Exogenous glycine partially attenuates homocysteine-induced apoptosis and membrane peroxidation in chick embryos

The effects of exogenous glycine on homocysteine (HoCys)-induced reductions in chick (Gallus gallus) embryo viability, HoCys-induced increases in brain and hepatic membrane lipid peroxidation, HoCys-induced apoptosis (caspase-3 activities) in brain and hepatic tissues, and HoCys-induced reductions in brain and hepatic S-adenosylemethionine (SAM)/S-adenosylhomocysteine (SAH) levels were studied. Exogenous HoCys caused reductions in percent living embryos and reductions in embryo masses. Exogenous glycine attenuated these HoCys-induced reductions in embryo viability. Brain and liver tissues of HoCys-treated embryos exhibited increased caspase-3 activities, increased lipid hydroperoxide (LPO) levels, and reduced levels of long-chain polyunsaturated membrane fatty acids. While exogenous glycine attenuated HoCys-induced changes in brain caspase-3 activities, brain LPO levels, and brain membrane PUFA levels, exogenous glycine was less effective in attenuating HoCys-induced changes in hepatic caspase-3 activities and hepatic membrane PUFA levels. HoCys-induced reductions in SAM/SAH ratios were observed in brains and livers. Exogenous glycine attenuated HoCys-induced reductions in brain SAM/SAH. However, glycine was unable to attenuate HoCys-induced reductions in hepatic SAM/SAH levels.     

Homocysteine-induced changes in brain membrane composition correlate with increased brain caspase-3 activities and reduced chick embryo viability

In adult systems, high homocysteine (HoCys) levels inhibit methylation reactions and can induce apoptosis in the central nervous system. In embryos, exogenous HoCys is teratogenic and is associated with neural tube defects. Because, methylation inhibitors and inducers of apoptosis can influence membrane composition, we have studied whether or not embryonic exposure to HoCys influenced membrane phospholipid levels, membrane fatty acid composition, and Caspase-3 activities in embryonic chick brains. Embryonic exposure to HoCys caused reduced brain phosphatidylcholine levels and increased levels of brain phosphatidylethanolamine. Exogenous HoCys also promoted decreased levels of long-chain, unsaturated membrane fatty acids and increased levels of saturated short-chain membrane fatty acids. These HoCys-induced brain membrane changes correlated with HoCys-induced increases in brain Caspase-3 activities, HoCys-induced reductions in brain mass, HoCys-induced reductions in embryo mass, and HoCys-induced reductions in the percentage of embryos that survived to 11 days of development (theoretical stage 37). Thus, HoCys-induced changes in brain membrane composition correlated with HoCys-induced apoptosis and reduced embryo viability.     

Reduced de novo synthesis of 5-methyltetrahydrofolate and reduced taurine levels in ethanol-treated chick brains

In previous studies, exogenous ethanol (3 mmol EtOH/kg egg) caused a 1.6-fold increase in chick brain homocysteine (HoCys) levels at 11 days of development and the mixture of 3 mmol EtOH/kg egg and 34 μmol folic acid/kg egg attenuated EtOH-induced increases in chick brain HoCys levels. Because HoCys is converted to methionine utilizing the methyl donor, 5-methyltetrahydrofolate (5-methyl THF), we studied whether exogenous ethanol (3 mmol EtOH/kg egg) or the mixture of 3 mmol EtOH/kg egg and 34 μmol 5-methyl THF/kg egg inhibited chick brain 10-formyltetrahydrofolate dehydrogenase (10-FTHF DH; EC 1.5.1.6) activities and brain N⁵, N¹⁰-methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) activities at 11 days of development. Three daily dosages of 3 mmol EtOH/kg egg (E_0–2) caused approximately a 7-fold reduction in brain 10-FTHF DH activities and approximately a 1.9-fold reduction in brain MTHFR activities as compared to controls at 11 days of development (p ≤ 0.05). Because HoCys is also removed by the transsulfuration pathway, which synthesizes taurine, we studied whether exogenous ethanol (3 mmol EtOH/kg egg) or the mixture of 3 mmol EtOH/kg egg and 34 μmol 5-methyl THF/kg egg influenced chick brain taurine levels. In EtOH-treated and EtOH and 5-methyl THF-treated embryos, brain taurine levels decreased by approximately 5.5-fold and 6.2-fold as compared to controls, respectively (p ≤ 0.05). Exogenous 5-methyl THF failed to attenuate EtOH-induced decreased brain taurine levels at 11 days of development.     

