Alternaria mycotoxins in foodstuffs current information for health risk assessment

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1 Alternaria mycotoxins in foodstuffs current information for health risk assessment Vladimír Ostry 1,2, Jarmila Skarkova 2, Jiri Ruprich 1 1 National Institute of Public Health in Prague, Department for Food Safety and Nutrition, CZ Brno, Czech Republic, ostry@chpr.szu.cz 2 National Institute of Public Health in Prague, Laboratory Department for Food Safety and Nutrition, CZ Brno, Czech Republic SUMMARY The genus Alternaria is ubiquitous and includes both plant pathogenic and saprophytic species that may damage crops in the field or cause post harvest decay. Certain species are also capable of producing mycotoxins which can contaminate plant products. A large number of Alternaria metabolites has been reported to occur naturally in food commodities (e.g. fruit, vegetables, cereals and oil plants). The major Alternaria mycotoxins belong to three structural classes: the tetramic acid derivative, tenuazonic acid (TeA); the dibenzopyrone derivatives, alternariol (AOL), alternariol monomethyl ether (ME) and altenuene (ALT); and the perylene derivatives, the altertoxins (ATX- I- III). The toxic effects of the Alternaria toxins have not yet received the same attention as the biological activities of other mycotoxins. However, the Alternaria mycotoxins should not be underestimated since they are produced by several Alternaria species. The aim of this paper is a processing of current informations ( ) about Alternaria mycotoxins (their producers, analytical methods, an occurrence in foodstuffs and toxicological data) for health risk assessment. The toxicity data of the Alternaria mycotoxins (AOH, AME, ALT and TeA) relevant for hazard characterization are always not available. The toxic capability of Alternaria mycotoxins and their hazard for human consumption is needed to better define eventual guidelines on Alternaria mycotoxin limits in food. Therefore, worldwide regulations for mycotoxins in food and feed do not currently consider Alternaria mycotoxins. Key words: Alternaria; mycotoxins; tenuazonic acid; alternariol; alternariol monomethyl ether; altenuene; altertoxin I-III; foodstuffs; food safety; health risk assessment. INTRODUCTION The genus Alternaria includes nearly 100 species of dematiaceous hyphomycetes that occur worldwide in a variety of habitats (Simmons 2007). Several species are saprobes commonly found in soil and/or on dead or dying plant tissue. The majority, however, are plant pathogens that, collectively, cause a range of economically important diseases on a variety of crops worldwide. The genus Alternaria may affect crops in the field or can cause harvest and postharvest decay of plant products (Logriego et al. 2009). Some Alternaria species produces a number of mycotoxins, including alternariol (AOH), alternariol monomethyl ether (AME), altertoxin I (ATX- I) and tenuazonic acid (TeA) in infected plants and/or in agricultural commodities. Alternaria species is a species of particular interest to mycotoxicologists. There are currently no statutory or guideline limits set for Alternaria mycotoxins by regulatory authorities. Current data on the natural occurrence of Alternaria mycotoxins point to low human dietary exposure. Further information about occurrence and hazard characterization might lead to consideration for the need for regulations, however. Monitoring of foods (e.g. total diet study) using LC-MS/MS method is desired in order to provide information on dietary intake of Alternaria mycotoxins. Monitoring of foods may give impetus to further toxicological studies (for hazard 1

2 characterisation e.g. of benchmark doses) if the occurrence of Alternaria toxins in food becomes a concern (Ostry 2008). CHEMICAL CHARACTERIZATION OF ALTERNARIA MYCOTOXINS More than 30 potentially toxic products have been isolated from Alternaria species. The major Alternaria mycotoxins belong to three structural classes: the tetramic acid derivative, tenuazonic acid (TeA); the dibenzopyrone derivatives, alternariol (AOH), alternariol monomethyl ether (AME) and altenuene (ALT); and the perylene derivatives, the altertoxins ATX- I-III.) (Ostry 2008, Logriego et al. 2009) (see Fig. 1-7). Alternariol (AOH) (3,7,9-trihydroxy-1-methyl-6H-dibenzo[b,d]pyran-6-one); CAS (Chemical Abstracts Services Registry No.): ; molecular weight (M.W.): 258; molecular formula: C 14 H 10 O 5. Chemical structure of AOH is shown in Figure 1. Figure 1 Chemical structure of AOH Alternariol monomethyl ether (AME) (3,7-dihydroxy-9-methoxy-1-methyl-6H-dibenzo[b,d]pyran-6-one); CAS (Chemical Abstracts Services Registry No.): (or ); M.W.: 272; molecular formula: C 15 H 12 O 5. Chemical structure of AME is shown in Figure 2. Figure 2 Chemical structure of AME Altenuene (ALT) (2α,3α,4aβ-tetrahydro-2,3,7-trihydroxy-9-methoxy-4a-methyl-6H-dibenzo[b,d]pyran-6-one); CAS (Chemical Abstracts Services Registry No.): ; M.W.: 292; molecular formula: C 15 H 16 O 6. Chemical structure of ALT is shown in Figure 3. 2

