Peroxisomal Marker Enzyme Research Articles

Peroxisomes during postnatal development of mouse endocrine and exocrine pancreas display cell-type- and stage-specific protein composition

Peroxisomal dysfunction unhinges cellular metabolism by causing the accumulation of toxic metabolic intermediates (e.g. reactive oxygen species, very -chain fatty acids, phytanic acid or eicosanoids) and the depletion of important lipid products (e.g. plasmalogens, polyunsaturated fatty acids), leading to various proinflammatory and devastating pathophysiological conditions like metabolic syndrome and age-related diseases including diabetes. Because the peroxisomal antioxidative marker enzyme catalase is low abundant in Langerhans islet cells, peroxisomes were considered scarcely present in the endocrine pancreas. Recently, studies demonstrated that the peroxisomal metabolism is relevant for pancreatic cell functionality. During the postnatal period, significant changes occur in the cell structure and the metabolism to trigger the final maturation of the pancreas, including cell proliferation, regulation of energy metabolism, and activation of signalling pathways. Our aim in this study was to (i) morphometrically analyse the density of peroxisomes in mouse endocrine versus exocrine pancreas and (ii) investigate how the distribution and the abundance of peroxisomal proteins involved in biogenesis, antioxidative defence and fatty acid metabolism change during pancreatic maturation in the postnatal period. Our results prove that endocrine and exocrine pancreatic cells contain high amounts of peroxisomes with heterogeneous protein content indicating that distinct endocrine and exocrine cell types require a specific set of peroxisomal proteins depending on their individual physiological functions. We further show that significant postnatal changes occur in the peroxisomal compartment of different pancreatic cells that are most probably relevant for the metabolic maturation and differentiation of the pancreas during the development from birth to adulthood.

Hormone Excretion and Peroxisomes of Human Immortalized Extravillous Trophoblast Cells (TCL-2) Derived from First-Trimester Placenta

We studied the hormone excretion of human immortalized extravillous trophoblast cells (TCL-2, first-trimester cells) and determined whether peroxisomes are present in TCL-2. The results of TCL-2 were compared with those of TCL-1 (third-trimester cells). Morphologically, TCL-2 cells were fibroblast-like, and the growth rate of TCL-2 was slower than that of TCL-1 during 3 days culture. Progesterone was detected in the medium of TCL-2, and its concentration was approximately one-tenth of that in TCL-1. The activity of the peroxisomal marker enzyme catalase was detected in the TCL-2 homogenate, and it was about one-third the level of that in TCL-1. Fatty acyl-CoA oxidase activity was detected in TCL-2, and it was about one-seventh the level of that in TCL-1. On the other hand, human chorionic gonadotropin (hCG) was detected in the medium of TCL-2, and its concentration after 3 days of culture was about 2-fold that in TCL-1. Using the diaminobenzidine (DAB) method, peroxisomes were found in TCL-2, but only a very small amount of catalase was detected. These results indicate that human immortalized extravillous trophoblast cells (TCL-2) synthesize, secrete hCG and progesterone, and may contain peroxisomes. Because extravillous trophoblast cells are difficult to obtain from the first-trimester placenta, TCL-2 cells are useful for the study of the physiologic functions (including peroxisomal function) of first-trimester cells.

