Unraveling Cecal Alterations in Clostridioides difficile Colonized Mice through Comprehensive Metabolic Profiling.

The disruption of gut microbiota caused by antibiotics favors the intestinal colonization of Clostridioides difficile - a Gram-positive, spore-forming anaerobic bacterium that causes potentially fatal gastrointestinal infections. In an endeavor to elucidate the complexities of the gut-brain axis in the context of Clostridium difficile infection (CDI), a murine model has been used to investigate the potential effects of antibiotic administration and subsequent colonization by C. difficile, as well as the impact of three different 10-day treatments (metronidazole, probiotics, and fecal microbiota transplantation), on the cecal metabolome for the first time. This follows our previous research which highlighted the metabolic effect of CDI and these treatments in the brain and employs the same four different metabolomics-based methods (targeted GC-MS/MS, targeted HILIC-MS/MS, untargeted RP-LC-HRMS/MS and untargeted GC-MS). A total of 286 unique metabolites have been identified in the mouse cecal profiles and statistical analysis revealed that CDI, as well as the subsequent treatments, significantly alters cecal metabolites and lipids implicated in various biochemical pathways centered around amino acid metabolism, glycerophospholipid metabolism, and central carbon metabolism. To our knowledge, this study represents the first exploration of the effects of C. difficile-induced colitis and potential treatments on the cecal tissue metabolome.

Redox Biomarkers Assessment after Oral Administration of Wine Extract and Grape Stem Extract in Rats and Mice.

Wine and by-products of the winemaking process, such as grape stems, are rich in bioactive polyphenolic compounds that might be beneficial for animal and human health. In recent years, the administration of dietary polyphenols with strong antioxidant and cytoprotective properties has constituted an emerging line of research interest toward disease prevention. However, in scientific literature, only a limited number of studies have investigated the safety and the toxicological risks of polyphenolic compounds in vivo. Based on the above, the purpose of the present study was two-fold: first, to examine the effects of oral administration of a grape stem extract, derived from the Greek red wine Mavrodaphne, on mice redox biomarkers; and second, to investigate the biological effects of oral administration of a wine extract, derived from the emblematic Greek red wine Xinomavro, on rats. Toward this purpose, body weight, growth rate, hematological, biochemical, and histopathological parameters, as well as a panel of redox biomarkers, were examined. According to our results, the administration of Mavrodaphne grape stem extract in mice induced alterations in redox homeostasis, preventing mice from the adverse effects of lipid peroxidation. Contrariwise, the administration of Xinomavro wine extract induced both beneficial and harmful outcomes on rat redox status determined by the examined tissue. Collectively, our study reports that the Mavrodaphne grape stem extract, a serious pollutant when disposed in environmental matrices, is an important source of bioactive polyphenolic compounds that could protect from oxidative damage and improve animal and human health. Finally, the Xinomavro wine extract exerts tissue-specific changes in redox balance, which are indicative of the complexity that characterizes the biological systems.

Metabolic Phenotyping Study of Mouse Brain Following Microbiome Disruption by C.difficile Colonization.

Clostridioides difficile infection (CDI) is responsible for an increasing number of cases of post-antibiotic diarrhea worldwide, which has high severity and mortality among hospitalized elderly patients. The disruption of gut microbiota due to antibacterial medication facilitates the intestinal colonization of C. difficile. In the present study, a murine model was used to investigate the potential effects of antibiotic administration and subsequent colonization by C. difficile, as well as the effects of three different 10-day treatments (metronidazole, probiotics, and fecal microbiota transplantation), on the brain metabolome for the first time. Four different metabolomic-based methods (targeted HILIC-MS/MS, untargeted RP-LC-HRMS/MS, targeted GC-MS/MS, and untargeted GC-MS) were applied, resulting in the identification of 217 unique metabolites in the brain extracts, mainly glycerophospholipids, glycerolipids, amino acids, carbohydrates, and fatty acids. Univariate and multivariate statistical analysis revealed that CDI, as well as the subsequent treatments, altered significantly several brain metabolites, probably due to gut dysbiosis, and affected the brain through the gut-brain axis. Notably, none of the therapeutic approaches completely restored the brain metabolic profile to the original, healthy, and non-infected phenotype, even after 10 days of treatment.

