Automation of thein vitromicronucleus and chromosome aberration assay for the assessment of the genotoxicity of the particulate and gas–vapor phase of cigarette smoke

Abstract

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.

The genotoxicological assessment of a tobacco heating product relative to cigarette smoke using the in vitro micronucleus assay

“…In these high throughput approaches 10,000 cells can be analysed in as little as 2 min vs. 600 cells in 15 min using traditional scoring approaches [ 48 ]. Furthermore, it was reported that automated scoring could reduce man hours per study by 70 %, increase data turn around by 50 % and due to the assessment of large cell numbers, the variability of the automated technique could be lowered too [ 50 ]. Due to the nature and applicability of these techniques with both suspension and attachment cell lines, such an approach could easily be combined with whole aerosol exposure methodologies, to create a modern version of the classic IVMN assay.…”

Chemical Composition and In Vitro Toxicity Profile of a Pod-Based E-Cigarette Aerosol Compared to Cigarette Smoke

“…5). 34,35 For example for 3R4F smoke, the values from the three test days were averaged and the effective concentration to achieve EC-MN3 was calculated using nonlinear regression (Table 6). No statistical comparison could be modeled for the myblu aerosol because it did not induce any significant increase in micronuclei frequencies in V79 cells (Table 6).…”

“…33 V79 cells show a good responsiveness to cigarette smoke extracts and that the use of V79 cells results in robust reproducible genotoxicity results. 35,51,52 Within this study, OECD guidelines were followed, although e-cigarette aerosol and cigarette smoke were tested with metabolic activation only (no ÀS9 condition within the study); although this is a potential study limitation, the treatment V79 cells do not express P450 enzymes and the inclusion of metabolic activation increases the human relevance of the assay. However, Thorne et al showed that V79 cells were most responsive to cigarette smoke constituents after an extended recovery/ expression period without S9.…”

High throughput toxicity screening and intracellular detection of nanomaterials

“…Different approaches have been proposed to increase the speed of this assay. Classically, the long and tedious visual scoring of slides has been relieved by using automated platforms scoring many slides in each run 134, 135, 136. Different commercial automatic scoring devices are now on the market and the time saving, based on person‐hours, due to the automation is approximately 70%.…”

“…Classically, the long and tedious visual scoring of slides has been relieved by using automated platforms scoring many slides in each run. [134][135][136] Different commercial automatic scoring devices are now on the market and the time saving, based on person-hours, due to the automation is approximately 70%. Another high-throughput change in the micronucleus assay includes the use of flow cytometry, based on the pioneering work of Nüsse and Kramer.…”