Acetaminophen-induced hepatotoxicity of gel entrapped rat hepatocytes in hollow fibers

An important application of primary hepatocyte cultures is for hepatotoxicity research. In this paper, gel entrapment culture of rat hepatocytes in miniaturized BAL system were evaluated as a potential in vitro model for hepatotoxicity studies in comparison to monolayer cultures. After exposure for 24 and 48 h to acetaminophen (2.5 mM), gel entrapped hepatocytes were more severely damaged than hepatocyte monolayer detected by methyl thiazolyl tetrazolium (MTT) reduction, intracellular glutathione (GSH) content, reactive oxygen species (ROS) levels, urea genesis and albumin synthesis. CYP 2E1 activities detected by 4-nitrocatechol (4-NC) formation were higher in gel entrapped hepatocytes than in hepatocyte monolayers while the addition of CYP 2E1 inhibitor, diethyl-dithiocarbamate (DDC), more significantly reduced acetaminophen-induced toxicity in gel entrapped hepatocytes. In addition, protective effects of GSH, liquorice extract and glycyrrhizic acid against acetaminophen hepatotoxicity were clearly observed in gel entrapped hepatocytes but not in hepatocyte monolayer at an incubation time of 48 h. Overall, gel entrapped hepatocytes showed higher sensitivities to acetaminophen-induced hepatotoxicity than hepatocyte monolayer by a mechanism that higher CYP 2E1 activities of gel entrapped hepatocytes could induce more severe acetaminophen toxicity. This indicates that gel entrapped hepatocytes in hollow fiber system could be a promising model for toxicological study in vitro.     

Culture duration alters the glutathione content and sensitivity to ethacrynic acid of rat hepatocyte monolayer cultures

Many of the differentiated functions of hepatocytes are lost in culture, yet addition of certain medium supplements can aid in the retention of differentiated character. Therefore, the effect of time in monolayer culture on rat hepatocyte glutathione (GSH) synthesis and sensitivity to the GSH detoxicated xenobiotic ethacrynic acid was examined in cultures with and without medium supplementation by transferrin and sodium selenite. GSH content was found to be about 12 nmol/µg DNA at 4 hr in culture and to approximately triple by 24 hr. Intracellular GSH levels continued to increase in transferrin/sodium selenite-supplemented cultures, from 32 to 41.6 nmol/µg DNA, while GSH levels in unsupplemented cultures declined to 18 nmol/µg DNA. However, the rate of GSH synthesis after diethylmaleate depletion was found to decrease from 4.2 to 2.8 nmol/hr/µg DNA at 4 and 24 hr after inoculation, respectively. GSH repletion rate increased to 3.9 nmol/hr/µg DNA at 48 hr. The GSH accumulation rate after depletion in supplemented cultures did not vary significantly over the initial 48 hr. Incubation for 3 hr with 100 µM ethacrynic acid (EA) did not elicit an increase in LDH leakage in hepatocyte monolayers after 4 or 48 hr in culture or in cultures with supplemented medium at any time point tested. Cultures 24 hr in medium without transferrin/sodium selenite supplementation exhibited significant LDH leakage after 3 hr of EA treatment. Over the 3 hr EA treatment, intracellular GSH content was decreased in all cultures. Only in the 24 hr unsupplemented cultures did GSH depletion exceed the 90% level previously associated with depletion of the mitochondrial pool of GSH and EA toxicity in hepatocytes. The experiments show that during the redifferentiation of hepatocytes in culture, a transient period occurs when apparent GSH synthesis is depressed and enhanced sensitivity to GSH-detoxicated compounds is observed. This period of increased sensitivity is prevented or at least delayed by inclusion of supplemental transferrin and sodium selenite, suggesting that redifferentiation can be regulated by extracellular influences.Abbreviations CYSSG cysteine-glutathione mixed disulfide - DEM diethyl maleate - EA ethacrynic acid - GSH reduced glutathione - GSSG oxidized glutathione - HBS HEPES buffered saline - HWME hepatocyte Williams' Medium E (WME with insulin, corticosterone and 0.5 mM methionine) - LDH lactate dehydrogenase - TS-HWME transferrin/sodium selenite-supplemented HWME - WME Williams' Medium E     

Co-culture of primary rat hepatocytes with rat liver epithelial cells enhances interleukin-6-induced acute-phase protein response

