Matrix metalloproteinase-1 production observed after solar-simulated radiation exposure is assumed by dermal fibroblasts but involves a paracrine activation through epidermal keratinocytes

Chronic exposure of human skin to solar UV radiation leads to serious dermal damages, a hallmark of photoaging. In vivo, acute UV radiation has been shown previously to induce various matrix-degrading proteases. Among them, matrix metalloproteinase-1 (MMP-1) has been suggested to be involved in skin photodamage. The purpose of this study was to investigate the effects of solar-simulated radiation (SSR) on MMP-1 production in normal human skin cells. SSR exposure of human skin reconstructed in vitro comprising both a differentiated epidermis and a fibroblast-populated dermal equivalent led to an increase in MMP-1 production, which was abolished when epidermis was removed immediately after SSR exposure. In addition, SSR exposure of differentiated keratinocytes grown on an acellular collagen gel did not induce MMP-1 production. Experiments on cell cultures grown on plastic confirmed that keratinocytes failed, in contrast with fibroblasts, to produce MMP-1 in response to SSR exposure. However, when conditioned medium from SSR-exposed keratinocytes was added to human fibroblasts in culture, MMP-1 production was induced. Altogether, these data show that MMP-1 production observed after SSR exposure involved the release of soluble epidermal factors, which could modulate its production by dermal fibroblasts.     

In Vitro Investigations on the Effect of Dermal Fibroblasts on Keratinocyte Responses to Ultraviolet B Radiation

Exposure to ultraviolet radiation is closely linked to the development of skin cancers in humans. The ultraviolet B (UVB) radiation wavelength (280–320 nm), in particular, causes DNA damage in epidermal keratinocytes, which are linked to the generation of signature premalignant mutations. Interactions between dermal fibroblasts and keratinocytes play a role in epidermal repair and regeneration after UVB‐induced damage. To investigate these processes, established two and three‐dimensional culture models were utilized to study the impact of fibroblast–keratinocyte crosstalk during the acute UVB response. Using a coculture system it was observed that fibroblasts enhanced keratinocyte survival and the repair of cyclobutane pyrimidine dimers (CPDs) after UVB radiation exposure. These findings were also mirrored in irradiated human skin coculture models employed in this study. Fibroblast coculture was shown to play a role in the expression and activation of members of the apoptotic cascade, including caspase‐3 and Bad. Interestingly, the expression and phosphorylation of p53, a key player in the regulation of keratinocyte cell fate postirradiation, was also shown to be influenced by fibroblast‐produced factors. This study highlights the importance of synergistic interactions between fibroblasts and keratinocytes in maintaining a functional epidermis while promoting repair and regeneration following UVB radiation‐induced damage.     

A study of protein synthesis in cells cultured from involved psoriatic skin

Using histochemical techniques an abnormal programme of epidermal differentiation has been well documented in psoriasis. In order to characterise further the biochemistry of this process we have cultured dermal fibroblasts and epidermal keratinocytes from involved psoriatic skin. This has facilitated metabolic radiolabelling of skin cells and analysis of protein synthesis by two-dimensional polyacrylamide gel electrophoresis. The expression of keratin and differentiation markers was identical to that of normal keratinocytes, suggesting that psoriatic epidermal differentiation is not truncated in vitro as has been postulated to be the case in vivo. Low molecular mass components (5-8.5 kDa), previously shown to be upregulated in suprabasal keratinocytes, were detected in epidermal fractions from psoriatic skin enriched for basal cells. Of special interest was a component of 26 kDa, pI 5.9, which was highly upregulated in psoriatic as compared to normal cultured keratinocytes and was not detected in fibroblasts. These findings are in accord with a qualitatively abnormal pattern of differentiation for keratinocytes in the involved psoriatic epidermis.     

