Héctor R. Rubinstein

Fingerprints for Main Varieties of Argentinean Wines: Terroir Differentiation by Inorganic, Organic, and Stable Isotopic Analyses Coupled to Chemometrics

Our main goal was to investigate if robust chemical fingerprints could be developed for three Argentinean red wines based on organic, inorganic, and isotopic patterns, in relation to the regional soil composition. Soils and wines from three regions (Mendoza, San Juan, and Córdoba) and three varieties (Cabernet Sauvignon, Malbec, and Syrah) were collected. The phenolic profile was determined by HPLC-MS/MS and multielemental composition by ICP-MS; (87)Sr/(86)Sr and δ(13)C were determined by TIMS and IRMS, respectively. Chemometrics allowed robust differentiation between regions, wine varieties, and the same variety from different regions. Among phenolic compounds, resveratrol concentration was the most useful marker for wine differentiation, whereas Mg, K/Rb, Ca/Sr, and (87)Sr/(86)Sr were the main inorganic and isotopic parameters selected. Generalized Procrustes analysis (GPA) using two studied matrices (wine and soil) shows consensus between them and clear differences between studied areas. Finally, we applied a canonical correlation analysis, demonstrating significant correlation (r = 0.99; p < 0.001) between soil and wine composition. To our knowledge this is the first report combining independent variables, constructing a fingerprint including elemental composition, isotopic, and polyphenol patterns to differentiate wines, matching part of this fingerprint with the soil provenance.

Subchronic mycotoxicoses in Wistar rats: Assessment of the in vivo and in vitro genotoxicity induced by fumonisins and aflatoxin B1, and oxidative stress biomarkers status

Some evidence suggests that fumonisin B(1) (FB(1)), a worldwide toxic contaminant of grains produced by Fusarium verticillioides, exhibits an oxidative stress mediated genotoxicity. We studied the DNA damage (by the alkaline comet and the micronucleus tests) and biomarkers of cellular oxidative stress (malondialdehyde, MDA; catalase, CAT; and superoxide dismutase, SOD) in spleen mononuclear cells of male Wistar rats subchronically (90 days) fed on a control experimental diet (CED) or poisoned with experimental diets contaminated with a culture material containing 100 ppm of FB(1) (FED), with 40 ppb of aflatoxin B(1) (a common toxic co-contaminant in cereals, AFB(1)ED), and with a mixture of both toxins (MED). The DNA damage was found in 13.7%, 81.7%, 98.0% and 99.3% (comet assay) and in 2.8%, 7.0%, 10.8% and 8.8% (micronucleus technique) in groups CED, FED, AFB(1)ED and MED, respectively. The MDA levels as well as the CAT and SOD activities were increased in all the poisoned animals. A similar behavior was observed in cells exposed in vitro to the toxins. These data support the hypothesis of an oxidative stress mediated genotoxicity induced by FB(1). Furthermore, the extent of DNA damage assessed by the comet assay suggests a possible protective effect of the fumonisins-AFB(1) mixtures in vitro against the genotoxicity induced individually by the toxins.

Reactive oxygen species sources and biomolecular oxidative damage induced by aflatoxin B1 and fumonisin B1 in rat spleen mononuclear cells.

Aflatoxin B1 (AFB(1)) and fumonisin B1 (FB(1)) are mycotoxins widely found as cereal contaminants. Their immunotoxicities predispose to infectious diseases and may alter the tumor immunosurveillance of human and animals, but the mechanisms underlying have not been fully elucidated, and the induction of oxidative stress has been proposed as a probable mechanism. This work was aimed at evaluating in spleen mononuclear cells (SMC) from Wistar rats the effects of the exposure, in vitro for up to 48 h, to 20 μM AFB(1), 10 μM FB(1) and AFB(1)-FB(1) mixture (MIX), over cellular oxidative status, as well as at elucidating the contribution of different reactive oxygen species (ROS) to biomolecular oxidative damage, the biochemical pathways involved, and the probable interaction of both toxins to induce oxidative stress. All the treatments increased total ROS and oxidation of biomolecules, with MIX having the greatest effects. However, only MIX increased superoxide anion radical. The main ROS involved in oxidation of proteins, lipids and DNA appear to be hydrogen peroxide and hydroxyl radical. The mitochondrial complex I and CYP450 were involved in the ROS generation induced by all treatments. The NADPH oxidase system was induced by FB1 and MIX. The arachidonic acid metabolism contributed to the ROS formation induced by AFB(1) and MIX. These results demonstrate that an interaction between AFB(1) and FB(1) occur in the oxidative stress induction, and show the biochemical pathways involved in ROS generation in SMC. The oxidative stress could mediate the AFB(1) and FB(1) individual and combined immunotoxicities.

