RESUMO
New agrochemicals must demonstrate safety to numerous ecological systems, including aquatic systems, and aquatic vertebrate toxicity is typically evaluated by using the in vivo acute fish toxicity (AFT) test. Here, we investigated two alternative in vitro assays using a cell line isolated from rainbow trout (Onchorhynchus mykiss) gill tissue: (i) adenosine triphosphate (ATP) luminescence and (ii) cell painting. The former assay measures cytotoxicity, while the latter measures changes in cellular morphology in response to chemical exposure. We assessed how well end points in these two assays predicted acute lethality (i.e., LC50 values) in independent in vivo AFT tests. When compared to results from OECD TG 249 (in vitro), we found that the ATP assay was not as predictive (R2 = 0.53) as the cell painting assay. Similarly, when compared to results from OECD TG 203 (in vivo), the cell painting was much more predictive (R2 = 0.67). Our results show that such in vitro assays are useful for fast and efficient screening alternatives to in vivo fish testing that can aid in the agrochemical discovery phase, where thousands of potential new actives are tested each year.
Assuntos
Agroquímicos , Oncorhynchus mykiss , Animais , Agroquímicos/toxicidade , Proteção de Cultivos/métodos , Testes de Toxicidade Aguda , Linhagem Celular , Poluentes Químicos da Água/toxicidade , Trifosfato de Adenosina/metabolismoRESUMO
Understanding the environmental impact of nanoparticle (NP) mixtures is essential to accurately assess the risk they represent for aquatic ecosystems. However, although the toxicity of individual NPs has been extensively studied, information regarding the toxicity of combined NPs is still comparatively rather scarce. Hence, this research aimed to investigate the individual and combined toxicity mechanisms of two widely consumed nanoparticles, zinc oxide (ZnO NPs) and titanium dioxide (TiO2 NPs), using an in vitro model, the RTgill-W1 rainbow trout gill epithelial cell line. Sublethal concentrations of ZnO NPs (0.1 µg mL-1) and TiO2 (30 µg mL-1) and a lethal concentration of ZnO NPs causing 10% mortality (EC10, 3 µg mL-1) were selected based on cytotoxicity assays. Cells were then exposed to the NPs at the selected concentrations alone and to their combination. Cytotoxicity assays, oxidative stress markers, and targeted gene expression analyses were employed to assess the NP cellular toxicity mechanisms and their effects on the gill cells. The cytotoxicity of the mixture was identical to the one of ZnO NPs alone. Enzymatic and gene expression (nrf2, gpx, sod) analyses suggest that none of the tested conditions induced a strong redox imbalance. Metal detoxification mechanisms (mtb) and zinc transportation (znt1) were affected only in cells exposed to ZnO NPs, while tight junction proteins (zo1 and cldn1), and apoptosis protein p53 were overexpressed only in cells exposed to the mixture. Osmoregulation (Na + /K + ATPase gene expression) was not affected by the tested conditions. The overall results suggest that the toxic effects of ZnO and TiO2 NPs in the mixture were significantly enhanced and could result in the disruption of the gill epithelium integrity. This study provides new insights into the combined effects of commonly used nanoparticles, emphasizing the importance of further investigating how their toxicity may be influenced in mixtures.
Assuntos
Brânquias , Oncorhynchus mykiss , Titânio , Óxido de Zinco , Animais , Óxido de Zinco/toxicidade , Titânio/toxicidade , Brânquias/efeitos dos fármacos , Linhagem Celular , Nanopartículas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Poluentes Químicos da Água/toxicidadeRESUMO
The aim of the experiment was to investigate the pharmacokinetics of oxytetracycline dihydrate after a single oral administration of 80 mg kg-1 day-1 in rainbow trout and assess its biosafety at concentration of 80, 240, 400, and 800 mg kg-1 day-1 over 30 days, focusing on various aspects such as effective feed consumption, physiological responses, drug tolerance, and detection of low drug concentrations in rainbow trout. The pharmacokinetics study spanned a duration of 5 days, while the assessment of biosafety extended for a 30-day safety margin, followed by a subsequent 10-day residual analysis. Pharmacokinetic analysis revealed slow absorption with low-rate constant in tissues. Absorption rates vary among tissues, with the gill showing the highest rate (0.011 h-1) and plasma exhibiting the slowest (0.0002 h-1). According to pharmacokinetic analysis, the highest concentration, Cmax (µg kg-1) was observed in the kidney (9380 µg kg-1) and gill (8710 µg kg-1), and lowest in muscle (2460 µg kg-1). The time (Tmax) to reach peak concentration (Cmax) varied among tissues, ranging from 3 h in the gill to 32 h in the muscle, with 24 h in plasma, 32 h in the kidney, and 16 h in both the liver and skin. The liver and kidney had the highest area under the concentration-time curve (AUC(0-128)), indicating widespread drug distribution. Prolonged elimination occurred at varying rates across tissues, with the gill showing the highest rate. The study found that OTC concentrations exceeded the LOD and LOQ values. Biosafety evaluation showed effective feed consumption, physiological responses, and low drug concentrations in muscle at the recommended dosage of 80 mg kg-1 fish day-1.