3 Figure 3 Chemical structure of ALT Altertoxin I (ATX-I) (1,2,7,8,12b-pentahydro-1,4,6b,10-tetrahydroxyperylene-3,9-dione);CAS (Chemical Abstracts Services Registry No.): ; M.W.: 352; molecular formula: C 20 H 16 O 6. Chemical structure of ATX-I is shown in Figure 4. Figure 4 Chemical structure of ATX-I Altertoxin II (ATX-II) [perylo(1,2-b)oxirene-7,11-dione,7a,8a,8b,8c,9,10-hexahydro-1,6,8c-trihydroxy-, (7aR,8aR,8bS,8cR)-]; CAS (Chemical Abstracts Services Registry No.): ; M.W.: 350; molecular formula: C 20 H Chemical structure of ATX-II is shown in Figure 5. Figure 5 Chemical structure of ATX-II Altertoxin III (ATX-III) [perylo(1,2-b:7,8-b')bisoxirene-5,10-dione, 1a,1b,5a,6a,6b,10a-hexahydro-4,9-dihydroxy-]; CAS (Chemical Abstracts Services Registry No.): ; M.W.: 348; molecular formula: C 20 H Chemical structure of ATX-III is shown in Figure 6. 3

4 Figure 6 Chemical structure of ATX-III Tenuazonic acid (TeA) (3-acetyl-5-sec-butyl-4-hydroxy-3-pyrrolin-2-one); CAS (Chemical Abstracts Services Registry No.): ; M.W.: 197; molecular formula: C 10 H 15 O 3 N. Chemical structure of TeA is shown in Figure 7. Figure 7 Chemical structure of TeA PRODUCERS OF ALTERNARIA MYCOTOXINS Alternaria mycotoxins produced by different Alternaria species are reported in Table 1. (Patriarca et al., 2007, Ostry 2008, Logriego et al. 2009) Table 1 The production of important Alternaria mycotoxins by Alternaria species Species Mycotoxin A. alternata(fr.) Keissler AOH, AME, ALT, ATX-I-III, TeA A. arborescens E.G. Simmons AOH, AME, ALT, ATX- I, TeA A. brassicae (Berk.) Sacc. AOH, AME, TeA A. capsici-anui Săvul. & Sandu AOH, AME,TeA A. cassiae Jurair & A. Khan ATX- I, -II A. citri Ell. & Pierce AOH, AME, ALT, TeA A. cucumerina (Ell. & Ev.) Elliott AOH, AME A. dauci (Kühn) Groves & Skolko AOH, AME A. gaisen Nagano AME, ATX- I A. helianthi (Hansf.) Tubaki & Nishih. TeA A. cheiranthi (Lib.) P.C. Bolle AOH, AME, TeA A. japonica Yoshii AOH, AME, TeA A. kikuchiana Tanaka AOH, AME, TeA A. longipes (Ell. & Ev.) AOH, AME, TeA, ATX- I A. mali Roberts AOH, AME, ATX-I,-II,-III, TeA 4

