Repair of chromatin damage in glutathione-depleted V-79 cells: comparison of oxic and hypoxic conditions

We have assessed the effects of two radiomodifying conditions, glutathione (GSH) depletion and hypoxia, on the formation and repair of radiation-induced chromatin damage, specifically DNA-protein cross-links (DPC). As measured by a nitrocellulose filter-binding assay, untreated V79 cells contain a low level of DPC (1-1.5% of the cellular DNA). The background level of DPC is elevated in cells treated with L-buthionine sulfoximine (BSO), in hypoxic cells, and in cells treated with BSO and made hypoxic (2.98%, 2.82%, and 7.71%, respectively). The dose response for production of radiation-induced DPC is approximately 6.0% DNA bound per 100 Gy for cells irradiated in air, and the dose response is not significantly different for BSO-treated cells but increases by a factor of about 1.4 for hypoxic cells and 1.7 for BSO-pretreated hypoxic cells. DPC were also assayed by alkaline elution with or without proteinase K treatment. By this analysis, the yield of DPC appears to be elevated in irradiated hypoxic and irradiated GSH-depleted cells. It is not possible to assay for background DPC alone in unirradiated cells by alkaline elution. Cells not exposed to BSO repair 70-80% of the radiation-induced DPC in 4 h. BSO-treated cells are considerably less efficient in repair of DPC. As analyzed by alkaline elution, GSH depletion had little or no effect on the yield of radiation-induced single-strand breaks (SSB) but slowed their repair. The data suggest that depletion of GSH impairs an enzyme system(s) responsible for the turnover of both background and radiation-induced DPC and that hypoxia elevates both the background level of DPC and the ratio of radiation-induced DPC to SSB.     

Effects of iron chelators and glutathione depletion on the induction and repair of chromosomal aberrations by tert-butyl hydroperoxide in cultured Chinese hamster cells

The effects of iron chelators and glutathione (GSH) depletion on the induction of chromosomal aberrations by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. t-BuOOH in a concentration range of 0.1-1.0 mM induced chromosomal structural aberrations, consisting mainly of chromatid gaps and breaks, in a dose-dependent fashion. The divalent iron chelator o-phenanthroline almost completely suppressed the formation of chromosomal aberrations while the trivalent chelator desferrioxamine was less effective. GSH depletion did not affect the formation of chromosomal aberrations and DNA single-strand breaks (ssb) by t-BuOOH. DNA ssb by 0.5 mM t-BuOOH were repaired within 60 min of treatment in both GSH-depleted (GSH-) and non-depleted (GSH+) cells. In contrast, chromosomal aberrations increased a little during the 60 min after treatment in both GSH- and GSH+ cells. The aberrations were then repaired in GSH+ cells but those in GSH- cells were maintained to a great extent during 20 h of post-treatment incubation. These results indicate that divalent iron mediates the induction of chromosomal aberrations by t-BuOOH. That t-BuOOH-induced chromosomal aberrations remain even after DNA ssb were repaired suggests involvement of other lesions than DNA ssb in the formation of chromosomal aberrations by the hydroperoxide.     

Differential effects of glutathione depletion and metallothionein induction on the induction of DNA single-strand breaks and cytotoxicity by tert-butyl hydroperoxide in cultured mammalian cells

Induction of DNA single-strand breaks (ssb), their resealing and cytotoxicity by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. The effect of the depletion of cellular glutathione (GSH), iron chelation and induction of metallothionein (MT) on these parameters was studied. t-BuOOH in a concentration range of 0.02-0.5 mM induced DNA ssb in a dose-dependent fashion. Strand breakage increased as a function of time up to 1 h. Divalent iron chelator o-phenanthroline suppressed markedly the induction of DNA ssb while the trivalent iron chelator desferrioxamine had no effect. GSH-depletion increased cytotoxicity of t-BuOOH. In contrast, the depletion of GSH did not affect the efficiency of formation of DNA ssb by t-BuOOH and the rate of resealing of the DNA damage. The induction of MT did not influence the efficiency of formation of DNA ssb by t-BuOOH. In summary, while GSH depletion and MT induction affected the formation of DNA ssb and cytotoxicity differently divalent iron was required for both. Therefore, appears likely that DNA breakage and cytotoxicity by t-BuOOH are caused by independent mechanisms.     

