Determination of submicron aerosols effective density using two-stage low-pressure impactor and aerosol electrometer based on pre-measured particle size distribution.

A novel methodology was presented for determining the representative effective density of aerosols of a given size distribution, using a lab-made two-stage low-pressure impactor and an aerosol electrometer. Electrical currents upstream (Imeasured, up) and downstream (Imeasured, down) of the 2nd stage of the impactor were measured using a corona charger and the aerosol electrometer. In addition, the electrical currents upstream (Icalculated, up) and downstream (Icalculated, down) of the 2nd stage of the impactor were calculated using the aerosol charging theory. Then, the difference between the ratio of Imeasured,down to Imeasured,up and the ratio of Icalculated,down to Icalculated,up was iterated with varying the presumed effective density until the difference was smaller than 0.001. The methodology was validated using poly-disperse sodium chloride (NaCl) particles. The effective densities of ambient aerosols were then obtained from indoor and outdoor environments and compared with those calculated from a relation between mobility (scanning mobility particle sizer (SMPS) measurement) and aerodynamic (electrical low-pressure impactor (ELPI) measurement) diameters. Compared to the effective densities obtained with SMPS and ELPI measurements, the effective densities obtained using the methodology introduced in this paper differed within 10 % deviation, depending on measurement location. After an averaged effective density for given size distribution is obtained at a measurement location, the number-based size distribution can be easily converted to mass-based size distribution using the representative effective density.

The Accomplishments of KoCVAM in the Development and Implementation of Alternative Methods in Korea.

The Korean Center for the Validation of Alternative Methods (KoCVAM), which promotes the Three Rs principles and the use of alternative methods in Korea, has been operating within the Toxicological Screening and Testing Division of the Ministry of Food and Drug Safety (MFDS) since 2009. KoCVAM has exchanged opinions and information on the development and validation of non-animal alternative test methods as part of the International Cooperation on Alternative Test Methods (ICATM), and provided input into draft OECD Test Guidelines (TGs). Several Korean laws (e.g. the Cosmetics Act) encourage the use of alternative test methods for chemical testing and assessment. To promote and support the use of alternative test methods in the country, KoCVAM has published information and provided training on the national guidelines, which are based on the OECD TGs. In addition, KoCVAM has held annual training workshops on alternative test methods, to help Korean research institutions (including GLP test facilities) to implement them. In addition, by helping to develop and validate alternative test methods that were adopted in OECD TG 442B, TG 492 and TG 439, KoCVAM has contributed to the enhanced competitiveness of Korean industry on the worldwide stage.

Size-classified monitoring of ATP bioluminescence for rapid assessment of biological distribution in airborne particulates.

The COVID-19 pandemic ignited massive research into the rapid detection of bioaerosols. In particular, nanotechnology-based detection strategies are proposed as alternatives because of issues in bioaerosol enrichment and lead time for molecular diagnostics; however, the practical implementation of such techniques is still unclear due to obstacles regarding the large research and development effort and investment for the validation. The use of adenosine triphosphate (ATP) bioluminescence (expressed as relative luminescence unit (RLU) per unit volume of air) of airborne particulate matter (PM) to determine the bacterial population as a representative of the total bioaerosols (viruses, bacteria, and fungi) has been raised frequently because of the high reponse speed, resolution, and compatibility with culture-based bioaerosol monitoring. On the other hand, additional engineering attempts are required to confer significance because of the size-classified (bioluminescence for different PM sizes) and specific (bioluminescence per unit PM mass) biological risks of air for providing proper interventions in the case of airborne transmission. In this study, disc-type impactors to cut-off aerosols larger than 1 µm, 2.5 µm, and 10 µm were designed and constructed to collect PM1, PM2.5, and PM10 on sampling swabs. This engineering enabled reliable size-classified bioluminescence signals using a commercial ATP luminometer after just 5 min of air intake. The simultaneous operations of a six-stage Andersen impactor and optical PM spectrometers were conducted to determine the correlations between the resulting RLU and colony forming unit (CFU; from the Andersen impactor) or PM mass concentration (deriving specific bioluminescence).

