Xiaomin Zhao, Xin Qu

Shanghai Technical Center, Ashland China Inc., China

Air pollution includes indoor and outdoor pollution.The harmful impact of outdoor pollution because of gas and industry absorbs a lot of concern. Actually, indoor pollution can be similarly or more harmful. In China, more than 70%of the households use solid fuels for cooking and heating.According to EPA (Environmental Protection Agency of USA), the indoor environment can be two to five times more toxic than the outdoor environment if with indoor open fire combustion or tobacco smoking. The combustion of coal and biomass indoors emits a substantial amount of toxic pollutants including particulate matter (PM), polycylic aromatic hydrocarbons (PAHs), carbon monoxide, nitrogen oxides and sulfur dioxide. Various studies showed that exposure to indoor air pollution causes pronounced skin aging.[1]Obviously, indoor pollution is harmful to children,especially to babies. Outdoor air pollutants everone concerns include particulate matter, volatile organic compounds, ozone, nitrogen dioxide and sulphur dioxide.Studies showed with AQI (Air Quality Index) increase, the incidence of various skin disease increase which related skin barrier dysfunctions.[2,3]Outdoor air pollutants also cause external skin aging.[3-6]Although the components between outdoor and indoor pollutants are some kind of different, they exert similar detrimental effect on skin.In this context, the mechanisms were discussed for how major pollutants including PM and PAHs impact on skin,then the study method of skin damage by pollutants were also discussed.

Mechanisms of pollution effects on skin

Besides respiratory organs, skin is an interface between the body and the surrounding atmosphere and is therefore the primary contact for ambient pollutants. Obviously, the protective ability of the skin in not unlimited, and problems arise when an abnormal exposure to environmental stressors exceeds the skin’s normal defensive potential.[4]Current research suggests that each individual air pollutant most probably has a specific, toxic action on the skin. For intact skin, ozone mainly acts on the stratum corneum.In contrast, organic compounds present on the surface of PM may penetrate into the skin and have direct effects on viable skin cells such us keratinocytes and melanocytes.For barrier deficient skin, particles itself might penetrate and cause oxidative stress and skin inflammation.[5]

AhR (aryl hydrocarbon receptor) activation pathway

PAHs are one of the most widespread organic pollutants.The main source of atmospheric PAH is residual of wood burning. It is also found in automobile exhaust fumes(especially the exhaust gases from diesel engines), and in all smoke resulting from the combustion of organic material (including cigarette smoke).[6]Notorious benzo(a) pyrene (BaP) and dioxin are the members of the PAHs family. AhR, a ligand-activated transcription factor, is an important regulator of drug metabolism that is activated by a broad spectrum of low-molecular-weight compounds,including dioxins, other PAHs, plant polyphenols, and tryptophan photoproducts.[7]AhR targets cytochrome P450 (CYP) 1A1, CYP1A2, and CYP1B1. Several studies on rodents have shown that, if not all, most of toxic effects caused by dioxin are mediated by the AhR pathway.[7]But overexpression of constitutively actived AhR was associated with inflammatory skin lesions. An increase in CYP activity leads to reactive oxygen species formation and CYP1 enzymes are critical for chemical induced skin carcinogenesis.[8,9]In the skin, experimental evidence from keratinocyte cultures exposed to Asian dust storm particles increased AhR expression and upregulated proinflammatory cytokines.[10]Also, AhR activation was found to induce transcriptional expression of genes which are of known functional relevance for both wrinkle formation and pigment spot formation.[5]

Tobacco-smoke similar mechanism

As shown in Table 1, tobacco smoke is a complex mixture of pollutants similarly to air pollution. For example, they both contain carbon monoxide, PAHs，heavy metals and PM. Tobacco smoke induces skin aging, mainly the risk of developing wrinkles.[11]And it has been shown that tobacco smoke extract affects collagen metabolism in cultured skin fibroblasts.[12]By exposing tobacco smoke extract in cultured human skin fibroblasts, the production of the collagen precursors procollagen types I and III was decreased significantly in the supernatants, whereas the matrix-metalloproteinases (MMPs)-1 and MMP-3 were induced, but the expression of MMPs inhibitors remained unchanged. In total, after tobacco smoke extract exposure the balance is shifted to collagen degradation from collagen production. Further, the tobacco smoke extract also induces the non-functional latent form of transforming growth factor-(TGF-β) in supernatants of cultured skin fibroblasts.[13]This results in decreased synthesis of extracellular matrix proteins. Moreover, PAHs included in tobacco smoke can trigger AhR signaling pathway.

