Proliferation of hepatic peroxisomes in rats following the intake of green or black tea.

Rats maintained on green, black or decaffeinated black tea (2.5%, w/v) as their sole drinking fluid displayed higher hepatic CN- insensitive palmitoyl CoA oxidase activity than controls; the extent of increase was similar with the three types of tea. Morphological examination of the liver using electron microscopy revealed an increase in the number of peroxisomes in the tea-treated animals. The same treatment of the animals with green and black tea resulted in a similar rise in hepatic microsomal lauric acid hydroxylation. Analysis by HPLC of the aqueous tea extracts employed in the current study showed that the total flavanol content of the green variety was much higher than the black varieties, and confirmed the absence of caffeine in the decaffeinated black tea. It may be concluded from the present studies that neither caffeine nor flavanoids are likely to be responsible for the proliferation of peroxisomes observed in rats treated with tea.

The Pex16p homolog SSE1 and storage organelle formation in Arabidopsis seeds.

Mature Arabidopsis seeds are enriched in storage proteins and lipids, but lack starch. In the shrunken seed 1 (sse1) mutant, however, starch is favored over proteins and lipids as the major storage compound. SSE1 has 26 percent identity with Pex16p in Yarrowia lipolytica and complements pex16 mutants defective in the formation of peroxisomes and the transportation of plasma membrane- and cell wall-associated proteins. In Arabidopsis maturing seeds, SSE1 is required for protein and oil body biogenesis, both of which are endoplasmic reticulum-dependent. Starch accumulation in sse1 suggests that starch formation is a default storage deposition pathway.

Isocitrate lyase of Ashbya gossypii--transcriptional regulation and peroxisomal localization.

The isocitrate lyase-encoding gene AgICL1 from the filamentous hemiascomycete Ashbya gossypii was isolated by heterologous complementation of a Saccharomyces cerevisiae icl1d mutant. The open reading frame of 1680 bp encoded a protein of 560 amino acids with a calculated molecular weight of 62584. Disruption of the AgICL1 gene led to complete loss of AgIcl1p activity and inability to grow on oleic acid as sole carbon source. Compartmentation of AgIcl1p in peroxisomes was demonstrated both by Percoll density gradient centrifugation and by immunogold labeling of ultrathin sections using specific antibodies. This fitted with the peroxisomal targeting signal AKL predicted from the C-terminal DNA sequence. Northern blot analysis with mycelium grown on different carbon sources as well as AgICL1 promoter replacement with the constitutive AgTEF promoter revealed a regulation at the transcriptional level. AgICL1 was subject to glucose repression, derepressed by glycerol, partially induced by the C2 compounds ethanol and acetate, and fully induced by soybean oil.

Peroxisome-proliferating effects of fenoprofen in mice.

We report on hepatic effects obtained in vivo by treating mice with different doses of fenoprofen, an arylpropionic acid previously shown to inhibit in vitro peroxisomal very long chain fatty acid oxidation. A strong and dose-related induction of peroxisomal palmitoyl-CoA oxidase, and of carnitine acyltransferase and acyl-CoA hydrolase activities was recorded in liver homogenates of mice fed diets supplemented with different contents [0.01, 0.05, 0.1, or 1% (w/w)] of fenoprofen for 6 d. Peroxisomal glycolate oxidase and mitochondrial butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA dehydrogenases were unaffected or increased. Hepatic catalase activity was significantly increased in mice fed the diet with 0.05 and 0.1% fenoprofen but, surprisingly, was not stimulated in mice fed the 1% fenoprofen-containing diet. A time-related but unequal induction of acyl-CoA oxidases and catalase was observed with the 0.1% fenoprofen diet: at 21 d of treatment, the induction of lignoceroyl-CoA and palmitoyl-CoA oxidase activities were five-fold stronger than that of catalase activity. In mice treated with 1% fenoprofen for up to 6 d, only acyl-CoA oxidase activities were found to be significantly increased. Morphometric analysis of the liver peroxisomes in mice treated with 0.1% fenoprofen evidenced an increase in size, volume density, and surface density along with a reduced ratio between perimeter and area of the peroxisomal profiles. No morphological marker for very long chain fatty acid deposition could be detected in livers from fenoprofen-treated animals. Our findings clearly demonstrate that fenoprofen acts as a peroxisome proliferator in the liver of mice and do not support the occurrence of in vivo reduction of very long chain fatty acid oxidation in liver from treated animals.

