Abstract
Ferroptosis is an iron-dependent regulated form of regulated cell death that is caused by excessive lipid peroxidation-mediated cell membrane damage. The present study compared the cytotoxic effects of nicotine- and tar-free cigarette smoke extract (CSE) prepared from heated tobacco products (HTPs) and a ferroptosis inducer erastin on vascular smooth muscle cells. Cigarette smoke of HTPs (Ploom X, IQOS 3, and IQOS ILUMA) was generated according to the HTP-259-CTR smoking regime (55 mL puff volume, 30 s puff interval, 2 s puff duration, bell-shaped puff profile, and not blocking the ventilation holes) using an analytical vaping machine LM5E (Körber Technologies Instruments GmbH). The cytotoxicity of CSE and erastin to rat vascular smooth muscle cells (A7r5 cells) was evaluated by measuring mitochondrial metabolic activity and lactate dehydrogenase (LDH) leakage. Erastin and CSE induced a decrease in mitochondrial metabolic activity and an increase in LDH leakage. The cytotoxic effects of erastin were almost completely inhibited by a radical trapping antioxidant UAMC-3203, an iron chelator deferoxamine mesylate (DFO), a 12/15-lipoxygenase (12/15-LOX) inhibitor baicalein, and a selective 15-LOX-1 inhibitor ML351. On the other hand, CSE-induced cell damage was partially attenuated by UAMC-3203, baicalein, and ML351, but not DFO. These results suggest that although erastin induces iron-dependent, 15-LOX-mediated ferroptosis, CSE primarily causes cell damage through a ferroptosis-independent mechanism..
