Skin penetration and metabolism of topically applied chemicals in six mammalian species, including man: An in vitro study with benzo[a]pyrene and testosterone

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

t&502-5

16 (1985)

Skin Penetration and Metabolism of Topically Applied Chemicals in Six Mammalian Species, Including Man: An in Vitro Study with Benzo[a]pyrene and Testosterone’ JOHN KAO,

FRANCES K. PATTERSON,’

AND JERRY HALL

Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 Received April 26, 1985; accepted July 22, I985 Skin Penetration and Metabolism of Topically Applied Chemicals in Six Mammalian Species, Including Man: An in Vitro Study with Benzo[a]pyrene and Testosterone. KAO, J., PATTERSON, F. K., AND HALL, J. (1985). Toxicol. Appl. Pharmacol. 81,502-5 16. Percutaneous absorption of chemicals is generally considered a diffusional process, with the rate-limiting barrier being the nonviable stratum comeum. Because viable skin possessesenzyme activities, including those involved in the metabolism of xenobiotics, the extent to which cutaneous metabolism may influence the percutaneous fate of topically applied chemicals in the skin was examined in mammalian skin maintained as short-term organ cultures. Skin samples from mouse, rat, rabbit, guinea pig, marmoset, and man were examined. The results from studies with benzo[a]pyrene (BP) and testosterone showed that, in all species, metabolic viability was a major factor involved in the in vitro skin permeation of surface-applied chemicals. Permeation was accompanied by extensive cutaneous “first pass” metabolism; both parent compounds and a full spectrum of metabolites were found in the receptor fluid from viable skin preparations. However, in previously frozen nonviable skin preparations, essentially only unchanged parent compounds were detected in the receptor fluid. Permeation of BP and testosterone was highest in mouse skin, and significant species variations in the metabolite profiles were observed. Studies with mourn skin also demonstrated that induction of cutaneous drug-metabolizing enzymes can result in a two- to threefold increase in the in vitro permeation of topical BP, and a significant reduction in permeation was observed when KCN was added to the perfusion medium. These results indicate that diffisional and metabolic processesare intimately involved in the percutaneous fate of surface-applied chemicals. The relative importance of these pmceasesis dependent upon the physicochemical properties of the compounds and the metabolic capabilities of the skin toward the compounds in question. Furthermore, these findings suggest that meaningful in vitro studies on skin absorption should consider both diffusion and cutaneous biotransformation of the applied compound. o 1985 Academic Press, Inc.

The major barrier to skin absorption is generally considered to be the stratum corneum (Marzulli, 1962; Scheuplein, 1977; Dugard, ’ Research was sponsored by the Othce of Health and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc. The U.S. Government’s right to retain a nonexclusive royalty-free license in and to the copyright covering this paper, for governmental purposes, is acknowledged. ’ Present address: University of Tennessee, Memorial Hospital Research Center, Knoxville, Tenn. 37920. 0041-008X/85 Co-t All r&ha

$3.00

0 1985 by Academic Pm% Inc. of reproduction in any form merwd.

502

198 1; Scheuplein and Bronaugh, 1983). This outermost layer of the skin is composed essentially of dead cells containing keratin; consequently, the skin has frequently been thought of as a passive, inert barrier covering the body, and percutaneous absorption of chemicals was thought to be essentially a diffusional process. Although cutaneous “first pass” metabolism of chemicals such as benzoyl peroxide (Nacht et al., 198 1) and nitroglycerin (Wester et al., 1983) has been suggested, in general, the con-

PERCUTANEOUS

ABSORPTION

sideration of the skin as merely a diffisional barrier persists, despite the fact that the skin is an organ system active in many metabolic functions, including those involved in the metabolism of xenobiotics (Pannatier et al., 1978; Noonan and Wester, 1983; Bickers, 1983). These metabolic processes in the skin could play an important role in the percutaneous fate of topically applied substances. However, to what extent cutaneous biochemical processesmay contribute to the physiological disposition and toxicity of chemicals following topical exposure remains to be investigated. Previous studies in our laboratory have demonstrated that in mouse skin maintained in organ culture, the in vitro penetration and cutaneous metabolic fate of topically applied benzo[a]pyrene (BP) was significantly influenced by the biochemical viability and metabolic status of the cultured skin (Smith and Holland, 1981; Holland et al., 1984; Kao et al., 1984). The results strongly implicated cutaneous metabolism as an important factor in the percutaneous fate of surface-applied chemicals. In this paper we continue to examine the interrelationship of the skin as a drug-metabolizing organ and a primary portal of entry for chemicals. The in vitro penetration and percutaneous fate of topically applied BP and testosterone in the skin of six mammalian species, including man, maintained as shortterm organ cultures, are described and compared. In addition, the influence of biological viability and the effects of a metabolic inhibitor and of enzyme inducers are reported. METHODS Animals and Preparation of Skin Samples Species used in these studies included mouse (C3H, C57BL/6, and DBA/2, male, 9 to 10 weeks old), rat (CDF, female, 12 to 16 weeks old), guinea pig (Hartley, male, 4 to 6 months old), rabbit (New Zealand white, male, 4 to 6 months old), and marmoset (male, 8 years old). The animals were housed under climate-controlled conditions (20°C 50% humidity, 0700 to 1900 hr light period), on conventional bedding with free accessto water and &andard laboratory chow. The backs of the animals were shaved with electric clippers prior to experimentation. For

