Changes In Glutathione Levels Research Articles

Effects of N-Acetyl-Cysteine on Endothelial Function and Inflammation in Patients with Type 2 Diabetes Mellitus

Endothelial dysfunction has been associated with premature vascular disease. There is increasing data that N-acetyl-cysteine (NAC) may prevent or improve endothelial dysfunction. The aim of this study was to assess the effects of NAC on endothelial function in patients with type 2 diabetes mellitus, a population at high risk for endothelial dysfunction. Twenty-four patients with diabetes mellitus were assigned randomly to initial therapy with either 900 mg NAC or placebo twice daily in a double-blind, cross-over study design. Flowmediated vasodilation (FMD) of the brachial artery was assessed at baseline, after four weeks of therapy, after a four-week wash-out period, and after another four weeks on the opposite treatment. Plasma and red blood cell glutathione levels and high-sensitivity C-reactive protein (CRP) were measured at all four visits. At baseline, FMD was moderately impaired (3.7±2.9%). There was no significant change in FMD after four weeks of NAC therapy as compared to placebo (0.1±3.6% vs. 1.2±4.2%). Similarly, there was no significant change in glutathione levels. However, median CRP decreased from 2.35 to 2.14 mg/L during NAC therapy (p=0.04), while it increased from 2.24 to 2.65 mg/L with placebo. No side effects were noted during the treatment period. In this double-blind, randomized cross-over study, four weeks of oral NAC therapy failed to improve endothelial dysfunction in patients with diabetes mellitus. However, NAC therapy decreased CRP levels, suggesting that this compound may have some efficacy in reducing systemic inflammation.

Detection of Leukemia in MLL-AF9 Transgenic Mice Using Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy

Standard methods to assess tumor progression and treatment response for leukemia patients rely on repeat sampling of bone marrow. These methods evaluate overall disease status, but are not sensitive to early biochemical or cellular responses to therapies. Identification and validation of additional pharmacodynamic endpoints that are useful for accurate prediction of therapeutic efficacy would allow for earlier assessment of clinical response and thereby facilitate the development of effective individualized treatment regimens. Ideally, these markers would be assessed using non-invasive methods. Toward this end, we used in vivo magnetic resonance imaging (MRI) and ex vivo magnetic resonance spectroscopy (MRS) to investigate changes in the bone marrow, spleen, and/or blood of leukemic MLL-AF9 transgenic (Tg) mice relative to wild-type littermates.MLL-AF9 Tg mice with leukemia exhibited a statistically significant 1.5-fold increase in bone marrow T1-weighted MRI signal intensity. Increased signal intensity preceded development of leukemia and is therefore likely to be due, at least in part, to increased bone marrow cellularity, initially as a result of pre-neoplastic myeloproliferation and later as a result of marrow infiltration with leukemic blasts. We are currently determining microvessel density in the bone marrow of leukemic MLL-AF9 Tg mice and littermate controls to determine whether an increase in blood supply may also contribute to changes in MRI signal intensity in leukemic mice. These studies suggest that T1-weighted MRI signal intensity may be useful as an indicator of bone marrow tumor burden.Leukemic MLL-AF9 Tg mice also exhibited statistically significant changes in metabolite levels in spleen, blood, and bone marrow. The Warburg effect, whereby cancer cells utilize aerobic glycolysis to meet their increased energy demands, was evident in all tissues examined as indicated by increased glycolysis rates, increased glucose utilization, and increased levels of lactate and alanine, the end-products of glycolysis. Additional changes in metabolite levels were observed in the bone marrow and/or spleen of leukemic mice. Absolute levels of glutathione were increased. Glutathione reduces reactive oxygen species that are generated as a result of increased glycolysis and high levels of glutathione are associated with chemoresistance and poor prognosis in patients with acute leukemia (Maung et al., 1994, Leukemia 8:1487–91; Kearns et al., 2001, Blood 97:393–8). Increased glycine levels were also observed and may be associated with increased pyridine and DNA synthesis in leukemia cells. Decreased glutamate and glutamine levels may reflect a decrease in utilization of the mitochondrial Krebs cycle in tumor cells. Decreased levels of myo-inositol and taurine, which function as osmoregulators, may occur as a result of osmotic stress due to increased cellularity in leukemic organs. Levels of aromatic acids, lysine and arginine, and creatine and phosphocreatine were also decreased. Many of these changes in metabolism are recapitulated in immunocompetent mice orthotopically transplanted with MLL-AF9 Tg leukemias.This study represents the first description of a comprehensive analysis of the “metabolomic” profile associated with development of acute leukemia and is, to our knowledge, the first description of changes in metabolism in an animal model of acute leukemia. The data presented here suggest novel metabolic targets for therapeutic intervention. In addition, because metabolic changes often precede detectable changes in tumor burden, they may be particularly useful as early indicators of therapeutic efficacy and may thereby allow for more rapid determination of clinical response, decreased exposure to toxic therapies in resistant patients, and more expedient conversion to effective therapies. Because changes in glucose metabolism are a central feature of tumorigenesis and changes in glutathione levels have been associated with clinical outcome in patients with acute leukemia, these metabolites are of particular interest. Future studies will use MLL-AF9 transgenic mice to investigate the roles of metabolic changes during de novo development of leukemia. Mice transplanted with MLL-AF9 Tg leukemias will also be used for studies investigating the utility of metabolic changes as indicators of therapeutic efficacy.

