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Chemico-biological Interactions Jul 2024Tris(2-butoxyethyl) phosphate (TBOEP) is an organophosphorus flame retardant ubiquitously present in the environment and even the human body. TBOEP is toxic in multiple...
Tris(2-butoxyethyl) phosphate (TBOEP) is an organophosphorus flame retardant ubiquitously present in the environment and even the human body. TBOEP is toxic in multiple tissues, which forms dealkylated and hydroxylated metabolites under incubation with human hepatic microsomes; however, the impact of TBOEP metabolism on its toxicity, particularly mutagenicity (typically requiring metabolic activation), is left unidentified. In this study, the mutagenicity of TBOEP in human hepatoma cell lines (HepG2 and C3A) and the role of specific CYPs were studied. Through molecular docking, TBOEP bound to human CYP1A1, 1B1, 2B6 and 3A4 with energies and conformations favorable for catalyzing reactions, while the conformations of its binding with human CYP1A2 and 2E1 appeared unfavorable. In C3A cells (endogenous CYPs being substantial), TBOEP exposing for 72 h (2-cell cycle) at low micromolar levels induced micronucleus, which was abolished by 1-aminobenzotriazole (inhibitor of CYPs); in HepG2 cells (CYPs being insufficient) TBOEP did not induce micronucleus, whose effect was however potentiated by pretreating the cells with PCB126 (CYP1A1 inducer) or rifampicin (CYP3A4 inducer). TBOEP induced micronucleus in Chinese hamster V79-derived cell lines genetically engineered for stably expressing human CYP1A1 and 3A4, but not in cells expressing the other CYPs. In C3A cells, TBOEP selectively induced centromere protein B-free micronucleus (visualized by immunofluorescence) and PIG-A gene mutations, and elevated γ-H2AX rather than p-H3 (by Western blot) which indicated specific double-strand DNA breaks. Therefore, this study suggests that TBOEP may induce DNA/chromosome breaks and gene mutations in human cells, which requires metabolic activation by CYPs, primarily CYP1A1 and 3A4.
Topics: Animals; Humans; Flame Retardants; Molecular Docking Simulation; Cricetinae; Cytochrome P-450 Enzyme System; Mutagens; Organophosphorus Compounds; Cricetulus; Organophosphates; Hep G2 Cells; Micronucleus Tests
PubMed: 38823534
DOI: 10.1016/j.cbi.2024.111088 -
Mutation Research. Genetic Toxicology... 2024The article by Ceppi and colleagues, Genotoxic Effects of Occupational Exposure to, Glass Fibres - A Human Biomonitoring Study, published in Mutation Research -Genetic...
Slovakian glass fibre factory genotoxicity biomonitoring study - unsupported adverse outcome pathway (AOP) from the toxicology and human epidemiological experience of synthetic vitreous fibres (SVFs).
The article by Ceppi and colleagues, Genotoxic Effects of Occupational Exposure to, Glass Fibres - A Human Biomonitoring Study, published in Mutation Research -Genetic Toxicology and Environmental Mutagenesis in 2023 was reviewed with great interest. The authors undertook a novel approach to conducting a biomonitoring study of genotoxicity markers among a population of glass fibre manufacturing workers in Slovakia. On the surface, the Ceppi et al. (2023) study provides an interesting application of genotoxicity markers among a human population of workers to explore potential markers of effect (DNA strand breaks) and potential risk of susceptibility (e.g., genetic damage, disease, death). However, limited data for exposure reconstruction, uncertain influences from smoking history, and lack of consideration of decades of human epidemiology research showing no increased risk of malignant or non-malignant respiratory disease and mortality among glass fibre manufacturing workers, reveals that the conclusions of the authors are overreaching and inconsistent with the existing science. The limitations of this study preclude the ability to draw causal inferences or conclusions about DNA strand breaks as a marker of exposure, effect, or susceptibility within this population of Slovakian glass fibre workers. Further longitudinal research is required (e.g., more robust temporal assessment of occupational exposures - fibres and other compounds - and smoking history) to support the study conclusions.
