Proteomic study of hepatic nuclear extracts in an adaptive acetaminophen tolerance model

Abstract

Introduction: Variability in response to acetaminophen (APAP)-induced aseptic inflammation and tolerance to the impending hepatic damage has been described. To understand the mechanism of adaptive tolerance, we investigated the proteomic profiles of crude nuclear lysates in a mouse model. We hypothesized that pretreatment with low doses of APAP prior to a toxic dose results in differential protein expression. Materials and Methods: Mice (BALB/C) were separated into three groups: the pretreated (PT) group received incremental doses of APAP while the last dose only (LD) and naïve groups were given saline vehicle. A toxic dose of APAP was administered on the seventh day to the PT and LD animals only and all groups were euthanized 3 h postdose. Total protein from crude hepatic nuclear lysates were applied to protein arrays and analyzed by immunoaffinity mass spectrometry. Results and Discussion: Comparative data analyses of protein peaks revealed a protein that was significantly increased at m/z of 60,030 (p60) in the LD animals vs the other two groups. The closest match for the preliminary identification of the p60 protein based on a Swiss-Prot/TagIdent database search using the approximate isoelectric point and molecular weight information was Ccr4-Not complex subunit-2. This protein is a subunit of a multiprotein complex and serves as a transcriptional suppressor involved in controlling mRNA synthesis and degradation. Preliminary identification was also supported by Western blot analysis using anti-CNOT2 antibody. Conclusion: Considering the APAP tolerance model, we conclude that toxicogenomic approaches such as nuclear profiling are useful tools in assessing differential expression of transcriptional factors involved in inflammatory response and adaptive tolerance to toxins. © 2009 Humana Press.