Oncogenic and tumor suppressor pathways in subchronic aflatoxicosis in rats: Association with serum and urinary aflatoxin exposure biomarkers.

In this study, the changes in oncogenic and tumor suppressor signaling pathways in liver and their association with serum and urinary biomarkers of aflatoxin exposure were evaluated in Wistar rats fed diets containing aflatoxin B1 (AFB1) for 90 days. Rats were divided into four groups (n = 15 per group) and assigned to dietary treatments containing 0 (control), 50 (AFB50), 100 (AFB100) and 200 µg AFB1 kg-1 diet (AFB200). Multiple preneoplastic foci of hepatocytes marked with glutathione-S-transferase-placental form (GST-P) were identified in AFB100 and AFB200 groups. Hepatocellular damage induced by AFB1 resulted in overexpression of cyclin D1 and ß-catenin. The liver expression of retinoblastoma (Rb) and p27Kip1 decreased in AFB100 and AFB200 groups, confirming the favorable conditions for neoplastic progression to hepatocellular carcinoma. All samples from rats fed AFB1-contaminated diets had quantifiable AFB1-lysine in serum or urinary AFM1 and AFB1-N7-guanine, with mean levels of 20.42-50.34 ng mL-1, 5.31-37.68 and 39.15-126.37 ng mg-1 creatinine, respectively. Positive correlations were found between AFB1-lysine, AFM1 or AFB1-N7-guanine and GST-P+, ß-catenin+ and cyclin D1+ hepatocytes, while Rb + cells negatively correlated with those AFB1 exposure biomarkers. The pathways evaluated are critical molecular mechanisms of AFB1-induced hepatocarcinogenesis in rats.

Mycotoxin occurrence in breast milk and exposure estimation of lactating mothers using urinary biomarkers in São Paulo, Brazil.

In this study, the occurrence of aflatoxins (AFs), fumonisins (FBs), ochratoxin A (OTA), deoxynivalenol (DON), zearalenone (ZEN) and some of their metabolites were assessed in breast milk and urine of lactating women (N = 74) from Pirassununga, São Paulo, Brazil. Exposure estimations through urinary mycotoxin biomarkers was also performed. Samples were collected in four sampling times (May and August 2018, February and July 2019) and analyzed by liquid chromatography coupled to tandem mass spectrometry. Aflatoxin M1 (AFM1) was not detected in breast milk. However, two samples (3%) presented FB1 at 2200 and 3400 ng/L, while 4 samples (5%) had OTA at the median level of 360 ng/L. In urine, AFM1 and aflatoxin P1 (AFP1) were found in 51 and 11% of samples, respectively (median levels: 0.16 and 0.07 ng/mg creatinine, respectively). Urinary DON (median level: 38.59 ng/mg creatinine), OTA (median level: 2.38 ng/mg creatinine) and ZEN (median level: 0.02 ng/mg of creatinine) were quantified in 18, 8 and 10% of the samples, respectively. Mean probable daily intake (PDI) values based on urinary biomarkers were 1.58, 1.09, 5.07, and 0.05 µg/kg body weight/day for AFM1, DON, OTA, and ZEN, respectively. Although a low mycotoxin occurrence was detected in breast milk, the PDI for the genotoxic AFs was much higher than those reported previously in Brazil, while PDI values obtained for OTA and DON were higher than recommended tolerable daily intakes. These outcomes warrant concern on the exposure of lactating women to these mycotoxins in the studied area.

Assessment of mycotoxin exposure and risk characterization using occurrence data in foods and urinary biomarkers in Brazil.