Hepatic Metabolic Profiling of Lifelong Exercise Training Rats.

Regular physical exercise has been investigated as a primary preventive measure of several chronic diseases and premature death. Moreover, it has been shown to synchronize responses across multiple organs. In particular, hepatic tissue has proven to be a descriptive matrix to monitor the effect of physical activity. In this study, we performed an untargeted metabolomics-based analysis of hepatic tissue extracts from rats that have undergone either lifelong or chronic exercise training. For this purpose, 56 hepatic samples were collected and were analyzed by UHPLC-TOF-MS in negative ionization mode. This approach involved untargeted metabolite detection on hepatic tissue extracts accompanied by an in-house retention time/accurate mass library enabling confident metabolite identification. Unsupervised (PCA) and supervised (OPLS-DA) multivariate analysis showed significant metabolic perturbation on a panel of 28 metabolites, including amino acids, vitamins, nucleotides, and sugars. The training regime employed in this study resulted in a probable acceleration of the bioenergetic processes (glycolysis, glycogen metabolism), promoted catabolism of purines, and supplied biosynthetic precursors via the pentose phosphate pathway and pentose and glucuronate interconversions. Overall, the applied methodology was able to discriminate the different training schedules based on the rat liver metabolome.

The addition of crocin in the freezing medium extender improves post-thaw semen quality.

Semen cryopreservation is arguably the most important method or technique contributing to the advancement of modern animal production. However, the quality of sperm after thawing is still highly variable. The addition of antioxidant compounds to the freezing medium has been used customarily to counteract the harmful effects of Reactive Oxygen Species (ROS) that are produced during the freeze/thaw process. Crocin, a potent antioxidant, improves the fertilizing capacity of spermatozoa. In this study, we evaluated the potential of crocin (0, 0.5 and 1 mM) as an extender additive to diminish the damaging effects of cryopreservation on bovine spermatozoa. Post-thaw semen quality was assessed by means of motility, viability and lipid peroxidation (LPO). We further investigated the effect of crocin supplementation upon freezing on sperm quality parameters during their incubation at 37°C for up to 2 hr. Overall, the data assessment indicates that crocin facilitated a general improvement of the quality of freeze/thawed spermatozoa, under the present experimental conditions. Crocin (1 mM) maintained a higher percentage of alive spermatozoa with intact acrosome with rapid and progressive motility, compared to the control extender. Moreover, the spermatozoa cryopreserved in the presence of crocin exhibited higher values in CASA kinematic parameters (VCL, VSL, VAP, ALH) immediately after thawing. Furthermore, the positive effect of crocin on motility parameters was also sustained over a period of 2 hr incubation at 37°C. This effect of crocin may be attributed to the observed inhibition of LPO during the incubation period. Thus, the results indicate that the addition of crocin (especially at a final concentration of 1 mM) in the freezing extender medium may benefit the preservation of the quality parameters of spermatozoa that are compromised by the freeze/thaw heat shock and the stress during handling for IVF or artificial insemination.

Effects of Aging, Long-Term and Lifelong Exercise on the Urinary Metabolic Footprint of Rats.

Life expectancy has risen in the past decades, resulting in an increase in the number of aged individuals. Exercise remains one of the most cost-effective treatments against disease and the physical consequences of aging. The purpose of this research was to investigate the effects of aging, long-term and lifelong exercise on the rat urinary metabolome. Thirty-six male Wistar rats were divided into four equal groups: exercise from 3 to 12 months of age (A), lifelong exercise from 3 to 21 months of age (B), no exercise (C), and exercise from 12 to 21 months of age (D). Exercise consisted in swimming for 20 min/day, 5 days/week. Urine samples collection was performed at 3, 12 and 21 months of life and their analysis was conducted by liquid chromatography-mass spectrometry. Multivariate analysis of the metabolite data did not show any discrimination between groups at any of the three aforementioned ages. However, multivariate analysis discriminated the three ages clearly when the groups were treated as one. Univariate analysis showed that training increased the levels of urinary amino acids and possibly protected against sarcopenia, as evidenced by the higher levels of creatine in the exercising groups. Aging was accompanied by decreased levels of urinary amino acids and signs of increased glycolysis. Concluding, both aging and, to a lesser degree, exercise affected the rat urinary metabolome, including metabolites related to energy metabolism, with exercise showing a potential to mitigate the consequences of aging.

