Quercetin protects cutaneous tissue-associated cell types including sensory neurons from oxidative stress induced by glutathione depletion: cooperative effects of ascorbic acid

Oxidation reactions are essential biological reactions necessary for the formation of high-energy compounds used to fuel metabolic processes, but can be injurious to cells when produced in excess. Cutaneous tissue is especially susceptible to damage mediated by reactive oxygen species and low-density lipoprotein oxidation, triggered by dysmetabolic diseases, inflammation, environmental factors, or aging. Here we have examined the ability of the flavonoid quercetin to protect cutaneous tissue-associated cell types from injury induced by oxidative stress, and possible cooperative effects of ascorbic acid. Human skin fibroblasts, keratinocytes, and endothelial cells were cultured in the presence of buthionine sulfoximine (BSO), an irreversible inhibitor of glutathione (GSH) synthesis. Depletion of intracellular levels of GSH leads to an accumulation of cellular peroxides and eventual cell death. Quercetin concentration-dependently (EC50: 30-40 microM) reduced oxidative injury of BSO to all cell types, and was also effective when first added after BSO washout. BSO caused marked decreases in the intracellular level of GSH, which remained depressed in quercetin-protected cells. Ascorbic acid, while by itself not cytoprotective synergized with quercetin, lowered the quercetin EC50 and prolonged the window for cytoprotection. The related flavonoids rutin and dihydroquercetin also decreased BSO-induced injury to dermal fibroblasts, albeit less efficaciously so than quercetin. The cytoprotective effect of rutin, but not that of dihydroquercetin, was enhanced in the presence of ascorbic acid. Further, quercetin rescued sensory ganglion neurons from death provoked by GSH depletion. Direct oxidative injury to this last cell type has not been previously demonstrated. The results show that flavonoids are broadly protective for cutaneous tissue-type cell populations subjected to a chronic intracellular form of oxidative stress. Quercetin in particular, paired with ascorbic acid, may be of therapeutic benefit in protecting neurovasculature structures in skin from oxidative damage.     

Direct evidence for glutathione as mediator of apoptosis in neuronal cells

Recent evidence has focused attention on the role of oxidative stress in various acute and chronic neurodegenerative diseases. Particularly, a decrease in the level of the powerful antioxidant glutathione (GSH) and death of dopaminergic neurons in substantia nigra are prominent features in Parkinson's disease. The mode of neuronal death is uncertain; however, apoptosis has been hypothesized to be mediated through the induction of free radicals via oxidative pathways. An approach to determine the role of GSH depletion in neurodegeneration and apoptosis was to create a selective modulation of this antioxidant by metabolic manipulations in a clonal cell line of neuronal origin (mouse neuroblastoma NS20Y). Intracellular GSH levels was lowered by inhibiting its biosynthesis with L-buthionine-(S,R)-sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase. This treatment led to a GSH depletion of 50% after 1 h and 98% after 24 h. A direct cause/effect relationship between GSH depletion and apoptosis was evidenced in this neuronal cell type. GSH depletion induced the death of NS20Y and promoted nuclear alterations of apoptosis as demonstrated by the in situ staining of DNA fragmentation after 5 days of BSO treatment (by terminal-deoxynucleotide transferase-mediated dUTP-nick end labeling), and the appearance of DNA laddering on agarose gel. These results suggested that redox desequilibrium induced by GSH depletion may serve as a general trigger for apoptosis in neuronal cells, and are consistent with the hypothesis that GSH depletion contribute to neuronal death in Parkinson's disease.     

Psychomotor effects of dopamine infusion under decreased glutathione conditions

Administration of buthionine sulfoximine (BSO) selectively inhibits glutathione (GSH) biosynthesis, thereby inducing a GSH deficiency. Because GSH plays a critical role in intracellular antioxidant defense, decreased GSH levels in the brain may result in less oxidative stress (OS) protection. Thus, the pro-oxidant effects of dopamine (DA), which rapidly oxidizes to form reactive oxygen species, may increase. In this study, the behavioral consequences of reduced OS protection were examined by administering BSO (3.2 mg in 30 microl Ringer's solution, intracerebroventricularly) every other day for 12 d to male Fischer 344 rats. In addition, DA (15 microl of 500 microM) was administered every day; when given on the same day as BSO, it was either 1 h after BSO (BSO + DA group) or 1 h before BSO (DA + BSO group). Tests of psychomotor behavior--rod walking, wire suspension, and plank walking--were performed five times during the experiment. BSO + DA administration, but not DA + BSO, impaired performance by decreasing latency to fall in the rod and plank walk tests compared to a vehicle only (Ringer's) group. Therefore, depletion of GSH with BSO, followed by DA treatment, produced deficits in psychomotor behavior. These deficits are similar to those seen in aged rats, suggesting that the oxidation of DA coupled with a reduced capacity to respond to OS may be responsible for the induction of age-related motor behavioral deficits.     