Three different primary rat hepatocyte culture methods were compared for their ability to allow the secretion of fibrinogen and albumin under basal and IL-6-stimulated conditions. These culture methods comprised the co-culture of hepatocytes with rat liver epithelial cells (CC-RLEC), a collagen type I sandwich culture (SW) and a conventional primary hepatocyte monolayer culture (ML). Basal albumin secretion was most stable over time in SW. Fibrinogen secretion was induced by IL-6 in all cell culture models. Compared with ML, CC-RLEC showed an almost three-fold higher fibrinogen secretion under both control and IL-6-stimulated conditions. Induction of fibrinogen release by IL-6 was lowest in SW. Albumin secretion was decreased after IL-6 stimulation in both ML and CC-RLEC. Thus, cells growing under the various primary hepatocyte cell culture techniques react differently to IL-6 stimulation with regard to acute-phase protein secretion. CC-RLEC is the preferred method for studying cytokine-mediated induction of acute-phase proteins, because of the pronounced stimulation of fibrinogen secretion upon IL-6 exposure under these conditions.     

Regulation of apoptosis by glutathione redox state in PC12 cells exposed simultaneously to iron and ascorbic acid

We previously reported that the levels of non-protein-bound iron (NPBI) and ascorbic acid (AA) are markedly increased in the cerebrospinal fluid of infants with perinatal asphyxia. The present study showed that FeSO4 and AA synergistically induced apoptosis of PC12 cells, which was prevented by alpha-tocopherol and glutathione (GSH) ethyl ester. Markers of free radical damage, such as ortho-tyrosine, meta-tyrosine, and F(2alpha)-isoprostane, showed a gradual increase. AA and ferrous NPBI disappeared rapidly from the culture medium, but exposure for only a few hours was sufficient to trigger apoptosis. Intracellular GSH decreased progressively along with a concomitant increase of glutathione disulfide (GSSG). The baseline half-cell reduction potential (Ehc) for GSSG, 2H+/2GSH couple was -246 mV and an Ehc of -200 mV was the critical level to switch on apoptosis, although some cells escaped this fate by transient increase of intracellular GSH. Once Ehc reached around -165 mV (81 mV oxidation from the baseline), all cells lost the ability to maintain an adequate intracellular GSH level and subsequently underwent apoptosis. These findings at least partly explain the mechanism of Fe-AA cytotoxicity, in that ferrous iron catalyzes hydroxyl radical generation and induces lipid peroxidation, after which subsequent depletion of GSH raises Ehc to the critical level for triggering or potentiating the apoptotic cascade.     

Depletion of Intracellular Glutathione Increases Susceptibility to Nitric Oxide in Mesencephalic Dopaminergic Neurons

Using primary neuronal cultures, we investigated the effects of GSH depletion on the cytotoxic effects of glutamate and NO in dopaminergic neurons. Intracellular GSH was depleted by 24-h exposure to L-buthionine-[S,R]-sulfoximine (BSO), an irreversible inhibitor of GSH synthase. BSO exposure caused concentration-dependent reduction of the viability of both dopaminergic and nondopaminergic neurons. In contrast, 24-h exposure of cultures to glutamate or NOC18, an NO-releasing agent, significantly reduced the viability of nondopaminergic neurons without affecting that of dopaminergic neurons. Pretreatment with N-acetyl-L-cysteine for 24 h ameliorated the NOC18-induced toxicity in nondopaminergic neurons. In dopaminergic neurons, sublethal concentrations of BSO reduced intracellular GSH content and markedly potentiated glutamate- and NOC18-induced toxicity. These results suggested that glutamate toxicity was enhanced in dopaminergic neurons by suppression of defense mechanisms against NO toxicity under conditions of GSH depletion. Under such conditions, free iron plays an important role because BSO-enhanced NO toxicity was ameliorated by the iron-chelating agent, deferoxamine. These results suggest that GSH plays an important role in the expression of NO-mediated glutamate cytotoxicity in dopaminergic neurons. Free iron may be related to enhanced NO cytotoxicity under GSH depletion.     

