ABSTRACT
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.
Subject(s)
Liver/drug effects , Microbodies/drug effects , Peroxisome Proliferators/pharmacology , Tea , Animals , Blotting, Western , Body Weight/drug effects , Dose-Response Relationship, Drug , Lauric Acids/metabolism , Liver/ultrastructure , Male , Microbodies/ultrastructure , Microscopy, Electron , Palmitoyl Coenzyme A/metabolism , Rats , Rats, Wistar , Tea/adverse effectsABSTRACT
Previously we demonstrated that peroxisomicine A1 (T-514), a plant toxin isolated from Karwinskia species, has a deteriorating effect on the integrity of peroxisomes of methylotrophic yeasts. Here we describe two strains of Hansenula polymorpha, affected in the normal utilization of methanol as sole source of carbon and energy due to peroxisomicine A1 treatment. The two strains isolated (L17 and RV31) grew poorly on methanol, apparently due to malfunctioning of their peroxisomes. Moreover, the cells displayed a high peroxisome turnover rate. We argue that the peroxisomicine A1 induced phenotype of both strains is due to a genomic mutation. Strain L17 was functionally complemented after transformation with a H. polymorpha genomic library. The complementing 2.8 kb DNA fragment did not contain a well-defined ORF and led us to speculate that it may contain regulatory sequences that, when present in multiple copies in the cell, result in a change of expression of specific genes, thus causing restoration of normal methylotrophic growth.
Subject(s)
Anthracenes/toxicity , Methanol/metabolism , Microbodies/drug effects , Pichia/metabolism , Plant Extracts/toxicity , Cloning, Molecular , Drug Interactions , Immunohistochemistry , Microbodies/chemistry , Microbodies/metabolism , Microscopy, Electron , Pichia/classification , Pichia/drug effects , Pichia/genetics , Pichia/ultrastructureABSTRACT
Estradiol administration induces peroxisome proliferation and the production of 3-hydroxy fatty acid pheromones in the uropygial glands of the duck, but not in the goose gland, which does not produce such pheromones. We isolated a peroxisome proliferator-activated receptor (PPAR)gamma1 cDNA from a duck uropygial gland cDNA library. Northern blots revealed two transcripts, PPAR gamma1 and gamma2, and showed that PPAR gamma was expressed at higher levels than PPAR alpha in the uropygial gland of the duck. Although PPAR gamma2 was expressed in both duck and goose uropygial gland, PPAR gamma1 was expressed only in the duck gland, which responds to estrogen by peroxisome proliferation. In NIH 3T3 transfected cells, PPAR gamma1 was activated by peroxisome proliferators such as Wy-14643, clofibric acid and Ly-171883 causing induction of the target marker gene. By cotransfection with a plasmid containing alpha-cis-retinoic acid receptor RXR alpha, the induction increased up to 9-fold. These results suggest that PPAR gamma1 may be involved in peroxisome proliferation while PPAR gamma2 may be involved in lipid metabolism.
Subject(s)
Estrogens/pharmacology , Microbodies/drug effects , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cloning, Molecular , DNA Primers , DNA, Complementary , Ducks , Humans , Mice , Microbodies/metabolism , Molecular Sequence Data , Receptors, Cytoplasmic and Nuclear/chemistry , Sebaceous Glands/metabolism , Sequence Homology, Amino Acid , Transcription Factors/chemistryABSTRACT
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.