Immunobiological effects of AFB1 and AFB1-FB1 mixture in experimental subchronic mycotoxicoses in rats.

Maize co-contamination with aflatoxin B1 (AFB1) and fumonisin B1 (FB1) is frequently found in several countries. Although the alterations on nutritional and immunologic parameters induced by these mycotoxins, when administered individually, are partially characterised, little is known about the effects induced in animals by a subchronic administration of both toxins mixtures. We have studied the nutritional and immunological alterations induced in rats fed during 90 days with a diet without mycotoxins, containing 40 ppb AFB1, and with a diet containing a mixture of 40 ppb AFB1 and 100 ppm FB1. Animals fed with the mixture of toxins obtained lower body weight than the control ones. The mitogenic response of spleen mononuclear cells (SMC) in vivo was higher in animals fed with AFB1. In in vitro studies, lower proliferations of SMC pre-exposed to AFB1 and to the mixture of toxins were detected. The SMC of animals fed with AFB1 produced lower levels of IL-2, higher of IL-4 and equal levels of IL-10. The SMC of animals fed with both toxins produced higher levels of IL-4, lower of IL-10 and equal levels of IL-2. The SMC preincubated with an AFB1-FB1 mixture produced higher concentrations of IL-4, lower of IL-10 and equal levels of IL-2. The peritoneal macrophages of animals that consumed AFB1 released less H(2)O(2), while animals fed with the mixture of toxins produced higher levels. In in vitro studies, macrophages pre-exposed to the mixture of toxins released less H(2)O(2). These results show different immunobiological effects produced by a mixture of mycotoxins in comparison to the individual action of the same toxins.

Antifumonisin activity of natural phenolic compounds A structure-property-activity relationship study.

Fumonisin B(1) (FB(1)) is a Fusarium mycotoxin that has received considerable attention from food regulatory agencies, since it shows immunotoxic, neurotoxic, hepatotoxic, nephrotoxic and carcinogenic properties in animals. Although several publications have reported that some natural phenolic compounds can cause a reduction in mycotoxin production, little is known about the molecular properties related to their antitoxigenic activities. The objective of this work was to evaluate which of these molecular properties are important in antifumonisin activity, with this being the first structure-activity relationship study concerning the antimyctoxigenic activity of natural phenolic compounds. The results of the experimental determination of the FB(1) inhibition capacity for ten natural phenolic compounds revealed thymol, carvacrol, and isoeugenol followed by eugenol to be the most active antifumonisin compounds. Lipophilicity, molar refractivity and saturated area were demonstrated to be the molecular properties or descriptors which best explained the antifumonisin activity of these phenolic compounds. A mathematical expression, obtained by QSAR analysis, was able to predict the antifumonisin activity of other structurally related molecules. These findings could provide an important contribution in the search for new compounds with antifumonisin activity.

Experimental subchronic mycotoxicoses in mice: individual and combined effects of dietary exposure to fumonisins and aflatoxin B1

We have used a murine model of subchronic mycotoxicoses produced by ingestion of mycotoxins. The five groups of animals studied were fed for 30, 60 and 90 days, respectively, with commercial diet (CD), experimental control diet (ECD), experimental with fumonisin B1 diet (EFD) and experimental with mixtures of mycotoxins diet (EMD). The animals fed EFD and EMD showed a significant increase in feed consumption/day with respect to the animals fed ECD (P < 0.005 for both groups). The biochemical measurements showed significant differences at 90 days in those animals fed EAD exhibiting a marked decrease in the values of alkaline phosphatase (ALP) and cholesterol (P < 0.05), along with a significant increase in calcium (P < 0.01). Differences in the decrease of the parameters studied were observed in mice fed EFD for triglycerides, cholesterol and calcium (P < 0.05 for all of them). The activity of aspartate transaminase (AST) increased significantly in animals fed EMD (P < 0.01). The tissue specimens at 60 days showed lesions in the livers of the animals fed EAD and EFD. At 90 days, and in those fed EAD, EFD and EMD, the lesions were intensified in the liver at 60 days in 80, 90 and 100% of the animals, respectively.

Involvement of excretion-secretion products from Fasciola hepatica inducing suppression of the cellular immune responses.

Normal rats i.p. injected with Fasciola hepatica excretor-secretor antigen (ESA) induced a population of spleen mononuclear (SpM) cells, which suppressed the delayed type hypersensitivity (DTH) response to parasite antigens as well as to non-related antigens (human serum albumin) by adoptive transfer. A similar effect was observed when the cell transfer was performed with SpM cells non-adherent to nylon wool. The DTH was not modified by cells transfer adherent to nylon wool in syngeneic receptor animals. The observed suppression depended on the concentration and inoculation moment of the antigen; 1.8 mg of protein ESA being enough to suppress the DTH response at the different days studied, before and after immunization with whole F. hepatica antigens. A marked suppression was observed when ESA was injected on day 7 pre-immunization. On the other hand, inoculation of ESA treated with 0.01 M sodium periodate (carbohydrate oxidant) diminished the suppressor effect found after the native ESA inoculation, indicating participation of ESA glucidic components in induced suppression. Inoculation of ESA fractions obtained from polyacrylamide gel elution with different MW range, showed that components between 12 and 23 kDa actively induced suppression to the DTH response to parasite antigens.

