Expression of peroxisome proliferator-activated receptors in zebrafish (Danio rerio) depending on gender and developmental stage.

Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors involved in embryo development and differentiation of several tissues in mammals. The aim of the present study was to investigate the possible differential expression of the three PPAR subtypes (PPARalpha, PPARbeta, and PPARgamma) in relation to gender and developmental stage in zebrafish. For this purpose PPAR expression was assessed by immunohistochemistry in 7-day-old larvae, 1-month-old juveniles, and 1-year-old adults. Additionally, the activity of peroxisomal acyl-CoA oxidase (AOX), a gene regulated by PPARs, and the volume density of catalase-immunolabeled liver peroxisomes (V(VP)) was examined. No significant gender-related differences were detected in the tissue distribution of the three PPAR subtypes or in peroxisomal AOX activity and V(VP). The percentage of PPARbeta-positive hepatocytes was significantly higher in females than in males suggesting a specific regulatory role of this subtype in female zebrafish. The three PPAR subtypes were already expressed at the larval stage, with a similar tissue distribution pattern to that found in adults. For all stages, PPARalpha and PPARgamma were expressed at higher levels than PPARbeta, and PPARbeta immunolabeling was stronger in juveniles than in larval or adult stages. The percentages of hepatocyte nuclei immunolabeled for PPARs was higher in early developmental stages than in adults, similarly to AOX activity and V(VP). In conclusion, our results indicate that PPAR expression, the activity of its target gene AOX, and peroxisomal biogenesis are developmentally modulated in zebrafish.

Expression of peroxisome proliferator-activated receptors in the liver of gray mullet (Mugil cephalus).

During the last decade, peroxisome proliferation has emerged as a novel biomarker of exposure to certain organic chemical pollutants in aquatic organisms. Peroxisome proliferation is mediated by nuclear receptors, peroxisome proliferator-activated receptors (PPARs). Three PPAR subtypes have been described in mammals: PPAR alpha, PPAR beta and PPAR gamma. PPARs have also been discovered in several fish species. The aim of the present study was to investigate the expression of PPAR subtypes and their cellular distribution patterns in the liver of gray mullet Mugil cephalus, a fish species widely distributed in estuaries and coastal areas in Europe and used as sentinel of environmental pollution. For this purpose, antibodies were generated against the three subtypes of mouse PPARs and different protocols of antigen retrieval were used. In western blots, main bands were detected of approximately 44 kDa for PPAR alpha, two bands of 44 and 58 kDa for PPAR beta and a single band of 56 kDa for PPAR gamma. Similar results were obtained in mouse liver and may indicate antibody recognition of two forms of the protein in certain cases. PPAR alpha was the subtype most markedly expressed in gray mullet liver, being expressed mainly in melanomacrophages, nuclei of hepatocytes and sinusoidal cells and connective tissue surrounding bile ducts. PPAR beta was expressed in the same cell types but immunolabeling was generally weaker than for PPAR alpha. PPAR gamma showed very weak expression; positivity was mainly found in melanomacrophages and connective tissue surrounding bile ducts. Our results demonstrate that all the three PPAR subtypes are expressed in gray mullet liver but in different intensities. The cellular distribution patterns of PPAR subtypes in gray mullet liver resembled partly those found in mouse liver with PPAR alpha as the main subtype expressed in hepatocytes. The fact that melanomacrophages, cells of the immune system in fish, show strong expression of both PPAR alpha and PPAR beta whereas PPAR gamma expression is almost restricted to this cell type suggest a significant role of PPAR-mediated regulation of cell function in melanomacrophages.

Peroxisome proliferation as a biomarker in environmental pollution assessment.

