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Determination of aflatoxin M1 in breast milk as a biomarker of maternal and infant exposure in Colombia a
b
Gonzalo J. Diaz & Marlib Paloma Sánchez a
Laboratorio de Toxicología, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Bogotá D.C., Colombia b
Departamento de Toxicología, Facultad de Medicina, Universidad Nacional de Colombia, Bogotá D.C., Colombia Accepted author version posted online: 11 May 2015.Published online: 03 Jun 2015.
Click for updates To cite this article: Gonzalo J. Diaz & Marlib Paloma Sánchez (2015): Determination of aflatoxin M1 in breast milk as a biomarker of maternal and infant exposure in Colombia, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2015.1049563 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1049563
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Food Additives & Contaminants: Part A, 2015 http://dx.doi.org/10.1080/19440049.2015.1049563
Determination of aflatoxin M1 in breast milk as a biomarker of maternal and infant exposure in Colombia Gonzalo J. Diaz
a
* and Marlib Paloma Sánchez
b
a
Laboratorio de Toxicología, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Bogotá D.C., Colombia; bDepartamento de Toxicología, Facultad de Medicina, Universidad Nacional de Colombia, Bogotá D.C., Colombia
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(Received 26 February 2015; accepted 5 May 2015) Chronic exposure to aflatoxins, and especially to aflatoxin B1 (AFB1), causes hepatocellular carcinoma with prevalence 16–32 times higher in developing compared with developed countries. Aflatoxin M1 (AFM1) is a monohydroxylated metabolite from AFB1 that is secreted in milk and which can be used as a biomarker of AFB1 exposure. This study aimed to determine AFM1 levels in human breast milk using immunoaffinity column clean-up with HPLC and fluorescence detection. Breast milk samples were obtained from 50 nursing mothers. Volunteers filled in a questionnaire giving their consent to analyse their samples as well as details of their socioeconomic, demographic and clinical data. The possible dietary sources of aflatoxins were assessed using a food frequency questionnaire. A total of 90% of the samples tested positive for AFM1, with a mean of 5.2 ng l−1 and a range of 0.9–18.5 ng l−1. The study demonstrated a high frequency of exposure of mothers and neonates to AFB1 and AFM1 in Colombia, and it points out the need to regulate and monitor continuously the presence of aflatoxins in human foods. Further research is needed in order to determine the presence of other mycotoxins in foods and in human samples as well as to devise protection strategies in a country where mycotoxins in human foods are commonly found. Keywords: aflatoxins; aflatoxin M1; exposure; breast milk; infant
Introduction Aflatoxins are a group of approximately 20 related fungal metabolites, although only four of them are actually produced by fungi, namely aflatoxins B1 (AFB1), B2 (AFB2), G1 and G2 (Da Rocha et al. 2014). Aflatoxins are produced primarily by the fungus Aspergillus flavus (produces only B aflatoxins) and A. parasiticus (produces both B and G aflatoxins) (Yates 1985). Aflatoxins are ubiquitous contaminants of the human food supply and their production is dependent in part on climatic conditions, being more prevalent in areas of the world with hot humid climates such as the tropical regions (Wild & Gong 2010). The major hosts of Aspergillus spp. among crop plants are corn, peanuts and cereal grains, in which invasion occurs primarily during storage, resulting in potentially high levels of aflatoxins (IARC 2012). Human exposure by dietary intake is especially important in populations that rely heavily on these staple foods (Wild & Gong 2010). Prevention of aflatoxin formation might be achieved by the implementation of good agricultural, manufacturing and storage practices (Codex Alimentarius 2005). Unfortunately, exposure to aflatoxins is almost unavoidable, particularly in developing countries such as Colombia, where access to quality products is limited, and policies needed to prevent food contamination are absent (Marroquín-Cardona et al. 2014). *Corresponding author. Email: [email protected] © 2015 Taylor & Francis
Naturally occurring aflatoxins negatively affect human health and AFB1 is the most potent chemical liver carcinogen known in humans (Wild & Turner 2002). The adverse health effects associated with acute exposure to AFB1 are numerous including vomiting, abdominal pain, pulmonary oedema, fatty liver, and necrosis and even death (Kensler et al. 2011). Chronic exposure causes human hepatocellular carcinoma (HCC), which is the third leading cause of cancer deaths worldwide, with prevalence 16–32 times higher in developing countries than in developed countries (WHO 2008). Further, specific epigenetic processes may be affected by aflatoxins, especially during a prolonged period of exposure, promoting tumorigenesis, angiogenesis, invasion and metastasis to other organs in cases of hepatocellular carcinoma (Bbosa et al. 2013). In a study conducted in Colombia, 10.5% of 202 HCC cases had the characteristic aflatoxin-induced mutation of codon 249 of the p53 tumour suppressor gene (Navas et al. 2011). Several studies describe a variety of other adverse health effects associated with aflatoxins, such as impaired growth in children (Khlangwiset et al. 2011), hepatomegaly (Gong et al. 2012), suppression of immune function, especially cell-mediated immune responses (Mehrzad et al. 2014), and reproductive health effects (Shuaib et al. 2010). Furthermore, aflatoxins can cross the
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placental barrier and be bioactivated in utero (Turner et al. 2007). Ingestion of contaminated food during pregnancy and lactation may expose the foetus or the newborn to the deleterious effects of aflatoxins during these critical development and growth periods (Abdulrazzaq et al. 2003). At least three monohydroxylated metabolites are produced by CYP450 from AFB1: aflatoxin M1 (AFM1), Q1 (AFQ1) and B2a (AFB2a) (Diaz & Murcia 2011). About 0.3–6.2% of the ingested AFB1 is eliminated as AFM1 in milk (Creppy 2002). Ingested AFB2 can also be monohydroxylated in the liver and eliminated in milk as aflatoxin M2 (AFM2) (Creppy 2002). Breast milk, therefore, constitutes a unique matrix for biomonitoring exposure in human populations (Needham & Wang 2002). Measurement of aflatoxins in breast milk is important to determine the true contribution of these mycotoxins to public health, especially to the health of the newborn. Moreover, breast milk is a convenient sample for biomonitoring programmes because relatively large volumes (50–100 ml) can be collected non-invasively. This makes it an ideal matrix easily sampled in a large population with easily identified subgroups. The aim of the present study was to determine for the first time AFM1 and AFM2 levels in breast milk from a select group of Colombian nursing mothers.
