Article pubs.acs.org/crt
Detection of Pyrrolizidine Alkaloid DNA Adducts in Livers of Cattle Poisoned with Heliotropium europaeum Peter P. Fu,*,† Qingsu Xia,† Xiaobo He,† Shimon Barel,‡ Nir Edery,§ Frederick A. Beland,† and Jakob A. Shimshoni*,‡ †
National Center for Toxicological Research, Jefferson, Arkansas 72079, United States Department of Toxicology, Kimron Veterinary Institute, 50250 Bet Dagan, Israel § Department of Pathology, Kimron Veterinary Institute, 50250 Bet Dagan, Israel ‡
S Supporting Information *
ABSTRACT: Pyrrolizidine alkaloids are among the most common poisonous plants affecting livestock, wildlife, and humans. Exposure of humans and livestock to toxic pyrrolizidine alkaloids through the intake of contaminated food and feed may result in poisoning, leading to devastating epidemics. During February 2014, 73 mixed breed female beef cows from the Galilee region of Israel were accidently fed pyrrolizidine alkaloid contaminated hay for 42 days, resulting in the sudden death of 24 cows over a period of 63 days. The remaining cows were slaughtered 2.5 months after the last ingestion of the contaminated hay. In this study, we report the histopathological analysis of the livers from five of the slaughtered cows and quantitation of pyrrolizidine alkaloid-derived DNA adducts from their livers and three livers of control cows fed with feed free of weeds producing pyrrolizidine alkaloids. Histopathological examination revealed that the five cows suffered from varying degrees of bile duct proliferation, fibrosis, and megalocytosis. Selected reaction monitoring HPLC−ES-MS/MS analysis indicated that (±)-6,7dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts were formed in all five livers. The livers from the three control cows did not have any liver damage nor any indication of DHP−DNA adduct formed. These results confirm that the toxicity observed in these cattle was caused by pyrrolizidine alkaloid poisoning and that pyrrolizidine alkaloid-derived DNA adducts could still be detected and quantified in the livers of the chronically poisoned cows 2.5 months after their last exposure to the contaminated feed, suggesting that DHP-derived DNA adducts can serve as biomarkers for pyrrolizidine alkaloid exposure and poisoning.
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INTRODUCTION There are more than 660 pyrrolizidine alkaloids and pyrrolizidine alkaloid N-oxides present in over 6000 plant species worldwide, and about half of the pyrrolizidine alkaloids are hepatotoxic.1−12 Approximately 3% of flowering plants in the world contain toxic pyrrolizidine alkaloids.12 Due to their widespread prevalence and highly cytotoxic and genotoxic properties, pyrrolizidine alkaloid-containing plants are probably the most common type of poisonous plants affecting livestock, wildlife, and humans.2,5,8,13−15 Pyrrolizidine alkaloids have been shown to induce tumors, primarily liver tumors, in experimental animals.5,7,8 The structures of several representative toxic pyrrolizidine alkaloids and their co-occurring N-oxides are shown in Figure 1. We have previously demonstrated that riddelliine, a representative tumorigenic pyrrolizidine alkaloid, induces liver © 2017 American Chemical Society
tumors in rodents through a genotoxic mechanism mediated by 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adduct formation.2 The DHP−DNA adduct levels correlated closely with the tumorigenic response in the rats fed different doses of riddelliine, and the metabolic pattern and DNA adduct profiles were highly relevant to humans.16,17 Partly because of these mechanistic findings, the U.S. National Toxicology Program (NTP) classified riddelliine as “reasonably anticipated to be a human carcinogen” in the 13th Report on Carcinogens.18 Our further mechanistic studies indicated that different types of tumorigenic pyrrolizidine alkaloids generated the same set of DNA adducts in vivo, but these DNA adducts were not formed by the nontumorigenic Received: December 16, 2016 Published: January 27, 2017 851
DOI: 10.1021/acs.chemrestox.6b00456 Chem. Res. Toxicol. 2017, 30, 851−858
Article
Chemical Research in Toxicology
Figure 1. Structures of heliotrine N-oxide, europine N-oxide, and lasiocarpine N-oxide, which were found in the contaminated hay involved in this present study, and several representative toxic pyrrolizidine alkaloids.
