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.     

Phenotyping of glutathione S-transferase M1 in the Estonian population by ELISA using GSTM1a and GSTM1b specific monoclonal antibodies

We examined the effects of stimulation on either postnatal days 1-7 or 21-27 on passive avoidance reaction (PAR) of young rats. Animals received tactile or visual stimulation for 10 min each day, and were trained on postnatal day 28 in a step-through apparatus using a footshock of 0.75 mA for 2 s. Retention was tested on five consecutive days beginning on day 29. Memory retention was measured for each rat 24, 48, 72, 96 and 120 h after the acquisition trial. Step-through latencies to enter the dark compartment, time spent in the illuminated compartment and number of crossings of the light beam were recorded up to 200 s. Rats that received tactile or visual stimulation during the 4th postnatal week displayed significantly lower PAR latencies, a shorter stay in the illuminated compartment and a higher number of crossings of the light beam compared to rats treated during the 1st postnatal week. The untreated control group showed a rapid decline of PAR latencies. All experimental groups remained in the illuminated compartment longer and showed PAR latencies well above those of the control group. The differences became more pronounced when visual stimulation in the first postnatal week was used. The number of crossings of the light beam was significantly reduced by the treatment, with the exception of the experimental group stimulated visually in the 4th week. The behavioural changes induced by tactile or visual stimulation have a long-lasting effect in coping with a stressful task.     

Effects of buthionine sulfoximine on the outcome of the in utero administration of alcohol on fetal development

The adverse effects of the maternal consumption of alcohol on the fetus have been recognized for centuries. Fetal alcohol syndrome is characterized by pre- and postnatal growth retardation, mental retardation, behavioral deficits, and facial deformities. Despite numerous animal studies, the biochemical mechanism(s) by which alcohol produces teratogenic effects on the developing fetus are not well understood. Several studies have shown that administration of alcohol to adult rats produces a decrease in hepatic levels of glutathione (GSH). In utero administration of alcohol has also been shown to produce a decrease in GSH levels, as well as prenatal growth retardation and intrauterine death. In an effort to determine if GSH may have a vital role in protecting the fetus against the teratogenic effects of alcohol, buthionine (SR)-sulfoximine (BSO) was used to deplete GSH levels in the mother and fetus. Timed pregnant Sprague-Dawley rats were placed on a liquid BioServ diet containing either 0%, 11%, 23%, 29%, 31%, 33%, or 35% ethanol-derived calories, with or without BSO (888 mg/kg/24 hr), starting on day 1 of pregnancy. Another set of mothers were fed lab chow and water as a control group for the liquid diet. The mothers were maintained on the diet until gestation day 21 when they were anesthetized with sodium pentobarbital and the pups delivered by cesarean section. The offspring were counted, weighed, killed, and the brain and liver weighed. The effects of BSO on the alcohol dose-response curves (body weights, brain weights, and litter number) were then determined to ascertain if a depletion in GSH potentiated the effects of alcohol. In utero administration of BSO, aside from the depletion of GSH in the liver and brain in the developing fetus, produced a shift to the left in the alcohol dose-response curve.     

Rotation of water in the Morris water maze interferes with path integration mechanisms of place navigation

The idea that place navigation in the Morris water maze is implemented by path integration between locations determined by landmark sighting was investigated in a 200-cm-diameter pool in which circular (7.2 degrees/s) motion of water could be induced by tangentially arranged water jets. The rats were trained at 8 trials per day to navigate to an erectable platform which was raised after the rat had spent a criterion time in the target annulus (30 cm in diameter) in the midpoint of the NW quadrant. Asymptotic escape latency of 7 s was reached after 9 days in moving water (n = 8) and after 6 days in stationary water (n = 8). The group overtrained for 13 days in stable water performed well even after it was transferred to moving water. Changing the sense of rotation of water from counterclockwise to clockwise did not affect the asymptotic performance. The above findings show that overtrained rats rely on landmark sighting rather than on path integration. The influence of water movement reappeared when place navigation to a new target (SW) was examined in alternating 2-s periods of light (L) and darkness (D). On the first day, the latencies were 15.2 +/- 1.2 and 22.8 +/- 1.9 s in stable and moving water, respectively, but dropped to 10 s on the following day. The tracks generated in the L period were more tortuous than those generated in the D period and this difference was more pronounced in moving than in stable water. It is concluded that path integration mechanisms supporting navigation during intervals of darkness are impaired in moving water but that this impairment disappears in overtrained animals.     