Anniversaries, peroxisomes and reactive oxygen species

In 2008, the annual symposium of the Society for Histochemistry was held in Interlaken, Switzerland (1–4 October). It was not only the 50th symposium of the Society for Histochemistry, but also the 50th anniversary of Histochemistry and Cell Biology. Initially, I was invited by Jurgen Roth and the organizers to give a seminar on peroxisomes in a session on cellular defense mechanisms. When I became aware that the session was supposed to be chaired by H. Dariush Fahimi (the former supervisor of my PhD thesis) who was about to celebrate his 75th anniversary in 2008, I communicated this coincidental observation to the organizers. We spontaneously decided to celebrate H. Dariush Fahimi`s anniversary as well (without telling him!), and the session was renamed “Peroxisomes, ROS and HDF 75”. Furthermore, colleagues from the “early days” of peroxisome research were invited to make a contribution to the session (Fig. 1). Although scheduled for 8:00 am on a cloudy and rainy Friday morning, the topic attracted many participants and congratulators and led to vivid discussions. Subsequently, the speakers of the session were encouraged by the editors of this journal to summarize their talks in the form of a brief report or mini-review and to give a short overview and/or perspective of their Weld of interest. As an introduction to those reports, H. Dariush Fahimi (Heidelberg, Germany; Fig. 1) delineates how he contributed to the introduction of the DAB-method for light and electron microscopic visualization of peroxisomes. Coincidentally, the 50th anniversary symposium also commemorated the 40th year of the introduction of this elegant method, which led to the discovery of peroxisomes as a ubiquitous eukaryotic organelle. It was revealed that the peroxidatic activity of catalase, an abundant peroxisomal marker enzyme, is responsible for that staining. The discovery of the co-localization of several H2O2-producing oxidases, together with catalase in peroxisomes, was the Wrst indication suggesting the participation of peroxisomes in the metabolism of oxygen metabolites. It also led Christian de Duve to propose the term “peroxisome” for the designation of that organelle. In our brief report (Delille et al. this issue), we address the classical and novel views on the formation of peroxisomes, which is again hotly debated. We contributed to the identiWcation of the Wrst components involved in the growth and division of peroxisomes. Interestingly, some of these components (e.g., dynamin-like protein 1, hFis1, MV) are shared by the division machinery of both peroxisomes and mitochondria. Recently, it became clear that this is a common strategy used by mammals, fungi and plants. We propose a closer interrelationship between peroxisomes and mitochondria, which might have an impact on functionality and disease conditions. Furthermore, peroxisomes and mitochondria have been suggested to contribute to pathological conditions associated with oxidative stress and have as well been linked to aging. The central role of peroxisomes in the generation and scavenging of H2O2 has been well known ever since their discovery almost Wve decades ago. Recent Wndings on the biochemical properties of catalase and the abundant peroxisomal oxidases (e.g., D-amino acid oxidase, urate oxidase, xanthine oxidase) are highlighted by Alfred Volkl et al. (Heidelberg, Germany; Fig. 1; Angermuller et al. this issue). The cerium technique and immunoelectron microscopy have been successfully used to localize these enzymes in peroxiM. Schrader (&) Centre for Cell Biology and Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal e-mail: mschrader@ua.pt

Presence and Some Characteristics of Peroxisomes in Immortalized Human Trophoblast Cells

Using morphological and biochemical (Western blot and cell fractionation) methods, we investigated whether peroxisomes are present in human extravillous trophoblast cells. Immortalized extravillous trophoblast cells (TCL-1) were incubated in the presence or absence of 0.5 mM clofibric acid for 3 d. In immunofluorescence staining of trophoblast cells with antibodies anti-catalase and anti-acyl-CoA oxidase (marker enzymes of peroxisomes), electron microscopy and immunoelectron microscopy, peroxisomes were detected in the cells. The size and number of peroxisomes in the trophoblast cells were smaller than those in rat liver. The number of peroxisomes was increased by clofibric acid. In Western blot experiment with antibodies anti-peroxisomal enzymes of beta-oxidation system, densitometric analysis revealed approximately two fold increase in staining by clofibric acid. When we performed cell fractionation experiment using catalase as one of the peroxisomal marker enzymes, the highest activity of catalase was found in the light mitochondrial fraction. Specific activity of catalase in the light mitochondrial fraction was significantly increased to about 1.3 times higher than the control value by clofibric acid treatment. Upon Nycodenz density gradient centrifugation, the catalase activity was concentrated in the density fraction around 1.14-1.15. These findings suggest that microperoxisomes, which have a density smaller than those of rat hepatic peroxisomes, do exist in human extravillous trophoblast cells. It may also be possible to proliferate human peroxisomes in limited quantities using peroxisome proliferator of rodents.

Expression of catalase mRNA and protein in adult rat brain: detection by nonradioactive in situ hybridization with signal amplification by catalyzed reporter deposition (ISH-CARD) and immunohistochemistry (IHC)/immunofluorescence (IF).

