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The Effects of an Alpha Hydroxy Acid (Glycolic Acid) on Hairless Guinea Pig Skin Permeability
Food and Chemical Toxicology 37 (1999) 1105±1111
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Research Section
The Eects of an Alpha Hydroxy Acid (Glycolic Acid) on Hairless Guinea Pig Skin Permeability H. L. HOOD1, M. E. K. KRAELING1, M. G. ROBL2 and R. L. BRONAUGH1* 1
Oce of Cosmetics & Colors and 2Oce of Beltsville Technical Operations, Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708, USA (Accepted 19 April 1999)
AbstractÐThe barrier integrity of hairless guinea pig skin after treatment with an alpha hydroxy acid was assessed through in vivo topical application of an oil-in-water emulsion containing 5 or 10% glycolic acid at pH 3.0. The control was a commercial moisturizing lotion, pH 7.8. A dosing regimen for the glycolic acid formulations that was tolerated by the hairless guinea pigs and signi®cantly decreased stratum corneum turnover time was determined using the dansyl chloride staining technique. Once-daily dosing of hairless guinea pig skin for 3 weeks with the glycolic acid formulations resulted in approximately a 36±39% decrease in stratum corneum turnover time compared with the control lotion. After this treatment, hairless guinea pigs were sacri®ced for the in vitro measurement of the percutaneous absorption of [14C]hydroquinone and [14C]musk xylol. No signi®cant dierences in the 24-hour absorption of either test compound were found for skin treated with the control lotion or the glycolic acid formulations. There were also no signi®cant dierences found in the absorption of [3H]water through skin from the dierent treatment groups. Although no increase in skin penetration occurred after treatment with the glycolic acid formulations, histology revealed approximately a twofold increase in epidermal thickness. Also the number of nucleated cell layers nearly doubled in skin treated with 5% and 10% glycolic acid compared with the control lotion and untreated skin. These studies demonstrate that substantial changes in the structure of hairless guinea pig epidermis can occur without signi®cant eect on skin permeability of two model compounds. Published by Elsevier Science Ltd. All rights reserved Keywords: glycolic acid; skin permeability; hairless guinea pig. Abbreviations: AHA = alpha hydroxy acid; BSA = bovine serum albumin; DC = dansyl chloride; GA = glycolic acid; HGP = hairless guinea pig; HHBSS = HEPES-buered Hanks' balanced salt solution; HQ = hydroquinone; VIC = Vaseline Intensive Care.
INTRODUCTION
In the past several years, alpha hydroxy acids (AHAs) have gained considerable attention for their alleged ability to minimize the eects of skin ageing. They are reported to moisturize the skin, stimulate skin turnover, exfoliate, and act as skin peels (Smith, 1993). Nevertheless, repeated use of AHA formulations has been demonstrated to alter the structure of the stratum corneum (Leyden et al., 1995), viable epidermis and dermis (Lavker et al., 1992), and may result in skin barrier alterations *Corresponding author.
that could cause changes in the percutaneous absorption of topically applied chemicals. In this paper, the eect of chronic application of an AHA on the permeability and structure of skin was examined in hairless guinea pigs (HGPs). HGPs were treated daily with glycolic acid (GA) formulations according to a protocol determined to decrease stratum corneum turnover time (which is an increase in stratum corneum turnover rate). After treatment, animals were sacri®ced and the skin from the GA-treated and control sites was excised to measure the barrier properties of the skin in diusion cells. Changes in skin structure were assessed through histological examination of the tis-
0278-6915/99/$ - see front matter Published by Elsevier Science Ltd. PII S0278-6915(99)00100-3
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sue. Hydroquinone (HQ) and musk xylol were selected as test compounds for the in vitro studies because their diering oil and water solubility properties suggested that they would be absorbed at dierent rates.
MATERIALS AND METHODS
Chemicals [14C(U)]Hydroquinone (sp. act. 22.9 mCi/mmol) and [5-14CH3]2,4,6-trinitro-1,3-dimethyl-5-tert-butylbenzene (musk xylol, sp. act. 19.8 mCi/mmol) were synthesized by Research Triangle Institute (Research Triangle Park, NC, USA). [3H]Water (sp. act. 55.5 mCi/mmol) was purchased from New England Nuclear Corp. (Boston, MA, USA). All compounds had a radiochemical purity of 97% or greater as determined by thin-layer chromatography. Receptor ¯uid media and GA lotions were all prepared in the laboratory. Prepackaged Hanks' balanced salts were purchased from Life Technologies (Grand Island, NY, USA). Bovine serum albumin (BSA), GA and dansyl chloride (5[dimethylamino]naphtahlene-1-sulfonyl chloride) were obtained from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals, unless otherwise stated, were purchased from Sigma Chemical Co. or J.T. Baker Inc. (Phillipsburg, NJ, USA). Animals HGPs were purchased from Charles River Laboratories (Wilmington, MA, USA). The animals were housed separately in polycarbonate cages and were given food and water ad lib. Lighting was maintained on a 12-hr cycle in a room regulated at 75±808C and 65% relative humidity. HGPs, 3±6 months of age, were used in preliminary studies to determine the in vivo dosing regimen and to measure skin cell renewal times. For the in vitro studies, the HGPs were 8±9 months old. All animals were sacri®ced by CO2 asphyxiation and prepared for use in a skin penetration study as described under percutaneous absorption. The Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources Council, National Academy of Sciences, 1996) was followed. Estimation of skin cell turnover (renewal) time The replacement time of HGP stratum corneum was measured using the DC staining technique described by Jansen et al. (1974). DC at 5% (w/w) was ®nely ground into white petrolatum. Approximately 150 mg ointment was applied to the centre of a 3 cm2 cotton patch. Just prior to application the animal was anaesthetized with a mixture of ketamine and xylazine (70.0:8.75 mg/kg). The coated patch was then fastened to HGP skin under 1 semi-occlusive tape with Skin-Bond cement (Smith & Nephew United, Inc., Largo, FL, USA) applied
