Induction Of Micronuclei Research Articles

Micronucleus Induction in Vicia faba Root Tips by Crude Oil-Polluted Soil from Ecuadorian Amazon

In the past four decades, the Amazon rainforest has emerged as a crucial zone for crude oil extraction in the South American region. In the Ecuadorian Amazon rainforest, hydrocarbon blocks (called “bloques”) cover vast zones, including agricultural and livestock farms, protected natural regions and the territories of uncontacted indigenous tribes. This study proposes a micronuclei assay on Vicia faba following a 24 h exposure to various soil samples collected from Bloque 57 in Ecuador. Sampling was conducted between the Dayuma and Aguarico zones, approximately 30 km from Nueva Loja city. The research aimed to assess the impact of different soil samples, particularly those from areas affected by crude oil spills, to induce micronuclei and mitotic index changes in V. faba roots. Results: The soil pollution caused by crude oil is not the sole factor contributing to cytotoxicity and genotoxicity in V. faba. Most samples from areas polluted by crude oil outside the small-scale farm showed no significant difference in micronuclei rate compared to negative control and Amazon unpolluted soil. Conversely, samples from the small-scale farm displayed a statistically significant genotoxic effect. Furthermore, samples collected from open-air wastewater pools demonstrated higher levels of cytotoxicity compared to the controls and those from small-scale farms. The mitotic index was lower in seedlings exposed to wastewater in open-air pools, especially for the 20 cm deep samples. This phenomenon could be linked to bitumen-like substances and oils floating on the surface, attaching to the small roots and causing suffocation.

Saxagliptin, a selective dipeptidyl peptidase-4 inhibitor, alleviates somatic cell aneugenicity and clastogenicity in diabetic mice.

Diabetes-related complications are becoming increasingly common as the global prevalence of diabetes increases. Diabetes is also linked to a high risk of developing cancer. This raises the question of whether cancer vulnerability is caused by diabetes itself or the use of antidiabetic drugs. Chromosomal instability, a source of genetic modification involving either an altered chromosomal number or structure, is a hallmark of cancer. Saxagliptin has been approved by the FDA for diabetes treatment. However, the detailed in vivo effects of prolonged saxagliptin treatment on chromosomal instability have not yet been reported. In this study, streptozotocin was used to induce diabetes in mice, and both diabetic and non-diabetic mice received saxagliptin for five weeks. Fluorescence in situ hybridization was conducted in combination with a bone marrow micronucleus test for measuring chromosomal instability. Our results indicated that saxagliptin is neither mutagenic nor cytotoxic, under the given treatment regimen. Diabetic mice had a much higher incidence of micronuclei formation, and a centromeric DNA probe was present inside the majority of the induced micronuclei, indicating that most of these were caused by chromosome nondisjunction. Conversely, diabetic mice treated with saxagliptin exhibited a significant decrease in micronuclei induction, which were centromeric-positive and centromeric-negative. Diabetes also causes significant biochemical changes indicative of oxidative stress, such as increased lipid peroxidation and decreased reduced/oxidized glutathione ratio, which was reversed by saxagliptin administration. Overall, saxagliptin, the non-mutagenic antidiabetic drug, maintains chromosomal integrity in diabetes and reduces micronuclei formation by restoring redox imbalance, further indicating its usefulness in diabetic patients.

Benzophenone-3 sunscreen causes phytotoxicity and cytogenotoxicity in higher plants.

