The microsomal metabolism of pentachlorophenol and its covalent binding to protein and DNA

The microsomal metabolism of pentachlorophenol (PCP) was investigated, with special attention to the conversion dependent covalent binding to protein and DNA. The two metabolites detected were tetrachloro-1,2- and tetrachloro-1,4-hydroquinone. Microsomes from isosafrole (ISF)-induced rats were by far the most effective in catalyzing the reaction: the rate of conversion was increased 7-fold over control microsomes. All other inducers tested (hexachlorobenzene (HCB), phenobarbital (PB) and 3-methylcholanthrene (3MC) gave 2--3-fold increases over control. There are indications that the 1,2- and 1,4-isomers are produced in different ratio's by various cytochrome P-450 isoenzymes: Microsomes from PB- and HCB-treated rats produced the tetrachloro-1,4- and tetrachloro-1,2-hydroquinone in a ratio of about 2, while microsomes from rats induced with 3 MC and ISF showed a ratio of about 1.3. When PCP was incubated with microsomes from rats treated with HCB, a mixed type inducer of P-450, the ratio between formation of the 1,4- and 1,2-isomers decreased with increasing concentration of PCP, suggesting the involvement of at least two P-450 isoenzymes with different Km-values. The overall apparent Km-value for HCB-microsomes was 13 microM both for the formation of the soluble metabolites and the covalent binding to microsomal protein, suggesting both stem from the same reaction. The covalent binding could be inhibited by ascorbic acid and this inhibition was accompanied by an increase in formation of tetrachlorohydroquinones (TCHQ). Although a large variation was observed in rates of conversion between microsomes treated with different (or no) inducers, the rate of covalent binding to microsomal protein was remarkably constant. A conversion-dependent covalent binding to DNA was observed in incubations with added DNA which was 0.2 times the amount of binding to protein (37 pmol/mg DNA).     

Quinone-induced inhibition of urease: elucidation of its mechanisms by probing thiol groups of the enzyme

In this work we studied the reaction of four quinones, 1,4-benzoquinone (1,4-BQ), 2,5-dimethyl-1,4-benzoquinone (2,5-DM-1,4-BQ), tetrachloro-1,4-benzoquinone (TC-1,4-BQ) and 1,4-naphthoquinone (1,4-NQ) with jack bean urease in phosphate buffer, pH 7.8. The enzyme was allowed to react with different concentrations of the quinones during different incubation times in aerobic conditions. Upon incubation the samples had their residual activities assayed and their thiol content titrated. The titration carried out with use of 5,5'-di-thiobis(2-nitrobenzoic) acid was done to examine the involvement of urease thiol groups in the quinone-induced inhibition. The quinones under investigation showed two distinct patterns of behaviour, one by 1,4-BQ, 2,5-DM-1,4-BQ and TC-1,4-BQ, and the other by 1,4-NQ. The former consisted of a concentration-dependent inactivation of urease where the enzyme-inhibitor equilibrium was achieved in no longer than 10min, and of the residual activity of the enzyme being linearly correlated with the number of modified thiols in urease. We concluded that arylation of the thiols in urease by these quinones resulting in conformational changes in the enzyme molecule is responsible for the inhibition. The other pattern of behaviour observed for 1,4-NQ consisted of time- and concentration-dependent inactivation of urease with a nonlinear residual activity-modified thiols dependence. This suggests that in 1,4-NQ inhibition, in addition to the arylation of thiols, operative are other reactions, most likely oxidations of thiols provoked by 1,4-NQ-catalyzed redox cycling. In terms of the inhibitory strength, the quinones studied formed a series: 1,4-NQ approximately 2,5-DM-1,4-BQ<1,4-BQ相似文献   

Effects of pentachlorophenol and some of its known and possible metabolites on different species of bacteria

The antibacterial activity of pentachlorophenol and 35 of its known or possible metabolites against 30 different species of bacteria was tested. In comparison with pentachlorophenol, no increase of inhibitory activity was found for any of the chlorinated anisoles tested (except for pentachloroanisole against Streptomyces spp.), 2-chlorophenol, 2,6-dichlorophenol, 2,3,6- and 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, tetrachloro-1,4- and -1,3-benzenediol (except for the 1,3-isomer against Streptomyces spp.), tetrachloro-1,3-dimethoxybenzene, and tetrachloro-1,3-benzenediol diacetate. Two chlorophenols, five dichlorophenols, four trichlorophenols, two tetrachlorophenols, and tetrachloro-1,2-benzenediol were more active than pentachlorophenol against some, but not all, of the strains tested.     