IN SIX MAMMALS

503

mice this was carried out at least 24 hr prior to termination, and only those mice in the resting phase of their hair cycle were used. For the other species, shaving was done after the animals were killed. Animals were killed by carbon dioxide asphyxiation, their dorsal skin was excised, the subcutis was carefully removed, and circular full-thickness skin disks (l-in. diameter) were cut out as described previously (Kao et al., 1983). One skin disk was cut from each mouse, 4 to 6 disks were cut from each rat and marmoset, and 10 to 15 disks were cut from each rabbit and guinea pig. The skin disks were placed, epidermal side up, in petri dishes (5 to 6 disks/dish) containing culture medium (1 to 2 ml) and kept at room temperature until needed (approx. 1 hr). The culture medium was minimum essential medium with Hepes buffer (25 mM) and Hanks’ salts without ~-&damine (Gibco, Grand Island, N.Y.) containing fetal calf serum (10% v/v. Gibco) and gentamicin (0.1 mg/ml, Gibco). Immediately following surgical amputation, a freshly excised sample of human skin from the leg was obtained from the Pathology Department of the University of Tennessee, Memorial Hospital Research Center, Knoxville, Tennessee. For transportation from the hospital to the laboratory, the skin was wrapped in surgical gauze saturated with the above culture medium and kept in a sterile container at ambient temperature. In the laboratory fullthickness skin disks were prepared and maintained as described above. The whole process of transporting and preparing the human skin disks was achieved in under 2 hr. In studies with mouse skin where the effectsof induction of cutaneous drug-metabolizing enzymes were examined, animals were pretreated in vivo by ip injection of the inducers. The enzyme inducers used were 3methylcholanthrene [3-MC, 25 mg/kg for 3 consecutive days (Sigma, St. Louis, MO.)] and 2,3,7,8-tetrachlorodibenzcqdioxin [TCDD, 40 &kg (KOR Inc., Cambridge, Mass.)]. Control animals were treated in parallel and given an equivalent amount of the vehicle ip (corn oil at 1% v/ w body weight). 3-MC- and TCDD-induced animals were killed 24 and 48 hr, respectively, following pretreatment, and skin disks were prepared as described above. Skin Organ Culture and in Vitro Viability Determination Two culture systemswere used: the “dynamic” system (Holland et al., 1984) for studies on the kinetics of in vitro skin absorption and the “static” system (Kao et al., 1984) primarily for studies in cutaneous metabolism. Briefly, in the dynamic system,the skin disks form the upper seal of wells of a compact, water-jacketed multisample skin penetration chamber. Freshly oxygenated culture medium was continuously perfused through the wells and the well effluents were collected at timed intervals with a fraction collector. In the static system, individual skin disks, sup ported on filterpaper circles (Whatman 40, 4.5 cm), were placed onto a stainless-steel supporting ring in a culture

504

KAO, PATTERSON,

dish (60 X 15 mm, Falcon 3017) containing culture medium (12.0 ml). The supporting fdter paper was saturated with culture medium and in contact with the medium throughout the culture period, also, the surface of the skin remained above the level of the culture medium. Both culture systemsused minimal essential medium containing Earle’s salts with L-glutamine and Dvaline in place of Lvaline (Gibco), fetal calf serum ( 10% w/v), and gentamicin (0.1 mg/ml). The cultures were kept at constant temperature (36 to 37’C) and the medium was gassed throughout the incubation period with a 5% CO*, 40% Or, and 55% N2 mixture. To ascertain the viability and structural integrity of the cultured skin samples histological sections were prepared, stained with hematoxylin and eosin, and examined by light microscopy. Additionally, the ability of the cultured skin to metabolize [14C]glucose to 14COr was examined and used as an indicator of in vitro biochemical viability. The details of this determination have been reported previously (Kao ef al., 1984). Kinetics of in Vitro Penetration of Topically Applied Chemicals: Studies with the Dynamic System For in vitro kinetics of skin penetration studies, [‘H]BP [[G-‘H]BP (40 Ci/mmol, Batch H14; Amersham, Arlington Heights, Ill.) diluted with BP (Gold Label, Aldrich Chemical Company, Milwaukee, Wise.)] and [3H]testosterone [[ 1,2,6,7-‘HItestosterone (92 Ci/fimmol, Batch 54, Ametsham) diluted with testosterone (Sigma)] were used. The radiochemical purity of these compounds as determined by thin-layer chromatography (TLC) was in excessof 95%. The dynamic system was set up with the appropriate skin preparation as described, and continuous perfusion of the culture medium was initiated. Following a period of acclimatization (approx. 1 hr), the radiolabeled compound (5 &skin disk, 7 to 10 pCi in acetone, 10 to 25 ~1) under investigation was applied topically to the cultured skin samples (available surface area 2 cm’). Aliquots of the effluent medium were collected at hourly intervals and assayedfor radioactivity. The cumulative radioactivity in the effluent medium provided a measure of the rate of in vitro skin penetration. Since skin samples which have been stored frozen are widely used in a multitude of in vitro skin penetration studies as acceptable tissue preparations (Franz, 1975; Bronaugh et al.. 1981; Swarbrick et al., 1982; Hawkins and Reifenrath, 1984), the influence of metabolic viability on percutaneous absorption was investigated by comparing the permeation of the selected tritiated compounds in freshly prepared skin samples with the corresponding skin preparations which had been previously frozen on dry ice and stored at -20°C for 24 to 48 hr. The frozen skin preparations were thawed and allowed to equilibrate at room temperature before use. To further assessthe metabolic aspect of skin absorption, in some experiments with mouse skin, KCN (0.8 mM) was introduced into the per-

AND HALL fusion medium a few hours after topical application of the selected compounds. The possible role of cutaneous biotransformation in skin penetration was examined by comparing the rate and overall penetration in skin samples obtained from control and induced mice. Skin penetration studies on control and test samples were routinely run in parallel and in duplicate. On completion of the experiments, the percent recovery of the applied dose was determined. The individual skin disks were washed in organic solvent; the skin samples were then solubilized by enzymatic digestion in pronase [lo m&5 ml in 0.1 mrvtTris buffer containing 0.2% w/v sodium dodecyl sulfate, pH 7.8, at 50°C (&lb&hem-Behring Corp., La Jolla, Calif.)] and the radioactivity associated with the skin digest was determined. Individual tissue weils of the penetration chamber were washed with 70% ethanol and residual radioactivity was determined. The sum of the radioactivity present in the various components (i.e., effluent medium, skin digest, and chamber wells) gave the total recovery of the applied radiochemical. A recovery in excessof 90% was routinely observed. In Vitro Cutaneous Metabolism and Disposition of Top ically Applied Chemicals: Studies with the Static System For metabolism studies, both freshly prepared and previously frozen skin samples from the selected species were examined. [7,10-‘4C]BP [29.7 mCi/mmol, Batch 38, (Amersham)] and [4-‘4C]testosterone [58 mCi/mmol, Batch 50, (Amersham)] were used. The radiochemical purity of these compounds was greater than 95% by thinlayer chromatography. Skin samples were treated topically with radiolabeled compounds (10 fig/20 ~1 acetone/skin disk) and cultured as described. After 24 hr in culture, the skin disks, filter-paper supports, and medium from individual cultures were analyzed for the distribution of radioactivity and the metabolite profiles of the compounds were determined. Generally, the radioactivity content of aliquots of the culture medium (0.2 to 1.Oml) and the filter-paper support were determined by liquid scintillation counting. The skin samples were solubilized by pronase digestion and analyzed for radioactivity; the sums of the 14Ccontent of the medium, filter paper, and skin digest were totaled for the percentage recovery of the applied dose. To determine the metabolite profile of the culture medium, amyl alcohol (200 ~1) was added to the medium and extracted with ethyl acetate (2 X 3 vol); radioactivity in the organic and aqueous phases was determined. The ethyl acetate extract was dried over anhydrous sodium sulfate, concentrated, and cochromatographed with authentic metabolites on HPTLC plates (silica gel G60 F254 (Merck, Darmstadt, Germany)] in the appropriate solvent system. Quantitative sample application to the HPTLC plates was achieved with a Linomat III applicator and chromatograms were developed in a Linear chamber (Camag, Mattenz, Switzerland). For BP metabolites, dihy-

