Insight into the pulmonary molecular toxicity of heated tobacco products using human bronchial and alveolar mucosa models at air-liquid interface

• Heated tobacco products (HTP) are novel nicotine delivery products with limited toxicological data. HTP uses heating instead of combustion to generate aerosol (HTP-smoke). Physiologically relevant human bronchial and alveolar lung mucosa models developed at air–liquid interface were exposed to HTP-smoke to assess broad toxicological response (n = 6–7; ISO puffing regimen; compared to sham; non-parametric statistical analysis; significance: p < 0.05). Elevated levels of total cellular reactive oxygen species, stress responsive nuclear factor kappa-B, and DNA damage markers [8-hydroxy-2′-deoxyguanosine, phosphorylated histone H2AX, cleaved poly-(ADP-Ribose) polymerase] were detected in HTP-smoke exposed bronchial and/or alveolar models. RNA sequencing detected differential regulation of 724 genes in the bronchial- and 121 genes in the alveolar model following HTP-smoke exposure (cut off: p ≤ 0.01; fold change: ≥ 2). Common enriched pathways included estrogen biosynthesis, ferroptosis, superoxide radical degradation, xenobiotics, and α-tocopherol degradation. Secreted levels of interleukin (IL)1ꞵ and IL8 increased in the bronchial model whereas in the alveolar model, interferon-γ and IL4 increased and IL13 decreased following HTP-smoke exposure. Increased lipid peroxidation was detected in HTP-smoke exposed bronchial and alveolar models which was inhibited by ferrostatin-1. The findings form a basis to perform independent risk assessment studies on different flavours of HTP using different puffing topography and corresponding chemical characterization.

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