Air pollution‐induced tanning of human skin*

British Journal of DermatologyVolume 185, Issue 5 p. 1026-1034 Translational ResearchOpen Access Air pollution-induced tanning of human skin* S. Grether-Beck, S. Grether-Beck orcid.org/0000-0002-8318-2625 IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: Conceptualization (lead), Formal analysis (equal), Software (equal), Visualization (equal), Writing - original draft (equal)Search for more papers by this authorI. Felsner, I. Felsner IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: ​Investigation (equal)Search for more papers by this authorH. Brenden, H. Brenden IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: ​Investigation (equal)Search for more papers by this authorA. Marini, A. Marini IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: Conceptualization (equal), ​Investigation (equal)Search for more papers by this authorT. Jaenicke, T. Jaenicke IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: ​Investigation (equal), Project administration (lead)Search for more papers by this authorN. Aue, N. Aue IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: ​Investigation (supporting)Search for more papers by this authorT. Welss, T. Welss Henkel Beauty Care, Düsseldorf, Germany Contribution: Conceptualization (supporting)Search for more papers by this authorI. Uthe, I. Uthe IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: ​Investigation (supporting)Search for more papers by this authorJ. Krutmann, Corresponding Author J. Krutmann jean.krutmann@iuf-duesseldorf.de orcid.org/0000-0001-8433-1517 IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany Correspondence Jean Krutmann. Email: jean.krutmann@iuf-duesseldorf.de Contribution: Conceptualization (equal), Funding acquisition (lead), Resources (lead), Writing - original draft (equal)Search for more papers by this author S. Grether-Beck, S. Grether-Beck orcid.org/0000-0002-8318-2625 IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: Conceptualization (lead), Formal analysis (equal), Software (equal), Visualization (equal), Writing - original draft (equal)Search for more papers by this authorI. Felsner, I. Felsner IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: ​Investigation (equal)Search for more papers by this authorH. Brenden, H. Brenden IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: ​Investigation (equal)Search for more papers by this authorA. Marini, A. Marini IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: Conceptualization (equal), ​Investigation (equal)Search for more papers by this authorT. Jaenicke, T. Jaenicke IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: ​Investigation (equal), Project administration (lead)Search for more papers by this authorN. Aue, N. Aue IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: ​Investigation (supporting)Search for more papers by this authorT. Welss, T. Welss Henkel Beauty Care, Düsseldorf, Germany Contribution: Conceptualization (supporting)Search for more papers by this authorI. Uthe, I. Uthe IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Contribution: ​Investigation (supporting)Search for more papers by this authorJ. Krutmann, Corresponding Author J. Krutmann jean.krutmann@iuf-duesseldorf.de orcid.org/0000-0001-8433-1517 IUF – Leibniz Research Institute of Environmental Medicine, Düsseldorf, Germany Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany Correspondence Jean Krutmann. Email: jean.krutmann@iuf-duesseldorf.de Contribution: Conceptualization (equal), Funding acquisition (lead), Resources (lead), Writing - original draft (equal)Search for more papers by this author First published: 15 May 2021 https://doi.org/10.1111/bjd.20483Citations: 2 †Funding sources The development of the ex␣vivo and in vivo models was supported by an unrestricted grant from Henkel AG & Co. KGaA, Düsseldorf. Germany. The ex␣vivo and in␣vivo studies with CE Ferulic® were partially supported by a grant from SkinCeuticals Inc., New York, NY, USA. ‡Conflicts of interest T.W. is an employee of Henkel AG & Co. KGaA. J.K. serves as an advisor for Henkel AG. §Data availability statement All data are available in the Supporting Information. * Plain language summary available online AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary Background Melanism is more frequent in animals living in polluted areas on urban–industrial sites. Given that an increasing number of people are exposed to elevated air pollution levels, it is possible that environmental pollutants affect melanogenesis in human skin. Epidemiological studies have shown that exposure to traffic-related air pollutants such as diesel exhaust particles (DEP) is associated with more clinical signs of hyperpigmentation. However, mechanistic