British Journal of Dermatology (1977) 9**) i7l-
Pharmacology and Treatment
The binding of topically applied glucocorticoids to rat skin J.R.J.BAKER, R.A.CHRISTIAN, P.SIMPSON AND A.M.WHITE Horsham Research Centre, CIBA Laboratories, Wimblehurst Road, Horsham West Sussex RH12 4AB Accepted for publication 18 July 1976
SUMMARY
The epidermis of rat skin was shown to be the major site of accumulation of label after the topical application of ["^H]-cortisol, [^H]-corticosterone and [^'H]-triamcinolone acetonide. However, incubation of the skin in vitro in a way which ensured contact only between the dermal side of the skin and the steroid showed preferential accumulation of label from [-'HJ-cortisol and ['Hl-corticosteronc in the epidermis whereas label from ['H]-triamcinolone acetonide appeared evenly spread in the dermis and epidermis. The affinity of corticosterone to soluble proteins in skin homogenates was greater than that of cortisol as measured by displacement binding and correspondingly glucose uptake into the skin in vitro was inhibited for i8 h after a single application of corticosterone but was not inhibited by cortisol.
The details of the mechanism of binding of glucocorticoids to whole skin have never been established with certainty although indirect evidence for the existence of a specific binding mechanism in the epidermis is available. Thus Vickers (1963), using vasoconstriction as the indicator of steroid effectiveness, demonstrated the existence of a reservoir of fiuocinolone acetonide. Feldman & Maibach (1965) demonstrated that the rate of excretion of ['"^Cl-cortisol was considerably lower after topical application than after either intradermal or intravenous injection and that there was less retention in skin from which the stratum corneum had been stripped than in skin which was intact. Liicker & Nowack (1968) showed the accumulation of fluprednylidene-21-acetate in the upper epidermis of pigs after topical application in cream. Similarly, in experiments with human skin made by Liicker et al. (1968) and Zesch & Schafer (1973) it was shown that there was a high concentration of various glucocorticoids in the stratum corneum when a technique was used in which consecutive layers of the epidermis were stripped off. The work described in this paper was designed to localize binding sites for [^H]-cortisoI, ["'H]corticosterone and ["'H]-triamcinolone acetonide through the use of autoradiography, to establish the relative strength of binding of these steroids to the binding sites and to relate this strength of binding to the duration of a biochemical effect, namely inhibition of glucose uptake. Corticosterone is the natural glucocorticoid in the rat and it was shown in previous studies by Bullock et al. (1972) to have a shorter duration of action on muscle than triamcinolone acetonide. 171
172
J.R.J.Baker et al. MATERIALS AND METHODS
Radioactive steroids. [i,2--'H2lcorticostcrone (spec. act. 36 Ci/mmol) and [i,2-'H2]cortisol (spec. act. 50 Ci/mmol) were obtained from the Radiochemical Centre, Amersham, Bucks and [i,2,4--'H]triamcinolone acetonide (spec. act. 9-5 Ci/mmol) was obtained from Centre fitude Nuclear, Department des Radio-isotopes, Mol—Donk, Belgium. The steroids were diluted with carrier for the experiments as required. Tissue culture medium. Basal medium (Eagle's) with Hanks' balanced salt solution and containing a-glutamine and 20 Mm HEPES buffer was obtained from Flow Laboratories Ltd., Irvine, Scotland. Chromatographic materials. Kieselgel (Merck) was used for column chromatography and was cleaned in the following way. Silica (50 g) was mixed with 160 ml of acid ethanol (80 ml HCl diluted to 480 ml with 95%, aqueous ethanol) and filtered. The gel was washed three times with 160 ml of ether, filtered again, and air dried at 100 C. Thin layer chromatography (TLC) was carried out on Eastman Silica gel plates with fluorescent indicator (No. 6000). Animals. Rats were male Wistar (250-300 g), CFHB strain from Carworth Europe, Alconbury, Huntingdon, U.K. Cream base. This contained the following ingredients (percent w/w). Stearic acid 5-0, liquid paraffin 20-0, emulsifying wax 90, glycerin 60, Methyl Butone 015, Propyl Butone 0 05, distilled water to 100%. Application of steroids to skin in vivo. Cream base (o-1 g) containing i ;(g of steroids (25 /iCi) was applied to an area of skin (2 25 cm^) about 20 min after shaving the animals. The cream was gently rubbed in over the whole area to ensure uniform coverage. Alternatively, solutions of the steroids in ethyl acetate (o-i ml) were applied to a similar area of skin in streaks with a small pipette. The solvent evaporated almost immediately. Application of steroids to skin in vitro. Pieces of shaved rat skin, measuring 2-25 cm^ and weighing approximately i g each were pinned across the shallow depression formed when molten paraffin wax was allowed to harden in a beaker. The depression was then filled with Eagle's Medium (25 ml) containing one of the tritiated steroids (25 //g, 250 /iCi) such that the inner (dermal) surface only of the skin was in contact with the medium. The apparatus was incubated under sterile conditions at 37 C for 2,6 or 18 h. At the end of these periods the skin was removed without the outer (epidermal) surface of this skin being allowed to touch the medium. Excess medium was removed from the dermal surface by placing the skin on filter paper. Measurement of concentration of steroids in skin samples. At various times after the topical application of steroids, animals were killed by decapitation. The areas of shaved skin were wrapped in aluminium foil and quickly frozen for storage in liquid nitrogen. Skin samples, after being milled in liquid nitrogen, were suspended in Krebs-Henseleit buffer and the suspensions were centrifuged at 105,000 g for 125 h. The supernatant (2 ml) was removed, extracted twice with ethyl acetate (50 ml) and the combined ethyl acetate extracts were either reduced to a small volume for analysis by thin layer or liquid chromatography, or dried down in a counting vial for the determination of radioactivity by liquid scintillation counting.