Materials and methods Study population and data collection Breast milk samples were obtained from 50 nursery mothers that voluntarily agreed to participate in the study and were collected from May to September 2013. The mothers were recruited at The Mercy Hospital Foundation (HOMI), a high-complexity children’s hospital where paediatric patients are referred from all over the country. Volunteers were questioned by a physician at the time of sample donation: they were asked details about socioeconomic, demographic and clinical data that included level of education, place of residence, age, gynaecobstetric history, mother’s health status and current medications, stage and details of lactation, baby’s weight at delivery and baby’s medical history. The potential intake of dietary sources of aflatoxins was also assessed using a structured food frequency questionnaire to record the mother’s intake of food items over the last 72 h before sample collection. The groups of food included grain products (corn, rice, wheat and derivatives of these), legumes (beans, lentils), dried fruits, and peanuts. The data on food intake were expressed as servings per day. A previously described protocol to collect the breast milk samples was followed (Lovelady et al. 2002).
Chemicals and consumables AFM1 and AFM2 standards of 2000 ng ml−1 in acetonitrile were purchased from Micotox Ltd (Bogota, Colombia). Immunoaffinity columns with antibodies against aflatoxins M1 and M2 (NeoColumn Aflatoxin wide bore) were purchased from Neogen Europe Ltd (Ayr, UK). Acetonitrile and methanol were HPLC grade from Merck KGaA (Darmstadt, Germany). Water was from a Millipore Milli-Q system (EMD Millipore, Billerica, MA, USA).
Determination of AFM1 The method used in the present study was based on an international standard developed for cow’s milk (ISO 14501, 1998) which was validated in-house for human milk samples. Breast milk was collected in a sterile plastic container by self-expression. Samples were kept at 4°C during transportation and processing within a day, otherwise they were kept at −72°C. Milk samples were analysed for the presence of AFM1 and AFM2 using immunoaffinity column for clean-up and HPLC with fluorescence detection for determination and quantitation, as follows. Milk samples (100 ml) were warmed to 37°C and centrifuged at 4000 rpm for 15 min. After centrifugation, the upper fat layer was discarded. The skimmed milk was filtered through qualitative filter paper and an aliquot of 50 ml was passed through the immunoaffinity clean-up column. After the sample had passed through, the column was washed with distilled water (2 volumes of 10 ml) to remove extraneous non-specific material. The column was then dried by gentle blowing of air and the AFM1 and AFM2 bound to the antibodies were released by elution with 1.25 ml acetonitrile-methanol (2:3, v/v) followed by 1.25 ml of water into a 4-ml silanised amber vial. Finally, 1 ml of the mixed eluate was taken to be transferred into a 1.5 ml amber vial followed by determination of AFM1 and AFM2 content by HPLC.
Chromatography HPLC was performed on a Shimadzu Prominence HPLC system consisting of a DGU-20A3 degassing unit, an LC-20AB dual pump, a SIL-20A HT autosampler, an RF-20A XS fluorescence detector, a CTO-20A column oven and a CBM-20A communications bus module, all controlled by Lab Solutions software (Shimadzu Scientific, Columbia, MD, USA). The mobile phase consisted of an isocratic mix of water–acetonitrile–methanol (50:30:20, v/v/v) at a flow rate of 0.6 ml min−1. The column used was a 250 × 4.6 mm and 5 µm particle size Phenomenex Prodigy ODS3 (Phenomenex, Inc. Torrance, CA, USA) at 30°C. The fluorescence detector was set at excitation and emission wavelengths of 346 and 430 nm, respectively. Metabolites were identified by comparison of retention times and co-injection of standards.
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In-house method validation An in-house validation of the ISO method for cow’s milk (ISO 14501, 1998) was carried out in order to verify the fitness of the method for human milk. The validation was performed using breast milk from volunteers that contained no detectable levels of aflatoxins M1 or M2. Linearity was determined by preparing calibration curves for AFM1 and AFM2 at five different levels corresponding to sample concentrations of 12.5, 25, 50, 100 and 200 pg ml–1. Three independent replicates were prepared for each level. Selectivity was evaluated by analysing six blank samples. The accuracy and repeatability of the method were assessed by spiking blank samples with three different levels of aflatoxins (25, 50 and 100 pg ml–1) in triplicate. LOD and LOQ were initially estimated using two different methods: by the calibration curves and by the detector signal-to-noise ratio. LOD was defined as the lowest concentration of aflatoxins that could be reliably detected (> 3:1 signal-to-noise ratio). Based on this initial qualitative approach, LOD and LOQ were determined using the EPA method (EPA 40 CRF Part 136, Appendix B), which is based in the analysis of eight independently spiked samples fortified at the lowest quantitation limits estimated previously (EPA 1984).
Statistical analysis Comparison between AFM1 content in milk and each of the socioeconomic and food intake parameters was evaluated by Pearson R Correlation Tests. R software programme version 3.0.2 (R Core Team 2013) was used for analysis of data. A value of p < 0.05 was considered to be statistically significant.
Ethical considerations The study was approved by the Ethics Committee of the School of Medicine of the National University of Colombia (document CE-011-12) and by The Mercy Hospital Foundation (document GCCI-008-13). All subjects were made aware of the content of the study, and if they agreed to participate, a written informed consent was obtained.
Results In-house validation The calibration curves had linear equations of y = 2435x + 169 for AFM1 and y = 2436x + 40 for AFM2. In both cases the correlation coefficient was 1.0. The selectivity tests showed no interferences eluting at the retention times of the analytes. Average recoveries for AFM1 and AFM2 were 92.3% and 90.2%, respectively. The relative standard deviations for repeatability (RSDr)
Figure 1. HPLC chromatogram: (1) aflatoxin M1 standard equivalent to 500 pg ml–1; (2) milk sample spiked with 200 pg ml–1 aflatoxin M1; and (3) type 1 water sample spiked with 200 pg ml–1 aflatoxin M1. In the spiked samples, the AFM1 signal overlaps.
of the three levels evaluated were 0.6, 2.4 and 0.85 for AFM1 and 0.6, 3.3 and 1.5 for AFM2. The estimated LOD and LOQ for AFM1 according to the EPA method were 0.6 and 1.8 pg ml–1, respectively. The same values for AFM2 were 0.4 and 1.2 pg ml–1, respectively. Figure 1 shows a typical chromatogram of an AFM1 standard, a milk sample and type 1 water spiked with 200 pg ml−1 AFM1.