Effect of glutathione depletion on exocyclic adduct levels in the liver DNA of F344 rats

The effects of glutathione (GSH) depletion on the in vivo formation of cyclic 1,N2- propanodexoxyguanosine adducts (AdG and CdG) as background lesions in the liver DNA of F344 rats were investigated. A group of 5 male F344 rats were given drinking water containing 30 mM L-buthionine (S,R)-sulfoximine (BSO) for 21 days, and another group of 8 rats were given only drinking water as controls. The BSO-treated rats had significantly lower weight gain than control rats. The hepatic GSH levels in the BSO-treated group were reduced by 84% as compared with the control group, from 4.43 to 0.72 mumol/g of tissue. The isomeric AdG3, CdG1, and CdG2 were detected by the 32P-postlabeling/HPLC method in the liver DNA of rats without carcinogen treatment, as we reported previously [Nath, R. G., and Chung, F.-L. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7491-7495. Nath, R. G., et al. (1996) Cancer Res. 56, 452-456]. The mean levels (mumol/mol of guanine) for AdG3, CdG1, and CdG2 were 0.57 +/- 0.25, 0.15 +/- 0.18, and 0.16 +/- 0.22 for the control group and 1.18 +/- 1.03, 3.16 +/- 3.26, and 2.50 +/- 2.59 for the BSO group, respectively. These increases correspond to approximately 2-fold for AdG and 15-21-fold for CdG adducts. The dramatic increase in the cyclic adduct levels in rat liver DNA could have resulted mainly from GSH depletion as a result of the BSO treatment, even though other unknown effects due to the toxicity of BSO cannot be ruled out. These results suggest that GSH plays an important role in protecting the liver against cyclic propano DNA adduction and provide further support for the endogenous origin of these adducts.     

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.     

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 morphine on striatal dopamine metabolism and ascorbic and uric acid release in freely moving rats

Recent ex vivo findings have shown that morphine increases dopamine (DA) and xanthine oxidative metabolism and ascorbic acid (AA) oxidation in the rat striatum. In the present study, we evaluated the effects of subcutaneous daily morphine (20 mg/kg) administration on DA, dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), AA and uric acid in the striatum of freely moving rats using microdialysis. Dialysates were assayed by high performance liquid chromatography with electrochemical detection. On the first day, morphine administration caused a significant increase in extracellular DA, DOPAC, HVA, AA and uric acid concentrations over a 3 h period after morphine. In all treated rats (n = 7), individual concentrations of DOPAC + HVA were directly correlated with individual AA and uric acid concentrations. Last morphine administration on the 4th day increased DOPAC, HVA, AA and uric acid concentrations but failed to increase those of DA. Individual DOPAC + HVA concentrations were still directly correlated with individual AA and uric acid concentrations. These results suggest that systemic morphine increases both striatal DA release and DA and xanthine oxidative metabolism. Only the former effect undergoes tolerance. The increase in DA oxidative metabolism is highly correlated with that of xanthine. The subsequent enhancement in reactive oxygen species production may account for the increase in extracellular AA.     