Anzer honey prevents N-ethylmaleimide-induced liver damage in rats

N-ethylmaleimide (NEM) is a sulphydryl blocker which impairs the sulphydryl dependent antioxidant system (mainly glutathione) in the body by alkylating endogenous sulphydryls. This study was designed to investigate the effects of Anzer honey on NEM-induced liver injury in rats. Thirty female Wistar albino rats were divided equally into three groups. Group 1: control; Group 2: NEM; Group 3: Anzer honey+NEM. NEM (0.075 mg kg −1) was given to both group 2 and 3 administered subcutaneously (s.c.) for 30 days. The animals in the Anzer honey+NEM group were treated with Anzer honey at a dose of 0.275 g kg −1, (p.o.) at 1 h prior to every NEM injection. At the end of the 30 day treatment period, liver samples were taken for determination of the glutathione levels and histological examination. NEM treatment alone caused a significant reduction of the liver glutathione levels in group 2. Furthermore, NEM treatment caused congestion and mononuclear cell infiltration in the liver when compared to the control group. In group 3, Anzer honey treatment reversed all the changes in glutathione level, as well as histopathological alterations, normally induced by NEM. The findings imply that depletion of glutathione concentration plays a causal role in NEM-induced liver injury, and that the hepatoprotective effect of Anzer honey may be mediated through sulfhydryl-sensitive processes. They further imply that it may also possess antioxidant properties.

N‐Acetylcysteine‐induced changes in susceptibility of transformed eukaryotic cells to bacterial invasion

The effect of N-acetylcysteine (NAC) on morphological and physiological properties of "normal" 3T3 and 3T3-SV40 fibroblasts was studied. Incubation of the cells with 10 and 20 mM NAC for 20 h resulted in a reversible increase in the intracellular level of reduced glutathione and disorganization of actin cytoskeleton. Surprisingly, upon removal of NAC, 3T3-SV40 fibroblasts demonstrated formation of well-adhered cells with structured 3T3-like stress-fibers. Neither changes in glutathione levels, nor cytoskeleton disorganization/assembly abolished resistance of 3T3 cells to invasion by the bacterium Escherichia coli A2. On the other hand, pretreatment with NAC converted bacteria-susceptible 3T3-SV40 cells into resistant ones. These results show that NAC can induce partial reversion of transformed phenotype. We suggest that this effect is due to NAC-induced modifications of cell surface proteins rather than to changes in the level of intracellular glutathione.

Fluorescence-based detection of thiols in vitro and in vivo using dithiol probes

Thiols play a central role in maintaining biological homeostasis. Their levels can change dramatically in response to oxidative stress associated with toxic insults, bacterial infection, and disease. Therefore, a reagent that can monitor thiol levels both in vitro and in vivo would be useful for assays and as a biomarker. Such a reagent should (i) be selective for thiols, (ii) be able to penetrate cell walls, and (iii) have a low reduction potential so as not to create oxidative stress in a cell. We have developed such a fluorescent reagent (DSSA) based on a dithiol linker: (i) the use of a dithiol linker makes it selective for thiols; (ii) the use of fluorophores that populate neutral states at physiological pH improves cell wall penetration; and (iii) because of the reagent’s low reduction potential (−0.60 V), it will not stress cells oxidatively. For example, 5 μM of reagent is responsive to changes in glutathione levels in the physiologically relevant range of 1 to 10 mM, yet this would oxidize less than 1% of cellular glutathione. In Escherichia coli, decreased thiol levels were detected in cells deficient in glutathione synthesis. In zebrafish embryos, the DSSA reagent permitted detection of unusually high thiol levels in the zebrafish chorion.

