Skin aging results from the cumulative effects of two superimposed mechanisms [1] . The first one is intrinsic aging. Genetically determined, it is due to the passage of time and affects all tissues, leading to relatively mild aging signs: fine lines, xerosis, and laxity [2] . Yet, the skin is constantly exposed to, and a major target of, environmental stressors that damage it in a process known as extrinsic aging. Extrinsic aging is characterized by more pronounced aging signs: coarse wrinkles, uneven pigmentation, and solar lentigos. Besides, pollutants can lead to several skin diseases, including skin cancer [3] [4] .

Among the widespread skin stressors, the deleterious effects of UV Radiation (UVR) on all skin compartments have long been recognized [5] . While UVB (290 - 315 nm) and UVA (315 - 400 nm) are of particular importance, near-infrared radiation (770 - 1400 nm) also plays a role [2] . Still, the impact of UVR depends on skin pigmentation, DNA repair, antioxidants, and other endogenous protective mechanisms. Furthermore, responses to acute and chronic exposures differ in the skin reaction they induce, and photoaging essentially results from daily exposure to low doses of radiation [6] .

Nevertheless, UVR is not the only skin stressor. Since first reported [7] , it is clear that air pollutants (smog, ozone, particulate matter, organic compounds, etc.) also have multiple deleterious effects, leading at the level of the skin to aging signs, pigmentary disorders, and a wide range of pathologies [8] . Among the most widespread pollutants is Benzo[a]pyrene (B[a]P), a Polycyclic Aromatic Hydrocarbon (PAH) mainly resulting from incomplete combustion of fossil fuels and other organic compounds, including automobile exhaust and cigarette smoke [9] . Besides being a potent carcinogenic compound [10] , skin exposure by inhalation or direct absorption activates detoxification mechanisms, results in oxidative stress, and exacerbates inflammation [11] [12] . It also impairs the barrier function, facilitating the entry of other pollutants [7] [13] .

It should be pinpointed that the results mentioned above are from the analysis of single stressors [12] . While useful, such an approach is far from real-life conditions in which the skin is subjected to the simultaneous assault of multiple stressors. Despite the awareness of this complexity and efforts to unravel how stressors interact, their synergistic effects are poorly understood, although there is evidence that they do. This is the case for specific PAHs such as B[a]P, which present increased toxicity upon UV irradiation [14] [15] . Considering the health threat of pollution, there is an urge to better apprehend the effects of pollutants and their mixtures.

A hallmark of intrinsic aging and stressor-induced deleterious effects is oxidative stress and inflammation, which ultimately result in cellular senescence, impaired tissue homeostasis, and phenotypic alterations ( Figure 1 ) [16] . Besides these direct effects, intrinsic aging and pollutant exposure have indirect impacts mediated by epigenetic alterations [17] - [25] . These epigenetic changes encompass DNA methylation, histone modification (mostly methylation or acetylation), and RNA interference [17] , which, without altering the DNA sequence, lead to heritable changes in gene expression. Several studies pinpoint the role of these changes in intrinsic aging and upon skin assault by pollutants, whether UVR or organic pollutants like B[a]P, and have started deciphering the underlying mechanisms [17] - [24] . Epigenetic modifications are not only induced by aging and pollutants; they are an integral part of the aging process [25] , and the changes they cause to the accessibility of DNA are critical in determining the proteins expressed and their levels of expression, including DNA repair pathways that preserve genomic integrity and cell survival [26] [27] .

Apart from its role in regulating gene expression, several reports also indicate that epigenetics is essential to control alternate splicing and polyadenylation, leading to alternate mRNA isoforms [28] . If alternate splicing/polyadenylation explains the massive proteomic complexity compared to the relatively limited number of genes, it is now clear that such mechanisms significantly contribute to the regulation of many biological processes [28] . Whether environmental stressors also induce alternate mRNA processing has never been addressed.

A previous mass-spectrometry-based proteomic analysis revealed that contrary to UVA alone, B[a]P and UVA co-exposure of ex vivo skin explants leads to the overexpression of proteins involved in many processes but to the downregulation of those implicated in epidermal development, keratinocyte differentiation, and redox processes [29] . To gain further insight into the skin reactions to environmental stressors, we evaluated two aspects of the modifications occurring upon B[a]P and UVA co-exposure. The first one is epigenetics. We analyzed the changes happening to global DNA methylation and histone H3 upon co-exposure, which are major epigenetic markers. Due to their crucial role in the proteome, we also assessed the expression of four splicing factors that were identified as showing differentially expressed upon co-exposure [29] . Finally, we evaluated the effect of an Arundo donax L. extract containing compounds known to influence epigenetic marks on some of the co-exposure-induced alterations evidenced.

While the individual impacts of environmental stressors are well-documented, there is a scarcity of studies exploring their impact when combined. However, we are constantly exposed to such mixtures, and they are accumulating evidence that they result in exacerbated reactions, heightened disease risks, and increased mortality [31] - [34] .

In a previous study focusing on the epidermal layer of skin explants, several processes were identified as being affected by B[a]P and UVA co-exposure compared to UVA alone [29] . Building upon these results, we used similar co-exposure conditions, primarily focusing on changes affecting epigenetic marks in a preliminary exploratory study. It is important to specify that our study exclusively evaluated the effects of B[a]P and UVA co-exposure. Without control exposures to UVA or B[a]P alone, we cannot exclude the influence of a single stressor or conclusively attribute results to co-exposure, nor can we ascertain a synergistic effect of both stressors.