to the edges of the tape. After 24 hr, the patch was removed and excess material wiped o with a moist cloth. As DC ¯uoresces under UV light, the ¯uorescence of skin was monitored daily with a short wavelength UV lamp. The number of days elapsed for ¯uorescence to disappear was taken as a measure of the stratum corneum turnover time. In measuring skin cell turnover after treatment with the control or test lotions, daily application of the lotions (with prewashing) was continued until DC ¯uorescence disappeared. With untreated skin, daily washing of the skin was also performed prior to ¯uorescence monitoring. In vivo topical exposure studies The test lotions for topical exposure consisted of an oil-in-water emulsion containing 5% GA or 10% GA at pH 3.0. The control lotion was Vaseline Intensive Care (VIC) Lotion for Sensitive Dry Skin (Chesebrough-Ponds USA, Greenwich, CT, USA), pH 7.8. 48 hr prior to dosing, HGPs were carefully washed with a 1% soap solution (Palmolive dish detergent). Two areas of application (8 cm 5 cm, one on either side of the spine) were outlined on the back with a permanent marker, and remarked as needed. Lotions were applied at 3 mg/ cm2 by determining the amount of vehicle remaining on the weighing paper and applicator following application. Lotions were applied once daily for 3 wk (Sundays excluded) with a gentle water rinse and drying step prior to each application. At 3 wk, HGPs were sacri®ced for the in vitro percutaneous absorption studies. Percutaneous absorption In vitro skin absorption studies were performed according to the methodology of Bronaugh and Stewart (1985, 1986). Before excising HGP skin, loose surface ¯akes on the GA-treated side were carefully removed (to facilitate dermatoming) and the skin gently cleaned with water and dried. All skin samples were prepared at a thickness of 250± 300 mm with a Padgett Electrodermatome (Padgett Instruments, Kansas City, MO, USA). Before experiments were conducted, the ¯ow-through diusion cell system was disinfected with 70% ethanol following by rinsing with receptor ¯uid. Viability of skin was maintained in the diusion cells throughout the 24-hr study with a physiological medium (Collier et al., 1989). HEPES-buered Hanks' balanced salt solution (HHBSS) served as receptor ¯uid for the polar HQ. For the musk xylol studies, 4% BSA was added to the HHBSS receptor ¯uid which was stirred to facilitate partitioning of this lipophilic compound from skin. The integrity of the barrier of pretreated and control HGP skin was assessed measuring [3H]water absorption in a 20min test (Bronaugh et al., 1986) prior to the HQ and musk xylol absorption studies.
Eects of glycolic acid on skin permeability
[14C]HQ and [14C]musk xylol were applied to skin in oil-in-water emulsion vehicles (3 mg/cm2) at a dose of approximately 2.5 mg/cm2 and 5.0 mg/cm2, respectively. Receptor ¯uid was collected in 6-hr fractions for a total of 24 hr at a ¯ow rate of 1.5 ml/hr. At 24 hr, the skin surface was washed three times with a 10% soap solution and then rinsed three times with distilled water to remove unabsorbed material. The combined liquids were collected separately for each cell and mixed with 1 Ultima Gold liquid scintillation cocktail (Packard Instruments, Meriden, CT, USA). Each piece of 1 skin was dissolved in Scintigest tissue solubilizer (Fisher Scienti®c, Philadelphia, PA) at 608C overnight and then mixed with cocktail. Aliquots of receptor ¯uid were sampled from each 6-hr fraction and also mixed with cocktail. The amount of radiolabelled material in the wash, the skin, and the receptor ¯uid was determined with a Packard Tri1 Carb 4000 Series Liquid Scintillation Counter. Oil-in-water emulsion formulations The 5% and 10% GA emulsions used for the in vivo topical exposure studies were prepared according to Kraeling and Bronaugh (1997). The emulsion vehicle used in studying the in vitro percutaneous absorption of musk xylol was from BASF (1991). For HQ application, a modi®ed formulation from Harry's Cosmeticology (Wilkinson and Moore, 1982) was prepared as listed in Table 1. HQ and musk xylol were ®rst dissolved in ethanol for incorporation into their respective emulsion vehicles.
the centre of the treatment site. For paran sections, one section was placed in 10% neutral buffered formalin for a minimum of 48 hr. The tissue was then processed into paran blocks for sectioning at a thickness of about 6 mm. The sections were stained with haematoxylin and eosin (H&E) for light microscopic evaluation. For frozen sections, the skin was placed into Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA, USA) ®lled in tissue cassettes. The tissue cassettes were assembled onto a ¯at stainless-steel surface that had been precooled by exposure to dry ice. Once the supporting compound hardened, the cassettes were placed in a freezer compartment at ÿ48C. Frozen sections were cut to a thickness of about 6 mm on a cryostat and stained with H&E for light microscopic evaluation. Morphometric analysis Thickness measurements of the stratum corneum 1 and viable epidermis were made by using a Nikon TM photo microscope (Labophot ) equipped with a Microimage automatic camcorder, Model CA2063 (Microimage Video, Inc., Boyertown, PA, USA) at 400 magni®cation. Imaging analysis was performed in real-time mode or with captured image graphic ®les using a Pentium computer equipped with a multifunction frame grabber board (Imaging Technologies, Inc., Bedford, MA). Bioquant/True Color WindowsTM software (R&M Biometrics, Inc., Nashville, TN, USA) was used to determine length measurements after calibration with a micrometer. Statistical analysis
Histology Thickness measurements were taken of the stratum corneum and viable epidermis from frozen sections of skin. The number of nucleated cell layers was counted by viewing paran sections of the viable epidermis. Six dierent sites (approx. 30± 50 mm intervals) were measured for each tissue sample from every treatment site. Full-thickness skin samples (1.0±1.5 cm in diameter) were taken from treatment sites on the HGP immediately following euthanasia. Each sample was divided into two equal sections by a cut through Table 1. Composition of hydroquinone cream g/100 g Phase A Polyoxyethylene (100) stearate Mineral oil Cetearyl alcohol Phase B Laureth-4 Propylene glycol Water, deionized Phase C Sodium metabisul®te Ascorbic acid Hydroquinone
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2.00 7.00 5.00 1.00 5.00 77.75 0.15 0.10 2.00
All data are represented as the mean2 SE for the dierent groups. A one-way analysis of variance (ANOVA, SigmaStatTM Statistical Software, Jandel Scienti®c Software, San Rafael, CA, USA) was employed when statistical signi®cance was determined. The Student±Newman±Keuls test was used for multiple pairwise comparisons at a signi®cance level of P < 0.05. RESULTS
The eect of GA formulations on skin turnover was determined by measuring the disappearance of DC ¯uorescence from the skin surface. HGPs were able to tolerate the once-a-day dosing with GA formulations for 2 wk, the DC application, and then the continued daily dosing until ¯uorescence disappeared. The stratum corneum turnover times were reduced by the 5 and 10% GA treatments compared with the VIC control (Fig. 1). Percent reduction in turnover for the 5 and 10% GA formulations was 39% and 36%, respectively. A 3wk treatment period was therefore determined to be sucient to cause a decrease in stratum corneum turnover time in HGPs used for the skin absorption studies. 3 wk of dosing with 5 and 10% GA
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H. L. Hood et al.
