Increased DNA Damage Research Articles

Regulation of DNA damage and transcriptional output in the vasculature through a cytoglobin-HMGB2 axis

Identifying novel regulators of vascular smooth muscle cell function is necessary to further understand cardiovascular diseases. We previously identified cytoglobin, a hemoglobin homolog, with myogenic and cytoprotective roles in the vasculature. The specific mechanism of action of cytoglobin is unclear but does not seem to be related to oxygen transport or storage like hemoglobin. Herein, transcriptomic profiling of injured carotid arteries in cytoglobin global knockout mice revealed that cytoglobin deletion accelerated the loss of contractile genes and increased DNA damage. Overall, we show that cytoglobin is actively translocated into the nucleus of vascular smooth muscle cells through a redox signal driven by NOX4. We demonstrate that nuclear cytoglobin heterodimerizes with the non-histone chromatin structural protein HMGB2. Our results are consistent with a previously unknown function by which a non-erythrocytic hemoglobin inhibits DNA damage and regulates gene programs in the vasculature by modulating the genome-wide binding of HMGB2.

PG545 sensitizes ovarian cancer cells to PARP inhibitors through modulation of RAD51-DEK interaction.

PG545 (Pixatimod) is a highly sulfated small molecule known for its ability to inhibit heparanase and disrupt signaling mediated by heparan-binding-growth factors (HB-GF). Previous studies indicated that PG545 inhibits growth factor-mediated signaling in ovarian cancer (OC) to enhance response to chemotherapy. Here we investigated the previously unidentified mechanisms by which PG545 induces DNA damage in OC cells and found that PG545 induces DNA single- and double-strand breaks, reduces RAD51 expression in an autophagy-dependent manner and inhibits homologous recombination repair (HRR). These changes accompanied the ability of PG545 to inhibit endocytosis of the heparan-sulfate proteoglycan interacting DNA repair protein, DEK, leading to DEK sequestration in the tumor microenvironment (TME) and loss of nuclear DEK needed for HRR. As a result, PG545 synergized with poly (ADP-ribose) polymerase inhibitors (PARPis) in OC cell lines in vitro and in 55% of primary cultures of patient-derived ascites samples ex vivo. Moreover, PG545/PARPi synergy was observed in OC cells exhibiting either de novo or acquired resistance to PARPi monotherapy. PG545 in combination with rucaparib also generated increased DNA damage, increased antitumor effects and increased survival of mice bearing HRR proficient OVCAR5 xenografts compared to monotherapy treatment in vivo. Synergistic antitumor activity of the PG545/rucaparib combination was likewise observed in an immunocompetent syngeneic ID8F3 OC model. Collectively, these results suggest that targeting DEK-HSPG interactions in the TME through the use of PG545 may be a novel method of inhibiting DNA repair and sensitizing cells to PARPis.

Abstract P3193: Cardiac Glutamine Metabolism As NAD+ Generating Pathway During Chronic Hypoxia

Cardiovascular disease morality relies in the inability of the adult mammalian heart to regenerate damaged tissue due to the inability of the adult cardiomyocytes to proliferate. This lack of proliferation in the adult heart is due to an increased DNA damage and activation of the DNA Damage response upon an increase of mitochondrial ROS generation. We have previously shown that modulation of mitochondrial metabolism towards glucose oxidation at expenses of fatty acid utilization can induce adult cardiomyocyte proliferation and support cardiac regeneration. Moreover, by modulating the oxygen levels and, thus, cardiomyocyte metabolism, by chronic hypoxia exposure, we were also able to induce cardiomyocyte proliferation and cardiac regeneration in adult mice. Remarkably, our metabolomic studies of the hypoxic hearts did not show increase of lactate production, a hallmark of hypoxic metabolism in order to regenerate NAD+ required for glycolysis and ATP production under low oxygen conditions. Interestingly, glutamine metabolism is one of the most compensatory pathways in these conditions. Glutamine can be metabolized to its conversion into glutamate, which can enter in the Krebs’ Cycle, but also feed the malate-aspartate shuttle. This shuttle has the ability to generate cytoplasmic NAD+. Here, we hypothesize that, during chronic hypoxia exposure, the heart uses glutamine as a fuel to regenerate cytoplasmic NAD+ as an alternative to lactate production. By means of carbon labeling and metabolic flux studies in vivo, we have proven that glutamine contribution to the malate-aspartate shuttle is enhanced during hypoxia in the heart. Moreover, we have determined that glutamine supplementation during hypoxia in cell culture increases cytoplasmid NAD+ levels, confirming the contribution of glutamine catabolism to NAD/NADH balance during hypoxia. Overall, we show that glutamine metabolism is an important adaptation to hypoxic metabolism in the heart and describe a new mechanism of metabolic rewiring under hypoxia beyond the classic lactate production upon glycolysis upregulation.

