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.
Lin Li,Xianwei Shen,Chaocheng Zhao,Qiyou Liu,Xuwei Liu,Yanan Wu
A potent bacterial strain capable of degrading dibenzothiophene (DBT) was isolated and evaluated for its characteristics. The strain, designated as LKY-5, is rod-shaped, gram-negative, and occurs mainly in clusters. It was identified as belonging to the Pseudomonas genus based on the 16S rDNA sequence and phylogenic analysis. Determination of its DBT depletion efficiency by gas chromatography revealed that the isolate was able to completely degrade up to 100 mg L −1 DBT within 144 h. The pH values, DBT concentrations, and biomasses in the medium varied significantly in the initial 24 h. A biosurfactant produced by LKY-5 was extracted and identified as a di-rhamnolipid with the formula Rha-Rha-C 8 -C 8:1 by HPLC-ESI-MS/MS. There were 26 metabolites in the DBT degradation process. Pseudomonas sp. LKY-5 exhibited unusually high DBT degradation efficiency via multiple metabolic pathways. Compared with the reported 4S and Kodama pathways, two more expanded metabolic pathways for the degradation of DBT are proposed. The polycyclic aromatic sulfur heterocycles (PASHs) in diesel , such as C 1 -DBT, C 2 -DBT, C 3 -DBT, 4,6-DMDBT, and 2,4,6-TMDBT, can also be degraded with 28.2–42.3% efficiency. The results showed that LKY-5 is an excellent bacterial candidate for the bioremediation of PASH-contaminated sites and sediments.
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