Effect of occupational exposure to antineoplastic drugs on DNA damage in nurses: a cross-sectional study

BackgroundAlthough the therapeutic effect of antineoplastic drugs is incontestable, these agents can also potentially act as carcinogens, mutagens and/or teratogens in people. The aim of this study was to assess the effect of occupational exposure to antineoplastic drugs on DNA damage, assessed by the comet assay and cytokinesis-block micronucleus (CBMN) assay, in nurses.MethodsThe cross-sectional study enrolled 305 nursing staff members from 7 public hospitals in Shenzhen who handled antineoplastic drugs, and 150 healthy nursing staff members who were not exposed to antineoplastic drugs as the control group. DNA damage was assessed by the comet and CBMN assay. Multiple linear regressions and logistic regressions models were used to analyse the effect of occupational exposure to antineoplastic drugs on DNA damage.ResultsAfter adjustment for confounding factors, compared with non-exposure to antineoplastic drugs, exposure to antineoplastic drugs was positively related to tail moment, olive moment, tail length and tail DNA per cent, and adjusted β or OR (95% CI) was 0.17 (0.08 to 0.26), 0.18 (0.10 to 0.27), 1.03 (0.47 to 1.60) and 1.16 (1.04 to 1.29) (all p<0.05). Moreover, similar significant relationships were observed for the biomarkers of the CBMN assay. Additionally, other than age, there was no interaction between antineoplastic drug exposure and other variables for the levels of biomarkers of the CBMN assay and the comet assay.ConclusionsThe present results showed that exposure to antineoplastic drugs was positively related to the risk of DNA damage in nurses. The results imply that occupational exposure to antineoplastic agents is an important global public health problem that requires urgent attention.

Could scoring tailed and dumbbell-shaped nuclei increase the sensitivity of micronucleus analysis as a biomarker of radiation exposure?

In the cytokinesis-block micronucleus (CBMN) assay, micronuclei (MNi), nucleoplasmic bridges (NPBs), and nuclear budding (NBUD) are the most commonly analysed morphological types of nuclear abnormalities. In contrast, tailed and dumbbell-shaped nucleus have historically received little attention in the CBMN assay. Interestingly, the incidence of tailed and dumbbell-shaped nuclei in lymphocytes is closely related with that of dicentric chromosomes or NPBs in the CBMN assay. To provide a better picture of the implications and significance of tailed and dumbbell-shaped nuclei as markers of radiation exposure, a literature review was performed in this study. Twenty articles were found in PubMed, PubMed Central, and manually searched. The articles were screened and those that met the inclusion criteria and did not meet the exclusion criteria were reviewed by all authors. At the end, nine articles were included. In conclusion, the assessment of in vivo tailed nuclei in blood smears and accounting for the occurrence of dumbbell-shaped nuclei in the CBMN assay can increase the sensitivity of the CBMN assay for biodosimetry involving a high dose exposure.

CytoRADx: A High-Throughput, Standardized Biodosimetry Diagnostic System Based on the Cytokinesis-Block Micronucleus Assay

In a large-scale catastrophe, such as a nuclear detonation in a major city, it will be crucial to accurately diagnose large numbers of people to direct scarce medical resources to those in greatest need. Currently no FDA-cleared tests are available to diagnose radiation exposures, which can lead to complex, life-threatening injuries. To address this gap, we have achieved substantial advancements in radiation biodosimetry through refinement and adaptation of the cytokinesis-block micronucleus (CBMN) assay as a high throughput, quantitative diagnostic test. The classical CBMN approach, which quantifies micronuclei (MN) resulting from DNA damage, suffers from considerable time and expert labor requirements, in addition to a lack of universal methodology across laboratories. We have developed the CytoRADx™ System to address these drawbacks by implementing a standardized reagent kit, optimized assay protocol, fully automated microscopy and image analysis, and integrated dose prediction. These enhancements allow the CytoRADx System to obtain high-throughput, standardized results without specialized labor or laboratory-specific calibration curves. The CytoRADx System has been optimized for use with both humans and non-human primates (NHP) to quantify radiation dose-dependent formation of micronuclei in lymphocytes, observed using whole blood samples. Cell nuclei and resulting MN are fluorescently stained and preserved on durable microscope slides using materials provided in the kit. Up to 1,000 slides per day are subsequently scanned using the commercially based RADxScan™ Imager with customized software, which automatically quantifies the cellular features and calculates the radiation dose. Using less than 1 mL of blood, irradiated ex vivo, our system has demonstrated accurate and precise measurement of exposures from 0 to 8 Gy (90% of results within 1 Gy of delivered dose). These results were obtained from 636 human samples (24 distinct donors) and 445 NHP samples (30 distinct subjects). The system demonstrated comparable results during in vivo studies, including an investigation of 43 NHPs receiving single-dose total-body irradiation. System performance is repeatable across laboratories, operators, and instruments. Results are also statistically similar across diverse populations, considering various demographics, common medications, medical conditions, and acute injuries associated with radiological disasters. Dose calculations are stable over time as well, providing reproducible results for at least 28 days postirradiation, and for blood specimens collected and stored at room temperature for at least 72 h. The CytoRADx System provides significant advancements in the field of biodosimetry that will enable accurate diagnoses across diverse populations in large-scale emergency scenarios. In addition, our technological enhancements to the well-established CBMN assay provide a pathway for future diagnostic applications, such as toxicology and oncology.

