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Title: Competing Mechanistic Hypotheses of Acetaminophen-Induced Hepatotoxicity Challenged by Virtual Experiments}
Article:
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.
Introduction:
Acetaminophen( APAP)-induced liver injury( AILI) in mice is the widely used model for drug-induced liver injury in humans. There has been dramatic progress in identifying involvement of a variety of molecular level events and pathways[1–3]. To use that knowledge effectively in predicting injury and developing new treatment strategies, we need more credible explanations of how, when, and where key injury features emerge within hepatic lobules, and why humans exhibit a wide diversity of responses. A characteristic early feature is that necrosis, proceeded by covalent adduct formation, begins perivenous, adjacent to the central vein( CV) of hepatic lobules, and then progresses( radially) outward[4, 5]. Given that the fraction of APAP converted to the reactive metabolite( NAPQI: N-acetyl-p-benzoquinone imine) within hepatocytes increases from lobule’s periportal( PP) space to CV( called zonation), the prevailing mechanistic explanations assume that zonation of NAPQI formation is a necessary and sufficient prerequisite to explain early perivenous necrosis. That NAPQI zonation hypothesis provides an explanatory foundation for current efforts to better understand downstream complexities leading to tissue regeneration or liver failure. However, challenging that explanation directly in mice is currently impracticable because it requires exercising precise control over zonation of many mechanism features along with methods to measure intralobular features sequentially within the same mouse. Those barriers have impeded progress. Here, we successfully circumvent those impediments using multi-attribute software-based experiments. We challenge the NAPQI zonation explanation by experimenting on a strongly analogous software mechanism( as distinct from being described mathematically), called NZ-Mechanism, that is instantiated within a virtual mouse. This software analog is engineered to be quantitatively and qualitatively biomimetic across all anatomical, hepatic zonation, and cell biology features currently believed relevant to challenging the NAPQI zonation hypothesis. Results from virtual experiments provide strong quantitative evidence that zonation of NAPQI formation alone is insufficient to explain early perivenous necrosis. A parsimoniously more complex explanatory mechanism is needed, one involving additional feature zonation. After testing several, we focused on two equally plausible mechanism zonation features, but they too failed to meet stringent tests of sufficiency. However, when the two zonation features that had failed were combined into a unified mechanism, sufficiency was achieved, thus demonstrating the potential power of the virtual experiment approach. Physiologically reasonable perturbations of the merged mechanism caused significant changes in both the numbers of hepatocytes killed and the intralobular patterns of dead hepatocytes for a fixed toxic APAP dose of 300 mg/kg. Injury diversity was similar to that reported for humans[6, 7] and to differences in 24-hour necrosis scores among 37 genetically different mouse strains[7]. For the latter, we hypothesized that variation of lobule location-dependent features within the combined mechanism, resulting from changing selected mechanism configurations, can be sufficient to account for such necrosis score diversity. We present results of virtual experiments supporting that operating hypothesis with the intent that these results will help formulate new wet-lab experiments to further improve explanatory mechanism insight( Fig 1).