Metabolic Phenotyping Study of Mouse Brains Following Acute or Chronic Exposures to Ethanol.

The chronic and acute effect of ethanol administration on the metabolic phenotype of mouse brain was studied in a C57BL/6 mouse model of ethanol abuse using both untargeted and targeted ultra performance liquid chromatography-tandem mass spectrometry. Two experiments based on either chronic (8 week) exposure to ethanol of both male and female mice or acute exposure of male mice for 11 days, plus 2 oral gavage doses of 25% ethanol, were undertaken. Marked differences were found in amino acids, nucleotides, nucleosides, and related metabolites as well as a number of different lipids. Using untargeted metabolite profiling, acute ethanol exposure found significant decreases in several metabolites including nucleosides, fatty acids, glycerophosphocholine, and a number of phospholipids, while chronic exposure resulted in increases in several amino acids with notable decreases in adenosine, acetylcarnitine, and galactosylceramides. Similarly, targeted metabolite analysis, focusing on the hydrophilic fraction of the brain tissue extract, identified significant decreases in the metabolism of amino acids and derivatives, as well as purine degradation especially after chronic exposure to ethanol.

Self-Nanoemulsifying Drug Delivery Systems (SNEDDS) Containing Rice Bran Oil for Enhanced Fenofibrate Oral Delivery: In Vitro Digestion, Ex Vivo Permeability, and In Vivo Bioavailability Studies.

Lipid-based drug delivery systems (LbDDS), such as self-nanoemulsifying drug delivery systems (SNEDDS), constitute a prominent formulation approach for enhancing the aqueous solubility and oral bioavailability of poorly water-soluble compounds. Utilization of biorefinery wastes, such as oil from rice bran, may prove advantageous to both improving drug solubilization and absorption and to achieving sustainable agri-food waste valorization. Here, we assessed the effect of four SNEDDS compositions differing in the oil (rice bran oil and corn oil) and surfactant type (Kolliphor RH40 and EL) on the oral bioavailability of fenofibrate, a BCS class II compound. Prior to the in vivo oral administration of the SNEDDS in rats, drug solubilization was tested in vitro using the static digestion model, followed by the ex vivo permeability study of the predigested SNEDDS using the non-everted gut sac model. No significant variation was observed in the solubilization capacity within the different SNEDDS formulations. On the other hand, the ex vivo permeability data of the predigested SNEDDS correlated well with the in vivo bioavailability data designating the superiority of rice bran oil with Kolliphor EL as the surfactant, to enhance the oral absorption of fenofibrate. Results indicated that valorization of agro-industrial waste such as rice bran oil may prove useful in enhancing the oral performance of LbDDS in the case of fenofibrate, while at the same time maximizing the use of agricultural by-products via the creation of new sustainable value chains in the pharmaceutical field.

Synbiotics administration leads to attenuated mucosal inflammatory neutrophil infiltration and increased hematocrit in experimental ulcerative colitis.

OBJECTIVE: The objective of the study was to estimate the effects of synbiotics on laboratory, macroscopic, and histopathologic features in dextran sulfate sodium (DSS) experimental colitis. MATERIALS AND METHODS: A total of 40 Wistar rats received 5% of DSS in their drinking water for 8 days to induce ulcerative colitis (UC). Eight rats were sacrificed to confirm the presence of UC. The remaining rats were randomly assigned to two groups: the synbiotics group, which received synbiotics once per day and the control group, which received tap water for another 8 days. RESULTS: On the 8th day of DSS administration animals developed UC with bloody diarrhea. In the majority of the hematologic variables studied (hemoglobin [HB], red blood cells, platelets, mean corpuscular volume, and mean corpuscular HB), in bodyweight and histopathologic colitis score there was no significant difference between groups. However, the synbiotics group, compared to control, presented a significantly greater colon length on the 4th day, significantly increased hematocrit (HT) on the 8th day, and a significantly decreased number of myeloperoxidase positive cells on the 8th day. Furthermore, there was a trend toward histopathological and clinical improvement. CONCLUSIONS: Administration of synbiotics in the experimental UC results in an attenuation of mucosal inflammatory neutrophil infiltration and an increase in HT.

