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Heme-binding protein 23 (HBP23), also termed peroxiredoxin (Prx) I, and heme oxygenase-1 (HO-1) are distinct antioxidant stress proteins that are co-ordinately induced by oxidative stress. HBP23/Prx I has thioredoxin-dependent peroxidase activity with high binding affinity for the pro-oxidant heme, while HO-1 is the inducible isoform of the rate-limiting enzyme of heme degradation. We investigated the cellular and subcellular localization of both proteins in rat liver. Whereas by immunohistochemistry (IHC) a uniformly high level of HBP23/Prx I expression was observed in liver parenchymal and different sinusoidal cells, HO-1 expression was restricted to Kupffer cells. By immunoelectron microscopy using the protein A-gold technique, HBP23/Prx I immunoreactivity was detected in cytoplasm, nuclear matrix, mitochondria, and peroxisomes of parenchymal and non-parenchymal liver cell populations. In contrast, the secretory pathway, i.e., the endoplasmic reticulum and Golgi complex, was free of label. As determined by immunocytochemical (ICC) studies in liver cell cultures and by Western and Northern blotting analysis, HBP23/Prx I was highly expressed in cultures of isolated hepatocytes and Kupffer cells. In contrast, HO-1 was constitutively expressed only in Kupffer cell cultures but was also inducible in hepatocytes. These data suggest that HBP23/Prx I and HO-1 may have complementary antioxidant functions in different cell populations in rat liver.

Catalase, the classical peroxisomal marker enzyme, decomposes hydrogen peroxide and is involved in the antioxidant defense mechanisms of mammalian cells. In addition, catalase can oxidize, by means of its peroxidatic activity, a variety of substrates such as methanol and ethanol, producing the corresponding aldehydes. The involvement of brain catalase in the oxidation of ethanol is well established, and severe afflictions of the CNS in hereditary peroxisomal diseases (e.g., Zellweger syndrome) are well known. Whereas the distribution of catalase in the CNS has been investigated by enzyme histochemistry and immunohistochemistry (IHC), very little is known about the exact localization of catalase mRNA in brain. Here we report the application of a tyramine/CARD (catalyzed reporter deposition)-enhanced nonradioactive in situ hybridization (ISH) protocol for detection of catalase mRNA in sections of perfusion-fixed, paraffin-embedded rat brain. Catalase mRNA could be demonstrated in a large number of neurons throughout the rat brain as a distinct cytoplasmic staining signal with excellent morphological resolution. Compared to our standard ISH protocol, the CARD-enhanced protocol for catalase mRNA detection in rat brain showed higher sensitivity and significantly better signal-to-noise ratio. In parallel IHC experiments, using an antigen retrieval method consisting of combined trypsin digestion and microwave treatment of paraffin sections, the catalase antigen was found as distinct cytoplasmic granules in most catalase mRNA-positive neurons. In addition, catalase-positive granules, presumably peroxisomes, were found by confocal laser scanning microscopy in glial cells, which were identified by double labeling immunofluorescence for GFAP and CNPase for astroglial cells and oligodentrocytes, respectively. The excellent preservation of morphology and sensitive detection of both mRNA and protein in our preparations warrant the application of the protocols described here for systematic studies of catalase and other peroxisomal proteins in diverse pathological conditions such as Alzheimer's disease and aging.

