Aflatoxins are toxic metabolites produced by certain fungi in/on foods and feeds . They are probably the best known and most intensively researched mycotoxins in the world. Aflatoxins have been associated with various diseases , such as aflatoxicosis , in livestock , domestic animals and humans throughout the world . The occurence of aflatoxins is influenced by certain environmental factors ; hence the extent of contamination will vary with geographic location , agricultural and agronomic practices, and the susceptibility of commodities to fungal invasion during preharvest , storage, and/or processing periods . Aflatoxins have received greater attention than any other mycotoxins because of their demonstrated potent carcinogenic effect in susceptible laboratory animals and their acute toxicological effects in humans . As it is realized that absolute safety is never achieved , many countries have attempted to limit exposure to aflatoxins by imposing regulatory limits on commodities intended for use as food and feed .
In the 1960 more than 100,000 young turkeys on poultry farms in England
died in the course of a few months from an apparently new disease that
was termed "Turkey X disease" . It was soon found that
the difficulty was not limited to turkeys . Ducklings and young pheasants
were also affected and heavy mortality was experienced .
A careful survey of the early outbreaks showed that they were all associated
with feeds, namely Brazilian peanut meal . An intensive investigation of
the suspect peanut meal was undertaken and it was quickly found that this
peanut meal was highly toxic to poultry and ducklings with symptoms typical
of Turkey X disease .
Speculations made during 1960 regarding the nature of the toxin suggested
that it might be of fungal origin. In fact, the toxin-producing fungus
was identified as Aspergillus flavus (1961) and
the toxin was given the name Aflatoxin by virtue of its origin (A.flavis-->
Afla).
This discovery has led to a growing awareness of the potential hazards
of these substances as contaminants of food and feed causing illness and
even death in humans and other mammals. Studies that are summarized in
the following sections revealed that aflatoxins are produced primarily
by some strains of A. Flavus and by most , if not all , strains
of A. parasiticus , plus related species, A. nomius and A.
niger . Moreover , these studies also revealed that there are four
major aflatoxins : B1 , B2
, G1 , G2 plus two
additional metabolic products , M1 and M2 , that
are of significance as direct contaminants of foods and feeds . The aflatoxins
M1 and M2 were first isolated from milk of lactating animals fed aflatoxin
preparations ; hence , the M designation . Whereas the B designation of
aflatoxins B1 and B2 resulted from the exhibition of blue fluorescence
under UV-light , while the G designation refers to the yellow-green fluorescence
of the relevant structures under UV-light . These toxins have closely similar
structures and form a unique group of highly oxygenated, naturally occuring
heterocyclic compounds . Their molecular formulas as established from elementary
analyses and mass spectrometric determinations are :
Aflatoxins B2 and G2 were established as the dihydroxy derivatives of B1
and G1 , respectively . Whereas , aflatoxin M1 is 4-hydroxy aflatoxin B1
and aflatoxin M2 is 4-dihydroxy aflatoxin B2 .
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Aflatoxins often occur in crops in the field
prior to harvest . Postharvest contamination can occur if crop drying is
delayed and during storage of the crop if water is allowed to exceed critical
values for the mold growth . Insect or rodent infestations facilitate mold
invasion of some stored commodities.
Aflatoxins are detected occasionally in milk, cheese, corn, peanuts, cottonseed,
nuts, almonds, figs, spices, and a variety of other foods and feeds . Milk,
eggs, and meat products are sometimes contaminated because of the animal
consumption of aflatoxin-contaminated feed . However, the commodities with
the highest risk of aflatoxin contamination are corn, peanuts, and cottonseed.
Corn is probably the commodity of greatest worldwide concern , because it is grown in climates that are likely to have perennial contamination with aflatoxins and corn is the staple food of many countries . However, procedures used in the processing of corn help to reduce contamination of the resulting food product . This is because although aflatoxins are stable to moderately stable in most food processes , they are unstable in processes such as those used in making tortillas that employ alkaline conditions or oxidizing steps . Aflatoxin-contaminated corn and cottonseed meal in dairy rations have resulted in aflatoxin M1 contaminated milk and milk products , including non-fat dry milk , cheese , and yogurt .
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Fungal growth and aflatoxin contamination are the consequence of interactions among the fungus, the host and the environment . The appropriate combination of these factors determine the infestation and colonization of the substrate , and the type and amount of aflatoxin produced . However , a suitable substrate is required for fungal growth and subsequent toxin production , although the precise factor(s) that initiates toxin formation is not well understood . Water stress , high-temperature stress , and insect damage of the host plant are major determinig factors in mold infestation and toxin production . Similarly , specific crop growth stages, poor fertility , high crop densities, and weed competition have been associated with increased mold growth and toxin production. Aflatoxin formation is also affected by associated growth of other molds or microbes . For example, preharvest aflatoxin contamination of peanuts and corn is favored by high temperatures, prolonged drought conditions, and high insect activity; while postharvest production of aflatoxins on corn and peanuts is favored by warm temperatures and high humidity.
