Detection of peroxisomes in human liver and kidney fixed with formalin and embedded in paraffin: the use of catalase and lipid beta-oxidation enzymes as immunocytochemical markers

Summary We describe the immunocytochemical localization of four peroxisomal enzymes by light microscopy in human liver and kidney processed routinely by formalin fixation and paraffin embedding. Monospecific antisera against catalase and three enzymes of peroxisomal lipid -oxidation (acyl-CoA oxidase, bifunctional protein (enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase) and 3-ketoacyl-CoA thiolase) were used in conjunction with either the indirect immunoperoxidase method or the protein A—gold technique followed by silver intensification. The sections of formalin-fixed paraffin-embedded tissue had to be deparaffinized and subjected to controlled proteolysis in order to obtain satisfactory immunostaining. Under the conditions employed, peroxisomes were distinctly visualized in liver parenchymal cells with no reaction in bile duct epithelial or sinusoidal lining cells. In the kidney, peroxisomes were confined to the proximal tubular epithelial cells with negative staining of glomeruli, distal tubules and collecting ducts. A positive immunocytochemical reaction was obtained even in paraffin blocks stored for several years. The method offers a simple approach for detection of peroxisomes and evaluation of their various enzyme proteins in material processed routinely in histopathology laboratories and should prove useful in the investigation of the role of peroxisomes in human pathology for both prospective and retrospective studies.     

Cytochemical localization of catalase and several hydrogen peroxide-producing oxidases in the nucleoids and matrix of rat liver peroxisomes

Synopsis The distribution of catalase, amino acid oxidase, -hydroxy acid oxidase, urate oxidase and alcohol oxidase was studied cytochemically in rat hepatocytes. The presence of catalase was demonstrated with the conventional diaminobenzidine technique. Oxidase activities were visualized with methods based on the enzymatic or chemical trapping of the hydrogen peroxide produced by these enzymes during aerobic incubations.All enzymes investigated were found to be present in peroxisomes. Catalase activity was found in the peroxisomal matrix, but also associated with the nucleoid. After staining for oxidase activities the stain deposits occurred invariably in the peroxisomal matrix as well as in the nucleoids. In all experiments the activity of both catalase and the oxidases was confined to the peroxisomes. The presence of a hydrogen peroxide-producing alcohol oxidase was demonstrated for the first time in peroxisomes in liver cells.The results imply that the enzyme activity of the nucleoids of rat liver peroxisomes is not exclusively due to urate oxidase. The nucleoids obviously contain a variety of other enzymes that may be more or less loosely associated with the insoluble components of these structures.     

Post-mortem visualization of peroxisomes in rat and in human liver

Summary This paper describes spontaneous post-mortem changes of peroxisomal staining in normal liver and kidney of rats and in human autopsy liver. At room temperature, regional staining loss is observed at 18h after death in rat kidney, at 24h in human liver and at 48 h in rat liver. Preservation at 4°C delays this phenomenon. In human liver, the peroxisomal volume density is decreased at both temperatures at 48 h. After freezing of fresh tissue in dry ice, peroxisomal staining is decreased homogeneously. Under the electron microscope, peroxisomal alterations suggest a loss of catalase activity. These changes do not necessarily preclude the study of peroxisomal features since, even after 48 h at room temperature, peroxisomes are still well stained in the less affected regions. Catalase and three -oxidation enzymes, namely acyl-CoA oxidase, bifunctional protein (with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase) and 3-oxoacyl-CoA thiolase, could be visualized immunocytochemically in human autopsy livers up to 48 h after death. However, the study of certain peroxisomal features such as catalase activity and peroxisomal distribution, may be hampered as the post-mortem period is prolonged.     

Insulin-degrading enzyme exists inside of rat liver peroxisomes and degrades oxidized proteins

Insulin-degrading enzyme (IDE) was detected by immunoblot analysis in highly purified rat liver peroxisomes. IDE in the peroxisomal fraction was resistant to proteolysis by trypsin and chymotrypsin under conditions where the peroxisomal membranes remained intact. After sonication of the peroxisomal fraction, IDE was recovered in the supernatant fraction. Further, the localization of IDE in the peroxisomes was shown by immunoelectron microscopy. In addition, IDE isolated from peroxisomes degraded insulin as well as oxidized lysozyme as a model substrate for oxidized proteins. These results suggest that IDE exists in an active form in the matrix of rat liver peroxisomes and is involved in elimination of oxidized proteins in peroxisomes.     