Alpha-tocopherol and gamma-tocopherol attenuate ethanol-induced changes in membrane fatty acid composition in embryonic chick brains

BACKGROUND: This project investigated whether or not EtOH-induced reductions in the levels of long-chain polyunsaturated membrane fatty acids could be attenuated by exogenous exposure to either alpha-tocopherol, gamma-tocopherol, or diallyl sulfide (DAS). METHODS: At 0 days of development, fertile chicken eggs were injected with a single dose of either saline supplemented with various concentrations of EtOH, alpha- or gamma-tocopherol and EtOH, or DAS and EtOH. At 18 days of development, brains were isolated and subjected to membrane analyses. RESULTS: When exposed to EtOH, concentrations ranging from 0-60.50 microm/Kg egg, dose-dependent decreases in the levels of brain 18:0, 18:1 (n-9), 18:2 (n-6), 18:3 (n-3), and 20:4 (n-6) were observed. These ethanol-induced changes in membrane fatty acid composition correlated with ethanol-induced reductions in brain mass, brain protein levels, acetylcholine esterase (AChE) activities and correlated with increased lipid hydroperoxide levels. Exposure to either 2.5 microm alpha-tocopherol/Kg egg and 6.050 mm EtOH/Kg egg, or 2.5 microm alpha-tocopherol/ Kg egg and 6.050 mm EtOH/Kg egg attenuated EtOH-induced changes in membrane fatty acid composition, brain mass, brain protein levels, AChE activities, and lipid hydroperoxide levels. Embryonic exposure to the cytochrome p450-2E1 inhibitor, diallyl sulfide (DAS), also attenuated EtOH-induced decreases in long-chain, unsaturated membrane fatty acids. However, embryonic exposure to DAS promoted abnormally low brain mass. CONCLUSION: EtOH-induced reductions in the levels of brain long-chain polyunsaturated fatty acid are caused by lipid peroxidation.     

Hyperglycemia-induced changes in hepatic membrane fatty acid composition correlate with increased caspase-3 activities and reduced chick embryo viability

Fertile chicken eggs were injected with various concentrations of either d-glucose or l-glucose during the first three days of embryonic development. The exogenous glucose concentrations ranged from 0 to 18.58 micromol/kg egg. At 18 days of development (theoretical stage 44), brains, livers, and blood from chorio-allantoic vessels were isolated from living embryos. Exogenous d-glucose and l-glucose caused increased plasma d-glucose levels, increased plasma alanine aminotransferase (ALT) activities, and decreased embryo viability. Embryo viability was monitored by a reduction in the percentage of living embryos at theoretical stage 44, reduced embryo masses, reduced brain masses, and reduced liver masses. When compared to controls, embryonic exposure to either exogenous d-glucose or l-glucose caused increased caspase-3 activities and increased lipid hydroperoxide (LPO) levels in both brain and liver tissues. Because lipid hydroperoxides are lipid peroxidation intermediates that result in the attack of any unsaturated neutral lipid or unsaturated phospholipid, the effect of exogenous glucose on hepatic membrane fatty acid composition was studied. Exogenous glucose (either d-glucose or l-glucose) promoted reduced levels of several unsaturated, long-chain fatty acids and increased levels of saturated, short-chain fatty acids within hepatic membranes. Exogenous-glucose induced decreases in the ratios of unsaturated/saturated fatty acids and long-chain/short-chain fatty acids within hepatic membranes which strongly correlated with glucose-induced increases in plasma ALT activities and moderately correlated to hepatic LPO levels. These observations are consistent with the hypothesis that embryonic hyperglycemia promotes hepatic membrane lipid peroxidation and hepatic cell death.     

