Biological effects of air pollution on the function of human skin equivalents.

The World Health Organization reports that 99% of the global population are exposed to pollution levels higher than the recommended air quality guidelines. Pollution-induced changes in the skin have begun to surface; however, the effects require further investigation so that effective protective strategies can be developed. This study aimed to investigate some of the aging-associated effects caused by ozone and particulate matter (PM) on human skin equivalents. Full-thickness skin equivalents were exposed to 0.01 µg/µL PM, 0.05 µg/µL PM, 0.3 ppm ozone, or a combination of 0.01 µg/µL PM and 0.3 ppm ozone, before skin equivalents and culture medium were harvested for histological/immunohistochemical staining, gene and protein expression analysis using qPCR, Western blotting, and ELISA. Markers include MMP-1, MMP-3, COL1A1, collagen-I, 4-HNE, HMGCR, and PGE2. PM was observed to induce a decrease in epidermal thickness and an enhanced matrix building phenotype, with increases in COL1A1 and an increase in collagen-I protein expression. By contrast, ozone induced an increase in epidermal thickness and was found to induce a matrix-degrading phenotype, with decreases in collagen-I gene/protein expression and increases in MMP-1 and MMP-3 gene/protein expression. Ozone was also found to induce changes in lipid homeostasis and inflammation induction. Some synergistic damage was also observed when combining ozone and 0.01 µg/µL PM. The results presented in this study identify distinct pollutant-induced effects and show how pollutants may act synergistically to augment damage; given individuals are rarely only exposed to one pollutant type, exposure to multiple pollutant types should be considered to develop effective protective interventions.

Adaptive responses to air pollution in human dermal fibroblasts and their potential roles in aging.

The damaging effects of air pollution on the skin are becoming increasingly researched and the outcomes of this research are now a major influence in the selection and development of protective ingredients for skincare formulations. However, extensive research has not yet been conducted into the specific cellular defense systems that are being affected after exposure to such pollutants. Research investigating the affected systems is integral to the development of suitable interventions that are capable of augmenting the systems most impacted by air pollutant exposure. The following studies involved exposing primary human dermal fibroblasts to different concentrations of particulate matter and analyzing its effects on mitochondrial complex activity, nuclear factor erythroid 2-related factor 2 localization using immunocytochemistry and protein expression of electron transport chain complex proteins, sirtuin-1 (SIRT1), and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) using western blotting. Particulate matter-induced alterations in both mitochondrial complex protein and activity, indicating oxidative stress, which was also complimented by increased expression of antioxidant proteins GSTP1/2 and SOD2. Particulate matter also seemed to modify expression of the proteins SIRT1 and PGC-1α which are heavily involved in the regulation of mitochondrial biogenesis and energy metabolism. Given the reported results indicating that particulate matter induces damage through oxidative stress and has a profound effect on mitochondrial homeostasis, interventions involving targeted mitochondrial antioxidants may help to minimize the damaging downstream effects of pollutant-induced oxidative stress originating from the mitochondria.

Mitochondrial DNA as a Sensitive Biomarker of UV-Induced Cellular Damage in Human Skin.

Mitochondrial DNA (mtDNA) has been demonstrated to be a reliable biomarker of UV-induced genetic damage in both animal and human skin. Properties of the mitochondrial genome which allow for its use as a biomarker of damage include its presence in multiple copies within a cell, its limited repair mechanisms, and its lack of protective histones. To measure UV-induced mtDNA damage (particularly in the form of strand breaks), real-time quantitative PCR (qPCR) is used, based on the observation that PCR amplification efficiency is decreased in the presence of high levels of damage. Here, we describe the measurement of UV-induced mtDNA damage which includes the extraction of cellular DNA, qPCR to determine the relative amount of mtDNA, qPCR to determine UV-induced damage within a long strand of mtDNA, and the verification of the amplification process using gel electrophoresis.

Microneedle-based devices for point-of-care infectious disease diagnostics.

