Development of an Eye Irritation Test Method Using an In-House Fabrication of a Reconstructed Human Cornea-like Epithelium Model for Eye Hazard Identification.

In a previous study, a novel human corneal-like epithelium model utilizing an immortalized human corneal epithelial cell line (iHCE-NY1) was developed as an alternative to animal models to identify chemicals not classified under the United Nations Globally Harmonized System of Classification and Labeling of Chemicals (GHS) and was evaluated following the criteria of Test Guideline 492 of the Organization for Economic Co-operation and Development (OECD). In the present study, our aim was to establish an eye irritation test protocol using the iHCE-NY1 model to classify liquid chemicals under the GHS ocular hazard categories: no effect, no classification (No Cat.), Category 2 (Cat. 2) reversible effects, and Category 1 (Cat. 1) irreversible eye damage. The protocol involved exposing the iHCE-NY1 model to 31 liquid test chemicals for 5 min, followed by observation at post-incubation periods (PIPs) to assess recovery. Classification was based on cell viability, and histopathological findings on PIP days 7, 14, and 21. The outcomes were compared with an established database of classifications. All Cat. 1 liquid chemicals, 62.5% of No Cat., and 63.2% of Cat. 2 were correctly categorized. This study demonstrates that the iHCE-NY1 model can not only distinguish No Cat. test liquid chemicals but also differentiate between Cat. 2 and Cat. 1 liquid chemicals.

Characterization of an L-α,ß-diaminopropionic acid polymer with comb-like structure isolated from a poly(ε-L-lysine)-producing Streptomyces sp.

Polymers of basic amino acids function as polycationic compounds under physiological conditions and exhibit intriguing biological properties, such as antimicrobial and antiviral activities, immunopotentiating ability, and DNA-binding activity. Poly(ε-L-lysine) (ε-PL) produced by some strains of Streptomyces spp. is a cationic homopolymer of L-lysine linking between ε-amino and α-carboxylic acid functional groups and has been used as a food preservative based on its biocompatibility and biodegradability. An ε-PL-producing strain of Streptomyces sp. USE-33 was found to secrete a novel polycationic substance into its culture broth along with ε-PL. High-performance liquid chromatography analyses and one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) experiments, accompanied by NMR titration studies, revealed that the secreted substance was poly[ß-(L-diaminopropionyl-L-diaminopropionic acid)], PAP, characterized by an isopeptide backbone linking between the ß-amino and α-carboxylic acid groups of L-α,ß-diaminopropionic acid (L-Dpr) with pendent L-Dpr residues. PAP had a molecular weight of 500 to 1400, and copolymers composed of the two amino acids L-Dpr and L-lysine were not detected in the producer strain USE-33. The strain coproduced high levels of the two poly(amino acid)s in the presence of glycerol, citrate, and ammonium sulfate at pH 4.0 in a two-stage cultivation procedure. PAP exhibited strong inhibitory activities against several yeasts and weaker activities against bacteria than ε-PL. PAP may share a number of biological functions with ε-PL, and the use of PAP along with ε-PL has potential as a specific and advanced material for technical applications in various fields.Key points• Novel cationic poly(amino acid) was found in an ε-PL-producing Streptomyces species.• The l-α,ß-diaminopropionic acid polymer was characterized by a comb-like structure.• The novel poly(amino acid), PAP, exhibited antibacterial and antifungal activities.

Establishment of a new immortalized human corneal epithelial cell line (iHCE-NY1) for use in evaluating eye irritancy by in vitro test methods.

In vitro test methods that use human corneal epithelial cells to evaluate the eye irritation potency of chemical substances do not use human corneal epithelium because it has been difficult to maintain more than four passages. In this study, we make a new cell line comprising immortalized human corneal epithelial cells (iHCE-NY1). The IC50 of iHCE-NY1 cells is slightly higher than that of Statens Seruminstitut Rabbit Cornea (SIRC) cells, which are currently used in some in vitro test methods. CDKN1A in iHCE-NY1 cells was used as a marker of gene expression to indicate cell cycle activity. This enabled us to evaluate cell recovery characteristics at concentrations lower than the IC50 of cytotoxic tests.

Evaluation of combinations of in vitro sensitization test descriptors for the artificial neural network-based risk assessment model of skin sensitization.

