Cytochrome P-450 dependent metabolic activation of 1-naphthol to naphthoquinones and covalent binding species

1-Naphthol was metabolised by a fully reconstituted cytochrome P-450 system in the presence of NADPH to methanol-soluble and covalently bound products. The formation of 1,4-naphthoquinone, the major methanol-soluble product at early time points, showed an almost total dependence on cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH, and to a lesser extent on dilauroylphosphatidylcholine. The metabolism was rapid and detectable levels of 1,4-naphthoquinone were formed within 30 sec. 1,4-Naphthoquinone formation was dependent on the concentration of both cytochrome P-450 (0.05-0.04 microM) and 1-naphthol (5-50 microM). Whereas 1,4-naphthoquinone was the major product observed at early time points, additional products were observed after prolonged incubation. In the absence of NADPH and NADPH-cytochrome P-450 reductase, 1-naphthol was metabolised, in a cumene hydroperoxide- and cytochrome P-450-dependent reaction, to 1,2- and 1,4-naphthoquinone and covalently bound products. Glutathione and ethylenediamine inhibited both the NADPH- and cumene hydroperoxide-dependent formation of covalently bound products. These data show that cytochrome P-450 catalyses the activation of 1-naphthol to naphthoquinone metabolites and covalently bound species, the latter most likely being derived from naphthoquinones.     

Metabolic activation of 1-naphthol by rat liver microsomes to 1,4-naphthoquinone and covalent binding species

1-Naphthol was metabolized by rat liver microsomes, in the presence of an NADPH-generating system, both to methanol-soluble metabolites including 1,4-naphthoquinone and an uncharacterized product(s) (X) and also to covalently bound products. NADH was much less effective as an electron donor than NADPH. Metyrapone, SKF 525-A and carbon monoxide all inhibited the metabolism of 1-naphthol to 1,4-naphthoquinone and to covalently bound products suggesting the involvement of cytochrome P-450 in at least one step in the metabolic activation of 1-naphthol to reactive products. Ethylene diamine, which reacts selectively with 1,2-naphthoquinone but not 1,4-naphthoquinone, did not affect the covalent binding whereas glutathione, which reacts with both naphthoquinones, caused an almost total inhibition of covalent binding. These and other results suggested that 1,4-naphthoquinone, or a metabolite derived from it, was responsible for most of the covalent binding observed and that little if any of the binding was due to 1,2-naphthoquinone.     

Metabolism of 1-naphthol by tyrosinase

1-Naphthol was metabolized by the polyphenol oxidase, tyrosinase, primarily to 1,2-naphthoquinone and to small amounts of 1,4-naphthoquinone as well as to covalently bound products. The inhibition of covalent binding by ethylenediamine, which reacts specifically with 1,2-naphthoquinone but not 1,4-naphthoquinone, suggested that most of the covalent binding was due to 1,2-naphthoquinone or a metabolite of similar structure. The activation by tyrosinase of 1-naphthol to covalently bound products suggested that it may alter the reaction kinetics of the enzyme. This was investigated by studying the effects of 1-naphthol on the tyrosinase-catalysed oxidation of 4-hydroxyanisole. Preincubation of tyrosinase with 1-naphthol increased the lag period of the oxidation of 4-hydroxyanisole, which may be due to a decrease in the amount of active enzyme, as well as to a reaction of 1-naphthol with 3,4-anisylquinone, an oxidation product of 4-hydroxyanisole. The metabolic activation of 1-naphthol by tyrosinase to covalently bound species suggests that 1-naphthol or a structurally related derivative may be of potential therapeutic application in the treatment of cells high in tyrosinase activity, such as certain melanomas.     

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.     

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)     

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.     

