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1
Fraser, L., Ł. Zasiadczyk, and C. S. Pareek. "Effects of boar variability on comet-detected sperm-DNA damage following cryopreservation." Animal Production Science 58, no. 2 (2018): 252. http://dx.doi.org/10.1071/an16274.
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Assessment of sperm-DNA integrity is a crucial issue in male fertility. In the present study, parameters derived from the image analysis of comets after single-cell gel electrophoresis were used to analyse the types of DNA damage of frozen–thawed boar spermatozoa. Semen, frozen in a cryoprotectant-free extender or in cryoprotectant-based extenders, was analysed for DNA fragmentation and with the following comet tail measures: percentage DNA in comet tail, comet tail length and olive tail moment. The percentages of sperm DNA damage in the comet tails were classified as Type 0 (no DNA damage), Type I (very low DNA damage), Type II (light DNA damage), Type III (medium DNA damage) and Type IV (heavy DNA damage). Sperm motility characteristics and membrane integrity were assessed in the pre-freeze and frozen–thawed semen samples. Assessment of sperm DNA fragmentation and comet tail measures showed marked inter-boar variability following cryopreservation. However, consistent differences among the boars, with respect to cryo-induced sperm DNA damage, were detected by the comet tail length and olive tail moment. Besides Type IV, all types of DNA damage were detected in the cryoprotectant-based extenders. It was found that the frequency of Type II and Type III of DNA damage of frozen–thawed spermatozoa was significantly greater in the cryoprotectant-based and cryoprotectant-free extenders respectively. Deterioration in the quality of the sperm DNA integrity was concomitant with a marked decline in sperm motility characteristics, reduced plasma membrane integrity and higher lipid peroxidation and aspartate aminotransferase activity after cryopreservation. It can be suggested that the comet-assay parameters, coupled with routine laboratory tests, are useful to improve the sperm evaluations of post-thaw quality of semen from individual boars and would offer more comprehensive information for a better understanding of the degree of cryo-induced sperm-DNA damage.
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Mohsin Bhat, Towseef, M. Y. K. Ansari, Sana Choudhary, Rumana Aslam, and Alka. "Synergistic Cytotoxic Stress and DNA Damage in Clover (Trifolium repens) Exposed to Heavy Metal Soil from Automobile Refining Shops in Kashmir-Himalaya." ISRN Toxicology 2011 (January 5, 2011): 1–7. http://dx.doi.org/10.5402/2011/109092.
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Coexposure to heavy metals occurs in many occupational settings, such as automobile refining shops, pigment, and batteries production. Heavy metals around automobile refining shops were tested for their ability to induce synergistic cytogenetic effects in Trifolium repens L. by using the chromosomal aberrasions (CAs), micronucleus (MN) and comet assay. A significant increase in micronucleus (MN), chromosomal abrations (CAs), percentage of nuclei with comet tails (NCTs), the relative comet tail length (CTL), comet tail DNA (CT, DNA), and tail moment (TM) were observed with increased concentration of three heavy metals, like Cd, Pb, Hg. The present result indicate that exposure of T. repens to soils contaminated by heavy metals around automobile refining shops shows clastogenicity, cytotoxicity, and DNA damage at higher concentrations.
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Han, Jun Song, Xin Lu Lv, Ya Li Gao, Xiang Gao, and De Qi Xiong. "Analysis of DNA Damages of Gonadal Cells of Hemicentrotus pulcherrimus in Petroleum Hydrocarbons." Applied Mechanics and Materials 522-524 (February 2014): 251–56. http://dx.doi.org/10.4028/www.scientific.net/amm.522-524.251.
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The single cell gel electrophoresis (SCGE) is a rapid and sensitive procedure for measuring strand breaks in DNA. In the present study, sea urchin (Hemicentrotus pulcherrimus) was chosen as the test organism and SCGE was applied to assess DNA damage of its gonadal cells exposed to petroleum hydrocarbon. The gonadal cells of sea urchin had been seriously damaged above 50 mg/L of Water Accommodated Fractions (WAFs), whileas no damages occurred in the lower concentrations. There were good linear relationships between exposure days and DNA damage rate, percentage of DNA in the comet tail (%TDNA) as well as comet tail length (TL).
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Ahbab, M. A., Ü. Ündeğer, N. Barlas, and N. Başaran. "In utero exposure to dicyclohexyl and di-n-hexyl phthalate possess genotoxic effects on testicular cells of male rats after birth in the comet and TUNEL assays." Human & Experimental Toxicology 33, no. 3 (July 8, 2013): 230–39. http://dx.doi.org/10.1177/0960327113494903.
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Phthalates are diester derivatives of phthalic acid widely used in many commercial applications. The aim of this study is therefore to evaluate possible genotoxicity of di- n-hexyl phthalate (DHP) and dicyclohexyl phthalate (DCHP) at different concentrations using single-cell gel electrophoresis (comet) and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labeling (TUNEL) assays in testes samples of male rat pups. DCHP and DHP in corn oil were administered to the pregnant rats by gavage at the doses of 0 (vehicle), 20, 100, and 500 mg kg−1 day−1 from gestational day 6 (GD6) to GD19. After delivery, male rats were allowed to grow until prepubertal, pubertal, and adulthood. At necropsy, the blood samples were collected from heart and were excised immediately. The apoptotic cells of prepubertal, pubertal, and adult testis were detected using TUNEL assay. The comet assay was performed on blood lymphocytes and testes samples of adult male rats. The comet assay results showed that tail length, tail intensity, olive tail moment (OTM), and percentage of DNA present in tail were higher when DHP content was increased. Judging from the values of OTM and percentage of DNA, DHP could significantly induce DNA breakage at doses of 100 and 500 mg kg−1 day−1 compared with the control group. An increase in TUNEL-positive cells of prepubertal, pubertal, and adult testicular cells was observed in the treated groups. In conclusion, prenatal exposure to DHP and DCHP may possess genotoxic risk to testicular cells of rats at all stages of development, even at adulthood.
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Wendisch, M., G. Wunderlich, R. Freudenberg, J. Kotzerke, and R. Runge. "DNA damage in lymphocytes after irradiation with 211At and 188Re." Nuklearmedizin 48, no. 06 (2009): 221–26. http://dx.doi.org/10.3413/nukmed-0262.
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Summary Aim: Ionising radiation produces many types of DNA lesions of different complexity. High linear energy transfer (LET) types of radiation are biological more effective than low LET radiation. In the present work we applied the single cell gel electrophoreses (comet assay) to study the induction of initial DNA damage, efficiency of repair and residual DNA damage in lymphocytes after treatment with 211At and 188Re. Methods: Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood of healthy donors and irradiated with 211At and 188Re at different doses. The comet assay was performed under alkaline and neutral conditions in order to detect the initial DNA damage and its repair. The measure of damage was % tail DNA (percentage of DNA in the tail). Results: After treatment of cells with 188Re the initial DNA damage (% tail DNA) detected with the alkaline comet assay was higher than the damage measured for 211At. The neutral comet assay estimated higher tail intensities for 211At in contrast to 188Re. Compared with the complete repair (10%) after irradiation with 188Re, the radiotoxicity of alpha particles indicated reduced rejoining of DNA strand breaks (60–80% residual damage). Rejoining of DNA damage measured by the neutral comet method detected about 70% unrepaired strand breaks for 211At and 188Re. Conclusions: There are major differences between the repair of strand breaks caused by 188Re and 211At detected by the alkaline comet assay. The DNA-damage induced by the high LET Emitter 211At remains nearly unrepaired detected by both alkaline and neutral comet assay. Represented data following irradiation of lymphocytes with alpha and beta particles demonstrated higher biological effectiveness of 211At by factors of 2.0–2.5.
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Ambreen, Faiza, and Muhammad Javed. "Nuclear Damage in Peripheral Erythrocytes of Cyprinus Carpio Exposed to Binary Mixture of Pesticides." Journal of Zoo Biology 2, no. 1 (December 22, 2019): 39–47. http://dx.doi.org/10.33687/zoobiol.002.01.1506.
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The present study was undertaken to examine the DNA damage in peripheral blood erythrocytes of Cyprinus carpio under the binary exposure of bifenthrin and chlorpyrifos by using single cell gel electrophoresis (SCGE). Limited efforts have been made to study the genotoxic effect for long duration period. Therefore, the present investigation was aimed to assess the genotoxicity of pesticide mixture to the freshwater carp, Cyprinus carpio at sub-lethal concentration exposure (33% LC50). At first 96-hr LC50 value of pesticide, the mixture was determined for Cyprinus carpio in a static system and then sub-lethal concentration was calculated and fish was exposed to this sub-lethal concentration of the mixture in glass aquaria for 70 days (five fortnights) at constant laboratory conditions. Peripheral blood erythrocytes were taken on a fortnightly basis for the time-dependent DNA damage assessment in-terms of percentage of damaged cells, genetic damage index and a cumulative tail length of comets. Concentration-dependent increase in the percentage of DNA damaged cells were observed up to a 4th fortnight, followed by a slight decrease in the 5th fortnight. Similarly, statistically significant time-dependent DNA damage was observed in terms of percentage of damaged cells, genetic damage index and a cumulative tail length of comets in treated fish (at 33% of LC50) as compared to control groups. The results supported the use of SCGE for evaluating the toxicity of pollutants which may be used as part of environmental monitoring programs.
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Jaenisch, Rodrigo B., Giuseppe P. Stefani, Camila Durante, Chalyne Chechi, Vítor S. Hentschke, Douglas D. Rossato, Anelise Sonza, Cláudia R. Rhoden, and Pedro Dal Lago. "Respiratory muscle training decreases diaphragm DNA damage in rats with heart failure." Canadian Journal of Physiology and Pharmacology 96, no. 3 (March 2018): 221–26. http://dx.doi.org/10.1139/cjpp-2017-0069.
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Respiratory muscle training (RMT) promotes beneficial effects on respiratory mechanics, heart and lung morphological changes, and hemodynamic variables in rats with heart failure (HF). However, the relation between RMT effects and diaphragm oxidative stress remains unclear. Therefore, the aim of this study was to evaluate the RMT effects on diaphragm DNA damage in HF rats. Wistar rats were allocated into 4 groups: sedentary sham (Sed-Sham, n = 8), trained sham (RMT-Sham, n = 8), sedentary HF (Sed-HF, n = 8), and trained HF (RMT-HF, n = 8). The animals underwent a RMT protocol (30 min/day, 5 days/week for 6 weeks), whereas sedentary animals did not exercise. Groups were compared by a two-way ANOVA and Tukey’s post hoc tests. In rats with HF, RMT promoted reduction in pulmonary congestion (p < 0.0001) and left ventricular end diastolic pressure (p < 0.0001). Moreover, RMT produced a decrease in the diaphragm DNA damage in HF rats. This was demonstrated through the reduction in the percentage of tail DNA (p < 0.0001), tail moment (p < 0.01), and Olive tail moment (p < 0.001). These findings showed that a 6-week RMT protocol in rats with HF promoted an improvement in hemodynamic function and reduces diaphragm DNA damage.