Proliferation of myocardial peroxisomes caused by several agents and conditions.

In view of considerable gaps in our knowledge of myocardial peroxisomes. the aim of the present study is, on the basis of extensive electron-microscopic investigations, to provide reliable results on the inducibility of a proliferation (increase in the number) of these organelles in rodents' heart by several agents and conditions. As far as possible, we compared the response of heart and liver peroxisomes. Morphometric investigations were performed to assess the effectiveness of the hypolipidemic agent HL 41, erucic acid, ethanol, nifedipine, chlorpromazine, two cardiotonic drugs, isoprenaline, adriamycin, and physical exercise. The study also included spontaneously hypertensive rats (SHR). A further objective was to determine synergistic or additive effects that might occur when two or three peroxisome-proliferating stimuli act simultaneously. In every case we observed a clear peroxisome proliferation, which was found to increase by between 10 and 97% under the influence of an additional inducer. The observed increase in peroxisome number ranged from almost 200% to nearly 400%. Our results suggest that very different agents and conditions can induce myocardial peroxisome proliferation when they lead to metabolic alterations associated with an increased need for a peroxisomal beta-oxidation of fatty acids as an energy source and/or for preventing toxic effects. Regulatory mechanisms of these adaptive processes are apparently also present in the heart via peroxisome proliferator-activated receptors (PPARs) and their activation by fatty acids, which can also stimulate the PPARs gene expression. The assumption that stimulated catalase gene expression might be responsible for the induction of peroxisome proliferation as a cellular response to an extraperoxisomal oxidative stress situation (isoprenaline, adriamycin, or physical exercise) poses some critical questions. These questions pertain especially to: (a) quantitative aspects with regard to the possible effectiveness of an increase in catalase activity by two-, three-, or four-fold enhanced peroxisome numbers; (b) the role of cytoplasmic catalase; (c) the existence and importance of a myocardial mitochondrial catalase; and (d) the co-operation between the two H2O2-destroying enzymes catalase and glutathione peroxidase.

Castor bean isocitrate lyase lacking the putative peroxisomal targeting signal 1 ARM is imported into plant peroxisomes both in vitro and in vivo.

To understand and manipulate plant peroxisomal protein targeting, it is important to establish the universality or otherwise of targeting signals. Contradictory results have been published concerning the nature and location of the glyoxysomal/peroxisomal targeting signal of isocitrate lyase (ICL). L.J. Olsen, W.F. Ettinger, B. Damsz, K. Matsudaira, A. Webb, and J.J. Harada ([1993] Plant Cell 5: 941-952) concluded that the last 5 amino acids (AKSRM) of Brassica napus ICL were sufficient and the last 37 amino acids were necessary for targeting to Arabidopsis leaf peroxisomes. In contrast, R. Behari and A. Baker ([1993]) J Biol Chem 268: 7315-7322) could find no requirement for the almost identical carboxy-terminal sequence AKARM for import of Ricinus communis ICL into isolated sunflower cotyledon glyoxysomes. To resolve this discrepancy, the import characteristics of a mutant R. communis ICL lacking the last 19 amino acids of the carboxy terminus was studied. ICL delta 19 was able to be imported by isolated sunflower glyoxysomes and by tobacco leaf peroxisomes when expressed transgenically. These results demonstrate that the in vitro import system faithfully reflects targeting in vivo, and that the source of the organelles (Arabidopsis versus sunflower, leaf peroxisomes versus seed glyoxysomes) is not responsible for observed differences between B. napus and R. communis ICL. The R. communis enzyme would therefore appear to possess an additional glyoxysome/peroxisome targeting signal that is lacking in the B. napus protein.

Dietary docosahexaenoic acid has little effect on peroxisomes in healthy mice.

