Comet Length Research Articles

Establishment of a cell culture from Daphnia magna as an in vitro model for (eco)toxicology assays: Case study using Bisphenol A as a representative cytotoxic and endocrine disrupting chemical

Bisphenol A (BPA) is a widely used industrial compound found in polycarbonate plastics, epoxy resin, and various polymer materials, leading to its ubiquitous presence in the environment. The toxicity of BPA to aquatic organisms has been well documented following in vivo exposure scenarios, with known cytotoxic and endocrine-disrupting effects. As such, BPA was used in this study as a well-characterized chemical to implement more ethical and resource-efficient scientific practices in toxicity testing through new approach methods (NAMs). Due to the frequent use of Daphnia spp. as a model organism in toxicology, we developed an in vitro cell culture system from Daphnia magna embryos, with optimized medium to support cell longevity. The cultures were maintained for up to two months, demonstrating their stability and suitability for cytotoxicity studies. Using this novel system, lethal concentration 50 (LC50) values were determined at the 24 and 48 h time points following BPA exposure. Subsequently, oxidative stress, endocrine disruption, and DNA damage were assessed through gene expression, activity assays, and a comet assay in BPA-exposed cells. LC50 values of 52 µM and 20 µM BPA were calculated after 24 and 48 h exposures, respectively. BPA cells exposed to 20 and 52 µM had significantly increased GSH, GPx, and GST activity levels. mRNA expression analysis revealed significant upregulations in the mRNA expression of hsp70, hsp90, gst, gpx, vtg1, and cyp4, with downregulations of sod, cat, and ecr following BPA exposure. Furthermore, comet assays showed a significantly higher level of DNA damage induced by BPA compared to controls, with greater comet and tail lengths. This study established a novel in vitro Daphnia model, using BPA as a case study for determining toxic effects, further highlighting the importance and applicability of utilizing alternative methods in ecotoxicological research through reducing animal use.

The thrombin receptor (PAR1) is associated with microtubules, mitosis and process formation in glioma cells

The cell surface protease-activated receptor 1 (PAR1) is overexpressed in glioblastoma multiforme (GBM). We studied the function and structure of intracellular microtubule (MT) and PAR1 in a tubulin-mediated process. We found that exposure to thrombin increased the percentage of proliferative, S, and M phases cells, affected morphology, and increased process elongation. PAR1 antagonist inversely affects these measures, increases tubulin end-binding protein 3 (EB3) mRNA expression in C6 cells, and reduces EB3 comet length, track length, and duration in neuroblastoma cells. In addition, immunofluorescence staining suggests that PAR1 is in close association with the MT α-tubulin and with coagulation cascade proteins during cell division stages. Our findings support PAR1 involvement in MT dynamics.

Biological effects of hydroxyl radical inactivation for typical red tide algae Alexandrium tamarense

Dinoflagellates are responsible for harmful algal blooms (HABs) worldwide and cause serious ecological security crises and economic losses to the aquaculture industry. In this study, hydroxyl radicals (•OH) were generated by the synergistic effect of strong ionization discharge and hydrodynamic cavitation and were injected into cells to inactivate Alexandrium tamarense within 9 s at a total reactive oxidant (TRO) concentration of 1.0 mg/L. The concentration and time (CT) value of •OH inactivating A. tamarense was 0.153 mg•min/L, which is 1/100 that of ClO2 inactivation. Scanning electron microscopy results demonstrated that the integrity of the algal cells remained without any leakage of intracellular organic matter post •OH inactivation. Furthermore, the chromosomes in the nucleus unwound and broke into fragments under a transmission electron microscope, indicating that the DNA was damaged. In comparison, ClO2 contributed to the severe destruction of cell membranes and walls, resulting in massive intracellular organic matter (IOM) leakage. Based on the single cell gel electrophoresis results, DNA comet length increased 329 % post •OH inactivation, demonstrating that •OH induced DNA fragmentation. Additionally, based on the transcriptomic analysis of A. tamarense, •OH induced a DNA repair response, indicating that DNA strand breakage occurred. In summary, DNA strand breakage and fragmentation were the major reasons for •OH rapidly inactivating A. tamarense with cell integrity retained, which provided critical advancements in controlling HAB safely and efficiently. This method avoids the risk of A. tamarense lysis and IOM release, while guaranteeing marine water security.

