Comparative evaluation of the mutagenicity and genotoxicity of smoke condensate derived from Korean cigarettes

Abstract

Objectives: Cigarette smoking is associated with carcinogenesis owing to the mutagenic and genotoxic effects of cigarette smoke. The aim of this study was to evaluate the mutagenic and genotoxic effects of Korean cigarettes using in vitro assays.

Methods: We selected 2 types of cigarettes (TL and TW) as benchmark Korean cigarettes for this study, because they represent the greatest level of nicotine and tar contents among Korean cigarettes. Mutagenic potency was expressed as the number of revertants per μg of cigarette smoke condensate (CSC) total particulate matter whereas genotoxic potency was expressed as a concentration-dependent induction factor. The CSC was prepared by the International Organization for Standardization 3308 smoking method. CHO-K1 cells were used in vitro micronucleus (MNvit) and comet assays. Two strains of Salmonella typhimurium (Salmonella enterica subsp.enterica; TA98 and TA1537) were employed in Ames tests.

Results: All CSCs showed mutagenicity in the TA98 and TA1537 strains. In addition, DNA damage and micronuclei formation were observed in the comet and MNvit assays owing to CSC exposure. The CSC from the 3R4F Kentucky reference (3R4F) cigarette produced the most severe mutagenic and genotoxic potencies, followed by the CSC from the TL cigarette, whereas the CSC from the TW cigarette produced the least severe mutagenic and genotoxic potencies.

Conclusions: The results of this study suggest that the mutagenic and genotoxic potencies of the TL and TW cigarettes were weaker than those of the 3R4F cigarette. Further study on standardized concepts of toxic equivalents for cigarettes needs to be conducted for more extensive use of in vitro tests.

Keywords: Ames test; Cigarette smoke condensate; Comet assay; Genotoxicity; Micronucleus assay; Mutagenicity.

Figure 1.

Viability of CHO-K1 cells exposed to 3 CSCs. The cells were incubated with 3R4F (green bar), TL (sky blue bar), and TW (red bar) CSCs. for 24 hours, after which the WST-1 assay was performed. Cell viability was expressed as a percentage of that of the control cells (0.0 μg TPM per mL). Each value represents the mean±standard deviation of 5 separate experiments. CSCs, cigarette smoke condensates; 3R4F, 3R4F Kentucky reference; TPM, total particulate matter. *p<0.05, **p<0.01 for values significantly different from those of the control group.

Figure 2.

DNA breakage in CHO-K1 cells exposed to 3 CSCs. The cells were treated with 3R4F (green bar), TL (sky blue bar), and TW (red bar) CSCs. for 3 hours in the presence of the S9 mix. The positive control cells were exposed to 10 μM b(a)p. DNA breakage was expressed as Olive tail moment (tail distance × %DNA in the tail), which was expressed as a foldinduction relative to the control group (0.0 μg TPM per mL). Each value represents the mean±standard deviation of 5 separate experiments. CSCs, cigarette smoke condensates; 3R4F, 3R4F Kentucky reference; b(a) p, benzo[a]pyrene; TPM, total particulate matter. *p<0.05, **p<0.01 for values significantly different from those of the control group.

Figure 3.

MN formation in CHO-K1 cells exposed to 3 CSCs. The cells were treated with 3R4F (green bar), TL (sky blue bar), and TW (red bar) CSCs for 3 hours in the presence of S9 mix, followed by a 21-hour recovery period under exposure to 0.75 μg/mL cytochalasin B. The positive control cells were exposed to 5 μM b(a)p. The CBPI of the cells treated with the 3R4F, TL, and TW CSCs at a concentration of 25.0 μg/mL were 1.75±0.03, 1.76±0.05, and 1.69±0.04, respectively. Treatment with CSCs did not significantly reduce the CBPI of treated cells in comparison with that of the control cells (1.77±0.04). The MN formation results are expressed as fold-induction relative to that of the control group (0.0 μg TPM per mL). Each value represents the mean±standard deviation of 5 separate experiments. MN, micronuclei; CSCs, cigarette smoke condensates; 3R4F, 3R4F Kentucky reference; b(a)p, benzo(a)pyrene; CBPI, cytokinesis-block proliferation index; TPM, total particulate matter. *p<0.05, **p<0.01 for values significantly different from those of the control group