OBJETIVO: Estimar los efectos de los simbióticos en la colitis experimental causada por dextrano sulfato de sodio (DSS). MATERIAL Y MÉTODOS: Cuarenta ratas Wistar recibieron DSS al 5% en su agua de beber por 8 días para inducir colitis ulcerosa (CU). Ocho ratas fueron sacrificadas para confirmar la presencia de CU. Las ratas restantes fueron asignadas aleatoriamente a dos grupos: un grupo que recibió simbióticos una vez al día y un grupo control que recibió agua del grifo por 8 días. RESULTADOS: En el octavo día de la administración de DSS los animales desarrollaron CU con diarrea sanguinolenta. En la mayoría de las variables hematológicas estudiadas (hemoglobina, glóbulos rojos, plaquetas, volumen corpuscular medio, hemoglobina corpuscular media), en el peso corporal y en la clasificación histopatológica de la CU no hubo diferencias significativas entre los grupos. Sin embargo, el grupo con simbióticos, en comparación con el grupo control, presentó una longitud del colon más larga en el cuarto día, un hematocrito muy aumentado en el octavo día y un número de células mieloperoxidasa positivas significativamente reducido en el octavo día. Además, hubo una tendencia hacia un mejoramiento histopatológico y clínico. CONCLUSIONES: La administración de simbióticos en la CU experimental tiene como resultado una atenuación de la infiltración inflamatoria de neutrófilos de la mucosa y un aumento del hematocrito.

Whey protein boosts the antioxidant profile of rats by enhancing the activities of crucial antioxidant enzymes in a tissue-specific manner.

Whey protein, a by-product of cheese industry, is harmful for the environment (i.e., surface and subterranean waters, soil) and, therefore, for humans due to its high polluting burden. Concomitantly, it has been reported that it is a mixture with potent antioxidant action since it is rich in cysteine residues, which are necessary for glutathione synthesis in vivo. On this basis, this study intended to examine the role of whey protein on the intensification of tissue antioxidant arsenal. To this end, a dose of sheep/goat whey protein equal to 1 g/kg of body weight/day dissolved in drinking water was administered to rats for 28 consecutive days. According to our findings, whey protein improved the antioxidant profile of liver, small intestine, lung and muscle whereas it did not affect the redox state of kidney. Our results were based on the alterations found in the protein expression of glutamate cysteine ligase, catalase and superoxide dismutase-1 measured in all tissues and the activity of glutathione S-transferase evaluated in muscle. Although tissue-specific, it is obvious that the action of whey protein is biologically beneficial and could serve as a biofunctional constituent for foods able to improve redox profile when administered against redox-related diseases.

Effects of lifelong exercise and aging on the blood metabolic fingerprint of rats.

Regular exercise is an important part of a healthy lifestyle, as it helps maintain a healthy weight and reduces the risk of chronic diseases. We explored the effects of lifelong exercise and aging on rat metabolism through a metabolomics approach. Thirty-six rats were divided into four equal groups: exercise during the 1st half of life (3-12 months), lifelong exercise (3-21 months), no exercise, and exercise during the 2nd half of life (12-21 months). Exercise consisted in swimming for 20 min, five times a week. Blood samples collected at 3, 12, and 21 months of life were analysed by 1H NMR spectroscopy. The groups that exercised during the 2nd half of life weighed less than the groups that did not. Exercise had an orexigenic effect during the 1st half and an anorexigenic effect during the 2nd half. Multivariate analysis showed a clear discrimination between ages when groups were treated as one and between the exercising and non-exercising groups at 12 months. Univariate analysis showed many effects of aging and some effects of exercise on metabolites involved in carbohydrate, lipid and protein metabolism. Especially during the 1st half, exercise had anabolic effects, whereas aging had catabolic effects on amino acid metabolism. In two cases (glycine and succinate), exercise (especially during the 1st half) mitigated potentially harmful effects of aging. The higher values of succinate and the lower values of lactate during the 1st half in the exercising groups suggest increased oxidative metabolism. In conclusion, moderate-intensity exercise for life or half-life had strong and potentially healthful effects on body weight and (partly) appetite, as well as on some blood metabolites. The effects of aging on the rat blood metabolome seemed to be stronger than those of exercise.

