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More than 20 years have passed since the early reports of acute hepatotoxicity with APAP overdose. During that period investigative research to discover the "mechanism" underlying the toxicity has been conducted in many species and strains of intact animals as well as in a variety of in vitro and culture systems. Such work has clarified the primary role of biotransformation and the protective role of GSH. Understanding the former provides explanations for the toxic interactions which may occur with alcohol or other xenobiotics, while understanding of the latter led to the development of antidotes for the treatment of acute poisoning. Acetaminophen (APAP)-induced hepatotoxicity: roles for protein arylation. Initiating events in toxicity require biotransformation of APAP to NAPQI followed by arylation of several important proteins with subsequent alteration of protein structure and function. The immediate consequence of the alterations is detectable in several organelles and these may represent multiple initiating events which are depicted as acting in concert to cause cell injury (large arrowheads). Arylation of cytosolic 58-ABP with subsequent translocation to the nucleus is depicted as a possible signaling mechanism for determining outcome at the cell or organ level (within dotted boundary). For simplicity NAPQI's potentials for oxidizing protein sulfhydryls and direct binding to DNA have been omitted. Significant light has also been shed on the biochemical and cellular events which accompany APAP-induced hepatotoxicity. However, such studies have not identified a unique mechanism of toxicity that is universally accepted. The recent identification of several protein targets which become arylated during toxicity--along with the findings that arylation of some of those target proteins results in loss of protein function--demonstrates that covalent binding does, indeed, have biological consequences and is not merely an indicator of the fleeting presence of reactive electrophiles. These observations further suggest that multiple independent insults to the cell may be involved in toxicity. it is now apparent that the concept of a multistage process that involves both initiation and progression events is appropriate for APAP toxicity, and it is unlikely that a unique initiating event will ever be identified. In light of recent findings it is more likely that a number of such cellular events occur very early after toxic overdosage, and that they collectively set in motion and perpetuate the biochemical, cellular, and molecular processes which will determine outcome. The importance of 58-ABP arylation with early, apparently selective, translocation to the nucleus remains to be elucidated. To date there is nothing to suggest that this represents an initiating event in toxicity. rather it is plausible that the translocation may play a role in signaling electrophile presence and in calling for cellular defense against electrophile insult. This is reflected in the hypothetical model presented in Fig. 3. Critical experimenta...

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Two-dimensional (2-D) gel electrophoresis is often used in toxicologic and metabolic studies to assess treatment- or stage-specific changes in protein synthesis, degradation or posttranslational modification. When combined with cell fractionation studies the detectability of low abundance proteins is enhanced, and changes in subcellular distribution of proteins can also be monitored. Detergent fractionation is a simpler alternative to differential pelleting, which partitions cellular constituents into functionally distinct populations while preserving cytoskeletal integrity. We defined and characterized a differential detergent fractionation (DDF) protocol to enable protein dynamics in cytoskeletal and noncytoskeletal compartments of isolated hepatocytes to be monitored simultaneously. Rat hepatocytes were maintained in suspension culture and fractionated by sequential extraction with detergent-containing buffers (digitonin/EDTA, Triton/EDTA, Tween/deoxycholate). DDF reproducibly yielded four electrophoretically distinct fractions enriched in cytosolic, membrane-organelle, nuclear membrane and cytoskeletal-matrix markers, respectively. Immunoblotting with over 20 different antibodies corroborated the selectivity of fractionation and was used to characterize the distribution profiles of cytoskeletal (actin, tubulins, cytokeratins, vinculin, myosin, desmoplakins, fodrin, nuclear lamins) and noncytoskeletal proteins (heat-shock 70 proteins, glutathione-S-transferase, calpains, carbamoyl phosphate synthetase, etc.), as well as to identify spots in 2-D gels. Detergent buffers were compatible with equilibrium or nonequilibrium 2-D gel electrophoretic analysis. Extensive 2-D maps of acidic and basic proteins in each fraction were generated along with a tabular listing of M(r) and pI. Thus, DDF reproducibly partitions hepatocytic proteins into functionally distinct cytoskeletal and noncytoskeletal compartments that are readily analyzed by 2-D gel electrophoresis. DDF is simple, applicable to use with other cell types or culture systems and is especially useful when biomaterial is limited (i.e., clinical studies).