From The British Journal of Dermatology

Clinical Evidence of Benefits of a Dietary Supplement Containing Probiotic and Carotenoids on Ultraviolet-induced Skin Damage
D. Bouilly-Gauthier; C. Jeannes; Y. Maubert; L. Duteil; C. Queille-Roussel; N. Piccardi; C. Montastier; P. Manissier; G. Pirard; J.-P. Ortonne Posted: 10/13/2010; The British Journal of Dermatology. 2010;163(3):536-543. 2010 Blackwell Publishing

Abstract and Introduction

Abstract

Background Lactobacillus johnsonii (La1) has been reported to protect skin immune system homeostasis following ultraviolet (UV) exposure. Objectives To assess the effects of a dietary supplement (DS) combining La1 and nutritional doses of carotenoids on early UV-induced skin damage. Methods Three clinical trials (CT1, CT2, CT3) were performed using different UV sources: nonextreme UV with a high UVA irradiance (UV-DL, CT1), extreme simulated solar radiation (UV-SSR, CT2) and natural sunlight (CT3). All three clinical trials were carried out in healthy women over 18 years of age with skin type IIIV. In CT1, early markers of UV-induced skin damage were assessed using histology and immunohistochemistry. In CT2, the minimal erythemal dose (MED) was determined by clinical evaluation and by chromametry. Chromametry was also used to evaluate skin colour. Dermatologists' and subjects' assessments were compiled in CT3. Results A 10-week DS intake prevented the UV-DL-induced decrease in Langerhans cell density and the increase in factor XIIIa+ type I dermal dendrocytes while it reduced dermal inflammatory cells. Clinical and instrumental MED rose by 20% and 19%, respectively, and skin colour was intensified, as shown by the increase in the E* parameter. The efficacy of DS was confirmed by dermatologists and subjects under real conditions of use. Conclusions Nutritional supplementation combining a specific probiotic (La1) and nutritional doses of carotenoids reduced early UV-induced skin damage caused by simulated or natural sun exposure in a large panel of subjects (n = 139). This latter result might suggest that DS intake could have a beneficial influence on the long-term effects of UV exposure and more specifically on skin photoageing.
Introduction

Sun exposure clearly damages skin. This is reflected by the condition of commonly exposed body areas (presence of deep wrinkling, loss of resilience, increased fragility, age spots etc.) compared with unexposed areas. Ultraviolet (UV) radiation (UVR) is responsible for both acute and long-term effects. [1] Acute effects are early events resulting from direct impact of UVR on biological chromophores such as DNA, leading to structural impairment [27] and release of inflammatory cytokines, enzymes and immunosuppressive factors.[36] Long-term effects result from cumulative damage and improper or incomplete cell repair possibly leading to skin photoageing and cancer. All these deleterious effects can be induced by both UVB and UVA exposure, by a direct impairment of cellular structures or by the generation of reactive oxygen species (ROS).[812] Approximately 50% of UVR-induced damage has been estimated to result from production of ROS. [13]