Effects of pentachlorophenol and some of its known and possible metabolites on different species of bacteria.

The antibacterial activity of pentachlorophenol and 35 of its known or possible metabolites against 30 different species of bacteria was tested. In comparison with pentachlorophenol, no increase of inhibitory activity was found for any of the chlorinated anisoles tested (except for pentachloroanisole against Streptomyces spp.), 2-chlorophenol, 2,6-dichlorophenol, 2,3,6- and 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, tetrachloro-1,4- and -1,3-benzenediol (except for the 1,3-isomer against Streptomyces spp.), tetrachloro-1,3-dimethoxybenzene, and tetrachloro-1,3-benzenediol diacetate. Two chlorophenols, five dichlorophenols, four trichlorophenols, two tetrachlorophenols, and tetrachloro-1,2-benzenediol were more active than pentachlorophenol against some, but not all, of the strains tested.     

Role of membrane charge and semiquinone structure on naphthosemiquinone derivatives and 1,4-benzosemiquinone disproportionation and membrane-buffer distribution coefficients.

Semiquinone membrane/buffer partition coefficients have been determined for 1,2-naphthosemiquinone (ONQ.-), 1,4-naphthosemiquinone (NQ.-) and two of its hydroxylated derivatives, 5,8-dihydroxy-1,4-naphthosemiquinone (NZQ.-) and 5-hydroxy-1,4-naphthosemiquinone (JQ.-) as a function of membrane charge in multilamellar vesicles of phosphatidylcholine (PC) and equimolar mixtures of this lipid and phosphatidic acid (PC:PA) and cetyltrimethylammonium bromide (PC:CTAB) at physiological pH with the exception of values corresponding to PC:PA mixtures which were obtained at pH 9. These coefficients follow the order PC:PA < PC < PC:CTAB in agreement with the negative charge of the semiquinones. The disproportionation equilibria of the naphthosemiquinone derivatives are shifted to the semiquinone in the presence of neutral and positive membranes, being more pronounced in the latter. However, very low partition coefficients as well as small shifts in the semiquinone disproportionation equilibrium were observed for ONQ.- as compared to the other semiquinones. No partition of 1,4-benzosemiquinone (BQ.-) into the lipid phase was detected for either charged or neutral lipid membranes. The presence of lipid membranes decreases the BQ.- equilibrium concentration in the presence of all the types of membranes considered here.     

Differential stereoselectivity on metabolism of triphenylene by cytochromes P-450 in liver microsomes from 3-methylcholanthrene- and phenobarbital-treated rats

Metabolism of triphenylene by liver microsomes from control, phenobarbital(PB)-treated rats and 3-methylcholanthrene(MC)-treated rats as well as by a purified system reconstituted with cytochrome P-450c in the absence or presence of purified microsomal epoxide hydrolase was examined. Control microsomes metabolized triphenylene at a rate of 1.2 nmol/nmol of cytochrome P-450/min. Treatment of rats with PB or MC resulted in a 40% reduction and a 3-fold enhancement in the rate of metabolism, respectively. Metabolites consisted of the trans-1,2-dihydrodiol as well as 1-hydroxytriphenylene, and to a lesser extent 2-hydroxytriphenylene. The (-)-1R,2R-enantiomer of the dihydrodiol predominated (70 to 92%) under all incubation conditions. Incubation of racemic triphenylene 1,2-oxide with microsomal epoxide hydrolase produced dihydrodiol which was highly enriched (80%) in the (-)-1R,2R-enantiomer. Experiments with 18O-enriched water showed that attack of water was exclusively at the allylic 2-position of the arene oxide, indicating that the 1R,2S-enantiomer of the oxide was preferentially hydrated by epoxide hydrolase. Thiol trapping experiments indicated that liver microsomes from MC-treated rats produced almost exclusively (greater than 90%) the 1R,2S-enantiomer of triphenylene 1,2-oxide whereas liver microsomes from PB-treated rats formed racemic oxide. The optically active oxide has a half-life for racemization of only approximately 20 s under the incubation conditions. This study may represent the first attempt to address stereochemical consequences of a rapidly racemizing intermediary metabolite.     