PERCUTANEOUS

ABSORPTION

drodiols, phenols, and quinones (NCI, standard chemical carcinogen reference repository), the solvent system was toluene/methanol(9/ 1, v/v) and the metabolites were localized by visualizing the developed chromatograms under uv light. For testosterone, the reference metabolites included 4-androstane-3,1 ‘I-dione, epiandrosterone, 5a-dihydrotestosterone, androsterone, Sfl-dihydrotestosterone, and 5cu-androstane-3,17dione (Sigma), and the solvent systemwas cyclohexane/ethylacetate (4/3, v/v). To localize testosterone metabolites, the developed chromatograms were sprayed with 5% v/v H,S04 in methanol and heated to 120°C for 5 to 10 min. For quantification, the areas on the chromatogram corresponding to the authentic metabolites were appropriately located; these areas were marked and scraped, and their radioactivity content was determined by liquid scintillation counting. The radioactivity associated with each of these areas was expressed as a percentage of the total radioactivity recovered from the radiochromatogram, and the metabolite profiles were determined as a percentage of the total radioactivity in the culture medium. In a similar manner, the metabolite profiles of [‘%]BP and [‘%]testosterone in the skin digests were determined and expressed as a percentage of the radioactivity in the skin digests.

RESULTS Viability of Full-Thickness in Organ Culture

Mammalian

Skins

Light microscopic examination of the mammalian skin explants, maintained under our culture conditions for 24 hr, showed the tissues, from all the species studied, to be viable, and the overall morphology was essentially unchanged. Species variations in the thicknesses of the cultured skin samples were evident; as estimated from the histologic preparations, the order of increasing thicknesses was mouse (0.34 * 0.0 I mm), marmoset (1.1 + 0.06 mm), rat (1.18 +- 0.03 mm), human (1.32 t- 0.05 mm), rabbit (1.52 rf: 0.06 mm), and guinea pig (1.81 f 0.04 mm). Also, in vitro topical exposure of these skin samples in culture to either BP or testosterone, at the quantities studied, did not result in any adverse visible morphological effects. As demonstrated by the liberation of 14C02 from [‘4C]glucose (Table l), freshly excised mammalian skin samples maintained in culture were biochemically viable. However, skin samples which had been previously frozen and

505

IN SIX MAMMALS TABLE 1

GLUCOSE UTILIZATIONBYCULTUREDSKINSAMPLES FROMVARIOUS SPECIESASANINDICATOROFBNXHEMICAL VIABILITY [‘4C]Glucose metabolism Species

Fresh, viable skin

Frozen, nonviable skin

Human Guinea pig Rabbit Rat Mouse

105.4 +_ 8.8 40.0 f 1.5 45.5 + 6.9 172.6 k 11.4 85 1.4 xk 60.3

0 8.3 * 4.1 4.9 k 0.5 0 12.8 f 1.6

Note. Skin samples maintained in the dynamic culture system for 16 hr were transferred individually to culture medium containing [‘4C]glucose. They were incubated for 3 hr and the liberated r4COz was trapped in hyamine hydroxide and subsequently assayed for radioactivity by liquid scintillation counting. Results are given as X + SE dpm/mg tissue from 5 to 10 individual skin samples.

stored at -20°C before being maintained in culture were biochemically nonviable; moreover, histological examination of these initially frozen and then cultured skin samples revealed extensive necrosis of the epidermis and adnexal structures. In Vitro Penetration of Topical BP and Testosterone in Mammalian Skin Maintained in the Dynamic Culture System With the recovery of radioactivity in the effluent medium as a measure of skin penetration, studies with the dynamic culture system showed significant species differences; more importantly, significant differences between biochemically viable and nonviable skin samples were observed. As shown in Fig. 1, the rate and overall translocation of in vitro topically applied [3H]BP (5 pg,/2 cm2) across rat and mouse skin were low and was dependent on the viability of the tissue. Permeation of [3H]BP was negligible in previously frozen nonviable skin preparations. In studies with rabbit and guinea pig skin, similar differences between viable and nonviable tissue trends

506

KAO, PATTERSON,

AND HALL

+

oa+

8

Od 6 iii

M

0

1

1

I

,

0

0510!52005lo1520

TlMEINCULTURE @ours)

TME INCULTURE (hows)

FIG. 1. Rate of in vitro penetration of topical [3H]BP in mammalian skin in organ culture: influence of tissue viability and the effect of KCN in the culture medium. Fresh (m) and previously frozen (0) full thickness skin samples maintained in the dynamic culture system were treated topically with [3H]BP (5 pg/ 2 cm*). In addition, for freshly prepared C3H mouse skin samples, 0.8 mM KCN (Cl) was added to the per&on medium 5; hr following topical application of [‘HJBP. Cumulative recovery of radioactivity in the perfusion medium, expressed as a percentage of the applied dose, was determined and results are given as i + SE (+) of 10 individual skin samples.

were observed. These differences may be the consequence of physical changes resulting from freezing and frozen storage. However, studies with C3H mouse skin have demonstrated that following the addition of KCN, a metabolic inhibitor, to the perfusion culture medium, the in vitro penetration of topically applied [3H]BP was significantly reduced (Fig. 1). Furthermore, in studies where skin preparations were obtained from inducible and noninducible strains of mice following ip pretreatment with 3-MC, the rate and overall in vitro permeation of topical [3H]BP were increased by two- to threefold in skin samples from the inducible C57BL/6 mice; whereas in skin samples from the noninducible DBA/2 mice, no changes were observed (Fig. 2). However, following pretreatment with the potent enzyme inducer TCDD, increased rates and overall in vitro penetration of i3H]BP were observed in the skin from both C57BL/6 and DBA/2 mice (data not shown). In contrast, similar penetration studies with [3H]testosterone (5 &2 cm2) showed extensive translocation of radioactivity across both viable and nonviable mammalian skin sam-

ples; moreover, as shown in Fig. 3, permeation through viable skin was lower than in nonviable skin samples. Also, the addition of KCN and the in vivo pretreatment with enzyme inducers (e.g., TCDD) produced no apparent changes in the in vitro penetration of topical [3H]testosterone in mouse skin (data not shown). Cutaneous Metabolic Fate of Topical BP and Testosterone in Mammalian Skin Maintained in the Static Organ Culture System Comparison between viable and previously frozen nonviable skin preparations. The cutaneous fate of [‘4C]BP and [‘4C]testosterone was examined by determining the distribution of radioactivity 24 hr after topical application to skin samples from six species. The total percutaneous translocation of the topically applied chemicals through the cultured skin was given by the combined radioactivity recovered from the culture medium and the filter paper saturated with culture medium. As shown in Fig. 4, the relative differences in penetration between viable and nonviable skin

