Impact of a Korean Behavior-Based Smoking Regimen on Cigarette Smoke Constituents and Cellular Changes

Abstract

Objectives

This study aims to compare a Korean Behavior-Based Smoking Regimen (KO-BSR), which reflects the actual smoking behavior of Korean smokers, with international standard smoking regimens of the International Organization for Standardization (ISO) and Health Canada Intense (HCI), and to evaluate differences in selected cigarette smoke constituents and cellular toxicity.

Methods

Whole cigarette smoke condensate (WCSC) was generated using a rotary smoking machine under ISO, HCI, and KO-BSR conditions. For each smoking regimen, total particulate matter (TPM) per cigarette was quantified, and four major smoke constituents, namely nicotine, acetone, triacetin, and glycerin, were measured by gas chromatography-mass spectrometry. Korean-derived oral squamous cell carcinoma cell lines YD-9 and YD-38 were exposed to WCSC at various concentrations for 5 days, and cell viability was assessed using a water-soluble tetrazolium salt assay. In addition, the concentration at which cell viability decreased by 20% (IC₂₀) was calculated.

Results

Compared with ISO, KO-BSR generated significantly higher levels of TPM and concentrations of nicotine, acetone, triacetin, and glycerin in the final WCSC stock (all p<0.05). Compared with HCI, KO-BSR generated significantly higher TPM levels and concentrations of acetone, triacetin, and glycerin, whereas nicotine concentration was numerically higher but did not differ significantly. In both cell lines, WCSC exposure reduced cell viability in a concentration-dependent manner, with the greatest reduction observed following exposure to WCSC generated under KO-BSR conditions. The IC₂₀ value was lowest under the KO-BSR condition in YD-9 cells, whereas in YD-38 cells, IC₂₀ values could only be calculated under the HCI and KO-BSR conditions.

Conclusions

KO-BSR generated higher levels of measured smoke constituents and induced greater cellular toxicity than the international standard smoking regimens. These findings support the potential use of KO-BSR as a Korean smoking behavior-based exposure scenario that may complement international standard smoking regimens. Further validation using representative distributions of smoking behavior, human biomarkers of exposure, and multiple toxicity endpoints is needed before KO-BSR can be incorporated into risk assessment frameworks.

Key messages

① What is known previously?

Toxicity assessments of cigarette smoke have generally relied on international standard smoking regimens, such as International Organization for Standardization (ISO) and Health Canada Intense (HCI). However, these regimens may not fully capture the smoking behavior observed among Korean smokers, and toxicity studies explicitly incorporating Korean Behavior-Based Smoking Regimen (KO-BSR) have been limited.

② What new information is presented?

Smoke generated under KO-BSR contained higher levels of total particulate matter (TPM) and the four measured constituents (nicotine, acetone, triacetin, and glycerin) than smoke generated under ISO. KO-BSR-based whole cigarette smoke condensate (WCSC) also contained higher levels of TPM, acetone, triacetin, and glycerin than HCI, whereas the difference in nicotine concentration between KO-BSR and HCI was not statistically significant. KO-BSR-based WCSC induced a more pronounced concentration-dependent reduction in cell viability in Korean-derived oral squamous cell carcinoma cell lines.

③ What are implications?

KO-BSR may produce higher levels of selected cigarette smoke constituents and greater cellular toxicity than international standard smoking regimens. These findings support the potential use of KO-BSR as a Korean smoking behavior-based exposure scenario that complements international standard regimens; however, further validation using human biomarkers and multiple toxicity endpoints is needed before its application in risk assessment frameworks.

Introduction

Although the negative effects of smoking are already widely known, people continue smoking. In addition, various novel tobacco products have emerged alongside conventional cigarettes. Recently, various types of tobacco products, such as liquid-type electronic cigarettes, heated tobacco products, and flavored tobacco products, have been introduced, but the use rate of conventional cigarettes remains the highest, and related health risk research continues to be conducted [1]. Existing smoking-related human risk assessments have been conducted based on international standard smoking regimens, such as International Organization for Standardization (ISO) and Health Canada Intense (HCI). However, these regimens do not sufficiently reflect actual smokers’ compensatory smoking or differences in smoking habits across cultures and populations [2].

