Caspase-3 Activation and DNA Damage in Pig Skin Organ Culture After Solar Irradiation

Written By: Sam Savage

Published Date: October 5, 2008 Last Edited: October 5, 2008

By Bacqueville, Daniel Mavon, Alain

ABSTRACT In the present study, a convenient and easy-to-handle skin organ culture was developed from domestic pig ears using polycarbonate Transwell(R) culture inserts in 12-well plate. This alternative model was then tested for its suitability in analyzing the shortterm effects of a single solar radiation dose (from 55 to 275 kJ m^sup -2^). Differentiation of the pig skin was maintained for up to 48 h in culture, and its morphology was similar to that of fresh human skin. Solar irradiation induced a significant release of the cytosolic enzymes lactate dehydrogenase and extracellular signal- related kinase 2 protein in the culture medium 24 h after exposure. These photocytotoxic effects were associated with the formation of sunburn cells, thymine dimers and DNA strand breaks in both the epidermis and dermis. Interestingly, cell death was dose dependent and associated with p53 protein upregulation and strong caspase-3 activation in the basal epidermis. None of these cellular responses was observed in non-irradiated skin. Finally, topical application of a broad-spectrum UVB + A sunfilter formulation afforded efficient photoprotection in irradiated explants. Thus, the ex vivo pig ear skin culture may be a useful tool in the assessment of solar radiation-induced DNA damage and apoptosis, and for evaluating the efficacy of sunscreen formulations.

INTRODUCTION

Ultraviolet (UV) radiation present in sunlight is a major environmental human carcinogen that also contributes to the photoaging process and remains a useful therapeutic agent for various skin diseases (psoriasis, vitiligo and atopic dermatitis) (1- 4). To overcome the risk of inducing skin cancer in humans, experimental models have been developed both in vitro and in vivo to study UV-induced cytotoxicity and mutagenicity. Accordingly, the HaCaT cells have been extensively used in vitro, but they are more susceptible to UVB-induced apoptosis than normal keratinocytes (KCs) and display aberrant signaling (5,6), suggesting that caution should be used in extrapolating the biological responses observed in cultured cells to those of normal human KCs. In addition, cell culture is not adapted to topical application of a formulation and does not mimic tissue microenvironment. Therefore, human epidermis models have been reconstructed in vitro by tissue engineering (7- 10). Although these three-dimensional models exhibit a weak barrier function, they are an innovative means of studying skin biology, UV- induced cytotoxicity and sunscreen photoprotection (11,12). Human skin culture following plastic surgery also represents an interesting alternative model, as this ex vivo approach allows the skin to be fully differentiated at the time of biopsy, and it maintains a good barrier function (12,13). Recently, human explants have been used to demonstrate that the topical application of a vitamin E prodrug improves resistance to UV radiation (14), as observed in vivo in volunteers (15). However, these studies can only be performed on a limited number of subjects, as the availability of human tissue remains limited.

Various animal species have also been used to describe UV- activated molecular pathways. Among them, pig skin has received much attention, owing to its high resemblance to human skin (histology, physiology and barrier function) (1619). In vivo experiments have highlighted the relevance of porcine skin as a surrogate to human skin in phototoxicologic studies. For example, it has been shown that UVB-induced apoptosis is enhanced in hyperproliferative skin (20) and that topically applied antioxidants protect the skin against solar irradiation-induced oxidative stress (21,22). A few research groups have developed an ex vivo approach, although porcine skin purchased from a slaughterhouse is available in large amounts (as a byproduct of the meat industry) and complies with the 3R (reduce, refine and replace) concept on animal experimentation and the ban on animal testing for final products in the European cosmetic industry (23-26). In this respect, the sensitivity of porcine skin to the UVA-induced DNA breaks is similar to that of human skin (27), and an isolated perfused porcine skin flap system is being used for percutaneous absorption and toxicology studies (28). Interestingly, Rijnkels et al. (29,30) have recently set up a full-thickness skin organ culture from the back of the domestic pig. This ex vivo culture system was developed on a nylon grid in a Petri dish. It revealed that UVB induces dose- and time-dependent tissue damage (30) and that a topical dose of antioxidants such as alpha- tocopherol reduces UVB-induced oxidative stress and lipid peroxidation, thereby decreasing apoptotic response (29). Unfortunately, these studies were limited to UVB exposure and did not address the location of apoptotic cells in the skin as the biochemical assays were performed from freshly isolated KCs.

