Increase In Micronucleus Formation Research Articles

Exploring the toxicity profile of coriander (C. sativum L.) essential oil: implications for translational toxicological research

The plant species C. sativum L. is a staple in cuisine and holds significant ethnopharmacological value. Its essential oil (EO) is of particular interest, yet its toxicity profile remains a subject of inquiry. This study aimed to elucidate the chemical constituents of C. sativum L. EO and evaluate its toxicity through various parameters, including cytotoxicity assays on HaCaT keratinocytes, in vivo toxicity tests on Galleria mellonella larvae, in vivo genotoxicity assessments on mice and cytotoxicity assays on human erythrocytes. Notably, major constituents such as 2-decen-1-ol, dec-(2E)-enal, and 1,6-octadien-3-ol were found to remain predominant. The IC50 value for the essential oil on the keratinocyte cell line was determined to be 60.13 ± 2.02 µg/mL. However, in vivo toxicity tests with G. mellonella larvae demonstrated safety at doses below 4.5 g/kg. Additionally, genotoxicity assessment revealed that a single dose of 20 mg/mL (5 mg/kg) did not induce a significant increase in micronuclei formation. EO concentrations above 250 µg/mL led to significant changes in human erythrocytes cell viability (p < 0.0001), resulting in over 60% hemolysis. These findings collectively suggest that the essential oil of C. sativum L. exhibits a suitable toxicity profile for conducting preclinical studies in vertebrate animal models.

Inhibition of key DNA double strand break repair protein kinases enhances radiosensitivity of head and neck cancer cells to X-ray and proton irradiation

Ionising radiation (IR) is widely used in cancer treatment, including for head and neck squamous cell carcinoma (HNSCC), where it induces significant DNA damage leading ultimately to tumour cell death. Among these lesions, DNA double strand breaks (DSBs) are the most threatening lesion to cell survival. The two main repair mechanisms that detect and repair DSBs are non-homologous end joining (NHEJ) and homologous recombination (HR). Among these pathways, the protein kinases ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR) and the DNA dependent protein kinase catalytic subunit (DNA-Pkcs) play key roles in the sensing of the DSB and subsequent coordination of the downstream repair events. Consequently, targeting these kinases with potent and specific inhibitors is considered an approach to enhance the radiosensitivity of tumour cells. Here, we have investigated the impact of inhibition of ATM, ATR and DNA-Pkcs on the survival and growth of six radioresistant HPV-negative HNSCC cell lines in combination with either X-ray irradiation or proton beam therapy, and confirmed the mechanistic pathway leading to cell radiosensitisation. Using inhibitors targeting ATM (AZD1390), ATR (AZD6738) and DNA-Pkcs (AZD7648), we observed that this led to significantly decreased clonogenic survival of HNSCC cell lines following both X-ray and proton irradiation. Radiosensitisation of HNSCC cells grown as 3D spheroids was also observed, particularly following ATM and DNA-Pkcs inhibition. We confirmed that the inhibitors in combination with X-rays and protons led to DSB persistence, and increased micronuclei formation. Cumulatively, our data suggest that targeting DSB repair, particularly via ATM and DNA-Pkcs inhibition, can exacerbate the impact of ionising radiation in sensitising HNSCC cell models.

Risk Characterization of Botanical Extracts Containing Hydroxyanthracenes as Determined by a Validated Micronucleus In Vitro Assay

Extracts of Rheum palmatum L., Rhamnus purshiana DC., Rhamnus frangula L., and Cassia senna L. are used in traditional medicine thanks to their beneficial properties. These species contain hydroxyanthracene derivatives, considered genotoxic and possibly related to colorectal cancer development. This research aimed to study, using a micronucleus assay in vitro, the genotoxic potential of Rheum palmatum L., Rhamnus purshiana DC., Rhamnus frangula L. (bark), and Cassia senna L. (leaves and fruits) extracts. The extracts were evaluated at different concentrations: from 0 to 2000 µg/mL for Rhamnus purshiana DC, from 0 to 2500 µg/mL for Rheum palmatum L. and Rhamnus frangula L., and from 0 to 5000 µg/mL for Cassia senna L. The cytokinesis-block proliferation index was calculated to analyse if the used concentrations showed cytotoxicity. The hydroxyanthracene content varied between 0.06% and 0.23% for aloe-emodin, and between 0.07% and 0.16% for emodin and rhein. No cytotoxic effect was detected at any of these concentrations. Micronucleus analyses showed a lack of genotoxicity for all the extracts tested. These results show that Rheum palmatum L., Rhamnus purshiana DC, Rhamnus frangula L., and Cassia senna L. extracts do not induce genotoxicity since no increase in micronuclei formation in human lymphocytes in vitro was detected.

