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In vitro micronucleus assay for cigarette smoke using a whole smoke exposure system: A comparison of smoking regimens
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Experimental and Toxicologic Pathology 62 (2010) 433–440 www.elsevier.de/etp
In vitro micronucleus assay for cigarette smoke using a whole smoke exposure system: A comparison of smoking regimens Kosuke Okuwa, Masahiro Tanaka, Yasuo Fukano, Hidenori Nara, Yosuke Nishijima, Tomoki Nishino Tobacco Science Research Center, Japan Tobacco Inc., 6-2, Umegaoka, Aoba Ward, Yokohama, Kanagawa 227-8512, Japan Received 22 April 2009; accepted 3 June 2009
Abstract Previous studies on the biological assessment of cigarette smoke (CS) mainly focused on the total particulate matter (TPM) collected using a Cambridge filter or gas vapor phase (GVP) bubbled through phosphate-buffered saline (PBS). To study the effects of native CS in vitro, direct exposure methods have been developed. Meanwhile, in vitro micronucleus (MN) assays have been reported to evaluate the mutagenicity of CS. The objective of this research is to investigate the MN-inducing activity of whole smoke (WS) and GVP using a whole smoke exposure system, CULTEXs, which allows direct exposure of cultured cells to native CS at the air/liquid interface (ALI). CS was generated according to the International Organization for Standardization (ISO; 35 ml, 2 s, once per 60 s) or the Health Canada Intensive (HCI; 55 ml, 2 s, once per 30 s, with complete ventilation block) regimens and Chinese hamster lung (CHL/IU) cells were then exposed to this smoke. Dosages were adjusted according to the amount of smoke entering the actual exposure position. Under both smoking regimens, WS and GVP from 2R4F reference cigarettes induced MN responses. The concept of the dosage and similar dose–response relationships between theoretical and monitored dosage values under the two regimens enabled us to compare the MN-inducing activities of cigarettes in the direct exposure assay, even in the case of various experimental settings or different TPM amounts. MN-inducing activities of 2R4F under the ISO regimen seemed to be higher than those under HCI estimated by the TPM equivalent calculated values. r 2009 Elsevier GmbH. All rights reserved. Keywords: Cigarette smoke; Direct exposure; CULTEXs system; Micronucleus assay; Whole smoke; Gas vapor phase; Air/liquid interface; Dosage
Introduction Cigarette smoke (CS) is a complex and dynamic mixture of gaseous, volatile and particle compounds of more than 4000 chemicals (Baker, 1999). The biological Corresponding author. Tel.: +81 45 345 5245; fax: +81 45 973 6781. E-mail address: [email protected] (Y. Fukano).
0940-2993/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.06.002
evaluation of CS in vitro has focused on collected compounds such as total particulate matter (TPM) trapped using a Cambridge filter or gas vapor phase (GVP) bubbled through phosphate-buffered saline (PBS) (Mizusaki et al., 1977; Nakayama et al., 1985; Andreoli et al., 2003). These indirect methods allow simple and high-throughput biological assessment. However, extracted fractions of CS may not completely reflect the biological effects of native smoke. To evaluate
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the biological effects of CS that is closer to fresh smoke, direct exposure methods have been developed (Bombick et al., 1997; Fukano et al., 2004; Maunders et al., 2007; Aufderheide and Gressmann, 2008; Clunes et al., 2008). The in vitro micronucleus (MN) assay is a mutagenicity test that detects both aneugens and clastogens. Spindle apparatus defects or structural chromosomal damage converted to lagging of whole chromosome distribution or chromosomal fragments cause MN formation. The MN assay was performed by a simple and rapid method that showed highly concordant results with the chromosomal aberration test in vitro (Matsushima et al., 1999). Using the MN assay, several studies have reported the mutagenicity of CS fractions (Baker et al., 2004; Pre´fontaine et al., 2006). One study reported that direct exposure of whole smoke induced MN formation (Massey et al., 1998). However, there is no information regarding the MN-inducing activities under different smoking regimens using the direct exposure method. In this study, we compare MN-inducing activities under both International Organization for Standardization (ISO) and Health Canada Intensive (HCI) smoking regimens. To accomplish these objectives, we used an original dosage unit that allowed us to compare different smoking conditions or different design of cigarettes.
Cell line and culture conditions The Chinese hamster lung (CHL/IU) cells (Dainippon Pharmaceutical Co. Ltd., Tokyo, Japan) were maintained in Eagle’s minimum essential medium supplemented with 10% heat-inactivated (56 1C, for 30 min) bovine serum, 25 mM Hepes buffer and 1 mg/ml gentamycin. Cells were grown in a humidified atmosphere of 95% air and 5% CO2 at 37 1C. In preparation for direct exposure, CHL/IU cells were trypsinized and seeded onto the microporous membranes (pore size 0.4 mm, growth area 0.9 cm2) of cell culture inserts (Becton Dickinson, Franklin Lakes, NJ) at a density of 1 105 cells/insert and cultured for 24 h.
Equipment for whole smoke exposure system The CULTEXs system 2006 (SIBATA, Japan) was used for the experiments. This experimental system is composed of a smoking machine, a dilution system, and a glass exposure module (Fig. 1A). The CS was generated by a smoking machine, diluted with clean air in the dilution system and sucked via negative pressure into the exposure module (Fig. 1B), where CHL/IU cells, cultured on the microporous membranes at the air/liquid interface (ALI), were exposed to the smoke. The exposure module was maintained at 37 1C by the warm water circulating through the device.
Materials and methods Exposure settings Cigarettes Kentucky standard reference cigarettes 2R4F were used for the experiments. The cigarettes were conditioned at 22 1C and 60% relative humidity for at least 48 h before use. Under the HCI smoking regimen, the filter ventilation was blocked with cellophane tape.