Evaluation of the anticancer activity of Origanum Marjoram as a safe natural drink for daily use

Background Chemotherapeutic agents have numerous side effects. There is a major interest in using natural and safe plants as food or drink to prevent from cancer. Origanum marjoram (OMAE) is a medicinal plant that can be used as a tea, food, and additive in traditional medicine. Objective This study aimed to evaluate the potential anticancer effects of OMAE as a soft drink for daily use against a model cancer, prevention and treatment. Method MCF-7 cells were chosen as model cancer cells. The MTT assay was used to assess the in vitro inhibitory effects of OMAE on cell growth. Moreover, quantitative real‐time PCR (qRT-PCR) was used to detect specific genes associated with cancer, such as ESR1, Bax, Bcl-2, and p53. Furthermore, the DNA damage was evaluated using the comet assay. Results OMAE has IC50 of 53.1 and IC90 of 97.5 μg/ml dependent inhibition of cell proliferation after 48 h of treatment toward MCF-7. Also, a significant decrease in the expression level of the ESR1 gene in the MCF-7 cell line. Furthermore, there was a significant increase in the comet length and comet-positive cells after treatment with OMAE (88.7%) compared with those in the untreated control cells (9.5%), suggesting a high induction of DNA damage by OMAE. Also, OMAE showed a modification in bcl-2, tumor suppressor gene (p53), and Bax levels and influenced the BAX/BCL-2 ratio via releasing the cytochrome C. Conclusion The results of the study were promising, suggesting that the reduced apoptotic rate of MCF-7 breast cancer cells in this work was correlated to the potential anticancer effect of OMAE which would be a suitable preventable drink against cancer. However, further studies are needed to fully understand the potential of OMAE as a cancer treatment.

Studies on triazophos-induced oxidative stress and genotoxicity in freshwater fish Cyprinus Carpio following sublethal exposures

Triazophos (TAP), an organophosphorus insecticide, is widely used in agricultural practice for controlling various insect pests. The present research work aimed to elucidate the impact of TAP on the antioxidant status and DNA content of the freshwater fish Cyprinus carpio. The fish were grouped into batches (n=6) and received a sublethal dose of 0.3mg/L for a duration of 1 (E1), 10 (E2), 20 (E3) and 30 days (E4). Another group, devoid of any toxicant, was maintained as the control (C). Changes in the enzymatic threshold of the selected antioxidants and malondialdehyde (MDA) levels suggested the conformation of oxidative stress in the livers of the freshwater fish C. carpio due to TAP exposure. Additionally, comet assays and micronucleus tests performed on the peripheral blood of the fish suggested increased damage in the form of the percentage of tail DNA formation and a high frequency of micronucleus as compared to the control. A positive correlation was seen between the decline in antioxidant activity, the elevation in MDA and the comet length and micronucleus frequency. The study thus highlights the impact of TAP on antioxidant levels in the livers and genotoxicity in the blood of the freshwater fish C. carpio. The findings of the study confirm that the antioxidant status, along with the comet assay and micronucleus tests could be used as tools in determining the potential genotoxicity due to the TAP impact. It is therefore suggested that extensive use of TAP should be avoided as it may contribute to the decline in the C. carpio population in its natural habitats.

Sperm telomere length is associated with sperm nuclear DNA integrity and mitochondrial DNA abnormalities among healthy male college students in Chongqing, China.

