Induced Micronucleus Formation Research Articles

A Novel Non-Psychoactive Fatty Acid from a Marine Snail, Conus inscriptus, Signals Cannabinoid Receptor 1 (CB1) to Accumulate Apoptotic C16:0 and C18:0 Ceramides in Teratocarcinoma Cell Line PA1.

The cannabinoid-type I (CB1) receptor functions as a double-edged sword to decide cell fate: apoptosis/survival. Elevated CB1 receptor expression is shown to cause acute ceramide accumulation to meet the energy requirements of fast-growing cancers. However, the flip side of continual CB1 activation is the initiation of a second ceramide peak that leads to cell death. In this study, we used ovarian cancer cells, PA1, which expressed CB1, which increased threefold when treated with a natural compound, bis(palmitoleic acid) ester of a glycerol (C2). This novel compound is isolated from a marine snail, Conus inscriptus, using hexane and the structural details are available in the public domain PubChem database (ID: 14275348). The compound induced two acute ceramide pools to cause G0/G1 arrest and killed cells by apoptosis. The compound increased intracellular ceramides (C:16 to 7 times and C:18 to 10 times), both of which are apoptotic inducers in response to CB1 signaling and thus the compound is a potent CB1 agonist. The compound is not genotoxic because it did not induce micronuclei formation in non-cancerous Chinese hamster ovarian (CHO) cells. Since the compound induced the cannabinoid pathway, we tested if there was a psychotropic effect in zebrafish models, however, it was evident that there were no observable neurobehavioral changes in the treatment groups. With the available data, we propose that this marine compound is safe to be used in non-cancerous cells as well as zebrafish. Thus, this anticancer compound is non-toxic and triggers the CB1 pathway without causing psychotropic effects.

Synthesis and Biological Toxicity Evaluation of Red Light-Emitting Carbon Quantum Dots

This study delineates the successful fabrication of red light-emitting carbon quantum dots (R-CQDs), approximately 3.5 nm in size, via hydrothermal methods. These carbon quantum dots (CQDs) display distinctive fluorescence properties, particularly a laser-dependency. The infrared and Raman spectra were subjected to a thorough investigation, revealing the presence of hydroxyl, amino, and carboxyl groups on the surface of the CQDs. Experimental findings indicate a significant correlation between the antibacterial effect of R-CQDs on E. coli and Yeast and their concentration. Subsequent research suggests that this antibacterial activity primarily stems from the CQDs’ disruption of cell membrane integrity, leading to the leakage of intracellular substances and consequently inhibiting the growth of these two microorganisms. The study also reveals that R-CQDs can trigger chromosomal aberrations in the root tip cells of broad beans and induce micronuclei formation. The frequency of micronuclei is directly proportional to the CQDs dosage, and an extended treatment duration results in an increased micronucleus rate. This suggests potential damage to the genetic material of broad beans by CQDs, which could adversely affect their growth and development. The study further identifies a significant impact of R-CQDs on the height of rice seedlings, causing a substantial reduction. Moreover, it was found that CQDs can infiltrate the rice body and instigate oxidative stress responses.

Formalin Induced Micronucleus Formation in the Buccal Mucosa of Pathology Laboratory Workers.

Formaldehyde is an irritating substance that is categorized as a definite carcinogen (Group A1), according to the International Agency for Research on Cancer (IARC). This study was conducted to determine the role of this substance in the frequency of micronuclei (MN) in the buccal mucosal cells due to long-term exposure of the pathology staff to formaldehyde. In this case-control study, 32 pathology laboratory staff members were assigned to the case group, and 32 staff members who were not exposed to formaldehyde were assigned to the control group. Buccal mucosa cells were collected with a wet spatula and stained with Papanicolaou stain. In each sample, 500 cells were counted; then, the frequency of MN and the average number of MN in the micronucleated cells were assessed and compared between the 2 groups using the independent t test. Furthermore, the relationship between gender and MN was evaluated using the independent t test. The relationship between years of exposure and time of exposure during the day (in hours) for the case group, as well as the relationship between age and frequency of MN was analyzed using the Pearson correlation coefficient. The mean frequency of MN in exfoliated buccal cells was 18.33±12.36 in the case group, which was significantly higher than the control group (10.55±6.22; P=0.003). The difference in the mean number of total MN in the micronucleated cells was not significant between the case and control groups (P=0.11). The relationship between sex, age, and years of exposure with the mean frequency of MN and the total number of MN in the micronucleated cells was not significant. The relationship between exposure time during the day and both the mean frequency of MN and the total number of MN in the micronucleated cells was significant (P=0.03). Formaldehyde exposure and extended time of exposure during the day can increase the frequency of MN, which can prognosticate the incidence of precancerous and cancerous lesions. Therefore, continuous exposure to formaldehyde can be considered an occupational health hazard, though further studies are needed to confirm this result.