Most studies on UV-induced skin damage use UVR spectra with a high erythemogenic activity. Such 'extreme' standard spectra (UV solar-simulated radiation, UV-SSR) reproduce summer, quasizenithal sunlight with a clear sky, near solar noon and at specific latitude ranging from 335N to 335S. In contrast, Christiaens et al. [14] have modelled a mean spectral irradiance representing 'nonextreme' exposure conditions referred to as UV daylight (UV-DL). UV-DL involves a high level of UVA, prevalent most of the time outside tropical areas, and is considered to be adequately simulated with a UVA/UVB irradiance ratio of 24 instead of close to 10 for zenithal sun.[14] Recent studies showed that suberythemal doses of UV-DL were able to generate biological damage causing dysfunction and irreversible deterioration of dermal tissue that can be prevented by an adapted photoprotection. [1517] Epidemiological studies in the U.S.A. have revealed that the predominant UVR exposure occurs under these conditions.[18,19] Adequate photoprotection, including sun avoidance, clothing and sunscreen, is essential to prevent UV-induced skin damage. Recently, dietary photoprotection with specific nutrients proved to be successful in preventing some UVR deleterious effects. Among these nutrients, carotenoids (i.e. carotene, lycopene) have been shown to be efficient in preventing photo-oxidative damage through scavenging ROS.[20,21] In addition to reducing erythema,[22,23] -carotene supplementation interferes with UVA-induced gene expression by multiple pathways,[24] thus inhibiting expression of matrix metalloproteinases [25] and protecting mitochondrial DNA.[26] Lycopene is efficient in quenching detrimental singlet oxygen[27] and contributes to skin protection against UV-induced erythema in humans.[28] In the last few years, probiotics have emerged as a new strategy in systemic photoprotection. Probiotics are known to modulate the immune system of the gut and to protect against infectious and inflammatory diseases of the gastrointestinal tract.[29,30] In 2006, oral supplementation for 10 days with a specific probiotic Lactobacillus johnsonii (La1) was reported to protect against UV-induced suppression of contact hypersensitivity, decrease of Langerhans cell (LC) density and increase of interleukin-10 serum levels.[31] In addition, La1 has been shown to accelerate the recovery of LC functionality after UVR exposure in humans.[32] The purpose of the present paper is to summarize the effects of a dietary supplement (DS) combining a specific probiotic (La1) with nutritional doses of carotenoids, i.e. -carotene and lycopene, on early damage induced by UVR exposure in humans. The three-step evaluation addressed the effects of DS on skin condition following exposure to UV-DL with a high UVA rate, UV-SSR close to summer zenithal sun and natural sunlight during summer holidays.

Materials and Methods

The DS provided a daily dose of 5 108 colony-forming units of La1 (Skin-Probiotic) and 72 mg carotenoids for 6, 6 and 34 weeks before UVR exposure in clinical trials 1, 2 and 3, respectively (CT1, CT2 and CT3). Placebo in CT2 was maltodextrin. In CT2 and CT3, the subjects were requested to return unused products and empty packs to the investigator in order to check for compliance.
Details of the Clinical Trials and Study Populations

CT1, CT2 and CT3 were performed using three different types of UVR exposure: nonextreme with high UVA level (CT1), extreme close to summer zenithal sun (CT2) and natural summer sunlight (CT3). In CT1, early markers of UV-induced skin damage were assessed using histology and immunohistochemistry. In CT2, the minimal erythemal dose (MED) was determined by clinical

evaluation and by chromametry. Chromametry was also used to evaluate skin colour. Dermatologists' and subjects' assessments were compiled in CT3. All three clinical trials were carried out on healthy women aged over 18 years. Sixteen subjects (mean SD age 31 3 years) with skin type II[33] were enrolled in CT1 (Fig. 1). CT2 was a randomized, double-blind vs. placebo study involving 43 subjects with skin type III or IV. Subjects were distributed into two groups (mean SD age 34 7 and 35 7 years, respectively, in DS and placebo groups) matched for age, skin type and colour (Fig. 2). CT1 and CT2 took place during the periods November March and MayJuly, respectively. In CT1 and CT2, subjects were required not to expose themselves to natural or artificial sunlight during the whole duration of the studies. Absence of tanning was verified at each visit.

Figure 1. Design of clinical trial 1. Volunteers were exposed for 18 days to 075 minimal erythema dose (MED) of ultraviolet daylight (UV-DL; UVA/UVB ratio = 24) before and after 6 weeks of receiving the dietary supplement (DS). Skin biopsy samples were collected before and after supplementation on both nonexposed and exposed areas (24 h after last exposure). Nonexposed and exposed areas were located close to each other on the same side of the body. Skin biopsy sites and UV exposure locations before supplementation (right or left buttock) were randomized at the beginning of the study. Skin biopsy and UV exposure after supplementation were performed on the other buttock.

