Tail DNA Research Articles

Cytotoxicity and genotoxicity of tributyltin in the early embryonic chick, Gallus gallus domesticus.

Tributyltin (TBT) is used in many commercial applications, including pesticides and antifouling paints, due to its biocidal properties. We examined the cytotoxicity and genotoxicity of TBT in the early chick embryo (Gallus gallus domesticus). Chick embryos (11 days) were treated with various doses of TBT to measure LD50 values for 24, 48, and 72h exposures, which were determined to be 110, 54, and 18μg/egg, respectively. The embryos were exposed to sub-lethal doses of TBT for evaluation of cytotoxicity and genotoxicity. An increase in the incidence of micronuclei (MN) was observed but it was not statistically significant. Induction of other nuclear abnormalities (ONA) after 72h TBT exposure was significant. A significant increase in comet assay tail DNA content was also detected in TBT-exposed embryos. Cytotoxicity was also evidenced by alteration in the polychromatic erythrocytes (PCE) to normochromatic erythrocytes (NCE) ratio and by an increase in the erythroblast population in treated organisms. The cytotoxicity and genotoxicity of TBT may have long-term complications in later stages of the life cycle.

Effect of electron beam irradiation on the pine wood nematode, Bursaphelenchus xylophilus

Purpose Reproduction inhibition of the pine wood nematode (PWN) by electron beam (e-beam) irradiation both in vitro and in vivo was tested to determine if ionizing radiation could control the PWN by reducing survival and preventing reproduction, thus reducing the risk of pine wilt disease (PWD) spread. Materials and methods E-beam (10 MeV) irradiation treatment at different doses (0–4 kGy) was applied to PWNs in a Petri dish. Treatment of pine wood logs infested with PWNs was performed at 10 kGy. Mortality was determined by comparing the survival rates before and after irradiation treatment. DNA damage by e-beam irradiation (0–10 kGy) in the PWN was determined using the comet assay. Results E-beam irradiation increased mortality and suppressed reproduction with increasing doses. The lethal dose (LD) values (kGy) were estimated as follows: LD50 = 2.32, LD90 = 5.03, and LD99 = 9.48. E-beam irradiation of pine wood logs significantly suppressed PWN reproduction. Comets of e-beam-irradiated cells showed an increased tail DNA level and moment with an increasing dose. Conclusion This study suggests that e-beam irradiation could be used as an alternative method for the management of pine wood logs infested with PWNs.

Estimation of antigenotoxic properties of Ginger (Zingibe officinale) for DNA Damage after Exposure to Patulin in rats

Patulin, a known fungal poison, is an important pollutant in apples and products derived from apples and fodder. Ginger improved the liver function and prevents hepatotoxicity against many toxins and used in treatment of various diseases. The genetic toxicity of patulin in the liver of rats using the comet assay in vivo was investigated and effect of ginger (Zingibe officinale) on DNA damage induced by patulin. Five groups of adult male Sprague Dawley rats were used. The rats were given 3.75 mg kg-1 intraperitoneal (i.p) patulin. The rats were treated with ginger at a dose (100 mg kg-1) for four weeks and eight weeks. DNA damage was measured in the liver of rats using a standard genetic toxicity tests comet assay. Tail length and percentage of tail DNA within the comet increased significantly (p <0.05) in patulin group (p =0.01), (p =0.000) respectively, compared with control group a time-dependent manner, indicating an increase in DNA damage. The liver tissue showed signs of improvement in DNA damage after treating rats with ginger. In conclusion, these results indicate that patulin is genotoxic at the injected dose and that liver is an important target organ. Instead, ginger therapy can reduce DNA damage in liver cells from the toxicity caused by patulin.

DNA strand break levels in cryopreserved mononuclear blood cell lines measured by the alkaline comet assay: results from the hCOMET ring trial.

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.

Long-term cryopreservation of potassium bromate positive assay controls for measurement of oxidatively damaged DNA by the Fpg-modified comet assay: results from the hCOMET ring trial.

The formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay is widely used for the measurement of oxidatively generated damage to DNA. However, there has not been a recommended long-term positive control for this version of the comet assay. We have investigated potassium bromate as a positive control for the Fpg-modified comet assay because it generates many Fpg-sensitive sites with a little concurrent generation of DNA strand breaks. Eight laboratories used the same procedure for the treatment of monocytic THP-1 cells with potassium bromate (0, 0.5, 1.5, and 4.5 mM) and subsequent cryopreservation in a freezing medium consisting of 50% foetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulphoxide. The samples were analysed by the Fpg-modified comet assay three times over a 3-year period. All laboratories obtained a positive concentration-response relationship in cryopreserved samples (linear regression coefficients ranging from 0.79 to 0.99). However, there was a wide difference in the levels of Fpg-sensitive sites between the laboratory with the lowest (4.2% Tail DNA) and highest (74% Tail DNA) values in THP-1 cells after exposure to 4.5 mM KBrO3. In an attempt to assess sources of inter-laboratory variation in Fpg-sensitive sites, comet images from one experiment in each laboratory were forwarded to a central laboratory for visual scoring. There was high consistency between measurements of %Tail DNA values in each laboratory and the visual score of the same comets done in the central laboratory (r = 0.98, P < 0.001, linear regression). In conclusion, the results show that potassium bromate is a suitable positive comet assay control.

ROLE OF VITAMIN C AND E ON GENOTOXICITY, HEMATOLOGICAL AND BIOCHEMICAL INVESTIGATION IN CYPRINUS CARPIO L. FOLLOWING ZINC OXIDE NANOPARTICLES EXPOSURE

This study was aimed to evaluate the role of vitamins C and E on genotoxicity, biochemical and hematological indices in common carp, Cyprinus carpio following zinc oxide nanoparticles exposure. Zinc oxide nanoparticles were synthesized (size was &lt; 34nm) and characterized using Fourier Transform Infrared Spectra (FTIR) and X-Ray Diffraction (XRD) analysis. About 120 common carp (weight 20.0-32.0 g) were randomly divided into 12 tanks at rate of 10 fish/tank (two (replicates/treatment); fish were fed diet as follows: Control (C) were fed basal diet; T1 fish were fed basal diet mixed with vitamins C and E (400 mg/kg); T2 and T3 fish were fed basal diet mixed with 10%and 15% ZnONPs respectively; T4 and T5 fish were fed basal diet mixed with ZnONPs 10% and 15% plus vitamins C and E (400 mg/kg dw) respectively. Post 40 d feeding trail, variable changes were registered in blood indices (“Hb content, PCV%, WBCs and RBCs numbers”) in all treated groups compared to C and T1 groups. The highest DNA damage (% tail DNA using Comet assay) was seen in T2 and T3 which asserted highly significant increased (P≤0.01) compared to C and to treated groups. As well as, glutathione peroxidase (GPx) activity exhibited highly significant increase (P≤0.01) in T2 and T3 groups relative to C, T4, T1, T5 respectively. This investigation clearly proved that sub-lethal doses (10 and 15% in diet) of ZnONPs were able to induce an oxidative stress in carp fish as reflected by significantly increase of DNA damage to erythrocytes and “the combination of vitamins C and E was able to alleviate the oxidative stress generated due to exposure to ZnONPs.”

Assessment of imidacloprid induced genotoxicity in Pethia conchonius (Rosy barb), a common freshwater fish of India