Effect of crocin on antioxidant gene expression, fibrinolytic parameters, redox status and blood biochemistry in nicotinamide-streptozotocin-induced diabetic rats.

BACKGROUND: Diabetes is regarded as an epidemiological threat for the twenty-first century. Phytochemicals with known pharmaceutical properties have gained interest in the field of alleviating secondary complications of diseases. Such a substance is crocin, a basic constituent of saffron (Crocus sativus). The present study aimed at examining the beneficial effects of per os crocin administration on the antioxidant status, blood biochemical profile, hepatic gene expression and plasminogen activator inhibitor-1 activity (PAI-1) in the liver, kidney and plasma (an important marker of pre-diabetic status and major factor of thrombosis in diabetes) of healthy rats, as well as of rats with nicotinamide-streptozotocin-induced diabetes. RESULTS: Diabetes disrupted the oxidation-antioxidation balance, while crocin improved the antioxidant state in the liver by significantly affecting SOD1 gene expression and/or by restoring SOD and total antioxidant capacity (TAC) levels. In the kidney, crocin improved hydrogen peroxide decomposing activity and TAC. In blood, hepatic transaminases ALT and AST decreased significantly, while there was a trend of decrease regarding blood urea nitrogen (BUN) levels. The expression of PAI-1 gene was affected in the liver by the dose of 50 mg kg-1. CONCLUSIONS: Crocin treatment contributed in restoring some parameters after diabetes induction, primarily by affecting significantly hepatic transaminases ALT and AST, SOD1 and PAI-1 gene expression and nephric H2O2 decomposing activity. In conclusion, crocin did contribute to the alleviation of some complications of diabetes.

Postoperative Administration of Alpha-tocopherol Enhances Osseointegration of Stainless Steel Implants: An In Vivo Rat Model.