Topics: Humans; Occupational Exposure; Glass; Slovakia; Biological Monitoring; DNA Damage; Mutagens; Mutagenicity Tests
PubMed: 38821677
DOI: 10.1016/j.mrgentox.2024.503769 -
Mutation Research. Genetic Toxicology... 2024N-Nitrosamines, known as drug impurities and suspected carcinogens, have drawn significant public concern. In response to drug regulatory needs, the European Medicines...
N-Nitrosamines, known as drug impurities and suspected carcinogens, have drawn significant public concern. In response to drug regulatory needs, the European Medicines Agency (EMA) has previously proposed a carcinogenic potency categorization approach based on the N-nitrosamine α-hydroxylation hypothesis, i.e., that N-nitrosamine mutagenicity increases with the number of α-hydrogen atoms. However, this structure-activity relationship has not been fully tested in vivo. NEIPA (N-nitrosoethylisopropylamine) and NDIPA (N-nitrosodiisopropylamine) are small N-Nitrosamines with similar structures, differing in that the former compound has an additional α-hydrogen atom. In this study, NEIPA and NEIPA doses, 25-100 mg/kg, were administered orally to C57BL/6 J mice for seven consecutive days, and their mutation and DNA damage effects were compared. Compared with NDIPA, the mutagenicity and DNA damage potencies of NEIPA (which contains one more α-hydrogen) were much greater. These differences may be related to their distinct metabolic pathways and target organs. This case study confirms the role of α-hydroxyl modification in the mutagenicity of nitrosamines, with oxidation at the α-hydrogen being a crucial step in the formation of mutagens from N-Nitrosamines, and can inform mutagenicity risk assessment and the formulation of regulatory standards for N-nitrosamine impurities.
Topics: Animals; Mice, Inbred C57BL; Mice; Nitrosamines; Mutagenicity Tests; DNA Damage; Mutagens; Male; Structure-Activity Relationship; Carcinogens; Diethylnitrosamine; Mutation; Administration, Oral
PubMed: 38821676
DOI: 10.1016/j.mrgentox.2024.503763 -
Mutation Research. Genetic Toxicology... 2024Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety...
Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants.
Topics: Humans; Hepatocytes; Mutagens; Micronucleus Tests; Mutagenicity Tests; Liver; Lab-On-A-Chip Devices; DNA Damage; Comet Assay; Cyclophosphamide; Methyl Methanesulfonate; Cell Line; Benzo(a)pyrene; Coculture Techniques; Ethyl Methanesulfonate; Mutation
PubMed: 38821675
DOI: 10.1016/j.mrgentox.2024.503762 -
Mutation Research. Genetic Toxicology... 2024Diabetes mellitus is a complex metabolic disorder resulting from the interplay of environmental, genetic, and epigenetic factors that increase the risk of cancer...
Diabetes mellitus is a complex metabolic disorder resulting from the interplay of environmental, genetic, and epigenetic factors that increase the risk of cancer development. However, it is unclear whether the increased cancer risk is due to poor glycemic control or the use of some antidiabetic medications. Therefore, we investigated the genetic and epigenetic changes in somatic cells in a mouse model of diabetes and studied whether multiple exposures to the antidiabetic medication dapagliflozin influence these changes. We also elucidated the mechanism(s) of these ameliorations. The micronucleus test and modified comet assay were used to investigate bone marrow DNA damage and methylation changes. These assays revealed that dapagliflozin is non-genotoxic in the tested regimen, and oxidative DNA damage and hypermethylation were significantly higher in diabetic mice. Spectrophotometry also evaluated oxidative DNA damage and global DNA methylation, revealing similar significant alterations induced by diabetes. Conversely, the dapagliflozin-treated diabetic animals significantly reduced these changes. The expression of some genes involved in DNA repair and DNA methylation was disrupted considerably in the somatic cells of diabetic animals. In contrast, dapagliflozin treatment significantly restored these disruptions and enhanced DNA repair. The simultaneous effects of decreased oxidative DNA damage and hypermethylation levels suggest that dapagliflozin can be used as a safe antidiabetic drug to reduce DNA damage and hypermethylation in diabetes, demonstrating its usefulness in patients with diabetes to control hyperglycemia and decrease the development of its subsequent complications.
