Comet Tail Moment Research Articles

Abstract Mo036: DNA Damage and Repair in Patients Undergoing Myocardial Perfusion Single-Photon Emission Computed Tomography

Background: As medical imaging has continuously grown, patient exposure to ionizing radiation and the risks associated with it are important issues. Myocardial perfusion single-photon emission computed tomography (MP-SPECT) is one of the most frequently performed noninvasive cardiac imaging tests, generating concern about the potentially excessive radiation exposure for patients, including the cumulative doses of lifetime radiation. even though the individual levels of radiation exposure are considered low. Aim: To evaluate DNA damage and DNA repair markers after MP-SPECT. Methods: Thirty-two patients undergoing Tc-99m sestamibi MP-SPECT (mean injected Tc-99m sestamibi dose: 15.1 mCi) were studied. Peripheral blood was collected before radiotracer injection at rest and 60-90 minutes after injection. The comet assay (single-cell gel electrophoresis) was performed with blood lymphocytes to detect DNA strand breaks, which appear as DNA fragmentation (the comet tail; Figure1). Images of fluorescence microscopy were analyzed using the Comet Assay Software Project Lab (CASPLab 1.2.3 beta 2). Three parameters were evaluated: the percentage of DNA in the comet tail, tail length, and tail moment (the measure of migratory DNA and number of fragments). Quantitative PCR (qPCR) was performed to evaluate the expression of five genes related to signaling pathways in response to DNA damage and repair (ATM, ATR, BRCA1, CDKN1A, XPC). Data are expressed as medians, and Wilcoxon or Mann-Whitney tests were employed for statistical analysis. A p-value <0.05 was considered statistically significant. Results: Figure 2 shows pre- and post- radiation exposure comet assay parameters. After radiotracer injection, there was a 10.7% increase of the DNA percentage in the tail (pre-exposure median 2.185 vs post-exposure median 2.418, p<0.0065). Tail length increased 6.7% (15.0 pre-exposure vs 16.0 post-exposure, p<0.0034). Tail moment increased 27.5% (0.3177 pre-exposure vs 0.4050 post-exposure, p<0.0001). qPCR did not reveal increased expression of any gene after radiation exposure. Conclusion: An increase in DNA damage was detected after a single radiotracer injection for MP-SPECT, yet unaccompanied by activation of repair genes. This may suggest that DNA damage is not large enough to trigger intense repair responses and might not result in biologically significant DNA damage.

DNA damage and repair in patients undergoing myocardial perfusion single-photon emission computed tomography

As patient exposure to ionizing radiation from medical imaging and its risks are continuing issues, this study aimed to evaluate DNA damage and repair markers after myocardial perfusion single-photon emission computed tomography (MPS). Thirty-two patients undergoing Tc-99m sestamibi MPS were studied. Peripheral blood was collected before radiotracer injection at rest and 60–90 min after injection. The comet assay (single-cell gel electrophoresis) was performed with peripheral blood cells to detect DNA strand breaks. Three descriptors were evaluated: the percentage of DNA in the comet tail, tail length, and tail moment (the product of DNA tail percentage and tail length). Quantitative PCR (qPCR) was performed to evaluate the expression of five genes related to signaling pathways in response to DNA damage and repair (ATM, ATR, BRCA1, CDKN1A, and XPC). Mann–Whitney’s test was employed for statistical analysis; p < 0.05 was considered significant. Mean Tc-99m sestamibi dose was 15.1 mCi. After radiotracer injection, comparing post-exposure to pre-exposure samples of each of the 32 patients, no statistically significant differences of the DNA percentage in the tail, tail length or tail moment were found. qPCR revealed increased expression of BRCA1 and XPC, without any significant difference regarding the other genes. No significant increase in DNA strand breaks was detected after a single radiotracer injection for MPS. There was activation of only two repair genes, which may indicate that, in the current patient sample, the effects of ionizing radiation on the DNA were not large enough to trigger intense repair responses, suggesting the absence of significant DNA damage.

CD276 enhances sunitinib resistance in clear cell renal cell carcinoma by promoting DNA damage repair and activation of FAK-MAPK signaling pathway.