Neurotoxicity from glutathione depletion is mediated by Cu-dependent p53 activation

Loss of intracellular neuronal glutathione (GSH) is an important feature of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The consequences of GSH depletion include increased oxidative damage to proteins, lipids, and DNA and subsequent cytotoxic effects. GSH is also an important modulator of cellular copper (Cu) homeostasis and altered Cu metabolism is central to the pathology of several neurodegenerative diseases. The cytotoxic effects of Cu in cells depleted of GSH are not well understood. We have previously reported that depletion of neuronal GSH levels results in cell death from trace levels of extracellular Cu due to elevated Cu(I)-mediated free radical production. In this study we further examined the molecular pathway of trace Cu toxicity in neurons and fibroblasts depleted of GSH. Treatment of primary cortical neurons or 3T3 fibroblasts with the glutathione synthetase inhibitor buthionine sulfoximine resulted in substantial loss of intracellular GSH and increased cytotoxicity. We found that both neurons and fibroblasts revealed increased expression and activation of p53 after depletion of GSH. The increased p53 activity was induced by extracellular trace Cu. Furthermore, we showed that in GSH-depleted cells, Cu induced an increase in oxidative stress resulting in DNA damage and activation of p53-dependent cell death. These findings may have important implications for neurodegenerative disorders that involve GSH depletion and aberrant Cu metabolism.     

Reduction of intracellular glutathione content and radiosensitivity

The intracellular glutathione (GSH) content of HeLa, CHO and V79 cells was reduced by incubating the cells in growth medium containing buthionine sulphoximine or diethyl maleate (DEM). Clonogenicity, single-strand DNA breaks (ssb) and double-strand DNA breaks (dsb) were used as criteria for radiation-induced damage after X- or gamma-irradiation. In survival experiments, DEM gave a slightly larger sensitization although it gave a smaller reduction of the intracellular GSH. In general, sensitization was larger for dsb than for ssb, also the reduction of the o.e.r. was generally larger for dsb than for ssb. This may be due to the higher dose rate in case of dsb experiments resulting in a higher rate of radiochemical oxygen consumption. In general, no effect was found on post-irradiation repair of ssb and dsb.     

Glutathione metabolism in cultured Sertoli cells and spermatogenic cells from hamsters

Isolated spermatocytes and spermatids from hamsters contained a large amount of glutathione (GSH) (approximately 40 and 30 nmol GSH/mg protein, respectively), but showed a spontaneous decrease of GSH content during prolonged incubation (t1/2 approximately 35 h). Incubation of the germ cells in the presence of the glutathione biosynthesis inhibitor buthionine sulphoximine (BSO) provided evidence that the cells can perform glutathione synthesis. This synthesis, however, was not sufficient to maintain the GSH content of the isolated cells, or to restore the cellular GSH pool after depletion caused by exposure of the cells to the glutathione S-transferase substrate, diethyl maleate (DEM). Cultured Sertoli cells, containing approximately 10 nmol GSH/mg protein, had a more active BSO-sensitive GSH synthesis system. The Sertoli cells, but also tubule fragments containing Sertoli cells and germ cells, were able to restore their GSH pool after DEM-induced depletion. DEM treatment of the tubule fragments resulted in a 90% decrease of the GSH content of the spermatocytes and spermatids present within the fragments. The GSH levels of the tubule fragments and the enclosed germ cells were restored during a subsequent incubation in the absence of DEM. As indicated above, such a recovery was not observed for isolated spermatocytes and spermatids. The results illustrate the importance of Sertoli cell-germ cell interaction, and point to a role of Sertoli cells in glutathione synthesis by the germ cells.     

