Exposure of the human population to physical agents such as radiation, biological agents, and chemicals can be very high, particularly when there is mishandling, technical malfunctions, or accident. Human biomonitoring offers important insights into exposure outcomes and aids in identifying health hazards, which is crucial for assessing the effects of environmental stressors on human health. Using several sources and routes, biomonitoring enables the thorough data gathering of an individual's total exposure. Assessing the combined risks of different exposures, as well as their interactions and corresponding levels, is still a challenging task (Gajski et al., 2024). An array of biological effects induced by different agents depends on its concentration, stability, and biological half-life, as well as the incident's circumstances (weather, terrain, time, and environment). Genetic alterations play a key role in the development and progression many disorders and disease including neoplasms. Investigating the effects of various genotoxic agents is vital, as these substances can harm genetic material through multiple mechanisms. Gaining insight into their specific actions, consequences, and associated risks is key to, a) understanding mechanism of action b) environmental and occupational health c) therapeutic development and d) risk assessment and regulation (Wu and Liu, 2022). Cytogenetic techniques are employed in human biomonitoring to assess the occurrence and degree of chromosomal damage in populations exposed to various genotoxic substances (Anwar, 1994; Nguyen et al., 2024). In addition to providing useful information on accumulated genetic damage during an individual's lifespan with effect biomarkers, spontaneous or baseline frequencies of various cytogenetic parameters can also complement exposure data with mechanistically-based information (Gnanasekaran, 2021; Tamizh Selvan and Venkatachalam, 2024a).

Peripheral blood lymphocytes were employed to analyse different genetic markers that enable the evaluation of genotoxic effects. These comprise sister chromosomal aberration analysis (Lee et al., 2019), cytokinesis blocked micronucleus (CBMN) assay (Nersesyan et al., 2016), gamma-H2AX (Ma et al., 2023), gene expression and transcriptomic approaches (Gollapalli et al., 2024; Sekaran et al., 2021) and comet also known as single-cell microgel electrophoresis, are used to identify initial DNA damage with higher sensitivity (Azqueta et al., 2020). While many assays are available to monitor the exposed individuals, limited tools could discriminate the nature of genotoxic agents. Such identification is an important measure, particularly in the absence of information on the nature of exposure during accidents where many peoples can get exposed. The CBMN cytome assay has the potential to differentiate the nature of DNA damaging agent, quantify the amount of induced damage and also the type of cell death (Fenech, 2006). At the cytogenetic level, micronuclei (MN) formation serves as a marker of chromosomal damage. These structures can arise from acentric fragments caused by clastogens or from lagging chromosomes due to spindle poisons, typically forming during nuclear envelope reassembly after mitosis. The cytokinesis-block technique, which uses cytochalasin B, enables the identification of dividing cells by generating binucleated cells and the MN detected in these cells provide a reliable indicator of clastogen or aneugen activity (Nersesyan et al., 2016). In human investigations, the assay serves as a biological dosimeter for ionising radiation exposure (Tamizh Selvan et al., 2021) an indication of mutagen and radiation sensitivity, a measure of genomic integrity and DNA repair capability (also in the context of other disorders) (Tamizh Selvan and Venkatachalam, 2024b), and a predictor of cancer susceptibility/risk. The CBMN assay using human lymphocytes is frequently utilised as an in vitro test in genotoxicity testing in addition to its application in human investigations (Sommer et al., 2020).

While conventional CBMN assay in uniformly stained binucleated cells did not permit to differentiate the clastogenic and aneugenic events (Rosefort et al., 2004), alternative approaches like, measuring the MN size (Nüsse et al., 1996), DNA content (Nüsse et al., 1996), antikinetochore antibody (CREST) staining (Rosefort et al., 2004), and fluorescence in-situ hybridisation (FISH) with DNA probes that bind specifically in the centromeric region of chromosomes (Salassidis et al., 1992) were used. The in vitro CBMN assay, when combined with FISH using pancentromeric probes can be effectively used to detect clastogens and anugens (Zgno et al., 1994). This CBMN assay combined with FISH was used extensively to study accidental overexposures (Jalil and Molla, 1992), industrial radiographers (Sari-Minodier et al., 2002), mining industries (Kryscio et al., 2001), occupational exposures (Thierens et al., 2000) and biodosimetry (Wojcik et al., 2000). Of late FISH approach has been applied for measuring chromosomal loss, through the detection of MN in cultured cells, in order to discriminate MN produced by chromosome breakage from those arising by spindle malfunction (Ng and Hande, 2024). In modern perspectives nanoparticles are being used for purpose of cancer treatment and drug delivery as well as interactions of nanoparticles with biological systems are reported (Khalilov et al., 2024; Nasibova et al., 2024). Due to their small size and large surface area, nanoparticles are capable of penetrating biological membranes. This allows them to interact directly with DNA or interfere with mitotic spindle formation during cell division, potentially resulting in mutagenesis or carcinogenesis (Shukla et al., 2021). To investigate nanoparticle-induced genotoxicity, CBMN assay, in combination with FISH using centromeric probes, serves as a valuable analytical approach. In this study, we present the results of the CBMN assay in combination with FISH to study the origin of MN induced by different DNA damaging agents namely X-ray, radio mimetic drug bleomycin (BLM), anti-cancer drug mitomycin-C (MMC) and spindle poison colchicine (COL) and discussed its potential application for bio-monitoring of exposed population.