Doramectin induced cytotoxic and genotoxic effects on bovine peripheral lymphocytes and cumulus cells in vitro

The effect of doramectin (DOR) was tested on two experimental somatic bovine cells in vitro: peripheral lymphocytes (PL) and cumulus cells (CC). The cytotoxicity and genotoxicity of DOR were assessed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, single cell gel electrophoresis assay (SCGE) and cytokinesis-block micronucleus cytome (CBMN Cyt) assay. Both cells were treated with three concentrations of DOR (20, 40, 60?ng?mL^–1) for 24?h. The results obtained from PL demonstrated that DOR was able to induce cytotoxic effect and DNA damage with all concentrations tested. Additionally, DOR increased micronuclei (MNi) frequency and nuclear buds (NBuds) with 20, 40, 60?ng?mL^–1, and nucleoplasmic bridges (NPBs) only with 40?ng?mL^–1. On the other hand, the three concentrations of DOR were not able to induce cytotoxic effect and DNA damage using SCGE in the bovine CC. Nevertheless, the two higher concentrations of DOR (20, 40?µg mL^–1) significantly increased the frequency of micronucleus formation in bovine CC. These results represent the first experimental evidence of genotoxic and cytotoxic effects exerted by DOR on bovine PL and CC.     

Atrazine effects on in vitro maturation and in vitro fertilization in the bovine oocyte

The effect of low levels of atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) on in vitro oocyte maturation, in vitro capacitation of sperm, or in vitro fertilization of bovine oocytes and on the quality of blastocyst formation was studied. Bovine oocytes collected from abattoir ovaries were matured, fertilized, and developed to the blastocyst stage in vitro. Embryos that reached a morula or blastocyst stage were stained with Hoechst 33258 stain to determine the number of blastomeres per embryo. Three bulls whose fertilization rates were proven consistent among straws were used for this study. Atrazine was tested at concentrations of 0.01, 0.1, 1, and 10 microM in either the maturation medium, sperm capacitation medium, or the fertilization medium. Because atrazine was dissolved in ethanol, an ethanol control was used to determine any possible effects of ethanol on the in vitro process. The addition of atrazine to both the maturation and fertilization media did not result in any significant difference in fertilization rates between the controls and the treatments. In the capacitation medium, a significant difference between the controls and the atrazine levels of 0.1, 1, and 10 microM was noted for one bull. Atrazine did not affect the number of blastomeres per embryo. There was not a significant difference (p>0.05) in the number of blastomeres per embryo between the controls and the different levels of atrazine in each medium. This study indicates that low levels of atrazine do not have an effect on in vitro fertilization rates or the number of blastomeres per embryo produced in vitro.     

Molecular,functional, and cellular alterations of oocytes and cumulus cells induced by heat stress and shock in animals

Environmental Science and Pollution Research - Global warming is considered as the main environmental stress affecting ecosystems as well as physiological and biochemical characteristics, and...     

Evaluation of potential genotoxic/cytotoxic effects induced by epoxiconazole and fenpropimorph-based fungicide in bovine lymphocytes in vitro

Potential genotoxic/cytotoxic effects of the epoxiconazole/fenpropimorph-based fungicide were investigated using single cell gel electrophoresis and cytogenetic assays: chromosomal aberrations, sister chromatid exchanges, micronuclei and fluorescence in situ hybridization in cultured bovine lymphocytes. No statistically significant elevations of DNA damage and increases in cytogenetic endpoints were seen. However, evident cytotoxic effect presented as a decrease in mitotic and proliferation indices were recorded after exposure of bovine lymphocytes to the fungicide for 24 and 48 h at concentrations ranging from 3 to 15 µg mL^?1 (P < 0.05, P < 0.01, P < 0.001). Similarly, for 24 h an inhibition in the cytokinesis block proliferation index (CBPI) was obtained after exposure to the fungicide at concentrations ranging from 1.5 to 15 µg mL^?1 (P < 0.01, P < 0.001) in each donor.     

