Liver Of Whitefish Research Articles

De Novo Profiling of Long Non-Coding RNAs Involved in MC-LR-Induced Liver Injury in Whitefish: Discovery and Perspectives.

Microcystin-LR (MC-LR) is a potent hepatotoxin for which a substantial gap in knowledge persists regarding the underlying molecular mechanisms of liver toxicity and injury. Although long non-coding RNAs (lncRNAs) have been extensively studied in model organisms, our knowledge concerning the role of lncRNAs in liver injury is limited. Given that lncRNAs show low levels of sequence conservation, their role becomes even more unclear in non-model organisms without an annotated genome, like whitefish (Coregonus lavaretus). The objective of this study was to discover and profile aberrantly expressed polyadenylated lncRNAs that are involved in MC-LR-induced liver injury in whitefish. Using RNA sequencing (RNA-Seq) data, we de novo assembled a high-quality whitefish liver transcriptome. This enabled us to find 94 differentially expressed (DE) putative evolutionary conserved lncRNAs, such as MALAT1, HOTTIP, HOTAIR or HULC, and 4429 DE putative novel whitefish lncRNAs, which differed from annotated protein-coding transcripts (PCTs) in terms of minimum free energy, guanine-cytosine (GC) base-pair content and length. Additionally, we identified DE non-coding transcripts that might be 3′ autonomous untranslated regions (3′UTRs) of mRNAs. We found both evolutionary conserved lncRNAs as well as novel whitefish lncRNAs that could serve as biomarkers of liver injury.

Molecular characterization of the cyclin-dependent protein kinase 6 in whitefish (Coregonus lavaretus) and its potential interplay with miR-34a

Cyclin-dependent protein kinase 6 (CDK6) plays a pivotal role in the regulation of the cell cycle and cell proliferation in mammals, and disruption of its expression by various microRNAs has been implicated in the pathogenesis of multiple human cancers. In mammals, miR-34a acts as a downstream effector of p53, and thus indirectly targets Cdk6, abrogating its effects. However, no studies have been done so far to examine the mechanistic involvement of miR-34a in the silencing of cdk6 in fish. In the present study, we found that the cDNA sequence of whitefish cdk6 has a 3′UTR region that contains a binding site for miR-34a. Using a luciferase reporter assay, we demonstrated that whitefish cdk6 is a direct target of miR-34a in vitro. In order to confirm this relationship in vivo, we measured the miR-34a and cdk6 mRNA expression patterns in the liver of whitefish after short-term (8, 24, and 48 h) and long-term (14 and 28 days) exposure to microcystin-LR (MC-LR), a known hepatotoxin and tumor promoter. In contrast to the in vitro findings, we noticed an up-regulation of miR-34a and cdk6 expression after long-term MC-LR treatment. While these results indicate that both, miR-34a and cdk6 are responsive to MC-LR treatment, they do not support the presence of a miR-34a:cdk6 mRNA regulatory pair in the MC-LR–challanged whitefish liver in vivo. On the other hand, our findings suggests that cell regulatory elements, partnering with either miR-34a or cdk6, are worthy of further screening to better understand the molecular mechanisms that underlie the physiological response of fish challenged with hepatotoxic environmental pollutants like microcystins.

In vivo miRNA delivery in whitefish: Synthetic MiR92b-3p uptake and the efficacy of gene expression silencing.

The delivery of short snippets of RNA, such as synthetic miRNA agents, is an essential step for achieving RNA-mediated knockdown, which has not been studied in sufficient detail in fish. Our results indicate that a MiR92b-3p mimic may be effectively delivered via intraperitoneal injection to the spleen and the liver of whitefish, and that it likely achieves functionality without causing any apparent toxic effects in the challenged animals. We report the novel finding that the MiR92b-3p mimic reduced the in vivo liver mRNA expression levels of its putative pro-apoptotic targets (p53, cdkn1a, and pcna), and important metabolic genes, e.g. cdo1. This shows that this methodology of MiR92b-3p mimic transfection in vivo may be a useful tool for studies that investigate the molecular pathways that confer pro-proliferative and anti-apoptotic phenotypes or those that regulate intracellular metabolism in fish and other vertebrates.