Cytochemical studies on the localization of methanol oxidase and other oxidases in peroxisomes of methanol-grown Hansenula polymorpha

The localization of methanol oxidase activity in cells of methanol-limited chemostat cultures of the yeast Hansenula polymorpha has been studied with different cytochemical staining techniques. The methods were based on enzymatic or chemical trapping of the hydrogen peroxide produced by the enzyme during aerobic incubations of whole cells in methanol-containing media. The results showed that methanol-dependent hydrogen peroxide production in either fixed or unfixed cells exclusively occurred in peroxisomes, which characteristically develop during growth of this yeast on methanol. Apart from methanol oxidase and catalase, the typical peroxisomal enzymes d-aminoacid oxidase and l--hydroxyacid oxidase were also found to be located in the peroxisomes. Urate oxidase was not detected in these organelles. Phase-contrast microscopy of living cells revealed the occurrence of peroxisomes which were cubic of form. This unusual shape was also observed in thin sections examined by electron microscopy. The contents of the peroxisomes showed, after various fixation procedures, a completely crystalline or striated substructure. It is suggested that this substructure might represent the in vivo organization structure of the peroxisomal enzymes.     

Effects of fixation and processing on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded liver tissue

We compared the effects of different fixatives and enzymatic-digestion procedures on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded normal human liver sections. None of the fixatives tested allowed the staining of these antigens without prior enzymatic digestion. The best results i.e. strong staining intensity and well-defined localization, were obtained when liver tissue was fixed in Bouin's fluid or in other solutions containing picric acid. Several other fixatives, including Carnoy's fluid, Lillie's AAF, 10% neutral formalin and 96% ethanol, gave unsatisfactory results. Pepsin was ineffective for unmasking type-I and -III collagen antigens, and was only partially effective for visualizing the type-IV collagen antigen. The best results were obtained when material fixed in Bouin's fluid was embedded in paraffin and digested with trypsin. Using this procedure, the results were comparable to those obtained in unfixed frozen sections with respect to the staining intensity, specificity and non-specific staining.     

Effects of fixation and processing on the immunohistochemical visualization of type-I, -III and -IV collagen in paraffin-embedded liver tissue

Summary We compared the effects of different fixatives and enzymatic-digestion procedures on the immunohistochemical visualization of type-I,-III and-IV collagen in paraffin-embedded normal human liver sections. None of the fixatives tested allowed the staining of these antigens without prior enzymatic digestion. The best results i.e. strong staining intensity and well-defined localization, were obtained when liver tissue was fixed in Bouin's fluid or in other solutions containing picric acid. Several other fixatives, including Carnoy's fluid, Lillie's AAF, 10% neutral formalin and 96% ethanol, gave unsatisfactory results. Pepsin was ineffective for unmasking type-I and-III collagen antigens, and was only partially effective for visualizing the type-IV collagen antigen. The best results were obtained when material fixed in Bouin's fluid was embedded in paraftin and digested with trypsin. Using this procedure, the results were comparable to those obtained in unfixed frozen sections with respect to the staining intensity, specificity and non-specific staining.     

Application of automatic image analysis for morphometric studies of peroxisomes stained cytochemically for catalase

Summary The feasibility of the application of a television-based image analyzer, the Texture Analysis System (TAS, Leitz Wetzlar, FRG) in conjunction with a light microscope for morphometric studies of hepatic peroxisomes has been investigated. Rat liver peroxisomes were stained with the alkaline-DAB method for localization of catalase and semi-thin (0.25 and 1 m) sections of plastic-embedded material were examined under an oil immersion objective. The TAS detected the peroxisomal profiles selectively and determined their morphometric parameters automatically. The same parameters were obtained also by morphometric analysis of electron micrographs from the same material. The volume density of peroxisomes determined by TAS in semithin sections of normal liver, after correction for section thickness, is quite close to the corresponding value obtained by morphometry of electron micrographs. The difference is approximately 20%. In animals treated with the hypolipidemic drug bezafibrate, which causes proliferation of peroxisomes, TAS detected readily the increase in volume density of peroxisomes in semithin sections. In comparison with electron microscopy, however, the light-microscopic approach seems to underestimate the proliferation. The lower resolution of the light microscope and overlapping of neighbouring particles in relatively thick sections used for lightmicroscopic analysis may account for the differences.The present study has demonstrated the usefulness of automatic image analysis in conjunction with selective cytochemical staining of peroxisomes for morphometry of this organelle in rat liver. The light-microscopic approach is not only faster but is also extremely economical by obviating the use of an electron microscope.     

