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Effects of UV irradiation on the sebaceous gland and sebum secretion in hamsters
Journal of Dermatological Science (2003) 31, 151 /159
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Effects of UV irradiation on the sebaceous gland and sebum secretion in hamsters Yasuchiyo Akitomoa,*, Hirohiko Akamatsub, Yuri Okanoc, Hitoshi Masakic, Takeshi Horioa a
Department of Dermatology, Kansai Medical University, 10-15, Humizono-cho, Moriguchi, Osaka 5708506, Japan b Department of Dermatology, Fujita Health University School of Medicine, 1-98, Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan c Cosmos Technical Center Co. Ltd., 3-24-3, Hasune, Itabashi-ku, Tokyo 174-0046, Japan Received 2 September 2002; received in revised form 2 December 2002; accepted 2 December 2002
KEYWORDS Hamster sebocyte; Ultraviolet irradiation; Lipid peroxide; Transepidermal water loss
Summary Background: Although an understanding of the photobiology of the skin has been extensively advanced recently, the effect of ultraviolet (UV) radiation on sebaceous glands is not well known. Objective: In this study, we examined the direct effect of UV radiation on cultured sebocytes from hamsters in vitro experimental system. Moreover, we examined whether UV-induced peroxidation of skin surface lipids may affect barrier function of horney layer. Methods: We irradiated cultured sebocytes from hamsters, which have similar biological characteristics to the human sebocytes, with UV radiation. Moreover, transepidermal water loss (TEWL) was examined after topical application of cholesterol or triglyceride (TG) and UV exposures on the back of hamsters. Results: The number of sebocytes were increased significantly (120 /140%) after 4 days as compared with the non-irradiated controls. Lipid production in sebocytes was also increased on day 7 in an irradiation-dependent manner up to 4.1 times of the pre-irradiated level. When UVB was irradiated to TG- or cholesterol-applied skin at the minimum ear-swelling dose, TEWL increased twice or more as compared with UVB irradiation to unapplied sites. When in vitro-irradiated TG, in vitro-irradiated cholesterol, TG-peroxide (TG-OOH), and cholesterol-peroxide (CHO-OOH) were applied to the skin, TEWL increased significantly. Conclusion: These results suggest that UVB may directly activate the functions of the sebaceous gland in vivo to produce increased amounts of sebum, which may undergo peroxidation by UV light and damage the barrier functions of the skin. – 2003 Japanese Society for Investigative Dermatology. Published by Elsevier Science Ireland Ltd. All rights reserved.
1. Introduction *Corresponding author. Tel.: /81-6-6992-1001; fax: /81-66992-5965. E-mail address: [email protected] (Y. Akitomo).
Much is known about the acute and chronic effects of UV radiation on epidermis and dermis [1 /4]. In contrast, only limited investigations have
0923-1811/03/$30.00 – 2003 Japanese Society for Investigative Dermatology. Published by Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0923-1811(03)00003-3
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been aimed at the effects on the skin appendage such as the sebaceous gland. The DNA, RNA, and protein syntheses of keratinocytes are depressed shortly after UV exposure, but recover and accelerate thereafter [3]. These findings may explain the epidermal hyperplasia after UV irradiation. Chronic exposures to the sunlight clinically cause sebaceous gland hyperplasia on the face as one of the manifestations of the photoaging in human. However, the dynamics of sebocytes after UV irradiation has not been fully understood. It has been reported that, when hairless mice or hamsters were exposed to UVB in vivo, the sebaceous glands showed hyperplasia and the number of sebocytes increased [5,6]. These studies suggest that UV radiation in the sunlight can directly or indirectly influence the sebaceous gland. In this study, we examined the direct effect of UV radiation on cultured sebocytes from hamsters in vitro experimental system. We also examine the effect of UV radiation on the sebum production because there are some reports that UVB phototherapy or PUVA photochemotherapy increased the amount of skin surface lipids [7,8]. It has been shown that hamsters are useful as an experimental animal to examine the functions of sebaceous gland because the hamster sebaceous gland is similar to the human gland with regard to its size, response to androgens, and turnover time in vivo [9]. Recently, we established a tissue culture system for hamster sebaceous glands without damaging their functions [10]. Furthermore, it has been demonstrated that these hamster sebocytes are similar to human sebocytes in the proliferation and lipid synthesis [11]. Moreover, we examined whether UV-induced peroxidation of skin surface lipids may affect barrier function of horney layer.
2. Materials and methods 2.1. In vitro experiment 2.1.1. Culture of sebocytes Earlobes removed from 5-week-old golden male hamsters were incubated in Dulbecco’s modified Eagle’s medium (DMEM; Nikken Biomedical Laboratory, Kyoto, Japan) with 100 IU/ml penicillin and 100 mg/ml streptomycin (Flow laboratories Ltd., Ayrshire, Scotland) for 3 h at 4 8C, and were successively washed with Ca2- and Mg2-free phosphate-buffered saline (PBS(/)), then cut into 5 /5 mm2 pieces. They were incubated for 14 h in 2.4 U/ml dispase (Godoshusei Co. Ltd., Tokyo, Japan) at 4 8C to separate epidermis from
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dermis. The epidermis was peeled from the dermis in PBS(/), and sebaceous glands were isolated from the dermis by using microsurgical instrument under microscopy. The isolated sebaceous glands were seeded in 60 mm culture dishes (Becton Dickinson, Tokyo, Japan) on a 3T3-cell feeder layer in the medium consisted of DMEM and Ham’s F12 medium (1:1; Gibco BRL, NY, USA), 8% heatdenatured fetal calf serum (Gibco BRL), 2% human serum, 10 ng/ml epidermal growth factor (Gibco BRL), 3.4 mM L-glutamine (Gibco BRL), 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37 8C with 5% CO2. DMEM and Ham’s F12 medium contain 1.0 mM CaCl2. The 3T3-cell proliferation has been blocked by the addition of mitomycin-C (Sigma, Deisenhofen, Germany) for 4 h, 24 h before gland seeding [12]. 3T3-cell feeder layer was used only for primary sebocyte cultures. All experiments were performed using secondary sebocyte cultures. 2.1.2. Culture of keratinocytes The epidermal sheets were processed to singlecell suspensions using 0.25% trypsin. The isolated keratinocytes were cultured on 3T3-cell feeder layer in the medium consisted of DMEM low glucose (Gibco BRL), 8% heat-denatured fetal calf serum, 3.4 mM L-glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin by 37 8C with 5% CO2. DMEM low glucose contains 1.8 mM CaCl2. 3T3-cell feeder layer was used only for primary cultures. All experiments were performed using secondary keratinocyte cultures. 2.1.3. Ultraviolet irradiation The UVB source utilized was a parallel bank of seven fluorescent sunlamps (FL20S.E30/DMR light bulbs, Toshiba Medical Supply, Tokyo, Japan) with an emission spectrum of 275 /375 nm peaking at 305 nm. The UVA source was a bank of 14 black lights (FL32S.BL, Toshiba Medical Supply, Tokyo, Japan) with an emission spectrum of 300 /430 nm peaking at 352 nm. The irradiance of UVA and UVB was measured by a UV radiometer, UVR-305/365D(II). 2.1.4. Cell proliferation On day 0, sebocytes and keratinocytes were seeded in 60 mm plates (Becton Dickinson) at concentration of 3.5 /103 cells/cm2 and were left to attach for 24 h at 37 8C with 5% CO2 in each culture medium. On day 1, medium was changed to PBS(/), then cultured cells were exposed to UVB at doses of 10 or 20 mJ/cm2, which did not influence cell viabilities determined by the exclusion of trypan blue
Effects of UVR on the sebaceous gland and sebum secretion in hamsters
