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High-content imaging technology for the evaluation of drug-induced steatosis using a multiparametric cell-based assay
M Teresa Donato1, Laia Tolosa, Nuria Jiménez
1Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Valencia, Spain. donato_mte@gva.es
Abstract:
In the present study, we developed a cell-based protocol for the identification of drugs able to induce steatosis. The assay measures multiple markers of toxicity in a 96-well plate format using high-content screening (HCS) technology. After treating HepG2 cells with increasing concentrations of the tested compounds, toxicity parameters were analyzed using fluorescent probes: BODIPY493/503 (lipid content), 2',7'-dihydrodichlorofluorescein diacetate (reactive oxygen species [ROS] generation), tetramethyl rhodamine methyl ester (mitochondrial membrane potential), propidium iodide (cell viability), and Hoechst 33342 (nuclei staining). A total of 16 drugs previously reported to induce liver steatosis through different mechanisms (positive controls) and six nonsteatotic compounds (negative controls) were included in the study. All the steatosis-positive compounds significantly increased BODIPY493/503 fluorescence in HepG2 cells, whereas none of the negative controls induced lipid accumulation. In addition to effects on fat levels, increased ROS generation was produced by certain compounds, which could be indicative of increased risk of liver damage. Our results suggest that this in vitro approach is a simple, rapid, and sensitive screening tool for steatosis-inducing drugs. This conclusion should be confirmed by testing a larger number of steatosis-positive and -negative inducers.
Insights
This study presents a new cell-based assay for identifying drugs that cause liver steatosis (fatty liver). The high-content screening method accurately detects steatosis-inducing compounds, offering a rapid and sensitive tool for drug development.
Area of Science:
- Hepatology
- Toxicology
- Drug Discovery
Background:
- Drug-induced liver injury (DILI) is a significant clinical concern.
- Steatosis, characterized by lipid accumulation in hepatocytes, is a common form of DILI.
- Accurate identification of steatogenic compounds early in drug development is crucial.
Purpose of the Study:
- To develop and validate a robust cell-based assay for identifying drugs that induce hepatic steatosis.
- To utilize high-content screening (HCS) technology for multiplexed toxicity parameter analysis.
- To establish a reliable in vitro method for early-stage detection of steatosis-inducing drug candidates.
Main Methods:
- A cell-based protocol using HepG2 cells in a 96-well plate format was established.
- High-content screening (HCS) technology was employed to analyze multiple toxicity markers.
- Fluorescent probes were used to quantify lipid content (BODIPY493/503), reactive oxygen species (ROS), mitochondrial membrane potential, cell viability, and nuclear morphology.
Main Results:
- The assay successfully distinguished between known steatosis-inducing drugs (positive controls) and non-steatotic compounds (negative controls).
- All positive controls significantly increased lipid accumulation (BODIPY493/503 fluorescence) in HepG2 cells.
- Negative controls did not induce lipid accumulation, demonstrating assay specificity. Certain compounds also showed increased ROS generation, indicating potential for liver damage.
Conclusions:
- The developed in vitro assay is a simple, rapid, and sensitive tool for screening steatosis-inducing drugs.
- This HCS-based approach facilitates early identification of potential hepatotoxicants in drug discovery pipelines.
- Further validation with a broader range of steatogenic and non-steatogenic compounds is recommended.
