Application of Spectro-DPRA, KeratinoSens™ and h-CLAT to estimation of the skin sensitization potential of cosmetics ingredients.

Ethical issues in animal toxicity testing have led to the search for alternative methods to determine the skin sensitization potential of cosmetic products. The emergence of ethical testing issues has led to the development of many alternative methods that can reliably estimate skin sensitization potentials. However, a single alternative method may not be able to achieve high predictivity due to the complexity of the skin sensitization mechanism. Therefore, several prediction assays, including both in chemico and in vitro test methods, were investigated and integrated based on the skin sensitization adverse outcome pathway. In this study, we evaluated three different integrated approaches to predict a human skin sensitization hazard using data from in vitro assays (KeratinoSens™ and human cell line activation test [h-CLAT]), and a newly developed in chemico assay (spectrophotometric direct peptide reactivity assay [Spectro-DPRA]). When the results of the in chemico and in vitro assays were combined, the predictivity of human data increased compared with that of a single assay. The highest predictivity was obtained for the approach in which sensitization potential was determined by Spectro-DPRA followed by final determination using the result of KeratinoSens™ and h-CLAT assays (96.3% sensitivity, 87.1% specificity, 86.7% positive predictive value, 96.4% negative predictive value and 91.4% accuracy compared with human data). While further optimization is needed, we believe this integrated approach may provide useful predictive data when determining the human skin sensitization potential of chemicals.

Alstonia scholaris R. Br. Significantly Inhibits Retinoid-Induced Skin Irritation In Vitro and In Vivo.

Topical retinoids inhibit matrix metalloproteinases and accelerate collagen synthesis, thereby triggering antiaging effects in the skin. However, topical retinoids can cause severe skin reactions, including scaling, erythema, papules, and inflammation. The present study demonstrates that the ethanolic bark extract of Alstonia scholaris R. Br. can significantly inhibit all-trans retinoic acid-induced inflammation in human HaCat keratinocyte cells. Furthermore, two representative retinoid-induced proinflammatory cytokines, monocyte chemoattractant protein-1 and interleukin-8, were significantly suppressed by A. scholaris extract (by 82.1% and 26.3% at 100 ppm, and dose-dependently across the tested concentrations) in vitro. In a cumulative irritation patch test, A. scholaris extract decreased retinol-induced skin irritation, while strengthening the ability of retinoids to inhibit matrix metalloproteinase-1 expression, which is strongly associated with aging effects. These results suggest that A. scholaris is a promising compound that may increase the antiaging function of retinoids while reducing their ability to cause skin irritation.

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.

Hybrid skin chips for toxicological evaluation of chemical drugs and cosmetic compounds.

Development of drugs and cosmetics for topical application require safety tests in skin models. However, current skin models, such as skin cell sheets and artificial tissue-engineered skin, do not allow sophisticated toxicological evaluations (e.g., sensory irritation, hepatotoxicity). Animal models are prohibited worldwide for testing cosmetics. Therefore, reliable human skin models that recapitulate physiological events in skin tissue need to be established under in vitro settings. In this study, hybrid human skin models that enable delicate toxicological evaluations of drugs and cosmetic compounds are demonstrated. To recapitulate skin cornification, keratinocytes in the top layer of a vertical microfluidic chip were cultured at the air-liquid interface. For the skin-nerve hybrid model, differentiated neural stem cells in 3D collagen were positioned adjacent to and right below the skin layer. This model enables real-time quantitative skin sensitization analysis following chemical treatments by detecting alterations in neuronal activity in combination with a calcium imaging technique. For the skin-liver model, hepatic cells derived from pluripotent stem cells were cultured in 3D collagen distant from the skin layer. Potential hepatotoxicity of cutaneously applied chemicals in this model can be evaluated by quantification of glutathione and reactive oxygen species. Our study suggests that 3D hybrid skin chips would provide useful human skin models in pharmaceutical and cosmetic industries.

Comparative study of the ocular irritation potential of various alkyl polyglucoside surfactants.