dye 24 h after UVB irradiation. After UVB irradiation, PBS(/) was changed to each culture medium, and cells were incubated at 37 8C with 5% CO2. On days 2, 4, and 7, the number of cells in suspensions was counted with a blood cell counter. 2.1.5. Analysis of intracellular lipid content Lipids (TG, free fatty acid (FFA), and cholesterol) were extracted from cultured cells according to the method of Zouboulis et al. [13]. Briefly, the culture medium was removed from the culture dishes. Remaining cells were washed three times with PBS(/) and were harvested after treatment with 0.25% trypsin and 0.02% ethylenediamine tetraacetic acid (EDTA) in PBS(/). The cells were suspended in a solution of chloroform /methanol (2:1) and sonicated. The filtrates from the solutions were evaporated at 40 8C under a stream of nitrogen. The lipids were solubilized in 100 ml chloroform /methanol (2:1, v/v) and spotted on high-performance thin-layer chromatography (HPTLC) plates (HPTLC silica gel 60, 10 /20 cm2, Merck, Darmstadt, Germany). TG, FFA, and cholesterol were resolved by using the following development system. The plates were developed twice in hexane:ethylether:acetic acid (56:14:0.3). After charring the chromatograms with 10% CuSO4 and 8% H3PO4 aqueous solution at 200 8C for 5 min, photodensitometry was performed and the isolated lipid fractions were quantified by comparison with commercially available lipid standards, run on the sample plates: triolein (Sigma), FFA (Sigma), and cholesterol (Sigma). 2.1.6. Lipid content of culture medium Culture medium was collected on days 4 and 7, and lipid contents were quantified by enzymatic assay described by Ito et al. [14]. TGs were degraded into glycerol and FFAs by lipase. Glycerol produces hydrogen peroxide under the catalytic activity of glycerol kinase and glycerol-3-phosphate oxidase. The hydrogen peroxide, with peroxidase catalysis, induces oxidative condensation of 4-aminoantipyrine (4-AA) and 3,5dimethoxy-N-ethyl-N-(2?-hydroxy-3?-sulfopropyl)aniline sodium (DAOS) (a chromogen), to produce a blue quinone pigment with a maximum absorbance wavelength of 600 nm. The absorbance at 600 nm was measured with a spectrophotometer and the amount of TGs was determined by standard curves. Measurements were performed using triglycerides E -test Wako (Wako Pure Chemical Industries, Osaka, Japan). FFAs produce acyl-CoA under the catalytic influence of acyl-CoA synthetase. Acyl-CoA in turn
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generates hydrogen peroxide, in a reaction catalyzed by acyl-CoA oxidase. Hydrogen peroxide, with peroxidase catalysis, quantitatively induces oxidative condensation of 4-AA and 3-methyl-Nethyl-N -(b-hydroxyethyl)aniline (a chromogen), to produce a reddish-purple quinone pigment with a maximum absorbance wavelength of 550 nm. The absorbance at 550 nm was measured with a spectrophotometer and the amount of FFAs was determined by standard curves. Measurements were performed using esterified fatty acid C -test Wako (Wako Pure Chemical Industries). Cholesterol esters were degraded into free cholesterol and FFAs catalyzed by cholesterol esterase. Free cholesterol subsequently generates hydrogen peroxide, in a reaction catalyzed by cholesterol oxidase. Hydrogen peroxide, with peroxidase catalysis, quantitatively induces oxidative condensation of 4-AA and DAOS, to produce a blue quinone pigment with a maximum absorbance wavelength of 600 nm. The absorbance at 600 nm was measured with a spectrophotometer and the amount of cholesterol was determined by standard curves. Measurements were performed using cholesterol E -test Wako (Wako Pure Chemical Industries).
2.2. In vivo experiment 2.2.1. Animals Six-week-old golden male hamsters were used (n /6). 2.2.2. Topical application of test chemicals and UVR Hamster’s hair on the back was removed 3 days before the beginning of the experiments. TEWL was measured on the day prior to topical application and UVR. Test solution (20 ml/cm2) was topically applied to the depilated dorsal area on day 0. Soon after the application, the back area was exposed to UVB at a dose of 150 (approximately one earswelling dose) or 300 mJ/cm2. 2.2.3. Transepidermal water loss Transepidermal water loss (TEWL) was measured on the back surface of the hamsters before and various time points after UV irradiation, using an evaporimeter (EP1, Servomed, Stockholm, Sweden). 2.2.4. Materials for topical application Cholesterol powder was dissolved in the 20% ethanol in 1,3-butyleneglycol and 0.5% cholesterol or cholesterol-peroxide (CHO-OOH) was used for each study.
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CHO-OOH was synthesized by photooxidation of cholesterol in the presence of methylene blue as a photosensitizer. Briefly, cholesterol dissolved in ethanol was irradiated with visible light at 4 8C overnight in the presence of 0.1% methylene blue. Methylene blue remained in the reaction solution was removed using silica gel column chromatography (Wakogel c-200, Wako Pure Chemical Industries). CHO-OOH production was confirmed for the hydroperoxide assay by high-performance liquid chromatography (HPLC) coupled with chemiluminescence detection (CL-HPLC method). Next, the ethanol solution was displaced with the hexane solution by evaporating under a nitrogen gas stream, and centrifuged to remove free cholesterol. Residual free cholesterol was removed from CHO-OOH hexane /acetic ether (7:3, v/v) solution three times using silica gel column. TG-peroxide (TG-OOH) was synthesized by photooxidation of TG in the presence of methylene blue. TG in ethanol was irradiated with visible light at 4 8C overnight in the presence of 0.1% methylene blue. Ethanol was removed by evaporating and the solvent was replaced with chloroform, then methylene blue was removed with silica gel column chromatography. Next, free TG was removed from TG-OOH with silica gel column chromatography using hexane /ether (56:14, v/v) solution as solvent.
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ing on the irradiated dose. The number of 10 or 20 mJ/cm2 UVB-irradiated cells was about 1.2 times (P B/0.05) or 1.4 times (P B/0.01) higher than that of non-irradiated cells, respectively. On day 7, however, there was no difference in the proliferation between irradiated and non-irradiated cells (Fig. 1a). Keratinocyte proliferation was inhibited with UVB in a dose-dependent manner during days 2 / 4. On day 4, the number of 20 mJ/cm2 UVBirradiated cells was about 2/3 (P B/0.01) of nonirradiated cells. On day 7, on the contrary, proliferation of cells tended to be accelerated depending on the irradiated dose, although there was no significant difference between irradiated and non-irradiated cells (Fig. 1b).
3.2. Effects of UVB on the lipid production of cultured sebocytes The amount of lipids in sebocytes increased depending on the irradiated dose on day 4. TG
2.2.5. In vitro UVR on cholesterol and TG TG and cholesterol in vehicle in Petri dish were exposed to UVB at a dose of 1200 mJ/cm2. As concerns UVA, cholesterol, and TG were exposed at a dose of 10 J/cm2 in the presence of coproporphyrin (Sigma) as a photosensitizer.
2.3. Statistical analysis Statistical analysis was performed using the analysis of variance (ANOVA). Statistical significance was defined as P B/0.05, 0.01, and 0.001.
3. Results 3.1. Effects of UVB on proliferation of cultured sebocytes and keratinocytes Proliferation of sebocytes was inhibited on day 2 depending on the dose of UVB. The number of 10 or 20 mJ/cm2 UVB-irradiated cells was about 2/3 (P B/0.001) or 1/2 (P B/0.001) of non-irradiated cells, respectively. On day 4, on the contrary, proliferation of the cells was accelerated depend-
Fig. 1 Effect of UVB on proliferation of cultured hamster cells. (a) The sebocyte proliferation was inhibited with UVB on day 2, but on day 4, it was accelerated in dose-dependent manner and on day 7, there were no significant differences in the proliferation between irradiated and non-irradiated cells. (b) The keratinocyte proliferation was inhibited with UVB in dose-dependent manner during days 2 /4. On day 7, the number of keratinocytes tended to be accelerated with UVB irradiation. The data represent the mean9/S.D. of 12 dishes. Significantly different from each day in 0 mJ/cm2 (*P B/ 0.05, **P B/0.01, and ***P B/0.001).