Sphincter of Oddi dysfunction is associated with chronic pancreatitis

To clarify whether the content of glutathione (GSH) in the brain can be estimated by the uptake of 99mTc-meso-HMPAO, we conducted the following in vivo and in vitro experiments. METHODS: We investigated the effect of diethyl maleate (DEM) and buthionine sulfoximine (BSO) administration on the brain uptake of 99mTc-meso-HMPAO in the mouse, rat and rabbit, and the chemical specificity of in vitro interaction of 99mTc-HMPAO to GSH using measurements of octanol-extractable radioactivity as an index of remaining intact tracer. RESULTS: The uptake of 99mTc-meso-HMPAO in the mouse and rat brain were reduced together with decreased content of GSH by preloading of DEM, a GSH depletor that acts through glutathione S-transferase. Neither 99mTc-meso-HMPAO uptake nor GSH content was affected in the rabbit brain. Similarly, the uptake of 99mTc-meso-HMPAO and GSH content in the mouse brain was reduced by preinjection of BSO, a GSH depletor that acts through gamma-glutamylcysteine synthetase. In an in vitro study, 99mTc-HMPAO showed reactivity to the molecules possessing a -SH group, but were not specific to GSH. The order of 99mTc-meso-HMPAO reactivity to the mouse brain homogenate agreed with the order of GSH concentration: normal > BSO > DEM. GSH was a major contributor to the conversion reaction of 99mTc-meso-HMPAO to hydrophilic complex in mouse brain homogenate. CONCLUSION: GSH may have a major responsibility for trapping 99mTc-HMPAO in the brain, suggesting the possibility of in vivo measurement of brain GSH with 99mTc-meso-HMPAO.     

Interactions between D1 and muscarinic receptors in the induction of striatal c-fos in rats depleted of dopamine as neonates

The contributions of striatal D1 receptors to the expression of sensorimotor behavior are qualitatively different in rats depleted of dopamine (DA) as neonates vs. as adults. In an effort to reveal neuronal mechanisms underlying these behavioral difference we determined the effects of the partial D1 agonist SKF 38393, the muscarinic antagonist scopolamine, and the combination of the two drugs on the induction of c-fos in the striatum and its projection sites, the globus pallidus and substantia nigra. Adult rats, given intracerebroventricular injections of 6-hydroxydopamine (6-OHDA, 50 micrograms/5 microliters/hemisphere) or its vehicle on postnatal day 3, were treated with SKF 38393 (1.5 mg/kg, i.p.), scopolamine (5.0 mg/kg, i.p.) or the combination of the two drugs. There was no significant induction of c-fos in vehicle-treated controls, regardless of drug administration. In DA-depleted rats, scopolamine also did not induce c-fos whereas SKF 38393 produced a significant increases in the number of FOS-positive cells in the dorsal, but not ventral, striatum. The combined administration of scopolamine and SKF 38393 resulted in a potent synergism in the number of FOS-positive cells in DA-depleted rats. These interactions between lesion condition and drugs on c-fos induction were not secondary to differences in drug-induced behavioral activity. Activity levels were no different in vehicle vs. DA-depleted rats following the combined administration of scopolamine + SKF 38393, yet the two groups of rats exhibited marked differences in the density of FOS-positive striatal neurons. The effects of scopolamine and SKF 38393 on c-fos induction in striatum are qualitatively similar to those reported in rats DA-depleted as adults and suggest that, at this single-label level of analysis, the ability of D1 and muscarinic receptors to influence striatal activity does not contribute to the marked age-related differences in the behavioral effects of DA depletions.     

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.     

Receptor mediated effects of adenosine and caffeine

The inhibition of glutathione (GSH) synthesis by L-buthionine-SR-sulfoximine (BSO) causes aggravation of hepatotoxicity of paraquat (PQ), an oxidative-stress inducing substance, in mice. On the other hand, synthesis of metallothionein (MT), a cysteine-rich protein having radical scavenging activity, is induced by PQ, and the induction by PQ is significantly enhanced by pretreatment of mice with BSO. The purpose of present study is to examine whether generation of reactive oxygens is involved in the induction of MT synthesis by PQ under inhibition of GSH synthesis. Administration of PQ to BSO-pretreated mice increased hepatic lipid peroxidation and frequency of DNA single strand breakage followed by manifestation of the liver injury and induction of MT synthesis. Both vitamin E and deferoxamine prevented MT induction as well as lipid peroxidation in the liver of mice caused by administration of BSO and PQ. In cultured colon 26 cells, both cytotoxicity and the increase in MT mRNA level caused by PQ were significantly enhanced by pretreatment with BSO. Facilitation of PQ-induced reactive oxygen generation was also observed by BSO treatment. These results suggest that reactive oxygens generated by PQ under inhibition of GSH synthesis may stimulate MT synthesis. GSH depletion markedly increased reactive oxygen generation induced by PQ, probably due to the reduced cellular capability to remove the radical species produced.     

