Sunscreens Inadequately Protect Against Ultraviolet-A-Induced Free Radicals in Skin: Implications for Skin Aging and Melanoma?
2003; Elsevier BV; Volume: 121; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2003.12498.x
ISSN1523-1747
AutoresRachel Haywood, Peter Wardman, Roy Sanders, Claire Linge,
Tópico(s)Photodynamic Therapy Research Studies
ResumoSunscreens are employed to mitigate the adverse effects of sunlight on skin but are primarily designed to prevent ultraviolet-B-associated burning and damage. The increasingly recognized role of ultraviolet A in aging, and possibly melanoma, highlights the need to include ultraviolet A screens; however, validation remains difficult. We have used a novel method to establish the efficacy of sunscreens, by measuring ultraviolet-A-induced free-radical production (thought to contribute towards ultraviolet-A-related aging and malignant change). Electron spin resonance spectroscopy was used to detect free radicals directly in human Caucasian skin during irradiation with levels of ultraviolet comparable to solar intensities. Using this system the protection afforded by three high factor sunscreens (sun protection factor 20+) that claim ultraviolet A protection was examined. Each sunscreen behaved similarly: at recommended application levels (≥2 mg per cm2) the ultraviolet-induced free radicals were reduced by only about 55%, and by about 45% at 0.5–1.5 mg per cm2 (0.5 mg per cm2 reported for common usage). A "free-radical protection factor" calculated on the basis of these results was only 2 at the recommended application level, which contrasts strongly with the erythema-based sun protection factors (mainly indicative of ultraviolet B protection) quoted by the manufacturers (20+). The disparity between these protection factors suggests that prolonged sunbathing (encouraged by use of these creams) would disproportionately increase exposure to ultraviolet A and consequently the risk of ultraviolet-A-related skin damage. Sunscreens are employed to mitigate the adverse effects of sunlight on skin but are primarily designed to prevent ultraviolet-B-associated burning and damage. The increasingly recognized role of ultraviolet A in aging, and possibly melanoma, highlights the need to include ultraviolet A screens; however, validation remains difficult. We have used a novel method to establish the efficacy of sunscreens, by measuring ultraviolet-A-induced free-radical production (thought to contribute towards ultraviolet-A-related aging and malignant change). Electron spin resonance spectroscopy was used to detect free radicals directly in human Caucasian skin during irradiation with levels of ultraviolet comparable to solar intensities. Using this system the protection afforded by three high factor sunscreens (sun protection factor 20+) that claim ultraviolet A protection was examined. Each sunscreen behaved similarly: at recommended application levels (≥2 mg per cm2) the ultraviolet-induced free radicals were reduced by only about 55%, and by about 45% at 0.5–1.5 mg per cm2 (0.5 mg per cm2 reported for common usage). A "free-radical protection factor" calculated on the basis of these results was only 2 at the recommended application level, which contrasts strongly with the erythema-based sun protection factors (mainly indicative of ultraviolet B protection) quoted by the manufacturers (20+). The disparity between these protection factors suggests that prolonged sunbathing (encouraged by use of these creams) would disproportionately increase exposure to ultraviolet A and consequently the risk of ultraviolet-A-related skin damage. electron spin resonance minimal erythemal dose sun protection factor It is clearly established that ultraviolet (UV) wavelengths of sunlight are carcinogenic, contributing towards the formation of skin malignancy in the form of squamous and basal cell carcinoma and melanoma. There is a general consensus that basal and squamous cell carcinomas are predominantly a result of direct damage to the DNA by interaction with UVB (solar wavelengths 280–320 nm) (Linge, 1996Linge C. Relevance of in vitro melanocytic cell studies to the understanding of melanoma.Cancer Surveys. 