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The fate of altertoxin II during tomato food processing1234Hannes Puntscher1, Doris Marko1, Benedikt Warth15617Währingerstr. 38, 1090 Vienna, AustriaDepartment of Food Chemistry and Toxicology, Faculty of Chemistry, University of Vienna,89101112131415161718192021CORRESPONDING AUTHOR: Benedikt Warth, University of Vienna, Department of Food Chemistry and22Toxicology, Währingerstr. 38, 1090 Vienna, Austria.23Phone: 43 1 4277 7080624E-mail: benedikt.w[email protected]: https://orcid.org/0000-0002-6104-07061
26Abstract27The emerging Alternaria mycotoxin altertoxin II demonstrated substantial genotoxicity in vitro. Ubiquitous28Alternaria ssp. frequently infest various agricultural crops, leading to economic losses and also potential29food safety issues caused by associated mycotoxin contaminations. Due to the lack of commercially30available reference standards, data on the general chemical behavior, the occurrence and the31biological/toxicological effects of altertoxin II are scarce. Since tomatoes are particularly prone to32Alternaria infestations, we simulated the storage and food processing of intact tomatoes and purees after33altertoxin II-addition. We observed significant decrease in altertoxin II concentrations during storage at34room temperature and particularly under thermal stress, by employing a validated LC-MS/MS method.35Moreover, the reduction to the compound’s epoxide group to the alcohol, i.e. the formation of altertoxin36I, was determined at considerable ratios in intact tomato fruits suggesting effective enzymatic s48Alternaria alternata, emerging contaminants, food safety, liquid chromatography, tandem mass49spectrometry, food processing, thermal treatment502
511. Introduction52Altertoxin II (ATX-II) is a toxic secondary metabolite produced by the fungal genus Alternaria (Fig.1). Also53known as “black molds”, Alternaria spp. are ubiquitously occurring saprophytes and plant pathogens,54often responsible for considerable economic losses due to infestations of a broad variety of agricultural55crops like cereals, tomatoes, and oil seeds (EFSA, 2016; Escrivá et al., 2017; Fraeyman et al., 2017; Lee et56al., 2015; Ostry, 2008). Due to the capability of Alternaria fungi to produce toxic secondary metabolites,57infested food and feed may imply a health risks for humans and animals. Moreover, in contrast to other58molds endemic to rather warm climates, this genus can proliferate even at lower temperatures, allowing59for infestations not only on the agricultural field, but also post-harvest during refrigerated storage and60transport (Juan et al., 2016; Ostry, 2008). The scientific report by the European Food Safety Authority61released in 2016 (EFSA, 2016) elaborated a detailed dietary exposure assessment including the four most62studied Alternaria toxins alternariol (AOH), alternariol monomethyl ether (AME), tentoxin (TEN) and63tenuazonic acid (TeA). However, due to the lack of comprehensive occurrence and toxicological data of64other emerging Alternaria toxins, a reliable risk assessment could not be conducted. Defining a threshold65of toxicological concern (TTC value) of 2.5 ng/kg body weight per day for the genotoxic compounds AOH66and AME, investigations indicated a possible health concern considering their estimated exposure data67(EFSA, 2011, 2016). AOH and AME are regularly found in food commodities intended for human68consumption (Hickert et al., 2017; López et al., 2016; Ostry, 2008; Puntscher et al., 2018b; Tölgyesi et al.,692015; Walravens et al., 2016; Zwickel et al., 2016) and proved to be quite stable even along the food70processing chain of tomato products (Estiarte et al., 2018), but also in fruit juices and wine (Scott and71Kanhere, 2001) and even during bread baking (Siegel et al., 2010).72Interestingly, AOH plays only a minor role with the respect to genotoxicity of Alternaria culture extracts,73while particularly ATX-II was identified to show a substantial genotoxic potential (Fleck et al., 2012;74Schuchardt et al., 2014; Schwarz et al., 2012a). However, ATX-II has not been reported in naturally75contaminated food samples so far. This might be due to the lack of commercially available reference76material for the determination of ATX-II and the consequence that it is not screened for in standard assays.77Only a few LC-MS based methods can determine and accurately quantify this potent toxin (Liu and Rychlik,782015; Puntscher et al., 2018b; Zwickel et al., 2016). To obtain reference standards it has been isolated79from fungal cultures in these studies.80The genotoxic and mutagenic effects of AOH described in vitro (Brugger et al., 2006) were linked to its81activity as a topoisomerase I and II poison (Fehr et al., 2009). The mechanism of action related to the even3