Alteration of intracellular GSH levels and its role in microcystin-LR-induced DNA damage in human hepatoma HepG2 cells

Microcystin-LR (MCLR) is a liver-specific toxin known as a tumour promoter in experimental animals. Its mechanisms of hepatotoxicity have been well documented; however, the mechanisms of other effects, in particular those related to its genotoxicity, are not well understood. In our previous studies, we showed that MCLR-induced DNA strand breaks are transiently present and that the damage is mediated by reactive oxygen species (ROS). In this study, we show that exposure of HepG2 cells to non-cytotoxic doses of MCLR-induced time-dependent alterations in the level of intracellular reduced glutathione (GSH). These comprised a rapid initial decrease followed by a gradual increase, reaching a maximum after 6h of exposure, before returning to the control level after 8h. During the first 4h, expression of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, increased, indicating an increased rate of de novo synthesis of GSH. The most important observation of this study, combined with the results of our previous studies is the correlation between the time course of alterations of intracellular GSH content and the formation and disappearance of MCLR-induced DNA damage. When the intracellular GSH level was reduced, MCLR-induced DNA damage was observed to increase. Later, when the level of intracellular GSH was normal or elevated, new DNA damage was not induced and existing damage was repaired. To confirm the role of GSH system in MCLR-induced genotoxicity, the intracellular GSH level was moderated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and with N-acetylcysteine (NAC), a GSH precursor. Pre-treatment with BSO dramatically increased the susceptibility of HepG2 cells to MCLR-induced DNA damage, while pre-treatment with NAC almost completely prevented MCLR-induced DNA damage. Thus, intracellular GSH is shown to play a critical role in the cellular defence against MCLR-induced DNA damage in HepG2 cells.     

Metabolic flux analysis of cultured hepatocytes exposed to plasma

Hepatic metabolism can be investigated using metabolic flux analysis (MFA), which provides a comprehensive overview of the intracellular metabolic flux distribution. The characterization of intermediary metabolism in hepatocytes is important for all biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. During BAL operation, hepatocytes are exposed to plasma or blood from the patient, at which time they are prone to accumulate intracellular lipids and exhibit poor liver-specific functions. In a prior study, we found that preconditioning the primary rat hepatocytes in culture medium containing physiological levels of insulin, as opposed to the typical supraphysiological levels found in standard hepatocyte culture media, reduced lipid accumulation during subsequent plasma exposure. Furthermore, supplementing the plasma with amino acids restored hepatospecific functions. In the current study, we used MFA to quantify the changes in intracellular pathway fluxes of primary rat hepatocytes in response to low-insulin preconditioning and amino acid supplementation. We found that culturing hepatocytes in medium containing lower physiological levels of insulin decreased the clearance of glucose and glycerol with a concomitant decrease in glycolysis. These findings are consistent with the general notion that low insulin, especially in the presence of high glucagon levels, downregulates glycolysis in favor of gluconeogenesis in hepatocytes. The MFA model shows that, during subsequent plasma exposure, low-insulin preconditioning upregulated gluconeogenesis, with lactate as the primary precursor in unsupplemented plasma, with a greater contribution from deaminated amino acids in amino acid-supplemented plasma. Concomitantly, low-insulin preconditioning increased fatty acid oxidation, an effect that was further enhanced by amino acid supplementation to the plasma. The increase in fatty acid oxidation reduced intracellular triglyceride accumulation. Overall, these findings are consistent with the notion that the insulin level in medium culture presets the metabolic machinery of hepatocytes such that it directly impacts on their metabolic behavior during subsequent plasma culture.     

Alteration of intracellular GSH levels and its role in microcystin-LR-induced DNA damage in human hepatoma HepG2 cells

Microcystin-LR (MCLR) is a liver-specific toxin known as a tumour promoter in experimental animals. Its mechanisms of hepatotoxicity have been well documented; however, the mechanisms of other effects, in particular those related to its genotoxicity, are not well understood. In our previous studies, we showed that MCLR-induced DNA strand breaks are transiently present and that the damage is mediated by reactive oxygen species (ROS). In this study, we show that exposure of HepG2 cells to non-cytotoxic doses of MCLR-induced time-dependent alterations in the level of intracellular reduced glutathione (GSH). These comprised a rapid initial decrease followed by a gradual increase, reaching a maximum after 6 h of exposure, before returning to the control level after 8 h. During the first 4 h, expression of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis, increased, indicating an increased rate of de novo synthesis of GSH. The most important observation of this study, combined with the results of our previous studies is the correlation between the time course of alterations of intracellular GSH content and the formation and disappearance of MCLR-induced DNA damage. When the intracellular GSH level was reduced, MCLR-induced DNA damage was observed to increase. Later, when the level of intracellular GSH was normal or elevated, new DNA damage was not induced and existing damage was repaired. To confirm the role of GSH system in MCLR-induced genotoxicity, the intracellular GSH level was moderated by pre-treatment with buthionine-(S,R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and with N-acetylcysteine (NAC), a GSH precursor. Pre-treatment with BSO dramatically increased the susceptibility of HepG2 cells to MCLR-induced DNA damage, while pre-treatment with NAC almost completely prevented MCLR-induced DNA damage. Thus, intracellular GSH is shown to play a critical role in the cellular defence against MCLR-induced DNA damage in HepG2 cells.     

