Melatonin immunoreactivity of epidermal skin is higher in the evening than morning but does not account for erythema sensitivity

ABSTRACT The skin is a site of melatonin synthesis, and melatonin has a role in protecting against ultraviolet radiation-induced damage. Ultraviolet B (UVB) induced erythema seems to vary between morning and evening. We investigated whether epidermal melatonin immunoreactivities in the morning differed from those in the evening, and whether UVB-induced erythema was associated with these melatonin immunoreactivities in healthy volunteers. Erythema sensitivity of the skin was determined in the morning and in the evening by scoring the Minimal Erythema Dose and quantifying the erythema index (EI). We took biopsies from the non-UVB-exposed skin of healthy volunteers (n = 39) in the morning and in the evening to study melatonin immunoreactivity with immunohistochemistry (IHC). In the IHC staining, there was more melatonin immunoreactivity in the evening than in the morning (p < .001). Erythema was more pronounced in the evening than in the morning irradiated skin (p < .001). The graded amount of melatonin immunoreactivity in the samples was not associated with the EI. We discovered melatonin immunoreactivity of the non-irradiated skin to vary diurnally. However, endogenous skin melatonin does not seem to be the reason why NB-UVB induces more erythema in the evening than in the morning.


Introduction
The central circadian clock in the suprachiasmatic nucleus (SCN) of hypothalamus adjusts the actions of human body physiology to 24 h a day. In addition, partially autonomous peripheral circadian clocks control the physiology of most cells and tissues (Brown et al. 2019). The central circadian clock in the SCN senses the time of the day via external signals and synchronizes clocks in the peripheral tissues by releasing, e.g., melatonin and glucocorticoids (Buhr et al. 2010;Pevet and Challet 2011;Tahara et al. 2017). The skin also has its inbuilt circadian clock (Wu et al. 2018) that affects e.g. UV radiation-induced erythema in human skin (Nikkola et al. 2018).
Ultraviolet radiation (UVR) in the skin induces oxidative stress, direct DNA damage, and acute skin inflammation, which manifests as erythema (Hönigsmann 2002). Solar UVR is subdivided into the following three sections: UVA (315-400 nm), UVB (280-315 nm), and UVC (100-280 nm) by the Commission Internationale de l'Eclairage (CIE) (CIE 2011). UVB is highly erythematogenic and is mainly responsible for epidermal carcinomas of the skin (D'Orazio et al. 2013). On the other hand, narrowband ultraviolet B (NB-UVB) is a common treatment for several skin diseases in dermatology clinics. For example, consensus-based European guidelines recommend phototherapy, especially NB-UVB, as a treatment for moderate-to-severe psoriasis and atopic eczema (Pathirana et al. 2009;Wollenberg et al. 2018). Although the UVB radiation penetration depth in the human skin is only millimeters, its implications extend to homeostasis of entire body (Slominski et al. 2018b).
The hormone melatonin (N-acetyl-5-methoxytryptamine) is well known for its influence on circadian rhythms (Pevet and Challet 2011). Secretion of melatonin from the pineal gland is controlled by the central circadian clock in the SCN (Moore 1996) and varies in individuals expressing different chronotypes (Claustrat et al. 2005). Some of the actions of melatonin are transmitted through receptors: the melatonin membrane-bound receptors MTNR1A and MTNR1B and the so-called third melatonin receptor NQO2, an enzyme that binds melatonin in the cytosol. In addition, melatonin can also function through non-receptor mediated mechanisms, and, for example, serve as a scavenger for reactive nitrogen and oxygen species (Rusanova et al. 2019;Slominski et al. 2012). It remains unknown whether there are nuclear receptors for melatonin (Slominski et al. 2018a). Melatonin is, in addition to the pineal gland, synthesized in several organs such as bone marrow (Tan et al. 1999), gut (Bubenik 2002;Kobayashi et al. 2005), ovaries (Itoh et al. 1999), and skin (Slominski et al. 2002).
In the skin, melatonin, its precursors and metabolites are endogenously produced in the keratinocytes (Kim et al. 2015). For strong antioxidants, melatonin and its metabolites may play a role in protecting against UVRinduced cell damage (Slominski et al. , 2017. In human epidermal keratinocytes, ex vivo, melatonin and its metabolites protect cells against UVB-induced oxidative stress via receptor-independent pathways and against DNA damage through activation of melatonin receptors . Of the melatonin receptors, MTNR1A and NQO2 are expressed in the epidermal keratinocytes of normal human skin (Fischer et al. 2008). Melatonin not only acts as an antioxidant but may also have UV-protective effects due to its anti-inflammatory properties. An oxidative stress-sensitive factor NFκB, important in skin inflammation, can be inhibited by melatonin, leading to reduction of pro-inflammatory cytokines (Fuchs et al. 2001;Sainz et al. 2008). Both antioxidant and anti-inflammatory actions could link melatonin with skin erythema induction.
Chronotype is a construct reflecting individual differences in the preference for the timing of daily activities (Adan et al. 2012;Horne and Östberg 1976). The chronotype is also related to the timing of rhythmic melatonin secretion from the pineal gland (Mongrain et al. 2006). While extrapineal melatonin synthesis has mostly been assumed not to be rhythmic , no results comparing melatonin expression in the skin with the chronotype have been presented. Melatonin secretion is also linked to seasonal affective disorder (SAD) which is a type of depression that results from a delay (or rarely, an advance) in the circadian rhythms (Rosenthal et al. 1984). The phase shift hypothesis holds that the abnormalities in the phase of circulating melatonin cause SAD, and that bright light exposure in turn suppresses the circulating levels of melatonin and shifts the phase and relieves symptoms of SAD (Lewy et al. 2009).
The effect of topically applied melatonin on UVinduced erythema has been studied earlier (Scheuer et al. 2016), but no research was available on the effect of the naturally occurring melatonin in the skin. Our aims were to study the association between epidermal melatonin immunoreactivity and narrow-band UVB (NB-UVB) induced skin erythema, and if the level of epidermal melatonin immunoreactivity differed between morning and evening. Due to the linkage between the chronotype and pineal melatonin, we also studied if the chronotype was associated with epidermal melatonin immunoreactivity.

