Neuroprotective effect of insulin-like growth factor I in immortalized hypothalamic cells.

The neuroprotective action of insulin-like growth factor I (IGF-I) was tested in immortalized hypothalamic GT1-7 cells exposed to reduced glutathione depleting agents, which cause oxidative stress and cell death. The extent of cell survival was assessed by either using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide cytotoxicity assay or counting at the fluorescence microscope GT1-7 cells prelabeled with fluorescent dyes selective for viable and dead cells. Treatments with buthionine sulfoximine (500 microns), diethylmaleate (1 mM), and ethacrynic acid (200 microns) caused diffuse GT1-7 cell death (40-60%). Exposure of the same cells to IGF-I (either before or concomitant to the toxic agent, depending on the drug used) significantly prevented neuronal death. This effect was rapid, concentration-dependent, maximal at concentrations of 25-50 ng/ml, and mimicked by IGF-II, fibroblast growth factor, and the potent antioxidant idebenone. In contrast, IGF-I, as well as idebenone, were completely ineffective in antagonizing the toxic effect produced by different concentrations of menadione. In conclusion, the present data demonstrate a protective role for IGF-I against glutathione depleting agents-induced damage in GT1-7 cells suggesting an antioxidant action of this growth factor in hypothalamic neurons.

Gastric injury and protection against alcohol and acid: influence of perturbations in glutathione metabolism.

This study assessed the role that inhibition of glutathione (GSH) synthesis and decreased GSH peroxidase (GPX) activity in the rat played in modulating gastric injury induced by ethanol and acid and its prevention by 16,16-dimethyl PGE2 (dmPGE2) and the mild irritant, 25% ethanol. Although numerous studies have proposed that GSH may be important in maintaining gastric mucosal defense, the exact role of this antioxidant in protecting the stomach from injury remains undefined. The present study addressed this consideration by blocking the synthesis of GSH and altering the major pathway by which it exhibits its antioxidant activity and determining the effect of these perturbations on gastric injury and protection. Four to six rats were used for each experimental group. GSH synthesis was blocked by the potent and specific inhibitor L-buthionine sulfoximine (BSO), 2 or 6 mmole/kg intraperitoneally. The activity of the major form of GPX, which is selenium dependent and utilizes GSH as a substrate to detoxify hydrogen peroxide and other hydroperoxides, was inhibited by placing animals on a selenium-deficient diet for 6 weeks. Gastric damage was induced by 100% ethanol, 50% ethanol in 150 mM HCl, and 0.75 M HCl. Prevention of such injury was accomplished with oral pretreatment using 25% ethanol or dmPGE2 (5 microgram/kg). The damaging effects of 100% ethanol, 50% ethanol/150 mM HCl, or 0.75% M HCl were not adversely affected by BSO pretreatment even though GSH synthesis was inhibited by as much as 80%. Similarly, inhibition of GPX activity by 58% in adult rats and 98% in weanling rats failed to potentiate the damaging effect of 100% ethanol. Furthermore, with both perturbations in GSH metabolism, the protective action of dmPGE2 and 25% ethanol was maintained. Our results indicate that profound alterations in gastric GSH metabolism by themselves do not aggrevate the injurious effects of ethanol or acid, nor do they prevent the protective action of a prostaglandin or mild irritant.

Generation of DNA base modification following treatment of cultured murine keratinocytes with benzoyl peroxide.

Benzoyl peroxide (BzPO) is a free radical generating compound that acts as a tumor promoter and progressor in mouse skin. BzPO is cleaved in the presence of copper to produce benzoyloxyl and phenyl radicals. Treatment of mutation reporter plasmids with BzPO and copper yields predominantly single-strand breaks and G-->T transversion mutations. To explore the role of base modifications in the possible mammalian mutagenicity of BzPO the formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) within the DNA of cultured murine keratinocytes was investigated. Treatment with 10 microM BzPO produced a maximum 3-fold increase in levels of 8-OHdG versus vehicle controls within 1-2 h, with significant levels of 8-OHdG persisting 6 h after initial exposure to BzPO. Pretreatment with the copper chelator bathocuproine disulfonic acid reduced the levels of 8-OHdG generated by BzPO to near background. However, treatment with the iron chelator desferal did not. The stable metabolic product of BzPO benzoic acid was ineffective in producing 8-OHdG. Depletion of cellular glutathione with L-buthionine-(S,R)-sulfoximine increased the amount of BzPO-generated 8-OHdG, while supplementation with glutathione monoethyl ester reduced the number of 8-OHdG molecules formed. Collectively, these results suggest that BzPO at non-cytotoxic concentrations undergoes copper-dependent activation to a reactive product to generate 8-OHdG within cultured murine keratinocytes.