Biogeochemistry of Cadmium: Anthropogenic Dispersion, Bioaccumulation, and Ecotoxicity

The article considers biogeochemical features of cadmium as one of the most dangerous elements. Data on its concentrations in waters of small lakes in the European Russia (from the tundra to deserts) and large river systems (Volga, Severnaya Dvina, Pechora) are given. It is shown that, despite the relatively low concentrations of the element in waters, its enrichment factor is hundreds and thousands of units, especially in the northern regions and regions subjected to the influence of non-ferrous metallurgy. Data are presented on the bioaccumulation of cadmium within fish body systems, which is determined by the concentration of the element in waters, as well as by the water pH and the calcium content. It is proved that the highest Cd concentrations are accumulated in kidneys and liver of whitefish and salmon, causing pathological disturbances. Data are reported on the accumulation of cadmium in kidneys and liver of human population of the industrially developed Northern region under impacts of copper–nickel smelters. The similarity in tendencies of the Cd accumulation and pathological disturbances in fish and the population consuming water from contaminated sources makes it possible to recommend fish as a bioindicator of the environmental pollution by cadmium.

C-myc in whitefish (Coregonus lavaretus): structure, expression, and insights into possible posttranscriptional regulatory mechanism.

c-myc has a crucial function in growth control, differentiation, and apoptosis of vertebrate cells. Despite the important role of c-myc in mediating the biological effects, studies of c-myc gene expression and factors that control it in organisms other than mammals, such as fish, have been rare. In the current study, we asked whether c-myc mRNA of whitefish, a feasible organism for pollution monitoring in aquatic systems and a model in toxicological research, contains activity sites for regulatory motifs in its 5'- and 3'-UTRs, similar to those found in mammals. We were particularly interested in whether miRNA-34, a known negative regulator of c-myc's in mammals, is able to regulate c-myc in fish. To answer these questions, we determined the mRNA sequence of whitefish c-myc and inferred the structure of the protein that it codes for. We found that the active sites of mRNA and structures of the inferred c-myc protein are similar to those found in mammals and other fish. Remarkably, levels of c-myc mRNA expression were very high in ovaries compared to other tissues of whitefish, thus corroborating previous data in fish. Using bioinformatic searches on c-myc 3'-UTR, we confirmed the presence of two miRNA-34a(miR-34a)response elements. Luciferase reporter assay showed that activity of reporters containing either the miR response elements or entire c-myc 3'-UTR was significantly reduced (p<0.001) by ectopic expression of miR-34a. Therefore, we further investigated possible involvement of miR-34a in c-myc gene silencing by profiling the expression of both genes in livers of whitefish treated for 8, 24, 48h with MC-LR, a potent c-myc inducer in mammals. Although the difference was only significant at p=0.08, the expression of c-myc mRNA in challenged whitefish after 24h of the treatment was notably higher than that in livers of control fish. Concurrently, we noticed slight but significant up-regulation of miR-34a after 24 and 48h of the challenge (p<0.05); however, we found no significant correlation of the c-myc mRNA levels and miR-34a expression. Together, these results suggest that miR-34a might regulate c-myc gene expression in whitefish liver; however, their involvement in MC-LR hepatotoxicity should be clarified in future studies.

Habitat coupling in a large lake system: delivery of an energy subsidy by an offshore planktivore to the nearshore zone of Lake Superior

Summary We hypothesised that the autumn spawning migration of Lake Superior cisco (Coregonus artedi) provides a resource subsidy, in the form of energy‐rich cisco eggs, from the offshore pelagic to the nearshore benthic community over winter, when alternate prey production is likely to be low. We tested this hypothesis using fish and macroinvertebrate surveys, fish population demographics, diet and stable isotope analyses, and bioenergetics modelling. The benthic, congeneric lake whitefish (C. clupeaformis) was a clear beneficiary of cisco spawning. Cisco eggs represented 16% of lake whitefish annual consumption in terms of biomass, but 34% of energy (because of their high energy density: &gt;10 kJ g wet mass−1). Stable isotope analyses were consistent with these results and suggest that other nearshore fish species may also rely on cisco eggs. The lipid content of lake whitefish liver almost doubled from 26 to 49% between November and March, while that of muscle increased from 14 to 26% over the same period, suggesting lake whitefish were building, rather than depleting, lipid reserves during winter. In the other Laurentian Great Lakes, where cisco populations remain very low and rehabilitation efforts are underway, the offshore‐to‐nearshore ecological link apparent in Lake Superior has been replaced by non‐native planktivorous species. These non‐native species spawn in spring have smaller eggs and shorter incubation periods. The rehabilitation of cisco in these systems should reinstate the onshore subsidy as it has in Lake Superior.