Materials and methods
Healthy adult volunteers presenting with anamnestic skin phototype II, III or IV were eligible to participate (Fitzpatrick 1988). Skin phototype II is susceptible to burning upon sun exposure, and afterward gets some tanning, phototype III tans more easily and burns only occasionally and phototype IV tans easily and rarely burns. The skin phototypes were pre-screened in a clinical interview eliciting skin burns and tanning and comparing the responses to clinical inspection of the skin. Individuals with photosensitizing, immune modulating or psychiatric medication, photosensitivity, pregnancy, lactation, history of skin cancer or extensive scarring, or marked UVR exposure in the three preceding months were not eligible to participate. During the study, birth control pills, thyroxin, hypercholesterolemia and hypertension medications were allowed. No other medication was allowed.

Study protocol (Groups A and B)
The first group of volunteers (Group A) consisted of 19 persons, who were investigated from January to April in 2016. To increase the sample size and to enable comparison between different times of the day, we recruited another group of volunteers (Group B) consisting of 20 volunteers, who were investigated in mid-winter in the years 2018 and 2019. In each individual case, the study protocol was implemented on two consecutive days. As regards photo-testing, both Groups A and B were treated equally. On day one, the photo-testing with NB-UVB was performed twice, once in the morning and once in the evening 12 h apart to determine the Minimal Erythema Dose (MED) for the morning and for the evening. On day two, the erythema produced in the 1 cm 2 photo-test squares was evaluated initially by the naked eye, and then the erythema was quantified objectively with a reflectance spectrometer.
As regards the skin biopsies taken on day two, the Groups A and B differed as follows: From group A, we took one non-UV-exposed skin biopsy (in the morning), whereas from group B, we took in total two non-UVexposed skin biopsies (one in the morning and one in the evening). The study protocols of the groups were otherwise identical.

