A I E U a G T a b c R A U S h 0 B n Bras Dermatol. 2020;95(6):684---690 Anais Brasileiros de Dermatologia www.anaisdedermatologia.org.br NVESTIGATION valuation of ex vivo melanogenic response to UVB, VA, and visible light in facial melasma and unaffected djacent skin�,�� iovana Piteri Alcantara a, Ana Cláudia Cavalcante Esposito b, hainá Oliveira Felicio Olivatti c, Melissa Mari Yoshida c, Hélio Amante Miot a,∗ Department of Dermatology and Radiotherapy, Faculty of Medicine, Universidade Estadual Paulista, Botucatu, SP, Brazil Graduate Program in Pathology, Faculty of Medicine, Universidade Estadual Paulista, Botucatu, SP, Brazil Faculty of Medicine, Universidade Estadual Paulista, Botucatu, SP, Brazil eceived 27 October 2019; accepted 27 February 2020 vailable online 17 September 2020 KEYWORDS Melanosis; Photobiology; Ultraviolet rays Abstract Background: The independent role of solar radiation in the differential melanogenesis between melasma and adjacent skin is unknown. Objectives: To assess the melanogenic responses of skin with facial melasma and of the adjacent skin to UVB, UVA, and visible light, in an ex vivo model. Methods: This was a quasi-experimental study involving 22 patients with melasma. Facial melasma and adjacent skin samples were collected and stored in DMEM medium, at room tem- perature. One fragment was placed under the protection from light, while another was exposed to UVB, UVA, and visible light (blue-violet component): 166 mJ/cm2, 1.524 J/cm2, and 40 J/cm2, respectively. Subsequently, all samples were kept for 72 hours in a dark environment and stained by Fontana-Masson to assess basal layer pigmentation, dendrites, and melanin granulation. Results: Effective melanogenesis was observed in the basal layer in melasma and in the nor- mal adjacent skin after all irradiations (p < 0.01), with the following median increment: UVB (4.7% vs. 8.5%), UVA (9.5% vs. 9.9%), and visible light (6.8% vs. 11.7%), with no significant dif- ference between anatomical sites. An increase in melanin granulation (coarser melanosomes) was observed only after irradiation with UVA and only in the skin with melasma (p = 0.05). An increase in the melanocyte dendrite count induced by UVB radiation was observed in both anatomical sites (p ≤ 0.05). � How to cite this article: Alcantara GP, Esposito ACC, Olivatti TOF, Yoshida MM, Miot HA. Evaluation of ex vivo melanogenic response to VB, UVA, and visible light in facial melasma and unaffected adjacent skin. An Bras Dermatol. 2020;95:784---790. �� Study conducted at the Department of Dermatology and Radiotherapy, Faculty of Medicine, Universidade Estadual Paulista, Botucatu, P, Brazil. ∗ Corresponding author. E-mail: heliomiot@gmail.com (H.A. Miot). ttps://doi.org/10.1016/j.abd.2020.02.015 365-0596/© 2020 Sociedade Brasileira de Dermatologia. Published by Elsevier España, S.L.U. This is an open access article under the CC Y license (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1016/j.abd.2020.02.015 http://www.abd.org http://crossmark.crossref.org/dialog/?doi=10.1016/j.abd.2020.02.015&domain=pdf https://orcid.org/0000-0001-6749-7160 https://orcid.org/0000-0001-9283-2354 https://orcid.org/0000-0002-4719-4821 https://orcid.org/0000-0001-8566-0523 https://orcid.org/0000-0002-2596-9294 mailto:heliomiot@gmail.com https://doi.org/10.1016/j.abd.2020.02.015 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Melanogenesis induced by UVB, UVA, and visible light in facial melasma 685 Study limitations: Use of an ex vivo model, with independent irradiation regimes for UVB, UVA, and visible light. Conclusions: Melanogenesis induced by UVB, UVA, and visible light was observed both in melasma and in the adjacent skin. The morphological patterns suggest that different irradiations promote individualized responses on the skin with melasma. © 2020 Sociedade Brasileira de Dermatologia. Published by Elsevier España, S.L.U. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 2 f t e t t o a o d c a s c t w e n t T p o s a M i p A w U 1 r s 1 f b d a i i i t Introduction Melasma is a chronic and acquired hypermelanosis resulting from a dysfunction of melanogenesis: increased produc- tion, transfer, and greater maturity of melanosomes.1 It is a frequent condition in the dermatological routine; accord- ing to a study by the Brazilian Society of Dermatology published in 2018, it accounts for 3.6% of dermatological consultations.2 It affects mainly women of childbearing age (25---50 years) and intermediate phototypes (III---V). The clini- cal lesions of melasma are macules, commonly symmetrical, hyperchromic, well-defined, and