Immunocytochemical localization of urate oxidase, fatty acyl-CoA oxidase, and catalase in bovine kidney peroxisomes

We investigated the localization of urate oxidase, peroxisomal fatty acyl-CoA oxidase, and catalase in bovine kidney by immunoblot analysis and protein A-gold immunocytochemistry, using the respective polyclonal monospecific antibodies raised against the enzymes purified from rat liver. By immunoblot analysis, these three proteins were detected in bovine kidney and bovine liver homogenates. Subcellular localization of these three enzymes in kidney was ascertained by protein A-gold immunocytochemical staining of Lowicryl K4M-embedded tissue. Peroxisomes in bovine kidney cortical epithelium possessed crystalloid cores or nucleoids, which were found to be the exclusive sites of urate oxidase localization. The limiting membrane, the marginal plate, and the matrix of renal peroxisomes were negative for urate oxidase staining. In contrast, catalase and fatty acyl-CoA oxidase were found in the peroxisome matrix. These results demonstrate that, unlike rat kidney peroxisomes which lack urate oxidase, peroxisomes of bovine kidney contain this enzyme as well as peroxisomal fatty acyl-CoA oxidase.     

Effect of ciprofibrate on the activation and oxidation of very long chain fatty acids

The effect of ciprofibrate, a hypolipidemic drug, was examined in the metabolism of palmitic (C_16:0) and lignoceric (C_24:0) acids in rat liver. Ciprofibrate is a peroxisomal proliferating drug which increases the number of peroxisomes. The palmitoyl-CoA ligase activity in peroxisomes, mitochondria and microsomes from ciprofibrate treated liver was 3.2, 1.9 and 1.5-fold higher respectively and the activity for oxidation of palmitic acid in peroxisomes and mitochondria was 8.5 and 2.3-fold higher respectively. Similarly, ciprofibrate had a higher effect on the metabolism of lignoceric acid. Treatment with ciprofibrate increased lignoceroyl-CoA ligase activity in peroxisomes, mitochondria and microsomes by 5.3, 3.3 and 2.3-fold respectively and that of oxidation of lignoceric acid was increased in peroxisomes and mitochondria by 13.4 and 2.3-fold respectively. The peroxisomal rates of oxidation of palmitic acid (8.5-fold) and lignoceric acid (13.4-fold) were increased to a different degree by ciprofibrate treatment. This differential effect of ciprofibrate suggests that different enzymes may be responsible for the oxidation of fatty acids of different chain length, at least at one or more step(s) of the peroxisomal fatty acid -oxidation pathway.     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Preparation of peroxisomes from carp liver by zonal rotor censity gradient centrifugation

Synopsis Peroxisomes from carp liver can be separated by isopycnic density gradient centrifugation in sucrose. Without reaching complete sedimentation equilibrium, the purification by this method is quite successful. There is a 40-fold enrichment of catalase, the peroxisomal marker, with a total yield of 27%. No pretreatment of animals is necessary for separation from lysosomes, which, besides high fragility, show lower buoyant densities than peroxisomes. The enzyme content of carp liver peroxisomes is similar to that of rat liver, with the exception of -glycerophosphate dehydrogenase, which in this tissue is a completely soluble cytoplasmic enzyme. Total activities are much lower than in the rat, for the characteristic peroxisomal oxidases the difference being in the range of one order of magnitude.     