Biopsies and immunoreactivity
We took all the biopsies on the second day of the study. As regards Group A, to study melatonin and its receptor immunoreactivity in the non-UV-exposed healthy skin, we took one punch biopsy from the skin of the buttocks a minimum of 10 cm away from the MED test skin area in the morning (between 07:00 h and 09:00 h).
As regards Group B, to study the impact of different time of day on immunoreactivity, we took a respective biopsy in the morning (between 07:00 h and 09:00 h) as in Group A, but in addition another biopsy in the evening (between 19:00 h and 21:00 h). In Group B, both the biopsies were also taken a minimum of 10 cm away from the MED testing area.
Biopsy protocols were identical for all biopsies. Lidocaine 1% without epinephrine was the local anesthetic used. The specimen was put in 4% formalin for fixation for one to three days, embedded in paraffin, and then sectioned at 4-μm thickness for further processing. For the processing, we used Ventana Benchmark immunostainer (Ventana Medical Systems, Tucson, Arizona, USA). Ventana CC1-buffer pH 9 was used as a pretreatment.
To assess the melatonin and the two melatonin receptors MTNR1A and NQO2 immunoreactivities, we performed immunohistochemical (IHC) staining according to the protocols of the manufacturer with antibodies described in more detail in Supplementary Table S1. The pineal gland (surgical specimen) was used as the positive control for melatonin antibody, and the suprachiasmatic nucleus (autopsy material) was used as the positive control for the receptors. A board certified dermatopathologist (R.H.-O.) and the first author (V. N.) assessed each slide under a microscope (Olympus BX51, Tokyo, Japan) at 100 and 200 magnifications. The settings of the microscope and the camera were held constant, and no corrective measures were taken to adjust the color balance. Melatonin, MTNR1A, and NQO2 immunoreactivities were evaluated initially using three grades: negligible, positive, and strongly positive. To analyze their connection with the erythema sensitivity, the positive findings were combined, and the level of immunoreactivity was presented as two grades: low, consisting of negligible findings, and high, consisting of positive and strongly positive findings.

UVB irradiations
For the NB-UVB photo-testing irradiations, a Waldmann UV 801KL phototherapy device equipped with four TL20W/01 narrow-band ultraviolet B (NB-UVB) tubes with peak emission at 311 nm was used. The irradiance of the device was measured before the study using an Ocean Optics S2000 spectroradiometer provided by the Nuclear Safety Authority of Finland (L. Y.), and the time to irradiate the MED series was based on the irradiance measurement. Uncertainty (2σ) of the measurement of the Ocean Optics S2000 was estimated to be approximately 14% (Ylianttila et al. 2005). The measurements are traceable to the National Institute of Standards and Technology (Gaithersburg, Maryland, USA). One Standard Erythema Dose (SED) is defined as the erythemal effective radiant exposure of 10 mJ/cm 2 (CIE, Commission Internationale de l'Eclairage, Vienna, Austria), being equivalent to a non-weighted physical dose of 172 mJ/cm 2 emitted from our NB-UVB lamps (CIE). Before testing, the tubes were preheated for 7 min to stabilize irradiation.
The erythema threshold of the skin, depicting photosensitivity of the skin, is conventionally assessed by defining the smallest UVR dose needed to induce faint, just perceptible erythema in the skin, i.e., the minimum erythema dose (MED) (Faurschou and Wulf 2009). In the present study, we used the geometric UVR dose series and defined the doses as SED series as follows: 1 SED, 1.4 SED, 2 SED, 2.8 SED, and 4 SED, where the incremental factor was √2. The doses were administered to the skin of the buttocks on five 1 cm 2 test squares while covering the rest of the skin with impermeable flexible plastic (Faurschou and Wulf 2009). All erythema testing irradiations were performed on day one. The first irradiation series was executed in the morning (between 07:00 h and 09:00 h) and the second in the evening (between 19:00 h and 21:00 h).

Measurement of erythema
Measurement of erythema in the photo-testing squares was identical for Groups A and B. On day two in the morning (between 07:00 h and 09:00 h) and in the evening (between 19:00 h and 21:00 h), i.e., 24 h after irradiations, we first evaluated the MED and graded erythema in the test squares by the naked eye as minus, i.e., "no erythema" to (+) "barely perceptible erythema" and +, ++ or +++, i.e., "definite erythema" separately for both irradiated areas. The consequent erythema threshold (MED) was defined according to the barely perceptible erythema and was evaluated by the naked eye (Faurschou and Wulf 2009). In cases where MED appeared higher than the maximal exposed dose of 4 SED, it was determined to be 5.6 SED (two persons).
Thereafter, we assessed erythema of the skin in another way using a DermaSpectrometer (Cortex Technology, Hadsund, Denmark), i.e., a reflectance spectrometer. The instrument was calibrated for white and black controls as suggested in the manual. The output Erythema Index (EI) represents the intensity of the reflected red and green lights (Ly et al. 2020). The low EI value reflects less erythema, whereas the high EI value reflects more erythema. The reflectance spectrometer even detects erythema at doses of UVR below MED (Farr and Diffey 1984). Here, we quantified erythema in both the non-irradiated skin and then in all the five test squares, each in triplicate, to use the arithmetic mean of the three readouts in the analyses.

