The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory ...acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test. Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation. In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed, but a consensus regarding acceptability for regulatory purposes could not be reached at that time. Subsequent validation efforts, combined with accumulated published data, demonstrate that blood-derived reticulocytes from rats as well as mice are acceptable when young reticulocytes are analyzed under proper assay protocol and sample size. The working group reviewed the results of micronucleus assays using target cells/tissues other than hematopoietic cells. We also discussed the relevance of the liver micronucleus assay using young rats, and the importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have been integrated into the data bases, enhancing confidence in the utility of these models. A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not dose-limiting.
Background and purpose: Cilostazol is a prescription medication for intermittent claudication in patients with peripheral artery disease. Previous research has demonstrated that this 2-oxoquinoline ...derivative possesses antithrombotic, vasodilator, antimitogenic, and antioxidant effects. Cilostazol exerts its products through the inhibition of PDE3 activity and the prevention of cAMP degradation. The present study aimed to examine the protective properties of cilostazol against carboplatin-induced cytotoxicity and genotoxicity in Beas-2B and human blood lymphocyte cells. Materials and methods: cells were pre-treated with different concentrations of cilostazol (5, 25, 50, and 100 μM) with carboplatin at an optimum cytotoxic dosage (9.2 µM). The MTT and micronucleus assays were used to assess cytotoxicity and genotoxicity. Statistical analysis was performed using the one-way ANOVA in Prism Ver. 8 software. Results: The cytotoxic effect of carboplatin was dose-dependent, as evidenced by the 48h culture treatment with concentrations of 0.3, 1, 3, 10, and 30 µM. Pre-treatment of cilostazol at 25, 50, and 100 µM with carboplatin at 9.2 µM enhanced cell viability in Beas-2B cells compared to the carboplatin alone as positive control. Additionally, cilostazol at 50 and 100 µM showed its potent genoprotective effects via micronucleus assay against carboplatin at IC50 at blood lymphocyte cells. Conclusion: Cilostazol provided conceivable protective effects by modulating cytotoxicity and genotoxicity induced by carboplatin in Beas-2B and human blood lymphocyte cells.
Oxygenated polycyclic aromatic hydrocarbons (oxy-PAHs) are a group of environmental pollutants found in complex mixtures together with PAHs. In contrast to the extensively studied PAHs, which have ...been established to have mutagenic and carcinogenic properties, much less is known about the effects of oxy-PAHs. The present work aimed to investigate the genotoxic potency of a set of environmentally relevant oxy-PAHs along with environmental soil samples in human bronchial epithelial cells (HBEC). We found that all oxy-PAHs tested induced DNA strand breaks in a dose-dependent manner and some of the oxy-PAHs further induced micronuclei formation. Our results showed weak effects in response to the oxy-PAH containing subfraction of the soil sample. The genotoxic potency was confirmed in both HBEC and HepG2 cells following exposure to oxy-PAHs by an increased level of phospho-Chk1, a biomarker used to estimate the carcinogenic potency of PAHs in vitro. We further exposed zebrafish embryos to single oxy-PAHs or a binary mixture with PAH benzoapyrene (BaP) and found the mixture to induce comparable or greater effects on the induction of DNA strand breaks compared to the sum of that induced by BaP and oxy-PAHs alone. In conclusion, oxy-PAHs were found to elicit genotoxic effects at similar or higher levels to that of BaP which indicates that oxy-PAHs may contribute significantly to the total carcinogenic potency of environmental PAH mixtures. This emphasizes further investigations of these compounds as well as the need to include oxy-PAHs in environmental monitoring programs in order to improve health risk assessment.
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•Genotoxic potency of oxy-PAHs and soil samples in HBECs and zebrafish embryos.•Oxy-PAHs induced DNA strand breaks and micronuclei in a dose-dependent manner.•Binary mixtures with BaP caused both additive and non-additive genotoxic effects.
Background: The present study aims to assess chromosomal instabilities between fertile couples and couples with recurrent pregnancy loss (RPL) and the causative relation of chromosomal instabilities ...with RPL. Methods: A case-control study was performed with a study sample of 27 couples with a history of RPL who attended the Department of Obstetrics and Gynecology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) and twenty-seven healthy fertile couples as controls. The procedure done was Cytokine Blocked Micronucleus Assay (CBMN). After obtaining consent and details of the couple, 3 ml of heparinized blood was collected from cases and controls. Cases comprising couples with RPL (gestational age ≤ 24 weeks) were included, and couples with a history of diabetes mellitus, thyroid disorders, and hypertension were excluded. Among cases, women with a history of two or more two spontaneous abortions ≤ 24 weeks of gestation were selected. Blood was collected and assessed from both male and female partners. The lymphocytes were cultured per the standard protocol and were screened as the number of micronuclei per 1000 binucleate cells under X 200 magnification in CBMN. Results: Chromosomal instabilities in the form of micronuclei in cases were found to be 7.52±3.99 and 0.07±0.26 in controls (p˂0.05). A statistically significant difference was revealed among those with and without chromosomal instability. Conclusion: Chromosomal instability serves as a significant causative factor for those couples leading to pregnancy losses
Freshwater fish Labeo rohita were exposed to 0.03, 0.06, 0.09, 0.12 and 0.15 mg/L fipronil concentrations for 9 days in triplicate groups. Among signs loss of coordination, increased opercular ...movement, and tremors of fins were noted. Erythrocytic indices, lymphocytes, and monocytes decreased while total leukocyte counts, and neutrophils increased significantly. Relative weight of kidneys, gills, heart, and brain were significantly reduced. Gills and kidneys showed severe lesions. Erythrocytes showed a variety of nuclear abnormalities. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) increased significantly in treated fish. It was concluded that fipronil induced severe clinical symptoms and adverse hemato-biochemical values in rohu fish, even at low concentrations.
During almost 40 years of use, the micronucleus assay (MN) has become one of the most popular methods to assess genotoxicity of different chemical and physical factors, including ionizing ...radiation-induced DNA damage. In this minireview, we focus on the position of MN among the other genotoxicity tests, its usefulness in different applications and visibility by international organizations, such as International Atomic Energy Agency, Organization for Economic Co-operation and Development and International Organization for Standardization. In addition, the mechanism of micronuclei formation is discussed. Finally, foreseen directions of the MN development are pointed, such as automation, buccal cells MN and chromothripsis phenomenon.
Abstract
At any moment, the continuous usage of medications can accompanied by DNA damage and the accumulation of such damages can cause serious consequences. Antidepressants are long-term used drugs ...and the incidence of their genotoxic impacts cannot be excluded. Therefore, this work was designed to investigate the possible genotoxic effects of the commonly used antidepressants (fluoxetine and amitriptyline) in adult male rats.
Detection of DNA damage in individual cells was assessed by comet and micronucleus assays in three different cell populations i.e. liver, testis and bone marrow tissues of 24 swiss albino adult male rats. The animals were randomly allocated into three groups of 8 rats each: Group I - rats orally-administered distilled water via gavage tube for four weeks as a negative control. Group II - rats orally-treated with fluoxetine hydrochloride solution (7.2mg/kg/day) via gavage tube for four weeks. Group III - rats orally-treated with amitriptyline hydrochloride solution (27mg/kg/day) via gavage tube for four weeks.
The results showed that both drugs (Group II and Group III) induced the same extent of DNA damage, as evidenced by a significantly higher DNA fragmentation in liver and testis tissues with increased frequencies of micronuclei formation in bone marrow tissues as compared with the negative control (Group I).
These findings indicates that both Fluoxetine and Amitriptyline have genotoxic potentials and can induce the same extent of cytogenetic damage in rats. Special precautions and medical supervision should be taken in consideration with their uses.