Biogenesis of peroxisomes in regenerating rat liver. I. Sequential changes of catalase and urate oxidase detected by ultrastructural cytochemistry

The biogenesis of peroxisomes has been investigated in the model of regenerating rat liver after partial hepatectomy using ultrastructural cytochemical staining methods: catalase as a marker of the peroxisomal matrix and uricase for the cores. The peroxisomes in regenerating rat liver showed several distinctive features: a) marked variation in shape and size, e.g., peroxisomes with tail-like extensions and tortuously elongated rod-shaped ones, b) formation of peroxisomal clusters and, c) interconnections between adjacent peroxisomes suggesting cleavage or budding. Whereas the reaction product for catalase was present at all intervals after hepatectomy in the matrix of all peroxisomes, the pattern of localization of uricase case varied with the time. It was confined to the cores in controls and at 10 days after the operation, while at 24 and 48 h it showed, in addition, a diffuse reaction in the matrix of some peroxisomes. In interconnected apparently dividing peroxisomes, the core with positive uricase reaction was present only in one half, while the other half was devoid of the reaction product. Similarly, the diffuse uricase staining was confined to the half which contained the core with the other half remaining unstained. These observations are consistent with the concept that new peroxisomes are formed from preexisting ones by budding and segmentation. While catalase is transferred uniformly to all new segments, uricase is compartmentalized in certain portions, of the apparently growing "peroxisomal reticulum".     

The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization

Summary Conventional solution-phase polymerase chain reaction (PCR) and in situ PCR/PCR in situ hybridization are powerful tools for retrospective analysis of fixed paraffin wax-embedded material. Amplification failure using these techniques is now encountered in some centres using archival fixed tissues. Such ailures may not only be due to absent target DNA sequences in the tissues, but may be a direct effect of the type of fixative, fixation time and/or fixation temperature used. The type of nucleic acid extraction procedure applied will also influence amplification results. This is particularly true with in situ PCR/PCR in situ hybridization.To examine these effects in solution-phase PCR, -globin gene was amplified in 100 mg pieces of tonsillar tissue fixed in Formal saline, 10% formalin, neutral buffered formaldehyde, Carnoy's, Bouin's, buffered formaldehyde sublimate, Zenker's, Helly's and glutaraldehyde at 0 to 4°C, room temperature and 37°C fixation temperatures and for fixation periods of 6, 24, 48 and 72 hours and 1 week. DNA extraction procedures used were simple boiling and 5 days' proteinase K digestion at 37°C. Amplified product was visible primarily yet variably from tissue fixed in neutral buffered formaldehyde and Carnoy's, whereas fixation in mercuric chloride-based fixatives produced consistently negative results. Room temperature and 37°C fixation temperature appeared most conducive to yielding amplifiable DNA template. Fixation times of 24 and 48 hours in neutral buffered formaldehyde and Carnoy's again favoured amplification.Fixed SiHa cells (containing 1–2 copies of HPV 16) were examined using PCR in situ hybridization for the amplification of HPV 16. Discrete and diffuse amplification signals were obtained. Neutral buffered formaldehyde fixation for 12–24 hours yielded amplifiable material suitable for use with PCR in situ hybridization. Overall amplification success within cellular preparations was 40%, with non-specific background staining also seen. Possible technical problems encountered with PCR in situ hybridization are discussed.     

Changes in membrane surface properties of hepatic peroxisomes of rats under several conditions as determined by partition in aqueous polymer two-phase systems

Changes in membrane surface properties of hepatic peroxisomes of rats under several conditions were observed by aqueous polymer two-phase systems, which contained 6% (w/w) dextran T 500, 6% (w/w) polyethyleneglycol 4000, 250 mmol sucrose/kg and various concentrations of sodium phosphate buffer. The partition of peroxisomes into the upper phase depended to a large extent on their membrane surface charge. The cross-points of peroxisomes shifted from 5.55 to 5.25 and 5.2 after the administration of clofibrate and aspirin for 2 weeks, respectively, although that of alloxan-diabetic rat peroxisomes was not altered. The hydrophobic properties of peroxisomes, examined by means of a partition containing polyethyleneglycol monostearate, were altered by diabetes and starvation, but no change occurred in rats treated with clofibrate or aspirin. In the liver of rats fed a high-fat diet, the partition of peroxisomes was the same as that of the control. These findings indicate that hypolipidemic drugs such as clofibrate and aspirin induce the proliferation of peroxisomes and lead to the alteration of the surface charge of peroxisomal membranes. Diabetes or fasting lead to an alteration mainly of the hydrophobic properties. Both changes are probably due to alteration of content and/or composition of the proteins and the phospholipids in peroxisomal membrane under the conditions used.     