Recent infectious disease outbreaks, such as COVID-19 and Ebola, have highlighted the need for rapid and accurate diagnosis to initiate treatment and curb transmission. Successful diagnostic strategies critically depend on the efficiency of biological sampling and timely analysis. However, current diagnostic techniques are invasive/intrusive and present a severe bottleneck by requiring specialist equipment and trained personnel. Moreover, centralised test facilities are poorly accessible and the requirement to travel may increase disease transmission. Self-administrable, point-of-care (PoC) microneedle diagnostic devices could provide a viable solution to these problems. These miniature needle arrays can detect biomarkers in/from the skin in a minimally invasive manner to provide (near-) real-time diagnosis. Few microneedle devices have been developed specifically for infectious disease diagnosis, though similar technologies are well established in other fields and generally adaptable for infectious disease diagnosis. These include microneedles for biofluid extraction, microneedle sensors and analyte-capturing microneedles, or combinations thereof. Analyte sampling/detection from both blood and dermal interstitial fluid is possible. These technologies are in their early stages of development for infectious disease diagnostics, and there is a vast scope for further development. In this review, we discuss the utility and future outlook of these microneedle technologies in infectious disease diagnosis.

Metabolic dysfunction in human skin: Restoration of mitochondrial integrity and metabolic output by nicotinamide (niacinamide) in primary dermal fibroblasts from older aged donors.

Alterations in metabolism in skin are accelerated by environmental stressors such as solar radiation, leading to premature aging. The impact of aging on mitochondria is of interest given their critical role for metabolic output and the finding that environmental stressors cause lowered energy output, particularly in fibroblasts where damage accumulates. To better understand these metabolic changes with aging, we performed an in-depth profiling of the expression patterns of dermal genes in face, forearm, and buttock biopsies from females of 20-70 years of age that encode for all subunits comprising complexes I-V of the mitochondrial electron transport chain. This complements previous preliminary analyses of these changes. "Oxidative phosphorylation" was the top canonical pathway associated with aging in the face, and genes encoding for numerous subunits had decreased expression patterns with age. Investigations on fibroblasts from older aged donors also showed decreased gene expression of numerous subunits from complexes I-V, oxidative phosphorylation rates, spare respiratory capacity, and mitochondrial number and membrane potential compared to younger cells. Treatment of older fibroblasts with nicotinamide (Nam) restored these measures to younger cell levels. Nam increased complexes I, IV, and V activity and gene expression of representative subunits. Elevated mt-Keima staining suggests a possible mechanism of action for these restorative effects via mitophagy. Nam also improved mitochondrial number and membrane potential in younger fibroblasts. These findings show there are significant changes in mitochondrial functionality with aging and that Nam treatment can restore bioenergetic efficiency and capacity in older fibroblasts with an amplifying effect in younger cells.

Exposing human primary dermal fibroblasts to particulate matter induces changes associated with skin aging.

With a large proportion of the world's population living in areas where air quality does not meet current WHO guidelines, combined with the knowledge that pollutants can interact with human skin, it is now of even greater importance that the effects of air pollutant exposure on human skin be investigated. To evaluate the damaging effects of a known component of air pollution (particulate matter) on human primary dermal fibroblasts. These studies were undertaken by exposing primary human dermal fibroblasts to different concentrations of particulate matter and analyzing the effects over time using resazurin reduction assays. Immunofluorescence microscopy was used to determine if particulate matter caused activation of the aryl hydrocarbon receptor, and phosphorylation of histone H2AX, a known marker of double-strand DNA breaks. Dot blotting was also used to analyze expression changes in secreted MMP-1, MMP-3, and TGFß. Particulate matter was found to dose-dependently increase cellular viability, activate the aryl hydrocarbon receptor, increase double-strand DNA breaks, and increase the expression of MMP-1, MMP-3, and TGFß. With the potential of air pollutants such as particulate matter to not only modulate the expression of proteins implicated in skin aging, but also affect cells at a genetic level, brings a pressing need for further investigation so protective strategies can be implemented.

Individual and combined effects of the infrared, visible, and ultraviolet light components of solar radiation on damage biomarkers in human skin cells.