Extracellular reduced glutathione increases neuronal vulnerability to combined chemical hypoxia and glucose deprivation

In addition to its intracellular antioxidant role, reduced glutathione (GSH) is released by CNS cells and may mediate or modulate excitatory neurotransmission. Although extracellular GSH levels rise in the ischemic cortex, its effect on the viability of energy-compromised neurons has not been defined. In this study, we tested the hypothesis that exogenous GSH would increase the vulnerability of cultured cortical neurons to azide-induced chemical hypoxia combined with glucose deprivation. Thirty minutes azide exposure in a glucose-free buffer was tolerated by most neurons, with release of less than 10% of neuronal LDH over the subsequent 21-25 h. Concomitant treatment with 10-100 microM GSH increased cell death in a concentration-dependent fashion, to 71.6+/-5.1% of neurons at 100 microM; GSH alone was nontoxic. Injury was blocked by the selective N-methyl-d-aspartate (NMDA) antagonist MK-801 but not by the AMPA/kainate antagonist NBQX. The sulfhydryl reducing agent mercaptoethanol (10-100 microM) mimicked the action of GSH; however, the zinc chelator ethylenediaminetetraacetic acid (EDTA) was ineffective. Two GSH analogues that lack a sulfhydryl group, S-hexylglutathione (SHG) and oxidized glutathione (GSSG), were inactive per se but attenuated the effect of both GSH and mercaptoethanol. These results suggest that micromolar concentrations of GSH enhance neuronal loss due to energy depletion by altering the extracellular redox state, resulting in increased NMDA receptor activation.     

Effect of buthionine sulfoximine, a synthesis inhibitor of the antioxidant glutathione, on the murine nigrostriatal neurons

This study analyzed the effects of acute systemic treatment with buthionine sulfoximine (BSO), a synthesis inhibitor of the antioxidant reduced glutathione (GSH), on dopaminergic neurons of the murine nigrostriatal pathway. Part 1 of the study established a dose-response curve and the temporal pattern of GSH loss and recovery in the substantia nigra and striatum following acute BSO treatment. Part 2 of the study determined the effect of acute BSO treatment on the morphology and biochemistry of nigrostriatal neurons. We found that decreases in GSH levels had profound morphological effects, including decreased catecholamine fluorescence per cell, increased levels of lipid peroxidation and lipofuscin accumulation, and increased numbers of dystrophic axons in dopaminergic neurons of the nigrostriatal pathway. However, no measurable effects were observed in biochemical levels of either dopamine or its metabolites. These changes mimic those that have been reported to occur in the nigrostriatal system of rodents with advancing age. Our data suggest that reduction of GSH via BSO treatment results in the same types of nigrostriatal degenerative effects that occur during the aging process and consequently is a good model system for examining the role of GSH in protecting this area of the brain against the harmful effects of age-related oxidative stress.     

Selective and synergistic activity of L-S,R-buthionine sulfoximine on malignant melanoma is accompanied by decreased expression of glutathione-S-transferase

L-buthionine-S,R-sulfoximine (BSO) selectivley inhibits glutathione (GSH) synthesis. Malignant melanoma may be uniquely dependent on GSH and its linked enzymes, glutathione S-transferase (GST) and GSH-peroxidase, for metabolism of reactive orthoquinones and peroxides produced during melanin synthesis. We compared the in vitro effects of BSO on melanoma cell lines and fresh melanoma specimens (n = 118) with breast and ovarian cell lines and solid tumors (n = 244). IC50 values (microM) for BSO on melanoma, breast and ovarian tumor specimens were 1.9, 8.6, and 29, respectively. The IC90 for melanoma was 25.5 microM, a level 20-fold lower than steady state levels achieved clinically. The sensitivity of individual specimens of melanoma correlated with their melanin content (r = 0.63). BSO synergistically enhanced BCNU activity against melanoma cell lines and human tumors. We followed GSH levels, GST enzyme activity, GST isoenzyme profiles and mRNA levels after BSO. BSO (50 microM) treatment for 48 hr resulted in a 95% decrease in ZAZ and M14 melanoma cell line GSH levels, and a 60% decrease in GST enzyme activity. GST-mu protein and mRNA levels were significantly reduced in both cell lines. GST-pi expression was unaffected. These data suggest that BSO action on melanoma may be related to GSH depletion, diminishing the capacity to scavenge toxic metabolites produced during melanin synthesis. We report here for the first time that BSO enhancement of alkylator action may be related in part to down regulation of GST. BSO may be a clinically useful adjunct in the treatment of malignant melanoma.     