Assuntos
Oncorhynchus mykiss , Oxitetraciclina , Animais , Oncorhynchus mykiss/metabolismo , Oxitetraciclina/farmacocinética , Oxitetraciclina/administração & dosagem , Antibacterianos/farmacocinética , Antibacterianos/administração & dosagem , Administração Oral , Distribuição Tecidual , Brânquias/metabolismoRESUMO
Kynurenic acid (KYNA) is a metabolite of tryptophan formed on the kynurenine pathway. Its pharmacological effects are relatively well characterized in mammals, whereas its role in fish is poorly understood. Therefore, the aim of the study was to expand the knowledge of KYNA's presence inside a fish's body and its impact on fish development and function. The study was performed on zebrafish larvae and adult rainbow trout. We provide evidence that KYNA is present in the embryo, larva and mature fish and that its distribution in organs varies considerably. A study of KYNA's effect on early larval development suggests that it can accelerate larval maturation, especially under conditions that are suboptimal for fish growth. Moreover, KYNA in concentrations over 1 mM caused morphological impairment and death of larvae. However, long-lasting exposure of larvae to subtoxic concentrations of KYNA does not affect the behavior of 5-day-old larvae kept under standard optimal conditions. We also show that ingestion of KYNA-supplemented feed can lead to KYNA accumulation, particularly in the pyloric caeca of mature trout. These results shed new light on the relevance of KYNA and provide new impulse for further research on the importance of the kynurenine pathway in fish.
Assuntos
Embrião não Mamífero , Ácido Cinurênico , Larva , Oncorhynchus mykiss , Peixe-Zebra , Animais , Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Ácido Cinurênico/metabolismo , Ácido Cinurênico/farmacologia , Oncorhynchus mykiss/metabolismo , Oncorhynchus mykiss/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Larva/metabolismo , Larva/crescimento & desenvolvimento , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismoRESUMO
Fish are exposed to increased water temperatures and aquatic pollutants, including endocrine-disrupting compounds (EDCs). Although each stressor can disturb fish liver metabolism independently, combined effects may exist. To unveil the molecular mechanisms behind the effects of EDCs and temperature, fish liver cell lines are potential models needing better characterisation. Accordingly, we exposed the rainbow trout RTL-W1 cells (72 h), at 18 °C and 21 °C, to ethynylestradiol (EE2), levonorgestrel (LNG), and a mixture of both hormones (MIX) at 10 µM. The gene expression of a selection of targets related to detoxification (CYP1A, CYP3A27, GST, UGT, CAT, and MRP2), estrogen exposure (ERα, VtgA), lipid metabolism (FAS, FABP1, FATP1), and temperature stress (HSP70b) was analysed by RT-qPCR. GST expression was higher after LNG exposure at 21 °C than at 18 °C. LNG further enhanced the expression of CAT, while both LNG and MIX increased the expressions of CYP3A27 and MRP2. In contrast, FAS expression only increased in MIX, compared to the control. ERα, VtgA, UGT, CYP1A, HSP70b, FABP1, and FATP1 expressions were not influenced by the temperature or the tested EDCs. The RTL-W1 model was unresponsive to EE2 alone, sensitive to LNG (in detoxification pathway genes), and mainly insensitive to the temperature range but had the potential to unveil specific interactions.
Assuntos
Etinilestradiol , Levanogestrel , Oncorhynchus mykiss , Animais , Etinilestradiol/toxicidade , Levanogestrel/farmacologia , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/metabolismo , Estrogênios/metabolismo , Linhagem Celular , Disruptores Endócrinos/toxicidade , Inativação Metabólica/genética , Regulação para Cima/efeitos dos fármacos , Progestinas/farmacologia , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Poluentes Químicos da Água/toxicidade , Temperatura , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genéticaRESUMO
Systemic inflammation elicits sickness behaviors and fever by engaging a complex neuronal circuitry that begins in the preoptic area of the hypothalamus. Ectotherms such as teleost fish display sickness behaviors in response to infection or inflammation, seeking warmer temperatures to enhance survival via behavioral fever responses. To date, the hypothalamus is the only brain region implicated in sickness behaviors and behavioral fever in teleosts. Yet, the complexity of neurobehavioral manifestations underlying sickness responses in teleosts suggests engagement of higher processing areas of the brain. Using in vivo models of systemic inflammation in rainbow trout, we find canonical pyrogenic cytokine responses in the hypothalamus whereas in the telencephalon and the optic tectum il-1b and tnfa expression is decoupled from il-6 expression. Polyamine metabolism changes, characterized by accumulation of putrescine and decreases in spermine and spermidine, are recorded in the telencephalon but not hypothalamus upon systemic injection of bacteria. While systemic inflammation causes canonical behavioral fever in trout, blockade of bacterial polyamine metabolism prior to injection abrogates behavioral fever, polyamine responses, and telencephalic but not hypothalamic cytokine responses. Combined, our work identifies the telencephalon as a neuronal substrate for brain responses to systemic inflammation in teleosts and uncovers the role of polyamines as critical chemical mediators in sickness behaviors.
