Significant Levels Of DNA Damage Research Articles

Abstract 3226: PRMT1 is a critical dependency in clear cell renal cell carcinoma with roles in post- transcriptional regulation and DNA damage response

Abstract The majority of sporadic renal cell carcinoma (RCC) cases present as the clear cell subtype (ccRCC) and are associated with the biallelic inactivation of the von Hippel-Lindau (VHL) gene, leading to constitutive activation of the cellular hypoxia response and deleterious alterations to gene expression, metabolism, and growth. Despite the near ubiquity of VHL loss, evidence suggests that it alone is insufficient to drive tumor establishment. Additional loss of function (LOF) mutations in epigenetic regulatory proteins including PBRM1, SETD2 and BAP1 have been identified as tumor drivers. The frequency and penetrance of these mutations in epigenetic regulators implicates a vulnerability in ccRCC that may be exploited to develop new therapies. To explore this vulnerability, the Ailles lab completed a screen of the Structural Genomics Consortium’s (SGC) epiprobe library - a panel of inhibitors targeting an array of epigenetic regulators in patient derived ccRCC models. Among the several promising targets identified, a particularly favorable inhibition profile was noted for MS023, an inhibitor of the type I protein arginine methyltransferase family (PRMT1/3/4/6 and 8). PRMTs transfer methyl groups to nuclear histones and cytoplasmic targets, influencing gene expression, mRNA processing and viability. This growth inhibition was corroborated using an additional type I PRMT compound, GSK3368715. CRISPR-mediated deletion of type I PRMT targets indicated that PRMT1 is the main enzyme responsible for growth inhibition in our ccRCC models. Furthermore, genetic depletion of PRMT1 via shRNA phenocopied pharmacological inhibition and proliferative growth was restored in the presence of MS023 by overexpressing PRMT1. Finally, MS023 treatment of mice bearing ccRCC cell line and patient derived xenografts led to significant inhibition of tumor growth in vivo. Transcriptomic profiling of chemical treatment and PRMT1 genetic depletion demonstrated that PRMT1 inhibition triggers a significant down regulation of genes related to cell cycle progression and DNA damage. ccRCC cells treated with MS023 accumulated significant levels of DNA damage before undergoing cell death, with several DDR-related proteins being down-regulated. Proximity ligation and proteomic analysis (Bio-ID) revealed that PRMT1’s interactors skew heavily towards RNA metabolism regulators and widespread alterations in mRNA processing were noted with MS023 treatment. Evidence continues to mount that PRMT1 is implicated in the biology of cancer, but to our knowledge, no thorough investigations have been performed in the context of ccRCC. Our data suggests that inhibition of PRMT1 inhibits processing of mRNA transcripts, compromises DDR mechanisms, leading to an accumulation of double stranded breaks, cytostasis and cell death. Thus PRMT1 represents a viable therapeutic target in ccRCC. Citation Format: Joseph Walton, Karen Arevalo, Angel SN Ng, Jalna Meens, Christina Karamboulas, Panagiotis Prinos, Brian Raught, Eric Chen, Cheryl Arrowsmith, Laurie Ailles. PRMT1 is a critical dependency in clear cell renal cell carcinoma with roles in post- transcriptional regulation and DNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3226.

Comparison of DNA damage in granulosa cells of women undergoing controlled ovarian stimulation in in vitro fertilization protocols with the recombinant human follicle-stimulating hormones Corneumon®, Gonal-F®, Pergoveris® and Puregon®: a randomized trial.

