Heliotropium europaeum Poisoning in Cattle and Analysis of

Article pubs.acs.org/JAFC

Heliotropium europaeum Poisoning in Cattle and Analysis of Its Pyrrolizidine Alkaloid Profile Jakob Avi Shimshoni,*,† Patrick P.J. Mulder,§ Arieli Bouznach,‡ Nir Edery,‡ Israel Pasval,∥ Shimon Barel,† Mohammed Abd-El Khaliq,⊥ and Samuel Perl‡ †

Department of Toxicology and ‡Department of Pathology, Kimron Veterinary Institute, Bet Dagan 50250, Israel RIKILT-Wageningen UR, P. O. Box 230, 6700 AE Wageningen, The Netherlands ∥ Hachaklait Veterinary Services, Caesarea Industrial Park 38900, Israel ⊥ Veterinary Services and Animal Health, Ministry of Agriculture and Rural Development, Bet Dagan 50259, Israel §

S Supporting Information *

ABSTRACT: Pyrrolizidine alkaloids (PAs) are carcinogenic and genotoxic phytochemicals found exclusively in angiosperms. The ingestion of PA-containing plants often results in acute and chronic toxicities in man and livestock, targeting mainly the liver. During February 2014, a herd of 15−18-month-old mixed-breed beef cattle (n = 73) from the Galilee region in Israel was accidently fed hay contaminated with 12% Heliotropium europaeum (average total PA intake was 33 mg PA/kg body weight/d). After 42 d of feed ingestion, sudden death occurred over a time period of 63 d with a mortality rate of 33%. Necropsy and histopathological examination revealed fibrotic livers and moderate ascites, as well as various degrees of hyperplasia and fibrosis of bile duct epithelial cells. Elevated γ-glutamyl-transferase and alkaline phosphatase levels were indicative of severe liver damage. Comprehensive PA profile determination of the contaminated hay and of native H. europaeum by LC−MS/MS revealed the presence of 30 PAs and PA-N-oxides, including several newly reported PAs and PA-N-oxides of the rinderine and heliosupine class. Heliotrine- and lasiocarpine-type PAs constituted 80% and 18% of the total PAs, respectively, with the N-oxides being the most abundant form (92%). The PA profile of the contaminated hay showed very strong resemblance to that of H. europaeum. KEYWORDS: pyrrolizidine alkaloids, Heliotropium europaeum, intoxication, beef cattle

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INTRODUCTION Pyrrolizidine alkaloids (PAs) are highly toxic carcinogenic and genotoxic phytochemicals exclusively biosynthesized by angiosperms that are utilized by the plants as a defense mechanism against herbivores. 1−4 The plant families of the most toxicological importance for their toxic PA content are the Asteraceae, Boraginaceae, and Fabaceae.1−5 Over 6000 plants have been estimated to contain PAs of which at present more than 600 PAs and PA-N-oxides have been identified, and more than half of them revealed genotoxic effects.3−5 In Israel the genus Heliotropium (Boraginaceae) is widely distributed and represented nationwide by 14 different species.6 Of these, Heliotropium europaeum is one of the most prevalent species, being previously reported to be implicated in numerous poisoning events in humans and livestock worldwide.7−11 The most common toxic PAs found in Heliotropium spp. are generally mono- or diesters of C1−C2 unsaturated necine bases (1,2-dehydro PAs) of the supinidine-type (1), heliotridine-type (2), and retronecine-type (3) (Figure 1).3−5 Many PAs frequently co-occur in two forms, as their N-oxide and as their tertiary PA base.3−5 1,2-Dehydro PAs are protoxins, requiring metabolic activation by cytochrome P450 to generate cytotoxic electrophilic dehydropyrrolizidine ester intermediates.12−14 Upon formation, the dehydropyrrolizidine esters react with DNA and proteins to generate dehydropyrrolic− DNA and −protein adducts as well as dehydropyrrolic−DNA and −protein cross-links, responsible for the genotoxic and carcinogenic properties of PAs.12−14 The primary site of insult © XXXX American Chemical Society

of the reactive dehydropyrrolizidine esters is the liver; however, pulmonary and renal toxicity was also observed following the ingestion of certain PAs, such as monocrotaline.1,2,11 PA poisoning of livestock caused by the accidental ingestion of feed contaminated with Heliotropium spp. is common worldwide.5,11 In general, grazing animals will avoid PAcontaining plants, although in conditions of overgrazed pastures or drought favoring the development of weeds, livestock has been observed to consume PA-containing weeds.11 Most reported intoxication events in farm animals resulted from ingestion of crops contaminated with PA-containing weeds, which in dried form were intermixed with hay and other feed ingredients, preventing animals from exercising discrimination.5,11,15 PA poisoning in livestock is clinically characterized by staggering, tremor, tenesmus, and sudden death, the latter usually occurs several weeks up to several months from the time of exposure.7−9 Macroscopically, the livers are pale and firm, histopathologicaly characterized by proliferation of bileduct epithelial cells, megalocytosis, veno-occlusive lesions, nodular regeneration, and fibrosis.9,16,17 Thus far, only one case study concerning a chronic PA toxicosis outbreak in cattle in Israel following ingestion of feed contaminated with H. europaeum has been reported in the Received: November 2, 2014 Revised: January 14, 2015 Accepted: January 15, 2015

A

DOI: 10.1021/jf5052199 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Classification of 1,2-unsaturated PA, commonly found in Heliotropium spp., into four subgroups according to their PA base-type and the number/type of ester bonds. The generic names are indicated in bold below each chemical structure.