DT-diaphorase as a quinone reductase: a cellular control device against semiquinone and superoxide radical formation

Rat liver microsomes incubated in the presence of NADPH catalyze the oxidation of menadione (2-methyl-1,4-naphthoquinone) by two pathways: NADPH-cytochrome P-450 reductase and DT-diaphorase. The former pathway gives rise to labile semiquinones which are readily autooxidized as revealed by a nonstoichiometric NADPH oxidation and a concomitant O₂ consumption. The reduction of menadione catalyzed by DT-diaphorase on the other hand results in a relatively stable hydroquinone accompanied by a stoichiometric oxidation of NADPH and no O₂ consumption. The total amount of NADPH oxidized by a given amount of menadione reflects the relative contributions of the two pathways which can be demonstrated by the addition of selective inhibitors of the two enzymes or by treatment of the rats with phenobarbital or 3-methylcholanthrene which preferentially induces NADPH-cytochrome P-450 reductase and DT-diaphorase, respectively. Addition of cytosol, which contains the bulk of cellular DT-diaphorase, minimizes the formation of semiquinones and the concomitant O₂ consumption. Data relating to other quinones are also presented. The results support the earlier proposal that DT-diaphorase serves as a cellular control device against quinone toxicity.     

Comparative metabolism of 7H-dibenzo[c,g]carbazole and dibenz[a,j]acridine by mouse and rat liver microsomes.

The comparative metabolism of the carcinogenic pollutants 7H-dibenzo[c,g]-carbazole (DBC) and dibenz[a,j]acridine (DBA) was investigated in vitro using 3-methylcholanthrene (3MC) induced Sprague-Dawley rat and Hsd:ICR(Br) mouse liver microsomal preparations with benzo[a]pyrene (BaP) as the positive control. Metabolites were isolated and separated by HPLC and identified by spectroscopic and co-chromatographic techniques using synthetic standards. The major metabolites of DBC were the phenols: the 5-OH-DBC, 3-OH-DBC, and 2-OH-DBC. Traces of 1-OH-DBC were also found yet no dihydrodiols were identified. The major metabolites of DBA were the 3,4-diol-DBA and 5,6-diol-DBA, 1,2-diol-DBA, DBA-5,6-oxide and 4-OH-DBA. Treatment of both mice and rats with 3MC resulted in significant (P less than or equal to 0.05) increases relative to control in the microsomal metabolism of DBA to dihydrodiol and phenol metabolites, similar to that observed for BaP. 3MC-induced rat liver microsomes significantly (P less than or equal to 0.05) increased DBC metabolism relative to control microsomes whereas DBC metabolism was not increased with 3MC-induced mouse liver microsomes. These data indicate that different enzymatic pathways are involved in the metabolic activation of DBC in the Hsd:ICR(Br) mouse and Sprague-Dawley rat.     

DNA adduction by polychlorinated biphenyls: adducts derived from hepatic microsomal activation and from synthetic metabolites

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants and complete carcinogens in rodents. Metabolism of lower chlorinated congeners with rat liver microsomes was investigated in earlier studies and DNA adduction was also reported. The current study was designed to compare DNA adducts formed after bioactivation of PCBs with rat, mouse and human hepatic microsomes, and to investigate the role of quinoid PCB metabolites in DNA adduct formation. Eight congeners ranging from mono- to hexachlorinated biphenyls were tested. Metabolites obtained through microsomal bioactivation as well as synthetic quinoid metabolites of 4-monochlorobiphenyl (4-CB) were incubated with calf-thymus DNA (CT-DNA), and the resulting adducts were analyzed by the 32P-post-labelling method. DNA adducts were formed with mono- di- and tri-chlorinated congeners, but not with higher chlorinated congeners. Similar adduct patterns were observed for 2-monochlorobiphenyl (2-CB) activated with hepatic microsomes from rat, mouse and human, while 4-CB, 3,4-dichlorobiphenyl (3,4-CB) and 3,4,5-trichlorobiphenyl (3,4,5-CB) showed similar patterns for two out of the three microsomal systems tested. 4,4' -trichlorobiphenyl (4,4' -CB) showed different adduct patterns in all microsomal systems. Higher adduct levels were obtained with the rodent microsomes compared with human microsomes and were related to higher cytochrome P450 activity. When adducts derived from microsomal activation of 4-CB were compared by co-chromatography with those derived from the incubation of DNA with synthetic 2-(4' -chlorophenyl)-1,4-benzoquinone (4-BQ), one adduct co-migrated in three different chromatography systems. This study demonstrates that rodents as well as human hepatic enzymes metabolize lower chlorinated biphenyl congeners to reactive intermediates that form DNA adducts in vitro and shows that the para-quinone metabolites of PCBs are, in part, involved in direct DNA adduction.     