5 A. oryzae Hara TeA A. porri (Ell.) Cif. AME, ALT, TeA A. radicina Meier, Drechsler & Eddy ATX-I,-II,-III, TeA A. raphani J.W. Groves & Skolko AME, AOH, TeA A. solani Sorauer AOH, AME, TeA A. tenuissima (Kunze) Wiltshire AOH, AME, ATX-I, TeA A. tomato (Cooke) Jones AOH, AME, ATX-I,-II,-III, TeA A correct identification of the Alternaria species combined with wider surveys on the potentially contaminable crops, is an important key for establishing the toxicological risk related to Alternaria contamination of fruit, vegetables and cereals (Logriego et al. 2009). OCCURRENCE OF ALTERNARIA MYCOTOXINS IN FOODSTUFFS Occurrence of Alternaria mycotoxins in foods and foodstuffs was last reviewed by Ostry (2008). AOH, AME and TeA were frequently detected in apples, apple products, apple juice concentrates, mandarins, olives, pepper, red pepper, tomatoes, tomato products, oilseed rape meal, sunflower seeds, sorghum, wheat and edible oils (olive oil, rapeseed oil, sesame oil, sunflower oil). AOH and AME were detected in prune nectar, raspberries, red currant, barley, oats, Japanese pears, citrus fruit and carrots. AME and TeA were detected in melon. AOH was found in blackberries, gooseberries and strawberries. Natural occurrence of AOH has been reported in apple juice, cranberry juice, grape juice, prune nectar, raspberry juice, red wine and lentils. ATX-I-II were detected in Alternaria-infected apples, sorghum and wheat. The maximum levels of Alternaria mycotoxins reported in marketed products were in the range μg/kg; higher levels were found in samples visibly infected by Alternaria rot, i.e. in products obviously not suitable for consumption. DETERMINATION OF ALTERNARIA MYCOTOXINS Alternaria mycotoxins have been determined recently after separation by LC-MS/MS and EIA. The determination of the seven Alternaria toxins (AOH, AME, ALT, ATX-I, TeA, TEN (tentoxin), TA 1 and 2 (AAL-toxins) in edible oils and oilseeds was developed. Limits of detection (LOD) for AOH, AME, ALT, ATX-I, TeA, TEN, TA 1 and 2 were 0.07, 0.05, 0.25, 0.05, 0.86, 0.11, 0.14 and 0.05 ng/g, respectively (Kocher, 2007). A multi-method was developed with which 33 mycotoxins (including AOH and AME) in various products (peanut, pistachio, wheat, maize, cornflakes, raisins and figs) could be analysed simultaneously. The mycotoxins were extracted with an acetonitrile/water mixture, diluted with water and then directly injected into a LC-MS/MS system. The mycotoxins were separated by reversed-phase LC and detected using an electrospray ionisation interface (ESI) and tandem MS, using MRM in the positive ion mode, to increase specificity for quality control (Spanjer et al., 2008). 5

6 A multi-analyte method for the LC-MS/MS determination of 20 mycotoxins (including AOH, AME and ALT) in food supplements was employed. Liquid-liquid partition with n- hexane was applied as partial clean-up step to remove excess of co-extracted non-polar components. Further clean-up was performed on Oasis HLB cartridges. Limits of detection (LOD) and quantification (LOQ) were in the range of ng/g and ng/g, respectively (Diana Di Mavungu et al., 2009). Stable isotope dilution assays (SIDAs) for the determination of the most important mycotoxins of the black mold Alternaria, namely, AOL and AME, have been developed. For this purpose, deuterated AOL and AME were synthesized by palladium catalyzed protiumdeuterium exchange from the unlabeled toxins. Reaction conditions were chosen in such a manner that the formation of the [(2)H(4)]-isotopologues was favored. The synthesized products were characterized by LC-MS, NMR, and UV-spectroscopy. On the basis of the use of [(2)H(4)]-alternariol and [(2)H(4)]-alternariol methyl ether as internal standards, SIDAs were developed and applied to the determination of AOL and AME in beverages using LC- MS/MS. Method validation revealed a high sensitivity, i.e., low limits of detection (LOD) (AOL, 0.03 ng/g; AME, 0.01 ng/g) and limits of quantitation (LOQ) (AOL, 0.09 ng/g; AME, 0.03 ng/g), respectively. Recovery from spiked apple juice was /- 3.4% for AOL (range ng/g) and /- 1.6% for AME (range ng/g). Interassay precision (expressed as coefficient of variation, CEV) for AOL was 4.0% (7.82 +/ ng/g; vegetable juice, naturally contaminated) and 4.6% (1.04 +/ ng/g; grape juice, naturally contaminated). For AME, a CEV of 2.3% (0.79 +/ ng/g; vegetable juice, naturally contaminated) was obtained. Analysis of fruit juices showed low contamination with alternariol and alternariol methyl ether in general, but higher values of both toxins were found in wine and vegetable juices. The values for AOL were higher than those for alternariol methyl ether in nearly any case. However, the developed SIDA has proven to be optimally suited for further studies on AOL and AME content in food samples to obtain further insight into possible health hazards for the consumer (Asam et al., 2009). A novel approach for the detection of TeA in cereals by liquid chromatography-ion-trap multistage mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine was described. The product of the derivatization reaction and its major MS(2) fragments were characterised by Fourier transform-ion cyclotron resonance tandem mass spectrometry. Without preconcentration, the established method features a limit of detection (LOD) of 10 ng/g using 2g of sample in a rapid workup procedure. Accuracy, precision and linearity were evaluated in the working range of ng/g. TeA was detected in 13 and quantified in 3 out of 27 cereal samples obtained from a local supermarket, the average content being 49 ng/g (highest incidence: 851+/-41 ng/g) (Siegel et al., 2009). Immunochemical method EIA (competitive indirect enzyme immunoassay) have been developed recently for AOH in food (Ackermann et al., 2009). The development of EIA method for the determination of TeA in foodstuffs can be expected (Gross et al., 2009). TOXICOLOGY OF ALTERNARIA MYCOTOXINS Dietary exposure to Alternaria toxins has been linked to a variety of adverse health effects. TeA is able to act as both phytotoxin and mycotoxin, and is acutely toxic for several animal species such as mice, chicken and dogs. In particular, increasing TeA in chicken feed from sublethal to lethal levels progressively reduced feed efficiency, suppressed weight gain and increased internal haemorrhaging. Moreover, TeA has been associated with human haematological disorders such as onyalai, a form of thrombocytopenia which only seems to 6