Role of glutathione redox cycle in TNF-alpha-mediated endothelial cell dysfunction.

Modulation of the glutathione redox cycle may influence tumor necrosis factor-alpha (TNF)-mediated disturbances of endothelial integrity. To test this hypothesis, normal endothelial cells or cells with either increased or decreased glutathione levels were exposed to 100 ng (500 U) TNF/ml. Increased glutathione levels were achieved by exposure to 0.2 mM N-acetyl-L-cysteine (NAC) and decreased glutathione levels by exposure to 25 microM buthionine sulfoximine (BSO). Several components of the glutathione redox cycle as well as markers of endothelial integrity, such as cytoplasmic free calcium and transendothelial albumin transfer, were measured in the treated cells. Exposure to TNF for 3 and 6 h decreased total glutathione levels, which was followed by an increase at later time points. Moreover, treatment with TNF resulted in an increase in the ratio of oxidized to reduced glutathione, intracellular free calcium, albumin transfer across endothelial monolayers and lipid hydroperoxides. However, an increase in lipid hydroperoxides was seen only when endothelial cell cultures were supplemented with iron. BSO treatment increased susceptibility of endothelial cells to TNF-mediated metabolic disturbances. On the other hand, NAC partially protected against TNF-induced injury to endothelial monolayers. Our results demonstrate the important role of the glutathione redox cycle in TNF-mediated disturbances of the vascular endothelium and indicate that modulation of glutathione levels may potentiate the injurious effects of this inflammatory cytokine.

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.

Liver and kidney necrosis in selenium-deficient rats depleted of glutathione.

BACKGROUND: Selenium and glutathione have interrelated oxidant defense roles in vivo. Experiments were carried out to determine the effect of glutathione depletion in selenium-deficient rats. EXPERIMENTAL DESIGN: Selenium-deficient and control rats were injected with phorone to deplete glutathione. Histologic assessment of liver and kidney injury was performed at 24 hours. In another experiment, glutathione depletion, lipid peroxidation, and liver injury were measured for 12 hours after phorone administration to determine their relationships with one another. In a final experiment, selenoproteins were correlated with protection against lipid peroxidation and liver necrosis. Selenium-deficient rats were injected with vehicle alone and with 5, 10, or 25 micrograms of selenium/kg. Twelve hours later, selenoproteins were measured in some of the rats, and phorone was injected into others. Liver injury and lipid peroxidation were assessed 6 hours after the phorone injection. RESULTS: Twenty-four hours after phorone administration (125 mg/kg), centrilobular hepatic necrosis and renal tubular necrosis were evident in selenium-deficient rats but not in controls. The time-course experiment revealed that phorone (250 mg/kg) caused sharp decreases in liver and kidney glutathione levels in both groups within 2 to 4 hours. Lipid peroxidation, as assessed by F2 isoprostane concentrations, in selenium-deficient animals. Liver necrosis, indicated by a rise in plasma ALT, took place in selenium-deficient rats but not in controls. Selenium injections into selenium-deficient rats increased selenoprotein P concentrations from 4% of control to as high as 39% but had little effect on glutathione peroxidase activities. Six hours after phorone administration, rats that had received selenium had no rise in ALT, and the rises in F2 isoprostanes were abolished or attenuated. CONCLUSIONS: We conclude that depletion of glutathione in selenium-deficient liver and kidney leads to necrosis in those organs associated with evidence of lipid peroxidation. Protection against this injury by selenium correlates with selenoprotein P concentration in plasma but not with glutathione peroxidase activity in tissues or in plasma. These findings raise the possibility that selenoprotein P protects cell membranes against oxidant injury and that glutathione is involved in that protection.

Polyunsaturated fatty acid enrichment increases ultraviolet A-induced lipid peroxidation in NCTC 2544 human keratinocytes.