Comparative sub-chronic toxicity studies in rats of two indistinguishable herbal plants, Cynanchum wilfordii (Maxim.) Hemsley and Cynanchum auriculatum Royle ex Wight.

Sub-chronic toxicity studies using rats have been conducted for Cynanchum wilfordii (Maxim.) Hemsley (CW) and Cynanchum auriculatum Royle ex Wight (CA). CW water extract didn't show any adverse effects whereas administering CW powder decreased body weights in complication with decreased food consumptions. In the case of CA water extract, triglyceride and absolute/relative liver weights were elevated and vacuolation was observed in liver. Treated CA powder in male rats increased alanine aminotransferase and aspartate aminotransferase and induced single cell necrosis and multinucleated hepatocyte in liver. As for female rats, increased absolute/relative weights and hypertrophy/vacuolation in adrenal glands and vacuolation in ovaries were observed when administered CA powder. In conclusion, no observed adverse effect level (NOAEL) of CW water extract was over 5000 mg/kg/day, while NOAEL of CW powder was 700 mg/kg/day for female and 150 mg/kg/day for male. In case of CA, NOAEL of water extract was 1500 mg/kg/day for male and 2000 mg/kg/day for female, while NOAEL of powder was 150 mg/kg/day for both gender. To the best of our knowledge, this is the first sub-chronic toxicity study on the adverse effects, target organs and its dose levels of C. wilfordii (Maxim.) Hemsley and C. auriculatum Royle ex Wight following GLP protocols.

Colony-Forming Efficiency Assay to Assess Nanotoxicity of Graphene Nanomaterials.

The nano-market has grown rapidly over the past decades and a wide variety of products are now being manufactured, including those for biomedical applications. Despite the widespread use of nanomaterials in various industries, safety and health effects on humans are still controversial, and testing methods for nanotoxicity have not yet been clearly established. Nanomaterials have been reported to interfere with conventional cytotoxicity tests due to their unique properties, such as light absorption or light scattering. In this regard, the colony-forming efficacy (CFE) assay has been suggested as a suitable test method for testing some nanomaterials without these color-interferences. In this study, we selected two types of GNPs (Graphene nanoplatelets) as test nanomaterials and evaluated CFE assay to assess the cytotoxicity of GNPs. Moreover, for further investigation, including expansion into other cell types, GNPs were evaluated by the conventional cytotoxicity tests including the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), Cell Counting Kit-8 (CCK-8), and Neutral red uptake (NRU) assay using MDCK, A549 and HepG2 cells. The results of CFE assay suggest that this test method for three cell lines can be applied for GNPs. In addition, the CFE assay was able to evaluate cytotoxicity regardless more accurately of color interference caused by residual nanomaterials.

Formation of cellular close-ended tunneling nanotubes through mechanical deformation.

Membrane nanotubes or tunneling nanotubes (TNTs) that connect cells have been recognized as a previously unidentified pathway for intercellular transport between distant cells. However, it is unknown how this delicate structure, which extends over tens of micrometers and remains robust for hours, is formed. Here, we found that a TNT develops from a double filopodial bridge (DFB) created by the physical contact of two filopodia through helical deformation of the DFB. The transition of a DFB to a close-ended TNT is most likely triggered by disruption of the adhesion of two filopodia by mechanical energy accumulated in a twisted DFB when one of the DFB ends is firmly attached through intercellular cadherin-cadherin interactions. These studies pinpoint the mechanistic questions about TNTs and elucidate a formation mechanism.

In Vivo Evaluation of the Oral Toxicity of the Chlorobutanol.

