Greater DNA Damage Research Articles

DNAR-03. ONCOMAGNETIC TREATMENT KILLS GLIOMA CELLS BY INHIBITING MITOCHONDRIAL ELECTRON TRANSPORT AND BY INDUCING OXIDATIVE STRESS AND DNA DAMAGE

Abstract A new noninvasive cancer therapeutic device developed in our laboratory called the Oncomagnetic device involves repeated stimulation with oscillating magnetic fields (sOMF) produced by spinning permanent magnets. It is technologically and mechanistically distinct from the Optune® device approved by the FDA and provides a novel approach for CNS tumor treatment. In 7 end-stage GBM patients, this device reversed the progression of recurrent tumors causing >30% reduction in their contrast-enhanced volumes within 4 – 8 weeks of treatment. Immune-competent mice with implanted mouse glioma cells in their brains also showed marked reduction in tumor size, increased survival (p< 0.05, n = 9) and greater DNA damage (γ-H2AX foci) in implanted tumor after sOMF treatment. Normal mice exposed to sOMF for 4 months had no adverse effects on the brain and other organs. In-vitro, sOMF inhibited mitochondrial ETC complex-I and markedly increased reactive oxygen species (ROS) levels in GBM cells. Detection of γ-H2AX and 53BP1 foci showed that sOMF caused significant DNA damage in GBM cells and diffuse intrinsic pontine glioma (DIPG) cells but not in normal astroglial SVGp12 cells. GBM cells exposed to sOMF for 4 h caused cell cycle arrest in the G1 phase and 30% - 40% loss of surviving colonies detected by clonogenic cell survival assay, while normal SVGp12 cells were not affected. This loss of survival in GBM cells was rescued by the antioxidant Trolox. These results indicate that sOMF stimulation has high anticancer potency comparable to low dose radiation therapy at the cellular level with an underlying mechanism of action that is substantially different from that proposed for Optune® TTF.

Different DNA Binding and Damage Mode between Anticancer Antibiotics Trioxacarcin A and LL-D49194α1.

Trioxacarcin A (TXN) is a highly potent cytotoxic antibiotic with remarkable structural complexity. The crystal structure of TXN bound to double-stranded DNA (dsDNA) suggested that the TXN interaction might depend on positions of two sugar subunits on the minor and major grooves of dsDNA. LL-D49194α1 (LLD) is a TXN analogue bearing the same polycyclic polyketide scaffold with a distinct glycosylation pattern. Although LLD was in a phase I clinical trial, how LLD binds to dsDNA remains unclear. Here, we solved the solution structures at high resolutions of palindromic 2″-fluorine-labeled guanine-containing duplex d(A1A2C3C4GFGFT7T8)2 and of its stable LLD and TXN covalently bound complexes. Combined with biochemical assays, we found that TXN-alkylated dsDNA would tend to keep DNA helix conformation, while LLD-alkylated dsDNA lost its stability more than TXN-alkylated dsDNA, leading to dsDNA denaturation. Thus, despite lower cytotoxicity in vitro, the differences of sugar substitutions in LLD caused greater DNA damage than TXN, thereby bringing about a completely new biological effect.

Lipoprotein(a) is associated with DNA damage in patients with heterozygous familial hypercholesterolemia

Heterozygous familial hypercholesterolemia (HeFH) is a common autosomal-dominant inherited disorder associated with atherosclerotic cardiovascular disease (ASCVD). HeFH subjects have a higher lipoprotein(a), i.e. Lp(a), concentration than the general population. Patients with FH are exposed to elevated levels of LDL from birth and ox-LDL may induce other oxidation pathways. The aim of the study was to determine the levels of markers of oxidative stress and DNA damage in patients with HeFH and describe the effect of Lp(a) on the resulting damage. Higher DNA damage was identified in patients with HeFH compared to the normolipidemic ones, and ASCVD was associated with greater damage. Oxidative stress markers were elevated in HeFH patients; however, only ox-LDL was higher in the ASCVD group and its level correlated with DNA damage. A positive correlation was found between DNA damage and Lp(a) concentration in the HeFH patients. Higher levels of Lp(a) were associated with greater DNA damage, especially in patients with HeFH and ASCVD. In HeFH patients, the optimal Lp(a) cut-off point associated with ASCVD is > 23.45 nmol/L, i.e. much lower than for the general population; however this cut-off point needs validation in a larger group of HeFH patients.

