Cigarette smoke induced genotoxicity and respiratory tract pathology: evidence to support reduced exposure time and animal numbers in tobacco product testing

Abstract

Many laboratories are working to develop in vitro models that will replace in vivo tests, but occasionally there remains a regulatory expectation of some in vivo testing. Historically, cigarettes have been tested in vivo for 90 days. Recently, methods to reduce and refine animal use have been explored. This study investigated the potential of reducing animal cigarette smoke (CS) exposure to 3 or 6 weeks, and the feasibility of separate lung lobes for histopathology or the Comet assay. Rats were exposed to sham air or CS (1 or 2 h) for 3 or 6 weeks. Respiratory tissues were processed for histopathological evaluation, and Alveolar type II cells (AEC II) isolated for the Comet assay. Blood was collected for Pig-a and micronucleus quantification. Histopathological analyses demonstrated exposure effects, which were generally dependent on CS dose (1 or 2 h, 5 days/week). Comet analysis identified that DNA damage increased in AEC II following 3 or 6 weeks CS exposure, and the level at 6 weeks was higher than 3 weeks. Pig-a mutation or micronucleus levels were not increased. In conclusion, this study showed that 3 weeks of CS exposure was sufficient to observe respiratory tract pathology and DNA damage in isolated AEC II. Differences between the 3 and 6 week data imply that DNA damage in the lung is cumulative. Reducing exposure time, plus analyzing separate lung lobes for DNA damage or histopathology, supports a strategy to reduce and refine animal use in tobacco product testing and is aligned to the 3Rs (replacement, reduction and refinement).

• Cigarette smoke
• comet assay
• DNA damage
• inhalation
• in vivo

Acknowledgements

The authors acknowledge the technical contribution of Ana Belén Sierra, Arantxa Saiz Bautista, Miguel López Angulo and Hernando Servello Ferreiro (AEC II isolation and COMET analysis), Jesús Illán Manero, Elena Fernández Alargunsoro, Ruth Micha Gavilán (inhalation studies), Javier Rivera Trillo, Sara Anca Óvilo, Tamara Domínguez Guerrero and Carlos Martín Llanos (necropsy). We thank Dorothea Torous at Litron Laboratories for support with sample collection, Pig-a mutation and micronucleus analysis data, and review of the manuscript. We would also like to thank Ian Crooks, Marianna Gaḉa and Marc Princivalle for review of the manuscript and Jason Adamson for support with Figure 2.

Declaration of interest

The authors report no declarations of interest, and are employees of British American Tobacco, were employees of British American Tobacco at the time of study initiation or were contracted by British American Tobacco.