Magda Carvajal

Aflatoxin M1 in pasteurized and ultrapasteurized milk with different fat content in Mexico.

High per capita milk consumption in Mexico indicated a strong need for documentation of aflatoxin M1 (AFM1) levels in milk. A survey of 580, 2-liter samples (n = 290), was conducted to quantify AFM1 using high-performance liquid chromatography, considering two maximum tolerance levels (0.05 and 0.5 microg/liter). We relate aflatoxin levels in the seven most consumed brands from different regions, with two processes (pasteurized and ultrapasteurized), different expiration dates, and different fat content: whole fat (28, 30, and 33 g), half-skimmed (10, 16, and 20 g), light (1, 2, and 4 g), and with vegetable oil. Pasteurization and ultrapasteurization did not diminish AFM1 contamination present at levels of 0 to 8.35 microg/liter in 40% of the milk samples at concentrations > or = 0.05 microg/liter and in 10% of the samples at > or = 0.5 microg/liter. Statistically significant relationships were AFM1 contamination with brand (P = 0.002 at the > or = 0.05 microg/liter level and P = 0.034 at the > or = 0.5 microg/ liter level) and higher AFM1 levels with mild or warm seasons of the year (P = 0.0003). Samples with greater fat content had slightly more probability (P = 0.067) of being contaminated by AFM1 at the > or = 0.5 microg/liter level. The milk with the lowest contamination of AFM1 was a brand imported as powder and rehydrated in Mexico.

Aflatoxin B1 and its interconverting metabolite aflatoxicol in milk: the situation in Mexico

Between 1996 and 1998, 580 litres of milk in Mexico were surveyed for aflatoxin B1 (AFB1) and its metabolite aflatoxicol (AFL), which are mutagenic and carcinogenic mycotoxins that interconvert AFB1–AFL–AFB1. The seven most consumed brands from different regions of Mexico included pasteurized and ultrapasteurized milk with four different fat levels: whole fat (28–33 g l−1), half-skimmed (10–20 g l−1), light (1–4 g l−1) and with vegetable oil (33 g l−1). Aflatoxins in each sample were concentrated with total aflatoxin immunoaffinity columns and quantitated by high-performance liquid chromatography. A milk sample was considered contaminated if it contained ≥0.05 μg l−1 AFL. Pasteurization and ultrapasteurization of milk did not control contamination with AFL, which was present in 13% of samples at ≥0.05 μg l−1 and in 8% at ≥0.5 μg l−1, with a range of AFL from 0 to 12.4 μg l−1. AFB1 was present mainly in traces (0–0.4 μg l−1). The safest milk in relation to AFL contamination was imported milk powder with vegetable oil. There was a significant correlation between contamination of milk with AFL and the autumn (p<0.0002); the fat content was not significant.

Aflatoxin (B1, B2, G1, and G2) contamination in rice of mexico and spain, from local sources or imported

Rice is an important cereal but it is often contaminated with aflatoxins (AFs). The purpose of this study was to identify and quantify AF (B1 , B2 , G1 , and G2 ) in 67 rice samples cultivated in Mexico and Spain, and from imported crops collected in 2008 and 2009. The methodology was validated, the rice samples were concentrated and purified with immunoaffinity columns and were quantified by high-pressure liquid chromatography (HPLC). The average total AF (AFt) in the Spanish rice was 37.3 μg/kg, the range was from 1.6 to 1383 μg/kg, the most contaminated samples being from San Juan de Aznalfarache, Sevilla (AFt = 138.6 μg/kg), from Tortosa, Tarragona (AFt = 104.6 μg/kg), and Calasparra, Murcia (AFt = 103.9 μg/kg). The rice imported from France to Spain had AFt of 26.6 μg/kg and from Pakistan AFt of 18.4 μg/kg, showing less AF contamination than the local one. The rice which originated from Mexico contained (AFt = 16.9 μg/kg), and those imported from the United States (AFt = 14.4 μg/kg) and Uruguay (AFt = 15.6 μg/kg). The imported rice had better quality in terms of the presence of AFs.