located in anatomical sites exposed to the sun, especially the face.3 Its pathophysiology is still not completely understood, but there is an interac- tion between genetics (autosomal dominant inheritance), hormonal factors, and exposure to solar radiation, leading to greater focal melanogenesis.3---7 Phenotypic changes in the skin with melasma result from both morphological (enlarged melanocytes and prominent dendrites) and dysfunctional epidermal alterations, resul- ting from the complex interaction between keratinocytes, melanocytes, and components of the upper dermis.8 There is a clear relationship between sun exposure and melasma worsening, due to its exclusive occurrence in photo exposed areas, prevalence in professionals who are exposed to the sun, and better therapeutic response associated with photoprotection.9,10 Even with evidence of the role of solar radiation in the clinical history of the disease, the indi- vidualized response pattern to the different bands of solar radiation (UVB, UVA, and visible light [VL]) of melasma in comparison with normal adjacent skin is not known. Knowledge of the different influences of solar radiation in the melanogenesis of melasma can lead to a better under- standing of the pathophysiology of the disease, as well as to the development of new treatment options and optimized photo protection strategies. This study aimed to determine the melanogenic response of facial melasma in comparison with the normal adjacent skin after irradiation with UVA, UVB, or VL in an ex vivo model. Material and methods This was a quasi-experimental ex vivo study of skin frag- ments of 22 patients with facial melasma, diagnosed by an experienced dermatologist. Participants were selected among patients attended at the dermatology service of Hos- pital das Clínicas, Faculty of Medicine de Botucatu, SP, Brazil, between September/2018 and January/2019. The project was approved by the research ethics committee (No. p q 1 ( ,700,889) and all participants signed an informed consent orm before inclusion. Adult patients (>18 years old), of both sexes, with pho- otypes III---V, with facial melasma and without treatment, xcept for sunscreen for at least 30 days, were eligible for he study. The phototype restriction was based on the fact hat phototypes I and II represent less than 10% of cases f melasma and do not show satisfactory melanogenesis fter experimental irradiation.3,11 There is no description f melasma in phototype VI. Patients with other facial dermatoses, photosensitive ermatoses, collagenoses, blood dyscrasias, users of anti- oagulant medication, those who were immunosuppressed, nd pregnant or lactating women were not included in the tudy. Sampling was carried out by convenience, recruiting onsecutive consenting patients during medical consulta- ions at the institution. Sample material collection occurred in a standardized ay and under a sterile procedure. Two samples from ach participant were collected: skin with melasma and ormal adjacent skin, up to 2 cm apart, under local infil- rative anesthesia with 2% lidocaine with vasoconstrictor. he removal included the reticular dermis, with a 3 mm unch and 6---0 nylon monofilament suture. Peripheral areas f the face were preferred, in order to minimize unsightly cars. Samples were sectioned longitudinally in two parts nd stored in 10 mL of DMEM (Dulbecco’s Modified Eagle’s edium, high glucose --- D5796, Sigma Inc., United Kingdom) n a sterile transparent plastic bottle and kept at room tem- erature, according to the protocol set forth by Olivatti.12 part of the fragment was placed immediately in the dark, hile the other was exposed to radiation, for a total UVB, VA, and VL dose (effective in the tissue) of 166 mJ/cm2, .524 J/cm2, and 40 J/cm2 (blue-violet light component), espectively. The fragments were irradiated with artificial ources of UVB (230 �W/cm2; source: FS72T12/UVB/HO) for 2 min, UVA (1270 �W/cm2; source: Phillips TL 100W/10R) or 6 min, and LED light (110 mW/cm2 in the blue-violet and; source: GBRLUX 200W) for 20 min, at a standardized istance of 10 cm. The absorbance of the plastic vials was round 50% for all radiations. The allocation of participants n each type of irradiation was consecutive (not random- zed). After irradiation, both fragments (irradiated and non- rradiated) were cultured in a dark setting. After 72 hours, he samples were removed from the culture medium and reserved in 10% buffered formalin for 36 hours. Subse- uently, they were dehydrated in 70% alcohol for at least 2 hours before embedding in paraffin and Fontana-Masson FM) staining. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ 6 t s i i w r f t m ( U d a g b g c m y p f d i u c I s R A m s d o ( c U c a V ( v a T v e i a u i ( m d U D E m q m t o e m 2 c r a b T u a a s c s p t c l l i b V t b v s b m t m m w i e o m V m e p U 86 To estimate the melanization of the fragments, four pho- ographs of central and interfolicular areas of the samples tained by FM were captured, favoring hotspot areas; the mages were saved in. JPG format (1264 × 681 pixels). Dur- ng the entire capture and analysis process, the investigators ere blinded to the anatomical sites or type and irradiation egime. The selected images were analyzed using ImageJ 1.43, rom the manual selection of the basal layer, followed by hresholding using the ‘‘red’’ channel.13 The main outcome of the study was the percentage of elanization of the basal layer, compared between groups melasma vs. adjacent normal skin) for each radiation (UVB, VA, or VL). The percentage of melanin in the superficial ermis, dendrite count, and melanosome granulation were lso evaluated (score: 1+, fine granulation; 2+, moderate ranulation; 3+, coarse granulation) in the sampled areas. The mean value of the percentage of melanin in the asal layer and in the upper dermis, dendrite count, and ranulation scores were tabulated for each specimen. The omparison groups were assessed by a generalized linear ixed effects model, adjusted for phototype (robust anal- sis) with an unstructured covariance matrix and Šidák’s ost-hoc correction.14 The probability distributions varied or the type of variable analyzed (ordinal and gamma). The sample was sized after a pretest with radiation osimetry that led to the expectation of a 10% increase n the percentage of melanization in the basal layer eval- ated by digital image analysis of the slides stained by FM, onsidering alpha of 5% and power of 20%.15 The data were tabulated in MSExcel 2013 and analyzed in BM SPSS v. 25. Two-tailed p-values ≤0.05 were considered ignificant. esults total of 89 skin fragments from 22 patients with facial elasma were evaluated (one participant volunteered four amples in order to test all irradiations). The mean age (stan- ard deviation) of the participants was 42.1 (10.4) years, 21 f whom were female, with the following phototypes: III 46%), IV (27%), and V (27%). Three culture fragments were ontaminated (UVA, normal adjacent skin, pre-irradiation; VB, melasmic skin, post-irradiation; and UVB, normal adja- ent skin, post-irradiation) and were not included in the nalysis of the results. Patients whose samples were submitted to UVB, UVA, and L irradiation had the following phototypes, respectively: III 33% vs. 37% vs. 0%), IV (44% vs. 37% vs. 71%), and VL (22% s. 25% vs. 28%) (p = 0.35). Melanogenesis was observed in melasma and in normal djacent skin (p < 0.01) after all irradiations (Figs. 1---3, able 1): median UVB increment (4.7% vs. 8.5%), UVA (9.5% s. 9.9%), and VL (6.8% vs. 11.7%), with no significant differ- nce in the comparison between anatomical sites. When adjusted for phototype, the dendrite count ncreased (68.8%) only after irradiation with UVB in both natomical sites (Table 1). An increase in melanin gran- lation (coarser melanosomes) was observed only after rradiation with UVA and only in the skin with melasma p = 0.05). An increase was observed in the percentage of a d t Alcantara GP et al. elanin in the upper dermis in the adjacent skin after irra- iation with UVB, and in melasmic skin after irradiation with VA (p ≤ 0.05). iscussion ffective epidermal melanogenesis was observed in skin odels (ex vivo) submitted to UVB, UVA, and VL, with no uantitative difference between the anatomical sites (facial elasma and normal adjacent skin), supporting the impor- ance of broad spectrum photoprotection in the treatment f pigmentary dermatoses, such as melasma.16 The radiation doses tested, although independent, were quivalent to approximately 6---12 min of sun exposure at idday, in the summer, in countryside Brazil (latitude: 2o53′09′′S; longitude: 48o26′42′′W; altitude: 804 m), on a loudless day, as measured on December 9, 2018, by the esearchers. However, the levels of solar UVA and VL reach n energy plateau from 9 am to 6 pm, while the UVB peaks etween 11 am and 3 pm, both in summer and in winter. his may justify the report of melasma worsening after brief nprotected exposures outside peak UVB hours, or even fter long exposures with a photoprotector (e.g., during traffic jam), since sunscreens do not completely block olar radiation.17 UVB, UVA, and VL radiation measured in losed environments (e.g., from cell phone and computer creens, or