Drug-Induced Liver Injury (DILI) is the main cause of acute liver failure in the USA and Europe and is one of the predominant reasons for market withdrawals and regulatory actions1.
Hepatic toxicity has accounted for 15 of the 47 drugs withdrawn from the market between 1975 - 20072. DILI is classified as dose-dependent or intrinsic, acetaminophen (paracetamol) being the most significant example of this class3 or idiosyncratic which is unpredictable and not directly reliant on dose4.
An individual’s susceptibility to developing idiosyncratic DILI includes numerous factors including age, sex, drug interactions, alcohol consumption, and genetic variability4. Although cellular and animal models to predict intrinsic (dose-dependent) DILI have been improved, idiosyncratic DILI is nearly impossible to predict.
Withdrawal of compounds at a late stage (or post-marketing) due to idiosyncratic DILI is expensive and can lead to a mistaken withdrawal of compounds that might be useful. Monocyte-derived hepatocyte-like (MH) cells have been developed as a tool to investigate long term hepatotoxicity, metabolism and drug interactions5. Additionally, patient-derived MH cells could deliver a tool for diagnosis or exclusion of idiosyncratic DILI1,6 and supply the causative agent in polymedicated patients.
In this study, MH cells (MetaHeps®) were used on the CardioExcyte 96 and variations in impedance, and therefore confluency was used as a toxicity measure. Intrinsic (dose-dependent) effects of paracetamol could be determined consistent with other means of liver injury detection. Therefore, the CardioExcyte 96 combined with patient-specific MH cells offers a new tool in the investigation of idiosyncratic and intrinsic DILI.
The impedance signal recorded on the CardioExcyte 96 varies due to alterations in confluency, the conductivity of adherent cells and cell contact (morphological shape) and therefore supplies a measure of toxicity. MH cells from MetaHeps® were grown on NSP-96 plates and base impedance was checked over time. Paracetamol demonstrated a dose-dependent reduction in base impedance (Figure 1A).
This result can be compared directly with a standard toxicity (lactate dehydrogenase (LDH) release) assay5,6 (Figure 1B). Significantly, the CardioExcyte 96 enables base impedance to be continuously monitored utilizing a single plate revealing that the greatest effect is attained within the initial few hours of exposure. Employing the standard toxicity assay, measurements of LDH in the supernatant and lysate are conducted only at 24-hour intervals or at several time points necessitating a number of plates.
Figure 1. (A) Increasing concentrations of paracetamol induce a decrease in base impedance of MH cells which can be monitored continuously. Tween (2%) induced 100% cell death and was used as a positive control. (B) Toxicity measurement of MH cells using an LDH release assay. Paracetamol showed a dose-dependent effect on toxicity in both assays.
The impact of low and intermediate concentrations of paracetamol and washout was studied in the following experiments. Figure 2A illustrates the effect on the base impedance of MH cells after exposure for 24 or 48 hours of 1 mM paracetamol. The results demonstrate that exposure to low doses of paracetamol results in only transient effects on toxicity parameters comparable with cellular “adaptation”. This phenomenon is reported in humans7 and animals8, even when further doses of paracetamol would typically lead to damage to the liver8. This effect is also consistent with toxicity assays that use LDH release experiments (Figure 2B).
Figure 2. (A) Normalized impedance versus time (in hours) after MH cells were exposed to 1 mM paracetamol for 24 or 48 hours. Repeat exposure to paracetamol causes adaptation and only transient toxicity is observed. (B) Toxicity measurement using LDH release assay also shows repeated exposure to low doses of paracetamol causes transient toxicity of hepatocytes.
Figure 3 illustrates the exposure of MH cells to an intermediate dose (5 mM) of paracetamol on the CardioExcyte 96 (A) and LDH release assay (B). In this example, washout of paracetamol after a day led to recovery from toxicity whereas continued exposure resulted in an increase in hepatoxicity. This is vital in order to determine compounds that may potentially cause liver damage even if treatment is stopped.
Figure 3. (A) Normalized impedance versus time (in hours) after MH cells were exposed to 5 mM paracetamol for 24 or 48 hours. Cells recovered, indicated by the increase in base impedance, when paracetamol was washed out after 24 hours but toxicity continued when 5 mM paracetamol was added again for a further 24 hours. (B) Toxicity measurement using the LDH release assay also shows that toxicity is reversed upon washout of paracetamol but continues with the 2nd dose of paracetamol after 24 hours.
To summarize, the CardioExcyte 96 can record numerous samples of hepatocyte-like cells simultaneously in real-time, a crucial feature in monitoring cytotoxicity. The data recorded on the CardioExcyte 96 is comparable with results obtained from the standard LDH release assay and therefore strengthens the scientific relevance of impedance-based toxicity assays. In order to stop the inappropriate withdrawal of potentially life-saving compounds, a dependable assay for the detection of DILI is urgently needed.
Monocyte-derived hepatocyte-like cells from MetaHeps® were used. MH cells were obtained from healthy donors and cryopreserved after generation.
Impedance measurements were performed in accordance with Nanion’s standard procedures for the CardioExcyte 96. MH cells were thawed, seeded on the Sensor Plate at a suitable density to supply the required confluency and cultured under propriety conditions for 24 hours before exposure to paracetamol.
Around two hours prior to drug application, the medium was completely taken away from the wells and 200 μl fresh medium was added. Every signal was normalized to a group of control measurements (n=5-11) on the same plate.
References and Further Reading
- Benesic, A., & Gerbes, A.L. 2015. Dig. Dis. 33: 486-491
- Stevens, J.L., & Baker, T.K. 2008. Drug Discovery Today. 14(3/4): 162-167
- McGill, M.R., & Jaeschke, H. 2013. Pharm Res. 30(9): 2174-2187
- Chalasani, N., & Björnsson, E. 2010. Gastroenterology. 38: 2246–2259
- Benesic, A., et al., 2012. Laboratory Investigation. 92: 926-936
- Benesic, A., et al., 2016. Gut. 65: 1555-1563
- Watkins, P.B. et al., 2006. JAMA. 296(1): 87-93
- Shayiq, R.M. et al., 1999. Hepatol. 29(2): 451-463
About Nanion Technologies
Nanion Technologies is a spin-off from the Center of Nanoscience (CeNS) of the University of Munich (LMU).
Nanion combines bio- and microtechnology in a company serving the life sciences industry by offering products and services which will dramatically increase the speed and efficiency of the drug discovery process in an important segment of the pharmaceutical market.
Nanion bases its business on a proprietary chip technology and will design and develop High Throughput Screening (HTS) systems for ion channel active drugs (ICADs). Ion channels are prime targets for innovative medicines aimed at many important diseases.
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