Effect of microplastics on oxytetracycline trophic transfer: Immune, gut microbiota and antibiotic resistance gene responses.

Microplastics and antibiotics are prevalent and emerging pollutants in aquatic ecosystems, but their interactions in aquatic food chains remain largely unexplored. This study investigated the impact of polypropylene microplastics (PP-MPs) on oxytetracycline (OTC) trophic transfer from the shrimp (Neocaridina denticulate) to crucian carp (Carassius auratus) by metagenomic sequencing. The carrier effects of PP-MPs promoted OTC bioaccumulation and trophic transfer, which exacerbated enterocyte vacuolation and hepatocyte eosinophilic necrosis. PP-MPs enhanced the inhibitory effect of OTC on intestinal lysozyme activities and complement C3 levels in shrimp and fish, and hepatic immunoglobulin M levels in fish (p < 0.05). Co-exposure of MPs and OTC markedly increased the abundance of Actinobacteria in shrimp and Firmicutes in fish, which caused disturbances in carbohydrate, amino acid, and energy metabolism. Moreover, OTC exacerbated the enrichment of antibiotic resistance genes (ARGs) in aquatic animals, and PP-MPs significantly increased the diversity and abundance of ARGs and facilitated the trophic transfer of teta and tetm. Our findings disclosed the impacts of PP-MPs on the mechanism of antibiotic toxicity in aquatic food chains and emphasized the importance of gut microbiota for ARGs trophic transfer, which contributed to a deeper understanding of potential risks posed by complex pollutants on aquatic ecosystems.

Effects of supplementing coated methionine in a high plant-protein diet on growth, antioxidant capacity, digestive enzymes activity and expression of TOR signaling pathway associated genes in gibel carp, Carassius auratus gibelio.

This study explored the impacts of supplementation of different levels of coated methionine (Met) in a high-plant protein diet on growth, blood biochemistry, antioxidant capacity, digestive enzymes activity and expression of genes related to TOR signaling pathway in gibel carp (Carassius auratus gibeilo). A high-plant protein diet was formulated and used as a basal diet and supplemented with five different levels of coated Met at 0.15, 0.30, 0.45, 0.60 and 0.75%, corresponding to final analyzed Met levels of 0.34, 0.49, 0.64, 0.76, 0.92 and 1.06%. Three replicate groups of fish (initial mean weight, 11.37 ± 0.02 g) (20 fish per replicate) were fed the test diets over a 10-week feeding period. The results indicated that with the increase of coated Met level, the final weight, weight gain (WG) and specific growth rate initially boosted and then suppressed, peaking at 0.76% Met level (P< 0.05). Increasing dietary Met level led to significantly increased muscle crude protein content (P< 0.05) and reduced serum alanine aminotransferase activity (P< 0.05). Using appropriate dietary Met level led to reduced malondialdehyde concentration in hepatopancreas (P< 0.05), improved superoxide dismutase activity (P< 0.05), and enhanced intestinal amylase and protease activities (P< 0.05). The expression levels of genes associated with muscle protein synthesis such as insulin-like growth factor-1, protein kinase B, target of rapamycin and eukaryotic initiation factor 4E binding protein-1 mRNA were significantly regulated, peaking at Met level of 0.76% (P< 0.05). In conclusion, supplementing optimal level of coated Met improved on fish growth, antioxidant capacity, and the expression of TOR pathway related genes in muscle. The optimal dietary Met level was determined to be 0.71% of the diet based on quadratic regression analysis of WG.

Intermolecular Interactions between Cysteine and Aromatic Amino Acids with a Phenyl Moiety in the DNA-Binding Domain of Heat Shock Factor 1 Regulate Thermal Stress-Induced Trimerization.