The influence of cell enrichment with fatty acids with increasing degree of unsaturation on the ultraviolet A-induced formation of lipid-peroxidation products (thiobarbituric acid reactive substances [TBARS]) has been investigated in NCTC 2544 human keratinocytes. A 48-h preculture of cells in controlled medium supplemented with unsaturated fatty acids resulted in a marked increase in TBARS appearance under ultraviolet A exposure. This effect was dependent upon the degree of unsaturation of the fatty acids, with the following order of efficiency: arachidonic > linolenic > linoleic > oleic acid. For arachidonic acid (AA), the potentiating effect on ultraviolet A-induced lipid peroxidation was dependent upon the fatty acid concentration, with about a 2.5-fold increase in TBARS formation in cells pre-cultured with 5 x 10(-5) M AA, then exposed to a UVA dose of 13 J/cm2. The increase in TBARS formation by AA was almost totally prevented by supplementation of cells with 5 x 10(-5) M vitamin E, whereas buthionine sulfoximine, a chemical which depletes cell glutathione, potentiated lipid peroxidation. These results suggest that the nature of the fatty acids of cellular lipids could influence the response of keratinocytes to ultraviolet A, and especially the ultraviolet A-induced lipid peroxidation.

Buthionine sulfoximine enhances glutathione-but attenuates glutamate-stimulated cell proliferation.

Buthionine sulfoximine (BSO) inhibits proliferation of human lung carcinoma A549 cells, and exogenous glutathione (GSH) overcomes the antiproliferative effect. The BSO antiproliferation may result from inhibition of cellular uptake of amino acids, and the antagonistic effect of GSH would result from supplementation of amino acids via the gamma-glutamyl cycle. To explore these possibilities, the present study was undertaken to determine effects of BSO on glutamate- and GSH-stimulated cell proliferation. A549 cells were cultured in a glutamine-deficient Dulbecco's modified Eagle's medium (Gln-(-)DMEM), in which they did not proliferate. Addition of glutamate or GSH in the medium to a concentration of 4 mM stimulated cell proliferation. BSO of 0.1 mM enhanced the GSH-stimulated cell proliferation and attenuated the glutamate-stimulated cell proliferation. This BSO effect correlated with changes in cellular glutamate levels; that is, BSO increased and decreased glutamate concentrations, respectively, in GSH- and glutamate-stimulated cells. GSH or glutamate alone significantly increased cellular GSH levels. BSO depleted cellular GSH in both GSH- and glutamate-stimulated cells to the same level. These changes in GSH levels did not correlate with the respective growth modulatory effect. Because BSO inhibits cellular uptake of some amino acids and the A549 cells contain high levels of gamma-glutamyl transpeptidase activity, the results suggest that the BSO inhibition of glutamate-stimulated cell proliferation may result from decreased glutamate uptake. GSH would supplement the cells with glutamate via the gamma-glutamyl pathway to bypass the inhibition of amino acid uptake and overcome the BSO-antiproliferative effect.

Glutathione stimulates A549 cell proliferation in glutamine-deficient culture: the effect of glutamate supplementation.

Extracellular glutathione (GSH) is degraded by an external cell-surface enzyme, gamma-glutamyltranspeptidase (gamma-GT). The products are transported into cells to participate in important cellular processes. In the present study, we tested the hypothesis that extracellular GSH is a source of glutamic acid for cells that express gamma-GT. Under a glutamine-deficient culture condition, the extracellular GSH-supplemented glutamic acid would enhance intracellular glutamine synthesis, thereby stimulating cell proliferation. Human lung carcinoma A549 cells were cultured in glutamine-deficient Dulbecco's modified Eagle medium, and they did not proliferate unless glutamine was supplemented. Extracellular GSH, however, provoked a partial proliferation. The GSH effect correlated with a high level of gamma-GT activity and an increased intracellular level of glutamic acid. A constituent amino acid of GSH, glutamic acid but not cysteine, produced the same growth-stimulatory effect as GSH. Furthermore, neither oxothiazolidine-4-carboxylate (OTC), a cellular cysteine-delivery compound, nor cysteinylglycine, a dipeptide released from the gamma-GT reaction, stimulated cell proliferation. Moreover, buthionine sulfoximine (BSO), a selective inhibitor of gamma-glutamylcysteine synthetase, enhanced the GSH growth stimulatory effect, suggesting that increased cellular GSH synthesis does not correlate with cell growth stimulation. The results obtained demonstrated that glutamine is required for A549 cell proliferation and exogenous GSH partially substitutes for the growth stimulatory action of glutamine. It also suggests that the glutamic acid rather than the cysteine released from the GSH is responsible for the cell proliferation.