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.

Existence of catalase-less peroxisomes in Sf21 insect cells

Catalase activity, a peroxisomal marker enzyme, was not detectable in any of the subcellular fractions of Spodoptera frugiperda (Sf) 21 insect cells, although marker enzymes in other organelles were distributed in the fractions in a manner similar to that seen in mammalian cells. When a green fluorescent protein fused with peroxisome targeting signal 1 at the C-terminal (GFP-SKL) was expressed in Sf21 cells, punctate fluorescent dots were observed in the cytoplasm. The fraction where GFP-SKL was concentrated exhibited long-chain and very-long-chain fatty acid β-oxidation activities in the presence of KCN and the density of this fraction was slightly higher than that of mitochondria. Immunoelectron microscopy studies with anti-SKL antibody demonstrated that Sf21 cells have immunoreactive peroxisome-like organelles which are structurally distinct from mitochondria, endoplasmic reticulum, and lysosomes. In contrast to peroxisomal matrix proteins, adrenoleukodystrophy protein, a peroxisomal membrane protein, was not located to peroxisomes. This suggests that the targeting signal for PMP in insect cells is distinct from that in mammalian cells. These results demonstrate that Sf21 insect cells have unique catalase-less peroxisomes capable of β-oxidation of fatty acids.

A 75-kDa plant protein isolated by tubulin-affinity chromatography is a peroxisomal matrix enzyme

The function of microtubules in plant cells relies on their interactions with various, largely unidentified, proteins. A 75-kDa polypeptide (p75) was isolated previously by tubulin affinity chromatography of tobacco BY-2 proteins and is further characterized here. We have obtained two peptide sequences of 13 and 8 amino acid residues from the p75, which have 77% and 87% similarity to two putative dTDP-glucose 4,6-dehydratase proteins of 74.9 and 75.3 kDa in Arabidopsis. The proteins contain a peroxisomal matrix targeting signal SKL or AKL near their carboxyl terminus, an epimerase-dehydratase motif, and a region of 321 amino acid residues sharing 41% similarity with a dTDP-glucose 4,6-dehydratase from Aneurinibacillus thermoaerophilus. A monoclonal antibody 4B9 against the tobacco p75 reacted with a 75-kDa polypeptide on immunoblots of tobacco BY-2 and Arabidopsis protein extracts, and in immunofluorescence microscopy it revealed small organelle-like structures in tobacco BY-2 and Arabidopsis root-tip cells. Double labelling with an antibody against the peroxisomal marker enzyme, catalase, showed that the organelles are indeed peroxisomes. The peroxisomes were in closer association with actin filaments than microtubules. This observation supports recent findings that plant peroxisomes move on actin filaments. We propose that the peroxisomal dTDP-glucose 4,6-dehydratases may be involved in plant defence responses to oxidative stress.Key words: actin, antibodies, microtubules.

Catalase immunocytochemistry allows automatic detection of lung type II alveolar cells.

In mammalian lung, type II pneumocytes are especially critical in normal alveolar functioning, as they are the major source of surfactant and the progenitors of type I alveolar cells. Moreover, they undergo proliferation and transformation into type I cells in most types of cellular injury, where flattened type I pneumocytes are selectively destroyed. Hyperplasia of alveolar type II cells has also been described in some human chronic lung diseases. In lung, type II pneumocytes and non-ciliated bronchiolar cells are the unique cell types that contain a considerable amount of peroxisomes. Due to the presence of dihydroxyacetone phosphate acyltransferase and non-specific lipid-transfer protein, these organelles have been suggested to be involved in the synthesis and/or transport of the lipid moiety of surfactant. In the present research, the peroxisomal marker enzyme catalase was immunolocalised at the light microscopic level, utilising the avidin-biotin complex method, in lung specimens excised from newborn, adult and aged rats. In all the examined stages the immunoreactivity was so selective for type II pneumocytes it allowed quantitation of these cells by an automated detection system. This was accomplished on specimens from newborn rat lung, in which labelled alveolar cells were counted by a grey level-based procedure and their main morphometric parameters were determined.

