Cytotoxicity of menadione and related quinones in freshly isolated rat hepatocytes: effects on thiol homeostasis and energy charge

The cytotoxic events in freshly isolated rat hepatocytes following exposure over 2 h to menadione (2-methyl-1,4-naphthoquinone) and two closely related quinones, 2,3-dimethyl-1,4-naphthoquinone (DMNQ) and 1,4-naphthoquinone (NQ), were examined. These quinones differ in their arylation capacity (NQ > menadione DMNQ) and in their potential to induce redox cycling (NQ menadione DMNQ). The glutathione status (reduced and oxidized glutathione) of the hepatocytes was determined using HPLC after derivatization with monobromobimane. Protein thiols were measured spectrophotometrically and the energy charge of the cells was determined with HPLC using ion pair chromatography. The leakage of lactate dehydrogenase was used as a marker for cell viability. All three quinones caused alterations of the glutathione status of the exposed cells but the effects were markedly different. Exposure to DMNQ resulted in a slow decrease of reduced glutathione and an increase of mixed disulfides. The other two quinones caused an almost complete depletion of reduced glutathione within 5 min. Hepatocytes exposed to NQ accumulated oxidized glutathione whereas menadione-exposed hepatocytes showed increased levels of mixed disulfides. We did not find any effects of DMNQ (200 M) on protein thiols, energy charge or cell viability. There was a clear difference in the effects of menadione and NQ on protein thiols, energy charge and cell viability: exposure to NQ resulted in a more extensive decrease of protein thiols and energy charge and an earlier onset of lactate dehydrogenase leakage. From our results we conclude that the arylation capacity of a quinone is a determining factor in the cytotoxic potential of such compounds and that the decrease of protein thiols and of the energy charge are critical events preceding loss of cell viability.     

TEMPERATURE-DEPENDENT QUINONE CYTOTOXICITY IN PLATELETS INVOLVES ARYLATION

Menadione (MEN), a representative quinone compound, produces cytotoxicity in many cells by arylation with protein thiols and oxidative stress due to redox cycling. Previously it was demonstrated that protein arylation appears to be a primary mechanism for MEN-induced toxicity in platelets. To test the hypothesis that temperature conditions may be important in MEN-induced cytotoxicity in noncancer cells, platelets were incubated with menadione at 25, 37, or 42°C. As temperature was increased, MEN significantly enhanced lactate dehydrogenase (LDH) leakage. MEN-induced depletion of protein thiol levels also increased as temperature was elevated. To investigate the mechanism of temperature-dependent MEN cytotoxicity, MEN-induced platelet toxicity was compared to two other quinone substances. Benzoquinone (BQ), which acts via arylation, produced cytotoxic effects similar to those of MEN. Dimethoxy-1,4-naphthoquinone (DMNQ), which exerts toxicity via oxidative radical generation, failed to produce cytotoxicity at all three temperatures. While MEN and DMNQ enhanced O 2 consumption in a temperature-dependent manner, BQ did not affect this parameter. MEN, which possesses an electrophilic 3-position, was found to react with thiols to form a thioether linkage, a direct indicator of arylation. In the case of MEN uptake kinetics, the amount of cellular uptake was not different at various temperatures, but concentration of MEN in extracellular medium decreased temperature dependently. This might be due to increased arylation capacity binding to cellular proteins as temperature rises. These data suggest that MEN-induced platelet cytotoxicity involves arylation that is temperature related.     