Thus, the present study was first undertaken in order to develop a convenient and easy-to-handle short-term skin organ culture from domestic pig ears using polycarbonate Transwell(R) culture inserts in 12-well plate. We then explored whether this ex vivo organ culture system is suitable for investigating solar radiation- induced cytotoxicity, DNA damage and apoptosis. Finally, a broad- spectrum UVB + A sunscreen formulation was applied to the skin to determine if this alternative model could be useful in the assessment of photoprotection.

MATERIALS AND METHODS

Pig ear skin organ culture and human akin. Skin organ cullure was developed from domestic pig ears (Pietrain breed, 6-month-old female) as they are easily obtained from a local abattoir and they constitute a little-known alternative model to human skin. After cleaning and shaving, the skin was immediately excised from the outer side with a scalpel, then sectioned at a thickness of 500 [mu]m using a dermatome (Aesculap, Tuttlingen, Germany) and punched into 12 mm diameter discs (1.1 cm^sup 2^). The punch areas were free of structural changes such as scratches, erosions and scars, as such skin damage could affect the organ culture and cellular responses to UV radiation. Before starting the organ culture, the explants were washed for 1 h in the culture medium at 37[degrees]C in a 5% CO2/ air incubator. The culture medium was Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1 mM pyruvate, 8 mM glutamine, 100 U penicillin, 100 [mu]g mL^sup -1^ streptomycin and 100 [mu]g mL^sup – 1^ gentamycin (all from Sigma, St. Quentin Fallavier, France), and finally 2.5 [mu]g mL^sup -1^ fungizone (Invitrogen, Cergy Pontoise, France). The skin was then seeded dermal side down in polycarbonate Transwell*(R) inserts (12 mm diameter, 12 [mu]m pore size. Corning Life Sciences, Avon, France) in 12-well plate prefilled with 1 mL culture medium. To improve tissue adhesion, the inserts were coated with 0.25% gelatin type A from porcine skin (Sigma) for 30 min at room temperature and dried overnight in the incubator. Thus, this ex vivo organ culture system maintained the explants at the air-liquid interface and fed the dermis and the epidermis by nutrient diffusion across the insert. The culture medium was changed 4 h after seeding and the skin was cultured for a maximum of 48 h for all irradiation experiments. Fresh human skin was obtained from two healthy adults undergoing abdominoplasty after informed consent and approval of the Institutional Review Board.

Solar-simulated radiation and sunscreen photoprotection. Solar- simulated radiation (SSR) was applied using a Suntest CPS^sup +^ chamber (ATLAS Material Testing Technology BV, Moussy le Neuf, France) equipped with an NXE 1500 Xenon lamp, and fitted with a UV filler to eliminate wavelengths less than 290 nm. The irradiance in UV spectra (from 290 to 400 nm) was 60.9 W m^sup -2^ measured with an MSS2040 spectroradiometer (MSS Elektronik GmbH, Bielefeld, Germany). The skin was exposed to various SSR doses (0, 55, 110 and 275 kJ.m^sup -2^), and the irradiation chamber was maintained at 37[degrees]C using ice-cold water and airflow. The explants were fed with 1 mL fresh culture medium immediately after irradiation and cultured for 24 h at 37[degrees]C. Each experiment was conducted at least three times and the explants were weighed before harvesting.

