Alfred Völkl

Reactive oxygen species and peroxisomes: struggling for balance.

Reactive oxygen species (ROS) can surely be considered as multifunctional biofactors within the cell. They are known to participate in regular cell functions, for example, as signal mediators, but overproduction under oxidative stress conditions leads to deleterious cellular effects, cell death and diverse pathological conditions. Peroxisomal function has long been linked to oxygen metabolism due to the high concentration of H2O2‐generating oxidases in peroxisomes and their set of antioxidant enzymes, especially catalase. Still, mitochondria have been very much placed in the centre of ROS metabolism and oxidative stress. This review discusses novel findings concerning the relationship between ROS and peroxisomes, as they revealed to be a key player in the dynamic spin of ROS metabolism and oxidative injury. An overview of ROS generating enzymes as well as their antioxidant counterparts will be given, exemplifying the precise fine‐tuning between the opposing systems. Various conditions in which the balance between generation and scavenging of ROS in peroxisomes is perturbed, for example, exogenous manipulation, ageing and peroxisomal disorders, are addressed. Furthermore, peroxisome‐derived oxidative stress and its effect on mitochondria (and vice versa) are discussed, highlighting the close interrelationship of both organelles.

Effects of 17a-ethinylestradiol on the expression of three estrogen-responsive genes and cellular ultrastructure of liver and testes in male zebrafish

In order to monitor the influence of estrogenic compounds on the reproductive physiology of fish, molecular markers for zebrafish vitellogenin, estrogen receptor and ZP2 were developed. For this purpose, sequence information about the zebrafish estrogen receptor and vitellogenin had to be obtained. By means of RT-PCR, a sequence fragment of the zebrafish estrogen receptor alpha was cloned and sequenced. Continuous cDNAs of two zebrafish vitellogenin-like gene products (zfvg1 and zfvg3) were constructed by the help of expressed sequence tags of zebrafish and completely sequenced. The sequences of the estrogen receptor and of the vitellogenins showed significant similarities to corresponding cDNAs of other fish species. Expression of these gene products was measured following exposure to 17alpha-ethinylestradiol and compared with histological endpoints. RT-PCR was used as a semiquantitative technique to record gene expression in adult male zebrafish, which were exposed to 17alpha-ethinylestradiol in time-and dose-response experiments. As for time-dependent expression, all hepatic genes investigated were expressed at considerable amounts from 24 h after onset of exposure to 50 ng/l 17alpha-ethinylestradiol to the end of experiment (17 days). In testes, expression of the estrogen receptor- as well as ZP2-mRNA remained unchanged for the entire experiment, except for the individuals exposed for 17 days, which displayed elevated expression levels of ZP2. In the dose-response experiment, male zebrafish were exposed to 17alpha-ethinylestradiol in concentrations from 0.25-85 ng/l for 4 and 21 days. LOECs for vitellogenin as well as estrogen receptor alpha expression were found to be 2.5 ng/l already after 4 d of exposure. Extension of the exposure time to 21 days resulted in enhanced transcription of vitellogenin-mRNAs at 2.5 ng/l 17alpha-ethinylestradiol, whereas the detection limit could not be lowered. In contrast, in testes no induction of both ZP2 as well as estrogen receptor expression was detected at any concentration tested. To examine estrogen-caused alterations at the ultrastructural level, liver and testes of males exposed to 25 ng/l 17alpha-ethinylestradiol were analysed. Male livers responded with a feminisation reflected by the proliferation of rough endoplasmatic reticulum and Golgi apparatus typical of female hepatocytes during vitellogenesis. However, in testes no signs of feminisation were detectable; rather, destructive phenomena like phagocytosis of sperm cells by Sertoli cells were observed. Thus, in sexually differentiated males no reorganisation of the gonadal tissue towards an ovary could be definitely detected at any level investigated.