This study aimed to explore the effect of CD276 expression on the sunitinib sensitivity of clear cell renal cell carcinoma (ccRCC) cell and animal models and the potential mechanisms involved. CD276 expression levels of ccRCC and normal samples were analyzed via online databases and real-time quantitative PCR (RT-qPCR). CD276 was knocked down in ccRCC cell models (sunitinib-resistant 786-O/R cells and sunitinib-sensitive 786-O cells) using shRNA transfection, and the cells were exposed to a sunitinib (2 µM) environment. Cells proliferation was then analyzed using MTT assay and colony formation experiment. Alkaline comet assay, immunofluorescent staining, and western blot experiments were conducted to assess the DNA damage repair ability of the cells. Western blot was also used to observe the activation of FAK-MAPK pathway within the cells. Finally, a nude mouse xenograft model was established and the nude mice were orally administered sunitinib (40mg/kg/d) to evaluate the in vivo effects of CD276 knockdown on the therapeutic efficacy of sunitinib against ccRCC. CD276 was significantly upregulated in both ccRCC clinical tissue samples and cell models. In vitro experiments showed that knocking down CD276 reduced the survival rate, IC50 value, and colony-forming ability of ccRCC cells. Knocking down CD276 increased the comet tail moment (TM) values and γH2AX foci number, and reduced BRCA1 and RAD51 protein levels. Knocking down CD276 also decreased the levels of p-FAK, p-MEK, and p-ERK proteins. Knocking down CD276 effectively improved the sensitivity of ccRCC cell and animal models to sunitinib treatment.

Evaluation of the Biological Effects of Exposures to Magnetic Resonance Imaging on Single-Strand DNA: An In-vivo Study

Introduction: Magnetic resonance imaging (MRI) is a powerful diagnostic technique used to acquire detailed information on the structure and function of the body’s organs. Data on the extent of genetic damage following exposure to electromagnetic fields in MRI is variable, necessitating further evidence. This study aims to examine the biological effect of exposure to MRI at various magnetic strengths on the DNA single-strand. Methods: The study was an in-vivo non-randomized controlled experiment involving New Zealand rabbits (n=39, males) scanned using three different MRI strengths (0.5, 1.5 and 3.0 T) and at different time intervals (10, 20, 30, and 40 minutes). The alkaline comet assay was used to study DNA damage by quantifying single-strand breaks. In addition, tail length (TL), tail moment (TM), and the fraction of total DNA in the tail were evaluated. Results: The DNA single-strand breaks were significant for all tested parameters in both MRI 1.5 T (p&lt;0.01) and 3.0 T (p&lt;0.001). In addition, 3.0 T for 40 minutes had the most comet tails and tail moment (13.87), resulting in greater %DNA damage (mean=22.37). Exposure to 0.5 T was found to be only significant at 30 and 40 minutes (p&lt;0.001). Conclusion: Higher MRI strength for a longer duration resulted in a significant increase in DNA single-strand breaks. Understanding the interaction between the magnetic fields generated by MRI and DNA will optimize safe and effective MRI scanning in both patients and healthy individuals.

Abstract 3369: Dual inhibition of ATR and PI3K pathways promotes cell death in platinum-resistant endometrial cancer cells by increasing DNA damage

