Citrulline-malate effect on microsome phospholipids and cytochrome P450 in Euglena grown with ethanol

This study indicates for the first time the presence of cytochrome P450 in the microsomes of Euglena grown in lactate medium and substantiates the use of Euglena as a hepatic cell model. Similar effects of ethanol on Euglena and on rat hepatic microsomes were demonstrated: (i) decrements in the quantities of FA per milligram of proteins; (ii) increases in the proportions of PE; (iii) decreases in the proportions of PC; and (iv) production of cytochrome P450, degraded in P420. The citrulline-malate reestablishes in the microsomes the phospholipid environment and the cytochrome P450 concentration. These findings illustrate that the complex acts on the lipid peroxidation via the changes in cytochrome P450 activity.     

Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain.

Polyclonal antibodies to the major beta-naphthoflavone (BNF)-inducible form of cytochrome P-450 (P450IA) and to the major phenobarbitone (PB)-inducible form (P450IIB) have been used to quantify the contribution of these subfamilies to the total amount of cytochrome P-450 in rat livers and rat hepatocyte cultures treated with PB, BNF and metyrapone for 24 and 72 h. The P450IA and IIB subfamilies were not detectable (less than 5 pmol/mg of microsomal protein) in the livers of control rats, but administration of BNF resulted in the P450IA subfamily comprising more than 80% of the total hepatic cytochrome P-450. Administration of PB and metyrapone to rats did not elevate the level of this subfamily but elevated the levels of the P450IIB subfamily to 60% and 30% respectively of the total. Thus metyrapone is a ''PB-like'' inducer. However, in contrast with their effects in vivo, treatment with PB and metyrapone of rat hepatocytes did not elevate the proportion of the P450IIB subfamily relative to that in untreated cells but rather, like BNF, increased the P450IA subfamily. This would account for the ability of metyrapone to produce in hepatocyte culture, like BNF, a pronounced induction of ethoxyresorufin O-de-ethylase activity, but it does not account for why of all inducers studied only metyrapone can maintain the total cytochrome P-450 content of cultured hepatocytes, or the activity of ethylmorphine N-demethylase. This activity is generally considered to be associated with the P450IIB subfamily, but the lack of effect of metyrapone on this subfamily in hepatocyte culture must suggest that metyrapone is able to prevent the loss of the total amount of the cytochrome by increasing the expression of other cytochromes P-450.     

Effect of mesitylene on ethanol metabolism in rat liver microsomes.

Increased catalase activity was observed in the liver microsomal fraction of ethanol-treated rats (10% v/v aqueous ethanol solution per os for 5 weeks). In contrast, cytochrome P-450 concentration and specific activity of NADPH-cytochrome c reductase remained at the same level as in the liver of control rats (drinking water). The ratio of microsomal H2O2-generation to catalase activity was lower in the "ethanol" group than in the control one. This phenomenon seems to be related to the increased contribution of the "peroxidatic" reaction (increased rate of ethanol oxidation). Administration of mesitylene (1,3,5-trimethylbenzene) by gastric tube for 3 days (5 mmoles per kg daily) increased cytochrome P-450 concentration, specific activity of NADPH-cytochrome c reductase and ethanol metabolism.     

The microsomal ethanol oxidizing system: its role in ethanol and xenobiotic metabolism

After chronic ethanol consumption, the activity of the microsomal ethanol-oxidizing system (MEOS) increases and contributes to ethanol tolerance, as most conclusively shown in alcohol-dehydrogenase-negative deermice. In man and animals, there is an associated rise in microsomal cytochrome P-450, including a specific form (P-450IIEI) with high affinity for ethanol and for the activation of some drugs (i.e. acetaminophen), carcinogens (i.e. N-nitrosodimethylamine) and hepatotoxic agents (i.e. CCl4), thereby contributing to the susceptibility of alcoholics to xenobiotics, including industrial solvents. In addition, a benzoflavone-inducible liver cytochrome P-450 isoenzyme distinct but catalytically similar to cytochrome P-450IIE1 was purified which may play a significant role in drinkers who also are heavy smokers. Cross-induction of other microsomal enzymes is associated with enhanced metabolism of various drugs, resulting in drug tolerance. Catabolism of retinol was also found to be accelerated, in part through activation of newly discovered vitamin A depletion and possibly toxicity. Thus, elucidation of the microsomal metabolism of ethanol explains a number of complications that develop in alcoholics.     

Immunochemical characterization of NADPH-cytochrome P-450 reductase from Jerusalem artichoke and other higher plants.