NMRI mice were fed diets supplemented with 0.05, 0.2, or 2% (w/w) docosahexaenoic acid (DHA), a polyunsaturated fatty acid present in fish oil, for 3 d, 3 wk, or 3 mon. The doses of DHA were chosen to supply the mice with concentrations of DHA which approximate those that have been reported to be beneficial to patients with peroxisomal disease. Diets containing 0.05 or 0.2% DHA did not change hepatic, myocardial, and renal catalase (EC 1.11.1.6) activity except for a slight but significant increase (to 120%) in myocardial catalase activity in mice treated with the 0.05% DHA diet for 3 mon. A diet with 2% DHA induced myocardial catalase activity to 150% after both 3 d and 3 wk of administration. In the liver of mice fed this diet for 3 wk, hepatic catalase activity was increased to 140% while no induction of palmitoyl-CoA oxidase (EC 1.3.99.3), urate oxidase (EC 1.7.3.3), and L-alpha-hydroxyisovalerate oxidase (EC 1.1.3.a) was observed. With the light microscope, no changes in peroxisomal morphology were visually evaluated in catalase stained sections of liver, myocardium, and kidney of mice fed either diet. Our results show that in healthy mice a low dietary DHA dose (< 0.2%; this corresponds to a dose prescribed to peroxisomal patients) has no effect on several hepatic peroxisomal H2O2-producing enzymes, including the rate-limiting enzyme of the peroxisomal fatty acid beta-oxidation. This may indicate that such a DHA dose will not add a strong load on the often disturbed fatty acid metabolism in the liver of patients with peroxisomal disorders.

Peroxisomes in mice fed a diet supplemented with low doses of fish oil.

The influence of low dietary doses (0.1 and 0.8% w/w) of a commercial fish oil preparation on peroxisomes in normal mice was studied and compared to the known strong inductive effects of high (10%) fish oil diets. Low fish oil doses were chosen to supply the mice with a concentration of docosahexaenoic acid, which was beneficial to patients with a peroxisomal disease. Peroxisomes were evaluated by cytochemical, morphometric, and enzymological techniques. The 0.1% fish oil diet had no effect on peroxisomes in liver, heart, and kidney even after prolonged treatment. The 0.8% diet did not change the peroxisomal number nor the catalase (EC 1.11.1.6) activity in the liver. Hepatic peroxisomal beta-oxidation, however, was increased by 50% after 14 d. This was accompanied by reduced peroxisomal size. The 0.8% diet also caused a small increase (+25%) in myocardial catalase activity. No effect was observed in kidneys. Our results indicate that in mice a low (< 0.8%) dietary fish oil dose has no or only a slight effect on hepatic peroxisomal beta-oxidation. This may be of particular interest to patients with a peroxisomal fatty acid beta-oxidation defect and who display a severe deficiency of docosahexaenoic acid--diets supplemented with low fish oil doses will improve the docosahexaenoic acid level without adding a strong load to the disturbed fatty acid metabolism.

Peroxisomes in liver, heart, and kidney of mice fed a commercial fish oil preparation: original data and review on peroxisomal changes induced by high-fat diets.

Male NMRI mice were fed a diet with 10% w/w Beromegan for up to three weeks. Beromegan is a commercial fish (salmon) oil preparation rich in eicosapentaenoic acid and docosahexaenoic acid. Peroxisomal beta-oxidation capacity, catalase activity, and ultrastructural morphometry of the hepatic peroxisomes were investigated. In myocardium and kidney, catalase activity, peroxisomal staining after catalase cytochemistry, peroxisomal morphology, and morphometry (in myocardium) were evaluated. In liver, we found a significant increase in peroxisomal beta-oxidation, catalase activity, and peroxisomal number already after 3 days of dietary treatment. These changes were more pronounced after 3 weeks. Peroxisomal size was not changed. Positive correlations were found between peroxisomal enzyme activities and the number but not the size of the peroxisomes, and between catalase activity and beta-oxidation capacity. The mean peroxisomal diameter per animal was inversely proportional to catalase activity measured in homogenate. In myocardium, catalase activity was increased with duration of fish oil feeding. Peroxisomal staining, number, and size were also increased when compared to controls. In kidney, no alterations were observed. Our results indicate a beneficial effect of a diet supplemented with fish oil on the peroxisomal metabolism in liver and myocardium; it differs from the changes induced by xenobiotic peroxisome proliferation.

Lack of aconitase in glyoxysomes and peroxisomes.