According to a study by the Korea Disease Control and Prevention Agency, the puff volume per cigarette among Korean smokers is higher than the values specified under the ISO and HCI regimens, making them more likely to be exposed to relatively greater amounts of cigarette smoke constituents. In addition, the concentrations of urinary smoking biomarkers—cotinine¹⁾, trans-3’-hydroxycotinine²⁾, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol³⁾—have been reported to increase significantly with increases in daily cigarette consumption and puff volume per cigarette (ml) [3]. These findings suggest that the amounts of major harmful substances in the smokers’ bodies, including nicotine, total particulate matter (TPM), nicotine-free dry particulate matter (tar), tobacco-specific nitrosamines, and polycyclic aromatic hydrocarbons, may increase depending on actual smoking behavior. Therefore, risk assessment based solely on international standard smoking regimens may not adequately reflect the real-world smoking environment, potentially leading to an underestimation of the associated risks.

To compare smoking-related risks at the cellular level, an appropriate cell model and cigarette smoke exposure method are required. Smoking-related risks are particularly pronounced in the mucosal tissues of the head and neck region of a smoker, and the oral cavity, pharynx, and larynx are known to be the sites most directly exposed to mainstream smoke. Indeed, smoking has been reported to be a major risk factor for head and neck cancer [4]. Considering the site specificity, head- and neck-derived cells are suitable cell models for the comparative evaluation of smoking-related risks.

For cigarette smoke exposure, a cell culture method using a cell culture medium containing diluted cigarette smoke extract was adopted in the present study. The whole cigarette smoke condensate (WCSC) used in this study contained both cigarette smoke condensate (CSC), an extract of particulate matter in cigarette smoke, and cigarette smoke extract (CSE), an extract of gas-phase matter. WCSC confers the advantage of enabling simultaneous exposure to major harmful substances found in cigarette smoke. WCSC is suitable for repeated experiments and comparative studies across conditions because of its consistent composition and ease of concentration control. It has also been proposed as a method that can address the limitations of conventional methods involving separate exposure to CSC and CSE [5].

To date, no study has evaluated cell-based risk using WCSC generated under the Korean Behavior-Based Smoking Regimen (KO-BSR). In particular, evidence regarding the effects of the KO-BSR, which has relatively more intensive characteristics than international standard regimens, on the amounts of harmful substances generated in mainstream smoke and on cellular-level toxicity is limited. Therefore, this study aimed to apply the KO-BSR, which experimentally reproduces the actual smoking behavior of Korean smokers, including puff volume and puff interval, to WCSC preparation and to evaluate the relative risk at the cellular level in comparison with the ISO and HCI regimens.

This study aimed to compare and evaluate the differences in relative risk among smoking regimens and establish a foundation for developing a Korean-specific smoking risk assessment model. In addition, rather than aiming to estimate the absolute amount of actual human exposure, this study used cell-based models to provide toxicological information for comparing and screening relative differences in the composition of mainstream smoke extracts generated under different smoking regimens.

Methods

1. WCSC Extraction Conditions and Methods

The Tobacco Harm Analysis Laboratory is equipped with a rotary smoking machine (RM20H) manufactured by Borgwaldt (currently Koerber Technologies Instruments GmbH), and the equipment specifications and cigarette smoke generation principles are based on ISO 3308:2012 (Routine analytical cigarette-smoking machine—Definitions and standard conditions). To extract WCSC, the RM20H was operated under three smoking regimens: ISO, HCI, and KO-BSR. The KO-BSR used in this study was established to reflect the average smoking behavior of Korean smokers [3]. Specifically, Korean smokers have been reported to have a greater puff volume and a shorter puff interval than those specified under the ISO and HCI regimens and to tend to block the filter ventilation holes by placing the cigarette filter deep in the mouth (ventilation blocking). Accordingly, the puff volume, puff duration, puff interval, and ventilation blocking rate were set to the values presented in previous study (Table 1) [3], and these parameters were defined as KO-BSR.

Table 1. Smoking condition parameters applied to the smoking machine.

Smoking regimens	ISO	HCI	KO-BSR
Puff volume (ml)	35	55	73
Puff interval (s)	60	30	9
Puff duration (s)	2	2	1.6
Ventilation blocking (%)	0	100	100

Puff volume: The volume of mainstream smoke generated by each puff. Puff interval: The time between successive puffs. Puff duration: The duration of each puff. Ventilation blocking: The extent to which the cigarette filter ventilation holes are blocked or occluded. ISO=International Organization for Standardization; HCI=Health Canada Intense; KO-BSR=Korean Behavior-Based Smoking Regimen.

