Defining the reactivity of nanoparticles to peptides through direct peptide reactivity assay (DPRA) using a high pressure liquid chromatography system with a diode array detector.

The possibility of inducing skin sensitization reactions following exposure to various chemicals can lead to skin diseases, and the evaluation of skin sensitivity to such substances is very important. However, as animal tests for skin sensitization are prohibited, the OECD Test Guideline 442 C was designated as part of an alternative testing method. Therefore, in this study, the reactivity of cysteine and lysine peptides to nanoparticle substrates was identified through HPLC-DAD analysis according to the skin sensitization animal replacement test method specified in the OECD Test Guideline 442 C. In this study, all criteria for skin sensitization experiments specified in OECD Test Guideline 442 C were satisfied. As a result of analyzing the disappearance rates of cysteine and lysine peptides for the five types of nanoparticle substrates (TiO2, CeO2, Co3O4, NiO, and Fe2O3) using the established analytical method, all were identified as positive. Therefore, our findings suggest that basic data from this technique can contribute to skin sensitization studies by providing the depletion percentage of cysteine and lysine peptides for nanoparticle materials that have not yet been tested for skin sensitization.

Pre-validation study of spectrophotometric direct peptide reactivity assay (Spectro-DPRA) as a modified in chemico skin sensitization test method.

Skin sensitization is induced when certain chemicals bind to skin proteins. Direct peptide reactivity assay (DPRA) has been adopted by the OECD as an alternative method to evaluate skin sensitization by assessing a substance's reaction to two model peptides. A modified spectrophotometric method, Spectro-DPRA, can evaluate skin sensitization, in a high throughput fashion, to obviate some limitations of DPRA. Pre-validation studies for Spectro-DPRA were conducted to determine transferability and proficiency, within- and between-laboratory reproducibility, and predictive ability based on GLP principles at three laboratories (AP, KTR, and KCL). All laboratories confirmed high (> 90%) concordance for evaluating the sensitivity induced by ten chemical substances. The concordance among the three tests performed by each laboratory was 90% for AP, 100% for KTR, and 100% for KCL. The mean accuracy of the laboratories was 93.3% [compared to the standard operating procedure (SOP)]. The reproducibility among the three laboratories was as high as 86.7%; the accuracy was 86.7% for AP, 100% for KTR, and 86.7% for KCL (compared to the SOP). An additional 54 substances were assessed in 3 separate labs to verify the prediction rate. Based on the result, 29 out of 33 substances were classified as sensitizers, and 19 out of 21 identified as non-sensitizers; the corresponding sensitivity, specificity, and accuracy values were 87.9%, 90.5%, and 88.9%, respectively. These findings indicate that the Spectro-DPRA can address the molecular initiating event with improved predictability and reproducibility, while saving time and cost compared to DPRA or ADRA.

A self-emulsifying omega-3 fatty acids delivery system for enhanced gastro-intestinal absorption in rats.

Omega-3 fatty acids have many health benefits as they help to prevent and treat coronary artery disease, hypertension, diabetes mellitus, arthritis, and autoimmune disorders. Omega-3 fatty acids miscible in lecithin were found to spontaneously form microemulsions in water. The particle sizes of emulsions ranged from 300 to 800 nm and their morphologies were observed by optical microscopy. In vitro testing showed that the amounts of omega-3 fatty acids released by self-emulsifying delivery (SED) formulations containing lecithin, were higher than that released by a commercial formulation without lecithin. The Cmax values of docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) were approximately 1.38-1.40-fold for the optimized SED formulation than for the control group (P < 0.01). Similarly, the mean AUC0 - 48 values of DHA or EPA in the SED group were 1.27-1.29-fold higher than in the control group (P < 0.05). Phospholipids and lecithin were found to have considerable potentials as bioavailability enhancing excipients for SED systems.

Grading criteria of histopathological evaluation in BCOP assay by various staining methods.

