The role of N-methyl-D-aspartate receptor activation in homocysteine-induced death of retinal ganglion cells.

PURPOSE: Elevated plasma homocysteine has been implicated in glaucoma, a vision disorder characterized by retinal ganglion cell death. The toxic potential of homocysteine to ganglion cells is known, but the mechanisms are not clear. A mechanism of homocysteine-induced death of cerebral neurons is via N-methyl-D-aspartate (NMDA) receptor overstimulation, leading to excess calcium influx and oxidative stress. This study examined the role of the NMDA receptor in homocysteine-mediated ganglion cell death. METHODS: Primary mouse ganglion cells were used for these experiments. NMDA receptor stimulation by homocysteine was determined by patch clamp analysis and fluorescent detection of intracellular calcium. NMDA receptor involvement in homocysteine-mediated cell death was determined through assessment of lactate dehydrogenase release and TUNEL analysis. These experiments used the NMDA receptor blocker MK-801. Induction of reactive species superoxide, nitric oxide, and peroxynitrite was measured by electron paramagnetic resonance spectroscopy, chemiluminescent nitric oxide detection, and immunoblotting for nitrotyrosine, respectively. RESULTS: 50 µM homocysteine stimulated the NMDA receptor in presence of 100 µM glycine. Homocysteine induced 59.67 ± 4.89% ganglion cell death that was reduced to 19.87 ± 3.03% with cotreatment of 250 nM MK-801. Homocysteine elevated intracellular calcium ∼7-fold, which was completely prevented by MK-801. Homocysteine treatment increased superoxide and nitric oxide levels by ∼40% and ∼90%, respectively, after 6 hours. Homocysteine treatment elevated peroxynitrite by ∼85% after 9 hours. CONCLUSIONS: These experiments provide compelling evidence that homocysteine induces retinal ganglion cell toxicity through direct NMDA receptor stimulation and implicate, for the first time, the induction of oxidative stress as a potent mechanism of homocysteine-mediated ganglion cell death.

Comparison of selenohomolanthionine and selenomethionine in terms of selenium distribution and toxicity in rats by bolus administration.

The distribution and metabolism of selenohomolanthionine (4,4'-selenobis[2-aminobutanoic acid], SeHLan), a newly identified selenoamino acid in selenized Japanese pungent radish, were compared with those of selenomethionine (SeMet) in rats. Either selenoamino acid was injected intravenously at a bolus dose of 1.0 mg Se/kg body weight. SeMet was preferably accumulated in the pancreas, increasing the serum amylase level, an index of pancreatic damage. SeHLan was preferably accumulated in the kidneys, raising the serum creatinine level, an index of kidney damage. On the other hand, the levels of two major urinary selenometabolites, i.e., Se-methylseleno-N-acetyl-galactosamine and trimethylselenonium, were comparable between SeHLan- and SeMet-administered rats, suggesting that there may be no differences in the efficiency of metabolism of these two selenoamino acids to the urinary selenometabolites despite the difference in distribution. SeHLan is expected to be a potential supplemental source of Se without inducing the onset of pancreatic damage. The specific toxicity of SeHLan to the kidneys may be avoided if its dose is lower than the one used in the present study.

Homocysteine induces cell death in H9C2 cardiomyocytes through the generation of peroxynitrite.

Homocysteine (HCY) is toxic on blood vessels, but a potential direct toxicity of HCY on the heart is unknown. We addressed this issue by exposing H9C2 cardiomyocytes to HCY (0.1-5 mM) for up to 6h. At these concentrations, HCY reduced cell viability, induced necrosis and apoptosis and triggered the cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP). This was associated with the intracellular generation of the potent oxidant peroxynitrite. Removing peroxynitrite by the decomposition catalyst FeTPPS considerably reduced LDH release, DNA fragmentation, cleavage of caspase-3 and PARP, and restored normal cell morphology. In additional experiments performed in primary rat ventricular cardiomyocytes, HCY (1 mM, 6h) activated the phosphorylation of the MAP kinases ERK and JNK, two essential stress signaling kinases regulating myocardial apoptosis, hypertrophy and remodeling. These results provide the first demonstration that HCY kills cardiomyocytes through the generation of peroxynitrite and can activate key signaling cascades in the myocardium.