An intracellular mechanism of aluminum tolerance associated with high antioxidant status in cultured tobacco cells

An aluminum (Al) tolerance mechanism, together with oxidative stress tolerance, was investigated in an Al tolerant cell line (ALT301) and the parental Al sensitive cell line (SL) of tobacco. During Al exposure in a simple calcium solution for 24 h, Al triggered the evolution of a reactive oxygen species (ROS) in SL much higher than ALT301 [Plant Physiol. 128 (2002) 63]. Under the conditions, Al enhanced comparable rates of citrate secretion from both cell lines to the same extent. Al enhanced the gene expression of manganese superoxide dismutase (MnSOD) in both cell lines, but at a significantly higher rate in SL than in ALT301, and also enhanced the enzyme activity of MnSOD in both cell lines to nearly the same level. These results suggest that the extracellular chelation of Al with organic acids and MnSOD is not involved in the mechanism of Al tolerance of ALT301. ALT301 contained ascorbate (ASA) and glutathione (GSH) levels that were higher than SL under normal growth conditions. During 24 h of post-Al treatment culture in growth medium, but not during 24-h Al exposure in a simple Ca(2+) solution, lipid peroxidation was enhanced in SL much higher than in ALT301, and the average SL amounts of ASA and GSH were exhausted compared to ALT301. Pre-loading of ASA prior to Al treatment improved the growth of SL during the post-Al treatment culture. ALT301 also exhibited cross-tolerance to H(2)O(2), Fe(2+) and Cu(2+). Under these oxidant exposures, ALT301 contained lower levels of intracellular H(2)O(2) or lipid peroxides, and maintained higher amounts of ASA and GSH than SL. Taken together, we conclude that the accumulation of Al in cells enhances the peroxidation of lipids exclusively under growing conditions, and that the higher content of ASA and GSH in ALT301 than in SL seems to be in part responsible for the tolerance mechanism of ALT301 to Al by protecting cells from either lipid peroxidation or H(2)O(2) commonly enhanced by Al or other oxidants.     

Multidrug resistance-associated protein 1 as a major mediator of basal and apoptotic glutathione release

The proteins responsible for reduced glutathione (GSH) export under both basal conditions and in cells undergoing apoptosis have not yet been identified, although recent studies implicate some members of the multidrug resistance-associated protein family (MRP/ABCC) in this process. To examine the role of MRP1 in GSH release, the present study measured basal and apoptotic GSH efflux in HEK293 cells stably transfected with human MRP1. MRP1-overexpressing cells had lower intracellular GSH levels and higher levels of GSH release, under both basal conditions and after apoptosis was induced with either Fas antibody or staurosporine. Despite the enhanced GSH efflux in MRP1-overexpressing cells, intracellular GSH levels were not further depleted when cells were treated with Fas antibody or staurosporine, suggesting an increase in GSH synthesis. MRP1-overexpressing cells were also less susceptible to apoptosis, suggesting that the stable intracellular GSH levels may have protected cells from death. Overall, these results demonstrate that basal and apoptotic GSH release are markedly enhanced in cells overexpressing MRP1, suggesting that MRP1 plays a key role in these processes. The enhanced GSH release, with a concurrent decrease of intracellular GSH, appears to be necessary for the progression of apoptosis.     

