The mechanism of cytogenetic genotoxicity of exogenous glutathione in V-79 cells in vitro—implication of hydrogen peroxide and general traits of oxidative chromosome damage

The mechanism of cytogenetic genotoxicity (clastogenicity, induction, cell cycle delay) of 10^–3 M glutathione in V79-E cells, as described by Thust and Bach (1985), was studied in detail by using different treatment conditions. It was found that I-cystine is the essential cofactor in the incubation system. Catalase, but not superoxide dismutase, abolished the genotoxic effect, and the iron chelator desferoxamine, as well as the hydroxyl radical scavenger mannitol, diminished the activity. It is suggested that glutathione, in combination with V79-E cells and cystine, forms a hydrogen peroxide-generating system which provokes the adverse effects. Glutathione as well as I-cysteine and 2-mercaptopropionylglycine, which were checked for comparison, show a paradoxic genotoxicity, i.e., at 10^–2 M the effects return almost to the level of controls. Concentration dependence and other criteria of cytogenetic genotoxicity observed with glutathione show obvious similarities to those of other oxidatively acting agents and reveal striking differences to the cytogenetic effects of typical genotoxins.Abbreviations BUdR 5-bromodeoxyuridine - EMEM Eagle's minimum essential medium - GSH glutathione - MPG 2-mercaptopropionylglycine - SCE sister chromatid exchange - SOD superoxide dismutase - TPA 12-0-tetradecanoylphorbol-13-acetate     

Induction of endoreduplication by hydrazine in Chinese hamster V 79 cells and reduced incidence of sister chromatid exchanges in endoreduplicated mitoses

Hydrazine in high concentrations very effectively induces endoreduplication in Chinese hamster V 79 cells. The addition of 5-bromodeoxyuridine (BrdU) for the duration of one cell cycle prior to the induction of endoreduplication produces diplochromosomes with sister chromatid differentiation (SCD) after differential chromatid staining. The fact that diplochromosomes with complete SCD are obtained shows that endoreduplication was induced in cells that were in G2-phase. The analysis of sister chromatid exchanges (SCEs) showed that hydrazine treatment rarely led to increased SCE frequencies in mitoses after endoreduplication, but that it caused a strong SCE induction in diploid second division metaphases in the same culture. Neither catalase nor cysteine had an effect on the induction of endoreduplication or the incidence of SCEs. Treatment of the cells with mitomycin C prior to addition of BrdU led to increased SCE frequencies. Compared with the normal mitoses from the same preparation, the mitoses after endoreduplication showed a significantly reduced induction of SCEs. In contrast to these findings, SCE induction was not reduced in the common tetraploid V 79 cells after colcemid-induced polyploidization.     

Effect of sucrose on polyploidization in early callus cultures of Solanum tuberosum

Leaf segments of a monohaploid, dihaploid and tetraploid genotype of the potato (Solanum tuberosum; x = 12) were cultured on callus-inducing medium with 10, 20, 30 or 40 gl^–1 sucrose. After 5 and 7 days of culture, metaphases contained the somatic or polyploidized number of mono- or diplochromosomes. The percentages of polyploidized metaphases were inversely correlated with the number of chromosome sets of the genotypes. In monohaploid leaf segments the percentages of polyploidized metaphases and of metaphases with diplochromosomes increased when the sucrose concentration was raised from 10 or 20 to 30 gl^–1 and remained constant or decreased from 30 to 40 gl^–1. Higher concentrations of sucrose but not higher osmolalities of the medium due to mannitol induced endoreduplication in more cells. The frequency of polyploidized metaphases and metaphases with diplochromosomes in dihaploid and tetraploid leaf segments remained constant through increases in sucrose concentrations.     

Do the frequencies of sister chromatid exchanges in endoreduplieated mitoses provide a measure for lesion persistence and repair?

