Species-specific adverse effects of phthalate esters: Key protein structures that cause human-specific TRPA1 activation

<p>Previous studies reported that exposure to phthalates exacerbated allergic symptoms. Regarding the mechanisms of their allergic effects, it has been suggested that the activation of transient receptor potential ankyrin 1 (TRPA1) is responsible for the adjuvant effects of phthalates. Our previous study reported that Mono(2-ethylhexyl) phthalate (MEHP), a representative metabolite of di(2-ethylhexyl)phthalate, activated human TRPA1 (hTRPA1), but not mouse TRPA1 (mTRPA1). Therefore, this study investigated the mechanisms underlying the species difference in TRPA1 activation by MEHP. First, we established several Flp-In 293 cell lines stably expressing human-mouse chimeric TRPA1. Next, to evaluate the human-mouse chimeric TRPA1 activation by MEHP, the intracellular Ca²⁺ levels were measured using the cell lines. Consequently, MEHP caused an increase in intracellular Ca²⁺ levels in a concentration-dependent manner (EC₅₀ value: 0.5 µM) in hTRPA1-expressing cells, whereas MEHP had no effects on mTRPA1. The activation efficacy and/or affinity of MEHP was decreased in chimeric hTRPA1-expressing cells containing N-terminal linker domain of mTRPA1 or C-terminal domain of mTRPA1. These results demonstrated that the N-terminal linker domain and C-terminal domain play an important role in the species-specific activation of TRPA1 by MEHP. Moreover, the species differences could be attributed to the "pocket region", composed of the linker domain and C-terminal domain.</p>