In the present work, we assessed the relationship between alkyl carbon chain length and ocular irritation potentials using the hen's egg test-chorioallantoic membrane (HET-CAM) and bovine corneal opacity and permeability (BCOP) assays using 5 commercial alkyl polyglucoside surfactants with different compositions of alkyl chain lengths (C(6)-C(16)). With HET-CAM, there was a good correlation between the proportion of C(10) alkyl polyglucoside and the eye irritation potential Q score (r(2) = 0.912, p = .011). There were no significant differences between the proportion of C(10) alkyl polyglucoside and corneal opacity in BCOP assays; however, there was a relatively high positive correlation between the proportion of C(10) alkyl carbon chain lengths and corneal permeability (r(2) = 0.736, p = .063).

A novel sphingosylphosphorylcholine and sphingosine-1-phosphate receptor 1 antagonist, KRO-105714, for alleviating atopic dermatitis.

BACKGROUND: Atopic dermatitis (eczema) is a type of inflammation of the skin, which presents with itchy, red, swollen, and cracked skin. The high global incidence of atopic dermatitis makes it one of the major skin diseases threatening public health. Sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P) act as pro-inflammatory mediators, as an angiogenesis factor and a mitogen in skin fibroblasts, respectively, both of which are important biological responses to atopic dermatitis. The SPC level is known to be elevated in atopic dermatitis, resulting from abnormal expression of sphingomyelin (SM) deacylase, accompanied by a deficiency in ceramide. Also, S1P and its receptor, sphingosine-1-phosphate receptor 1 (S1P1) are important targets in treating atopic dermatitis. RESULTS: In this study, we found a novel antagonist of SPC and S1P1, KRO-105714, by screening 10,000 compounds. To screen the compounds, we used an SPC-induced cell proliferation assay based on a high-throughput screening (HTS) system and a human S1P1 protein-based [35S]-GTPγS binding assay. In addition, we confirmed the inhibitory effects of KRO-105714 on atopic dermatitis through related cell-based assays, including a tube formation assay, a cell migration assay, and an ELISA assay on inflammatory cytokines. Finally, we confirmed that KRO-105714 alleviates atopic dermatitis symptoms in a series of mouse models. CONCLUSIONS: Taken together, our data suggest that SPC and S1P1 antagonist KRO-105714 has the potential to alleviate 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.

High-throughput screening (HTS)-based spectrophotometric direct peptide reactivity assay (Spectro-DPRA) to predict human skin sensitization potential.

Some cosmetic ingredients can act as a chemical hapten to induce an immune response; therefore, evaluating the sensitizing potential of cosmetic ingredients is essential. We previously developed a novel in chemico direct peptide reactivity assay involving a spectrophotometric evaluation (Spectro-DPRA) for animal skin sensitization tests (local lymph node assay; LLNA). Based on previous research, we expanded the test materials to confirm the effectiveness of the Spectro-DPRA method for predicting the animal skin sensitization potential, and further determined the feasibility of the method for estimating the human skin sensitization potential. Spectro-DPRA showed 83.1% or 89.1% accuracy compared to a conventional LLNA or prediction based on human data, respectively, with a combination model using both a cysteine peptide and lysine peptide cut-off. To identify the effect of the lipophilicity of a chemical on predicting the skin sensitization potential, we applied our prediction model to chemicals with a Log Pow range of -1 to 4. Overall predictability was increased, and the accuracy compared to the LLNA and human data was 91.5% and 94.9%, respectively, in the combination cut-off prediction model. In conclusion, Spectro-DPRA serves as an easy, rapid, and high-throughput in chemico screening method with high accuracy to predict the human skin sensitization potential of chemicals.

Gene discovery analysis from mouse embryonic stem cells based on time course microarray data.