p-Dichlorobenzene-induced hepatotoxicity in mice depleted of glutathione by treatment with buthionine sulfoximine.

p-Dichlorobenzene (p-DCB) is widely used as a moth repellent and a space deodorant. In mice pretreated with DL-buthionine sulfoximine (BSO; 2 mmol/kg or higher doses, i.p.), an inhibitor of glutathione (GSH) synthesis, administration of p-DCB (100-400 mg/kg, p.o.) resulted in dose-dependent hepatotoxicity as judged by increased serum alanine aminotransferase (ALT) activities and liver calcium concentrations and by histological examination of the liver, p-DCB alone (up to 1200 mg/kg) resulted in no hepatotoxicity. Administration of GSH monoethyl ester, which is known as a useful means for increasing organ GSH levels, protected against the hepatotoxicity caused by p-DCB in combination with BSO. Treatment with inhibitors of hepatic cytochrome P-450-dependent monooxygenases, carbon disulfide, metyrapone and piperonyl butoxide also prevented the hepatotoxicity. These results suggest that p-DCB is activated by a cytochrome P-450-dependent metabolic reaction and that the hepatotoxicity is caused by inadequate rates of detoxification of the resulting metabolite in mice depleted of hepatic GSH by BSO treatment. The liver injury was preceded by an extensive depletion of hepatic GSH but not accompanied by significant changes in hepatic contents of lipid peroxides and protein thiols.

Exercise-induced oxidative stress: glutathione supplementation and deficiency.

Glutathione (GSH) plays a central role in coordinating the synergism between different lipid- and aqueous-phase antioxidants. We documented 1) how exogenous GSH and N-acetylcysteine (NAC) may affect exhaustive exercise-induced changes in tissue GSH status, lipid peroxides [thiobarbituric acid-reactive substances (TBARS)], and endurance and 2) the relative role of endogenous GSH in the circumvention of exercise-induced oxidative stress by using GSH-deficient [L-buthionine-(S,R)-sulfoximine (BSO)-treated] rats. Intraperitoneal injection of GSH remarkably increased plasma GSH; exogenous GSH per se was an ineffective delivery agent of GSH to tissues. Repeated administration of GSH (1 time/day for 3 days) increased blood and kidney total GSH [TGSH; GSH+oxidized GSH (GSSG)]. Neither GSH nor NAC influenced endurance to exhaustion. NAC decreased exercise-induced GSH oxidation in the lung and blood. BSO decreased TGSH pools in the liver, lung, blood, and plasma by approximately 50% and in skeletal muscle and heart by 80-90%. Compared with control, resting GSH-deficient rats had lower GSSG in the liver, red gastrocnemius muscle, heart, and blood; similar GSSG/TGSH ratios in the liver, heart, lung, blood, and plasma; higher GSSG/TGSH ratios in the skeletal muscle; and more TBARS in skeletal muscle, heart, and plasma. In contrast to control, exhaustive exercise of GSH-deficient rats did not decrease TGSH in the liver, muscle, or heart or increase TGSH of plasma; GSSG of muscle, blood, or plasma; or TBARS of plasma or muscle. GSH-deficient rats had approximately 50% reduced endurance, which suggests a critical role of endogenous GSH in the circumvention of exercise-induced oxidative stress and as a determinant of exercise performance.

Effect of selenium deficiency and glutathione-modulating agents on diquat toxicity and lipid peroxidation in rats.

