumulation of the drug in the normal dermis was significantly higher than in the scar one. In the in vivo cutaneous distribution, the epidermis and dermis of the normal skin contained higher amounts of RU 58841 than the scar skin (ninefold with the solution and 16-fold with liposomes). An autoradiography study showed that with the solution, the drug was mainly localized in the stratum corneum/epidermis, and with the liposomes, the drug was mainly localized in the sebaceous glands. We concluded that the sebaceous glands constituted the main pathway for RU 58841. The alcoholic solution encouraged the localization of the drug into the stratum corneum, whereas liposomes targeted the sebaceous glands.

Barrier function of reconstructed epidermis at the air-liquid interface: influence of dermal cells and extracellular components.

To evaluate the epidermal barrier function of in vitro reconstructed epidermis, we measured the penetration of estradiol and water across human keratinocytes cultured in defined medium (DM), in the presence of proliferative fibroblasts (pF) or conditioned medium derived from pF, at the air-liquid interface on synthetic porous membrane, noncoated or coated with laminin, fibronectin, type I collagen or type IV collagen. Ultrastructural analysis showed a well-developed stratum corneum whatever the culture conditions. The permeability of reconstructed epidermis in DM on a noncoated porous membrane was 5- to 10-fold higher than human native epidermis, with both tracers. No significant change in barrier function was observed whatever the culture conditions.

Comparison of six different methods to assess UVA cytotoxicity on reconstructed epidermis. Relevance of a fluorimetric assay (the calcein-AM) to evaluate the photoprotective effects of alpha-tocopherol.

A three-dimensional culture of human keratinocytes exposed at the air-liquid interface has been developed and used in conjunction with fluorimetric, colorimetric and radioligand incorporation assays to assess the in vitro toxicity of UVA. The aims of the study were: (1) to compare the relevance of the neutral red uptake (NR), MTT metabolism, (35)S-methionine incorporation, IL1-alpha release and calcein-AM esterification assays for the evaluation of UVA injury; (2) to test the preventive protective effect of an emulsion containing 3% of tocopherol applied on the reconstructed epidermis, in comparison with an application of tocopherol 3% diluted in culture medium either on the apical compartment or in the underneath compartment of the skin culture insert. Viability measurement methods are based on different endpoints. None of the five endpoints measured produced LD(50) values (40 J/cm(2)) that differed significantly from the others. However, calcein-AM assay was relatively more reproducible and easier to handle than the others, and seemed to be a better choice for the evaluation of the protective effects of the tocopherol emulsion. Tocopherol diluted in culture medium under the epidermis 24 hr before irradiation failed to protect the epidermis against UVA damage, whereas diluted in culture medium or in oily emulsion and applied to the epidermis reduced cellular death (cellular recovery values are, respectively, 24% and 21%). Since cosmetic or pharmaceutical formulations can be directly applied on the reconstructed epidermis as in vivo, this model in combination with a fluorescent viability assay appears to be a suitable approach for pharmaco-toxicological evaluations.

Human epidermis reconstructed on synthetic membrane: influence of experimental conditions on terminal differentiation.

Cell suspensions of human keratinocytes seeded onto cell culture inserts may undergo terminal differentiation in the absence of fibroblasts. Among the parameters that control these morphogenic events, exposure to air and the composition of the culture medium were investigated. In the latter case, three media were considered DMEM:Ham's F12, MCDB 153, and keratinocyte SFM medium at equivalent calcium (1.5 mM) and fetal calf serum (5%) concentrations. Immunochemical methods and transmission electron microscopy show that cells cultured in DMEM:Ham's F12 medium, and then raised at the air-liquid interface, form a basal layer plus suprabasal cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasal keratinocyte layers show morphologies that resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-filaggrin granules. When the cultures are kept submerged, the keratinocytes show occasional keratohyalin granules and are connected by fewer desmosomes. Additionally, no proper stratum corneum is formed. In keratinocyte SFM medium and MCDB 153, cultures raised at the air-liquid interface are not able to form an epithelium of normal architecture and do not express terminal differentiation markers. Differentiation is initiated, however, since desmosomes and bundles of keratin filaments appear; on the other hand, filaggrin is not expressed even after 28 d in culture. Membrane-bound transglutaminase is expressed throughout the entire suprabasal compartment in MCDB153 and DMEM:Ham's F12 media but never appears in keratinocyte SFM medium. These studies show the relative independence of epidermal differentiation program to the composition (including the calcium concentration) of the media contacting the dermis and filling the extracellular space.(ABSTRACT TRUNCATED AT 250 WORDS)

A new three-dimensional culture of human keratinocytes: optimization of differentiation.