PERCUTANEOUS

ABSORPTION

5,

IN SIX MAMMALS

507

lo

9 k 5

S” fi ii

0

0

051025200

TME INCULTwiE@w-s)

lMEsN aJLbRE (Eousg20

FIG. 2. Rate of in vitro penetration of topical [‘H]BP in skin samples from different mouse strains: influence of systemic induction with 3-MC. Inducible (C57BL/6) and noninducible (DBA/Z) mice were pretreated in vivo by ip injection with either corn oil (W) or 25 mg/kg 3-MC in corn oil (Cl) for 3 consecutive days. In vitro penetration of topical [3H]BP (5 pg/2 cm’) was determined by the dynamic system and results are given as described in Fig. 1.

samples were also apparent in the static system. Permeation of testosterone through mammalian skin was higher in nonviable tissue than in viable tissue; for BP, the converse was observed and penetration in nonviable skin preparations was found to be negligible. The overall translocation of surface-applied radioactivity from [ “C]testosterone in viable tissue was highest in mouse skin; about 70%

of the applied dose permeated through the skin in 24 hr. In rabbit and human skin samples, permeation was approximately 50%; while in rat, guinea pig, and marmoset skin samples, penetration was much lower, being less than 25% of the applied dose in 24 hr. For [‘4C]BP, permeation in viable tissue was again highest in mouse skin, being 10% of the surface-applied dose in 24 hr. In rabbit, rat, and mar-

20

lo

0 0

0

TitvtEkl Cd&E

(k~)~~

20

TiMEid C”LbRE &urs)

FIG. 3. Rate of in vitro penetration of topical [‘HItestosterone in mammalian skin in organ culture: influence of tissue viability. [3H]Testosterone (5 &2 cm*) was applied topically to full-thickness fresh (B) and previously frozen (0) skin samples in the dynamic system and results are given as described in Fig. 1.

KAO, PATTERSON,

508

TESTOSTERONE

AND HALL

KNZO(a)PYREM

I

#

0

x O~rn~~~!O~

&

INc&RED &Ju (+fib pcper;

FIG. 4. In vitro permeation of topical [‘%]Testosterone and [“C]BP in mammalian skin in organ culture: species differences and influence of tissue viability. Fresh (0) and previously frozen (@ full-thickness skin samples were treated topically with the “C-radiolabeled chemicals (10 &5 cm*) and maintained in the static culture system. Permeation, as judged by the percentage recovery of the surface-applied radioactivity in the culture medium plus filter paper, was determined 24 hr following topical application. The results are given as the X f SE of 4 individual skin samples. Permeation in previously frozen marmoset skin was not determined.

moset skin samples, permeation was 1 to 3% and only 0.1% of the applied dose permeated guinea pig skin preparations. The remainder of the applied radioactivity for both 14C-compounds was found in the cultured tissue. Total recovery of the applied dose in all six species was 83 to 99%. On examining the nature and distribution of the radioactivity recovered in the culture medium and the skin samples, major differences were observed between viable and nonviable tissue preparations. For example, analysis of the culture medium from human skin exposed topically to [14C]testosterone for 24 hr showed that essentially all of the radioactivity present in the culture medium from nonviable skin samples can be attributed to unmetabolized testosterone (Fig. 5). In contrast, testosterone accounted for only about 50% of the radioactivity in the culture medium from viable skin samples; various metabolites of testosterone made up the remaining 50% of the radioactivity present. Similar analysis of the human skin digest showed some 90% of the radioactivity present in the nonviable skin sample exposed topically to [‘4C]testosterone was the unmetabolized parent compound;

while in the viable tissue, 40% of the radioactivity was unchanged [ 14C]testosterone, and metabolites of testosterone accounted for the remaining 60%. Studies with human skin exposed topically to [14C]BP for 24 hr resulted in the same observations (Fig. 6). In the medium from nonviable skin samples, 90% of the radioactivity was extracted into ethyl acetate, and this was shown to be essentially unchanged BP. On the other hand, less than 50% of the radioactivity in the culture medium from viable skin samples was extractable into the organic solvent. Moreover, in the organic extract, all classes of metabolites of BP were found, with unchanged BP accounting for only 18% of the total radioactivity present in the culture medium. In the pronase digest of the nonviable human skin preparation, 87% of the recovered radioactivity was shown to be unmetabolized BP; whereas in the viable skin preparation, unchanged BP accounted for 57% of the radioactivity, the remainder being attributed to BP metabolites. Similar studies comparing viable and nonviable skin preparations from other species resulted in the same observations (data

PERCUTANEOUS 3 "1

ABSORPTION

HUN4N:abemedi1m

IN SIX MAMMALS

509

into organic solvent (Table 2). Approximately 50% of the radioactivity in the media from human and rabbit skin preparations remained in the aqueous residues following ethyl acetate extraction. From the media of rat, mouse, and marmoset skin preparations, the radioactivity retained in the aqueous residue amounted to approximately 70%. Chromatographic analysis of the ethyl acetate extracts showed that, in all six species, all major classes of BP metabolites were represented. BPdiols and “polar metabolites” predominated; a small amount (-2%) of BP phenols and quinones was found and, except human and perhaps rabbit skin preparations, negligible amounts of unmetab-

OL

FIG. 5. Metabolite profile of [‘%]testosterone in culture medium and skin digest from human skin in organ culture 24 hr after in vitro topical application of 10 pg to fresh (0) and previously frozen (M) skin samples. AQ, aqueous residue, radioactivity of which remained in the aqueous culture medium or skin digest following ethyl acetate extraction; POL, polar metabolites, radioactivity in the ethyl acetate extract which remained at or near the origin and migrated with R, values less than testosterone (T) on HPTLC plates (silica gel G60 F254, Merck) with cyclohexane/ethyl acetate (4/3 v/v) as solvent system;A-C, are radioactive peaks which migrated with R, values greater than testosterone (T) and cochromatographed with authentic metabolites of testosterone (see text). Results are expressed as a percentage of the radioactivity recovered in the culture medium and skin digest and are given as X f SE from 4 individual skin samples.