Chlorobutanol (CB) is used as a preservative in cosmetics and has antibacterial activity. This study investigated the single- and repeated-dose 28-day oral toxicity of a CB solvent in Sprague Dawley (SD) rats. For the single-dose oral toxicity study, a dose of 62.5, 125, or 250 mg per kg of body weight (mg/kg b.w.) of CB was given once orally via gavage. For the repeated-dose 28-day toxicity study, the high dose was set as 100 mg/kg b.w./day, and the middle, middle-low, and low doses were set to 50, 25, and 12.5 mg/kg b.w./day, respectively. Body weight was not significantly changed in the repeated-dose toxicity study. Relative liver and kidney weights were significantly increased in both sexes of the 100 mg/kg b.w./day treatment group. However, there were histopathological changes in liver and kidney for females and males, respectively. These data suggested that the approximate lethal dose (ALD) of CB was over 250 mg/kg b.w./day in the single-dose study, and the no adverse effect level (NOAEL) for CB was over 50 and 12.5 mg/kg b.w./day for female and male rats in the repeated-dose toxicity study.

Effect of Pulmonary Inflammation by Surface Functionalization of Zinc Oxide Nanoparticles.

Zinc oxide nanoparticles (ZnO NPs) are used in various industries such as food additives, cosmetics, and biomedical applications. In this study, we evaluated lung damage over time by three types of ZnO NPs (L-serine, citrate, and pristine) following the regulation of functional groups after a single intratracheal instillation to rats. The three types of ZnO NPs showed an acute inflammatory reaction with increased LDH and inflammatory cell infiltration in the alveoli 24 h after administration. Especially in treatment with L-serine, citrate ZnO NPs showed higher acute granulocytic inflammation and total protein induction than the pristine ZnO NPs at 24 h. The acute inflammatory reaction of the lungs recovered on day 30 with bronchoalveolar fibrosis. The concentrations of IL-4, 6, TNF-α, and eotaxin in the bronchoalveolar lavage fluid (BALF) decreased over time, and the levels of these inflammation indicators are consistent with the following inflammatory cell data and acute lung inflammation by ZnO NP. This study suggests that single inhalation exposure to functionalized ZnO NPs may cause acute lung injury with granulocytic inflammation. Although it can recover 30 days after exposure, acute pulmonary inflammation in surface functionalization means that additional studies of exposure limits are needed to protect the workers that produce it.

The Research of Toxicity and Sensitization Potential of PEGylated Silver and Gold Nanomaterials.

Polyethylene glycol (PEG) is a polymer used for surface modification of important substances in the modern pharmaceutical industry and biopharmaceutical fields. Despite the many benefits of PEGylation, there is also the possibility that the application and exposure of the substance may cause adverse effects in the body, such as an immune response. Therefore, we aimed to evaluate the sensitization responses that could be induced through the intercomparison of nanomaterials of the PEG-coated group with the original group. We selected gold/silver nanomaterials (NMs) for original group and PEGylated silver/gold NMs in this study. First, we measured the physicochemical properties of the four NMs, such as size and zeta potential under various conditions. Additionally, we performed the test of the NM's sensitization potential using the KeratinoSens™ assay for in vitro test method and the LLNA: 5-bromo-2-deoxyuridine (BrdU)-FCM for in vivo test method. The results showed that PEGylated-NMs did not lead to skin sensitization according to OECD TG 442 (alternative test for skin sensitization). In addition, gold nanomaterial showed that cytotoxicity of PEGylated-AuNMs was lower than AuNMs. These results suggest the possibility that PEG coating does not induce an immune response in the skin tissue and can lower the cytotoxicity of nanomaterials.

Determination of Residual Triflumezopyrim Insecticide in Agricultural Products through a Modified QuEChERS Method.