Polyclonal antibodies were prepared against NADPH-cytochrome P-450 reductase purified from Jerusalem artichoke. These antibodies inhibited efficiently the NADPH-cytochrome c reductase activity of the purified enzyme, as well as of Jerusalem artichoke microsomes. Likewise, microsomal NADPH-dependent cytochrome P-450 mono-oxygenases (cinnamate and laurate hydroxylases) were efficiently inhibited. The antibodies were only slightly inhibitory toward microsomal NADH-cytochrome c reductase activity, but lowered NADH-dependent cytochrome P-450 mono-oxygenase activities. The Jerusalem artichoke NADPH-cytochrome P-450 reductase is characterized by its high Mr (82,000) as compared with the enzyme from animals (76,000-78,000). Western blot analysis revealed cross-reactivity of the Jerusalem artichoke reductase antibodies with microsomes from plants belonging to different families (monocotyledons and dicotyledons). All of the proteins recognized by the antibodies had an Mr of approx. 82,000. No cross-reaction was observed with microsomes from rat liver or Locusta migratoria midgut. The cross-reactivity generally paralleled well the inhibition of reductase activity: the enzyme from most higher plants tested was inhibited by the antibodies; whereas Gingko biloba, Euglena gracilis, yeast, rat liver and insect midgut activities were insensitive to the antibodies. These results point to structural differences, particularly at the active site, between the reductases from higher plants and the enzymes from phylogenetically distant plants and from animals.     

Peroxidative Activity in Euglena gracilis

Cell-free homogenates of Euglena gracilis contain very low levels of catalase activity as compared to higher plants and some other algae. Purified Euglena cytochrome c acts catalytically as a peroxidase. The observed catalytic activity of cytochrome c in extracts from heterotrophically grown cells was more than enough to account for the observed rates of hydrogen peroxide destruction. The peroxidative activity of Euglena cytochrome c was completely inhibited by 20 mm 3-amino-1,2,4-triazole.     

The markers of pig heart mitochondrial sub-fractions : I. - The dual location of NADPH-cytochrome c reductase in outer membrane and microsomes.

Evidence is presented about the dual location of NADPH-cytochrome c reductase in mitochondrial outer membranes as well as in microsomes, from pig heart. A high specific activity, was found in both fractions, even after their purification by washing, digitonin treatments, or passages on sucrose gradients. A large fraction of the total activity was associated with both mitochondria and microsomes. Mitochondrial outer membrane differs from microsomes by a low choline phosphotransferase activity and the absence of cytochrome P-450. The properties of mitochondrial and microsomal rotenone-insensitive NADH- and NADPH-cytochrome c reductases were studied. In microsomes, both activities have the same optimum pH (8.5) ; in contrast, in mitochondria they have a different one. The Km-NADPH were always much higher than those for NADH. In mitochondria the Km for NAD(P)H were dependent on cytochrome c concentration. The results show that the rotenone-insensitive NADH- and NADPH-cytochrome c reductases of mitochondria and microsomes have quite different behavior and do not appear to be supported by the same enzyme.     

The role of cytochrome P-450 in the hydroperoxide-catalyzed oxidation of alcohols by rat-liver microsomes.

The organic hydroperoxide cumene hydroperoxide is capable of oxidizing ethanol to acetaldehyde in the presence of either catalase, purified cytochrome P-450 or rat liver microsomes. Other hemoproteins like horseradish peroxidase, cytochrome c or hemoglobin were ineffective. In addition to ethanol, higher alcohols like 1-propanol, 1-butanol and 1-pentanol are also oxidized to their corresponding aldehydes to a lesser extent. Other organic hydroxyperoxides will replace cumene hydroperoxide in oxidizing ethanol but less effectively. The cumene-hydroperoxide-dependent ethanol oxidation in microsomes was inhibited partially by cytochrome P-450 inhibitors but was unaffected by catalase inhibitors. Phenobarbital pretreatment of rats increased the specific activity of the cumene-hydroperoxide-dependent ethanol oxidation per mg of microsomes about seven-fold. The evidence suggests that cytochrome P-450 rather than catalase is the enzyme responsible for hydroperoxide-dependent ethanol oxidation. However, when H2O2 is used in place of cumene hydroperoxide, the microsomal ethanol oxidation closely resembles the catalase system.     