To verify the suitability of the cigarette smoke extraction process of the rotary smoking machine, cigarette smoke was generated in accordance with ISO 3308, and the measured values were compared with the certified values (University of Kentucky) for the 1R6F certified reference cigarette under the ISO smoking regimen (Table 2) [6]. The mean puff count measured in this study was 7.76±0.31 puffs (n=7), and the TPM yield was 9.87±0.58 mg/cigarette (n=7). These values fell within the valid comparison ranges for the certified reference cigarette, and no significant differences were found. Therefore, the rotary smoking machine used in this study was considered to have sufficient reproducibility and reliability.

Table 2. Verification of smoking machine performance reproducibility based on University of Kentucky reference values [6].

	Certified value (University of Kentucky)	This study
Puff count (puff/cigarette)	7.53±0.56 (k=2.55)	7.76±0.31 (n=7)
TPM (mg/cigarette)	9.99±0.78 (k=2.04)	9.87±0.58 (n=7)

Puff count (puff/cigarette): The number of puffs taken to burn a cigarette to the predetermined endpoint. Total particulate matter (TPM, mg/cigarette): The total mass of particulate matter collected from a single cigarette. Certified value: Reference value for the 1R6F certified reference cigarette provided in the Certificate of Analysis issued by the University of Kentucky Center for Tobacco Reference Products. k (coverage factor): A numerical factor used to multiply the combined standard uncertainty to obtain the expanded uncertainty. Under an approximately normal distribution, k≈2 corresponds to an approximate 95% coverage interval.

During a single generation of cigarette smoke, CSC and CSE were simultaneously collected to prepare WCSC. For each smoking regimen, five 1R6F reference cigarettes per run were combusted using the smoking machine to generate mainstream smoke, and the generated particulate matter was collected on a 44-mm Cambridge filter pad (CFP; Borgwaldt). The CFP through which mainstream smoke had passed was completely immersed in 5 ml of methanol and then gently agitated at 150 rpm for 30 minutes to dissolve the particulate matter in methanol. Thereafter, the methanol solution was concentrated by slowly evaporating it with high-pressure nitrogen gas for approximately 2 hours, and CSC was prepared by adding 320 µl of dimethyl sulfoxide (DMSO) and redissolving the concentrate.

CSE collection was performed simultaneously with CSC preparation. The system was designed so that cigarette smoke passed through 10 ml of Dulbecco’s phosphate-buffered saline (DPBS) via a specially fabricated impinger to collect gas-phase substances, which were then used as CSE. The prepared CSC and CSE were aliquoted into amber vials and stored frozen at −20℃ (Figure 1).

Figure 1. Schematic diagram of the preparation of whole cigarette smoke condensate (WCSC).

DPBS=Dulbecco’s phosphate buffered saline; CSE=cigarette smoke extract; TPM=total particulate matter; DMSO=dimethyl sulfoxide; CSC=cigarette smoke condensate.

Immediately before cell exposure, CSC and CSE were completely thawed at room temperature, after which 20 μl of CSC and 560 μl of CSE were mixed to prepare the final WCSC stock solution (580 µl). In each batch wherein mainstream smoke was collected, the mean volumes of CSC and CSE recovered after the combustion of five cigarettes were approximately 340 μl and 9,500 μl, respectively. The mixing ratio of WCSC for cell exposure was established to reflect this relative recovery ratio. Based on the volume required on the day of the experiment, treatment solutions for cell exposure were prepared by directly adding the WCSC stock solution to 5% fetal bovine serum (FBS)-Dulbecco’s modified Eagle medium (DMEM) to achieve final WCSC concentrations of 0.0625%, 0.125%, 0.25%, or 0.5% (v/v). For example, the 0.5% WCSC dilution was prepared by adding 25 μl of the WCSC stock solution to 5 ml of 5% FBS-DMEM. Intermediate dilutions were not used, and the treatment solutions were freshly prepared at each medium change. This method was based on a previous study [5], with some modifications made in consideration of the reproducibility and ease of handling of the cell exposure experiments.