Fig. 1. Turnover times of stratum corneum for untreated HGP (n = 8) and HGP skin pretreated for 2 wk with VIC (n = 3), 5% GA (n = 2) or 10% GA (n = 2). Bars are mean2 SE. GA = glycolic acid; HGP = hairless guinea pig; VIC = Vaseline Intensive Care.
resulted in some mild redness and/or ¯aking of the skin whereas the VIC-treated skin remained normal in appearance. The eect of the various treatment formulations on the barrier integrity of HGP skin was initially determined by measuring [3H]water absorption through skin in diusion cells set up for the HQ and musk xylol in vitro studies. As seen in Table 2, no signi®cant dierence in the percent of the applied [3H]water absorbed was detected between the treatment groups in cells for each test compound. The percutaneous absorption of HQ was virtually unaected by treatment applications. No signi®cant dierence was found between the partial (receptor ¯uid or skin) or total absorption values measured for the dierent treatment groups (Table 3). In general, musk xylol absorption was also unaected by treatment applications (Table 3). Total absorption values for VIC, 5% GA and 10% GA, however, were signi®cantly dierent from untreated skin; the VIC and GA lotions were not dierent from each other. Although no increase in penetration of HQ or musk xylol was observed following a 3-wk treatment with GA lotions to HGP skin, the structure of skin was substantially altered. Full-thickness sections of untreated skin, and skin treated with VIC,
5% GA and 10% GA were obtained immediately after excision. Comparison of viable epidermal cell layers revealed a similarity in thickness of untreated and VIC-treated skin and a thickening of the epidermis in the skin treated with 5% and 10% GA (Table 4). For example, the number of epidermal cell layers increased approximately twofold following treatment with the 5% GA formulation compared with untreated skin as seen in Plate 1. There was no signi®cant dierence between the number of cell layers measured following use of the 5% and 10% GA formulations. In addition, there appeared to be hypertrophy of the epithelium lining the hair follicles. The stratum corneum is important in barrier function but its thickness could not be accurately measured because of its partial destruction in the preparation of the paran sections. Therefore, two additional HGPs were treated with the test formulations for 3 wk and frozen sections of skin were prepared for thickness measurements. Statistical analysis of results could not be conducted because of the small sample size. The thickness of the stratum corneum was comparable between untreated and VIC-treated skin (Table 4). Stratum corneum thickness measurements in 10% GA-treated skin appeared to increase nearly twofold compared with untreated or VIC-treated skin. Eects on epidermal thickness were similar to those seen from the number of cell layers in the paran sections. Values for the viable epidermal thickness following 5 and 10% GA treatments were slightly more than twice the thickness values of untreated and VIC-treated HGP skin (Table 4).
DISCUSSION
HGPs were used as an animal model for human skin because they have a thicker epidermis than the haired variety (Mershon et al., 1990) and they do not require shaving in topical studies. It is well recognized that dierences do exist in the structure of HGP and human skin. In fact, the thickness of untreated HGP epidermis measured in this study (about 29 mm) is much less than the 100 mm value determined for human skin (Schaefer and Redelmeier, 1996).
Table 2. Eect of pretreatment on barrier integrity of hairless guinea pig skin Pretreatment formulations
HQ cells Musk xylol cells
Untreated
VIC
5% GA
10%GA
0.09 20.05 0.06 20.02
0.08 20.03 0.06 20.03
0.142 0.01 0.122 0.04
0.172 0.04 0.162 0.05
Values are percent of applied dose of [3H]water absorbed and are the mean 2SE of determinations in three animals (usually three replicates per animal). One-way ANOVA (P < 0.05) found no signi®cant dierences between treatment groups in cells used for the HQ and musk xylol studies. GA = glycolic acid; HQ = hydroquinone; VIC = Vaseline Intensive Care.*Evaluated by the 20 min [3H]water test (Bronaugh et al., 1986) prior to application of 14C-HQ or 14C-musk xylol.
Eects of glycolic acid on skin permeability
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Plate 1. Cross-section of formalin ®xed hairless guinea pig skin, H&E stain (100 magni®cation). The number of cell layers was measured from individual photographs at six locations approximately 30± 50 mm apart. (a) Untreated skin; (b) 5% glycolic acid-treated skin.