This study was performed to provide information on classifying eye irritating chemicals using the BCOP assay. After the BCOP assay, bovine corneas were classified by IVIS presented in OECD test guideline 437, and three special staining methods (H&E, MT, and PAS) were performed for histopathological evaluation. Non-irritant chemicals (IVIS ≤ 3), showed intact structures. In the 3 < IVIS ≤ 55 group, epithelial cell edema was observed by H&E staining, and loose collagen bundles were confirmed by MT staining. In PAS staining, bleaching of the epithelium and reduced visibility of the basement membrane were observed. Severe irritant chemicals (IVIS > 55) showed large increases edema and nuclear condensation by H&E staining. Loose collagen bundles and vacuoles around keratocytes were also observed by MT staining. Bleaching of the epithelial layer, reduction in visibility, and thickness of the basement membrane were confirmed by PAS staining. Based on the stepwise histopathological analysis, we set the criteria and grades for histopathological evaluation and found that eye irritation was increased following the irritation degree of test chemicals. Further histopathological study will support and lead to improvements in the BCOP assay.

Therapeutic Effects of Dipterocarpus tuberculatus with High Antioxidative Activity Against UV-Induced Photoaging of NHDF Cells and Nude Mice.

To investigate the therapeutic effects of methanol extracts of Dipterocarpus tuberculatus Roxb. (MED) against UV-induced photoaging, we assessed for alterations in the antioxidant activity, anti-apoptotic effects, ECM modulation, skin appearances, and anti-inflammatory response in normal human dermal fibroblast (NHDF) cells and nude mice orally treated with MED. High levels of tannin content and high free radical scavenging activity to DPPH were determined in MED, while seven active components, namely, gallic acid, bergenin, ellagic acid, ε-viniferin, asiatic acid, oleanolic acid, and 2α-hydroxyursolic acid, were identified using LC-MS analyses. UV-induced alterations in the NO concentration, SOD activity, and Nrf2 expression were remarkably recovered in MED-treated NHDF cells. Moreover, the decreased number of apoptotic cells and G2/M phase arrest were observed in the UV + MED-treated groups. Similar recoveries were detected for ß-galactosidase, MMP-2/9 expression, and intracellular elastase activity. Furthermore, MED treatment induced suppression of the COX-2-induced iNOS mediated pathway, expression of inflammatory cytokines, and inflammasome activation in UV-radiated NHDF cells. The anti-photoaging effects observed in NHDF cells were subsequently evaluated and validated in UV + MED-treated nude mice through skin phenotypes and histopathological structure analyses. Taken together, these results indicate that MED exerts therapeutic effects against UV-induced photoaging and has the potential for future development as a treatment for photoaging.

Beet root (Beta vulgaris) protects lipopolysaccharide and alcohol-induced liver damage in rat.

The purpose of this study was to investigate the protective effects of beet root (Beta vulgaris var. rubra) in lipopolysaccharide (LPS) and alcohol induced liver damage. Beta vulgaris ethanol extract (BVEE) showed good antioxidant activity in the contents of polyphenol and flavonoid compounds, and the electron-donating ability and ABTS+ radical scavenging activity. As for anti-inflammatory effect in RAW 264.7 cells, inhibition rate of nitric oxide production was increased in dose dependent manner. In hepatotoxicity model induced by LPS and alcohol in rat, BVEE significantly decreased serum AST, ALT and γ-GTP concentrations in a dose-dependent manner. The histopathological changes after H&E staining showed that fat accumulation and inflammatory cell infiltration were decreased by BVEE. The collagen fibers around the central lobule observed by Masson's trichrome staining were also decreased by BVEE. In addition, as for the immunohistochemical staining and Transmission electron microscopy, BVEE improved morphological characteristics of damaged liver lesion. The increased mRNA expressions of NF-κB, MAPK1, MAPK3, CYP2E1, and α-SMA were significantly decreased in BVEE treated group. These results indicated that BVEE would have protective effects in hepatotoxicity by altering various indicators related to the liver damage induced by LPS or alcohol.

Correction to: Beet root (Beta vulgaris) protects lipopolysaccharide and alcohol­induced liver damage in rat.

[This corrects the article DOI: 10.1007/s43188-019-00030-4.].

Importin-11 is Essential for Normal Embryonic Development in Mice.