Dose imbalance of DYRK1A kinase causes systemic progeroid status in Down syndrome by increasing the un-repaired DNA damage and reducing LaminB1 levels.

People with Down syndrome (DS) show clinical signs of accelerated ageing. Causative mechanisms remain unknown and hypotheses range from the (essentially untreatable) amplified-chromosomal-instability explanation, to potential actions of individual supernumerary chromosome-21 genes. The latter explanation could open a route to therapeutic amelioration if the specific over-acting genes could be identified and their action toned-down. Biological age was estimated through patterns of sugar molecules attached to plasma immunoglobulin-G (IgG-glycans, an established "biological-ageing-clock") in n=246 individuals with DS from three European populations, clinically characterised for the presence of co-morbidities, and compared to n=256 age-, sex- and demography-matched healthy controls. Isogenic human induced pluripotent stem cell (hiPSCs) models of full and partial trisomy-21 with CRISPR-Cas9 gene editing and two kinase inhibitors were studied prior and after differentiation to cerebral organoids. Biological age in adults with DS is (on average) 18.4-19.1 years older than in chronological-age-matched controls independent of co-morbidities, and this shift remains constant throughout lifespan. Changes are detectable from early childhood, and do not require a supernumerary chromosome, but are seen in segmental duplication of only 31 genes, along with increased DNA damage and decreased levels of LaminB1 in nucleated blood cells. We demonstrate that these cell-autonomous phenotypes can be gene-dose-modelled and pharmacologically corrected in hiPSCs and derived cerebral organoids. Using isogenic hiPSC models we show that chromosome-21 gene DYRK1A overdose is sufficient and necessary to cause excess unrepaired DNA damage. Explanation of hitherto observed accelerated ageing in DS as a developmental progeroid syndrome driven by DYRK1A overdose provides a target for early pharmacological preventative intervention strategies. Main funding came from the "Research Cooperability" Program of the Croatian Science Foundation funded by the European Union from the European Social Fund under the Operational Programme Efficient Human Resources 2014-2020, Project PZS-2019-02-4277, and the Wellcome Trust Grants 098330/Z/12/Z and 217199/Z/19/Z (UK). All other funding is described in details in the "Acknowledgements".

Evaluation of the cytotoxicity and genotoxicity of glufosinate-ammonium at technical and commercial grades in HepG2 cells

Exposure to genotoxic agents is associated with the development of cancer and related diseases. For this reason, assessing the genotoxicity of chemical compounds is necessary. In this line, information about the genotoxic effect of glufosinate-ammonium (GLA) has been reported only for the technical grade. However, humans are frequently exposed to commercial formulations of pesticides. Commercial formulations are characterized by using inner agents that increase toxicity compared to pesticides in technical grade. This study aimed to determine the cytotoxic and genotoxic effects of GLA on HepG2 cells. MTT and comet assays were performed to evaluate cell viability and DNA damage, respectively. HepG2 cells were exposed for 24 h to different concentrations of GLA (at 0.01 µg/mL; 0.04 µg/mL; 0.1 µg/mL; 0.24 µg/mL; 0.52 µg/mL; 1.25 µg/mL; 2.62 µg/mL and 13.12 µg/mL) in commercial- (Finale Ultra®) or technical-grade (GLAT). The results indicated that only Finale Ultra® induced a reduction in cell viability at 13.12 µg/mL. Furthermore, exposure to Finale Ultra® or GLAT was associated with increased DNA damage at concentrations from 0.52–13.12- µg/mL. This study shows the genotoxic effect of GLA on HepG2 cells.

Cohesin maintains replication timing to suppress DNA damage on cancer genes.