Figure 2. Design of clinical trial 2. Clinical and colorimetric minimal erythema dose (MED) UV-SSR; (UVA/UVB ratio = 10) were determined on each volunteer's back before and after intake of dietary supplement (DS). Location of MED determination was randomly assigned on one side of the back before DS intake. After DS intake, MED was measured on the other side. Volunteers were exposed for four consecutive days to 09 MED before and after DS intake. Chronic exposures were located close to the site of MED determination. Skin colour change was followed by chromametry for 10 days after each series of UV exposures. CT3 was an open study performed on 80 subjects (mean age SD 42 12 years) for the most part with skin type III. The study took place between July and October and subjects were asked not to change their habits in terms of sun bathing and sunscreen use. The main inclusion criterion for CT1 and CT2 was women accepting not to eat dairy products containing bacteria or other living organisms during the study period. Main exclusion criteria were pregnancy or breastfeeding, intake of other nutritional supplements or vitamins and particular diet (e.g. vegetarian). For CT3 main exclusion criteria were pregnancy or breastfeeding, a history of skin cancer or intake of any other nutritional supplements.

All the subjects participating in these three trials gave written informed consent before starting the study. CT1 and CT2 protocols were approved by the Ethics Committee of Nice, France.
Ultraviolet Radiation Sources and Exposure

A sun simulator with a 1000 W xenon lamp (Oriel, Stratford, CT, U.S.A.) equipped with a dichroic mirror and Schott WG 320/15 mm (CT2) or 2 mm (CT1) filter was used, providing after adjustment an UVA/UVB irradiance ratio of 24 (CT1) and 10 (CT2).[14] The spectral power distribution at the skin level was measured with a calibrated spectroradiometer (Macam 9910; Macam, Livingston, U.K.). The daily output was monitored with the Solar Light PMA 2100 radiometer (Solar Light, Philadelphia, PA, U.S.A.) equipped with UVA and erythemal UV sensors. CT1: individual MED was first determined on the buttock of each subject as previously described.[16,34] Two 5 5 cm areas were selected on each buttock, one used as unexposed control while the other one was exposed to 075 MED per day for 18 days. After 6 weeks of DS intake, the other buttock was similarly exposed while continuing daily DS intake. Each subject was, therefore, her own control (Fig. 1). CT2: individual MED was determined at day 1 on the upper back of each subject. A 5 5 cm area on one side of the back of each subject was then exposed daily to 09 MED for four consecutive days before DS intake and a symmetrical area of the back was similarly exposed after 6 weeks of DS intake. Beside each exposed area, an unexposed area served as a control (Fig. 2). CT3: natural sunlight during summer holidays.
Histology and Immunohistochemistry (Clinical Trial 1)

Biopsy samples (4 mm) were collected from unexposed and exposed buttock areas before and after supplementation (Fig. 1). They were fixed with 10% buffered formaldehyde, dehydrated, embedded in paraffin and cut in 7-m thick sections. A FontanaMasson argentaffin stain was used to reveal intraepidermal melanin. The density of silver granules was evaluated by digital image analysis of the vertical cut surface of the Malpighian layer. For immunohistochemistry, paraffin sections were immunolabelled with antisera diluted in phosphate-buffered saline as follows: 1 : 50 for factor XIIIa (Biogenex, San Ramon, CA, U.S.A.), 1 : 50 for tyrosinase (Novocastra, Newcastle upon Tyne, U.K.), 1 : 200 for leucocyte common antigen or CD45 (Dako, Glostrup, Denmark) and 3 drops mL1 for protein S100 (Dako). Antiprotein S100 antibody was used to count the LC number. Positive cells were counted, excluding those in the basal layer of epidermis (melanocytes). Antityrosinase antibody was used to evaluate the number of active melanocytes. Antifactor XIIIa antibody was used to detect type I dermal dendrocytes. An anti-CD45 antibody was used to count inflammatory cells carrying the leucocyte common antigen.
Assessment of Skin Sensitivity and Colour (Clinical Trial 2)

