Peroxisomal enoyl-CoA Hydratase Research Articles

Renal Fanconi Syndrome Is Caused by a Mistargeting-Based Mitochondriopathy.

We recently reported an autosomal dominant form of renal Fanconi syndrome caused by a missense mutation in the third codon of the peroxisomal protein EHHADH. The mutation mistargets EHHADH to mitochondria, thereby impairing mitochondrial energy production and, consequently, reabsorption of electrolytes and low-molecular-weight nutrients in the proximal tubule. Here, we further elucidate the molecular mechanism underlying this pathology. We find that mutated EHHADH is incorporated into mitochondrial trifunctional protein (MTP), thereby disturbing β-oxidation of long-chain fatty acids. The resulting MTP deficiency leads to a characteristic accumulation of hydroxyacyl- and acylcarnitines. Mutated EHHADH also limits respiratory complex I and corresponding supercomplex formation, leading to decreases in oxidative phosphorylation capacity,mitochondrial membrane potential maintenance, and ATP generation. Activity of the Na(+)/K(+)-ATPase is thereby diminished, ultimately decreasing the transport activity of the proximal tubule cells.

Suppressor Screen and Phenotype Analyses Revealed an Emerging Role of the Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 in Compensated Cell Enlargement.

Efficient use of seed nutrient reserves is crucial for germination and establishment of plant seedlings. Mobilizing seed oil reserves in Arabidopsis involves β-oxidation, the glyoxylate cycle, and gluconeogenesis, which provide essential energy and the carbon skeletons needed to sustain seedling growth until photoautotrophy is acquired. We demonstrated that H+-PPase activity is required for gluconeogenesis. Lack of H+-PPase in fugu5 mutants increases cytosolic pyrophosphate (PPi) levels, which partially reduces sucrose synthesis de novo and inhibits cell division. In contrast, post-mitotic cell expansion in cotyledons was unusually enhanced, a phenotype called compensation. Therefore, it appears that PPi inhibits several cellular functions, including cell cycling, to trigger compensated cell enlargement (CCE). Here, we mutagenized fugu5-1 seeds with 12C6+ heavy-ion irradiation and screened mutations that restrain CCE to gain insight into the genetic pathway(s) involved in CCE. We isolated A#3-1, in which cell size was severely reduced, but cell number remained similar to that of original fugu5-1. Moreover, cell number decreased in A#3-1 single mutant (A#3-1sm), similar to that of fugu5-1, but cell size was almost equal to that of the wild type. Surprisingly, A#3-1 mutation did not affect CCE in other compensation exhibiting mutant backgrounds, such as an3-4 and fugu2-1/fas1-6. Subsequent map-based cloning combined with genome sequencing and HRM curve analysis identified enoyl-CoA hydratase 2 (ECH2) as the causal gene of A#3-1. The above phenotypes were consistently observed in the ech2-1 allele and supplying sucrose restored the morphological and cellular phenotypes in fugu5-1, ech2-1, A#3-1sm, fugu5-1 ech2-1, and A#3-1; fugu5-1. Taken together, these results suggest that defects in either H+-PPase or ECH2 compromise cell proliferation due to defects in mobilizing seed storage lipids. In contrast, ECH2 alone likely promotes CCE during the post-mitotic cell expansion stage of cotyledon development, probably by converting indolebutyric acid to indole acetic acid.

Proteomic and metabolomic analysis of H 2O 2-induced premature senescent human mesenchymal stem cells

Stress induced premature senescence (SIPS) occurs after exposure to many different sublethal stresses including H 2O 2, hyperoxia, or tert-butylhydroperoxide. Human mesenchymal stem cells (hMSCs) exhibit limited proliferative potential in vitro, the so-called Hayflick limit. According to the free-radical theory, reactive oxygen species (ROS) might be the candidates responsible for senescence and age-related diseases. H 2O 2 may be responsible for the production of high levels of ROS, in which the redox balance is disturbed and the cells shift into a state of oxidative stress, which subsequently leads to premature senescence with shortening telomeres. H 2O 2 has been the most commonly used inducer of SIPS, which shares features of replicative senescence (RS) including a similar morphology, senescence-associated β-galactosidase activity, cell cycle regulation, etc. Therefore, in this study, the senescence of hMSC during SIPS was confirmed using a range of different analytical methods. In addition, we determined five differentially expressed spots in the 2-DE map, which were identified as Annexin A2 (ANXA2), myosin light chain 2 (MLC2), peroxisomal enoyl-CoA hydratase 1 (ECH1), prosomal protein P30-33K (PSMA1) and mutant β-actin by ESI-Q-TOF MS/MS. Also, proton ( 1H) nuclear magnetic resonance spectroscopy (NMR) was used to elucidate the difference between metabolites in the control and hMSCs treated with H 2O 2. Among these metabolites, choline and leucine were identified by 1H-NMR as up-regulated metabolites and glycine and proline were identified as down-regulated metabolites.