Many attempts have been made to obtain reconstructed human epidermis comprised of keratinocytes and extracellular-matrix constituents (essentially collagen) in the presence or absence of fibroblasts. A simple model of cultured human keratinocytes, grown at the air-liquid interface of a noncoated artificial membrane, has been developed. This culture system offers many advantages: easy control of environmental factors and routine examination using optical or electronic microscopy, immunohistochemistry and indirect immunofluorescence techniques. This model enables the analysis of well-known differentiation markers and also integrins, a family of cell-surface molecules involved in cell-cell and cell-extracellular matrix interactions, whose receptors are expressed on all basal keratinocytes. In our culture system, the expression of the different integrin subunits (alpha 2, alpha 3, alpha 5, alpha 6, beta 1) was studied as a function of the differentiation state in two different media (K-SFM or DMEM/Ham's F12) supplemented with 5% fetal calf serum and adjusted to 1.5 mmol/L calcium. The most significant data are the preponderant expression of the alpha 2 and alpha 3 subunits in the basal and suprabasal layers, with membrane expression differing according to the culture medium; terminal differentiation was obtained in DMEM/Ham's F12. The use of membrane inserts represents a significant technological advance in culturing keratinocytes and is an easy-to-handle and valid model for determining the influence of physiological or pharmacological factors on cell proliferation or differentiation.

Influence of fibroblasts on epidermization by keratinocytes cultured on synthetic porous membrane (insert) at the air-liquid interface.

Culture of keratinocytes on a noncoated porous synthetic membrane maintained at the air-liquid interface allows the establishment of a fibroblast/keratinocyte co-culture, without direct cell-cell contact between the two cellular layers. The influence of fibroblasts (proliferating, confluent or blocked by mitomycin C) on epidermization (i.e., expression of integrins and markers of epidermal differentiation) was studied by immunohistochemistry in two culture media. In the medium supplemented with FCS or Ultroser G and in the absence of fibroblasts, alpha 2, alpha 3, alpha 5 and alpha 6 subunits of integrins are expressed by the basal keratinocytes, except alpha 5 which does not appear with the medium supplemented with Ultroser G. During stratification, the alpha 3 subunit is the only one to persist on suprabasal cells and all the markers of epidermal differentiation studied (filaggrin, involucrin, transglutaminase, keratins K1/K10) are expressed at the 14th day of emerged culture. The presence of fibroblasts modifies the expression profile of integrins: when they are proliferative, the expression of alpha 2 and alpha 6 chains is delayed in the medium supplemented with FCS, and the alpha 6 chain is absent in the medium supplemented with Ultroser G; when they are confluent or blocked by mitomycin C, greater changes are observed only in the medium supplemented with Ultroser G and lead to inhibition or delay of the expression of alpha 2 and alpha 6.(ABSTRACT TRUNCATED AT 250 WORDS)

Percutaneous absorption of estradiol and progesterone in normal and appendage-free skin of the hairless rat: lack of importance of nutritional blood flow.

Percutaneous absorption occurs after passive diffusion through the different layers of the skin and its appendages. Thereafter, a resorption process into the cutaneous microcirculation brings the compounds into the systemic circulation. The objective of this in vivo study in the hairless rat was to compare the percutaneous absorption of two steroids on normal and appendage-free (scar) skin and to show if differences in absorption result only from the lack of hair follicles and sebaceous glands and/or from a modification of local blood flow. Percutaneous absorption was evaluated with estradiol and progesterone after 30 min, 2 and 6 h. Except after 30 min, the reservoir function of the stratum corneum of scar skin was approximately twice as high as in normal skin. Eighty to ninety percent of the estradiol and progesterone found in the stratum corneum were located in its superficial layers. Inversely, whatever the application time was, the concentrations of both steroids in the epidermis and dermis were significantly higher in normal skin than in scar skin with maximal difference between about 40 and 400 microns, the area of sebaceous gland localization. Cutaneous blood flow in full-thickness skin, assessed by the thallium-201 method, was globally identical in normal and in scar skin. In scar skin, at the level of papillary dermis, a decrease in blood flow due to the thicker viable epidermis and the flat dermoepidermal junction has been shown without implying an accumulation of drug in the epidermis and superficial dermis. Under these conditions, our results clearly demonstrate that the nutritional blood flow does not interfere with the percutaneous absorption of estradiol and progesterone in normal and scar skin. Thus, they confirm the significant contribution of hair follicles and sebaceous glands to drug penetration into the skin and subsequently the systemic circulation.

