The active centre of triose phosphate isomerase

1. Cystamine (2,2'-diaminodiethyl disulphide) caused an unmasking of mitochondrial adenosine triphosphatase and a leakage of Mg(2+) from the mitochondria, and decreased the stimulation of adenosine triphosphatase by 2,4-dinitrophenol. When Mg(2+) was added, cystamine potentiated the activation of adenosine triphosphatase by 2,4-dinitrophenol. 2. Cystamine was without effect on the adenosine triphosphatase of disrupted mitochondria. 3. Cystamine was moderately potent as an uncoupling agent and as an inhibitor of the [(32)P]P(i)-ATP exchange reaction. 4. Cysteamine (2-aminoethanethiol) was without the above effects, when special precautions were taken to counteract its autoxidation. 5. The effects of cystamine should probably be ascribed to its disulphide group, since the diamine cadaverine protected slightly against the loss of Mg(2+) and the decrease of 2,4-dinitrophenol-stimulated adenosine-triphosphatase activity caused by aging of the mitochondria. It is suggested that cystamine acts by a breakdown of mitochondrial permeability barriers.     

The inhibitory effect of reduced glutathione on the lipid peroxidation of the microsomal fraction and mitochondria

1. GSH efficiently inhibited the ascorbate-stimulated lipid peroxidation of the unsaturated fatty acids in the fresh microsomal fraction and mitochondria of rat liver, whereas the peroxidation in heat-denatured particles was little inhibited. 2. Cysteamine and diethyldithiocarbamate inhibited the peroxidation in both fresh and boiled particles. Thioglycollate and 2-mercaptoethanol had no inhibiting effect. Cysteine and homocysteine both stimulated the lipid peroxidation even in the absence of ascorbate. 3. The added GSH disappeared at nearly the same rate in the presence of fresh and of boiled particles to which ascorbate had been added, although considerably more malonaldehyde was formed in the boiled particles. In the absence of ascorbate little GSH disappeared. 4. It is suggested that the protective effect of GSH against lipid peroxidation depends on the preservation of heat-labile structures in the microsomal fraction and mitochondria.     

Cysteamine supplementation during in vitro maturation and embryo culture: a useful tool for increasing the efficiency of bovine in vitro embryo production

Cysteamine when added during in vitro maturation (IVM) or in vitro embryo culture (IVC) stimulates glutathione (GSH) synthesis and improves embryo developmental rates. This suggests that GSH synthesis is decreased in the in vitro produced embryo. The present study was carried out to evaluate if addition of cysteamine to culture medium at the same time, during IVM and IVC of bovine oocytes, may promote an overall improvement on the developmental rate and embryo quality. Oocytes were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 or 100 microM of cysteamine for 24 hr. After IVM, the oocytes were fertilized (day 0). Day 2 embryos (2-8 cell) were washed and transferred to fresh IVC medium supplemented with 0, 25, 50, or 100 microM of cysteamine and cultured for 48 hr. After this, embryos were cultured in IVC medium without cysteamine until day 8 of IVC. In the present study, we confirmed our previous results by demonstrating that the percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.05) when 100 microM of cysteamine was added during IVM, and this was further improved when 100 and 50 microM of cysteamine where present during IVM and IVC, respectively (P < 0.05). After cryopreservation, no differences were observed on embryo development, but a significant increase on embryo hatching was found between unsupplemented and supplemented oocytes with 100 and 50 microM of cysteamine during IVM and IVC, respectively (P < 0.05). We can conclude that GSH synthesis stimulation during bovine IVM with cysteamine, concomitant with GSH stimulation during IVC, will be a useful and simple tool for increasing the efficiency of in vitro bovine embryo production.     

Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content

Cysteamine and beta-mercaptoethanol supplementation of in vitro maturation (IVM) medium has been found to increase intracellular glutathione (GSH) content in oocytes and to improve embryo development and quality in several species. The objective of this experiment was to study the effect of cysteamine and beta-mercaptoethanol added during IVM of sheep oocytes on GSH synthesis and embryo development. Furthermore, we examined if cysteamine addition (hence GSH production) had an effect on the reduction of the intracellular peroxide content. We matured oocytes obtained from ovaries collected at a slaughterhouse in vitro in the presence of 0, 50, 100, and 200 microM cysteamine (Experiment 1) or with 0, 50, 100, and 200 microM beta-mercaptoethanol (Experiment 2). Following fertilization and embryo development, there was a increasing level of morula and blastocyst development in the presence of cysteamine, reaching significance in the presence of 200 microM (P < 0.05). However, beta-mercaptoethanol did not influence on the rate of embryo development. GSH levels were measured in oocytes matured in the presence or absence of 200 microM cysteamine (Experiment 3) or 50 microM beta-mercaptoethanol (Experiment 4), with or without buthionine sulfoximide (BSO), an inhibitor of GSH synthesis. Results demonstrated that for both cysteamine and beta-mercaptoethanol, intracellular GSH levels increased against control values (P < 0.01), which was abolished in the presence of BSO. Finally, we reduced intracellular peroxide levels, as measured by the relative fluorescence of the intracellular peroxide probe, carboxy-H2DCFDA, in the presence of either 200 microM cysteamine or 50 microM beta-mercaptoethanol (Experiment 5). These results demonstrate that cysteamine, but not beta-mercaptoethanol, when present during IVM, stimulates sheep embryo development; both cysteamine and beta-mercaptoethanol stimulate GSH synthesis; the increase in intracellular GSH is associated with a decrease in peroxide levels within oocytes.     