Is sperm telomere length (STL) associated with sperm nuclear DNA damage and mitochondrial DNA abnormalities? Sperm telomere length is related to sperm nuclear DNA integrity and mitochondrial DNA abnormalities in healthy young college students. Many studies have revealed the correlations between sperm genetic alterations in both the nucleus and mitochondria and sperm functionality, however, the possible associations between the telomere, an important component of chromosome, and conventional indicators of mitochondrial DNA and nuclear DNA changes have not been investigated. A prospective cohort study, Male Reproductive Health in Chongqing College Students (MARHCS), was conducted from June 2013 to June 2015. We pooled data collected from the follow-up study in 2014 and a total of 444 participants were included. STL was measured by quantitative (Q)-PCR. Sperm nuclear DNA integrity was determined using sperm chromatin structure assay (SCSA) and comet assay. Mitochondrial DNA damage was assessed by mitochondrial DNA copy number (mtDNAcn) evaluated with Q-PCR, and mtDNA integrity was determined with long PCR. The univariable-linear regression analysis revealed that STL was significantly positively correlated with markers of sperm nuclear DNA damage including the DNA fragmentation index (DFI) and comet parameters (the percentage of DNA in the tail, tail length, comet length, and tail moment). Additionally, STL was also significantly positively correlated with mtDNAcn and significantly negatively correlated with mtDNA integrity. After adjustment for potential confounders, these relationships remained appreciable. Moreover, we investigated potential effects of biometric factors, including age, parental age at conception, and BMI on STL and found that STL was increased with paternal age at conception. A mechanistic explanation of the correlation between STL, sperm nuclear DNA integrity, and mtDNA abnormalities cannot be provided with a cross-sectional study design, so well-designed longitudinal studies are still necessary. In addition, a single semen samples were provided and were not all obtained at the same time point, which may increase the intraindividual bias in this study. The findings extend the literature including assessment of mitochondrial dysfunction, sperm nuclear DNA damage, and telomere length and provide new insights into the relevance of STL in male reproduction. This work was supported by the National Natural Science Foundation of China (No. 82073590), the National Natural Science Foundation of China (No. 81903363), the National Natural Science Foundation of China (No. 82130097), and the National Key R&D Program of China (2022YFC2702900). The authors declare no conflicts of interest. N/A.

The effect of granulocyte colony stimulating factor on genotoxicity in allogeneic peripheral blood stem cell transplantation donors: a prospective case-control study.

Every year, thousands of donors are exposed to granulocyte-colony stimulating factor (G-CSF) for stem cell mobilization in hematopoietic stem cell transplantations (HSCT). Previous studies about the genotoxicity of G-CSF were inconclusive. In this study, the genotoxic effects of G-CSF in peripheral blood stem cell (PBSC) donors were evaluated prospectively by using three different validated and reliable methods for the first time in the literature to the best of our knowledge. Donors of PBSC transplantation (n=36), who received G-CSF were evaluated for genotoxicity by micronucleus test (MNT), nuclear division index (NDI), and comet assay (CA). Genotoxic effects are expected to cause an increase in MNT and CA values and decrease in NDI. Blood samples were collected at three timepoints (TP): before starting G-CSF (TP1), after G-CSF for five days (TP2), and one month after the last dose (TP3). Sixteen controls were included for baseline comparison of genotoxicity tests. CD34 cell counts and hemograms were also analyzed. MNT and CA parameters; comet and tail length, tail DNA%, and tail moment, showed no change in time whereas another CA parameter, Olive`s tail moment (OTM) was increased significantly at TP3 compared to both baseline and TP2 (p=0.002 and p=0.017, respectively). Nuclear division index decreased significantly at TP2 (p < 0.001), then increased above baseline at TP3 (p=0.004). Baseline comparison with controls showed higher MN frequency in donors without statistical significance (p=0.059). Whereas, CA results were significantly higher in controls. CD34 cell count showed moderate positive correlation with white blood cell count at TP2 (Pearson R=0.495, p=0.004). Our results showed the genotoxic effect of G-CSF in healthy donors, in two of the three tests performed, short-term effect in NDI, and long-lasting effect in OTM. So, this study provides novel information for the debate about the genotoxicity of G-CSF and supports the need for further studies with a larger sample size and longer follow-up.