Sunscreen photoprotection was achieved using a commercial suncare product (Laboratoires Avene, Boulogne, France), a water-inoil emulsion containing water, glycerin, ethylhexyl methoxycinnamate, bis-ethylhexyloxyphenol methoxyphenyl triazine, methylene bis benzotriazolyl tetramethylbutylphenol, decyl glucoside, cyclopentadecamethyl siloxane, glyceryl stearate, C12-15 alkyl benzoate, ethylhexyl palmitate, potassium cetyl phosphate, cetylic alcohol, stearic alcohol, PVP/eicosene copolymer, magnesium aluminum silicate, xanthan gum, preservatives and fragrance (INCI nomenclature). This sunscreen was a broad-spectrum UV blockcr measured using a Labsphere(R) UV1000S ultraviolet transmittance analyzer after being spread (1.2 mg cm^sup -2^) on a polymethylmethacrylate plate (Fig. 1). The sunfilter was applied 4 h after culturing and each skin sample received 2 mg cm^sup -2^ of product checked by double weighing. After overnight incubation, a second application was performed 60 min before irradiation. Figure 1. UV absorption spectrum of sunscreen formulation.

Lactate dehydrogenase assays. The viability of the explants was evaluated by measuring the leakage of the cytosolic enzyme lactate dehydrogenase (LDH) into the culture medium, using a LDH-based in vitro toxicology assay kit according to the supplier’s recommendations (Sigma). This colorimetric test is based on lhc reduction of NAD by LDH. The resulting reduced NAD (NADH) is then utilized in the stoichiometric conversion of a yellow tetrazolium dye into formazan. A calibration curve from 25 to 800 mU mL^sup -1^ was prepared, using LDH from porcine heart (Sigma). Assays were performed in triplicate in 96-well plate, and absorbance was measured at 490 nm using a Synergy HT microplate reader equipped with KC4 software (Bio-Tek(R) Instruments, Inc., Winooski, VT).

Epidermal extracts and western blot analysis. The epidermis was separated from the dermis by treatment with 500 [mu]g mL^sup -1^ thermolysin (Sigma) for 2 h at 37[degrees]C, as previously detailed by Rijnkels et al. (29,30). The epidermal sheets were then hydrolyzed at 4[degrees]C for 1 h with a homogenization buffer (50 mM Tris-HCl, pH 7.6, 200 mM NaCl, 2 mM EGTA, 1 mM DTT containing 1% Triton X-100, 100 [mu]M PMSF, 1 mM Na^sub 3^VO^sub 4^, 1 mM NaF and 10 [mu]g mL^sup -1^ each of leupeptin and aprotinin). Then, the extracts were centrifuged al 10 000 g for 20 min, and proteins were quantified using the bicinchoninic acid method (Uptima lnterchim, Montlucon, France). Proteins from the culture medium (20 [mu]L) and epidermal extracts (30 [mu]g) were separated using 12% SDS-PAGE (Bio- Rad, Marnes-la-Coquette, France), transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Saclay, France) and immunoblotted as previously described (31). The membranes were incubated overnight at 4[degrees]C with the following primary antibodies: anti-extracellular signal-related kinase 2 (ERK2, 1/ 2000; Santa Cruz Biotechnology, Inc., TEBU, Le Perray en Yvelines, France), anti-caspase-3 (1/1000; Cell Signaling Technology, Ozyme, St. Quentin Yvelines, France) rabbit polyclonal antibodies and anti- p53 (DO-7, 1/500; Dako, Trappes, France) mouse monoclonal antibodies. For internal control, the blots were probed with an anti- betaactin mouse monoclonal antibody (AC-15, 1/1000; Sigma). Finally, horseradish peroxidase-conjugated secondary antibodies (1/5000) were incubated for 1 h, and immunoreactive proteins were observed with an enhanced chemiluminescence-linked detection system (Amersham Pharmacia Biotech).

Sunburn cell counting. The skin was embedded in an OCT compound (VWR, Val de Fontenay, France), frozen in liquid nitrogen, and cut into 6 [mu]m thin sections using the HM 500 OM cryostat (Microm, Francheville, France). Skin morphology and sunburn cells (SBC) were analyzed using hematoxylin staining according to the supplier’s recommendations (Dako)- Light microscope examination was carried out using an Eclipse E600 microscope equipped with a digital camera (Nikon DXM1200, Champigny sur Marne, France). The SBC number was determined by counting four representative fields of epidermis for each section, and images were collected under 50x magnification using Lucia G software.