Analysis of UV-B-induced DNA damage and its repair in heat-shocked skin cells

The heat-shock response is a cellular defence mechanism against environmental stresses that is evolutionarily conserved from bacteria to man. Numerous reports demonstrate the beneficial effects of heat-shock protein induction on cell survival under toxic or oxidative stress, e.g., in cardiac and cerebral ischemia or prior to organ transplantation. However, there is little data on the effects of heat treatment on damage caused by UV irradiation. Applying three independent techniques, we have tested the influence of thermal pretreatment of skin cells (1 h, 43 degrees C) on the initial extent of UV-B-induced DNA damage and its subsequent repair. For cultured human epidermal keratinocytes and dermal fibroblasts we can show reduced levels of nucleotide-excision-repair-associated DNA strand incision in the comet assay. Moreover, immunostaining and flow cytometric quantitation of thymidine dimers immediately and one day after irradiation, respectively, reveal that the initial DNA damage is not (keratinocytes) or only moderately (fibroblasts) lower in heat-shocked cells as compared to untreated controls. However, excision repair of dimers is significantly attenuated during the first 24 h in both cell types. Furthermore, using a modified host-cell reactivation assay, we are able to demonstrate that repair of UV-B-damaged plasmid DNA is lower if the transfected cells are previously heat shocked. In summary, heat treatment (1 h, 43 degrees C) inducing heat-shock proteins reduces nucleotide excision repair of UV-B-mediated DNA lesions in fibroblasts and keratinocytes during the following 24 h. This is not necessarily caused by elevated heat-shock protein levels themselves. Possibly the direct thermal damage of repair enzymes is more severe than the potential protective effects of heat-shock proteins.     

TWO GENES CONTRIBUTE TO DIFFERENT EXTENTS TO THE HEME OXYGENASE ENZYME ACTIVITY MEASURED IN CULTURED HUMAN SKIN FIBROBLASTS AND KERATINOCYTES: IMPLICATIONS FOR PROTECTION AGAINST OXIDANT STRESS

Abstract— Activation of expression of the heme oxygenase (HO) gene appears to be involved in a cellular defense system in mammalian cells. We now demonstrate that while HO-1 mRNA levels are strongly inducible in dermal fibroblasts they are barely inducible in human epidermal keratinocytes following oxidative stress (UVA radiation and hydrogen peroxide). Paralleling this result was the observation that HO-2 mRNA levels were low in dermal fibroblasts but were high in epidermal keratinocytes. In neither case was the HO-2 gene inducible. The expression of the two HO genes led to enzymatic activity in both types of skin cells with an approximately 2.5-fold higher level of enzymatic activity present in keratinocytes compared with fibroblasts derived from the same biopsy. In addition, ferritin levels, which have been found to be augmented via the HO-dependent release of iron from endogenous heme sources, were two- to three-fold higher in keratinocytes compared with matching fibroblasts. This higher ferritin pool would result in an enhancement of cellular iron sequestering capacity that may confer increased resistance to oxidative stress. Indeed, keratinocytes showed less UVA radiation-dependent cell membrane damage than fibroblasts. These results are consistent with the hypothesis that HO expression in human epidermis and dermis is related to cellular defense mechanisms that operate in human skin.     

Lack of correlation between DNA strand breakage and p53 protein levels in human fibroblast strains exposed to ultraviolet lights

The contribution of DNA strand breaks accumulating in the course of nucleotide excision repair to upregulation of the p53 tumor suppressor protein was investigated in human dermal fibroblast strains after treatment with 254 nm ultraviolet (UV) light. For this purpose, fibroblast cultures were exposed to UV and incubated for 3 h in the presence or absence of l-beta-D-arabinofuranosylcytosine (araC) and/or hydroxyurea (HU), and then assayed for DNA strand breakage and p53 protein levels. As expected from previous studies, incubation of normal and ataxia telangiectasia (AT) fibroblasts with araC and HU after UV irradiation resulted in an accumulation of DNA strand breaks. Such araC/HU-accumulated strand breaks (reflecting nonligated repair-incision events) following UV irradiation were not detected in xeroderma pigmentosum (XP) fibroblast strains belonging to complementation groups A and G. Western blot analysis revealed that normal fibroblasts exhibited little upregulation of p53 (approximately 1.2-fold) when incubated without araC after 5 J/m2 irradiation, but showed significant (three-fold) upregulation of p53 when incubated with araC after irradiation. AraC is known to inhibit nucleotide excision repair at both the damage removal and repair resynthesis steps. Therefore, the potentiation of UV-induced upregulation of p53 evoked by araC in normal cells may be a consequence of either persistent bulky DNA lesions or persistent incision-associated DNA strand breaks. To distinguish between these two possibilities, we determined p53 induction in AT fibroblasts (which do not upregulate p53 in response to DNA strand breakage) and in XP fibroblasts (which do not exhibit incision-associated breaks after UV irradiation). The p53 response after treatment with 5 J/m2 UV and incubation with araC was similar in AT, XPA, XPG and normal fibroblasts. In addition, exposure of XPA and XPG fibroblasts to UV (5, 10 or 20 J/m2) followed by incubation without araC resulted in a strong upregulation of p53. We further demonstrated that HU, an inhibitor of replicative DNA synthesis (but not of nucleotide excision repair), had no significant impact on p53 protein levels in UV irradiated and unirradiated human fibroblasts. We conclude that upregulation of p53 at early times after exposure of diploid human fibroblasts to UV light is triggered by persistent bulky DNA lesions, and that incision-associated DNA strand breaks accumulating in the course of nucleotide excision repair and breaks arising as a result of inhibition of DNA replication contribute little (if anything) to upregulation of p53.     