Effects of UVR on the sebaceous gland and sebum secretion in hamsters
increased to about 3.7 times (P B/0.05) and 4.1 times (P B/0.05) higher in the 10 and 20 mJ/cm2 UVB-irradiated group, respectively, than that in the non-irradiated group, cholesterol increased to about 1.3 times (P B/0.05) and 2.0 times (P B/ 0.05) higher, respectively, and FFA increased to about 3.2 times (P B/0.05) higher in the 20 mJ/cm2 UVB-irradiated group than that in the non-irradiated cells (Fig. 2a). On day 7, the amount of TG and cholesterol increased in all groups of cultured sebocytes including non-irradiated cells. However, those in the UVB-irradiated sebocyte were significantly higher than in non-irradiated cells (Fig. 2b). There was no difference in the lipid amounts in the cultured supernatants of sebocytes in the UVBirradiated and non-irradiated groups on day 4 (Fig. 3a). On day 7, TG, FFA, and cholesterol in the cultured supernatants increased depending on the irradiated dose. The 20 mJ/cm2 UVB exposure increased the amounts of TG and FFA to approximately two times (P B/0.001 and 0.01, respectively), but cholesterol to 1.3 times (P B/0.001; Fig. 3b).
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When the skin was irradiated with 150 mJ/cm2 UVB after topical application of TG, the TEWL began to increase significantly on day 1, reached a peak on day 5 (about 3.1 times of the control), and subsequently recovered (Fig. 4, days 1, 3, and 4: P B/0.001 and day 2: P B/0.01). When the skin was irradiated with 300 mJ/cm2 UVB after topical application of TG, the TEWL further markedly increased to about 5.4 times of the control on day 1, about 6.0 times higher at a peak on day 3, and subsequently recovered (Fig. 4, days 1 /3: P B/ 0.001). From days 1 /4 of 150 mJ/cm2 UVB irradiation after topical application of TG and from days 1 /3 of 300 mJ/cm2 UVB irradiation after topical application of TG, the TEWL markedly increased compared with that after an exposure of UVB at 150 and 300 mJ/cm2. Topical application of TG and cholesterol had no effects on the TEWL without UVB irradiation (data not shown).
3.3. Effects of UVB irradiation to the lipidapplied skin on TEWL After an exposure of UVB at 150 mJ/cm2, the TEWL on irradiated skin began to increase on day 2 (P B/0.01), increased on day 4, reached a peak on day 5 (2.8 times of the control, days 4 and 5: P B/ 0.001), and subsequently recovered (Fig. 4). When the skin was irradiated with 300 mJ/cm2 UVB, the TEWL began to increase on day 1, significantly increased on day 2 (day 2: P B/0.001 and day 3: P B/0.01), reached a peak on day 4 (about 4.6 times of the control, P B/0.001) and subsequently recovered (Fig. 4). When the skin was irradiated with 150 mJ/cm2 UVB after topical application of cholesterol, the TEWL showed no significant increase on days 1 and 2, increased on day 3 (P B/0.01), reached a peak on day 5 (about 3.5 times of the control, P B/0.001) and subsequently recovered (Fig. 4). When the skin was irradiated with 300 mJ/cm2 UVB after topical application of cholesterol, the TEWL significantly increased on day 1, reached a peak on day 4 (about 5.1 times of the control), and subsequently recovered (Fig. 4, days 1 /5: P B/0.001). From days 3 /5 of 150 mJ/cm2 UVB irradiation after topical application of cholesterol and on days 1 and 5 of 300 mJ/cm2 UVB irradiation after topical application of cholesterol, the TEWL markedly increased compared with that after an exposure of UVB at 150 and 300 mJ/cm2.
Fig. 2 Effect of UVB on the amount of lipids in cultured hamster sebocytes. (a) On day 4, the amount of TG, FFA, and CHO in sebocytes increased with UVBR in dosedependent manner. (b) On day 7, the amount of TG and CHO in sebocytes significantly increased with UVBR in dose-dependent manner. The data represent the mean9/ S.D. of 12 dishes. Significantly different from each composition in 0 mJ/cm2 (*P B/0.05, **P B/0.01, and ***P B/0.001). Sum of each composition; TG, triglyceride; FFA, free fatty acid; CHO, cholesterol.
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Fig. 3 Effect of UVB on the amount of lipid in the supernatant of cultured hamster sebocytes. (a) On day 4, there were no differences in the lipid amounts in the cultured supernatants in the UVB- and non-irradiated groups. (b) On day 7, the amounts of TG, FFA, and CHO in cultured supernatants increased in dose-dependent manner. The data represent the mean9/S.D. of 12 dishes. Significantly different from each composition in 0 mJ/cm2 (**P B/0.01 and ***P B/0.001). Sum of each composition; TG, triglyceride; FFA, free fatty acid; CHO, cholesterol.
3.4. Effects of in vitro-irradiated cholesterol and TG on TEWL 2
When 1200 mJ/cm UVB-irradiated cholesterol was applied to the back of hamsters, the TEWL increased on day 1 but thereafter showed no marked change (Fig. 5a). After the application of 10 J/cm2 UVA-irradiated cholesterol, the TEWL increased about 2.7 times higher than that of the control on day 1 and subsequently recovered (Fig. 5b, days 1 /3: P B/0.01). When CHO-OOH was applied, on the other hand, the TEWL significantly increased on day 3 after application, reached a peak on day 5 (about 2.5 times of the control), and subsequently recovered (Fig. 5c, day 3: P B/0.01 and days 4 and 5: P B/0.001). In vitro-irradiated TG showed a similar but more significant effects on TEWL (Fig. 5a: UVB, day 1: P B/0.001 and day 2: P B/0.01; Fig. 5b: UVA, day 1: P B/0.001 and days 2 and 3: P B/0.01). Topical application of TG-OOH and in vitro-irradiated TG showed almost similar changes on the TEWL.
4. Discussion The sebaceous gland is a holocrine gland. The sebocytes produce lipid droplets with differentiation and finally rupture to excrete sebum to the skin surface. The amount of excreted sebum changes according to the degrees of proliferation and differentiation of sebocytes. The functions of sebaceous gland are controlled by endocrine factors such as several hormones produced in pituitary gland, adrenal cortex, and gonad [15 /17]. Among them, androgen is the most important hormone. It is known that androgen increases the size of sebaceous gland [18 /20], cell division [18,19,21], and sebum production [20,22,23]. With respect to UV light, it has been reported that in vivo UVB radiation induced hyperplasia of sebaceous glands of hairless mice and hamsters [5,6]. Clinically, it is said that UV light influences on the pathology of acne development [24 /28]. However, the detailed effects of UV light on sebaceous gland have not been clarified yet. We therefore examined the
Effects of UVR on the sebaceous gland and sebum secretion in hamsters
Fig. 4 Effects of UVB irradiation to the lipid-applied skin on TEWL. On day 4, after 150 mJ/cm2 UVBR, TEWL increased twofold compared with untreated control, reached a peak on day 5. On day 2, TEWL increased significantly after 300 mJ/cm2 UVBR, reached a peak on day 4 (4.6-fold). From days 3 /5 of CHO/150 mJ/cm2 UVBR and on days 1 and 5 of CHO/300 mJ/cm2 UVBR, TEWL increased greater than that after a UVB exposure, respectively. From days 1 /4 of TG/150 mJ/cm2 UVBR and from days 1 /3 of TG/300 mJ/cm2 UVBR, TEWL markedly increased compared with that after a UVB exposure, respectively. Values represent the mean9/S.D. of six animals. *P B/0.01 and **P B/0.001.
direct effects of UV light on hamster sebocytes to exclude influences of hormones and other constituent cells of sebaceous glands. Hamster sebocytes seem to be useful as an in vitro model to examine the functions of sebaceous gland [10,11], because cell proliferation and lipid synthesis were similar to those of human sebocytes. With respect to lipid synthesis, we analyzed the amount of lipids in cells and also in the cultured supernatant since the sebaceous gland is a holocrine gland. Epstein et al. [3] examined the effect of UV radiation on the proliferation of keratinocytes in hairless mice. The DNA, RNA, and protein syntheses of keratinocytes were inhibited at 2 /3 h after UVB irradiation, and began to recover at 24 h, and then, increased until day 7, peaking at 48 /72 h [3]. In the present experiment, hamster sebocytes and keratinocytes similarly responded to UVB radiation. Shortly after the UVB irradiation, the proliferations of sebocytes and keratinocytes were transiently suppressed and accelerated thereafter.