Immunohistochemistry. The skin cryosections were fixed in acetone at -200C for 10 min and air dried for 15 min. For thymine dimers, samples were fixed for 20 min in 4% p-formaldehyde, placed in a citrate buffer, pH 6.0 (Dako), heated in a microwave oven for 3 min at 750 W and cooled at room temperature for 20 min. The slides were then washed with Tris-buffered saline (TBS) (Sigma) and blocked for 30 min in TBS containing 0.1% Triton X-100 and 5% goat serum (from Sigma and VWR, respectively). Immunostainings were performed overnight at 4[degrees]C as previously described (31). The following primary antibodies were used: anti-involucrin (SY5, 1/25; Santa Cruz Biotechnology, Inc.), anti-cytokeratin 10 (DE-K10, 1/50; Dako), antilaminin (LAM-89, 1/25; Novocastra Laboratories Ltd, Newcastle, UK), anti-collagen I (COL-1, 1/2000; Sigma), anti-thymine dimer (KTM53, 1/60; Kamiya Biomedical Company, Seattle, WA) mouse monoclonal antibodies and finally anli-cleaved caspase-3 (Asp175) (1/ 100; Cell Signaling Technology) rabbit polyclonal antibody. Fluorescent detection was achieved after 1 h of incubation with Alexa Fluor(R) 594-coupled F(ab’)^sub 2^ fragment anti-rabbit/mouse IgG (H + L) antibodies (1/500; Invitrogen). As a positive control, immunolabelings were performed using either irrelevant normal mouse IgG (5 [mu]g mL^sup -1^; Santa Cruz Biotechnology, Inc.) or omitting the primary antibody. Finally, slides were mounted using ProLong(R) Gold Antifade Reagent containing DAPl (Invitrogen), and the fluorescence signal was observed using a Nikon microscope.

TUNEL and caspase-3 assays. DNA fragmentation was identified using an in situ cell death detection kit according to the manufacturer’s instructions (Roche Diagnostics, Meylan, France). This assay is based on specific labeling of DNA strand breaks by terminal deoxynueleotidyl transferase (TdT), which catalyzes Ihe polymerization of fluorescent dUTP to free 3′-OH ends (TdT-mediated dUTP nick-end labeling [TUNEL] reaction). Briefly, the skin sections were fixed in 4% p-formaldehyde, washed in TBS and microwavcd for 1 min at 750 W in target retrieval solution pH 6.0 from DakoCytomation. Afler permeabilization, TUNEL assays were performed for 60 min at 37[degrees]C in a CO2 incubator. A negative control was generated by omitting the TdT enzyme from the labeling mixture. As a positive control, the skin sections were treated with 2000 U mL^sup -1^ Recombinant Grade I DNAse (Roche) for 10 min at room temperature. Finally, slides were mounted using a ProLong(R) medium as mentioned above and TUNEL-positive cells were observed in green under epifluorescent illumination. Caspase-3 activity was measured using the CaspACE(TM) Colorimetric Assay System according to the supplier’s protocol (Promega, Charbonnieres, France). This assay is based on spectrometric detection of the chromophore p-nitroaniline (pNA) after cleavage from the labeled substrate acetyl-Asp-Glu-Val- Asp-p-nitroaniline (Ac-DEVD-pNA). Caspase-3 specific activation was demonstrated by pretreating the epidermal extracts (50 [mu]g protein) with 100 [mu]M Z-VAD-FMK, a broad-range caspase inhibitor, 1 h before substrate addition. All assays were incubated overnight at 37[degrees]C before absorbance reading at 405 nm.

Statistical analysis. All values are expressed as mean +- SD. Statistical analysis was calculated using a two-tailed Student’s t- lest and differences were considered statistically significant from P

RESULTS

Pig skin differentiation is maintained in organ culture

We first checked whether the pig skin organ culture system maintains skin differentiation for 48 h (Fig. 2). Immunohistochemical analysis revealed that the epidermis preserved its multilayered epithelium composed of a basal layer (cytokeratin [CK] 10- and involucrin-negative staining) and several suprabasal layers (CK10-positive staining) comprising granular (involucrin- positive staining) and cornified layers. Type I collagen and laminin immunostainings showed that the epidermis was separated from the dermis by the basement membrane. Laminin labeling also clearly identified the fibrovascular tract in the dermis. All stainings were specific, as no fluorescence signal was detected when the primary antibody was omitted (data not shown). In addition, the expression profile of the differentiation markers was similar for both cultured pig skin and fresh human skin (Fig. 2). These data confirm that pig skin is closely related to its human counterpart and that the short- term organ culture does not affect skin differentiation.