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Joshi, Mayur S., Yogendra Verma, Anil K. Gautam, Vijay K. Shivgotra, Girish Parmar, and Sunil Kumar. "Assessment of Genetic Damage Among Chewers of Mixture Containing Mainly Areca Nut and Tobacco." Asia Pacific Journal of Public Health 23, no. 6 (September 13, 2011): 852–60. http://dx.doi.org/10.1177/1010539511419838.
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Chewing mixture containing areca nut and tobacco is believed to be associated with oral cancer. Habit of chewing such mixture is prevalent among South Asian countries. This study aimed to evaluate the genotoxic effect of areca nut and tobacco on human lymphocytes. Peripheral blood from 107 subjects (nonchewers, 48; chewers, 59, including 20 subjects with oral submucous fibrosis [OSMF]) analyzed by cytokinesis-block micronucleus (CBMN) and alkaline comet assay. Nuclear anomalies, namely, binucleated cells with micronuclei (BN MN), total MN, nucleoplasmic bridge, and nuclear buds were higher in chewers whereas elevation in BN MN and total MN were significant among subjects with OSMF than nonchewers. DNA damage assessed by comet assay showed increased percentage of Tail DNA, Tail moment, and Olive tail moment among chewers as well as OSMF subjects. Significant positive correlation was observed between induction of CBMN and consumption of quids per day ( r = .280, P = .033). Results suggested cytotoxic and genotoxic potential of mixture containing areca nut and tobacco.
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Buchynska, L. G., O. V. Brieieva, N. P. Iurchenko, V. V. Protsenko, and S. V. Nespryadko. "DNA DAMAGE IN TUMOR CELLS AND PERIPHERAL BLOOD LYMPHOCYTES OF ENDOMETRIAL CANCER PATIENTS ASSESSED BY THE COMET ASSAY." Experimental Oncology 39, no. 4 (December 22, 2017): 299–303. http://dx.doi.org/10.31768/2312-8852.2017.39(4):299-303.
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To date, genome instability is considered to be a common feature not only of tumor cells, but also of non-malignant cells of cancer patients, including peripheral blood lymphocytes (PBLs). The issue of the association between genome instability in tumor cells and PBLs, as well as of its relationship with tumor progression remains poorly understood. Aim: To evaluate the level DNA damage in tumor cells and PBLs of endometrial cancer (EC) patients with regard to clinical and morphological characteristics of the patients. Materials and Methods: DNA damage was assessed in 106 PBLs samples and 42 samples of tumor cell suspension from EC patients by comet assay. PBLs from 30 healthy women were used as control. The level of DNA damage was expressed as the percentage of DNA in the comet tails (% tail DNA). Results: It was revealed that the amount of DNA damage in PBLs of EC patients was 2.2 times higher in comparison with that of healthy donors (8.3 ± 0.7 and 3.7 ± 0.4% tail DNA, respectively) (p < 0.05). In this study, no association between the levels of DNA damage in endometrial tumor cells and PBLs was observed (r = 0.11; p > 0.05). The amounts of DNA damage both in tumor cells and PBLs were not related to the degree of tumor differentiation as well as the depth of myometrial invasion, but depended on the body mass index (BMI) of EC patients: high level of lesions was observed in patients with elevated BMI values. Furthermore, the level of DNA damage in tumor cells was associated to familial aggregation of cancer and was significantly higher in endometrial cells from patients with family history of cancer vs that from EC patients with sporadic tumors (32.3 ± 2.9 and 22.8 ± 1.8% tail DNA, respectively) (p < 0.05). It was also found that for women who had high level of DNA damage in PBLs, the risk of EC was greater (odds ratio value of 3.5) compared to those with low level of such lesions. Conclusion: Genome instability that appears as an increased level of DNA damage in tumor cells and PBLs of EC patients is associated with BMI and family history of cancer and can reflect a predisposition to cancer.
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Chen, Yan, Xiaodong Liu, Christopher H. K. Cheng, Tony Gin, Kate Leslie, Paul Myles, and Matthew T. V. Chan. "Leukocyte DNA Damage and Wound Infection after Nitrous Oxide Administration." Anesthesiology 118, no. 6 (June 1, 2013): 1322–31. http://dx.doi.org/10.1097/aln.0b013e31829107b8.
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Abstract Background: Nitrous oxide inactivates methionine synthase and may lead to DNA damage and wound infection. By using single-cell gel electrophoresis (comet assay), the authors determined the effect of nitrous oxide on DNA damage in circulating leukocytes. Methods: In this double-blind, randomized controlled trial, 91 patients undergoing major colorectal surgery were randomized to receive 70% nitrous oxide (n = 31) or nitrous oxide-free anesthesia using 30 (n = 30) or 80% (n = 30) oxygen. Venous blood was collected before and 24 h after surgery. The primary outcome was extent of DNA damage, quantified as the percentage of DNA staining intensity in the comet tail using digital fluorescence microscopy. Incidence of postoperative wound infection was also recorded. Results: Nitrous oxide exposure was associated with a two-fold increase in the percentage of DNA intensity in tail (P = 0.0003), but not in the 30 (P = 0.181) or 80% oxygen groups (P = 0.419). There was a positive correlation between the duration of nitrous oxide exposure and extent of DNA damage, r = 0.33, P = 0.029. However, no correlation was observed in nitrous oxide-free patients. The proportions of postoperative wound infection, using the Centers for Disease Control and Prevention criteria, were 19.4% (6 of 31) in the 70% nitrous oxide group and 6.7% (2 of 30) in both the 30 and 80% oxygen groups, P = 0.21. An increase in DNA damage was associated with a higher risk of wound infection, adjusted odds ratio (95% CIs): 1.19 (1.07–1.34), P = 0.003. Conclusions: Nitrous oxide increased DNA damage compared with nitrous oxide-free anesthesia and was associated with postoperative wound infection.
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Garaj-Vrhovac, Verica, and Goran Gajski. "Evaluation of the Cytogenetic Status of Human Lymphocytes After Exposure to a High Concentration of Bee Venom In Vitro." Archives of Industrial Hygiene and Toxicology 60, no. 1 (March 1, 2009): 27–34. http://dx.doi.org/10.2478/10004-1254-60-2009-1896.
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Evaluation of the Cytogenetic Status of Human Lymphocytes After Exposure to a High Concentration of Bee Venom In VitroSeveral studies have reported radioprotective, antimutagenic, anti-inflammatory, antinociceptive, and anticancer effects of bee venom both in the cell and the whole organism. The aim of this study was to assess the effects of a single high dose of 100 μg mL-1 of whole bee venom in human lymphocytes in vitro over a variety of time spans (from 10 min to 24 h). After the treatment, we used the comet assay and micronucleus test to see the effect of bee venom on the cell. The comet assay confirmed that the venom damaged the DNA molecule. Tail length, tail intensity, tail moment showed a significant increase (P<0.05). The percentage of long-tailed nuclei (LTN) with the tail length exceeding the 95th percentile also increased in a time-dependent manner. The micronucleus parameters (number of micronuclei, nucleoplasmic bridges, and nuclear buds) also showed a significant time-dependent increase (P<0.05). This research indicates that high concentrations of bee venom can lead to cellular instability. Further research is needed to understand the mechanism of action of bee venom and its components in human cells and to see if this natural product may find application in medicine.
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Ramadhani, Dwi, Devita Tetriana, and Viria Agesti Suvifan. "OPTIMALISASI TES KOMET UNTUK PENENTUAN TINGKAT KERUSAKAN PADA DNA AKIBAT PAPARAN RADIASI." Jurnal Sains dan Teknologi Nuklir Indonesia 17, no. 1 (March 18, 2016): 37. http://dx.doi.org/10.17146/jstni.2016.17.1.2405.
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Tes komet dapat digunakan untuk mengukur tingkat kerusakan asam deoksiribonukleat (DNA) pada sel limfosit darah tepi akibat paparan radiasi. Aspek yang harus diperhatikan saat melakukan tes komet antara lain adalah konsentrasi agarose yang digunakan, waktu inkubasi pada alkali, kondisi elektroforesis (waktu, temperatur serta gradien voltase yang digunakan), serta parameter yang digunakan dalam analisis. Parameter yang sangat disarankan dalam menganalisis citra komet adalah persentase DNA ekor (% DNA ekor). Persentase DNA ekor dapat dikonversi menjadi frekuensi lesion per 106 pasangan basa (bp) DNA dengan menggunakan kurva yang menggambarkan hubungan antara dosis radiasi pengion dengan besarnya % DNA ekor. Untuk mendapatkan hasil analisis tes komet yang akurat perlu dilakukan pembuatan kurva kalibrasi yang menggambarkan hubungan antara dosis radiasi pengion dengan besarnya % DNA ekor. Analisis citra komet sebaiknya dilakukan dengan menggunakan perangkat lunak pengolahan citra sehingga dapat meningkatkan akurasi dan presisi serta mengurangi subjektivitas dalam menganalisis citra komet. OPTIMIZATION ON COMET ASSAY FOR ASSESSMENT OF DNA DAMAGE BECAUSE RADIATION EXPOSURE.Comet assay can be used to measure deoxyribonucleic acid (DNA) damage level caused by natural radiation exposure in peripheral blood lymphocytes. The principle of the comet assay is based on the amount of denatured DNA fragments that migrated out of the cell nucleus during electrophoresis. There are several aspects that must be concerned when doing the comet assay. For example the agarose concentration, duration of alkaline incubation, electrophoresis conditions (time, temperature, and voltage gradient), and the measurement parameters that used in analyze the comet. Percentage of DNA in the comet tail (% tail DNA) is strongly recommended as a parameter when analyze the comet because it can be converted to lesions per 106 base pairs (bp) using calibration curve that show relationship between the dose of ionizing radiation and % tail DNA. To obtain an accurate result, the calibration curve must be made and comet should be analyzing using image processing analysis software since it can be increase the precision and reduce the subjectivity of the measurement process.
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Atha, Donald H., Omobola Cole, Breece Clancy, Alessandro Tona, and Vytas Reipa. "Cellular Reference Materials for DNA Damage Using Electrochemical Oxidation." Journal of Nucleic Acids 2020 (January 30, 2020): 1–9. http://dx.doi.org/10.1155/2020/2928104.
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Reference materials are needed to quantify the level of DNA damage in cells, to assess sources of measurement variability and to compare results from different laboratories. The comet assay (single cell gel electrophoresis) is a widely used method to determine DNA damage in the form of strand breaks. Here we examine the use of electrochemical oxidation to produce DNA damage in cultured mammalian cells and quantify its percentage using the comet assay. Chinese hamster ovary (CHO) cells were grown on an indium tin oxide electrode surface and exposed 12 h to electrochemical potentials ranging from 0.5 V to 1.5 V (vs Ag/AgCl). The resulting cells were harvested and analyzed by comet and a cell viability assay. We observed a linear increase in the percentage (DNA in tail) of strand breaks along with a loss of cell viability with increasing oxidation potential value. The results indicate that electrochemically induced DNA damage can be produced in mammalian cells under well-controlled conditions and could be considered in making a cellular reference material for the comet assay.