Topics: Animals; Benzhydryl Compounds; Glucosides; DNA Methylation; DNA Damage; Diabetes Mellitus, Experimental; Mice; Oxidative Stress; Male; Hypoglycemic Agents; Micronucleus Tests; DNA Repair; Comet Assay
PubMed: 38821673
DOI: 10.1016/j.mrgentox.2024.503765 -
Mutation Research. Genetic Toxicology... 2024Human epidemiological studies with biomarkers of effect play an invaluable role in identifying health effects with chemical exposures and in disease prevention. Effect... (Review)
Review
Human epidemiological studies with biomarkers of effect play an invaluable role in identifying health effects with chemical exposures and in disease prevention. Effect biomarkers that measure genetic damage are potent tools to address the carcinogenic and/or mutagenic potential of chemical exposures, increasing confidence in regulatory risk assessment decision-making processes. The micronucleus (MN) test is recognized as one of the most successful and reliable assays to assess genotoxic events, which are associated with exposures that may cause cancer. To move towards the next generation risk assessment is crucial to establish bridges between standard approaches, new approach methodologies (NAMs) and tools for increase the mechanistically-based biological plausibility in human studies, such as the adverse outcome pathways (AOPs) framework. This paper aims to highlight the still active role of MN as biomarker of effect in the evolution and applicability of new methods and approaches in human risk assessment, with the positive consequence, that the new methods provide a deeper knowledge of the mechanistically-based biology of these endpoints.
Topics: Humans; Risk Assessment; Micronucleus Tests; Biomarkers; DNA Damage; Mutagens; Animals
PubMed: 38821670
DOI: 10.1016/j.mrgentox.2024.503768 -
Mutation Research. Genetic Toxicology... 2024Gene therapies have emerged as promising treatments for various conditions including inherited diseases as well as cancer. Ensuring their safe clinical application... (Review)
Review
Gene therapies have emerged as promising treatments for various conditions including inherited diseases as well as cancer. Ensuring their safe clinical application requires the development of appropriate safety testing strategies. Several guidelines have been provided by health authorities to address these concerns. These guidelines state that non-clinical testing should be carried out on a case-by-case basis depending on the modality. This review focuses on the genome safety assessment of frequently used gene therapy modalities, namely Adeno Associated Viruses (AAVs), Lentiviruses, designer nucleases and mRNAs. Important safety considerations for these modalities, amongst others, are vector integrations into the patient genome (insertional mutagenesis) and off-target editing. Taking into account the constraints of in vivo studies, health authorities endorse the development of novel approach methodologies (NAMs), which are innovative in vitro strategies for genotoxicity testing. This review provides an overview of NAMs applied to viral and CRISPR/Cas9 safety, including next generation sequencing-based methods for integration site analysis and off-target editing. Additionally, NAMs to evaluate the oncogenicity risk arising from unwanted genomic modifications are discussed. Thus, a range of promising techniques are available to support the safe development of gene therapies. Thorough validation, comparisons and correlations with clinical outcomes are essential to identify the most reliable safety testing strategies. By providing a comprehensive overview of these NAMs, this review aims to contribute to a better understanding of the genome safety perspectives of gene therapies.
Topics: Genetic Therapy; Humans; Gene Editing; Animals; Dependovirus; Genetic Vectors; CRISPR-Cas Systems; Lentivirus; Endonucleases; Mutagenicity Tests; Nucleotides
PubMed: 38821669
DOI: 10.1016/j.mrgentox.2024.503767 -
Mutation Research. Genetic Toxicology... 2024In this paper, we studied the potential genotoxic effects of human plasma from healthy volunteers, as well as patients with gastro-oesophageal reflux disease, Barrett's...