Subject(s)
Cyclooxygenase Inhibitors/pharmacology , Fenoprofen/pharmacology , Liver/drug effects , Microbodies/drug effects , Animals , Body Weight/drug effects , Carnitine Acyltransferases/metabolism , Liver/enzymology , Liver/ultrastructure , Male , Mice , Mice, Inbred Strains , Microbodies/enzymology , Microbodies/ultrastructure , Organ Size/drug effects , Oxidoreductases/metabolism , Palmitoyl-CoA Hydrolase/metabolism , Time FactorsABSTRACT
A cDNA fragment which encodes salmon peroxisome proliferator activated receptor y (sPPARgamma) was amplified by PCR from the liver of Atlantic salmon (Salmo salar L.). The fragment was 627 bp long. The sequence of the amplified PCR product was similar to the PPARgamma of mouse and hamster. 59% of the bases were identical. Northern blot analysis of salmon liver mRNA showed that the amplified sPPARgamma fragment hybridised to three specific transcripts of lengths 1.6, 2.4 and 3.3 kb. Clofibric acid and bezafibrate, administered to salmon hepatocytes in culture, resulted in a 1.7-fold increase of the 1.6 kb sPPARgamma transcript. The activity of acyl-CoA oxidase also increased approx. 1.7-fold after administration of fibrates. These results indicate that PPAR is an important factor in mediating enzymatic response to fibrates in fish.
Subject(s)
Fatty Acids/pharmacology , Liver/drug effects , Microbodies/drug effects , Oxidoreductases/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Salmon , Transcription Factors/genetics , Acyl-CoA Oxidase , Animals , Base Sequence , Bezafibrate/pharmacology , Blotting, Northern , Clofibrate/pharmacology , DNA, Complementary/analysis , DNA, Complementary/chemistry , Humans , L-Lactate Dehydrogenase/metabolism , Liver/metabolism , Liver/ultrastructure , Microbodies/enzymology , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Analysis, DNA , Sequence Homology , Transcription, Genetic/drug effectsABSTRACT
BACKGROUND: In mice, fibrates induce mdr2 gene expression, and its encoded P-glycoprotein in the canalicular domain of hepatocytes, as well as increasing biliary phospholipid output. It is not known whether this effect is restricted to fibrates or is a common property of peroxisome proliferators. AIMS: To test the effect of structurally unrelated peroxisome proliferators on mdr2 gene expression and biliary phospholipid output, and to explore the molecular mechanism(s) of mdr2 gene induction. METHODS: Male CFI mice were fed on a diet supplemented with several peroxisome proliferators: phenoxyacetic acid herbicides, plasticizers, acetylsalicylic acid and partially hydrogenated fish oil. RESULTS: Increased levels of mdr2 mRNAs, assessed by Northern blot analysis, were observed in the liver of mice treated with phenoxyacetic acid herbicides: 2,4,5-trichlorophenoxyacetic acid 570+/-133%, 2,4-dichlorophenoxyacetic acid 233+/-54% (p<0.005); plasticizers: di-(2-ethylhexyl)phthalate 282+/-78%, di-(isoheptyl)phthalate 163+/-40%, phthalic acid dinonyl ester 225+/-48% (p<0.01); and partially hydrogenated fish oil 372+/-138% (p<0.005). P-glycoprotein traffic ATPase content increased in the canalicular domain of hepatocyte of mice treated with the herbicide 2,4,5-trichlorophenoxyacetic acid and with partially hydrogenated fish oil (108% and 87%, respectively, p<0.05) as well as biliary phospholipid output (106% and 74%, respectively, p<0.05). In 2,4,5-trichlorophenoxyacetic acid-fed mice we found five-fold increase on mdr2 transcription rate, assessed by nuclear run-off assay. CONCLUSIONS: Peroxisome proliferators induce mdr2 gene, its encoded P-gp in the canalicular domain of hepatocytes and increase biliary phospholipid output. The modulation of mdr2 gene might be part of the pleiotrophic response of peroxisome proliferation in mice liver and seems to be regulated mainly at a transcriptional level.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , ATP Binding Cassette Transporter, Subfamily B , ATP-Binding Cassette Transporters/biosynthesis , Drug Resistance, Multiple/genetics , Fish Oils/pharmacology , Herbicides/pharmacology , Liver/physiology , Microbodies/drug effects , Plasticizers/pharmacology , Transcription, Genetic/drug effects , 2,4,5-Trichlorophenoxyacetic Acid/pharmacology , 2,4-Dichlorophenoxyacetic Acid/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Adenosine Triphosphatases/metabolism , Animals , Bile/chemistry , Bile/metabolism , Bile Acids and Salts/metabolism , Cholesterol/metabolism , Hydrogenation , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred Strains , Phospholipids/metabolism , Phthalic Acids/pharmacology , RNA, Messenger/biosynthesisABSTRACT