Smoke generation was computer controlled using the ISO or HCI smoking regimens. Freshly generated smoke was pressed into a dilution system and diluted by a constant flow of clean air controlled by a mass-flow controller and valves. Diluted smoke was sucked into the exposure module at a velocity of 5 mL/min via
Fig. 1. The diagram shows (A) direct exposure system, (B) dilution system interior: 1, generated smoke; 2, dilution air; 3, flow rate and (C) calculating formula of percentage of CS.
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negative pressure from a vacuum pump. To generate the GVP, a Cambridge filter (44 mm diameter) was inserted into the smoke path between the cigarette port and the cylinder of the smoking machine. Under the ISO smoking regimen, 2, 4, 6 and 8 cigarettes were smoked by taking a puff of 35 ml volume over 2 s every 60 s. Smoke was released into the dilution system in 2.8 s puffs. In the dilution system, smoke was diluted by dilution air at a velocity of 4.5 L/min for whole smoke (WS) exposure and 2.25 L/min for GVP exposure. Under the HCI smoking regimen, 1, 2, 3 and 4 cigarettes with completely blocked ventilation were smoked by taking a 55 ml puff volume over 2 s every 30 s. Smoke was released into the dilution system in 2.8 s puffs. In the dilution system, smoke was diluted by dilution air at a velocity of 3.5 L/min for WS exposure and 1.25 L/min for GVP exposure. CHL/IU cells were cultivated and exposed at the ALI that means medium supply from the basal part of the membrane, whereas the apical part with the cells is in direct contact with the test atmosphere. As a negative control, cells were exposed to clean air at a velocity of 5 mL/min.
Dosage setting Dosages were adjusted to the amount of smoke entering the exposure module and expressed as a percentage of CS (% of cig.) according to the formula (Fig. 1C). The numerator in the calculating formula is the flow rate that represents the amount of smoke finally entering the exposure module. The denominator part of the formula, the sum of the velocities of generated smoke and dilution air, show the total amount of smoke that passed through the dilution system. The values of the % of cig. are expressed as a percentage and this indicates how much generated smoke entered the exposure module. The values of the % of cig. can be converted to TPM equivalent values by multiplying the % of cig. values by the TPM amount per cigarette. Dry particle matter (DPM) equivalent values can also be calculated by multiplying the % of cig. values by waterfree TPM amount per cigarette.
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variations from the photometer were translated into TPM values. Monitored TPM values were divided by the TPM amount per cigarette under each smoking regimen to calculate the ‘‘monitored’’ % of cig. values. The ‘‘theoretical’’ % of cig. values were calculated according to the formula.
Smoke analyses TPM was measured gravimetrically using a Cambridge filter. Nicotine and water were determined from isopropanol extracts of TPM using the HP-6890 gas chromatography system (Hewlett Packard, Palo Alto, CA) according to the relevant ISO standards (ISO Standard 10315, 2000; ISO Standard 10362-1, 1999).
MN assay The MN assay was carried out without cytochalasin B and without metabolic activation. CHL/IU cells cultured on microporous membranes were exposed to WS or GVP. After smoke exposure, cell culture inserts were filled with fresh culture medium without washing, followed by a 24 h recovery period. After the recovery period, cells were trypsinized and centrifuged. The cell pellet was subjected to hypotonic treatment with 75 mM KCl at 37 1C for 5 min and fixed with acetic acid– methanol (1:3). Fixed cells were suspended with 1% acetic acid in methanol and air dried. Preparations were stained with acridine orange. Two thousand cells per dosage were scored for MN cell counting using a fluorescent microscope.
Cytotoxicity assessment For cytotoxicity assessment, the trypsinized cell suspension after the recovery period was scored by Coulter Counter (Beckman Coulter Inc., Fullerton, CA). Counts of air-exposed samples were regarded as 100% and the percentage of cell counts in each dosage were calculated as a percentage of viable cells.
Results
Particle phase monitoring
Particle phase monitoring
The amount of smoke entering the exposure module was monitored under ISO and HCI smoking regimen conditions. Monitoring was carried out using a light scattering photometer (Vitrocell, Germany) in the smoke path extended into the exposure module. Prior to the monitoring, we calibrated the photometer by measuring the voltage variations and actual TPM values at the same time. Using this calibration curve, voltage
To use the % of cig. as a dosage unit, we had to confirm whether the ‘‘monitored’’ % of cig. is consistent with the ‘‘theoretical’’ one. Fig. 2 shows the plots of the actual value of ‘‘monitored’’ vs. ‘‘theoretical’’ % of cig. across various exposure conditions. The regression formula was y ¼ 1.24x and the correlation coefficient was 0.998 under the ISO smoking regimen. Under the HCI smoking regimen,
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the regression formula was y ¼ 1.09x and the correlation coefficient was 0.983. It was confirmed that the ‘‘monitored’’ and ‘‘theoretical’’ % of cig. were consistent and in good correlation under both ISO and HCI smoking regimens and the slopes of linear regression were similar under these two regimens.
Smoke analyses TPM, nicotine and water content under experimental conditions are summarized in Table 1. The HCI smoking regimen produced an increase in the TPM amount of about 2.8-fold that of ISO. The water content in TPM was about 11% under the ISO smoking regimen and about 30% under the HCI smoking regimen.
MN-inducing activity and cytotoxicity of 2R4F: ISO WS and GVP from 2R4F showed dose-related MN responses under the ISO smoking regimen (Fig. 3A). The highest average percentage of the MN frequency was 4.98% and the dosage was 0.57% of cig. in WS exposure. In GVP exposure, the highest average percentage of the MN frequency was 5.42% and the dosage was 1.00% of cig. With the increase in the % of cig. values, the cytotoxicity, the decrease of viable cell count, was also observed (Fig. 3B).
