Risk assessment of exposure to aflatoxin B1 and ochratoxin A through consumption of different Pistachio (Pistacia vera L.) cultivars collected from four geographical regions of Iran
Introduction
Pistachio tree (Pistacia vera) belongs to the Anacardiaceae family. Pistachio kernels are often consumed as a snack (roasted and salted) and used in ice creams and confections. Strains of Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius, under certain conditions of temperature and humidity, can grow on crops such as nuts, cereals, cottonseed, corn, and produce mycotoxins and different carcinogenic secondary metabolites that may affect human health (Neamatallah and Serdar, 2013; Vlata et al., 2005).
These organisms grow under aerobic and anaerobic conditions (Nidhina et al., 2017). Detection of ochratoxin A (OTA) and aflatoxins (AFs) in food commodities is critical since their adverse effects on human health have been shown in several studies (Rastegar et al., 2017).
OTA has been found in various agricultural products, such as cereals including wheat, maize and barley, horticultural crops such dried vine fruits (Pfohl- Leszkowicz and Manderville, 2007; Polisenska et al., 2010), dried figs and dried apricots or all dried fruit (10 μg/kg), mixture of species (15 μg/kg), sunflower and pumpkin seeds, pistachios, hazelnuts or all tree nuts (5 μg/kg), liquorice placed on the market for the final consumer (10 μg/kg), herbs and herbal teas (10 μg/kg) and cocoa powder (2 μg/kg) (EFSA, 2017).
According to the Joint FAO/WHO Expert Committee on Food Additives, OTA exposure may cause neurotoxicity, immunotoxicity, nephrotoxicity and genotoxicity (Joint FAO/WHO, 2010) ; also, numerous studies correlated OTA with DNA damage, lipid peroxidation and cytotoxicity (Sorrenti et al., 2013).
According to The European Food Safety Authority limits, the maximum levels (MLs) for OTA in sunflower and pumpkin seeds, pistachios, hazelnuts or all tree nuts is 5 μg/kg and 10 μg/kg for liquorice placed on the market for the final consumer (EFSA, 2017).
Eighteen aflatoxins are known, but only AFB1, AFB2, AFG1 and AFG2 have been found in agricultural crops (Torre et al., 2015). Among them, aflatoxin B1 (AFB1) is the most toxic metabolite (Tsakiris et al., 2013). The International Agency for Research on Cancer (IARC) has also noted that AFB1 as a lipophilic molecule, accumulates in the liver inducing acute hepatitis, that following long-term exposure, may result in liver cancer [(available at http://monographs.iarc.fr/ENG/Classification/List_of_Classifications.pdf and http://monographs.iarc.fr/ENG/Classification/)]. However, so far, no carcinogenic properties have been demonstrated for AFB2, AFG1 and AFG2 in humans (Joint FAO/WHO, 2001). Also, for other mycotoxins (e.g. zearalenone), carcinogenicity and teratogenicity have been reported (Alegakis et al., 1999; Vlata et al., 2006). According to the IARC, OTA and aflatoxins have been classified as “possibly carcinogenic to humans (Group 2B)” and “carcinogenic to humans (Group 1)”, respectively (available at http://monographs.iarc.fr/ENG/Classification/List_of_Classifications.pdf and http://monographs.iarc.fr/ENG/Classification/).
The volume of food consumed over a specified period of time and the level of xenobiotic residues are the two critical parameters required for risk assessment. According to the risk assessment principals, the estimated human exposure to xenobiotics should be compared with the reference values such as Acceptable Daily Intake, Acute Reference value, etc. In case of genotoxic carcinogens such as AFB1, the non-threshold approach of “as low as reasonably achievable” (ALARA) (EFSA, 2007a), or the Margin of Exposure (MoE) approach are usually used (EFSA, 2007b)
Determination of AFB1 levels, along with elimination of AFB1 or reduction its concentration in pistachio have attracted considerable attention in major pistachio-producing countries such as Iran. During cultivation period, depending on the weather conditions or/and insufficient plant protection practices, pistachios may become contaminated with mycotoxins produced by fungi. During storage, water activity (aw) and temperature crucially affect fungal growth and aflatoxins production (Liu et al., 2017). Traditional harvesting and processing methods combined with storage under high humidity and temperature, favor microorganisms development. Moreover, soil, animals, insects and humans are other potential sources of contamination (Campbell et al., 2003).