Abstract Background: Endometrial cancer (EC) is the most common gynecologic malignancy with increasing incidence and mortality in the U.S. Among the four subtypes characterized by the Cancer Genome Atlas, the serous-like copy number high (CNH) subtype which occurs in 15-25% of EC is associated with poor prognosis and platinum resistance, requiring alternative treatment options. ~85% CNH EC cases have impaired G1/S checkpoint regulation, making cells rely on ATR-mediated G2/M cell cycle checkpoints for optimal DNA replication and repair. Additionally, over 80% of ECs exhibit PI3K pathway activation (e.g., PIK3CA mutations), linked to enhanced DNA repair and drug resistance. Therefore, we hypothesized that dual ATR and PI3Kα inhibition would enhance DNA damage, increasing cell death in EC. Methods: CNH EC cell lines included platinum-resistant (KLE, MFE280, HEC1A, ARK2) as well as platinum-sensitive (ARK1) lines used to evaluate the activity of ATR inhibitor (ATRi) camonsertib and PI3Kα inhibitor (PI3Kαi) inavolisib. Cell growth was assessed by 3-day XTT and 9-12-day colony-forming assays. The degree of combination synergy, additivity, or antagonism was calculated using SynergyFinder with a reference highest single agent (HSA) model. An HSA synergy score &amp;gt; 10 indicates synergy, -10 to 10 suggests additivity and &amp;lt; -10 represents antagonism. DNA damage endpoints were measured by alkaline comet assay and immunoblotting for γ-H2AX. Data were repeated in triplicate, analyzed using one-way ANOVA test, and shown as mean ± SD. P &amp;lt; 0.05 was statistically significant. Results: All tested EC cell lines showed sensitivity to clinically attainable doses of ATRi monotherapy (IC50 0.09 - 0.98 μM), but varying sensitivity to PI3Kαi monotherapy (IC50 0.03 - 67.01 μM) unrelated to their PIK3CA mutation status. Combination treatment using clinically attainable concentrations (camonsertib &amp;lt; 6 μM; inavolisib &amp;lt; 10 μM) yielded additivity in all cell lines independent of platinum sensitivity (HSA synergy scores -0.8-10.0). Notably, increased cytotoxic effects were observed in both PIK3CA wild-type (KLE) and PIK3CA-mutant (MFE280 and HEC1A) platinum-resistant lines by colony-forming assays (4.8- to 15.4-fold decrease in colony-forming ability relative to ATRi; 1.5- to 3.5-fold decrease relative to PI3Kαi). ATRi and PI3Kαi combination induced greater DNA damage, evidenced by elevated mean comet tail moment relative to ATRi (increased 2.5- to 6.7-fold; P &amp;lt; 0.01) or PI3Kαi (increased 2.1- to 5.6-fold; P &amp;lt; 0.01) regardless of PIK3CA mutation status. Increased γ-H2AX levels were found in HEC1A cells with combination treatment (3.2-fold increase relative to ATRi; 16-fold increase relative to PI3Kαi). Conclusion: Our results suggest that dual inhibition of ATR and PI3K pathways induces greater cell death by increasing DNA damage in platinum-resistant EC cells independent of PIK3CA mutation status. Citation Format: Kristen R. Ibanez, Tzu-Ting Huang, Sotirios Sotiriou, Yvonne G. Lin, Jung-Min Lee. Dual inhibition of ATR and PI3K pathways promotes cell death in platinum-resistant endometrial cancer cells by increasing DNA damage [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 3369.

ANTIOXIDANTS ENHANCE CHROMIUM (VI) MEDIATED TOXICITY IN NEUROSPORA CRASSA

Chromium can be found in the environment in two main valence states: hexavalent Cr (VI) and trivalent Cr (III). Reduction activation of carcinogenic Cr (VI) is required for the induction of DNA damage and mutations. There is a controversy over antioxidants’ role in Cr (VI) toxicity and cell death in mammalian systems. In the present study we compared the effect of antioxidants and insulin on Cr (VI) induced genotoxicity in wall-less mutant of Neurospora crassa (slime), which contains insulin receptor on its cell surface as a model system. Analysis by comet assay indicated Potassium dichromate (K2 Cr2 O7 ) induced DNA damage in slime cells, as increase in comet tail moment and the effect was dose dependent. Simultaneous addition of N-acetyl cysteine (NAC) &amp; Sodium ascorbate (ASC) increased the extent of DNA damage whereas insulin decreased the extent of DNA damage induced by Cr (VI). The simultaneous exposure of antioxidants and insulin in presence of Cr (VI) showed enhanced cytotoxicity as observed by MTT assay. The results obtained suggest that N-acetyl cysteine and Sodium ascorbate reduce Cr (VI) to Cr (III) enhancing the levels of reactive oxygen species. Absence of protective role of insulin in presence of antioxidants indicate that a differential route may be adopted by the cells in response to metallic stress.

The optimum concentration of soya-lecithin for replacing egg yolk in INRA-82-modified semen extenders improved the cryopreserved semen quality and DNA integrity of Arabian stallions

The cryopreservation of stallion spermatozoa varies greatly between animals. Different equine semen extender supplements were invented to maximize the post-thaw semen quality. Soy lecithin is one of those additives that contain plant origin phospholipids used to replace egg yolk to minimize disease transmission. This study aimed to determine which concentrations of soy lecithin can replace egg-yolk in modified INRA-82 semen extenders for the cryopreservation of horse semen. Semen was collected from six Arabian stallions and extended with modified INRA-82 semen extender supplemented with 0.0, 1.25, 2.50, and 5.0% soya lecithin. Frozen-thawed sperm motility was measured at 0, 1, 2, and 3 h. Viability index, sperm plasma membrane integrity (SPMI), and acrosome integrity in addition to the comet assay non-fragmented DNA, head DNA, tail DNA, tail length, and tail moment were statistically analyzed. The addition of 2.5% soya lecithin had the highest post-thaw sperm motility at 0, 1, 2 h (P &lt; 0.001) and 3 h (P &lt; 0.0001), the optimum viability index (P &lt; 0.0001), SPMI (P &lt; 0.05), acrosome integrity (P &lt; 0.0001), non-fragmented DNA (P &lt; 0.01), DNA in the comet head (P &lt; 0.05), minimum DNA in the comet tail (P &lt; 0.05), shortest comet tail length (P &lt; 0.0001), and lowest comet tail moment (P &lt; 0.01). Soy lecithin at concentrations other than 2.5% deteriorated all post-thaw semen parameters. Soy lecithin (2.5%) can replace egg yolk in the modified INRA-82 semen extender for the cryopreservation of good-quality stallion semen.