Detection of peroxisomal proteins and their mRNAs in serial sections of fetal and newborn mouse organs.

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)

Chemical, biochemical and cellular responses in the digestive gland of the mussel Mytilus galloprovincialis from the Spanish Mediterranean coast

Mussels (Mytilus galloprovincialis) were sampled in March 1996 from five stations along the Western Mediterranean coast (Barcelona, Ebro Delta, Alboraya, Cullera, Denia) corresponding to urban, industrial and agricultural areas. Different biochemical and cellular markers were determined in the mussels in order to assess the effects and/or exposure to pollutants. The cytochrome P450 system, acetylcholinesterase and metallothioneins were among the biochemical markers selected for the study. Histochemical analysis of ß-glucuronidase and catalase activity were performed as marker enzymes for lysosomes and peroxisomes. Chemical analyses indicated that mussels from Barcelona and Denia as highly exposed to polycyclic aromatic hydrocarbons (PAHs)(1.8-2.7 µg g-1 w.w. against 0.02-0.10 µg g-1 w.w.), and polychlorinated biphenyls (PCBs)(132-260 ng g-1 w.w. against 8-24 ng g-1 w.w.). This was in agreement with changes in lysosome structure and higher number of peroxisomes in those organisms. High levels of metals (particularly Cr and Cu) were recorded in the digestive gland of Alboraya mussels, which also had elevated metallothionein content (28 nmol g-1 w.w.) in comparison with the other stations (15-20 nmol g-1 w.w.). Benzo(a)pyrene hydroxylase (BPH) activity indicated Cullera and Barcelona as possibly polluted sites. The results support the usefulness of the biomarker approach to assess and diagnose environmental pollution. The use of a battery of biomarkers at different levels of biological organization coupled with chemical analysis is highlighted.

Induction of the Peroxisomal Glycerolipid-synthesizing Enzymes during Differentiation of 3T3-L1 Adipocytes: ROLE IN TRIACYLGLYCEROL SYNTHESIS

The glycerophosphate backbone for triglyceride synthesis is commonly believed to be created through the conversion of dihydroxyacetone phosphate (DHAP) by glycerophosphate dehydrogenase (GPD) to sn-glycerol 3-phosphate (GP), which is then converted by glycerophosphate acyltransferase (GPAT) to 1-acyl-GP. Consistent with this, GPD and GPAT are highly induced during differentiation of mouse 3T3-L1 preadipocytes. While the acyl dihydroxyacetone phosphate (acyl-DHAP) pathway for glycerolipid synthesis is commonly believed to be involved only in glycerol ether lipid synthesis, we report here that during conversion of 3T3-L1 preadipocytes to adipocytes, the specific activity of peroxisomal DHAP acyltransferase (DHAPAT) is increased by 9-fold in 6 days, while acyl-DHAP:NADPH reductase is induced by 5-fold. A parallel increase in the catalase (the peroxisomal marker enzyme) activity is also seen. In contrast, the specific activity of alkyl-DHAP synthase, the enzyme catalyzing the synthesis of the ether bond, is decreased by 60% during the same period. Unlike microsomal GPAT, the induced DHAPAT is found to have high activity at pH 5.5 and is resistant to inhibition by sulfhydryl agents, heat, and proteolysis. On subcellular fractionation, DHAPAT is found to be associated with microperoxisomes whereas GPAT activity is mainly present in microsomes. Northern blot analyses reveal that induction of DHAPAT can be largely explained through increases in DHAPAT mRNA. A comparison of microsomal and peroxisomal glycerolipid synthetic pathways, using D-[3-(3)H, U-(14)C]glucose as the precursor of the lipid glycerol backbone shows that about 40-50% of triglyceride is synthesized via the acyl-DHAP pathway. These results indicate that the acyl-DHAP pathway is important not only for the synthesis of ether lipids, but also for the synthesis of triacylglycerol and other non-ether glycerolipids.