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literature.9 A mortality rate of about 30% was recorded over a time period of 26 weeks. The surviving animals were eventually slaughtered after it was shown that some clinically normal calves had striking pathological lesions. Between March 14 and May 15, 2014, in a herd of 15−18-month-old replacement mixed-breed beef cattle (n = 73) from the Galilee region in Israel, a 33% mortality rate occurred, following ingestion of total mixed ration (TMR) containing hay that turned out to be contaminated with H. europaeum. The aims of the present work were to provide a detailed description of the chronic PA poisoning in cattle, including clinical signs, pathology, and histopathology in association with the total PA levels found in the feed, and to characterize the PA/PA-N-oxide composition in the contaminated hay in comparison to the PA and PA-Noxide profile of the aerial portions of H. europaeum collected under similar conditions.

MATERIALS AND METHODS

Toxicosis Event. History of the herd: the affected cattle consisted of 73 mixed-breed beef cattle (15−18 months old, weighting 340−390 kg), which were reared in Kibbutz Gazit located in the Galilee region in Israel. The herd was held in a semiopen pen with a yard. The herd was fed from the beginning of February 2014 a TMR supplied by a local private company composed of 50% hay accidently contaminated with H. europaeum, 47.5% poultry litter, and 2.5% Stevia rebaudiana. Neither the feed producer, the farmer, nor the farm veterinarian considered the dried green plant material present in the hay a noxious weed. After 42 days of contaminated TMR consumption, followed by 1 week of rearing on a nearby pasture, as commonly practiced at the beginning of spring, two cows were found dead (March 14, 2014), and 2 days later two additional cows were found dead (Figure 2). The mortality cases were immediately reported to the Israeli Veterinary Institute, and of the four dead cows, three were submitted to the Department of Pathology for full necropsy and histopathological as well as parasitological and virological examination. Furthermore, B


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30 V, desolvation gas temperature at 450 °C, source block temperature at 120 °C, and the argon collision gas pressure at 4.0 × 10−3 mbar. Dwell time of the selected transitions was 10 ms with an interscan delay of 0.5 ms. The column used was a 150 × 2.1 mm i.d., 1.7 μm Acquity BEH RP-18 (Waters, Milford, MA), kept at 50 °C and run at 0.4 mL/min. A mobile phase consisting of 6.5 mM NH4OH in water and 6.5 mM NH4OH in acetonitrile was used. The gradient started at 100% water and was changed linearly to 50% acetonitrile in 12 min. After 0.2 min the composition was returned to the starting condition and the column was allowed to equilibrate for another 2.5 min. Mass spectrometric data were processed using Masslynx 4.1 software (Waters, Milford, MA). Representative H. europaeum samples were screened for the presence of known and new PAs using parent ion scanning. Typical product fragments were selected, such as m/z 120, 122, 138, 156, 172, and 254. The mass scanning range was from m/z 200 to 500 with a mass resolution of 0.1 Da and a scan time of 800 ms. Spectra were recorded using a fixed cone voltage of 30 V and fixed collision energy of 30 eV. Compounds producing a protonated molecular ion with an even mass and displaying fragmentation behavior typical for specific types of PAs were tentatively identified as PAs. These potential PAs were further identified by collecting individual fragmentation spectra at a collision energy range of 20−40 eV. On the basis of their fragmentation spectra, tentative structures could be derived. These compounds have been included in the MRM method, by selecting two product ions, typically the most abundant ones. See Table 1 for the mass spectrometric settings selected for each compound. PAs were quantitated against a range of calibrant samples (0−500 ng/mL) containing available PA standards [heliotrine (4), europine (5), lasiocarpine (6), their corresponding N-oxides (7−9), lycopsamine, and echimidine (Phytoplan, Heidelberg, Germany)]. Echimidine-Noxide was synthesized in-house by N-oxidation of echimidine. For a number of PAs, no reference standard was available. For those PAs, a semiquantitative result was obtained by comparison of the peak area with that of a structurally related standard, as indicated in Table 1. The available PA standards accounted for approximately 90% of the PA content in the hay and plant material. The limit of quantification in the H. europaeum plant material was 1 μg/g and in the hay approximately 0.1 μg/g. Aflatoxin B1, B2, G1, and G2 Analysis by HPLC with Postcolumn Photochemical Derivatization and Fluorescence Detection. Homogenized hay was extracted and analyzed according to a previously published method with slight modifications.22 In brief, a 5 g sample was extracted with 15 mL of acetonitrile:water 80:20 (v/v) and shaken for 1 h. The sample was centrifuged for 10 min at 4000g and 13 mL of organic supernatant was transferred into a new 15 mL Falcon tube and evaporated to dryness in a water bath (40 °C) under a stream of N2. The dried residue was reconstituted with 0.5 mL of methanol:water 60:40 (v/v) in water and subjected to centrifugation for 10 min at 14 000g and finally injected into a HPLC-FLD equipped with a PHRED photochemical derivatization system (AURA Industries, New York, NY) installed before the fluorescence detector. The method utilized a model 1100 (Agilent Technologies, Waldbronn, Germany) liquid chromatography system (equipped with binary pump, degasser, column compartment, and autosampler) combined with a fluorescence detector with excitation and emission wavelengths of 360 and 455 nm, respectively. The column used was a 150 × 4.5 mm i.d., 3.5 μm, ZORBAX RP-18 (Agilent Technologies, Santa Clara, CA), and the following gradient program was utilized: separation was started with 70% water, 17% methanol, and 13% acetonitrile (0−7 min), followed by an increase of the organic phase to 55% water, 30% methanol, and 15% acetonitrile (7−10 min), holding at the latter conditions for an additional 6 min, and eventually returning to the initial mobile phase conditions (16−19 min). The flow rate was 1.0 mL/min and the injection volume 40 μL. A limit of quantitation of 1 and 0.3 μg/kg was achieved for AFB1 and AFG1, and AFB2 and AFG2, respectively. Blood Samples. Blood samples were collected from the jugular vein of 27 randomly chosen live cattle during the intoxication event, counted from the first mortality event, over a time period of 63 d. Samples for complete blood count were collected in potassium-EDTA

