Induction of the hepatic microsomal and nuclear cytochrome P-450 system by hexachlorobenzene, pentachlorophenol and trichlorophenol.

The application of hexachlorobenzene (HCB), pentachlorophenol (PCP) and 2,4,5-trichlorophenol (TCP) to female rats led to an induction of both the microsomal and the nuclear cytochrome P-450 system in the liver. The increase of th mixed-function hydroxylase activities examined (7-ethoxycoumarin deethylase, 7-ethoxyresorufin deethylase, NADPH-dependent cytochrome c reductase, aminopyrine demethylase, benzpyrene hydroxylase) did not correlate strictly with the cytochrome P-450 content. Depending on the inducers and the substrates used, the content and the activity of the cytochrome P-450 were essentially smaller in the nuclei than in the microsomes. It was striking that in the nuclei those activities (benzpyrene hydroxylase, 7-ethoxyresorufin deethylase, 7-ethoxycoumarin deethylase) were preferably induced which can be attributed to the methyl-cholanthrene-induced form of the cytochrome P-450 (cytochrome P-448). These results suggest, also in the light of findings of other authors, the induction of different species of cytochrome P-450 in the nuclei and microsomes.     

Ribose. An oxidation product of glucose 6-phosphate in microsomal fraction

An oxidative metabolism of glucose 6-phosphate was studied in rat liver microsomal fraction. Although radioactive 14CO2 was formed from [1-14C]glucose 6-phosphate in the microsomal fraction (Hino, Y., and Minakami, S. (1982) J. Biochem. (Tokyo) 92, 547-557), the formation was negligible when [2-14C]glucose 6-phosphate was used as a starting substrate. These results indicated an inability of the microsomal fraction to rearrange [2-14C]glucose 6-phosphate to form [1-14C] glucose 6-phosphate, and it was expected that a certain compound derived from glucose 6-phosphate accumulated as an end-product of the reaction. We, therefore, have tried to identify the product by high performance liquid chromatography, and found that ribose accumulated as the end-product. The formation of ribose was inhibited in the same manner as that of 14CO2 by antibodies against rat liver microsomal hexose-6-phosphate dehydrogenase, and the ratios of ribose to 14CO2 formed in the reaction were 0.5-0.8 on a molar basis. The finding of ribose formation further suggested the involvement of ribose phosphate isomerase and phosphatase activities in the reaction.     

天然产物莪术醇单克隆抗体的制备

为制备小分子化合物莪术醇的单克隆抗体,先将莪术醇(curcumol)与载体蛋白牛血清蛋白(BSA)偶联形成完全抗原,用基质辅助激光解吸飞行时间质谱法(MALDI-TOF-MS)鉴定莪术醇人工抗原的偶联率,然后采用杂交瘤技术获得杂交瘤株,并对其进行小鼠腹水的制备与纯化.结果表明:莪术醇半抗原与载体的偶联比为19.6,单克...     

p-Hydroxyphenylacetaldehyde, the major product of tyrosine oxidation by the activated myeloperoxidase system can act as an antioxidant in LDL

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. HOCl generated by the myeloperoxidase/H2O2/Cl- system of activated neutrophils may be operative in vivo making LDL atherogenic. Tyrosine has been found to be oxidized by HOCl to p-hydroxyphenylacetaldehyde (p-HA) capable of modifying phospholipid amino groups in LDL. As an amphiphatic phenolic compound, p-HA may have the potential to act as an antioxidant in the lipid phase of LDL. The present results show that (a) tyrosine exerts a protective effect on LDL modification by HOCl, (b) p-HA could act as antioxidant associated with the lipoprotein preventing cell- and transition metal ion-mediated LDL oxidation and (c) p-HA was able to scavenge free radicals.     

