title: 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).
authors.affiliation: a British American Tobacco, R&D , Southampton , Hampshire , UK .
authors.forename: Annette
authors.initials: A
authors.lastname: Dalrymple
authors.affiliation: b Vivotecnia Research S.L., Parque Científico de Madrid , Tres Cantos , Madrid , Spain , and.
authors.forename: Patricia
authors.initials: P
authors.lastname: Ordoñez
authors.affiliation: a British American Tobacco, R&D , Southampton , Hampshire , UK .
authors.forename: David
authors.initials: D
authors.lastname: Thorne
authors.affiliation: a British American Tobacco, R&D , Southampton , Hampshire , UK .
authors.forename: David
authors.initials: D
authors.lastname: Walker
authors.affiliation: a British American Tobacco, R&D , Southampton , Hampshire , UK .
authors.forename: Oscar M
authors.initials: OM
authors.lastname: Camacho
authors.affiliation: c Histovia GmbH , Overath , Germany.
authors.forename: Ansgar
authors.initials: A
authors.lastname: Büttner
authors.affiliation: a British American Tobacco, R&D , Southampton , Hampshire , UK .
authors.forename: Debbie
authors.initials: D
authors.lastname: Dillon
authors.affiliation: a British American Tobacco, R&D , Southampton , Hampshire , UK .
authors.forename: Clive
authors.initials: C
authors.lastname: Meredith
mesh_terms: D000818:Animals; D020552:Comet Assay; D004249:DNA Damage; D005260:Female; D008168:Lung / Q000187:drug effects* / Q000473:pathology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D015162:Micronucleus Tests; D009154:Mutation; D017207:Rats, Sprague-Dawley; D012107:Research Design; D012906:Smoke / Q000009:adverse effects*; D062789:Tobacco Products / Q000633:toxicity; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D008565:Membrane Proteins; D012906:Smoke; C080086:phosphatidylinositol glycan-class A protein
keywords: Cigarette smoke; DNA damage; comet assay; in vivo; inhalation