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Fig. 5 Effects of lipid peroxides on TEWL (a: topical application of UVB-irradiated lipids in vitro, b: topical application of UVA-irradiated lipids in vitro, and c: topical application of lipid peroxides). (a) When 1200 mJ/cm2 UVB-irradiated CHO was applied, the TEWL increased on day 1 but thereafter showed no marked change. (b) When 10 J/cm2 UVA-irradiated CHO was applied, the TEWL increased on day 1 and subsequently recovered. (c) When CHO-OOH was applied, the TEWL significantly increased on day 3, reached a peak on day 5. (a, b) In vitro-irradiated TG showed more significant effects on the TEWL. (a /c) In vitro-irradiated TG and topical application of TG-OOH showed almost similar changes. Values represent the mean9/S.D. of six animals. *P B/0.01 and **P B/0.001.
It has been reported that the amount of skin surface lipid was altered after irradiation of UV light [7,8]. The rates of cholesterol and FFA in sebum increased, while those of wax ester, TG, and squalene in sebum decreased [7]. However, in the present experiment, the amount of TG, FFA, and cholesterol in sebocytes increased depending on the irradiated UVB dose. Therefore, there are differences in TG changes among these experiments. TG is hydrolyzed in vivo with bacterial lipase produced by microorganisms such as Propionibacterium acnes , Staphylococcus epidermidis ,
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and Pityrosporum to produce FFA. Increased TG by UVB irradiation in sebaceous glands may be hydrolyzed to FFA before reaching at skin surface. If so, the apparently different data described above are explainable. The skin surface lipid is derived from sebocytes and forms the skin surface film, which prevents invasions from outside and inhibits water loss from inside the skin. It also has a bactericidal and emollient effect [29]. The skin surface lipid is always exposed to air and sunlight, and can be oxidized to form lipid peroxides. For instance, UV radiation produces squalene peroxide, which can reduce the skin conductance [30], become comedogenic substances [27,31], and induces various injuries in the skin. Furthermore, Yamazaki et al. [32] reported that cholesterol 7-hydroperoxide, which is a marker for lipid peroxidation, increased in rat skin after UVB irradiation. They also reported the increase of cholesterol hydroperoxides in human skin lipid by sunlight exposure, and they suggested that hydroperoxides of other lipid components could exist in human skin and these hydroperoxides may be increased by sunlight exposure [33]. It is possible that cholesterol and TG are peroxidized due to UV irradiation since these molecules have unsaturated bonds which undergo photooxidation to form highly reactive peroxides. Clinically, it has been reported that seborrhoeic dermatitis of the face was induced by PUVA therapy [34]. Furthermore, it is said that squalene peroxide produced by sunlight exposure irritates skin and causes seborrhoeic dermatitis [35]. The lipid peroxidation on the skin by UV radiation may damage the barrier function of the skin. To address this issue, TEWL was examined after topical application of cholesterol or TG and UV exposures on the back of hamsters. Similar to the previous report in hairless mice [36], UVB exposures alone increased the TEWL until day 7. Moreover, it was found that, UVB irradiation to the TG- or cholesterol-applied skin increases the TEWL significantly. Next, we examined the effects of in vitro-irradiated TG, in vitro-irradiated cholesterol, TG-OOH, and CHOOOH on TEWL. All the materials applied on the skin significantly increased TEWL. The TEWL showed the time course after application of in vitro-irradiated TG almost similar to that after application of TG-OOH. From these results, it was strongly suggested that TG-OOH was synthesized due to UVA and UVB irradiation, which might increase the TEWL. With respect to cholesterol, the change in the TEWL after application of CHOOOH reached a peak on day 5 as observed in UVB irradiation to the cholesterol-applied skin. After application of in vitro-irradiated cholesterol, how-
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ever, the TEWL showed a slight increase on day 1 and subsequently recovered. From these results, it was suggested that CHO-OOH was synthesized due to UVA and UVB irradiation, which might also increase the TEWL. However, the change in the TEWL was small after application of in vitroirradiated cholesterol. It is possible that unreacted free cholesterol existed in in vitro-irradiated cholesterol and this unreacted free cholesterol might protect the dual membrane structure and prevent intercellular permeation of CHO-OOH. From the above in vivo results, it was suggested that sebaceous components such as cholesterol and TG could undergo peroxidation due to irradiation of UV light. These lipid peroxides could permeate into the intercellular spaces to change the dual membrane structure and injure the barrier function of the skin. In conclusion, it was suggested that UV radiation in the sunlight could directly activate the function of sebaceous gland to secrete increased amounts of sebum on the skin surface. Further exposures to UV may peroxidate the skin surface lipids, which damage the barrier functions of the skin. This may explain the dry skin after exposure to UV light in summer and dysfunction of the chronically UVexposed skin.
References [1] Gilchrest BA, Stoff JF, Soter NA. Chronologic aging alters the response to UV-induced inflammation in human skin. J Invest Dermatol 1982;79:11 /5. [2] Danno K, Horio T, Takigawa M, Imamura S. Role of oxygen intermediates in UV-induced epidermal cell injury. J Invest Dermatol 1984;83:166 /8. [3] Epstein JH, Fukuyama K, Fye K. Effects of ultraviolet radiation on the mitotic cycle and DNA, RNA, and protein synthesis in mammalian epidermis in vivo. Photochem Photobiol 1970;12:57 /65. [4] Kupper TS. Mechanisms of cutaneous inflammation. Interactions between epidermal cytokines, adhesion molecules, and leukocytes. Arch Dermatol 1989;125:1406 /12. [5] Lesnik RH, Kligman LH, Kligman AM. Agents that cause enlargement of sebaceous glands in hairless mice. II. Ultraviolet radiation. Arch Dermatol Res 1992;284:106 /8. [6] Dachs U, Plewig G. Effecte des UV-Lichtes auf Hautadnexe am Beispiel des Syrischen Hamsters. Hautartzt 1977;28(Suppl. 2):237 /8. [7] Gloor M, Karenfeld A. Effect of ultraviolet light therapy, given over a period of several weeks, on the amount and composition of the skin surface lipids. Dermatologica 1977;154:5 /13. [8] Weissman A, Noble WC. Photochemotherapy of psoriasis. Br J Dermatol 1980;102:185 /93. [9] Plewig G, Luderschmidt C. Hamster ear model for sebaceous glands. J Invest Dermatol 1977;68:171 /6. [10] Ito A, Sakiguchi T, Kitamura K, Akamatsu H, Horio T. Establishment of a tissue culture system for hamster sebaceous gland cells. Dermatology 1998;197:238 /44.
Effects of UVR on the sebaceous gland and sebum secretion in hamsters [11] Sato T, Imai N, Akimoto N, Sakiguchi T, Kitamura K, Ito A. Epidermal growth factor and 1a,25-dihydroxyvitamin D3 suppress lipogenesis in hamster sebaceous gland cells in vitro. J Invest Dermatol 2001;117:965 /70. [12] Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 1975;6:331 /44. [13] Zouboulis ChC, Korge B, Akamatsu H, Xia L, Schiller S, Gollnick H, Orfanos CE. Effects of 13-cis -retinoic acid, alltrans -retinoic acid, and acitretin on the proliferation, lipid synthesis, and keratin expression of cultured human sebocytes in vitro. J Invest Dermatol 1991;96:792 /7. [14] Ito A, Kitamura K, Sato K, Akamatsu H. A novel enzymatic assay for the quantification of skin surface lipids. J Int Med Res 1996;24:69 /83. [15] Akamatsu H, Zouboulis ChC, Orfanos CE. Control of human sebocyte proliferation in vitro by testosterone and 5-alphadihydrotestosterone is dependent on the localization of the sebaceous glands. J Invest Dermatol 1992;99:509 /11. [16] Zouboulis ChC, Akamatsu H, Stephanek K, Orfanos CE. Androgens affect the activity of human sebocytes in culture in a manner dependent on the localization of the sebaceous glands and their effect is antagonized by spironolactone. Skin Pharmacol 1994;7:33 /40. [17] Akamatsu H, Zouboulis ChC, Orfanos CE. Spironolactone directly inhibits proliferation of cultured human facial sebocytes and acts antagonistically to testosterone and 5a-dihydrotestosterone in vitro. J Invest Dermatol 1993;100:660 /2. [18] Ebling FJ, Ebling E, McCaffery V, Skinner J. The response of the sebaceous glands of the hypophysectomized-castrated male rat to 5-dihydrotestosterone, androstenedione, dehydroepiandrosterone, and androsterone. J Endocrinol 1971;51:181 /90. [19] Ebling FJ, Ebling E, McCaffery V, Skinner J. The responses of the sebaceous glands of the hypophysectomized-castrated male rat to 5-androstanedione and 5-androstane-3,17-diol. J Invest Dermatol 1973;60:183 /7. [20] Thody AJ, Cooper MF, Bowden PE, Meddis D, Shuster S. Effect of alpha-melanocyte-stimulating hormone and testosterone on cutaneous and modified sebaceous glands in the rat. J Endocrinol 1976;71:279 /88. [21] Sauter LS, Loud AV. Morphometric evaluation of sebaceous gland volume in intact, castrated, and testosterone-treated rats. J Invest Dermatol 1975;64:9 /13.
