Copper-induced diurnal hepatic toxicity is associated with Cry2 and Per1 in mice

This study aimed to investigate diurnal variations in copper-induced hepatic toxicity and the molecular mechanisms underlying this chronotoxicity. Male C57BL/6J mice were intraperitoneally injected with copper chloride (CuCl ) at zeitgeber time 2 (ZT2) or 14 (ZT14), twice per week for 5 or 8 weeks. Seventy-two hours after the final CuCl injection, the mice were euthanized, and plasma samples were collected. The livers and kidneys were collected and weighed. In vitro experiments were performed to assess cell viability and fluctuations in clock gene expression levels in Hepa1-6 cells after CuCl treatment. We examined copper homeostasis- and apoptosis-related genes under clock genes overexpression. Repeated CuCl administration for 8 weeks resulted in more severe toxicity at ZT14 compared to ZT2. CuCl administration at ZT14 elevated plasma aspartate aminotransferase, hepatic tumor necrosis factor-α, and interleukin-6 for 5 weeks, whereas the toxic effects of CuCl administration at ZT2 were weaker. Moreover, CuCl treatment inhibited Hepa1-6 cell viability in a dose-dependent manner. We observed increased expression of three clock genes (Ciart, Cry2, and Per1) after CuCl treatment. Among them, overexpression of Cry2 and Per1 accelerated CuCl -induced inhibition of Hepa1-6 cell viability. Moreover, we found that the overexpression of Cry2 and Per1 regulates cleaved caspase-3 by modulating the copper transporter genes ATP7B and CTR1. These results suggest that CuCl -induced diurnal toxicity is associated with Cry2 and Per1 expression through the regulation of copper transporter genes in mice.