Sister chromatid cohesion establishment during DNA replication termination.

Newly copied sister chromatids are tethered together by the cohesin complex, but how sister chromatid cohesion coordinates with DNA replication is poorly understood. Prevailing models suggest that cohesin complexes, bound to DNA before replication, remain behind the advancing replication fork to keep sister chromatids together. By visualizing single replication forks colliding with preloaded cohesin complexes, we find that the replisome instead pushes cohesin to where a converging replisome is met. Whereas the converging replisomes are removed during DNA replication termination, cohesin remains on nascent DNA and provides cohesion. Additionally, we show that CMG (CDC45-MCM2-7-GINS) helicase disassembly during replication termination is vital for proper cohesion in budding yeast. Together, our results support a model wherein sister chromatid cohesion is established during DNA replication termination.

Sister chromatid cohesion is mediated by individual cohesin complexes.

Eukaryotic genomes are organized by loop extrusion and sister chromatid cohesion, both mediated by the multimeric cohesin protein complex. Understanding how cohesin holds sister DNAs together, and how loss of cohesion causes age-related infertility in females, requires knowledge as to cohesin's stoichiometry in vivo. Using quantitative super-resolution imaging, we identified two discrete populations of chromatin-bound cohesin in postreplicative human cells. Whereas most complexes appear dimeric, cohesin that localized to sites of sister chromatid cohesion and associated with sororin was exclusively monomeric. The monomeric stoichiometry of sororin:cohesin complexes demonstrates that sister chromatid cohesion is conferred by individual cohesin rings, a key prediction of the proposal that cohesion arises from the co-entrapment of sister DNAs.

The meta-analysis of sister chromatid exchange as a biomarker in healthcare workers with occupational exposure to antineoplastic drugs.

BACKGROUND: Sister chromatid exchange (SCE) can be used to identify early occupational health status in health care workers. Our aim is to comprehensively assess the relationship between long-term exposure to antineoplastic drugs (ADs) and SCE in health care workers via meta-analysis. METHODS: Five databases were systematically searched for relevant articles published from inception to November 30, 2022. Literature data are expressed as mean difference and 95% confidence intervals (CI) or relative risk and 95% CI. For I2 > 50% trials, random effect model is used for statistical analysis, otherwise fixed effect model is used. This review was registered in the International Prospective Register of Systematic Reviews (identifier CRD42023399914). RESULTS: Fourteen studies were included in this study. Results showed the level of SCE in healthcare workers exposed to ADs was significantly higher than in controls. The mean difference of the SCE trial was 0.53 (95% CI: 0.10-0.95, P = .01) under a random-effects model. CONCLUSIONS: The findings suggested a significant correlation between occupational exposure to ADs in health care workers and SCE, requiring the attention of health care workers in general.

Using Sister Chromatid Exchange Assay to Detect Homologous Recombination Deficiency in Epigenetically Deregulated Urothelial Carcinoma Cells.

Sister chromatid exchange (SCE) is the process of exchanging regions between two sister chromatids during DNA replication. Exchanges between replicated chromatids and their sisters can be visualized in cells when DNA synthesis in one chromatid is labelled by 5-bromo-2'-deoxyuridine (BrdU). Homologous recombination (HR) is considered as the principal mechanism responsible for the sister chromatid exchange (SCE) upon replication fork collapse, and therefore SCE frequency upon genotoxic conditions reflects the capacity of HR repair to respond to replication stress. During tumorigenesis, inactivating mutations or altered transcriptome can affect a plethora of epigenetic factors that participate in DNA repair processes, and there are an increasing number of reports which demonstrate a link between epigenetic deregulation in cancer and homologous recombination deficiency (HRD). Therefore, the SCE assay can provide valuable information regarding the HR functionality in tumors with epigenetic deficiencies. In this chapter, we provide a method to visualize SCEs. The technique outlined below is characterized by high sensitivity and specificity and has been successfully applied to human bladder cancer cell lines. In this context, this technique could be used to characterize the dynamics of HR repair in tumors with deregulated epigenome.

Antigenotoxic effect of hyperoside against Mitomycin C and hydrogen peroxide-induced genotoxic damage on human lymphocytes.