The aim of this work was to find out whether aconitase [citrate (isocitrate) hydro-lyase, EC 4.2.1.3] which is rapidly inactivated by H2O2, is present in the microbodies from plant cells. The separation of intact organelles from castor-bean (Ricinus communis) endosperm and potato (Solanum tuberosum) tuber indicated that aconitase activity is essentially limited to the mitochondria and cytosol fraction, but was not detected in highly purified castor-bean endosperm and potato tuber peroxisomes. An isotropic e.p.r. signal of the type expected for the 3Fe cluster of oxidized aconitase was not detected in microbodies. In immunoblot analyses, antibodies raised against potato tuber mitochondrial aconitase did not cross-react with any glyoxysomal or peroxisomal protein. Positive reactions were found for cytosol fraction and mitochondria of castor-bean endosperm. The operation of the full glyoxylate cycle in isolated glyoxysomes requires the presence of aconitase in the incubation medium. It is concluded that glyoxysomes are probably devoid of aconitase and that the glyoxylate cycle requires a detour via the cytosol, which contains a powerful aconitase activity.

31P-NMR of liver peroxisome membranes from normal and clofibrate-treated rats.

Normal and clofibrate-induced rat liver peroxisomes were studied in order to correlate the drug-elicited modifications of membrane permeability and fluidity with changes of the phospholipid component. The phospholipid composition and membrane fluidity of purified peroxisomal fractions from control and test animals were evaluated by 31P-NMR and fluorescence polarization spectroscopic techniques. The ultrastructural, enzymatic and electrophoretic analyses confirmed that the drug was able to induce: i) peroxisome proliferation; ii) increase of the catalase and fatty acyl CoA beta-oxidation system (FAOS) activities, and iii) neosynthesis of the 69 kDa peroxisomal membrane protein (PMP). The distribution pattern of the peroxisomal enzymes and the fluorescence polarization values of anilinonaphthalene-8-sulfonate (ANS)-labelled peroxisomes, suggests that the membrane permeability and fluidity are altered, while 31P-NMR spectroscopy seems to indicate that the phospholipid composition and the phospholipid/lysophospholipid ratio do not vary between test and control samples.

Rapid effects of dietary fish oil on peroxisomes in mouse liver.

We investigated hepatic catalase activity, and morphologic and morphometric alterations of hepatocellular peroxisomes after catalase cytochemistry, in mice given a diet supplemented with 10% Beromegan, a commercial fish oil preparation, for up to three days. Fish oil is rich in docosahexaenoic acid (C22:6 (n-3)) and in eicosapentaenoic acid (20:5 (n-3)). Hepatic catalase activity showed a gradual increase in mice fed this diet, being significantly increased (136 +/- 10 UB/g liver) after three days when compared top controls (85 +/- 11 UB/g liver). Light microscopy indicated an increase in peroxisomal staining and peroxisomal proliferation. The latter observation was confirmed by ultrastructural morphometry: number, volume density and surface density of the peroxisomes were more than doubled after a three day diet containing fish oil. Peroxisomal size was not changed. These alterations are suggestive for an increased peroxisomal metabolism induced by a diet rich in poly-unsaturated fatty acids.

The involvement of selenium in peroxisome proliferation caused by dietary administration of clofibrate to rats.

The effects of dietary treatment with clofibrate (0.5% w/w for 10 days) on the livers of selenium-deficient male rats were examined. The peroxisome proliferation (as determined by electron microscopy) in the livers of selenium-deficient animals was much less pronounced than in the case of selenium-adequate rats and no increase in peroxisomal fatty acid beta-oxidation (assayed both as antimycin-insensitive palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity) was observed in the deficient animals. On the other hand, in selenium-deficient rats clofibrate caused increases in the specific activity of microsomal lauric acid omega- and omega-1-hydroxylation and an apparent change in mitochondrial size, seen as a redistribution of mitochondria from the 600 x g(av) pellet to the 10,000 x g(av) pellet, which were approximately 50% as great as the corresponding effects on control animals. Obviously, then, these three different effects of clofibrate are not strictly coupled and may involve at least partially distinct underlying mechanisms. Initial experiments demonstrated that peroxisome proliferation could be obtained by exposing primary hepatocyte cultures derived from selenium-deficient rats to clofibric acid (an in vivo hydrolysis product of clofibrate which is the proximate peroxisome proliferator), nafenopin or mono(2-ethylhexyl)phthalate. This finding suggests that selenium deficiency does not have a direct influence on the basic process(es) underlying peroxisome proliferation, but rather has indirect effects, influencing, for example, the pharmacokinetics of clofibrate and/or hormonal factors.