The dipyridyl herbicide diquat undergoes redox cycling in vivo resulting in superoxide generation. Diquat administration causes hepatic and renal toxicity in rodents. Selenium deficiency worsens this injury and lipid peroxidation is a prominent feature of the toxicity. However, there is limited data regarding the role of lipid peroxidation in diquat-induced toxicity in selenium-adequate animals. In addition, little is known about the effect of glutathione-modulating agents on diquat-induced toxicity and lipid peroxidation in vivo. F2-isoprostanes are novel prostanoids which, both free in plasma and esterified to phospholipids in tissues, are markers of lipid peroxidation in vivo. By using F2-isoprostane quantitation, we examined the effects of selenium deficiency and modulation of glutathione status with 1,3-bis (2-chloroethyl)-1-nitrosourea, phorone or buthionine sulfoximine on diquat-induced toxicity and lipid peroxidation. F2-isoprostanes increased 2- to 9-fold in plasma, liver, kidney and lung in selenium-adequate Fischer 344 rats with liver injury after receiving 100 mumol of diquat per kg. Selenium deficiency or modulation of glutathione status increased diquat toxicity. This was accompanied by 10- to 100-fold increases in plasma and kidney F2-isoprostane levels. Liver F2-isoprostanes were increased 2- to 5-fold. These studies suggest that glutathione, in addition to selenium, is an important defense against diquat-induced toxicity and lipid peroxidation.

Differential effect of cyanohydroxybutene on glutathione synthesis in liver and pancreas of male rats.

1-Cyano-2-hydroxy-3-butene (CHB), an aliphatic nitrile found in cruciferous vegetables, causes a two- and sevenfold elevation in reduced glutathione (GSH) in rat liver and pancreas, respectively, after oral administration of 200 mg/kg. While this dose is also associated with pancreatotoxicity, a single 100 mg/kg dose or multiple lesser doses show the same effect, although somewhat reduced in magnitude, with no concomitant toxicity. In an attempt to identify the mechanism of this increase, we investigated the effect of CHB on GSH synthesis by examining the effect of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, on CHB-induced GSH elevation. Male Fischer 344 rats received 3 mmol BSO/kg ip 24 and 34 hr following CHB or corn oil. The CHB-mediated elevation in hepatic and pancreatic GSH was eradicated by BSO, suggesting that increased synthesis was responsible. The rate-limiting step in synthesis is gamma-glutamyl cysteine synthetase (GCS); the limiting substrate is cysteine. Therefore, CHB effects on GCS activity and hepatic and pancreatic cysteine equivalents were investigated. When rats were treated by gavage with CHB (100 mg/kg), hepatic GCS mRNA concentrations were increased 24 hr after treatment and hepatic cysteine equivalents were significantly elevated 4 hr following CHB. No significant elevation in hepatic GCS activity was observed, however, even 24 hr following CHB. Pancreatic cysteine equivalents were elevated at both 4 and 8 hr after CHB treatment. However, there was no detectable GCS mRNA or activity in pancreas, in either control or treated animals. Furthermore, CHB had no direct effect on the activity of GCS purified from kidney, regardless of whether GSH was present or absent. These results suggest that the mechanism of CHB-mediated induction of GSH may involve early increases in GSH precursors as well as a later increase in GCS mRNA. The mechanism of GSH elevation identified in these studies may hold therapeutic or prophylactic implications.

Lethal damage to endothelial cells by oxidized low density lipoprotein: role of selenoperoxidases in cytoprotection against lipid hydroperoxide- and iron-mediated reactions.

Oxidized low density lipoprotein (LDLox) is believed to be an important contributor to endothelial cytodamage and atherogenesis. The purpose of this study was to examine the role of glutathione (GSH) and GSH-dependent selenoperoxidases in cytoprotection against the damaging effects of LDLox. When irradiated in the presence of a phthalocyanine sensitizing dye, human LDL accumulated chromatographically detectable and iodometrically measurable lipid hydroperoxides (LOOHs). Photogenerated LDLox caused lethal damage to bovine aortic endothelial (BAE) cells in vitro, as determined by lactate dehydrogenase release and inhibition of thiazolyl blue reduction. When depleted of GSH by buthionine sulfoximine treatment, BAE cells became more sensitive to LDLox. Cells grown in 2% serum/DME-HAM's F-12 medium without added selenium [Se(-) cells] exhibited far lower GSH-peroxidase and phospholipid hydroperoxide GSH-peroxidase activities than selenium-supplemented controls [Se(+) cells], and were much more sensitive to oxidative injury induced by t-butyl hydroperoxide, liposomal cholesterol hydroperoxides, and LDLox. Preincubation of LDLox with GSH and Ebselen (a selenoperoxidase mimetic) resulted in a dramatic reduction in both LOOH content and cytotoxicity. Moreover, treating Se(-) cells themselves with Ebselen substantially restored their resistance to LDLox-induced damage. LDLox toxicity to Se(-) cells was strongly inhibited by desferrioxamine and stimulated by ferric-8-hydroxyquinoline (a lipophilic chelate), indicating that iron is an active participant in oxidative damage. These results demonstrate that the GSH-dependent selenoperoxidase(s) play(s) an important role in cellular defense against oxidized low density lipoprotein, presumably by detoxifying lipid hydroperoxides and thereby preventing their iron-catalyzed decomposition to damaging free radical intermediates.