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Otterstrom, Jason, Alvaro Castells-Garcia, Chiara Vicario, Pablo A. Gomez-Garcia, Maria Pia Cosma, and Melike Lakadamyali. "Super-resolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo." Nucleic Acids Research 47, no. 16 (July 9, 2019): 8470–84. http://dx.doi.org/10.1093/nar/gkz593.
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Abstract Chromatin organization is crucial for regulating gene expression. Previously, we showed that nucleosomes form groups, termed clutches. Clutch size correlated with the pluripotency grade of mouse embryonic stem cells and human induced pluripotent stem cells. Recently, it was also shown that regions of the chromatin containing activating epigenetic marks were composed of small and dispersed chromatin nanodomains with lower DNA density compared to the larger silenced domains. Overall, these results suggest that clutch size may regulate DNA packing density and gene activity. To directly test this model, we carried out 3D, two-color super-resolution microscopy of histones and DNA with and without increased histone tail acetylation. Our results showed that lower percentage of DNA was associated with nucleosome clutches in hyperacetylated cells. We further showed that the radius and compaction level of clutch-associated DNA decreased in hyperacetylated cells, especially in regions containing several neighboring clutches. Importantly, this change was independent of clutch size but dependent on the acetylation state of the clutch. Our results directly link the epigenetic state of nucleosome clutches to their DNA packing density. Our results further provide in vivo support to previous in vitro models that showed a disruption of nucleosome-DNA interactions upon hyperacetylation.
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Yanuaryska, Ryna Dwi, Afit Aditya Atmoko, Isti Rahayu Suryani, and Ratna Shantiningsih. "Viability and DNA Damage of Buccal Mucosa Cells in Patients Exposed to Panoramic X-ray." Archives of Orofacial Sciences 16, Supp. 1 (September 22, 2021): 43–49. http://dx.doi.org/10.21315/aos2021.16.s1.8.
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Panoramic X-ray is well known to cause DNA damage and induces cellular death. The aim of the present study was to evaluate the cytotoxicity of radiation exposure from panoramic radiography on human buccal mucosa cells by assessing the cell viability using the simple-trypan blue exclusion test. The genotoxicity effect was evaluated by assessing comet assay score. This research included a total of 20 healthy patients who had panoramic radiography for a routine dental examination. Buccal mucosa cells were collected from all participants before X-ray exposure and at 30 min or 24 h after exposure in Groups 1 and 2, respectively, and subjected to a comet assay and trypan blue exclusion test to assess cell viability and DNA damage. Cell viability was calculated as the ratio of live (translucent) to total counted cells. Comet assay output images were analysed using OpenComet software and a visual score by measuring the percentages of tail DNA and summing the visual score, respectively. A statistically significant (p < 0.05) reduce in cell viability was observed at 30 min after exposure, furthermore there is no more reduction after 24 h. Both comet assay measurements showed a significant (p < 0.05) increase in the percentage of tail DNA and visual score at 30 min after exposure, then tend to decrease after 24 h of exposure, although it was not significant (p > 0.05). The results showed that panoramic radiography interfered cell viability and induced DNA damage in buccal mucosa cells within 30 min after exposure, but these effects were ceased after 24 h.
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Alotaibi, Saqer S. "Salinity Stress Alerts Genome Stability and Genotoxicity of Ocimum basilicum Cultivars." International Journal of Agriculture and Biology 25, no. 06 (June 1, 2021): 1311–20. http://dx.doi.org/10.17957/ijab/15.1793.
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Salinity is an important abiotic stress that greatly influences growth, secondary product content and genotoxicity in plants. Ocimum basilicum L. (family Lamiaceae) produces a volatile oil, which is used in many pharmaceutical industries, but the oil biosynthesis is affected by salt stress. The aim of this study was to evaluate the effect of salinity stress on genome stability and genotoxicity of three basil cultivars (Gigante, Gralissimum and Verde) using comet assays to study the genotoxic impact of salinity stress (0, 50, 100 and 200 mM NaCl) and a semi-quantitative real time polymerase chain reaction to study terpene gene expression. Both analyses revealed considerable genetic effects of salinity stress on the O. basilicum genome, detected by a regular increase in DNA damage and by diversity in the transcript levels of terpene biosynthesis and inhibitor genes. Our findings confirmed that basil plants were affected by NaCl salinity stress and that exposure to 200 mM NaCl resulted in significant DNA damage in the form of tail moment, DNA tail percentage and tail length. The accumulation of linalool synthase enzyme (LS) and hexokinase synthase (HK) gene transcripts was greatly increased in response to salinity, whereas FPPS, GPPS and DXR gene transcription was suppressed in all three basil cultivars. © 2021 Friends Science Publishers
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Thomas, Emilia G., Maja Šrut, Anamaria Štambuk, Göran I. V. Klobučar, Alfred Seitz, and Eva Maria Griebeler. "Effects of Freshwater Pollution on the Genetics of Zebra Mussels (Dreissena polymorpha) at the Molecular and Population Level." BioMed Research International 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/795481.
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Revealing long-term effects of contaminants on the genetic structure of organisms inhabiting polluted environments should encompass analyses at the population, molecular, and cellular level. Following this concept, we studied the genetic constitution of zebra mussel populations from a polluted (Dp) and reference sites (Cl) at the river Drava, Croatia, and applied microsatellite and DNA damage analyses (Comet assay, micronucleus test (MNT)). Additionally, mussels from both populations were exposed to polluted wastewater in the laboratory for three days, and DNA damage was analyzed to evaluate acclimatization and genetic adaptation of the investigated populations to the polluted environment. The two populations differed in their genetic constitution. Microsatellite analysis suggested that Dp had undergone a genetic bottleneck. Comet assay did not indicate any difference in DNA damage between the two populations, but MNT revealed that Dp had an increased percentage of micronuclei in hemocytes in comparison to Cl. The laboratory experiment revealed that Dp had a lower percentage of tail DNA and a higher percentage of micronuclei than Cl. These differences between populations were possibly caused by an overall decreased fitness of Dp due to genetic drift and by an enhanced DNA repair mechanism due to acclimatization to pollution in the source habitat.
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Yagubova, Svetlana, Aliy Zhanataev, Rita Ostrovskaya, Еlena Anisina, Тatiana Gudasheva, Аndrey Durnev, and Sergey Seredenin. "Dimeric NGF Mimetic Attenuates Hyperglycaemia and DNA Damage in Mice with Streptozotocin-Induced Early-Stage Diabetes." Endocrine, Metabolic & Immune Disorders - Drug Targets 20, no. 3 (March 24, 2020): 453–63. http://dx.doi.org/10.2174/1871530319666190806115623.
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Background: NGF deficiency is one of the reasons for reduced β-cells survival in diabetes. Our previous experiments revealed the ability of low-weight NGF mimetic, GK-2, to reduce hyperglycaemia in a model of advanced diabetes. The increase in DNA damage in advanced diabetes was repeatedly reported, while there were no data about DNA damage in the initial diabetes. Aim: The study aimed to establish whether DNA damage occurs in initial diabetes and whether GK-2 is able to overcome the damage. Methods: The early-stage diabetes was modelled in Balb/c mice by streptozotocin (STZ) (130 mg/kg, i.p.). GK-2 was administered at a dose of 0.5 mg/kg, i.p., subchronically. The evaluation of DNA damage was performed using the alkaline comet assay; the percentage of DNA in the tail (%TDNA) and the percentage of the atypical DNA comets (“ghost cells”) were determined. Results: STZ at this subthreshold dose produced a slight increase in glycemia and MDA. Meanwhile, pronounced DNA damage was observed, concerning mostly the percentage of “ghost cells” in the pancreas, the liver and kidneys. GK-2 attenuated the degree of hyperglycaemia and reduced the % of “ghost cells” and %TDNA in all the organs examined; this effect continued after discontinuation of the therapy. Conclusion: Early-stage diabetes is accompanied by DNA damage, manifested by the increase of “ghost cells” percentage. The severity of these changes significantly exceeds the degree of hyperglycaemia and MDA accumulation. GK-2 exerts an antihyperglycaemic effect and attenuates the degree of DNA damage. Our results indicate that the comet assay is a highly informative method for search of antidiabetic medicines.
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García, Omar, Ivonne Romero, Jorge Ernesto González, Damaris L. Moreno, Elizabeth Cuétara, Yesenia Rivero, Ariadne Gutiérrez, et al. "Visual estimation of the percentage of DNA in the tail in the comet assay: Evaluation of different approaches in an intercomparison exercise." Mutation Research/Genetic Toxicology and Environmental Mutagenesis 720, no. 1-2 (February 2011): 14–21. http://dx.doi.org/10.1016/j.mrgentox.2010.11.011.
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Cho, Su-Youn, Wi-Young So, and Hee-Tae Roh. "Effect of C242T Polymorphism in the Gene Encoding the NAD(P)H Oxidase p22phox Subunit and Aerobic Fitness Levels on Redox State Biomarkers and DNA Damage Responses to Exhaustive Exercise: A Randomized Trial." International Journal of Environmental Research and Public Health 17, no. 12 (June 12, 2020): 4215. http://dx.doi.org/10.3390/ijerph17124215.
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NAD(P)H oxidases (NOXs) constitute a principal source of cellular reactive oxygen species (ROS) and contribute to exercise-induced ROS production in the skeletal muscle. Here, we aimed to investigate the effect of single-bout exhaustive exercise on redox state biomarkers and oxidative DNA damage based on the C242T polymorphism in the gene encoding NOXs subunit p22phox (CYBA) and aerobic fitness levels. We enrolled 220 healthy adults in their 20s (men, n = 110; women, n = 110), who were divided into CC genotype and T allele groups through the analysis of the CYBA C242T polymorphism. Furthermore, maximum oxygen uptake (VO2max) was evaluated to divide subjects into high fitness (HF; 70th percentile for aerobic fitness) and mid-range fitness (MF; 40–60th percentile for aerobic fitness) groups, with a total of 32 subjects assigned to four groups (eight subjects per group): CC genotype and HF group (CC + HF), CC genotype and MF group (CC + MF), T allele and HF group (T + HF), and T allele and MF group (T + MF). All subjects performed treadmill running exercise at 85% of VO2max until exhaustion. Plasma lactate, malondialdehyde (MDA), superoxide dismutase (SOD), and lymphocyte DNA damage (tail DNA percentage [TD], tail length [TL], and the tail moment [TM]) were measured in the blood samples obtained immediately before (IBE), immediately after (IAE), and 30 min after exercise (30 MAE). Plasma lactate levels, SOD activities, and lymphocyte DNA damage markers (TD, TL, and TM) were significantly increased at IAE than that at IBE and significantly decreased at 30 MAE (p < 0.05). All groups displayed increased plasma MDA levels at IAE rather than at IBE, with CC + MF being significantly higher than T + HF (p < 0.05); only the CC + HF and T + HF groups exhibited a significant reduction at 30 MAE (p < 0.05). Moreover, TL at IAE was significantly higher in the CC + MF group than in the T + HF group (p < 0.05), and significantly higher in the CC + MF and CC + HF groups than in the T + HF group at 30 MAE (p < 0.05). TM was significantly higher in the T + MF than in the T + HF group at IAE (p < 0.05) and that of CC + MF was significantly higher than CC + HF and T + HF values at IAE and 30 MAE (p < 0.05). These results suggest that single-bout exhaustive exercise could induce peripheral fatigue and the accumulation of temporary redox imbalance and oxidative DNA damage. Moreover, high aerobic fitness levels combined with the T allele may protect against exercise-induced redox imbalance and DNA damage.
