Addition Of Menadione Research Articles

Reconstruction of reverse transsulfuration pathway enables cysteine biosynthesis and enhances resilience to oxidative stress in Chinese Hamster Ovary cells

Cysteine is a critically important amino acid necessary for mammalian cell culture, playing key roles in nutrient supply, disulfide bond formation, and as a precursor to antioxidant molecules controlling cellular redox. Unfortunately, its low stability and solubility in solution make it especially problematic as an essential medium component that must be added to Chinese hamster ovary and other mammalian cell cultures. Therefore, CHO cells have been engineered to include the capacity of endogenously synthesizing cysteine by overexpressing multiple enzymes, including cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CTH) and glycine N-methyltransferase (GNMT) to reconstruct the reverse transsulfuration pathway and overcome a key metabolic bottleneck. Some limited cysteine biosynthesis was obtained by overexpressing CBS and CTH for converting homocysteine to cysteine but robust metabolic synthesis from methionine was only possibly after incorporating GNMT which likely represents a key bottleneck step in the cysteine biosynthesis pathway. CHO cells with the reconstructed pathway exhibit the strong capability to proliferate in cysteine-limited and cysteine-free batch and fed-batch cultures at levels comparable to wildtype cells with ample cysteine supplementation, providing a selectable marker for CHO cell engineering. GNMT overexpression led to the accumulation of sarcosine byproduct, but its accumulation did not affect cell growth. Furthermore, pathway reconstruction enhanced CHO cells’ reduced and glutathione levels in cysteine-limited conditions compared to unmodified cells, and greatly enhanced survivability and maintenance of redox homeostasis under oxidative stress induced by addition of menadione in cysteine-deficient conditions. Such engineered CHO cell lines can potentially reduce or even eliminate the need to include cysteine in culture medium, which not only reduces the cost of mammalian media but also promises to transform media design by solving the challenges posed by low stability and solubility of cysteine and cystine in future mammalian biomanufacturing processes.

Cyclosporin A Inhibits the Activation of Membrane-Bound Guanylate Cyclase GC-A of Atrial Natriuretic Factor via NAD(P)H Oxidase.

Cyclosporin A (CsA) is a common immunosuppressant wildly used in patients with organ transplant and autoimmune diseases; however, it can cause several adverse effects, such as nephrotoxicity and hypertension. The detailed mechanisms have not been completely understood. Atrial natriuretic factor (ANF) and its receptor (mGC-A) have been shown to play a crucial role in the regulation of blood pressure. Here, we investigated the effects of CsA on the activation of mGC-A in ANF-treated LLC-PK1 cells. In our study, ANF-induced mGC-A activities and superoxide generation in LLC-PK1 cells were measured by guanosine 3',5'-cyclic monophosphate (cGMP) radioimmunoassay and lucigenin-dependent chemiluminescence, respectively. We found that CsA can reduce about 60% of mGC-A activities in ANF-treated LLC-PK1 cells. CsA is known to induce superoxide. Addition of superoxide generators menadione and diamide mimicked the effects of CsA, whereas DPI (a reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor) and Tiron (a superoxide quencher) blocked the suppressive effects of CsA on ANF-induced mGC-A activities. We previously showed that the catalytic domain of GC-A (GC-c) expresses guanylate cyclase activities. Addition of menadione, diamide, or peroxynitrite or transfection of Nox-4 NAD(P)H oxidase abolished GC-c activities. In conclusion, CsA inhibits ANF-stimulated mGC-A activities through superoxide and/or peroxynitrite generated by an NAD(P)H oxidase by interacting with the catalytic domain of mGC-A.

Enzymatic Recording of Local Hydrogen Peroxide Generation Using Genetically Encodable Enzyme.

Reactive oxygen species (ROS) are endogenously generated in live cells and essential for cell signaling. However, excess ROS generation can cause oxidative damage to biomolecules, which are implicated in various human diseases, including aging. Here, we developed an in vivo hydrogen peroxide monitoring method using a genetically encodable peroxidase (APEX2)-based system. We confirmed that APEX2 is activated by endogenous H2O2 and generates phenoxyl radicals to produce biotinylated signals (i.e., biotin-phenol) and fluorescent signals (i.e., AmplexRed), which can be detected using a fluorescence microscope. We observed that all subcellular targeted APEX2s were activated by local H2O2 generation by menadione treatment. Among them, the endoplasmic reticulum lumen and lysosome-targeted APEX2 showed the highest response upon addition of menadione which implies that local H2O2 levels in those spaces are highly increased by menadione treatment. Using APEX2, we also found that a minimum amount of menadione (>10 μM) is required to generate detectable levels of H2O2 in all subcellular compartments. We also checked the local H2O2-quenching effect of N-acetylcysteine using our system. As APEX2 can be genetically expressed in diverse live organisms (e.g., cancer cell lines, mice, fly, worm, and yeast), our method can be effectively used to detect local generation of endogenously produced H2O2 in diverse live models.

