Milk is the major nutrient for the rapidly growing infants, who are theoretically at their most vulnerable stage of development, as far as the induction of carcinogenesis and susceptibility to other toxic effects are concerned . Despite all the importance and benefits of breast milk, it was observed that a wide range of contaminants can be secreted in breast milk [2,3]. This raises important health issues, as the consequences of such exposures on the developing infants are poorly understood . Monitoring maternal and early infant exposure and identifying the exposure patterns are thus important for a number of highly toxic contaminants that are potentially transferred to breast milk [4,5].
One of these toxins is aflatoxins, produced by Aspergillus fungi. Aflatoxins are highly toxic, mutagenic, teratogenic, and carcinogenic compounds . Aflatoxin-producing members of Aspergillus are common and widespread in nature. They can contaminate many foodstuffs at various stages. Poor harvesting practices, improper drying, handling, packaging, storage, and transport conditions contribute to fungal growth and increase the risk of mycotoxin production. Aflatoxins have a high presence in tropical and subtropical regions where humidity and temperature conditions are optimal for toxin production. Until now, nearly 18 different types of aflatoxins have been identified; aflatoxin B1 (AFB1) is considered as one of the most toxic . Mammals who ingest AFB1-contaminated diets eliminate into milk amounts of the principal 4-hydroxylated metabolite known as ‘milk toxin’ or aflatoxin M1 (AFM1) . There is a protein called breast cancer resistance protein (BCRP, Abcg2) that mediates the transfer of aflatoxins into breast milk. This protein reduces systemic exposure to dietary carcinogens as aflatoxins by decreasing their net uptake from intestine and by increasing their hepatobiliary, intestinal, and renal elimination. BCRP is also highly expressed in lactating mammary glands in humans and cows mediating aflatoxins excretion in milk .
Milk has the greatest demonstrated potential for introducing aflatoxin residues from edible animal tissues into the human diet [10,11]. Moreover, as milk is the main dietary component for children, the presence of AFM1 in human breast milk and in commercially available milk and milk products is one of the most serious problems of food safety. To reduce the risk of exposure, many countries have established maximum levels of permissible AFM1 in fluid milk and in other milk products varying from 0.05 to 1.0 μg/l . The European Community and Codex Alimentarius prescribe that the maximum level of AFM1 in liquid milk should not exceed 0.05 μg/l .
The health risk of AFM1 on the Egyptian breast-fed infants has not been adequately evaluated. The aim of our study is to identify the degree of breast milk contamination by AFM1, together with assessment of the growth of these infants and measuring the liver enzymes of mothers and infants. We chose to measure AFM1 in breast milk as an excellent biomarker of the dietary exposure to aflatoxins by the mothers and as a predictor of the likely exposure of their breast-fed infants.
Materials and methods
This was a cross-sectional study conducted in the period between January 2008 and July 2009. This study was approved by the Cairo University Children's Hospital and was conducted in accordance with the University Bylaws for Human Research. One hundred and fifty mother–infant dyads were chosen from the lactation clinic in the Cairo University Children's Hospital. All of them attended the clinic after birth and were properly instructed and followed for the correct breastfeeding practice as recommended by the WHO. All the infants were exclusively breastfed, taking no food or drink other than breast milk. Infant weights were documented at birth and at 6 months of age. Standard deviation scores (SDS) for the infant weights were obtained using Tanner et al.  curves.
Samples were taken from the mothers and their infants at 6 months postpartum (±2 weeks) just before the introduction of any supplements.
The following laboratory investigations were performed:
- (1) AFM1 levels in breast milk were measured using enzyme-linked immunosorbent assay (ELISA).
- (2) Liver enzymes such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured by the Olympus AU800 procedure. They were measured in all the mothers and in their babies to assess the impact of aflatoxins on the liver. The normal range of ALT in adults is 10–35 U/l and in infants is 7–40 U/l. The normal range of AST in adults and in infants is 5–34 U/l. Mothers taking drugs that might affect their liver enzymes were excluded from the study. Hepatitis markers were measured for mothers having higher than normal liver enzymes to exclude positive cases.
AFM1 sample preparation
Analysis of AFM1 was performed by competitive ELISA, Helica Biosystem, International Company Catalogue Number 961AFLM01M). Breast milk samples were collected by hand expression into glass tubes; the milk samples were stored at −20°C. Samples were thawed gradually to 4°C and then centrifuged at 10°C. Aflatoxins are water-soluble; hence, the upper creamy layer was discarded and the lower phase was used for the quantitative test.
The monoclonal antibody for ELISA was obtained using an aflatoxin–bovine serum albumin conjugate as the immunogen, which was coated to a microwell. Aflatoxins–horseradish peroxidase conjugate (AF-HRP) was mixed and added to the antibody-coated microwell. The AF in the sample and the AF-HRP conjugate competed to bind with the antibody in the microwell. After the removal of the nonspecific reactants by washing the microwell, an enzyme substrate tetramethylbenzidine was added and a blue color develops. The blue color intensity is proportional to the amount of AF-HRP conjugate and is inversely proportional to the concentration of AF in the sample.
