Metabolomics-on-a-Chip of Hepatotoxicity Induced by Anticancer Drug Flutamide and Its Active Metabolite Hydroxyflutamide Using HepG2/C3a Microfluidic Biochips

Abstract

We used the recently introduced "metabolomics-on-a-chip" approach to test secondary drug toxicity in bioartificial organs. Bioartificial organs cultivated in microfluidic culture conditions provide a beneficial environment, in which the cellular cytoprotective mechanisms are enhanced, compared with Petri dish culture conditions. We investigated the metabolic response of HepG2/C3a cells exposed to flutamide, an anticancer prodrug, and hydroxyflutamide (HF), its active metabolite, in a microfluidic biochip. The cellular response was analyzed by (1)H nuclear magnetic resonance spectroscopy to identify cell-specific molecule-response markers. The metabolic response to flutamide results in a disruption of glucose homeostasis and in mitochondrial dysfunctions. This flutamide-specific metabolic response was illustrated by a reduction of the extracellular glucose and fructose consumptions and a general reduction of the tricarboxylic acid cycle activity leading to the reduction of the consumption of several amino acids. We also found a higher production of 3-hydroxybutyrate and lactate, and the reduction of the albumin production compared with controls. The toxic metabolic signature associated with the active metabolite HF was illustrated by a high-energy demand and an increase in several amino acid metabolism. Finally, for both molecules, the hepatotoxicity was correlated to the glutathione (GSH) metabolism illustrated by the levels of the 2-hydroxybutyrate and pyroglutamate productions and the increase of the glutamate and glycine productions. Thus, the entire set of results contributed to extract specific mechanistic toxic signatures and their relation to hepatotoxicity, which appeared consistent with literature reports. As new finding of HepG2/C3a cells hepatotoxicity, we propose a metabolic network with a related list of metabolite variations to describe the GSH depletion when followed by a cell death for the HepG2/C3a cells cultivated in our polydimethylsiloxane microfluidic biochips. Our findings illustrate the potential of metabolomics-on-a-chip as an in vitro alternative method for predictive toxicology.

Organs-on-chips at the frontiers of drug discovery

“…The proof of principle for this approach has been demonstrated in recent studies that utilized human liver-on-a-chip devices for metabolomic analysis of hepatotoxicity. For example, microfluidic culture of human hepatocytes was coupled with NMR spectroscopy to develop a bioanalytical platform for monitoring metabolic responses of hepatocytes to the anticancer drug flutamide and its active metabolite hydroxyflutamide, both of which are known to possess hepatotoxic properties 42 . Through quantitative analysis and mapping of metabolic and mitochondrial activities, the investigators identified metabolic signatures of toxic drug responses in this model, and delineated metabolic pathways involved in the induction of hepatotoxicity due to flutamide and hydroxyflutamide.…”

Drug discovery for remyelination and treatment of MS

“…These new in vitro models, termed “organs‐on‐chips” may not necessarily mimic the function of an entire organ, but rather recapitulate specific aspects of its function on a single mm‐sized device. Through reproducing organ‐specific 3D structure, microenvironment and physiology, a number of devices have been generated to mimic the lung, heart, kidney, liver, intestine and brain (Agarwal, Goss, Cho, McCain, & Parker, ; Choucha Snouber et al, ; Huh et al, ; Kim & Ingber, ; Schwartz et al, ). These platforms not only allow visualization of dynamic, organ‐level responses that cannot normally be observed in conventional 2D cell culture modes, but can also be used to facilitate drug development.…”

Engineered Liver Platforms for Different Phases of Drug Development

“…Similar concentration gradients were established in another biochip designed to mimic the liver sinusoid [38]. The Leclerc group has designed biochips for characterizing the metabolic profiles of rat hepatocytes [39,40], evaluating drug metabolism in PHHs [41,42], and investigating drug-induced ROS formation and glutathione depletion in rat hepatocytes [43] and HepG2/C3a cells [44]. Chao et al cultured PHH monolayers in a microfluidic platform for evaluating drug clearance [45], which was extended by Novik et al to PHH-NPC co-cultures [46].…”