CSE was prepared in complete cell culture medium using a pump-assisted bubbling method and either the tip of a 5 mL pipette (L=large, 1.85 mm diameter bore opening) or a narrow gel-loading pipette tip (S=small, 0.61 mm bore). Only one cigarette was used per extract to avoid potential occlusion of the orifice by tar, and no visible occlusion was observed. CSE was then added at 40% or 80% concentration to cultures of the U937 promonocytic cell line (equal starting number of cells). After 48 h, cell counts and viability were assessed. Two trials were conducted with three replicates each.
As assessed by statistical analysis of viable and dead cell densities (concentrations), treatment with CSE significantly (adjusted p <0.0001) reduced cell counts compared with untreated control at each dose and with each pipette type/bore size (Fig. 1A and B). The tip type also had a significant (adjusted p <0.01) effect on viable cell concentration within each dose comparison. Indeed, the only viability comparisons without significant (corrected p <0.05) differences were between the two doses of CSE produced with the small-bore tip (Trial 1) and 80% large-bore versus 40% small-bore (Trial 2). A two-factor analysis of the treatment data by dose and pipette type also revealed that both variables contributed significantly to variation (adjusted p of 0.0001 or less).
Dose-dependent toxicity (0%, 40%, and 80% CSE) was also observed throughout by examining the percentage of viable cells (Fig. 1C and 1D). The possible exception was 40% CSE prepared with the large-bore pipette tip, which did not demonstrate a significant difference compared to the control (0%) CSE. In this condition, viability was slightly (4-5% on average) lower than control in both trials, but the difference was not statistically significant following adjustment for multiple comparisons. Again, two-factor analysis of the treatment data by dose and bore size also revealed that both variables contributed significantly to variation (adjusted p of 0.0001 or less).
These results lead us to several conclusions. First, switching from a large- to a small- bore pipette for CSE generation achieved similar (Trial 1) or greater (Trial 2) increases in toxicity and/or growth suppression compared with doubling the CSE concentration. Second, the lowest toxicity condition (40% CSE generated with a large-bore tip) appeared to suppress growth without a significant increase in toxicity, suggesting that multiple mechanisms could explain the observed results. Third, despite highly similar results between the two trials, substantial variability was also evident: viable cell percentage after the most toxic treatment (with 80% CSE generated with the small-bore tip) ranged from a negligible 1% (Trial 2) to around 39% (Trial 1). However, total cell counts in the untreated condition also differed substantially, by more than two-fold.
Several factors might contribute to these results. To us, the most obvious explanation is a gas exchange, which varies by the bore size of the pipette inserted into the aqueous medium. A narrow-bore pipette gives rise to smaller bubbles than a wider-bore pipette, achieving more gas exchange through greater surface-to-volume ratio per bubble as well as more bubbles per given volume of gas. As a result, the transfer of soluble toxins from the smoke into the medium would be expected to be more efficient for smaller bubbles, consistent with our results.
However, other explanations should be considered, as well. First, the flow rate of the apparatus might vary across pipette types despite our use of the same pump setting in all experiments. We investigated this possibility with a water displacement assay. Setting the flow rate of the apparatus without a cigarette (free intake) and with a large tip to 100.0% (standard deviation 0.5%), we found that the flow rate through the small tip was slightly decreased (94.6%+/-1.0%). Repeating these experiments with a lit cigarette did not change the flow rate: 99.9%+/-0.9% (large tip) versus 94.1%+/-1.3% (small tip; incidentally, flow rate did not decrease over time for the small tip, suggesting that occlusion of the orifice is not significant for one cigarette). In any case, the small percent reduction in flow rate through the smaller bore tip would tend to reduce, not increase, the toxicity of CSE. A second explanation could be simple aeration; that is, greater gas exchange during small-tip bubbling would result in the medium that more closely approaches atmospheric oxygen levels (around 21%). However, we tend to discount this explanation due to the limited area of exchange and length of bubbling and, perhaps more importantly, the rapid rate of oxygen diffusion in cell culture as compared with the 48 h length of our culture experiments. Finally, different materials were used for the large-bore (polystyrene) and small-bore pipettes (polypropylene). It is possible that the passage of gas through different plastics could result in different absorption or release of compounds, even though these materials are both used widely for the labware due to their relatively inert nature. Polypropylene, which can in many cases resist autoclaving, is even more resistant to high temperatures than polystyrene. In any case, all conditions included exposure to both polypropylene (gel-loading tip and/or insertion of the cigarette into a 1 mL pipette tip; 50 mL conical) and polystyrene (5 mL pipette and/or culture vessels). Thus, while we admit the possibility of other explanations and restrict our claims here to differences associated with two different pipettes, we maintain that the most likely contributor to our findings is the greater gas exchange achieved with a narrow-bore pipette, resulting in higher concentrations of cigarette smoke-derived chemicals with cytostatic and/or cytotoxic effects.