Quantitative analysis of the relative mutagenicity of five chemical constituents of tobacco smoke in the mouse lymphoma assay

Abstract

Quantifying health-related biological effects, like genotoxicity, could provide a way of distinguishing between tobacco products. In order to develop tools for using genotoxicty data to quantitatively evaluate the risk of tobacco products, we tested five carcinogens found in cigarette smoke, 4-aminobiphenyl (4-ABP), benzo[a]pyrene (BaP), cadmium (in the form of CdCl2), 2-amino-3,4-dimethyl-3H-imidazo[4,5-f]quinoline (MeIQ) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in the mouse lymphoma assay (MLA). The resulting mutagenicity dose responses were analyzed by various quantitative approaches and their strengths and weaknesses for distinguishing responses in the MLA were evaluated. L5178Y/Tk (+/-) 3.7.2C mouse lymphoma cells were treated with four to seven concentrations of each chemical for 4h. Only CdCl2 produced a positive response without metabolic activation (S9); all five chemicals produced dose-dependent increases in cytotoxicity and mutagenicity with S9. The lowest dose exceeding the global evaluation factor, the benchmark dose producing a 10%, 50%, 100% or 200% increase in the background frequency (BMD10, BMD50, BMD100 and BMD200), the no observed genotoxic effect level (NOGEL), the lowest observed genotoxic effect level (LOGEL) and the mutagenic potency expressed as a mutant frequency per micromole of chemical, were calculated for all the positive responses. All the quantitative metrics had similar rank orders for the agents' ability to induce mutation, from the most to least potent as CdCl2(-S9) > BaP(+S9) > CdCl2(+S9) > MeIQ(+S9) > 4-ABP(+S9) > NNK(+S9). However, the metric values for the different chemical responses (i.e. the ratio of the greatest value to the least value) for the different chemicals ranged from 16-fold (BMD10) to 572-fold (mutagenic potency). These results suggest that data from the MLA are capable of discriminating the mutagenicity of various constituents of cigarette smoke, and that quantitative analyses are available that can be useful in distinguishing between the exposure responses.

Limited mutagenicity of electronic cigarettes in mouse or human cells in vitro

“…To demonstrate the competency of mouse embryonic fibroblasts to metabolize inert carcinogens and convert them to DNA reactive agents capable of inducing mutation, we similarly exposed these cells to two prominent tobacco carcinogens, including B[ a ]P and 4-ABP, which require metabolic activation to yield promutagenic DNA adducts with distinct mutagenic potencies [45, 46]. In both cases, we detected significant mutagenic effects as reflected by the 2.5- and 4-fold increases, respectively, in relative cII mutant frequency in the 4-ABP- and B[ a ]P-treated cells, respectively.…”

“…, day 7 post-treatment), all cultures had undergone 3–4 population doublings, a requisite for fixation of mutations into the genome [44]. As a positive control, counterpart cell cultures were treated with two tobacco carcinogens, benzo[ a ]pyrene (B[ a ]P) and 4-aminobiphenyl (4-ABP), both of which require metabolic activation to exert mutagenic effects [45, 46]. B[ a ]P and 4-ABP have different mutagenic potencies, with the former being a stronger mutagen [45, 46].…”

“…As a positive control, counterpart cell cultures were treated with two tobacco carcinogens, benzo[ a ]pyrene (B[ a ]P) and 4-aminobiphenyl (4-ABP), both of which require metabolic activation to exert mutagenic effects [45, 46]. B[ a ]P and 4-ABP have different mutagenic potencies, with the former being a stronger mutagen [45, 46]. To keep a consistent cytotoxicity threshold, control cultures were treated with 5 μM of B[ a ]P and 10 μM of 4-ABP that resulted in 80% cell viability post-treatment.…”

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

“…All WSSs were tested in the absence or presence of S9 metabolic activation. An S9 cofactor mixture was prepared by mixing the S9 liver fraction with a reduced nicotinamide adenine dinucleotide phosphate (NADPH)‐generating system at a ratio of 1:4 (Guo et al, ). The standard S9 mix contained 50 mM sodium phosphate buffer (pH 8.0), 30 mM KCl, 10 mM MgCl 2 , 4 mM NADP, 5 mM glucose‐6‐phosphate, 10 mM CaCl 2 , and 2 mg/ml of S9 protein.…”

“…Because tobacco smoke is classified as a Group 1 human carcinogen by IARC (), we previouly modelled the genotoxicity outcomes from different cigarette smoke condensates in order to measure the varying toxicity of individual tobacco products (Guo et al, ). A subsequent study applied point‐of‐departure (PoD) and other quantitative metrics to mouse lymphoma assay (MLA) data with the aim of ranking and quantitatively evaluating select chemicals representing different chemical classes found in tobacco smoke on the basis of their genotoxicity (Guo et al, ). The quantitative metrics we used were the benchmark dose (BMD), the no observed genotoxic effect level (NOGEL), the lowest observed genotoxic effect level (LOGEL), and mutagenic potency expressed as mutant frequency (MF) per µM chemical.…”