High pollution levels were initially identified in urban areas, however they became a global concern and the world’s largest single environmental health risk factor associated with millions of deaths annually (WHO, 2021). The anti-pollution movement in cosmetic and personal care industry began in the Asia Pacific region and it has now reached the western markets, launching hundreds of commercially successful products addressing this global concern (Pitman, 2017). One of the targets for these products is skin hyperpigmentation as the production of melanin is a very sensitive marker to changes induced by stress factors, including pollution. Integration of the complex biological pathway of melanin biosynthesis in the world wide context of skin tone uniqueness combined with geographically specific pollution concerns makes the research of the molecular mechanisms involved even more important.

The source and composition of pollutants are important aspects to take into account when conducting research focused on their effects on skin pigmentation. Air pollutants are emitted directly from the source (volcanic eruptions, forest fires, biological decay, power plants, industries, car emissions, fossil fuel burning, etc.) or following exposure to heat or UV (Krutmann et al., 2014). PM is a key component of air pollution that has been thoroughly investigated. The particle size, concentration and chemical properties of PM vary widely geographically and in time (Kampa and Castanas, 2008). Because of their textured structure, the particles have an ideal surface for the attachment of other toxicants such as PAHs, which originate from wood burning, combustion of organic material including coal burning, automobile exhaust fumes, and cigarette smoke. PAHs are highly lipophilic and therefore can easily penetrate through the skin barrier (Jux et al., 2011) and reach the melanocytes, the pigment producing cells resident in the skin. Multiple studies have shown that exposure to other air pollutants such as DEPs is associated with clinical signs of hyperpigmentation (Krutmann et al., 2020; Grether-Beck et al., 2021). Last but not least, PCBs are highly persistent environmental pollutants known to have accidentally contaminated rice oil alongside polychlorinated dibenzofurans (PCDFs) and polychlorinated quaterphenyls (PCQs) (Yusho accident, Japan, 1968 and Yu-Cheng accident, Taiwan, 1979) (Masuda, 1985). One of the symptoms for the poisoning that occurred during these incidents, though not the most impactful in terms of toxic effects, was increased skin pigmentation.

Impact of air pollution on animal species has been the subject of observations in populated areas and provided more evidence into the long-term effects on skin. For example, increased levels of pigmentation were observed to be common in several animal species (e.g., pigeons and moths) found in urban industrial environments (Chatelain et al., 2014; Van’t Hof et al., 2016). Observations on Emydocephalus annulatus showed that overproduction of melanin occurred more frequently in skin of sea snakes living in polluted water compared to unpolluted environments (Goiran et al., 2017). These observations support the contribution of pollution to skin hyperpigmentation.

The ability of particles to penetrate the skin layers is still debatable especially since studies report conflicting results likely due to having used a variety of particle types and different experimental models and designs (Toll et al., 2004; Baroli et al., 2007). Ambient PM was shown to penetrate the skin either following the route of hair follicles and sweat ducts (Lademann et al., 2004; Koohgoli et al., 2017) or transepidermally (Jin et al., 2018). Thus, they may be able to reach the melanocytes and release the highly lipophilic and toxic surface-bound PAHs. Air pollutants are also capable to affect directly the function of skin cells by hijacking classic signaling pathways otherwise known to be UV’s routes of action on skin pigmentation. It was recently demonstrated that particles can also penetrate the stratum corneum (Burke, 2018). Further mechanistic studies are necessary to elucidate the contribution of the particles and other attached environmental stress factors to extrinsic skin aging in order to find reliable means to counteract their actions.

Of an equally complex composition to air pollutants is the tobacco smoke, an aerosolized by-product of tobacco combustion during the smoking of cigarettes and related products and consisting of a particulate and a gas phase. While nicotine is known to be absorbed through skin (Benowitz and Hukkanen, 2009), details regarding the penetration of other fractions of the tobacco smoke through dermal layers is lacking. Even though nicotine is easy to use on its own in research experiments, the reality indicates that the most harmful toxicants are found primarily in the gas phase. It is therefore of the utmost importance to perform hazard and risk assessment using a combination of both phases to better represent the in-use exposure and to take into consideration any synergistic effects based on the interactions between the two phases. The particulate phase may include PAHs and tobacco specific byproducts, while the gas phase may contain dioxins among other harmful radicals. Of the dioxins, 2,3,7,8-tetrachlorodibenzo para-dioxin (TCDD) was demonstrated to be the most toxic molecule and one of the first environmental-related molecule shown to stimulate skin pigmentation (Luecke et al., 2010), thus opening up the investigations into premature skin aging induced by tobacco (Kennedy et al., 2003; Morita et al., 2009).

Melanocytes are highly specialized cells responsible for melanin biosynthesis through a complex pathway hosted by membrane-bound organelles known as melanosomes (Hearing, 1997). Following four stages of maturation, the melanosomes are transferred through the dendrites of the melanocytes to the surrounding keratinocytes where they play a critical role in photo-protection. The established “epidermal melanin unit” is comprised of a ratio of one melanocyte to 40 keratinocytes in the basal and suprabasal layers (Ostrowski and Fisher, 2019). The epidermal melanin unit responds rapidly to a multitude of environmental stimuli, of which UV was identified early as one of the main contributing factors to stimulate the melanin production. Melanocytes respond directly to UV exposure and through their close communication with the keratinocytes.

Melanin biosynthesis is tightly regulated by three melanogenic enzymes, Tyrosinase (TYR), Tyrosinase-related protein 1 (TYRP-1), and Dopachrome Tautomerase (DCT, also referred to as Tyrosinase-related protein 2 - TRP-2), located in melanosomes (Basrur et al., 2003). Tyrosinase is a key enzyme of the melanogenic pathway and primarily responsible for melanin biosynthesis. It catalyzes the oxidation of L-Tyrosine to L-DOPA which subsequently is oxidized to L-DOPAquinone that ultimately produces DOPAchrome, which is further converted into 5,6-dihidroxyindole-2carboxylic acid (DHICA) by DCT. TYRP-1 can oxidize DHICA to indole-5-6quinone carboxylic acid, and further to DHI-melanin (Figure 1—Melanin Synthesis); the DHI and DHICA moieties are part of eumelanin. Pheomelanin synthesis is based on the production of Cysteinyl-DOPA conjugates from DOPAquinone and consists of benzothiazine and benzothiazole groups. TYRP-1 plays a key role in the correct trafficking of TYR to melanosomes (Toyofuku et al., 2001), while DCT is involved in crucial detoxification processes within melanosomes (Milac and Negroiu, 2018). Activation of TYR, TYRP-1, and DCT is controlled by Microphthalmia Transcription Factor (MITF), a key transcriptional regulator of the pathway (D’Mello et al., 2016), which has been subjected to detailed studies looking into mechanisms of hyperpigmentation (Figure 1).

