DNA Damage Assays Research Articles

Volatile profiling, elemental composition and biological activities of aerial parts of seven Poaceae species

Chemical constituents and elemental studies of seven Poaceae species were investigated using fourier-transformed infrared spectroscopy (FTIR), gas chromatography-mass spectrometry (GC-MS) and laser-induced breakdown spectroscopy (LIBS) techniques. FTIR spectroscopy revealed almost similar functional groups in all species and GC-MS showed existence of maximum number (34) of compounds in Cymbopogon citratus. Hexadecanoic acid- methyl ester was present most abundantly (up to 38%) in these species. LIBS spectra indicated the presence of 16 elements and the intensity of these elements was high in Cenchrus ciliaris. Extracts were prepared in ethanol and acetone and total phenolic and flavonoid contents (TPC and TFC) were determined. Antioxidant activity was checked by six assays and antimicrobial activity was tested against eleven microorganisms. Among all species, C. citratus exhibited maximum antioxidant potential (51.16% and 45.71%) and also significantly inhibited the growth of tested microorganisms. Further, DNA damage assay revealed that the extracts of C. ciliaris and C. citratus effectively protected p1391Z plasmid DNA from damage caused by Fenton’s reagent. Overall, C. citratus and C. ciliaris extracts are valuable source of compounds and elements that could have applications in the prevention and treatment of diseases caused by microbial infections and oxidative damage.

Abstract 3054: Synergistic activity of DNA damage response kinase inhibitors in combination with the targeted alpha therapy radium-223 dichloride for metastatic castration-resistant prostate cancer

Abstract Radium-223 dichloride (Ra-223, Xofigo®) is the first approved α-particle-emitting radionuclide for the treatment of symptomatic bone metastases in patients with castration-resistant prostate cancer (CRPC) with no known evidence of visceral metastases. Ra-223 is a calcium-mimetic that preferentially binds with the bone mineral hydroxyapatite at areas of high bone turnover, such as bone metastases. This highly localized radiotherapy causes a high frequency of unrepairable double-stranded DNA breaks (DSBs), resulting in a potent antitumor effect on bone metastases. Recent evidence has suggested that patients with mutations in the DNA damage response pathway (DDR), may have differential outcomes to Ra-223 treatment (1).DDR comprises a dynamic network of signaling pathways for the maintenance of genomic integrity. Ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) are critical proteins which orchestrate the DDR and their activation is dependent on the type of DNA lesion. ATM is the primary responder to DSBs whilst ATR is activated by a range of lesions including single strand DNA structures at resected ends of DBSs and after replication fork stalling. In this study, we evaluated the impact of combining DDR kinase inhibitors with Ra-223 to investigate whether a greater radiosensitization response occurs in comparison to standard X-rays in PC3 and DU145 human prostate cell lines and normal prostate epithelial RWPE-1 cells. Cell assays including clonogenic survival, DNA damage assays and flow cytometry were used to assess the effect of DDR kinase inhibitors in combination with ionizing radiation. Cells were pre-treated with DDR inhibitors one-hour before exposure to 2Gy X-rays or an equivalent dose of 0.25Gy Ra-223. Our data show that, in all prostate models, DDR kinase inhibitors in combination with Ra-223 significantly enhanced radiosensitivity (p<0.005) response in comparison to combined treatment with X-rays. Furthermore, a greater quantity of residual DSBs at 24 hours post combination treatment was observed after Ra-223 exposure in comparison to X-ray exposure (p<0.001). Promisingly, this combined treatment had minimal effect on RWPE-1 normal cells. Our findings strongly support the combination of DNA damage induction by Ra-223 with DDR kinase inhibitors as a novel potential treatment option for mCRPC patients in order to improve clinical outcome. {{References}}:1.Velho PI, Qazi F, Hassan S, et al. Efficacy of Radium-223 in Bone-metastatic Castration-resistant Prostate Cancer with and Without Homologous Repair Gene Defects. European Urology. 2019; 76: 170-176. Citation Format: Victoria L. Dunne, Timothy Wright, Francisco Liberal, Joe M. O'Sullivan, Kevin M. Prise. Synergistic activity of DNA damage response kinase inhibitors in combination with the targeted alpha therapy radium-223 dichloride for metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3054.