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells cultivated aerobically or anaerobically

Cytotoxic effects of menadione on normal and cytochrome c-deficient yeast cells were examined on the basis of the cell growth rate, NAD(P)H concentration, reactive oxygen production, plasma membrane H+-ATPase activity, and ethanol production. In aerobically or anaerobically cultured yeast cells, NAD(P)H concentration decreased with increasing concentration of menadione, and the recovery of NAD(P)H concentration was proportional to the cell growth rate. However, there was no relationship among the inhibition of the cell growth and reactive oxygen production, plasma membrane H+-ATPase activity, and ethanol production. Among them, ethanol production showed resistance to the cytotoxicity of menadione, suggesting the resistance of glycolysis to menadione. The growth inhibitory effect of menadione depended on the rapid decrease and the recovery of NAD(P)H rather than production of reactive oxygen species regardless of aerobic culture or anaerobic culture and presence or absence of mitochondrial function. The recovery of NAD(P)H concentration after the addition of menadione might depend on menadione-resistant glycolytic enzymes.

NFE2L2 activator RS9 protects against corneal epithelial cell damage in dry eye models.

Oxidative stress may cause ocular surface damage during the development of dry eye. Mammalian cells have defense systems against oxidative stress. A central regulator of the stress response is nuclear factor-erythroid 2-related factor 2 (NFE2L2). NFE2L2 is activated by the novel triterpenoid RS9 (a biotransformation compound of RTA 402). The purpose of this study was to assess the efficacy of RS9 against dry eye using in vitro and in vivo models. Bioactivity was estimated by the induction of mRNAs for two NFE2L2-targeted genes: NQO1 (prevents radical species) and GCLC (glutathione synthesis), using a corneal epithelial cell line (HCE-T). Protection against oxidation and cell damage was tested in vitro by culturing cells under hyperosmotic stress or by the addition of menadione, a generator of reactive oxygen species (ROS). Dry eye in vivo was induced by the injection of scopolamine into rats. Then, 930 nM of RS9 was applied to both eyes for 2 weeks. Oxidative stress was measured by the accumulation of 8-hydroxy-2’-deoxyguanosine (8-OHdG). Corneal wound healing was measured by scoring for superficial punctate keratitis (SPK). Corneal epithelial cell densities were evaluated histologically. RS9 and RTA 402 induced the expression of NQO1 and GCLC mRNAs in HCE-T cells. And both compounds suppressed hyperosmotic-ROS generation and menadione induced cellular damage. However RS9 had a stronger protective effect than RTA 402. Ocular instillation of RS9 also significantly upregulated the expression of Nqo1 mRNA in the corneal epithelium. Accumulation of 8-OHdG, increase of SPK scores and decrement of basal cell density were observed in corneal epithelium from scopolamine-injected rats. These changes were significantly ameliorated by the topical administration of RS9. RS9 induced Nfe2l2 activation and Nfe2l2-targeted genes, reduced oxidation, and ameliorated symptoms of dry eye using in vitro and in vivo models. Thus, RS9 might be a potent candidate agent against dry eye disease.