Fig. 2. The graph shows the relationship between ‘‘monitored’’ % of cig. values and ‘‘theoretical’’ % of cig. values under the ISO smoking regimen and HCI smoking regimen. Symbol: K ISO smoking regimen, J HCI smoking regimen.
Table 1. Yields of TPM, nicotine and water of 2R4F reference cigarette under experimental settings. Smoking regimen ISO HCI
TPM (mg/ cigarette)
Nicotine (mg/ cigarette)
Water content (mg/cigarette)
11.3 32.0
0.61 2.12
1.2 9.7
MN-inducing activity and cytotoxicity of 2R4F: HCI WS and GVP from 2R4F showed dose-related MN responses under the HCI smoking regimen (Fig. 4A). The highest average percentage of the MN frequency was 5.22% and the dosage was 0.32% of cig. in WS exposure. In GVP exposure, the highest average percentage of the MN frequency was 5.7% and the dosage was 0.62% of cig. With the increase in the % of cig. values, the cytotoxicity, the decrease of viable cell count, was also observed (Fig. 4B).
Comparison of ISO and HCI smoking regimens Under the ISO smoking regimen, the experimental setting that showed the highest MN frequency was 6
Fig. 3. The graphs show (A) MN-inducing activities and (B) cytotoxicity under the ISO smoking regimen. 2, 4, 6 and 8 cigarettes were smoked and diluted by dilution air at a velocity of 4.5 L/min for WS exposure and 2.25 L/min for GVP exposure. Diluted smoke was introduced into the exposure module at a velocity of 5 mL/min. The results represent means and S.D. (n ¼ 3 for each condition). Symbol: K WS exposure, GVP exposure.
Figures represent mean values (n ¼ 3) for each condition.
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Fig. 4. The graphs show (A) MN-inducing activities and (B) cytotoxicity under the HCI smoking regimen. 1, 2, 3 and 4 cigarettes were smoked and diluted by dilution air at a velocity of 3.5 L/min for WS exposure and 1.25 L/min for GVP exposure. The results represent means and S.D. (n ¼ 3 for each condition). Symbol: WS exposure, & GVP exposure.
Fig. 5. The graphs show (A) WS MN-inducing activities based on TPM equivalent values, (B) GVP MN-inducing activities based on TPM equivalent values, (C) WS MN-inducing activities based on DPM equivalent values and (D) GVP MN-inducing activities based on DPM equivalent values. The results represent means and S.D. (n ¼ 3 for each condition). Symbol: K WS exposure under ISO regimen, GVP exposure under ISO regimen, WS exposure under HCI regimen, & GVP exposure under HCI regimen.
cigarettes with 4.5 L/min dilution rate in WS exposure and 6 cigarettes with 2.25 L/min dilution rate in GVP exposure. Under the HCI smoking regimen, the experimental setting showed that the highest MN frequency was 3 cigarettes with 3.5 L/min dilution rate in WS exposure and 3 cigarettes with 1.25 L/min dilution rate in GVP exposure. To compare ISO with HCI smoking regimens, TPM equivalent values were applied because the total amount of smoke under the two smoking
regimens were different and it was impossible to use the % of cig. for a qualitative comparison. TPM equivalent values, composed of tar, nicotine and water, were calculated by multiplying the % of cig. values by TPM amount per cigarette. The TPM equivalent values of the ISO MN peaks were 64.4 mg in WS exposure and 113.0 mg in GVP exposure. The TPM equivalent values of the HCI MN peaks were 102.4 mg in WS exposure and 198.4 mg in GVP exposure. ISO MN peaks resulted in
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lower TPM equivalent values than those of HCI under both WS and GVP exposure (Fig. 5A, B). To exclude the effect of water content difference caused by different smoking regimens, DPM equivalent values, composed of tar and nicotine, were calculated by multiplying the % of cig. by water-free TPM amount per cigarette. The DPM equivalent values of the ISO MN peaks were 57.6 mg in WS exposure and 101.0 mg in GVP exposure. The DPM equivalent values of the HCI MN peaks were 71.4 mg in WS exposure and 138.3 mg in GVP exposure. In the DPM equivalent evaluation, MN-inducing activities under both smoking regimens were closer than in the case of the TPM equivalent evaluation (Fig. 5C, D).
Discussion Biological evaluation of CS in vitro mainly focuses on collected compounds such as TPM collected using a Cambridge filter or GVP bubbled through PBS. In these cases, separating the particle phase and GVP as fractions may fail to reflect the biological effects of native aerosol. Furthermore, chemicals that are impossible to collect by the above-mentioned methods may be present, or there may be changes in the chemical composition compared with fresh smoke. It is important to detect the effects of CS as integral to the particle phase and GVP without modification by the collecting method. Therefore we used a whole smoke exposure system, CULTEXs, which allows direct exposure of complex mixtures such as native CS at the ALI of cultured cells, supplying fresh culture medium through the membranes of cell culture inserts (Aufderheide and Mohr, 1999; Aufderheide, 2008). The CULTEXs system has been introduced into many laboratories and has revealed clues to some biological phenomena in vitro (Wolz et al., 2002; Fukano et al., 2006; Olivera et al., 2007; Diabate´ et al., 2008). Numerous studies have reported the biological effects of CS in vitro. Most of them were conducted with a standard smoke generation procedure such as ISO. Meanwhile, some authorities originally recommended more intensive smoking regimens than the standard procedures because there are many reports that some smokers appear to be taking larger puffs and/or increased smoking frequency and/or a higher ratio of vent blocking in human smoking behavior (Borgerding and Klus, 2005). There are some reports in which those regimens have been applied to toxicity evaluation of CS (Foy et al., 2004). However, there is no information regarding the in vitro MN assay, a mutagenicity assay, under different smoking regimens using a direct exposure system. For this reason, we attempted to compare MN-inducing activities under both ISO and HCI smoking regimens using a direct exposure system.