In vitro toxicity of naphthalene, 1-naphthol, 2-naphthol and 1,4-naphthoquinone on human CFU-GM from female and male cord blood donors

In animal models, naphthalene toxicity has been studied in different target organs and has been shown to be gender-dependent and metabolism related. In humans, it is readily absorbed and is metabolised by several cytochrome P450's. Naphthalene and its metabolites can cross the placental barrier and consequently may affect foetal tissues. The aim of this study was to compare the in vitro toxicity of naphthalene and its metabolites, 1-naphthol, 2-naphthol and 1,4-naphthoquinone, on human haematopoietic foetal progenitors (CFU-GM) derived from newborn male and female donors. The mRNA expression of Cyp1A2 and Cyp3A4 was also evaluated. Naphthalene did not affect CFU-GM proliferation, while 1-naphthol, 2-naphthol and particularly 1,4-naphthoquinone strongly inhibited the clonogenicity of progenitors, from both male and female donors. mRNA of Cyp1A2 and Cyp3A4 was not expressed neither at the basal level, nor after naphthalene treatment, while treatment with 1,4-naphthoquinone induced expression of both enzymes in both genders, with Cyp1A2 being expressed four times more than Cyp3A4. Female CFU-GM was significantly more sensitive to 1,4-naphthoquinone than male and after treatment both enzymes were expressed twice as much as in the male precursors. These results suggest that a gender-specific 1,4-naphthoquinone metabolic pathway may exist, which gives rise to unknown toxic metabolites.     

Protective effect of aloe extract against the cytotoxicity of 1,4-naphthoquinone in isolated rat hepatocytes involves modulations in cellular thiol levels

Aloe is a familiar ingredient in a wide range of health care and cosmetic products and has been reported to possess various physiological effects, antioxidative, anticarcinogenic, antiinflammatory and laxative. Aloe has also been reported to have an effect on liver function. The cytoprotective effect of aloe extract against 1,4-naphthoquinone-induced hepatotoxicity was evaluated in primary cultured rat hepatocytes. After exposure to 1,4-naphthoquinone (100 microM), a decrease in cell viability measured as >60% lactate dehydrogenase depletion was induced. Cellular glutathione (GSH) and protein-SH levels were also significantly decreased in a time-dependent manner. However addition of aloe extract resulted in a dose-dependent improvement of these effects. This cytoprotective effect of aloe could be attributed to its inhibition of GSH and protein-SH depletions. The effect of the aloe extracts were also dose-dependent. Addition of diethyl maleate (1 mM), a cellular glutathione-depleting agent, to hepatocytes treated with both 1,4-naphthoquinone and aloe extract, induced depletion of GSH, but did not affect protein-SH or lactate dehydrogenase. These results suggest that the 1,4-naphthoquinone-induced toxicity in rat hepatocytes was inhibited by aloe extract, and that this protective effect was due to the maintenance of cellular thiols, especially protein-SH.     

Haemolytic activity and nephrotoxicity of 2-hydroxy-1,4-naphthoquinone in rats

The short-term toxicity of 2-hydroxy-1,4-naphthoquinone (lawsone) and 2-methyl-1,4-naphthoquinone (menadione) has been compared in rats. 2-Methyl-1,4-naphthoquinone has been shown previously to cause haemolytic anaemia in animals, and this was confirmed in the present experiment. 2-Hydroxyl-1,4-naphthoquinone was found also to cause haemolysis, in a dose-dependent manner, as reflected by decreased blood packed cell volumes and haemoglobin levels and by histopathological changes in spleen, liver and kidney. With both naphthoquinones, the haemolysis was of the oxidative type, characterized by the presence of Heinz bodies within erythrocytes. Haemolysis was the only toxic change identified in rats dosed with 2-methyl-1,4-naphthoquinone. In contrast, 2-hydroxyl-1,4-naphthoquinone was not only a haemolytic agent but also a nephrotoxin, causing renal enlargement, elevated plasma levels of urea and creatinine and histologically-identified tubular necrosis, largely confined to the distal segment of the proximal convoluted tubules. The relationship between the in vivo toxic effects of these naphthoquinones and previously-reported data on their in vitro cytotoxic action is discussed.     