Importin-11 (Ipo11) is a novel member of the human importin family of transport receptors (karyopherins), which are known to mediate the nucleocytoplasmic transport of protein and RNA cargos. Despite its role in the transport of protein, we found that knockout of Ipo11 nuclear import factor affects normal embryonic development and govern embryo-lethal phenotypes in mice. In this study, we for the first time produced a mouse line containing null mutation in Ipo11 gene utilized by gene trapping. The Ipo11-/- embryos showed an embryonic lethal phenotype. The Ipo11-/- embryos showed a reduced size at embryonic day 10.5 (E10.5) when compared with Ipo11+/+ or Ipo11+/- embryos and died by E11.5. Whereas Ipo11+/- mice were healthy and fertile, and there was no detectable changes in embryonic lethality and phenotype when reviewed. In the X-gal staining with the Ipo11-/- or Ipo11+/- embryos, strong X-gal staining positivity was detected systematically in the whole mount embryos at E10.5, although almost no X-gal positivity was detected at E9.5, indicating that the embryos die soon after the process of Ipo11 expression started. These results indicate that Ipo11 is essential for the normal embryonic development in mice.

Therapeutic Effects of Cold-Pressed Perilla Oil Mainly Consisting of Linolenic acid, Oleic Acid and Linoleic Acid on UV-Induced Photoaging in NHDF Cells and SKH-1 Hairless Mice.

Positive physiological benefits of several plant oils on the UV-induced photoaging have been reported in some cell lines and model mice, but perilla oil collected from the seeds of Perilla frutescens L. has not been investigated in this context. To study the therapeutic effects of cold-pressed perilla oil (CPO) on UV-induced photoaging in vitro and in vivo, UV-induced cellular damage and cutaneous photoaging were assessed in normal human dermal fibroblasts (NHDFs) and HR-1 hairless mice. CPO contained five major fatty acids including linolenic acid (64.11%), oleic acid (16.34%), linoleic acid (11.87%), palmitic acid (5.06%), and stearic acid (2.48%). UV-induced reductions in NHDF cell viability, ROS production, SOD activity, and G2/M cell cycle arrest were remarkably improved in UV + CPO treated NHDF cells as compared with UV + Vehicle treated controls. Also, UV-induced increases in MMP-1 protein and galactosidase levels were remarkably suppressed by CPO. In UV-radiated hairless mice, topical application of CPO inhibited an increase in wrinkle formation, transepidermal water loss (TEWL), erythema value, hydration and melanin index on dorsal skin of UVB-irradiated hairless mice. CPO was observed to similarly suppress UV-induced increases in epidermal thickness, mast cell numbers, and galactosidase and MMP-3 mRNA levels. These results suggest CPO has therapeutic potential in terms of protecting against skin photoaging by regulating skin morphology, histopathology and oxidative status.

7­MEGA™ 500 regulates the expression of COX­2, MMP­3 and type 1 procollagen in UVB­irradiated human keratinocytes and dermal fibroblasts.

AlaskOmega® Omega 7 500, also known as Omega­7 fatty acid or 7­MEGA™, is a highly concentrated palmitoleic acid (C16:1). Little is known about how 7­MEGA regulates skin inflammation and wrinkle formation in cultured skin cells. The present study aimed to investigate the effects of 7­MEGA on the expression of cyclooxygenase­2 (COX­2), matrix metallopeptidase (MMP)­1/3 and type 1 procollagen, which are markers of skin inflammation and wrinkle formation, in ultraviolet B (UVB)­irradiated human dermal fibroblasts (HDFs) and keratinocytes (HaCaT). No toxicity was observed upon treatment of HDFs and HaCaT cells with 0.5­2.5 µl/ml 7­MEGA. The exposure of HaCaT cells to 10 mJ/cm2 UVB for 6 h resulted in increased protein and/or mRNA expression of COX­2 and MMP­3. Treatment of HaCaT cells with 2.5 µl/ml 7­MEGA suppressed the UVB­induced expression of COX­2 and MMP­3 in these cells. In addition, treatment with 2.5 µl/ml 7­MEGA attenuated the UVB­induced expression and phosphorylation levels of c­Fos and c­Jun, two components of the activator protein­1 (AP­1) transcription factor, in HaCaT cells. Exposure of HDFs to 60 mJ/cm2 UVB for 6 h significantly decreased the expression of type 1 procollagen protein, whereas treatment with 2.5 µl/ml 7­MEGA partially reversed the effects of UVB on the expression of type 1 procollagen protein. These results demonstrated for the first time that 7­MEGA regulated the expression of COX­2, MMP­3 and type 1 procollagen in UVB­irradiated skin cells. The present study suggested that 7­MEGA may serve as a novel agent against UVB­induced skin inflammation and damage.