There are two types of skin pigmentation: constitutive and facultative. The constitutive skin color is determined genetically, is not directly affected by sun exposure (or other factors) and has photo-protective properties (Ostrowski and Fisher, 2019). Facultative skin color (tan) is induced by UV exposure, hormonal changes, disease or other stress factors (Ostrowski and Fisher, 2019). Dark skin was shown to have large and numerous melanosomes that are individually bound within a membrane compared to lighter skin, which traditionally produces less melanin and contains smaller melanosomes (Alaluf et al., 2002) aggregated and bound in groups as complexes (Thong et al., 2003; Coelho et al., 2009). These specific characteristics generate the tremendous variation of skin tones in relation to ethnicity and response to toxicants (pollutants) and to products designed to counteract their deleterious effects on skin pigmentation. The complexity of pollutants in terms of composition needs to be superimposed on the melanogenic pathway and its governing factors in order to understand the impacts and to address them at the cellular and molecular level.

MAPK signaling plays a key role in cellular proliferation, cell metabolism, and inflammation. It is also known to regulate the transcription of cyclic adenosine monophosphate (cAMP) responsive-element binding protein (CREB) (Estrada et al., 2022) and MITF, which is the master regulator of the melanogenic enzymes in charge of pigments synthesis (D’Mello et al., 2016). Of the multiple promoters of MITF gene, M promoter is selectively used in melanocytes and targeted by several transcriptional factors that impact the melanogenic pathway: paired box 3 (PAX3) protein, SRY-related High Mobility Group (HMG) box 9 and 10 (SOX9, SOX10), lymphoid enhancer factor 1 (LEF-1), one cut domain 2 (ONECUT-2), MITF itself (Steingrímsson et al., 2004; Passeron et al., 2007), and CREB. Through a ligand-receptor system, melanocytes respond to increased levels of hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), steel factor (SLF/SCF), leukemia inhibitory factor (LIF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by keratinocytes following UV exposure. They ultimately activate the MAPK pathway (Figure 1), thereby phosphorylating MITF and upregulating the expression of TYR, TYRP-1 and DCT (Yarden et al., 1987; Bonaventure et al., 2013).

Of the signaling systems that act upon the MAPK cascade, the one governed by SLF/SCF/c-KIT plays a key role in melanocyte homeostasis. c-KIT has functional xenobiotic-responsive elements (XREs) in its promoter, which are recognized by the Aryl hydrocarbon Receptor (AhR) (Jux et al., 2011), the key player of one of the signaling pathways pollutants act upon, subsequently impacting skin pigmentation. The MAPK-AhR connection is one of the many cross talking points discussed in this part of the manuscript. For simplicity, the direct connection between c-KIT and AhR pathway is not marked on Figure 1.

AhR is a highly conserved cytosolic transcription factor activated by a broad variety of exogenous and endogenous ligands (Nebert, 2017; Rothhammer and Quintana, 2019). In the absence of a ligand, AhR exists in an inactive/latent state as part of a cytosolic multi-protein complex, consisting of two heat-shock protein 90 molecules (HSP90), immunophilin-like hepatitis B virus X-associated Protein (XAP)2, the co-chaperone protein p23, and pp60src (Nebert, 2017). Activation of the receptor following the interaction with a ligand follows either a canonical or a non-canonical pathway, as further explained.

In the canonical signaling pathway, binding of a ligand to the AhR induces conformational changes that facilitate the nuclear translocation of the AhR-ligand complex, releasing hsp90, XAP2 and p23 (Salminen, 2022). AhR subsequently dimerizes with its partner, a structurally related nuclear protein, aryl hydrocarbon receptor nuclear translocator (ARNT) (Salminen, 2022). The complex binding to XREs induces the transcription of genes involved in the response to toxicants such as those encoding for both phase I and phase II xenobiotic metabolism enzymes [cytochromes P450 (CYP) 1A1, CYP1A2, CYP1B1 and UDP glucuronosyltransferase 1 family polypeptide A6 (UGT1A6)] (Ikuta et al., 2009). The activity of these enzymes can be deleterious because they generate reactive oxygen species (ROS). The contribution of ROS to melanin production is discussed in Section 3.5 as one key cross talking point between several intracellular pathways affecting melanin production. As the canonical pathway unfolds, the dissociated pp60src activates the epidermal growth factor receptor (EGFR) and induces its internalization and nuclear translocation (Agostinis et al., 2007). As a result, the downstream MAPK signal transduction is activated (Noakes, 2015), with direct impact on skin pigmentation (Figure 1). This also results in the transcriptional induction of cyclooxygenase-2 (COX-2) which is responsible for the production of Prostaglandin E2 (PGE2) (Matsumura, 2009), a key player in two complex pathways that crosstalk with the melanogenesis pathway: PGE2 induces downstream inflammatory events that lead to hyperpigmentation (as discussed in Section 3.6) and acts directly on the Protein Kinase C (PKC) pathway (Section 3.3).

As part of the non-canonical AhR signaling pathway, AhR binds to non-consensus XRE (NC-XRE) sites (Huang and Elferink, 2012) that share marked homology with the DNA binding sequence of the Krüppel-like factors (KLFs) family (Wilson et al., 2013). Of this family, KLF6 regulates expression of the p21Cip1 cyclin-dependent kinase inhibitor (Bieker, 2001), which is known to lead to ROS accumulation (crosstalk). RelB gene was shown to mediate another non-canonical pathway by interaction with AhR and binding to an unrecognized genomic RelB/AhR responsive element (RelBAhRE) (Vogel et al., 2007). As a result, production of inflammatory markers was shown to stimulate melanin production (Khmaladze et al., 2020; Fernández-Martos et al., 2021; Nkengne et al., 2021; Moon et al., 2022 - detailed in Table 1), another cross talking point in the complex pathways network governing melanogenesis (see Section 3.6).