Our earlier electron microscopic observations revealed that prolonged exposure of glutaraldehyde-fixed rat liver sections to buffer solutions induced focal membrane disruptions of peroxisomes with catalase diffusion as shown cytochemically. Recently, it was suggested that 15-lipoxygenase (15-LOX) might be involved in natural degradation of membrane-bound organelles in reticulocytes by integrating into and permeabilizing the organelle membranes, leading to the release of matrix proteins. We have now investigated the localization of 15-LOX and its role in degradation of peroxisomal membranes in rat liver. Aldehyde-fixed liver slices were incubated in a medium that conserved the 15-LOX activity, consisting of 50 mM HEPES-KOH buffer (pH 7.4), 5 mM mercaptoethanol, 1 mM MgCl(2), 15 mM NaN(3), and 0.2 M sucrose, in presence or absence of 0.5-0.05 mM propyl gallate or esculetin, two inhibitors of 15-LOX. The exposure of aldehyde-fixed liver sections to this medium induced focal disruptions of peroxisome membranes and catalase diffusion around some but not all peroxisomes. This was significantly reduced by both 15-LOX inhibitors, propyl gallate and esculetin, with the latter being more effective. Double immunofluorescent staining for 15-LOX and catalase revealed that 15-LOX was co-localized with catalase in some but not all peroxisomes in rat hepatocytes. By postembedding immunoelectron microscopy, gold labeling was localized on membranes of some peroxisomes. These observations suggest that 15-LOX is involved in degradation of peroxisomal membranes and might have a physiological role in programmed degradation and turnover of peroxisomes in hepatocytes. (J Histochem Cytochem 49:613-621, 2001)

We present a protocol for detection of peroxisomal proteins and their corresponding mRNAs on consecutive serial sections of fetal and newborn mouse tissues by immunohistochemistry (IHC) and nonradioactive in situ hybridization (ISH). The use of perfusion-fixation with depolymerized paraformaldehyde combined with paraffin embedding and digoxigenin-labeled cRNA probes provided a highly sensitive ISH protocol, which also permitted immunodetection with high optical resolution by light and/or fluorescence microscopy. Signal enhancement was achieved by the addition of polyvinyl alcohol (PVA) for ISH color development. For IHC, signal amplification was obtained by antigen retrieval combined with biotin-avidin-HRP and Nova Red as substrate or by the catalyzed reporter deposition of fluorescent tyramide. Using this protocol, we studied the developmental changes in localization of the peroxisomal marker enzymes catalase (CAT) and acyl-CoA oxidase 1 (AOX), the key regulatory enzyme of peroxisomal beta-oxidation, at the protein and mRNA levels in mice from embryonic Day 14.5 to birth (P0.5). The mRNA signals for CAT and AOX were detected in sections of complete fetuses, revealing organ- and cell-specific variations. Here we focus on the localization patterns in liver, intestine, and skin, which showed increasing mRNA amounts during development, with the strongest signals in newborns (P0.5). Immunolocalization of the corresponding proteins revealed, in close correlation with the mRNAs, a distinct punctate staining pattern corresponding to the distribution of peroxisomes. (J Histochem Cytochem 49:155-164, 2001)

The past decade has witnessed unprecedented progress in elucidation of the complex problems of the biogenesis of peroxisomes and related human disorders, with further deepening of our understanding of the metabolic role of this ubiquitous cell organelle. There have been many recent reviews on biochemical and molecular biological aspects of peroxisomes, with the morphology and cytochemistry receiving little attention. This review focuses on the state-of-the-art cytochemical techniques available for investigation of peroxisomes. After a brief introduction into the use of the 3,3'-diaminobenzidine method for localization of catalase, which is still most commonly used for identification of peroxisomes, the cerium technique for detection of peroxisomal oxidases is discussed. The influence of the buffer used in the incubation medium on the ultrastructural pattern obtained in rat liver peroxisomes in conjunction with the localization of urate oxidase in their crystalline cores is discussed, particularly since Tris-maleate buffer inhibits the enzyme activity. In immunocytochemistry, quantitation of immunogold labeling by automatic image analysis enables quantitative assessment of alterations of proteins in the matrix of peroxisomes. This provides a highly sensitive approach for analysis of peroxisomal responses to metabolic alterations or to xenobiotics. The recent evidence suggesting the involvement of ER in the biogenesis of "preperoxisomes" is mentioned and the potential role of preembedding immunocytochemistry for identification of ER-derived early peroxisomes is emphasized. The use of GFP expressed with a peroxisomal targeting signal for the investigation of peroxisomes in living cells is briefly discussed. Finally, the application of in situ hybridization for detection of peroxisomal mRNAs is reviewed, with emphasis on a recent protocol using perfusion-fixation, paraffin embedding, and digoxigenin-labeled cRNA probes, which provides a highly sensitive method for detection of both high- and low-abundance mRNAs encoding peroxisomal proteins. (J Histochem Cytochem 47:1219-1232, 1999)