Oxidation pathway

Oxidative cascade pathway is main mechanism how air pollutants works on the skin. Some indoor air pollutants, such as tobacco smoke will firstly attack the unsaturated skin lipids by oxidation. Polyunsaturated fatty acids, glycolipids, phospholipids, and cholesterol are well-known targets of damaging. Under low lipid peroxidation rates (subtoxic conditions), the cells stimulate their maintenance and survival through constitutive antioxidants defense systems, by contrast, under medium or high lipid peroxidation rates, the extrent of oxidative damage overwhelms repair capacity, and the cells induce apoptosis or necrosis, both processes eventually lead to molecular cell damage which may facilitate development of accelerated aging or even pathological states.[14-16]And during lipid peroxidation, a wide variety of oxidation products will be produces, different aldehydes as secondary products are among of them. Malondialdehyde(MDA), propanal, hexanal and 4-hydroxynonenal (4-HNE) have been extensively studied. MDA appears to be the most mutagenic product, whereas 4-HNE is the most toxic.[17]MDA is an important biomarker to determine the level of lipid peroxidation. The lipid peroxidation-derived aldehydes can easily diffuse across cell membranes and can covalently modify any protein or DNA in the cytoplasm and nucleus, far from their site of origin. This will result in protein oxidation, carbonylation,and forming Advanced Glycation End products (AGEs),then finally leads to accelerating aging.[18]Because of the electrophilicity of these aldehydes, they are high reactivity.A recent review shows a list of up to thirty-three proteins known to be modified by MDA and including enzymatic proteins, carrier proteins, cytoskeletal proteins, and mitochondrial and antioxidant proteins.[19]And the protein damage, especially protein carbonylation can result in decreasing water-holding capacity of stratum corneum, and changing skin transparency.[20-22]

Besides, another transcription factor—Nrf2 (nuclear factor erythroid-2 related factor 2) is the master regulator of cellular antioxidant response. Under normal or unstressed conditions, Nrf2 is kept in the cytoplasm by a cluster of proteins that degrade it quickly. Under oxidative stress, Nrf2 is not degraded, but instead travels to the nucleus where it binds to DNA promoter and initiates transcription of antioxidative genes and their proteins. In this mode, Nrf2 finished its cytoprotection function.

As mentioned above, dioxin, polycylic aromatic hydrocarbons activate AhR pathway, this results in the CYP-mediated formation of reactive oxygen species (ROS)and elctrophilic metabolites. And this create oxidative stress and activated Nrf2 pathway. The interaction between AhR and Nrf2 signalling is probably a crucial enhancer of cytoprotection against toxic by-products of the AhR-dependent detoxification process. Some study proposed that compound both attenuate ligand-activated AhR signaling and stimulate the Nrf2 system are probably the most potent candidates to prevent the severe health effects provoked by environmental noxae.[23]

Epigenetics and air pollutants

Epigenetics investigates heritable changes in gene expression occurring without changes in DNA sequence.As shown in Figure 1, because of different environment,the human or flower physical appearance changed based on epigenetic.[24]Several epigenetic mechanisms,including DNA methylation, histone modification, and microRNA (miRNA) expression, can change genome function under exogenous influence. Several studies have established an association between DNA methylation and environmental metals, including nickel, cadmium,lead, and particularly arsenic.[25-27]PAHs, PM and other pollutants are associated with epigenetics changes. PAHs are associated with DNA methylation levels of repeated elements as well as changes in levels of gene-specific DNA methylation of the repeated elements, the long interspersed nucleotide element-1 (LINE-1) and the short interspersed nucleotide element (Alu) are the most studied in epidemiological studies. Investigations of PM2.5 exposure and DNA methylation have also focused on candidate genes of interest, primarily in the context of cardiovascular or respiratory disease. For example, several studies have evaluated the effect of PM on regulation of the inducible nitric oxide gene (iNOS), a gene responsible for production of nitric oxide, which is important for both cardiovascular and respiratory health.[28]

In short, to prevent skin from pollution damage, it’s necessary to enhance skin barrier function, to attenuate AhR signaling and increase Nrf2 activity, to increase the capability of cell detoxifying, to neutralize ROS, to repair the wrinkles, facial sagging, skin pigmentation by different biofunctional ingredient or peptides. In the following context, the skin damage by pollution were studied.

Figure 1. Human or flower physical appearance differences based on epigenetics

Study methods on skin damage by pollution

The study methods discussion will cover the evaluation based on cell level, through human reconstructed 3D skin to in vivo methods.