LPS-induced ROS generation and changes in glutathione level and their relation to the maturation of human monocyte-derived dendritic cells

Lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) generation and the concomitant decline in the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were demonstrated in human monocyte-derived dendritic cells (DC). Further, their relation to the maturation of DC, characterized by the production of cytokines, up-regulation of cell surface molecules and allo-stimulatory capacity, was examined. The LPS-induced ROS generation was demonstrated using electron paramagnetic resonance spectroscopy in intact cells, and was also confirmed using laser scanning confocal microscopy. The GSH/GSSG was assesed using a glutathione assay kit. When the DC were treated with α-phenyl- tert-butylnitrone, the ROS generation was attenuated, but the declined GSH/GSSG was not attenuated, and only cytokine production was suppressed among the above-mentioned maturation characteristics. When the DC were treated with glutathione monoethyl ester, both the ROS generation and the declined GSH/GSSG were attenuated, and the maturation characteristics were all suppressed. These findings suggest that the LPS-induced ROS generation and the concomitant decline in GSH/GSSG occur in human monocyte-derived DC and that the former is involved in cytokine production, while the latter is involved in the up-regulation of cell surface molecules and allo-stimulatory capacity. Since the cytokine production and the allo-stimulatory capacity of DC play an important role in inflammatory and immune responses, differential regulation of the ROS generation and the declined GSH/GSSG may be useful as therapeutic tools in diseases where both responses become entangled, such as sepsis and graft-versus-host disease.

Alterations in glutathione levels of brain structures caused by acute restraint stress and by nitric oxide synthase inhibition but not by intraspecific agonistic interaction

Glutathione is the major non-protein thiol to which many different roles in the central nervous system (CNS) are attributed. To further investigate the glutathione response in the CNS, we tested the effect of three stress models on glutathione levels in the brain. We tested the effect of two models of repeated intraspecific agonistic interaction in mice. No influence was observed over the glutathione levels in the mice cerebral cortex, cerebellum, liver, and blood. Acute restraint stress in rats was found to induce an increase in glutathione levels in the cerebellum after 2 and 4 h of immobilization, an effect not observed in the cerebral cortex, striatum, and hippocampus. To investigate the interference of an inhibitor of nitric oxide synthase (NOS), N ωNitro- l-arginine-methyl-ester ( l-NAME, 50 mg/kg) was applied i.p. at the beginning of restraint stress. l-NAME alone did not lead to a change in glutathione levels although, in combination with restraint stress, it induced an increase in such levels. This effect was observed in all four structures studied, i.e. cortex, hippocampus, striatum, and cerebellum. The values returned to basal levels after 6 h of immobilization. In conclusion, the pattern of dominance, after repeated intraspecific agonistic interaction, was ineffective in producing alterations in brain glutathione, whereas acute restraint stress led to an increase in glutathione levels within a window of 2–4 h, and the inhibition of NOS increased glutathione levels in all studied rat brain structures, suggesting a specificity interference of acute restraint stress with the glutathione system.

Safety and Efficacy of Weekly Oral Oltipraz in Chronic Smokers

Cigarette smoking is thought to contribute to carcinogenesis by formation of DNA adducts of tobacco smoke constituents leading to genotoxic damage. The dithiolethione, oltipraz, is a putative cancer chemopreventive agent that induces phase II detoxifying enzymes in preclinical models and reduces aflatoxin adducts in humans living in areas with high dietary levels. To determine if oltipraz could reduce adduct levels of tobacco smoke constituents in the lungs and other target organs, chronic smokers were enrolled to one of three arms: 400 or 200 mg/wk oral oltipraz or placebo. Endobronchial tissue and bronchoalveolar lavage were done before and after 12 weeks of drug treatment; peripheral blood, urine, and oral saline rinse were also collected. Toxicity was assessed every 4 weeks. Fifty-nine of the 77 enrolled subjects completed the study. Of those receiving oltipraz, 15% experienced grade 2/3 toxicity, which was predominantly gastrointestinal. All subject withdrawals occurred in the oltipraz groups. There was no significant difference between pre- and post-polycyclic aromatic hydrocarbon-DNA adduct levels in lung epithelial cells measured by immunoperoxidase staining between treatment and placebo groups. Likewise, no significant differences were found in polycyclic aromatic hydrocarbon or benzo(a)pyrene-7,8-diol-9,10-epoxide adducts measured in blood, oral lining cells, or bladder lining cells. There was also no increase in mRNA or enzymatic activity of phase II enzymes and no change in glutathione levels. Thus, despite moderate drug-related toxicity, there was no significant effect on pharmacodynamic or surrogate risk biomarkers. Other agents with lower toxicity and greater activity to induce phase II enzymes are needed to definitively test the detoxification-induction paradigm in smokers.

