Toxicon 114 (2016) 65e74

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Toxicon

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A novel containing armillatus

Dahai Shao a, Shusheng Tang b, Rosanne A. Healy c, 1, Paula M. Imerman a, * Dwayne E. Schrunk a, Wilson K. Rumbeiha a, a Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, 50011, Ames, IA, USA b College of Veterinary Medicine, China Agriculture University, 100193, Beijing, China c Department of Organismic and Evolutionary Biology, Harvard University, 02138, Cambridge, MA, USA article info abstract

Article history: Orellanine (3,30,4,40-tetrahydroxy-2,20-bipyridine-1,10-dioxide) is a tetrahydroxylated di-N-oxidized Received 4 October 2015 bipyridine compound. The toxin, present in certain species of Cortinarius , is structurally Received in revised form similar to herbicides and . and are the major 5 February 2016 orellanine-containing mushrooms. Cortinarius mushrooms are widely reported in Europe where they Accepted 11 February 2016 have caused human poisoning and deaths through accidental ingestion of the poisonous species Available online 23 February 2016 mistaken for the edible ones. In North America, Cortinarius orellanosus was recently reported to cause renal failure in a Michigan patient. Cortinarius mushroom poisoning is characterized by Keywords: Chromatography delayed acute renal failure, with some cases progressing to end-stage kidney disease. There is debate Mass spectrometry whether other Cortinarius mushroom contain orellanine or not, especially in North America. Currently, Orellanine there are no veterinary diagnostic laboratories in North America with established test methods for detection and quantitation of orellanine. We have developed two diagnostic test methods based on HPLC and LC-MSMS for identification and quantitation of orellanine in mushrooms. Using these methods, we have identified as a novel orellanine-containing mushroom in North America. The mean toxin concentration of 145 ug/g was <1% of that of the more toxic C. rubellus. The HPLC method can detect orellanine at 17 mgg 1 while the LC-MSMS method is almost 2000 times more sensitive and can detect orellanine at 30 ng g 1. Both tests are quantitative, selective and are now available for veterinary diagnostic applications. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Among these, Cortinarius orellanus and Cortinarius rubellus are recognized as the most frequent causes of human poisoning. Some Fruiting bodies in the Cortinarius include both edible and of these human poisoning cases have resulted in mortality (Denal toxic mushrooms. Toxic species are in the subgenus Cortinarius, et al., 2001; Prast et al., 1988). Toxic Cortinarius mushrooms are section (formerly subgenus Leprocybe, section Orellani mistaken for edible species and Cortinarius Bresinsky and Besl, 1990). Toxic species contain orellanine praestans because species in Cortinarius are notoriously difficult to (3,30,4,40-tetrahydroxy-2,20-bipyridine-1,10-dioxide), a tetrahy- morphologically identify with certainty (Niskanen et al., 2009, droxylated di-N-oxidized bipyridine molecule, which is a highly 2011; Peintner et al., 2004). They have also been mistaken for potent nephrotoxin. This toxin is similar in molecular structure to more easily distinguished edible species such as chantherelles due herbicides Paraquat and Diquat, and to the dopaminergic neuro- to lack of knowledge and experience on the part of amateur har- toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Toxic vesters (Lincoff, 2011; Phillips, 2006; Waring, 2007). So far, studies Cortinarius mushrooms have been widely reported in Europe. have confirmed C. orellanus (Cantin et al., 1989; Koller et al., 2002; Oubrahim et al., 1997; Richard et al., 1988), C. rubellus (Cantin et al., 1989; Koller et al., 2002; Judge et al., 2010; Oubrahim et al., 1997), Cortinarius orellanosus (Judge et al., 2010), and Cortinarius rain- * Corresponding author. Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, 50011, Ames, IA. USA. ierensis (Oubrahim et al., 1997) as some of the species with sig- E-mail address: [email protected] (W.K. Rumbeiha). nificant amounts of orellanine. In the opinion of Robertson et al. 1 Current address: Department of Plant Pathology, University of Florida, 32611, (2006). C. rainierensis is a synonym of C. rubellus. Other synonyms Gainesville, FL, USA. http://dx.doi.org/10.1016/j.toxicon.2016.02.010 0041-0101/© 2016 Elsevier Ltd. All rights reserved. 66 D. Shao et al. / Toxicon 114 (2016) 65e74 include Cortinarius orellanoides and varieties thereof, Cortinarius to test Cortinarius mushroom specimens collected from North speciosissimus and varieties thereof, Cortinarius speciosus J. Favre, America, for presence of orellanine. Confirmation of mushroom and orellanoides (Kirk, 2016). species was done by genetic methods. In North America, C. rubellus is found in Northern areas of the east and west coasts, while C. orellanosus has been reported in 2. Materials and methods Michigan. There is one reported case of human poisoning in North America involving Cortinarius orellanosus, in which a definitive 2.1. Chemicals, reagents, and materials identification of Cortinarius mushrooms was made (Judge et al., 2010). Although there are other cases previously reported in High performance liquid chromatography (HPLC)-grade Meth- North America, it is debatable whether the mushrooms involved in anol, HPLC-grade acetonitrile, concentrated hydrochloric acid, those cases were actually Cortinarius since neither a definitive ammonium hydroxide, ammonium acetate, o-phosphoric acid identification of species, nor a demonstration of presence of orel- (85%), formic acid, and 0.45 mm nylon syringe filters were pur- lanine was done (Bronstein et al., 2008, 2010; Denal et al., 2001; chased from Thermo Fisher Scientific (Waltham, MA). Orellanine Judge et al., 2010; Karlson-Stiber and Persson, 2003; Litovitz standard (95%) was purchased from Ramidus AB (Lund, Scania. et al., 1996; Moore et al., 1991; Raff