Buthionine Sulphoximine Research Articles

Relationship between melanogenesis, glutathione levels and melphalan toxicity in human melanoma cells.

Resistance to alkylating agents has been correlated with cellular levels of reduced glutathione (GSH) and glutathione-S-transferase (GST). GSH is also involved in regulation of melanin synthesis. Therefore, we examined sensitivity to melphalan as a function of differentiation and GSH/GST levels in three human melanoma cell lines. The Me8 cell line, classified as undifferentiated on the basis of cell shape, absence of pigment, insignificant dopa oxidase activity and presence of inhibitors of dopa-melanin formation, showed the lowest GST activity among the cell lines investigated. GLL19 cells exhibited normal differentiation as indicated by the presence of dendrites, typical eumelanosomes, melanin granules and dopa oxidase activity. These cells showed the highest GSH content and the highest GST activity. The JUSO cell line showed incomplete differentiation, and its dopa oxidase and GST activities were intermediate between the Me8 and GLL19 cell lines. The sensitivity of melanoma cell lines to melphalan increased with their degree of differentiation; it was lowest for Me8, intermediate for JUSO and highest for GLL19. Dibutyryl cyclic AMP (dbcAMP) enhanced melphalan toxicity against Me8 cells. Depletion of intracellular GSH with buthionine sulphoximine (BSO) resulted in a three-fold increase in melphalan sensitivity in all three cell lines. Our results indicate that melphalan toxicity is related to cell differentiation and GSH status of melanoma cells. Based on the observed relationship between dopa oxidase, GSH/GST levels and drug toxicity, it is proposed that competition for the GSH pool between quinonoid melanin intermediates and melphalan could diminish drug conjugation and increase cytotoxicity.

Relationships between thermotolerance, oxidative stress responses and induction of stress proteins in human tumour cell lines

Thermotolerance, resistance to oxidative stress and induction of stress proteins were examined in a panel of 10 human tumour cell lines. An inverse relationship was indicated between intrinsic thermotolerance (cell survival after treatment at 43.5°r 3hr) and thermotolerance induced by pretreatment at 42.5° 30 min. Similar levels of induction of hsp 70 were found in cell lines with high or low levels of intrinsic thermotolerance; induction of other stress proteins could not be detected. Cell survival following treatment with H 2O 2correlated with that following streptonigrin treatment (P < 0.05). Pretreatment with buthionine sulphoximine or diamide synergistically increased the toxicity of heat, H 2O 2 and streptonigrin whereas reduced glutathione had the reverse effect. No direct correlation was found, however, between tolerance to heat and to oxidative stress, and hsp 70 was not induced by the latter. The stress protein heme oxygenase, detected by immunoblotting with the monoclonal antibody HO, was induced by H 2O 2 in melanoma cell lines but not in HeLa. Cadmium and arsenite ions, however, readily induced heme oxygenase in HeLa, indicating that in these cells induction of heme oxygenase by oxidative stress involves a different mechanism. Overall, the results suggest that tolerance to heat or oxidative stress in these cell lines may not necessarily be associated with the induction of heat shock proteins or heme oxygenase but that cell survival after both types of stress depends to a certain extent on cellular sulphydryls.

Evidence against a role for phytochelatins in naturally selected increased cadmium tolerance in Silene vulgaris (Moench) Garcke

summaryThe production of phytochelatins in roots of both cadmium–sensitive and cadmium–tolerant plants of Silene vulgaris (Moench) Garcke in response to Cd supply was recorded over a 7–day growth period. At the same level of Cd, sensitive plants produced more phytochelatins than tolerant ones. In sensitive roots this higher production of PCs was associated with a higher Cd content within one day. From the third day, however, Cd contents were similar, but the difference in PC production persisted. Exposure to buthionine sulphoximine strongly inhibited the production of phytochelatins in roots during cadmium treatment in both sensitive and tolerant plants, but only affected root growth of sensitive plants. Sensitive plants exhibited a higher Cd uptake, which was manifested in a higher Cd accumulation in the shoot. It is concluded that differential cadmium tolerance mechanism in Silene vulgaris is not due to differential production of phytochelatins.