Failure of homocysteine to induce neural tube defects in a mouse model.

BACKGROUND: Folate deficiencies have been associated with many adverse congenital abnormalities. It is not clear, however, whether these defects are due to a folate deficiency or to an increase in homocysteine. Homocysteine has been shown to be teratogenic in the chicken-embryo model and it has been suggested that homocysteine-induced defects are mediated by inhibiting the N-methyl-D-aspartate (NMDA) receptor on neural crest cells. The majority of the teratology studies have been carried out using the chicken embryo model. In an effort to develop a murine model of homocysteine-induced neural tube defects, several inbred mouse strains were treated with homocysteine or the NMDA inhibitor MK801 and the fetuses examined for any induced-NTD. METHODS: Several in-bred mouse strains were administered homocysteine once on gestational day (GD) E8.5 or once daily on GD 6.5-10.5. Additionally, because homocysteine was been reported to mediate its effects through the NMDA receptor, the effect of MK801, an antagonist of this receptor, was also investigated. RESULTS: Regardless of the mouse treatment time, homocysteine failed to induce neural tube defects in our in-bred mouse strains. Homocysteine also failed to increase the number of neural tube defects in the splotch strain, regardless of the genotype. CONCLUSIONS: Irrespective of the mouse strain or treatment, homocysteine failed to induce neural tube defects in our mouse models, which is in contrast to what has been reported in the chicken embryo models.

Excitotoxic neuronal injury in chronic homocysteine neurotoxicity studied in vitro: the role of NMDA and group I metabotropic glutamate receptors.

Elevated homocysteine is a risk factor in cardiovascular diseases and neurodegeneration. Among the putative mechanisms of homocysteine-evoked neurotoxicity, disturbances in methylation processes and NMDA receptor-mediated excitotoxicity have been suggested. Our previous studies demonstrated that group I metabotropic glutamate receptors along with NMDA receptors participate in acute homocysteine-induced neuronal damage. In this study, using propidium iodide staining, we tested whether the same mechanism may mediate chronic homocysteine neurotoxicity. Our results confirmed that the application of D,L-homocysteine in micromolar concentrations for 3 days induces neurodegeneration in primary cultures of cerebellar granule neurons. Uncompetitive NMDA receptor antagonist MK-801, and mGlul or mGlu5 receptor antagonists (LY367385 and MPEP, respectively), given alone provided very limited neuroprotection. However, simultaneous application of the NMDA receptor antagonists MK-801, memantine or amantadine and MPEP almost completely prevented chronic homocysteine neurotoxicity. These findings suggest a novel therapeutic strategy to combat neurodegeneration induced by hyperhomocysteinemia comprising a combination of antagonists of group 1 metabotropic glutamate receptors and NMDA receptors.

[Inhibition on the endocytosis of early visceral yolk sac induced by homocysteine in rats].

The effect of homocysteine (HCY) on the development and endocytosis of rat visceral yolk sac (VYS) was investigated. A preliminary method on early (GD9.5) rat yolk-sac placenta cultured in vitro was established by techniques using microdissection and transplantation. The results showed that HCY did damage VYS development and blood vessel formation, the damages were characterized by shrunk surface, small or defective blood island and significant reduction of diameter (P < 0.01). HCY also inhibited the process of VYS transport, specifically the endocytosis (as measured by [U-14C] sucrose uptake), the rate of endocytosis was significantly decreased when HCY was included in the medium (P < 0.01). These results suggested that the inhibition of sanguimotor development and the reduction of endocytosis induced by HCY may directly correlate with the teratologic and embryopathologic events.

Excess levels of cysteine and homocysteine induce tibial dyschondroplasia in broiler chicks.