Transforming growth factor beta (TGF-beta) inhibits hepatocyte DNA synthesis independently of EGF binding and EGF receptor autophosphorylation

Subpicomolar concentrations of human platelet-derived transforming growth factor beta (TGF-beta) inhibited growth factor-stimulated DNA synthesis in primary cultures of adult rat hepatocytes. This inhibition was not the result of changes in the size of intracellular pools of 3H-thymidine and was not dependent on the state of confluence of the cells. A 24-hr exposure to TGF-beta either before or after insulin/EGF stimulation was as inhibitory on DNA synthesis between 48 and 72 hr of culture as was TGF-beta present throughout 72 hr of culture. From 12 hr in culture to 24 hr, hepatocyte EGF binding sites dropped from about 230,000 to 85,000 per cell with no significant change in Kd, but with a loss in capacity for EGF-induced receptor down-regulation. Maximally inhibitory concentrations of TGF-beta did not compete with EGF for the EGF receptor, and a 4- to 24-hr exposure to TGF-beta did not alter subsequent EGF binding. Coincubation of hepatocytes with TGF-beta and EGF did not influence the 60% reduction in EGF binding sites produced by EGF alone. In addition, TGF-beta did not prevent EGF-induced autophosphorylation of the 170,000 dalton EGF receptor in membranes from whole liver. Our studies suggest that TGF-beta regulates hepatocyte growth independently of changes in EGF receptor number, ligand affinity, or postbinding autophosphorylation.     

Effects of intracellular cyclic AMP and cyclic GMP levels on DNA synthesis of young-adult rat hepatocytes in primary culture.

Possible roles of dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) and dibutyryl-guanosine 3',5'-cyclic monophosphate (cGMP) in regulation of hepatocyte DNA synthesis were examined using primary cultures of young-adult rat hepatocytes maintained in arginine-free medium. Throughout the experimental period, nonparenchymal cells were hardly observed in the selective medium. When epidermal growth factor (EGF) was added to the cultures, a transient increase in the intracellular cAMP level preceded the elevation of hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was remarkably enhanced by the elevation of the intracellular cAMP level induced by treatment with cAMP alone or a combination of cAMP and theophylline, an inhibitor of cyclic nucleotide phosphodiesterase. Furthermore, the early elevation of intracellular cAMP alone, which was induced by treatment with the combination of cAMP and theophylline, caused a remarkable increase in hepatocyte DNA synthesis. On the other hand, addition of EGF to the cultures caused a rapid decrease in the intracellular cGMP level followed by an increase in hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was severely suppressed or completely inhibited by the elevation of the intracellular cGMP level induced by treatment with cGMP alone or a combination of cGMP and dipyridamole, a specific inhibitor of cGMP phosphodiesterase. These findings indicate that cAMP and cGMP act oppositely on the regulation of DNA synthesis of young-adult rat hepatocytes in primary culture: cAMP plays a positive role, whereas cGMP plays a negative role. Also it is strongly suggested that an early elevation of the intracellular cAMP level is essential for the onset of DNA synthesis in hepatocyte primary cultures.     

A novel 3D liver organoid system for elucidation of hepatic glucose metabolism

Hepatic glucose metabolism is a key player in diseases such as obesity and diabetes as well as in antihyperglycemic drugs screening. Hepatocytes culture in two-dimensional configurations is limited in vitro model for hepatocytes to function properly, while truly practical platforms to perform three-dimensional (3D) culture are unavailable. In this work, we present a practical organoid culture method of hepatocytes for elucidation of glucose metabolism under nominal and stress conditions. Employing this new method of culturing cells within a hollow fiber reactor, hepatocytes were observed to self-assemble into 3D spherical organoids with preservation of tight junctions and display increased liver-specific functions. Compared to both monolayer culture and sandwich culture, the hepatocyte organoids displayed higher intracellular glycogen content, glucose consumption, and gluconeogenesis and approached the in vivo values, as also confirmed by gene expression of key enzymes. Moreover, hepatocyte organoids demonstrated more realistic sensitivity to hormonal challenges with insulin, glucagon, and dexamethasone. Finally, the exposure to high glucose demonstrated toxicities including alteration of mitochondrial membrane potential, lipid accumulation, and reactive oxygen species formation, similar to the in vivo responses, which was not captured by monolayer cultures. Collectively, hepatocyte organoids mimicked the in vivo functions better than hepatocyte monolayer and sandwich cultures, suggesting suitability for applications such as antihyperglycemic drugs screening.     