Originally, AhR was identified as a TCDD-binding receptor protein and was later demonstrated to also recognize benzo[a]pyrene (BaP) (Tsuji et al., 2011; Nakamura et al., 2016), other dioxins, PAHs (Schecter et al., 2006), benzanthrone (BA) (Abbas et al., 2017), and related persistent organic pollutants (POPs) (Nebert, 2017) as ligands leading to hyperpigmentation (Luecke et al., 2010; Jux et al., 2011; Esser et al., 2013). TCDD, BaP, and benzanthrone are also components of tobacco and research by Tsuji et al., 2011; van den Bogaard et al., 2013; Nakamura et al., 2016; Abbas et al., 2017 supported their involvement in stimulation of melanogenesis. Furthermore, AhR also mediates melanoblast to melanocyte maturation and plays wider roles in skin melanogenesis (Esser et al., 2013; Nakamura et al., 2013). The contribution of AhR to melanin production in response to environmental stimuli is further supported by evidence provided by UV exposure as another proof of pollutants using established intracellular pathways leading to hyperpigmentation. For example, genes targeted by AhR were shown to be induced by UVB irradiation (Noakes, 2015). In sum, skin’s exposure to environmental stimuli can activate hyperpigmentation via AhR activation and subsequent increase of TYR activity, melanin synthesis, and upregulation of TYRP-1, DCT, and MITF expression (Krutmann et al., 2008; Luecke et al., 2010; Nakamura et al., 2013; Nakamura et al., 2016; Abbas et al., 2017; Leijs et al., 2018; Chan et al., 2021; Shi et al., 2021; Ahn et al., 2022) (Table 1).

The activation of adenylate cyclase (AC) upon exposure to UV as primary stimulus or to other factors catalyzes the conversion of ATP to the second messenger cAMP, which attaches to PKA leading to its activation. PKA subsequently phosphorylates CREB that can enhance MITF’s overexpression (Shin et al., 2015), with direct contribution to melanin production. Proteolytic cleavage of the proopiomelanocortin (POMC) protein results in the formation of adrenocorticotropic hormone (ACTH) and alpha-melanocyte-stimulating hormone (α-MSH). Both these factors are produced by keratinocytes in response to UV exposure and act as agonists to melanocortin 1 receptor (MC1R) on melanocytes, thereby increasing cAMP levels (Yamaguchi and Hearing, 2009) (Figure 1). At this point, the PKA and MAPK cascades crosstalk and contribute to melanin production through the common factor CREB (Figure 1). Evidence of tobacco or air pollutants acting directly on this signaling pathway are lacking. Given the common players among the MAPK, PKA and AhR pathways, it is conceivable to believe that the PKA could be chosen by environmental stimuli as direct or indirect port of entry to melanocytes. The only link between the AhR and PKA signaling routes was identified in the study by Bahraman et al., 2021 showing that the increase of α-MSH-induced expression of AhR may be due to the influence of cAMP directly or indirectly via the Wingless-related integration site (Wnt)/β-catenin pathway or both pathways. The participation of β-catenin to skin pigmentation in response to stimuli is discussed later (Section 3.4). Last but not least, there is another intersection point that could be of interest in the context discussed herein: between the PKA and adrenergic signaling pathways (Section 3.9).

The PKC pathway regulates melanogenesis (Gordon and Gilchrest, 1989) following the binding of Endothelin (EDN) 1 to its receptor (EDNRB) and of PGE2/PGF2 to their respective receptors, EP1/EP3/FP, in response to UV exposure (Park et al., 2006) (Figure 1). After the complex formation, phospholipase C β (PLCβ) is activated and catalyzes the production of inositol triphosphate (IP₃) and diacylglycerol (DAG). IP₃ induces the production of cytosolic Ca²⁺ response in melanocyte dendrites (Stanisz et al., 2012; Belote and Simon, 2020), whereas DAG activates PKC, which enhances the expression of MITF directly or indirectly via the MAPK cascade (Serre et al., 2018) (crosstalk) (Figure 1). In addition, the EP3 receptor controls the effects of PGE2 on cAMP in melanocytes that stimulates TYR activity and proliferation (Scott et al., 2004; Scott et al., 2005) (cross talk with PKA signaling pathway - see Section 3.2). The signaling of the fourth receptor of PGE2, EP4, stimulates cAMP production in melanocytes and subsequently TYR and the formation of dendrites (Starner et al., 2010), with direct implications in melanin biosynthesis (for simplicity, not displayed in Figure 1).

PKC usually resides in an inactive state in the cytoplasm but is activated by DAG generated from UV-irradiated cell membranes (Bae-Harboe and Park, 2012). DAG induces PKC translocation from the cytoplasm to membranes where PKC catalyzes the phosphorylation of serine residues on the cytoplasmic domain of TYR, thus activating it (Park et al., 1999). This is also another cross talking point with ROS-generation pathways that increase oxidative stress in response to toxicants, leading to high production of DAG and subsequent stimulation of melanin production (Costin and Hearing, 2007) (Figure 1). Finally, the cholinergic (muscarinic) and adrenergic (α1) signaling pathways crosstalk with the PKC cascade in response to stimuli (Figure 1) and have been speculated or demonstrated to stimulate the melanin production in skin (discussed later, in Sections 3.8 and 3.9, respectively). Direct impact of tobacco and environmental toxicants on PKC signaling is also lacking, however this is a pathway of much interest to investigate further as it already offers multiple communication points with other routes directly taken by pollutants to act on skin pigmentation.

The Wnt signaling pathway mediates melanogenesis by enhancing MITF’s expression. As part of this cascade, Frizzled (FZD) receptors bind to the transmembrane molecule Low-Density Lipoprotein Receptor-related Protein (LRP5/6) to form the LRP-FZD dimer complex that modulates cell differentiation and proliferation (Nusse and Clevers, 2017). Wnt’s expression is upregulated by exposure to UV (Yamada et al., 2019) and in response it connects with the LPR-FZD complex. This binding stabilizes the cytoplasmic β-catenin which then translocates to the nucleus where it binds with the T Cell/Lymphoid Enhancing (TCF/LEF) transcription factor to synergistically upregulate MITF gene expression (Takeda et al., 2000; Steingrímsson et al., 2004; Pillaiyar et al., 2017) (Figure 1), with direct downstream upregulating effects on melanogenic enzymes. This pathway cross talks with multiple others to impact melanin production: AhR signaling pathway (Bahraman et al., 2021) (Section 3.1), PKA cascade through α-MSH (Yamaguchi and Hearing, 2009) (Section 3.2), and PKB pathway through the Glycogen Synthase Kinase (GSK)-3β route (detailed herein).

The intricacies of AhR and Wnt/β-catenin pathways crosstalk are still not fully understood. Several studies using animal test systems or liver cells supported the impact of TCDD from tobacco smoke on AhR activation regulated by the Wnt/β-catenin signaling (Mathew et al., 2008; Procházková et al., 2011). Previous publications reported on a direct connection between the components of this intracellular route whereby β-catenin’s expression was strongly induced in melanocytes after exposure to AhR ligands (Nakamura et al., 2013). Another entry of AhR to the Wnt/β-catenin pathway is governed by the α-MSH-induced PKA cascade (Bahraman et al., 2021). These studies providing direct or indirect links between multiple signaling routes exemplify the complexity of the intracellular pathways contributing to melanin production as a response of melanocytes to stress factors.

