The importance of selecting a proper biological milieu for protein corona analysis in vitro: Human plasma versus human serum

Nanoparticle (NP) exposure to biological fluids in the body results in protein binding to the NP surface, which forms a protein coating that is called the “protein corona”. To simplify studies of protein–NP interactions and protein corona formation, NPs are incubated with biological solutions, such as human serum or human plasma, and the effects of this exposure are characterized in vitro. Yet, how NP exposure to these two different biological milieus affects protein corona composition and cell response has not been investigated. Here, we explore the differences between the protein coronas that form when NPs are incubated in human serum versus human plasma. NP characterization indicated that NPs that were exposed to human plasma had higher amounts of proteins bound to their surfaces, and were slightly larger in size than those exposed to human serum. In addition, significant differences in corona composition were also detected with gel electrophoresis and liquid chromatography–mass spectrometry/mass spectrometry, where a higher fraction of coagulation proteins and complement factors were found on the plasma-exposed NPs. Flow cytometry and confocal microscopy showed that the uptake of plasma-exposed NPs was higher than that of serum-exposed NPs by RAW 264.7 macrophage immune cells, but not by NIH 3T3 fibroblast cells. This difference is likely due to the elevated amounts of opsonins, such as fibrinogen, on the surfaces of the NPs exposed to plasma, but not serum, because these components trigger NP internalization by immune cells. As the human plasma better mimics the composition of the in vivo environment, namely blood, in vitro protein corona studies should employ human plasma, and not human serum, so the biological phenomena that is observed is more similar to that occurring in vivo.     

Daily rhythms of lipid metabolic gene expression in zebra fish liver: Response to light/dark and feeding cycles

Despite numerous studies about fish nutrition and lipid metabolism, very little is known about the daily rhythm expression of lipogenesis and lipolysis genes. This research aimed to investigate the existence of daily rhythm expressions of the genes involved in lipid metabolism and their synchronization to different light/dark (LD) and feeding cycles in zebra fish liver. For this purpose, three groups of zebra fish were submitted to a 12:12?h LD cycle. A single daily meal was provided to each group at various times: in the middle of the light phase (ML); in the middle of the dark phase (MD); at random times. After 20 days of acclimation to these experimental conditions, liver samples were collected every 4?h in one 24-h cycle. The results revealed that most genes displayed a significant daily rhythm with an acrophase of expression in the dark phase. The acrophase of lipolytic genes (lipoprotein lipase – lpl, peroxisome proliferator-activated receptor – pparα and hydroxyacil CoA dehydrogenase – hadh) was displayed between ZT 02:17?h and ZT 18:31?h. That of lipogenic genes (leptin-a – lepa, peroxisome proliferator-activated receptor – pparγ, liver X receptor – lxr, insulin-like growth factor – igf1, sterol regulatory element-binding protein – srebp and fatty acid synthase – fas) was displayed between ZT 15:25?h and 20:06?h (dark phase). Feeding time barely influenced daily expression rhythms, except for lxr in the MD group, whose acrophase shifted by about 14?h compared with the ML group (ZT 04:31?h versus ZT 18:29?h, respectively). These results evidence a strong synchronization to the LD cycle, but not to feeding time, and most genes showed a nocturnal acrophase. These findings highlight the importance of considering light and feeding time to optimize lipid metabolism and feeding protocols in fish farming.     

Comparative skin mucus and serum humoral defence mechanisms in the teleost gilthead seabream (Sparus aurata)

Mucosal surfaces of fish, including skin, gill and gut, contain numerous immune substances poorly studied that act as the first line of defence against a broad spectrum of pathogens. This study aimed to identify and characterize for the first time different constitutive humoral defence mechanisms of the skin mucus of gilthead seabream (Sparus aurata). To do this, the levels of total immunoglobulin M, several enzymes and proteins (peroxidase, lysozyme, alkaline phosphatase, esterases, proteases and antiproteases), as well as the bactericidal activity against opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum, Photobacterium damselae) and non-pathogenic bacteria (Escherichia coli, Bacillus subtilis) were measured in the skin mucus and compared with those found in the serum. This study demonstrates that gilthead seabream skin mucus contains lower levels of IgM, similar levels of lysozyme, alkaline phosphatase and proteases, and higher esterase, peroxidase and antiprotease activities than serum. In addition, skin mucus revealed stronger bactericidal activity against tested fish pathogen bacteria compared to the serum activity, while human bacteria can even grow more in the presence of mucus. The results could be useful for better understanding the role of the skin mucus as a key component of the innate immune system with potential application for the aquaculture.     

