Comparative electrophysiological characterization of ammodytoxin A, a ß-neurotoxin from the nose-horned viper venom, and its enzymatically inactive mutant.

Presynaptic- or ß-neurotoxicity of secreted phospholipases A2 (sPLA2) is a complex process. For full expression of ß-neurotoxicity, the enzymatic activity of the toxin is essential. However, it has been shown that not all toxic effects of a ß-neurotoxin depend on its enzymatic activity, for example, the inhibition of mitochondrial cytochrome c oxidase. The main objective of this study was to verify whether it is possible to observe and study the phospholipase-independent actions of ß-neurotoxins by a standard ex vivo twitch-tension experimental approach. To this end, we compared the effects of a potent snake venom ß-neurotoxin, ammodytoxin A (AtxA), and its enzymatically inactive mutant AtxA(D49S) on muscle contraction of the mouse phrenic nerve-hemidiaphragm preparation. While AtxA significantly affected the amplitude of the indirectly evoked isometric muscle contraction, the resting tension of the neuromuscular (NM) preparation, the amplitude of the end-plate potential (EPP), the EPP half decay time and the resting membrane potential, AtxA(D49S) without enzymatic activity did not. From this, we can conclude that the effects of AtxA independent of enzymatic activity cannot be studied with classical electrophysiological measurements on the isolated NM preparation. Our results also suggest that the inhibition of cytochrome c oxidase activity by AtxA is not involved in the rapid NM blockade by this ß-neurotoxin, but that its pathological consequences are rather long-term. Interestingly, in our experimental setup, AtxA upon direct stimulation reduced the amplitude of muscle contraction and induced contracture of the hemidiaphragm, effects that could be interpreted as myotoxic.

Beneficial effects of water-soluble chestnut (Castanea sativa Mill.) tannin extract on chicken small intestinal epithelial cell culture.

Feed and water supplementation with powdered hydrolyzable tannins from chestnut represents a valuable alternative strategy to antibiotics in animal nutrition. In this study, we evaluated the effects and safety of a water-soluble form of chestnut tannin (WST) in an in vitro model of chicken small intestinal epithelial cells (CSIEC). A chicken cell culture was established, and WST in concentrations of 0.025, 0.05, 0.1, and 0.2% were tested for cytotoxicity, cell proliferation, metabolic activity, production of reactive oxygen species, intracellular antioxidative potential, genotoxicity, and influence on the epithelia cell cycle. The tested concentrations showed a significant (P < 0.05) greater proliferative effect on CSIEC than the control medium (maximal proliferation at 0.1% WST as determined by optical density measurements). The 0.2% concentration of WST was cytotoxic, causing significantly higher (P < 0.05) nitric oxide and hydrogen peroxide production but with no short-term genotoxicity. Although increasing the concentration caused a decline in the metabolism of challenged cells (the lowest at 0.1% WST), metabolic activity remained higher than that in control cells. The antioxidant potential was 75% better and significantly (P < 0.05) higher in the 0.1% WST cultured cells compared to control. In conclusion, the cultured CSIEC are useful tools in basic and clinical research for the study of intestinal physiology, as they retain physiological and biochemical properties and epithelial morphology close to the original tissue and, in many ways, reflect the in vivo state. Our results indicate that WST exert a beneficial effect on intestinal epithelia, since they: i) stimulate proliferation of enterocytes; ii) increase antioxidative potential; iii) have no genotoxic effect; and iv) do not affect cellular metabolism. Our results reinforce the importance of WST as promising candidates for further evaluation and use in commercial broiler farm production.

Serum cortisol and haematological, biochemical and antioxidant enzyme variables in horse blood sampled in a slaughterhouse lairage, immediately before stunning and during exsanguination.

