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Methods in Molecular Biology (Clifton,... 2024Across eukaryotes, genome stability is essential for normal cell function, physiology, and species survival. Aberrant expression of key genes or exposure to genotoxic...
Across eukaryotes, genome stability is essential for normal cell function, physiology, and species survival. Aberrant expression of key genes or exposure to genotoxic agents can have detrimental effects on genome stability and contribute to the development of various diseases, including cancer. Chromosome instability (CIN), or ongoing changes in chromosome complements, is a frequent form of genome instability observed in cancer and is a driver of genetic and cell-to-cell heterogeneity that can be rapidly detected and quantitatively assessed using surrogate markers of CIN. For example, single cell quantitative imaging microscopy (QuantIM) can be used to simultaneously identify changes in nuclear areas and micronucleus formation. While changes in nuclear areas are often associated with large-scale changes in chromosome complements (i.e., ploidy), micronuclei are small extra-nuclear bodies found outside the primary nucleus that have previously been employed as a measure of genotoxicity of test compounds. Here, we present a facile QuantIM approach that allows for the rapid assessment and quantification of CIN associated phenotypes and genotoxicity. First, we provide protocols to optimize and execute CIN and genotoxicity assays. Secondly, we present the critical imaging settings, optimization steps, downstream statistical analyses, and data visualization strategies employed to obtain high quality and robust data. These approaches can be easily applied to assess the prevalence of CIN associated phenotypes and genotoxic stress for a myriad of experimental and clinical contexts ranging from direct tests to large-scale screens of various genetic contexts (i.e., aberrant gene expression) or chemical compounds. In summary, this QuantIM approach facilitates the identification of novel CIN genes and/or genotoxic agents that will provide greater insight into the aberrant genes and pathways underlying CIN and genotoxicity.
Topics: Chromosomal Instability; Single-Cell Analysis; Humans; DNA Damage; Microscopy; Mutagenicity Tests; Cell Nucleus; Mutagens; Micronucleus Tests
PubMed: 38913318
DOI: 10.1007/978-1-0716-3946-7_18 -
Biomedicine & Pharmacotherapy =... Jun 2024Cannabidiol (CBD), a naturally occurring cyclic terpenoid found in Cannabis sativa L., is renowned for its diverse pharmacological benefits. Marketed as a remedy for...
Exploring the safety of cannabidiol (CBD): A comprehensive in vitro evaluation of the genotoxic and mutagenic potential of a CBD isolate and extract from Cannabis sativa L.
Cannabidiol (CBD), a naturally occurring cyclic terpenoid found in Cannabis sativa L., is renowned for its diverse pharmacological benefits. Marketed as a remedy for various health issues, CBD products are utilized by patients as a supplementary therapy or post-treatment failure, as well as by healthy individuals seeking promised advantages. Despite its widespread use, information regarding potential adverse effects, especially genotoxic properties, is limited. The present study is focused on the mutagenic and genotoxic activity of a CBD isolate (99.4 % CBD content) and CBD-rich Cannabis sativa L extract (63.6 % CBD content) in vitro. Both CBD samples were non-mutagenic, as determined by the AMES test (OECD 471) but exhibited cytotoxicity for HepG2 cells (∼IC 26 µg/ml, ∼IC 6-8 µg/ml, MTT assay). Noncytotoxic concentrations induced upregulation of genes encoding metabolic enzymes involved in CBD metabolism, and CBD oxidative as well as glucuronide metabolites were found in cell culture media, demonstrating the ability of HepG2 cells to metabolize CBD. In this study, the CBD samples were found non-genotoxic. No DNA damage was observed with the comet assay, and no influence on genomic instability was observed with the cytokinesis block micronucleus and the γH2AX and p-H3 assays. Furthermore, no changes in the expression of genes involved in genotoxic stress response were detected in the toxicogenomic analysis, after 4 and 24 h of exposure. Our comprehensive study contributes valuable insights into CBD's safety profile, paving the way for further exploration of CBD's therapeutic applications and potential adverse effects.
PubMed: 38908200
DOI: 10.1016/j.biopha.2024.116969 -
International Journal of Molecular... May 2024Micro-sized particles of synthetic polymers (microplastics) are found in all parts of marine ecosystems. This fact requires intensive study of the degree of danger of...
