The impacts of DUVR exposures were determined in several studies using DUVR spectrum in in vivo clinical studies and in in vitro experiments.
3.1. Effects of DUVR in Human Skin in Vivo
In order to investigate the impact of a non-extreme solar exposure in human skin, the first clinical and biochemical study was conducted in 12 volunteers exposed to acute, and in 22 volunteers exposed to repeated sub-erythemal doses, of DUVR or to UV-SSR [
36].
Individual minimal erythema doses (MED) of DUVR and of UV-SSR were determined. For skin phototypes II and III, the average MED of DUVR and UV-SSR was found to be 12 ± 2.1 and 3.4 ± 0.55 J/cm2, respectively.
With regards to the biological effects induced by acute exposure to DUVR, most significant changes were obtained using 1 or 1.5 MED of DUVR, with the formation of sunburn cells (SBC), the accumulation of nuclear p53, thymine dimers, fibroblast apoptosis, a decrease in number and size of Langerhans cells, as well as an increased number of melanocytes. UV-SSR was more efficient than DUVR to induce SBC and p53 accumulation, in agreement with the known contribution of UVB in these effects. The dose of 0.5 MED of DUVR did not lead to any significant alteration of the tested endpoints but interestingly, a linear dose-response effect of DUVR was evidenced for p53 accumulation and the induction of dermal apoptotic cells.
One single exposure to a sub-erythemal DUVR dose had no significant effect, but assuming that the harmful consequences of daily UV exposures mostly result from chronic exposure, the cumulative effects of DUVR exposure were investigated. Volunteers were submitted to 9 repeated exposures to sub-erythemal doses of DUVR (0.25, 0.5 and 0.75 MED), or to 19 repeated exposures to 0.5 MED DUVR (
Table 2). Exposure to 9 repeated sub-erythemal doses of DUVR led to significant changes in skin pigmentation, as assessed by colorimetric measurement using the Commission Internationale de l’Eclairage CIE lab 1976 color system, with L* expressing Luminance (from black to white), a* red-green component and b* yellow-blue component. The absolute values of L*, a*, b* are used to define the color of the skin. Δa*, Δb*, ΔL* are the differences between exposed and non-exposed sites of a *, b * and L * values, respectively. This exposure also led to significant changes in skin hydration, elasticity and microtopography, such as loss of skin density (
Table 2). Biological alterations and damage were also observed, including an increase in the epidermal thickness, a decrease in number of Langerhans cells together with an increase of their size, urocanic acid isomerization [
23], an increase in number and size of melanocytes and melanin deposition, an increase in keratinocyte proliferation, as well as SBC formation and p53 accumulation. The dermis was also affected with the induction of tenascin, a decrease in fibrillin and pro-collagen I, and a reduction of glycosaminoglycan deposition (
Table 2). Importantly, most of the skin changes evidenced following 9 repeated exposures occurred at the lowest dose of 9 × 0.25 MED that did not induce any erythema reaction [
36]. This 0.25 MED dose corresponds to 5% of the UV daylight dose received on a horizontal surface, during the day-time in mid-April (6:00 am–08:00 pm) in Paris, France (
Table 1). Exposure to 19 repeated doses of 0.5 MED DUVR led to most of the skin changes cited above (
Table 2) [
36].
Table 2.
Summary of alterations induced in human skin by repeated exposures to Daily UV radiation (DUVR) [
23,
36].
Table 2.
Summary of alterations induced in human skin by repeated exposures to Daily UV radiation (DUVR) [23,36].