Assessment of chronotype and seasonality
The chronotypes of the participants were assessed with a shortened version of Horne and Östberg's Morningness-Eveningness Questionnaire (MEQ) (Horne and Östberg 1976). The questionnaire includes six items (4,7,9,15,17 and 19 from the original MEQ, thus MEQ-6) and the sum yields of the Morningness-Eveningness Score (MES), ranging from 5 to 27 (Hätönen et al. 2008). The lowest scores indicate definite eveningness, whereas the highest scores indicate definite morningness. The presence of SAD was screened for using the Seasonal Pattern Assessment Questionnaire (SPAQ) (Rosenthal et al. 1984).

Statistical analysis
The descriptive statistics are presented as means with SDs or as counts with percentages. A statistical comparison between the groups was performed using the permutation test and the Fisher-Freeman-Halton test. Repeated measures were analyzed using a permutation approach for analyzing repeated measures analysis of variance (ANOVA) for continuous variables and exact symmetry test for categorical variables. Stata 16.0 (StataCorp LP; College Station, Texas, USA) statistical package was used for the analysis.

Ethics
The Regional Ethics Committee of Tampere University Hospital District approved the study protocol (R16001) and all volunteers gave their written informed consent. The study was carried out at the Department of Dermatology, Päijät-Häme Central Hospital, Lahti, Finland.

Results
Altogether 39 healthy volunteers completed the study. In Group A (n = 19) three were men and 16 were women, the mean age being 42 y (range 22-64 y). Eight volunteers presented with anamnestic skin phototype II and 11 with phototype III (Fitzpatrick 1988). Their phototested MED values for NB-UVB ranged from 1.4 SED to 5.6 SED (one person). In Group B (n = 20) all were women, the mean age 43 y (range 22-65 y). Five volunteers presented with anamnestic skin phototype II, 12 with phototype III and three with phototype IV (Fitzpatrick 1988). Their photo-tested MED values for NB-UVB also ranged from 1.4 SED to 5.6 SED (one person).
In assessing melatonin immunoreactivity there were a total of 38 skin samples (Group A + B) obtained from the skin in the morning, and respectively 20 samples (Group B) obtained in the evening. One of the morning samples was not eligible due to damage while processing the biopsy. Melatonin immunoreactivity in the skin was nuclear, and there was also some cytoplasmic positivity. Melatonin immunoreactivity in the pineal gland specimen (positive control) was both nuclear and cytoplasmic ( Figure 3). We did not find the phototype (II, III or IV) to correlate with melatonin immunoreactivity.
As regards the melatonin receptors, the study material of immunohistochemical staining consisted of 39 morning (Group A + B) and 20 evening (Group B) skin samples. MTNR1A immunoreactivity was membranous and slightly cytoplasmic, whereas the NQO2 immunoreactivity was cytoplasmic with some nuclear positivity and showed more intense positivity in the basal cell layer than MTNR1A. Levels of immunoreactivity of melatonin, MTNR1A and NQO2 are presented in Table 1.
There was more melatonin immunoreactivity in the evening samples (Group B) than in the morning samples (Group A + B), when graded as negligible, positive or strongly positive in the IHC staining (p < .001). We found erythema, as measured with reflectance spectrometer in EI, to be more pronounced in the evening irradiated MED test squares (Group A + B) than in the morning irradiated MED test squares (Group A + B) (p < .001). The mean difference between the evening and morning EIs among all volunteers (n = 39) taking advantage of all the EI readings of the whole MED test series was 1.4 EI units (95% CI: 0.9 to 1.9).
We divided the 39 study subjects into groups according to their level of melatonin immunoreactivity. Figure 1 shows the average dose-response curves for the irradiations delineating the EI scores (i.e., amount of redness of the skin) as classified according to the low or high melatonin immunoreactivity. The EI of the two groups, subjects with either low or high melatonin immunoreactivity, did not differ significantly (p = .08). The graded amount (low versus high) of melatonin immunoreactivity in the samples did not explain the difference in EI in the morning versus evening (Figure 1). Melatonin immunoreactivity was not associated with erythema as detected by the naked eye or the defined MED value.
The MTNR1A and NQO2 immunoreactivities were rather similar in the morning versus evening. There was no correlation between the NQO2 receptors ( Figure 2) or MTNR1A receptor immunoreactivity (equal finding, figure not shown) versus the NB-UVB induced EI in the morning or in the evening. Neither did the amount of MTNR1A nor NQO2 immunoreactivity explain the difference in EI in the morning versus evening samples. No significant correlations were detected between the graded visually assessed erythema or the defined MED value and melatonin receptor immunoreactivity either.
Of the 39 participants, 21 displayed morning, eight evening and 10 intermediate chronotype (Horne and Östberg 1976). Their total Morningness-Eveningness score (MES) ranged from 9 to 25 points. There was no association of the melatonin, MTNR1A or NQO2 immunoreactivity with the MES or with the chronotype.  Chronotype was not associated with erythema sensitivity. According to SPAQ, none of the participants suffered from SAD.