In this paper, we studied the potential genotoxic effects of human plasma from healthy volunteers, as well as patients with gastro-oesophageal reflux disease, Barrett's oesophagus (BO) and oesophageal adenocarcinoma (OAC) using the oesophageal adenocarcinoma cell line (OE33) and the lymphoblastoid cell line (TK6). Both TK6 and OE33 cells were treated with plasma (10 % volume, replacing foetal bovine serum (FBS) or horse serum (HS)) at different time points of 4 h (for the micronucleus (Mn) assay and the invasion assay) and 24 h (for the cell cycle studies). Plasma-induced effects on DNA damage levels, cell viability and the cell cycle were studied by the micronucleus assay, cytokinesis block proliferation index (CBPI) and flow cytometry respectively. The expression of IL-8 in supernatants of TK6 cells and IFN-β in OE33 cells was also analysed by enzyme-linked immunosorbent assay (ELISA). Finally, we carried out an assessment of cellular invasion of OE33 cells following plasma treatment. The results of the micronucleus assay confirmed the genotoxicity of direct plasma treatment from some participants through the increase in DNA damage in TK6 cells. Conversely, some individual patient plasma samples reduced background levels of TK6 cell Mn frequency, in an anti-genotoxic fashion. In TK6 cells, (on average) plasma samples from patients with Barrett's oesophagus induced higher micronucleus levels than healthy volunteers (p= 0.0019). There was little difference in Mn induction when using plasma versus serum to treat the cells in vitro. Cell cycle results showed that direct plasma treatment had a marked impact on OE33 cells at 24 h (p=0.0182 for BO and p=0.0320 for OAC) by decreasing the proportion of cells in the S phase, while plasma exposure was less impactful on the cell cycle of TK6 cells. Invasion of OE33 cells was also seen to be non-significantly affected by plasma treatment of OE33 cells. The addition of N-acetyl cysteine NAC in a dose-dependent matter did not alter the formation of Mn in TK6 cells, suggesting that reactive oxygen species (ROS) are not the root cause of plasma's genotoxicity. The concentration of IL-8 in TK6 cells and IFN-β in OE33 cells was significantly higher in cells treated with OAC-derived plasma than in the untreated negative control. Collectively, our results demonstrate that plasma-specific effects are detectable which helps us better understand some important aspects of the biology of blood-based biomarkers under development.
Topics: Humans; Micronucleus Tests; DNA Damage; Barrett Esophagus; Adenocarcinoma; Esophageal Neoplasms; Plasma; Interleukin-8; Cell Line, Tumor; Cell Cycle; Male; Middle Aged; Adult; Cell Survival; Female; Micronuclei, Chromosome-Defective; Interferon-beta; Aged
PubMed: 38821668
DOI: 10.1016/j.mrgentox.2024.503766 -
Mutation Research. Genetic Toxicology... 2024Hairdressers are constantly occupationally exposed to many chemicals have the potential to cause allergies and carcinogenic effects, act as skin and eye irritants and...
Hairdressers are constantly occupationally exposed to many chemicals have the potential to cause allergies and carcinogenic effects, act as skin and eye irritants and induce oxidative stress and DNA damage. This study aimed to evaluate occupation-induced genotoxicity based on the presence of micronucleus (MN) and other nuclear anomalies in urothelial cells and measure oxidative DNA damage based on the 8-hydroxy-2'-deoxyguanosine level in the urine of Turkish hairdressers. Originality of this study comes from that there was no study on MN and other nuclear anomalies frequencies and oxidative DNA damage in urine samples of hairdressers in the literature. The mean±standard deviation frequency (‰) of micronucleated (MNed) cells was higher in the hairdresser group (n=56) (4.81±7.87, p<0.001) than in the control group (n=56) (0.93±1.85). Nuclear buds were not observed in either group. While the frequency of basal cells was higher in the control group (446.6±106.21) than in the hairdresser group (367.78±101.51, p<0.001), the frequency of binuclear, karyolytic, pycnotic and karyorrhectic cells were higher in the hairdresser group (0.41±0.80, p<0.001; 438.02±118.27, p<0.001; 0.43±0.76, p<0.001; and 47.27±28.40, p<0.001) than in the control group (0.04±0.27, 358.57±95.71, 0.05±0.23 and 24.41±14.50). Condensed chromatins were observed only in the hairdresser group. Specific gravity adjusted 8-hydroxy-2'-deoxyguanosine level was statistically lower in the hairdresser group (908.21±403.25 ng/mL-SG) compared to the control group (1003.09±327.09 ng/mL-SG) (p=0.024). No significant correlation was found between the 8-hydroxy-2'-deoxyguanosine level and the frequency MN. The amount of formaldehyde released during Brazilian keratin treatment was higher than the American Conference of Governmental Industrial Hygienists -Threshold Limit Value (ACGIH-TLV; 0.1 ppm). Similarly, the amount of ethyl acetate released in three salons was above the recommended limit (400 ppm). These findings suggest that hairdressers have an increased risk of genotoxicity and cytotoxicity owing to occupational exposure, regardless of age, working hours, smoking and alcohol consumption.
