Particulate Matter 2.5 Induced Hyperpigmentation in Reconstructed Human Epidermis Model (MelaKutis®)

Epidemiological studies have established a link between fine particulate matter (PM2.5) and skin hyperpigmentation, but experimental evidence has been lacking. This study aimed to investigate the impact of PM2.5 on skin melanogenesis using normal human epidermal keratinocytes (NHEKs), melanocytes (NHEMs), and a reconstructed human epidermis model known as MelaKutis®. MelaKutis® is a three-dimensional (3D) skin model composed of human keratinocytes and melanocytes, which closely mimics the structure and metabolic features of natural human skin. The model allows for the stimulation of melanocytes located in the basal layer by external factors, leading to melanin production and hyperpigmentation.

To simulate the effect of PM2.5 on human skin, PM2.5 samples were collected in Beijing, China, during haze days from November 2018 to March 2019. An HY-1000 intelligent large-flow TSP sampler equipped with a PM2.5 cutter was used for quartz filter sampling at an average flow rate of 1000 L/min. The collected samples were immersed in 75% ethanol and ultrasonically shaken for 60 minutes in a water bath to elute particles. A high-concentration stock solution was prepared using sterile water and stored at -20°C. Benzo[a]pyrene (BAP), a toxic component of PM2.5, was purchased from Sigma-Aldrich Chemical and used as a positive control.

The effects of PM2.5 and BAP on the viability of NHEKs and NHEMs were determined using the MTT assay. Cells were incubated with various concentrations of PM2.5 (3.13, 6.25, 12.50, 25.00, 50.00, 100.00, 200.00, and 400.00 mg/mL) or BAP (0.50, 1.00, 2.50, 3.50, 5.00, 10.00, 20.00, and 50.00 mmol/L) at 37°C in 5% CO2 for 24 hours. After treatment with MTT dye for another 4 hours in the dark, absorbance was measured at 490 nm using a spectrophotometer. Cell morphology was observed under an inverted microscope at 200x magnification. All cell experiments were conducted in triplicate.

MelaKutis® models were maintained in M-TA medium at 37°C in 5% CO2. Different concentrations of PM2.5 (7.50 and 12.50 mg/mL) or BAP (3.00 and 5.00 mmol/L) were added to the surface of each model, and the medium was changed daily. After 7 days of treatment, the models were subjected to appearance observation, apparent brightness (L value) measurement, melanin content analysis, melanin distribution analysis, and tissue morphology examination using Hematoxylin-Eosin (H&E) staining. The models were divided into two groups: Group A for appearance observation, immunohistochemistry, and histology examinations, and Group B for L value and melanin content measurements.

The results showed that lower concentrations of PM2.5 (≤12.50 mg/mL) and BAP (≤5.00 mmol/L) did not significantly decrease cell viability or alter cell morphology in NHEKs and NHEMs. Higher concentrations, however, led to cell death and deformation in a dose-dependent manner. Based on these findings, PM2.5 = 12.50 mg/mL and BAP = 5.00 mmol/L were defined as the maximum safe concentrations (Cmax) for NHEKs and NHEMs. Subsequent experiments were conducted using PM2.5 concentrations of 7.50 and 12.50 mg/mL and BAP concentrations of 3.00 and 5.00 mmol/L.

Continuous stimulation of MelaKutis® with PM2.5 or BAP for 7 days did not cause obvious abnormalities in tissue morphology. However, compared to the control groups, the 12.50 mg/mL PM2.5-treated and 5.00 mmol/L BAP-treated MelaKutis® models became darker, with increased melanin particles mainly in the lower parts of the sections. Fissures were observed in the 5.00 mmol/L BAP group. The L* values of MelaKutis® treated with 12.50 mg/mL PM2.5 and 5.00 mmol/L BAP decreased significantly (P = 0.000), while the changes in the 7.50 mg/mL PM2.5 and 3.00 mmol/L BAP groups were not statistically significant (P = 1.000). The melanin content in the 12.50 mg/mL PM2.5 and 5.00 mmol/L BAP groups increased significantly (P = 0.001 and P = 0.047, respectively), but the changes in the 7.50 mg/mL PM2.5 and 3.00 mmol/L BAP groups were not significant (P = 0.948 and P = 1.000, respectively).

The study concluded that low concentrations of PM2.5 may induce hyperpigmentation in the reconstructed human epidermis model. Since no significant change in the number of keratinocytes or melanocytes was observed, it was speculated that PM2.5 induced hyperpigmentation mainly by increasing melanin synthesis. The stratum corneum, the strongest barrier against environmental stressors, likely prevented PM2.5 from directly contacting melanocytes. Instead, the proposed mechanism involves PM2.5 stimulating keratinocytes, which in turn activate melanocytes through paracrine and autocrine pathways, leading to increased melanin production.

The epidermal-melanin unit, composed of keratinocytes and melanocytes, plays a crucial role in melanogenesis. This unit responds rapidly to external stimuli, leading to melanin production. PM2.5 exposure has been shown to activate the aryl hydrocarbon receptor (AhR) signaling pathway, induce oxidative stress, and trigger an inflammatory cascade in keratinocytes. AhR, expressed in all skin cell types, induces the expression of target genes such as cytochrome P450 family enzymes, which metabolize polycyclic aromatic hydrocarbons (PAHs) and produce reactive oxygen species (ROS). PM2.5 also triggers ROS production through various pathways, promoting melanin production by melanocytes.

Inflammatory factors such as melanocyte-stimulating hormone (α-MSH), interleukin (IL)-1α, IL-1β, IL-6, IL-8, matrix metalloproteinase (MMP)-1, MMP-2, MMP-9, and tumor necrosis factor-α (TNF-α) are produced by keratinocytes in response to PM exposure. These factors are partly related to ROS and contribute to melanogenesis. α-MSH is a key paracrine cytokine secreted by keratinocytes that promotes melanin production. IL-1 enhances the secretion of endothelin-1 in keratinocytes, which increases melanocyte migration and differentiation.

The study also observed that the increased melanin was mainly distributed in the lower parts of the MelaKutis® sections, consistent with the natural metabolism of melanin. Melanosomes are synthesized in melanocytes and transferred to surrounding keratinocytes via endocytosis/exocytosis. As keratinocytes move upward during epidermal turnover, melanosomes are gradually digested and absorbed. The experimental duration of 7 days was shorter than the typical epidermal turnover time of 28 days, which may explain the predominant distribution of melanin in the lower sections.

Fissures observed in the 5.00 mmol/L BAP group may be related to the inflammatory response and increased dendritic degree of melanocytes. PM2.5 has been shown to damage the skin barrier by disrupting tight junctions in epithelial cells. The inflammatory factors produced by keratinocytes in response to PM2.5 can further induce the secretion of cytokines and adhesion molecules by epithelial cells, fibroblasts, and endothelial cells, leading to skin inflammation and barrier disruption. The presence of fissures in the BAP-treated group resembles the acantholysis observed in pemphigus vulgaris (PV), which is associated with MMPs, particularly MMP-9. PM2.5 upregulates MMP-1, MMP-2, and MMP-9 in keratinocytes, suggesting a possible role of MMPs in the observed fissures.

In summary, this study provides experimental evidence that low concentrations of PM2.5 can induce skin hyperpigmentation in a reconstructed human epidermis model. The proposed mechanism involves PM2.5 stimulating keratinocytes, which activate melanocytes through paracrine and autocrine pathways, leading to increased melanin production. Further in vitro and in vivo studies are needed to fully elucidate the mechanisms involved in PM2.5-induced hyperpigmentation.

doi.org/10.1097/CM9.0000000000001934

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