7 occur in central southern Africa. Finally, TeA exhibited cellular activity toward three mammalian cell lines and it was considered to be able to inhibit the protein biosynthesis since TeA reduced the rate of total protein contents in all three mammalian cell lines (Zhou and Qiang, 2008). On the other hand, the Ames Salmonella test showed no mutagenic activity of TeA (Ostry 2008, Logriego et al. 2009). AOH, AME and ALT are not very acutely toxic. AOH and AME have been reported to be genotoxic, however, AOH was revealed in several assays to be more genotoxic than AME in human colon carcinoma cells (Ostry 2008, Logriego et al. 2009). As shown by Fehr et al. (2007), AOH has cytotoxic, genotoxic, and mutagenic properties in vitro. In particular, AOH reduced the viability of porcine endometrial cells by affecting gene expression on the translational level (Wollenhaupt et al., 2008). AOH could inhibit cell proliferation of human endometrial adenocarcinoma (Ishikawa) and Chinese hamster V79 cell lines by interfering with the cell life. Moreover, AOH have proved to be estrogenic toward both cell lines by increasing the level of alkaline phosphatase (ALP) mrna and the enzymatic activity of ALP in the Ishikawa cell line. Finally, AOH and AME have shown carcinogenic properties, since they induced squamous cell carcinoma in mice (Ostry 2008, Logriego et al. 2009). AOH and AME were identified as potent inhibitors of topoisomerase IIα, which might at least contribute to the DNA strand breaking properties of these mycotoxins (Marko 2007, Marko et al., 2009, Fehr et al., 2009, Bächler et al., 2009). AOL and AME negatively affect progesterone synthesis in porcine granulosa cells in vitro. AOL and AME contaminated feed may therefore affect reproductive performance in pig and other mammalian species (Tiemann et al., 2009). The main biological activity of ATXs is related to their mutagenic capability in the Salmonella Ames Test, which is even higher than alternariol toxins in mice. Among them, ATX-I proved to be acutely toxic in mice and highly mutagenic in mammalian cell lines, while both ATX-I and ATX-III were related to a potential role in cell transformation. Finally, the Alternaria genus includes species which can produce a class of sphinganine analogue mycotoxins (SAMs), the A. alternata toxins (AAL-TA and AAL-TB). These toxins, which are structurally similar to the fumonisins, harmful mycotoxins produced by Fusarium species, have been shown to affect the viability of mammalian cells, in particular dog kidney, rat liver hepatoma and mouse fibroblast cell lines (Ostry 2008, Logriego et al. 2009). CONCLUSIONS The toxicity data of the Alternaria mycotoxins (AOH, AME, ALT and TeA) relevant for hazard characterization are always not available. NOAEL (no-observed-adverse-effectlevels), LOAEL (lowest-observed-adverse-effect-levels) and BMD (benchmark doses) could not be established for Alternaria mycotoxins (AOH, AME, ALT, ATX-I-II) for different endpoints in the studies described above. Exposure limits as TDI (Tolerable Daily Intake) or PMTDI (Provisional Maximum Tolerable Daily Intake) in the terminology of JECFA for Alternaria mycotoxins have not been derived by JECFA and EFSA. However, data on the occurrence of Alternaria mycotoxins in foodstuffs are too limited to make a reliable assessment of dietary exposure to these mycotoxins. Together with such an assessment, more information on the toxic capability of Alternaria mycotoxins and their hazard for human consumption is needed to better define eventual guidelines on Alternaria mycotoxin limits in food. Therefore, worldwide regulations for mycotoxins in food and feed do not currently consider Alternaria mycotoxins. 7