evidence linking DEP exposure to pigmentation has been elusive. Objectives To develop an ex␣vivo skin model to allow for repetitive topical application of relevant ambient DEP, and to provide proof of concept in humans. Methods We measured skin pigmentation, melanin and pigmentation-associated gene expression, and evaluated oxidative stress. Results Repetitive exposure of ex␣vivo skin to DEP at nontoxic concentrations increased skin pigmentation. This increase was visible to the naked eye, time dependent, and associated with an increase in melanin content and the transcription of genes involved in de novo melanin synthesis. Similarly, in healthy participants (n = 76), repetitive topical application of DEP at nontoxic concentrations increased skin pigmentation. DEP-induced pigmentation was mediated by an oxidative stress response. After the application of DEP, epidermal antioxidants were depleted, lipid peroxidation and oxidative DNA damage were enhanced, and in a vehicle-controlled, double-blind clinical study DEP-induced pigmentation was prevented by the topical application of an antioxidant mixture. Conclusions Similar to solar radiation, air pollutants cause skin tanning. As eumelanin is an antioxidant, it is proposed that this response serves to protect human skin against air pollution-induced oxidative stress. Melanism has been found to be more common in several animal species (e.g. pigeons and moths) living in urban–industrial environments.1, 2 In the sea snake Emydocephalus annulatus, it has been shown that melanic snakes occur more frequently if they live in polluted water vs. unpolluted environments and that increased melanin in the snake’s skin serves to bind pollutant-associated trace elements, which are then disposed of by skin sloughing.3 These observations link pollution to skin pigmentation and indicate that pigmentation represents a defence mechanism against environmental toxicants. An increasing number of people live in heavily polluted urban environments. Therefore, we decided to investigate whether exposure to air pollutants increases human skin pigmentation. Epidemiological studies have found significant associations between the clinical signs of skin hyperpigmentation and exposure to traffic-related air pollutants. The strongest associations (with odds ratios > 1·2) exist between traffic-related soot containing diesel exhaust particles (DEP) and facial pigment spots (solar or senile lentigines), in which melanin synthesis is increased.4-6 However, mechanistic evidence providing a direct link between exposure to DEP and skin pigmentation has been elusive. We developed a model system to allow us to assess directly if and how DEP can modulate pigmentation of human skin. We reasoned that this model should meet the following prerequisites: (i) it should allow for topical application of DEPs as there is currently no evidence to suggest that air pollution-related skin effects are mediated by a systemic mechanism (which, theoretically, might be initiated by lung inflammation as a consequence of DEP inhalation); (ii) DEPs used in this model should be of known ambient relevance and their toxicology well characterized; and (iii) in order to be as close as possible to physiologically relevant exposure scenarios, DEPs should be applied repetitively at nontoxic concentrations. By following these preconditions, we succeeded in developing a robust, standardized ex␣vivo human skin model, which – in a second step – was validated in␣vivo in healthy human skin and named the ‘Düsseldorf pollution patch test’ (DPPT). The results obtained with these two models were congruent and showed that the topical application of nontoxic concentrations of ambient relevant DEPs increases the pigmentation of human skin by inducing melanogenesis. Mechanistic studies revealed that DEP-induced melanogenesis involved the generation of oxidative stress. Materials and methods Preparation of ex␣vivo skin Ex vivo human skin was obtained from aesthetic surgery (‘tummy tuck’) after informed consent and approval of the local ethics committee of the Heinrich-Heine University, Düsseldorf, Germany (Appendix S1; see Supporting Information). Standard reference material 1650b was obtained from the National Institute of Standards and Technology (NIST; Gaithersburg, MD, USA)7 and used as ambient relevant DEP.8 Stock suspensions with a concentration of 1000 μg mL–1 were prepared in Dulbecco’s phosphate-buffered saline (Thermo Fisher Scientific, Waltham, MA, USA). The suspensions were ultrasonicated on ice twice for 30 s (Branson Sonifier B-12; Emerson, Danbury, CT, USA) and vortexed. Final concentrations of 6 µg cm–² in 6·8 µL and 40 µg cm–2 in 150 µL were applied to ex␣vivo skin or to in␣vivo skin on filter paper discs in 18-mm Finn Chambers