Biomedical subjects

A I Cederbaum

Publications and source records attributed to A I Cederbaum.

At least 73 records · Page 4Linked to original sources

PubMed1996

Expression of cytochrome P4502E1 in rat fetal hepatocyte culture.

Cytochrome P450 (CYP) 2E1 is present at very low levels or cannot be detected in rat fetal liver. Experiments were carried out to develop an ex vivo model of CYP2E1 expression in fetal liver. Fetal hepatocytes were prepared from pregnant rats on gestation days ranging from 12 to 19 and placed into culture for 2 days. Expression of CYP2E1 was observed at all gestational periods as evident from immunoblots and oxidation of paranitrophenol and N,N-dimethylnitrosamine by fetal liver microsomes. Northern blot analysis indicated production of CYP2E1 mRNA by the fetal hepatocytes cultured for 2 days but not by freshly isolated fetal rat hepatocytes. The addition of ethanol to the hepatocyte cultures did not have a significant effect on CYP2E1 catalytic oxidation of substrates or CYP2E1 mRNA levels. The content of CYP2E1, CYP2E1 mRNA levels, and CYP2E1 catalytic activity was greater in the fetal cultures grown in the presence of 2.5% fetal calf serum than in that grown with 15% fetal calf serum, suggesting that factors present in the serum limit expression or stability of CYP2E1. CYP2E1 was not detectable in two human fetal livers; however, expression did occur when human fetal hepatocytes were placed into culture for 4 days. These results suggest that cultures of rat and human fetal hepatocytes may be a valuable model with which to study factors that regulate expression of CYP2E1 and the influence of ethanol and other inducers on expression and stabilization of CYP2E1.

Animals↗

PubMed1996

1-Hydroxyethyl radical formation during NADPH- and NADH-dependent oxidation of ethanol by human liver microsomes.

Ethanol can be oxidized to the 1-hydroxyethyl radical (HER) by rat and deer mice liver microsomal systems. Experiments were carried out to evaluate the ability of human liver microsomes to catalyze this reaction, compare the effectiveness of NADH with that of NADPH, and assess the possible role of cytochrome b5 in HER formation. HER was detected as the alpha-(4-pyridly-1 -oxide)-N-t-butylnitrone/HER adduct. Human liver microsomes catalyzed HER formation with either NADPH or NADH as cofactor; rates with NADH were approximately 50% those found with NADPH. Chelex-100 treatment of the reaction mixture produced marked inhibition of HER formation, suggesting that a transition metal, such as iron, was required to catalyze the reaction. The addition of ferric chloride restore HER formation. Catalase (2600 units/ml) and superoxide dismutases (500 units/ml) nearly completely inhibited the reaction with either NADPH or NADH. The NADH-dependent rates of superoxide production, detected as 5,5-dimethyl-1-pyrroline-N-oxide-O2H, were approximately 50% the NADPH-dependent rates, which is consistent with the rates of HER formation. Anti-cytochrome b5 IgG decreased NADPH- and NADH-dependent HER formation, and this was associated with inhibition of superoxide formation with both reductants. These results indicate that human liver microsomes can catalyze the oxidation of ethanol of HER with either NADPH or NADH as reductant. The effectiveness of NADH may be significant in view of the increased NADH/NAD+ redox ratio in the liver as a consequence of ethanol oxidation by alcohol dehydrogenase. HER formation by human liver microsomes seems to be catalyzed by an oxidant derived from the interaction of iron with superoxide or H2O2, and a close association exists between HER formation and superoxide production. Cytochrome b5 seems to play a role in HER formation, most likely due to its effect on superoxide production.

Adult↗

PubMed1995

Role of cytochrome b5 in NADH-dependent microsomal reduction of ferric complexes, lipid peroxidation, and hydrogen peroxide generation.

