The oxidation of NADH by vanadate plus sugars

Sugars and sugar phosphates enable vanadate to catalyze the oxidation of NADH. Superoxide dismutase inhibits this oxidation. Incubation of sugars with vanadate, prior to addition of NADH, accelerates this oxidation of subsequently added NADH and eliminates the lag phase otherwise noted. Incubation of sugars with vanadate also results in the reduction of vanadate to vanadyl, with appearance of a blue-green color probably associated with a vanadyl-vanadate complex. It appears that sugars reduce vanadate to vanadyl which, in turn, reduces O2 to O2- and that vanadate plus O2- then catalyzes the oxidation of NAD(P)H by a free radical chain reaction. Such oxidation of NAD(P)H may account for several of the biological effects of vanadate.     

Vanadate-dependent NAD(P)H oxidation by microsomal enzymes

Vanadate-dependent NAD(P)H oxidation, catalyzed by rat liver microsomes and microsomal NADPH-cytochrome P450 reductase (P450 reductase) and NADH-cytochrome b5 reductase (b5 reductase), was investigated. These enzymes and intact microsomes catalyzed NAD(P)H oxidation in the presence of either ortho- or polyvanadate. Antibody to P450 reductase inhibited orthovanadate-dependent NADPH oxidation catalyzed by either purified P450 reductase or rat liver microsomes and had no effect on the rates of NADH oxidation catalyzed by b5 reductase. NADPH-cytochrome P450 reductase catalyzed orthovanadate-dependent NADPH oxidation five times faster than NADH-cytochrome b5 reductase catalyzed NADH oxidation. Orthovanadate-dependent oxidation of either NADPH or NADH, catalyzed by purified reductases or rat liver microsomes, occurred in an anaerobic system, which indicated that superoxide is not an obligate intermediate in this process. Superoxide dismutase (SOD) inhibited orthovanadate, but not polyvanadate-mediated, enzyme-dependent NAD(P)H oxidation. SOD also inhibited when pyridine nucleotide oxidation was conducted anaerobically, suggesting that SOD inhibits vanadate-dependent NAD(P)H oxidation by a mechanism independent of scavenging of O2-.     

Superoxide is responsible for the vanadate stimulation of NAD(P)H oxidation by biological membranes

Vandate augments the oxidation of NAD(P)H, but not of NMNH, by rat liver microsomes. Paraquat increases the vanadate effect on NADPH, but not on NADH, oxidation. Substoichiometric levels of NADPH caused the co-oxidation of NADH or NMNH and SOD inhibited in all cases. The ratio of NADH or NMNH co-oxidized per NADPH added allowed estimation of average chain length, which increased as the pH was lowered from 8.0 to 7.1. The initial rate of this co-oxidation of NMNH was a saturating function of the concentration of microsomes, reflecting a decrease in chain length with an increase in number of concomitant reaction chains, and due to increasing radical-radical termination reactions. Mitochondrial outer membranes behaved like the microsomal membranes, but mitochondrial inner membranes catalyzed a rapid oxidation of NADH which could be augmented by vanadate, whose action was enhanced by paraquat and inhibited by antimycin or rotenone. These and related observations support the view that vanadate stimulates NAD(P)H oxidation by biological membranes, not by virtue of interacting with enzymes, but rather by interacting with O-2.     

Hydroxyl Radical Generation in the Nadh/Microsomal Reduction of vanadate

ESR spin trapping measurements demonstrate generation of hydroxyl (.OH) radical from reduction of vanadate by rat liver microsomes/NADH without exogenous H₂O₂. Catalase decreases the .OH signal while increasing a vanadium(4+) signal. Addition of superoxide dismutase (SOD) or measurements under an argon atmosphere show decreased .OH radical production. The results suggest that during the one-electron vanadate reduction process by microsomes/NADH, molecular oxygen is reduced to H₂O₂, which then reacts with vanadium (4+) to generate .OH radical via a Fenton-like mechanism.     