Inhibitory effects of Aristotelia chilensis water extract on 2,4-Dinitrochlorobenzene induced atopic-like dermatitis in BALB/c Mice.

BACKGROUND: Maqui berry (Aristotelia chilensis) has been reported to have anti-glycation, anti-inflammation, lipogenesis-inhibiting activities highly related to its anti-oxidation function, but practical efficacy studies on immunological mechanisms for atopic dermatitis, have not been reported yet. OBJECTIVE: This study investigated the immune regulation mechanism of Aristotelia chilensis water extract (ACWE) related to atopic-like dermatitis . METHODS: Antioxidant and anti-inflammatory effects of ACWE was assayed. Atopy inhibitory effect was evaluated using in vitro cell study and in vivo 2,4-dinitrochlorobenzene (DNCB)-induced mouse atopic-like dermatitis model. RESULTS: ACWE has good antioxidant activities, and atopic indications were improved in ACWE group in DNCB-induced atopic-like dermatitis model of BALB/c mice. In spleen cells from mice, ACWE increased interferon-gamma (IFN-γ) levels, and decreased interleukin-4 (IL-4) levels compared with the DNCB control. CONCLUSIONS: ACWE was efficacious for atopic dermatitis which indicates that ACWE might have potential as an agent for atopic dermatitis.

Two tiered approaches combining alternative test methods and minimizing the use of reconstructed human cornea-like epithelium tests for the evaluation of eye irritation potency of test chemicals.

In order to overcome the limitations of single in vitro eye irritation tests, Integrated Approaches to Testing Assessment strategies have been suggested for evaluating eye irritation. This study developed two tiered approaches combining alternative test methods. They were designed in consideration of the solubility property of test chemicals and to use the RhCE tests at final steps. The tiered approach A is composed of the STE, BCOP, HET-CAM or RhCE tests, whereas the tiered approach B is designed to perform simultaneously two in vitro test methods at the first stage and the RhCE test at the final stage. The predictive capacity of the two tiered approaches was estimated using 47 chemicals. The accuracy, sensitivity, and specificity value of the tiered approach A were 95.7% (45/47), 100% (34/34), and 84.6% (11/13), respectively, whereas those of the tiered approach B were 95.7% (45/47), 97.1% (33/34), and 92.3% (12/13), respectively. The approach A and B were considered to be available methods for distinguishing test chemicals of Category 1 (all 73.3%) and No Category (84.6% and 92.3%), respectively. Especially, the approach B was considered as an efficient method as the Bottom-Up approach, because it predicted correctly test chemicals classified as No Category.

Alleviation of Ultraviolet B-Induced Photoaging by 7-MEGATM 500 in Hairless Mouse Skin.

The purpose of this study was to investigate the effect of 7-MEGA™ 500 on the improvement of skin aging in an UVB-induced photo-aging model of hairless mice. The dorsal skin of hairless mice was exposed to UVB three times a week for 12 weeks to induce skin wrinkle. After inducing the wrinkle, 7-MEGA™ 500 was orally administered once a day for 4 weeks. Skin thickness, skin barrier function, and wrinkle indicators were improved by treatment with 7-MEGA™ 500. Both gene and protein expression levels of MMP-3 and c-Jun in skin were significantly decreased by 7-MEGA™ 500. Therefore, the intake of 7-MEGA™ 500 is thought to have a positive effect on the improvement of skin aging, although further studies are needed.

