BJD

British Journal of Dermatology

PHOTOBIOLOGY

Phototoxicity of narrowband ultraviolet (UV) B (311 nm) compared with broadband UVB in the photo hen’s egg test P.R. Bafteh, M. Siegesmund, V. Kuerten and N.J. Neumann Department of Dermatology, Porphyria Specialist Center, Heinrich Heine University Duesseldorf, Duesseldorf, Germany

Summary Correspondence Pascal Robert Bafteh. E-mails: [email protected], pascal.bafteh@ uni-duesseldorf.de

Accepted for publication 19 June 2014

Funding sources No external funding.

Conflicts of interest None declared. DOI 10.1111/bjd.13221

Background Broadband ultraviolet B (BB-UVB) is a well-established treatment option in dermatology. However, during the last decade BB-UVB has increasingly been replaced by narrowband UVB 311 nm (NB-UVB), especially in the therapy of psoriasis, atopic eczema and vitiligo. Several studies have indicated a better therapeutic response for almost all indications compared with BB-UVB. Objectives The aim of our study was to investigate the phototoxic effects of NBUVB in comparison with BB-UVB in vivo. Methods Therefore, we employed the photo hen’s egg test (PHET), an established phototoxic model, based on the yolk sac blood vessel system of incubated hen’s eggs. NB-UVB and BB-UVB dosages increasing from 30 up to 1200 mJ cm 2 were applied on 17 test groups (each n = 12 eggs) and two unirradiated test groups served as controls. Twenty-four hours after irradiation we observed the following test parameters: lethality, membrane discoloration and haemorrhages. Results Following our results, the lethal half dose (LD50) was 60 and 720 mJ cm 2 for BB-UVB and NB-UVB, respectively. These LD50 dosages provoked severe membrane discoloration and haemorrhaging. Summarizing our results, the LD50 of NB-UVB was 12-fold higher than BB-UVB. Conclusions Interestingly, these findings are in good accordance with the literature, where the minimal erythema dose (MED) of NB-UVB in human skin is up to 14 times higher than the MED of BB-UVB. These results show that the PHET is a valid test model to evaluate the phototoxic effects of various UVB wavelengths. Moreover, our results indicate that regarding the investigation of phototoxic effects the PHET might serve as a model representative for human skin, which might reduce the extent of photoprovocation in humans in the future.

What’s already known about this topic?

• •

Broadband ultraviolet B (BB-UVB) has been further replaced by narrowband UVB 311 nm (NB-UVB), especially in the therapy of psoriasis, atopic eczema and vitiligo. Several studies have indicated a better therapeutic response for almost all indications compared with BB-UVB.

What does this study add?

• •

462

This study shows the phototoxic effects of NB-UVB in comparison with BB-UVB in an in vivo setting. Furthermore, it shows that the photo hen’s egg test is a valid model for examining phototoxicity.

British Journal of Dermatology (2015) 172, pp462–466

© 2014 British Association of Dermatologists

Phototoxicity of NB-UVB compared with BB-UVB in the PHET, P.R. Bafteh et al. 463 Table 1 Test groups Test group

Irradiation (mJ cm 2)

BB-UVB NB-UVB

30, 60, 90, 120, 180, 240 30, 60, 90, 120, 180, 240, 480, 720, 960, 1080, 1200

UV, ultraviolet; BB-UVB, broadband UVB; NB-UVB, narrowband UVB.

Table 2 Photo hen’s egg test classification of MD and HR Level

Description

0 1 2 3

No visible MD or HR Just visible MD or HR Visible MD or HR; structures covered partially Visible MD or HR; structures covered entirely

MD, membrane discoloration; HR, haemorrhage.

As an alternative to the rabbit’s eye test (Draize test), the hen’s egg test (HET) was originally introduced by toxicologists as a screening model for mucocutaneous toxicity.1 As a modification of the HET, the photo hen’s egg test (PHET) was established as an alternative model to phototoxicity in vitro models, mostly based on cell cultures.2 Meanwhile, the PHET is proven to be a useful, inexpensive and valid method to screen the phototoxic potentials of drugs or photoirritants and the capacity of photoprotective agents.2–6 Furthermore, the PHET shows good accuracy in the discrimination of strong, moderate, weak or nonphototoxic chemicals.2,3 While phototherapy has been used since more than a century ago by Niels Finsen to treat lupus vulgaris (1903), just about 30 years ago, Parrish and Jaenicke pointed out that wavelengths around 311 nm provoke the least erythema while being the most effective for clearing psoriasis.7,8 Subsequently, a fluorescent bulb was developed (TL-01), emitting a major peak at 311 nm and a minor peak at 305 nm. The treatment based on this device was then called narrowband ultraviolet B (NB-UVB). Later on, numerous studies showed its superior efficacy in the phototherapy of psoriasis, eczematous conditions, pruritus, cutaneous T-cell lymphoma and vitiligo.9–16 This treatment option replaced the use of broadband (BB) UVB (290–320 nm) as a first-line therapy for psoriasis as there were shown to be fewer side-effects and a lower carcinogenic potential.17,18 UVB induces several biological effects such as erythema, pigmentation or hyperplasia.19 UVB also induces inflammation and DNA damage.20,21 In fact, the treatment of UVB light is mainly thought to be based on its potential to induce apoptosis.22 Prolonged UVB exposure results in irreparable DNA damage, which leads to the decision to undergo programmed cell death.23 By activating caspase enzymes, UVB induces apoptosis in both keratinocytes and lymphocytes.24,25 While cutaneous carcinogenesis could not be linked to NB-UVB or BB-UVB therapy and these findings should be followed up in © 2014 British Association of Dermatologists