WS and GVP under both smoking regimens showed dose-related MN responses. In our results, the dosages that indicated the highest MN frequency in WS exposure were about one half those of GVP under both ISO and HCI smoking regimens. These results suggest that the contribution of GVP in MN-inducing activities of 2R4F WS under both smoking regimens seems to be approximately half that estimated by the % of cig. at the highest MN frequency. The evaluation methods for CS are quantity or quality based. In indirect methods, both have been applied to the biological evaluation of CS (Tewes et al., 2003). In contrast, only quantity-based evaluation has been applied in direct exposure so far in which the dosage setting was mainly determined by the parameters such as the dilution rate and the number of cigarettes (Bombick et al., 1998; Massey et al., 1998). This methodology creates some difficulties in comparing cigarettes qualitatively because the appropriate dose ranges for detecting the better biological responses often differ among cigarette designs or smoking regimens. To adapt the direct exposure system to not only quantitybased evaluation but also quality-based evaluation of CS, we proposed the % of cig., based on how much generated smoke is introduced into the exposure module. This allows us to convert the values to TPM equivalent easily and flexibly by multiplying the TPM amount per cigarette to conduct the quality-based evaluation. It was confirmed that the % of cig. values are linearly correlated with the amount of smoke entering the exposure module under ISO and HCI smoking regimens and the slopes of regression curves were similar under both smoking regimens. The results, similar dose–response relationships between ‘‘theoretical’’ and ‘‘monitored’’ % of cig. values confirmed under both smoking regimens, respectively, gave us the verification for the conversion of the dosages to the TPM equivalent values, which enabled us to conduct quality-based evaluation in the direct exposure MN assay. Adjusting dosage is often a critical point in toxicological studies and we offer a new way to solve this problem for cigarette smoke in the direct exposure assay. Our results show that the MN-inducing activities of 2R4F under the HCI smoking regimen seemed to be lower than those under the ISO regimen estimated by the TPM equivalent values calculated from the % of cig. In the experiments with collected CS condensate, similar phenomena were reported. A more intensive regimen gave higher TPM yields on a per cigarette basis, showing less cytotoxicity and mutagenicity of the TPM per unit basis (Roemer et al., 2004) and TPM under the more intensive smoking regimen showed relatively less potency in MN-inducing activities (DeMarini et al., 2008). TPM collected under the HCI smoking regimen contains more water than that collected under the ISO
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smoking regimen. Increased water content under the HCI smoking regimen could be a key factor in the differences in MN activities measured by TPM equivalent figures. We adjusted the difference in water content and reevaluated the MN-inducing activities based on DPM base equivalent. Nevertheless, MN-inducing activities under the HCI smoking regimen still appeared to be less than under the ISO regimen in DPM figures, suggesting the change of chemical composition between ISO and HCI. To which extent the chemical composition alters and affects biological responses under different smoking regimens is interesting to note. The abundance of chemical compounds in CS may differ due to larger puff volume or greater ventilation blocking causing increased air volume to pass through a tobacco column and higher coal temperature may affect the combustion or pyrolysis at the burning cone under an intensive regimen. One recent study suggested that the chemical changes in the CS compounds caused by different smoking regimens might affect the cytotoxicity (Rickert et al., 2007). Mainstream smoke component variations caused by changing smoking regimens and cigarette designs have been described previously, indicating that alterations in chemical compositions were not uniform (Counts et al., 2005). In this report, we used 2R4F reference cigarettes that correspond to the low-ventilation group (filter ventilation o30%, Chen and Moldoveanu, 2003) according to the classification of Counts et al. (2005). Although the chemical changes in the low-ventilation group were relatively less than those in the highventilation groups, Counts et al. (2005) reported that constituent/tar yield ratios were relatively greater under the HCI smoking regimen than under the ISO smoking regimen for pyridine and, to a lesser extent, formaldehyde in particulate-phase chemicals. In vapor-phase chemicals, constituent/tar yield ratios were relatively greater under the HCI smoking regimen than under the ISO smoking regimen for hydrogen cyanide and styrene and, to a lesser extent, acrolein and methyl ethyl ketone. In contrast, other chemicals that include those known to possess the MN-inducing activities such as hydroquinone, catechol, phenol and acetaldehyde (Bird et al., 1982; Yager et al., 1990) showed a quite opposite reaction, with greater constituent/tar yield ratios under the ISO smoking regimen than under the HCI smoking regimen. In our results, the MN-inducing activities were closer in the DPM figures, by which we excluded the effects of water content, than in the TPM figures. However, the MN-inducing activities of WS and GVP under ISO and HCI smoking regimens in the DPM figures did not match completely. The lower abundance of chemicals that are considered to possess the MN-inducing activities may contribute to the lesser MN-inducing activities under the HCI smoking regimen.
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In conclusion, we describe a direct exposure system that can detect and evaluate the biological responses of fresh CS qualitatively as well as quantitatively in vitro. We established a method for quality-based evaluation that enables comparison of CS showing a different TPM value in a direct exposure assay. Using these techniques, we showed the possibility of comparing the MN-inducing activities under different smoking regimens using a direct exposure system.
Acknowledgements We are grateful to Dr. Hiroki Shikata and Dr. Kunio Iwata for their technical suggestions and for reviewing the manuscript.
