MALDI-TOF mass spectrometry profiling of bovine skim milk for subclinical mastitis detection.

Introduction: Mastitis is one of most impacting health issues in bovine dairy farming that reduces milk yield and quality, leading to important economic losses. Subclinical forms of the disease are routinely monitored through the measurement of somatic cell count (SCC) and microbiological tests. However, their identification can be tricky, reducing the possibilities of early treatments. In this study, a MALDI-TOF mass spectrometry approach was applied to milk samples collected from cows classified according to the SCC, to identify differences in polypeptide/protein profiles. Materials and methods: Twenty-nine raw milk samples with SCC >200,000 cell/ml (group H) and 91 samples with SCC lower than 200,000 (group L) were randomly collected from 12 dairy farms. Spectral profiles from skim milk were acquired in the positive linear mode within the 4,000-20,000 m/z mass acquisition range. Results and discussion: Based on signal intensity, a total of 24 peaks emerged as significant different between the two groups. The most discriminant signals (4,218.2 and 4,342.98 m/z) presented a ROC curve with AUC values higher than 0.8. Classification algorithms (i.e., quick classifier, genetic algorithm, and supervised neural network) were applied for generating models able to classify new spectra (i.e., samples) into the two classes. Our results support the MALDI-TOF mass spectrometry profiling as a tool to detect mastitic milk samples and to potentially discover biomarkers of the disease. Thanks to its rapidity and low-cost, such method could be associated with the SCC measurement for the early diagnosis of subclinical mastitis.

Effects of Turmeric Powder on Aflatoxin M1 and Aflatoxicol Excretion in Milk from Dairy Cows Exposed to Aflatoxin B1 at the EU Maximum Tolerable Levels.

Due to the climatic change, an increase in aflatoxin B1 (AFB1) maize contamination has been reported in Europe. As an alternative to mineral binders, natural phytogenic compounds are increasingly used to counteract the negative effects of AFB1 in farm animals. In cows, even low dietary AFB1 concentrations may result in the milk excretion of the genotoxic carcinogen metabolite aflatoxin M1 (AFM1). In this study, we tested the ability of dietary turmeric powder (TP), an extract from Curcuma longa (CL) rich in curcumin and curcuminoids, in reducing AFM1 mammary excretion in Holstein-Friesian cows. Both active principles are reported to inhibit AFM1 hepatic synthesis and interact with drug transporters involved in AFB1 absorption and excretion. A crossover design was applied to two groups of cows (n = 4 each) with a 4-day washout. Animals received a diet contaminated with low AFB1 levels (5 ± 1 µg/kg) for 10 days ± TP supplementation (20 g/head/day). TP treatment had no impact on milk yield, milk composition or somatic cell count. Despite a tendency toward a lower average AFM1 milk content in the last four days of the treatment (below EU limits), no statistically significant differences with the AFB1 group occurred. Since the bioavailability of TP active principles may be a major issue, further investigations with different CL preparations are warranted.

Curcumin Supplementation Protects Broiler Chickens Against the Renal Oxidative Stress Induced by the Dietary Exposure to Low Levels of Aflatoxin B1.

Aflatoxin B1 (AFB1) causes hepatotoxicity, immunotoxicity, and kidney damage, and it is included in group I of human carcinogens. The European Commission has established maximum limits of AFB1 in feed, ranging from 5 to 20 µg/kg. Chicken is moderately sensitive to AFB1, which results in reduced growth performance and economic losses. Oxidative stress triggered by AFB1 plays a crucial role in kidney damage and the antioxidant activity of Curcumin (CURC) could help in preventing such adverse effect. Twenty-days-old broilers were treated for 10 days with AFB1 (0.02 mg/kg feed), alone or in combination with CURC (400 mg/kg feed), to explore the effects on the renal tissue. Animals exposed to AFB1 alone displayed alterations of the oxidative stress parameters compared with controls: serum antioxidant capacity, and enzymatic activity of kidney superoxide dismutase, catalase and glutathione peroxidase were decreased, while renal malondialdehyde levels and NADPH oxidase complex expression were increased. The administration of CURC attenuates all the oxidative stress parameters modified by AFB1 in the chicken kidney, opening new perspectives in the management of aflatoxicosis.

