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

Abstract

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.

Functional and proliferative effects of repeated low‐dose oral administration of furan in rat liver

“…These changes were consistent with the small increase in cholesterol after 4-wk treatment and suggest that furan may alter hepatobiliary function. Although we cannot conclude at present whether these effects are due to impaired hepatobiliary transport, interference with bile acid biosynthesis or depletion of free amino acids due to reaction with cis-2-butene-1,4-dial, it is interesting to note that teucrin A, a hepatotoxic furancontaining diterpenoid found in the herb germander, was recently shown to form covalent protein adducts with bile acid CoA:amino acid N-acyltransferase [24], an enzyme responsible for catalyzing the conjugation of bile acids with both glycine and taurine. Considering that biotransformation of teucrin A gives rise to an 1,4-enedial derivative structurally similar to cis-1,4-butene-dial, it is tempting to speculate that bile acid CoA:amino acid N-acyltransferase may also be a target protein of furan reactive metabolites, leading to reduced ability of furan treated rat liver to conjugate and hence excrete bile acids.…”

Role of Reactive Metabolites in Drug-Induced Hepatotoxicity

“…Nevertheless, the structure of the metabolite-protein adduct has been determined in only a few cases (Baer et al 2007;Bambal and Hanzlik 1995;Damsten et al 2007;Sleno et al 2007;Yukinaga et al 2007;Zhang et al 2003), and identification of the modified amino acid residue in vivo remains a major analytical challenge (Koen et al 2006). Greater progress has been made in identification of the cellular proteins that are modified in vivo (Druckova et al 2007;Ikehata et al 2008;Koen et al 2007;Qiu et al 1998;Shipkova et al 2004). Potential targets within individual organelles can now be identified by using model electrophiles in cell fractions (Shin et al 2007;Wong and Liebler 2008).…”

Hepatotoxicity of Reactive Aldehydes☆

“…The compound responsible for hepatotoxicity was NCD. NCD is metabolized by CYP3A to form a reactive enedial presented in Figure 6 which is involved in the mechanism of toxicity (Druckova et al, 2007;Lekehal et al, 1996).…”

“…The toxicity of teucrin A has been attributed to the metabolic activation of the 3-substituted furan ring to an electrophilic metabolite (Druckova et al, 2007). Oral administration of germander extracts or teucrin A results in depletion of cellular GSH and damage to protein thiols which results in elevated ALT activity and significant centrilobular necrosis in mice (Druckova et al, 2007).…”

“…Oral administration of germander extracts or teucrin A results in depletion of cellular GSH and damage to protein thiols which results in elevated ALT activity and significant centrilobular necrosis in mice (Druckova et al, 2007). GSH depletion appears to be the major initial event that leads to toxicity, and toxicity was shown to be enhanced when depletion of GSH was aggravated due to the feeding of a diet deficient in sulfur-containing amino acids (Lekehal et al, 1996).…”