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Experimental and Toxicologic Pathology 62 (2010) 433–440 www.elsevier.de/etp
In vitro micronucleus assay for cigarette smoke using a whole smoke exposure system: A comparison of smoking regimens Kosuke Okuwa, Masahiro Tanaka, Yasuo Fukano, Hidenori Nara, Yosuke Nishijima, Tomoki Nishino Tobacco Science Research Center, Japan Tobacco Inc., 6-2, Umegaoka, Aoba Ward, Yokohama, Kanagawa 227-8512, Japan Received 22 April 2009; accepted 3 June 2009
Abstract Previous studies on the biological assessment of cigarette smoke (CS) mainly focused on the total particulate matter (TPM) collected using a Cambridge filter or gas vapor phase (GVP) bubbled through phosphate-buffered saline (PBS). To study the effects of native CS in vitro, direct exposure methods have been developed. Meanwhile, in vitro micronucleus (MN) assays have been reported to evaluate the mutagenicity of CS. The objective of this research is to investigate the MN-inducing activity of whole smoke (WS) and GVP using a whole smoke exposure system, CULTEXs, which allows direct exposure of cultured cells to native CS at the air/liquid interface (ALI). CS was generated according to the International Organization for Standardization (ISO; 35 ml, 2 s, once per 60 s) or the Health Canada Intensive (HCI; 55 ml, 2 s, once per 30 s, with complete ventilation block) regimens and Chinese hamster lung (CHL/IU) cells were then exposed to this smoke. Dosages were adjusted according to the amount of smoke entering the actual exposure position. Under both smoking regimens, WS and GVP from 2R4F reference cigarettes induced MN responses. The concept of the dosage and similar dose–response relationships between theoretical and monitored dosage values under the two regimens enabled us to compare the MN-inducing activities of cigarettes in the direct exposure assay, even in the case of various experimental settings or different TPM amounts. MN-inducing activities of 2R4F under the ISO regimen seemed to be higher than those under HCI estimated by the TPM equivalent calculated values. r 2009 Elsevier GmbH. All rights reserved. Keywords: Cigarette smoke; Direct exposure; CULTEXs system; Micronucleus assay; Whole smoke; Gas vapor phase; Air/liquid interface; Dosage
Introduction Cigarette smoke (CS) is a complex and dynamic mixture of gaseous, volatile and particle compounds of more than 4000 chemicals (Baker, 1999). The biological Corresponding author. Tel.: +81 45 345 5245; fax: +81 45 973 6781. E-mail address: [email protected] (Y. Fukano).
0940-2993/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2009.06.002
evaluation of CS in vitro has focused on collected compounds such as total particulate matter (TPM) trapped using a Cambridge filter or gas vapor phase (GVP) bubbled through phosphate-buffered saline (PBS) (Mizusaki et al., 1977; Nakayama et al., 1985; Andreoli et al., 2003). These indirect methods allow simple and high-throughput biological assessment. However, extracted fractions of CS may not completely reflect the biological effects of native smoke. To evaluate
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the biological effects of CS that is closer to fresh smoke, direct exposure methods have been developed (Bombick et al., 1997; Fukano et al., 2004; Maunders et al., 2007; Aufderheide and Gressmann, 2008; Clunes et al., 2008). The in vitro micronucleus (MN) assay is a mutagenicity test that detects both aneugens and clastogens. Spindle apparatus defects or structural chromosomal damage converted to lagging of whole chromosome distribution or chromosomal fragments cause MN formation. The MN assay was performed by a simple and rapid method that showed highly concordant results with the chromosomal aberration test in vitro (Matsushima et al., 1999). Using the MN assay, several studies have reported the mutagenicity of CS fractions (Baker et al., 2004; Pre´fontaine et al., 2006). One study reported that direct exposure of whole smoke induced MN formation (Massey et al., 1998). However, there is no information regarding the MN-inducing activities under different smoking regimens using the direct exposure method. In this study, we compare MN-inducing activities under both International Organization for Standardization (ISO) and Health Canada Intensive (HCI) smoking regimens. To accomplish these objectives, we used an original dosage unit that allowed us to compare different smoking conditions or different design of cigarettes.
Cell line and culture conditions The Chinese hamster lung (CHL/IU) cells (Dainippon Pharmaceutical Co. Ltd., Tokyo, Japan) were maintained in Eagle’s minimum essential medium supplemented with 10% heat-inactivated (56 1C, for 30 min) bovine serum, 25 mM Hepes buffer and 1 mg/ml gentamycin. Cells were grown in a humidified atmosphere of 95% air and 5% CO2 at 37 1C. In preparation for direct exposure, CHL/IU cells were trypsinized and seeded onto the microporous membranes (pore size 0.4 mm, growth area 0.9 cm2) of cell culture inserts (Becton Dickinson, Franklin Lakes, NJ) at a density of 1 105 cells/insert and cultured for 24 h.
Equipment for whole smoke exposure system The CULTEXs system 2006 (SIBATA, Japan) was used for the experiments. This experimental system is composed of a smoking machine, a dilution system, and a glass exposure module (Fig. 1A). The CS was generated by a smoking machine, diluted with clean air in the dilution system and sucked via negative pressure into the exposure module (Fig. 1B), where CHL/IU cells, cultured on the microporous membranes at the air/liquid interface (ALI), were exposed to the smoke. The exposure module was maintained at 37 1C by the warm water circulating through the device.
Materials and methods Exposure settings Cigarettes Kentucky standard reference cigarettes 2R4F were used for the experiments. The cigarettes were conditioned at 22 1C and 60% relative humidity for at least 48 h before use. Under the HCI smoking regimen, the filter ventilation was blocked with cellophane tape.
