Improved Protection Against Solar-Simulated Radiation-Induced Immunosuppression by a Sunscreen with Enhanced Ultraviolet A Protection
2000; Elsevier BV; Volume: 114; Issue: 4 Linguagem: Inglês
10.1046/j.1523-1747.2000.00946.x
ISSN1523-1747
AutoresA. Fourtanier, Audrey Guéniche, Delphine Compan, Susan L. Walker, Antony R. Young,
Tópico(s)Effects of Radiation Exposure
ResumoUltraviolet radiation-induced immunosuppression is thought to play a part in skin cancer. Several studies have indicated that sunscreens that are designed to protect against erythema failed to give comparable protection against ultraviolet radiation-induced immunosuppression. One possible reason for this discrepancy is inadequate ultraviolet A protection. This study evaluated the level of immunoprotection in mice afforded by two broad-spectrum sunscreens with the same sun protection factor, but with different ultraviolet A protection factors. Both sunscreens contained the same ultraviolet B and ultraviolet A filters, in the same vehicle, but at different concentrations. Solar simulated radiation dose–response curves for erythema, edema, and systemic suppression of contact hypersensitivity were generated and used to derive protection factors for each end-point. The results of three different techniques for determining immune protection factor were compared. A comparison of the two sunscreens showed that the protection factor for erythema in mice was similar to that determined in humans (sun protection factor) but the protection factor for edema in mice was lower. Both sunscreens protected against suppression of contact hypersensitivity but the product with the higher ultraviolet A-protection factor showed significantly greater protection. The three techniques for determining immunoprotection gave very similar results for a given sunscreen, but immune protection factor was always lower than sun protection factor. These data suggest that sun protection factor may not predict the ability of sunscreens to protect the immune system and that a measure of ultraviolet A protection may also be necessary. Ultraviolet radiation-induced immunosuppression is thought to play a part in skin cancer. Several studies have indicated that sunscreens that are designed to protect against erythema failed to give comparable protection against ultraviolet radiation-induced immunosuppression. One possible reason for this discrepancy is inadequate ultraviolet A protection. This study evaluated the level of immunoprotection in mice afforded by two broad-spectrum sunscreens with the same sun protection factor, but with different ultraviolet A protection factors. Both sunscreens contained the same ultraviolet B and ultraviolet A filters, in the same vehicle, but at different concentrations. Solar simulated radiation dose–response curves for erythema, edema, and systemic suppression of contact hypersensitivity were generated and used to derive protection factors for each end-point. The results of three different techniques for determining immune protection factor were compared. A comparison of the two sunscreens showed that the protection factor for erythema in mice was similar to that determined in humans (sun protection factor) but the protection factor for edema in mice was lower. Both sunscreens protected against suppression of contact hypersensitivity but the product with the higher ultraviolet A-protection factor showed significantly greater protection. The three techniques for determining immunoprotection gave very similar results for a given sunscreen, but immune protection factor was always lower than sun protection factor. These data suggest that sun protection factor may not predict the ability of sunscreens to protect the immune system and that a measure of ultraviolet A protection may also be necessary. butylmethoxydibenzoylmethane dinitrofluorobenzene edema protection factor erythema protection factor immune protection factor 50% immune protection factor global immune protection factor 50% immunosuppressive dose minimal erythema dose minimal edema dose minimal immunosuppressive dose octocrylene persistent pigment darkening sun protection factor UVA protection factor Animal studies have shown that ultraviolet radiation (UVR)-induced suppression of cell-mediated immunity plays an important part in