Ethanol- and Fe(+2)-induced membrane lipid oxidation is not additive in developing chick brains

In order to study the effects of exogenous EtOH and/or Fe(+2) on membrane lipid peroxidation, exogenous EtOH, FeCl(2), FeCl(2) & EtOH, NaCl and NaCl & EtOH were injected into fertile chicken eggs. Controls were either shams or injected with saline. These injections were made at 0 days or 0-2 days of development and tissue removed at stage 37 (11 days of development). Embryonic exposure to exogenous EtOH and/or Fe(+2) promoted decreased brain mass, decreased levels of brain membrane polyunsaturated fatty acids, elevated levels of brain lipid hydroperoxides, and elevated levels of Fe(+2) within embryonic brain and liver. These alterations were more severe in triple-injected embryos (E0-2/E11) as compared to single-injected embryos (E0/E11). While exogenous treatments of either EtOH and/or FeCl(2) promoted increased levels of endogenous brain Fe(+2), the effects were not additive. These observations are consistent with the hypothesis that embryonic exposure to exogenous EtOH and/or Fe(+2) promotes brain membrane lipid peroxidation.     

钙调蛋白及其调节酶PDE在鸡脑胚胎发育过程中的变化

钙调蛋白(CaM)参与脑中多种细胞过程的调节,推测它与大脑的分化发育有关。本实验研究了鸡脑从胚胎早期(原基)至大脑成熟各发育阶段可溶部分的CaM及其调节酶——环核苷酸磷酸二酯酶(PDE)的含量或活力变化。未经孵育的鸡胚中检测不出CaM,在孵育3—14天的胚脑中CaM含量逐渐增加:总CaM增加了3倍,活性CaM增加了4倍;而且在鸡脑细胞分裂分化最活跃的期间(皮质和髓质形成期间,即孵育8—14天),活性CaM的增加尤为显著(增加近3倍)。提示CaM与脑细胞的分裂分化有关。PDE的活力出现晚于CaM5天,随着胚龄增加,不断上升,提示它与脑细胞的分裂分化无直接关系,可能与大脑的功能活动有关。本实验还研究了鸡脑发育期中可逆性钙结合蛋白的变化。     

Hyperglycemia-induced membrane lipid peroxidation and elevated homocysteine levels are poorly attenuated by exogenous folate in embryonic chick brains

Injection of L-glucose (9.29 micromol/kg egg) into the air sac of fertile chicken eggs during the first 3 days of embryonic development (E(0-2)) has been reported to cause hyperglycemia and membrane lipid peroxidation in embryonic chick hepatic membranes. These observations have now been extended into embryonic chick brains at 11 days of development (theoretical stage 37). L-glucose caused a 1.7-fold increase in serum D-glucose levels (p< or =0.05), a 1.4-fold decrease in the % living embryos (p< or =0.05), a 1.1-fold decrease in embryonic masses (p< or =0.05), and a 1.4-fold decrease in embryonic brain masses (p< or =0.05) as compared to controls. L-glucose also caused a 3.8-fold increase in brain lipid hydroperoxide (LPO) levels (p< or =0.05) and complex changes in the relative fatty acid composition of brain membranes. Consistent with the hypothesis of hyperglycemia-induced increases in lipid peroxidation were decreased docosahexaenoic acid (DHA: 22: 6, n-3) levels as compared to controls (p< or =0.05). However, hyperglycemia-induced increased docosapentaenoic acid (DPA: 22:5, n-6) levels, decreased arachidonic acid (20; 4, n-6) levels, decreased linoleic acid (18:2, n-6) levels, and increased levels of several saturated short-chain membrane fatty acids were also observed as compared to controls (p< or =0.05). l-glucose caused a 12-fold increase in brain homocysteine levels, a 2.5-fold decrease in S-adenosylmethionine levels, and a 2-fold increase in S-adenosylhomocysteine levels as compared to controls (p< or =0.05). These hyperglycemia-induced alterations were poorly attenuated by exogenous folic acid (181.2 micromol/kg egg).     