The binding of glucocorticoids to rat skin
173
Silica gel column chromatography. The column contained 6 g cleaned Kieselgel (100-120 mesh), which had been deactivated by suspension in water (20% w/w), suspended in redistilled chloroform and packed in a 25 ml column. The samples were applied in CHCl, (i ml) and steroids were eluted with successive 10 ml portions of CHCI3 containing 10,15, 20, 25, 30, 35 and 50" „ acetone. Successive 10 ml fractions were collected and dried under nitrogen. Triamcinolone acetonide was eluted in fraction 2, corticosterone in fraction 3 and cortisol in fraction 6. Thin layer chromatography. Solvent system I (dichloroethane: methanol; water; 95:5:0 2). Solvent system II (benzene:acetone; 50:50). Estimation of steroid binding. The Sephadex batch method of Simpson & White (1973) was used on supematants in Krebs-Henseleit medium, prepared as described above. Competitive binding of steroids. Each steroid to be tested for displacement by competitors was added to a portion of skin supernatant prepared from non-treated animals as described above to give a concentration of i x 10"^ M. This included sufficient labelled steroid to give a radioactivity of 005 /(Ci/ml. The competing unlabelled steroids were added to the supernatant to give a final concentration of 2 X 10"** M. The system was allowed to equilibrate at 4'G for 2 h, with occasional shaking, and then the amount of bound labelled steroid remaining was determined by the Sephadex batch method of Simpson & White (1973) referred to above. Preparation of tissue for autoradiography. The tissue was cut into small pieces (3x5 mm) and frozen by immersion in either Arcton 12 (dichlorodifluoromethane) cooled in liquid nitrogen, or hexane cooled in a carbon dioxide/acetone mixture. If not sectioned immediately, the specimens were stored in a liquid nitrogen refrigerator. Sections were cut on a Slee cryostat at — 30 C and at a 5 /im setting. They were prepared for autoradiography, developed and stained with haematoxylin and eosin according to the method of Applcton (1972) with the exception that pre-emulsioned microscope slides were used in preference to cover-slips. A 3-week expostire was allowed in all cases. Glucose uptake into skin. Animals were shaved in the abdominal region, the area of shaving being marked with a square template (43 X4 3 cm). Care was taken to avoid the epididymal fat deposits in the lower abdominal region. Rats were kept after shaving for at least 30 min before application of the corticosteroid. The steroid solution (o-i ml, 1 mg/ml) in ethyl acetate was streaked across the shaved area with a pipette to ensure even distribution. Controls had an application of ethyl acetate only. The rats were left in separate cages for 18-24 ^ with food and water ad Hb. They were killed by decapitation and the shaved areas of skin, after dissection, were submerged in Krebs Henseleit buffer previously gassed with 95",, O2/5",, CO;. Each piece of skin was blotted dry, weighed on filter paper and cut into six smaller pieces. For incubating the skin, wax moulds were made by allowing melted wax poured into 100 ml beakers to cool slowly forming a hollowed surface. The pieces of skin were pinned on to the surface, ail the skin from one rat being incubated in one beaker. Krebs-Henseleit medium (10 ml) was pipetted into the hollow completely submerging the skin. The medium contained o-i mg/ml D-gtucose, 30 /Jg/ml penicillin and 50/ig/ml streptomycin besides the normal ingredients. The samples were incubated for 4 h at 37 C in a shaking water bath, with gassing to maintain the pH of the buffer. Hourly samples (100 /d) were taken from each beaker for glucose assay. Initial samples were taken from the medium. The uptake of glucose was linear over the 4 h period. Results were calculated as /ig glucose uptake/g skin/min.