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[22] Burgess TL, Wilson JD. Studies on hormonal regulation of squalene synthesis in preputial gland and skin of the rat. Proc Soc Exp Biol Med 1963;113:747 /50. [23] Sansone G, Davidson W, Cummings B, Reisner RM. Sebaceous gland lipogenesis induced by testosterone: early metabolic events. J Invest Dermatol 1971;57:144 /8. [24] Cunliffe WJ. Treatment of acne. In: Marks R, editor. Acne. UK: Martin Dunitz, 1989:252 /87. [25] Hjorth N, Sjolin KE, Sylvest B, Thomsen K. Acne aestivalis / Mallorca acne. Acta Derm Venereol 1972;52:61 /3. [26] Jones C, Bleehen SS. Acne induced by PUVA treatment. Br Med J 1977;2:866. [27] Motohashi K. Enhanced comedo formation in rabbit ear skin by squalene and oleic acid peroxides. Br J Dermatol 1983;109:191 /8. [28] Mills OH, Kligman AM. Ultraviolet phototherapy and photochemotherapy of acne vulgaris. Arch Dermatol 1978;114:221 /3. [29] Pochi PE. Sebum: its nature and physiopathologic responses. In: Moschella SL, Hurley HJ, editors. Dermatology, 3rd ed.. USA: Saunders, 1992:88 /93. [30] Chiba K, Sone T, Kawakami K, Onoue M. Skin roughness and wrinkle formation induced by repeated application of squalene-monohydroperoxide to the hairless mouse. Exp Dermatol 1999;8:471 /9. [31] Mills OH, Porte M, Kligman AM. Enhancement of comedogenic substances by ultraviolet radiation. Br J Dermatol 1978;98:145 /50. [32] Yamazaki S, Ozawa N, Hiratsuka A, Watabe T. Cholesterol 7hydroperoxides in rat skin as a marker for lipid peroxidation. Biochem Pharmacol 1999;58:1415 /23. [33] Yamazaki S, Ozawa N, Hiratsuka A, Watabe T. Increases in cholesterol 7-hydroperoxides in lipids of human skin by sunlight exposure. Free Radic Biol Med 1999;26:1126 /33. [34] Tegner E. Seborrhoeic dermatitis of the face induced by PUVA treatment. Acta Derm Venereol (Stockholm) 1983;63:335 /9. [35] Nishioka K. Seborrhoeic dermatitis. In: Ikeda S, editor. Standard Text Book of Dermatology, 5th ed. (in Japanese). Tokyo: Igaku-syoin, 1997:104 /5. [36] Holleran WM, Uchida Y, Halkier-Sorensen L, Haratake A, Hara M, Epstain JH, Elias PM. Structural and biochemical basis for the UVB-induced alterations in epidermal barrier function. Photodermatol Photoimmunol Photomed 1997;13:117 /28.
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Effects of UV irradiation on the sebaceous gland and sebum secretion in hamsters Yasuchiyo Akitomoa,*, Hirohiko Akamatsub, Yuri Okanoc, Hitoshi Masakic, Takeshi Horioa a
Department of Dermatology, Kansai Medical University, 10-15, Humizono-cho, Moriguchi, Osaka 5708506, Japan b Department of Dermatology, Fujita Health University School of Medicine, 1-98, Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan c Cosmos Technical Center Co. Ltd., 3-24-3, Hasune, Itabashi-ku, Tokyo 174-0046, Japan Received 2 September 2002; received in revised form 2 December 2002; accepted 2 December 2002
KEYWORDS Hamster sebocyte; Ultraviolet irradiation; Lipid peroxide; Transepidermal water loss
Summary Background: Although an understanding of the photobiology of the skin has been extensively advanced recently, the effect of ultraviolet (UV) radiation on sebaceous glands is not well known. Objective: In this study, we examined the direct effect of UV radiation on cultured sebocytes from hamsters in vitro experimental system. Moreover, we examined whether UV-induced peroxidation of skin surface lipids may affect barrier function of horney layer. Methods: We irradiated cultured sebocytes from hamsters, which have similar biological characteristics to the human sebocytes, with UV radiation. Moreover, transepidermal water loss (TEWL) was examined after topical application of cholesterol or triglyceride (TG) and UV exposures on the back of hamsters. Results: The number of sebocytes were increased significantly (120 /140%) after 4 days as compared with the non-irradiated controls. Lipid production in sebocytes was also increased on day 7 in an irradiation-dependent manner up to 4.1 times of the pre-irradiated level. When UVB was irradiated to TG- or cholesterol-applied skin at the minimum ear-swelling dose, TEWL increased twice or more as compared with UVB irradiation to unapplied sites. When in vitro-irradiated TG, in vitro-irradiated cholesterol, TG-peroxide (TG-OOH), and cholesterol-peroxide (CHO-OOH) were applied to the skin, TEWL increased significantly. Conclusion: These results suggest that UVB may directly activate the functions of the sebaceous gland in vivo to produce increased amounts of sebum, which may undergo peroxidation by UV light and damage the barrier functions of the skin. – 2003 Japanese Society for Investigative Dermatology. Published by Elsevier Science Ireland Ltd. All rights reserved.
1. Introduction *Corresponding author. Tel.: /81-6-6992-1001; fax: /81-66992-5965. E-mail address: [email protected] (Y. Akitomo).
Much is known about the acute and chronic effects of UV radiation on epidermis and dermis [1 /4]. In contrast, only limited investigations have
0923-1811/03/$30.00 – 2003 Japanese Society for Investigative Dermatology. Published by Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0923-1811(03)00003-3
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been aimed at the effects on the skin appendage such as the sebaceous gland. The DNA, RNA, and protein syntheses of keratinocytes are depressed shortly after UV exposure, but recover and accelerate thereafter [3]. These findings may explain the epidermal hyperplasia after UV irradiation. Chronic exposures to the sunlight clinically cause sebaceous gland hyperplasia on the face as one of the manifestations of the photoaging in human. However, the dynamics of sebocytes after UV irradiation has not been fully understood. It has been reported that, when hairless mice or hamsters were exposed to UVB in vivo, the sebaceous glands showed hyperplasia and the number of sebocytes increased [5,6]. These studies suggest that UV radiation in the sunlight can directly or indirectly influence the sebaceous gland. In this study, we examined the direct effect of UV radiation on cultured sebocytes from hamsters in vitro experimental system. We also examine the effect of UV radiation on the sebum production because there are some reports that UVB phototherapy or PUVA photochemotherapy increased the amount of skin surface lipids [7,8]. It has been shown that hamsters are useful as an experimental animal to examine the functions of sebaceous gland because the hamster sebaceous gland is similar to the human gland with regard to its size, response to androgens, and turnover time in vivo [9]. Recently, we established a tissue culture system for hamster sebaceous glands without damaging their functions [10]. Furthermore, it has been demonstrated that these hamster sebocytes are similar to human sebocytes in the proliferation and lipid synthesis [11]. Moreover, we examined whether UV-induced peroxidation of skin surface lipids may affect barrier function of horney layer.
