(629e) Circadian Regulation of Cytochromes P450 in 3D Hepatocarcinoma Spheroids

Circadian rhythms characterized by 24 h oscillations, also known as the circadian clock, is universally present in all organisms and regulate physiological functions based on the 24 h structure of a day. Circadian disruption has been implicated in the pathogenesis of aging and disease, including diabetes, cancer and neurodegenerative disorders such as Alzheimer’s Disease and Parkinson’s Disease. While the internal clock synchronizes gene expression with the guidance of external cues like light, a similar synchronization of gene expression can be induced in vitro by incubating the cells with an increased percentage of serum followed by rapid removal. This ability to mimic gene synchronization in vitro provides an opportunity to understand the influence of the circadian clock on expression of drug metabolizing enzymes, most specifically the cytochromes P450. Previous studies have suggested that synchronization of HepG2 cell line induces the rhythmic gene and protein expression of CYP3A4, CYP2D6 and CYP2E1. However, further insight on the role played by the 3D microenvironment on the rhythmicity of these genes and the corresponding enzymatic activity is lacking. To understand this interplay, gene expression of the circadian machinery and CYP450s were compared using the model human hepatocarcinoma cell line, HepG2. Upon cell synchronization by serum shock, gene and protein expression of core clock regulators and CYP450 enzyme activity were assessed using RT-PCR, western blots, and selective enzyme assays. Rhythmic expression of these genes was demonstrated. Further insight into the influence of the 3D microenvironment on circadian regulation of the CYP450s were then obtained. This work provides a better understanding of chronopharmacokinetic events in humans by using physiologically relevant 3D culture systems.