A systems approach reveals species differences in hepatic stress response capacity.

To minimize the occurrence of unexpected toxicities in early phase preclinical studies of new drugs, it is vital to understand fundamental similarities and differences between preclinical species and humans. Species differences in sensitivity to acetaminophen (APAP) liver injury have been related to differences in the fraction of the drug that is bioactivated to the reactive metabolite N-acetyl-p-benzoquinoneimine (NAPQI). We have used physiologically based pharmacokinetic modeling to identify oral doses of APAP (300 and 1000 mg/kg in mice and rats, respectively) yielding similar hepatic burdens of NAPQI to enable the comparison of temporal liver tissue responses under conditions of equivalent chemical insult. Despite pharmacokinetic and biochemical verification of the equivalent NAPQI insult, serum biomarker and tissue histopathology analyses revealed that mice still exhibited a greater degree of liver injury than rats. Transcriptomic and proteomic analyses highlighted the stronger activation of stress response pathways (including the Nrf2 oxidative stress response and autophagy) in the livers of rats, indicative of a more robust transcriptional adaptation to the equivalent insult. Components of these pathways were also found to be expressed at a higher basal level in the livers of rats compared with both mice and humans. Our findings exemplify a systems approach to understanding differential species sensitivity to hepatotoxicity. Multiomics analysis indicated that rats possess a greater basal and adaptive capacity for hepatic stress responses than mice and humans, with important implications for species selection and human translation in the safety testing of new drug candidates associated with reactive metabolite formation.

Detection of Hepatic Drug Metabolite-Specific T-Cell Responses Using a Human Hepatocyte, Immune Cell Coculture System.

Drug-responsive T-cells are activated with the parent compound or metabolites, often via different pathways (pharmacological interaction and hapten). An obstacle to the investigation of drug hypersensitivity is the scarcity of reactive metabolites for functional studies and the absence of coculture systems to generate metabolites in situ. Thus, the aim of this study was to utilize dapsone metabolite-responsive T-cells from hypersensitive patients, alongside primary human hepatocytes to drive metabolite formation, and subsequent drug-specific T-cell responses. Nitroso dapsone-responsive T-cell clones were generated from hypersensitive patients and characterized in terms of cross-reactivity and pathways of T-cell activation. Primary human hepatocytes, antigen-presenting cells, and T-cell cocultures were established in various formats with the liver and immune cells separated to avoid cell contact. Cultures were exposed to dapsone, and metabolite formation and T-cell activation were measured by LC-MS and proliferation assessment, respectively. Nitroso dapsone-responsive CD4+ T-cell clones from hypersensitive patients were found to proliferate and secrete cytokines in a dose-dependent manner when exposed to the drug metabolite. Clones were activated with nitroso dapsone-pulsed antigen-presenting cells, while fixation of antigen-presenting cells or omission of antigen-presenting cells from the assay abrogated the nitroso dapsone-specific T-cell response. Importantly, clones displayed no cross-reactivity with the parent drug. Nitroso dapsone glutathione conjugates were detected in the supernatant of hepatocyte immune cell cocultures, indicating that hepatocyte-derived metabolites are formed and transferred to the immune cell compartment. Similarly, nitroso dapsone-responsive clones were stimulated to proliferate with dapsone, when hepatocytes were added to the coculture system. Collectively, our study demonstrates the use of hepatocyte immune cell coculture systems to detect in situ metabolite formation and metabolite-specific T-cell responses. Similar systems should be used in future diagnostic and predictive assays to detect metabolite-specific T-cell responses when synthetic metabolites are not available.