Assuntos
Inflamação , Oncorhynchus mykiss , Poliaminas , Telencéfalo , Animais , Telencéfalo/metabolismo , Poliaminas/metabolismo , Inflamação/metabolismo , Oncorhynchus mykiss/metabolismo , Oncorhynchus mykiss/imunologia , Neurônios/metabolismo , Hipotálamo/metabolismo , Espermina/metabolismo , Putrescina/metabolismo , Comportamento de Doença/fisiologia , Espermidina/metabolismoRESUMO
Copper (Cu) is recognized as an essential trace elements for the body; However, excessive levels of Cu can lead to toxic effects. We investigated the effects of Cu2+(75⯵g/L, 150⯵g/L, and 300⯵g/L) on the rainbow trout liver. Combination of transcriptome and metabolome analyses, the regulatory mechanisms of the liver under Cu stress were elucidated. The results showed that Cu affected the antioxidant levels, leading to disruptions in the normal tissue structure of the liver. Combined transcriptome and metabolome analyses revealed significant enrichment of the insulin signaling pathway and the adipocytokine signaling pathway. Additionally, Cu2+ stress altered the amino acid metabolism in rainbow trout by reducing serine and arginine levels while increasing proline content. Apoptosis is inhibited and autophagy and lipid metabolism are suppressed; In summary, Cu2+ stress affects energy and lipid metabolism, and the reduction of serine and arginine represents a decrease in the antioxidant capacity, whereas the increase in proline and the promotion of apoptosis potentially serving as crucial strategies for Cu2+ resistance in rainbow trout. These findings provided insights into the regulatory mechanisms of rainbow trout under Cu2+ stress and informed the prevention of heavy metal pollution and the selection of biomarkers under Cu pollution.
Assuntos
Cobre , Fígado , Metabolômica , Oncorhynchus mykiss , Transcriptoma , Poluentes Químicos da Água , Animais , Oncorhynchus mykiss/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Cobre/toxicidade , Transcriptoma/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Metabolismo dos Lipídeos/efeitos dos fármacos , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Metaboloma/efeitos dos fármacosRESUMO
The same elements can yield disparate nanoproducts that may elicit different harmful effects in cells and organisms. This study aimed to compare the effects of copper (Cu NPs) and copper oxide (CuO NPs) nanoparticles and Cu2+ (from CuSO4) on the physico-biochemical variables of rainbow trout spermatozoa. The cell death assay, along with the activation of caspases 8 and 9, the level of reactive oxygen species (ROS), and the percentage of cells exhibiting a high mitochondrial membrane potential (MMP) were quantified over 24-hour incubation. Interestingly, during exposure, all copper products induced cell apoptosis. However, Cu NPs had a stronger effect than CuO NPs, while the impact of the Cu in ionic form was found to be between the other two compounds. The extrinsic and intrinsic apoptotic pathways were activated, as evidenced by the activation of caspases 8 and 9. Initially, caspase activation increased without a corresponding decrease in MMPs but prolonged exposure resulted in a significant decrease in MMP levels. In all treated cells, the ROS levels increased over time. Further studies are needed to confirm the lower CuO NPs' toxicity compared to Cu NPs because their effect on cells also depends on many other parameters such as size or shape.