For cutoff value, we used the European Communities and Codex Alimentarius limit of 0.05 μg/l for AFM1 as maximum tolerated levels in liquid milk . Mothers having levels above 0.05 μg/l were considered to be the aflatoxin-positive group.
The Statistical Package for Social Science program (SPSS, Chicago, Illinois, USA) version 9.0 was used for analysis of data. Data were summarized as mean, standard deviation, and median. The nonparametric test (Mann–Whitney U-test) was used for the analysis of the difference in quantitative data between two groups. The χ2-test was used for the analysis of qualitative data. P value was considered significant if it was less than 0.05. Simple linear correlation (Pearson's correlation for quantitative data) was performed to detect the relation between variables.
After analyzing the breast milk samples of the 150 mothers, 98 of them (65.33%) had AFM1 levels above 0.05 μg/l. These mothers together with their infants were considered the AFM1-positive group. The remaining 52 mothers (34.67%) together with their infants were considered the AFM1-negative group. The prevalence of AFM1 positivity in breast milk samples is shown as pie chart in Fig. 1.
In AFM1-positive mothers, the AFM1 levels ranged between 0.2 and 19.0 μg/l (mean: 7.1±5.0 μg/l, median: 5.6 μg/l). In AFM1-negative mothers, the AFM1 levels ranged between 0.01and 0.05 μg/l (mean: 0.04±0.01 μg/l, median: 0.037 μg/l).
The age of AFM1-positive mothers ranged between 16 and 36 years (mean: 23.9±5.5 years) and the age of AFM1-negative mothers ranged between 18 and 32 years (mean: 24.6±3.7 years); P value=0.2. Correlation between the breast milk AFM1 levels and the age of the mothers showed nonsignificant results. In AFM1-positive mothers, r=0.1 and P value=0.3 and in AFM1-negative mothers r=−0.2 and P value=0.1.
Liver enzymes in the form of ALT and AST were assessed in the mothers and in the infants of both groups. Comparisons were performed between both groups and the results are shown in Table 1.
ALT and AST were significantly higher in AFM1-positive mothers than in AFM1-negative mothers (P value=0.0001 in both). In AFM1-positive mothers, ALT was above the normal values stated for the kits in 37 cases (37.76%) as compared with only two cases (3.8%) in AFM1-negative mothers and AST was above the normal values for the kits in 43 cases (43.88%) as compared with only four cases (7.69%) in AFM1-negative mothers.
None of the infants of AFM1-positive mothers or AFM1-negative mothers had elevated ALT or AST. Despite this, infants of the AFM1-positive mothers had statistically significant higher levels of ALT (P=0.03).
Correlations were carried out between the AFM1 levels and the liver enzymes of the mothers and the infants in both groups (Table 2). None of these were statistically significant.
The birth weights of the infants of AFM1-positive mothers ranged between 1.9 and 3.6 kg (mean: 2.8±0.4 kg) and their weights at 6 months ranged between 3.8 and 7.6 kg (mean: 5.5±0.9 kg). The birth weights of the infants of AFM1-negative mothers ranged between 2.0 and 3.9 kg (mean: 3.0±0.4 kg) and their weights at 6 months ranged between 5.0 and 7.5 kg (mean: 6.1±0.6 kg). Infant weights' SDS were documented at birth and at 6 months. The difference between both was calculated as a reflection of the rate of growth of the infants. Comparisons between infant weights' SDS at birth, at 6 months, and their difference were carried out between both groups and the results are shown in Table 3. In all of the three comparisons, the weights' SDS were lower in the infants of the AFM1-positive mothers than in the infants of AFM1-negative mothers with statistically significant results (P=0.04, 0.0001, and 0.03, respectively).
Correlation between AFM1 levels and the different weights' SDS was carried out in both groups and the results are shown in Table 4. Only infants of AFM1-positive mothers showed statistically significant negative correlations between the AFM1 levels and the weights' SDS at birth, at 6 months, and their difference(s) were statistically significant. (r=−0.3,−0.6, and −0.4 and P=0.002, 0.0001, and 0.0001, respectively).
Assessment of the AFM1 levels in the breast milk of 150 mothers living in Cairo revealed that 65.33% had AFM1-contaminated breast milk samples. This AFM1-positive group of mothers had higher liver enzymes and their infants had compromised growth when compared with the AFM1-negative group of mothers.
Our study revealed higher incidence of breast milk contamination than studies previously conducted in Egypt. In Qalyubiyah Governorate, 36% of 388 breastfeeding mothers were positive for aflatoxins , whereas in Mansoura and Zagazig Governorates, 48% of 50 breastfeeding mothers were positive for aflatoxins but without renal or hepatic dysfunction . Previous studies revealed that breast milk contamination with aflatoxins also represents a health problem in countries other than Egypt, for example, Sudan , UAE , Turkey [19,20], and Iran , but not in Brazil .