To compare the DNA damage in granulosa cells (GCs) of women undergoing ovarian-stimulated cycles with four widely used recombinant human follicle-stimulating hormones (rhFSH) in in vitro fertilization (IVF) protocols (Corneumon®, Gonal-F®, Pergoveris® and Puregon®). A randomized trial was carried out at a Mexican hospital. GCs were isolated from 18 women with infertility undergoing assisted reproductive techniques (ART). Four controlled ovarian stimulation (COS) protocols including Corneumon®, Gonal-F®, Pergoveris® or Puregon® were used. GCs DNA damage was assessed by the Comet assay. Two parameters were measured: comet tail length (CTL), and Olive tail moment (OTM, the percentage of DNA in the tail multiplied by the distance between the center of the tail and head). Use of the different hrFSH in COS caused variable and statistically significant levels of DNA damage in GCs of infertile women. CTL was similar in the Corneumon® and Pergoveris® groups (mean values of 48.73 and 55.18, respectively) and Corneumon® CTL was significantly lower compared to the Gonal-F® and Puregon® groups (mean values of 61.98 and 91.17, respectively). Mean OTM values were significantly lower in Corneumon® and Pergoveris® groups, compared to Gonal-F® and Puregon® groups (25.59, 27.35, 34.76, and 47.27, respectively). Use of Corneumon® and Pergoveris® in COS caused statistically significantly lower levels of DNA damage in GCs of infertile women undergoing ART, which could potentially correlate with better reproductive outcomes.

Genotoxicity of Novel Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides in Normal and Cancer Cells In Vitro

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides constitute a novel group of heterocyclic compounds with broad biological activities including anticancer properties. The compounds investigated in this study (MM134, -6, -7, and 9) were found to have antiproliferative activity against BxPC-3 and PC-3 cancer cell lines in micromolar concentrations (IC50 0.11-0.33 µM). Here, we studied the genotoxic potential of the tested compounds with alkaline and neutral comet assays, accompanied by immunocytochemical detection of phosphorylated γH2AX. We found that pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides induce significant levels of DNA damage in BxPC-3 and PC-3 cells without causing genotoxic effects in normal human lung fibroblasts (WI-38) when used in their respective IC50 concentrations (except for MM134) and showed a dose-dependent increase in DNA damage following 24 h incubation of tested cancer cells with these agents. Furthermore, the influence of MM compounds on DNA damage response (DDR) factors was assessed using molecular docking and molecular dynamics simulation.

Wavelength-dependent DNA Photodamage in a 3-D human Skin Model over the Far-UVC and Germicidal UVC Wavelength Ranges from 215 to 255 nm.

ABSTRACTThe effectiveness of UVC to reduce airborne‐mediated disease transmission is well established. However, conventional germicidal UVC (~254 nm) cannot be used directly in occupied spaces because of the potential for damage to the skin and eye. A recently studied alternative with the potential to be used directly in occupied spaces is far UVC (200–235 nm, typically 222 nm), as it cannot penetrate to the key living cells in the epidermis. Optimal far‐UVC use is hampered by limited knowledge of the precise wavelength dependence of UVC‐induced DNA damage, and thus we have used a monochromatic UVC exposure system to assess wavelength‐dependent DNA damage in a realistic 3‐D human skin model. We exposed a 3‐D human skin model to mono‐wavelength UVC exposures of 100 mJ/cm2, at UVC wavelengths from 215 to 255 nm (5 nm steps). At each wavelength, we measured yields of DNA‐damaged keratinocytes, and their distribution within the layers of the epidermis. No increase in DNA damage was observed in the epidermis at wavelengths from 215 to 235 nm, but at higher wavelengths (240–255 nm) significant levels of DNA damage was observed. These results support use of far‐UVC radiation to safely reduce the risk of airborne disease transmission in occupied locations.

Acridine–O6-benzylguanine hybrids: Synthesis, DNA binding, MGMT inhibition and antiproliferative activity