The effect of vitamin A supplementation on stearoyl-CoA desaturase gene 1 expression in mouse liver was characterized. Normal BALB/c mice were fed 0.01% and 0.1% retinol palmitate as components of nonpurified diets. This treatment resulted in a 3-fold and a 7-fold induction of SCD1 mRNA levels, respectively, as determined by RNase protection analysis. Vitamin A-deficient animals were also fed diets containing 0.01% and 0.1% retinol palmitate, resulting in a similar pattern of SCD1 mRNA induction. Fatty acid synthase and beta-actin mRNA levels did not respond consistently or significantly to retinoic acid treatment. Dietary and hormonal studies were carried out to investigate the role of the retinoid X receptor in the regulation of SCD1 by type II steroid hormones. A receptor-saturating dose of thyroid hormone, triiodothyronine, repressed vitamin A-elevated SCD1 mRNA levels in vivo. Peroxisome proliferator-elevated SCD1 mRNA levels were unaffected by administration of thyroid hormone. This suggests that the retinoic acid receptor transcriptionally regulates SCD1 through a traditional mechanism of heterodimerization with the retinoid X receptor.
Subject(s)
Gene Expression Regulation, Enzymologic , Liver/enzymology , Stearoyl-CoA Desaturase/genetics , Vitamin A/analogs & derivatives , Actins/genetics , Animals , Clofibrate/pharmacology , Diterpenes , Dose-Response Relationship, Drug , Fatty Acid Synthases/genetics , Male , Mice , Mice, Inbred BALB C , Microbodies/drug effects , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Retinoic Acid/metabolism , Retinoid X Receptors , Retinyl Esters , Transcription Factors/metabolism , Triiodothyronine/pharmacology , Vitamin A/pharmacology , Vitamin A Deficiency/enzymologyABSTRACT
Oxidative stress is discussed as a possible hepatocarcinogenic mechanism of peroxisome proliferators (PP) in rodents and is suggested to result from the induction of peroxisomal beta-oxidation (PBOX) by PP. The induced PBOX is assumed to produce excessive H2O2 from the degradation of fatty acids, ultimately leading to oxidative stress and lipid peroxidation. In the present short term-study, we attempted to stimulate lipid peroxidation in male Wistar rats by (1) inducing PBOX enzymes with the peroxisome proliferator nafenopin at 90 mg/kg body weight per day in the diet for 10-11 days, and (2) by supplying the induced PBOX with an abundant amount of fatty acid as substrate, using a corn oil gavage at 20 ml/kg body weight. The corn-oil gavage alone, i.e. without preceding nafenopin treatment, enhanced liver triacylglycerol nine- to tenfold and hepatic lipid peroxidation, measured as thiobarbituric acid reactive substances (TBARS), was increased 50% compared with controls. Both observations were made after 18 h when the peak elevations occurred. Upon pretreatment with nafenopin, associated with a sevenfold induction of PBOX, the corn oil gavage however caused only a threefold maximal increase in hepatic triacylglycerol, also at the 18 h time-point; TBARS remained almost at control levels, as monitored at seven time points over 24-25 h. These results suggest that nafenopin reduces rather than enhances lipid peroxidation, despite the provision, in a short term study, of high doses of substrate to the induced enzyme system that is hypothetically causing oxidative stress in the liver.
Subject(s)
Carcinogens/pharmacology , Lipid Peroxidation/drug effects , Liver/drug effects , Microbodies/drug effects , Nafenopin/pharmacology , Animals , Body Weight/drug effects , Carnitine O-Acetyltransferase/metabolism , Corn Oil , Drug Therapy, Combination , Eating/drug effects , Liver/metabolism , Male , Microbodies/enzymology , Rats , Rats, Wistar , Triglycerides/analysisABSTRACT
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.
