The volatilome refers to the complete set of volatile organic compounds (VOCs) emitted by an organism [1] . In healthy humans, VOCs are released from breath, skin, urine, saliva, breast milk, and blood, collectively forming a complex mixture comprising thousands of compounds [2] . Breath is the most significant of these sources, with 1488 VOCs identified in 2021, followed by the skin, which emits 623 VOCs [3] .

The skin volatilome originates from three endogenous sources: eccrine, sebaceous, and apocrine glands [4] . The distribution of these glands, varying across body regions, results in distinct VOC profiles. These VOCs are produced by endogenous biochemical reactions, and the composition of the volatilome varies between individuals, reflecting genetic background, diet, and lifestyle [2] [5] - [7] . In addition, VOCs are generated by skin-resident microorganisms, some of which metabolise sweat and sebum [8] . As the skin is the interface with the external environment, its volatilome also reflects environmental exposures and the use of cosmetic products [9] [10] . Consequently, the skin volatilome constitutes a complex mixture of VOCs from both endogenous and exogenous origins.

Similarly, to the breath volatilome analysis [11] [12] , the study of the skin volatilome offers a non-invasive method to disease diagnosis [13] . It has also proven valuable in monitoring psychological stress [14] - [17] . Initial works identified a limited number of differentially emitted compounds under stress [14] - [16] . More recently, 17 VOCs were found to be increasingly emitted in response to psychological stress, several of which had not been previously reported [17] .

Stress not only alters the skin’s VOC profile but also directly affects the skin itself. Beyond its involvement in various dermatological conditions and pathologies [18] - [20] , stress has also been implicated in skin ageing [21] . It rapidly triggers the production of glucocorticoids via the hypothalamic-pituitary-adrenal (HPA) axis and induces catecholamine release by the autonomic nervous system [18] . The skin, possessing functional peripheral HPA and catecholaminergic systems, responds with impaired barrier function [22] , compromised stratum corneum integrity [23] , and diminished innate and adaptive immune responses [24] [25] . However, the nature of the stress—acute or chronic—modulates its effects. While acute stress responses are tightly regulated through feedback mechanisms, chronic stress leads to habituation, attenuating HPA axis activity and heightening sensitivity to new stimuli [26] . This ultimately weakens immunoprotection, increasing susceptibility to infection and exacerbating allergic and inflammatory conditions [27] .

While the impact of glucocorticoids and the autonomic nervous system on the skin’s stress response is well-documented, the production of specific VOCs in this context raises questions about their potential role. In studies of extrinsic ageing, many VOCs—alongside UV radiation—have been shown to promote oxidative stress, leading to macromolecular damage, cellular senescence, and visible signs of ageing [28] [29] . This suggests that stress-induced VOC production may represent an unrecognised mechanism contributing to skin damage.

To explore this hypothesis, the effects of physiological doses of three stress-modulated VOCs were evaluated: heptadecane, 2,6,10,14-tetramethylpentadecane, and 2-hydroxyethyl acetate [17] . Besides their increased release under stress, the rationale behind this selection was the commercial availability of these compounds and, for heptadecane and 2-hydroxyethyl acetate, a prior identification within the skin volatilome [15] . The impacts of these three VOCs on reconstituted human epidermis (RHE) were evaluated by assessing the expression of genes and proteins involved in inflammation, epidermal differentiation, and oxidative stress.

By assessing the effects of a single, relatively brief exposure to physiological doses of three VOCs emitted by the skin under psychological stress, we evidenced that each compound distinctly impacted the expression of genes and proteins involved in inflammation, epidermal differentiation, and oxidative stress. For instance, heptadecane induced the early upregulation of the inflammation marker IL-1α, whereas tetramethylpentadecane upregulated it after five hours. In contrast, heptadecane and hydroxyethyl acetate markedly reduced IL-1α expression after five-hours. Similarly, genes whose products are involved in epidermal differentiation and oxidative stress displayed varying patterns of up- or downregulation depending on the VOC. These changes were not restricted to gene expression but also occurred at the protein level in most cases. Finally, none of the VOCs exhibited a cytotoxic effect (data not shown).

However, as a preliminary analysis, limitations must be acknowledged. Analysing only IL-1α gene is insufficient to draw robust conclusions about inflammation, necessitating further experiments assessing additional genes and cytokines. Moreover, some inconsistencies were observed between RT-qPCR data and protein quantification by immunohistochemistry. While these differences could be explained by temporal delays between transcription and translation, a deeper understanding of these relations is required. In particular, assessing protein levels at later time points would help establish whether the observed mRNA variations translate into corresponding protein changes. Despite these limitations, our results reasonably suggest that the three stress-modulated VOCs impact skin functions, albeit through distinct molecular mechanisms. Consequently, an attempt to discuss their effects should consider them individually.