Lack of effect of glutathione depletion on cytotoxicity, mutagenicity and DNA damage produced by doxorubicin in cultured cells

Since endogenous glutathione (GSH), the main non-protein intracellular thiol compound, is known to provide protection against reactive radical species, its depletion by diethylmaleate (DEM) was used to assess the role of free radical formation mediated by doxorubicin in DNA damage, cytotoxicity and mutagenicity of the anthracycline. Subtoxic concentrations of DEM that produced up to 75% depletion of GSH did not increase doxorubicin cytotoxicity in a variety of cell lines, including Chinese hamster ovary (CHO) and lung (V-79) cells, LoVo human carcinoma cells and P388 murine leukemia cells. Similarly, the number of doxorubicin-induced DNA single strand breaks in CHO cells and the mutation frequency in V-79 cells were not affected by GSH depletion. The results obtained suggest that mechanisms other than free radical formation are responsible for DNA damage, cytotoxicity and mutagenicity of anthracyclines.     

The effect of glutathione depletion on sister-chromatid exchange induction by cytostatic drugs

Using sister-chromatid exchanges (SCEs) as an indicator for DNA damage, we investigated the role of glutathione (GSH) as a determinant of cellular sensitivity to the DNA-damaging effects of the cytostatic drugs adriamycin (AM) and cyclophosphamide (CP). Exposure of V79 cells to buthionine sulfoximine (BSO) resulted in a complete depletion of cellular GSH content without toxicity and without increasing the SCE frequency. Subsequent 3-h treatment of GSH-depleted cells with AM or S9-mix-activated CP caused a potentiation of SCE induction. In Chinese hamster ovary (CHO) cells, which showed a higher GSH level compared to V79 cells, BSO treatment led to a depletion of GSH to about 5% of the control and increased SCE induction by AM and CP. Compared to V79 cells, the effect of AM on SCE frequencies was less distinct in CHO cells, while CP exerted a similar effect in both cell lines. Pretreatment of V79 cells with GSH increased the cellular GSH content, but had no effect on the induction of SCEs by AM, and pretreatment with cysteine influenced neither GSH levels nor SCE induction by AM. The study shows that SCEs are a suitable indicator for testing the modulation of of drug genotoxicity by GSH. The importance of different GSH contents of cell lines for their response to mutagens is discussed.     

Implications of altered glutathione metabolism in aspirin-induced oxidative stress and mitochondrial dysfunction in HepG2 cells

We have previously reported that acetylsalicylic acid (aspirin, ASA) induces cell cycle arrest, oxidative stress and mitochondrial dysfunction in HepG2 cells. In the present study, we have further elucidated that altered glutathione (GSH)-redox metabolism in HepG2 cells play a critical role in ASA-induced cytotoxicity. Using selected doses and time point for ASA toxicity, we have demonstrated that when GSH synthesis is inhibited in HepG2 cells by buthionine sulfoximine (BSO), prior to ASA treatment, cytotoxicity of the drug is augmented. On the other hand, when GSH-depleted cells were treated with N-acetyl cysteine (NAC), cytotoxicity/apoptosis caused by ASA was attenuated with a significant recovery in oxidative stress, GSH homeostasis, DNA fragmentation and some of the mitochondrial functions. NAC treatment, however, had no significant effects on the drug-induced inhibition of mitochondrial aconitase activity and ATP synthesis in GSH-depleted cells. Our results have confirmed that aspirin increases apoptosis by increased reactive oxygen species production, loss of mitochondrial membrane potential and inhibition of mitochondrial respiratory functions. These effects were further amplified when GSH-depleted cells were treated with ASA. We have also shown that some of the effects of aspirin might be associated with reduced GSH homeostasis, as treatment of cells with NAC attenuated the effects of BSO and aspirin. Our results strongly suggest that GSH dependent redox homeostasis in HepG2 cells is critical in preserving mitochondrial functions and preventing oxidative stress associated complications caused by aspirin treatment.     