Imidacloprid is one of the highly efficient, globally used neonicotinoid groups of insecticides. The indiscriminate use of imidacloprid is contaminating large water bodies affecting not only the target organisms but also non-target organisms including fish. The present study aimed to assess the extent of nuclear DNA damage by imidacloprid in Pethia conchonius a freshwater fish in India using comet and micronucleus assays. The LC50 value of imidacloprid was estimated to be 227.33 mg L−1. Based on the LC50-96 h value, three sub-lethal concentrations of imidacloprid, SLC I −18.94 mg L−1, SLC II −28.41 mg L−1 and SLC III −56.83 mg L−1 were used to detect its genotoxic effect at DNA and cellular level. The imidacloprid exposed fishes exhibited higher DNA damage and nuclear abnormalities (p < 0.05) than the control. The %head DNA, %tail DNA, tail length and the frequency of micronuclei with other nuclear abnormalities like blebbed and notched nuclei were significantly higher than the control in a time and concentration-dependent manner. The DNA damage parameters such as %head DNA (29.107 ± 1.843), %tail DNA (70.893 ± 1.843), tail length (361.431 ± 8.455) micronucleus (1.300 ± 0.019), notched (0.844 ± 0.011) and blebbed (0.811 ± 0.011) nuclei were found to be highest for SLC III (56.83 mg L−1) at 96 h. The findings indicate that IMI is highly genotoxic in fish and other vertebrates leading to mutagenic/clastogenic effects. The study will be helpful in optimization of the imidacloprid use.

Ethanolic Extract of Garcinia kola Stem Bark Inhibits LDL-Uptake and LPS-Induced Anxa-1 and ICAM-1 Expression in Human Umbilical Vein Endothelial Cells (HUVECS)

The endothelial cells’ dysfunction linked to the development of atherosclerosis plays an important role in the regulation of inflammatory responses. Previous studies demonstrated the antiatherogenic effects of Garcinia kola seed extracts based on their lipid lowering effects. Our recent studies showed the in vitro antioxidant and anti-inflammatory activities of the ethanolic extract of Garcinia kola stem barks (EE). For more insight on the antiatherogenic effect of EE, we investigated its activity on some key points of the atherosclerosis process. The cytotoxicity of EE as well as its effects on LDL-uptake, LPS-induced InterCellular Adhesion Molecule (ICAM-1) and Annexin-1 (Anxa-1) expression and LPS-induced DNA damage were evaluated in Human Umbilical Vein Endothelial Cells (HUVECs). EE significantly (p&lt;0.0001) increased the cell viability in both naive and LPS-treated HUVECs. At the concentrations of 50 and 100 µg/ml, EE significantly reduced LDL-uptake by endothelial cells stimulated with LPS. The immunohistochemistry results showed a significant (p&lt;0.01) decrease in the ICAM-1 expression at the EE concentration of 250 µg/ml. EE also showed a significant (p&lt;0.0001) concentration-dependent reduction of Annexin-1 expression in LPS-treated HUVECs. Besides, EE exhibited significant inhibition on LPS-induced genotoxicity marked by a decrease in tail DNA expression (p&lt;0.0001) and tail movement expression (p&lt;0.001) for the concentrations of 50 and 100 µg/ml. These findings showed that EE may mitigate the atherogenic process by reducing LDL-uptake and the expression of adhesion molecules. Thus, the EE of Garcinia kola turns out to be a potential candidate for the treatment of atherosclerosis with a lower risk of toxicity.&#x0D; Keywords: Garcinia kola, LDL-uptake, LPS-induced inflammation, comet assay.

Potential toxic effects of titanium oxide (TiO2) nanoparticles on the biological, biochemical, and histological aspects of the land snail Helix aspersa

Nanotechnology has come a long way in our lives. However, it maintains some negative effects on the environment. This study aims to use the land snail Helix aspersa as a bioindicator. Titanium dioxide nanoparticles (TiO2NPs) had been used at 70 and 140 µg/L for two weeks by the spraying method. The oxidative biomarkers, condition index (CI), DNA damage, hemocyte count, and phagocytic activity were estimated. The toxicity of TiO2NPs was determined (LC50 = 544 µg/L). The exposure to TiO2NPs caused a significant reduction of the activities of superoxide dismutase (SOD) and catalase (CAT) in the digestive gland of Helix aspersa (the activity of CAT was 3.4 ± 0.1 (P = 0.001), SOD was 11 ± 1 (P = 0.0002) at concentration 140 µg/L after two weeks). The activity of glutathione peroxidase (GPX) was (1.13 ± 0.01 µ/mg protein at 140 µg/L compared with controls (5.47 ± 0.01 µ/mg protein). The treatment caused DNA damage in the hemocytes (tail DNA % = 8.66 ± 0.02 and tail moment = 52.99 ± 0 at140 µg/L (P = 0.002)). In the digestive gland, both tail DNA % and tail moment increased (tail moment = 78.38 ± 0.08 compared with control = 2.29 ± 0.09 (P = 0.0001)). The total count of hemocytes significantly decreased after two weeks (the average number was 71 ± 1.5 compared with controls 79 ± 1.1 at 140 µg/L). Furthermore, TiO2NPs caused histological alterations in the digestive gland of Helix aspersa. It can be concluded that the Helix aspersa can be used as environmental pollution bioindicator. A comprehensive evaluation of toxic effects induced by TiO2NPs in vivo assays must be investigated.