Heptadecane is the VOC that most rapidly affects the expression of key genes involved in epidermal terminal differentiation, including KRT1, KRT10, FLG, and LOR, with corresponding reductions in filaggrin and loricrin protein levels. Filaggrin, keratin, and loricrin are essential structural components of the stratum corneum [30] [31] . Their interaction results in the collapse and flattening of corneocytes, providing mechanical resistance to the skin. Additionally, filaggrin proteolysis during terminal differentiation releases hygroscopic amino acids essential for stratum corneum hydration [32] . Collectively, filaggrin, keratin, and loricrin are crucial for maintaining the skin’s barrier function. Interestingly, murine studies have shown that disruption of this barrier induces inflammation through upregulation of several cytokines, including IL-1α [33] [34] , as we observed.

Tetramethylpentadecane also downregulates KRT1/10 expression, later upregulating IL-1α, possibly through mechanisms similar to those mentioned above for heptadecane. It also downregulates AQP3, a gene encoding the primary epidermal transporter of water, glycerol, and small solutes in the epidermis. Aquaporin-3 also contributes to cell proliferation, migration, and skin barrier maintenance [35] . Although tetramethylpentadecane also downregulates oxidative stress-related genes (CAT, SOD1, SOD2) early on, no decrease in the corresponding protein levels was detected within five hours. While SOD proteins, essential to protect cells against oxidative stress, are typically constitutively expressed [36] [37] , environmental stressors such as air pollution can modulate their expression [38] [39] . Therefore, the decreased gene expression could result in decreased SOD levels, and whether such a decrease occurs after five-hour time remains to be determined. If it does, it should lead to diminished antioxidant defences, weakening resistance to oxidative stress.

Hydroxyethyl acetate elicits an entirely different effects compared to the two other VOCs. It is characterised by a marked upregulation of SOD1 and SOD2 after five hours, partially reflected at the protein level. Although poorly responsive to external stimuli, as already mentioned, SOD gene expression is triggered by the NF-κB transcription factor under oxidative stress conditions [36] , suggesting that hydroxyethyl acetate could induce oxidative stress. However, the simultaneous downregulation of IL-1α and upregulation of differentiation markers (CALML5, KRT1, KRT10, LOR) runs counter to typical oxidative stress gene expression profiles, usually involving inflammation and impaired epidermal development [40] . Thus, if oxidative stress were the main driver of the hydroxyethyl acetate response, one would expect reduced KRT, LOR, and CALML5 expression. Other mechanisms must, therefore, be at play and remain to be identified.

Future research should expand this preliminary analysis by including additional genes/markers and functional assays, particularly to assess the impact on inflammation and the skin barrier function. Nevertheless, all three stress-modulated VOCs seem to affect the skin, at least the epidermis, inducing changes that can also be evidenced in response to exogenous stressors such as environmental VOCs. However, we only analysed three VOCs out of the 17 recently identified [17] . Whether the remaining 14 compounds also impact the skin remains to be determined. Moreover, the effects of stress-modulated VOCs are likely not only due to their individual impact, but rather from their interactions. Besides, we only assessed single, relatively short exposure to VOCs. Analysing longer and repeated exposures to acute stress will be of interest. Although more complex, it will also be interesting to assess the effects of chronic stress, which results in habituation, to determine its impact on the skin volatilome and the effects of this volatilome on the skin.

Environmental VOCs pollutants are well known to compromise skin integrity, contributing to barrier dysfunction, premature ageing, and visible signs of senescence [28] [29] . It is therefore unsurprising that stress-modulated VOCs may similarly affect the skin. Stress itself is already recognised for affecting the skin through the rapid release of glucocorticoids via the hypothalamic-pituitary-adrenal (HPA) axis and catecholamines from the autonomic nervous system [18] . These responses impair barrier function, compromise stratum corneum integrity, and weaken both innate and adaptive immunity. However, the role of stress-modulated VOCs has never been considered before, and our results suggest that it may represent an additional mechanism linking stress to skin dysfunction.

This work was financed by Clarins.

The authors would like to thank Laura Gilbert for her collaboration in the design of the study. They also wish to thank Dr. Philippe Crouzet, Estium-Concept, for scientific writing services.

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