Abstract 2406: Targeting prmt5 inhibits DNA repair and enhances the radiosensitivity of GBM cells

Abstract Glioblastoma (GBM) is considered highly radioresistant. To improve the efficacy of GBM radiotherapy, one approach is the addition of a targeted radiosensitizer, which requires an understanding of the molecules that determine GBM radioresponse. Along these lines, PRMT5 is a type II arginine methyltransferase that symmetrically dimethylates histone and non-histone substrates to regulate chromatin structure and mRNA splicing, two parameters that can influence radiosensitivity. As an initial test of PRMT5 as a target for GBM radiosensitization, PRMT5 knockdown using siRNA was shown to increase the in vitro radiosensitivity of U251 GBM cells. PRMT5 was then targeted using the small molecule inhibitor LLY-283, which penetrates the blood brain barrier. Exposure of U251 cells to LLY-283 (24h) reduced the levels of symmetric dimethylated arginine as determined by western blot, indicating an inhibition of PRMT5 activity. Based on clonogenic analysis, LLY-283 exposure beginning 1h prior to irradiation significantly enhanced the radiosensitivity of U251 cells. Whereas treatment of the normal fibroblast cell line MRC9 with LLY-283 reduced PRMT5 activity, no effect was detected on radiosensitivity, suggesting tumor cell selective radiosensitization. As an initial investigation of the radiosensitizing mechanism, U251 were treated with LLY-283 1h prior irradiation (2Gy) and the number of γH2AX foci, marker for DNA double strand breaks (DSBs), was determined. Whereas LLY-283 had no effect on the initial level of radiation-induced γH2AX foci, the dispersal of foci was significantly delayed in LLY-283 treated cells. These results suggest that LLY-283 inhibits the repair of radiation-induced DSBs. The neutral comet assay, an alternative measure of radiation-induced DSBs showed that LLY-283 delivered 1h prior irradiation (10Gy) significantly increased the comet tail moment detected at 6 and 24h as compared to 10Gy only, consistent with an inhibition of DSB repair. Based on these two approaches LLY-283-induced radiosensitization appears to be mediated by an inhibition of DSB repair. Extending these studies to GBM stem-like cell lines (GSCs) showed that LLY-283 inhibited PRMT5 activity and when delivered 1h prior to irradiation enhanced radiosensitivity. Analysis of γH2AX foci showed a delay in foci dispersal in LLY-283 treated cells consistent with an inhibition of DSB repair. Because a 1h LLY-283 pretreatment was sufficient for the inhibition of DSB repair and enhanced radiosensitivity, it appears that the radiosensitization was primarily the result of altered chromatin function rather than changes in gene expression due to alternative mRNA splicing. Altogether, these results suggest targeting PRMT5 as a potential strategy for enhancing the radiosensitivity of GBMs. Citation Format: Charlotte M. Degorre, Steven Lohard, Philip J. Tofilon. Targeting prmt5 inhibits DNA repair and enhances the radiosensitivity of GBM cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2406.

Effects of cysteamine supplementation on cryopreserved buffalo bull semen quality parameters