Micro-sized particles of synthetic polymers (microplastics) are found in all parts of marine ecosystems. This fact requires intensive study of the degree of danger of such particles to the life activity of hydrobionts and needs additional research. It is evident that hydrobionts in the marine environment are exposed to microplastics modified by biotic and abiotic degradation. To assess the toxic potential of aging microplastic, comparative studies were conducted on the response of cytochemical and genotoxic markers in hemocytes of the mussel (Gould, 1850) after exposure to pristine and photodegraded (UV irradiation) polystyrene microparticles (µPS). The results of cytochemical tests showed that UV-irradiated µPS strongly reduced metabolism and destabilized lysosome membranes compared to pristine µPS. Using a Comet assay, it was shown that the nuclear DNA of mussel hemocytes showed high sensitivity to exposure to both types of plastics. However, the level of DNA damage was significantly higher in mussels exposed to aging µPS. It is suggested that the mechanism of increased toxicity of photo-oxidized µPS is based on free-radical reactions induced by the UV irradiation of polymers. The risks of toxic effects will be determined by the level of physicochemical degradation of the polymer, which can significantly affect the mechanisms of toxicity.
Topics: Animals; Mytilus; Microplastics; Polystyrenes; DNA Damage; Hemocytes; Water Pollutants, Chemical; Ultraviolet Rays; Comet Assay
PubMed: 38891928
DOI: 10.3390/ijms25115740 -
Cells May 2024Glufosinate-ammonium (GLA), an organophosphate herbicide, is released at high concentrations in the environment, leading to concerns over its potential genotoxic...
Glufosinate-ammonium (GLA), an organophosphate herbicide, is released at high concentrations in the environment, leading to concerns over its potential genotoxic effects. However, few articles are available in the literature reporting the possible cellular and nuclear effects of this compound. We assessed, by in vitro and in vivo micronucleus assays, the genotoxicity of GLA on cultured human lymphocytes and hemocytes at six concentrations: 0.010 (the established acceptable daily intake value), 0.020, 0.050, 0.100, 0.200, and 0.500 µg/mL. In human lymphocytes, our results reveal a significant and concentration-dependent increase in micronuclei frequency at concentrations from 0.100 to 0.500 μg/mL, while in hemocytes, significant differences were found at 0.200 and 0.500 μg/mL. A significant reduction in the proliferation index was observed at all tested concentrations, with the only exception of 0.010 μg/mL, indicating that the exposure to GLA could lead to increased cytotoxic effects. In a significant reduction in laid eggs and body growth was also observed at all concentrations. In conclusion, we provided evidence of the genomic and cellular damage induced by GLA on both cultured human lymphocytes and a model organism's hemocytes; in addition, we also demonstrated its effects on cell proliferation and reproductive health in .
Topics: Herbicides; Aminobutyrates; Humans; Animals; Genomic Instability; Lymphocytes; Hemocytes; Micronucleus Tests; Cell Proliferation
PubMed: 38891041
DOI: 10.3390/cells13110909 -
Journal of Materials Science. Materials... Jun 2024Pyrophyllite is the least studied natural clay in terms of its potential in biomedical applications, although there are many deposits of this aluminosilicate around the...
Pyrophyllite is the least studied natural clay in terms of its potential in biomedical applications, although there are many deposits of this aluminosilicate around the world. Genotoxicity study was performed in vitro for this mineral. Subsequently, Wister rats were exposed to the pyrophyllite micronized to below 100 µm. After the exposure period, histology of the lung, liver, kidney and gastric tissues were performed, followed by the stereological and hematological analysis. The physicochemical analyses revealed typical XRD characteristics of pyrophyllite clay with particle-size distribution ranging 50 nm-100 μm with stable mineral composition and unique buffering property to pH around 8. The results showed that there were no cytotoxic effects on to THP-1 cells, or genotoxicity of pyrophyllite measured by the Comet assay. In vivo studies are accompanied by the thorough physicochemical characterization of the micronized pyrophyllite. Histology of the lung tissue proved presence of an inflammatory reaction. On the other hand, gastric tissue has shown the selective accumulation of nanoparticles in enterocytes of the stomach only, as supported by ultrastructural analysis. Liver and kidney tissues have shown tolerability for pyrophyllite particles. The results give directions for further comprehensive studies of potential biomedical applications of the pyrophyllite.
