Effect of acute and chronic ethanol treatment on rat brain phospholipid turnover

Phospholipid turnover was studied in the rat brain after treatment in vitro with (1) an acute dose of ethanol, (2) after the development of tolerance to ethanol, (3) of physical dependence on ethanol, and (4) of withdrawal from dependence. It was found that the turnover of phosphatidylcholine, of phosphatidylinosotol/phosphatidylserine and of phosphatidylethanolamine was increased in tolerant animals and that tolerance developed to these increases in dependent animals. A drastic decrease in ³²P-labeled phosphatidylinositol/phosphatidylserine of microsomal fractions was observed in animals withdrawing from dependence.     

Effect of ethanol on calcium-uptake and phospholipid turnover by stimulation of adrenoceptors and muscarinic receptors in mouse brain and heart synaptosomes

The effect of ethanol treatment on mouse brain and heart synaptosomal 45Ca uptake and the incorporation of [3H]inositol and [14C]choline into phosphatidylinositol (PI) and phosphatidylcholine (PC) were investigated. Ethanol in drinking water (15%) was given to mice for 3 weeks. The consumption of ethanol increased gradually during treatment but food intake was almost the same as control. The body weight of ethanol-treated mice was slightly less than that of control. The synaptosomal lipid peroxidation level of ethanol-treated mice was almost the same as control in the brain and heart. On the other hand, the synaptosomal glutathione level of ethanol-treated mice was higher than control in both brain and heart. The 45Ca uptake of brain and heart from ethanol-treated mice was 87% and 216% of control mice, respectively. Not only ethanol but also norepinephrine (NE), carbachol (Carb), or isoproterenol (IsoPro) added in vitro increased 45Ca uptake in all cases. The incorporation of [3H]inositol into PI in the brain and heart synaptosomes of ethanol-treated mice was 150% and 113% of control, respectively. The incorporation of [14C]choline into PC in the brain and heart of ethanol-treated mice was 104% and 125% of control, respectively. In vitro addition of ethanol, NE, Carb or IsoPro to brain synaptosomes increased the incorporation of [3H]inositol and [14C]choline into PI and PC, respectively, in both control and ethanol-treated mice. In the case of heart synaptosomes, NE and Carb increased the incorporation of [3H]inositol and [14C]choline into phospholipids in control mice but not ethanol-treated mice. However, IsoPro increased the incorporation by both control and ethanol-treated heart synaptosomes. These results suggest that alpha-adrenoceptors and the cholinergic system of the heart play important roles in modulating the toxic effects of ethanol.     

Effect of butylidenephthalide on calcium mobilization in isolated rat aorta

Butylidenephthalide (Bdph), an antispasmodic compound originally isolated from the rhizome of Ligusticum chuaxiong, has a selective anti-anginal effect without changing blood pressure. Experiments have been performed to determine the mechanism of this action. Synthetic Z-butylidenephthalide concentration-dependently relaxed phenylephrine (1 microM)- or KCl (60 mM)-induced precontractions of intact and denuded rat aorta rings. The relaxation induced by Bdph was endothelium-independent. Bdph (30-300 microM) concentration-dependently reduced cumulative phenylephrine- and KCl-induced contractions of intact rat aortic rings and non-competitively inhibited their log concentration-response curves. The pD2' values of Bdph for phenylephrine- and KCl-induced contraction were 3.66+/-0.13 (n = 8) and 3.71+/-0.07 (n = 8), respectively, which were not significantly different from each other. Bdph also concentration-dependently reduced cumulative Ca2+-induced contractions of intact rat aortic rings in high-KCl (60 mM) Ca2+-free physiological salt solution and non-competitively inhibited its log concentration-response curve. The pD2' value of Bdph for the Ca2+-induced contractions was 3.21+/-0.01 (n = 7) which was significantly different from the pD2' value obtained from the cumulative KCl-induced contractions. These results suggest that Bdph inhibits calcium release from calcium stores more selectively than calcium influx from extracellular space via voltage-dependent calcium channels. The inhibition by Bdph of calcium release from KCl-sensitive calcium stores might be similar to its inhibition of calcium release from phenylephrine-sensitive calcium stores. However, because phenylephrine generates inositol-1,4,5-trisphosphate (IP3) whereas KCl does not, the inhibitory effect of Bdph might not be related to IP3 production.     