Proteomic changes in the heart of diet-induced pre-diabetic mice

The development of type 2 diabetes (T2D) is strongly associated with obesity. In humans, T2D increases the risk for end organ complications. Among these, heart disease has been ranked as the leading cause of death. We used a proteomic methodology to test the hypothesis that a pre-diabetic state generated by high-fat diet leads to changes in proteins related to heart function and structure. Over 300 protein spots were resolved by two-dimensional gel electrophoresis (2-DE). Fifteen protein spots were found to be altered (7 decreased and 8 increased) in pre-diabetic hearts. The protein spots were then identified by mass spectrometry and immunoblots. Among the decreased proteins, 3 are involved in heart structure (one isoform of desmin, troponin T2 and α-cardiac actin), 3 are involved in energy metabolism (mitochondrial ATP synthase β subunit, adenylate kinase and creatine kinase) and one is a component of the citric acid cycle (isocitrate dehydrogenase 3). In contrast, proteins involved in fatty acid oxidation (two isoforms of peroxisomal enoyl-CoA hydratase) and the citric acid cycle (three isoforms of malate dehydrogenase) were increased in pre-diabetic hearts. The results suggest that changes in the levels of several heart proteins may have implications in the development of the cardiac phenotype associated to T2D.

Analysis of Enoyl-Coenzyme A Hydratase Activity and Its Stereospecificity Using High-Performance Liquid Chromatography Equipped with Chiral Separation Column

Enoyl-coenzyme A (CoA) hydratase catalyzes the hydration of trans-2-enoyl-CoA to yield 3-hydroxyacyl-CoA during fatty acid degradation (β-oxidation). Although much research has focused on the stereospecificities of 2-enoyl-CoA hydratases, a direct quantification of the production of 3(R)- and 3(S)-hydroxyacyl-CoA has not yet been established. Therefore, we developed a method of concurrently quantifying 3(R)- and 3(S)-hydroxyacyl-CoA using high-performance liquid chromatography (HPLC) equipped with a chiral separation column. The optimized conditions for the separation of 3(R)-, 3(S)-hydroxyhexadecanoyl-CoA and trans-2-hexadecenoyl-CoA, were determined to be as follows: mobile phase of 35/65 (v/v) of 50 mM phosphate buffer (pH 5.0)/methanol; flow rate of 0.5 mL/min; detection at 260 nm; and column temperature of 25°C. This method was applied to subcellular fractions of rat liver; the results directly confirmed that 3(S)-hydroxyhexadecanoyl-CoA is the dominant product obtained from the heat-stable enoyl-CoA hydratase-catalyzed reaction of trans-2-hexadecenoyl-CoA. Finally, the stereospecificities of L-bifunctional protein (L-BP) and D-bifunctional protein (D-BP) were reinvestigated using this method, and it was confirmed that L- and D-BP yielded 3(S)- and 3(R)-hydroxyhexadecanoyl-CoA were yielded from trans-2-hexadecenoyl-CoA, respectively. 3(R)-Hydroxyacyl-CoA is a peroxisomal β-oxidation-specific intermediate. Therefore, this method is potentially useful not only studies regarding the stereochemistry of enoyl-CoA hydratase but also for the diagnosis of diseases caused by defects of peroxisomal enoyl-CoA hydratase.

Protein profile of human hepatocarcinoma cell line SMMC-7721: Identification and functional analysis

To investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis (2DE). The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed. Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

Identification and Functional Characterization of a Monofunctional Peroxisomal Enoyl-CoA Hydratase 2 That Participates in the Degradation of Even cis-Unsaturated Fatty Acids in Arabidopsis thaliana

A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high beta-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the beta-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over- or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core beta-oxidation cycle.