Changes in endogenous tissue glutathione level in relation to murine ascites tumor growth and the anticancer activity of cisplatin.

Changes in glutathione levels were determined in tissues of 11- to 12-week-old Swiss albino mice at different stages of Dalton's lymphoma tumor growth and following cisplatin (8 mg/kg body weight, ip) treatment for 24-96 h, keeping 4-5 animals in each experimental group. Glutathione levels increased in spleen of tumor-bearing compared to normal mice (9.95 +/- 0.14 vs 7.86 +/- 1.64 micromol/g wet weight, P<or=0.05) but decreased in blood (0.64 +/- 0.10 vs 0.85 +/- 0.09 mg/ml) and testes (9.28 +/- 0.15 vs 10.16 +/- 0.28 micromol/g wet weight, P<or=0.05). Dalton's lymphoma cells showed an increase in glutathione concentration (4.43 +/- 0.26 micromol/g wet weight) as compared to splenocytes, their normal counterpart (3.62 +/- 0.41 micromol/g wet weight). With the progression of tumor in mice, glutathione levels decreased significantly in testes (approximately 10%) and bone marrow cells (approximately 13%) while they increased in Dalton's lymphoma cells (28-46%) and spleen (15-27%). Glutathione levels in kidney, Dalton's lymphoma cells and bone marrow cells (8.50 +/- 1.22, 4.43 +/- 0.26 and 3.28 +/- 0.17 micromol/g wet weight, respectively) decreased significantly (6.04 +/- 0.42, 3.51 +/- 0.32 and 2.17 +/- 0.14 micro mol/g wet weight, P<or=0.05) after in vivo cisplatin treatment for 24 h. Along with a decrease in glutathione level, the glutathione-S-transferase (GST) activity also decreased by 60% in tumor cells after cisplatin treatment. The elevated drug uptake by the tumor cells under the conditions of reduced glutathione concentration and GST activity after treatment could be an important contributory factor to cisplatin's anticancer activity leading to tumor regression. Furthermore, lower doses of cisplatin in combination with buthionine sulfoximine (an inhibitor of glutathione synthesis) may be useful in cancer chemotherapy with decreased toxicity in the host.

Dihydroisotanshinone I protects against menadione-induced toxicity in a primary culture of rat hepatocytes.

Dihydroisotanshinone I is a phenanthrenequinone derivative isolated from the roots of Salvia trijuga Diels. The present study demonstrated the hepatoprotective effect of dihydroisotanshinone I against menadione-induced cytotoxicity in a primary culture of rat hepatocytes. Pretreating the cells with dihydroisotanshinone I at concentrations ranging from 2.5 microM to 20 microM for 24 hours caused dose-dependent protection against hepatotoxicity induced by menadione. Intracellular glutathione level and activity of DT-diaphorase have been suggested to play important roles in menadione-induced cytotoxicity. However, treating the hepatocytes with 20 microM dihydroisotanshinone I for 24 hours did not cause a significant change in glutathione level and DT-diaphorase activity. On the contrary, adding dihydroisotanshinone I to freshly isolated hepatocytes at concentrations between 50 nM to 200 nM inhibited NADH-induced superoxide production dose-dependently as indicated by the decrease of lucigenin-amplified chemiluminescence. In addition, dihydroisotanshinone I at concentrations ranging from 5 microM to 20 microM inhibited tert-butyl hydroperoxide-induced lipid peroxidation dose-dependently in isolated hepatocytes as indicated by the level of malondialdehyde. These results suggest that the protective action of dihydroisotanshinone I against menadione-induced hepatotoxicity is attributed to its antioxidant properties including the free radical scavenging activity and inhibition of lipid peroxidation. Abbreviations. DTD:DT-diaphorase GSH:glutathione LDH:lactate dehydrogenase MDA:malondialdehyde MTT:3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide TBHP: tert-butyl hydroperoxide