Cytoskeletal injury induced by hexavalent chromate

To understand the mechanisms of toxicity of hexavalent chromate (K 2CrO 4, Cr 6+), the effects of this metal ion on the organization of microtubules (MTs) and microfilaments (MFs), DNA synthesis, cytoskeletal protein synthesis, cytoskeletal protein sulphhydryls (-SH groups), and cellular glutathione (GSH) levels in 3T3 cells were examined. Exposure of cells to Cr 6+ for 16 hr resulted in a dose-dependent inhibition of DNA synthesis with 50% inhibition occurring at 16.6 μ m. Treatment of cells with 3.13 μ m-Cr 6+ for 16 hr resulted in a slight cell retraction and, in some cells, MT bundling, without much effect on the morphology of dense MFs. At 25 μ m, Cr 6+ caused disruption of the cell sheet, depolymerization of MTs, particularly those in the peripheral areas, and redistribution of MFs, which assumed a smearing morphology. Exposure to 100 μ m-Cr 6+ induced severe thinning of MTs and loss of MFs. Although doses of 3.13 μ m-Cr 6+ or less slightly enhanced the level of cytoskeletal protein synthesis (e.g. 38% increase at 3.13 μ m-Cr 6+), Cr 6+ at 6.25 μ m or more produced a dose-dependent inhibition of cytoskeletal protein synthesis (50% inhibition at 11.25 μ m). Exposure of cells to Cr 6+ for 16 hr resulted in a dose- and time-dependent increase of cellular GSH level. Furthermore, the elevated cellular GSH induced by Cr 6+ was diminished by treatment with buthionine sulphoximine (BSO), a potent inhibitor of GSH biosynthesis. In addition, depletion of GSH by BSO increased cell sensitivity to Cr 6+ insult and aggravated Cr 6+-induced cytoskeletal perturbation. However, Cr 6+ treatment of cells did not significantly affect the amount of cytoskeletal protein sulphhydryls. These results suggest that cytoskeletal injury may be an important part of the mechanism for Cr 6+ toxicity. The cytoskeletal damage may result directly from the inhibition of cytoskeletal protein synthesis rather than from the interaction between Cr 6+ and cytoskeletal protein sulphhydryls.

Relationship between glutathione content in liver and glutathione conjugation rate in the rat in vivo. Effect of buthionine sulphoximine pretreatment on conjugation of the two 2-bromoisovalerylurea enantiomers during intravenous infusion.

The relationship between hepatic glutathione content and hepatic glutathione conjugation rate in the rat in vivo was investigated. As substrate for glutathione conjugation, racemic (R,S)-2-bromoisovalerylurea (BIU) was used which gives rise to the biliary excretion of two diastereoisomeric glutathione conjugates and the urinary excretion of two diastereoisomeric mercapturates. The excretion rate of the glutathione conjugate in bile reflects hepatic conjugation exclusively. An intravenous infusion of BIU was given and the excretion rates of the metabolites in bile and urine were determined. The glutathione concentration in the liver was followed by taking biopsies every hour. Glutathione was depleted by the infused substrate; in rats that were pretreated with the inhibitor of glutathione biosynthesis, buthionine sulphoximine (BSO), the depletion of the glutathione content was more rapid. The rate of excretion of the glutathione conjugate in bile was plotted against hepatic glutathione content. These results indicate that the 'organ Km' for glutathione in the liver is approximately 0.5 mumol/g of liver, so that the hepatic glutathione conjugation rate is decreased only at severe glutathione depletion.

Influence of modulators of epoxide metabolism on the cytotoxicity of trans-anethole in freshly isolated rat hepatocytes