Main characteristics of the study population The average age of the mothers was 25 years (range 15–41 years). Twenty-nine mothers (58%) were less than 27 years old. Most mothers (64%) belonged to a low socioeconomic status (SES, 1 and 2). The formula used to categorise the population in socioeconomic groups is country specific, where 1 is the lowest and 6 the highest. This is based on where the person resides, access to/ownership of consumer durable goods, and the existence of public services. The highest academic qualification obtained by the lactating mothers was a bachelor’s degree (14%), while 48% had secondary or technical education and 8% had no elementary education. The majority of lactating women (84%) lived in urban areas. All mothers were healthy. For 28 mothers (56%) this was their first child, while the rest had two to five children. The average infant age (same as lactation time) was 2 months; 75.5% of the infants weight less than 3300 g. A lower infant body weight at birth was associated with a higher AFM1 content in milk (< 2500 g for a 3.9 pg ml−1 AFM1 mean versus > 3500 g for a 2.2 pg ml−1 AFM1 mean).
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Table 1.
Aflatoxin M1 content in milk of nursing mothers in Colombia (total and grouped by selected socio-economic characteristics).
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Median AFM1 Minimum– (pg ml–1) maximum (pg ml–1)
η
% Positive
Mean AFM1 (pg ml–1)
Total
50
90
5.2
3.2
0.9–18.5
Age (years)
15–18 19–22 23–26 27–30 31–34 35–38 39–42
3 16 10 8 7 4 2
4 32 18 12 12 8 4
3.5 5.7 4.8 8.2 2.8 4.8 3.1
3.5 3.1 3.3 6.1 2.7 2.6 3.1
n.d.–4.6 1.1–18.5 n.d.–15.4 n.d.–18.2 n.d.–4.2 1.2–6.5 3.0–3.3
Socioeconomic class
1 2 3 4
12 20 17 1
20 38 30 2
8.7 3.7 4.6 6.2
4.1 2.6 3.1 6.2
n.d.–18.5 n.d.–10.4 n.d.–15.4 6.2–6.2
Area
Rural Urban
8 42
14 76
6.8 4.9
3.3 3.0
n.d.–15.4 n.d.–18.5
Number of children
1 2 3 4 5
28 13 5 3 1
52 24 8 4 2
4.5 4.7 9.8 7.8 3.1
3.5 2.9 9.3 7.8 3.1
0.9–17.3 n.d.–18.5 n.d.–18.2 n.d.–6.5 3.1–3.1
Infant body weight at birth (g)
3500
6 18 19 6
11 33 35 11
3.9 5.4 5.1 2.2
4.0 2.8 3.1 2.7
n.d.–6.0 n.d.–18.5 1.7–18.2 n.d.–3.4
Education
No school Primary Unfinished secondary education Preparatory Secondary technical Less than university University Graduate
1 4 6 12 12 6 7 2
2 8 10 20 22 12 12 4
8.0 5.3 10.9 5.3 3.5 5.0 2.9 5.2
8.0 2.9 15.4 3.2 2.9 4.7 2.4 5.2
8.0–8.0 2.6–6.5 n.d.–18.2 n.d.–18.5 0.9–6.0 2.0–10.4 n.d.–3.1 4.2–6.2
Mother
Note: η = Number of samples analysed; % positive = percentage of positive samples (mean and median values were calculated for the positive samples only); minimum-maximum = minimum and maximum values; n.d. = not detected (detection limit = 0.62 pg ml−1).
Grouped socio-demographic characteristics of the participants as compared with the mean concentration of AFM1 levels found are presented in Table 1.
Aflatoxin M1 levels and food consumption AFM1 was detected in 90% of the milk samples with a mean of 5.2 pg ml–1 and a median of 3.2 pg ml–1. The contamination level of AFM1 in milk ranged from 0.9 to 18.5 pg ml–1. No detectable levels of AFM2 were found in any sample. Figure 2 shows the chromatogram of a positive AFM1 sample; Figure 3 shows the frequency distribution of the contamination levels found. Aflatoxin levels did not correspond to a normal (Gaussian) distribution. The 25th, 50th
and 75th percentiles of AFM1 were 2.1, 3.2 and 6.1 pg ml–1, respectively. Analysis of the food questionnaire revealed that rice was the most commonly consumed food, with 94% of the participants reporting the intake of at least one serving and 54% the intake of three servings in the last 72 h. Rice was followed by bread with 76% of the mothers consuming one serving and 42% three servings; pasta followed with 62% taking at least one serving. The food frequency intake of mothers with levels of AFM1 above the 75th percentile (5.0 pg ml–1) showed the intake of corn arepa (corn cakes made from pre-cooked corn flour) and peanuts by 41.6% and 33.3% of the participants, respectively (data not shown). Higher mean AFM1 levels were seen in mothers from low SES (1 and 2) and those who ate at least three
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Figure 2. HPLC chromatogram: (dark line) breast milk sample positive for aflatoxin M1 (18.5 pg ml–1); and (grey line) aflatoxin M2 and M1 standards, each equivalent to 200 pg ml–1 in the sample.
35 30 25 Frequency
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Food Additives & Contaminants: Part A
20 15 10 5 0
Figure 3. milk.
‹ LOQ
LOQ – 5 5 – 10 10 – 15 Aflatoxin M1 concentration (pg ml–1)
15 – 20
Distribution of aflatoxin M1 concentration in breast
servings of corn arepa and at least three servings of rice and pasta in the last 72 h (data not shown). The same was observed in samples of mothers from rural areas. However, neither univariate nor bivariate analysis showed a significant correlation between the amount of AFM1 in milk and the ingestion of any of the foods.
Discussion Contamination of cereals intended for human and animal consumption is a serious food safety issue worldwide. In Colombia the presence of aflatoxigenic Aspergillus spp. as well as aflatoxins in foods have been reported in previous studies (Diaz et al. 2001, 2009). As AFB1 and AFB2 can be eliminated as AFM1 and AFM2 in lactating humans and other
mammals, it was important to determine the extent and amount of this transfer in lactating mothers. The present study demonstrated a high prevalence of AFM1 in human milk in Colombia, and confirms frequent exposure to AFB1 in the population. However, no levels of AFM2 were detected in any sample. This finding is not surprising since AFB2 levels are usually much lower than those of AFB1 and previous studies have not been able to report AFM2 in human milk (Creppy 2002). The prevalence of AFM1 detected in breast milk (90%) was close to the 92% reported in the United Arab Emirates (UAE) (Abdulrazzaq et al. 2003). In contrast, the prevalence was much higher than the 24.6% reported in Turkey (Atasever et al. 2014), the 35.5% found in Egypt (Polychronaki et al. 2006), and the 22% reported in Iran (Mahdavi et al. 2010). The higher prevalence of AFM1 may be due to tropical weather conditions found in Colombia compared with other countries. It has been reported that fungal action for producing aflatoxins is high in hot and humid environments (MarroquínCardona et al. 2014). Whatever the reason, the high prevalence of AFM1 found in human milk in the present study shows the need to assess further the extent of exposure to aflatoxins in Colombia and the impact that this has on public health. The mean AFM1 levels found in the present study (5.2 pg ml−1) were lower than those reported in other surveys. For instance, Polychronaki et al. (2006) found a median AFM1 content of 13.5 pg ml−1 in breast milk in a selected group of Egyptian mothers, whereas Mahdavi et al. (2010) reported mean AFM1 levels of 7.0 pg ml−1 (ranging from 5.1 to 8.1 pg ml−1) in Iranian women milk samples. Differences in the amount of AFM1 detected in milk samples are obviously due to differences in AFB1 content in the food ingested by the different human populations.