Reduction of Quinones by NADH Catalyzed by Organoiridium Complexes

Reduction of Quinones by NADH Catalyzed by Organoiridium Complexes

Effect of Oxidative Stress on Homer Scaffolding Proteins

Homer proteins are a family of multifaceted scaffolding proteins that participate in the organization of signaling complexes at the post-synaptic density and in a variety of tissues including striated muscle. Homer isoforms form multimers via their C-terminal coiled coil domains, which allows for the formation of a polymeric network in combination with other scaffolding proteins. We hypothesized that the ability of Homer isoforms to serve as scaffolds would be influenced by oxidative stress. We have found by standard SDS-PAGE of lysates from adult mouse skeletal muscle exposed to air oxidation that Homer migrates as both a dimer and monomer in the absence of reducing agents and solely as a monomer in the presence of a reducing agent, suggesting that Homer dimers exposed to oxidation could be modified by the presence of an inter-molecular disulfide bond. Analysis of the peptide sequence of Homer 1b revealed the presence of only two cysteine residues located adjacent to the C-terminal coiled-coil domain. HEK 293 cells were transfected with wild-type and cysteine mutant forms of Homer 1b and exposed to oxidative stress by addition of menadione, which resulted in the formation of disulfide bonds except in the double mutant (C246G, C365G). Exposure of myofibers from adult mice to oxidative stress resulted in decreased solubility of endogenous Homer isoforms. This change in solubility was dependent on disulfide bond formation. In vitro binding assays revealed that cross-linking of Homer dimers enhanced the ability of Homer 1b to bind Drebrin, a known interacting partner. Our results show that oxidative stress results in disulfide cross-linking of Homer isoforms and loss of solubility of Homer scaffolds. This suggests that disulfide cross-linking of a Homer polymeric network may contribute to the pathophysiology seen in neurodegenerative diseases and myopathies characterized by oxidative stress.

The molecular mechanism of menadione resistance in the Synechocystis sp. PCC 6803 cyanobacterium

The effect of mutations in the genes encoding dehydrogenases and oxidases on the resistance of the Synechocystis sp. PCC 6803 cyanobacterium to menadione, an oxidative stress inducer, was studied. An enhanced sensitivity to menadione was observed in the mutants carrying inserts in the drgA gene encoding the NAD(P)H:quinone oxidoreductase (NQR) and in the ndhB gene encoding the subunit of NDH-1 complex. The menadione resistance in the mutants lacking oxidases (Ox), succinate dehydrogenase (SDH), and NDH-2 dehydrogenase do not differ from those in wild-type cells. An additional mutation in the drgA gene increased the sensitivity to menadione in the NDH-2 and Ox mutants. The double mutant that lacks both SDH and NQR was not viable. The expression of the drgA gene decreased during cell incubation in the dark but increased in the presence of glucose both in the dark and in light. Under photoautotrophic growth conditions, the dehydrogenase activity of the cells mainly depends on the NQR and NDH-1 functions. The re-reduction rate of the photosystem I reaction center (P700+) increased in wild-type and NDH-1 mutants after its oxidation with white light in the presence of DCMU after addition of menadione, and it decreased in the NQR mutant. The reduction of P700+ was accelerated in the presence of menadiol in all the strains studied. These results suggest that NQR provides defense of cyanobacterium cells from the toxic effect of menadione via its two-electron reduction to menadiol. An increased sensitivity of the NDH-1 mutant to menadione may result from the inhibition of respiration and the cyclic electron transport in photosystem I.

Oxidative stress induces heme oxygenase 1 in rat lung microvascular endothelial cells (RLMVECs)

Heme oxygenase 1 (HO-1) plays an important role in mitigating the deleterious effects of oxidative stress. To examine the anti-oxidant effect of HO-1, we studied the effect of reactive oxygen species (ROS) on HO-1 levels in RLMVECs. Intracellular ROS levels were increased by addition of menadione, which undergoes cellular redox cycling. Cultured, quiesced RLMVEC were incubated with menadione (10nM–100μM) from 30 min to 12 hrs and HO-1 protein expression determined by Western blot analysis. Incubation of RLMVECs with menadione (10nM–100μM) for 30 min or 1 hr failed to induce HO-1 expression. However, a 10-fold increase in HO-1 expression was observed at 2 and 6 hrs incubation with 10 μM menadione. To prevent/reverse menadione-induced ROS production, tempol (0.5 to 500μM), a cell permeable free radical scavenger, was preincubated with RLMVECs for 1hr prior to addition of 10μM menadione. Menadione-induced HO-1 levels (140 ±10% over control; P< 0.01) was reversed by tempol (0.5–5 μM) to 27±2% and 29±3 (P< 0.02), respectively. Higher concentrations of tempol (50–500μM) were ineffective in reducing menadione-induced HO-1 expression (124±40%). Thus, low dose tempol may be effective in mitigating oxidative stress which has been implicated in the development of pulmonary vascular pathologies such as hypoxia- induced pulmonary hypertension Supported by Philip Morris USA Inc. and by Philip Morris International

Role of NAD(P)H:quinone oxidoreductase encoded by drgA gene in reduction of exogenous quinones in cyanobacterium Synechocystis sp. PCC 6803 cells.