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Mathialagan, Ramya Dewi, Zariyantey Abd Hamid, Qing Min Ng, Nor Fadilah Rajab, Salwati Shuib, and Siti Razila Binti Abdul Razak. "Bone Marrow Oxidative Stress and Acquired Lineage-Specific Genotoxicity in Hematopoietic Stem/Progenitor Cells Exposed to 1,4-Benzoquinone." International Journal of Environmental Research and Public Health 17, no. 16 (August 13, 2020): 5865. http://dx.doi.org/10.3390/ijerph17165865.
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Hematopoietic stem/progenitor cells (HSPCs) are susceptible to benzene-induced genotoxicity. However, little is known about the mechanism of DNA damage response affecting lineage-committed progenitors for myeloid, erythroid, and lymphoid. Here, we investigated the genotoxicity of a benzene metabolite, 1,4-benzoquinone (1,4-BQ), in HSPCs using oxidative stress and lineage-directed approaches. Mouse bone marrow cells (BMCs) were exposed to 1,4-BQ (1.25–12 μM) for 24 h, followed by oxidative stress and genotoxicity assessments. Then, the genotoxicity of 1,4-BQ in lineage-committed progenitors was evaluated using colony forming cell assay following 7–14 days of culture. 1,4-BQ exposure causes significant decreases (p < 0.05) in glutathione level and superoxide dismutase activity, along with significant increases (p < 0.05) in levels of malondialdehyde and protein carbonyls. 1,4-BQ exposure induces DNA damage in BMCs by significantly (p < 0.05) increased percentages of DNA in tail at 7 and 12 μM and tail moment at 12 μM. We found crucial differences in genotoxic susceptibility based on percentages of DNA in tail between lineage-committed progenitors. Myeloid and pre-B lymphoid progenitors appeared to acquire significant DNA damage as compared with the control starting from a low concentration of 1,4-BQ exposure (2.5 µM). In contrast, the erythroid progenitor showed significant damage as compared with the control starting at 5 µM 1,4-BQ. Meanwhile, a significant (p < 0.05) increase in tail moment was only notable at 7 µM and 12 µM 1,4-BQ exposure for all progenitors. Benzene could mediate hematological disorders by promoting bone marrow oxidative stress and lineage-specific genotoxicity targeting HSPCs.
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Shetty, Vinaya, NJ Shetty, SR Ananthanarayana, SK Jha, and RC Chaubey. "Evaluation of gamma radiation-induced DNA damage in Aedes aegypti using the comet assay." Toxicology and Industrial Health 33, no. 12 (October 9, 2017): 930–37. http://dx.doi.org/10.1177/0748233717733599.
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The study was undertaken to evaluate gamma radiation-induced DNA damage in Aedes aegypti. The comet assay was employed to demonstrate the extent of DNA damage produced in adult male A. aegypti exposed to seven different doses of gamma radiation, ranging from 1 Gy to 50 Gy. DNA damage was measured as the percentage of comet tail DNA. A significant linear increase in DNA damage was observed in all samples; the extent of damage being proportional to the dose of gamma radiation the organism received, except in those treated with 1 Gy. The highest amount of DNA damage was noticed at 1 h postirradiation, which decreased gradually with time, that is, at 3, 6 and 12 h postirradiation. This may indicate repair of the damaged DNA and/or loss of heavily damaged cells as the postirradiation time increased. The comet assay serves as a sensitive and rapid technique to detect gamma radiation-induced DNA damage in A. aegypti. This could be used as a potential biomarker for environmental risk assessment.
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Gonzalez-Castro, Raul A., Fabio Amoroso-Sanches, JoAnne E. Stokes, James K. Graham, and Elaine M. Carnevale. "Localisation of phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (WBP2 N-terminal like) on equine spermatozoa and flow cytometry quantification of PLCZ1 and association with cleavage in vitro." Reproduction, Fertility and Development 31, no. 12 (2019): 1778. http://dx.doi.org/10.1071/rd19217.
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Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal piece of the tail. Anti-WBP2NL antibody identified two WBP2NL bands (~28 and ~32kDa) in the postacrosomal region, midpiece and principal piece of the tail. PLCZ1 and WBP2NL expression was positively correlated (P=0.04) in sperm heads. Flow cytometry evaluation of PLCZ1 revealed large variations in fluorescence intensity and the percentage of positively labelled spermatozoa among stallions. PLCZ1 expression was significantly higher in viable than non-viable spermatozoa, and DNA fragmentation was negatively correlated with PLCZ1 expression and the percentage of positively labelled spermatozoa (P<0.05). The use of equine sperm populations considered to have high versus low PLCZ1 content resulted in significantly higher cleavage rates after ICSI of bovine and equine oocytes, supporting the importance of PLCZ1 for oocyte activation.
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Iriti, M., S. Guarnieri, and F. Faoro. "Responsiveness of Lycopersicon pimpinellifolium to acute UV-C exposure: histo-cytochemistry of the injury and DNA damage." Acta Biochimica Polonica 54, no. 2 (May 23, 2007): 273–80. http://dx.doi.org/10.18388/abp.2007_3247.
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The in vivo and in vitro effects of UV-C (254 nm) exposure (0.039 watt . m(-2) . s for 2 h) of currant tomato (Lycopersicon pimpinellifolium), indigenous to Peru and Ecuador, were assayed. H(2)O(2) deposits, dead cells and DNA damage were localized, 12/24 h after irradiation, mainly in periveinal parenchyma of the 1st and 2nd order veins of the leaves, and before the appearance of visible symptoms, which occurred 48 h after irradiation. Cell death index was of 43.5 +/- 12% in exposed leaf tissues, 24 h after treatment. In currant tomato protoplasts, the percentage of viable cells dropped 1 h after UV-C irradiation from 97.42 +/- 2.1% to 43.38 +/- 4.2%. Afterwards, the protoplast viability progressively decreased to 40.16 +/- 7.25% at 2 h, to 38.31 +/- 6.9% at 4 h, and to 36.46 +/- 1.84% at 6 h after the exposure. The genotoxic impact of UV-C radiation on protoplasts was assessed with single cell gel electrophoresis (SCGE, or comet assay). UV-C treatment greatly enhanced DNA migration, with 75.37 +/- 3.7% of DNA in the tail versus 7.88 +/- 5.5% in the case of untreated nuclei. Oxidative stress by H(2)O(2) used as a positive control, induced a similar damage on non-irradiated protoplasts, with 71.59 +/- 5.5% of DNA in the tail, whereas oxidative stress imposed on UV-C irradiated protoplasts slightly increased the DNA damage (85.13 +/- 4.1%). According to these results, SCGE of protoplasts could be an alternative to nuclei extraction directly from leaf tissues.
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Dominic, Shini, and Padmaja V. "AMLODIPINE-INDUCED TESTICULAR DNA DAMAGE AND ITS MODIFICATION BY A HERBAL PREPARATION IN WISTAR ALBINO RATS." INDIAN DRUGS 58, no. 06 (August 17, 2021): 42–48. http://dx.doi.org/10.53879/id.58.06.11610.
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The present study originated from the reports of adverse effects on male reproductive potential caused by the dihydropyridine derivative type calcium channel blockers, which are widely used agents for different cardiovascular disorders. The study was conducted to verify whether the repeated administration of this type of calcium channel blockers can cause any testicular DNA damage and if so, whether the damage can be modified by the co-administration of a herbal protective agent. The study was conducted in male Wistar albino rats. The testicular DNA damage of different groups of rats was evaluated by comet assay taking percentage DNA in comet tail as the main parameter. It was found that oral administration of the widely used dihydropyridine type calcium channel blocker amlodipine produced a duration- dependent and significant (P<0.001) DNA damage in the testes of Wistar albino rats. The co- administration of the alcoholic extract of seeds of Asteracantha longifolia produced a dose dependent and significant ((P<0.001) protective effect against this amlodipine-induced testicular DNA damage.
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Dördü, Tuba C., Rüştü Hatipoğlu, Mehmet Topaktaş, and Erman S. İstifli. "In Vitro Genotoxicity and Molecular Docking Study of Ellagic Acid." Current Bioactive Compounds 16, no. 7 (October 28, 2020): 1072–82. http://dx.doi.org/10.2174/1573407215666191102130417.
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Background: Ellagic Acid (EA) is a polyphenolic compound that is classified in the natural antioxidants group. Polyphenolic compounds that exert antioxidant activity possess particular importance for scientists, food producers and consumers due to their positive effects on human health. However, despite considerable evidence that EA shows antigenotoxic activity by binding to DNA, there is no systematic genotoxicity study of this substance, which can covalently bind to DNA. This study aims to reveal the possible genotoxic activity of EA using widely accepted assays for the assessment of DNA clastogenic activity: sister chromatid exchange, chromosome aberration, micronucleus and comet assays as well as to predict the interactions among EA and DNA through molecular docking. Methods: Different assays were carried out to identify the clastogenic activity of EA on human lymphocyte DNA using Sister Chromatid Exchange (SCE), Chromosome Aberration (CA), Micronucleus (MN) and single-cell gel electrophoresis (SCGE/comet) assays. For this aim, human peripheral blood lymphocytes were treated with EA (60, 80 and 100 μg/ml) for 24 and 48 hrs in the SCE, CA and MN assays and for 1 hr in the comet assay. Furthermore, molecular docking experiments were also performed to calculate the binding energy of EA on human B-DNA structure (B-DNA dodecamer) as well as to predict noncovalent interactions among these macromolecules. Results: At the concentrations and treatment times (24- or 48-hr) tested, EA did not induce either SCE or Chromosome Aberrations (CAs) as compared to the negative and solvent controls. Although EA slightly increased the percentage of Micronucleated Binuclear (%MNBN) cells as well as the percentage of Micronucleus (%MN) in 24 or 48-hr treatment periods at all concentrations, this increase was not statistically significant as compared to both controls. The effect of EA on DNA replication (nuclear division) was determined by the Proliferation Index (PI), the Nuclear Division Index (NDI) and the Mitotic Index (MI). No statistically significant differences were observed in the PI or NDI in 24- or 48-hr treatment periods in human lymphocyte cultures treated with EA at various concentrations. EA generally had no significant effect on the MI, as observed with the PI and NDI. Discussion: Although the concentrations of 60 and 80 μg/mL at a 24-hr treatment period and the concentrations of 60 μg/mL and 100 μg/mL at 48-hr treatment period generally decreased the MI, those decreases were not statistically significant when compared to negative and solvent controls. Moreover, none of the concentrations of EA tested in this study were able to increase DNA damage determined by the tail DNA length, %DNA in tail and tail moment parameters in the comet assay. Although the amount of DNA damage in the comet assay decreased with increasing concentrations of EA, this decrease was not statistically significant as compared to both controls. However, molecular docking experiments interestingly showed that the binding free energy of EA with B-DNA was -7.84 kcal/mol-1, indicating a strong interaction between the two molecules. Conclusion : Although the findings of our study show that EA does not have genotoxic potential in human chromosomes, molecular docking experiments revealed strong hydrogen bonding between EA and B-DNA molecules. Therefore, it has been proposed that the prevailing information suggesting that the molecules that bind to DNA cause genotoxic effects should be reconsidered from a wider perspective.