Effects of ageing on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxic effects on striatum and brainstem in the rat

In 3- and 18-month-old male Wistar rats, levels of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), ascorbic acid (AA), dehydroascorbic acid (DHAA), noradrenaline (NA), uric acid, glutathione (GSH) and 1-methyl-4-phenylpyridinium ion (MPP+) were determined by HPLC in the striatum and/or in the brainstem 24 h after single injections of MPTP (12-35 mg/kg i.p.). Aged rats had lower baseline levels of AA and GSH, compared to young rats. In aged rats, MPTP 35 mg/kg induced a 70% death rate and a decrease in striatal DOPAC/DA ratio which was significantly correlated to MPP+ concentrations (r = -0.840, P < 0.005); in addition, MPTP did not increase AA oxidation. In the brainstem, the MPTP-induced decrease in NA levels and increase in uric acid levels were significantly correlated to the MPP+ concentrations (r = -0.709, P < 0.05, and r = +0.888, P < 0.001, respectively). In conclusion, evidence is given of a mechanism of toxicity of MPTP involving oxidative stress produced by xanthine oxidase; in addition, in aged rats the neuronal antioxidant system (levels of AA and GSH) is considerably lower than in young rats and may play an enabling role in the MPTP age-related neurotoxic effects on striatum and brainstem.     

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.     

Newborn organ weight and spontaneous hypertension: recombinant inbred strain study

This study investigated the effects of neonatal hippocampal ablation on the development of spatial learning and memory abilities in rats. Newborn rats sustained bilateral electrolytic lesions of the hippocampus or were sham-operated on postnatal day 1 (PN1). At PN20-25, PN50-55, or PN90-95, separate groups of rats were tested in a Morris water maze on a visible "cue" condition (visible platform in a fixed location of the maze), a spatial "place" condition (submerged platform in a fixed location), or a no-contingency "random" condition (submerged platform in a random location). Rats were tested for 6 consecutive days, with 12 acquisition trials and 1 retention (probe) trial per day. During acquisition trials, the rat's latency to escape the maze was recorded. During retention trials (last trial for each day, no escape platform available), the total time the rat spent in the probe quadrant was recorded. Data from rats with hippocampal lesions tested as infants (PN20-25) or as adults (PN50-55 and PN90-95) converged across measures to reveal that 1) spatial (place) memory deficits were evident throughout developmental testing, suggesting that the deficits in spatial memory were long-lasting, if not permanent, and 2) behavioral performance measures under the spatial (place) condition were significantly correlated with total volume of hippocampal tissue damage, and with volume of damage to the right and anterior hippocampal regions. These results support the hypothesis that hippocampal integrity is important for the normal development of spatial learning and memory functions, and show that other brain structures do not assume hippocampal-spatial memory functions when the hippocampus is damaged during the neonatal period (even when testing is not begun until adulthood). Thus, neonatal hippocampal damage in rats may serve as a rodent model for assessing treatment strategies (e.g., pharmacological) relevant to human perinatal brain injury and developmental disabilities within the learning and memory realm.     

Learning deficits in congenitally hydrocephalic rats and prevention by early shunt treatment

Shunt surgery is the usual treatment for infantile hydrocephalus; however, the extent to which it avoids subsequent neurological deficits is uncertain. The effect of early-onset hydrocephalus was tested in H-Tx rats using the Morris water maze. Spatial learning was assessed at 21 days after birth in control (n = 18), hydrocephalic (n = 18) and hydrocephalic rats shunt-treated at 4-5 (n = 7) or at 10-12 days of life (n = 13). The time taken to find a hidden platform was measured in five trials on 2 consecutive days and the data analyzed by one- and two-way ANOVA and t-tests. The latencies of the control rats decreased significantly between the first and second trial on the 1st day, and learning was retained until the 2nd day. The hydrocephalic group had longer latencies than controls on both days, with no significant decrease between any trials. Performance was not significantly different between the two shunt groups. Overall, the shunted rats had latencies which were not significantly different from controls but were significantly lower than hydrocephalics. Despite this, the shunted rats did not perform as well as the controls. It is concluded that, although shunt treatment improved learning, some effects of early-onset hydrocephalus may not be reversible and/or a longer recovery time is required.     