Anticancer and Antimutagenic Properties of Pogonatherum paniceum on Colorectal Cancer Cells.

Pogonatherum paniceum (P. paniceum) (Lam.) Hack. plays an important role in detoxification. However, its anticancer activity has not yet been elucidated. The aim of our study was to examine the suppressive proliferation, anti-migration and mutagenic/antimutagenic properties of P. paniceum. Moreover, we set out to determine the cellular mechanism underlying its antiproliferation. To investigate P. paniceum's anticancer ability, HCT116 and HT29 cell lines were treated with a water extract containing P. paniceum, and then the cell viability was examined using the trypan blue exclusion method which were compared to HEK293 (non-cancerous cells). The anticancer effects were investigated by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and colony formation assay. Apoptosis induction, cell cycle distribution, and migration abilities were assessed by cell death detection enzyme-linked immunoassay (ELISA), flow cytometry, and wound healing assay. Finally, the mutagenicity and antimutagenicity were evaluated using the micronucleus assay. Treatment with P. paniceum caused a loss of cell viability in HCT116 and HT29 cells (not found in HEK293), which had an IC50 (half-maximal inhibitory concentration) of 1,156.2 and 1,207.0 µg/mL, respectively. We found that P. paniceum significantly inhibited the proliferative function of HCT116 and HT29 cells. To find the mechanism that exerts a suppressive proliferation effect on P. paniceum, we determined the DNA fragmentation and cell cycle distribution. We also found that P. paniceum treatment increased apoptosis and arrested of the cell cycle at G0/G1 remarkably when compared with the control group. Moreover, P. paniceum could decrease the migration of HCT116 and HT29 cancer cells. Finally, the treatment of P. paniceum did not induce micronucleus formation but did decrease the micronucleus frequency against mutagen-mitomycin C. P. paniceum did not possess any toxicity (cytotoxic and mutagenic) but has the potential for anticancer activity against human colorectal cells by increasing apoptosis, which leads to the suppression of cell proliferation. P. paniceum also inhibits cell migration and exerts antimutagenicity, thereby suggesting that P. paniceum might be useful for colorectal cancer treatment.

Induction of clastogenesis and gene mutations by carbamazepine (at its therapeutically effective serum levels) in mammalian cells and the dependence on human CYP2B6 enzyme activity

Carbamazepine (CBZ, an antiepileptic) is metabolized by multiple CYP enzymes to its epoxide and hydroxides; however, whether it is genotoxic remains unclear. In this study, molecular docking (CBZ to CYPs) and cytogenotoxic toxicity assays were employed to investigate the activation of CBZ for mutagenic effects, in various mammalian cell models. Docking results indicated that CBZ was valid as a substrate of human CYP2B6 and 2E1, while not for CYP1A1, 1A2, 1B1 or 3A4. In the Chinese hamster (V79) cell line and its derivatives genetically engineered for the expression of human CYP1A1, 1A2, 1B1, 2E1 or 3A4 CBZ (2.5 ~ 40μM) did not induce micronucleus, while in human CYP2B6-expressing cells CBZ significantly induced micronucleus formation. In a human hepatoma C3A cell line, which endogenously expressed CYP2B6 twofold higher than in HepG2 cells, CBZ induced micronucleus potently, which was blocked by 1-aminobenzotriazole (inhibitor of CYPs) and ticlopidine (specific CYP2B6 inhibitor). In HepG2 cells CBZ did not induce micronucleus; however, pretreatment of the cells with CICTO (CYP2B6 inducer) led to micronucleus formation by CBZ, while rifampicin (CYP3A4 inducer) or PCB126 (CYP1A inducer) did not change the negative results. Immunofluorescent assay showed that CBZ selectively induced centromere-free micronucleus. Moreover, CBZ induced double-strand DNA breaks (γ-H2AX elevation, by Western blot) and PIG-A gene mutations (by flowcytometry) in C3A (threshold being 5μM, lower than its therapeutic serum concentrations, 17 ~ 51μM), with no effects in HepG2 cells. Clearly, CBZ may induce clastogenesis and gene mutations at its therapeutic concentrations, human CYP2B6 being a major activating enzyme.