Peroxisomes (POs) are a heterogenous population of cell organelles which, in mammals, are most abundant in liver and kidney. Although they are usually isolated by differential and density gradient centrifugation, isolation is hampered by their high fragility, sensitivity to mechanical stress, and their sedimentation characteristics, which are close to those of other major organelles, particularly microsomes. Consequently, until now only the so-called "heavy" POs with a buoyant density of 1.22-1.24 g/cm(3) have been highly purified from rat liver, whereas the other subpopulations also present in that tissue have escaped adequate characterization. The purification of these subpopulations has become an essential task in view of the functional significance of POs in humans, and the putative importance of peroxisomal subpopulations in the biogenesis of this organelle. Here we used an alternative novel approach to density gradient centrifugation, called immune free flow electrophoresis (IFFE). IFFE combines the advantages of electrophoretic separation with the high selectivity of an immune reaction. It makes use of the fact that the electrophoretic mobility of a subcellular particle complexed to an antibody against the cytoplasmic domain of one of its integral membrane proteins is greatly diminished, provided that the pH of the electrophoresis buffer is adjusted to pH approximately 8.0, the pI of IgG molecules. Because of this reduced electrophoretic mobility, IgG-coupled particles can be separated in an electric field from those that are noncoupled and hence more mobile. The IFFE technique has been recently applied for isolation of regular POs (rho = 1.22-1.24 g/cm(3)) from a light mitochondrial fraction of rat liver. We succeeded in isolating different subpopulations of POs by applying IFFE to heavy, light, and postmitochondrial fractions separated by differential centrifugation of a rat liver homogenate. The PO subfractions obtained differed in their composition of matrix and membrane proteins, as revealed by immunoblotting. This indicates that they indeed represent distinct subpopulations of rat hepatic POs.

Peroxisomes in the human hepatoblastoma cell line HepG2 exhibit a high degree of plasticity. Whereas in confluent cultures they appear as small (0.1-0.3 micrometer) spherical particles, they undergo dramatic changes, forming elongated tubules measuring up to 5 micrometer on separation of cells and cultivation at low density. We recently showed that several growth factors, including nerve growth factor (NGF), induce the formation of tubular peroxisomes and that this induction is sensitive to K 252b, a specific tyrosine kinase inhibitor, suggesting the involvement of this signal transduction pathway. Because tyrosine kinase is also involved in signal transduction via the reactive oxygen species (ROS), we have analyzed in this study the effects of UV irradiation, H(2)O(2), and oxygen on tubulation of peroxisomes. UVC irradiation induced a significant increase in formation of tubular peroxisomes (40-50% of cells) and this effect was dose-dependently inhibited by pretreatment with N-acetyl cysteine, confirming the involvement of ROS in the UV effect. Furthermore, H(2)O(2) also directly induced the tubulation of peroxisomes, although to a lesser extent. Finally, cultivation under hypoxic conditions (1.5% O(2)) drastically reduced the inducing effect of fetal calf serum on tubulation of peroxisomes, suggesting the involvement of oxygen-mediated signaling. Taken together, our observations indicate that ROS induce the tubulation of peroxisomes in HepG2 cells. Because peroxisomes harbor most of the enzymes for catabolism of ROS, the tubulation and expansion of the peroxisome compartment could have a cell rescue function against the destructive effects of ROS.

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.

