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Alternaria Toxins

Richard Lawley, Leatherhead Food International, Randalls Road, Leatherhead, Surrey KT22 7RY, England

Introduction

Alternaria is a common genus with a number of species that can invade crops at the pre- and post-harvest stage and cause considerable losses due to rotting of fruits and vegetables. Under suitable conditions it may lead to production of a range of mycotoxins as well as other less-toxic metabolites. Some species are fairly specific to particular crops. A. alternata is probably the most important mycotoxin-producing species and occurs on cereals, sunflower seeds, oilseed rape, olives, various fruits etc. A large number of metabolites have been reported so that the situation is similar to the Fusarium mycotoxins in that, in the present state of knowledge, only a few occur naturally in food commodities or are of major toxicological significance. The principle Alternaria mycotoxins that have been shown to occur naturally are tenuazonic acid, alternariol monomethyl ether, alternariol, altenuene, and altertoxin I. Iso-altenuene and altertoxin II have not been found in crops to date. AAL-toxins are produced by A. alternata f. sp. Lycopersici, a rarely occurring pathotype of A. alternata, and are structurally related to fumonisins. There is only one report on their natural occurrence in hay silage.

Chemical and Physical Properties

Tenuazonic acid is a colourless, viscous oil and is a monobasic acid with pKa 3.5. It is soluble in methanol and chloroform. On standing, heating or treatment with a base, optical activity is lost and crystallisation may occur as a result of formation of isotenuazonic acid. It forms complexes with calcium, magnesium, copper, iron and nickel ions. Tenuazonic acid is usually stored as its copper salt. Alternariol and alternariol monomethyl ether crystallise from ethanol as colourless needles, and melting points with decomposition are 350 Deg C and 267 Deg C respectively. They sublime in a high vacuum without decomposing at 250 Deg C and 180-200 Deg C. They are soluble in most organic solvents and give a purple colour reaction with ethanolic ferric chloride. Altenuene crystallises as colourless prisms melting at 190-191 Deg C. Altertoxin I is an amorphous solid melting at 180 Deg C and fluoresces bright yellow under UV light.

Toxicity and Importance

Alternaria toxins exhibit both acute and chronic effects. The LD50 values for alternariol monomethyl ether, alternariol, altenuene, and altertoxin I in mice is reported as 400, 400, 50 and 200 mg/kg b. w. respectively. Those for tenuazonic acid are 162 and 115 mg/kg b.w. (i.v.) for male and female mice respectively.

Alternaria toxins have been implicated in animal and in human health disorders. During investigation into outbreaks of suspected mycotoxicoses, it was shown that cereal samples collected from affected farms in Germany were more frequently contaminated with Alternaria mycotoxins than samples from farms with healthy animals. Cases of death in rabbits and poultry have been reported as a result of toxic action of Alternaria spp. found in the fodder and feed. Alternaria spp. were also detected in cereal samples in which Fusarium spp. were implicated as the likely cause for the outbreak of alimentary toxic aleukia in Russia

Tenuazonic acid has been most studied. Its principle mode of action appears to be the inhibition of protein synthesis by suppression of the release of newly formed proteins from the ribosomes into the supernatant fluid. It exhibits antitumor, antiviral and antibacterial activity. Alternariol and alternariol monomethyl ether show foetotoxic and teratogenic effects in mice, including a synergistic effect when a combination of the toxins was administered. Most Alternaria mycotoxins exhibit considerable cytotoxic activity, including mammalian toxicity. and the altertoxins are of a particular concern due to their mutagenic activity. Altertoxin III exhibits mutagenic activity that is approximately one tenth of that of aflatoxin B1, while altertoxins I and II show lower mutagenicity.

Products Affected and Natural Occurrence

Contamination with alternariol, alternariol monomethyl ether, tenuazonic acid, and, in some cases, altenuene and altertoxin I, is normally associated with fruits, vegetables and oilseeds visibly infected by Alternaria rot, including tomatoes, olives, mandarins, peppers, and apples. Occurrence of Alternaria spp. and their mycotoxins in oilseeds has been reported by a number of workers (e.g. discoloured pecan nuts, sunflower seed, sunflower seed meal and oil seed rape). Mycotoxins produced by Alternaria spp. have also been reported in cereals such as sorghum, wheat, rye, diseased rice and tobacco. Alternariol and alternariol monomethy ether have also been produced in artificially mould-infested building materials in a study aimed at detecting mycotoxins produced by common fungi growing on such materials

Sampling and Analysis

Alternaria mycotoxins can be extracted by methods based on methanol or similar polarity solvents followed by defatting with hexane and further clean-up procedures, although the precise conditions and solvents will depend on the foodstuff and the mycotoxin being sought. Different extraction and clean-up methods are usually required for tenuazonic acid. Methods for Alternaria mycotoxins have been reported using TLC, HPLC, GC, GC/MS and HPLC/MS. GC/MS, HPLC/MS and HPLC-MS/MS methods have been reported for confirmation of identity of these mycotoxins in naturally contaminated samples whereas HPLC with UV or fluorometric detection is commonly used for routine analysis of food and feed commodities.

Stability and Persistence

Few studies on the stability and reactions have been carried out on Alternaria mycotoxins although in common with other mycotoxins they are probably quite stable. A major proportion of the toxins survived the autoclaving of tomatoes in producing tomato paste. Alternariol, alternariol monomethyl ether, and altertoxin I were stable in fruit juices and wine over 20 days or at 80 Deg C after 20 min. When olives are pressed only a very small percentage is transferred to olive oil. However, heat treatment at 121 Deg C for 60 minutes significantly reduced the concentration of alternariol, alternariol monomethyl ether and tenuazonic acid in A. alternata-contaminated sunflower flour. The heat-treated material produced toxic effects when fed to rats.

Legislation and Control

There are currently no statutory or guideline limits set for Alternaria mycotoxins because surveys to date have shown that their natural occurrence in foods is very low and the possibility for human exposure is very limited. The need for regulation is kept under review as new information becomes available.

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