Cohesin loss-of-function mutations are frequently observed in tumors, but the mechanism underlying its role in tumorigenesis is unclear. Here, we found that depletion of RAD21, a core subunit of cohesin, leads to massive genome-wide DNA breaks and 147 translocation hotspot genes, co-mutated with cohesin in multiple cancers. Increased DNA damages are independent of RAD21-loss-induced transcription alteration and loop anchor disruption. However, damage-induced chromosomal translocations coincide with the asymmetrically distributed Okazaki fragments of DNA replication, suggesting that RAD21 depletion causes replication stresses evidenced by the slower replication speed and increased stalled forks. Mechanistically, approximately 30% of the human genome exhibits an earlier replication timing after RAD21 depletion, caused by the early initiation of >900 extra dormant origins. Correspondingly, most translocation hotspot genes lie in timing-altered regions. Therefore, we conclude that cohesin dysfunction causes replication stresses induced by excessive DNA replication initiation, resulting in gross DNA damages that may promote tumorigenesis.

MiR-653-3p promotes genomic instability of colorectal cancer cells via targeting SIRT1/TWIST1 signaling pathway

Development of colorectal cancer (CRC) accompanied with genomic instability. Genomic instability was promoted by microRNAs (miRNAs) inhibiting key genes in DNA damage repair and spindle assembly processes. Whether miR-653-3p affects genomic instability is unknown. The aim of this study is to explore the effect of miR-653-3p on genomic instability in CRC cells. Based on RT-qPCR analysis, miR-653-3p was highly expressed in CRC cells. Through single-cell electrophoresis assay and chromosome karyotype analysis, we determined ectopic expression of miR-653-3p induced increased DNA damage but inhibited apoptosis by promoting chromosomal instability. Mechanistically, luciferase assay identified the direct interaction of miR-653-3p with the 3′ UTR of SIRT1, and western blot analysis indicated miR-653-3p inhibited SIRT1 and then promoted STAT3 phosphorylation and TWIST1 expression. The results of karyotype analysis showed that the upregulation of SIRT1 and the downregulation of TWIST1 caused by the downregulation of miR-653-3p suppressed chromosomal instability. Additionally, our evidence showed that miR-653-3p promoted CRC cell proliferation, migration, and 5-FU resistance, and miR-653-3p induced the development of CRC in the xenograft mice model. Altogether, our evidence suggests that miR-653-3p regulates SIRT1/TWIST1 signaling pathway and plays an important role in promoting genomic instability, proliferation, migration, and chemoresistance of CRC cells, which may serve as a promising therapeutic target for CRC therapy.

Disturbed Antioxidant Capacity in Patients with Systemic Sclerosis Associates with Lung and Gastrointestinal Symptoms.

The correct balance between reactive oxygen species and antioxidant defense in an organism is disturbed in oxidative stress. To assess oxidative balance in 36 SSc patients and 26 healthy controls (HCs), we measured reactive oxidative metabolites (ROMs), total antioxidant capacity (TAC), lipid peroxidation (measuring 4-HNE), and DNA oxidative damage (measuring 8-OHdG) in serum. Furthermore, DNA breaks in leukocytes of 35 SSc patients and 32 HCs were evaluated using COMET. While we report high ROMs for both SSc patients and age/sex matched HC samples, there was a significant increase in TAC in SSc patients as compared to HCs, and thus also a significantly higher oxidative stress index in SSc patients. TAC was significantly higher in SSc patients with ILD and gastrointestinal involvement, as well as in patients with anti-topoisomerase antibodies. We observe no difference in serum lipid peroxidation status or oxidative DNA damage. However, SSc patients had significantly more leukocyte DNA breaks than HCs; the most damage was observed in patients treated with immunosuppressives. Thus, our study confirms presence of oxidative stress and increased DNA damage in leukocytes of SSc patients; however, it points toward increased antioxidant capacity, which needs to be further studied.