Temperature-dependent quinone cytotoxicity in platelets involves arylation

Menadione (MEN), a representative quinone compound, produces cytotoxicity in many cells by arylation with protein thiols and oxidative stress due to redox cycling. Previously it was demonstrated that protein arylation appears to be a primary mechanism for MEN-induced toxicity in platelets. To test the hypothesis that temperature conditions may be important in MEN-induced cytotoxicity in noncancer cells, platelets were incubated with menadione at 25, 37, or 42 degrees C. As temperature was increased, MEN significantly enhanced lactate dehydrogenase (LDH) leakage. MEN-induced depletion of protein thiol levels also increased as temperature was elevated. To investigate the mechanism of temperature-dependent MEN cytotoxicity, MEN-induced platelet toxicity was compared to two other quinone substances. Benzoquinone (BQ), which acts via arylation, produced cytotoxic effects similar to those of MEN. Dimethoxy-1,4-naphthoquinone (DMNQ), which exerts toxicity via oxidative radical generation, failed to produce cytotoxicity at all three temperatures. While MEN and DMNQ enhanced O(2) consumption in a temperature-dependent manner, BQ did not affect this parameter. MEN, which possesses an electrophilic 3-position, was found to react with thiols to form a thioether linkage, a direct indicator of arylation. In the case of MEN uptake kinetics, the amount of cellular uptake was not different at various temperatures, but concentration of MEN in extracellular medium decreased temperature dependently. This might be due to increased arylation capacity binding to cellular proteins as temperature rises. These data suggest that MEN-induced platelet cytotoxicity involves arylation that is temperature related.     

Mechanisms of toxicity of 2- and 5-hydroxy-1,4-naphthoquinone; absence of a role for redox cycling in the toxicity of 2-hydroxy-1,4-naphthoquinone to isolated hepatocytes

The mechanisms of toxicity to isolated rat hepatocytes of two structurally related naphthoquinones have been studied. Both 5-OH-1,4-naphthoquinone (5-OH-1,4-NQ; juglone) and 2-OH-1,4-naphthoquinone (2-OH-1,4-NQ; lawsone) caused a concentration-dependent cytotoxicity to hepatocytes which was preceded by a depletion of intracellular glutathione. 5-OH-1,4-NQ caused a depletion of intracellular glutathione when incubated either at 4 degrees C or 37 degrees C whereas 2-OH-1,4-NQ caused a depletion of intracellular glutathione when the hepatocytes were incubated at 37 degrees C but not at 4 degrees C. 5-OH-1,4-NQ but not 2-OH-1,4-NQ reacted with glutathione in buffered solution. These results suggested that the depletion of intracellular glutathione by 2-OH-1,4-NQ is enzyme mediated whereas in the case of 5-OH-1,4-NQ the direct chemical reaction with gluathione may be largely responsible for the depletion. A critical role for depletion of protein thiols in menadione-induced cytotoxicity has been proposed. In agreement with earlier work, menadione caused a decrease in protein sulphydryls prior to cell death, however, at cytotoxic concentrations of both 2-OH-1,4-NQ and 5-OH-1,4-NQ this decrease only accompanied rather than preceeded cell death. The mechanism of toxicity of 5-OH-1,4-NQ is similar to that of other naphthoquinones and involves formation of its corresponding naphthosemiquinone, active oxygen species and redox cycling as it stimulated a disproportionate increase in both microsomal NADPH oxidation and oxygen consumption.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of toxicity of naphthoquinones to isolated hepatocytes

