On the Relevance of Ochratoxin A Contamination in Coffee from a Toxicological Point of View

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On the Relevance of Ochratoxin A

Contamination in Coffee from a Toxicological Point of View

Abschlussarbeit

Postgradualstudium Toxikologie der Universität Leipzig

eingereicht von

Dipl.-Biochemiker Michael Haack geb. am 1. Dezember 1978 in Halle/Saale

eingereicht am

7. Januar 2008

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Abbreviations

AA aristolochic acid

AOAC Association of Official Analytical Chemists API atmospheric pressure ionisation

BEN Balkan endemic nephropathy CAS chemical abstract specification

CE-LIF capillary electrophoresis with laser-induced fluorescence (detection)

CIT citrinin

CYP cytochrome P450 EC European Commission EFSA European Food Safety Authority ELISA enzyme-linked immuno-sorbent assay ESI electrospray ionisation

HPLC high-performance liquid chromatography IAC immunoaffinity column

IARC International Agency for Research on Cancer LC liquid chromatography

LC-FD liquid chromatography with fluorescence detection LC-MS/MS liquid chromatography with tandem mass spectrometry LLE liquid-liquid extraction

LOEL lowest observed effect level LOX lipoxygenase

LPO lipid peroxidation

MAFF UK Ministry of Agriculture, Fisheries and Food NTP US National Toxicology Program

OAT organic anion transporter OM-OTA O-methylated ochratoxin A OP-OTA opened form of ochratoxin A OTA ochratoxin A

OTAHQ ochratoxin A-derived hydroquinone OTAQ ochratoxin A-derived quinone

OTα ochratoxin α

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PGSH prostaglandin synthase

PTWI provisional tolerable weekly intake ROS reactive oxygen species

SCOOP scientific cooperation (on questions relating to food) SPE solid-phase extraction

TLC thin-layer chromatography UTT urinary tract tumour WHO World Health Organization

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1 Mycotoxins: An Introduction

Being responsible for large financial losses in conjunction with contaminated and thus unsafe food, beverages and feed as well as being linked to the genesis of several disease states, mycotoxins have been recognised as potential threat to both human and animal health and are under surveillance of international agencies attempting to define safe legal limits for concentrations in foodstuffs, processed food and in animal fodder [1, 2].

Mycotoxins are secondary metabolites that have no biochemical significance in fungal growth and development but have been associated with the incidence of adverse effects in animals and humans. Moreover, mycotoxins are produced by the mycelial structure of filamentous fungi including members of the Fusarium, Aspergillus, Claviceps, Alternaria and Penicillium families but may also be present in the spores of these organisms [3]. These slime moulds grow under a wide range of climatic conditions on agricultural commodities, such as grains, spices, fruits, coffee, nuts etc., both in the field and during storage.

Predominantly offering both a considerable thermal and chemical stability as well as exhibiting a great structural diversity, some hundred different mycotoxin species have been described so far. Unfortunately, they cannot or can be in part removed by food processing or other suitable decontamination processes. Today, aflatoxins, ochratoxins, trichothecenes, zearalenone, fumonisins, tremorgenic toxins and ergot alkaloids are the mycotoxins of greatest public and agro-economic significance due to their frequent occurrence and their severe effects on animal and human health [4-6]. Examples of fungal species and mycotoxins that are of biological and economical relevance in animal agriculture are presented in Table 1 [7]. In this context it should be noted that some moulds are able to produce more than one mycotoxin, while several mycotoxins may be produced by more than one fungal species.

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Table 1. Main toxigenic fungal species and their mycotoxins (D’Mello and MacDonald, 1997 [7])

Fungal species Mycotoxin Deleterious effects

Aspergillus flavus and A.

parasticus

Aflatoxins carcinogenic and hepatotoxic in humans [8]

A. flavus Cyclopiazonic acid

A. ochraceus, Penicillium

viridicatum and P. cyclopium Ochratoxin A nephrotoxic effects (renal damage) in poultry, acutely toxic in rats and mice, tumorgenic in humans [9-13]

P. expansum Patulin

Fusarium culmorum, F.

graminearum and F. sporotrichoides

Deoxynivalenol potent feed intake inhibitor in pigs, teratogenic, ruminants tolerant [14]

F. sporotrichoides and F. poae T-2 toxin F. sporotrichoides, F.

graminearum and F. poae Diacetoxyscirpenol

implicated in field cases of mycotoxicosis in ruminants F. culmorum, F. graminearum

and F. sporotrichoides

Zearalenone infertility, reduced milk production and hyperoestrogenism in cows [15], estrogenic effects commonly found in farm animals [16]

F. proliferatum and F.

verticillioides Fumonisins hepatic lesions in pigs and cattle, equine leukoencephalomalacia, porcine pulmonary oedema, implicated in oesophageal cancer in humans [17- 20]

Acremonium coenophialum Ergopeptine alkaloids reduced growth, reproductive performance and milk production in ruminants; increased susceptibility to heat stress [21-24]

A. lolii Lolitrem alkaloids neurological effects: shaking of the head, incoordination, staggering and collapse in ruminants [25, 26]

Phomopsis leptostromiformis Phomopsins lupinosis: ill-thrift, liver damage, jaundice, photosensitisation and death in sheep [27, 28]

Pithomyces chartarum Sporidesmins facial eczema in sheep: liver damage, urinary lesions, photosensitisation [29]

The presence or production of mycotoxins in food and feed is caused by several factors, including environmental conditions related to storage that can be controlled. Other extrinsic factors such as climate, especially temperature and relative humidity, and extent of insect infestation or intrinsic factors such as fungal strain specificity, strain variation and instability of toxigenic properties are more difficult to control [4, 30]. All in all the interaction between all these factors not yet well understood. Hence, toxin production cannot reasonably be predicted and is beyond human control. In addition, the effect of fungicides is of particular interest, since these are widely used to control fungal diseases in crops. Whereas the risks from mycotoxin contamination are low when fungicides are used successfully, several studies

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show that sub-lethal concentrations of the fungicides may enhance mycotoxin production [31, 32].

The toxic effect of mycotoxins on animal and human health is referred to as mycotoxicosis, the severity of which depends on the toxicity of the mycotoxin, the extent of exposure, age and nutritional status of the individual and possible synergistic effects of other chemicals to which the individual is exposed [33]. Mycotoxicoses are characterised as food or feed related, non-contagious, non-transferable, non-infectious and non-traceable to microorganisms other than fungi. Mycotoxins that adversely affect human or animal health are found mainly in post-harvest crops such as cereal grains or forages. These toxins are produced by saprophytic fungi during storage or by endophytic fungi during plant growth. With the exception of fumonisin B, mycotoxins are generally lipophilic and therefore accumulate in the fat fraction of plants and animals [4]. Moreover, mycotoxins exhibit severe nephrotoxic, neurotoxic, carcinogenic, immunosuppressive and estrogenic effects, and diseases are either caused by direct contamination of plant materials or products thereof [5, 6, 34], or by spreading of mycotoxins and their metabolites into animal tissues, milk and eggs after intake of contaminated feed [7]. Chronic intake of small amounts of mycotoxins as well as less critical compounds may reduce weight gain in animals and cause diarrhoea in humans [4, 35]. In addition, several endemic diseases in Asia, Africa and Europe are thought to be related to acute mycotoxin intoxication, such as Balkan Endemic Nephropathy (BEN), tumours of the urinary tract as well as Kwarshiorkor and Reye’s syndrome [36-40].