Lipid dynamics in goldfish (Carassius auratus) during a period of gonadal recrudescence: Effects of beta-sitosterol and 17beta-estradiol exposure

The potential for contaminants to alter lipid or cholesterol dynamics in fish is rarely investigated and may include critical physiological endpoints that are impacted by exposure to endocrine-active substances. The current study investigated plasma and tissue lipid dynamics over a period of recrudescence in goldfish, while also examining the potential for beta-sitosterol (beta-sit), a phytosterol and 17beta-estradiol (E(2)), an endogenous estrogen, to alter lipid homeostasis. Goldfish were exposed to 0 microg/g (no chemical; control), 200 microg/g beta-sit (72.3% sitosterol mixture) or 10 microg/g 17beta-estradiol (E2) via Silastic implants for a period of five months. Plasma lipids peaked in control fish coincident with maximum liver size, while gonadal cholesterol concentration was highest concomitant with maximum gonad size. Plasma lipid concentrations were highly affected by E2 but not beta-sit exposure; E2 elevated total cholesterol (p<0.001) and triglyceride (TG; p<0.001) and decreased high-density lipoprotein (HDL) concentration (p<0.001) in male fish. Tissue cholesterol concentrations were minimally affected by beta-sit exposure, while hepatic cholesterol concentrations were increased in E2 exposed females (p=0.041), indicating elevated liver lipogenesis in response to E2, but not beta-sit, exposure. The present study demonstrates differential effects by beta-sit and E2 on plasma lipoprotein profile and TG concentration and indicates estrogen-specific effects on hepatic lipid metabolism during gonadal development.     

Effects of Increased CO2 on Fish Gill and Plasma Proteome

Ocean acidification and warming are both primarily caused by increased levels of atmospheric CO₂, and marine organisms are exposed to these two stressors simultaneously. Although the effects of temperature on fish have been investigated over the last century, the long-term effects of moderate CO₂ exposure and the combination of both stressors are almost entirely unknown. A proteomics approach was used to assess the adverse physiological and biochemical changes that may occur from the exposure to these two environmental stressors. We analysed gills and blood plasma of Atlantic halibut (Hippoglossus hippoglossus) exposed to temperatures of 12°C (control) and 18°C (impaired growth) in combination with control (400 µatm) or high-CO₂ water (1000 µatm) for 14 weeks. The proteomic analysis was performed using two-dimensional gel electrophoresis (2DE) followed by Nanoflow LC-MS/MS using a LTQ-Orbitrap. The high-CO₂ treatment induced the up-regulation of immune system-related proteins, as indicated by the up-regulation of the plasma proteins complement component C3 and fibrinogen β chain precursor in both temperature treatments. Changes in gill proteome in the high-CO₂ (18°C) group were mostly related to increased energy metabolism proteins (ATP synthase, malate dehydrogenase, malate dehydrogenase thermostable, and fructose-1,6-bisphosphate aldolase), possibly coupled to a higher energy demand. Gills from fish exposed to high-CO₂ at both temperature treatments showed changes in proteins associated with increased cellular turnover and apoptosis signalling (annexin 5, eukaryotic translation elongation factor 1γ, receptor for protein kinase C, and putative ribosomal protein S27). This study indicates that moderate CO₂-driven acidification, alone and combined with high temperature, can elicit biochemical changes that may affect fish health.     

Effects of cadmium on the reproductive axis of Japanese medaka (Oryzias latipes)

Cadmium (Cd) is a ubquitous element and a significant inorganic pollutant that has previously been found to bioaccumulate in reproductive organs of fish and disrupt important endocrine processes, especially those involved in synthesis, release and metabolism of hormones. Clearly, there is potential for reproductive effects in fish populations exposed to Cd, however, few studies have investigated the non-lethal consequences of Cd in fish. To this extent, adult male and female Japanese medaka were exposed to 0-10 ppb Cd for 7 weeks. Reproductive endpoints were monitored during weeks 6 and 7 of exposure and compared to physiological responses along the hypothalamus-pituitary-gonadal (HPG) axis, including plasma vitellogenin (VTG), hepatic estrogen receptor (ER), plasma steroids, gonadal-somatic indices (GSI), and gonadal steroid release. There were no observed effects on VTG and ER by long-term Cd exposure. However, gonadal steroid release was significantly decreased in males and females at all exposure concentrations and female plasma estradiol levels were significantly altered at concentrations higher than 5 ppb Cd. Overall, responses along the HPG axis were more sensitive to Cd exposure than the reproductive and developmental endpoints, which were not affected in this study, indicating that higher level impairment in fish might be relatively protected.     