The dangerous link between coal dust exposure and DNA damage: unraveling the role of some of the chemical agents and oxidative stress

Exposure to coal mining dust poses a substantial health hazard to individuals due to the complex mixture of components released during the extraction process. This study aimed to assess the oxidative potential of residual coal mining dust on human lymphocyte DNA and telomeres and to perform a chemical characterization of coal dust and urine samples. The study included 150 individuals exposed to coal dust for over ten years, along with 120 control individuals. The results revealed significantly higher levels of DNA damage in the exposed group, as indicated by the standard comet assay, and oxidative damage, as determined by the FPG-modified comet assay. Moreover, the exposed individuals exhibited significantly shorter telomeres compared to the control group, and a significant correlation was found between telomere length and oxidative DNA damage. Using the PIXE method on urine samples, significantly higher concentrations of sodium (Na), phosphorus (P), sulfur (S), chlorine (Cl), potassium (K), iron (Fe), zinc (Zn), and bromine (Br) were observed in the exposed group compared to the control group. Furthermore, men showed shorter telomeres, greater DNA damage, and higher concentrations of nickel (Ni), calcium (Ca), and chromium (Cr) compared to exposed women. Additionally, the study characterized the particles released into the environment through GC–MS analysis, identifying several compounds, including polycyclic aromatic hydrocarbons (PAHs) such as fluoranthene, naphthalene, anthracene, 7H-benzo[c]fluorene, phenanthrene, pyrene, benz[a]anthracene, chrysene, and some alkyl derivatives. These findings underscore the significant health risks associated with exposure to coal mining dust, emphasizing the importance of further research and the implementation of regulatory measures to safeguard the health of individuals in affected populations.Graphic abstract

Aging results in DNA damage and telomere dysfunction that is greater in endothelial versus vascular smooth muscle cells and is exacerbated in atheroprone regions.

Aging increases the risk of atherosclerotic cardiovascular disease which is associated with arterial senescence; however, the mechanisms responsible for the development of cellular senescence in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) remain elusive. Here, we study the effect of aging on arterial DNA damage and telomere dysfunction. Aging resulted in greater DNA damage in ECs than VSMCs. Further, telomere dysfunction-associated DNA damage foci (TAF: DNA damage signaling at telomeres) were elevated with aging in ECs but not VMSCs. Telomere length was modestly reduced in ECs with aging and not sufficient to induce telomere dysfunction. DNA damage and telomere dysfunction were greatest in atheroprone regions (aortic minor arch) versus non-atheroprone regions (thoracic aorta). Collectively, these data demonstrate that aging results in DNA damage and telomere dysfunction that is greater in ECs than VSMCs and elevated in atheroprone aortic regions.

Parathyroid Hormone Reduces Persistent Proton Induced DNA Double-Strand Breaks in Osteocytes

<h3>Purpose/Objective(s)</h3> Healthy bone is commonly irradiated during radiotherapy for many cancers, and bone fractures can be a late complication. A recent study revealed a higher incidence of rib fractures after breast irradiation with protons (7%) compared to photons (1%) at the same dose, but the underlying biological mechanisms are unknown. Here we used <i>in vitro</i> experiments, supported by Monte Carlo track structure simulations, to investigate differences in bone cell responses to proton and photon radiation, and to identify potential therapeutic interventions. <h3>Materials/Methods</h3> We used TOPAS-nBio, a radiobiology simulation toolkit, to simulate a segment of bone using geometric models of bone cells (osteoblasts, osteoclasts, and osteocytes). Photon and proton tracks were simulated through bone matrix to investigate energy distributions received by each cell. For <i>in vitro</i> experiments, we utilized the murine osteocytic cell line Ocy454. Cells were irradiated with different doses of protons or photons. Surviving fraction as a function of dose was determined using colony formation assays. DNA double-strand breaks were assessed by 53BP1 foci assay. For PTH treatments, cells were treated with 10 nM human PTH(1-34) for 24 hours prior to irradiation. All experiments were repeated independently three or more times. Statistical analysis was performed by unpaired t-test with Bonferroni correction. <h3>Results</h3> Monte Carlo track simulations showed osteocytes are the bone cell most likely to receive radiation damage, particularly with protons. In the case of large multi-nucleated osteoclasts, both photons and protons spared some of the nuclei, suggesting functional sparing of osteoclasts from radiation damage. We therefore focused our <i>in vitro</i> experiments on Ocy454 osteocytes. Interestingly, the surviving fraction of osteocytes was significantly lower after protons compared to photons for all investigated doses of 1 to 8 Gy. The dose for 10% surviving fraction was 5.38 Gy for protons and 7.46 Gy for photons (Relative Biological Effect 1.39). Although protons and photons induced similar numbers of 53BP1 foci at 1 hour (79% vs. 72% cells with foci), a significantly higher number of foci was observed at 24 hours after protons compared to photons (22% vs. 6.5%, p=0.0078), suggestive of persistent DNA damage after protons. Remarkably, we found that pre-treatment with parathyroid hormone (PTH) decreased 53BP1 foci at 24 hours in proton-irradiated cells to levels comparable to that of non-irradiated cells (22% without PTH vs. 6.3% with PTH, p=0.014). <h3>Conclusion</h3> Surviving fraction of osteocytes was lower after proton compared to photon irradiation, suggesting a greater sensitivity of osteocytes to protons. Protons result in greater DNA damage in osteocytes, but this damage may be mitigated with the addition of PTH. Further study is needed to investigate the potential of PTH to reduce bone damage induced by proton irradiation.