2. Analysis of Four Volatile Organic Compounds in WCSC under Different Smoking Regimens Using GC-MS

To identify the major constituents in WCSC under each smoking regimen, four constituents were analyzed: nicotine, triacetin, and glycerol, which are semi-volatile organic compounds, and acetone, which is a volatile organic compound. After five cigarettes were combusted using a single CFP, WCSC was prepared by mixing approximately 340 μl of the recovered CSC with approximately 9,500 μl of the recovered CSE. WCSC was then diluted 20-fold with methanol hypergrade for liquid chromatography-mass spectrometry (Supelco) and filtered through a 0.45 μm syringe filter to prepare the sample. For quantitative analysis, standards of acetone (acetone for analysis, Supelco, 1.00014), glycerol (glycerol≥99.5%, Sigma-Aldrich, G9012), nicotine ((–)-Nicotine≥99% (GC), Sigma-Aldrich, N3876), and triacetin (triacetin 99%, Sigma-Aldrich, 525073) were used. Calibration curves were constructed using each standard at five concentrations.

Gas chromatography-mass spectrometry (GC-MS, Agilent 7890B GC/5977A MSD) was used to analyze the major constituents. A DB-WAX column (60 m×0.25 mm×0.25 µm) was used, with helium as the carrier gas. The flow rate was set to 2 ml/min, the inlet temperature to 230℃, and the sample injection volume to 1 µl. The oven temperature was maintained at 40℃ for 5 minutes, increased to 230℃ at a rate of 5℃/min, and then maintained at 230℃ for 10 minutes. The split ratio was set to 10:1, the split flow rate to 20 ml/min, and the detector temperature was maintained at 230℃. Quantitative analysis was performed in selected ion monitoring mode, with m/z 43, 61, 84, and 103 for acetone, glycerol, nicotine, and triacetin, respectively.

3. Culture of Korean-Derived Oral Cancer Cell Lines

In this study, the Korean-derived oral cancer cell lines YD-9 and YD-38, obtained from the Korea Cell Line Bank, were used. YD-9, derived from a squamous cell carcinoma of the buccal mucosa in a 56-year-old male, consists of slightly elongated, adherent cells, whereas YD-38, originating from a squamous cell carcinoma of the lower gingiva in a 67-year-old male, consists of polygonal, adherent cells [7].

The cell culture medium was prepared by mixing DMEM (Welgene) containing 4,500 mg/L glucose with 2% antibiotic-antimycotic solution (Welgene), 1% L-glutamine (Welgene), and 1% HEPES buffer (Welgene). The medium used for cell culture was supplemented with 10% FBS (R&D Systems), whereas the medium used for cell viability assessment was supplemented with 5% FBS. Both cell lines were cultured in an incubator at 37℃ with 5% CO₂ and subcultured once weekly, and the cell culture medium was replaced every 2–3 days.

4. Method for WCSC Exposure by Smoking Regimen and Cell Viability Assessment

Cell viability was assessed using a water-soluble tetrazolium salt assay (WST assay) (EZ-Cytox; DoGENBio), and the entire procedure from cell seeding to absorbance measurement took 7 days. On Day 1, YD-9 and YD-38 cells were seeded in 96-well plates at densities of 2×10³ cells/well and 1×10³ cells/well, respectively, each with 100 µl of culture medium. On Day 3 (approximately 48 hours later), WCSC prepared under each of the three smoking regimens was diluted to different concentrations in 5% FBS-DMEM. All existing medium was then removed from the 96-well plates and replaced with medium containing WCSC at a final concentration of 0.5% or lower. On Day 5 (approximately 72 hours later), the medium was replaced in the same manner. On Day 7 (approximately 48 hours later), all medium was removed and replaced with cell culture medium containing 10% WST assay solution. After incubation for approximately 1 hour in an incubator at 37℃ with 5% CO₂, absorbance was measured at 450 nm using a SpectraMax i3x microplate reader (Molecular Devices) to assess cell viability.