2. Materials and methods 2.1. In vitro experiment 2.1.1. Culture of sebocytes Earlobes removed from 5-week-old golden male hamsters were incubated in Dulbecco’s modified Eagle’s medium (DMEM; Nikken Biomedical Laboratory, Kyoto, Japan) with 100 IU/ml penicillin and 100 mg/ml streptomycin (Flow laboratories Ltd., Ayrshire, Scotland) for 3 h at 4 8C, and were successively washed with Ca2- and Mg2-free phosphate-buffered saline (PBS(/)), then cut into 5 /5 mm2 pieces. They were incubated for 14 h in 2.4 U/ml dispase (Godoshusei Co. Ltd., Tokyo, Japan) at 4 8C to separate epidermis from
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dermis. The epidermis was peeled from the dermis in PBS(/), and sebaceous glands were isolated from the dermis by using microsurgical instrument under microscopy. The isolated sebaceous glands were seeded in 60 mm culture dishes (Becton Dickinson, Tokyo, Japan) on a 3T3-cell feeder layer in the medium consisted of DMEM and Ham’s F12 medium (1:1; Gibco BRL, NY, USA), 8% heatdenatured fetal calf serum (Gibco BRL), 2% human serum, 10 ng/ml epidermal growth factor (Gibco BRL), 3.4 mM L-glutamine (Gibco BRL), 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37 8C with 5% CO2. DMEM and Ham’s F12 medium contain 1.0 mM CaCl2. The 3T3-cell proliferation has been blocked by the addition of mitomycin-C (Sigma, Deisenhofen, Germany) for 4 h, 24 h before gland seeding [12]. 3T3-cell feeder layer was used only for primary sebocyte cultures. All experiments were performed using secondary sebocyte cultures. 2.1.2. Culture of keratinocytes The epidermal sheets were processed to singlecell suspensions using 0.25% trypsin. The isolated keratinocytes were cultured on 3T3-cell feeder layer in the medium consisted of DMEM low glucose (Gibco BRL), 8% heat-denatured fetal calf serum, 3.4 mM L-glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin by 37 8C with 5% CO2. DMEM low glucose contains 1.8 mM CaCl2. 3T3-cell feeder layer was used only for primary cultures. All experiments were performed using secondary keratinocyte cultures. 2.1.3. Ultraviolet irradiation The UVB source utilized was a parallel bank of seven fluorescent sunlamps (FL20S.E30/DMR light bulbs, Toshiba Medical Supply, Tokyo, Japan) with an emission spectrum of 275 /375 nm peaking at 305 nm. The UVA source was a bank of 14 black lights (FL32S.BL, Toshiba Medical Supply, Tokyo, Japan) with an emission spectrum of 300 /430 nm peaking at 352 nm. The irradiance of UVA and UVB was measured by a UV radiometer, UVR-305/365D(II). 2.1.4. Cell proliferation On day 0, sebocytes and keratinocytes were seeded in 60 mm plates (Becton Dickinson) at concentration of 3.5 /103 cells/cm2 and were left to attach for 24 h at 37 8C with 5% CO2 in each culture medium. On day 1, medium was changed to PBS(/), then cultured cells were exposed to UVB at doses of 10 or 20 mJ/cm2, which did not influence cell viabilities determined by the exclusion of trypan blue
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dye 24 h after UVB irradiation. After UVB irradiation, PBS(/) was changed to each culture medium, and cells were incubated at 37 8C with 5% CO2. On days 2, 4, and 7, the number of cells in suspensions was counted with a blood cell counter. 2.1.5. Analysis of intracellular lipid content Lipids (TG, free fatty acid (FFA), and cholesterol) were extracted from cultured cells according to the method of Zouboulis et al. [13]. Briefly, the culture medium was removed from the culture dishes. Remaining cells were washed three times with PBS(/) and were harvested after treatment with 0.25% trypsin and 0.02% ethylenediamine tetraacetic acid (EDTA) in PBS(/). The cells were suspended in a solution of chloroform /methanol (2:1) and sonicated. The filtrates from the solutions were evaporated at 40 8C under a stream of nitrogen. The lipids were solubilized in 100 ml chloroform /methanol (2:1, v/v) and spotted on high-performance thin-layer chromatography (HPTLC) plates (HPTLC silica gel 60, 10 /20 cm2, Merck, Darmstadt, Germany). TG, FFA, and cholesterol were resolved by using the following development system. The plates were developed twice in hexane:ethylether:acetic acid (56:14:0.3). After charring the chromatograms with 10% CuSO4 and 8% H3PO4 aqueous solution at 200 8C for 5 min, photodensitometry was performed and the isolated lipid fractions were quantified by comparison with commercially available lipid standards, run on the sample plates: triolein (Sigma), FFA (Sigma), and cholesterol (Sigma). 2.1.6. Lipid content of culture medium Culture medium was collected on days 4 and 7, and lipid contents were quantified by enzymatic assay described by Ito et al. [14]. TGs were degraded into glycerol and FFAs by lipase. Glycerol produces hydrogen peroxide under the catalytic activity of glycerol kinase and glycerol-3-phosphate oxidase. The hydrogen peroxide, with peroxidase catalysis, induces oxidative condensation of 4-aminoantipyrine (4-AA) and 3,5dimethoxy-N-ethyl-N-(2?-hydroxy-3?-sulfopropyl)aniline sodium (DAOS) (a chromogen), to produce a blue quinone pigment with a maximum absorbance wavelength of 600 nm. The absorbance at 600 nm was measured with a spectrophotometer and the amount of TGs was determined by standard curves. Measurements were performed using triglycerides E -test Wako (Wako Pure Chemical Industries, Osaka, Japan). FFAs produce acyl-CoA under the catalytic influence of acyl-CoA synthetase. Acyl-CoA in turn
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generates hydrogen peroxide, in a reaction catalyzed by acyl-CoA oxidase. Hydrogen peroxide, with peroxidase catalysis, quantitatively induces oxidative condensation of 4-AA and 3-methyl-Nethyl-N -(b-hydroxyethyl)aniline (a chromogen), to produce a reddish-purple quinone pigment with a maximum absorbance wavelength of 550 nm. The absorbance at 550 nm was measured with a spectrophotometer and the amount of FFAs was determined by standard curves. Measurements were performed using esterified fatty acid C -test Wako (Wako Pure Chemical Industries). Cholesterol esters were degraded into free cholesterol and FFAs catalyzed by cholesterol esterase. Free cholesterol subsequently generates hydrogen peroxide, in a reaction catalyzed by cholesterol oxidase. Hydrogen peroxide, with peroxidase catalysis, quantitatively induces oxidative condensation of 4-AA and DAOS, to produce a blue quinone pigment with a maximum absorbance wavelength of 600 nm. The absorbance at 600 nm was measured with a spectrophotometer and the amount of cholesterol was determined by standard curves. Measurements were performed using cholesterol E -test Wako (Wako Pure Chemical Industries).
2.2. In vivo experiment 2.2.1. Animals Six-week-old golden male hamsters were used (n /6). 2.2.2. Topical application of test chemicals and UVR Hamster’s hair on the back was removed 3 days before the beginning of the experiments. TEWL was measured on the day prior to topical application and UVR. Test solution (20 ml/cm2) was topically applied to the depilated dorsal area on day 0. Soon after the application, the back area was exposed to UVB at a dose of 150 (approximately one earswelling dose) or 300 mJ/cm2. 2.2.3. Transepidermal water loss Transepidermal water loss (TEWL) was measured on the back surface of the hamsters before and various time points after UV irradiation, using an evaporimeter (EP1, Servomed, Stockholm, Sweden). 2.2.4. Materials for topical application Cholesterol powder was dissolved in the 20% ethanol in 1,3-butyleneglycol and 0.5% cholesterol or cholesterol-peroxide (CHO-OOH) was used for each study.