The possible mechanisms of naphthoquinone-induced toxicity to isolated hepatocytes were investigated using three structurally-related naphthoquinones, 1,4-naphthoquinone (1,4-NQ), 2-methyl-1,4-naphthoquinone (2-Me-1,4-NQ) and 2,3-dimethyl-1, 4-naphthoquinone (2,3-diMe-1,4-NQ). 1,4-NQ was more toxic than 2-Me-1,4-NQ whereas 2,3-diMe-1,4-NQ did not cause cell death at the solubility-limited concentrations used. All three naphthoquinones extensively depleted intracellular glutathione (GSH). However, the depletion of GSH induced by 1,4-NQ and 2-Me-1,4-NQ prior to cell death was more rapid and extensive than that induced by the nontoxic 2,3-diMe-1,4-NQ. Further studies demonstrated that 2,3-diMe-1,4-NQ was cytotoxic in the presence of dicoumarol, a compound which also potentiates the cytotoxicity of 1,4-NQ and 2-Me-1,4-NQ. To investigate the differential cytotoxicity of these three naphthoquinones, their relative capacities to redox cycle and to bind covalently to cellular nucleophiles were assessed. Redox cycling was investigated using rat liver microsomes where the order of potency for quinone-stimulated redox cycling was 1,4-NQ approximately 2-Me-1,4-NQ much greater than 2,3-diMe-1,4-NQ as indicated by nonstoichiometric amounts of NADPH oxidation and O2 consumption. NADPH-cytochrome P-450 reductase was implicated as the enzyme primarily responsible for naphthoquinone-stimulated redox cycling. The reactivity of the naphthoquinones with glutathione and, by implication, with other cellular nucleophiles was 1,4-NQ greater than 2-Me-1,4-NQ much greater than greater than 2,3-diMe-1,4-NQ. Overall, these studies indicate that 2,3-diMe-1,4-NQ is not cytotoxic (except in the presence of dicoumarol) and this lack of toxicity may be related either to its lesser capacity to redox cycle and/or its inability to react directly with cellular nucleophiles.     

Quinone-induced DNA single strand breaks in rat hepatocytes and human chronic myelogenous leukaemic K562 cells.

In rat hepatocytes exposed to the quinones menadione and 2,3-dimethoxy-1,4-naphthoquinone (2,3-diOMe-1,4-NQ) a decrease in NAD+ is observed. DNA damage and activation of poly(ADP-ribose)polymerase are often associated with a decrease in NAD+. Using rat hepatocytes and human myeloid leukaemic cells (K562), we examined the extent of DNA damage induced by these quinones at non-toxic concentrations, i.e. at concentrations at which the cells completely exclude the dye trypan blue. Both quinones caused significant DNA damage at very low concentrations (5-100 microM). With 2,3-diOME-1,4-NQ (15 microM) or menadione (15 microM) single strand breaks (SSB) were observed at very early time points (less than 5 min), reaching a maximum between 20 and 30 min. Most SSB were repaired within 45 min of the removal of the quinones. Whilst extensive repair was observed within 4 hr of the removal of 2,3-diOMe-1,4-NQ (15 microM), only partial repair was observed following exposure to menadione (15 microM). SSB induced by 2,3-diOMe-1,4-NQ (15 microM) were completely inhibited by the iron chelator 1,10-phenanthroline (25 microM), whereas in cells exposed to menadione (15 microM) they were only partially inhibited. Finally, although the membrane integrity of K562 cells was unaffected by exposure to high concentrations of both quinones (less than or equal to 400 microM), cytostasis was observed at much lower concentrations (50 microM). Our results demonstrate that at very low concentrations these quinones induce extensive DNA damage possibly caused by hydroxyl radicals. The DNA damage was accompanied by an early cytostasis but no loss of membrane integrity.     

Important role of glutathione in protecting against menadione-induced cytotoxicity in rat platelets

Our previous studies demonstrate that menadione (MEN) is cytotoxic to platelets of rats by depleting glutathione (GSH). In order to clarify whether GSH has a role in protecting against menadione-induced cytotoxicity, the effect of GSH depletors as well as GSH precusors on menadione-induced cytotoxicity was investigated. Cysteine and dithiothreitol (DTT) prevent MEN-induced cytotoxicity in a dose-dependent manner, as determined by LDH leakage and change in turbidity. When platelets were treated with 1-chloro-2,4-dinitrobenzene (CNDB) and diethylmaleate (DEM), both of which deplete intracellular GSH, MEN-induced cytotoxicity was potentiated in the CDNB-treated platelets, but not in the DEM-treated platelets. These data suggest that the GSH in platelets plays an important role in protecting against cytotoxicity induced by menadione.     

Induction of cell damage by menadione and benzo(a)pyrene-3,6-quinone in cultures of adult rat hepatocytes and human fibroblasts

The cytotoxicity of menadione (2-methyl-1,4-naphthoquinone) and benzo(a)pyrene-3,6-quinone (BP-3,6-Q) was tested in cultures of adult rat hepatocytes and human fibroblasts. Menadione induced DNA strand breaks, cell membrane damage and depletion of reduced glutathione (GSH) in both hepatocytes and fibroblasts. In fibroblasts, effects on both DNA and membrane integrity were potentiated by the presence of dicoumarol, a specific inhibitor of the 2-electron reduction of quinones by DT-diaphorase, whereas in hepatocytes only the cell membrane damage was sensitive to dicoumarol. Results indicate that menadione toxicity is mediated via 1-electron reduction, although in hepatocytes different reactive species may be responsible for damage to DNA and to the membrane. BP-3,6-Q induced DNA strand breaks in fibroblasts at concentrations as low as 1 microM. The extent of DNA damage was insensitive to dicoumarol. Even after GSH depletion and inhibition of glucuronidation and sulphate conjugation, BP-3,6-Q caused no DNA damage in hepatocytes. In contrast to menadione, BP-3,6-Q did not induce cell membrane leakage or decrease in GSH levels in either hepatocytes or fibroblasts. These studies show the complexity of the metabolic pathways involved, in terms of activation and detoxification processes, in the toxicity of quinones.     

Diethyldithiocarbamate (DEDC) enhances quinone mediated oxidative stress cytotoxicity in isolated hepatocytes by forming toxic quinone conjugates

The copper-chelating thiol drug, diethyldithiocarbamate (DEDC) had previously been used to inhibit superoxide dismutase (SOD) and enhance oxidative stress mediated cytotoxicity. Using isolated rat hepatocytes, it was confirmed that DEDC enhances oxidative stress cytotoxicity induced by 1,4-naphthoquinone (1,4-NQ) and 1,4-naphthoquinone-2-sulphonate (1,4-NQ-2S). However, equimolar concentrations of DEDC also enhances cytotoxicity induced by benzoquinone, previously shown to cause cytotoxicity as a result of alkylation and not oxidative stress. Higher DEDC concentrations on the other hand protected against benzoquinone-induced cytotoxicity. Finally, the susceptibility of hepatocytes to quinone mediated oxidative stress cytotoxicity was not enhanced if the DEDC was removed before incubating the hepatocytes with naphthoquinone or benzoquinone. Enhanced oxidative stress cytotoxicity was only observed if the DEDC was present when hepatocytes were treated with quinones. It was concluded that DEDC forms conjugates with quinones which undergo futile redox cycling in the hepatocyte and form H2O2 as well as increase the susceptibility of hepatocytes to H2O2.     

Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as alpha-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.     

Role of glutathione reductase during menadione-induced NADPH oxidation in isolated rat hepatocytes

Metabolism of menadione (2-methyl-1,4-naphthoquinone) results in the rapid oxidation of NADPH within isolated rat hepatocytes. The glutathione redox cycle is thought to play a major role in the consumption of NADPH during menadione metabolism, chiefly through glutathione reductase (GSSG-reductase). This enzyme reduces oxidized glutathione (GSSG), formed via the glutathione-peroxidase reaction, with the concomitant oxidation of NADPH. To explore the relationship between GSSG-reductase and the consumption of NADPH during menadione metabolism, isolated rat hepatocyte suspensions were exposed to non-lethal and lethal menadione concentrations (100 and 300 microM respectively) following the inhibition of GSSG-reductase with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Menadione produced a concentration-related depletion of GSH (measured as non-protein sulfhydryl content) which was potentiated markedly by BCNU. Menadione toxicity was potentiated at either concentration by BCNU based on lactate dehydrogenase leakage at 2 hr. In addition, the NADPH content of isolated hepatocytes rapidly declined following exposure to either concentration of menadione. However, at the lower menadione concentration (100 microM), the NADPH content returned to control values or above by 60 min, whereas the NADPH content of cells exposed to 300 microM menadione with or without BCNU remained depressed for the duration of the incubation. These data suggest that, although NADPH is required by GSSG-reductase for the reduction of GSSG to GSH during quinone-induced oxidative stress, this pathway does not appear to be the major route by which NADPH is consumed during the metabolism of menadione in isolated hepatocytes.     