In vitro metabolism of 3-t-butyl-4-hydroxyanisole and its irreversible binding to proteins

A method for the preparation of methyl-labelled 3-t-butyl-4-hydroxyanisole (BHA) is described. Metabolism of [14C]BHA using four different enzyme systems (liver microsomes + NADPH; liver microsomes + cumene hydroperoxide (CHP); sheep seminal vesicle (SSV) microsomes (as a source of prostaglandin synthetase) + arachidonic acid (AA); horseradish peroxidase (HRP) + hydrogen peroxide) was investigated. In all systems, BHA was oxidized to a variety of products including formaldehyde, a dimer di-BHA, polar and water soluble metabolites as well as a reactive intermediate(s) that binds irreversibly to proteins. With liver microsomes and NADPH, phenobarbital (PB) induction gave increased yields of all products while 3-methylcholanthrene (MC) induction specifically increased protein binding but decreased other metabolite formation. BHA addition effectively discharged the activated oxygen complex of cytochrome P-450 (liver microsomes) as well as Comp. I and Comp. II of HRP suggesting that it is a good one electron peroxidase donor. BHA addition also increased the net rate of NADPH oxidation in the presence of liver microsomes suggesting uncoupling. It is proposed that in all system investigated BHA is oxidized predominantly via a one electron oxidation process to yield first the BHA free radical which then dimerizes, forms more products or binds to proteins.     

Novel stereoselectivity of rat liver cytochrome(s) P450 toward enantiomers of the trans-1,2-dihydrodiol of triphenylene

Metabolism of 3H-labeled (+)-(S,S)- and (-)-(R,R)-1,2-dihydrodiols of triphenylene by rat liver microsomes and 11 purified isozymes of cytochrome P450 in a reconstituted monooxygenase system has been examined. Although both enantiomers were metabolized at comparable rates, the distribution of metabolites between phenolic dihydrodiols and bay-region, 1,2-diol 3,4-epoxide diastereomers varied substantially with the different systems. Treatment of rats with phenobarbital (PB) or 3-methylcholanthrene (MC) caused a slight reduction or less than a twofold increase, respectively, in the rate of total metabolism (per nanomole of cytochrome P450) of the enantiomeric dihydrodiols compared to microsomes from control rats. Among the 11 isozymes of cytochrome P450 tested, only cytochromes P450c (P450IA1) and P450d (P450IA2) had significant catalytic activity. With either enantiomer of triphenylene 1,2-dihydrodiol, both purified cytochrome P450c (P450IA1) and liver microsomes from MC-treated rats formed diol epoxides and phenolic dihydrodiols in approximately equal amounts. Purifed cytochrome P450d (P450IA2), however, formed bay-region diol epoxides and phenolic dihydrodiols in an 80:20 ratio. Interestingly, liver microsomes from control or PB-treated rats produced only diol epoxides and little or no phenolic dihydrodiols. The diol epoxide diastereomers differ in that the epoxide oxygen is either cis (diol epoxide-1) or trans (diol epoxide-2) to the benzylic 1-hydroxyl group. With either purified cytochromes P450 (isozymes c or d) or liver microsomes from MC-treated rats, diol epoxide-2 is favored over diol epoxide-1 by at least 4:1 when the (-)-enantiomer is the substrate, while diol epoxide-1 is favored by at least 5:1 when the (+)- enantiomer is the substrate. In contrast, with liver microsomes from control or PB-treated rats, formation of diol epoxide-1 relative to diol epoxide-2 was favored by at least 2:1 regardless of the substrate enantiomer metabolized. This is the first instance where the ratio of diol epoxide-1/diol epoxide-2 metabolites is independent of the dihydrodiol enantiomer metabolized. Experiments with antibodies indicate that a large percentage of the metabolism by microsomes from control and PB-treated rats is catalyzed by cytochrome P450p (P450IIIA1), resulting in the altered stereoselectivity of these microsomes compared to that of the liver microsomes from MC-treated rats.     