Figure 2. Daily mortality and cumulative mortality curve of mixedbreed beef cattle (15−18 months old) poisoned with H. europaeum. The time scale begins with the first mortality events (day 0) following 42 days of consumption of H. europaeum-contaminated feed and 7 days after cessation of feeding the affected herd with the contaminated hay. leftovers of the suspected feed material were submitted to the Department of Toxicology and Bacteriology for toxicological and bacteriological evaluation. The sudden deaths occurred without any previously noticeable clinical signs according to the farmer’s testimony. Noticeable clinical signs appeared only about 1 week after the first sudden death events and affected only a small proportion of the herd and included lethargy, staggering with dragging of the hind quarters, and tenesmus, invariably accompanied by varying degrees of rectal prolapse. In some cases, restlessness was a marked feature, sometimes coupled with circling within the perimeter of the yard. The course of all these manifestations was short, with affected animals invariably dying within 48 h with a significant loss of weight during this period. Over a period of 63 d counted from the first mortality event, 24 cows were found dead, yielding a mortality rate of 33%. As clinically normal cows revealed marked pathological liver lesions, the remaining 49 cows were slaughtered. The Israeli Veterinary Services decided to incinerate the meat of the affected cattle, due to an unknown risk associated with meat products possibly contaminated with PA residues. The suspected contaminated hay was stored for inspection in a closed confinement within the kibbutz. Laboratory Investigations. Total Mixed Ration (TMR) Analysis. Samples of the TMR ingredients (hay, poultry litter, and Stevia) were screened for a wide range of pesticides, including organophosphates, carbamates, pyrethroids, and organochlorides as well as PAs by GC− MS (Agilent Technologies, Santa Clara, CA), according to an inhouse-validated screening method published recently.18 The TMR ingredients were also analyzed for doxycycline and oxytetracycline, as well as the ionophores monensin, lasalocid, salinomycin, maduramicin, semduramicin, and narasin by LC−MS/MS according to the previously described method.19 The analysis of the TMR ingredients for aflatoxins B1, B2, G1, and G2 was performed by HPLC with postcolumn photochemical derivatization and fluorescence detection. Moreover, the TMR ingredients were analyzed for the elements As, Cd, Co, Zn, Cu, Fe, Pb, Mn, Hg, Mo, Se, Tl, and Zn by utilizing an ARCOS ICP-AES (Spectro Analytical Instruments, Kleve, Germany) according to the EPA method 6010c.20 LC−MS/MS Analysis for PA and PA-N-Oxides in Animal Feed and H. europaeum. Dried, ground plant material was extracted using a slightly adapted procedure.21 Powdered material was extracted with 2% formic acid (Merck, Darmstadt, Germany) solution in a 1:100 ratio (w/v). The extract was then filtered and 5 μL was diluted 200 times with 2 mM NH4OH solution (Merck). The contaminated hay material was extracted the same way, but in this case, 50 μL was diluted 20 times with 20 mM NH4OH solution. PA composition and content of the samples were determined using an Acquity chromatographic system coupled to a Quattro Premier XE tandem mass spectrometer (Waters, Milford, MA), run in multiple reaction monitoring mode (MRM) with positive electrospray ionization. Cone voltage was set at C