To assess the possible impact of AFM1 on hepatocytes, ALT and AST were assessed in all the mothers and their infants. Both ALT and AST levels were significantly higher in AFM1-positive mothers than in AFM1-negative mothers (P=0.0001 for both enzymes). The ALT levels of infants of AFM1-positive mothers were significantly higher than that of infants of AFM1-negative mothers (P value=0.03).
The statistically significant higher levels of ALT and AST might be due to the hepatotoxic effect of AFM1. It might also represent an alarm to a more intense future liver involvement. AFM1 can cause DNA damage, gene mutation, chromosomal anomalies, and cell transformation in mammalian cells in vitro . Hepatocellular carcinoma (HCC) is the third-leading cause of cancer deaths worldwide, with a prevalence of 16–32 times higher in developing than developed countries. Of the 550 000–600 000 new HCC cases worldwide per year, approximately 25 200–155 000 may be attributable to aflatoxin exposure . In Egypt, the burden of HCC has been increasing with a double incidence in the past 10 years. This has been attributed to several factors including increased exposure to aflatoxins . A study conducted in Mansoura University in Egypt revealed that the serum level of AFB1 was significantly higher in patients with HCC as compared with the control group (P≤0.0001) and was significantly associated with higher incidence of hepatitis C virus .
An association between aflatoxin levels and impaired growth even during the fetal life has been reported . Assessment of aflatoxins in human cord blood and sera of women after birth demonstrated the transplacental transfer and concentration of aflatoxin by the fetoplacental unit. There is a strong negative correlation between aflatoxin levels in maternal sera and birth weights of their infants [27–28]. This was evidenced in our study by the statistically significant lower birth weights' SDS in infants of AFM1-positive mothers than in infants of AFM1-negative mothers (P=0.04). These infants also had lower weights' SDS at 6 months (P=0.0001) as well as a lower rate of growth as evidenced by the difference between the 6 months weights' SDS and birth weights' SDS (P=0.03). Only infants of the AFM1-positive mothers showed statistically significant negative correlation between AFM1 levels and the three recorded weights' SDS: birth weights' SDS (r=−0.3, P=0.002), 6 months weights' SDS (r=−0.6, P=0.0001), and their difference (r=−0.4, P=0.0001).
Previous studies have shown the association between aflatoxin levels and protein energy malnutrition, especially kwashiorkor [1,29,30]. In kwashiorkor infants, aflatoxins were significantly found in liver biopsies , liver autopsies , lung autopsies , and kidney autopsies , which might have relevance to the pathogenesis of this disease. In Egypt, aflatoxins were detected in the serum and in the urine of 80% of infants having kwashiorkor and in 46.7% of infants having marasmus .
After weaning, infants and children are still at risk of exposure to aflatoxins containing food. Urinary levels of aflatoxins were found to be high in the Egyptian children . A major source of aflatoxins is the consumption of the milk of cattle eating contaminated corn . The BCRP protein plays a role in the excretion of AFM1 in cattle milk as in humans . Infants can be exposed to aflatoxins through breast milk if their mothers eat contaminated dairy products or by direct exposure in artificial milk or weaning food. The presence of AFM1 in milk is a concern as it is resistant to thermal inactivation, pasteurization, autoclaving, and other varieties of food-processing procedures . Thus, in our study, the high level of AFM1 in breast milk does not provide any superiority to formula feeds.
Many foodstuffs can be also contaminated with aflatoxins. All factors that accentuate water stress increase AFM1 accumulation . The use of plastic or synthetic bags was found to promote humidity, mold growth, and toxin production . In Egypt, citizens depend a lot on legumes, for example, beans and lentils, as a major source of cheap proteins. They are commonly stored in plastic bags, which might contribute to aflatoxin production. The Food and Drugs Administration's goal has been to minimize contamination by implementing regulations that could not realistically be used in developing countries. Small-scale industries, subsistence production, and food insecurity usually make the economics and enforcement of regulations impractical. The result is a ‘divide’ in the prevalence of aflatoxicosis exposure between people living in developed and developing countries .
Conclusion and recommendations
Aflatoxins represent a real threat in Egypt. It might be related to the compromised growth of the infants and the hepatic affection of the exposed individuals. This necessitates cooperation of ministries to combat this problem. The public should be educated about proper food storage and about the hazards of aflatoxin ingestion. In this study, we used the maximum tolerance limit of AFM1 in liquid milk (0.05 μg/l) accepted by the European Union and Codex Alimentarius Commission. We need a new maximum tolerance limit appropriate for infants who still have immature organ function and who might have compromised metabolism and excretion of aflatoxins.
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