Effect of triazole pesticide formulation on bovine culture cells

To date, most data about the possible genotoxic effect of triazole pesticides are focused on laboratory animals resulting in limited information on further non-target organisms such as cattle. The objective of the present study was to investigate the effect of triazole (tebuconazole/prothioconazole) fungicide formulation on the induction of chromosomal aberrations (CAs), sister chromatid exchanges (SCEs) and DNA fragmentation in bovine cultured lymphocytes. Our results showed that the fungicide formulation did not induce significant number of CAs in bovine cells after 24 h treatment. Nevertheless, the dose-dependent reduction of mitotic division was observed, with the strongest effect at 30.0 μg mL^?1 in both donors (P < 0.01 and P < 0.001, respectively). Prolonged 48 h exposure caused the increased level of breaks in treated cultures (3.0?15.0 μg mL^?1; P < 0.05) and significant decrease in mitotic index (MI). The tested fungicide failed to produce any statistical changes in the SCE frequency neither after 24 h nor 48 h treatment. However, the significant decline of the proliferation index (PI) was observed after 24 h indicating the fungicide influence on cell cycle kinetics. Prolonged 48 h exposure caused cytotoxicity reflecting in lower PI value relative to control mainly at the highest fungicide concentrations (30.0 μg mL^?1, P < 0.001). Using painting probes for bovine chromosomes 1, 5 and 7 (BTA1, BTA5 and BTA7) only low levels of aneuploidies were detected. Significant increase of polyploidy cells (P < 0.05) was induced by a 3.0 μg mL^?1 dose of the fungicide after 48 h. DNA fragmentation assay didn't reveal the presence of DNA nucleosome ladder in cell cultures at any time (24 h and 48 h) and fungicide concentration.     

Carbon black nanoparticles promote the maturation and function of mouse bone marrow-derived dendritic cells

Particulate matter including carbon black (CB) nanoparticles can enhance antigen-related inflammation and immunoglobulin production in vivo. Dendritic cells (DC) as antigen-presenting cells (APC) are the most capable inducers of immune responses. The present study was designed to determine whether CB nanoparticles affect the maturation/activation and function of DC in vitro. DC were differentiated from bone marrow (BM) cells of BALB/c mice by culture with granulocyte macrophage colony stimulating factor (GM-CSF). At day 8 of culture, BM-derived DC (BMDC) were exposed to CB nanoparticles with a diameter of 14nm or 56nm for 24h. The expression of major histocompatibility complex (MHC) class II, DEC205, CD80, and CD86 (maturation/activation markers of BMDC) was measured by flow cytometry. BMDC function was evaluated by an allogeneic mixed lymphocyte reaction (MLR) assay. CB nanoparticles significantly increased the expression of DEC205 and CD86 in BMDC and tended to increase MHC class II and CD80 expression; however, a size-dependent effect was not observed. On the other hand, BMDC-mediated MLR was significantly enhanced by the CB nanoparticles and the enhancement was greater by 14nm CB nanoparticles than by 56nm CB nanoparticles. Taken together, CB nanoparticles can promote the maturation/activation and function of BMDC, which could be related to their effects on allergic diseases and/or responses. In addition, BMDC-mediated MLR might be useful assay for in vitro screening for adjuvant activity of environmental toxicants.     

Genotoxic and cytotoxic effects of pethoxamid herbicide on Allium cepa cells and its molecular docking studies to unravel genotoxicity mechanism

Environmental Science and Pollution Research - Pethoxamid is chloroacetamide herbicide. Pethoxamid is commonly used to kill different weeds in various crops. Pethoxamid can leach in the water and...     

The effect of thiacloprid formulation on DNA/chromosome damage and changes in GST activity in bovine peripheral lymphocytes