We investigated the immunoreactivity of the peroxisomal lipid beta-oxidation enzymes acyl-CoA oxidase, trifunctional protein, and thiolase in guinea pig liver and compared it with that of homologous proteins in rat, using immunoblotting of highly purified peroxisomal fractions and monospecific antibodies to rat proteins. In addition, the immunocytochemical localization of beta-oxidation enzymes in guinea pig liver was compared with that of catalase. All antibodies showed crossreactivity between the two species, indicating that these peroxisomal proteins have been well conserved, although all exhibited some differences with respect to molecular size and, in the case of acyl-CoA oxidase, in frequency of the immunoreactive bands. In the latter case, a distinct second band in the 70 KD range was observed in guinea pig, in addition to the regular band due to subunit A present in rat liver. This novel band could be due either to trihydroxycoprostanoyl-CoA oxidase or to the non-inducible branched chain fatty acid oxidase described recently. All three beta-oxidation enzymes were immunolocalized by light and electron microscopy to the matrix of peroxisomes, in contrast to catalase, which is also found in the cytoplasm and the nucleus of hepatocytes in guinea pig liver.

L-alpha-hydroxyacid oxidase (HAOX), a peroxisomal marker enzyme in mammals, exists in two isozymic forms, HAOX A (EC 1.1.3.1) and HAOX B (EC 1.3.4.2), which differ in their substrate specificity. In rat tissues HAOX A is found exclusively in hepatocyte peroxisomes and HAOX B in renal peroxisomes. Recently we found enzymatic evidence that highly purified peroxisome preparations from beef and sheep kidney cortex contain both isozymes. In situ, the peroxisomes in the proximal tubule cells of both species exhibit peculiar angular outlines apparently due to the presence of multiple marginal plates. Marginal plates are plate-like crystalline matrix inclusions which are apposed to the inner aspect of the peroxisomal membrane. In this study monospecific antibodies against HAOX A and B proteins purified from rat liver and kidney, respectively, were raised in rabbits and used to study the intraorganellar localization of each isozyme in beef and sheep kidney cortex peroxisomes. Incubation of ultra-thin sections of LR White-embedded tissue with anti-HAOX A or B followed by protein A-gold revealed that in both species HAOX A is present diffusely in the peroxisomal matrix, whereas HAOX B is localized almost exclusively in the membrane associated marginal plates. This is the first report on the in situ immunocytochemical characterization of marginal plates, which are the most common inclusions in the matrix of renal peroxisomes.

The cytochemical localization of endogenous peroxidase activity in sinus lining cells of mouse liver has been investigated. Kupffer cells, as identified by their exclusive ability to phagocytize large (0.8 micron) latex particles, exhibited strong peroxidase activity in nuclear envelope and endoplasmic reticulum. In addition, weak to moderate peroxidase activity was found in 57% of all endothelial cells. The enzyme in endothelial cells was also localized in nuclear envelope and endoplasmic reticulum, with a negative reaction in the Golgi apparatus. These observations indicate that peroxidase staining, as a marker for identification of Kupffer cells in mouse liver, is only of limited value and should be used in conjunction with other methods (e.g., latex phagocytosis).

The intracellular localization of beta-NADPase in rat hepatocytes and Kupffer cells has been studied and compared with the pattern of TPPase in these cells. The reaction product for beta-NADPase is present in some but not all hepatocytes in two cisternae on the trans aspect of the Golgi apparatus. It is absent from the trans-most lamella and the GERL of hepatocytes. TPPase, on the other hand, is limited to the first Golgi cisterna on the trans aspect with sprinkles of reaction product in the second lamella. Considering that TPPase is a marker of the trans Golgi lamella and hepatocyte Golgi stacks contain usually 2-4 lamellae, our observations suggest that beta-NADPase is localized in the trans as well as in the intermediate Golgi lamellae of liver parenchymal cells. In Kupffer cells, the reaction product for both beta-NADPase and TPPase was found in some but not in all cells. The enzyme beta-NADPase was localized in the rigid lamella and the tubulovacuolar system of GERL. This pattern differed significantly from that for TPPase, which was found in 2-3 cisternae at the trans aspect of the Golgi complex in Kupffer cells. These observations demonstrate the difference in the localization of beta-NADPase in hepatocytes and Kupffer cells. Such differences should be taken into consideration in studies of Golgi fractions, when phosphatase reactions are used as specific markers of Golgi components.