Abstract 5408: Targeting ATR and AKT pathways promotes cell death in PARP inhibitor-resistant ovarian cancer cells by increasing DNA damage and lethal replication stress

Abstract Poly (ADP-ribose) polymerase inhibitors (PARPis) have changed the treatment paradigm in the management of high-grade serous ovarian cancer (HGSOC). However, most HGSOC ultimately develops resistance to PARPi, leading to unmet medical needs for better therapeutic strategies. ATR-mediated G2/M cell cycle checkpoints are important for cell survival and DNA repair, especially in HGSOC due to its dysfunctional p53-mediated G1/S checkpoint, making them an attractive therapeutic target. However, only modest monotherapy activity of ATR inhibitors (ATRis) has been reported in patients with HGSOC, highlighting the need for combination treatment. We conducted a high-throughput drug combination screen of ATRi (ceralasertib) with 1,912 drugs in two PARPi-resistant BRCA2-mutant PEO1 (PEO1/OlaR and PEO1/OlaJR) HGSOC cell lines. In this screen, 234 drugs showed synergistic cytotoxicity with ATRi, including 31 (13.2%) cell cycle checkpoint inhibitors, 27 (11.5%) chemotherapeutic drugs, and 13 (5.6%) PI3K/AKT pathway inhibitors. We prioritized the PI3K/AKT pathway inhibitors for ATRi combination because the PI3K/AKT pathway is upregulated in &amp;gt;70% of HGSOC and associated with an aggressive phenotype. Moreover, PI3K/AKT signaling is found to be activated in HGSOC cells with PARPi resistance. We separately validated that an ATRi (ceralasertib &amp;lt; 1 μM) in combination with an AKT inhibitor (AKTi; capivasertib &amp;lt; 10 μM) yielded additive/synergistic cytotoxic effects (combination index &amp;lt; 1) in various HGSOC cell lines, including acquired and de novo PARPi-resistant (PEO1/OlaR, PEO1/OlaJR, and PEO4) and platinum-resistant HGSOC cell lines (OVCAR3), by XTT and colony formation assays. With clinically attainable concentrations of ATRi (1 μM) and AKTi (10 μM), the combination treatment increased cell apoptosis compared to ATRi (increased 2.1 to 95.2-fold; P &amp;lt; 0.001) or AKTi alone (increased 3.3 to 92.2-fold; P &amp;lt; 0.001) in both PARPi-sensitive and -resistant HGSOC cells. ATRi+AKTi treatment also showed greater DNA damage as evidenced by increased percentage of cells with ≥ 5 γH2AX foci relative to ATRi (increased 1.1 to 7.6-fold; not significant in PEO1 and P &amp;lt; 0.001 in PARPi-resistant cells) or AKTi (increased 2.3 to 6.2-fold; P &amp;lt; 0.001) in both PARPi-sensitive and -resistant cells. Furthermore, ATRi+AKTi treatment augmented replication stress as measured by increased phospho-RPA+/γH2AX+ populations compared with ATRi (increased 1.7 to 4.7-fold; P &amp;lt; 0.001) or AKTi (increased 3.7 to 12.0-fold; P &amp;lt; 0.001). Overall, our results suggest that dual inhibition of ATR and AKT pathways results in greater cell death by increasing DNA damage and replication stress in HGSOC cells with PARPi resistance. Citation Format: Tzu-Ting Huang, Chih-Yuan Chiang, Nitasha Gupta, Kelli Wilson, Ken Chih-Chien Cheng, Jung-Min Lee. Targeting ATR and AKT pathways promotes cell death in PARP inhibitor-resistant ovarian cancer cells by increasing DNA damage and lethal replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5408.

