Mechanistic Tox Assays

APPLICATION & SPECIALITY USES:ADME-Toxicology Products and Services
Keywords:Cytotoxicity AssessmentHepaticin vitro ModelsMechanistic Tox

We use HEPATOPAC Technology to create stable hepatocyte cultures that can remain viable for up to 28 days. Because of their long-term stability, we recommend HEPATOPAC cultures for the following mechanistic toxicity studies:

  • Chronic and Acute Toxicity: ATP, ALT, Albumin, and Urea assessment
  • Mitochondrial Dysfunction: MTT and ATP assessment
  • Reactive Metabolites: GSH depletion and metabolite formation
  • Cholestasis: Transporter activity and BSEP inhibition assessment
  • Steatosis: Lipid and phospholipid accumulation
  • Apoptosis: Caspase 3 activity assessment
  • Innate Immune-Mediated Toxicity: Cytokine secretion, ATP, and ALT assessment.
Case Study: Reactive Metabolite Toxicity

Many types of drugs can be bioactivated leading to formation of reactive metabolites. This metabolic activation is often the initial event in many chemically-induced toxicities. In a study of compounds withdrawn from the US market due to hepatotoxicity, there was evidence of reactive metabolite formation in 5 out of 6 drugs that were withdrawn. Acetaminophen (APAP) is a commonly used antipyretic and analgesic compound that has hepatotoxic potential. It is primarily metabolized by glucuronidation and sulfation. However, its hepatotoxicity is mediated by its toxic metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which is generated by liver cytochrome P450s (CYP2E1 and CYP1A2) and is detoxified by conjugation with hepatic glutathione (GSH). During an overdose with APAP, cellular GSH is depleted, and cellular functions are impaired by binding of NAPQI to macromolecules, which can lead to cell death. Using APAP as a model toxicant and mimicking cellular glutathione (GSH) depletion by addition of L-buthionine (S,R)-sulfoximine (BSO) an inhibitor of GSH synthesis to cultures, we have shown the utility of human HEPATOPAC cultures for detecting reactive metabolite toxicities. Addition of 200 µM BSO to human HEPATOPAC cultures reduces GSH content by about 80% and potentiated acetaminophen-induced ATP depletion in these cultures.

Data from these studies is illustrated in the charts below:

HepatoPac treated with APAP shows GSH depletionTreatment of HepatoPac with APAP shows GSH depletion