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Table 1. Mass Spectrometric Conditions Used and Indicative Retention Times of Utilized Standards and Pyrrolizidine Alkaloids Detected in H. europaeum. pyrrolizidine alkaloid supinine supinine-N-oxide heleurine heleurine-N-oxide lycopsaminea lycopsamine-N-oxideb echinatine echinatine-N-oxide rinderine rinderine-N-oxide heliotrine heliotrine-N-oxide 5′-hydroxyrinderinec 5′-hydroxyrinderine-Noxidec europine europine-N-oxide 3′-acetylrinderinec 3′-acetylrinderine-Noxidec 5′-acetyleuropine 5′-acetyleuropine-N-oxide 7-angeloylheliotrine-Noxide echimidine echimidine-N-oxide heliosupine heliosupine-N-oxide lasiocarpine lasiocarpine-N-oxide iso-lasiocarpinec iso-lasiocarpine-N-oxidec 3′-acetylheliosupine 3′-acetylheliosupine-Noxide 5′-acetyllasiocarpine 5′-acetyllasiocarpine-Noxide iso-acetyllasiocarpinec iso-acetyllasiocarpine-Noxidec

precursor ion (m/z)

fragment 1 (m/z)

collision energy (eV)

fragment 2 (m/z)

collision energy (eV)

retention time (min)

standard used for quantification

284.2 300.2 298.2 314.2 300.2 316.2 300.2 316.2 300.2 316.2 314.2 330.2 316.2 332.2

122 120 122 120 94 94 138 111 138 111 138 111 94 111

20 30 30 30 35 40 30 40 30 40 25 35 40 35

140 156 140 156 156 172 156 172 156 172 156 172 138 172

25 30 25 25 30 30 30 30 30 30 25 25 30 30

8.95 5.55 10.75 6.65 6.35 4.76 6.95 4.89 7.05 4.95 8.32 5.95 5.63 3.85

lycopsamine lycopsamine-N-oxide heliotrine heliotrine-N-oxide lycopsamine lycopsamine-N-oxide lycopsamine lycopsamine-N-oxide lycopsamine lycopsamine-N-oxide heliotrine heliotrine-N-oxide lycopsamine lycopsamine-N-oxide

330.2 346.2 342.2 358.2

94 172 120 172

35 30 30 30

138 256 138 298

30 25 20 25

6.72 4.81 8.16 5.92

europine europine-N-oxide lycopsamine lycopsamine-N-oxide

372.2 388.2 412.2

156 172 136

25 30 30

254 328 330

25 20 25

8.19 6.18 9.30

europine europine-N-oxide lasiocarpine-N-oxide

398.2 414.2 398.2 414.2 412.2 428.2 412.2 428.2 440.2 456.2

120 254 120 94 120 138 120 138 120 254

25 30 25 30 25 30 25 30 25 30

220 352 220 254 220 254 220 254 220 338

20 25 20 30 20 25 20 25 20 25

9.92 7.27 9.74 7.08 11.10 7.92 10.96 7.76 10.95 7.95

echimidine echimidine-N-oxide echimidine echimidine-N-oxide lasiocarpine lasiocarpine-N-oxide lasiocarpine lasiocarpine-N-oxide echimidine echimidine-N-oxide

454.2 470.2

120 352

30 20

336 410

25 20

12.56 9.18

lasiocarpine lasiocarpine-N-oxide

454.2 470.2

120 352

30 20

336 410

25 20

12.41 9.02

lasiocarpine lasiocarpine-N-oxide

a

Coelutes with its structural isomer intermedine. bCoelutes with its structural isomer intermedine-N-oxide. cTentative identification, based on fragmentation spectra, MW, retention time, and the presence/absence of N-oxides. The prefix iso indicates the presence of a structural isomer (e.g., 7-tigloyl or 7-senecioyl) instead of the 7-angeloyl ester moiety. tubes and analyzed within 2 h of collection using an Advia 120 hematology analyzer (Siemens Medical Solutions Diagnostics, Erfurt, Germany). Samples for serum chemistry were collected in plain tubes with gel separators. Samples were centrifuged within 2 h of collection, and harvested sera were analyzed immediately using a Cobas Integra 400 Plus wet chemistry analyzer (Roche, Mannheim, Germany) at 37 °C, and reagents were supplied by the manufacturer. The serum was assayed for the enzymes creatine phosphokinase (CK), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and γ-glutamyltransferase (GGT) and for the concentrations of albumin, total protein, globulin, calcium, phosphate, magnesium, and urea. Whole blood was examined to determine the values of the red blood-cell count (RBC), mean cell volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and white blood cell count (WBC). Bacteriology and Virology. Fresh samples of lung, liver, spleen, and kidney from three dead cows were examined for evidence of

bacteriological and viral infections. The analysis for botulism neurotoxin from feed, stomach content, and plasma was performed by the Israeli Reference Laboratory, Department of Bacteriology, The Kimron Veterinary Institute, Israel, utilizing in-house-validated mouse lethality assay.23 Brain tissues were tested for rabies virus (RABV) at the Israeli Reference Laboratory, Department of Rabies, The Kimron Veterinary Institute, Israel, utilizing the direct fluorescent antibody test according to an in-house-validated method.24 Necropsy and Gross Pathology. Three cows were submitted to the Kimron Veterinary Institute for full necropsy on the first week of the intoxication event. Histopathology. Tissues collected for histopathological examination from three cows included the entire heart, lung, liver, kidney, spleen, brain, both ventricular walls and the interventricular septum, and small intestine and were preserved in 10% buffered formalin. In addition, liver samples from 49 slaughtered cows were collected for D