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2 Ochratoxins

Ochratoxins are fungal secondary metabolites of Aspergillus, Penicillium and Fusarium species in subtropical and temperate climate [5]. They can chemically be described as 3,4- dihydromethylisocoumarin derivatives linked via an amide bond to the amino group of L-β- phenylalanine [41]. Ochratoxin A (OTA) is chlorinated, which is unusual for natural products.

Both the unchlorinated ochratoxin B as well as the ethyl ester of OTA, ochratoxin C, are less toxic and less common [42-44]. Most studies on ochratoxins have therefore focused on OTA [45]. The systematic chemical nomenclature for OTA is (R)-N-[(5-chloro-3,4-dihydro-8- hydroxy-3-methyl-1-oxo-1H-2-benzopyran-7-yl)-carbonyl]-L-phenylalanine (CAS No. 303- 47-9). The chemical structures of ochratoxins are shown in Figure 1.

2.1 Ochratoxin A

First isolated from Aspergillus ochraceus in South Africa in 1965 [42], ochratoxin A is the most toxic compound of this group and has received particular attention because of its cancer- promoting activity. OTA is a mycotoxin produced by several fungal species in the Penicillium and Aspergillus genera, primarily Penicillium verrucosum, Aspergillus ochraceus and A.

carbonarius together with a low percentage of isolates of the closely related A. niger as well as by Aspergilli in the sections Nigri, Circumdati and Flavi [46-57]. Since these species of Penicillium and the four sections of Aspergillus differ in their ecological niches, different commodities will be affected. The frequency of their occurrence depends on the geographical region. A. carbonarius grows at high temperatures and has been found to be responsible for OTA contamination in maturing fruits, especially fresh grapes, wine and dried vine fruits [58, 59], but it is also a source of OTA in coffee. Due to its black spores, A. carbonarius is highly resistant to sunlight and survives sun-drying. In contrast, A. ochraceus grows at moderate temperatures and at a water activity above 0.8. It is the source of OTA in green coffee beans but may also infect coffee beans during sun-drying, cocoa, cereals and edible nuts. Being found only sporadically in a wide range of stored food commodities, A. ochraceus is seldom the source of substantial OTA concentrations. In contrast, contamination of grain in cooler regions of Northern Europe and Canada is mainly caused by P. verrucosum, which grows at temperatures below 30 °C and at a water activity above 0.8 [60, 61]. As P. verrucosum does not occur in the tropics and subtropics, cereals from those regions are unlikely to contain OTA from this source [62].

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In temperate climate the natural occurrence of OTA in food is widespread and generally associated with a variety of products such as cereals, cereal-based products, grape juices, wine, dried fruits, cocoa and coffee beans, beer, spices and products of animal origin (for a review on EU occurrence data see [63]).

Ochratoxin A is a colourless crystalline compound with blue fluorescence under UV light (fluorescence emission maximum in absolute ethanol is at 428 nm) and weak acidic character.

OTA is highly soluble in polar organic solvents, slightly soluble in water and soluble in aqueous hydrogen carbonate. The dissociation constants (pKa) are in the range of 4.2–4.4 for the carboxyl group and 7.0–7.3 for the phenolic group [64, 65]. Since temperatures above 250 °C for several minutes are required to reduce toxin concentration, OTA is not destroyed by common food preparation procedures [66, 67]. Baking and roasting reduce the toxin content by only 20 %, while boiling has no effect [68]. Therefore, raw and processed food commodities may be contaminated with OTA.

Figure 1. Ochratoxins A, B and C.

The list of ochratoxin A producing fungi as well as the list of foods that can be contaminated with ochratoxins has expanded in the last years. The worldwide occurrence of OTA as a natural contaminant of raw agricultural products has been repeatedly reported. It has been found in a variety of plant products such as cereal grains (barley, wheat, rye, maize, oat and rice), coffee and cocoa beans, pulses, dried vine fruit, spices and liquorice [69-75].

Naturally occurring OTA as a contamination of corn from commercial markets in the United States was reported for the first time by Shotwell and coworkers in 1969 [76]. Moreover, OTA has also been detected in beverages such as grape juice, wine and beer [77-79]. Due to its ability to move up the food chain, OTA can be found in the tissues and organs of animals, especially in liver and kidneys of pigs, and subsequently in humans, including blood and breast milk. Recently, the occurrence of OTA in food for human consumption has been extensively reviewed by Clark and Snedeker [80].

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2.2 Ochratoxin A Toxicity

The occurrence of OTA in food and feed has been reported worldwide (for review see [5, 69, 71, 81]). OTA binds almost completely to plasma proteins, is metabolised very slowly and accumulates in kidney and liver. Therefore, the World Health Organisation (WHO) has set a provisional tolerable weekly intake (PTWI) for OTA of 112 ng/kg body weight based on a LOEL of 8 µg/kg body weight per day for deterioration of renal function in pigs to which a safety factor of 500 was applied [82]. In the meantime the PTWI value has been confirmed and rounded off to 100 ng/kg body weight [83]. The European Food Safety Authority (EFSA) suggested an intake level of 120 ng/kg body weight. Recent analyses of the dietary exposure of adult European consumers to OTA revealed that the weekly exposure at present ranges from 15 to 60 ng OTA per kg body weight per week for average and high consumers, respectively [84]. Although it cannot be excluded that infants and children as well as distinct segments of the population, representing high consumers of certain locally-produced food specialities, may experience higher rates of exposure, this rate is even below the PTWI value of 100 ng/kg body weight set by the WHO.

Besides cereal products that have been found to be the most contaminated products in a 2.5- year study of the German Federal Ministry of Health published in 1999, OTA can also be frequently detected in animal products as a result of direct contamination of the products with toxigenic moulds or indirect transmission by feeding animals with naturally contaminated feed. Taking into account the strict regulations on maximum levels of OTA as well as its high toxicity and persistence in the food chain, accurate and sensitive detection of OTA in a variety of food, feed and biological matrices at the sub ppb range is needed.

OTA is known to be a potent nephrotoxin and hepatotoxin with teratogenic, mutagenic, carcinogenic and immunosuppressive effects. In some Eastern European countries OTA has been associated with a high incidence of Balkan endemic nephropathy in human, which is a disease characterised by severe kidney damage [62, 85-87]. It has been suggested that the consumption of OTA-contaminated food during pregnancy and/or childhood may lead to testicular DNA damage, which in turn could give rise to testicular cancer later on [88]. This toxin has been classified as a possible human carcinogen (category 2B) by the International Agency for Research on Cancer (IARC) [89-91].

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Changes in the concentration of OTA in the organism over time (toxicokinetics) as well as the dynamic interaction of OTA with biological targets and their downstream biological effects (toxicodynamics) determine the toxicity of OTA and have been recently reviewed by Ringot et al. as well as Pfohl-Leszkowicz and Manderville in more detail [92, 93]. A brief account of both the toxicokinetics of OTA as well as the mechanisms of OTA toxicity will be given.

