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"Competing Mechanistic Hypotheses of Acetaminophen-Induced Hepatotoxicity Challenged by Virtual Experiments" Andrew K. Smith, Brenden K. Petersen, Glen E. P. Ropella, Ryan C. Kennedy, Neil Kaplowitz, Murad Ookhtens, C. Anthony Hunt. PLoS Comput Biol. 2016 Dec 16;12(12):e1005253 (2016)  View
Abstract

Acetaminophen-induced liver injury in mice is a model for drug-induced liver injury in humans. A precondition for improved strategies to disrupt and/or reverse the damage is a credible explanatory mechanism for how toxicity phenomena emerge and converge to cause hepatic necrosis. The Target Phenomenon in mice is that necrosis begins adjacent to the lobule's central vein (CV) and progresses outward. An explanatory mechanism remains elusive. Evidence supports that location dependent differences in NAPQI (the reactive metabolite) formation within hepatic lobules (NAPQI zonation) are necessary and sufficient prerequisites to account for that phenomenon. We call that the NZ-mechanism hypothesis. Challenging that hypothesis in mice is infeasible because 1) influential variables cannot be controlled, and 2) it would require sequential intracellular measurements at different lobular locations within the same mouse. Virtual hepatocytes use independently configured periportal-to-CV gradients to exhibit lobule-location dependent behaviors. Employing NZ-mechanism achieved quantitative validation targets for acetaminophen clearance and metabolism but failed to achieve the Target Phenomenon. We posited that, in order to do so, at least one additional feature must exhibit zonation by decreasing in the CV direction. We instantiated and explored two alternatives: 1) a glutathione depletion threshold diminishes in the CV direction; and 2) ability to repair mitochondrial damage diminishes in the CV direction. Inclusion of one or the other feature into NZ-mechanism failed to achieve the Target Phenomenon. However, inclusion of both features enabled successfully achieving the Target Phenomenon. The merged mechanism provides a multilevel, multiscale causal explanation of key temporal features of acetaminophen hepatotoxicity in mice. We discovered that variants of the merged mechanism provide plausible quantitative explanations for the considerable variation in 24-hour necrosis scores among 37 genetically diverse mouse strains following a single toxic acetaminophen dose.

Related Publications
"A Model Mechanism-Based Explanation of an In Vitro-In Vivo Disconnect for Improving Extrapolation and Translation" Andrew K. Smith, Yanli Xu, Glen E. P. Ropella C. Anthony Hunt. J Pharmacol Exp Ther. 2018 Apr;365(1):127-138 (2018)  View
Abstract

An improved understanding of in vivo-to-in vitro hepatocyte changes is crucial to interpreting in vitro data correctly and further improving hepatocyte-based in vitro-to-in vivo extrapolations to human targets. We demonstrate using virtual experiments as a means of helping to untangle plausible causes of inaccurate extrapolations. We start with virtual mice that use biomimetic software livers. Previously, using these mice, we discovered model mechanisms that enabled achieving quantitative validation targets while also providing plausible causal explanations for temporal characteristics of acetaminophen hepatotoxicity. We isolated virtual hepatocytes, created a virtual culture, and then conducted dose-response experiments in both culture and mice. We expected to see differences between the two dose-response curves but were somewhat surprised that they crossed because it evidenced that simulated acetaminophen metabolism and toxicity are different for virtual culture and mouse contexts even though individual hepatocyte mechanisms were unchanged. Differences in dose-response curves provide a virtual example of an in vivo-to-in vitro disconnect. We use detailed results of experiments to explain this disconnect. Individual hepatocytes contribute differently to system-level phenomena. In liver, hepatocytes are exposed to acetaminophen sequentially. Relative production of the reactive acetaminophen metabolite is largest (smallest) in pericentral (periportal) hepatocytes. Because that sequential exposure is absent in culture, hepatocytes from different lobular locations do not respond the same. A virtual culture-to-mouse translation can stand as a scientifically challengeable hypothesis explaining an in vivo-to-in vitro disconnect. It provides a framework to develop more reliable interpretations of in vitro observations, which then may be used to improve extrapolations.

Mechanistic Agent-based Damage and Repair Models as Hypotheses for Patterns of Necrosis Caused by Drug Induced Liver Injury Andrew K Smith, Glen E.P. Ropella, Neil Kaplowitz, Murad Ookhtens, and C. Anthony Hunt. (2014)  View
Abstract

Increasing model reuse and facilitating repurposing is expected to expand simulation use for better understanding biological phenomena. We demonstrate doing so in the context of liver diseases caused by toxic exposure to xenobiotics. A clinical goal is improved mechanistic explanations of how damage is generated, which can lead to new strategies to block and/or reverse injury. A goal for this work is to provide concrete, plausible explanations for acetaminophen induced liver injury (AILI) in mice. We instantiate mechanistic hypotheses that map to cellular damage and repair pathways and begin identifying plausible simulated causal cascades capable of generating the characteristic AILI spatial and temporal patterns. We use discrete event simulation of agent-based, multiscale, biomimetic models and Monte Carlo sampling. We use an Iterative Refinement protocol for implementing and validating/falsifying mechanistic hypotheses on a previously validated In Silico Liver. We simulated an observed necrosis pattern. Further approach improvement will yield new methods that combine iterations of in-silico and wet-lab experiments.

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