Hyperoside is a flavonol glycoside isolated from various plant genera such as Hypericum and Crataegus. It has an important place in the human diet and is used medically to relieve pain and ameliorate cardiovascular functions. However, a comprehensive profile of the genotoxic and antigenotoxic effects of hyperoside is not known. The current study aimed to investigate the genotoxic and antigenotoxic effects of hyperoside against genetic damages induced by two genotoxins (MMC and H2O2) using chromosomal aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) assays in human peripheral blood lymphocytes in vitro. Blood lymphocytes were incubated with 7.8-62.5 µg/mL concentrations of hyperoside alone and simultaneously with 0.20 µg/mL Mitomycin C (MMC) or 100 µM Hydrogen peroxide (H2O2). Hyperoside did not exhibit genotoxic potential in the CA, SCE, and MN assays. Moreover, it did not cause a decrease in mitotic index (MI) which is an indicator of cytotoxicity. On the other hand, hyperoside significantly decreased CA, SCE, and MN (except for MMC treatment) frequencies induced by MMC and H2O2. Hyperoside, increased mitotic index against both mutagenic agents at 24-h treatment when compared to positive control. Our results demonstrate that hyperoside exhibited antigenotoxic effects rather than genotoxic in vitro human lymphocytes. Therefore, hyperoside may be a potential preventive agent in inhibiting chromosomal and oxidative damage induced by genotoxic chemicals.

The meta-analysis of cytogenetic biomarkers as an assessment of occupational risk for healthcare workers exposed to antineoplastic drugs.

OBJECTIVE: Antineoplastic drugs (ADs) are widely used in clinical practice and have been demonstrated to be effective in treating malignant tumors. However, they carry a risk of cytogenotoxicity for healthcare workers. Studies have reported that genotoxic biomarkers can be applied to assess the occupational health status of healthcare workers at an early stage, but results of different studies are variable. The objectives of the review were examine the association between long-term exposure to ADs and cytogenetic damage to healthcare workers. METHODS: We systematically reviewed studies between 2005 and 2021 using PubMed, Embase and Web of Science databases that used cytogenetic biomarkers to assess occupational exposure to ADs in healthcare workers. We used RevMan5.4 to analyze the tail length parameters of the DNA, frequency of the chromosomal aberrations, sister chromatid exchanges and micronuclei. A total of 16 studies were included in our study. The studies evaluate the quality of the literature through the Agency for Healthcare Research and Quality. RESULTS: The results revealed that under the random-effects model, the estimated standard deviation was 2.37 (95% confidence interval [CI] 0.92-3.81, P = 0.001) for the tail length parameters of the DNA, 1.48 (95% CI 0.71-2.25, P = 0.0002) for the frequency of chromosomal aberrations, 1.74 (95% CI 0.49-2.99, P = 0.006) for the frequency of sister chromatid exchanges and 1.64 (95% CI 0.83-2.45, P < 0.0001) for the frequency of micronuclei. CONCLUSIONS: The results indicate that there is a significant association between occupational exposure to ADs and cytogenetic damage, to which healthcare workers should be alerted.

Helical coiling of metaphase chromatids.

Chromatids of mitotic chromosomes were suggested to coil into a helix in early cytological studies and this assumption was recently supported by chromosome conformation capture (3C) sequencing. Still, direct differential visualization of a condensed chromatin fibre confirming the helical model was lacking. Here, we combined Hi-C analysis of purified metaphase chromosomes, biopolymer modelling and spatial structured illumination microscopy of large fluorescently labeled chromosome segments to reveal the chromonema - a helically-wound, 400 nm thick chromatin thread forming barley mitotic chromatids. Chromatin from adjacent turns of the helix intermingles due to the stochastic positioning of chromatin loops inside the chromonema. Helical turn size varies along chromosome length, correlating with chromatin density. Constraints on the observable dimensions of sister chromatid exchanges further supports the helical chromonema model.

Investigation of genotoxic effect of octyl gallate used as an antioxidant food additive in in vitro test systems.

Several antioxidant food additives are added to oils, soups, sauces, chewing gum, potato chips, and so on. One of them is octyl gallate. The purpose of this study was to evaluate the potential genotoxicity of octyl gallate in human lymphocytes, using in vitro chromosomal abnormalities (CA), sister chromatid exchange (SCE), cytokinesis block micronucleus cytome (CBMN-Cyt), micronucleus-FISH (MN-FISH), and comet tests. Different concentrations (0.031, 0.063, 0.125, 0.25, and 0.50 µg/ml) of octyl gallate were used. A negative (distilled water), a positive (0.20 µg/ml Mitomycin-C), and a solvent control (8.77 µl/ml ethanol) were also applied for each treatment. Octyl gallate did not cause changes in chromosomal abnormalities, micronucleus, nuclear bud (NBUD), and nucleoplasmic bridge (NPB) frequency. Similarly, there was no significant difference in DNA damage (comet assay), percentage of centromere positive and negative cells (MN-FISH test) compared to the solvent control. Moreover, octyl gallate did not affect replication and nuclear division index. On the other hand, it significantly increased the SCE/cell ratio in three highest concentrations compared to solvent control at 24 h treatment. Similarly, at 48 h treatment, the frequency of SCE raised significantly compared to solvent controls at all the concentrations (except 0.031 µg/ml). An important reduction was detected in mitotic index values in the highest concentration at 24 h treatment and almost all concentrations (except 0.031 and 0.063 µg/ml) at 48 h treatment. The results obtained suggest that octyl gallate has no important genotoxicological action on human peripheral lymphocytes at the concentrations applied in this study.

Genotoxicity of nedaplatin in cultured lymphocytes: modulation by vitamin E.

Nedaplatin is a chemotherapeutic agent used widely in cancer therapy. Nedaplatin has been shown to cause DNA damage to cells via the induction of oxidative stress. Vitamin E (Vit E) has an anti-mutagenic activity that can protect cells from DNA damaging agents. The objective of this study is to examine the genotoxic and cytotoxic effects of nedaplatin in human cultured lymphocytes. In addition, modulation of such effects by Vit E was also examined. The frequencies of sister chromatid exchange (SCE) and chromosomal aberrations (CAs) were used as an indicator for genotoxicity. The mitotic and proliferative indices were used to examine the cytotoxic effects of nedaplatin. The results showed that nedaplatin significantly elevated SCE and CA frequencies in human lymphocytes (p Ë‚ 0.01). The increases in the frequencies of SCE and CA caused by nedaplatin were lowered by pretreatment treatment with Vit E (p < 0.05). Nedaplatin significantly lowered mitotic index but Vit E pretreatment did not modulate this effect. These results suggest that Vit E has the potential to ameliorate the genotoxicity of nedaplatin in cultured lymphocytes.

In vitro cytogenotoxic evaluation of aripiprazole on human peripheral lymphocytes and computational molecular docking analysis.

Two different drug groups, typical (classic) and atypical (new), are used in the treatment of schizophrenia. Aripiprazole, an atypical antipsychotic chemical, is the active ingredient of the drug Abilify. This study was conducted to determine the possible genotoxic effect of aripiprazole. For this purpose, four different doses of aripiprazole (5; 10; 20, and 40 µg/mL) were examined with Chromosome Abnormality (CA), Sister Chromatid Exchange (SCE), Micronucleus (MN) tests. Based on these tests, Proliferation Index (PI), Percent Abnormal Cells (AC), Mitotic Index (MI), Micronuclear Binuclear Cell (MNBN), and Nuclear Division Index (NDI) levels were determined in human peripheral lymphocytes treated for 24 and 48 hours. Also, to determine possible binding sites of Aripiprazole on B-DNA molecular docking analysis was performed using AutoDock 4.0 (B-DNA dodecamer, PDB code: 1BNA). Aripiprazole binds to B-DNA with a very significant free binding energy (-11.88 Kcal/mol). According to our study, aripiprazole did not significantly change SCE, CA, AC percentage, MN frequencies when compared with control. According to these results, aripiprazole does not have a genotoxic effect. At the same time, no significant change was observed in the PI, MI, and NDI frequencies when compared with the control. In line with these results, it was observed that the use of aripiprazole in the treatment of schizophrenia did not pose any acute genotoxic and cytotoxic risk.

Analysis for Sister Chromatid Exchange.