The effect of modulating epoxide metabolism by inhibiting microsomal and cytosolic epoxide hydrolases and depleting glutathione, on the cytotoxicity of trans-anethole has been examined in freshly isolated rat hepatocytes in suspension. Hepatocytes derived from female Sprague-Dawley CD rats by collagenase perfusion were incubated in suspension and sampled at intervals over a 6-hr period. Cytotoxicity was assessed by the leakage of lactate dehydrogenase into the culture medium and in the cells after lysis. Glutathione was determined by fluorimetry. Anethole showed a dose-dependent cytotoxicity at concentrations ranging from 5 × 10 −4 to 5 × 10 −3 m, with concentrations of 10 −3 m and above causing greater than 63% leakage of lactate dehydrogenase in 6 hr. Microsomal epoxide hydrolase was inhibited by trichloropropene oxide (10 −4 m) and cyclohexene oxide (10 −3 m), and cytosolic epoxide hydrolase by 4-fluorochalcone oxide (5 × 10 −4 m). Cellular glutathione was depleted by diethyl maleate (5 × 10 −4 m), and its synthesis inhibited by 2.5 × 10 −3 m-l-buthionine ( S, R)-sulphoximine. Suspensions treated with a sub-cytotoxic concentration of anethole (5 × 10 −4 m) showed a rapid increase in cytotoxicity when 4-fluorochalcone oxide was present (complete loss of viability within 2 hr), while pretreatment of hepatocytes with diethyl malcate in combination with buthionine sulphoximine, to deplete glutathione, slowly increased the cytotoxic response at later times (after 4 hr of incubation). The association of the effects of 4-fluorochalcone oxide with the inhibition of cytosolic epoxide hydrolase is strengthened by the inability of chalcone oxide, a close structural analogue of 4-fluorochalcone oxide, which has no effect on epoxide hydrolase or glutathione conjugation, to influence the effects of anethole on hepatocytes. These data are discussed in terms of the role of anethole epoxide in the cytotoxicity of trans-anethole.

Comparison of the oxidant damage induced by phosphine and the uptake and tracheal exchange of 32P-radiolabelled phosphine in the susceptible and resistant strains of Rhyzopertha dominica (F.) (Coleoptera: Bostrychidae)

The activity of antioxidant enzymes and the effects of phosphine on them were compared in a susceptible and two resistant strains of Rhyzopertha dominica (F.). Phosphine treatment lowered the activity of catalase and peroxidase and raised the activity of superoxide dismutase and the content of lipid peroxides in the susceptible insects. The generation of toxic oxyradical species appeared to be taking place in insects as a consequence of the interaction of phosphine with the mitochondrial respiration. Compared to the extent of oxidant damage in the susceptible insects, phosphine treatment caused only minor effects in a laboratory-selected strain while it produced no significant effects in a highly resistant field strain, even when treated at higher doses. The dietary administration of buthionine sulphoximine (BSO) lowered the content of glutathione in the resistant insects but caused no appreciable change in the susceptible strain. There were no appreciable effects on the susceptibility of BSO-treated strains to phosphine or on the uptake of 32P when treated with 32P-radiolabelled phosphine. In a “pulse-chase” style experiment, the resistant insects showed a much lower uptake of 32P and a significantly greater exchange of gaseous [ 32P]PH 3 compared to the susceptible strain. The mechanisms of insect resistance to phosphine are discussed in the light of these results.

Regulation of total ovarian glutathione content in the rat

Glutathione (GSH) is an important intracellular thiol capable of altering metabolism following exposure to certain important biologic toxicants including radiation and cyclophosphamide. In order to evaluate the inhibition of glutathione synthesis in the ovary, 30-day-old Sprague Dawley rats were treated with either saline or 0.6 μmol/kg (0.133 mg/kg), 6.0 μmol/kg (1.33 mg/kg), or 4.5 mmol/kg (1000 mg/kg) buthionine sulphoximine (BSO) IP and sacrificed at 0, 1, 2, 4, 6, 8, and 24 h. There was an inhibition of glutathione synthesis with 4.5 mmol/kg (1000 mg/kg) BSO with a nadir at 8 h ( P < 0.001) and complete recovery at 24 h. In the subsequent experiments rats were divided into four groups. All animals received either saline or BSO 4.5 mmol/kg/day (1000 mg/kg/day) from day 27 to 30 of life and either saline or PMSG 5 IU IP on day 29 of life. BSO reduced ovarian content of GSH (saline-saline compared with BSO-saline, P<0.0001), which was countered by the prior administration of PMSG (BSO-saline compared with BSO-PMSG, P<0.005). Glutathione levels were as follows: saline-saline 4.3 ± 0.04; saline-PMSG 5.0 ± 0.4; BSO-saline 2.13 ± 0.2 ; BSO-PMSG 3.24 ± 0.2 nmol/mg ovary. These findings suggest the ovary is susceptible to GSH depletion by in vivo administration of BSO. Gonadotropin (PMSG) is capable of effecting a partial return of total ovarian GSH content.