Smoke generation was computer controlled using the ISO or HCI smoking regimens. Freshly generated smoke was pressed into a dilution system and diluted by a constant flow of clean air controlled by a mass-flow controller and valves. Diluted smoke was sucked into the exposure module at a velocity of 5 mL/min via
Fig. 1. The diagram shows (A) direct exposure system, (B) dilution system interior: 1, generated smoke; 2, dilution air; 3, flow rate and (C) calculating formula of percentage of CS.
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negative pressure from a vacuum pump. To generate the GVP, a Cambridge filter (44 mm diameter) was inserted into the smoke path between the cigarette port and the cylinder of the smoking machine. Under the ISO smoking regimen, 2, 4, 6 and 8 cigarettes were smoked by taking a puff of 35 ml volume over 2 s every 60 s. Smoke was released into the dilution system in 2.8 s puffs. In the dilution system, smoke was diluted by dilution air at a velocity of 4.5 L/min for whole smoke (WS) exposure and 2.25 L/min for GVP exposure. Under the HCI smoking regimen, 1, 2, 3 and 4 cigarettes with completely blocked ventilation were smoked by taking a 55 ml puff volume over 2 s every 30 s. Smoke was released into the dilution system in 2.8 s puffs. In the dilution system, smoke was diluted by dilution air at a velocity of 3.5 L/min for WS exposure and 1.25 L/min for GVP exposure. CHL/IU cells were cultivated and exposed at the ALI that means medium supply from the basal part of the membrane, whereas the apical part with the cells is in direct contact with the test atmosphere. As a negative control, cells were exposed to clean air at a velocity of 5 mL/min.
Dosage setting Dosages were adjusted to the amount of smoke entering the exposure module and expressed as a percentage of CS (% of cig.) according to the formula (Fig. 1C). The numerator in the calculating formula is the flow rate that represents the amount of smoke finally entering the exposure module. The denominator part of the formula, the sum of the velocities of generated smoke and dilution air, show the total amount of smoke that passed through the dilution system. The values of the % of cig. are expressed as a percentage and this indicates how much generated smoke entered the exposure module. The values of the % of cig. can be converted to TPM equivalent values by multiplying the % of cig. values by the TPM amount per cigarette. Dry particle matter (DPM) equivalent values can also be calculated by multiplying the % of cig. values by waterfree TPM amount per cigarette.
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variations from the photometer were translated into TPM values. Monitored TPM values were divided by the TPM amount per cigarette under each smoking regimen to calculate the ‘‘monitored’’ % of cig. values. The ‘‘theoretical’’ % of cig. values were calculated according to the formula.
Smoke analyses TPM was measured gravimetrically using a Cambridge filter. Nicotine and water were determined from isopropanol extracts of TPM using the HP-6890 gas chromatography system (Hewlett Packard, Palo Alto, CA) according to the relevant ISO standards (ISO Standard 10315, 2000; ISO Standard 10362-1, 1999).
MN assay The MN assay was carried out without cytochalasin B and without metabolic activation. CHL/IU cells cultured on microporous membranes were exposed to WS or GVP. After smoke exposure, cell culture inserts were filled with fresh culture medium without washing, followed by a 24 h recovery period. After the recovery period, cells were trypsinized and centrifuged. The cell pellet was subjected to hypotonic treatment with 75 mM KCl at 37 1C for 5 min and fixed with acetic acid– methanol (1:3). Fixed cells were suspended with 1% acetic acid in methanol and air dried. Preparations were stained with acridine orange. Two thousand cells per dosage were scored for MN cell counting using a fluorescent microscope.
Cytotoxicity assessment For cytotoxicity assessment, the trypsinized cell suspension after the recovery period was scored by Coulter Counter (Beckman Coulter Inc., Fullerton, CA). Counts of air-exposed samples were regarded as 100% and the percentage of cell counts in each dosage were calculated as a percentage of viable cells.
Results
Particle phase monitoring
Particle phase monitoring
The amount of smoke entering the exposure module was monitored under ISO and HCI smoking regimen conditions. Monitoring was carried out using a light scattering photometer (Vitrocell, Germany) in the smoke path extended into the exposure module. Prior to the monitoring, we calibrated the photometer by measuring the voltage variations and actual TPM values at the same time. Using this calibration curve, voltage
To use the % of cig. as a dosage unit, we had to confirm whether the ‘‘monitored’’ % of cig. is consistent with the ‘‘theoretical’’ one. Fig. 2 shows the plots of the actual value of ‘‘monitored’’ vs. ‘‘theoretical’’ % of cig. across various exposure conditions. The regression formula was y ¼ 1.24x and the correlation coefficient was 0.998 under the ISO smoking regimen. Under the HCI smoking regimen,
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the regression formula was y ¼ 1.09x and the correlation coefficient was 0.983. It was confirmed that the ‘‘monitored’’ and ‘‘theoretical’’ % of cig. were consistent and in good correlation under both ISO and HCI smoking regimens and the slopes of linear regression were similar under these two regimens.
Smoke analyses TPM, nicotine and water content under experimental conditions are summarized in Table 1. The HCI smoking regimen produced an increase in the TPM amount of about 2.8-fold that of ISO. The water content in TPM was about 11% under the ISO smoking regimen and about 30% under the HCI smoking regimen.
MN-inducing activity and cytotoxicity of 2R4F: ISO WS and GVP from 2R4F showed dose-related MN responses under the ISO smoking regimen (Fig. 3A). The highest average percentage of the MN frequency was 4.98% and the dosage was 0.57% of cig. in WS exposure. In GVP exposure, the highest average percentage of the MN frequency was 5.42% and the dosage was 1.00% of cig. With the increase in the % of cig. values, the cytotoxicity, the decrease of viable cell count, was also observed (Fig. 3B).