Effect of acute and chronic ethanol administration on serotonin turnover in rat brain

In rats treated acutely and chronically with ethanol, brain serotonin (5-HT) turnover was determined by measuring in the same animals the rate of accumulation of 5-HT and the rate of decline of 5-hydroxyindoleacetic acid (5-HIAA) in the brain after administration of the MAO inhibitor pargyline-HCl. In the acute study, 5-HT turnover was not altered after a single 4 g/kg oral dose of ethanol. In two separate studies, it was shown that chronic administration of ethanol in the form of a liquid diet for 32 days also did not alter brain 5-HT turnover, even when the rats had developed a significant degree of tolerance to the motor-impairing effects of ethanol. These results suggest that tolerance to ethanol is unrelated to any change in brain 5-HT turnover.     

Effect of a cyclohexenonic long-chain fatty alcohol on calcium mobilization

Cyclohexenonic long-chain fatty alcohols constitute a family of synthetic compounds with trophic, secretagogue and antioxidant properties. Despite their multiple biological actions in neuronal and non-neuronal tissues, the intracellular mechanisms underlying CFA activity remain unknown. In the present study, we show that 3-(15-hydroxypentadecyl)-2,4,4-trimethyl-2-cyclohexen-1-one (tCFA15) directly mobilizes Ca(2+) in the pituitary neural lobe synaptosomes and in primary sensory neurons from dorsal root ganglia. This effect is dependent on the presence of extracellular Ca(2+), but does not involve transmembrane voltage-operated calcium channels. Using a combination of pharmacological agents that block or deplete intracellular Ca(2+) stores, our results suggest the implication of a calcium induced-calcium release mechanism evoked by tCFA15-induced Ca(2+) influx. To our knowledge, these findings constitute the first attempt towards the comprehension of the biological actions of cyclohexenonic long-chain fatty alcohols at a molecular level.     

Effects of delta9-tetrahydrocannabinol and ethanol on body weight protein and nucleic acid synthesis in chick embryos.

Delta9-Tetrahydrocannabinol (THC) and/or ethanol were administered into the yolk sac of chicken eggs at various times of incubation. Repetitive injections of 1 or 2 mg THC and/or 10 mul of alcohol were fiven. With a schedule of injections on days 2, 4, 7, and 9 with sacrifice on day 11, or on days 2, 4, 7, 9, and 11 with sacrifice on day 15, the body weight was reduced and the liver weight increased. The latter schedule also decreased the weight of the forebrain. Injections of labeled leucine and uridine were also given 1 hr before sacrifice to determine the rate of incorporation of these precursors into protein and RNA. Apparent changes were found in the specific activities of protein and RNA in different parts of the brain and liver. No overall metabolic trends emerged, however. No teratogenic effects were visible with the dosage schedules used.     

Effect of ethanol on intracellular ionized calcium concentrations in synaptosomes and hepatocytes

The effect of ethanol on intracellular ionized calcium concentrations (Cai) was studied in synaptosomes isolated from mouse whole brain and in hepatocytes isolated from rat liver. The fluorescent calcium chelator, fura-2, was used to quantitate Cai. Incubation of synaptosomes with ethanol (350-700 mM) increased resting Cai and decreased the effectiveness of KCl to raise Cai in a concentration-dependent manner. Ethanol produced an initial rapid (less than 10 sec) increase in resting Cai that remained elevated for at least 14 min in the presence of the drug. The increase in resting Cai was correlated with the inhibitory effect of ethanol on depolarization-induced increases in Cai. Resting Cai was dependent on the external calcium concentration (0-1 mM). However, the ethanol-induced increase in resting Cai (expressed as percent of control) did not differ in the presence of several extracellular calcium concentrations (0.01, 0.1, and 1 mM). Incubation of synaptosomes in a Na-free buffer resulted in a higher resting Cai and slightly enhanced the effect of ethanol to increase resting Cai. In contrast to these results in brain tissue, ethanol (30-600 mM) did not alter resting Cai or vasopressin-stimulated increases in Cai in hepatocytes. Our results suggest that the anesthetic effects of alcohols may be mediated, in part, by increased resting Cai and by decreased calcium influx through voltage-sensitive calcium channels. In addition, our findings suggest possible mechanisms by which ethanol increases resting Cai in neuronal tissue.     