Interrelation between the analgesic and ulcerogenic action of cysteamine

Interrelationship was studied between the influence of cysteamine on pain threshold and ulcerogenic effect on the duodenum. Cysteamine (350 mg/kg) induced analgesia in mice which was prevented by naloxone (1.5 mg/kg). In rats, cysteamine produced duodenal ulcers with concomitant analgesia. The intensity of ulceration was higher in animals with lower basal pain threshold. The correlation between central and peripheral effects of endogenous opioids in the development of experimental duodenal ulcers is discussed.     

Radioprotection by glutathione esters and cysteamine in normal and glutathione-depleted mammalian cells

Monoethyl (MEE) and diethyl (DEE) esters of glutathione (GSH) had the capacity to provide some protection of normal and buthionine sulfoximine (BSO) pretreated cells against X-irradiation. Both compounds appeared to be transported through the cell membrane into the cells. MEE was intracellularly partly hydrolysed to GSH and caused a limited rise of intracellular GSH. DEE was intracellularly mainly converted into MEE and partly into GSH. DEE caused a larger rise of the intracellular GSH content than MEE; it also provided a better radioprotection. Radioprotection by the GSH esters may be explained by an increase of intracellular GSH as well as by the presence of the esters themselves. Cysteamine caused no rise of the intracellular GSH content, thus its radioprotection could not be mediated by an increase of intracellular GSH. When the radiosensitivity of GSH-depleted cells protected by cysteamine was compared with the radiosensitivity of non-GSH-depleted cells similarly protected by cysteamine, it appeared that the GSH-depleted cells remained more sensitive to irradiation. Thus, it seems that in this respect cysteamine cannot fully substitute for endogenous GSH.     

Antioxidant Effect of Cysteamine in Brain Cortex of Young Rats

Cysteamine is a cystine-depleting drug used in the treatment of cystinosis, a metabolic disorder caused by deficiency of the lysosomal cystine carrier. As a result, cystine accumulates within lysosomes in many tissues and organs, including the nervous system. Studies with cystine dimethyl ester loaded cells suggest that cystine might induce apoptosis through oxidative stress. Our objective was to investigate the effects of co-administration of cysteamine with the oxidant cystine dimethyl ester on several parameters of oxidative stress in the brain cortex of rats. Animals were injected with 1.6 μmol/g cystine dimethyl ester and/or 0.26 μmol/g body weight cysteamine. Cystine dimethyl ester induced lipoperoxidation, protein carbonylation, and stimulated superoxide dismutase, glutathione peroxidase and catalase activities, probably through the formation of free radicals. Cysteamine prevented those effects, possibly increasing cellular thiol pool and acting as a scavenger of free radicals. These results suggest that the antioxidant effect of cysteamine may be important in the treatment of cystinosis.     

Oxidation of reduced glutathione by subcellular fractions of rat liver

1. A new method was used to diminish the autoxidation of GSH. 2. The oxidation of GSH by liver homogenates was studied with regard to concentration of homogenate, concentration of GSH, time, pH and anaerobiosis. 3. GSH was oxidized by recombinations of the supernatant with microsomes and with mitochondria. Each fraction alone caused little oxidation. 4. Proteins in the supernatant were required to obtain the effect, and low-molecular-weight compounds in the same fraction increased its effect. 5. GSH diminished the formation of malonaldehyde in homogenates. 6. GSH prevented a stimulating effect of the supernatant on the formation of malonaldehyde in microsomes and in mitochondria. 7. The malonaldehyde formation in microsomes together with the supernatant did not start until the concentration of endogenous low-molecular-weight thiols had decreased to a low level. 8. It is suggested that part of the oxidation of GSH in homogenates is coupled to a mechanism that counteracts the peroxidation of membrane lipids.     

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.     