Skin colour was monitored using a Minolta CR 300 Chromameter (Konica Minolta, Osaka, Japan), before (from day 2 to day 12) and after supplementation (from day 58 to day 68) (Fig. 2). Instrumental measurements included the three coordinates of the CIE L*a*b* system[35] which defines colour from L* (lightness), a* (the chromatic red-green component) and b* (the chromatic yellow-blue component). The colour difference, termed E*, can be calculated from the following equation: E* = [(L*) 2 + (a*) 2 + (b*) 2]/2. L*, a* and b* represent the difference between UV-exposed and unexposed areas, respectively, for parameters L*, a* and b*. The greater the E* value the more

intense the skin colour. The MED was determined on the upper back, both by a clinician and with a Chromameter (Fig. 2).
Dermatologists' and Subjects' Assessments (Clinical Trial 3)

Subjects went to the dermatologists' office 34 weeks before their summer holidays and again 68 weeks later, after their holidays. Two different questionnaires were filled in at each visit, one by the subject and one by the dermatologist, mainly to assess skin resistance to sun exposure.
Statistical Analysis

CT1: the ShapiroWilk test was used to check for normal distribution. For the efficacy analysis, a Student's paired t-test was used for variables with normal distribution and a Wilcoxon test for variables with asymmetrical distribution. Analysis of variance was used to study factor interactions (supplementation, exposure). The level of significance was set at 5% using a bilateral approach. CT2: repeated-measures analysis of variance was used for quantitative variables and weighted least square approach or generalized estimating equations for qualitative variables. The level of significance was set at 5% using a bilateral approach. Concerning the change of skin colour (E* parameter), the effect of the product was compared using a mixed model for repeated measurements [fixed effects: treatment, period (before and after intake), day within the period and all interactions; random effect: subject] and followed by a Tukey test comparing the periods for each supplementation group and each day.

Results
Clinical Trial 1

As shown in Table 1, before DS intake UV-DL induced a statistically significant decrease in LC density. After 10 weeks of supplementation, this decrease was statistically less significant. Before DS intake, UV-DL tended to increase the number of type I dermal dendrocytes while no significant UV effect was noticed after DS intake. On exposed areas, the density of CD45+ dermal inflammatory cells was statistically less significant after DS intake. A moderate but statistically significant increase in active melanocytes was found after UV-DL exposure both before and after DS intake, with no difference as a result of supplementation. Also, chronic suberythemal UV-DL exposure augmented melanin content. After DS intake, the increase in melanin density was significantly lower than before.
Table 1. Effect of suberythemal doses of ultraviolet daylight (UV-DL) on skin immune cells, inflammatory infiltrate and pigmentation (clinical trial 1) Before DS intake Nonexposed skin (n = 16) UV-DL-exposed skin (n = 16) After DS intake Nonexposed skin (n =16) UV-DL-exposed skin (n = 16)

Langerhans cells (protein S100) Dermal dendrocytes (factor XIIIa+) Dermal inflammatory cells (CD45+)

623 299 4245 2038 44 26

416 148a 4915 2144 84 40a

613 270 4215 1996 41 22

545 215bc 4118 1982c 59 33ab

Active melanocytes (tyrosinase) Melanin content (FontanaMasson)

296 115 2809 1166

331 83a 3801 1355a

321 109 2795 1133

363 154a 3296 1197abc

DS, dietary supplement. Results are shown as mean SD. The square of the cell count mm1 length of stratum corneum represents the density of cells mm2 of skin surface. Dermal dendrocyte count was expressed as the number mm2 of factor XIIIa+ cells in the perivascular compartment of the upper dermis. CD45+ cell count was expressed mm2 of skin surface area. Antityrosinase cells were counted in the basal layer of the epidermis. Melanin content was evaluated using digital image analysis after FontanaMasson staining. aExposed vs. nonexposed area, P < 005 (Student's t-test), bexposed areas before vs. after supplementation, P < 005 (Student's t-test), ccomparison of exposed vs. unexposed areas before vs. after supplementation, P < 005 (Wilcoxon test).
Clinical Trial 2

Instrumental measurements showed a significant increase in MED (+19%, P < 005) after DS intake, while no change was evidenced after placebo intake (Fig. 3). This result was confirmed by clinical determination (+20%, P < 005, data not shown). A follow-up of E*, for 10 days, evidenced a statistically significant increase of this parameter after DS intake while no significant change was noticed in the placebo group (Fig. 4).