Subject(s)
Heart/drug effects , Hypertension/pathology , Microbodies/drug effects , Animals , Calcium Channel Blockers/pharmacology , Cardiotonic Agents/pharmacology , Drug Evaluation, Preclinical , Erucic Acids/pharmacology , Hypolipidemic Agents/pharmacology , Liver/drug effects , Liver/enzymology , Liver/ultrastructure , Male , Mice , Microbodies/ultrastructure , Microscopy, Electron , Myocardium/enzymology , Myocardium/ultrastructure , Physical Exertion/physiology , Rats , Rats, Inbred Strains , Reproducibility of ResultsSubject(s)
Docosahexaenoic Acids/pharmacology , Eicosapentaenoic Acid/pharmacology , Liver/drug effects , Triglycerides/metabolism , Animals , Cholesterol/metabolism , Corn Oil/metabolism , Corn Oil/pharmacology , Docosahexaenoic Acids/metabolism , Eicosapentaenoic Acid/metabolism , Liver/cytology , Liver/metabolism , Microbodies/drug effects , Microbodies/metabolism , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , RatsABSTRACT
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.
Subject(s)
Dietary Fats, Unsaturated/administration & dosage , Docosahexaenoic Acids/administration & dosage , Microbodies/drug effects , Acyl-CoA Oxidase , Animals , Catalase/biosynthesis , Enzyme Induction/drug effects , Heart/drug effects , Hydrogen Peroxide/metabolism , Kidney/drug effects , Kidney/enzymology , Liver/drug effects , Liver/enzymology , Male , Mice , Microbodies/metabolism , Microbodies/ultrastructure , Myocardium/enzymology , Oxidoreductases/metabolism , Peroxisomal Disorders/diet therapy , Peroxisomal Disorders/enzymology , Time Factors , Urate Oxidase/metabolismABSTRACT
Peroxisome proliferators induce stearoyl-CoA desaturase activity (EC 1.14.99.5) in liver [Kawashima, Y., Hanioka, N., Matsumura, M. & Kozuka, H. (1983) Biochim. Biophys. Acta 752, 259-264]. We analyzed the changes in stearoyl-CoA desaturase 1 (SCD1) mRNA to further define the molecular mechanism for the induction of stearoyl-CoA desaturase by peroxisome proliferators. SCD1 mRNA was analyzed from the livers of BALB/c mice that had been fed diets supplemented with clofibrate or gemfibrozil. Clofibrate was found to induce liver SCD1 mRNA levels 3-fold within 6 hr to a maximum of 22-fold in 30 hr. Gemfibrozil administration resulted in a similar induction pattern. This induction is primarily due to an increase in transcription of the SCD1 gene, as shown by nuclear run-on transcription assays and DNA deletion analysis of transfected SCD1-chloramphenicol acetyltransferase fusion genes. The cis-linked response element for peroxisome proliferator-activated receptor (PPAR) was localized to an AGGTCA consensus sequence between base pairs -664 to -642 of the SCD1 promoter. Clofibrate-mediated induction of SCD1 mRNA was shown to be independent of polyunsaturated fatty acids, with peroxisome proliferators and arachidonic acid having opposite effects on SCD1 mRNA levels. Additionally, the activation of SCD1 mRNA by clofibrate was inhibited 77% by cycloheximide administration. Levels of liver beta-actin and albumin mRNAs were unchanged by these dietary manipulations. Our data show that hepatic SCD1 gene expression is regulated by PPARs and suggest that peroxisome proliferators and poly-unsaturated fatty acids act through distinct mechanisms.