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The carryover of dietary AFB1 to AFM1 in milk ranges from 2.0% to 6.2% in dairy cows (FinkGremmels 2008). Assuming a similar carryover in lactating women, the mean AFM1 levels found in the present study (5.2 pg ml−1) would correspond to dietary levels ranging from 0.08 to 0.26 µg kg−1 and the highest concentration found (18.5 pg ml−1) would correspond to dietary levels ranging from 0.3 to 0.9 µg kg−1. These levels are lower than those reported in several Colombian foods in which AFB1 was found at concentrations from 1.0 to 103.3 µg kg−1 (Diaz et al. 2001). Either the AFB1 exposure of the mothers sampled in the present study was lower than the levels reported by Diaz et al. (2001) or the carryover of AFB1 into maternal milk is lower in women than in cows. This issue needs to be further investigated. Even though neither univariate nor bivariate analysis showed significant correlations between the content of AFM1 and the different socioeconomic factors studied, a higher concentration of AFM1 was found in samples of mothers from rural areas. These results are in agreement with those found in Tabriz (Iran) where a prevalence of 22% of AFM1 contamination was found in breast milk samples from lactating women from rural areas while no levels of AFM1 were detected in breast milk of mothers from urban areas (Mahdavi et al. 2010). This might be explained by the fact that populations in rural areas of developing countries generally depend on locally produced foods and can face issues related to food security and food quality. On the other hand, the lack of positive samples in mothers from urban areas in the Iranian study can be related to the detection limit of their analytical technique (5 pg ml−1), which was eight times higher compared with that of the present study (0.62 pg ml−1). It was interesting to observe a tendency to higher infant body weight at birth associated with lower AFM1 in milk. This finding substantiates previous studies demonstrating growth retardation in children exposed to AFM1 from the maternal milk and foetal growth retardation in experimental animals exposed prenatally to AFB1 (Mahdavi et al. 2010). In the present study, no significant correlations were found between AFM1 presence in breast milk and the consumption of any specific food. This might be due to high levels of food contamination rather than high frequency of contaminated food consumption or can also be due to inaccuracies in the reported foods ingested by the mothers. Nevertheless, it draws attention to the higher levels of AFM1 found in participants who reported frequent consumption (at least three servings) of corn arepa or peanuts. In Colombia these foods can be made and commercialised informally, without proper quality standards during production, packaging and storage. This lack of oversight and regulation would increase the risk of aflatoxin contamination. Even though the levels of AFM1 recorded in this study were lower than those of other studies, it is important to
note that there is no threshold for the carcinogenic effects of aflatoxins. Existing research demonstrates that maternal exposure to environmental chemicals during the perinatal period alters the expression and function of metabolic and transport proteins in the progeny later in life (JiménezChillarón et al. 2012). Additionally, early exposure to xenobiotics potentially affects the ability of the liver to metabolise and excrete chemicals because xenobiotic metabolism during the perinatal and postnatal periods is in maturation (Peng et al. 2013). Given that the foetus and infants are particularly sensitive to the adverse effects of aflatoxins, it is important to investigate this exposure further and take measures to decrease it. The results of the present study – the first conducted in Colombia – indicate a high prevalence of AFB1 exposure in mothers that results in a high prevalence of AFM1 in milk. The exposure to AFB1 can affect the mother herself, the developing foetus and, later on, the lactating infant. Given the fact that both AFB1 and AFM1 are considered human carcinogens by the IARC (Group 1), more control is needed by the Colombian authorities to prevent foods from containing AFB1 levels above the maximum allowed in grains (10 µg kg−1 of sum of aflatoxins) that find their way into the human food chain. Further research is needed in order to determine the presence of other mycotoxins in foods and biological fluids as well as to devise protection strategies in a country where mycotoxin monitoring is not common because regulation on this matter has only been recently released (October 2013).
Acknowledgements The authors thank Dr Herman Boermans for constructive comments made on the manuscript.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was partially funded by the College of Medicine, National University of Colombia [grant number 202010018822].
ORCID G.J. Diaz http://orcid.org/0000-0002-9858-0845 http://orcid.org/0000-0002-5717-8585 M.P. Sánchez
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Khlangwiset P, Shephard GS, Wu F. 2011. Aflatoxins and growth impairment: A review. Crit Rev Toxicol. 41:740–755. Lovelady CA, Dewey KG, Picciano MF, Dermer A. 2002. Guidelines for collection of human milk samples for monitoring and research of environmental chemicals. J Toxicol Environ Health A. 65:1881–1891. Mahdavi R, Nikniaz L, Arefhosseini SR, Vahed Jabbari M. 2010. Determination of aflatoxin M1 in breast milk samples in Tabriz-Iran. Matern Child Health J. 14:141–145. Marroquín-Cardona AG, Johnson NM, Phillips TD, Hayes AW. 2014. Mycotoxins in a changing global environment – A review. Food Chem Toxicol. 69:220–230. Mehrzad J, Devriendt B, Baert K, Cox E. 2014. Aflatoxin B1 interferes with the antigen-presenting capacity of porcine dendritic cells. Toxicol In Vitro. 28:531–537. Navas MC, Suarez I, Carreño A, Uribe D, Rios WA, Cortesmancera F, Martel G, Vieco B, Lozano D, Jimenez C, et al. 2011. Hepatitis B and hepatitis C infection biomarkers and TP53 mutations in hepatocellular carcinomas from Colombia. Hepat Res Trea. Article ID 582945:10 p. Needham LL, Wang RY. 2002. Analytic considerations for measuring environmental chemicals in breast milk. Environ Health Perspect. 110:A317–A324. Peng l, Cui JY, Yoo B, Gunewardena SS, Lu H, Klaassen CD, Zhong XB. 2013. RNA-sequencing quantification of hepatic ontogeny of phase-I enzymes in mice. Drug Metab Dispos. 41:2175–2186. Polychronaki N, Turner PC, Mykkänen H, Gong Y, Amra H, Abdel-Wahhab M, El-Nezami H. 2006. Determinants of aflatoxin M1 in breast milk in a selected group of Egyptian mothers. Food Addit Contam. 23:700–708. Shuaib FM, Ehiri J, Abdullahi A, Williams JH, Jolly PE. 2010. Reproductive health effects of aflatoxins: A review of the literature. Reprod Toxicol. 29:262–270. Turner PC, Collinson AC, Cheung YB, Gong Y, Hall AJ, Prentice AM, Wild CP. 2007. Aflatoxin exposure in utero causes growth faltering in Gambian infants. Int J Epidemiol. 36:1119–1125. Wild CP, Gong YY. 2010. Mycotoxins and human disease: a largely ignored global health issue. Carcinogenesis. 31:71–82. Wild CP, Turner PC. 2002. The toxicology of aflatoxins as a basis for public health decisions. Mutagenesis. 17:471–481. [WHO] World Health Organization. 2008. The global burden of disease: 2004 update. Geneva: World Health Organization; p. 29–30. Yates IE. 1985. Cytotoxicity and mutagenicity of aflatoxins B1, B2, G1 and G2 in Photobacterium phosphoreum. J Microbiol Meth. 3:181–186.