Assuntos
Apoptose , Cobre , Potencial da Membrana Mitocondrial , Nanopartículas Metálicas , Oncorhynchus mykiss , Espécies Reativas de Oxigênio , Espermatozoides , Animais , Cobre/toxicidade , Oncorhynchus mykiss/metabolismo , Masculino , Espécies Reativas de Oxigênio/metabolismo , Espermatozoides/efeitos dos fármacos , Espermatozoides/metabolismo , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Apoptose/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacosRESUMO
Identifying pathogenic microorganisms causing disease is important for epidemiological research, antimicrobial therapy, and control. The current study was carried out to use different methods for the identification of Vibrio anguillarum from diseased rainbow trout (Oncorhynchus mykiss) obtained from Türkiye (Mugla-Fethiye), the damage caused by the pathogenic microorganism in the tissues and organs, and the determination of the antibiotic effective against the pathogen. Hemorrhagic and ulcerative skin lesions and diffuse petechial hemorrhage in the internal organs were clinically detected in diseased fish obtained from the rainbow trout farm. Bacteria isolated from diseased fish were subjected to analysis using conventional bacteriological methods, a commercial bacterial identification test kit (API), an automated bacteria identification system known as Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), BD Phoenix™, and 16S rRNA sequence analysis. All isolated bacteria were identified as V. anguillarum by API 20E and conventional bacteriological method. These results have been confirmed with 16S rRNA sequence analysis. However, the isolated bacteria were identified as Grimontia hollisae (syn. Vibrio holisae) with the BD Phoenix system. Histologically, tissue damage such as melano-macrophage centers and necrosis in the kidney and spleen, hyperemia and mononuclear cell infiltration in the liver, as well as mononuclear cell infiltration on muscles, talengectiasis in the gill tissue was observed. In addition, it has been determined that the most effective antibiotic against the pathogen was enrofloxacin. When comparing all identification methods used for this pathogen causing tissue damage, it was demonstrated that the MALDI-TOF MS provides better results than other methods in terms of cost and identification time, and it could be used as an alternative to the conventional method to the identification of V. anguillarum.
Assuntos
Doenças dos Peixes , Oncorhynchus mykiss , RNA Ribossômico 16S , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vibrioses , Vibrio , Animais , Vibrio/isolamento & purificação , Vibrio/patogenicidade , Vibrio/efeitos dos fármacos , Oncorhynchus mykiss/microbiologia , Vibrioses/veterinária , Vibrioses/microbiologia , Doenças dos Peixes/microbiologia , RNA Ribossômico 16S/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Antibacterianos/farmacologia , FilogeniaRESUMO
Objective: The aim of this study was to determine the plasma pharmacokinetics of oxytetracycline (OTC) in rainbow trout (Oncorhynchus mykiss) of different body sizes. Methods: The research was carried out on three groups as small (30-50 g), medium (90-110 g) and large (185-215 g) body sizes at 8 ± 0.5 °C. OTC was administered orally at a dose of 60 mg/kg to all groups. Blood samples were taken at 19 different sampling times until the 384 h after oxytetracycline administration. The plasma concentrations of OTC were measured using high pressure liquid chromatography-ultraviolet and pharmacokinetic parameters were evaluated using non-compartmental analysis. Results: OTC was detected in small-body sized fish until the 336 h and in medium and large-body sized fish until the 384 h. The elimination half-life of OTC was 85.46, 87.24 and 86.98 h in the small, medium and large body size groups, respectively. The peak plasma concentration increased from 0.66 to 1.11 µg/mL, and the area under the plasma concentration-versus time curve from zero (0) h to infinity (∞) increased from 87.86 to 151.52 h*µg/mL, in tandem with the increase in fish body size. As fish body size increased, volume of distribution and total body clearance decreased. Conclusion: These results show that the pharmacokinetics of OTC vary depending on fish size. Therefore, there is a need to reveal the pharmacodynamic activity of OTC in rainbow trout of different body sizes.
Assuntos
Antibacterianos , Tamanho Corporal , Oncorhynchus mykiss , Oxitetraciclina , Animais , Oncorhynchus mykiss/metabolismo , Oxitetraciclina/farmacocinética , Oxitetraciclina/sangue , Oxitetraciclina/administração & dosagem , Administração Oral , Antibacterianos/farmacocinética , Antibacterianos/administração & dosagem , Antibacterianos/sangue , Meia-Vida , Cromatografia Líquida de Alta PressãoRESUMO
In the present study, we aimed to determine the parameters of oxidative stress markers, motility and kinematics of rainbow trout (Oncorhynchus mykiss) sperm cells exposed to different doses (0.001, 0.01, 0.1, 1.0, and 10 mg/L, in vitro 4 h) of tire rubber based microplastic particles (TRMP-Ps) the leachates procedure of rubber pieces. First of all, TRMP-Ps were prepared by abrasion method in accordance with the literature. Structural and morphological features of TRMP-Ps were determined by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) methods, respectively. Energy dispersive X-ray (EDX) analysis technique was used to characterize the elemental composition of TRMP-Ps. Particle size of microplastic structures was measured hydrodynamically with dynamic light scattering analysis (DLS). After exposure, the effect of TRMP-Ps was defined by the observations of kinematics and antioxidant activities in sperm cells. Our findings showed that the straight line velocity, the curvilinear velocity, the angular path velocity, and the amplitude of lateral displacement of sperm cells decreased. Moreover, while the level of superoxide dismutase decreased dose-dependently against the toxicity of TRMP-Ps, no significant change was observed in the levels of malondialdehyde and total glutathione. The 4-h median effective concentrations (EC50) of TRMP-Ps based on mobility parameters of sperm ranged from 0.31 mg/L for reduced straight line velocity of sperm cells to 0.51 mg/L for reduced amplitude of lateral displacement of the spermatozoa head. Therefore, we concluded that TRMP-Ps can be a risk for the reproduction cycle of fish in aquatic environments.