This work aimed to determine the effect of cysteamine (25, 50, 100 and 200 μM) incorporated during dilution on frozen thawed buffalo semen quality. Semen was collected twice weekly for 7 consecutive weeks from three Egyptian buffalo bulls using an artificial vagina. Semen samples were pooled and extended with a Tris-based extender, cooled, equilibrated and finally frozen in liquid nitrogen. The diluted semen was evaluated for motility, viability, morphology, plasma membrane and DNA integrity, in addition to oxidative stress and in vitro fertilizing capability. The post thaw motility and velocity parameters noticeably increased with different concentrations of cysteamine (mainly 100 μM) during different incubation periods. The post thaw sperm viability and normality significantly (p < 0.05) improved with concentrations of 50 and 100 μM. Plasma membrane integrity substantially increased at all concentrations of cysteamine. Cysteamine reduced alanine aminotransferase (at all concentrations), aspartate aminotransferase (at 25–100 μM), and creatine kinase (at 100 and 200 μM). Cysteamine at a concentration of 100 μM noticeably enhanced the total antioxidant capacity and glutathione peroxidase and decreased nitric oxide production. Cysteamine, at concentrations of 100 and 200 μM, increased the DNA intensity in the comet head (%) and decreased the DNA % in the comet tail. The comet tail length and moment substantially decreased at concentrations of 50–200 μM. Cysteamine did not affect the in vitro fertilizing capability of sperm. In conclusion, cysteamine incorporation (mainly at a concentration of 100 μM) in buffalo semen extender showed varying protective effects on different sperm parameters against cryo-damage; however, it did not affect the in vitro fertilizing capacity of sperm.

Determination of genotoxic damages of picloram and dicamba with comet assay in Allium cepa rooted in tissue culture and distilled water.

Many genotoxicity tests allow us to understand the mechanism of damages on genetic material occurring in living organisms against various physical and chemical agents. One of them is the Comet test. The current study aimed to evaluate genotoxic caused by picloram and dicamba to root meristems of Allium cepa utilizing comet assay. Two different protocols were used for rooting and auxin/pesticide application. (i) A. cepa bulbs were rooted in MS medium and then treated with Murashige and Skoog (MS) medium (control) and 0.67, 1.34, 2.01, 2.68, 3.35, 4.02, and 8.04mg/L of picloram and dicamba using aseptic tissue culture techniques. (ii) A. cepa bulbs were then rooted in bidistilled water and treated with 0 (control), 0.67, 1.34, 2.01, 2.68, 3.35, 4.02, and 8.04mg/L of picloram and dicamba in distilled water. The A. cepa root tip cells in both treatment groups were examined using comet test to find the possible DNA damaging effects of picloram and dicamba. The results obtained at all the concentrations were statistically compared with their control groups. Almost at all the concentrations of Picloram and dicamba increased comet tail intensity (%) and tail moment in roots treated in MS medium. Two highest concentrations revealed toxic effect. On the other hand, DNA damaging effect of both auxins was only noted on the highest (> 4.02mg/L) in roots treated in distilled water. This study approve and confirm genotoxic effects of how growth regulators on plants. These findings give an evidence of DNA damage in A. cepa. Therefore, both picloram and dicamba should only be used in appropriate and recommended concentrations in agriculture to conserve ecosystem and to pose minimum threat to life.

EVALUATION OF CYTOTOXIC AND GENOTOXIC EFFECTS OF SAXAGLIPTIN

Saxagliptin (SAX) is an oral drug that is a hypoglycemic (between an anti-diabetic agent) dipeptidyl peptidase-4 inhibitor. In this study, cytotoxic, genotoxic effects and DNA damage of SAX on human lymphocytes were investigated. For this purpose, Single Cell Gel Electrophoresis (SCGE), Micronucleus (MN) and Mitotic Index (MI) tests were used. Based on the daily doses of SAX, 0.017, 0.035, 0.07, 0.14 µg/mL concentrations used for the study. SAX significantly reduced the MI only at the highest concentration (0.14 µg/mL) for 24 hours, and 0.07- and 0.14-µg/mL concentrations for 48 hours. SAX did not cause a statistically significant change in MN frequency (except for concentration 0.14 µg/mL). In the SCGE test, a statistically significant increase of comet tail length was observed at 0.07- and 0.14-µg/mL concentrations. SAX did not cause a statistically significant change in comet tail moment and tail intensity (except for concentration 0.14 µg/mL). As a result, SAX caused statistically differences in the SCGE, MI and MN tests only at the highest concentrations that are not recommended commercial use (except for tail length, 0.035 µg/mL). When the results of all these studies are evaluated together, it can be said that SAX has no aneugenic, mutagenic and clastogenic effects at daily doses in in vitro studies on human lymphocytes.

