Effects of the PPARα agonist WY-14,643 on plasma lipids, enzymatic activities and mRNA expression of lipid metabolism genes in a marine flatfish, Scophthalmus maximus.

Fibrates and other lipid regulator drugs are widespread in the aquatic environment including estuaries and coastal zones, but little is known on their chronic effects on non-target organisms as marine fish. In the present study, turbot juveniles were exposed to the PPARα model agonist WY-14,643 for 21 days by repeated injections at the concentrations of 5mg/kg (lo-WY) and 50mg/kg (hi-WY), and samples taken after 7 and 21 days. Enzyme activity and mRNA expression of palmitoyl-CoA oxidase and catalase in the liver were analyzed as first response, which validated the experiment by demonstrating interactions with the peroxisomal fatty acid oxidation and oxidative stress pathways in the hi-WY treatment. In order to get mechanistic insights, alterations of plasma lipids (free cholesterol, FC; HDL associated cholesterol, C-HDL; triglycerides, TG; non-esterified fatty acids, NEFA) and hepatic mRNA expression of 17 genes involved in fatty acid and lipid metabolism were studied. The exposure to hi-WY reduced the quantity of plasma FC, C-HDL, and NEFA. Microsomal triglyceride transfer protein and apolipoprotein E mRNA expression were higher in hi-WY, and indicated an increased formation of VLDL particles and energy mobilization from liver. It is speculated that energy depletion by PPARα agonists may contribute to a higher susceptibility to environmental stressors.

Normalization strategies for gene expression studies by real-time PCR in a marine fish species, Scophthalmus maximus.

Thorough evaluation of normalization approaches is a fundamental aspect in real-time quantitative RT-PCR experiments to avoid artificial introduced intergroup variations. In our study, we tested three normalization strategies in an experimental data set derived from a toxicological exposure of Scophthalmus maximus to the peroxisome proliferator-activated receptor alpha (PPARα) agonist WY-14643. Juvenile turbots were exposed by repeated injections to 5 mg or 50 mg WY-14643/kg, and liver samples were taken at day 1, 7 and 21. Specifically, the mRNA expression of peroxiredoxin 5 (prdx5) was normalized to the cDNA content, to the mRNA expression of single reference genes (b-actin, b-act; elongation factor 1 α, ef1a; glyceraldehyde-3-phosphate dehydrogenase, gapdh; ribosomal protein L8, rpl8; tata-box binding protein, tbp; tubulin beta 2C chain, tubb2c; ubiquitin-conjugating enzyme E2L 3, ub2l3) or to a combination of multiple reference genes using geNorm, BestKeeper or NormFinder algorithms. Four single reference genes (ef1a, rpl8, tubb2c, tbp) did not show any significant differences between the treatment groups over time, while significant intergroup variations were observed for cDNA content, gapdh, b-act and ub2l3. The normalization of prdx5 to the valid (not altered) single reference genes led to significant up-regulated (prdx5/rpl8), not-regulated (prdx5/ef1a; prdx5/tbp) or down-regulated (prdx5/tubb2c) mRNA expression pattern. The multiple reference gene approaches resulted in different rankings and combinations of the most stable expressed reference genes (geNorm: ef1a>rpl8>b-act; BestKeeper: ub2l3>gapdh>ef1a; NormFinder: b-act>ef1a). However, the normalization with the three multiple reference gene procedures demonstrated consistent expression pattern with a significant up-regulation of prdx5 in response to the higher concentration after 21 days. Concluding, even if not yet established as "gold" standard for expression profiling in environmental toxicology or physiology using freshwater or marine fish models, the multiple reference gene approach is recommended, since it eliminates any biased results, which represented the major flaw of single reference genes.

Dynamics of PPARs, fatty acid metabolism genes and lipid classes in eggs and early larvae of a teleost.

Dietary long chain polyunsaturated fatty acids (FA) have been recognized of crucial importance in early development of vertebrates, contributing to the impressive morphological and physiological changes both as building blocks and to energy production. The importance of lipids along development depends on ontogenetic, phylogenetic and environmental parameters. The expression patterns of FA metabolism genes have not been characterized in developing fish embryos nor compared to lipid class profiles. Full lipid metabolism only occurred after hatching, as revealed by alterations in lipid profiles and FA gene expression. Nonetheless, transcriptional changes of some FA genes were already present in embryos at notochord formation. Many genes displayed an expression profile opposed to the decrease of lipids along the development, while others responded solely to starvation. Transcription of most genes involved in FA metabolism had a strong correlation to PPARs' mRNA levels (α1, α2, ß, γ). The comparison of mRNA expression of the genes with the lipid profiles produced new insights into the FA metabolism and regulation during the development of turbot larvae, providing the basis for future studies including comparative approaches with other vertebrate species.

Cytological, immunocytochemical, ultrastructural and growth characterization of the rainbow trout liver cell line RTL-W1.

Despite its wide use in toxicology, a detailed characterization of RTL-W1 cell line lagged behind leaving ambiguities about its cell origin. We aimed to better characterize the line regarding cell phenotype and tumorigenic state. We studied RTL-W1 cells in monolayers and in (4-22-week-old) aggregates considering: (a) morphology (light and electron microscopy); (b) immunophenotype using AE1/AE3, vimentin, Cam5.2, CK7 and CK19 and e-cadherin antibodies and (c) growth behavior. RTL-W1 organelle content is constituted basically by mitochondria and abundant free ribosomes, with no (cytochemically) detectable peroxisomes and lysosomes. Immunocytochemistry showed a strong marking for AE1/AE3 and vimentin (in a cell subset). Since AE1/AE3 stained biliary epithelial ducts in trout liver, and considering the morphological characteristics and long term culture, RTL-W1 cells seem more similar to bile preductular epithelial cells (considered as stem cells in teleost liver). Also, we observed abnormal nuclear features described for both malignant cell lines and stem cells, so we could not conclude about tumorigenicity. Cell aggregates had signs of hepatocytic differentiation, such as the development of RER and microvillus-like projections into intercellular spaces. The morphological resemblance to the original tissue suggests that aggregates could have an added value in metabolic as well as in cell-to-cell interaction studies.

Tissue expression of PPAR-α isoforms in Scophthalmus maximus and transcriptional response of target genes in the heart after exposure to WY-14643.

Peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of lipid and carbohydrate metabolism and can be activated either by natural ligands as fatty acids or by synthetic ligands including several environmental chemicals. In this study, two PPARα isoforms (α1 and α2) were analyzed in turbot (Scophthalmus maximus) for a different tissue distribution. PPARα1 was ubiquitously expressed, while the PPARα2 was predominantly expressed in the heart. Following this result, turbot juveniles were exposed by injection to a synthetic selective PPARα agonist, WY-14643, for 14 days. Suppression subtractive hybridization (SSH) was performed with pools of heart samples of control and exposed fish to get insights into PPARα-regulated genes in the heart of juvenile turbot. Four genes were positively identified in the forward-subtracted and 12 genes in the reverse-subtracted cDNA SSH library, corresponding to the down-regulated and up-regulated genes in response to the WY-14643 treatment, respectively. The confirmation of these results in individual samples of juvenile turbot exposed to WY-14643 revealed a statistically significant mRNA induction of two cardiac muscle proteins (myosin light chain 2 and tropomyosin 4), which were shown to be involved in heart contraction and heartbeat regulation in other teleost species. Herewith, we showed for the first time that PPARα2 is predominantly expressed in the heart and that a PPARα agonist can induce the mRNA expression of cardiac muscle proteins in teleosts.

Effects of ethinylestradiol and of an environmentally relevant mixture of xenoestrogens on steroidogenic gene expression and specific transcription factors in zebrafish.

In natural environments fish are exposed to endocrine disrupting compounds (EDCs) present at low concentrations and with different modes of actions. Here, adult zebrafish of both sexes were exposed for 21 days to an estrogenic mixture (Mix) of eleven EDCs previously quantified in Douro River estuary (Portugal) and to 100 ng/L 17α-ethinylestradiol (EE2) as positive control. Vitellogenin mRNA and HSI in males confirmed both exposure regimes as physiologically active. Potential candidates for estrogenic disturbance of steroidogenesis were identified (StAR, 17ß-HSD1, cyp19a1), but Mix only affected cyp19a1 in females. Significant differences in the response of FSHß, cypa19a2, 20ß-HSD were observed between EE2 and Mix. Mtf-1 and tfap2c transcription factor binding sites were discovered in the putative promoter regions and corresponding transcription factors were found to be differentially expressed in response to Mix and EE2. The results suggest that "non-classical effects" of estrogenic EDC in fish are mediated via transcription factors.