The role of non-protein sulphydryls in determining the chemical repair rates of free radical precursors of DNA damage and cell killing in Chinese hamster V79 cells.

Chinese hamster V79 fibroblasts were irradiated in the gas explosion apparatus and the chemical repair rates of the oxygen-dependent free radical precursors of DNA double-strand breaks (dsb) and lethal lesions measured using filter elution (pH 9.6) and a clonogenic assay. Depletion of cellular GSH levels, from 4.16 fmol/cell to 0.05 fmol/cell, by treatment with buthionine sulphoximine (50 mumol dm-3; 18 h), led to sensitization as regards DNA dsb induction and cell killing. This was evident at all time settings but was particularly pronounced when the oxygen shot was given 1 ms after the irradiation pulse. A detailed analysis of the chemical repair kinetics showed that depletion of GSH led to a reduction in the first-order rate constant for dsb precursors from 385 s-1 to 144 s-1, and for lethal lesion precursors from 533 s-1 to 165 s-1. This is generally consistent with the role of GSH in the repair-fixation model of radiation damage at the critical DNA lesions. However, the reduction in chemical repair rate was not proportional to the severe thiol depletion (down to approximately 1% for GSH) and a residual repair capacity remained (approximately 30%). This was found not to be due to compartmentalization of residual GSH in the nucleus, as the repair rate for dsb precursors in isolated nuclei, washed virtually free of GSH, was identical to that found in GSH-depleted cells (144 s-1), also the OER remained substantially above unity. This suggests that other reducing agents may have a role to play in the chemical repair of oxygen-dependent damage. One possible candidate is the significant level of protein sulphydryls present in isolated nuclei.

Toxicity of coumarin and various methyl derivatives in cultures of rat hepatocytes and V79 cells

The toxicity of coumarin and various simple methyl derivatives in rat hepatocyte and V79 cell cultures was studied to investigate further the mechanism of coumarin hepatotoxicity. Coumarin was six times more toxic in hepatocyte cultures from phenobarbitone (PB)-treated rats than in those from untreated rats. At concentrations below 3 m m, coumarin did not affect the survival of V79 lung fibroblasts. SKF-525A inhibited coumarin-induced toxicity in hepatocytes cultured from PB-treated rats, whereas depletion of hepatocyte glutathione (GSH) levels with buthionine sulphoximine (BSO) significantly increased toxicity. Dihydrocoumarin (DHC) had little effect on the survival of cultured hepatocytes, indicating that the 3,4-double bond is an important determinant of coumarin toxicity. In general, the toxicity of coumarin in hepatocyte cultures was reduced by substitution with one or more methyl groups. 3-Methylcoumarin (MeC), however, was more toxic than coumarin itself in hepatocyte cultures from untreated rats. Except for 3,4-diMeC, the methyl derivatives were markedly more toxic in rat hepatocytes than in V79 cell cultures. The data obtained for coumarin and 4-MeC, and possibly 6-MeC and 7-MeC, are consistent with hepatocyte toxicity being due to the cytochrome P-450-dependent formation of one or more toxic metabolites that may be detoxified by reacting with GSH. This was less apparent for 3-MeC and 3,4-diMeC, although depletion of GSH levels significantly increased the hepatocyte toxicity of both compounds.

Caffeic acid causes metal-dependent damage to cellular and isolated DNA through H2O2 formation.