Effects of fixation on the preservation of peroxisomal structures for immunofluorescence studies using HepG2 cells as a model system

Summary The immunofluorescence technique has become an important tool for the investigation of peroxisomes in cell culture. We have used this method for the study of peroxisomes in the human hepatoblastoma cell line HepG2. A marked heterogeneity of peroxisomal forms was detected. Besides spherical (about 100 nm) and rod-shaped structures (about 300 nm) many elongated, undulating tubular forms (up to 5 m) were found. Further observations indicate that the appearance of the peroxisomal forms in immunofluorescence is dependent on the fixation procedure used. Whereas the fixation with methanol-acetone (–20°C) or ethanol results in a punctate pattern with spherical particles, the use of formaldehyde/Triton X-100 fixation shows well-preserved tubules and rods. These observations may be of special importance for studies on the biogenesis of peroxisomes.     

Biogenesis of peroxisomes: sequential biosynthesis of the membrane and matrix proteins in the course of hepatic regeneration

The biogenesis of peroxisomes was investigated in the model of regenerating rat liver after partial hepatectomy (PH), using analytical differential centrifugation in combination with immunoblotting and in vivo pulse labeling as well as immunoelectron microscopy. The total activity of catalase decreased sharply after PH, returning gradually over several days to normal levels. In the 16 to 32-h period the enzyme activity started to increase first in the heavy mitochondrial fraction, shifting at 28 h to the crude peroxisomal and at 32 h to the microsomal fraction, suggesting de novo formation of peroxisomes by budding or fragmentation from larger aggregates. Whereas most peroxisomal matrix proteins were reduced during the 16 to 32-h period after PH, the 26 and 70 kDa peroxisomal membrane proteins were increased. Moreover, in vivo pulse labeling studies with radioactive leucine showed significantly higher levels of specific activity in the peroxisomal membrane than in the matrix subfractions at 16 h with increasing labeling of the matrix at 32 h after PH. These findings suggest that de novo formation of peroxisomes in regenerating rat liver is initiated by the synthesis of membrane proteins and is followed by that of the matrix components.     

Determination of the cross-points of rat liver peroxisomes,peroxisomal core and the core components by cross-partition

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same crosspoint, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, d-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2:1. The theoretical value of cross-point of the core calculated from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

Determination of the cross-points of rat liver peroxisomes, peroxisomal core and the core components by cross-partition.

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same cross-point, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, D-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2 : 1. The theoretical value of cross-point of the core calculated from from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.     

Changes to the integral membrane protein composition of mouse liver peroxisomes in response to the peroxisome proliferators clofibrate,Wy-14,643 and Di(2-ethyl-hexyl)phthalate

Peroxisomes were purified from livers of control mice and from mice treated with three agents which induce proliferation of hepatic peroxisomes — namely two structurally unrelated hypolipidemic drugs, clofibrate (ethyl--p-chlorophenoxyisobutyrate) and Wy-14,643 (4-chloro-6[2,3-xylidino)-2-pyrimidinylthio] acetic acid), and a plasticizer, DEHP (di-(2-ethylhexyl)phthalate).Membranes were isolated from these purified peroxisomes and analysed by SDS-polyacrylamide gel electrophoresis. All membranes which were tested, displayed two predominant integral membrane proteins of apparent molecular weights of 68 kDa and 70 kDa respectively, as well as a number of minor components. Treatment of animals with clofibrate, Wy-14,643 and DEHP was observed to result in each case in an increased proportion of the 70 kDa protein in the peroxisomal membranes. These treatments also resulted in increased peroxisomal fatty acid oxidation in livers and an increase in the proportion of catalase activity in the cytosolic fraction of liver cells.These results have been discussed in relation to alterations in the molecular composition of the membranes, the mechanisms of peroxisome proliferation and the inducibility of peroxisomal membrane proteins.