Cytokinesis-block Micronucleus Assay Research Articles

Assessment of genotoxicity biomarkers in gasoline station attendants due to occupational exposure.

Gasoline station attendants are exposed to numerous chemicals that might have genotoxic and carcinogenic potential, such as benzene in fuel vapor and particulate matter and polycyclic aromatic hydrocarbons in vehicle exhaust emission. According to IARC, benzene and diesel particulates are Group 1 human carcinogens, and gasoline has been classified as Group 2A "possibly carcinogenic to humans." At gas stations, self-service is not implemented in Turkey; fuel-filling service is provided entirely by employees, and therefore they are exposed to those chemicals in the workplace during all working hours. Genetic monitoring of workers with occupational exposure to possible genotoxic agents allows early detection of cancer. We aimed to investigate the genotoxic damage due to exposures in gasoline station attendants in Turkey. Genotoxicity was evaluated by the Comet, chromosomal aberration, and cytokinesis-block micronucleus assays in peripheral blood lymphocytes. Gasoline station attendants (n = 53) had higher tail length, tail intensity, and tail moment values than controls (n = 61). In gasoline station attendants (n = 46), the frequencies of chromatid gaps, chromosome gaps, and total aberrations were higher compared with controls (n = 59). Increased frequencies of micronuclei and nucleoplasmic bridges were determined in gasoline station attendants (n = 47) compared with controls (n = 40). Factors such as age, duration of working, and smoking did not have any significant impact on genotoxic endpoints. Only exposure increased genotoxic damage in gasoline station attendants independently from demographic and clinical characteristics. Occupational exposure-related genotoxicity risk may increase in gasoline station attendants who are chronically exposed to gasoline and various chemicals in vehicle exhaust emissions.

RABiT-III: an Automated Micronucleus Assay at a Non-Specialized Biodosimetry Facility.

Micronuclei, detected through the cytokinesis-block micronucleus assay, are valuable indicators of ionizing radiation exposure, especially in short-term lymphocyte cultures. The peripheral human blood lymphocyte assay is recognized as a prime candidate for automated biodosimetry. In a prior project at the Columbia University Center for Radiological Research, we automated this assay using the 96-well ANSI/SLAS microplate standard format and relied on established biotech robotic systems named Rapid Automated Biodosimetry Tool (RABiT). In this study, we present the application of a similar automated biotech setup at an external high-throughput facility (RABiT-III) to implement the same automated cytokinesis-block micronucleus assay. Specifically, we employed the Agilent BRAVO liquid-handling system and GE IN Cell Analyzer 6000 imaging system in conjunction with the PerkinElmer Columbus image data storage and analysis system. Notably, this analysis system features an embedded PhenoLOGIC machine learning module, simplifying the creation of cell classification algorithms for CBMN assay image analysis and enabling the generation of radiation dose-response curves. This investigation underscores the adaptability of the RABiT-II CBMN protocol to diverse RABiT-III biotech robotic platforms in non-specialized biodosimetry centers. Furthermore, it highlights the advantages of machine learning in rapidly developing algorithms crucial for the high-throughput automated analysis of RABiT-III images.

Exploring cytokinesis block micronucleus assay in Croatia: A journey through the past, present, and future in biomonitoring of the general population

In this study, we used the cytokinesis-block micronucleus (CBMN) assay to evaluate the background frequency of cytogenetic damage in peripheral blood lymphocytes of the general population concerning different anthropometric data and lifestyle factors. The background frequency of CBMN assay parameters was analysed in 850 healthy, occupationally non-exposed male and female subjects (average age, 38±11 years) gathered from the general Croatian population from 2000 to 2023. The mean background values for micronuclei (MNi) in the whole population were 5.3±4.3 per 1000 binucleated cells, while the mean frequency of nucleoplasmic bridges (NPBs) was 0.7±1.3 and of nuclear buds (NBUDs) 3.1±3.2. The cut-off value, which corresponds to the 95th percentile of the distribution of 850 individual values, was 14 MNi, 3 NPBs, and 9 NBUDs. Results from our database also showed an association of the tested genomic instability parameters with age and sex but also with other lifestyle factors. These findings underscore the importance of considering several anthropometric and lifestyle factors when conducting biomonitoring studies. Overall, the normal and cut-off values attained here present normal values for the general population that can later serve as baseline values for further human biomonitoring studies either in Croatia or worldwide.