The aim of the study was to determine changes of serum cortisol and biochemical, haematological and antioxidant enzyme variables in the blood of horses sampled during the pre-slaughter period (in the lairage and in the stunning box) and during exsanguination. A total of 24 Slovenian warm-blooded horses were observed. Blood samples for determination of serum cortisol and biochemical, haematological (red blood cell count, haematocrit, haemaglobin concentration) and antioxidant enzyme (whole blood superoxide dismutase and glutathione peroxidase) variables were collected by venipuncture of the left jugular vein in the lairage pen, 60 min before stunning (lairage) and immediately before stunning (stunning box). At exsanguination, blood samples were collected from the wound at the time of jugular vein sticking. During blood collection in the lairage pen and in the stunning box, horses were gently restrained with a halter. They were stunned using a penetrating captive bolt pistol impelled by air and were bled by jugular vein sticking. Horses were physically active in the lairage pen and in the race before entering the stunning box. After stunning, the horses showed paddling movements with their legs. In horses, the plasma lactate and glucose concentrations, the serum potassium concentration, the activities of the serum muscle enzymes aspartate aminotransferase and creatine kinase, and values of most of the other biochemical (Table 1) and haematological variables (Table 2), were significantly (P < 0.05) higher at exsanguination, than in blood sampled while they were in the lairage and in the stunning box. The serum concentrations of cortisol and chloride and the activities of alanine aminotransferase and antioxidant enzymes were not significantly different between the pre-slaughter period and exsanguination. All selected blood variables were not significantly different between the lairge and the stunning box sampling time, indicating no physiological stress responses of the investigated horses to stressors, such as novelty of the pre-slaughter environment and handling, present in the slaughterhouse between the lairage and the stunning box. However, the significantly higher values, at exsanguination, for the plasma lactate and glucose concentrations, serum muscle enzyme activities and haematological variables, than during the pre-slaughter period, might partially be attributed to stimulation of the sympathetic nervous system, caused by stunning and bleeding.

A functional cytochrome P450 lanosterol 14 alpha-demethylase CYP51 enzyme in the acrosome: transport through the Golgi and synthesis of meiosis-activating sterols.

Mammalian lanosterol 14 alpha-demethylase (CYP51) is a microsomal cytochrome P450 that demethylates lanosterol to FF-MAS, an oocyte meiosis-activating sterol and late intermediate of cholesterol biosynthesis. Herein we report CYP51 unequivocally localized to acrosomal membranes of male germ cells in mouse, bull, and ram, in which it synthesizes FF-MAS in the presence of the acrosomal form of nicotinamide adenine dinucleotide phosphate reduced-P450 reductase. In the mouse, CYP51 (53 kDa) resides in endoplasmic reticulum (ER) and Golgi during all phases of acrosome development, indicating an intracellular transport from ERs through the Golgi to the acrosome. CYP51 (50 kDa) also resides on acrosomal membranes of bull- and ram-ejaculated sperm. In mouse liver, a 53-kDa CYP51 is no longer detected in trans Golgi, suggesting retrieval back to the ER and no further transport to other organelles. Glycosylated high-molecular-mass CYP51-immunoreactive proteins in acrosomal membranes of bull and ram and Golgi-enriched fractions of mouse liver indicate that mammalian CYP51s are subjected to posttranslational modifications in the Golgi. In conclusion, CYP51 is the first cytochrome P450 enzyme to be detected on acrosomal membranes. It exhibits a unique, cell-type-specific intracellular transport that is in agreement with its cell-type-specific physiological role: production of cholesterol in the liver and sterols with signaling properties in sperm. Demethylation of lanosterol to FF-MAS by the acrosomal lanosterol 14 alpha-demethylase enzyme complex demonstrates for the first time the ability of ejaculate sperm to synthesize meiosis-activating sterols.

Microcystin-LR affects cytoskeleton and morphology of rabbit primary whole embryo cultured cells in vitro.

Microcystin-LR is the most frequently studied cyclic heptapeptide produced by different genera of cyanobacteria and is hepatotoxic to livestock and human populations. The adverse effects of microcystin-LR on morphology and cytoskeletal elements in different stages of early embryonal development have been studied in vitro. Embryos and whole embryo cultures have been exposed to microcystin-LR (10-100 microM). Actin filaments were visualized by fluorescence staining and the microtubular network labelled by immunostaining. Growth, development and cytoskeleton organization of the embryos embedded in zona pellucida are not affected by microcystin-LR in concentrations up to 100 microM, while whole embryo cell cultures are affected by the presence of microcystin-LR in the culture medium. High microcystin-LR concentrations (100 microM) cause cells to be detached and destroyed, while lower concentrations (10-20 microM) profoundly affect actin and microtubule organization. These effects are confirmed also by the presence of transformed microcystin-LR in all the media at the lowest concentrations. It seems that the changes to the cells are far more serious than that expressed in cell morphology. From our experiments we conclude that the presence of zona pellucida is an effective way of embryo protection against xenobiotics like microcystin-LR.