82more potent genotoxic ATX-II has not been elucidated so far. The rather reactive epoxide functionality of83ATX-II is likely to be involved in its toxicological effects. However, even altertoxin I (ATX-I, Fig. 1),84structurally the same scaffold but lacking the epoxide group, was reported to be mutagenic to a certain85extent in vitro (Schrader et al., 2006). While ATX-II did not show estrogenic effects in Ishikawa cells86(Dellafiora et al., 2018), chemical degradation reactions of the compound were suggested in the presence87of the anthocyanin delphinidin (Aichinger et al., 2018). Little is known about metabolic pathways of ATX-88II. In several cell lines (Caco-2, HCT 116, HepG2 and V79), it has been reported that the epoxide group of89ATX-II was reduced to an alcohol resulting in ATX-I (Fleck et al., 2014a; Fleck et al., 2014b). In contrast,90ATX-I seemed not to be further metabolized in Caco-2 cells. Xenobiotic pathways also found for AOH and91AME like hydroxylation (Burkhardt et al., 2011; Pfeiffer et al., 2008; Pfeiffer et al., 2007; Tiessen et al.,922017) or glucuronidation (Burkhardt et al., 2012; Burkhardt et al., 2009; Burkhardt et al., 2011; Pfeiffer et93al., 2009), were not determined neither for ATX-I nor for ATX-II (Fleck et al., 2014b).94Please insert Fig. 1 here95In the study at hand, we aimed to gain insights into the fate of a simulated ATX-II contamination in intact96tomato fruits and tomato products during food processing at a laboratory scale. Given that tomatoes are97frequently infested by Alternaria spp., the stability and persistence of this highly genotoxic compound is98of general interest and might be a considerable, yet under-investigated health issue for consumers.994
1002. Material and Methods1012.1 Reagents, solvents and chemicals102ATX-II was isolated from Alternaria alternata cultures grown on rice by an optimized protocol based on103Schwarz et al. (2012b) and confirmed by NMR. ATX-I was purchased from Romer Labs (Tulln, Austria).104Methanol (MeOH), water and acetonitrile (all LC-MS grade) were purchased from Honeywell (Seelze,105Germany) and the eluent additives ammonia solution (25 % in water, for LC-MS) and ammonium acetate106(LC-MS grade) from Sigma Aldrich. For sample preparation Milli-Q water, MeOH (HPLC grade) and acetic107acid (p.a.) from Sigma Aldrich (Steinheim, Germany) were used.108The stock solution of ATX-II (250 µg/mL in MeOH) was diluted for the preparation of the working solution109(25 µg/mL MeOH) needed for the tomato puree experiments and the preparation of calibration solutions.110A multi component calibration solution (also including alterperylenol (ALP) and stemphyltoxin-III111(STTX-III), both isolated from rice cultures) was used for external calibration of additional Alternaria toxins.112All solutions were demonstrated to be stable during storage at -20 C and measurement at 10 C over up113to 72 h.1142.2 Sample preparation115Cherry tomatoes were purchased from a retail market in Vienna, Austria, in May 2018. Twelve fruits, taken116from the same truss, were checked for the absence of visible fungal infections to minimize the chance of117natural contamination. The tomatoes were thoroughly rinsed with water and subsequently dried on paper118towels. All experiments were performed in triplicate. The study design is presented in Fig. 2. Six randomly119picked tomatoes (“Intact” tomato samples) were stored at room temperature until the start of the120experiment. The remaining six tomatoes were cut into pieces using a scalpel on a petri dish and121homogenized in 15 mL tubes at room temperature using a FastPrep-24 5G High Speed Homogenizer (MP122Biomedicals Life Sciences Division Santa Ana, CA, United States). The resulting tomato purees were123combined (“Puree” samples), before transferring 24 representative aliquots of 1 g to plastic tubes (15 mL,124Sarstedt). Six of these tubes were heated up to 100 C under constant magnetic stirring for 30 min (“Pre-125heated puree” samples) using a water bath. As a solvent control, nine tubes were filled with 1 mL Milli-Q126water (“Solvent” samples).127Please insert Fig. 2 here5