The oxidatin of NADH by vanadate plus sugars

Sugars and sugar phosphates enable vanadate to catalyze the oxidation of NADH. Superoxide dismutase inhibits this oxidation. Incubation of sugars with vanadate, prior to addition of NADH, accelerates this oxidation of subsequently added NADH and eliminates the lag phase otherwise noted. Incubation of sugars with vanadate also results in the reduction of vanadate to vanadyl, with appearance of a blue-green color probably associated with a vanadyl-vanadate complex. It appears that sugars reduce vanadate to vanadyl which, in turn, reduces O₂ to O₂⁻ and that vanadate plus O₂⁻ then catalyzes the oxidation of NAD(P)H by a free radical chain reaction. Such oxidation of NAD(P)H may account for several of the biological effects of vanadate.     

The vanadate-stimulated oxidation of NAD(P)H by biomembranes is a superoxide-initiated free radical chain reaction

Rat liver microsomes catalyze a vanadate-stimulated oxidation of NAD(P)H, which is augmented by paraquat and suppressed by superoxide dismutase, but not by catalase. NADPH oxidation was a linear function of the concentration of microsomes in the absence of vanadate, but was a saturating function in the presence of vanadate. Microsomes did not catalyze a vanadate-stimulated oxidation of reduced nicotinamide mononucleotide (NMNH), but gained this ability when NADPH was also present. When the concentration of NMNH was much greater than that of NADPH a minimal average chain length could be calculated from 1/2 the ratio of NMNH oxidized per NADPH added. The term chain length, as used here, signifies the number of molecules of NMNH oxidized per initiating event. Chain length could be increased by increasing [vanadate] and [NMNH] and by decreasing pH. Chain lengths in excess of 30 could easily be achieved. The Km for NADPH, arrived at from saturation of its ability to trigger NMNH oxidation by microsomes in the presence of vanadate, was 1.5 microM. Microsomes or the outer mitochondrial membrane was able to catalyze the vanadate-stimulated oxidation of NADH or NADPH but only the oxidation of NADPH was accelerated by paraquat. The inner mitochondrial membrane was able to cause the vanadate-stimulated oxidation of NAD(P)H and in this case paraquat stimulated the oxidation of both pyridine coenzymes. Our results indicate that vanadate stimulation of NAD(P)H oxidation by biomembranes is a consequence of vanadate stimulation of NAD(P)H or NMNH oxidation by O-2, rather than being due to the existence of vanadate-stimulated NAD(P)H oxidases or dehydrogenases.     

Activation of rabbit liver high affinity cAMP (type IV) phosphodiesterase by a vanadyl-glutathione complex. Characterization of the role of the sulfhydryl.

Activation of rabbit liver microsomal high affinity cAMP phosphodiesterase (Type IV PDE) by vanadyl-glutathione complexes was studied as a possible model of insulin stimulation of the enzyme in a cell-free system. The effect of VO.2GSH activation of PDE was a 21-fold decrease in the IC50 value for cGMP inhibition and a 2.6-fold increase in the Vmax of the higher affinity cAMP catalytic site. Cyclic AMP and cGMP substrate affinities and cGMP hydrolysis were unaffected by VO.2GSH activation. Selective Type IV PDE inhibitors and cGMP analogs indicated that VO.2GSH complexes activated the cGMP-inhibitable form of the Type IV PDE activities which co-localized in hepatic microsomes. The Type IV PDE activating complex appears to consist minimally of vanadyl ion and 2 oxidized electron donor compounds. The components of the electron donor required to achieve an enzyme activation complex are: 1) a free -SH group as the electron donor for vanadate reduction and 2) a minimum structure of cysteamine (NH2-CH2-CH2-SH). Maximal activation of the enzyme required near 2:1 molar ratios of either glutathione or cysteamine mixed with sodium orthovanadate. Active vanadyl-cysteamine complexes were isolated by reverse- phase high performance liquid chromatography. Tungsten, niobium, and tantalum, but not manganese, chromium, or molybdenum, substituted for vanadium to form enzyme-activating complexes with glutathione. VO.RSH complex activation occurred rapidly upon addition to microsomes and was reversible. We conclude from these studies that VO.RSH complexes and insulin activate the same form of Type IV PDE in rabbit liver microsomes; our findings are discussed with respect to the involvement of a possible electron transfer enzyme oxidation in the activation mechanism.     

