SIRT5 rs12216101 T>G variant is associated with liver damage and mitochondrial dysfunction in patients with non-alcoholic fatty liver disease.

BACKGROUND & AIMS: Sirtuin 5, encoded by the SIRT5 gene, is a NAD+-dependent deacylase that modulates mitochondrial metabolic processes through post-translational modifications. In this study, we aimed to examine the impact of the SIRT5 rs12216101 T>G non-coding single nucleotide polymorphism on disease severity in patients with non-alcoholic fatty liver disease (NAFLD). METHODS: The rs12216101 variant was genotyped in 2,606 consecutive European patients with biopsy-proven NAFLD. Transcriptomic analysis, expression of mitochondrial complexes and oxidative stress levels were measured in liver samples from a subset of bariatric patients. Effects of SIRT5 pharmacological inhibition were evaluated in HepG2 cells exposed to excess free fatty acids. Mitochondrial energetics in vitro were investigated by high-performance liquid chromatography. RESULTS: In the whole cohort, the frequency distribution of SIRT5 rs12216101 TT, TG and GG genotypes was 47.0%, 42.3% and 10.7%, respectively. At multivariate logistic regression analysis adjusted for sex, age >50 years, diabetes, and PNPLA3 rs738409 status, the SIRT5 rs12216101 T>G variant was associated with the presence of non-alcoholic steatohepatitis (odds ratio 1.20, 95% CI 1.03-1.40) and F2-F4 fibrosis (odds ratio 1.18; 95% CI 1.00-1.37). Transcriptomic analysis showed that the SIRT5 rs12216101 T>G variant was associated with upregulation of transcripts involved in mitochondrial metabolic pathways, including the oxidative phosphorylation system. In patients carrying the G allele, western blot analysis confirmed an upregulation of oxidative phosphorylation complexes III, IV, V and consistently higher levels of reactive oxygen species, reactive nitrogen species and malondialdehyde, and lower ATP levels. Administration of a pharmacological SIRT5 inhibitor preserved mitochondrial energetic homeostasis in HepG2 cells, as evidenced by restored ATP/ADP, NAD+/NADH, NADP+/NADPH ratios and glutathione levels. CONCLUSIONS: The SIRT5 rs12216101 T>G variant, heightening SIRT5 activity, is associated with liver damage, mitochondrial dysfunction, and oxidative stress in patients with NAFLD. IMPACT AND IMPLICATIONS: In this study we discovered that the SIRT5 rs12216101 T>G variant is associated with higher disease severity in patients with non-alcoholic fatty liver disease (NAFLD). This risk variant leads to a SIRT5 gain-of-function, enhancing mitochondrial oxidative phosphorylation and thus leading to oxidative stress. SIRT5 may represent a novel disease modulator in NAFLD.

(+)-Lipoic Acid Reduces Lipotoxicity and Regulates Mitochondrial Homeostasis and Energy Balance in an In Vitro Model of Liver Steatosis.

Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids within hepatocytes, which compromises liver functionality following mitochondrial dysfunction and increased production of reactive oxygen species (ROS). Lipoic acid is one of the prosthetic groups of the pyruvate dehydrogenase complex also known for its ability to confer protection from oxidative damage because of its antioxidant properties. In this study, we aimed to investigate the effects of lipoic acid on lipotoxicity and mitochondrial dynamics in an in vitro model of liver steatosis. HepG2 cells were treated with palmitic acid and oleic acid (1:2) to induce steatosis, without and with 1 and 5 µM lipoic acid. Following treatments, cell proliferation and lipid droplets accumulation were evaluated. Mitochondrial functions were assessed through the evaluation of membrane potential, MitoTracker Red staining, expression of genes of the mitochondrial quality control, and analysis of energy metabolism by HPLC and Seahorse. We showed that lipoic acid treatment restored membrane potential to values comparable to control cells, as well as protected cells from mitochondrial fragmentation following PA:OA treatment. Furthermore, our data showed that lipoic acid was able to determine an increase in the expression of mitochondrial fusion genes and a decrease in mitochondrial fission genes, as well as to restore the bioenergetics of cells after treatment with palmitic acid and oleic acid. In conclusion, our data suggest that lipoic acid reduces lipotoxicity and improves mitochondrial functions in an in vitro model of steatosis, thus providing a potentially valuable pharmacological tool for NAFLD treatment.

