Food Control 31 (2013) 525e529
Contents lists available at SciVerse ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Occurrence of Ochratoxin A and Aflatoxin M1 in human breast milk in Sari, Iran P. Afshar a, M. Shokrzadeh b, *, S. Kalhori c, Z. Babaee d, S.S. Saeedi Saravi e,1 a
Mycology Laboratory of the Referral Laboratory of the Mazanadaran University of Medical Sciences, Sari, Iran Department of Toxicology-Pharmacology, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Mazandaran Pharmaceutical Research Center, Sari, Iran c Mazanadaran University of Medical Sciences, Sari, Iran d Food Quality Control Laboratory, Mazandaran University of Medical Sciences, Sari, Iran e Department of Pharmacology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 September 2012 Received in revised form 1 December 2012 Accepted 8 December 2012
Mycotoxins are important risks for human health due to their widespread presence in food and environment. However, feeding of infants with safe breast milk without contamination by different pollutants including mycotoxins is essential. The aim of this study is assessing the presence and values of the mycotoxins Ochratoxin A and Aflatoxin M1 in human milk in Sari, Northern Iran, and to evaluate the potential risk for the newborn derived from this mycotoxins ingestion. The study has been carried out on 136 lactating women randomly selected in seven hospitals in Sari, Northern Iran. The breast milk samples after delivery were tested to determine Ochratoxin A and Aflatoxin M1 values. The enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography (HPLC) methods with a fluorescence detector following an extraction procedure were used for analysis of Ochratoxin A and Aflatoxin M1. In final scrutiny, of a total of 136 samples analyzed, only one was contaminated with Aflatoxin M1, at 20 ng/l, and two with Ochratoxin A, at 90 and 140 ng/l. These results pointed out the exposure of mothers and neonates to Aflatoxin M1 and Ochratoxin A and although the incidence observed was low, it is recommended for further investigations on mycotoxin contamination both in food and biological fluids as well as protection strategies. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Ochratoxin A Aflatoxin M1 Human breast milk ELISA HPLC
1. Introduction Humans are exposed to different chemicals including carcinogenic substances during their life (El-Nezami, Nicoletti, Neal, Donohue, & Ahokas, 1995; Galvano et al., 2008; Navas, Sabino, & Rodriguez-Amaya, 2005). One of them is a family of naturally occurring compounds, mycotoxins that have aroused significant public concern worldwide. The occurrence of mycotoxins in human, animal and milk products is one of the most serious problems of food hygiene since milk is important food for adults, and the unique nutrient for infant. The major concerns about human milk contamination by mycotoxins are Ochratoxin A (OTA) and aflatoxin
* Corresponding author. Tel.: þ98 9111263448. E-mail addresses:
[email protected] (P. Afshar), m_ali_shokrzadeh@ yahoo.com (M. Shokrzadeh),
[email protected] (S. Kalhori),
[email protected] (Z. Babaee),
[email protected], s-saeedi@ razi.tums.ac.ir (S.S. Saeedi Saravi). 1 Tel.: þ98 9113537724. 0956-7135/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2012.12.009
M1 (AFM1) (Galvano, Ritieni, Piva, Pietri, & Diaz, 2005). Ochratoxin A produced mainly Aspergillus ochraceus and Penicillium verrucosum and it is found in cereals, legumes, coffee, peanuts, meat transport from feed and other plant substrate (Galvano, Galofaro, & Galvano, 1996; Jrgensen, Rasmussen, & Thorup, 1996; Speijers, van Egmond, Creppy, Castegnaro, & Dirheimer, 1993), and can grow when the temperature is a low as 5 C (WHO, 2002, pp. 70). Aflatoxins are extremely potent cancerogenous substances in all animal species, classified by the IARC as “cancerogenous substances for man” (IARC, 1993, p. 245e395) and are mostly produced by strains of Aspergillus flavus and Aspergillus parasiticus, growing on corn, wheat, rice, peanuts and dried fruits. The disease caused by OTA exposure is known as ochratoxicosis, and the primary target is the kidney. Epidemiological studies show that OTA may be involved in the pathogenesis of different forms of human nephropathies, including kidney cancer (IARC, 1993, p. 245e395; Marquardt & Frohlich, 1992; Ringot, Chango, Schneider, & Larondelle, 2002). Tumor incidence data from long-term animal studies also provides reasons for concern about the effect of OTA
526
P. Afshar et al. / Food Control 31 (2013) 525e529
exposure on the human population. Thus, OTA was classified as a possible carcinogen (Group 2B) to humans by The International Agency for Research on Cancer (IARC) (Pfohl-Leszkowicz & Manderville, 2007). The mechanism of action of OTA is unclear. Recent reports suggest that oxidative pathway and genotoxicity are the key points for both nephrotoxicity and carcinogenicity (Ringot et al., 2002). Degenerative changes of the epithelial cells of the kidneys and the liver could be explained by the route of elimination of OTA via the kidneys and partly via the liver (Koynarski et al., 2007). Infant exposure to OTA can initiate during prenatal life since the fetoplacental unit can constitute a site concentration due to their ability to cross the human placenta (Galvano et al., 1996). Aflatoxins are one of the major etiological factors in the development of hepatocellular carcinoma (IARC, 1993, p. 245e395, 2002, p. 1e556), and more recently associations between childhood aflatoxin exposure and both growth faltering (Gong et al., 2002; Gong et al., 2004), and salivary IgA levels (Turner, Moore, Hall, Prentice, & Wild, 2003) have been reported. Aflatoxin B1 (AFB1) is the most potent of the dietary aflatoxins, and its major