Glutathione regulates interleukin-2 activity on cytotoxic T-cells

In this study, we examined whether and how the cellular activity of interleukin-2 (IL-2) is affected by glutathione (GSH), an important tripeptide existing in most cells. Cell culture and thymidine incorporation assay showed that addition of GSH enhanced the effect of IL-2 on the proliferation and thymidine incorporation of IL-2-dependent cytotoxic T-cells such as CTLL-2 and CT-4R. Treatment of the cells with GSH resulted in a 2-fold increase in the amount of IL-2 bound to the cells and a rapid internalization of the bound IL-2. In addition, the degradation of IL-2 in the cells was enhanced by GSH treatment. These effects of GSH were accompanied by an increase in the intracellular GSH level. L-Buthionine-(S,R)-sulfoximine, an inhibitor of de novo GSH synthesis, blunted the increase of intracellular GSH level and modulated the effect of GSH on IL-2 activity. These results suggest that GSH regulates the binding, internalization, degradation, and T-cell proliferative activity of IL-2; alterations of cellular GSH concentration may thus affect the growth and replication of IL-2-sensitive cytotoxic T-cells.     

Oxidative stress potentially enhances FcεRI-mediated leukotriene C4 release dependent on the late-phase increase of intracellular glutathione in mast cells

Cysteinyl leukotrienes (cysLTs), which include leukotriene C₄ (LTC₄), are the predominant class of LTs synthesized by mast cells. CysLTs can induce many of the abnormalities seen in asthma. LTC₄ is generated by the conjugation of LTA₄ with reduced glutathione (GSH) by LTC₄ synthase. During screening of the effects of prostanoids on high-affinity IgE receptor (FcεRI)-mediated LTC₄ release from mast cells, we realized that some prostanoids, including ONO-AE1-259-01 and ONO-AE-248, inhibited LTC₄ release, which was associated with a decrease in the amount of intracellular total GSH. We ascertained that l-buthionine-S,R-sulfoximine (BSO), a selective inhibitor of glutamate-cysteine ligase, inhibited LTC₄ release. In addition, cell-permeable GSH, the glutathione reduced form ethyl ester (GSH-OEt), enhanced LTC₄ release in accordance with the change in intracellular total GSH. Depletion of intracellular total GSH induced by ONO-AE-248 or BSO enhanced FcεRI-mediated LTB₄ release in contrast to LTC₄. Oxidative stress contributes to many pathological conditions including asthma. GSH is a major soluble antioxidant and a cofactor for several detoxifying enzymes including GSH peroxidase. Exposure of mast cells to hydrogen peroxide (H₂O₂) or diamide to mimic oxidative stress unexpectedly increased rather than decreased the intracellular reduced GSH content as well as total GSH in the late phase (i.e., 24 or 48 h after exposure), which was accompanied by an increase in LTC₄ release. In conclusion, FcεRI-mediated LTC₄ release from mast cells is mainly regulated by the amount of intracellular GSH. In some cases, oxidative stress may induce a late-phase increase in intracellular GSH, resulting in enhanced LTC₄ release from mast cells.     

Hormone- and substrate-regulated intracellular degradation of cytochrome P450 (2E1) involving MgATP-activated rapid proteolysis in the endoplasmic reticulum membranes.

We have examined differences in post-translational regulation between rat liver ethanol-inducible cytochrome P450 2E1 (CYP2E1) and phenobarbital-inducible CYP2B1 using hepatocyte cultures and subcellular fractions, prepared from starved and acetone-treated rats. The intracellular degradation of CYP2E1 was rapid (approximate t1/2 = 9 h) and increased by glucagon treatment of the cells in an isozyme-specific manner, whereas CYP2B1 degradation in the same cells, was slower (t1/2 = 21 h). The glucagon effect on CYP2E1 degradation was abolished by either cycloheximide treatment of cells, indicating the involvement of protein components with rapid turnover, or by lowering of the culture temperature to 23 degrees C. The rapid phase of CYP2E1 degradation was not influenced by inhibitors of the autophagosomal/lysosomal pathway. In vitro experiments with isolated liver microsomes revealed the presence of a Mg(2+)-ATP-activated proteolytic system active on CYP2E1, previously modified by phosphorylation on Ser-129 or denatured by reactive metabolites formed from carbon tetrachloride. Imidazole, a CYP2E1 substrate, specifically inhibited the rapid intracellular degradation of CYP2E1 and also prevented phosphorylation and subsequent proteolysis in isolated microsomes. In contrast, no proteolysis of CYP2B1 occurred under the conditions used. The microsomal Mg(2+)-ATP-dependent CYP2E1 proteolysis could not be solubilized with high salt and 0.05% sodium cholate, indicating the action of membrane-integrated protease(s). Subfractionation of microsomes revealed that the Mg(2+)-ATP-dependent proteolytic system active on CYP2E1 was present in both rough and smooth endoplasmic reticulum. It is suggested that hepatic cytochromes P450 are degraded both in a bulk process, according to the autophagosomal/lysosomal pathway and more rapidly, in a hormone- and substrate-regulated fashion, by a specific proteolytic system in the endoplasmic reticulum, active on physiologically or exogenously modified molecules.     