Subject(s)
Clofibrate/pharmacology , Gemfibrozil/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Liver/enzymology , Receptors, Cytoplasmic and Nuclear/metabolism , Stearoyl-CoA Desaturase/genetics , Transcription Factors/metabolism , Animals , Arachidonic Acid/pharmacology , Base Sequence , Chloramphenicol O-Acetyltransferase/genetics , Cycloheximide/pharmacology , Fatty Acids, Unsaturated/pharmacology , Mice , Mice, Inbred BALB C , Microbodies/drug effects , Molecular Sequence Data , Promoter Regions, Genetic , RNA, Messenger/metabolism , Transcription, Genetic/drug effectsABSTRACT
We present a nonradioactive in siru hybridization (ISH) protocol for detection of mRNAs in rat liver encoding for three peroxisomal proteins: catalase and urate oxidase as representatives of high-level abundance mRNAs and trifunctional protein (PH) as that of low-level abundance mRNAs. In addition to normal rats, animals treated for 24 hr with a single dose of bezafibrate were studied. The use of perfusion-fixation with 4% depolymerized paraformaldehyde/0.05% glutaraldehyde combined with paraffin embedding and the application of digoxigenin-labeled cRNA probes provided optimal cytological resolution and high sensitivity comparable to that of radioactive ISH. In parallel experiments, the same digoxigenin-labeled cRNA probes were used for Northern and semiquantitative dot-blot analysis of isolated RNAs. In control animals, the mRNAs for catalase and urate oxidase were uniformly distributed across the liver lobule and were confined to liver parenchymal cells. The bile duct epithelial and the sinusoidal cells remained negative. The specificity and the high resolution of our protocol were further substantiated by reciprocal localization of transcripts for albumin and glyceraldehyde-3-phosphate dehydrogenase in different regions of the liver lobule and for catalase in the proximal tubules of the renal cortex. Whereas in control livers the transcripts for PH were barely detectable, a strong signal was found in pericentral hepatocytes of bezafibratetreated animals, corresponding to an 8-10-fold increase of mRNA detected in dot-blots. In contrast, the urate oxidase mRNA was reduced by more than 50%, with diminution of staining in pericentral regions of the liver lobule. The mRNA encoding for catalase was only slightly affected. Further applications of this protocol should be helpful in elucidation of the cell-specific transcriptional regulation of peroxisomal proteins in various organs under normal and pathological conditions.
Subject(s)
Bezafibrate/pharmacology , Hypolipidemic Agents/pharmacology , In Situ Hybridization/methods , Liver/chemistry , Microbodies/chemistry , Animals , Catalase/isolation & purification , Digoxigenin , Liver/anatomy & histology , Liver/drug effects , Liver/enzymology , Male , Microbodies/drug effects , Microbodies/enzymology , RNA, Complementary , RNA, Messenger/isolation & purification , Rats , Rats, Sprague-Dawley , Tissue Distribution , Tissue Fixation/methods , Urate Oxidase/isolation & purificationABSTRACT
We studied the response of male DBA/2N mouse liver monooxygenases to acute (one-day) and subacute (7-day) exposure to clofibrate, gemfibrozil, and corn oil. The day following a single treatment with clofibrate (200 mg/kg), coumarin 7-hydroxylase (COH) activity decreased significantly (by 70%) with a concomitant decrease in the CYP2A4/5 protein and mRNA levels. The 7-day treatment schedule also decreased COH activity by only by 30%, though the levels of CYP2A4/5 protein and mRNA were still low. Treatment 1 and 7-day with clofibrate decreased 7-pentoxyresorufin O-dealkylase (PROD) activity by 40%. No changes were seen in testosterone 15 alpha-hydroxylase (T15 alpha OH) activity after 1 day of treatment with clofibrate but, after 7 days, it was decreased by 50%. Clofibrate treatment had no significant effects on testosterone 7 alpha-hydroxylase (T7 alpha OH), 7-ethoxyresorufin O-deethylase (EROD), or benzphetamine N-demethylase (BZDM) activities. Gemfibrozil (200 mg/kg) did not alter COH activity or CYP2A4/5 protein content after a single treatment, but a slight decrease was seen in the mRNA level. Treatment for 7 days significantly increased (2.5-fold) the activity and mRNA content but the amount of protein remained unchanged. Gemfibrozil enhanced (2-2.7-fold PROD and EROD (2-2.5-fold) activities by both treatments, whereas T15 alpha OH, T7 