O6-Methylguanine-DNA-methyltransferase (MGMT) is a key DNA repair enzyme involved in chemoresistance to DNA-alkylating anti-cancer drugs such as Temozolomide (TMZ) through direct repair of drug-induced O6-methylguanine residues in DNA. MGMT substrate analogues, such as O6-benzylguanine (BG), efficiently inactivate MGMT in vitro and in cells; however, these drugs failed to reach the clinic due to adverse side effects. Here, we designed hybrid drugs combining a BG residue covalently linked to a DNA-interacting moiety (6-chloro-2-methoxy-9-aminoacridine). Specifically, two series of hybrids, encompassing three compounds each, were obtained by varying the position of the attachment point of BG (N9 of guanine vs. the benzyl group) and the length and nature of the linker. UV/vis absorption and fluorescence data indicate that all six hybrids adopt an intramolecularly stacked conformation in aqueous solutions in a wide range of temperatures. All hybrids interact with double-stranded DNA, as clearly evidenced by spectrophotometric titrations, without intercalation of the acridine ring and do not induce thermal stabilization of the duplex. All hybrids, as well as the reference DNA intercalator (6-chloro-2-methoxy-9-aminoacridine 8), irreversibly inhibit MGMT in vitro with variable efficiency, comparable to that of BG. In a multidrug-resistant glioblastoma cell line T98G, benzyl-linked hybrids 7a–c and the N9-linked hybrid 19b are moderately cytotoxic (GI50 ≥ 15 μM after 96 h), while N9-linked hybrids 19a and 19c are strongly cytotoxic (GI50 = 1–2 μM), similarly to acridine 8 (GI50 = 0.6 μM). Among all compounds, hybrids 19a and 19c, similarly to BG, display synergic cytotoxic effect upon co-treatment with subtoxic doses of TMZ, with combination index (CI) values as low as 0.2–0.3. In agreement with in vitro results, compound 19a inactivates cellular MGMT but, unlike BG, does not induce significant levels of DNA damage, either alone or in combination with TMZ, as indicated by the results of γH2AX immunostaining experiments. Instead, and unlike BG, compound 19a alone induces significant apoptosis of T98G cells, which is not further increased in a combination with TMZ. These results indicate that molecular mechanisms underlying the cytotoxicity of 19a and its combination with TMZ are distinct from that of BG. The strongly synergic properties of this combination represent an interesting therapeutic opportunity in treating TMZ-resistant cancers.

Mechanistic Evaluation of Black Cohosh Extract-Induced Genotoxicity in Human Cells.

Black cohosh extract (BCE) is marketed to women as an alternative to hormone replacement therapy for alleviating menopausal symptoms. Previous studies by the National Toxicology Program revealed that BCE induced micronuclei (MN) and a nonregenerative macrocytic anemia in rats and mice, likely caused by disruption of the folate metabolism pathway. Additional work using TK6 cells showed that BCE induced aneugenicity by destabilizing microtubules. In the present study, BCE-induced MN were confirmed in TK6 and HepG2 cells. We then evaluated BCE-induced DNA damage using the comet assay at multiple time points (0.5-24 h). Following a 0.5-h exposure, BCE induced significant, concentration-dependent increases in %tail DNA in TK6 cells only. Although DNA damage decreased in TK6 cells over time, likely due to repair, small but statistically significant levels of DNA damage were observed after 2 and 4 h exposures to 250 µg/ml BCE. A G1/S arrest in TK6 cells exposed to 125 µg/ml BCE (24 h) was accompanied by apoptosis and increased expression of γH2A.X, p-Chk1, p-Chk2, p53, and p21. Conditioning TK6 cells to physiological levels of folic acid (120 nM) did not increase the sensitivity of cells to BCE-induced DNA damage. BCE did not alter global DNA methylation in TK6 and HepG2 cells cultured in standard medium. Our results suggest that BCE induces acute DNA strand breaks which are quickly repaired in TK6 cells, whereas DNA damage seen at 4 and 24 h may reflect apoptosis. The present study supports that BCE is genotoxic mainly by inducing MN with an aneugenic mode of action.

A Prospective Phase II Single-arm Study of Niraparib Plus Dostarlimab in Patients With Advanced Non–small-cell Lung Cancer and/or Malignant Pleural Mesothelioma, Positive for PD-L1 Expression and Germline or Somatic Mutations in the DNA Repair Genes: Rationale and Study Design