Amino acid and nucleotide sequences of human peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase cDNA.

Deficiency of enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase (peroxisomal bifunctional enzyme), one of the enzymes of the peroxisomal beta-oxidation system, leads to clinical manifestations resembling Zellweger syndrome with hypotonia, psychomotor delay, hepatomegaly, typical facial appearance and accumulation of very long-chain fatty acids. The nucleotide sequence of the human peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase cDNA has been reported by Hoefler and colleagues; however, we have found some amino acid differences from our originally isolated cDNA. Contrary to the findings described in a previous paper, we report here the cDNA sequence of human peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase in which there are 9 authenticated amino acid alterations.

Isolation and Characterization of Rat and Human cDNAs Encoding a Novel Putative Peroxisomal Enoyl-CoA Hydratase

We have used a PCR-based subtractive hybridization method to identify upregulated cDNAs in the livers of rats treated with a peroxisome proliferator [clofibrate or di(2-ethylhexyl) phthalate]. After four rounds of subtractive hybridization 62 differentially hybridizing clones were partially sequenced and analyzed by sequence homology searching. Of 62, 49 were identical to 14 different upregulated rat sequences in the databank (mostly genes encoding microsomal or peroxisomal enzymes), 4 of 62 were fragments of three previously unknown genes, and 9 of 62 were false positives. Two of the unknown fragments hybridized to a single novel cDNA that was found to be more than 20-fold induced by both peroxisome proliferators. The 36-kDa predicted protein product of this cDNA shows a high degree of sequence homology to enoyl-CoA hydratases of several different species and has a C-terminal peroxisomal targeting sequence. An epitope-tagged protein product of a full-length cDNA was targeted to peroxisomes in a human cell line. We named this gene, which encodes an apparent peroxisomal enoyl-CoA hydratase, ECH1. We have also identified human ECH1 cDNA and mapped its structural gene to 19q13, 3′ to the ryanodine receptor, by hybridization to somatic cell hybrid DNA and chromosome 19-specific cosmid arrays. Possible roles for the ECH1 protein product in peroxisomal β-oxidation are discussed.

Induction of enoyl-CoA hydratase by LD 50 exposure to perfluorocarboxylic acids detected by two-dimensional electrophoresis

The effect of in vivo exposure to perfluoro- n-octanoic and perfluoro- n-decanoic acids was examined in the rat liver by two-dimensional electrophoresis (2DE). Using nonequilibrium pH-gradient electrophoresis in the first dimension separation, proteins associated with the mitochondrial/peroxisomal cell fraction were observed and immunologically identified. Conspicuous inductions in peroxisomal enoyl-CoA hydratase and other proteins of the peroxisomal β-oxidative pathway were observed following single-dose exposure to each compound. The abundance of the tentatively-identified mitochondrial equivalent, crotonase, was not altered by these intoxications. These results confirm previous observations of perfluorocarboxylic acid toxicity and support the use of 2D protein-pattern alterations in biomarker research. The ability to identify this type of alteration via 2DE, in association with specific toxic effects by chemically related compounds, may provide new and additional markers for chemical-induced tissue damage.

CDNA Cloning of the Human Peroxisomal Enoyl-CoA Hydratase: 3-Hydroxyacyl-CoA Dehydrogenase Bifunctional Enzyme and Localization to Chromosome 3q26.3-3q28: A Free Left Alu Arm Is Inserted in the 3′ Noncoding Region

Enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme is one of the four enzymes of the peroxisomal β-oxidation pathway. Here, we report the full-length human cDNA sequence and the localization of the corresponding gene on chromosome 3q26.3-3q28. The cDNA sequence spans 3779 nucleotides with an open reading frame of 2169 nucleotides. The tripeptide SKL at the carboxy terminus, known to serve as a peroxisomal targeting signal, is present. DNA sequence comparison of the coding region showed an 80% homology between human and rat bifunctional enzyme cDNA. The 3′ noncoding sequence contains 117 nucleotides homologous to an Alu repeat. Based on sequence comparison, we propose that these nucleotides are a free left Alu arm with 86% homology to the Alu-J family. RNA analysis shows one band with highest intensity in liver and kidney. This cDNA will allow in-depth studies of molecular defects in patients with defective peroxisomal bifunctional enzyme. Moreover, it will also provide a means for studying the regulation of peroxisomal β-oxidation in humans.