not shown). In nonviable skin samples, surface-applied BPand testosterone permeated through the skin preparation and appeared in the culture medium unchanged; while in viable tissue, both metabolites and the unchanged parent compound were found to be present in both the culture medium and skin digest 24 hr after topical exposure. Species variations in metabolite profiles. In all species studied, the majority of the radioactivity in the culture medium from skin samples dosed with [14C]BP was not extractable

FIG. 6. Metabolite profile of [“‘C]BP in culture medium and skin digest from human skin in organ culture 24 hr after in vitro topical application of IO pg to fresh (Cl) and previously frozen (M) skin samples. AQ, aqueous residue, radioactivity of which remained in the aqueous culture medium or skin digest following ethyl acetate extraction; POL, polar metabolites, radioactivity in the ethyl acetate extract which remained at or near the origin and migrated with Rrvalues lessthan BP dihydrodiols on HPTLC plates with toluene/methanoI (9/l v/v) assolvent system;DIOLS, BP dihydrodiols; OH, BP phenols; Q, BP quinones; BP, benzo[a]pyrene. Results are expressed as a percentage of the radioactivity recovered in the culture medium and skin digest and are given as 2 + SE for 4 individual skin samples.

Culture medium Skin Culture medium Skin Culture medium Skin

Culture medium Skin Culture medium Shin

Sample

8.30 k 0.60 13.16 + 2.40

+ 3.50

5.80 rt 1.95

1.21 + 0.52

72.20

5.79 -t 0.78

-t 3.20

8.39 f 0.27 5.45 + 0.46

10.63 zt 1.03

69.26

55.01 rk 2.18 20.15 5~ 2.69

1.27 f 0.17

3.39 + 0.45

8.07 -+ 0.81 6.63 + 0.66 6.33 + 0.25

-e 6.16'

POL

14.69 + 1.71 77.10 z!z0.93

51.78

AQ

5.13 f 0.68

15.40 + 1.40

4.14 k 0.42

10.99 + 0.93

15.29 rt 1.44 7.88 + 1.37

1.81 f 0.42

7.98 + 1.19

16.86 + 1.94 8.57 + 1.12

Diols

1.64 z!z 0.12

0.70 + 0.10

1.11 * 0.22

1.79 -+ 0.28

1.53 + 0.30 1.75 +_ 0.28

0.62 + 0.08

0.82 f 0.17

1.23 + 0.28 1.52 + 0.25

OH

Ethyl acetate extract

Metabolite urofile”

3.22 f 0.32

0.60 _+ 0.20

2.09 + 0.21 2.96 i 0.73

2.57 +_ 0.18

1.70 + 0.40

1.31 + 0.39

2.52 + 0.25 2.01 + 0.20 1.23 k 0.28

Q

+ 1.74

70.70

88.66

1.04

+ 4.93

0

+ 5.20

2.94 f

6.66 + 3.20 49.54 f 4.34

90.44

0.24 k 0.04

17.74 f 6.83 56.75 + 1.42

BW

102.56

97.18

108.61

92.86

88.57 87.34

98.84

93.69

98.13 90.18

1.82

samples,

+ 1.45

+ 4.93

+ 7.04

f

+ 1.30 f 2.77

f 0.33

f 1.57

f 2.11 + 5.12

Recovery

PROFILE OF [r4C]Benzo[a]pyrene IN SKIN AND CULTURE MEDIUM 24 hr FOLLOWING TOPICAL APPLICATION TO MAMMALIAN SKIN IN ORGAN CULTURE

a For explanation of abbreviations see legend to Fig. 6. * Results are expressed as a percentage of the radioactivity recovered in the culture medium and the skin 24 hr following topical application of 10 &skin and are given as the X k SE from 4 individual cultured skin samples.

Mouse

Rat

Rabbit

Marmoset

Human

Species

METABOL~TE

TABLE 2

/z

F

zi 0

wz

3

g

$ tl

F 0

PERCUTANEOUS

ABSORPTION

olized BP were present. The relatively large percentage ( 18%) of unmetabolized BP found in the medium from human skin samples suggests the possibility of inherent differences in the permeability of human skin compared with skin from other species studied. However, it is more probable that the increased penetration of unchanged BP in human skin can be attributed to the loss of some barrier function due to the extensive washing and scrubbing of the skin with antimicrobial disinfectant prior to surgical excision. Following pronase digestion, solvent partition of the skin digest showed that the majority of the radioactivity in the skin was extractable into ethyl acetate. A small percentage remained in the aqueous residues and accounted for a low of 3% from the marmoset skin to a high of 20% from the rabbit skin. Chromatography of the ethyl acetate extracts showed that the major fraction of the radioactivity in the skin digest from all species was attributable to unmetabolized BP, however, in all cases, various amounts of different classes of BP metabolites were detected. In studies with [‘4C]testosterone (Table 3) more than 80% of the radioactivity in the culture media from treated skin samples was extractable into organic solvent. Examination of the ethyl acetate extracts by thin-layer radiochromatography with cyclohexane/ethyl acetate (4/3, v/v) as the solvent system detected the presence of at least five radioactive peaks. A poorly resolved radioactive region was found at or near the origin of the chromatogram and is being described as constituting “polar metabolites” (POL). Peak T was identified as testosterone (R,-= 0.32). Peak A comigrated with 4-androstene-3,17-dione (RI- = 0.43); however, epiandrosterone and 5@-dihydrotestosterone also migrated with the same Rf values. Peak B comigrated with androsterone and 5a-dihydrotestosterone (Rf = 0.5 1) and peak C corresponded to 5a-androstane-3,17-dione (Rf = 0.66). The metabolite profile of [‘4C]testosterone in the culture medium showed considerable species variation. The percentage of radioac-