Abrogated GADD45b-Mediated Integrity Control of Hematopoietic Stem Cells upon ER Stress and DNA Damage Triggers Lymphopoiesis over Granulopoiesis in Congenital Neutropenia

In patients with severe congenital neutropenia (CN) with maturation arrest of bone marrow granulopoiesis, hematopoietic and myeloid progenitor cells exhibit elevated endoplasmic reticulum (ER) stress and unfolded protein response (UPR) due to inherited mutations in ELANE (neutrophil elastase, NE) or HAX1 (HCLS1-associated protein X-1). UPR and ER stress may induce genetic instability in hematopoietic stem and progenitor cells (HSPCs). Indeed, we recently reported severe changes in cell cycle control and DNA repair pathways (e.g. CHEK1 and BRCA1) in HSPCs of CN patients, as compared to G-CSF treated healthy individuals. We further studied the molecular mechanism of the hyper-sensitivity of CN HSPC to stress conditions. We found that expression of Growth Arrest and DNA-Damage-inducible, beta (GADD45b), which functions as stress sensor regulating cell cycle, survival and apoptosis in response to ER stress or DNA damage, was markedly reduced in myeloid progenitors of CN patients, as compared to G-CSF-treated healthy individuals. GADD45b is essential in stress-induced murine hematopoiesis and myeloid differentiation is severely compromised in gadd45b knockout mice (Gupta S. et al., 2006). Less is known about the functions of GADD45b in the DNA damage response and myeloid differentiation of human HSPCs. To study GADD45b functions in human hematopoiesis, we generated human induced pluripotent stem cells (iPSC) of healthy individuals haplo-insufficient for GADD45b using CRISPR/Cas9-mediated knockout of GADD45b . Human iPSCs were transfected with GADD45b -specific CRISPR/Cas9-gRNA RNP and gene-edited clones were selected for further study. We could not detect any iPSCs clones with frameshift homozygote or compound heterozygote mutations in exon 1 of GADD45b leading to the complete knockout of GADD45b . Most probably GADD45b-deficient iPSCs are lethal. GADD45b+/- iPSCs also presented a profound phenotype. While these cells retained pluripotency, we detected elevated γH2AX levels in GADD45b+/- iPSCs upon UV light exposure, as compared to GADD45b-WT cells. In line with up-regulated γH2AX levels, we measured strongly induced DNA damage, as quantified by Lord-Q DNA damage assay. We further analyzed myeloid differentiation of GADD45b+/- iPSCs using embryoid body (EB)-based method and found that GADD45b+/- iPSCs cells completely lost the capacity to differentiate into hematopoietic cells. We were able to make EBs of GADD45b+/- cells, these EBs survived and showed no signs of degradation or cell death, but they produced no floating (hematopoietic) cells. To study the effects of GADD45b inhibition in hematopoietic cells we transfected bone marrow CD34+ cells with GADD45b -specific CRISPR/Cas9-gRNA RNP. Similar to iPSCs, we detected markedly elevated UV-induced DNA damage and diminished G-CSF-mediated myeloid differentiation of GADD45b-edited CD34+cells in colony-forming units (CFU) assay. These data strongly support the essential role of GADD45b in hematopoietic and myeloid differentiation. Diminished GADD45b expression in CN HSPCs may increase susceptibility to stress and may contribute to malignant transformation.In order to prevent the appearance and clonal outgrowth of stressed HSPCs with inherited disease-causing mutations in combination with acquired oncogenic mutations, intrinsic DNA damage must be tightly controlled. Failures in the control mechanisms may affect the fate of HSPCs as for instance altered proliferation and wrongly biased differentiation. Therefore, we further evaluated the composition of the HSPC pool in GADD45b-deficient bone marrow cells of CN patients (CN- ELANE n=5, CN- HAX1 n=5), in comparison to healthy individuals treated (n=5) or not (n =7) with G-CSF using multicolor FACS panel of J. Dick. Treatment of healthy individuals with G-CSF induces increase of common myeloid progenitors (CMP, 7-fold) and decrease of multi-lymphoid progenitors (MLP, 10-fold). At the same time, the hematopoietic differentiation in CN patients is substantially shifted from CMP (14-fold decrease) to MLP (36-fold increase) lineage with dramatically reduced numbers of GMPs (3-fold) and increased numbers of B/NK progenitors (5-fold). These data suggest that deregulated integrity control of HSPC pool due to elevated UPR and ER stress and reduced GADD45b expression in hematopoietic cells of CN patients may change composition of HSPC pool and G-CSF response. DisclosuresNo relevant conflicts of interest to declare.