Lanosterol 14alpha-demethylase and MAS sterols in mammalian gametogenesis.

The lanosterol 14alpha-demethylase protein complex is composed of a cytochrome P450 enzyme CYP51 and its redox partner NADPH cytochrome P450 reductase. The complex participates in cholesterol biosynthesis and produces folicular fluid meiosis activating sterol (FF-MAS) from lanosterol. FF-MAS is metabolized further by sterol Delta14-reductase to testis-meiosis activating sterol (T-MAS). Additional enzymatic steps are needed before cholesterol is produced. Using the anti-human CYP51 antibody we have studied CYP51 protein expression by confocal microscopy in male and female mouse gonads. Leydig cells and acrosomes of spermatids express the highest levels of the CYP51 protein. CYP51 protein is also detected in primary mouse oocytes of non-treated mice and in some granulosa cells. While regulatory mechanisms responsible for FF-MAS accumulation in the ovary are not yet established, two mechanisms contributing to production the of T-MAS in the testis have been found. Potential in vivo roles of FF-MAS and T-MAS in fertilization are discussed.

Cardiovascular effects of equinatoxin III from the sea anemone Actinia equina (L.).

Equinatoxin III is the most hemolytic, and the least lethal of the three basic proteins isolated from the sea anemone Actinia equina (L.). Its LD50 in mice is 83 microg/kg. Preliminary results on Wistar rats have suggested cardiorespiratory arrest as a putative cause of death, but the mechanism of its action has not yet been studied. So far only equinatoxin II has been investigated more thoroughly. As equinatoxin II is less lythic, but more toxic, than equinatoxin III (its LD50 in mice=35 microg/kg), it may be assumed that haemolysis with a consequent rise in plasma potassium level is not the major factor in the lethality of equinatoxins. To assess the relative contribution of hyperkalemia in the lethality of the toxin in rat, the effects of equinatoxin III were compared to the effects of hyperkalemia caused by the injection of KCl giving the same final concentration of K+ in the plasma as that observed after an i.v. injection of 3LD50 of equinatoxin III. As coronary vasoconstriction may be an important mechanism of the cardiotoxic action of equinatoxins, the effect of EqT III on isolated porcine coronary arteries was studied by measurements of smooth muscle tension in the presence of 1-100 nM equinatoxin III. The results revealed that animals survive the elevated K+ plasma concentration caused by an i.v. application of KCl. This suggests that equinatoxin III induced haemolysis is not the major mechanism of equinatoxin III lethality. However, equinatoxin III increases the potassium induced contractions of coronary smooth muscle for 289+/-29%, suggesting that coronary vasoconstriction may be an important factor in the cardiotoxic effects of equinatoxin III.

Specific absorption rate study for radiofrequency current density imaging using a two-dimensional finite element model.

Radiofrequency current density imaging is an MR technique that images tissue conductivity contrast. Compared to conventional MRI, RF-CDI uses two additional sources of RF power to be absorbed and that must be evaluated in terms of proper parameter optimization to prevent excessive tissue heating and effects on the nervous system. In view of possible future clinical use of RF-CDI, a simple 2D finite element model of a rat brain was built to simulate current density distribution and distribution of absorbed RF power, i.e., SAR and related tissue heating. Current density in the rat brain was also evaluated qualitatively and quantitatively in an in vivo RF-CDI experiment. The results demonstrate that a numerical model can predict SAR and tissue temperature changes. The study also shows that substantial sensitivity and resolution of RF-CDI can be achieved using imaging parameters that produce SAR and temperature changes within allowed limits.

Effects of ammodytin L on miniature and endplate potentials in neuromuscular junction of frog m. cutaneus pectoris.