In this study, we investigated the trimerization mechanism and structure of heat shock factor 1 (HSF1) using western blotting, tryptophan (Trp) fluorescence spectroscopy, and molecular modeling. First, we examined the DNA-binding domains of human (Homo sapiens), goldfish (Carassius auratus), and walleye pollock (Gadus chalcogrammus) HSF1s by mutating key residues (36 and 103) that are thought to directly affect trimer formation. Human, goldfish, and walleye pollock HSF1s contain cysteine at residue 36 but cysteine (C), tyrosine (Y), and phenylalanine (F), respectively, at residue 103. The optimal trimerization temperatures for the wild-type HSF1s of each species were found to be 42, 37, and 20 °C, respectively. Interestingly, a mutation experiment revealed that trimerization occurred at 42 °C when residue 103 was cysteine, at 37 °C when it was tyrosine, and at 20 °C when it was phenylalanine, regardless of the species. In addition, it was confirmed that when residue 103 of the three species was mutated to alanine, trimerization did not occur. This suggests that in addition to trimerization via disulfide bond formation between the cysteine residues in human HSF1, trimerization can also occur via the formation of a different type of bond between cysteine and aromatic ring residues such as tyrosine and phenylalanine. We also confirmed that at least one cysteine is required for the trimerization of HSF1s, regardless of its position (residue 36 or 103). Additionally, it was shown that the trimer formation temperature is related to growth and survival in fish.

Roles of ß-catenin in innate immune process and regulating intestinal flora in Qi river crucian carp (Carassius auratus).

In mammals, ß-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of ß-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). ß-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, ß-catenin increased in gill, intestine, liver and kidney, indicating that ß-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the ß-catenin pathway using FH535, an inhibitor of Wnt/ß-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3, defb3, LYZ-C, INF-γ), upregulation of inflammatory factors (NF-κB, TNF-α, IL-1, IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that ß-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp.

Ginger polysaccharide alleviates the effects of acute exposure to carbonate in crucian carp (Carassius auratus) by regulating immunity, intestinal microbiota, and intestinal metabolism.

Alkaline stress poses a significant challenge to the healthy growth of fish. Ginger polysaccharide (GP) is one of the main active substances in ginger and has pharmacological effects, such as anti-oxidation and immune regulation. However, the physiological regulatory mechanism of GP addition to diet on alkalinity stress in crucian carp remains unclear. This study aimed to investigate the potential protective effects of dietary GP on antioxidant capacity, gene expression levels, intestinal microbiome, and metabolomics of crucian carp exposed to carbonate (NaHCO3). The CK group (no GP supplementation) and COG group (NaHCO3 stress and no GP supplementation) were set up. The GPCS group (NaHCO3 stress and 0.4% GP supplementation) was stressed for seven days. Based on these data, GP significantly increased the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), acid phosphatase (ACP), and alkaline phosphatase (AKP) in carp under alkalinity stress (p < 0.05) and decreased the activity of malon dialdehyde (MDA) (p < 0.05). GP restored the activity of GSH-PX, ACP, and AKP to CK levels. The expression levels of tumor necrosis factor ß (TGF-ß), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), and interleukin 8 (IL-8) genes were decreased, and the expression levels of determination factor kappa-B (NF-κB) and interleukin 10 (IL-10) genes were increased (p < 0.05). Based on 16 S rRNA high-throughput sequencing, GP improved the changes in the intestinal microbial diversity and structural composition of crucian carp caused by NaHCO3 exposure. In particular, GP increased the relative abundance of Proteobacteria and Bacteroidetes and decreased the relative abundance of Actinobacteria. The metabolic response of GP to NaHCO3 exposed crucian carp guts was studied using LC/MS. Compared to the COG group, the GPCS group had 64 different metabolites and enriched 10 metabolic pathways, including lipid metabolism, nucleotide metabolism, and carbohydrate metabolism. The addition of GP to feed can promote galactose metabolism and provide an energy supply to crucian carp, thus alleviating the damage induced by alkalinity stress. In conclusion, GP can mitigate the effects of NaHCO3 alkalinity stress by regulating immune function, intestinal flora, and intestinal metabolism in crucian carp. These findings provide a novel idea for studying the mechanism of salt-alkali tolerance in crucian carp by adding GP to feed.

Evidence of binding between diethylstilbestrol (DES) and the goldfish (Carassius auratus) membrane progesterone receptor α.