Combined effects of buthionine sulfoximine and cepharanthine on cytotoxic activity of doxorubicin to multidrug-resistant cells

We studied the potentiation of doxorubicin (DOX) activity in multidrug-resistant (MDR) cells by buthionine sulfoximine (BSO), a specific inhibitor of gamma-glutamylcysteine synthetase, and by cepharanthine (CE), which interacts with P-glycoprotein. The glutathione (GSH) of MDR cells was approximately 1.5-fold greater than that of the parental cell line. BSO reduced GSH content of MDR cells compared to that of the sensitive ones. The BSO treatment (50 microM) enhanced the effect of DOX by 1.8-fold, while CE caused a greater reversal of drug resistance. The combination of BSO with CE produced further potentiation of DOX activity in an antiproliferative effect. Pretreatment of cells with BSO did not alter the cellular accumulation of DOX in the absence or presence of CE. The addition of BSO (30 mM) to the drinking water of mice reduced the tissue levels of GSH in tumor cells, suggesting that the marked decrease in GSH might diminish the ability of that tumor to resist DOX. Combined administration of CE and DOX resulted in enhancement of DOX antitumor activity and prolongation of survival time. The survival of mice treated with BSO and CE as a supplement to DOX treatment was superior that of mice receiving DOX alone. These studies demonstrated that the combinations of BSO with CE may be useful for killing drug-resistant tumor cells.     

Effect of ovariectomy on magnetic resonance T2* in rat femur

Glutathione (GSH) is an important factor involved in the resistance of tumor cells to anticancer agents. Buthionine sulfoximine (BSO), a specific inhibitor of GSH synthesis, effectively decreases cellular GSH concentrations both in vitro and in vivo. Depletion of GSH by BSO sensitizes a variety of cancer cells to chemotherapeutic agents. Therefore, BSO has been on clinical trial as an anticancer adjuvant. For this purpose, it is important to understand the effect of BSO treatment not only on the sensitivity of tumor cells to anticancer agents, but also on the metabolism and function of normal tissues. The present study was undertaken to determine the effect of BSO treatment on GSH concentrations in the blood, liver, and ovary, and changes in concentrations of ovarian hormones and other important components in plasma. Female Sprague-Dawley rats, 90 days of age, were treated with 2.0 mmol/kg BSO in saline by intraperitoneal injection, twice daily for 7 days. This treatment depressed GSH concentrations in the blood, liver and ovary by 95, 75, and 85%, respectively. Several blood components were measured. These included red blood cells, hemoglobin, ceruloplasmin, hematocrit, mean corpuscular volume and hemoglobin concentration, alkaline phosphatase, urea nitrogen, creatine and creatinine, glucose, cholesterol, triglycerides, triiodothyronine (T3), thyroxine (T4), and hormones including estradiol, progesterone, and prolactin. BSO treatment significantly (P < 0.05) elevated and lowered plasma concentrations of ceruloplasmin and urea nitrogen, respectively, More importantly, plasma concentrations of estradiol and progesterone were decreased markedly (P < 0.05) in the BSO-treated animals. The hormonal results suggest that investigations on the role of BSO-induced GSH depletion in the treatment of malignancies both with and without hormone dependence in women should be undertaken.     

