Artificial reproduction of the indoor-cultured brackish form of maraena whitefish (Coregonus maraena) under recirculated aquaculture system (RAS) conditions.

Maraena whitefish (Coregonus maraena) is a promising coldwater aquaculture species, especially in terms of meat quality. This versatile whitefish species, which inhabits both fresh and brackish waters, including the Baltic Sea, offers promising prospects for aquaculture breeding and cultivation. However, the main problem is the lack of availability of spawners of this species. Therefore, in this study, an attempt was made to raise the spawning stock under controlled conditions and then artificially reproduce the fish to obtain high-quality gametes and then larvae. The conducted research showed that successful culture of maraena whitefish spawners is possible under recirculating aquaculture system (RAS) conditions. The application of proper nutrition and manipulation of environmental conditions allowed the breeding of sexually mature whitefish spawners in less than two years in captivity (meat weight 476 g). Through stimulation of environmental conditions (temperature and photoperiod), it was possible to stimulate the final maturation of gametes of the F1 generation of cultured fish. The use of Ovaprim hormonal stimulation induced 100% of females to ovulate within 10 days of injection, compared to 60% of females in the control group. Additionally, the other examined parameters, such as the pseudogonado-somatic index (PGSI: 11.46% vs. 6.80%), fertilization rate (94.2% vs. 60.1%), embryo survival rate (92.6% vs. 41.1%) and embryo survival rate to the eyed-egg-stage (90.0% vs. 28.0), were significantly higher in the hormonally induced group, finally two times and three times, respectively. The mortality rate of spawners, after spawning and within three months of artificial spawning, was extremely low. The results obtained from the study demonstrate the feasibility of culturing the spawning stock of maraena whitefish in captivity and successfully inducing artificial spawning.

Cyclopenta[c]phenanthrenes--chemistry and biological activity.

Despite cyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs) having been detected in the environment, the ability of these compounds to induce cellular and tissue responses remains poorly characterized. In this review, we look at the chemistry and biological activity of the cyclopenta[c]phenanthrenes (CP[c]Phs) as potential chemicals of concern in the process of risk assessment. The first part of the review deals with the environmental occurrence and chemistry of CP-PAHs, focusing on available methods of CP[c]Ph chemical synthesis. The most interesting structural feature of the CP[c]Ph is the presence of a pseudo fjord-region constructed by the cyclopentane ring. This compound can be treated either as a structurally similar one to B[c]Ph, or as a phenanthrene skeleton with an electrodonating alkyl substituent in the bay-region of the molecule. The second thread, providing available data on the adverse effects of CP[c]Ph compounds on cells and tissues of living organisms, mainly fish, improves our understanding of these possible environmental hazards. The data show that CP[c]Ph is less potent at inducing CYP1A gene expression in rainbow trout than benzo[a]pyrene (B[a]P), a well-known Ah-receptor agonist. Interestingly, the CP[c]Ph dependent up-regulation of CYP1A mRNA is positively correlated with the incidences of clastogenic changes in rainbow trout erythrocytes. CP[c]Ph has, comparably to B[a]P, a potential to repress expression of tumor suppressor p53, in the head kidney of rainbow trout. Furthermore, estrogen responsive genes in fish liver, ERα and VTG, are not induced by CP[c]Ph, suggesting that the compound has no endocrine disrupting potential. However, some CP[c]Phs show mutagenic activity when investigated in the Ames test, and exhibit genotoxic properties in in vitro micronucleus assay. The above characteristics suggest that CP-PAHs are chemicals of concern for which potential pathways of exposure should be further identified.

CYP1A expression in liver and gills of roach (Rutilus rutilus) after waterborne exposure to two phenanthrene derivatives, 1-methylphenanthrene and 4-methylphenanthrene.