The benzophenone-3 (BP-3) sunscreen is recurrently released into the environment from different sources, however, evaluations of its adverse effects on plants do not exist in the literature. In this study, BP-3 was evaluated, at concentrations 2; 20, and 200µg/L, regarding phytotoxicity, based on germination and root elongation in seeds, in Lactuca sativa L., Cucumis sativus L. and Allium cepa L., and phytotoxicity, cytogenotoxicity and oxidative stress in A. cepa bulb roots. The BP-3 concentrations, except for the 200µg/L concentration in L. sativa, caused no significant reduction in seed germination. All concentrations tested significantly reduced the elongation of roots from seeds and roots from bulbs. The 20 and 200µg/L concentrations caused oxidation in cells, disturbances in the cell cycle, and alterations in prophase and metaphase, as well as the induction of micronuclei, in A. cepa root meristems. Furthermore, the three concentrations induced a high number of prophases in root tips. Such disorders were caused by excess H2O2 and superoxide produced in cells due to exposure to BP-3, which triggered significant phytotoxicity, cytotoxicity, and genotoxicity in root meristems. Thus, the recurrent contamination of agricultural and non-agricultural soils with BP-3, even at a concentration of 2µg/L, represents an environmental risk for plants. These results point to the impending need to set limits for the disposal of this sunscreen into the environment since BP-3 has been used in industry for several decades.

Acetaldehyde as a Food Flavoring Substance: Aspects of Risk Assessment.

The Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) has reviewed the currently available data in order to assess the health risks associated with the use of acetaldehyde as a flavoring substance in foods. Acetaldehyde is genotoxic in vitro. Following oral intake of ethanol or inhalation exposure to acetaldehyde, systemic genotoxic effects of acetaldehyde in vivo cannot be ruled out (induction of DNA adducts and micronuclei). At present, the key question of whether acetaldehyde is genotoxic and mutagenic in vivo after oral exposure cannot be answered conclusively. There is also insufficient data on human exposure. Consequently, it is currently not possible to reliably assess the health risk associated with the use of acetaldehyde as a flavoring substance. However, considering the genotoxic potential of acetaldehyde as well as numerous data gaps that need to be filled to allow a comprehensive risk assessment, the SKLM considers that the use of acetaldehyde as a flavoring may pose a safety concern. For reasons of precautionary consumer protection, the SKLM recommends that the scientific base for approval of the intentional addition of acetaldehyde to foods as a flavoring substance should be reassessed.

Genotoxicity of bisphenol AF in rats: Detrimental to male reproductive system and probable stronger micronucleus induction potency than BPA.

Bisphenol AF (BPAF), as one of structural analogs of BPA, has been increasingly used in recent years. However, limited studies have suggested its adverse effects similar to or higher than BPA. In order to explore the general toxicity and genotoxicity of subacute exposure to BPAF, the novel 28-day multi-endpoint (Pig-a assay + micronucleus [MN] test + comet assay) genotoxicity evaluation platform was applied. Male rats were randomly distributed into seven main experimental groups and four satellite groups. The main experimental groups included BPAF-treated groups (0.5, 5, and 50 μg/kg·bw/d), BPA group (10μg/kg·bw/d), two solvent control groups (PBS and 0.1% ethanol/99.9% oil), and one positive control group (N-ethyl-N-nitrosourea, 40 mg/kg bw). The satellite groups included BPAF high-dose recovery group (BPAF-HR), oil recovery group (oil-R), ENU recovery group (ENU-R), and PBS recovery group (PBS-R). All groups received the agents orally via gavage for 28 consecutive days, and satellite groups were given a recovery period of 35 days. Among all histopathologically examined organs, testis and epididymis damage was noticed, which was further manifested as blood-testis barrier (BTB) junction protein (Connexin 43 and Occludin) destruction. BPAF can induce micronucleus production and DNA damage, but the genotoxic injury can be repaired after the recovery period. The expression of DNA repair gene OGG1 was downregulated by BPAF. To summarize, under the design of this experiment, male reproductive toxicity of BPAF was noticed, which is similar to that of BPA, but its ability to induce micronucleus production may be stronger than that of BPA.

Genotoxicity assessment in HepaRG™ cells as a new approach methodology follow up to a positive response in the human TK6 cell micronucleus assay: Naphthalene case study.