The Antibacterial and Larvicidal Potential of Bis-(2-Ethylhexyl) Phthalate from Lactiplantibacillus plantarum.

Lactic acid bacteria produce a variety of antibacterial and larvicidal metabolites, which could be used to cure diseases caused by pathogenic bacteria and to efficiently overcome issues regarding insecticide resistance. In the current study, the antibacterial and larvicidal potential of Bis-(2-ethylhexyl) phthalate isolated from Lactiplantibacillus plantarum BCH-1 has been evaluated. Bioactive compounds were extracted by ethyl acetate and were fractionated by gradient column chromatography from crude extract. Based on FT-IR analysis followed by GC-MS and ESI-MS/MS, the active compound was identified to be Bis-(2-ethylhexyl) phthalate. Antibacterial potential was evaluated by disk diffusion against E. coli (12.33 ± 0.56 mm inhibition zone) and S. aureus (5.66 ± 1.00 mm inhibition zone). Larvicidal potency was performed against Culex quinquefasciatus Say larvae, where Bis-(2-ethylhexyl) phthalate showed 100% mortality at 250 ppm after 72 h with LC50 of 67.03 ppm. Furthermore, after 72 h the acetylcholinesterase inhibition was observed as 29.00, 40.33, 53.00, 64.00, and 75.33 (%) at 50, 100, 150, 200, and 250 ppm, respectively. In comet assay, mean comet tail length (14.18 ± 0.28 μm), tail DNA percent damage (18.23 ± 0.06%), tail movement (14.68 ± 0.56 µm), comet length (20.62 ± 0.64 µm), head length (23.75 ± 0.27 µm), and head DNA percentage (39.19 ± 0.92%) were observed at 250 ppm as compared to the control. The current study for the first time describes the promising antibacterial and larvicidal potential of Bis-(2-ethylhexyl) phthalate from Lactiplantibacillus plantarum that would have potential pharmaceutical applications.

Sequential evaluation of DNA damage in patients with head and neck carcinoma receiving radiotherapy

Background: Head and neck cancers account for about 30% of all cancers in India. Studies showed that there is an increased primary DNA damage even before the commencement of any modality of treatment in cancer patients which is further increased by the treatment. Chemo-radiation induced DNA damage is not repaired so effectively in patients with carcinoma which might pave way for secondary carcinoma. Aims and Objectives: The aim of this study was to assess the degree of DNA damage by comet assay technique in patients with head and neck carcinoma receiving radiotherapy. The degree of DNA damage was compared according to the age, gender, and associated risk factors of the patients. Materials and Methods: 35 patients with Stages II, III, and IVA, histopathologically confirmed Squamous cell carcinoma of head and neck with Karnofsky Performance Status &gt;70 attending radiotherapy OPD for treatment were included in this study.1 ml of heparinized blood was collected from the study participants during various doses of radiation treatment. All the samples were processed immediately and analyzed for DNA damage by single cell gel electrophoresis assay - Comet assay technique. Results: The comet length parameter, head diameter, and tail length were found to be increased when compared to baseline sample. The percentage of DNA in head parameter of post-RT sample was decreased when compared to baseline sample All these findings are indicative of DNA damage following radiotherapy. Conclusion: Patients with locally advanced head and neck carcinoma following radiotherapy showed a sequential increase in the DNA damage. The co-existing risk factors and old age may increase the baseline DNA damage in the patients with head and neck cancers.