Pilot clinical trial and phenotypic analysis in chemotherapy-pretreated, metastatic triple-negative breast cancer patients treated with oral TAK-228 and TAK-117 (PIKTOR) to increase DNA damage repair deficiency followed by cisplatin and nab paclitaxel

BackgroundA subset of triple-negative breast cancers (TNBCs) have homologous recombination deficiency with upregulation of compensatory DNA repair pathways. PIKTOR, a combination of TAK-228 (TORC1/2 inhibitor) and TAK-117 (PI3Kα inhibitor), is hypothesized to increase genomic instability and increase DNA damage repair (DDR) deficiency, leading to increased sensitivity to DNA-damaging chemotherapy and to immune checkpoint blockade inhibitors.Methods10 metastatic TNBC patients received 4 mg TAK-228 and 200 mg TAK-117 (PIKTOR) orally each day for 3 days followed by 4 days off, weekly, until disease progression (PD), followed by intravenous cisplatin 75 mg/m2 plus nab paclitaxel 220 mg/m2 every 3 weeks for up to 6 cycles. Patients received subsequent treatment with pembrolizumab and/or chemotherapy. Primary endpoints were objective response rate with cisplatin/nab paclitaxel and safety. Biopsies of a metastatic lesion were collected prior to and at PD on PIKTOR. Whole exome and RNA-sequencing and reverse phase protein arrays (RPPA) were used to phenotype tumors pre- and post-PIKTOR for alterations in DDR, proliferation, and immune response.ResultsWith cisplatin/nab paclitaxel (cis/nab pac) therapy post PIKTOR, 3 patients had clinical benefit (1 partial response (PR) and 2 stable disease (SD) ≥ 6 months) and continued to have durable benefit in progression-free survival with pembrolizumab post-cis/nab pac for 1.2, 2, and 3.6 years. Their post-PIKTOR metastatic tissue displayed decreased mismatch repair (MMR), increased tumor mutation burden, and significantly lower levels of 53BP1, DAG Lipase β, GCN2, AKT Ser473, and PKCzeta Thr410/403 compared to pre-PIKTOR tumor tissue.ConclusionsPriming patients’ chemotherapy-pretreated metastatic TNBC with PIKTOR led to very prolonged response/disease control with subsequent cis/nab pac, followed by pembrolizumab, in 3 of 10 treated patients. Our multi-omics approach revealed a higher number of genomic alterations, reductions in MMR, and alterations in immune and stress response pathways post-PIKTOR in patients who had durable responses.Trial RegistrationThis clinical trial was registered on June 21, 2017, at ClinicalTrials.gov using identifier NCT03193853.

Genotoxic, mutagenic, and cytotoxic analysis in bats in mining area.

Pollution generated by the mining industry can cause harm to wildlife. This study aimed to evaluate the cytotoxicity, genotoxicity and mutagenicity in bats environmentally exposed to open pit mining. Thus, 62 bats of the following species, Carollia perspicillata, Glossophaga soricina, Phyllostomus hastatus, and Desmodus rotundus exposed to mining activities (ferronickel) were used in the analysis. The animals were obtained in samplings in July and November of 2021, totaling 8days of sampling in the field. The results indicated that species differ in the frequency of genotoxic damage between sampling points within the mining landscape. Cytotoxicity was observed by scoring of karyorrhexis, pyknosis and karyolysis. The most captured species, C. perspicillata, showed differences in DNA damage between exposed and unexposed populations, but no differences were observed between males (n = 14) and females (n = 20). G. soricina was also a sensitive species for indicating a high frequency of DNA damages compared to the omnivore P. hastatus. Elements such as Mn, Cr, Pb, and Zn observed in water samples were at high levels in the mining area. We conclude that bats in mining areas are susceptible to increased DNA damage as already identified for other species.

TP5: A Novel Therapeutic Approach Targeting Aberrant and Hyperactive CDK5/p25 for the Treatment of Colorectal Carcinoma.