Another crosstalk is based upon Wnt’s activity on the PKB pathway: when Wnt acts on its cell surface receptor LRP/FZD (He et al., 2004), Dishevelled (Dvl) cytoplasmic phosphoprotein acting directly downstream to FZD induces the accumulation of β-catenin in the cytoplasm by inhibiting the GSK-3β-dependent phosphorylation of β-catenin (Kishida et al., 2001). Accumulated β-catenin is then translocated into the nucleus where it follows the path described above to stimulate MITF and subsequently the melanogenic pathway. Also acting at this crossroads is TCDD, the high-affinity ligand of AhR, which was shown to be able to increase the phosphorylation level of PKB and GSK-3β (Xu et al., 2014) and to activate β-catenin (Al-Dhfyan, 2017). This crosstalk between AhR and PKB/GSK-3β/β-catenin pathway remains not thoroughly understood and likely not fully explored. To complicate the mechanistic profile even further, it has been reported that AhR could activate PKB in a ROS-dependent manner (Liao et al., 2014) or through binding Src kinase in the cell membrane (Ye et al., 2018). All these interactions between factors and intracellular routes have been supported by experiments in various cells lines, with no direct relation to keratinocytes or melanocytes. Therefore, work to verify them in skin lineages should be conducted to determine any direct implications on skin pigmentation in response to pollutants and tobacco. Other mechanisms are also possible and further investigations are surely needed to clarify the complexity of communication between these pathways.

Air pollutants become bioavailable to the skin layers via nanoparticles and play a key role in the synthesis of quinones which are responsible for the production of ROS. Increased amounts of ROS and free radicals within the cells overcome skin’s innate enzymatic (glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase) and non-enzymatic antioxidant defense systems (vitamin E, vitamin C, and glutathione) (Thiele et al., 1997). The interaction of ROS and free radicals with the lipid-rich plasma membranes initiates a lipid peroxidation reaction that ultimately triggers an increase of metalloproteinases (MMPs) (Farage et al., 2008), which represent a key cross talking point between multiple pathways. ROS also stimulate the release of pro-inflammatory mediators, which results in a vicious cycle of inflammation and metabolic impairments, with downstream damaging activities on skin pigmentation (cross talking, see Section 3.6). Therefore, ROS are at the crossroads of multiple physiological pathways directly or indirectly impacted by environmental stimuli.

Epidermal melanocytes are especially susceptible to high ROS concentrations due to their specialized function to synthesize melanin through a process that itself induces oxidative stress. ROS are generated in the skin from sources that can be extrinsic (UV, pollution, tobacco) and intrinsic (metabolically generated pro-oxidants), and were shown to accelerate skin pigmentation in experiments using antioxidants. Keratinocytes adjacent to melanocytes have a significant contribution to UV-induced skin pigmentation. Furthermore, H₂O₂ (generated by UVB irradiation) (Schallreuter et al., 2004) activates epidermal phenylalanine hydroxylase, which produces L-Tyrosine from L-Phenylalanine, and thus contributes to melanogenesis by increasing the concentration of L-Tyrosine, the initial substrate of TYR (Figure 1—Melanin Synthesis Pathway). Additional studies have also demonstrated another mechanism involving H₂O₂ in the regulation of TYR via p53 through transcription of the hepatocyte nuclear factor (HNF)-1α, which in turn can also affect the POMC response (Schallreuter et al., 2008), with downstream action on the PKA pathway (see Section 3.2). Many studies reported that UV can induce the formation of ROS and Reactive Nitrogen Species (RNS) that act as signaling messengers to stimulate melanin production (Mastore et al., 2005; Dong et al., 2010; Kim and Lee, 2013) by increasing the amount of TYR and TYRP-1 (Sasaki et al., 2000). While the molecular mechanisms are still subject to investigation, studies showed that nitric oxide (NO) may play a vital role in the activation of TYR through guanylate cyclase and guanosine 3′,5′-cyclic monophosphate (cGMP)-dependent protein kinase (PKG) or by increasing its expression in melanocytes (Sasaki et al., 2000).

Through crosstalk, ROS activate the MAPK family subsequently inducing the activator protein 1 (AP-1), a transcription factor which plays an essential role in the transcriptional regulation of MMP-1, MMP-3, MMP-9, and MMP-12, found to be elevated in aged human skin (Shin et al., 2015; Mujahid et al., 2017). Nuclear factor-κB (NF-κB) is another transcription factor activated by ROS (Cui et al., 2007) and known to mediate the responses to UV irradiation and photo-aging, primarily by upregulation of MMP-1 and MMP-3 in dermal fibroblasts (Pittayapruek et al., 2016). In relation to pigmentation, upon melanocytes activation, their migration is initiated by the decoupling from the basement membrane (Haass and Herlyn, 2005) and further from keratinocytes (Birlea et al., 2017). MMPs induce the decoupling of melanocytes from keratinocytes and coordinate the attachment of melanocytes to the next epidermal unit. Therefore, it is conceivable that pollutants could impact this pathway and ultimately play a role in the associated stimulation of melanin synthesis, however more research in this regard is needed. For simplicity, these possible pathways are not included in Figure 1.

Of the pollutants known to impact the skin, BaP and hazardous dioxins activate AhR with a robust ROS generation, leading to acne and chloracne (Tsuji et al., 2011; Furue et al., 2014). The BaP/AhR signal also induces CYP1A1 (Li et al., 2003; Tsuji et al., 2011; Mescher et al., 2019), which further converts PAHs to quinones capable to upregulate ROS synthesis (Li et al., 2003). Furthermore, PM2.5 contains environmental metals and PAHs (Nel et al., 2006) that may increase the PAH-induced ROS generation (Chen and Lippmann, 2009; Magnani et al., 2016). Further supporting evidence was reported by Murphy et al., 2004 showing that the AhR pathway is activated by both TCDD and all-trans retinoic acid which increase MMP-1 expression in normal human keratinocytes. TCDD-induced expression of MMP-1 through AP-1 route (Murphy et al., 2004). Human melanocytes and some melanoma cell lines are activated by TCDD, and were shown to express both AhR and ARNT, and to increase MMP expression and activity following the exposure to toxicants (Villano et al., 2006). All these interactions are cross talking points between complex pathways that pollutants follow to impact melanin production in the skin.