BACKGROUND: In a previous study, diethylstilbestrol (DES) was shown to induce oocyte maturation in fish. In the present study, the interaction of DES on goldfish membrane progesterone receptor α (GmPRα) was investigated using a competitive binding assay with radiolabeled steroids. The results indicate that DES exerts its effects on membrane progesterone receptor alpha (mPRα) and induces oocyte maturation through nongenomic steroid mechanisms. This study provides empirical data that demonstrate the binding between DES and GmPRα. METHODS: Binding of DES to GmPRα was achieved by using radiolabeled DES and recombinant GmPRα expressed in culture cells or purified GmPRα proteins that coupled to graphene quantum dots (GQDs). Additionally, the competitive binding of fluorescently labeled progesterone to GmPRα-expressing cells was evaluated. RESULTS: Although significant nonspecific binding of radiolabeled DES to the cell membrane that expresses GmPRα has been observed, specific binding of DES to GmPRα has been successfully identified in the presence of digitonin. Furthermore, the specific binding of DES to GmPRα was confirmed by a binding assay using GQD-GmPRα. The radiolabeled DES was shown to bind to GQD-GmPRα. Additionally, the competition for the binding of fluorescently labeled progesterone to GmPRα-expressing cells was achieved with the DES. CONCLUSIONS: The results of the experiments revealed that DES binds to GmPRα. Thus, it can be concluded that DES induces goldfish oocyte maturation by binding to GmPRα.

Functional, structural, and molecular remodelling of the goldfish (Carassius auratus) heart under moderate hypoxia.

The goldfish (Carassius auratus) is known for its physiologic ability to survive even long periods of oxygen limitation (hypoxia), adapting the cardiac performance to the requirements of peripheral tissue perfusion. We here investigated the effects of short-term moderate hypoxia on the heart, focusing on ventricular adaptation, in terms of hemodynamics and structural traits. Functional evaluations revealed that animals exposed to 4 days of environmental hypoxia increased the hemodynamic performance evaluated on ex vivo cardiac preparations. This was associated with a thicker and more vascularized ventricular compact layer and a reduced luminal lacunary space. Compared to normoxic animals, ventricular cardiomyocytes of goldfish exposed to hypoxia showed an extended mitochondrial compartment and a modulation of proteins involved in mitochondria dynamics. The enhanced expression of the pro-fission markers DRP1 and OMA1, and the modulation of the short and long forms of OPA1, suggested a hypoxia-related mitochondria fission. Our data propose that under hypoxia, the goldfish heart undergoes a structural remodelling associated with a potentiated cardiac activity. The energy demand for the highly performant myocardium is supported by an increased number of mitochondria, likely occurring through fission events.

Gibberellic acid induced oxidative stress, endoplasmic reticulum stress, and apoptosis in the livers of gibel carp (Carassius auratus gibelio).

Gibberellic acid (GA3), one of the most plant growth stimulator, is widely applied in agricultural regions and in beer industry. However, GA3 residue remained in soil and water can cause toxicity to all organisms. In this study, we investigated the mechanisms of GA3-induced hepatic injury in gibel carp (Carassius auratus gibelio). We found that GA3 exposure caused oxidative stress, endoplasmic reticulum stress (ERS), and apoptosis. The gibel carp exposed to GA3 exhibited significant alteration in erythrocyte nuclei. GA3 induced liver damage, as indicated by increasing the aminopherase activities. GA3 led to oxidative stress by increasing malondialdehyde content and decreasing the activities of CAT and GPx. GA3 stimulated ERS and increased the expression of grp78, perk, eif2s1α, chop, atf4, ire1α, xbp1, and atf6. Additionally, GA3 down-regulated the level of anti-apoptotic gene Bcl-2 and up-regulated the levels of pro-apoptotic genes bax and caspase-3. Overall results demonstrated that GA3 caused hepatic injury in gibel carp by increasing oxidative stress, ERS, and apoptosis.

Effects of environmental hypoxia on the goldfish skeletal muscle: Focus on oxidative status and mitochondrial dynamics.