FLIP(C1orf112)-FIGNL1 complex regulates RAD51 chromatin association to promote viability after replication stress

Homologous recombination (HR) plays critical roles in repairing lesions that arise during DNA replication and is thus essential for viability. RAD51 plays important roles during replication and HR, however, how RAD51 is regulated downstream of nucleofilament formation and how the varied RAD51 functions are regulated is not clear. We have investigated the protein c1orf112/FLIP that previously scored in genome-wide screens for mediators of DNA inter-strand crosslink (ICL) repair. Upon ICL agent exposure, FLIP loss leads to marked cell death, elevated chromosomal instability, increased micronuclei formation, altered cell cycle progression and increased DNA damage signaling. FLIP is recruited to damage foci and forms a complex with FIGNL1. Both proteins have epistatic roles in ICL repair, forming a stable complex. Mechanistically, FLIP loss leads to increased RAD51 amounts and foci on chromatin both with or without exogenous DNA damage, defective replication fork progression and reduced HR competency. We posit that FLIP is essential for limiting RAD51 levels on chromatin in the absence of damage and for RAD51 dissociation from nucleofilaments to properly complete HR. Failure to do so leads to replication slowing and inability to complete repair.

Determination of micronuclei frequency in Danio rerio for assessing genotoxicity induced by propineb

The aim of this study was to investigate the genotoxic effect of Propineb fungicide at different concentrations (0.167, 0.335 and 0.670 mg L−1) and different treatment times (24, 48, 72 and 96 h) on Danio rerio. At the end of the treatment periods, blood was collected from the fish with a heparin injector; smear preparations were prepared, fixed and stained. In the prepared preparations, the numbers of cells with MN and erythrocyte nucleus abnormalities were examined. It was found that propineb increased micronucleus formation at all treatment times and concentrations and induced the formation of erythrocytes with morphological abnormal nuclei such as segmented, kidney-shaped, notched, vacuolated nuclei and binucleated. The increase in micronucleus formation and the number of erythrocytes with abnormal nuclei were found to be concentration and treatment time-dependent. In conclusion, in this study, Danio rerio erythrocytes were used to evaluate the genotoxic effects of propineb fungicide on aquatic organisms, which have an important place in environmental risk assessment criteria. Since fungicides used in agricultural control such as propineb may have the potential to be genotoxic to aquatic organisms, the results of toxicity tests should be taken into consideration in the selection and use of concentrations of these chemicals.

Effect of Melissa officinalis L. leaf extract on manganese-induced cyto-genotoxicity on Allium cepa L.

Although the antioxidant properties of Melissa officinalis extract (Mox) are widely known, little work has focused on its protective capacity against heavy metal stress. The primary objective of this study was to determine the potential of Mox to mitigate manganese (II) chloride (MnCI2)-induced cyto-genotoxicity using the Allium and comet assays. Physiological, genotoxic, biochemical and anatomical parameters as well as the phenolic composition of Mox were examined in Allium cepa (L.). Application of 1000 µM MnCl2 reduced the rooting percentage, root elongation, weight gain, mitotic index and levels of chlorophyll a and chlorophyll b pigments compared to the control group. However, it increased micronuclei formation, chromosomal abnormality frequencies, tail DNA percentage, proline amount, lipid peroxidation level and meristematic damage severity. The activities of superoxide dismutase and catalase also increased. Chromosomal aberrations induced by MnCl2 were fragment, sticky chromosome, vagrant chromosome, unequal distribution of chromatin and bridge. Application of 250 mg/L Mox and 500 mg/L Mox along with MnCl2 significantly alleviated adverse effects dose dependently. The antioxidant activity bestowed by the phenolic compounds in Mox assisted the organism to combat MnCl2 toxicity. Consequently, Mox exerted remarkable protection against MnCl2 toxicity and it needs to be investigated further as a potential therapeutic option.