Sister chromatid exchange (SCE) is the exchange event of genetic material between two identical sister chromatid. Elevation of SCE frequency is considered as a result of replication stress from genetic defects, ROS stress, and genomic damages. SCE staining needs extra processes compared to regular Giemsa staining. Usually two rounds of cell cycle progress are required to observe SCE under microscope. SCE can be visualized with the fluorescence plus Giemsa (FPG) staining method or fluorescence staining methods with immunocytochemistry to BrdU or Click reaction to EdU which provide more clear images of SCE. This chapter will provide the detailed method for the SCE staining and measurement for the traditional FPG staining, BrdU monoclonal antibody staining method, and newly developed EdU Click reaction staining method.

In vitro genotoxic and antigenotoxic effects of an exopolysaccharide isolated from Lactobacillus salivarius KC27L.

Exopolysaccharide isolated from Lactobacillus salivarius (new genus name Ligilactobacillus) KC27L strain (EPSKC27L) exhibits antioxidant properties with 1,1-diphenyl-2-picrylhydrazase (DPPH) radical and superoxide anion radical (O2-.) scavenging effect and iron ion (Fe2+) chelating activity. This study aimed to investigate the in vitro genotoxic effects of EPSKC27L alone (12.50, 25.00, 50.00, and 100.00 µg/mL) and its antigenotoxic activity against DNA damage induced by mitomycin-C (MMC; 0.20 µg/mL), methyl methanesulfonate (MMS; 5.00 µg/mL), and hydrogen peroxide (H2O2; 100 µM). For this purpose, chromosome aberration (CA), sister chromatid exchange (SCE), micronucleus (MN), and comet assays were performed in human peripheral lymphocytes. In addition, the structure of EPSKC27L was investigated in the scanning electron microscope (SEM). EPSKC27L alone did not cause a significant genotoxic effect in CA, SCE, MN, and comet tests. EPSKC27L significantly decreased the frequency of CA, SCE, and MN induced by MMC and MMS. EPSKC27L also significantly reduced DNA damage induced by H2O2. This study showed that the EPSKC27L alone has no genotoxic risk at these concentrations and shows antigenotoxic activity against MMC, MMS, and H2O2. Consequently, EPSKC27L was found to exhibit chemopreventive activity against genotoxic agents. This effect is believed to be due to the antioxidant properties of EPSKC27L.

Sister chromatid exchanges induced by perturbed replication can form independently of BRCA1, BRCA2 and RAD51.

Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, SCE induction upon irradiation requires the canonical HR factors BRCA1, BRCA2 and RAD51. In contrast, replication-blocking agents, including PARP inhibitors, induce SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs are enriched at difficult-to-replicate genomic regions, including common fragile sites (CFSs). PARP inhibitor-induced replication lesions are transmitted into mitosis, suggesting that SCEs can originate from mitotic processing of under-replicated DNA. Proteomics analysis reveals mitotic recruitment of DNA polymerase theta (POLQ) to synthetic DNA ends. POLQ inactivation results in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Accordingly, analysis of CFSs in cancer genomes reveals frequent allelic deletions, flanked by signatures of POLQ-mediated repair. Combined, we show PARP inhibition generates under-replicated DNA, which is processed into SCEs during mitosis, independently of canonical HR factors.

Single-sister labeling in the C. elegans germline using the nucleotide analog EdU.

Reciprocal exchanges between genetically identical sister chromatids (sister chromatid exchanges or SCEs) have been challenging to study. Here, we describe a protocol that utilizes a pulse/chase of the thymidine analog 5-ethyl-3'-deoxyuridine (EdU) in combination with click chemistry and antibody labeling to selectively label sister chromatids in the C. elegans germline. Labeling has no discernable effects on meiosis, allowing for cytological quantification of SCEs. This protocol can be combined with a variety of imaging approaches, including STED, confocal and super-resolution. For complete details on the use and execution of this protocol, please refer to Almanzar et al. (2021).

Mapping of sister chromatid exchange events and genome alterations in single cells.