Depletion of glutathione after gamma irradiation modifies survival

The relationship between the intracellular glutathione (GSH) concentration and the aerobic radiation response was studied in Chinese hamster ovary cells. Various degrees of GSH depletion were produced by exposure to buthionine sulfoximine (BSO) and/or diethyl maleate (DEM). Diethyl maleate did not act as a classical radiosensitizer under the experimental conditions employed, nor did exposure to DEM/BSO nonspecifically affect protein thiols as measured by thiol blotting. Dose-response curves were obtained using cells irradiated in the absence or presence of DEM/BSO, which decreased GSH levels by 90-95%. Exposure to DEM/BSO did not affect the formation of DNA single-strand breaks or DNA-protein crosslinks measured immediately after irradiation performed at ice temperatures. Analysis of survival curves indicated that the Dq was decreased by 18% when GSH depletion occurred prior to, during, and after irradiation. The DEM/BSO exposure did not affect D0. To study postirradiation conditions, cells were exposed to 10 microM DEM prior to and during irradiation, which was performed at ice temperatures. Levels of GSH were depleted by 75% by this protocol. Immediately after irradiation, the cells were rapidly warmed by the addition of 37 degrees C growth medium containing either 10 or 90 microM DEM. Addition of 10 microM DEM after irradiation did not affect the degree of depletion, which remained constant at 75%. In contrast, GSH depletion was increased to 90% 10 min after addition of the 90 microM DEM. Addition of 90 microM DEM after irradiation produced a statistically significant difference in survival compared to addition of 10 microM DEM. In a second depletion protocol, cells were exposed to 100 microM DEM at room temperature for 5 min, irradiated, incubated at 37 degrees C for 1 h, washed, and then incubated in 50 microM BSO for 24 h. This depletion protocol reduced survival by a factor of 2.6 compared to cells not exposed to the combination of DEM/BSO. Survival was not affected if the cells were exposed to the DEM or BSO alone. This was interpreted to indicate that survival was not affected by GSH depletion occurring after irradiation unless depletion was rapid and sustained. The rate of repair of sublethal and potentially lethal damage was measured and found to be independent of the DEM/BSO exposure. These experimental results in addition to previous ones (Freeman and Meredith, Int. J. Radiat. Oncol. Biol. Phys. 13, 1371-1375, 1987) were interpreted to indicate that under aerobic conditions GSH depletion may alter the expression of radiation damage by affecting metabolic fixation.     

Glutathione modulates the toxicity of, but is not a biologically relevant reductant for, the Pseudomonas aeruginosa redox toxin pyocyanin

Pyocyanin is an important redox toxin produced by the common human pathogen Pseudomonas aeruginosa. It generates reactive oxygen species (ROS) that alter intracellular redox status and cell function. Reducing equivalents for pyocyanin are provided by intracellular NAD(P)H and, it has been reported, glutathione (GSH). Cellular GSH levels are at least 1-2 orders of magnitude greater than NAD(P)H; therefore GSH should represent the major reductant for pyocyanin and potentiate its toxicity. Paradoxically, GSH has been found to inhibit pyocyanin toxicity in cellular models. This study was undertaken to evaluate the potential of GSH as a biologically relevant reductant for pyocyanin. As observed using spectrophotometry, under aerobic conditions pyocyanin readily oxidized NADPH, whereas oxidation of GSH could not be detected. Under anaerobic conditions pyocyanin was reduced by NADPH, but reduction by GSH could not be detected. Reduction of molecular oxygen and the formation of ROS readily proceeded in the presence of pyocyanin and NADPH, whereas GSH was without effect. Finally, exposure of normal human dermal fibroblasts to subcytotoxic concentrations of pyocyanin did not lead to depletion of endogenous GSH, but exogenous GSH provided protection against the senescence-inducing effects of the toxin. In summary, GSH does not reduce pyocyanin under physiologically relevant conditions or contribute to pyocyanin toxicity. However, GSH does provide protection against the deleterious effects of this important bacterial toxin on mammalian cells.