Discussion
Our pilot study shows that the melatonin immunoreactivity of the non-UV-irradiated skin on the buttocks is different in the morning from that in the evening. To the best of our knowledge, this is the first human in vivo study to show such a difference. This study also confirmed our earlier findings: exposure to NB-UVB induced erythema differently by the time of the day (Nikkola et al. 2018). However, it was not due to the difference in levels of melatonin immunoreactivity in the skin. We found that melatonin immunoreactivity of the non-irradiated skin of the buttocks varied between morning and evening. There was more melatonin immunoreactivity in the evening samples than in the morning samples. Pineal expression of melatonin begins in the evening and peaks at night. Some melatonin remains in blood in the morning as it fades and remains undetectable during the day (Reiter 1991). According to earlier studies, extra-pineal melatonin expression may be steady, oscillating or fluctuating in a circadian manner (Acuña-Castroviejo et al. 2014). As regards the extra-pineal localizations, rhythmic melatonin expression has been detected in retina and thymus, both in melatonin content peaking at night (Naranjo et al. 2007;Tosini et al. 2007). However, extra-pineal sites can express melatonin oscillation that does not vary in a circadian manner; this occurs in the liver and cerebral cortex (Venegas et al. 2012). Pineal melatonin has only limited access from blood to the skin due to its degradation in the liver (Slominski et al. 2017). However, without any external cues, the human skin is known to have its own inbuilt circadian rhythm (Spörl et al. 2012;Wu et al. 2018). According to our findings, skin melatonin content may follow the pattern of fluctuation of circulating melatonin. The melatonin content of the skin should next be investigated at several time points on consecutive days using a quantitative method like mass spectrometry to ascertain if epidermal daily fluctuation of melatonin is circadian or not.
Erythema index (EI), i.e. redness of the skin, was significantly lower in skin irradiated in the morning than in skin irradiated in the evening. Our results suggest that endogenous skin melatonin seems not to be the reason behind this variation, because melatonin immunoreactivity   in the skin was not associated with the change in erythema sensitivity. We have shown earlier that NB-UVB induces more intense erythema in healthy human skin in the evening than in the morning (Nikkola et al. 2018). Several studies have shown that topical melatonin significantly prevents both UVB and natural sunlight-induced erythema when applied before UV exposure (Scheuer et al. 2014(Scheuer et al. , 2016. However, topically applied melatonin seems to work specifically in the stratum corneum, a non-viable topmost layer of the skin (Marto et al. 2016). We did not detect melatonin in the stratum corneum ( Figure 3). Contradictory findings from our present study and previously published topical melatonin administration studies may be due to different locations of melatonin in the skin. Based on our present study, endogenous skin melatonin does not seem to protect skin from NB-UVB to any significant extent.
Furthermore, with the use of ICH staining, we demonstrated that melatonin immunoreactivity was mainly nuclear and to a small extent cytoplasmic, MTNR1A immunoreactivity was mainly membranous, and NQO2 immunoreactivity was mainly cytoplasmic. Melatonin immunoreactivity was earlier found to be cytoplasmic in keratinocytes localized in the skin of a human scalp (Slominski et al. 2005b). Immunolocalizations of MTNR1A and serotonin, a precursor of melatonin, were likewise cytoplasmic and membranous in the same anatomical location (Slominski et al. 2020). MTNR1A is a membrane receptor, while NQO2 is located in cytosol . Although NQO2 gene expression has been detected in human skin cells (Slominski et al. 2005a), immunolocalization of NQO2 in human keratinocytes in vivo has not so far been reported. Melatonin has a lipophilic structure, which enables it to cross all biological barriers and exert its influence on the mitochondria, cytosol and nucleus of a cell. Different immunolocalizations of melatonin as found in our study versus the earlier findings of Slominski and colleagues (Slominski et al. 2005b) may be due to different anatomical locations of the skin biopsies taken. In the skin of a human scalp, melatonin may have actions related to hair growth via mitochondria (Rusanova et al. 2019) which may explain the more cytoplasmic location. Immunolocalizations of investigated melatonin receptors MTNR1A and NQO2 are in line with earlier research. We posit that melatonin responsible for cellular functions in human buttocks skin is located mainly in the nucleus. Being located in different parts of the cell, it is likely that the actions of epidermal melatonin are not mediated, at least to a large extent, by MTNR1A and NQO2 receptors.