A scanning transmission electron microscope (STEM) was used to examine ultrathin sections of rabbit white skeletal muscle. Lactic dehydrogenase (LDH) activity was localized in the tissue using the tetra-nitro blue tetrazolium (TNBT) method. For most specimens postfixation was omitted in order to avoid reoxidation and solubilization of the formazan by osmium tetroxide. The STEM image revealed sufficient contrast of the intracellular structures and apparently electron-dense reaction product in the sarcoplasmic reticulum and mitochondria. Substantially less contrast was obtained when the same areas were observed by conventional transmission electron microscopy (CTEM). In material postfixed with osmium tetroxide, although the tissue contrast was improved, the TNBT reaction product was focally leached out, exhibiting lower contrast than in unosmicated sections. These results indicate that the fine structural visualization of dehydrogenases with TNBT, the STEM technique as used in the present study is superior to that obtained by CTEM.

We investigated the immunocytochemical localization of urate oxidase by light and electron microscopy. Rabbits were immunized with urate oxidase prepared from rat liver and the resulting antibody was further purified by affinity chromatography. Immunoblotting of the antigen revealed a single band of Mr 32,500 daltons, consistent with a subunit of uricase. The same band was observed in immunoblots prepared from a total peroxisome fraction and in its subfraction containing the cores, but not in the matrix portion. Immunostaining of 1-micron sections with the antibody against uricase followed by protein A-gold-silver showed fine granules in hepatocytes, which exhibited distinct fluorescence when examined in a microscope equipped with epifluorescence illumination. Incubation of ultra-thin sections of rat liver, embedded in Lowicryl K4M, LR White, or Epon, with the anti-uricase antibody followed by protein A-gold showed prominent labeling of the crystalline cores, with no reaction in the surrounding peroxisomal matrix. In contrast, the core region was spared whereas the matrix was heavily labeled in sections incubated with an antibody against catalase. Direct incubation of cores, isolated by centrifugation, with the anti-uricase antibody followed by protein A-gold revealed gold particles on the surface of isolated cores, with rare particles within the lumen of the polytubular structures that make up the cores. Specificity of the immunolabeling was established in sections incubated with an IgG fraction from pre-immunized rabbits. These observations demonstrate that in normal rat liver urate oxidase is exclusively associated with the crystalline cores in peroxisomes.

The reaction product of cerium used for localization of peroxisomal oxidases is highly electron-dense but lacks sufficient contrast at the light microscopic level. We describe two methods for converting the reaction product of cerium to colored compounds visible by light microscopy. The first method is based on 3,3'-diaminobenzidine (DAB) amplification of transition metal compounds, of which cerium is one. Sections of glutaraldehyde-fixed rat liver or kidney are incubated first in media for various oxidases containing CeCl3, followed by treatment with DAB in Na acetate buffer, pH 5.3. To prevent any interference by the peroxidatic activity of catalase, NaN3 or Na pyruvate is added to the DAB amplification medium. Staining with DAB can be further intensified with NiCl2 or CoCl2. The second method is based on the conversion of the cerium reaction product with alkaline lead citrate and the final visualization of the lead compound with ammonium sulfide. These methods allow the evaluation of large sections for peroxisomal oxidases by light microscopy, making close correlation between light and electron microscopy possible.