UV-induced degradation of collagen I is mediated by soluble factors released from keratinocytes

Photoaging is a complex condition but its hallmark is the destruction of dermal collagen. This has been attributed to the direct activation of fibroblast matrix metalloproteinases by solar UV. However, we report here that unirradiated fibroblasts increase metalloproteinase production and digest collagen when exposed to cell culture media from irradiated keratinocytes. Enhanced DNA repair in the keratinocytes ameliorates this response. This suggests that soluble factors induced by DNA damage in UV-exposed epidermal keratinocytes signal collagen degradation by fibroblasts in the dermis. This motif of DNA damage in keratinocytes producing effects on other cell types mediated by soluble factors was first identified by Kripke and colleagues in studying UV-induced immune suppression.     

Development of Effective Skin Cancer Treatment and Prevention in Xeroderma Pigmentosum

Xeroderma pigmentosum (XP) is a rare, recessively transmitted genetic disease characterized by increasingly marked dyspigmentation and xerosis (dryness) of sun‐exposed tissues, especially skin. Skin cancers characteristically develop in sun‐exposed sites at very much earlier ages than in the general population; these are often multiple and hundreds or even thousands may develop. Eight complementation groups have been identified. Seven groups, XP‐A…G, are associated with defective genes encoding proteins involved in the nucleotide excision DNA repair (NER) pathway that recognizes and excises mutagenic changes induced in DNA by sunlight; the eighth group, XP‐V, is associated with defective translesion synthesis (TLS) bypassing such alterations. The dyspigmentation, xerosis and eventually carcinogenesis in XP patients appear to be due to their cells’ failure to respond properly to these mutagenic DNA alterations, leading to mutations in skin cells. A subset of cases, especially those in some complementation groups, may develop neurological degeneration, which may be severe. However, in most XP patients, in the past the multiple skin cancers have led to death at an early age due to either metastases or sepsis. Using either topical 5‐fluorouracil or imiquimod, we have developed a protocol that effectively prevents most skin cancer development in XP patients.     

XERODERMA PIGMENTOSUM FIBROBLASTS INCLUDING CELLS FROM XP VARIANTS ARE ABNORMALLY SENSITIVE TO THE MUTAGENIC AND CYTOTOXIC ACTION OF BROAD SPECTRUM SIMULATED SUNLIGHT

Abstract— The cytotoxic and mutagenic effects of broad spectrum simulated sunlight, as delivered by a Westinghouse Sun Lamp FS 20 filtered to eliminate wavelengths below 290 nm, were determined in diploid human skin fibroblasts which differ in their ability to repair pyrimidine dimers, and compared with results obtained with UV 254 nm radiation. The cell strains tested included normal fibroblasts; excision repair-deficient xeroderma pigmentosum (XP) cells from patients XP12BE (complementation group A). XP7BE (group D). and XP2BI (group G): and an XP variant patient (XP4BE) whose cells excise pyrimidinc dimers at a normal rate, but exhibit abnormal replication of DNA containing unexcised lesions. Cytotoxicity was assayed from loss of colony-forming ability. The group A cells were most sensitive to the killing effect of the Sun Lamp; the group D and G cells were slightly less sensitive; the XP variant cells showed intermediate sensitivity; and normal cells were most resistant. When the Sun Lamp survival curves for the group A, group D, the XP variant and normal cells were compared with their respective UV 254 nm survival curves, the relationships between the strains were virtually identical (i. e. the curves were related by a constant fluence modification factor). suggesting a common lesion for cell killing. The marker for mutagenesis was resistance to 6-thioguanine. The group A XP cells proved most sensitive to mutations induced by the simulated sunlight: the variant cells were intermediate; and the normal cells were the most resistant. Again, when the curves for mutations induced in these cell strains by simulated sunlight were compared with their respective 254 nm UV mutation curves, these were related by a constant fluence modification factor. suggesting a common lesion for mutagenesis. These results. taken together with published data indicating that at equicytotoxic levels of UV254 nm radiation and the filtered Sun Lamp. the number of pyrimidine dimers in the DNA of XP12BE cells was equal. support the hypothesis that the dimer is the lesion principally involved in both effects. Our data also support the hypothesis that mutations are involved in the sunlight-induced skin cancer of XP patients.     