The skin sensitization potential of chemicals has been determined with the use of the murine local lymph node assay (LLNA). However, in recent years public concern about animal welfare has led to a requirement for non-animal risk assessment systems for the prediction of skin sensitization potential, to replace LLNA. Selection of an appropriate in vitro test or in silico model descriptors is critical to obtain good predictive performance. Here, we investigated the utility of artificial neural network (ANN) prediction models using various combinations of descriptors from several in vitro sensitization tests. The dataset, collected from published data and from experiments carried out in collaboration with the Japan Cosmetic Industry Association (JCIA), consisted of values from the human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA), SH test and antioxidant response element (ARE) assay for chemicals whose LLNA thresholds have been reported. After confirming the relationship between individual in vitro test descriptors and the LLNA threshold (e.g. EC3 value), we used the subsets of chemicals for which the requisite test values were available to evaluate the predictive performance of ANN models using combinations of h-CLAT/DPRA (N = 139 chemicals), the DPRA/ARE assay (N = 69), the SH test/ARE assay (N = 73), the h-CLAT/DPRA/ARE assay (N = 69) and the h-CLAT/SH test/ARE assay (N = 73). The h-CLAT/DPRA, h-CLAT/DPRA/ARE assay and h-CLAT/SH test/ARE assay combinations showed a better predictive performance than the DPRA/ARE assay and the SH test/ARE assay. Our data indicates that the descriptors evaluated in this study were all useful for predicting human skin sensitization potential, although combinations containing h-CLAT (reflecting dendritic cell-activating ability) were most effective for ANN-based prediction.

Test battery with the human cell line activation test, direct peptide reactivity assay and DEREK based on a 139 chemical data set for predicting skin sensitizing potential and potency of chemicals.

To develop a testing strategy incorporating the human cell line activation test (h-CLAT), direct peptide reactivity assay (DPRA) and DEREK, we created an expanded data set of 139 chemicals (102 sensitizers and 37 non-sensitizers) by combining the existing data set of 101 chemicals through the collaborative projects of Japan Cosmetic Industry Association. Of the additional 38 chemicals, 15 chemicals with relatively low water solubility (log Kow > 3.5) were selected to clarify the limitation of testing strategies regarding the lipophilic chemicals. Predictivities of the h-CLAT, DPRA and DEREK, and the combinations thereof were evaluated by comparison to results of the local lymph node assay. When evaluating 139 chemicals using combinations of three methods based on integrated testing strategy (ITS) concept (ITS-based test battery) and a sequential testing strategy (STS) weighing the predictive performance of the h-CLAT and DPRA, overall similar predictivities were found as before on the 101 chemical data set. An analysis of false negative chemicals suggested a major limitation of our strategies was the testing of low water-soluble chemicals. When excluded the negative results for chemicals with log Kow > 3.5, the sensitivity and accuracy of ITS improved to 97% (91 of 94 chemicals) and 89% (114 of 128). Likewise, the sensitivity and accuracy of STS to 98% (92 of 94) and 85% (111 of 129). Moreover, the ITS and STS also showed good correlation with local lymph node assay on three potency classifications, yielding accuracies of 74% (ITS) and 73% (STS). Thus, the inclusion of log Kow in analysis could give both strategies a higher predictive performance.

CJ-21,058, a new SecA inhibitor isolated from a fungus.

A new equisetin derivative, CJ-21,058 (I) was isolated from the fermentation broth of an unidentified fungus CL47745. It shows antibacterial activity against Gram-positive multi-drug resistant bacteria by inhibiting ATP-dependent translocation of precursor proteins across a bacterial cell membrane.

Thielavins as glucose-6-phosphatase (G6Pase) inhibitors: producing strain, fermentation, isolation, structural elucidation and biological activities.

High-throughput screening of microbial extracts using rat hepatic microsomal glucose-6-phosphatase (G6Pase) led us to find thielavin B as a G6Pase inhibitor with inhibition of glucose output from glucagon-stimulated hepatocytes. Further searching for more potent analogs identified 11 new thielavins F-P in addition to the known thielavins A and B from a fungus Chaetomium carinthiacum ATCC 46463. Thielavin G showed the strongest activity as a G6Pase inhibitor (IC50=0.33 microM), while the IC50 of thielavin B was 5.5 microM. According to the structure-activity relationship, including authentic thielavins C, D and 3 partial hydrolysates from thielavins A and B, 3 benzoic acid-units and carboxylic acid functions are essential for G6Pase inhibition.