Insertion mutant Ins2 of the cyanobacterium Synechocystis sp. PCC 6803, lacking NAD(P)H:quinone oxidoreductase (NQR) encoded by drgA gene, was characterized by higher sensitivity to quinone-type inhibitors (menadione and plumbagin) than wild type (WT) cells. In photoautotrophically grown cyanobacterial cells more than 60% of NADPH:quinone-reductase activity, as well as all NADPH:dinoseb-reductase activity, was associated with the function of NQR. NQR activity was observed only in soluble fraction of cyanobacterial cells, but not in membrane fraction. The effects of menadione and menadiol on the reduction of Photosystem I reaction center (P700(+)) after its photooxidation in the presence of DCMU were studied using the EPR spectroscopy. The addition of menadione increased the rate of P700(+) reduction in WT cells, whereas in Ins2 mutant the reduction of P700(+) was strongly inhibited. In the presence of menadiol the reduction of P700(+) was accelerated both in WT and Ins2 mutant cells. These data suggest that NQR protects the cyanobacterial cells from the toxic effect of exogenous quinones by their reduction to hydroquinones. These data may also indicate the probable functional homology of Synechocystis sp. PCC 6803 NQR with mammalian and plant NAD(P)H:quinone oxidoreductases (DT-diaphorases).

Superoxide dismutase and catalase activities in carotenoid-synthesizing fungi Blakeslea trispora and Neurospora crassa under the oxidative stress

The addition of menadione into the medium during cultivation of Neurospora crassa in the dark activated its constitutive superoxide dismutase. Exposure to light not only activated superoxide dismutase and catalase, but also increased the content of neurosporaxanthin. Superoxide dismutase activity in the mixed (+/-) mycelium of Blakeslea trispora synthesizing beta-carotene in the dark was much lower than that in Neurospora crassa. The superoxide dismutase activity further decreased in oxidative stress. The catalase activity decreased with an increase in the content of beta-carotene. Our results indicate that neurosporaxanthin possesses photoprotective properties in Neurospora crassa. In Blakeslea trispora (+/-) fungi, this compound acts as a major antioxidant during inactivation of enzymes that detoxify reactive oxygen species.

Menadione-catalyzed O2- production by Escherichia coli cells: application of rapid chemiluminescent assay to antimicrobial susceptibility testing.

This study proposes a novel chemiluminescent assay of bacterial activity. Luminol chemiluminescence (LC) was amplified on addition of menadione to Escherichia coli suspension, and it was effectively inhibited by addition of superoxide dismutase rather than catalase. This fact suggests that H2O2 produced from O2 by superoxide dismutase is decomposed by catalase of E. coli. NAD(P)H:menadione reductase activities in periplasm and cytosol corresponded to the amplification of menadione-catalyzed LC, and outer and cytoplasmic membranes were only slightly involved in the LC. The total activity and Vmax of NAD(P)H:menadione reductase in the cytoplasm were greater than those in the periplasm. A transient increase in menadione-catalyzed LC was observed in the exponential phase and the LC decreased in the stationary phase during growth of E. coli. Menadione-catalyzed LC was sensitive to antibiotic action. A decrease in menadione-catalyzed LC by the impairment of membrane functions and by the inhibition of protein synthesis was observed at 5 min and 3 hr, respectively. These findings suggest the possibility that menadione-catalyzed luminol chemiluminescent assay is applicable to rapid antimicrobial assay because LC is sensitive to the change in growth and cytotoxic events caused by antimicrobial agents.

Scavenging effect of benzophenones on the oxidative stress of skeletal muscle cells

Benzophenone is an ultraviolet (UV)-absorbing agent that has been used in industry and medicine for more than 30 years. Consumers of cosmetics and sunscreens containing UV-absorbers are exposed to benzophenones on a daily basis, owing to the widespread use of these compounds. However, the efficacy of these compounds as scavengers of oxidative stress is still not well established. In the present study, we investigate the antioxidative capacity of six sunscreen benzophenone compounds. A primary myoblast culture was mixed in vitro with 100 μM menadione. The cytotoxic effect by menadione-induced oxidative stress was monitored by the lucigenin- or luminol-amplified chemiluminescence, methylthiotetrazole (MTT) assay, and the antioxidative effects of various benzophenone compounds were evaluated. The results showed that the addition of menadione can induce oxidative stress on myoblasts by superoxide and hydrogen peroxide production, which can be eradicated by superoxide dismutase (SOD) and catalase, respectively, in a dose-dependent mode. The catalase has a protective effect on the cytotoxicity induced by menadione as measured by the MTT assay, while the SOD does not. The selected benzophenones also have a significant scavenging effect on the menadione-induced cell death on the myoblasts. The ortho-dihydroxyl structure and other hydroxy groups in the same ring have a stronger scavenging effect on the superoxide anion on myoblasts; thus, a stable penoxy radical may be formed. The mechanism of this effect remains to be clarified.

