Quantitative analysis of the concentration of nano­carbon black originating from tire-wear particles in the road dust.

Exhaust and non-exhaust particulate matter (PM) is regarded as the most significant airborne during driving. Among the source of non-exhaust PM, the tire-wear particles (TWP) can be quantified using pyrolysis-gas chromatography/mass spectrometry (Py-GC-MS). TWPs are fragmented by continuous weathering once exposed to the road. Approximately 5 wt% of carbon black (CB) bound in the rubber matrix of TWPs tends to detach from it, and thus some portion of free-bound CB could be co-existed in the road dust. Although there are existing methods for analyzing pure CB and TWPs, only few analysis techniques on the amount of free-bound CB in contaminant samples have been discovered. Herein, we propose a method for quantifying the total and free-bound CB in road dust using a combination of four analytical tools: a semi-continuous carbon analyzer, element analyzer, thermogravimetric analyzer, and Py-GC-MS. This study is the first attempt in quantifying the concentration of nano-CB derived from TWPs in road dust. The proposed methodology was applied to the samples collected from five open sites, three closed sites, and four types of air conditioner (AC) filters in passenger vehicles. Compared to the samples obtained in open sites, the road dust in the closed sites exhibited 21.5 times higher TWP content (59,747 mg/kg) and 5.1 times higher free-bound CB content (14,632 mg/kg). In addition, unintentional driver respiratory exposure to PM fixed in the vehicle filters was discovered owing to the increase in CB and TWP contents in aged AC filters.

Estimation of the concentration of nano-carbon black in tire-wear particles using emission factors of PM10, PM2.5, and black carbon.

Particulate matter (PM) from automobile exhaust has drastic effects on human health. The enforcement of environmental laws has controlled vehicle emissions and reduced the total PM. However, another significant source of PM is debris from tire wear, break wear, and road wear. In particular, tire-wear particles (TWPs) are further fragmented into nanoparticles, similar to the PMx or black carbon (BC) sources. As approximately 30 wt% of carbon black (CB) is used as filler in tires, TWPs can fragment into free-bound nano-CB. This study evaluates the emission factors of BC from the ternary plots of PMx and BC to estimate the concentration of nano-CB in TWPs. Based on the emission factors of BC for TWP, approximately 500 monitoring data points were acquired at four different sites. Semi-closed sites in a field measurement test have 2.9-4.0 times larger BC concentration than open sites. The mass concentration of nano-CB evaluated with the BC data and emission factors at the open sites is 22.47-23.96 ng/m3, whereas that at the semi-closed sites is 66.32-90.33 ng/m3. Transmission electron microscopy analysis with scanning mobility particle sizer and selected-area electron diffraction reveals grape-like aggregated nanoparticles, which is considered as CB. To compare the effect of the washing out of airborne particulates by rain, further analysis is conducted on the interior and exterior of the tunnel on a rainy day. While the concentration of PMs was effectively reduced by rainfall, the amount of BC and CB in the interior of a tunnel was not changed. Namely, even under rainfall, nano-CB still exists in the tunnels and thus free-bound CB and nanoparticles released from TWP will be effected on the human health.

Development of an Aged Full-Thickness Skin Model Using Flexible Skin-on-a-Chip Subjected to Mechanical Stimulus Reflecting the Circadian Rhythm.

The skin is subject to both intrinsic aging caused by metabolic processes in the body and extrinsic aging caused by exposure to environmental factors. Intrinsic aging is an important obstacle to in vitro experimentation as its long-term progression is difficult to replicate. Here, we accelerated aging of a full-thickness skin equivalent by applying periodic mechanical stimulation, replicating the circadian rhythm for 28 days. This aging skin model was developed by culturing a full-thickness, three-dimensional skin equivalent with human fibroblasts and keratinocytes to produce flexible skin-on-a-chip. Accelerated aging associated with periodic compressive stress was evidenced by reductions in the epidermal layer thickness, contraction rate, and secretion of Myb. Increases in ß-galactosidase gene expression and secretion of reactive oxygen species and transforming growth factor-ß1 were also observed. This in vitro aging skin model is expected to greatly accelerate drug development for skin diseases and cosmetics that cannot be tested on animals.