Seasonal Biodistribution of Some Trace Elements (Cd, Pb, Cr, Hg) and "Blood Biomarkers" Response in Mugil cephalus (Linnaeus, 1758).

This study had a purpose to evaluate the seasonal biodistribution of some trace elements (cadmium, Cd; Lead, Pb; chrome, Cr; and mercury, Hg) in different tissues (muscle, gills, liver, stomach, and intestine) of striped mullet Mugil cephalus (Linnaeus, 1758) and the hematological and biochemical responses of this species to aquatic pollution by trace metals. For this purpose, 80 M. cephalus (20 for each season) were captured in three different stations of Faro Lake, Messina, Sicily. Biometric indices (weight, fork length, and total length) of each fish were registered. The physico-chemical parameters of the water of the study area were also measured, and the content of trace elements in water and sediment was determined. The hematological (white blood cell, WBC; red blood cell, RBC; hemoglobin concentration, Hb; hematocrit, Hct; mean corpuscular volume, MCV; mean corpuscular hemoglobin, MCH; mean corpuscular hemoglobin concentration, MCHC; thrombocytes count, TC), biochemical parameters (aspartate aminotransferase, AST; alanine aminotransferase, ALT; alkaline phosphatase, ALP; lactate dehydrogenase, LDH), and the seasonal concentration of the trace elements in the different tissues of M. cephalus were assessed. Our results showed a different biodistribution of the trace elements in M. cephalus and significant variations of the blood parameters in the different seasons. This research provides a valid contribute to environmental biomonitoring techniques useful in aquatic pollution control and water management. It also contributes to broadening the studies on the improvement of the health and sustainability of aquatic environments.

Effects of heavy pollution in different water bodies on male rainbow trout (Oncorhynchus mykiss) reproductive health.

This research aimed to investigate the influence of heavy metals on the reproductive health of rainbow trout (Oncorhynchus mykiss) captured from two locations, Verinag (S1) and Panzath (S2). Sixty (n. 60) mature rainbow trout samples (30 from each site) with body weights ranging from 400 to 650 g were collected from Verinag hatchery, which was less polluted (S1), and Panzath hatchery, which was more polluted (S2). The findings revealed significant differences between the two sites, S1 (less polluted) and S2 (more polluted), as well as significant variations within the tissues. Iron (Fe) and Zinc (Zn) were found to be highest in sampling water as well as in fish tissues sampled from S2 sites. Similarly, concerning S1 captured fishes, histopathological examination of testes from S2 captured fish was found. Testicular abnormalities that included disorganization of the seminiferous tubules, reduction in the number of germ cells (sperm cells, spermatozoa), vacuolization, and large empty areas in the seminiferous epithelium were found. In testicular cells, the frequency of apoptotic cells collected from S2 water increased significantly (P < 0.05). SOD, catalase, and glutathione peroxidase activity increased in S1 captured fishes but decreased in S2 captured fishes. MDA levels gradually increased in S2 captured fish, and the degree of heavy metal stress was positively correlated (P < 0.05). In male rainbow trout, testosterone and 11-ketotestosterone levels increased substantially in S1 captured fishes. While the fatty acids of testes in S2 fishes decreased with respect to S1 fishes. In conclusion, S2 captured fish suffered more damage due to heavy metals including cellular damage, apoptosis, oxidative damage, and altered steroid hormones when compared to fish from S1 waters.

Nickel and cadmium tissue bioaccumulation and blood parameters in Chelon auratus and Mugil cephalus from Anzali free zone in the south Caspian Sea (Iran) and Faro Lake (Italy): A comparative analysis.