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Samples were assayed for glucose using a standard method (Bergmeyer, 1963). RESULTS
At various times up to 18 h after application of the steroids to the skin 80-85/0 of the total radioactivity in the extract of milled skin made in Krebs-Henseleit buffer was extractable into ethyl acetate and of this radioactivity more than 90" „ was shown, through the use of column and thin layer chromatography, to be associated with unchanged steroid. From these results it was concluded that direct counting of the radioactivity in the ethyl acetate extracts would give a measure of the unchanged steroid accurate enough for the purpose of the experiments. Ethyl acetate had to be used as a solvent in which to apply topically the labelled steroids because topical application in cream base resulted in concentrations of label in the skin which were inadequate for autoradiography. The amount of radioactivity absorbed by the skin and attributable to unchanged steroid was increased by a factor of more than 10 by the use of ethyl acetate. In separate experiments this solvent was shown not to have any irritant or hyperplastic effects. Binding of corticosteroids to the soluble fraction of rat skin. As seen in Table i the three steroids tested were in the following order with respect to their extent of binding to soluble macromolecules: corticosterone > triamcinolone acetonide > cortisol. TABLE I. Concentration and extent of steroid binding in tbe 105,000 g supernatant 2 h after their topical application in ethyl acetate. The homogenates and 105,000 g supernatants were made as described in the Experimental section. The quantity of radioactivity used varied between 25 and 42 fiCi. Each result is the mean of 5 experiments (+ s.e.m,)
Steroid Cortisol Corticosterone Triamcinolone acetonide
Amount applied per animal O'g)
Measured molarity of steroid in skin supernatant (X io-«)
Percentage binding per mg tissue protein
O'i8 0'2I
1-59 1-34
21 9 - H 2 1
O>27
2'l8
147 ± 1-8
8-5 ± 0 7
Competitive binding. From Table 2 it can be seen that there was more corticosterone bound in the soluble fraction than either of the other two steroids and that corticosterone lowered the binding of both the other steroids when incubated in their presence. From these data it appears that corticosterone is a powerful competitor for binding sites occupied by cortisol and triamcinolone acetonide. When the labelled steroids were diluted 2oo-fo!d by the addition of the same non-labelled steroid the amounts of labelled corticosterone and cortisol bound were reduced to 13 and 60",, of the control values, respectively, indicating that the binding sites were becoming saturated. On the other hand the amount of labelled triamcinoione acetonide bound was not reduced showing that even at a concentration of 2 X 10"^ M all the labelled steroid was still bound and therefore the binding was proportional to the total steroid concentration over the concentration range tested. The binding, therefore, appeared
The binding of glucocorticoids to rat skin
I75
TABLE 2. The amounts of different labelled steroids bound in the 105,000 S supernatant in the presence of steroid competitors Amount of labelled steroid bound (ng/mg proteinj Test steroid
Corticosterone Cortisol Triamcinolone acetonide
Compering cortisol
Competing triamcinolone aceconido
Competing corticosterone
2 X 10-"M
2X 10"" M
2 X 10"" M
0-87 0-54
0-40 0-32
076 0 36
O-U
0-39
041
038
No competing steroid
032
to be non-saturable over this concentration range. The behaviour of triamincolone acetonide was clearly different from that of the other two steroids. Location of binding sites by autoradiography after topical application of [^H]corticosterone and \^H]-
triamcinohne acetonide. The majority of the label, and therefore unchanged steroid, 2 h after application of triamcinolone acetonide in ethyl acetate was found over the stratum corneum and to some extent over the shaft and epithelium of some hair follicles (Fig. i). The results for corticosterone and cortisol are not shown but were similar. Eighteen hours after the application of these steroids the silver grains were much reduced in number but those remaining were still mainly in infoldings of the epidermis. Autoradiography after incubation of skin in the presence of [^H}corticosterone [^H]cortisol and [•'//]-
triamcinolone acetonide in vitro. In the case of corticosterone and cortisol the major sites of labelling are shown in Figs 2 and 3 which illustrate the situation after 6 and 2 h incubation of skin respectively in vitro. There was no obvious change in the amount of label over the tissue during the time-period examined, i.e. between 2 and 18 h. It can be seen that there was pronounced concentration of the label in the epidermis and other epithelial tissues in the case of both steroids. The time course of labelling up to 18 h was the same for corticosterone and cortisol. Detailed descriptions of the autoradiographs are given in the legends but one main conclusion emerged, namely that the concentration of radioactivity was maintained largely over the epithelia. The binding location of triamcinolone acetonide differed from that occupied by cortisol and corticosterone. Only the 6 h point is shown (Fig. 4) since the other autoradiographs taken after 2 and 18 h of incubation had the same general appearance. The densest labelling was in the lower corium and there was a gradation in labelling density from lower to upper dermis with only a few grains overlying the epidermis. Glucose ttptake into skin in vitro. The results given in Table 3 show the biochemical effects of topically applied steroids. Cortisol had no effect on glucose uptake 18 and 24 h after application; the effect of triamcinolone acetonide persisted up to 24 h while the effect of corticosterone persisted only up to 18 h and was no longer apparent at 24 h. DISCUSSION
All the techniques used are well established and in the case of the autoradiography the methodology
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J.R.J.Baker et al.
