ADME / Tox >> In Vitro Toxicity >> Hepatotoxicity

Determine your drug’s hepatotoxicity potential early in discovery

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Hepatotoxicity is one of the major causes of failure during drug development and the major reason for removal of approved drugs from the market. In vivo toxicity testing is an essential part of drug safety testing. However, because of the time and cost involved, along with the high quantity of compound required, it is impractical to test a large number of compounds that are produced during early lead optimization in animals. Thus, in practice, in vivo studies are limited to a small amount of late stage compounds, when a considerable investment into the program has already occurred.

To address the need for early stage hepatotoxicity testing, Eurofins has developed a multiplexed hepatotoxicity assay that provides an effective in vitro model to assess hepatotoxicity potential in cells derived directly from human liver tissue. In vitro cell-based toxicity assays mimic in vivo tissue studies and in turn provide a reliable tool for safety evaluation in early stages of drug discovery. Eurofins offers four image-based High Content Analysis (HCA) assays using cryopreserved human primary hepatocytes or HepG2 (human liver hepatocellular carcinoma) cells. These multi-endpoint analyses provide a thorough assessment of hepatotoxicity while supplying detailed mechanistic information underlying the observed toxicity.

Advantages of Eurofins' Hepatotoxicity Assays:

  • Pre-validated cryopreserved human primary hepatocytes or HepG2 cells are used for reproducible assays
  • High Content Analysis (HCA) provides objective and more consistent data analysis compared to other methods
  • Low compound requirement - as little as 300 μl of a 20 mM solution is needed for testing at a final assay concentration of 100 μM
  • Each test compound is evaluated in full dose response with 10 concentrations tested in triplicate at half log dilutions
  • Relevant reference compound and vehicle controls are included on each plate

Mechanistic cytotoxicity assessment with human primary hepatocytes

A three parameter multiplexed study using cryopreserved human primary hepatocytes (HPH) is used to assess compound induced hepatotoxicity in vitro. A decrease in cell viability is a very sensitive marker to detect general toxicity. An increased in reactive oxygen species (ROS) level may result in significant damage to cell structures and cause oxidative stress. The reduction in electrochemical gradient across the mitochondrial membrane is an indicator of poor respiratory capacity and cell health.

Features of Eurofins' cytotoxicity assessment

  • High Content Analysis, cytotoxicity parameters:
    • Cell viability - decrease in Hoechest (nuclear dye) stained cells
    • ROS - increase in fluorescence with 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DFFDA).
    • Mitochondrial membrane potential - decrease in staining with a TMRE dye
  • 24 hr compound incubation

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Figure 1. Effect of mitoxantrone treatment on the health of human primary hepatocytes as assessed by cell number (black), ROS, reactive oxygen species, (blue) and mitochondrial membrane potential (red)

General cytotoxicity assessment with human primary hepatocytes

A two parameter multiplexed study using cryopreserved human primary hepatocytes (HPH) is used to assess compound induced hepatotoxicity in vitro. A decrease in cell viability is a very sensitive marker to detect general toxicity. Activation of caspase 3 indicates an apoptotic mechanism.

Features of Eurofins' general HPH cytotoxicity assessment

  • Cryopreserved human primary hepatocytes
  • High Content Analysis, cytotoxicity parameters:
    • Cell viability - decrease in DAPI (nuclear dye) stained cells
    • Apoptosis - increased staining with an anti-active caspase antibody
  • 72 hr compound incubation


Cellular stress assessment with human primary hepatocytes

A two parameter multiplexed study using cryopreserved human primary hepatocytes (HPH) is used to assess compound induced cellular stress in vitro. A decrease in cell viability is a very sensitive marker to detect general toxicity. Heat shock protein 27 (HSP 27) is expressed in response to cellular stress in an attempt to block the apoptosis pathway.

Features of Eurofins' cellular stress assessment

  • High Content Analysis, cellular stress parameters:
    • Cell viability - decrease in DAPI (nuclear dye) stained cells
    • Cellular stress - increased staining using an anti-HSP27 antibody
  • 24 hr compound incubation


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Figure 2. Cellular stress in human primary heptatocytes induced by Chlorpromazine treatment as demonstrated by a decrease in viable cells (black) and increase in HSP27 expression (blue)


Complete cytotoxicity assessment using HepG2 cells

A five parameter multiplexed study using human hepatocellular carcinoma (HepG2) cells is used to assess compound induced hepatotoxicity in vitro. A decrease in cell viability is a very sensitive marker to detect general toxicity. An uncontrolled increase in cytoplasmic calcium is a common pathophysiological event in cellular toxicity. Nuclear shrinkage, a result of chromatin condensation, is one of the hallmarks of apoptotic cell death. Disruption of the cytoplasmic membrane, resulting in an increase in membrane permeability, is a common and late event in cellular toxicity. Finally, the reduction in electrochemical gradient across the mitochondrial membrane is an indicator of poor respiratory capacity and cell health.

Features of Eurofins' HepG2 cytotoxicity assessment

  • High Content Analysis, cytotoxicity parameters:
    • Cell viability - decrease in Hoechst (nuclear dye) stained cells
    • Cytoplasmic calcium - increase in fluorescence measured with Fluo-4 calcium sensitive dye
    • Apoptosis - decreased nuclear size by Hoechst (nuclear dye) stained cells
    • Cytoplasmic membrane disruption - increase cytoplasmic staining with TOTO-3 dye
    • Mitochondrial membrane potential - decrease in staining with a TMRM dye
  • 72 hr compound incubation



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Figure 3. Multi-parametric analysis of the cytotoxic effect of cerivastatin on HepG2 Cells