Role of glutathione in augmenting the anticancer activity of pyrroloquinoline quinone (PQQ)

Abstract

Pyrroloquinoline quinone (PQQ), a bacterial redox co-factor and antioxidant, is highly reactive with nucleophilic compounds present in biological fluids. PQQ induced apoptosis in human promonocytic leukemia U937 cells and this was accompanied by depletion of the major cellular antioxidant glutathione and increase in intracellular reactive oxygen species (ROS). Treatment with glutathione (GSH) or N-acetyl-L-cysteine (NAC) did not spare PQQ toxicity but resulted in a 2–5-fold increase in PQQ-induced apoptosis in U937 cells. Cellular GSH levels increased following treatment by NAC alone but were severely depleted by co-treatment with NAC and PQQ. This was accompanied by an increase in intracellular ROS. Alternatively, depletion of glutathione also resulted in increased PQQ cytotoxicity. However, the cells underwent necrosis as evidenced by dual labeling with annexin V and propidium iodide. PQQ-induced cytotoxicity is thus critically regulated by the cellular redox status. An increase in GSH can augment apoptosis and its depletion can switch the mode of cell death to necrosis in the presence of PQQ. Our data suggest that modulation of intracellular GSH can be used as an effective strategy to potentiate cytotoxicity of quinones like PQQ.     

Induction and Rejoining of DNA Single-Strand Breaks in Relation to Cellular Growth in Human Cells Exposed to Three Hydroperoxides at 0°C and 37°C

There was a 5-fold increase in cytotoxicity for cumene hydroperoxide, 10-fold for tert-butyl hydroperoxide and 25-fold for hydrogen peroxide, under metabolizing conditions (37°C) in comparison to nonmetabolizing conditions (0°C), when human P31 cells were exposed for 60 min. The induction of DNA single-strand breaks correlated poorly with cytotoxicity. Hydrogen peroxide was by far the most effective agent inducing single-strand breaks irrespective of temperature. Cumene hydroperoxide produced fewer strand breaks than tert-butyl hydroperoxide despite its greater cytotoxicity at either 37°C or at 0°C. The pattern of single-strand break induction did not change with temperature. The number of breaks, however, increased when the cells were exposed at 37°C. The pattern of rejoining was similar for hydrogen peroxide- and tert-butyl hydroperoxide-induced breaks at both temperatures whereas the rejoining of cumene hydroperoxide-induced breaks deviated somewhat from this pattern. The results indicate that there is no clear-cut relationship between induction of DNA single-strand breaks and cytotoxicity after hydroperoxide exposure.     

Immunochemical detection of DNA damage induction and repair at different cellular stages of spermatogenesis of the hamster after in vitro or in vivo exposure to ionizing radiation

An immunochemical method has been used to detect quantitatively DNA damage caused by ionizing radiation in germ cells. With this method, DNA strand breaks as well as lesions converted into breaks in alkaline medium are measured as a function of controlled partial unwinding of the DNA, a time-dependent process starting at each breakage site, followed by the determination of the relative amount of single-stranded regions by use of a single-strand specific monoclonal antibody. With this method the induction and repair of DNA damage in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids) of the hamster were investigated. Germ cells were irradiated in vitro with 60Co-gamma-rays, at doses between 0 and 5 Gy. A linear dose-response relationship was observed. Spermatocytes and round spermatids had normal, fast repair of the lesions when compared with the repair of these sites in cultured V79 or CHO cells and human lymphocytes. The elongated spermatids, however, showed hardly any repair. Similar results were obtained after the in vivo gamma-irradiation of hamsters with doses of 0. 4, and 8 Gy and subsequent isolation of germ cells. The damage was still detectable in the elongated spermatids at 24 h after exposure. The results of the experiments show substantial differences in repair capacity between different stages of germ cell development. Because DNA is the major target for mutation induction, this assay may be useful for assessment of the genetic risk of exposure of male germ cells to ionizing radiation, in relation to the stage of development.     