Figure 3. Dietary supplement (DS) intake for 6 weeks induced a statistically significant increase in minimal erythema dose (MED), while placebo had no effect on this parameter (clinical trial 2). *P < 005 after vs. before DS intake (contrast resulting from a mixed model with repeated measurements). Results are expressed as mean SEM in arbitrary units (AU). One minimal erythema dose expressed in AU corresponds to 210 J m2.

Figure 4. Change in skin colour following ultraviolet exposure over 10 days (from day 3 to day 12), before and after intake of (a) dietary supplement (DS) or (b) placebo (clinical trial 2). Results are expressed as mean SEM E* for all subjects. *P < 005 after vs. before DS intake (Tukey test).
Clinical Trial 3

According to dermatologists, DS intake was able to prevent sunburn, sun intolerances (benign summer light eruption, labial herpes) as well as the appearance of sunspots in most of the subjects usually experiencing these phenomena. Dermatologists also reported a homogeneous UV-induced pigmentation and no or mild UV-induced skin dryness after DS supplementation. In accordance with the dermatologists' opinion, subjects reported an improvement of skin resistance to sun exposure. Furthermore, they noticed an improved skin colour (intensity, evenness) and a better skin condition.

Discussion
Deterioration of fibrillar structures of dermal tissue and clinical symptoms related to photoageing such as wrinkling, folding, shrivelling, patchy/mottled pigmentation, telangiectasia and sagging are the outcome of a long process and a progressive decline in skin function associated with cumulative UVinduced injury.[6] As the alterations of dermal components and clinical signs require prolonged exposure over years to become apparent, it seemed more appropriate to focus our studies on early UV-induced alterations such as immune cell damage[36] and inflammatory responses as well as the development of pigmentation, the control of which is essential to prevent the appearance of sunspots and dyschromia. It also appeared interesting to study three types of exposure, i.e. 'extreme' (UV-SSR), 'moderate' (UVDL) involving a far higher UVA content, and natural summer sunlight which corresponds to real conditions of sun exposure. Sunburn related to UVR exposure can easily be quantified by noninvasive methods and represents one of the earliest effects of sun damage. The MED is defined by the COLIPA recommendations as the first perceptible unambiguous redness with clearly defined border,[37,38] and sun protection factor measurement is based on the determination of the MED. In the present study, DS intake resulted in a 19% increase in the UV dose required to produce erythema. This increase could be perceived as small regarding the sun protection factor provided by sunscreens, but the tested nutritional supplement exhibited this increase of MED without the absorbing or reflecting properties of sunscreens. Moreover, this result complies with previous papers reporting the effect of nutritional supplementation on skin erythema. [39,40] Lower skin erythema could result in less infiltration by neutrophils which release proteolytic enzymes such as elastase and metalloproteases that could play a role in skin photoageing.
[41]

Investigating the effects of DS on the biological consequences induced by standard and cumulative exposure to UV-DL (i.e. nonextreme exposure conditions) reflects a more realistic situation for most human populations[14] compared with extreme UV-SSR. Moreover, it is impossible to study the effects of systemic photoprotection on the long-term effects of UVR exposure in humans. In this paper, we have addressed this question by studying early skin biomarkers biologically relevant for long-term damaging effects of UVR.