1996; 26: 71-87PubMed Google Scholar). Epidemiologic data links melanoma to intense sunlight exposure in childhood, and provides support for a role of UVA (Moan et al., 1999Moan J. Dahlback A. Setlow R.B. Epidemiological support for an hypothesis for melanoma induction indicating a role for UVA radiation.Photochem Photobiol. 1999; 70: 243-247Crossref PubMed Scopus (194) Google Scholar). Although there is agreement that UV radiation is the cause, however, the precise wavelengths and mechanisms involved are not clear.Setlow et al., 1993Setlow R.B. Grist E. Thompson K. Woodhead A.D. Wavelengths effective in induction of malignant melanoma.Proc Natl Acad Sci USA. 1993; 90: 6666-6670Crossref PubMed Scopus (598) Google Scholar showed the induction of melanoma in the fish model Xiphophorus by UVA, UVB, and blue-visible wavelengths;Ley, 1997Ley R.D. Ultraviolet radiation A-induced precursors of cutaneous melanoma in Monodelphis domestica.Cancer Res. 1997; 57: 3682-3684PubMed Google Scholar demonstrated equal effectiveness of UVA and UVB in inducing melanocytic hyperplasia in the opossum Monodelphis domestica; andNoonan et al., 2001Noonan F.P. Recio J.A. Takayama H. et al.Neonatal sunburn and melanoma in mice.Nature. 2001; 413: 271Crossref PubMed Scopus (311) Google Scholar, Noonan et al., 2003Noonan F.P. Dudek J. Merlino G. De Fabo E. Animal models of melanoma: An HGF/SF transgenic mouse model may facilitate experimental access to UV initiating events.Pigment Cell Res. 2003; 16: 16-25Crossref PubMed Scopus (75) Google Scholar, using combined UVB and UVA wavelengths (ratio 2:1), recently demonstrated the induction of melanoma in a transgenic neonatal mouse model.Berking et al., 2001Berking C. Takemoto R. Satyamoorthy K. Elenitsas R. Herlyn M. Basic fibroblast growth factor and ultraviolet B transform melanocytes in human skin.Am J Pathol. 2001; 158: 943-953Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar showed that UVB in combination with basic fibroblast growth factor could transform human melanocytes. The role of UVA in human melanoma is still inconclusive (Wang et al., 2001Wang S.Q. Setlow R. Berwick M. Polsky D. Marghoob A.A. Kopf A.W. Bart R.S. Ultraviolet A and melanoma: A review.J Am Acad Dermatol. 2001; 44: 837-846Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Whereas UVB is believed to interact directly with DNA to initiate signature mutations of basal and squamous cell carcinomas, UVA wavelengths (320–400 nm) are believed to interact indirectly, inducing the production of free radicals (Packer, 1994Packer L. Ultraviolet radiation (UVA, UVB) and skin antioxidants.in: Rice-Evans C.A. Burdon R.H. Free Radical Damage and its Control. 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UV-A induces persistent genomic instability in human keratinocytes through an oxidative stress mechanism.Free Rad Biol Med. 2002; 32: 474-480Crossref PubMed Scopus (95) Google Scholar), and immunosuppression (Dumay et al., 2001Dumay O. Karam A. Vian L. et al.Ultraviolet AI exposure of human skin results in Langerhans cell depletion and reduction of epidermal antigen-presenting cell function: Partial protection by a broad spectrum sunscreen.Br J Dermatol. 2001; 144: 1161-1168Crossref PubMed Scopus (79) Google Scholar) have been demonstrated. Fas expression has also been shown to result from UVA as well as UVB (Bang et al., 2002Bang B. Rygaard J. Baadsgaard O. Skov L. Increased expression of Fas on human epidermal cells after in vivo exposure to single-dose ultraviolet (UV) B or long-wave UVA irradiation.Br J Dermatol. 2002; 147: 1199-1206Crossref PubMed Scopus (28) Google Scholar). Despite the extensive use of sunscreens during the last two decades, the incidence of skin cancers is still increasing, and the role of sunscreens in protecting against skin cancers is controversial. Sunscreen use has been shown to decrease the formation of actinic keratoses, which are linked to squamous cell carcinomas (Thompson et al., 1993Thompson S.C. Jolley D. Marks R. Reduction of solar keratoses by regular sunscreen use.N Engl J Med. 1993; 329: 1147-1151Crossref PubMed Scopus (661) Google Scholar; Naylor et al., 1995Naylor M.F. Boyd A. Smith D.W. Cameron G.S. Hubbard D. Neldner K.H. High sun protection factor sunscreens in the suppression of actinic neoplasia.Arch Dermatol. 