A DNA-damage immune response assay combined with PET biomarkers predicts response to neo-adjuvant chemotherapy and survival in oesophageal adenocarcinoma

18F-fluorodeoxyglucose PET-CT may guide treatment decisions in patients with oesophageal adenocarcinoma (OAC). This study evaluated the added value of maximum standardised uptake value (SUVmax) to a novel DNA-damage immune response (DDIR) assay to improve pathological response prediction. The diagnostic accuracy of PET response and the prognostic significance of PET metrics for recurrence-free survival (RFS) and overall survival (OS) were assessed. This was a retrospective, single-centre study of OAC patients treated with neo-adjuvant chemotherapy from 2003 to 2014. SUVmax was recorded from baseline and repeat PET-CT after completion of pre-operative chemotherapy. Logistic regression models tested the additional predictive value of PET metrics combined with the DDIR assay for pathological response. Cox regression models tested the prognostic significance of PET metrics for RFS and OS. In total, 113 patients were included; 25 (22.1%) were DDIR positive and 88 (77.9%) were DDIR negative. 69 (61.1%) were PET responders (SUVmax reduction of 35%) and 44 (38.9%) were PET non-responders. After adding PET metrics to DDIR status, post-chemotherapy SUVmax (hazard ratio (HR) 0.75, p = 0.02), SUVmax change (HR 1.04, p = 0.003) and an optimum SUVmax reduction of 46.5% (HR 4.36, p = 0.021) showed additional value for predicting pathological response. The optimised SUVmax threshold was independently significant for RFS (HR 0.47, 95% CI 0.26–0.85, p = 0.012) and OS (HR 0.51, 95% CI 0.26–0.99, p = 0.047). This study demonstrated the additional value of PET metrics, when combined with a novel DDIR assay, to predict pathological response in OAC patients treated with neo-adjuvant chemotherapy. Furthermore, an optimised SUVmax reduction threshold for pathological response was calculated and was independently significant for RFS and OS.

VRK1 Depletion Facilitates the Synthetic Lethality of Temozolomide and Olaparib in Glioblastoma Cells.

BackgroundGlioblastomas treated with temozolomide frequently develop resistance to pharmacological treatments. Therefore, there is a need to find alternative drug targets to reduce treatment resistance based on tumor dependencies. A possibility is to target simultaneously two proteins from different DNA-damage repair pathways to facilitate tumor cell death. Therefore, we tested whether targeting the human chromatin kinase VRK1 by RNA interference can identify this protein as a novel molecular target to reduce the dependence on temozolomide in combination with olaparib, based on synthetic lethality.Materials and MethodsDepletion of VRK1, an enzyme that regulates chromatin dynamic reorganization and facilitates resistance to DNA damage, was performed in glioblastoma cells treated with temozolomide, an alkylating agent used for GBM treatment; and olaparib, an inhibitor of PARP-1, used as sensitizer. Two genetically different human glioblastoma cell lines, LN-18 and LN-229, were used for these experiments. The effect on the DNA-damage response was followed by determination of sequential steps in this process: H4K16ac, γH2AX, H4K20me2, and 53BP1.ResultsThe combination of temozolomide and olaparib increased DNA damage detected by labeling free DNA ends, and chromatin relaxation detected by H4K16ac. The combination of both drugs, at lower doses, resulted in an increase in the DNA damage response detected by the formation of γH2AX and 53BP1 foci. VRK1 depletion did not prevent the generation of DNA damage in TUNEL assays, but significantly impaired the DNA damage response induced by temozolomide and olaparib, and mediated by γH2AX, H4K20me2, and 53BP1. The combination of these drugs in VRK1 depleted cells resulted in an increase of glioblastoma cell death detected by annexin V and the processing of PARP-1 and caspase-3.ConclusionDepletion of the chromatin kinase VRK1 promotes tumor cell death at lower doses of a combination of temozolomide and olaparib treatments, and can be a novel alternative target for therapies based on synthetic lethality.

Kinetics of γH2AX and phospho-histone H3 following pulse treatment of TK6 cells provides insights into clastogenic activity

The desire for in vitro genotoxicity assays to provide higher information content, especially regarding chemicals' predominant genotoxic mode of action, has led to the development of a novel multiplexed assay available under the trade name MultiFlow®. We report here on an experimental design variation that provides further insight into clastogens' genotoxic activity. First, the standard MultiFlow DNA Damage Assay-p53, γ H2AX, phospho-histone H3 was used with human TK6 lymphoblastoid cells that were exposed for 24 continuous hours to each of 50 reference clastogens. This initial analysis correctly identified 48/50 compounds as clastogenic. These 48 compounds were then evaluated using a short-term, 'pulse' treatment protocol whereby cells were exposed to test chemical for 4 h, a centrifugation/washout step was performed, and cells were allowed to recover for 20 h. MultiFlow analyses were accomplished at 4 and 24 h. The γ H2AX and phospho-histone H3 biomarkers were found to exhibit distinct differences in terms of their persistence across chemical classes. Unsupervised hierarchical clustering analysis identified three groups. Examination of the compounds within these groups showed one cluster primarily consisting of alkylators that directly target DNA. The other two groups were dominated by non-DNA alkylators and included anti-metabolites, oxidative stress inducers and chemicals that inhibit DNA-processing enzymes. These results are encouraging, as they suggest that a simple follow-up test for in vitro clastogens provides mechanistic insights into their genotoxic activity. This type of information will contribute to improve decision-making and help guide further testing.