Food Additives & Contaminants: Part A Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfac20
Determination of aflatoxin M1 in breast milk as a biomarker of maternal and infant exposure in Colombia a
b
Gonzalo J. Diaz & Marlib Paloma Sánchez a
Laboratorio de Toxicología, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Bogotá D.C., Colombia b
Departamento de Toxicología, Facultad de Medicina, Universidad Nacional de Colombia, Bogotá D.C., Colombia Accepted author version posted online: 11 May 2015.Published online: 03 Jun 2015.
Click for updates To cite this article: Gonzalo J. Diaz & Marlib Paloma Sánchez (2015): Determination of aflatoxin M1 in breast milk as a biomarker of maternal and infant exposure in Colombia, Food Additives & Contaminants: Part A, DOI: 10.1080/19440049.2015.1049563 To link to this article: http://dx.doi.org/10.1080/19440049.2015.1049563
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Food Additives & Contaminants: Part A, 2015 http://dx.doi.org/10.1080/19440049.2015.1049563
Determination of aflatoxin M1 in breast milk as a biomarker of maternal and infant exposure in Colombia Gonzalo J. Diaz
a
* and Marlib Paloma Sánchez
b
a
Laboratorio de Toxicología, Facultad de Medicina Veterinaria y de Zootecnia, Universidad Nacional de Colombia, Bogotá D.C., Colombia; bDepartamento de Toxicología, Facultad de Medicina, Universidad Nacional de Colombia, Bogotá D.C., Colombia
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(Received 26 February 2015; accepted 5 May 2015) Chronic exposure to aflatoxins, and especially to aflatoxin B1 (AFB1), causes hepatocellular carcinoma with prevalence 16–32 times higher in developing compared with developed countries. Aflatoxin M1 (AFM1) is a monohydroxylated metabolite from AFB1 that is secreted in milk and which can be used as a biomarker of AFB1 exposure. This study aimed to determine AFM1 levels in human breast milk using immunoaffinity column clean-up with HPLC and fluorescence detection. Breast milk samples were obtained from 50 nursing mothers. Volunteers filled in a questionnaire giving their consent to analyse their samples as well as details of their socioeconomic, demographic and clinical data. The possible dietary sources of aflatoxins were assessed using a food frequency questionnaire. A total of 90% of the samples tested positive for AFM1, with a mean of 5.2 ng l−1 and a range of 0.9–18.5 ng l−1. The study demonstrated a high frequency of exposure of mothers and neonates to AFB1 and AFM1 in Colombia, and it points out the need to regulate and monitor continuously the presence of aflatoxins in human foods. Further research is needed in order to determine the presence of other mycotoxins in foods and in human samples as well as to devise protection strategies in a country where mycotoxins in human foods are commonly found. Keywords: aflatoxins; aflatoxin M1; exposure; breast milk; infant
Introduction Aflatoxins are a group of approximately 20 related fungal metabolites, although only four of them are actually produced by fungi, namely aflatoxins B1 (AFB1), B2 (AFB2), G1 and G2 (Da Rocha et al. 2014). Aflatoxins are produced primarily by the fungus Aspergillus flavus (produces only B aflatoxins) and A. parasiticus (produces both B and G aflatoxins) (Yates 1985). Aflatoxins are ubiquitous contaminants of the human food supply and their production is dependent in part on climatic conditions, being more prevalent in areas of the world with hot humid climates such as the tropical regions (Wild & Gong 2010). The major hosts of Aspergillus spp. among crop plants are corn, peanuts and cereal grains, in which invasion occurs primarily during storage, resulting in potentially high levels of aflatoxins (IARC 2012). Human exposure by dietary intake is especially important in populations that rely heavily on these staple foods (Wild & Gong 2010). Prevention of aflatoxin formation might be achieved by the implementation of good agricultural, manufacturing and storage practices (Codex Alimentarius 2005). Unfortunately, exposure to aflatoxins is almost unavoidable, particularly in developing countries such as Colombia, where access to quality products is limited, and policies needed to prevent food contamination are absent (Marroquín-Cardona et al. 2014). *Corresponding author. Email: [email protected] © 2015 Taylor & Francis
Naturally occurring aflatoxins negatively affect human health and AFB1 is the most potent chemical liver carcinogen known in humans (Wild & Turner 2002). The adverse health effects associated with acute exposure to AFB1 are numerous including vomiting, abdominal pain, pulmonary oedema, fatty liver, and necrosis and even death (Kensler et al. 2011). Chronic exposure causes human hepatocellular carcinoma (HCC), which is the third leading cause of cancer deaths worldwide, with prevalence 16–32 times higher in developing countries than in developed countries (WHO 2008). Further, specific epigenetic processes may be affected by aflatoxins, especially during a prolonged period of exposure, promoting tumorigenesis, angiogenesis, invasion and metastasis to other organs in cases of hepatocellular carcinoma (Bbosa et al. 2013). In a study conducted in Colombia, 10.5% of 202 HCC cases had the characteristic aflatoxin-induced mutation of codon 249 of the p53 tumour suppressor gene (Navas et al. 2011). Several studies describe a variety of other adverse health effects associated with aflatoxins, such as impaired growth in children (Khlangwiset et al. 2011), hepatomegaly (Gong et al. 2012), suppression of immune function, especially cell-mediated immune responses (Mehrzad et al. 2014), and reproductive health effects (Shuaib et al. 2010). Furthermore, aflatoxins can cross the
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G.J. Diaz and M.P. Sánchez
placental barrier and be bioactivated in utero (Turner et al. 2007). Ingestion of contaminated food during pregnancy and lactation may expose the foetus or the newborn to the deleterious effects of aflatoxins during these critical development and growth periods (Abdulrazzaq et al. 2003). At least three monohydroxylated metabolites are produced by CYP450 from AFB1: aflatoxin M1 (AFM1), Q1 (AFQ1) and B2a (AFB2a) (Diaz & Murcia 2011). About 0.3–6.2% of the ingested AFB1 is eliminated as AFM1 in milk (Creppy 2002). Ingested AFB2 can also be monohydroxylated in the liver and eliminated in milk as aflatoxin M2 (AFM2) (Creppy 2002). Breast milk, therefore, constitutes a unique matrix for biomonitoring exposure in human populations (Needham & Wang 2002). Measurement of aflatoxins in breast milk is important to determine the true contribution of these mycotoxins to public health, especially to the health of the newborn. Moreover, breast milk is a convenient sample for biomonitoring programmes because relatively large volumes (50–100 ml) can be collected non-invasively. This makes it an ideal matrix easily sampled in a large population with easily identified subgroups. The aim of the present study was to determine for the first time AFM1 and AFM2 levels in breast milk from a select group of Colombian nursing mothers.