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Aherne, S. Aisling, Joseph P. Kerry, and Nora M. O'Brien. "Effects of plant extracts on antioxidant status and oxidant-induced stress in Caco-2 cells." British Journal of Nutrition 97, no. 2 (February 2007): 321–28. http://dx.doi.org/10.1017/s0007114507250469.
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Experimental evidence suggests that most herbs and spices possess a wide range of biological and pharmacological activities that may protect tissues against O2-induced damage. The objectives of the present study were: first, to determine the effects of plant extracts on the viability, membrane integrity, antioxidant status and DNA integrity of Caco-2 cells and second, to investigate the cytoprotective and genoprotective effects of these plant extracts against oxidative stress in Caco-2 cells. The plant extracts examined were rosemary (Rosmarinus officinalis L.), oregano (Origanum vulgare L.), sage (Salvia officinalis L.) and echinacea (Echinacea purpurea L.). Cell membrane integrity was assessed by the lactate dehydrogenase release assay. Viability was determined by the neutral red uptake assay (NRUA) and the concentration of compound that resulted in 50 % cell death (IC50) was calculated. Antioxidant status of the cells was assessed by measuring GSH content, catalase activity and superoxide dismutase activity. To examine their cytoprotective and genoprotective effects, Caco-2 cells were pre-treated with each plant extract for 24 h followed by exposure to H2O2. DNA damage was assessed by the comet assay and cell injury was determined by the NRUA. Rosemary was the most toxic (IC50 123 μg/ml) and echinacea the least toxic (IC50 1421 μg/ml). Sage was the only plant extract to affect the antioxidant status of the cells by increasing GSH content. Sage, oregano and rosemary protected against H2O2-induced DNA damage (olive tail moment and percentage tail DNA), whereas protection against H2O2-induced cytotoxicity was afforded by sage only.
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Chakrabarti, Srijita, Danswrang Goyary, Sanjeev Karmakar, and Pronobesh Chattopadhyay. "Exploration of cytotoxic and genotoxic endpoints following sub-chronic oral exposure to titanium dioxide nanoparticles." Toxicology and Industrial Health 35, no. 9 (September 2019): 577–92. http://dx.doi.org/10.1177/0748233719879611.
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Health hazards of titanium dioxide nanoparticles (TiO2-NPs) have raised severe concerns because of the paucity of information regarding the toxic effects among the population. In the present research, the in vitro and in vivo cytotoxic potential of TiO2-NPs were evaluated using flow cytometric techniques. Further, in vitro and in vivo genotoxic endpoints were estimated by means of comet, micronucleus (MN), and chromosomal aberration (CA) assays. In vitro analysis was performed at the concentration range of 10–100 µg/mL using murine RAW 264.7 cells. In vivo experiments were conducted on Albino mice (M/F) by exposing them to 200 and 500 mg/kg TiO2-NPs for 90 days. Decreased percentage of cell viability with higher doses of TiO2-NPs was evident in both in vitro and in vivo flow cytometric analysis. Further, an impaired cell cycle (G0/G1, S, and G2/M) was reflected in the present investigation following the exposure to TiO2-NPs. Increased comet scores such as tail length, % DNA in tail, tail moment, and olive moment were also observed with the higher doses of TiO2-NPs in vitro and in vivo comet assays. Finally, the in vivo MN and CA assays revealed the formation of MN and chromosomal breakage following the exposure to TiO2-NPs.
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Gomes, M., A. Gonçalves, E. Rocha, R. Sá, A. Alves, J. Silva, A. Barros, M. L. Pereira, and M. Sousa. "Effect of in vitro exposure to lead chloride on semen quality and sperm DNA fragmentation." Zygote 23, no. 3 (February 13, 2014): 384–93. http://dx.doi.org/10.1017/s0967199413000671.
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SummaryExposure to lead may cause changes in the male reproductive system. We evaluated the effect of lead chloride (PbCl2) in vitro on semen quality from 31 individuals. Samples were incubated at room temperature for two exposure times (4 h and 8 h) and with two concentrations of PbCl2 (15 μg/ml or 30 μg/ml). Results showed that PbCl2 significantly inhibited rapid progressive motility and caused an increase in the percentage of tail anomalies in both times and concentrations assessed, as well as a decrease in vitality in the group exposed to 30 μg/ml PbCl2. A significant increase in immotile sperm was also observed between the group control and the groups submitted to lead. Total motility and DNA fragmentation also showed a significant decrease and increase, respectively, after 4 h of incubation in the group exposed to 30 μg/ml and in both groups after 8 h of incubation. In conclusion, PbCl2 affected sperm parameters and DNA integrity, which are essential for male fertility.
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Šobot, Ana Valenta, Dunja Drakulić, Gordana Joksić, Jadranka Miletić Vukajlović, Jasmina Savić, Jelena Potočnik, and Jelena Filipović Tričković. "Yellow gentian root extract provokes concentration- and time-dependent response in peripheral blood mononuclear cells." Archives of Industrial Hygiene and Toxicology 71, no. 4 (December 1, 2020): 320–28. http://dx.doi.org/10.2478/aiht-2020-71-3476.
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Abstract Yellow gentian (Gentiana lutea L.), a medicinal plant widely used in traditional medicine, displays multiple biological effects, ranging from beneficial to toxic. Since many promising applications have been reported so far, our aim was to evaluate its potential concentration- and time- dependent cytotoxic and genotoxic effects in vitro. To that end we exposed human peripheral blood mononuclear cells to 0.5, 1, and 2 mg/mL of yellow gentian root extract (YGRE) to determine its effects on oxidative stress parameters [pro/antioxidant balance (PAB) and lipid peroxidation], DNA damage (alkaline comet assay and chromosome aberrations), and cell viability (trypan blue exclusion test). Cell viability decreased with increasing concentrations and treatment duration. Only the lowest YGRE concentration (0.5 mg/mL) increased oxidative stress but produced minor DNA damage and cytotoxicity. At higher concentrations, redox parameters returned to near control values. The percentage of chromosome aberrations and percentage of DNA in the comet tail increased with increased YGRE concentration after 48 h and declined after 72 h of treatment. This points to the activation of DNA repair mechanism (homologous recombination), evidenced by the formation of chromosomal radial figures after 72 h of treatment with the highest YGRE concentration of 2 mg/mL. Our results suggest that YGRE, despite induction of cytotoxic and genotoxic effects, activates cell repair mechanisms that counter oxidative and DNA lesions and induce cell death in highly damaged cells. Therefore, observed protective effects of yellow gentian after longer exposure could be a result of activated repair and removal of cells with irreparable damage.
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Hamid, Asmah, Nor Fadilah Rajab, Tay Shu Shen, and Mohd Nazir Nasrom. "Cytotoxic and Genotoxic Effects of Zerumbone on WEHI 7.2 Wild Type Murine Thymoma Cells." Journal of Agricultural Science 9, no. 13 (December 24, 2017): 1. http://dx.doi.org/10.5539/jas.v9n13p1.
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Zerumbone (ZER), a sesquiterpene in the rhizomes of Zingiber zerumbet Smith, was shown to exhibit antiproliferative activities on various cancer cells. This study was carried out to determine the cytotoxic and genotoxic effects of ZER on WEHI 7.2 wild type murine thymoma cells through the employment of standard MTT assay, alkaline comet assay and flow cytometry Annexin V/PI. Results from the MTT assay demonstrated that ZER has a dose-dependent but not a time-dependent cytotoxic effect towards WEHI 7.2 wild type cells with IC50 values at 24, 48 and 72 hours were 3.02±0.20 µg/ml (13.832 µM), 2.73±0.13 µg/ml (12.503 µM) and 2.65±0.13 µg/ml (12.137 µM) respectively. Using IC10 and IC25 values obtained from the MTT assay, alkaline comet assay was carried out to detect DNA damage in ZER treated cells at three different time points (1/2 h, 1 h and 2 h). From the results, it was found that ZER induced significant DNA damage at all three time points for both concentrations (p < 0.05). Comparison of DNA damage levels at both concentrations suggested a concentration-dependent genotoxicity, as significantly higher values of tail DNA percentage and tail moment were obtained for cells treated with IC25 concentration (p < 0.05). Furthermore, to understand the mode of cell death induced by ZER, flow cytometry Annexin V/PI was performed and it was found that cytotoxicity was achieved primarily via apoptosis. Collectively, ZER is able to induce genotoxicity in treated cells which subsequently leads to cytotoxicity via apoptosis and these presented characteristics suggest the compound as a potential anticancer drug.
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Giovannelli, Lisa, Andrea Cozzi, Ilaria Guarnieri, Piero Dolara, and Flavio Moroni. "Comet Assay as a Novel Approach for Studying DNA Damage in Focal Cerebral Ischemia: Differential Effects of NMDA Receptor Antagonists and Poly(ADP-Ribose) Polymerase Inhibitors." Journal of Cerebral Blood Flow & Metabolism 22, no. 6 (June 2002): 697–704. http://dx.doi.org/10.1097/00004647-200206000-00008.
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The single-cell gel electrophoresis (comet assay) was used to evaluate the possibility of detecting single-strand breaks of brain DNA in the early phase of ischemia. Four hours after occlusion of the middle cerebral artery (MCAO) in rats, the percentage of DNA migrating into the comet tail (indicating the presence of breaks) increased from 11.4 ± 4.70 to 34.7 ± 9.2 (means ± SD) in the caudate and from 9.9 ± 4.3 to 42.8 ± 14.1 in the cortex. Interestingly, a subpopulation of cells exhibiting higher resistance to the ischemic insult was present in the caudate putamen, but not in the cortex. Administration of MK801, an N-methyl-d-aspartate (NMDA) glutamate receptor antagonist, (1 mg/kg subcutaneously, 10 minutes before MCAO), reduced the ischemia-induced DNA breaks and the infarct volume, suggesting that excessive stimulation of NMDA receptors contributes to the formation of both DNA damage and infarct volume. In contrast, DPQ, an inhibitor of poly(ADP-ribose) polymerase (PARP) (10 mg/kg intraperitoneally, 2 hours before and 1 hour after MCAO), reduced the infarct volume but not DNA damage, suggesting that the neuroprotective actions of PARP inhibitors occur at a later step of the processes leading to postischemic neuronal death.