Evidence of peroxisomes and peroxisomal enzyme activities in the oleaginous yeast Apiotrichum curvatum.

The presence of peroxisomes and peroxisomal enzyme activities were investigated in the oleaginous yeast Apiotrichum curvatum ATCC 20509 (formerly Candida curvata D.) Catalase, a marker enzyme for peroxisomes, was measured in cell-free extracts prepared by sonication. The nature of the carbon and nitrogen sources in the growth medium greatly affected catalase activity. Cells grown on corn oil had high specific activity of catalase, but those grown on glucose, sucrose, or maltose had low specific activity. High specific activity of catalase was measured in cultures grown on media that supported poor growth (with soluble starch as carbon source or with methylamine, urea, or asparagine as nitrogen source). Peroxisomes from cells grown on corn oil were separated from other subcellular fractions in a discontinuous sucrose gradient. Major peaks of activity of fatty acid beta-oxidation and of two key enzymes in the glyoxylate cycle were found in fractions containing peroxisomes, but not in fractions corresponding to the mitochondria. Peroxisomal beta-oxidation showed equivalent activity with palmitoyl CoA or n-octanoyl CoA as substrate. Mitochondria did not seem to contain NAD-linked glutamate dehydrogenase. Peroxisomes with a homogeneous matrix and core surrounded by a single-layer membrane were observed with an electron microscope in cells grown on corn oil, but not in those grown on glucose. Staining with 3,3'-diaminobenzidine revealed that catalase activity was located in peroxisomes. Peroxisomes in this oleaginous yeast play important roles in lipid metabolism.

Erucic acid metabolism in rat heart. A combined biochemical and radioautographical study.

Metabolism of Erucic Acid was studied in rat heart in comparison with that of oleic acid, particularly in relation with diet lipids. Rats were fed for 3 or 60 days a diet containing 30% of the calories of either Rapessed Oil, rich in erucic acid or sunflower seed oil rich in linoleic acid. They were I.V. injected with tritiated erucic or oleic acid. After 1 or 15 min the radioactivity recovered in heart lipids was very low whatever the diet (1 to 2%). One minute after injection of erucic acid the radioactivity was mainly recovered in the free fatty acid fraction and as untransformed erucic acid. After 15 min the major part of radioactivity was recovered in the triacylglycerol fraction which contained a high proportion of labelled oleic acid formed by shortening of erucic acid. When oleic was injected, the radioactivity was principally recovered in triacylglycerols as untransformed oleic acid whatever the experimental conditions. Electron microscopy showed that a much higher proportion of peroxisomes, was present in heart cells, following sunflower seed oil diet as compared to rapeseed oil diet. In all cases mitochondria supported the greater part of radioactivity, especially when erucic acid was injected in rats fed rapeseed oil. After sunflower seed oil, a noticeable radioactivity was observed in peroxisomes, most of them containing silver grains, especially when oleic acid was injected. According to the data reported, peroxisomes do not seem more implicated than mitochondria in the metabolism of erucic acid in myocardium.

Electron cytochemical studies of Cryptococcus neoformans grown on uric acid and related sources of nitrogen.

Cells of Cryptococcus neoformans grown on xanthine or urate as the sole sources of nitrogen produced numerous, single membrane-bound organelles, deemed to be microbodies. Electron images of these structures showed positive cytochemical staining for catalase and alpha-hydroxy acid oxidase, known marker enzyme activities for microbodies. Microbodies in xanthine and urate-grown cells were cytochemically reactive for the presence of the hydrogen peroxide-producing xanthine and urate oxidases. Molybdenum and phosphorus (elements associated with the cofactor common to nitrogen scavenging enzymes) were detected in the substrate-induced microbodies by X-ray dispersive microanalysis. The single limiting membrane of the substrate-induced microbody was stained by a modified Gomori reaction for the presence of alkaline phosphatase, thereby suggesting the participation of this enzymic activity in the events associated with microbody chemistry.

Morphometric cytochemistry of diminution of catalase-containing peroxisomes in copper-loaded liver.