Spatial learning and memory deficits induced by dopamine administration with decreased glutathione

Administration of buthionine sulfoximine (BSO) selectively inhibits glutathione (GSH) biosynthesis and induces a GSH deficiency. Decreased GSH levels in the brain may result in less oxidative stress (OS) protection, because GSH contributes substantially to intracellular antioxidant defense. Under these conditions, administration of the pro-oxidant, dopamine (DA), which rapidly oxidizes to form reactive oxygen species, may increase OS. To test the cognitive behavioral consequences of decreased GSH, BSO (3.2 mg in 30 microliters, intracerebroventricularly) was administered to male Fischer 344 rats every other day for 4 days. In addition, DA (15 microliters of 500 microM) was administered every day [either 1 h after BSO (BSO + DA group) or 1 h before BSO (DA + BSO group), when given on the same day as BSO] and spatial learning and memory assessed (Morris water maze, six trials/day). BSO + DA rats, but not DA + BSO rats, demonstrated cognitive impairment compared to a vehicle group, as evidenced by increased latencies to find the hidden platform, particularly on the first trial each day. Also, the BSO + DA group utilized non-spatial strategies during the probe trials (swim with no platform): i.e., less time spent in the platform quadrant, fewer crossings and longer latencies to the previous platform location, and more time spent in the platform quadrant, fewer crossings and longer latencies to the previous platform location, and more time spent around the edge of the pool rather than in the platform zone. Therefore, the cognitive behavioral consequences of decreasing GSH brain levels with BSO in conjunction with DA administration depends on the order of administration. These findings are similar to those seen previously on rod and plank walking performance, as well as to those seen in aged rats, suggesting that the oxidation of DA coupled with a reduced capacity to respond to oxidative stress may be responsible for the induction of age-related cognitive deficits.     

Nigral glutathione deficiency is not specific for idiopathic Parkinson's disease

The consistent findings of decreased levels of the major antioxidant glutathione in substantia nigra of patients with idiopathic Parkinson's disease (PD) has provided most of the basis for the oxidative stress hypothesis of the etiology of PD. To establish whether a nigral glutathione deficiency is unique to PD, as is generally assumed, or is present in other Parkinsonian conditions associated with nigral damage, we compared levels of reduced glutathione (GSH) in postmortem brain of patients with PD to those with progressive supranuclear palsy (PSP) and multiple system atrophy (MSA). As compared with the controls, nigral GSH levels were decreased in the PD and PSP patient groups (P < 0.05 for PD [-30%], PSP [-21%]), whereas a similar decrease in the MSA patient group did not reach statistical significance (P = 0.078, MSA [-20%]). GSH levels were normal in all examined normal and degenerating extra-nigral brain areas in PSP and MSA. A trend for decreased levels of uric acid (antioxidant and product of purine catabolism) also was observed in nigra of all patient groups (-19 to -30%). These data suggest that glutathione depletion, possibly consequent to overutilisation in oxidative stress reactions, could play a causal role in nigral degeneration in all nigrostriatal dopamine deficiency disorders, and that antioxidant therapeutic approaches should not be restricted to PD.     

Mitochondrial respiratory enzyme function and superoxide dismutase activity following brain glutathione depletion in the rat

In substantia nigra from patients with Parkinson's disease, there are decreased levels of reduced glutathione (GSH) and diminished activities of mitochondrial complex I and alpha-ketoglutarate dehydrogenase (alpha-KGDH), along with increased activity of superoxide dismutase (SOD). However, the interrelationship among these events is uncertain. We now report the effect of decreased brain GSH levels on SOD and mitochondrial respiratory enzyme activity in rat brain. In addition, we have investigated the ability of thioctic acid, an endogenous antioxidant, to alter these parameters. Unilateral or bilateral intracerebroventricular (ICV) administration of buthionine sulphoximine (BSO; 1 x 3.2 mg or 2 x 1.6 mg) over a 48-hr period reduced cortical GSH by 55-70%. There was no change in the activity of complex I, II/III, or IV or of citrate synthase in cortex. Similarly, there was no alteration of mitochondrial or cytosolic SOD activity. Thioctic acid (50 or 100 mg/kg IP) alone had no effect on cortical GSH levels in control animals and did not reverse the decrease in GSH levels produced by unilateral or bilateral ICV BSO administration. Thioctic acid (50 or 100 mg/kg IP) had no overall effect on complex I, II/III, or IV or on citrate synthase activity in control animals. Thioctic acid also did not alter cortical mitochondrial respiratory enzyme activity in BSO-treated rats. At the lower dose, thioctic acid tended to increase mitochondrial and cytosolic SOD activity in control animals and in BSO-treated rats. However, at the higher dose, thioctic acid tended to decrease mitochondrial SOD activity. Overall, there was no consistent effect of thioctic acid (50 or 100 mg/kg IP) on SOD activity in control or BSO-treated animals. This study shows that BSO-induced glutathione deficiency does not lead to alterations in mitochondrial respiratory enzyme activity or to changes in SOD activity. GSH depletion in Parkinson's disease therefore may not account for the alterations occurring in complex I and mitochondrial SOD in substantia nigra. Thioctic acid did not alter brain GSH levels or mitochondrial function. Interestingly, however, it did produce some alterations in SOD activity, which may reflect either its antioxidant activity or its ability to act as a thiol-disulphide redox couple.     