We are evaluating the use of metabolically competent HepaRG™ cells combined with CometChip® for DNA damage and the micronucleus (MN) assay as a New Approach Methodology (NAM) alternative to animals for follow up genotoxicity assessment to in vitro positive genotoxic response. Naphthalene is genotoxic in human TK6 cells inducing a nonlinear dose-response for the induction of micronuclei in the presence of rat liver S9. of naphthalene. In HepaRG™ cells, naphthalene genotoxicity was assessed using either 6 (CometChip™) or 12 concentrations of naphthalene (MN assay) with the top dose used for assessment of genotoxicity for the Comet and MN assay was 1.25 and 1.74 mM respectively, corresponding to approximately 45% cell survival. In contrast to human TK6 cell with S9, naphthalene was not genotoxic in either the HepaRG™ MN assay or the Comet assay using CometChip®. The lack of genotoxicity in both the MN and comet assays in HepaRG™ cells is likely due to Phase II enzymes removing phenols preventing further bioactivation to quinones and efficient detoxication of naphthalene quinones or epoxides by glutathione conjugation. In contrast to CYP450 mediated metabolism, these Phase II enzymes are inactive in rat liver S9 due to lack of appropriate cofactors causing a positive genotoxic response. Rat liver S9-derived BMD10 over-predicts naphthalene genotoxicity when compared to the negative genotoxic response observed in HepaRG™ cells. Metabolically competent hepatocyte models like HepaRG™ cells should be considered as human-relevant NAMs for use genotoxicity assessments to reduce reliance on rodents.

Polyalthic Acid from Copaifera lucens Demonstrates Anticariogenic and Antiparasitic Properties for Safe Use.

This study aimed at evaluating the potential of Copaifera lucens, specifically its oleoresin (CLO), extract (CECL), and the compound ent-polyalthic acid (PA), in combating caries and toxoplasmosis, while also assessing its toxicity. The study involved multiple assessments, including determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against cariogenic bacteria. CLO and PA exhibited MIC and MBC values ranging from 25 to 50 μg/mL, whereas CECL showed values equal to or exceeding 400 μg/mL. PA also displayed antibiofilm activity with minimum inhibitory concentration of biofilm (MICB50) values spanning from 62.5 to 1000 μg/mL. Moreover, PA effectively hindered the intracellular proliferation of Toxoplasma gondii at 64 μg/mL, even after 24 h without treatment. Toxicological evaluations included in vitro tests on V79 cells, where concentrations ranged from 78.1 to 1250 μg/mL of PA reduced colony formation. Additionally, using the Caenorhabditis elegans model, the lethal concentration (LC50) of PA was determined as 1000 μg/mL after 48 h of incubation. Notably, no significant differences in micronucleus induction and the NDI were observed in cultures treated with 10, 20, or 40 μg/mL of CLO. These findings underscore the safety profile of CLO and PA, highlighting their potential as alternative treatments for caries and toxoplasmosis.

Magnesium oxyhydroxide nanoparticles: Synthesis, characterization and evaluation of their genotoxicity in Vicia faba L.

Magnesium oxyhydroxide nanoparticles (MgO/Mg(OH)2-NPs) were synthesized by a chemical route and their toxicity were evaluated using Vicia faba L. The obtained solids were characterized using visible infrared and ultraviolet spectroscopy, X-ray diffraction (XRD), electron microscopy and surface area by the BET method. The results indicated the presence of two phases: periclase (MgO) and brucite (Mg(OH)2). These MgO/Mg(OH)2 were <50 nm in size, with band gap energy between 5.95 and 6.12 eV and Urbach energy of ∼340 meV. To determine toxicological endpoints, such as mitotic index (MI) and micronucleus induction (MN), Vicia faba L. root tip cells were treated with suspensions containing concentrations of 12.5, 25, 50 and 100 mg/L of MgO/Mg(OH)2-NPs. The results of the toxicological evaluation showed a decrease in MI in the roots of Vicia faba L. exposed to NPs and a positive correlation between the percentage of cells with MN and the concentrations of MgO/Mg(OH)2-NPs used.