Topics: Humans; 8-Hydroxy-2'-Deoxyguanosine; Occupational Exposure; Adult; Turkey; Urothelium; Deoxyguanosine; Male; Micronucleus Tests; Micronuclei, Chromosome-Defective; DNA Damage; Oxidative Stress; Middle Aged; Female; Young Adult; Case-Control Studies; Cell Nucleus
PubMed: 38821667
DOI: 10.1016/j.mrgentox.2024.503754 -
Mutation Research. Genetic Toxicology... 2024Cytogenetic studies have shown that human chromosomes 1, 9, and 16, with a large heterochromatic region of highly methylated classical satellite DNA, are prone to...
Cytogenetic studies have shown that human chromosomes 1, 9, and 16, with a large heterochromatic region of highly methylated classical satellite DNA, are prone to induction of chromatid breaks and interchanges by mitomycin C (MMC). A couple of studies have indicated that material from chromosome 9, and possibly also from chromosomes 1 and 16, are preferentially micronucleated by MMC. Here, we further examined the chromosome-specific induction of micronuclei (MN; with and without cytochalasin B) and chromosomal aberrations (CAs) by MMC. Cultures of isolated human lymphocytes from two male donors were treated (at 48 h of culture, for 24 h) with MMC (500 ng/ml), and the induced MN were examined by a pancentromeric DNA probe and paint probe for chromosome 9, and by paint probes for chromosomes 1 and 16. MMC increased the total frequency of MN by 6-8-fold but the frequency of chromosome 9 -positive (9) MN by 29-30-fold and the frequency of chromosome 1 -positive (1) MN and chromosome 16 -positive (16) MN by 12-16-fold and 10-17-fold, respectively. After treatment with MMC, 34-47 % of all MN were 9, 17-20 % 1, and 3-4 % 16. The majority (94-96 %) of the 9 MN contained no centromere and thus harboured acentric fragments. When MMC-induced CAs aberrations were characterized by using the pancentromeric DNA probe and probes for the classical satellite region and long- and short- arm telomeres of chromosome 9, a high proportion of chromosomal breaks (31 %) and interchanges (41 %) concerned chromosome 9. In 83 % of cases, the breakpoint in chromosome 9 was just below the region (9cen-q12) labelled by the classical satellite probe. Our results indicate that MMC specifically induces MN harbouring fragments of chromosome 9, 1, and 16. CAs of chromosome 9 are highly overrepresented in metaphases of MMC-treated lymphocytes. The preferential breakpoint is below the region 9q12.
Topics: Humans; Mitomycin; Male; Chromosome Aberrations; Micronuclei, Chromosome-Defective; Chromosomes, Human, Pair 9; Chromosomes, Human, Pair 1; Chromosomes, Human, Pair 16; Lymphocytes; Adult; Micronucleus Tests; Cells, Cultured; Cytochalasin B; In Situ Hybridization, Fluorescence
PubMed: 38821666
DOI: 10.1016/j.mrgentox.2024.503753