A simple and rapid method for standard preparation of gas phase extract of cigarette smoke

Abstract

Cigarette smoke consists of tar and gas phase: the latter is toxicologically important because it can pass through lung alveolar epithelium to enter the circulation. Here we attempt to establish a standard method for preparation of gas phase extract of cigarette smoke (CSE). CSE was prepared by continuously sucking cigarette smoke through a Cambridge filter to remove tar, followed by bubbling it into phosphate-buffered saline (PBS). An increase in dry weight of the filter was defined as tar weight. Characteristically, concentrations of CSEs were represented as virtual tar concentrations, assuming that tar on the filter was dissolved in PBS. CSEs prepared from smaller numbers of cigarettes (original tar concentrations ≤ 15 mg/ml) showed similar concentration-response curves for cytotoxicity versus virtual tar concentrations, but with CSEs from larger numbers (tar ≥ 20 mg/ml), the curves were shifted rightward. Accordingly, the cytotoxic activity was detected in PBS of the second reservoir downstream of the first one with larger numbers of cigarettes. CSEs prepared from various cigarette brands showed comparable concentration-response curves for cytotoxicity. Two types of CSEs prepared by continuous and puff smoking protocols were similar regarding concentration-response curves for cytotoxicity, pharmacology of their cytotoxicity, and concentrations of cytotoxic compounds. These data show that concentrations of CSEs expressed by virtual tar concentrations can be a reference value to normalize their cytotoxicity, irrespective of numbers of combusted cigarettes, cigarette brands and smoking protocols, if original tar concentrations are ≤15 mg/ml.

Figure 1. Schematic diagram of an apparatus for preparation of gas phase extracts of cigarette smoke.

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.

Figure 2. Quantification of the weight of the tar of cigarette smoke trapped on the Cambridge filter.

(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.

Figure 3. Effects of the bubbling condition on gas phase extracts of cigarette smoke.

Effects of the temperature of phosphate-buffered saline (PBS) (A) and pore size of a glass ball filter for bubbling the gas phase of cigarette smoke into PBS (B) on the cytotoxic activities of the gas phase extract were examined. The gas phase extract of cigarette smoke (designated cCSE) was prepared as described in the legend for Fig. 1, by continuous smoking of four cigarettes of Hi-Lite brand which gave the virtual tar concentration of approximately 10 mg/ml PBS. In panel A, the temperature of PBS for extraction of cigarette smoke was kept at either 25°C (open circle) or 0°C (closed circle), and in panel B, the pore size of the glass ball filter was either normal (pore size, 20–30 µm; open circle) or rough (pore size, 100–120 µm; closed circle). For evaluation of the cytotoxicity of cCSE, C6 glioma cells were incubated for 4 h with various concentrations of cCSE and MTS reduction activity was determined as described in Materials and methods. MTS reduction activity in the absence of cCSE was represented as 100%. Values represent means ± SE of three experiments, each in triplicate.

Figure 4. Cytotoxic activities of gas phase extracts of cigarette smoke and the number of cigarette.

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.

Figure 5. Relationship between cytotoxic activities of gas phase extracts of cigarette smoke and cigarette brand.

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 Fig. 1. The cCSEs were subjected to MTS reduction assay for evaluation of their cytotoxic activities, as described in Fig. 3. 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).

Figure 6. Cytotoxic activities of gas phase extracts of cigarette smoke and smoking methods.

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 Fig. 1 or puff smoking (pCSE) as described in Materials and Methods. 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.

Figure 7. Pharmacological properties of cytotoxic activities of two types of gas phase extracts of cigarette smoke.

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.

Figure 8. Sensitivities of various cultured cells to the gas phase extracts of cigarette smoke.

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.