The ability of solar ultraviolet (UV) to induce skin cancer and photoaging is well recognized. The effect of the infrared (IR) and visible light (Vis) components of solar radiation on skin and their interaction with UV is less well known. This study compared the effects of physiologically relevant doses of complete (UV + Vis + IR) solar-simulated light and its individual components on matched primary dermal fibroblasts and epidermal keratinocytes from human donors on three biomarkers of cellular damage (reactive oxygen species (ROS) generation, mitochondrial DNA (mtDNA), and nuclear DNA (nDNA) damage). There was a greater induction of ROS, mtDNA, and nDNA damage with the inclusion of the visible and IR components of solar-simulated light in primary fibroblast cells compared to primary keratinocytes (P < .001). Experiments using exposure to specific components of solar light alone or in combination showed that the UV, Vis, and IR components of solar light synergistically increased ROS generation in primary fibroblasts but not primary keratinocytes (P < .001). Skin cell lines were used to confirm these findings. These observations have important implications for different skin cell type responses to the individual and interacting components of solar light and therefore photodamage mechanisms and photoprotection interventions.

Optimised detection of mitochondrial DNA strand breaks.

Intrinsic and extrinsic factors that induce cellular oxidative stress damage tissue integrity and promote ageing, resulting in accumulative strand breaks to the mitochondrial DNA (mtDNA) genome. Limited repair mechanisms and close proximity to superoxide generation make mtDNA a prominent biomarker of oxidative damage. Using human DNA we describe an optimised long-range qPCR methodology that sensitively detects mtDNA strand breaks relative to a suite of short mitochondrial and nuclear DNA housekeeping amplicons, which control for any variation in mtDNA copy number. An application is demonstrated by detecting 16-36-fold mtDNA damage in human skin cells induced by hydrogen peroxide and solar simulated radiation.

UVA-induced carbon-centred radicals in lightly pigmented cells detected using ESR spectroscopy.

Ultraviolet-A and melanin are implicated in melanoma, but whether melanin in vivo screens or acts as a UVA photosensitiser is debated. Here, we investigate the effect of UVA-irradiation on non-pigmented, lightly and darkly pigmented melanocytes and melanoma cells using electron spin resonance (ESR) spectroscopy. Using the spin trap 5,5 Dimethyl-1-pyrroline N-oxide (DMPO), carbon adducts were detected in all cells. However, higher levels of carbon adducts were detected in lightly pigmented cells than in non-pigmented or darkly pigmented cells. Nevertheless, when melanin levels were artificially increased in lightly pigmented cells by incubation with L-Tyrosine, the levels of carbon adducts decreased significantly. Carbon adducts were also detected in UVA-irradiated melanin-free cell nuclei, DNA-melanin systems, and the nucleoside 2'-deoxyguanosine combined with melanin, whereas they were only weakly detected in irradiated synthetic melanin and not at all in irradiated 2'-deoxyguanosine. The similarity of these carbon adducts suggests they may be derived from nucleic acid- guanine - radicals. These observations suggest that melanin is not consistently a UVA screen against free-radical formation in pigmented cells, but may also act as a photosensitizer for the formation of nucleic acid radicals in addition to superoxide. The findings are important for our understanding of the mechanism of damage caused by the UVA component of sunlight in non-melanoma and melanoma cells, and hence the causes of skin cancer.

What is the role of mitochondrial dysfunction in skin photoaging?

Skin ageing is a complex process involving both internal and external factors, which leads to a progressive loss of cutaneous function and structure. Solar radiation is the primary environmental factor implicated in the development of skin ageing, and the term photoaging describes the distinct clinical, histological and structural features of chronically sun-exposed skin. The changes that accompany photoaging are undesirable for aesthetic reasons and can compromise the skin and make it more susceptible to a number of dermatological disorders. As a result, skin ageing is a topic that is of growing interest and concern to the general population, illustrated by the increased demand for effective interventions that can prevent or ameliorate the clinical changes associated with aged skin. In this viewpoint essay, we explore the role that mitochondria play in the process of skin photoaging. There is continuing evidence supporting the proposal that mitochondrial dysfunction and oxidative stress are important contributing factors in the development of skin photoaging. Further skin-directed mitochondrial research is warranted to fully understand the impact of mitochondrial status and function in skin health. A greater understanding of the ageing process and the regulatory mechanisms involved could lead to the development of novel preventative interventions for skin ageing.

Aware, motivated and striving for a 'safe tan': an exploratory mixed-method study of sun-protection during holidays.