BACKGROUND: Alpha-tocopherol, a well-known antioxidative agent, may have a positive effect on bone formation during the remodeling phase of secondary fracture healing. Fracture healing and osseointegration of implants share common biological pathways; hence, alpha-tocopherol may enhance implant osseointegration. QUESTIONS/PURPOSES: This experimental study in rats assessed the ability of alpha-tocopherol to enhance osseointegration of orthopaedic implants as determined by (1) pull-out strength and removal torque and (2) a histomorphological assessment of bone formation. In addition, we asked, (3) is there a correlation between the administration of alpha-tocopherol and a reduction in postoperative oxidative stress (as determined by malondialdehyde, protein carbonyls, reduced and oxidized glutathione and their ratio, catalase activity and total antioxidant capacity) that develops after implantation of an orthopaedic implant? METHODS: This blinded study was performed in study and control groups, each consisting of 15 young adult male Wistar rats. On Day 0, a custom-designed stainless-steel screw was implanted in the proximal metaphysis of both tibias of all rats. On Day 1, animals were randomized to receive either alpha-tocopherol (40 mg/kg once per day intraperitoneally) or saline (controls). Animals were treated according to identical perioperative and postoperative protocols and were euthanized on Day 29. All animals completed the study and all tibias were suitable for evaluation. Implant pullout strength was assessed in the right tibias, and removal torque and histomorphometric evaluations (that is, volume of newly formed bone surrounding the implant in mm, percentage of newly formed bone, percentage of bone marrow surrounding the implant per optical field, thickness of newly formed bone in µm, percentage of mineralized bone in newly formed bone, volume of mature newly formed bone surrounding the implant in mm and percentage of mineralized newly formed bone per tissue area) were performed in the left tibias. The plasma levels of alpha-tocopherol, malondialdehyde, protein carbonyls, glutathione, glutathione disulfide, catalase, and the total antioxidant capacity were evaluated, and the ratio of glutathione to oxidized glutathione was calculated. RESULTS: All parameters were different between the alpha-tocopherol-treated and control rats, favoring those in the alpha-tocopherol group. The pullout strength for the alpha-tocopherol group (mean ± SD) was 124.9 ± 20.7 newtons (N) versus 88.1 ± 12.7 N in the control group (mean difference -36.7 [95% CI -49.6 to -23.9]; p < 0.001). The torque median value was 7 (range 5.4 to 8.3) versus 5.2 (range 3.6 to 6 ) N/cm (p < 0.001). The newly formed bone volume was 29.8 ± 5.7 X 10 versus 25.2 ± 7.8 X 10 mm (mean difference -4.6 [95% CI -8.3 to -0.8]; p = 0.018), the percentage of mineralized bone in newly formed bone was 74.6% ± 8.7% versus 62.1% ± 9.8% (mean difference -12.5 [95% CI -20.2 to -4.8]; p = 0.003), the percentage of mineralized newly formed bone per tissue area was 40.3 ± 8.6% versus 34.8 ± 9% (mean difference -5.5 [95% CI -10.4 to -0.6]; p = 0.028), the glutathione level was 2 ± 0.4 versus 1.3 ± 0.3 µmol/g of hemoglobin (mean difference -0.6 [95% CI -0.9 to -0.4]; p < 0.001), the median glutathione/oxidized glutathione ratio was 438.8 (range 298 to 553) versus 340.1 (range 212 to 454; p = 0.002), the catalase activity was 155.6 ± 44.6 versus 87.3 ± 25.2 U/mg Hb (mean difference -68.3 [95% CI -95.4 to -41.2]; p < 0.001), the malondialdehyde level was 0.07 ± 0.02 versus 0.14 ± 0.03 µmol/g protein (mean difference 0.07 [95% CI 0.05 to 0.09]; p < 0.001), the protein carbonyl level was 0.16 ± 0.04 versus 0.27 ± 0.08 nmol/mg of protein (mean difference -0.1 [95% CI 0.05 to 0.15]; p = 0.002), the alpha-tocopherol level was 3.9 ± 4.1 versus 0.9 ± 0.2 mg/dL (mean difference -3 [95% CI -5.2 to -0.7]; p = 0.011), and the total antioxidant capacity was 15.9 ± 3.2 versus 13.7 ± 1.7 nmol 2,2-diphenyl-1-picrylhydrazyl radical/g of protein (mean difference -2.1 [95% CI -4.1 to -0.18]; p = 0.008). CONCLUSIONS: These results using an in vivo rat model support that postoperatively administered alpha-tocopherol can enhance the osseointegration of an orthopaedic implant, although a cause and effect relationship between the administration of alpha-tocopherol and a reduction in postoperative stress cannot be securely established. CLINICAL RELEVANCE: These findings suggest that postoperative administration of alpha-tocopherol is a promising approach to enhance osseointegration of orthopaedic implants in patients. Further studies with different animal models and/or different implants and those evaluating the alpha-tocopherol dose response are needed before performing clinical trials that will examine whether these promising, preliminary results can be extrapolated to the clinical setting as well.

A mixture of routinely encountered xenobiotics induces both redox adaptations and perturbations in blood and tissues of rats after a long-term low-dose exposure regimen: The time and dose issue.

Exposure of humans to xenobiotic mixtures is a continuous state during their everyday routine. However, the majority of toxicological studies assess the in vivo effects of individual substances rather than mixtures. Therefore, our main objective was to evaluate the impact of the 12- and 18-month exposure of rats to a mixture containing 13 pesticides, food, and life-style additives in three dosage levels (i.e. 0.0025 × NOAEL, 0.01 × NOAEL, and 0.05 × NOAEL), on redox biomarkers in blood and tissues. Our results indicate that the exposure to the mixture induces physiological adaptations by enhancing the blood antioxidant mechanism (i.e., increased glutathione, catalase and total antioxidant capacity and decreased protein carbonyls and TBARS) at 12 months of exposure. On the contrary, exposure to the 0.05 × NOAEL dose for 18 months induces significant perturbations in blood and tissue redox profile (i.e., increased carbonyls and TBARS). This study simulates a scenario of real-life risk exposure to mixtures of xenobiotics through a long-term low-dose administration regimen in rats. The results obtained could support, at least in part, the necessity of introducing testing of combined stimuli at reference doses and long term for the evaluation of the risk from exposure to chemicals.