Treatment with poly ADP ribose polymerase (PARP)1/2 inhibitors represents a novel opportunity to selectively kill a subset of cancer cell types by exploiting their deficiencies in DNA repair, thus leading to synthetic lethality. Treatment of homologous recombination deficient (HRD)-tumors with PARP inhibitors generates significant levels of DNA damage, which has the potential to further increasing tumor mutational burden, promoting neoantigen release, and upregulating both interferons and programmed death ligand-1 (PD-L1) expression, suggesting a potential complementary and synergistic role with immune checkpoint inhibitors in cancer treatment. Here we present the design and rationale of a prospective, phase II, single-arm study aiming to investigate the safety and antitumor activity of the combination of niraparib and dostarlimab in patients with HRD-positive and PD-L1 ≥ 1% advanced non–small-cell lung cancer (NSCLC) and/or malignant pleural mesothelioma (MPM).Considering the prevalence of pathogenetic germline mutations in DNA repair genes, reported to be around 5% to 10% in patients with MPM and NSCLC, a total of 700 to 1000 cases will be screened to identify 70 patients who are HRD-positive/PD-L1 ≥ 1% (N = 35 NSCLC; N = 35 MPM) to be included. Patients will receive the combination of niraparib orally once daily and dostarlimab intravenously. The primary endpoint is progression-free survival. Secondary endpoints are objective response, duration of response, overall survival, and safety.The results of this study will provide evidence on the safety and antitumor activity of niraparib and dostarlimab combination in patients with advanced, HRD-positive and PD-L1 ≥ 1% NSCLC and/or MPM.

Analiza DNK oštećenja izazvanog tiazofurinom u humanim ćelijama pune krvi primenom in vitro komet testa

Objective. Inosine 5'-monophosphate dehydrogenase (IMPDH) activity in cancer cells is increased. Tiazofurin selectively inhibits the activity of IMPDH, and it has been granted for the treatment of different cancers and new viral diseases. Its widespread use was limited because exposure to tiazofurin under certain circumstances was found to have a higher frequency of severe non-hematologic toxicity. Therefore, the objective of this study was to examine genotoxic action and inducement of DNA damage of tiazofurin using the comet assay. Methods. The ability of tiazofurin to induce DNA damage was evaluated using single-cell gel electrophoresis (SCGE) technique/comet assay. Human whole blood cells were exposed to three final concentrations of tiazofurin (1µM/mL, 2 µM/mL, and 5 µM/mL) for 30 min in vitro. Results. Our results indicate that tiazofurin produced a significant level of DNA damage on whole blood cells after 30 min of exposure vs. control. All tested concentrations were significantly comet-forming, in a concentration-dependent manner. Conclusion. Our investigation on the tiazofurin-treated cells and their relationship to the formation of DNA damage demonstrated that the genotoxic effect was induced after exposure to tiazofurin under described conditions.

Abstract 1756: Electronic cigarettes induce DNA damage in the oral epithelium of users

Abstract Background: The use of electronic cigarettes (e-cigs) has skyrocketed among adolescents and young adults. E-cigs are promoted as safer than combustible cigarettes but their long term effects on human health are still unknown. E-cig aerosols contain significantly lower number of chemicals than tobacco smoke, however they also contain known carcinogens, reactive oxygen species (ROS), and unique chemicals poorly studied. In vitro and animal studies revealed that e-cig aerosols increases DNA damage and reduces DNA repair. There have been no studies assessing e-cig-induced DNA damage in human subjects. This reflects, at least in part, technical limitations that make it quite challenging to measure the in vivo steady-state levels of DNA damage, particularly for relatively low levels of genotoxic exposure. Our lab has previously developed and validated a novel quantitative primer anchored DNA damage detection assay (q-PADDA) to quantify in vivo DNA damage. We have also reported that, in an in vitro setting, q-PADDA has high sensitivity to detect e-cig-induced DNA damage. Here, we take advantage of this assay to start assessing the potential long-term impact of e-cig use on human health. Aim: To determine whether e-cig users have higher levels of DNA damage on oral epithelial cells than non-users. Methods: Ethics Committee approval was obtained. Participants were recruited through internet ads, leaflets, flyers and word-of-mouth. After written informed consent, a total of 35 individuals were included in this study: 20 exclusive e-cigarette users and 15 individuals reporting no e-cig or tobacco use. Self-report of tobacco use was biochemically confirmed by salivary cotinine and by measuring exhaled carbon monoxide (CO). Oral mucosa samples were collected by cytobrush and used for DNA extraction and damage quantification. DNA damage was quantified by q-PADDA within p53, the most frequently mutated gene in human cancer. Plasma nicotine levels were analyzed by liquid chromatography-mass spectrometry. Data was analyzed by Student’s t test. Results: The study including participants using high and low voltage e-cig devices. E-cig users (33 ± 8) and non-users (33 ± 7) had similar age distribution. We observed that oral epithelial cells collected from e-cig users have significantly higher levels of DNA damage in both transcribed and non-transcribed DNA strands of the p53gene when compared to never users. Conclusion: Our work is the first study to measure DNA damage in e-cig users. Our study shows for the first time that e-cig users have significant levels of DNA damage in their oral mucosa epithelium. These data has major clinical implications and suggest that e-cig users might have an increased cancer risk. Further studies are urgently needed to expand these kind of studies and to characterize the molecular mechanisms underlying e-cig induced DNA damage which may leads to cancer. Citation Format: Vengatesh Ganapathy, Theodore Wagener, Lurdes Queimado. Electronic cigarettes induce DNA damage in the oral epithelium of users [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1756.