Parameters | DUVR Spread over 2 Weeks | DUVR Spread over 4 Weeks |
---|
9 × 0.25 MED | 9 × 0.50 MED | 9 × 0.75 MED | 19 × 0.5 MED |
---|
Clinical Parameters |
Pigmentation |
Δa* | + | ++ | +++ | ++ |
Δb* | ns | + | ++ | + |
ΔL* | − | −−− | −−− | − |
Erythema | ns | + | ++ | + |
Hydration | − | − | − | ns |
Biomechanical properties |
Elasticity | ns | − | − | ns |
Residual deformation | ns | ns | ns | ND |
Microtopography |
Number of wrinkles | ns | ns | − | + |
Coefficient of developed profile | ns | ns | − | ns |
Loss of skin density (densiscore) § | ND | ND | + | ND |
Biological parameters |
Epidermis |
Histology |
Epidermal thickness | ns | ns | + | + |
Langerhans cells |
Number of Langerhans cells | − | −− | −−− | −− |
Size of Langerhans cells | + | ++ | +++ | ns |
Urocanic acid isomerization | + | ND | ND | ND |
Melanocytes |
Number of melanocytes | + | + | + | + |
Size of melanocytes | + | ++ | +++ | + |
Melanin deposition | + | ++ | +++ | + |
Proliferation |
Ki-67 + cells | + | ++ | +++ | ns |
Cellular damage |
sunburn cell formation | ns | + | + | + |
p53 accumulation | ns | ++ | +++ | + |
Dermis |
Tenascin | ns | ns | ++ | + |
Elastin | ns | ns | ns | ns |
Fibrillin | ns | − | − | ND |
Lyzozyme/elastin | ns | ns | ns | + |
Pro-collagen I | − | −− | −−− | ns |
Pro-collagen III/Pro-collagen I | ns | ns | + | ns |
Glycosaminoglycan deposition | − | − | − | −− |
These results indicate that under repeated exposures to a realistic DUVR dose that does not lead to any sunburn reaction, several significant clinical and biological skin alterations can be induced in both epidermal and dermal compartments. The study also evidenced that some biological endpoints were more sensitive to UV-SSR such as SBC formation, whereas activation of melanocytes was more sensitive to DUVR, indicating that UV spectrum is of high importance regarding the biological and clinical impacts of UV rays on skin, as shown in previous studies [
37,
38]
The impact of DUVR on skin pigmentation was further investigated regarding ethnic origin (
Figure 2). Ten Caucasian volunteers and 8 Asian volunteers with similar constitutive pigmentation were enrolled (mean individual typologic angle (ITA°) value of 34° and 35° for Caucasian and Asian volunteers respectively, [
39]). Volunteers were exposed four times to 0.75 MED DUVR daily, from day 0 to day 3. Skin color was assessed by colorimetric measurement using L*a*b* color system and by visual scoring, before each DUVR exposure and at different time points until day 32,
i.e., 29 days after the last DUVR exposure.
Figure 2.
Pigmentation induced in human skin exposed to DUVR. Variation of skin pigmentation (ΔE) and luminance (ΔL*) induced by four exposures of 0.75 MED DUVR (from day 0 to day 3) in Caucasian and Asian skin. The evolution of the color of the skin expresses itself through the combination of changes of the coordinates L * a * b * as follows: ΔE = [(ΔL*)2 + (Δa*)2 + (Δb*)2]1/2, where Δa*, Δb*, ΔL* are the differences between exposed and non-exposed sites of a*, b* and L* values, respectively.
Figure 2.
Pigmentation induced in human skin exposed to DUVR. Variation of skin pigmentation (ΔE) and luminance (ΔL*) induced by four exposures of 0.75 MED DUVR (from day 0 to day 3) in Caucasian and Asian skin. The evolution of the color of the skin expresses itself through the combination of changes of the coordinates L * a * b * as follows: ΔE = [(ΔL*)2 + (Δa*)2 + (Δb*)2]1/2, where Δa*, Δb*, ΔL* are the differences between exposed and non-exposed sites of a*, b* and L* values, respectively.
A significant increase in skin pigmentation was detected for both populations (decrease in luminance ΔL* and increase in ΔE) after DUVR exposure, from day 1 to day 32, compared to day 0 (Tukey test,
p < 0.001). Seventy-two hours after the last DUVR exposure (day 7), pigmentation was stable and persistent (
Figure 2). Results also clearly showed that pigmentation induced by DUVR was significantly higher in Asian skin compared to Caucasian skin (Tukey test,
p < 0.05) (
Figure 2). Colorimetric measurements were confirmed by visual assessment using a scale scoring from “absence of pigmentation” to “darker brown pigmentation”.
To summarize, in human skin in vivo, acute or repeated exposures to DUVR can modulate detectable short term clinical parameters such as pigmentation, hydration, and microtopography, and can induce biological and biophysical alterations in the dermis and the epidermis, that could be linked to long term adverse clinical effects such as photo-aging including pigmentary disorders and photo-cancers.
3.2. In Vitro Effects of DUVR in Reconstructed Human Skin Model
In order to better characterize the cellular and molecular impact and the early events induced by non-extreme exposure conditions,
in vitro studies were performed using a three dimensional (3D) reconstructed human skin model composed of a dermal equivalent including living adult fibroblasts covered by a fully differentiated epidermis. The 3D architecture of the model enables UV penetration properties, depending on wavelength, to be taken into account. The model has been shown to be a useful tool for studying the responses of fibroblasts and keratinocytes to solar UV exposure
in vitro and can reproduce sunburn related markers and dermal damage associated with the photo-aging process [
40,
41].