Aflatoxin contamination also depends on the form of pistachio shell, which is related to cultivar type. Early splitting and hull cracking are two main factors allowing the entrance of fungus into the pistachio (Tajabadipour and Sheibani Tezerji, 2011). In early summer, hull is attached to shell and then shell splits. Shell splitting facilitates the consumption of pistachio for consumers, but the hull does not split and protects the kernel against penetration of insects and fungi. In some pistachio cultivars known as "early split", hull splitting can increase the chance of aflatoxin contaminations (Doster and Michailides, 1995).
The European Food Safety Authority (EFSA) has set the MLs of 4 μg/kg for total aflatoxins (B1+ B2+ G1 + G2) in groundnuts, nuts, dried fruits, cereals and processed products and 2 μg/kg for AFB1 (EFSA, 2007b). Moreover, the EFSA has established an ML of 5 μg/kg for OTA (EFSA, 2017).
This study aimed to (1) measure AFB1 and OTA levels in five pistachio cultivars collected from four sites of Iran, (2) assess the health risk posed by consumption of pistachio based on hazard index (HI), and (3) introduce the best cultivar and site of cultivation in which the aflatoxin content is near the standard levels.
Section snippets
Chemicals and reagents
Aflatoxin B1 and ochratoxin A with 98% purity were procured from Sigma (St. Louis, MO, USA). Ethanol, methanol, acetonitrile, phosphate-buffered saline (PBS) were supplied from Sigma (Sigma-Aldrich, Steinheim, Germany). Solvents were filtered and degassed prior to application. All other chemicals were of analytical grade and supplied by Merck (Darmstadt, Germany) and Sigma (St. Louis, MO, USA). Immuno-affinity columns (IAC) for AFB1 and OTA were prepared from Libios (Pontcharra-Sur-Turdine,
Method validation
The recovery value for AFB1 and OTA was calculated by analyzing the AFB1 and OTA concentrations in the spike samples and comparing them with those of standard solutions (2, 4, and 6 ng/g for AFB1 and 4, 8, and 12 ng/g for OTA solutions). All the experiments were performed in triplicate. Limit of detection (LOD) for AFB1 and OTA were 0.066 and 0.27 ng/g, respectively, and limit of quantification (LOQ) for AFB1 and OTA were 0.2 and 0.81 ng/g, respectively, (Table 3). In this study, we determined
Discussion
In our study, the mean AFB1 level was 1.14 ng/g, which was lower than the modified maximum level of AFB1 in pistachio kernels (2 ng/g) proposed for direct human consumption. The mean level of OTA content was 0.413 (ng/g), which was also below the level established by the European Union for OTA (i.e. 5 ng/g) (EFSA, 2007a, 2007b). In this experiment, aflatoxin contamination was only observed in pistachios with discolored shell. In our study, in all cultivation sites, the highest content of AFB1
Conclusion
Measurement of the levels of two mycotoxins in five commercial pistachio cultivars collected from four main cultivation sites of Iran, showed that in all samples, AFB1 and OTA levels were below the maximum limit recommended by the European Commission regulations. Moreover, the HI value was below 1 which indicates that consumption of these cultivars pose no significant health risk to humans. It is highly suggested to conduct similar studies on other nuts cultivated in Iran and exported to other
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgments
The authors thank Pharmaceutical Research Center, Medical Toxicology Research Center, and Pharmacy School Mashhad University of Medical Sciences, Iran.
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These authors contributed equally to this work.