larger studies,26 we know that long-term UVB irradiation (especially wavelengths ranging from 310 to 320 nm) induces mutation formation.27 The aim of the present study was to investigate the phototoxic effects of NB-UVB in comparison with BB-UVB in vivo.

Materials and methods Fertile ‘White Leghorn’ eggs (333 Shaver Starcross 288A; Lohmann, Cuxhaven, Germany) were incubated in a horizontal position using a commercial incubator at 37
8 °C and 65% relative humidity. At day 4 of incubation, 5 mL of egg white was sucked out in order to lower the embryo level to shape a 1
5 9 2
5 cm window out of the eggshell. Only eggs with normally developed embryos and blood vessel systems were used for provocation. Nineteen test groups were employed. To investigate the photoirritant range of UVB, six groups were exposed to increasing BB-UVB dosages from 30 to 240 mJ cm 2 and 11 test groups were exposed to NB-UVB with dosages from 30 to 1200 mJ cm 2. Two additional unirradiated groups served as controls. Each group consisted of 12 eggs. To evaluate more subtle changes to the yolk sac blood vessel system, we determined smaller ranges between the irradiation intensities of the NB-UVB test groups (see Table 1). In a double-blind investigation, embryo lethality was recorded and the morphological parameters membrane discoloration (MD) and haemorrhage (HR) were monitored and graded following a 4-point-scale (Table 2) using a microscope (M 420; Leitz, Wetzlar, Germany) during an observation period of 24 h. The contingency table test for ordered categories was employed for the parameters MD and HR and Fisher’s contingency table test for the parameter lethality.

Results Broadband ultraviolet B Dependent on the irradiation intensities, the morphological parameters MD and HR were observed in increasing form. Groups exposed to 30 or 60 mJ cm 2 showed distinctly lesser pronounced MD and HR than groups exposed to 90 mJ cm 2 or more (see Table 3). The lethality scale showed an exponential increase starting with 16
7% fatalities at an irradiation intensity of 30 mJ cm 2. Groups exposed to 120 mJ cm 2 or higher showed a lethal rate of 100% (see Figs 1 and 3c). Narrowband ultraviolet B With higher irradiation levels, the morphological parameters MD and HR were observed in an increasing form. Groups exposed to 30–240 mJ cm 2 showed no severe MD and HR. Groups exposed to 480 mJ cm 2 or more showed more intense photodamage, including severe MD and HR (see Table 3). Fatalities were observed starting with a rate below British Journal of Dermatology (2015) 172, pp462–466

464 Phototoxicity of NB-UVB compared with BB-UVB in the PHET, P.R. Bafteh et al. Table 3 Effects of BB-UVB and NB-UVB on the yolk sac blood vessel system Membrane discoloration (%)

Haemorrhage (%)

Substance and irradiation

Non

Slight

Moderate

Severe

Non

Slight

Moderate

Severe

Lethality (%)

BB-UVB (mJ cm 2) 30 60 90 120 180 240 Control

16
7 8
3 25
0 0 0 0 83
3

25
0 33
3 50
0 8
3 0 8
3 8
3

41
6 33
3 16
7 58
3 58
3 25
0 8
3

16
7 25
0 8
3 33
0 41
6 66
7 0

16
3 0 0 0 0 0 75
0

50
0 75
0 25
0 8
3 0 0 16
3

25
0 16
7 33
3 50
0 25
0 16
3 8
3

8
3 8
3 41
6 41
6 75
0 83
7 0

16
7 50
0 83
7 100 100 100 0

NB-UVB (mJ cm 2) 30 60 90 120 180 240 480 720 960 1080 1200 Control

91
7 83
7 75
0 50
0 33
3 33
3 16
7 8
3 0 0 0 91
7

0 8
3 16
7 41
6 50
0 33
3 41
6 50
0 25
0 16
7 16
7 8
3

8
3 8
3 16
7 8
3 16
7 33
3 25
0 33
3 41
6 50
0 33
3 0

0 0 0 0 0 0 16
7 8
3 33
3 33
3 50
0 0

83
7 83
7 66
7 50
0 33
3 41
6 16
7 8
3 8
3 0 0 83
3

8
3 8
3 33
3 33
3 41
6 41
6 41
6 41
6 50
0 33
3 25
0 16
7

8
3 8
3 0 16
7 25
0 16
7 25
0 41
6 25
0 33
3 41
6 0

0 0 0 0 0 0 16
7 8
3 16
7 33
3 33
3 0

0 0 0 0 0 8
3 33
3 50
0 58
3 83
7 100 0

UV, ultraviolet; BB-UVB, broadband UVB; NB-UVB, narrowband UVB.