The Effect of Inactivating Heterozygous Mutation in NBS1 Gene on DNA Damage and Repair Markers and Apoptosis Markers in Mice.

We studied the state of the DNA repair system and apoptosis in young mice carrying heterozygous inactivating mutation in the NBS1 gene (c.1971insT, p.Arg658Stop). In the peripheral blood cells of 4-month-old NBS1insT males, the %DNA in the comet tail was higher by 10% than in wild-type mice (wt) (p<0.05). In hepatocytes of NBS1insT mice, the proportion of γH2AX+ nuclear regions marking DNA double-strand breaks was lower by 2 times than in wt mice (p<0.05), which can be an indicator of less efficient DNA repair. In the kidney tissue of NBS1insT mice, a tendency towards the proapoptotic ratio of Bax and Bcl-2 protein markers was revealed against the background of their reduced expression. Thus, the disturbances detected NBS1insT mice in young age suggest that this model is promising for further studies of carcinogenesis.

Photoactive conjugated microporous polymer@C60 with quencher on tailed Y-triangular DNA structure for high-performance signal-off photoelectrochemical biosensing

Herein, we synthesized a conjugated microporous polymer (CMP) decorated C60 (CMP@C60) with high photoelectric conversion efficiency, in which continuously repeated donor-acceptor (D-A) π electron unit within one molecule of CMP on C60 could not only effectively increase the mobility of photogenerated carriers with improved electron transmission, but also constitute the cascade energy band matching with reduced electron-hole recombination. Based on the high-performance of CMP@C60 for producing exciting initial photoelectrochemical (PEC) signal, a sensitive signal-off sensing platform was designed for lead ion (Pb2+) assay by coupling with quencher methylene blue (MB) interacting on efficient long tailed Y-triangular DNA structure (LYTD). The proposed LYTD with a tripod structure could generate six long tails in situ on its side at the same time via a simple hybridization chain reaction (HCR), providing notably grooves on electrode to accommodate quencher MB to significantly depress the signal for sensitive detection of Pb2+. As a result, the proposed PEC biosensor revealed excellent analysis capability with a low detection limit of 0.3 fM (S/N = 3). Additionally, it also showed satisfactory stability in the detection of tap water samples, lake water samples and clinical serum samples, manifesting great application prospect in the areas of environmental pollutant detection.

Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial.

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) in % Tail DNA units indicates the importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern.

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.

Evaluation of Primary DNA Damage in Young Healthy Females Based on Their Dietary Preferences

