Significant Increase In Micronucleus Frequency Research Articles

Genotoxicity assessment of eight nitrosamines using 2D and 3D HepaRG cell models.

N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates. There are only limited data on the genotoxicity of nitrosamines in human cell systems. In this study, we used metabolically competent human HepaRG cells, whose metabolic capability is comparable to that of primary human hepatocytes, to evaluate the genotoxicity of eight nitrosamines [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. Under the conditions we used to culture HepaRG cells, three-dimensional (3D) spheroids possessed higher levels of CYP activity compared to 2D monolayer cells; thus the genotoxicity of the eight nitrosamines was investigated using 3D HepaRG spheroids in addition to more conventional 2D cultures. Genotoxicity was assessed as DNA damage using the high-throughput CometChip assay and as aneugenicity/clastogenicity in the flow-cytometry-based micronucleus (MN) assay. Following a 24-h treatment, all the nitrosamines induced DNA damage in 3D spheroids, while only three nitrosamines, NDBA, NDEA, and NDMA, produced positive responses in 2D HepaRG cells. In addition, these three nitrosamines also caused significant increases in MN frequency in both 2D and 3D HepaRG models, while NMBA and NMPA were positive only in the 3D HepaRG MN assay. Overall, our results indicate that HepaRG spheroids may provide a sensitive, human-based cell system for evaluating the genotoxicity of nitrosamines.

Evaluating the role of cytokinesis-block micronucleus assay as a biomarker for oxidative stress-inducing DNA damage in type 2 diabetes mellitus patients

BackgroundType 2 Diabetes Miletus (T2DM) is a common metabolic and lifestyle disorder leading to increased oxidative stress and DNA damage. The present study aims to evaluate the feasibility of utilizing the cytokinesis-block micronucleus assay (CBMN) as a biomarker for assessing the DNA damage induced due to variations in oxidative stress.MethodologyThe study group includes diabetic (n = 50) and non-diabetic (n = 50) subjects. The assays for the diabetes-like fasting blood sugar, postprandial glucose and hemoglobin A1c (HbA1c), lipid profiling, and serum ferritin level along with c-reactive protein (CRP) were applied. Further, the CBMN assay was performed to evaluate the micronuclei present in the lymphocytes of control and T2DM groups.ResultsSignificant imbalance in the glycaemic index, dyslipidemia, increased ferritin levels, and CRP levels, with a significant increase of micronucleus frequency, was found in T2DM patients compared with the control group. Results suggest a trend of positive correlation between HbA1c and the micronuclei, indicating the assay’s potential importance as a biomarker for T2DM-induced risk assessment.ConclusionFrom the observed results, it can be suggested that the CBMN assay could be used to assess the risk of oxidative stress-induced DNA damage in high glycaemic index diabetic patients.

Does cone-beam computed tomography examination increase the micronuclei frequency in the oral mucosa exfoliated cells? A systematic review and meta-analysis

ObjectiveThis systematic review (SR) with meta-analysis aimed to evaluate the frequency of micronuclei in the oral mucosa exfoliated cells after cone-beam computed tomography (CBCT) examination.MethodsWe performed language-independent computer-assisted data searches using PubMed databases, Cochrane, Embase, Web of Science all databases, and Google Scholar. The literature on micronucleus (MN) frequency of clinical trials before and after CBCT examination was included. The frequency of MN in exfoliated cells of the human oral mucosa was the primary outcome of the study. All statistical analyses were performed with R (version 4.1.0), RStudio (version 2022.02.2 + 485) software, and Meta packages (version 5.2–0). Two reviewers independently assessed the quality of the included studies by the EPHPP (Effective Public Health Practice Project) Modified scale with minor modifications. The heterogeneity of the data was analyzed using I2 statistics, in which I2 > 50% was considered substantial heterogeneity.ResultsA total of 559 articles were selected through the search strategy. After screening titles and abstracts, nine full-text manuscripts were assessed for eligibility, and six observational studies were included in the meta-analysis. The present study showed a significant increase in MN frequency of human oral mucosal exfoliated cells 10 days after CBCT examination compared to baseline (SMD = − 0.56, 95%-CI = − 0.99 ~ − 0.13, p = 0.01). Because of the high heterogeneity among the studies (I2 = 72%), after removing one study that was the main source of heterogeneity, excluding the study (I2 = 47%), the common-effect model was chosen, and the meta-analysis also showed that the frequency of MN in human oral mucosa exfoliated cells increased significantly 10 days after CBCT examination (SMD = − 0.35, 95%-CI = − 0.59 ~ − 0.11, p = 0.004).ConclusionThis review suggested that CBCT examination increases the frequency of micronuclei in oral mucosal exfoliated cells.

