Quantitative adverse outcome pathway modeling for cigarette smoke-inducible airway mucus hypersecretion. Part 1: AOP-based in vitro assessment with repeated exposure to whole cigarette smoke Authors

Sakuya Ichikawa Shugo Muratani Keigo Sano Kazuo Erami Akina Mori Risa Matsumoto Shigeaki Ito ^*

• Japan Tobacco (Japan), Tokyo, Japan

Adverse outcome pathway (AOP)-based chemical risk assessment, a promising tool for regulatory decision-making, is typically used in toxicological assessments. However, it also holds potential for pharmacological and disease-related evaluations. This study focused on an AOP for decreased lung function. While lung function is normally robustly maintained by homeostatic capacity, repeated and chronic stimulation can disrupt this capacity, leading to impaired lung function and mucus hypersecretion. We developed an AOP-based in vitro method to test the disease-related state reproduced by exposing three-dimensionally cultured human bronchial epithelial cells (3D-HBECs) to whole cigarette smoke (WCS). Over 2 weeks, we conducted six repeated exposures of 3D-HBECs from six different donors to WCS, observing both acute phase responses (oxidative stress, epidermal growth factor receptor activation, and SP1 activation) and chronic phase responses (intracellular mucus production, goblet cell metaplasia/hyperplasia, and mucus hypersecretion) along the AOP. Our results demonstrated that, although repeated exposure to WCS induced biological responses along the AOP in all donors, there were inter-donor differences, particularly in the timing and amplitude of chronic phase responses. Because not all smokers exhibit phenotypic changes with the same smoking duration, this variability likely reflects individual differences. We anticipate that our AOP-based assessment method, combined with computational quantitative AOP modeling in Part 2, will become a valuable tool for assessing the disease risk of airborne materials and inhalable products.