Colorectal carcinoma (CRC) is a prevalent cancer worldwide with a high mortality rate. Evidence suggests that increased expression of Cyclin-dependent kinase 5 (CDK5) contributes to cancer progression, making it a promising target for treatment. This study examined the efficacy of selectively inhibiting CDK5 in colorectal carcinoma using TP5, a small peptide that selectively inhibits the aberrant and hyperactive CDK5/p25 complex while preserving physiological CDK5/p35 functions. We analyzed TP5's impact on CDK5 activity, cell survival, apoptosis, the cell cycle, DNA damage, ATM phosphorylation, and reactive oxygen species (ROS) signaling in mitochondria, in CRC cell lines, both alone and in combination with chemotherapy. We also assessed TP5's efficacy on a xenograft mouse model with HCT116 cells. Our results showed that TP5 decreased CDK5 activity, impaired cell viability and colony formation, induced apoptosis, increased DNA damage, and led to the G1 phase arrest of cell cycle progression. In combination with irinotecan, TP5 demonstrated a synergy by leading to the accumulation of DNA damage, increasing the γH2A.X foci number, and inhibiting G2/M arrest induced by Sn38 treatment. TP5 alone or in combination with irinotecan increased mitochondrial ROS levels and inhibited tumor growth, prolonging mouse survival in the CRC xenograft animal model. These results suggest that TP5, either alone or in combination with irinotecan, is a promising therapeutic option for colorectal carcinoma.

Therapeutic effect of thymoquinone on brain damage caused by nonylphenol exposure in rats.

Nonylphenol (NP), causes various harmful effects such as cognitive impairment and neurotoxicity. Thymoquinone (TQ), has antioxidant, anti-inflammatory, and neuroprotective properties. In this study, our aim is to investigate the effects of TQ on the brain damage caused by NP. Corn oil was applied to the control group. NP (100 mg/kg/day) was administered to the NP and NP + TQ groups for 21 days. TQ (5 mg/kg/day) was administered to the NP + TQ and TQ groups for 7 after 21 days. At the end of the experiment, the new object recognition test was applied to the rats and the rats were killed and their brain tissues were removed. Sections taken from brain tissues were stained with hematoxylin-eosin for histopathological evaluation. In addition, neuronal nuclei (NeuN), glial fibrillary acidic protein(GFAP), Cas-3, and nervegrowth factor (NGF) immunoreactivities were evaluated in brain tissue sections. In addition,malondialdehyde (MDA), superoxide dismutase(SOD), and catalase(CAT) activities were determined. Comet assay was applied to determine DNA damage in cells. The results of our study showed that NP, caused behavioral disorders and damage to the cerebral cortex in rats. This damage in the form of neuron degeneration seen in the cortex was associated with apoptosis involving Cas-3 activation, increased DNA damage, and free oxygen radicals. NP, SOD, and CAT caused a decrease in enzyme activities. In addition, the cellular protein NeuN was decreased, astrocytosis-associated GFAP was increased, and growth factor NGF was decreased. When all our evaluations are taken together, treatment with TQ showed an ameliorative effect on the behavioral impairment and brain damage caused by NP exposure.

FDFT1 repression by piR-39980 prevents oncogenesis by regulating proliferation and apoptosis through hypoxia in tongue squamous cell carcinoma

AimTongue squamous cell carcinoma (TSCC) is one of the most aggressive tumors whose underlying molecular mechanism remains elusive. Previous studies have identified piR-39980, a non-coding RNA, as a tumour suppressor or oncogene in different malignancies and the cholesterogenic protein, Farnesyl-Diphosphate Farnesyltransferase 1 (FDFT1) playing critical roles in cancer. The present study investigates the role of piR-39980, and its target FDFT1, in regulating the malignancy of TSCC. Main methodsWe performed qRT-PCR to determine the expression of FDFT1, piR-39980 and validated FDFT1 as a target of piR-39980 by dual luciferase assay. Then, to investigate the role of FDFT1 overexpression and piR-39980's inhibitory effect on FDFT1 in TSCC oncogenesis, we carried out MTT, migration, ROS estimation, and flow cytometric cell cycle assays. In addition to the above experiments, we also carried out flow cytometric apoptosis assay, chromatin condensation, γ-H2AX accumulation, and phalloidin staining assays upon overexpression and silencing of piRNA to unveil its mechanism of actions in TSCC malignancy. Key findingsFDFT1 promotes the oncogenesis of TSCC cells. Further, transient overexpression of piR-39980 significantly inhibited proliferation, migration, ROS generation, and colony formation and increased DNA damage and chromatin condensation causing cell death by repressing FDFT1. We conjectured that FDFT1 repression induces hypoxia, which slows DNA repair and accumulates damaged DNA, causing death of TSCC cells. SignificanceOur study showed FDFT1 acts as an oncogene in TSCC, unlike other cancers, whose repression by a piRNA could prevent oncogenesis by regulating proliferation and apoptosis through hypoxia. This study reveals novel gene-regulatory mechanistic insights into TSCC oncogenesis.

