Early redox homeostasis disruption contributes to the differential cytotoxicity of imiquimod on transformed and normal endothelial cells.

The off-label use of imiquimod (IQ) for hemangioma treatment has shown clinical benefits. We have previously reported a selective direct IQ-cytotoxic effect on transformed (H5V) vs. normal (1G11) endothelial cells (EC). In the present study, we investigated the mechanism underlying this selective cytotoxicity in terms of TLR7/8 receptor expression, NF-κB signalling and time-dependent modifications of oxidative stress parameters (ROS: reactive oxygen species, catalase and superoxide dismutase activities, GSH/GSSG and lipid peroxidation). TLR7/8 level was extremely low in both cell lines, and IQ did not upregulate TLR7/8 expression or activate NF-κB signalling. IQ significantly induced ROS in H5V after 2 h and strongly affected antioxidant defenses. After 12 h, enzyme activities were restored to baseline levels but a robust drop in GSH/GSSG persisted together with increased lipid peroxidation levels and a marked mitochondrial dysfunction. Although in normal IQ-treated EC some oxidative stress parameters were affected after 4 h, mitochondrial health and GSH/GSSG ratio remained notably unaffected after 12 h. Therefore, the early alterations (0-2 h) in transformed EC breached redox homeostasis as strongly as to enhance their susceptibility to IQ. This interesting facet of IQ as redox disruptor could broaden its therapeutic potential for other skin malignancies, alone or in adjuvant schemes.

Endogenous formaldehyde scavenges cellular glutathione resulting in redox disruption and cytotoxicity.

Formaldehyde (FA) is a ubiquitous endogenous and environmental metabolite that is thought to exert cytotoxicity through DNA and DNA-protein crosslinking, likely contributing to the onset of the human DNA repair condition Fanconi Anaemia. Mutations in the genes coding for FA detoxifying enzymes underlie a human inherited bone marrow failure syndrome (IBMFS), even in the presence of functional DNA repair, raising the question of whether FA causes relevant cellular damage beyond genotoxicity. Here, we report that FA triggers cellular redox imbalance in human cells and in Caenorhabditis elegans. Mechanistically, FA reacts with the redox-active thiol group of glutathione (GSH), altering the GSH:GSSG ratio and causing oxidative stress. FA cytotoxicity is prevented by the enzyme alcohol dehydrogenase 5 (ADH5/GSNOR), which metabolizes FA-GSH products, lastly yielding reduced GSH. Furthermore, we show that GSH synthesis protects human cells from FA, indicating an active role of GSH in preventing FA toxicity. These findings might be relevant for patients carrying mutations in FA-detoxification systems and could suggest therapeutic benefits from thiol-rich antioxidants like N-acetyl-L-cysteine.