Fig. 2. The graph shows the relationship between ‘‘monitored’’ % of cig. values and ‘‘theoretical’’ % of cig. values under the ISO smoking regimen and HCI smoking regimen. Symbol: K ISO smoking regimen, J HCI smoking regimen.
Table 1. Yields of TPM, nicotine and water of 2R4F reference cigarette under experimental settings. Smoking regimen ISO HCI
TPM (mg/ cigarette)
Nicotine (mg/ cigarette)
Water content (mg/cigarette)
11.3 32.0
0.61 2.12
1.2 9.7
MN-inducing activity and cytotoxicity of 2R4F: HCI WS and GVP from 2R4F showed dose-related MN responses under the HCI smoking regimen (Fig. 4A). The highest average percentage of the MN frequency was 5.22% and the dosage was 0.32% of cig. in WS exposure. In GVP exposure, the highest average percentage of the MN frequency was 5.7% and the dosage was 0.62% of cig. With the increase in the % of cig. values, the cytotoxicity, the decrease of viable cell count, was also observed (Fig. 4B).
Comparison of ISO and HCI smoking regimens Under the ISO smoking regimen, the experimental setting that showed the highest MN frequency was 6
Fig. 3. The graphs show (A) MN-inducing activities and (B) cytotoxicity under the ISO smoking regimen. 2, 4, 6 and 8 cigarettes were smoked and diluted by dilution air at a velocity of 4.5 L/min for WS exposure and 2.25 L/min for GVP exposure. Diluted smoke was introduced into the exposure module at a velocity of 5 mL/min. The results represent means and S.D. (n ¼ 3 for each condition). Symbol: K WS exposure, GVP exposure.
Figures represent mean values (n ¼ 3) for each condition.
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Fig. 4. The graphs show (A) MN-inducing activities and (B) cytotoxicity under the HCI smoking regimen. 1, 2, 3 and 4 cigarettes were smoked and diluted by dilution air at a velocity of 3.5 L/min for WS exposure and 1.25 L/min for GVP exposure. The results represent means and S.D. (n ¼ 3 for each condition). Symbol: WS exposure, & GVP exposure.
Fig. 5. The graphs show (A) WS MN-inducing activities based on TPM equivalent values, (B) GVP MN-inducing activities based on TPM equivalent values, (C) WS MN-inducing activities based on DPM equivalent values and (D) GVP MN-inducing activities based on DPM equivalent values. The results represent means and S.D. (n ¼ 3 for each condition). Symbol: K WS exposure under ISO regimen, GVP exposure under ISO regimen, WS exposure under HCI regimen, & GVP exposure under HCI regimen.
cigarettes with 4.5 L/min dilution rate in WS exposure and 6 cigarettes with 2.25 L/min dilution rate in GVP exposure. Under the HCI smoking regimen, the experimental setting showed that the highest MN frequency was 3 cigarettes with 3.5 L/min dilution rate in WS exposure and 3 cigarettes with 1.25 L/min dilution rate in GVP exposure. To compare ISO with HCI smoking regimens, TPM equivalent values were applied because the total amount of smoke under the two smoking
regimens were different and it was impossible to use the % of cig. for a qualitative comparison. TPM equivalent values, composed of tar, nicotine and water, were calculated by multiplying the % of cig. values by TPM amount per cigarette. The TPM equivalent values of the ISO MN peaks were 64.4 mg in WS exposure and 113.0 mg in GVP exposure. The TPM equivalent values of the HCI MN peaks were 102.4 mg in WS exposure and 198.4 mg in GVP exposure. ISO MN peaks resulted in
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lower TPM equivalent values than those of HCI under both WS and GVP exposure (Fig. 5A, B). To exclude the effect of water content difference caused by different smoking regimens, DPM equivalent values, composed of tar and nicotine, were calculated by multiplying the % of cig. by water-free TPM amount per cigarette. The DPM equivalent values of the ISO MN peaks were 57.6 mg in WS exposure and 101.0 mg in GVP exposure. The DPM equivalent values of the HCI MN peaks were 71.4 mg in WS exposure and 138.3 mg in GVP exposure. In the DPM equivalent evaluation, MN-inducing activities under both smoking regimens were closer than in the case of the TPM equivalent evaluation (Fig. 5C, D).
Discussion Biological evaluation of CS in vitro mainly focuses on collected compounds such as TPM collected using a Cambridge filter or GVP bubbled through PBS. In these cases, separating the particle phase and GVP as fractions may fail to reflect the biological effects of native aerosol. Furthermore, chemicals that are impossible to collect by the above-mentioned methods may be present, or there may be changes in the chemical composition compared with fresh smoke. It is important to detect the effects of CS as integral to the particle phase and GVP without modification by the collecting method. Therefore we used a whole smoke exposure system, CULTEXs, which allows direct exposure of complex mixtures such as native CS at the ALI of cultured cells, supplying fresh culture medium through the membranes of cell culture inserts (Aufderheide and Mohr, 1999; Aufderheide, 2008). The CULTEXs system has been introduced into many laboratories and has revealed clues to some biological phenomena in vitro (Wolz et al., 2002; Fukano et al., 2006; Olivera et al., 2007; Diabate´ et al., 2008). Numerous studies have reported the biological effects of CS in vitro. Most of them were conducted with a standard smoke generation procedure such as ISO. Meanwhile, some authorities originally recommended more intensive smoking regimens than the standard procedures because there are many reports that some smokers appear to be taking larger puffs and/or increased smoking frequency and/or a higher ratio of vent blocking in human smoking behavior (Borgerding and Klus, 2005). There are some reports in which those regimens have been applied to toxicity evaluation of CS (Foy et al., 2004). However, there is no information regarding the in vitro MN assay, a mutagenicity assay, under different smoking regimens using a direct exposure system. For this reason, we attempted to compare MN-inducing activities under both ISO and HCI smoking regimens using a direct exposure system.