Assessment of Cytogenetic Damage in Flexographic Workers: Micronucleus Assay in Exfoliated Buccal Epithelial Cells.

Literature review revealed that chemicals used in the printing industry show an association between genotoxicity and occupational exposure. Flexography is one type of printing technique recently becoming popular because of its fast, cost-efficient, and high-quality label printing. The micronucleus (MN) is considered to be a reliable marker for genotoxic damage, and it has a close association with cancer incidences by determining the presence and the extent of the chromosomal damage. Because there are no studies on flexographic workers (FWs), this study was intended to analyze and evaluate the effect of occupational exposure on the MN frequency of buccal epithelial cells. The study comprised 100 FWs and 100 age-matched healthy controls with and without smoking habits. Buccal epithelial cells were collected from all subjects by using a cytobrush, followed by staining with Feulgen fast green. The MN frequency was recorded for each individual using the Tolbert et al. criteria. Data was statistically analyzed by using one-way analysis of variance and the posthoc test. FWs with smoking habits showed a significant increase in MN frequency (1.86 ± 1.77) than workers without the habit (1.02 ± 1.08) and controls (with the habit 1.26 ± 1.33 and without the habit 0.62 ± 0.92). However, there was no significant increase of MN in FWs without the habit when compared with controls. This study observed the cytogenetic damage in FWs and concludes that these workers are at greater risk for genotoxicity, and the MN assay can serve as a useful biomarker.

Evaluation of Clastogenic/Aneugenic Damage Using the FISH Micronucleus Assay in Mice Exposed to Chromium (VI).

Exposure to chromium (VI) [Cr(VI)] has been postulated to be associated with the induction of cancer. In vivo studies utilizing biomarkers of genotoxic damage could aid in elucidating the mechanisms underlying the genotoxic effects of Cr(VI) and their relationship with carcinogenesis. In this study, the origin (clastogenic and/or aneugenic damage) and kinetics of micronuclei (MN) induced by Cr(VI) were investigated. Hsd:ICR female mice were divided into groups of five individuals each. MN kinetics were measured in groups treated with 20 or 25 mg/kg CrO3 intraperitoneally using acridine orange-coated slides in peripheral blood obtained from the caudal vein 0, 12, 24, 36, 48, 60, and 72 h after treatment. Whereas identification of MN with centromeric DNA (MNK+) was measured at the dose of 20 mg/kg of CrO3, using fluorescence in situ hybridization (FISH) with a centromere-specific probe in peripheral blood obtained at 0, 12, and 48 h after treatment. Control groups were administered vehicle only. Total MN were quantified and the clastogenic/aneugenic effects of Cr(VI) were evaluated based on the proportion of MNK+ versus micronuclei without centromeric DNA (MNK-). There was a significant increase in MN frequencies beginning at 12 h in the Cr(VI)-treated groups demonstrating its genotoxicity. When calculating the MNK+ as a percentage of the total MN, the increase was significant beginning 12 h after treatment. The fact that the MNK+ and MNK- were observed at both evaluation times corroborates Cr(VI) as a genotoxic agent and demonstrates that both clastogenic and aneugenic damages are involved in the formation of MN.