et al., 1992). The mix up is due in Sweden). All aqueous solutions were prepared in 18.2 MU $cm de- part to misidentifications, a poor understanding of species ionized (DI) water (Aries High Purity Water System, Aries Filter geographic limits of the fungi, and a failure to conduct confirmatory Network, West Berlin, NJ). Bond Elut Jr Mycotoxin clean-up col- analytical tests for orellanine in suspect mushrooms. Until recently, umns were purchased from Agilent Technologies (Santa Clara, CA). North America and Europe were thought to share the same species A solution of 3 M hydrochloric acid was prepared by adding 24.9 mL of fungi. While it is true that there are some species that are broadly concentrated hydrochloric acid into DI water and bringing the dispersed among continents, molecular phylogenetic analyses have volume to 100 mL by water. A solution of 3 M hydrochloric acid in demonstrated that some fungi, particularly ectomycorrhizal fungi was prepared by adding 24.9 mL concentrated hydro- like Cortinarius, are endemic (Bonito et al., 2013; Matheny et al., chloric acid into HPLC-grade methanol and bringing the volume to 2009; O'Donnell et al., 2011). 100 mL by methanol. A 4 mM ammonium acetate aqueous solution Lack of awareness of species complexes have also contributed to was prepared by dissolving 0.308 g ammonium acetate in DI water erroneous identifications. Outdated mushroom field guides use a and bringing the volume to 1 L. A 1% formic acid aqueous solution single name to erroneously label multiple species, and yet these was prepared by diluting 5 mL formic acid to a total volume of species may not share the same genetic, chemical or ecological 500 mL with DI water. Orellanine standards were prepared by properties (Harrington and Wingfield, 1995; Jaklitsch et al., 2013; dissolving a corresponding amount of orellanine pure standard in Zhang et al., 2015). Mycologists are now more aware of these po- 3 M hydrochloric acid. They were stored in aluminum-foil-wrapped tential problems, and as a result, many groups of mushrooms are vials for protection from photo-decomposition. currently undergoing revision, with new species being discovered (Folz et al., 2013; Justo et al., 2014; Liimatainen et al., 2014). 2.2. Cortinarius mushrooms analyzed for orellanine Therefore, it is important not to assume that toxins or lack thereof in European species are similarly present or absent in North All mushrooms studied are from the genus Cortinarius. Corti- American species, even if they appear morphologically similar. narius distans, C.alboviolaceus, C. glaucopus, C. cinnabarinus, and C. Moreover, because some Cortinarius mushrooms are edible, it is castaneus were provided by the Ada Hayden Herbarium (ISC) of advisable to conduct additional studies to identify other Cortinarius Iowa State University (Ames, IA). Cortinarius alboviolaceus, C. cor- species that may contain the toxin. Lastly, failure to test suspect rugatus, C. coalescens, and C. armillatus were collected in Harvard mushrooms to confirm presence of the orellanine toxin has also Forest, Petersham, Massachusetts and were accessioned at the contributed to erroneously attributing intoxication to orellanine. Farlow Herbarium (FH) of the Harvard University Herbaria (Cam- Chemical confirmation of the presence of orellanine is a better way bridge, MA). Cortinarius rubellus, a positive control specimen of diagnosing intoxication and adds value to morphological known to contain significant quantities of orellanine, was a gift identification. from Dr. Otto Miettinen, Finnish Museum of Natural History e Mushroom-induced toxicity is also commonly encountered in LUOMUS Botanical Museum, University of Helsinki (Helsinki, pets, particularly dogs that eat raw mushrooms in yards. The most Finland). (Accession number: H 6835:3623, H 6694-310, H 6693- widely recognized mushroom-induced toxicity in pets is the 310, H 6869 446, H 6689 398, H 6705 352, and H 6900 335.) An amatoxin-induced hepatotoxicity (Puschner et al., 2007; Puschner , known to be free of orellanine, Shiitake (Lenti- and Wegenast, 2012). There are no reports of Cortinarius mush- nula edodes), was purchased from a local grocery store in Ames, IA room poisoning reported in pets. However, because these mush- and served as a negative control sample. The photographs of the rooms are present in North America and poisoning has been above-mentioned mushrooms are shown in Figs. 1 and 2. All reported in humans, we suspect that animal poisoning by these voucher specimens used in this study are listed along with their mushrooms occurs. A lack of awareness among veterinarians about collector numbers, date of collection, and collection sites in Table 1, the toxicity of these mushrooms and coupled with a lack of except for C. rubellus for which collection site information was not confirmatory diagnostic tests for orellanine may limit diagnosis of available. poisoning by these mushrooms. Until now, there was no veterinary diagnostic laboratories set up for detection and quantitation of 2.3. Sample preparation orellanine in mushrooms for confirmation of orellanine poisoning in people and pets in North America. The sample preparation procedure was adapted from a pub- There were two objectives for this study: 1) to develop selective, lished method (Herrmann et al., 2012). Two major changes were sensitive and quantitative in-house diagnostic test methods for made. A clean-up step was added to further remove the in- detection and quantitation of orellanine in mushrooms. These terferences from orellanine. This cleanup step gave good selectivity methods will be used for definitive confirmation of the presence of for orellanine. Because the pH of mobile phases was essential in orellanine for quick unequivocal diagnosis of orellanine poisoning stabilizing orellanine in the HPLC analysis, we replaced formic acid in animals. The test can also be used for research purposes; and 2) with o-phosphoric acid because the latter is less volatile and able to D. Shao et al. / Toxicon 114 (2016) 65e74 67

