Relationship between cytotoxic activities of gas phase extracts of cigarette smoke and cigarette brand.

<p>The gas phase extracts of cigarette smoke (designated cCSE) at the original tar concentration of 10 mg/ml were prepared from cigarettes of various brands by continuous smoking protocol as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107856#pone-0107856-g001" target="_blank">Fig. 1</a>. The cCSEs were subjected to MTS reduction assay for evaluation of their cytotoxic activities, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107856#pone-0107856-g003" target="_blank">Fig. 3</a>. MTS reduction activity in the absence of the gas phase extract was represented as 100%. Values represent means ± SE of three experiments, each in triplicate. P, Peace (JT, Japan; 28 mg tar, 2.3 mg nicotine), HL, Hi-Lite (JT, Japan; 17 mg tar, 1.4 mg nicotine), SS, Seven Stars (JT, Japan; 14 mg tar, 1.2 mg nicotine), M, Mevius (JT, Japan; 10 mg tar, 0.8 mg nicotine), MSL, Mevius Super Light (JT, Japan; 6 mg tar, 0.5 mg nicotine), Ma, Marlboro (Phillip Morris, USA; 12 mg tar, 1.0 mg nicotine), LS, Lucky Strike (British American Tobacco, UK; 11 mg tar, 0.9 mg nicotine), K9, Kent 9 mg (British American Tobacco, UK; 9 mg tar, 0.8 mg nicotine).</p

Pharmacological properties of cytotoxic activities of two types of gas phase extracts of cigarette smoke.

<p>The gas phase extracts of cigarette smoke at the virtual tar concentration of 10 mg/ml PBS were prepared from Hi-Lite brand cigarettes by either continuous (cCSE) or puff smoking protocol (pCSE), and they were subjected to MTS reduction assay (A) and LDH leakage assay (B). For determination of the effects of inhibitors of protein kinase C or NADPH oxidase, 5 µM BIS I or 1 µM DPI was added to the culture medium of C6 glioma cells, respectively, 30 min before the start of 4-h incubation with cCSE or pCSE. In panel A, MTS reduction activity was represented as a percentage of the control value in the absence of CSEs (PBS) within the vehicle-treated group. In panel B, LDH activity leaked into culture medium was represented as a percentage of total activity in the medium of cells lysed by 0.2% Triton X-100. Values represent means ± SE of three experiments, each in triplicate. **P<0.01 vs PBS-treated cells within either of three groups (Vehicle-, BIS I- and DPI-treated groups); ^##P<0.01 vs cCSE- or pCSE-treated cells within the vehicle-treated group.</p

Schematic diagram of an apparatus for preparation of gas phase extracts of cigarette smoke.

<p>A standard method for preparation of the gas phase extract of cigarette smoke is as follows. Four cigarettes of Hi-Lite brand, unless otherwise specified, were sequentially combusted and the main-stream smoke was continuously sucked through a Cambridge filter at a constant flow rate of 1.050 l/min by an aspirator, to remove the tar phase. The remaining gas phase was bubbled through a glass ball filter (pore size: 20–30 µm) into phosphate buffered saline (PBS, 15 ml) in a graduated cylinder kept at 25°C. After combustion of cigarette, the filter was dried in air for 12 h at 25°C, and the dry weight of the tar phase trapped on the Cambridge filter was obtained by subtracting the weight of filter before use from that after use. The concentration of the gas phase extract was expressed as the virtual tar concentration (mg tar/ml PBS), assuming that the tar phase trapped on the Cambridge filter is dissolved in the PBS. Four cigarettes of Hi-Lite brand gave the dry tar weight of approximately 150 mg. Notably, cytotoxicity of the gas phase extracts depends not on cigarette brands but on the virtual tar concentration.</p

Sensitivities of various cultured cells to the gas phase extracts of cigarette smoke.

<p>The gas phase extracts of cigarette smoke (cCSE) at the virtual tar concentration of 10 mg/ml were prepared from Hi-Lite brand cigarettes by continuous smoking protocol, and they were subjected to MTS reduction assay using various cultured cells. MTS reduction activity was represented as a percentage of the control value in the absence of cCSE. Values represent means ± SE of three experiments, each in triplicate. (A) C6, rat glioma cells; HEK293T, human embryonic kidney cells; CHO, Chinese hamster ovary cells; HeLa, human cervical carcinoma cells; U937, human monocytes; RAW264.7, mouse macrophages; HUVEC, immortalized human umbilical vein endothelial cells; A7r5, rat aorta smooth muscle cells. (B) SBC-3, human lung small cell carcinoma; H1299, human lung squamous cell carcinoma; A549, human lung adenocarcinoma.</p

Cytotoxic activities of gas phase extracts of cigarette smoke and the number of cigarette.

<p>Concentration-response curves of the gas phase extracts of cigarette smoke prepared from varying numbers of cigarettes (Hi-Lite brand) (A) and of the phosphate buffered saline (PBS) in the second graduated cylinder (B) for inhibition of MTS reduction activity. (A) The gas phase extract of cigarette smoke (designated cCSE) was prepared from varying numbers (2–40) of cigarettes (Hi-Lite brand) based on continuous smoking protocol, while a new Cambridge filter was used every 4 cigarettes. Inset: Concentration-response curves of the cCSE prepared from 20 or 40 cigarettes with a change in scale of concentrations on x axis. (B) In the apparatus for preparation of cCSE, the second graduated cylinder with 15 ml of PBS was incorporated downstream of the first one, and cCSE was prepared from either 4 or 14 cigarettes (Hi-Lite brand). The cytotoxicity of PBS in the original and second graduated cylinders was evaluated using MTS reduction assay. MTS reduction activity in the absence of the gas phase extract was represented as 100%. Values represent means ± SE of three experiments, each in triplicate. 4-1 (14-1) and 4-2 (14-2) represent the cytotoxic activities of the PBS in the first (original) and second graduated cylinders prepared from 4 (14) cigarettes, respectively.</p

The EC₅₀ and maximal values for inhibition of MTS reduction activity of the cCSEs prepared from varying numbers (2–40) of cigarettes (Hi-Lite brand).

a<p>The cCSEs at the original tar concentration of 10 mg/ml were subjected to MTS reduction assay in C6 glioma cells.</p><p>The concentration-response curves for inhibition of MTS reduction activity were constructed, and the EC₅₀ values and maximal inhibition were determined. The concentrations of cCSEs were represented by the virtual tar concentrations. MTS reduction activity in the absence of the cCSEs was represented as 100%. Values represent means ± SE of three experiments, each in triplicate.</p><p>**P<0.01 vs the value for 2 cigarettes.</p><p>The EC₅₀ and maximal values for inhibition of MTS reduction activity of the cCSEs prepared from varying numbers (2–40) of cigarettes (Hi-Lite brand).</p

Cytotoxic activities of gas phase extracts of cigarette smoke and smoking methods.

<p>The gas phase extracts of cigarette smoke were prepared from Hi-Lite brand cigarettes by either continuous smoking protocol (cCSE) as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107856#pone-0107856-g001" target="_blank">Fig. 1</a> or puff smoking (pCSE) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107856#s2" target="_blank">Materials and Methods</a>. The original gas phase extracts at the virtual tar concentration of 10 mg/ml PBS were prepared, and they were subjected to MTS reduction assay (A), LDH leakage assay (B), DNA fragmentation assay (C) and PI uptake assay (D, E) in cultured C6 glioma cells for evaluation of their cytotoxic activities. In MTS reduction assay, substrate reduction activity was represented as a percentage of the value in the absence of the gas phase extract; in LDH leakage assay, LDH activity leaked into culture medium was represented as a percentage of total activity in the medium of cells lysed by 0.2% Triton X-100; in PI uptake assay, the number of the cells positive for PI uptake was represented as a percentage of total number of cells identified by Hoechst 33342 for nuclear staining. Values in panels A, B and E represent means ± SE of three experiments, each in triplicate. The results in panels C and D are representative of three separate experiments.</p

Comparison of concentrations of carbonyl compounds in cCSE and pCSE.

a<p>The cCSE and pCSE at the original tar concentration of 10 mg/ml were prepared from Hi-Lite brand cigarettes by either continuous (cCSE) or puff smoking protocol (pCSE).</p><p>cCSE and pCSE were fractionated by HPLC and each fraction was analyzed for cytotoxic activities using PI uptake assay. The positive fractions were analyzed by GC/MS after derivatization with a carbonyl reagent PFBOA. Values represent means ± SD of three experiments.</p><p>Comparison of concentrations of carbonyl compounds in cCSE and pCSE.</p

Quantification of the weight of the tar of cigarette smoke trapped on the Cambridge filter.

<p>(A) Time-course of a decrease in the weight of the tar phase of cigarette smoke trapped on the Cambridge filter after drying at 25°C (open circle) or 55°C (closed circle). Four cigarettes of Hi-Lite brand were sequentially combusted and the main-stream smoke was sucked through a Cambridge filter at a constant flow rate of 1.050 l/min by an aspiration pump. After combustion of cigarette, the filter was dried for various lengths of time at 25°C (open circle) or 55°C (closed circle), and the weight of the tar phase of cigarette smoke trapped on the Cambridge filter was obtained by subtracting the filter weight before combustion of cigarette from the weight after combustion. (B) The relationship between the number of combusted cigarettes and the dry weight of the tar phase trapped on the Cambridge filter. Various numbers of Hi-Lite brand cigarettes were sequentially combusted as described in A. After combustion, the filter was dried for 12 h at 25°C, and the dry weight of the tar phase on the Cambridge filter was determined as described in A. Values represent means ± SD of three experiments. *, P<0.05; **, P<0.01 versus 25°C.</p

Tar content per cigarette of various brands, the dry weight of tar trapped on the Cambridge filter after combustion of one cigarette and the EC₅₀ values of cCSEs for inhibition of MTS reduction activity.

a<p>Nicotine and tar content per cigarette is the value reported by its manufacturer and determined by puff smoking based on ISO regulation.</p>b<p>For determination of dry tar weight per cigarette, smoke of one cigarette from either brand was continuously sucked through the Cambridge filter, and the increase in the dry weight of the filter was determined (represented as means ± SD of three experiments).</p>c<p>For determination of the EC₅₀ values, cCSEs at the original tar concentration of 10 mg/ml were prepared from cigarettes of various brands by continuous smoking, and subjected to MTS reduction assay in C6 glioma cells for construction of concentration-response curves from which the EC₅₀ values (means ± SE of three experiments, each in triplicate) were determined.</p><p>Tar content per cigarette of various brands, the dry weight of tar trapped on the Cambridge filter after combustion of one cigarette and the EC₅₀ values of cCSEs for inhibition of MTS reduction activity.</p