DNA damage is known to be associated with many adverse health outcomes, including cancer and chronic diseases, but also with the process of aging. Empirical evidence has shown that environmental exposures, such as certain lifestyle factors, can affect a variety of health-related biomarkers and also impact the stability of DNA through the upregulation of the antioxidant defense system and alteration of its repair capacity. In addition to exercising, diet is an important lifestyle factor that can affect the development of a variety of chronic diseases and growing evidence suggests that plant-based diets, including vegetarianism, may promote health, longevity, and well-being. Therefore, we aimed to assess the primary DNA damage in 32 young healthy females from Zagreb, Croatia, based on their dietary preferences. The participants were divided into two groups: vegetarians and non-vegetarians, where the non-vegetarian group was further divided into omnivores (traditional mixed diet) and pescatarians (consumption of fish and seafood). According to statistical analysis, the DNA damage measured in whole blood cells expressed as the % tail DNA was significantly (p &lt; 0.05) higher in vegetarians (3.6 ± 1.1%) compared to non-vegetarians (2.8 ± 1.0%). When further dividing the participants into specific sub-groups, lower DNA damage was observed amongst omnivorous subjects (3.2 ± 0.8%) compared to vegetarians, with the lowest DNA damage found in females practicing a pescatarian diet (2.4 ± 1.1%). Although a vegetarian diet can lead to a higher intake of specific vitamins and micronutrients, it can also lead to a deficiency of iron, calcium, and total proteins, which may affect genome stability and induce oxidative stress. Even though our results have shown that the pescatarian diet would be more beneficial in terms of maintaining DNA integrity, further research should be carried out to assess how specific dietary preferences affect DNA integrity on a larger scale.

Genotoxicity evaluation of orally administered styrene monomer in mice using comet, micronucleus, and Pig-a endpoints.

Male B6C3F1 mice were administered styrene monomer by oral gavage for 29 consecutive days at dose levels of 0, 75, 150, or 300 mg/kg/day. The highest dose level represented the maximum tolerated dose based on findings in a 28-day dose range-finding study, in which the bioavailability of orally administered styrene was also confirmed. The positive control group received ethyl nitrosourea (ENU; 51.7 mg/kg/day) on Study Days 1-3 and ethyl methanesulfonate (EMS; 150 mg/kg/day) on Study Days 27-29 by oral gavage. Approximately 3 h following the final dose, blood was collected to assess erythrocyte Pig-a mutant and micronucleus frequencies. DNA strand breakage was assessed in glandular stomach, duodenum, kidney, liver, and lung tissues using the alkaline comet assay. The %tail DNA for stomach, liver, lung, and kidney in the comet assay among the styrene-treated groups was neither significantly different from the respective vehicle controls nor was there any dose-related increasing trend in any of the tissues; results for duodenum were interpreted to be inconclusive because of technical issues. The Pig-a and micronucleus frequencies among styrene-treated groups also did not show significant increases relative to the vehicle controls and there was also no evidence for a dose-related increasing trend. Thus, orally administered styrene did not induce DNA damage, mutagenesis, or clastogenesis/aneugenesis in these Organization of Economic Co-operation and Development test guideline-compliant genotoxicity studies. Data from these studies can contribute to the overall assessment of genotoxic hazard and risk posed to humans potentially exposed to styrene.

Baseline and oxidatively damaged DNA in end-stage renal disease patients on varied hemodialysis regimens: a comet assay assessment.

Global estimates exhibit that one million people have end-stage renal disease, a disease-state characterized by irreversible loss of kidney structure and function, thus necessitating renal replacement therapy. The disease-state, oxidative stress, inflammatory responses, as well as the treatment procedure can have damaging effects on the genetic material. Therefore, the present study was carried out to investigate DNA damage (basal and oxidative) using the comet assay in peripheral blood leukocytes of patients (n = 200) with stage V Chronic Kidney Disease (on dialysis and those recommended but yet to initiate dialysis) and compare it to that in controls (n = 210). Basal DNA damage was significantly elevated (1.13x, p ≤ 0.001) in patients (46.23 ± 0.58% DNA in tail) compared to controls (40.85 ± 0.61% DNA in tail). Oxidative DNA damage was also significantly (p ≤ 0.001) higher in patients (9.18 ± 0.49 vs. 2.59 ± 0.19% tail DNA) compared to controls. Twice-a-week dialysis regimen patients had significantly elevated % tail DNA and Damage Index compared to the non-dialyzed and to the once-a-week dialysis group implying dialysis- induced mechanical stress and blood-dialyzer membrane interactions as probable contributors to elevated DNA damage. The present study with a statistically significant power implies higher disease-associated as well as maintenance therapy (hemodialysis)-induced basal and oxidatively damaged DNA, which if not repaired has the potential to initiate carcinogenesis. These findings mark the need for improvement and development of interventional therapies for delaying disease progression and associated co-morbidities so as to improve life expectancy of patients with kidney disease.