ATR signaling controls the bystander responses of human chondrosarcoma cells by promoting RAD51-dependent DNA repair

Purpose Radiation-induced bystander effect (RIBE) frequently is seen as DNA damage in unirradiated bystander cells, but the repair processes initiated in response to that DNA damage are not well understood. RIBE-mediated formation of micronuclei (MN), a biomarker of persistent DNA damage, was previously observed in bystander normal fibroblast (AG01522) cells, but not in bystander human chondrosarcoma (HTB94) cells. The molecular mechanisms causing this disparity are not clear. Herein, we investigate the role of DNA repair in the bystander responses of the two cell lines. Methods Cells were irradiated with X-rays and immediately co-cultured with un-irradiated cells using a trans-well insert system in which they share the same medium. The activation of DNA damage response (DDR) proteins was detected by immunofluorescence staining or Western blotting. MN formation was examined by the cytokinesis-block MN assay, which is a robust method to detect persistent DNA damage. Results Immunofluorescent foci of γH2AX and 53BP1, biomarkers of DNA damage and repair, revealed a greater capacity for DNA repair in HTB94 cells than in AG01522 cells in both irradiated and bystander populations. Autophosphorylation of ATR at the threonine 1989 site was expressed at a greater level in HTB94 cells compared to AG01522 cells at the baseline and in response to hydroxyurea treatment or exposure to 1 Gy of X-rays. An inhibitor of ATR, but not of ATM, promoted MN formation in bystander HTB94 cells. In contrast, no effect of either inhibitor was observed in bystander AG01522 cells, indicating that ATR signaling might be a pivotal pathway to preventing the MN formation in bystander HTB94 cells. Supporting this idea, we found an ATR-dependent increase in the fractions of bystander HTB94 cells with pRPA2 S33 and RAD51 foci. A blocker of RAD51 facilitated MN formation in bystander HTB94 cells. Conclusion Our results indicate that HTB94 cells were likely more efficient in DNA repair than AG01522 cells, specifically via ATR signaling, which inhibited the bystander signal-induced MN formation. This study highlights the significance of DNA repair efficiency in bystander cell responses.

Phytochemical Profile and Evaluation of the Antioxidant, Cyto-Genotoxic, and Antigenotoxic Potential of Salvia verticillata Hydromethanolic Extract.

This study comprises the phytochemical characterization, the evaluation of the total phenolic content (TPC) and antioxidant activity (AA), and the investigation of the cyto-genotoxic and antigenotoxic potential of hydromethanolic extract derived from Salvia verticillata L. leaves. HPLC-DAD-ESI-MS and HPLC-DAD were used for the characterization of the extract and determination of the major ingredients. Afterwards, the TPC and AA were determined. The cytotoxic and genotoxic effect of the extract on cultured human lymphocytes at concentrations of 10, 25, and 50 μg mL-1 was investigated via the Cytokinesis Block MicroNucleus (CBMN) assay. Moreover, its antigenotoxic potential against the mutagenic agent mitomycin C (MMC) was assessed using the same assay. The hydromethanolic extract comprises numerous metabolites, with rosmarinic acid being the major compound. It had a high value of TPC and exerted significant AA as shown by the results of the Ferric Reducing Antioxidant Power (FRAP) and Radical Scavenging Activity by DPPH• assays. A dose-dependent cytotoxic potential was recorded, with the highest dose (50 μg mL-1) exhibiting statistically significant cytotoxicity. None of the tested concentrations induced significant micronuclei (MN) frequencies, indicating a lack of genotoxicity. All tested concentrations reduced the MMC-mediated genotoxic effects, with the two lowest showing statistically significant antigenotoxic potential.

The Micronucleus Assay on Cryopreserved Whole Blood.

