Related Experiment Videos
Mechanism for isaxonine hepatitis. I. Metabolic activation by mouse and human cytochrome P-450
Abstract:
The metabolism of isaxonine was first investigated in mice. Incubation, under air, of [2-14C] isaxonine (1 mM) with mouse liver microsomes and an NADPH-generating system resulted in the irreversible binding of a [14C] isaxonine metabolite to microsomal proteins; binding required active microsomes, NADPH and oxygen, it was inhibited by 4 mM piperonyl butoxide or by a CO-O2 (80:20) atmosphere. In the presence of various concentrations of isaxonine (0.125-2 mM), binding followed Michaelis-Menten kinetics; the Vmax was increased by both phenobarbital and 3-methylcholanthrene pretreatments. In vivo, 2.5 hr after the administration of [2-14C] isaxonine (4 mmol X kg-1 i.p.), a [14C] isaxonine material was irreversibly bound to mouse liver proteins; this binding was decreased by piperonyl butoxide and increased by phenobarbital or 3-methylcholanthrene pretreatments. Irreversible binding also occurred in the kidney. Unlike their effects in the liver, piperonyl butoxide and phenobarbital did not modify significantly in vitro metabolic activation by kidney microsomes and in vivo covalent binding to kidney proteins; pretreatment with 3-methylcholanthrene increased both in vitro and in vivo binding in the kidney. In a second series of experiments, in vitro metabolic activation was demonstrated with human liver microsomes; as in mice, covalent binding required NADPH and was markedly inhibited by piperonyl butoxide. We conclude that isaxonine is activated by mouse and human cytochromes P-450 into a reactive metabolite. In vivo covalent binding to mouse liver and kidney proteins appears to result mainly from the in situ binding of the metabolite formed in each organ.
Insights
Isaxonine is metabolized by cytochrome P-450 enzymes in mice and humans, forming reactive metabolites that bind irreversibly to liver and kidney proteins. This metabolic activation is crucial for understanding isaxonine
Area of Science:
- Biochemistry
- Pharmacology
- Toxicology
Background:
- Isaxonine metabolism and its potential for covalent binding to proteins were not well understood.
- Cytochrome P-450 enzymes are known to metabolize xenobiotics and can produce reactive intermediates.
- Understanding drug metabolism is critical for assessing safety and efficacy.
Purpose of the Study:
- To investigate the in vitro and in vivo metabolism of isaxonine in mice.
- To determine if isaxonine is activated into reactive metabolites that bind covalently to proteins.
- To compare the metabolic activation of isaxonine in mouse and human liver microsomes.
Main Methods:
- Incubation of [2-14C] isaxonine with mouse liver microsomes and NADPH-generating system.
- Assessment of covalent binding to microsomal proteins under various conditions (e.g., inhibitors, atmosphere).
- In vivo administration of [2-14C] isaxonine to mice and analysis of protein binding in liver and kidney.
Main Results:
- Isaxonine undergoes metabolic activation by NADPH-dependent enzymes, likely cytochromes P-450, leading to irreversible binding to liver and kidney proteins in mice.
- This binding is inhibited by piperonyl butoxide and CO, suggesting a role for cytochrome P-450.
- Phenobarbital and 3-methylcholanthrene pretreatments altered the Vmax and binding, indicating induction of P-450 enzymes.
- Similar in vitro metabolic activation and covalent binding were observed with human liver microsomes.
- Piperonyl butoxide and phenobarbital had different effects on kidney versus liver binding.
Conclusions:
- Isaxonine is activated by mouse and human cytochromes P-450 into a reactive metabolite.
- In vivo covalent binding to mouse liver and kidney proteins results from the in situ formation of this metabolite.
- The findings highlight the potential for isaxonine-induced protein adducts and warrant further toxicological evaluation.