Materials and methods Study population and data collection Breast milk samples were obtained from 50 nursery mothers that voluntarily agreed to participate in the study and were collected from May to September 2013. The mothers were recruited at The Mercy Hospital Foundation (HOMI), a high-complexity children’s hospital where paediatric patients are referred from all over the country. Volunteers were questioned by a physician at the time of sample donation: they were asked details about socioeconomic, demographic and clinical data that included level of education, place of residence, age, gynaecobstetric history, mother’s health status and current medications, stage and details of lactation, baby’s weight at delivery and baby’s medical history. The potential intake of dietary sources of aflatoxins was also assessed using a structured food frequency questionnaire to record the mother’s intake of food items over the last 72 h before sample collection. The groups of food included grain products (corn, rice, wheat and derivatives of these), legumes (beans, lentils), dried fruits, and peanuts. The data on food intake were expressed as servings per day. A previously described protocol to collect the breast milk samples was followed (Lovelady et al. 2002).
Chemicals and consumables AFM1 and AFM2 standards of 2000 ng ml−1 in acetonitrile were purchased from Micotox Ltd (Bogota, Colombia). Immunoaffinity columns with antibodies against aflatoxins M1 and M2 (NeoColumn Aflatoxin wide bore) were purchased from Neogen Europe Ltd (Ayr, UK). Acetonitrile and methanol were HPLC grade from Merck KGaA (Darmstadt, Germany). Water was from a Millipore Milli-Q system (EMD Millipore, Billerica, MA, USA).
Determination of AFM1 The method used in the present study was based on an international standard developed for cow’s milk (ISO 14501, 1998) which was validated in-house for human milk samples. Breast milk was collected in a sterile plastic container by self-expression. Samples were kept at 4°C during transportation and processing within a day, otherwise they were kept at −72°C. Milk samples were analysed for the presence of AFM1 and AFM2 using immunoaffinity column for clean-up and HPLC with fluorescence detection for determination and quantitation, as follows. Milk samples (100 ml) were warmed to 37°C and centrifuged at 4000 rpm for 15 min. After centrifugation, the upper fat layer was discarded. The skimmed milk was filtered through qualitative filter paper and an aliquot of 50 ml was passed through the immunoaffinity clean-up column. After the sample had passed through, the column was washed with distilled water (2 volumes of 10 ml) to remove extraneous non-specific material. The column was then dried by gentle blowing of air and the AFM1 and AFM2 bound to the antibodies were released by elution with 1.25 ml acetonitrile-methanol (2:3, v/v) followed by 1.25 ml of water into a 4-ml silanised amber vial. Finally, 1 ml of the mixed eluate was taken to be transferred into a 1.5 ml amber vial followed by determination of AFM1 and AFM2 content by HPLC.
Chromatography HPLC was performed on a Shimadzu Prominence HPLC system consisting of a DGU-20A3 degassing unit, an LC-20AB dual pump, a SIL-20A HT autosampler, an RF-20A XS fluorescence detector, a CTO-20A column oven and a CBM-20A communications bus module, all controlled by Lab Solutions software (Shimadzu Scientific, Columbia, MD, USA). The mobile phase consisted of an isocratic mix of water–acetonitrile–methanol (50:30:20, v/v/v) at a flow rate of 0.6 ml min−1. The column used was a 250 × 4.6 mm and 5 µm particle size Phenomenex Prodigy ODS3 (Phenomenex, Inc. Torrance, CA, USA) at 30°C. The fluorescence detector was set at excitation and emission wavelengths of 346 and 430 nm, respectively. Metabolites were identified by comparison of retention times and co-injection of standards.
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In-house method validation An in-house validation of the ISO method for cow’s milk (ISO 14501, 1998) was carried out in order to verify the fitness of the method for human milk. The validation was performed using breast milk from volunteers that contained no detectable levels of aflatoxins M1 or M2. Linearity was determined by preparing calibration curves for AFM1 and AFM2 at five different levels corresponding to sample concentrations of 12.5, 25, 50, 100 and 200 pg ml–1. Three independent replicates were prepared for each level. Selectivity was evaluated by analysing six blank samples. The accuracy and repeatability of the method were assessed by spiking blank samples with three different levels of aflatoxins (25, 50 and 100 pg ml–1) in triplicate. LOD and LOQ were initially estimated using two different methods: by the calibration curves and by the detector signal-to-noise ratio. LOD was defined as the lowest concentration of aflatoxins that could be reliably detected (> 3:1 signal-to-noise ratio). Based on this initial qualitative approach, LOD and LOQ were determined using the EPA method (EPA 40 CRF Part 136, Appendix B), which is based in the analysis of eight independently spiked samples fortified at the lowest quantitation limits estimated previously (EPA 1984).
Statistical analysis Comparison between AFM1 content in milk and each of the socioeconomic and food intake parameters was evaluated by Pearson R Correlation Tests. R software programme version 3.0.2 (R Core Team 2013) was used for analysis of data. A value of p < 0.05 was considered to be statistically significant.
Ethical considerations The study was approved by the Ethics Committee of the School of Medicine of the National University of Colombia (document CE-011-12) and by The Mercy Hospital Foundation (document GCCI-008-13). All subjects were made aware of the content of the study, and if they agreed to participate, a written informed consent was obtained.