Pulsed field gel electrophoresis showed that caffeic acid induced DNA strand breaks in cultured human cells in the presence of Mn(II). With alkali treatment, DNA single-strand breaks were observed. The strand breakage was increased by the treatment of buthionine sulphoximine (a GSH synthesis inhibitor) and 3-aminotriazol (a catalase inhibitor) and decreased by catalase, indicating the involvement of H2O2. The DNA damage was decreased by o-phenanthroline, indicating the involvement of transition metal ion. Damage to isolated DNA from c-Ha-ras-1 protooncogene was investigated by a DNA sequencing technique. Caffeic acid caused DNA damage in the presence of Cu(II) but not in the presence of either Mn(II) or Fe(III). Caffeic acid plus Cu(II) induced piperidine-labile sites frequently at thymine residues, especially of the 5'-GTC-3' and 5'-CTG-3' sequences. Typical OH scavengers showed no inhibitory effects. The inhibitory effects of bathocuproine and catalase on Cu(II)-mediated DNA damage suggest that Cu(I) and H2O2 have important roles in the production of active species causing DNA damage. The Cu(II)-mediated DNA damage was enhanced by pre-incubation of caffeic acid with Mn(II). Mn(II)- or Cu(II)-catalyzed autoxidation of caffeic acid produced H2O2 with efficiency of Mn(II) greater than Cu(II). These results suggest that in the presence of Mn(II) or Cu(II), caffeic acid produces H2O2, which is activated by transition metals to cause damage to DNA in vitro and probably in cultured cells.

Glutathione content of human malignant melanoma cell lines

We measured the glutathione content of a panel of human malignant melanoma cell lines by flow cytometry after staining with mercury orange, using visible light (488 nm) excitation, and compared the values obtained with those measured biochemically using a modified Tietze assay. Glutathione levels were depleted by culturing cells with various concentrations of buthionine sulphoximine to provide a suitable spread of glutathione concentrations. The two assays showed good correlation (r = 0.93). We found a number of technical factors to be critically important. In particular, the conditions of staining, cell number, and method of mixing media, cells and stain were responsible for wide variations in fluorescence intensity. We applied the flow cytometric technique to cell suspensions obtained by fine needle aspiration biopsy of human malignant melanoma deposits, and observed a proportion of cells with high glutathione levels in many samples. The results confirm the feasibility of measuring glutathione content by visible light flow cytometry, and raise the possibility of monitoring glutathione content as an indicator of drug resistance in clinical therapy of human malignant melanoma.

Non-myelotoxic antitumour effects of L-dopa, buthionine sulphoximine and tamoxifen on neuroblastoma cells in vitro and in vivo.

The effects of three non-myelotoxic cancer drugs on the growth of neuroblastoma cells were investigated in vitro and in vivo: dihydroxyphenylalanine (L-dopa, a drug with selective toxicity for melanoma cells), DL-buthionine sulphoximine (BSO, a drug with radiosensitizing effects), and tamoxifen (a drug used in the treatment of human mammary carcinoma). In vivo these substances significantly reduced the weight of neuroblastoma tumour transplants in the mice (nude/nude) (P less than 0.05). A dose/effect relationship could be established. In vitro, the D50 was determined, using fibroblasts as controls. The growth of neuroblastoma tumours was inhibited by different mechanisms: L-dopa and its metabolite dopamine reduced the activity of tyrosinase, BSO reduced glutathione levels, and L-dopa and tamoxifen raised cAMP concentrations.

Mechanism of toxicity of the antimelanoma drug 4-hydroxyanisole in mouse hepatocytes

To elucidate the mechanism of the hepatotoxicity of 4-hydroxyanisole (4-HA), its effect on the viability of mouse hepatocytes in suspension was investigated. Cell viability was assessed by measurement of release of lactate dehydrogenase into the medium. 4-HA was cytotoxic in a concentration-dependent and time-dependent fashion with an IC 50 of 0.26 mmol/l after 4 h incubation. Almost all cells were killed after exposure to 4-HA for 4 h at 0.5 mmol/l or for 2 h at 1.0 mmol/l. At 5 and 10 mmol/l, 4-HA caused less cytotoxicity than 1 mmol/l or below. On coincubation with the P450 inhibitor octylamine, 4-HA cytotoxicity was reduced, which suggests the involvement of cytochrome P450 in the hepatocytotoxicity of this drug. Induction of P450 isoenzymes IA, IIB and IIE1 by pretreatment of mice with phenobarbitone, 3-methylcholanthrene or acetone had no significant effect on the toxicity of 4-HA towards hepatocytes. Depletion of hepatic glutathione by pretreatment of mice with buthionine sulphoximine (1.6 g/kg, intraperitoneally) 4 h before cell isolation led to an increase in 4-HA cytotoxicity. Incubation with N-acetylcysteine (10 mmol/l) abolished the cytotoxicity of 4-HA (1 mmol/l). Both these results are consistent with the intermediacy of a reactive metabolite of 4-HA. Production of hydroquinone by oxidative demethylation of 4-HA as toxication mechanism can be excluded as formation of formaldehyde was not observed on incubation of 4-HA with mouse liver microsomes. 3,4-diacetoxyanisole, a prodrug of the known 4-HA metabolite 3,4-dihydroxyanisole, was not more cytotoxic towards hepatocytes than 4-HA. This result suggests that 3,4-dihydroxyanisole is unlikely to mediate the hepatotoxicity of 4-HA.