Suitability of the In Vitro Cytokinesis-Block Micronucleus Test for Genotoxicity Assessment of TiO2 Nanoparticles on SH-SY5Y Cells

Standard toxicity tests might not be fully adequate for evaluating nanomaterials since their unique features are also responsible for unexpected interactions. The in vitro cytokinesis-block micronucleus (CBMN) test is recommended for genotoxicity testing, but cytochalasin-B (Cyt-B) may interfere with nanoparticles (NP), leading to inaccurate results. Our objective was to determine whether Cyt-B could interfere with MN induction by TiO2 NP in human SH-SY5Y cells, as assessed by CBMN test. Cells were treated for 6 or 24 h, according to three treatment options: co-treatment with Cyt-B, post-treatment, and delayed co-treatment. Influence of Cyt-B on TiO2 NP cellular uptake and MN induction as evaluated by flow cytometry (FCMN) were also assessed. TiO2 NP were significantly internalized by cells, both in the absence and presence of Cyt-B, indicating that this chemical does not interfere with NP uptake. Dose-dependent increases in MN rates were observed in CBMN test after co-treatment. However, FCMN assay only showed a positive response when Cyt-B was added simultaneously with TiO2 NP, suggesting that Cyt-B might alter CBMN assay results. No differences were observed in the comparisons between the treatment options assessed, suggesting they are not adequate alternatives to avoid Cyt-B interference in the specific conditions tested.

The Potential Risk of Electronic Waste Disposal into Aquatic Media: The Case of Personal Computer Motherboards

Considering that electronic wastes (e-wastes) have been recently recognized as a potent environmental and human threat, the present study aimed to assess the potential risk of personal computer motherboards (PCMBs) leaching into aquatic media, following a real-life scenario. Specifically, PCMBs were submerged for 30 days in both distilled water (DW) and artificial seawater (ASW). Afterwards, PCMBs leachates were chemically characterized (i.e., total organic carbon, ions, and trace elements) and finally used (a) for culturing freshwater (Chlorococcum sp. and Scenedesmus rubescens) and saltwater (Dunaliella tertiolecta and Tisochrysis lutea) microalgae for 10 days (240 h), (b) as the exposure medium for mussel Mytilus galloprovincialis (96 h exposure), and (c) for performing the Cytokinesis Block Micronucleus (CBMN) assay in human lymphocytes cultures. According to the results, PCMBs could mediate both fresh- and marine algae growth rates over time, thus enhancing the cytotoxic, oxidative, and genotoxic effects in the hemocytes of mussels (in terms of lysosomal membrane impairment, lipid peroxidation, and NO content and micronuclei formation, respectively), as well as human lymphocytes (in terms of MN formation and CBPI values, respectively). The current findings clearly revealed that PCMBs leaching into the aquatic media could pose detrimental effects on both aquatic organisms and human cells.