Toxicity of styrene and styrene oxide on chick embryos

Styrene and styrene oxide were injected into the air space of fertilized chicken eggs at different times during an incubation period of 14 days. The toxicity of styrene and styrene oxide when injected on the fourth day of incubation revealed an LD50 of 40 mumol/egg and 1.5 mumol/egg, respectively. Malformations were found in 0-20% of the embryos, but never in the controls. The results obtained point to a need for further experimental, and possibly epidemiologic, studies on the consequences of styrene exposure.     

Teratogenicity of ochratoxin A in chick embryos

Experiments were conducted to evaluate the teratogenic potential of ochratoxin A given to developing chick embryos. Ochratoxin A was dissolved in propylene glycol and injected into embryonating chicken eggs at doses ranging from 0.0005 to 0.007 mg/egg. The injections were made into the air sacs of eggs after 48, 72, and 96 hr of incubation (Groups A, B, and C). Control eggs were injected with an equivalent volume of propylene glycol (0.1 ml/egg). In all, 720 chicken eggs were used for this study. All embryos were examined at Day 8. The LD₅₀ for embryos in Groups A, B, and C was 0.0007, 0.003, and 0.007 mg/egg, respectively. The following malformations were observed: short and twisted limbs, short and twisted neck, microphthalmia, exencephaly, everted viscera, and reduced body size. Microscopic examination of whole embryos treated at 48 hr (Group A) showed ventricular septal defects, aortic stenosis, and malformations of the valves. The results of the present study indicate that ochratoxin A is teratogenic.     

Endpoints in the development of chick embryos

Although the use of mammalian embryos must remain the ideal for most teratological studies, non-mammalian embryos offer certain advantages. Their independence from the mother makes it possible to study the effects of reagents directly on the embryo without having to take possible placental effects into consideration, and it enables manipulations to be carried out more easily. In the early embryonic stages the cells of Xenopus and the chick are larger than those of the mouse, and these non-mammalian embryos tend to be bigger than those of the mouse at comparable stages of development. Attention is focused on the chick embryo as a useful experimental animal in the study of teratological effects. Consideration is given to some of the critical stages in embryogenesis as well as to the culture techniques available.     

Cadmium effects on early development of chick embryos

The toxic potential of cadmium (Cd) is well-documented for young and adult vertebrates, but it is still poorly understood in the early stages of development. In this study, cadmium effects were investigated on Gallus gallus embryos after injection of CdCl(2) (5μM and 50μM) within the egg air chamber, and incubation for 48 and 72h. After exposure, morphological and enzymatic analyses for glucose-6-phosphate dehydrogenase (G6PDH) and glutathione S-transferase (GST) were performed. Critical morphological abnormalities occurred after exposure to the highest concentration of cadmium, mainly in the cephalic region, indicating the powerful teratogenic effect of Cd to chick embryos. Cd exposure did not alter enzymatic activities when compared to the control group, but the levels of G6PDH activity were highest in older embryos at stage 19, indicating that antioxidant defenses are not so robust in the earliest embryo stages.     