Regulation of Ca2+ homeostasis by glucose metabolism in rat brain

Dopamine (DA) neurons are uniquely vulnerable to damage and disease. Their loss in humans is associated with diseases of the aged, most notably, Parkinson's Disease (PD). There is now a great deal of evidence to suggest that the destruction of DA neurons in PD involves the accumulation of harmful oxygen free radicals. Since the antioxidant hormone, melatonin, is one of the most potent endogenous scavengers of these toxic radicals, we tested its ability to rescue DA neurons from damage/death in several laboratory models associated with oxidative stress. In the first model, cells were grown in low density on serum-free media. Under these conditions, nearly all cells died, presumably due to the lack of essential growth factors. Treatment with 250 microM melatonin rescued nearly all dying cells (100% tau+ neurons), including tyrosine hydroxylase immunopositive DA neurons, for at least 7 days following growth factor deprivation. This effect was dose and time dependent and was mimicked by other antioxidants such as 2-iodomelatonin and vitamin E. Similarly, in the second model of oxidative stress, 250 microM melatonn produced a near total recovery from the usual 50% loss of DA neurons caused by neurotoxic injury from 2.5 microM 1-methyl-4-phenylpyridine (MPP+). These results indicate that melatonin possesses the remarkable ability to rescue DA neurons from cell death in several experimental paradigms associated with oxidative stress.     

Induction of reactive oxygen species in neurons by haloperidol

Haloperidol (HP) is widely prescribed for schizophrenia and other affective disorders but has severe side effects such as tardive dyskinesia. Because oxidative stress has been implicated in the clinical side effects of HP, rat primary cortical neurons and the mouse hippocampal cell line HT-22 were used to characterize the generation of reactive oxygen species (ROS) and other cellular alterations caused by HP. Primary neurons and HT-22 cells are equally sensitive to HP with an IC50 of 35 microM in the primary neurons and 45 microM in HT-22. HP induces a sixfold increase in levels of ROS, which are generated from mitochondria but not from the metabolism of catecholamines by monoamine oxidases. Glutathione (GSH) is an important antioxidant for the protection of cells against HP toxicity because (1) the intracellular GSH decreases as the ROS production increases, (2) the exogenous addition of antioxidants, such as beta-estradiol and vitamin E, lowers the level of ROS and protects HT-22 cells from HP, and (3) treatments that result in the reduction of the intracellular GSH potentiate HP toxicity. The GSH decrease is followed by the increase in the intracellular level of Ca2+, which immediately precedes cell death. Therefore, HP causes a sequence of cellular alterations that lead to cell death and the production of ROS is the integral part of this cascade.     

A role for 12-lipoxygenase in nerve cell death caused by glutathione depletion

An early and highly specific decrease in glutathione (GSH) in the substantia nigra is associated with Parkinson's disease, and low levels of GSH lead to the degeneration of cultured dopaminergic neurons. Using immature cortical neurons and a clonal nerve cell line, it is shown that a decrease in GSH triggers the activation of neuronal 12-lipoxygenase (12-LOX), which leads to the production of peroxides, the influx of Ca2+, and ultimately to cell death. The supporting evidence includes: 1) inhibitors of arachidonate metabolism and 12-LOX block cell death induced by GSH depletion; 2) there is an increase in 12-LOX activity and a membrane translocation in HT22 cells, and an induction of the enzyme in primary cortical neurons following the reduction of GSH; 3) 12-LOX is directly inhibited by GSH; and 4) exogenous arachidonic acid potentiates cell death. These data show that the LOX pathway is a critical intermediate in at least some forms of neuronal degeneration.     