Abstract B069: Targeting vulnerabilities in double strand break repair in cancer with polymerase theta inhibitors

Abstract Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in approximately 80% of secondary glioblastomas and low-grade gliomas and to a lesser extent in AML, cholangiocarcinoma, colorectal cancer, and chondrosarcomas. Mutations in IDH1/2 lead to the overproduction of 2-hydroxyglutarate (2HG), which has been named an “oncometabolite” and is implicated in cancer progression. Work by our group has shown that 2HG induced hypermethylation suppresses homologous recombination repair. As a result, cells with mutations in IDH1/2 accumulate double strand breaks (DSBs) that must be addressed by other modes of DSB repair, such as canonical NHEJ or polymerase theta mediated end joining (TMEJ). Furthermore, TMEJ is understudied in IDH1/2-mutant cancers but has been studied extensively in other HR-deficient cancer contexts, such as BRCA-mutant cancers. Conflicting reports remain on whether TMEJ promotes genomic stability or instability in these contexts, due to its error prone repair. To better understand how IDH1/2-mutant cells utilize TMEJ and polymerase theta’s impact on genomic stability, we studied the effects of polymerase theta inhibitors on isogenic cell line models of the IDH1R132H mutation. We find that mRNA levels of polymerase theta are increased in IDH1-mutant cell lines. Using a polymerase theta inhibitor, ART558, we also observed reduced viability in IDH1-mutant cells compared to parental cells. This loss of viability in IDH1-mutant cells is likely a result of the observed increase of gamma H2AX subnuclear foci in IDH1-mutant cells treated with ART558, which is a marker of unresolved DNA damage. Preliminary data suggests that this unrepaired damage is contributing to an overall increase in micronuclei formation in IDH1-mutant cells treated with ART558 as well. Taken together, these results suggest that IDH1-mutant cells upregulate polymerase theta expression to promote genomic stability through repair of persistent DSBs. This study on TMEJ in IDH1/2 mutant cancers has identified a therapeutic vulnerability in these HR-deficient cancers that can be targeted with polymerase theta inhibitors. Citation Format: Katelyn Noronha, Samantha Liu, Karlie Lucas, Ranjit S. Bindra. Targeting vulnerabilities in double strand break repair in cancer with polymerase theta inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B069.

TIPRL1 and its ATM-dependent phosphorylation promote radiotherapy resistance in head and neck cancer.

TIPRL1 (target of rapamycin signaling pathway regulator-like 1) is a known interactor and inhibitor of protein phosphatases PP2A, PP4 and PP6 - all pleiotropic modulators of the DNA Damage Response (DDR). Here, we investigated the role of TIPRL1 in the radiotherapy (RT) response of Head and Neck Squamous Cell Carcinoma (HNSCC). TIPRL1 mRNA (cBioportal) and protein expression (immunohistochemistry) in HNSCC samples were linked with clinical patient data. TIPRL1-depleted HNSCC cells were generated by CRISPR/Cas9 editing, and effects on colony growth, micronuclei formation (microscopy), cell cycle (flow cytometry), DDR signaling (immunoblots) and proteome (mass spectrometry) following RT were assessed. Mass spectrometry was used for TIPRL1 phosphorylation and interactomics analysis in irradiated cells. TIPRL1 expression was increased in tumor versus non-tumor tissue, with high tumoral TIPRL1 expression associating with lower locoregional control and decreased survival of RT-treated patients. TIPRL1 deletion in HNSCC cells resulted in increased RT sensitivity, a faster but prolonged cell cycle arrest, increased micronuclei formation and an altered proteome-wide DDR. Upon irradiation, ATM phosphorylates TIPRL1 at Ser265. A non-phospho Ser265Ala mutant could not rescue the increased radiosensitivity phenotype of TIPRL1-depleted cells. While binding to PP2A-like phosphatases was confirmed, DNA-dependent protein kinase (DNA-PKcs), RAD51 recombinase and nucleosomal histones were identified as novel TIPRL1 interactors. Histone binding, although stimulated by RT, was adversely affected by TIPRL1 Ser265 phosphorylation. Our findings underscore a clinically relevant role for TIPRL1 and its ATM-dependent phosphorylation in RT resistance through modulation of the DDR, highlighting its potential as a new HNSCC predictive marker and therapeutic target.