fluorescent lamps) are negligible for the pur- oses of melanogenesis (data not shown). VL has a wide spectrum of wavelengths (400---700 nm) hat do not act homogeneously on pigmentation. A study arried out in France tested the spectrum of blue-violet ight (400---500 nm) in comparison with the spectrum of red ight (620---750 nm); those authors observed no pigmentation nduced by the latter, justifying the choice of the blue-violet and for the present study.18 Only opaque sunscreens have effective protection against L; the majority of them also perform better in UVA protec- ion. In a Brazilian survey with 41 opaque sunscreens, 63% locked >99.9% of UVA, and 63% blocked >99.9% of blue- iolet light transmittance. While the percentage was the ame, it was not overlapped: 31% of opaque filters that locked >99.9% of the VL did not present the same perfor- ance for UVA.17 The present study is the first to highlight he role of VL in the melanogenesis of patients with facial elasma. No differential melanogenesis was observed between elasma and adjacent normal skin at the doses tested, hich does not exclude the possibility that melasmic skin s sensitive to lower doses of irradiation or responds differ- ntly to repeated exposures. The greater basal pigmentation f melasma itself can also present some resistance to elanogenesis induced by chronic exposure to UVR and L. Furthermore, there is greater epidermal retention of elanin in melasma, and its melanogenesis depends on sev- ral other dermal changes, which favor the maintenance of igmentation to the detriment of melanogenic induction by VR and VL.4 Melanogenesis occurs unevenly, depending on the type nd dose of radiation. UVB causes erythema that persists for ays to weeks (depending on the phototype) and pigmenta- ion that evolves to tanning. UVA causes erythema in four Melanogenesis induced by UVB, UVA, and visible light in facial melasma 687 Figure 1 Histological sections of skin with melasma and normal adjacent skin samples stained with Fontana-Masson before and after irradiation with 166 mJ/cm2 UVB, revealing an increase in the basal melanin density in the different samples. (A) Normal adjacent skin before UVB irradiation; (B) normal adjacent skin after UVB irradiation; (C) skin with melasma before UVB irradiation; (D) skin with melasma after UVB irradiation. Figure 2 Histological sections of skin with melasma and normal adjacent skin samples stained with Fontana-Masson before and after irradiation with 1.524 mJ/cm2 UVB, revealing an increase in the basal melanin density in the different samples. (A), normal adjacent skin before UVB irradiation; (B), normal adjacent skin after UVB irradiation; (C), skin with melasma before UVB irradiation; (D), skin with melasma after UVB irradiation. Figure 3 Histological sections of skin with melasma and normal adjacent skin samples stained with Fontana-Masson before and after irradiation with 40 mJ/cm2 visible light (VL), revealing an increase in the basal melanin density between the samples. (A), nor- mal adjacent skin before VL irradiation; (B), normal adjacent skin aft (D), skin with melasma after VL irradiation. er VL irradiation; (C), skin with melasma before VL irradiation; 688 Alcantara GP et al. Table 1 Median and quartiles (p25---p75) of the percentage of melanin in the basal layer and in the upper dermis, dendrite count, and melanin granulation in the histological sections of skin with melasma and normal adjacent skin, before and after (pre and post) irradiation with UVB, UVA, and visible light (VL). Data analysis adjusted by phototype. Irradiation Variable Facial melasma Normal adjacent skin p (time × group)** Pre Post p* Pre Post p* UVB MelBasal 64 (60---65) 65 (65---68) <0.01 54 (53---56) 56 (55---62) <0.01 0.09 MelUD 44 (30---77) 45 (38---97) 0.24 44 (32---71) 67 (41---87) 0.04 0.84 Dendrites 6 (2---8) 10 (8---18) <0.01 6 (3---14) 11 (7---16) 0.05 0.59 GranMelanos 2.0 (1.5---2.1) 1.6 (1.3---2.0) 0.11 1.9 (1.4---2.0) 1.9 (1.6---2.0) 0.80 0.98 UVA MelBasal 62 (54---66) 70 (57---72) <0.01 56 (49---61) 57 (50---63) <0.01 0.06 MelUD 41 (18---88) 86 (36---144) 0.05 45 (27---90) 74 (34---204) 0.50 0.88 Dendrites 8 (4---11) 8 (2---22) 0.92 3 (2---5) 5 (3---9) 0.26 0.53 GranMelanos 1.0 (1.0---1.8) 2.0 (1.8---2.1) 0.05 1.3 (1.3---2.0) 1.3 (1.1---1.9) 0.34 0.03 Visible light MelBasal 73 (59---74) 75 (69---80) <0.01 64 (56---66) 70 (61---75) <0.01 0.09 MelUD 62 (54---72) 52 (45---83) 0.11 66 (59---83) 75 (53---127) 0.34 0.21 Dendrites 2 (1---4) 4 (2---14) 0.11 1 (0---3) 4 (1---5) 0.06 0.38 GranMelanos 1.4 (1.0---2.0) 1.5 (1.0---2.3) 0.11 1.5 (1.0---2.0) 1.5 (1.0---2.0) 0.80 