Stress hormones induce DNA damage in ovarian cancer cells

Activation of the sympathetic nervous system (SNS) is associated with cancer progression. Recent data suggest that adrenergic signaling alters the genetic integrity of cancer cells by damaging the DNA and/or influencing the DNA‐maintenance machinery composed of the DNA Damage Response Pathway and DNA Repair Pathways. Consequently, accumulation of genomic alterations can lead to genomic instability and the acquisition of other cancer hallmarks that drive tumor progression. Still, the exact mechanisms of how sustained adrenergic signaling influences genomic stability is an area that needs to be further studied. We hypothesize that increased adrenergic stimuli will induce significant levels of DNA damage. To assess the extent of DNA damage after catecholamine exposure, we determined p‐H2AX (sensitive marker for double‐stranded DNA damage) expression by western blot and immunofluorescence analyses in SKOV3 and other high‐grade serous ovarian cancer cell lines. Neutral Comet Assays were performed to assess double‐stranded DNA damage after catecholamine exposure. SKOV3 cells that were exposed to epinephrine (Epi) and norepinephrine (Ne) at 1h, 3h, 6h, 12h, and 24h displayed increased expression of p‐H2AX. Moreover, immunofluorescence analyses for p‐H2AX in SKOV3 and OV90 showed increased foci formation (reflecting 1:1 foci/double‐strand break) when compared to the control group. Lastly, Neutral Comet Assay analyses in catecholamine‐treated SKOV3 and COV362 cells displayed higher levels of double‐stranded DNA breaks in comparison to untreated cells. Lastly, to determine if DNA damage is due to adrenergic stimuli, cells were pre‐treated with propranolol (non‐specific β‐blocker) and results showed a significant reduction in the levels of DNA damage. These results suggest that catecholamines can induce double‐stranded DNA damage in ovarian cancer cells.Support or Funding InformationNIMHHD (G12MD007579), NIGMS‐RISE R25GM082406, NCI (U54CA163071 and U54CA163068), U54CA163071‐06, and Ponce Research Institute.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 3593: Electronic cigarettes induce significant DNA damage and reduce cellular antioxidant levels