Among these damaging effects, sun exposure induces local and systemic immune suppression that might play a role in skin cancer in the long term.[42,43] This process is partly related to direct LC damage. [44,45] In a previous study, La1 intake was shown to accelerate the recovery of LC functionality after extreme UVR exposure.[32] Confirming previous results,[15] we observed that cumulative exposures to suberythemal doses of UV-DL resulted in a significant decrease in LC density. DS intake significantly prevented this decrease, suggesting a positive effect of DS in sustaining the cutaneous immune system following UVR exposure. Photoageing refers to the effects of long-term UVR exposure and sun damage superimposed on intrinsically aged skin. Inflammation plays an important role in photoageing of human skin in vivo.[11] In CT1, we have shown that repeated exposure to suberythemal doses of UV-DL induces a significant increase in CD45+ cells. After supplementation, this increase was statistically lower, suggesting a beneficial effect of DS intake on UV-induced inflammation and probably on photoageing. These data are of importance with regard to a previous report [16] emphasizing a marked increase in inflammatory cells as a prominent dermal change induced by repeated suberythemal exposure to UVR. Photoaged skin is characterized by pigmentary alterations such as lentigines and hyperpigmentation. Dermal dendrocytes share common features with macrophages, [46] and their density is increased in inflammatory dermatoses as well as in photoexposed areas.[47] They may be involved in photoageing[48] and hyperpigmented spots.[49] Controlling this cell population after UVR exposure might help to limit UV-induced hyperpigmentation. Repeated exposure to UV-DL had a tendency to increase numbers of type I dermal dendrocytes, and DS intake prevented this phenomenon. Taken together, the effects of DS on early biomarkers of the UV signature suggest that nutritional supplementation with La1 associated with low doses of carotenoids may have beneficial effects on long-term UV-induced clinical features. Another interesting point emerging from CT2 refers to the effect of DS intake on skin colour which is clearly more intense and more even in the DS group. Six weeks of DS intake induced a change in the colour of unexposed skin which is further mirrored in the colour change of tan developing after UVSSR exposure. This result is probably not related to an effect on melanin synthesis but rather to a direct action of DS on skin colour, as we have shown a reduction of melanin content after DS intake, whereas the level of UV-DL-stimulated melanocytes in the exposed zone was similar before and after supplementation. To our knowledge, there is no published study describing the effects of a nutritional supplement in real conditions of use (i.e. natural sunbathing during summer holidays). CT3 is thus representative of the most frequently encountered use of oral photoprotectants. In this study, both dermatologists and subjects reported the beneficial effects of DS intake on skin condition, skin colour, various types of sun intolerance and skin resistance to sun exposure. Bioavailability of the nutritional supplement was not considered in this three-step study because serum and skin bioavailability of carotenoids is well documented [50,51] and La1 is speculated to be somehow able to modulate local or systemic cytokine levels that favour skin immune system homeostasis through interaction with the gut. These three clinical trials demonstrate for the first time the effects of a DS combining probiotic La1 and nutritional doses of carotenoids in reducing early UV-induced skin damage as well as in modulating early skin biomarkers of UV effects. This latter result seems to suggest that DS intake may have a

beneficial influence on the long-term effects of UVR exposure and more specifically on skin photoageing. This nutritional supplement thus represents a complementary strategy to sun avoidance and sunscreen use for a global approach to photoprotection.

Sidebar
What's Already Known About This Topic?

Supplementation with high doses of carotenoids is effective in providing protection against sunburn reaction (among other effects). Nutritional supplementation with Lactobacillus johnsonii (La1) is able to protect against ultraviolet radiation (UVR)-induced decrease of epidermal Langerhans cell density and to accelerate the recovery of skin immune homeostasis after UVR-induced immunosuppression.

What Does This Study Add?

Combination of nutritional doses of carotenoids and La1:

increases minimal erythemal dose and 'modulates' early skin biomarkers relevant for long-term UVR-damaging effects.

Efficacy perceived by dermatologists and consumers under real conditions of use.

References

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

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Conflicts of interest D.B.-G., C.J., N.P. and P.M. are employees of Laboratoires innov. C.M. was an employee of L'Oral at the time of the studies. G.P. and J.-P.O. received consultancy fees from Laboratoires innov for their contribution to clinical trial 1 and/or clinical trial 2. The three studies were funded by Laboratoires innov. The other authors do not have any conflict of interest to declare. Acknowledgments We gratefully thank M. Claude Bouillon for his active contribution in the writing of this paper, Mrs Anny Fourtanier, Mrs Dominique Moyal and Mrs Sophie Sit for their help in the interpretation of the results, all the C.P.C.A.D. team for evaluations, measurements and analyses of clinical trials 1 and 2 and the Effistat team for statistical analysis of clinical trial 2. The three studies were funded by Laboratoires The British Journal of Dermatology. 2010;163(3):536-543. 2010 Blackwell Publishing