Calpain Inhibition Prevents Ethanol-Induced Alterations in Spinal Motoneurons

Long-term exposure of ethanol (EtOH) alters the structure and function in brain and spinal cord. The present study addresses the mechanisms of EtOH-induced damaging effects on spinal motoneurons in vitro. Altered morphology and biochemical changes of such damage were demonstrated by in situ Wright staining and DNA ladder assay. EtOH at low to moderate (25–50 mM) concentrations induced damaging effects in the motoneuronal scaffold which involved activation of proteases like μ-calpain and caspase-3. Caspase-8 was seen only at higher (100 mM) EtOH concentration. Further, pretreatment with calpeptin, a potent calpain inhibitor, confirmed the involvement of active proteases in EtOH-induced damage to motoneurons. The lysosomal enzyme cathepsin D was also elevated in the motoneurons by EtOH, and this effect was significantly attenuated by inhibitor treatment. Overall, EtOH exposure rendered spinal motoneurons vulnerable to damage, and calpeptin provided protection, suggesting a critical role of calpain activation in EtOH-induced alterations in spinal motoneurons.     

The Regional Concentrations of S-Adenosyl-l-Methionine, S-Adenosyl-l-Homocysteine, and Adenosine in Rat Brain

Abstract: The concentrations of S -adenosyl- l -methionine (SAM), S -adenosyl- l -homocysteine (SAH), and adenosine (Ado) were determined in whole brain and rat brain regions by HPLC. The whole brain contains, respectively, 22 nmol, 1 nmol, and 64 nmol of SAM, SAH, and Ado per g of wet tissue. Their distribution indicated that SAM and SAH levels are highest in brainstem, whereas the Ado level is highest in cortex. With aging the SAM concentrations decrease in whole brain, brainstem, and hypothalamus (–25%) and SAH levels increase by 90% in striatum and by 160% in cerebellum, while Ado levels are increased in all regions by 100–180%.     

Utilization of ketone bodies by chick brain and spinal cord during embryonic and postnatal development

Lipid synthesis from acetoacetate and 3-hydroxybutyrate was studied in chick embryo from 15 to 21 days and in chick neonate from 1 to 21 days. Embryonic spinal cord showed higher ability than brain to incorporate acetoacetate into total lipids, although a sharp decrease was found at hatching. 3-Hydroxybutyrate incorporation into total lipids was also higher in spinal cord than in brain, especially during the embryonic period. Phospholipids were the main lipids formed in both tissues from both precursors. An appreciable percentage of radioactivity was also recovered as free cholesterol, especially during the embryonic phase. The developmental patterns of amino acid synthesis from acetoacetate and 3-hydroxybutyrate were similar in both tissues: a clear increase after hatching was followed by a decrease at day 4 of neonatal life. Acetoacetate was a better substrate for amino acid synthesis than 3-hydroxybutyrate during the embryonic development in both tissues. Oxidation of both precursors to CO₂ strongly decreased between 15 and 21 days of embryonic development both in brain and spinal cord.     

CHICK BRAIN CREATINE KINASE ONTOGENY BEFORE THE ADVENT OF CREATINE AND INSULIN

The ontogeny of brain creatine kinase (CK) was studied during chick embryo development. The cytosolic activity increased 270% in 10 h from the 2nd to the 3rd days of incubation; this was followed by a plateau phase throughout development and at the end of incubation there appeared to be another increase of cytosolic and mitochondrial CK activities. Therefore, early embryonic chick brain CK is another‘constitutive’enzyme like the early embryonic chick heart CK since creatine has not been enzymatically detected in the embryo until day 4 of incubation. Insulin does not appear to stimulate the early increase of brain CK activity since the hormone is not present in the embryo until day 5 of incubation. It is likely that CK increase is associated with neuronal multiplication at early stages and possibly to neuronal maturation before hatching.     

Changes in caspase expression in Alzheimer's disease: comparison with development and aging

Changes in caspase-2 and -3 protein levels in Alzheimer's disease (AD) brains were assessed in comparison with their expression in development and aging. The protein levels of caspase-2 and -3 were significantly increased in AD brains, caspase-2 in the particulate fraction and caspase-3 in both the cytosolic and particulate fractions, compared with controls. Immunoblot analysis of brain extracts from embryonic day 19 (E19) to postnatal 96-week-old rats indicated that caspase-3 levels were high from E19 to 2 weeks, while caspase-2 remained high from 4 to 96 weeks, indicating that the expression of caspase-2 and -3 is differentially regulated during development and aging. These results suggest that caspase-2 and -3 are upregulated in AD and that the changes in AD are different from those observed in senescent brains.     