0.98 MelBasal, percentage of melanin in the basal layer of the epidermis; MelUD, percentage of melanin in the upper dermis (×100); Gran- Melanos: granulation score of melanosomes of the epidermis (1+, fine granulation; 2+, moderate granulation; 3+, coarse granulation), mean of four fields. espo h t e g m w s ( t t e e f s f a a m a G m i p a o p m m m U o s a A m r m o t m m I o a m t a l i b o s a F v o p t i t t * p-value, pre vs. post (adjusted by phototype). ** p-value, melasma vs. normal adjacent skin depending on the r ours and immediate pigmentation that evolves with lasting anning. In VL, erythema is less prominent and may occur arly (within two hours). Pigmentation is sustained with pro- ression to tanning, but only in higher phototypes.19---21 Every melanogenic response occurs in a dose-dependent anner. However, it is essential to consider the phototypes hen analyzing the performance of different radiations, ince melanogenesis is different in the higher phototypes III---V), causing greater pigmentation with evolution to anning in a dose-dependent manner. In turn, in lower pho- otypes (I---I) irradiation promotes less stimulation. In an xperimental study, UVA and VL did not induce melanogen- sis in patients with phototype II.11,22 Human melanogenesis is a complex process in which dif- erent interactions occur in an organized manner to protect kin structures and to regulate cycles that are essential or survival, such as the circadian cycle. Melanocytes are network with their own sense of ambient light, responding ccording to the aforementioned objectives.23 Different characteristics were observed in the elanogenic response when comparing anatomical sites nd radiations. The formation of dendrites occurs in the 2 phase of melanocyte growth and is an important sign of elanogenic response; in the present study, it was mainly nduced by the irradiation with the greatest mutagenic otential (UVB). Although melanogenesis is perceived in similar way in both anatomical sites, the formation f dendrites can indicate a greater persistence of the henomenon and greater efficiency in the transfer of elanosomes in the epidermis.11 Melasmic skin is also characterized by the presence of ore mature melanosomes, equivalent to what occurs in ore pigmented populations. In the present experiment, VA irradiation promoted more evident granulation than the ther tested radiations exclusively on skin with melasma, howing that, in melasma, melanogenesis is more intense m U a T nse to irradiation (adjusted by phototype). nd resembles the most pigmented phenotypes. In fact, frican ancestry was associated with an increased risk of elasma in the Brazilian population, and ancestral genes elated to melanogenesis may justify the differentiated pig- ent response between anatomical sites.7 The role of dermal melanin remains unclear in melasma; ne of the hypotheses is that the architectural dysfunc- ion of the basement membrane and the autophagy of the elanocytes of the dermis may cause the presence of der- al melanophages, similar to what occurs in photoaging.24,25 n the present study, UVA and UVB promoted a higher rate f melanin in the upper dermis; however, this density was pproximately 100 times smaller than that of the epider- is in both anatomical sites, which likely little contributes o the clinical phenotype, but may signal underlying alter- tions in the upper dermis, basement membrane, and basal ayer. Finally, despite the fundamental role of solar radiation n worsening melasma, no differential expressions of p53 etween the melasma and the normal adjacent skin were bserved, suggesting that the maintenance of melanogene- is may be due to underlying changes in the upper dermis nd epidermis, which induce melanocytic hyperfunction.4 urthermore, the exclusive use of sunscreen, despite its pre- entive effect, is not sufficient for the complete remission f the condition, which indicates the need for a continuous athophysiological study in search of treatments that lead o the reversal of the entire process.9,26 The present study promoted a preliminary analysis of the ndependent effects of UVB, UVA, and VL radiation. Fur- her investigations should be conducted in order to elucidate he effect of combinations and dosages of irradiations that irror environmental conditions. The interaction between VA and VL is still unknown; they are believed to inter- ct in the same melanin precursor during melanogenesis. his association may result in a more evident behavior of al m C N R 1 1 1 1 1 1 1 1 Melanogenesis induced by UVB, UVA, and visible light in faci melanogenesis in natural situations, where