In vitro hyperspectral biomarkers of human chondrosarcoma cells in nanoparticle-mediated radiosensitization using carbon ions

New therapeutic approaches are needed for the management of the highly chemo- and radioresistant chondrosarcoma (CHS). In this work, we used polyethylene glycol-encapsulated iron oxide nanoparticles for the intracellular delivery of the chemotherapeutic doxorubicin (IONPDOX) to augment the cytotoxic effects of carbon ions in comparison to photon radiation therapy. The in vitro biological effects were investigated in SW1353 chondrosarcoma cells focusing on the following parameters: cell survival using clonogenic test, detection of micronuclei (MN) by cytokinesis blocked micronucleus assay and morphology together with spectral fingerprints of nuclei using enhanced dark-field microscopy (EDFM) assembled with a hyperspectral imaging (HI) module. The combination of IONPDOX with ion carbon or photon irradiation increased the lethal effects of irradiation alone in correlation with the induction of MN. Alterations in the hyperspectral images and spectral profiles of nuclei reflected the CHS cell biological modifications following the treatments, highlighting possible new spectroscopic markers of cancer therapy effects. These outcomes showed that the proposed combined treatment is promising in improving CHS radiotherapy.

Evaluation of genotoxicity of (4-fluorophenyl) thiazolidin-4-one in CHO-K1 cells

4-thiazolidinones are five-membered heterocyclic ring compounds with diverse pharmacological impacts. In a previous study, we reported a series of newly synthesized derivatives of 4-thiazolidinones with different functional groups, which exhibited anticancer activity against ovarian (SKOV3) and cervical (HeLa) cancer cell lines. Among these derivatives, (4-fluorophenyl) thiazolidin-4-one (4-TH) demonstrated potent cytotoxic activity against SKOV3, with an IC50 value of 12.3 &amp;mu;M. However, it was also found to be extremely toxic to normal cells (CHO-K1) with an IC50 of 7.5 &amp;mu;M. Before considering its use in cancer research, it is crucial to gain a comprehensive understanding of its potential genotoxic effects on normal cells. In this study, we aimed to assess the in vitro cytogenetic toxicity of 4-TH using normal Chinese hamster ovary cells (CHO-K1). Referring to the IC50 of 4-TH, we selected three sub-lethal concentrations (2, 5, and 7.5 &amp;mu;M) and treated CHO-K1 cells for 24 h (one cell cycle duration) to estimate its dose-dependent induction of chromosome aberrations, and examine the effect of 4-TH on cell division, micronucleus induction potential and cell cycle arrest properties following standard protocols. The results showed that 4-TH was highly toxic to normal cells, as all three sublethal concentrations caused a statistically significant increase in the number of chromosomal aberrations (P &lt; 0.001), formation of micronuclei (P &lt; 0.01), and changes in the rate of cell division (mitotic index) (P &lt; 0.05) compared to control. In addition, there was a significant increase in the number of cells in the G1 phase, indicating that all concentrations of 4-TH tested induced apoptosis. The evaluation of the cytotoxic, clastogenic, and aneugenic properties of 4-TH, a potent cytotoxic agent, will undoubtedly provide critical information for determining its safety and potential as an anticancer drug.

Role of Chemical Reduction and Formulation of Graphene Oxide on Its Cytotoxicity towards Human Epithelial Bronchial Cells.

Graphene-based materials may pose a potential risk for human health due to occupational exposure, mainly by inhalation. This study was carried out on bronchial epithelial 16HBE14o- cells to evaluate the role of chemical reduction and formulation of graphene oxide (GO) on its cytotoxic potential. To this end, the effects of GO were compared to its chemically reduced form (rGO) and its stable water dispersion (wdGO), by means of cell viability reduction, reactive oxygen species (ROS) generation, pro-inflammatory mediators release and genotoxicity. These materials induced a concentration-dependent cell viability reduction with the following potency rank: rGO > GO >> wdGO. After 24 h exposure, rGO reduced cell viability with an EC50 of 4.8 μg/mL (eight-fold lower than that of GO) and was the most potent material in inducing ROS generation, in contrast to wdGO. Cytokines release and genotoxicity (DNA damage and micronucleus induction) appeared low for all the materials, with wdGO showing the lowest effect, especially for the former. These results suggest a key role for GO reduction in increasing GO cytotoxic potential, probably due to material structure alterations resulting from the reduction process. In contrast, GO formulated in a stable dispersion seems to be the lowest cytotoxic material, presumably due to its lower cellular internalization and damaging capacity.