FIGURE I. Autoradiograph of skin section excised and frozen 2 h after the application ol' ["'H]triamcinolone acetonide in ethyl acetate. The dense region of silver grains is located mostly over the stratum corneum and part of the shaft and epithelium of some hair follicles (hf) ( x 200). FIGURE 2. Autoradiograph of a frozen section of skin after 6 h incubation sub-dermally in the presence of [•'H]-corticosterone from which the section has been removed. The low power profile of silver grains reveals that most label corresponds to epithelial regions such as epidermis (e) sebaceous glands (sg) and connective tissue nuclei (n). Also labelled is the epithelium of hair follicle sheath (hf) ( x 200). FIGURE 3. Autoradiograph of a frozen section of skin after sub-dermal incubation for 2 h in the presence of [^H]-cortisol. The sebaceous glands (sg) are less heavily radio-labelled than was the case after treatment with corticosterone. Other labels as in Fig. 2 ( x 200). FIGURE 4. Autoradiograph of a frozen section of skin after sub-dermal incubation in the presence of [•*H]-triamcinolone acetonide for 6 h. Intensity of label is greater in the dermis than in the epidermis (e) ( x 200). of Appleton (1972) was strictly adhered to, including as many precautions as possible against the relocation of the steroids during processing of the samples. With ethyl acetate as the vehicle the epidermis was the major site of labelling after topical application of all the tritiated steroids. These results agree with those of Liicker et al. (1968) who showed
The binding of glucocorticoids to rat skin
177
TABLE 3. Amount of glucose (/ig glucose/g skin/min) taken up by skin over a 4 h incubation period in vitro following its removal from animals which were killed 18 or 24 h after topical application of different steroids. Each result is the mean of 4 test incubations ( ± SEM} derived from 4 animals and carried our at the same time as incubations of control skin
Glucose uptake
Treatment
Steroid
Time after application
Control
Steroid-treated
18 24 18 24 18
0-95+0-08
0-96 ± 003
I-2I + O-O9 1-75 +0-23 1-34 + 0-27
24
I-37±O'I5
1-12 + 0-06 1-03 + 0-05* o-62 + O'O8* r-23+o-ii* 1-45 ±0-17
(h) Cortisol Triamcinolone acetonide Corticosterone
I-72±OT6
that the lower layer of human epidermis hound the label from topically applied [-^HJ-fluprednylidene2i-acetate and the results of Scott & Kalz (1956) who showed that most of the label, after the application of ['"^CJ-cortisol to human skin, was concentrated in the lower part of the epidermis after 2 h. Label was not detectable, however, after 16 h, an observation which was also consistent with the present study. After in vitro labelling of skin with tritiated steroids ('sub-dermal' incubation) autoradiographic techniques gave results which distinguished triamcinolone acetonide from both cortisol and corticosterone. For the latter two steroids there appeared to be binding sites of high aflinity in the epidermis capable of binding and concentrating these steroids even when they were presented on the dermal side of the incubated skin pieces. This was not the case for triamcinolone acetonide, which was seen to bind fairly evenly over the dermis and the epidermis. These results mean that only in the case of corticosterone and cortisol do the results of binding studies reflect relative affinities for epidermal binding sites. In the case of triamcinolone acetonide more dermal, possibly nonsaturable, binding sites were present in the homogenates, and therefore a ranking order with respect to all three steroids in respect of their relative aflinity for the epidermis was not possible. The stronger binding of corticosterone to the epidermis compared with cortisol was confirmed in the experiments involving glucose uptake where it was shown that inhibition persisted after topical application of corticosterone but not in the case of cortisol. The fact that the inhibition of glucose uptake persisted longest with triamcinolone acetonide could have been due to its strong binding aflinity to both dermal and epidermal sites, although pharmacologically active levels following topical application are unlikely to be detected by the low magnification autoradiography used in this study. An indirect result suggesting that this may be the case for this steroid was previously obtained by Gerard & Kozub (1967). These workers showed that after the subcutaneous injection of ['"'Cj-triamcinolone acetonide in niice, label was detected in the dermis and epidermis up to 5 days after injection. It would appear, therefore, that the long-term effectiveness of triamcinolone acetonide in these studies (and possibly the chnical efl"ectiveness of this and other topically active steroids) could be due mainly to retention in the non-cellular regions of the dermis
j ^
J.R.J.Baker et al.
following release from the epidermal binding sites, thus being locally available on a much more widespread basis than their naturally occurring counterparts. ACKNOWLEDGMENT
The authors express their thanks to Miss B. Craig for technical assistance. REFERENCES APPLETON, T . C . (1972) Auroradiography of diffusible substances. In: Autoradiography for Biologists (Ed. by P.B. Gahan), p. 51. Academic Press, London and New York. BERGMEYER, H.V. (1963) Methods of enzymatic analysis. Academic Press, London and New York. BULLOCK, G.R., CARTER, E.E., ELLIOTT, P., PETERS, R.F., SIMPSON, P. & WHITE, A.M. (1972) Relative changes
in the function of muscle ribosomes and mitochondria during the early phase of steroid-induced catabolism. Biochemical Journal, 127, 881. FELDMAN, R.J. & MAIBACH, H.I. (1965) Penetration of ['"^C] hydrocortisone through normal skin. Archives of Dermatology, 91, 661. GERARD, A.G. & KOZUB, W.R. (1967) Relationship of subdermally injected ['*C]-triamcinolone acetonide to the overlying skin. A radioautographic study in mice. Journal of Investigative Dermatology, 49, 256. LtiCKER, VAN P. & NOWAK, H . (1968) Tier experimentelle Untersuchungen zur Penetrations kinetik eines 9«fluor-i6-methylen-prednisolonesters (FIuprednyliden-21-acetat) am Schwein. Arzneimittel-Forschung, 18, 24-26. LtJCKEB, VAN P., NOWAK, H.J STIJTTGEN, G. & WEBMER, G . (1968) Penetrations-kinetic eines Tritium—markierten 9a-fluor-i6-methylen-prednisolonesrers nach epicutaner Applikation beim Menschen. ArzneimiitelForschungi 18, 27. SCOTT, A. & KALZ, F . (1956) The penetration and distribution of [ '*C]-hydrocortisone in human skin after its topical application. Journal of Investigative Dermatology, 26, 149. SIMPSON, P. & WHITE, A.M. (1973) Binding of glucocorticoids to a soluble fraction from rat skeletal muscle. Biochemical Pharmacology, 19, 1195. ViCKERS, C.H.F. (1963) Existence of a reservoir in the stratum corneum. Archives of Dermatology, 88, 72. ZESCH, VAN A. & SCHAFER, H . (1973) Penetrations verhalten verschieden radiomerkierter Steroid in der menschlichen Haut. Arzneimittel-Forschung, 23, 415.