Triangular gold nanoparticles modify shell characteristics and increase antioxidant enzyme activities in the clam Ruditapes decussatus

Context: Nanoparticles may cause adverse environmental effects but there is limited information on their interactions with marine organisms.

Objective: Our aim was to examine the effects of triangular gold nanoparticles (Tr-Au NPs) on the clam, Ruditapes decussatus.

Materials and methods: Clams were exposed to Tr-Au1?=?5?µg/L and Tr-Au2?=?10?µg/L for 2 and 7?days. Effects on shell structure were investigated. Superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST) activities, protein carbonyl levels and malondialdehyde content were used to assess biochemical status.

Results: Transmission electron microscopy (TEM) and electron dispersive X-ray microanalysis (EDX) showed that Tr-Au NPs modified shell structure and morphology. Tr-Au NPs size increased forming aggregate particles. Tr-Au NPs increased SOD, CAT and GST activities in gill and digestive gland in a concentration- and time-dependent manner indicating defence against oxidative stress. Enhanced lipid peroxidation and protein carbonyl levels confirmed oxidative stress.

Conclusion: Tr-Au NPs cause oxidative stress and affect shell structure of clams. These findings may have relevance to other marine species.     

Quantitative Cross-Species Extrapolation between Humans and Fish: The Case of the Anti-Depressant Fluoxetine

Fish are an important model for the pharmacological and toxicological characterization of human pharmaceuticals in drug discovery, drug safety assessment and environmental toxicology. However, do fish respond to pharmaceuticals as humans do? To address this question, we provide a novel quantitative cross-species extrapolation approach (qCSE) based on the hypothesis that similar plasma concentrations of pharmaceuticals cause comparable target-mediated effects in both humans and fish at similar level of biological organization (Read-Across Hypothesis). To validate this hypothesis, the behavioural effects of the anti-depressant drug fluoxetine on the fish model fathead minnow (Pimephales promelas) were used as test case. Fish were exposed for 28 days to a range of measured water concentrations of fluoxetine (0.1, 1.0, 8.0, 16, 32, 64 µg/L) to produce plasma concentrations below, equal and above the range of Human Therapeutic Plasma Concentrations (H_TPCs). Fluoxetine and its metabolite, norfluoxetine, were quantified in the plasma of individual fish and linked to behavioural anxiety-related endpoints. The minimum drug plasma concentrations that elicited anxiolytic responses in fish were above the upper value of the H_TPC range, whereas no effects were observed at plasma concentrations below the H_TPCs. In vivo metabolism of fluoxetine in humans and fish was similar, and displayed bi-phasic concentration-dependent kinetics driven by the auto-inhibitory dynamics and saturation of the enzymes that convert fluoxetine into norfluoxetine. The sensitivity of fish to fluoxetine was not so dissimilar from that of patients affected by general anxiety disorders. These results represent the first direct evidence of measured internal dose response effect of a pharmaceutical in fish, hence validating the Read-Across hypothesis applied to fluoxetine. Overall, this study demonstrates that the qCSE approach, anchored to internal drug concentrations, is a powerful tool to guide the assessment of the sensitivity of fish to pharmaceuticals, and strengthens the translational power of the cross-species extrapolation.     

Fatty acid alterations caused by PCBs (Aroclor 1242) and copper in adipose tissue around lymph nodes of mink

Fatty acid composition was determined in adipose tissue surrounding the mesenteric lymph nodes of mink (Mustela vison) exposed to polychlorinated biphenyls (PCBs: 1 mg Aroclor 1242 in food day⁻¹ for 28 days) and/or copper (62 mg kg⁻¹ food). These specific adipose tissues are known to have functional relationships with lymphocytes, and proliferation of cultured lymphocytes is influenced by the quality of fatty acids available in media. In six experimental groups the diet was based on freshwater fish, and in two groups it was based on marine fish. These basal diets differed in terms of fatty acid composition and content of fat-soluble vitamins A₁ and E. The fatty acid composition of membrane phospholipids (PL) responded to PCBs more than that of triacylglycerols (TG). The effects of copper were small. In female minks fed a diet of freshwater fish, the proportion of highly unsaturated fatty acids in PL decreased by 5 wt.% due to PCBs, and the acids seemed to be replaced by monounsaturated fatty acids (9 wt.% increase of total). This decrease of highly unsaturated fatty acids in PL was milder in minks on the marine fish diet rich in fat-soluble vitamins. In TG of minks on the marine diet, however, PCBs decreased the proportion of docosahexaenoic acid (22:6n-3). The possibility that these alterations in the fatty acid metabolism of adipose tissue supporting the lymph nodes affect immune function during PCB exposure should be studied further. Interestingly, the quality of the fish diet affected the magnitude of the alterations. The fatty acid responses may also differ between males and females.     

