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The unfolded protein response (UPR) events triggered by the accumulation of unfolded protein in endoplasmic reticulum (ER) activate the three UPR signaling pathways mediated by IRE1, ATF6 and PERK. Spliced XBP1 mRNA induced by activated IRE1 is translated to the protein, a potent transcription factor that induces BiP expression. XBP1 is also induced by activated ATF6. It is thus thought to be an important marker reflecting both IRE1 and ATF6 signaling in response to ER stress. For quantitative measurement of XBP1 gene expression, it is important to distinguish between the spliced and non-spliced form of XBP1 mRNA. We have developed a new method to detect the spliced XBP1 mRNA by means of real-time PCR and we compared the result with measurements of the expression of the ER stress inducible gene BiP. A good correlation was found between spliced XBP1 expression and BiP expression. Thus, our method may be useful for simple and quantitative evaluation of ER stress.

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.