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CHO-OOH was synthesized by photooxidation of cholesterol in the presence of methylene blue as a photosensitizer. Briefly, cholesterol dissolved in ethanol was irradiated with visible light at 4 8C overnight in the presence of 0.1% methylene blue. Methylene blue remained in the reaction solution was removed using silica gel column chromatography (Wakogel c-200, Wako Pure Chemical Industries). CHO-OOH production was confirmed for the hydroperoxide assay by high-performance liquid chromatography (HPLC) coupled with chemiluminescence detection (CL-HPLC method). Next, the ethanol solution was displaced with the hexane solution by evaporating under a nitrogen gas stream, and centrifuged to remove free cholesterol. Residual free cholesterol was removed from CHO-OOH hexane /acetic ether (7:3, v/v) solution three times using silica gel column. TG-peroxide (TG-OOH) was synthesized by photooxidation of TG in the presence of methylene blue. TG in ethanol was irradiated with visible light at 4 8C overnight in the presence of 0.1% methylene blue. Ethanol was removed by evaporating and the solvent was replaced with chloroform, then methylene blue was removed with silica gel column chromatography. Next, free TG was removed from TG-OOH with silica gel column chromatography using hexane /ether (56:14, v/v) solution as solvent.
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ing on the irradiated dose. The number of 10 or 20 mJ/cm2 UVB-irradiated cells was about 1.2 times (P B/0.05) or 1.4 times (P B/0.01) higher than that of non-irradiated cells, respectively. On day 7, however, there was no difference in the proliferation between irradiated and non-irradiated cells (Fig. 1a). Keratinocyte proliferation was inhibited with UVB in a dose-dependent manner during days 2 / 4. On day 4, the number of 20 mJ/cm2 UVBirradiated cells was about 2/3 (P B/0.01) of nonirradiated cells. On day 7, on the contrary, proliferation of cells tended to be accelerated depending on the irradiated dose, although there was no significant difference between irradiated and non-irradiated cells (Fig. 1b).
3.2. Effects of UVB on the lipid production of cultured sebocytes The amount of lipids in sebocytes increased depending on the irradiated dose on day 4. TG
2.2.5. In vitro UVR on cholesterol and TG TG and cholesterol in vehicle in Petri dish were exposed to UVB at a dose of 1200 mJ/cm2. As concerns UVA, cholesterol, and TG were exposed at a dose of 10 J/cm2 in the presence of coproporphyrin (Sigma) as a photosensitizer.
2.3. Statistical analysis Statistical analysis was performed using the analysis of variance (ANOVA). Statistical significance was defined as P B/0.05, 0.01, and 0.001.
3. Results 3.1. Effects of UVB on proliferation of cultured sebocytes and keratinocytes Proliferation of sebocytes was inhibited on day 2 depending on the dose of UVB. The number of 10 or 20 mJ/cm2 UVB-irradiated cells was about 2/3 (P B/0.001) or 1/2 (P B/0.001) of non-irradiated cells, respectively. On day 4, on the contrary, proliferation of the cells was accelerated depend-
Fig. 1 Effect of UVB on proliferation of cultured hamster cells. (a) The sebocyte proliferation was inhibited with UVB on day 2, but on day 4, it was accelerated in dose-dependent manner and on day 7, there were no significant differences in the proliferation between irradiated and non-irradiated cells. (b) The keratinocyte proliferation was inhibited with UVB in dose-dependent manner during days 2 /4. On day 7, the number of keratinocytes tended to be accelerated with UVB irradiation. The data represent the mean9/S.D. of 12 dishes. Significantly different from each day in 0 mJ/cm2 (*P B/ 0.05, **P B/0.01, and ***P B/0.001).
Effects of UVR on the sebaceous gland and sebum secretion in hamsters
increased to about 3.7 times (P B/0.05) and 4.1 times (P B/0.05) higher in the 10 and 20 mJ/cm2 UVB-irradiated group, respectively, than that in the non-irradiated group, cholesterol increased to about 1.3 times (P B/0.05) and 2.0 times (P B/ 0.05) higher, respectively, and FFA increased to about 3.2 times (P B/0.05) higher in the 20 mJ/cm2 UVB-irradiated group than that in the non-irradiated cells (Fig. 2a). On day 7, the amount of TG and cholesterol increased in all groups of cultured sebocytes including non-irradiated cells. However, those in the UVB-irradiated sebocyte were significantly higher than in non-irradiated cells (Fig. 2b). There was no difference in the lipid amounts in the cultured supernatants of sebocytes in the UVBirradiated and non-irradiated groups on day 4 (Fig. 3a). On day 7, TG, FFA, and cholesterol in the cultured supernatants increased depending on the irradiated dose. The 20 mJ/cm2 UVB exposure increased the amounts of TG and FFA to approximately two times (P B/0.001 and 0.01, respectively), but cholesterol to 1.3 times (P B/0.001; Fig. 3b).
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When the skin was irradiated with 150 mJ/cm2 UVB after topical application of TG, the TEWL began to increase significantly on day 1, reached a peak on day 5 (about 3.1 times of the control), and subsequently recovered (Fig. 4, days 1, 3, and 4: P B/0.001 and day 2: P B/0.01). When the skin was irradiated with 300 mJ/cm2 UVB after topical application of TG, the TEWL further markedly increased to about 5.4 times of the control on day 1, about 6.0 times higher at a peak on day 3, and subsequently recovered (Fig. 4, days 1 /3: P B/ 0.001). From days 1 /4 of 150 mJ/cm2 UVB irradiation after topical application of TG and from days 1 /3 of 300 mJ/cm2 UVB irradiation after topical application of TG, the TEWL markedly increased compared with that after an exposure of UVB at 150 and 300 mJ/cm2. Topical application of TG and cholesterol had no effects on the TEWL without UVB irradiation (data not shown).
3.3. Effects of UVB irradiation to the lipidapplied skin on TEWL After an exposure of UVB at 150 mJ/cm2, the TEWL on irradiated skin began to increase on day 2 (P B/0.01), increased on day 4, reached a peak on day 5 (2.8 times of the control, days 4 and 5: P B/ 0.001), and subsequently recovered (Fig. 4). When the skin was irradiated with 300 mJ/cm2 UVB, the TEWL began to increase on day 1, significantly increased on day 2 (day 2: P B/0.001 and day 3: P B/0.01), reached a peak on day 4 (about 4.6 times of the control, P B/0.001) and subsequently recovered (Fig. 4). When the skin was irradiated with 150 mJ/cm2 UVB after topical application of cholesterol, the TEWL showed no significant increase on days 1 and 2, increased on day 3 (P B/0.01), reached a peak on day 5 (about 3.5 times of the control, P B/0.001) and subsequently recovered (Fig. 4). When the skin was irradiated with 300 mJ/cm2 UVB after topical application of cholesterol, the TEWL significantly increased on day 1, reached a peak on day 4 (about 5.1 times of the control), and subsequently recovered (Fig. 4, days 1 /5: P B/0.001). From days 3 /5 of 150 mJ/cm2 UVB irradiation after topical application of cholesterol and on days 1 and 5 of 300 mJ/cm2 UVB irradiation after topical application of cholesterol, the TEWL markedly increased compared with that after an exposure of UVB at 150 and 300 mJ/cm2.
Fig. 2 Effect of UVB on the amount of lipids in cultured hamster sebocytes. (a) On day 4, the amount of TG, FFA, and CHO in sebocytes increased with UVBR in dosedependent manner. (b) On day 7, the amount of TG and CHO in sebocytes significantly increased with UVBR in dose-dependent manner. The data represent the mean9/ S.D. of 12 dishes. Significantly different from each composition in 0 mJ/cm2 (*P B/0.05, **P B/0.01, and ***P B/0.001). Sum of each composition; TG, triglyceride; FFA, free fatty acid; CHO, cholesterol.
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Fig. 3 Effect of UVB on the amount of lipid in the supernatant of cultured hamster sebocytes. (a) On day 4, there were no differences in the lipid amounts in the cultured supernatants in the UVB- and non-irradiated groups. (b) On day 7, the amounts of TG, FFA, and CHO in cultured supernatants increased in dose-dependent manner. The data represent the mean9/S.D. of 12 dishes. Significantly different from each composition in 0 mJ/cm2 (**P B/0.01 and ***P B/0.001). Sum of each composition; TG, triglyceride; FFA, free fatty acid; CHO, cholesterol.
