Reduced tumorigenicity of murine hepatoma cells after treatment with antioxidants and melatonin

The tumor growth of murine hepatoma cells MH22a treated with N-acetylcysteine (NAC, 10 mM) and alpha-lipoic acid (ALA, 1.25 mM) antioxidants or hormone melatonin (1 μM) and transplanted into syngeneic (C3HA) mice has been studied. NAC, ALA, or melatonin treatment for 20 h reduced the tumor development and the number of dead mice. Melatonin produced the most pronounced effect. Tumors appeared in 10 days in 100% of control mice injected with untreated cells; the injection of cells pretreated by NAC or ALA generated tumors in 40 and 53% of mice, respectively. Cells pretreated with melatonin produced tumors 18–20 days after injection; 67% of control mice died in 36 days (the observation period). The mortality rate was 20 and 53% if the injected cells were treated with NAC or ALA, respectively. No mice died during this period with melatonin-pretreated cells. We found that treatment with antioxidants delayed (NAC) or completely inhibited (ALA) the progression of the cell cycle of murine hepatoma cells. After the antioxidant removal, the cell cycle was restored. Melatonin did not affect the cell cycle phase distribution. We conclude that there is no direct correlation between the loss of tumorigenic properties and the altered proliferative activity of hepatoma cells. Different mechanisms of antioxidants and melatonin action that underlie the transient normalization of the tumor phenotype are discussed.     

Increased actin nucleating activity in tumorigenic cells

The kinetics of actin polymerization has been used to quantitate the relative levels of actin nucleating activity in extracts from a number of related tumorigenic and non-tumorigenic cells. The level of nucleating activity was significantly elevated in the tumorigenic compared with the non-tumorigenic cell extracts whether the results were expressed on the basis of per protein (2-3 fold increase) or per total endogenous cellular actin (3-4 fold increase). It is concluded that this activity is probably due to an actin filament capping/severing regulatory protein(s) and that this protein(s) may be, at least partially, responsible for the microfilament disruption observed in transformed cells.     

Decrease of acid phosphatase activity in murine peritoneal macrophages infected with Leishmania donovani

The effect of infection with Leishmania donovani on the activity and isoenzyme composition of acid phosphatase within individual murine peritoneal macrophages maintained in vitro was studied. Concentrations of acid phosphatase activity and number of intracellular parasites were quantitated using a computer-assisted cytospectrophotometry system. Changes in the isoenzyme composition of macrophages during infection with L. donovani were detected by comparing the patterns of acid phosphatase levels between macrophages treated in the absence and presence of an enzyme inhibitor. It was observed that the concentration levels of acid phosphatase activity in macrophages were decreased significantly by infection with L. donovani. An inverse relation existed between concentration of acid phosphatase activity and the number of intracellular L. donovani. Reduced concentrations of acid phosphatase activity were also observed in macrophages uninfected but exposed to L. donovani. The isoenzyme composition in macrophages did not change during the course of infection with L. donovani. These results demonstrate that L. donovani reduces the acid phosphatase activity of macrophages.     

Changes in the lectin binding capacities of hepatoma cells after treatment with chondroitinase.

Binding capacities of cells of ascites hepatoma, AH 130 FN, towards lectins were examined before and after treatment with chondroitinase AC. Chondroitin sulfate A was removed from the cells by the enzyme treatment, and the binding capacity of the cells towards $\text{Ricinus communis}$ agglutinin (RCA) increased remarkably while the binding constant was unchanged, whereas that towards Concanavalin A (ConA) remained practically unchanged.     

Decrease of serotonin N-acetyltransferase activity in rat pineal organs after treatment with prostaglandin synthesis inhibitor indomethacin

Administration of indomethacin to rats abolished the cyclic AMP dependent, dark induced rise in serotonin N-acetyltransferase, presumably by inhibiting prostaglandin synthesis.     

Decrease of saturation density of cells of hamster cell lines after treatment with dextran sulfate

Dextran sulfates of various molecular weights were added to cultures of 3 transformed cell lines of hamster, 3T6 cells and embryonic fibroblastic cells. Dextran sulfate of high molecular weight reduced the saturation densities of all the cell lines of hamster and 3T6 cells, but those of low molecular weight did not. The mitotic rate of the treated cells decreased at stationary cell density. Dextran sulfate had no effect on the growth of normal fibroblastic cells derived from mouse and hamster embryos. Viability of treated cells was indicated by the following results. Cells of cultures seeded at different cell densities grew at almost the same rate in the presence of dextran sulfate. Treated cells remaining in the monolayer stage began to grow after removal of dextran sulfate. The colony formation rate of treated cells was the same as that of untreated cells. With the exception of one cell line, the morphology of cells treated with dextran sulfate of high molecular weight was more flattened and there was less overlapping than in untreated cells. Treated cells were less agglutinable to concanavalin A than were untreated cells. These results suggest that dextran sulfate affects the cell surface, resulting in the decrease of saturation density of cell lines.     