2.2.1 Toxicokinetics

Absorption

Ochratoxin A is readily absorbed from the upper gastrointestinal tract, whereby the small intestine, particularly the proximal jejunum, has been found to be the major site of absorption [94]. Absorption from the duodenum can take place against a concentration gradient [94], highlighting the presence of organic anion transporter (OAT) proteins for OTA transport [95- 97]. The acidic moieties (the carboxylic and the phenolic group) play an important role in OTA absorption. In most animal species OTA is passively absorbed in both its non-ionised and monoanion (OTA⁻) forms from the stomach and, particularly, from the proximal duodenum, which, in addition, is favoured by the high binding of OTA to plasma proteins [94, 98, 99]. The overall percentage of OTA found to be absorbed were 66 % in pigs, 56 % in rats and rabbits as well as 40 % in chickens [100, 101]. OTA is subsequently transported via the portal vein to the liver and thereafter distributed to different tissues and organs. The biological half-life of OTA in male Wistar rats was estimated to be 127 h [102]. The toxicokinetics of the mycotoxin when given intratracheally, intravenously and orally were comparable. Interestingly, simultaneous administration of OTA and phenylalanine to mice appeared to increase the absorption of OTA from the stomach and intestine and to increase gastrointestinal transit [99]. The bioavailability of OTA after oral administration was very low in fish, but ranged from 44 to 97 % in rat and mouse [103]. Moreover, bioavailability may vary in the presence of food components as well as inhibitors. OTA absorption and cellular accumulation is increased by some flavonoids and resveratrol [104].

Distribution

In the bloodstream more than 99% of OTA is bound to serum albumin and other macromolecules. The pronounced binding of OTA to plasma proteins retards its elimination by limiting its transfer from the bloodstream to hepatic and renal cells; however, OTA is only

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detected in traces in erythrocytes [101, 103, 105-109]. In contrast, more specific binding of the toxin to small serum proteins (20 kDa), which can be filtered through the glomerular membrane, could be relevant for the nephrotoxic effects in mammals [110]. In vivo studies reveal that the lifetime of OTA is dependent on the presence of serum albumin and that this binding is species- and sex-dependent [108, 111]. Considerable variations in the serum half- life of OTA after oral administration have been reported: 4.1 h in chickens, 6.7 h in quail, 24–

39 h in mice, 77 h in pre-ruminant calves, 55–120 h in rats, 72–120 h in pigs, 456–504 h in vervet monkeys (Cercophithecus aethiops) and 510 h in rhesus macaques (Macaca mulata);

these are even higher after intravenous administration [98, 101, 103, 112-115]. In one human volunteer the half-life of OTA was 840 h [89]. The wide species difference in serum half-life of OTA was recently reviewed by Petzinger and Ziegler [89].

The concentration of ochratoxin A and its metabolites in tissues and plasma is dependent on the animal species, the dose and form of OTA administered (crystalline or naturally occurring in feed), the diet composition as well as the health status of the animals [93]. In general, the elimination half-life of OTA in blood is longer than in tissues, which may at least in part be attributed to the higher binding affinity of the mycotoxin to blood proteins [43, 103, 116]. In pigs, rats, chickens and goats OTA was mainly distributed to the kidneys with lower concentrations found in liver, muscle and fat [114, 117-119]. Moreover, it has been shown that tissue distribution is dependent on dose, sex and duration of OTA exposure (ingestion, injection) [120-123]. In mice, rats, pigs and humans it has been shown that OTA can readily cross the placenta, depending on the day of gestation; these suggest that the transfer is influenced by the developmental stage of the placenta, which is considered to be completely developed after day 9 of gestation [112, 124-129]. Moreover, a very efficient transfer of OTA from the blood to the milk and subsequently to the offspring has been demonstrated in lactating rats and rabbits [127, 130, 131]. Consistent with these observations is the fact that the amount of OTA found in kidney and blood of the offspring exposed both through the placenta and lactation were four- to five-fold higher when compared to offspring exposed only through the placenta or through lactation [127].

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Elimination

Ochratoxin A shows a rapid absorption and a slow elimination. In all species studied both biliary and renal excretion have been shown to be involved in the plasma clearance of ochratoxin A. The relative contribution of each excretory route is influenced by the route of administration, the dose, the degree of serum macromolecular binding as well as the differences in the degree of enterohepatic recirculation of OTA [43, 69, 103, 132, 133]. The major excretory products are ochratoxin α (OTα), ochratoxin A itself as well as the 4R- Hydroxy-OTA epimer [134]. All the metabolites are considered to be less toxic than OTA itself. In addition to the urinary und faecal routes of excretion, the mammalian milk excretion appears to be relatively effective.

In humans and non-human primates (vervet monkeys) the parent OTA is mainly excreted via the kidneys [115, 116]. Due to the high plasma protein binding potential of OTA its glomerular filtration is limited. Instead, OTA rather undergoes tubular secretion into the urine and is also reabsorbed in all nephron segments [135-137]. The involvement of several transporter proteins carrying OTA across tubular membranes as well as their sex-dependent regulation has been recently reviewed by Pfohl-Leszkowicz and Manderville [93]. The reabsorption of filtered and secreted OTA retards its excretion and may lead to the accumulation of the toxin in the renal tissue and thus contribute to its renal toxicity.

Faecal excretion of OTA is mainly due to biliary excretion and very efficient [94, 99, 138].

Furthermore, the amount of OTA secreted by the intestinal epithelial cells into the lumen of the intestine must also be taken into account and is comparable to the amount of OTA transported via the bile into the intestine [94, 100, 139]. In the intestine the conjugated compounds are hydrolysed by the intestinal microflora, and the released OTA may undergo enterohepatic circulation. The microbial microflora has also been shown to hydrolyse OTA to OTα in the large intestine [92].

Another important route of excretion of OTA is the elimination via the milk of lactating mammals. Several studies have been carried out in different mammalian species to evaluate the transfer of orally or intravenously administered OTA and of OTA present in contaminated feed into milk [127, 130, 131, 140-143]. Depending on the dose originally administered or present in the feed OTA and OTα have been identified in milk. Several authors have reported on the OTA levels in human milk, thereby concluding that OTA amounts in human milk show

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significant inter-individual and geographic variations [143-145]. More recently, a correlation between OTA contamination of human milk and its dietary intake has been shown by Skaug et al. [146].

2.2.2 Biotransformation

Accumulation of ochratoxin A occurs in blood, liver and kidney. The latter organs are also the major sites of OTA biotransformation. However, metabolism of OTA has not been elucidated in detail, and at the present time data regarding OTA biotransformation are controversial [92]. A schematic view of biotransformation reactions is given in Figure 2.

Figure 2. In vivo metabolism of ochratoxin A.

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Hydrolysis

The hydrolysis of OTA to the much less toxic compound OTα, which represents the major metabolic pathway, is a detoxication reaction catalysed by the bacterial microflora in the intestine, particularly the caecum [107, 133, 147-149]. Moreover, OTA has been shown to be hydrolysed by carboxypeptidase A, trypsin, α-chymotrypsin and cathepsin C in vitro [150- 153]. Whereas duodenum, ileum and pancreas also have a high capacity to carry out this reaction, the activity in liver and kidney was low or absent [100, 154, 155]. The relative resistance of ruminants, such as cows and sheep, to the effects of OTA in feed is based on ruminant protozoa that hydrolyse OTA to OTα with a remarkable velocity before it reaches the blood [156-158]. The capacity of the rumen to hydrolyse OTA is strongly dependent on the animal diet [159-161].