Effect of cysteamine on amino acid pools of cultured fibroblasts and the uptake of L-proline

Cysteamine when incubated with human normal and cystinotic fibroblasts produces significant alterations in cellular amino acid pools with a decrease in proline, glycine, histidine, and methionine as well as branch chain and dibasic amino acids. Serine content was increased while aspartate, glutamate and glutamine were unchanged. The uptake of L-proline by cysteamine treated cells was impaired suggesting that altered uptake of amino acids may be a factor in the cysteamine effect.     

Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender

The aim of this study was to evaluate the effects of ergothioneine and cysteamine as antioxidant supplements in a soybean lecithin extender for freezing ram semen. Twenty-four ejaculates were collected from four rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and cysteamine or ergothioneine (2, 4, 6 or 8 mM). Motility by CASA, viability, plasma membrane functionality (HOS test), total abnormality, lipid peroxidation, glutathione peroxidase (GPx) activity and capacitation status (CTC staining) were assessed after thawing. Using 6 mM of either antioxidant improved total motility. Cysteamine at 6 mM and ergothioneine at 4 and 6 mM improved viability and reduced lipid peroxidation (malondialdehyde concentration). Both antioxidants improved membrane functionality significantly, except at 8 mM. Progressive motility, kinematic parameters, GPx activity, capacitation status and sperm abnormalities were not influenced by the antioxidant supplements. In conclusion, cysteamine at 6 mM and ergothioneine at 4 or 6 mM seem to improve the post-thawing quality of ram semen cryopreserved in a soybean lecithin extender.     

Combined chemical and hypoxic protection against whole-body X-irradiation in the nestling rat

Litters of 8- to 10-day-old nestling rats were protected against whole-body x-irradiation by cysteamine, AET, and deep hypothermia, singly, and by a combination of cysteamine pretreatment and deep hypothermia. The different treatments were randomized among littermates to minimize animal variation. A comparison of the LD50(30) values obtained for the different treatments gave a dose-reduction factor of 3.3 for the combined treatment, which was significantly greater than the factor for hypothermia alone, 2.6, or that for cysteamine alone, 1.4. Cysteamine therefore provided additional protection in the fully hypoxic (hypothermic) animal. Judged from the histological appearance of the gonads of the surviving male nestling rats, killed 30 days after irradiation, hypothermia afforded some protection to the germinal epithelium. Cysteamine, although injected when the testes were abdominal, had no demonstrable protective effect.     

Myeloperoxidase-oxidase oxidation of cysteamine.

Cysteamine oxidation was shown to be catalysed by nanomolar concentrations of myeloperoxidase in a peroxidase-oxidase reaction, i.e. an O2-consuming oxidation of a compound catalysed by peroxidase without H2O2 addition. When auto-oxidation of the thiol was prevented by the metal-ion chelator diethylenetriaminepenta-acetic acid, native, but not heat-inactivated, myeloperoxidase induced changes in the u.v.-light-absorption spectrum of cysteamine. These changes were consistent with disulphide (cystamine) formation. Concomitantly, O2 was consumed and superoxide radical anion formation could be detected by Nitro Blue Tetrazolium reduction. Both superoxide dismutase and catalase inhibited the reaction, whereas the hydroxyl-radical scavengers mannitol and ethanol did not. O2 consumption increased with increasing pH (between pH 6.0 and 8.0), and 50% inhibition was exhibited by about 3 mM-NaCl at pH 7.0 and by about 100 mM-NaCl at pH 8.0. Cysteamine was about 5 times as active (in terms of increased O2 consumption at pH 7.5) as the previously reported peroxidase-oxidase substrates NADPH, dihydroxyfumaric acid and indol-3-ylacetic acid. A possible reaction pathway for the myeloperoxidase-oxidase oxidation of cysteamine is discussed. These results indicate that cysteamine is a very useful substrate for studies on myeloperoxidase-oxidase activity.     

Dose-related effects of cysteamine treatment on hypothalamic and striatal dopamine, noradrenaline and serotonin neurotransmission

The dose-related effects of cysteamine treatment on hypothalamic and striatal neurotransmission were investigated. Cysteamine pretreatment with a dose of 150 mg/kg slightly increased the dopamine, and markedly decreased the noradrenaline, content of the hypothalamus in a dose-related manner. The serotonin levels of the hypothalamus and striatum were not affected. Cysteamine pretreatment with a higher dose (300 mg/kg sc) slightly increased the uptake of noradrenaline into hypothalamic slices. The drug did not influence dopamine and serotonin uptake into hypothalamus and striatal slices. These results suggest that cysteamine decreases rather selectively the noradrenaline content of the hypothalamus.     