A rapid and simple analytical method for triflumezopyrim, a new class of mesoionic insecticides and commercialized molecules from DuPont, was developed with a modified QuEChERS method. The pH adjustment was used to improve the extraction efficiency of acetonitrile solvent, and dispersive solid-phase extraction was employed for the clean-up process. The five selected food commodities were used to verify the present optimized method, which displayed good linearity with an excellent correlation coefficient (R2 = 0.9992-0.9998) in the 0.003-0.30 mg/kg calibration range. The method limits of detection (LOD) and quantification (LOQ) were determined to be a value of 0.003 and 0.01 mg/kg, respectively. The mean recovery for the triflumezopyrim was in the 89.7-104.3% range. The relative standard deviations were ≤9.8% for intra- (n = 5) and inter-day (n = 15) precisions at concentrations of 0.01, 0.1, and 0.5 mg/kg in the five representative samples. The matrix effect has been calculated to confirm the effect during ionization of the analyte in the UPLC-MS/MS. The matrix effects of the instrumental analysis showed that triflumezopyrim was less susceptible to matrices. The proposed analytical method in this study has effectively improved the accuracy, selectivity, and sensitivity for the determination of triflumezopyrim in agricultural commodities; therefore, it can serve as a reference method for the establishment of maximum residue limits (MRLs).

Corrigendum: Copper and Cobalt Ions Released From Metal Oxide Nanoparticles Trigger Skin Sensitization.

[This corrects the article DOI: 10.3389/fphar.2021.627781.].

Skin Sensitization Evaluation of Carbon-Based Graphene Nanoplatelets.

Graphene nanoplatelets (GNPs) are one of the major types of carbon based nanomaterials that have different industrial and biomedical applications. There is a risk of exposure to GNP material in individuals involved in their large-scale production and in individuals who use products containing GNPs. Determining the exact toxicity of GNP nanomaterials is a very important agenda. This research aimed to evaluate the skin sensitization potentials induced by GNPs using two types of alternative to animal testing. We analyzed the physicochemical characteristics of the test material by selecting a graphene nanomaterial with a nano-size on one side. Thereafter, we evaluated the skin sensitization effect using an in vitro and an in vivo alternative test method, respectively. As a result, we found that GNPs do not induce skin sensitization. In addition, it was observed that the administration of GNPs did not induce cytotoxicity and skin toxicity. This is the first report of skin sensitization as a result of GNPs obtained using alternative test methods. These results suggest that GNP materials do not cause skin sensitization, and these assays may be useful in evaluating the skin sensitization of some nanomaterials.

Copper and Cobalt Ions Released from Metal Oxide Nanoparticles Trigger Skin Sensitization.

Human skins are exposed to nanomaterials in everyday life from various sources such as nanomaterial-containing cosmetics, air pollutions, and industrial nanomaterials. Nanomaterials comprising metal haptens raises concerns about the skin sensitization to nanomaterials. In this study, we evaluated the skin sensitization of nanomaterials comparing metal haptens in vivo and in vitro. We selected five metal oxide NPs, containing copper oxide, cobalt monoxide, cobalt oxide, nickel oxide, or titanium oxide, and two types of metal chlorides (CoCl2 and CuCl2), to compare the skin sensitization abilities between NPs and the constituent metals. The materials were applied to KeratinoSensTM cells for imitated skin-environment setting, and luciferase induction and cytotoxicity were evaluated at 48 h post-incubation. In addition, the response of metal oxide NPs was confirmed in lymph node of BALB/C mice via an in vivo method. The results showed that CuO and CoO NPs induce a similar pattern of positive luciferase induction and cytotoxicity compared to the respective metal chlorides; Co3O4, NiO, and TiO2 induced no such response. Collectively, the results implied fast-dissolving metal oxide (CuO and CoO) NPs release their metal ion, inducing skin sensitization. However, further investigations are required to elucidate the mechanism underlying NP-induced skin sensitization. Based on ion chelation data, metal ion release was confirmed as the major "factor" for skin sensitization.

Evaluation of the Skin Sensitization Potential of Carbon Nanotubes Using Alternative In Vitro and In Vivo Assays.

Carbon nanotubes (CNTs) are one of the major types of nanomaterials that have various industrial and biomedical applications. However, there is a risk of accidental exposure to CNTs in individuals involved in their large-scale production and in individuals who use products containing CNTs. This study aimed to evaluate the skin sensitization induced by CNTs using two alternative tests. We selected single-wall carbon nanotubes and multi-walled carbon nanotubes for this study. First, the physiochemical properties of the CNTs were measured, including the morphology, size, and zeta potential, under various conditions. Thereafter, we assessed the sensitization potential of the CNTs using the ARE-Nrf2 Luciferase KeratinoSens™ assay, an in vitro alternative test method. In addition, the CNTs were evaluated for their skin sensitization potential using the LLNA: BrdU-FCM in vivo alternative test method. In this study, we report for the first time the sensitization results of CNTs using the KeratinoSens™ and LLNA: BrdU-FCM test methods in this study. This study found that both CNTs do not induce skin sensitization. These results suggest that the KeratinoSens™ and LLNA: BrdU-FCM assay may be useful as alternative assays for evaluating the potential of some nanomaterials that can induce skin sensitization.

Orally Administered 6:2 Chlorinated Polyfluorinated Ether Sulfonate (F-53B) Causes Thyroid Dysfunction in Rats.

The compound 6:2 chlorinated polyfluorinated ether sulfonate (F-53B), a replacement for perfluorooctanesulfonate (PFOS) in the electroplating industry, has been widely detected in numerous environmental matrices, human sera, and organisms. Due to regulations that limit PFOS use, F-53B use is expected to increase. Therefore, in this study, we performed a subchronic oral toxicity study of F-53B in Sprague Dawley (SD) rats. F-53B was administered orally once daily to male and female rats for 28 days at doses of 5, 20, and 100 mg/kg/day. There were no toxicologically significant changes in F-53B-treated rats, except in the thyroid gland. However, F-53B slightly reduced the serum concentrations of thyroid hormones, including triiodothyronine and thyroxine, compared with their concentrations in the vehicle group. F-53B also induced follicular hyperplasia and was associated with increased thyroid hormone biosynthesis-associated protein expression. These results demonstrate that F-53B is a strong regulator of thyroid hormones in SD rats as it disrupts thyroid function. Thus, caution should be exercised in the industrial application of F-53B as an alternative for PFOS.

Comparison of Sample Preparation and Determination of 60 Veterinary Drug Residues in Flatfish Using Liquid Chromatography-Tandem Mass Spectrometry.

This study was performed to optimize the analytical method for multi-residues of 60 compounds in flatfish samples. Three sample preparation methods were tested to identify the optimal recovery conditions for target analytes. As a result, 10 mL of water/acetonitrile (1:4, v/v) was used to extract analytes from fish samples. For purification, C18 and 10 mL of acetonitrile saturated hexane were used to treat the samples. After evaporation and reconstitution, the fish samples were analyzed by ultra-performance liquid chromatography-tandem mass spectrometry. The proposed method was validated according to the CODEX guidelines (CAC/GL-71). Our results showed the recoveries of 73.2%-115% and coefficients of variation of 1.6%-22.1%. The limit of quantification was 0.0005-0.005 mg/kg in the fishery products. In analysis of real samples, no samples exceeded the limit of quantification. This analytical method can be used for multi-residue screening and confirmation of the residues of veterinary drugs in fishery products.

Convolutional neural network model to predict causal risk factors that share complex regulatory features.

Major progress in disease genetics has been made through genome-wide association studies (GWASs). One of the key tasks for post-GWAS analyses is to identify causal noncoding variants with regulatory function. Here, on the basis of >2000 functional features, we developed a convolutional neural network framework for combinatorial, nonlinear modeling of complex patterns shared by risk variants scattered among multiple associated loci. When applied for major psychiatric disorders and autoimmune diseases, neural and immune features, respectively, exhibited high explanatory power while reflecting the pathophysiology of the relevant disease. The predicted causal variants were concentrated in active regulatory regions of relevant cell types and tended to be in physical contact with transcription factors while residing in evolutionarily conserved regions and resulting in expression changes of genes related to the given disease. We demonstrate some examples of novel candidate causal variants and associated genes. Our method is expected to contribute to the identification and functional interpretation of potential causal noncoding variants in post-GWAS analyses.

Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis.

Hematogenous metastasis is a multistep, selectin-regulated process whose mechanisms remain poorly understood. To investigate this biological pathway of cancer dissemination and better understand circulating cancer cells, we developed a high-throughput methodology that integrates organ-on-chip-like microfluidic and photoconvertible protein technologies. Our approach can ascribe single-cell velocity as a traceable cell property for off-chip analysis of the direct relationships between cell molecular profiles and adhesive phenotypes in the context of physiologically relevant fluid flow. We interrogate how natively expressed selectin ligands relate to colon cancer cell rolling frequencies and velocities and provide context for previously reported disparities in in vitro and in vivo models of selectin-mediated adhesion and metastasis. This integrated methodology represents a versatile approach for the development of anti-metastatic therapeutics as well as to generate and test mechanistic hypotheses regarding spatiotemporal processes that occur over timescales of seconds to hours with single-cell resolution.

Selection on the regulation of sympathetic nervous activity in humans and chimpanzees.

Adrenergic α2C receptor (ADRA2C) is an inhibitory modulator of the sympathetic nervous system. Knockout mice for this gene show physiological and behavioural alterations that are associated with the fight-or-flight response. There is evidence of positive selection on the regulation of this gene during chicken domestication. Here, we find that the neuronal expression of ADRA2C is lower in human and chimpanzee than in other primates. On the basis of three-dimensional chromatin structure, we identified a cis-regulatory region whose DNA sequences have been significantly accelerated in human and chimpanzee. Active histone modification marks this region in rhesus macaque but not in human and chimpanzee; instead, repressive marks are enriched in various human brain samples. This region contains two neuron-restrictive silencer factor (NRSF) binding motifs, each of which harbours a polymorphism. Our genotyping and analysis of population genome data indicate that at both polymorphic sites, the derived allele has reached fixation in humans and chimpanzees but not in bonobos, whereas only the ancestral allele is present among macaques. Our CRISPR/Cas9 genome editing and reporter assays show that both derived nucleotides repress ADRA2C, most likely by increasing NRSF binding. In addition, we detected signatures of recent positive selection for lower neuronal ADRA2C expression in humans. Our findings indicate that there has been selective pressure for enhanced sympathetic nervous activity in the evolution of humans and chimpanzees.

P-, but not E- or L-, selectin-mediated rolling adhesion persistence in hemodynamic flow diverges between metastatic and leukocytic cells.

The ability of leukocytic cells to engage selectins via rolling adhesion is critical to inflammation, but selectins are also implicated in mediating metastatic dissemination. Using a microfluidic- and flow-based cell adhesion chromatography experimental and analytical technique, we interrogated the cell-subtype differences in engagement and sustainment of rolling adhesion on P-, E-, and L-selectin-functionalized surfaces in physiological flow. Our results indicate that, particularly at low concentrations of P-selectin, metastatic but not leukocytic cells exhibit reduced rolling adhesion persistence, whereas both cell subtypes exhibited reduced persistence on L-selectin and high persistence on E-selectin, differences not revealed by flow cytometry analysis or reflected in the extent or velocity of rolling adhesion. Conditions under which adhesion persistence was found to be significantly reduced corresponded to those exhibiting the greatest sensitivity to a selectin-antagonist. Our results suggest that potentially therapeutically exploitable differences in metastatic and leukocytic cell subtype interactions with selectins in physiological flow are identifiable through implementation of functional assays of adhesion persistence in hemodynamic flow utilizing this integrated, flow-based cell adhesion chromatography analytical technique.