128ATX-II was added to six intact tomato fruits (“Intact” tomato samples) by an injection of the stock solution129using a pipette (35-45 µL per tomato fruit). For “puree”, “pre-heated puree” and “solvent” control130samples, the diluted working solution (25 µg/mL) was used: twelve aliquots of “puree”, all six “pre-heated131puree” and nine “solvent” control samples were fortified with ATX-II. The added volume of ATX-II132solutions was adjusted for both, tomato fruits and tomato puree samples, to reach a final concentration133of 1 µg ATX-II per 1 g of sample. No ATX-II solution was added to the remaining six aliquots of “puree”134samples at this point. Three of these were providing for blank matrix samples (“Blank” samples) to135investigate potential natural contaminations. The other three blank matrix samples were spiked right136before their extraction (“Spike” samples) to reach the same final concentration of ATX-II solution as the137other samples. These samples were used to determine the concentration of ATX-II at the formal time138point 0 h (considered as 100 %). All samples (apart form the intact tomato fruits) were vortexed gently139after the addition of ATX-II to allow for appropriate homogenization. Subsequently, six “puree” samples140and three “solvent” control samples were heated up to 100 C for 30 min after adding ATX-II. Mimicking141the thermal processing step allowed for the investigation of thermal stability. The prepared samples were142divided in two batches: batch 1, which was extracted 1.5 h after ATX-II addition, and batch 2, which was143extracted after 24 h (see Fig. 2). Batch 1 included three “intact” tomato fruits, six “blank” samples144including the three intended for spiking right before the extraction, three “puree” samples kept at room145temperature, three “puree” samples heated after ATX-II addition, three “pre-heated puree” samples, as146well as three “solvent” control samples kept at room temperature and three heated after ATX-II addition.147Batch 2 included the remaining samples.1482.3 Sample extraction149Tomato samples were extracted according to the sample preparation protocol described in Puntscher et150al. (2018b). Tomato puree and solvent control samples were directly extracted, while whole tomatoes151fruits were chopped and homogenized before (same procedure as described for the preparation of the152“puree” samples, see above). All homogenized samples (1.000 0.005 g) were extracted by adding 5 mL153extraction solvent (methanol/water/acidic acid, 79/20/1, v/v/v) and shaking for 60 min using an over-154head shaker (Roto-Shake Genie, Scientific Industries, NY, USA). These extracts were subsequently diluted1551:1 with methanol/water (10/90, v/v), centrifuged at 20.000 rcf and 4 C for 15 min and stored at -20 C156until LC-MS/MS measurement.1576
1582.4 Mass spectrometric quantitation159LC-MS/MS analysis was performed on a high-performance liquid chromatography (HPLC) system160(UltiMate3000, Thermo Scientific) coupled to a triple-quadrupole mass spectrometer (MS, TSQ Vantage,161Thermo Scientific) applying a quantitation method validated for tomato matrix as described by Puntscher162et al. (2018b). Briefly, the mass spectrometric system was operated in multiple reaction monitoring163(MRM) mode using negative electrospray ionization. The target analyte ATX-II was quantified by matrix-164matched calibration. Therefore, blank matrix extracts were spiked with the ATX-II stock solution. The same165solution was also used for the tomato samples in the experiment. Calibration solutions were prepared for166final concentrations of 0.1, 0.3, 1, 3, 10, 30 and 100 ng/mL. For quality control, solvent-matched167calibration solutions were also measured (diluted with 10% methanol in water). Furthermore, tomato168matrix-matched multi-analyte solutions, including other perylene quinones (ATX-I, STTX-III and ALP) were169injected at the beginning and the end of a measurement sequence. These served to quantify ATX-I in the170unknown samples. Moreover, the general integrity of the instrument was confirmed by the frequent171measurement of solvent blanks, and a reference standard mix of known small molecules before and after172each sequence. Chromeleon Chromatography Data System Software (version 6.80 SR13 Build 3818),173Xcalibur Software (version 3.0, Thermo Scientific), and TraceFinder (version 3.3) were used for174instrument control, data acquisition and data evaluation, respectively.1757
1763. Results and discussion177The detection and quantitation of ATX-II and ATX-I was conducted by LC-MS/MS analysis. The performed178spiking experiment confirmed a satisfying extraction efficiency of 102-104 % for ATX-II in the tomato179matrix. Natural contamination of Alternaria toxins has been excluded by the analysis of blank matrix180extractions. The concentrations of ATX-II were decreasing in all prepared samples over time. Surprisingly,181after 1.5 h at room temperature, the ATX-II levels were reduced very similarly to 87-90 % (Fig. 3) in both182tomato puree types, e.g. the “puree” just homogenized at room temperature and the “pre-heated puree”183(heated up to 100 C for 30 min before adding ATX-II at room temperature), as well as in the “solvent”184control samples. The comparable decrease in the solvent control samples suggests a generally limited185chemical