In preliminary experiments, HGP skin was found to be sensitive to topically administered AHAs at 5 and 10% concentrations. Studies conducted with twice daily application of the formulations had to be curtailed after 7 to 10 days because of irritation of the dorsal skin and subsequent scratching by the animals. Their skin became red and contained scratch marks indicating possible damage to the
skin barrier. Once-a-day application of the AHA formulations, however, was tolerated with only occasional reddened areas on the skin. The average turnover of the stratum corneum in untreated HGP skin was determined to be 26.5 days (Fig. 1). This turnover time was reduced by daily application of VIC to 16.5 days. The daily washing and application of a lotion appears to have
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H. L. Hood et al. Table 3. In vitro skin absorption of two cosmetic Ingredients following pretreatment of hairless guinea pig skin Pretreatment formulations
HQ receptor ¯uid skin total Musk xylol receptor ¯uid skin total
Untreated
VIC
5% GA
10% GA
4.25 2 0.56 15.02 0.67 19.32 0.43
5.89 20.30 13.2 21.07 19.0 21.03
6.442 0.88 15.42 1.02 21.82 1.87
4.402 0.52 16.12 1.82 20.52 2.10
30.3 2 2.49 18.02 2.55 48.32 1.73
23.6 20.17 18.8 21.21 42.4 21.07
20.72 4.23 16.12 3.20 36.72 1.31
21.62 3.56 18.82 2.12 40.42 1.72
Values are percent of applied dose absorbed in 24 hr and are the mean 2SE of determinations in three animals (usually three replicates per animal). One-way ANOVA (P < 0.05) found no signi®cant dierences between treatment groups except for total absorption of musk xylol. VIC, 5% GA and 10% GA were not dierent from each other but each was signi®cantly dierent from untreated skin. GA = glycolic acid; HQ = hydroquinone; VIC = Vaseline Intensive Care.
enhanced the removal of DC or possibly the VIC lotion may have had some eect on turnover time. The further decrease in stratum corneum turnover time by the 5 and 10% GA formulations of 39 and 36% (compared with VIC) is similar to the 29% decrease in human stratum corneum turnover observed after application of 3% GA, pH 3.0 (Smith, 1994). We observed a similar decrease in turnover with our pH 3.0 emulsion with or without the addition of GA (data not shown). Clearly, an acidic pH is important in enhancing desquamation of the stratum corneum that occurs with the use of AHA products. Epidermal hypertrophy was observed in skin treated with both 5 and 10% GA in agreement with previously reported human studies (Lavker et al., 1992). Data compiled from frozen-skin sections indicated greater changes in viable epidermal thickness of HGP skin than the average 19% increases reported in human skin by Lavker et al. (1992). A recent study by Fartasch et al. (1997), which evaluated the eects of GA on human stratum corneum, contradicted our ®ndings. After a twice-daily application of GA for 3 wk, they report a more compact appearing stratum corneum with no increased thickness. With the same conditions of use but only once-a-day application, our data suggests a thickening of the stratum corneum in HGPs. Our studies were conducted at pH 3.0, not pH 3.8, which was used in the human study. It is possible that the thickening of the stratum corneum induced by GA
may represent a response to the irritation of HGP skin. The eect of AHA treatment on human stratum corneum thickness is not clear. Studies on subjects with dry skin conditions such as xerosis or ichthyosis (Van Scott and Yu, 1974) have shown that stratum corneum (as well as viable epidermal) thickness is reduced following AHA use. However, these subjects have a thickened stratum corneum that may simply be reduced to normal thickness by AHA treatment. The eect of AHAs on normal stratum corneum thickness may be minimal if product use does not lead to skin irritation (Fartasch et al., 1997; Leyden et al., 1995). Leyden et al. (1995) have observed removal of only a few outer cell layers of stratum corneum in clinical studies with 2±10% formulations of AHAs, with no evidence of exfoliation of the deeper layers. The percutaneous absorption of water (initial 20min test), HQ and musk xylol was not signi®cantly changed after treatment with GA although substantial alterations in stratum corneum turnover and thickness were observed. It appears that the skin barrier has not been altered by the AHA administered to HGPs in these studies. The thicker stratum corneum may be constructed more porously during hyperproliferation of the epidermis after GA treatment resulting in no change in permeation of test compounds compared with VIC-treated skin. Thus, the studies presented herein have found no alteration of epidermal barrier function in hairless
Table 4. Number of epidermal cell layers, stratum corneum thickness and epidermal thickness following pretreatment of hairless guinea pig skin Paran sections Pretreatment Untreated VIC 5% GA 10% GA
Cell layers 5.0 20.2a,b (12) 5.02 0.2c,d (11) 9.8 20.5a,c (6) 8.1 2 1.3b,d (5)
Frozen sections Stratum corneum (mm) 13.8 23.7 14.3 24.3 20.3 20.8 26.3 23.8
(2) (2) (2) (2)
Epidermis (mm) 15.42 1.6 15.42 0.3 36.72 1.5 36.92 1.4
(2) (2) (2) (2)
Values are the mean 2 SE from the number of animals in parentheses (one section/animal). Reported values for cell layers with similar superscripts are signi®cantly dierent from each other (P < 0.05).
Eects of glycolic acid on skin permeability
guinea pig skin after daily application of either 5% or 10% glycolic acid formulation. AcknowledgementÐThe authors appreciate the contribution of Dr Dennis Hinton in making the skin thickness measurements. REFERENCES
BASF (1991) Cosmetics and Toiletries. December 106, 105. Bronaugh R. L. and Stewart R. F. (1985) Methods for in vitro percutaneous absorption studies IV: the ¯owthrough diusion cell. Journal of Pharmaceutical Sciences 74, 64±67. Bronaugh R. L. and Stewart R. F. (1986) Methods for in vitro percutaneous absorption studies VI: preparation of the barrier layer. Journal of Pharmaceutical Sciences 75, 487±491. Bronaugh R. L., Stewart R. F. and Simon M. (1986) Methods for in vitro percutaneous absorption studies VII: Use of excised human skin. Journal of Pharmaceutical Sciences 75, 1094±1097. Collier S. W., Sheikh N. M., Sakr A., Lichtin J. L., Stewart R. F. and Bronaugh R. L. (1989) Maintenance of skin viability during in vitro percutaneous absorption/metabolism studies. Toxicology and Applied Pharmacology 99, 522±533. Fartasch M., Teal J. and Menon G. K. (1997) Mode of action of glycolic acid on human stratum corneum: ultrastructural and functional evaluation of the epidermal barrier. Archives of Dermatological Research 289, 404±409.