Proliferation and tumorigenity of murine hepatoma cells irradiated with polychromatic visible and infrared light

In experiments in vitro, the effects of polychromatic visible (VIS) light combined with polychromatic infrared light (VIS-IR, 480–3400 nm) and the effects of the entire spectrum of VIS radiation (385–750 nm) on viability and proliferative activity of the murine hepatoma cells MH22a were studied. In experiments in vivo, changes in the tumorigenic properties of cells MH22a were studied after the same kinds of light exposure. It was shown that irradiation of hepatoma cells with two kinds of polychromatic light at a wide range of doses (4.8–38.4 J/cm²) did not lead to an increase in the number of dead cells for 24–72 h of cultivation and did not cause deceleration of the hepatoma cell proliferation; moreover, the VIS-IR light at a dose of 4.8 J/cm² and the VIS light at a dose 38.4 J/cm² even promoted more intense cell proliferation after 24 h. In cells irradiated with VIS-IR and VIS light, the proliferation index rose by 1.6 and 1.4 times, respectively, and the time of the cells’ number doubling decreased as compared with control. Studying the tumorigenic properties of irradiated tumor cells has shown that, for 30 days after transplantation to syngenic mice C3HA of hepatoma cells 24 h after their irradiation with VIS-IR light at a dose of 4.8 J/cm², the tumor volume decreased significantly (2.6–4.1 times) at all periods of observation, while the incidence of tumor formation decreased, whereas the survival of the tumor-bearing mice did not change. Transplantation of cells irradiated with the same light at a dose of 9.6 J/cm² did not lead to significant changes in the tumor volume, the tumor formation incidence, and animal survival. The main contribution to the antitumor effect of VIS-IR light seems to be made by the VIS component, as transplantation into mice of cells irradiated with VIS light alone at a dose of 38.4 J/cm² also stimulating proliferation of hepatoma cells in vitro resulted in a decrease of their tumorigenic properties. However, the IR component in the combined VIS-IR radiation enhanced the antitumor effect of the VIS light; as a result, it was manifested after use of doses eight times lower (4.8 J/cm²) than in the case of VIS light alone (38.4 J/cm²). Mechanisms of the decrease of tumorigenic properties of hepatoma cells after irradiation with polychromatic light at doses stimulating their proliferation in vitro are studied.     

Protective effects of antioxidants on deoxynivalenol-induced damage in murine lymphoma cells

As contradictory results have been reported on the immunotoxic properties of deoxynivalenol (DON) in animal studies, we introduced a lymphoblast cell culture model in order to examine the effects of DON on lymphoblastic cell growth and metabolism as well as the preventive properties of free radical scavenger molecules against the DON-induced cell damage. Murine YAC-1 lymphoma cells were used because lymphoblasts have been shown to be sensitive to DON-induced immunotoxicity. Cells were quantified and their proliferative activity was measured by a proliferation test. Lipid peroxidation and protein oxidation were determined using assays quantifying thiobarbituric acid reactive substances (TBARS) and carbonylated proteins. Severely reduced cell counts were detected in DON-treated samples, confirmed by a 5–10 times lower proliferative activity. Significant increases in lipid peroxidation and protein oxidation were found in parallel incubated samples. The pre-incubation with free radical scavengers significantly reduced DON-induced changes to proteins and lipids as well as the tarnished cell viability and cell proliferation. These results suggest that YAC-1 lymphoma cells are a suitable model to investigate and elucidate the basic molecular and cellular mechanisms for possible immunotoxic effects of DON. With regard to the impact of free radical scavengers, the applied in-vitro model might enable the investigation of potential prophylactic and therapeutic effects before or even without harmful animal experiments and cost- and time-intensive expenses.     

Decrease in serum dehydroepiandrosterone level after fenofibrate treatment in males with hyperlipidemia

The influence of steroid hormones on plasma lipids and lipoproteins was confirmed by many studies. On the other hand, the effect of plasma lipids on metabolism of steroid hormones has so far not been examined. The objective of this research project was to determine (1) the levels of cortisol, testosterone, estradiol, dehydroepiandrosterone (DHEA), its sulfate (DHEAS), 7-hydroxylated DHEA, and SHBG in men suffering from mixed hyperlipidemia (HPL) (n=23, age 46.1+/-7.9 years) in comparison with healthy male volunteers (n=17, age 45.1+/-15.6 years); (2) whether therapy with fenofibrate influences the levels of the above mentioned steroids and SHBG; (3) what are the correlations between lipids and steroids in healthy males and HPL patients before and after therapy. Compared to controls, untreated patients had significantly higher estradiol and free testosterone index (IFT) levels (p<0.0003 and p<0.02, respectively) and significantly lower SHBG (p<0.02). Due to fenofibrate therapy, a significant decrease of TC, TG, and DHEA levels occurred (mean decrease: 14 %, 52 % and 21 %, respectively). Triglycerides correlated negatively with testosterone and SHBG in healthy subjects. HDL-C correlated positively and consequently, atherogenic index correlated negatively with 7-hydroxylated epimers of DHEA in treated patients. This is the first study dealing with the influence of fenofibrate administration on the steroid levels. Taking together, the most important is the finding of decrease DHEA levels after fenofibrate therapy. It could be explained, at least in part, by the effect of the fenofibrateon on the biosynthesis of DHEA and its regulation.     

