Quantitative differentiation of whole smoke solution-induced mutagenicity in the mouse lymphoma assay

Abstract

In vitro genotoxicity dose-response data have been investigated for their utility in modeling and assessing potential differences in mutagenic responses between machine-generated whole smoke solutions (WSSs) from combusted cigarette tobacco products. Our previous study observed that potency ranking by benchmark dose (BMD) analysis was a useful modeling approach for quantitative assessment of differences between the mutagenicity of several structurally diverse chemical constituents of cigarette smoke. To follow-up on these observations, we used the mouse lymphoma assay (MLA) to evaluate the mutagenicity of WSSs prepared from two commercial cigarettes smoked under two different smoking machine regimens. L5178Y cells were exposed to ≥5 concentrations of each WSS for 4 hr ± S9 activation. S9 reduced the cytotoxicity and enhanced the mutagenicity of the WSSs. The resulting S9-mediated mutagenicity dose-responses were compared between test articles using BMD analysis, the lowest dose exceeding the Global Evaluation Factor, the no observed or lowest observed genotoxic effect level, and the mutagenic potency. The BMD₁₀ , BMD₅₀ , BMD₁₀₀ , and BMD₂₀₀ , indicating a 10%, 50%, 100%, or 200% increase in the background mutant frequency, respectively, were calculated using the PROAST software package. Overall, the quantitative approaches resulted in a similar rank order of mutagenic potency for the MLA tested WSSs, with potency increasing with the level of tar. The BMD approach using covariate analysis produced the most informative comparisons. Differences in potency were associated with the number of cigarettes smoked, the cigarette product smoked, and the smoking machine protocol used to prepare the sample. Under the conditions of this study, these results suggest that our hypothesis of modeling MLA data using the BMD approach to quantitatively discriminate between the mutagenic potential of WSSs from combustible cigarettes might be an useful method. Environ. Mol. Mutagen. 59:103-113, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA.

Keywords: benchmark dose; mouse lymphoma assay; quantitative analysis; whole smoke solution.

Fig. 1.

Relative mutagenicity of six whole smoke solutions (WSSs) in the mouse lymphoma assay. (A) BMDL₁₀, calculated from the exponential model of the PROAST Software using whole smoke solution as covariate approach, is the lower limit of the BMD₁₀ that is a dose that produces a predetermined change in the response of an adverse effect over control [in this case, 10% increase in mutant frequency (MF)]; NOGEL and LOGEL, determined by one way ANOVA followed by Dunnett’s test; GEF, Lowest positive response based on the global evaluation factor (GEF). A lower value indicates a higher mutagenic potential. No values were generated for NOGEL, LOGEL, and GEF in C1-HCI-20, as no positive response was observed over the tested concentrations. (B) Mutagenic potency, calculated from the slope of the linear regression of the dose–response curve.

Fig. 2.

Comparison of BMD values for WSSs in the mouse lymphoma assay. The BMD (BMD₁₀, BMD₅₀, BMD₁₀₀, and BMD₂₀₀) estimates producing a 10%, 50%, 100%, or 200% increase in the background frequency were calculated using exponential model of PROAST both without (A,B,C,D) and with covariate analysis approach (E,F,G,H). The bars represent the calculated 95% confidence interval of each value. The lower and upper limits derived from the BMD estimates were used to differentiate between responses based on non-overlapping confidence intervals.

Fig. 3.

Comparison of cytotoxic and mutagenic effects in mouse lymphoma cells treated with WSSs from cigarettes machine-smoked under different regimens. (A,C) Cytotoxicity is presented as relative total growth (RTG, %); (B,D) mutagenicity is presented as mutant frequencies (MF) per 10⁶ cells. All data are expressed as the mean ± 1 SD from 3 or 4 independent experiments.