The in vitro cytokinesis-block micronucleus (CBMN) assay is a widely used technique in radiobiology research, biological dosimetry, genotoxicity studies, and in vitro radiosensitivity testing. This cytogenetic method is based on the detection of micronuclei in binucleated cells resulting from chromosomal fragments lagging during cell division. Fresh whole blood samples are the most preferred sample type for the CBMN assay. However, the disadvantages of working with fresh blood samples include immediate processing after blood collection and the limited number of repeated analyses that can be performed without extra blood sampling. As the need for fresh blood samples can be logistically challenging, CBMN assay on cryopreserved whole blood samples would be of great advantage, especially in large-scale patient studies. This paper describes a protocol to freeze whole blood samples and to perform the CBMN assay on these frozen blood samples. Blood samples from healthy volunteers have been frozen and thawed at different time points and then, subjected to a modified micronucleus assay protocol. The results demonstrate that this optimized procedure allows the performance of the CBMN assay on frozen blood samples. The described cryopreservation protocol may also be very useful for other cytogenetic assays and a variety of functional assays requiring proliferating lymphocytes.

New insights into the size-independent bioactive potential of pristine and UV-B aged polyethylene microplastics

The present study investigates the morphological, physicochemical, and structural changes occurred by the UV-B aging process of low-density polyethylene microplastics (LDPE MPs), as well as the bioactive potential of both pristine and UVaged MPs towards healthy peripheral blood lymphocytes. Specifically, LDPE MPs (100–180 μm) prepared by mechanical milling of LDPE pellets, were UV-B irradiated for 120 days (wavelength 280 nm; temperature 25 °C; relative humidity 50 %) and further examined for alterations in their particle size and surface, their functional groups, thermal stability, and crystallinity (by means of SEM, FTIR spectroscopy, XRD patterns, and TGA measurements, respectively). In parallel, isolated human peripheral blood lymphocytes were treated with different concentrations (25–500 μg mL−1) of either pristine or aged MPs (UVfree and UV120d LDPE MPs) for assessing the cytogenotoxic (by means of trypan blue exclusion test and the cytokinesis-block micronucleus assay using cytochalasin-B) and oxidative effects (using the DCFH-DA staining) in both cases. According to the results, UVfree and UV120d-LDPE MPs, with a size ranging from 100 to 180 μm, can differentially promote cytogenotoxic and oxidative alterations in human lymphocytes. In fact, UVfree LDPE MPs not being able to be internalized by cells due to their size, could indirectly promote the onset of mild oxidative and cytogenotoxic damage in human peripheral lymphocytes, via a dose-dependent but size-independent manner. The latter is more profound in case of the irregular-shaped UV120d-LDPE MPs, bearing improved dispersibility and sharp edges (by means of cracks and holes), as well as oxygen-containing and carbonyl groups. To our knowledge, the present findings provide new data regarding the bioactive behavior of pristine and UV-B aged LDPE MPs, at least in the in vitro biological model tested, thus giving new evidence for their size-independent and/or indirect mode of action.

Measurement of chromosomal instability and level of DNA damage in peripheral blood mononuclear cells of endometrial cancer patients.

Endometrial cancer is one of the most common invasive gynecologic malignancies in developed countries. The aim of this study was to evaluate chromosomal instability and level of DNA damage in peripheral blood mononuclear cells (PBMCs) of newly diagnosed endometrial cancer patients in relation to health status (diagnosis), age, histological grade of cancer, residence, smoking, number of pregnancies, miscarriages, and abortions. The analyzed sample consisted of 60 individuals, 30 endometrial cancer patients with an average age of 64.37 ± 7.08, and 30 healthy control women with an average age of 60.23 ± 11.55. Chromosomal instability was evaluated by the cytokinesis-block micronucleus (CBMN) assay, and the level of DNA damage by the single-cell gel electrophoresis (comet) assay in PBMCs. The average frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) as well as nuclear buds (NBUDs) were significantly higher in cancer patients compared to controls (P < .0005). There was no difference in the nuclear division index (NDI) among the analyzed samples. The comet assay showed that the patients had a significantly increased genetic damage index (GDI) compared with controls (P < .0005). Using linear regression analysis, we found that health status (diagnosis) had the strongest influence on the MN frequency as well as GDI (P < .0005). Our results indicated that there is a high level of genetic damage in both the level of DNA and the level of chromosomes in the PBMCs of newly diagnosed patients with endometrial cancer, where the frequency and level of damage were significantly affected by health status, grade of cancer, residence, number of pregnancies, miscarriages, and abortions.

