The 3D reconstructed skin micronucleus assay using imaging flow cytometry and deep learning: A proof-of-principle investigation.

The reconstructed skin micronucleus (RSMN) assay was developed in 2006, as an in vitro alternative for genotoxicity evaluation of dermally applied chemicals or products. In the years since, significant progress has been made in the optimization of the assay, including publication of a standard protocol and extensive validation. However, the diverse morphology of skin cells makes cell preparation and scoring of micronuclei (MN) tedious and subjective, thus requiring a high level of technical expertise for evaluation. This ultimately has a negative impact on throughput and the assay would benefit by the development of an automated method which could reduce scoring subjectivity while also improving the robustness of the assay by increasing the number of cells that can be scored. Imaging flow cytometry (IFC) with the ImageStream®X Mk II can capture high-resolution transmission and fluorescent imagery of cells in suspension. This proof-of-principle study describes protocol modifications that enable such automated measurement in 3D skin cells following exposure to mitomycin C and colchicine. IFC was then used for automated image capture and the Amnis® Artificial Intelligence (AAI) software permitted identification of binucleated (BN) cells with 91% precision. On average, three times as many BN cells from control samples were evaluated using IFC compared to the standard manual analysis. When IFC MNBN cells were visually scored from within the BN cell images, their frequency compared well with manual slide scoring, showing that IFC technology can be applied to the RSMN assay. This method enables faster time to result than microscope-based scoring and the initial studies presented here demonstrate its capability for the detection of statistically significant increases in MNBN frequencies. This work therefore demonstrates the feasibility of combining IFC and AAI to automate scoring for the RSMN assay and to improve its throughput and statistical robustness.

Can a digital slide scanner and viewing technique assist the visual scoring for the cytokinesis-block micronucleus cytome assay?

The cytokinesis-block micronucleus cytome (CBMNcyt) assay is a comprehensive method to measure DNA damage, cytostasis and cytotoxicity caused by nutritional, radiation and chemical factors. A slide imaging technique has been identified as a new method to assist with the visual scoring of cells for the CBMNcyt assay. A NanoZoomer S60 Digital Pathology slide scanner was used to view WIL2-NS cells treated with hydrogen peroxide (H2O2) and measure CBMNcyt assay biomarkers using a high-definition desktop computer screen. The H2O2-treated WIL2-NS cells were also scored visually using a standard light microscope, and the two visual scoring methods were compared. Good agreement was found between the scoring methods for all DNA damage indices (micronuclei, nucleoplasmic bridges and nuclear buds) and nuclear division index with correlation R values ranging from 0.438 to 0.789, P < 0.05. Apoptotic and necrotic cell frequency was lower for the NanoZoomer scoring method, but necrotic frequency correlated well with the direct visual microscope method (R = 0.703, P < 0.0001). Considerable advantages of the NanoZoomer scoring method compared to direct visual microscopy includes reduced scoring time, improved ergonomics and a reduction in scorer fatigue. This study indicates that a digital slide scanning and viewing technique may assist with visual scoring for the CBMNcyt assay and provides similar results to conventional direct visual scoring.

RABiT-II: A Fully-Automated Micronucleus Assay System with Shortened Time to Result.

In this work, we describe a fully automated cytokinesis-block micronucleus (CBMN) assay with a significantly shortened time to result, motivated by the need for rapid high-throughput biodosimetric estimation of radiation doses from small-volume human blood samples. The Rapid Automated Biodosimetry Tool (RABiT-II) currently consists of two commercial automated systems: a PerkinElmer cell::explorer Workstation and a GE Healthcare IN Cell Analyzer 2000 Imager. Blood samples (30 µl) from eight healthy volunteers were gamma-ray irradiated ex vivo with 0 (control), 0.5, 1.5, 2.5, 3.5 or 4.5 Gy and processed with full automation in 96-well plates on the RABiT-II system. The total cell culture time was 54 h and total assay time was 3 days. DAPI-stained fixed samples were imaged on an IN Cell Analyzer 2000 with fully-automated image analysis using the GE Healthcare IN Cell Developer Toolbox version 1.9. A CBMN dose-response calibration curve was established, after which the capability of the system to predict known doses was assessed. Various radiation doses for irradiated samples from two donors were estimated within 20% of the true dose (±0.5 Gy below 2 Gy) in 97% of the samples, with the doses in some 5 Gy irradiated samples being underestimated by up to 25%. In summary, the findings from this work demonstrate that the accelerated CBMN assay can be automated in a high-throughput format, using commercial biotech robotic systems, in 96-well plates, providing a rapid and reliable bioassay for radiation exposure.