An embryonic stem cell is a powerful tool for investigation of early development in vitro. The study of embryonic stem cell mediated neuronal differentiation allows for improved understanding of the mechanisms involved in embryonic neuronal development. We investigated expression profile changes using time course cDNA microarray to identify clues for the signaling network of neuronal differentiation. For the short time course microarray data, pattern analysis based on the quadratic regression method is an effective approach for identification and classification of a variety of expressed genes that have biological relevance. We studied the expression patterns, at each of 5 stages, after neuronal induction at the mRNA level of embryonic stem cells using the quadratic regression method for pattern analysis. As a result, a total of 316 genes (3.1%) including 166 (1.7%) informative genes in 8 possible expression patterns were identified by pattern analysis. Among the selected genes associated with neurological system, all three genes showing linearly increasing pattern over time, and one gene showing decreasing pattern over time, were verified by RT-PCR. Therefore, an increase in gene expression over time, in a linear pattern, may be associated with embryonic development. The genes: Tcfap2c, Ttr, Wnt3a, Btg2 and Foxk1 detected by pattern analysis, and verified by RT-PCR simultaneously, may be candidate markers associated with the development of the nervous system. Our study shows that pattern analysis, using the quadratic regression method, is very useful for investigation of time course cDNA microarray data. The pattern analysis used in this study has biological significance for the study of embryonic stem cells.

Risk assessment of the organophosphate pesticides isazofos and pyraclofos using a 21-day dietary toxicity study in Japanese quail.

Six-week-old male and female Japanese quails (Coturnix japonica) received two organophosphate pesticides, isazofos and pyraclofos, for a 21-day dietary toxicity test, based on the OECD workshop report. During the treatment period, body weight and food consumption of the quail decreased with exposure to either isazofos or pyraclofos. Using the up-and-down procedure to determine the 50% mortality value, we found that the 21-day LC(50) of isazofos and pyraclofos were 40 and 87 mg/kg body weight, respectively. Ataxia, salivation, diarrhea, ruffled feathers, and convulsions at a dead point were observed with both pesticides. The tips of the villi were necrotic in the high dosage groups of isazofos- and pyraclofos-treated quail. Based on these results, body weight, food consumption, clinical signs, and histopathological findings may be useful parameters for detecting the dietary toxicity associated with isazofos and pyraclofos exposure. In addition, Japanese quail could be an excellent bird model for monitoring the toxicological risks of pesticides in Korea.

Untargeted metabolite profiling for koji-fermentative bioprocess unravels the effects of varying substrate types and microbial inocula.

Untargeted metabolomics unraveled the effects of varying substrates (soybean, wheat, and rice) and inocula (Aspergillus oryzae and Bacillus amyloliquefaciens) on metabolite compositions of koji, a starter ingredient in various Asian fermented foods. Multivariate analyses of the hyphenated mass spectrometry datasets for different koji extracts highlighted 61 significantly discriminant primary metabolites (sugars and sugar alcohols, organic acids, amino acids, fatty acids, nucleosides, phenolic acids, and vitamins) according to varying substrates and inocula combinations. However, 59 significantly discriminant secondary metabolites were evident for koji-types with varying substrates only, viz., soybean (flavonoids, soyasaponins, and lysophospholipids), wheat (flavones and lysophospholipids), and rice (flavonoids, fatty acids derivatives, and lysophospholipids). Independently, the substrates influenced primary metabolite compositions in koji (soybean > wheat, rice). The inocula choice of A. oryzae engendered higher carbohydrates, organic acids, and lipid derivative levels commensurate with high α-amylase and ß-glucosidase activities, while B. amyloliquefaciens affected higher amino acids levels, in respective koji types.

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.

Evaluating the Headspace Volatolome, Primary Metabolites, and Aroma Characteristics of Koji Fermented with Bacillus amyloliquefaciens and Aspergillus oryzae.