Detoxification and Bioactivation by Phase I Reactions

A small percentage of absorbed OTA is hydroxylated to hydroxyl-OTA through phase I detoxification reactions [64]. These reactions occur in the hepatocytes after OTA has reached the liver via the hepatic portal vein (Vena portae) [94]. Uptake and secretion of OTA by hepatocytes may be carrier-mediated, but only limited data have been published on this transport mechanism [162, 163]. Studies using liver microsomes from human, pig and rat revealed the formation of both epimers of 4-hydroxy-OTA, (4R)- and (4S)-hydroxy-OTA [164-166]. The formation of 10-hydroxy-OTA has been described after incubation of OTA with rabbit liver microsomes in vitro [155, 167, 168], whereas up to now this metabolite has not been shown to be formed in vivo. In addition, this metabolite has also been found in in vitro studies using human bronchial epithelial cells as well as in rabbit kidney microsomes and appears not to be genotoxic [169, 170].

The dechlorinated derivative of OTA, ochratoxin B (OTB), may occur together with OTA in cereal products and was identified in an in vitro study as a metabolite of OTA after incubation of monkey kidney cells with the mycotoxin [171, 172]. OTB is tenfold less toxic than OTA in 1-day-old chicks and not toxic in ducklings and rats as well as in in vitro studies using human-derived liver cells; this less pronounced toxicity may be due to the absence of the chlorine atom [173-175].

The naturally co-occurring ochratoxin C (OTC), which in the body may be converted into OTA and is also found as a metabolite of OTA in rumenal fluid, was shown to be as toxic as

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OTA for 1-day-old duckling and chicks [171, 173, 176]. Moreover, it was found to be cytotoxic in prokaryotic Bacillus brevis cells and human cervix epithelial cells as well as in human embryonic and renal carcinoma cell lines in vitro [177, 178].

The large variety of toxic effects that are caused by OTA, particularly its nephrotoxic and carcinogenic effects, cannot only be explained by its biotransformation products. Numerous studies have established that an oxidative mechanism is involved in OTA-mediated DNA damage and lipid peroxidation. Thus, several hypotheses are put forward and have been reviewed in detail by Ringot et al. [92]. Briefly, Hoehler et al. showed that OTA and its analogues mediate the generation of reactive oxygen species (ROS) and suggested that OTA induces oxidative damage rather indirectly [179, 180]. Furthermore, Xiao et al. showed that O-methylated ochratoxin A (OM-OTA, Figure 3) is toxic in mice and suggested that its toxicity is related to its lactone carbonyl group [177]. More recently it has been hypothesised that OTA-derived quinones (OTAQ/OTAHQ, Figure 3) might be the ultimate reactive metabolites. The quinones have been shown to be generated by oxidative dechlorination in vitro and this fact is in accordance with the previous observations that chlorine is essential for OTA genotoxicity and that chlorinated compounds inducing an oxidative stress are known to undergo bioactivation to benzoquinones before they cause DNA damage [181-186]. The opened form of OTA (OP-OTA, Figure 3), which can be obtained by basic hydrolysis of OTA, has also been detected in bile and urine of rats [177, 187]. OP-OTA is highly toxic and eliminated much more slowly than OTα and 4-OH-OTA [43].

Figure 3. Structures of O-methylated ochratoxin A (OM-OTA), the opened form of ochratoxin A (OP-OTA) and the OTA-derived quinone/hydroquinone (OTAQ/OTAHQ)

Enzymes that have been shown to be involved in OTA biotransformation comprise isoforms of cytochromes P450 (CYP), prostaglandin synthase (PGSH) as well as lipoxygenase (LOX).

Basically, phase I biotransformation reactions are catalysed by the cytochrome P450 system;

cytochromes P450 are predominantly found in liver, but are also expressed in the intestinal mucosa, lung, nasal epithelium, skin, kidney, sertoli cells of the testis, ovary and placenta.

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Reviews describing the role of diverse P450 isoforms in OTA metabolism have been published by Ringot et al. and Pfohl-Leszkowicz and Manderville [92, 93].

2.2.3 Effects on Enzymes and Other Biochemical Parameters

In order to explain OTA toxicity, several hypotheses regarding specific and non-specific interactions of ochratoxin A and its metabolites with endogenous molecules have been postulated.

Ochratoxin A is thought to have a diabetogenic effect by inhibiting insulin synthesis and/or its release from pancreatic cells, thereby suppressing glycolysis and glycogenesis and enhancing gluconeogenesis and glycogenolysis [188]. Moreover, OTA specifically inhibits protein synthesis at the post-transcriptional level. OTA competitively inhibits phenylalanine- tRNA^Phe synthetase, so that amino acylation and peptide elongation are stopped. This effect can be prevented by simultaneous administration of phenylalanine [189, 190]. OTA may thereby be regarded as a structural analogue of phenylalanine, whereas OTα, which lacks the phenylalanine moiety, showed no effect on the translation step [191, 192]. Furthermore, since OTA disrupts protein synthesis, it indirectly impairs the activity of several cellular enzymes, and particularly the activity of cytosolic phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme of the gluconeogenic pathway [193-196]. In addition, OTA inhibits liver phenylalanine hydroxylase and could act as a substrate for this enzyme, which is tightly controlled [197-199]. Whereas a too rapid degradation of phenylalanine will lead to its depletion, the accumulation of this amino acid will lead to impairment of production of compounds derived from phenylalanine as seen in the phenylketonuria.

Further studies have shown that OTA leads to a competitive inhibition of succinate dehydrogenase and cytochrome C oxidase activities in the mitochondria of rats [200], to the alteration of the mitochondrial membrane transport system and to a competitive inhibition of inner membrane ATPase activity [201] as well as to the competitive inhibition of enzyme activities involved in the metabolism of phenylalanine [190, 192, 197, 202]. In addition, in vitro studies with human and canine kidney cells have demonstrated a stimulatory effect of OTA on extracellular protein kinase and caspase, which results in apoptosis [203, 204].

Exposure of human urothelial cells to OTA leads to the induction of unscheduled DNA synthesis [205].

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2.2.4 Toxicological Studies

Acute and Subacute Toxicity

The toxicity of ochratoxin A varies widely and depends on animal species, sex and route of administration. The LD50 values after oral administration range from 0.2 mg/kg body weight in dogs to 30.3 mg/kg body weight in male rats. OTA is even more toxic when administered intravenously or intraperitoneally. The mycotoxin is very toxic to numerous animal species, the kidneys thereby being the main target organ [64, 71]. Further toxic effects observed include cardiac and hepatic histological abnormalities, aberration of coagulation factors in the rat, accompanied by haemorrhage and thrombosis in the spleen, brain, liver, kidney and heart, lesions of the gastro-intestinal tract and lymphoid tissues in the hamster, myelotoxicity in mice as well as intestinal fragility and kidney lesions in chickens [103, 206-209]. Whereas exposure of poultry to feed contaminated with 0.5 ppm OTA only resulted in a decreased food intake, contamination with 2 ppm OTA led to weight loss, decreased egg production, increase of water intake, diarrhoea and excessive urine excretion, all signs indicative of a renal disorder, as well as changes in haematological parameters [210-216]. A dramatic increase in the mortality rate was observed with 4 ppm OTA [212, 213].

Nephrotoxicity

Ochratoxin A is nephrotoxic to birds and mammals, but not to adult ruminants [217-220].

Several studies in Scandinavia, Hungary and Poland have shown that OTA is involved as an aetiological agent in porcine nephropathy and dealt with the morphological changes of renal target cells in detail [221-224]. These studies show a high similarity between the nephropathy occurring in pigs and BEN observed in humans [87, 225, 226]. In both cases the main kidney cells being affected were the epithelial cells of the proximal tubules. Moreover, many cell organelles in the damaged epithelial cells decreased in number and exhibited loss of membrane integrity; furthermore, the brush border was reduced in height and density. A large number of apical vesicles, lysosomes and peroxisomes with granular material were seen and many nuclei showed condensation of chromatin and disappearance of the nuclear envelope.