Cytokinesis-Block Micronucleus Cytome Assay Evolution into a More Comprehensive Method to Measure Chromosomal Instability

This review describes the cytokinesis-block micronucleus (CBMN) cytome assay and its evolution into a molecular cytogenetic method of chromosomal instability (CIN). Micronuclei (MNi) originate from whole chromosomes or chromosome fragments that fail to segregate to the poles of the cell during mitosis. These lagging chromosomes are excluded from the daughter nuclei and are enveloped in their own membrane to form MNi. The CBMN assay was developed to allow MNi to be scored exclusively in once-divided binucleated cells, which enables accurate measurement of chromosome breakage or loss without confounding by non-dividing cells that cannot express MNi. The CBMN assay can be applied to cell lines in vitro and cells such as lymphocytes that can be stimulated to divide ex vivo. In the CBMN assay, other CIN biomarkers such as nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) are also measured. Use of centromere, telomere, and chromosome painting probes provides further insights into the mechanisms through which MNi, NPBs and NBUDs originate. Measurement of MNi is also important because entrapment within a micronucleus may cause chromosomes to shatter and, after nuclear reintegration, become rearranged. Additionally, leakage of DNA from MNi can stimulate inflammation via the cyclic GMP-AMP Synthase—Stimulator of Interferon Genes (cGAS-STING) DNA sensing mechanism of the innate immune system.

The Cytokinesis-Block Micronucleus Assay on Human Isolated Fresh and Cryopreserved Peripheral Blood Mononuclear Cells

The cytokinesis-block micronucleus (CBMN) assay is a standardized method used for genotoxicity studies. Conventional whole blood cultures (WBC) are often used for this assay, although the assay can also be performed on isolated peripheral blood mononuclear cell (PBMC) cultures. However, the standardization of a protocol for the PBMC CBMN assay has not been investigated extensively. The aim of this study was to optimize a reliable CBMN assay protocol for fresh and cryopreserved peripheral blood mononuclear cells (PBMCS), and to compare micronuclei (MNi) results between WBC and PBMC cultures. The G0 CBMN assay was performed on whole blood, freshly isolated, and cryopreserved PBMCS from healthy human blood samples and five radiosensitive patient samples. Cells were exposed to 220 kV X-ray in vitro doses ranging from 0.5 to 2 Gy. The optimized PBMC CBMN assay showed adequate repeatability and small inter-individual variability. MNi values were significantly higher for WBC than for fresh PBMCS. Additionally, cryopreservation of PBMCS resulted in a significant increase of MNi values, while different cryopreservation times had no significant impact. In conclusion, our standardized CBMN assay on fresh and cryopreserved PBMCS can be used for genotoxicity studies, biological dosimetry, and radiosensitivity assessment.

The use of a centrifuge-free RABiT-II system for high-throughput micronucleus analysis

ABSTRACT The cytokinesis-block micronucleus (CBMN) assay is considered to be the most suitable biodosimetry method for automation. Previously, we automated this assay on a commercial robotic biotech high-throughput system (RABiT-II) adopting both a traditional and an accelerated micronucleus protocol, using centrifugation steps for both lymphocyte harvesting and washing, after whole blood culturing. Here we describe further development of our accelerated CBMN assay protocol for use on high-throughput/high content screening (HTS/HCS) robotic systems without a centrifuge. This opens the way for implementation of the CBMN assay on a wider range of commercial automated HTS/HCS systems and thus increases the potential capacity for dose estimates following a mass-casualty radiological event.

Estimating genotoxic effects of anticancer drugs using cytokinesis-block micronucleus assay on human lymphocytes

Here we review the current experience of using cytokinesis-block micronucleus (CBMN) assay on cultures of human lymphocytes to evaluate genotoxic effects of anticancer drugs. Having performed search in PubMed, Scopus, Web of Science, TOXLINE, and the Cochrane Library, we identified a total of 172 relevant studies. Out of them, 89 were conducted in vitro, and 41 were published within the last decade. The mentioned studies concordantly demonstrated a significant increase in micronuclei, protrusions, nucleoplasmic bridges, and a decrease in proliferation in cells treated with anticancer drugs in a time- and dose-dependent manner. Notably, the results of CBMN assay are consistent with the data obtained from other cytogenetic techniques (comet assay, chromosomal aberration analysis, analysis of mutations in housekeeping genes, and fluorescence in situ hybridisation). Conclusion. CBMN assay permits a reliable evaluation of the mutagenic effects related to anticancer drugs.