Importance of hydroxyl radical in the vanadium-stimulated oxidation of NADH

Vanadium compounds are known to stimulate the oxidation of NAD(P)H, but the mechanism remains unclear. This reaction was studied spectrophotometrically and by electron spin resonance spectroscopy (ESR) using vanadium in the reduced state (+4, vanadyl) and the oxidized state (+5, vanadate). In 25 mM sodium phosphate buffer at pH 7.4, vanadyl was slightly more effective in stimulating NADH oxidation than was vanadate. Addition of a superoxide generating system, xanthine/xanthine oxidase, resulted in a marked increase in NADH oxidation by vanadyl, and to a lesser extent, by vanadate. Decreasing the pH with superoxide present increased NADH oxidation for both vanadate and vanadyl. Addition of hydrogen peroxide to the reaction mixture did not change the NADH oxidation by vanadate, regardless of concentration or pH. With vanadyl however, addition of hydrogen peroxide greatly enhanced NADH oxidation which further increased with lower pH. Use of the spin trap DMPO in reaction mixtures containing vanadyl and hydrogen peroxide or a superoxide generating system resulted in the detection by ESR of hydroxyl. In each case, the hydroxyl radical signal intensity increased with vanadium concentration. Catalase was able to inhibit the formation of the DMPO--OH adduct formed by vanadate plus superoxide. These results show that the ability of vanadium to act in a Fenton-type reaction is an important process in the vanadium-stimulated oxidation of NADH.     

Increase in alpha-glycerophosphate dehydrogenase and other oxidoreductase activities of hepatic mitochondria on administration of vanadate to the rat

Liver mitochondria isolated from vanadate-administered rats showed increased (20-25%) rates of oxidation of both NAD(+)-linked substrates and succinate. Respiratory control index and ADP/O were unaffected by the treatment. Dormant and uncoupler-stimulated ATPase activity also was not affected by vanadate administration. Membrane-bound, electron-transport-linked dehydrogenase activities (both NAD(+)- and succinate-dependent) increased by 15-20% on vanadate treatment. Mitochondrial alpha-glycerophosphate dehydrogenase activity increased by 50% on vanadate administration. The above effects of vanadate on oxidoreductase activities could be prevented by the prior administration of antagonists to alpha-adrenergic receptors. Substrate-dependent H2O2 generation by mitochondria also showed an increase on vanadate administration.     

Metabolism of added orthovanadate to vanadyl and high-molecular-weight vanadates by Saccharomyces cerevisiae

The effect of vanadium oxides on living systems may involve the in vivo conversion of vanadate and vanadyl ions. The addition of 5 mM orthovanadate (VO4(3-), V(V)), a known inhibitor of the (Na,K)-ATPase, to yeast cells stopped growth. In contrast, the addition of 5 mM vanadyl (VO2+, V(IV) stimulated growth. Orthovanadate addition to whole cells is known to stimulate various cellular processes. In yeast, both ions inhibited the plasma membrane Mg2+ ATPase and were transported into the cell as demonstrated with [48V]VO4(3-) and VO2+. ESR spectroscopy has been used to measure the cell-associated paramagnetic vandyl ion, while 51V NMR has detected cell-associated diamagnetic vanadium (e.g. V(V)). Cells were exposed to both toxic (5 mM) and nontoxic (1 mM) concentrations of vanadate in the culture medium. ESR showed that under both conditions, vanadate became cell associated and was converted to vanadyl which then accumulated in the cell culture medium. 51V NMR studies showed the accumulation of new cell-associated vanadium resonances identified as dimeric vanadate and decavanadate in cells exposed to toxic amounts of medium vanadate (5 mM). These vanadate compounds did not accumulate in cells exposed to 1 mM vanadate. These studies confirm that the inhibitory form of vanadium usually observed in in vitro experiments is vanadate, in one or more of its hydrated forms. These data also support the hypothesis that the stimulatory form of vanadium usually observed in whole cell experiments is the vanadyl ion or one or more of its liganded derivatives.     