Rat liver peroxisomes after fibrate treatment: A survey using quantitative mass spectrometry

Fibrates are known to induce peroxisome proliferation and the expression of peroxisomal β-oxidation enzymes. To analyze fibrate-induced changes of complex metabolic networks, we have compared the proteome of rat liver peroxisomes from control and bezafibrate-treated rats. Highly purified peroxisomes were subfractionated, and the proteins of the matrix, peripheral, and integral membrane subfractions thus obtained were analyzed by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry after labeling of tryptic peptides with the iTRAQ reagent. By means of this quantitative technique, we were able to identify 134 individual proteins, covering most of the known peroxisomal proteome. Ten predicted new open reading frames were verified by cDNA cloning, and seven of them could be localized to peroxisomes by immunocytochemistry. Moreover, quantitative mass spectrometry substantiated the induction of most of the known peroxisome proliferator-activated receptor α-regulated peroxisomal proteins upon treatment with bezafibrate, documenting the suitability of the iTRAQ procedure in larger scale experiments. However, not all proteins reacted to a similar extent but exerted a fibrate-specific induction scheme showing the variability of peroxisome proliferator-activated receptorα-transmitted responses to specific ligands. In view of our data, rat hepatic peroxisomes are apparently not specialized to sequester very long chain fatty acids (C22–C26) but rather metabolize preferentially long chain fatty acids (C16–18).

TNF-α downregulates the peroxisome proliferator activated receptor-α and the mRNAs encoding peroxisomal proteins in rat liver

We have studied the effects of TNF‐α on the mRNAs coding for the peroxisome proliferator activated receptor α (PPAR‐α), and for catalase (Cat), acyl‐CoA oxidase (AOX), multifunctional enzyme (PH), and β‐actin in rat liver. Total RNA was isolated from livers of male SD‐rats 16 h after administration of a single dose of 25 μg TNF‐α and mRNAs were analyzed by a novel dot blot RNase protection assay. The mRNAs for PPAR‐α and for Cat, AOX and PH were significantly reduced by TNF‐treatment. In addition, the level of PPAR‐α protein was also decreased after TNF. In contrast, the mRNA for β‐actin was markedly increased implying that the effect of TNF on PPAR‐α and the peroxisomal mRNAs is highly selective. This effect may have important implications in perturbation of the lipid metabolism induced by TNF‐α.

L-Lactate Dehydrogenase A- and AB Isoforms Are Bona Fide Peroxisomal Enzymes in Rat Liver EVIDENCE FOR INVOLVEMENT IN INTRAPEROXISOMAL NADH REOXIDATION

The subcellular localization of L-lactate dehydrogenase (LDH) in rat hepatocytes has been studied by analytical subcellular fractionation combined with the immunodetection of LDH in isolated subcellular fractions and liver sections by immunoblotting and immunoelectron microscopy. The results clearly demonstrate the presence of LDH in the matrix of peroxisomes in addition to the cytosol. Both cytosolic and peroxisomal LDH subunits have the same molecular mass (35.0 kDa) and show comparable cross-reactivity with an anti-cytosolic LDH antibody. As revealed by activity staining or immunoblotting after isoelectric focussing, both intracellular compartments contain the same liver-specific LDH-isoforms (LDH-A > LDH-AB) with the peroxisomes comprising relatively more LDH-AB than the cytosol. Selective KCl extraction as well as resistance to proteinase K and immunoelectron microscopy revealed that at least 80% of the LDH activity measured in highly purified peroxisomal fractions is due to LDH as a bona fide peroxisomal matrix enzyme. In combination with the data of cell fractionation, this implies that at least 0.5% of the total LDH activity in hepatocytes is present in peroxisomes. Since no other enzymes of the glycolytic pathway (such as phosphoglucomutase, phosphoglucoisomerase, and glyceraldehyde-3-phosphate dehydrogenase) were found in highly purified peroxisomal fractions, it does not seem that LDH in peroxisomes participates in glycolysis. Instead, the marked elevation of LDH in peroxisomes of rats treated with the hypolipidemic drug bezafibrate, concomitantly to the induction of the peroxisomal β-oxidation enzymes, strongly suggests that intraperoxisomal LDH may be involved in the reoxidation of NADH generated by the β-oxidation pathway. The interaction of LDH and the peroxisomal palmitoyl-CoA β-oxidation system could be verified in a modified β-oxidation assay by adding increasing amounts of pyruvate to the standard assay mixture and recording the change of NADH production rates. A dose-dependent decrease of NADH produced was simulated with the lowest NADH value found at maximal LDH activity. The addition of oxamic acid, a specific inhibitor of LDH, to the system or inhibition of LDH by high pyruvate levels (up to 20 mM) restored the NADH values to control levels. A direct effect of pyruvate on palmitoyl-CoA oxidase and enoyl-CoA hydratase was excluded by measuring those enzymes individually in separate assays. An LDH-based shuttle across the peroxisomal membrane should provide an efficient system to regulate intraperoxisomal NAD/NADH levels and maintain the flux of fatty acids through the peroxisomal β-oxidation spiral.

Induction of biotransformation in the liver of eel (Anguilla anguilla L.) by sublethal exposure to dinitro-o-cresol: An ultrastructural and biochemical study

Structural and functional alterations in hepatocytes of the European eel, Anguilla anguilla, following a 4-week-exposure to 5, 50, and 250 micrograms/liter dinitro-o-cresol (DNOC) were investigated by means of electron microscopy and biochemistry and compared to liver pathology in eels exposed to the chemical spill into the Rhine river at Basle in November 1986. Whereas phenological parameters (growth, condition factor) are unaffected, ultrastructural and biochemical alterations are detectable at greater than or equal to 50 and 5 micrograms/liter DNOC, respectively. Structural modifications include: rounding-up of the nuclei; fractionation and reduction of the rough endoplasmic reticulum; proliferation of the smooth endoplasmic reticulum (SER), mitochondria, peroxisomes, and lysosomes; bundles of rod-shaped SER profiles; annulate lamellae; membrane whorls within mitochondria; crystallization of the peroxisomal matrix and glycogen bodies; glycogen depletion and lipid augmentation. Structural changes can be correlated to an increase in hepatic lipid and protein contents as well as stimulation of mitochondrial (cytochrome c oxidase), peroxisomal (catalase, allantoinase, uricase), lysosomal (arylsulfatase), and microsomal (esterase) enzymes. An increase in NADPH-cytochrome c reductase and cytochrome P450 as well as UDP-glucuronyltransferase and arylsulfotransferase activities in the microsomal fraction document an induction of hepatic biotransformation as a functional correlate to SER proliferation. Maximum inducibility of biotransformation enzymes at 50 micrograms/liter indicates a biphasic, concentration-dependent reaction of eel liver. Comparison of DNOC-induced effects with liver pathology in eel exposed to the chemical spill in 1986 reveals striking similarities so that DNOC may not be excluded as a possible factor in the fish kill in the Rhine river.

Measurement of vitellogenin-mRNA expression in primary cultures of rainbow trout hepatocytes in a non-radioactive dot blot/RNAse protection-assay

The induction of vitellogenin synthesis both in vivo and in vitro has proven to be a reliable biomarker for assessing the estrogenic activity of individual substances and the more complex effluents of sewage treatment plants. However, due to the requirement of radioactively labelled nucleotides, the measurement of vitellogenin-mRNA has not been widely used in routine testing--even though this technique promises elevated sensitivity. In order to develop a practicable, reliable and cost-effective bioassay suitable for routine testing, a combined dot-blot/RNAse protection assay, utilising digoxigenin-labelled cRNA transcripts of plasmid psg5Vg1.1 was used for the quantification of vitellogenin-mRNA in isolated rainbow trout (Oncorhynchus mykiss) hepatocytes. By re-cloning the Vg1.1 insert into a pGemZf7(-)-vector, the sense-transcript of Vg1.1 was utilized as a standard for the quantification of vitellogenin-mRNA concentrations. Male rainbow trout hepatocytes were cultured as monolayers in pure M199 medium. The addition of serum supplements did not result in increased expression of vitellogenin-mRNA following 17 beta-estradiol administration. This indicates that for this assay no supplementation of the culture medium is necessary. After addition of 17 beta-estradiol, hepatocytes exhibited an exponential time-dependent expression of vitellogenin-mRNA over a period of 144 h. The dot blot system was sufficiently sensitive to detect vitellogenin-mRNA following addition of 1 microM 17 beta-estradiol after 6 h of incubation. However, the amount of vitellogenin-mRNA expressed was found to be a function of both incubation time and inducer concentration. Prolonged incubation times were therefore required to enhance the sensitivity of the system. After a 96-h incubation, detection limits for 17 beta-estradiol were between 100 pM and 1 nM. Vitellogenin-mRNA could not be detected in untreated hepatocytes. The vitellogenin-mRNA dot blot/RNAse protection assay was further used as a tool for assessing the estrogenic potential of the xenoestrogens nonylphenol and bisphenol A, which exhibited estrogenic activities approximately 2000-fold less than the natural inducer 17 beta-estradiol. The vitellogenin-mRNA response to 17 alpha-ethinylestradiol reached maximum efficacy down to the lowest tested concentration of 10(-9) M. The assay also successfully identified estrogenic activity in selected waste water samples.