Glutathione requirement for the rejoining of radiation-induced DNA breaks in misonidazole-treated cells

The role of glutathione (GSH) in the rejoining of radiation-induced single-strand DNA breaks (ssb) was studied in human fibroblast cultures sensitized to radiation by a 30 min treatment with 1 mM misonidazole (MISO). Hypoxically irradiated cells, deficient in GSH, either inherently, or due to a 16 h incubation with 1 mM buthionine sulphoximine (BSO), rejoined the breaks after MISO treatment at a lower rate and to a lesser extent than did GSH-proficient cells. Without MISO treatment, the hypoxically induced ssb were rejoined in the GSH-deficient cells as effectively as in the proficient cells. It is concluded that a large proportion of the breaks which arise after hypoxic irradiation in the presence of MISO are of a different type to those which arise in the absence of the drug, and require a particular GSH-dependent, enzymatic repair system. This requirement for rejoining in hypoxically irradiated, MISO-treated cells is similar to that seen earlier in MISO-untreated, oxically irradiated cells, and suggests that the ssb induced by radiation in the presence of MISO or oxygen are of a similar nature.     

Radiation-induced damage to DNA in drug- and radiation-resistant sublines of a human breast cancer cell line.

An Adriamycin-resistant subline of a human breast cancer cell line, MCF-7 ADRR, has been shown to exhibit radioresistance associated with an increase in the size of the shoulder on the radiation survival curve. In the present study, damage to DNA of MCF-7 sublines WT and ADRR by 60Co gamma radiation was measured by filter elution techniques. The initial amount of DNA damage, measured by both alkaline and neutral filter elution, was lower in ADRR cells, suggesting that these cells are resistant to radiation-induced single- and double-strand DNA breaks. In the case of double-strand breaks the difference between WT and ADRR cells was significant only at the lower radiation doses studied (up to 100 Gy). In cells depleted of glutathione (GSH) by L-buthionine sulfoximine (BSO) treatment, ADRR cells were sensitized to radiation-induced DNA damage, while WT cells were unaffected. The rate of repair of single- and double-strand DNA breaks following radiation was the same for both sublines, and repair of radiation damage was not affected by BSO treatment in either cell line. The relative resistance of ADRR cells to initial DNA damage by radiation is the only difference so far detected at the molecular level which reflects radiation survival, and it is possible that other factors are involved in the resistance of ADRR cells to killing by radiation. Sensitization of ADRR cells to radiation-induced DNA damage by GSH depletion, although not likely to involve inhibition of GSH-dependent detoxification enzymes per se (irradiation was done at 4 degrees C), suggests that at the molecular level radioresponse in this subline is related to maintenance of GSH/GSSG redox equilibrium.     

Possible involvement of oxidative stress in trichloroethylene-induced genotoxicity in human HepG2 cells

Trichloroethylene (TCE) is an environmental and industrial pollutant whose hepatotoxicity has been demonstrated in experimental animals. However, the mechanisms of the effects, in particular those related to its genotoxicity in humans, are not well understood. The aim of this study was to assess the genotoxic effects of TCE and to identify and clarify the mechanisms, using human hepatoma HepG2 cells. Exposure of the cells to TCE caused significant increase of DNA migration in comet assay and of micronuclei (MN) frequencies at all tested concentrations (0.5-4mM), respectively, which suggests that TCE caused DNA strand breaks and chromosome damage. The involvement of lipid peroxidation in the genotoxic properties of TCE was confirmed by using immunoperoxidase staining for 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS). To elucidate the role of glutathione (GSH) in these effects, the intracellular GSH level was modulated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and by co-treatment with N-acetylcysteine (NAC), a GSH precursor. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the susceptibility of HepG2 cells to TCE-induced cytotoxicity and DNA damage, while when the intracellular GSH content was elevated by NAC, the DNA damage induced by TCE was almost completely prevented. These results indicate that TCE exerts genotoxic effects in HepG2 cells, probably through DNA damage by oxidative stress; GSH, as a main intracellular antioxidant, is responsible for cellular defense against TCE-induced DNA damage.     