To minimize the edge effect that may occur due to differences in cell growth rates between the edges and center of the plate during long-term culture, only 60 wells, excluding the edge wells, were used in each 96-well plate. Because the WCSC treatment groups were prepared using the WCSC stock solution directly diluted in the medium, the final concentrations of DMSO and DPBS were proportional to the WCSC treatment concentration. The WCSC stock solution was prepared by mixing 20 μl of CSC with 560 μl of CSE. In the 0.5% WCSC treatment group, the final concentrations of DMSO and DPBS were approximately 0.017% and 0.483%, respectively. For the control group, a control stock solution was prepared by mixing 20 μl of DMSO with 560 μl of DPBS to match the vehicle conditions of the group treated with the highest WCSC concentration. The control stock solution was then diluted in the medium and used under the same conditions as those applied to the group treated with the highest WCSC concentration. Accordingly, the control group contained 0.02% DMSO and 0.5% DPBS.

5. Statistical Analysis

All experiments were performed at least three times independently, and the results were expressed as mean±standard deviation. Statistical analyses were performed using GraphPad Prism 5 (version 5.01; GraphPad). The results of the WCSC constituent analysis were analyzed using one-way analysis of variance (ANOVA) to compare differences among the groups for a single factor, followed by Tukey’s multiple comparisons test as the post hoc test.

Cell viability data were analyzed using two-way ANOVA to simultaneously account for smoking regimen and WCSC concentration. The Bonferroni post-test was used to compare the differences in mean values across different conditions. One-way ANOVA was used to analyze the differences in cell viability across different WCSC concentrations within the same smoking regimen, and Dunnett’s multiple comparisons test was used to evaluate the effect of each concentration relative to the control group. The statistical significance level for all analyses was set at p<0.05.

Additionally, to quantitatively compare cytotoxicity among smoking regimens, the concentration resulting in a 20% reduction in cell viability (IC20) was also calculated. IC20 values were estimated through nonlinear regression analysis using a four-parameter logistic model fitted to cell viability data across the different WCSC concentrations. Under some conditions, the reduction in cell viability was limited, resulting in low reliability of the regression curve. In such cases, the IC20 value was estimated through linear interpolation using cell viability values from adjacent concentration intervals.

Results

1. WCSC Analysis Results by Extraction Criteria

The puff counts under the ISO, HCI, and KO-BSR regimens were 7.77±0.33, 9.25±0.48, and 8.65±0.47 puffs (n=5 each), respectively. Puff counts under the HCI and KO-BSR regimens were significantly higher than under the ISO regimen (p<0.05). TPM also differed significantly among the smoking regimens (p<0.05). It was the lowest under the ISO regimen at 9.40±0.71 mg/cigarette, whereas it was 31.19±3.46 and 41.43±2.55 mg/cigarette under the HCI and KO-BSR regimens. In particular, TPM under the KO-BSR regimen was significantly higher than those under both the ISO and HCI regimens (one-way ANOVA, p<0.05).

The concentrations of the major chemical constituents (nicotine, acetone, triacetin, and glycerin) in WCSC obtained under the ISO, HCI, and KO-BSR regimens are presented in Table 3. The nicotine concentration was 252.83±39.54, 629.37±115.69, and 782.46±120.48 μg/ml under the ISO, HCI, and KO-BSR regimens, respectively, with the latter two being significantly higher than that under the ISO regimen (p<0.05). The nicotine concentration under the KO-BSR regimen was higher than that under the HCI regimen, but the difference was not statistically significant. The concentrations of acetone, triacetin, and glycerin under the HCI and KO-BSR regimens were significantly higher than those under the ISO regimen, and the concentrations under the KO-BSR regimen were also significantly higher than those under the HCI regimen (all p<0.05). TPM under the KO-BSR regimen was significantly higher than that under the ISO and HCI regimens (p<0.05).

Table 3. Comparison of puff count, total particulate matter, and major constituent concentration in final whole cigarette smoke condensate.

Parameter	ISO	HCI	KO-BSR
Puff count (puff/cigarette)	7.77±0.33	9.25±0.48a)	8.65±0.47a)
TPM (mg/cigarette)	9.40±0.71	31.19±3.46a)	41.43±2.55a,b)
Nicotine (μg/ml)	252.83±39.54	629.37±115.69a)	782.46±120.48a)
Acetone (μg/ml)	29.31±4.68	127.83±11.74a)	143.91±3.57a,b)
Triacetin (μg/ml)	56.82±18.78	357.68±49.20a)	497.87±55.18a,b)
Glycerin (μg/ml)	220.80±11.47	577.89±44.18a)	668.31±69.94a,b)

Values are presented as mean±standard deviation. Chemical constituents are reported as concentrations in the final whole cigarette smoke condensate stock (μg/ml). Puff count (puff/cigarette): The number of puffs taken to burn a cigarette to the predetermined endpoint. Total particulate matter (TPM, mg/cigarette): The total mass of particulate matter extracted from a single cigarette. TPM is reported as mg/cigarette. ISO=International Organization for Standardization; HCI=Health Canada Intense; KO-BSR=Korean Behavior-Based Smoking Regimen. ^a)Significantly different from the ISO smoking regimen (p<0.05). ^b)Significantly different from the HCI smoking regimen (p<0.05).

