Identification of the Hepatic Protein Targets of Reactive Metabolites of Acetaminophen in Vivoin Mice Using Two-dimensional Gel Electrophoresis and Mass Spectrometry

189

270

Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress. Diffusible electrophilic R, -unsaturated aldehydes, such as 4-hydroxynonenal (HNE), have been shown to modify proteins that mediate cell signaling (e.g., IKK and Keap1) and alter gene expression pathways responsible for inducing antioxidant genes, heat shock proteins, and the DNA damage response. To fully understand cellular responses to HNE, it is important to determine its protein targets in an unbiased fashion. This requires a strategy for detecting and isolating HNE-modified proteins regardless of the nature of the chemical linkage between HNE and its targets. Azido or alkynyl derivatives of HNE were synthesized and demonstrated to be equivalent to HNE in their ability to induce heme oxygenase induction and induce apoptosis in colon cancer (RKO) cells. Cells exposed to the tagged HNE derivatives were lysed and exposed to reagents to effect Staudinger ligation or copper-catalyzed Huisgen 1,3 dipolar cycloaddition reaction (click chemistry) to conjugate HNE-adducted proteins with biotin for subsequent affinity purification. Both strategies yielded efficient biotinylation of tagged HNE-protein conjugates, but click chemistry was found to be superior for the recovery of biotinylated proteins from streptavidincoated beads. Biotinylated proteins were detected in lysates from RKO cell incubations with azido-HNE at concentrations as low as 1 µM. These proteins were affinity purified with streptavidin beads, and proteomic analysis was performed by linear ion trap mass spectrometry. Proteomic analysis revealed a dose-dependent increase in labeled proteins with increased sequence coverage at higher concentrations. Several proteins involved in stress signaling (heat shock proteins 70 and 90 and the 78-kDa glucoseregulated protein) were selectively adducted by azido-and alkynyl-HNE. The use of azido and alkynyl derivatives in conjunction with click chemistry appears to be a valuable approach for the identification of the protein targets of HNE.

mentioning

confidence: 70%

“…Both halothane and bromobenzene were shown to adduct GRP-78 in rat liver (49,51). Glutathione-S-transferase-Pi was adducted by acetaminophen and BHT in mouse liver (52,53). Hsp-90R was adducted by BHT in mouse lung (53), and peroxiredoxin-1 was adducted by acetaminophen in mouse liver (52).…”

mentioning

confidence: 99%

Identification of Protein Targets of 4-Hydroxynonenal Using Click Chemistry for ex Vivo Biotinylation of Azido and Alkynyl Derivatives

Vila

Tallman

Jacobs

et al. 2008

189

270

“…Some 30 proteins modified by the reactive metabolite of acetaminophen have been identified (53); however, it is very difficult to determine which binding is responsible for toxicity. The covalent binding of the meta isomer of acetaminophen, 3-hydroxyacetanilide, is comparable to that of acetaminophen; yet, it is not significantly hepatotoxic (it is possible that the meta isomer would cause IDRs, but its potential in this regard is unknown because it has not been used in humans).…”

Section: Other Advancesmentioning

confidence: 99%

Idiosyncratic Drug Reactions: Past, Present, and Future

Uetrecht

2007

238

190

Although the major working hypothesis for the mechanism of idiosyncratic drug reactions (IDRs), the hapten hypothesis, has not changed since 1987, several hypotheses have been added, for example, the danger hypothesis and the pharmaceutical interaction hypothesis. Genetic studies have found that several IDRs are linked to specific HLA genes, providing additional evidence that they are immune-mediated. Evidence that most IDRs are caused by reactive metabolites has led pharmaceutical companies to avoid drug candidates that form significant amounts of reactive metabolites; however, at least one IDR, ximelagatran-induced liver toxicity, does not appear to be caused by a reactive metabolite. It is possible that there are biomarkers such as those related to cell stress that would predict that a drug candidate would cause a significant incidence of IDRs; however, there has been no systematic study of the changes in gene expression induced by drugs known to cause IDRs. A major impediment to the study of the mechanisms of IDRs is the paucity of valid animal models, and if we had a better mechanistic understanding, it should be easier to develop such models. There is growing evidence that these adverse reactions are more varied and complex than previously recognized, and it is unlikely that a quick fix will be achieved. However, IDRs are an important cause of patient morbidity and mortality and markedly increase the uncertainty of drug development; therefore, continued basic research in this area is essential.

“…The loss of membrane potential can cause the release of the contents into the cytosol, leading to necrosis. Analogous mechanisms operate in the toxicity of APAP, whose reactive electrophilic metabolite N-acetyl-para-benzoquinone imine (NAP-QI) adducts preferentially mitochondrial proteins involved in respiration (107). Among other NAPQI mitochondrial targets found in this study were aldehyde dehydrogenase and glutamate dehydrogenase.…”

Section: Discussionmentioning

confidence: 92%

Identification of the Protein Targets of the Reactive Metabolite of Teucrin A in Vivo in the Rat

Druckova

Mernaugh

Ham

et al. 2007

Covalent modification of proteins is associated with the toxicity of many electrophiles, and the identification of relevant in vivo protein targets is a desirable but challenging goal. Here, we describe a strategy for the enrichment of adducted proteins utilizing single-chain fragment variable (ScFv) antibodies selected using phage-display technology. Teucrin A is a furan-containing diterpenoid found in the herb germander that is primarily responsible for the herb's hepatotoxicity in rodents and humans following metabolic activation by cytochrome P450 enzymes. Conjugates of the 1,4-enedial derivative of teucrin A, its presumed toxic metabolite, with lysine-and cysteine-containing peptides were synthesized and used to select ScFvs from a rodent phage-displayed library, which recognized the terpenoid moiety of the teucrin-derived adducts. Immunoaffinity isolation of adducted proteins from rat liver homogenates following administration of a toxic dose of teucrin A afforded a family of proteins that were identified by liquid chromatography/tandem mass spectrometry. Of the 46 proteins identified in this study, most were of mitochondrial and endoplasmic reticulum origin. Several cytosolic proteins were found, as well as four peroxisomal and two secreted proteins. Using Ingenuity Pathway Analysis software, two significant networks involving the target genes were identified that had major functions in gene expression, small molecule biochemistry, and cellular function and maintenance. These included proteins involved in lipid, amino acid, and drug metabolism. This study illustrates the utility of chemically synthesized biological conjugates of reactive intermediates and the potential of the phage display technology for the generation of affinity reagents for the isolation of adducted proteins.