The Impact of Re-Mating Interval and Genotype on Physiological Response of Rabbits after First Kindling

This is an experiment aimed to study the effect of re-mating interval on rabbit does after first kindling on hormonal (insulin, leptin, and T3) and metabolites (triglycerides, urea, and glucose) levels. DNA damage in ovary cells of rabbit does during the 2nd parity was also studied. Two varieties were used: APRI (synthetic line) and Baladi Black (BB, Egyptian breed). A total number of 120 mature rabbit does (60 does for each breed) were 6 months of age and were used at the beginning of the breeding season. Does of each breed were divided into three equal groups according to reproductive rhythm. The 1st group was postpartum (PP). The 2nd group was 11 days after parturition (P11). The 3rd group was post-weaning (PW). There were significant (P&amp;ge;0.05) differences in plasma leptin concentration during 1st parity. The highest value of plasma leptin concentration was recorded by the PW group at mating. Also, there were significant differences in plasma insulin and T3 hormones concentrations of doe rabbits. The highest value of plasma insulin concentration was recorded by the PW group at mating in 1st parity and the highest value of plasma T3 hormone concentration was recorded for the PS group at mating. While there were insignificant differences during 2nd parity in T3 hormone concentration in rabbits, the differences of plasma glucose and triglyceride concentrations of doe rabbits during 1st parity and 2nd parity were significant. However, the highest significant value of plasma glucose concentration was recorded by the PW group at mating. On the other hand, there were insignificant differences in plasma urea concentration of doe rabbits during 1st parity and 2nd parity. Finally, no significant effects were observed on comet length, head diameter, tail length, or DNA % tail.

On the Relationship Between EB-3 Profiles and Microtubules Growth in Cultured Cells.

Microtubules are dynamic structures undergoing rapid growth and shrinkage in living cells and in vitro. The growth of microtubules in vitro was analyzed with subpixel precision (Maurer et al., Current Biology, 2014, 24 (4), 372–384); however, to what extent these results could be applied for microtubules growing in vivo remains largely unknown. Particularly, the question is whether microtubule growth velocity in cells could be sufficiently approximated by a Gaussian distribution or its variability requires a more sophisticated description? Addressing this question, we used time-lapse microscopy and mathematical modeling, and we analyzed EB-3 comets forming on microtubules of cultured cells with subpixel precision. Parameters of comets (shape, form, and velocity) were used as topological characteristics of 3D voxel objects. Using regression analysis, we determined the real positions of the microtubule tips in time-lapse sequences. By exponential decay fitting of the restored comet intensity profile, we found that in vivo EB-3 rapidly exchanges on growing microtubule ends with a decoration time ∼ 2 s. We next developed the model showing that the best correlation between comet length and microtubule end growth velocity is at time intervals close to the decoration time. In the cells, EB comet length positively correlates with microtubule growth velocity in preceding time intervals, while demonstrating no correlation in subsequent time intervals. Correlation between comet length and instantaneous growth velocity of microtubules remains under nocodazole treatment when mean values of both parameters decrease. Our data show that the growth of microtubules in living cells is well-approximated by a constant velocity with large stochastic fluctuations.

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

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

Comet Parameters and Plasma 8-Iso-Prostaglandins F2α

Background: Major depression can be characterized by isolated episodes of disturbance in psychosomatic functions for a duration of at least two weeks along with intermediate non-symptomatic periods. The factors playing important role in the etiology of major depression were family history, genetic factors, decreased levels of serotonin and norepinephrine; increased levels of interleukins or any other medical illness having an increased cytokine production. The pathogenesis of major depression involves oxidative stress, which consists of synthesis of free radicals causing damage to nucleic acids, lipids, carbohydrates, and proteins present in the cells. Oxidative stress – induced DNA damage consists of numerous types of lesions which can be assessed by the comet assay method. Prostaglandin F2-alpha is produced because of oxidative stress-induced peroxidation of cell membrane lipids and has elevated plasma levels in patients with major depression. Fluoxetine is the drug of choice for major depression and found to have antioxidant properties. The present study was done to assess the correlation between plasma 8-iso-PGF2α levels and the comet parameters, namely comet length (CL), head diameter (HD), % of DNA in head (%DNA Head), tail length (TL) and % of DNA in tail (%DNA Tail), before starting and after finishing the eight-week fluoxetine therapy. Methodology: The prospective clinical study was conducted in the Department of Anatomy in collaboration at, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry. The study group consisted of drug naïve, newly diagnosed major depression patients belonging to age-group of ≥ 18 to 50 years and followed up after eight weeks of fluoxetine therapy. Results: The correlation was found to be positive between the levels of 8-iso-PGF2α in the plasma and all the comet parameters except %DNA Tail, which showed a negative (inverse) relationship with the levels of 8-iso-PGF2α in the plasma. Conclusion: The current study suggested the role of oxidative stress in causing DNA damage and lipid peroxidation in major depression patients and the antioxidant role of fluoxetine in causing decrease in the levels of parameters of oxidative stress and subsequent DNA repair.

