Pirfenidone is approved for the treatment of idiopathic pulmonary fibrosis. Idiosyncratic drug reactions, due to clinical application of pirfenidone, have been documented, even along with death cases resulting from acute liver failure. The present study aimed at the investigation of metabolic activation of pirfenidone possibly participating in the reported adverse reactions. Pirfenidone-derived glutathione/N-acetylcysteine (GSH/NAC) conjugates were detected in microsomal/primary hepatocyte incubations after exposure to pirfenidone. The GSH/NAC conjugates were also observed in bile and urine of rats given pirfenidone, respectively. The observation of the conjugates suggests the formation of a quinone methide intermediate derived from pirfenidone. The intermediate was possibly generated through two pathways. First, pirfenidone was directly metabolized to the quinone methide intermediate via dehydrogenation; second, pirfenidone was oxidized to 5-hydroxymethyl pirfenidone, followed by sulfation to a benzyl alcohol-sulfate derivative. The findings facilitate the understanding of the mechanisms of pirfenidone-induced idiosyncratic toxicity and assist medicinal chemists to minimize toxicities in the development of new pharmaceutical agents.

Furanoid 8-epidiosbulbin E acetate (EEA) is one of the most abundant diterpenoid lactones in herbal medicine Dioscorea bulbifera L. (DB). Our early work proved that EEA could be metabolized to EEA-derived cis-enedial (EDE), a reactive intermediate, which is required for the hepatotoxicity observed in experimental animals exposed to EEA. Also, we found that EDE could modify hepatic protein by reaction with thiol groups and/or primary amines of protein. The present study was inclined to develop polyclonal antibodies to detect protein modified by EDE. An immunogen was prepared by reaction of EDE with keyhole limpet hemocyanin (KLH), and polyclonal antibodies were raised in rabbits immunized with the immunogen. Antisera collected from the immunized rabbits demonstrated high titers evaluated by enzyme-linked immunosorbent assays (ELISAs). Immunoblot analysis showed that the polyclonal antibodies recognized EDEmodified bovine serum albumin (BSA) in a hapten load-dependent manner but did not cross-react with native BSA. Competitive inhibition experiments elicited high selectivity of the antibodies toward EDE-modified BSA. The antibodies allowed us to detect and enrich EDE-modified protein in liver homogenates obtained from EEA-treated mice. The developed immunoprecipitation technique, along with mass spectrometry, enabled us to succeed in identifying multiple hepatic proteins of animals given EEA. We have successfully developed polyclonal antibodies with the ability to recognize EDE-derived protein adducts, which is a unique tool for us to define the mechanisms of toxic action of EEA.

Dioscorea bulbifera L. (DBL), a traditional Chinese medicine, is a well-known herb with hepatotoxicity, and the biochemical mechanisms of the toxic action remain unknown. Diosbulbin B (DSB), a major component of DBL, can induce severer liver injury which requires cytochrome P450-catalyzed oxidation of the furan ring. It is reported that a cis-enedial reactive intermediate resulting from metabolic activation of DSB can react with thiols and amines to form pyrrole or pyrroline derivatives. In this study, we investigated the interaction of the reactive intermediate with polyamines, biogenic amines, and amino acids involved in the polyamine metabolic pathway, including putrescine, spermidine, spermine, histamine, arginine, ornithine, lysine, glutamine, and asparagine. Seven DSB-derived amine adducts were detected in microsomal incubations supplemented with DSB and individual amines. Six adducts were observed in cultured rat primary hepatocytes after exposure to DSB. DSB was found to induce apoptosis and cell death in time-and concentration-dependent manners. Apparently, the observed apoptosis was associated with the detected amine adduction. The findings facilitate the understanding of the mechanisms of toxic action of DSB.

Dioscorea Bulbifera L. (DBL), an effective traditional Chinese medicine, has been restricted because of multiple reports that it can cause severe hepatotoxicity. 8-Epidiosbulbin E acetate (EEA), one of the main components of DBL, can induce severe liver injury. It has been reported that EEA can be metabolized by CYP3A to the corresponding cis-enedial intermediate which alkylates the lysine residues of proteins to form pyrroline derivatives. The present study unexpectedly found that the reactive intermediate reacted with the amide groups of asparagine (Asn) and glutamine (Gln) residues of hepatic proteins of mice treated with EEA. The amide-derived protein modification increased with the increase in the dose administered. Like the adduction of the primary amine of lysine residues, the electrophilic metabolite reacted with the amide groups of Asn and Gln residues to offer the corresponding pyrrolines. The structures of the pyrrolines were confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy.

Diosbulbin B (DSB), a major component of herbal medicine Dioscorea bulbifera L. (DB), can be metabolized to an electrophilic intermediate, DSB-derived cis-enedial (DDE). DDE was suggested to contribute to the hepatotoxicity observed in experimental animals and humans after their exposure to DSB. Our previous work found that DDE reacted with primary amino and/or sulfhydryl groups of hepatic protein. The objective of the study was to develop polyclonal antibodies that can recognize DDE-derived protein adducts. Immunogens synthesized from DDE and keyhole limpet hemocyanin were employed to raise polyclonal antibodies in rabbits. An enzyme-linked immunosorbent assay (ELISA) demonstrated high titers of antisera obtained from immunized rabbits. Immunoblot analysis showed that DDE-modified bovine serum albumin (BSA) was recognized by the obtained polyclonal antibodies in a concentration-dependent manner and without cross-reaction to native BSA. Competitive ELISA and competitive immunoblot analyses defined the specificity of the antibodies to recognize BSA modified by DDE. Immunoblot analysis also detected a multitude of chemiluminescent bands with a variety of molecular weights in liver homogenates that were harvested from mice treated with DSB. In summary, we have successfully raised polyclonal antibodies to detect protein adducts derived from DDE.