Vitamin C and vitamin E restore the resistance of GSH-depleted lens cells to H2O2

A decline in reduced glutathione (GSH) levels is associated with aging and many age-related diseases. The objective of this study was to determine whether other antioxidants can compensate for GSH depletion in protection against oxidative insults. Rabbit lens epithelial cells were depleted of > 75% of intracellular GSH by 25-200 microM buthionine sulfoximine (BSO). Depletion of GSH by BSO alone had little direct effect on cell viability, but resulted in an approximately 30-fold increase in susceptibility to H(2)O(2)-induced cell death. Experimentally enhanced levels of nonprotein sulfhydryls other than GSH (i.e., N-acetylcysteine) did not protect GSH-depleted cells from H(2)O(2)-induced cell death. In contrast, pretreatment of cells with vitamin C (25-50 microM) or vitamin E (5-40 microM), restored the resistance of GSH-depleted cells to H(2)O(2). However, concentrations of vitamin C > 400 microM and vitamin E > 80 microM enhanced the toxic effect of H(2)O(2). Although levels of GSH actually decreased by 10-20% in cells supplemented with vitamin C or vitamin E, the protective effects of vitamin C and vitamin E on BSO-treated cells were associated with significant ( approximately 70%) decreases in oxidized glutathione (GSSG) and concomitant restoration of the cellular redox status (as indicated by GSH:GSSG ratio) to levels detected in cells not treated with BSO. These results demonstrate a role for vitamin C and vitamin E in maintaining glutathione in its reduced form. The ability of vitamin C and vitamin E in compensations for GSH depletion to protect against H(2)O(2)-induced cell death suggests that GSH, vitamin C, and vitamin E have common targets in their actions against oxidative damage, and supports the preventive or therapeutic use of vitamin C and E to combat age- and pathology-associated declines in GSH. Moreover, levels of these nutrients must be optimized to achieve the maximal benefit.     

Modulation of DNA single-strand breaks by intracellular glutathione in human lung cells exposed to asbestos fibers

We investigated the role of glutathione and nitric oxide synthase (NOS) in fiber-induced cell and DNA toxicity using alkaline (pH 13) single-cell gel electrophoresis (the Comet assay). Transformed cultured human pleural mesothelial (MeT-5A) cells and alveolar epithelial cells (A549) were exposed to crocidolite asbestos fibers (1-10 microg/cm(2)) in the presence of buthionine sulfoximine (BSO) or L-arginine-methyl ester (L-NAME). BSO inhibits gamma-glutamylcysteine synthetase (gamma-GCS) and causes glutathione depletion, and L-NAME inhibits nitric oxide generation. Studies were also conducted to assess the expression of the heavy and light subunits of gamma-GCS in human pleural mesothelium and bronchial epithelium in vivo and the induction of inducible NOS (iNOS) by asbestos fibers. Asbestos fibers caused DNA single-strand breaks, and the process was significantly enhanced by BSO (69% compared to the non-treated cells). A549 cells had a 3.5-fold glutathione content compared to MeT-5A cells, which was consistent with the higher resistance of these cells against oxidants and fibers. Flow cytometry of iNOS showed no change of iNOS by the fibers in either cell type in vitro. L-NAME had no effects on the DNA single-strand breaks in the Comet assay, either. Studies on lung biopsies showed that the immunoreactivities of both gamma-GCS subunits were very low in healthy human mesothelium in vivo. We conclude that glutathione may play an essential role in protecting intact cells against fiber-induced oxidative DNA alterations, and low gamma-GCS reactivity in pleural mesothelium may be associated with the high sensitivity of mesothelial cells to fiber-induced toxicity.     