Contribution of reductase activity to quinone toxicity in three kinds of hepatic cells

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle, and the arylation of intracellular nucleophiles. The redox cycle is catalyzed by intracellular reductases, and therefore the toxicity of redox cycling quinone is considered to be closely associated with the reductase activity. This study examined the relationship between quinone toxicity and the intracellular reductase activity using 3 kinds of hepatic cells; rat primary hepatocytes, HepG2 and H4IIE. The intracellular reductase activity was; primary hepatocyte >HepG2>H4IIE. The three kinds of cells showed almost the same vulnerability to an arylating quinone, 1,4-naphthoquinone (NQ). However, the susceptibility to a redox cycling quinone, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) was; primary hepatocyte>HepG2>H4IIE. In addition, the cytotoxicity elicited by DMNQ was significantly attenuated in HepG2 cells and almost completely suppressed in primary hepatocytes by diphenyleneiodonium chloride, a reductase inhibitor. These data suggest that cells with a high reductase activity are susceptible to redox cycling quinones. This study provides essential evidence to assess the toxicity of quinone-based drugs during their developmental processes.     

Menadione-induced oxidative stress in hepatocytes isolated from fed and fasted rats: the role of NADPH-regenerating pathways

Isolated hepatocytes were prepared from fed and fasted rats and exposed to a range of menadione (2-methyl-1,4-naphthoquinone) concentrations. Menadione (300 microM) caused a rapid decline in the (NADPH)/(NADPH + NADP+) ratio from 0.85 to 0.39 within 15 min, with further decreases over the 90-min incubation period in cells isolated from fed animals. This decrease of NADPH resulted from oxidation to NADP+ since there was no loss of total pyridine nucleotide (NADP+ + NADPH) content. In addition, menadione (100 microM) caused a five-fold stimulation of the hexose monophosphate shunt by 30 min as indicated by the oxidation of [1-14C]glucose. LDH leakage was slightly but significantly elevated (30% of total) following exposure of cells to 300 microM menadione for 2 hr. Menadione caused a concentration-dependent GSH depletion: 100 microM menadione caused no depletion and 200 and 300 microM menadione caused a 75 and 95% decrease, respectively. Intracellular NADPH was significantly reduced within 30 min by 100 and 200 microM menadione but then returned to values equivalent to or greater than control by 60 min. In contrast, a sustained decrease of NADPH was produced by 300 microM menadione (5% of control after 2 hr). A marked potentiation of the oxidative cell injury produced by menadione was observed in hepatocytes prepared from 24-hr-fasted rats. LDH leakage was 50 and 95% when these cells were exposed to 100 and 200 microM menadione, respectively. Menadione (100 and 200 microM) also caused a marked GSH depletion (95% of control) by 90 min. In contrast to cells isolated from fed animals, menadione (100 and 200 microM) caused an 85% depletion of NADPH by 60 min in cells isolated from fasted rats. This potentiation of menadione-induced oxidative injury was not related to the decreased GSH content produced by fasting since menadione toxicity was not potentiated in control cells partially depleted of GSH by diethyl maleate. A further comparison was made between cells isolated from fasted rats and incubated either with or without supplemental glucose in order to determine a possible protective effect by glucose. In this comparison a significant (p less than 0.05) glucose effect was indeed observed in the direction of preventing GSH and NADPH depletion, as well as attenuating LDH leakage, when hepatocytes were exposed to either 50 or 100 microM menadione.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytotoxic action of juglone and plumbagin: a mechanistic study using HaCaT keratinocytes