The density of hepatocellular catalase-containing peroxisomes was quantified, utilizing a computer-aided image analysing technique, on 1-micron thick diaminobenzidine-stained sections. Hepatic copper accumulation following intraperitoneal injection of cupric chloride resulted in a dose-dependent reduction in the density of catalase-containing peroxisomes. A significant correlation between the density of peroxisomes and the activity of hepatic catalase indicated that computer-aided image analysis of peroxisomes stained by the diaminobenzidine technique provided a useful estimate of catalase activity in liver injured by copper. Slight treatment-related differences in the mean diameter of peroxisomes were detected in high-dose but not low-dose rats.

Reversible phenotypic modulation induced by deprivation of exogenous essential fatty acids.

Essential fatty acid deficiency, produced by deprivation of omega-6 and omega-3 fatty acids, is a condition characterized by renal disease, dermatitis, and infertility. Although many of the biochemical aspects of this disorder have been investigated, little is known about the ultrastructural changes induced by essential fatty acid deficiency. Using a unique fatty acid-deficient cell line (EFD-1), which demonstrates the in vivo fatty acid changes of essential fatty acid deficiency, and the prostaglandin E2-producing mouse fibrosarcoma line from which it was derived (HSDM1C1), we correlated ultrastructural and biochemical changes induced by prolonged deprivation of all exogenous lipids and subsequent repletion of selected essential fatty acids. We found that in cells deprived of all exogenous lipids, there was dilation of rough endoplasmic reticulum and an associated defect in protein secretion; these changes were specifically reversed by arachidonate. There was also an accumulation of secondary lysosomes containing degraded membranes in these cells with an associated increase in phospholipids relative to parent HSDM1C1 cells. Cytoplasmic lipid bodies present in parent cells disappeared, with an associated decrease in triacylglycerol. After just 2 days in lipid-free medium, all these changes were apparent, and prostaglandin E2 production was markedly impaired despite normal amounts of cellular arachidonate. Incubation of EFD-1 cells with arachidonate, the major prostaglandin precursor fatty acid, induced a reversion to the HSDM1C1 phenotype, whereas other fatty acids were totally ineffective. These results indicate changes in fatty acid metabolism in essential fatty acid deficiency are associated with marked alterations in ultrastructure and secretion of protein from cells.

Kinetics of the assembly of peroxisomes after fusion of complementary cell lines from patients with the cerebro-hepato-renal (Zellweger) syndrome and related disorders.

We have recently identified four complementation groups in fibroblasts from patients deficient in peroxisomes. Here we describe a kinetic analysis of the complementation process. The kinetics of peroxisome assembly was assessed in heterokaryons of complementary cell lines by measuring the rate of incorporation of catalase, initially present in the cytosol, into particles. In two combinations of cell lines assembly was rapid and insensitive to cycloheximide. Thus the components required for peroxisome assembly must have been present in the parental cell lines, at least one of which presumably contained peroxisomal ghosts. In three other combinations of cell lines assembly of peroxisomes was slow and sensitive to cycloheximide.

Peroxisomal proliferation in heart and liver of mice receiving chlorpromazine, ethyl 2(5(4-chlorophenyl)pentyl) oxiran-2-carboxylic acid or high fat diet: a biochemical and morphometrical comparative study.

Chlorpromazine and related drugs including trifluoperazine, clopenthixol, and fluphenazine are in vitro inhibitors of mitochondrial carnitine palmitoyltransferase and cytochrome c oxidase and of peroxisomal carnitine octanoyltransferase from mouse heart and liver. By contrast with 0.1% ethyl 2(5(4-chlorophenyl)pentyl) oxiran-2-carboxylic acid or 0.1% clofibrate-containing diets, the treatment of mice with 0.1% chlorpromazine-containing diet fails to induce peroxisomal proliferation in liver and heart. An 0.5% chlorpromazine-containing diet did induce peroxisomal proliferation. Inhibition of peroxisomal beta-oxidation presumably via the reduction of carnitine octanoyltransferase by chlorpromazine elicits the appearance in liver of lamellar structures resembling those seen in human peroxisomal disorders and induces accumulation of very long-chain fatty acids in plasma. The peroxisomal proliferation induced by administration of high dose chlorpromazine is ascribed to its ability to depress mitochondrial fatty acid oxidation by impairing cytochrome c oxidase and carnitine palmitoyltransferase activities.