3.4. Effects of in vitro-irradiated cholesterol and TG on TEWL 2
When 1200 mJ/cm UVB-irradiated cholesterol was applied to the back of hamsters, the TEWL increased on day 1 but thereafter showed no marked change (Fig. 5a). After the application of 10 J/cm2 UVA-irradiated cholesterol, the TEWL increased about 2.7 times higher than that of the control on day 1 and subsequently recovered (Fig. 5b, days 1 /3: P B/0.01). When CHO-OOH was applied, on the other hand, the TEWL significantly increased on day 3 after application, reached a peak on day 5 (about 2.5 times of the control), and subsequently recovered (Fig. 5c, day 3: P B/0.01 and days 4 and 5: P B/0.001). In vitro-irradiated TG showed a similar but more significant effects on TEWL (Fig. 5a: UVB, day 1: P B/0.001 and day 2: P B/0.01; Fig. 5b: UVA, day 1: P B/0.001 and days 2 and 3: P B/0.01). Topical application of TG-OOH and in vitro-irradiated TG showed almost similar changes on the TEWL.
4. Discussion The sebaceous gland is a holocrine gland. The sebocytes produce lipid droplets with differentiation and finally rupture to excrete sebum to the skin surface. The amount of excreted sebum changes according to the degrees of proliferation and differentiation of sebocytes. The functions of sebaceous gland are controlled by endocrine factors such as several hormones produced in pituitary gland, adrenal cortex, and gonad [15 /17]. Among them, androgen is the most important hormone. It is known that androgen increases the size of sebaceous gland [18 /20], cell division [18,19,21], and sebum production [20,22,23]. With respect to UV light, it has been reported that in vivo UVB radiation induced hyperplasia of sebaceous glands of hairless mice and hamsters [5,6]. Clinically, it is said that UV light influences on the pathology of acne development [24 /28]. However, the detailed effects of UV light on sebaceous gland have not been clarified yet. We therefore examined the
Effects of UVR on the sebaceous gland and sebum secretion in hamsters
Fig. 4 Effects of UVB irradiation to the lipid-applied skin on TEWL. On day 4, after 150 mJ/cm2 UVBR, TEWL increased twofold compared with untreated control, reached a peak on day 5. On day 2, TEWL increased significantly after 300 mJ/cm2 UVBR, reached a peak on day 4 (4.6-fold). From days 3 /5 of CHO/150 mJ/cm2 UVBR and on days 1 and 5 of CHO/300 mJ/cm2 UVBR, TEWL increased greater than that after a UVB exposure, respectively. From days 1 /4 of TG/150 mJ/cm2 UVBR and from days 1 /3 of TG/300 mJ/cm2 UVBR, TEWL markedly increased compared with that after a UVB exposure, respectively. Values represent the mean9/S.D. of six animals. *P B/0.01 and **P B/0.001.
direct effects of UV light on hamster sebocytes to exclude influences of hormones and other constituent cells of sebaceous glands. Hamster sebocytes seem to be useful as an in vitro model to examine the functions of sebaceous gland [10,11], because cell proliferation and lipid synthesis were similar to those of human sebocytes. With respect to lipid synthesis, we analyzed the amount of lipids in cells and also in the cultured supernatant since the sebaceous gland is a holocrine gland. Epstein et al. [3] examined the effect of UV radiation on the proliferation of keratinocytes in hairless mice. The DNA, RNA, and protein syntheses of keratinocytes were inhibited at 2 /3 h after UVB irradiation, and began to recover at 24 h, and then, increased until day 7, peaking at 48 /72 h [3]. In the present experiment, hamster sebocytes and keratinocytes similarly responded to UVB radiation. Shortly after the UVB irradiation, the proliferations of sebocytes and keratinocytes were transiently suppressed and accelerated thereafter.
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Fig. 5 Effects of lipid peroxides on TEWL (a: topical application of UVB-irradiated lipids in vitro, b: topical application of UVA-irradiated lipids in vitro, and c: topical application of lipid peroxides). (a) When 1200 mJ/cm2 UVB-irradiated CHO was applied, the TEWL increased on day 1 but thereafter showed no marked change. (b) When 10 J/cm2 UVA-irradiated CHO was applied, the TEWL increased on day 1 and subsequently recovered. (c) When CHO-OOH was applied, the TEWL significantly increased on day 3, reached a peak on day 5. (a, b) In vitro-irradiated TG showed more significant effects on the TEWL. (a /c) In vitro-irradiated TG and topical application of TG-OOH showed almost similar changes. Values represent the mean9/S.D. of six animals. *P B/0.01 and **P B/0.001.
It has been reported that the amount of skin surface lipid was altered after irradiation of UV light [7,8]. The rates of cholesterol and FFA in sebum increased, while those of wax ester, TG, and squalene in sebum decreased [7]. However, in the present experiment, the amount of TG, FFA, and cholesterol in sebocytes increased depending on the irradiated UVB dose. Therefore, there are differences in TG changes among these experiments. TG is hydrolyzed in vivo with bacterial lipase produced by microorganisms such as Propionibacterium acnes , Staphylococcus epidermidis ,
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and Pityrosporum to produce FFA. Increased TG by UVB irradiation in sebaceous glands may be hydrolyzed to FFA before reaching at skin surface. If so, the apparently different data described above are explainable. The skin surface lipid is derived from sebocytes and forms the skin surface film, which prevents invasions from outside and inhibits water loss from inside the skin. It also has a bactericidal and emollient effect [29]. The skin surface lipid is always exposed to air and sunlight, and can be oxidized to form lipid peroxides. For instance, UV radiation produces squalene peroxide, which can reduce the skin conductance [30], become comedogenic substances [27,31], and induces various injuries in the skin. Furthermore, Yamazaki et al. [32] reported that cholesterol 7-hydroperoxide, which is a marker for lipid peroxidation, increased in rat skin after UVB irradiation. They also reported the increase of cholesterol hydroperoxides in human skin lipid by sunlight exposure, and they suggested that hydroperoxides of other lipid components could exist in human skin and these hydroperoxides may be increased by sunlight exposure [33]. It is possible that cholesterol and TG are peroxidized due to UV irradiation since these molecules have unsaturated bonds which undergo photooxidation to form highly reactive peroxides. Clinically, it has been reported that seborrhoeic dermatitis of the face was induced by PUVA therapy [34]. Furthermore, it is said that squalene peroxide produced by sunlight exposure irritates skin and causes seborrhoeic dermatitis [35]. The lipid peroxidation on the skin by UV radiation may damage the barrier function of the skin. To address this issue, TEWL was examined after topical application of cholesterol or TG and UV exposures on the back of hamsters. Similar to the previous report in hairless mice [36], UVB exposures alone increased the TEWL until day 7. Moreover, it was found that, UVB irradiation to the TG- or cholesterol-applied skin increases the TEWL significantly. Next, we examined the effects of in vitro-irradiated TG, in vitro-irradiated cholesterol, TG-OOH, and CHOOOH on TEWL. All the materials applied on the skin significantly increased TEWL. The TEWL showed the time course after application of in vitro-irradiated TG almost similar to that after application of TG-OOH. From these results, it was strongly suggested that TG-OOH was synthesized due to UVA and UVB irradiation, which might increase the TEWL. With respect to cholesterol, the change in the TEWL after application of CHOOOH reached a peak on day 5 as observed in UVB irradiation to the cholesterol-applied skin. After application of in vitro-irradiated cholesterol, how-
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ever, the TEWL showed a slight increase on day 1 and subsequently recovered. From these results, it was suggested that CHO-OOH was synthesized due to UVA and UVB irradiation, which might also increase the TEWL. However, the change in the TEWL was small after application of in vitroirradiated cholesterol. It is possible that unreacted free cholesterol existed in in vitro-irradiated cholesterol and this unreacted free cholesterol might protect the dual membrane structure and prevent intercellular permeation of CHO-OOH. From the above in vivo results, it was suggested that sebaceous components such as cholesterol and TG could undergo peroxidation due to irradiation of UV light. These lipid peroxides could permeate into the intercellular spaces to change the dual membrane structure and injure the barrier function of the skin. In conclusion, it was suggested that UV radiation in the sunlight could directly activate the function of sebaceous gland to secrete increased amounts of sebum on the skin surface. Further exposures to UV may peroxidate the skin surface lipids, which damage the barrier functions of the skin. This may explain the dry skin after exposure to UV light in summer and dysfunction of the chronically UVexposed skin.
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