This study aimed to assess the exposure of Brazilian residents (N = 86) from rural areas to multiple mycotoxins and characterize the associated risk in two sampling periods (SP) (April-May and December/2016). Mycotoxins in food and urine samples were determined by liquid chromatography coupled to tandem mass spectrometry. Mean probable daily intake (PDI) values based on occurrence data in foods in both SP varied from 0.007 to 0.013, 0.069 to 1.002, 0.119 to 0.321 and 0.013-0.156 µg kg-1 body weight (bw) day-1 for aflatoxins (AFs), deoxynivalenol (DON), fumonisins (FBs) and zearalenone (ZEN), respectively. Mean PDI values based on urinary biomarkers were 0.001, 84.914, 0.031, 0.377 and 0.002 µg kg-1 bw day-1 for AFB1, DON, ochratoxin A (OTA), FB1 and ZEN, respectively. Hazard quotient (HQ) calculated using food data revealed a potential health concern for ZEN in 2nd SP. HQ > 1 based on urinary biomarkers were observed for DON in the two SP. Although OTA was not detected in any food sample, the HQ based on urinary OTA levels was >1 in the 1st SP. Margin of exposure values for AF from food and urine data in the 1st SP were below 10,000, indicating potential health risks.

Co-occurrence of mycotoxins in maize food and maize-based feed from small-scale farms in Brazil: a pilot study.

A limited survey was conducted to assess the co-occurrence of aflatoxins (AF) B1, B2, G1, and G2; fumonisins (FB) B1 and B2; ochratoxin A (OTA); zearalenone (ZEN); and deoxynivalenol (DON) in maize food (N = 26) and animal feed (N = 45) collected from 21 small-scale farms from the states of São Paulo (SP) and Santa Catarina (SC), Brazil. Samples evaluated were maize meal and maize flour for human consumption available in the farm households, and maize-based feed intended for broiler chicks, laying hens, and dairy cows. Analyses of mycotoxins were performed by ultra-performance liquid chromatography coupled with tandem mass spectrometry. The median levels of mycotoxins found in maize food were 2.5 µg/kg (total AF), 120 µg/kg (total FB), 13 µg/kg (ZEN), and 57 µg/kg (DON). All values were below the Brazilian tolerance limits, except for total FB in one sample of maize flour. In feed samples, median levels of total AF, total FB, ZEN, and DON were 100 µg/kg, 680 µg/kg, 160 µg/kg, and 200 µg/kg, respectively. The co-occurrence of two or more mycotoxins was confirmed in 35% and 51% of maize food and feed, respectively. Results indicate a low human exposure to mycotoxins in the small-scale farms evaluated and a higher exposure of farm animals to mycotoxins in the feed.

Comprehensive high-resolution multiple-reaction monitoring mass spectrometry for targeted eicosanoid assays.

Eicosanoids comprise a class of bioactive lipids derived from a unique group of essential fatty acids that mediate a variety of important physiological functions. Owing to the structural diversity of these lipids, their analysis in biological samples is often a major challenge. Advancements in mass spectrometric have been helpful for the characterization and quantification of these molecular lipid species in complex matrices. However, there are technical limitations to this approach, including low-abundant and/or poorly ionizable lipids. Using high-resolution multiple-reaction monitoring (MRMHR), we were able to develop a targeted bioanalytical method for eicosanoid quantification. For this, we optimized the LC-MS/MS conditions and evaluated several parameters, including linearity, limits of quantification, matrix effects and recovery yields. For validation purposes, we looked at the method's precision and accuracy. A library of high-resolution fragmentation spectra for eicosanoids was developed. Our comprehensive dataset meets benchmark standards for targeted analysis, having been derived using best-practice workflows and rigorous quality assessments. As such, our method has applications for determining complex eicosanoid profiles in the biomedical field.

Aflatoxin B1 residues in human livers and their relationship with markers of hepatic carcinogenesis in São Paulo, Brazil.