Characterization of a glutathione conjugate of the 1,4-benzosemiquinone-free radical formed in rat hepatocytes

Rat hepatocytes treated with 1,4-benzoquinone formed 1,4-benzosemiquinone and 2-S-glutathionyl-1,4-benzosemiquinone radicals as detected by ESR spectroscopy. The 2-S-glutathionyl-1,4-benzosemiquinone radical was first obtained from the reaction of 1,4-benzoquinone with glutathione. Glutathione both reduced benzoquinone to form benzosemiquinone and conjugated benzoquinone to form 2-S-glutathionyl-1,4-benzosemiquinone radical. The ratio of these two radicals depended upon the ratio of 1,4-benzoquinone to glutathione. At near equimolar ratios, the 2-S-glutathionyl-1,4-benzosemiquinone radical was predominantly formed. This radical was characterized by computer simulation of the experimental spectra and identified by comparison of its hyperfine coupling constants with those of chemical analogues. The 2-S-glutathionyl-1,4-benzosemiquinone radicals formed inside hepatocytes, and then crossed the plasma membrane into the media.     

Oxygen activation during oxidation of methoxyhydroquinones by laccase from Pleurotus eryngii

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH(2)) and 2, 6-dimethoxy-1,4-benzohydroquinone (DBQH(2)) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH(2) more efficiently than it oxidized MBQH(2); both the affinity and maximal velocity of oxidation were higher for DBQH(2) than for MBQH(2). Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q(*-) + O(2) <--> Q + O(2)(*-)). As this reaction is reversible, its existence was first noted in studies of the effect of systems consuming and producing O(2)(*-) on quinone formation rates. Then, the production of H(2)O(2) in laccase reactions, as a consequence of O(2)(*-) dismutation, confirmed that semiquinones autoxidized. The highest H(2)O(2) levels were obtained with DBQH(2), indicating that DBQ(*-) autoxidized to a greater extent than did MBQ(*-). Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation and laccase oxidation. Two ways of favoring semiquinone autoxidation over dismutation and laccase oxidation were increasing the rate of O(2)(*-) consumption with superoxide dismutase (SOD) and recycling of quinones with diaphorase (a reductase catalyzing the divalent reduction of quinones). These two strategies made the laccase reaction conditions more natural, since O(2)(*-), besides undergoing dismutation, reacts with Mn(2+), Fe(3+), and aromatic radicals. In addition, quinones are continuously reduced by the mycelium of white-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 microM concentrations of MBQ(*-) and DBQ(*-) from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ(*-) autoxidation rose to 13.8% and that of DBQ(*-) increased to 39.9%.     

Effects of 1-ethynylpyrene and related inhibitors of P450 isozymes upon benzo[a]pyrene metabolism by liver microsomes

The effects of three aryl acetylenes, 1-ethynylpyrene (EP), 2-ethynylnaphthalene (EN) and 3-ethynylperylene (EPE), upon the metabolism of benzo[a]pyrene (BaP) by microsomes isolated from rat liver were investigated. These aryl acetylenes all inhibited the total metabolism of BaP. Formation of BaP 7,8-dihydrodiol and BaP tetrol products by microsomal preparations from rats that had been pretreated with 3-methylcholanthrene (3MC) were preferentially inhibited. The effects of EP upon the metabolism of BaP 7,8-dihydrodiol by microsomes from rat liver were also studied. This aryl acetylene strongly inhibited the formation of BaP tetrols from BaP 7,8-dihydrodiol by liver microsomes both from untreated rats and from rats pretreated with 3MC, but enhanced the conversion of the BaP dihydrodiol into other metabolites.     

On the inhibition of microsomal drug metabolism by polychlorinated biphenyls (PCB) and related phenolic compounds.