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Sivasangari, S., L. Asaikumar, and L. Vennila. "Arbutin prevents alterations in mitochondrial and lysosomal enzymes in isoproterenol-induced myocardial infarction: An in vivo study." Human & Experimental Toxicology 40, no. 1 (August 6, 2020): 100–112. http://dx.doi.org/10.1177/0960327120945790.
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The present study demonstrated the protective effects of arbutin (ARB) on hyperlipidemia, mitochondrial, and lysosomal membrane damage and on the DNA damage in rats with isoproterenol (ISO)-induced myocardial infarction (MI). Rats were pretreated with ARB (25 and 50 mg/kg body weight (bw)) for 21 days. After pretreatment with ARB, MI was induced by subcutaneous injection of ISO (60 mg/kg bw) for two consecutive days at an interval of 24 h. The levels of TC, TG, and FFA were increased and decreased the level of PL in the heart tissue of ISO-induced MI rats. Very-low-density lipoprotein cholesterol and low-density lipoprotein cholesterol were increased while high-density lipoprotein cholesterol was decreased in the plasma of ISO-administered rats. A heart mitochondrial fraction of the ISO rats showed a significant decrease in the activities of mitochondrial enzymes isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase, and malate dehydrogenase. The activities of lysosomal enzymes (β-glucosidase, β-glucuronidase, α-galactosidase, β-galactosidase, cathepsin-B, and cathepsin-D) were increased significantly in the heart tissue homogenate of disease control rats. In ISO-induced MI, rat’s significant increase in the percentage of tail DNA and tail length, and a decrease in the level of head DNA were also observed. ARB administration to MI rats brought all these parameters to near normality, showing the protective effect of ARB against MI in rats. The results of this study demonstrated that the 50 mg/kg bw of ARB shows higher protection than 25 mg/kg bw against ISO-induced damage.
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Villar, Silvia, Noelia Kandratavicius, Stefanie Martinez, and Pablo Muniz. "Single cell gel electrophoresis as a tool to assess genetic damage in Heleobia cf. australis (Mollusca: Gastropoda) as sentinel for industrial and domestic pollution in Montevideo bay (Uruguay)." Brazilian Journal of Oceanography 63, no. 3 (September 2015): 347–54. http://dx.doi.org/10.1590/s1679-87592015090906303.
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AbstractThe knowledge of the extent of DNA damage in aquatic organisms in polluted areas is an important issue because contamination may alter their health at sublethal levels. Although molluscs have been widely used to monitor water pollution, there are no records of in vivo genotoxicity studies. Heleobia cf. australis, is distributed in almost all Uruguayan coastal ecosystems, including highly polluted sites. The comet assay is a damage genetic biomarker based on the migration of negatively charged DNA fragments produced by mutagenic agents in individual cells. Live individuals were collected in the Montevideo Bay (impacted area) and Laguna Garzón (control) to analyze the presence of mutagenic agents in the former site through comet assay. Cells from organisms of the impacted area showed significantly higher levels of genetic damage than those obtained in the control population, measured by percentage of DNA in the tail. Although preliminary, this approach supports the idea that H. cf. australis could be used as a sentinel to evaluate the presence of mutagenic agents in estuarine environments, alerting to the impact of contamination in its early stages.
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Iqbal, Tariq, Maosheng Cao, Zijiao Zhao, Yun Zhao, Lu Chen, Tong Chen, Chunjin Li, and Xu Zhou. "Damage to the Testicular Structure of Rats by Acute Oral Exposure of Cadmium." International Journal of Environmental Research and Public Health 18, no. 11 (June 4, 2021): 6038. http://dx.doi.org/10.3390/ijerph18116038.
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Cadmium (Cd) is one of the most important heavy metal toxicants, used throughout the world at the industrial level. It affects humans through environmental and occupational exposure and animals through the environment. The most severe effects of oral exposure to Cd on the male reproductive system, particularly spermatogenesis, have not been discussed. In this study, we observed the damage to the testes and heritable DNA caused by oral exposure to Cd. Adult male Sprague–Dawley rats were divided into four groups: a control group and three groups treated with 5, 10, and 15 mg Cd/kg/day for 17 days by oral gavage. Our results revealed that Cd significantly decreases weight gain in 10 and 15 mg/kg groups, whereas the 5 mg/kg groups showed no difference in weight gain. The histopathology showed adverse structural effects on the rat testis by significantly reducing the thickness of the tunica albuginea, the diameter of the tubular lumen, and the interstitial space among seminiferous tubules and increasing the height of the epithelium and the diameter of the seminiferous tubules in Cd treated groups. Comet assay in epididymal sperms demonstrated a significant difference in the lengths of the head and comet in all the 3 Cd treated groups, indicating damage in heritable DNA, although variations in daily sperm production were not significant. Only a slight decrease in sperm count was reported in Cd-treated groups as compared to the control group, whereas the tail length, percentage of DNA in head, and tail showed no significant difference in control and all the experimental groups. Overall, our findings indicate that Cd toxicity must be controlled using natural sources, such as herbal medicine or bioremediation, with non-edible plants, because it could considerably affect heritable DNA and induce damage to the reproductive system.
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Palacka, Patrik, Zuzana Sestakova, Andrea Holickova, Katarina Kalavska, Katarina Rejlekova, Boris Kollárik, Michal Chovanec, et al. "Endogenous DNA damage levels in peripheral blood mononuclear cells in patients with muscle-infiltrating urothelial bladder carcinoma." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e16020-e16020. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e16020.
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e16020 Background: Cisplatin-based combination chemotherapy is used as a standard neoadjuvant treatment of patients with muscle-infiltrating urothelial bladder carcinoma (MIUBC). DNA damage represents one of the most important factors contributing to its toxicity. The objective of this prospective study is to evaluate the prognostic value of the endogenous DNA damage level in peripheral blood mononuclear cells (PBMCs) from MIUBC patients before treatment with neoadjuvant chemotherapy. Methods: PBMCs isolated from 25 consecutive MIUBC patients (16 men, 64%) were included into this study. Karnofsky index < 80% was present in 1 patient (4%). DNA damage levels in PBMCs were evaluated by the Comet assay and scored as percentage of DNA in tail by the Metafer-MetaCyte analyzing software. Cut-off of 5.25 was used to dichotomize DNA damage level as “high” or “low” based on the previous study. Results: At the median follow-up 12.1 months, 13 patients progressed (52%) and 8 patients (32%) died. The median and IQR (interquartile range) of endogenous DNA damage level was 7.52 (4.07-27.9). Patients with low DNA damage levels had non-significantly better progression free survival (HR = 0.33, 95% CI: 0.09-1.29) and overall survival (HR = 0.64, 95% CI: 0.11-3.87) compared to patients with high DNA damage levels. Conclusions: These data suggest that endogenous DNA damage levels in PBMCs from MIUBC patients may serve as a prognostic marker early identifying patients with poor outcome. This study is supported by VEGA 2/0053/19 and APVV-17-0384.
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Delisma, Cici, Sri Peni Fitrianingsih, and Suwendar Suwendar. "UJI AKTIVITAS ANALGETIKA EKSTRAK n-HEKSANA DAUN AFRIKA (Vernonia amygdalina Delile) TERHADAP MENCIT SWISS WEBSTER JANTAN." Jurnal Ilmiah Farmasi Farmasyifa 1, no. 1 (October 10, 2017): 26–34. http://dx.doi.org/10.29313/jiff.v1i1.3109.
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ABSTRAKNyeri merupakan persepsi sensorik mengganggu yang dapat ditangani dengan analgetika. Daun afrika (Vernonia amygdalina Delile) secara tradisional digunakan untuk mengobati nyeri seperti sakit gigi. Tujuan penelitian ini untuk menentukan aktivitas analgetika ekstrak n-heksana daun afrika dengan 2 metode pengujian dan menentukan dosis efektifnya. Metode Tail Flick Test untuk menguji aktivitas analgetika sentral dan metode Writhing Test untuk menguji aktivitas analgetika perifer. Mencit dibagi ke dalam 5 kelompok. Kelompok kontrol yang diberi CMC Na, kelompok uji yang diberi ekstrak n-heksana daun afrika dosis 100, 200 dan 400 mg/kg BB serta kelompok pembanding yang diberi tramadol (metode Tail Flick Test) dan aspirin (metode Writhing Test). Analisis data dilakukan dengan ANOVA dan dilanjutkan dengan LSD pada taraf kepercayaan 95% (p ≤ 0,05). Pada metode Tail Flick Test mencit diinduksi nyeri dengan panas suhu 50±2oC dan parameter yang diamati adalah total waktu yang dibutuhkan mencit untuk menjentikkan ekor. Hasil menunjukkan bahwa ekstrak dengan dosis 400 mg/kg BB secara signifikan memperpanjang waktu mencit menjentikkan ekor dibandingkan terhadap kontrol (p=0,006), tetapi aktivitasnya tidak sebanding dengan tramadol dosis 6,5 mg/kg BB (p=0,000). Pada metode Writhing Test mencit diinduksi nyeri dengan asam asetat 0,6%(v/v) dan parameter yang diamati adalah total geliat mencit selama pengamatan. Hasil menunjukkan bahwa ekstrak dengan dosis 100, 200 dan 400 mg/kg BB secara signifikan menurunkan total geliat mencit dibandingkan terhadap kontrol (p=0,000), dengan nilai persen efektivitas sebesar 32,01%, 51,60% dan 82,41% yang lebih lemah dibandingkan aspirin dosis 65 mg/kg BB dengan persen efektivitas 100%.Kata kunci: Daun afrika, Vernonia amygdalina Delile, analgetika, Tail Flick Test, Writhing Test. ABSTRACTPain is a disturbing sensory perception that can be treated with analgesics. Bitter leaf (Vernonia amygdalina Delile) has traditionally noun to treat pain such as toothache. This study aims to evaluate the analgesic activity of n-hexane extract of bitter leaf with 2 testing methods and determine the effective dose. The first method to test central analgesic activity is Tail Flick Test method and the second method is Writhing Test method to test peripheral analgesic activity. The test was done on mice were divided into 5 groups. The control group that was administered to CMC Na, the test group was administered n-hexane extract of bitter leaf dose 100, 200 and 400 mg/kg BW and the comparable group was administered tramadol (for Tail Flick Test method) and aspirin (for Writhing Test method). Data were analyzed by ANOVA test, followed by LSD test at 95% confidence level (p ≤ 0,05). In the Tail Flick Test method, the mice were induced by pain by heat at 50 ± 2℃ and the observed parameters were the total time required of the mice to flick the tail. The results showed that the extract at dose 400 mg/kg BW significantly prolonged the mice's flicking time compared to the control (p=0,006), but the activity was not comparable with tramadol dose of 6,5 mg/kg BW (p=0,000). In the Writhing Test method, the mice were induced by pain by acetic acid 0,6%(v/v) and the observed parameters were the total writhing of mice. The results showed that extracts with doses of 100, 200 and 400 mg/kg BW significantly decrease the total writhing of mice compared to control (p=0,000), with the effectivity percentage of 32,01%, 51,60% and 82,41% which are weaker than 65 mg/kg BW dose aspirin effectivity percentage 100%.Keywords: Bitter leaf, Vernonia amygdalina Delile, analgesics, Tail Flick Test, Writhing Test.