Assessment of DNA Damage in Chick Embryo Brains Exposed to 2G and 3G Cell Phone Radiation using Alkaline Comet Assay Technique

Introduction: The cellular phones/mobile phones have emerged as the fastest growing man-made phenomenon ever discovered in the history. Controversies still exist among the scientific community regarding the ill-effects of Radiofrequency Radiation (RFR) exposure from cell phones on biological tissues. The present study will provide an insight into the basic mechanisms by which RF fields interact with developing brain in an embryo. Aim: To assess the possible Deoxyribonucleic Acid (DNA) damage in developing brain of chick embryo following chronic exposure to Ultra-High Frequency/Radiofrequency Radiation (UHF/RFR) emitted from 2G and 3G cell phone. Materials and Methods: Fertilised hen eggs were divided into three groups. Experimental Group A (exposed to 2G radiation, 24 eggs), Experimental Group B (exposed to 3G radiation, 24 eggs) and Group C sham exposed control group (24 eggs). After the completion of scheduled duration of exposure (72 minutes per day), the chick embryos were sacrificed from 9th-12th day and the brains were dissected out. The chick embryo brains were then subjected to alkaline comet assay technique to assess the DNA damage. The results were statistically compared using one-way Analysis of Variance (ANOVA). Results: In the present study, the exposure of chick embryo brains to 2G and 3G cell phone radiation caused increased mean comet length (p&lt;0.001), mean tail length (p&lt;0.001), mean percentage of DNA in the tail (p&lt;0.001) and mean tail moment (p&lt;0.01) suggestive of increased DNA damage. Conclusion: The present study concludes that the RFR exposure caused significant increase in DNA damage in developing brain of chick embryos with changes more pronounced in 3G exposure group.

Epigallocatechin-3-gallate (EGCG) addition as an antioxidant in a cryo-diluent media improves microscopic parameters, and fertility potential, and alleviates oxidative stress parameters of buffalo spermatozoa

The disparity between the endogenous antioxidants concentration and free radicals in spermatozoa results in reactive oxygen species (ROS) generation. In this prospect, epigallocatechin-3-gallate (EGCG) preserves vigorous antioxidant features. Current study explored the influence of EGCG in a cryo-diluent media on microscopic parameters, oxidative stress parameters, and fertility potential of buffalo spermatozoa during cryopreservation. Concisely, collected semen from three donor bulls for four times were then evaluated for volume, motility, concentrations and then dilution in a cryo-diluent media with different concentrations of EGCG (EGCG-0 = control; EGCG-50 = 50 μM, EGCG-100 = 100 μM, EGCG-200 = 200 μM, and EGCG-300 = 300 μM) at 37 °C, cooled to 4 °C in 2 h, equilibrated for 4 h at 4 °C, and cryopreserved. At post-thawing, Computer-Assisted Sperm motion Analysis motilities (total and progressive, %) and rapid velocity (%), plasma membrane functionality, supravital plasma membrane integrity, and mitochondrial potential (%) were found higher (P < 0.05) in EGCG-200, and EGCG-300 than control, whereas average-path, straight-line, and curved-linear velocities (μm/sec), and acrosome integrity (%) were recorded higher in EGCG-300 than control. Further, comet length (μm), and tail length (μm), LPO (lipid peroxidation, μM/mL), and apoptosis-like changes (%) in spermatozoa were significantly decreased in EGCG-300 than control. Seminal plasma antioxidant enzymes activities (glutathione peroxidase, U/mL, and superoxide dismutase, U/mL) were increased with EGCG-300 than control. Moreover, EGCG-300 addition in a cryo-diluent media improves the fertility potential (%) of buffalo spermatozoa. In a nutshell, the inclusion of EGCG-300 in a cryo-diluent media enhances post-thaw microscopic parameters, and fertility potential, whereas decreases oxidative stress parameters in buffalo spermatozoa.