Fig. 1. A. Cortinarius armillatus RH1535; B. Cortinarius armillatus RH1557; C. Cortinarius armillatus RH1557; D. Cortinarius cf distans (recorded as C. brunneus, ISC 437745); E. Cor- tinarius cinnabarinus (ISC 443350); F. Cortinarius rubellus drawing by Belinda Y Mahama. All bars ¼ 2 cm. maintain the pH for an extended period of time. control, and a negative control orellanine-spiked mushroom sam- Briefly, a 15 mg mushroom sample was mixed with 4 mL 3 M ple were included. A calibration curve was established with 0 mg, hydrochloric acid and then extracted for 3 h with magnetic stirring. 0.25 mg, 0.50 mg, 1.0 mg, 1.5 mg, 2.0 mg, and 2.5 mg of orellanine in The mixture was then centrifuged at 3000 rpm for 30 min solvent for HPLC analyses. Because the LC-MSMS method was more (1744 g). The pH of the supernatant was neutralized with 1 mL sensitive than the HPLC method, a calibration curve with 0 mg, ammonium hydroxide and then added onto clean-up columns. 0.1 mg, and 0.2 mg of orellanine in solvent was used for LC-MSMS After washing the columns with 20 mL DI water and 20 mL analyses. These extracts were used for both HPLC and LC-MSMS methanol (both adjusted to pH 1.5 with formic acid), the orellanine analyses. In order to protect orellanine from photo- was eluted with 5 mL 3 M hydrochloric acid in methanol at a flow decomposition, orellanine-containing solutions were kept in rate of 10e20 mL/min. The eluent was concentrated to 0.2 mL by aluminum-foil-wrapped tubes throughout the procedure. nitrogen gas. The negative control mushroom, Shiitake (L. edodes) was forti- 2.4. HPLC conditions fied with orellanine at a level of 50 mgmL 1. The extracts were prepared in the same way as described above and subjected to Measurements were performed with an Ultimate 3000 HPLC analysis by HPLC or liquid chromatography-tandem mass spec- system (Dionex, now Thermo Fisher Scientific, Waltham, MA) trometry (LC-MSMS). This was the approach used for determina- equipped with a Chromeleon program for the system manipulation, tion of spike recovery. The orellanine-positive control mushroom, data acquisition and analysis, a photodiode array UVeVis detector, C. rubellus was also analyzed as a reference. All mushroom extracts and an HPLC column (PLRP-S C18, 3 mm, 150 mm 4.6 mm, Agilent fi were quanti ed by the HPLC method and those that tested positive Technologies, Santa Clara, CA). The mobile phase consisting of fi for orellanine were subject to LC-MSMS con rmation. In all test 4 mM ammonium acetate aqueous solution (A) and methanol (B) procedures, a positive control C. rubellus extract and a negative (Herrmann et al., 2012) was pumped at a flow rate of 0.3 mL min 1. 68 D. Shao et al. / Toxicon 114 (2016) 65e74

Fig. 2. AeB Cortinarius corrugatus FH RH1522; C-D. Cortinarius cf alboviolaceus FH RH1546; E-F Cortinarius cf coalescens FH RH1552; All bars ¼ 2 cm.

Table 1 Voucher information for Cortinarius tested for orellanine in this study.

Species Herbarium Accession no. Collector no. Locality Date

armillatus FH RH1535 RH1535 USA, MA: Harvard Forest 5-Sep-13 armillatus FH RH1577 RH1577 USA, MA: Harvard Forest 6-Sep-13 cf coalescens FH RH1552 RH1552 USA, MA: Harvard Forest 5-Sep-13 corrugatus FH RH1522 RH1522 USA, MA: Harvard Forest 5-Sep-13 corrugatus FH RH1545 RH1545 USA, MA: Harvard Forest 5-Sep-13 cf alboviolaceus FH RH1546 RH1546 USA, MA: Harvard Forest 5-Sep-13 cf alboviolaceus FH RH1574 RH1574 USA, MA: Harvard Forest 6-Sep-13 cf distans ISC 437745 RH and LHT sn USA, IA: Mann Wilderness Area State Preserve 13-Jul-04 alboviolaceus, ISC 437743 RH sn USA, IA: Mann Wilderness Area State Preserve 18-Aug-04 glaucopus ISC 438732 HD Thiers 53,156 USA, CA: Lincoln Creek Campground, Sierra County 5-June-90 cinnabarinus ISC 443350 RH66 USA, IA: Woodman Hollow State Preserve 30-Aug-06 castaneus ISC 438439 RH sn USA, IA: Fallen Rock State Preserve 6-Nov-04 rubellus H 6835:3623 2009 rubellus H 6694-310 2000 rubellus H 6693-310 2000 rubellus H 6869 446 1996 rubellus H 6689 398 2000 rubellus H 6705 352 3-Oct-03 rubellus H 6900 335 1997 D. Shao et al. / Toxicon 114 (2016) 65e74 69

Both mobile phases were adjusted to pH 1.5 with o-phosphoric Table 3 acid. A gradient elution was used to give an optimum separation. A summary of LC-MSMS specifications for orellanine analysis. The details of the gradient protocol were tabulated in Table 2.A Q1, m/z Q3, m/z Capillary voltage, V Collision voltage, V Dwell time, ms total running time of 20 min was used. The injection volume was 253 163 36 20 0.5 50 mL. The separation was performed at room temperature. Orel- 253 191 36 25.5 0.5 lanine was detected spectroscopically at the wavelength 295 nm in 253 219 36 20 0.5 the UVeVis detector. 253 236 36 14 0.5