DNA damage in peripheral blood mononuclear cells in patients with melanoma

Introduction. The incidence and mortality of malignant melanoma have increased steadily over the last decades; therefore, the development of novel diagnostic markers for malignant melanoma is of great importance. The purpose of the study was to assess whether the development of melanoma before any treatment is accompanied by the body changes and, in particular, DNA damage in the mononuclear cells of the peripheral blood of patients. Material and Methods. In 93 patients (26 men and 67 women) admitted to the N.N. Petrov National Medical Research Center of Oncology for surgical treatment of stage T1c-2a-b-3a-b4a-bN0-1 cutaneous malignant melanoma, and in 118 healthy people as a comparison group, the level of damage to DNA in peripheral blood mononuclear cells was studied using the “comet” method. All patients were divided into two groups: group 1 included 45 patients (13 men and 32 women) who were examined before a decision on treatment was made and group 2 consisted of 48 patients (13 men and 35 women) who previously underwent excision biopsy for melanoma. Results. The level of DNA damage in peripheral blood mononuclear cells, assessed by the comet assay, was found to be signifcantly higher in patients with melanoma than in the comparison group. Moreover, the increase in the level of DNA damage was similar both in patients with a primary tumor before starting any treatment and in those who previously underwent excision biopsy for melanoma. The relationship between the level of DNA damage in peripheral blood mononuclear cells and the morphological characteristics of the tumor cells was revealed. The Spearman correlation analysis showed that all parameters that determined DNA damage positively correlated with the thickness of melanoma according to the Breslow’s depth, and the percentage of DNA in the comet and the comet tail moment correlated with the stage of the disease. Conclusion. The development of cutaneous melanoma is accompanied by an increase in the level of DNA damage in peripheral blood mononuclear cells. The level of DNA damage in peripheral blood mononuclear cells refects the changes that occur in the patient’s body under the infuence of the tumor process, which may allow using this indicator as an additional criterion for the diagnosis and aggressiveness of melanoma.

A novel and potent dihydroorotate dehydrogenase inhibitor suppresses the proliferation of colorectal cancer by inducing mitochondrial dysfunction and DNA damage

AbstractDihydroorotate dehydrogenase (DHODH) is a quite attractive target in cancer therapy. Nevertheless, the antitumor effects of DHODH inhibitors against colorectal cancer (CRC) and the underlying mechanism have seldom been studied. Here, we explored the anti‐CRC efficacy of M62, a novel and potent DHODH inhibitor. In the study, M62 significantly inhibited the proliferation of CRC cells and induced S phase arrest. The antiproliferative effects caused by M62 were rescued by uridine supplementation. Mechanistically, messenger RNA sequencing results showed that M62‐triggered gene changes related to mitochondrial dysfunction, DNA damage, and DNA damage repair. Further validation showed that M62 treatment induced the generation of reactive oxygen species and decreased ΔΨm and ATP production. Meanwhile, M62 induced the accumulation of γ‐H2AX and longer comet tail moment, which were both markers of DNA damage. The downregulated DNA repair proteins and PI3K/ATK pathway were observed after M62 treatment. Furthermore, M62 significantly inhibited CRC xenograft tumor growth without detectable toxicity. Therefore, we conclude that M62 inhibits CRC growth both in vitro and in vivo by causing mitochondrial dysfunction and DNA damage, suggesting that DHODH inhibitors are potential therapeutic strategies for treating CRC.

Polycyclic aromatic hydrocarbons, long non-coding RNA expression, and DNA damage in coke oven workers.

Exposure to polycyclic aromatic hydrocarbons (PAHs) was associated with DNA damage, while the roles of long non-coding RNAs (lncRNAs) in the associations were unclear. We aimed to assess the association of lncRNA NR_024564 with urinary monohydroxy PAHs (OH-PAHs) and DNA damage among 332 coke oven workers. We determined 12 OH-PAHs by gas chromatography-mass spectrometry, and the expression level of NR_024564 by droplet digital RT-PCR and DNA damage by the comet assay. In total participants, we found that NR_024564 was not significantly associated with OH-PAHs or comet parameters. However, among workers with ≥ 20 working years, multiple OH-PAHs including urinary 1-hydroxyphenanthrene (1-OHPh), 2-OHPh, 3-OHPh, 9-OHPh, 1‑hydroxypyrene, and total PAH metabolites were related to increased comet parameters. Moreover, NR_024564 was significantly associated with 2-OHPh and four comet parameters. Each 1% increase in 2-OHPh was associated with 0.35% reduction (95% CI: 0.16%, 0.55%) in NR_024564 (P-FDR = 0.005), and 2-OHPh was marginally interacted with working years in relation to NR_024564 decrease. Also, each 1% increment of NR_024564 was related to 0.04-0.13% decrease of Olive tail moment, percent DNA in the comet tail, tail length, and tail moment (all P-FDR < 0.05). Furthermore, low NR_024564 level combined with high levels of 1-OHPh and 2-OHPh or ≥ 20 working years was positively associated with the comet parameters among the total participants. Our results indicated that NR_024564 might be linked to the adverse associations of PAHs with the DNA damage of coke oven workers who worked for ≥ 20years.