Binding of vanadate to human serum transferrin

Human serum transferrin specifically and reversibly binds 2 equiv of vanadate at the two metal-binding sites of the protein. The vanadium(V)-transferrin complex can be formed either by the addition of vanadate to apotransferrin or by the air oxidation of the vanadyl(IV)-transferrin complex. The formation of the vanadium complex can be blocked by loading the apotransferrin with iron(III), and bound vanadium can be displaced from the protein by the subsequent addition of either gallium(III) or iron(III). The binding constant for the second equiv of vanadate is 10(6.5) in 0.1 M hepes, pH 7.4 at 25 degrees C. The binding constant for the first equiv of vanadate is probably very similar, although no quantitative value could be determined. Although transferrin reacts with the vanadate anion, studies on the transferrin model compound ethylenebis(o-hydroxyphenylglycine) indicate that at pH 9.5, the vanadium is binding at the metal-binding site as a dioxovanadium(V) cation coordinated to two phenolic residues at each binding site. This bound cation appears to be protonated over the pH range 9.5-6.5, as shown by changes in the difference uv spectrum of the transferrin complex, to produce an oxohydroxo species. Further decreases in the pH lead to dissociation of the vanadium-transferrin complex.     

Vanadate-stimulated NADH oxidation in microsomes

Addition of vanadate, stimulated oxidation of NADH by rat liver microsomes. The products were NAD⁺ and H₂O₂. High rates of this reaction were obtained in the presence of phosphate buffer and at low pH values. The yellow-orange colored polymeric form of vanadate appears to be the active species and both ortho- and meta-vanadate gave poor activities even at mM concentrations.The activity as measured by oxygen uptake was inhibited by cyanide, EDTA, mannitol, histidine, ascorbate, noradrenaline, adriamycin, cytochrome c, Mn²⁺, superoxide dismutase, horseradish peroxidase and catalase. Mitochondrial outer membranes possess a similar activity of vanadate-stimulated NADH oxidation. But addition of mitochondria and some of its derivative particles abolished the microsomal activity. In the absence of oxygen, disappearance of NADH measured by decrease in absorbance at 340 nm continued at nearly the same rate since vanadate served as an electron acceptor in the microsomal system. Addition of excess catalase or SOD abolished the oxygen uptake while retaining significant rates of NADH disappearance indicating that the two activities are delinked. A mechanism is proposed wherein oxygen receives the first electron from NAD radical generated by oxidation of NADH by phosphovanadate and the consequent reduced species of vanadate (V^iv) gives the second electron to superoxide to reduce it H₂O₂. This is applicable to all membranes whereas microsomes have the additional capability of reducing vanadate.     

Vanadate-induced cell growth regulation and the role of reactive oxygen species

While vanadium compounds are known as potent toxicants as well as carcinogens, the mechanisms of their toxic and carcinogenic actions remain to be investigated. It is believed that an improper cell growth regulation leads to cancer development. The present study examines the effects of vanadate on cell cycle control and involvement of reactive oxygen species (ROS) in these vanadate-mediated responses in a human lung epithelial cell line, A549. Under vanadate stimulation, A549 cells generated hydroxyl radical (*OH), as determined by electron spin resonance (ESR), and hydrogen peroxide (H2O2) and superoxide anion (O2*-), as detected by flow cytometry using specific dyes. The mechanism of ROS generation involved the reduction of molecular oxygen to O2*- by both a flavoenzyme-containing NADPH complex and the mitochondria electron transport chain. The O2*- in turn generated H2O2, which reacted with vanadium(IV) to generate *OH radical through a Fenton-type reaction (V(IV) + H2O2 --> V(V) +*OH + OH-). The ROS generated by vanadate induced G2/M phase arrest in a time- and dose-dependent manner as determined by measuring DNA content. Vanadate also increased p21 and Chk1 levels and reduced Cdc25C expression, leading to phosphorylation of Cdc2 and a slight increase in cyclin B1 expression as analyzed by Western blot. Catalase, a specific antioxidant for H2O2, decreased vanadate-induced expression of p21 and Chk1, reduced phosphorylation of Cdc2Tyr15, and decreased cyclin B1 levels. Superoxide dismutase, a scavenger of O2*-, or sodium formate, an inhibitor of *OH, had no significant effects. The results obtained from the present study demonstrate that among ROS, H2O2 is the species responsible for vanadate-induced G2/M phase arrest. Several regulatory pathways are involved: (1) activation of p21, (2) an increase of Chk1 expression and inhibition of Cdc25C, which results in phosphorylation of Cdc2 and possible inactivation of cyclin B1/Cdc2 complex.     