Selenite-induced inhibition of colony formation by buthionine sulfoximine-sensitive and resistant cell lines.

We previously demonstrated that treatment of HeLa cells with buthionine sulfoximine (BSO), which decreases the level of cellular glutathione, resulted in a decrease in the potency of selenite in inhibiting cell colony formation. We have now examined the effect of selenite on normal human lung fibroblast (CCL-210) cells, which resemble HeLa cells in their sensitivity to BSO, and on human lung adenocarcinoma (A549) cells, which are relatively insensitive to BSO. We have found that BSO treatment caused an approximately fourfold decrease in selenite potency in the CCL-210 cells, but had no significant effect on its potency in A549 cells. These results support the hypothesis that for selenite to exert its cytotoxic effect, it must undergo the reaction with an SH compound to form the selenotrisulfide. As a result of the lower sensitivity of the tumor cells to BSO, it was possible to achieve a large differential sensitivity to the cytotoxic effect of selenite.

In vivo formation of chromium(V) in chick embryo liver and red blood cells.

In order to understand the possible role of reactive intermediates in the formation of tissue-specific DNA damage by chromium(VI), electron paramagnetic resonance spectroscopy was used to study the in vivo formation of chromium(V) in the liver and red blood cells of 14 day chick embryos following treatment with chromium(VI). In vivo administration of sodium dichromate onto the inner shell membrane of 14 day chick embryos resulted in the formation of a persistent chromium(V) species in liver cells (g = 1.987). The intensity of the chromium(V) signal in liver cells plateaued at 70 min and persisted for 240 min after treatment with chromium(VI). The dependence of chromium(V) formation on the dose of sodium dichromate administered to the embryo was clearly different in liver versus red blood cells. Chromium(V) was detected in red blood cells only at high doses of sodium dichromate (0.50-0.60 mmol/kg), whereas chromium(V) was undetectable in red blood cells at lower doses of sodium dichromate (0.10-0.30 mmol/kg) which produced clear evidence for chromium(V) in liver. Uptake studies showed that total chromium levels in red blood cells were 10-fold greater than in liver cells, and that up to 10% of the total chromium existed as chromium(V) in liver and red blood cells in vivo. Depletion of glutathione by pretreatment of embryos with L-buthionine-S,R-sulfoximine (BSO) for 24 h prior to treatment with a high dose of sodium dichromate (0.60 mmol/kg) caused both a decrease in the levels of chromium(V) species produced and a decrease of chromium uptake into red blood cells 50 min after treatment. At this high dose of chromium(VI), BSO pre-treatment had no effect on the level of the chromium(V) or on chromium uptake into liver cells after a 70 min incubation period. Thus, the concentration of chromium(V) inside the cell correlated with the levels of chromium taken up into the cell. Chromium(V) may be the form of chromium which is responsible for induction of DNA damage following in vivo administration of sodium dichromate.

A scientific basis for proposed quality assurance of a new screening method for tumor-like growths in the planarian, Dugesia dorotocephala.