Acute Diarrhea in Dogs: Current Management and Potential Role of Dietary Polyphenols Supplementation.

Acute diarrhea is one of the most common reasons why pet owners seek veterinary care for their canine companions. In many cases, signs resolve spontaneously or with symptomatic therapy without a specific cause being discovered. However, life-threatening cases can occur. The etiology is complex, including infectious diseases (endoparasites, virus, bacteria, protozoa, fungal agents) by both zoonotic and non-zoonotic pathogens, dietary indiscretion, endocrine diseases, and stress (e.g., travel or environmental changes). In the last years, the role played by oxidative stress in the pathogenesis of acute and chronic enteropathies, independently from the initial noxa, has been highlighted by many researches in both humans and animals. As a result, a series of dietary antioxidant compounds have been studied for their potential use in the treatment of intestinal inflammation. This review summarizes the traditional therapeutic and nutritional options to manage canine acute diarrhea, highlighting the need to explore the role of oxidative stress and potential antioxidant supplementation, especially polyphenols, during acute diarrheic episodes.

Modulation of aflatoxin B1 cytotoxicity and aflatoxin M1 synthesis by natural antioxidants in a bovine mammary epithelial cell line.

Aflatoxin (AF) B1, a widespread food and feed contaminant, is bioactivated by drug metabolizing enzymes (DME) to cytotoxic and carcinogenic metabolites like AFB1-epoxide and AFM1, a dairy milk contaminant. A number of natural antioxidants have been reported to afford a certain degree of protection against AFB1 (cyto)toxicity. As the mammary gland potentially participates in the generation of AFB1 metabolites, we evaluated the role of selected natural antioxidants (i.e. curcumin, quercetin and resveratrol) in the modulation of AFB1 toxicity and metabolism using a bovine mammary epithelial cell line (BME-UV1). Quercetin and, to a lesser extent, resveratrol and curcumin from Curcuma longa (all at 5 µM) significantly counteracted the AFB1-mediated impairment of cell viability (concentration range: 96-750 nM). Moreover, quercetin was able to significantly reduce the synthesis of AFM1. The quantitative PCR analysis on genes encoding for DME (phase I and II) and antioxidant enzymes showed that AFB1 caused an overall downregulation of the detoxifying systems, and mainly of GSTA1, which mediates the GSH conjugation of the AFB1-epoxide. The negative modulation of GSTA1 was efficiently reversed in the presence of quercetin, which significantly increased GSH levels as well. It is suggested that quercetin exerts its beneficial effects by depressing the bio-transformation of AFB1 and counterbalancing its pro-oxidant effects.

In vitro interactions of malachite green and leucomalachite green with hepatic drug-metabolizing enzyme systems in the rainbow trout (Onchorhyncus mykiss).

Malachite green (MG) has been widely used in aquaculture to treat a number of microbial and parasitic diseases. It is currently banned in the EU because of the high cytotoxicity and carcinogenic activity, which is also shared by leucomalachite green (LMG), a reduced MG metabolite that can persist in fish tissues for months. There is scant information about the ability of either compound to interact with drug metabolizing enzymes in fish. Therefore we evaluated the in vitro effects of MG and LMG (25, 50 and 100µM) on some DMEs and glutathione (GSH) content in rainbow trout liver subfractions. LMG did not affect any of the examined parameters. In contrast, MG proved to deplete GSH and to depress to a various extent the activities of NAD(P)H cytochrome c reductase, 7-ethoxycoumarin O-deethylase, 1-naphthol uridindiphosphoglucuronyl-transferase and maximally those of 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) accepting 1-chloro2,4-dinitrobenzene (CDNB) as substrate. The inhibition mechanisms of EROD and GST were investigated by means of non-linear Michaelis-Menten kinetics and Lineweaver-Burk plots using 0.175-8µM MG. The calculated IC50 for EROD was 7.1µM, and the inhibition appeared to be competitive (Ki 2.78±0.24µM). In the case of GST, the calculated IC50 was 0.53µM. The inhibition was best described as competitive toward GSH (Ki 0.39±0.02µM) and of mixed-type toward CDNB (Ki 0.64±0.06µM). Our findings indicate that, contrary to LMG, MG behaves as a relatively strong inhibitor of certain liver DMEs and can reversibly bind GSH.