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and intoxicated cows by Student’s t test, with a level of significance of p < 0.05. The biochemical values of the healthy mixed breed cattle were provided by the Veterinarian Hospital, The Hebrew University, BeitDagan, Israel. The biochemical reference values were determined from dozens of healthy mixed-breed cattle over a time period of 5 years, weighting 300−400 kg.

histopathological evaluation. Tissues were embedded in paraffin, sectioned at a 5 μm thickness, and stained with hematoxylin and eosin. Ethical Animal Use. Blood sampling for diagnostic purpose was performed by the farm veterinarian in compliance with the Animal Disease Order, Article 11, 1985.25 Cattle slaughtering was performed according to the Israeli Livestock Slaughter Regulations, Article 30, 1964.26 Statistical Analysis. The results of the blood tests were analyzed for differences between healthy (standard reference values) (Table 2)

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Necropsy and Gross Pathology. Necropsy was performed on three cows that were found dead 7−9 d after being allocated to a nearby grazing field free of any visible Heliotropium weeds. The findings in the three dead cows were consistent. Pathological abnormalities were mostly confined to the liver, which was pale-yellowish, firm, and smaller than normal, with fibrotic streaks in most cases (Figure 3). Furthermore, mild to moderate ascites and mesenteric edema were observed. The kidneys were mildly congested but retained normal architecture. The rest of the necropsy revealed no gross abnormalities within the rest of the body. Histopathology. Histopathological examination of the livers revealed similar changes in all animals, with hyperplasia of bile ductulus and prominent periportal fibrosis. Megalocytosis was not consistently seen in all liver samples. Cirrhosis was observed in all livers, with fibroblasts infiltrating the liver parenchyma (Figure 4). Nodules of regeneration of hepatocytes were also recorded. No pathological changes were observed in the other organs examined. Blood Analysis. Biochemical tests of blood samples collected from 27 live cows during the mortality event revealed extraordinarily high CK levels in all animals tested (125−4128 U/L), whereas the enzyme activities for AST (68−811 U/L), GGT (17−232 U/L), and ALP (51−439 U/L) were significantly elevated in more than 47% of all animals examined (Table 2). Serum urea, Ca2+, Mg2+, phosphate, albumin, total protein, and globulin were within the normal range (Table 2). Blood hemoglobin, hematocrit, red blood-cell count (RBC),

Table 2. Serum Biochemical Markers of Intoxicated MixedBreed Beef Cattle in Comparison to Reference Values of Healthy Mixed-Breed Beef Cattle of the Same Age blood values outside the normal rangeb (%)

biochemical markera

mean (range) values of intoxicated cows

AST

180 (68−811) U/Lc

48

CK

100

GGT ALP

749 (124−4128) U/Lc 58 (17−232) U/Lc 224 (51−439) U/Lc

albumin

3.2 (2.3−4.5) g/dL

33

total protein globulin urea

8 (6.1−10.7) g/dL 5.3 (2.7−6.6) g/dL 13 (6−28) mg/dL

62.5 66 18.7

Ca2+

9 (8−10.7) mg/dL

12.5

phosphate

6.9 (3−9.5) mg/dL

37.5

Mg2+

2.4 (2−3.6) mg/dL

13

70 62

RESULTS

mean (range) values of healthy cows 75 (45−110) U/L 57 (14−107) U/L 12 (5−25) U/L 80 (17−152) U/L 2.9 (2.5−4) g/dL 6.8 (6−8) g/dL 3.6 (3−5) g/dL 15 (8−24) mg/dL 10.2 (8−11) mg/dL 5.6 (4−7) mg/dL 2.3 (1.7−3) mg/dL

AST = aspartate aminotransferase, CK = creatinine kinase, GGT = γglutamyl transferase, and ALP = alkaline phosphatase. bThe percent values are related to the number of cattle being outside the normal range. cMean enzyme activity values of healthy vs intoxicated cows are significantly different according to the Student’s t test (p < 0.05). a

Figure 3. Macroscopic depiction of a liver of mixed-breed cattle (15 months old) following chronic PA toxicity due to H. europaeum ingestion. Depicted is a pale-yellowish, firm liver with diffuse fibrosis. E


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Figure 4. Histopathological liver sample of mixed-breed cattle (15 months old) following chronic PA toxicity due to H. europaeum ingestion (A) revealing extensive hepatocellular necrosis and moderate megalocytosis and fibrosis and (B) extensive biliary epithelial hyperplasia (paraffin embedded, hematoxylin and eosin stained, ×100).