Methanol extract of Artemisia brevifolia as a curative agent against CCl4 induced nephrotoxicity in albino rats

Carbon tetrachloride (CCl4) is a Potential nephrotoxin that causes severe and prolonged chemical toxicity. Investigations have been accomplished on numerous species of genus Artemisia, but the plant Artemisia brevifolia has hardly been used in this respect. The present study was considered to ascertain the effects of “Artemisia brevifolia plant extract” against CCl4 prompted nephrotoxicity in male albino rats. Forty-eight male albino rats were allocated into eight groups containing 6 rats in each. Group I was the control group. Group II was given dimethyl sulfoxide (DMSO) (1 ml/kg) orally. Group III was treated with dissolved CCl4 in olive oil (1 ml/kg). Group IV was given CCl4 + Silymarine. Group V and VI were administered with Artemisia brevifolia extract (150 and 300 mg/kg respectively) + CCl4. Group VII and VIII were provided with only 150 and 300 mg/kg extract of Artemisia brevifolia respectively. Results displayed that CCl4 administration significantly (p < 0.05) reduced the activity of antioxidant enzymes like catalase (CAT), peroxidase (POD), Superoxide dismutase (SOD), glutathione (GSH), glutathione reductase (GSR), glutathione-S-transferase (GST), albumin, creatinine clearance and Comet parameters (head length, % in head) while increased the level of thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H2O2), urea, creatinine, urobilinogen, urinary proteins and comet parameters (number of comets, tail length, comet length, tail moment, % in tail). The administration of Artemisia brevifolia significantly (p < 0.05) improved the histopathology of renal tissues. The results revealed that Artemisia brevifolia has protective consequences against CCl4 induced kidney damage.

Redox-dependent regulation of end-binding protein 1 activity by glutathionylation.

Cytoskeletal proteins are susceptible to glutathionylation under oxidizing conditions, and oxidative damage has been implicated in several neurodegenerative diseases. End-binding protein 1 (EB1) is a master regulator of microtubule plus-end tracking proteins (+TIPs) and is critically involved in the control of microtubule dynamics and cellular processes. However, the impact of glutathionylation on EB1 functions remains unknown. Here we reveal that glutathionylation is important for controlling EB1 activity and protecting EB1 from irreversible oxidation. In vitro biochemical and cellular assays reveal that EB1 is glutathionylated. Diamide, a mild oxidizing reagent, reduces EB1 comet number and length in cells, indicating the impairment of microtubule dynamics. Three cysteine residues of EB1 are glutathionylated, with mutations of these three cysteines to serines attenuating microtubule dynamics but buffering diamide-induced decrease in microtubule dynamics. In addition, glutaredoxin 1 (Grx1) deglutathionylates EB1, and Grx1 depletion suppresses microtubule dynamics and leads to defects in cell division orientation and cell migration, suggesting a critical role of Grx1-mediated deglutathionylation in maintaining EB1 activity. Collectively, these data reveal that EB1 glutathionylation is an important protective mechanism for the regulation of microtubule dynamics and microtubule-based cellular activities.

