Integrated metabolomic insights and biochemical profiling of indole-3-butyric acid hydrazones in mitigating heavy metal-induced oxidative and metabolic dysregulation through Nrf2 pathway activation

Indole-3-butyric acid (IBA), a synthetic auxin analogue, has been extensively studied in plant systems; however, its therapeutic role in mammalian models remains unexplored. In this study, two novel IBA-based hydrazone derivatives: 4-indole-3-butane-(thiophen-2-ylmethylene)hydrazide hydrazone (TIBH- featuring a thiophene moiety) and 1-(4-amino-3,5-dichlorophenyl)ethylidene-4-indole-3-butane hydrazide hydrazone (DIBH- bearing a dichlorophenyl group), were evaluated for their protective efficacy against cadmium (Cd)-induced metabolic and oxidative toxicity using Swiss albino mice. Cd, a known environmental pollutant, induces multi-organ dysfunction primarily through redox imbalance, mitochondrial disruption, and metabolic dysregulation. Ascorbic acid (AA) served as a reference antioxidant. Cd exposure led to significant elevations in blood glucose, HbA1c, and pro-inflammatory cytokines, along with altered liver and kidney function biomarkers. Molecular analyses revealed suppression of key antioxidant genes, including superoxide dismutase 2 (SOD2), catalase (CAT), heme oxygenase-1 (HO1), and nuclear factor erythroid 2–related factor 2 (NRF2) and depletion of glutathione (GSH), consistent with histological evidence of necrosis, vascular congestion, and cellular hypertrophy in liver, pancreas, and brain tissues. Comprehensive metabolomic profiling using liquid chromatography- tandem mass spectrometry (LC-MS/MS) revealed that Cd exposure significantly altered lipid and amino acid metabolism. Elevated levels of lipid peroxidation products such as myristic acid, N-oleoyl serine, and phosphatidylserine (PS) (m/z 790.21) indicated oxidative membrane damage. Essential phospholipids, including lyso-phosphatidylserine (LysoPS) (m/z 718.03), were downregulated, suggesting compromised membrane integrity. Additionally, key amino acids, serine and methionine, central to redox and one-carbon metabolism, were markedly reduced. These metabolic perturbations aligned with biochemical markers of oxidative stress, supporting a strong correlation between LC-MS/MS findings and systemic toxicity. TIBH treatment markedly restored metabolic homeostasis. It significantly reduced peroxidation-associated lipids, partially recovered serine and methionine levels, and reversed the biochemical and molecular alterations induced by Cd. The detection of a unique TIBH-derived metabolite (m/z 327.30) confirmed hepatic biotransformation and systemic bioactivity, supporting its role as an active antioxidant agent. Conversely, DIBH treatment showed moderate efficacy, with a chlorinated derivative (m/z 419.10) identified in serum, suggesting different pharmacodynamics. AA provided partial protection but was less effective than TIBH in restoring metabolic and gene expression profiles. Mechanistically, TIBH reactivated the Nrf2/Keap1-antioxidant response element (ARE) signaling axis, restoring antioxidant gene expression, redox balance, and mitochondrial function. This integrative biochemical, histological, molecular, and metabolomic analysis positions TIBH as a potent therapeutic candidate for mitigating heavy metal-induced metabolic disruption.