Preventive effects of indole-3-carbinol against alcohol-induced liver injury in mice via antioxidant,anti-inflammatory,and anti-apoptotic mechanisms: Role of gut-liver-adipose tissue axis

Indole-3-carbinol (I3C), found in Brassica family vegetables, exhibits antioxidant, anti-inflammatory, and anti-cancerous properties. Here, we aimed to evaluate the preventive effects of I3C against ethanol (EtOH)-induced liver injury and study the protective mechanism(s) by using the well-established chronic-plus-binge alcohol exposure model. The preventive effects of I3C were evaluated by conducting various histological, biochemical, and real-time PCR analyses in mouse liver, adipose tissue, and colon, since functional alterations of adipose tissue and intestine can also participate in promoting EtOH-induced liver damage. Daily treatment with I3C alleviated EtOH-induced liver injury and hepatocyte apoptosis, but not steatosis, by attenuating elevated oxidative stress, as evidenced by the decreased levels of hepatic lipid peroxidation, hydrogen peroxide, CYP2E1, NADPH-oxidase, and protein acetylation with maintenance of mitochondrial complex I, II, and III protein levels and activities. I3C also restored the hepatic antioxidant capacity by preventing EtOH-induced suppression of glutathione contents and mitochondrial aldehyde dehydrogenase-2 activity. I3C preventive effects were also achieved by attenuating the increased levels of hepatic proinflammatory cytokines, including IL1β, and neutrophil infiltration. I3C also attenuated EtOH-induced gut leakiness with decreased serum endotoxin levels through preventing EtOH-induced oxidative stress, apoptosis of enterocytes, and alteration of tight junction protein claudin-1. Furthermore, I3C alleviated adipose tissue inflammation and decreased free fatty acid release. Collectively, I3C prevented EtOH-induced liver injury via attenuating the damaging effect of ethanol on the gut-liver-adipose tissue axis. Therefore, I3C may also have a high potential for translational research in treating or preventing other types of hepatic injury associated with oxidative stress and inflammation.     

Evidence for altered methionine methyl-group utilization in the diabetic rat's brain

The methionine (MET) derivative, S-adenosylmethionine (SAM), provides methyl-groups for methylation reactions in many neural processes. In rats made diabetic with streptozotocin (SZ), brain SAM levels were generally lower (10–20%) than in controls, with a constant decrease being observed five weeks after onset of diabetes. This decrease in SAM levels may be due to reduced precursor (MET) availability because greatly elevating plasma MET concentrations in SZ diabetic rats by dietary manipulation increased their neural SAM concentrations to be approximately or even greater than (5–20%) those of controls. In contrast, neural levels of SAM's demethylated product, S-adenosylhomocysteine (SAH), were reduced to a greater extent (17–44%) than SAM levels in all groups of SZ diabetic rats independent of their plasma MET concentrations or brain SAM levels. This indicates that the decrease in SAH levels is not simply due to substrate (SAM) restriction. These changes in MET metabolites appear to be a general effect of diabetes rather than a non-pancreatic side-effect of SZ, because genetically diabetic BB Wistar rats also exhibited reduced brain SAM (25%) and brain SAH (46%) levels. These results indicate that methyl-groups from MET are handled differently in the brain of the diabetic rat, which considering the variety and importance of neural methylation reactions, could have important consequences for the diabetic.Abbreviations MET methionine - SAM S-adenosylmethionine - SAH S-adenosylhomocysteine - SZ streptozotocin - BBW BB Wistar - LNAA large neutral amino acids - BCAA branchedchain amino acids - MET:BCAA methionine to branched-chain amino acid ratio - MET:LNAA methionine to large neutral amino acid ratio     

Influence of acute heat stress on the development of GABAergic neurons in HPA-axies of mouse embryos