Identification and quantification of aflatoxins and aflatoxicol from poultry feed and their recovery in poultry litter

Aflatoxins (AF) are toxic fungal secondary metabolites and are known mycotoxins pathological to animals and humans. Poultry litter is frequently used as a food supplement for ruminants, and when poultry feed contains AF, the litter becomes contaminated as well, thus having an effect on livestock health. This study identified and quantified AF (AFB(1), AFB(2), AFG(1), and AFG(2)) from poultry feed and their recovery, together with their metabolites (AFM(1), AFM(2), AFP(1), and aflatoxicol) in litter. An experiment with 25 Hy-Line W-36 hens, in their second production stage, 121 wk old, was carried out. Hens were distributed in 3 groups placed in individual cages and 1 ration of 250 g of feed was given to each hen daily. Nine hens of the control group were fed with clean feed, without AFB(1); the other 2 experimental groups, with 8 hens each, were fed with 2 AFB(1) concentrations: 30 and 500 microg.kg(-1). The feed was replaced and weighed daily throughout a 7-d period to register the amount of feed consumed by the hens. Litter from each hen was collected, weighed, and dried individually. The chemical analysis of 40 g of each one of the 200 feed and 200 litter samples was chemically extracted and concentrated with immunoaffinity columns for total AF. To quantify AF, calibration curves for each AF were done by HPLC. Feed samples of the 3 groups presented significant difference with AFB(2) and AFG(2), whereas in litter samples, there were significant differences for AFG(2) in the 500 microg.kg(-1) group. Poultry litter had traces of AFM(1), AFM(2), AFP(1), and AFL with no significant differences among treatments. Aflatoxin B(1) prevalence in litter samples can cause damages in livestock because this mycotoxin reduces the digestibility of ruminant feed up to 67%.

The presence of aflatoxin B1-FAPY adduct and human papilloma virus in cervical smears from cancer patients in Mexico

The carcinogenic biomarker aflatoxin B1-formamidopyrimidine 2,3-dihydro-2-(N-formyl)-2′,5′,6′-triamino-4′-4′-oxy-N-pyrimidyl-3-hydroxy-AFB1 called AFB1-FAPY adduct, and Human Papilloma Virus (HPV) types 16 and 18 were quantified from DNA cervical scrapes from 40 women with cervical cancer (CC) and 14 healthy women as controls. The relationship between the AFB1-FAPY adduct and HPV types 16 and 18 was determined. Competitive inhibitory indirect ELISA was validated with 94% inhibition to quantify the AFB1-FAPY adducts in picograms per milligram of DNA (limit of detection = 0.1 pg/mg, and limit of quantification = 10 pg/mg), polymerase chain reaction and DNA sequencing to identify HPV types. The average concentration of AFB1-FAPY adducts/mg DNA in the CC cases was 1025 pg, 1420 pg with HPV16 and 630 pg sharing HPV18 (p = 0.03). In comparison, healthy controls had ≤2.6 pg/mg DNA, a statistically significant difference (p = 0.00006). The presence of AFB1-FAPY adduct increased six-fold the risk for CC between cases and controls, the odds ratio was 6.1 (95% CI = 1.4–25.4). There was a close relationship between the AFB1-FAPY adducts and HPV16 in CC samples.

Survey of aflatoxins in maize tortillas from Mexico City

In Mexico, maize tortillas are consumed on a daily basis, leading to possible aflatoxin exposure. In a survey of 396 2-kg samples, taken over four sampling days in 2006 and 2007 from tortilla shops and supermarkets in Mexico City, aflatoxin levels were quantified by HPLC. In Mexico, the regulatory limit is 12 µg kg−1 total aflatoxins for maize tortillas. In this survey, 17% of tortillas contained aflatoxins at levels of 3–385 µg kg−1 or values below the limit of quantification (12 µg kg−1 and 87% were below the regulatory limit. Average aflatoxin concentrations in 56 contaminated samples were: AFB1 (12.1 µg kg−1); AFB2 (2.7 µg kg−1); AFG1 (64.1 µg kg−1) and AFG2 (3.7 µg kg−1), and total AF (20.3 µg kg−1).