An overall assessment of the effects of antidepressant paroxetine on aquatic organisms and human cells

Paroxetine (PRX) is one of the most used antidepressants and an emerging contaminant with potential harmful effects to the environment and human health. The present study investigates in detail the toxic potential of PRX using a battery of bioassays on fresh- and marine species, marine bacteria, and human lymphocytes. All the tested organisms and human lymphocytes were exposed at concentrations ranging from μg L−1 to mg L−1. It was found that PRX can cause toxic effects to aquatic organisms at environmental relevant concentrations (μg L−1 level). A significant effect of PRX was observed in all tested algal species especially at the first 24 h. However, differences in responses and sensitivities among the tested algal species were observed. The most sensitive organism was found to be Dunaliella tertiolecta with IC50 = 0.092 mg L−1 (72 h). In the case of Aliivibrio fischeri, EC50 values were determined to be 16.65, 14.31 and 14.41 mg L−1 for 5, 15 and 30 min of exposure, respectively. PRX also induced cytotoxic and genotoxic effects in human lymphocytes. A dose-dependent increase in micronucleus frequencies was occurred at all tested concentrations with a statistically significant increase in micronucleus frequencies at the medium to high PRX tested concentrations. The findings of the present study expand the available toxicity profile of PRX on aquatic organisms and the knowledge about the potential risk of PRX to induce genotoxic effects in cultured human lymphocytes.

Genotoxicity evaluation of nitrosamine impurities using human TK6 cells transduced with cytochrome P450s.

Many nitrosamines are recognized as mutagens and potent rodent carcinogens. Over the past few years, nitrosamine impurities have been detected in various drugs leading to drug recalls. Although nitrosamines are included in a 'cohort of concern' because of their potential human health risks, most of this concern is based on rodent cancer and bacterial mutagenicity data, and there are little data on their genotoxicity in human-based systems. In this study, we employed human lymphoblastoid TK6 cells transduced with human cytochrome P450 (CYP) 2A6 to evaluate the genotoxicity of six nitrosamines that have been identified as impurities in drug products: N-nitrosodiethylamine (NDEA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutanoic acid (NMBA), N-nitrosomethylphenylamine (NMPA), N-nitrosodiisopropylamine (NDIPA), and N-nitrosodibutylamine (NDBA). Using flow cytometry-based assays, we found that 24-h treatment with NDEA, NEIPA, NMBA, and NMPA caused concentration-dependent increases in the phosphorylation of histone H2A.X (γH2A.X) in CYP2A6-expressing TK6 cells. Metabolism of these four nitrosamines by CYP2A6 also caused significant increases in micronucleus frequency as well as G2/M phase cell-cycle arrest. In addition, nuclear P53 activation was found in CYP2A6-expressing TK6 cells exposed to NDEA, NEIPA, and NMPA. Overall, the genotoxic potency of the six nitrosamine impurities in our test system was NMPA > NDEA ≈ NEIPA > NMBA > NDBA ≈ NDIPA. This study provides new information on the genotoxic potential of nitrosamines in human cells, complementing test results generated from traditional assays and partially addressing the issue of the relevance of nitrosamine genotoxicity for humans. The metabolically competent human cell system reported here may be a useful model for risk assessment of nitrosamine impurities found in drugs.

Protective action of polysaccharides from Laurencia papillose (Rhodophyta) against imidacloprid induced genotoxicity and oxidative stress in male albino rats.

Imidacloprid (IMI), the main component of neonicotinoid insecticides, promotes oxidative stress and genotoxicity in mammals. The aim of this experiment is to assess oxidative stress in liver cells and genotoxicity of erythrocytes for rats exposed to sub-lethal doses of IMI and the protective effects for Rhodophyta as antioxidant material versus imidacloprid. A total of 30 adult male albino rats (average body weight, 190–200 g) were divided into six groups (n=5) as follows: group 1 served as the control, group 2 received 200 mg/kg red algae, group 3 received 45 mg/kg IMI (high-dose group), group 4 received 22.5 mg/kg IMI (low-dose group), group 5 received 200 mg/kg red algae +45 mg/kg IMI, and group 6 received 200 mg/kg red algae +22.5 mg/kg IMI. After 28 d of treatment, the antioxidant activity of the crude extract of red algae was assessed in terms of free radical scavenging activity and found to be higher in TCA (75.57%) followed by DPPH (50.08%) at concentration 100 μg extract and a significant increase in lipid peroxidation and reductions in glutathione were observed in liver cells were intoxicated with high and low doses of IMI. Moreover decreases in catalase and glutathione peroxidase parameters in same previous groups which indicated oxidative stress. In addition significant increases in micronucleus frequency (MN) in the bone marrow of the rats as a genotoxicity marker which indicated DNA damage in erythrocytes cells with alterations in the histopathology of liver cells were also noted such as necrosis, inflammatory cells, infiltration, and necrobiotic changes. Whereas Rhodophyta succeeded in alleviation the oxidative damage and genotoxicity induced by the insecticide. In conclusion, IMI demonstrates hazardous effects, such as alterations in antioxidant status and mutagenicity of erythrocytes and polysaccharides from Rhodophyta has good antioxidant activity in vivo model systems against imidacloprid.

