Metabolism of Diosbulbin B In Vitro and In Vivo in Rats: Formation of Reactive Metabolites and Human Enzymes Involved

Abstract

Diosbulbin B (DB), a major constituent of the furano-norditerpenes in Dioscorea bulbifera Linn, exhibits potential antineoplasmic activity and hepatotoxicity. The metabolism and reactive metabolites of DB in vitro (with human and animal liver microsomes) and in vivo in rats were investigated. The human enzymes involved in DB metabolism were identified. DB was first catalyzed into reactive metabolites of 2-butene-1,4-dial derivatives dependent on NADPH and then trapped by Tris base or oxidized to hemiacetal lactones (M12 and M13) in microsomal incubations. Tris base was used as buffer constituent and as a trapping agent for aldehyde. Methoxylamine and glutathione (GSH) were also used as trapping agents. DB metabolism in vivo in rats after oral administration was consistent with that in vitro. The structures of M12 and M13, as well as mono-GSH conjugates of DB (M31), were confirmed by nuclear magnetic resonance spectroscopy of the chemically synthesized products. The bioactivation enzymes of DB were identified as CYP3A4/5, 2C9, and 2C19. CYP3A4 was found to be the primary enzyme using human recombinant cytochrome P450 enzymes, specific inhibitory studies, and a relative activity factor approach for pooled human liver microsomes. Michaelis-Menten constants K m and V max were determined by the formation of M31. The reactive metabolites may be related to the hepatotoxicity of DB. The gender difference in CYP3A expression in mice and rats contributed to the gender-related liver injury and pharmacokinetics in mice and rats, respectively.

Detection of Emodin Derived Glutathione Adduct in Normal Rats Administered with Large Dosage of Polygoni Multiflori Radix

“…Further study is to synthesize sufficient amounts of the chemical entity of emodin-GSH adduct that enables a dynamic quantification analysis to reflect the status and severity of injury. Notably, the emodin-GSH adduct was not detected in the plasma and urine, a similar observation was made in another research (Yang et al, 2014), this could be explained by the fact that the conjugated emodin was mainly excreted via bile (Bachmann and Schlatter, 1981) or the undetectable concentration of emodin-GSH adduct in blood or urine. On the basis of the detection of emodin-GSH adduct, the derivatization and detection of emodin-GSH conjugates or emodin-protein adducts in blood or urine deserve further investigation for clinical detection.…”

Chemical Identity of Interaction of Protein with Reactive Metabolite of Diosbulbin B In Vitro and In Vivo

“…Toxicological studies consistently showed that pretreatment with ketoconazole (KTC, inhibitor of P450 3A) protected the animals from hepatotoxicities and hepatic GSH depletion induced by DIOB. The chemically reactive cis -enedial intermediate was found to react with the sulfhydryl group of glutathione (GSH) and the primary amine group of N -acetyl lysine (NAL) to produce a chemically stable pyrrole derivative [ 21 , 22 , 23 ]. We hypothesized that the reactive metabolite generated in situ reacts covalently with cysteine (Cys) thiol and lysine (Lys) primary amines of hepatic proteins to generate covalent binding.…”

Discovery of Hepatotoxic Equivalent Combinatorial Markers from Dioscorea bulbifera tuber by Fingerprint-Toxicity Relationship Modeling

“…In contrast, diterpenoid lactones in DBT are found to be hepatotoxic. Actually, the parent diterpenoid lactones do not appear to be hepatotoxic, while the metabolic activation of the furan ring by cytochromes P450 (CYP450) is the key procedure of acute liver injury 25 , 41 – 43 . Considering low CYP expression in cell lines 44 , the in vivo animal experiments were therefore conducted to evaluate the potential hepatotoxicity of the DBT extracts.…”