dietary pro bio tics and carotenoids for uv damaged skin

8/2/2019 Dietary Pro Bio Tics and Carotenoids for UV Damaged Skin

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From The British Journal of Dermatology

Clinical Evidence of Benefits of a Dietary Supplement

Containing Probiotic and Carotenoids on Ultraviolet-induced

Skin DamageD. 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

BackgroundLactobacillus 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]
http://www.medscape.com/index/list_4739_0http://www.medscape.com/index/list_4739_0


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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/UVBirradiance 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 requestedto 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


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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 tochange 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.


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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 anUVA/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


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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 thesubject 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), theeffect 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 wasnoticed 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 After DS intake

Nonexposed skin(n = 16)

UV-DL-exposedskin (n = 16)

Nonexposed skin(n =16)

UV-DL-exposedskin (n = 16)

Langerhans cells(protein S100)

623 299 416 148a 613 270 545 215bc

Dermal dendrocytes(factor XIIIa+)

4245 2038 4915 2144 4215 1996 4118 1982c

Dermal inflammatorycells (CD45+)

44 26 84 40a 41 22 59 33ab


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Active melanocytes(tyrosinase)

296 115 331 83a 321 109 363 154a

Melanin content(FontanaMasson)

2809 1166 3801 1355a 2795 1133 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

dietary pro bio tics and carotenoids for uv damaged skin

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