The skeletal muscle is a highly plastic tissue. Its ability to respond to external stimuli and challenges allows it to face the functional needs of the organism. In the goldfish Carassius auratus, a model of hypoxia resistance, exposure to reduced oxygen is accompanied by an improvement of the swimming performance, relying on a sustained contractile behavior of the skeletal muscle. At the moment, limited information is available on the mechanisms underlying these responses. We here evaluated the effects of short- (4 days) and long- (20 days) term exposure to moderate water hypoxia on the goldfish white skeletal muscle, focusing on oxidative status and mitochondrial dynamics. No differences in lipid peroxidation, measured as 2-thiobarbituric acid-reacting substances (TBARS), and oxidatively modified proteins (OMP) were detected in animals exposed to hypoxia with respect to their normoxic counterparts. Exposure to short-term hypoxia was characterized by an enhanced SOD activity and expression, paralleled by increased levels of Nrf2, a regulator of the antioxidant cell response, and HSP70, a chaperone also acting as a redox sensor. The expression of markers of mitochondrial biogenesis (TFAM) and abundance (VDAC) and of the mtDNA/nDNA ratio was similar under normoxia and under both short- and long-term hypoxia, thus excluding a rearrangement of the mitochondrial apparatus. Only an increase of PGC1α (a transcription factor involved in mitochondrial dynamics) was detected after 20 days of hypoxia. Our results revealed novel aspects of the molecular mechanisms that in the goldfish skeletal muscle may sustain the response to hypoxia, thus contributing to adequate tissue function to organism requirements.

Toxic effects of polystyrene microbeads and benzo[α]pyrene on bioaccumulation, antioxidant response, and cell damage in goldfish Carassius auratus.

Microplastics (MP) are harmful, causing stress in aquatic species and acting as carriers of hydrophobicity. In aquatic environments, benzo[α]pyrene (BaP) is an endocrine-disrupting chemical that accumulates in the body and causes toxic reactions in living organisms. We investigated the effects of single and combined microbead (MB) and BaP environments on goldfish antioxidant response and apoptosis. For 120 h, goldfish were exposed to single (MB10, MB100, and BaP5) and combined (MB10+BaP5 and MB100+BaP5) environments of 10 and 100 beads/L of 0.2 µm polystyrene MB and 5 µg/L BaP. We measured MB and BaP bioaccumulation as well as plasma parameters including ALT, AST, and glucose. The level of oxidative stress was determined by evaluating lipid peroxidation (LPO) and total antioxidant capacity (TAC) in plasma, as well as antioxidant-related genes for superoxide dismutase and catalase (SOD and CAT) and caspase-3 (Casp3) mRNA expression in liver tissue. The TUNEL assay was used to examine SOD in situ hybridization and apoptosis in goldfish livers. Except for the control group, plasma LPO levels increased at the end of the exposure period in all experimental groups. TAC increased up to 24 h of exposure and then maintained a similar level until the trial ended. SOD, CAT, and Casp3 mRNA expression increased substantially up to 120 h as the exposure concentration and time increased. The TUNEL assay revealed more signals and apoptotic signals in the combined exposure environments as a consequence of SOD in situ hybridization than in single exposure environments. These results suggest that combined exposure to toxic substances causes oxidative stress in organisms, which leads to apoptosis.

Study on the acute toxicity of trichlorfon and its breakdown product dichlorvos to goldfish (Carassius auratus) based on 1H NMR metabonomics.

Trichlorfon, one of the most widely used organophosphate insecticides, is commonly employed in aquaculture and agriculture to combat parasitic infestations. However, its inherent instability leads to rapid decomposition into dichlorvos (DDVP), increasing its toxicity by eightfold. Therefore, the environmental effects of trichlorfon in real-world scenarios involve the combined effects of trichlorfon and its degradation product, DDVP. In this study, we systematically investigated the degradation of trichlorfon in tap water over time using HPLC and LC-MS/MS analysis. Subsequently, an experiment was conducted to assess the acute toxicity of trichlorfon and DDVP on goldfish (Carassius auratus), employing a 1H NMR-based metabolic approach in conjunction with serum biochemistry, histopathological inspection, and correlation network analysis. Exposure to trichlorfon and its degradation product DDVP leads to increased lipid peroxidation, reduced antioxidant activity, and severe hepatotoxicity and nephrotoxicity in goldfish. Based on the observed pathological changes and metabolite alterations, short-term exposure to trichlorfon significantly affected the liver and kidney functions of goldfish, while exerting minimal influence on the brain, potentially due to the presence of the blood-brain barrier. The changes in the metabolic profile indicated that trichlorfon and DDVP influenced several pathways, including oxidative stress, protein synthesis, energy metabolism, and nucleic acid metabolism. This study demonstrated the applicability and potential of 1H NMR-based metabonomics in pesticide environmental risk assessment, providing a feasible method for the comprehensive study of pesticide toxicity in water environments.