MicroRNA-222 alleviates radiation-induced apoptosis by targeting BCL2L11 in cochlea hair cells.

Radiation-induced hair cell injury is detrimental for human health but the underlying mechanism is not clear. MicroRNAs (miRNAs) have critical roles in various types of cellular biological processes. The present study investigated the role of miR-222 in the regulation of ionizing radiation (IR)-induced cell injury in auditory cells and its underlying mechanism. Real-time PCR was performed to identify the expression profile of miR-222 in the cochlea hair cell line HEI-OC1 after IR exposure. miRNA mimics or inhibitor-mediated up- or down-regulation of indicated miRNA was applied to characterize the biological effects of miR-222 using MTT, apoptosis and DNA damage assay. Bioinformatics analyses and luciferase reporter assays were applied to identify an miRNA target gene. Our study confirmed that IR treatment significantly suppressed miR-222 levels in a dose-dependent manner. Up-regulation of miR-222 enhances cell viability and alleviated IR-induced apoptosis and DNA damage in HEI-OC1 cells. In addition, BCL-2-like protein 11 (BCL2L11) was validated as a direct target of miR-222. Overexpression of BCL2L11 abolished the protective effects of miR-222 in IR-treated HEI-OC1 cells. Moreover, miR-222 alleviated IR-induced apoptosis and DNA damage by directly targeting BCL2L11. The present study demonstrates that miR-222 exhibits protective effects against irradiation-induced cell injury by directly targeting BCL2L11 in cochlear cells.

Unraveling the biomechanistic role of Rac1/TWEAK/Fn14/NF-κB intricate network in experimentally doxorubicin-induced cardiotoxicity in rats: The role of curcumin.

Doxorubicin (DOX) is an important chemotherapeutic drug. Cardiotoxicity diminishes its clinical efficacy. We aimed to focus on the mechanism of DOX-induced cardiotoxicity, in addition, to evaluate curcumin's protective effect against it. Twenty-eight rats were divided into the normal control group I, curcumin-treated (200 mg/kg body weight [b.w.]) group II, DOX-treated (4 mg/kg b.w.) group III, and DOX + curcumin group IV. Cardiac injury markers, heart tissue oxidative stress indices, interferon-gamma (INF-γ), tumor necrosis factor-like weak inducer of apoptosis (TWEAK), upregulated modulator of apoptosis (PUMA), p53 and nuclear factor kappa-B p65 (NF-κB p65) levels as well as messenger RNA gene expression of Rac1 and fibroblast growth factor-inducible protein 14 (Fn14) were assayed, besides the assay of DNA damage, histopathological changes, survivin immunohistochemistry and electron microscopic examination. Curcumin significantly downregulated Rac1 and Fn14 gene expression and significantly decreased p53, NF-κB p65, INF-γ, and PUMA levels in the cardiac tissue. In addition, curcumin improved oxidative stress indices, DNA damage, and cardiac toxicity markers in the form of lactate dehydrogenase (LD), creatine kinase isoenzyme-MB (CK-MB), and cardiac troponin-I (cTn-I). Meanwhile, upregulated antiapoptotic marker survivin was observed. Light and electron microscopic findings confirmed our biochemical and molecular outcomes. The current study established the antioxidant, anti-inflammatory, and antiapoptotic roles of curcumin against DOX cardiotoxicity.

Caffeine disrupts ataxia telangiectasia mutated gene-related pathways and exacerbates acetaminophen toxicity in human fetal immortalized hepatocytes

Preterm infants are at greater risk for adverse drug effects due to hepatic immaturity. Multiple interventions during intensive care increases potential for drug interactions. In this setting, high-dose caffeine used for apnea in premature infants may increase acetaminophen toxicity by inhibiting ataxia telangiectasia mutated (ATM) gene activity during DNA damage response. To define caffeine and acetaminophen interaction, we modeled infantile prematurity in late-gestation fetal stage through human immortalized hepatocytes and liver organoids. The acute toxicity studies included assays for cell viability, mitochondrial dysfunction and ATM pathway-related DNA damage. Fetal cells expressed hepatobiliary properties, albeit with lower metabolic, synthetic and antioxidant functions than more mature hepatocytes. Acetaminophen in IC50 amount of 7.5 millimolar caused significant oxidative stress, mitochondrial membrane potential impairments, and DNA breaks requiring ATM-dependent repair. Caffeine markedly exacerbated acetaminophen toxicity by suppressing ATM activity in otherwise nontoxic 2.5 millimolar amount. Similarly, the specific ATM kinase antagonist, KU-60019, reproduced this deleterious interaction in 5 micromolar amount. Replicative stress from combined acetaminophen and caffeine toxicity depleted cells undergoing DNA synthesis in S phase and activated checkpoints for G0/G1 or G2/M restrictions. Synergistic caffeine and acetaminophen toxicity in liver organoids indicated these consequences should apply in vivo. The antioxidant, N-acetylcysteine, decreased oxidative damage, mitochondrial dysfunction and ATM pathway disruption to mitigate caffeine and acetaminophen toxicity. We concluded that hepatic DNA damage, mitochondrial impairment and growth-arrest after combined caffeine and acetaminophen toxicity will be harmful for premature infants. Whether caffeine and acetaminophen toxicity may alter outcomes in subsequently encountered hepatic disease needs consideration.