Differential accumulation of cadmium and changes in glutathione levels as a function of symbiotic state in the sea anemone Anthopleura elegantissima

Coastal ecosystems are increasingly impacted by anthropogenic activities and contaminant inputs, including heavy metals. This paper investigates the responses to laboratory cadmium exposures in the North American Pacific coast temperate sea anemone Anthopleura elegantissima. Anemones were exposed to cadmium chloride (control, 20 and 100 μg l −1 Cd) for 14 days (sampled at days 0, 2, 7 and 14) and analyzed for Cd content using inductively coupled plasma mass spectroscopy (ICP-MS). Higher levels of Cd were demonstrated in symbiotic anemones compared to symbiont-free (aposymbiotic) anemones (e.g. 100 μg l −1 Cd exposure at day 14; 12.4±2.4 and 4.5±1.9 ng Cd mg −1 dry weight, respectively; t-test; p<0.001). These higher levels in symbiotic anemones were not due to increasing uptake of Cd by the algal symbionts (levels in algal-containing pellet fractions were constant over dose and time; t-test; p>0.05) but rather to increased accumulation by both partners. No changes in algal cell density were observed in symbiotic anemone tentacle clips at any dose or time point (ANOVA; p>0.05). Changes in glutathione (GSH) levels have been proposed as a useful biomarker in other marine invertebrates in response to heavy metal stress, but GSH studies have not been reported for this species. We demonstrate the presence of GSH in A. elegantissima and its depletion (58.2% of control values) following 1-week exposure to buthionine sulfoximine (BSO), a nontoxic chemical known to deplete GSH. Levels of GSH were higher in symbiotic compared to aposymbiotic anemones (control averages, 563±81 and 485±58 nmol GSH mg −1 wet weight, respectively; t-test; p<0.001). Following Cd exposure, significant reductions ( t-test; p<0.05) in GSH levels were observed only in the aposymbiotic anemones (exposure to 100 μg l −1 Cd resulted in a 27% reduction from control levels at day 14). Our results demonstrate that the accumulation of Cd depends upon symbiotic state and provides the first identification of a GSH response in anemones and indeed in any cnidarian in response to heavy metals.

Roles of endogenous glutathione levels on 6-hydroxydopamine-induced apoptotic neuronal cell death in human neuroblastoma SK-N-SH cells

We investigated the roles of endogenous glutathione on 6-hydroxydopamine (6-OHDA)-induced apoptosis in human neuroblastoma SK-N-SH cells using DNA fragmentation enzyme-immunoassay and the DNA dye Hoechst 33,258 staining. We observed that exogenous reduced glutathione (GSH), but not oxidized glutathione (GSSG), protected 6-OHDA (25 μM)-induced apoptosis in a dose-dependent manner. Preincubation (18 h) with the glutathione synthesis inhibitor DL-buthionine-( S, R)-sulfoximine (BSO) significantly potentiated the toxic effects of 6-OHDA (12.5 or 25 μM). In contrast to BSO, N-acetylcysteine (NAC) blocked, and L-(-)-cystine, the glutathione precursor, significantly attenuated 6-OHDA (25 μM)-induced apoptosis, respectively. No alterations in endogenous glutathione concentrations were detected at 5, 15, 30, 60 min, 1 hour, 3 hours, or 6 hours after 6-OHDA (25 μM) treatment. However, we found a 3.5-fold increase of intracellular glutathione levels 24 hours later. On the contrary, higher concentration (100 μM) of 6-OHDA treatment, which caused more severe cell death, showed no changes of glutathione levels. These results suggest that delayed induction of endogenous glutathione might play an important role in the neuroprotective mechanism against dopamine cell death. In addition, we found that NAC might work as a beneficial catecholaminergic neuron-survival factor more efficiently than exogenous glutathione or L-cystine.

Role of glutathione in the response of Escherichia coli to osmotic stress.