A microarray to measure repair of damaged plasmids by cell lysates

DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.     

γH2AX,an Accurate Marker That Analyzes UV Genotoxic Effects on Human Keratinocytes and on Human Skin

The phosphorylated form of histone H2AX, γH2AX, is a component of the DNA repair system. Most studies have focused on the role of γH2AX during cell transformation and human cancer, but little is known about its role in keratinocytes and the skin during UV irradiation. We analyzed the response to UV irradiation focusing on the phosphorylation of histone H2AX both in vitro, in keratinocyte cultures and in artificial epidermis, and then in vivo, in human skin. Acute UVB irradiation of human keratinocytes increased the phosphorylation of H2AX in a dose-dependent manner; two types of γH2AX response were observed either in vitro or in vivo. After a low nonapoptotic UVB irradiation, cells contained phosphorylated H2AX and arrested their cell cycle to repair the DNA damages. For a stronger and proapoptotic UVB irradiation, keratinocytes dramatically increased the phosphorylation of H2AX and committed apoptosis. Our results indicate that γH2AX constitutes a highly sensitive marker relevant for studying subapoptotic doses as well as proapoptotic doses of UVB in human skin.     

Photobiological information obtained from XPA gene-deficient mice

The XPA gene-deficient mouse, an animal model of xeroderma pigmentosum (XP), develops enhanced photobiologic reactions including acute inflammation, immunosuppression and skin carcinogenesis, because of the defect in the excision repair of ultraviolet-induced DNA lesions. The results strongly suggest that nuclear DNA is an important chromophore to initiate acute and chronic skin damages. The model mouse is a useful experimental animal not only to investigate the mechanisms of photosensitivity in XP, but also to study physiological photobiology in humans, because photobiologic reactions are greatly intensified in this mouse.     

Damage to UV-Sensitive Cells by Short UV in Photographic Flashes

Abstract— Light emitted by electronic photographic flash units is shown to damage bacteria and human skin fibroblasts deficient in repair systems, with survival curves very similar to those produced by 254 nm short UV. The lesions induced by these flashes are as photorepairable by the photolyase enzyme as those induced by 254 nm UV and result in equivalent survival rates. Biological dosimetry performed with microorganisms highly sensitive to UV ( Escherichia coli K12 AB2480, deficient in excision and recombinational-dependent repair systems and Bacillus subtilis UVSSP spores, deficient in excision and in a specific spore repair process) revealed that each 1 ms flash of light from the photographic unit used in this work contained the equivalent of 0.25 J m⁻² of 254 nm UV, when measured at a distance of 7.0 cm. This dose of UV was found to be lethal to both repair-deficient E. coli bacteria and repair-deficient human skin fibroblasts obtained from xeroderma pigmentosum donors, as well as mutagenic in B/r wild-type and HCR-mutant bacteria.     

A Practical Approach for the In Vitro Safety and Efficacy Assessment of an Anti-Ageing Cosmetic Cream Enriched with Functional Compounds

(1) Background: Cosmeceuticals are topical products applied to human skin to prevent skin ageing and maintain a healthy skin appearance. Their effectiveness is closely linked to the compounds present in a final formulation. In this article, we propose a panel of in vitro tests to support the efficacy assessment of an anti-ageing cream enriched with functional compounds. (2) Methods: biocompatibility and the irritant effect were evaluated on reconstructed human epidermis (RHE) and corneal epithelium (HCE) 3D models. After a preliminary MTT assay, normal human dermal fibroblasts (NHDF) and keratinocytes (HaCaT) were used to evaluate the extracellular matrix (ECM) protein synthesis, and interleukin-6 (IL-6) and metalloproteinase-1 (MMP-1) production. (3) Results: data collected showed good biocompatibility and demonstrated the absence of the irritant effect in both 3D models. Therefore, we demonstrated a statistical increase in collagen and elastin productions in NHDF cells. In HaCaT cells, we highlighted an anti-inflammatory effect through a reduction in IL-6 levels in inflammatory stimulated conditions. Moreover, the reduction of MMP-1 production after UV-B radiation was demonstrated, showing significant photo-protection. (4) Conclusion: a multiple in vitro assays approach is proposed for the valid and practical assessment of the anti-ageing protection, anti-inflammatory and biocompatible claims that can be assigned to a cosmetic product containing functional compounds.     