At a later stage thickening of the basement tubular membranes of the proximal tubules and a large number of collagen fibres in the interstitium were predominantly observed [87]. OTA also induces glomerulonephrosis, tubulonephrosis, focal tubular epithelial cell proliferation and multiple adenoma-like structures in the renal parenchyma in broiler chicks [227].

Nephropathy may also be induced in rats, chickens and poultry in general [209, 228, 229].

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OTA has also been found to be hepatotoxic in chickens [230]. The most prominent feature of OTA toxicity –even after a short-term exposure– is nuclear enlargement and polyploidy of proximal tubular cells, which is a consequence of nuclear division without cytokinesis [231- 233]. Most recently, the functional and pathological characteristics of chronic OTA nephrotoxicity compared to the clinical presentation of endemic nephropathy have been summarised by Mally et al. [234]. All in all the pathological lesions observed in kidneys of rats treated with OTA appear to differ from those described for BEN.

Immunotoxicity and Myelotoxicity

The mechanisms of OTA immunotoxicity are multiple and not yet fully understood, mainly due to relatively few and partially contradictory reports. Al-Anati and Petzinger recently reviewed immunotoxic studies mainly carried out in poultry, particularly in chickens, which makes interpretation of the data much more difficult [235]. However, it is clear that OTA alters different immune functions in man, mice, rats, pigs and chicken. The authors conclude that there is a general agreement in the scientific literature that OTA causes a decrease in relative weights of immune organs such as thymus, spleen, lymph nodes and the Bursa fabricii. The immunosuppressing activity is further characterised by the depression of antibody responses, alteration in the number and functions of immune cells and the modulation of cytokine production [236-239]. Thus, the immunotoxic activity of OTA probably results from degenerative changes and cell death following necrosis and apoptosis in combination with a slow replacement of the affected immune cells due to inhibition of protein synthesis. Cell death would thereby lead to a reduction in the number of antibody-producing cells in lymphoid organs as well as to a decrease in serum levels of immunoglobulins.

Teratogenicity

Ochratoxin A is considered to be teratogenic in mice, rats, hamsters, chickens and rabbit, but not in pigs [240-247]. As already mentioned in Chapter 2.2.1 (Toxicokinetics), maternal OTA is able to cross the placenta and thus may accumulate in foetal tissues and induce malformation, mainly in the central nervous system after administration to pregnant animals (reviewed in [69]). Teratogenicity studies have demonstrated a direct fetotoxic effect of OTA [126, 242, 248-251].

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2.2.5 Carcinogenicity

The first studies on OTA carcinogenicity were reported in rodents and trout, in which OTA was administered orally and intraperitoneally [252-254]. Induction of tumours in the kidney of mice and rats as well as of hepatoma in the trout was only observed after oral administration. In the meantime and after IARC had decided that the evidence for the carcinogenicity of OTA was either inconclusive or limited two further studies in mice demonstrated the carcinogenicity of OTA [255, 256]. Based on the results of the two previously mentioned studies and on a study by the US National Toxicology Program (NTP) [257], IARC finally found the experimental evidence for carcinogenicity to be sufficient and classified OTA as ‘possibly carcinogenic to humans’ [258]. More recently, a commission of the Deutsche Forschungsgemeinschaft (Commission for the investigation of health hazards of chemical compounds in the work area) has classified OTA in category 2 as a substance which is considered to be carcinogenic for man [259].

2.2.6 Molecular Mechanisms

Genotoxicity and Mutagenicity

Ochratoxin A has long been considered non-genotoxic. OTA was found to be not mutagenic in the Ames test, even in the presence of liver-based metabolic activation systems [260-262].

Increased revertant numbers were only observed when the Ames test was performed with mouse kidney microsomes [263, 264]. Furthermore, genotoxic effects such as DNA strand breaks, sister chromatid exchanges, chromosomal aberrations and induction of micronuclei have been observed in some mammalian cell systems in response to OTA exposure. These studies have recently been summarised by Mally and Dekant [265].

Based on structure-activity relationship studies, several groups proposed that the presence of the C-5 chlorine atom as well as the phenylalanine moiety and the para-chlorophenolic group are important for in vitro and in vivo genotoxicity [167, 177, 183, 266]. Whereas Rahimtula et al. [267] proposed that iron chelation may be necessary for toxicity, Xiao and coworkers [167] showed that an OP-OTA derivative that lacks metal chelating properties was equally efficient in generating ROS and suggested that the toxicity of OTA is not linked to the chelation of Fe²⁺. Moreover, the OTA-derived quinone OTAQ can undergo either a two- electron reduction by action of the NAD(P)H:quinone reductase to form hydroquinone

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OTAHQ or a one-electron reduction to yield a semiquinone radical, which in turn could induce DNA breaks, LPO and exocyclic adducts [268].

Formation of DNA Adducts

Covalent binding of carcinogens or their reactive metabolites to DNA is believed to be the initial step in chemical carcinogenesis [269]. Hence, metabolic activation of most chemical carcinogens is required for the interaction with cellular macromolecules. Lutz et al. found a strong correlation between DNA adduct formation and the frequency of mutations [269]. As recently reviewed by Mally and Dekant [265], genotoxicity studies do not suggest that OTA is mutagenic or a potent genotoxin, consistent with the lack of formation of reactive metabolites. Nevertheless, based on ³²P-labeling analyses some authors have postulated that a time- and dose-dependent formation of DNA adducts in different organs in both mice and rats does in fact occur and proposed that genotoxicity plays a major role in OTA-induced tumourigenesis [85, 270-272]. All adducts disappeared in liver and spleen 5 days after OTA administration, whereas some adducts persisted up to 16 days in the kidney. However, these putative DNA adducts have not been shown to contain OTA or a part of the OTA molecule and it remains unclear whether they are covalent DNA adducts. Moreover, the data are in contrast to results obtained by using radiolabeled OTA or accelerator mass spectrometry, which clearly demonstrate that OTA does not form covalent DNA adducts [273-276].

Furthermore, OTA-related DNA adducts were not detected in recent studies using sensitive

32P-postlabeling and LC-MS/MS techniques [274, 277]. Thus, the EFSA Scientific Panel on Contaminants in the Food Chain concluded that there was no evidence for the existence of specific OTA-DNA adducts [84].

Effects on Apoptosis and Signal Transduction

Mally et al. have shown that the repeated administration of OTA causes unusual histopathological alterations and changesin the kidney [232]. The authors proposed an OTA- specific mechanism of kidney damage, involving disruption of mitotic and apoptotic signalling pathways, which turn could lead to the perturbation of cytoskeletal organisation or loss of cell adhesion [278-280]. This specific mechanism of toxicity is in accordance with the absence of covalent binding of OTA to DNA, the lack of mutagenicity in bacteria and the weak (biotransformation-independent) genotoxicity of OTA in mammalian cells. Recently, Mally et al. investigated the effect of OTA treatment on canine kidney epithelial cells [281].