Anti-tuberculosis drugs used in a directly observed treatment short course (DOTS) schedule alter endocrine patterns and reduce the ovarian reserve and oocyte quality in the mouse.

Tuberculosis is one of the major infectious diseases, with people of reproductive age group having a high risk of infection. The present study was designed to understand the consequences of anti-tuberculosis drugs (ATDs) used in DOTS (directly observed treatment short course) schedule on ovarian function. Adult female Swiss albino mice were orally administered with combinations of ATDs used in the DOTS schedule every day for 4weeks. At 2weeks after the cessation of ATDs administration, the endocrine changes and ovarian function were assessed in mice. Administration of ATDs to mice resulted in a prolonged estrous cycle, reduced ovarian follicle reserve, alteration in FSH, LH, and progesterone level, and decreased the number of ovulated oocytes. Further, the degree of fragmentation, degeneration, abnormal distribution of cytoplasmic organelles, abnormal spindle organisation, and chromosomal misalignment were higher in oocytes that were ovulated following superovulation. Blastocysts derived from ATDs treated mice had significantly lower total cell numbers and greater DNA damage. A marginal increase in the number of resorbed fetuses was observed in all the ATDs treated groups except in the multidrug resistance treatment group. Male progeny of ATDs treated mice had decreased sperm count and lower progressive motility, while female progeny exhibited a non-significant reduction in the number of oocytes ovulated. Theresults of this study suggest that ATDs can have significant adverse effects on the ovarian reserve, cytoplasmic organisation of oocytes, and can potentially cause transgenerational changes. The findings of the present study indicate ovarian toxicity of ATDs and warrant further research in the direction of identifying alternate drugs with minimal toxicity, and strategies to mitigate the ovarian toxicity induced by these drugs.

In Utero Exposure to Metformin Reduces the Fertility of Male Offspring in Adulthood.

Metformin is a drug used for the treatment of type 2 diabetes and disorders associated with insulin resistance. Metformin is also used in the treatment of pregnancy disorders such as gestational diabetes. However, the consequences of foetal exposure to metformin on the fertility of exposed offspring remain poorly documented. In this study, we investigated the effect of in utero metformin exposure on the fertility of female and male offspring. We observed that metformin is detectable in the blood of the mother and in amniotic fluid and blood of the umbilical cord. Metformin was not measurable in any tissues of the embryo, including the gonads. The effect of metformin exposure on offspring was sex specific. The adult females that had been exposed to metformin in utero presented no clear reduction in fertility. However, the adult males that had been exposed to metformin during foetal life exhibited a 30% reduction in litter size compared with controls. The lower fertility was not due to a change in sperm production or the motility of sperm. Rather, the phenotype was due to lower sperm head quality – significantly increased spermatozoa head abnormality with greater DNA damage – and hypermethylation of the genomic DNA in the spermatozoa associated with lower expression of the ten-eleven translocation methylcytosine dioxygenase 1 (TET1) protein. In conclusion, while foetal metformin exposure did not dramatically alter gonad development, these results suggest that metabolic modification by metformin during the foetal period could change the expression of epigenetic regulators such as Tet1 and perturb the genomic DNA in germ cells, changes that might contribute to a reduced fertility.

Hypothyroidism induces oxidative stress and DNA damage in breast.