The growth of Escherichia coli mutants deficient in glutathione synthesis (gshA) and in glutathione reductase (gor) was suppressed in medium of elevated osmolarity. A mutant in gamma-glutamyl transpeptidase (ggt) displayed better ability for osmoadaptation than the parental strain. The unfavorable effect of the gsh mutation on osmoadaptation of growing E. coli cells was more pronounced at low concentrations of K+ in the medium. An increase in osmolarity caused an increase in the intracellular content of glutathione. Changes in the extracellular glutathione level were biphasic: the glutathione level rapidly decreased during the first stage of the response and increased during the second stage. The changes in glutathione levels suggest that under hyperosmotic shock the glutathione transport from the medium into the cell can contribute to the intracellular glutathione accumulation. Changes in the level of intracellular K+ were similarly biphasic: a rapid increase in the K+ level during the first stage of the response to hyperosmotic shock changed to a gradual decrease during the second stage. In mutant gshA cells adapted to osmotic shock, the intracellular K+ level was markedly higher than in the parental strain cells. The possible role of glutathione in the response of E. coli to osmotic shock is discussed.

The effect of eucalyptus oils on the erythrocytes of koalas

The oxidant effects of two fractions of eucalyptus oil; monoterpenes (C10) and the larger sesquiterpenes (C15), were investigated in the erythrocytes of the koala (Phascolarctos cinereus). The effects studied included the degree of haemolysis and changes in the intracellular levels of glutathione (GSH) and adenosine triphosphate (ATP) and in the membrane phospholipids. The results indicate that koala erythrocytes are susceptible to eucalyptus oil-induced oxidative damage. The two types of eucalyptus oils have different effects on the erythrocytes; monoterpenes appear to induce haemolysis through oxidative damage to the intracellular constituents, whereas sesquiterpenes may attack the red cell membrane.

Glutathione modulates rat and mouse hepatocyte sensitivity to tumor necrosis factor α toxicity

Background & Aims: Tumor necrosis factor (TNF)-α causes much of the hepatocellular injury and cell death that follows toxin-induced liver damage. The mechanism by which toxic liver injury sensitizes hepatocytes to TNF-α cytotoxicity is unknown. The aim of this study was to determine the role of the antioxidant glutathione in this process. Methods: A rat hepatocyte cell line and primary hepatocytes sensitized to TNF-α toxicity by the addition of actinomycin D were examined for changes in glutathione levels and for the effects of glutathione depletion or supplementation on cell death. The in vivo effects of glutathione depletion were determined in mice treated with galactosamine plus lipopolysaccharide. Results: Treatment of hepatocytes with actinomycin D and TNF-α induced apoptotic cell death without affecting cellular glutathione levels or production of the reactive oxygen intermediate H 2O 2. Glutathione depletion induced by diethyl maleic acid significantly increased TNF-α–induced cell death even when this agent was administered 2 hours after TNF-α treatment. Hepatocyte cell death was not affected by glutathione supplementation. In mice treated with galactosamine plus lipopolysaccharide, glutathione depletion increased mortality from liver injury from 32% to 72%. Conclusions: TNF-α–induced cytotoxicity in hepatocytes occurs in the absence of glutathione depletion. However, a preexisting reduction in glutathione levels can significantly increase cell death from TNF-α. GASTROENTEROLOGY 1998;115:1229-1237

Toxicity of fumonisin B 1 to B6C3F 1 mice: A 14-day gavage study

Fumonisin B 1 (FB 1) is a fungal toxin produced by members of the genus Fusarium. Ingestion of FB 1 causes species-specific neurotoxic, nephrotoxic, hepatotoxic and pulmonary effects. The clinical, haematological and pathological responses of adult male and female B6C3F 1 mice to FB 1 were assessed following 14 daily gavage doses ranging from 1 to 75 mg FB 1/kg body weight/day. There were no consistent sex-related changes. Although all responses were modest, the most notable effects of FB 1 were on the liver, bone marrow, adrenals and kidneys. In the liver, hepatocellular single cell necrosis, mitosis and anisokaryosis were observed, accompanied by elevated serum ALT. In the kidneys, minor histopathological changes were confined to female mice, while mild decreases in ion transport and increases in blood urea nitrogen were seen only in males. Small changes in glutathione levels were observed in the kidneys and livers of male mice. Adrenal cortical cell vacuolation was observed at 15 mg FB 1/kg and higher in females and from 35 mg FB 1/kg in males. Serum cholesterol was elevated in both male and female mice, possibly due to FB 1-induced changes in lipid metabolism in the liver and adrenals. Although bone marrow cell numbers were unchanged, increases in vacuolated myeloid cells and lymphocytes were observed in female mice. In general, the degree of changes observed indicate that mice are not as sensitive a model of FB 1 toxicity as rats.