Decoding the Link Between XPC and Lung Cancer Susceptibility: A Study of Cigarette Smoke-Induced DNA Damage in the Setting of XPC Deficiency

Background/Objective:&#x0D; Despite the known mutagenicity of cigarette smoke, only 10-15% of smokers will develop lung cancer in their lifetimes. What determines a smoker’s susceptibility to lung cancer is poorly understood. We identify the nucleotide excision repair (NER) protein Xeroderma pigmentosum Complementation Group C (XPC) as a tumor suppressor that may contribute to lungtumorigenesis when mutated and combined with cigarette smoke. Micronuclei, which are chromosome fragments and/or lagging chromosomes separated from the main nucleus, occur in many cancers, and indicate genomic instability. We hypothesize that cigarette smoke and XPC knockdown will cause genomic instability that will activate the DNA Damage Response (DDR) and increase the frequency of micronuclei and nuclear aberrancies.&#x0D; Methods:&#x0D; A human bronchial epithelial cell line (Beas-2B), and two lung adenocarcinoma cell lines (H1299 and A549) with stable XPC lentiviral knock-down (shXPC) or control shRNA (shCtrl) were treated with cigarette smoke extract (CSE) or air control (AC). DNA Damage Response (DDR) proteins were analyzed via immunoblot (Western). Micronuclei and nuclear aberrancies were quantified through cytokinesis-block micronucleus assay (CBMT) using immunofluorescence microscopy (DAPI).&#x0D; Results:&#x0D; Both CSE and XPC knockdown independently amplify expression of phospho-ATM (pATM), a DDR protein, in H1299 cells. Nuclear aberrancies increased significantly (p&lt;0.05) with CSE in all three cell lines. Micronucleus frequency increased significantly with CSE in H1299 and Beas-2B cells (p&lt;0.05) and with XPC knockdown in Beas-2B cells compared to shCtrl (p&lt;0.001).&#x0D; Scientific Impact and Implications:&#x0D; We identified a previously uncharacterized role of XPC deficiency in augmenting cigarette smoke induced chromosomal breaks manifesting as micronuclei, particularly in non-cancerous Beas-2B cells. These findings offer insight into tumorigenesis in cigarette smoking and shed light on mechanisms of continued DNA damage in cancer cells. Future research should clarify the mechanisms of micronucleus formation in human translational specimens and pinpoint additional functions of XPC beyond NER, including in replication repair.

Evaluation of Cytogenetic Damage Induced during Hysterosalpingography Procedure: A Cross-sectional Study

Introduction: Hysterosalpingography (HSG) is a radiographic procedure used to visualise the fallopian tubes and uterine cavity by injecting a contrast medium, commonly employed for diagnosing infertility cases. However, due to the associated radiographic and fluoroscopic exposure, there is an increased risk of chromosomal damage to the gonads. Aim: To investigate the impact of radiographic and fluoroscopic exposure on chromosomal integrity in peripheral blood lymphocytes using the Cytokinesis Blocked Micronucleus (CBMN) assay. Materials and Methods: This cross-sectional time-bound study included 10 patients (n=10) scheduled for HSG procedures at the Department of Radiology, Justice KS Hegde Hospital, Mangaluru, Karnataka, India from April 2021 to March 2022. Following radiation exposure, the CBMN assay was conducted to evaluate chromosomal damage in both test and control group blood samples. Blood samples from healthy donors were divided into three cryo vials, with one serving as the control and the remaining two exposed to radiation at the entrance and exit areas during the HSG procedure. The data were expressed as mean±Standard Deviation (SD). A p-value &lt;0.05 was considered statistically significant. The analysis was performed using Statistical Package for Social Sciences (SPSS) version 20.0. Results: A statistically significant (p&lt;0.05) increase in biological damage was observed when comparing the Micronucleus (MN) frequency of the test groups (entrance=0.022±0.005 and exit group=0.0172±0.005) with the control group (0.006±0.003). Additionally, a decrease in MN frequency was noticed within the test groups, particularly in the exit group, although the results were not statistically significant (p=0.066). Conclusion: The HSG is an effective clinical diagnostic technique. However, the present study indicates a potential risk of chromosomal damage associated with the procedure, emphasising the need for judicious use of radiation exposure during HSG.