nonmelanoma skin cancer and a similar role is suspected in humans (Nishigori et al., 1996Nishigori C. Yarosh D.B. Donawho C. Kripke M.L. The immune system in ultraviolet carcinogenesis.J Invest Dermatol Symp Proceedings of The. 1996; 1: 143-146PubMed Google Scholar). Sunscreen use is widely advocated to reduce skin cancer risk, so it is important to know if a given reduction of erythemogenic UVR by a sunscreen is associated with a comparable reduction of skin cancer related photodamage. UVR-induced suppression of cell-mediated immunity can be evaluated in vivo by measuring the impairment of the contact hypersensitivity response (CHS) to chemical haptens in mouse and humans (Noonan et al., 1981Noonan F.P. De Fabo E.C. Kripke M.L. Suppression of contact hypersensitivity to UV radiation and its relationship to UV-induced suppression of tumor immunity.Photochem Photobiol. 1981; 34: 683-689Crossref PubMed Scopus (187) Google Scholar;De Fabo and Noonan, 1983De Fabo E.C. Noonan F.P. Mechanism of immune suppression by ultraviolet irradiation in vivo. I. Evidence for the existence of a unique photoreceptor in skin and its role in photoimmunology.J Exp Med. 1983; 157: 84-98Crossref Scopus (432) Google Scholar;Cooper et al., 1992Cooper K.D. Oberhelman L. Hamilton T.A. et al.UV exposure reduces immunization rates and promotes tolerance to epicutaneous antigens in humans: relationship to dose, CDla–DR+ epidermal macrophage induction, and Langerhans cell depletion.Proc Natl Acad Sci USA. 1992; 89: 8497-8501Crossref PubMed Scopus (334) Google Scholar;LeVee et al., 1997LeVee G.J. Oberhelman L. Anderson T. Koren H. Cooper K.D. UvA Il exposure of human skin results in decreased immunization capacity, increased induction of tolerance and a unique pattern of epidermal antigen-presenting cell alteration.Photochem Photobiol. 1997; 65: 622-629Crossref PubMed Scopus (62) Google Scholar;Kelly et al., 1998Kelly D.A. Walker S.L. McGregor J.M. Young A.R. A single exposure of solar simulated radiation suppresses contact hypersensitivity responses both locally and systemically in humans: quantitative studies with high frequency ultrasound.J Photochem Photobiol B Biol. 1998; 44: 130-142https://doi.org/10.1016/s1011-1344(98)00136-5Crossref PubMed Scopus (0) Google Scholar). This end-point has been widely used to evaluate sunscreen immunoprotection and make comparisons with protection from inflammation (erythema/edema). Comparisons of a given sunscreen's protection efficacies against immunosuppression and inflammation, however, have given conflicting results (Reeve et al., 1991Reeve V.E. Bosnic M. Boehm-Wilcox C. Ley R.D. Differential protection by two sunscreens from UV radiation-induced immunosuppression.J Invest Dermatol. 1991; 97: 624-628Abstract Full Text PDF PubMed Google Scholar;Ho et al., 1992Ho K.K. Halliday G.M. Barnetson R.S. Sunscreens protect epidermal Langerhans cells and Thy-1+ cells but not local contact sensitization from the effects of ultraviolet light.J Invest Dermatol. 1992; 98: 720-724Crossref PubMed Scopus (41) Google Scholar;Bestak et al., 1995Bestak R. Barnetson R. St C. Nearn M.R. Halliday G.M. Sunscreen protection of contact hypersensitivity responses from chronic solar-simulated ultraviolet irradiation correlates with the absorption spectrum of the sunscreen.J Invest Dermatol. 1995; 105: 345-351Crossref PubMed Scopus (93) Google Scholar;Wolf et al., 1993aWolf P. Donawho C.K. Kripke M.L. Analysis of the protective effect of different sunscreens on ultraviolet radiation-induced local and systemic suppression of contact hypersensitivity and inflammatory responses in mice.J Invest Dermatol. 1993; 100: 254-259Abstract Full Text PDF PubMed Google ScholarWolf et al., 1993bWolf P. Yarosh D.B. Kripke M.L. Effects of sunscreens and a DNA repair enzyme on ultraviolet radiation-induced inflammation, immune suppression and cyclobutane pyrimidine dimer formation in mice.J Invest Dermatol. 1993; 106: 523-527Crossref Scopus (108) Google Scholar;Roberts and Beasley, 1995Roberts L.K. Beasley D.G. Commercial sunscreen lotions prevent ultraviolet-radiation-induced immune suppression of contact hypersensitivity.J Invest Dermatol. 