Modulation of intracellular glutathione and cysteine metabolism in bovine oviduct epithelial cells cultured In vitro

The objective of this study was to determine how alterations in intracellular thiol levels of oviduct epithelium occur in response to chemical or environmental conditions that could result in oxidative stress. Bovine oviducts were classified as follicular (F) or luteal (L) according to the reproductive stage of the ovary. Epithelial cells were harvested from the ampulla (AMP) and isthmus (ISTH) region of each oviduct, suspended in culture medium, and then plated into collagen-coated culture plates and grown to confluency. Basal levels of intracellular cysteine (Cys) and glutathione (GSH) were determined in oviduct epithelial cells and found to range from 0.36 to 0.46 pmol/ microg protein for Cys and from 5.3 to 6.4 pmol/ microg protein for GSH. Oxidized Cys values ranged from 21% to 39% of total Cys, whereas the oxidized GSH levels observed were from 21% to 28% of total GSH except in luteal ISTH, where they were significantly lower (6%). Confluent cells were exposed to GSH-depleting agents, L-buthionine-S,R-sulfoximine (BSO) or diethyl maleate (DEM), at doses ranging from 10 to 5000 microM. Both compounds depleted GSH in a dose-dependent manner, and 500 microM concentrations were chosen for subsequent studies with each compound. Cys levels in BSO (500 microM)-treated oviduct epithelial cells were transiently elevated over control values during the initial 5-h incubation; there was then a decrease in Cys levels by AMP but not ISTH oviduct epithelial cells. BSO-treated oviduct epithelial cells displayed a continued depletion of GSH over the incubation period and by 24 h were depleted by 38% to 61%. These results demonstrate a difference in GSH turnover in oviduct epithelial cells associated with reproductive stage. Exposure to DEM (500 microM) caused a decline in both Cys and GSH levels, which were partially restored after DEM removal. In general, L-staged oviduct epithelial cells were observed to be more competent at replenishing thiol stores than F-staged oviduct epithelial cells. Results from this study suggest that reproductive stage and region influence intracellular oviduct epithelium thiol status and therefore may affect how this tissue responds to chemicals or environmental conditions leading to oxidative stress.     

Esophagitis in Sprague-Dawley rats is mediated by free radicals

Free radical-mediated esophagitis was studied during duodenogastroesophageal reflux (mixed reflux) or acid reflux in rats. The influence of reflux on esophageal glutathione levels was also examined. Mixed reflux caused more gross mucosal injury than acid reflux. Gross mucosal injury occurred in the mid-esophagus. Total glutathione (GSH) in the esophageal mucosa of control rats was highest in the distal esophagus. The time course of esophageal GSH in rats treated by mixed reflux showed a significant decrease 4 hr after initiation of reflux, followed by a significant increase from the 12th hour on. Mucosal GSH was increased in both reflux groups after 24 hr but significantly more so in the mixed than in the acid reflux group. The free radical scavenger superoxide dismutase (SOD) prevented esophagitis and was associated with decreased GSH levels. GSH depletion by buthionine sulfoximine (BSO) prevented esophagitis and stimulated SOD production in the esophageal mucosa. It is concluded that gastroesophageal reflux is associated with oxidative stress in the esophageal mucosa. The lower GSH levels in the mid-esophagus may predispose to damage in this area. Duodenogastroesophageal reflux causes more damage than pure acid reflux. Oxidative stress leads to GSH depletion of the esophageal mucosa in the first few hours following damage but then stimulates GSH production. GSH depletion by BSO does not worsen esophagitis since it increases the esophageal SOD concentration.     

Catecholamines potentiate amyloid beta-peptide neurotoxicity: involvement of oxidative stress, mitochondrial dysfunction, and perturbed calcium homeostasis

