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.

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.

Assessment of aflatoxin exposure using serum and urinary biomarkers in São Paulo, Brazil: A pilot study.

The aim of this study was to evaluate the human exposure of individuals from Pirassununga, Brazil, to dietary aflatoxins B1 (AFB1) and M1 (AFM1) by determination of serum AFB1-lysine and urinary aflatoxin biomarkers (AFM1 and AFB1-N(7)-guanine). The participants were recruited among employees from a Campus of the University of São Paulo, which provided food samples from their homes, as well as serum and urine samples four times every three months, from June 2011 until March 2012. The probable daily intake (PDI) of aflatoxin was estimated by using the results from analysis of food products collected by the time of samples collection, and data from a 24-hour dietary recall questionnaire. Analyses of AFB1 and AFM1 in food samples were conducted by high-performance liquid chromatography with fluorescence detection. Biomarkers in serum and urine were determined by tandem mass spectrometry. AFB1 and AFM1 were detected in 38 samples of cereals (28%, N=136) and 31 milk products (36%, N=86), respectively. AFB1-lysine and AFB1-N(7)-guanine and were not detected in serum or urine samples, respectively. However, AFM1 was found in 74 urine samples (65%), at mean levels in the 4 sampling times ranging from 0.37±0.23 to 1.70±2.88pg/mg creatinine. The mean PDI varied among different sampling times, ranging from 0.09±0.09 to 1.35±5.98ng/kg body weight/day. A modest though significant correlation (r=0.45; p=0.03; N=23) was found for the first time in Brazil between the AFM1 concentration in urine and the PDI for total aflatoxins (AFB1+AFM1) in sampling 1 (June 2011). Urinary AFM1 was confirmed as very sensitive for monitoring the human exposure to dietary aflatoxin. Further studies using serum and urinary biomarkers are needed to estimate the aflatoxin exposure of populations in higher risk areas in Brazil.

Determination of urinary biomarkers for assessment of short-term human exposure to aflatoxins in São Paulo, Brazil.

In the present study, a longitudinal assessment was carried out to evaluate the short-term human exposure to aflatoxins in Pirassununga region, São Paulo, Brazil, by determination of urinary aflatoxins by a liquid chromatography coupled to mass sprectrometry (UPLC-MS/MS) method. Sixteen volunteers with ages ranging from 14 to 55 years old were instructed to collect the early morning first urine four times every three months, from June 2011 to March 2012, totaling 64 samples. Aflatoxin M1 (AFM1) was found in 39 samples (61%) at levels ranging from 0.19 to 12.7 pg·mg-1 creatinine (mean: 1.2 ± 2.0 pg·mg-1 creatinine). Residues of aflatoxins B1, B2, G1, G2 and aflatoxicol were not identified in any urine sample. No significant difference was found among the AFM1 mean levels in urine samples collected in the four sampling periods. The levels of AFM1 found in urine samples indicate a low short-term exposure of the population studied to aflatoxins through the diet, although further investigations are needed to assess other long-term biomarkers of exposure to AFB1.

Comparative evaluation of extraction procedures and method validation for determination of carbohydrates in cereals and dairy products by capillary electrophoresis.

This work describes the development and application of two optimized electrolytes: 15 mmol/L sorbate, 0.2 mmol/L CTAB, and 35 mmol/L sodium hydroxide for the determination of fructose, glucose, maltose, maltotriose, and sucrose in cereals and 15 mmol/L sorbate, 0.3 mmol/L CTAB, and 55 mmol/L sodium hydroxide for the determination of fructose, glucose, galactose, lactose, and sucrose in dairy products. Both methods were validated with respect to linearity, limits of detection (using both signal-to-noise ratio and the Eurachem approach), recovery tests, and intra- and inter-day precision exhibiting adequate performance. Additionally, statistically similar results were obtained in a comparative study of extraction procedures for carbohydrates using the AOAC protocol, ultrasound extraction, magnetic stirring, and sample dissolution.

Optimization of the separation of flavonoids using solvent-modified micellar electrokinetic chromatography.

In this work the separation of eighteen flavonoids was attempted using reduced-flow micellar elektrokinetic chromatography (RF-MEKC) electrolytes modified by selected solvents with differing properties: methanol (MeOH), acetonitrile (ACN) and tetrahydrofuran (THF). Structural aspects such as unsaturation of the C ring, number and position of OH groups, methylation and glycosylation as well as solvent effects and their impact on the electrophoretic behavior of flavonoids were addressed. By evaluating the electropherograms obtained from mixture-designed electrolytes and searching for changes in the critical pairs, a favorable separation condition was achieved using 20 mmol/L phosphate buffer at pH 2.5 containing 50 mmol/L sodium dodecyl sulfate (SDS), 15% ACN and 5% THF (one critical pair) in less than 12 min with 1.5% coefficient of variation (CV) for retention factor and 3% CV for peak area (n = 5). The applicability of the proposed separation condition was demonstrated by the inspection of flavonoids in herbal extracts of Neem.

Evaluation of diabetic ketoacidosis-related ionic metabolites in human blood serum by capillary electrophoresis.

Three methodologies are presented to contemplate the analysis of sodium, potassium, chloride, bicarbonate, lactate, acetoacetate, and beta-hydroxybutyrate in blood serum samples of diabetic patients using free solution capillary electrophoresis with indirect detection (214 nm for cations and 254 nm for anions). The cations were analyzed in less than 6 min in an electrolyte comprised of 15 mmol x L(-1) imidazole, 5 mmol x L(-1) lactate and 1 mmol x L(-1) 18-crown-6-ether, adjusted to pH 4.5. Chloride and bicarbonate were analyzed in 2 min in a 5 mmol x L(-1) chromate, 0.1 mmol.L(-1) cetyltrimethylammonium bromide (CTAB), pH 9.0 electrolyte solution. Ketoacids and lactate were analyzed in less than 11 min in an electrolyte composed of 15 mmol x L(-1) 3,5-dinitrobenzoate, 0.1 mmol x L(-1) CTAB, at pH 3.5. All methodologies were validated with respect to linearity, selectivity, sensitivity, precision and accuracy performing adequately for clinical purposes.