Figure 2. Immunohistochemical characterization of pig skin organ culture. Pig skin was processed from ears and cultured 48 h in DMEM before harvesting. Serial cryosections were then prepared, fixed and stained. Red immunofluorcscence labeling showed the expression of keratinocyte-specific differentiation markers involucrin and cytokeratin 10. The dermis and the basement membrane were respectively identified by type I collagen and laminin labelings. The nuclei were stained with DAPI in blue. A similar expression pattern was obtained using fresh human skin. Scale bar: 20 [mu]m.

Solar-simulated radiation is cytotoxic in pig skin organ culture

We next tested the cytotoxic effects of SSR on porcine explants. Using a colorimetric assay, we measured the activity of the cytosolic enzyme LDH released into the culture medium in response to various UV doses (Fig. 3a). LDH activity increased in a dose- dependent manner 24 h after irradiation, and reached 7559 +- 1170 mU g^sup -1^ tissue (n = 3) in pig skin exposed to a 275 kJ-m^sup -2^ dose. This increase was significant, and the LDH activity level was about 6.5-fold higher than that of non-irradiated skin. To ascertain that the increase in LDH activity was specific to UV treatment, we investigated the effects of a broad-spectrum sunscreen formulation (Fig. 3a). Topical application of a sunfilter was not toxic for the skin and efficiently blocked SSR-induced LDH activity even after an acute UV dose of 275 kJ.m^sup -2^. The presence of the protein kinase ERK2, another cytosolic protein unrelated to LDH, was also analyzed in the culture medium 24 h post-irradiation (Fig. 3b). These immunoblot experiments showed that a high dose of SSR induced strong ERK2 leakage from the explants and that sunscreen application provided good photoprotection without affecting skin viability. Thus, LDH activity was well correlated to ERK2 protein release in the culture medium following irradiation.

Figure 3. Solar-simulated radiation induces the release of lactate dehydrogenase and ERK2 protein into the medium of pig skin organ culture. Pig skin was pretreated with or without sunscreen, and then exposed to solar-simulated radiation. Culture media were harvested 24 h later and tested for the presence of cytosolic proteins released from the explants, (a) LDH activity was measured by colorimetry after exposition to 0, 55, 110 and 275 kJ.m^sup -2^ UV doses. Results represent mean +- SD (n = 3) and are expressed as mU/g tissue. (b) ERK2 expression was analyzed by immunoblotting after a 275 kj.m^sup -2^ UV exposure. Results are representative of three independent experiments. Note that topical application of sunscreen reduced the leakage of both LDH and ERK2 from UV- irradiated pig skin (*P

Solar-simulated radiation induces DNA damage in pig skin organ culture

The above data suggest that the pig skin organ culture is a suitable model for studying SSR-induced cellular damage. Therefore, we next investigated the DNA damage induced by an SSR exposure at a 275 kJ.m^sup -2^ dose. We first focused on SBC formation, which corresponds to apoptosis induction in KCs (Fig. 4). SBCs were easily detected by hematoxylin staining in the epidermis 24 h after irradiation. They were characterized by a round shape, a loss of connection with surrounding KCs and a typically contracted nucleus (Fig. 4a). Moreover, the SBCs were located mainly in the basal layers, and their number significantly decreased under irradiation in sunscreen-protected pig skin (Fig. 4b). Using immunohistochcmistry, we also demonstrated that SSR stimulates the formation of thymine dimers in both the epidermal and dermal compartments (Fig. 5a). These DNA lesions were not detected in non- irradiated skin, and the application of the sunscreen almost completely protected against these alterations. DNA staining with the blue dye DAPI also revealed that the nuclei of KCs become fragmented and condensed after irradiation. In contrast. CK 10 and involucrin staining were unaffected upon SSR exposure (data not shown). Finally, we performed TUNEL assays to visualize UV-induced DNA fragmentation in situ (Fig. 5b). Rare TUNEL-positive nuclei were restricted to the uppermost layers of the viable epidermis underneath the stratum corneum in non-irradiated skin. No signal was observed in the dermal compartment in non-irradiated pig skin. The assays were specific as the pretreatment of samples with DNAse I caused an intensive TUNEL staining whereas TdT enzyme omission yielded no staining (data not shown). In contrast, SSR induced a strong positive staining. As observed for SBCs, DNA strand breaks were mainly detected in the basal layers of the porcine epidermis but also in the dermis. The TUNEL signal remained at the basal level in sunscreentreated skin. Thus, pig skin organ culture can be used to detect SSR-dependent genomic alterations.

