Size- and time-dependent alteration in metabolic activities of human hepatic cytochrome P450 isozymes by gold nanoparticles via microsomal coincubations
Nano-sized particles are known to interfere with drug-metabolizing cytochrome P450 (CYP) enzymes, which may lead to unintended adverse reactions. However, the mechanisms behind this inhibition are not yet fully understood. In this study, we systematically investigated the effects of gold nanoparticles (AuNPs) on five major CYP isozymes during in vitro incubations with human liver microsomes (HLMs) and tannic acid (TA)-stabilized AuNPs, ranging in size from 5 to 100 nm. Our results show that smaller AuNPs exert more pronounced inhibitory effects on CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in a dose-dependent manner, while CYP1A2 is the least affected by AuNPs. The size- and dose-dependent inhibition of CYP activity, as well as the nonspecific binding of drugs to nanogold in the incubation medium, can be significantly reduced by increasing the microsomal protein-to-AuNP ratio, likely through a noncompetitive inhibition mechanism. Additionally, AuNPs were found to induce slow, time-dependent inactivation of CYP2D6 and CYP3A4 in an NADPH-independent manner. Various techniques, including UV-vis spectroscopy, dynamic light scattering, and zeta potential measurements, were employed to monitor changes in the properties of AuNPs during the AuNP/HLM/CYP incubations. The results suggest that AuNPs exhibit improved stability when mixed with HLM, indicating that AuNP-HLM interactions may stabilize the particles more rapidly than salt-induced aggregation in phosphate buffer. The findings suggest that the CYP inhibition induced by AuNPs could be partly due to the nanoparticles binding to the enzymes, altering their structure, or binding to the HLM membrane, thereby impairing integral membrane proteins. Moreover, AuNPs may block the substrate binding pocket on the CYP enzymes, with the extent of inhibition dependent on both the particle characteristics and the structural diversity of the CYP isozymes. These results provide insights into additional mechanisms behind the differential inhibitory effects of AuNPs on the metabolic activities of NADPH tetrasodium salt hepatic CYP isozymes.