Mitigation of Iron Irradiation-Induced Genotoxicity and Genomic Instability by Postexposure Dietary Restriction in Mice.

Background and Purpose. Postexposure onset of dietary restriction (DR) is expected to provide therapeutic nutritional approaches to reduce health risk from exposure to ionizing radiation (IR) due to such as manned space exploration, radiotherapy, or nuclear accidents as IR could alleviate radiocarcinogenesis in animal models. However, the underlying mechanisms remain largely unknown. This study is aimed at investigating the effect from postexposure onset of DR on genotoxicity and genomic instability (GI) induced by total body irradiation (TBI) in mice. Materials and Methods. Mice were exposed to 2.0 Gy of accelerated iron particles with an initial energy of 500 MeV/nucleon and a linear energy transfer (LET) value of about 200 keV/μm. After TBI, mice were either allowed to free access to a standard laboratory chow or treated under DR (25% cut in diet). Using micronucleus frequency (MNF) in bone marrow erythrocytes, induction of acute genotoxicity and GI in the hematopoietic system was, respectively, determined 1 and 2 months after TBI. Results and Conclusions. TBI alone caused a significant increase in MNF while DR alone did not markedly influence the MNF. DR induced a significant decrease in MNF compared to the treatment by TBI alone. Results demonstrated that postexposure onset of DR could relieve the elevated MNF induced by TBI with high-LET iron particles. These findings indicated that reduction in acute genotoxicity and late GI may be at least a part of the mechanisms underlying decreased radiocarcinogenesis by DR.

Heavy metal accumulation and genotoxic effect of long-term wastewater irrigated peri-urban agricultural soils in semiarid climate

Water scarcity is becoming an alarming issue in the Mediterranean countries. Therefore, using the treated wastewater in the irrigation is considered as a valuable option. However, uncontrolled and long-term irrigation by wastewater leads to human health and environmental damages, mainly related to some specific pollutants. The assessment of the availability and toxicity of the heavy metals after long term irrigation, under semi-arid climate, is not yet well documented. In this study, physicochemical properties, genotoxicity (Vicia faba micronucleus test), total and available (CaCl2-extractable) concentrations of Cr, Pb, Cu, Zn, Co and Cd in eight soils of peri-urban farms irrigated with wastewater were examined to evaluate their accumulation.The results indicated that long-term irrigation with wastewater induced significant increase of electrical conductivity, organic matter, calcium carbonate equivalent and nutrient availability. Total and available concentration of heavy metals were significantly higher (P < 0.05) in irrigated soils by wastewater. The total concentrations of Zn, Pb, Cu, Cr, Cd and Co in irrigated soils by wastewater at 0–40 cm depth were 85.69, 43.94, 34.86, 14.62, 9.94 and 7.17 mg kg−1, respectively. Furthermore, the increase of the available metal fraction in irrigated soils by wastewater at 0–40 cm depth followed the following order: Co (1270.1%) > Cd (914.5%) > Cu (881.5%) > Cr (471.2%) > Pb (230.8%) > Zn (223.8%). The micronucleus assay indicated significant increase of micronucleus frequencies (41.25‰, 35.48‰, 21.66‰, 16.23‰ and 13.62‰ respectively for P1, P2, P3, P4 and P7) which were higher than the negative control (0‰) and the irrigated soil by fresh water (3.29‰). The micronucleus induction was significantly correlated with the high available fraction of Cd, Co and Zn at P1, P2 and P7. The genotoxicity can be a powerful test to assess the ecological effects associated with the interactions of heavy metals with other pollutants.

Micronucleus Formation Induced by Glyphosate and Glyphosate-Based Herbicides in Human Peripheral White Blood Cells.

Glyphosate is the most commonly used herbicide around the world, which led to its accumulation in the environment and consequent ubiquitous human exposure. Glyphosate is marketed in numerous glyphosate-based herbicide formulations (GBHs) that include co-formulants to enhance herbicidal effect of the active ingredient, but are declared as inert substances. However, these other ingredients can have biologic activity on their own and may interact with the glyphosate in synergistic toxicity. In this study, we focused to compare the cytogenetic effect of the active ingredient glyphosate and three marketed GBHs (Roundup Mega, Fozat 480, and Glyfos) by investigating cytotoxicity with fluorescent co-labeling and WST-1 cell viability assay as well as genotoxicity with cytokinesis block micronucleus assay in isolated human mononuclear white blood cells. Glyphosate had no notable cytotoxic activity over the tested concentration range (0–10,000 μM), whereas all the selected GBHs induced significant cell death from 1,000 μM regardless of metabolic activation (S9). Micronucleus (MN) formation induced by glyphosate and its formulations at sub-cytotoxic concentrations (0–100 μM) exhibited a diverse pattern. Glyphosate caused statistically significant increase of MN frequency at the highest concentration (100 μM) after 20-h exposure. Contrarily, Roundup Mega exerted a significant genotoxic effect at 100 μM both after 4- and 20-h exposures; moreover, Glyfos and Fozat 480 also resulted in a statistically significant increase of MN frequency from the concentration of 10 μM after 4-h and 20-h treatment, respectively. The presence of S9 had no effect on MN formation induced by either glyphosate or GBHs. The differences observed in the cytotoxic and genotoxic pattern between the active principle and formulations confirm the previous concept that the presence of co-formulants in the formulations or the interaction of them with the active ingredient is responsible for the increased toxicity of herbicide products, and draw attention to the fact that GBHs are still currently in use, the toxicity of which rivals that of POEA-containing formulations (e.g., Glyfos) already banned in Europe. Hence, it is advisable to subject them to further comprehensive toxicological screening to assess the true health risks of exposed individuals, and to reconsider their free availability to any users.

Assessment of Peripheral Blood Lymphocytes in Parents of Autistic Children by Cytokinesis Block Micronucleus Assay.

Autism spectrum disorders are neurodevelopmental complex diseases with causative de-novo and inherited genetic factors. They include a range of cognitive and behavioral conditions such as pervasive developmental disorder, Asperger's syndrome, and autism. Cytokinesis-block micronucleus assay, as a cytogenetic study has been considered as one of the indicators of chromosomal damage in peripheral blood. This study aimed to investigate the frequency of micronucleus (MN) in peripheral blood lymphocytes of parents with autistic children. The study was case-control and the cases were parents of autistic children referring to the psychiatric department of the Ali-Asghar Hospital of Tehran. The total number of samples was 60 cases and 30 controls. The results showed that autistic children's parents had a significant increase in MN frequency in binucleated lymphocytes. Further researches are suggested to analyze the environmental and genetic reasons for MN increase in autistic children parents.

In vivo experimental study to investigate cytogenotoxicity of a contaminated estuary from Southeastern Brazilian Coast

The aim of this study was to evaluate cytogenotoxicity in mammalian cells induced by ingestion of superficial water from SESS. For this purpose, surface water was collected from two points of SESS: São Vicente Channel (SVC) and Piaçaguera Channel (PIC). Four groups (n = 5) of adult male Wistar (8 weeks old) received for 5 days: (a) filtered tap water (water control), (b) tap water with 2.4% of NaCl (saline control), (c) estuarine water from PIC and (d) estuarine water from SVC. Results demonstrated that Ki67 immunoexpression was higher in hepatocytes exposed to both sampling site, while caspase-3 demonstrated downregulation in rat liver exposed to estuarine water. There was also significant increase in micronuclei frequency in bone marrow cells and hepatocytes, and DNA damage in blood and liver of rats exposed to estuarine water from SVC and PIC. In summary, studies with complex mixtures, such as contaminated estuarine water are important since this work confirmed by experiments using in vivo mammalian cells of rats that SESS water are genotoxic, mutagenic and cytotoxic, denoting concern for environmental health.