In terms of tobacco’s participation to skin pigmentation, significantly increased levels of MMP-1 mRNA were observed in the dermal connective tissue of smokers compared to nonsmokers in a clinical study (Lahmann et al., 2001). Tobacco smoke extract induced the production of MMP-1 and MMP-3 and resulted in abnormal regulation of extracellular matrix deposition in human cultured skin fibroblasts (Yin et al., 2003). Moreover, tobacco smoke is a major source of PAHs exposure in humans. In this regard, it has been shown that tobacco smoke extract induced MMP-1 expression via activation of AhR signaling pathway in human fibroblasts and keratinocytes (Ono et al., 2013). Supportive evidence regarding the contribution of pollutants and tobacco to skin hyperpigmentation via ROS is also provided by multiple other studies (Vierkötter et al., 2010; Cho et al., 2012; Ishiwata et al., 2013; Delijewski et al., 2014; Peng et al., 2017; Leijs et al., 2018; Milani et al., 2019; Khmaladze et al., 2020; Krutmann et al., 2020; Suo et al., 2020; Yang et al., 2020; Chan et al., 2021; Grether-Beck et al., 2021; Moon et al., 2022) discussed in detail in Table 1.

Skin exposure to air pollution toxicants induces mechanisms of cell detoxification which are active over extended periods of time and lead to elevated ROS levels and lipid peroxidation, resulting in skin alterations, including hyperpigmentation (Vierkötter et al., 2010; Patatian et al., 2021). Air pollution also induces inflammation, activates the AhR pathway, and leads to multiple alterations in skin’s physiology (Cho et al., 2012; Mancebo and Wang, 2015; Damevska et al., 2021; Nkengne et al., 2021). It seems that AhR is at the crossroads of these multiple pathways as it was shown to determine the severity of symptoms in chronic skin inflammation (Di Meglio et al., 2014). Its activation is essential for melanocyte survival and melanogenesis, which can be linked to the appearance of senile lentigines (Nakamura et al., 2015).

Recent research has shown that IL-33 stimulates phosphorylation of p38 and CREB, and consequently increases the expression of TYR, TYRP-1 and DCT through MITF, resulting in augmentation of melanogenesis (Zhou et al., 2014). Abundant IL-33 mRNA was found to be induced by UVB in human keratinocytes (Meephansan et al., 2012) and fibroblasts (Arend et al., 2008; Byrne et al., 2011), suggesting that skin’s response to stress factors leads to IL-33 release and ultimately to skin pigmentation. By involving mast cells (Ali et al., 2007; Moulin et al., 2007), macrophages (Schmitz et al., 2005), CD 4+T cells, basophils, dendritic cells and neutrophils, IL-33 is likely promoting Th2-skewed skin inflammation, which is another indirect effect on pigmentation (Hsu et al., 2020). Through activation of NF-kB, IL-33 may be able to regulate outcome of diseases such as atopic dermatitis, which can ultimately trigger pigmentation (Cevikbas and Steinhoff, 2012).

IL-18 is known to modulate both innate and adaptive immunity, and its dysregulation can cause autoimmune or inflammatory disease. Recent studies suggest its participation in regulation of pigmentation based on its activity to promote melanogenesis through upregulation of TYRP-1 and DCT expression (Zhou et al., 2013; Zhou et al., 2016), and by activating pathways that ultimately increase MITF’s expression. Furthermore, Chen et al., 2010 showed that the combination of keratinocyte growth factor (KGF) and IL-1α increased melanin deposition and is responsible for initial stage of human solar lentigines.

PGE2 is released in significant amounts by keratinocytes exposed to UV and in inflammatory conditions such as wound healing, and stimulates the formation of dendrites in melanocytes (Pentland et al., 1990; Pentland and Mahoney, 1990). PGE2 is released by keratinocytes and melanocytes through a process controlled by phospholipase A2 (PLA2), COX and prostaglandin E synthase (Gledhill et al., 2010). PGF2α is produced by fibroblasts and keratinocytes and was shown to stimulate melanocyte dendrite formation and TYR activation (Scott et al., 2005). Therefore, prostaglandins are potent inflammatory mediators with demonstrated activity on the melanogenic pathway and offer an established route for any pollutants to act upon. Further targeted experiments in this regard should be conducted to confirm this hypothesis.

Besides lentigines, post-inflammatory hyperpigmentation (PIH) is another skin condition typified by dark and flattened spots on the body (Markiewicz et al., 2022). Uneven skin tone is caused by exaggerated melanin production, often further aggravated by UV exposure. Inflammatory markers can also trigger PIH associated with skin conditions such as acne, atopic dermatitis, etc. (Kaufman et al., 2018). The PIH profile is characterized by production of cytokines, chemokines, and ROS during the inflammation that ultimately stimulates melanocyte growth, melanin production and its transfer to keratinocytes. Factors promoting these inflammatory events are EGF, IL-1, IL-6, tumor necrosis factor (TNF), leukotrienes (LT)C4 and LTD4, prostaglandins E2 and D2, and thromboxane-2 (Taylor et al., 2009). All these factors intersect on multiple other pathways that directly or indirectly augment the production of melanin in the skin and open wide the gates for environmental stimuli to enhance the pigmentation and accelerate skin aging. The direct connections however still await confirmation through thorough research that can be accomplished by NAMs that cast a wide mechanistic net and provide needed clarifications.

A significant body of evidence is reported in the scientific literature highlighting important aspects of the relationship between nicotine and melanin, primarily based on animal studies (Tsujimoto et al., 1975; Leeds and Turner, 1977). Nicotine itself and other tobacco constituents, specifically the carcinogens N′-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and BaP, have been shown to accumulate in tissues containing melanin (Domellöf et al., 1987). Although many studies demonstrated that tobacco smoke causes skin aging mediated by its effects on fibroblasts and keratinocytes (Morita, 2007; Morita et al., 2009; Ono et al., 2013), narrowing of blood vessels (Argacha et al., 2008) and loss of vitamin C (reviewed by Carr and Rowe, 2020), only recently several studies reported on the mechanisms underlying smoker’s pigmentation.