Investigating the Role of MK2 in Head and Neck Squamous Cell Carcinoma Growth, Metastasis and STING Pathway Activation

Our prior work demonstrated that inhibition of MAPKAPK2 (MK2) can enhance radiation (RT)-mediated in vivo head and neck squamous cell carcinoma (HNSCC) tumor control and survival in preclinical immune incompetent models. The cytosolic DNA sensor cyclic GMP-AMP synthetase (cGAS) and its downstream adaptor protein, stimulator of interferon genes (STING), are conserved proteins within the innate immune signaling pathways and are important for mediating host defense against microbial infection and can play a role in anti-cancer immunity. We hypothesized that loss of MK2 enhances radiation-induced cGAS-STING pathway activation leading to improved tumor control and survival. MK2 shRNA knockdown human (Tu167, CAL27) and MK2 Cas9/CRISPR knockout (KO) syngeneic murine (Ly2, MLM3) HNSCC cell lines were treated with 10 Gy irradiation. Micronuclei were quantitated by DAPI-immunofluorescence (IF). Protein changes in cGAS-STING were evaluated by immunoblot. Inflammatory cytokine production including Type I IFNβ1 were evaluated by RT-qPCR. Ly2 and MLM3 cells were orthotopically or flank engrafted into immune competent mice (Balb/c, C57Bl/6, respectively) for animal tumor control-survival studies. Tumor immune cell infiltrate was examined using FACS and immunohistochemistry. Selected drug studies using the MK2 inhibitor, ATI-450, were performed with RT. Loss of MK2 in HNSCC (Tu167, CAL27, Ly2, MLM3) treated with RT led to a significant increase in micronuclei formation compared to control cells. MK2-enhanced micronuclei generation following RT could be inhibited with the actin filament polymerization inhibitor, cytochalasin B. RT treatment of MK2 shRNA cells led to increased cGAS and phospho-STING levels compared to either treatment alone. IFNβ1 levels were significantly higher in HNSCC cell lines treated with RT and with MK2 inhibited by an MK2 inhibitor (ATI-450) or genetic reduction compared to either treatment alone. In-vivo implantation of MLM3 cells into C57Bl/6 comparing control vs MK2 KO tumors treated with ±8 Gy RT demonstrated improved mouse survival favoring RT+MK2 KO over RT, MK2 KO or parental (63, 58, 58.5, 35 days, respectively). FACS analysis of MLM3 WT v KO tumors 3 days post RT showed an overall increase in the number of CD3/CD8 T-cells infiltrating into the tumor in all groups except for parental tumors. Further analysis demonstrated that loss of MK2 reversed CD8 T-cell exhaustion and when combined with radiotherapy led to increased CD8 T-cell activation. Furthermore, activated CD4 and CD8 T cells were reduced in WT+RT cells compared to WT tumors whereas no reduction was seen in the KO or KO+RT. HNSCC tumor MK2 inhibition enhances RT-mediated micronuclei formation and subsequent cGAS-STING-IFNβ1 levels. Loss of HNSCC MK2 leads to increased CD4-CD8 T-cell infiltration into the tumor and this effect is enhanced following RT. Targeting tumor MK2 may facilitate improved tumor control.

Combination of BET Inhibition with Radiation Induces Anti-Tumor Immune Response in Pre-Clinical Models of ER-Positive Breast Cancer