Acute exposure of Cr and Cu induces oxidative stress, genotoxicity and histopathological alterations in snakehead fish Channa punctatus

Aim: This study aimed to investigate the oxidative stress, genotoxicity and histopathology after sub-lethal exposure of chromium (Cr6+) and copper (Cu+2) to common food fish Channa punctatus. Metal exposure to fishes of River Ganga is a very common phenomenon due to their ubiquitous nature and through discharge of domestic and industrial waste into the river water. Methodology: An in-vitro experiment was performed to study ill effects of metallic stressors on fish. Fishes were divided into seven groups. Group I served as control, Group II, III, IV as 1/20th ,1/10th and 1/5th of 96 hr-LC50 of Cr6+. Group V, VI and VII were similarly treated with Cu at 1/20th, 1/10th and 1/5th of 96 hr LC50 of Cu+2, respectively. All the treatments were done for 15, 30 and 45 days of exposure period. Genotoxicity was evaluated by micronuclei (MN) induction. Enzymatic and non-enzymatic oxidative stress markers, including reactive oxygen species (ROS), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione reductase (GR), and glutathione peroxidase (GPx) were evaluated. Histopathological studies of liver and kidney tissue were also performed. Results: Stress biomarkers were more profound in fishes of chromium treated groups in comparison to copper. Particularly, SOD and CAT showed a significant hike in the activity levels. ROS levels in blood cells increased significantly (p &lt; 0.05) in all the treated groups (Group II, III, and IV) in a dose dependent manner as compared with control (Group I). There was significant induction in micronuclei (MN) frequency in all the treated groups for both the metals. The highest frequency of MN induction was recorded in Group IV after 96 hr of the exposure period.Significant histological alterations were observed in liver and kidney of treated fish, changes were more pronounced in chromium treated fish than copper. Interpretation: Acute exposure of chromium and copper induces ROS and generates oxidative stress mediated genotoxicity and histopathological perturbations in the prime organs of fish.Cr and Cu induces the changes in the production and accumulation of enzymatic antioxidant system for the regulation and scavenging of ROS induced by Cr and Cu. Key words: Channa punctatus, Genotoxicity, Histology, Heavy metals, Micronuclei, Oxidative stress

Genotoxic and cytotoxic effects of thiomersal and paracetamol combination

Background: Thiomersal is used as a preservative of some vaccines or as a trace from the pathogen inactivation process in vaccine production. Prophylactic use of paracetamol upon vaccination is still common, even though paracetamol decreases immune response on some vaccines. Considering the cytotoxic and genotoxic potential of thiomersal and paracetamol and possible interaction in vivo, in vitro study was performed. Methods: The genotoxic and cytotoxic effects of thiomersal and paracetamol combination were examined on human lymphocyte cultures by using two methods: analysis of chromosomal aberrations and cytokinesis-block micronucleus cytome assay. Blood samples of three healthy donors were analyzed with the following concentrations of tested substances: thiomersal 1 µg/ml and 0.5 µg/ml, paracetamol 20 µg/ml, thiomersal 0.5 µg/ml with paracetamol 20 µg/ml and thiomersal 1.0 µg/ml with paracetamol 20 µg/ml. Results: The analysis of structural chromosomal aberrations was significantly increased in all treated cultures. In cells treated with the combination of thiomersal 1 µg/ml and 20 µg/ml of paracetamol, the number of aberrations was significantly decreased. Cytokinesis-block micronucleus cytome assay analyses showed significantly increased micronucleus frequency in lymphocytes cultivated with thiomersal 1 µg/ml compared to lymphocytes cultures exposed to thiomersal 0.5 µg/ml. Conclusions: Induction of structural chromosome aberrations and micronucleus is shown as a sign of genotoxicity for the examined concentrations of thiomersal and paracetamol. The suppressing effect of paracetamol on thiomersal genotoxicity in lymphocytes culture treated with thiomersal was shown to be indicative of further examination of paracetamol use in the prevention of genotoxicity.