Effect of cysteamine on suckling-induced prolactin secretion in the rat

We have examined the effects of the thiol agent cysteamine on physiological prolactin secretion in the female rat. Administration of cysteamine completely abolishes suckling-induced prolactin secretion in a dose-dependent manner. Cysteamine treatment does not alter nursing behavior of the mothers. Further, we have found that the prolactin-depleting ability of cysteamine is not altered by a prior suckling stimulus. These results indicate that cysteamine administration inhibits physiologically-induced prolactin secretion with similar potency and efficacy as previously reported for cysteamine effects on basal and pharmacologically-induced prolactin secretion. Furthermore, the effect of cysteamine is not compromised by a previous suckling stimulus, suggesting that "depletion-transformation" of pituitary prolactin stores does not protect against the effect of cysteamine.     

Oxidative cell injury in the killing of cultured hepatocytes by allyl alcohol

The killing of cultured hepatocytes by allyl alcohol depended on the metabolism of this hepatotoxin by alcohol dehydrogenase to the reactive electrophile, acrolein. An inhibitor of alcohol dehydrogenase, pyrazole, prevented both the toxicity of allyl alcohol and the rapid depletion of GSH. Treatment of the hepatocytes with a ferric iron chelator, deferoxamine, or an antioxidant, N,N'-diphenyl-p-phenylenediamine (DPPD), prevented the cell killing but not the metabolism of allyl alcohol and the resulting depletion of GSH. Inhibition of glutathione reductase by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) sensitized the hepatocytes to allyl alcohol, an effect that was not attributable to the reduction in GSH with BCNU. The cell killing with allyl alcohol was preceded by the peroxidation of cellular lipids as evidence by an accumulation of malondialdehyde in the cultures. Deferoxamine and DPPD prevented the lipid peroxidation in parallel with their protection from the cell killing. These data indicate that acrolein produces an abrupt depletion of GSH that is followed by lipid peroxidation and cell death. Such oxidative cell injury is suggested to result from the inability to detoxify endogenous hydrogen peroxide and the ensuing iron-dependent formation of a potent oxidizing species. Oxidative cell injury more consistently accounts for the hepatotoxicity of allyl alcohol than does the covalent binding of acrolein to cellular macromolecules.     

Inactivation of 2-oxoglutarate dehydrogenase in rat liver mitochondria by its substrate and t-butyl hydroperoxide

Ketoacid oxidation in rat liver mitochondria was very sensitive to t-butyl hydroperoxide (t-BuOOH). Furthermore, 2-oxoglutarate and pyruvate each enhanced t-BuOOH-induced oxidative stresses of mitochondria, such as oxidation of pyridine nucleotides and GSH, inhibition of respiration with the other NAD-linked substrates, and peroxidation of mitochondrial lipids. We provide evidence that the t-BuOOH and ketoacid-induced effects are due to the failure of supply of NADH by 2-oxoglutarate dehydrogenase, and report the inactivation of the dehydrogenase in mitochondria by simultaneous addition of 2-oxoglutarate and t-BuOOH. Using the purified enzyme, we confirmed that t-BuOOH-induced inactivation of 2-oxoglutarate dehydrogenase was enhanced by its substrate and thiamine pyrophosphate protected the dehydrogenase from the inactivation. In contrast, succinate-dependent oxidation of mitochondria was not only scarcely affected by t-BuOOH, but also succinate protected against inactivation of 2-oxoglutarate dehydrogenase by t-BuOOH in mitochondria.     

Effect of clofibrate treatment on lipid peroxidation in rat liver homogenate and subcellular fractions

1. A study was made of the effect of hypolipidemic drug clofibrate on the level of lipid peroxidation in homogenates and subcellular fractions of rat liver. The intensity of lipid peroxidation was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that under the action of clofibrate the levels of Fe/ADP-ascorbate-, as well as t-butyl hydroperoxide (Bu'OOH)-induced lipid peroxidation were decreased in the whole and "post-nuclear" liver homogenates. Dilution of the homogenates prevented depressing effect of clofibrate on lipid peroxidation. 3. Clofibrate significantly decreased the level of the Bu'OOH-dependent lipid peroxidation, but did not affect the activity of the Fe/ADP-ascorbate-induced reaction in rat liver mitochondria and microsomes. 4. Peroxidative alteration of membrane lipids in vivo was evaluated by determining the extent of conjugated dienes formation (absorption at 233 nm). It was shown that clofibrate did not increase the level of ultraviolet absorption of lipids from rat liver subcellular fractions. 5. The data obtained indicate that cytosol from the clofibrate treated rat liver contains a factor(s) which prevents lipid peroxidation in the mitochondria and microsomes.     

New mechanistic explanation for the localization of ulcers in the rat duodenum: role of iron and selective uptake of cysteamine

Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis.