Peroxisome proliferation is a phenomenon occurring when responsive animals are exposed to certain compounds so-called peroxisome proliferators and is regulated through a nuclear receptor named ...peroxisome proliferator-activated receptor (PPAR). PPAR family members exhibit a strong binding affinity for both saturated and unsaturated fatty acids. Activators of PPARα include a variety of endogenously present fatty acids, leukotrienes and hydroxyeicosatetraenoic acids (HETEs) and clinically used drugs, such as fibrates. PPARβ activators include fatty acids, prostaglandin A
2 (PGA
2) and prostacyclin (PGI
2). PPARγ is the most selective receptor and, among others, 15-deoxy-Δ
12,14 prostaglandin J
2 (PGJ
2) has been described to be a PPARγ-specific ligand. The aim of the present study was to determine if known PPARα and PPARγ ligands were able to alter the expression of these subtypes in an in vitro model of zebrafish primary hepatocyte culture. With this purpose, a PPARα specific ligand (8S-HETE), a PPARγ specific ligand (PGJ
2) and a peroxisome proliferator of the fibrate class (clofibrate) were selected. In addition, the female hormone 17β-estradiol was also used as it is known to interact with PPARs. After cell exposure for 24 h, cells were immunohistochemically stained for both PPARs and immunolabeling was quantified as percentage of positive nuclei and cells. Levels of expression of PPARs were also measured by image analysis as grey level per cell. Expression was induced for both PPARα and PPARγ by clofibrate (at 0.5 mM for PPARα and at 1 and 2 mM for PPARγ), by HETE (1 μM), and by PGJ
2 (0.3 and 1 μM for PPARα and 0.3 μM for PPARγ). Expression of PPARγ was also induced at 10 μM by 17β-estradiol. The percentage of PPARα positive nuclei increased significantly at 1 μM HETE and the percentage of PPARγ positive cells decreased at 10 μM 17β-estradiol. As a conclusion, clofibrate, HETE and PGJ
2 are able to induce expression of both PPARα and PPARγ in zebrafish primary hepatocyte cultures. Further studies are needed to identify how the expression of different PPAR subtypes is regulated and to elucidate the implication of PPAR subtypes in zebrafish cell functions.
Mussels Mytilus galloprovincialis were collected monthly in 2 localities in the Northern Iberian Peninsula, Oia (Galicia) and Mundaka (Basque Coast), from April 2005 to May 2006, to investigate ...whether seasonal variability in biomarkers follows the same pattern at both geographical areas. The following battery of biomarkers was analysed: lysosomal membrane stability and structural changes, intracellular neutral lipid accumulation, cell type composition in the digestive gland epithelium, structural changes of digestive alveoli and digestive gland histopathology. Additionally, gonad index was measured as a supporting parameter. Overall, results demonstrate that most studied biomarkers showed similar seasonal variability pattern in both localities. Lysosomal membrane stability (labilisation period LP) and most histopathological conditions showed similar seasonal variability patterns in both localities, although values of LP and prevalences of some parasites were higher in Mundaka. Data provide baseline values to be used as reference values for the biomonitoring programmes carried out in coastal areas from Galicia to the Basque Coast. It is concluded that for monitoring programmes based on biomarkers, samples must be taken at least twice per year (spring and autumn) to avoid the effect on biomarkers of the late stages of each reproductive cycle.
The aim of the present work was to study the effects of the peroxisome proliferator dibutylphthalate (DBP) and the xenoestrogen 17α‐ethynylestradiol (EE2) on liver peroxisomes, reproduction, and ...development of zebrafish (Danio rerio). In experiment 1, newly fertilized zebrafish eggs were exposed for five weeks, covering the entire period of sexual determination, to nominal concentrations of 25 and 100 μg/L of DBP and 5 μg/L of EE2. In experiment 2, adult female zebrafish were exposed for 15 d to 100 and 500 μg/L of DBP and 5 μg/L of EE2, and afterward, they were paired with untreated males to study the effects in the resultant offspring. Ethynylestradiol provoked marked mortality (∼50%) and delayed development of larvae as well as sterility of adult females, possibly related to alterations in aromatase gene expression. Ethynylestradiol up‐regulated vitellogenin expression in the early life stages and increased vitellogenin synthesis and accumulation in adult females. Ethynylestradiol caused liver peroxisome proliferation in early life stages but not in adult females. Dibutylphthalate caused teratogenic effects in early life stages and mortality of the larvae obtained from exposed females. Dibutylphthalate provoked liver peroxisome proliferation and up‐regulation of cytochrome P450A1 in early life stages at the end of the exposure and in adult females. Dibutylphthalate also up‐regulated the expression of aromatase genes. In conclusion, the xenoestrogen EE2 caused liver peroxisome proliferation in early life stages of zebrafish, but the peroxisome proliferator DBP did not behave as a typical xenoestrogen. Overall, changes in gene expression were more marked during early life stages than in adult female zebrafish.
The epithelium of the digestive tubules of the mussel Mytilus galloprovincialis is comprised of two cell types, namely digestive and basophilic cells. In basophilic cells, the secretory granules are ...beta-glucuronidase immunoreactive, a fact that enhances the hypothesis that beta-glucuronidase is synthesized in basophilic cells. A novel observation at the ultrastructural level is the pinocytic activity associated with the formation of coated pits. This observation constitutes direct evidence for endocytic processes taking place in basophilic cells. The use of cryostat sections from the same digestive tubules reveals, in many instances, a very pronounced neutral lipid accumulation in the same structures giving a positive reaction for N-acetyl-beta-hexosaminidase, indicating the association of those lipids with lysosomes. In some mussels, a high content of lipofuscin was observed in the lysosomes of the digestive cells. In these cases, the lysosomal structures show a limited neutral lipid content, and a weaker N-acetyl-beta-hexosaminidase reaction. In the digestive cells, the carbohydrate content of the lysosomes, and very well-developed canal system in the apical part of cells are discussed in relation to their function.
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.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Haemocytes play an essential role in the internal defence of molluscs. It has been reported that organic xenobiotics commonly found as pollutants in the marine environment impair defence capabilities ...of haemocytes. The purpose of the present study was to investigate the effects of benzo(a)pyrene B(a)P on the integrity of the actin cytoskeleton and on endocytosis in haemocytes and to see if these effects are related to generation of reactive oxygen species. Haemocytes were exposed in vitro to B(a)P (0.5–40 μg/ml) for 1 h. Cell viability (using 2,3-bis2-methoxy-4-nitro-5-sulfophenyl-2H-tetrazolium-5-carboxanilide or XTT assay) indicated that selected doses were sublethal. Uptake of neutral red was significantly decreased in a dose-dependent manner in B(a)P-treated haemocytes. Distribution of actin filaments, labeled with rhodamine-conjugated phalloidin, was altered in haemocytes treated with 20 or 40 μg/ml B(a)P. These effects could be related to an increased production of superoxide anion during B(a)P metabolism, as detected by the nitroblue tetrazolium (NBT) reduction assay in haemocytes treated with ⩾10 μg/ml B(a)P.
Variations in structure and function of peroxisomes in digestive tissues of marine mussels have been proposed to be valid biomarkers of environmental contamination by organic xenobiotics. The aim of ...the present work was to study the seasonal variations in peroxisomal enzyme activities, catalase, palmitoyl-CoA oxidase (AOX) and
d-amino acid oxidase (DAOX) and peroxisomal structure in mussels. Peroxisomal changes were related to the seasonal variation in the contents of neutral lipids in the digestive gland as the lipid metabolism in mussels is subjected to seasonal variations linked to the reproductive cycle and food intake. Significant higher catalase activities were recorded from April to June when compared to the rest of the year and AOX activity was also markedly induced during the late winter and spring (February to May) with maximal activities in April. DAOX activity did not vary seasonally, the highest activities being measured in November, February and May. Stereological studies of peroxisomes in digestive tubule cells revealed significantly higher volume, surface and numerical densities in animals collected in spring and summer. Changes in peroxisomal volume density were found to be significantly and positively correlated with changes in AOX and catalase activities. The peroxisomal structure and enzyme activities were negatively correlated with the lipid contents of digestive tubules. In April, the volume density occupied by neutral lipids was higher in duct epithelia while previously it was higher in digestive tubule epithelia. This trend was maintained during the period in which peroxisomes were more abundant until July. In the following September, digestive tubules recovered their lipid load. It is concluded that seasonal changes related to food intake and reproductive cycle induce changes in peroxisomal parameters that can be compared to typical peroxisome proliferation, with a 25-fold increase in AOX activity and an 8-fold increase in the peroxisomal numerical density in early spring.
Peroxisome proliferation has been proposed as novel biomarker of exposure to organic pollutants in aquatic organisms. Peroxisome proliferator compounds comprise a heterogeneous group of substances ...known for their ability to cause massive proliferation of peroxisomes and liver carcinogenesis in sensitive species such as rodents. Recently, several marine organisms (mussels and fish) have been shown as target species of peroxisome proliferators. In the present work, we aimed to investigate the specificity of the peroxisome proliferation response in mussels. For this purpose, mussels (
Mytilus edulis) were exposed for three weeks to North Sea crude oil (NSO), a mixture of NSO, alkylphenols and extra PAHs (MIX), diallylphthalate (DAP), bisphenol-A (BPA) and tetrabromodiphenylether (TBDE), or transplanted for three weeks to four stations showing different copper concentrations in a copper mine. Peroxisome proliferation was assessed by measuring the activity of the peroxisomal β-oxidation enzyme acyl-CoA oxidase (AOX) and the volume density occupied by peroxisomes (
V
VP) in the digestive gland. Mussels exposed to NSO and MIX showed significantly increased AOX activities and
V
VP compared to control animals. Significantly higher
V
VP was also found in DAP and TBDE exposed mussels.
V
VP did not vary in mussels transplanted into a copper concentration gradient. Our results confirm the usefulness and specificity of peroxisome proliferation as a suitable biomarker of exposure to organic contaminants such as oil derived hydrocarbons, phthalate plasticizers and polybrominated flame retardants in mussels.
The aim of this work was to determine the immunolocalization of the antioxidant enzymes catalase, Cu,Zn-superoxide dismutase (SOD), Mn-SOD, and glutathione peroxidase (GPX) in the bivalve mollusks ...Mytilus galloprovincialis and Crassostrea sp., the crab Carcinus maenas, and the teleostean fish Mugil cephalus. By immunoblotting, crossreactivity between antibodies and the corresponding proteins in the digestive gland/hepatopancreas of invertebrates and the fish liver was demonstrated. Immunohistochemical studies showed that the stomach epithelium was strongly immunostained for catalase in mollusks. In crabs, ducts showed stronger immunostaining than tubules and in mullet hepatocytes the reaction appeared in discrete granules corresponding to peroxisomes. With regard to Cu,Zn-SOD, the apex of the tubule cells in mussels and crabs was distinctly immunostained, whereas in oysters the reaction was more marked in ducts and in mullet liver a uniform diffuse cytoplasmic staining was found. Mn-SOD was strongly positive in mollusk and crab ducts and in mullet periportal hepatocytes. Finally, GPX was not detected in mussels while in oysters a slight reaction was noted in all cell types. In crabs, connective tissue cells and the apex of duct cells were immunostained, but in mullet liver only erythrocytes appeared reactive. Immunoelectron microscopy revealed that catalase was localized in peroxisomes with a dense labeling in fish and less intense labeling in invertebrates. Cu,Zn-SOD was mainly a cytosolic protein although additional positive subcellular sites (peroxisomes, nuclei) were also observed, while Mn-SOD was restricted to mitochondria. GPX was localized in the cytosol, nucleus, and lysosomes, occurring also in peroxisomes of the fish liver. The results presented here provide a basis for future application of the immunodetection techniques to study the possible differential induction of antioxidant enzymes in aquatic organisms subjected to oxidative stress as a result of exposure to environmental pollutants.
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