Automated Hematological Approach and Protein Electrophoretic Pattern in Tilapia (Oreochromis niloticus): An Innovative and Experimental Model for Aquaculture.

This study aimed to assess the usefulness of two innovative automated methods (automated blood count counters and flow cytometry) for hematological investigation in Tilapia to make a contribution to the clinical diagnostics of this farmed species. Moreover, serum total proteins and their electrophoretic fractions (prealbumin, albumin, α-, ß-, and γ-fraction), as health condition indicators, were assessed. The analysis of serum total proteins and electrophoretic fraction showed a normal and typical electrophoretic pattern of healthy fish (serum total proteins, 3.70 ± 0.62 g/dL; prealbumin, 0.44 ± 0.20 g/dL; albumin, 1.17 ± 0.66 g/dL; α-fraction, 1.49 ± 0.64 g/dL; ß-fraction, 0.32 ± 0.16 g/dL; and γ-fraction, 0.29 ± 0.13 g/dL). The relationships between the values of red blood cells (RBCs), white blood cells (WBCs), and thrombocytes (TCs) obtained with the two automated methods were determined using Pearson correlation analysis. The results showed a significant positive correlation between automatic blood cell counting and flow cytometry analysis for RBCs (r = 0.97, p < 0.0001) and WBCs (r = 0.91, p < 0.0001), whereas no correlation was found for TCs (r = -0.11, p = 0.66). The preliminary results gathered in this study seem to highlight the usefulness of the new analytical techniques herein investigated in tilapia, suggesting their application in the hematological investigation of farmed fish species and their usefulness for monitoring the health and well-being of fish reared in aquaculture.

Toxicity of Titanium Dioxide-Cerium Oxide Nanocomposites to Zebrafish Embryos: A Preliminary Evaluation.

The widespread use of metal nanoparticles in different fields has raised many doubts regarding their possible toxicity to living organisms and the accumulation and discharge of metals in fish species. Among these nanoparticles, titanium dioxide (TiO2) and cerium oxide (CeO2) nanoparticles have mainly been employed in photocatalysis and water depuration. The aim of this research was to evaluate the potential toxic effects, after a co-exposure of TiO2-3%CeO2 nanoparticles, on zebrafish development, using an acute toxicity test. Increasing concentrations of TiO2-3%CeO2 nanoparticles were used (0.1-1-10-20 mg/L). The heartbeat rate was assessed using DanioscopeTM software (version 1.2) (Noldus, Leesburg, VA, USA), and the responses to two biomarkers of exposure (Heat shock proteins-70 and Metallothioneins) were evaluated through immunofluorescence. Our results showed that the co-exposure to TiO2-3%CeO2 nanoparticles did not affect the embryos' development compared to the control group; a significant difference (p < 0.05) at 48 hpf heartbeat for the 1, 10, and 20 mg/L groups was found compared to the unexposed group. A statistically significant response (p < 0.05) to Heat shock proteins-70 (Hsp70) was shown for the 0.1 and 1 mg/L groups, while no positivity was observed in all the exposed groups for Metallothioneins (MTs). These results suggest that TiO2-3%CeO2 nanocomposites do not induce developmental toxicity; instead, when considered separately, TiO2 and CeO2 NPs are harmful to zebrafish embryos, as previously shown.

Helminths of the Bluntnose Sixgill Shark, Hexanchus griseus (Bonnaterre, 1788), from the Strait of Messina (Sicily, Southern Italy).

Bluntnose sixgill shark, Hexanchus griseus (Bonnaterre, 1788), is a little-known elasmobranch in the Mediterranean Sea. Given the lack of information about H. griseus, the aim of our study was to describe the helminth fauna of this species. In March 2023, one H. griseus juvenile female specimen was found off the coast of Messina (Italy) and referred by the Italian Coast Guard to our laboratory for necropsy and parasitological evaluation. After necropsy, the specimen's gills, stomach and spiral valve were investigated for parasite presence. All collected parasites were stored in 70% ethanol for routine parasitological analysis. No lesions due to parasites were found in the gills or skin. Three species of helminths were found in one studied female specimen of Hexanchus griseus, namely, two cestode species (Phyllobothrium sinuosiceps and Nybelinia sp., larvae) and one trematode (Otodistomum veliporum). Among them, five Trypanorhyncha plerocercoid larvae were found attached to the stomach mucosa, and six adult cestodes and one digenean trematode were collected from the spiral valve. No other parasite taxa were found in the celomic organs. This study reports new information regarding the parasitic fauna of H. griseus from the central Mediterranean Sea.