These results show that the concentrations of major constituents generated in cigarette smoke differed markedly across smoking regimens. In particular, the amounts of major constituents generated under the KO-BSR regimen showed an overall increasing trend.

2. Changes in Cell Viability Following WCSC Exposure by Smoking Regimen

Following the exposure of each of the Korean-derived oral cancer cell lines YD-9 and YD-38 to WCSC prepared under three smoking regimens (ISO, HCI, and KO-BSR) for 5 days, both the smoking regimen and WCSC exposure concentration were identified as factors that significantly affected cell viability (two-way ANOVA, p<0.05). Changes according to WCSC concentration under each smoking regimen were concentration-dependent in both cell lines.

When YD-9 cells were exposed to WCSC prepared under the three smoking regimens, a significant decrease was observed in the 0.25% exposure group. Among the 0.5% exposure groups, the measured value was lower under the HCI regimen (approximately 40%) than under the ISO regimen (approximately 50%) and was the lowest under the KO-BSR regimen (approximately 20%) (Figure 2). Nonlinear regression analysis using a four-parameter logistic model estimated the IC20 values for YD-9 cells to be approximately 0.24%, 0.19%, and 0.15% under the ISO, HCI, and KO-BSR regimens, respectively. A characteristic finding in YD-9 cells was that exposure under the KO-BSR regimen resulted in a clear decrease in cell viability beginning at the intermediate concentration (0.25%), and changes in cell viability were observed even in a relatively low concentration range compared with the ISO and HCI regimens at the same concentration.

Figure 2. Changes in the viability of YD-9 human oral squamous cell carcinoma cells following exposure to WCSC generated under different smoking regimes.

WCSC was applied at final concentrations of 0.0625%, 0.125%, 0.25%, and 0.5% (v/v). The final concentrations of DMSO and DPBS in the WCSC-treated groups were proportional to the WCSC concentrations. The control group contained approximately 0.02% DMSO and 0.5% DPBS, corresponding to the highest WCSC treatment concentration (0.5%). Asterisks indicate p<0.05 versus the control within the same smoking regimen. ISO=International Organization for Standardization; HCI=Health Canada Intense; KO-BSR=Korean Behavior-Based Smoking Regimen; WCSC=whole cigarette smoke condensate; DMSO=dimethyl sulfoxide; DPBS= Dulbecco’s phosphate buffered saline.

In YD-38 cells, no significant changes were observed at most concentrations under the ISO smoking regimen. In YD-38 cells, the HCI 0.125% treatment group and the KO-BSR 0.0625% and 0.125% treatment groups showed significant increases in cell viability compared with the control group (p<0.05). However, these findings could not be conclusively regarded as a cell viability-promoting effect and were interpreted in this study as a non-monotonic concentration-response pattern. At the high concentration (0.5%), cell viability was significantly lower under the HCI and KO-BSR regimens than under the ISO regimen. Nonlinear regression analysis estimated the IC20 values for YD-38 cells to be approximately 0.35% and 0.33% under the HCI and KO-BSR regimens, respectively. Under the ISO regimen, no significant cellular changes were observed even in the high-concentration 0.5% exposure group; therefore, the IC20 value could not be calculated. YD-38 cells exhibited a threshold-like response pattern, with no changes in the low-concentration range followed by a rapid decrease in cell viability at the high concentration (0.5%). Nevertheless, the magnitude of the decrease in cell viability in the high-concentration range was the greatest under the KO-BSR regimen, showing a trend similar to the results obtained for YD-9 (Figure 3).

Figure 3. Changes in the viability of YD-38 human oral squamous cell carcinoma cells following exposure to WCSC generated under different smoking regimens.