Urine and fecal samples targeted metabolomics of carobs treated rats.

Ceratonia siliqua, known as the carob, is considered to be of high nutritional value and of great economic significance due to its unique composition. The beneficial effects of carob against cancer, metabolic syndrome, diabetes, diarrhea, hyperlipidemia and gastro esophageal reflux disease are only a few of its therapeutic actions. Metabolomics-based analysis provides an ultimate tool, for the deciphering of nutritional intervention derived metabolic alterations. In the present study, 16 male Wistar rats were treated with carob powder for a 15-day period. Fecal and urine samples were collected at 5 time points (0, 1, 5, 10 and 15 days). By the applied HILIC-MS/MS method, 63 and 67 hydrophilic metabolites were detected in the fecal and urine samples, respectively, including amino acids, organic acids, sugars, vitamins and other endogenous compounds. A clear group separation based on fecal metabolome was observed after 1 day and 15 days treatment, while only a mild differentiation at day 1 was observed based on urine metabolome. Twenty-one fecal metabolites were responsible for the separation including amino acids and their derivatives, vitamins and organic acids. However, only 7 metabolites were altered in rat urine samples. Metabolic alterations in fecal samples could be attributed to physiological and biochemical adaptations derived from the nutritional intervention. Fecal targeted metabolomics were proven to be suitable for uplifting and highlighting such alterations.

The Strong Antioxidant Sheep/Goat Whey Protein Protects Against mTOR Overactivation in Rats: A Mode of Action Mimicking Fasting.

Whey protein, a by-product of the cheese industry, can be putatively used as a functional food due to its beneficial health properties. The main objective of the present study was to assess in vivo the effect of a sheep/goat whey protein on the plasma amino acid profile and mammalian target of rapamycin (mTOR), a regulator of skeletal myogenesis. A control group was fed with a standard commercial diet while the experimental group received a standard commercial diet plus sheep/goat whey protein for 28 days. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was conducted to determine plasma amino acid levels while the expression of p70-S6 Kinase 1 (p70-S6K1) in liver and quadriceps muscles was quantified and used as a biomarker of mTOR activity. The results obtained showed a decrease in the levels of essential and branched-chain amino acids (BCAAs) in the experimental group. Furthermore, p70-S6K1 expression was decreased in the liver of rats consumed whey protein. In conclusion, the reduction of amino acid levels and the concomitant inactivation of mTOR imply that whey could potentially act protectively against disorders induced by mTOR overactivation. Intriguingly, this mode of action mimics fasting, an approach with established advantageous health effects.

A lightly roasted coffee extract improves blood and tissue redox status in rats through enhancement of GSH biosynthesis.

Coffee is a highly consumed beverage with many putative beneficial health effects, however these often come from observational studies. In the current work, a lightly roasted coffee extract that has previously been reported to exhibit potent antioxidant properties was administered for two weeks in rats to examine the potential improvement of blood and tissue redox status. The dose was equivalent to a moderate human daily consumption. According to our results, coffee exerted beneficial effects in all tissues mainly by increasing reduced glutathione (GSH) levels. Interestingly, the brain was the most significantly affected tissue, while the gastrointestinal tract, the main metabolic organs and the quadriceps were also benefited. In addition, protein and lipid oxidation was reduced in several tissues. The observed increase in GSH was attributed to increased levels of the rate-limiting enzyme in its biosynthesis pathway, namely γ-glutamylcysteine ligase both in the protein and gene levels. Overall, moderate coffee consumption showed beneficial short term effects in rat tissues by stimulating parts of the endogenous antioxidant mechanisms.

Six months exposure to a real life mixture of 13 chemicals' below individual NOAELs induced non monotonic sex-dependent biochemical and redox status changes in rats.