The potential genotoxic effect of thiacloprid formulation on bovine peripheral lymphocytes was evaluated using the comet assay and the cytogenetic endpoints: chromosome aberrations (CAs), sister chromatid exchanges (SCEs) and micronuclei (MNi). Whole blood cultures were treated with the insecticide at concentrations of 30, 60, 120, 240 and 480 μg mL^?1 for 24, 48 h and/or 2 h of incubation. A statistically significant increase in the frequency of DNA damage, as well as in unstable chromosome aberrations (% breaks) were found after exposure to the insecticide at concentrations ranging from 120 to 480 μg mL^?1 (P < 0.05, P < 0.01, P < 0.001). For the detection of stable structural chromosome aberrations (e.g., translocations) and numerical aberrations by the FISH method, three whole chromosome painting probes for bovine chromosomes 1, 5 and 7 (BTA1, BTA5 and BTA7) were used in our experiments. We observed numerical aberrations, but without any statistical significance. Regarding the sister chromatid exchanges, no significant elevation in the SCE frequencies was found after 24-h exposure to the insecticide. A dose-related response in the SCE induction was obtained in bovine cultures after the prolonged time of exposure (48 h) to thiacloprid formulation at concentrations ranging from 120 to 480 μg mL^?1 in each donor (P < 0.05, P < 0.01), which was associated with a reduction of the PI (P < 0.05, P < 0.01). The insecticide failed to produce MNi; however, a significant reduction of CBPI was observed. Using real-time PCR, a decrease in the expression of bovine glutathione S-transferase M3 (GSTM3) was detected at the lowest dose. The higher concentrations of thiacloprid formulation caused an increase in the mRNA expression.     

Correction to: Genotoxic and cytotoxic effects of pethoxamid herbicide on Allium cepa cells and its molecular docking studies to unravel genotoxicity mechanism

Environmental Science and Pollution Research -     

Neurotoxic effect and metabolic responses induced by a mixture of six pesticides on the earthworm Aporrectodea caliginosa nocturna

The effects of a mixture of insecticides and/or fungicides at different environmental concentrations were investigated on a Aporrectodea caliginosa nocturna population. This laboratory experiment was carried out in order to reproduce Gaillac (France) vineyard conditions. Neurotoxicity (cholinesterase), metabolisation (glutathione-S-transferase) and oxidative stress (catalase) enzymes were studied as biomarkers in earthworms after short-term exposure in terraria. The aim was to observe the global effects of pesticide exposure, as in a vineyard, rather than focus on each isolated biomarker variation, or on each compound's impact. ChE activity was inhibited after a few days of insecticide and/or fungicide exposure, indicative of a neurotoxic effect in earthworms. The significant increase in GST and CAT activities revealed the metabolisation of these products resulting in the production of reactive oxygen species. After a long period of exposure or high concentrations, earthworms were physiologically damaged: they could not cope with the high toxicity (cellular dysfunction, protein catabolism...). Chemical analysis showed that pesticide bioaccumulation in earthworm tissues, even in those exposed to the highest concentrations and for the longest periods, was very low (under LOD) or absent. However, the study of pesticide residues in terraria after 34 days in a climate chamber suggested that earthworms participate in soil pesticide breakdown.     

Low dose exposure of patulin and protective effect of epicatechin on blood cells in vitro

In the present study, we aimed to assess antioxidant status in erythrocytes in vitro after patulin (PAT) and epicatechin exposure by measuring antioxidant enzymes (superoxide-dismutase – SOD, glutathione peroxidase – GPx and catalase – CAT) and parameters associated with oxidative stress (malondialdehyde – MDA and ROS). We also investigated the effect of PAT on viability and count of lymphocytes and lymphocyte subpopulations in rabbit blood in vitro. Whole blood of rabbits was used for analysis of antioxidant changes in rabbit erythrocytes after epicatechin and PAT treatment (separately or in combination, at concentrations of 0.2; 2; 20; 200?µg mL^–1 of epicatechin and 0.5; 5; 10?µg mL^–1 of PAT). Whole blood of rabbits was also used for analysis of count and viability of lymphocytes after PAT treatment at concentrations of 10; 25 and 50?µg mL^–1. Results from our experiment confirmed the ability of epicatechin to protect cells against oxidative stress and lipoperoxidation. Our findings indicate that mycotoxin PAT in low concentrations did not affect the activity of antioxidant enzymes in erythrocytes of rabbits significantly. Only slight non-significant changes in lymphocytes count after treatment with low doses of PAT in rabbit blood were observed.     

The cytotoxic effect of wastewater from the phosphoric gypsum depot on common oak (Quercus robur L.) and shallot (Allium cepa var. ascalonicum)

The effect of wastewater from a phosphoric gypsum depot on common oak, Quercus robur L., at cytogenetical level was studied. Allium-test was used as a control. The treatment of common oak seedlings with wastewater under laboratory conditions caused mitodepressive effect. Chromosome aberrations and mitotic irregularities were found. Cytogenetic analysis of common oak seedlings grown from acorns collected near the depot did not show changes in mitotic activity in comparison to control but the number of aberrations was higher than in control. In comparison to Alliumtest common oak was found to be more tolerant to wastewater from the phosphoric gypsum depot.     