Safety profile, antimicrobial and antibiofilm activities of a nanostructured lipid carrier containing oil and butter from Astrocaryum vulgare: in vitro studies

Ethnopharmacological relevance: Tucumã (Astrocaryum vulgare)is a fruit native to the Amazon region. Extracts from the peel and pulp are thought of as promising treatments for bacterial infections. The primary constituents of Tucumã oil and butter possess unsaturated carbon chains that are susceptible to oxidation by light or heat. The oils have high volatility and low aqueous solubility that limits their use without a vehicle. Nanotechnology refers to techniques to solve these problems. Nanostructured lipid carriers (NLC), for example, protect fixed oils degradation by heat or light, as well as from oxidation and evaporation, ensuring greater stability and function, thereby prolonging the useful life of the final product. Study objectives: The objective of this study was to evaluate the hemolytic, cytotoxic, antimicrobial and antibiofilm properties of an NLC containing Tucumã butter and oil soasto improve the solubility and photosensitivity of the compounds, generating better pharmacological efficacy. Materials and methods: The NLC was assessed for stability for 60 days. The cytotoxicity of nanoparticles in peripheral blood mononucleated cells was determined in culture using assays for cell viability, DNA damage, oxidative metabolism and damage to human erythrocytes. Antimicrobial activity was determined using the broth microdilution technique and antibiofilm activity according to standardized protocols. Results: The Tucumã NLC remained stable throughout the evaluated period, with pH between 5.22–5.35, monodisperse distribution (PDI<0.3) and average particle size of 170.7 ± 3nm. Cytotoxicity studies revealed that the NLC is safe and modulates inflammatory processes, demonstrated by increased cell viability and nitric oxide levels. There was low hemolytic activity of the NLC against human erythrocytes almost concentrations tested. Conclusion: Taken together, the data suggest that NLC containing Tucumã oil and butter showed antimicrobial and antibiofilm activity against organisms that cause morbidity and mortality in humans. They may be alternative solutions to public health problems related to bacterial infections.

Role of Oxidative Stress in La2O3 Nanoparticle-Induced Cytotoxicity and Apoptosis in CHANG and HuH-7 Cells.

IntroductionNanoparticles are extensively applied in pharmaceutical, agriculture, food processing industries, and in many other fields. In the current experiment, we have determined the mechanism of toxicity of lanthanum oxide nanoparticles (La2O3 NPs) on human liver cell lines.MethodsBefore the investigation, we have characterized the size and shape of La2O3 NPs using dynamic light scattering (DLS) and transmission electron microscope (TEM). The mean size of the La2O3 NPs was found 32 ±1.6 nm with a sheet-like shape. The cytotoxicity effect of La2O3 NPs for 24 h on CHANG and HuH-7 cells was determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays.ResultsThe cytotoxicity was observed in a concentration-dependent manner in both cells but NPs were more toxic to HuH-7 than CHANG cells. Generation of reactive oxygen species (ROS) was determined using fluorescent dye 2′,7′-dichlorofluorescin diacetate (DCFDA) and high green fluorescence was observed in HuH-7 cells than CHANG cells. Oxidative stress biomarker such as glutathione (GSH) was decreased and antioxidant enzyme superoxide dismutase (SOD) was increased but SOD level was decreased in HuH-7 cells than CHANG cells. Apoptotic cells were determined by using fluorescence-activated cell sorting (FACS) analysis. Maximum percentage of the apoptotic cell was observed at 300 µg/mL in HuH-7 cells. DNA double-stranded breakage was observed by comet assay and maximum DNA damage was found in CHANG cells than HuH-7 cells at 300 µg/mL La2O3 NPs for 24 h.ConclusionThus, this study demonstrated that La2O3 NPs were toxic to human liver cells and induced more toxicity in HuH-7 cells than CHANG cells.