IN SIX MAMMALS

511

tivity remaining in the aqueous residues following ethyl acetate extraction ranged from a high of 18% for marmoset to a low of 4% for human skin. Unmetabolized testosterone constituted a significant fraction of the radioactivity in the culture medium from all six species. In the media from mouse and human skin preparations, the parent compound accounted for as much as 50% of the radioactivity while, in other species, less than 30% of the radioactivity can be attributed to testosterone. The remaining radioactivity in the medium was associated with biotransformation products of testosterone. In the media from marmoset and guinea pig skin preparations, peak A was shown to be the major metabolite(s), accounting for 39 and 55% of the radioactivity, respectively. In the media from human, rabbit, and rat skin samples, polar metabolites constituted the major metabolites and accounted for 3 1,52, and 50%, respectively. In the mouse, the relative proportions of polar metabolites and peak A were similar and accounted for 15% of the radioactivity present in the medium. Metabolites corresponding to peaks B and C accounted for the rest of the radioactivity and, depending on the species, ranged from 2 to 14% of the radioactivity recovered in the culture medium. Solvent partition of the skin following pronase digestion showed that approximately 90% of the radioactivity to be in the ethyl acetate fractions with the major component demonstrated by chromatography to be unchanged testosterone. The proportion of unchanged [‘4C]testosterone found in the skin digest ranged from a low of 22% in mouse skin to a high of 60% in rat skin; the remaining radioactivity was associated with various metabolites of testosterone. In human skin, the major metabolite, accounting for 24% of the radioactivity, corresponded to peak B; whereas peak A constituted the major metabolite(s) in the skin preparations from marmoset and guinea pig, and accounted for, respectively, 25 and 2 1% of the radioactivity in the skin digest. Major metabolites in mouse skin included peak A, 19%, and peak C, 24%, while, in rat

Skin

Culture

Skin

Culture

Skin

Culture

medium

medium

medium

k 1.52

7.36 rt_ 0.76

9.81 + 1.52

0.51 _+ 0.13

14.01 + 0.79

13.32 + 1.93

13.38 + 0.64 I? 2.28

+ 2.41

+ 3.68

7.39 * 0.13

15.27 + 1.65

10.78 + 0.61

49.71

24.95

51.87

8.25 + 0.48

5.20 + 1.08

8.28 f 2.26

4.52 f 0.49 6.67 f 0.15

18.46 + 1.69

f 2.71

14.25 f 1.05

30.72

2.05 + 0.27

18.29

3.71 + 0.21

3.71 f O.lOb

POL

IN SKIN AND CULTURE

’ For explanation of abbreviations see legend to Fig. 5. b Results are expressed as a percentage of the radioactivity recovered are given as X f SE from 4 individual cultured skin samples.

Mouse

Rat

Rabbit

Skin

medium

Guinea

pig

medium

Skin Culture

medium

Culture

Skin

Culture

AQ

OF ~‘%Z~TESTOSTERONE

Sample

PROFILE

Marmoset

Human

Species

METABOLITE

+ 3.15

+ 1.32

T

Metabolite

+ 4.70

+ 2.91

f 2.36

+ 2.71

f 6.17

in the culture

22.42

45.49

60.89

medium

+ 5.25

k 3.27

+ 3.09

14.24 k 1.66

38.96

25.21

56.76

27.33

46.24 f 4.08

k 1.91

-t_ 2.99

-t 3.25

19.23

+ 0.65

1.15

topical

8.39 t 0.83

6.04 + 0.99

3.23 f 0.72

2.00 a 0.24

2.40 f 0.24

1.25 f 0.12

3.51 + 0.39

10.42 f

8.35 k 0.75

5.33 Ik 0.59

23.71

24 hr following

2 1.25

15.41 + 0.63

10.83 + 1.04

15.69 + 1.33

8.25 +_ 1.01

6.22 2 0.96

20.78

54.91

25.40

39.16

16.07 + 0.37

B

C

application

23.91

of 10 &skin

f 2.13

7.64 + 0.46

0.38 -I- 0.13

0.47 * 0.05

1.41 k 0.51

0.20 + 0.04

1.48 + 0.11

1.02 -+ 0.10

6.75 k 1.51

2.48 -t 0.78

2.62 -t 0.10

3.72 2.78 0.82 1.72

and

+ 2.32

+ 1.97

+ 2.36 + 1.78 -+ 1.50

& 0.93 + 1.70

+ 5 t f

sample,

88.71

99.62

89.30 96.10 92.64

97.62 98.32

99.64 98.91 97.07 103.40

+ 1.35

Recovery

CULTURE

106.96

SKIN IN ORGAN

1.43 + 0.16

TO MAMMA~WN

5.74 f 0.56

APPLICATION

extract

13.78 + 1.31

A

acetate

TOPICAL

and the skin,

Ethyl

profile”

24 hr FOLLOWING

3

15.20 + 1.23

39.27

51.52

MEDIUM

TABLE

F F

z

3 3 B 8 “2

PERCUTANEOUS

ABSORPTION

skin, the relative proportion of polar metabolites and peak A was similar and each one accounted for 11% of the radioactivity present. DISCUSSION The importance of cutaneous metabolism and its relationship to skin absorption of topically applied chemicals were examined by comparing the percutaneous fate of two model lipophilic compounds in skin samples maintained as short-term organ cultures. BP and testosterone were chosen as model compounds because of their similarity in molecular weight, size, and structure and also because information dealing with the biotransformation of these chemicals in skin in vivo, in cultured cells, and in subcellular fractions derived from skin is readily available in the literature (Berry et al., 1977; Phillips et al., 1978; Ashurst and Cohen, 1981; Bickers et al., 1983; Wortiz et al., 1956; Rongone, 1966; Mauvais-Jarvis et al., 1969; Mulay et al., 1972). Previous experiments in our laboratory demonstrated that mouse skin can be maintained metabolically viable and structurally intact in organ culture (Kao et al., 1983; Holland et al., 1984). In the present studies, the culture systems developed for mouse skin were found applicable to use with skin samples from five other mammalian species, including man, and they provide a means whereby species variation in skin penetration and metabolism of topical xenobiotics may be studied under defined conditions. Studies with the dynamic system showed that biological viability of the tissue has a profound effect on the in vitro penetration of surface-applied chemicals. With BP, the results from different species supported and confirmed our previous observations with mouse skin (Smith and Holland, 198 1; Holland et al., 1984); that is, permeation of BP in previously frozen, nonviable skin preparations was negligible, and biological viability plays an essential role in the translocation of topical BP through the skin. Testosterone, on the other hand, readily permeated both viable and