Nicotine was shown to have affinity for melanin partly due to its precursor role in the pigment synthesis (Mizuno et al., 1997) and to its irreversible binding to the complex melanin molecules (King et al., 2009; Delijewski et al., 2014; Tamai et al., 2014). Nicotine and tobacco-specific toxic byproducts may also be trapped in tissues containing melanin (see also Section 3.10 regarding the possible contribution of adipose tissue), which results in extended exposure and associated health issues noticed in smokers (Szüts et al., 1978; Szparaga et al., 2021). The activity of melanogenesis enzymes may be a factor contributing to the release or degradation of toxic tobacco compounds (Brittebo and Tjälve, 1981). The role of melanin in tissue uptake of nicotine and tobacco-specific toxicants may be of particular concern to individuals with highly pigmented skin (Yerger and Malone, 2006). It was also shown that nicotine has the ability to form adducts with DOPAquinone, a precursor of melanin production (Dehn et al., 2001). Furthermore, nicotine can be incorporated in melanin molecules during hair formation (Stout and Ruth, 1999).

The cholinergic signaling system (nicotinic and muscarinic receptors) plays an important role in keratinocytes and melanocytes physiology (Kurzen et al., 2007; Wu et al., 2020a; Wu et al., 2020). Nicotine and related byproducts are known to impact the skin primarily by activating nicotinic acetylcholine receptors (nAChRs) (Grando et al., 2006) and downstream by mediating their complex actions in tobacco users (Benowitz and Hukkanen, 2009). Nicotine was shown to induce pigmentation in oral mucosal lesions following application of sublingual tablets (2 mg) for 3–6 months in a smoking cessation study (Wallström et al., 1999). Nicotine was also found to induce melanosome dispersion in dermal melanocytes (melanophores) of a teleostean fish (Visconti et al., 1984).

Within the cholinergic system, α7 nAChR plays a key regulatory role in modulating melanosome uptake in keratinocytes being induced by UV exposure (Figure 1). The exposure to UV is a stimulus to release acetylcholine from keratinocytes (Wu et al., 2020a; Wu et al., 2020), which further triggers the activation of α7 nAChR, localized on the surface of keratinocytes, leading to the intracellular influx of calcium. The mobilization of calcium ultimately initiates phagocytosis of melanosomes by keratinocytes (Guo et al., 2023).

While melanocytes appear to lack α7 nAChR, primary human sebocytes in vitro and sebaceous glands were reported to express the receptor (Li et al., 2013), which leaves room for speculation in terms of the contribution of adipose tissue to pigmentation when exposed to tobacco or other pollutants (see Section 3.10). Therefore, the evidence available thus far indicates that the impact of nicotine on pigmentation is mediated primarily by keratinocytes and their response to stimuli. Interesting reports showed some possibilities of signaling mediated by α7 nAChR however provided by cell lineages other than melanocytes. For example, the receptor was shown to transduce signals through the PI3K cascade in rat neuronal cells (Kihara et al., 2001), a path that in melanocytes acts directly onto MITF (see Section 3.4). Liu et al., 2017a showed using dopaminergic neurons that α7 nAChR mechanisms play a key role in a Parkinson’s disease mouse model via regulation of the Wnt/β-catenin signaling pathway, known to also impact pigmentation through the PKA and AhR synergistic effects. Since melanocytes derive from neural crests, this association can be explored from the perspective of the nicotinic receptors to determine if tobacco and pollutants can directly join the pathway and impact the melanin production in skin as a result.

The autocrine adrenergic intra- and intercellular signal transduction network in the human epidermis contributes significantly to the regulation of vital functions within the epidermal melanin unit. The epidermal adrenergic signals control multiple cellular processes such as cell growth and differentiation, motility, calcium homeostasis, and pigmentation via the α1-and β2-adrenoceptors.

It was established that human epidermis has the capacity to synthesize catecholamines (norepinephrine and epinephrine) from the essential amino acid L-phenylalanine. The first step leads to the production of L-Tyrosine catalyzed by phenylalanine hydroxylase in the presence of iron, molecular oxygen, and the cofactor/electron donor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin, followed by the rate-limiting step via tyrosine hydroxylase controlled by the same cofactor (Nagatsu et al., 1964) (Figures 1 – Catecholamines Synthesis). The binding of catecholamines to the β2-adrenergic receptor triggers the activation of cAMP production and the downstream PKA signaling pathway (cross talk, see Section 3.2) (Figure 1). Gillbro et al., 2004 demonstrated that a specific functional β2-adrenergic signal exists in human melanocytes leading to pigmentation through the cAMP/PKA pathway, which is now considered the main axis for the catecholamine control of melanogenesis. In a cross talking manner, the epidermal β2-adrenergic signaling also increases cAMP in response to release of epinephrine by keratinocytes, which results in calcium increase via activated PKC (Schallreuter et al., 1992; Koizumi et al., 1997). Therefore, cAMP can be considered the liaison between the PKC and PKA pathways.

It was also established that human melanocytes express α1-adrenoceptors, which are induced by norepinephrine yielding the IP₃ and DAG signals (Figure 1). While the direct link between nicotine and adrenergic receptors in the skin is not supported currently by publications, indirect evidence comes to make the possibility worth investigating it further. For example, Choi et al., 2020 demonstrated the anti-melanogenic potential of carvedilol, a nonselective beta blocker with weak α1-blocking activities. Carvedilol reduced melanin content and cellular TYR activity without impact on cellular viability of human and mouse melanocytes. The compound also downregulated MITF, TYRP-1, DCT, and CREB.

Furthermore, the catecholamine synthesis pathway (Figure 1) uses Dopamine which is also involved directly in the melanin synthesis; therefore, increase in Dopamine in response to stimuli could increase the melanin production directly or indirectly.

PCBs and similar derivatives such as dioxins, polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD) etc. are often grouped under the umbrella of POPs. Included in this group even though it is not itself a POP is the endocrine disruptor bisphenol A (BPA) which acts as a POP. By sequestering POPs, the adipose tissue can protect other organs and tissues from toxic overload, however this storage may prove in long term detrimental to the body. A relatively recent study (Liu et al., 2017b) showed that PM2.5 inhibited sebocyte proliferation, reduced lipid synthesis and induced inflammatory cytokines IL-1α, IL-6, and IL-8. Additionally, the expression of AhR, ARNT, cytochrome P450 1A1 was significantly increased following PM2.5 exposure.