WS and GVP under both smoking regimens showed dose-related MN responses. In our results, the dosages that indicated the highest MN frequency in WS exposure were about one half those of GVP under both ISO and HCI smoking regimens. These results suggest that the contribution of GVP in MN-inducing activities of 2R4F WS under both smoking regimens seems to be approximately half that estimated by the % of cig. at the highest MN frequency. The evaluation methods for CS are quantity or quality based. In indirect methods, both have been applied to the biological evaluation of CS (Tewes et al., 2003). In contrast, only quantity-based evaluation has been applied in direct exposure so far in which the dosage setting was mainly determined by the parameters such as the dilution rate and the number of cigarettes (Bombick et al., 1998; Massey et al., 1998). This methodology creates some difficulties in comparing cigarettes qualitatively because the appropriate dose ranges for detecting the better biological responses often differ among cigarette designs or smoking regimens. To adapt the direct exposure system to not only quantitybased evaluation but also quality-based evaluation of CS, we proposed the % of cig., based on how much generated smoke is introduced into the exposure module. This allows us to convert the values to TPM equivalent easily and flexibly by multiplying the TPM amount per cigarette to conduct the quality-based evaluation. It was confirmed that the % of cig. values are linearly correlated with the amount of smoke entering the exposure module under ISO and HCI smoking regimens and the slopes of regression curves were similar under both smoking regimens. The results, similar dose–response relationships between ‘‘theoretical’’ and ‘‘monitored’’ % of cig. values confirmed under both smoking regimens, respectively, gave us the verification for the conversion of the dosages to the TPM equivalent values, which enabled us to conduct quality-based evaluation in the direct exposure MN assay. Adjusting dosage is often a critical point in toxicological studies and we offer a new way to solve this problem for cigarette smoke in the direct exposure assay. Our results show that the MN-inducing activities of 2R4F under the HCI smoking regimen seemed to be lower than those under the ISO regimen estimated by the TPM equivalent values calculated from the % of cig. In the experiments with collected CS condensate, similar phenomena were reported. A more intensive regimen gave higher TPM yields on a per cigarette basis, showing less cytotoxicity and mutagenicity of the TPM per unit basis (Roemer et al., 2004) and TPM under the more intensive smoking regimen showed relatively less potency in MN-inducing activities (DeMarini et al., 2008). TPM collected under the HCI smoking regimen contains more water than that collected under the ISO
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smoking regimen. Increased water content under the HCI smoking regimen could be a key factor in the differences in MN activities measured by TPM equivalent figures. We adjusted the difference in water content and reevaluated the MN-inducing activities based on DPM base equivalent. Nevertheless, MN-inducing activities under the HCI smoking regimen still appeared to be less than under the ISO regimen in DPM figures, suggesting the change of chemical composition between ISO and HCI. To which extent the chemical composition alters and affects biological responses under different smoking regimens is interesting to note. The abundance of chemical compounds in CS may differ due to larger puff volume or greater ventilation blocking causing increased air volume to pass through a tobacco column and higher coal temperature may affect the combustion or pyrolysis at the burning cone under an intensive regimen. One recent study suggested that the chemical changes in the CS compounds caused by different smoking regimens might affect the cytotoxicity (Rickert et al., 2007). Mainstream smoke component variations caused by changing smoking regimens and cigarette designs have been described previously, indicating that alterations in chemical compositions were not uniform (Counts et al., 2005). In this report, we used 2R4F reference cigarettes that correspond to the low-ventilation group (filter ventilation o30%, Chen and Moldoveanu, 2003) according to the classification of Counts et al. (2005). Although the chemical changes in the low-ventilation group were relatively less than those in the highventilation groups, Counts et al. (2005) reported that constituent/tar yield ratios were relatively greater under the HCI smoking regimen than under the ISO smoking regimen for pyridine and, to a lesser extent, formaldehyde in particulate-phase chemicals. In vapor-phase chemicals, constituent/tar yield ratios were relatively greater under the HCI smoking regimen than under the ISO smoking regimen for hydrogen cyanide and styrene and, to a lesser extent, acrolein and methyl ethyl ketone. In contrast, other chemicals that include those known to possess the MN-inducing activities such as hydroquinone, catechol, phenol and acetaldehyde (Bird et al., 1982; Yager et al., 1990) showed a quite opposite reaction, with greater constituent/tar yield ratios under the ISO smoking regimen than under the HCI smoking regimen. In our results, the MN-inducing activities were closer in the DPM figures, by which we excluded the effects of water content, than in the TPM figures. However, the MN-inducing activities of WS and GVP under ISO and HCI smoking regimens in the DPM figures did not match completely. The lower abundance of chemicals that are considered to possess the MN-inducing activities may contribute to the lesser MN-inducing activities under the HCI smoking regimen.
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In conclusion, we describe a direct exposure system that can detect and evaluate the biological responses of fresh CS qualitatively as well as quantitatively in vitro. We established a method for quality-based evaluation that enables comparison of CS showing a different TPM value in a direct exposure assay. Using these techniques, we showed the possibility of comparing the MN-inducing activities under different smoking regimens using a direct exposure system.
Acknowledgements We are grateful to Dr. Hiroki Shikata and Dr. Kunio Iwata for their technical suggestions and for reviewing the manuscript.
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