Mammalian genomes encode over a hundred different helicases, many of which are implicated in the repair of DNA lesions by acting on DNA structures arising during DNA replication, recombination or transcription. Defining the in vivo substrates of such DNA helicases is a major challenge given the large number of helicases in the genome, the breadth of potential substrates in the genome and the degree of genetic pleiotropy among DNA helicases in resolving diverse substrates. Helicases such as WRN, BLM and RECQL5 are implicated in the resolution of error-free recombination events known as sister chromatid exchange events (SCEs). Single cell Strand-seq can be used to map the genomic location of individual SCEs at a resolution that exceeds that of classical cytogenetic techniques by several orders of magnitude. By mapping the genomic locations of SCEs in the absence of different helicases, it should in principle be possible to infer the substrate specificity of specific helicases. Here we describe how the genome can be interrogated for such DNA repair events using single-cell template strand sequencing (Strand-seq) and bioinformatic tools. SCEs and copy-number alterations were mapped to genomic locations at kilobase resolution in haploid KBM7 cells. Strategies, possibilities, and limitations of Strand-seq to study helicase function are illustrated using these cells before and after CRISPR/Cas9 knock out of WRN, BLM and/or RECQL5.

Dose response to methylating agents in the γH2AX, SCE and colony formation assays: Effect of MGMT and MPG overexpression.

Cells have developed diverse protective mechanisms that enable them to tolerate low doses of genotoxic compounds. DNA repair processes attenuate the mutagenic and carcinogenic effects of alkylating agents, and multiple studies indicate a key role of specific DNA repair factors and pathways in establishing non-linear dose response relationships. Using an overexpression approach, we investigated the impact of O6-methylguanine-DNA-methyltransferase (MGMT), which repairs O6-methylguanine (O6MeG) in a damage reversal reaction, and N-methylpurine-DNA glycosylase (MPG), which acts as an apical enzyme in the BER pathway, on the DNA damage response to the alkylating agents MNNG and MMS. Our data indicate a clear protective effect of MGMT against MNNG-induced nuclear γH2AX foci formation, sister chromatid exchanges (SCE) and cytotoxicity, as determined in the colony formation assay. MGMT protected with similar efficiency against MMS-induced cytotoxicity and γH2AX foci formation, but suppressed SCE induction only weakly, which indicates that recombination events induced by MMS result from other lesions than O6MeG. In contrast, overexpression of MPG had only a very mild protective effect on the cellular defense against MMS and MNNG. Collectively, our data indicate that overexpression of MGMT results in non-linear DNA damage responses to O6MeG inducers. In contrast, MPG overexpression has only minor impact on the DNA damage response to alkylating drugs, indicating that other downstream enzymes in the BER pathway are limiting.

Genotoxicity of Glyphosate on Cultured Human Lymphocytes.

Glyphosate-based herbicides are the most used herbicides in the world. Despite being widely used, a dispute exists whether glyphosate-based herbicides have a negative effect on human health, particularly genotoxic effects. Therefore, the aim of this study was to investigate glyphosate genotoxicity on cultured human lymphocytes. Cultured human lymphocytes were treated with different concentrations of glyphosate (20, 40, and 200 µmol/L). Four toxicity measures were examined: frequency of chromosomal aberrations (CAs), frequency of sister-chromatid exchange (SCE), production of 8-OHdG, and cell kinetics analysis. The results show that glyphosate induced significant (P < 0.05) increases in the levels of SCE at the highest used concentration (200 µmol/L). However, no significant elevation in SCE levels was observed at the lower examined concentrations (20 and 40 µmol/L). No significant changes in CA were detected at all examined concentrations (P = 0.86). Also, glyphosate did not induce changes to the normal level of 8-OHdG at all examined concentrations (P = 0.98). Last, no significant changes in either mitotic index or proliferative index were observed at any examined concentrations (P > 0.05). The results collectively indicate a lack of genotoxicity and cytotoxicity of glyphosate in cultured human lymphocytes when dealing with environmentally relevant concentrations (20 and 40 µmol/L). However, being exposed to higher concentrations (200 µmol/L) led to slightly higher level of SCE. Therefore, we recommend cautionary measures when dealing with glyphosate-based herbicides for individuals, such as farmers, who may be extensively exposed to high concentrations of these herbicides.

Sister chromatids separate during anaphase in a three-stage program as directed by interaxis bridges.