Occupational exposure to radiation among health workers: Genome integrity and predictors of exposure

The current study aimed to investigate genomic instabilities in healthcare workers who may experience varying levels of radiation exposure through various radiological procedures. It also sought to determine if factors related to the work environment and dosimeter reading could effectively explain the observed genomic instabilities. Utilizing the cytokinesis-block micronucleus assay (CBMN) on peripheral blood lymphocytes, we assessed a spectrum of genomic aberrations, including nucleoplasmic bridge (NPB), nuclear budding (NBUD), micronucleus (MN) formation, and total DNA damage (TDD). The study uncovered a statistically significant increase in the occurrence of distinct DNA anomalies among radiology workers (with a significance level of P < 0.0001 for all measurements). Notably, parameters such as total working hours, average work duration, and time spent in projection radiography exhibited significant correlations with MN and TDD levels in these workers. The dosimeter readings demonstrated a positive correlation with the frequency of NPB and NBUD, indicating a substantial association between radiation exposure and these two genomic anomalies. Our multivariable models identified the time spent in projection radiography as a promising parameter for explaining the overall genomic instability observed in these professionals. Thus, while dosimeters alone may not fully explain elevated total DNA damage, intrinsic work environment factors hold potential in indicating exposure levels for these individuals, providing a complementary approach to monitoring.

Marketable 1,3-dimethylamylamine and caffeine-based thermogenic supplements: Regulatory genotoxicity assessment through in vitro and in silico approaches

ABSTRACT The consumption of dietary supplements to enhance physical performance has increased significantly in the last century, especially thermogenic pre-workout supplements. Nevertheless, this industry has faced criticism for inadequate safety measures surveillance in regulatory issues regarding their products. The aims of our study were to investigate two pre-workout supplements with respect to (1) mutagenicity utilizing Salmonella/microsome assay; (2) genotoxicity employing cytokinesis-block micronucleus (CBMN) assay protocols; and (3) hepatocytoxicity using WST cell proliferation, activities of lactate dehydrogenase (LDH) and alkaline phosphatase using human liver carcinoma (HepG2) and mouse fibroblast (F C3H) cells. Oxidative stress was determined through glutathione (GSH) measurement and in silico for predictions of pharmacokinetics and toxicity for the most abundant isolated substances present in these supplements. Both supplements induced mutagenicity in all examined bacterial strains, especially in the presence of exogenous metabolism. Further, tested supplements significantly elevated the formation of micronuclei (MN) as well as other cellular phenomena. Concentration- and time-dependent curves were observed for hepatotoxicity in both studied cell lines. In addition, both supplements decreased levels of intracellular and extracellular GSH. In silico predictions showed that the isolated individual compounds failed to induce the observed outcomes. Our findings provide contributions to the molecular mechanisms underlying two pre-workout supplement-induced toxicity and the need for surveillance.

Biocompatibility of bulk-fill resins in vitro.

This study aims to evaluate the cytotoxicity and genotoxicity of three different extracts obtained from Filtek™ One Bulk Fill, Tetric Evoceram® Bulk Fill and Coltene Fill-Up! resins. The cytotoxicity was determined on 3T3 fibroblast cells using the MTT and crystal violet assays. The genotoxicity was determined using a cytokinesis-block micronucleus assay. The cytotoxicity of the resin extracts on 3T3 mouse fibroblasts was found to be dose-dependent with both the MTT and crystal violet assays. Extracts concentrated above 1% were cytotoxic according to the MTT assay. The Filtek™ One Bulk Fill, Tetric Evoceram® Bulk Fill, and Coltene Fill-Up! resins reached the LD50 at concentrations of 60%, 50%, and 20%, respectively, and showed genotoxicity rates that were 2-5 times, 3-8 times, and 4-15 times higher than the negative control, respectively. Coltene Fill-Up! resin extracts were the most cytotoxic and genotoxic, followed by Tetric Evoceram® Bulk Fill and Filtek™ One Bulk Fill. The analyzed bulk-fill resins showed differences in in vitro biocompatibility, and the Filtek™ One Bulk Fill was found to be the safest for clinical use.