In this study, hepatic biopsies from autopsy cases in São Paulo, Brazil, showing hepatocellular carcinoma (HCC, n = 8), cirrhosis associated with viral hepatitis (VC, n = 20), cirrhosis associated with alcoholism (AC, n = 20), and normal livers (NL or controls, n = 10) were subjected to determination of aflatoxin B1 (AFB1) and its main metabolites, and of markers of hepatic carcinogenesis Only non-metabolized AFB1 was detected in 13 samples (27.1%, N = 48) of liver disorders (HCC, VC and AC), at levels between 10.0 and 418.0 pg/g (mean: 76.6 ± 107.7 pg/g). Immuno-labeling of p53, cyclin D1, p21, ß-catenin, and Prohibitin (PB) increased mainly in HCC patients, in relation to the controls. AFB1+ samples of HCC presented higher expressions of p53, cyclin D1, p21, and ß-catenin compared with AFB1-livers. In contrast, p27, p16, and Rb immuno-labeling decreased in HCC, VC, and AC samples, compared with NL, with lowest values in AFB1+ samples for all liver disorders. Compared with NL, gene expression of cyclin D1 and PB in AFB1+ samples of HCC and AC were also higher, along with higher gene expression of p21 in VC and AC AFB1+ livers. Results indicated that patients with liver disorders were exposed to dietary aflatoxins, and that residual AFB1 in liver negatively affected the p53 and protein Rb pathways in HCC. Moreover, the presence of AFB1 in cirrhotic livers warrants concern about the potential contribution of dietary aflatoxin to disease progression during VC and AC.

Assessment of multiple mycotoxins in breakfast cereals available in the Portuguese market.

Mycotoxins are secondary metabolites of fungi that cause toxic and carcinogenic effects. Human exposure to multiple mycotoxins constitutes an increasing health concern due to potential mycotoxins combined effects. The presence of mycotoxins mixtures in foodstuffs as cereals has been reported over the last years, but few studies are available concerning its occurrence in cereals primarily marketed for children, a particular vulnerable population group. The present study aims to assess the co-occurrence of twenty-one mycotoxins and metabolites present in breakfast cereals primarily marketed for children in Portugal. Results showed that 96% of the analysed breakfast cereal samples were contaminated with several mycotoxins. Twenty-two combinations were identified including two to seven different mycotoxins. Conclusions pointed out an urgent need to review legislative limits in food matrices consumed by children and to perform a more accurate risk assessment of children's exposure to mycotoxins mixtures in food.

Determination of fumonisin B1 levels in body fluids and hair from piglets fed fumonisin B1-contaminated diets.

The levels of fumonisin B1 (FB1) residues in plasma, urine, feces and hair from 24 piglets fed FB1-contaminated diets containing 3.1, 6.1 or 9.0 µg FB1.g-1 for 28 days were determined using liquid chromatography coupled to mass spectrometry (LC-MS/MS). The levels of FB1 in plasma, urine, feces and pooled hair (n = 3) samples varied from 0.15 to 1.08 µg.L-1, 16.09-75.01 µg.L-1, 1.87-13.89 µg.g-1 and 2.08-8.09 ng.g-1, respectively. Significant correlations (r = 0.808-0.885; P < 0.001; N = 18) were found between FB1 intake and plasma FB1 on days 7, 14, 21 and 28. However, urinary FB1 correlated with FB1 intake only on days 7 and 14 (r = 0.561-572; P = 0.02; N = 18). A significant correlation (r = 0.509; P = 0.02; N = 24) was also found for the first time between FB1 in hair samples and FB1 intake. Plasma and urinary FB1 are good biomarkers of early exposure of pigs to low dietary FB1 levels, although plasma is recommended to assess prolonged exposure (>14 days). The possibility to evaluate hair as a biomarker of fumonisin exposure was established, although further studies are needed to provide physiologically based toxicokinetics of residual FB1 in the pig hair.

Determination of Aflatoxin B1-Lysine in Pig Serum and Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