The results found support the hypothesis that disruption of cell-cell signalling due to

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interference with cell adhesion, cell-cell communication and the cytoskeleton may contribute to OTA toxicity and carcinogenicity [281]. The same group could further show that treatment of immortalised human kidney epithelial cells with OTA resulted in a time- and dose- dependent increase of apoptosis [282]. The authors suggest that the mechanism by which OTA promotes tumour formation involves interference with microtubule dynamics and mitotic spindle formation, resulting in apoptosis or premature exit from mitosis. Induction of apoptosis has also been shown in MDCK-C7 and OK cells by Gekle et al. and Sauvant et al., respectively [135, 204, 283], whereas in other in vitro systems OTA did not induce apoptosis or only did it at a very low rate [68].

Cytosolic Ca²⁺ is a modulator of numerous cellular events such as metabolism, transport and gene expression. Since OTA induces Ca²⁺ influx into cells, this could lead to apoptosis, but may also explain the activation of diverse intracellular cell signalling pathways [284, 285].

OTA has also been shown to induce oxidative stress, which may then lead to initiation of apoptotic processes as observed in rat kidney [286]. Oxidative stress could arise during metabolic transformation of OTA and thus could be a consequence of biotransformation.

Evidence for its role in mediating at least in part the toxic effects of OTA is derived from animal studies, which describe the attenuation of OTA-dependent toxic effects and reduced formation of DNA lesions in kidney and other organs by coadministration of various antioxidants [287].

Increased apoptotic rates in kidney may lead to polycystic kidney disease, glomerular sclerosis or interstitial fibrosis [288-290].

2.2.7 Ochratoxin A in Human Diseases

OTA was linked with an outbreak of porcine nephropathy in Scandinavian countries [291].

Subsequently, the still unproven hypothesis of the involvement of OTA in the development of the Balkan endemic nephropathy (BEN) was postulated. BEN is a familial, chronic, non- inflammatory tubulointerstitial disease with insidious onset and slow progression to terminal renal failure that was first described in Bulgaria, former Yugoslavia and Romania [292, 293].

It affects people that live in the alluvial deposits along the tributaries of the Danube river in Bosnia and Herzegovina, Croatia, Romania, Bulgaria as well as Serbia [86]. Thus, the main characteristics of BEN are not only its focused and limited geographical distribution, but also

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its occurrence in farming households, and a high mortality due to uraemia. The disease usually affects adults in their fourth to fifth decade and leads to tubular atrophy, periglomerular fibrosis and cortical cysts, inevitably progressing to degenerative and necrotic renal epithelia, hyperplastic arteriopathy and end-stage renal failure in their sixth decade [86, 294, 295]. BEN is often accompanied by upper urinary tract urothelial cancer, which is extremely aggressive in nature, and in spite of its endemic incidence in Balkan areas a similar clinical entity has been described throughout Europe, Asia and North America [295-297]. The incidence of urinary tract tumours (UTT) has been found to be much higher in regions with endemic nephropathy than in other regions [297-299]. The accompanying functional deficits in the early stages of the disease, which begins without an acute episode, include increased urinary concentrations of glucose, proteins, leucine aminopeptidase and γ-glutamyl transferase, coupled with a decrease in serum cholesterol and protein concentration.

Creatinine clearance rates and urinary specific gravity are markedly reduced [68].

Studies on OTA involvement in the aetiology of BEN and UTT were performed in Bulgaria and Croatia, where the mycotoxin was measured in various commodities of plant and animal origin as well as in human blood [300-307]. In endemic regions high OTA blood concentrations were a frequent finding in patients with chronic renal insufficiency. However, low concentrations of OTA have frequently been detected both in the blood of people from non-endemic regions and in the blood of healthy inhabitants from other countries where endemic nephropathy does not occur (cited in [300]). Hence, it is still not possible to link the exposure to OTA to BEN and UTT.

While there is a general agreement in the scientific literature that at least one environmental toxin must be involved, the responsible agent has still not been identified. It has been proposed that materials derived from the plant Aristolochia clematitis and related species as well as mycotoxins such as OTA might play a role in the outbreak of BEN and UTT [308]. A third hypothesis, the Pliocene lignite hypothesis, proposes that a long-term exposure to polycyclic aromatic hydrocarbons and other toxic organic compounds, which leak into the well drinking water from low-rank coals in the vicinity of the endemic settlements, is responsible for the onset of BEN [309-311]. At an international symposium held in Zagreb (Croatia) in 2006 Grollman and Jelakovic favoured the long-term exposure to aristolochic acid (AA) as the cause for BEN [296]. At the same symposium, Long et al. postulated that neither OTA nor AA can be firmly linked to Balkan endemic nephropathy [312]. Their

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analysis revealed environmental and health sciences issues that need to be adressed in order to understand the role of OTA and/or AA in the etiology of BEN. In contrast, Pfohl-Leszkowicz et al. recently reported on a comparative investigation of the mycotoxins OTA and citrinin (CIT) as well as AA and highlighted the implication of OTA and CIT in BEN and UTT, whereas AA derivatives were not implicated either in BEN or in slimming regimens [294].

Nikolic et al. demonstrated that the very intimate link between BEN and UTT can be explained by the insult induced by an environmental contaminant [313]. The intake of the agent at high doses causes nephropathy and early appearance of renal failure (BEN) during the third and the fourth decade of a patient’s life. Moreover, at low doses of the potential causative agent, the nephropathy is not recognised, but UTT still develops. Under these conditions, the patient may die from UTT even though kidney damage is minimal and subclinical, so that most of the patients (75 %) in a BEN settlement may show no symptoms of renal failure at the time of nephrectomy.

Interestingly, BEN rather affects people living in rural areas but not those living in towns in the vicinity [93]. This could be explained by the fact that rural populations consume more home-grown and home-stored food, whereas city dwellers consume predominantly commercially available and industrially produced food. The disease often affects many members of one family, while neighbouring families may be free from the disease over several generations. This may be due to the family’s dietary habits as well as conditions of food storage and, consequently, conditions promoting mould contamination and growing.

The predominant occurrence of BEN in specific families suggested that a genetic predisposition may also be involved. Toncheva et al. proposed that alterations in the gene coding for transforming growth factor-β (TGF-β), the genetic heterogeneity of xenobiotic- metabolising enzymes or defects in the immune system of the host could predispose to BEN [315]. Recently, genetic polymorphisms of some xenobiotic-metabolising enzymes have been associated with BEN [314-317]. Genetic variants of enzymes involved in the uptake, biotransformation and elimination of xenobiotics determine individual levels of detoxification and are modifiers of an increased/decreased risk for chronic diseases and cancer. Furthermore, genetic alterations predisposing BEN patients to UTT could be germline mutations in tumour- suppressor genes and/or acquired somatic mutations in oncogenes [318]. Genetic

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polymorphisms related to specific transporters may also contribute to inter-individual differences in the response to environmental toxins such as OTA [316, 319, 320].

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3 Ochratoxin A in Coffee

3.1 Source of Ochratoxin A in Coffee

Starting in the 1970s various studies have shown that contamination of coffee with OTA is predominantly caused by Aspergillus ochraceus [321-323]. However, further studies have concluded that other Aspergilli species may also be responsible for the contamination of coffee beans with OTA (Table 2). At the present time it is not clear, which species are responsible for the OTA contamination of coffee. Levels of contamination as well as the species that produces OTA vary from region to region. Most studies have focused on the yellow spored A. ochraceus, since this species has frequently been proposed to be the major source of OTA in green coffee. More recently, the black spored Aspergilli A. niger and A.

carbonarius have been isolated from coffee by several groups, which indicates that these two species might also be potential sources of OTA in coffee [323-326].

Table 2. Examples of fungal species found in coffee samples.