This study assessed the potential adverse health effects of long-term low-dose exposure to chemical mixtures simulating complex real-life human exposures. Four groups of Sprague Dawley rats were administered mixtures containing carbaryl, dimethoate, glyphosate, methomyl, methyl parathion, triadimefon, aspartame, sodium benzoate, calcium disodium ethylene diamine tetra-acetate, ethylparaben, butylparaben, bisphenol A, and acacia gum at doses of 0, 0.25, 1 or 5 times the respective Toxicological Reference Values (TRV): acceptable daily intake (ADI) or tolerable daily intake (TDI) in a 24 weeks toxicity study. Body weight gain, feed and water consumption were evaluated weekly. At 24 weeks blood was collected and biochemistry parameters and redox status markers were assessed. Adverse effects were observed on body weight gain and in hepatotoxic parameters such as the total bilirubin, alanine aminotransferase (ALT) and alkaline phosphatase (ALP), especially in low dose and affecting mainly male rats. The low dose group showed increased catalase activity both in females and males, whereas the high dose group exhibited decreased protein carbonyl and total antioxidant capacity (TAC) levels in both sex groups. Non-monotonic effects and adaptive responses on liver function tests and redox status, leading to non-linear dose-responses curves, are probably produced by modulation of different mechanisms.

Effects of sheep/goat whey protein dietary supplementation on the redox status of rats.

The purpose of the present study is to estimate the effects of sheep/goat whey protein dietary supplementation on the redox status of blood and tissues of rats. Twelve male Wistar rats were divided into the control group (standard commercial diet) and whey group [standard commercial diet + sheep/goat whey protein (1 g kg b.w/day)] (6 rats/group). The animals were maintainted on their respective diet for 28 days. At the end of the experimental period, reduced glutathione, catalase activity, total antioxidant capacity, thiobarbituric reactive substances, protein carbonyls and the decomposition rate of H2O2 were measured in blood and tissues of rats. According to the results, the rats fed with the sheep/goat whey protein exhibited improved antioxidant status and decreased free radical­induced toxic effects on lipids and proteins. Specifically, in blood, GSH and CAT levels were significantly increased while TBARS and protein carbonyl levels were significantly decreased compared to the control group. Regarding the effects on tissues, it was observed that GSH levels were significantly increased in small intestine, quadriceps muscle, pancreas and lung tissue compared to the control group. The decomposition rate of H2O2 was significantly decreased in liver, brain and quadriceps muscle, but was significantly increased in spleen tissue compared to the control group. TBARS levels were significantly decreased in liver, brain, quadriceps muscle, pancreas, lung and spleen tissue compared to the control group. Finally, protein carbonyl levels were significantly decreased in brain, small intestine, kidney, pancreas and spleen tissue compared to the control group. Thus, the present findings show the beneficial effects of sheep/goat whey protein, a by­product of cheese manufacturing, on the redox status in an in vivo model.

Silver Nanoparticles Alter Cell Adhesion and Proliferation of Sheep Primary Mesothelial Cells.

BACKGROUND: Human exposure to engineered nanoparticles has been linked to pleural effusion, inflammation and fibrosis. Silver nanoparticles (AgNPs) are widely used in medical and domestic products, increasing the risk of occupational and domestic exposure. We assessed the influence of AgNPs on adhesion and proliferation of sheep primary pleural mesothelial cells. MATERIALS AND METHODS: Cells were used for cell adhesion (90 min) and proliferation experiments (3 days) while exposed to 20 nm and 60 nm AgNPs (0.2 µg/ml and 2 µg/ml) using colorimetric assays. RESULTS: Exposure to 0.2 µg/ml of 20 nm and 60 nm AgNPs significantly increased cell adhesion, while at 2 µg/ml this effect was not elicited. Cell proliferation was significantly increased by both 20 nm and 60 nm AgNPs at 0.2 µg/ml, while at 2 µg/ml this effect was only elicited by the 60 nm AgNPs. CONCLUSION: AgNPs alter the adhesive and proliferative properties of primary pleural mesothelial cells.