Flavoenzymes reduce vanadium(V) and molecular oxygen and generate hydroxyl radical.

ESR spectroscopic evidence is presented for the formation of vanadium(IV) in the reduction of vanadium(V) by three typical, NADPH-dependent, flavoenzymes: glutathione reductase, lipoyl dehydrogenase, and ferredoxin-NADP+ oxidoreductase. The vanadium(V)-reduction mechanism appears to be an enzymatic one-electron reduction process. Addition of superoxide dismutase (SOD) showed that the generation of vanadium(IV) does not involve the superoxide (O2-) radical significantly. Measurements under anaerobic atmosphere showed, however, that the enzymes-vanadium-NADPH mixture can cause the reduction of molecular oxygen to generate H2O2. The H2O2 and vanadium(IV) thus formed react to generate hydroxyl (.OH) radical. The .OH formation is inhibited strongly by catalase and to a lesser degree by SOD, but it is enhanced by exogenous H2O2, suggesting the occurrence of a Fenton-like reaction. The inhibition of vanadium(IV) formation by N-ethylmaleimide indicates that the SH group on the flavoenzyme's cystine residue plays an important role in the enzyme's vanadium(V) reductase function. These results thus reveal a new property of the above-mentioned, NADPH-dependent flavoenzymes--their function as vanadium(V) reductases, as well as that as generators of .OH radical in the vanadium(V) reduction mechanism.     

Glutathione reduces cytoplasmic vanadate. Mechanism and physiological implications

The mechanism by which cells reduce cytoplasmic vanadium(V) (vanadate) to vanadium(IV) was investigated using the human red cell as a model system. Vanadate uptake by red cells occurs with a rapid phase involving chemical equilibration across the plasma membrane and a slower phase resulting in a high concentration of bound vanadium(IV). The slow phase was inhibited in glucose-starved cells and restored upon addition of glucose indicating an energy requirement for this process. The time course of vanadium(IV) appearance (monitored by EPR spectroscopy of intact cells) paralleled the slow phase of uptake indicating that this phase involves vanadium reduction. The reduction of intracellular vanadate to vanadium(IV) was nearly quantitative after 23 h. The intracellular reduction is not enzymatic, since a similar time course of vanadium reduction and binding to hemoglobin was observed when glutathione was added to a hemoglobin + vanadate solution in vitro. Vanadium(IV) binding to hemoglobin was reduced by addition of ATP, 2,3-diphosphoglycerate or EDTA, probably through chelation of the cation. The stability constant of the ATP-vanadium (IV) complex was determined to be 150 M-1 at pH 4.9. The time course of red cell vanadate uptake and reduction was followed in the concentration range in which approximately 60% inhibition of the (Na+ + K+)-ATPase is observed. It is concluded that vanadate is reduced by cytoplasmic glutathione in this concentration range and that the reduction explains the resistance of the (Na+ + K+)-ATPase to vanadium in intact cells.     

Interaction of heme nonapeptide derived from cytochrome c with microsomal reductases

The interaction of heme nonapeptide (a proteolytic product of cytochrome c) with purified NADH:cytochrome b5 (EC 1.6.2.2) and NADPH:cytochrome P-450 (EC 1.6.2.4) reductases was investigated. In the presence of heme nonapeptide, NADH or NADPH were enzymatically oxidized to NAD+ and NADP+, respectively. NAD(P)H consumption was coupled to oxygen uptake in both enzyme reactions. In the presence of carbon monoxide the spectrum of a carboxyheme complex was observed during NAD(P)H oxidation, indicating the existence of a transient ferroheme peptide. NAD(P)H oxidation could be partially inhibited by cyanide, superoxide dismutase and catalase. Superoxide and peroxide ions (generated by enzymic xanthine oxidation) only oxidized NAD(P)H in the presence of heme nonapeptide. Oxidation of NAD(P)H was more rapid with O2- than O2-2. We suggest that a ferroheme-O2 and various heme-oxy radical complexes (mainly ferroheme-O-2 complex) play a crucial role in NAD(P)H oxidation.     

