Improved performance of the SH test as an in vitro skin sensitization test with a new predictive model and decision tree.

Demands for the elimination and replacement of animal experiments for cosmetic safety assessment have increased in recent years. Evaluation of skin sensitization, however, is a critical issue in cosmetic safety assessment. The SH test is an in vitro skin sensitization test method that evaluates protein binding of chemical substances, which is an important event in skin sensitization. We previously verified the technical transferability and between-laboratory reproducibility of the SH test, a domestic test method for which no scientific research has been conducted, and improved the protocol, but also noted some unresolved issues. Therefore, in the present study, we successfully improved the operational efficiency and clarity of the final judgment of the SH test by (i) developing a new decision-making system that can make a final judgment without statistical processing, (ii) changing the statistical method, and (iii) evaluating and determining the maximum number of repetitions necessary for optimal efficiency. The improved SH test was verified by comparing it with existing test methods already adopted by the Organization for Economic Cooperation and Development. The results of this study demonstrated excellent performance of the improved SH test, with high reproducibility, reliable predictability, and good operational efficiency. The predictive performance of the improved method does not differ significantly from that of the conventional method, although it is clearer and more efficient. Therefore, the results of the present improved method are consistent with those obtained using the conventional method, with higher efficiency.

Enhancing between-facility reproducibility of the SH test as an in vitro skin sensitization test by the improved test method.

There has been an increased demand to eliminate animal experiments and to replace the experiments with alternative tests for assessing the safety of cosmetics. The SH test is an in vitro skin sensitization test that evaluates the protein binding abilities of a test substance. Skin sensitization must be evaluated by multiple test methods. The SH test uses the same cell line and measuring instruments as the human Cell-Line Activation Test (h-CLAT), which is one of the test methods used to evaluate different key events and is listed in the OECD test guidelines. There are cost advantages to usher the SH test into facilities that are already running the h-CLAT. The SH test is conducted only at a facility that has developed the SH test because studies on the between-facility reproducibility and validity have not been performed. Therefore, to verify the transferability of the SH test and the between-facilities reproducibility, we evaluated the reproducibility of the SH test results at three facilities, including the development facility. After an initial round of testing, the protocol was refined as follows to improve reproducibility among the three facilities: i) determine the optimum pH range, ii) change the maximum applicable concentration of water-soluble substances, and iii) define the appropriate dispersion conditions for evaluating hydrophobic substances. These refinements markedly enhanced the between-facility reproducibility (from 76.0% to 96.0%) for the 25 substances evaluated in this study. This study confirmed that the SH test is an effective skin sensitization test method with high technical transferability and between-facility reproducibility.

Glucagon regulates intracellular distribution of adipose differentiation-related protein during triacylglycerol accumulation in the liver.

Cellular lipid droplets (LD) are organelles involved in cellular lipid metabolism. When liver cellular components were fractionated using sucrose density gradient centrifugation, adipose differentiation-related protein (ADRP) was distributed in both the top and bottom fractions, which correspond to the LD and membranous fractions, respectively, in the mouse liver under normal feeding conditions. After overnight fasting, triacylglycerol and ADRP increased nearly 2.5-fold in the mouse liver, and a portion appeared in the intermediate-density LD (iLD) fractions. ADRP in the iLD fractions was also increased in a mouse nonalcoholic steatohepatitis model induced by methione/choline-deficient diet. When HuH-7 human hepatoma cells were incubated with oleic acid for 24 h, the amount of ADRP increased, and it was distributed in both the LD and membrane fractions. However, ADRP appeared in the iLD fractions upon treatment of HuH-7 cells with glucagon. This behavior of ADRP was cAMP-dependent, as the ADRP-positive iLD fractions were induced by dibutylyl cAMP and were blocked by protein kinase A inhibitors. A portion of ADRP colocalized microscopically with calnexin, which is present in the iLD fractions, by treatment of HuH-7 cells or human primary hepatocytes with oleic acid and glucagon, but not by treatment with oleic acid alone. Glucagon has a role in the reorganization of endoplasmic reticulum membranes to generate ADRP-associated lipid-poor particles in hepatic cells, which is related to LD formation during lipid storage.