stability or reactivity of ATX-II at room temperature per se. This has also been reported by Zwickel186et al. (2016). After 24 h at room temperature, the levels further declined to 47-49 % in the tomato puree187samples (“puree”, “pre-heated puree”) and to 18 % in water (“solvent”). This might give an indication that188interactions with the polar solvent water do not favor a stable condition of the compound. ATX-II seemed189to degrade/react slower in tomato matrix, potentially related to stabilizing pH conditions or by matrix190interactions.191Please insert Fig. 3 here192Generally, thermal stress reduces the enzymatic activities of living tissue, which includes the plant193metabolism of xenobiotics. Nevertheless, the ATX-II concentrations for the puree kept at room194temperature and the thermally processed “pre-heated puree” were almost identical after 1.5 and after19524 hours, respectively. This raises the question, whether the enzymatic activity in the puree homogenized196at room temperature was also reduced significantly by the applied mechanical stress. To prevent or197minimize thermal stress already during homogenization, a pause of three minutes was included between198two short homogenization steps of 40 s. However, by disrupting the natural texture of the tomato tissue199and even damaging cell structures, enzymes might be inactivated to some extent, too. Thermal processing200of the tomato puree samples after ATX-II addition clearly led to the most efficient reduction of ATX-II ( 95201%). These samples, heated up to 100 C for 30 min right after adding ATX-II, contained just 4 % and 2.5 %202of the added concentration after 1.5 h and 24 h, respectively. Surprisingly, this is not the case for ATX-II203in the solvent control samples after 1.5 h, which were heated identically right after the ATX-II addition.204These levels were much higher (31 %) and might indicate that interactions with the matrix can play a205different role at higher temperatures by allowing for adsorption, absorption effects, as well as covalent206binding. Finally, ATX-II levels in intact tomato fruits were reduced to 23 % after 1.5 h and therefore much8
207more efficiently as in all other samples at room temperature. After 24 h, less than 1 % of the added amount208was recovered. This strongly indicates active plant metabolism as an effective tool to deal with the209xenobiotic ATX-II. Interestingly, 7 % of the ATX-II amount added to the intact tomato fruit was recovered210as ATX-I after 1.5 h and 12 % after 24 h (for these calculations the molar masses of the compounds were211taken into account). This clearly suggests that the tomato tissue is capable of reducing the epoxide group212of ATX-II to the corresponding hydroxyl-group of ATX-I. This metabolic pathway has already been reported213in in vitro experiments in the human cell lines Caco-2, HCT 116, HepG2 and the Chinese hamster cell line214V79 (Fleck et al., 2014a; Fleck et al., 2014b), but not in plant metabolism. However, de-epoxidation is also215known as a metabolic detoxification pathway for other epoxide-holding mycotoxins, including the216trichothecene deoxinivalenol (DON). De-epoxy DON (DOM-1) was identified as a product of intestinal or217rumen microbe activity and detected in aminal excreta (Pestka, 2010; Yoshizawa et al., 1986). In this case,218the epoxide of DON is converted to a carbon-carbon double bond, instead of being reduced to the alcohol219like in the case of ATX-II. While sulfation and glucuronidation of DON is known for the hydroxyl groups on220positions C3 and C15 preserving the epoxide, glutathione conjugates were identified in naturally221contaminated grain primarily linked via the epoxide group (Uhlig et al., 2016). Mammalian epoxide222hydrolases were reported to play a major role in converting a large number of structurally different223epoxides to the corresponding less reactive vicinal diols and are therefore considered as important224detoxification enzymes (Decker et al., 2009). In an in vivo study, we conducted very recently, a complex225Alternaria culture extract containing high concentrations of ATX-I and ATX-II (among other Alternaria226toxins) was administered to rats via gavage. While ATX-I was recovered in both urine and fecal samples,227ATX-II was not (Puntscher et al., 2018a).228In the presented study, much smaller amounts (in the lower ng range) of ATX-I were also determined in229other tomato matrix samples, but not in the solvent controls. As it was observed for ATX-II, ATX-I230concentrations were nearly the same for the “puree” kept at room temperature and the “pre-heated231puree” (0.7-0.9 ng/g sample after 1.5 h and 3.1-5.0 ng/g sample after 24 h). These amounts correspond232to less than 0.1 % and 0.3 % of the added ATX-II. Heating the sample after ATX-II addition led to slightly233higher ATX-I concentrations. They were similar for both time points (13.4-14.7 ng/g), corresponding to2341.3-1.4 % of the added ATX-II. These results suggest that the tomato matrix contains components235catalyzing chemical reduction of the ATX-II epoxide. However, this conversion reaction is much slower236compared to intact tomato tissue (by a factor of up to 100). Since not 100 % of the decreased ATX-II was237converted to ATX-I, further decomposing products might be identified in the future, as neither238alterperylenol, nor stemphyltoxin-III was determined in any sample.9