Production of good Koji primarily depends upon the selection of substrate materials and fermentative microflora, which together influence the characteristic flavor and aroma. Herein, we performed comparative metabolomic analyses of volatile organic compounds (VOCs) and primary metabolites for Koji samples fermented individually with Bacillus amyloliquefaciens and Aspergillus oryzae. The VOCs and primary metabolites were analyzed using headspace solid phase microextraction (HS-SPME) followed by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). In particular, alcohols, ketones, and furans were mainly detected in Bacillus-fermented Koji (Bacillus Koji, BK), potentially due to the increased levels of lipid oxidation. A cheesy and rancid flavor was characteristic of Bacillus Koji, which is attributable to high content of typical 'off-flavor' compounds. Furthermore, the umami taste engendered by 2-methoxyphenol, (E,E)-2,4-decadienal, and glutamic acid was primarily detected in Bacillus Koji. Alternatively, malty flavor compounds (2-methylpropanal, 2-methylbutanal, 3-methylbutanal) and sweet flavor compounds (monosaccharides and maltol) were relatively abundant in Aspergillus-fermented Koji (Aspergillus Koji, AK). Hence, we argue that the VOC profile of Koji is largely determined by the rational choice of inocula, which modifies the primary metabolomes in Koji substrates, potentially shaping its volatolome as well as the aroma characteristics.

12(S)-HPETE induces itch-associated scratchings in mice.

The itch-associated responses evoked by intradermal injection of 12(S)-HPETE and leukotriene B4 were compared in ICR-mice. 12(S)-HPETE and leukotriene B4 (0.01-0.2 nmol/site) induced scratching of the injected site, respectively; the dose-responses were a peak at 0.05 nmol/site (12(S)-HPETE) or 0.03 nmol/site (leukotriene B4). The scratching response by 12(S)-HPETE (0.05 nmol/site) started within 1 min, peaked in the first 10 min period, had almost subsided by 25 min whereas the effect of leukotriene B4 peaked in the second 10 min. The effect of leukotriene B4 is slightly stronger than that of 12(S)-HPETE in 40 min of count. The scratching induced by 12(S)-HPETE was inhibited by capsaicin, naltrexon, and LY255283. These results suggest the possibility that 12-lipoxygenase product can be added to a new member of an endogenous itch mediator in the skin.

Evaluation of phoP and rpoS mutants of Salmonella enterica serovar Typhi as attenuated typhoid vaccine candidates: virulence and protective immune responses in intranasally immunized mice.

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.

Gene expression profile related to prognosis of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous group of diseases with respect to biology and clinical course. Through genome-wide scanning, we can have an improvement of the diagnosis and assay system of AML. Microarray was performed for the identification of acute myeloid leukemia prognosis. We divided patients into two groups (good prognosis group, GPG and poor prognosis group, PPG) based on differences in the individual reactions to treatment. Gene expression profiles were analyzed using microarray. Among genes up-regulated at least two-fold and down-regulated at least 0.5-fold in HL-60, we chose three up-regulated genes (PPP2CA, ME3, and CCDN2) and three down-regulated genes (GLO1, ANXA2, and BMI1) and confirmed the expression of these six genes by RT-PCR. We created a leukemia-specific subclass microarray, based on the gene expression profiles. Clinical samples from the bone marrow of four patients were hybridized on this microarray. Among the genes selected by the microarray technology, NB4, silenced TRIB3 and overexpressed XRN2 were not differentiated in spite of treatment with ATRA. This indicates that XRN2 and TRIB3 play an important role in cell differentiation. These data provided an expression profile for the diagnosis and prognosis of AML patients and identified candidate genes that might allow the prognosis of AML through the relative comparison of the expression level of genes between GPG and PPG.

Prevalidation trial for a novel in vitro eye irritation test using the reconstructed human cornea-like epithelial model, MCTT HCE™.