Phenanthrenes (Phs) substituted with alkyl groups are a class of compound present in the environment, and they appear to be toxic to developing fish. The present study aimed to investigate the effect of waterborne exposure to two monomethyl derivatives of phenanthrene, 1-methylphenanthrene (1M-Ph) and 4-methylphenanthrene (4M-Ph), on cytochrome P450 1A (CYP1A) gene expression in fish gills and liver. Juvenile common roaches (Rutilus rutilus) were exposed to water with dimethyl sulfoxide (DMSO) solutions of 1M-Ph, 4M-Ph, benzo[a]pyrene (BaP; positive control), each at a dose of 100 µg/L, or to water with DMSO alone (negative control group) for 2 d and 7 d. Significant CYP1A responses with regard to treatment and exposure duration were noted (2-way analysis of variance [ANOVA]) in gills (p = 0.013 and p = 0.003, respectively) and liver (p < 0.001). The 2 monomethyl Phs did not induce consistent gene expression changes, except for 4-MPh, which elevated the CYP1A messenger ribonucleic acid (mRNA) level in the liver at the end of the treatment (almost 4-fold; p < 0.05; 7 d). As was expected, exposure to BaP resulted in elevation of CYP1A mRNA expression in treated fish compared with the control group. Expressions after 2 d and 7 d were approximately 220- and 180-fold higher in liver and 8- and 6-fold higher in gills respectively. The CYP1A protein levels remained stable in both tissues, with one notable exception in roach liver treated for 2 d with BaP (∼ 6-fold increase; p < 0.05). The different effects of the 1- and 4-methylphenanthrenes on CYP1A gene expression in roach liver suggest a relationship between chemical or 3-D structure of the differentially substituted monomethyl Phs and their biological activity.

Differential gene expression in rainbow trout (Oncorhynchus mykiss) liver and ovary after exposure to zearalenone.

Zearalenone (ZEA) is a mycotoxin of worldwide occurrence, and it has been shown to produce numerous adverse effects in both laboratory and domestic animals. However, regardless of recent achievements, the molecular mechanisms underlying ZEA toxicity remain elusive, and little is known about transcriptome changes of fish cells in response to ZEA occurrence. In the present study, differential display PCR was used to generate a unique cDNA fingerprint of differentially expressed transcripts in the liver and ovary of juvenile rainbow trout after either 24, 72, or 168 h of intraperitoneal exposure to ZEA (10 mg/kg of body mass). From a total of 59 isolated cDNA bands (ESTs), 5 could be confirmed with Real-Time qPCR and their nucleotide sequences were identified as mRNAs of: acty (ß-centractin), the cytoskeleton structural element; bccip, responsible for DNA repair and cell cycle control; enoa (α-enolase), encoding enzyme of the glycolysis process; proc (protein C), that takes part in the blood coagulation process; and frih, encoding the heavy chain of ferritin, the protein complex important for iron storage. Further qPCR analysis of the confirmed ESTs expression profiles revealed significant mRNA level alterations in both tissues of exposed fish during the 168 h study. The results revealed a complex network of genes associated with different biological processes that may be engaged in the cellular response to ZEA exposure, i.e. blood coagulation or iron-storage processes.

CYP1A expression in liver and gills of rainbow trout (Oncorhynchus mykiss) after short-term exposure to dibenzothiophene (DBT).

Dibenzothiophene (DBT), a common component of crude oil, is a widespread environmental pollutant of known adverse effects to aquatic vertebrates. However, the molecular mechanism by which DBT exerts its effects still remains unknown. Our goal for this study was to examine DBT effects on CYP1A expression in liver and gills of rainbow trout after short-term exposure. Juvenile trout individuals were injected intraperitoneally with two doses of DBT (10 or 50mgkg(-1)) and were kept in tanks for 8 and 24h (T=14 degrees C), then their gene expression levels were evaluated by Real-Time qPCR and Western-blot analysis. Treatment with DBT at either dose decreased CYP1A mRNA levels through the exposure period, which resulted in the final decrease of CYP1A protein levels in liver and gills on the end of experiment (24h). Thus, our results showing significant depletion of CYP1A molecules in metabolic tissues upon DBT treatment correlate with those previous reports that indicate a role of DBT in reducing CYP1A activity in fish.