3.2.1. Biological Efficient Dose and Histologic Changes
Since the MED determination could not be achieved in such
in vitro experimental conditions, the biological efficient dose (BED) has been previously defined as the minimal dose able to induce morphological alterations after acute UV exposure [
42,
43]. Histological analysis of this reconstructed skin model exposed to increasing doses of DUVR established the DUVR BED at 13 J/cm
2. At this dose, observed alterations were mostly located in the dermal compartment and were characterized by the disappearance of fibroblasts. Such changes have also been observed following exposure to UVA alone [
42,
43] (
Figure 3). Some alterations were also detected in the epidermis. These included slight alterations in the granular layer resembling those observed after UVA exposure, as well as thinning of the epidermis and thickening of the cornified layer. Moreover, at this BED of DUVR, few sunburn cells and p53 positive keratinocytes could be detected. The histological damage induced by DUVR was correlated with the release of the well-known matrix metalloproteinase 1 (MMP-1), a photo-aging marker in the culture medium of reconstructed skin [
44,
45]. To summarize, the BED of 13 J/cm
2 DUVR induced histological alterations mostly in the dermis, as observed after UVA and some alterations in the epidermis that were similar to those induced by UV-SSR or UVB (
Figure 3) [
46]. The lower dose of 7 J/cm
2 DUVR was not sufficiently high to induce any of the cited histological damage. Repetitive exposures to DUVR for five consecutive days showed drastic alterations in the dermis and in the epidermis, even with the sub-BED dose of 7 J/cm
2, attesting that chronic exposure to low DUVR dose may account for long term harmful consequences [
45].
The determined BED of DUVR in a reconstructed skin model (13 J/cm
2) corresponded to a realistic dose since it represented 20% of the daily dose of UV received in Paris on mid-April (
Table 2) and was correlated with human
in vivo data that established an average MED of 12 ± 2.1 J/cm
2 DUVR for skin phototypes II and III [
25,
36].
Figure 3.
Morphological changes induced by the biologically efficient doses of DUVR (13 J/cm
2), of UVA (25 J/cm
2) or of UV-SSR (5.4 J/cm
2) in reconstructed human skin [
43,
44,
45,
46]. Black arrows indicate the zone where the incidence of fibroblasts has decreased. White arrows indicate sunburn cells.
Figure 3.
Morphological changes induced by the biologically efficient doses of DUVR (13 J/cm
2), of UVA (25 J/cm
2) or of UV-SSR (5.4 J/cm
2) in reconstructed human skin [
43,
44,
45,
46]. Black arrows indicate the zone where the incidence of fibroblasts has decreased. White arrows indicate sunburn cells.
3.2.2. Modulation of Gene Expression
To further characterize DUVR induced changes, gene expression was studied in reconstructed skin exposed to DUVR using cDNA arrays and quantitative PCR. The expression of more than 200 genes related to skin biology and stress response was studied in fibroblasts and keratinocytes separately. DUVR induced the modulation of expression of numerous genes in both cell types. In the cDNA arrays profiling, the biological efficient dose of 13 J/cm
2 DUVR induced the modulation of 27% and 31% of the genes analyzed in fibroblasts and keratinocytes respectively [
47]. In the study using QPCR arrays 16% and 27% of the genes analyzed were found modulated by 12 J/cm
2 DUVR, in fibroblasts and keratinocytes respectively [
48]. These results confirmed the impact of DUVR at the surface and in deeper layers of skin, as already described in
in vivo and
in vitro histological analyses.
DUVR modulated genes were related to several functional families. In the epidermis, DUVR affected the expression of keratinocytes markers involved in the differentiation/proliferation balance. Several members of the epidermal differentiation complex (filaggrin, loricrin, involucrin, CRCT1, SPPR1A, SPRR1B, SPRR2A, LCE2B, LCE2D) and other differentiation markers such as corneodesmosin, calmodulin-like 5, transgutaminase 1, stratifin, serpinB2 transcripts had their expression modulated by DUVR exposure. In addition, DUVR affected expression of markers related to epidermal proliferation and markers expressed in basal keratinocytes (keratin 5, keratin 6B, Ki67 and ornithine decarboxylase 1 ODC1) [
48]. These changes can be linked to
in vivo skin surface alterations following DUVR exposure such as perturbations in hydration, skin microtopography, epidermal proliferation and thickening (
Table 2, [
36]).