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Aflatoxicosis is primarily a hepatic disease
. The susceptibility of individual animals to aflatoxins varies considerably
depending on species, age, sex, and nutrition . In fact, aflatoxins cause
liver damage, decreased milk and egg production, recurrent infection as
a result of immunity suppression (eg. salmonellosis), in addition to embryo
toxicity in animals consuming low dietary concentrations. While the young
of a species are most susceptible, all ages are affected but in different
degrees for different species. Clinical signs of aflatoxicosis in animals
include gastrointestinal dysfunction, reduced reproductivity, reduced feed
utilization and efficiency , anemia, and jaundice. Nursing animals may
be affected as a result of the conversion of aflatoxin B1 to the metabolite
aflatoxin M1 excreted in milk of dairy cattle.
The induction of cancer by aflatoxins has been extensively studied. Aflatoxin
B1, aflatoxin M1, and aflatoxin G1 have been shown to cause various types
of cancer in different animal species. However, only aflatoxin B1 is considered
by the International Agency for Research on Cancer (IARC) as having
produced sufficient evidence of carcinogenicity
in experimental animals to be identified as a carcinogen.
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Humans are exposed to aflatoxins by consuming foods contaminated with
products of fungal growth . Such exposure is difficult to avoid because
fungal growth in foods is not easy to prevent . Even though heavily contaminated
food supplies are not permitted in the market place in developed countries,
concern still remains for the possible adverse effects resulting from long-term
exposure to low levels of aflatoxins in the food supply .
Evidence of acute aflatoxicosis in humans has been reported from many parts
of the world , namely the Third World Countries, like Taiwan, Ouganda,
India, and many others . The syndrome is characterized by vomiting, abdominal
pain, pulmonary edema, convulsions, coma, and death with cerebral edema
and fatty involvment of the liver , kidneys , and heart.
Conditions increasing the likelihood of acute aflatoxicosis in humans include
limited availability of food, environmental conditions that favor fungal
development in crops and commodities, and lack of regulatory systems for
aflatoxin monitoring and control.
Because aflatoxins, especially aflatoxin B1, are potent carcinogens in
some animals, there is interest in the effects of long-term exposure to
low levels of these important mycotoxins on humans . In 1988, the IARC
placed aflatoxin B1 on the list of human carcinogens. This is supported
by a number of epidemiological studies done in Asia and Africa that have
demonstrated a positive association between dietary aflatoxins and Liver
Cell Cancer (LCC) . Additionally , the expression of aflatoxin-related
diseases in humans may be influenced by factors such as age, sex, nutritional
status, and/or concurrent exposure to other causative agents such as viral
hepatitis (HBV) or parasite infestation.
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Sampling and sample preparation remain a considerable source of error in the analytical identification of aflatoxins. Thus, systematic approaches to sampling, sample preparation, and analysis are absolutely necessary to determine aflatoxins at the parts-per-billion level . In this regard, specific plans have been developed and tested rigorously for some commodities such as corn, peanuts, and tree nuts; sampling plans for some other commodities have been modeled after them. A common feature of all sampling plans is that the entire primary sample must be ground and mixed so that the analytical test portion has the same concentration of toxin as the original sample.
All analytical procedures include three steps: extraction, purification,
and determination. The most significant recent improvement in the purification
step is the use of solid-phase extraction.
Test extracts are cleaned up before instrumental analysis(thin layer or
liquid chromatography) to remove coextracted materials that often interfere
with the determination of target analytes.
Thin layer chromatography (TLC) , also known as flat bed chromatography or planar chromatography is one of the most widely used separation techniques in aflatoxin analysis. Since 1990, it has been considered the AOAC official method and the method of choice to identify and quantitate aflatoxins at levels as low as 1 ng/g. The TLC method is also used to verify findings by newer, more rapid techniques .
Liquid chromatography (LC) is similar to TLC in many respects, including
analyte application, stationary phase, and mobile phase. Liguid chromatography
and TLC complement each other. For an analyst to use TLC for preliminary
work to optimize LC separation conditions is not unusual.
Liquid chromatography methods for the determination of aflatoxins in foods
include normal-phase LC (NPLC), reversed-phase LC (RPLC) with pre- or before-column
derivatization (BCD), RPLC followed by postcolumn derivatization (PCD),
and RPLC with electrochemical detection.
Thin layer chromatography and LC methods for determining aflatoxins in food are laborious and time consuming . Often, these techniques require knowledge and experience of chromatographic techniques to solve sepatation and and interference problems. Through advances in biotechnology, highly specific antibody-based tests are now commercially available that can identify and measure aflatoxins in food in less than 10 minutes. These tests are based on the affinities of the monoclonal or polyclonal antibodies for aflatoxins. The three types of immunochemical methods are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), and immunoaffinity column assay (ICA).