pyrrolizidine alkaloids.14 This set of DHP−DNA adducts contains four DHP-derived DNA adducts: (i) a pair of epimers of 7-hydroxy-9-(deoxyguanosin-N2-yl)dehydrosupinidine adducts (termed DHP-dG-3 and DHP-dG-4), which were the predominant DNA adducts; and (ii) a pair of epimers of 7hydroxy-9-(deoxyadenosin-N6-yl)dehydrosupinidine adducts (termed DHP-dA-3 and DHP-dA-4 adducts), which were relatively minor adducts (Figure 2).2,14,19 The overall results of the previous studies indicate that this set of DNA adducts can serve as a common biological biomarker of pyrrolizidine alkaloid exposure and tumorigenicity.14 Exposure of humans and livestock to toxic pyrrolizidine alkaloids through intake of contaminated food and feed has resulted in numerous acute poisoning epidemics, with high mortality and morbidity rates.1,7,8,20−25 The Senecio plant genus, which belongs to the Compositae (Asteraceate) family, has been frequently involved in pyrrolizidine alkaloid-associated intoxications.8 The first reported livestock poisoning, which was caused by grazing upon the pyrrolizidine alkaloid-containing plant tansy ragwort (Senecio jacobaea), occurred in 1787 in Great Britain.24 The species Heliotropium europaeum has also been implicated in many poisoning events in humans and livestock, including in cattle, sheep, chickens, ducks, and pigs.8,21−26
During February 2014, a herd of 73 mixed-breed beef cattle (15−18 months old) from the Galilee region of Israel were fed for 6 weeks a total mixed ration containing hay contaminated with 15% Heliotropium europaeum, resulting in a mortality rate of 33% over a period of 63 days.26 All of the remaining cows were slaughtered 2.5 months after the last ingestion of the contaminant hay. Detailed feed and histopathological liver analysis indicated that the cattle poisoning was due to the exposure to pyrrolizidine alkaloids present in Heliotropium europaeum.26 Mattocks8 reported that the pattern of pyrrolizidine alkaloidinduced liver injury to livestock and experimental animals was similar in nature. Thorpe and Ford27 found that five calves fed Senecio ragwort-containing diet developed veno-occlusive disease, fibrosis, and megalocytosis in their livers. The aims of the present study were to quantify levels of DHP−DNA adducts by selected reaction monitoring (SRM) HPLC−ES-MS/MS analysis of liver samples from cows slaughtered 2.5 months after the last exposure to the contaminated hay and to explore the potential correlation with the corresponding histopathological profile. Additionally three liver samples obtained from cows (matched by age and weight to the intoxicated cows) fed total feed free of weeds producing pyrrolizidine alkaloids were used as matched 852
DOI: 10.1021/acs.chemrestox.6b00456 Chem. Res. Toxicol. 2017, 30, 851−858
Article
Chemical Research in Toxicology
Three control liver samples of healthy cows (designated #32, #37, and #42) were obtained from a slaughter house. The healthy cows from which the livers were obtained were matched by age and weight to the intoxicated cows. Liver DNA Isolation and Enzymatic Hydrolysis. DNA samples from cow livers were extracted using a Blood & Cell Culture DNA Isolation kit (QIAGEN Inc., Valencia, CA) following the manufacturer’s instructions. The concentrations of the DNA were determined spectrophotometrically. Cow liver DNA samples were enzymatically hydrolyzed to nucleosides as previously described,28 with minor modifications. Briefly, DNA (200 μg) in 100 μL of water was mixed with 30 μL of 0.1 M succinate buffer and 30 μL of micrococcal nuclease/spleen phosphodiesterase II solution and incubated for 5 h at 37 °C. Nuclease P1 (8 μL) was then added, and the incubation was continued for 2 h. The resulting nucleotides were stored at −20 °C before analysis. Histopathology. Necropsy of the five slaughtered cows was performed