Import of human bifunctional enzyme into peroxisomes of human hepatoma cells [formula omitted

A polypeptide containing the carboxyl-terminal fragment of human peroxisomal enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme was synthesized in vitro from its cDNA clone. This expression polypeptide was transported into purified rat liver peroxisomes. When the expression polypeptide was incubated with postnuclear supernatant fractions of human hepatoma cells and analyzed by Nycodenz gradient SDS-PAGE and fluorography, it was imported specifically into peroxisomes as indicated by its resistance to proteinase K degradation. A deletion of the last nine amino acid residues at the carboxyl-terminus of this polypeptide prevents its peroxisomal import. A tripeptide sequence, SKL, located at the carboxyl-terminus of human bifunctional enzyme appears to be the targeting signal for the peroxisomal importation of bifunctional enzyme in human cells.

Isolation of peroxisomal enoyl-CoA hydratase in rainbow trout and immunochemical identification with the bifunctional enzyme

Peroxisomes are the sites for β-oxidation of long-chain fatty acids. The peroxisomal bifunctional enzyme (PBE) enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase catalyzes the second and third reactions of the β-oxidation system. Originally termed PPA-80 for peroxisome-proliferation associated 80,000 MW polypeptide, PBE levels are monitored to measure peroxisome proliferation in rodents and other species. The quantity of a 79,000 MW polypeptide in the light mitochondrial fraction of the liver, as analyzed by SDS-PAGE, increases when rainbow trout are exposed to peroxisome proliferating agents. This correlates with increases in acyl-CoA oxidase activity and peroxisome volume density. In the present study, peroxisomal enoyl-CoA hydratase was purified from trout liver and analyzed by immunoblotting with anti-PBE. A positive reaction with the 79,000 MW polypeptide band was observed providing strong evidence that this is the bifunctional enzyme.

Molecular cloning and sequence analysis of the cDNA for rat mitochondrial enoyl-CoA hydratase. Structural and evolutionary relationships linked to the bifunctional enzyme of the peroxisomal beta-oxidation system.

To elucidate structural relationships between the mitochondrial and peroxisomal isozymes of beta-oxidation systems, cDNA of the mitochondrial enoyl-CoA hydratase was cloned and sequenced. The 1454-bp cDNA sequence contained a 870 bp of open reading frame, encoding a polypeptide of 290 amino acid residues. When compared with the amino-terminal sequence of the mature enzyme, the predicted sequence contained a 29-residue presequence at the amino terminus. This presequence had characteristics typical of a mitochondrial signal peptide. The primary structure of this enzyme showed significant similarity with the amino-terminal portion of sequence of the peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. The carboxy-terminal part of the latter enzyme has sequence similarity with mitochondrial 3-hydroxyacyl-CoA dehydrogenase [Ishii, N., Hijikata, M., Osumi, T. & Hashimoto, T. (1987) J. Biol. Chem. 262, 8144-8150]. These findings suggest that the peroxisomal bifunctional enzyme has the hydratase and dehydrogenase functions on the amino- and carboxy-terminal sides, respectively. The mitochondrial beta-oxidation enzymes and the peroxisomal bifunctional enzyme may have common evolutionary origins.

Evidence that the enoyl-CoA hydratase bifunctional protein of mouse liver peroxisomes is identical with the 70,000 dalton peroxisomal membrane protein

Peroxisomal enoyl-CoA hydratase was purified from livers of mice treated with di-(2-ethylhexyl)phthalate and its properties compared with those of the 70 kDa protein present in the membranes prepared by carbonate extraction of peroxisomes. The two proteins had identical subunit molecular masses, of about 70,000 daltons. Limited proteolysis of these proteins using the V8 proteinase of S. aureus yielded identical peptide maps, with these peptides crossreacting with antiserum raised against the 70 kDa membrane protein. These data are consistent with the proposal that the peroxisomal 70 kDa membrane protein and the peroxisomal enoyl-CoA hydratase are the same protein.