alpha OH, or BZDM activities were not significantly affected. Treatment with corn oil for 7 days significantly decreased (65%) COH activity and CYP2A4/5 protein and mRNA levels. PROD (55%) and T15 alpha OH (65%) activities were significantly decreased even after a single dose although injection for 7 days had no effect. Neither of the corn oil schedules had any marked effect on T7 alpha OH, EROD, or BZDM activities. These results demonstrate: 1. a decrease in the expression of CYP2A4/5 gene by clofibrate and corn oil; 2. substantial differences within the CYP2A subfamily in their responses to corn oil, clofibrate, and gemfibrozil; and 3. distinct responses of other xenobiotic metabolizing CYP subfamily enzymes to clofibrate and gemfibrozil.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Corn Oil/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Microbodies/drug effects , Microsomes, Liver/drug effects , Steroid Hydroxylases , Animals , Clofibrate/pharmacology , Gemfibrozil/pharmacology , Male , Mice , Mice, Inbred DBA , Microsomes, Liver/enzymologyABSTRACT
The activity of selenium-dependent glutathione peroxidase is known to be reduced in the liver of both rats and mice after exposure to nafenopin, as well as other peroxisome proliferators. The mechanism for this down-regulation is not known, but might involve changes in incorporation of selenium into selenoproteins. In this paper we show that both incorporation of selenium into selenoproteins and the level of selenium in liver is reduced in mice treated with nafenopin. The activity of selenium dependent glutathione peroxidase (GPx), as well as incorporation of selenium into its 23 kD subunit were found to be decreased. Contrary to what might have been expected, the decreased GPx activity was detected concomitantly with a slight increase in mRNA levels after 10 days of treatment, while a small decrease in mRNA levels was detected in treated animals after 26 weeks, together with the decrease in GPx-activity. Incorporation of selenium into liver fatty acid binding protein (L-FABP) was also decreased, even though large increases in protein and mRNA levels were detected. Taken together these data suggest that the decrease in GPx-activity in response to nafenopin is due to post-transcriptional mechanisms, involving changes in selenium metabolism.
Subject(s)
Glutathione Peroxidase/metabolism , Nafenopin/pharmacology , Neoplasm Proteins , Nerve Tissue Proteins , Selenium/metabolism , Animals , Blotting, Northern , Body Weight/drug effects , Carrier Proteins/metabolism , Fatty Acid-Binding Protein 7 , Fatty Acid-Binding Proteins , Kidney/chemistry , Kidney/drug effects , Kidney/metabolism , Liver/chemistry , Liver/drug effects , Liver/metabolism , Male , Mice , Mice, Inbred Strains , Microbodies/drug effects , Microbodies/metabolism , Myelin P2 Protein/metabolism , Organ Size/drug effects , Oxidation-Reduction , Proteins/metabolism , RNA, Messenger/metabolism , Testis/chemistry , Testis/drug effects , Testis/metabolismABSTRACT
Preclinical safety studies with the leukotriene D4 antagonist RG 12525 were conducted by the oral route in mice, rats, and monkeys. Oral administration of RG 12525 was repeated daily in studies up to 6 months in duration. RG 12525 was shown to have limited high-dose toxicity after repeated oral administration. The effects of RG 12525 were strongly dependent upon the species considered. High doses of RG 12525 caused significant increases in liver weight in mice, rats, and monkeys that were associated with diffuse hepatocellular hypertrophy in mice and rats but not in monkeys. No related clinical chemistry changes were observed in any of the species and hepatic activities of peroxisomal enzymes or cytochrome P450 were increased only slightly. Proliferation of brown adipose tissue (BAT) was observed in rats and mice but not in monkeys. The BAT reaction was more pronounced in the interscapular area but it was also observed in other subcutaneous locations as well as in mediastinal and bone marrow fat. In all locations, the RG 12525-induced BAT had some morphological similarities with cold-adapted BAT. Repeated administration of RG 12525 at high doses to female rats resulted in a lack of progression to the luteal phase of the estrous cycle that was reversible after discontinuation of treatment. Finally, RG 12525 was nephrotoxic in mice with males being more sensitive than females.
