Pollyana Cristina Maggio de Castro Souto

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.

Assessment of aflatoxin exposure using serum and urinary biomarkers in Sao 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.

Comparative biotransformation of aflatoxin B1 in swine, domestic fowls, and humans

Abstract Aflatoxin B1 (AFB1) is primarily biotransformed in the liver by cytochrome P450 (CYP) enzymes, which can yield either the genotoxic metabolite AFB1-8,9 epoxide that causes liver carcinogenicity or less toxic compounds. The biotransformation of AFB1 is better understood in humans, including gene expression of CYPs involved in the detoxification process. Studies on farm animals have demonstrated genes homologous to human CYPs that play similar roles in AFB1 biotransformation. This review compares the activities of the most important CYPs related to the biotransformation of AFB1 in humans, swine and domestic fowls (chickens, quail, turkeys and ducks), as well the main detoxification mechanisms in these species.

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 individuals exposure of swine to AFB1.

Ganho de peso, consumo de ração e histologia de órgãos de leitões alimentados com rações contendo baixos níveis de fumonisina B1

Fumonisin B1 (FB1) is a secondary metabolite produced mainly by Fusarium verticilioides in several types of foods, particularly corn, which is the basis for composition of feed for several domestic animals. FB1 is particularly toxic to pigs, being the clinical manifestations evident in animals exposed to high concentrations of FB1 in the diet (generally above 30mg/kg). However, there are few studies on the effects of FB1 on pigs fed rations containing low concentrations of fumonisin, which are most probably found under field conditions. The aim of the study was to evaluate the effects of a 28-day exposure of piglets to low levels of FB1 in the feed on the weight gain, feed consumption, organ weights and histological aspects of the spleen, liver, lungs, kidneys and heart. Twenty-four pigs were assigned into 4 experimental groups and fed diets containing 0mg (control), 3.0mg, 6.0mg or 9.0mg FB1/kg diet. The different diets did not affect (P>0.05) the weight gain or the weight of organs examined. There were no macroscopic or histological lesions in the spleen, liver, kidneys and heart. However, histological lesions were found in the lungs from all animals fed rations containing fumonisin, hence indicating that none of the FB1 levels used in the experiment could be considered as safe for piglets. Further studies on the mechanisms of toxic action of FB1 in pigs are needed, particularly under conditions of prolonged exposure to low contamination levels in the diet.

Relação entre níveis de fumonisina B1 e ácido fólico em farinha de milho e a concentração de ácido fólico no soro humano

Neste trabalho foram determinados os niveis de acido folico e de fumonisina B1 (FB1) em farinha de milho consumida por 24 voluntarios residentes em um campus universitario no estado de Sao Paulo, bem como sua relacao com as concentracoes de acido folico serico nos individuos. As analises de acido folico e de FB1 em farinha de milho foram realizadas por cromatografia liquida de alta eficiencia (CLAE), enquanto a determinacao de acido folico serico foi feita por kit de imunoensaio. Detectou-se a FB1 em 100% das amostras de farinha de milho, em niveis que variaram de 142 a 3.037 µg kg-1 (media: 738 ± 591 µg kg-1). As concentracoes de acido folico nas amostras de farinha de milho ficaram entre < 0,3 µg kg-1 (limite de quantificacao) e 1.705 µg kg-1, com media de 713 ± 435 µg kg-1, o que representa 47% do limite minimo exigido pela Agencia Nacional de Vigilância Sanitaria (ANVISA) para farinhas de milho comercialmente disponiveis. Nas amostras de soro humano, os niveis de acido folico variaram de 6,7 a 24,0 ng mL-1 (media: 13,4 ± 5,4 ng mL-1). Nao houve correlacao (p < 0,05) entre os niveis de acido folico no soro dos individuos e as concentracoes de FB1 ou acido folico nas amostras de farinha de milho. Outros estudos sao necessarios para estimar a ingestao total de FB1 por meio da dieta para averiguar os efeitos das fumonisinas sobre a absorcao de acido folico nos individuos avaliados.