Effects of thermal stress responses in goldfish (Carassius auratus): growth performance, total carotenoids and coloration, hematology, liver histology, and critical thermal maximum.

The present study aimed to investigate the effect of thermal stress on growth, feed utilization, coloration, hematology, liver histology, and critical thermal maximum (CTmax) in goldfish (Carassius auratus) cultured at three different acclimation temperatures including 27 °C, 30 °C, and 34 °C for 10 weeks. Goldfish were assigned randomly to tanks with a quadruplicate setup, accommodating 20 fish per tank. The result showed that fish acclimated to different temperatures did not significantly differ in weight gain (WG) and specific growth rate (SGR). However, increasing temperature significantly decreased feed efficiency ratio (FER), protein efficiency ratio (PER), and protein productive value (PPV), but significantly increased feed conversion ratio (FCR) (P < 0.05). The coloration parameters significantly decreased by high temperature in the trunk region with increasing temperature (L* and a* at week 5; L*, a*, and b* at week 10; P < 0.05). Total carotenoid contents in serum, fin, muscle, and skin also significantly decreased with increasing temperature (P < 0.05). Total protein, albumin, and globulin levels exhibited a notable decrease, while the albumin: globulin ratio showed a slight insignificant increase, with increasing temperature. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol, and triglycerides significantly increased with increasing temperature (P < 0.05). While, high-density lipoprotein cholesterol (HDL-c) decreased linearly (P < 0.05). Glucose and cortisol levels linearly increased with increasing temperature, the highest levels being observed in the 34 °C group. Liver histology showed swollen hepatocytes, nuclei displacement, and infiltration of inflammation in fish cultured at 34 °C. Goldfish acclimated to 34 °C displayed a higher CTmax of 43.83 °C compared to other groups. The present study showed that temperature should be kept below 34 °C for goldfish culture to prevent high FCR, fading coloration, and liver damages.

α-lipoic acid ameliorates hepatotoxicity induced by chronic ammonia toxicity in crucian carp (Carassius auratus gibelio) by alleviating oxidative stress, inflammation and inhibiting ERS pathway.

High environment ammonia (HEA) poses a deadly threat to aquatic animals and indirectly impacts human healthy life, while nutritional regulation can alleviate chronic ammonia toxicity. α-lipoic acid exhibits antioxidative effects in both aqueous and lipid environments, mitigating cellular and tissue damage caused by oxidative stress by aiding in the neutralization of free radicals (reactive oxygen species). Hence, investigating its potential as an effective antioxidant and its protective mechanisms against chronic ammonia stress in crucian carp is highly valuable. Experimental fish (initial weight 20.47 ± 1.68 g) were fed diets supplemented with or without 0.1% α-lipoic acid followed by a chronic ammonia exposure (10 mg/L) for 42 days. The results revealed that chronic ammonia stress affected growth (weight gain rate, specific growth rate, and feed conversion rate), leading to oxidative stress (decreased the activities of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase; decreased total antioxidant capacity), increased lipid peroxidation (accumulation of malondialdehyde), immune suppression (decreased contents of nonspecific immune enzymes AKP and ACP, 50% hemolytic complement, and decrease of immunoglobulin M), impaired ammonia metabolism (reduced contents of Glu, GS, GSH, and Gln), imbalance of expression of induced antioxidant-related genes (downregulation of Cu/Zu SOD, CAT, Nrf2, and HO-1; upregulation of GST and Keap1), induction of pro-apoptotic molecules (transcription of BAX, Caspase3, and Caspase9), downregulation of anti-apoptotic gene Bcl-2 expression, and induction of endoplasmic reticulum stress (upregulation of IRE1, PERK, and ATF6 expression). The results suggested that the supplementation of α-lipoic acid could effectively induce humoral immunity, alleviate oxidative stress injury and endoplasmic reticulum stress, and ultimately alleviate liver injury induced by ammonia poisoning (50-60% reduction). This provides theoretical basis for revealing the toxicity of long-term ammonia stress and provides new insights into the anti-ammonia toxicity mechanism of α-lipoic acid.