Melphalan induced germ cell toxicity and dose-dependent effects of β-aminoisobutyric acid in experimental rat model: Role of oxidative stress, inflammation and apoptosis.

The success of chemotherapy regimens has led to an increase in cancer survival rate over the last decades. Melphalan has been widely used for the treatment of several types of cancers despite its gonadotoxic effects. Due to its ability to cause mutations in the spermatogonial stem cells and spermatids, melphalan can exert a negative impact on male reproductive health in young cancer survivors. β-aminoisobutyric acid (BAIBA), a myokine released by skeletal muscles, has been reported to have beneficial effects in diabetic nephropathy, cardiomyopathy and hepatic toxicity. However, the exact role of BAIBA in chemotherapy-induced germ cell toxicity is still unexplored. The present study aims to determine the dose-dependent (25, 50, and 100 mg/kg) effects of BAIBA on melphalan-induced (1.5 mg/kg) germ cell toxicity in sprague-dawley (SD) rats. The evaluation parameters included quantification of oxidative stress biomarkers, sperm count, sperm motility and head morphology, sperm and testicular DNA damage, sperm mitochondrial membrane potential, ultrastructural changes in sperms, histological and protein expression studies in testes. Melphalan treatment significantly altered all the above-mentioned parameters and the high dose (100 mg/kg) of BAIBA restored melphalan-induced toxicity in a significant manner by exerting antioxidant, anti-inflammatory and antiapoptotic effects. However, the medium dose (50 mg/kg) of BAIBA decreased the toxicity of melphalan and the low dose (25 mg/kg) of BAIBA failed to counteract the melphalan-induced male germ cell toxicity as well as the peripheral blood micronucleus induction. The antioxidant, anti-inflammatory and antiapoptotic role of BAIBA in melphalan-induced gonadal damage is a novel finding in an experimental rat model.

Areca nut and smokeless tobacco exposure induces micronucleus, other nuclear abnormalities and cytotoxicity in early chick embryo.

Areca nut (AN) and smokeless tobacco (SLT) are indiscriminately consumed among the populations of Southeast and South Asian countries, even by women during the gestational period. This study aimed to investigate the genotoxic and cytotoxic potentials of AN and Sadagura (SG), a unique homemade SLT preparation, alone and in combination in early chick embryos. Fertile white leghorn chicken eggs were randomly divided into five treatment groups: vehicle control, positive control (Mitomycin C, 20 μg/egg), AN, SG, and AN+SG. AN, SG, and AN+SG were given at dosages of 0.125, 0.25, and 0.5 mg/egg. The hen's egg test for micronucleus induction (HET-MN) was performed in chick embryos to evaluate the genotoxic potential of the test agents. Furthermore, the cytotoxic potential was assessed by studying erythroblast cell populations and the polychromatic erythrocytes (PCEs) to normochromatic erythrocytes (NCEs) ratio. Our results indicated a significant increase (p < .001) in MN frequency and other nuclear abnormalities, suggesting the potential of AN and SG to cause genotoxicity. Also, AN and SG exposure alone and in combination considerably altered the erythroblast cell population (%) and the PCE to NCE ratio in all the treatment periods. Our findings established the genotoxic and cytotoxic potential of both AN and SG alone and in combination during early embryonic development in the chick embryo.

Phytochemical Analysis and Genotoxicological Evaluation of Prickly Pear Peel Extracts.