Oxidative stress and mitochondrial dysfunction are implicated in the neuronal cell death that occurs in physiological settings and in neurodegenerative disorders. In Alzheimer's disease (AD) degenerating neurons are associated with deposits of amyloid beta-peptide (A beta), and there is evidence for increased membrane lipid peroxidation and protein oxidation in the degenerating neurons. Cell culture studies have shown that A beta can disrupt calcium homeostasis and induce apoptosis in neurons by a mechanism involving oxidative stress. We now report that catecholamines (norepinephrine, epinephrine, and dopamine) increase the vulnerability of cultured hippocampal neurons to A beta toxicity. The catecholamines were effective in potentiating A beta toxicity at concentrations of 10-200 microM, with the higher concentrations (100-200 microM) themselves inducing cell death. Serotonin and acetylcholine were not neurotoxic and did not modify A beta toxicity. Levels of membrane lipid peroxidation, and cytoplasmic and mitochondrial reactive oxygen species, were increased following exposure to neurons to A beta, and catecholamines exacerbated the oxidative stress. Subtoxic concentrations of catecholamines exacerbated decreases in mitochondrial energy charge and transmembrane potential caused by A beta, and higher concentrations of catecholamines alone induced mitochondrial dysfunction. Antioxidants (vitamin E, glutathione, and propyl gallate) protected neurons against the damaging effects of A beta and catecholamines, whereas the beta-adrenergic receptor antagonist propanolol and the dopamine (D1) receptor antagonist SCH23390 were ineffective. Measurements of intracellular free Ca2+ ([Ca2+]i) showed that A beta induced a slow elevation of [Ca2+]i which was greatly enhanced in cultures cotreated with catecholamines. Collectively, these data indicate a role for catecholamines in exacerbating A beta-mediated neuronal degeneration in AD and, when taken together with previous findings, suggest roles for oxidative stress induced by catecholamines in several different neurodegenerative conditions.     

Women and menopause: beliefs, attitudes, and behaviors. The North American Menopause Society 1997 Menopause Survey

It has been demonstrated that exposure to mercury or cadmium compounds causes alterations in the glutathione system in a model glial cell line, C6. Here we report that two organic tin compounds, triethyltin (TET) and trimethyltin (TMT), are also toxic to these cells with EC50 values for cell death of c. 0.02 microM and 0.8 microM respectively. Exposure for 24 h to either of these compounds at sub-toxic concentrations caused increases in the amount of reduced glutathione (GSH) per cell. Increases in glutathione-S-transferase enzyme activity were also demonstrated after TET or TMT exposure. This suggests that glutathione increases occur in glial cells after toxic insults below that required to cause cell death, possibly acting as a protective mechanism. To test whether GSH plays a role in organotin-induced cell death we manipulated GSH in the culture media or via intracellular GSH and looked at the effects on sensitivity to TET or TMT toxicity. Adding GSH to the culture media did not protect the cells. Depletion of intracellular GSH with buthionine-[S,R] sulphoximine did not alter cytotoxicity of TET or TMT. However, pre-treatment with (-)-2-oxo-4-thiazolidine carboxylic acid (OTC), which increases intracellular GSH levels, protected the cells against both compounds. The EC50 for TMT was increased from 0.77 to 1.8 microM, a 2.3-fold shift, whereas the EC50 for TET was increased > 20-fold, from 0.022 to 0.47 microM. One interpretation of these results is that GSH protects cells against the toxicity of organic tin compounds without reacting directly with them to any significant extent. Under conditions where GSH is depleted, additional protective mechanisms may be active.     

Conjugates of catecholamines with cysteine and GSH in Parkinson's disease: possible mechanisms of formation involving reactive oxygen species

Oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) and dopamine (DA) to generate semiquinones/quinones, oxygen radicals, and other reactive oxygen species may play a role in neuronal cell death in Parkinson's disease (PD). In particular, semiquinones/quinones can form conjugates with thiol compounds such as GSH and cysteine. Exposure of L-DOPA, DA, and other catecholamines to a system generating O2.- radical led to O2(.-)-dependent depletion of added GSH (or cysteine), accompanied by the formation of thiol-DA or -DOPA adducts as detected by HPLC. Superoxide could additionally cause destruction of these adducts. Iron or copper ions could also promote conjugate formation between GSH or cysteine and DA and L-DOPA, especially if H2O2 was present. We applied HPLC to measure glutathionyl and cysteinyl conjugates of L-DOPA, DA, and 3,4-dihydroxyphenylacetic acid (DOPAC) in postmortem brain samples from PD patients and normal control subjects. Conjugates were detected in most brain areas examined, but levels were highest in the substantia nigra and putamen. In most regions, adduct levels were lower in PD, but there were significant increases in cysteinyl adducts of L-DOPA, DA, and DOPAC in PD substantia nigra, suggesting that acceleration of L-DOPA/DA oxidation occurs in PD, although we cannot say if this is a primary feature of the disease or if it is related to therapy with L-DOPA. In vitro, conjugate formation could be inhibited by the dithiol dihydrolipoate but not by its oxidised form, lipoic acid.