Breast cancer and thyroid dysfunctions have been associated for decades. Although many studies suggest a biological correlation, the mechanisms linking these two pathologies have not been elucidated. Reactive oxygen species (ROS) can oxidize lipids, proteins, and DNA molecules and may promote tumor initiation. Hence, we aimed at evaluating the mammary redox balance and genomic instability in a model of experimental hypothyroidism. Female Wistar rats were treated with 0.03% methimazole for 7 or 21 days to evaluate ROS generation, antioxidant enzyme activities, and oxidative stress biomarkers, as well as genomic instability. After 7 days, lower catalase, GPX, and DUOX activities were detected in the breast of hypothyroid group compared to the control while the levels of 4-hydroxynonenal (HNE) were higher. In addition, hypothyroid group showed an increase in γH2Ax/H2Ax ratio. Twenty-one days hypothyroid group had increased catalase and SOD activities, without significant differences between groups in the levels of oxidative stress biomarkers and DNA damage. TSH-treated MCF10A cells showed a higher extracellular, intracellular, and mitochondrial ROS production. Additionally, greater DNA damage was observed in these cells, demonstrated by a higher comet tail DNA percentage and increased 53BP1 foci. Finally, we found that TSH treatment was not able to alter cell viability. The Genome Cancer Atlas (TGCA) data showed that high TSHR expression is associated with more invasive breast cancer types. In conclusion, we demonstrate that oxidative stress and DNA damage in breast are early events of experimental hypothyroidism. Moreover, high TSH levels induce oxidative stress and genomic instability in mammary cells.

Ethanol consumption synergistically increases ultraviolet radiation induced skin damage and immune dysfunction

BackgroundExcessive UV radiation disrupts skin homeostasis by multiple mechanisms that extend beyond the simple erythema associated with sunburns including reduction of antioxidants, increased DNA damage, and impairment of skin immune responses. Recreational UV exposure frequently occurs concurrently with excessive ethanol (EtOH). Epidemiological studies suggest a harmful, dose-dependent impact of EtOH in the setting of high UV exposure, leading to increased severity of sunburns relative to those generated in the absence of EtOH. Furthermore, EtOH consumption and UV radiation have multiple overlapping effects on the skin that could account for the epidemiological association. ObjectiveTo elucidate the relationship between excessive EtOH ingestion and UV exposures on early skin damage and downstream immune dysfunction. MethodsWe examined the impact of UVB on local skin damage, including inflammation, sunburned cells, apoptotic cells, melanin and antioxidant levels, DNA damage and immune dysfunction in the presence or absence of EtOH ingestion by combining standard mouse models of EtOH consumption and UVB exposure models. To confirm that the observed changes in mouse skin were relevant to human skin, we investigated the effects of EtOH on UV-induced skin damage with human skin explants. ResultsWe demonstrated that EtOH consumption and UV exposure act synergistically to increase the severity of local skin damage resulting in impaired melanin responses, reduced antioxidants, greater DNA damage, and immune dysfunction as measured by reduced contact hypersensitivity. ConclusionsThe results support incorporation of the risks of combined UV exposure and excessive alcohol consumption into public health campaigns.

MicroRNA-191 modulates cisplatin-induced DNA damage response by targeting RCC2.

Cisplatin is a first-line chemotherapeutic agent for the treatment of many types of cancer, but the emergence of chemoresistance hinders its application. Thus, a better understanding of cisplatin-induced DNA damage response (DDR) would help to overcome this problem. Previously, we have identified a panel of microRNAs with altered expression after cisplatin treatment in HeLa cells. In the current study, we focused on one of them, miR-191, and investigated its function in cisplatin-induced DDR. We found that overexpression of miR-191 sensitized HeLa cells to the cytotoxic effects of cisplatin, resulted in decreased viable cells. However, overexpression of miR-191 did not cause changes in apoptotic cell ratio but rather induced significant G2/M arrest in HeLa cell treated with cisplatin. Additionally, enhanced cisplatin-induced DNA damage was observed. Through bioinformatic analysis and verified by dual-luciferase assay, it was demonstrated that the chromosome condensation 2 regulator (RCC2) gene is a target for miR-191 regulation. Furthermore, the downregulation of RCC2 by siRNA mimics the effects of miR-191, in which greater DNA damage was observed upon cisplatin treatment. Taken together, our study indicates that miR-191 may be an important player in cisplatin-induced DDR, and it elicits its function, at least partially, through the regulation of RCC2.