Decrease of body temperature after aglepristone treatment in bitches

Body temperature responses and the timing of abortions were evaluated in pregnant bitches with the anti-progestin aglepristone. Fifteen purebred and crossbred, 25-45 days pregnant, were included in this study and seven untreated bitches at the same stage of pregnancy served as controls. Treated bitches were administered two applications of aglepristone (10 mg/kg SC) 24 h apart for pregnancy termination. Pregnancy termination was confirmed by ultrasonographic assessment. Body temperature was rectally measured three times a day for 6 days beginning 24 h before treatment or pregnancy diagnosis in the treated and control bitches, respectively. Additionally, serum progesterone concentrations were assessed at time points during the study in the treated bitches. Pregnancy was terminated in 14 treated bitches in a mean+/-S.E.M. of 4.3+/-0.7 days after treatment. Control bitches remained pregnant. In the treated bitches, but not in the controls, body temperature significantly decreased 24 h after the beginning of the treatments (P < 0.01) and then gradually returned to pre-treatment values. Correlation between the day of mean minimum body temperature and the day of pregnancy termination was low (0.07; > 0.05). Progesterone did not show significant change throughout the study. Body temperature does not seem to be a suitable variable to clinically monitor the aborting effect of aglepristone. Decrease of body temperature after aglepristone treatment could represent further evidence of its hypothalamic effects.     

Cryopreservation of murine testicular Leydig cells by modified solid surface vitrification with supplementation of antioxidants

Reports on the vitrification of somatic cells are scarce. Here, we show that Leydig cells (murine cell line TM3) could be successfully vitrified by both open vitrification [plastic straw (PS) and plastic vials (PV)] and closed ultravitrification [microdrop (MD) and solid surface vitrification (SSV)], after protocol optimization. However, open ultravitrification resulted in better post-warming viability than closed systems of vitrification with highest success obtained in modified SSV (84.8 ± 1.86%; p < 0.05). Leydig cells vitrified-warmed by modified SSV also showed superior (p < 0.05) cell growth, mitochondrial activity and cytoplasmic esterase enzyme activity, than MD, PS and PV, respectively. It was also observed that vitrified-warmed cells had higher level of ROS activity than non-vitrified control cells (41.6 ± 4.0 vs. 16.7 ± 1.0; p < 0.05). Treatment of cells with glutathione (GSH) or 2-mercaptoethanol (2-ME) (0, 10, 50, 100 μM) significantly (p < 0.05) reduced the ROS activity but had no significant (p > 0.05) effect on post-warm viability. Nevertheless, antioxidant-treated cells had improved mitochondrial activity, cytoplasmic esterase activity and cell growth during in vitro culture (p < 0.05). In conclusion, our results suggest that modified SSV offers a viable method for vitrifying single cell suspension of Leydig cells. To the best of our knowledge, this is the first report on cryopreservation of Leydig cells by vitrification.     

褪黑素对小鼠MFC前胃癌细胞的survivin、caspase-3表达的影响

目的探讨褪黑素（Melatonin,MLT）对小鼠前胃癌细胞（murine foregastric carcinoma,MFC）的生存素survivin和半胱天冬酶-3（cysteinyl aspartate-specific protease-3,caspase-3）表达的影响。方法使用不同浓度褪黑素处理培养的胃癌细胞,运用细胞免疫组化、实时荧光定量PCR、免疫印迹法、分光光度法等方法检测褪黑素在抑制小鼠MFC前胃癌细胞增殖过程中对细胞survivin、caspase-3的表达影响。结果与空白对照组相比,6mmol/L浓度褪黑素干预组的胃癌细胞survivin表达下调,caspase-3蛋白表达上调,并呈剂量依赖性。结论褪黑素能够降低胃癌细胞survivin的表达,进而激活caspase-3的活性,是褪黑素体外抑制肿瘤细胞增殖的作用机制之一。     

Correlation of phenylalanine hydroxylase activity with cell density in cultured hepatoma cells.