Table 3. Total PA Profile of Hay Contaminated with H. europaeum in Comparison to the Total PA Profile of Aerial Plant Parts of H. europaeum pyrrolizidine alkaloid-type classificationa supinine-type (1)

heliotrine-type (2)

lasiocarpine-type (3)

pyrrolizidine alkaloidb

contaminated hay (μg/g dry wt)c ± ± ± ± ±

0.2 0.7 0.1 20 45

pyrrolizidine alkaloid-type classificationa

H. europaeum (μg/g dry wt) 10.5 66 35 540 1180

± ± ± ± ±

supinine supinine-N-oxide heleurine heleurine-N-oxide heliotrine (4)

0.5 9.4 1.4 75 170

6.9 24 10 100 290

europine (5) heliotrine-N-oxide (7) europine-N-oxide (8) rinderine (10) 5′-hydroxyrinderined (11) 3′-acetylrinderined (12) rinderine-N-oxide (13) 5′-hydroxyrinderineN-oxided (14) 3′-acetylrinderine-Noxided (15) 5′-acetyleuropine 5′-acetyleuropine-Noxide echinatine-N-oxide 7-angeloylheliotrineN-oxide lasiocarpine (6) lasiocarpine-N-oxide (9) iso-lasiocarpined

90 ± 33 950 ± 54

830 ± 470 9340 ± 1050

990 ± 210

9680 ± 3400

1.5 ± 0.8 1.4 ± 0.8

8.4 ± 4.4 6.0 ± 2.6

0.2 ± 0.1

0.6 ± 0.3

31 ± 16

260 ± 110

17.1 ± 0.4

100 ± 74

0.5 ± 0.4

3.6 ± 2.5

1.6 ± 0.9 60 ± 20

11.4 ± 3.1 760 ± 250

5.9 ± 2.5 4.4 ± 0.7

37 ± 13 9.1 ± 7.0

56 ± 5 715 ± 43

240 ± 200 3800 ± 1740

4.9 ± 1.1

18 ± 21

pyrrolizidine alkaloidb iso-lasiocarpine-Noxided 5′-acetyllasiocarpine 5′-acetyllasiocarpineN-oxide isoacetyllasiocarpined isoacetyllasiocarpineN-oxided heliosupine (16) 3′-acetylheliosupine (17) heliosupine-N-oxide (18) 3′-acetylheliosupineN-oxide (19)

total PA content % PA bases % PA-N-oxides % supinine-type % heliotrine-type % lasiocarpinetype

contaminated hay (μg/g dry wt)c

H. europaeum (μg/g dry wt)

51 ± 1

180 ± 190

3.8 ± 0.0 100 ± 10

22 ± 15 440 ± 270

0.4 ± 0.1

1.9 ± 2.1

9.7 ± 0.8

41 ± 42

1.1 ± 0.2 0.3 ± 0.1

8.3 ± 4.1 1.6 ± 1.6

10.7 ± 1.6

92 ± 52

4.1 ± 0.2

13.8 ± 8.1

3370 9.8 90.2 2.5 68.8 28.7

± ± ± ± ± ±

410 1.4 1.4 0.2 3.0 2.2

27700 8.4 91.6 2.3 80.0 17.7

± ± ± ± ± ±

5900 1.4 1.4 0.4 7.9 7.7

a

Classification according to the PA necine base and the number and type of esterifying necic acids (Figure 1). bThe chemical structure of the PAs without a bold compound number can be found in ref 5. cn = 3. dTentative identification. The prefix iso indicates the presence of a structural isomer (e.g., 7-tigloyl or 7-senecioyl) instead of the 7angeloyl ester moiety.

in the analysis.19 The antibiotics and ionophores analyzed by LC−MS/MS were below the detection limit of 60 μg/kg. The feed ingredients analyzed for aflatoxins by HPLC-FLD were below the limit of quantitation (1 μg/kg). Our routine qualitative multiresidue screening analysis utilizing GC−MS of the TMR ingredients did not reveal any pesticide contamination. However, in the hay samples analyzed, GC−MS clearly indicated the presence of three PAs, heliotrine (4), europine (5), and lasiocarpine (6). Hay inspection by the Department of Toxicology and by a certified botanist revealed the presence of aerial parts of dried H. europaeum, and therefore, a subsequent qualitative and quantitative PAs analysis of the contaminated hay samples together with the aerial parts

packed cell volume (PCV), and white blood cell count (WBC) were within normal limits. Liver Analysis for Trace Elements. The elements analyzed in the liver samples by ICP-AES (As, Cd, Co, Zn, Cu, Fe, Pb, Mn, Hg, Mo, Se, Tl, and Zn) were within the normal limits.27 Bacteriology and Virology. No pathogenic bacteria (aerobic, anaerobic and Salmonella, Clostridium) and viruses of epidemiological significance were detected in the tissues examined. Feed Analysis. The feed ingredients were concomitantly analyzed for ionophores, tetracycline antibiotics, pesticides, and aflatoxins. Only ionophore and tetracyclines, approved by the Ministry of Agriculture for use in farm animals, were included F