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Jansen L. H., Hojyo-Tomoko M. T. and Kligman A. M. (1974) Improved ¯uorescence staining technique for estimating turnover of the human stratum corneum. British Journal of Dermatology 90, 9±12. Kraeling M. E. K. and Bronaugh R. L. (1997) In vitro percutaneous absorption of alpha hydroxy acids in human skin. Journal of the Society of Cosmetic Chemists 48, 187±197. Lavker R. M., Kaidbey K. and Leyden J. J. (1992) Eects of topical ammonium lactate on cutaneous atrophy from a potent topical corticosteroid. Journal of the American Academy of Dermatology 26, 535±544. Leyden J. J., Lavker R. M., Grove G. and Kaidbey K. (1995) Alpha hydroxy acids are more than moisturizers. Journal of Geriatric Dermatology 3 (Suppl. A), 33A±37A. Mershon M. M., Mitcheltree L. W., Petrali J. P., Braue E. H. and Wade J. V. (1990) Hairless guinea pig bioassay model for vesicant vapor exposures. Fundamental and Applied Toxicology 15, 622±630. Schaefer H. and Redelmeier T. E. (1996) Structure and dynamics of the skin barrier. In Skin Barrier: Principles of Percutaneous Absorption, ed. H. Schaefer and T. E. Redelmeier, pp. 1±42. Karger, Basel. Smith W. P. (1993) Hydroxy acids and skin aging. Soap/ Cosmetics/Chemical Specialties September, 54±58 & 76. Smith W. P. (1994) Hydroxy acids and skin aging. Cosmetics and Toiletries 109, September, 41±48. Van Scott E. J. and Yu R. J. (1974) Control of keratinization with alpha hydroxy acids and related compounds. Archives of Dermatology 110, 586±590. Wilkinson J. B. and Moore R. J. (1982) Skin lighteners or bleaches. In Harry's Cosmeticology, ed. J. B. Wilkinson and R. J. Moore, 7th edn, pp. 264±275. Chemical Publishing, New York.
www.elsevier.com/locate/foodchemtox
Research Section
The Eects of an Alpha Hydroxy Acid (Glycolic Acid) on Hairless Guinea Pig Skin Permeability H. L. HOOD1, M. E. K. KRAELING1, M. G. ROBL2 and R. L. BRONAUGH1* 1
Oce of Cosmetics & Colors and 2Oce of Beltsville Technical Operations, Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708, USA (Accepted 19 April 1999)
AbstractÐThe barrier integrity of hairless guinea pig skin after treatment with an alpha hydroxy acid was assessed through in vivo topical application of an oil-in-water emulsion containing 5 or 10% glycolic acid at pH 3.0. The control was a commercial moisturizing lotion, pH 7.8. A dosing regimen for the glycolic acid formulations that was tolerated by the hairless guinea pigs and signi®cantly decreased stratum corneum turnover time was determined using the dansyl chloride staining technique. Once-daily dosing of hairless guinea pig skin for 3 weeks with the glycolic acid formulations resulted in approximately a 36±39% decrease in stratum corneum turnover time compared with the control lotion. After this treatment, hairless guinea pigs were sacri®ced for the in vitro measurement of the percutaneous absorption of [14C]hydroquinone and [14C]musk xylol. No signi®cant dierences in the 24-hour absorption of either test compound were found for skin treated with the control lotion or the glycolic acid formulations. There were also no signi®cant dierences found in the absorption of [3H]water through skin from the dierent treatment groups. Although no increase in skin penetration occurred after treatment with the glycolic acid formulations, histology revealed approximately a twofold increase in epidermal thickness. Also the number of nucleated cell layers nearly doubled in skin treated with 5% and 10% glycolic acid compared with the control lotion and untreated skin. These studies demonstrate that substantial changes in the structure of hairless guinea pig epidermis can occur without signi®cant eect on skin permeability of two model compounds. Published by Elsevier Science Ltd. All rights reserved Keywords: glycolic acid; skin permeability; hairless guinea pig. Abbreviations: AHA = alpha hydroxy acid; BSA = bovine serum albumin; DC = dansyl chloride; GA = glycolic acid; HGP = hairless guinea pig; HHBSS = HEPES-buered Hanks' balanced salt solution; HQ = hydroquinone; VIC = Vaseline Intensive Care.
INTRODUCTION
In the past several years, alpha hydroxy acids (AHAs) have gained considerable attention for their alleged ability to minimize the eects of skin ageing. They are reported to moisturize the skin, stimulate skin turnover, exfoliate, and act as skin peels (Smith, 1993). Nevertheless, repeated use of AHA formulations has been demonstrated to alter the structure of the stratum corneum (Leyden et al., 1995), viable epidermis and dermis (Lavker et al., 1992), and may result in skin barrier alterations *Corresponding author.
that could cause changes in the percutaneous absorption of topically applied chemicals. In this paper, the eect of chronic application of an AHA on the permeability and structure of skin was examined in hairless guinea pigs (HGPs). HGPs were treated daily with glycolic acid (GA) formulations according to a protocol determined to decrease stratum corneum turnover time (which is an increase in stratum corneum turnover rate). After treatment, animals were sacri®ced and the skin from the GA-treated and control sites was excised to measure the barrier properties of the skin in diusion cells. Changes in skin structure were assessed through histological examination of the tis-
0278-6915/99/$ - see front matter Published by Elsevier Science Ltd. PII S0278-6915(99)00100-3
1106
H. L. Hood et al.
sue. Hydroquinone (HQ) and musk xylol were selected as test compounds for the in vitro studies because their diering oil and water solubility properties suggested that they would be absorbed at dierent rates.