Despite advances in multidisciplinary care, the risk of late recurrence in Estrogen Receptor-positive (ER+) breast cancer (BC) patients following standard-of-care treatment ranges from 20-52% at 20 years. While immune checkpoint inhibitors (CPI), have shown enormous potential in inducing long term remission in some cases, patients with ER+ BC show poor response to these agents. The objective of this study is to develop effective radiation therapy (RT)-based combination regimens that induce anti-tumor immunity against immunologically cold ER+ breast tumors. We have previously shown that pharmacological inhibition of bromodomain and extraterminal domain (BET) family of epigenetic regulator proteins overcomes both endocrine therapy resistance and radiation resistance in pre-clinical models of ER+ BC by reversing pathological reprogramming of transcriptional and DNA repair pathways, respectively. In this study, we explore the potential of RT+BET inhibition combination to elicit anti-tumor immunity against ER+ BC. A unique ER+ BC model, MXT+, was used to generate tumors in female, BDF1 mice. PD-L1 expression following RT or RT+OTX015 (a BET inhibitor) was assessed by Western blot analysis. Comet assay was used to assess the degree of unrepaired DNA damage following treatment with RT alone, OTX015 alone and RT+OTX015. Multiplex IHC was used to assess changes in tumor microenvironment after various treatments. Tumor growth characteristics were established following treatment with vehicle, RT alone (8 Gy x 3), OTX015 alone (100 mg/kg x 3 days) and RT+OTX015. Using murine ER+ BC model, MXT+, we show that RT is a potent inducer of PD-L1 expression. Combination of RT+OTX015 completely ablated RT-mediated PD-L1 induction at the transcription level. OTX015 also accentuated RT-induced DNA damage through inhibition of NHEJ pathway, resulting in increased micronuclei formation (which activate cGAS/STING pathway, a critical bridge between innate and adaptive immunity). Combination of OTX015 with RT also caused a statistically significant (on unpaired t test) increase tumor infiltrating lymphocytes. In in vivo therapeutic studies, combination of OTX015 with RT (8 Gy x 3) resulted in extraordinary synergy and caused complete inhibition of long-term tumor growth. Treatment with RT alone (8 Gy x 3) or OTX015 alone (on 3 consecutive days) only caused modest inhibition of tumor growth. ANOVA with Dunnett's test was used to adjust for multiple comparisons and showed statistically significant difference in tumor growth between OTX015+RT arm and OTX alone as well as OTX015+RT arm and RT alone. Patients with ER+ BC face high risk of late recurrence after definitive treatment and show poor response to immune CPIs. We show that combination of BET inhibition with RT induces anti-tumor immunity and completely prevents tumor growth. Clinical validation of this regimen is warranted to prevent late recurrences in ER+ BC patients.

Exploring the anticancer properties of the gum of Ferula gummosa: impact on cytotoxicity, caspase 3/7 activity and apoptosis, and gene expression in SW-480 cells

ABSTRACT Ferula gummosa Boiss. is a well-known Iranian endemic plant that grows in the north and northeast regions of Iran. In Iranian traditional medicine, its gum is utilized to treat inflammation, pain, and infections of the gastrointestinal system. However, no studies have been conducted to investigate the anticancer potential of its gum against colorectal cancer cells. This study aimed to identify the chemical components of the gum of F. gummosa and investigate its effects on SW-480 cells. The experiments included MTT, clonogenic, micronucleus formation, acridine orange/ethidium bromide stain, DNA degradation, caspase 3/7 activity assay, and in vitro wound-healing experiment and investigating the expression of BAX, BCL2, MTOR, and PTEN genes. Chemical analysis using GC/MS identified 102 compounds. The gum had a significant cytotoxic effect on SW-480 cells, with an IC50 value of 1.8 µg/ml for 48 hours. The gum induced apoptosis. Microscopic observations revealed a decrease in cell proliferation, as evidenced by nuclear condensation, increased micronucleus formation, and inhibition of colony formation. Additionally, the gum suppressed cell migration, induced the expression of PTEN and BAX, and down-regulated MTOR and BCL2 genes. These findings suggest that Ferula gummosa has strong cytotoxic properties and warrants further investigation.

Reduction of oral pathogens and oxidative damage in the CAL 27 cell line by Rosmarinus officinalis L. and Taraxacum officinale Web. extracts