Comparative sensitivity of liver monoamine oxidases of frog and whitefish to some tricyclic compounds

There is performed a comparative analysis of action of four acridine derivatives and of one xanthene derivative (pyronine G) on activity of liver monoamine oxidase (MAO) of two species of poikilothermal freshwater animals: a representative of amphibians--the common frog Rana temnporaria and a representative of the order Salmonidae--the European whitefish Coregonus lavaretus. The studied synthetic hexamerous tricyclic compounds show the irreversible character of inhibition of intermediate potency towards the enzyme from both biological sources. There are obtained qualitative and quantitative differences in the reactional ability and selectivity of action of the studied inhibitors for liver MAO of frog and whitefish. The obtained data of the inhibitory analysis with use of specific substrates are an indirect proof for the existence in liver of the studies frog species of two molecular forms, whereas in the whitefish liver--single molecular MAO form.

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 μg/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 μg/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 24 h of exposure to MC-LR (100 μg/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).

Special traits of tissue lipids of whitefish Coregonus lavaretus living in water bodies with different anthropogenous load

The impact of water pollution on the content of lipid components of the liver and eggs of whitefish Coregonus lavaretus is investigated. The content of triacylglycerols (TAG), sphingomyelin (SPM), phosphatidylinositol (PI), and phosphatidylserine (PS) is higher in the liver of whitefish from polluted waters being a part of adaptogenous reactions (modification of permeability of biomembranes, of activity of membrane enzymes, etc.) providing a possibility of the survival of fish under unfavorable ecological factors. The lipid spectra of whitefish gonads are more stable in comparison with the liver. This may be related to protection of the reproductive system from external influences.

Confirmation of in situ exposure of fish to secondary treated bleached‐kraft mill effluent using a laboratory simulation

AbstractTo corroborate the responses in whitefish (Coregonus Iavaretus L.) exposed to elemental chlorine free (ECF) bleached‐kraft pulp mill effluent (BKME) in situ, a 30‐d laboratory exposure was carried out at concentrations simulating the field conditions. The flow‐through exposures were conducted at four secondary (activated sludge) treated effluent (STE) concentrations: 1.3, 2.3, 3.5, and 7%. To evaluate the role of the secondary treatment, fish were also exposed to one concentration (3.5%) of pretreated effluent (PTE) from the mill. Compared to the control, whitefish liver 7‐ethoxyresorufin‐O‐deethylase (EROD) activity was twofold in fish exposed to 3.5% STE, which was similar to monooxygenase induction in the field at the same effluent dilution. The exposure to 3.5% PTE caused a 12‐fold relative induction in whitefish. The activity of pentoxyresorufin dealkylase showed a high correlation with EROD activity (r2 = 0.85, p &lt; 0.01). The plasma concentration of 17β‐estradiol was reduced by 37% (p &lt; 0.05) in fish exposed to 3.5% STE, whereas testosterone was reduced by about 40% (p &lt; 0.05) in fish in both 3.5% STE and PTE groups. The accumulation of chlorophenolics (CPs) and resin acids (RAs) in the bile of the fish was negligible at the three lowest STE concentrations, reflecting the nearly nondetectable levels of CPs and RAs in secondary treated whole effluent. The measured blood parameters plasma immunoglobulin M, glucose, hemoglobin, and hematocrit were not affected by effluent exposure. The responses obtained from the laboratory simulation well accorded with the exposures in the field, although signs of reproductive impairment could be detected in the laboratory. Overall, however, it is evident that the improvements to mill processes and wastewater treatment have substantially reduced the load of harmful constituents in bleached‐kraft mill effluent and biological impacts in the receiving environment.