Here, we report the results of a prevalidation trial for an in vitro eye irritation test (EIT) using the reconstructed human cornea-like epithelium, MCTT HCE™. The optimal cutoff to determine irritation in the prediction model was established at 35% with the receiver operation characteristics(ROC) curve for 126 substances. Within-lab(WL) and between-lab(BL) reproducibility was tested for 20 reference substances by 3 participating laboratories. Viability data described by mean±SD or ±1/2 difference between duplicate wells, and scatter plots, demonstrated the WL/BL consistency. WL/BL concordance with the binary decision, whether non-irritant or irritant was estimated to be 85-95% and 95%, respectively. WL/BL reproducibility of viability data was further supported by a strong correlation(ICC, r>0.9). WL/BL agreement of binary decisions was also examined by Fleiss' Kappa statistics, which showed a strong level of agreement (>0.78), nevertheless weaker than the reproducibility of the viability. The EIT with MCTT HCE™ exhibited a sensitivity of 82.2% (60/73), a specificity of 81.1% (43/53), and an accuracy of 81.8% (103/126) for 126 reference substances (for liquids; a sensitivity of 100% (47/47), a specificity of 70.6% (24/34), and an accuracy of 87.7% (71/81), and for solids, a sensitivity of 50% (13/26), a specificity of 100% (19/19), and an accuracy of 71.1% (32/45), suggesting that the accuracy is satisfactory but the sensitivity needs improvement, which shall be addressed through correcting the poor sensitivity for solid substances in future full validation trials.

Effect of granulocyte macrophage-colony stimulating factor (GM-CSF) on 5-FU-induced ulcerative mucositis in hamster buccal pouches.

The present study was performed to investigate the protective effects of granulocyte macrophage-colony stimulating factor (GM-CSF) against ulcerative mucositis in hamster buccal pouch. GM-CSF was topically administered to the buccal pouches of hamsters with two different doses of 5 and 20 microg/ml. The treatment of GM-CSF led to rapid healing effects in gross and histopathological findings. It decreased expression of pro-inflammatory cytokine mRNA levels in the mucosal tissue of buccal pouches. Also GM-CSF-treated animals showed high numbers of Ki-67 positive cells in basal cell layer. These results suggest that GM-CSF provided excellent healing effects to ulcerative mucositis in the buccal pouch of hamster.

Development and evaluation of the Web-based wound care course for undergraduate nursing students.

PURPOSE: This study developed a web-based wound care course for undergraduate nursing students and evaluated the course's content, system, and student-satisfaction. METHODS: This study was done in three stages, the development of the web-based wound care course, the implementation and evaluation of the course. The course was developed based on the ARCS model. 80 undergraduate nursing students to Y University in Korea used the web-based wound care program during four weeks. After that, they completed questionnaires, evaluating the contents, system, and their satisfaction. RESULTS: Eighteen learning objectives were used to create the web-based wound care course and the course was developed with 7 chapters and 20 subsections. The analysis of the questionnaires showed a mean score for content and system-related items of 3.04 each, out of a possible 4 points. Student satisfaction items had a mean score of 2.89. CONCLUSIONS: The web-based course allowed students access to the course anytime and anywhere, and according to their own learning abilities. However this advantage would only be possible when nurse educators develop qualitative web-based course to meet the demand of a complex health care system as well as the needs of the students and the effectively incorporate it into traditional lectures.

Antimicrobial effect of lactic acid producing bacteria culture condensate mixture (LCCM) against Salmonella enteritidis.

The antimicrobial effects of a lactic acid producing bacteria culture condensate mixture (LCCM) were assessed against Salmonella enteritidis. In the presence of LCCM, bacterial growth was assessed in vitro by the measurement of optical density (OD) and viable bacterial counting. At concentrations of 1.25 and 2.5% LCCM, OD values were significantly lower than that of the control broth, and at concentrations of 5 and 10% LCCM, OD values did not increase for the entire period of experiment. At 8 h after incubation, the viable bacterial numbers in 5% and 10% LCCM-containing broths were remarkably lower than that in the control broth. This antimicrobial ability of the LCCM was fundamentally attributed to causing cell death rather than inhibiting growth. Even when the pH of LCCM-containing broth was adjusted to 7.2, the number of viable bacteria was significantly lower in the broths containing LCCM over 2.5% than that in control broth at 8 h after incubation. However, the OD value of each culture in the presence of each concentration of the LCCM increased over 1.0 at the completion of the experiment. The in vivo antimicrobial effects of the LCCM against S. enteritidis were also assessed. In S. enteritidis-infected mice, the LCCM decreased both the viable bacteria found in the feces and the mortality rate of the mice. These findings showed that the LCCM might have an antimicrobial ability against S. enteritidis.