Treatment of unresectable hepatocellular carcinoma with lipiodol chemoembolization: a multicenter randomized trial. Groupe CHC

We investigated the effect of successive administrations of SA4503 (1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride), a novel cognitive enhancer with high affinity and selectivity for the sigma1 receptor subtype, on the cortical cholinergic dysfunction-induced impairment of the spatial learning performance in the Morris water maze (MWM) task in rats. The impairment of the spatial learning performance was produced by the ibotenic acid-induced lesion of the basal forebrain (BF) area in rats. Escape latencies to find the platform during the training trials of the MWM task were significantly prolonged in the BF-lesioned rats compared with the sham-operated rats. Daily treatment with SA4503 (0.1-0.5 mg/kg, P.O./day) for 13 days ameliorated this learning deficit. In the probe trial, BF-lesioned rats reduced the number of times each rat crossed the former platform location during the training trials (goal area) in comparison with sham-operated rats. Successive administrations of SA4503 (0.25 mg/kg, P.O./day) also significantly increased the BF lesion-induced reduction of the number of times each rat crossed the goal area. These results suggest that the successive administrations of SA4503 attenuate the impairment of the spatial learning performance in rats with cortical cholinergic dysfunction, and that SA4503 is useful as a therapeutic drug for Alzheimer's disease.     

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.     

Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability

Glutathione (GSH) has been shown to play an important role in embryo development. In a previous study, we demonstrated that cysteamine supplementation of in vitro maturation (IVM) medium increased the intracellular GSH content in bovine oocytes and improved subsequent embryo development to the blastocyst stage. The present study was carried out to evaluate the effect of inhibition by buthionine sulfoximide (BSO) of GSH synthesis during IVM in the presence of cysteamine, on subsequent embryo development, and the effect of cysteamine during IVM on the survival of blastocysts following freezing. The effect of beta-mercaptoethanol and cysteine added to the maturation medium on GSH levels in bovine oocytes, as well as the effect of these compounds on de novo GSH synthesis by oocytes during in vitro maturation, was also studied. The inhibitory effect of BSO during in vitro maturation on GSH synthesis was also evaluated. Evidence was found confirming that GSH synthesis occurs intracellularly during IVM of oocytes and is stimulated by cysteamine, beta-mercaptoethanol and cysteine. Moreover, the present results suggest that the increase in the rate of embryo development exerted by cysteamine, when present during IVM, was due to its stimulatory effect on GSH synthesis. This increase in GSH levels during IVM improves embryo development and quality, producing more embryos reaching the blastocyst stage on day 6, those most suitable for freezing.     

Modulation of cisplatin chronotoxicity related to reduced glutathione in mice

Intracellular reduced glutathione (GSH) concentrations were measured according to the tissue sampling-time along the 24 h scale in male B6D2F1 mice. A significant circadian rhythm in GSH content was statistically validated in liver, jejunum, colon and bone-marrow (P < or = 0.02) but not in kidney. Tissue GSH concentration increased in the dark-activity span and decreased in the light-rest span of mice. The minimum and maximum of tissue GSH content corresponded respectively to the maximum and minimum of cisplatin (CDDP) toxicity. The role of GSH rhythms with regard to CDDP toxicity was investigated, using a specific inhibitor of GSH biosynthesis, buthionine sulfoximine (BSO). Its effects were assessed on both tissue GSH levels and CDDP toxicity at three circadian times. BSO resulted in a 10-fold decrease of the 24 h-mean GSH in kidney. However a moderate GSH decrease characterized liver (-23%) and jejunum (-30%). BSO pretreatment largely enhanced CDDP toxicity which varied according to a circadian rhythm. Although BSO partly and/or totally abolished the tissue GSH rhythms, it did not modify those in CDDP toxicity. We conclude that GSH have an important influence on CDDP toxicity but not in the circadian mechanism of such platinum chronotoxicity.