The NADH-dependent microsomal electron transfer system consists of NADH-cytochrome b5 reductase and cytochrome b5, which donates reducing equivalents to fatty acyl desaturase, cytochrome P450, and other reactions. A study was carried out to investigate the interaction of NADH with several ferric complexes and to evaluate the role of cytochrome b5 in these interactions. NADH-dependent microsomal lipid peroxidation was stimulated by ferric-ATP, ferric-histidine, and ferric-ammonium sulfate, but not by ferric-EDTA. Anti-cytochrome b5 IgG produced a concentration-dependent inhibition of lipid peroxidation catalyzed by all three ferric complexes. Addition of purified cytochrome b5 to the microsomes increased the rate of lipid peroxidation with all three ferric complexes. Lipid peroxidation in control and the cytochrome b5-fortified microsomes was not sensitive to superoxide dismutase, catalase, or DMSO and was completely inhibited by trolox and propylgallate. Ferric-EDTA stimulated NADH-dependent microsomal production of H2O2 and NADH consumption. Anti-cytochrome b5 IgG had only a small inhibitory effect on this stimulation by ferric-EDTA. NADH supported microsomal reduction of ferric complexes in the order ferric-ATP > ferric-histidine approximately ferric-ammonium sulfate > ferric-EDTA. Anti-cytochrome b5 IgG inhibited, whereas added cytochrome b5 stimulated, the reduction of ferric-ATP, ferric-histidine, and ferric-ammonium sulfate, whereas reduction of ferric-EDTA was not affected by these additions. Ferric-ATP, at high concentrations, was more effective than ferric-histidine or ferric-ammonium sulfate in stimulating lipid peroxidation and in becoming reduced by NADH-dependent microsomal electron transport; anti-cytochrome b5 IgG was less inhibitory and added b5 was less stimulatory at 50 microM ferric-ATP compared to 5 microM ferric-ATP or 50 microM ferric-histidine or 50 microM ferric-ammonium sulfate. It is concluded that cytochrome b5 is required for reduction of low and high concentrations of ferric-histidine and ferric-ammonium sulfate and low concentrations of ferric-ATP and for the lipid peroxidation catalyzed by these ferric complexes. The reductase, not cytochrome b5, is involved in interaction with ferric-EDTA. Higher concentrations of ferric-ATP can also interact with the reductase, as well as with cytochrome b5.

Animals↗

PubMed1995

Increased production of hydroxyl radical by pericentral microsomes compared to periportal microsomes after pyrazole induction of cytochrome P4502E1.

Cytochrome P4502E1 is localized in the pericentral (PC) zone of the liver acinus to a greater extent than in the periportal (PP) zone. After pyrazole treatment, PC microsomes were more active in oxidizing typical substrates of CYP2E1 than PP microsomes and had an increased content of CYP2E1. The ability of PC and PP microsomes from pyrazole-treated rats to interact with iron and generate reactive oxygen species such as the hydroxyl radical (.OH) was evaluated. A sensitive DNA strand cleavage assay was used to detect .OH; supercoiled plasmid DNA is compact but is converted by .OH-induced single strand breaks to the relaxed open circular state. Microsomes from PC hepatocytes of pyrazole-treated rats were several fold more reactive than PP microsomes in promoting NADPH-dependent DNA strand cleavage with a variety of iron catalysts, including ferric-ATP, ferric-histidine, ferric-citrate, ferric ammonium sulfate, and ferric-EDTA. DNA strand cleavage was inhibited by superoxide dismutase, catalase, and .OH scavengers such as DMSO and ethanol. Rates of H2O2 production were higher with the PC microsomes. These results indicate that rates of .OH production are higher with PC microsomes than PP microsomes after pyrazole treatment to induce cytochrome P4502E1 and suggest the possibility that elevated production of reactive oxygen species may play a role in ethanol toxicity to the PC zone of the liver acinus.

Animals↗

PubMed1995

Increased cytotoxicity of 3-morpholinosydnonimine to HepG2 cells in the presence of superoxide dismutase. Role of hydrogen peroxide and iron.