Acetaminophen Induces DNA Damage and ATM Mediated DNA Damage Repair in Mouse Hepatocytes

Acetaminophen (APAP) is a common analgesic/antipyretic that is widely used throughout the world. While safe at therapeutic doses, it presents a risk for acute liver failure at higher dosages. Accounting for 50% of the cases of liver failure, drug-induced liver toxicity is the most common cause of liver damage in western societies. With the only efficacious form of treatment being liver transplant, it is imperative to find alternative forms of treatment. Gaining a greater understanding of the molecular pathology contributing to the progression of liver failure, we allow insights on methods to enhance patient care and quality of life. Acute liver injury from APAP is associated with increased levels of inflammation, oxidative stress, and hepatic cell death. High levels of oxidative stress damages multiple systems within the cell, most notability the DNA. In response to DNA damage, DNA damage response (DDR) markers are activated leading to cell cycle arrest, DNA repair and the eventual death of the cell if damage is unable to be repaired. Our hypothesis is that the accumulation of DNA damage results in the activation of the DDR, which then contributes to the further progression of APAP-induced liver injury. Methods Mouse hepatocytes (FL86B cells) were treated with APAP (1mM-100mM) and viability was assessed. FL83B cells were then treated for 24 hours with 5mM APAP, a dose that gave 70% of viability after 24 hr. A DNA comet assay was performed on the treated cells to assay DNA damage. ATM, phospho-ATM, phospho-Chk2 and gH2AX were assessed via immunofluorescence. ATM, Chk2, and BP53 were assess via qPCR. To assess the effects that the removal of ATM has on APAP exposure, the FL86B cells were transfected with ATM siRNA for 48 hours prior to 24-hour exposure of 5mM APAP. ATM and Chk2 were assessed via qPCR and ATM, phospho-ATM, Chk2 and phospho-Chk2 were assessed via immunofluorescence. Results APAP exposure to mouse hepatocytes showed a marked increase in the accumulation of DNA damage when compared to untreated hepatocytes. An increase in the markers for DNA double strand breaks (DSB), gH2AX and 53BP, as well the comet assay confirmed the accumulation of DNA damage after exposure to APAP. An increase of ATM and phosphor-ATM and its downstream effector Chk2 and active form phospho-Chk2 were increased after exposure to APAP. The removal of ATM attenuated the phosphorylation of ATM and Chk2 and enhanced cell viability with exposure to APAP. Conclusion Exposure to APAP induces DNA damage within hepatocytes. This elevated amount of DNA damage results in the activation of ATM and the DDR through Chk2. ATM-mediated signaling may be a potential driver for hepatocellular death and ALF. Agents designed to inhibit ATM-mediated signaling or the DDR response need to be assessed as potential therapeutic targets for the treatment of APAP-induced liver damage.

Complex Mechanisms of Antimony Genotoxicity in Budding Yeast Involves Replication and Topoisomerase I-Associated DNA Lesions, Telomere Dysfunction and Inhibition of DNA Repair.

Antimony is a toxic metalloid with poorly understood mechanisms of toxicity and uncertain carcinogenic properties. By using a combination of genetic, biochemical and DNA damage assays, we investigated the genotoxic potential of trivalent antimony in the model organism Saccharomyces cerevisiae. We found that low doses of Sb(III) generate various forms of DNA damage including replication and topoisomerase I-dependent DNA lesions as well as oxidative stress and replication-independent DNA breaks accompanied by activation of DNA damage checkpoints and formation of recombination repair centers. At higher concentrations of Sb(III), moderately increased oxidative DNA damage is also observed. Consistently, base excision, DNA damage tolerance and homologous recombination repair pathways contribute to Sb(III) tolerance. In addition, we provided evidence suggesting that Sb(III) causes telomere dysfunction. Finally, we showed that Sb(III) negatively effects repair of double-strand DNA breaks and distorts actin and microtubule cytoskeleton. In sum, our results indicate that Sb(III) exhibits a significant genotoxic activity in budding yeast.

Biotransformation of soy whey into a novel functional beverage by Cordyceps militaris SN-18

Soy whey, a liquid nutritional by-product of soybean manufacture, is rich in proteins, oligosaccharides and isoflavones. Soy whey can be used to produce functional beverages, instead of discarding it as a waste. In this study, unfermented soy whey (USW) and Cordyceps militaris SN-18-fermented soy whey (FSW) were investigated and compared for their physicochemical and functional properties by high performance liquid chromatography (HPLC) and DNA damage assay. Results show that C. militaris SN-18 fermentation could increase the contents of essential amino acids, total phenolic and flavonoid and isoflavone aglycones and eliminate the oligosaccharides in soy whey. Furthermore, C. militaris SN-18 could significantly enhance the ABTS radical scavenging ability, reducing power and ferric reducing power of soy whey, and its fermented products could prominently attenuate Fenton reaction-induced DNA damage. These findings indicate that soy whey can potentially be converted into a novel soy functional beverage by C. militaris SN-18 fermentation.Graphical abstract

High-Throughput Screening of Compound Neurotoxicity Using 3D-Cultured Neural Stem Cells on a 384-Pillar Plate.