Solar-simulated radiation induces caspase-3 activation in basal layers of the epidermis in pig skin organ culture

To better characterize the molecular pathways involved in SSR- induced apoptosis in the ex vivo skin model, caspase-3 activation was assessed 24 h after UV exposure. Western blotting analysis of epidermal extracts clearly showed that irradiation induces dose- dependent caspase-3 processing from its 35 kDa inactive zymogen into an active fragment of 17 kDa (Fig. 6a). This response was correlated with the upregulation of the p53 protein. Caspase-3 activation was confirmed by enzymatic assays with a specific substrate, the chromophore Ac-DEVD-pNA (Fig. 6b). These tests indicated that protease activity increases about seven-fold under radiation compared to non-irradiated skin. The assays were specific, as a caspase inhibitor (Z-VAD-FMK) blocked caspase-3 activation in response to SSR exposure (data not shown). Furthermore, sunscreen application completely abrogated caspase-3 induction for all SSR doses tested. Immunohistochemical experiments using an anti-cleaved caspase-3 antibody indicated that the protease is strongly activated in the basal layers of the epidermis 24 h after a 275 kJ.m^sup -2^ irradiation (Fig. 7). Rare basal ceils were stained in non- irradiated skin and sunscreen afforded efficient photoprotection. Altogether, these findings demonstrate that SSR-mediated apoptosis is a caspase-3 dependent process in pig skin organ culture.

Figure 5. Solar-simulated radiation induces DNA damage in pig skin organ culture. Pig skin was pretreated with or without sunscreen, and then exposed to a 275 kJ.m^sup -2^ dose of solar- simulated radiation. DNA damage was analyzed in situ 24 h post- irradiation. (a) Thymine dimers were identified by immunohistochemistry (red labeling). (b) DNA strand breaks were identified by TUNEL reaction (green labeling). Note that topical application of sunscreen prevented the formation of DNA lessons in both dermal fibroblast and keratinocyte nuclei stained with DAPI in blue. Analysis of skin from three different ears showed similar results. Dashed lines correspond to the dermal-epidermal junction. Scale bar: 50 [mu]m.

Figure 6. Solar-simulated radiation activates caspase-3 in pig skin organ culture. Pig skin was pretreated with or without sunscreen, and then exposed to solar-simulated radiation (0, 55, 110 and 275 kJ.m^sup -2^ UV doses). Skin was harvested 24 h post- irradiation and caspase-3 was analyzed. (a) Caspase-3 expression and p53 upregulation were studied by immunoblotting from epidermal extracts. beta-Actin represents a loading control. Results are representative of three independent experiments, (b) Caspase-3 activity was measured from the epidermis using a colorimetric substrate. Results are mean +- SD (n = 3, *P

Figure 7. Solar-simulated radiation activates caspase-3 in the basal epidermis in pig skin organ culture. Pig skin was pretreated with or without sunscreen, and then exposed to a 275 kJm^sup -2^ dose of solarsimulated radiation. The active form of caspase-3 was detected by immunohistochemistry 24 h post-irradiation (red labeling). The nuclei were stained with DAPI in blue. Analysis of skin from three different ears showed similar results. Scale bar: 50 [mu]m. Note that topical application of sunscreen prevented caspase- 3 activation in response to UV exposure.