Even though the impact of pollutants on various cell lines was investigated, experiments targeting the adipose tissue are rather rare. Going further, from a pigmentation perspective, the contribution of adipose tissue is often overlooked and seldom researched with few exceptions. For example, Baranova et al., 2005 reported a statistically significant overexpression of TYR, DCT, melanosome transport protein RAB27a, and melan-A (MLANA) in visceral adipose tissue of morbidly obese patients. A later follow-up study by Randhawa et al., 2009 confirmed the expression of TYR, DCT, TYRP-1, and MITF and subsequent melanin biosynthesis in adipose tissue. Furthermore, TYR activity in the adipose tissue samples was much higher compared to samples collected from non-obese subjects. One of the limitations of investigating the impact of pollutants on pigmentation in adipose tissues is given by the incapacity of cultured adipocytes to sustain the enzymatic activity of TYR or the production of melanin, nor adequate post-translational modifications or the correct folding of TYR. We consider the participation of the adipose tissue to the melanin synthesis and its significant role in storing POPs with demonstrated impact on melanogenesis an avenue yet to be fully explored (see also Section 3.8 for further speculations on sebocytes possible contribution to pigmentation via the cholinergic pathway as they express the α7 nAChR).

Proteins’ accumulation in the endoplasmic reticulum (ER) is a trigger of the unfolded protein response (UPR). Inositol-requiring protein 1 (Ire1), Protein Kinase R (PKR)-like endoplasmic reticulum kinase (Perk), and activating transcription factor (Atf)-6 are components of the UPR (Bertolotti et al., 2000). One very recent study showed that PM can increase melanin production in melanocyte, mouse skin, and human skin models. The expressions of unfolded protein response molecules was also increased in response to PM exposure. The IRE1α signaling pathway was consistently upregulated and it was shown to be further related to PM-triggered melanogenesis (Ahn et al., 2022).

PM treatment of melanocytes induced cAMP and the expressions of phosphorylated Calcium-Calmodulin-dependent Protein Kinase II (CAMKII) and CREB. In parallel, PKA was shown to induce IRE1α phosphorylation (Mao et al., 2011; Asada et al., 2015) which in turn phosphorylates CAMKII and CREB (Kim et al., 2010; Vo et al., 2019). Based on these discoveries, it was hypothesized that PM induced melanogenesis through PKA signaling in a cross talking manner with the IRE1α signaling pathway (Ahn et al., 2022). These preliminary results make the investigation of the folding pathways appealing in the attempt to determine if a direct impact of pollutants and tobacco exists, with downstream effects on skin hyperpigmentation. For simplicity this speculative pathway is not presented in Figure 1.

Investigation of Tyr folding in mouse melanoma cells was conducted using N-butyldeoxynojirimycin (NB-DNJ), an N-alkylated imino-sugar analog of glucose which inhibits α-glucosidase I and II activity. As a result, most glycans on Tyr molecule are arrested as glycosylated structures and prevented from undergoing any further processing (Branza-Nichita et al., 1999). This inhibition of early N-glycan processing in the ER may impact the maturation of the amino acid chain by preventing the correct association with ER chaperones calnexin/calreticulin responsible for folding. This study showed that NB-DNJ dramatically impacts the folding and maturation of Tyr. As a consequence, its enzymatic activity was almost entirely abolished, resulting in a complete loss of pigmentation in the treated cells. However, the enzyme was correctly transported to the melanosomes. It should be pointed out that NB-DNJ is the active ingredient in Zavesca (miglustat), which is an oral drug used to inhibit the biosynthesis of macromolecular substrates that accumulate pathologically in glycosphyinolipidoses such as type I Gaucher disease. While repurposing of drugs is a common practice in pharma, allowing an existing licensed drug to be reused for a different indication, this step has never been taken for cosmetic purposes. Since imino-sugar derivatives may hold potential as depigmenting agents and they could be considered as cosmetic candidates for the treatment of various pigmentary conditions such as senile lentigines, melasma, etc. (Costin et al., 2002), this possibility of repurposing is intriguing and appealing.

Our analysis of the studies reporting data regarding the impact of pollution and tobacco on skin pigmentation revealed two key aspects: 1) there is more research conducted and/or published on pollution compared to tobacco, with a visible focus on PM; and 2) overall, the NAMs are used more frequently compared to animal or clinical studies, which is an approach we encourage to continue and be further explored.

For example, we identified 16 manuscripts reporting on pollution’s contribution to skin hyperpigmentation of which 11 addressed the PM (and 2 of those were specifically on DEPs), 4 were on PAHs and 1 for PCBs (Table 1) in terms of the type of toxicant. Regarding the study design, the manuscripts reported a total number of 32 experiments conducted as follows (determined using the length of treatment column): 7 clinical studies, 15 cell-based methods, 2 based on reconstructed tissue models, 4 on ex vivo explants and 4 using animal test systems. As already mentioned, PM was thoroughly investigated using a comprehensive portfolio of in vitro, in vivo, ex vivo and clinical approaches, thus providing a fuller picture on how PM impacts skin pigmentation, at a macroscopic and molecular, mechanistic level. This approach based primarily on NAMs should be expanded and applied for the research of other pollutants in order to obtain fast, human-relevant, reliable mechanistic insights. The cell-based assays used to investigate pollution’s contribution to hyperpigmentation were based on primary or immortalized human keratinocytes or melanocytes of various degrees of pigment producing capacity; in rare cases mouse melanocytes were used (Krutmann et al., 2008; Luecke et al., 2010; Abbas et al., 2017). In general, the endpoints of interest were conducted for safety assessment (viability, apoptosis) or to gain mechanistic knowledge on pollution’s impact on the key players of the melanogenic pathway. Of these assays, many were biochemical (enzymatic activity, melanin content) (Suo et al., 2020; Shi et al., 2021), histological, based on gene and protein expression (Suo et al., 2020; Yang et al., 2020; Shi et al., 2021; Yang et al., 2022), including key markers of various intracellular pathways. Of similar versatility in terms of endpoints and data output were the ex vivo platforms and those based on reconstructed tissue models (Krutmann et al., 2020; Grether-Beck et al., 2021). The animal studies usually employed histological endpoints or assessed melanin content, melanocyte density (Krutmann et al., 2008; Abbas et al., 2017), while clinical studies were focused primarily on the impact of pollutants on skin color at a macroscopic level (Vierkötter et al., 2010; Peng et al., 2017; Nkengne et al., 2021) (refer to Section 4.3; Subsection 4.3.3. Endpoints for more details on endpoints).

For tobacco research analyzed in our manuscript (Table 1), the majority of studies were clinical (8), with a small number of pre-clinical studies (3) based on cell culture models using human melanocytes to address the effect of cigarette smoke (Nakamura et al., 2013) and nicotine (Delijewski et al., 2014) on enzymatic activity relating to melanin production. While the majority of the clinical studies address skin pigmentation on a surface level, few others have collected more than “skin deep” measurements by analyzing bodily fluids for enzymes (King et al., 2009; Dalrymple et al., 2022) and metabolites (Liakoni et al., 2019).