The induction of liver peroxisomal proliferation by β,β′-methyl-substituted hexadecanedioic acid (MEDICA 16)

Treatment of rats by β,β'-methyl-substituted hexadecanedioic acid (MEDICA 16) resulted in a dose- and time-dependent increase in liver peroxisomal enoyl-CoA hydratase and cyanide-insensitive palmitoyl-CoA oxidation with a concomitant increase in the volume density of peroxisomes as determined by morphometry. The induced peroxisomal proliferation was sustained as long as treatment was maintained and was accompanied by an increase in liver weight. Incubation of cultured rat hepatocytes in the presence of MEDICA 16 added to the culture medium resulted in a dose-dependent increase in peroxisomal β-oxidation activities with a concomitant elevation of the volume density of peroxisomes. The induction of peroxisomal proliferation by MEDICA 16 in culture could be prevented in the presence of carnitine palmitoyltransferase inhibitors added to the culture medium, e.g. 2-bromopalmitate, 2-tetradecylglycidic acid or 2-[5-(4-chlorophenyl)-pentyl]oxirane-2-carboxylate. The induction of liver peroxisomes by MEDICA 16 conforms to the previously denned requirement for an amphipathic carboxylate in initiating peroxisomal proliferation. The prevention of peroxisomal proliferation by carnitine acyltransferase inhibitors may implicate the involvement of this acyltransferase in the induction of peroxisomal proliferation by xenobiotic or native amphipathic carboxylates.

Cloning and expression of the rat liver cDNA for peroxisomal enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase in lambda GT11. Transcriptional regulation of enzyme activity by Wy-14643 in primary cultures of rat hepatocytes.

Proliferation of rat liver peroxisomes by the hypolipidemic drug Wy-14643 is associated with a concomitant induction of peroxisomal enzymes involved in the beta-oxidation of fatty acids. In order to explore the molecular mechanism of this induction process we have cloned the cDNA for the peroxisomal bifunctional enzyme enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase (ECH) in the lambda gt11 expression vector. The library was screened with the monospecific rabbit antiserum to ECH. Hybrid-selected-mRNA translation established that the immunoreactive clones contain the cDNA sequences of the ECH bifunctional enzyme. The cloned cDNA was used to define the early events associated with enzyme induction in primary cultures of rat hepatocytes. Dot-blot hybridization of the total hepatocyte RNA with the ECH cDNA probe showed that the ECH mRNA begins to rise at about 10-15 h following incubation with Wy-14643. At 24 h and 48 h of incubation the stimulation of the ECH mRNA over the vehicle-treated control reached 26-fold and 47-fold respectively. Run-off experiments in the isolated nuclei of hepatocytes showed no increase in the transcription rate of the ECH gene at 5 h after drug treatment and a 2-fold and 11-fold increase at 10 h and 20 h of drug treatment. From these results we conclude that the increase in ECH activity by Wy-14643 is due to an enhancement of the rate of transcription of the ECH gene. However, the relatively long lag period of about 10-15 h after exposure of hepatocytes to Wy-14643 suggests that the induction of the ECH mRNA may involve an indirect effect of the drug on the transcription of this gene.

Deficient activities and proteins of peroxisomal β-oxidation enzymes in infants with Zellweger syndrome

The activities and amounts of enzyme proteins of peroxisomal β-oxidation in Japanese children with Zellweger syndrome were investigated. Cyanide-insensitive fatty acid oxidation, peroxisomal enoyl-CoA hydratase and 3-oxoacyl-CoA thiolase activities were not detectable in liver tissue at autopsy, whereas the activities of mitochondrial enoyl-CoA hydratase, 3-oxoacyl-CoA thiolase and carnitine palmitoyltransferase were similar to those in the healthy controls. On immunoblot analysis, immunoreactive proteins of peroxisomal acyl-CoA oxidase, bifunctional protein and 3-oxoacyl-CoA thiolase were not detected in the livers, kidneys and fibroblasts from the patients. Proteins of catalase and some enzymes of mitochondrial fatty acid oxidation were similar as in normal controls. These data indicate that increased levels of very-longchain fatty acids in Zellweger syndrome are due to the lack of the enzyme proteins of peroxisomal β-oxidation.

Induction of peroxisomal enzymes in livers of neonatal rats exposed to lactating mothers treated with hypolipidaemic drugs. Role of drug metabolite transfer in milk.