Background: This article presents an exploratory study, aiming to explore the correspondence between knowledge, motivation and sun-protection practices during holidays. Methods: Seventeen participants aged 21-62 years old, recruited from community settings took part in individual face-to-face semi-structured interviews, completed sun sensitivity questions and an objective assessment of sunscreen use. Holidaymakers' knowledge about sun-safe messages, intentions and perceptions of barriers and facilitators for sun-protection were assessed. Qualitative data were analysed using thematic analysis and integrated with quantitative data, using a pragmatic theory-informed approach to synthesise the findings. Results: Participants were well informed about sun-safe messages, highly motivated to protect themselves from solar UV radiation (UVR) and they perceived themselves as well protected. However, they did not seem to use effective protective practices. Sunscreen was the preferred method of sun-protection, but most participants used considerably less than the recommended amount and significantly overestimated the amount of time they could be safely exposed. Seeking shade was the least used method of sun-protection and covering-up strategies were mostly implemented as a partial protection (i.e. hats or sunglasses). The desire to reach an optimal balance between getting a tan and using sun-protection to avoid sunburns was preeminent. Several additional barriers and facilitators for sun-protection were identified. Conclusions: Holidaymakers might have a false sense of security when it comes to sun-exposure. They are aware of the need to protect from solar UVR, but the motive for a safe tan, the overreliance on sunscreen, the overestimation of the safe sun-exposure time for their skin type and the insufficient application of sunscreen leaves holidaymakers motivated to protect their skin at significant risk of overexposure, sunburn and skin cancer. Public health messages need to address how to implement effective sun-safe strategies.

Systematic and Iterative Development of a Smartphone App to Promote Sun-Protection Among Holidaymakers: Design of a Prototype and Results of Usability and Acceptability Testing.

BACKGROUND: Sunburn and intermittent exposure to ultraviolet rays are risk factors for melanoma. Sunburn is a common experience during holidays, making tourism settings of particular interest for skin cancer prevention. Holidaymakers are a volatile populations found at different locations, which may make them difficult to reach. Given the widespread use of smartphones, evidence suggests that this might be a novel, convenient, scalable, and feasible way of reaching the target population. OBJECTIVE: The main objective of this study was to describe and appraise the process of systematically developing a smartphone intervention (mISkin app) to promote sun-protection during holidays. METHODS: The iterative development process of the mISkin app was conducted over four sequential stages: (1) identify evidence on the most effective behavior change techniques (BCTs) used (active ingredients) as well as theoretical predictors and theories, (2) evidence-based intervention design, (3) co-design with users of the mISkin app prototype, and (4) refinement of the app. Each stage provided key findings that were subsequently used to inform the design of the mISkin app. RESULTS: The sequential approach to development integrates different strands of evidence to inform the design of an evidence-based intervention. A systematic review on previously tested interventions to promote sun-protection provided cues and constraints for the design of this intervention. The development and design of the mISkin app also incorporated other sources of information, such as other literature reviews and experts' consultations. The developed prototype of the mISkin app was evaluated by engaging potential holidaymakers in the refinement and further development of the mISkin app through usability (ease-of-use) and acceptability testing of the intervention prototype. All 17 participants were satisfied with the mISkin prototype and expressed willingness to use it. Feedback on the app was integrated in the optimization process of the mISkin app. CONCLUSIONS: The mISkin app was designed to promote sun-protection among holidaymakers and was based on current evidence, experts' knowledge and experience, and user involvement. Based on user feedback, the app has been refined and a fully functional version is ready for formal testing in a feasibility pilot study.

Bad air gets under your skin.

Air pollution is increasing beyond previous estimates and is viewed as the world's largest environmental health risk factor. Numerous clinical and epidemiological studies have highlighted the adverse effects of environmental pollutants on health. Although there is comparatively less research investigating the cutaneous effects of ambient pollution, there is growing recognition of the adverse effects on skin. In this article, we provide an overview of the nature of environmental pollution and highlight the current evidence detailing the effects on cutaneous health. There is convincing evidence demonstrating that air pollution has a detrimental impact on skin and can exacerbate skin disease. Further epidemiological and experimental studies are required to assess the short- and long-term deleterious effects of ambient pollutant exposure on skin. The future challenge would be to use this evidence to develop specific strategies to protect against pollution-induced damage and prevent the effects of "bad air getting under our skin."