Juglone (5-hydroxy-1,4-naphthoquinone) and plumbagin (5-hydroxy-3-methyl-1,4-naphthoquinone) are yellow pigments found in black walnut (Juglans regia). Herbal preparations derived from black walnut have been used as hair dyes and skin colorants in addition to being applied topically for the treatment of acne, inflammatory diseases, ringworm, and fungal, bacterial, or viral infections. We have studied the cytotoxicity of these quinones to HaCaT keratinocytes. Exposure to juglone or plumbagin (1-20 microM) resulted in a concentration-dependent decrease in cell viability. The cytotoxicity of these quinones is due to two different mechanisms, namely, redox cycling and reaction with glutathione (GSH). Redox cycling results in the generation of the corresponding semiquinone radicals, which were detected by electron paramagnetic resonance. Incubation of keratinocytes with the quinones generated hydrogen peroxide (H(2)O(2)) and resulted in the oxidation of GSH to GSSG. Depletion of GSH by buthionine sulfoximine enhanced semiquinone radical production, increased H(2)O(2) generation, and produced greater cytotoxicity, suggesting that GSH plays an important protective role. Both quinones decreased the intracellular levels of GSH. However, plumbagin stoichiometrically converted GSH to GSSG, indicating that redox cycling is its main metabolic pathway. In contrast, much of the GSH lost during juglone exposure, especially at the higher concentrations (10 and 20 microM), did not appear as GSSG, suggesting that the cytotoxicity of this quinone may also involve nucleophilic addition to GSH. Our findings indicate that topical preparations containing juglone and plumbagin should be used with care as their use may damage the skin. However, it is probable that the antifungal, antiviral, and antibacterial properties of these quinones are the result of redox cycling.     

Discriminating redox cycling and arylation pathways of reactive chemical toxicity in trout hepatocytes.

The toxicity of four quinones, 2,3-dimethoxy-1,4-naphthoquinone (DMONQ), 2-methyl-1,4-naphthoquinone (MNQ), 1,4-naphthoquinone (NQ), and 1,4-benzoquinone (BQ), which redox cycle or arlyate in mammalian cells, was determined in isolated trout (Oncorhynchus mykiss) hepatocytes. More than 70% of cells died in 3 h when exposed to BQ or NQ; 50% died in 7 h when exposed to MNQ, with no mortality compared to controls after 7 h DMONQ exposure. A suite of biochemical parameters was assessed for ability to discriminate these reactivity pathways in fish. Rapid depletion of glutathione (GSH) with appearance of glutathione disulfide (GSSG) and increased dichlorofluoroscein fluorescence were used as indicators of redox cycling, noted with DMONQ, MNQ, and NQ. Depletion of GSH with no GSSG accumulation, and loss of free protein thiol (PrSH) groups (nonreducible) indicated direct arylation by BQ. All toxicants rapidly oxidized NADH, with changes in NADPH noted later (BQ, NQ, MNQ) or not at all (DMONQ). Biochemical measures including cellular energy status, cytotoxicity, and measures of reactive oxygen species, along with the key parameters of GSH and PrSH redox status, allowed differentiation of responses associated with lethality. Chemicals that arylate were more potent than redox cyclers. Toxic pathway discrimination is needed to group chemicals for potency predictions and identification of structural parameters associated with distinct types of reactive toxicity, a necessary step for development of mechanistically based quantitative structure-activity relationships (QSARs) to predict chemical toxic potential. The commonality of reactivity mechanisms between rodents and fish was also demonstrated, a step essential for species extrapolations.     