Various abnormal growths appear on planarians, Dugesia dorotocephala, during and after exposure to polychlorinated biphenyls (PCBs) 28, 110, and 126; Aroclor 1254; cadmium sulfate; and L-buthionine-(R,S)-sulfoximine (BSO). Daily observations under magnification were used to describe the location, development, and morphology of three different types of tumor-like growths ("tumors"). "Post-head tumors" were found to be highly invasive, progressive, and lethal to the animal depending on concentrations and combinations of the compounds used. Survivors from post-head tumors exhibited aberrant morphogenesis, but developmental abnormalities were eventually shed. Post-head tumors occurred within 2 weeks of initial exposure, while "round tail tip tumors" appeared after 2-3 weeks. The rate of progression and invasiveness was greater for the round tail tip tumors. "Pigmented rose thorn tail tumors" occurred in low incidence (4-20%) and appeared to be harmless and noninvasive, requiring months to develop from the first appearance of pigmentation. The aggressive, proliferative, and invasive characteristics of post-head and round tail tip tumors are analogous to those of malignant tumors, while pigmented rose thorn tumors were benign. High dose of cadmium alone were sufficient to initiate the post-head and round tail tip tumors. PCBs potentiated the tumorigenicity of low cadmium doses and enhanced the very low spontaneous incidence of pigmented rose thorn tumors. PCBs also impaired motor activity, causing the graceful gliding locomotion to be replaced by a twisting serpentine movement accompanied by muscular dystrophy. In addition, high (50 micrograms) doses of PCB 110 depressed activity, while lower (5 micrograms) doses and 50 micrograms Aroclor 1254 induced restlessness and enhanced locomotion. These data provide the basis for quality assurance.

Roles of glutathione and glutathione peroxidase in the protection against endothelial cell injury induced by 15-hydroperoxyeicosatetraenoic acid.

We investigated the role of the glutathione redox cycle in endothelial cell injury induced by 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), an arachidonate lipoxygenase product. Pretreatment of endothelial monolayers with reduced glutathione (GSH) markedly suppressed 15-HPETE-induced cellular injury, which was determined by the 51Cr-release assay. 15-HPETE-induced cytotoxicity was modified by several GSH-modulating agents such as buthionine sulfoximine and 2-oxothiazolidine-4-carboxylate, indicating that this cyto-protective action of GSH was correlated with the intracellular GSH level. These GSH-modulating agents also modified the conversion of 15-HPETE to 15(S)-hydroxyeicosatetraenoic acid by endothelial cells. On the other hand, the exposure of endothelial cell monolayers to 15-HPETE did not deplete intracellular GSH levels but decreased GSH peroxidase activity. In addition, sodium selenite and ebselen, a stimulator and mimic of GSH peroxidase activity, respectively, displayed remarkable protective effects against 15-HPETE-induced cytotoxicity. These results suggest that intracellular GSH plays a pivotal role in the protection against 15-HPETE-induced endothelial cell injury, and that the decreased activity of GSH peroxidase activity is involved in 15-HPETE-induced cytotoxicity.

Effects of glutathione or polyamine depletion on in vivo thermosensitization.

Investigations with the melphalan-sensitive and -resistant human rhabdomyosarcoma xenografts TE-671 and TE-671 MR were performed to examine the effect of glutathione and polyamine modulation on thermosensitivity. Regimens of intraperitoneally injected and orally administered buthionine sulfoximine were utilized to achieve glutathione depletion to 8.7% and 13% of control levels in TE-671 and TE-671 MR, respectively. Animals treated with L-buthionine-S,R-sulfoximine and 42 degrees C or 43 degrees C hyperthermia for 70 min showed no detectable growth delays beyond those observed for hyperthermia alone. Hyperthermia at 42 degrees C of disaggregated TE-671 and TE-671 MR xenografts following growth in short-term culture was performed following preincubation with buthionine sulfoximine or 0.9% saline. Buthionine sulfoximine-mediated glutathione depletion produced a significant increase in hyperthermia-induced cytotoxicity only with TE-671 MR at 43 degrees C. Polyamine depletion was achieved with a 7-day orally administered course of MDL 72.175DA [(2R,5R)-6-heptyne,5-diamine dihydrochloride], an irreversible inhibitor of ornithine decarboxylase. Although this treatment caused significant depletion of intracellular putrescine and spermidine levels, spermine levels remained relatively unaffected. No significant growth delays were observed in either xenograft line for animals treated with MDL 72.175DA or MDL 72.175DA plus hyperthermia as compared with untreated controls. These results contrast with previous work performed in vitro showing synergism between glutathione or polyamine depletion and hyperthermia, and indicate that further studies are needed.