Targeting and Subcellular Localization of Toxoplasma gondii Catalase: IDENTIFICATION OF PEROXISOMES IN AN APICOMPLEXAN PARASITE

We sought to identify and characterize peroxisomes in the apicomplexan parasite Toxoplasma gondii. To initiate this process, we first cloned and sequenced the gene for T. gondii catalase (EC 1. 11.1.6), a marker enzyme for peroxisomes in eukaryotic cells. The gene predicts a protein of 57.2 kDa and 502 amino acids and has a strong homology to other eukaryotic catalases. A polyclonal antiserum raised against a glutathione S-transferase fusion protein recognized a single band with a molecular mass of 63 kDa by immunoblot. By immunofluorescence T. gondii catalase is present primarily in a punctate staining pattern anterior to the parasite nucleus. This compartment is distinguishable from other parasite organelles, namely micronemes, rhoptries, dense granules, and the apicoplast. Cytochemical visualization of catalase using diaminobenzidine precipitation gives a vesicular staining pattern anterior to the nucleus at the light level and round, vesicular structures with an estimated diameter of 100-300 nm by electron microscopy. T. gondii catalase has a putative C-terminal peroxisomal targeting signal in the last 3 amino acids (-AKM). Expression of T. gondii catalase in mammalian cells results in peroxisomal localization, whereas a construct lacking the targeting signal remains in the cytosol. Furthermore, addition of -AKM to the C terminus of chloramphenicol acetyltransferase is sufficient to target this protein to peroxisomes. These results provide the first evidence for peroxisomes in Apicomplexan parasites.

Isolation and biochemical characterization of peroxisomes from cultured rat glial cells.

Peroxisomes are now recognized to play important cellular functions and its dysfunction leads to a group of neurological disorders. This study reports peroxisomal enzyme activities in cultured glial cells and peroxisomes isolated from cultured oligodendrocytes and C6 glial cells. Peroxisomal enzyme activities were found to be higher in oligodendroglial cells than in astrocytes or mixed glial cells. We also developed a method for the isolation of peroxisomes from glial cells by a combination of differential and density gradient centrifugation techniques. Peroxisomes from oligodendrocytes in nycodenz gradient were isolated at a density of 1.165 g/ml +/- 0.011. Activities of dihydroxyacetone phosphate acyl transferase, beta-oxidation of lignoceric acid and alpha-oxidation of phytanic acid were almost exclusively associated with the distribution of catalase activity (a marker enzyme for peroxisomes) in the gradient. This protocol should be a resource for studies designed to investigate the structure and function of peroxisomes in brain cells.

Immunocytochemical investigation of catalase and peroxisomal lipid β-oxidation enzymes in human hepatocellular tumors and liver cirrhosis

A significant reduction of catalase activity, a peroxisomal marker enzyme, occurs in human hepatic neoplasias, but no information is available on other peroxisomal proteins. We have studied by means of immunohistochemistry four specific proteins of peroxisomes (catalase and three enzymes of lipid beta-oxidation) in human hepatocellular tumors of various differentiation grades from adenoma to anaplastic carcinoma. In all tumors, except the adenomas, the tumor cells contained fewer peroxisomes than extrafocal hepatocytes and the reduction of antigenic sites in the tumor types generally correlated with the degree of tumor dedifferentiation as assessed by classical histopathological criteria. Two poorly differentiated tumors had no detectable peroxisomes at all. There were no major differences in the intensities of the immunocytochemical staining for all four studied peroxisomal antigens in different tumors, suggesting that the neoplastic transformation affects the biogenesis of the entire organelle and not merely the individual peroxisomal enzyme proteins. Some tumors exhibited a distinct peripheral distribution of peroxisomes. In cases with associated liver cirrhosis, the hepatocytes in the adjacent liver showed marked peroxisome proliferation, forming large perinuclear aggregates, occupying occasionally the entire cytoplasm. Taken together, our observations indicate that peroxisomes are significantly altered in both hepatocellular tumors and liver cirrhosis and, thus, could be responsible for some of the metabolic derangements observed in those disease processes.