Constitutive expression of the AHR signaling pathway in a bovine mammary epithelial cell line and modulation by dioxin-like PCB and other AHR ligands.

Environmental pollutants, such as dioxin-like (DL) PCBs, benzo(a) pyrene (B[a]P), and flavonoids are aryl hydrocarbon receptor (AHR) ligands and may be excreted in dairy milk. The expression of AHR-target genes, particularly those involved in xenobiotic biotransformation, and their modulation by two DL-PCBs, B[a]P, and ß-naphthoflavone was investigated in a bovine mammary epithelial cell line (BME-UV). As assessed by quantitative PCR, BME-UV cells expressed a functional AHR signaling pathway. All the AHR ligands induced a concentration-related increase in the transcription of cytochrome P450 1A1 and 1B1, known to be implicated in the bioactivation of several xenobiotics. Conversely, genes encoding for antioxidant and detoxifying enzymes, like quinone oxidoreductase or glutathione S-transferase A2, were not affected or even depressed. This study demonstrates the occurrence and the modulation by different AHR-ligands of genes involved in xenobiotic metabolism in BME-UV cells, with the potential generation of (re) active metabolites that may damage mammary tissue and/or affect animal or human health via the contaminated milk.

Modulation of aryl hydrocarbon receptor target genes in circulating lymphocytes from dairy cows bred in a dioxin-like PCB contaminated area.

Animal productions (i.e. fish, eggs, milk and dairy products) represent the major source of exposure to dioxins, furans, and dioxin-like (DL) polychlorobiphenyls for humans. The negative effects of these highly toxic and persistent pollutants are mediated by the activation of the aryl hydrocarbon receptor (AHR) that elicits the transcriptional induction of several genes, including those involved in xenobiotic metabolism. Previously we demonstrated the presence and functioning of the AHR signaling pathway in primary cultures of bovine blood lymphocytes. The aim of the present study was to investigate by real time PCR the expression and the inducibility of selected target genes (i.e. AHR, AHR nuclear translocator (ARNT), AHR repressor, CYP1A1 and CYP1B1) in uncultured cells from dairy cows naturally exposed to DL-compounds. The study was carried out on two groups of animals bred in a highly polluted area and characterized by a different degree of contamination, as assessed by bulk milk TEQ values, and a control group reared in an industry free area. Bovine lymphocytes expressed only AHR, ARNT and CYP1B1 genes to a detectable level; moreover, only CYP1B1 expression appeared to be correlated to TEQ values, being higher in the most contaminated group, and decreasing along with animal decontamination. Finally, lymphocytes from exposed cows displayed a lower inducibility of both CYP1A1 and CYP1B1 after the in vitro treatment with a specific AHR ligand. In conclusion, our results indicate that DL-compound contaminated cows may display significant changes in AHR-target gene expression of circulating lymphocytes.

Gene expression and inducibility of the aryl hydrocarbon receptor-dependent pathway in cultured bovine blood lymphocytes.