Hydroxyl radical formation as a result of the interaction between primaquine and reduced pyridine nucleotides. Catalysis by hemoglobin and microsomes

Kinetic, circular dichroism, and NADH and NADPH fluorescence quenching studies indicate that these compounds interact with the antimalarial drug primaquine (PQ). The affinity of both pyridine nucleotides for PQ is similar. The data are in contrast with a previous report (Thornalley et al. (1983) Biochem. Pharmacol. 32, 3571-3575) suggesting specificity for the interaction with NADPH. The complex was seen to facilitate electron transfer from NAD(P)H to oxygen, generating oxygen-free radicals which were detected by the spin-trapping technique and to flavin nucleotides, giving rise to flavin semiquinone radicals which were demonstrated by direct ESR spectroscopy under anaerobic conditions. A twofold increase in oxygen uptake and hydroxyl radical generation by the NAD(P)H-PQ complex was observed in the presence of hemoglobin. This effect was independent of heme concentration (in the range 1 X 10(-5)-1 X 10(-4) M) and oxidation state of the iron. Under anaerobic conditions, the NAD(P)H-PQ complex reduces Fe-III to Fe-II hemoglobin, and under aerobic conditions about 65% of the heme chromophore is irreversibly destroyed. Superoxide dismutase inhibits hydroxyl radical generation by the NAD(P)H-PQ pair; this effect is not observed in the presence of hemoglobin. In the presence of microsomes there is a 10-fold increase in both oxygen consumption and hydroxyl radical generation by the NAD(P)H-PQ pair. The fact that both pyridine nucleotides are active, and the inability of SKF 525A in decreasing hydroxyl radical generation, suggests that microsomal reductases are involved in the catalysis.     

The uptake and fate of vanadyl ion in ascidian blood cells and a detailed hypothesis for the mechanism and location of biological vanadium reduction. A visible and X-ray absorption spectroscopic study

Vanadium K-edge X-ray absorption spectroscopy (XAS) has been used to track the uptake and fate of VO(2+) ion in blood cells from Ascidia ceratodes, following exposure to dithiothreitol (DTT) or to DTT plus VO(2+). The full range of endogenous vanadium was queried by fitting the XAS of blood cells with the XAS spectra of model vanadium complexes. In cells exposed only to DTT, approximately 0.4% of a new V(III) species was found in a site similar to Na[V(edta)(H(2)O)]. With exposure to DTT and VO(2+), average intracellular [VO(aq)](2+) increased from 3% to 5%, and 6% of a new complexed form of vanadyl ion appeared evidencing a ligand array similar to [VO(edta)](2-). At the same time, the relative ratio of blood cell [V(H(2)O)(6)](3+) increased at the expense of [V(H(2)O)(5)(SO(4))](+) in a manner consistent with a significant increase in endogenous acidity. In new UV/Visible experiments, VO(2+) could be reduced to 7-coordinate [V(nta)(H(2)O)(3)] or [V(nta)(ida)](2-) with cysteine methyl ester in pH 6.5 solution. Ascorbate reduced [VO(edta)](2-) to 7-coordinate [V(edta)(H(2)O)](-), while [VO(trdta)](2-) was unreactive. These results corroborate the finding that the reductive EMF of VO(2+) is increased by the availability of a 7-coordinate V(III) product. Finally, a new and complete hypothesis is proposed for an ascidian vanadate reductase. The structure of the enzyme active site, the vanadate-vanadyl-vanadic reduction mechanism, the cellular locale, and elements of the regulatory machinery governing the biological reduction of vanadate and vanadyl ion by ascidians are all predicted. Together these constitute the new field of vanadium redox enzymology.     