Neurotoxic effects of ammodytin L (AtnL), a potent phospholipase A2 homologue, was studied in frog neuromuscular preparation m. cutaneous pectoris by measuring the influence of the toxin on the amplitude and the frequency of miniature and endplate potentials (MEPPs, EPPs). AtnL, in 100 nM concentration, significantly increases spontaneous quantal acetylcholine release from the motor nerve endings, observed as the increase in MEPPs frequency. At 100 nM or higher concentration the toxin decreases EPPs amplitude and the membrane potential (MP) simultaneously. No significant effect of AtnL on EPPs was observed at any concentration bellow 100 nM. Our results indicate that in frog AtnL shows the typical myotoxic effects, but it also exerts presynaptic effects.

Subchronic liver injuries caused by microcystins.

The subchronic effects of cyanobacterial lyophilizate (CL) containing microcystins on liver were investigated in female New Zealand rabbits. Sterilised CL containing microcystins was injected i.p. Liver toxicity was assessed by histological examination of liver samples. Non-invasive magnetic resonance imaging (MRI) of liver was also performed in order to assess changes in the homogeneity of liver tissue. Subchronical intoxication with microcystins caused morphological changes of liver tissue that were also detected by use of MRI. Histological analysis showed that changes seen on MRI represent liver injury characterised with fatty infiltration and periportal fibrosis. This demonstrates that subchronic exposure to microcystins can lead to liver degeneration, which can easily be detected in vivo by use of MRI.

Assessment of kainate toxicity using contrast enhanced magnetic resonance imaging.

The purpose of this study was to test the capability of contrast enhanced magnetic resonance imaging (MRI) in assessing lesion formation in rat brain after systemic (i.v.) administration of kainate. MRI was performed with T1-weighted spin echo sequence before and after the administration of kainate and contrast media. Contrast media used were based on paramagnetic gadolinium (III) ion: Gd-DTPA (gadoliniumdiethylenetriaminepentaacetic acid) and prototype agents for blood-pool enhancement. Gadomer-17 and polylysine-Gd-DTPA. Enhancement of lesion rims and other brain tissue abnormalities due to kainate with Gd-DTPA, Gadomer-17 and polylysine-Gd-DTPA were observed mainly in the region of hippocampus and in the areas not protected by the blood-brain-barrier (BBB).

Decreased cortisol secretion by adrenal glands perfused with the P-glycoprotein inhibitor valspodar and mitotane or doxorubicin.

The aim of this study was to investigate the role of P-glycoprotein (P-gp) in the adrenal gland. It has been presumed that P-gp, rather than being involved in physiological cortisol secretion, plays a role in protecting the adrenacortical cells from xenobiotics. To explore this a study was performed on perfused bovine adrenal glands. Individual experimental groups were perfused with either a selective P-gp blocker (valspodar) alone, with a xenobiotic (mitotane or doxorubicin) alone or with both valspodar and a xenobiotic. The cumulative amounts of cortisol secreted in each individual group were calculated and the two-sample t-test was used to compare the mean values of cumulative amounts. The mean value of cortisol secreted from the group of adrenals perfused with the P-gp blocker was not significantly different from that of the control group. Treatment with either mitotane or doxorubicin decreased the amount of cortisol secreted but not significantly when compared to the amount of cortisol secreted in basal conditions. However, treatment with the P-gp blocker valspodar in addition to either mitotane or doxorubicin significantly decreased cortisol secreted compared to the amount of cortisol secreted by the glands treated with either mitotane (p=0.009) or doxorubicin (p=0.017) alone. The regressive changes discovered in all experimental groups were most prominent when valspodar was used with either mitotane or doxorubicin. We found that P-gp blockade increases by xenobiotic (mitotane and doxorubicin)-induced damage of adrenocortical cells, which points to a role of P-gp in the protection of adrenal gland from xenobiotics.

Ca(2+) and Na(+) contribute to the swelling of differentiated neuroblastoma cells induced by equinatoxin-II.