Pharmacology and Treatment
The binding of topically applied glucocorticoids to rat skin J.R.J.BAKER, R.A.CHRISTIAN, P.SIMPSON AND A.M.WHITE Horsham Research Centre, CIBA Laboratories, Wimblehurst Road, Horsham West Sussex RH12 4AB Accepted for publication 18 July 1976
SUMMARY
The epidermis of rat skin was shown to be the major site of accumulation of label after the topical application of ["^H]-cortisol, [^H]-corticosterone and [^'H]-triamcinolone acetonide. However, incubation of the skin in vitro in a way which ensured contact only between the dermal side of the skin and the steroid showed preferential accumulation of label from [-'HJ-cortisol and ['Hl-corticosteronc in the epidermis whereas label from ['H]-triamcinolone acetonide appeared evenly spread in the dermis and epidermis. The affinity of corticosterone to soluble proteins in skin homogenates was greater than that of cortisol as measured by displacement binding and correspondingly glucose uptake into the skin in vitro was inhibited for i8 h after a single application of corticosterone but was not inhibited by cortisol.
The details of the mechanism of binding of glucocorticoids to whole skin have never been established with certainty although indirect evidence for the existence of a specific binding mechanism in the epidermis is available. Thus Vickers (1963), using vasoconstriction as the indicator of steroid effectiveness, demonstrated the existence of a reservoir of fiuocinolone acetonide. Feldman & Maibach (1965) demonstrated that the rate of excretion of ['"^Cl-cortisol was considerably lower after topical application than after either intradermal or intravenous injection and that there was less retention in skin from which the stratum corneum had been stripped than in skin which was intact. Liicker & Nowack (1968) showed the accumulation of fluprednylidene-21-acetate in the upper epidermis of pigs after topical application in cream. Similarly, in experiments with human skin made by Liicker et al. (1968) and Zesch & Schafer (1973) it was shown that there was a high concentration of various glucocorticoids in the stratum corneum when a technique was used in which consecutive layers of the epidermis were stripped off. The work described in this paper was designed to localize binding sites for [^H]-cortisoI, ["'H]corticosterone and ["'H]-triamcinolone acetonide through the use of autoradiography, to establish the relative strength of binding of these steroids to the binding sites and to relate this strength of binding to the duration of a biochemical effect, namely inhibition of glucose uptake. Corticosterone is the natural glucocorticoid in the rat and it was shown in previous studies by Bullock et al. (1972) to have a shorter duration of action on muscle than triamcinolone acetonide. 171
172
J.R.J.Baker et al. MATERIALS AND METHODS
Radioactive steroids. [i,2--'H2lcorticostcrone (spec. act. 36 Ci/mmol) and [i,2-'H2]cortisol (spec. act. 50 Ci/mmol) were obtained from the Radiochemical Centre, Amersham, Bucks and [i,2,4--'H]triamcinolone acetonide (spec. act. 9-5 Ci/mmol) was obtained from Centre fitude Nuclear, Department des Radio-isotopes, Mol—Donk, Belgium. The steroids were diluted with carrier for the experiments as required. Tissue culture medium. Basal medium (Eagle's) with Hanks' balanced salt solution and containing a-glutamine and 20 Mm HEPES buffer was obtained from Flow Laboratories Ltd., Irvine, Scotland. Chromatographic materials. Kieselgel (Merck) was used for column chromatography and was cleaned in the following way. Silica (50 g) was mixed with 160 ml of acid ethanol (80 ml HCl diluted to 480 ml with 95%, aqueous ethanol) and filtered. The gel was washed three times with 160 ml of ether, filtered again, and air dried at 100 C. Thin layer chromatography (TLC) was carried out on Eastman Silica gel plates with fluorescent indicator (No. 6000). Animals. Rats were male Wistar (250-300 g), CFHB strain from Carworth Europe, Alconbury, Huntingdon, U.K. Cream base. This contained the following ingredients (percent w/w). Stearic acid 5-0, liquid paraffin 20-0, emulsifying wax 90, glycerin 60, Methyl Butone 015, Propyl Butone 0 05, distilled water to 100%. Application of steroids to skin in vivo. Cream base (o-1 g) containing i ;(g of steroids (25 /iCi) was applied to an area of skin (2 25 cm^) about 20 min after shaving the animals. The cream was gently rubbed in over the whole area to ensure uniform coverage. Alternatively, solutions of the steroids in ethyl acetate (o-i ml) were applied to a similar area of skin in streaks with a small pipette. The solvent evaporated almost immediately. Application of steroids to skin in vitro. Pieces of shaved rat skin, measuring 2-25 cm^ and weighing approximately i g each were pinned across the shallow depression formed when molten paraffin wax was allowed to harden in a beaker. The depression was then filled with Eagle's Medium (25 ml) containing one of the tritiated steroids (25 //g, 250 /iCi) such that the inner (dermal) surface only of the skin was in contact with the medium. The apparatus was incubated under sterile conditions at 37 C for 2,6 or 18 h. At the end of these periods the skin was removed without the outer (epidermal) surface of this skin being allowed to touch the medium. Excess medium was removed from the dermal surface by placing the skin on filter paper. Measurement of concentration of steroids in skin samples. At various times after the topical application of steroids, animals were killed by decapitation. The areas of shaved skin were wrapped in aluminium foil and quickly frozen for storage in liquid nitrogen. Skin samples, after being milled in liquid nitrogen, were suspended in Krebs-Henseleit buffer and the suspensions were centrifuged at 105,000 g for 125 h. The supernatant (2 ml) was removed, extracted twice with ethyl acetate (50 ml) and the combined ethyl acetate extracts were either reduced to a small volume for analysis by thin layer or liquid chromatography, or dried down in a counting vial for the determination of radioactivity by liquid scintillation counting.