2.5. LC-MSMS conditions technique, and each placed into 250 ml of 2x CTAB where it was stored at 24 C until extraction. Samples were pulverized to a fine Analyses were performed using a triple-quadruple Varian 310 grain in CTAB, and 250 ml additional 2x CTAB added, mixed well, LC-MSMS system (Agilent Technologies, Santa Clara, CA) with an and incubated at 60 C for 1 h. DNA was extracted with an equal electrospray ionization (ESI) chamber in the positive mode, a 410 volume of 24:1 :isoamyl alcohol followed by centrifu- Varian Prostar autosampler (Agilent Technologies, Santa Clara, CA), gation at 13,000 rpm for 12 min. Samples were incubated with 50 ml and two 210 Varian Prostar HPLC pumps (Agilent Technologies, RNAase A at 37 C for 30 min, followed by another extraction and Santa Clara, CA). Needle voltage was 3000 V, CID gas pressure was centrifugation as before. A 2/3 volume of isopropanol was added to 0.20 Pa, drying gas temperature was 150 C, nebulizer gas pressure each extract, chilled at 20 C overnight, then centrifuged at was 3.45 105 Pa, drying gas pressure was 6.89 104 Pa, detector 13,000 rpm for 7 min. Supernatant was removed and the pellet was was biased at 1500 V, and acquisition time was 10 min. The mobile washed with chilled 70% ethanol followed by centrifugation at phase consisted of solvent A: 1% formic acid, and solvent B: 13,000 rpm for 5 min, then ethanol pipetted off, and the pellet dried acetonitrile, at an isocratic elution with a ratio of 5:95, v:v. The in a laminar flow hood for 1 h. DNA was reconstituted in 50 mlTE mobile phase was modified from a study (Herrmann et al., 2012) for buffer, and an aliquot diluted to 10x. The internal transcribed spacer use with PRP-1 column (Hamilton PRP-1, 10 mm, 250 4.1 mm I.D. (ITS) and 50 end of the 28S (LSU) regions of nuclear ribosomal DNA purchased from Phenomenex, Torrance, CA). The flow rate was were amplified using primer pairs ITS5/LR5 (Bertini et al., 1999; 0.4 mL/min. This also was modified to speed up the analysis. Vilgalys and Hester, 1990) with the following PCR cycling Orellanine was detected using the following parent to daughter schedule: 1) 94 C for 2 min 2) 94 C for 1 min 3) 50 C for 45s 4) transitions: 253 > 163, 253 > 191, 253 > 219, 253 > 236 which were 72 C for 1.5 min 5) repeat steps 2e4 35 times 6) final elongation at obtained by direct infusion of orellanine. A detailed summary of LC- 72 C for 5 min. Sanger sequencing was done with the same primers MSMS parameters is listed in Table 3. Samples were diluted 1:10 in at Beckman Coulter Genomics Facility. Sequences were viewed and 90:10, v:v, methanol: 3 M HCl, and 20 ml was injected on the column trimmed to include only high quality bases in Geneious Pro v 5.6.7 (Herrmann et al., 2012). (Drummond et al., 2012). Sequences were hand aligned with highly similar sequences in SeAl v 2.0a11 (Rambaut, 2007). Maximum 2.6. Genetic confirmation of Cortinarius armillatus Likelihood analysis was performed on the final alignment using RAxML (Stamatakis, 2014) with a GTR þ gamma nucleotide sub- Cortinarius armillatus mushrooms (Fig. 1 AeB) were collected on stitution model and 1000 bootstrap iterations on the Cipres Portal Sept 6e7, 2013 in Harvard Forest, in Petersham, located in central (Miller et al., 2010). Sequences have been deposited in GenBank Massachusetts. This was part of a study of ectomycorrhizal under the numbers KT750146-KT750155. (mutually beneficial root symbionts) fungi in the Harvard Forest. Collections used in this study included RH1577 and RH1593, which came from the Prospect Hill tract, and RH1535, which came from 3. Results the Tom's Swamp tract. These tracts were moist, well drained sites with acidic soil (pH 3.6) supporting a mixed forest dominated by a 3.1. HPLC canopy of red oak, paper birch, white pine, red maple, and eastern hemlock with an understory of American . All Cortinarius HPLC analysis was performed on all species of mushrooms listed basidiomata were collected under red oak tree canopies, but C. in Section 2.2. Results show that of all the mushrooms analyzed, armillatus was sequenced from birch root tips. Spores were exam- only C. armillatus and the well-known orellanine-containing spe- ined and measured in a water mount, and color change noted with cies, C. rubellus, tested positive for orellanine. Since this is the first Melzer's reagent (iodine solution). Images of C. armillatus used in time orellanine was found in C. armillatus, this mushroom was this study are shown in Fig. 1 A and B. subsequently re-extracted and tested on a separate day for confir- For a definitive identification of the mushrooms, the ITS locus, mation of the presence and quantification of orellanine. which is the barcoding region for fungi was amplified and In Fig. 3, Chromatographs of orellanine are shown for (a) from sequenced. The ITS region of DNA is the official barcode locus for top to bottom line, C. armillatus, negative control mushroom (L. fungi (Schoch et al., 2009), and is a good indicator of species edodes), and orellanine standard solution and (b) from top to bot- identity for most fungi, including Cortinarius (Niskanen et al., 2009, tom line, C. rubellus (positive control), negative control mushroom 2011). For isolation of genomic DNA, a 3 mm2 piece of clean gill (L. edodes), and orellanine standard solution. with spores was excised from each specimen using sterile A summary of orellanine results for C. armillatus and C. rubellus

Table 2 A summary of HPLC gradient for orellanine analysis.

Time, min 4 mM ammonium acetate aqueous solution (A), % Methanol (B), %

0e2.0 98 2 2.0e4.0 15 85 4.0e20.0 15 85 70 D. Shao et al. / Toxicon 114 (2016) 65e74

Fig. 3. Chromatographs of orellanine in: (a) Test mushroom C. armillatus, negative control mushroom (Lentinula edodes), and orellanine standard solution; (b) Positive control C. rubellus, negative control mushroom (Lentinula edodes), and orellanine standard solution.

are shown in Table 4. The spike recovery results are shown as well. 137 mgg 1 for the two separate analyses, respectively. The mean The chromatographs of these samples as well as the ones for concentration from the two analyses was 145 mgg 1. The calibra- negative control Shiitake mushrooms (L. edodes) are shown in tion curve for testing orellanine in C. rubellus gave a linearity of Fig. 3. Calibration curves showed a good linearity of 0.996 and 0.999 0.997 (R2) in the range of 0.25e2.5 mg, indicating reliable calibra- for the ranges of 0.25e2.5 mg and 1e10 mg respectively for cali- tion. The concentration of orellanine in C. rubellus was bration curves established when testing orellanine in C. armillatus. 24,000 mgg 1 [2.4% (w/w)], two orders of magnitude higher than The dry-weight orellanine concentration was 153 mgg1 and that in C. armillatus, consistent with the fact that it is a potent

Table 4 A summary of HPLC analyses of orellanine in C. armillatus, C. rubellus, and a spiked control sample.