The effect of glutathione depletion by buthionine sulphoximine on 1-cyano-3,4-epithiobutane toxicity

The effect of glutathione (GSH) depletion by buthionine sulphoximine (BSO) on the nephrotoxicity and GSH-enhancing effect of the naturally occurring, crucifer-derived nitrile 1-cyano-3,4-epithiobutane (CEB), was investigated. Male Fischer 344 rats were administered 50 or 125 mg CEB/kg body weight by gavage with or without prior ip treatment with 550 mg/kg body weight l-BSO. One group of control animals was treated with water only by gavage, while another group was pretreated with BSO and then given water by gavage. Liver and kidney samples were taken 48 hr after CEB treatment for GSH determinations and histological examination. The high-dose CEB without BSO resulted in increased GSH in liver and kidney, marked karyomegaly in the pars recta of renal proximal tubules and tubular epithelial necrosis, which was limited to a few renal tubules. The low-dose CEB alone resulted in increased hepatic GSH and mild karyomegaly. Pretreatment with BSO abrogated the tubular necrosis and karyomegaly induced by either CEB dose. BSO pretreatment inhibited low-dose CEB-induced GSH enhancement in the liver. The combined BSO and high-dose CEB treatment still resulted in increased hepatic GSH, although the increase was less than that observed with high-dose CEB alone. In the kidney, BSO pretreatment abrogated the high-dose CEB-induced increase in GSH, but GSH content was not significantly different from that with high- or low-dose CEB alone. These results provide evidence that CEB conjugation may be a bioactivation reaction with the conjugate involved in nephrotoxicity. The conjugate may also be involved in increasing renal and hepatic GSH.

Enhancement of SR 2508 (etanidazole) radiosensitization by buthionine sulphoximine at low-dose-rate irradiation.

SR 2508 (etanidazole) (1 mM) or buthionine sulphoximine (BSO, 50 microM) or both drugs together did not radiosensitize oxic V79 Chinese hamster cells irradiated at either an acute dose rate (2.35 Gy/min) or at a low dose rate (0.117 Gy/min). BSO pretreatment (15 h at 37 degrees C) depleted cellular glutathione (GSH) to less than or equal to 1% of control level and radiosensitized hypoxic cells at both dose rates with an enhancement ratio (ER) of 1.2. SR 2508 alone radiosensitized hypoxic cells equally at both dose rates with an ER of 1.5. However, ER values of 2.2 and 2.5 were obtained with 1 mM SR 2508 in GSH-depleted cells at acute and low dose rate, respectively, with no significant difference between the two, i.e. there is no dose rate dependence for this potentiation. Since BSO increases SR 2508 radiosensitization and the combined BSO + SR 2508 treatment is extremely cytotoxic to hypoxic cells, our results suggest that combining BSO with SR 2508 will be useful in brachytherapy as well as external-beam therapy if the toxicity from both drugs in vivo is less than the gain in radiosensitization achieved.