Assuntos
Microplásticos , Oncorhynchus mykiss , Borracha , Espermatozoides , Poluentes Químicos da Água , Animais , Masculino , Oncorhynchus mykiss/fisiologia , Poluentes Químicos da Água/toxicidade , Borracha/toxicidade , Microplásticos/toxicidade , Espermatozoides/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Motilidade dos Espermatozoides/efeitos dos fármacosRESUMO
BACKGROUND: The salmonid pathogen Flavobacterium psychrophilum poses a significant economic threat to global aquaculture, yet our understanding of its genetic and phenotypic diversity remains incomplete across much of its geographic range. In this study, we characterise the genetic and phenotypic diversity of 70 isolates collected from rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta m. fario) from fish farms in the Czech Republic between 2012 and 2019 to compare their genomic content with all draft or complete genomes present in the NCBI database (n = 187). RESULTS: The Czech isolates underwent comprehensive evaluation, including multiplex PCR-based serotyping, genetic analysis, antimicrobial resistance testing, and assessment of selected virulence factors. Multiplex PCR serotyping revealed 43 isolates as Type 1, 23 as Type 2, with sporadic cases of Types 3 and 4. Multi-locus sequence typing unveiled 12 sequence types (ST), including seven newly described ones. Notably, 24 isolates were identified as ST329, a novel sequence type, while 22 were classified as the globally-distributed ST2. Phylogenetic analysis demonstrated clonal distribution of ST329 in the Czech Republic, with these isolates lacking a phage sequence in their genomes. Antimicrobial susceptibility testing revealed a high proportion of isolates classified as non-wild type with reduced susceptibility to oxolinic acid, oxytetracycline, flumequine, and enrofloxacin, while most isolates were classified as wild type for florfenicol, sulfamethoxazole-trimethoprim, and erythromycin. However, 31 isolates classified as wild type for florfenicol exhibited minimum inhibitory concentrations at the susceptibility breakpoint. CONCLUSION: The prevalence of the Czech F. psychrophilum serotypes has evolved over time, likely influenced by the introduction of new isolates through international trade. Thus, it is crucial to monitor F. psychrophilum clones within and across countries using advanced methods such as MLST, serotyping, and genome sequencing. Given the open nature of the pan-genome, further sequencing of strains promises exciting discoveries in F. psychrophilum genomics.
Assuntos
Doenças dos Peixes , Infecções por Flavobacteriaceae , Flavobacterium , Variação Genética , Tipagem de Sequências Multilocus , Oncorhynchus mykiss , Filogenia , Animais , Flavobacterium/genética , Flavobacterium/isolamento & purificação , Flavobacterium/classificação , Flavobacterium/efeitos dos fármacos , República Tcheca , Doenças dos Peixes/microbiologia , Infecções por Flavobacteriaceae/microbiologia , Infecções por Flavobacteriaceae/veterinária , Oncorhynchus mykiss/microbiologia , Antibacterianos/farmacologia , Sorotipagem , Aquicultura , Fenótipo , Fatores de Virulência/genética , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana/genética , Genoma Bacteriano/genética , Truta/microbiologiaRESUMO
Aggregation is a fundamental feature of parasite distribution in the host population, but the biological implications of the aggregation indices used to describe the relationships between the populations of parasites and hosts are not evident. It is speculated that the form of distribution in each case is predicated on the host's varying resistance to the infection, which is hard to control, making it difficult to adequately interpret the index values. This paper examines several cases from trout farms in Russian Karelia to explore the monogenean Gyrodactylus spp. infection in rainbow trout of varying ages. The genetic homogeneity of cage-reared fish and the direct life cycle of the helminths make the relationship between the species more lucid than in natural host-parasite systems. The results give no ground to speak of any specific patterns: as well as in the natural systems, the infection rates in trout vary widely, i.e., the helminth distribution has not become more uniform; the observed distributions in all cases are adequately approximated by the negative binomial model; the positive abundance-occupancy relationships (AORs) and abundance-variance relationships (AVRs) common for parasitic systems apply to the basic infection parameters. The form of the negative binomial distribution is shaped by two parameters-k and θ, the former being a metric of the infection variability, which depends on the host's individual resistance, and the latter representing the parasites' reproduction and establishment success rates. A rise in the parameter k indicates increased aggregation and a higher parameter θ points to a more uniform frequency distribution. These parameters can be used as a representative tool for monitoring the parasite communities in salmonid fishes, including in aquaculture.
