In vitro biological assessment of the stability of cigarette smoke aqueous aerosol extracts
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RESEARCH NOTE
In vitro biological assessment of the stability of cigarette smoke aqueous aerosol extracts Mark Taylor, Simone Santopietro* , Andrew Baxter, Nicole East, Damien Breheny, David Thorne and Marianna Gaça
Abstract Objectives: Cigarette smoke aqueous aerosol extracts (AqE) have been used for assessing tobacco products, particularly with in vitro models such as oxidative stress and inflammation. These test articles can be generated easily, but there are no standardised methods for the generation and characterisation or stability. We investigated the effects of pro-oxidant smoke-derived chemicals by using 3R4F AqE generated under standardised conditioning and smoking regimes and assessed the stability over 31-week timeframe. Twenty batches generated from ten puffs per cigarette bubbled through 20 ml cell culture media were used fresh and thawed from frozen aliquots stored at – 80 ºC. Results: Nicotine levels quantified by gas chromatography/mass spectrometry and optical density at 260 nm showed chemical and physical stability from week 0 (fresh sample) to weeks 1, 4, 8 and 31 (frozen samples). No significant change in H292 human bronchial epithelial cell viability or oxidative stress were observed between fresh AqE at week 0 and frozen AqE at 31 weeks. AqEs generated by our protocol were stable for up to 31 weeks for all tested end points, suggesting that it may not be necessary to use freshly generated AqE for each study, thus reducing batch-tobatch variability. Keywords: In vitro, AqE, Aqueous extract, Cigarette smoke, Stability Introduction Tobacco smoke is a mixture of more than 7000 constituents [1–3] partitioned in vapour and particulate phases of cigarette smoke aerosol. In vitro toxicological assessments of cigarette smoke have been conducted extensively in total particulate matter (TPM), whole aerosol, or aqueous aerosol extracts (AqE) and by a range of biological techniques [4–7]. TPM is well characterised and shows stability over 2 years [4]. Stability of whole aerosol approaches is less important for cigarettes because they are designed to generate and deliver the aerosol in seconds [5, 6]. Although the use of cigarette smoke AqE has been well documented, including the effective capturing of semi-volatiles, such as carbonyls [8–11], shortterm and long-term stability of volatile and semi-volatile *Correspondence: [email protected] British American Tobacco, R&D, Southampton SO15 8TL, Hampshire, UK
fractions remain uncharacterised. The generation of fresh extracts can be time consuming and subjected to a risk of batch-to-batch variability. Mitigation of variability might be possible if pooled batches are generated, stored and used throughout the lifecycle of a study. AqE generation is further confounded by the lack of a standardised approach. A cigarette smoke aqueous test matrix consists of one or more cigarettes smoked into a specified volume of solution and the resulting aqueous soluble fraction of the cigarette smoke (AqE) is diluted and applied
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