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Peixoto, Milena Simões, Andressa de Vasconcelos e Souza, Iris Soares Andrade, Carolina de Carvalho el Giusbi, Caroline Coelho Faria, Fabio Hecht, Leandro Miranda-Alves, Andrea Claudia Freitas Ferreira, Denise Pires Carvalho, and Rodrigo S. Fortunato. "Hypothyroidism induces oxidative stress and DNA damage in breast." Endocrine-Related Cancer 28, no. 7 (July 1, 2021): 505–19. http://dx.doi.org/10.1530/erc-21-0010.
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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.
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Zhu, Liqian, Xiaotian Fu, Chen Yuan, Xinyi Jiang, and Gaiping Zhang. "Induction of Oxidative DNA Damage in Bovine Herpesvirus 1 Infected Bovine Kidney Cells (MDBK Cells) and Human Tumor Cells (A549 Cells and U2OS Cells)." Viruses 10, no. 8 (July 26, 2018): 393. http://dx.doi.org/10.3390/v10080393.
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Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle that causes lesions in mucosal surfaces, genital tracts and nervous systems. As a novel oncolytic virus, BoHV-1 infects and kills numerous human tumor cells. However, the mechanisms underlying the virus-induced cell damages are not fully understood. In this study, we demonstrated that virus infection of MDBK cells induced high levels of DNA damage, because the percentage of comet tail DNA (tailDNA%) determined by comet assay, a direct indicator of DNA damage, and the levels of 8-hydroxyguanine (8-oxoG) production, an oxidative DNA damage marker, consistently increased following the virus infection. The expression of 8-oxoguanine DNA glycosylase (OGG-1), an enzyme responsible for the excision of 8-oxoG, was significantly decreased due to the virus infection, which corroborated with the finding that BoHV-1 infection stimulated 8-oxoG production. Furthermore, the virus replication in human tumor cells such as in A549 cells and U2OS cells also induced DNA damage. Chemical inhibition of reactive oxidative species (ROS) production by either ROS scavenger N-Acetyl-l-cysteine or NOX inhibitor diphenylene iodonium (DPI) significantly decreased the levels of tailDNA%, suggesting the involvement of ROS in the virus induced DNA lesions. Collectively, these results indicated that BoHV-1 infection of these cells elicits oxidative DNA damages, providing a perspective in understanding the mechanisms by which the virus induces cell death in both native host cells and human tumor cells.
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Lu, Xu, and Jeffrey C. Hansen. "Revisiting the structure and functions of the linker histone C-terminal tail domain." Biochemistry and Cell Biology 81, no. 3 (June 1, 2003): 173–76. http://dx.doi.org/10.1139/o03-041.
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Linker histones stabilize folded chromatin, acting through their long C-terminal tails. The C-termini contain high percentages of evenly distributed lysine and arginine residues and have no secondary structure in solution. Hence, it has generally been believed that the C-termini function by shielding negative charges on the DNA backbone. However, recent evidence supports a mechanism of action of the linker histone C-terminus that involves formation of specific secondary structure(s) upon interaction with other components of the chromatin fiber.Key words: linker histones, chromatin folding, charge neutralization, secondary structure.
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Narumi, K., H. Takasawa, W. Ohyama, and K. Kaneko. "In vivo comet assay of a novel galacto-oligosaccharide in rats." Human & Experimental Toxicology 33, no. 5 (October 15, 2013): 488–95. http://dx.doi.org/10.1177/0960327113506236.
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A novel galacto-oligosaccharide (GOS) manufactured by a two-step enzyme reaction of lactose was examined in a comet assay for its potential to induce DNA damage in vivo by estimating the DNA fragmentation level in the cellular nuclei of the glandular stomach, colon, and peripheral blood. GOS was orally administered at doses of 0 (vehicle alone), 500, 1000, and 2000 mg/kg/day to five male Crl: CD(Sprague Dawley) rats per group three times (48, 24, and 3 h before the animals were terminated). The specimens were prepared in accordance with the standard protocol (version 14.2) of the “International Validation of the In Vivo Rodent Alkaline Comet Assay for the Detection of Genotoxic Carcinogens” organized by the Japanese Center for the Validation of Alternative Methods. No significant differences in the percentage of DNA in the tail were observed between the GOS-treated groups and vehicle controls in any of the organs evaluated. Additionally, no GOS-related clinical signs or effects on body weight were seen. Based on these results, the comet assay of GOS in the glandular stomach, colon, and peripheral blood using rats was judged negative. Therefore, it is concluded that GOS did not induce DNA damage in vivo under the conditions employed in this study.
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AL-Huqail, Asma A., and Ekram Abdelhaliem. "Evaluation of Genetic Variations in Maize Seedlings Exposed to Electric Field Based on Protein and DNA Markers." BioMed Research International 2015 (2015): 1–15. http://dx.doi.org/10.1155/2015/874906.
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The current study analyzed proteins and nuclear DNA of electric fields (ELF) exposed and nonexposed maize seedlings for different exposure periods using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isozymes, random amplified polymorphic DNA (RAPD), and comet assay, respectively. SDS-PAGE analysis revealed total of 46 polypeptides bands with different molecular weights ranging from 186.20 to 36.00 KDa. It generated distinctive polymorphism value of 84.62%. Leucine-aminopeptidase, peroxidase, and catalase isozymes showed the highest values of polymorphism (100%) based on zymograms number, relative front (Rf), and optical intensity while esterase isozyme generated polymorphism value of 83.33%. Amino acids were analyzed using high-performance liquid chromatography, which revealed the presence of 17 amino acids of variable contents ranging from 22.65% to 28.09%. RAPD revealed that 78 amplified DNA products had highly polymorphism value (95.08%) based on band numbers, with variable sizes ranging from 120 to 992 base pairs and band intensity. Comet assay recorded the highest extent of nuclear DNA damage as percentage of tailed DNA (2.38%) and tail moment unit (5.36) at ELF exposure of maize nuclei for 5 days. The current study concluded that the longer ELF exposing periods had genotoxic stress on macromolecules of maize cells and biomarkers used should be augmented for reliable estimates of genotoxicity after exposure of economic plants to ELF stressors.
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Borowiec, K., D. Szwajgier, A. Olejnik, K. Kowalska, and Z. Targoński. "Effects of a bilberry preparation on selected cell lines of the digestive system." Czech Journal of Food Sciences 34, No. 4 (September 5, 2016): 300–305. http://dx.doi.org/10.17221/375/2015-cjfs.
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Bilberry is a valuable wild forest fruit harvested in many countries in Europe. The biological activities of bilberry include antioxidant, anticancer, antiviral, antibacterial, and anticholinesterase activities. This study examines the protective effects of a bilberry (BB) preparation on IEC-6, Caco-2, and HepG2 cell lines. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to study the cytotoxicity of BB. The genotoxicity was determined using single-cell microgel electrophoresis. The Ames test was employed to assay bilberry mutagenicity. No significant effects of BB (12.5–100 µg dry mass/ml) were observed on the viability of IEC-6, Caco-2, and HepG2 cells. There were no differences in the percentage of DNA in the comet tail between the cells treated with BB (100 µg dry mass/ml) and the control cells. However, a significant reduction of oxidative DNA damage in the HepG2 cells was found. BB exhibited neither mutagenic nor promutagenic effects. Our results suggest that bilberry can be a potential tool in the prevention of chronic diseases, without any undesired effects on the cells of the gastrointestinal tract.
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Arif, Mohammad, MM Towhidul Islam, and Hossain Uddin Shekhar. "Lead induced oxidative DNA damage in battery-recycling child workers from Bangladesh." Toxicology and Industrial Health 34, no. 4 (March 8, 2018): 213–18. http://dx.doi.org/10.1177/0748233717754163.
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Lead exposure can damage cells directly by effecting DNA or indirectly by modifying proteins and enzymes. In Bangladesh, many working children are exposed to a very high level of lead during their early life due to their involvement with lead-oriented professions. This imposes a severe threat to the growth and development of the children. Therefore to study the effect of lead, we enrolled 60 age-matched male children, from an area of old Dhaka city, where battery-recycling shops are located, depending on their blood lead concentration. If the children had a plasma lead concentration above the WHO recommended threshold level of 10 µg/dl, we grouped them as test subjects and others as control subjects to determine the effect of lead on different biochemical parameters of the body. Compared to the controls, acculumlation of the lipid peroxidation product, malondialdehyde, increased significantly in test subjects ( p < 0.01). Lead exposure also increased the protein carbonyl content ( p < 0.05) and significantly decreased the plasma glutathione levels of test subjects compared to the controls ( p < 0.05). While comparing the lead-exposed group against controls, it was found that the percentage of damaged DNA, as measured using the Comet assay, significantly increased in tail ( p < 0.01) and decreased in head regions. All of these results suggest that high-plasma lead content may induce an oxidative stress to the study population, which may lead to DNA damage.
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Mattana, C. M., M. A. Cangiano, L. E. Alcaráz, A. Sosa, F. Escobar, C. Sabini, L. Sabini, and A. L. Laciar. "Evaluation of Cytotoxicity and Genotoxicity ofAcacia aromaLeaf Extracts." Scientific World Journal 2014 (2014): 1–6. http://dx.doi.org/10.1155/2014/380850.
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Acacia aroma, native plant from San Luis, Argentina, is commonly used as antiseptic and for healing of wounds. The present study was conducted to investigate thein vitrocytotoxicity and genotoxicity of hot aqueous extract (HAE) and ethanolic extract (EE) ofA. aroma. The cytotoxic activity was assayed by neutral red uptake assay on Vero cell. Cell treatment with a range from 100 to 5000 μg/mL of HAE and EE showed that 500 μg/mL and 100 μg/mL were the maximum noncytotoxic concentrations, respectively. The CC50was 658 μg/mL for EE and 1020 μg/mL for HAE. The genotoxicity was tested by the single-cell gel electrophoresis comet assay. The results obtained in the evaluation of DNA cellular damage exposed to varied concentrations of the HAE showed no significant genotoxic effect at range of 1–20 mg/mL. The EE at 20 mg/mL showed moderate genotoxic effect related to the increase of the DNA percentage contained in tail of the comet; DNA was classified in category 2. At concentrations below 5 mg/mL, the results of cytotoxicity and genotoxicity of aqueous and ethanolic extracts ofAcacia aromaguarantee the safety at cell and genomic level. However further studies are needed for longer periods including animal models to confirm the findings.