Effects of reserpine and l-cysteine and glutathione depletion on 2-bromoethylamine hydrobromide-induced tubular necrosis in Swiss ICR mice

Female Swiss ICR mice were injected ip with 100 or 300 mg 2-bromoethylamine hydrobromide (BEA)/kg body weight. Male Swiss ICR mice were subjected to water deprivation, or treated with 5% dextrose in water, dimethylsulphoxide, piperonyl butoxide, SKF-525A, sodium phenobarbital, β-naphthoflavone, probenecid, reserpine, diethyl maleate, buthionine sulphoximine or l-cysteine. Urine collected sequentially from male Swiss ICR mice given 300 mg BEA/kg body weight was analysed for glucose, protein, pH and specific gravity. Female mice were less sensitive to BEA than were male mice. Diuresis, antidiuresis, treatment with cytochrome P-450 inducers and inhibitors, and the antioxidant dimethylsulphoxide had no effect on the incidence or severity of tubular necrosis (TN) induced by BEA. Probenecid and l-cysteine decreased the severity, but they had no effect on the incidence of TN. Glutathione depletion by diethyl maleate and inhibition of glutathione synthesis by buthionine sulphoximine decreased the dose of BEA necessary to cause TN; buthionine sulphoximine was more effective than diethyl maleate. Reserpine decreased both the incidence and severity of TN. Glycosuria, aciduria and decreased urinary specific gravity occurred before morphological changes were seen under the microscope, indicating that the functional changes precede the morphological changes. These data indicate that glutathione is important in protecting against BEA-induced TN, that BEA or a metabolite is concentrated in the tubule epithelium by way of anion transport, and that vasoconstriction contributes to the development of BEA-induced TN.

Spatial Characterization of Glutathione Depletion in the KHT Sarcoma Using Flow Cytometry

Intravenous administration of the fluorescent DNA stain Hoechst 33342 to tumour-bearing mice was used to label cells proportionally to their proximity from the vasculature. Flow cytometry was used to sort cells from the tumour into populations based on their Hoechst 33342-derived fluorescence. The cell populations were then assayed for their glutathione (GSH) content and their radiosensitivity. Tumours from mice pretreated with buthionine sulphoximine (BSO) were compared with untreated animals. The major findings of this study suggest that the cellular GSH concentration within tumours decreases with distance from the vasculature, and that the GSH concentration within cells from all locations in the tumour can be depleted by enzymatically inhibiting its synthesis using BSO. This depletion of GSH resulted in a small degree of hypoxic radiosensitization of cells both distal and proximal to the vasculature.

Chemoresistance in rat ovarian tumours

In a cisplatin resistant subline (O-342/DPP) of an intraperitoneally growing transplantable rat ovarian tumour (O-342), intracellular glutathione (GSH) was approximately doubled (mean [S.E.] 1.5 [0.26] vs. 0.8 [0.2] nmol/10 6 cells). GSH reductase activity was higher (30.64 [4.07] vs. 20 [0.92] nmol/min per mg protein), although no difference was found for GSH-S-transferase. 24 h after exposure to cisplatin, formation of DNA interstrand cross-links was at a maximum in both lines and significantly higher in O-342 (162 [23] vs. 88 [22] rad eq). Combination treatment of O-342/DDP with buthionine sulphoximine plus cisplatin resulted in a marginal increase in survival compared with cisplatin treatment; treatment of this line with 3-aminobenzamide plus cisplatin was also superior to cisplatin alone. In the sensitive line both combinations were likewise superior to cisplatin alone. In vitro, at equimolar concentration, a new platinum complex (CTDP) was at least as active as cisplatin in both lines, which suggests a superior therapeutic index because its LD 50 in mice is threefold higher than that of cisplatin. A ruthenium complex (ICR) had a higher activity in the resistant line. A titanium complex (budotitane) was not active.

Role of glutathione in vanadate reduction in young and mature rats: Evidence for direct participation of glutathione in vanadate inactivation

The influence of renal glutathione content, modulated by the glutathione synthesis inhibitor buthionine sulphoximine or by glutathione infusion, on the polyuric and natriuretic effects of i.v. administered vanadate was investigated in 20- and 55-day-old rats. The modulation of renal glutathione content led to significant changes in urine volume and sodium excretion, independently of age. A decrease in the renal glutathione level led to intensification and prolongation of the diuretic effects of vanadate in 55-day-old animals. Treatment with glutathione abolished and treatment with buthionine sulphoximine increased the polyuric effect of vanadate. These observations indicate a role for renal glutathione in vanadate inactivation. Age-dependent differences in the polyuric and natriuretic effectiveness of vanadate are caused by differences in renal glutathione content during maturation of the kidney.