Cladonia rangiformis Acetone Extract— New Insight into the Chemical Composition and Biological Activity

Introduction Cladonia rangiformis, also known as reindeer lichen, has been used for various remedies in folk medicine. The rich nutritional value of this lichen is reflected in the fact that it contains atranorin, which has been proven to have numerous biological activities. Objective The chemical composition, antioxidant, anticholinesterase, and antigenotoxic activities of the C. rangiformis acetone extract were investigated. The novelty of this work is that anticholinesterase activity, protective effect on human lymphocytes, and antioxidant activity by the cupric-reducing antioxidant capacity (CUPRAC) method of C. rangiformis extract were determined for the first time, as well as gas chromatography - mass spectrometry (GC-MS) profile. Methods Chemical composition was carried out by high-performance liquid chromatography (HPLC)-diode array detector and GC-MS. Antigenotoxic effect was evaluated in human lymphocytes by cytokinesis-block micronucleus assay. Total phenolic content, DPPH and ABTS radical scavenging capacities, CUPRAC, and total reducing power were determined spectrophotometrically. Determination of acetylcholinesterase activity was performed by Ellman's colorimetry assay. The statistical analysis of variance (One-way ANOVA) was performed using Origin software package version 7.0. Results HPLC method was used to identified 3 compounds with atranorin as predominant constituent (97.2%). Fumarprotocetraric and atraric acid were represented in a much smaller mutually approximate amount, 0.3 and 0.5%, respectively. Rangiformic acid was the most abundant volatile compound. Acetone extract of C. rangiformis (2 μg/mL) decreased the frequency of micronuclei by 15.5% while the radioprotectant amifostine reduced the frequency of occurrence of micronuclei by 11.4%. On the contrary, at a concentration of 1.0 mg/mL the extract exhibited an activating effect on cholinesterase (2.0%), while at a concentration of 10.0 mg/mL it showed a weak inhibitory effect (7.3%). The total phenol content was 132.71 μg gallic acid equivalents per mg of dry extract. The IC50 value for the DPPH experiment was 16.19 mg/mL and for ABTS was 11.80 mg/mL. Cupric reducing capacity and total reducing power were 17.81 μg Trolox equivalents per mg of dry extract and 0.45 μg ascorbic acid equivalents per mg of dry extract, respectively. Conclusion In addition to previously identified compounds in the acetone extract, atraric acid was identified for the first time by using HPLC method, and orcinol, β-orcinol, lauric alcohol, atranol, and rangiformic acid by using GC-MS method. The results of biological activity proved that acetone extract had antioxidant capacity and weak anticholinesterase activity. Reduction of the number of micronuclei in human lymphocytes, classified C. rangiformis as promising substances source with beneficial effects on DNA damage.

AZD-7648, a DNA-PK Inhibitor, Induces DNA Damage, Apoptosis, and Cell Cycle Arrest in Chronic and Acute Myeloid Leukemia Cells.

The non-homologous end joining pathway is vital for repairing DNA double-strand breaks (DSB), with DNA-dependent protein kinase (DNA-PK) playing a critical role. Altered DNA damage response (DDR) in chronic (CML) and acute myeloid leukemia (AML) offers potential therapeutic opportunities. We studied the therapeutic potential of AZD-7648 (DNA-PK inhibitor) in CML and AML cell lines. This study used two CML (K-562 and LAMA-84) and five AML (HEL, HL-60, KG-1, NB-4, and THP-1) cell lines. DDR gene mutations were obtained from the COSMIC database. The copy number and methylation profile were evaluated using MS-MLPA and DDR genes, and telomere length using qPCR. p53 protein expression was assessed using Western Blot, chromosomal damage through cytokinesis-block micronucleus assay, and γH2AX levels and DSB repair kinetics using flow cytometry. Cell density and viability were analyzed using trypan blue assay after treatment with AZD-7648 in concentrations ranging from 10 to 200 µM. Cell death, cell cycle distribution, and cell proliferation rate were assessed using flow cytometry. The cells displayed different DNA baseline damage, DDR gene expressions, mutations, genetic/epigenetic changes, and p53 expression. Only HEL cells displayed inefficient DSB repair. The LAMA-84, HEL, and KG-1 cells were the most sensitive to AZD-7648, whereas HL-60 and K-562 showed a lower effect on density and viability. Besides the reduction in cell proliferation, AZD-7648 induced apoptosis, cell cycle arrest, and DNA damage. In conclusion, these results suggest that AZD-7648 holds promise as a potential therapy for myeloid leukemias, however, with variations in drug sensitivity among tested cell lines, thus supporting further investigation to identify the specific factors influencing sensitivity to this DNA-PK inhibitor.