Cytochrome P-450-mediated oxidation of substrates by electron-transfer; role of oxygen radicals and of 1- and 2-electron oxidation of paracetamol

The mechanism by which the hepatic cytochrome P-450 (Cyt. P-450) containing mixed-function oxidase system oxidizes the analgesic drug paracetamol (PAR) to a hepatotoxic metabolite was studied. Since previous studies excluded the possibility of oxygenation of PAR, three other mechanisms, namely direct 1-electron oxidation by a Cyt. P-450-ferrous-dioxygen complex under concomitant formation of H2O2 to N-acetyl-p-semiquinone imine (NAPSQI), direct 2-electron oxidation by a Cyt. P-450-ferric-oxene complex to N-acetyl-p-benzoquinone imine (NAPQI) and indirect oxidation by active oxygen species released from Cyt. P-450, were considered. Indirect oxidation by active oxygen species was not involved, as active oxygen scavengers such as superoxide dismutase, catalase and DMSO did not affect the oxidation of PAR in hepatic microsomes. No reaction products characteristic for a direct 1-electron oxidation of PAR by Cyt. P-450 were observed: neither NAPSQI radical formation was detectable by ESR, nor PAR-dimer formation, nor stimulation of the microsomal H2O2 production was found to occur. In fact, PAR inhibited the spontaneous microsomal H2O2 formation. Studies on the reactions of NAPSQI with glutathione (GSH) revealed that NAPSQI hardly conjugated with GSH to a 3-glutathionyl-paracetamol conjugate (PAR-GSH) conjugate. The reactions of the elusive reactive metabolite formed during microsomal oxidation of PAR in the presence of GSH closely resembled those of synthetic NAPQI: both PAR-GSH and oxidized glutathione (GSSG) formation occurred. Furthermore, in agreement with a 2-electron oxidation hypothesis, iodosobenzene-dependent oxidation of PAR by cyt. P-450 in the presence of GSH resulted in the formation of the PAR-GSH conjugate. It is concluded that bioactivation of PAR by the Cyt. P-450 containing mixed-function oxidase system consists of a direct 2-electron oxidation to NAPQI.     

Mechanisms of 1,3-butadiene oxidations to butadiene monoxide and crotonaldehyde by mouse liver microsomes and chloroperoxidase

NADPH-dependent oxidation of 1,3-butadiene by mouse liver microsomes or H2O2-dependent oxidation by chloroperoxidase produced both butadiene monoxide and crotonaldehyde; methyl vinyl ketone and 2,3- and 2,5- dihydrofuran were not detected. The crotonaldehyde to butadiene monoxide ratio remained constant over time in both the microsomal and the chloroperoxidase reactions; however, much more crotonaldehyde was produced by chloroperoxidase than microsomes; crotonaldehyde was not detected when reference samples of butadiene monoxide were used in control incubations containing NADPH and microsomes or H2O2 and chloroperoxidase. Moreover, incubations of 1,3-butadiene with horseradish peroxidase and H2O2, or microsomes and H2O2 or arachidonic acid did not result in the oxidation of 1,3-butadiene. In microsomes, metabolite formation was dependent on incubation time, NADPH, and protein concentrations and did not change when the 1,3-butadiene pressure was varied between 24 and 52 cm Hg. Inclusion of the cytochrome P450 inhibitor 1-benzylimidazole inhibited 1,3-butadiene metabolism, but inclusion of KCN, catalase, or superoxide dismutase had no effect. These results support the role of cytochrome P450 in 1,3-butadiene oxidation by mouse liver microsomes. The formation of crotonaldehyde but not methyl vinyl ketone by cytochrome P450 or chloroperoxidase indicates regioselectivity in the oxygen transfer from the hemoproteins to 1,3-butadiene. The intermediates formed may undergo either ring closure to form butadiene monoxide or a hydrogen shift to form 3-butenal which tautomerizes to produce crotonaldehyde. Evidence for this tautomerization was obtained by the finding that 3-buten-1-ol, an alternative precursor of 3-butenal, was oxidized to crotonaldehyde under incubation conditions similar to that used for 1,3-butadiene.