The binding of glucocorticoids to rat skin
173
Silica gel column chromatography. The column contained 6 g cleaned Kieselgel (100-120 mesh), which had been deactivated by suspension in water (20% w/w), suspended in redistilled chloroform and packed in a 25 ml column. The samples were applied in CHCl, (i ml) and steroids were eluted with successive 10 ml portions of CHCI3 containing 10,15, 20, 25, 30, 35 and 50" „ acetone. Successive 10 ml fractions were collected and dried under nitrogen. Triamcinolone acetonide was eluted in fraction 2, corticosterone in fraction 3 and cortisol in fraction 6. Thin layer chromatography. Solvent system I (dichloroethane: methanol; water; 95:5:0 2). Solvent system II (benzene:acetone; 50:50). Estimation of steroid binding. The Sephadex batch method of Simpson & White (1973) was used on supematants in Krebs-Henseleit medium, prepared as described above. Competitive binding of steroids. Each steroid to be tested for displacement by competitors was added to a portion of skin supernatant prepared from non-treated animals as described above to give a concentration of i x 10"^ M. This included sufficient labelled steroid to give a radioactivity of 005 /(Ci/ml. The competing unlabelled steroids were added to the supernatant to give a final concentration of 2 X 10"** M. The system was allowed to equilibrate at 4'G for 2 h, with occasional shaking, and then the amount of bound labelled steroid remaining was determined by the Sephadex batch method of Simpson & White (1973) referred to above. Preparation of tissue for autoradiography. The tissue was cut into small pieces (3x5 mm) and frozen by immersion in either Arcton 12 (dichlorodifluoromethane) cooled in liquid nitrogen, or hexane cooled in a carbon dioxide/acetone mixture. If not sectioned immediately, the specimens were stored in a liquid nitrogen refrigerator. Sections were cut on a Slee cryostat at — 30 C and at a 5 /im setting. They were prepared for autoradiography, developed and stained with haematoxylin and eosin according to the method of Applcton (1972) with the exception that pre-emulsioned microscope slides were used in preference to cover-slips. A 3-week expostire was allowed in all cases. Glucose uptake into skin. Animals were shaved in the abdominal region, the area of shaving being marked with a square template (43 X4 3 cm). Care was taken to avoid the epididymal fat deposits in the lower abdominal region. Rats were kept after shaving for at least 30 min before application of the corticosteroid. The steroid solution (o-i ml, 1 mg/ml) in ethyl acetate was streaked across the shaved area with a pipette to ensure even distribution. Controls had an application of ethyl acetate only. The rats were left in separate cages for 18-24 ^ with food and water ad Hb. They were killed by decapitation and the shaved areas of skin, after dissection, were submerged in Krebs Henseleit buffer previously gassed with 95",, O2/5",, CO;. Each piece of skin was blotted dry, weighed on filter paper and cut into six smaller pieces. For incubating the skin, wax moulds were made by allowing melted wax poured into 100 ml beakers to cool slowly forming a hollowed surface. The pieces of skin were pinned on to the surface, ail the skin from one rat being incubated in one beaker. Krebs-Henseleit medium (10 ml) was pipetted into the hollow completely submerging the skin. The medium contained o-i mg/ml D-gtucose, 30 /Jg/ml penicillin and 50/ig/ml streptomycin besides the normal ingredients. The samples were incubated for 4 h at 37 C in a shaking water bath, with gassing to maintain the pH of the buffer. Hourly samples (100 /d) were taken from each beaker for glucose assay. Initial samples were taken from the medium. The uptake of glucose was linear over the 4 h period. Results were calculated as /ig glucose uptake/g skin/min.
174
J.R.J.Baker ct al.