Geno-cytotoxicity and congenital malformations produced by relevant environmental concentrations of aluminum, diclofenac and their mixture on Cyprinus carpio. An interactions study

Several studies highlight the presence of aluminum and diclofenac in water bodies around the world and their ability to induce oxidative stress and a negative effect on biomolecules in several aquatic species. However, studies evaluating the toxic effect of mixtures of these contaminants are scarce. The objective of this work was to determine the genotoxic, cytotoxic and embryotoxic effect of the mixture of aluminum and diclofenac at environmentally relevant concentrations on Cyprinus carpio.Juveniles of Cyprinus carpio were exposed to 0.31 μg L−1 of diclofenac, 24.45 mg L-1 of aluminum, and a mixture of both contaminants at the same concentrations for 12, 24, 48, 72 and 96 h. After the exposure time the liver, gills and blood were extracted and the following biomarkers were evaluated: micronucleus frequency, comet assay, caspase activity and TUNEL test. On the other hand, Cyprinus carpio embryos were exposed to diclofenac (0.31 μg L−1), aluminum (0.06 mg L−1) and their mixture at the same concentrations and exposure time. Microscopic observation was performed to evaluate embryonic development at 12, 24, 48, 72 and 96 h.Diclofenac (0.31 μg L−1) induces significant increases in micronucleus frequency with respect to control (p < 0.05), in all tissues. Aluminum (24.45 mg L−1) significantly increases DNA damage index in liver and blood cells with respect to control (p < 0.05). All treatments increase caspases activity in all tissues with respect to control (p < 0.05). Diclofenac increases the percentage of TUNEL-positive cells in liver and blood; while aluminum and the mixture increases it significantly in gills and blood with respect to the control (p < 0.05). The mixture significantly delays embryonic development, while aluminum and the mixture significantly increase teratogenic index with respect to control (p < 0.05). In conclusion, exposure to environmental concentrations of aluminium, diclofenac and their mixture induces genotoxic damage, cell death by apoptosis and negative effects on the development of Cyprinus carpio and the toxic response is modified by the interaction of the xenobiotics.

Assessment of In Vitro Antigenotoxic Effect of Nigella Sativa Oil.

Cyclophosphamide (CP) is an alkylating agent widely used as an antineoplastic and immunosuppressive agent. The genotoxicity of CP has been studied in a variety of in vivo and in vitro systems and is routinely used as a positive control in genotoxicity tests. Traditional medicine Nigella sativa L., (N. sativa), Ranunculaceae family, especially in the Eastern Mediterranean countries, especially in many countries, and is widely used in many countries as a spice and folk medicine since the time of Dioscorides used as a plant. In this study, it was aimed to show the protective effects of N. sativa oil at different concentrations against the genotoxic effects of CP by micronucleus test. For this purpose, healthy cells were treated in vitro with N. sativa oil at concentrations of 1, 5, 10 μg/mL and CP as positive control for 68 hours. The micronuclei were then counted. No significant increase in micronucleus frequency was observed when the application of N. sativa oil at concentrations of 1, 5, 10 μg/mL compared with the negative control. There was a decrease in the number of micronucleus in all three concentrations (1, 5, 10 μg/mL) compared to the CP group in the groups treated with N. sativa oil and CP. It has been shown that N. sativa oil may have protective effects against genotoxicity agents in vitro. But more work is needed to understand the mechanism of the genotoxicity effects of N. sativa oil.

Assessing a Freshwater Ecosystem Using Tradescantia Model Test Object

The clastogenicity of the water of a lake was investigated using the Tradescantia micronuclei test (Trad-MCN). Genomic damage in the plant’s generative sphere is manifested by a significant increase in micronuclei frequency in all samples. An integrative statistical analysis linked the test endpoints with metals present in low concentration in the lake’s water. Thorough comparisons of the results obtained from Tradescantia stamen hair mutation test (Trad-SHM) were conducted for each of the seven study stations. The performances of both tests used for indicating the genotoxic potential of the lake’s water were compared. Although both tests demonstrated significant genetic disturbances (in pollen mother cells of the plant and during active cell division in the stamen hair), a similar indication of the level of toxicity per site has been produced by the endpoints of Trad-MCN and the non-surviving stamen hair endpoint of Trad-SHM. The integration of the results of both Tradescantia-based assays could be recommended for improving the assessment of the genotoxic potential of natural freshwater.

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry.