Results In-house validation The calibration curves had linear equations of y = 2435x + 169 for AFM1 and y = 2436x + 40 for AFM2. In both cases the correlation coefficient was 1.0. The selectivity tests showed no interferences eluting at the retention times of the analytes. Average recoveries for AFM1 and AFM2 were 92.3% and 90.2%, respectively. The relative standard deviations for repeatability (RSDr)
Figure 1. HPLC chromatogram: (1) aflatoxin M1 standard equivalent to 500 pg ml–1; (2) milk sample spiked with 200 pg ml–1 aflatoxin M1; and (3) type 1 water sample spiked with 200 pg ml–1 aflatoxin M1. In the spiked samples, the AFM1 signal overlaps.
of the three levels evaluated were 0.6, 2.4 and 0.85 for AFM1 and 0.6, 3.3 and 1.5 for AFM2. The estimated LOD and LOQ for AFM1 according to the EPA method were 0.6 and 1.8 pg ml–1, respectively. The same values for AFM2 were 0.4 and 1.2 pg ml–1, respectively. Figure 1 shows a typical chromatogram of an AFM1 standard, a milk sample and type 1 water spiked with 200 pg ml−1 AFM1.
Main characteristics of the study population The average age of the mothers was 25 years (range 15–41 years). Twenty-nine mothers (58%) were less than 27 years old. Most mothers (64%) belonged to a low socioeconomic status (SES, 1 and 2). The formula used to categorise the population in socioeconomic groups is country specific, where 1 is the lowest and 6 the highest. This is based on where the person resides, access to/ownership of consumer durable goods, and the existence of public services. The highest academic qualification obtained by the lactating mothers was a bachelor’s degree (14%), while 48% had secondary or technical education and 8% had no elementary education. The majority of lactating women (84%) lived in urban areas. All mothers were healthy. For 28 mothers (56%) this was their first child, while the rest had two to five children. The average infant age (same as lactation time) was 2 months; 75.5% of the infants weight less than 3300 g. A lower infant body weight at birth was associated with a higher AFM1 content in milk (< 2500 g for a 3.9 pg ml−1 AFM1 mean versus > 3500 g for a 2.2 pg ml−1 AFM1 mean).
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Table 1.
Aflatoxin M1 content in milk of nursing mothers in Colombia (total and grouped by selected socio-economic characteristics).
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Median AFM1 Minimum– (pg ml–1) maximum (pg ml–1)
η
% Positive
Mean AFM1 (pg ml–1)
Total
50
90
5.2
3.2
0.9–18.5
Age (years)
15–18 19–22 23–26 27–30 31–34 35–38 39–42
3 16 10 8 7 4 2
4 32 18 12 12 8 4
3.5 5.7 4.8 8.2 2.8 4.8 3.1
3.5 3.1 3.3 6.1 2.7 2.6 3.1
n.d.–4.6 1.1–18.5 n.d.–15.4 n.d.–18.2 n.d.–4.2 1.2–6.5 3.0–3.3
Socioeconomic class
1 2 3 4
12 20 17 1
20 38 30 2
8.7 3.7 4.6 6.2
4.1 2.6 3.1 6.2
n.d.–18.5 n.d.–10.4 n.d.–15.4 6.2–6.2
Area
Rural Urban
8 42
14 76
6.8 4.9
3.3 3.0
n.d.–15.4 n.d.–18.5
Number of children
1 2 3 4 5
28 13 5 3 1
52 24 8 4 2
4.5 4.7 9.8 7.8 3.1
3.5 2.9 9.3 7.8 3.1
0.9–17.3 n.d.–18.5 n.d.–18.2 n.d.–6.5 3.1–3.1
Infant body weight at birth (g)
3500
6 18 19 6
11 33 35 11
3.9 5.4 5.1 2.2
4.0 2.8 3.1 2.7
n.d.–6.0 n.d.–18.5 1.7–18.2 n.d.–3.4
Education
No school Primary Unfinished secondary education Preparatory Secondary technical Less than university University Graduate
1 4 6 12 12 6 7 2
2 8 10 20 22 12 12 4
8.0 5.3 10.9 5.3 3.5 5.0 2.9 5.2
8.0 2.9 15.4 3.2 2.9 4.7 2.4 5.2
8.0–8.0 2.6–6.5 n.d.–18.2 n.d.–18.5 0.9–6.0 2.0–10.4 n.d.–3.1 4.2–6.2
Mother
Note: η = Number of samples analysed; % positive = percentage of positive samples (mean and median values were calculated for the positive samples only); minimum-maximum = minimum and maximum values; n.d. = not detected (detection limit = 0.62 pg ml−1).
Grouped socio-demographic characteristics of the participants as compared with the mean concentration of AFM1 levels found are presented in Table 1.
Aflatoxin M1 levels and food consumption AFM1 was detected in 90% of the milk samples with a mean of 5.2 pg ml–1 and a median of 3.2 pg ml–1. The contamination level of AFM1 in milk ranged from 0.9 to 18.5 pg ml–1. No detectable levels of AFM2 were found in any sample. Figure 2 shows the chromatogram of a positive AFM1 sample; Figure 3 shows the frequency distribution of the contamination levels found. Aflatoxin levels did not correspond to a normal (Gaussian) distribution. The 25th, 50th
and 75th percentiles of AFM1 were 2.1, 3.2 and 6.1 pg ml–1, respectively. Analysis of the food questionnaire revealed that rice was the most commonly consumed food, with 94% of the participants reporting the intake of at least one serving and 54% the intake of three servings in the last 72 h. Rice was followed by bread with 76% of the mothers consuming one serving and 42% three servings; pasta followed with 62% taking at least one serving. The food frequency intake of mothers with levels of AFM1 above the 75th percentile (5.0 pg ml–1) showed the intake of corn arepa (corn cakes made from pre-cooked corn flour) and peanuts by 41.6% and 33.3% of the participants, respectively (data not shown). Higher mean AFM1 levels were seen in mothers from low SES (1 and 2) and those who ate at least three
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Figure 2. HPLC chromatogram: (dark line) breast milk sample positive for aflatoxin M1 (18.5 pg ml–1); and (grey line) aflatoxin M2 and M1 standards, each equivalent to 200 pg ml–1 in the sample.
35 30 25 Frequency
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Food Additives & Contaminants: Part A
20 15 10 5 0
Figure 3. milk.