Samples were assayed for glucose using a standard method (Bergmeyer, 1963). RESULTS
At various times up to 18 h after application of the steroids to the skin 80-85/0 of the total radioactivity in the extract of milled skin made in Krebs-Henseleit buffer was extractable into ethyl acetate and of this radioactivity more than 90" „ was shown, through the use of column and thin layer chromatography, to be associated with unchanged steroid. From these results it was concluded that direct counting of the radioactivity in the ethyl acetate extracts would give a measure of the unchanged steroid accurate enough for the purpose of the experiments. Ethyl acetate had to be used as a solvent in which to apply topically the labelled steroids because topical application in cream base resulted in concentrations of label in the skin which were inadequate for autoradiography. The amount of radioactivity absorbed by the skin and attributable to unchanged steroid was increased by a factor of more than 10 by the use of ethyl acetate. In separate experiments this solvent was shown not to have any irritant or hyperplastic effects. Binding of corticosteroids to the soluble fraction of rat skin. As seen in Table i the three steroids tested were in the following order with respect to their extent of binding to soluble macromolecules: corticosterone > triamcinolone acetonide > cortisol. TABLE I. Concentration and extent of steroid binding in tbe 105,000 g supernatant 2 h after their topical application in ethyl acetate. The homogenates and 105,000 g supernatants were made as described in the Experimental section. The quantity of radioactivity used varied between 25 and 42 fiCi. Each result is the mean of 5 experiments (+ s.e.m,)
Steroid Cortisol Corticosterone Triamcinolone acetonide
Amount applied per animal O'g)
Measured molarity of steroid in skin supernatant (X io-«)
Percentage binding per mg tissue protein
O'i8 0'2I
1-59 1-34
21 9 - H 2 1
O>27
2'l8
147 ± 1-8
8-5 ± 0 7
Competitive binding. From Table 2 it can be seen that there was more corticosterone bound in the soluble fraction than either of the other two steroids and that corticosterone lowered the binding of both the other steroids when incubated in their presence. From these data it appears that corticosterone is a powerful competitor for binding sites occupied by cortisol and triamcinolone acetonide. When the labelled steroids were diluted 2oo-fo!d by the addition of the same non-labelled steroid the amounts of labelled corticosterone and cortisol bound were reduced to 13 and 60",, of the control values, respectively, indicating that the binding sites were becoming saturated. On the other hand the amount of labelled triamcinoione acetonide bound was not reduced showing that even at a concentration of 2 X 10"^ M all the labelled steroid was still bound and therefore the binding was proportional to the total steroid concentration over the concentration range tested. The binding, therefore, appeared
The binding of glucocorticoids to rat skin
I75
TABLE 2. The amounts of different labelled steroids bound in the 105,000 S supernatant in the presence of steroid competitors Amount of labelled steroid bound (ng/mg proteinj Test steroid
Corticosterone Cortisol Triamcinolone acetonide
Compering cortisol
Competing triamcinolone aceconido
Competing corticosterone
2 X 10-"M
2X 10"" M
2 X 10"" M
0-87 0-54
0-40 0-32
076 0 36
O-U
0-39
041
038
No competing steroid
032
to be non-saturable over this concentration range. The behaviour of triamincolone acetonide was clearly different from that of the other two steroids. Location of binding sites by autoradiography after topical application of [^H]corticosterone and \^H]-
triamcinohne acetonide. The majority of the label, and therefore unchanged steroid, 2 h after application of triamcinolone acetonide in ethyl acetate was found over the stratum corneum and to some extent over the shaft and epithelium of some hair follicles (Fig. i). The results for corticosterone and cortisol are not shown but were similar. Eighteen hours after the application of these steroids the silver grains were much reduced in number but those remaining were still mainly in infoldings of the epidermis. Autoradiography after incubation of skin in the presence of [^H}corticosterone [^H]cortisol and [•'//]-
triamcinolone acetonide in vitro. In the case of corticosterone and cortisol the major sites of labelling are shown in Figs 2 and 3 which illustrate the situation after 6 and 2 h incubation of skin respectively in vitro. There was no obvious change in the amount of label over the tissue during the time-period examined, i.e. between 2 and 18 h. It can be seen that there was pronounced concentration of the label in the epidermis and other epithelial tissues in the case of both steroids. The time course of labelling up to 18 h was the same for corticosterone and cortisol. Detailed descriptions of the autoradiographs are given in the legends but one main conclusion emerged, namely that the concentration of radioactivity was maintained largely over the epithelia. The binding location of triamcinolone acetonide differed from that occupied by cortisol and corticosterone. Only the 6 h point is shown (Fig. 4) since the other autoradiographs taken after 2 and 18 h of incubation had the same general appearance. The densest labelling was in the lower corium and there was a gradation in labelling density from lower to upper dermis with only a few grains overlying the epidermis. Glucose ttptake into skin in vitro. The results given in Table 3 show the biochemical effects of topically applied steroids. Cortisol had no effect on glucose uptake 18 and 24 h after application; the effect of triamcinolone acetonide persisted up to 24 h while the effect of corticosterone persisted only up to 18 h and was no longer apparent at 24 h. DISCUSSION
All the techniques used are well established and in the case of the autoradiography the methodology
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J.R.J.Baker et al.