Cellular level experiment

As mentioned above, air pollutants like diesel exhaust particles, particulate materials collected from ambient environment, etc., were added in the human keratinocyte or melanocyte cultures, and was cultivated for a period of time. And then the AhR expression status, inflammatory factors or other signaling proteins were determined. Based on this, the cell damage and how the pollutants act on cells can be analyzed. In the French lab, different concentration of PM10 or PM2.5 purchased from Institute for reference material and measurement were exposed to the keratinocytes cultures for 48 h, which carried many kinds of PAHs, heavy metals and dioxins and so on, and then the release of lactate dehydrogenase (LDH) to represent the cell stress was tested.If biofunctional ingredients were added as treated side, the ability that the ingredient protect cell from damage can be evaluated.

3D Reconstructed human skin level and animal experiment.

Apply pollutants like benzo (a) pyrene (BaP) — one of the PAHs on the 3D reconstructed human epidermis (Rhe), or reconstructed full thickness skin with melanocytes, and then skin discoloration, skin barrier function can be evaluated.In the French lab, certain concentration of biofunctional ingredients and placebo respectively on 3D Rhe for 10 days were applied, then SDS (sodlium dodecyl sulfonate) which dissolving in the Lucifier yellow fluorescene dye solution was applied, fluorescene penetration associated with skin barrier function can be analyzed as shown in Figure 2.

Figure 2. Skin barrier function

In vivo methods

Short-term test

As Figure 4 shows, the subjects’ back skin[29]or forearm skin was exposed to air pollutants contained tobacco smoke in a pollution simulatio device for a short period of time, about 15 mins to several hours. Then skin lipids were collected in order to analyze the lipid oxidation level, for example, MDA (malondialdehyde), SQOOH (Squalene monohydroperoxide) with HPLC. In our lab, stratum corneum was collected by tape-stripping using D-squame,and then protein carbonylation level of forearm skin was analyzed after several hours' exposure as shown in Figure 3.The protein carbonylation level increased after long-term exposure as shown in Figure 4. Moreover, inflammatory factors, vitamin E, ceramide, and so on can be used to evaluate the skin condition.

Figure 3. Pollution simulating device, (a) used by Proderm,(b) used by Ashland Shanghai lab

L’Oreal conducted a compare study in two of Mexico cities. The first group included 96 volunteers from Mexico City subjected to high level of pollution. A second group included 93 volunteers from Cuernavaca considered as poorly exposed to pollution. Forehead sebum and facial tape-stripping stratum corneum sample were collected from the above two groups. Tocopherol, squalene, lactic acid and cholesterol were analyzed. The results showed that tocopherol and squalene level were statistically significantly lower in Mexico City population (P ＜ 0.001),Lactic acid level was significantly higher in Mexico City population(P ＜ 0.001). But no statistical difference was showed on cholesterol level.[16]

Long-term test

Long-term exposure of skin to air pollutants has been associated with skin barrier damage, collagen breakdown,loss of skin elasticity, and growth of pigmentation. Based on that, skin erythema, skin wrinkles, elasiticity, AGEs(advanced glycation end products) level which represent the cross-linked collagen and make skin get stiff, skin tone, skin spots can be analyzed.

Figure 4. Protein carbonylation level changes with time

Vierkotter etc., studied the impacts of airborne particles on skin aging. 211 volunteers from Ruhr Germany (polluted area, with higher exposure of soot and traffic-related particles) and 189 volunteers from rural area were studied.All the volunteers aged between 70 and 80 years. The SCINEXA (score of intrinsic and extrinsic skin aging) were used to assess skin aging. This score is suitable to determine and differentiate between extrinsic and intrinsic skin aging.A significant association was found between traffic-related airbone particles and signs of extrinsic skin aging, namely,more pigment spots and more nasolabial folds were shown on Ruhr area.[3]

Ashland lab chose winter to conduct the pollution.The air pollution is heavy in Shanghai every winter.Smoker and non-smoker volunteers aged between 35 and 65 were chosen. Smokers smoked more than 3 years, and more than 10 cigarettes per day. Non-smokers had not smoked for 3 years, and barely exposed second hand smoking. Then the following parameters were determined.

As shown in Table 2, the test method covered skin protein damage, skin sensitivity, skin elasticity, skin aging and skin tone. Based on double-blind split face method,this study determined the effect of a bioflavone (Trade name: Elixiance) incorporating in a cream versus its placebo. The results showed that the bioflavone worked significantly better on almost all the above parameters than placebo. Skin tone parameters except b★ value worked better on smoker group than on non-smoker group,protein damage (SCCP and AGEs) showed better effect on non-smoker group than on smoker group. It indicated that smokers possess more damages proteins to be repaired. It’s reported that smoking, which is a significant risk factor for skin aging, can accelerate formation of AGEs and increase their deposition in various tissues including skin.[18]

Table 2. Test parameters and methods used in Ashland lab

conclusion

In conclusion, air pollution leds to detrimental effect on skin with a wide range of aspects. But few studies in this field has been done.

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