MATERIALS AND METHODS
Chemicals [14C(U)]Hydroquinone (sp. act. 22.9 mCi/mmol) and [5-14CH3]2,4,6-trinitro-1,3-dimethyl-5-tert-butylbenzene (musk xylol, sp. act. 19.8 mCi/mmol) were synthesized by Research Triangle Institute (Research Triangle Park, NC, USA). [3H]Water (sp. act. 55.5 mCi/mmol) was purchased from New England Nuclear Corp. (Boston, MA, USA). All compounds had a radiochemical purity of 97% or greater as determined by thin-layer chromatography. Receptor ¯uid media and GA lotions were all prepared in the laboratory. Prepackaged Hanks' balanced salts were purchased from Life Technologies (Grand Island, NY, USA). Bovine serum albumin (BSA), GA and dansyl chloride (5[dimethylamino]naphtahlene-1-sulfonyl chloride) were obtained from Sigma Chemical Co. (St Louis, MO, USA). All other chemicals, unless otherwise stated, were purchased from Sigma Chemical Co. or J.T. Baker Inc. (Phillipsburg, NJ, USA). Animals HGPs were purchased from Charles River Laboratories (Wilmington, MA, USA). The animals were housed separately in polycarbonate cages and were given food and water ad lib. Lighting was maintained on a 12-hr cycle in a room regulated at 75±808C and 65% relative humidity. HGPs, 3±6 months of age, were used in preliminary studies to determine the in vivo dosing regimen and to measure skin cell renewal times. For the in vitro studies, the HGPs were 8±9 months old. All animals were sacri®ced by CO2 asphyxiation and prepared for use in a skin penetration study as described under percutaneous absorption. The Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources Council, National Academy of Sciences, 1996) was followed. Estimation of skin cell turnover (renewal) time The replacement time of HGP stratum corneum was measured using the DC staining technique described by Jansen et al. (1974). DC at 5% (w/w) was ®nely ground into white petrolatum. Approximately 150 mg ointment was applied to the centre of a 3 cm2 cotton patch. Just prior to application the animal was anaesthetized with a mixture of ketamine and xylazine (70.0:8.75 mg/kg). The coated patch was then fastened to HGP skin under 1 semi-occlusive tape with Skin-Bond cement (Smith & Nephew United, Inc., Largo, FL, USA) applied
to the edges of the tape. After 24 hr, the patch was removed and excess material wiped o with a moist cloth. As DC ¯uoresces under UV light, the ¯uorescence of skin was monitored daily with a short wavelength UV lamp. The number of days elapsed for ¯uorescence to disappear was taken as a measure of the stratum corneum turnover time. In measuring skin cell turnover after treatment with the control or test lotions, daily application of the lotions (with prewashing) was continued until DC ¯uorescence disappeared. With untreated skin, daily washing of the skin was also performed prior to ¯uorescence monitoring. In vivo topical exposure studies The test lotions for topical exposure consisted of an oil-in-water emulsion containing 5% GA or 10% GA at pH 3.0. The control lotion was Vaseline Intensive Care (VIC) Lotion for Sensitive Dry Skin (Chesebrough-Ponds USA, Greenwich, CT, USA), pH 7.8. 48 hr prior to dosing, HGPs were carefully washed with a 1% soap solution (Palmolive dish detergent). Two areas of application (8 cm 5 cm, one on either side of the spine) were outlined on the back with a permanent marker, and remarked as needed. Lotions were applied at 3 mg/ cm2 by determining the amount of vehicle remaining on the weighing paper and applicator following application. Lotions were applied once daily for 3 wk (Sundays excluded) with a gentle water rinse and drying step prior to each application. At 3 wk, HGPs were sacri®ced for the in vitro percutaneous absorption studies. Percutaneous absorption In vitro skin absorption studies were performed according to the methodology of Bronaugh and Stewart (1985, 1986). Before excising HGP skin, loose surface ¯akes on the GA-treated side were carefully removed (to facilitate dermatoming) and the skin gently cleaned with water and dried. All skin samples were prepared at a thickness of 250± 300 mm with a Padgett Electrodermatome (Padgett Instruments, Kansas City, MO, USA). Before experiments were conducted, the ¯ow-through diusion cell system was disinfected with 70% ethanol following by rinsing with receptor ¯uid. Viability of skin was maintained in the diusion cells throughout the 24-hr study with a physiological medium (Collier et al., 1989). HEPES-buered Hanks' balanced salt solution (HHBSS) served as receptor ¯uid for the polar HQ. For the musk xylol studies, 4% BSA was added to the HHBSS receptor ¯uid which was stirred to facilitate partitioning of this lipophilic compound from skin. The integrity of the barrier of pretreated and control HGP skin was assessed measuring [3H]water absorption in a 20min test (Bronaugh et al., 1986) prior to the HQ and musk xylol absorption studies.
Eects of glycolic acid on skin permeability
[14C]HQ and [14C]musk xylol were applied to skin in oil-in-water emulsion vehicles (3 mg/cm2) at a dose of approximately 2.5 mg/cm2 and 5.0 mg/cm2, respectively. Receptor ¯uid was collected in 6-hr fractions for a total of 24 hr at a ¯ow rate of 1.5 ml/hr. At 24 hr, the skin surface was washed three times with a 10% soap solution and then rinsed three times with distilled water to remove unabsorbed material. The combined liquids were collected separately for each cell and mixed with 1 Ultima Gold liquid scintillation cocktail (Packard Instruments, Meriden, CT, USA). Each piece of 1 skin was dissolved in Scintigest tissue solubilizer (Fisher Scienti®c, Philadelphia, PA) at 608C overnight and then mixed with cocktail. Aliquots of receptor ¯uid were sampled from each 6-hr fraction and also mixed with cocktail. The amount of radiolabelled material in the wash, the skin, and the receptor ¯uid was determined with a Packard Tri1 Carb 4000 Series Liquid Scintillation Counter. Oil-in-water emulsion formulations The 5% and 10% GA emulsions used for the in vivo topical exposure studies were prepared according to Kraeling and Bronaugh (1997). The emulsion vehicle used in studying the in vitro percutaneous absorption of musk xylol was from BASF (1991). For HQ application, a modi®ed formulation from Harry's Cosmeticology (Wilkinson and Moore, 1982) was prepared as listed in Table 1. HQ and musk xylol were ®rst dissolved in ethanol for incorporation into their respective emulsion vehicles.