Assessing the neurotoxicity of test chemicals has typically been performed using two-dimensionally (2D)-cultured neuronal cell monolayers and animal models. The in vitro 2D cell models are simple and straightforward compared to animal models, which have the disadvantage of being relatively low throughput, expensive, and time consuming. Despite their extensive use in this area of neurotoxicology research, both models often do not accurately recapitulate human outcomes. To bridge this gap and attempt to better replicate what happens in vivo, three-dimensionally (3D) cultured neural stem cells (NSCs) encapsulated in hydrogels on a 384-pillar plate have been developed via miniature 3D bioprinting. This technology allows users to print NSCs on a pillar plate for rapid 3D cell culture as well as high-throughput compound screening. For this, the 384-pillar plate with bioprinted NSCs is sandwiched with a standard 384-well plate with growth medium for 3D culture, allowing researchers to expose the cells to test compounds and stain them with various fluorescent dyes for a suite of high-content imaging assays, including assays for DNA damage, mitochondrial impairment, cell membrane integrity, intracellular glutathione levels, and apoptosis. After acquiring cell images from an automated fluorescence microscope and extracting fluorescence intensities, researchers can obtain the IC50 value of each compound to evaluate critical parameters in neurotoxicity. Here, we provide a detailed description of protocols for cell printing on a 384-pillar plate, 3D NSC culture, compound testing, 3D cell staining, and image acquisition and analysis, which altogether will allow researchers to investigate mechanisms of compound neurotoxicity with 3D-cultured NSCs in a high-throughput manner. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Three-dimensional neural stem cell culture on a 384-pillar plate Basic Protocol 2: Compound treatment and cell staining Basic Protocol 3: Image acquisition, processing, and data analysis.

DNA repair and cholesterol-mediated drug efflux induce dose-dependent chemoresistance in nutrient-deprived neuroblastoma cells

SummaryNeuroblastoma is a solid, heterogeneous pediatric tumor. Chemotherapy is widely used to treat neuroblastoma. However, dose-dependent responses and chemoresistance mechanisms of neuroblastoma cells to anticancer drugs remain challenging. Here, we investigated the dose-dependent effects of topotecan on human neuroblastoma cells (SK-N-SH, SH-SY5Y, and SK-N-BE) under various nutrient supply conditions. Serum-starved human neuroblastoma cells showed reduced toxicity. Their survival rate increased upon treatment with a high concentration (1 μM) of topotecan. Quantitative profiling of global and phosphoproteome identified 12,959 proteins and 48,812 phosphosites, respectively, from SK-N-SH cells. Network analysis revealed that topotecan upregulated DNA repair and cholesterol-mediated topotecan efflux, resulting in topotecan resistance. Results of DNA damage assay, cell cycle, and quantitative analyses of membrane cholesterol supported the validity of these resistance factors and their applicability to all neuroblastoma cells. Our results provide a model for high dose-dependent chemoresistance in neuroblastoma cells that could enable a patient-dependent chemotherapy screening strategy.

Preclinical Evaluation of Artesunate as an Antineoplastic Agent in Ovarian Cancer Treatment.

Background: Ovarian cancer is the deadliest gynecologic malignancy despite current first-line treatment with a platinum and taxane doublet. Artesunate has broad antineoplastic properties but has not been investigated in combination with carboplatin and paclitaxel for ovarian cancer treatment. Methods: Standard cell culture technique with commercially available ovarian cancer cell lines were utilized in cell viability, DNA damage, and cell cycle progression assays to qualify and quantify artesunate treatment effects. Additionally, the sequence of administering artesunate in combination with paclitaxel and carboplatin was determined. The activity of artesunate was also assessed in 3D organoid models of primary ovarian cancer and RNAseq analysis was utilized to identify genes and the associated genetic pathways that were differentially regulated in artesunate resistant organoid models compared to organoids that were sensitive to artesunate. Results: Artesunate treatment reduces cell viability in 2D and 3D ovarian cancer cell models. Clinically relevant concentrations of artesunate induce G1 arrest, but do not induce DNA damage. Pathways related to cell cycle progression, specifically G1/S transition, are upregulated in ovarian organoid models that are innately more resistant to artesunate compared to more sensitive models. Depending on the sequence of administration, the addition of artesunate to carboplatin and paclitaxel improves their effectiveness. Conclusions: Artesunate has preclinical activity in ovarian cancer that merits further investigation to treat ovarian cancer.

Comparative anticancer activities of Ficus carica and Ficus salicifolia latex in MDA-MB-231 cells.