‹ LOQ
LOQ – 5 5 – 10 10 – 15 Aflatoxin M1 concentration (pg ml–1)
15 – 20
Distribution of aflatoxin M1 concentration in breast
servings of corn arepa and at least three servings of rice and pasta in the last 72 h (data not shown). The same was observed in samples of mothers from rural areas. However, neither univariate nor bivariate analysis showed a significant correlation between the amount of AFM1 in milk and the ingestion of any of the foods.
Discussion Contamination of cereals intended for human and animal consumption is a serious food safety issue worldwide. In Colombia the presence of aflatoxigenic Aspergillus spp. as well as aflatoxins in foods have been reported in previous studies (Diaz et al. 2001, 2009). As AFB1 and AFB2 can be eliminated as AFM1 and AFM2 in lactating humans and other
mammals, it was important to determine the extent and amount of this transfer in lactating mothers. The present study demonstrated a high prevalence of AFM1 in human milk in Colombia, and confirms frequent exposure to AFB1 in the population. However, no levels of AFM2 were detected in any sample. This finding is not surprising since AFB2 levels are usually much lower than those of AFB1 and previous studies have not been able to report AFM2 in human milk (Creppy 2002). The prevalence of AFM1 detected in breast milk (90%) was close to the 92% reported in the United Arab Emirates (UAE) (Abdulrazzaq et al. 2003). In contrast, the prevalence was much higher than the 24.6% reported in Turkey (Atasever et al. 2014), the 35.5% found in Egypt (Polychronaki et al. 2006), and the 22% reported in Iran (Mahdavi et al. 2010). The higher prevalence of AFM1 may be due to tropical weather conditions found in Colombia compared with other countries. It has been reported that fungal action for producing aflatoxins is high in hot and humid environments (MarroquínCardona et al. 2014). Whatever the reason, the high prevalence of AFM1 found in human milk in the present study shows the need to assess further the extent of exposure to aflatoxins in Colombia and the impact that this has on public health. The mean AFM1 levels found in the present study (5.2 pg ml−1) were lower than those reported in other surveys. For instance, Polychronaki et al. (2006) found a median AFM1 content of 13.5 pg ml−1 in breast milk in a selected group of Egyptian mothers, whereas Mahdavi et al. (2010) reported mean AFM1 levels of 7.0 pg ml−1 (ranging from 5.1 to 8.1 pg ml−1) in Iranian women milk samples. Differences in the amount of AFM1 detected in milk samples are obviously due to differences in AFB1 content in the food ingested by the different human populations.
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G.J. Diaz and M.P. Sánchez
The carryover of dietary AFB1 to AFM1 in milk ranges from 2.0% to 6.2% in dairy cows (FinkGremmels 2008). Assuming a similar carryover in lactating women, the mean AFM1 levels found in the present study (5.2 pg ml−1) would correspond to dietary levels ranging from 0.08 to 0.26 µg kg−1 and the highest concentration found (18.5 pg ml−1) would correspond to dietary levels ranging from 0.3 to 0.9 µg kg−1. These levels are lower than those reported in several Colombian foods in which AFB1 was found at concentrations from 1.0 to 103.3 µg kg−1 (Diaz et al. 2001). Either the AFB1 exposure of the mothers sampled in the present study was lower than the levels reported by Diaz et al. (2001) or the carryover of AFB1 into maternal milk is lower in women than in cows. This issue needs to be further investigated. Even though neither univariate nor bivariate analysis showed significant correlations between the content of AFM1 and the different socioeconomic factors studied, a higher concentration of AFM1 was found in samples of mothers from rural areas. These results are in agreement with those found in Tabriz (Iran) where a prevalence of 22% of AFM1 contamination was found in breast milk samples from lactating women from rural areas while no levels of AFM1 were detected in breast milk of mothers from urban areas (Mahdavi et al. 2010). This might be explained by the fact that populations in rural areas of developing countries generally depend on locally produced foods and can face issues related to food security and food quality. On the other hand, the lack of positive samples in mothers from urban areas in the Iranian study can be related to the detection limit of their analytical technique (5 pg ml−1), which was eight times higher compared with that of the present study (0.62 pg ml−1). It was interesting to observe a tendency to higher infant body weight at birth associated with lower AFM1 in milk. This finding substantiates previous studies demonstrating growth retardation in children exposed to AFM1 from the maternal milk and foetal growth retardation in experimental animals exposed prenatally to AFB1 (Mahdavi et al. 2010). In the present study, no significant correlations were found between AFM1 presence in breast milk and the consumption of any specific food. This might be due to high levels of food contamination rather than high frequency of contaminated food consumption or can also be due to inaccuracies in the reported foods ingested by the mothers. Nevertheless, it draws attention to the higher levels of AFM1 found in participants who reported frequent consumption (at least three servings) of corn arepa or peanuts. In Colombia these foods can be made and commercialised informally, without proper quality standards during production, packaging and storage. This lack of oversight and regulation would increase the risk of aflatoxin contamination. Even though the levels of AFM1 recorded in this study were lower than those of other studies, it is important to
note that there is no threshold for the carcinogenic effects of aflatoxins. Existing research demonstrates that maternal exposure to environmental chemicals during the perinatal period alters the expression and function of metabolic and transport proteins in the progeny later in life (JiménezChillarón et al. 2012). Additionally, early exposure to xenobiotics potentially affects the ability of the liver to metabolise and excrete chemicals because xenobiotic metabolism during the perinatal and postnatal periods is in maturation (Peng et al. 2013). Given that the foetus and infants are particularly sensitive to the adverse effects of aflatoxins, it is important to investigate this exposure further and take measures to decrease it. The results of the present study – the first conducted in Colombia – indicate a high prevalence of AFB1 exposure in mothers that results in a high prevalence of AFM1 in milk. The exposure to AFB1 can affect the mother herself, the developing foetus and, later on, the lactating infant. Given the fact that both AFB1 and AFM1 are considered human carcinogens by the IARC (Group 1), more control is needed by the Colombian authorities to prevent foods from containing AFB1 levels above the maximum allowed in grains (10 µg kg−1 of sum of aflatoxins) that find their way into the human food chain. Further research is needed in order to determine the presence of other mycotoxins in foods and biological fluids as well as to devise protection strategies in a country where mycotoxin monitoring is not common because regulation on this matter has only been recently released (October 2013).
Acknowledgements The authors thank Dr Herman Boermans for constructive comments made on the manuscript.
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was partially funded by the College of Medicine, National University of Colombia [grant number 202010018822].
ORCID G.J. Diaz http://orcid.org/0000-0002-9858-0845 http://orcid.org/0000-0002-5717-8585 M.P. Sánchez
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