Mushroom species Sample Calibration range of orellanine, mg Linearity (R2) Concentration, mgg 1 Average concentration, mgg 1 Spike recovery, %

C. armillatus 1 0.25e2.5 0.996 153 145 e 21e10 0.999 137 C. rubellus 1 0.25e2.5 0.997 2.4 104 2.4 104 e (2.4% (w/w)) (2.4% (w/w)) Negative control (L. edodes) 1 0.25e2.5 0.997 ee 78.3 D. Shao et al. / Toxicon 114 (2016) 65e74 71 mushroom. The spike recovery of the negative control specimen further analyses. RH1535 and RH1577 sequences were highly was 78.3%. The lowest quantitation standard was 0.25 mg, equiva- similar to the sequence from the neotype specimen for C. armillatus lent to 17 mgg1 orellanine based on this method. All other (DQ114744) with one bp difference. Since we did not sequence the mushrooms tested negative for orellanine at the lowest quantifi- ITS2 region, the length was 334 bp rather than the full 605 bp, so able standard of 17 mgg 1 of orellanine. there could be more differences. However, the nearly perfect match of the 346 nucleotides in the ITS1 and 5.8S region helps to 3.2. LC-MSMS molecularly identify this taxon as C. armillatus. Phylogenetic anal- ysis of our ITS sequences in an alignment with highly similar se- The chromatographs of orellanine in negative control Shittake quences downloaded from a BLAST search against the nucleotide (L. edodes) mushrooms, spiked L. edodes mushrooms, C. armillatus database in GenBank, and other species delimited in the armillati mushrooms, and C. rubellus mushrooms by LC-MSMS are shown in section of Cortinarius (Niskanen et al., 2011) inferred our sequences Fig. 4. The slightly off retention time of the orellanine peak in the as unambiguously consistent with that of the neotype specimen C. rubellus mushroom may be due to vast difference in the amounts sequence for C. armillatus (Supplementary Fig. 1). The 28s locus was of toxin being chromatographed. LC-MSMS analyses for orellanine sequenced for RH1577 (770 bp) and RH1535 (819 bp). This locus is in C. armillatus showed a concentration of 130 mgg 1. The difference well conserved in Cortinarius, and though the 28S sequences of our between this and the HPLC results was only 10%. By HPLC, the mean specimens were a perfect match to C. armillatus, they were also 99% orellanine concentration was 145 mgg1. The concentration for similar to Cortinarius luteo-ornatus and Cortinarius pinigaudis. fi orellanine in C. rubellus was 26,000 mgg 1[2.6% (w/w)]. This was Morphologically, these specimens t the description for e m also comparable to HPLC results, with only 7% difference noted C. armillatus. Spore size was 9.6-10-10.4 6 6.4 m, they were between the two results. The spike recovery was 85.0% and very dextrinoid in Melzer's reagent, and the spore wall was relatively m comparable to that of HPLC (78.3%). A linearity with a good corre- thick, ~0.4 m. Color of annulus was initially whitish (Fig. 1 A) but lation coefficient of 0.989 (R2) was obtained in the range of became rust-colored due to spore deposit. fi 0.1 mge0.2 mg for calibration curves. A summary of these results is Both morphology and molecular analyses con rmed the iden- shown in Table 5. Results of LC-MSMS provide definitive confir- tity of the Cortinarius samples used in this study as C. armillatus.It mation of the identity of the toxin in C. armillatus as orellanine. appears that this species has a widespread distribution, being found in both the North Eastern US and in Europe, where it is known to associate with birch trees (Niskanen et al., 2011). We fi 3.3. Genetic identi cation of Cortinarius armillatus sequenced C. armillatus from birch roots at the site of the collec- tions of fruit bodies in Harvard Forest. It was essential to have definitive genetic identification of C. armillatus. The post trimmed ITS sequences for RH1535 (345 bp, Genbank accession KT750146) and RH1577 (346 bp, Genbank 4. Discussion accession KT750150) included ITS1 and 5.8s. The sequence for RH1593 (141 bp) was too short and therefore not included in Orellanine-containing mushrooms are present in North America

Fig. 4. Chromatographs of orellanine in (a) negative control Shiitake (Lentinula edodes) mushrooms, (b) spiked L. edodes mushrooms, (c) C. armillatus, and (d) C. rubellus (positive control) by LC-MSMS analysis. 72 D. Shao et al. / Toxicon 114 (2016) 65e74

Table 5 A summary of LC-MSMS analyses of orellanine in C. armillatus, C. rubellus, and a spiked control sample.