1995; 131: 170-175Crossref PubMed Scopus (349) Google Scholar). Animal models have shown that sunscreens reduce the incidence of basal and squamous cell tumors (Sekura Snyder and May, 1975Sekura Snyder D. May M. Ability of PABA to protect mammalian skin from ultraviolet light-induced skin tumours and actinic damage.J Invest Dermatol. 1975; 65: 543-546Crossref PubMed Scopus (74) Google Scholar;Kligman et al., 1980Kligman L.H. Akin F.J. Kligman A.M. Sunscreens prevent ultraviolet photocarcinogenesis.J Am Acad Dermatol. 1980; 3: 30-35Abstract Full Text PDF PubMed Scopus (165) Google Scholar; Forbes et al., 1989Forbes P.D. Davies R.E. Sambuco C.P. Urbach F. Inhibition of ultraviolet radiation-induced skin tumours in hairless mice by topical application of the sunscreen 2-ethyl hexyl-p-methoxycinnamate.J Toxicol - Cut Ocular Toxicol. 1989; 8: 209-226Crossref Scopus (27) Google Scholar; Reeve et al., 1990Reeve V.E. Bosnic M. Boehm-Wilcox C. Effect of ultraviolet (UV) radiation and UVB-absorbing sunscreen ingredients on 7,12-dimethylbenz(a)anthracene-initiated skin tumorigenesis in hairless mice.Photodermatol Photoimmunol Photomed. 1990; 7: 222-227PubMed Google Scholar), which are UVB related; however, there have been several studies to suggest that sunscreen use is associated with increased risk of melanoma (Autier, 1995Autier P. et al.Melanoma and the use of sunscreens: An EORTC case-control study in Germany, Belgium and France.Int J Cancer. 1995; 61: 749-755Crossref PubMed Scopus (185) Google Scholar; Azizi, 2000Azizi E. et al.Use of sunscreen is linked with elevated naevi counts in Israeli school children and adolescents.Melanoma Res. 2000; 10: 491-498Crossref PubMed Scopus (42) Google Scholar; Vainio and Bianchini, 2000Vainio H. Bianchini F. Cancer-preventative effects of sunscreens are uncertain.Scand J Work Environ Health. 2000; 26: 529-531Crossref PubMed Scopus (31) Google Scholar). This may reflect inadequate sunscreen application (Stokes and Diffey, 1997Stokes R. Diffey B. How well are sunscreen users protected?.Photodermatol Photoimmunol Photomed. 1997; 13: 186-188Crossref PubMed Scopus (116) Google Scholar;Wulf et al., 1997Wulf H.C. Stender I.M. Lock-Andersen J. Sunscreens used at the beach do not protect against erythema: A new definition of SPF is proposed.Photodermatol Photoimmunol Photomed. 1997; 13: 129-132Crossref PubMed Scopus (126) Google Scholar; Gaughan and Padilla, 1998Gaughan M.D. Padilla R.S. Use of a topical fluorescent dye to evaluate effectiveness of sunscreen application.Arch Dermatol. 1998; 134: 515-517Crossref PubMed Scopus (14) Google Scholar); lack of durability of the application; the lack of, or inadequacy of, UVA filters in sunscreen preparations combined with prolonged sunbathing (Autier, 1995Autier P. et al.Melanoma and the use of sunscreens: An EORTC case-control study in Germany, Belgium and France.Int J Cancer. 1995; 61: 749-755Crossref PubMed Scopus (185) Google Scholar); the photo-instability of sunscreen filters that results in reduced protection; or the production of reactive free radicals or mutagens within the cream (Flindt-Hansen et al., 1988Flindt-Hansen H. Nielsen C.J. Thune P. Measurements of the photodegradation of PABA and some PABA derivatives.Photodermatol. 1988; 5: 257-261PubMed Google Scholar; Shaw et al., 1992Shaw A.A. Wainschel L.A. Shetlar M.D. Photoaddition of p-aminobenzoic acid to thymine and thymidine.Photochem Photobiol. 1992; 55: 657-663Crossref PubMed Scopus (27) Google Scholar; Gasparro, 1993Gasparro F.P. The molecular basis of UV-induced mutagenicity of suncreens.FEBS Lett. 1993; 336: 184-185Abstract Full Text PDF PubMed Scopus (7) Google Scholar; Knowland et al., 1993Knowland J. McKenzie E.A. McHugh P.J. Cridland N.A. Sunlight-induced mutagenicity of a common sunscreen ingredient.FEBS. 1993; 324: 309-313Abstract Full Text PDF PubMed Scopus (87) Google Scholar; Dunford et al., 1997Dunford R. Salinaro A. Cai L. Serpone N. Horikoshi S. Hidaka H. Knowland J. Chemical oxidation and DNA damage catalysed by inorganic sunscreen ingredients.FEBS Lett. 1997; 418: 87-90Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar). A link between sunscreen use and melanoma, however, is still debated (Huncharek and Kupelnick, 2002Huncharek M. Kupelnick B. Use of topical sunscreens and the risk of malignant melanoma: A meta-analysis of 9067 patients from 11 case-control studies.Am J Public Health. 