Role of transepidermal and transfollicular routes in percutaneous absorption of steroids: in vitro studies on human skin.

Percutaneous absorption theoretically comprises two components: the transepidermal and the transfollicular routes. The aim of the present work was to confirm this hypothesis in the human skin by comparing the in vitro percutaneous absorption of four steroids through scar skin without hair follicles and sebaceous glands and through normal adjacent skin from abdominal or mammary plasties. In all cases, the absorption of the four steroids was significantly higher in normal skin than in scar skin. The cumulative percentages of progesterone and testosterone after 8 h of application were, respectively, 3.1- and 2.4-fold higher in normal skin than in scar skin. After 24 h of application, the cumulative percentages of estradiol and hydrocortisone were 1.7- and 2.4-fold higher in normal skin than in scar skin. At the end of the experiments, the quantities of drugs remaining in the skin after 8 or 24 h of application were the same in normal skin and in scar skin except for progesterone for which they were 2-fold greater in normal than in scar skin. In each case, a histological characterization of the scar skin was made in comparison with the normal adjacent skin. The main modifications observed on scar skin were the following: absence of hair follicles and sebaceous glands, thinning of the collagenous fibers with parallel orientation to the dermoepidermal junction and decrease in the number or disappearance of the elastic fibers. These experiments confirmed that human skin appendages, hair follicles and sebaceous glands, constitute a route of penetration for steroids and thus probably for other chemicals of similar molecular weight and properties.

Activity of Vittel water on proliferation of human fibroblasts, proliferation and differentiation of human keratinocytes.

Synopsis The effects of Vittel water on the proliferation of fibroblasts, proliferation and differentiation of keratinocytes from human origin were studied. To determine the relative importance of calcium and other elements of the mineral water, cultures were raised in a low-calcium medium (low-Ca medium), in a medium prepared with Vittel water (Vittel medium) and in a medium containing an identical calcium concentration to that of Vittel medium (Ca medium). The fibroblasts and keratinocytes were cultured in immersion for proliferation assays and on a reconstructed epidermis at the air-liquid interface to evaluate keratinocyte differentiation. Vittel medium decreased proliferation of keratinocytes when compared to low-Ca medium. The effect was similar to that of Ca medium at the beginning of the experiment, but significantly higher at day 7. A stratified epithelium appeared with the three types of media when keratinocytes were incubated at the air-liquid interface; however the number of sheets was more regular and greater in Vittel medium and Ca medium than in low-Ca medium. Filaggrin and transglutaminase expression appeared earlier with Vittel medium than with the other media. After 2 weeks, expression of markers was similar in the three media. After 3 weeks culture in Vittel medium, there was a greater expression of filaggrin. Proliferation of young fibroblasts was significantly higher in Vittel medium than in Ca medium. It was lower in low- Ca medium. With old fibroblasts the degree of proliferation was lower than with young fibroblasts. The augmentation of proliferation happen earlier in Vittel medium than in low-Ca medium and Ca medium. Vittel medium regulated the growth rate of old fibroblasts, rendering it identical to that of young fibroblasts in low-Ca medium. The effects of Vittel water were not linked to the sole presence of calcium since, with medium at an equimolecular concentration in calcium, the medium containing Vittel water had a better activity. One explanation of these effects of Vittel water might be the presence of magnesium.

Free-radical damage by UV or hypoxanthine-xanthine oxidase in cultured human skin fibroblasts: Protective effects of two human plasma fractions.