IN

SIX

MAMMALS

513

nonviable skin preparations in vitro; skin permeation was significantly greater than for BP, and enhanced penetration was also apparent in nonviable skin preparations. In addition, the results of studies in mouse skin with metabolic inhibitors and inducers of drug-metabolizing enzymes further supported our contention that skin viability and metabolism are important determinants in percutaneous absorption of certain chemicals. The addition of cyanide to the perfusion medium and the modulation of the drug-metabolizing enzyme systems in the skin greatly influenced the in vitro percutaneous permeation of topical BP, but had no apparent effect on the in vitro rate and overall permeation of topical testosterone. These relative differences in skin penetration between BP and testosterone are, in part, a reflection of the differences in the physicochemical properties, such as lipophilicity, of these chemicals. The importance of such physicochemical properties and the empirical correlation between lipid/water partition coefficients and permeability constants as they pertain to the diffusional aspect of skin absorption of chemicals have been discussed by others (Katz and Shaikh, 1965; Scheuplein, 1977; Scheuplein et al., 1969; Franz, 1975: Michaels et al., 1975; Bronaugh and Congdon, 1984; Bronaugh and Stewart, 1984). Our results, however, indicated that metabolic transformation can also have important influences on the translocation of certain surface-applied chemicals through the skin. In all six species, examination of the metabolite profiles of BP and testosterone in the receptor fluid from the static cultures showed that essentially only unchanged parent compound was found in the medium from nonviable skin; while in the medium from viable skin, a full spectrum of metabolites, as well as unchanged parent compound, was present. These results indicate that, in addition to the stratum comeum, which acts as a passive, inert diffusion barrier limiting percutaneous absorption, the viable epidermis, acting as a metabolizing membrane which can provide a metabolic barrier to skin penetration, should

514

KAO,

PATTERSON,

also be considered. Indeed, pharmacokinetic models of percutaneous absorption in which cutaneous metabolism was included as an integral part of the absorption process have been described (Ando et al., 1977; Hadgraft, 1980). For the highly lipid-soluble compound BP, our results suggested that biotransformation by the viable epidermis to more polar metabolites may be the more important rate-limiting process in the translocation of BP through the skin to a location from which it can enter systemic circulation via the dermal microvasculature or lymphatics. For testosterone our results indicated that skin absorption in all six species is dictated primarily by passive diffusion, but is accompanied also by a significant degree of first pass cutaneous metabolism. Cutaneous biotransformation of testosterone to metabolites with different physicochemical properties, and therefore different penetration characteristics, could account for the observed differences in permeation between viable and previously frozen nonviable tissue. It has been reported that in vitro skin permeation of dihydrotestosterone, a metabolite of testosterone, was considerably lower than testosterone (Schaefer et al., 1982). It is possible that the observed in vitro metabolism of these compounds may be attributed to the contaminating skin microorganisms. However, during the development of a method for pregraft viability determination for skin based on the ability of the excised tissue to metabolize various substrates, it was demonstrated that the metabolic contributions due to skin microorganisms were negligible (May and DeClement, 1982). Also, our results with human skin, which has been thoroughly scrubbed with antimicrobial disinfectant, would support the conclusion that it is cutaneous metabolism rather than microbial metabolism which is responsible for the observed biotransformations. The rate and overall extent of in vitro skin permeation of these compounds showed considerable species variation. Permeation of both compounds was highest in mouse skin, but no other correlation between the thickness of the

AND

HALL

cultured skin and the extent of penetration as assessed in terms of the percentage of the applied radioactivity recovered in the receptor fluid 24 hr after topical application was ap parent. For BP, the order of extent of penetration was mouse > marmoset and human > rat and rabbit % guinea pig. Permeation of BP in guinea pig skin was essentially negligible. For testosterone, the order was mouse > rabbit and human > rat > guinea pig and marmoset. It is recognized that the thickness of the stratum corneum is an important factor in skin absorption and contributes to species variation (Scheuplein and Bronaugh, 1983; Bronaugh et al., 1982). However, species differ considerably in the structure and functions of their skin, and species variations in the skin absorption of chemicals may not be reflected entirely by variations in the thickness of their stratum corneum; differences may, in part, reflect the cutaneous metabolic differences between species. Although complete characterization of individual metabolites was not performed, an examination of the metabolite profiles in the skin and receptor fluid from our studies indicate that the pattern of metabolites found differs greatly from species to species. Moreover, relative differences in the metabolite profiles between the skin digest and the corresponding receptor fluid were observed. Since metabolism can have an important role in the translocation of surface-applied chemicals, differences in the metabolic capabilities of the skin from different species can contribute to species variation in the absorption and cutaneous fate of topically applied substances. Appreciation of these differences would greatly facilitate our understanding of the relevance and the interrelationship of cutaneous metabolism in percutaneous absorption of topically applied chemicals. The results of these investigations showed that, in all species studied, the translocation of surface-applied BP and testosterone through the skin was accompanied by varying degrees of cutaneous first pass metabolism. This result demonstrated that, in addition to tissue viability, the metabolic status and capabilities of

PERCUTANEOUS

ABSORPTION

the skin are important determining factors in modulating skin absorption. This is contrary to the currently accepted view that diffusion through the stratum corneum dictates percutaneous absorption and determines the systemic bioavailability of topically applied chemicals. Our results support the concept that both diffusional and metabolic processes are intimately involved in percutaneous absorption of chemicals. The relative importance of these processes is a function of the physicochemical properties of the compounds and the ability of the epidermal cells to metabolize the compound in question. Furthermore, it is evident that meaningful in vitro studies of the cutaneous fate of topically applied chemicals should include not only a measure of the diffusion of the compound through the skin but also an assessment of the cutaneous biotransformation of the applied compound. REFERENCES ANDO, Y. W., Ho, N. F. H., AND HIGUCHI, W. I. (1977). Skin as an active metabolizing barrier. 1. Theoretical analysis of topical bioavailability. J. Pharm. Sci. 66, 1525-1528. ASHURST, S. W., AND COHEN, G. M. (1981). In vivo formation of benzo(a)pyrene diol epoxide-deoxyadenosine adducts in skin of mice susceptible to benzo(a)pyrene induced carcinogenesis. Int. J. Cancer 21, 357-364. BERRY. D. L., BRACKEN, W. R., AND SLAGA, T. J. (1977). Benzo(a)pyrene metabolism in mouse epidermis: Analysis by high pressure liquid chromatography and DNA binding. Chem. Biol. Interact. 18, 129-142. BICKERS, D. R. (1983). Drug, carcinogen and steroid hormone metabolism in the skin. In Biochemistry nnd Physiology of the Skin (L. A. Goldsmith, ed.), Vol. 2, pp. 1169-l 186. Oxford Univ. Press, New York. BICKERS,D. R., MUKHTAR, H., AND YANG, S. K. (1983). Cutaneous metabolism of benzo(a)pyrene: Comparative studies in C57BL/GN and DBA/GN mice and neonatal Sprague-Dawley rats. Chem. Biol. Interact. 43, 263270. BRONAUGH, R., ANDCONGDON, E.(l984). Percutaneous absorption of hair dyes: Correlation with partition coefficients. J. Invest. Dermatol. 83, 124-127. BRONAUGH, R. L., CONGDON, E. R., AND SCHEUPLEIN, R. J. (198 1). The effect of cosmatic vehicles on penetration of N-nitroscdiethanolamine through excised human skin. J. Invest. Dermatol. 76, 94-96. BRONAUGH, R. L., AND STEWART, R. (1984). Methods for in vitro percutaneous absorption studies. III. Hydrophobic compounds J. Pharm. Sci. 73, 1255-1258.