Endoreduplication was induced in V 79 cells using Colcemid. The concentration of Colcemid necessary to induce endoreduplication is about 1000 times higher than that needed to arrest mitoses or to induce ordinary tetraploid cells. Diplochromosomes with sister chromatid differentiation were obtained by adding BrdU for the duration of one cell cycle prior to the induction of endoreduplication. The induction of endoreduplication with Colcemid had no influence on the frequency of sister chromatid exchanges (SCEs). Treating the cultures with mitomycin C (MMC) before adding BrdU increased the percentage of endoreduplieated mitoses and also led to marked SCE induction. In the diplochromosomes, the frequencies of both twin SCEs (first cycle) as well as single SCEs (second cycle) were increased. It was also found that the SCE frequencies in mitoses after endoreduplication were lower than the values found in diploid and ordinary tetraploid metaphases of the same preparation. The possible conclusions concerning the lifetime of SCE-inducing lesions and the influence of repair processes are discussed.     

Induction of sister chromatid exchanges by inhibitors of topoisomerases

To investigate the role of topoisomerases in the production of sister chromatid exchanges, the effects of inhibitors of type I and II topoisomerases on baseline and mutagen-induced sister chromatid exchanges were compared. V79 cells were treated with VM-26 and m-AMSA, known inhibitors of type II topoisomerase, or with camptothecin, the only known inhibitor of type I topoisomerase. We observed that inhibitors of both type I and II topoisomerases induced high levels of sister chromatid exchanges at 10^–6 M, and that the dose-response curves of these drugs were very similar. A clear heterogeneity in the distribution patterns of exchanges induced by inhibitors of topoisomerases was observed. We believe that this heterogeneity in response to these compounds is due to variation in sensitivity within the cell cycle. We also studied interactions of these agents with mitomycin-C and with PUVA (8-methoxypsoralen + UVA), both cross-linking agents and potent sister chromatid exchange inducers, and with x-rays, an agent that induces high levels of DNA strand breaks. No significant change in exchange levels was observed in interactions between topoisomerase inhibition and the levels induced by the agents studied. We conclude that double-strand break prevalence, known to be increased through inhibition of type II topoisomerase, is not the primary mechanism for induction of sister chromatid exchanges. We further conclude that acute inhibition of type I and type II topoisomerases does not influence substantially the induction of exchanges by other agents.Abbreviations MMC mitomycin C - 8-MOP 8-methoxypsoralen - SCE sister chromatid exchange - SFM serum-free medium     

Phloem transport of sulfur in Ricinus

Mature leaves of Ricinus communis fed with ³⁵SO ₄ ^2- in the light export labeled sulfate and reduced sulfur compounds by phloem transport. Only 1–2% of the absorbed radiosulfur is exported to the stem within 2–3 h, roughly 12% of ³⁵S recovered was in reduced form. The composition of phloem translocate moving down the stem toward the root was determined from phloem exudate: 20–40% of the ³⁵S moved in the form of organic sulfur compounds, however, the bulk of sulfur was transported as inorganic sulfate. The most important organic sulfur compound translocated was glutathione, carrying about 70% of the label present in the organic fraction. In addition, methionine and cysteine were involved in phloem sulfur transport and accounted for roughly 10%. Primarily, the reduced forms of both, glutathione and cysteine are prsent in the siever tubes.Abbreviations CySH cysteine - GSH glutathione - GSSG glutathione disulfide - NEM N-ethylmaleimide - CyS-SCy cystine     

Accumulation of ubiquitinated proteins in mouse neuronal cells induced by oxidative stress

Ubiquitin protein conjugates are commonly detected in neuronal brain inclusions of patients with neurodegenerative disorders. The failure to eliminate the ubiquitin-protein deposits in the degenerating neurons may result from changes in the activity of the ubiquitin/ATP-dependent proteolytic pathway. This proteolytic pathway plays a major role in the degradation of short lived, abnormal and denatured proteins. Cadmium is a potent cell poison and is known to affect the ubiquitin pathway and to cause oxidative stress. Increases in protein mixed-disulfides (Pr-SSG) and decreases in glutathione (GSH) are often used as markers of oxidative stress. To investigate the relationship between the ubiquitin pathway and cellular glutathione (GSH), we treated HT4 cells (a mouse neuronal cell line) and rat mesencephalic primary cultures with different concentrations of the heavy metal. We observed marked increases in Pr-SSG as well as decreases in GSH, after exposure of HT4 cells or primary mesencephalic cultures to Cd²⁺. Furthermore, our results show that Cd²⁺ induced the accumulation of ubiquitinated proteins. Detection was by Western blotting of total cell extracts probed with antibodies that recognize ubiquitin-protein conjugates. These results suggest that the ubiquitin-pathway is closely involved in the cell response to cadmium-mediated oxidative stress. Abbreviations: GSH – glutathione; GSSG – glutathione disulfide; Pr-SSG – protein mixed disulfides.     