The in vitro metabolism of p-nitroanisole, aminopyrine, and aniline by rat liver microsomal monoxygenases were studied in the presence of different polychlorinated biphenyl (PCB) mixtures and some related hydroxybiphenyls. The tested PCB mixtures contained preferably dichloro- (di-CB), tetrachloro- (tetra-CB), or hexachlorobiphenyls (hexa-CB). All PCB were competitive inhibitors of only aminopyrine demethylation by normal microsomes (Ki 22-39 micron). In microsomes of PCB-pretreated rats the aminopyrine demethylation was inhibited noncompetitively by di-CB and hexa-CB whereas tetra-CB remained a competitive inhibitor (Ki 12 micron). Moreover, after PCB pretreatment all PCB were competitive inhibitors of p-nitroanisole demethylation. 2-OH-biphenyl and 4-OH-biphenyl caused competitive inhibition of aminopyrine demethylation and aniline hydroxylation but failed to inhibit p-nitroanisole metabolism by normal microsomes. Chlorinated 4-hydroxybiphenyls inhibited competitively the metabolism of both type I and type II substrates. However, after PCB pretreatment all phenolic compounds caused uncompetitive inhibition of aniline hydroxylation.     

Enantioselective aliphatic hydroxylations of racemic 1-hydroxy-3-methylcholanthrene by rat liver microsomes

Enantiomeric pairs of 1-hydroxy-3-hydroxymethylcholanthrene (1-OH-3-OHMC), 3-methylcholanthrene (3MC) trans- and cis-1,2-diols, and 1-hydroxy-3-methylcholanthrene (1-OH-3MC) were resolved by HPLC using a covalently bonded (R)-N-(3,5-dinitrobenzoyl)phenylglycine chiral stationary phase (Pirkle type 1A) column. The absolute configuration of an enantiomeric 3MC trans-1,2-diol was established by the exciton chirality CD method following conversion to a bis-p-N,N-dimethylaminobenzoate. Incubation of an enantiomeric 1-OH-3MC with rat liver microsomes resulted in the formation of enantiomeric 3MC trans- and cis-1,2-diols; the absolute configurations of the enantiomeric 1-OH-3MC and 3MC cis-1,2-diol were established on the basis of the absolute configuration of an enantiomeric 3MC trans-1,2-diol. Absolute configurations of enantiomeric 1-OH-3-OHMC were determined by comparing their CD spectra with those of enantiomeric 1-OH-3MC. The relative amount of three aliphatic hydroxylation products formed by rat liver microsomal metabolism of racemic 1-OH-3MC was 1-OH-3-OHMC greater than 3MC cis-1,2-diol greater than 3MC trans-1,2-diol. Enzymatic hydroxylation at C2 of racemic 1-OH-3MC was enantioselective toward the 1S-enantiomer over the 1R-enantiomer (approximately 3/1); hydroxylation at the C3-methyl group was enantioselective toward the 1R-enantiomer over the 1S-enantiomer (approximately 58/42). Rat liver microsomal C2-hydroxylation of racemic 1-OH-3MC resulted in a 3MC trans-1,2-diol with a (1S,2S)/(1R,2R) ratio of 63/37 and a 3MC cis-1,2-diol with a (1S,2R)/(1R,2S) ratio of 12/88, respectively.     

The interactions of 9,10-phenanthrenequinone with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a potential site for toxic actions