IN SIX MAMMALS

515

BRONAUGH, R. L., STEWART, R. F., AND CONGDON, E. R. ( 1982). Method for in vitro percutaneous absorption studies. II. Animal models for human skin. Toxicol. Appl. Pharmacol. 62,48 l-488. DUGARD, P. H. (198 1). Skin pemeabihty theory in relation to measurements of percutaneous absorption in toxicology. In Dermatology (F. N. Marzulli and H. I. Maibath, eds.), 2nd ed.. pp. 91-129. Hemisphere, New York. FRANZ, T. J. (1975). Percutaneous absorption on the relevance of in vitro data. J. Invest. Dermatol. 64, 190195. HADCRAFT, J. (1980). Theoretical aspect of metabolism in the epidermis. Int. J. Pharm. 4,229-239. HAWKINS, G. S., AND REIFENRATH, W. G. (1984). Development of an in vitro model for determining the fate of chemicals applied to the skin. Fundam. Appl. Toxicol. 4, 5 133-5 144. HOLLAND, J. M., KAO, J. Y., AND WHITAKER, M. J. (1984). A multisample apparatus for kinetic evaluation of skin penetration in vitro: The influence of viability and metabolic status of the skin. Toxicol. Appl. Pharmacol.

12,272-280.

KAO, J. Y., HALL, J., AND HOLLAND, J. M. (1983). Quantitation of cutaneous toxicity: An in vitro approach using skin in organic culture. Toxicol. Appl. Pharmacol. 68, 206-217. KAO, J. Y., HALL, J., SHUGART, L. R., AND HOLLAND, J. M. (1984). An in vitro approach to studying cutaneous metabolism and disposition of topically applied xenobiotics. Toxicol. Appl. Pharmacol. 75, 289-298. KATZ, M., AND SHAIKH, Z. I. (1965). Percutaneous corticosteroid absorption correlated to partition coefficient. J. Pharm.

Sci. 54,591-594.

MARZULLI. F. N. (1962). Barrier to skin penetration. J. Pharm.

Sci. 39,337-353.

MAUVAIS-JARVIS, P., BERCOVICI, J. P., AND GAUTHIER, F. ( 1969). In vivo studies on testosterone metabolism by skin of normal male and patients with the syndrome of testicular terminization. J. Clin. Endocrinol. Metab. 29,417-421. MAY, S. R., AND DECLEMENT, F. A. (1982). Development of a radiometric metabolic viability testing method for human and porcine skin. Cryobiology 19, 362-37 1. MICHAELS, A. S., CHANDRASEKARAN, S. K., AND SHAW, J. E. (1975). Drug permeation through human skin: Theory and in vitro experimental measurement. A. I. Ch. E. 21, 985-996. MULAY, S., FINKELBERG,R., PINSKY, L., AND SOLOMON, S. (1972). Metabolism of 4-‘%-testosterone by serially subcultured human skin fibroblasts. J. Clin. Endocrinol. 34, 133-143. NACHT, S., YEUNG, D., BEASLEY, J. N., ANJO, M. D., AND MAIBACH, H. I. (198 1). Benzoylperoxide: Percutaneous penetration and metabolic disposition. Amer. Acad.

Dermatol.

4, 3 1-37.

NOONAN, P. K., AND WESTER, R. C. (1983). Cutaneous biotransformation: Some pharmacological and toxico-

516

KAO,

PATTERSON,

logical implications. In Dermafotoxicology (F. N. Marzulli and H. I. Maibach, eds.), 2nd ed., pp. 7 l-90. Hemisphere, New York. PANNATIER, A., JENNER,P., TESTA, B., AND ELTER, J. C. (1978). The skin as a drug metabolizing organ. Drug Metab. Rev. 8, 3 19-343. PHILLIPS, D. H., GROVER, P. L., AND SIMS, P. (1978). The covalent binding of polycyclic hydrocarbons to DNA in skin of mice of different strains. Int. J. Cancer 22,487-494.

RONCONE, E. L. (1966). Testosterone metabolism by human male mammary skin. Steroid 7,489-504. SCHAEFER,H., ZESCH, A., AND STUT~GER, G. (1982). Skin permeability. Springer-Verlag, New York. SCHEUPLEIN, R. J. (1977). Permeability of the skin. In Handbook ofPhysiology, Sect. 9, Reactions to Environmental Agents (D. H. Lee, ed.), pp. 299-322. Amer. Physiol. Sot., Washington, D.C. SCHEUPLEIN,R. J., BLANK, I. H., BRAUNER, G. J., AND

AND

HALL

MACFARLANE, D. J. (1969). Percutaneous absorption of steroids. J. Invest. Dermatol. 52, 63-70. SCHEUPLEIN, R. J., AND BRONAUGH, R. L. (1983). Percutaneous absorption. In Biochemistry and Physiology of the Skin (L. A. Goldsmith, ed.), Vol. 2, pp. 12% 1295. Oxford Univ. Press, New York. SMITH, L. H., AND HOLLAND, J. M. (198 1). Interaction between benzo[a]hpyrene and mouse skin in organ culture. Toxicology 21,47-57. SWARBRICK,J.. LEE, G., AND BROM, J. (1982). Drug permeation through human skin. I. Effect of storage condition of skin. J. Invest. Dermatol. 78, 63-66. WESTER, R. C., NOONAN, P. K., &EACH, S., AND KosoBUD, L. (1983). Pharmacokinetics and bioavailability of intravenous and topical nitroglycerin in the rhesus monkey. Estimate of percutaneous first passmetabolism. J. Pharm. Sci. 72,745-748. WORTIZ, H., MESCON, H., DOPPEL, H., AND LEMON, H. (1956). The in vitro metabolism of testosterone by human skin. J. Invest. Dermatol. 26, 113-120.