Paweł Brzuzan

Critical partial pressures of oxygen causing precocious hatching in Coregonus lavaretus and C. albula embryos

Abstract Embryos of whitefish ( Coregonus lavaretus ) and vendace ( C. albula ) were exposed to various hypoxic conditions at constant temperatures of 8°C and 11°C at the developmental stages of “eye movement visible” and “first embryos hatched”. Eggs exposed to hypoxia responded with precocious hatching and the response depended on the degree of hypoxia, test temperature, and developmental phase. The calculated critical partial pressures of oxygen ( p O 2 ) causing precocious hatching at 8°C were 40 mm Hg (3.0 ppm dissolved oxygen concentration—DO) for whitefish and 28 mm Hg (2.1 ppm DO) for vendace embryos. The sensitivity of embryos to hypoxic stress increased rapidly as development progressed. Eventually, the critical p O 2 for vendace eggs increased to 81 mm Hg (6.0 ppm DO) at the stage of “first embryos hatched”. Higher temperatures caused stronger response of embryos to hypoxia: exposure of whitefish embryos for 60 min to p O 2 of 3 mm Hg (0.2 ppm DO) at 8°C resulted in hatching of 43% of eggs, whereas at 11°C, hatching increased to 95% (at the same oxygen concentration). Adequate DO concentrations must be provided in incubation to prevent early hatching and increased mortality.

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).

Expression profiling in vivo demonstrates rapid changes in liver microRNA levels of whitefish (Coregonus lavaretus) following microcystin-LR exposure.

At present, little is known about the role of miRNAs in liver response of fish to the cyanobacterial hepatotoxin microcystin-LR (MC-LR) treatment, despite the fact that the exposure is thought to underlie multiple acute and chronic effects. To address this question, we used the Real-Time PCR method to examine the differential expression of 6 miRNAs putatively playing roles in signal transduction (let-7c, miR-9b), apoptosis and cell cycle (miR-16a, miR-21a, miR-34a) and fatty acid metabolism (miR-122) in whitefish (Coregonus lavaretus) liver, during the first 48h after intraperitoneal injection of MC-LR (100 μg/kg body weight). In addition, we analyzed expression levels of 8 mRNAs and p53 protein, known to be involved in the cell response on the exposure to environmental stressors. Following the challenge we observed a rapid and transient increase in the mean (n=5) levels of individual miRNA expression (from 2.7-fold for miR-122 to 6.8-fold for let-7c), compared to the respective levels in control fish, which mostly peaked at 24h of the experiment. This increase was correlated with a reduction in the expression of mRNAs of genes coding for ferritin H (frih) and HNK Ras -like protein (p-ras) and an overexpression of mRNAs of genes coding for bcl2-associated X protein (bax), cyclin dependent kinase inhibitor 1a (cdkn1a), dicer (dcr), histone 2A (h2a) and p53. Expression of the remaining caspase 6 (cas6) mRNA did not change over 48 h of the treatment. Moreover, exposure to MC-LR did not alter whitefish p53 protein levels. Bearing in mind a variety of likely silencing targets for, and the onset of, the aberrant miRNA expression it may be concluded that they are involved in molecular pathways, such as liver cell metabolism, cell cycle regulation and apoptosis, and may contribute to the early phase of MC-LR induced hepatotoxicity.

Effects of cyclopenta[c]phenanthrene and its derivatives on zona radiata protein, ERα, 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.

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 (β-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.

Solvent-free condensations of ketones with malononitrile catalysed by methanesulfonic acid/morpholine system

The preparation of ylidenemalononitriles via Knoevenagel condensations of ketones with malononitrile under solvent-free conditions is described. Good yields and short reaction time are the features observed with methanesulfonic acid (MSA)/morpholine used as the catalyst. The wide applicability of the protocol is shown by the fact that not only unconjugated, but also aryl-alkyl ketones gave satisfactory yields.

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.

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.

MOLECULAR GEOMETRY, CYP1A GENE INDUCTION AND CLASTOGENIC ACTIVITY OF CYCLOPENTA[c]PHENANTHRENE IN RAINBOW TROUT

Cyclopenta[c]phenanthrene (CP[c]Ph) is a PAH member that shows similarities to bay-and fjord region possessing PAHs. On the basis of X-ray measurements it was found that the molecule of this hydrocarbon is planar. In this case, intramolecular strains caused by repulsion between protons in the pseudo fjord-region are balanced by both the shortening of some bonds which acquire more double bond character, and by the enlargement of exocyclic angles within the pseudo fjord-region. The activity of CP[c]Ph was investigated in vivo in rainbow trout, Oncorhynchus mykiss. The CYP1A mRNA levels following 48h-treatment with CP[c]Ph or benzo[a]pyrene (B[a]P; positive control) were determined and compared with incidences of clastogenic changes observed in the peripheral blood erythrocytes. We have found that the ability to induce CYP1A by these PAH compounds is positively correlated with the incidences of clastogenic changes in rainbow trout erythrocytes.

Illumina Sequencing Reveals Aberrant Expression of MicroRNAs and Their Variants in Whitefish (Coregonus lavaretus) Liver after Exposure to Microcystin-LR

Molecular analyses show that challenging fish with microcystin-LR (MC-LR) causes perturbations of microRNA (miRNA) signaling. However, the significance and scope of these alterations is currently unknown. To address this issue, we studied miRNA gene expression in the liver of juvenile whitefish, C. lavaretus, during 28 days of exposure to a subacute dose of MC-LR (100 μg·kg-1 body mass). Using genomic resources of Atlantic salmon (AGKD03), the mature miRNA library of Atlantic salmon (miRBase-21) and bioinformatics tools (sRNAbench), we discovered and annotated a total of 377 distinct mature miRNAs belonging to 93 families of evolutionary conserved miRNAs, as well as 24 novel mature miRNA candidates that were mapped to 14 distinct S. salar miRNA precursors. miRNA-Seq transcriptome profiling of liver tissues revealed differential miRNA expression in control and treated fish at 14 days (73 miRNAs were modulated) and at 28 days (83 miRNAs) of the treatment, subsequently validated by qPCR for nine selected differentially expressed miRNAs. Additional qPCR study confirmed the miRNA-Seq data and revealed consistent, aberrant miRNAs expression profile in the later phase of MC-LR hepatotoxicity (7–28 d). Functional annotation analysis revealed that the aberrantly expressed miRNAs have target genes involved in cytoskeletal remodeling, cell metabolism, cell cycle regulation and apoptosis; dysregulation of these processes in liver cells leads to cirrhosis and hepatocellular carcinoma in humans. To enable deeper insight into the molecular responses of liver cells in fish exposed to MC-LR, we expanded the miRNAome analysis by inclusion of miRNA variants (isomiRs) profiles, and we showed that the isomiR profiles of liver specific MiR122, and a few other miRNAs, correlated with MC-LR treatment. Given the importance of isomiRs for disease biology in mammals, we believe that further research focused on the miRNA isoforms will bring us closer to better understanding the molecular mechanisms of MC-LR hepatotoxicity.