Abstract Background: Cigarette smoking is the leading preventable cause of mortality in the world and the main risk factor for lung and head and neck cancer. E-cigarettes (ECs) are battery-operated devices that deliver nicotine via inhaled aerosols. Although ECs are marketed as a less harmful alternative to tobacco cigarettes and a smoking cessation aid, the health risks posed by exposure to EC aerosols are unknown. Nonetheless, the use of ECs has increased exponentially since 2003, with EC users reporting inhaling on average 200 puffs a day. EC aerosols have been reported to contain variable levels of genotoxins, including carcinogenic substances and reactive oxygen species (ROS). Some toxins in EC aerosols have been reported to reach the levels of similar to those in tobacco smoke. However, the genotoxicity of EC aerosols has not been characterized. Aims: (1) To determine the cytotoxicity and genotoxicity of short and long-term exposure to EC aerosols on human epithelial normal and cancer cell lines. (2) To evaluate whether exposure of EC aerosols modifies the cellular total antioxidant capacity (TAC). Methods: EC extracts were prepared from NJOY (12 or 18 mg/ml of nicotine) and Oakley eGo-T (0, 12 or 18 mg/ml of nicotine). Standard tobacco extracts were used for comparison. To assess the effects of short-term exposure, human epithelial normal (NuLi-1) and cancer (UD-SCC1) cell lines were exposed for 1 hour to various EC extract concentrations. To assess the effects of long-term exposure, cells lines were exposed every other day for 2 weeks to EC extracts. Cytotoxicity, DNA damage and TAC were evaluated at 1 h and 2 weeks. Cell viability was determined by MTT assay. DNA damage was quantified using the primer anchored DNA damage detection assay (PADDA). TAC was determined by the Antioxidant Assay Kit (Cayman). Data were analyzed by Student's t-test. Results: At the range of EC extract concentrations used in this study and expected to occur in EC users (1 to 100 puffs/5 L of blood), no cytotoxicity was observed for either normal or cancer cells. However, significant levels of DNA damage were observed in cancer cells exposed to 10 or more puffs/5 L of EC extracts and in normal cells exposed to100 puffs/5 L. Long-term exposure to EC extracts resulted in a significant decrease in TAC, a measure of free radical scavengers. Conclusion: Even short-term exposure to low levels of EC aerosols can cause significant DNA damage. Our study emphasizes the need to further investigate the carcinogenic potential of EC aerosols and highlights the importance of regulating EC use. Grant support: This work was supported by the Oklahoma Tobacco Research Center (LQ). Dr. Queimado holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: Vengatesh Ganapathy, Jimmy Manyanga, Lacy Brame, David Rubenstein, Theodore L. Wagener, Ilangovan Ramachandran, Lurdes Queimado. Electronic cigarettes induce significant DNA damage and reduce cellular antioxidant levels. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3593.

From Immunity and Vaccines to Mammalian Regeneration

Our current understanding of major histocompatibility complex (MHC)-mediated antigen presentation in self and nonself immune recognition was derived from immunological studies of autoimmunity and virus-host interactions, respectively. The trimolecular complex of the MHC molecule, antigen, and T-cell receptor accounts for the phenomena of immunodominance and MHC degeneracy in both types of responses and constrains vaccine development. Out of such considerations, we developed a simple peptide vaccine construct that obviates immunodominance, resulting in a broadly protective T-cell response in the absence of antibody. In the course of autoimmunity studies, we identified the MRL mouse strain as a mammalian model of amphibian-like regeneration. A significant level of DNA damage in the cells from this mouse pointed to the role of the cell cycle checkpoint gene CDKN1a, or p21(cip1/waf1). The MRL mouse has highly reduced levels of this molecule, and a genetic knockout of this single gene in otherwise nonregenerating strains led to an MRL-type regenerative response, indicating that the ability to regenerate has not been lost during evolution.

Methamphetamine induces DNA damage in specific regions of the female rat brain.

Methamphetamine (METH) is a highly addictive psychostimulant that has been shown to produce neurotoxicity. Methamphetamine increases the release of dopamine by reversing the direction of monoamine transporter proteins, leading to the formation of reactive oxygen species in the brain. In this study, we examined the effect of METH on DNA damage in vivo using the single cell gel electrophoresis assay (comet assay) under two different conditions. Rats treated with multiple doses of METH (10 mg/kg × 4) showed significant levels of DNA damage in the nucleus accumbens and striatum, both dopamine-rich areas. In contrast, a single dose of METH did not lead to significant levels of DNA damage in any of the dopamine-rich brain regions that were tested. Overall, the results of our study demonstrate that METH produces greater oxidative DNA damage in brain areas that receive greater dopamine innervation.

The rate of X-ray-induced DNA double-strand break repair in the embryonic mouse brain is unaffected by exposure to 50 Hz magnetic fields

Purpose: Following in utero exposure to low dose radiation (10–200 mGy), we recently observed a linear induction of DNA double-strand breaks (DSB) and activation of apoptosis in the embryonic neuronal stem/progenitor cell compartment. No significant induction of DSB or apoptosis was observed following exposure to magnetic fields (MF). In the present study, we exploited this in vivo system to examine whether exposure to MF before and after exposure to 100 mGy X-rays impacts upon DSB repair rates.Materials and methods: 53BP1 foci were quantified following combined exposure to radiation and MF in the embryonic neuronal stem/progenitor cell compartment. Embryos were exposed in utero to 50 Hz MF at 300 μT for 3 h before and up to 9 h after exposure to 100 mGy X-rays. Controls included embryos exposed to MF or X-rays alone plus sham exposures.Results: Exposure to MF before and after 100 mGy X-rays did not impact upon the rate of DSB repair in the embryonic neuronal stem cell compartment compared to repair rates following radiation exposure alone.Conclusions: We conclude that in this sensitive system MF do not exert any significant level of DNA damage and do not impede the repair of X-ray induced damage.