Effect of combustion condition on cytotoxic and inflammatory activity of residential wood combustion particles

Residential heating is an important local source of fine particles and may cause significant exposure and health effects in populations. We investigated the cytotoxic and inflammatory activity of particulate emissions from normal (NC) and smouldering (SC) combustion in one masonry heater. The PM_1–0.2 and PM_0.2 samples were collected from the dilution tunnel with a high-volume cascade impactor (HVCI). Mouse RAW 264.7 macrophages were exposed to the PM-samples for 24 h. Inflammatory mediators, (IL-6, TNFα and MIP-2), and cytotoxicity (MTT-test), were measured. Furthermore, apoptosis and cell cycle of macrophages were analyzed. The HVCI particulate samples were characterized for ions, elements and PAH compounds. Assays of elemental and organic carbon were conducted from parallel low volume samples. All the samples displayed mostly dose-dependent inflammatory and cytotoxic activity. SC samples were more potent than NC samples at inducing cytotoxicity and MIP-2 production, while the order of potency was reversed in TNFα production. SC-PM_1–0.2 sample was a significantly more potent inducer of apoptosis than the respective NC sample. After adjustment for the relative toxicity with emission factor (mg MJ^?1), the SC-PM emissions had clearly higher inflammatory and cytotoxic potential than the NC-PM emissions. Thus, operational practice in batch burning of wood and the resultant combustion condition clearly affect the toxic potential of particulate emissions.     

Effects of lime amendment on availability of heavy metals and maturation in sewage sludge composting

A batch composting study was performed to evaluate the feasibility of co-composting sewage sludge with lime, aiming at reducing the availability of heavy metals in the sludge compost. Sewage sludge with sawdust as bulking agent was amended with lime at 0, 0.63, 1.0, and 1.63% w/w, and composted for 100 days in laboratory batch reactors. The changes in temperature, pH, electrical conductivity (EC), and extractable heavy metal contents were measured while compost maturity was determined by C/N(organic) and cress seed germination during the composting period. Liming raised pH of compost effectively at the initial stage of composting and caused a decrease in EC through precipitation of soluble ions. Lime amendment also significantly reduced water-soluble and Diethylene triamine pentracetic acid (DTPA)-extractable metal contents. The maximum reductions were 60 and 40% for Cu, 80 and 40% for Mn, 55 and 10% for Zn, and 20 and 25% for Ni at the end of the composting period for the lime-amended sludge as compared to the control. In spite of the inhibitory effect of lime amendment on the decomposition activity of sewage sludge, all treatments reached maturation after 63 days of composting as indicated by the results of C/N(organic) ratio and cress seed germination test results. A lime amendment of < or =1.0% is recommended to co-compost with sewage sludge.     

利用新型高温好氧堆肥器提高鸡粪谷壳有机肥肥效

针对传统堆肥器存在堆肥时间长、环境污染严重等问题,开发快速、无臭、环保型堆肥器对促进粪污肥料化具有重要意义.研发的新型高温好氧堆肥器主要包括4个部分:控制面板、发酵罐(50 L有效容积)、空压机通风系统(0.15 m3·s-1排气量和40 L气容量)、氨气吸收系统(188 L容积及内部成阶梯环形吸收模式).利用新型高温...     