VL and UVA are expressed together.20 This study has limitations inherent to the technique in an ex vivo model, which may differ depending on collection and storage. However, this limitation did not prevent the identification of a melanogenic response. Another limitation can be pointed out by the fact that irradiations in ex vivo skin models occur with independent wavelengths (UVB, UVA and VL), which is not the case in environmental exposure. In addition to the factors presented above, ex vivo models are dissociated from the organism, limiting the interaction between the hormonal, neural, and vasomotor systems. New experiments should consider low-intensity repeated irradiation models, as well as associated irradiation, to bet- ter reproduce the daily exposure of patients. In addition, the inclusion of substances such as tranexamic acid and antiox- idants in the prevention of melanogenesis in melasma can be tested. Conclusions An epidermal melanogenic response induced by UVB, UVA, or VL was observed both in skin with melasma and in nor- mal adjacent skin. The morphological patterns related to the photobiology of melanogenesis suggest that different radiations promote individualized responses in the skin with melasma. Financial support CNPq (PIBIC No. 148501/2018-4); FUNADERM (001/2019). Authors’ contributions Giovana Piteri Alcantara: Approval of the final version of the manuscript; conception and planning of the study; elab- oration and writing of the manuscript; obtaining, analyzing, and interpreting the data; critical review of the literature; critical review of the manuscript. Ana Cláudia Cavalcante Esposito: Approval of the final version of the manuscript; conception and planning of the study, elaboration and writing of the manuscript; effective participation in research orientation; critical review of the literature; critical review of the manuscript. Thainá Oliveira Felicio Olivatti: Approval of the final version of the manuscript; obtaining, analyzing, and inter- preting the data; critical review of the manuscript. Melissa Mari Yoshida: Elaboration and writing of the manuscript; obtaining, analyzing, and interpreting the data; critical review of the manuscript. Hélio Amante Miot: Statistical analysis; approval of the final version of the manuscript; conception and planning of the study; elaboration and writing of the manuscript; obtaining, analyzing, and interpreting the data; effective participation in research orientation; intellectual participa- tion in propaedeutic and/or therapeutic conduct of studied cases; critical review of the literature; critical review of the manuscript. 1 elasma 689 onflicts of interest one declared. eferences 1. Miot LDB, da Silva MG, Miot HA, Marques MEA. Morphological and functional comparative study of melanocytes in melasma lesions. An Bras Dermatol. 2007;82:529---34. 2. Sociedade Brasileira de Dermatologia, Miot HA, Penna GO, Ramos AMC, Penna MLF, Schmidt SM, et al. Profile of der- matological consultations in Brazil (2018). An Bras Dermatol. 2018;93:916---28. 3. Tamega AA, Miot LD, Bonfietti C, Gige TC, Marques ME, Miot HA. Clinical patterns and epidemiological characteristics of facial melasma in Brazilian women. J Eur Acad Dermatology Venereol. 2013;27:151---6. 4. Espósito ACC, Brianezi G, de Souza NP, Miot LDB, Marques MEA, Miot HA. Exploring pathways for sustained melanogenesis in facial melasma: an immunofluorescence study. Int J Cosmet Sci. 2018;40:420---4. 5. Miot LDB, Miot HA, da Silva MG, Marques MEA. Physiopathology of melasma. An Bras Dermatol. 2009;84:623---35. 6. Holmo NF, Ramos GB, Salomão H, Werneck RI, Mira MT, Miot LDB, et al. Complex segregation analysis of facial melasma in Brazil: evidence for a genetic susceptibility with a dominant pattern of segregation. Arch Dermatol Res. 2018;310:827---31. 7. D’Elia MPB, Brandão MC, de Andrade Ramos BR, Silva MG, Miot LDB, Santos SEB, et al. African ancestry is associated with facial melasma in women: A cross-sectional study. BMC Med Genet. 2017;18:1---7. 8. Kang WH, Yoon KH, Lee ES, Kim J, Lee KB, Yim H, et al. 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http://refhub.elsevier.com/S0365-0596(20)30227-0/sbref0130 http://refhub.elsevier.com/S0365-0596(20)30227-0/sbref0130 http://refhub.elsevier.com/S0365-0596(20)30227-0/sbref0130 http://refhub.elsevier.com/S0365-0596(20)30227-0/sbref0130 Evaluation of ex vivo melanogenic response to UVB, UVA, and visible light in facial melasma and unaffected adjacent skin Introduction Material and methods Results Discussion Conclusions Financial support Authors’ contributions Conflicts of interest References