WCSC was applied at final concentrations of 0.0625%, 0.125%, 0.25%, and 0.5% (v/v). The final concentrations of DMSO and DPBS in the WCSC-treated groups were proportional to the WCSC concentrations. The control group contained approximately 0.02% DMSO and 0.5% DPBS, corresponding to the highest WCSC treatment concentration (0.5%). Asterisks indicate p<0.05 versus the control within the same smoking regimen. ISO=International Organization for Standardization; HCI=Health Canada Intense; KO-BSR=Korean Behavior-Based Smoking Regimen; WCSC=whole cigarette smoke condensate; DMSO=dimethyl sulfoxide; DPBS= Dulbecco’s phosphate buffered saline.

In summary, comparison of cell viability and IC20 values in YD-9 and YD-38 cells demonstrated that cell viability was markedly more inhibited under the KO-BSR regimen than under the ISO and HCI regimens.

Conclusion

In this study, we established a smoking regimen reflecting the actual smoking behavior of Korean smokers and prepared WCSC based on this regimen to evaluate its chemical constituents and cellular-level risk. Comparison with the ISO and HCI regimens confirmed that the amounts of TPM and major harmful constituents (nicotine, acetone, triacetin, and glycerin) generated per cigarette increased significantly under the KO-BSR regimen. TPM is an indicator of the total amount of particulate matter generated in mainstream smoke and is directly affected by smoking regimen parameters, including puff volume, puff interval, puff duration, and ventilation blocking. Our results showed that the short puff intervals and high puff volumes of Korean smokers constitute a smoking regimen that generates more harmful substances than international standard regimens. However, these increases refer to increases in the amounts of harmful substances generated in mainstream smoke under experimental conditions. Actual human exposure levels may also vary depending on individual smoking behavior, including inhalation depth, smoking interval, and compensatory smoking. Nevertheless, the finding that the amounts of TPM and major harmful constituents increased under an intensive smoking regimen suggests that the potential exposure of Korean smokers to harmful substances may increase under this regimen.

When the Korean-derived oral cancer cell lines YD-9 and YD-38 were exposed to WCSC prepared under each smoking regimen, both the smoking regimen and WCSC concentration significantly affected cell viability in both cell lines. In particular, WCSC prepared under the KO-BSR regimen reduced cell viability to a greater extent than WCSC prepared under the ISO and HCI regimens, and both cell lines showed concentration-dependent decreases in viability. The decrease in cell viability can be interpreted as the result of cellular damage and physiological stress responses caused by exposure to harmful substances contained in CSE. Accordingly, the decrease in cell viability can reasonably be understood as a biological indicator of cellular-level susceptibility to harmful substances. Cancer cell lines, such as the head and neck cancer cell lines used in this study, are sensitive test models suitable for quantitatively evaluating concentration-response relationships at various concentrations and under various conditions because of their high responsiveness to external stimuli, rapid viability rates, and ease of culture. Many studies have used these cell lines as cellular models for comparing and screening relative differences in toxicity among chemicals [8].

The findings of this study are also significant because they address the limitations of previous studies that investigated CSC and CSE separately and, therefore, did not sufficiently reflect the characteristics of actual cigarette smoke exposure. Because the WCSC used in this study contains both TPM and gas-phase substances, it can more comprehensively simulate the characteristics of mainstream smoke exposure in actual smoking environments and can be regarded as an approach that addresses the limitations of conventional separate extraction methods [9]. In addition, although smoking risk studies have so far been conducted primarily based on the ISO smoking regimen, the recent increase in studies applying the intensive regimen proposed by HCI has highlighted the need for assessment methods that reflect more realistic smoking regimens [10]. In addition to this international research trend, this study is important because it scientifically proposed a regimen reflecting the unique smoking characteristics of Koreans.

Rather than proposing KO-BSR as an independent Korean-specific risk assessment standard, this study presents its potential use as an exposure scenario based on Korean smoking behavior that complements international standard smoking regimens. The same WCSC volumetric concentration in this study does not represent the same TPM equivalent or the same cigarette equivalent across smoking regimens and should be interpreted as a concentration-response range for comparing the relative cellular responses to WCSC generated under different smoking regimens. Future studies should verify the validity and scope of the application of KO-BSR by integrating representative smoking behavior distributions, a broader range of major harmful constituents, human biomarkers, and various toxicity endpoints, including inflammation, oxidative stress, and genotoxicity.