Pyridoxine stimulates filaggrin production in human epidermal keratinocytes.

Pyridoxine (PN), one of the vitamers of vitamin B6, plays an important role in the maintenance of epidermal function and is used to treat acne and rough skin. Clinical studies have revealed that PN deficiency causes skin problems such as seborrheic dermatitis and stomatitis. However, the detailed effects of PN and its mechanism of action in epidermal function are poorly understood. In this study, we examined the effects of PN on epidermal function in normal human epidermal keratinocytes and found that PN specifically causes an increase in the expression of profilaggrin mRNA, among marker genes of terminal epidermal differentiation. In addition, PN treatment caused an increase in the production of filaggrin protein in a concentration-dependent manner. Treatment with P2x purinoceptor antagonists, namely, pyridoxal phosphate-6-azo (benzene-2,4-disulfonic acid) tetrasodium salt hydrate and TNP-ATP hydrate, induced an increase in the filaggrin protein levels. Moreover, we showed that elevated filaggrin production induced upon PN treatment was suppressed by ATP (known as P2x purinoceptor agonist). This study is the first to report that PN causes an increase in filaggrin transcription and production, and these results suggest that PN-induced filaggrin production may be a useful target as a daily care component in atopic dermatitis, wherein filaggrin levels are specifically reduced.

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.

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.

In vitro Evaluation Method of UV Protecting Ability of Sunscreens: Clarifying and Overcoming Problems to Develop New Method.

Factors influencing on in vitro evaluation of UV protecting ability of sunscreens were analyzed. It was found that any factors making the sunscreen layer spatially inhomogeneous, such as directional viscous fingering during the sunscreen application, dewetting of applied sunscreen layer, and the surface roughness of the standard PMMA plate, alter the UV transmittance. New application procedure and new type of flat hydrophilic plate were developed for inhibiting the generation of spatial inhomogeneity in applied sunscreen layer. The method created by the combination of these newly developed technologies succeeded in providing reliable and reproducible in vitro evaluation of UV protecting ability.

Investigation on the Validity of in vitro Sun Protection Factor and Ultraviolet-A Protection Factor Evaluation Method for Sunscreen Samples.

Factors affecting the in vitro Sun Protection Factor (SPF) and Ultraviolet-A Protection Factor (UVA-PF) of sunscreens were analyzed for verifying the validity and reliability of the ISO24443 evaluation method. UV absorbance measurements by different spectrophotometers did not lead to the large difference in in vitro SPF/UVA-PF, although the UV absorbance determined by each spectrophotometer exhibited relatively large difference when it was larger than 2. On the other hand, relatively large difference was found in in vitro SPF/UVA-PF by utilizing European Cosmetic and Perfumery Association (Colipa) 1994 or UV-solar simulated radiation (UV-SSR) for the spectral irradiance. Appropriateness of employing the coefficient of adjustment for the determination of in vitro UVA-PF was also found to be reexamined.

New in vitro SPF Evaluation Method for Hydrophilic Sunscreen Samples.

A new method was developed for the in vitro sun protection factor (SPF) evaluation of sunscreen samples. A new type of substrate, a hydroxyalkyl cellulose-coated plate, was also prepared specifically for hydrophilic samples. This new substrate was required because hydrophilic samples would be unlikely to wet the surface of the standard cosmetic PMMA UV evaluation plate. A super-hydrophilic quartz plate was prepared by corona-discharge treatment before an aqueous solution of hydroxyalkyl cellulose was spread on it. A flat and uniform hydroxyalkyl cellulose film was subsequently formed through the evaporation of water. Special care was taken to inhibit the generation of spatial non-uniformity. Six hydrophilic sunscreen samples with in vivo SPF values of 56, 55, 52, 25, 15, and 4, were then applied to the prepared hydroxyalkyl cellulose-coated plate, as well as a super-hydrophilic quartz plate and a flat hydrophobic PMMA plate. The thicknesses of the applied layers were determined using a wheel-shaped wet film thickness gauge immediately after the application, and UV transmission was measured using an SPF analyzer. The value of in vitro SPF was calculated from the UV absorbance and the thickness of the layer. For two out of the six samples, PMMA plate could not be available, as the samples were unable to wet the PMMA surface. Relatively small differences were observed between the in vitro SPF values when the super-hydrophilic and hydroxyalkyl cellulose-coated plates were used. Samples exhibiting higher in vivo SPF were also associated with higher in vitro SPF values, although a linear relationship was not observed. In contrast to the super-hydrophilic plate whose half-life of the super-hydrophilicity is only approximately five days, the hydrophilicity of the hydroxyalkyl cellulose-coated plate scarcely varied during six months of storage. Finally, a simplified evaluation method was also proposed. The validity of the method was verified through a ring test where three researchers employed this method in different laboratories at three independent organizations.