DNA damage in gill cells of Corbicula japonica exposed to natural and anthropogenic stressors

DNA damage in gill cells of Corbicula japonica exposed to natural and anthropogenic stressors

Mouse Spermatocytes Express CYP2E1 and Respond to Acrylamide Exposure

Metabolism of xenobiotics by cytochrome P450s (encoded by the CYP genes) often leads to bio-activation, producing reactive metabolites that interfere with cellular processes and cause DNA damage. In the testes, DNA damage induced by xenobiotics has been associated with impaired spermatogenesis and adverse effects on reproductive health. We previously reported that chronic exposure to the reproductive toxicant, acrylamide, produced high levels of DNA damage in spermatocytes of Swiss mice. CYP2E1 metabolises acrylamide to glycidamide, which, unlike acrylamide, readily forms adducts with DNA. Thus, to investigate the mechanisms of acrylamide toxicity in mouse male germ cells, we examined the expression of the CYP, CYP2E1, which metabolises acrylamide. Using Q-PCR and immunohistochemistry, we establish that CYP2E1 is expressed in germ cells, in particular in spermatocytes. Additionally, CYP2E1 gene expression was upregulated in these cells following in vitro acrylamide exposure (1 µM, 18 h). Spermatocytes were isolated and treated with 1 µM acrylamide or 0.5 µM glycidamide for 18 hours and the presence of DNA-adducts was investigated using the comet assay, modified to detect DNA-adducts. Both compounds produced significant levels of DNA damage in spermatocytes, with a greater response observed following glycidamide exposure. A modified comet assay indicated that direct adduction of DNA by glycidamide was a major source of DNA damage. Oxidative stress played a small role in eliciting this damage, as a relatively modest effect was found in a comet assay modified to detect oxidative adducts following glycidamide exposure, and glutathione levels remained unchanged following treatment with either compound. Our results indicate that the male germ line has the capacity to respond to xenobiotic exposure by inducing detoxifying enzymes, and the DNA damage elicited by acrylamide in male germ cells is likely due to the formation of glycidamide adducts.

Pesticide Residues, Heavy Metals, and DNA Damage in Sentinel Oysters Crassostrea gigas From Sinaloa and Sonora, Mexico

Pesticides and heavy metals were analyzed in sentinel Crassostrea gigas oysters placed in six aquaculture sites close to a contaminated agricultural region. Each site was sampled twice. Tests revealed the presence of organochlorine (OC) pesticides in the oysters at concentrations varying from 31.8 to 72.5 μg/kg for gamma-hexachlorocyclohexane (γ-HCH); from 1.2 to 3.1 μg/kg for dichlorodiphenyldichloroethylene (4,4-DDE); from 1.6 to 2.3 μg/kg for endosulfan I; and from 1.4 to 41.2 μg/kg for endosulfan II, as well as heavy metals in concentrations that exceeded Mexican tolerance levels (405.5 to 987.8 μg/g for zinc; 4.2 to 7.3 μg/g for cadmium; and 7.2 to 9.9 μg/g for lead). Significant levels of DNA damage in oyster hemocytes were also detected. There was a significant, positive correlation between genotoxic damage and concentration of nickel or the presence of endosulfan II. Cellular viability evaluated by cytotoxic analyses was found to be high at 80%. Marked inhibition in activity of acetylcholinesterase (AChE ) and induction of glutathione S-transferase (GST) activity was noted. Data demonstrated a significant relation between AChE activity inhibition and presence of endosulfan II, γ-HCH, copper, lead, and 4,4-DDE, as well as between AChE and GST activity at different sites.