Titanium Dioxide Nanoparticles: Effects on Development and Male Reproductive System.

Titanium dioxide nanoparticles (TiO2-NPs) are used intensively. Thanks to their extremely small size (1-100 nm), TiO2-NPs are more absorbable by living organisms; consequently, they can cross the circulatory system and then be distributed in various organs including the reproductive organs. We have evaluated the possible toxic effect of TiO2-NPs on embryonic development and the male reproductive system using Danio rerio as an organism model. TiO2-NPs (P25, Degussa) were tested at concentrations of 1 mg/L, 2 mg/L, and 4 mg/L. TiO2-NPs did not interfere with the embryonic development of Danio rerio, however, in the male gonads the TiO2-NPs caused an alteration of the morphological/structural organization. The immunofluorescence investigation showed positivity for biomarkers of oxidative stress and sex hormone binding globulin (SHBG), both confirmed by the results of qRT-PCR. In addition, an increased expression of the gene responsible for the conversion of testosterone to dihydrotestosterone was found. Since Leydig cells are mainly involved in this activity, an increase in gene activity can be explained by the ability of TiO2-NPs to act as endocrine disruptors, and, therefore, with androgenic activity.

Toxic Effects of Gemcitabine and Paclitaxel Combination: Chemotherapy Drugs Exposure in Zebrafish.

Pharmaceuticals are widely recognized as potentially hazardous to aquatic ecosystems. In the last two decades, the constant intake of biologically active chemicals used in human healthcare has been related to the growing release of these agents into natural environments. As reported by several studies, various pharmaceuticals have been detected, mainly in surface water (seas, lakes, and rivers), but also in groundwater and drinking water. Moreover, these contaminants and their metabolites can show biological activity even at very low concentrations. This study aimed to evaluate the developmental toxicity of exposure to the chemotherapy drugs gemcitabine and paclitaxel in aquatic environments. Zebrafish (Danio rerio) embryos were exposed to doses of gemcitabine 15 µM in combination with paclitaxel 1 µM from 0 to 96 h post-fertilization (hpf) using a fish embryo toxicity test (FET). This study highlights that both gemcitabine and paclitaxel exposure at single non-toxic concentrations affected survival and hatching rate, morphology score, and body length after exposure in combination. Additionally, exposure significantly disturbed the antioxidant defense system and increased ROS in zebrafish larvae. Gemcitabine and paclitaxel exposure caused changes in the expression of inflammation-related, endoplasmic reticulum stress-related (ERS), and autophagy-related genes. Taken together, our findings underline that gemcitabine and paclitaxel increase developmental toxicity in zebrafish embryos in a time-dependent manner.

Effects of carnosine on the embryonic development and TiO2 nanoparticles-induced oxidative stress on Zebrafish.

Oxidative stress is due to an unbalance between pro-oxidants, such as reactive oxygen (ROS) and nitrogen (RNS) species, and antioxidants/antioxidant system. Under physiological conditions these species are involved in different cellular processes such as cellular homeostasis and immune response, while an excessive production of ROS/RNS has been linked to the development of various diseases such as cancer, diabetes, and Alzheimer's disease. In this context, the naturally occurring dipeptide carnosine has shown the ability to scavenge ROS, counteract lipid peroxidation, and inhibit proteins oxidation. Titanium dioxide nanoparticles (TiO2-NPs) have been widely used to produce cosmetics, in wastewater treatment, in food industry, and in healthcare product. As consequence, these NPs are often released into aquatic environments. The Danio rerio (commonly called zebrafish) embryos exposure to TiO2-NPs did not affect the hatching rate, but induced oxidative stress. According to this scenario, in the present study, we first investigated the effects of carnosine exposure and of a sub-toxic administration of TiO2-NPs on the development and survival of zebrafish embryos/larvae measured through the acute embryo toxicity test (FET-Test). Zebrafish larvae represent a useful model to study oxidative stress-linked disorders and to test antioxidant molecules, while carnosine was selected based on its well-known multimodal mechanism of action that includes a strong antioxidant activity. Once the basal effects of carnosine were assessed, we then evaluated its effects on TiO2-NPs-induced oxidative stress in zebrafish larvae, measured in terms of total ROS production (measured with 2,7-dichlorodihydrofluorescein diacetate probe) and protein expression by immunohistochemistry of two cellular stress markers, 70 kDa-heat shock protein (Hsp70) and metallothioneins (MTs). We demonstrated that carnosine did not alter the phenotypes of both embryos and larvae of zebrafish at different hours post fertilization. Carnosine was instead able to significantly decrease the enhancement of ROS levels in zebrafish larvae exposed to TiO2-NPs and its antioxidant effect was paralleled by the rescue of the protein expression levels of Hsp70 and MTs. Our results suggest a therapeutic potential of carnosine as a new pharmacological tool in the context of pathologies characterized by oxidative stress such as neurodegenerative disorders.