The damaging effects of acute heat stress in pregnant mice is well known, however, very little is known about the impact of heat on embryonic neurodevelopment and its dependence on the dam's physiological response to the stress. To study the changes in GABAergic neurons expression on the hypothalamo-pituitary-adrenocortical axis (HPA-axis), superoxide dismutase (SOD) activities and malondialdehyde (MDA) levels were measured in mouse embryos on E13-17 following acute, maternal heat stress. Blood samples and amniotic fluid from pregnant mice, and homogenates of whole embryos and embryo brains were collected for SOD and MDA analyses. SOD and MDA activities were measured in embryo and the sections by staining with anti-γ-aminobutyric acid-A- α1 receptor (GABAA receptor- α1), anti-γ-aminobutyric acid-B-1 receptor (GABAB receptor-R1) and anti-glutamate decarboxylase-65(GAD-65) antibodies. After the pregnant mice underwent acute heat stress on E13, the embryonic GABAergic neurons and GABA receptor expression were triggered immediately as a result of induction of the HPA-axis. This expression recovered to normal levels consistent with the control groups. However, the expression of the GABAA receptor in embryonic adrenal gland decreased continually. The SOD activity decreased in the embryonic brain and increased in the amniotic fluid after the heat stress, whereas the MDA levels increased in the maternal plasma only. Therefore, the GABAergic neurons in the developing HPA-axis of the embryos are susceptible to heat stress and the enhancement of SOD activities in the amniotic fluid might be a protective mechanism.     

Cigarette smoke extract increases S-adenosylmethionine and cystathionine in human lung epithelial-like (A549) cells

The effect of cigarette smoke extract (CSE) on S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and sulfur amino acid metabolism was examined in human lung epithelial-like (A549) cells exposed to various CSE concentrations (2.5-100%) for 24 or 48 h. Intracellular SAM and SAM/SAH ratio were elevated after exposure to CSE for 48 h. Cell SAH content decreased, but the effect was not consistent. Cellular cystathionine, cysteine, and methionine levels were increased after CSE exposure for 48h. Sub-acute exposure to CSE induced increases in cellular SAM and SAM/SAH ratio. The transsulfuration pathway was likely activated by CSE since cystathionine increased, potentially contributing to the increased total intracellular GSH content.     

Synthesis of sterols by chick brain and liver during embryonic development

The evolution throughout embryonic development of the rate at which acetate was converted into sterols was studied in chick brain and liver. Acetate incorporation (nmol/h/g tissue) was clearly higher in brain than in liver and sharply decreased with the age of embryo. Cholesterol and desmosterol were the major sterols formed from acetate by chick embryo brain, followed by lanosterol and squalene. No desmosterol was found in chick embryo liver, organ where cholesterol was the major sterol synthesized. In brain, the relative percentage of cholesterol increased throughout embryonic development reaching more than 50% at hatching, while the percentage of desmosterol decreased during the same period and represented at hatching only about 10–15% of the total nonsaponifiable fraction. The relative percentages of lanosterol and squalene did not change significantly throughout the period assayed. In liver, the percentage of cholesterol increased until 19 days but sharply decreased at hatching.     

Glutamine Transaminase K and ω-Amidase Activities in Primary Cultures of Astrocytes and Neurons and in Embryonic Chick Forebrain: Marked Induction of Brain Glutamine Transaminase K at Time of Hatching

Abstract: Glutamine transaminase K and ω-amidase activities are present in the chick brain and in the brains of adult mice, rats, and humans. However, the activity of gluta-mine transaminase K in adult mouse brain is relatively low. In the chick embryo, cerebral glutamine transaminase K activity is low between embryonic days 5 and 17, but by day 23 (day of hatching) activity rises dramatically (< 15-fold). Cerebral ω-amidase activity is relatively high at embryonic day 5 but lower between days 5 and 17; at embryonic day 23 the activity rises to a maximum. Both glutamine transaminase K and ω-amidase are present in cultured chick, rat, and mouse astrocytes and neurons. For each species, the activity of glutamine transaminase K is higher in the astrocytes than in the neurons. The activity of ω-amidase is about the same in the cultured chick astrocytes and neurons but significantly higher in rat astrocytes than in rat neurons. The data suggest that the rise in brain glutamine transaminase K activity in the chick embryo at hatching correlates with maturation of astrocytes. Glutamine transaminase K may be involved in glutamine cycling in astrocytes. Glutamine transaminase K appears to be a major cysteine S-conjugate β-lyase of the brain and may play a role in the neurotoxicity associated with exposure to dichloroacetylene and perhaps to other toxins.