Peroxisome proliferators comprise a heterogeneous group of compounds known for their ability to cause massive proliferation of peroxisomes and liver carcinogenesis in rodents. In recent years it has become evident that other animals may be threatened by peroxisome proliferators, in particular aquatic organisms living in coastal and estuarine areas. These animals are exposed to a variety of pollutants of industrial, agricultural and urban origin which are potential peroxisome proliferators. Both laboratory and field studies have shown that phthalate ester plasticizers, PAHs and oil derivatives, PCBs, certain pesticides, bleached kraft pulp and paper mill effluents, alkylphenols and estrogens provoke peroxisome proliferation in different fish or bivalve mollusc species. The response appears to be mediated by peroxisome-proliferator activated receptors, members of the nuclear receptor family, recently cloned in fish. Based on these results it is proposed that peroxisome proliferation could be used as a biomarker of exposure to a variety of pollutants in environmental pollution assessment. This is illustrated by a case study in which mussels, used worldwide as sentinels of environmental pollution, were transplanted from reference to contaminated areas and vice versa. In mussels native to an area polluted with PAHs and PCBs, peroxisomal acyl-CoA oxidase (AOX) activity and peroxisomal volume density were 2-3 fold and 5-fold higher, respectively, compared to the reference site. When animals were transplanted to the polluted station, with increased concentration of organic xenobiotics, a concomitant significant increase of AOX was recorded. Conversely, in animals transplanted to the cleaner station, AOX activity and peroxisomal volume density decreased significantly. These results indicate that peroxisome proliferation is a rapid (i.e., two days) and reversible response to pollution in mussels. Before peroxisome proliferation can be implemented as a biomarker in biomonitoring programs, a well-defined protocol should be established and validated in intercalibration and quality assurance programmes. Furthermore, the influence of biotic and abiotic factors, some of which are known to affect peroxisome proliferation (season, tide level, interpopulation and interindividual variability), should be taken into consideration. The possible hepatocarcinogenic effects as well as the potential adverse effects on reproduction, development, and growth of peroxisome proliferators are unknown in aquatic organisms, thus providing a challenge for future investigations.

Expression of peroxisome proliferator-activated receptors in zebrafish (Danio rerio).

Peroxisomes increase in size and number in responsive animals ranging from mammals to marine mussels and fish species when treated with certain compounds named peroxisome proliferators. This phenomenon, known as peroxisome proliferation, is mediated by nuclear receptors termed peroxisome proliferator-activated receptors (PPARs). Three PPAR subtypes have been described (alpha, beta, and gamma) and in mammals PPARalpha is mainly expressed in tissues that catabolize fatty acids, PPARbeta is ubiquitously distributed, and PPARgamma is mainly expressed in the adipose tissue and immune system. The aim of this study was to analyze the tissue distribution of different PPAR subtypes in zebrafish Danio rerio using commercially available antibodies against PPARalpha, PPARbeta, and PPARgamma. In western blots, specific bands were detected at about 58 kDa for PPARalpha and PPARbeta. For PPARgamma the band was detected at 56 kDa. Similar results were obtained in mouse liver homogenates used as positive control, indicating the specificity of the antibodies. Immunohistochemistry was performed in paraformaldehyde-fixed tissue using either microwave or microwave plus trypsin pretreatment for antigen retrieval. In zebrafish, PPARalpha was expressed mainly in liver parenchymal cells, proximal tubules of kidney, enterocytes, and pancreas. PPARbeta showed a widespread distribution and was expressed in the liver, proximal and distal tubules and glomeruli of the kidney, pancreas, enterocytes and smooth muscle of the intestine, skin epithelium, lymphocytes, and male and female gonads. PPARgamma expression was weak in pancreatic cells, intestine, and gonads for both pretreatments. Most of the signal detected was cytoplasmic; only in the cases of PPARalpha and PPARbeta was some nuclear labeling detected in the liver. In mouse tissues, the distribution of PPAR subtypes was similar to that described previously for rats. Our results demonstrate that all three distinct PPAR subtypes are present in zebrafish. The tissue and cellular distribution of PPAR subtypes in zebrafish resembled partly that described before in mammals. Further studies are needed to decipher the functions of PPAR subtypes in zebrafish and other aquatic organisms and particularly their role in regulation of metabolic responses to xenobiotic exposure.