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the oxidative phosphorylation of glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate, one of the precursors for glycolytic ATP biosynthesis. The enzyme contains an active site cysteine thiolate, which is critical for its catalytic function. As part of a continuing study of the interactions of quinones with biological systems, we have examined the susceptibility of GAPDH to inactivation by 9,10-phenanthrenequinone (9,10-PQ). In a previous study of quinone toxicity, this quinone, whose actions have been exclusively attributed to reactive oxygen species (ROS) generation, caused a reduction in the glycolytic activity of GAPDH under aerobic and anaerobic conditions, indicating indirect and possible direct actions on this enzyme. In this study, the effects of 9,10-PQ on GAPDH were examined in detail under aerobic and anaerobic conditions so that the role of oxygen could be distinguished from the direct effects of the quinone. The results indicate that, in the presence of the reducing agent DTT, GAPDH inhibition by 9,10-PQ under aerobic conditions was mostly indirect and comparable to the direct actions of exogenously-added H2O2 on this enzyme. GAPDH was also inhibited by 9,10-PQ anaerobically, but in a somewhat more complex manner. This quinone, which is not considered an electrophile, inhibited GAPDH in a time-dependent manner, consistent with irreversible modification and comparable to the electrophilic actions of 1,4-benzoquinone (1,4-BQ). Analysis of the anaerobic inactivation kinetics for the two quinones revealed comparable inactivation rate constants (k(inac)), but a much lower inhibitor binding constant (K(i)) for 1,4-BQ. Protection and thiol titration studies suggest that these quinones bind to the NAD+ binding site and modify the catalytic thiol from this site. Thus, 9,10-PQ inhibits GAPDH by two distinct mechanisms: through ROS generation that results in the oxidization of GAPDH thiols, and by an oxygen-independent mechanism that results in the modification of GAPDH catalytic thiols.     

Are quinones producers or scavengers of superoxide ion in cells?

The effects of quinones (benzoquinone, menadione, and doxorubicin) on the superoxide production in cell free systems (xanthine oxidase and rat liver microsomes) and of polycationic electrolyte- and latex-stimulated rat peritoneal macrophages have been studied. Contradictory results were obtained in cell free systems when two traditional assays for detection of superoxide ion, the cytochrome c reduction and the lucigenin-dependent chemiluminescence (CL), were used: all quinones inhibited the lucigenin-dependent CL at sufficiently large concentrations, but they did not inhibit at all the reduction of cytochrome c. It was proposed that the cytochrome c assay gave erroneous results due to the reversibility of the interaction of semiquinones with dioxygen. The effect of quinones on the superoxide production by peritoneal macrophages was biphasic: all quinones stimulated the O2-. formation at low concentrations and inhibited it at elevated concentrations. It was concluded that among the quinones studied, only menadione was capable of stimulating the superoxide production via a one-electron transfer mechanism in cell free systems, while the stimulatory effect of small concentrations of quinones on the O2-. production in macrophages was possibly due to their action on the activation of NADPH oxidase.     

Metabolism of aflatoxin B1 by rat hepatic microsomes induced by polyhalogenated biphenyl congeners

The metabolism of aflatoxin B1 to aflatoxins M1 and Q1 by rat liver microsomes from animals pretreated with polychlorinated or polybrominated biphenyl congeners depended on the structure of the halogenated biphenyl inducers. Microsomes from rats treated with phenobarbital (PB) or halogenated biphenyls that exhibit PB-type activity preferentially enhanced the conversion of aflatoxin B1 to aflatoxin Q1. In contrast, microsomes from rats treated with 3-methylcholanthrene (MC) or halogenated biphenyls that exhibit MC-type induction activity increased the metabolism of aflatoxin B1 to aflatoxin M1. The coadministration of PB and MC produced microsomes that exhibited both types of induction activity (mixed type) in catalyzing the oxidative metabolism of diverse xenobiotic agents. However, PB-plus-MC-induced hepatic microsomes from immature male Wistar rats preferentially increased the metabolism of aflatoxin B1 to aflatoxin M1 but did not enhance the conversion of aflatoxin B1 to aflatoxin Q1. Comparable results were observed with microsomes from rats pretreated with halogenated biphenyls classified as mixed-type inducers; moreover, in some cases there was a significant decrease in the conversion of aflatoxin B1 to aflatoxin Q1 (compared with that of controls treated with corn oil).     

New cytotoxic furoquinones obtained from terpenyl-1,4-naphthoquinones and 1,4-anthracenediones

A series of new furoterpenyl-1,4-naphtho(anthra)quinones have been prepared via oxidative cyclization of the corresponding 2-hydroxy-3-butenyl-1,4-naphtho(anthra)quinones. Depending on the reaction conditions the 1,2-quinones or the 1,4-quinones were obtained. Several new furo-1,4-anthraquinones were also obtained by condensation of 2,3-dichloroquinones with 1,3-dicarbonyls. The compounds synthesized have been evaluated for their cytotoxicity against neoplastic cell lines, some of them being effective below the micromolar level.