2002; 92: 1173-1177Crossref PubMed Scopus (149) Google Scholar;Rigel, 2002Rigel D.S. The effect of sunscreen on melanoma risk.Dermatol Clin. 2002; 20: 601-606Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and unclear (Bigby, 1999Bigby M. The sunscreen and melanoma controversy.Arch Dermatol. 1999; 135: 1526-1527PubMed Google Scholar). The sun protection factor (SPF) of sunscreens is an internationally accepted standard by which the efficacy of sunscreens is assessed: it is based solely on prevention of erythema (sunburn), which is principally induced by UVB (Cole, 2001Cole C. Sunscreen protection in the ultraviolet A region: How to measure effectiveness.Photodermatol Photoimmunol Photomed. 2001; 17: 2-10Crossref PubMed Scopus (53) Google Scholar), and it is erythema (a downstream inflammatory response to the direct damage itself) that is the criterion by which people usually limit their sun exposure (Autier et al., 1999Autier P. Pedeux R. Doré J.F. Sunscreen use and duration of sun exposure: A double-blind randomised trial.J Natl Cancer Inst. 1999; 91: 1304-1309Crossref PubMed Scopus (226) Google Scholar). Whereas SPF may indicate protection against UVB-induced carcinogenesis, it cannot be used as an indicator of the "indirect" damage resulting from UVA exposure, as erythema is predominantly a response to UVB. As skin carcinogenesis is highly complex, the use of a range of markers for damage in skin itself is likely to be necessary to complement SPF (an indicator of UVB protection) for use in evaluation of total skin cancer risk. Other studies have been published that assess "direct" DNA damage, p53 formation, and protection against UV-induced immunosuppression (Freeman et al., 1988Freeman S.E. Ley R.D. Ley K.D. Sunscreen protection against UV-induced pyrimidine dimers in DNA of human skin in situ.Photodermatol. 1988; 5: 243-247PubMed Google Scholar; Ley and Fourtanier, 1997Ley R.D. Fourtanier A. Sunscreen protection against ultraviolet radiation-induced pyrimidine dimers in mouse epidermal DNA.Photochem Photobiol. 1997; 65: 1007-1011Crossref PubMed Scopus (39) Google Scholar;Ananthaswamy et al., 1998Ananthaswamy H.N. Loughlin S.M. Ullrich S.E. Kripke M.L. Inhibition of UV-induced p53 mutations by sunscreens: Implications for skin cancer prevention.J Invest Dermatol Symp Proc. 1998; 3: 52-56PubMed Scopus (38) Google Scholar; Burren et al., 1998Burren R. Scaletta C. Frenk E. Panizzon R.G. Applegate L.A. Sunlight and carcinogenesis: Expression of p53 and pyrimidine dimers in human skin following UVA I,+II and solar simulating radiations.Int J Cancer. 1998; 76: 201-206Crossref PubMed Scopus (101) Google Scholar; Bykov et al., 1998Bykov V.J. Marcusson J.A. Hemminki K. Ultraviolet B-induced DNA damage in human skin and its modulation by a sunscreen.Cancer Res. 1998; 58: 2961-2964PubMed Google Scholar). There are several methods to measure the protection afforded by sunscreens against UVA damage to skin (reviewed byLim et al., 2001Lim H.W. Naylor M. Honigsmann H. et al.American Academy of Dermatology Consensus Conference on UVA protection of sunscreens: Summary and recommendations.J Am Acad Dermatol. 2001; 44 (Washington, DC, Feb 4, 2000): 505-508Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar); however, these methods are not validated (Cole, 2001Cole C. Sunscreen protection in the ultraviolet A region: How to measure effectiveness.Photodermatol Photoimmunol Photomed. 