The influence of various commercial preparations of horseradish peroxidase upon the arterial pressure has been investigated using direct intra-arterial pressure recordings in Nembutal anesthetized rats of different strains. A marked hypotension was noted in Wistar and Sprague-Dawley rats when sufficiently high doses (5-10 mg/100 g body weight) were injected intravenously. This effect was observed with both highly purified (Sigma type VI, Boehringer Reinheitsgrad I) as well as less pure preparations (Sigma type II, Boehringer Reinheitsgrad II). Prior injection of promethazin, an antihistamine, prevented the drop in arterial pressure. Wistar/Furth rats appeared almost resistant to the hypotensive effect of Sigma preparations but developed moderate to marked hypotension with both types of Boehringer horseradish peroxidase. When instead of Nembutal, ether anesthesia was used only a transient mild hypotension developed and the animals recovered within 7-10 min. No effect was noted in Syrian hamsters receiving injections with the same doses of horseradish peroxidase. Electrophoresis revealed marked variations in the isozyme composition of different horseradish peroxidase preparations. Since the exact nature of the hypotensive agent remains unknown, it is suggested that the arterial pressure be monitored before extensive systematic application of this tracer in vascular permeability studies in rats.

Ultrastructural localization of uricase (urate: oxygen oxidoreductase, E.C.1.7.3.3.) in rat liver parenchymal cells has been studied with the cerium technique. The cerous ions react with H2O2 generated by the activity of the enzyme in the presence of urate, forming the electron-dense reaction product of cerous perhydroxide. Tissue fixation is carried out by perfusion for 5 min with a low concentration (0.25%) of glutaraldehyde. Since in a biochemical assay it was found that the activity of uricase determined in Trismaleate buffer is substantially weaker than in the Pipes buffer, the classical medium of Briggs et al. (6) was modified, and the latter buffer was substituted for the Trismaleate. Vibratome sectons are incubated at 37 degrees C for 60 min in 0.1 M Pipes buffer, pH 7.8, containing 3 mM cerium chloride and 0.1 mM sodium urate. Under these conditions, the reaction product is localized in the crystalline cores of hepatic peroxisomes. The intensity of the staining is dependent on the concentration of the substrate and the incubation time. In control preparations incubated without urate or with 2,6,8-trichloropurine, a specific inhibitor of uricase, staining is almost completely abolished. In sections incubated with 5 mM cerium and 0.1 mM sodium urate, fine granules with a distribution corresponding to peroxisomes are also visible at the light microscopic level. This latter observation is invaluable for correlative light and electron microscopic studies.

The intracellular localization of catalase has been studied using monospecific Fab fragments against rat liver catalase (RLC) and preembedding immunoelectron microscopy. Purified RLC, exhibiting a single band on sodium dodecyl sulfate gel electrophoresis, was used for the immunization of rabbits. The anti-RLC IgG was purified by affinity chromatography. Fab fragments were obtained by papain digestion and were labeled with horseradish peroxidase (HRP) using a modified two-step procedure and glutaraldehyde as coupling agents. Livers were perfused with 4% depolymerized paraformaldehyde and chopper sections were incubated with HRP-labeled Fab fragments against RLC. Because of the limited penetration of labeled Fab fragments into chopper sections a simple method for preparation of a cell suspension from aldehyde-fixed livers was devised. Adequate staining of more than 90% of cell was obtained by incubation of cell suspensions for 12--18 hr with the labeled antibody. By light microscopy specific staining was present in fine granules in the cytoplasm of hepatocytes. By electron microscopy the electron-dense reaction product was localized in the matrix of peroxisomes with no reaction in the endoplasmic reticulum and the Golgi complex. In some hepatocytes, positively reacted peroxisomes were seen side by side with unstained particles. Although focal diffusion was noted around a few peroxisomes, no evidence of cytoplasmic catalase independent of peroxisomes was found. These observations indicate that in rat liver peroxisomes are the only organelle containing substantial amounts of catalase antigen and rule out any involvement of the endoplasmic reticulum and the Golgi complex in the sequestration of this protein.