Mushroom species Calibration range of orellanine, mg Linearity (R2) Concentration, mgg 1 Spike recovery, %

C. armillatus 0.1e0.2 0.989 130 e C. rubellus 0.1e0.2 0.989 2.6 104 e (2.6% (w/w)) Negative control (L. edodes) 0.1e0.2 0.989 e 85.0

and Europe. Some of these mushrooms are edible, while others are also a danger to other animal species which can consume especially those belonging to the subgenus Cortinarius section mushrooms. No single diagnosis of Cortinarius mushroom Orellani are toxic (Bresinsky and Besl, 1990; as subgenus Leprocybe). poisoning in animals including pets has ever been reported in In North America, C. rubellus is found in Northern areas of the east North America, yet toxic Cortinarius mushrooms including and west coasts, while C. orellanosus has been reported in Michigan. C. rubellus and C. orellanosus are reported to exist in North America. These toxic Cortinarius mushroom species contain orellanine, a C. rubellus is found in northern areas of both the east and west bipyridyl compound similar to herbicides Paraquat and Diquat. coasts. C. orellanosus has only recently been identified reported in Both people and animals are susceptible to orellanine, a toxin with Michigan but its entire geographic distribution is currently un- potentially multiple toxic outcomes, of which nephrotoxicity is the known. Veterinarians generally are not aware of dangers posed by most widely recognized. All previously known orellanine- these mushrooms. It is possible that a lack of awareness among containing mushroom species belonging to the subgenus Corti- veterinarians, coupled with a lack of suitable diagnostic tests may narius section Orellani are toxic. Toxicosis is notably characterized account for a lack of recognition of Cortinarius mushroom poisoning by a gradually developing acute renal failure over a period of days in animals in North America. Cortinarius mushroom intoxication to up to 2 weeks following a onetime toxic exposure. should be considered as part of the differentials of causes of acute Orellanine poisoning is well recognized in Europe (Denal et al., renal failure in pets. Cortinarius mushroom poisoning in small ru- 2001; Prast et al., 1988; Schumacher and Høiland, 1983). There is minants was reported in (Overås et al., 1979). In that case 4 one reported case of human poisoning in North America involving sheep died after grazing pastures containing Cortinarius mush- C. orellasosus, in which a definitive identification of Cortinarius rooms. The disease was experimentally reproduced confirming that mushrooms was made (Judge et al., 2010). Since neither measure- Cortinarius mushrooms were responsible for the deaths (Overås ment of orellanine in mushrooms nor definitive genetic identifi- et al., 1979). cation of mushrooms were done, there is doubt that other In this collaborative study, we developed highly sensitive and previously reported cases of orellanine poisoning in North America specific analytical test methods for detection and quantitation of were actually caused by Cortinarius mushrooms (Bronstein et al., orellanine in mushrooms. We used these analytical test methods to 2008, 2010; Denal et al., 2001; Judge et al., 2010; Karlson-Stiber analyze several Cortinarius mushroom species collected from the and Persson, 2003; Litovitz et al., 1996; Moore et al., 1991; Raff East Coast to the Midwest USA. Our results indicated that among et al., 1992). Lack of awareness of species complexes and use of several North American Cortinarius mushrooms analyzed for outdated mushroom field guides have also contributed to erro- presence of orellanine, only C. armillatus was found to contain neous identifications. Outdated mushroom field guides use a single orellanine. This is an important finding. Danel et al. indicated that it name to erroneously label multiple species, and yet these species is impossible to establish an extensive list of toxic Cortinarius may not share the same chemical or ecological properties mushroom species and recommended avoiding all Cortinarius (Harrington and Wingfield, 1995; Jaklitsch et al., 2013; Zhang et al., species (Denal et al., 2001). This study suggests that most of the 2015). Mycologists are now more aware of these potential prob- mushrooms studied did not contain orellanine. Although the con- lems, and as a result, many groups of mushrooms are currently centration of orellanine in C. armillatus was low compared to that of undergoing revision, with new species being discovered (Folz et al., C. rubellus, more work is needed to determine the concentration 2013; Justo et al., 2014; Liimatainen et al., 2014). Establishment of range of this toxin in this mushroom species. The content of natural selective, sensitive analytical tests for testing mushrooms as those toxins in plants and mushrooms varies depending on environ- described here will add value by leading to accurate diagnosis of mental conditions. It is possible that the concentration of orellanine Cortinarius mushroom poisoning. in C. armillatus is variable due to genetic differences, but may also Although our taxon is unambiguously C. armillatus as delimited be affected by environmental factors such as soils, and weather. by Niskanen et al. (2011), it is possible that there are variations Thus more studies are needed to establish the concentration of within populations of this species that could be parsed out using orellanine in C. armillatus. It is important that clinicians are aware methods of genotyping more refined than the ITS barcoding region. that C. armillatus contains orellanine because there are many fac- In this regards, it would be of interest to test the orellanine content tors which influence a toxic outcome. Whereas ingesting low of European C. armillatus to compare with the North American amounts of orellanine may not cause renal failure in young in- C. armillatus, as well as testing other species in the armillati section. dividuals or animals at the prime of their health, low amounts of Considering that some of the Cortinarius mushrooms are edible, it orellanine in C. armillatus may push patients with pre-existing may be of interest to assess the potential toxicity of other Corti- renal disease over the edge to cause renal failure. Besides, mush- narius mushrooms as well. Assessing the entire genus of an esti- room hunters should be aware of this information so that they mated 2000 species is not necessary, however. It is known that make personal decisions whether or not to consume these orellanine-containing tissue will fluoresce under UV light, so mushrooms. choosing species that fluoresce would filter out the species that do The other significance of this study is that the analytical test not have the toxin. Fluorescence alone, however, does not defini- methods for orellanine we have established in our laboratory can tively identify orellanine (Keller-Dilitz et al., 1985). be extended and used for diagnostic purposes. Toxic-induced acute Besides being hazardous to people, toxic Cortinarius mushrooms renal failure is reported in pets and there are multiple causes are also a potential danger to pets, especially dogs which are known (Rumbeiha, 2000). There are no pathognomonic signs for orellanine to have indiscriminate eating habits. Toxic Cortinarius mushrooms poisoning. Orellanine poisoning causes a severe renal disease. D. Shao et al. / Toxicon 114 (2016) 65e74 73