Microcystin-LR induced apoptosis and mRNA expression of p53 and cdkn1a in liver of whitefish (Coregonus lavaretus L.).

There is growing evidence that adverse effects of microcystin-LR (MC-LR) are closely related to oxidative stress processes, free radicals and DNA damage, and involve major gene transcript changes. This study, utilizing gene expression analysis and plasma chemistries was the first to measure the effects of MC-LR in whitefish (Coregonus lavaretus L.), a feasible organism for pollution monitoring in aquatic systems. Fish were injected with different concentrations of MC-LR (0, 10 and 100 microg/kg of body weight) and then sacrificed at either 0, 8, 24, 48 or 72 h later, and their liver tissue were harvested for detailed investigation. Specifically, we were interested whether MC-LR is capable of: (i) modulating expression of two genes, tumor suppressor gene p53 and cdkn1a, p53 direct transcription target, and (ii) inducing apoptosis in whitefish liver. To study these effects, we developed a real-time qPCR assays useful for measuring both p53 and cdkn1a gene transcript levels in liver. To obtain necessary information for the study, either full-length p53 cDNA of whitefish (Wf-p53) was determined, using molecular cloning and rapid amplification of cDNA ends (RACE), or as for Wf-cdkn1a, specific primers were designed based on highly conserved regions of cdkn1a in fish. The Wf-p53 was found to share the same characteristics with a known p53 mRNA sequence of other vertebrates. Whitefish p53 amino acid sequence showed a high degree of homology with the sequences from fishes, amphibians, and mammals. The injection study showed that MC-LR at a higher dose, i.e. 100 microg/kg body weight, up-regulated expression of p53 and cdkn1a genes in whitefish liver, as reflected by the continuous increase in their mRNA levels through the whole experiment. Furthermore, DNA fragmentation was observed in liver cells of whitefish after 24h of exposure to MC-LR (100 microg/kg) that suggests the possibility of apoptosis. Finally, the study confirmed previous observations of severe injury of the liver and loss of normal organ functions as revealed by elevated levels of blood AspAT, AlaAT, and hepatosomatic index (HSI).

Effects of cyclopenta[c]phenanthrene and its derivatives on zona radiata protein, ERalpha, and CYP1A mRNA expression in liver of rainbow trout (Oncorhynchus mykiss Walbaum).

Despite cyclopenta-fused polycyclic aromatic hydrocarbons (CP-PAHs) have been detected in the environment, the ability of CP-PAH to induce cellular and tissue responses remains poorly characterized. In this study, xenoestrogen-associated responses (mRNA levels of estrogen receptor alpha, ERalpha, and zona radiata protein, Zrp) and xenobiotic effects (CYP1A mRNA) have been investigated in liver of juvenile rainbow trout after short-term treatment (8 and 24 h) with following compounds administered singly: cyclopenta[c]phenanthrene (CP[c]Ph); its derivatives, 5A-CP[c]Ph; 5A6M-CP[c]Ph; 5A9M-CP[c]Ph; B[c]Ph, a structurally similar polycyclic aromatic hydrocarbon; B[a]P, a model CYP1A inducer; and zearalenone (ZEA), naturally occurring ligand for ER. The CYP1A mRNA expression after 24 h of exposure with CP[c]Ph or its derivatives, except 5A9M-CP[c]Ph, was 3-9-fold higher compared to controls (P<0.05), but it was less than that caused by B[a]P (65-fold up regulation; P<0.01). Moreover, neither of the CP-PAH compounds modulated liver ERalpha or Zrp mRNA levels as compared to effects associated with ZEA. Interestingly, a treatment with this ER-ligand, caused moderate but significant increase of CYP1A mRNA expression (about 2.5-fold; P<0.05). The finding that ZEA is capable of acting as either estrogenic and xenobiotic compound, should be further explored in a more detailed and differently designed experiment.