Optimized High-Content Imaging Screening Quantifying Micronuclei Formation in Polymer-Treated HaCaT Keratinocytes.

Research on nano- and micro-plastic particles (NMPPs) suggests their potential threat to human health. Some studies have even suggested genotoxic effects of NMPP exposure, such as micronuclei (MN) formation, while others found the opposite. To clarify the ability of NMPP to induce MN formation, we used non-malignant HaCaT keratinocytes and exposed these to a variety of polystyrene (PS) and poly methyl methacrylate (PMMA) particle types at different concentrations and three different sizes. Investigations were performed following acute (one day) and chronic exposure (five weeks) against cytotoxic (amino-modified NMPPs) and genotoxic (methyl methanesulfonate, MMS) positive controls. An optimized high-content imaging workflow was established strictly according to OECD guidelines for analysis. Algorithm-based object segmentation and MN identification led to computer-driven, unsupervised quantitative image analysis results on MN frequencies among the different conditions and thousands of cells per condition. This could only be realized using accutase, allowing for partial cell detachment for optimal identification of bi-nucleated cells. Cytotoxic amino-modified particles were not genotoxic; MMS was both. During acute and long-term studies, PS and PMMA particles were neither toxic nor increased MN formation, except for 1000 nm PS particles at the highest concentration of unphysiological 100 µg/mL. Interestingly, ROS formation was significantly decreased in this condition. Hence, most non-charged polymer particles were neither toxic nor genotoxic, while aminated particles were toxic but not genotoxic. Altogether, we present an optimized quantitative imaging workflow applied to a timely research question in environmental toxicity.

Triphenyl phosphate induces clastogenic effects potently in mammalian cells, human CYP1A2 and 2E1 being major activating enzymes

As a new-type flame retardant and toxic substance, triphenyl phosphate (TPP) is a ubiquitous pollutant present even in human blood. TPP is transformed by human CYP enzymes to oxidized/dealkylated metabolites. The impact of TPP metabolism on its toxicity, however, remains unclear. In this study, the genotoxicity of TPP in several mammalian cell lines and its relevance to CYP/sulfortransferase (SULT) activities were investigated. The results indicated that TPP induced micronucleus formation at ≥1 μM concentrations in a human hepatoma (C3A, endogenous CYPs being substantial) cell line, which was abolished by 1-aminobenzotriazole (CYPs inhibitor). In cell line HepG2 (parental to C3A with lower CYP expression) TPP was inactive up to 10 μM, while pretreatment with ethanol (CYP2E1 inducer), PCB 126 (CYP1A inducer), or rifampicin (CYP3A inducer) led to micronucleus formation by TPP. In V79-Mz and V79-derived cells expressing human CYP1A1 TPP was inactive (up to 32 μM), and in cells expressing human CYP1B1, 2B6 and 3A4 it induced micronucleus weakly (positive only at 32 μM). However, TPP induced micronucleus potently in V79-derived cells expressing human CYP1A2, while this effect was drastically reduced by human SULT1A1 co-expression; likewise, TPP was inactive in cells expressing both human CYP2E1 and SULT1A1, but became positive with pentachlorophenol (inhibitor of SULT1) co-exposure. Moreover, in C3A cells TPP selectively induced centromere-free micronucleus (immunofluorescent assay), and TPP increased γ-H2AX (by Western blot, indicating double-strand DNA breaks). In conclusion, this study suggests that TPP is potently clastogenic, human CYP1A2 and 2E1 being major activating enzymes while SULT1A1 involved in detoxification.

Beta Human Papillomavirus 8 E6 Induces Micronucleus Formation and Promotes Chromothripsis.