The effect of excessive levels of cysteine and homocysteine on tibial dyschondroplasia (TD) in broiler chicks was studied. In the first experiment, graded levels of L-cysteine as well as one level of L-homocysteine were supplemented to a corn-soybean-based diet adequate in sulfur amino acids. Levels equal to or above 0.5% supplemental cysteine increased the incidence of TD, and levels equal to or above 0.75% supplemental cysteine increased the severity of TD above that found in chicks fed the basal diet. Also, L-homocysteine at 0.5% induced TD. In the second experiment, graded levels of DL-homocystine were added to the basal diet to determine the threshold value of homocystine needed to induce TD, and a level of ammonium sulfate isosulfurous to 0.45% homocystine was added to a basal diet. The results showed that 0.45% DL-homocystine was the lowest level that increased the severity of TD above that found in chicks fed the basal diet and that sulfate did not induce TD. In the third experiment, a 2 x 2 factorial design was used to investigate the interaction between DL-homocystine and copper. Copper supplementation lessened the severity of TD caused by DL-homocystine. Copper supplementation also tended to improve growth, especially in birds fed DL-homocystine.

Growth support and toxicity of homocysteine and its effects on methionine metabolism in non-transformed and chemically transformed C3H/10T1/2 cells.

The effects of homocysteine (Hcy) on one non-transformed (Cl 8) and two malignant clones (Cl 16 and Cl T422) of the C3H/10T1/2 mouse embryo fibroblasts, were examined with regard to toxicity, ability to support growth and effects on methionine (Met) metabolism and glutathione level. Homocysteine in its reduced form (Hcy-SH) was toxic to all cell lines, and the LD90 was estimated to be 1.0 X 10(-4) M for Cl 8 and Cl 16 cells measured by plating efficiency, 0.8 X 10(-4) M for Cl 8 and 0.3 X 10(-4) M for Cl 16 when measured by total cell growth. At toxic concentrations, Hcy-SH showed a drastic effect on cell morphology both in the presence and absence of Met. The same effect was demonstrated with L-cysteine. No toxic effect was seen with homocystine (Hcy-SS-Hcy) or homocysteine thiolactone (Hcy-tl) at similar concentrations. Hcy-tl supported growth of both the non-transformed and malignant cells in Met-deficient medium but with decreasing efficiency in the order Cl 8, Cl 16 and Cl T422. The growth rate constant compared to that of Met-supplemented medium was 0.62 for Cl 8, 0.44 for Cl 16 and 0.38 for Cl T422 cells. The intracellular level of S-adenosylhomocysteine (AdoHcy) increased in all three cell lines in Hcy-tl-supplemented medium. The S-adenosylmethionine (AdoMet) content increased in Cl 8 cells, was constant in Cl 16 cells and decreased in Cl T422 cells under the same conditions. This resulted in a constant ratio of AdoMet/AdoHcy in the non-transformed cells (Cl 8) whereas this ratio decreased by 40% in Cl 16 and by 72% in Cl T422 cells when Hcy-tl replaced Met in the medium. The ability of Hcy-tl to support growth thus seemed to correlate well with alteration in Met metabolism in this cell culture system. The intracellular level of glutathione (GSH) was measured during exponential growth, but showed small variations between non-transformed cells and Cl 16 cells. However, Cl T422 cells showed a distinct lower level of GSH in Met-supplemented medium, and this increased 3- to 4-fold when Met was replaced with Hcy-tl.

Methionine toxicity in chicks and poults.

In feeding experiments with poults, 2% DL-methionine caused a marked growth depression which could be alleviated by the addition of glycine. Homocystine at an equimolar level depressed growth to a lesser degree than methionine, and this growth depression could be alleviated by glycine. Betaine could alleviate the growth depression of homocystine but not that of methionine. Methionine-fed poults developed a cervical paralysis similar to that of a folic acid deficiency, but the addition of this vitamin at several times the requirement was ineffective in counteracting the toxicity of methionine in either chicks or poults.