Ethnopharmacological relevanceDandelion (Taraxacum officinale Web.) and rosemary (Rosmarinus officinalis L.) are treasured botanicals with a long usage history in traditional herbal practices worldwide. Dandelion was used to treat kidney, spleen, and liver disease, as well as cardiovascular disease, diabetes, and bacterial infections, whereas rosemary was used to treat pain, spasms, and to improve blood circulation. Aim of the studyThe aim of this study was to determine the influence of rosemary and dandelion leaves aqueous extracts on the human tongue epithelial carcinoma cell line (CAL 27) at the level of interaction between oral microbiota and tongue epithelial cells, genomic damage, and H2O2 - induced oxidative damage protection. Materials and methodsThe polyphenolic composition of the extracts was determined by spectrophotometric and HPLC analyses. After extract treatment, cytotoxic impact and ROS generation in CAL 27 cells were measured using the MTT assay and the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay, respectively. Microdilutions were applied to investigate the antimicrobial and adhesive properties against representatives of the oral microbiota. The single-cell gel electrophoresis (comet assay) and cytokinesis-blocked micronucleus cytome assay (CBMN cyt) were used to detect induced genomic damages. ResultsBoth extracts increased the adhesion of the lactic acid bacteria L. plantarum but decreased the adhesion of the bacterial pathogens S. enterica serovar Typhimurium LT21 and E. coli K-12 MG1655 adhesion onto CAL 27 cells. 1 h treatment with 5x concentrated dandelion extract and 1x, 2.5x, and 5x of rosemary extract caused an increase in comet tail intensity. CBMN cyt results demonstrated a significant increase in micronucleus formation even at concentrations several times lower than the usual bioactive compound concentrations found in a cup of beverage, with higher concentrations also inducing cell apoptosis and necrosis. Rosemary extract showed a protective effect against H2O2 - induced oxidative damage by decreasing the apoptotic cell number, probably preventing mutations leading to tumor aggressiveness, invasion, and metastasis. ConclusionsBoth tested extracts demonstrated their usefulness in maintaining good oral bacteria balance and their protective capability as powerful antitumor agents by causing a protective apoptotic effect in tumor cell line already at the dosage of an average daily cup.

An Evaluation of the Cytotoxic and Genotoxic Effects of the Marine Toxin C17-SAMT in Human TK6 and HepaRG Cell Lines.

This study investigates the genotoxicity and cytotoxicity of C17-sphinganine analog mycotoxin (C17-SAMT) using in vitro assays. C17-SAMT was previously identified as the cause of unusual toxicity in cultured mussels from the Bizerte Lagoon in northern Tunisia. While a previous in vivo genotoxicity study was inconclusive, in vitro results demonstrated that C17-SAMT induced an increase in micronucleus formation in human lymphoblastoid TK6 cells at concentrations of 0.87 µM and 1.74 µM. In addition, multiparametric cytotoxicity assays were performed in the human hepatoma HepaRG cell line, which showed that C17-SAMT induced mitochondrial dysfunction, decreased cellular ATP levels, and altered the expression of various proteins, including superoxide dismutase SOD2, heme oxygenase HO-1, and NF-κB. These results suggest that C17-SAMT is mutagenic in vitro and can induce mitochondrial dysfunction in HepaRG cells. However, the exact mode of action of this toxin requires further investigation. Overall, this study highlights the potential toxicity of C17-SAMT and the need for further research to better understand its effects.

Abstract 302: Loss of head and neck squamous cell carcinoma MK2 increases STING activation through increased micronuclei formation

Abstract Our previous work has shown that the phosphorylation of MK2, a downstream kinase of the p38 pathway, is a prognostic feature of head and neck cancer (HNC) where patients with higher MK2 phosphorylation had lower overall survival. Studies with a MK2 inhibitor demonstrate control of HNC growth in a mouse model that was synergistic with radiotherapy (RT) in inhibiting tumor growth. The mechanism of action that MK2 inhibition combined with RT has on modulating HNC growth is unknown. A better understanding of this mechanism might reveal how to effectively deploy MK2 inhibitors in HNC treatment. Here we describe our studies in which we suppressed MK2 expression in HNC cell lines and found increased genomic instability in response to radiotherapy. Tu167 and Cal27 HNC cells were transfected with a stable shRNA construct that suppressed ~90% of the MK2 expression compared to a control shRNA construct (SCR) as determined by immunoblot. Loss of MK2 did not significantly affect cell growth nor did it affect cells from forming colonies. However, exposing the cells to 10 Gy radiation caused the formation of micronuclei that was increased in the shRNA cells compared to SCR at 48 hours (Cal 27 SCR + 10 Gy = 0.5 ± 0.3 Micronuclei/cell v Cal27 shRNA + 10 Gy = 1.6 ± 0.1. Micronuclei/cell P&amp;lt;0.01; Tu167 SCR + 10 Gy = 1.1 ± 0.2 Micronuclei/cell; Tu167 shRNA + 10 Gy = 2.3 ± .3; Micronuclei/cell p&amp;lt;0.001). Immunocytochemical techniques were used to image cells at 60x magnification. In conjunction with increased micronuclei formation, we saw an increase expression in both the DNA sensor cGAS, and phosphorylation of STING in the shRNA cells compared to SCR. The increase in micronuclei caused by radiation was largely inhibited by cytochalasin B which also inhibited the increase in STING phosphorylation. There was no difference in the staining of γH2AX after 2 hours following RT but after 48 hours the γH2AX staining was concentrated in the micronuclei which were more numerous in the shRNA cells. These data provide evidence that MK2 plays a role in controlling the DNA damage response, and we show for the first time that loss of MK2 increases the number of micronuclei formed causing an enhanced activation of the cGAS-STING pathway, an innate immune DNA sensing pathway. This suggests that inhibition of MK2 may enhance RT effectiveness by activating STING and facilitating an anti-tumor immune response. Citation Format: Colby Spiess, Grace Millington, Hannah Smith, Dakota D. Okwuone, Kiersten Berggren, Deri Morgan, Chris Lominska, Sufi M. Thomas, Jingxin Wang, Mary Markiewicz, Gregory N. Gan. Loss of head and neck squamous cell carcinoma MK2 increases STING activation through increased micronuclei formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 302.