Peroxisome-Proliferator Regulates Key Enzymes of the Tryptophan-NAD+ Pathway

Structually diverse peroxisome-proliferators (PPs) were investigated regarding their effects on NAD+ level and two key enzyme activities in the tryptophan (Trp)–NAD+ pathway in the liver of rats (Sprague–Dawley male) fed PP-containing diets freely for 2 weeks. All PPs, except for thyroxine, significantly increased hepatic NAD+ level in concert with hepatic hypertrophy. Activity of quinolinate phosphoribosyltransferase (QAPRTase), one of the key enzymes in the Trp–NAD+ pathway, was increased by the PPs which caused significant increase in the hepatic NAD+. On the other hand, α-amino-β-carboxymuconate-ϵ-semialdehyde decarboxylase (ACMSDase), another key enzyme in the Trp–NAD+ pathway, was drastically inhibited by all PPs except for linolenic acid, which was only slightly inhibitory. Most PPs investigated activated peroxisomal marker enzymes such as palmitoyl-CoA oxidase, catalase, and PPAR-α(peroxisome-proliferator activated receptor-α)-dependent enzymes, such as malic enzyme and l-3-glycerophosphate dehydrogenase. NAD+ was also increased in the rat hepatocytes cultured in the medium supplemented with PPs. These data suggested that regulation of the key enzymes in the Trp–NAD+ pathway was associated with PPAR-α directly or indirectly, and as a consequence the hepatic NAD+ was increased by PPs.

Structure of peroxisomes and activity of the marker enzyme catalase in digestive epithelial cells in relation to PAH content of mussels from two Basque estuaries (Bay of Biscay): seasonal and site-specific variations.

The aim of the present work was to study the seasonal as well as the site-specific variations in the structure of peroxisomes and in the activity of the peroxisomal marker enzyme catalase in digestive epithelial cells of mussels to validate the potential use of these parameters as early biomarkers of environmental organic pollution in estuarine ecosystems. For this purpose, mussels were sampled monthly for 14 months in two Basque estuaries (Bay of Biscay) with different degrees of pollution. Stereological procedures were applied to detect changes in peroxisome structure, and microspectrophotometry was used to quantify changes in catalase activity. The animals from the two studied sampling sites presented differences in polycyclic aromatic hydrocarbon (PAH) burdens, mussels from Plentzia generally showing lower total PAH contents than mussels from Galea. The peroxisome structure of the animals from the two estuaries suffered seasonal variations that were of different kind and intensity in both sites. In this way, a strong peroxisome proliferatory response was found in mussels sampled in Plentzia during the summer months, while mussels from Galea presented few variations along the year. Catalase activity behaved similarly in the animals sampled in the two estuaries, with higher values in spring. It appeared that mussels exposed chronically to PAHs and other pollutants, such as those from Galea, lost their ability to respond to this exposure in terms of peroxisome proliferation. In contrast, mussels collected in Plentzia effectively responded to an increased bioavailability of organic pollutants during the summer by increasing peroxisome volume and surface and numerical densities in digestive epithelial cells. However, these increases were transient because elevated PAH body burdens detected in mussels sampled in Plentzia in autumn were not accompanied by a peroxisome proliferatory response. Further studies are needed before changes in peroxisomal structure and in the activity of catalase could be used as early biomarkers to assess environmental quality in pollution monitoring programs like the Mussel Watch.

Effects of peroxisome-proliferators on the TRP-NAD pathway.

The mechanism of the elevation of hepatic NAD level in the rats administered clofibrate and other peroxisome-proliferators were investigated. In the hepatocytes from the clofibrate-fed rats, NAD biosynthesis from Trp, but not from nicotinic acid, was specifically stimulated. The activities of peroxisomal marker enzymes, the peroxisome-proliferator activated receptors (PPAR)-dependent enzymes and key enzymes in the Trp-NAD pathway changed in parallel with the hepatic NAD increase; the activity of quinolinate phosphori-bosyltransferase (QPRT) was increased whereas that of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) was drastically reduced. The results strongly suggest that the hepatic NAD increase might be caused by transcription of genes coding the key enzymes of the Trp-NAD pathway via PPAR.