To our surprise, we did not find any correlation between epidermal melatonin immunoreactivity fluctuation and the chronotype. Liu et al. have earlier shown that a late-time peak of melatonin in the circulation, that is mainly pineal in origin, was associated with eveningness (Liu et al. 2000). Taking into account the limited access of melatonin from the circulation to the skin and that melatonin is endogenously produced in keratinocytes (Slominski et al. 2017), the melatonin we detected in keratinocytes can be a mixture from both origins, the blood and the skin. Unfortunately, we did not take blood samples in our present study, but in future this could be done. While a connection between the circulating melatonin and chronotype is well documented, not enough is known so far about the relationship between the circulating and epidermal levels of melatonin (Slominski et al. 2018a). As far as we know, no studies connecting chronotype and melatonin in the skin have so far been presented. Based on our findings that chronotype was not associated with melatonin immunoreactivity of the skin, the expression of melatonin in the skin seems different from the pineal melatonin expression. Studies on the relationship between the circulating and epidermal levels of melatonin are needed to ascertain if they are directly connected with each other. In future studies, chronotype could also be assessed by different methods, such as dim light melatonin onset (DLMO).
The melatonin immunoreactivity we detected in ICH was located mostly in different sites than receptor immunoreactivity. While melatonin has effects on skin homeostasis via melatonin receptors, the strong protective effects of melatonin against UV damage in the human skin have been thought to be mediated by receptor-free direct radical scavenging (Fischer et al. 2013(Fischer et al. , 2008Gómez-Moreno et al. 2010) and antiinflammatory inhibition of NLRP3 inflammasome and reduction of NF-κB expression (Ortiz et al. 2015). The fact that melatonin immunoreactivity of epidermal skin was not significantly connected to erythema sensitivity in this study does not rule out UV protective receptor-free actions of melatonin. However, it seems that there are diverse mechanisms of action which prevent erythema and work more effectively in the morning hours. This needs to be studied in more detail, e.g. with mRNA transcriptome sequencing in the morning compared to corresponding analysis in the evening and by measuring the number of reactive oxygen and nitrogen species.
A limitation of our pilot study is that we did not take serum samples from the volunteers to assess melatonin and its derivatives in circulation. However, the participants were not suffering from SAD, but had only mild symptoms of SAD, which leads us to presume that their phases of circulating melatonin were close to average. The invasive parts of the study, such as taking biopsies, limited the number of volunteers. The actions of melatonin through non-receptor mediated mechanisms, e.g., by measuring the number of reactive oxygen and nitrogen species could likewise not be evaluated in this clinical human study. Melatonin seems to have differential modes of actions which may vary according to the site of functioning, and this must be taken into account in further studies.
In conclusion, our study showed for the first time that melatonin immunoreactivity of the non-irradiated buttocks skin varied in a diurnal manner between morning and evening. The outcome adds information to explain the physiology of epidermal melatonin in human skin in vivo. The fluctuation could be related to circulating melatonin levels. Epidermal melatonin does not seem to be the reason why NB-UVB induces more erythema in the evening than in the morning. Epidermal melatonin did not reflect individual chronotypes assessed by the six-item MEQ. Melatonin and UVR connections remain an interesting target for further study using different sophisticated methods in parallel.