¹H NMR spectroscopic characterisation of HepG2 cells as a model metabolic system for toxicology studies

The immortalised human hepatocellular HepG2 cell line is commonly used for toxicology studies as an alternative to animal testing due to its characteristic liver-distinctive functions. However, little is known about the baseline metabolic changes within these cells upon toxin exposure. We have applied ¹H Nuclear Magnetic Resonance (NMR) spectroscopy to characterise the biochemical composition of HepG2 cells at baseline and post-exposure to hydrogen peroxide (H₂O₂). Metabolic profiles of live cells, cell extracts, and their spent media supernatants were obtained using ¹H high-resolution magic angle spinning (HR-MAS) NMR and ¹H NMR spectroscopic techniques. Orthogonal partial least squares discriminant analysis (O-PLS-DA) was used to characterise the metabolites that differed between the baseline and H₂O₂ treated groups. The results showed that H₂O₂ caused alterations to 10 metabolites, including acetate, glutamate, lipids, phosphocholine, and creatine in the live cells; 25 metabolites, including acetate, alanine, adenosine diphosphate (ADP), aspartate, citrate, creatine, glucose, glutamine, glutathione, and lactate in the cell extracts, and 22 metabolites, including acetate, alanine, formate, glucose, pyruvate, phenylalanine, threonine, tryptophan, tyrosine, and valine in the cell supernatants. At least 10 biochemical pathways associated with these metabolites were disrupted upon toxin exposure, including those involved in energy, lipid, and amino acid metabolism. Our findings illustrate the ability of NMR-based metabolic profiling of immortalised human cells to detect metabolic effects on central metabolism due to toxin exposure. The established data sets will enable more subtle biochemical changes in the HepG2 model cell system to be identified in future toxicity testing.