Effects of di-isononyl phthalate (DINP) on peroxisomal markers in the marmoset-DINP is not a peroxisome proliferator.

In the present study the systemic toxic potential of di-isononyl phthalate (DINP) was assessed in a 13-week study in marmosets. Particular attention was given to its potential for hepatic peroxisome proliferation. Three groups of four male and four female marmosets received DINP, by oral gavage administration, at dosages of 100, 500 or 2500 mg/kg/day for 13 weeks. A fourth group served as a concurrent Control group and received the vehicle (1% methylcellulose and 0.5% Tween) only. A fifth group received clofibrate at a dosage of 500 mg/kg/day to provide a positive Control for liver peroxisome activity. At the end of the treatment period, the animals were killed and their livers were removed. 3000 x g supernatant and microsomal subcellular fractions were prepared from homogenised liver by differential centrifugation. The peroxisomal marker enzyme activity, cyanide-insensitive palmitoyl CoA oxidase, was assayed in the former, while cytochrome P450 concentration and lauric acid 11- and 12-hydroxylase activities (selective for CYP2E1 and 4A, respectively) were assayed in the microsomes. No statistically significant changes were seen in any of these parameters measured following DINP treatment, compared with the Control. Clofibrate treatment resulted in an approximately 100% increase (p < 0.01) in both male and female marmoset cyanide-insensitive palmitoyl CoA oxidase activity and a similar increase (p < 0.05) in male (only) lauric acid 11-hydroxylase activity. No other changes were statistically significant at the 5% level. These data provide no evidence that DINP was acting as a peroxisome proliferator when administered to marmosets under the conditions of the study.

Peroxisomal localization of d-aspartate oxidase and development of peroxisomes in the yeast Cryptococcus humicolus UJ1 grown on d-aspartate

The peroxisomal localization of d-aspartate oxidase (EC. 1.4.3.1) was demonstrated in the yeast Cryptococcus humicolus UJ1 cells grown in the medium containing d-aspartate as a nitrogen source. The conclusion is based on the identical behavior of the enzyme with those of peroxisomal marker enzymes, catalase and urate oxidase, during all steps of subcellular fractionations. Supporting evidence was provided by the morphometric analysis of the peroxisomes with electron microscopy, showing that the cells grown on d-aspartate contained more and larger peroxisomes than those grown on l-aspartate, consistent with the 500-fold and 3-fold higher contents of d-aspartate oxidase and catalase activities, respectively, in the former cells than the latter.

Copper–zinc superoxide dismutase is a constituent enzyme of the matrix of peroxisomes in the cotyledons of oilseed plants

The number and type of isoforms of superoxide dismutase (SOD) and their activities were compared in mitochondria and peroxisomes isolated from cotyledons of three different oilseed seedlings. Mitochondrial and peroxisomal isoforms of SOD could be distinguished in nondenaturing polyacrylamide gels by their differential sensitivities to KCN and/or H2 O2 . The type of SOD was not the same for each organelle in each of the three oilseed species. For example, a single Mn-SOD was found in cotton and cucumber mitochondria, whereas four CuZn-SODs were present in mitochondria from sunflower. At least one CuZn-SOD isoform was found in the peroxisomes of all three species. Cucumber peroxisomes contained both a CuZn-SOD and a Mn-SOD, cotton peroxisomes contained a single CuZn-SOD, whilst four separate CuZn-SODs, but no Mn-SOD were found in sunflower peroxisomes. Using antibodies against CuZn-SOD from watermelon peroxisomes or from chloroplasts of Equisetum, a single polypeptide of c. 16·5 kDa was detected on immunoblots of peroxisomal fractions from the three species. Post-embedment, electron-microscopic double immunogold-labelling showed that CuZn-SOD, with malate synthase used as marker enzyme of peroxisomes, was localized in the matrix of these organelles of all three species. These results suggest that CuZn-SOD is a characteristic matrix enzyme of peroxisomes in oilseed cotyledons.