Epigenetics in Canine Mammary Tumors: Upregulation of miR-18a and miR-18b Oncogenes Is Associated with Decreased ERS1 Target mRNA Expression and ERα Immunoexpression in Highly Proliferating Carcinomas.

The expression of miRNAs is one of the main epigenetic mechanisms responsible for the regulation of gene expression in mammals, and in cancer, miRNAs participate by regulating the expression of protein-coding cancer-associated genes. In canine mammary tumors (CMTs), the ESR1 gene encodes for ERα, and represents a major target gene for miR-18a and miR-18b, previously found to be overexpressed in mammary carcinomas. A loss in ERα expression in CMTs is commonly associated with poor prognosis, and it is noteworthy that the downregulation of the ESR1 would appear to be more epigenetic than genetic in nature. In this study, the expression of ESR1 mRNA in formalin-fixed, paraffin-embedded (FFPE) canine mammary tumors (CMTs) was evaluated and compared with the expression levels of miR18a and miR18b, both assessed via RT-qPCR. Furthermore, the possible correlation between the miRNA expression data and the immunohistochemical prognostic factors (ERα immunoexpression; Ki67 proliferative index) was explored. A total of twenty-six FFPE mammary samples were used, including 22 CMTs (7 benign; 15 malignant) and four control samples (three normal mammary glands and one case of lobular hyperplasia). The obtained results demonstrate that miR-18a and miR-18b are upregulated in malignant CMTs, negatively correlating with the expression of target ESR1 mRNA. Of note, the upregulation of miRNAs strictly reflects the progressive loss of ERα immunoexpression and increased tumor cell proliferation as measured using the Ki67 index. The results suggest a central role of miR-18a and miR-18b in the pathophysiology of canine mammary tumors as potential epigenetic mechanisms involved in ERα downregulation. Moreover, as miRNA expression reflects ERα protein status and a high proliferative index, miR-18a and miR-18b may represent promising biomarkers with prognostic value. More detailed investigations on a larger number of cases are needed to better understand the influence of these miRNAs in canine mammary tumors.

Morphological and Molecular Identification of Mullet Helminth Parasite Fauna from Ganzirri Lagoon (Sicily, Southern Italy).

Mullets (Osteichthyes: Mugilidae) are a euryhaline species widely distributed all over the world, thus representing an excellent study model for host-parasite interactions. From March to June 2022, 150 mullets, belonging to Chelon labrosus (n = 99), Chelon auratus (n = 37), and Oedalechilus labeo (n = 14) species, were caught to identify the helminth parasite fauna of the different mullet species present in the Ganzirri Lagoon (Messina, Sicily, Italy). A parasitological evaluation of the gastrointestinal tract (GIT) was carried out with a total worm count technique (TWC) to detect helminth presence. All collected parasites were stored in 70% ethanol until morphological evaluation, and frozen at -80 °C for subsequent molecular analysis, using 28S, ITS-2, 18S primers. The morphological evaluation allowed for the identification Acanthocephalan parasites (Neoechinorhynchus agilis) from two C. labrosus specimens. Sixty-six samples were positive for adult digenean trematodes (C. labrosus, 49.5 %; C. auratus, 27%, and O. labeo, 50%), molecularly identified as Haploporus benedeni. This study represents the first survey of helminthic parasite fauna of mullets from the south of Italy. The presence of Hydrobia sp. in the stomach contents of mullets allowed us to infer the H. benedeni life cycle in the Ganzirri lagoon.

Early Exposure to Environmental Pollutants: Imidacloprid Potentiates Cadmium Toxicity on Zebrafish Retinal Cells Death.

In the present study, we analyzed the combination of non-toxic concentrations per se, of Cd and a pesticide the imidacloprid (IMI) (10 and 50 µM for Cd and 195 µM for IMI), to highlight early developmental toxicity and possible damage to retinal cells. Co-exposure to Cd and IMI showed a toxic effect in zebrafish larval development, with lowered degrees of survival and hatching, and in some cases the induction of structural alterations and edema. In addition, co-exposure to 50 and 195 µM, respectively, for Cd and IMI, also showed increased apoptosis in eye cells, accompanied by up regulation of genes associated with antioxidant markers (cat, sod1, nrf2 and ho-1). Thus, the present study aims to highlight how the presence of multiple contaminants, even at low concentrations, can be a risk factor in a model of zebrafish (Danio rerio). The presence of other contaminants, such as IMI, can cause an enhancement of the toxic action of Cd on morphological changes in the early life stage of zebrafish, but more importantly disrupt the normal development of the retina, eventually triggering apoptosis.

Impact of Mycotoxin Contaminations on Aquatic Organisms: Toxic Effect of Aflatoxin B1 and Fumonisin B1 Mixture.

(1) Background: Multiple contaminations of several mycotoxins have been detected in human and veterinary food and feed worldwide. To date, a number of studies on the combined effects of mycotoxins have been conducted on cell and animal models, but very limited studies have been done on aquatic organisms. (2) The purpose of the present study was to evaluate the combined toxic effects of Aflatoxin B1 (AFB1) and Fumonisin B1 (FB1) on zebrafish (Danio rerio) embryos. (3) Results: Our results showed that the combination of AFB1 and FB1 at nontoxic concentrations exerted a negative effect on the lethal endpoints analyzed, such as survival, hatching, and heart rate. In addition, the mixture of mycotoxins caused an increase in the levels of enzymes and proteins involved in the antioxidant process, such as superoxide dismutase (SOD) and catalase (CAT), both in terms of protein levels and gene expression, as well as an increase in the levels of the detoxification enzymes glutathione s-transferases (GST) and cytochromes P450 (CYP450). Furthermore, we showed that the mycotoxin mixture induced an increase in pro-apoptotic proteins such as bax and caspase 3, and at the same time reduced the gene expression of the anti-apoptotic bcl-2 protein. Finally, a significant decrease in thyroid function was observed in terms of triiodothyronine (T3), thyroxine (T4), and vitellogenin (VTG) levels. (4) Conclusion: We can say that the mixture of mycotoxins carries a greater risk factor than individual presences. There is a greater need for effective detoxification methods to control and reduce the toxicity of multiple mycotoxins and reduce the toxicity of multiple mycotoxins in feed and throughout the food chain.

Postmortem Electrical Conductivity Changes of Dicentrarchus labrax Skeletal Muscle: Root Mean Square (RMS) Parameter in Estimating Time since Death.

Electric impedance spectroscopy techniques have been widely employed to study basic biological processes, and recently explored to estimate postmortem interval (PMI). However, the most-relevant parameter to approximate PMI has not been recognized so far. This study investigated electrical conductivity changes in muscle of 18 sea bass specimens, maintained at different room temperatures (15.0 °C; 20.0 °C; 25.0 °C), during a 24 h postmortem period using an oscilloscope coupled with a signal generator, as innovative technology. The root mean square (RMS) was selected among all measured parameters, and recorded every 15 min for 24 h after death. The RMS(t) time series for each animal were collected and statistically analyzed using MATLAB®. A similar trend in RMS values was observed in all animals over the 24 h study period. After a short period, during which the RMS signal decreased, an increasing trend of the signal was recorded for all fish until it reached a peak. Subsequently, the RMS value gradually decreased over time. A strong linear correlation was observed among the time series, confirming that the above time-behaviour holds for all animals. The time at which maximum value is reached strongly depended on the room temperature during the experiments, ranging from 6 h in fish kept at 25.0 °C to 14 h in animals kept at 15.0 °C. The use of the oscilloscope has proven to be a promising technology in the study of electrical muscle properties during the early postmortem interval, with the advantage of being a fast, non-destructive, and inexpensive method, although more studies will be needed to validate this technology before moving to real-time field investigations.