Composite-induced toxicity in human gingival and pulp fibroblast cells

Objective. The most important requirement for a material to be used in medical applications is its biocompatibility. Dental composite materials come into direct contact with oral tissues, especially gingival and pulpal cells. This study was performed to evaluate possible DNA damage in cells of human origin exposed to dental composites in vitro using a cytogenetic assay. Materials and methods. Two composite resins (Vertise Flow, Kalore) were tested on human gingival and pulp fibroblasts using the acridine orange/ethidium bromide viability staining and alkaline comet assay. Cultures were treated with polymerized composites in two different concentrations (20 mg/ml, 40 mg/ml) for 14 days. Chi-square and Kruskall-Wallis non-parametric test were used for the statistical analysis (p < 0.05). Results. Significant cytotoxicity was observed for 40 mg/ml of Vertise Flow in both cultures, while Kalore (40 mg/ml) showed cytotoxic effect only on human pulp fibroblasts. A significant level of DNA damage was detected for both materials and concentrations, in both cell cultures. Conclusion. If the two cell cultures are compared, the pulp cells were more sensitive to the cyto/genotoxic effects of dental composites. Based on the results, one can conclude that the use of tested materials may cause cellular damage in gingival and pulp fibroblasts in vitro.

Oxidative Stress and DNA Damage Induced by Chromium in Liver and Kidney of Goldfish, Carassius auratus

Chromium (Cr) is an abundant element in the Earth’s crust. It exhibits various oxidation states, from divalent to hexavalent forms. Cr has diverse applications in various industrial processes and inadequate treatment of the industrial effluents leads to the contamination of the surrounding water resources. Hexavalent chromium (Cr (VI)) is the most toxic form, and its toxicity has been associated with oxidative stress. The present study was designed to investigate the toxic potential of Cr (VI) in fish. In this research, we investigated the role of oxidative stress in chromium-induced genotoxicity in the liver and kidney cells of goldfish, Carassius auratus. Goldfish were acclimatized to the laboratory conditions and exposed them to 5% and 10% of 96 hr-LC50 (85.7 mg/L) of aqueous Cr (VI) in a continuous flow through system. Fish were sampled every 7 days for a period of 28 days to analyze the lipid hydroperoxides (LHP) levels and genotoxic potentials in the liver and kidney. LHP levels were analyzed by spectrophotometry while genotoxicity was assessed by single cell gel electrophoresis (comet) assay. LHP levels in the liver increased significantly at week 1, followed by a decrease. LHP levels in the kidney increased significantly at weeks 1, 2, and 3, and decreased at week 4 compared to the control. The percentage of DNA damage increased in both liver and kidney at both test concentrations. The results clearly indicate that Cr (VI) induces significant levels of DNA damage in liver and kidney cells of goldfish. The induced LHP levels in both organs were concentration-dependent and were directly correlated with the levels of DNA damage. The two tested Cr (VI) concentrations induced significant levels of oxidative stress in both organs, however the kidney appears to be more vulnerable and sensitive to Cr-induced toxicity than the liver.

DNA Damage in Patients of Carcinoma Breast a Clinical Study by using Comet Assay

35 cases of 27-68years old breast cancer individuals and 15 female individuals of FDFRS formed the material for the current study. An equal number of normal healthy females controls were also included to investigate the extent of DNA damaged in cases, FDFRs and controls. 'Comet assay' was done by conventional methods with slight modifications. For Comet metrics, a Trinocular research microscope, Nikkon Optiphot model with automatic photomicrograph attachment was used. Thus quantification of the DNA damage was done by measuring comet tail length in all the three groups (cases, FDFRs and controls).There was significant increase in the mean comet tail length from controls to FDFRs (p<0.0001) and from FDFRs to cases (p<0.0001). In other words the DNA damage significantly increased from controls to FDFRs and from FDFRs to cases. It was also observed that among various stages of cases the mean comet tail length increased significantly from stage II A to stage III B.Mean comettail length wasfound to be increased significantly in the advanced stage of carcinomas, i.e. stage III B followed by stage III A and II A. The FDFRs of breast carcinoma individuals showed significant level of DNA damage. This may be used as a marker/tool forthe identification of the diseased condition which gets manifested infamilies.