on Scanpor (SmartPractice, Dr. Ebeling & Assoc., Hamburg, Germany). As the DEP solution was dark and could have potentially contributed to any change in skin colour, skin pieces in the ex␣vivo experiments were thoroughly rinsed (Figure 1a), and in the in␣vivo experiments, to remove any residual DEP from the skin surface, which might otherwise create the impression of increased skin pigmentation, DEP-treated skin areas were cleaned with a swab. Figure 1Open in figure viewerPowerPoint Repetitive, topical application of diesel exhaust particles (DEPs) induces melanogenesis in ex␣vivo human skin models. (a) Scheme␣of repetitive DEP application. DEP (6 µg cm–2) was topically applied at days 1, 4 and 7, as indicated. ITA, individual topology angle; PBS, phosphate-buffered saline. (b) Repetitive application of DEP did not affect the viability of human skin ex␣vivo [n = 3; anova on ranks, Tukey’s test, median (twenty-fifth and seventy-fifth percentiles)]. *P < 0·05 vs. time-matched untreated control. (c) DEP-induced darkening of ex␣vivo skin. Photographs show a representative ex␣vivo skin model experiment with time-dependent skin colour darkening after DEP treatment. *On day 1 (D1), photos were taken prior to the application of DEP. (d) Chromametric assessment of skin darkening in DEP-treated ex␣vivo skin. DEP treatment caused a time-dependent decrease in individual topology angle (ΔITA°), indicating skin darkening [n = 13; anova on ranks Student–Newman–Keuls (SNK), median (fifth and ninety-fifth percentiles)]. *P < 0·001 vs. time-matched untreated control. (e) Fontana–Masson staining of ex␣vivo human skin models. Melanin is increased in DEP-treated skin vs. corresponding control skin after three DEP treatments (320-fold magnification). Arrows indicate black melanin granules. (f) Melanin content in human ex␣vivo skin models. Normalized melanin content was significantly increased after DEP treatment (n = 4; t-test, SNK, boxplot, fifth and ninety-fifth percentiles]. The red line indicates the mean. (g) Tyrosinase mRNA expression in ex␣vivo human skin models. TYR mRNA expression was significantly increased after two and three DEP treatments [n = 13; anova on ranks, Dunn’s test, median (fifth and ninety-fifth percentiles)]. *P < 0·05 vs. time-matched controls. (h) Tyrosinase protein expression in ex␣vivo human skin models. Tyrosinase staining was increased vs. a corresponding control skin after three treatments with DEP (320-fold magnification, one representative experiment of three). Arrows indicate brownish tyrosinase expression. (i) Transcriptional expression of melanogenesis-related genes in human ex␣vivo skin models. Expression of TRP1, POMC and EDN1 mRNA was significantly increased after three DEP treatments [n = 13; anova on ranks, Dunn’s test, median (fifth and ninety-fifth percentiles)]. *P < 0·05 vs. time-matched control skin (dashed vertical line). Untr, untreated. Supporting data for the figure are available in Appendix S2 (see Supporting Information) Viability test The cytotoxicity of DEP was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in skin explants after double and triple applications of DEPs after 6 and 9 days, respectively. The percentage cell viability for each tissue sample was expressed relative to a negative control as: 100 × (mean ODDEP treated)/(mean ODuntreated), where OD refers to the optical density. By default, the viability of the untreated control samples was set at 100%. For each experiment the mean (SD) cell viability of the three tissue samples was calculated. Determination of skin colour Skin colour was assessed by chromametry ex␣vivo and in␣vivo. All measurements were done by the same investigator in an air-conditioned examination room (in␣vivo) or in a biological safety cabinet (ex␣vivo), and were carried out according to the Efficacy Measurement of Cosmetics and other Topical Products (EEMCO) guidelines.9 A tristimulus colorimeter CR-400 (Konica Minolta, Tokyo, Japan) was used, as described in Appendix S1. In addition, the melanin index of in␣vivo skin was assessed with a DSM III Color Meter (Cortex Technology, Hadsund, Denmark), which is an index-calculating, narrowband, simple reflectance spectrophotometric instrument composed of light-emitting diodes, which emit light in two narrow wavebands corresponding to red and green.10 Antioxidant levels Carotenoids such as β-carotene or lycopene served as marker substances for the antioxidant network of human skin.11 They were measured (as previously described) with multiple spatially resolved reflection spectroscopy (Biozoom Skin Scanner; Biozoom Services GmbH, Kassel, Germany)12 by their absorption in human skin. For full details, see Appendix S1. Photographs The ex␣vivo skin samples were photographed using a digital microscope