环境相关四环素长期暴露对吉富罗非鱼生长性能、肝脏脂肪代谢和抗氧化反应的影响

为研究水环境浓度四环素(Tetracycline, TC)长期暴露对鱼类的影响, 实验以吉富罗非鱼(Oreochromis niloticus)为实验模型, 将其暴露于0、400和800 ng/L三种四环素水平下10周。结果表明, 相较于对照组, 四环素暴露显著提高了生长性能, 但增加了肝脏中甘油三酯含量和四环素浓度。脂肪生成基因表达(fas、scd、accα、srebp1和pparγ)被上调, 但降低了与脂肪分解相关基因的表达(atgl、hsl、cpt1和pparα)。此外, 6-磷酸葡萄糖脱氢酶(6PGD)、葡萄糖-6-磷酸脱氢酶(G6PD)、异柠檬酸脱氢酶(ICDH)、苹果酸脱氢酶(ME)及脂肪酸合成酶(FAS)的活性显著增加, 但肉碱棕榈酰转移酶1(CPT1)活性则被显著抑制, 这与基因表达相一致。而且, 四环素暴露显著降低过氧化氢酶、总超氧化物歧化酶的活性及总抗氧化能力, 并诱导了鱼类的氧化应激, 导致丙二醛水平显著增加。因此, 与环境相关四环素浓度可促进吉富罗非鱼的生长性能、上调产脂代谢、降低抗氧化能力并诱导脂质过氧化。     

Changes in Atlantic salmon (Salmo salar) epidermal mucus protein composition profiles following infection with sea lice (Lepeophtheirus salmonis)

The mucus protein profile of Atlantic salmon (Salmo salar) and changes due to infection with sea lice (Lepeophtheirus salmonis) were examined. Two-dimensional gel electrophoresis was performed on salmon skin mucus and comparisons between control and infected fish mucus were made. LC MS/MS identified intracellular proteins, calmodulin, actin, and hemopexin and plasma proteins, such as apolipoproteins, lectin, plasminogen and transferrin. Plasma proteins in the mucus may result from either direct expression by epidermal cells, leakage of plasma or via a secondary circulation system. Therefore, RT-PCR was used to measure mRNA of transferrin and lectin in Atlantic salmon skin. Transferrin expression was observed suggesting direct expression by the epidermis. Lectin expression was not detected suggesting another mechanism of entry into mucus, either leakage from plasma or secondary circulation. The lack of observable albumin on 2D gels, suggests that mucus lectin may arise from the secondary circulation route. Interestingly, β-actin was a significant component of Atlantic salmon mucus. Cleaved actin and transferrin fragments were observed and positively correlated with sea lice infection suggestive of proteolytic activity. Increased levels of cleaved transferrin during sea lice infection may activate the nitrous oxide response of salmon macrophages, as part of the fish's immune response to sea lice infection.     

Ultrastructural study of skin and eye of UV‐B irradiated ayu Plecoglossus altivelis

A scanning electron microscopic study of the skin and eye of UV‐B radiated ayu Plecoglossus altivelis (age 30 days, mean ±  s . e . total length: 16·25 ± 0·11 mm) under laboratory condition showed marked changes when compared with the control fish without UV‐B radiation. The exposure of the fish to the radiation resulted in the destruction of microridges in the epidermis and exposed neuromast cells of the skin. Domed protrusions were also more common in the skin of UV‐B radiated fish than in the control fish. The appearance of mucus in both groups was different. In the control skin the mucus was spread over a wide area whereas in the treated fish the mucus was concentrated in a small area. The anastomozing structures of the microridges of the eyes were lost in UV‐B radiated fish and the microridges themselves were fewer in number, fragmented, and aggregated. Mucus cells, prominently visible in the control fish, were distorted in the treated fish. Cell contours were irregular in UV‐B radiated fish and cell to cell contacts had been lost in this group.     