HPRT gene mutation, sister chromatid exchange and comet assay in peripheral lymphocytes of radiation laborers at Al-Amal Cancer Hospital in Baghdad

A Study performed on 30 Iraqi radiation Labors had been exposed tо a low dose tо ionizing radiation at Al-Amal Cancer Hospital in Baghdad the use of three genetic endpoints. Covered 12 females and 18 males between the aged tо (22-57) years. In addition to 20 Healthy individuals from the population, dwelling Baghdad, covered, 7 females and 13 males, aged (19 - 55) years which are non-smokers, non- alcoholic. The cytogenetic analysis, including, HPRT gene mutation¸ comet assay and SCE have been carried out on peripheral blood lymphocytes of labors and control groups. The HPRT gene mutation assay showed a significant increase (p&lt;0.05) in the radiation labors. There were found a significant increase (p&lt;0.05) in comet tail length and tail moment values in thе human lymphocyte in these radiation Labors compared with thе control group. Also the SCE in human lymphocyte for radiation Laborers was significantly (p&lt;0.05) higher than in a control group. Results obtained confirmed the cytogenetic analysis; including, HPRT gene mutation¸ comet assay and SCE are useful as biodosimetric markers for the detection of human exposure to low doses of ionizing radiation. It reflects the simultaneous exposure and actual levels of DNA damage present in radiological laborers' peripheral blood leukocytes by detecting temporary DNA damage and / or repair activity.

Investigation of cyto-genotoxic effects of a food sweetener Acesulfame potassium

Acesulfame potassium (ACE-K) is an artificial sweetener widely used in many foods. This investigation assessed the cytotoxic effect of ACE-K using MTT assay in human hepatocellular carcinoma (HepG2) cell line and the genotoxic effect using chromosomal aberrations (CAs), micronucleus (MN), and comet assays in human lymphocytes. 7.5-240 μg/mL concentrations of ACE-K were applied to cells. ACE-K notably decreased the cell viability on HepG2 cells, especially at 120 and 240 μg/mL at 24 and 48 h. It also significantly reduced the mitotic index (MI) at 60, 120, and 240 μg/mL at both treatments (24 and 48 h) in human lymphocytes. The frequency of the CAs significantly increased at 60, 120, and 240 μg/mL for 48 h treatment compared to control. However, no difference was observed in the frequency of MN and nuclear division index (NDI) at all the treatments. ACE-K also induced comet tail length, tail intensity, and moment at 15 μg/mL in isolated human lymphocytes. Therefore, ACE-K showed a cytotoxic effect in HepG2 cells as well as human lymphocytes at higher concentrations. It also exhibits a mild genotoxic effect by increasing the frequency of CAs at long-term treatment and DNA damaging effect only at 15 μg/mL.

Geraniol, a natural monoterpene, identifications of cytotoxic and genotoxic effects in vitro

ABSTRACT Geraniol (GER) is an acyclic monoterpene alcohol. This study aimed to investigate the cytotoxic and genotoxic effect of GER in human lymphocytes using chromosomal aberrations (CAs) micronucleus (MN) assays and DNA damaging effect using comet assay in isolated human lymphocytes as well as cytotoxic effect in MCF-7 cells using MTT and Neutral red uptake (NRU) assays. Cells were treated with between 4 and 500 µg/mL of GER. GER significantly reduced the cell viability at 4, 20, 100 and 500 µg/mL at 48 hours in MCF-7 cells. GER also had a cytotoxic effect by lowering the mitotic index at 20–100 μg/mL in human lymphocytes. GER significantly induced the frequency of CAs and CA/cell at both treatments. GER did not significant affect the MN frequency. GER significantly increased the comet tail intensity and moment at 50, 100, and 500 µg/mL. GER exerted cytotoxic and genotoxic effects especially at higher concentrations.