Assuntos
Doenças dos Peixes , Interações Hospedeiro-Parasita , Oncorhynchus mykiss , Trematódeos , Infecções por Trematódeos , Animais , Oncorhynchus mykiss/parasitologia , Doenças dos Peixes/parasitologia , Infecções por Trematódeos/veterinária , Infecções por Trematódeos/parasitologia , Trematódeos/fisiologia , Trematódeos/genética , Trematódeos/classificação , Trematódeos/isolamento & purificação , Federação Russa , Platelmintos/fisiologia , Platelmintos/genética , Platelmintos/classificaçãoRESUMO
There is a consensus that electroneutral Na+/H+ exchangers (NHEs) are important in branchial Na+ uptake in freshwater fish. There is also widespread belief, based on mammalian data, that EIPA [5-(N-ethyl-N-isopropyl)-amiloride]], and HMA [5-(N,N-hexamethylene)-amiloride)] are more potent and specific in blocking Na+ uptake than amiloride. We evaluated this idea by testing the three drugs at 10-7 to 10-4 M, i.e. 0.1 to 100 µM in two model species, rainbow trout (Oncorhynchus mykiss) and goldfish (Carassius auratus), using 22Na+ to measure unidirectional Na+ influx and efflux rates. In both species, the potency order for inhibiting unidirectional Na+ influx was HMA > amiloride > EIPA (IC50 values in the 10-70 µM range), very different from in mammals. At 100 µM, all three drugs inhibited Na+ influx by >90% in both species, except for amiloride in goldfish (65%). However, at 60-100 µM, all three drugs also stimulated unidirectional Na+ efflux rates, indicating non-specific effects. In trout, HMA and EIPA caused significant increases (2.1- to 2.3-fold) in efflux rates, whereas in goldfish, significant efflux elevations were greater (3.1- to 7.2-fold) with all three drugs. We conclude that the inhibitory potency profile established in mammals does not apply to the NHEs in fish gills, that non-specific effects on Na+ efflux rates are a serious concern, and that EIPA and HMA offer no clear benefits in terms of potency or specificity. Considering its much lower cost, we recommend amiloride as the drug of choice for in vivo experiments on freshwater fishes.
Assuntos
Amilorida , Carpa Dourada , Sódio , Animais , Amilorida/farmacologia , Amilorida/análogos & derivados , Carpa Dourada/metabolismo , Sódio/metabolismo , Brânquias/metabolismo , Brânquias/efeitos dos fármacos , Oncorhynchus mykiss/metabolismo , Água Doce , Trocadores de Sódio-Hidrogênio/metabolismo , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Transporte de Íons/efeitos dos fármacos , Truta/metabolismoRESUMO
Aeromonas salmonicida is an opportunistic pathogen with relevance for aquaculture. Fish epithelia are covered by a mucus layer, composed mainly by highly glycosylated mucins, which are the first point of contact between fish and pathogens. Quorum sensing (QS), a bacterial communication mechanism through secreted autoinducer signals that governs gene expression, influences bacterial growth and virulence. The main A. salmonicida autoinducers are mediated by the luxS and asaI genes, corresponding to inter- and intraspecies communication, respectively. The aim of this study was to determine the effect of the mucins that pathogens encounter during colonization of the gill and skin on A. salmonicida QS. We found that expression of A. salmonicida asaI, but not luxS, was increased after culture at 20 °C compared to 10 °C. Rainbow trout gill and skin mucins up-regulated asaI expression 2-fold but down-regulated luxS 10-fold. The downregulation of luxS was reflected by a reduction in autoinducer-2 secretion. Mucins isolated from skin had a stronger inhibitory effect than mucins isolated from gills on both luxS expression and A1-2 secretion, consistent with a higher relative abundance of N-Acetylneuraminic acid on skin mucins than on gill mucins. Reduction of AI-2 production by mucins or luxS-deletion lead to a reduced A. salmonicida auto-aggregation. Furthermore, after colonization of the gill, luxS was down regulated whereas asaI expression was upregulated. Both in vivo and in vitro, the expression of luxS and asaI were thus differentially regulated, frequently in an inverse manner. The strong AI-2 inhibiting effect of the skin mucins is likely part of the mucin-based defense against pathogens.