system DMS1000 (Leica Microsystems, Wetzlar, Germany) with a constant camera setting (complementary metal oxide semiconductor camera) within a single experiment. Melanin quantification Quantification of the melanin content of ex␣vivo skin samples was achieved using the SOLVABLE™ melanin assay, as described by Bachelor et␣al. in a modification of the method of Costin and Raabe.13, 14 Melanin quantification was done in ex␣vivo skin models, which were harvested at days 6 and 9 of treatment. Data were obtained as ng melanin per mg protein and untreated samples were set equal to one. Gene expression was studied after double and triple application of DEPs and incubation for 6 and 9 days, respectively. For details and primers, see Table S1 (see Supporting Information). Gene expression was normalized to 18S rRNA, and differences between samples were quantified based on the ΔΔCt method.15 In vivo studies The Düsseldorf pollution patch test (study 1) This study were conducted at the Leibniz Research Institute for Environmental Medicine (IUF) between 5 March 2018 and 1 August 2019 in accordance with the principles of the Declaration of Helsinki, and adherence to the International Conference on Harmonisation’s Good Clinical Practice (ICH GCP) guidelines, as applicable. The studies were approved by the local ethics committee of the Heinrich-Heine University, Düsseldorf, Germany (registration ID 2016014913). Seventy-six healthy participants [63 females and 13 males; mean age 46·24 (10·92) years (range 22–63)], as assessed by their medical histories and standard medical examinations, were enrolled. Sixty-four patients had Fitzpatrick skin type II and 12 had Fitzpatrick skin type III. See Appendix S1 for the exclusion criteria. For pollution patch testing on day 1, two fields measuring approximately 5 × 5 cm were marked on skin for either DEP or phosphate-buffered saline (PBS) application in Finn chambers, which were covered with occlusive patches. On day 4, the patches were removed and the skin was cleaned with a dry cellulose swab. After a recovery period of 1 h, skin measurements were performed and new Finn chambers were used and covered with occlusive patches. These procedures were repeated twice (Figure S1; see Supporting Information). In all 76 participants, skin colour was measured by chromametry and by determination of the melanin index. In 20 participants, 4-mm punch biopsies were obtained for further analysis. Vehicle-controlled, double-blind clinical study (study 2) A randomized, vehicle-controlled, blinded, intraindividual comparative study to assess the efficacy of a commercially available cosmetic product to modulate DEP-induced skin pigmentation by means of the DPPT was conducted at IUF between 2 September 2019 and 6 December 2019. This study also followed the principles of the Declaration of Helsinki, and ICH GCP guidelines were adhered to, as applicable. The study was approved by the local ethics committee of Heinrich-Heine University, Düsseldorf, Germany (registration ID 2019-459). Twenty healthy participants [16 females and four males; mean age 44·35 (13·01) years (range 25–64)], as assessed by their medical histories and standard medical examinations, were enrolled. All had Fitzpatrick skin type II. Exclusion criteria were the same as for study 1 (see Appendix S1). Study 2 consisted of a screening and a baseline visit, six interim visits and a final visit within a timeframe of 14 days (Figure S2; see Supporting Information). Four skin areas of 25 cm2 were identified on the backs of the participants. The DPPT was carried out as described above. There were four test areas per volunteer: PBS only, DEP only, vehicle + DEP and CE Ferulic® (CEF) + DEP. Vehicle and CEF were applied at a dose of 2 mg cm–2. For randomization, the treatment areas were allocated according to an online randomization program (https://www.sealedenvelope.com/simple-randomiser/v1/). For the composition of the vehicle, see Appendix S1. Vehicle and active products were obtained from SkinCeuticals (New York, NY, USA). As the primary endpoint, changes in skin colour were measured by chromametry and determination of the melanin index. Immunohistochemical staining Full-thickness skin specimens from study participants or from ex␣vivo human skin were fixed overnight in ROTI Histofix 4% (Carl Roth, Karlsruhe, Germany), embedded in paraffin and sectioned at 7 µm. For details of the kits and antibodies used, see Appendix S1. Statistical analysis SigmaPlot 14·0 (Systat Software, Erkrath, Germany) was used for statistical analysis and graph design. Normality of the data was tested using the Shapiro–Wilk test. For comparison of significant differences, a paired test, Student’s t-test or anova was used. In case of the failure of normality, the