at the slaughter house by the Veterinary Officer for the slaughter house, and the livers were delivered on ice on the same day to the Pathology Department, Kimron Veterinary Institute, for further investigation. Liver tissues from the intoxicated as well as from control cows were collected for histopathological examination and immediately preserved in 10% buffered formalin, embedded in paraffin, sectioned at a 5 μm thickness, and stained with hematoxylin and eosin (H&E). Toxicosis Event. A herd of mixed-breed beef cattle (15−18 months old, weighing 340−390 kg), reared in Kibbutz Gazit located in the Galilee region of Israel, was fed for 42 days, from the beginning of February 2014, a total mixed ration composed of 50% hay contaminated with Heliotropium europaeum, 47.5% poultry litter, and 2.5% Stevia rebaudiana. The first deaths occurred 1 week after the cessation of contaminated hay consumption. Over a period of 63 days from the first death, 24 cows were found dead, yielding a mortality rate of 33%. Since the apparently clinically normal cows revealed marked pathological liver lesions, the remaining 49 cows were slaughtered. Noticeable clinical signs appeared only about a week after the first sudden deaths, affected only a small proportion of the herd, and included lethargy, staggering, and varying degrees of rectal prolapse. The course of all these manifestations was short and affected animals that invariably died within 48 h, with a significant loss of weight during this period. Semiquantitative Liver Cirrhosis Evaluation. Evaluation of liver cirrhosis was made by the following qualitative grading classification of the degrees of bile duct proliferation, fibrosis, and megalocytosis: − = no change, ± = dubious change, + = slight change (≤25% change), ++ = marked change (25−50% change), and +++ = severe change (>50%). Quantitation of DHP-dG and DHP-dA Adducts by LC/MS/MS Analysis. HPLC Lipid Chromatography. A Finnigan Surveyor HPLC system was coupled with the TSQ mass spectrometer. The samples were loaded onto a reverse phase column (ACE 3 C18, 4.6 mm × 150 mm, 3 μm, MAC-MOD Analytical, Chadds Ford, PA) with a gradient of methanol and water (containing 2 mM ammonium acetate, pH = 5), with a flow rate of 0.3 mL/min. The gradient began with 15% methanol for 5 min, followed by a linear gradient up to 65% methanol over the next 35 min, and then methanol was increased to 95% in 1 min. After holding at 95% methanol for 6 min, the gradient was reset to 15% methanol in 1 min. The column was equilibrated for 15 min before the next injection. The total run time between injections was 63 min. The samples were maintained at 5 °C in the autosampler during the entire analysis. Mass Spectrometry. A TSQ Quantum Ultra Triple Stage Quadrupole MS/MS System (ThermoFinnigan, San Jose, CA, USA), equipped with an atmospheric pressure ionization (API) electrospray (ESI) interface, was used to perform the MS−MS analyses. For DHP− DNA adduct assays, the spray voltage was 3000 V, vaporizer temperature 400 °C, capillary temperature 280 °C. The nitrogen pressure of sheath and auxiliary gas was 30 and 5 (arbitrary units), respectively. The argon collision gas pressure was 1.5 mTorr, the
Figure 2. Structures of the four DHP−DNA adducts, DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts, that can potentially serve as common biological biomarkers of pyrrolizidine alkaloid exposure and tumorigenicity. In the nomenclatures of these DHPderived DNA adducts, dehydrosupinidine is a trivial name for 6,7dihydro-1-hydroxymethyl-5H-pyrrolizine. Compared with DHP, dehydrosupinidine lacks the 7-hydroxyl group. It is the hydroxyl group at C7 that is epimerized in DHP-dG-3 and DHP-dG-4 and in DHP-dA-3 and DHP-dA-4.
controls, as well as for determining the background levels of DHP−DNA adducts in the livers of healthy cows.
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EXPERIMENTAL PROCEDURES
Chemicals. 2′-Deoxyguanosine, 2′-deoxyadenosine, micrococcal nuclease, spleen phosphodiesterase II, and nuclease P1 were purchased from Sigma-Aldrich (St. Louis, MO). [15N5]-2′-Deoxyguanosine (98% isotopically pure) and [15N5,13C10]-2′-deoxyadenosine (96−98% isotopically pure) were purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA). All solvents used were HPLC grade. For SRM HPLC−ES-MS/MS quantitation of DHP-dG and DHPdA adducts formed in the cow livers, DHP-dG-1, DHP-dG-2, DHPdG-3, DHP-dG-4, DHP-dA-1, DHP-dA-2, DHP-dA-3, DHP-dA-4, isotopically labeled DHP-[15N5]dG-1, DHP-[15N5]dG-2, DHP[ 15 N 5 ]dG-3, DHP-[ 15 N 5 ]dG-4, DHP-[ 15 N 5 , 13 C 10 ]dA-1, DHP[15N5,13C10]dA-2, DHP-[15N5,13C10]dA-3, and DHP-[15N5,13C10]dA-4 were prepared as previously described.2,19 In Vivo Cow Liver Samples. A herd of 73 mixed-breed beef cattle (15−18 months old) from the Galilee region of Israel was accidently fed for 6 weeks a total mixed ration containing hay contaminated with 15% Heliotropium europaeum. Based upon estimated feed intake and body weights, the cows were exposed to 34 mg total pyrrolizidine alkaloid/kg body weight/day. The main pyrrolizidine alkaloids found in the hay were heliotrine N-oxide (950 μg/g dry weight), lasiocarpine N-oxide (715 μg/g dry weight), and europine N-oxide (990 μg/g dry weight). These three pyrrolizidine alkaloids constituted about 80% of the total pyrrolizidine alkaloids found in the hay.26 Five cow liver samples (designated #1, #3, #481, #484, and #199112) were randomly selected for the present study. 853
DOI: 10.1021/acs.chemrestox.6b00456 Chem. Res. Toxicol. 2017, 30, 851−858
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Chemical Research in Toxicology
no visible megalocytosis (Table 1, Figure 3). Sample #481 displayed the highest levels of fibrosis and a similar degree of bile duct proliferation as samples #3 and #199112. Megalocytosis was observed to a similar degree in samples #481, #3, and #199112, whereas in samples #484 and #1, megalocytes could not be unequivocally detected (Table 1, Figure 3). All three control livers revealed no histopathological changes and were considered normal (data not shown). LC−MS/MS Analysis of DHP-dG and DHP-dA Adducts in Vehicle and Positive Control. We have previously determined that epimeric DHP-dG-3 and DHP-dG-4 adducts, as well as epimeric DHP-dA-3 and DHP-dA-4 adducts, are interconvertible19 The biological activity of these epimers is expected to be similar. Hence, for convenience purposes, the combined levels of each epimer pair were selected for quantification. The LC/MS SRM chromatograms of synthetic standards of the adducts, DHP-dG-1, DHP-dG-2, DHP-dG-3/4, DHP-dA-1, DHP-dA-2, and DHP-dA3/4, as well as the isotope labeled internal standards (IS) DHP-[15N5]-dG and DHP-[15N5,13C10]dA, are shown in Figure S1. In this study, we employed the previously developed LC/ MS/MS method2,14 for the detection and quantitation of DHPdG and DHP-dA adducts. The LC/MS SRM chromatograms shown in Figure 4 are the results of a negative vehicle control sample (left panel) and of a positive control sample, liver DNA from a rat dosed daily with 24 μmol riddelliine/kg bw/day for three consecutive days (right panel). In Figure 4, DHP-[15N5]dG and DHP-[15N5,13C10]-dA are labeled internal standards (IS). LC−MS/MS Analysis of DHP-dG and DHP-dA Adducts Formed in Cow Livers in Vivo. DNA samples were isolated from the cow livers for the quantitation of DHP-dG and DHPdA adducts by LC−MS/MS using SRM mode with the use of DHP-[15N5]dG-1, DHP-[15N5]dG-2, DHP-[15N 5]dG-3/4, DHP-[15N5,13C10]dA-1, DHP-[15N5,13C10]dA-2, and DHP[15N5,13C10]dA-3/4 as internal standards. There were totally eight cow liver DNA samples, five liver samples from the cows that were fed pyrrolizidine alkaloid-contaminated hay and three liver samples from the cows not exposed to pyrrolizidine alkaloid producing weeds. By LC−MS/MS analysis, in all eight cow liver samples, DHP-dG-1, DHP-dG-2, DHP-dA-1, or DHP-dA-2 were below the limit of detection (LOD) (Table 2). Furthermore, DHPdG-3/4 or DHP-dA-3/4 were below the LOD in the three control cow liver samples #32, #37, and #42 (Table 2). In