10% (240 mJ cm 2) rising up to 100% (1200 mJ cm 2) (see Figs 2 and 3b). Controls The unirradiated control groups showed low-grade morphological changes but no fatalities (see Table 3, Fig. 3a). Statistical analysis The morphological parameters of all UV-exposed groups were statistically significant in comparison with the control group (P < 0
05). Lethality rates showed no statistical significance.

Discussion UV radiation shows several biological effects on human skin, of which erythema and hyperpigmentation are the most commonly known ones. The intensity of these effects mainly depends on the wavelength ranges of the UV light, the dose of irradiation and the Fitzpatrick skin type in relation to the melanin content of the skin.28–32 UVB, particularly at 307 nm, is the most effective wavelength for eliciting erythema in the human skin. UVB-induced erythema is a delayed response. It reaches a peak at 6–24 h depending on the dose, with erythema, pruritus and pain in sun-exposed areas.33,34

100% 100% 12

100%

12

100%

No. of eggs

No. of eggs

10 8

50%

6 4

83·3%

10

83·3%

16·7%

8 6

0%

33·3% 8·3%

0%

l ro nt Co

m Jc m–

2

2

24 0

2

m Jc m–

m Jc m– 18 0

2

12 0

2

m Jc m– 90

m Jc m– 60

30

m Jc m–

2

–2

British Journal of Dermatology (2015) 172, pp462–466

0%

0

0

Fig 1. Lethality rates broadband ultraviolet B.

58·3%

4 2

2

50%

80 –1 30

m Jc m

–2

0 24

m Jc m

–2

0 48

m Jc m

0 72

–2

–2

–2

m Jc m

0 96

m Jc m

80 10

m Jc m

–2

00 12

m Jc m

Co

ro nt

l

Fig 2. Lethality rates narrowband ultraviolet B. © 2014 British Association of Dermatologists

Phototoxicity of NB-UVB compared with BB-UVB in the PHET, P.R. Bafteh et al. 465

(a)

(b)

(c)

Fig 3. (a–c) Unirradiated healthy yolk sac blood vessel systems (a), embryos exposed to narrowband ultraviolet B (b) and broadband ultraviolet B (c), showing strong and lesser pronounced morphological changes. Table 4 Lethal half dose/lethal dose Test group

LD50

LD100 (mJ cm ²)

BB-UVB NB-UVB

60 720

120 1200

LD50, lethal half dose; LD100, lethal dose; UV, ultraviolet; BBUVB, broadband UVB; NB-UVB, narrowband UVB.

In photodermatology, the patient’s minimal erythema dose (MED), defined as the minimal dose in producing just-perceptible erythema determined 24 h after irradiation is a wellestablished measurement method.33 This erythema, which is considered as sunburn,35 is induced due to vasodilation. Additionally, several other events occur in the skin as a result of UV exposure, e.g. inflammatory reactions, increased vascular permeability, blood flow changes and damage to epidermal cells.36 © 2014 British Association of Dermatologists

According to the literature, mean MED doses are significantly higher for NB-UVB compared with BB-UVB, 10
8 times higher in hairless mice and ranging from 2
8 up to 10 times higher in healthy volunteers.9,37–40 Walters et al.41 investigated the mean MED for BB-UVB 45 mJ cm 2 (range 15– 75 mJ cm 2) and NB-UVB 293 mJ cm 2 (range 135– 540 mJ cm 2) with NB-UVB. Interestingly, both values, the recorded lethal half doses in our study and the mean MED seen in healthy volunteers, are comparable. Moreover, the MED/LD of NB-UVB is about 10–12 times higher than that of BB-UVB (see Table 4). Therefore, the mean MED values for human skin resemble those that are considered as the LD in the PHET. Following our results, the PHET, as an inexpensive and valid screening method, has again shown its capacity especially to differentiate phototoxic potentials of moderate or weak photoirritants and various UV wavelengths. Moreover, our findings indicate that the yolk sac blood vessel system seems to be comparable with the biological system British Journal of Dermatology (2015) 172, pp462–466

466 Phototoxicity of NB-UVB compared with BB-UVB in the PHET, P.R. Bafteh et al.

of the human skin, particularly related to the time course of the skin changes and may therefore serve as a functioning in vivo test model for phototoxicity caused by UV irradiation.

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