In Vitro Approaches for Assessing the Genotoxicity of Nanomaterials.

Genotoxicity is associated with serious health effects and includes different types of DNA lesions, gene mutations, structural chromosome aberrations involving breakage and/or rearrangements of chromosomes (referred to as clastogenicity) and numerical chromosome aberrations (referred to as aneuploidy). Assessing the potential genotoxic properties of chemicals, including nanomaterials (NMs), is a key element in regulatory safety assessment. State-of-the-art genotoxicity testing includes a battery of assays covering gene mutations, structural and numerical chromosome aberrations. Typically various in vitro assays are performed in the first tier. It is not very likely that NMs may induce as yet unknown types of genotoxic damage beyond what is already known for chemicals. Thus, principles of genotoxicity testing as established for chemicals should be applicable to NMs as well. However, established test guidelines (i.e., OECD TG) may require adaptations for NM testing, as currently under discussion at the OECD. This chapter gives an overview of genotoxicity testing of NMs in vitro based on experiences from various research projects. We recommend a combination of a mammalian gene mutation assay (at either Tk or HPRT locus), the in vitro comet assay, and the cytokinesis-block micronucleus assay, which are discussed in detail here. In addition we also include the Cell Transformation Assay (CTA) as a promising novel test for predicting NM-induced cell transformation in vitro.

Evaluation of Genotoxicity of Nanoparticles in Mouse Models.

Owing to new and unique properties, engineered nanoparticles (NPs) likely pose different risks than their constituent chemicals and these risks need to be understood. In particular, it is important to assess genotoxicity, since genotoxicity is a precursor to carcinogenicity. Here we describe a battery of tests for the assessment of genotoxicity of NPs in vivo in mice. Mice can be exposed to NPs for various exposure durations and by any route of exposure, provided NPs are absorbed into the systemic blood circulation. The testing battery measures three well-established markers of DNA damage: oxidative DNA damage, double strand breaks (DSBs) and chromosomal damage. These markers are measured in peripheral blood cells by microscopic techniques. 8-oxo-7,8-dihydro-2-deoxyguanine (8-oxoG), indicative of oxidative DNA damage, and phosphorylated histone 2AX (γ-H2AX) foci, indicative of DSBs, are determined in white blood cells by immunofluorescence. Micronuclei, indicative of chromosomal damage, are examined in erythrocytes on Giemsa-stained peripheral blood smears. This testing battery can be easily integrated in general toxicology studies or studies examining carcinogenic potential of NPs.

Impact of Anthropogenic Activities on Water Quality of The Paranhana River, Southern Brazil

Abstract: The Paranhana River, located in Southern Brazil, is one of the major tributaries of the Sinos River basin and receives mainly industrial and domestic effluents. In the present study, water physicochemical and microbiological analyses, condition factor, micronucleus test, gill histopathology and metal bioaccumulation in the muscle of the native fish Bryconamericus iheringii collected at two sites (S1 and S2) of the Paranhana River under different degrees of anthropogenic pressures were assessed in four sampling campaigns. Data from water quality parameters, condition factor, mucous cells proliferation in fish gills and bioaccumulation of chromium and manganese in muscle evidenced higher impacts at S2, whereas a higher genotoxic potential was observed at S1. Gill histopathological alterations were found in fish captured at both sampling sites. Temporal variations in all biomarkers analyzed and bioaccumulation of manganese and nickel were observed at S1, whereas only variations in condition factor, gill alterations and bioaccumulation of manganese and aluminum were found at S2. Our study evidences that S1 is under minor anthropogenic impacts and that the high urbanization at S2 reflects in a poor water quality. Nonetheless, the human consumption of fish from the Paranhana River should be avoided given the high concentrations of cadmium, chromium and lead.

Automation of the in vitro micronucleus assay using the Imagestream® imaging flow cytometer.