Lactating rats were administered by gavage 100 mg/kg body wt. twice a day of either nafenopin or Wy-14,643, two hypolipidaemic drugs with hepatic peroxisome proliferative property. Neonatal rats, after feeding from the drug-treated mothers for 8-14 days, showed sustained increases in both the proliferation of hepatic peroxisomes, as well as in levels of the peroxisome-associated enzymes catalase (3-fold), carnitine acetyltransferase (15-35-fold), peroxisomal enoyl-CoA hydratase (29-46-fold), and palmitoyl-CoA oxidation (12-14-fold). These increases in enzyme activities in suckling rats were similar to those seen in the livers of the drug-treated, lactating mothers after 14 days of treatment. After administering [3H]nafenopin or [3H]Wy-14,643 to lactating rats, significant levels of drug-derived radioactivity were observed in suckling rat gastric milk curds by 2-4 h with significant radioactivity seen in suckling rat livers by 4-6 h. T.l.c. analysis of organic extracts of milk samples from [3H]Wy-14,643 treated animals indicated no detectable levels of the parent drug, only more-polar metabolites. Wy-14,643 metabolites preparatively purified from a rat liver microsomal fraction incubation induced peroxisome proliferation when injected into a neonatal rat. Preparative high pressure liquid chromatography purification and mass spectral analysis has allowed preliminary assessment of the structures of the Wy-14,643 microsomal metabolites. It is concluded that one or more of the metabolite fractions of Wy-14,643 transferred in milk exert the biological ability to induce peroxisome proliferation and peroxisomal enzymes in neonatal livers.

Induction, immunochemical identity and immunofluorescence localization of an 80 000-molecular-weight peroxisome-proliferation-associated polypeptide (polypeptide PPA-80) and peroxisomal enoyl-CoA hydratase of mouse liver and renal cortex.

The hypolipidaemic drugs methyl clofenapate, BR-931, Wy-14643 and procetofen induced a marked proliferation of peroxisomes in the parenchymal cells of liver and the proximal-convoluted-tubular epithelium of mouse kidney. The proliferation of peroxisomes was associated with 6-12-fold increase in the peroxisomal palmitoyl-CoA oxidizing capacity of the mouse liver. Enhanced activity of the peroxisomal palmitoyl-CoA oxidation system was also found in the renal-cortical homogenates of hypolipidaemic-drug-treated mice. The activity of enoyl-CoA hydratase in the mouse liver increased 30-50-fold and in the kidney cortex 3-5-fold with hypolipidaemic-drug-induced peroxisome proliferation in these tissues, and over 95% of this induced activity was found to be heat-labile peroxisomal enzyme in both organs. Sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis of large-particle and microsomal fractions obtained from the liver and kidney cortex of mice treated with hypolipidaemic peroxisome proliferators demonstrated a substantial increase in the quantity of an 80000-mol.wt. peroxisome-proliferation-associated polypeptide (polypeptide PPA-80). The heat-labile peroxisomal enoyl-CoA hydratase was purified from the livers of mice treated with the hypolipidaemic drug methyl clofenapate; the antibodies raised against this electrophoretically homogeneous protein yielded a single immunoprecipitin band with purified mouse liver enoyl-CoA hydratase and with liver and kidney cortical extracts of normal and hypolipidaemic-drug-treated mice. These anti-(mouse liver enoyl-CoA hydratase) antibodies also cross-reacted with purified rat liver enoyl-CoA hydratase and with the polypeptide PPA-80 obtained from rat and mouse liver. Immunofluorescence studies with anti-(polypeptide PPA-80) and anti-(peroxisomal enoyl-CoA hydratase) provided visual evidence for the localization and induction of polypeptide PPA-80 and peroxisomal enoyl-CoA hydratase in the liver and kidney respectively of normal and hypolipidaemic-drug-treated mice. In the kidney, the distribution of these two proteins is identical and limited exclusively to the cytoplasm of proximal-convoluted-tubular epithelium. The immunofluorescence studies clearly complement the biochemical and ultrastructural observations of peroxisome induction in the liver and kidney cortex of mice fed on hypolipidaemic drugs. In addition, preliminary ultrastructural studies with the protein-A-gold-complex technique demonstrate that the heat-labile hepatic enoyl-CoA hydratase is localized in the peroxisome matrix.