Type and concentration of redox reagents influencing nitrile formation upon myrosinase (Brassica carinata)-catalyzed hydrolysis of glucosibarin

The ratio of isothiocyanates (ITCs) to nitriles formed in the myrosinase-catalyzed hydrolysis of glucosinolates is a key factor determining the physiological effect of glucosinolate containing plants and materials. In this context, the mechanism by which nitrile formation occurs is not well understood. In the present paper we have studied the effect of three redox reagents – Fe²⁺, glutathione (GSH) and ascorbic acid – on the profile of products obtained upon the hydrolysis of a model glucosinolate (glucosibarin ((2R)-2-hydroxy-2-phenylethylglucosinolate)) catalyzed by Brassica carinata myrosinase. A Micellar Electrokinetic Capillary Chromatography method that allows following on-line the hydrolysis of the glucosinolate, the formation of the degradation products and the oxidation of GSH was used. Increasing the concentration of Fe²⁺ and GSH (from 0.25- to 2-fold molar excess with respect to the glucosinolate) increased the ratio of nitrile ((2R)-2-hydroxy-2-phenylethylcyanide) to oxazolidine-2-thione ((5S)-5-phenyloxazolidine-2-thione), whereas increasing the concentration of ascorbic acid decreased this ratio. Low concentrations of ascorbic acid favored nitrile formation. A mechanism for nitrile formation involving a disulfide bond in the myrosinase complex is proposed.     

Analysis of bromodeoxyuridine-induced single and twin sister chromatid exchanges in tetraploid Chinese hamster ovary cells

Culture of cells in high exogenous levels (>10^–4 M) of bromodeoxyuridine (BrdUrd) or thymidine will increase the baseline sister chromatid exchange (SCE) frequency. The effect is thought to be related to the balance of the DNA precursors thymidine and deoxycytidine. Exogenous addition of deoxycytidine will reverse this effect. Single and twin SCEs were analysed in Colcemid-induced tetraploid Chinese hamster ovary cells exposed to different concentrations of BrdUrd to determine at what stage SCEs are induced by high levels of BrdUrd. In cells exposed to low concentrations of BrdUrd (10^–5 M), equal numbers of SCEs were induced in each of the two cell cycles. With increasing concentrations of BrdUrd (10^–4 to 2×10^–4 M), SCE frequency increased in both cell cycles, but far more SCEs were induced in the second cell cycle. Deoxycytidine (2×10^–4 M) reduced the frequency of SCEs primarily by reducing the frequency of SCEs induced in the second cell cycle. Treatment with 3-aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) polymerase, produced effects similar to exposure to high levels of BrdUrd including inducing SCEs in the second replication cycle. This suggests a similar mechanism of action. Deoxycytidine had no effect on 3AB-induced SCEs, however, and there was no interaction between 3AB and high exogenous levels of BrdUrd in SCE induction. Thus these two agents probably act through different mechanisms.     

Rapid irradiation procedure for obtaining permanent differential staining of sister chromatids and aspects of its underlying mechanism

Summary When fixed metaphase preparations of lymphocytes cultured in the presence of BrdU during two cell cycles are subjected to a 1-min simple irradiation treatment with near-ultraviolet light (radiation dose 3×10⁵ J/m²), subsequent Giemsa staining produces differential staining of sister chromatids irrespective of previous exposure to a photosensitizer. The effects of this procedure were analyzed by irradiating single metaphases under the microscope, thus allowing precise dosage of radiation: Metaphase were subsequently stained with Giemsa and then subjected to the Feulgen-Schiff procedure. Whereas in the presence of DAPI as a photosensitizer a differential breakdown of BrdU-containing DNA in the chromatids under the influence of irradiation appeared to be the cause of sister chromatid differentiation, alterations presumably in the higher oeder structure of chromatin, not accompanied by removal of DNA, induced sister chromatid differentiation without DAPI.     