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

Micronucleus evaluation of exfoliated buccal epithelial cells from automobile spray painters – A preliminary study

Automobile spray painters in the Mechanic village at Coal Camp, Enugu State, Nigeria are exposed to genotoxic agents such as the automobile paints and spent engine lubricants which contaminate the soil of their working environment. The present study applied the micronucleus (MN) test on exfoliated oral mucosal cells as a means to assess the genotoxicity risk associated with occupational exposure to genotoxic agents. Two (2) groups of subjects which comprised 30 automobile spray painters and a control group of 30 apparently healthy unexposed volunteers were enrolled in the study. The study participants were all males. Moistened wooden spatulas were used to obtain buccal smears of the participating individuals, and the smears were stained. The presence of MN was assessed under light microscopy and a total of 1000 cells per individual were scored. The results obtained showed that statistically significant increase in MN frequency in buccal epithelial cells of automobile spray painters when compared with the control group (p&lt;0.05). Elevated MN frequency was also observed withincreased age, smoking and alcohol consumption habits. MN frequency was significantly affected by the duration of working experience (years) of the spray painters whereas no difference was observed with number of workinghours/day. In conclusion, the present study has revealed that automobile spray painters in the Mechanic village of Coal Camp, Enugu State could be under risk of cytogenetic damage from exposure to genotoxic chemicals.Key words: Micronuclei frequency; Exfoliated buccal cells; Smoking; Alcohol consumption, Spray Painters; Genotoxicity;

The Role of Micronucleus Assay to Detect Genetic Instability in Respiratory Cancer Patients.

Asbestos represents the main risk factor for malignant pleural mesothelioma (PM) even if only a minority of exposed people develop this tumor, suggesting a significant role for genetic susceptibility. This study aims to evaluate micronuclei (MN) frequency as a biomarker of genome instability in peripheral blood lymphocytes of PM and lung cancer (LC) patients when compared with healthy controls (HCs) and patients with nonneoplastic respiratory diseases (RDs). Lymphocyte cytokinesis-block MN assay was carried out on 317 subjects. Mutagen sensitivity, measured by quantifying MN frequency after an in vitro challenge with ionizing radiation, was evaluated in 252 subjects. A significant increase in MN frequency was observed in cancer patients compared to HCs, with a mean ratio (MR) of 1.35 and 1.36 at baseline and 1.43 and 1.38 after irradiation in PM and LC patients, respectively. A positive (synergistic) interaction between asbestos exposure and disease status was observed for MN frequency after irradiation in PM patients with possible exposure to asbestos (MR = 1.62). The evidence of increased genetic damage in cancer cases confirms lymphocyte cytokinesis-block MN assay as a sensitive predictor of the role of genetic instability in carcinogenic processes.

Genotoxic and Chemopreventive Effects of Vochysia divergens Leaves (Pantanal, Brazil).

The medicinal plant Vochysia divergens is a colonizing tree species of the Pantanal, a unique and little explored wetland region in Brazil. This species is used in folk medicine as syrups and teas to treat respiratory infections, digestive disorders, asthma, scarring, and skin diseases. The objectives of this study were to evaluate the antioxidant, cytotoxic, and genotoxic potential of the ethanolic extract of Vochysia divergens leaves (VdE), as well as the influence of VdE and its major component (the flavone 3′,5-dimethoxy luteolin-7-O-β-glucopyranoside; 3′5 DL) on MMS-induced genotoxicity. The extract significantly reduced the viability of V79 cells in the colorimetric XTT assay at concentrations ≥ 39 μg/mL. A significant increase in micronucleus frequencies was observed in V79 cell cultures treated with VdE concentrations of 160 and 320 μg/mL. However, animals treated with the tested doses of VdE (500, 1000, and 2000 mg/kg b.w.) exhibited frequencies that did not differ significantly from those of the negative control group, indicating the absence of genotoxicity. The results also showed that VdE was effective in reducing MMS-induced genotoxicity at concentrations of 20, 40, and 80 μg/mL in the in vitro test system and at a dose of 15 mg/kg b.w. in the in vivo test system. Its major component 3′5 DL exerted no protective effect, suggesting that it is not responsible for the effect of the extract. The results of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay showed that VdE was able to scavenge 92.6% of free radicals. In conclusion, the results suggest that the protective effect of VdE may be related, at least in part, to the antioxidant activity of its chemical constituents.