collision energy 17 eV for DHP-dG and its internal standard, and 21 eV for DHP-dA and its internal standard. Positive ions were acquired in the SRM mode (dwell time of 100 ms for each analyte and internal standard). DHP-dG adducts were monitored at the [M + H]+ m/z 403 to m/z 269 transition and DHP[15N5]dG internal standards at the [M + H]+ m/z 408 to m/z 274 transition. DHP-dA adducts were monitored at the [M + H]+ m/z 387 to m/z 253 transition and DHP-[15N5, 13C10]dA internal standards at the [M + H]+ m/z 402 to m/z 263 transition. Samples were quantified by comparing the areas of the unlabeled chromatogram peaks to those of the corresponding labeled internal standard chromatogram peaks. Data acquisition and reprocessing were performed using Thermo Xcalibur 2.0 SR2 software. Standard Characterization and Calibration Curve. Standard curves were generated by plotting the amounts of standard compounds against peak area. Each sample was tested with a 10 μL injection volume containing 100 fmol of DHP-[15N5]dG and 25 fmol of DHP-[15N5,13C10]dA as internal standards. The calibration curves were linear over the concentration range of 1−20 fmol for DHP-dG-1, DHP-dG-2, DHP-dA-1, and DHP-dA-2, respectively; 0.5−40 fmol for DHP-dG-3/4; and 0.4−20 fmol for DHP-dA-3/4. The best linear fit and least variability for the calibration curve were achieved with a weighting factor of 1/X, with the correlation coefficients (R2) for all analyses above 0.99.
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RESULTS Pathological and Histopathological Examination. Histopathological examination of liver samples #481, #484, #3, and #199112 revealed similar changes, but with varying degrees of bile duct hyperplasia, periportal fibrosis, and megalocytosis (Table 1, Figure 3). Liver sample #1 displayed Table 1. Histopathological Findings and Their Gradings in Liver Samples from Heliotropium europaeum-Poisoned Cows and Control Liversa liver sample
bile duct proliferation
fibrosis
megalocytosis
481 484 1 3 199112 control: 32, 37, 42
+++ ++ ++ +++ +++ −
+++ + + ++ ++ −
+ − − + + −
For the symbols: − = no change, ± = dubious change, + = slight change (≤25% change), ++ = marked change (25−50% change), and +++ = severe change (>50%).
a
Figure 3. Histopathology of liver sample of mixed breed cattle (15 months old) following chronic exposure to H. europaeum; paraffin embedded; H&E × 100. Note the extensive biliary epithelial hyperplasia (A) and hepatocellular necrosis and fibrosis (B) (arrowhead). 854
DOI: 10.1021/acs.chemrestox.6b00456 Chem. Res. Toxicol. 2017, 30, 851−858
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Chemical Research in Toxicology
Figure 4. LC/MS/MS SRM chromatograms of (left panel) a negative vehicle control sample and (right panel) a positive control sample, liver DNA from a rat dosed daily with 24 μmol riddelliine/kg bw/day for three consecutive days.
Table 2. Levels of DHP-dG and DHP-dA Adducts Formed in the Livers of Female Cows Fed Hay Containing and Not Containing Pyrrolizidine Alkaloids for Six Consecutive Weeksa levels of DHP-dG and DHP-dA/108 nucleotides cow liver sample
DHP-dG-1
DHP-dG-2
DHP-dG-3/4
DHP-dA-1
DHP-dA-2
negative controlb sample #32c sample #37c sample #42c sample #1d sample #199112d sample #3d sample #481d sample #484d riddelliinee