While the effects of environmental stressors on DNA methylation and their consequences on embryonic development or human health are well documented [35] - [39] , their impact on the skin remains largely unexplored. Nevertheless, a few studies have shown that UV radiation alters the methylation profile of epidermal cells, resulting in global and site-specific modifications [23] . Changes in DNA methylation profiles were also evidenced in isolated primary fibroblasts, but only after repeated exposure and after a few days [40] . Our study revealed that co-exposure to B[a]P and UVA increases total DNA methylation in HaCaT cells and primary fibroblasts. A single exposure and 24 hours are sufficient to highlight these changes.

Histone modification is another factor exerting genome-wide control. The only modification we evidenced upon B[a]P and UVA co-exposure is the phosphorylation of Ser10 in the tail of histone H3, a prominent modification [41] [42] . This increased frequency is consistent with its higher frequency upon UV radiation, via the MAP kinase cascade [43] . Besides, in mice, lung exposure to cigarette smoke leads to the activation of IκB kinase α, which also results in increased S10P frequency [44] . Yet, such a modification has never been evidenced in epidermal cells subjected to stressors. Besides, it is generally associated with S28 phosphorylation and closely linked to the methylation of K9 and K14 [41] [42] , modifications we did not highlight in our analyses. Thus, the biological effects of this increased S10P frequency are difficult to establish without further studies, especially since it promotes two opposite processes: chromosome condensation during mitosis and gene transcription during interphase [41] [42] [45] .

Changes in mRNA splicing represent another process that significantly affects the proteome by modifying the mRNA isoforms produced [28] . A previous study on epidermal cells of skin explants co-exposed to B[a]P and UVA identified four differentially expressed splicing factors [29] . The analysis of HaCaT cells subjected to the same treatment revealed that only SRSF1 and SFPQ are overexpressed, a discrepancy probably due to the different experimental systems. Thanks to their ability to bind nucleic acid and numerous proteins, these two proteins are implicated in mRNA splicing and several processes related to RNA metabolism [45] - [49] . Best studied for their involvement in disease development, they are also stress-response elements. Both relocate to the cytoplasm under stress, being involved in alternate splicing and ultimately participating in stress response. The fact that SRSF1 is overexpressed under stress, as we have evidenced, has never been proven to the best of our knowledge. Yet, using lung epithelial cells, Zhao et al. recently showed that small particulate matter induces several proteins involved in mRNA splicing, including SRSF1 [50] .

These major genome-wide changes in epigenetic marks induced by environmental stressors would be expected to lead to substantial modifications in gene expression. While such changes have been observed in skin explants after repeated exposure over seven days [29] , our study identified limited changes in the expression of keratinocyte-/fibroblast-relevant genes following a single co-exposure period and 24 hours. Most notably, inflammation genes were induced, but other affected genes showed only mild downregulation.

In addition to assessing the effects of co-exposure, we evaluated the impact of an Arundo donax L. extract. This rhizomatous tall grass, native to Asia, thrives almost worldwide thanks to its highly efficient clonal reproduction. Thus, it exhibits low genetic variability but is believed to have successfully adapted to diverse environments thanks to its epigenetic variability [51] [52] . The partial characterization of the extract we used revealed its richness in polyphenol. Besides common plant flavonoids, these polyphenols also encompass unusual ones. This is the case of catechins, compounds best studied in green tea. They are not only potent antioxidants but also modulate DNA methylation [53] - [55] , as do luteolins, another flavonoid found in the extract [56] - [58] .

In our model system, whether HaCaT cells or primary fibroblasts, the extract prevents the hypermethylation of DNA induced by co-exposure. It also hinders the overexpression of SRSF1 and SFPQ. While the extract did not affect Ser10 phosphorylation of histone H3 induced by co-exposure, it led to an increased methylation frequency of several residues. This is the case for K9me, with which S10P is generally associated, which could favor gene transcription [41] [42] [45] . In HaCaT cells, the extract has little influence on gene induction/repression induced by co-exposure. Yet, it leads to increased expression of some markers (ELN, PLOD3, and TNC), which seems related to the extract itself, possibly indicating a reinforcement of the extracellular matrix. While the full impact of the extract requires further work, it still seems of interest to partially reverse pollutant-induced changes in epigenetic marks.

The findings collectively demonstrate that co-exposure to B[a]P and UVA affects gene expression, as previously evidenced [29] , and modifies epigenetic marks as well as mRNA processing. These changes are induced by a single co-exposure and manifest within 24 hours after cells are subjected to stressors. Some of these modifications can be reverted by the Arundo donax L. extract evaluated. However, a limitation of this study is that we cannot ascertain whether the changes observed result from a synergistic effect of co-exposure. This point will be of great interest for further analysis, possibly using chemometric-related statistical tools. Another point that will be of great interest to elucidate is the consequences of these alterations.

The authors would like to thank Dr. Philippe Crouzet, Estium-Concept, for scientific writing services.

This work was financed by Clarins.

All authors are full-time employees of Clarins, a major company specializing in the design, manufacturing, and marketing of cosmetic products, which financed this study.