8 REFERENCES Ackermann, I., Curtui, V., Usleber, E Immunochemical assessment of alternariol in food. In: 31 th Mycotoxin Workshop, Münster, Germany, Society for Mycotoxin Research, 2009, s. 56. Asam, S., Konitzer, K., Schieberle, P., Rychlik, M Stable isotope dilution assays of alternariol and alternariol monomethyl ether in beverages. J. Agric. Food Chem., 57(12): Bächler, S., Fehr, M., Pahlke, G., Marko, D Impact of alternariol and alternariol monomethyl ether on Nrf-2 translocation in human tumor cells. In: 31 th Mycotoxin Workshop, Münster, Germany, Society for Mycotoxin Research, 2009, s Diana Di Mavungu, J., Monbaliu, S., Scippo, M.L., Maghuin-Rogister, G., Schneider, Y.J., Larondelle, Y., Callebaut, A., Robbens, J., Van Peteghem, C., De Saeger, S LC-MS/MS multianalyte method for mycotoxin determination in food supplements. Food Addit. Contam. Part A,, 26(6): Fehr, M., Burkart, J., Pahlke, G., Marko, D Impact of oxidative stress on alternariol- and alternariol monomethyl ether-induced toxicity in human tumor cells of different origin. In: 31 th Mycotoxin Workshop, Münster, Germany, Society for Mycotoxin Research, 2009, s. 98. Fehr, M., Pahlke, G., Fritz, J. and Marko, D., Alternariol acts as a topoisomerase poison. In: Gesellschaft für Mycotoxin Forschung (ed.) Proceedings of the 29th mycotoxin workshop, May, 2007, Stuttgart-Fellbach, Germany, p Gross, M., Curtui, V., Usleber, E Production snd chracterization of antibodies against tenuazonic acid. In: 31 th Mycotoxin Workshop, Münster, Germany, Society for Mycotoxin Research, 2009, s. 59. Kocher, U., Determination of 7 Alternaria-Toxins in edible oil and oilseeds by LC-MS/MS. In: 29 th Mycotoxin Workshop. May 14-16, Society for Mycotoxin research, Stuttgart-Fellbach, Germany, p. 72. Logrieco, A., Moretti, A. and Solfrizzo, M., Alternaria toxins and plant diseases: an overview of origin, occurrence and risks. World Mycotoxin Journal, 2 (2): Marko, D., Mechanisms of the genotoxic effect of Alternaria toxins. In: 29 th Mycotoxin Workshop, Society for Mycotoxin research, Stuttgart-Fellbach, Germany, p. 48. Marko, D., Bächler, S., Pahlke, G., Fehr, M TDP 1 modulates the DNA damaging properties of the topoisomerase poison alternariol. In: 31 th Mycotoxin Workshop, Münster, Germany, Society for Mycotoxin Research, 2009, s Ostry, V., Alternaria mycotoxins An overview on chemical characterization, producers, toxicity, analysis and occurrence in foodstuffs. World Mycotoxin Journal, 1 (1): Patriarca, A., Azcarate, M.P., Terminiello, L. and Fernandez Pinto, V., Mycotoxin production by Alternaria strains isolated from Argentinean wheat. International Journal of Food Microbiology 119: Siegel, D., Rasenko, T., Koch, M., Nehls, I Determination of the Alternaria mycotoxin tenuazonic acid in cereals by high-performance liquid chromatography-electrospray ionization ion-trap multistage mass spectrometry after derivatization with 2,4-dinitrophenylhydrazine. J Chromatogr A. 1216(21): Simmons, E.G., Alternaria: An Identification Manual. The American Phytopathological Society Press, 775 pp. Spanjer, M.C., Rensen, P.M. and Scholten, J.M., LC-MS/MS multi-method for mycotoxins after single extraction, with validation data for peanut, pistachio, wheat, maize, cornflakes, raisins and figs. Food Additives and Contaminants 25:

9 Tiemann, U., Tomek, W., Schneider, F., Müller, M., Pöhland, R., Vanselow, J The mycotoxins alternariol and alternariol methyl ether negatively affect progesterone synthesis in porcine granulosa cells in vitro. Toxicol Lett. 186(2): Wollenhaupt, K., Schneider, F. and Tiemann, U., Influence of alternariol (AOH) on regulator proteins of cap-dependent translation in porcine endometrial cells. Toxicology Letters 182: Zhou, B. and Qiang, S., Environmental, genetic and cellular toxicity of tenuazonic acid isolated from Alternaria alternata. African Journal of Biotechnology 7: