Nephrotoxicity and innovative risk assessment of cinnabar-containing An-Gong-Niu-Huang Wan based on bioavailability and speciation using on an IPBET/small intestinal organ-on-a-chip

An-Gong-Niu-Huang Wan (AGNHW) is a traditional Chinese medicine preparation containing cinnabar (HgS). However, its clinical use has been controversial due to concerns regarding mercury nephrotoxicity. To evaluate the renal toxicity, mice were administered AGNHW, cinnabar, methylmercury (MeHg), and mercuric chloride (HgCl₂) for 28 days. Blood biochemistry, histopathology, and the expression of nephrotoxicity-sensitive genes were assessed. To assess the health risks associated with AGNHW, we established an innovative Physiologically Based Extraction Test (IPBET) /small intestinal organ-on-a-chip model for the first time to simulate gastrointestinal digestion and absorption of mercury from AGNHW. This organ-on-a-chip model with HPLC-ICP-MS enabled the first determination of bioavailable mercury species in AGNHW. Urinary creatinine (UCr), total protein (TP), kidney injury molecule-1 (Kim-1), α1-microglobulin (a1-MG), N-cadherin levels, and histological findings in the MeHg and HgCl₂ groups showed significant differences compared to the control, cinnabar, and AGNHW groups. The expression of F4/80, CD11b, and MMP7 was markedly altered in the MeHg group. Our organ-on-a-chip model revealed that bioavailable MeHg in the MeHg group was 298-fold and 27-fold higher compared to the control and AGNHW groups. Our refined risk assessment indicated that, when adjusted by bioavailability and speciation, the health risks associated with mercury exposure from AGNHW consumption were acceptable. Collectively, considering bioavailability and mercury species, our IPBET/small intestinal organ-on-a-chip provides a novel approach for assessing cinnabar exposure risks in AGNHW. Our study provides a scientific basis that AGNHW and cinnabar are much less nephrotoxic than HgCl 2 and MeHg, and does not pose a significant threat to human health clinically.