Automation of the in vitro micronucleus and chromosome aberration assay for the assessment of the genotoxicity of the particulate and gas-vapor phase of cigarette smoke.

Total particulate matter (TPM) and the gas-vapor phase (GVP) of mainstream smoke from the Reference Cigarette 3R4F were assayed in the cytokinesis-block in vitro micronucleus (MN) assay and the in vitro chromosome aberration (CA) assay, both using V79-4 Chinese hamster lung fibroblasts exposed for up to 24 h. The Metafer image analysis platform was adapted resulting in a fully automated evaluation system of the MN assay for the detection, identification and reporting of cells with micronuclei together with the determination of the cytokinesis-block proliferation index (CBPI) to quantify the treatment-related cytotoxicity. In the CA assay, the same platform was used to identify, map and retrieve metaphases for a subsequent CA evaluation by a trained evaluator. In both the assays, TPM and GVP provoked a significant genotoxic effect: up to 6-fold more micronucleated target cells than in the negative control and up to 10-fold increases in aberrant metaphases. Data variability was lower in the automated version of the MN assay than in the non-automated. It can be estimated that two test substances that differ in their genotoxicity by approximately 30% can statistically be distinguished in the automated MN and CA assays. Time savings, based on man hours, due to the automation were approximately 70% in the MN and 25% in the CA assays. The turn-around time of the evaluation phase could be shortened by 35 and 50%, respectively. Although only cigarette smoke-derived test material has been applied, the technical improvements should be of value for other test substances.

Comet assay and air-liquid interface exposure system: a new combination to evaluate genotoxic effects of cigarette whole smoke in human lung cell lines.

Over the past three decades, the genotoxic effects of cigarette smoke have generally been evaluated in non-human cell models after exposure to particulate phase, gas phase, or cigarette smoke condensate, rather than the whole smoke aerosol itself. In vitro setups using human cell lines and whole smoke exposure to mimic actual aerosol exposure should more accurately reflect human cigarette smoke exposure. We investigated the VITROCELL® 24 air-liquid interface exposure system in combination with the comet assay to assess DNA damage in two different human lung epithelial cell lines exposed to whole smoke. Results showed a repeatable and reproducible dose-response relationship between DNA damage and increased whole smoke dose in both cell lines. Thus, the combination of the comet assay with the VITROCELL® 24 represents a valuable new in vitro test system to screen and assess DNA damage in human lung cells exposed to whole smoke.

Cigarette smoke-induced morphological transformation of Bhas 42 cells in vitro.

In vitro cell transformation assays detect transformed cells that have acquired the distinct characteristics of malignant cells and thus model one stage of in vivo carcinogenesis. These assays have been proposed as surrogate models for predicting the non-genotoxic carcinogenic potential of chemicals. The Bhas 42 cell transformation assay, a short-term assay that uses v-Ha-ras-transfected Balb/c 3T3 cells, can detect the tumour promoter-like activities of chemicals, but has not previously been used with cigarette smoke. The particulate phase of cigarette smoke (total particulate matter [TPM]) is known to induce tumours in vivo in the mouse skin painting assay. Therefore, we investigated the ability of this Bhas cell assay to form morphologically transformed foci in vitro when repeatedly challenged with TPM from a standard research cigarette. TPM induced a dose-dependent increase in Type III foci, and a significant increase (up to 20-fold) in focus formation at moderately toxic concentrations between 5 and 60µg TPM/ml, with a peak at 20µg/ml. Three batches of TPM were tested in three independent experiments. Precision (repeatability and reproducibility) was calculated by using 0, 5, 10, and 20µg TPM/ml. Repeatability and reproducibility, expressed as the relative standard deviation obtained from the normalised slopes of the dose-response curves, were 17.2% and 19.6%, respectively; the slopes were 0.7402 ± 0.1247, 0.9347 ± 0.1316, and 0.8772 ± 0.1767 (increase factor∗ml/mg TPM; mean ± SD) ; and the goodness of fit (r2) of the mean slopes, each derived from n = 6 repeats, was 0.9449, 0.8198, and 0.8344, respectively. This in vitro assay with Bhas 42 cells, which are regarded as already initiated in the two-stage paradigm of carcinogenesis (initiation and promotion), is able to detect cell transformation induced by cigarette smoke in a dose-dependent manner with a high sensitivity and good precision. Because this assay is fast and yields reliable results, it may be useful in product assessment, as well as for further investigation of the non-genotoxic carcinogenic activity of tobacco smoke-related test substances.

Characterization of a gap-junctional intercellular communication (GJIC) assay using cigarette smoke.

Inhibition of gap-junctional intercellular communication (GJIC) via exposure to various toxic substances has been implicated in tumor promotion. In the present study, cigarette smoke total particulate matter (TPM), a known inhibitor of GJIC, were used to characterize a new GJIC screening assay in three independent experiments. The main features of this assay were automated fluorescence microscopy combined with non-invasive parachute technique. Rat liver epithelial cells (WB-F344) were stained with the fluorescent dye Calcein AM (acetoxymethyl) and exposed to TPM from the Kentucky Reference Cigarette 2R4F (a blend of Bright and Burley tobaccos) and from two single-tobacco cigarettes (Bright and Burley) for 3h. Phorbol-12-myristate-13-acetate (TPA) was used as positive control and 0.5% dimethyl sulfoxide (DMSO) as solvent control. The transfer of dye to adjacent cells (percentage of stained cells) was used as a measure of cellular communication. A clear and reproducible dose-response of GJIC inhibition following TPM exposure was seen. Reproducibility and repeatability measurements for the 2R4F cigarette were 3.7% and 6.9%, respectively. The half-maximal effective concentration values were 0.34ng/ml for TPA, 0.050mg/ml for the 2R4F, 0.044mg/ml for the Bright cigarette, and 0.060mg/ml for the Burley cigarette. The assay was able to discriminate between the two single-tobacco cigarettes (P<0.0001), and between the single-tobacco cigarettes and the 2R4F (P=0.0008, 2R4F vs. Burley and P<0.0001, 2R4F vs. Bright). Thus, this assay can be used to determine the activity of complex mixtures such as cigarette smoke with high throughput and high precision.