1995; 105: 339-344Crossref PubMed Scopus (70) Google Scholar,Roberts and Beasley, 1997Roberts L.K. Beasley D.G. Sunscreens prevent local and systemic immunosuppression of contact hypersensitivity in mice exposed to solar-simulated ultraviolet radiation.J Photochem Phiotobiol B Biol. 1997; 39: 121-129Crossref PubMed Scopus (19) Google Scholar;Whitmore and Morison, 1995Whitmore S.E. Morison W.L. Prevention of UVB-induced immunosuppression in humans by a high sun protection factor sunscreen.Arch Dermatol. 1995; 131: 1128-1133Crossref PubMed Scopus (96) Google Scholar;Serre et al., 1997Serre I. Cano J.P. Picot M.C. Meynadier J. Meunier L. Immunosuppression induced by acute solar-simulated ultraviolet exposure in humans: Prevention by a sunscreen with a sun protection factor of 15 and high UVA protection.J Am Acad Dermatol. 1997; 37: 187-194Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar;Hayag et al., 1997Hayag M.V. Chartier T. DeVoursney J. Tie C. Machler B. Taylor J.R. A high SPF sunscreen's effects on UVB-induced immunosuppression of DNCB contact hypersensitivity.J Dermatol Sci. 1997; 16: 31-37https://doi.org/10.1016/s0923-1811(97)00617-8Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar;Moyal et al., 1997Moyal D. Courbière C. Le Corre Y. Lacharrière O. Hourseau C. Immunosuppression induced by chronic solar-simulated irradiation in humans and its prevention by sunscreens.Eur J Dermatol. 1997; 7: 223-225Google Scholar;Walker and Young, 1997Walker S.L. Young A.R. Sunscreens offer the same UVB protection factors for inflammation and immunosuppression in the mouse.J Invest Dermatol. 1997; 108: 133-138Crossref PubMed Scopus (35) Google Scholar). Poor immunoprotection, in comparison with protection from inflammation, has been reported in the majority of these studies and this has raised doubts about the benefits of suncreen use in the prevention of skin cancer. In many cases, however, the discrepancies between protection from inflammation and immunosuppression may be attributed, in part, to experimental design flaws. For example, very few studies have assessed immunoprotection with experimental conditions comparable with those recommended for sun protection factor (SPF) testing. SPF, a measure of acute protection from erythema, is highly dependent on the UVR source and the sunscreen's application density (Farr and Diffey, 1985Farr P.M. Diffey B.L. How reliable are sunscreen protection factors?.Brit J Dermatol. 1985; 112: 113-118Crossref PubMed Scopus (33) Google Scholar) but gives no indication of a product's protection against chronic exposure. Furthermore (as shown in the results), products with the same SPF may have quite different spectral profiles. It is therefore essential to determine SPF and immune protection factor (IPF) using similar experimental conditions.Roberts and Beasley, 1995Roberts L.K. Beasley D.G. Commercial sunscreen lotions prevent ultraviolet-radiation-induced immune suppression of contact hypersensitivity.J Invest Dermatol. 1995; 105: 339-344Crossref PubMed Scopus (70) Google Scholar demonstrated that IPF was 2-fold greater than SPF when a solar simulator was used and sunscreens were applied at 2 mg per cm2, but IPF was lower than SPF with nonsolar sources, or at a lower sunscreen application density. Repeated UVR exposure has a cumulative effect on suppression of CHS in the mouse (Noonan et al., 1981Noonan F.P. De Fabo E.C. Kripke M.L. Suppression of contact hypersensitivity to UV radiation and its relationship to UV-induced suppression of tumor immunity.Photochem Photobiol. 1981; 34: 683-689Crossref PubMed Scopus (187) Google Scholar), yet most investigators determine immunoprotection using multiple rather than single exposures (Reeve et al., 1991Reeve V.E. Bosnic M. Boehm-Wilcox C. Ley R.D. Differential protection by two sunscreens from UV radiation-induced immunosuppression.J Invest Dermatol. 1991; 97: 624-628Abstract Full Text PDF PubMed Google Scholar;Ho et al., 1992Ho K.K. Halliday G.M. Barnetson R.S. Sunscreens protect epidermal Langerhans cells and Thy-1+ cells but not local contact sensitization from the effects of ultraviolet light.J Invest Dermatol. 1992; 98: 720-724Crossref PubMed Scopus (41) Google Scholar;Bestak et al., 1995Bestak R. Barnetson R. St C. Nearn M.R. Halliday G.M. Sunscreen protection of contact hypersensitivity responses from chronic solar-simulated ultraviolet irradiation correlates with the absorption spectrum of the sunscreen.J Invest Dermatol. 