BACKGROUND: Analysis of heavy metal concentrations in fish blood is a valuable tool in environmental pollution monitoring. Among different type of fish, detritivorous fish are a very good indicator for monitoring pollution and environmental stress, along with hematological studies, which are an important indicator in eco-toxicological and biological studies. AIM: The aim of this study was to evaluate the influence of environment on bioaccumulation of Ni and Cd and on blood parameters in Chelon auratus and Mugil cephalus (a detritivorous fish which is widely distributed in the world) captured in Caspian Sea and Faro Lake. METHODS: For the research blood and tissues samples were collected from 40 mullets (20 Mugil cephalus from Italy and 20 Chelon auratus from Iran) in 2019. The hematological (white blood cell, WBC; red blood cell, RBC; thrombocyte count, TC; hematocrit, Hct; hemoglobin concentration, Hb; mean corpuscular volume, MCV; mean corpuscular hemoglobin, MCH and mean corpuscular hemoglobin concentration, MCHC) and biochemical parameters (aspartate aminotransferase, AST; alanine aminotransferase, ALT; alkaline phosphatase, ALP; lactate dehydrogenase, LDH and creatine phosphokinase, CPK were assessed. RESULTS: Although the concentration of Ni and Cd in the muscle of fish have no significant health risks and were low in both regions, the most elevated concentration was found in the liver of Caspian Sea mullet. In all cases, the results obtained for all biochemical and most hematological parameters of individual Faro Lake, were considerably lower than the Caspian Sea, demonstrating that habitats and environmental conditions affect the blood metabolites. CONCLUSIONS: The results of this study show that these measurements can be used as criteria for the quantitative evaluation of fish /health and provide information on the extent of potential poisoning and the risks posed to the populations and fisheries.

Cadmium Sub-Lethal Concentration Effect on Growth, Haematological and Biochemical Parameters of Mystus seenghala (Sykes, 1839).

The current study aims to assess the cadmium sub-lethal concentration influence on growth and haematological and biochemical parameters of Mystus seenghala. A total of 60 fish of three different length groups (20 each) were collected from Head Qadirabad, Pakistan. The fish were treated to the sub-lethal concentration viz. one-third of LC50, for 16 weeks except for the control groups. Water quality parameters were kept constant during the entire course of the research, and the major parameters were measured as temperature (28.03 ± 0.03 °C), DO (5.82 ± 0.14 mg L-1), pH (8.00 ± 0.01) and total hardness (249.98 ± 0.01 mg L-1). Findings revealed that the growth of three treated variant length groups was affected negatively by cadmium exposure and showed significantly (P < 0.05) lower average wet weight, body length and condition factor as compared to control groups, while the feed conversion ratio (FCR) increases by increasing the exposure duration. The haematological parameters including values of Hct, Hb and MCHC were significantly (P < 0.05) reduced in all Cd-treated groups than control groups, whereas the level of MCH and MCV were significantly higher, but no significant difference was found in the value of RBCs in all treated groups. Biochemical parameters such as ALT, AST, total lipid and glucose level in Cd exposure groups were significantly higher, while the total protein level was significantly (P < 0.05) reduced in all treated groups as compared to control groups. From the current study, it has been concluded that the growth, haematology and biochemical parameters are important indicators of ecotoxicology particularly contamination of the cadmium and health of the fish.

Intra-variability of some biochemical parameters and serum electrolytes in rainbow trout (Walbaum, 1792) bred using a flow-through system.

This study aims to evaluate some biochemical parameters and serum electrolytes in cultured rainbow trout Oncorhynchus mykiss (Walbaum, 1792) to evaluate a potential correlation with biometric parameters (weight and length). For this purpose, 100 cultured trout (300-700 g weight range, 25-38 cm length range) bred on a fish-farm were used for the study. Physico-chemical characteristics of water were measured on the farm. Blood samples were collected from each fish to analyze the following parameters: glucose, triglycerides, cholesterol, aspartate aminotransferase (AST); alanine aminotransferase (ALT), calcium (Ca2+), chlorine (Cl-), iron (Fe2+), phosphorus (P), magnesium (Mg2+), potassium (K+), sodium (Na+) and urea. Statistical data analysis showed a significant correlation between size and glucose and cholesterol. No correlation was found between size and other parameters studied. These results represent a contribution to the study of fish size leading to better understanding some biochemical parameters and serum electrolyte profiles in cultured rainbow trout. This research contributes to understanding the intra-individual variability of some blood parameters in cultured rainbow trout O. mykiss offering reliable information on chronic stress status, metabolic disorders and deficiencies in relation to different sizes. These could help in improve the health monitoring in trout fish farms.