Glutathione oxidation by hypochlorous acid in endothelial cells produces glutathione sulfonamide as a major product but not glutathione disulfide

Treatment of cells with hypochlorous acid (HOCl) at sublethal doses causes a concentration-dependent loss in reduced glutathione (GSH) levels. We have investigated the products of the reaction of HOCl with GSH in human umbilical vein endothelial cells. Despite a complete loss of GSH, there were only very small increases in intracellular and extracellular glutathione disulfide and glutathione sulfonic acid after exposure to HOCl. (35)S labeling of the GSH pool showed only a minimal increase in protein-bound GSH, suggesting that S-thiolation was not a major contributor to HOCl-mediated loss of GSH in endothelial cells. Rather, the products of the reaction were mostly exported from cells and included a peak that co-eluted with the cyclic sulfonamide that is a product of the reaction of GSH with reagent HOCl. Evidence of this species in endothelial cell supernatants after HOCl treatment was also obtained using electrospray mass spectrometry. In conclusion, exposure to HOCl causes the irreversible loss of cellular GSH with the formation of novel products that are rapidly exported from the cell, and resynthesis of GSH will be required to restore levels. The loss of GSH would alter the redox state of the cell and compromise its defenses against further oxidative stress.     

Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids

During the NADPH-Fe induced peroxidation of liver microsomal lipids, products are formed which show various cytopathological effects including inhibition of microsomal glucose-6-phosphatase. The major cytotoxic substance has been isolated and identified as 4-hydroxy-2,3-trans-nonenal. The structure was ascertained by means of ultraviolet, infrared and mass spectrometry and high-pressure liquid chromatographic analysis. Moreover, 4-hydroxynonenal, prepared by chemical synthesis, was found to reproduce the biological effects brought about by the biogenic aldehyde. Preliminary investigations suggest that as compared to 4-hydroxynonenal very low amounts of other 4-hydroxyalkenals, namely 4-hydroxyoctenal, 4-hydroxydecenal and 4-hydroxyundecenal are also formed by actively peroxidizing liver microsomes. In the absence of NADPH-Fe liver microsomes produced only minute amounts of 4-hydroxyalkenals. The biochemical and biological effects of synthetic 4-hydroxyalkenals have been studied in great detail in the past. The results of these investigations together with the finding that 4-hydroxyalkenals, in particular 4-hydroxynonenal, are formed during NADPH-Fe stimulated peroxidation of liver microsomal lipids, may help to elucidate the mechanism by which lipid peroxidation causes deleterious effects on cells and cell constituents.     

Protein hydroperoxides are a major product of low density lipoprotein oxidation during copper, peroxyl radical and macrophage-mediated oxidation

Damage to apoB100 on low density lipoprotein (LDL) has usually been described in terms of lipid aldehyde derivatisation or fragmentation. Using a modified FOX assay, protein hydroperoxides were found to form at relatively high concentrations on apoB100 during copper, 2,2'-azobis(amidinopropane) dihydrochloride (AAPH) generated peroxyl radical and cell-mediated LDL oxidation. Protein hydroperoxide formation was tightly coupled to lipid oxidation during both copper and AAPH-mediated oxidation. The protein hydroperoxide formation was inhibited by lipid soluble alpha-tocopherol and the water soluble antioxidant, 7,8-dihydroneopterin. Kinetic analysis of the inhibition strongly suggests protein hydroperoxides are formed by a lipid-derived radical generated in the lipid phase of the LDL particle during both copper and AAPH mediated oxidation. Macrophage-like THP-1 cells were found to generate significant protein hydroperoxides during cell-mediated LDL oxidation, suggesting protein hydroperoxides may form in vivo within atherosclerotic plaques. In contrast to protein hydroperoxide formation, the oxidation of tyrosine to protein bound 3,4-dihydroxyphenylalanine (PB-DOPA) or dityrosine was found to be a relatively minor reaction. Dityrosine formation was only observed on LDL in the presence of both copper and hydrogen peroxide. The PB-DOPA formation appeared to be independent of lipid peroxidation during copper oxidation but tightly associated during AAPH-mediated LDL oxidation.     

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

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

Role of -NADH for microsomal ethanol oxidation

α-NADH¹ was found to serve as electron donor for microsomal ethanol oxidation of rat liver. Almost no ethanol oxidation was observed with β-NADH. The α-NADH-dependent ethanol oxidation was almost completely inhibited by 0.1 mM cyanide or azide and strongly abolished in the presence of formate. α-NADH-dependent ethanol oxidation was increased by 1 mM SKF-525A, an inhibitor of microsomal mixed-function oxidase, to about 200%.These results suggested that hydrogen peroxide generated from α-NADH and molecular oxygen in microsomes might be a prerequisite step in the over-all reaction, eventually leading to the peroxidatic ethanol oxidation by catalase to acetaldehyde.     

Inhibition of microsomal oxidation by o-benzoquinone derivatives

It was found that o-benzoquinone derivatives inhibit the hydroxylation of aniline without any appreciable influence on the spectral changes which are due to cytochrome P-450 binding to the substrate. A comparative kinetic study of inhibition of aniline hydroxylation and NADPH-dependent reduction of o-benzoquinones in liver microsomes revealed that the inhibition is due to the ability of these compounds to shunt microsomal electron transport pathways.     

Measurement of adrenolutin as an oxidation product of catecholamines in plasma

Using the reverse phase high-performance liquid chromatography (HPLC) with mobile phases composed of simple acids, we have developed an assay technique for the measurement of adrenolutin, one of the oxidation products of catecholamines, in rat plasma. Ion-pairing chromatography permits the separation and quantitation of plasma adrenolutin (M) in a linear manner. Sample preparation involved the precipitation of plasma proteins with perchloric acid and it is easier to handle a large number of samples at a time. However, we were unable to demonstrate the presence of adrenochrome, another oxidation product of catecholamines, in plasma since adrenochrome was rapidly destroyed in acid as well as in blood and was quickly changed, into adrenolutin. Adrenolutin peak in HPLC was confirmed by 1) the retention time; 2) co-injection of adrenolutin and; 3) the appearance of ³H-adrenolutin after injection of ³H-norepinephrine. Administration of different catecholamines as well as adrenochrome and adrenolutin in rats also increased the level of adrenolutin in plasma. Adrenolutin was found to be present in plasma in other species including dog, rabbit and pig. High level of adrenolutin, which may represent total concentration of aminolutin in plasma, suggests the presence of an efficient mechanism for the oxidation of catecholamines under in vivo conditions.     

Effect of thiourea on microsomal oxidation of alcohols and associated microsomal functions.

Thiourea and diethylthiourea, two compounds which react with hydroxyl radicals, inhibited NADPH-dependent microsomal oxidation of ethanol and 1-butanol. Inhibition by both compounds was more effective in the presence of the catalase inhibitor, azide. Inhibition by thiourea was noncompetitive with respect to ethanol in the absence of azide but was competitive in the presence of azide. Urea, a compound which does not react with hydroxyl radicals or H2O2, was without effect. Thiourea had no effect on NADH- and NADH-cytochrome c reductase, NADPH oxidase, and NADH- and NADPH-dependent oxygen uptake. Thiourea inhibited the activities of aniline hydroxylase and aminopyrine demethylase. Thiourea, but no other hydroxyl radical scavengers, e.g., dimethyl sulfoxide, mannitol, and benzoate, reacted directly with H202 and decreased H2O2 accumulation in the presence of azide. Therefore the actions of thiourea are complex because it can react with both hydroxyl radicals and H2O2. Differences between the actions of thiourea and those previously reported for dimethyl sulfoxide, mannitol, and benzoate, e.g., effects on drug metabolism, effectiveness of inhibition in the absence of azide, or kinetics of the inhibition, probably reflect the fact that thiourea reacts directly with H2O2 whereas the other agents do not. The current results remain consistent with the concept that microsomal oxidation of alcohols involves interactions of the alcohols with hydroxyl radicals generated from microsomal electron transfer.     