DISCUSSION

The pig has been recognized for decades as an experimental animal in biomedical research, thanks to its morphological and physiological similarities to the human (18,19). However, ethical considerations and the ban on animal testing require that alternative models be found to perform phototoxicological studies. In this context, we have developed an ex vivo skin organ culture model from domestic pig ears. In addition, we have investigated whether this culture system might be useful in the assessment of SSR- induced cellular damage and the evaluation of sunscreen efficacy.

First, we determined whether pig skin differentiation was maintained in short-term organ culture (Fig. 2). Experiments revealed that the localization of both epidermal and dermal differentiation markers was well preserved in both human and pig skin, reflecting the high degree of similarity between the two species. In agreement with Rijnkels et al. (29,30), a culture medium containing DMEM supplemented with pyruvate and glutamine was sufficient to maintain skin differentiation for at least 48 h in gelatin-coated inserts. We did not conduct studies beyond a 48 h period. However, it seems obvious that culture conditions will have to be improved in order to preserve pig skin homeostasis and develop long-term organ culture. In this respect, it could be of interest to culture the explants over a type I collagen gel to better mimic the extracellular matrix found in the dermis (32,33). The addition of growth factors into the culture medium may also greatly increase skin survival. It is of note that insulin and hydrocortisone have been used successfully in constructing human epidermis in vitro (34) and in maintaining the viability of pig skin explants (29,30).

Next, we tested the cytotoxic effects of a single dose of SSR on porcine explants by performing LDH assays (Fig. 3). This type of assay measures cell membrane integrity and detects the leakage of LDH from damaged skin cells (35,36). It is a simple, rapid, accurate and non-destructive test since samples are pipetted directly from the culture medium. Results showed that LDH activity increased in a dose-dependent fashion following irradiation. Western-blot experiments using an antiERK2 antibody confirmed that an acute SSR dose was toxic for pig skin. We also performed 3-(4,5- dimethylthiazol)-2,5diphenyltetrazolium bromide (MTT) assays on the explants. Although this cell viability assay is commonly used in vitro to reflect mitochondrial metabolism (37), it was not appropriate for analyzing cell death in our ex vivo organ culture model (D. Bacqueville, unpublished data). In fact, MTT diffusion across the dermis could be compromised by the presence of a very dense extracellular matrix. In any case, LDH assay and ERK2 release can be used as viability markers in skin organ culture and seem to represent good endpoint parameters in the investigation into the harmful effects of solar radiation. Finally, these cytotoxicity tests could be complemented by analyzing the production of proinflammatory cytokines in the culture medium following SSR exposure. We also investigated SSR-induced DNA damage and apoptosis in pig explants. Analysis of SBC formation showed that apoptotic KCs were located in the mid-epidermal layers (Fig. 4). These observations coincided with the presence of thymine dimers in both epidermis and dermis 24 h after solar irradiation (Fig. 5). These lesions result from the direct absorption of UVB by genomic DNA and are thought to be crucial for the initiation of skin cancer because they are closely linked to the generation of mutations (1). UVB radiation also triggers signaling pathway activation in KCs (38- 42). In this regard, preliminary results suggest that SSR activates the epidermal growth factor receptor and mitogen-activated protein kinase pathways in pig explants (D. Bacqueville, personal communication), as observed in vivo in humans following UVB exposure (43). There is also increasing evidence that UVA plays a pivotal role in mutagenesis and skin cancer development (1). Although UVA is very poorly absorbed by DNA, its genotoxic effects have been attributed to the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine. However, recent studies have demonstrated that UVB and UVA light induce similar mutations in human skin cells (44) and that thymine dimers are predominant DNA lesions in whole human skin exposed to UVA radiation (45). The mechanisms involved in UVAinduced thymine dimer generation remain to be explored, even if oxidative stress seems to be essential through a triplet energy transfer photosensitization process (46). Furthermore, DNA strand breaks were present in the uppermost layers of the epidermis in non-irradiated skin, in agreement with findings previously reported by Gavrieli et al. (47) relative to the disintegration of nuclei during the keratinization process. In contrast, SSR induced the generation of DNA strand breaks in both the epidermal basal layers and the dermis (Fig. 5). In this context, it might be of interest to study the individual effects of UVB and UVA radiations on both thymine dimer and DNA strand break formation in the ex vivo pig skin organ culture model.