corresponding rank test, Wilcoxon signed rank test, Mann–Whitney rank sum test or Kruskal–Wallis one-way anova on ranks were employed. For the post-hoc analysis, the Student–Newman–Keuls or Tukey tests were used. A probability level of P < 0·05 was considered to be statistically significant. Data are presented as bar charts, box plots or point plots with median and/or mean and corresponding percentiles, as indicated in the figure legends. Results Diesel exhaust particles induce melanogenesis in ex␣vivo human skin models We first assessed whether topical application of DEPs would affect the pigmentation of ex␣vivo human skin models.16 Ambient relevant DEPs were repetitively applied to the surface of ex␣vivo human skin,8 which was obtained from abdominal reduction surgeries in healthy individuals with white skin (Figure 1a). As shown in Figure 1(b), DEP concentrations up to 6 µg cm–2 did not decrease cell viability, even when DEPs were applied repetitively (i.e. three times) over a 9-day culture period. Therefore, this concentration was used in subsequent experiments. DEP treatment of ex␣vivo skin models (n = 13) increased skin colour in a time-dependent manner. This tanning response was visible with the naked eye (Figure 1c), and quantifiable by chromametry, as shown by a significant decrease in individual topology angles (ΔITA°)17 (Figure 1d). The increase in skin colour was associated with an increase in black melanin, as shown by Fontana–Masson staining in DEP-treated skin models (Figure 1e), and by measuring total melanin content (Figure 1f). In addition, DEP treatment increased the expression of tyrosinase (encoded by TYR), which catalyses the rate-limiting step in melanogenesis in human skin,18 both at the mRNA (Figure 1g) and protein levels (Figure 1h). Other melanogenic genes whose transcriptional expression was upregulated in DEP-treated skin models included POMC, EDN1 and TRP1 (Figure 1i). These results demonstrate that repetitive treatments with ambient relevant DEP at nontoxic concentrations increased melanogenesis in human ex␣vivo skin models. Diesel exhaust particles induce a tanning response in␣vivo in human skin In order to assess the in␣vivo relevance of these findings, we next transferred the ex␣vivo protocol (Figure 1a) to the in␣vivo situation (Figure S1). Specifically, DEPs dissolved in PBS were topically applied to back skin of healthy human volunteers [n = 76; 63 females and 13 males (mean age 46·51 (10·09)] with Fitzpatrick skin type II (n = 64) or III (n = 12) by means of Finn chambers. As shown in Figure 2(a), repetitive application (i.e. three applications within a 9-day testing period) increased constitutive skin pigmentation in a time-dependent manner. In DEP-treated skin, but not in PBS-treated control skin areas, the melanin index increased (as measured by a narrowband spectrophotometer) and ΔITA° values decreased (Figure 2b; as measured with a tristimulus colorimeter). In biopsies taken from the DEP-treated skin of 20 volunteers, we observed an increase in melanin staining and in the transcriptional expression of genes important for de novo melanin synthesis such as POMC, EDN1 and MITF, as well as TYR, TRP1, MLANA and PMEL (Figure 2c, d). These results show that repetitive topical treatments with ambient relevant DEPs at nontoxic concentrations increased melanogenesis in human skin in␣vivo. The corresponding test protocol proved to be robust when used in a large number of volunteers (n = 76) and was termed the DPPT. Figure 2Open in figure viewerPowerPoint The Duesseldorf pollution patch test. Repetitive topical application of diesel exhaust particles (DEPs) induced skin pigmentation in␣vivo in human skin. Human skin was repetitively exposed in␣vivo to DEP, as described in the ‘Materials and Methods’. DEPs (40 µg cm–2) were dissolved in phosphate-buffered saline (PBS) and topically applied in Finn chambers on days 1, 4 and 7. DEP treatment induced a tanning response in␣vivo human skin (n = 76). (a) The melanin index significantly increased (indicating skin darkening) in DEP-treated skin areas (anova and Tukey’s test). The red line indicates the mean and the black line the median (fifth and ninety-fifth percentiles). *P < 0·001 vs. time-matched PBS-treated controls. (b) Individual topology angle (ITA°) values significantly decreased in DEP-treated skin areas [anova on rank Student–Newman–Keuls, median (fifth and ninety-five percentiles)]. *P < 0·001 vs. time-matched PBS-treated controls. (c, d) Analysis of biopsies obtained from 20 participants 24 h after the third DEP application. (c) Fontana–Masson staining of human skin sections. Melanin deposition was increased after three DEP treatments vs. PBS-treated control skin (320-fold magnification). Arrows indicate black melanin