Although analytical methods might consist of different extraction, clean-up, and quantitation steps, the results of the analyses by such methods should be similar when the methods are applied properly. Since the reliability of the quantitative data is not in question, the problem still to be solved is the confirmation of identity of the aflatoxins. The confirmation techniques used involve either chemical derivatization or mass spectrometry (MS).
Safety is a key issue for scientists working in the aflatoxin area. Steps must be taken to minimize exposure to the toxins as well as to the producing microorganisms, Aspergillus flavus and Aspergillus parasiticus. A safety program should be established that meets the requirements of the Laboratory Standard of the Occupational Safety and Health Administration (1990) and the guidelines of the National Institutes of Health (1981) covering use of chemical carcinogens .
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In the last few years, new technologies have been developed that more accurately monitor individual exposures to aflatoxins. Particular attention has been paid to the analysis of aflatoxin DNA adducts and albumin adducts as surrogates for genotoxicity in people. Autrup et al.(1983) pioneered the use of synchronous fluorescence spectroscopy for the measurement of aflatoxin DNA adducts in urine. Urine samples collected after exposure to alfatoxins were found to contain 2,3-dihydroxy-2-(N7-guanyl)-3-hydroxyaflatoxin B1, trivially known as AFB-Gual . Wild et al.(1986) used highly sensitive immunoassays to quantitate aflatoxins in human body fluids. An enzyme linked immunosorbent assay (ELISA) was used to quantitate aflatoxin B1 over the range of 0.01 ng /ml to 10 ng/ml , and was validated in human urine samples. Using this method, aflatoxin-DNA adduct excretion into urine was found to be positively correlated with dietary intake, and the major aflatoxin B1-DNA adduct excreted in urine was shown to be an appropriate dosimeter for monitoring aflatoxin dietary exposure.
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Aflatoxins are considered unavoidable contaminants of food and feed, even where good manufacturing practices have been followed. The FDA has established specific guidelines on acceptable levels of aflatoxins in human food and animal feed by establishing action levels that allow for the removal of violative lots from commerce. The action level for human food is 20 ppb total aflatoxins, with the exception of milk which has an action level of 0.5 ppb for aflatoxin M1. The action level for most feeds is also 20 ppb. However, it is very difficult to accurately estimate aflatoxins concentration in a large quantity of material because of the variability associated with testing procedures; hence, the true aflatoxin concentration in a lot cannot be determined with 100% certainty.
Because aflatoxin contamination is unavoidable, numerous strategies for their detoxification have been proposed. These include physical methods of separation, thermal inactivation, irradiation, solvent extraction, adsorption from solution, microbial inactivation, and fermentation. Chemical methods of detoxification are also practiced as a major strategy for effective detoxification :
A diverse group of chemicals has been tested for the ability to degrade
and inactivate aflatoxins. A number of these chemicals can react to destroy
(or degrade) aflatoxins effectively but most are impractical or potentially
unsafe because of the formation of toxic residues or the perturbation of
nutrient content and the organoleptic properties of the product. Two chemical
approaches to the detoxification of aflatoxins that have received considerable
attention are ammoniation and reaction with sodium bisulfite.
Many studies provide evidence that chemical treatment via ammoniation may
provide an effective method to detoxify aflatoxin-contaminated corn and
other commodities. The mechanism for this action appears to involve hydrolysis
of the lactone ring and chemical conversion of the parent compound aflatoxin
B1 to numerous products that exhibit greatly decreased toxicity.
On the other hand, sodium bisulfite has been shown to react with aflatoxins
(B1, G1 , and M1) under various conditions of temperature, concentration,
and time to form water-soluble products.
The toxicity of mycotoxins may be strongly influenced by dietary chemicals that alter the normal responses of mammalian systems to these substances. A variable array of chemical factors, including nutritional components (e.g. dietary protein and fat, vitamins, and trace elements), food and feed additives (e.g. antibiotics and preservatives), as well as other chemical factors may interact with the effects of aflatoxins in animals.
A new approach to the detoxification of aflatoxins is the addition of inorganic sorbent materials, known as chemisorbents, such as hydrated sodium calcium aluminosilicate (HSCAS) to the diet of animals. HSCAS possesses the ability to tightly bind and immobilize aflatoxins in the gastrointestinal tract of animals, resulting in a major reduction in aflatoxin bioavailability.
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The economic impact of aflatoxins derive directly from crop and livestock losses as well as indirectly from the cost of regulatory programs designed to reduce risks to animal and human health. The Food and Agriculture Organization (FAO) estimates that 25% of the world's food crops are affected by mycotoxins, of which the most notorious are aflatoxins. Aflatoxin losses to livestock and poultry producers from aflatoxin-contaminated feeds include death and the more subtle effects of immune system suppression, reduced growth rates, and losses in feed efficiency. Other adverse economic effects of aflatoxins include lower yields for food and fiber crops .
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