The importance of membrane-based compartmentalization in eukaryotic cell function has become broadly appreciated, and a number of studies indicate that these eukaryotic cell membranes contain coexisting liquid-ordered (L(o)) and liquid-disordered (L(d)) lipid domains. However, the current evidence for such phase separation is indirect, and so far there has been no direct demonstration of differences in the ordering and dynamics for the lipids in these two types of regions or their relative amounts in the plasma membranes of live cells. In this study, we provide direct evidence for the presence of two different types of lipid populations in the plasma membranes of live cells from four different cell lines by electron spin resonance. Analysis of the electron spin resonance spectra recorded over a range of temperatures, from 5 to 37 degrees C, shows that the spin-labeled phospholipids incorporated experience two types of environments, L(o) and L(d), with distinct order parameters and rotational diffusion coefficients but with some differences among the four cell lines. These results suggest that coexistence of lipid domains that differ significantly in their dynamic order in the plasma membrane is a general phenomenon. The L(o) region is found to be a major component in contrast to a model in which small liquid-ordered lipid rafts exist in a 'sea' of disordered lipids. The results on ordering and dynamics for the live cells are also compared with those from model membranes exhibiting coexisting L(o) and L(d) phases.

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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.