2001; 17: 2-10Crossref PubMed Scopus (53) Google Scholar). The protection against free radicals induced by UVA, to date, has not been measured. In this study we have adapted an electron spin resonance (ESR) method to measure UV-induced free-radical production in human skin, and assessed the protection against free-radical production provided by commercial sunscreens. The free radicals formed upon UV irradiation of skin (and that are associated with DNA and protein damage) are not usually directly detectable at room temperature. An exception to this, however, is the ascorbate radical, which is formed when ascorbate (vitamin C – a cellular antioxidant) reacts with free radicals. The ascorbate radical is readily detected using ESR spectroscopy in skin biopsies exposed to UV irradiation (Jurkiewicz and Buettner, 1996Jurkiewicz B.A. Buettner G.R. EPR detection of free radicals in UV-irradiated skin: Mouse versus human.Photochem Photobiol. 1996; 64: 918-922Crossref PubMed Scopus (120) Google Scholar) and is accepted as a reliable marker for cellular free-radical production and oxidative stress (Buettner and Jurkiewicz, 1995Buettner G.R. Jurkiewicz B.A. Ascorbate radical: A valuable marker of oxidative stress.in: Favier A.E. Cadet J. Kalyanaraman B. Fontecave M. Pierre J.-L. Anal of Free Radicals in Biological Systems. Birkhauser Verlag, Basel1995: 146-163Crossref Google Scholar; Jurkiewicz and Buettner, 1996Jurkiewicz B.A. Buettner G.R. EPR detection of free radicals in UV-irradiated skin: Mouse versus human.Photochem Photobiol. 1996; 64: 918-922Crossref PubMed Scopus (120) Google Scholar). We have used the relative quantification of this radical in the same skin sample, both before and after application of sunscreen, in order to estimate the level of protection against UVA irradiation afforded by three popular sunscreens that claim UVA protection, over a range of application densities. This study using consented skin from informed patients, was approved by the West Hertfordshire NHS Trust (EC2002-20) and was conducted in accordance with the Declaration of Helsinki. Caucasian skin was obtained from consenting patients undergoing breast reduction surgery. Skin was stored in normal-saline-soaked gauze at 4°C and used within 24 h. Prior to ESR spectroscopic analysis, skin was trimmed to remove subcutaneous fat and cut to approximately 1 cm2. The surface area of the skin held flat and undeformed between the silica plates of the flat cell was measured. Sample sizes could not exceed 1 cm2 due to technical limitations associated with tuning the spectrometer. Three popular brands of sunscreens, which claimed UVA protection, were randomly chosen for evaluation: brands 1, 2, and 3 were SPF 30 (containing the UVA filters titanium dioxide and terephthalylidene dicamphor sulfonic acid), SPF 25 (containing butylmethoxyldibenzoyl methane), and SPF 20+ (containing octocrylene and titanium dioxide), respectively (filter concentrations were unspecified). Two of the brands chosen had a four star UVA rating (highest UK rating), and the third was obtained outside the UK and did not have this rating (although it claimed UVA protection and anti-cell-aging effects). All creams were evaluated within 3 mo of purchase. ESR experiments were carried out using a Bruker EMX spectrometer (Rheinstetten/Karlsruhe, Germany) equipped with an ER 4103TM cavity and a Wilmad Glass tissue cell (WG 806-B-Q) (Buena, NJ). Typical ESR settings were 40 mW microwave power, 0.075 mT modulation amplitude, 2×105 receiver gain, and sweep time 20 s with repeated scanning (five scans) unless otherwise indicated. UV irradiation was carried out in situ in the spectrometer (with the cavity completely shielded by black plastic sheeting) using a super high pressure 100 W Nikon mercury lamp (model LH-M1100CB-1) focused on the cavity transmission window. The emission spectrum of the lamp is shown in Figure 1a. A 5 cm water filter was used to remove infrared radiation together with two optical glass filters having a combined thickness of 0.7 cm Barr and Stroud filtering (available from Andover Corporation, Salem, NH) wavelengths below 300 nm and having a 1% transmittance of UVB radiation at 300 nm and 19% at 320 nm (transmission spectrum shown in Figure 1b). Visible wavelengths were not filtered. The UV fluence incident upon the sample within the spectrometer was measured using a potassium ferrioxalate actinometer (Valenzeno et al., 1991Valenzeno D.P. Pottier R.H. Mathis P. Douglas R.H. Photobiological Techniques. Plenum Press, New York1991: 50-54Google Scholar), which was slightly modified as follows: 0.006 M stock actinometer solution (0.25 mL) was