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Developing a High Content Imaged-Based Method for Evaluating Hepatotoxicity

Authors:
Poster Author(s)
King, Emily, FDA/NCTR (Student); Arefin, Ayesha, FDA/NCTR (Mentor); Phanavanh, Bounleut, FDA/NCTR (Co-author); Papineau, Katy, FDA/NCTR (Co-author); Shi, Qiang, FDA/NCTR (Co-author); Schnackenberg, Laura, FDA/NCTR (Co-author);
Center:
Contributing Office
National Center for Toxicological Research

Abstract

Poster Abstract

Drug induced liver injury is a major concern in the development of new pharmaceuticals and results in many drugs being removed from the market, given a black box warning, or development being halted during the preclinical or clinical phases. In vitro methods are commonly employed in liver toxicity studies as a non-invasive model for evaluating severity and mechanisms of cellular damage following exposure to hepatotoxic drugs. Current methods for evaluating the toxicity of drugs include the use of biochemical assays to track changes in protein expression, mitochondrial function, cell viability, and other biological parameters after drug exposure. However, these assays have high costs and cannot provide detailed data across multiple time points for in vitro studies. High content microscopy combined with non-cytotoxic fluorescent staining can be utilized to non-invasively monitor cellular changes, such as nuclear damage and mitochondrial function. This project aims to develop an image-based assay for evaluating hepatotoxicity in a real time in vitro imaging system using liver hepatocellular carcinoma derived HepG2 cells, ImageXpress Micro 4 High Content Imaging system (IMX4), and MetaXpress software. This assay will be developed in both two-dimensional (2D) and three-dimensional (3D) culture conditions to detect toxicity-induced changes in phenotypic response - such as live cell count, nuclear morphology, and fluorescent intensity of actin and nuclei.  HepG2 cells will be cultured in a 96 well plate for 24 hours and will be exposed to one of three known hepatotoxicants - acetaminophen, nefazodone, or diclofenac - for 24, 48, and 72 hours with predetermined drug dosages based on previous studies. A vehicle-only control of 0.1% DMSO, as well as a Triton X control will be incorporated to validate the drug exposure and live cell analysis. After the incubation period, treated cells will be stained with DAPI and F-Actin dyes for nuclear and cytoplasmic staining, respectively. Cells will be imaged using the IMX4, with four images captured per well per timepoint, to track changes in nuclear count, cell morphology, and fluorescent intensity. The MetaXpress Cell Scoring module will be utilized to analyze the images and compare these parameters across time and between groups. Release of lactate dehydrogenase activity will be measured to compare cell cytotoxicity and death with the output from the image based live cell quantification. This preliminary method will then be applied to a three-dimensional liver microphysiological system along with a non-cytotoxic nuclear staining dye to develop a real time hepatoxicity screening assay using human primary hepatocytes. The proposed image-based method will contribute to the development of a phenotypic assay for a deeper understanding of cytotoxicity in real time while offering advantages over other biochemical assays.

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