Effect of α-Lipoic Acid on the Development of Human Skin Equivalents Using a Pumpless Skin-on-a-Chip Model.

Owing to the prohibition of cosmetic animal testing, various attempts have recently been made using skin-on-a-chip (SOC) technology as a replacement for animal testing. Previously, we reported the development of a pumpless SOC capable of drug testing with a simple drive using the principle that the medium flows along the channel by gravity when the chip is tilted using a microfluidic channel. In this study, using pumpless SOC, instead of drug testing at the single-cell level, we evaluated the efficacy of α-lipoic acid (ALA), which is known as an anti-aging substance in skin equivalents, for skin tissue and epidermal structure formation. The expression of proteins and changes in genotyping were compared and evaluated. Hematoxylin and eosin staining for histological analysis showed a difference in the activity of fibroblasts in the dermis layer with respect to the presence or absence of ALA. We observed that the epidermis layer became increasingly prominent as the culture period was extended by treatment with 10 µM ALA. The expression of epidermal structural proteins of filaggrin, involucrin, keratin 10, and collagen IV increased because of the effect of ALA. Changes in the epidermis layer were noticeable after the ALA treatment. As a result of aging, damage to the skin-barrier function and structural integrity is reduced, indicating that ALA has an anti-aging effect. We performed a gene analysis of filaggrin, involucrin, keratin 10, integrin, and collagen I genes in ALA-treated human skin equivalents, which indicated an increase in filaggrin gene expression after ALA treatment. These results indicate that pumpless SOC can be used as an in vitro skin model similar to human skin, protein and gene expression can be analyzed, and it can be used for functional drug tests of cosmetic materials in the future. This technology is expected to contribute to the development of skin disease models.

Differentiation of carbon black from black carbon using a ternary plot based on elemental analysis.

Carbon black (CB) is composed of engineered nanoparticles that are commercially produced by partial combustion of hydrocarbons. It is mainly used as a reinforcing agent in vehicle tires. Although the potential health effects of CB have been investigated extensively, some toxicological reports interchange CB with black carbon (BC), which has similar features, thereby misusing the term. BC is an undesirable byproduct of the incomplete combustion of fuels. Therefore, there is a need to differentiate CB from the unintentionally produced nanomaterials (BC) in nano-toxicity, environmental, and human health studies. To distinguish clearly CB from BC, it is important to find the key parameters from several characteristics of two substances. The fundamental physicochemical properties of commercial CB and naturally formed BC were conducted. Based on the elemental analysis, we found three key factors, which could be used to differentiate the CB from BC. And thus, herein, we propose a ternary plot of the aH/C-log(C/b)-1/H combination for use in differentiating CB from BC. The plot of the 100H/C-log(C/10)-1/H combination of elemental ratios separated the CB domain from the BC domain symmetrically. The effectiveness of the ternary chart was validated using 37 samples (nine samples in this work, 25 sample results taken from references studies, and three samples from the field). Therefore, the ternary plot could be used as a prescreening tool for distinguishing CB from BC.

Coenzyme Q10 Efficacy Test for Human Skin Equivalents Using a Pumpless Skin-On-A-Chip System.

A human skin equivalent (HSE) composed of the epidermis and dermis is cultured using a pumpless skin-on-a-chip system to supply cultures the desired flow rate using gravity flow without a pump or an external tube connection. Coenzyme Q10 efficacy is tested by adjusting its concentration, as it is known to have anti-aging and antioxidant effects in culture solutions. The relationship between the contraction rate of a full-thickness human skin equivalent and secreted transforming growth factor (TGF) ß-1 is analyzed via enzyme-linked immunosorbent assay (ELISA). Following hematoxylin and eosin (H&E) staining, an image of the skin equivalent is analyzed to measure the epidermal layer's thickness. The cell density and differentiation of the dermis layer are investigated. Gene and protein expression in the dermal and epidermal layers are quantitatively analyzed using quantitative real time polymerase chain reaction (qPCR) and immunohistochemical staining. As the coenzyme Q10 treatment concentration increased, the number of cells per unit area and the thickness of the epidermal layer increased, the expression level of filaggrin increased, and the contraction rate of full-thickness HSE was proportional to the amount of TGF ß-1 secreted.