GSH/APE1 Cascade-Activated Nanoplatform for Imaging Therapy Resistance Dynamics and Enzyme-Mediated Adaptive Ferroptosis.

Ferroptosis, as a type of programmed cell death process, enables effective damage to various cancer cells. However, we discovered that persistent oxidative stress during ferroptosis can upregulate the apurinic/apyrimidinic endonuclease 1 (APE1) protein that induces therapeutic resistance ("ferroptosis resistance"), resulting in an unsatisfactory treatment outcome. To address APE1-induced therapeutic resistance, we developed a GSH/APE1 cascade activated therapeutic nanoplatform (GAN). Specifically, the GAN is self-assembled by DNA-functionalized ultrasmall iron oxide nanoparticles and further loaded with drug molecules (drug-GAN). GSH-triggered GAN disassembly can "turn on" the catalysis of GAN to induce efficient lipid peroxidation (LPO) for ferroptosis toward the tumor, which could upregulate APE1 expression. Subsequently, upregulated APE1 can further trigger accurate drug release for overcoming ferroptosis resistance and inducing the recovery of near-infrared fluorescence for imaging the dynamics of APE1. Importantly, adaptive drug release can overcome the adverse effects of APE1 upregulation by boosting intracellular ROS yield and increasing DNA damage, to offset APE1's functions of antioxidant and DNA repair, thus leading to adaptive ferroptosis. Moreover, with overexpressed GSH and upregulated APE1 in the tumor as stimuli, the therapeutic specificity of ferroptosis toward the tumor is greatly improved, which minimized nonspecific activation of catalysis and excessive drug release in normal tissues. Furthermore, a switchable MRI contrast from negative to positive is in sync with ferroptosis activation, which is beneficial for monitoring the ferroptosis process. Therefore, this adapted imaging and therapeutic nanoplatform can not only deliver GSH/APE1-activated lipid peroxide mediated adaptive synergistic therapy but also provided a switchable MRI/dual-channel fluorescence signal for monitoring ferroptosis activation, drug release, and therapy resistance dynamics in vivo, leading to high-specificity and high-efficiency adaptive ferroptosis therapy.

A Syx-RhoA-Dia1 signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in glioblastoma.

Glioblastomas (GBM) are aggressive tumors that lack effective treatments. Here, we show that the Rho family guanine nucleotide exchange factor Syx promotes GBM cell growth both in vitro and in orthotopic xenografts derived from patients with GBM. Growth defects upon Syx depletion are attributed to prolonged mitosis, increased DNA damage, G2/M cell cycle arrest, and cell apoptosis, mediated by altered mRNA and protein expression of various cell cycle regulators. These effects are phenocopied by depletion of the Rho downstream effector Dia1 and are due, at least in part, to increased phosphorylation, cytoplasmic retention, and reduced activity of the YAP/TAZ transcriptional coactivators. Furthermore, targeting Syx signaling cooperates with radiation treatment and temozolomide (TMZ) to decrease viability in GBM cells, irrespective of their inherent response to TMZ. The data indicate that a Syx-RhoA-Dia1-YAP/TAZ signaling axis regulates cell cycle progression, DNA damage, and therapy resistance in GBM and argue for its targeting for cancer treatment.

Saikosaponin-D induces the pyroptosis of lung cancer by increasing ROS and activating the NF-κB/NLRP3/caspase-1/GSDMD pathway.

Saikosaponin-D (SSD), an active ingredient in Bupleurum chinense, exerts anticancer effects in various cancers by inhibiting cancer proliferation and inducing apoptosis. However, whether SSD can induce other forms of cell death is unknown. The current study aims to demonstrate that SSD can induce pyroptosis in non-small-cell lung cancer. In this study, HCC827 and A549 non-small-cell lung cancer cells were treated with different concentrations of SSD for 1.5 h. HE and TUNEL staining were used to verify cell damage caused by SSD. Immunofluorescence and western blotting were performed to verify the effect of SSD on the NF-κB/NLRP3/caspase-1/gasdermin D (GSDMD) pathway. Changes in inflammatory factors were detected by ELISAs. Finally, the reactive oxygen species (ROS) scavenger N-acetylcysteine (NAC) was introduced to verify that SSD induces pyroptosis through the ROS/NF-κB pathway. The results of the HE and TUNEL staining showed that SSD resulted in balloon-like swelling of NSCLC cells accompanied by increased DNA damage. Immunofluorescence and western blot assays confirmed that SSD treatment activated the NLRP3/caspase-1/GSDMD pathway, stimulated an increase in ROS levels and activated NF-κB in lung cancer cells. The ROS scavenger N-acetylcysteine significantly attenuated SSD-induced NF-κB/NLRP3/caspase-1/GSDMD pathway activation and inhibited the release of the inflammatory cytokines IL-1β and IL-18. In conclusion, SSD induced lung cancer cell pyroptosis by inducing ROS accumulation and activating the NF-κB/NLRP3/caspase-1/GSDMD pathway. These experiments lay the foundation for the application of SSD in the treatment of non-small-cell lung cancer and regulation of the lung cancer immune microenvironment.

Individual and Combined Effects of Pesticides with Active Ingredients of Mancozeb and Methomyl on the DNA Damage of Daphnia magna (Straus, 1820; Cladocera, Daphniidae)

Mancozeb and methomyl are active ingredients commonly contained in pesticides applied in shallot farming. Surface runoff can carry pesticide residues that enter water bodies and affect non-target organisms, such as Daphnia magna. This study evaluated the genotoxicity effects of individual and combined mancozeb and methomyl on the DNA damage of D. magna. Organisms at 24 h old and 48 h old were exposed to individual and combined concentrations of mancozeb and methomyl for 24 h to obtain the LC50-24 h values. These values were used to evaluate DNA damage by calculating the tail intensity (TI) (%), tail moment (TM), and tail factor (TF). Results showed that based on the LC50-24 h values, methomyl has the highest toxicity level, followed by the mancozeb:methomyl combination, and then mancozeb. The combination index of mancozeb:methomyl for both D. magna ages (24 h and 48 h) indicated that the two pesticides antagonistically interact (CI>1). However, based on TI%, TM, and TF values, the level of damage was almost the same between the individual and combined pesticide concentrations, and the DNA damage was more massive with increased pesticide concentration. The DNA damage of 24 h old and 48 h old D. magna did not significantly differ. Increased DNA damage in D. magna indicated that this parameter was sensitive to the presence of pesticides. In application, DNA damage can be used as a biomarker for biomonitoring pesticide pollution in the aquatic ecosystem.

New Insights into the Phototoxicity of Anthracene-Based Chromophores: The Chloride Salt Effect†.

Unraveling the causes underlying polycyclic aromatic hydrocarbon phototoxicity is an essential step in understanding the harmful effects of these compounds in nature. Toward this end, we have studied the DNA interactions and photochemistry of N1-(anthracen-9-ylmethyl)ethane-1,2-diaminium dichloride in the presence and absence of NaF, KF, NaCl, KCl, NaBr, KBr, NaI, and KI (350 nm hν, pH 7.0). Exposing pUC19 plasmid to UV light in solutions containing 400 mM KCl formed significantly more direct strand breaks in DNA compared to no-salt control reactions. In contrast, NaCl increased DNA damage moderately, while the sodium(I) and potassium(I) fluoride, bromide, and iodide salts generally inhibited cleavage (I- > Br- > F-). A halide anion-induced heavy-atom effect was indicated by monitoring anthracene photodegradation and by employing the hydroxyl radical (•OH) probe hydroxyphenyl fluorescein (HPF). These studies revealed that among no-salt controls and the eight halide salts, only NaCl and KCl enabled the anthracene to photosensitize the production of high levels of DNA-damaging reactive oxygen species (ROS). Pre-irradiation of N1-(anthracen-9-ylmethyl)ethane-1,2-diaminium dichloride at 350 nm increased the amounts of chloride salt-induced •OH detected by HPF in subsequent anthracene photoactivation experiments. Taking into consideration that •OH and other highly reactive ROS are extremely short-lived, this result suggests that the pre-irradiation step might lead to the formation of oxidized anthracene photoproducts that are exceedingly redox-active. The fluorometric probes HPF and Singlet Oxygen Sensor Green revealed that KCl concentrations ranging from 150 to 400 mM and from 100 to 400 mM, respectively, enhanced N1-(anthracen-9-ylmethyl)ethane-1,2-diaminium dichloride photosensitized •OH and singlet oxygen (1O2) production over no-salt controls. Considering the relatively high levels of Na+, K+, and Cl- ions that exist in the environment and in living organisms, our findings may be relevant to the phototoxic effects exhibited by anthracenes and other polycyclic hydrocarbons in vivo.