irradiated (5 min) directly in the flat cell held in the cavity of the spectrometer and then washed out of the flat cell into a 5 mL flask with distilled water; 2 mL of this solution was added to 0.4 mL 1% 1,10-phenanthralene, 1 mL pH 4.5 buffer (Sigma), and diluted to 5 mL with d-H2O. The optical densities of irradiated and unirradiated solutions were compared at 510 nm. The UV fluence is within levels of solar irradiation (the UVA component of the total UV radiation penetrating the earth's atmosphere is 90%) measured between 11.00 a.m. and 3.00 p.m. with the same UV actinometer (irradiated with natural sunlight through an aperture cut in black card to the same dimensions as the ESR cavity window), June–September, London, UK (direct sunlight). The photon flux was calculated in mol quanta per second (Calvert and Pitts, 1966Calvert J.G. Pitts J.N. Photochemistry. John Wiley and Sons, New York1966Google Scholar, page 781, equation 7.6) and converted to radiant flux in joules per second by multiplying by the Avogadro constant, Planck's constant, and the radiation frequency at 350 nm (mean frequency 300–400 nm over which the actinometer absorbs radiation). The molar absorptivity of potassium ferrioxalate (1.1 × 103 per mol dm3 cm) is also required for this calculation. The radiant flux incident upon the actinometer was calculated to be 3 mW (3 mJ per s) and the irradiance at the cavity window (area 2.3 cm2) was then calculated to be 1.3 mW per cm2 (mJ per s per cm2). The irradiance is lower than levels of UVA that have been employed for solar-simulated irradiation (reported levels of solar-simulated UVA are 35 and 60–80 mW per cm2) (Ley et al, 1997;Burren et al., 1998Burren R. Scaletta C. Frenk E. Panizzon R.G. Applegate L.A. Sunlight and carcinogenesis: Expression of p53 and pyrimidine dimers in human skin following UVA I,+II and solar simulating radiations.Int J Cancer. 1998; 76: 201-206Crossref PubMed Scopus (101) Google Scholar). ESR methodology was used to detect the ascorbate radical directly in human skin on UV irradiation as previously published (Jurkiewicz and Buettner, 1996Jurkiewicz B.A. Buettner G.R. EPR detection of free radicals in UV-irradiated skin: Mouse versus human.Photochem Photobiol. 1996; 64: 918-922Crossref PubMed Scopus (120) Google Scholar). The skin specimen (unprotected) was held in a Wilmad tissue cell, placed directly into the ESR cavity, and subjected to 100 s UV irradiation to establish the background levels of ascorbate radical. The skin sample, when mounted in a tissue cell, was held flat and undeformed between two silica plates comprising the tissue cell. The UV source was then blocked and the skin area was marked precisely on the covering silica slide and measured. The measured area is that of the skin held flat, and undeformed, between the silica slides. The sunscreen was applied to the measured area at a range of application levels (quantified by weighing) centered around that recommended in the sunscreen industry (2 mg per cm2). The slide was then placed with the cream side directly against the skin and again UV irradiated, and the free-radical signal intensity measured. The skin area was restricted to 0.5–1 cm2 and the amounts of cream that were applied to the skin were not lower than 0.5 mg to minimize errors due to weighing. This set a lower limit for application of approximately 0.5 mg per cm2. Given the results of preliminary experiments and that the manufacturers' recommended application level is 2 mg per cm2, an upper limit for the quantitative analysis of 4 mg per cm2 was chosen. Nine measurements performed in this way were taken for each sunscreen across this range of application. Quantification of the ascorbate radical spectrum was by measurement of the height of the low field absorbance peak relative to the midpoint (marked on Figure 2a). To verify that the signal of any radical species either already present in the sunscreen or formed as a result of UV irradiation of the sunscreen alone did not interfere with the signal of the ascorbate radical formed in the skin, comparison with the ESR spectra obtained from illumination of sunscreen alone was made. Two methods were used to measure protection: (1) using the same skin sample (reducing possible intersample variation in ascorbate levels) or (2) comparing two different samples of the same skin both unprotected and protected with cream. Method (1) was used for the majority of the study, not only to reduce intersample variation of ascorbate, but also to ensure adequate levels of ascorbate in each skin sample studied (which occasionally could be low in some specimens, believed to be for dietary reasons). This method required that ascorbate (1) is sufficiently stable in the skin over the 100 s UV irradiation period taken to obtain a measurement, and (2) is stable over the period between the measurements when skin was removed from the spectrometer, coated with cream, and then reinserted in the spectrometer. Stability over the 100 s irradiation period was verified using fresh skin by continuous measurement of the free-radical signal intensity over approximately 1400 s UV irradiation (Figure 2c). Stability between measurements was also verified (not shown). As skin was used up to 24 h post surgical excision, it was necessary to verify that ascorbate was sufficiently stable with refrigerated storage over this time period. Ascorbate levels in the skin were compared by comparison of the ascorbate radical signal intensity upon irradiation at different time periods of storage: at 0 (in practical terms approximately 2 h post excision) (n=3); 1 d (n=9); and 3 d (n=3) (Figure 2d). To verify that the ascorbate radical signal did not originate from irradiation scattered through the tissue cell (whose etched lower surface decreases light transmittance considerably compared to that through the transparent cover slip) skin was irradiated both protected at the front, and then at the back, by black tape of similar dimensions to the skin sample. The ascorbate radical signal was abolished when protective tape (greater than the skin area) was between the incident irradiation and the skin, but when the tape was placed behind the skin (to prevent entry into the skin of scattered radiation) the ascorbate radical signal was clearly detected at a similar signal intensity to skin not shielded by tape at the dermal aspect (not shown). If the tape was the exact dimensions of the skin sample, and placed in front of the skin, then a weak signal due to the ascorbate radical could occasionally be observed (up to about 10% the unprotected signal), suggesting entry of irradiation through the cut edge of the skin. As a further check of the primary method used (method (1)), the protection at 2 mg per cm2 application was also determined using method (2) for one brand of sunscreen. Paired samples of skin were cut from the same piece of skin (n=5, separate patients) to similar dimensions. One of these samples was mounted in the tissue cell, as described above, and irradiated for 100 s to determine the free-radical signal intensity in unprotected skin. This sample was then removed from the cell and replaced by the second skin sample, which was then covered with a film of factor 25 sunscreen applied to the upper silica slide, to an application density of 2 mg per cm2, to an area that exceeded that of the skin sample (to ensure blocking of the edge effect). This sunscreen-protected skin was irradiated for 100 s and the free-radical signal intensity was measured. Both sunscreen-protected and unprotected samples, in this experiment, were shielded at the dermal aspect with black tape. The protection provided by a commonly used dermatologic cream, lacking UV filters, was also measured. The dose–response of the ascorbate radical signal intensity to the UVA irradiation was verified using neutral-density filters to reduce the UVA dose to the skin by increasing amounts. Neutral-density filters were added sequentially to the water and two glass filter combination of the UV lamp, and for each filter system the absorbance at 510 nm of UV-irradiated actinometer solution was measured, relative to unirradiated solution. For each filter system, the ascorbate radical signal intensity, upon 100 s UV irradiation, was also measured in different samples of skin, cut to similar dimensions. C
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