Topics: Animals; Kidney; Rats; Rats, Wistar; Biocompatible Materials; Liver; Aluminum Silicates; Particle Size; Nanoparticles; Humans; Materials Testing; Gastric Mucosa; Male; X-Ray Diffraction; Comet Assay; Clay
PubMed: 38884813
DOI: 10.1007/s10856-024-06793-z -
Bioorganic Chemistry Jun 2024Hyperpigmentation disorders may result from inappropriate melanin deposition and/or excessive melanin synthesis. They are classified mainly as aesthetic problems, but...
Hyperpigmentation disorders may result from inappropriate melanin deposition and/or excessive melanin synthesis. They are classified mainly as aesthetic problems, but they can significantly affect human health by decreasing self-esteem. There are available only limited treatment options for hyperpigmentation disorder, among others, cosmetic products applied topically. Depigmenting ingredients were found to be ineffective and characterized by various side effects. As a result, many efforts are made to discover novel, potent, and safe melanogenesis inhibitors for possible use in topical cosmetic depigmenting formulations. Cinnamic acid derivatives constitute a widely tested group for that purpose. This article reports research in the group of N-alkyl cinnamamide derivatives (un)substituted in phenyl ring. Among tested series, (E)-3-(4-chlorophenyl)-N-(5-hydroxypentyl)acrylamide (compound 21) showed the most promising inhibitory properties in mushroom tyrosinase assay (IC = 36.98 ± 1.07 µM for monophenolase activity, IC = 146.71 ± 16.82 µM for diphenolase activity) and melanin production inhibition in B16F10 mouse melanoma cell line at concentration 6.25 µM resulting probably from decreasing of Tyr, Mitf, Tyrp-1, and Tyrp-2 genes expression. This compound also showed melanin production inhibitory properties in pigmented reconstructed human epidermis when used in 1 % and 2 % solutions in 50 % PEG400. In vitro evaluation of its safety profile showed no cytotoxicity to human keratinocytes HaCaT, human skin fibroblasts BJ, and human primary epidermal melanocytes HEMa, no mutagenicity in the Ames test, no genotoxicity in micronucleus test, no phototoxicity, as well as no skin irritation potential tested in PEG400 solution. This compound was also shown to penetrate across the epidermis to reach the possible site of action. The performed research led to classify (E)-3-(4-chlorophenyl)-N-(5-hydroxypentyl)acrylamide as a novel potential depigmenting cosmetic ingredient.
PubMed: 38878750
DOI: 10.1016/j.bioorg.2024.107533 -
Computers in Biology and Medicine Jun 2024Non-sugar sweeteners (NSSs) or artificial sweeteners have long been used as food chemicals since World War II. NSSs, however, also raise a concern about their...
Non-sugar sweeteners (NSSs) or artificial sweeteners have long been used as food chemicals since World War II. NSSs, however, also raise a concern about their mutagenicity. Evaluating the mutagenic ability of NSSs is crucial for food safety; this step is needed for every new chemical registration in the food and pharmaceutical industries. A computational assessment provides less time, money, and involved animals than the in vivo experiments; thus, this study developed a novel computational method from an ensemble convolutional deep neural network and read-across algorithms, called DeepRA, to classify the mutagenicity of chemicals. The mutagenicity data were obtained from the curated Ames test data set. The DeepRA model was developed using both molecular descriptors and molecular fingerprints. The obtained DeepRA model provides accurate and reliable mutagenicity classification through an independent test set. This model was then used to examine the NSSs-related chemicals, enabling the evaluation of mutagenicity from the NSSs-like substances. Finally, this model was publicly available at https://github.com/taraponglab/deepra for further use in chemical regulation and risk assessment.
PubMed: 38870727
DOI: 10.1016/j.compbiomed.2024.108731 -
Journal of Toxicology and Environmental... Sep 2024is one of the most predominant freshwater bloom-forming cyanobacterium found globally which is capable of producing toxic secondary metabolites including microcystins...
is one of the most predominant freshwater bloom-forming cyanobacterium found globally which is capable of producing toxic secondary metabolites including microcystins that might intoxicate animals and humans when contaminated water or food is ingested. Aubl is one of the plants that might possess bioactive compounds capable of controlling growth and reproduction of . The present study aimed to determine the presence of bioactive compounds in extracts and determine alterations occurred in growth and reproduction of when exposed to these plant extracts. In addition, this investigation aimed to examine the influence of on antioxidant enzymes detected in . The results obtained demonstrated that the aqueous and ethanolic extracts of presented potential for control of cyanobacteria populations, exhibiting algicidal action on as well as interfering in antioxidant enzymes activities and parameters associated with oxidative stress. Phytochemical analyses demonstrated the presence of polyphenols and flavonoids content in both extracts. In addition, application of extracts did not produce cytogenotoxicity and/or mutagenicity utilizing test. Therefore, further studies are needed in order to identify and characterize the compounds responsible for these effects on and provide information regarding the possible application of in the treatment of drinking water.