Transcriptome analysis reveals the mechanism of the effect of perfluorocaproic acid exposure on brain injury in Carassius auratus.

Perfluorocaproic acid (PFHxA) has received much attention as an emerging pollutant linked to neurological problems in humans and fish. However, the potential mechanism remains unknown. In this study, the pathological damage to tissue sections demonstrated that perfluorocaproic acid caused brain tissue damage, and the increased antioxidant index malondialdehyde (MDA) and decrease in superoxide Dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (AKP), glutathione peroxidase (GSH-Px), Catalase (CAT), and Lysozyme (LZM) that perfluorocaproic acid activated antioxidant stress and caused brain damage. Transcriptome sequencing discovered 1,532 divergent genes, 931 upregulated, and 601 down-regulated. Furthermore, according to GO enrichment analysis, the differently expressed genes were shown to be involved in biological processes, cellular components, and molecular functions. The MAPK, calcium, and Neuroactive ligand-receptor interaction were considerably enriched in the KEGG enrichment analysis. We then analyzed qRT-PCR and chose ten essential differentially expressed genes for validation. The qRT-PCR results followed the same pattern as the RNA-Seq results. In conclusion, our study shows that perfluorocaproic acid exposure causes oxidative stress in the brain. It establishes a theoretical foundation for future research into genes linked to perfluorocaproic acid toxicity.

Disruption of sex steroid hormones biosynthesis by short-term enrofloxacin antibiotic exposure in Carassius auratus var. Pengze.

BACKGROUND: It has been reported that antibiotic enrofloxacin can impair reproductive function of mammals, induces multi-generational oscillatory effects on reproduction of Caenorhabditis elegans, and disturbes endocrine system in grass carp. OBJECTIVES: This study aims to explore the effect of short-term enrofloxacin exposure on sex steroid hormones biosynthesis in Carassius auratus var. Pengze through assessing the contents of growth hormone (GH), thyroid hormone 4 (T4), estradiol (E2) and testosterone (T) in plasma, and investigating sex steroid hormones biosynthesis based on targeted metabonomics analysis, and determining expression level of some important genes, gonadotropin-releasing hormone (gnrh), gonadotropin hormone 1-ß (gth1-ß), gonadotropin hormone 2-ß (gth2-ß) and cyp19a1a in hypothalamus-pituitary-ovary axis (HPOA). RESULTS: We found that short-term exposure of enrofloxacin disordered contents of E2 and T in plasma of fish determined by ELISA detection, T content elevation and E2 content decline, which was confirmed by the following data from targeted metabonomics analysis of plasma. The metabonomic results showed that both T and its upstream intermediate products during the process of sex steroid hormones biosynthesis in fish were increased significantly, but E2 content was decreased markedly. At the exposure 24 h of enrofloxacin, expression of gnrh in hypothalamus, gth1-ß and gth2-ß in pituitary were promoted. Meanwhile GH and T4 contents in plasma, two inducers of sex steroid hormones synthesis, were augmented, which indicated that sex steroid hormones biosynthesis was improved. However cyp19a1a expression in ovary was repressed, and content of estriol (E3) was upregulated. These data suggested that enrofloxacin promoted sex steroid hormones biosynthesis and conversion of E2 to estriol (E3), but inhibited the conversion of T to E2. Finally, content of E2 was declined sharply. DISCUSSION: Animal specific antibacterial enrofloxacin is widely detectable in aquatic ecosystem, exposure of the agent can induce adverse effects on plants and animals. This study firstly evidenced induction of disruption of sex steroid hormones by enrofloxacin in fish, which indicates enrofloxacin is an endocrine disruption compound that can induce endocrine disruption of animals, including fish.