Hitchhiking of Cu/Zn Superoxide Dismutase to Peroxisomes – Evidence for a Natural Piggyback Import Mechanism in Mammals

Most newly synthesized peroxisomal proteins are imported in a receptor‐mediated fashion, depending on the interaction of a peroxisomal targeting signal (PTS) with its cognate targeting receptor Pex5 or Pex7 located in the cytoplasm. Apart from this classic mechanism, heterologous protein complexes that have been proposed more than a decade ago are also to be imported into peroxisomes. However, it remains still unclear if this so‐called piggyback import is of physiological relevance in mammals. Here, we show that Cu/Zn superoxide dismutase 1 (SOD1), an enzyme without an endogenous PTS, is targeted to peroxisomes using its physiological interaction partner ‘copper chaperone of SOD1’ (CCS) as a shuttle. Both proteins have been identified as peroxisomal constituents by 2D‐liquid chromatography mass spectrometry of isolated rat liver peroxisomes. Yet, while a major fraction of CCS was imported into peroxisomes in a PTS1‐dependent fashion in CHO cells, overexpressed SOD1 remained in the cytoplasm. However, increasing the concentrations of both CCS and SOD1 led to an enrichment of SOD1 in peroxisomes. In contrast, CCS‐mediated SOD1 import into peroxisomes was abolished by deletion of the SOD domain of CCS, which is required for heterodimer formation. SOD1/CCS co‐import is the first demonstration of a physiologically relevant piggyback import into mammalian peroxisomes.

Ultrastructure of hepatocytes in golden ide (Leuciscus idus melanotus L.; Cyprinidae: Teleostei) during thermal adaptation

SummaryThe morphological alterations of hepatocytes of golden ide, Leuciscus idus melanotus, following adaptation to low and high temperatures (14 and 28°C) were investigated by means of light and electron microscopy. The temperature-dependent behaviour of peroxisomes was visualized cytochemically with the alkaline diaminobenzidine medium; the morphological studies were supplemented by the biochemical determination of catalase activity.Cold adaptation of ide hepatocytes is manifested by proliferation and stacking of endoplasmic reticulum, an enhanced secretory activity of Golgi fields and a higher number of peroxisomes as compared with the warmadapted animals. The latter organelles are characterized by a marked heterogeneity in size, shape and catalase activity, and by a more intimate association with mitochondria and endoplasmic reticulum. The occurrence of small peroxisomal profiles is restricted to lower temperature. Catalase activity can be shown both cytochemically and biochemically to increase during cold adaptation.Whereas the number of mitochondria seems to be unaffected by thermal adaptation, stacking of mitochondria as well as the formation of intramitochondrial membrane piles indicate cold-adaptive processes.A feature typical of warm-adaptation is the formation of membrane-glycogen complexes, which may represent the morphological expression of enhanced carbohydrate metabolism documented in a decreased storage of glycogen at 28°C. At 28°C lipid is the predominant storage product.These findings indicate that fish liver is well-suited to serve as a model for the analysis of the interaction of environmental temperature conditions and hepatic morphology.

Peroxisomes and reactive oxygen species, a lasting challenge.

Oxidases generating and enzymes scavenging H2O2 predestine peroxisomes (PO) to a pivotal organelle in oxygen metabolism. Catalase, the classical marker enzyme of PO, exhibits both catalatic and peroxidatic activity. The latter is responsible for the staining with 3,3′-diamino-benzidine, which greatly facilitated the visualization of the organelle and promoted further studies on PO. d-Amino acid oxidase catalyzes with strict stereospecificity the oxidative deamination of d-amino acids. The oxidase is significantly more active in the kidney than in liver and more in periportal than pericentral rat hepatocytes. Peroxisomes in these tissues differ in their enzyme activity and protein concentration not only in adjacent cells but even within the same one. Moreover, the enzyme appears preferentially concentrated in the central region of the peroxisomal matrix compartment. Urate oxidase, a cuproprotein catalyzing the oxidation of urate to allantoin, is confined to the peroxisomal core, yet is lacking in human PO. Recent experiments revealed that cores in rat hepatocytes appear in close association with the peroxisomal membrane releasing H2O2 generated by urate oxidase to the surrounding cytoplasma. Xanthine oxidase is exclusively located to cores, oxidizes xanthine thereby generating H2O2 and O2– radicals. The latter are converted to O2 and H2O2 by CuZn superoxide dismutase, which has been shown recently to be a bona fide peroxisomal protein.