This study investigated the beneficial properties of prickly pear peel (PPP) extracts from Opuntia ficus-indica (L.) Mill. Extracts were obtained via the Soxhlet extraction method using methanol (P1), ethanol (P2) and ethanol-water (P3) as extraction solvents. Their total phenolic and flavonoid content (TPC and TFC, respectively) and their antioxidant activity (AA) were determined. The PPP extracts were characterized in detail using mass spectrometry techniques. Their cyto-genotoxic effect and antigenotoxic potential against mitomycin C were evaluated via the cytokinesis block micronucleus (CBMN) assay on human lymphocytes. Enhanced TPC, TFC and AA values were recorded for all the extracts. Moreover, P1 and P2 were cytotoxic only at the highest concentrations, whereas P3 was found to be cytotoxic in all cases. No significant micronucleus induction was observed in the tested extracts. The PPP extracts contain bioactive compounds such as flavonoids, carboxylic acids, alkaloids, fatty acids and minerals (mainly K, Si, Mg, Ca, P and Zn). The results showed that all three extracts exerted high antigenotoxic activity. Our findings confirm the beneficial and genoprotective properties of PPP extracts and further studies on the bioactive compounds of Opuntia ficus-indica (L.) Mill. are recommended, as it constitutes a promising plant in pharmaceutical applications.

Realistic scenarios of pesticide exposure alters multiple biomarkers in BOANA PULCHELLA (ANURA) Adult Frogs

Imazethapyr, a post-emergent herbicide used in worldwide soybean and corn crops, induces genetic and biochemical alterations in aquatic vertebrates. This study examined the relationship between biomarkers at different organization levels and imazethapyr real-life route exposure in Boana pulchella adults. Frogs were exposed to imazethapyr-based formulation Pivot® H (10.59%) at concentrations representing possible acute routes: field runoff (S1:10 mg.L-1), exposure after direct foliar application (S2:100 mg.L-1) and during direct foliar application (S3:1000 mg.L-1). Post-exposure, endpoints levels were evaluated: organism alterations, biochemical activities and cytogenetic assays. Forty-eight hours post-exposure, antioxidant enzymes decrease, micronuclei induction and DNA damage were observed in all scenarios, while cholinesterase activity increase and body condition reduction were observed in frog-exposed to S3. Ninety-six hours post-exposure, frogs showed glutathione-S-transferase inhibition in S1, micronuclei induction in S2 and S3, and DNA-damage increase in S3. Herbicides routes of exposures in real-life could indicate that authorized applications have a risk to amphibian populations.

Evaluation of the herbicide glyphosate, (aminomethyl)phosphonic acid, and glyphosate-based formulations for genotoxic activity using in vitro assays.

Glyphosate, the most heavily used herbicide world-wide, is applied to plants in complex formulations that promote absorption. The National Toxicology Program reported in 1992 that glyphosate, administered to rats and mice at doses up to 50,000 ppm in feed for 13 weeks, showed little evidence of toxicity, and no induction of micronuclei was observed in the mice in this study. Subsequently, mechanistic studies of glyphosate and glyphosate-based formulations (GBFs) that have focused on DNA damage and oxidative stress suggest that glyphosate may have genotoxic potential. However, few of these studies directly compared glyphosate to GBFs, or effects among GBFs. To address these data gaps, we tested glyphosate, glyphosate isopropylamine (IPA), and (aminomethyl)phosphonic acid (AMPA, a microbial metabolite of glyphosate), 9 high-use agricultural GBFs, 4 residential-use GBFs, and additional herbicides (metolachlor, mesotrione, and diquat dibromide) present in some of the GBFs in bacterial mutagenicity tests, and in human TK6 cells using a micronucleus assay and a multiplexed DNA damage assay. Our results showed no genotoxicity or notable cytotoxicity for glyphosate or AMPA at concentrations up to 10 mM, while all GBFs and herbicides other than glyphosate were cytotoxic, and some showed genotoxic activity. An in vitro to in vivo extrapolation of results for glyphosate suggests that it is of low toxicological concern for humans. In conclusion, these results demonstrate a lack of genotoxicity for glyphosate, consistent with observations in the NTP in vivo study, and suggest that toxicity associated with GBFs may be related to other components of these formulations.