Long‐term Genoprotection Effect of Sechium edule Fruit Extract Against UVA Irradiation in Keratinocytes

Photoprotection is essential to prevent the long‐term deleterious effects of ultraviolet (UV ), including skin cancer and photoaging. So far, there has been an increase in the use of natural bioactive phytochemicals for the development of more effective skin photoprotective agents. However, the molecular mechanisms underlying the photochemoprotection activity of such compounds remain largely unknown. The objective of this study was to investigate the effects of a Sechium edule fruit extract (SEE ) in terms of photoprotection against UVA in primary human keratinocytes. We found that SEE protected keratinocytes against UVA ‐induced cytotoxicity, decreased the intracellular amounts of reactive oxygen species, and reduced oxidatively induced DNA lesions after UVA exposure. Furthermore, SEE decreased the induction of CPD lesions in UVA ‐irradiated keratinocytes and exhibited increased DNA repair of such photoproducts at 24 h postexposure. Finally, using DNA repair biochips, we demonstrated that SEE ‐treated keratinocytes had DNA enzymatic repair activities more efficient for abasic sites, CPD and thymine glycols. Therefore, the benefits of SEE against UVA could be explained by a combination of antioxidant activity, the reduction in DNA damage, and the enhancement of DNA repair capacities.     

Interexperimental and interindividual variations of DNA repair capacities after UV-B and UV-C irradiations of human keratinocytes and fibroblasts

DNA repair plays a central role in the cellular response to UV. In this work we have studied the response of skin cells (i.e. fibroblasts and keratinocytes) from the same or from different individuals after both ultraviolet-B (UV-B) and ultraviolet-C (UV-C) irradiations using the comet assay to characterize the specific cellular response to UV-induced DNA damage. Cells were irradiated with increasing doses of UV-B or UV-C. To study the UV dose dependency of initial steps of DNA repair, namely recognition and incision at DNA damage level, the comet assay was performed, under alkaline conditions, 60 min after UV irradiation to allow detection of DNA strand breaks. Comparative analysis of tail moment values after UV exposure of cells from the same or from different individuals showed interexperimental and interindividual variations, implying that repeated assays are necessary to characterize the individual DNA repair capacity. With increasing doses of UV in keratinocytes, a plateau was rapidly reached after irradiation, whereas in fibroblasts a linear dose-effect relationship was observed. These interindividual variations associated with cellular specificity in DNA response may be of significance in skin cell and individual susceptibility toward UV-induced carcinogenesis.     

LETHALITY AND THE INDUCTION AND REPAIR OF DNA DAMAGE IN FAR, MID OR NEAR UV-IRRADIATED HUMAN FIBROBLASTS: COMPARISON OF EFFECTS IN NORMAL, XERODERMA PIGMENTOSUM AND BLOOM'S SYNDROME CELLS

Abstract Using normal human fibroblasts we have determined the ability of far (254 nm), mid (310 nm) or near (365 nm) UV radiation to: (i) induce pyrimidine dimers (detected as UV endonuclease sensitive sites) and DNA single-strand breaks (detected in alkali); (ii) elicit excision repair, monitored as unscheduled DNA synthesis (UDS); and (iii) reduce colony-forming ability. Unscheduled DNA synthesis studies were also performed on dimer excision-defective xeroderma pigmentosum (XP) cells, and the survival studies were extended to include XP and Bloom's syndrome (BS) strains. UV-induced cell killing in normal, BS and XP cells was found to relate to an equivalent dimer load per genome after 254 or 310 nm exposure, whereas at 365 nm the lethal effects of non-dimer damage appeared to predominate. Lethality could not be correlated with DNA strand breakage at any wavelength. The two XP strains examined showed the same relative UDS repair deficiency at the two shorter wavelengths in keeping with a predominant role for pyrimidine dimer repair in the expression of UDS. However, UDS was not detected in 365 nm UV-irradiated normal and XP cells despite dimer induction; this effect was due to the inhibition of DNA repair functions since 365 nm UV-irradiated normal cells showed reduced capacity to perform UDS subsequent to challenge with 254 nm UV radiation.
In short, the near UV component of sunlight apparently induces biologically important non-dimer damage in human cells and inhibits DNA repair processes, two actions which should be considered when assessing the deleterious actions of solar UV.     