Causes of DNA single-strand breaks during reduction of chromate by glutathione in vitro and in cells

Carcinogenic chromates induce DNA single-strand breaks (SSB) that are detectable by conventional alkali-based assays. However, the extent of direct breakage has been uncertain because excision repair and hydrolysis of Cr-DNA adducts at alkaline pH also generate SSB. We examined mechanisms of SSB production during chromate reduction by glutathione (GSH) and assessed the significance of these lesions in cells using genetic approaches. Cr(VI) reduction was biphasic and the formation of SSB occurred exclusively during the slow reaction phase. Catalase or iron chelators completely blocked DNA breakage, as did the use of GSH purified by a modified Chelex procedure. Thus, the direct intermediates of GSH-chromate reactions were unable to cause SSB unless activated by H2O2. SSB repair-deficient XRCC1(-/-) and proficient XRCC1+ EM9 cells had identical survival at doses causing up to 60% clonogenic death and accumulation of 1 mM Cr(VI). However, XRCC1(-/-) cells displayed higher lethality in the more toxic range and the depletion of GSH made them hypersensitive even to moderate doses. Elevation of cellular catalase or GSH levels eliminated survival differences between XRCC1(-/-) and XRCC1+ cells. In summary, formation of toxic SSB in cells occurs at relatively high chromate doses, requires H2O2, and is suppressed by high GSH concentrations.     

Direct evidence of intercellular sharing of glutathione via metabolic cooperation

Intracellular glutathione (GSH) content and cell density are known to be two important determinants of cell sensitivity to free radicals and radiation. We have investigated intercellular sharing of GSH via metabolic cooperation (MC) by measuring the GSH content of Chinese hamster V79 cells under conditions that varied MC among cells. GSH was measured by flow cytometry with monochlorobimane, which becomes fluorescent after conjugation to GSH by GSH-S-transferase. High-performance liquid chromatography was used to confirm the accuracy of GSH measurements by flow cytometry. Several lines of evidence indicate sharing of GSH or its precursor gamma-glutamylcysteine via MC. These include a cell density-dependent heterogeneity in GSH content, reconstitution of GSH in GSH-depleted cells by coculture with nondepleted cells (except when the depleted cells were MC deficient), and decreased equilibration of GSH among GSH-depleted cells and nondepleted cells when an inhibitor of MC (phorbol myristate acetate) was present. The equilibration of GSH among GSH-depleted cells and nondepleted cells in coculture was not inhibitable by acivicin, suggesting that this form of intercellular sharing of GSH does not rely on gamma-glutamyltransferase-mediated extracellular transport of GSH.     

Immunochemical detection of DNA damage induction and repair at different cellular stages of spermatogenesis of the hamster after in vitro or in vivo exposure to ionizing radiation

An immunochemical method has been used to detect quantitatively DNA damage caused by ionizing radiation in germ cells. With this method, DNA strand breaks as well as lesions converted into breaks in alkaline medium are measured as a function of controlled partial unwinding of the DNA, a time-dependent process starting at each breakage site, followed by the determination of the relative amount of single-stranded regions by use of a single-strand specific monoclonal antibody. With this method the induction and repair of DNA damage in different cellular stages of spermatogenesis (spermatocytes, round and elongated spermatids) of the hamster were investigated. Germ cells were irradiated in vitro with ⁶⁰Co-γ-rays, at doses between 0 and 5 Gy. A linear dose-response relationship was observed. Spermatocytes and round spermatids had normal, fast repair of the lesions when compared with the repair of these sites in cultured V79 or CHO cells and human lymphocytes. The elongated spermatids, however, showed hardly any repair. Similar results were obtained after the in vivo γ-irradiation of hamsters with doses of 0, 4, and 8 Gy and subsequent isolation of germ cells. The damage was still detectable in the elongated spermatids at 24 h after exposure. The results of the experiments show substantial differences in repair capacity between different stages of germ cell development. Because DNA is the major target for mutation induction, this assay may be useful for assessment of the genetic risk of exposure of male germ cells to ionizing radiation, in relation to the stage of development.