granules. (d) mRNA expression of melanogenesis-related genes in human skin. After three DEP treatments, mRNA expression of POMC, EDN1, MITF, TYR, DCT, MLANA, PMEL and TRP1 was increased vs. PBS-treated control skin [mean (paired t-test) or median (Wilcoxon signed rank test) PBS vs. DEP]. *P < 0·05 vs. PBS. Supporting data for the figure are available in Appendix S2 (see Supporting Information) Diesel exhaust particle-induced skin pigmentation is mediated by oxidative stress We next studied the signalling pathways underlying DEP-induced skin pigmentation. We found that DEP-induced skin pigmentation was mediated by an oxidative stress response.19 Accordingly, in the ex␣vivo models, topical application of DEP increased the formation of 4-hydroxynonenal adducts [visible as orange/brownish colouring; Figure S3 (see Supporting Information)] and of oxidative DNA damage (shown here by means of anti-8-hydroxyguanosine antibody staining; Figure 3a). In the in␣vivo model, resonance Raman spectroscopy revealed that epidermal carotenoids, which are regarded as markers for the skin’s antioxidant capacity,20 were slightly, but significantly, depleted in human epidermis after the in␣vivo application of DEP (Figure 3b). DEP-induced oxidative stress was of functional relevance for skin pigmentation as it was significantly reduced in ex␣vivo skin models (n = 7) if the surface had been pretreated with a commercially available cosmetic product containing three antioxidants (vitamin C, vitamin E and CEF, as the only active ingredients) and which has previously been shown to prevent ultraviolet radiation (UVR)-induced oxidative stress responses in skin (Figure 3c–e).21, 22 These ex␣vivo results were corroborated and extended in a vehicle-controlled, double-blind clinical in␣vivo study of 20 human volunteers (16 females and our males; mean age 43·62 (13·12) years) with Fitzpatrick skin type II. Pretreatment with the antioxidant cocktail, but not with the vehicle control, significantly inhibited DEP-induced pigmentation of human skin (Figure 3e). Figure 3Open in figure viewerPowerPoint Topical application of diesel exhaust particles (DEPs) induces an oxidative stress response in human skin ex␣vivo and in␣vivo. Topical application of DEP induced oxidative stress in human skin, which mediated the DEP-induced skin-tanning response. (a) DEP treatment increased oxidative DNA damage in ex␣vivo human skin. (a) Oxidative DNA damage was detected by 8-hydroxyguanosine staining. Arrows indicate brownish 8OHdG staining. (b) The antioxidant capacity of human skin was reduced in␣vivo after the topical application of DEP. Skin carotenoid content, which was assessed by resonance Raman spectroscopy in␣vivo in 76 participants, was significantly reduced in DEP-treated skin areas compared with phosphate-buffered saline (PBS)-treated control skin, median (Wilcoxon signed rank test). *P < 0·05 (PBS vs. DEP). (c–e) Antioxidant treatment inhibited DEP-induced skin tanning ex␣vivo and in␣vivo. (c) DEP induced a decrease in individual topology angle (ΔITA) values and was inhibited in ex␣vivo skin (n = 7) by antioxidants (anova on ranks, Student–Newman–Keuls test). *P < 0·05 vs. untreated or as indicated. (d) Photographs of a representative experiment depicting inhibition of DEP-induced skin tanning in ex␣vivo human skin by topical treatment with a cosmetic product containing the antioxidants vitamin C, vitamin E and CE Ferulic® (CEF). (e) DEP-induced increase of the melanin index was significantly inhibited in human skin in␣vivo (n = 20) by topical treatment with a cosmetic preparation containing the antioxidants vitamin C, vitamin E and CEF compared with vehicle-treated control skin (anova Tukey). P < 0·05 vs. PBS or as indicated. AO, antioxidants; ns, not significant; Untr, untreated. Supporting data for the figure are available in Appendix S2 (see Supporting Information) Discussion Repetitive topical application of noncytotoxic concentrations of ambient relevant DEPs cause a tanning response in human skin. This conclusion is based on results obtained ex␣vivo (Figure 1c, d, f) and in␣vivo (Figure 2a, b). This study thus identified traffic-related air pollution as a previously unrecognized environmental factor which, similar to natural sunlight, is capable of increasing melanogenesis in human skin. Our studies extend previous observations in other species, which have suggested that living in polluted, urban environments may increase skin pigmentation.1-3 They might also provide a possible mechanistic explanation of previously published epidemiological studies that reported significant associations between exposure to traffic-related air pollutants and facial pigment spots,4-6 and of darker skin colour in white and East Asian populations.6 The DEPs used in