The biochemical and molecular markers of Cyprenus carpio L. after chronic exposure to lead

This study was designed to detect the effects of lead on biochemical and molecular characteristics of common carp, Cyprenus carpio L. Two concentrations (10 and 20 ppm) were selected for lead respectively in one week. Biochemical markers such as acetylcholinesterase, superoxide dismutase and catalase were used to assess lead exposure cytotoxic effects, while lead-induced DNA damage was used to determine the effect of lead on molecular level. Fish samples were treated with two selected concentrations of lead solution (10 and 20 ppm) for one-week duration, as exposure period, to detect the impacts of lead exposure on this species. Various biochemical markers such as acetylcholinesterase, superoxide dismutase, and catalase were applied to determine the cytotoxic impacts of lead exposure at the cellular level, while the lead-induced DNA damages were identified to reveal the influences of lead exposure at the molecular level. The results represent that the highest concentration of lead solution (20 ppm) had more effects on the antioxidant enzymes activities such as superoxide dismutase (SOD) level (655.17 ± 21.76 mg U-1) followed by acetyl cholinesterase and catalase (655.17 ± 21.76 U L-1 and 87.93 ± 7.22 mg U-1) respectively. The DNA damages were estimated by the Comet assay technique and the highest level of DNA damages were documented when lead concentration was 20 ppm using different parameters of Comet assay technique such as Comet length, tail length, and tail moment (4021 ± 56.11 µL, 523 ± 55.80 µL, and 91.208 ± 9.45 µL respectively). Therefore, this study confirms that the exposure to high lead concentrations would cause harmful effects on aquatic organisms at both cellular and molecular levels.

Image Mosaic Algorithm-Based Analysis of Effects of Low-Dose X-Rays on Blood Immune Cells

This work aimed to explore the effects of image mosaic algorithm- (IMA-) based low-dose X-rays on deoxyribonucleic acid (DNA) damage of peripheral blood lymphocytes in cardiovascular patients. 84 patients with interventional cardiovascular surgeries were selected as the research objects and divided into coronary intervention surgery (A) group, coronary angiography (B) group, radiofrequency ablation (C) group, and permanent cardiac pacemaker implantation (D) group. They were treated with IMA-based low-dose X-rays, DNA damage indicators of lymphocytes were detected, and the relationship was analyzed between those indicators and radiation dose. Results showed that the cumulative dose (CD) and dose area product (DAP) of group A were the lowest, and those of group D were obviously higher than those of the other three groups ( P &lt; 0.01 ). Besides, the results also showed that the values of CD and DAP of patients from group A and group D were 0.38 ± 0.08 (Gy) and 0.38 ± 0.08 (Gy/cm2) and 1.37 ± 0.21 (Gy) and 1.37 ± 0.21(Gy/cm2), respectively. The comet tail DNA content (TDNA) and tail moment (TM) of each group after surgery increased dramatically compared with before surgery ( P &lt; 0.01 ), and the TDNA and TM of group D were greatly higher than those of the other three groups ( P &lt; 0.01 ). Therefore, TDNA and TM were markedly positively correlated with DAP and CD ( P &lt; 0.01 ). IMA-based X-ray radiotherapy caused reliable DNA damage to peripheral blood lymphocytes of cardiovascular patients, and the greater the radiation dose, the more serious the DNA damage.

Phytochemical and In Vitro Genotoxicity Studies of Standardized Ficus deltoidea var. kunstleri Aqueous Extract

The present study was carried out to assess the genotoxicity potential of Ficus deltoidea var. kunstleri aqueous extract (FDAE) using standard in vitro assays. The DNA damage of V79B cells was measured using the alkaline comet assay treated at 0.1 mg/mL (IC10) and 0.3 mg/mL (IC25) of FDAE together with positive and negative controls. For in vitro micronucleus assay, the V79B cells were treated with FDAE at five different concentrations (5, 2.5, 1.25, 0.625, and 0.3125 mg/mL) with and without S9 mixture. The bacteria reverse mutation assay of FDAE was performed on Salmonella typhimurium strains TA98, 100, 1535, 1537, and Escherichia coli strain WP2uvrA using pre-incubation method in the presence or in the absence of an extrinsic metabolic system (S9 mixture). FDAE at 0.1 and 0.3 mg/mL significantly increased DNA damage in both comet tail and tail moment (p < 0.05). No significant changes were detected in the number of micronucleated cell when compared to control. Tested at the doses up to 5000 µg/plate, the FDAE did not increase the number of revertant colonies for all strains. In conclusion, further investigation needs to be conducted in animal model to confirm the non-genotoxicity activities of FDAE.