FIGURE I. Autoradiograph of skin section excised and frozen 2 h after the application ol' ["'H]triamcinolone acetonide in ethyl acetate. The dense region of silver grains is located mostly over the stratum corneum and part of the shaft and epithelium of some hair follicles (hf) ( x 200). FIGURE 2. Autoradiograph of a frozen section of skin after 6 h incubation sub-dermally in the presence of [•'H]-corticosterone from which the section has been removed. The low power profile of silver grains reveals that most label corresponds to epithelial regions such as epidermis (e) sebaceous glands (sg) and connective tissue nuclei (n). Also labelled is the epithelium of hair follicle sheath (hf) ( x 200). FIGURE 3. Autoradiograph of a frozen section of skin after sub-dermal incubation for 2 h in the presence of [^H]-cortisol. The sebaceous glands (sg) are less heavily radio-labelled than was the case after treatment with corticosterone. Other labels as in Fig. 2 ( x 200). FIGURE 4. Autoradiograph of a frozen section of skin after sub-dermal incubation in the presence of [•*H]-triamcinolone acetonide for 6 h. Intensity of label is greater in the dermis than in the epidermis (e) ( x 200). of Appleton (1972) was strictly adhered to, including as many precautions as possible against the relocation of the steroids during processing of the samples. With ethyl acetate as the vehicle the epidermis was the major site of labelling after topical application of all the tritiated steroids. These results agree with those of Liicker et al. (1968) who showed
The binding of glucocorticoids to rat skin
177
TABLE 3. Amount of glucose (/ig glucose/g skin/min) taken up by skin over a 4 h incubation period in vitro following its removal from animals which were killed 18 or 24 h after topical application of different steroids. Each result is the mean of 4 test incubations ( ± SEM} derived from 4 animals and carried our at the same time as incubations of control skin
Glucose uptake
Treatment
Steroid
Time after application
Control
Steroid-treated
18 24 18 24 18
0-95+0-08
0-96 ± 003
I-2I + O-O9 1-75 +0-23 1-34 + 0-27
24
I-37±O'I5
1-12 + 0-06 1-03 + 0-05* o-62 + O'O8* r-23+o-ii* 1-45 ±0-17
(h) Cortisol Triamcinolone acetonide Corticosterone
I-72±OT6
that the lower layer of human epidermis hound the label from topically applied [-^HJ-fluprednylidene2i-acetate and the results of Scott & Kalz (1956) who showed that most of the label, after the application of ['"^CJ-cortisol to human skin, was concentrated in the lower part of the epidermis after 2 h. Label was not detectable, however, after 16 h, an observation which was also consistent with the present study. After in vitro labelling of skin with tritiated steroids ('sub-dermal' incubation) autoradiographic techniques gave results which distinguished triamcinolone acetonide from both cortisol and corticosterone. For the latter two steroids there appeared to be binding sites of high aflinity in the epidermis capable of binding and concentrating these steroids even when they were presented on the dermal side of the incubated skin pieces. This was not the case for triamcinolone acetonide, which was seen to bind fairly evenly over the dermis and the epidermis. These results mean that only in the case of corticosterone and cortisol do the results of binding studies reflect relative affinities for epidermal binding sites. In the case of triamcinolone acetonide more dermal, possibly nonsaturable, binding sites were present in the homogenates, and therefore a ranking order with respect to all three steroids in respect of their relative aflinity for the epidermis was not possible. The stronger binding of corticosterone to the epidermis compared with cortisol was confirmed in the experiments involving glucose uptake where it was shown that inhibition persisted after topical application of corticosterone but not in the case of cortisol. The fact that the inhibition of glucose uptake persisted longest with triamcinolone acetonide could have been due to its strong binding aflinity to both dermal and epidermal sites, although pharmacologically active levels following topical application are unlikely to be detected by the low magnification autoradiography used in this study. An indirect result suggesting that this may be the case for this steroid was previously obtained by Gerard & Kozub (1967). These workers showed that after the subcutaneous injection of ['"'Cj-triamcinolone acetonide in niice, label was detected in the dermis and epidermis up to 5 days after injection. It would appear, therefore, that the long-term effectiveness of triamcinolone acetonide in these studies (and possibly the chnical efl"ectiveness of this and other topically active steroids) could be due mainly to retention in the non-cellular regions of the dermis
j ^
J.R.J.Baker et al.
following release from the epidermal binding sites, thus being locally available on a much more widespread basis than their naturally occurring counterparts. ACKNOWLEDGMENT
The authors express their thanks to Miss B. Craig for technical assistance. REFERENCES APPLETON, T . C . (1972) Auroradiography of diffusible substances. In: Autoradiography for Biologists (Ed. by P.B. Gahan), p. 51. Academic Press, London and New York. BERGMEYER, H.V. (1963) Methods of enzymatic analysis. Academic Press, London and New York. BULLOCK, G.R., CARTER, E.E., ELLIOTT, P., PETERS, R.F., SIMPSON, P. & WHITE, A.M. (1972) Relative changes
in the function of muscle ribosomes and mitochondria during the early phase of steroid-induced catabolism. Biochemical Journal, 127, 881. FELDMAN, R.J. & MAIBACH, H.I. (1965) Penetration of ['"^C] hydrocortisone through normal skin. Archives of Dermatology, 91, 661. GERARD, A.G. & KOZUB, W.R. (1967) Relationship of subdermally injected ['*C]-triamcinolone acetonide to the overlying skin. A radioautographic study in mice. Journal of Investigative Dermatology, 49, 256. LtiCKER, VAN P. & NOWAK, H . (1968) Tier experimentelle Untersuchungen zur Penetrations kinetik eines 9«fluor-i6-methylen-prednisolonesters (FIuprednyliden-21-acetat) am Schwein. Arzneimittel-Forschung, 18, 24-26. LtJCKEB, VAN P., NOWAK, H.J STIJTTGEN, G. & WEBMER, G . (1968) Penetrations-kinetic eines Tritium—markierten 9a-fluor-i6-methylen-prednisolonesrers nach epicutaner Applikation beim Menschen. ArzneimiitelForschungi 18, 27. SCOTT, A. & KALZ, F . (1956) The penetration and distribution of [ '*C]-hydrocortisone in human skin after its topical application. Journal of Investigative Dermatology, 26, 149. SIMPSON, P. & WHITE, A.M. (1973) Binding of glucocorticoids to a soluble fraction from rat skeletal muscle. Biochemical Pharmacology, 19, 1195. ViCKERS, C.H.F. (1963) Existence of a reservoir in the stratum corneum. Archives of Dermatology, 88, 72. ZESCH, VAN A. & SCHAFER, H . (1973) Penetrations verhalten verschieden radiomerkierter Steroid in der menschlichen Haut. Arzneimittel-Forschung, 23, 415.