1995; 105: 345-351Crossref PubMed Scopus (93) Google Scholar;Roberts and Beasley, 1995Roberts L.K. Beasley D.G. Commercial sunscreen lotions prevent ultraviolet-radiation-induced immune suppression of contact hypersensitivity.J Invest Dermatol. 1995; 105: 339-344Crossref PubMed Scopus (70) Google Scholar,Roberts and Beasley, 1997Roberts L.K. Beasley D.G. Sunscreens prevent local and systemic immunosuppression of contact hypersensitivity in mice exposed to solar-simulated ultraviolet radiation.J Photochem Phiotobiol B Biol. 1997; 39: 121-129Crossref PubMed Scopus (19) Google Scholar;Whitmore and Morison, 1995Whitmore S.E. Morison W.L. Prevention of UVB-induced immunosuppression in humans by a high sun protection factor sunscreen.Arch Dermatol. 1995; 131: 1128-1133Crossref PubMed Scopus (96) Google Scholar;Hayag et al., 1997Hayag M.V. Chartier T. DeVoursney J. Tie C. Machler B. Taylor J.R. A high SPF sunscreen's effects on UVB-induced immunosuppression of DNCB contact hypersensitivity.J Dermatol Sci. 1997; 16: 31-37https://doi.org/10.1016/s0923-1811(97)00617-8Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar;Moyal et al., 1997Moyal D. Courbière C. Le Corre Y. Lacharrière O. Hourseau C. Immunosuppression induced by chronic solar-simulated irradiation in humans and its prevention by sunscreens.Eur J Dermatol. 1997; 7: 223-225Google Scholar). Discrepancies may also arise because of differing methods in the calculation of IPF.Bestak et al., 1995Bestak R. Barnetson R. St C. Nearn M.R. Halliday G.M. Sunscreen protection of contact hypersensitivity responses from chronic solar-simulated ultraviolet irradiation correlates with the absorption spectrum of the sunscreen.J Invest Dermatol. 1995; 105: 345-351Crossref PubMed Scopus (93) Google Scholar, who found that IPF was lower than SPF, used the ratio obtained from the highest UVR dose at which the sunscreen protected from immunosuppression vs. the minimal immunosuppressive dose (MISD).Roberts and Beasley, 1995Roberts L.K. Beasley D.G. Commercial sunscreen lotions prevent ultraviolet-radiation-induced immune suppression of contact hypersensitivity.J Invest Dermatol. 1995; 105: 339-344Crossref PubMed Scopus (70) Google Scholar,Roberts and Beasley, 1997Roberts L.K. Beasley D.G. Sunscreens prevent local and systemic immunosuppression of contact hypersensitivity in mice exposed to solar-simulated ultraviolet radiation.J Photochem Phiotobiol B Biol. 1997; 39: 121-129Crossref PubMed Scopus (19) Google Scholar) used the ratio of UVR doses causing approximately 50% inhibition of CHS response with and without sunscreen, and found IPF higher than SPF.Walker and Young, 1997Walker S.L. Young A.R. Sunscreens offer the same UVB protection factors for inflammation and immunosuppression in the mouse.J Invest Dermatol. 1997; 108: 133-138Crossref PubMed Scopus (35) Google Scholar used the ratio of UVB (311 nm) doses where the CHS response was at 50% of maximal, and compared dose–response curves, with and without sunscreen. To date there have been no attempts to compare these approaches and to assess the best method of IPF determination. Several studies have demonstrated that broad-spectrum sun-screens (290–400 nm) afford better protection than preparations that absorb mainly in the UVB region (Bestak et al., 1995Bestak R. Barnetson R. St C. Nearn M.R. Halliday G.M. Sunscreen protection of contact hypersensitivity responses from chronic solar-simulated ultraviolet irradiation correlates with the absorption spectrum of the sunscreen.J Invest Dermatol. 1995; 105: 345-351Crossref PubMed Scopus (93) Google Scholar;Damian et al., 1997Damian D.L. Halliday G.M. Barnetson R. St C. Broad-spectrum sunscreens provide greater protection against ultraviolet-radiation-induced suppression of contact hypersensitivity to a recall antigen in humans.J Invest Dermatol. 1997; 109: 146-151Crossref PubMed Scopus (118) Google Scholar;Gueniche et al., 1997Gueniche A. Fourtanier A. Mexoryl SX protects against photoimmunosuppression.in: Altmeyer P. Hoffmann K. Stucker M. Skin Cancer and UV Radiation. Springer-Verlag, Berlin1997: 249-262Crossref Google Scholar;Moyal et al., 1997Moyal D. Courbière C. Le Corre Y. Lacharrière O. Hourseau C. Immunosuppression induced by chronic solar-simulated irradiation in humans and its prevention by sunscreens.Eur J Dermatol. 