To better understand the molecular pathways involved in SSR- induced apoptosis in pig skin, we analyzed caspase-3 activation. The caspase-3 protease is one of the key executioners of apoptosis and serves as a convergence point for different signaling pathways (48,49). SSR activated caspase-3 in a dose-dependent fashion (Fig. 6). The caspase-3 activation was well correlated with LDH leakage in the culture medium and was associated with the upregulation of the p53 protein. This tumor suppressor plays a major role in protecting genome integrity and its gene is commonly mutated in human cancer, especially in nonmelanoma skin cancers (50-52). Moreover, Decraene et al. (53) have recently reported that a low UVB dose, with the potential to trigger a protective p53-dependent gene program, increases the resilience of KCs against future UVB insults, suggesting distinct signaling pathway activation in chronic versus acute responses following UV irradiation. Our data are consistent with results recently obtained in vivo in SSR-exposed pig skin (21). They also confirm those obtained from a UVB-irradiated whole human skin organ culture and epidermal equivalent (54). Interestingly, caspase-3 activation was detected mainly in the basal epidermis after irradiation (Fig. 7), suggesting that basal ICCs are more sensitive to UV exposure than suprabasal KCs and dermal fibroblasts. Thus, it is tempting to speculate that skin compartments are affected differently by SSR radiation. This hypothesis is supported by a high amount of DNA strand breaks in porcine basal epidermis and by the recent findings that the basal layer epidermis harbors more UVA than UVB fingerprint mutations in human squamous tumors (55). In this study, we focused on the caspase-3 activation as a specific biochemical marker of apoptosis. It is of note that this protease is not sufficient to cause apoptosis by itself and that it acts in concert with both other caspases (initiator caspases-2, -8, -9, – 10, effector caspases-6 and -7) and players (cytochrome c, Bcl-2 family proteins…) to induce cell death. Therefore, an important goal in the future will be to specify the molecular mechanisms involved in SSR-induced apoptosis (mitochondria and death receptor- mediated apoptotic pathways) and to determine the impact of UVB and UVA radiations in pig skin organ culture.

The reliability of the ex vivo pig skin organ culture model for testing a sunscreen formulation was evaluated by using a commercial broad-spectrum UVB + A sunfilter. Topical sunscreen application protected against SSR-induced DNA damage. Coincidentally, SBC formation, cytotoxicity and caspase-3 activation were all inhibited in sunfilter-treated pig skin. These data agree with previous studies obtained in vivo from UVB + A and UVB alone sunscreen- treated volunteers (56,57). Moreover, they confirm those obtained in vivo by Lin et al. (21), who demonstrated that topical application of antioxidants protects pig skin against UV-induced thymine dimer formation. Finally, Young et al. (58) recently reported that the detrimental effects of daily suberythemal exposure can be prevented by daily sunscreen application to human skin in vivo. Thus, it will be interesting to develop a long-term pig skin expiant culture model in order to assess photoprotection in response to chronic SSR exposure. These considerations are currently under investigation.

In summary, our findings suggest that the short-term culture of pig ear skin in Transwell(R) inserts in 12-well plate is a relevant alternative model for use in the study of the deleterious effects of solar irradiation, and may be a useful tool to determine the photoprotective capacity of sunscreen formulation.

Acknowledgement-We are grateful to Ms. G. Magnusson for her excellent critical reading of the manuscript.

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Daniel Bacqueville and Alain Mavon*

Laboratoire de Pharmacocinetique Cutanee, Institut de Recherche Pierre Fabre, Vigoulet-Auzil, France

Received 30 October 2007, accepted 13 December 2007, DOI: 10.1111/ j.1751-1097.2008.00297.x

* Corresponding author email: alain.mavomu pierre-fabre.com (Alain Mavon)

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