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Journal of Agricultural and Food Chemistry of H. europaeum collected in the fields used for the aforementioned hay harvest was carried out by LC−MS/MS. The PA and PA-N-oxide composition and relative concentrations of the individual PAs in the contaminated hay and in H. europaeum overlapped strongly, yielding in both sample types 30 structurally different PAs [16 N-oxides and 14 tertiary amines containing supinidine (1) and heliotridine (2) as necine bases; see Table 3 and Figure 1]. PAs containing a heliotridine necine base (2) were classified into two subtypes, according to the number of esterifying necic acids [monoester heliotrinetype PAs (4) and diester lasiocarpine-type PAs (6); Figure 1]. The total PA content in the contaminated hay was 3.4 mg/g dried hay, while the aerial plant parts of H. europaeum contained 27.7 mg PA/g dried plant (Table 3). The most abundant tertiary PA bases found in H. europaeum and the contaminated hay were heliotrine (4) (170 and 1180 μg/g, respectively), followed by europine (5) (90 and 830 μg/g, respectively) and lasiocarpine (6) (56 and 240 μg/g, respectively) (Table 3). The PA-N-oxides constituted the major part of the total PA composition in the hay and in H. europaeum (90 and 91%, respectively), with heliotrine-N-oxide (7) and europine-N-oxide (8) being the most prevalent Noxides (28−29% and 33−34%, respectively), followed by lasiocarpine-N-oxide (9) (21% and 13.6%, respectively) (Table 3). The LC−MS/MS analysis of H. europaeum revealed the presence of trace amounts of 10 PAs containing rinderine (10− 15) and heliosupine structures (16−19) (Figure 1). Putative assignments were made on the basis of retention time, protonated precursor mass, fragmentation behavior (such as the loss of characteristic fragments), the oxidation state of the nitrogen atom; and comparison with literature data and with additional plant extracts available at RIKILT. In this way, rinderine (10), 5′-hydroxyrinderine (11), 3′-acetylrinderine (12), rinderine-N-oxide (13), 5′-hydroxyrinderine-N-oxide (14), 3′-acetylrinderine-N-oxide (15), heliosupine (16), 3′acetylheliosupine (17), heliosupine-N-oxide (18), and 3′acetylheliosupine-N-oxide (19) could be identified (Figure 1 and Table 3). The supinidine-type PAs (1) exhibited low abundances in both the hay and the H. europaeum samples (Table 3).

suspected hay, eventually revealing the presence of H. europaeum. Subsequent analysis of the contaminated hay and native H. europaeum by LC−MS/MS resulted in the identification of 30 different PA and PA-N-oxides, at a total level of 3.4 mg/g dry weight in the hay (Table 3). In the literature there have been a number of descriptions of the histopathological changes of PA toxicosis, among which megalocytosis was often stated to be pathognomonic. However, the histopathological results of the present study indicate that megalocytosis was not a prominent and consistent feature, appearing only in one liver specimen and at a very low intensity, in agreement with the experimental trial results on cattle published previously.17,28 The differences reported regarding the prominence of megalocytosis might arise due to intra- and interspecies variation in PA composition and duration of exposure, as well as varying degrees of susceptibility to PA toxicities among different cattle breeds. From the results of this study together with previous studies, the inconsistent appearance of megalocytosis in a biopsy specimen does not rule out PA toxicosis in a clinical situation.17,28 Measurement of specific serum enzyme activities, in conjunction with markers of renal activity and complete blood count, is a very useful tool in the diagnosis of hepatobilliary injury due to hepatotoxic xenobiotics such as PAs and aflatoxins. Concomitant increases of ALP with GGT raises the suspicion of hepatobilliary disease, as was demonstrated in more than 70% of all cows tested in the present study (n = 27) (Table 2). Elevated serum GGT activity can be found in liver, biliary system, and pancreas disorders. In this respect, it is similar to ALP in indicating biliary tract diseases. The main value of GGT over ALP is in verifying that ALP elevations are due to biliary disease, as ALP may increase in certain bone diseases, while GGT does not.17 Increased AST activity in the serum is indicative of liver damage; however, in the present study only 48% of all examined cows revealed values above the normal AST range (Table 2); hence, according to the results presented herein, GGT and ALT are potentially more sensitive biomarkers for hepatobilliary injury due to PA toxicity. CK is generally very useful in diagnosing skeletal or cardiac muscle degeneration.17 In all examined cows, CK levels were substantially above the normal range over a time period of 63 d, indicating muscle trauma, most probably due to prolonged recumbancy, as histologically no marked myocardial damage was observed (Table 2). Measuring serum GGT and ALP as diagnostic enzymes could be useful as a first screening test in suspected cases of PA toxicosis.17 These enzymes, together with a histopathological examination of the liver, provide important information in diagnosing lethal exposure to hepatotoxic compounds such as PAs or aflatoxins. However, a differentiation between various hepatotoxic xenobiotics is only possible by direct determination of the noxious compound and/or its metabolites in the feed and/or organ tissues, such as the liver. The main obstacle in establishing a diagnosis of PA or aflatoxin intoxication is that in both cases the clinical symptoms and sudden death occur weeks or several months after chronic exposure, depending on the exposure level and animal species, age, and sex, so that often no samples of contaminated feed are available for chemical analysis.9,29 Moreover, since PAs and aflatoxins are quickly metabolized to their corresponding reactive intermediates, the ability even of highly sensitive instruments such as LC−MS/ MS to identify parent compounds as well as major metabolites