The exposure to dioxin-like (DL) compounds, an important class of persistent environmental pollutants, results in the altered expression of target genes. This occurs through the binding to the aryl hydrocarbon receptor (AhR), the subsequent dimerization with the AhR nuclear translocator (ARNT), and the binding of the complex to DNA responsive elements. A number of genes are up-regulated, including, among others, the AhR repressor (AHRR) and several biotransformation enzymes, such as the members of CYP1 family and NAD(P)H-quinone oxidoreductase (NOQ1). The expression and the inducibility of the above genes were investigated in mitogen-stimulated cultured blood lymphocytes from cattle, which represent a notable source of DL-compound human exposure through dairy products and meat. As assessed by real-time PCR, all the examined genes except CYP1A2 and NQO1 were detected under basal conditions. Cell exposure to the DL-compounds PCB126 or PCB77 in the 10(-6)-10(-9)M concentration range resulted in a 2-4-fold induction of CYPIA1 and CYP1B1, which was antagonized by α-naphthoflavone or PCB153. This study demonstrates for the first time the presence and inducibility of the AhR pathway in easily accessible cells like bovine peripheral lymphocytes and prompts further investigations to verify whether similar changes could occur under in vivo conditions.

Induction of MET by ionizing radiation and its role in radioresistance and invasive growth of cancer.

BACKGROUND: Ionizing radiation (IR) is effectively used in cancer therapy. However, in subsets of patients, a few radioresistant cancer cells survive and cause disease relapse with metastatic progression. The MET oncogene encodes the hepatocyte growth factor (HGF) receptor and is known to drive "invasive growth", a regenerative and prosurvival program unduly activated in metastasis. METHODS: Human tumor cell lines (MDA-MB-231, MDA-MB-435S, U251) were subjected to therapeutic doses of IR. MET mRNA, and protein expression and signal transduction were compared in treated and untreated cells, and the involvement of the DNA-damage sensor ataxia telangiectasia mutated (ATM) and the transcription factor nuclear factor kappa B (NF-κB) in activating MET transcription were analyzed by immunoblotting, chromatin immunoprecipitation, and use of NF-κB silencing RNA (siRNA). Cell invasiveness was measured in wound healing and transwell assays, and cell survival was measured in viability and clonogenic assays. MET was inhibited by siRNA or small-molecule kinase inhibitors (PHA665752 or JNJ-38877605). Combinations of MET-targeted therapy and radiotherapy were assessed in MDA-MB-231 and U251 xenografts (n = 5-6 mice per group). All P values were from two-sided tests. RESULTS: After irradiation, MET expression in cell lines was increased up to fivefold via activation of ATM and NF-κB. MET overexpression increased ligand-independent MET phosphorylation and signal transduction, and rendered cells more sensitive to HGF. Irradiated cells became more invasive via a MET-dependent mechanism that was further enhanced in the presence of HGF. MET silencing by siRNA or inhibition of its kinase activity by treatment with PHA665752 or JNJ-38877605 counteracted radiation-induced invasiveness, promoted apoptosis, and prevented cells from resuming proliferation after irradiation in vitro. Treatment with MET inhibitors enhanced the efficacy of IR to stop the growth of or to induce the regression of xenografts (eg, at day 13, U251 xenografts, mean volume increase relative to mean tumor volume at day 0: vehicle = 438%, 5 Gy IR = 151%, 5 Gy IR + JNJ-38877605 = 76%; difference, IR vs JNJ-38877604 + IR = 75%, 95% CI = 59% to 91%, P = .01). CONCLUSION: IR induces overexpression and activity of the MET oncogene through the ATM-NF-κB signaling pathway; MET, in turn, promotes cell invasion and protects cells from apoptosis, thus supporting radioresistance. Drugs targeting MET increase tumor cell radiosensitivity and prevent radiation-induced invasiveness.

Inhibition of Src impairs the growth of met-addicted gastric tumors.

PURPOSE: We examined whether inhibition of Src tyrosine kinase, a downstream effector of the MET oncogene, can hinder the malignant properties of gastric tumors dependent on Met for growth and survival. EXPERIMENTAL DESIGN: Sensitivity to Src inhibition was determined in vitro by measuring clonogenic survival (anchorage-independent growth) and in vivo by establishing xenograft models. Four "Met-addicted" gastric carcinoma cell lines (GTL16, MKN45, HS746T, and SNU5) and three Met-independent gastric carcinoma cell lines (KATO III, AGS, and NCI-N87) were treated with the Src inhibitor saracatinib (AZD0530). In GTL16 and KATO III, Src neutralization was also achieved by dasatinib and RNA interference. The biochemical and transcriptional consequences of Src inhibition were explored using anti-phosphoprotein antibodies and oligonucleotide microarrays. RESULTS: Inhibition of Src in Met-addicted gastric carcinoma cell lines (a) decreased the phosphorylation/activation levels of signaling intermediates involved in cell proliferation and protection from apoptosis and down-modulated the expression of several cell cycle regulators; (b) reduced anchorage-independent growth; (c) enhanced impairment of cell viability produced by Met inhibition; and (d) delayed tumorigenesis in xenotransplantation models. Immunohistochemical analysis of tumor xenograft tissues following systemic treatment with saracatinib showed a reduction of tumor cell proliferation index, increased apoptosis, and diminished phospho-focal adhesion kinase and phospho-paxillin staining. Tumor stroma parameters such as angiogenesis or inflammatory infiltration were unaffected. In clonogenic survival assays, gastric carcinoma cells without addiction to Met were less sensitive than Met-addicted cells to Src inhibition. CONCLUSIONS: Src is as a key downstream transducer of Met-driven tumor growth. Targeting Src might provide therapeutic benefit in Met-addicted tumors.

Time-dependent acetylsalicylic acid effects on liver CYP1A and antioxidant enzymes in a rat model of 7,12-dimethylbenzanthracene (DMBA)-induced mammary carcinogenesis.

7,12-Dimethylbenzanthracene (DMBA) is an abundant environmental contaminant, which undergoes bioactivation, primarily by the CYP1 family, both in liver and extra-hepatic tissues. Dietary acetylsalicylic acid (ASA) has been recently reported to inhibit DMBA-mediated mammary tumour formation in rats. Chemopreventive substances may reduce the risk of developing cancer by decreasing metabolic enzymes responsible for generating reactive species (phase I enzymes) and/or increasing phase II enzymes that can deactivate radicals and electrophiles. To test these hypotheses, Sprague-Dawley female rats were orally administered ASA as lysine acetylsalicylate (50 mg per capita/day for 21 days in water), DMBA (10 mg per capita in olive oil on day 7, 14, and 21), ASA and DMBA in combination, and vehicles only, respectively. Six rats for each group were sacrificed on day 8, 15, and 22. The DMBA-mediated increase in hepatic CYP1A expression and related activities was not significantly affected by ASA, which, conversely, enhanced in a time-dependent manner the liver reduced glutathione content (up to 52%) and the activity of NAD(P)H-quinone oxidoreductase (up to 34%) in DMBA-treated rats. It is proposed that the positive modulation of the hepatic antioxidant systems by ASA may play a role in the chemoprevention of mammary tumourigenesis induced by DMBA in the female rat.

The MET oncogene drives a genetic programme linking cancer to haemostasis.

The close relationship between activation of blood coagulation and cancer is an old enigma. In 1865, migrans trombophlebitis ('a condition of the blood that predisposes it to spontaneous coagulation') was described as a forewarning of occult malignancy (Trousseau's sign). This pioneering observation emphasized the existence of haemostasis disorders associated with cancer onset; this phenomenon has since been extensively reported in clinical and epidemiological studies, but has so far resisted a mechanistic explanation. Here we report a mouse model of sporadic tumorigenesis based on genetic manipulation of somatic cells. Targeting the activated, human MET oncogene to adult liver caused slowly progressing hepatocarcinogenesis. This was preceded and accompanied by a syndrome manifesting first with blood hypercoagulation (venous thromboses), and then evolving towards fatal internal haemorrhages. The pathogenesis of this syndrome is driven by the transcriptional response to the oncogene, including prominent upregulation of plasminogen activator inhibitor type 1 (PAI-1) and cyclooxygenase-2 (COX-2) genes. In vivo analysis showed that both proteins support the thrombohaemorrhagic phenotype, thus providing direct genetic evidence for the long-sought-after link between oncogene activation and haemostasis.