Mitochondrial damage and ageing using skin as a model organ.

Ageing describes the progressive functional decline of an organism over time, leading to an increase in susceptibility to age-related diseases and eventually to death, and it is a phenomenon observed across a wide range of organisms. Despite a vast repertoire of ageing studies performed over the past century, the exact causes of ageing remain unknown. For over 50 years it has been speculated that mitochondria play a key role in the ageing process, due mainly to correlative data showing an increase in mitochondrial dysfunction, mitochondrial DNA (mtDNA) damage, and reactive oxygen species (ROS) with age. However, the exact role of the mitochondria in the ageing process remains unknown. The skin is often used to study human ageing, due to its easy accessibility, and the observation that the ageing process is able to be accelerated in this organ via environmental insults, such as ultra violet radiation (UVR). This provides a useful tool to investigate the mechanisms regulating ageing and, in particular, the role of the mitochondria. Observations from dermatological and photoageing studies can provide useful insights into chronological ageing of the skin and other organs such as the brain and liver. Moreover, a wide range of diseases are associated with ageing; therefore, understanding the cause of the ageing process as well as regulatory mechanisms involved could provide potentially advantageous therapeutic targets for the prevention or treatment of such diseases.

Age-Dependent Decrease of Mitochondrial Complex II Activity in Human Skin Fibroblasts.

The mitochondrial theory of aging remains one of the most widely accepted aging theories and implicates mitochondrial electron transport chain dysfunction with subsequent increasing free radical generation. Recently, complex II of the electron transport chain appears to be more important than previously thought in this process, suggested predominantly by nonhuman studies. We investigated the relationship between complex II and aging using human skin as a model tissue. The rate of complex II activity per unit of mitochondria was determined in fibroblasts and keratinocytes cultured from skin covering a wide age range. Complex II activity significantly decreased with age in fibroblasts (P = 0.015) but not in keratinocytes. This was associated with a significant decline in transcript expression (P = 0.008 and P = 0.001) and protein levels (P = 0.0006 and P = 0.005) of the succinate dehydrogenase complex subunit A and subunit B catalytic subunits of complex II, respectively. In addition, there was a significant decrease in complex II activity with age (P = 0.029) that was specific to senescent skin cells. There was no decrease in complex IV activity with increasing age, suggesting possible locality to complex II.

Mitochondria-targeted antioxidants.

Redox homeostasis is maintained by the antioxidant defense system, which is responsible for eliminating a wide range of oxidants, including reactive oxygen species (ROS), lipid peroxides, and metals. Mitochondria-localized antioxidants are widely studied because the mitochondria, the major producers of intracellular ROS, have been linked to the cause of aging and other chronic diseases. Mitochondria-targeted antioxidants have shown great potential because they cross the mitochondrial phospholipid bilayer and eliminate ROS at the heart of the source. Growing evidence has identified mitochondria-targeted antioxidants, such as MitoQ and tiron, as potentially effective antioxidant therapies against the damage caused by enhanced ROS generation. This literature review summarizes the current knowledge on mitochondria-targeted antioxidants and their contribution to the body's antioxidant defense system. In addition to addressing the concerns surrounding current antioxidant strategies, including difficulties in targeting antioxidant treatment to sites of pathologic oxidative damage, we discuss promising therapeutic agents and new strategic approaches.

Determination of the Action Spectrum of UVR-Induced Mitochondrial DNA Damage in Human Skin Cells.

Biological responses of human skin to UVR including cancer and aging are largely wavelength-dependent, as shown by the action spectra of UVR-induced erythema and nuclear DNA (nDNA) damage. A molecular dosimeter of UVR exposure is therefore required. Although mitochondrial DNA (mtDNA) damage has been shown to be a reliable and sensitive biomarker of UVR exposure in human skin, its wavelength dependency is unknown. The current study solves this problem by determining the action spectrum of UVR-induced mtDNA damage in human skin. Human neonatal dermal fibroblasts and primary human adult keratinocyte cells were irradiated with increasing doses of UVR. Dose-response curves of mtDNA damage were produced for each of the UVR sources and cell types, and an action spectrum for each cell type was determined by mathematical induction. Similarities between these mtDNA damage action spectra and previously determined nDNA damage were observed, with the most detrimental effects occurring over the shorter UVR wavelengths. Notably, a statistically significant (P<0.0001) greater sensitivity to mtDNA damage was observed in dermal fibroblasts compared with keratinocytes at wavelengths >300 nm, possibly indicating a wider picture of depth dependence in sensitivity. This finding has implications for disease/photodamage mechanisms and interventions.

