In vitro neurotoxicity comparison: Colistimethate induces higher toxicity than colistin via formaldehyde accumulation

Colistin as a last-line of defence for treating multidrug-resistant Gram-negative bacterial pathogens is most commonly administered as its inactive prodrug, colistimethate (CMS), for its reduced nephorotoxity. This study addresses the critical gap in understanding neurotoxicity differences between colistin and CMS. Specifically, we aimed to compare the cytotoxicity of CMS and colistin in human neural SH-SY5Y cells and identify the key mediators of CMS-induced neurotoxicity and their underlying mechanisms.

We used a multi-experimental approach to link phenotypic toxicity to molecular mechanisms: firstly, measured cell viability of SH-SY5Y cells with MTT when treated with 0.3 mM CMS against equimolar colistin (CMS concentration = 6× average human in vivo steady-state levels). Subsequently, quantified colistin release from CMS hydrolysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS) and measured formaldehyde production using a commercial kit, and finally Employed RNA sequencing (RNA-seq) to analyze global gene expression changes in CMS- vs. colistin-treated cells.

CMS induced significantly higher cytotoxicity in SH-SY5Y cells than colistin at the same concentration. Formaldehyde was confirmed as a colistin-independent driver of CMS neurotoxicity. RNA-seq revealed CMS disrupted DNA damage repair and cell cycle regulation pathways-findings that aligned with prior formaldehyde toxicity studies. LC-MS/MS confirmed sustained colistin release from CMS, while RNA-seq validated pathway specificity.

CMS is more neurotoxic to human neural cells than colistin, raising concerns about its potential to impair central nervous system (CNS) function. Given this, extreme caution is warranted when administering high-dose CMS directly to the CNS via intrathecal or intraventricular routes.