Maximum yield of foreign lipase in Escherichia coli HB101 limited by duration of protein expression

When a microbial lipase was overexpressed in Escherichia coli HB101, the expression kinetics as represented by the expression rate, duration, and maximum yield of lipase were studied. Lipase synthesis, controlled by the tac promoter, continued for about 4h after IPTG induction. The duration of the expression phase was similar, irrespective of expression rate and yield, which were manipulated by using α-methyl glucose (α-MG), a competitive inhibitor of glucose. By measuring the specific oxygen uptake rate, specific CO 2 evolution rate, specific glucose uptake rate, intracellular protease level and the acetate concentration in the culture, the limited duration of the expression phase was found to be caused by metabolic stress arising from the rapid and massive production of the foreign protein under the strong promoter. Neither the total cell number nor the number of living cells increased substantially after induction, whereas the optical density of the culture gradually increased. The duration of the expression phase was reduced to less than 2 h by the addition of menadione, a redox cycling agent, seemingly due to an acceleration of the energetic flow of the host cells after induction. In contrast, the duration of the expression phase was extended to 8 h in the glucose-starved condition, although the maximum expression yield was much lower than that in the glucose-surplus condition. Therefore, it was suggested that the expression rate after induction determined the maximum expression yield of the foreign lipase gene in E. coli HB101 because of the restrained capacity of foreign protein production.

Role of hydrogen peroxide in hypoxia-induced erythropoietin production.

The addition of exogenous H2O2 inhibited hypoxia-induced erythropoietin (Epo) production in the human hepatoma cell line HepG2. Likewise, elevation of endogenous H2O2 levels by the addition of menadione or the catalase inhibitor, aminotriazole, dose-dependently lowered Epo production. The inhibitory effect of exogenous H2O2 on Epo formation could be completely overcome by co-incubation with catalase. When GSH levels in HepG2 cells were lowered, Epo production was more susceptible to H2O2-induced inhibition, indicating that H2O2 might affect thiol groups in regulatory proteins. Endogenous production of H2O2 in HepG2 cells was dependent on the pericellular O2 tension, being lowest under conditions of hypoxia. Our results support the hypothesis that an H2O2-generating haem protein might be part of the O2 sensor that controls Epo production. High H2O2 levels under conditions of normoxia suppress, whereas lower levels in hypoxic cells allow epo gene expression.

Ferritin Stimulation of Hydroxyl Radical Production by Rat Liver Nuclei

Iron mobilized from ferritin has been shown to catalyze production of potent reactive oxygen intermediates. Experiments were carried out to evaluate the ability of ferritin to catalyze nuclear generation of hydroxyl radical in the presence of either NADPH or NADH. In the absence of redox cycling agents, ferritin did not catalyze nuclear oxidation of hydroxyl radical scavenging agents (2-keto-4-thiomethylbutyric acid, dimethylsulfoxide, ethanol) even if EDTA was added to chelate any released iron. The addition of menadione or paraquat resulted in a ferritin-dependent oxidation of chemical scavengers; menadione promoted the catalysis by ferritin with either NADPH or NADH, whereas paraquat was much more reactive with NADPH as the nuclear reductant. The presence of an externally added iron chelator was required for elevated rates of scavenger oxidation, with EDTA and DTPA being more reactive than ATP or citrate and desferrioxamine being inhibitory. The ferritin-catalyzed hydroxyl radical scavenger oxidation was sensitive to superoxide dismutase, catalase, and competitive scavengers. In the absence or presence of ferritin, rates of NADPH- or NADH-dependent H 2O 2 production were low; menadione increased H 2O 2 production with both NADPH and NADH, whereas paraquat was mostly effective with NADPH. Depending on the nature of the added chelating agent (e.g., EDTA, ATP) and the reductant, rates of nuclear production of ·OH in the presence of redox cycling agent plus ferritin were 10 to 70% as high as rates found with redox cycling agent plus ferric-chelate (e.g., ferric-EDTA, ferric-ATP). Since reactive oxygen intermediates such as the hydroxyl radical can alter the structural integrity of the nucleus and interact with DNA, the ability of ferritin to promote nuclear generation of hydroxyl radical may play a role in the toxicity associated with iron as well as redox cycling agents.