Genotoxicity and endocrine disruption potential of haloacetic acids in human placental and lung cells

Chlorination of water results in the formation of haloacetic acids (HAAs) as major disinfection byproducts (DBPs). Previous studies have reported some HAAs species to act as cytotoxic, genotoxic, and carcinogenic. This work aimed at further exploring the toxicity potential of the most investigated HAAs (chloroacetic (CAA), bromoacetic (BAA), iodoacetic (IAA) acid) and HAAs species with high content of bromine (tribromoacetic acid (TBAA)), and iodine in their structures (chloroiodoacetic (CIAA) and diiodoacetic acid (DIAA)) to human cells. Novel knowledge was generated regarding cytotoxicity, oxidative stress, endocrine disrupting potential, and genotoxicity of these HAAs by using human placental and lung cells as in vitro models, not previously used for DBP assessment. IAA showed the highest cytotoxicity (EC50: 7.5 μM) and ability to generate ROS (up to 3-fold) in placental cells, followed by BAA (EC50: 20–25 μM and 2.1-fold). TBAA, CAA, DIAA, and CIAA showed no significant cytotoxicity (EC50 > 250 μM). All tested HAAs decreased the expression of the steroidogenic gene hsd17b1 up to 40 % in placental cells, and IAA and BAA (0.01–1 μM) slightly inhibited the aromatase activity. HAAs also induced the formation of micronuclei in A549 lung cells after 48 h of exposure. IAA and BAA showed a non-significant increase in micronuclei formation at low concentrations (1 μM), while BAA, CAA, CIAA and TBAA were genotoxic at exposure concentrations above 10 μM (100 μM in the case of DIAA). These results point to genotoxic and endocrine disruption effects associated with HAA exposure at low concentrations (0.01–1 μM), and the usefulness of the selected bioassays to provide fast and sensitive responses to HAA exposure, particularly in terms of genotoxicity and endocrine disruption effects. Further studies are needed to define thresholds that better protect public health.

ATR inhibition overcomes platinum tolerance associated with ERCC1- and p53-deficiency by inducing replication catastrophe.

ERCC1/XPF is a heterodimeric DNA endonuclease critical for repair of certain chemotherapeutic agents. We recently identified that ERCC1- and p53-deficient lung cancer cells are tolerant to platinum-based chemotherapy. ATR inhibition synergistically re-stored platinum sensitivity to platinum tolerant ERCC1-deficient cells. Mechanistically we show this effect is reliant upon several functions of ATR including replication fork protection and altered cell cycle checkpoints. Utilizing an inhibitor of replication protein A (RPA), we further demonstrate that replication fork protection and RPA availability are critical for platinum-based drug tolerance. Dual treatment led to increased formation of DNA double strand breaks and was associated with chromosome pulverization. Combination treatment was also associated with increased micronuclei formation which were capable of being bound by the innate immunomodulatory factor, cGAS, suggesting that combination platinum and ATR inhibition may also enhance response to immunotherapy in ERCC1-deficient tumors. In vivo studies demonstrate a significant effect on tumor growth delay with combination therapy compared with single agent treatment. Results of this study have led to the identification of a feasible therapeutic strategy combining ATR inhibition with platinum and potentially immune checkpoint blockade inhibitors to overcome platinum tolerance in ERCC1-deficient, p53-mutant lung cancers.

