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The aim of the work was to determine monensin, narasin hepatotoxicity and the nature of cell death. Rat hepatocyte model cell line (FAO) was used to investigate two ionophore antibiotic cytotoxic effects estimated by MTT, NRU and KB tests approved by INVITTOX. Additionally, the apoptotic/necrotic nature of cell death was determined by propiodine iodide and HO 342 staining of the cultured hapatocytes. IC₅₀ indices for monensin and narasine estimated by using the MTT test during a 24 hour incubation period were at a level of 0,027 ± 0,001 µM and 0,037 ± 0,001 µM, respectively. However, an incubation period of 48 hrs yielded an equal value - 0,02 µM - for both ionophores. Contrary to the MTT test, NRU and KB estimations demonstrated lower IC₅₀ values for narasine than for monensin. These results correlated to in-vivo acute toxicity and LD₅₀ indices in rats (data from references). The apoptotic nature of hepatocyte death dominated in the cultures. The article also discussed the mechanisms of ionophore induced cytotoxicity.
The aims of the study were determining the median cytotoxicity indicate (IC50), nature of cell death (apoptosis/ necrosis), assessment and morphology of changes observed in FAO cell line culture of hepatocytes subjected to ionophore antibiotics, salinomycin and lasalocid, incubations. INVTTOX recommended MTT, NRU and KB cytotoxicity tests were used to research mitochondrial, protein synthesis and cell proliferation. In addition cell staining in order to reveal membrane destruction that established cell death character May-Grunwald & Giemsa staining were also conducted. Cytotoxicity indices (IC50) estimated by the 24 hour MTT test were at a level 2.41 ± 0.29 mM and 7.93 ± 0.01 mM; however, after a 48 hour incubation the values lowered to 0.112739 ± 0.01 mM and 0.59 ± 0.01 mM for salinomycin and lasalocid, respectively. In contrast to the MTT data, that of NRU and KB tests were higher, indicating mitochondria as the main subcellular target for the antibiotic action. The determined IC50 values were positively related to DL50 (the data from references). Hepatocytes death were established to be of an apoptosis nature. Cell morphology was changed from IC50 depending on manner; the lower value of the indicate corresponded to more pronounced cytopathologic findings. Summarizing, monolayer cell cultures of rat hepatocytes proved to serve as a useful model for cytotoxicity studies enabling to indicate subcellular targets for ionophore antibiotics
Lasalocid is an ionophore coccidiostatic agent frequently used in poultry. Its extensive use causes the formation of residues in edible tissues and eggs which may pose a risk to consumers. Silybin is the main compound extracted from the herb milk thistle Silybum marianum and its hepatoprotective effect has been reported in literature. The aim of the study was to compare lasalocid and silybin cytotoxic effects followed by their combined use in HepG2 cell line. A cytoprotective effect resulting from the interaction of both pharmacologically active substances was measured. In this study, an MTT test, coomassie brillant blue binding test, and LDH release test determined the effective concentration (EC₅₀) of the compounds. The isobolograms and combination index were used to assess the nature of interaction. The lowest EC₅₀-value for lasalocid was established via the MTT test. This study revealed a lack of silybin cytotoxic effect on the cells. Co-actions of the two drugs led to a significant decrease of lasalocid cytotoxicity. The isobolograms and combination index showed a remarkable antagonistic effect in the course of silybin and lasalocid interaction. The results indicate that silybin revealed a cytoprotective effect when incubated with lasalocid since its cytotoxic impact on HepG2 cells has been significantly diminished.