Allopurinol-insensitive oxygen radical formation by milk xanthine oxidase systems.

Oxygen radical generation in the xanthine- and NADH-oxygen reductase reactions by xanthine oxidase, was demonstrated using the ESR spin trap 5,5'-dimethyl-1- pyrroline-N-oxide. No xanthine-dependent oxygen radical formation was observed when allopurinol-treated xanthine oxidase was used. The significant superoxide generation in the NADH-oxygen reductase reaction by the enzyme was increased by the addition of menadione and adriamycin. The NADH-menadione and -adriamycin reductase activities of xanthine oxidase were assessed in terms of NADH oxidation. From Lineweaver-Burk plots, the Km and Vmax of xanthine oxidase were estimated to be respectively 51 microM and 5.5 s-1 for menadione and 12 microM and 0.4 s-1 for adriamycin. Allopurinol-inactivated xanthine oxidase generates superoxide and OH.radicals in the presence of NADH and menadione or adriamycin to the same extent as the native enzyme. Adriamycin radicals were observed when the reactions were carried out under an atmosphere of argon. The effects of superoxide dismutase and catalase revealed that OH.radicals were mainly generated through the direct reaction of H2O2 with semiquinoid forms of menadione and adriamycin.

Superoxide-mediated release of iron from ferritin by some flavoenzymes

NADH-lipoamide dehydrogenase mobilized iron from ferritin under aerobic conditions. Superoxide dismutase strongly inhibited this mobilization, indicating that the superoxide radical is generated by the enzymatic reaction and releases iron from ferritin. Addition of lipoamide as an electron acceptor to NADH-lipoamide dehydrogenase increased the release of iron from ferritin and this release was partially inhibited by superoxide dismutase. Similarly, addition of menadione (2-methy-1, 4-naphthoquinone) as an electron acceptor to xanthine-xanthine oxidase promoted the release of iron from ferritin and this release was strongly inhibited by superoxide dismutase. These results suggest that dihydrolipoamide and semiquinone of menadione can react with oxygen to form the superoxide radical that mediates release of iron from ferritin.

Effects of carotenoids on Aflatoxin B1‐induced mutagenesis inS. typhimuriumTA 100 and TA 98

The effects of beta-carotene, canthaxanthin, and extracts of tomato paste (containing lycopene) and orange juice (containing cryptoxanthin) on aflatoxin B1 (AFB1)-induced mutagenesis in S. typhimurium TA 100 and TA 98 were investigated. Inhibition of mutagenesis was studied during and following completion of AFB1 metabolism (i.e., after the addition of menadione), thereby permitting separate examination of the metabolic activation and phenotypic expression phases. Each experimental carotenoid, except lycopene, inhibited AFB1-induced mutagenesis in both tester strains. Cryptoxanthin was the most potent inhibitor, being at least an order of magnitude more potent than the other carotenoids. Inhibition by beta-carotene and canthaxanthin was more prominent during the activation phase, whereas cryptoxanthin was more effective during the subsequent phenotypic expression phase. These inhibitory effects were not dependent on conversion to retinol.

Increased oxygen radical-dependent inactivation of metabolic enzymes by liver microsomes after chronic ethanol consumption.

Enzymatic and nonenzymatic mixed-function oxidase systems have been shown to generate an oxidant that catalyzes the inactivation of glutamine synthetase and other metabolic enzymes. Recent studies have shown that microsomes isolated from rats chronically fed ethanol generate reactive oxygen intermediates at elevated rates compared with controls. Microsomes from rats fed ethanol were found to be more effective than control microsomes in catalyzing the inactivation of enzymes added to the incubation system. The enzymes studied were alcohol dehydrogenase, lactic dehydrogenase, and pyruvate kinase. The inactivation process by both types of microsomal preparations was sensitive to catalase and glutathione plus glutathione peroxidase, but was not affected by superoxide dismutase or hydroxyl radical scavengers. Iron was required for the inactivation of the added enzymes; microsomes from the rats fed ethanol remained more effective than control microsomes in catalyzing the inactivation of enzymes in the absence or presence of several ferric complexes. The inactivation of enzymes was enhanced by the addition of menadione or paraquat to the microsomes, and rates of inactivation were higher with the microsomes from the ethanol-fed rats. The enhanced generation of reactive oxygen intermediates and increased inactivation of enzymes by microsomes may contribute toward the hepatotoxic effects associated with ethanol consumption.