Co-production of S-adenosyl-l-methionine and glutathione from spent brewer’s yeast cells

Both S-adenosyl-l-methionine (AdoMet) and glutathione (GSH) are important small molecules with pharmaceutical importance. The co-production of AdoMet and GSH using abundant spent brewer’s yeast cells from the beer industry and with l-methionine supplement was successfully realized. Experimental data showed that improvement of GSH productivity was accompanied by AdoMet accumulation. AdoMet productivity of 40–45 mg g⁻¹ (DCW) was successfully achieved and an additional 13–18 mg g⁻¹ (DCW) GSH was synthesized in spent brewer’s yeast cells.     

Glutathionylation of the Active Site Cysteines of Peroxiredoxin 2 and Recycling by Glutaredoxin

Peroxiredoxin 2 (Prx2) is a thiol protein that functions as an antioxidant, regulator of cellular peroxide concentrations, and sensor of redox signals. Its redox cycle is widely accepted to involve oxidation by a peroxide and reduction by thioredoxin/thioredoxin reductase. Interactions of Prx2 with other thiols are not well characterized. Here we show that the active site Cys residues of Prx2 form stable mixed disulfides with glutathione (GSH). Glutathionylation was reversed by glutaredoxin 1 (Grx1), and GSH plus Grx1 was able to support the peroxidase activity of Prx2. Prx2 became glutathionylated when its disulfide was incubated with GSH and when the reduced protein was treated with H₂O₂ and GSH. The latter reaction occurred via the sulfenic acid, which reacted sufficiently rapidly (k = 500 m⁻¹ s⁻¹) for physiological concentrations of GSH to inhibit Prx disulfide formation and protect against hyperoxidation to the sulfinic acid. Glutathionylated Prx2 was detected in erythrocytes from Grx1 knock-out mice after peroxide challenge. We conclude that Prx2 glutathionylation is a favorable reaction that can occur in cells under oxidative stress and may have a role in redox signaling. GSH/Grx1 provide an alternative mechanism to thioredoxin and thioredoxin reductase for Prx2 recycling.     

Proliferation of PHA-stimulated lymphocytes measured by combined autoradiography and sister chromatid differential staining

Lymphocyte proliferation in culture was studied by combined [³H]TdR incorporation and sister chromatid differential staining. The majority of 1st division metaphases in a 72 h culture commenced DNA synthesis after 48 h and had a cell cycle of less than 24 h. A small proportion of cells from some donors commenced DNA synthesis between 24–30 h and had cell cycle times of up to 48 h. Although many cells entered DNA synthesis at the same time, they showed marked asynchrony in the length of their cell cycle, with some completing one, some two and others three cell cycles in the 72 h culture period. The time taken for cells to enter S following stimulation with PHA ranged from 24 to 48 h and there was considerable variation between donors in the number of fast and slow responding cells.     

Modeling of the mechanism of nucleophilic aromatic substitution of fungicide chlorothalonil by glutathione

Chlorothalonil (2,4,5,6-tetrachloroisophthalonitrile, TCIN, CAS 1897-45-6) is a broad range spectrum fungicide whose fungitoxic action has been associated with the rapid formation of conjugated chlorothalonil–cellular thiol derivatives, specifically with thiol-rich enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and with glutathione (GSH). The biotransformation reaction sequence between enzyme-activated glutathione (GSH) and chlorothalonil depletes cellular glutathione reserves. The conjugation of glutathione with chlorothalonil via nucleophilic aromatic substitution was modeled for an isolated reacting species using semiempirical self-consistent field molecular orbital (SCF-MO) theory at the PM3 level. The potential energy hypersurface at each of the three possible chlorinated attack sites on chlorothalonil was elaborated using a thiolate (CH₃S^–) anion as a model for an enzyme-activated glutathione molecule. Calculated free energies of activation for formation of mono-RSH conjugates suggest that the order of nucleophilic attack on chlorine positions in TCIN is 2>4, 6>5 although energy differences are small (on the order of 1–2 kcal mol^–1). Meisenheimer or -complexes have been isolated as true intermediates on the hypersurface for each reaction, suggesting that the mechanism follows a two-step pathway.Electronic Supplementary Material available.     