Assuntos
Aeromonas salmonicida , Homosserina , Mucinas , Oncorhynchus mykiss , Percepção de Quorum , Animais , Oncorhynchus mykiss/imunologia , Aeromonas salmonicida/fisiologia , Mucinas/genética , Mucinas/metabolismo , Homosserina/análogos & derivados , Liases de Carbono-Enxofre/genética , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária , Proteínas de Bactérias/genética , Lactonas , Pele/imunologia , Pele/microbiologia , Brânquias/imunologia , Brânquias/metabolismoRESUMO
Changes to ivermectin (IVM [22,23-dihydro avermectin B1a + 22,23-dihydro avermectin B1b]) toxicokinetics (TK) with and without P-glycoprotein (P-gp) inhibition by cyclosporin A (CsA) were examined in rainbow trout (Oncorhynchus mykiss). Rainbow trout were injected with 175 µg/kg 3H-IVM (8.6 µCi/mg IVM) with or without co-administration of 480 µg/kg CsA into the caudal vasculature. Fish were sacrificed at various time points (0.25, 0.5, 1, 3, 24, 48, 96, and 168 h) for organ and tissue sampling (blood, liver, kidney, gill, intestines, brain [5 regions], eye, gonad, and fat) which were analyzed for IVM-derived radioactivity. The IVM concentration decreased over time in blood, liver, kidney, and gill, while concentrations in other tissues remained constant. The highest maximum IVM concentration (Cmax) was found in kidney, followed by liver; the lowest Cmax was found in eye, followed by brain and adipose tissue. The highest % of the administered dose was found in the blood 15 min post-IVM administration, followed by the intestine at 60 min post-IVM administration. P-gp inhibition by CsA did not significantly affect calculated TK parameters (AUC [7.33 ± 0.73 - 11.5 ± 2.5 mgâ¢h/kg], mean residence time [84.7 ± 21 - 125 ± 55 h], T1/2 [58.7 ± 15 - 86.8 ± 38 h], clearance rate [0.0152 ± 0.0033 - 0.0239 ± 0.0024 L/kgâ¢h], or volume of distribution [1.91 ± 0.47 - 2.02 ± 0.33 L/kg]), but resulted in small but significant changes in the % administered dose found in blood and medulla. These results suggest that P-gp plays a limited role in overall IVM TK, and that its role in xenobiotic protection may be much less robust in fish than it is in mammals.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Ciclosporina , Ivermectina , Oncorhynchus mykiss , Animais , Oncorhynchus mykiss/metabolismo , Ivermectina/análogos & derivados , Ivermectina/farmacocinética , Ivermectina/toxicidade , Ivermectina/sangue , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Ciclosporina/farmacocinética , Toxicocinética , Antiparasitários/farmacocinética , Distribuição TecidualRESUMO
The growth hormone (GH)-insulin-like growth factor-1 (IGF-1) system regulates skeletal muscle growth and function. GH has a major function of targeting the liver to regulate IGF-1 production and release, and IGF-1 mediates the primary anabolic action of GH on growth. However, skeletal muscle is a target tissue of GH as evidenced by dynamic GH receptor expression, but it is unclear if GH elicits any direct actions on extrahepatic tissues as it is difficult to distinguish the effects of IGF-1 from GH. Fish growth regulation is complex compared to mammals, as genome duplication events have resulted in multiple isoforms of GHs, GHRs, IGFs, and IGFRs expressed in most fish tissues. This study investigated the potential for GH direct actions on fish skeletal muscle using an in vitro system, where rainbow trout myogenic precursor cells (MPCs) were cultured in normal and serum-deprived media, to mimic in vivo fasting conditions. Fasting reduces IGF-1 signaling in the muscle, which is critical for disentangling the roles of GH from IGF-1. The direct effects of GH were analyzed by measuring changes in myogenic proliferation and differentiation genes, as well as genes regulating muscle growth and proteolysis. This study provides the first in-depth analysis of the direct actions of GH on serum-deprived fish muscle cells in vitro. Data suggest that GH induces the expression of markers for proliferation and muscle growth in the presence of serum, but all observed GH action was blocked in serum-deprived conditions. Additionally, serum deprivation alone reduced the expression of several proliferation and differentiation markers, while increasing growth and proteolysis markers. Results also demonstrate dynamic gene expression response in the presence of GH and a JAK inhibitor in serum-provided but not serum-deprived conditions. These data provide a better understanding of GH signaling in relation to serum in trout muscle cells in vitro.
Assuntos
Hormônio do Crescimento , Fator de Crescimento Insulin-Like I , Músculo Esquelético , Oncorhynchus mykiss , Animais , Oncorhynchus mykiss/metabolismo , Oncorhynchus mykiss/genética , Hormônio do Crescimento/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Transdução de Sinais , Células Cultivadas , Proteínas de Peixes/metabolismo , Proteínas de Peixes/genética , Receptores da Somatotropina/metabolismo , Receptores da Somatotropina/genéticaRESUMO
The aim of our study was to investigate the effects of cyanobacterial metabolites: microcystin-LR (MC-LR) anabaenopeptin-A (ANA-A), cylindrospermopsin (CYL), their binary and ternary mixtures on rainbow trout (Oncorhynchus mykiss) gill (RTgill-W1) cell line. We determined the following cell parameters: Hoechst and propidium iodide (PI) double staining, intracellular ATP level with luminometric assay, glutathione level with ThiolTracker Violet®- glutathione detection reagent and cytoskeletal F-actin fluorescence. The results showed that although reduction of Hoechst fluorescence was observed in both binary and ternary combinations of cyanobacterial metabolites, the mixture of MC-LR + ANA-A + CYL was the most potent inhibitor (EC50 = 148 nM). PI fluorescence and ATP levels were more increased in the cells exposed to the mixtures than those exposed to the individual metabolites with synergistic toxic changes suggesting apoptosis as the mechanism of cell death. Reduced glutathione level was also decreased in cells exposed both to single metabolites and their mixtures with the highest decrease and synergistic effects at 334 nM MC-LR+334 nM ANA-A+ 334 nM CYL suggesting induction oxidative stress by the tested compounds. Reduction of F-actin fluorescence was found in the cells from all of the groups exposed to individual metabolites and their mixtures, however the highest level of inhibition showed the binary MC-LR + CYL and the ternary MC-LR + ANA-A + CYL with synergistic interactions. The study suggests that in natural conditions fish gill cells may be very sensitive to individual cyanobacterial metabolites and more prone to their binary and ternary mixtures.
Assuntos
Alcaloides , Toxinas de Cianobactérias , Cianobactérias , Toxinas Marinhas , Microcistinas , Oncorhynchus mykiss , Uracila , Microcistinas/metabolismo , Animais , Alcaloides/farmacologia , Uracila/análogos & derivados , Linhagem Celular , Cianobactérias/metabolismo , Oncorhynchus mykiss/metabolismo , Brânquias/metabolismo , Brânquias/efeitos dos fármacos , Glutationa/metabolismo , Toxinas Bacterianas , Trifosfato de Adenosina/metabolismo , Apoptose/efeitos dos fármacos , Peptídeos Cíclicos/farmacologiaRESUMO
Triploid Oncorhynchus mykiss is an important economic fish worldwide. Fishing stress can affect its growth and meat quality. This study first explored the effects of fishing stress on fatty acid and amino acid in triploid O. mykiss. Results showed fishing stress significantly reduced the content of docosadienoic acid, Gly, Arg, and DAA (P < 0.05). Targeted lipidomics analysis furthered suggested that some lipid molecules belonging to TG, DG, PC, Cer, ChE, and So were significantly up-regulated; while some lipid molecules belonging to Cer, LPE, LPC, PS, PC, and SM were significantly down-regulated, suggesting an accelerated glycolipid metabolism. Eventually, the glycolipid metabolism-related enzyme activity and gene expressions were examined, and the results indicated that O. mykiss was anti-oxidative stress by affecting relevant glycolipid metabolism signaling pathways and participating in cellular redox homeostasis. Findings of this study provide a theoretical foundation for further investigation into the mechanisms through which fishing stress affects O. mykiss.
Assuntos
Aminoácidos , Ácidos Graxos , Glicolipídeos , Oncorhynchus mykiss , Animais , Oncorhynchus mykiss/metabolismo , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/crescimento & desenvolvimento , Aminoácidos/metabolismo , Aminoácidos/análise , Glicolipídeos/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos/química , Triploidia , Estresse Fisiológico , Metabolismo dos Lipídeos , PesqueirosRESUMO
The frequency of detection and concentrations of bifenthrin, a pyrethroid insecticide, in the waterways inhabited by the endangered species, steelhead trout (Oncorhynchus mykiss), has become a significant concern for regulatory agencies. Endocrine disruption has been observed with estrogenic and anti-estrogenic responses in fish species at different life stages. Since several studies have indicated alterations in dopaminergic signaling associated with endocrine responses, juvenile steelhead were exposed to environmentally relevant concentrations of 60 or 120 ng/L bifenthrin for two weeks. Fish brains were assessed for dopamine levels and the expression of genes involved in dopaminergic and estrogenic processes, such as catechol-o-methyltransferase (comt) and monoamine oxidase (mao). Vitellogenin (vtg) and estrogenic receptors (ERα1, ERß1, and ERß2) were also evaluated in livers of the animals. Dopamine concentrations were significantly higher in fish brains following bifenthrin exposure. Consistent with a reduction in dopamine clearance, there was a significant decrease in the mRNA expression of comt with increased bifenthrin concentration. Hepatic expression of ERα1 and ERß2 mRNA was significantly decreased with increased bifenthrin concentration. These data support the possible mechanism of bifenthrin altering the dopaminergic pathway at low ng/L concentrations, in juvenile steelhead, which could interfere with endocrine feedback loops. These findings support the need for and importance of identifying species and life stage differences in pesticide modes of action to reduce uncertainties in risk assessments.