The expression of genes encoding extracellular matrix (ECM) and dermal-epidermal components as well as proteins of ECM maturation and remodeling was also affected following DUVR exposure. For instance, while the expression of collagens and fibronectin ECM components was down-regulated, the expression of remodeling genes MMP1, MMP3 and members of the plasminogen activator system serpin1, serpinB2 and plasminogen activator tissue PLAT, was up-regulated [
48]. Alterations of ECM components and homeostasis have been widely described after UV exposure [
49]. These changes, especially MMPs induction and collagen synthesis and repression, represent hallmarks of the photo-aging process and the formation of solar elastosis [
50,
51]. These data may therefore emphasize the role of such low DUVR doses in the development of photo-aging clinical signs
in vivo (
Table 2, [
36]).
Genes encoding growth factors, receptors and hormones also had their expression modulated by DUVR exposure in fibroblasts as well as in keratinocytes. In this family, the expression of Heparin-Binding EGF-like Growth Factor HBEGF, Growth Differentiation Factor 15 GDF15, Transforming Growth Factor α TGFA, granulocyte/macrophage colony-stimulating factor (GMCSF/CSF2) and Fibroblast Growth Factor 7 FGF7 (also known as Keratinocyte Growth Factor KGF) was strongly up-regulated [
48]. Interestingly, FGF7 and CSF2 proteins have been shown to be positive regulators of skin pigmentation. Chronic solar exposure has been linked to pigmentary disorders [
52]. The formation of actinic lentigines or “age-spots”, only found in sun-exposed anatomical sites, brings irrefutable proof of this link. Up-regulation of genes related to skin pigmentation by DUVR evidenced a contribution of such non-extreme exposures to these clinical signs [
36,
53,
54,
55].
DUVR exposure also has an impact in skin immunity related markers: it strongly increased the expression of genes encoding cytokines and inflammation markers such as interleukins (IL1B, IL6, IL8), chemokines (CCL2), ICAM1, CSF2, TNF and PTGS2 (also called COX2), confirming the immune-competence of keratinocytes and fibroblasts. In contrast, several members of the innate immunity gene family had their expression down-regulated by DUVR, such as TLR1, TLR3 or TNFSF10. Again, this data reinforced the fact that daily UV exposure may also be implicated in the UV-induced immunological response of skin [
56,
57].
Response to stress was particularly enriched after DUVR exposure, attesting that DUVR represents a stress for skin cells. DUVR induced expression of genes encoding heat shock proteins (HSP27, HSPA1A/HSP70, HSP90, DNAJB1/HSP40, HSPA2, HSPA5), and of genes involved in cellular response to oxidative stress (this functional family will be emphasized further in this review). UV induction of HSP has already been described and is considered to be part of a natural defense mechanism against UV exposure [
58,
59]. In such a context, HSP70 plays a particular role in photo-aging. HSP70 and members of the HSP70 family are induced by UVB, by UVA, and by UVA1 [
60,
61,
62]. It was recently shown that the over-expression of HSP70 in mice led to the suppression of UV-induced skin damage and resulting inflammatory responses as well as UV-induced wrinkle formation [
63,
64].
3.2.3. Contribution of UVA Wavelengths to DUVR Biological Effects
As UV daylight includes a high and constant proportion of UVA wavelengths, with a UVA/UVB ratio around 27, corresponding to 96.5% UVA and 3.5% UVB, the biological contribution of UVA wavelengths included in the DUVR spectrum was assessed. Accordingly, gene expression profiling using cDNA arrays was performed following exposure of in vitro reconstructed skins to DUVR and to UVA at their respective BED (13 J/cm2 DUVR and 25 J/cm2 UVA). In fibroblasts, the expression of 225 genes was studied. Sixty genes were modulated by UVA or DUVR. Out of them 55/60 (92%) were common to DUVR and to UVA. In keratinocytes, the expression of 241 genes was studied. The vast majority (59/74, 80%) of the modulated genes were identical in DUVR or UVA exposure conditions. These results showed that both types of exposures share biological targets therefore attesting to a strong contribution of UVA wavelengths to the DUVR biological response.