Genome Stability Profiles of Titania Nanosurface Technology for Biomedical Applications

The emergence of nano-based products for advanced biomedical applications has underlined the need for toxicogenomic studies which govern genome stability endpoints. In the present study, the genome stability profiles on cell-TNA interaction were assessed via nuclear staining profile by a fluorescent microscope, DNA ploidy profile by flow-cytometry, metaphase chromosome analysis, DNA topoisomerase II alpha (TOP2A) profiling by chromogenic in situ hybridization (CISH), cytokinesis-blocked micronucleus assay (CBMN), reactive oxygen species (ROS) measurement and gene expression profile by real-time polymerase chain reaction (RT-PCR). Findings from this study demonstrated that no significant genomic instability risk was observed from cell-TNA interaction, especially at clastogenic and aneugenic levels. Findings also suggest that cell-TNA interaction could involve the regulation of ribonucleoprotein complex and cell metabolism associated with the cellular adaptation process towards the nanosurface. Further comprehensive studies involving in vivo models are needed to support this work.

Baseline micronucleus frequencies and 60Co cytokinesis-block micronucleus assay dose-response curve for biodosimetry in Vietnam.

This study aims to establish baseline micronucleus (MN) frequencies from various populations of residents in Vietnam and develop a 60Co dose-response curve for the cytokinesis-block micronucleus (CBMN) assay. Blood samples were exposed in vitro to a 60Co source at a dose rate of 275mGy per min in a range of 0.1 to 4.0Gy. MN background frequencies were 4.5±3.2, 7.3±4.6, 7.0±3.8 and 13.1±6.7 in 1000 binucleated (BN) cells for 96 healthy donors, 22 male radiation workers and 12 breast cancer patients, respectively. Blood samples from three healthy donors were used to generate the MN dose-response curve: y=C + (0.0496±0.0069)D + (0.0143±0.0026)D2. This curve was verified through an inter-laboratory comparison (RENEB ILC 2021). Our findings highlight the significance of the CBMN assay as an additional essential tool for biodosimetry in Vietnam.

Peripheral blood lymphocytes differ in DNA damage response after exposure to X-rays with different physical properties

Introduction: In radiology, low X-ray energies (<140 keV) are used to obtain an optimal image while in radiotherapy, higher X-ray energies (MeV) are used to eradicate tumor tissue. In radiation research, both these X-ray energies being used to extrapolate in vitro research to clinical practice. However, the energy deposition of X-rays depends on their energy spectrum, which might lead to changes in biological response. Therefore, this study compared the DNA damage response (DDR) in peripheral blood lymphocytes (PBLs) exposed to X-rays with varying beam quality, mean photon energy (MPE) and dose rate. Methods: The DDR was evaluated in peripheral blood lymphocytes (PBLs) by the ɣ-H2AX foci assay, the cytokinesis-block micronucleus assay and an SYTOX-based cell death assay, combined with specific cell death inhibitors. Cell cultures were irradiated with a 220 kV X-ray research cabinet (SARRP, X-Strahl) or a 6 MV X-ray linear accelerator (Elekta Synergy). Three main physical parameters were investigated: beam quality (V), MPE (eV) and dose rate (Gy/min). Additional copper (Cu) filtration caused variation in the MPE (78 keV, 94 keV, 118 keV) at SARRP; dose rates were varied by adjusting tube current for 220 kV X-rays (0.33–3 Gy/min) or water-phantom depth in the 6 MV set-up (3–6 Gy/min). Results: The induction of chromosomal damage and initial (30 min) DNA double-stranded breaks (DSBs) were significantly higher for 220 kV X-rays compared to 6 MV X-rays, while cell death induction was similar. Specific cell death inhibitors for apoptosis, necroptosis and ferroptosis were not capable of blocking cell death after irradiation using low or high-energy X-rays. Additional Cu filtration increased the MPE, which significantly decreased the amount of chromosomal damage and DSBs. Within the tested ranges no specific effects of dose rate variation were observed. Conclusion: The DDR in PBLs is influenced by the beam quality and MPE. This study reinforces the need for consideration and inclusion of all physical parameters in radiation-related studies.

