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Updated: Jan 12, 2026
Human Liver Microphysiological System for Assessing Drug-Induced Liver Toxicity In Vitro
Published on: January 31, 2022
In Vitro Metabolite Profiling of 4,4-Diaminodiphenylmethane Using Human Liver S9 Fractions and LC-Orbitrap HRMS: In
Sridhar Chinthakindi1,2, Radhika Kawathe1,2, Leela Sairam Andhela1,2
1Manipal Academy of Higher Education, Manipal, Karnataka, India.
Rationale:
4,4-Diaminodiphenylmethane (MDA) is an aromatic amine classified as a Group 2B carcinogen. MDA is widely used as a key ingredient in industrial and consumer products such as epoxy resins, polyurethanes, and dyes. Given its widespread use and potential for occupational and environmental exposure, understanding its hepatic metabolism is crucial in elucidating the toxicological profiles of MDA in humans. However, a limited number of metabolites have been reported in the literature. To address this gap, we conducted a comprehensive metabolite profiling study to identify and characterize all possible in vitro metabolites of MDA.
Methodology:
In vitro hepatic metabolism studies were conducted using pooled human liver S9 fractions to stimulate the metabolism of MDA in humans. An untargeted metabolite profiling approach was employed using an LC-Orbitrap-HRMS. Data processing and identification of metabolites were performed using the Compound Discoverer software. A targeted LC-MS/MS method was employed to find the relative abundances of metabolites at various incubation times. In silico toxicity prediction of MDA-derived metabolites was conducted using the ProTox 3.0 online tool.
Results:
A total of twelve MDA-derived metabolites were successfully identified, reflecting both Phase I (hydroxylation, desaturation, oxidative deamination) and Phase II (acetylation, sulfation, glucoside conjugation) biotransformation pathways. Among these, N-acetylated and desaturation intermediate-related metabolites were the most abundant. Based on these findings, an in silico toxicological report for the metabolites was proposed.
Conclusions:
The Orbitrap LC-HRMS platform enabled comprehensive identification and characterization of in vitro hepatic metabolic profiles of MDA. The predicted toxicological profiles and the discovery of additional metabolite formation beyond the known acetylation pathway provided new insights that can enhance toxicological risk assessment of MDA.