Effect of Fortified Feed with Phyto-Extract on the First Physical Barrier (Mucus) of Labeo rohita.

The aim of the current study was to assess the effect of two different fortified feeds with different concentrations of two important medicinal plants (Withania coagulans and Zingiber officinale) on the mucosal immunity of Labeo rohita. After a dietary intervention, mucus was tested against five pathogenic bacteria (in-vitro), while experimental fish were tested against the ectoparasite (Lernaea) (in-vivo). Our results revealed that all fish groups fed with different concentrations (1, 1.5, and 2%) of Z. officinale had low molecular weight proteins and did not develop any significant signs of parasitic infection, with low mortality rate; whereas the groups that were fed with W. coagulans (particularly with 1% and 2%), including a control group, developed rapid signs of infection with high mortality rate. The highest hemagglutination titer value was recorded for the fish fed with 1% and 1.5% of Z. officinale. The lowest value was found for the fish fed with 2% of W. coagulans. The mucus of all fish of fortified groups was active and inhibited the growth of tested bacterial pathogens as compared to the control group. Further, Z. officinale groups showed greater efficacy against bacteria as compared to the W. coagulans groups. In conclusion, Z. officinale can be considered as a potential and functional ingredient in aquaculture feed. Furthermore, future studies should be conducted to investigate more details on the subject.

Environmental Investigations and Tissue Bioaccumulation of Heavy Metals in Grey Mullet from the Black Sea (Bulgaria) and the Ionian Sea (Italy).

The environmental monitoring of chemical toxicants has been a widely studied topic in the last few decades. The main aim of the present study was to determine the total concentration of nine elements (Cd, Cr, Pb, Ni, Al, Cu, Fe, Mn, and Zn) in the fish species grey mullet (M. cephalus) and in the coastal marine waters collected from various sampling points along the Black Sea (Bulgaria) and the Ionian Sea (Italy). Further, those results were applied to predict the pollution degree in those coastal marine environments. The fish samples were subject to acid digestion followed by appropriate analytical determination. The metal concentrations in marine water samples collected from the Black Sea (Bulgaria) and the Ionian Sea (Italy) were also analyzed. Unpaired Student's t-test and the one-way ANOVA were applied for the statistical analysis of the data. The statistical results revealed a significant variation (p < 0.0001) in the concentration of various fish tissues. The accumulation of toxic and essential elements differs significantly in grey mullet species caught from the Black Sea (Bulgaria) and the Ionian Sea (Italy). The results from this study may serve as a convenient approach during marine pollution programs set by both countries (Italy and Bulgaria).

Response of vanadium bioaccumulation in tissues of Mugil cephalus (Linnaeus 1758).

Vanadium accumulation levels in different tissues (muscle and organs) of the striped mullet Mugil cephalus (Linnaeus, 1758) and possible relationships with blood parameters were evaluated in a Natural Protected Area (Lake Faro, Sicily, Italy), during the winter of 2017. Hematological parameters (red blood cell, RBC; white blood cell, WBC; hemoglobin concentration, Hb; hematocrit, Hct; mean corpuscular volume, MCV; mean corpuscular hemoglobin, MCH; mean corpuscular hemoglobin concentration, MCHC; thrombocytes, TC), biometric indices (weight, total and fork length), and vanadium levels in muscles and organs (gills, liver, stomach and intestine) were determined. Statistical analyses showed significant differences in concentrations of vanadium of the analyzed tissue of M. cephalus and a positive relationship between vanadium concentration in the liver and some hematological parameters (RBC, Hb and Hct) and biometric indices. Our results underline the importance of fish blood parameters as sensitive indicators of toxic impact of environmental factors such as metals. This study, focusing on an ongoing topic, represents a valuable contribution to research concerning the monitoring and prevention of vanadium pollution in aquatic organisms and environments.