Two in vitro methods to investigate free radical damage to cultured human skin fibroblasts have been used: irradiation with UVA or UVB, producing intracellular free radicals and DNA damage, or free radical production by the enzymatic system hypoxanthine-xanthine oxidase, releasing free radicals into the culture medium. These methods differ not only in the location of the free radicals generated but also in their nature and kinetics. The antioxidant properties of two human plasma extracts A and B (derived from Cohn's fraction IV and Cohn's fraction I + III) were investigated before, during and/or after the oxidative stress. Protection was observed when the fractions were added concomitantly with the enzymatic system (at 5 g/litre, fractions A and B exhibited, respectively, 77 and 50% activity) or during UVB irradiation (37 and 68% activity for fractions A and B, respectively at 5 g/litre). A small degree of protection was observed against UVA damage. No preventive or restorative effect was observed with the UVB system. The two fractions prevented UVA damage (at 2.5 g/litre, fraction A and B elicited 22 and 23% activity, respectively) but only fraction A also exhibited a restorative effect (at 5 g/litre, activity was 26%). One of the protective mechanisms could be the enhancement of intracellular antioxidant enzyme activity by incubation of cells with fractions A and B (after 24 hr of contact with fraction B, total glutathione peroxidase and glutathione peroxidase Se-dependent activities were significantly increased by 60 and 42%, respectively, compared with control values).

Role of the appendageal pathway in the percutaneous absorption of pyridostigmine bromide in various vehicles.

We studied the percutaneous absorption of [14C]-labelled pyridostigmine bromide mixed into various vehicles through normal and appendage-free scar rat skin, in vitro during 72 h. At the end of the experiment, the percentages of the drug absorbed were higher for nerol 8% in ethanol (respectively 78.4 +/- 3.6% and 72.8 +/- 4.5% on normal and scar skin) and azone 5% in ethanol-propylene glycol (90:10) (respectively 76.4 +/- 4.4% and 57.2 +/- 7.1% on normal and scar skin). Propylene glycol 10% in ethanol inhibits pyridostigmine absorption: 9.9 +/- 2.6% and 2.2 +/- 1.2% vs 14.7 +/- 3.8% and 5.5 +/- 5.1% with ethanol on control and scar skin. The transappendageal pathway seems to be less important for nerol (55% to 82% of the absorption routes between 4 h and 32 h) and azone (60% to 79% of the absorption routes until 32 h) than for propylene glycol (63% to 96% of the absorption pathways during the whole experiment), dimethylsulfoxide (about 78% during the first 32 h) and ethanol (more than 50% during most of the time). These results show that it is possible to increase or decrease the percutaneous absorption, as well as to modulate the relative importance of the transepidermal route and the transfollicular pathway.

Cutaneous bioavailability in hairless rats of tretinoin in liposomes or gel.

Topical bioavailability of drugs incorporated in liposomes is not well known. We compared the skin penetration of tretinoin in liposomes and in a classical alcoholic gel. [3H]Phosphatidylcholine dipalmitoyl (DPPC) and [14C]tretinoin (0.14%) were incorporated in the phospholipidic phase of the liposomes, and [14C]tretinoin was incorporated in a gel for comparison. Skin absorption was studied in vitro with Franz cells. In vivo distribution in cutaneous structures was studied according to Schaefer's method. Liposomes impregnated the stratum corneum, with a partial dissociation between tretinoin and phosphatidyl-choline dipalmitoyl. In dermis, tretinoin diffused alone. Tretinoin release seemed to be controlled, and steady state was reached later with liposomes than with gel. This phenomenon was linked with a significantly reduced absorption (1.60% for liposomes versus 3.09% for the gel) and higher retention in epidermis (mainly stratum corneum) and dermis (41 and 13%, respectively, with liposomal form versus 18 and 8%, respectively, with gel form). This study clearly shows that, compared with the gel, the liposome formulation tends to improve the local effect of tretinoin in the skin and decrease the systemic absorption.

Role of transepidermal and transfollicular routes in percutaneous absorption of hydrocortisone and testosterone: in vivo study in the hairless rat.

The importance of the transappendageal route on percutaneous absorption was assessed in the hairless rat. Skin permeation of two steroids, hydrocortisone and testosterone, was evaluated in vivo on normal and artificially damaged skin in which follicles and sebaceous glands disappeared during healing. The test compounds were applied for periods of 0.5, 2 and 6 h. Thereafter, the stratum corneum reservoir function, the epidermal and dermal distribution profiles, and systemic absorption were determined for both molecules. The results presented here show that the reservoir function of the stratum corneum of appendage-free (scar) skin is more pronounced than that of normal skin, whereas the concentration appearing in the epidermis and dermis was greater in normal skin. Moreover, sebaceous glands probably contribute to the penetration of hydrocortisone and testosterone. We show that the relative importance of the skin appendages depends on the intrinsic physical properties of the molecules tested, and the time of application.