Topics: Microcystis; Plant Extracts; Antioxidants; Oxidative Stress
PubMed: 38865103
DOI: 10.1080/15287394.2024.2366320 -
Food Chemistry Jun 2024The natural vanilla market, which generates millions annually, is predominantly dependent on Vanilla planifolia, a species characterized by low genetic variability and...
The natural vanilla market, which generates millions annually, is predominantly dependent on Vanilla planifolia, a species characterized by low genetic variability and susceptibility to pathogens. There is an increasing demand for natural vanilla, prized for its complex, authentic, and superior quality compared to artificial counterparts. Therefore, there is a necessity for innovative production alternatives to ensure a consistent and stable supply of vanilla flavors. In this context, vanilla crop wild relatives (WRs) emerge as promising natural sources of the spice. However, these novel species must undergo toxicity assessments to evaluate potential risks and ensure safety for consumption. This study aimed to assess the non-mutagenic and non-carcinogenic properties of ethanolic extracts from V. bahiana, V. chamissonis, V. cribbiana, and V. planifolia through integrated metabolomic profiling, in vitro toxicity assays, and in silico analyses. The integrated approach of metabolomics, in vitro assays, and in silico analyses has highlighted the need for further safety assessments of Vanilla cribbiana ethanolic extract. While the extracts of V. bahiana, V. chamissonis, and V. planifolia generally demonstrated non-mutagenic properties in the Ames assay, V. cribbiana exhibited mutagenicity at high concentrations (5000 μg/plate) in the TA98 strain without metabolic activation. This finding, coupled with the dose-dependent cytotoxicity observed in WST-1 (Water Soluble Tetrazolium) assays, a colorimetric method that assesses the viability of cells exposed to a test substance, underscores the importance of concentration in the safety evaluation of these extracts. Kaempferol and pyrogallol, identified with higher intensity in V. cribbiana, are potential candidates for in vitro mutagenicity. Although the results are not conclusive, they suggest the safety of these extracts at low concentrations. This study emphasizes the value of an integrated approach in providing a nuanced understanding of the safety profiles of natural products, advocating for cautious use and further research into V. cribbiana mutagenicity.
PubMed: 38852444
DOI: 10.1016/j.foodchem.2024.139948 -
Journal of Hazardous Materials Aug 2024Titanium dioxide nanoparticles (nTiO) have been considered a possible carcinogen to humans, but most existing studies have overlooked the role of human enzymes in...
Titanium dioxide nanoparticles (nTiO) have been considered a possible carcinogen to humans, but most existing studies have overlooked the role of human enzymes in assessing the genotoxicity of nTiO. Here, a toxicogenomics-based in vitro genotoxicity assay using a GFP-fused yeast reporter library was employed to elucidate the genotoxic potential and mechanisms of nTiO. Moreover, two new GFP-fused yeast reporter libraries containing either human CYP1A1 or CYP1A2 genes were constructed by transformation to investigate the potential modulation of nTiO genotoxicity in the presence of human CYP enzymes. This study found a lack of appreciable nTiO genotoxicity as indicated by the yeast reporter library in the absence of CYP expression but a significantly elevated indication of genotoxicity in either CYP1A1- or CYP1A2-expressing yeast. The intracellular reactive oxygen species (ROS) measurement indicated significantly higher ROS in yeast expressing either enzyme. The detected mitochondrial DNA damage suggested mitochondria as one of the target sites for oxidative damage by nTiO in the presence of either one of the CYP enzymes. The results thus indicated that the genotoxicity of nTiO was enhanced by human CYP1A1 or CYP1A2 enzyme and was associated with elevated oxidative stress, which suggested that the similar mechanisms could occur in human cells.
Topics: Humans; Titanium; Reactive Oxygen Species; Saccharomyces cerevisiae; Cytochrome P-450 CYP1A1; DNA Damage; Mutagenicity Tests; Cytochrome P-450 CYP1A2; Mutagens; Oxidative Stress; Genes, Reporter; Nanoparticles; Metal Nanoparticles; Green Fluorescent Proteins
PubMed: 38850947
DOI: 10.1016/j.jhazmat.2024.134850