Aromatic plants essential oils activity on Fusarium verticillioides Fumonisin B1 production in corn grain

The minimum inhibitory concentration (MIC) of Origanum vulgare, Aloysia triphylla, Aloysia polystachya and Mentha piperita essential oils (EOs) against Fusarium verticillioides M 7075 (F. moniliforme, Sheldon) were assessed, using the semisolid agar antifungal susceptibility (SAAS) technique. O. vulgare, A. triphylla, A. polystachya and M. piperita EOs were evaluated at final concentrations of 10, 20, 40, 50, 100, 200, 250, 500, 1000 and 1500 εl per litre (εl/l) of culture medium. A. triphylla and O. vulgare EOs showed the highest inhibitory effects on F. verticillioides mycelial development. This inhibition was observed at 250 and 500 εl/l for EOs coming from Aloysia triphylla and O. vulgare, respectively. Thus, the effects of EOs on FB1 production were evaluated using corn grain (Zea mays) as substrate. The EOs were inserted on the 5th, 10th, 15th and 20th day of maize postinoculation with a conidia suspension of F. verticillioides. O. vulgare and A. triphylla were applied to give final concentrations of 30 ppm and 45 ppm, respectively. Different effects were observed in the toxicogenicity at the 20th day treatment. The O. vulgare EO decreased the production level of FB1 (P < 0.01) while A. triphyla EO increased it (P < 0.001) with respect to those obtained in the inoculated maize, not EOs treated. Results obtained in the present work indicate that fumonisin production could be inhibited or stimulated by some constituents of EOs coming from aromatic plants. Further studies should be performed to identify the components of EOs with modulatory activity on the growth and fumonisins production of Fusarium verticillioides.

Apoptosis induction by glucuronoxylomannan of Cryptococcus neoformans

We studied the ability of glucuronoxylomannan (GXM), the major constituent of Cryptococcus neoformans capsular polysaccharide, to induce apoptosis in lymphocytes from normal rats. Spleen mononuclear cells (Smc) from normal rats treated with GXM for 24 h exhibited, in comparison with controls, an increased hypodiploidy in the DNA profile after staining with propidium iodide, as well as increased ladder-type DNA fragmentation in agarose gel electrophoresis and a high number of positive cells in the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) reaction. Furthermore, increased hypodiploidy in the DNA profile was also observed in Smc expressing T-cell receptor (TCR +). We also studied the induction of apoptosis in lungs and spleens from rats in the immunosuppressor period of disseminated cryptococcosis. TUNEL labeling of lungs and spleens from rats obtained 14 days after infection with C. neoformans showed a large number of apoptotic cells. Our results provide strong cytometric, molecular and morphological evidence that apoptosis could be a previously unrecognized immunosuppressive property of GXM in vitro. This programmed cell death may be involved in the immunosuppression observed during C. neoformans infection.

Immunosuppression, interleukin-10 synthesis and apoptosis are induced in rats inoculated with Cryptococcus neoformans glucuronoxylomannan

Glucuronoxylomannan (GXM) is the major Cryptococcus neoformans capsular polysaccharide and represents the main virulence factor of this fungus. In in vitro studies we have demonstrated previously that this acidic and high‐molecular‐weight polysaccharide suppresses lymphoproliferation, modulates cytokine production and promotes apoptosis in spleen mononuclear (Spm) cells from rats. In this study we demonstrate that these phenomena also occur in vivo after the intracardiac inoculation of GXM into normal Wistar rats. The results of this study show suppression of the proliferative response Spm cells to concanavalin A (Con A) or heat‐killed C. neoformans (HKCn) in the first 2 weeks after polysaccharide administration. In addition, increased levels of interleukin (IL)‐10 were produced by Con A‐stimulated Spm cells, coinciding with immunohistochemical GXM detection in the white pulp of spleen. In particular, high production of IL‐10 with diminution of IL‐2, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α synthesis were detected 14 days after GXM administration. In situ cell death detection by TdT‐mediated biotin–dUTP nick‐end labelling (TUNEL) reaction in sections of spleen, lung and liver demonstrates apoptosis in tissues with deposits of GXM. These data demonstrate the in vivo ability of GXM to modify cytokine synthesis by Spm cells and to promote host cell apoptosis.