Equinatoxin-II (EqTx-II), a cytotoxic protein (mol.wt 20 kDa) isolated from the sea anemone Actinia equina, was found to consistently increase the three-dimensional projected area of differentiated neuroblastoma (NG108-15) cells provided Ca(2+) was present in the medium. No swelling was detected when external NaCl was replaced by sucrose, but replacement of NaCl by Na-isethionate did not prevent the swelling, as revealed by confocal laser scanning microscopy. In addition, microspectrofluorometric measurements in cells preloaded with the Ca(2+) indicator fura-2/AM revealed that EqTx-II (100 nM) markedly increased the fluorescence (F(340)/F(380)) ratio indicating a rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). The elevation of [Ca(2+)](i) exhibited two components that seem to be related to the kinetics of EqTx-II-induced Ca(2+) entry since pretreatment of cells with Ca(2+)-ATPase inhibitors (thapsigargin), Ca(2+) channel blockers (nifedipine and Gd(3+)) or prolonged exposure to a high K(+) (75 mM) medium did not alter EqTx-II-induced Ca(2+) signals. As far as we know, this is the first demonstration that EqTx-II causes swelling of neuroblastoma cells and that this effect is correlated both with an increase of [Ca(2+)](i) and needs the presence of extracellular Na(+). It is suggested that EqTx-II has the ability to insert into the plasma membrane of neuroblastoma cells and to form pores altering the membrane permeability and the intracellular osmolality, inducing a marked influx of water into the cells.

Equinatoxin II increases intracellular Ca2+ in NG 108-15 cells.

Equinatoxin II (EqT II) is a basic 20 kD protein isolated from the sea anemone Actinia equina. Intravenous injection of 3 LD50 of EqT II causes cardiorespiratory arrest. The aim of our study was to check the effects of EqT II on neuronal cells to assess the role of neuronal mechanisms in respiratory arrest after intravenous injection of the toxin. Effects of EqT II on mouse neuroblastoma x rat glioma NG108-15 cell were studied using confocal laser scanning microscopy and by Fura-2 fluorescence measurements. The results show that EqT II applied in nanomolar range increases intracellular Ca2+ activity significantly, which is possibly responsible for the morphological changes of NG108-15 cells after the exposure to 10 nM EqT II. Intracellular increase in Ca2+ activity can not be prevented by use of the various pharmacological substances (e.g. Ca2+ channels blocker Verapamil and Bekanamycin). Swelling of the NG108-15 cells after the exposure to the EqT II also can not be blocked with the sodium channel blocker tetrodotoxin. Increase in the intracellular Ca2+ activity is probably a result of Ca2+ entry through pores produced by the toxin, which has been shown by other authors on other cells and on phospholipid bilayer. Respiratory arrest after intravenous injection of the toxin can be caused by the action of the toxin on neuronal cells in medulla oblongata provided that EqT II can damage blood brain barrier thus enabling access to the neuronal cells. The results allow the conclusion that EqT II can affect normal calcium homeostasis and cell morphology of neuronal cells that can disturb cell physiology and its function thus affecting normal respiratory pattern.

The effects of equinatoxin II on respiration--possible mechanism of the toxin lethality.

In rat an intravenous application (i.v.) of a lethal dose of the equinatoxin II (EqT II) provoked a respiratory arrest. It is not known whether the respiratory arrest is a result of a direct actions of the toxin on lung tissue, on neuromuscular junctions or the on the central nervous system. The influences of the toxin on the neuromuscular transmission and on muscular contraction were studied in isolated rat diaphragm. The effect of EqT II on end plate potentials of m. cutaneus pectoris was measured in the frog Rana esculenta. To monitor equinatoxin II effects on the central nervous system of a rat, the toxin was injected directly into the forth brain ventricle. The respiratory arrest was not the result of the toxin action on the neuromuscular junctions and peripheral nerves. The cessation of respiratory activity was most probably a result of equinatoxin II direct actions on lung tissue and on brain microcirculation.

Radiofrequency current density imaging of kainate-evoked depolarization.

The purpose of this study was to examine whether radiofrequency current density imaging (RF-CDI) can quantitatively monitor depolarizations evoked by excitatory amino acids in a rat's brain. To evoke depolarization, a glutamate receptor agonist, kainate, was administered into the right lateral ventricle. First, electroencephalographic activity was recorded in a basal condition and after the application of kainate. Complex behavioral patterns were observed. Second, impedance measurements were performed to assess the change in conductivity of the brain due to kainate at the Larmor frequency of the imager. Calculated changes were about 17%. Third, a set of current density images was obtained with RF-CDI before and after the administration of kainate. Kainate-induced excitatory changes were observed on current density images as brighter regions, mainly in the hippocampal area compared with the same area in the basal condition.