Abstract 621: Combined inhibition of checkpoint kinase 1 (CHK1) and phosphoinositide 3-kinase (PI3K) pathways induces greater replication stress and DNA damage in high-grade serous ovarian cancer (HGSOC)

Abstract HGSOC is characterized by universal TP53 mutations and consequent G1 cell cycle dysfunction, thus relies on ATR/CHK1-mediated G2/M arrest for survival. CHK1 is also necessary for chromosome stability by mitigating replication stress (RS) and promoting homologous recombination (HR) DNA repair following DNA damage. However, clinical responses to CHK1 inhibitor (CHK1i) monotherapy have been variable in HGSOC, requiring new treatment strategies. We thus conducted a high throughput drug combination screening of a CHK1i, prexasertib (Prex) with 1,912 drugs in HGSOC cell lines (BRCA wild-type [BRCAwt: OVCAR5 and OVCAR8] and BRCA2 mutant [BRCA2m: PEO1]). In this screen, 24 drugs showed synergistic cytotoxicity with Prex, including 14 (58%) chemotherapies, 6 (25%) PI3K pathway inhibitors and 4 (17%) targeted drugs. We prioritized the PI3K pathway inhibitors given the PI3K pathway is activated in &amp;gt;45% of HGSOC and associated with increased cell survival, DNA repair and chemo-resistance. We confirmed the combination of Prex and a dual PI3K/mTOR inhibitor LY3023414 (LY302) yielded synergistic cytotoxicity (combination index&amp;lt;1) in a panel of HGSOC cell lines (OVCAR3, OVCAR5, OVCAR8, OV90 and PEO4 [all BRCA-proficient] and PEO1) by XTT and colony formation assays. OVCAR8 and PEO1 were used for further mechanistic studies. With clinically attainable concentrations of Prex (5 nM) and LY302 (200 nM), Prex+LY302 increased caspase 3/7 activation compared to Prex (increased 2,407% and 39%, respectively; P&amp;lt;0.01) or LY302 (increased 2,457% and 57%, respectively; P&amp;lt;0.001) in both cell lines. Prex+LY302 also enhanced the percentage of cells with ≥5 γH2AX foci relative to Prex (increased 39% and 22%, respectively; P&amp;lt; 0.001) or LY302 (increased 48% and 32%, respectively; P&amp;lt;0.001) in both cells, indicating greater DNA damage. We found Prex+LY302 induced a significant HR deficiency as manifested by decreased DR-GFP reporter activity compared to Prex (decreased 37% and 28%, respectively; P&amp;lt;0.01) or LY302 (decreased 44% and 63%, respectively; P&amp;lt;0.01), suggesting Prex+LY302 causes DNA damage likely via reducing HR functionality. Cell cycle analysis showed an imposed S phase arrest in cells treated with Prex+LY302 compared to those treated with Prex (increased 30% and 35%, respectively; P&amp;lt;0.05) or LY302 (increased 38% and 51%, respectively; P&amp;lt;0.01), implying Prex+LY302 increases RS or DNA damage during S phase. Supporting this notion, Prex+LY302 augmented RS as evidenced by increased phospho-RPA+/γH2AX+ populations compared with Prex (increased 37% and 38%, respectively; P&amp;lt;0.05) or LY302 (increased 80% and 79%, respectively; P&amp;lt;0.001). Overall, our results suggest that dual inhibition of CHK1 and PI3K pathways results in greater RS, DNA damage and subsequent cell death in HGSOC cells independent of BRCA mutation status. Citation Format: Tzu-Ting Huang, Jayakumar Nair, Ethan Brill, Xiaohu Zhang, Kelli Wilson, Lu Chen, Craig J. Thomas, Jung-Min Lee. Combined inhibition of checkpoint kinase 1 (CHK1) and phosphoinositide 3-kinase (PI3K) pathways induces greater replication stress and DNA damage in high-grade serous ovarian cancer (HGSOC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 621.

Acrylamide Decreases Cell Viability, and Provides Oxidative Stress, DNA Damage, and Apoptosis in Human Colon Adenocarcinoma Cell Line Caco-2.

Acrylamide (AA) toxicity remains an interesting subject in toxicological research. The aim of the research performed in this paper was to determine mechanisms of cyto- and genotoxic effects of AA on the human colon adenocarcinoma cell line Caco-2, to estimate the inhibitory concentration (IC)50 values in cell viability assays, to measure the basal and oxidative DNA damage as well as the oxidative stress leading to apoptosis, and to assess the morphological changes in cells using microscopic methods. It has been proven that AA induces cytotoxic and genotoxic effects on Caco-2 cells. Higher cytotoxic activity was gained in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay compared with the PrestoBlue assay, with IC50 values of 5.9 and 8.9 mM after 24 h exposure, respectively. In the single-cell gel electrophoresis assay, the greatest DNA damage was caused by the highest concentration of acrylamide equal to 12.5 mM (89.1% ± 0.9%). AA also induced oxidative DNA damage and generated reactive oxygen species (ROS), which was concentration dependent and correlated with the depletion of mitochondrial membrane potential and apoptosis induction. In the microscopic staining of cells, AA in the dosage close to the IC50 induced morphological changes typical for apoptosis. Taken together, these results demonstrate that AA has a pro-oxidative effect on Caco-2 cells, leading to apoptotic cell death.

DIBI, a novel 3-hydroxypyridin-4-one chelator iron-binding polymer, inhibits breast cancer cell growth and functions as a chemosensitizer by promoting S-phase DNA damage.

Breast cancer is a leading cause of cancer-related death in women; however, chemotherapy of breast cancer is often hindered by dose-limiting toxicities, demonstrating the need for less toxic approaches to treatment. Since the rapid growth and metabolism of breast cancer cells results in an increased requirement for iron, withdrawal of bioavailable iron using highly selective iron chelators has been suggested to represent a new approach to breast cancer treatment. Here we show that the recently developed iron-binding polymer DIBI inhibited the growth of five different breast cancer cell lines (SK-BR3, MDA-MB-468, MDA-MB-231, MCF-7, and T47D). In cultures of MDA-MB-468 breast cancer cells, which were most sensitive to DIBI-mediated growth inhibition, iron withdrawal was associated with increased expression of transferrin receptor 1 and ferritin H mRNA but decreased expression of ferroportin mRNA. MDA-MB-468 cells that were exposed to DIBI experienced double-strand DNA breaks during the S phase of the cell cycle. DNA damage was not mediated by reactive oxygen species (ROS) since DIBI-treated MDA-MB-468 cells exhibited a reduction in intracellular ROS. DIBI-treated MDA-MB-468 cells also showed increased sensitivity to growth inhibition by the chemotherapeutic drugs cisplatin, doxorubicin, and 4-hydroperoxy cyclophosphamide (active metabolite of cyclophosphamide). Combination treatment of MDA-MB-468 cells with DIBI and cisplatin caused greater DNA damage than either treatment alone, which was also associated with an increase in apoptotic cell death. Taken together, these findings suggest that DIBI-mediated iron withdrawal may enhance the effect of chemotherapeutic agents used in breast cancer treatment.

Organophosphorus flame retardant (tricresyl phosphate) trigger apoptosis in HepG2 cells: Transcriptomic evidence on activation of human cancer pathways

Tricresyl phosphate (TCP) is one of the organophosphorus flame retardants (OPFRs) used as plasticizer in consumer products and mixed as a lubricant in commercial jet engine oil, reportedly induce neurotoxicity and aerodynamic syndrome. No studies have been attempted so far on TCP to induce hepatotoxicity in human cells. This study for the first time confirms the hepatotoxic potential and activation of cancer pathways in TCP treated human hepatocellular cells (HepG2). MTT and NRU data showed 39.3% and 49.85% decline in HepG2 survival when exposed to the highest concentration of TCP (400 μM) for 3 days. Comet assay showed 27.1-fold greater DNA damage in cells treated with TCP (400 μM). Flow cytometric analysis revealed an upsurge in the intracellular reactive oxygen species (ROS) and nitric oxide (NO) production in cells, affirming oxidative stress. TCP (400 μM) exposure resulted in 27% reduction in Rh123 fluorescence, indicating dysfunction of mitochondrial membrane potential (ΔΨm). Cell cycle analysis exhibited 62.53% cells in the subG1 apoptotic phase after TCP (400 μM) treatment, also a massive increase in Ca2+ influx validate the on-set of apoptosis in cells. Immunofluorescence of TCP exposed cells showed activation of p53, caspase3, caspase9 reaffirming the involvement of mitochondrial-dependent intrinsic apoptotic signaling. qPCR array of 84 genes unravel the transcriptomic alterations in HepG2 cells after TCP treatment. mRNA transcripts of ATP5A1, GADD45A, IGFBP5, SOD1, STMN1 genes were prominently upregulated providing candid evidence on TCP mediated activation of human cancer pathways to orchestrate the apoptotic death of HepG2 cells, specifying hepatotoxic potential of TCP.

Autophagy is a new protective mechanism against the cytotoxicity of platinum nanoparticles in human trophoblasts

Nanoparticles are widely used in commodities, and pregnant women are inevitably exposed to these particles. The placenta protects the growing fetus from foreign or toxic materials, and provides energy and oxygen. Here we report that autophagy, a cellular mechanism to maintain homeostasis, engulfs platinum nanoparticles (nPt) to reduce their cytotoxicity in trophoblasts. Autophagy was activated by nPt in extravillous trophoblast (EVT) cell lines, and EVT functions, such as invasion and vascular remodeling, and proliferation were inhibited by nPt. These inhibitory effects by nPt were augmented in autophagy-deficient cells. Regarding the dynamic state of nPt, analysis using ICP-MS demonstrated a higher accumulation of nPt in the autophagosome-rich than the cytoplasmic fraction in autophagy-normal cells. Meanwhile, there were more nPt in the nuclei of autophagy-deficient cells, resulting in greater DNA damage at a lower concentration of nPt. Thus, we found a new protective mechanism against the cytotoxicity of nPt in human trophoblasts.

Ruthenium complex delivery using liposomes to improve bioactivity against HeLa cells via the mitochondrial pathway.

The aim of this study was to encapsulate a ruthenium complex [Ru(ttbpy)2PIP](ClO4)2 (Ru) in liposomes to enhance their antitumor effect on human cervical cancer. The Ru-loaded PEGylated liposomes (Ru-Lip) were prepared using thin-film hydration method. The mechanism of action was studied. A novel Ru was successfully synthesized. Ru-Lip showed stronger cytotoxic activity against HeLa cells than Ru. Ru-Lip demonstrated a more significant increase in apoptosis, reactive oxygen species production and apoptosis-associated processes (intracellular calcium concentration, cytochrome c release and activation of Bax and caspase-3) than Ru. Ru-Lip exhibited greater blockade efficacy in the cell cycle G1 phase and greater DNA damage than Ru. Ru-Lip significantly elevates the anticancer effect via reactive oxygen species-mediated mitochondrial dysfunctional pathway.

6-OHBDE-47 induces transcriptomic alterations of CYP1A1, XRCC2, HSPA1A, EGR1 genes and trigger apoptosis in HepG2 cells

In this study, HepG2 cells were exposed to 6-hydroxy- 2,2′,4,4′-tetrabromodiphenyl ether (6-OH-BDE-47) for 3 and 6 days for monitoring cytotoxic effects and alterations in its transcriptomic profile. MTT assay showed that cells exposed to 6-OH-BDE-47 (50 nM) exhibited 48.5% and 53.7% decline in cell survival after 3 and 6 days. Neutral red uptake (NRU) assay also demonstrated 47.1% and 56% reduction in cell survival at 50 nM, indicating lysosomal toxicity. The flow cytometric data confirmed an increase in intracellular reactive oxygen species (ROS) and mitochondrial dysfunction (ΔΨm). In comet assay, HepG2 cells exposed to 6-OH-BDE-47 (50 nM) showed 7.6-fold greater DNA damage. Cell cycle data revealed G2/M arrest at 10 and 25 nM after 3 days of exposure, while 50 nM induced mild apoptotic effect. The intensity of apoptosis increased after 6 days of exposure with 21.5%, 47% and 99.1% of cells recorded in subG1 apoptotic phase vis-à- vis the control showed 14.5% background apoptotic cells. Transcriptome analysis of 6-OH-BDE-47 (25 nM, 3 days) treated cells revealed cross talk between vital pathways. Especially, the genes involved in oxidative or metabolic stress, heat shock, growth arrest and senescence were differentially up- and down regulated to orchestrate the cellular toxicity and triggering apoptosis in HepG2 cells.

Genotoxicity Biomonitoring Along a Coastal Zone Under Influence of Offshore Petroleum Exploration (Southeastern Brazil).

Offshore oil exploration creates threats to coastal ecosystems, including increasing urbanization and associated effluent releases. Genotoxicity biomarkers in mussels were determined across a gradient of coastal zone influences of offshore petroleum exploration in southeastern Brazil. Coastal ecosystems such as estuaries, beaches and islands were seasonally monitored for genotoxicity evaluation using the brown mussel Perna perna. The greatest DNA damage (5.2% ± 1.9% tail DNA and 1.5‰ ± 0.8‰ MN) were observed in urban estuaries, while Santana Archipelago showed levels of genotoxicity near zero and is considered a reference site. Mussels from urban and pristine beaches showed intermediate damage levels, but were also influenced by urbanization. Thus, mussel genotoxicity biomarkers greatly indicated the proposed oil exploration and urbanization scenarios that consequently are genetically affecting coastal organisms.