In recent years, the genotoxicity of graphene-related materials (GRMs) and other 2D materials has been evaluated using different models to ensure their safety. The OECD TG 487 in vitro micronucleus ...(MNu) test is one of the most widely used methods to assess the genotoxicity of nanomaterials (NMs). However, the nature of NMs interferes with the standard MNu process, thus requiring an adaptation to assess this kind of compound. Improvements have been incorporated into the standard method to solve the interferences and build a modified, reliable protocol that can be used to test NMs further. The main interference observed was NM agglomeration, which hinders the correct visualization of micronuclei. Addition of 10% dextran to the culture medium reduced agglomeration but interfered with toxicity by reducing NM-induced cell death. Mechanical shaking during cell incubation with NMs minimized the size and number of agglomerates and avoided their rapid sedimentation and deposition onto cells, thus allowing accurate visualization of the micronucleate cells. Once set up, the proposed method was tested with graphene oxide (GO), few-layer graphene (FLG), molybdenum disulfide (MoS2), and boron nitride (hBN). The novel in vitro MNu test for NMs showed that GO (0.05–5 μg/ml) and hBN (0.05 μg/ml) were genotoxic at sublethal doses, inducing irreparable chromosomal damage. With these adaptations, the modified in vitro MNu test is strongly recommended to test the genotoxicity of NMs. The results obtained with this protocol indicate that GO and hBN represent a risk that should be considered.
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•Cyto-b interferes with the MNu assay with graphene-related materials but not with other 2D materials.•NM agglomeration interferes with the assay but it can be solved by introducing mechanical shaking during cell incubation.•Shaking improves the effective concentration of NMs delivered to the cells.•GO and hBN are genotoxic at low concentrations and cause irreparable DNA damage.
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•The CBMN cytome assay is typically performed by visual microscopy.•The assay includes three biomarkers of DNA damage (MN, NPB, NBUD).•The assay also includes three biomarkers of ...cytotoxicity (NDI, necrosis, apoptosis).•Automated microscopy or flow cytometry cannot score the full range of biomarkers.•Performing the assay by imaging flow cytometry may overcome these limitations.
The lymphocyte Cytokinesis-Block Micronucleus (CBMN) assay was originally developed for the measurement of micronuclei (MN) exclusively in binucleated (BN) cells, which represent the population of cells that can express MN because they completed nuclear division. Recently the assay has evolved into a comprehensive cytome method to include biomarkers that measure chromosomal instability and cytotoxicity by quantification of nuclear buds (NBUDs), nucleoplasmic bridges (NPBs) and apoptotic/necrotic cells. Furthermore, enumeration of mono- and polynucleated cells allows for computation of the nuclear division index (NDI) to assess mitotic activity. Typically performed by manual microscopy, the CBMN cytome assay is laborious and subject to scorer bias and fatigue, leading to inter- and intra-scorer variability. Automated microscopy and conventional flow cytometry methods have been developed to automate scoring of the traditional and cytome versions of the assay. However, these methods have several limitations including the requirement to create high-quality microscope slides, lack of staining consistency and sub-optimal nuclear/cytoplasmic visualization. In the case of flow cytometry, stripping of the cytoplasmic membrane makes it impossible to measure MN in BN cells, calculate the NDI or to quantify apoptotic or necrotic cells. Moreover, the absence of cellular visualization using conventional flow cytometry, makes it impossible to quantify NBUDs and NPBs. In this review, we propose that imaging flow cytometry (IFC), which combines high resolution microscopy with flow cytometry, may overcome these limitations. We demonstrate that by using IFC, images from cells in suspension can be captured, removing the need for microscope slides and allowing visualization of intact cytoplasmic membranes and DNA content. Thus, mono-, bi- and polynucleated cells with and without MN can be rapidly and automatically identified and quantified. Finally, we present high-resolution cell images containing NBUDs and NPBs, illustrating that IFC possesses the potential for completely automated scoring of all components of the CBMN cytome assay.
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