Modifications in cellular viability, DNA damage and stress responses inflicted in cancer cells by copper-64 ions.

Due to combined therapeutical emissions, a high linear energy transfer Auger-electrons with the longer ranged β- particles, 64Cu-based radiopharmaceuticals raise particular theragnostic interest in cancer, by joined therapeutic and real-time PET imaging properties. The in vitro study aimed to investigate the biological and molecular background of 64CuCl2 therapy by analyzing the damages and stress responses inflicted in various human normal and tumor cell lines. Colon (HT29 and HCT116) and prostate carcinoma (DU145) cell lines, as well as human normal BJ fibroblasts, were treated up to 72 h with 2-40 MBq/mL 64CuCl2. Radioisotope uptake and retention were assessed, and cell viability/death, DNA damage, oxidative stress, and the expression of 84 stress genes were investigated at various time points after [64Cu]CuCl2 addition. All the investigated cells incorporated 64Cu ions similarly, independent of their tumoral or normal status, but their fate after exposure to [64Cu]CuCl2 was cell-dependent. The most striking cytotoxic effects of the radioisotope were registered in colon carcinoma HCT116 cells, for which a substantial decrease in the number of metabolically active cells, and an increased DNA damage and oxidative stress were registered. The stress gene expression study highlighted the activation of both death and repair mechanisms in these cells, related to extrinsic apoptosis, necrosis/necroptosis or autophagy, and cell cycle arrest, nucleotide excision repair, antioxidant, and hypoxic responses, respectively. The in vitro study indicated that 40 MBq/mL [64Cu]CuCl2 delivers a therapeutic effect in human colon carcinoma, but its use is limited by harmful, yet lower effects on normal fibroblasts. The exposure of tumor cells to 20 MBq/mL [64Cu]CuCl2, might be used for a softer approach aiming for a lower radiotoxicity in normal fibroblasts as compared to tumor cells. This radioactive concentration was able to induce a persistent decrease in the number of metabolically active cells, accompanied by DNA damage and oxidative stress, associated with significant changes in stress gene expression in HCT116 colon cancer cells.

The Over-Irradiation Metabolite Derivative, 24-Hydroxylumister-ol3, Reduces UV-Induced Damage in Skin.

The hormonal form of vitamin D3, 1,25(OH)2D3, reduces UV-induced DNA damage. UV exposure initiates pre-vitamin D3 production in the skin, and continued UV exposure photoisomerizes pre-vitamin D3 to produce "over-irradiation products" such as lumisterol3 (L3). Cytochrome P450 side-chain cleavage enzyme (CYP11A1) in skin catalyzes the conversion of L3 to produce three main derivatives: 24-hydroxy-L3 [24(OH)L3], 22-hydroxy-L3 [22(OH)L3], and 20,22-dihydroxy-L3 [20,22(OH)L3]. The current study investigated the photoprotective properties of the major over-irradiation metabolite, 24(OH)L3, in human primary keratinocytes and human skin explants. The results indicated that treatment immediately after UV with either 24(OH)L3 or 1,25(OH)2D3 reduced UV-induced cyclobutane pyrimidine dimers and oxidative DNA damage, with similar concentration response curves in keratinocytes, although in skin explants, 1,25(OH)2D3 was more potent. The reductions in DNA damage by both compounds were, at least in part, the result of increased DNA repair through increased energy availability via increased glycolysis, as well as increased DNA damage recognition proteins in the nucleotide excision repair pathway. Reductions in UV-induced DNA photolesions by either compound occurred in the presence of lower reactive oxygen species. The results indicated that under in vitro and ex vivo conditions, 24(OH)L3 provided photoprotection against UV damage similar to that of 1,25(OH)2D3.