1997; 7: 223-225Google Scholar), but no studies have specifically compared different levels of UVA protection. In order to answer some of these questions we have compared the immunoprotective efficacy of two broad-spectrum sunscreens with the same sun protection factor, but with quite different UVA protection factors (UVA-PF). We have used methods based on those recommended for sunscreen SPF testing. UVR-induced immunosuppression was assessed in hairless mice by the inhibition of the systemic CHS response to dinitrofluorobenzene (DNFB) after a single exposure to solar-simulated radiation (SSR). In addition, we have compared three methods of assessing IPF. Unlike several published studies, we have determined UVR dose–responses for all end-points with and without sunscreens. Outbred pathogen-free female Skh-1/hairless albino mice, aged 8–10 wk were housed individually with free access to food (standard laboratory mouse pellets) and water, in a room with controlled temperature (23°C ± 2) and relative humidity (50% ± 20) and a 12 h on/12 h off light–dark cycle room lighting provided with gold lamps. The solar simulator used for all mouse studies was a 1000 W xenon arc lamp including a dichroic mirror (Oriel, Stratford, USA) equipped with a WG320/1 mm thick filter and a UG11 filter/1 mm thick filter (Schott, Clichy, France). This filtered source provided a simulated solar UVR spectrum (290–400 nm) that almost eliminated all visible and infrared radiation. Irradiance was routinely measured before each exposure session with a Centra ARCC 1600 radiometer (Osram, Berlin, Germany). The integrated irradiance, measured before the beginning of the experiment with a calibrated Bentham DM150 double monochromator spectroradiometer (Bentham, Reading, U.K.), was 2.16 mW per cm2 for UVB (290–320 nm) and 16 mW per cm2 for UVA (320–400 nm) at skin level. For the determination of SPF and UVA-PF in humans, a Multiport solar simulator, Model 601 (Solar Light, Philadelphia, PA) was used. This simulator included a 150 W Xenon lamp and a dichroic mirror. It was fitted with the same Schott filters as those used in the mouse experiments, except that the WG320/1 mm thick filter was replaced with a WG335/3 mm thick filter (Schott, Clichy, France) for UVA-PF determinations. The emission beam of this simulator is focused and passed through six liquid light guides. The integrated irradiances measured spectroradiometrically at the skin level were 6.6 mW per cm2 for UVB and 55 mW per cm2 for UVA with WG320/1 mm filter and 50 mW per cm2 of UVA with WG335/3 mm filter. The emission spectra of the SSR sources and of the UVA source are shown in Figure 1. Two prototype preparations (5-A and 5-B) were formulated in the same oil-in-water vehicle. Both contained octocrylene, a UVB absorber (OC or Uvinul® N539, BASF Ludwigshafen, Germany) and butyl methoxy-dibenzoylmethane, a UVA absorber (BMDM or Parsol 1789®, Givaudan Roure, Vernier Geneva, Switzerland) but at different concentrations as shown in Table 1.Table 1Characteristics of sunscreens and human in vivo protection factorsSunscreenUVB filter OCaOC, octocrylene,UVA filter BMDMbBMDM, butyl methoxydibenzoylmethane.SPFcMean ± SD.UVA-PFcMean ± SD.Product 5-A (high UVA protection)7%3%7.1 ± 1.27.8 ± 0.5Product 5-B (low UVA protection)10%0.5%8.2 ± 2.53.1 ± 0.6a OC, octocrylene,b BMDM, butyl methoxydibenzoylmethane.c Mean ± SD. Open table in a new tab The UVR transmission spectra (T) of the sunscreen products were obtained using a modifiedDiffey and Robson, 1989Diffey B.L. Robson J. A new substrate to measure sunscreen protection factors throughout the ultraviolet spectrum.J Soc Cosmet Chem. 1989; 40: 127-133Google Scholar method. In this method the UVR transmitted through a roughened quartz plate (instead of Transpore® tape as in the original method), with and without the sunscreen applied (1 mg per cm2, instead of 1.5 or 2 μl per cm2 in the original method), was measured spectroradiometrically and the monochromatic protection factors (mPF) were calculated (mPF = 1/T). SPF were determined on 10 human volunteers (two skin type II, four skin type II, four skin type III) following the The European Cosmetic Toiletry & Perfumery Association (COLIPA), 1994The European Cosmetic Toiletry & Perfumery Association (COLIPA) SPF Test Method. 