Sebum, inflammasomes and the skin: current concepts and future perspective.

Increasing evidence has identified ultraviolet radiation (UVR) as the skins most potent mutagen as over exposure results in sunburn, inflammation and DNA damage, thus contributing to a photo-ageing phenotype and possibly skin carcinogenesis. The lipid-rich sebum secreted onto the surface of the skin plays an important physiological role in protecting the skin against external challenges. When skin is photosensitised by UVR, the lipid constituents of sebum are easily oxidised, generating several lipid photo-oxidative products (e.g. squalene peroxides). These photo-oxidative products have been shown to exert diverse toxicological, biological and immunological effects in the skin and have therefore been implicated in several detrimental skin alterations including premature skin ageing. The involvement of lipid peroxidation products in UVR-induced inflammatory responses has been inadequately studied and highly controversial. Furthermore, it is unclear to what extent these oxidative products contribute to the underlying mechanisms of skin photo-ageing. Therefore, this viewpoint essay will discuss the current knowledge on the effect of UVR exposure on skin surface lipids and how these may mediate UVR-induced inflammatory responses which may be key contributors to photo-damage in skin. This essay will also examine the potential role of inflammasomes (innate immune complexes) in the inflammatory response associated with UVR-induced lipid peroxidation. Limited evidence is available on the interactions between sebaceous lipids, downstream mediators and concomitant immune response in sun-exposed skin and clearer elucidation may lead to novel biomarkers of photo-ageing and the incorporation of new molecules into current skin therapies which better target oxidised lipids and or downstream mediators/pathways.

Mitochondrial DNA as a biosensor of UV exposure in human skin.

Mitochondrial DNA (mtDNA) has been demonstrated to be a reliable biomarker of UV-induced genetic damage in both animal and human skin. Properties of the mitochondrial genome which allow for its use as a biomarker of damage include its presence in multiple copies within a cell, its limited repair mechanisms, and its lack of protective histones. To measure UV-induced mtDNA damage (particularly in the form of strand breaks), real-time quantitative PCR (qPCR) is used, based on the observation that PCR amplification efficiency is decreased in the presence of high levels of damage. Here, we describe the measurement of UV-induced mtDNA damage, including the extraction of cellular DNA, qPCR to determine the relative amount of mtDNA, qPCR to determine UV-induced damage within a long strand of mtDNA, and the verification of the amplification process using gel electrophoresis.

Impact of hyperpigmentation on superoxide flux and melanoma cell metabolism at mitochondrial complex II.

Melanogenesis is a highly conserved process of cytophotoprotection from UV radiation present in many species. Although both mitochondrial function and UV radiation insults are well-documented promoters of increased cellular stress, their individual molecular relationships with skin pigmentation have not been clearly resolved. This study provides evidence for a direct relationship between cellular melanin content, superoxide flux, and mitochondrial function at complex II. Direct and significant correlation between increased pigmentation and complex II turnover was observed in genetically different melanoma cell lines of varied basal pigmentation states (P < 0.01). The same trend was also observed when comparing genetically identical cell cultures with increasing levels of induced pigmentation (P < 0.005). The observation of increased steady-state levels of the catalytic complex II succinate dehydrogenase subunit A alongside hyperpigmentation suggested coregulation of activity and pigment production (P < 0.01). The study also presents novel evidence for a relationship between hyperpigmentation and increased superoxide-generating capacity at complex II. By amperometrically monitoring superoxide flux from differently pigmented FM55 melanocytes and their isolated mitochondria, a dynamic and responsive relationship between pigmentation, complex II function, and intracellular superoxide generation was observed (P < 0.005). The data support hyperpigmentation as a protective antioxidant mechanism in response to complex II-mediated reactive oxygen species generation.