the centre of the treatment site. For paran sections, one section was placed in 10% neutral buffered formalin for a minimum of 48 hr. The tissue was then processed into paran blocks for sectioning at a thickness of about 6 mm. The sections were stained with haematoxylin and eosin (H&E) for light microscopic evaluation. For frozen sections, the skin was placed into Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA, USA) ®lled in tissue cassettes. The tissue cassettes were assembled onto a ¯at stainless-steel surface that had been precooled by exposure to dry ice. Once the supporting compound hardened, the cassettes were placed in a freezer compartment at ÿ48C. Frozen sections were cut to a thickness of about 6 mm on a cryostat and stained with H&E for light microscopic evaluation. Morphometric analysis Thickness measurements of the stratum corneum 1 and viable epidermis were made by using a Nikon TM photo microscope (Labophot ) equipped with a Microimage automatic camcorder, Model CA2063 (Microimage Video, Inc., Boyertown, PA, USA) at 400 magni®cation. Imaging analysis was performed in real-time mode or with captured image graphic ®les using a Pentium computer equipped with a multifunction frame grabber board (Imaging Technologies, Inc., Bedford, MA). Bioquant/True Color WindowsTM software (R&M Biometrics, Inc., Nashville, TN, USA) was used to determine length measurements after calibration with a micrometer. Statistical analysis
Histology Thickness measurements were taken of the stratum corneum and viable epidermis from frozen sections of skin. The number of nucleated cell layers was counted by viewing paran sections of the viable epidermis. Six dierent sites (approx. 30± 50 mm intervals) were measured for each tissue sample from every treatment site. Full-thickness skin samples (1.0±1.5 cm in diameter) were taken from treatment sites on the HGP immediately following euthanasia. Each sample was divided into two equal sections by a cut through Table 1. Composition of hydroquinone cream g/100 g Phase A Polyoxyethylene (100) stearate Mineral oil Cetearyl alcohol Phase B Laureth-4 Propylene glycol Water, deionized Phase C Sodium metabisul®te Ascorbic acid Hydroquinone
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2.00 7.00 5.00 1.00 5.00 77.75 0.15 0.10 2.00
All data are represented as the mean2 SE for the dierent groups. A one-way analysis of variance (ANOVA, SigmaStatTM Statistical Software, Jandel Scienti®c Software, San Rafael, CA, USA) was employed when statistical signi®cance was determined. The Student±Newman±Keuls test was used for multiple pairwise comparisons at a signi®cance level of P < 0.05. RESULTS
The eect of GA formulations on skin turnover was determined by measuring the disappearance of DC ¯uorescence from the skin surface. HGPs were able to tolerate the once-a-day dosing with GA formulations for 2 wk, the DC application, and then the continued daily dosing until ¯uorescence disappeared. The stratum corneum turnover times were reduced by the 5 and 10% GA treatments compared with the VIC control (Fig. 1). Percent reduction in turnover for the 5 and 10% GA formulations was 39% and 36%, respectively. A 3wk treatment period was therefore determined to be sucient to cause a decrease in stratum corneum turnover time in HGPs used for the skin absorption studies. 3 wk of dosing with 5 and 10% GA
1108
H. L. Hood et al.
Fig. 1. Turnover times of stratum corneum for untreated HGP (n = 8) and HGP skin pretreated for 2 wk with VIC (n = 3), 5% GA (n = 2) or 10% GA (n = 2). Bars are mean2 SE. GA = glycolic acid; HGP = hairless guinea pig; VIC = Vaseline Intensive Care.
resulted in some mild redness and/or ¯aking of the skin whereas the VIC-treated skin remained normal in appearance. The eect of the various treatment formulations on the barrier integrity of HGP skin was initially determined by measuring [3H]water absorption through skin in diusion cells set up for the HQ and musk xylol in vitro studies. As seen in Table 2, no signi®cant dierence in the percent of the applied [3H]water absorbed was detected between the treatment groups in cells for each test compound. The percutaneous absorption of HQ was virtually unaected by treatment applications. No signi®cant dierence was found between the partial (receptor ¯uid or skin) or total absorption values measured for the dierent treatment groups (Table 3). In general, musk xylol absorption was also unaected by treatment applications (Table 3). Total absorption values for VIC, 5% GA and 10% GA, however, were signi®cantly dierent from untreated skin; the VIC and GA lotions were not dierent from each other. Although no increase in penetration of HQ or musk xylol was observed following a 3-wk treatment with GA lotions to HGP skin, the structure of skin was substantially altered. Full-thickness sections of untreated skin, and skin treated with VIC,
5% GA and 10% GA were obtained immediately after excision. Comparison of viable epidermal cell layers revealed a similarity in thickness of untreated and VIC-treated skin and a thickening of the epidermis in the skin treated with 5% and 10% GA (Table 4). For example, the number of epidermal cell layers increased approximately twofold following treatment with the 5% GA formulation compared with untreated skin as seen in Plate 1. There was no signi®cant dierence between the number of cell layers measured following use of the 5% and 10% GA formulations. In addition, there appeared to be hypertrophy of the epithelium lining the hair follicles. The stratum corneum is important in barrier function but its thickness could not be accurately measured because of its partial destruction in the preparation of the paran sections. Therefore, two additional HGPs were treated with the test formulations for 3 wk and frozen sections of skin were prepared for thickness measurements. Statistical analysis of results could not be conducted because of the small sample size. The thickness of the stratum corneum was comparable between untreated and VIC-treated skin (Table 4). Stratum corneum thickness measurements in 10% GA-treated skin appeared to increase nearly twofold compared with untreated or VIC-treated skin. Eects on epidermal thickness were similar to those seen from the number of cell layers in the paran sections. Values for the viable epidermal thickness following 5 and 10% GA treatments were slightly more than twice the thickness values of untreated and VIC-treated HGP skin (Table 4).
DISCUSSION
HGPs were used as an animal model for human skin because they have a thicker epidermis than the haired variety (Mershon et al., 1990) and they do not require shaving in topical studies. It is well recognized that dierences do exist in the structure of HGP and human skin. In fact, the thickness of untreated HGP epidermis measured in this study (about 29 mm) is much less than the 100 mm value determined for human skin (Schaefer and Redelmeier, 1996).
Table 2. Eect of pretreatment on barrier integrity of hairless guinea pig skin Pretreatment formulations
HQ cells Musk xylol cells
Untreated
VIC
5% GA
10%GA
0.09 20.05 0.06 20.02
0.08 20.03 0.06 20.03
0.142 0.01 0.122 0.04
0.172 0.04 0.162 0.05
Values are percent of applied dose of [3H]water absorbed and are the mean 2SE of determinations in three animals (usually three replicates per animal). One-way ANOVA (P < 0.05) found no signi®cant dierences between treatment groups in cells used for the HQ and musk xylol studies. GA = glycolic acid; HQ = hydroquinone; VIC = Vaseline Intensive Care.*Evaluated by the 20 min [3H]water test (Bronaugh et al., 1986) prior to application of 14C-HQ or 14C-musk xylol.