In vivo murine studies on the biochemical mechanism of naphthalene cataractogenesis

The polycyclic aromatic hydrocarbon naphthalene is bioactivated by cytochromes P450 to an electrophilic epoxide intermediate, which subsequently is metabolized to naphthoquinones (NQ) and possibly to a free radical intermediate. These reactive intermediates may bind covalently to lenticular tissues, cause oxidant stress and/or lipid peroxidation, thereby initiating cataracts. To evaluate this hypothesis, male C57BL/6 or DBA/2 mice were treated with naphthalene or one of several naphthoquinone and naphthol metabolites, in the presence or absence of modulators of chemical bioactivation and detoxification. In C57BL/6 mice, cataracts were caused by naphthalene (500-2000 mg/kg ip) in a dose-dependent fashion. The incidence of naphthalene-induced cataracts was decreased by pretreatment with the P450 inhibitors SKF 525A and metyrapone, the antioxidants caffeic acid and vitamin E, the glutathione (GSH) precursor N-acetylcysteine, and the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (p less than 0.05). Naphthalene cataractogenicity was enhanced by pretreatment with the cytochrome P450 inducer phenobarbital and the GSH depletor diethyl maleate (DEM) (p less than 0.05), and was unaffected by pretreatment with the prostaglandin synthetase inhibitors aspirin or naproxen, or the epoxide hydrolase inhibitor trichloropropene oxide. Cataracts were initiated by 1,2-NQ and 1,4-NQ (5-250 mg/kg ip) in a dose-dependent fashion, with a molar potency about 10-fold higher than that for naphthalene. NQ cataractogenicity was enhanced by pretreatment with DEM (p less than 0.05). 1-Naphthol (56 to 562 mg/kg ip) demonstrated a cataractogenic potency intermediary to that for naphthalene and NQ. DBA/2 mice treated with naphthalene (2000 mg/kg ip), 1,4-NQ (65-250 mg/kg ip), 1,2-NQ (30-250 mg/kg ip), or DEM followed by 1,4-NQ (125 mg/kg ip) did not develop cataracts. These results suggest that naphthalene cataractogenesis in C57BL/6 mice requires P450-catalyzed bioactivation to a reactive intermediate, which may be the NQ and/or a free radical derivative, either of which is dependent upon GSH for detoxification.     

Structure-activity relationships in the haemolytic activity and nephrotoxicity of derivatives of 1,2- and 1,4-naphthoquinone

Naphthoquinone derivatives are under investigation as potential therapeutic agents. Some such compounds are known, however, to be toxic to both animals and humans. Many naphthoquinone derivatives are haemolytic agents, while others cause necrosis of tubular epithelial cells. In the present study, the short-term toxicity of 16 derivatives of 1,2- and 1,4-naphthoquinone has been examined in rats in order to give information on structure-activity relationships. All the naphthoquinones except one caused haemolytic anaemia, but only hydroxy and amino derivatives were nephrotoxic. Among derivatives of 2-amino-1,4-naphthoquinone, substitution in the 3-position decreased haemolytic activity and abolished nephrotoxicity. Methylation of the hydroxyl group of 2-hydroxy-1,4-naphthoquinone had a similar effect. In contrast, methylation of the amino group of 2-amino-1,4-naphthoquinone increased the severity of both haemolysis and renal damage. Among the 1,2-naphthoquinones tested, the 4-methoxy and 4-amino derivatives were more toxic than the corresponding 1,4-isomers, although 4-methyl-1,2-naphthoquinone was less toxic than 2-methyl-1,4-naphthoquinone. At present, the toxicity of naphthoquinone derivatives cannot accurately be predicted on the basis of their chemical structure. In developing naphthoquinone derivatives for use in humans, toxicological studies in animals should be conducted at an early stage, bearing in mind that clinical studies have shown that humans appear to be particularly vulnerable to the nephrotoxic action of these compounds, and that certain individuals are unusually susceptible to their haemolytic action.     

Conjugation of organic compounds in isolated hepatocytes from a marine fish, the plaice, Pleuronectes platessa

A method is described for the preparation of viable hepatocytes from a marine fish, the plaice. Their ability to detoxify organic compounds was measured by the formation of glucuronic acid and sulphate conjugates with the model substrates 1-naphthol and phenolphthalein. 1-Naphthol was conjugated three- to four-fold faster than phenolphthalein and glucuronidation predominated with both substrates. Strong substrate inhibition of glucuronidation was observed with 200 microM 1-naphthol or phenolphthalein. No measurable sulphate conjugation was detected with phenolphthalein. Treatment of fish with 3-methylcholanthrene induced formation of both glucuronide and sulphate conjugates by two- to three-fold. Compared with rat hepatocytes, the extent of sulphation was 100-fold lower in plaice hepatocytes whereas glucuronide formation was only 10-fold lower. The observations indicate that isolated plaice hepatocytes provide a suitable system for studies of the detoxication of xenobiotic pollutants in fish liver.     