Combined Effects of Potassium Perchlorate and a Neonicotinoid on Zebrafish Larvae (Danio rerio).

Imidacloprid (IMI) is part of the neonicotinoids family, insecticides widely used by humans and also found in wastewater. This class of compounds, if present in the environment, can cause toxicity to different species such as bees and gammarids, although little is known about vertebrates such as fish. In addition, several substances have been reported in the environment that can cause damage to aquatic species, such as potassium perchlorate (KClO4), if exposed to high concentrations or for long periods. Often, the co-presence of different contaminants can cause a synergistic action in terms of toxicity to fish. In the present study, we first analyzed different concentrations of IMI (75, 100 and 150 mg/L) and KClO4 (1, 1.5 and 5 mM) to highlight the morphological effects at 96 hpf and, subsequently, chose two nontoxic concentrations to evaluate their co-exposure and the pathway involved in their co-toxicity. Morphological alteration, mucus production, messenger RNA (mRNA) expression related to intestinal function and oxidative stress were measured. These results suggest that co-exposure to IMI and KClO4 could affect zebrafish embryo development by increasing gut toxicity and the alteration of antioxidative defense mechanisms.

Environmental Toxicity Assessment of Sodium Fluoride and Platinum-Derived Drugs Co-Exposure on Aquatic Organisms.

Pharmaceuticals are widely acknowledged to be a threat to aquatic life. Over the last two decades, the steady use of biologically active chemicals for human health has been mirrored by a rise in the leaking of these chemicals into natural environments. The aim of this work was to detect the toxicity of sodium fluoride (NaF) exposure and platinum-derived drugs in an ecological setting on aquatic organism development. From 24 to 96 h post-fertilization, zebrafish embryos were treated to dosages of NaF 10 mg/L-1 + cisplatin (CDDP) 100 µM, one with NaF 10 mg/L-1 + carboplatin (CARP) 25 µM, one with NaF 10 mg/L-1 + CDDP 100 µM + CARP 25 µM. Fluoride exposure in combination with Cisplatin and Carboplatin (non-toxic concentration) had an effect on survival and hatching rate according to this study. Additionally, it significantly disturbed the antioxidant defense system and increased ROS in zebrafish larvae. NaF 10 mg/L-1 associated with CDDP 100 µM and CARP 25 µM, increased the production of apoptosis-related proteins (caspase 3, bax, and bcl-2) and the downregulation of acetylcholinesterase (AChE) activity, while no effect was seen for the single exposure.

Chemical Characterization, Antibacterial Activity, and Embryo Acute Toxicity of Rhus coriaria L. Genotype from Sicily (Italy).

This study reports a full characterization of the Sicilian sumac, Rhus coriaria L. This fruit represents a potential source of fiber (33.21 ± 1.02%) and unsaturated fatty acids, being the contents of linoleic and α-linolenic acids, 30.82 ± 1.21% and 1.85 ± 0.07%, respectively. In addition, the content of phenolic and total anthocyanin was 71.69 ± 1.23 mg/g as gallic acid equivalents, and 6.71 ± 0.12 mg/g as cyanidin-3-O-glucoside equivalents, respectively. The high content in mineral elements, consisting mainly of potassium, calcium, magnesium, and phosphorus, followed by aluminum, iron, sodium, boron, and zinc, was detected by inductively coupled plasma mass spectrometry (ICP-MS). Moreover, its antimicrobial activity was evaluated against multidrug resistant (MDR) microorganisms, represented by Escherichia coli and Klebsiella pneumoniae strains isolated from poultry. The activity of seven different sumac fruit extracts obtained using the following solvents-ethanol (SE), methanol (SM), acetone (SA), ethanol and water (SEW), methanol and water (SMW), acetone and water (SAW), water (SW)-was evaluated. The polyphenol profile of SM extract, which showed better activity, was analyzed by ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS). The major component identified was gallic acid, followed by quercetin, methyl digallate, pentagalloyl-hexoside, and kaempferol 3-O-glucoside. The non-toxicity of Sicilian R. coriaria was confirmed by testing the effect of the same extract on zebrafish embryos.