Effects of microfiber and bead microplastic exposure in the goldfish Carassius auratus: Bioaccumulation, antioxidant responses, and cell damage.

We confirmed antioxidant-related gene expression, bioaccumulation, and cell damage following exposure to various microplastics in vivo and in vitro in the goldfish Carassius auratus. Exposure of C. auratus to a 500 µm fiber-type microplastic environment (MF; 10 and 100 fibers/L) and two sizes (0.2 and 1.0 µm) of beads (MB; 10 and 100 beads/L) for 120 h increased superoxide dismutase (SOD) mRNA expression in the liver until 24 h followed by a decrease. Whereas, catalase (CAT) mRNA expression increased from 12 h to the end of the in vivo experiment. In vitro experiments were conducted with diluted microfibers (1 and 5 fibers/L) and microbeads (1 and 5 beads/L) using cultured liver cells. The results of SOD and CAT mRNA expression analysis conducted in vitro showed a tendency similar to those of experiments conducted in vivo. The H2O2 level increased in the high-concentration experimental groups compared with that in the low-concentration groups of 0.2-µm beads. In addition, the H2O2 level increased in both MF and MB groups from 12 h of exposure. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in plasma were used as indicators of liver damage in fish. The ALT and AST levels increased up to 120 h after exposure. Caspase-3 (casp-3) mRNA expression was higher in the MB group than in the MF group. We visually confirmed liver casp-3 mRNA signals using in situ hybridization. The degree of DNA damage in the MF and MB high-concentration groups increased with the exposure time. The tail length and percent of DNA in the tail of the MB group were significantly higher than those of the MF group, confirming that DNA damage was greater in the MB group. Both fiber- and bead-type microplastics induced oxidative stress in C. auratus, but the bead-type induced greater stress than the fiber-type.

Roundup® disrupts tissue architecture, attenuates Na+/K+-ATPase expression, and induces protein oxidation/nitration, cellular apoptosis, and antioxidant enzyme expressions in the gills of goldfish, Carassius auratus.

Extensive agricultural activities to feed the growing population are one major driving force behind aquatic pollution. Different types of pesticides are used in farmlands to increase crop production and wash up into water bodies. Glyphosate-based herbicide Roundup® is one of the most used pesticides in the United States; however, its effects on teleost species are still poorly understood. This study focused on the effects of environmentally relevant concentrations of Roundup exposure (low- and high-dose: 0.5 and 5 µg/L for 2-week) on Na+/K+-ATPase (NKA, a biomarker for sodium­potassium ion pump efficacy), cytochrome P450-1A (CYP1A, a monooxygenase enzyme), 2,4-dinitrophenyl protein (DNP, a biomarker for protein oxidation), 3-nitrotyrosine protein (NTP, a biomarker for protein nitration), superoxidase dismutase (SOD, an antioxidant enzyme), catalase (CAT, an antioxidant enzyme) expressions, and cellular apoptosis in the gills of goldfish. Histopathological and in situ TUNEL analyses showed widespread tissue damage, including lamellar fusion, loss of gill architecture, club shape of primary lamellae, mucous formation, and distortion in the epithelium layer, as well as apoptotic nuclei in gills. Immunohistochemical and qRT-PCR analyses provided insights into the expressions of molecular indicators in gills. Fish exposed to Roundup exhibited a significant (P < 0.05) downregulation of NKA expression in gills. Additionally, we observed upregulation of CYP1A, DNP, NTP, SOD, and CAT expressions in the gills of goldfish. Overall, our results suggest that exposure to Roundup causes disruption of gill architecture, induces protein oxidation/nitration and cellular apoptosis, and alters prooxidant-antioxidant homeostasis in tissues, which may lead to reduced fitness and survivability of teleost species.

Insulin Controls Clock Gene Expression in the Liver of Goldfish Probably via Pi3k/Akt Pathway.