Effects of 2,5-hexanedione on angiogenesis and vasculogenesis in chick embryos

n-Hexane is widely used in industry and its metabolite, 2,5-hexanedione (2,5-HD), has been implicated as a neural toxin in the developing fetus. Using the chick embryo model, we have previously revealed the neurotoxicity of 2,5-HD during development and established that high dose of 2,5-HD was embryo lethal. In view of the close linkage in biology for neurogenesis and angiogenesis, we speculated that it was most likely caused by cardiovascular dysplasia, therefore in this study, we investigated the effects of 2,5-HD on the development of the vasculature, which involves vasculogenesis and angiogenesis. Using gastrulating chick embryos as a model, we demonstrated that the hemangioblasts (precursor of hematopoietic and endothelial cells) migrated to the area opaca where they form the blood islands. However, this process was impaired when the embryos were treated with 2,5-HD, suggesting that 2,5-HD is capable of impairing vasculogenesis. To study the effect of 2,5-HD exposure on angiogenesis, we used the chick yolk-sac membrane (YSM) and chorioallantoic membrane (CAM) models. We found that, at low (0.02 M) concentration, 2,5-HD stimulated angiogenesis while at higher concentrations (>0.1 M) it inhibited this process. This biphasic response of angiogenesis to 2,5-HD exposure was found to be associated with altered expression of the VEGF-R, FGF-2 and angiogenin. Moreover, we also determined that 2,5-HD exposure increased the reactive oxygen species (ROS) production. In conclusion, 2,5-HD could induce dysplasia in the developing vasculature, which in turn could cause extravascular hemolysis and the embryos to die.     

The effect of ethanol on phospholipid metabolism in rat pancreas

The phospholipid effect involves agonist-induced breakdown of phosphatidyl inositol (or polyinositides) generating second messengers followed by increased incorporation of 32P during the resynthetic phase of the cycle. Ethanol, an aetiological factor in pancreatitis, has been shown to have various effects on pancreatic secretion. In this study ethanol decreased the incorporation of 32P into phosphatidyl inositol but had no effect on the stimulated breakdown of prelabelled phosphatidyl inositol. However, in addition to recycling of phosphatidyl inositol stimulation of pancreatic tissue results in increased incorporation of precursors into other phospholipids. Cholecystokinin increased the incorporation of both [U-14C] glucose and 32P into phosphatidyl ethanolamine 3-fold but had no effect on 32P incorporation into phosphatidyl choline. As well as increased incorporation of 32P into phosphatidyl inositol (8-fold) cholecystokinin also increased the incorporation of [U-14C] glucose into phosphatidyl inositol (4-5-fold) implying significant de novo synthesis of 1,2 diacyl glycerol in addition to the currently accepted recycling of the 1,2 diacyl glycerol back to phosphatidyl inositol. Ethanol caused an inhibition of 32P incorporation into total phospholipid of rat pancreas during basal and stimulated conditions. When individual phospholipids were separated ethanol was found to decrease the incorporation of 32P into phosphatidyl choline under basal conditions and into all phospholipids during cholecystokinin stimulation. With [U-14C] glucose as the precursor, ethanol inhibited its incorporation into phosphatidyl choline only. Ethanol did not alter the total 32P radioactivity in the aqueous phase of the pancreatic extract nor the percent incorporated into nucleotides. This excluded decreased uptake of 32P and incorporation into nucleotides as a mechanism for the differential inhibition of 32P versus [U-14C] glucose incorporation into phospholipids other than phosphatidyl choline under stimulated conditions.     

Effects of ethanol on turnover and function of striatal dopamine

Acute oral administration of ethanol increased the rate of depletion of dopamine in the striata of rats injected with -methyl-p-tyrosine. This effect was eliminated by pretreatment with atropine or by lesioning of the striato-nigral tract. Ethanol also attenuated the inhibitory effect of apomorphine on turnover of striatal dopamine. Unilateral injection of ethanol into the neostriatum of rats followed by intraperitoneal injection of either apomorphine or amphetamine elicited marked ipsilateral head-to-tail body turning. This turning was blocked by pretreatment with haloperidol. Chronic intubation of ethanol to rats enhanced contralateral body turning elicited by unilateral intrastriatal injection of dopamine. Injection of 6-hydroxydopamine into the substantia nigra led to denervation supersensitivity of dopaminergic functions in the neostriatum. This effect was not seen in rats that were given ethanol postinjection of 6-hydroxydopamine. These results suggested that ethanol has an inhibitory effect on the nigrostriatal dopaminergic system.     