Menadione-induced cytotoxicity in rat platelets: Absence of the detoxifying enzyme,quinone reductase

The elevation of intracellular Ca²⁺ in various tissue through oxidative stress induced by menadione has been well documented. Increase of Ca²⁺ level in platelets results in aggregation of platelets. To test the hypothesis that menadione-induced Ca²⁺ elevations can play a role in platelet aggregation, we have studied the effect of menadione on aggregation of platelets isolated from female rats. Treatment with menadione to platelet rich plasma (PRP), which proved to be an adequate system, appeared to induce dose-dependent turbidity changes of platelets up to 60%, as determined by aggregometry. However, exposure of PRP to menadione leads to a loss of cell viability, as measured by lactate dehydrogenase (LDH) leakage, suggesting that menadione might induce cell lysis rather than aggregation of platelets. Turbidity changes induced by menadione were unaffected by addition of dicoumarol, which is a quinone reductase (QR) inhibitor. Consistent with these findings, no activity of QR was detected in any subcellular fractions of platelets. These data, which indicate an absence of the QR detoxifying pathway, suggest that platelets may be more susceptible to menadione-induced cytotoxicity than certain other cell, such as hepatocytes.     

Sulfhydryl binding and topoisomerase inhibition by PCB metabolites

Polychlorinated biphenyls (PCBs) are highly persistent contaminants in our environment. Their persistence is due to a general resistance to metabolic attack. Lower halogenated PCBs, however, are metabolized to mono- and dihydroxy compounds, and the latter may be further oxidized to quinones with the formation of reactive oxygen species (ROS). We have shown that PCB metabolism generates ROS in vitro and in cells in culture and this leads to oxidative DNA damage, like DNA strand breaks and 8-oxo-dG formation. In the present study, we have evaluated the reactivity of PCB metabolites with other nucleophiles, like glutathione (GSH), by assessing (1) quantitative GSH binding in vitro, (2) GSH and thiol (sulfhydryl) depletion in HL-60 cells, (3) the associated cytotoxicity, and (4) the inhibition of topoisomerase II activity in vitro. PCB quinones were found to bind GSH in vitro at a ratio of 1:1.5 and to deplete GSH in HL-60 cells as measured by both spectrophotometric and spectrofluorometric methods. By flow cytometry analysis, we confirmed that there was intracellular GSH depletion in HL-60 cells by PCB quinones and this is associated with cytotoxicity. On the other hand, the PCB hydroquinone metabolites did not bind GSH or other thiols within 1 h of exposure. However, by spectral analyses we found that the PCB hydroquinones could be oxidized enzymatically to the quinones, which could then bind GSH. The resulting hydroquinone-glutathione addition product(s) could undergo a second and third cycle of oxidation and GSH addition with the formation of di- and tri-GSH-PCB adducts. The effect of the PCB metabolites was also tested on a sulfhydryl-containing enzyme, topoisomerase II. PCB quinones inhibited topoisomerase II activity while the PCB hydroquinone metabolites did not. Hence, the oxidation of PCB hydroquinone metabolites to quinones in cells followed by the binding of quinones to GSH and to protein sulfhydryl groups and the resulting oxidative stress may be important aspects of the toxicity of these compounds.     

Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl-1,4-naphthoquinone)

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) resulted in a dose-dependent depletion of intracellular reduced glutathione (GSH), most of which was oxidized to glutathione disulfide (GSSG). Menadione metabolism was also associated with a dose- and time-dependent inhibition of glutathione reductase, impairing the regeneration of GSH from GSSG produced during menadione-induced oxidative stress. Inhibition of glutathione reductase by pretreatment of hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) greatly potentiated both GSH depletion and GSSG formation during the metabolism of low concentrations of menadione. Concomitant with GSH oxidation, mixed disulfides between glutathione and protein thiols were formed. The amount of mixed disulfides produced and the kinetics of their formation were dependent on both the intracellular GSH/GSSG ratio and the activity of glutathione reductase. The mixed disulfides were mainly recovered in the cytosolic fraction and, to a lesser extent, in the microsomal and mitochondrial fractions. The removal of glutathione from protein mixed disulfides formed in hepatocytes exposed to oxidative stress was dependent on GSH and/or cysteine and appeared to occur predominantly via a thiol-disulfide exchange mechanism. However, incubation of the microsomal fraction from menadione-treated hepatocytes with purified glutathione reductase in the presence of NADPH also resulted in the reduction of a significant portion of the glutathione-protein mixed disulfides present in this fraction. Our results suggest that the formation of glutathione-protein mixed disulfides occurs as a result of increased GSSG formation and inhibition of glutathione reductase activity during menadione metabolism in hepatocytes.     

