Cigarette smoking is associated with diseases including cardiovascular disease. Key events from exposure to cigarette smoke to the development of disease related endpoints can be mapped out in the form of an adverse outcome pathway (AOP). An AOP is a framework that documents a chain of biological effects induced by exposure (cigarette smoke) and describes molecular and biological responses at the cellular, tissue, organ, whole body and population level. AOPs offer promise for researchers, regulators and risk assessors, forming toxicological and biological knowledge frameworks to aid risk assessment based on mechanistic reasoning and supporting biomarker discovery and validation.

We have mapped out an AOP focussing on key events associated with the development of hypertension initiated by cigarette smoke-induced vascular endothelial oxidative stress. Oxidative stress contributes to endothelial dysfunction and can lead to impairment of endothelium-dependent vasodilation and hypertension having an inhibitory effect on endothelial nitric oxide (NO) production, critical for the maintenance of healthy vascular tone.

Using bovine aortic endothelial cells (BAECs) and aqueous extracts (AqE) from a 3R4F reference cigarette, we were able to model and further assess these endpoints in vitro. Exposure of BAECs to AqE resulted in significant changes to key events including a decrease in NO production with an increase in superoxide, and generation and accumulation of 4-HNE protein adducts. AqE exposure led to depletion of tetrahydrobiopterin (BH4) and total biopterin levels. Importantly, exposure of BAECs to AqE indicated a central role of the ubiquitin proteasome system (UPS) in AqE-induced eNOS dysfunction.

In conclusion, our results provide strong in vitro evidence to support an AOP of vascular endothelial oxidative stress leading to hypertension. These endpoints combined with clinical data can serve as potential biomarkers of cigarette smoke-induced vascular endothelial dysfunction and help to provide comparative data supporting the assessment of novel tobacco and nicotine products.