During mitosis, from late prophase onward, sister chromatids are connected along their entire lengths by axis-linking chromatin/structure bridges. During prometaphase/metaphase, these bridges ensure that sister chromatids retain a parallel, paranemic relationship, without helical coiling, as they undergo compaction. Bridges must then be removed during anaphase. Motivated by these findings, the present study has further investigated the process of anaphase sister separation. Morphological and functional analyses of mammalian mitoses reveal a three-stage pathway in which interaxis bridges play a prominent role. First, sister chromatid axes globally separate in parallel along their lengths, with concomitant bridge elongation, due to intersister chromatin pushing forces. Sister chromatids then peel apart progressively from a centromere to telomere region(s), step-by-step. During this stage, poleward spindle forces dramatically elongate centromere-proximal bridges, which are then removed by a topoisomerase IIα­dependent step. Finally, in telomere regions, widely separated chromatids remain invisibly linked, presumably by catenation, with final separation during anaphase B. During this stage increased separation of poles and/or chromatin compaction appear to be the driving force(s). Cohesin cleavage licenses these events, likely by allowing bridges to respond to imposed forces. We propose that bridges are not simply removed during anaphase but, in addition, play an active role in ensuring smooth and synchronous microtubule-mediated sister separation. Bridges would thereby be the topological gatekeepers of sister chromatid relationships throughout all stages of mitosis.

Genotoxic effects of gadobutrol and gadoversetamide active substances used in magnetic resonance imaging in human peripheral lymphocytes in vitro.

Gadobutrol and gadoversetamide are gadolinium-based contrast agents (GBCAs) widely used during magnetic resonance imaging examination. In this study, the genotoxicity of two GBCAs, gadobutrol and gadoversetamide, was investigated by using different endpoints: chromosome aberration (CAs), sister chromatid exchange (SCEs), and micronucleus (MNi). Human peripheral lymphocytes (PBLs) were treated with five concentrations (7 000, 14 000, 28 000, 56 000, and 112 000 µg/mL) of both agents. While a few concentrations of gadobutrol significantly increased abnormal cell frequency and CA/Cell, nearly all the concentrations of gadoversetamide significantly elevated the same aberrations. Similarly, the effect of gadoversetamide on the formation of SCEs was higher than those of gadobutrol. Only one concentration of gadoversetamide significantly increased MN% but no gadobutrol. The comet assay was applied for the only gadobutrol which induced a significant increase in tail intensity at the highest concentration only. On the other hand, significantly decreased mitotic index (MI) was observed following both substances, again gadoversetamide was slightly higher than those of the gadobutrol. The results revealed that both the contrast agents are likely to induce genotoxic risk in PBLs. However, different concentrations and treatment periods should be examined in vitro and specifically in vivo with different test systems for the safer usage of these contrast agents.

The formation of SCEs as an effect of occupational exposure to formaldehyde.

Formaldehyde (FA) is a ubiquitous toxic chemical employed worldwide due to its disinfectant and preservative properties. Despite being classified as a human carcinogen, FA is still employed as formalin in pathology wards as standard fixative. We evaluated its relationship with the formation of sister-chromatid exchanges (SCEs) in cultured peripheral blood lymphocytes on 57 pathologists and 48 controls and the risk/protective role played by several genetic polymorphisms. All subjects were assessed for SCEs and genotyped for the most common cancer-associated gene polymorphisms: CYP1A1 exon 7 (A > G), CYP1A1*2A (T > C), CYP2C19*2 (G > A), GSTT1 (presence/absence), GSTM1 (presence/absence), GSTP1 (A > G), XRCC1 (G399A), XRCC1 (C194T), XRCC1 (A280G), XPC exon 15 (A939C), XPC exon 9 (C499T), TNFα - 308 G > A), IL10 - 1082 (G > A), and IL6 - 174 (G > C). Air-FA concentration was assessed through passive personal samplers. Pathologists, exposed to 55.2 µg/m3 of air-FA, showed a significantly higher SCEs frequency than controls, exposed, respectively, to 18.4 µg/m3. Air-FA was directly correlated with SCEs frequency and inversely with the replication index (RI). Regression models showed FA exposure as a significant predictor in developing SCEs, while did not highlight any role of the selected polymorphisms. Our study confirms the role of low air-FA levels as genotoxicity inductor, highlighting the importance to define exposure limits that could be safer for exposed workers.