2394. Conclusion and outlook240We demonstrated that concentrations of the highly genotoxic Alternaria toxin ATX-II added to tomato241products are decreasing when mimicking food processing steps at a laboratory scale. Generally decreasing242concentrations due to degradation/reaction over time was determined to be comparatively slow, but243clearly a factor in both intact tomato fruits as well as processed tomato purees. Thereby, interactions with244matrix components (chemical reactions, ad- or absorptions) may play a considerable role. Compared to245the respective tomato samples, after 1.5 h at room temperature, lower concentrations of ATX-II were246determined in the water control samples. However, when applying thermal stress, they were higher in247the control samples, suggesting temperature-dependent matrix interactions between ATX-II and the248tomato components. Interestingly, intact fruits showed a much higher efficiency in reducing ATX-II249concentrations at room temperature (up to 100-times more efficient compared to homogenized or250heated tomato products). This and the increasing ATX-I concentrations suggest effective enzymatic251activities promoting the reduction of the epoxide group of ATX-II. The formation of ATX-I traces of up to2520.3 % was also determined for all other tomato based samples. However, intact tomato fruits clearly253showed the most efficient conversion rates to up to 12 % after 24 h. In conclusion, our results indicate a254limited persistence of free ATX-II in tomato based food commodities (particularly during heated food255processing) and the presence of efficient enzymatic detoxification strategies in living tomato tissue.256However, potential health concerns caused by degradation/reaction products cannot be excluded.257Moreover, large-scale food surveys are required to investigate the occurrence of perylene quiones in food258commodities.25910
260Acknowledgements261We would like to acknowledge Iva Cobankovic and the staff of the Mass Spectrometry Center (Faculty of262Chemistry, University of Vienna) for skillful technical support and Prof. Hanspeter Kählig (NMR Center,263Faculty of Chemistry, University of Vienna) for structural characterization. This work was financed by the264University of Vienna.265Conflict of interest266The authors declare no conflict of interest.267Abbreviations268LC, liquid chromatography; MRM, multiple reaction monitoring; MS, mass spectrometry;269Alternaria toxins: ALP, alterperylenol; AME, alternariol monomethyl ether; AOH, alternariol; ATX-I,270altertoxin I; ATX-II, altertoxin II; STTX-III, stemphyltoxin III; TeA, tenuazonic acid; TEN, tentoxin;271Solvents and chemicals: HAc, acetic acid; MeOH, Methanol; NH4Ac, ammonium acetate27211
273Figures274275276Fig. 1 Chemical structures of the Alternaria toxins altertoxin-I and altertoxin-II277278279280281282Fig. 2 Study design for investigating the fate of altertoxin II in tomato commodities, also considering mechanicaland thermal processing12
ree,eh24Pu 5hIntSpikConcentration [µg/g]1.00.82840.60.40.20.00.2ATX-IIATX-I283Fig. 3 Altertoxin I and II concentrations in the experimental tomato and solvent control samples13
285ESI286Electronic supplementary information287Table S1 Sample overview listing all ATX-II and ATX-I concentrationsTime points0 h 1.5 h 24 hSpikeBlankIntact fruitsHeatingPrePostaddition additionATX-IIxxxNon-heated pureexxxxxxPre-heated pureexxSolvent 8 0.0090100 %0%000%0%0.234 0.0130.004 0.00423 %0.4 %0.077 0.0160.127 0.027%12 %0.906 0.0210.510 0.0300.040 0.0020.025 0.00687 %49 %4%2%0.001 00.003 0.0010.015 0.0010.013 0.0010.1 %0.3 %1.4 %1.3 %0.899 0.0210.491 0.02387 %47 %0.001 00.005 0.0010.1 %0.5 %0.933 0.0710.184 0.0310.318 0.03690 %18 %31 %0000%0%0%
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3 51 1. Introduction 52 Altertoxin II (ATX-II) is a toxic secondary metabolite produced by the fungal genus Alternaria (Fig.1). Also 53 known as "black molds", Alternaria spp. are ubiquitously occurring saprophytes and plant pathogens, 54 often responsible for considerable economic losses due to infestations of a broad variety of agricultural