We have found that the specific activity of phenylalanine hydroxylase varies over at least a forty-fold range during the growth cycle of Reuber hepatoma (H4) cells growing in monolayer culture. The variation has three phases: (1) a very rapid drop in specific activity upon subculturing to a low cell density; (2) a region of low specific activity and (3) after confluency, a rise to a high specific activity. All the results indicate that the cell density in the culture dish is primarily responsible for this fall and rise in activity. Neither conditioning of the growth medium, the rate of cell division, nor enzyme leakage from the cells appear to play a major role in the changes observed. Lactic dehydrogenase specific activity was determined in all experiments; a much smaller, but still cell density-dependent variation was observed for this enzyme.     

Expression and functional activity of pro-oxidants and antioxidants in murine heart exposed to acute hypobaric hypoxia

Under hypobaric hypoxia, antioxidant defenses of the heart are stressed by the enhanced production of ROS. Mammalian heart acclimatizes to hypoxia through altered gene expression, which we studied in murine heart exposed to 10h of acute hypobaric hypoxia (AHH), equivalent to 15000ft, using cDNA arrays. Functional classification of genes with a > or =2-fold change revealed a number of pro-oxidants like Cyba, Xdh, Txnip, Ppp1r15b and antioxidants like Cat, Gpx1, Mt1, Mgst1. Interestingly, the protein level of Cyba, a subunit of NADPH oxidase, was markedly decreased in AHH exposed heart, suggesting the involvement of some stress response pathways. The AHH exposure also caused a significant reduction (50%) in the level of GSH (P<0.05). The present study provides a retrospective insight on the cellular antioxidant defense mechanisms under AHH.     

Production of an antiviral factor by murine spleen cells after treatment with Corynebacterium parvum.

We have previously shown that injection of Corynebacterium parvum (CP) in mice protected them against lethal encephalitis induced by herpes simplex virus, (HSV). It is shown here that spleen cells of CP-injected mice in vitro produce a factor capable of inhibiting the replication of HSV in mouse embryo fibroblasts (MEF). A similar activity was produced after in vitro exposure of spleen cells from untreated mice to CP. CP was only slightly mitogenic in contrast to phytohemagglutinin (PHA), concanavalin A (Con A), and bacterial lipopolysaccharide (LPS) which were strongly mitogenic but did not induce antiviral activity high enough to be detected in the HSV-MEF system. The activity produced by CP-treated spleen cells appeared to be interferon since it was trypsin sensitive and species specific and not virus specific, and since preincubation of the cells was required to demonstrate an antiviral effect. However, the identity of CP-induced interferon with any of the previously described subclasses of interferon remains to be determined.     

Migration of bone marrow-derived cells and improved perfusion after treatment with erythropoietin in a murine model of myocardial infarction

Erythropoietin (EPO) was shown to have protective effects after myocardial infarction (MI) by neovascularization and antiapoptotic mechanisms. Beside direct receptor-dependent mechanisms, mobilization and homing of bone marrow-derived cells (BMCs) may play a pivotal role in this regard. In this study, we intended to track different subpopulations of BMCs and to assess serially myocardial perfusion changes in EPO-treated mice after MI. To allow tracking of BMCs, we used a chimeric mouse model. Therefore, mice (C57BL/6J) were sublethally irradiated, and bone marrow (BM) from green fluorescent protein transgenic mice was transplanted. Ten weeks later coronary artery ligation was performed to induce MI. EPO was injected for 3 days with a total dose of 5000 IU/kg. Subpopulations (CD31, c-kit, CXCR-4 and Sca-1) of EGFP(+) cells were studied in peripheral blood, bone marrow and hearts by flow cytometry. Myocardial perfusion was serially investigated in vivo by pinhole single-photon emission computed tomography (SPECT) at days 6 and 30 after MI. EPO-treated animals revealed an enhanced mobilization of BMCs into peripheral blood. The numbers of these cells in BM remained unchanged. Homing of all BMCs subpopulations to the ischaemic myocardium was significantly increased in EPO-treated mice. Among the investigated subpopulations, EPO predominantly affected migration of CXCR-4(+) (4.3-fold increase). Repetitively SPECT analyses revealed a reduction of perfusion defects after EPO treatment over time. Our study shows that EPO treatment after MI enhances the migration capacity of BMCs into ischaemic tissue, which may attribute to an improved perfusion and reduced size of infarction, respectively.     

Biochemical changes in cultured murine fibroblasts after treatment with hydrazinophthalazines

Fibroblast cultures exposed to the drugs inducing a collagen-like syndrome (hydralazine and binazine) displayed growth inhibition and decrease in cellular protein content in a dose-dependent manner compared with control cultures. This was accompanied by the inhibitory effect of the drugs on DNA synthesis. The changes in the basic biochemical parameters of fibroblasts testify to the toxicity of hydrazinophthalazines in the connective tissue.