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DISCUSSION Chronic PA poisoning of cattle in Israel due to consumption of hay contaminated with H. europaeum was first reported about 30 years ago.9 The clinical manifestations and histopathological findings were similar to those described previously.9 In general, the clinical manifestation of PA toxicosis is not pathognomonic and might have been indicative of aflatoxicosis, lead poisoning, or rabies. Rabies was eliminated by examination of brain slices, and lead poisoning was ruled out by liver lead determination; neither latter poisonings are associated with characteristic liver lesions. Aflatoxicosis, which in cattle often displays histopathological signs similar to those of PA toxicosis, was ruled out by the results of the feed analysis by HPLC-FLD. Routine bacteriological examinations eliminated possible causative infective bacteria, while chemical analysis of TMR ingredients excluded the involvement of common causes of poisoning previously reported in Israel (organophosphorus, carbamate, organochlorine pesticides). However, the inspection of the TMR ingredients by GC−MS led to the identification of three PAs in the hay: heliotrine (4), europine (5), and lasiocarpine (6). The discovery of PAs in the hay led us to examine the G


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Journal of Agricultural and Food Chemistry thereof diminishes rapidly within a few days.12,30 On the other hand, protein and/or DNA adducts of PAs and aflatoxins have far longer tissue half-lives and therefore are currently the most promising biomarkers for establishing a conclusive diagnosis, even after several months of washout period.12 The average feed intake of 15−18-month-old mixed-breed cattle weighting 350 kg was estimated to be 7 kg feed/d, hence 20 g feed/d/kg body weight (BW). Taking into account an average total PA content of 3.4 mg/g hay and a dilution ratio of 1:2 with the other feed ingredients, the total mean PA intake leading to the observed high mortality rate and clinical and pathological signs was 34 mg/kg BW/d. This value is about twice as high as the lethal doses (15 mg PAs/kg BW/d) given as dried Senecio jacobaea for 14 d to four lactating cows and administered for 20 d to three calves in the form of dried Senecio riddellii (17 mg PAs/kg BW/d).11,31 LD50 values reported for various PAs vary widely, but macrocyclic and diester PAs tend to be somewhat more toxic than monoester PAs.5 Careful inspection of weed species likely to thrive in pastures and meadows under certain environmental conditions, such as drought, could prevent future cases of PA intoxications both in livestock and humans. An inspection protocol must include collection and retention specimens of the suspected feed and feed analysis, preferably by utilizing highly sensitive instruments such as GC−MS or LC−MS/MS. Furthermore, the establishment of a future database of MS spectra and corresponding retention times together with the availability of sufficient PAs standards would facilitate rapid diagnosis. To the best of our knowledge, this is the first study utilizing LC−MS/MS for qualitative and quantitative analysis of the PA content in H. europaeum. The high sensitivity of the LC−MS/ MS analysis utilized for the determination of the PA and PA-Noxide composition in the contaminated hay, as well as the possibility of measuring both forms of PAs simultaneously and the ease of sample preparation, enabled us to markedly expand the known PA profile of H. europaeum from 12 to 30 different PA and PA-N-oxides (Table 3).3 A total of 10 PAs of the rinderine and heliosupine classes are reported for the first time to be present in this species. To the best of our knowledge, PAs derivatives of rinderine (10−16, Figure 1) and heliosupine (16−19, Figure 1) have so far been reported only in Heliotropium indicum, Heliotropium peruvianum, Heliotropium transalpinum, and Heliotropium supinum.3 The definite assignment of the rinderine derivatives could not be made, because the obtained mass fragmentation spectra do not exclude the presence of echinatine structures. However, on the basis of the fact that (+)-trachelanthic acid, the necic acid in rinderine, has the same stereochemistry as heliotric acid, the necic acid in heliotrine, its presence was considered more likely than the echinatine structure, which is esterified with (−)-viridifloric acid and for which no heliotrine counterpart is known. Utilizing LC−MS/MS analysis techniques is expected to expand substantially the PA composition of known PAproducing plants.33 In agreement with previous studies, the most abundant PAs found in H. europaeum were heliotrine (4), europine (5), and lasiocarpine (6).3,32 In the investigated H. europaeum, the total PA level was 2.7%, being comparable to the levels found in H. europaeum grown in southeastern Australia at the same flowering stage.34 The total PA profiles of H. europaeum and of the contaminated hay strongly overlapped in terms of composition

and relative abundance, with heliotrine-N-oxide (7) and europine-N-oxide (8) being the most abundant alkaloids in both samples (Table 3). This finding suggests that a selection of abundant PA and PA-N-oxides may be used as a fast and reliable tool for species identification. In conclusion, a mortality rate of 33% in mixed-breed cattle was recorded, following ingestion of TMR contaminated with H. europaeum at a level of 12% dry weight. The toxicosis event was characterized by typical clinical and histopathological signs of PA toxicosis. LC−MS/MS analysis of the PA and PA-Noxide profile in H. europaeum enabled a comprehensive composition determination, including 10 PAs of the rinderine and heliosupine classes reported for the first time in H. europaeum.

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ASSOCIATED CONTENT

S Supporting Information *

Blood parameters from intoxicated mixed-breed beef cattle (Table S1) and additional information about the elemental composition, molecular weight, and chemical structures of PAs determined in the present study (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel. +972-3-9688911. E-mail: [email protected]. Fax: +972-3-9681730. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully thank Dr. Yotam Zipper at the Hebrew University of Jerusalem, for the assistance in determining the Heliotropium species. The authors thank Mr. Yossi Hofi and David Ochana, for their excellent technical assistance.

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REFERENCES

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Heliotropium europaeum Poisoning in Cattle and Analysis of

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