Assessment of Micronuclei Frequency in the Peripheral Blood of Adult and Pediatric Patients Receiving Fractionated Total Body Irradiation

The cytokinesis-block micronucleus (CBMN) assay is an established method for assessing chromosome damage in human peripheral blood lymphocytes resulting from exposure to genotoxic agents such as ionizing radiation. The objective of this study was to measure cytogenetic DNA damage and hematology parameters in vivo based on MN frequency in peripheral blood lymphocytes (PBLs) from adult and pediatric leukemia patients undergoing hematopoietic stem cell transplantation preceded by total body irradiation (TBI) as part of the conditioning regimen. CBMN assay cultures were prepared from fresh blood samples collected before and at 4 and 24 h after the start of TBI, corresponding to doses of 1.25 Gy and 3.75 Gy, respectively. For both age groups, there was a significant increase in MN yields with increasing dose (p < 0.05) and dose-dependent decrease in the nuclear division index (NDI; p < 0.0001). In the pre-radiotherapy samples, there was a significantly higher NDI measured in the pediatric cohort compared to the adult due to an increase in the percentage of tri- and quadri-nucleated cells scored. Complete blood counts with differential recorded before and after TBI at the 24-h time point showed a rapid increase in neutrophil (p = 0.0001) and decrease in lymphocyte (p = 0.0006) counts, resulting in a highly elevated neutrophil-to-lymphocyte ratio (NLR) of 14.45 ± 1.85 after 3.75 Gy TBI (pre-exposure = 4.62 ± 0.49), indicating a strong systemic inflammatory response. Correlation of the hematological cell subset counts with cytogenetic damage, indicated that only the lymphocyte subset survival fraction (after TBI compared with before TBI) showed a negative correlation with increasing MN frequency from 0 to 1.25 Gy (r = −0.931; p = 0.007). Further, the data presented here indicate that the combination of CBMN assay endpoints (MN frequency and NDI values) and hematology parameters could be used to assess cytogenetic damage and early hematopoietic injury in the peripheral blood of leukemia patients, 24 h after TBI exposure.

Blood molecular profile to predict genotoxicity from exposure to antineoplastic drugs.

Genotoxicity is an important information that should be included in human biomonitoring programmes. However, the usually applied cytogenetic assays are laborious and time-consuming, reason why it is critical to develop rapid and economic new methods. The aim of this study was to evaluate if the molecular profile of frozen whole blood, acquired by Fourier Transform Infrared (FTIR) spectroscopy, allows to assess genotoxicity in occupational exposure to antineoplastic drugs, as obtained by the cytokinesis-block micronucleus assay. For that purpose, 92 samples of peripheral blood were studied: 46 samples from hospital professionals occupationally exposed to antineoplastic drugs and 46 samples from workers in academia without exposure (controls). It was first evaluated the metabolome from frozen whole blood by methanol precipitation of macromolecules as haemoglobin, followed by centrifugation. The metabolome molecular profile resulted in 3 ratios of spectral bands, significantly different between the exposed and non-exposed group (p<0.01) and a spectral principal component-linear discriminant analysis (PCA-LDA) model enabling to predict genotoxicity from exposure with 73 % accuracy. After optimization of the dilution degree and solution used, it was possible to obtain a higher number of significant ratios of spectral bands, i.e., 10 ratios significantly different (p<0.001), highlighting the high sensitivity and specificity of the method. Indeed, the PCA-LDA model, based on the molecular profile of whole blood, enabled to predict genotoxicity from the exposure with an accuracy, sensitivity, and specificity of 92 %, 93 % and 91 %, respectively. All these parameters were achieved based on 1μL of frozen whole blood, in a high-throughput mode, i.e., based on the simultaneous analysis of 92 samples, in a simple and economic mode. In summary, it can be conclude that this method presents a very promising potential for high-dimension screening of exposure to genotoxic substances.