Ficus latex is rich in polyphenolic compounds and hence considered as an antioxidant and anti-proliferative. Many studies are available on Ficus carica (common fig) whereas Ficus salicifolia is less studied. F. salicifolia grows in a harsh dry environment, therefore its latex was selected in the current study along with the F. carica for their comparative anti-cancer potential and the involved molecular mechanism. Triple-negative breast cancer (TNBC) derived MDA-MB-231 cells were used in the study. MTT and morphological studies indicated that the latex of both plants has anti-proliferative effects. To know their anti-metastatic effects, a wound-healing assay was performed. Both species were able to maintain the wound size compared to the untreated cells indicating their anti-metastatic effects. Using a DNA damage assay kit, we found that both fig species have genotoxic and cytotoxic effects in MDA-MB-231 cells compared to the untreated control. To know the potential molecular mechanism involved, we used a human kinase array kit. We found that ERK2, CREB, and AKT2 were downregulated after treatment the MDA-Mb-231 cells with the latex of F. carica. We assumed that F. salicifolia will also affect the same pathways, however after confirmation through real-time (RT)-PCR, downregulations of the above mentioned pathways were confirmed in cells treated with F. carica latex, however, in cells treated with F. salicifolia the selected genes were upregulated at the transcriptional level. We conclude that latex of both species of ficus have anti-cancer effects in MDA-MB-231 cells, however differ in their level of toxicity and the mechanism of action at the molecular level.

Rare deleterious germline variants and risk of lung cancer

Recent studies suggest that rare variants exhibit stronger effect sizes and might play a crucial role in the etiology of lung cancers (LC). Whole exome plus targeted sequencing of germline DNA was performed on 1045 LC cases and 885 controls in the discovery set. To unveil the inherited causal variants, we focused on rare and predicted deleterious variants and small indels enriched in cases or controls. Promising candidates were further validated in a series of 26,803 LCs and 555,107 controls. During discovery, we identified 25 rare deleterious variants associated with LC susceptibility, including 13 reported in ClinVar. Of the five validated candidates, we discovered two pathogenic variants in known LC susceptibility loci, ATM p.V2716A (Odds Ratio [OR] 19.55, 95%CI 5.04–75.6) and MPZL2 p.I24M frameshift deletion (OR 3.88, 95%CI 1.71–8.8); and three in novel LC susceptibility genes, POMC c.*28delT at 3′ UTR (OR 4.33, 95%CI 2.03–9.24), STAU2 p.N364M frameshift deletion (OR 4.48, 95%CI 1.73–11.55), and MLNR p.Q334V frameshift deletion (OR 2.69, 95%CI 1.33–5.43). The potential cancer-promoting role of selected candidate genes and variants was further supported by endogenous DNA damage assays. Our analyses led to the identification of new rare deleterious variants with LC susceptibility. However, in-depth mechanistic studies are still needed to evaluate the pathogenic effects of these specific alleles.

Mode of action assessment of the genotoxic properties of antimony and its compounds evaluated in the ToxTracker assay

Antimony (Sb) and its compounds are negative in gene mutation assays in bacteria and cultured mammalian cells but positive in some assays for clastogenicity and/or DNA damage. In order to better understand the modes of action for antimony genotoxicity, we assessed reporter gene activation by antimony and antimony compounds in the new expanded ToxTracker assay. ToxTracker evaluates the activation of biomarkers for different cellular defense mechanisms using a series of green fluorescent protein reporters inserted into mouse embryonic stem cell lines. The assay responds to: 1) DNA damage and inhibition of DNA replication; 2) oxidative stress; 3) unfolded protein response (protein damage); and 4) p53-dependent cellular stress. Sb metal powder, six trivalent (Sb(III)) compounds, and five pentavalent antimony (Sb(V)) compounds were assessed. Sb powder and all six Sb(III) compounds activated oxidative stress ToxTracker reporters at non-toxic doses. Of the five Sb(V) compounds, antimony pentachloride and potassium hexahydroantimonate induced a robust oxidative stress response while sodium antimonate induced only a weak oxidative stress response. At higher concentrations (up to either 75 % toxicity or the highest dissolved concentration tested), Sb powder and all Sb(III) compounds except for antimony trichloride induced the unfolded protein response. Of the five Sb(V) compounds tested, only potassium hexahydroantimonate induced weak activation of the unfolded protein response and was also the only pentavalent compound to yield modest (30 %) cytotoxicity. None of the compounds tested activated the DNA damage/inhibition of DNA replication reporters, nor did they activate the p53-dependent response. All Sb(III) compounds, Sb powder, and three of the five Sb(V) compounds activated the oxidative stress reporters, but there was no activation of reporters associated with DNA damage and repair or p53-dependent cellular stress. The consistent activation of reporters for oxidative stress suggests this mode of action may underlie genotoxicity responses for antimony and its compounds.