Supervising programme of aflatoxins in Mexican corn

Contamination of maize with aflatoxins was detected in Tamaulipas in 1989 and the Mexican government organized a Supervising Programme to check the maize crop of Tamaulipas from 1989 to 1992. The monoclonal immunoaffinity column method quantified by fluorometry was chosen as the official one. After detecting, in 1989, the first lots of contaminated maize, in 1990 87% of the crop was quarantined and the Supervising Programme was implemented. Laboratories were constructed in each warehouse that received the maize; one chemical analysis for aflatoxins was performed for every 100 tons of maize received and there were around 60,000 analyses in the whole Programme (1989-1993). A random sampling method was applied, taking 2 kg from each truck. In 1991, the contaminated maize was dyed and separated as feed. Decontaminating methods with aluminosilicates and with ammonia were tested, but the cost was high and these practices were abandoned. A Technological Package for the farmers was organized by different institutions: the Ministry of Health, the Ministry of Agriculture, Conasupo, and the government of the State of Tamaulipas. This specified sowing time, insecticides, fungicides, fertilizers, irrigation periods, maize cultivars, etc. and had successful results in the field, in 1992, when lower quantities of total aflatoxins in maize were detected. On the other hand, the aflatoxin combination of maize persisted in storage areas, especially in the last week of July and the first week of August. The aflatoxin maize problem will be solved only when both field and storage contaminations are controlled.

Comparison between inhibitory indirect ELISA and HPLC methods to quantify free and adducted aflatoxins in human urine.

HPLC and Inhibitory Indirect ELISA (I.I. ELISA) methods for quantitation of aflatoxins (AF) in human urine were compared in terms of specificity, sensitivity, easiness and cost. I.I. ELISA was optimized in kind of antibody in use, type of plastic plate, adduct synthesis technique, peroxidase and antibody dilutions, etc. Both polyclonal (Cuban) and monoclonal (British) anti-AF antibodies were statistically studied and the process was standardized. HPLC and electrophoresis were performed while synthetizing AFB(1)-DNA and AFB(1)-Cl-Ovalbumin (AFB(1)-Cl-Ov) adducts. Costar polystyrene plate had the best adherence. Optimum coating dilution was 10 ng of AFB(1)-Cl-Ov per well. Dilutions of 1:1000 of monoclonal antibody from purified culture or 1:300 from monoclonal antibody from tissue culture and 1:1000 of peroxidase anti-mouse conjugate were the best. Optimum separation with HPLC was obtained isocratically with 60% MeOH and 40% distilled water mobile phase. ELISA had a sensitivity of 1 pg mL(-1) AFB(1) and HPLC sensitivity was 0.1 ng mL(-1) AFB(1) with fluorescence detector and 4.5 ng mL(-1) with UV detector. Monoclonal antibody gave more accurate results for determination of free and adducted AFB(1) in urine analysis.

Transformación de la aflatoxina B1 de alimentos, en el cancerígeno humano, aducto AFB1-ADN

Aflatoxins (AF) are secondary toxic metabolites formed mainly by the molds Aspergillus flavus and A. parasiticus; they are potent mutagens and carcinogens of foods, and human exposure to them is continuous. AF bind to DNA, RNA and proteins forming AFB1-DNA adducts that accumulate for years and damage from viruses to humans. To identify and quantify the chemical reactions and molecular biology of these adducts is of primary importance because they are long-term biomarkers validated as a source of mutagenicity and risk of cancer in animals and humans. The cytochrome P450 activates AF as an unstable molecule called 8,9 AFB1 epoxide, that binds mainly to the N7 of the guanine nucleotide forming adducts that are the active carcinogens themselves and, as biomarkers, an objective measure of human exposure to environmental carcinogens. Adducts represent an integration of exposure, absorption, distribution, metabolism, DNA repair, and cell turnover. The different issues presented here are the formation of AFB1-DNA adducts, in vitro and in vivo studies, dietary AFB1 exposure, effects, development of human cancer and mutations in the p53 tumor suppressor gene, the effect of diet, routes of exposure, vitamins, kinds of AFB1-DNA adducts, methodology used for their study and their control.