Aflatoxin B1 (AFB1) is a hepatocarcinogen produced by certain Aspergillus species growing on crops. After biotransformation in the liver, AFB1 generates several metabolites, one of which is AFB1 bound to lysine on serum albumin. AFB1-lysine (AFB1-lys) is a digest product of AFB1-albumin and is considered a biomarker of exposure to AFB1 in humans and animals. The objectives of this paper were to evaluate the performance characteristics of a new analytical method for determination of AFB1-lys levels in pig serum, heparinized and ethylenediaminetetraacetic acid (EDTA) plasma and to evaluate the interference of these anticoagulants in AFB1-lys quantification. Blank blood samples were obtained from eight crossbreed 91-day-old barrows fed AFB1-free diets. Pooled samples (n = 3) and individual samples of serum, EDTA and heparinized plasma collected from five pigs were enzymatically digested with pronase at 37°C for 4 h. AFB1-lys was isolated by solid-phase extraction and quantified by liquid chromatography coupled to tandem mass spectrometry. The analytical method was applied for determination of AFB1-lys in serum and EDTA plasma collected from five 49-day-old crossbreed barrows fed ad libitum diets containing 1.1 mg of AFB1 per kg of feed during 7 days (three animals) or 42 days (two animals). Samples of heparinized plasma were only available from animals intoxicated for 42 days. All animals had lower levels of AFB1-lys in EDTA plasma samples (24.78-37.40 ng/mL), when compared to serum (49.32-252.07 ng/mL-1) or heparinized plasma (176.81 and 264.24 ng/mL-1). EDTA did not interfere in AFB1-lys standard detection, but our findings suggest that EDTA should be avoided during blood collection since it affects the pronase activity in AFB1-albumin adduct digestion and, consequently, causes a reduction in the AFB1-lys levels. Hence, determination of AFB1-lys in serum and heparinized plasma is an approach to assess an individual's exposure of swine to AFB1.

Plasma eicosanoid profiles determined by high-performance liquid chromatography coupled with tandem mass spectrometry in stimulated peripheral blood from healthy individuals and sickle cell anemia patients in treatment.

Eicosanoids play an important role in homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca(2+) ionophores and Ca(2+)-ATPase inhibitors, as well as natural agonists such as formylmethionine-leucyl-phenylalanine (fMLP), can stimulate eicosanoid biosynthesis. The aims of this work were to develop a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was partially validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient ≥0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of ≤15%, except for the lower limit of quantification, where these values were ≤20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were tested. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli for the production or liberation of eicosanoids. We next compared the eicosanoid profiles of stimulated whole blood samples of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients and those of healthy subjects, mainly for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method can detect significant changes in eicosanoid profiles in stimulated whole blood, which will contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.

Erythropoietin Exacerbates Inflammation and Increases the Mortality of Histoplasma capsulatum-Infected Mice.

Erythropoietin (EPO) is a key hormone involved in red blood cell formation, but its effects on nonerythroid cells, such as macrophages, have not been described. Macrophages are key cells in controlling histoplasmosis, a fungal infection caused by Histoplasma capsulatum (Hc). Considering that little is known about EPO's role during fungal infections and its capacity to activate macrophages, in this study we investigated the impact of EPO pretreatment on the alveolar immune response during Hc infection. The consequence of EPO pretreatment on fungal infection was determined by evaluating animal survival, fungal burden, activation of bronchoalveolar macrophages, inflammatory mediator release, and lung inflammation. Pretreatment with EPO diminished mononuclear cell numbers, increased the recruitment of F4/80(+)/CD80(+) and F4/80(+)/CD86(+) cells to the bronchoalveolar space, induced higher production of IFN-γ, IL-6, MIP-1α, MCP-1, and LTB4, reduced PGE2 concentration, and did not affect fungal burden. As a consequence, we observed an increase in lung inflammation with extensive tissue damage that might account for augmented mouse mortality after infection. Our results demonstrate for the first time that EPO treatment has a deleterious impact on lung immune responses during fungal infection.