Disulfiram augments oxidative stress in rat brain following bilateral carotid artery occlusion

We examined the brain oxidative stress which accompanies 30 min of bilateral carotid artery ligation (BCAL) in terms of changes in brain levels of glutathione; reduced (GSH) and oxidized (GSSG) forms and the exacerbation of oxidative stress by disulfiram (DSF). These results indicate that BCAL alone decreases GSH content and limits glutathione reductase (GR) activity, and these changes were enhanced by DSF pretreatment. Similar observations were recorded with DSF alone. GR activity (74.3±4.0 µmol min^–1 mg^–1 tissue; p<0.001) and GSH content (1.23±0.06 µmol min^–1g^–1 tissue; p<0.001) was attenuated in rats subjected to synergistic effect of BCAL and DSF with a concomitant increase of GSSG (0.006±0.006 µmol min^–1 g^–1 tissue; p<0.001). Recovery of GSH/GSSG level and GR activity during reperfusion following 30 min BCAL was considerably delayed (96 h) in the BCAL and DSF group as compared to the recovery time of 24 h in the group subjected to BCAL-reperfusion alone. Perturbation of GSH/GSSG homeostasis as a result of BCAL was augmented by DSF. These findings clearly demonstrate central nervous system oxidative stress due to a BCAL-DSF synergistic effect. Based on the results obtained with this model, we conclude that DSF increases brain oxidative stress and this may be detrimental to alcoholics who might drink and develop an acetaldehyde-induced hypotension while taking DSF.     

Differential spiralization along mammalian mitotic chromosomes

Morphology of chromosomes replicating in the presence of 5-bromodeoxyuridine was studied using long-term cultures of Chinese hamster cells (line Blld-ii-FAF28). The cytological effect of the analog administered in various concentrations, at different stages of the S period, and during one and two successive mitotic cycles was studied. — The main cytological manifestation of the BUdR action consisted in spiralization delay of certain chromosome regions. The degree of the delay was dependent on the time interval between the introduction of the agent and mitosis, as well as on the agent's concentration. With prolongation of the interval, the spiralization delay diminished and disappeared being therefore always observable only in late replicating chromosome regions. Increased concentration of BUdR (in the range of 25 to 400 g/ml) produced enhancement of the delay of chromosome spiralization. — After two successive reproduction cycles in the presence of BUdR, a great number of metaphases contained chromosomes the sister chromatids of which showed unequal spiralization delay. Autoradiography of ³H-BUdR distribution showed that the sister chromatid with a more pronounced underspiralization corresponds to the chromatid incorporating BUdR into both strands of the DNA molecule. — Mechanisms of the effect observed, as well as chemical influence on chromosome spiralization as a usefull tool of displaying linear chromosome differentiation, are discussed.     

Effects of phorbol myristate acetate,phorbol dibutyrate,ethanol, dimethylsulfoxide,phenol, and seven metabolities of phenol on metabolic cooperation between chinese hamster v79 lung fibroblasts

The effect of phorbol myristate acetate, phorbol dibutyrate, ethanol, dimethylsulfoxide, phenol, and seven metabolites of phenol on metabolic cooperation were assessed as a function of mutant cell recovery from populations of cocultivated hypoxanthine-guanine phosphoribosyl transferase-deficient mutant (HGPRT–) and wild-type (HGPRT+) Chinese hamster V79 lung fibroblasts. Phorbol myristate acetate and phorbol diputyrate, two established tumor promoters, were potent inhibitors of metabolic cooperation. Ethanol and dimethylsulfoxide, solvents commonly used to prepare chemicals for testing, weakly inhibited metabolic cooperation. Phenol and phenylglucuronide had no effect on metabolic cooperation. Four oxidative metabolites (1,4-benzoquinone, catechol, hydroxyquinol and quinol) inhibited metabolic cooperation. Phenylsulfate weakly inhibited metabolic cooperation. Conversely, 2-methoxyphenol, a methylated derivative of catechol, appeared to enhance metabolic cooperation. These results generallyAbbreviations CAS Chemical Abstracts Service - DMSO dimethylsulfoxide - ETOH ethanol - HGPRT hypoxanthine-guanine phosphoribosyl transferase - HGPRT+ HGPRT-competent - HGPRT– HGPRT-te]deficient - MC metabolic cooperation - MC+ metabolic cooperation-competent - MC– metabolic cooperation-deficient - MEM minimum essential medium - PDBu phorbol dibutyrate - PMA phorbol myristate acetate - 6TG 6-thioguanine - 6TG^r 6-thioguanine-resistant - 6TG^s 6-thioguanine-sensitive - V79/MC assay Chinese hamster V79 lung fibroblast assay for metabolic cooperation     