Environmental Risk Assessment of Oxaliplatin Exposure on Early Life Stages of Zebrafish (Danio rerio).

Pharmaceuticals are actually identified as a threat to the ecosystem. Nowadays, the growing consumption of antineoplastic agents has been related to their continuous input in natural environments. These substances can interfere with physiological and biochemical processes of aquatic species over their entire life cycle. Oxaliplatin (OXA) is a widely used chemotherapeutic agent to treat colon or rectal cancer. This study was aimed to evaluate the developmental toxicity of the OXA exposure. To this end, zebrafish embryos were incubated with 0.001, 0.1, 0.5 mg/L OXA. At different timepoints mortality rate, hatching rate, developmental abnormalities, histological analysis, oxidative stress and mRNA expression of gene related to oxidative stress were evaluated. Our results showed that OXA exposure can induce increased mortality and developmental abnormalities reducing the hatching rate. Histological analysis demonstrated that OXA induced liver, intestine, muscle and heart injury. Superoxide dismutase and catalase activities were significantly increased after OXA exposure demonstrating its oxidative effects. The mRNA expression levels of apoptosis-related genes (caspase-3, bax and bcl-2) were significantly upregulated by OXA exposure. In conclusion, we highlighted that OXA exposure led to a dose-related developmental toxicity, oxidative stress and apoptosis.

Embryotoxicity of polystyrene microplastics in zebrafish Daniorerio.

In the recent years, increasing scientific and societal concern has been raised over the presence and accumulation of plastic debris in the environment and the effects of microplastics (MPs) that can easily interact with biota. In order to elucidate the impact of MPs at the critical development stages of freshwater fish species, a fish embryo toxicity test was herein performed on the zebrafish Danio rerio, exposed to 10 µm polystyrene MPs at 200 particles/mL for 120 hpf. After exposure, accumulation of MPs in larvae was measured, survival, hatching and larvae development were monitored and the oxidant/anti-oxidant responses and cellular detoxification evaluated. No impact on survival of developing zebrafish was revealed, but a moderate delay in hatching was observed. Alterations in larvae development were recorded with zebrafish exhibiting serious deformities, mainly at the level of column and tail, as well as a compromised integrity of the visual structure of the eyes. Moreover, increased levels of gene transcription involved in the oxidative stress (sod1, sod2 and cat) and in cellular detoxification (gst and cyp) were also detected in MPs-exposed zebrafish larvae. Overall, this research work provides new insights on the ecotoxicological impact of polystyrene MPs on the critical developmental stages of a freshwater fish species, therefore enhancing the current knowledge of the environmental risk posed by MPs to the aquatic ecosystem.

Combined Toxicity of Xenobiotics Bisphenol A and Heavy Metals on Zebrafish Embryos (Danio rerio).

Environmental pollutants may cause adverse effects on the immune system of aquatic organisms. This study revealed that combination of environmental pollutants and Bisphenol A(BPA) could cause an acute inflammatory response in zebrafish larvae as shown by body alterations, which may imply a common immunotoxicity mechanism for most environmental pollutants. In the present study we evaluated the toxicity after co-exposure of BPA and Cd or Cr (III) in zebrafish embryos and larvae, and the oxidative stress pathway involved. Evaluation of lethal and developmental endpoints such as hatching, edema, malformations, abnormal heart rate and survival rate were evaluated after 96 h of exposure. Combination of BPA at 10 µM with Cd or Cr at 0.5 µM exposure induce malformations at 96 hpf in zebrafish larvae, as well as significantly increases oxidative stress and induce apoptosis on larvae. Our study suggested how environmental pollutant showed a synergistic effect at common not-effective doses, promoting decrease of antioxidant defense and contrasted fish development.

Aflatoxin B1 Toxicity in Zebrafish Larva (Danio rerio): Protective Role of Hericium erinaceus.