The in vitro micronucleus (MN) assay is a well-established test for evaluating genotoxicity and cytotoxicity. The use of manual microscopy to perform the assay can be laborious and often suffers from user subjectivity and interscorer variability. Automated methods including slide-scanning microscopy and conventional flow cytometry have been developed to eliminate scorer bias and improve throughput. However, these methods possess several limitations such as lack of cytoplasmic visualization using slide-scanning microscopy and the inability to visually confirm the legitimacy of MN or storage of image data for re-evaluation using flow cytometry. The ImageStreamX® MK II (ISX) imaging flow cytometer has been demonstrated to overcome all of these limitations. The ISX combines the speed, statistical robustness, and rare event capture capability of conventional flow cytometry with high resolution fluorescent imagery of microscopy and possesses the ability to store all collected image data. This paper details the methodology developed to perform the in vitro MN assay in human lymphoblastoid TK6 cells on the ISX. High resolution images of micronucleated mono- and bi-nucleated cells as well as polynucleated cells can be acquired at a high rate of capture. All images can then be automatically identified, categorized and enumerated in the data analysis software that accompanies the ImageStream, allowing for the scoring of both genotoxicity and cytotoxicity. The results demonstrate that statistically significant increases in MN frequency when compared with solvent controls can be detected at varying levels of cytotoxicity following exposure to well-known aneugens and clastogens. This work demonstrates a fully automated method for performing the in vitro micronucleus assay on the ISX imaging flow cytometry platform. © 2018 The Author. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of ISAC.

RABiT-II: Implementation of a High-Throughput Micronucleus Biodosimetry Assay on Commercial Biotech Robotic Systems.

We demonstrate the use of high-throughput biodosimetry platforms based on commercial high-throughput/high-content screening robotic systems. The cytokinesis-block micronucleus (CBMN) assay, using only 20 µl whole blood from a fingerstick, was implemented on a PerkinElmer cell::explorer and General Electric IN Cell Analyzer 2000. On average 500 binucleated cells per sample were detected by our FluorQuantMN software. A calibration curve was generated in the radiation dose range up to 5.0 Gy using the data from 8 donors and 48,083 binucleated cells in total. The study described here demonstrates that high-throughput radiation biodosimetry is practical using current commercial high-throughput/high-content screening robotic systems, which can be readily programmed to perform and analyze robotics-optimized cytogenetic assays. Application to other commercial high-throughput/high-content screening systems beyond the ones used in this study is clearly practical. This approach will allow much wider access to high-throughput biodosimetric screening for large-scale radiological incidents than is currently available.

Evaluation of the automated MicroFlow® and Metafer™ platforms for high-throughput micronucleus scoring and dose response analysis in human lymphoblastoid TK6 cells.

The use of manual microscopy for the scoring of chromosome damage in the in vitro micronucleus assay is often associated with user subjectivity. This level of subjectivity can be reduced by using automated platforms, which have added value of faster with high-throughput and multi-endpoint capabilities. However, there is a need to assess the reproducibility and sensitivity of these automated platforms compared with the gold standard of the manual scoring. The automated flow cytometry-based MicroFlow® and image analysis-based Metafer™ were used for dose response analyses in human lymphoblastoid TK6 cells exposed to the model clastogen, methyl methanesulfonate (MMS), aneugen, carbendazim, and the weak genotoxic carcinogen, ochratoxin A (OTA). Cells were treated for 4 or 30 h, with a 26- or 0-h recovery. Flow cytometry scoring parameters and the Metafer™ image classifier were investigated, to assess any potential differences in the micronucleus (MN) dose responses. Dose response data were assessed using the benchmark dose approach with chemical and scoring system set as covariate to assess reproducibility between endpoints. A clear increase in MN frequency was observed using the MicroFlow® approach on TK6 cells treated for 30 h with MMS, carbendazim and OTA. The MicroFlow®-based MN frequencies were comparable to those derived by using the Metafer™ and manual scoring platforms. However, there was a potential overscoring of MN with the MicroFlow® due to the cell lysis step and an underscoring with the Metafer™ system based on current image classifier settings. The findings clearly demonstrate that the MicroFlow® and Metafer™ MN scoring platforms are powerful tools for automated high-throughput MN scoring and dose response analysis.

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.