Relationship between arsenic accumulation in tissues and hematological parameters in mullet caught in Faro Lake: a preliminary study.

The authors investigated the arsenic (As) accumulation in different tissues (muscle, gill, liver, stomach, and intestine) and the possible correlation between tissue concentration and hematological parameters in mullet (Mugil cephalus Linnaeus, 1758) caught in Faro Lake (Messina, Sicily, Italy). On all fish, hematological analyses of blood samples, measurement of biometric indices, and the removal of the muscles, gills, liver, stomach, and intestine for the determination of arsenic concentration were performed. A hemogram was performed to find effects of arsenic concentration in tissues on hematological variables. One-way analysis of variance showed significant differences of arsenic concentration in different tissues, with higher values in the gill. The correlation between hematological parameters and tissue arsenic concentration showed a statistical significance for red blood cell (RBC), hemoglobin concentration (Hb), and hematocrit (Hct) with the liver As concentration. Biometric indices (weight, length, and fork length) showed a significant correlation with As concentration of the muscle and liver also. Our results indicate the role of some hematological parameters as biomarkers useful to monitoring anthropogenic load of arsenic in water and sediment, because variations of these parameters represent one of the effects that arsenic exposure can have on fish.

Occurrence of diseases in fish used for experimental research.

The objective of the present study was to evaluate the occurrence of pathogens and diseases in laboratory fish over a 10-year period at the Centre for Experimental Fish Pathology of Sicily, University of Messina. This report also emphasizes the adverse effects of subclinical infections on research endpoints, as well as the importance of animal health with respect to welfare. Infections in fish used for research can alter experimental outcomes, increase the variability of data, and impede experimental reproducibility. For this purpose, 411 diseased fish of different species (out of a total of 2820 fish) that belonged to four marine species (Dicentrarchus labrax, Sparus aurata, Argyrosomus regius and Mugil cephalus) and to four fresh water species (Danio rerio, Carassius auratus, Xiphophorus variatus and Poecilia reticulata) were examined in this study. Our results showed that mycobacteriosis and myxosporidiosis were the most important diseases found in our research fish, and the results represent a useful tool to obtain wider knowledge on the incidence of various diseases in different fish species. Further studies in this field are necessary to improve knowledge on the state of the health of fish used for research.

Effects of Different Levels of Pomegranate Seed Oil on Some Blood Parameters and Disease Resistance Against Yersinia ruckeri in Rainbow Trout.

This study is aimed to assess the effects of pomegranate seed oil (PSO) supplementation on growth performance, some hematological, biochemical and immunological parameters, and disease resistance against Yersinia ruckeri in cultured rainbow trout Oncorhynchus mykiss (Walbaum, 1792). 240 fish in total were randomly assigned into four triplicate groups (20 fish/per aquarium) corresponding to four dietary treatments: control (PSO0; no addition of PSO), 0.5% (PSO5), 1.00% (PSO10), and 2.00% (PSO20) of PSO, respectively. After the 60 day-feeding trial, fish blood samples were collected and compared. Statistical analysis (one-way ANOVA) showed a significant (P < 0.05) effect of PSO on red blood cell count, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin concentration, cholesterol, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase parameters in PSO5 and PSO10 with regard to control. Moreover, a pronounced (P < 0.05) increased in weight gain, growth and feed conversion was found in fish fed with PSO supplemented diets. After the feeding trial, fish were challenged with Y. ruckeri and survival recorded for 20 days. Cumulative survival was 45.10% in fish fed with the control diet, whereas in fish fed with PSO5, PSO10, and PSO20 supplemented diets, survival was 58.82, 56.86, and 56.86%, respectively. In conclusion, dietary administration of PSO induced a reduction in mortality of rainbow trout infected with Y. ruckeri, intercalary significant differences occurred on growth performance and some blood values among treated groups. These positive effects of PSO could be considered for new applications in aquaculture.