The cvtoprotective effect of silibinin in course of cytotoxicity induced by lasalocid had been measured in rat hepatoma FaO cell line. In the course of the study, MTT test (cellular metabolism), coomassie brillant blue binding test - CBB (total cellular proteins), and LDH release test (membrane integrity) were applied. In addition, changes in the cell morphology after 24 h treatment were observed by light microscopy. The effective concentrations, EC₅₀ were quantified for each compound alone, whereas the nature of their co-action was assessed by isobologram plotting. Lasalocid EC₅₀ ranged from 4 to 10 µM and microphotographs showed significant morphological changes of the cells after 24 h exposure. Silibinin EC₅₀ ranged from 40 to 42 µM for MTT and CBB assays, and 63 µM for LDH assay, and no significant morphological changes occurred. When lasalocid EC₅₀ was used in combination with silibinin in 1-250 µM concentrations, the EC₅₀ values were plotted at 36 µM and 72 µM in MTT and LDH assays, respectively. Thus co-actions of the two drugs led to significant diminishing of lasalocid cytotoxicity in respect to cellular metabolism and membrane integrity. The isobolograms showed remarkable antagonistic effect of silibinin in the course of lasalocid cytotoxic action. Although a considerable interaction was concisely relevant to hepatoma cell line FaO, the promising results incline to extend the study on other in vitro models (primary hepatocytes), as well as to investigate in vivo the cytoprotective effect of silibinin against lasalocid in target animals.
The goal of this study was to evaluate the effect of various concentrations of interferon-tau (IFN-τ) with or without steroid hormones, 17ß estradiol or progesterone, on the proliferation of bovine endometrial cells in vitro. Endometrial epithelial and stromal cells were isolated from the uterus of cows during the early estrus cycle (2-3 days) and incubated with different doses of IFN-τ with or without steroid hormones. The proliferation was determined by the MTT test in 48, 96, and 144 h of incubation. An antiproliferative activity of IFN-τ was observed both in epithelial and stromal cells cultured in RPMI 1640 medium supplemented with 10% FBS or serum replacement. However, epithelial cells were more sensitive to antiproliferative action of interferon-tau. It;s activity was dose- and time-dependent. The inhibition of epithelial cell proliferation by 50% (ED50) was achieved at concentrations of 500 U/ml, 340 U/ml, and 8.8 U/ml of IFN-τ after 48, 96, and 144 h of incubation, respectively. None of the doses of IFN-τ (10-10.000 U/ml) used inhibited stromal cell proliferation in 50%. The most effective dose of IFN-τ inhibiting stromal cell proliferation was 10.000 U/ml, which decreased cell growth by 17.08%, 22.87%, and 2.6% after 48, 96, and 144 h of incubation, respectively. Steroid hormones, 17ß estradiol and progesterone, added to the culture of stromal cells with or without IFN-τ did not significantly modulate stromal cell growth. In contrast, a high concentration of progesterone (10-5M) alone significantly enhanced stromal cell growth. Progesterone at low, physiological concentrations (107 - 10-9 M) ameliorated the antiproliferative activity of IFN-τ, especially at the 109 M concentration. At this concentration, the stimulatory effect on stromal cell growth was observed. The mechanisms of such response are not entirely clear but may arise from the influence of IFN-τ on progesterone down regulation of its own receptor. Depicted activity of IFN-τ may find usefulness in therapy of neoplastic disorders.
Quercetin, one of the major flavonoids, exhibits many beneficial effects on human organism as antihistamine, antioxidant, anti-inflammatory, anticancer and antiviral drug. It is recommended as suplement of healthy diet but still the knowledge of its beneficial effect on normal cells is not satisfactory. We decided to examine the effect of flavonoid on neurons morphology and their susceptibility to cell death. Fractal analysis of rat neurons revealed that 24 hours long incubation with quercetin diminished neuronal arborisation in cortical neurons. Neurons also appeared to be very sensitive to cell death after flavonoid treatment in concentration dependent manner. Over 50% of cells died after incubation with 15 μg/ml of flavonoid while 1 μg/ ml of quercetin induced cell death only in 5%. Staining with Hoechst 33342 and propidium ioidide revealed the two types of cell death: apoptosis and necrosis. The number of apoptotic cells was comparable with necrotic ones. These results suggest toxic effect of quercetin on neurons what should be taken into consideration in further studies on using quercetin as therapeutic agent.
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