Versatility of tandem mass spectrometry for focused analysis of oxylipids.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the multiple reaction monitoring (MRM) scan mode has been the primary MS method applied for the target identification of specific and minor oxylipids in complex matrices, such as eicosanoids and docosanoids, which are potent lipid mediators derived from polyunsaturated fatty acid oxygenation. However, the high specificity of MRM can limit the detection of species with m/z MRM transitions not covered by the method. In addition to MRM, tandem-quadrupole mass analyzers enable other experiments to be conducted, by fragmenting ions via collision-induced dissociation process (CID). This paper presents the potential of tandem mass spectrometry for the focused analysis of oxylipids. We have successfully developed an LC-MS/MS method for the identification of precursor ions of m/z 115, a diagnostic product ion of 5-hydroxy- and 5-epoxy-fatty acids. As a proof of concept, the developed method was used to discover several oxylipids oxidized at C5 derived from arachidonic acid (C20 : 4) oxygenation in a hypothalamus rat extract that were not identified using the target MRM methodology. The proposed focused MS/MS-based approach in a tandem mass analyzer has proven to be a powerful strategy to accelerate the identification of oxylipids with structural similarities and assist the field of lipidomic research.

Novel binuclear µ-oxo diruthenium complexes combined with ibuprofen and ketoprofen: Interaction with relevant target biomolecules and anti-allergic potential.

This work presents the synthesis and characterization of two novel binuclear ruthenium compounds of general formula [Ru2O(carb)2(py)6](PF6)2, where py=pyridine and carb are the non-steroidal anti-inflammatory drugs ibuprofen (1) and ketoprofen (2). Both complexes were characterized by ESI-MS/MS spectrometry. The fragmentation patterns, which confirm the proposed structures, are presented. Besides that, compounds 1 and 2 present the charge transfer transitions within 325-330nm; and the intra-core transitions around 585nm, which is the typical spectra profile for [Ru2O] analogues. This suggests the carboxylate bridge has little influence in their electronic structure. The effects of the diruthenium complexes on Ig-E mediated mast cell activation were evaluated by measuring the enzyme ß-hexosaminidase released by mast cells stimulated by antigen. The inhibitory potential of the ketoprofen complex against mast cell stimulation suggests its promising application as a therapeutic agent for treating or preventing IgE-mediated allergic diseases. In addition, in vitro metabolism assays had shown that the ibuprofen complex is metabolized by the cytochrome P450 enzymes.

Inhibition of inflammatory response in LPS-induced macrophages by 9-KOTE and 13-KOTE produced by biotransformation.

Lipid mediators such as the leukotrienes, resolvins and protectins have been considered excellent models for the development of new anti-inflammatory drugs, due to their high potentiality. Nevertheless, only tiny amounts are available from natural sources and they have to be prepared by total synthesis. It is known that besides chemical reagents, microorganisms can also promote fatty acid oxygenation, via enzymatic reactions. In this context, the aim of this work was to produce oxylipids analogues in structure to lipid mediators employing microbial biotransformation. To this end, α-linolenic acid (ALA) was biotransformed by the fungi Aspergillus niger into oxylipids with different levels of oxygenation within 24h or 48h. The anti-inflammatory potential of products were evaluated by means of NO and TNF-α quantification in LPS-stimulated RAW264.7 macrophage cell line which guided the isolation of the regioisomers at m/z [M-H](-) 291, 9-keto-10E,12Z,15Z-octadecatrienoic acid (9-KOTE) and 13-keto-9Z,11E,15Z-octadecatrienoic acid (13-KOTE). We showed that biotransformation represents a powerful strategy for the production of potentially interesting candidates for development of anti-inflammation therapies.

Bioassay-guided isolation of a low molecular weight PHB from Burkholderia sp. with phytotoxic activity.

This work reports on the bioassay-guided isolation and identification of the macrocyclic pentolide 1, a cyclic polyhydroxybutyrate (PHB) with low molecular weight. This metabolite is produced by Burkholderia sp. and it exhibited phytotoxic activity in a Lemna minor bioassay. Its structure was determined by (1)H and (13)C NMR, heteronuclear multiple quantum correlation, heteronuclear multiple bond correlation, IR, and electrospray ionization tandem mass spectrometry analyses. The period for maximum production of the pentolide was optimized and determined on the basis of multiple reaction monitoring experiments at 15 days. The potential of Burkholderia sp. as a producer of higher biopolymers of PHB was also investigated. The methodology employed here accelerated the isolation and characterization of a phytotoxic metabolite whose structure can serve as a model for the synthesis of new classes of herbicides.