Our analysis draws encouraging conclusions regarding the importance and position of NAMs in cosmetic research focused on environmental toxicants and provides an up-to-date view to support the advances made by this field relying less and less on animal testing. Using NAMs provides several advantages over animal or clinical studies to evaluate the effects of air pollutants and tobacco on skin pigmentation and aging. Multiple alternative methods based on the 3Rs were developed in response to increasing ethical concerns regarding animal experimentation (Russell and Burch, 1959). The 3Rs principles were first used in 1998 when the United Kingdom implemented the first total ban on animal-tested cosmetic products. Besides overcoming ethical concerns related to animal test systems, in vitro methodologies offer other scientific advantages over animal studies such as reproducibility and high throughput, which support complex mechanistic studies to investigate the activity of toxicants on skin and the design of actives to counteract them. Animal or clinical studies are reliant on availability of animal species and human volunteers, which can increase the time to recruit and conduct these lengthy studies that provide limited molecular insights about cellular pathways. Animal testing can be performed on a variety of species, however the results are not entirely reflective of a human skin exposure mainly because of the differences in architectures and immune responses between animal and human skin. With careful selection, proper training and skillful handling, NAMs are now indispensable for modern dermatology and skin aging research. In vitro anti-pollution tests facilitate a deeper understanding of changes occurring in the skin cells. One of the biggest advantages of these assays is the control of specified conditions and the endless possibilities to customize the testing parameters to fit the purpose of the experiments. In an animal test system or in clinical studies, this flexibility is not always possible, thus limiting the mechanistic data output.

We integrated in Table 1 the existing studies that took advantage of the knowledge reported on mechanisms of action used by pollution and tobacco on skin pigmentation to design efficacious actives to counteract their deleterious effects. We identified a total of 5 reports and all of those only addressed the efficacy of actives on pollution exposure and impact on skin pigmentation. Of the total of 14 experiments these studies reported on, 1 was a clinical study, 9 were cell-based and 4 used ex vivo human explants. Even though based on a relatively small number of studies, our analysis draw several important conclusions as basis for future directions discussed in more details later: 1) efficacy of raw ingredients and finished products to counteract tobacco’s impact on skin pigmentation is either not researched or published on; 2) it is encouraging to see that no animal studies and a limited number (1) of clinical studies were conducted while the vast majority of the results were generated using cell- and explant-based experiments. The limited number of publications is somewhat understood or justified given the competitive market and the need to maintain a reasonable margin without disclosing confidential intellectual property information. However, to the benefit of the consumer, of the science supporting safe and efficacious products, encouraging the scientists to publish will avoid duplicating efforts and delaying launching of important products on the market.

There exist several active compounds, natural extracts and vitamins that have been investigated and showed capacity to inhibit or reverse the effects of pollution on skin pigmentation. Cultural preference plays a major role in the design and priority testing of anti-pollution products. For example, while tanned skin is often considered attractive in the United States, many Asian countries consider fair skin to be preferable (Khmaladze et al., 2020) and hyperpigmentation due to pollution is a concern. Highly populated cities in China, India and Pakistan have a correspondingly high level of pollution which is a growing consumer concern when considering cosmetic products (Mistry, 2017). This growing global awareness of pollution affecting the skin generated an increased cultural demand for anti-pollution products.

As outlined in Table 1, in vitro, ex vivo and clinical studies have all been used to assess the efficacy of these products. Clinical studies can address changes in skin color and epidermal morphological changes. In vitro methods (cell-based) can address pigmentation changes the human eye cannot identify such as gene expression, cell viability or death, cytokine activity and melanin content. Ex vivo models can also address many of these “below the surface” activities but can be more difficult to obtain as a reliable test system, in addition to the population variability that should be considered. In this context, science advanced tremendously and provides nowadays commercially available pigmented reconstructed tissue models that can not only eliminate the sourcing issue and population variability, but also offer the possibility to compare models of various phenotypes and explore their responses to toxicants in terms of skin pigmentation (see Section 4.3; Subsection 4.3.1. Test system).

Natural extracts from plants such as Deschampsia Antarctica (Milani et al., 2019; Fernández-Martos et al., 2021) and Korean Red Ginseng (Moon et al., 2022) were evaluated for anti-pollution claims. In a clinical study, Deschampsi Antarctica reduced dark spots (Milani et al., 2019); the study by Fernández-Martos et al., 2021 supported mechanistically the claims on this natural extract and identified the AhR and inflammatory pathways as those on which the extract is effective. Furthermore, the same study used an ex vivo human skin model that was pre-treated with Deschampsia Antarctica and then exposed to different pollutants. The pre-treatment prevented downregulation of E-cadherin receptors and overexpression of AhR which were shown to be in response to pollutants (Fernández-Martos et al., 2021). Pre-treatment of human keratinocytes, co-culture of human keratinocytes and melanocytes and murine macrophages with Korean Red Ginseng mitigated the increase of melanin in response to PM10 treatment (Moon et al., 2022). These datasets indicate another key aspect of NAMs: they allow the design of the experiments accommodate a preventive (pre-treatment) or therapeutic (post-exposure treatment) approach which may be difficult to execute in a clinical or animal setting. In this way, the potential of these ingredients to prevent or to treat the effects of pollutants can be addressed and is supported with mechanistic justification.

Besides botanical extracts which are very appealing given their natural origin (for a comprehensive review see Mistry, 2017), various chemically derived actives were of interest and tested using cell-based assays or as formulations using explants. For example, actives A, B, C and D (see Table 1 for definitions) were tested on human keratinocytes, human dermal fibroblasts, human melanocytes and a co-culture of melanocytes and keratinocytes (Khmaladze et al., 2020). In general, a pre-treatment or treatment with the actives upregulated genes involved in the regulation of melanogenic pathway, and inhibited melanin and cytokine expression. A formulation of the active ingredients applied to an ex vivo human skin model also inhibited genes related to expression of pigmentation. Vitamins C and E formulated with active ingredients (see Table 1) applied onto an ex vivo human skin model reduced hyperpigmentation and gene expression of MMP-1 (Krutmann et al., 2020).

Overall, the products currently offered on the market address several mechanisms of action in terms of routes by which pollution impacts skin pigmentation: oxidative stress, inflammation, direct action on the melanin pathway (through MITF, Moon et al., 2022), and the AhR governed pathway (Table 1). The fact that the knowledge gained din academic research is now applied to design efficacious products is a huge step, which should be further encouraged, supported and published.

Based on our analysis of the pollutants reported to impact skin pigmentation, on the current status of test systems, endpoints and mechanisms of action, we summarize below several key findings and points in support of future directions research could consider in this field.