The role of central corticotrophin-releasing factor receptor signalling in plasma glucose maintenance through ghrelin secretion in calorie-restricted mice.

Under severe calorie restriction (CR), the ghrelin-growth hormone axis in mice is involved in the maintenance of plasma glucose levels. Ghrelin, a stomach-derived acylated peptide, is up-regulated by the sympathetic nerve in the negative energy status. Central corticotrophin-releasing factor receptor (CRF-R) signalling stimulates the sympathetic tone. The present study aimed to examine the effect of central CRF-R signalling on the maintenance of plasma glucose concentrations in severe calorie-restricted mice with the involvement of ghrelin. Intracerebroventricular injections of urocorin-1 and urocorin-2, which are natural ligands for CRF-R1 and CRF-R2, elevated plasma ghrelin concentrations and ghrelin elevation with an i.c.v. injection of urocorin-1 was cancelled by atenolol (ß1 adrenergic receptor antagonist) administration. We then established a mice model of 60% CR and found that the administration of [d-Lys3]-GHRP-6 (a ghrelin receptor antagonist) in mice under 60% CR reduced the plasma glucose concentration more compared to the vehicle mice. Similarly, the atenolol injection in mice under 60% CR significantly reduced the plasma glucose concentration, which was rescued by the co-administration of ghrelin. An i.c.v. injection of the alpha helical CRH, a non-selective corticotrophin-releasing factor receptor antagonist, in mice under 60% CR significantly reduced the plasma glucose concentration, although the co-administration of α-helical CRH with ghrelin maintained plasma glucose levels. These results suggest that central CRF-R signalling is involved in the maintenance of plasma glucose levels in mice under severe CR via the sympathetic-ghrelin pathway.

Comparative assessment of 24-hr primary skin irritation test and human patch test data with in vitro skin irritation tests according to OECD Test Guideline 439 (for quasi-drugs in Japan).

The Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 439 is an in vitro test method of reconstructed human epidermis (RhE), which was developed for hazard identification of irritating chemicals in accordance with a primary skin irritation test using rabbits with 4-hr exposure. A regulation for quasi-drugs in Japan requires data from primary skin irritation tests using rabbits to undergo 24-hr exposure, and this is used as an evidence for 24-hr closed patch tests in humans. In this study with the same chemicals, primary skin irritation test data using rabbits undergoing 24-hr exposure and a 24-hr occlusive human patch test data were analyzed by comparing the results obtained with four test methods adopted in OECD TG 439. The performances of in vitro test methods showed a positive predictive value of 72.7-85.7% to predict the results of 24-hr primary rabbit skin irritation test knowing that its positive predictive value was 57.1% against humans only. The prediction factors of in vitro test methods were higher for the human patch test data with a sensitivity reaching 60 to 80%. Three surfactants gave false negatives in some of the RhE methods evaluated with the human patch test, but in each case, they were correctly classified as positive when evaluated at double concentration. Therefore, the approach of setting the margin to 2 was effective in eliminating false negatives. This suggests that in vitro test methods are useful for assessing skin irritation potential without animal testing for the application of quasi-drugs in Japan.