Prediction of eye irritancy potential of surfactants by cytotoxicity tests in vitro on cultures of human skin fibroblasts and keratinocytes.

The cytotoxicity of surfactants was evaluated on cultures of human skin fibroblasts and keratinocytes in order to predict their eye irritancy potential taking into account both immediate cytotoxicity after 2 hr of incubation and delayed cytotoxicity 24 hr after such incubation. The immediate cytotoxicity ranking of the surfactants, evaluated by MTT or neutral red assay after 2 hr of exposure in minimum Eagle's medium (MEM) without or with 10% foetal calf serum (FCS), was identical for both cell types. Keratinocytes were less sensitive than fibroblasts to all surfactants apart from Tween; however, cytotoxicity ranking remained the same for both cell types. The addition of 10% FCS to MEM reduced the cytotoxicity of surfactants, without modifying the ranking. Comparison of immediate and delayed cytotoxicity enabled the reversibility of cellular functions to be assessed by calculation of cell recovery rate (CRR). CRR values were positively related to cytotoxicity ranking for all surfactants except Brij. No correlation was found between EC(50) values of immediate or delayed cytotoxicity, under various experimental conditions, and ocular irritation scores in vivo. Brij surfactants, reported not to be irritant in vivo, showed a high degree of toxicity in the cell culture systems; however, a good correlation was found when analysis was carried out excluding these surfactants. In contrast, there was excellent correlation between CRR and ocular irritation scores in vivo, for all surfactants tested, including Brij.

Effect of biophysical changes on propidium iodide access to DNA during oxidative stress of cultured human skin cells.

The sensitive single-cell analytical techniques of flow cytometry and propidium iodide-binding have been used to examine the molecular effect of oxidative stress on cultured human skin fibroblasts. Cells synchronized by limited time attachment were exposed to a hypoxanthine-xanthine oxidase (HX/XO) system at different intervals after subculture. The characteristic feature of the treated population was a variation of the amount of nuclear DNA propidium iodide (PI)-fluorescence staining. Increased fluorescence intensity was observed with a shift of the G1/G0 and G2/M peak, which is dependent on both cell cycle stage and treatment level. When scavenger molecules (catalase, silybin) were added to the oxidative reaction, the nuclear DNA histogram of HX/XO-treated cells was similar to that obtained from untreated cells. In parallel, UV absorbance studies in vitro have shown that PI is capable of binding extensively to DNA when isolated from HX/XO-exposed cells, compared with control cells or HX/XO-exposed cells in the presence of scavengers. These results indicate that free radicals are responsible for the increase in fluorescence intensity in the HX/XO-exposed cells. This change in DNA stainability would be due to an opening of the DNA strands in situ, leading to an unmasking of new PI-binding sites on DNA. The strand separation may facilitate access on the fluorochrome to DNA, thereby enhancing dye binding. This flow cytometric assay based on DNA biophysical changes caused by free radicals is a useful means of measuring pro- and/or anti-oxidant potential.

Follicles play an important role in percutaneous absorption.

The relative importance of stratum corneum and follicles in percutaneous absorption is not fully understood. In order to quantitatively investigate the importance of the transappendageal route, we have previously developed a model of skin without follicles regrown dorsally on the hairless rat. Percutaneous absorption was compared, using a diffusion cell, in appendage-free skin relative to normal skin, and a predominant role of follicles for in vitro diffusion of [3H]hydrocortisone was noted. Results presented here of in vitro diffusion of tritiated hydrocortisone, niflumic acid, caffeine, and p-aminobenzoic acid, applied in acetone, confirm that appendageal diffusion is the major pathway in hairless rat skin. In the absence of follicles, the steady-state flux and the amounts diffusing in 24 or 48 h are 2-4 times lower than in normal skin. These results were confirmed in a second model in which diffusion of [3H]hydrocortisone was studied on skin samples taken one day after birth, at which time rat skin is still devoid of follicles, relative to five-day postnatal skin samples, in which follicles are fully developed. The steady-state flux and the total diffusion in 24 h were fivefold lower in follicle-free skin. These results support the view that follicles may have a far greater importance in percutaneous absorption than is generally assumed.