1994Google Scholar. UVA-PF was determined on eight additional volunteers (six skin type II, two skin type III) using the persistent pigment darkening (PPD) method. This technique is based on the minimal PPD dose and has been adopted by theJapan Cosmetic Industry Association (JCIA)., January 1, 1996Japan Cosmetic Industry Association (JCIA) Measurements standard for UVA protection efficacy. 1996Google Scholar. PPD, first described byHausser, 1938Hausser I. Uber spezifische Wirkungen des Langwelligen ultravioletten lichts auf die menschliche haut.Strahlentherapie. 1938; 62: 315-322Google Scholar, is the stabilized brownish-gray skin discoloration that follows the immediate pigment darkening response at about 2 h postirradiation. The minimal PPD dose of unprotected skin ranges from 15 to 25 J per cm2 of UVA with a mean value of about 20 J per cm2. The UVA-PF is the ratio of doses needed to obtain the minimal 2 h PPD reaction with and without sunscreen (Chardon et al., 1997Chardon A. Moyal D. Hourseau C. Persistent pigment darkening response as a method for evaluation of ultraviolet A protection assays.in: Lowe N. Shaath N. Pathak M. Sunscreens Development, Evaluation and Regulatory Aspects. Marcel Dekker, New York1997: 559-582Google Scholar). SPF were also determined in the mouse, using a modification of the human protocol, with the same SSR source used in the CHS studies Animals were lightly anesthetized and covered with a masking template with four openings. The mean minimal erythema dose (MED), that produced a uniform pale pink color at 24 h after a single SSR exposure, was determined to be 3.0 J per cm2 (total SSR spectrum) using a standard protocol. One-hundred microliters of product was applied by gloved finger massage over the dorsal and flank regions (approximately 40 cm2 resulting in an application density of about 2.5 mg per cm2) 15 min prior to SSR exposure. Use of templates allowed four SSR exposure doses per mouse. These were in 1 MED (group mean) increments ranging from 4 to 9 MED. SPF were determined for each product using at least 10 mice. SPF was the ratio of MED with and without sunscreen. Mice were lightly anesthetized, covered with a black masking template with one opening (3 cm × 1.5 cm) over the dorsal skin area. All other body sites (i.e., ears, tail) were protected. Sunscreen products, or vehicle, were applied to the irradiated site 15 min before a single SSR exposure as described for SPF testing. Mice were exposed to increasing SSR doses (0.5 MED increments) between 0.5 MED and 3 MED for untreated animals, 1.5 and 2.5 MED for vehicle treated animals and 4–16 MED (with 2 MED increments) for sunscreen-treated mice. Control groups included untreated unexposed mice (absolute controls), or mice treated with sunscreens/vehicle but not SSR exposed. During the CHS studies, the inflammatory response was also assessed by erythema and edema evaluation 24 h after the single SSR exposures. Erythema was graded with a standard scale ranging from 0 (no erythema) to 4 (very intense erythema) with a value of 1 for the reaction corresponding to 1 MED. The MED was the dose that produced a uniform pale pink color with clearly defined borders 24 h postirradiation. Edema was assessed by measuring the skin-fold thickness (mean of three different dorsal sites) of each mouse with a spring-loaded micrometer accurate to 0.01 mm (Käfler, Germany). The mean dorsal skin-fold thickness for each mouse and for each experimental group was calculated and the minimal edema dose (MEdD) statistically determined. This was the lowest dose at which the skin-fold thickness became significantly greater than that of unirradiated untreated controls as detailed in the statistical methods section. Five days after SSR exposure, each group of 20 mice was divided in two subgroups of 10. The mice were either treated with 50 μl of acetone or sensitized with 50 μl acetone containing 0.3% vol/vol DNFB (Sigma, St Louis, MO) on nonirradiated ventral skin. The application was repeated 24 h later. Six days after the last ventral application, 5 μl of acetone was applied to the left ear and 5 μl of acetone containing 0.2% vol/vol DNFB to the right ear of each mouse. Twenty-four hours after this challenge, the thickness of left (acetone treated) ear and right (DNFB challenged) ear were measured by