Hepatic cytochrome P-450-dependent monooxygenase systems of the trout, frog and snake--I. Components

1. Components of the hepatic monooxygenase systems (cytochrome P-450, cytochrome b5, NADPH cytochrome P-450- or c-reductase) of the brown trout (Salmo trutta), leopard frog (Rana pipiens) and garter snake (Thamnophis) were considerably lower than those found in the rat. 2. Reactivity of snake NADPH-cytochrome P-450-reductase with cytochrome P-450 was about twice that of the rat reductase; reactivities of trout and frog reductases were similar, but lower than that of the rat. The optimal temperature for the rat, frog and snake reductase activity was 37 degrees C, but 26 C for the trout reductase, regardless of whether cytochrome P-450 or cytochrome c was the electron acceptor for the reaction. 3. A type I substrate (benzphetamine) and a type II substrate (aniline) were less reactive with P-450 cytochrome from the trout, frog and snake than with P-450 cytochrome from the rat. 4. Qualitative differences were seen in the ethylisocyanide spectrum of microsomes from the rat, trout, frog and snake; these differences reflect qualitative differences in the populations of P-450 cytochromes among each of the four species.     

Effect of monooxygenase reactions catalyzed by cytochrome P-450 on the microsomal membrane

Hydroxylation of dimethylaniline in rabbit liver microsomes is accompanied by inactivation of cytochrome P-450 and the formation of products inhibiting the catalytic activity of non-inactivated cytochrome P-450. Other enzymes and electron carriers of microsomal membrane (cytochrome b5, NADH-ferricyanide reductase, NADPH-cytochrome c and NADPH-cytochrome P-450 reductases) as well as glucose-6-phosphatase were not inactivated in the course of the monooxygenase reactions. Phospholipids and microsomal membrane proteins were also unaffected thereby. Consequently, the changes in the microsomal membrane during cytochrome P-450 dependent monooxygenase system functioning are confined to the inactivation of cytochrome P-450.     

Detoxification effect of iron-encaging zeolite-processed water in tributyltin-intoxicated Euglena gracilis Z

In our previous paper, we reported the restoration promoting effects of mineral-encaging zeolite-processed water, especially of a Fe-encaging one, on tributyltin chloride (TBTCl)-intoxicated Euglena gracilis. This present study extends the investigation on the behavior of TBTCl and a xenobiotic enzyme, cytochrome P-450, in Euglena cells incubated with or without Fe-encaging zeolite-processed water (FeZW). Subcellular fractionation of TBTCl-intoxicated Euglena cells, atomic absorption spectrophotometry, and GC analyses showed that TBTCl was rapidly incorporated into the cells to halt cell motility. GC-MS showed that FeZW promoted conversion of TBTCl to dibutyltin (DBT) as the major metabolite in the microsomal fraction of the cells. An in vitro incubation system with heat-treated microsomes did not convert TBTCl to DBT. The contribution of cytochrome P-450 in the microsomal fraction was suggested by an immunochemical method. The results suggest that the improvement of detoxification by FeZW in the TBT-intoxicated Euglena cells should be due to activation of biotransformation system of the Euglena cells by FeZW.     

Oxidative phosphorylation during glycollate metabolism in mitochondria from phototrophic Euglena gracilis.

Mitochondria were isolated by gradient centrifugation on linear sucrose gradients from broken cell suspensions of phototrophically grown Euglena gracilis. An antimycin A-sensitive but rotenone-insensitive glycollate-dependent oxygen uptake was demonstrated in isolated mitochondria. The partial reactions of glycollate-cytochrome c oxidoreductase and cytochrome c oxidase were demonstrated by using Euglena cytochrome c as exogenous electron acceptor/donor. Isolated mitochondria contain glycollate dehydrogenase and glyoxylate-glutamate aminotransferase and oxidize exogenous glycine. A P:O ratio of 1.7 was obtained for glycollate oxidation, consistent with glycollate electrons entering the Euglena respiratory chain at the flavoprotein level. The significance of these results is discussed in relation to photorespiration in algae.     

Inducers of cytochrome P-450d: influence on microsomal catalytic activities and differential regulation by enzyme stabilization