Determination of cellular thiols and glutathione-related enzyme activities: versatility of high-performance liquid chromatography–spectrofluorimetric detection

A high-performance liquid chromatography (HPLC) method to determine the most important cellular thiols [reduced glutathione (GSH), cysteine, γ-glutamylcysteine and cysteinylglycine] is described. Separation relies upon isocratic ion-pairing reversed-phase chromatography and detection is operated by spectrofluorimetry coupled with post-column derivatization reactions using either N-(1-pyrenyl)maleimide (NPM) or ortho-phthalaldehyde (OPA). When OPA is used without co-reagent, only GSH and γ-glutamylcysteine are detected (heterobifunctional reaction). However, either the OPA reaction in the presence of glycine in the mobile phase (thiol-selective reaction) or NPM allows the detection of all the cited thiols. The HPLC system has been validated as concerning linearity, accuracy and precision. The low detection limits reached (in the pmol range for each thiol injected) allow the screening and the quantification of thiols (as NPM derivatives) in V79cl and V79HGGT cells as well as the measurement of two cytosolic enzymes related to the glutathione synthesis, using the heterobifunctional OPA reaction.     

Relationship Between Oxidation of Glutathione and Reactive Nitrogen Species During the Early-Reperfusion Phase of Cerebral Ischemia

A timed profile of glutathione oxidation and reactive nitrogen species during reperfusion after cerebral ischemia in rat was obtained. Dialysate was collected every 25 min from a microdialysis probe inserted into the cerebral cortex before and after cerebral ischemia. NO₂ ^–, NO₃ ^–, and reduced and oxidized glutathione (GSH, GSSG) were detected by high-performance liquid chromatography. GSH and GSSG increased and reached a peak: 3408 ± 1710% (mean ± SE) at 25 min of reperfusion (P < 0.0001) and 329 ± 104% at 50 min of reperfusion (P = 0.06), respectively. Oxidation ratio decreased from 0.82 ± 0.04 to 0.42 ± 0.07 (P < 0.0001) at 25 min of reperfusion. NO₃ ^– levels significantly decreased (68.3 ± 9.1%) (P < 0.01) during ischemia and remained lower than the control value during reperfusion. NO₂ ^– levels did not significantly change. These data suggest that GSH releases during early phase of reperfusion and that its rapid oxidation contributes to prevent an increase in reactive nitrogen species.     

THE FREQUENCY OF SISTER CHROMATID EXCHANGES FOLLOWING EXPOSURE TO VARYING DOSES OF H3-THYMIDINE OR X-RAYS

In the Chinese hamster cell line CHEF-125, sister chromatid exchanges occurred at a rate of a little higher than one per three chromosomes for each cell cycle. The exchanges were detectable by labeling with H³-thymidine and autoradiographic analyses of chromosomes at the second and subsequent metaphases after labeling had occurred. To test the hypothesis that sister chromatid exchanges are caused by radiation, cells were incubated in media with different amounts of H³-thymidine. No statistically significant change in the exchange rate was detected over 100-fold range of variation in the amount of incorporated H³-thymidine (determined by grain counts of autoradiographs). We have concluded that sister chromatid exchanges are not caused by tritium radiation and therefore are spontaneous events. Cultures were also irradiated with acute doses of x-rays up to 200 r and scored for sister chromatid exchanges. Between zero and 50 r there was a statistically significant increase in the rate of exchanges. This is interpreted as evidence that x-rays can induce some exchanges, although the majority of these events are probably spontaneous.