Fungal species Maturation of coffee Origin References A. ochraceus

green coffee beans

Brazil Vietnam

[323, 327, 328]

[329]

A. niger

green coffee beans green coffee beans

Brazil Vietnam Vietnam

[323, 327, 328]

[330]

[331]

A. steynii green coffee beans India [332]

A. westerdijkiae green coffee beans India [332]

A. lacticoffeatus green Robusta beans Venezuela [55]

A. sclerotioniger green Arabica beans India [55]

A. carbonarius green coffee beans green Robusta beans

green coffee beans green coffee beans

Thailand Brazil Vietnam

[325]

[326]

[327, 328]

[333]

Several national and international surveys on the presence of OTA in green and roasted coffee as well as in coffee-containing products have been conducted. Table 3 gives an overview of the published studies. Usually, neither the origin of the beans, the Coffea species (Coffea arabica vs. Coffea robusta) nor the way green coffee was processed (dry vs. wet processing) were taken into account. Romani et al. could show that coffee beans from Africa are generally contaminated with higher levels of OTA than coffee beans from Central or South America [324]. Moreover, a survey by the UK Ministry of Agriculture, Fisheries and Food (MAFF) indicated that Robusta green coffee was much more likely to contain OTA than

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Arabica coffee [334]. Bucheli et al. [337] further stated that strip-picking of coffee cherries, which tends to produce more defected beans, together with the use of cherry sun-drying (‘natural processing’) might explain the higher levels of OTA in green Robusta coffee.

Recently, Leong et al. [338] reported that in the case of Vietnamese green coffee beans significantly more Robusta than Arabica beans were infected with fungi. Interestingly, A.

niger was found to be the dominant species infecting Vietnamese coffee beans, but A.

carbonarius most probably was the source of OTA contamination [335].

Table 3. Systematic investigations on the occurrence of OTA in coffee.

Year of survey

Number of

samples Maturation Contamination rate

OTA

concentration References

1988 68 roasted coffee 7 % 3.2 – 17 µg/kg [336]

1989 29 green coffee 58 % 0.2 – 15 µg/kg [70]

1988- 1993

47 green coffee 30 % 0.1 – 17.4 µg/kg [325]

1995 25

40 green coffee

roasted coffee 52 %

35 % 1.2 – 56 µg/kg

0.3 – 2.3 µg/kg [337]

1995 23 roasted coffee 61 % 0.05 – 0.44 µg/kg

1996 30 roasted coffee 66 % 0.3 – 7.5 µg/kg [338]

1996 101 soluble coffee 74 % 0.2 – 6.5 µg/kg [339]

1997 20

80 roasted coffee

soluble coffee 85 %

80 % 0.2 – 2.1 µg/kg

0.1 – 8 µg/kg [340]

1998 11 roasted coffee 100% < 3.2 µg/kg [341]

1995-

1999 82

419 71 41

green coffee roasted coffee decaffeinated and

low-acid roasted coffee soluble coffee

27 % 46 % 37 % 71 %

0.2 – 24.5 µg/kg 0.2 – 12.1 µg/kg 0.2 – 2.7 µg/kg 0.3 – 4.8 µg/kg

[342]

2000 67 52

roasted coffee soluble coffee

32 % 46 %

0.3 – 3.3 µg/kg 0.3 – 9.5 µg/kg

[343]

2000 162 green coffee 65 % 0 – 48 µg/kg [322]

2000 34

16 roasted coffee soluble coffee (2 decaffeinated)

68 %

100 % 0.3 – 6.5 µg/kg

0.5 – 5.1 µg/kg [344]

2002 50 roasted coffee

and coffee blend 66% 0.17 – 1.3 µg/kg [345]

2003 40 green coffee 22 % 0.47 – 4.8 µg/kg [346]

2007 110 roasted and

grounded coffee 99 % < 4 µg/kg [347]

Due to the legislative pressure of industrialised nations, several surveys not only for the presence of OTA in green and roasted coffee as well as in final coffee products have been undertaken by the importers and coffee-producing countries. The fate of OTA during the processing of green coffee beans and during coffee manufacturing is also being investigated

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with increasing interest, since the identification of the stage and conditions under which OTA is produced during green coffee production and handling might give indications about the precautions that have to be taken to reduce the incidence of OTA contamination [348].

Infection with ochratoxin A-producing moulds and subsequent formation of OTA could occur on the coffee tree, during the ripening process of the cherries or may also take place within the developing coffee bean during cherry maturation [348]. In addition, it has also been suggested that a direct contamination of coffee beans may occur by OTA generated by saprophytic moulds in the soil and translocated directly via the plant into the coffee beans [349, 350].

Several authors concluded that OTA formation takes place during harvest and post-harvest processing of coffee beans. Moisture contents vary from 50–70 % in ripe cherries, 35–50 % in coffee raisins to 16–30 % in cherries that are dried on the plant [351]. In order to prevent mould contamination and fermentation the moisture content must be less than 12 % (water activity aw of 0.65–0.7) at the end of the drying step. Since coffee loses flavour at less than 9 % moisture content, while at more than 13 % there is an increasing risk of OTA contamination, drying of coffee beans, subsequent storage as well as transport and environmental conditions are critical to coffee quality and potential OTA contamination [352]. It has been shown in several studies that the presence of OTA in coffee is normally a result of insufficiently controlled harvesting procedures, precarious drying and inadequate storage conditions, which allows the growth of toxigenic fungi [323, 328, 353]. Coffee is a very hygroscopic material that is able to reabsorb moisture from the environment during storage and transport. In this context Palacios-Cabrera et al. [357] showed that cycling environmental conditions may indirectly favour OTA production. Since in a real coffee transport situation there is always a risk of increasing moisture content as a consequence of condensation, it became evident in recent studies that the presence of OTA in coffee is always an indication of serious failures in the practices of harvesting and storage. Moreover, recent studies investigated the growing requirements and potential OTA production of the three most important Aspergilli species on coffee raw material [326, 354, 355]. It has also been shown that OTA contamination could derive not only from inappropriate storage and transport of coffee, but also from coffee cherry drying as demonstrated by Bucheli et al. and Joosten et al.

[326, 356]. Their findings suggested that drying cherries under humid conditions is a rather

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risky route for OTA contamination, since cherries always contain plenty of water in the initial stage of drying, which in turn can support growth of A. carbonarius.

The contamination by OTA is reduced during the ongoing processing of green beans and the manufacturing of coffee. Although a small proportion of OTA is eliminated during the cleaning of green coffee, the most significant reduction is achieved during roasting, when OTA is either eliminated with chaff or destroyed, and by subsequent brewing processes during the production of soluble coffee [357, 358].

3.2 Analysis of Ochratoxin A in Coffee

During the last decade the analytical methods for ochratoxin A have been considerably improved with the use of immunoaffinity columns and high-performance liquid chromatography [359]. The first report (SCOOP-1) of the Scientific cooperation on questions relating to food (SCOOP), which was initiated by the European Commission (EC) in 1995, stated that 19% of the commodities on the European market were contaminated with OTA [360]. After initiation of a second SCOOP task in 1999, the SCOOP-2 report showed that 49 % of the commodities were contaminated with OTA, which is the consequence of the improvement of the analytical methods used [361].