The interaction of isosafrole, 3,4,5,3',4',5'-hexabromobiphenyl (HBB) and hexachlorobiphenyl (HCB) with cytochrome P-450d was evaluated by characterization of estradiol 2-hydroxylase activity. Displacement of the isosafrole metabolite from microsomal cytochrome P-450d derived from isosafrole-treated rats resulted in a 160% increase in estradiol 2-hydroxylase. The increase was fully reversed by incubation with 1 microM HBB. Although isosafrole is capable of forming a complex with many different cytochrome P-450 isozymes, it appears to bind largely to cytochrome P-450d in vivo as was demonstrated by measuring the enzymatic activity of microsomal cytochromes P-450b, P-450c, and P-450d from isosafrole-treated rats. When estradiol 2-hydroxylase was measured in rats treated with increasing doses of HCB, there was a gradual decrease in microsomal enzyme activity despite a 20-fold increase in cytochrome P-450d. The ability of cytochrome P-450d ligands to stabilize the enzyme was investigated in two ways. First, cytochromes P-450c and P-450d were quantitated immunochemically in microsomes from rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a dose which maximally induced total cytochrome P-450, followed by a single dose of a second inducer. The specific content of cytochrome P-450d was significantly increased when isosafrole or HCB was the second inducer but not when 3-methylcholanthrene was the second inducer. Second, the relative turnover of cytochrome P-450d was measured by the dual label technique. Following TCDD treatment, microsomal protein was labeled in vivo with [3H]leucine, the second inducer was given and protein was again labeled 3 days later with [14C]leucine. A higher ratio of 3H/14C in the cytochrome P-450d from isosafrole + TCDD- and HCB + TCDD-treated rats relative to TCDD (control)-treated rats suggested that isosafrole and HCB were able to retard the degradation of cytochrome P-450d, presumably by virtue of being tightly bound to the enzyme.     

The role of cytochrome b5 in adrenal microsomal steroidogenesis.

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.     

Treatment with poly I.C. enhances lipid peroxidation and the activity of xanthine oxidase, and decreases hepatic P-450 content and activities in mice and rats

Treatment of mice and rats with polyriboinosinic acid-polyribocytidylic acid (poly I.C., 5 mg/kg i.p.), a potent interferon inducer, decreased hepatic cytochrome P-450 system content and activities without influencing P-450-independent xenobiotic metabolizing enzymes. Treatment with poly I.C. decreased the content of P-450 by 28% in mice (P less than 0.05) and 30% in rats (P less than 0.05) but did not alter the activity of cytochrome c reductase. With treatment of poly I.C., the activity of XO increased 87% in mice (P less than 0.01) and 30% in rats (P less than 0.01). Lipid peroxidation was enhanced by 82% in mice (P less than 0.01) and 95% in rats (P less than 0.05). These results raise the possibility that a part of the depression of P-450 system content and activities by poly I.C. might be caused by enhanced lipid peroxidation associated with increased activity of XO.     

Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture. Thereafter the concentration decreased, reaching zero at a late-stationary phase. When the yeast was grown on a medium that contained lactose or pentoses (L-arabinose, L-rhamnose, D-ribose and D-xylose), cytochrome P-450 did not occur. When a non-fermentable energy source (glycerol, lactate or ethanol) was used, no cytochrome P-450 was detectable. Transfer of cells from D-glucose medium to ethanol medium caused a slow disappearance of cytochrome P-450, although the amount of the haemoprotein still continued to increase in the control cultures. Cytochrome P-450 appeared thus to accumulate in conditions where the rate of growth was fast and fermentation occurred. Occurrence of this haemoprotein is not necessarily linked, however, with the repression of mitochondrial haemoprotein synthesis.     

Effects of phenobarbitone on hepatic microsomal enzyme activity and liver blood flow in spontaneously hypertensive rats.

Hepatic microsomal enzyme activity, liver blood flow and pentobarbitone sleeping time were determined in spontaneously hypertensive rats (SHR) and normotensive Wistar rats (NR) after pretreatment with saline or phenobarbitone. In NR and SHR the increases in total liver blood flow produced by phenobarbitone were sufficient to maintain liver perfusion despite the increase in liver weight and in both strains of rat the increase was entirely due to increased portal venous return. Saline pretreated SHR had shorter pentobarbitone sleeping times than control NR and their livers had greater total cytochrome c reductase activities and total microsomal protein than those of NR but cytochrome P-450 contents were not significantly different. Phenobarbitone significantly shortened sleeping times in both strains but NR still slept longer than SHR. Total microsomal protein, cytochrome P-450 content and cytochrome c reductase activity were increased by phenobarbitone in both SHR and NR but the increases in cytochrome P-450 and cytochrome c reductase were greater in the hypertensive rats.     

Induction of cytochrome P-450 and ethanol oxidation in Yarrowia lipolytica

The study of the effect of different ethanol concentrations in the medium on the growth and the activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. But when the initial concentration of ethanol in the medium was 3 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity.

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.     

The Induction of Cytochrome P-450 and Ethanol Oxidation in Yarrowia lipolytica Cells

The study of the effect of different ethanol concentrations in the medium on the growth and activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. However, when the initial concentration of ethanol in the medium was 4 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.