The liver circadian clock plays a pivotal role in driving metabolic rhythms, being primarily entrained by the feeding schedule, although the underlying mechanisms remain elusive. This study aimed to investigate the potential role of insulin as an intake signal mediating liver entrainment in fish. To achieve this, the expression of clock genes, which form the molecular basis of endogenous oscillators, was analyzed in goldfish liver explants treated with insulin. The presence of insulin directly increased the abundance of per1a and per2 transcripts in the liver. The dependency of protein translation for such insulin effects was evaluated using cycloheximide, which revealed that intermediate protein translation is seemingly unnecessary for the observed insulin actions. Furthermore, the putative interaction between insulin and glucocorticoid signaling in the liver was examined, with the results suggesting that both hormones exert their effects by independent mechanisms. Finally, to investigate the specific pathways involved in the insulin effects, inhibitors targeting PI3K/AKT and MEK/ERK were employed. Notably, inhibition of PI3K/AKT pathway prevented the induction of per genes by insulin, supporting its involvement in this process. Together, these findings suggest a role of insulin in fish as a key element of the multifactorial system that entrains the liver clock to the feeding schedule.

Differential regulation of hepatic SH3 domain binding kinase 1 (SBK1) expression in mouse and goldfish.

SH3 domain binding kinase 1 (SBK1) is a serine/threonine kinase that belongs to the new kinase family (NFK) with limited information on its function. Previous studies reported that SBK1 plays a role in memory formation, lipid metabolism, and cancer cell progression. Nevertheless, the regulatory mechanism of Sbk1 expression in various tissues remains unknown. We report here that Sbk1 expression in mouse hepatocytes was downregulated by glucocorticoid, whereas saturated and unsaturated fatty acids were stimulators of Sbk1 expression. The regulatory role of glucocorticoid and fatty acid was further confirmed by the Sbk1 promoter assay, which aligned with the presence of several glucocorticoid-response elements (GRE) and peroxisome proliferator responsive elements (PPRE) in the mouse Sbk1 promoter. The inhibitory effect of glucocorticoids on hepatic Sbk1 expression and protein content could also be demonstrated in vivo after prednisolone injection. Moreover, the expression of SBK1 in goldfish (gfSBK1) was also sensitive to glucocorticoid suppression as their mouse orthologues. In contrast, insulin had a differential action on SBK1 expression that it promoted the expression of all SBK1 isoforms in the goldfish hepatocytes but inhibited Sbk1 expression in the mouse hepatocytes. Together, our findings indicate that SBK1 expression is hormone- and nutrient-sensitive with a species-specific response.

Micro-polystyrene plastic and benzo[α]pyrene exposure affects the endocrine system and causes physiological stress in Carassius auratus.

Microplastics, owing to their hydrophobic properties and the various chemicals used in their production, can act as carriers of persistent organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs). In this study, we exposed the goldfish Carassius auratus to benzo[α]pyrene (BaP, 10 µg/L), a representative PAH, and micro-polystyrene plastic (MP; 10 and 100 beads/L), of size 1.0 µm, as a single or complex environmental stressor, and evaluated the stress response and the resulting DNA damage. The expression of CRH and ACTH mRNA in the pituitary gland and hypothalamus, of the hypothalamus-pituitary-interrenal (HPI) axis, increased significantly after 6 h of exposure. Plasma cortisol levels showed a similar trend to the expression of stress-regulating genes along the HPI axis, and a significant increase was observed in the combined exposure groups (BaP + LMP [low-concentration MP] and BaP + HMP [high-concentration MP]) compared to those in the single exposure group. H2O2 concentration and CYP1A1 and MT mRNA expression levels in the liver were significantly higher in the combined exposure groups compared with in the single exposure groups. In situ hybridization revealed a similar pattern of MT mRNA expression, and many signals were observed in the BaP + HMP group. Furthermore, the BaP + HMP group showed more DNA damage, and the degree of DNA damage increased with exposure time for all experimental groups, except for the control group. Therefore, exposure to BaP and MP alone can induce stress in goldfish; however, when a combination of both substances is provided, their synergistic effect leads to increased stress and DNA damage. MP was confirmed to be a more serious stress-inducing factor in goldfish than BaP, based on the expression levels of stress-regulating genes along the HPI axis.