Subject(s)
Leukotriene D4/antagonists & inhibitors , Quinolines/toxicity , Tetrazoles/toxicity , Animals , Corpus Luteum/drug effects , Eating/drug effects , Erythrocyte Count/drug effects , Estrus/drug effects , Female , Hematocrit , Kidney Diseases/chemically induced , Kidney Diseases/pathology , Liver/drug effects , Liver/pathology , Macaca mulatta , Male , Mice , Mice, Inbred ICR , Microbodies/drug effects , Microbodies/metabolism , Organ Size/drug effects , Rats , Rats, Sprague-Dawley , Sex Characteristics , Weight Gain/drug effectsABSTRACT
The effects on blood and liver lipids after feeding rats with concentrated fractions from fish oil consisting of mono-unsaturated fatty acids (80% C20:1 and 22:1) or n-3 polyunsaturated fatty acids (85% C20:5 and 22:6 n-3) were examined. Mono-unsaturated fat had no effects on plasma triacylglycerol, total cholesterol phospholipids or unesterified fatty acid as compared to controls (lard). However, n-3 polyunsaturated fatty acid-fed animals showed a significant decrease in plasma triacylglycerol (74%), phospholipids (40%) and unesterified fatty acids (52%). The concentrated fractions had no effects on liver lipids. While the n-3 diet increased peroxisomal beta-oxidation 2.5-fold, there was only a slight increase with the mono-unsaturated diet. The fatty acid composition in plasma and liver phospholipids was changed with the various diets; 20:4 n-6 was significantly reduced in plasma and liver with the mono-unsaturated diet, and with the n-3 diet in liver. The mono-unsaturated diet, and especially the n-3 diet, increased the 20:5 n-3 level in both plasma and liver. Our results indicate that long-chain mono-unsaturated fatty acids in fish oil do not change the levels of plasma lipids. The beneficial role of fish oil on the level of blood lipids, may therefore be mostly attributed to the effects of long-chain n-3 fatty acids. However, the low 20:4 n-6 and high 20:5 n-3 levels in plasma and liver phospholipids with the concentrated mono-unsaturated fatty acid diet may be of importance for a favourable haemostatic balance with regard to cardiovascular diseases.
Subject(s)
Dietary Fats/pharmacology , Fatty Acids, Monounsaturated/pharmacology , Fatty Acids, Omega-3/pharmacology , Lipids/blood , Liver/chemistry , Liver/drug effects , Animals , Dietary Fats/administration & dosage , Dietary Fats/metabolism , Fatty Acids, Monounsaturated/administration & dosage , Fatty Acids, Omega-3/administration & dosage , Lipid Metabolism , Male , Microbodies/drug effects , Microbodies/metabolism , Oxidation-Reduction , Rats , Rats, WistarABSTRACT
We have previously described an aging-related decrease in the peroxisomal polyunsaturated fatty acid oxidizing system in mouse liver. In order to determine whether peroxisome synthesis is involved in this phenomenon, we focused our work on different peroxisomal enzyme activities during aging in the liver of mice fed for 5 days with either a control or a clofibrate supplemented diet which enhanced peroxisome biogenesis. Liver peroxisomal acyl-CoA oxidase (AOX), catalase (CAT) and urate oxidase (UOX) activities per gram of liver were determined. In control mice, UOX activity was not affected by aging whereas CAT and AOX activities were significantly decreased. At day 300 the clofibrate treatment increased all activities although UOX was not significantly increased. Thereafter, enzyme activities after clofibrate treatment were severely depressed at day 680. CAT and UOX were not induced in very old clofibrate-treated animals, whereas AOX was induced 7 fold in such mice compared to an 11 fold induction in day 300 animals. The present results suggest that: 1- Aging decreased the peroxisomal polyunsaturated fatty acid oxidizing system. 2- This took place via a specific decrease in AOX activity. 3- Since clofibrate treatment triggers the peroxisomal proliferation, the aging-related decrease in peroxisomal activities might be due to an alteration in peroxisome synthesis.