Aflatoxin B1 (AFB1), a secondary metabolite produced by fungi of the genus Aspergillus, has been found among various foods as well as in fish feed. However, the effects of AFB1 on fish development and its associated toxic mechanism are still unclear. In the present study, we confirmed the morphological alterations in zebrafish embryos and larvae after exposure to different AFB1 doses as well as the oxidative stress pathway that is involved. Furthermore, we evaluated the potentially protective effect of Hericium erinaceus extract, one of the most characterized fungal extracts, with a focus on the nervous system. Treating the embryos 6 h post fertilization (hpf) with AFB1 at 50 and 100 ng/mL significantly increased oxidative stress and induced malformations in six-day post-fertilization (dpf) zebrafish larvae. The evaluation of lethal and developmental endpoints such as hatching, edema, malformations, abnormal heart rate, and survival rate were evaluated after 96 h of exposure. Hericium inhibited the morphological alterations of the larvae as well as the increase in oxidative stress and lipid peroxidation. In conclusion: our study suggests that a natural extract such as Hericium may play a partial role in promoting antioxidant defense systems and may contrast lipid peroxidation in fish development by counteracting the AFB1 toxicity mechanism.

Anti-Atherogenic Effect of 10% Supplementation of Anchovy (Engraulis encrasicolus) Waste Protein Hydrolysates in ApoE-Deficient Mice.

Fish protein consumption exerts beneficial metabolic effects on human health, also correlating with a decreased risk for cardiovascular disease. Fish waste contains high amount of proteins and utilization may offer the opportunity for generating compounds advantageous for human health. Especially, fish waste protein hydrolysates beneficially influence pathways involved in body composition, exerting anti-inflammatory and antioxidant activities, making their potential supplementation in human disorders of increased interest. This study assessed the effect of a 10% (w/w) anchovy waste protein hydrolysate (APH) diet for 12 weeks in reducing atherosclerosis in ApoE-/- mice, through histological and immunohistochemical methods. In addition, monitoring of plaque development was performed, using high-frequency ultrasound and magnetic resonance imaging. Overall, the APH diet attenuated atherosclerotic plaque development, producing a regression of arterial lesions over time (p < 0.05). Twelve weeks on an APH diet had an anti-obesity effect, improving lipid metabolism and reducing hepatic enzyme activity. A significant reduction in plaque size and lipid content was observed in the aortic sinus of APH-fed mice, compared to the control (p < 0.001), whereas no differences in the extracellular matrix and macrophage recruitment were observed. Supplementation of APH significantly attenuates atherosclerosis in ApoE-/- mice, exerting a lipid-lowering activity. The opportunity to use fish waste protein hydrolysates as a nutraceutical in atherosclerosis is worthy of future investigations, representing a low cost, sustainable, and nutritional strategy with minimal environmental impact.

Administration of Protein Hydrolysates from Anchovy (Engraulis Encrasicolus) Waste for Twelve Weeks Decreases Metabolic Dysfunction-Associated Fatty Liver Disease Severity in ApoE-/-Mice.

Metabolic dysfunction-associated fatty liver disease (MAFLD) includes several diseases, ranging from simple steatosis to steatohepatitis, fibrosis and cirrhosis. Fish-rich diets are considered helpful in the prevention of MAFLD, and the enzymatic hydrolysis of fish waste has been explored as a means of obtaining high-value protein hydrolysates, which have been proven to exert beneficial bioactivities including anti-obesity and hypocholesterol effects. This study aimed to assess the effect of the administration of protein hydrolysates from anchovy waste (APH) for 12 weeks on attenuated high-fat diet-induced MAFLD in apolipoprotein E-knockout mice (ApoE-/-). Thirty ApoE-/- mice were divided into two groups (n = 15/group) and fed a high-fat diet (HFD), with and without the addition of 10% (w/w) APH. After 12 weeks, serum and hepatic lipid profiles, hepatic enzyme activities, liver histology and immunohistochemistry were analyzed to assess hepatic steatosis, inflammation and fibrosis. Twelve-weeks on a 10% (w/w) APH diet reduces total cholesterol and triglyceride serum levels, hepatic enzyme activity and hepatic triacylglycerol content (p < 0.0001), and results in a reduction in hepatic fat accumulation and macrophage recruitment (p < 0.0001). The results suggest that a 10% APH diet has an anti-obesity effect, with an improvement in lipid metabolism, hepatic steatosis and liver injury as a result of a high-fat diet. Protein hydrolysates from fish waste may represent an efficient nutritional strategy in several diseases, and their use as nutraceuticals is worthy of future investigation.