Untargeted LC–MS–Based Metabolomics Reveals Metabolic Fate and Cellular Biochemical Disruptions Induced by 4,4‐Diaminodiphenylmethane in Human Skin Fibroblasts

Human exposure to 4,4'-diaminodiphenylmethane (MDA) occurs predominantly through dermal contact, yet its extra-hepatic biotransformation pathways and the metabolomic disturbances it induces remain poorly characterized. In this study, human skin fibroblasts were incubated with MDA, and untargeted LC-HRMS metabolomics was employed to elucidate the fate of MDA and its associated metabolomic signatures using an integrated data analysis workflow. Metabolite identification (MET ID) results revealed biotransformation products similar to those previously reported in liver microsomes, underscoring the dermal toxicity potential of MDA. Untargeted metabolomics identified 93 and 113 significantly altered features in cell lysates and suspension media, respectively. Multivariate statistical analysis demonstrated pronounced metabolic disruptions, with most features markedly downregulated in MDA-treated cells. Pathway enrichment analysis indicated perturbations in several key metabolic pathways, including strong enrichment of valine, leucine, and isoleucine biosynthesis in cell lysates. In contrast, arginine biosynthesis was predominantly enriched in the suspension media. Colony-formation assays and glutathione measurements were performed to assess cellular toxicity and to support metabolomics interpretations. Collectively, these integrated data analysis findings map the biotransformation profile of MDA exposure and highlight its capacity to induce coordinated metabolic dysfunctions in human skin fibroblasts.