Association of quinone-induced platelet anti-aggregation with cytotoxicity.

Various anti-platelet drugs, including quinones, are being investigated as potential treatments for cardiovascular disease because of their ability to prevent excessive platelet aggregation. In the present investigation 3 naphthoquinones (2,3-dimethoxy-1,4-naphthoquinone [DMNQ], menadione, and 1,4-naphthoquinone [4-NQ]) were compared for their abilities to inhibit platelet aggregation, deplete glutathione (GSH) and protein thiols, and cause cytotoxicity. Platelet-rich plasma, isolated from Sprague-Dawley rats, was used for all experiments. The relative potency of the 3 quinones to inhibit platelet aggregation, deplete intracellular GSH and protein thiols, and cause cytotoxicity was 1,4-NQ > menadione > DMNQ. Experiments using 2 thiol-modifying agents, dithiothreitol (DTT) and 1-chloro-2,4-dintrobenzene (CDNB), confirmed the key roles for GSH in quinone-induced platelet anti-aggregation and for protein thiols in quinone-induced cytotoxicity. Furthermore, the anti-aggregative effects of a group of 12 additional quinone derivatives were positively correlated with their ability to cause platelet cytotoxicity. Quinones that had a weak anti-aggregative effect did not induce cytotoxicity (measured as LDH leakage), whereas quinones that had a potent anti-aggregative effect resulted in significant LDH leakage (84-96%). In one instance, however, p-chloranil demonstrated a potent anti-aggregative effect, but did not induce significant LDH leakage. This can be explained by the inability of p-chloranil to deplete protein thiols, even though intracellular GSH levels decreased rapidly. These results suggest that quinones that deplete GSH in platelets demonstrate a marked anti-aggregative effect. If this anti-aggregative effect is subsequently followed by depletion of protein thiols, cytotoxicity results.     

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.     

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.     

Major differences in the extent of conjugation with glucuronic acid and sulphate in human peripheral lung

Short-term organ cultures of human peripheral lung metabolise benzo(α)pyrene to water-soluble metabolites. Enzymic hydrolysis of these water-soluble metabolites with arylsurphatase (with saccharic acid 1,4-lactone) but not with β-glucuronidase released significant amounts of ethyl acetate-soluble radioactivity, which co-chromatographed with metabolites of benzo(α)pyrene. Similar studies of human peripheral lung showed that 1-naphthol, a model phenolic substrate, was metabolised extensively to its sulphate conjugate with little or no glucuronic acid conjugate being formed. In marked contrast to this, with short-term organ culture of rat lung, 1-naphthol is mainly conjugated with UDP-glucuronic acid. Thus, in human but not in rat lung, phenolic substrates such as monohydroxybenzo(α)pyrenes or 1-naphthol are metabolised predominantly to their respective sulphate conjugates with little or no glucuronide conjugates being formed.     

Reactions of naphthols with halogenmethanes under alkaline conditions

Reactions of Naphthols with Halogenmethanes under Alkaline Conditions α-Naphthol reacts in alkaline medium with chloroform to the blue anion I of 1,4-naphthoquinone-(4′-hydroxynaphthyl)-methide-(4). Carbon tetrachloride reacts with α-naphthol to the anion of 1,4-naphthoquinone-bis(4′-hydroxy-naphthyl)-methide-(4) III. β-Naphthol reacts with chloroform to the blue anion IX of 1,2-naphthoquinone-(2′-hydroxy-naphthyl)-methide-(2). The coloured products' structures are proved by conversion to dibenzo-[a;j]-10-oxonia-anthracene cation XII as well as dibenzo-[a;j]-xanthene XIII and 2,2′-dihydroxy-dinaphthylmethane XIV. β-Naphthol reacts with carbon tetrachloride in alkaline medium to dibenzo-[a;j]-xanthone XVII without colour.     

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.     

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.