Dietary administration of probiotic Bacillus coagulans and Mentha piperita can protect histological architecture and DNA damage in Catla catla (Hamilton, 1822)

The present experimental investigation was executed to evaluate the combined effect of probiotic bacterium Bacillus coagulans along with herb Mentha piperita on histological architecture; and extent of DNA damage after challenge trial with pathogenic bacterium Aeromonas hydrophila in Catla catla. In this experiment, six dietary treatments in triplicate were formulated; C0, M3 and M6 in which M. piperita was supplemented at 0, 3, 6 g kg−1 of feed, while in dietary treatments CP, MP3 and MP6; supplementation of probiotic bacterium B. coagulans at the rate of 3000 CFU g−1 was done along with different inclusion level of M. piperita (0, 3, 6 g kg−1 of feed). Fishes were fed for 60 days on different dietary treatments. Growth performance, histological study and extent of DNA damage using comet assay was examined. Significantly (p < 0.05) high growth % gain in BW was observed in treatment MP6 followed by treatment M6. The histological analysis clearly revealed that dietary M. piperita without or with probiotic bacterium can protect the histoarchitecture of intestinal layers and liver even after challenge with A. hydrophila. Analysis of various comet parameters (tail length, olive moment, tail DNA, head DNA) affirmed that M. piperita supplemented diet can reduce the extent of DNA damage in fishes after challenge trial with A. hydrophila, however dietary supplementation of probiotic bacterium along with M. piperita can reduce the DNA damage up to larger extent confirming its combined protective effect of supplemented diets.

Cryopreservation of sperm in the spotted scat, Scatophagus argus (Linnaeus, 1766) - Conservation and aquaculture perspectives

This study emphasizes the need for sperm quality assessment and cryopreservation strategy in the spotted scat Scatophagus argus as the occurrence of male population of this species is limited. Analyses of sperm quality were made including ionic content and osmolality of seminal plasma and blood serum. Sodium chloride (0.9%) was optimized as the extender with a dilution ratio of 1:3 (milt:cryomedium) for all experiments. Cryoprotectants including dimethyl sulfoxide (DMSO), dimethyl acetamide (DMA), glycerol (GLY), propylene glycol (PG), ethylene glycol (EG) and methanol (MeOH) were screened at various concentrations and equilibration times. Cryopreservation was performed using the cryoprotectants DMSO, GLY, EG, and MeOH individually as well as in combinations. Experiment 1 was done with DMSO, GLY, EG and MeOH (5, 10 and 15%); experiment 2 was done with DMSO (5%) + GLY (5%), DMSO (5%) + EG (5%), DMSO (5%) + MeOH (5%). Simultaneously, experiment 3 was done with both cryoprotectants and co-cryoprotectants viz. DMSO (5%) + egg yolk (5%), DMSO (5%) + coconut water (5%), DMSO (5%) + tomato water (5%) and DMSO (5%) + honey (5%). The cooling sample was optimized and performed with a one-step freezing protocol of −5 °C/min from 25 °C to −150 °C. The results indicated that samples subjected to experiment 2 using DMSO (5%) + EG (5%) for 60 days of storage registered maximum motility (82.33 ± 2.51%), duration time of sperm motility (10.45± 1.01 min) and viability (84.14 ± 5.29%) of post-thaw cryopreserved sperm. Morphology and ultrastructure of post-thaw sperm showed smooth surface topography and normal architecture of cellular organelles. Biochemical analysis indicated a reduction in the protein content of post-thaw cryopreserved sperm. DNA integrity (Comet assay) showed 6.30 ± 1.00% of tail DNA in post-thaw sperm, whereas fresh sperm registered 2.41 ± 1.85%. Functional integrity using the HOST of post-thaw sperm showed a maximum of 82.66 ± 1.15% with DMSO (5%) + EG (5%). Finally, fertility of post-thaw sperm using mature eggs of wild-caught females indicated the entry of sperm by breaking the vitelline membrane. Overall it was demonstrated that the DMSO (5%) + EG (5%) was amenable to the long-term preservation of sperm. Therefore, this protocol can be used as an alternative for male broodstock development under captive breeding program.