Comparison of Lens Biochemistry and Structure between BSO-treated and Glucocorticoid-treated Developing Chick Embryos

In this paper various changes in glutathione level, which were influenced by balance of its synthesis, degradation, transport and utilization, were analysed in chick embryos administered with glucocorticoid (GC) or buthionine sulfoximine (BSO; an inhibitor of glutathione synthesis). When BSO (30 μmol egg −1) was administered twice to chick embryos on day 14 and 15, the GSH in both the lens and the liver decreased to 15–20% and 30–40% of the age-matched control level, respectively, between 24 and 48 hr after the second treatment, then began to recover. Although this decline in the GSH level in these tissues was greater and more prolonged in embryos treated with BSO than with GC, the former embryos maintained lens transparency even up to 144 hr by a visual examination. However, histological changes in the lens occurred after 96 hr and more significantly 144 hr after second administration of BSO. The changes mainly consisted of pale epithelial cells on the anterior peripheral surface of the lens, irregular height of the epithelial cells at the equator, clefts between the epithelium and the cortex and swelling of almost all the cortical fibers. These observations may suggest that BSO treatment could produce the beginning of a cataract. Embryos with GC-cataract revealed the following changes at 48 hr: loss of transparency, elevation of LPO (TBA-reacting substance) in the lens, the blood and the liver. These were not observed in BSO-treated embryos during the experimental period. The GC-cataract may well depend on the generation of LPO. BSO cataract, having a distinct mechanism compared to that caused by GC, develops more slowly in GSH-depleted lenses. The BSO-treated chick embryos will be a useful model to screen the risk factors which accelerate cataract formation.

Cytotoxicity of Benzo[c]phenanthridinium Alkaloids in Isolated Rat Hepatocytes

Sanguinarine, chelerythrine and fagaronine were examined for hepatotoxic effect. Administration of sanguinarine and chelerythrine (10 mg/kg/day; i.p.) produced marked parenchymal injury not found in the livers of fagaronine treated rats. In chronic administration of sanguinarine (0.2 mg/kg/56 days; i.p.) degeneration and necrosis were not observed in the liver. The effect of the alkaloids was tested by enzyme leakage (LDH and ASAT) and changes in glutathione levels in isolated rat hepatocytes. Sanguinarine produced the greatest toxicity followed by lesser toxicity in the case of chelerythrine while no toxicity was displayed with fagaronine. These effects correlate with pK R values for pseudobase formation. Finally, no increased lipid peroxidation or mitochondrial membrane damage were found.

Cytotoxicity of Benzo[c]phenanthridinium Alkaloids in Isolated Rat Hepatocytes

Sanguinarine, chelerythrine and fagaronine were examined for hepatotoxic effect. Administration of sanguinarine and chelerythrine (10 mg/kg/day; i.p.) produced marked parenchymal injury not found in the livers of fagaronine treated rats. In chronic administration of sanguinarine (0.2 mg/kg/56 days; i.p.) degeneration and necrosis were not observed in the liver. The effect of the alkaloids was tested by enzyme leakage (LDH and ASAT) and changes in glutathione levels in isolated rat hepatocytes. Sanguinarine produced the greatest toxicity followed by lesser toxicity in the case of chelerythrine while no toxicity was displayed with fagaronine. These effects correlate with pKR+ values for pseudobase formation. Finally, no increased lipid peroxidation or mitochondrial membrane damage were found.

Induction of phytochelatins in Hydrilla verticillata (l.f.) royle under cadmium stress.

Plants tolerate Cd by sequestering them through synthesizing phytochelatins with the general structure (t-Glu-cys)n-gly where n= 2-11 depending upon the species from which these peptides are isolated. Recent biochemical evidence suggests that these peptides are synthesized via posttranslationally activated, metal-dependent enzymatic pathways from the precursor glutathione. However, most of these studies are confined to terrestrial species and only a few studies have been made on higher aquatic plants. Recently H. verticillata and other aquatic higher plants have been reported to be hyperaccumulators of Cd and have demonstrated the ability to remove many toxic metals, including Cd, from wastewater. It is hypothesized that cadmium hyperaccumulating ability of the macrophyte is associated with induction of the metal chelating peptides, the phytochelatins (PCs), to copeup with high cellular Cd levels. In view of this, it was considered worthwhile to examine the induction of phytochelatins and changes in levels of glutathione and related metabolites in H. verticillata under Cd stress.