Testing suspect mushrooms for orellanine as early as possible Ethical statements before clinical signs set in gives patients the best chances of survival and also minimizes cost of treatment. This test is now available for This study did not involve any human or animal studies. The analyzing suspect ingested mushrooms to determine if they mushroom species studied were obtained from museums or from a contain orellanine. Orellanine causes irreversible renal failure and forest where permission for collecting was granted to RH. so it is important that an early diagnosis is made. The test methods we have developed will be helpful in confirming or ruling out Acknowledgments orellanine poisoning, among other toxin-induced causes of acute renal failure. Both HPLC and LC-MSMS analytical tests are available The authors thank an anonymous reviewer for helpful com- and can be used to detect and quantify orellanine in mushrooms. ments that improved this manuscript. The authors are grateful to Both methods are specific for orellanine. Taking into account the Deborah A. Lewis of Ada Hayden Herbarium, Iowa State University variations between instrument and detection techniques, the two for her generous assistance in providing mushroom samples. methods are complimentary for assessment of orellanine in Healy's work was funded through a Sargent Award from the Har- mushrooms. vard Arnold Arboretum with matching support from Harvard For- As expected, the LC-MSMS method is more sensitive than the est. This work was also partially funded by Iowa State University HPLC method. The LOQ for the two methods was 30 ng g 1 and Startup funds to Wilson K Rumbeiha. The authors would also like to 17 mgg 1 respectively. For routine diagnosis, we recommend the acknowledge Dr. Otto Miettinen of Finnish Museum of Natural HPLC method first and the LC-MSMS for confirmation. The LC- History, Botany Unit, University of Helsinki for providing C. rubellus MSMS is specific for orellanine because this method looks at the mushroom specimen. They also express their gratitude to Dr. Tobias specific product from the parent orellanine ions and is rec- Guldberg Frøslev of Natural History Museum of Denmark and Dr. ommended as a confirmatory method. Other methods, including Matthew E. Smith of the University of Florida for enlightening thin layer chromatography, have limitations of causing false posi- discussion about identification and classification of mushroom tives, lack of sensitivity, and are difficult to perform (Danel, 2001). species. Ours is the only veterinary diagnostic laboratory which has estab- lished a diagnostic test for orellanine in the US. This method can be Transparency document utilized both for veterinary diagnostic purposes and for research to test for presence of orellanine in suspect mushrooms. For best re- Transparency document related to this article can be found sults we recommend that suspect mushrooms be submitted to the online at http://dx.doi.org/10.1016/j.toxicon.2016.02.010 lab in paper bags, shipped overnight to prevent decomposition of specimens. Appendix A. Supplementary data In conclusion, we have established two tests for detection and quantitation of orellanine in mushrooms using HPLC and LC-MSMS Supplementary data related to this article can be found at http:// methods. Using these tests, we have identified C. armillatus as a dx.doi.org/10.1016/j.toxicon.2016.02.010 novel orellanine containing mushroom. The concentration of orel- lanine was low compared to that found in the more potent mush- References rooms such as C. rubellus. However, more research is recommended on C. armillatus to determine the range of toxin concentration. Bertini, L., Amicucci, A., Agostini, D., Polidori, E., Guidi, C., Stocci, V., 1999. 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