Aromatase mRNA expression in the brain of adult Xenopus laevis exposed to Lambro river water and endocrine disrupting compounds.

Aromatase P450 (P450 arom; Cyp19) is a key enzyme for vertebrate reproduction and brain development that catalyzes the conversion of androgens to estrogens. The aim of this study was to improve the knowledge on EDC effects by analysing their potential impact on brain P450 arom in adult Xenopus laevis exposed for 4 weeks to an environmental sample, the water of the river Lambro (LAM), the most polluted tributary of the Po river in North Italy. Other groups were exposed to individual compounds 10(-8) M tamoxifen (TAM), ethinylestradiol (EE2), flutamide (FLU) and methyldihydrotestosterone (MDHT) known for their (anti)estrogenic and (anti)androgenic modes of action. Expression of CYP19 was evaluated in brain extracts by quantitative RT-PCR, using a pair of primers located in the open reading frame (ORF) that allowed the simultaneous amplification of all transcripts (Aro-ORF) and a pair of primers specific for brain aromatase (Aro-B). Significant increase in Aro-ORF and Aro-B mRNA levels were observed in both females and males exposed to LAM. Different changes were observed for the model compounds using two pairs of primers. Aro-ORF mRNA expression was significantly increased in EE2 and MDHT exposed males and in FLU-exposed females, while it was significantly decreased in TAM exposed females. Aro-B mRNA was significantly increased in both sexes exposed to FLU and decreased in TAM exposed females. In conclusion, aromatase mRNA in the brain of X. laevis was regulated differentially in a gender specific manner by certain (anti)estrogenic and (anti)androgenic EDCs, supporting previous hypotheses that diverse compounds present in the river Lambro may induce feminization and demasculinization effects.

Expression profiles of LHbeta, FSHbeta and their gonadal receptor mRNAs during sexual differentiation of Xenopus laevis tadpoles.

The gonadotropins, luteinising hormone (LH) and follicle stimulating hormone (FSH), are important hormones regulating reproductive biology in vertebrates, especially the processes of steroidogenesis and gamete maturation. Despite the role of gonadotropins during the reproductive cycle in amphibians is well established, much less is known about the functional maturation of the hypothalamus-pituitary-gonad axis during larval development. Therefore, the present study aimed to analyze the expression profiles of hypophyseal LHbeta and FSHbeta mRNA and of their corresponding gonadal receptors (LH-R, FSH-R) in Xenopus laevis tadpoles during their ontogeny and sexual differentiation. The first significant elevation of LHbeta and FSHbeta mRNA was observed at late premetamorphosis. A clear raise of LHbeta mRNA was present during prometamorphic stages especially in males, while the LH-R only slowly increased during ontogeny with highest levels during metamorphic climax. In contrast, FSHbeta mRNA expression only slightly increased during ontogeny, however in both sexes the FSH-R mRNA was considerably elevated at prometamorphosis and further at metamorphic climax. Our results suggest that LHbeta and LH-R mRNA expression might be involved in initial maturation events of gametes, at least in males, while the gradually increase of FSH-R mRNA coincided with the advancing process of gamete maturation in both sexes. The present study provides for the first time evidence based on expression of gonadotropins and their corresponding gonadal receptors that the hypothalamus-pituitary-gonad axis evolves already at early stages of ontogeny and sexual differentiation in amphibians.

Exposure of Xenopus laevis tadpoles to finasteride, an inhibitor of 5-alpha reductase activity, impairs spermatogenesis and alters hypophyseal feedback mechanisms.