3.2.1 Sampling and Sample Preparation

Since fungal contamination of raw materials, such as cereals, coffee and fruits, occurs randomly, it is very difficult to obtain representative samples, particularly in the case of cereals, since these are handled in bulk amounts [362]. Based on statistical sampling plans, multiple sub-samples are taken and pooled. Thereafter, this single sample has to be thoroughly ground to a fine powder (coffee, cereals) or minced (dried fruits) and slurred prior to analysis [363, 364]. In contrast, sampling of retail products is less problematic, since potentially contaminated commodities are already mixed and homogenised during food processing. But again, large numbers of individual samples need to be bulked together.

Therefore, sampling that encompasses not only the choice of sample size and source but also sample extraction techniques and subsequent clean-up are of fundamental importance and determine length of the analytical procedure, accuracy, recovery and achievable detection

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limits. Moreover, a wrong sampling plan can greatly affect the reliability of the measured levels of mycotoxin and can, in turn, even result in legal disputes and trade restrictions.

Miraglia et al. [368] have given a holistic view of sampling for mycotoxins in general, assuming that given considerations could be adapted to OTA in most cases. Therein, primary sampling schemes (‘why, where and when’ to collect samples), purposes of sample collection (monitoring, surveillance and targeted sampling) as well as secondary sampling schemes (‘how’ to collect samples) are discussed. In addition, Vargas et al. [369] reported on the design of a sampling plan for OTA detection in green coffee.

Replacing conventional liquid-liquid extraction (LLE) techniques, preparation of samples for subsequent analysis is achieved by several efficient clean-up and preconcentration procedures based on immunoaffinity columns (IAC) and solid-phase extraction (SPE), typically using underivatised silica, C8, C18 and CN stationary phases [325, 358]. In fact, underlying interactions in SPE are relatively unselective and the resulting clean-up levels might be insufficient for some challenging matrices. Therefore, sample clean-up and enrichment with immobilised antibodies that exclusively retain OTA became increasingly popular for samples that can be directly loaded on IAC, including coffee, beer and wine [365, 366]. Application of IACs results in the production of cleaner extracts with a minimum level of interfering matrix components and excellent signal-to-noise ratios compared to less selective SPE sorbent materials [365, 367]. However, Castegnaro et al. investigated the advantages and drawbacks of IAC in mycotoxin analyses and found OTA to be underestimated in more complex matrices such as breakfast cereals and coffee [368]. Indeed, roasted coffee tends to be the most problematic foodstuff regarding OTA analysis, and in some protocols an additional clean-up step is included prior to the affinity column step [369].

3.2.2 Analytical Methods

Several methods for the determination of OTA in foods have been established and make use of the strong native fluorescence of this mycotoxin. The methods include spectrofluorometry [370], thin-layer chromatography (TLC) [371-373] and high-performance liquid chromatography (HPLC) [325, 358, 374-378]. HPLC is nowadays the preferred routine analysis technique for ochratoxins and their metabolites, since it offers better selectivity and sensitivity compared to other detection methods [30]. Positive results are sometimes confirmed by methylation of OTA and a second HPLC experiment [65]. Due to its robustness

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and its easy and cost-effective handling TLC in combination with fluorescence detection is still routinely used in countries outside North America and Europe. Moreover, the official OTA analysis method of the Association of Official Analytical Chemists (AOAC) in green coffee beans is based on TLC for separation [379]. Mono-dimensional TLC has two main sources of error; the supposed mycotoxin spot might be a co-extracted impurity or the amount of mycotoxin present might be incorrectly assessed because of background interference. Both errors could be overcome by use of two-dimensional TLC [372]. Nevertheless this method is inapplicable to roasted coffee beans and coffee products since the detection in coffee products requires a much higher selectivity and sensitivity, which could be achieved by the establishment of HPLC for the determination of OTA in coffee beans and coffee products [380]. Most HPLC methods use a reversed-phase column and an acidic mobile phase, so that the carboxyl group of the toxin is in the undissociated form. Post-column addition of a 10 % ammonia solution may increase the fluorescence emission of OTA ten-fold at alkaline pH [381]. Recently, Fujii et al. reported on an indirect competitive and monoclonal antibody- based ELISA (ic-ELISA) for OTA-screening in green coffee that does not need a clean-up or concentration step but results in a fourfold increased detection limit as compared to HPLC [382]. New and innovative approaches based on immunochemistry and surface plasmon resonance, that are capable of simultaneous detection up to four mycotoxins, have been developed [383]. Additional sensitive methods for OTA detection based on mass spectrometry in combination with liquid chromatography (LC-MS) as well as capillary electrophoresis coupled with laser-induced fluorescence (CE-LIF) have been developed [384].

Results obtained therewith are comparable with those obtained by using a conventional LC- fluorescence (LC-FD) method. Becker et al. and Lau et al. reported on the development of a LC-ESI-MS/MS method for OTA detection in several foods and feeds, which turned out to be especially helpful to confirm doubtful ‘OTA positive’ results obtained by LC-FD [385, 386].

The problem of coelution of interfering compounds could be overcome by the structural information provided by tandem mass spectrometry. Recently, Zöllner et al. reviewed the application of LC-API-MS in the analysis of frequently occurring and highly toxic mycotoxins [30]. There are additional official and validated methods available for OTA analysis in several food matrices besides coffee, that have been recently reviewed by Visonti et al. [387].

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3.3 Risk Assessment

Coffee is one of the most common beverages and its contamination with OTA could therefore represent an important risk factor for human health. The natural occurrence of OTA in green coffee beans has been reported to be within the range of 0.2 to 360 µg/kg.

Subsequent roasting and brewing processes are known to influence the final OTA content [358]. However, considerable inconsistencies are found in the literature regarding this [70, 321, 388-392]. In 1998 Blanc et al. showed that more than 80 % of OTA originally present in green coffee beans is destroyed during soluble coffee manufacture under industrial conditions [357]. This finding was confirmed by Romani et al. [398], which also studied the elimination of OTA during the roasting process and found that both in high and low contaminated samples the OTA content could be reduced by more than 90 %. In addition and depending on the roasting conditions, van der Stegen et al. achieved OTA reductions in the range from 69 % to 96 % [393]. In a survey of 633 samples of final coffee products drawn from the markets of different European countries, the overall mean values of OTA contamination have been found to be 0.8 µg/kg for roasted and ground coffees and 1.3 µg/kg for instant coffees [394]. The EU maximum limits since April 2005 are 5 µg/kg for roasted coffee and 10 µg/kg for instant coffee [395]. By applying the brewing methods frequently used in Europe OTA is almost fully extracted. Recently, Mounjouenpou et al. found out that the OTA extraction parameters recommended by the European Union are not optimal and need to be modified [396]. Assuming a coffee consumption of four cups per day (24 g roasted and ground coffee or 8 g instant coffee), which is above the average per caput consumption level in most European countries, the daily intake of OTA amounts to 19 ng/day in the case of roasted/ground coffee and 10 ng/day in the case of instant coffee. The contribution of OTA in coffee represents less than 2 % of the PTWI set by the Joint FAO/WHO Expert Committee on Food Additives (i. e. 100 ng/kg body weight per week, calculated for a person weighing 70 kg). In 2001 the WHO reported on the contribution of different foods to the overall OTA intake within Europe [397]. Whereas cereals and cereal products account for about 60 % and wines for about 25 % of the overall OTA intake, grape juice and coffee each contributed by about 5 – 7 % to the total amount of OTA taken up; all other foods accounted for less than 1 % of OTA ingestion.

More recently, Napolitano et al. investigated the composition changes during the processing steps “from field to the cup” in coffee from seven different geographic regions [398].

On the Relevance of Ochratoxin A Contamination in Coffee from a Toxicological Point of View