Aluminum as an endocrine disruptor in female Nile tilapia (Oreochromis niloticus)

The effects of aluminum on plasma ion, lipid, protein and steroid hormone concentration were evaluated in Oreochromis niloticus broodstock females. Lipid and protein concentrations from the gonads and liver were also measured. Experiments were performed at neutral and acidic water pH. Four groups of fish were tested for 96 h: 1) control conditions at neutral water pH; 2) control conditions at acidic water pH (CTR-Ac); 3) aluminum at neutral water pH (Al-N); and 4) aluminum at acidic water pH (Al-Ac). Aluminum and acidic water pH exposure caused no ionoregulatory disturbances. Total lipid concentration increased in the mature gonads and decreased in the liver, suggesting an acceleration of lipid mobilization to the ovaries in animals exposed to aluminum. However, a decreased protein concentration in ovaries was also observed. Exposure of control fish to acidic water pH caused an increased concentration of plasma 17α-hydroxyprogesterone. However, females exposed to aluminum at acidic water pH showed a decreased of plasma 17α-hydroxyprogesterone and cortisol. No differences in plasma 17β-estradiol were observed. The physiological mechanisms underlying the disturbances observed are discussed focusing on reproduction. We suggest that aluminum can be considered an endocrine disrupting compound in mature O. niloticus females.     

Assessment the using of silica nanoparticles (SiO2NPs) biosynthesized from rice husks by Trichoderma harzianum MF780864 as water lead adsorbent for immune status of Nile tilapia (Oreochromis niloticus)

Rice husks (RHs) was used as a substrate for biosynthesis of high-value Silica nanoparticles (SiO₂NPs). An isolate of Trichoderma harzianum MF780864 (T. harzianum) was isolated and identified based on the Internal Transcribed Spacers (ITS) sequences; it showed the potentiality to induce SiO₂NPs in the process of RHs biotransformation. SiO₂NPs were produced extracellularly and their size was of about 89 nm. SiO₂NPs characterized by oval, rod and cubical particles by using Transmission Electron Microscope (TEM).The Fourier transform infrared spectroscopy (FTIR) confirmed the presence of various functional groups of biomolecules and capping protein, encapsulating SiO₂NPs. Water and fish samples were collected from private fish farms in El-Sharkia Governorate, Egypt. Lead (Pb) was detected from water and fish samples at its highest concentration at about 0.088 mg/L. The adsorption capacity of Pb by SiO₂NPs was evaluated by testing different concentrations of SiO₂NPs viz. 1, 2, and 3 mg/L, wherein 1 mg/L revealed the highest Pb adsorption efficiency. Within laboratory trials, the results indicated that highest Pb adsorption efficiency revealed through the increasing of SiO₂NPs concentrations until 120 h. In vivo trial that lasted for 8 weeks, Nile tilapia (Oreochromis niloticus) (29.78 ± 0.36 g body weight) supplemented with 0.088 mg/L Pb was divided into four experimental groups having three replicates (15 fish/replicate; 45 fish/group). The results showed that SiO₂NPs supplementation through water revealed significant increase in growth and hematological parameters of O. niloticus. Moreover, enhancement of antioxidant capacity (TAC), and immune related gene expression of IL-1β were increased in the presence of SiO₂NPs compared with the groups of Pb exposure. Moreover, Pb residue level in fish muscles was noticeably decreased in the SiO₂NPs treated groups. Thus, this research opens up other possibilities in the field of using SiO₂NPs as a lead adsorbent for water bioremediation.     

Antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to Chattonella marina and hydrogen peroxide: Implications on the cause of fish kills

Chattonella marina, a red tide or harmful algal bloom species, has caused mass fish kills and serious economic loss worldwide, and yet its toxic actions remain highly controversial. Previous studies have shown that this species is able to produce reactive oxygen species (ROS), and therefore postulated that ROS are the causative agents of fish kills. The present study investigates antioxidant responses and lipid peroxidation in gills and erythrocytes of fish (Rhabdosarga sarba) upon exposure to C. marina, compared with responses exposed to equivalent and higher levels of ROS exposure. Even though C. marina can produce a high level of ROS, gills and erythrocytes of sea bream exposed to C. marina for 1 to 6 h showed neither significant induction of antioxidant enzymes nor lipid peroxidation. Antioxidant responses and oxidative damage did not occur as fish mortality began to occur, yet could be induced upon exposure to artificially supplied ROS levels an order of magnitude higher. The result of this study implies that ROS produced by C. marina is not the principal cause of fish kills.