Electrochemical Investigation of Intracellular NADH: PASHs Effect on Mitochondrial Function

Abstract

Nicotinamide adenine dinucleotide (NADH) is a crucial biomolecule that serves as an essential cofactor in numerous biocatalytic reactions and a pivotal regulator of mitochondrial function and cellular physiology. This study developed a SECM platform utilizing a double-mediator system for the real-time, in situ, and non-invasive detection of intracellular NADH at the single living cell level. The system employs 1-methoxy-5-methylphenazinium methyl sulfate (mPMS), which permeates the cell membrane to oxidize intracellular NADH, and ferricyanide ([Fe(CN)₆]³⁻), which shuttles electrons from the reduced mPMS to the SECM tip. This methodology was applied to investigate mitochondrial impairment in human hepatoma (HepG2) cells induced by polycyclic aromatic sulfur heterocycles (PASHs). The metabolic response was corroborated by analyzing poly(ADP-ribose) polymerase (PARP) activity, adenosine triphosphate (ATP) content, and mitochondrial membrane potential (MMP). Results demonstrated that exposure to dibenzothiophene (DBT) activated PARP, leading to severe depletion of NADH and ATP and a significant decline in MMP, thereby confirming comprehensive mitochondrial dysfunction. Comparative analysis with its non-sulfur analog fluorene (Flu) revealed that the sulfur atom in DBT's structure is a critical determinant of its enhanced cytotoxicity. This work establishes a reliable and innovative analytical approach for assessing the subcellular metabolic effects of environmental pollutants, providing a new platform for in-depth cytotoxicity mechanistic studies.