3-Morpholinosydnonimine (SIN-1) is widely used to generate nitric oxide (NO(x).) and superoxide radical (O2-.). The effect of SOD on the toxicity of SIN-1 is complex, depending on what is the ultimate species responsible for toxicity. SIN-1 (< 1 mM) was only slightly toxic to HepG2 cells. Copper, zinc superoxide dismutase (Cu,Zn-SOD) or manganese superoxide dismutase (Mn-SOD) increased the toxicity of SIN-1. Catalase abolished, while sodium azide potentiated, this toxicity, suggesting a key role for H2O2 in the overall mechanism. Depletion of GSH from the HepG2 cells also potentiated the toxicity of SIN-1 plus SOD. Although Me2SO, sodium formate, and mannitol had no protective effect, iron chelators, thiourea and urate protected the cells against the SIN-1 plus Cu,Zn-SOD-mediated cytotoxicity. The cytotoxic effect of Cu,Zn-SOD but not Mn-SOD, showed a biphasic dose response being most pronounced at lower concentrations (10-100 units/ml). In the presence of SIN-1, Mn-SOD increased accumulation of H2O2 in a concentration-dependent manner. In contrast, Cu,Zn-SOD increased H2O2 accumulation from SIN-1 at low but not high concentrations of the enzyme, suggesting that high concentrations of the Cu,Zn-SOD interacted with the H2O2. EPR spin trapping studies demonstrated the formation of hydroxyl radical from the decomposition of H2O2 by high concentrations of the Cu,Zn-SOD. The cytotoxic effect of the NO donors SNAP and DEA/NO was only slightly enhanced by SOD; catalase had no effect. Thus, the oxidants responsible for the toxicity of SIN-1 and SNAP or DEA/NO to HepG2 cells under these conditions are different, with H2O2 derived from O2-. dismutation playing a major role with SIN-1. These results suggest that the potentiation of SIN-1 toxicity by SOD is due to enhanced production of H2O2, followed by site-specific damage of critical cellular sites by a transition metal-catalyzed reaction. These results also emphasize that the role of SOD as a protectant against oxidant damage is complex and dependent, in part, on the subsequent fate and reactivity of the generated H2O2.

Catalysis↗

PubMed1995

Production of nitric oxide and other iron-containing metabolites during the reductive metabolism of nitroprusside by microsomes and by thiols.

Sodium nitroprusside is used as a hypotensive agent because of its ability to produce nitric oxide (NO), although direct demonstration of this has not been reported in a biological system. Nitroprusside (NP) nitroxide radical anion, the first reduction product of NP generated in the presence of microsomes and NADPH, was found to undergo further metabolism. One of the products produced during this reductive metabolism was shown to be NO. By using N-methylglucamine dithiocarbamate-FeCl2 complex [(NMGD)2Fe(II)] as the NO trap, we have detected and characterized the mononitroso bis(N-methylglucamine dithiocarbamato) iron (II) complex (MNBI) (g = 2.040, and A(14N) = 13.3 G) as the product of NO trapping. The production of NO during the reductive metabolism of NP by submitochondrial particles and a human HepG2 hepatoblastoma cell line was also demonstrated using (NMGD)2Fe(II). In addition to MNBI, two other mononitrosyl iron complexes, the NP nitroxide radical anion and a second species designated as Fe(NO)(X)(Y) (g = 2.032, and A(14N) = 14.3 G), and additional unidentified paramagnetic products containing iron were also detected. Thiol compounds such as glutathione, cysteine, and cysteamine reduce NP to generate NP nitroxide radical anion and a paramagnetic species characterized as a dithiolated dinitroso iron complex (DDIC), Fe(NO)2(RS)2 g = 2.030, A(14N) = 2.2 G(2N), and A(1H) = 1.1 G(4H). At 77 K, DDIC generated from cysteine and NP has an axial symmetry, with g perpendicular = 2.040, and g parallel = 2.014. Two additional paramagnetic products, designated as species C (g = 2.020, linewidth = 4.8 G) and species D (g = 2.008, linewidth = 6.1 G), were also formed during NP reduction by thiol compounds. The characterization of these complexes has been hampered by the lack of hyperfine features in the ESR spectra. The production of NO during cysteamine reduction of NP was demonstrated by using (NMGD)2-Fe(II) as the spin-trap. These results directly demonstrate the production of NO during the reduction of NP by microsomes plus NADPH or by thiols. (NMGD)2-Fe(II) is a particularly useful spin-trap for the detection of NO in a strong reducing environment.

Animals↗

PubMed1995

Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the microsomal reductant. Interactions between rifamycin SV, iron and NADH generating hydroxyl-radical-like species may play a role in some of the hepatotoxic effects associated with the use of this antibacterial antibiotic.

Animals↗

PubMed1995

Increased oxidation of ethylene glycol to formaldehyde by microsomes after ethanol treatment: role of oxygen radicals and cytochrome P450.

The production of ferryl-type oxidants by microsomes from ethanol-fed rats and pair-fed controls was determined by assaying for the production of formaldehyde from ethylene glycol. Microsomes from the ethanol-fed rats were more reactive than controls in oxidizing ethylene glycol. Catalase was a powerful inhibitor for this reaction, superoxide dismutase was slightly inhibitory and hydroxyl radical scavengers had no effect. These results suggest an important role for H2O2, but not O2-. or .OH in the overall pathway for oxidizing ethylene glycol to formaldehyde. The production of H2O2 by microsomes was increased after ethanol treatment, the extent of increase corresponding to the increase in oxidation of ethylene glycol. A variety of inhibitors and ligands of cytochrome P450, including miconazole, diethyldithiocarbamate, tryptamine, and 4-methylpyrazole, inhibited formaldehyde production by both microsomal preparations. Anti-cytochrome P4502E1 IgG also inhibited the reaction with both microsomal preparations and prevented the increase caused by ethanol treatment. These results indicate that microsomes from ethanol-treated rats are more reactive than pair-fed controls in generating ferryl-type oxidants and that increased production of H2O2 by cytochrome P4502E1 plays a role in the elevated oxidation of ethylene glycol to formaldehyde.

Animals↗

PubMed1995

Boldine prevents human liver microsomal lipid peroxidation and inactivation of cytochrome P4502E1.

Boldine, an alkaloid found in the leaves and bark of boldo, prevented the ferric-ATP catalyzed peroxidation of human liver microsomes. Lipid peroxidation, dependent upon electron transfer from NADPH or NADH, was comparably inhibited by boldine, with a K(I) value of about 5 microM. Inactivation and decreased content of human cytochrome P4502E1 as a consequence of incubating microsomes with ferric-ATP and reductant was completely prevented by boldine. However, inactivation of cytochrome P4502E1 by CCl4 was not prevented by boldine, although the alkaloid prevented CCl4-catalyzed lipid peroxidation. This suggests that the CCl4 inactivation of P4502E1 may be independent of CCl4-mediated lipid peroxidation. In view of its low toxicity, lack of effect on P450 activity, and strong inhibition of peroxidation of human liver microsomes, boldine may be valuable as an antioxidant and hepatoprotective agent.

Antioxidants↗

PubMed1995

Cytotoxicity of acetaminophen in human cytochrome P4502E1-transfected HepG2 cells.

Acetaminophen (APAP) when administered in excess can cause severe hepatic necrosis in vivo. To study the mechanism of APAP toxicity and the role of cytochrome P450, a previously established human hepatoma HepG2 subline, MVh2E1-9, that constitutively expresses human CYP2E1 was used as a model. At high concentrations (above 5 mM) and when intracellular reduced glutathione (GSH) was depleted, APAP caused severe cytotoxicity in MVh2E1-9, but not in MV-5 cells which lack CYP2E1. The APAP cytotoxicity was dependent on the concentration of APAP and time of exposure, and could be blocked by 4-methylpyrazole, ethanol, diallyl sulfide, N-acetylcysteine and N-t-butyl-alpha-phenylnitrone, but not by propylgallate, an inhibitor of lipid peroxidation. Significantly more 14C-labeled APAP protein adduct was detected in MVh2E1-9 cells than MV-5 cells, especially after depletion of GSH. The formation of the APAP adducts could be inhibited by the same agents which prevent APAP cytotoxicity. At a lower concentration (1-2 mM), APAP inhibited proliferation in both MVh2E1-9 and the control MV-5 cells to similar extents. This antiproliferative action of APAP did not require depletion of GSH as did the cytotoxic action of APAP. These data suggest that APAP has a dual toxic effect on MVh2E1-9 cells: a P450-independent antiproliferative effect and the CYP2E1-dependent cytotoxic effect. These results demonstrate the ability of human CYP2E1 to activate APAP to reactive metabolites which form covalent protein adducts and cause toxicity to a hepatoma cell line.

Acetaminophen↗

PubMed1995

Inactivation and degradation of human cytochrome P4502E1 by CCl4 in a transfected HepG2 cell line.

