Gas phase extract of mainstream smoke derived from heated tobacco products causes iron-dependent, ferroptosis-independent cell injury in vascular endothelial cells.

Abstract

Ferroptosis is defined as an iron-dependent regulated necrosis that is caused by massive lipid peroxidation-mediated cell membrane damage. The present study examined whether ferroptosis is involved in endothelial dysfunction by nicotine- and tar-free cigarette smoke extract (CSE) prepared from heated tobacco products (HTPs). The CSE of HTPs (Ploom X, IQOS 3, and IQOS ILUMA) and a combustion cigarette (1R6F) was prepared according to Health Canada Intense smoking method (55 mL puff volume, 2 sec puff duration, and 1 puff every 30 sec) using an analytical vaping machine LM5E (Borgwaldt KC GmbH). The cytotoxicity of CSE of HTPs and 1R6F to human umbilical vein endothelial EA.hy926 cells was evaluated by measuring mitochondrial metabolic activity and lactate dehydrogenase (LDH) leakage. CSE from cigarettes except for Ploom X, and erastin, a ferroptosis inducer by inhibiting cystine-glutamate exchange transporter (system XC-), triggered a decrease in mitochondrial metabolic activity and an increase in LDH leakage. The cytotoxic effects of CSE of IQOS 3 and 1R6F were reduced by an iron chelator deferoxamine mesylate (DFO), but not by a ferroptosis inhibitor UAMC-3203, which scavenges lipid reactive oxygen species (ROS). On the other hand, erastin cytotoxicity was inhibited by both DFO and UAMC-3203. These results suggest that erastin-induced, iron-dependent ferroptosis leads to cell damage characterized by a decrease in mitochondrial metabolic activity and an increase in LDH leakage, in EA.hy926 cells. CSE of IQOS 3 and 1R6F causes iron-dependent mitochondrial and cell membrane damage, both of which are independent of lipid peroxidation by ROS.