Skin irritation and sensitization potential of oxidative hair dye substances evaluated with in vitro, in chemico and in silico test methods.

Permanent oxidative hair dyes are widely used but their toxicity is not well-established. Here we aimed to evaluate the skin sensitization and irritation of nine hair dye substances (MAP, MRP-N, RS, PAOX, 2,4-DAPE, 2,6-PYR, PPD, Grey HED and PM) permitted for use in EU and Korea, using in vitro and in chemico and in silico test methods. Skin sensitization was evaluated by the KeratinoSens™ assay, Direct Peptide Reactivity Assay (DPRA) and DEREK. Six of nine dyes tested were determined as sensitizers in common. However, the decision for MAP, RS or PAOX was diverged across assays showing 2 positives and 1 negative. Skin irritation of hair dye substances was assessed with or without 6% H2O2 on a reconstructed human epidermis, Epiderm™, which demonstrated that H2O2 increased the skin irritation potential of some hair dyes. PPD and PM were determined to be irritants with H2O2. Epidermal damages by hair dye and H2O2 could be further confirmed through the histology of tissue remaining after MTT assay. Collectively, our study demonstrated that hair dyes possess potential skin sensitization and irritation issues which could be further aggravated by H2O2.

Oral Wound Healing Effects of Acai Berry Water Extracts in Rat Oral Mucosa.

The objective of this study was to determine the oral wound healing effects of acai berry water extracts (ABWE) in rat oral mucosa. To estimate the anti-oxidative effects of ABWE, the contents of phenolic compounds, and DPPH (1,1-diphenyl-2-picryl hydrazyl) and ABTS (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) abilities were evaluated. Wound healing effects of ABWE were tested following 6-day exposure after induction of wound by applying 50% acetic acid to oral mucosa of Sprague-Dawley rats. Macroscopic and histopathological analyses were performed to determine wound healing effects of ABWE. Sodium fusidate (20 mg/g) was used as positive control. ABWE showed significantly high antioxidant effects in all assays, although its potency was weaker than the positive control. From day 3 after treatment, wound healing effects of ABWE were observed in oral mucosa. These wound healing effects were also consistent with histopathological evaluation results. Taken together, these results indicate that ABWE might have potential as an oral wound healing agent in the future.

Skin Wound Healing Effects and Action Mechanism of Acai Berry Water Extracts.

The purpose of this study was to investigate the wound healing effect of acai berry water extracts (ABWE) and a possible underlying mechanism involved in its action using various in vitro and in vivo models. The wound healing effect of ABWE was evaluated by migration assay using HS68 fibroblast cells. In addition, its effect on mRNA expression of procollagen, fibronectin, and MMP-1 was determined. Moreover, the wound healing effect of ABWE was evaluated in in vivo wound models through macroscopic and microscopic observation. In addition, mRNA expression levels of wound related genes were determined. Results revealed that ABWE was not cytotoxic. It increased migration of HS68 fibroblast cells. ABWE increased mRNA expression levels of fibronectin but decreased the mRNA expression levels of MMP-1. ABWE also showed significantly potent wound healing effect in vivo based on macroscopic and histopathological observation and mRNA expression evaluation for wound related genes. Taken together, our results indicated that ABWE might have potential as a wound healing agent.

In vitro skin absorption tests of three types of parabens using a Franz diffusion cell.

The objective of this study was to evaluate the permeation of paraben derivatives - methylparaben (MP), propylparaben (PP), and butylparaben (BP) - in hairless mouse full skin and human cadaver epidermis using a Franz diffusion cell method, which is proposed as a reliable alternative method to an skin absorption test. Parabens, esterified hydroxybenzoic acid compounds, are widely used as preservatives in food, cosmetics, and pharmaceutical products. The skin permeation rate showed dose dependency, and the hairless mouse full skin showed a higher flux value than human cadaver epidermis. Among the permeability coefficient (Kp) values of three parabens, MP showed a higher Kp value than PP or BP. Hence, according to the definitions of Marzulli et al., parabens would be classified as "moderate" penetrants.