Methionine oxidation as a major cause of the functional impairment of oxidized actin

A significant specific increase in the actin carbonyl content has been recently demonstrated in human brain regions severely affected by the Alzheimer's disease pathology, in postischemic isolated rat hearts, and in human intestinal cell monolayers following incubation with hypochlorous acid (HOCl). We have very recently shown that exposure of actin to HOCl results in the immediate loss of Cys-374 thiol, oxidation of some methionine residues, and, at higher molar ratios of oxidant to protein, increase in protein carbonyl groups, associated with filament disruption and inhibition of filament formation. In the present work, we have studied the effect of methionine oxidation induced by chloramine-T (CT), which at neutral or slightly alkaline pH oxidizes preferentially Met and Cys residues, on actin filament formation and stability utilizing actin blocked at Cys-374. Methionines at positions 44, 47, and 355, which are the most solvent-exposed methionyl residues in the actin molecule, were found to be the most susceptible to oxidation to the sulfoxide derivative. Met-176, Met-190, Met-227, and Met-269 are the other sites of the oxidative modification. The increase in fluorescence associated with the binding of 8-anilino-1-naphtalene sulfonic acid to hydrophobic regions of the protein reveals that actin surface hydrophobicity increases with oxidation, indicating changes in protein conformation. Structural alterations were confirmed by the decreased susceptibility to proteolysis and by urea denaturation curves. Oxidation of some critical methionines (those at positions 176, 190, and 269) causes a complete inhibition of actin polymerization and severely affects the stability of actin filaments, which rapidly depolymerize. The present results would indicate that the oxidation of some critical methionines disrupts specific noncovalent interactions that normally stabilize the structure of actin filaments. We suggest that the process involving formation of actin carbonyl derivatives would occur at an extent of oxidative insult higher than that causing the oxidation of some critical methionine residues. Therefore, methionine oxidation could be a damaging event preceding the appearance of carbonyl groups on actin and a major cause for the functional impairment of the carbonylated protein recently observed both in vivo and in vitro.     

Different patterns of hepatic microsomal enzyme activity produced by administration of pure hexachlorobiphenyl isomers and hexachlorobenzene

Three hexachlorobiphenyl isomers, 2,2′,4,4′,5,5′-hexachlorobiphenyl (I), 2,2′,3,3′,4,4′-hexachlorobiphenyl (II) and 2,2′,3,4,4′,5′-hexachlorobiphenyl (III), have been administered to rats and the effects of these three compounds upon hepatic microsomal drug metabolism and upon hepatic porphyrins have been studied. Comparisons have been made with hexachlorobenzene and a commercial polychlorinated biphenyl mixture, Aroclor 1254. From measurements of activities of microsomal drug oxidations in vitro, the durations of pharmacological actions of certain drugs in vivo and spectral shifts associated with cytochrome P-450 it is shown that the three pure hexachlorobiphenyl isomers initially produce changes in hepatic microsomal activity which resemble those seen after treatment with phenobarbitone (PB). In contrast, following chronic feeding of the isomers, compounds II and III but not I produce a pattern of hepatic microsomal enzyme activity which shows some characteristics of the 3-methylcholanthrene (3-MC) and some characteristics of the phenobarbitone classes of inducer. Also, compounds II and III, but not I, cause accumulation in the liver of porphyrins containing either seven or eight carboxyl groups. These two responses are similar to those observed following hexachlorobenzene treatment and suggest that a relationship may exist between the mixed pattern of enzyme induction and the onset of hepatic porphyrin accumulation.     

Identification of the Escherichia coli recN gene product as a major SOS protein.

The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis. In addition to the 38-kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen. The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response. The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination. The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains. The 12-kDa protein has yet to be identified.