micrometer on lightly anesthetized animals. The difference in ear thickness between right and left ears represented the CHS response, expressed in mm × 10-2, for each mouse. The percentage of suppression of CHS response was determined for each SSR-treated mouse using the following equation and averaged for each irradiated group %suppression=[1-(ESssr/EScon)]×100 where ESssr is the individual ear swelling (elicitation) response for SSR treated (± topical treatment) DNFB sensitized mice and EScon is the mean ear swelling (elicitation) response for non-SSR and nontopically treated but sensitized mice. For a given treatment, the MISD was defined as the dose at which the mean ear swelling became significantly different (p < 0.05) from that of unirradiated unexposed sensitized controls. The dose that induced 50% suppression of CHS response (ISD50), compared with the control response, was assessed graphically from the dose–response curves. In addition to the mouse and human SPF and human UVA-PF, five different protection factors were determined from the dose–response curves generated from the mouse CHS experiments. These were: (i) the erythema protection factor (EryPF) obtained by exposing each mouse of a given group to a single SSR dose (unlike the SPF determination already described in which each mouse receives a series of SSR doses via a template); (ii) the edema protection factor (EdPF); (iii) the immune protection factor (IPF) that is based on MISD ratio with and without sunscreen; (iv) the 50% immune protection factor (IPF50) that is based on ISD50 ratio with and without sunscreen; and (v) the global immune protection factor (IPFG). A global protection factor is not based on a specific level of biologic response, e.g., minimal or 50% maximal, but instead is a measure of protection across the entire SSR dose–response range. Human SPF and UVA-PF were compared by a nonparametric Wilcoxon signed ranks test. Mouse SPF were compared by a nonparametric Wilcoxon–Mann–Whitney test. For each response of the CHS study (i.e., erythema, edema, and suppression of CHS), variance–covariance analysis, with treatment as the factor and SSR dose as covariate, was used to compare the dose–response results. Analysis of variance, followed byTukey, 1994Tukey J.W. The problem of multiple comparisons, Mimeographed Notes, Princeton University, Reprinted.in: Braun H.I. The Collected Works Of John W. Tukey, Vol VIII–-Multiple Comparisons: 1948-83. Chapman & Hall, New-York1994Google Scholar procedure, was performed to compare means between groups. TheDunnett, 1955Dunnett C.W. A multiple comparison procedure for comparing several treatments with a control.J Am Stat Assoc. 1955; 50: 1096-1121Crossref Scopus (4641) Google Scholar test was used to determine the minimum significant difference between sensitized unexposed untreated groups and sensitized exposed groups, for ear swelling. Then, the associated MISD observed for each treatment, were graphically determined from the respective dose–response curves of CHS inhibition. IPF was thus estimated. For each treatment, from the curve representing CHS inhibition as a function of SSR dose, ISD50 was assessed graphically as the dose inducing a 50% inhibition of CHS of control group. IPF50 was then calculated, as the ratio of ISD50. In addition, nonlinear regressions were generated to determine global protection factors from the dose–response curves. EryPF and EdPF were estimated assuming a global model: γ=f[D/PF] in which y is the level of erythema or edema response at D, a given SSR dose, PF is EryPF or EdPF (with a value of 1 for the nonsunscreen group), and f a linear function fitting the dose–response curves. IPFG was calculated similarly from the overall SSR dose range using the same global model, in which y is the percentage CHS inhibition, PF is IPFG, with a value of 1 for the nonsunscreen group and f a sigmoidal function. Further details of this calculation are given in the Appendix. All tests and comparisons were performed at a 5% two-sided significance level. All these analyses were performed using SAS software release 6.12 (SAS Institute Inc., Cary, NC). Table 1 and Table 2 show that the SPF of the two products were very similar (7–8) in humans (p = 0.07) or mice (p = 0.6). The
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