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.

Characterization of the genotoxic profile of antineoplastic drugs using the cytokinesis-block micronucleus cytome assay

Since antineoplastic agents are frequently used in cancer therapy and able to affect the patient’s DNA, it is important to know the genotoxic consequences on non-cancerous tissue. Therefore, we aimed to characterize the genotoxic profile of antineoplastic drugs belonging to different classes, using the cytokinesis-block micronucleus cytome assay in a human monocytic cell line (THP-1). All tested antineoplastic agents resulted in increased micronucleus formation. Exposure to anthracyclines led to an increased number of vacuolated cells and cell death, while for mitotic spindle inhibitors, (different stages of) cell death and an increased nuclear bud formation was observed. Alkylating agents induce a high proportion of vacuolated cells and increased nuclear bud formation. No striking differences of nuclear division index or nucleoplasmic bridge formation were observed between exposed and non-exposed cells. The here presented class-specific aberrations may facilitate interpretation of genotoxic aberrations when evaluating clinical samples from patients treated with these antineoplastic agents.

In vitro genotoxicity assessment of graphene quantum dots nanoparticles: A metabolism-dependent response

Nanomaterials are progressively being applied in different areas, including biomedical uses. Carbon nanomaterials are relevant for biomedical sciences because of their biocompatibility properties. Graphene quantum dots (GQD) have a substantial potential in drug-delivery nanostructured biosystems, but there is still a lack of toxicological information regarding their effects on human health and the environment. We thus evaluated the mutagenicity, cytotoxicity and genotoxicity of this nanomaterial using alternative methods applied in regulatory toxicology guidelines. The Ames test was carried out in the presence and absence of exogenous metabolization. Salmonella enterica serovar Typhimurium strains TA97a, TA98, TA100, TA102, TA104, and TA1535 were exposed to GQD with concentrations ranging from 1 to 1000 μg/plate. The mammal cell viability assays were carried out with HepG2 and 3T3BalbC cell lineages and the in vitro Cytokinesis-Block Micronucleus assay (CBMN) was applied for 24 h of exposure in non-cytotoxic concentrations. Mutagenicity was induced in the TA97a strain in the absence of exogenous metabolization, but not in its presence. Mutagenicity was also detected in the TA102 strain in the assay with exogenous metabolization, suggesting redox misbalance mutagenicity. The WST-1 and LDH assays demonstrated that GQD decreased cell viability, especially in 3T3BalbC cells, which showed more sensitivity to the nanomaterial. GQD also increased micronuclei formation in 3T3BalbC and caused a cytostatic effect. No significant impact on HepG2 micronuclei formation was observed. Different metabolic systems interfered with the mutagenic, cytotoxic, and genotoxic effects of GQD, indicating that liver metabolism has a central role in the detoxification of this nanomaterial.

In vitro cytogenetic analysis of two different anti-phosphates (sevelamer hydrochloride and calcium carbonate) agents used by patients with hyperphosphatemia

Sevelamer hydrochloride (SH) and calcium carbonate (CaCO3) are two agents included in the phosphate-binding group which are frequently prescribed in the treatment of patients with hyperphosphatemia. However, there are no satisfactory studies on the genotoxic effects of SH in vitro. This study was conducted to reveal the genotoxic and/or cytotoxic potential of these two drugs in cultured human peripheral lymphocytes. Human peripheral lymphocytes were treated with SH and CaCO3 at sublethal concentrations for 24 or 48 h for micronucleus assay and 1 h in the comet assay. CaCO3 and SH stimulated a slight increase in micronucleus formation however this increase was not significant compared to the control group. According to the findings of the comet test, only one concentration of the SH caused significant DNA damage (2 mg/ml, 48 h) whereas CaCO3 did not cause important DNA breakage. No significant oxidative damage or anti-radical effect caused by test substances was observed on the pure pBR322 plasmid DNA in a cell-free medium. Also, it was found that the drugs were devoid of mutagenic activity in the Ames test, but had a weak cytotoxic effect. Both test substances, particularly SH, significantly reduced the nuclear division index compared to the control group. In conclusion, the cytotoxic effect of SH was evident on the basis of in vitro tests and slightly higher than CaCO3.