Effect of protein kinase C inhibitors on calcium ionophore-induced arachidonic acid mobilization in human leukocytes

Arachidonic acid mobilization in human polymorphonuclear leukocytes stimulated with calcium ionophore A23187 was amplified by synthetic diacylglycerol and, to a much lesser extent, by phorbol ester. The effect was synergistic and dependent upon influx of calcium ions. Thin layer chromatographic analysis of phospholipids of stimulated cells revealed a loss of arachidonic acid associated with phosphatidylinositol and phosphatidylcholine. The synergistic response was unaffected by treatment of cells with two inhibitors of protein kinase C, namely, polymyxin B and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. Moreover, both agents consistently augmented the cellular response to A23187 alone. These findings suggest that A23187-induced arachidonic acid mobilization in leukocytes is independent of protein kinase C activity.     

Teratogenicity of metals to chick embryos

The present study examines the effects of heavy metals on chick embryogenesis. The metals included were cadmium, arsenic, cobalt, copper, indium, iron, manganese, and molybdenum. Salts of each of the metals were dissolved in saline and injected into the air sacs on d 2 of incubation. Dose levels varied with the metal to be tested. Control eggs were injected with an equivalent volume of saline (0.1 ml/egg). On d 14, the live embryos were removed from the eggs and examined for gross malformations. From the LD50 values, the toxicity relationship between the metals is cadmium greater than arsenic greater than cobalt greater than copper greater than indium greater than molybdenum greater than manganese greater than iron. The LD50 values were 3, 9, 38, 58, 121, 333, 765, and 1185 micrograms/egg, respectively. The gross malformations observed were reduced body size, micromelia, twisted neck, hemorrhage, everted viscera, and microphthalmia. Arsenic and cobalt were observed to be more teratogenic than other metals. This study showed that the metals tested were both toxic and teratogenic to varying degrees in chick embryogenesis.     

Effect of bolesatine on phospholipid/calcium dependent protein kinase in vero cells and in rat thymus

 Bolesatine, a glycoprotein from Boletus satanas Lenz, has previously been shown to be mitogenic to rat and human lymphocytes at very low concentrations, whereas higher concentrations inhibit protein synthesis in vitro and in several in vivo systems. The mechanism whereby this mitogenic activity occurs was previously unknown. To elucidate this mechanism, the effects of bolesatine have been studied in a cell-free system, VERO cells in culture, and in rat thymus. Bolesatine was found to activate PKC, in vitro (cell free system), in VERO cells, and in vivo in rat thymus. In a cell-free system, bolesatine appears to be a direct effector of PKC. The activation is concentration dependent for 1 – 10 ng/ml. At the same time, VERO cells significantly proliferate when incubated with bolesatine (3, 5 and 10 ng/ml), since the DNA synthesis increases by 27, 48 and 59%, for respectively, 3, 5 and 10 ng/ml compared with control. Moreover, Bolesatine (5 and 10 ng/ml) induces InsP₃ release in a concentration-dependent manner (114 and 142%) as compared to control. In vivo, 24 h after oral administration of bolesatine to rats (20, 100 and 200 μg/kg), PKC activity is significantly increased in thymus. The most effective doses (100 and 200 μg/kg) give 590 – 620% increase in cytosolic PKC activity and 85 – 91% increase in total PKC activity as compared to control. This PKC activation by bolesatine in rat thymus is directly linked to the mitogenic activity observed in vivo. Bolesatine is thus capable of activating the PKC directly and/or indirectly (via InsP₃ release) during its mitogenic process. Received: 6 February 1995 / Accepted: 20 March 1995