Protective effects of the antioxidant extract collected from Styela clava tunics on UV radiation­induced skin aging in hairless mice.

Ultraviolet (UV) radiation is considered a primary cause of skin damage, which is characterized by deep wrinkles, roughness, laxity and pigmentation through oxidative stress and oxidative photodamage. To examine the therapeutic effects of ethanol extract of Styela clava tunics (EtSCT) on UV radiation-induced skin aging in hairless mice, alterations in skin phenotype, histological structures, inflammation, endoplasmic reticulum (ER) stress, oxidative conditions and toxicity were investigated during 13 weeks of UV irradiation and topical application of EtSCT. EtSCT showed high reducing power (3.1%), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (92.7%) and NO scavenging activity (15.6%) due to its high total flavonoids (15.3 mg/ml) and total phenolics (36.8 mg/ml). The topical application of EtSCT suppressed photoaging of the skin of UV-irradiated mice, and this was demonstrated by the inhibition of wrinkle formation, the suppression of the erythema index as well as the prevention of transepidermal water loss. Additionally, the epidermal thickness and adipocytes number were recovered to a similar level as that in the no radiation group in the UV + EtSCT­treated groups compared with the UV + vehicle­treated group, and the expression of collagen I increased. The attenuation of mitogen­activated protein kinase and ER stress signaling pathways activated by reactive oxygen species was also detected in the UV + EtSCT­treated group. Inflammatory responses including the infiltration of mast cells, CD31 expression and interleukin-6 secretion were significantly lower in the UV + EtSCT-treated groups. Moreover, the concentration of malondialdehyde was reduced and the activity of superoxide dismutase was effectively recovered in the UV + EtSCT-treated groups compared with that in the vehicle-treated groups. Liver and kidney toxicity factors were maintained at a constant level. These results suggest that EtSCT has the potential for use as therapeutic drug which protects against skin aging by regulating the skin morphology, histopathological structures, ER stress, inflammation and oxidative conditions.

A Web-based Alternative Non-animal Method Database for Safety Cosmetic Evaluations.

Animal testing was used traditionally in the cosmetics industry to confirm product safety, but has begun to be banned; alternative methods to replace animal experiments are either in development, or are being validated, worldwide. Research data related to test substances are critical for developing novel alternative tests. Moreover, safety information on cosmetic materials has neither been collected in a database nor shared among researchers. Therefore, it is imperative to build and share a database of safety information on toxicological mechanisms and pathways collected through in vivo, in vitro, and in silico methods. We developed the CAMSEC database (named after the research team; the Consortium of Alternative Methods for Safety Evaluation of Cosmetics) to fulfill this purpose. On the same website, our aim is to provide updates on current alternative research methods in Korea. The database will not be used directly to conduct safety evaluations, but researchers or regulatory individuals can use it to facilitate their work in formulating safety evaluations for cosmetic materials. We hope this database will help establish new alternative research methods to conduct efficient safety evaluations of cosmetic materials.

Adipose-derived mesenchymal stem cells reduce MMP-1 expression in UV-irradiated human dermal fibroblasts: therapeutic potential in skin wrinkling.

Adipose-derived mesenchymal stem cells (AdMSCs) have been reported to have therapeutic benefit in skin. The aim of this study was to examine the effects of AdMSCs in UV-irradiated human dermal fibroblasts (HDFs) for therapeutic potential in skin wrinkling. UV irradiation, a model naturally mimic skin wrinkle formation, is known to increase matrix metalloproteinase-1 (MMP-1), making MMP-1 a target for skin photoaging. Our findings identified that AdMSCs reduce MMP-1 level in UV-irradiated HDFs and increase type 1 procollagen in HDFs. A dose-dependent increase in type 1 procollagen was confirmed by AdMSC-conditioned medium. Importantly, our current findings showing the effects of AdMSCs on the induction of MMP-1 in UV-radiated HDFs and the expression of collagen in HDFs can provide an evidence of relationship between MMP-1 and procollagen production for the protection against wrinkle formation. Collectively, AdMSCs may contribute to anti-wrinkle effects in skin but further experiments are needed to identify the mechanism.