Sexual steroids have major regulatory functions in gonadal development, maturation of gametes and sexual differentiation in vertebrates. Previous studies in amphibians provided evidence that dihydrotestosterone and activity of 5-alpha reductases might play a significant role in androgen-mediated reproductive biology. To test the involvement of 5-alpha reductases in maturation of gametes in amphibians, Xenopus laevis was exposed to finasteride (FIN), a known inhibitor of 5-alpha reductase enzyme activity. In a long-term exposure from stage 46 to 66, severe disruption of spermatogenesis was observed in histological analysis of testes as detected by occurrence of empty spermatocysts, while ovaries remained unaffected. Real-time PCR analyses of male and female brain revealed an increase of LHbeta mRNA and a decrease of FSHbeta mRNA in males, suggesting a signalling on testes that could result in increased steroidogenesis and reduced Sertoli cell proliferation. Accordingly, the mRNA expression of P450 side chain cleavage enzyme and 5-alpha reductase type 2 was increased in testes, while no effects could be observed on steroidogenic genes in ovaries. A short-term exposure to testosterone, FIN and testosterone+FIN showed that transient effects of FIN targeted males selectively and, in particular, interfered with the hypothalamus-pituitary-gonad axis. Furthermore, a negative feedback of testosterone on LHbeta was observed on males and females. This study provides evidence that exposure of X. laevis to FIN, an inhibitor of 5-alpha reductases, impaired spermatogenesis and involved sex-specific hypophyseal feedback mechanisms.

Aromatase, steroid-5-alpha-reductase type 1 and type 2 mRNA expression in gonads and in brain of Xenopus laevis during ontogeny.

The key enzymes involved in the production of endogenous sex steroids are steroid-5-alpha-reductase and aromatase converting testosterone (T) into dihydrotestosterone (DHT) and into estradiol (E2), respectively. To gain more insights into the molecular mechanisms of sexual differentiation of amphibians, we determined the mRNA expression of steroid-5-alpha-reductase type1 (Srd5a1), type2 (Srd5a2) and aromatase (Aro) during ontogeny starting from the egg and ending after completion of metamorphosis in Xenopus laevis. Expression of all three enzymes was measured by means of semi-quantitative RT-PCR, determining for the first time Srd5a1 and Srd5a2 mRNA expression in amphibians. mRNA was analyzed in whole body homogenates from stage 12 to 48, while brain and gonads with kidney were studied separately from stage 48 to 66. Different ontogenetic mRNA expression patterns were observed for all genes analyzed, revealing early mRNA expression of Srd5a1 already in the egg at stage 12 whereas Srd5a2 and Aro was detected at stage 39. Sex-specific mRNA expressions of Srd5a2 and of Aro were determined in the gonads with kidney but not in brain. Srd5a2 was two-fold higher expressed in testes than in ovaries while Aro mRNA was ten-fold higher in ovaries. No gender-specific mRNA expression was observed for Srd5a1 in gonads and in brain. The ontogenetic patterns of Aro, Srd5a1 and Srd5a2 suggest that these genes are involved in sexual differentiation of gonads and brain already in early developmental stages. Especially in gonads Srd5a2 seems to be important for physiological regulation of testis development while Aro is associated with the development of ovaries.

Luteinizing hormone, follicle stimulating hormone, and gonadotropin releasing hormone mRNA expression of Xenopus laevis in response to endocrine disrupting compounds affecting reproductive biology.

Environmental pollutants can interfere with the endocrine system of a variety of animals and are suggested to contribute to the worldwide decline of amphibians. In this study, the effects of endocrine disrupting compounds (EDC) on the hypothalamus-pituitary-gonad axis, regulating reproduction, were investigated in Xenopus laevis by determining their potential impact on gene expression of gonadotropin releasing hormone (GnRH), luteinizing hormone beta-subunit (LHbeta) and follicle-stimulating hormone beta-subunit (FSHbeta) in brain and pituitary using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). One environmental sample and four model compounds, ethinylestradiol (EE2), tamoxifen (TAM), methyldihydrotestosterone (MDHT), and flutamide (FLU), corresponding to (anti)estrogenic and (anti)androgenic modes of action were used at 10(-8)M during a four weeks exposure of adults of both sexes. In general, males had a higher LHbeta mRNA level compared to females, while the mRNA expression of FSHbeta and GnRH did not differ between both sexes. EE2 and MDHT treatment decreased LHbeta mRNA expression in the brain of male X. laevis, while only EE2 but not MDHT reduced LHbeta mRNA in females indicating classical negative feed-back mechanisms on hypophyseal gonadotropin expression. TAM increased LHbeta mRNA and FSHbeta mRNA expression in female X. laevis while none of the other treatments showed an effect on FSHbeta mRNA expression. GnRH expression was not changed by any treatment and exposure of X. laevis to Lambro river water had no significant effect on any of the genes examined. It is reported for the first time in amphibians that gonadotropin mRNA expression is differentially regulated by (anti)estrogenic and (anti)androgenic EDC and that gender-specific patterns of gene expression exist.