Discussion:
Our intent is that parallel virtual and wet-lab experimentation will integrate into a faster and more effective scientific approach[17]( Fig 1). Most researchers seem to prefer including ample realistic detail in explanatory models. However, mechanisms like those employed in Mouse Analogs are scientifically preferable and more informative because they are only as detailed as necessary for explaining a particular phenomenon or challenging a particular hypothesis. A validated model Mechanism with limited detail subsumes many different yet equally plausible more detailed variants. When we falsify such a Mechanism( show that it is inadequate for its Target Attributes), we significantly shrink the space of equally plausible variants by eliminating whole classes of more detailed variants. The results in Fig 5 document the first use of virtual experiments to challenge competing biomimetic Mechanism hypotheses. The fact that three were falsified demonstrates how virtual experiments can shrink the space of plausible explanatory Mechanisms and facilitate the more fundamental work of wet-lab experimentation. When we started, we expected Mouse Analogs using NZ-Mechanism to show Hepatocyte Deaths occurring first near CV. We acquired new knowledge by examining why it, as well as MNZ- and GNZ-Mechanisms, failed to mimic that phenomenon. Explanatory insight was further improved by examining how MGNZ-Mechanism successfully achieved the Target Phenomenon. An example is the importance of zonated repair pathways in mitigating damage( Fig 6). We anticipate that other comparably parsimonious yet somewhat different Mouse Analog Mechanisms can do the same. The Iterative Refinement Protocol( described under Materials and Methods) outlines how to identify and challenge comparably parsimonious explanations of target phenomena. Mouse Analog concreteness, coupled with the ability to observe and measure propagating causal events, makes it easy for experts and non-experts to form and discuss opinions about the credibility of similarities between virtual experiment results and those from real or envisioned wet-lab counterparts. NZ-Mechanism was falsified because a small fraction of Hepatocytes in Zones 1 and 2 experienced an early Death trigger event( Fig 5) and consequently will experience an early Death event. The same phenomenon may occur in mice, but it has escaped detection because experiments were focused elsewhere. If deemed important, that phenomenon—that prediction—could be challenged directly using wet-lab experiments. During execution, MGNZ-Mechanism is a concrete hypothesis for how key features of APAP hepatotoxicity in mice are generated( Fig 3A). Suppose that strong analogies do exist between virtual and real mice. It is clear from concurrent differences within the three regions( Fig 6C–6H) that explanatory inferences drawn from whole liver measurements or hepatic biopsies will at best be flawed and likely misleading. Differences in zonal predictions can guide design of wet-lab experiments intended to challenge and possibly falsify the MGNZ-Mechanism hypothesis. For example, 1) at 30 min post-dose, GSH Depletion in the Midzonal region is significantly greater than in either CV or PP regions; and 2) at 10 min post-dose, counts of mitoD objects( maps to mitochondrial damage) in CV Hepatocytes are an order of magnitude greater than in PP Hepatocytes. Such experiments will be win-win: no matter the outcome, we will have useful new knowledge, and the new results are expected to motivate additional rounds of virtual experiments[17]( Fig 1). Note that, absent the data in Fig 6 there would be no basis to undertake such narrowly focused experiments. Within individuals, expressions of hepatic proteins are known to change significantly in response to a variety of environmental and health factors. We simulated possible examples( Fig 7A). Genetic differences among mouse strains that influence hepatic zonation more than they influence particular pathways or molecular level events may account for the diversity of differences in necrosis scores( Fig 7B). However, considerable uncertainty remains. As illustrated by the green bars in Fig 7A, for each Mouse strain explanation, there will exist a sizable set of Mechanism variants that would be equally explanatory. Further, MGNZ-Mechanism is just one realization drawn from a plausible yet finite Mechanism space. Automated methods, as they become available, can be used to more systematically explore that space, prior to or in parallel with new wet-lab experiments to challenge MGNZ-Mechanism. The data in Fig 7 demonstrate that, when targeting just one attribute, such as total Hepatocyte Death 24 hours post-dose, modest configuration changes for two or more Mouse Analog features can have counterbalancing consequences resulting in altered event details yet essentially no significant change in the measured feature. On the other hand, modest changes can have synergistic consequences resulting in a 3-fold change or more. Toxicity enhancing synergistic changes in cascade zonation, possibly initiated by changes in environmental and health factors, may be a contributor to idiosyncratic drug induced liver injury. In combination with the adaptation hypothesis[18], such change may help explain how and why only a small percentage of susceptible individuals develop overt liver injury.
Acetaminophen-induced liver injury in mice is a model for drug-induced liver injury in humans . Challenging an explanatory mechanism in mice is problematic because variables determining causes and effects cannot be controlled adequately . We circumvent that impediment by performing virtual experiments that challenge the prevailing scientific explanation for the characteristic spatiotemporal pattern of early acetaminophen-induced hepatic necrosis . Virtual mice utilize a biomimetic software liver . Results of virtual experiments provide compelling evidence that the prevailing explanation is insufficient . Without further studies in mice , we discovered a new , marginally more complex explanatory mechanism that met stringent tests of sufficiency . We argue that this virtual causal mechanism and the actual mechanism in mice are strongly analogous within and across multiple biological levels . Variants of the virtual mechanism provide possible explanations for the considerable variation in 24-hour necrosis scores among 37 genetically diverse mouse strains following a single toxic acetaminophen dose .