In Vivo Genotoxicity Assessment of Gold Nanoparticles of Different Doses by Comet Assays

Gold nanoparticles were synthesis by green method using the soaked orange peels (citrus Sinensis) as reducing and stabilizing agent. The gold nanoparticles were diagnosed using the approved method, including the transmission electron microscope and the visible UV spectroscopy. Thus, the results of the diagnosis showed the formation of spherical particles with a size of 36 nanometers. Their biological effects on the bone marrow cells of albino mice were studied. Male albino mice were used in this study and randomly divided into seven groups. The first group was the control group injected with the physiological solution (Normal Saline) and the other six groups were injected with different doses of the solution of the gold nanoparticles (1, 2, 4, 6, 8 and 10) Mg/kg. The effect of gold nanoparticles on the DNA damage by bone marrow cells was studied using comet assay according to the following criteria: (DNA in Head%, Comet Length, Tail Length, DNA in Tail%, Olive Moment), the results showed a significant decrease (P?0.01) in the rate of DNA in Head%, while it showed a significant increase in Comet Length, Tail Length, DNA in Tail%, Olive Moment compared to the control group and all Doses studied. The effect of gold nanoparticles was directly proportional to the increase in the studied doses. The dose was 10 mg/kg, which was the highest toxicity, followed by the dose of 8 mg/kg, then the rest of the doses was descending.

Changes in the levels of comet parameters before and after fluoxetine therapy in major depression patients.

Major depression belongs to mood disorders and characterized by worthlessness, no interest or happiness in any activity; lasting for atleast two weeks. Etio-pathological changes of major depression include oxidative stress leading to free radical synthesis which causes damage to carbohydrates, proteins, lipids and nucleic acids. Nucleic acid damage can be identified by either single or double strand breaks and for quantitative estimation of the same, neutral or alkaline comet assay is performed. Fluoxetine is the drug of choice for treatment of major depression having antioxidant function. In the current study eighty drug naïve major depression patients were recruited and comet parameters namely total comet length, head diameter and tail length were measured before starting the treatment and after completion of eight week fluoxetine therapy. The levels of comet parameters were higher in females than males suggesting higher prevalence of major depression among females. On categorizing into three age groups, the numbers of major depression patients belonging to 18–30 year age group were higher than 31–40 and 41–50 year age groups. All the parameters of deoxyribonucleic acid damage were reduced after eight week of fluoxetine therapy indicating that fluoxetine has anti-oxidant action along with its antidepressant properties, which cause reversal of oxidative stress induced damage occurring during major depression.

Algicidal Efficiency and Genotoxic Effects of Phanerochaete chrysosporium against Microcystis aeruginosa.

Eutrophication has become a severe environmental problem. This study evaluated the algicidal efficiency and genotoxic effects of Microcystis aeruginosa co-cultured with Phanerochaete chrysosporium for 48 h under the optimum conditions of 250 mg/L of P. chrysosporium at 25 °C with dissolved oxygen content of 7.0 mg/L. The results showed that the activity of algal dehydrogenase, superoxide dismutase, and peroxidase were all decreased and the malondialdehyde content increased after co-culturing. Fourier transform infrared spectroscopy and scanning electron microscopy observations showed that the functional group and structure of algal cells were significantly changed. Compared with those of control tadpoles, blood cells of Fejervarya multistriata tadpoles had increased micronucleus frequency (from 1.05 ± 0.09 to 1.99 ± 0.05) and abnormal nuclei (from 2.45 ± 0.06 to 5.83 ± 0.07). The tail length of M. aeruginosa co-cultured with P. chrysosporium increased from 1.12 ± 0.21 to 21.68 ± 0.34, and the comet length increased from 6.45 ± 0.09 to 36.45 ± 0.67 within 48 h. Micronucleus assay and Comet assay results demonstrated that P. chrysosporium might effectively remove algae and reduce genotoxic effects and may be safe for aquatic ecosystems.