Cutaneous beta genus human papillomaviruses (β-HPVs) are suspected to promote the development of nonmelanoma skin cancer (NMSC) by destabilizing the host genome. Multiple studies have established the genome destabilizing capacities of β-HPV proteins E6 and E7 as a cofactor with UV. However, the E6 protein from β-HPV8 (HPV8 E6) induces tumors in mice without UV exposure. Here, we examined a UV-independent mechanism of HPV8 E6-induced genome destabilization. We showed that HPV8 E6 reduced the abundance of anaphase bridge resolving helicase, Bloom syndrome protein (BLM). The diminished BLM was associated with increased segregation errors and micronuclei. These HPV8 E6-induced micronuclei had disordered micronuclear envelopes but retained replication and transcription competence. HPV8 E6 decreased antiproliferative responses to micronuclei and time-lapse imaging revealed HPV8 E6 promoted cells with micronuclei to complete mitosis. Finally, whole-genome sequencing revealed that HPV8 E6 induced chromothripsis in nine chromosomes. These data provide insight into mechanisms by which HPV8 E6 induces genome instability independent of UV exposure. IMPORTANCE Some beta genus human papillomaviruses (β-HPVs) may promote skin carcinogenesis by inducing mutations in the host genome. Supporting this, the E6 protein from β-HPV8 (8 E6) promotes skin cancer in mice with or without UV exposure. Many mechanisms by which 8 E6 increases mutations caused by UV have been elucidated, but less is known about how 8 E6 induces mutations without UV. We address that knowledge gap by showing that 8 E6 causes mutations stemming from mitotic errors. Specifically, 8 E6 reduces the abundance of BLM, a helicase that resolves and prevents anaphase bridges. This hinders anaphase bridge resolution and increases their frequency. 8 E6 makes the micronuclei that can result from anaphase bridges more common. These micronuclei often have disrupted envelopes yet retain localization of nuclear-trafficked proteins. 8 E6 promotes the growth of cells with micronuclei and causes chromothripsis, a mutagenic process where hundreds to thousands of mutations occur in a chromosome.

Abstract 6277: Identification of ATR inhibitors as therapeutic opportunities in Desmoplastic small round cell tumors

Abstract Introduction: Desmoplastic small round cell tumor (DSRCT) is a rare and very aggressive subtype of sarcoma, affecting predominantly young males. DSRCT is molecularly characterized by the t(11;22)(q24;q12) chromosomal translocation, which fuses EWSR1 to WT1 and encodes an aberrant chimeric transcription factor. A few additional secondary mutations have been reported, notably in genes encoding proteins involved in chromatin remodeling and DNA repair. No therapeutic advance have been done in the past 20 years, and the five-year overall survival is still below 15% in metastatic cases, despite aggressive poly-chemotherapy and extensive surgical debulking. Thus, we aimed to identify actionable targeted dependencies to propose novel therapeutic approaches, using drug screening and molecular biology. Material and Methods: Drug sensitivity screen evaluating 80 cancer-relevant small molecule inhibitors in dose-response was performed on the JN-DSRCT-1 (JN1) cell line. Revalidation was performed on JN1 and in a Champions Oncology PDX-derived in-house-generated DSRCT cell line (R) using multiple clinical compounds. Mechanistic dissection was performed using flow cytometry, immunofluorescence, western blotting, DNA fiber assay, RT-qPCR, and dot blot to evaluate markers of DNA damage response, replication stress and cell-autonomous innate immune signaling. Results: The drug screen identified that JN1 cells were particularly sensitive to one ATR inhibitor (ATRi) and multiple PARPi inhibitors (PARPi), which scored #6, 7, 9 and 20 of the hits. Sensitivity to ATRi was revalidated in the JN1 and R cell lines, whereas sensitivity to PARPi was only revalidated in the JN1 cell line - consistent with the very low PARP1 expression in that cell line. Sensitivity to ATRi and PARPi was partially reversed upon EWS-WT1 silencing. Combination experiments revealed that both drugs were synergistic in the JN1 cell line. Exposure to PARP inhibitor increased the number of γH2AX and RAD51 foci, suggesting that JN1 cells were not deficient in homologous recombination. Further, PARPi or ATRi exposure decreased replication fork speed and increased R-loops accumulation, whereas EWS-WT1 silencing increased replication fork speed. Resistance to PARPi and to ATRi was restored upon exogenous expression of the R-loop resolution enzyme RNAseH1. The combination of PARPi and ATRi also induced micronuclei formation and activated the cGAS-STING cytosolic innate immune sensing pathway, together with increased expression of CCL5, CXCL10 and PD-L1. Conclusion: DSRCT cells are sensitive to ATR and PARP inhibitors, through a mechanism that, at least in part, may involve enhanced replication stress and increased R-loop formation. Our data provide the preclinical rationale for assessing ATRi combined with PARPi in patients with DSRCT, as a cytotoxic therapy or as an immunomodulatory approach to enhance cell-autonomous immunogenicity. Citation Format: Asuka KAWAI-KAWACHI, Madison LENORMAND, Clémence HENON, Thomas EYCHENNE, Leo COLMET-DAAGE, Nicolas DORVAULT, Marlene GARRIDO, Clémence ASTIER, Carine NGO, Helen PEMBERTON, Aditi GULATI, Stephen PETTITT, Roman CHABANON, Christopher J LORD, Sophie POSTEL-VINAY. Identification of ATR inhibitors as therapeutic opportunities in Desmoplastic small round cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6277.