In keratinocytes, 20% of genes were specifically modulated by DUVR and not by UVA. They mostly included genes involved in the differentiation/proliferation balance, such as genes of the epidermal differentiation complex. In fibroblasts, only 3% of the analysed genes were specifically modulated by DUVR. The DUVR spectrum includes wavelength ranges from UVB and shortwave UVA (UVA2, 320–340 nm) to longwave UVA (UVA1, 340–400 nm), having different and increasing penetration properties. For this reason, keratinocytes, due to their surface location, receive photons of the whole DUVR spectrum, whereas fibroblasts, in deeper layers of the skin, are mostly exposed to UVA of the DUVR spectrum. Therefore, it may be hypothesized that the 20% of genes specifically modulated by DUVR may be attributed to UVB wavelengths included in the DUVR spectrum. In contrast, fibroblasts receive the same wavelengths from the DUVR spectrum as from the UVA spectrum resulting in the same changes in gene expression.
Altogether the results established that DUVR biological impact was mostly imputable to UVA wavelengths included in the DUVR spectrum, especially for dermal fibroblasts, located in skin depth. Photo-aging due to chronic exposure to UVA was particularly well illustrated by cases of unilateral dermatoheliosis occurring on the side of face that is chronically exposed to UVA through a glass window (e.g., truck or taxi drivers) showing skin thickening, roughness, wrinkling and laxity associated with an accumulation of elastotic material within dermis [
65].
3.2.4. Focus on Oxidative Stress Induced by DUVR and Characterization of the Fibroblast and Keratinocyte Response
Since (1) DUVR spectrum includes a high and constant proportion of UVA wavelengths, that are well-known stimulators of ROS production and (2) it was shown that UVA wavelengths particularly contributed to DUVR biological impact, oxidative stress induced by physiological doses of DUVR was carefully studied in reconstructed human skin model [
45]. DUVR induced the generation of ROS in both epidermis and dermis of reconstructed skin, with a significant dose effect (
Figure 4a). Cellular response to DUVR induced oxidative stress was analyzed by studying the expression of 24 genes encoding proteins involved in oxidative stress response in fibroblasts and keratinocytes of reconstructed skin, respectively. DUVR mostly altered the expression of four gene families: target genes of the cytoprotective to oxidative and electrophilic stress NF-E2-related factor 2 (Nrf2)-pathway, sestrins that participate in the regeneration of over-oxidized peroxiredoxins, metallothioneins that scavenge ROS and metal ions, and methionine sulfoxide reductase (MSRA), that is involved in the maintenance of protein structure and function. A differential response to oxidative stress between fibroblasts and keratinocytes was revealed, with regard to kinetics, direction or levels of modulation and nature of modulated genes. In dermal fibroblasts, oxidative stress response occurred as early as two hours post exposure, with a majority of the genes up-regulated; whereas in keratinocytes gene modulations were mostly detected six hours post DUVR exposure, with a higher proportion of down-regulations (
Figure 4b). Nrf2 target genes (
HO-1,
TXNR,
NQO1,
gammaGCS-L) were significantly up-regulated in dermal fibroblasts by DUVR, while in keratinocytes, only
NQO1 gene expression was significantly induced. Genes encoding metallothioneins were also differently modulated in fibroblasts and in keratinocytes, with a down-regulation by DUVR of MT1X, MT1E and MTE2A found only in keratinocytes. For the sestrin family and MSRA, the responses were quite similar between fibroblasts and keratinocytes. Most of the studied sestrins and MSRA, whose decline has been shown to be associated with aging and photo-aging, had their gene expression level decreased [
66,
67,
68] (
Figure 4b).
It was important to note that the low dose of 7 J/cm
2 DUVR, which did not lead to any detectable histologic changes, was sufficient to generate ROS, even in deeper layers of the dermis, and to modulate the expression of genes related to several functional families described above. This reveals the insidious impact of DUVR, even in the absence of any detectable tissue damage and shows that the dermal compartment is highly susceptible to DUVR [
45,
47].
Figure 4.
(a) DUVR induced ROS; (b) Cellular response to DUVR induced oxidative stress. Exposure to DUVR induced the modulation of the expression of genes involved in response to oxidative stress, in fibroblasts (F) and keratinocytes (K) of reconstructed human skin. White dotted line indicates dermal epidermal junction. White brackets indicate epidermal positive layer. White arrows indicate examples of positive dermal fibroblasts.
Figure 4.
(a) DUVR induced ROS; (b) Cellular response to DUVR induced oxidative stress. Exposure to DUVR induced the modulation of the expression of genes involved in response to oxidative stress, in fibroblasts (F) and keratinocytes (K) of reconstructed human skin. White dotted line indicates dermal epidermal junction. White brackets indicate epidermal positive layer. White arrows indicate examples of positive dermal fibroblasts.