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Nishimura, R. C., I. R. Bessa, M. A. N. Dode, and M. M. Franco. "241 METHYLATION PROFILE OF XIST GENE EXON 1 IN BOVINE SPERMATOGENIC CELLS." Reproduction, Fertility and Development 25, no. 1 (2013): 268. http://dx.doi.org/10.1071/rdv25n1ab241.
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Mammalian embryos undergo two waves of genome reprogramming, the first in the zygote, when paternal and maternal genomes are demethylated and, shortly thereafter, remethylated, and the second in the primordial germ cells. It is believed that remethylation is reestablished in male germ cells before birth; however, it is not clear when and how this process occurs in the bovine. Thus, this work aimed to verify whether epigenetic reprogramming occurs during spermatogenesis by evaluating the methylation profile of XIST gene exon 1 in different spermatogenic cells. Spermatocytes, elongated spermatids, and spermatozoa from the epididymis head and tail were collected from bovine testes obtained from a slaughterhouse. The DNA was extracted from the collected cells and treated with bisulfite. The treated DNA was amplified through nested PCR, and the product was inserted on a vector and cloned in bacteria. The plasmid DNA was extracted and sequenced. The sequences of the experiment were compared with the sequence of XIST gene from GenBank, and those that presented a bisulfite conversion rate of ≥95% and a homology of ≥97% with the sequence from GenBank were used. To compare the methylation pattern among groups, the total percentage of methylated CpG was calculated in each replicate and each group. The methylation pattern was compared using Kruskal-Wallis and Mann–Whitney tests because the data did not show normality. All data were compared using the Prophet Program, version 5.0 (1996; BBN Systems and Technologies, Cambridge, MA, USA), and are shown as the mean ± SEM. The significance level used was P < 0.05. The spermatocytes, elongated spermatids, and spermatozoa from the epididymis head and tail groups presented 15.70 ± 14.54%, 1.96 ± 1.96%, 74.09 ± 6.78%, and 45.09 ± 20.19% of methylation, respectively. The epididymis head group was more methylated than the spermatocyte and elongated spermatid groups (P < 0.01). When arranged in two groups, one with spermatocytes and elongated spermatids, and the other with epididymidal spermatozoa, and compared, it was observed that the methylation level was different (P < 0.01) between them. These results suggest that epigenetic reprogramming is still occurring during spermatozoa formation.
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Ng, Ka Lam Nelson, Claire Lynn, Thomas Wing Yan Leung, Eric Chi Wai So, and Anskar Yu Hung Leung. "Targeting DNA Damage and Repair in Acute Myeloid Leukemia Carrying Internal Tandem Duplication of Fms-like Tyrosine Kinase 3 (FLT3-ITD) - a Mechanistic Study." Blood 134, Supplement_1 (November 13, 2019): 1261. http://dx.doi.org/10.1182/blood-2019-129472.
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Objective Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) is one of the most common mutations in acute myeloid leukemia (AML), occurring in nearly 30% of cases. FLT3-ITD involves in-frame duplication of 3-400 base-pairs at the juxta-membrane domain, resulting in ligand-independent activation of FLT3 signaling. Downstream effectors include activation of ERK/STAT5 via SRC kinase, activation of PI3K/AKT, phosphorylation of FOXO3A, down-regulation of equilibrative nucleoside transporter 1 (ENT1) for cytarabine, and induction of reactive oxygen species (ROS) that may lead to increased DNA damage and defective repair. The present study investigated if the latter can be effectively targeted for the treatment of this AML subtype. Methods Primary samples from patients with FLT3-ITD AML, human cell line carrying FLT3-ITD (MOLM-13) as well as mouse B-lymphoid Ba/F3 cells transduced with human FLT3-ITD were used in this study. Traffic Light Reporter (TLR) assay was used to measure fidelity of double-strand breaks (DSB) repair, either via error-free homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Percentage of repair events by HR or NHEJ were quantified by flow cytometry. DNA DSB were examined by γ-H2AX foci staining using confocal microscopy. Single-cell DSB were quantified by neutral comet assay and analysed by OpenComet software. The tail moment was calculated as the length of comet tail multiplied by the tail DNA %. MOLM-13 was transplanted into NOD/SCID/IL2Rg-/- (NSG) mice by tail vein injection. Treatment comprised cytarabine (25mg/kg, i.p., day 1-5) and doxorubicin (1.5mg/kg, i.v., day 1-3), with or without olaparib (25mg/kg, i.p., day 1-5). Comparisons between groups of numerical data were evaluated using Student's t-test. P-values less than 0.05 were considered statistically significant. Results To investigate the link between FLT3-ITD AML and DNA damage response (DDR), expression of critical DDR genes in primary AML samples was examined by real-time quantitative PCR. The panel of genes included apical kinase ATM, ATR and DNA-PKcs; DNA damage mediators BRCA1,BRCA2 and PARP1; downstream response kinase CHEK1 and CHEK2 and effectors TP53. Among them, BRCA2 was significantly down-regulated in FLT3-ITD AML(Wild-type FLT3=18 samples; FLT3-ITD=13 samples; Average dCt 9.7 in WT vs. 10.6 in ITD; p<0.05). Down-regulation of BRCA2 in FLT3-ITD AML was further validated in a microarray database GSE15434 from a multicenter study investigating gene expression profiles of normal karyotype AML( FLT3-WT=148; FL3-ITD=86; ; log2 gene expression 5.61 in WT vs 5.45 in ITD; p<0.001). As BRCA2 is an important protein mediating HR, a DSB DNA repair TLR assay was performed. HR was significantly down-regulated in Ba/F3-FLT3-ITD as compared with parental Ba/F3 line by flow cytometry(1.21% HR repair in control vs. 0.44% in ITD; p<0.05) while NHEJ was unaffected (1.90% NHEJ repair in control vs. 2.38% in ITD; p=0.50). Down-regulation of BRCA2 expression and defective HR in FLT3-ITD AML was reminiscent of BRCA mutant breast and ovarian cancers. Therefore, the effects of poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) olaparib were examined. Ba/F3-FLT3-ITD were more sensitive to olaparib than parental line. Olaparib inhibited base excision repair and increased DSB as indicated by increased number of γ-H2AX foci and increased tail moment in Ba/F3-FLT3-ITD cells when compared to parental line. Olaparib-induced DSB in Ba/F3-FLT3-ITD cells was mainly repaired by NHEJ as shown by colocalization of γ-H2AX foci with 53BP1. Combination of chemotherapy (cytarabine and doxorubicin) and olaparib synergistically reduced leukemic cell growth of MOLM-13 in NSG murine xenograft model. Conclusion Results from the present study supported the use of PARPi in the treatment of FLT3-ITD AML. Its therapeutic benefits in combination with chemotherapy would have to be further evaluated. Acknowledgements: Health and Medical Research Fund (Project number: 04152326) and Li Shu Fan Medical Foundation, LKS Faculty of Medicine, University of Hong Kong. Disclosures No relevant conflicts of interest to declare.
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Kheirallah, Dalia Abdel Moneim, Lamia Mostafa El-Samad, El Hassan Mostafa Mokhamer, Karolin Kamel Abdul-Aziz, and Noura Abdel Haleem Toto. "DNA damage and oogenesis anomalies in Pimelia latreillei (Coleoptera: Tenebrionidae) induced by heavy metals soil pollution." Toxicology and Industrial Health 35, no. 11-12 (November 2019): 688–702. http://dx.doi.org/10.1177/0748233719893200.
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The present study used Pimelia latreillei as a biomonitoring insect for heavy metals soil pollution in a populated industrial area at Zawya Abd El-Qader, Alexandria, Egypt. Comet assay and histological analysis were applied to evaluate the potential risk of heavy metals. X-ray analysis of the soil samples collected from the polluted site revealed significantly increased metal percentages compared with the reference site. Moreover, a significant increase in metal percentages was detected by the X-ray analysis in insect ovaries collected from the polluted site. The Tail DNA length was significantly greater in the insects collected from the polluted site—47.6% compared with 11.4% at the reference site. Pronounced disruptions in oogenesis were observed through histological and ultrastructure investigations in insects collected from the polluted site. The study summarized the potential utility of insect biomonitors in predicting the effect of heavy metals soil pollution on occupational health.
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Ahmed, Rehab, Eman Aly, Sherif Mahmoud, Sahar Awad, and Gehan Kamal. "Ifosfamide toxicity to the retina and the possible roles of lecithin and quercetin in albino rats." International Journal of Engineering & Technology 7, no. 2 (May 23, 2018): 800. http://dx.doi.org/10.14419/ijet.v7i2.12483.
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Background: During cancer chemotherapy, drug-induced oxidative stress can limit therapeutic efficiency and cause a number of side effects. Objectives: Our study aimed to characterize the side effects of an alkylating agent chemotherapy ifosfamide to the retina and if the supplementation of lecithin and or quercetin can diminish its oxidative stress by means of comet assay and FTIR.Methods: Seventy female albino rats divided as control, rats given orally quercetin or lecithin, rats injected with ifosfamide, rats given quercetin or lecithin and in combination of them with ifosfamide injection.Results: Lecithin and quercetin groups indicate a normal comet parameters and distribution of protein secondary structure components content of β-turn, α-helix and β-sheet. After Ifosfamide injection, all comet parameters and β-Turns content were significant increase (p˂0.05) with the same context significant decrease (p˂0.05) of α-helix was observed. Lecithin or quercetin reduces the effect of ifosfamide injection in tail length and percentage tailed DNA. Combined treatment gives more protection against DNA damage. Lecithin role is cleared in returning the normal distribution of β-turn, α-helix, β-sheet and lack of protective effect of quercetin regarding the protein secondary structure of retina was observed.Conclusion: We suggest using lecithin and quercetin in combined treatment to reduce the oxidative stress due to ifosfamide.
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Smoller, J. W., G. E. Truett, J. Hirsch, and R. L. Leibel. "A molecular genetic method for genotyping fatty (fa/fa) rats." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 264, no. 1 (January 1, 1993): R8—R11. http://dx.doi.org/10.1152/ajpregu.1993.264.1.r8.
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After three decades of physiological research, the precise nature of the genetic lesion in Zucker fatty (fa/fa) rats remains unknown. Several methods have been used to identify preobese rats to detect the earliest phenotypic effects of the fa mutation. Most of these methods have used phenotypic characteristics that are not reliable until the second week of life, when increased adiposity is already evident. We have used a restriction fragment length polymorphism (RFLP) for a human genomic DNA probe (VC85) that is tightly linked to the fa locus on rat chromosome 5 to genotype the F2 progeny of a Zucker (13M) x Brown Norway (BN) fa/+ F1 intercross. Sixty-four rats, comprising five litters, were killed at 5-6 wk of age. DNA was isolated either from tail at age 4-7 days (36 rats) or from organs at the time of death (28 rats). Adiposity was scored using inguinal fat pad weight as a percentage of body weight. RFLP analysis was > 99% accurate in identifying obese (fa/fa) rats. This molecular genetic method can be used to genotype fatty rats from an appropriate genetic cross at any age, even prenatally. Moreover, this method can distinguish heterozygous from homozygous littermates, enabling an analysis of gene dosage effects.