Subject(s)
Aging/metabolism , Clofibrate/pharmacology , Fatty Acids/metabolism , Liver/drug effects , Microbodies/drug effects , Acyl-CoA Oxidase , Animals , Catalase/analysis , Female , Liver/metabolism , Liver/ultrastructure , Mice , Oxidation-Reduction , Oxidoreductases/analysis , Urate Oxidase/analysisABSTRACT
In order to understand the relationship between peroxisomal dysfunction and clinical manifestations of peroxisomal disorders, the effect of thioridazine, a peroxisomal beta-oxidation antagonist, on the differentiation, membrane lipid composition and membrane fluidity of C-6 glial cells was examined. In our study, induction of 2',3'-cyclic-nucleotide 3'-phosphohydrolase (CNP), which was considered to be a membrane-associated enzyme closely associated with myelination, was inhibited with supplementation of thioridazine, followed by an increase in the relative concentration of longer chain fatty acids in cell membrane lipids, indicating that thioridazine causes impaired differentiation in the glial stem cell system. Membrane fluidity of C-6 glial cells was examined using a fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The DPH anisotropy value was decreased in the glial cells treated with thioridazine. These results indicate that the alteration of the membrane lipid composition caused by thioridazine affects the differentiation of glial cells via the changes in membrane properties.
Subject(s)
2',3'-Cyclic-Nucleotide Phosphodiesterases/antagonists & inhibitors , Membrane Fluidity/drug effects , Membrane Lipids/analysis , Microbodies/drug effects , Neuroglia/metabolism , Phosphoric Diester Hydrolases , Thioridazine/pharmacology , 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase , Cell Line , Cholesterol/analysis , Diphenylhexatriene , Fatty Acids/analysis , Fluorescence Polarization , Microbodies/metabolism , Neuroglia/ultrastructure , Oxidation-Reduction , Phospholipids/analysisABSTRACT
By feeding rats a diet containing 20% (w/w) partially hydrogenated fish oil (PHFO), an apparent 6.3-fold increase in the cyanide insensitive palmitoyl-CoA-dependent NAD+ reduction was observed for the heart peroxisomal fractions. This finding was confirmed by a 7.6-fold and 7.9-fold increase in the specific activity of fatty acyl-CoA oxidase, with palmitoyl-CoA and erucoyl-CoA as the substrates, respectively. Immunoblots after SDS-PAGE of rat heart peroxisomal fractions revealed a 12-fold increase in the 52 kDa fatty acyl-CoA oxidase (FAO) subunit for PHFO-fed rats, whereas the 72 kDa subunit of FAO and several other peroxisomal proteins (including the trifunctional enzyme delta 3,delta 2-enoyl-CoA isomerase, 2-enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) increased only 2- to 3-fold. The increase in the 52 kDa subunit was markedly higher than the increase in the steady-state mRNA level of FAO (2.0-fold), and is most likely caused by a rather selective stabilization of the 52 kDa FAO subunit. Interestingly, PHFO feeding caused a larger increase in fatty acyl-CoA oxidase and catalase activities than did clofibrate in the heart. The opposite was the case in the liver, especially for fatty acyl-CoA oxidase. Rats fed a semisynthetic diet containing 6% (w/w) erucic acid (C22:1(n - 9), cis) or brassidic acid (C22:1(n - 9), trans) revealed a 5-fold and 3-fold increase vs. the control (pellet fed) rats in heart FAO activity, respectively, as well as a proportional and selective increase in the specific content of 52 kDa FAO subunit. Thus, the relatively high content of C22 monoene fatty acids appears to be one of the main factors responsible for the increase in rat heart peroxisomal FAO activity during PHFO feeding. However, the PHFO diet increased the heart peroxisomal FAO activity more than diets containing a similar amount of C22:1 in the form of erucic or brassidic acid, and additional compounds of lipid or a more xenobiotic nature may also play a role. SDS-PAGE electrophoresis of highly purified rat liver peroxisomes revealed that the specific content of polypeptides with mobilities corresponding to that of the beta-oxidation enzyme system, increased by a factor of < 2 as a result of feeding the PHFO diet. The 3.1-fold increase in cyanide insensitive palmitoyl-CoA-dependent NAD+ reduction was comparable to the increase (4.1-fold) in the acyl-CoA oxidase activity.(ABSTRACT TRUNCATED AT 400 WORDS)