Hepatotoxicity of copper sulfide nanoparticles towards hepatocyte spheroids using a novel multi-concave agarose chip method.

To explore the hepatotoxicity of copper sulfide nanoparticles (CuSNPs) toward hepatocyte spheroids. Other than the traditional agarose method to generate hepatocyte spheroids, we developed a multi-concave agarose chip (MCAC) method to investigate changes in hepatocyte viability, morphology, mitochondrial membrane potential, reactive oxygen species and hepatobiliary transporter by CuSNPs. The MCAC method allowed a large number of spheroids to be obtained per sample. CuSNPs showed hepatotoxicity through a decrease in spheroid viability, albumin/urea production and glycogen deposition. CuSNPs also introduced hepatocyte spheroid injury through alteration of mitochondrial membrane potential and reactive oxygen species, that could be reversed by N-acetyl-l-cysteine. CuSNPs significantly decreased the activity of BSEP transporter by downregulating its mRNA and protein levels. Activity of the MRP2 transporter remained unchanged. We observed the hepatotoxicity of CuSNPs with associated mechanisms in an advanced 3D culture system.