Evaluation of oxidative stress-mediated cytotoxicity and genotoxicity of copper and flubendiamide: amelioration by antioxidants in vivo and in vitro.

Present study was designed to evaluate toxic effects of copper (Cu) (@ 33mg/kg b.wt.) and flubendimide (Flb) (@ 200mg/kg b.wt.) alone and/or in combination on blood-biochemical indices, oxidative stress, and drug metabolizing enzymes (DMEs) in vivo in male Wistar rats following oral exposure continuously for 90days and their immunotoxic (cyto-genotoxic and apoptotic) potential in vitro on thymocytes. In in vivo study, ameliorative potential of α-tocopherol was assessed, whereas α-tocopherol, curcumin, resveratrol, and catechin were evaluated for protective effect in vitro. Significantly (P<0.05) increased AST activity and increment in total bilirubin, uric acid, creatinine, and BUN levels; however, reduction in total protein, GSH content, reduced activities of SOD and GST, and increased lipid peroxidation and GPx activity with severe degenerative changes in histopathological examination of liver and kidney in group of Cu and Flb were observed. Treatment with α-tocopherol improved biochemical variables, redox status, and histoarchitecture of liver and kidney tissues. Reduced hepatic CYP450, CYPb5, APH, UGT, and GST activities observed in both Cu and α-tocopherol alone and their combination groups, whereas significant increment in Flb alone, while α-tocopherol in combination with xenobiotics improved the activities of hepatic DMEs. Primary cell culture of thymocytes (106 cells/ml) exposed to Cu and Flb each @ 40μM increased TUNEL+ve cells, micronuclei induction, DNA shearing, and comet formation establishes their apoptotic and genotoxic potential, whereas treatment with antioxidants showed concentration-dependent significant reduction and their order of potency on equimolar concentration (10μM) basis is: curcumin > resveratrol > catechin=α-tocopherol.

High-throughput micronucleus assay using three-dimensional HepaRG spheroids for in vitro genotoxicity testing.

The in vitro micronucleus (MN) assay is a component of most test batteries used in assessing potential genotoxicity. Our previous study adapted metabolically competent HepaRG cells to the high-throughput (HT) flow-cytometry-based MN assay for genotoxicity assessment (Guo et al. in J Toxicol Environ Health A 83:702-717, 2020b, https://doi.org/10.1080/15287394.2020.1822972 ). We also demonstrated that, compared to HepaRG cells grown as two-dimensional (2D) cultures, 3D HepaRG spheroids have increased metabolic capacity and improved sensitivity in detecting DNA damage induced by genotoxicants using the comet assay (Seo et al. in ALTEX 39:583-604, 2022, https://doi.org/10.14573/altex.22011212022 ). In the present study, we have compared the performance of the HT flow-cytometry-based MN assay in HepaRG spheroids and 2D HepaRG cells by testing 34 compounds, including 19 genotoxicants or carcinogens and 15 compounds that show different genotoxic responses in vitro and in vivo. 2D HepaRG cells and spheroids were exposed to the test compounds for 24h, followed by an additional 3- or 6-day incubation with human epidermal growth factor to stimulate cell division. The results demonstrated that HepaRG spheroids showed generally higher sensitivity in detecting several indirect-acting genotoxicants (require metabolic activation) compared to 2D cultures, with 7,12-dimethylbenzanthracene and N-nitrosodimethylamine inducing higher % MN formation along with having significantly lower benchmark dose values for MN induction in 3D spheroids. These data suggest that 3D HepaRG spheroids can be adapted to the HT flow-cytometry-based MN assay for genotoxicity testing. Our findings also indicate that integration of the MN and comet assays improved the sensitivity for detecting genotoxicants that require metabolic activation. These results suggest that HepaRG spheroids may contribute to New Approach Methodologies for genotoxicity assessment.