Therapeutic and immunomodulatory potential of pazopanib in malignant phyllodes tumor

Malignant phyllodes tumors (PT) are rare aggressive fibroepithelial neoplasms with high metastatic potential and lack effective therapy. We established a patient-derived xenograft (PDX) and cell line model (designated MPT-S1) of malignant PT which demonstrated clinical response to pazopanib. Whole exome sequencing identified somatic mutations in TP53, RB1, MED12, and KMT2D. Immunohistochemistry and genomic profiles of the tumor, PDX and cell line were concordant. In keeping with clinical observation, pazopanib reduced cell viability in a dose-dependent manner and evoked apoptosis, and led to significant abrogation of in vivo tumor growth. Whole transcriptomic analysis revealed that pazopanib decreased expression of genes involved in oncogenic and apoptosis signaling. We also observed decreased expression of ENPP1, with known roles in cancer invasion and metastasis, as well as STING pathway upregulation. Accordingly, pazopanib induced micronuclei formation, and evoked phospho-TBK1 and PD-L1 expression. In an additional cohort of malignant PT (n = 14), six (42.9%) showed comparable or higher levels of ENPP1 relative to MPT-S1, highlighting its potential role as a therapeutic target. In conclusion, we established MPT-S1, a new PDX and cell line model, and provided evidence for the clinical efficacy of pazopanib in malignant PT.

Optimisation of an automated high-throughput micronucleus (HiTMiN) assay to measure genotoxicity of environmental contaminants

Anthropogenic contaminants can have a variety of adverse effects on exposed organisms, including genotoxicity in the form of DNA damage. One of the most commonly used methods to evaluate genotoxicity in exposed organisms is the micronucleus (MN) assay. It provides an efficient assessment of chromosomal impairment due to either chromosomal rupture or mis-segregation during mitosis. However, evaluating chromosomal damage in the MN assay through manual microscopy is a highly time-consuming and somewhat subjective process. High-throughput evaluation with automated image analysis could reduce subjectivity and increase accuracy and throughput. In this study, we optimised and streamlined the HiTMiN assay, adapting the MN assay to a miniaturised, 96-well plate format with reduced steps, and applied it to both primary cells from green turtle fibroblasts (GT12s-p) and a freshwater fish hepatoma cell line (PLHC-1). Image analysis using both commercial (Columbus) and freely available (CellProfiler) software automated the scoring of MN, with improved precision and drastically reduced time compared to manual scoring and other available protocols. The assay was validated through exposure to two inorganic (chromium and cobalt) and one organic (the herbicide metolachlor) compounds, which are genotoxicants of concern in the marine environment. All compounds tested induced MN formation below cytotoxic concentrations. The HiTMiN assay presented here greatly increases the suitability of the MN assay as a quick, affordable, sensitive and accurate assay to measure genotoxicity of environmental samples in different cell lines.

Evaluation of the Antigenotoxic Potential of Methanolic Leaves Extract of Triticum aestivum in Mice

Mutations are changes to the nucleotide sequence of the genetic material of an organism. Reactive oxygen species (ROS) play an important role in process like mutagenesis, carcinogenesis and aging by their ability to damage cellular DNA. Inhibition of mutagenesis or carcinogenesis is generally not based on one specific mechanism. Protection against cancer can occur at different stages of the complicated processes of carcinogenesis. Naturally occurring antioxidants have been extensively studied for their capacity to protect organisms and cells from oxidation. Triticum aestivum (T. aestivum) have revealed its medicinal potential for some human diseases; therefore, this study aimed to evaluate the genotoxic and antigenotoxic potential of methanolic extract. To accomplish this, the methanolic extract of T. aestivum was evaluated for its antigenotoxic effect using the chromosomal aberrations and micronucleus assay of bone marrow cells of mice.T. aestivum extract has shown significant protection against Cyclophosphamide induced micronucleus formation and chromosomal aberrations.

In vitro cytogenotoxic and mutagenic effects of Commiphora myrrha essential oil

Commiphora myrrha, located in the tropical zone, is a widely used tree for medicinal purposes in the Arabian Peninsula and a large part of Africa. In this research, cytogenotoxic effects of the commercially available Commiphora myrrha essential oil (myrrh) were studied using micronucleus (MN), comet, and total oxidant (TOS), and total antioxidant (TAS) assays on human peripheral lymphocytes under in vitro conditions. In addition, pure pBR322 plasmid DNA was used to investigate DNA damaging/protecting activity of the essential oil. Finally, a bacterial reversion (Ames) test was performed using Salmonella typhimurium mutant strains TA98 and TA100 to determine the potential effect of the agent in the induction of gene mutations. The high concentration of Commiphora myrrha (0.125 µL/mL) induced MN formation significantly compared to the untreated control in both treatment times (24 or 48 h). Only at the highest concentration, nuclear division index (NDI) values were found lower than the controls. In the Comet test performed on healthy lymphocytes, only the highest concentration of myrrh caused significant increases in the percentage of damaged cells and genetic damage index (GDI) values. Myrrh oil showed no significant mutagenic effect on mutant Salmonella strains. In addition, the substance did not directly damage plasmid DNA but also protected DNA against damaging factors such as H2O2 and UV. Finally, in the TAS and TOS assays, no significant differences on the oxidative stress parameters were found in cell culture compared to the control. The results of this study showed that myrrh oil exerts cytogenotoxic risk only at higher concentrations.

A MATLAB-based program for three-dimensional quantitative analysis of micronuclei reveals that neuroinflammation induces micronuclei formation in the brain

The micronucleus is known to be a biomarker for genomic instability, which is a hallmark of tumors and aging. Normally, micronuclei are produced by segregation errors and mechanical stresses arising from dividing or migrating cells, leading to activation of the innate immune response pathway. Although micronuclei often emerge in damaged tissues, the quantitative procedure for analyzing micronuclei accurately has been problematic. Here, we introduce a novel MATLAB-based program for quantifying micronuclei (CAMDi: calculating automatic micronuclei distinction) in vitro and in vivo. CAMDi is adaptable to various experimental imaging techniques and is useful for obtaining reproducible data. CAMDi enables us to measure the accurate size of micronuclei from the three-dimensional images. Using CAMDi, we revealed a novel link between the emergence of micronuclei and neuroinflammation. We found that inflammatory stimulation does not increase the number of micronuclei in primary neurons. On the other hand, the administration of lipopolysaccharide into mice slightly increases micronuclei formation in neurons of the hippocampus region. These findings demonstrate that neuronal micronuclei formations are induced by an inflammatory response in a non-cell-autonomous manner. We provide a novel tool, CAMDi, to quantify micronuclei and demonstrate that neuronal micronuclei are produced not only by the cell-autonomous process but also by the intercellular communication associated with neuroinflammation in vivo.

Abstract 1234: G4 binders as potential immunostimulatory compounds for cancer therapy

Abstract G-quadruplex (G4) is non-canonical nucleic acid structure involved in a plethora of fundamental biological processes. In past decades, it has been studied as a promising pharmaceutical target for anticancer therapy. However, no G4-targeting agent has shown significant clinical effects up to date. Pyridostatin (PDS), a well-known G4 binder, can induce double-stranded DNA breaks and genome instability. We have recently shown that PDS and other G4 binders can trigger micronuclei formation in cancer cells at non-cytotoxic concentrations [1]. As micronuclei can play a crucial role in linking genome instability to innate immunity, we have here wondered whether G4 binders can induce immune gene activation in human MCF-7 breast cancer cells. By using RNA-Seq technology, we show that non-cytotoxic doses of PDS can activate Interferon beta- and IRF3-dependent genes leading to an innate immune gene response in human cancer cells. In addition, immune gene activation follows the induction of micronuclei formation supporting cytoplasmic DNA as a trigger of immune gene activation. Immunofluorescence assays showed that cGAS (a cytoplasmic DNA sensor) can bind to and is activated by micronuclei. In addition, cytostatic PDS concentrations activate STING and IRF3 factors leading to activated immune gene expression. STING inhibition with siRNAs, CRISPR knockout or a chemical inhibitor resulted in a suppression of immune gene expression and Interferon-beta (IFN-B) production in human MCF-7 and murine B16 cell lines, showing that STING is required for PDS-induced immune gene activation in cancer cells. We have then extended the study to a structurally-unrelated G4 binder, PhenDC3, and the results show that PhenDC3 can induce micronuclei formation and IFN-B production in a STING-dependent manner. Then, using TCGA-BRCA dataset, we show that specific gene expression patterns mediated by PDS correlate with immunological hot features of human tumors, such as anti-tumor immune cell infiltration. We could also define a specific PDS gene signature that can predict a better survival of breast cancer patients, suggesting an impact of immunomodulation potential of PDS or other G4 binders in human patients. Thus, our results provide strong evidence that G4 binders can activate innate immune genes in human and murine cancer cells with therapeutic potentials, and that G4 binder-induced micronuclei accumulation can trigger the cGAS-STING signaling pathway. The findings may open to the development of novel immunotherapeutic anticancer strategies based on G4-targeting compounds.

Cytotoxicity and genotoxicity of salicylate- and calcium silicate-based root canal sealers on primer human periodontal ligament fibroblasts.

The aim of this study was to evaluate the biocompatibility of epoxy-resin-based AHPlus, salicylate-based MTA-Fillapex and calcium silicate-based iRootSP root canal sealers. Cytotoxicity was assessed by XTT test. The extracts from sealers of different setting times were serially diluted. Cell viability was calculated as the percentage of the control group (100%). The optimal concentration of each sealer was used at genotoxicity test, and micronuclei formations were detected. Statistical analyses were done by using Kruskal-Wallis and Dunn post hoc test with Bonferroni correction. AHPlus and MTA-Fillapex showed the lowest percentage of cell viability at higher concentrations (1:1, 1:2, 1:4), especially at first 12 h. iRootSP showed higher viability at all concentrations and times than AHPlus and MTA-Fillapex. At genotoxicity assay, AHPlus increased the number of micronuclei. MTA-Fillapex slightly induced micronucleus formation (not significant) and iRootSP was not increased. In conclusion, calcium silicate-based iRootSP had lowest cytotoxic and genotoxic potential and can be considered as a highly biocompatible material.

The Genotoxic and Pro-Apoptotic Activities of Advanced Glycation End-Products (MAGE) Measured with Micronuclei Assay Are Inhibited by Their Low Molecular Mass Counterparts.

An association between the cancer invasive activities of cells and their exposure to advanced glycation end-products (AGEs) was described early in some reports. An incubation of cells with BSA–AGE (bovine serum albumin–AGE), BSA–carboxymethyllysine and BSA–methylglyoxal (BSA–MG) resulted in a significant increase in DNA damage. We examined the genotoxic activity of new products synthesized under nonaqueous conditions. These were high molecular mass MAGEs (HMW–MAGEs) formed from protein and melibiose and low molecular mass MAGEs (LMW–MAGEs) obtained from the melibiose and N-α-acetyllysine and N-α-acetylarginine. We have observed by measuring of micronuclei in human lymphocytes in vitro that the studied HMW–MAGEs expressed the genotoxicity. The number of micronuclei (MN) in lymphocytes reached 40.22 ± 5.34 promille (MN/1000CBL), compared to 28.80 ± 6.50 MN/1000 CBL for the reference BSA–MG, whereas a control value was 20.66 ± 1.39 MN/1000CBL. However, the LMW–MAGE fractions did not induce micronuclei formation in the culture of lymphocytes and partially protected DNA against damage in the cells irradiated with X-ray. Human melanoma and all other studied cells, such as bronchial epithelial cells, lung cancer cells and colorectal cancer cells, are susceptible to the genotoxic effects of HMW–MAGEs. The LMW–MAGEs are not genotoxic, while they inhibit HMW–MAGE genotoxic activity. With regard to apoptosis, it is induced with the HMW–MAGE compounds, in the p53 independent way, whereas the low molecular mass product inhibits the apoptosis induction. Further investigations will potentially indicate beneficial apoptotic effect on cancer cells.

Radiation mitigating activities of Psidium guajava L. against whole-body X-ray-induced damages in albino Wistar rat model

In the present study, we investigated radiation mitigating activities of Psidium guajava L. (P. guajava) against whole-body X- ray induced damages in albino Wistar rat model. The animals were orally administered with 200 mg/kg bodyweight of hydroalcoholic leaf extract of P. guajava for five consecutive days and on the fifth day, after the last dose of extract administration, animals were exposed to 4 Gy of X-rays. Rats were sacrificed 24 h post X–ray irradiation. The radiomitigating activity of the herb extract was assessed by micronucleus assay, histopathology of the small intestine and hematological parameters. Hepatic cyclooxygenase–2 (COX-2), interleukin–6 (IL-6) and interleukin –10 (IL-10) levels were assayed to validate the anti-inflammatory property. Biochemical estimations were also performed in RBC lysates to corroborate antioxidant properties in the leaf extract. HPLC analysis of crude extract confirmed the presence of standard flavonoid quercetin. Our results indicated that radiation elevated COX-2, IL-6 and decreased IL-10 levels and also induced micronucleus formation in polychromatic erythrocytes, simultaneously impairing hematological parameters along with erythrocyte antioxidants. The animals pre-treated with P. guajava exhibited a significant decrease in the COX-2 (P ≤ 0.01), IL-6 levels (P ≤ 0.05) and also displayed significant increase in the hepatic IL-10 levels (P ≤ 0.01). Pre-treatment with plant extract improved antioxidant enzyme activities, hematological parameters and reduced the intestinal damage by recovering the architecture of the small intestine. Moreover, extract also rendered protection against radiation induced DNA damage, as evidenced by the significant (P ≤ 0.01) decrease in the percentage of radiation-induced micronucleus in polychromatic erythrocytes. Furthermore, the herb extract treatment increased radiation LD50/30 from 6.6 Gy to 9.0 Gy, offering a dose reduction factor (DRF) of 1.36. Our findings for the first time propose the beneficial use of P. guajava as a radioprotector against X-ray induced damage.

Discovery of a CNS penetrant small molecule SMN2 splicing modulator with improved tolerability for spinal muscular atrophy

Spinal muscular atrophy (SMA) is a motor neuron disease, typically resulting from loss-of-function mutations in the survival motor neuron 1 (SMN1) gene. Nusinersen/SPINRAZA, a splice-switching oligonucleotide that modulates SMN2 (a paralog of SMN1) splicing and consequently increases SMN protein levels, has a therapeutic effect for SMA. Previously reported small-molecule SMN2 splicing modulators such as risdiplam/EVRYSDI and its analog SMN-C3 modulate not only the splicing of SMN2 but also that of secondary splice targets, including forkhead box protein M1 (FOXM1). Through screening SMA patient-derived fibroblasts, a novel small molecule, designated TEC-1, was identified that selectively modulates SMN2 splicing over three secondary splice targets. TEC-1 did not strongly affect the splicing of FOXM1, and unlike risdiplam, did not induce micronucleus formation. In addition, TEC-1 showed higher selectively on galactosylceramidase and huntingtin gene expression compared to previously reported compounds (e.g., SMN-C3) due to off-target effects on cryptic exon inclusion and nonsense-mediated mRNA decay. Moreover, TEC-1 significantly ameliorated the disease phenotype in an SMA murine model in vivo. Thus, TEC-1 may have promising therapeutic potential for SMA, and our study demonstrates the feasibility of RNA-targeting small-molecule drug development with an improved tolerability profile.

Micronuclei assay as biomarker for ethyl methane sulphonate (EMS) genotoxicity in Eclipta alba (L.) Hassk.

The eukaryotic cells are complemented with a single nucleus. However, several cells show abnormal small nuclei known as micronuclei, with addition to the main nucleus. These anomalous small nuclei along with main nucleus which may be the result of exposure of cell to cytotoxic agents (Chemicals/physical mutagens). These mutagens also cause structural or numerical chromosomal aberrations. The treatment of EMS, a chemical mutagen, induces micronuclei formation and nuclear as well as chromosomal peculiarities in Eclipta alba. The effect of micronuclei is discussed in terms of cytotoxicity of genotoxins and several evidences of nuclear polymorphism illustrated due to the mutagenic effect of EMS. Pollen viability was also examined through pollen fertility. Micronuclei causes the genomic instability and elimination of genome which is effectively put to use in breeding programmes for the creation of aneuploids and also for haploid lines.