The effect of deoxynivalenol on the secretion activity, proliferation and apoptosis of porcine ovarian granulosa cells in vitro

The aim of this in vitro study was to examine the secretion activity, markers of proliferation and apoptosis in porcine ovarian granulosa cells (GCs) after deoxynivalenol (DON) addition. Ovarian granulosa cells were incubated with DON for 24h: 10, 100 and 1000 ng/mL, while the control group received no DON. The secretion of insulin-like growth factor I (IGF-I) and progesterone was determined by radioimmunoassay (RIA) and expression of cyclin B1, PCNA and caspase-3 by immunocytochemistry. IGF-I release by GCs was inhibited by DON, while progesterone release and the expression of cyclin B1 was stimulated by DON (at 1000 ng/mL but not at 10 and 100 ng/mL). PCNA expression was stimulated by DON (at 100 and 1000 ng/mL but not at 10 ng/mL). Caspase-3 expression was not influenced by DON treatment (at all doses). In conclusion, our results indicate, (1) a direct effect of DON on secretion of growth factor IGF-I and steroid hormone progesterone, (2) expression of markers of proliferation (cyclin B1 and PCNA) but not on the (3) expression of marker of apoptosis (caspase-3) in porcine ovarian granulosa cells. This in vitro study suggests the dose-dependent association of DON on porcine ovarian functions.     

The effect of deoxynivalenol on the secretion activity,proliferation and apoptosis of porcine ovarian granulosa cells in vitro

The aim of this in vitro study was to examine the secretion activity, markers of proliferation and apoptosis in porcine ovarian granulosa cells (GCs) after deoxynivalenol (DON) addition. Ovarian granulosa cells were incubated with DON for 24h: 10, 100 and 1000 ng/mL, while the control group received no DON. The secretion of insulin-like growth factor I (IGF–I) and progesterone was determined by radioimmunoassay (RIA) and expression of cyclin B1, PCNA and caspase-3 by immunocytochemistry. IGF–I release by GCs was inhibited by DON, while progesterone release and the expression of cyclin B1 was stimulated by DON (at 1000 ng/mL but not at 10 and 100 ng/mL). PCNA expression was stimulated by DON (at 100 and 1000 ng/mL but not at 10 ng/mL). Caspase-3 expression was not influenced by DON treatment (at all doses). In conclusion, our results indicate, (1) a direct effect of DON on secretion of growth factor IGF-I and steroid hormone progesterone, (2) expression of markers of proliferation (cyclin B1 and PCNA) but not on the (3) expression of marker of apoptosis (caspase-3) in porcine ovarian granulosa cells. This in vitro study suggests the dose-dependent association of DON on porcine ovarian functions.     

Investigation on the interaction between triclosan and bovine serum albumin by spectroscopic methods

Abstract

Multi-spectroscopic and molecular docking methods were used to study the interaction between triclosan (TCS) and bovine serum albumin (BSA). The results indicated that the fluorescence quenching of BSA by TCS was due to the formation of TCS–BSA complex through static quenching. This result was also demonstrated by time-resolved fluorescence experiment. The binding constants and number of binding sites between TCS and BSA were 1.30?×?10⁵ M^?1 and 1.17 at 298?K, respectively. The thermodynamic parameters were studied in detail which suggested that hydrophobic forces and hydrogen bond played major roles in the TCS–BSA interaction. Moreover, the site marker competitive experiments and docking studies revealed that TCS could bind BSA into site I in subdomain IIA. All the results of UV–vis spectrophotometry, circular dichroism spectroscopy and synchronous fluorescence spectroscopy showed that interaction between TCS and BSA induced conformation changes of BSA.     

Estrogenic and antiestrogenic effect of in vitro treatment of follicular cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin

OBJECTIVE: Two types of follicular cells from preovulatory ovary were cultured in vitro separately and in co-culture to test difference in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) action on particular cell types. METHODS: The accumulation of TCDD in follicular wall was analysed using coupled capillary gas chromatography mass spectrometry. Whole preovulatory follicles were isolated from ovary and incubated with prolonged exposure to 0.1 nM TCDD or single exposure to 10 nM TCDD for four days. In the second part of experiments direct effects of TCDD on steroidogenesis were investigated in porcine theca cells (Tc) and granulosa cells (Gc) cultured alone and in co-culture (GT). The media were collected after four days for steroid analysis. RESULTS: 59.3% and 81.2% of TCDD added to the culture medium was accumulated after 0.1 and 10 nM, respectively. TCDD in a dose-dependent manner increased estradiol secretion with concomitant progesterone secretion by theca interna cells. On the other hand decrease of both progesterone and estradiol secretion by granulosa cells cultured alone and in co-culture with theca cells was noted. CONCLUSION: Different cell-specific estrogenic or antiestrogenic effect of TCDD were found in ovarian follicles.