Treatment with CCl4 in vivo labilizes cytochrome P4502E1, inactivating the enzyme and enhancing its degradation. To investigate the mechanism of CCl4-induced degradation of human CYP2E1, a recently-established MVh2E1-9 cell line, which constitutively expresses the human CYP2E1 in HepG2 cells was used. CCl4 inhibited oxidation of p-nitrophenol in isolated microsomes from MVh2E1-9 cells suggesting that CCl4 could be metabolized in vitro by the system; however, CCl4 did not promote lipid peroxidation under these conditions. Treatment of the MVh2E1-9 cells in situ with 2 mM CCl4 for 24 hr caused a 30 to 50% loss of both enzyme activity and 2E1 protein. Treatment with cycloheximide at the same time to inhibit constitutive protein synthesis showed a more prominent loss of 2E1 activity and protein. CCl4-induced degradation of CYP2E1 could be prevented by ligands and substrates of 2E1. N-acetylcysteine, N-t-butyl-alpha-phenylnitrone or propylgallate did not significantly prevent CCl4-induced inactivation or degradation of 2E1. After treatment with 14C-labeled CCl4, there was increased radioactive adduct formation in MVh2E1-9 cells compared to control cells lacking CYP2E1. This increase was completely prevented by 4-methylpyrazole and ethanol indicating its dependence on CYP2E1. These results suggest that the human CYP2E1 expressed in the MVh2E1-9 cell line metabolizes CCl4, generating reactive species at the active site that directly inactivate the enzyme and also labilize P450 for degradation by proteases present in the HepG2 cells. Lipid peroxidation is not required for the CCl4-induced inactivation and degradation of CYP2E1 in these cells.

Acetaminophen↗

PubMed1994

DNA strand cleavage as a sensitive assay for the production of hydroxyl radicals by microsomes: role of cytochrome P4502E1 in the increased activity after ethanol treatment.

There is increasing interest in the role of reactive oxygen radicals in the hepatotoxicity associated with ethanol consumption. Reactive oxygen intermediates interact with DNA and can cause single-strand breaks of supercoiled DNA. Experiments were carried out to evaluate the utility of this system as a sensitive assay for the detection of potent oxidants generated by rat liver microsomes isolated from pair-fed control rats and rats treated chronically with ethanol. DNA strand cleavage was assayed by monitoring the migration of the supercoiled and open circular forms in agarose. Microsomes catalysed DNA strand breakage with either NADPH or NADH as cofactors; iron was required to catalyse the reaction and various ferric complexes were effective in promoting the reaction. DNA strand cleavage was prevented by catalase, superoxide dismutase, GSH and hydroxyl-radical-scavenging agents, suggesting that a hydroxyl-radical-like species was the oxidant responsible for the breakage. This assay system proved to be much more sensitive in detecting hydroxyl radicals than are other methods, such as e.s.r. spectroscopy or oxidation of chemical scavenging agents with respect to the amount of microsomal protein and the nature and concentration of the iron catalyst required. Microsomes from ethanol-treated rats were more reactive than control microsomes in catalysing the DNA strand cleavage with either NADPH or NADH; increased catalytic activity was observed with various ferric complexes and was sensitive to the above antioxidants. Compared with preimmune IgG, anti-(cytochrome P4502E1) IgG had no effect on DNA strand cleavage by the control microsomes, but completely prevented the NADPH- and the NADH-dependent increased activity found with microsomes from the ethanol-treated rats. Inhibitors of cytochrome P4502E1, such as diethyl dithiocarbamate and tryptamine, also lowered the extent of increase of DNA strand cleavage produced by microsomes from the ethanol-treated rats. These results indicate that DNA strand cleavage is a very sensitive assay for detecting the production of hydroxyl radicals by microsomes and to demonstrate increased activity by microsomes after chronic ethanol treatment. This increased activity with NADPH and NADH is due, at least in part, to induction of cytochrome P4502E1.

Animals↗

PubMed1994

Ethanol increases content and activity of human cytochrome P4502E1 in a transduced HepG2 cell line.

Using recombinant retroviral expression, a HepG2 cell line which stably and constitutively expresses the coding sequences of the human cytochrome P4502E1 was previously established. Addition of ethanol (2 to 100 mM) to the culture medium of this cell line for two days resulted in an increase in the content of P4502E1 as determined by immunoblotting and an increase in HepG2 microsomal oxidation of p-nitrophenol, aniline, and N,N-dimethylnitrosamine. The ethanol-induced increase in microsomal oxidation of these substrates was prevented by ligands and inhibitors of P4502E1 as well as anti-human P4502E1 IgG and corresponded to the increase in P4502E1 content. Several other agents including pyrazole, 4-methylpyrazole, isoniazid, pyridine, and DMSO also increased the content of P4502E1 in this cell line but not oxidation of substrates, presumably a reflection of remaining tightly bound to the active site of P4502E1. Slot blot analysis indicated that ethanol addition did not increase P4502E1 mRNA levels. These results indicate that ethanol can increase the content of P4502E1 as well as catalytic oxidation of substrates dependent on P4502E1 in this experimental model, perhaps by stabilization of the protein against degradation.

Blotting, Western↗

PubMed1994

Oxidation of glycerol to formaldehyde by microsomes: are glycerol radicals produced in the reaction pathway?

Microsomes and reconstituted systems containing cytochrome P450 can oxidize glycerol to formaldehyde in a reaction catalyzed by an oxidant produced from the interaction of nonheme iron with H2O2. To evaluate the mechanism for this oxidation, the generation of glycerol radicals by various systems was compared to rates of formaldehyde production from glycerol. Photolysis of H2O2, oxidation of xanthine by xanthine oxidase in the presence of iron catalysts, or NADPH-dependent microsomal electron transfer in the presence of ferric-EDTA produced hydroxyl radicals. In the presence of glycerol these reaction systems produced DMPO-glycerol radical adducts which were detected by ESR spectroscopy. Despite the production of .OH and glycerol spin-trapped adducts by these reaction systems, very low amounts or nondetectable amounts of formaldehyde were produced from the glycerol. However, significant amounts of formaldehyde were observed when microsomes were incubated in the presence of ferric ammonium sulfate or ferric-ATP, although .OH production was lower with these iron catalysts than with ferric-EDTA. These results fail to support correlation between .OH production and oxidation of glycerol to formaldehyde. Under conditions in which glycerol was oxidized to formaldehyde, no glycerol radical species could be observed with DMPO as the spin-trapping agent. These results suggest the oxidant (not .OH) derived from the interaction of H2O2 with iron apparently cleaves glycerol to formaldehyde without the formation of a radical intermediate. Alternatively, the radical intermediate may be produced at a too low concentration to be detected or the radical intermediate may not be formed as a free species and therefore cannot be spin-trapped.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Ferritin-dependent inactivation of microsomal glucose-6-phosphatase.

Glucose-6-phosphatase (G6Pase) is a microsomal enzyme which is very sensitive to inactivation by lipid peroxidation. Experiments were carried out to evaluate whether ferritin, which is the major storage form of iron within cells, could catalyze inactivation of G6Pase and to determine the mechanism responsible for this effect of ferritin. Incubation of microsomes with NADPH in the absence of ferritin led to decreased activity of G6Pase. Ferritin stimulated this inactivation of G6Pase in a time- and concentration-dependent manner. Ferritin did not stimulate G6Pase inactivation when NADH replaced NADPH as the microsomal reductant. Superoxide dismutase but not catalase or DMSO prevented the ferritin-stimulated inactivation of G6Pase suggesting a role for superoxide, but not H2O2 or hydroxyl radical, in the overall mechanism. Trolox, at concentrations which prevent lipid peroxidation, also prevented the ferritin-catalyzed inactivation of G6Pase. Inhibition of G6Pase by ferritin was further enhanced in the presence of ATP but was inhibited in the presence of EDTA or desferrioxamine; ferric-ATP stimulates, whereas ferric-EDTA inhibits microsomal lipid peroxidation. The redox cycling agent paraquat increased the ability of ferritin to inactivate G6Pase by a reaction prevented by superoxide dismutase, trolox, EDTA, and desferrioxamine, but not by catalase or DMSO. Ferritin stimulated microsomal light emission, a reaction reflecting lipid peroxidation, with time and concentration dependence, and sensitivity to scavengers (trolox, superoxide dismutase), iron chelators and paraquat, identical to the inactivation of G6Pase. These results indicate that one possible toxicological consequence of ferritin-catalyzed lipid peroxidation is inhibition of microsomal enzymes such as G6Pase.

Animals↗

PubMed1994

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 H2O2 production were low; menadione increased H2O2 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.

Animals↗

PubMed1994

Increased production of reactive oxygen species by rat liver mitochondria after chronic ethanol treatment.

Rat liver microsomes and, to a lesser extent, nuclei were previously shown to produce reactive oxygen species at elevated rates after chronic ethanol treatment. The ability of intact rat liver mitochondria to interact with iron and either NADH or NADPH, and the effects of ethanol treatment, on production of reactive oxygen intermediates was determined. In the presence of ferric-ATP, NADH or NADPH catalyzed mitochondrial lipid peroxidation. Rates were elevated two- to threefold with mitochondria from ethanol-fed rats with both reductants. Mitochondrial lipid peroxidation was insensitive to superoxide dismutase, catalase, or hydroxyl radical scavengers but was sensitive to GSH and anti-oxidants such as trolox. Mitochondrial generation of hydroxyl radical-like species (assayed by oxidation of chemical scavengers) was increased after chronic ethanol treatment, as was H2O2 production. Modifiers of mitochondrial metabolism such as rotenone, cyanide, or an uncoupling agent, had no effect on mitochondrial production of reactive oxygen intermediates. The membrane-impermeable thiol reagent, p-chloromercuribenzoate, was complete inhibitory with both mitochondrial preparations. The activity of the rotenone-insensitive NADH-cytochrome c reductase, an enzyme of the outer mitochondrial membrane, was increased 40 to 60% by the ethanol treatment. These results suggest that NADH acting via the outer membrane NADH reductase can catalyze an iron-dependent production of oxygen radicals by rat liver mitochondria. The outer mitochondrial membrane fraction, prepared by digitonin fractionation, displayed increased rotenone-insensitive NADH-cytochrome c reductase activity after ethanol treatment and was more reactive in catalyzing scission of pBR322 DNA from the supercoiled form to the open circular forms. Rates of oxygen radical production by mitochondria and the extent of increase produced by chronic ethanol treatment are similar to those previously found with microsomes when NADH is the cofactor. Oxidation of ethanol by alcohol dehydrogenase generates NADH, and NADH-dependent production of reactive oxygen species by various organelles is increased after chronic ethanol treatment. These acute metabolic interactions coupled to induction by chronic ethanol treatment may play an important role in the development of a state of oxidative stress in the liver by ethanol.

Animals↗

PubMed1994

Fractionation of liver microsomes with polyethylene glycol and purification of NADH-cytochrome b5 oxidoreductase and cytochrome b5.

A simplified, rapid procedure for the purification of NADH-cytochrome b5 oxidoreductase and cytochrome b5 from either rat or rabbit liver is described. Microsomes were prepared by fractionation with polyethylene glycol and solubilized with Triton X-100. Cytochrome b5 was purified by a two-column procedure, anion exchange chromatography using DEAE-cellulose, and hydrophobic chromatography on phenyl-Sepharose. The final preparation of cytochrome b5 was purified more than a 120-fold from rat or rabbit liver microsomes, with specific content of about 50 nmol per mg protein, and overall yield of 22 to 32%. Only a single band with mol wt of 18,600 was found on sodium dodecyl sulfate (SDS)-gels or on Western blots using a polyclonal antibody raised against the purified b5. NADH-cytochrome b5 oxidoreductase was purified by a three-column procedure, DEAE-cellulose, hydroxylapatite, and ADP-agarose. The final product was purified more than 400-fold from rat or rabbit liver microsomes with a yield of about 25% and final specific activity of about 1600 mumol ferricyanide reduced per minute per milligram of protein. A single band with mol wt of 33, 100 was found on SDS-gels. The reductase catalyzed reduction of ferricyanide, dichlorophenol-indophenol, and cytochrome b5. Cytochrome c was reduced in the presence of reductase plus cytochrome b5, and this was inhibited by the anti-b5 IgG. The reductase catalyzed a rapid rate of reduction of ferric-ATP, which was slightly elevated by cytochrome b5. Ferric-histidine and ferric-ammonium sulfate were slowly reduced by reductase; addition of cytochrome b5 markedly stimulated reduction of these ferric complexes but inhibited reduction of ferric-EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