Eects of glycolic acid on skin permeability
1109
Plate 1. Cross-section of formalin ®xed hairless guinea pig skin, H&E stain (100 magni®cation). The number of cell layers was measured from individual photographs at six locations approximately 30± 50 mm apart. (a) Untreated skin; (b) 5% glycolic acid-treated skin.
In preliminary experiments, HGP skin was found to be sensitive to topically administered AHAs at 5 and 10% concentrations. Studies conducted with twice daily application of the formulations had to be curtailed after 7 to 10 days because of irritation of the dorsal skin and subsequent scratching by the animals. Their skin became red and contained scratch marks indicating possible damage to the
skin barrier. Once-a-day application of the AHA formulations, however, was tolerated with only occasional reddened areas on the skin. The average turnover of the stratum corneum in untreated HGP skin was determined to be 26.5 days (Fig. 1). This turnover time was reduced by daily application of VIC to 16.5 days. The daily washing and application of a lotion appears to have
1110
H. L. Hood et al. Table 3. In vitro skin absorption of two cosmetic Ingredients following pretreatment of hairless guinea pig skin Pretreatment formulations
HQ receptor ¯uid skin total Musk xylol receptor ¯uid skin total
Untreated
VIC
5% GA
10% GA
4.25 2 0.56 15.02 0.67 19.32 0.43
5.89 20.30 13.2 21.07 19.0 21.03
6.442 0.88 15.42 1.02 21.82 1.87
4.402 0.52 16.12 1.82 20.52 2.10
30.3 2 2.49 18.02 2.55 48.32 1.73
23.6 20.17 18.8 21.21 42.4 21.07
20.72 4.23 16.12 3.20 36.72 1.31
21.62 3.56 18.82 2.12 40.42 1.72
Values are percent of applied dose absorbed in 24 hr and are the mean 2SE of determinations in three animals (usually three replicates per animal). One-way ANOVA (P < 0.05) found no signi®cant dierences between treatment groups except for total absorption of musk xylol. VIC, 5% GA and 10% GA were not dierent from each other but each was signi®cantly dierent from untreated skin. GA = glycolic acid; HQ = hydroquinone; VIC = Vaseline Intensive Care.
enhanced the removal of DC or possibly the VIC lotion may have had some eect on turnover time. The further decrease in stratum corneum turnover time by the 5 and 10% GA formulations of 39 and 36% (compared with VIC) is similar to the 29% decrease in human stratum corneum turnover observed after application of 3% GA, pH 3.0 (Smith, 1994). We observed a similar decrease in turnover with our pH 3.0 emulsion with or without the addition of GA (data not shown). Clearly, an acidic pH is important in enhancing desquamation of the stratum corneum that occurs with the use of AHA products. Epidermal hypertrophy was observed in skin treated with both 5 and 10% GA in agreement with previously reported human studies (Lavker et al., 1992). Data compiled from frozen-skin sections indicated greater changes in viable epidermal thickness of HGP skin than the average 19% increases reported in human skin by Lavker et al. (1992). A recent study by Fartasch et al. (1997), which evaluated the eects of GA on human stratum corneum, contradicted our ®ndings. After a twice-daily application of GA for 3 wk, they report a more compact appearing stratum corneum with no increased thickness. With the same conditions of use but only once-a-day application, our data suggests a thickening of the stratum corneum in HGPs. Our studies were conducted at pH 3.0, not pH 3.8, which was used in the human study. It is possible that the thickening of the stratum corneum induced by GA
may represent a response to the irritation of HGP skin. The eect of AHA treatment on human stratum corneum thickness is not clear. Studies on subjects with dry skin conditions such as xerosis or ichthyosis (Van Scott and Yu, 1974) have shown that stratum corneum (as well as viable epidermal) thickness is reduced following AHA use. However, these subjects have a thickened stratum corneum that may simply be reduced to normal thickness by AHA treatment. The eect of AHAs on normal stratum corneum thickness may be minimal if product use does not lead to skin irritation (Fartasch et al., 1997; Leyden et al., 1995). Leyden et al. (1995) have observed removal of only a few outer cell layers of stratum corneum in clinical studies with 2±10% formulations of AHAs, with no evidence of exfoliation of the deeper layers. The percutaneous absorption of water (initial 20min test), HQ and musk xylol was not signi®cantly changed after treatment with GA although substantial alterations in stratum corneum turnover and thickness were observed. It appears that the skin barrier has not been altered by the AHA administered to HGPs in these studies. The thicker stratum corneum may be constructed more porously during hyperproliferation of the epidermis after GA treatment resulting in no change in permeation of test compounds compared with VIC-treated skin. Thus, the studies presented herein have found no alteration of epidermal barrier function in hairless
Table 4. Number of epidermal cell layers, stratum corneum thickness and epidermal thickness following pretreatment of hairless guinea pig skin Paran sections Pretreatment Untreated VIC 5% GA 10% GA
Cell layers 5.0 20.2a,b (12) 5.02 0.2c,d (11) 9.8 20.5a,c (6) 8.1 2 1.3b,d (5)
Frozen sections Stratum corneum (mm) 13.8 23.7 14.3 24.3 20.3 20.8 26.3 23.8
(2) (2) (2) (2)
Epidermis (mm) 15.42 1.6 15.42 0.3 36.72 1.5 36.92 1.4
(2) (2) (2) (2)
Values are the mean 2 SE from the number of animals in parentheses (one section/animal). Reported values for cell layers with similar superscripts are signi®cantly dierent from each other (P < 0.05).
Eects of glycolic acid on skin permeability
guinea pig skin after daily application of either 5% or 10% glycolic acid formulation. AcknowledgementÐThe authors appreciate the contribution of Dr Dennis Hinton in making the skin thickness measurements. REFERENCES
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