metabolite, Aflatoxin M1, is frequently observed in the urine of aflatoxin exposed individuals, and additionally in the breast milk of nursing mothers. Aflatoxin M1 toxicity in this respect is important as it is known that within aflatoxin exposed nursing mothers it can provide a source of aflatoxin exposure to the infant (El-Nezami et al., 1995; Wild, Pionneau, Montesano, Mutiro, & Chetsanga, 1987; Zarba et al., 1992). The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2) and M2 (AFM2) in breast milk, has also been reported (IARC, 1993, p. 245e395). There is increased awareness of the link between growth and health of the fetus and infant, and disease risk in later life (Barker, 2004; Delisle, 2002; Firestone & Amler, 2003; Wild & Kleinjans, 2003). Long term pre and postnatal exposure to aflatoxins could be one of the factors contributing to growth faltering and/or the early onset of hepatocellular carcinoma (HCC) in countries with a high incidence of the disease (Wild et al., 1991). As human breast milk is main nutrient for infants who are considered to be more susceptible to adverse effects of mycotoxins (Sadeghi, Oveisi, Jannat Hajimahmoodi, Bonyani, & Jannat, 2009). Unfortunately, there is no data about Ochratoxin A incidence on human milk in our country. As the presence of Ochratoxin A and Aflatoxin M1 in human breast milk is of concern, the purpose of this survey was to determine occurrence and values of Ochratoxin A and Aflatoxin M1 in human breast milk in Sari, Iran. The probable correlation between the mycotoxins values in positive samples and potential risk factors such as type of job, dietary pattern and personal habits was evaluated. 2. Materials and methods
diseases of the breast or central nervous system, malnutrition, maternal allergy, alcohol and addiction. Other exclusion criteria were newborns with any malformation, cardiac or hemolytic disease. The diseases were diagnosed by history, physical examination, etc. Based on data collected by the dietary questionnaire, we classified the subjects according to the frequency of consumption into moderate consumers group (up to 7 times a week) and habitual consumers group (more than 7 times a week) (European Commission, 1997). 2.3. Extraction procedure Milk samples were extracted according to method of Chang and DeVries (1983) and Jonsyn, Maxwell, and Hendrickse (1995) with slight modifications. Briefly, saturated NaCl solution (0.2 ml) and chloroform (2.4 ml) were added to breast milk (1 ml) in a stoppered glass tube and warmed to 37 C in a water bath. Then, all samples were thawed gradually, and centrifuged at 13,000 rpm for 15 min at 4 C. After separation, the chloroform layer was removed and evaporated to dryness under steam of nitrogen. The residue was dissolved in acetonitrile (0.6 ml), and the solution was extracted twice with petroleum ether by vigorous shaking for 1 min to remove contaminating lipids. Ochratoxin A and Aflatoxin M1 are water soluble, so the upper creamy layers were completely discarded and the lower phase was used. 2.4. Analysis of AFM1and OTA in samples by competitive ELISA and HPLC The quantitative analysis of OTA in the samples was initially based on enzyme immunoassay (ELISA) test and then the all positive samples with HPLC method. ELISA test procedure was performed according to the instructions of the test kit (NEOGEN, USA: veratox for Ochratoxin #8610 and EuroProxima B.V, for Aflatoxin M1 #5121). HPLC systems used in this project were Waters HPLC system (Waters 2690, USA) equipped with an auto sampler and a Waters fluorescence detector 474; excitation and emission wavelengths were 365 and 430 nm, respectively. The HPLC column and guard were Capital HPLC 150 4.6 mm, 3 mm (Capital HPLC Ltd.) and 0.5 cm chromolith, respectively. Temperature in column oven was 40 C. The mobile phase prepared by methanol, acetonitrile, water (3:2:6, v/v/v), KBr (0.12 g) and HNO3 4 N (0.35 ml), in isocratic mode with a flow rate of 2 ml/min. Ochratoxin A and Aflatoxin M1 standards were obtained from Sigma Aldrich (St. Louis, MO, USA). Nine Ochratoxin A and seven Aflatoxin M1 standards of between 0.5 and 15 ng/ml and 0.1e10 ng/ ml were injected, respectively.
2.1. Sample collection 2.5. Statistical analysis During May to August 2011, a total of 136 breast milk samples were collected from 7 hospitals in Sari, Iran. The studied groups include lactating women and newborn aged less than 6 months. Breast milk samples (5e10 ml) were collected into sterile plastic container by self expression before nursing the baby. The samples were kept at 4 C and frozen within one day at 80 C before extraction in Referral Laboratory of Mazandaran University of Medical Sciences. 2.2. Clinical evaluation and exclusion criteria All volunteers were informed about the aim of the investigation and the procedure. Exclusion criteria for the subjects were fever, diabetes, infections or metabolic disease, gestational hypertension,
Statistical analysis was performed by the SPSS 16 software followed by student t-test for comparison of OTA concentrations in breast milk samples. Also, variable inter groups comparison was analyzed by means of non-parametric test ManneWhitney. p < 0.05 was considered statistically significant. 3. Results and discussion The average age of the mothers was 26.6 years (ranged from 16 to 39 years). 91.2% of the studied mothers were housewife and 8.8% were employed. The number of children was studied and we observed that 90 mothers (66.2%) undergo their first delivery, 39 mothers (28.7%) delivered their second neonate and the rest (5.1%)
P. Afshar et al. / Food Control 31 (2013) 525e529 Table 1 Ochratoxin A and aflatoxin M1 values in all 136 breast milk samples determined by ELISA and HPLC methods. Method
Mycotoxin
Positive samples (%)a
Range