Radiofrequency Irradiation Attenuates High-Mobility Group Box 1 and Toll-like Receptor Activation in Ultraviolet B–Induced Skin Inflammation

Ultraviolet B (UVB) exposure activates various inflammatory molecules of keratinocytes in the epidermis layer. Such UVB-mediated skin inflammation leaves post-inflammatory hyperpigmentation (PIH). Reports show a close relationship between PIH and high-mobility group box 1 (HMGB1) and its receptors. General clinical treatments of PIH, such as oral medication and laser treatment, have reported side effects. Recent studies reported the effects of radiofrequency (RF) irradiation on restoring dermal collagen, modulating the dermal vasculature, and thickening the basement membrane. To validate how RF regulates the inflammatory molecules from UVB-irradiated keratinocytes, we used UVB-radiated keratinocytes and macrophages, as well as animal skin. In addition, we examined two cases of RF-irradiated skin inflammatory diseases. We validated the effects of RF irradiation on keratinocytes by measuring expression levels of HMGB1, Toll-like receptors (TLRs), and other inflammatory factors. The results show that the RF modulates UVB-radiated keratinocytes to secrete fewer inflammatory factors and also modulates the expression of macrophages from HMGB1, TLRs, and inflammatory factors. RF irradiation could alleviate inflammatory skin diseases in patients. RF irradiation can regulate the macrophage indirectly through modulating the keratinocyte and inflammatory molecules of macrophages reduced in vitro and in vivo. Although the study is limited by the low number of cases, it demonstrates that RF irradiation can regulate skin inflammation in patients.     

Measurements of the Protective Effect of Topically Applied Sunscreens using in vitro Three-dimensional Dermal and Skin Equivalents

Abstract— For preventing or minimizing acute and chronic skin damage caused by UV radiation, the use of sunscreens is probably the most important measure. To screen the protective efficacy of new sunscreen molecules or formulations against UV rays, we evaluated as in vitro testing methods the use of two three-dimensional models, a dermal equivalent (DE) and a skin equivalent (SE). The DE is composed of a porous collagen-glycosaminoglycans-chitosan matrix populated by normal human fibroblasts. The SE is comprised of a fully differentiated epidermis realized by seeding keratinocytes onto the DE. In this study, we demonstrated that the DE and SE models react to the deleterious effects of UVA and UVB. Then, we extended our research to the evaluation of their usefulness for photoprotection trials. Sunscreen agents (Euso-lex 8020 and 6300) and commercially available sunscreens (chemical and physical filter formulations) that protect the skin against either UVA or UVB were evaluated. The tested products were applied (n = 6) topically (10 μL) and incubated for 30 min prior to irradiation over a range of UVA (0-50 J/cm²) or UVB (0-5 J/cm²). The photoprotection provided by the tested sunscreen molecules and formulations was evaluated by measurement of residual cellular viability 24 h postirradiation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) test and assessment of the inflammation response by interleukin-la release assay. When sunscreens were applied prior to UV exposure, a higher residual cellular viability versus control was obtained, demonstrating the photoprotective effects of the tested products. These in vitro models could be used for screening tests to evaluate the protective effects of sunscreen molecules and formulations, especially for UVA trials because there is a lack of consensus for an in vivo method.     

LLLT Effects on Oral Keratinocytes in an Organotypic 3D Model

Several in vitro studies evaluated the cellular and molecular events related to interactions between phototherapy and target tissues, including oral keratinocytes and fibroblasts, providing elucidative data about phototherapy‐induced healing. However, these interactions were limited to the application of a bidimensional cell culture model of oral mucosal cells. Thus, thisstudy evaluated the use of an organotypic oral epithelium model to elucidate the morphological and phenotypic responses of cells subjected to low‐level laser therapy (LLLT). Oral keratinocytes were seeded in the ex vivo ‐produced oral mucosal equivalent (EVPOME ) model, with a porcine acellular dermal matrix. LLLT was applied by means of the LaserTABLE device (780 nm, 25 mW ) at 0.5, 1.5 and 3 J cm⁻². After three irradiations, morphology, proliferation and gene expression of growth factors were assessed. LLLT and control groups presented similar morphological features, characterized by the formation of a stratified, differentiated and keratinized epithelium. LLLT enhanced the cell proliferation and gene expression of keratinocytes (hKGF ) as well as epidermal (hEGF ) growth factors. In general, analysis of these data shows that the three‐dimensional cell culture model can be applied for phototherapy studies and that the positive effects of LLLT were confirmed by the use of an organotypic model.