Methanolic fraction of Cassia fistula L. bark exhibits potential to combat oxidative stress and possess antiproliferative activity

ABSTRACT Cassia fistula L. is well known for its traditional medicinal properties as an anti-inflammatory, hepatoprotective, antifungal, antibacterial, antimutagenic, and wound healing agent. The aim of the present study was to determine antioxidant, genoprotective, and cytotoxic potential of different fractions of C. fistula bark including hexane (CaMH), chloroform (CaMC), ethyl acetate (CaME), and methanol (CaMM). Among all the fractions studied, CaMM exhibited maximal radical scavenging activity in antioxidant DPPH assay, Superoxide anion radical scavenging assay and nitric oxide radical scavenging assay displayed an IC50 value of 18.95, 29.41, and 13.38 µg/ml, respectively. CaMM fraction possessed the highest phenolic (130.37 mg gallic acid equivalent/g dry weight of extract) and flavonoid (36.96 mg rutin equivalent/g dry weight of fraction) content. Data demonstrated significant positive correlation between polyphenol levels and radical scavenging activity. Single cell gel electrophoresis (Comet assay) exhibited genoprotective potential of C. fistula bark fractions against DNA damage induced by hydrogen peroxide (H2O2) in human lymphocytes. CaMM fraction displayed highest protective ability against H2O2 induced-toxicity as evidenced by significant decrease in % tail DNA content from 30 to 7% at highest concentration (200 µg/ml). CaMM was found to be rich in catechin, gallic acid, chlorogenic acid, and kaempferol. The phenolic content and antioxidant ability of the fractions was markedly negatively correlated with H2O2- induced DNA damage in human lymphocytes. Cytotoxic potential was evaluated against dermal epidermoid carcinoma (A431), pancreatic (MIA PaCa-2) and brain glioblastoma (LN-18) cancer cell lines using MTT assay. Results showed that C. fistula bark fractions possessed highest toxicity against the skin carcinoma cells. CaMM fraction reduced over 50% cell growth at the concentration of 76.72 µg/ml in A431 cells. These findings suggest that fractions of C. fistula bark exhibit potential to be considered as therapeutic agents in various carcinomas.

Oxidative stress and DNA fragmentation in frozen/thawed common Carp Cyprinus carpio sperm with and without supplemental proteins

Using cryopreservation techniques can increase the effectiveness of reproducing cultured fish species by ensuring a dependable supply of sperm, although the quality of the sperm could be impacted by the procedures involved. The goal of this study was to investigate the effect of purified seminal plasma transferrin (Tf), bovine serum albumin (BSA), and antifreeze protein (AFP) types I and III at 1 µg mL−1 on relevant characteristics of cryopreserved sperm from common carp Cyprinus carpio. We compared oxidative stress indices, antioxidant activity, and DNA fragmentation of fresh sperm to that frozen with extender only or with Tf, BSA, or AFP types I and III. Fresh sperm had significantly lower levels of thiobarbituric acid reactive substances (TBARS) compared to samples that underwent cryopreservation without protein treatment, which resulted in 0.54 ± 0.06 nmol/108 cells of TBARS. Carbonyl derivatives of proteins (CP) decreased significantly (ANOVA; P > 0.05) in carp sperm with addition of Tf, AFPI, and AFPIII. Significant differences in superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) activity were seen in sperm supplemented with Tf, BSA, AFPI, and AFPIII from those without. Significantly less DNA damage, expressed as percent tail DNA (11.56 ± 1.34) and olive tail moment (0.59 ± 0.13), was recorded in samples cryopreserved with Tf. The findings indicated that addition of Tf, BSA, AFPI, or AFPIII to cryopreservation medium is beneficial to sperm preservation. The mechanisms through which these proteins act positively on sperm need to be further investigated.