Differences in the localization and extent of the renal proximal tubular necrosis caused by mercapturic acid and glutathione conjugates of 1,4-naphthoquinone and menadione

We have previously demonstrated that administration of various benzoquinol-glutathione (GSH) conjugates to rats causes renal proximal tubular necrosis and the initial lesion appears to lie within that portion of the S3 segment within the outer stripe of the outer medulla (OSOM). The toxicity may be a consequence of oxidation of the quinol conjugate to the quinone followed by covalent binding to tissue macromolecules. We have therefore synthesized the GSH and N-acetylcysteine conjugates of 2-methyl-1,4-naphthoquinone (menadione) and 1,4-naphthoquinone. The resulting conjugates have certain similarities to the benzoquinol-GSH conjugates, but the main difference is that reaction with the thiol yields a conjugate which remains in the quinone form. 2-Methyl-3-(N-acetylcystein-S-yl)-1,4-naphthoquinone caused a dose-dependent (50-200 mumol/kg) necrosis of the proximal tubular epithelium. The lesion involved the terminal portion of the S2 segment and the S3 segment within the medullary ray. At the lower doses, that portion of the S3 segment in the outer stripe of the outer medulla displayed no evidence of necrosis. In contrast, 2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone (200 mumol/kg) caused no apparent histological alterations to the kidney. 2-(Glutathion-S-yl)-1,4-naphthoquinone and 2,3-(diglutathion-S-yl)-1,4-naphthoquinone (200 mumol/kg) were relatively weak proximal tubular toxicants and the lesion involved the S3 segment at the junction of the medullary ray and the OSOM. A possible reason(s) for the striking difference in the toxicity of the N-acetylcysteine conjugate of menadione, as opposed to the lack of toxicity of the GSH conjugate of menadione, is discussed. The basis for the localization of the lesion caused by 2-methyl-3-(N-acetylcystein-S-yl)-1,4-naphthoquinone requires further study.     

Interconversion of NAD(H) to NADP(H). A cellular response to quinone-induced oxidative stress in isolated hepatocytes

Quinones may be toxic by a number of mechanisms, including oxidative stress caused by redox cycling and arylation. This study has compared the cytotoxicity of four quinones, with differing abilities to arylate cellular nucleophiles and redox cycle, in relation to their effects on cellular pyridine nucleotides and ATP levels in rat hepatocytes. Non-toxic concentrations (50 microM) of menadione (redox cycles and arylates), 2-hydroxy-1,4-naphthoquinone (neither arylates nor redox cycles via a one electron reduction) and 2,3-dimethoxy-1,4-naphthoquinone (a pure redox cycler) all caused markedly similar changes in cellular pyridine nucleotides. An initial decrease in NAD+ was accompanied by a small, transient increase in NADP+ and followed by a larger, prolonged increased in NADPH and total NADP+ + NADPH. At toxic concentrations (200 microM), the quinones caused an extensive depletion of NAD(H), an increase in levels of NADP+ and an initial rise in total NADP+ + NADPH, prior to a decrease in ATP levels and cell death. Nucleotide changes were not observed with non-toxic (20 microM) or toxic (100 microM) concentrations of p-benzoquinone (a pure arylator) and ATP loss accompanied or followed cell death. A novel mechanism for the activation of 2-hydroxy-1,4-naphthoquinone has been implicated. Our findings also suggest that a primary event in the response of the cell to redox cycling quinones is to bring about an interconversion of pyridine nucleotides, possibly mediated by an NAD+ reduction, in an attempt to combat the effects of oxidative stress.