Integrated proteomic and transcriptomic investigation of the acetaminophen toxicity in liver microfluidic biochip

Abstract

Microfluidic bioartificial organs allow the reproduction of in vivo-like properties such as cell culture in a 3D dynamical micro environment. In this work, we established a method and a protocol for performing a toxicogenomic analysis of HepG2/C3A cultivated in a microfluidic biochip. Transcriptomic and proteomic analyses have shown the induction of the NRF2 pathway and the related drug metabolism pathways when the HepG2/C3A cells were cultivated in the biochip. The induction of those pathways in the biochip enhanced the metabolism of the N-acetyl-p-aminophenol drug (acetaminophen-APAP) when compared to Petri cultures. Thus, we observed 50% growth inhibition of cell proliferation at 1 mM in the biochip, which appeared similar to human plasmatic toxic concentrations reported at 2 mM. The metabolic signature of APAP toxicity in the biochip showed similar biomarkers as those reported in vivo, such as the calcium homeostasis, lipid metabolism and reorganization of the cytoskeleton, at the transcriptome and proteome levels (which was not the case in Petri dishes). These results demonstrate a specific molecular signature for acetaminophen at transcriptomic and proteomic levels closed to situations found in vivo. Interestingly, a common component of the signature of the APAP molecule was identified in Petri and biochip cultures via the perturbations of the DNA replication and cell cycle. These findings provide an important insight into the use of microfluidic biochips as new tools in biomarker research in pharmaceutical drug studies and predictive toxicity investigations.

Figure 1. Morphology of the HepG2/C3a cells after 96 hours of culture.

(A) biochip without APAP; (B) biochip treated with 1 mM of APAP; (C) Petri dish without APAP; (D) Petri dish treated with 1 mM of APAP.

Figure 2. APAP effect on cell proliferation and cell cycle repartition.

(A) Comparison of the cell growth in biochip and Petri dishes in untreated and treated conditions with 1 mM of APAP after 96 h of cultures (n = 6, * P<5%); (B) DNA repartition in biochip and Petri dishes after 96 hours of culture. The DNA repartition show for both culture conditions a disruption of the cell cycle repartition compared to control (n = 6* P<5%).

Figure 3. Principal Component Analysis.

(A) Proteomic analysis; (B) Transcriptomic analysis; (circles denote Petri data, triangles denote biochips data, black symbols are control data, white symbols are APAP data).

Figure 4. Mean genes and proteins affected by the culture condition.

(A) Mean genes differentially expressed by the environment condition and involved in hepatic differentiated function; (B) Mean proteins differentially expressed by the environment condition and involved in hepatic differentiated function.

Figure 5. Experimental design description.

(A) Perfusion setup and peristaltic pump setup with 6 individual biochips and medium reservoirs; (B) Microfluidic biochip; (C) Microchannel design inside of the biochip; (D) Experimental procedure.