Identifying exposures to ribosome-inactivating proteins in blood samples: amplification of ricin-induced ribosomal damage products enables sensitive detection of active toxin and circulating depurinated 28S rRNA

  • Reut Falach
  • Ofir Israeli
  • Yoav Gal
  • Anita Sapoznikov
  • Ohad Shifman
  • Sharon Ehrlich
  • Moshe Aftalion
  • Adi Beth-Din
  • Tamar Sabo
  • Chanoch Kronman
Original Article

Abstract

Purpose

Ricin, a highly potent plant-derived toxin, operates by site-specific depurination of ribosomes, which in turn leads to protein synthesis arrest. Recently, we presented the TRISOL [Truncated RNA Identification by Synthetic Oligonucleotide Ligation] method for the highly sensitive detection of exposures to type II ribosome-inactivating proteins such as ricin in clinical samples, based on the specific amplification of truncated cDNA molecules formed on ricin-dependent depurinated 28S rRNA templates. In the present study, we demonstrate the application of the TRISOL method to detect ricin exposure in blood samples.

Methods

Sera collected from ricin-intoxicated animals was tested for the presence of either active ricin or ricin-induced depurinated 28S rRNA. Active ricin in serum samples from mice intranasally or intraperitoneally exposed to ricin was detected by the TRISOL method following incubation with a ribosomal-rich reticulocyte lysate preparation. For the detection of depurinated 28S rRNA in the serum, cell-free RNAs were isolated from the sera of mice and pigs at different time points following ricin intoxication by different exposure routes and analyzed for the presence of depurinated 28S rRNA.

Results and conclusions

The TRISOL method allowed sensitive detection of both active ricin and host ribosomal damage in blood samples. Active ricin was detected at both early and late time points after ricin intoxication by all routes of exposure tested. Depurinated 28S rRNA, detected at later time points after systemic and oral intoxications, enables the determination of ricin-induced damage to cells of the exposed subject.

Keywords

Ricin Depurinated 28S rRNA Truncated cDNA TRISOL assay Circulating cell-free rRNA Blood sample 

Notes

Acknowledgements

We thank Dr. Avital Tidhar for his useful remarks that contributed to the success of this work.

Compliance with ethical standards

Conflict of interest

R. Falach, O. Israeli, O. Shifman, A. Beth-Din, T. Sabo and C. Kronman are inventors on a patent application related to this work (detection of exposure to RIP II toxins—IL 252188).

Ethical approval

All applicable international, national and institutional guidelines for the care of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution in which the studies were conducted.

References

  1. 1.
    Audi J, Belson M, Patel M, Schier J, Osterloh J (2005) Ricin poisoning a comprehensive review. J Am Med Assoc 294:2273–2386CrossRefGoogle Scholar
  2. 2.
    Kumar O, Sugendran K, Vijayaraghavan R (2003) Oxidative stress associated hepatic and renal toxicity induced by ricin in mice. Toxicon 41:333–338CrossRefPubMedGoogle Scholar
  3. 3.
    Brinkworth CS (2010) Identification of ricin in crude and purified extracts from castor beans using on-target tryptic digestion and MALDI mass spectrometry. Anal Chem 82:5246–5252CrossRefPubMedGoogle Scholar
  4. 4.
    Rutenber E, Ready M, Robertus JD (1987) Structure and evolution of ricin B chain. Nature 326:624–626CrossRefPubMedGoogle Scholar
  5. 5.
    Olsnes S, Fernandez-Puentes C, Carrasco L, Vazquez D (1975) Ribosome inactivation by the toxic lectins abrin and ricin. Eur J Biochem 60:281–288CrossRefPubMedGoogle Scholar
  6. 6.
    Eiklid K, Olsens S, Pihl A (1980) Entry of lethal doses of abrin, ricin modeccin into the cytosol of HeLa cells. Exp Cell Res 126:321–326CrossRefPubMedGoogle Scholar
  7. 7.
    Becher F, Duriez E, Volland H, Tabet JC, Ezan E (2007) Detection of functional ricin by immunoaffinity and liquid chromatography-tandem mass spectrometry. Anal Chem 79:659–665CrossRefPubMedGoogle Scholar
  8. 8.
    Israeli O, Falach R, Sapoznikov A, Gal Y, Shifman O, Erlich S, Aftalion M, Beth-Din A, Kronman C, Sabo T (2017) Determination of ricin intoxication in biological samples by monitoring depurinated 28S rRNA in a unique reverse transcription-ligase-polymerase chain reaction assay. Forensic Toxicol 36:72–80CrossRefGoogle Scholar
  9. 9.
    Zhou H, Xu W, Qian H, Yin Q, Zhu W, Yan Y (2008) Circulating RNA as a novel tumor marker: an in vitro study of the origins and characteristics of extracellular RNA. Cancer Lett 259:50–60CrossRefPubMedGoogle Scholar
  10. 10.
    Lodde M, Fradet Y (2008) The detection of genetic markers of bladder cancer in urine and serum. Curr Opin Urol 18:499–503CrossRefPubMedGoogle Scholar
  11. 11.
    Taback B, Hoon DS (2004) Circulating nucleic acids and proteomics of plasma/serum: clinical utility. Ann N Y Acad Sci 1022:1–8CrossRefPubMedGoogle Scholar
  12. 12.
    Zhang L, Farrell JJ, Zhou H, Elashoff D, Akin D, Park N-H, Chia D, Wong DT (2010) Salivary transcriptomic biomarkers for detection of resectable pancreatic cancer. Gastroenterology 138:949–957CrossRefPubMedGoogle Scholar
  13. 13.
    Stroun M, Anker P, Maurice P, Lyautey J, Lederrey C, Beljanski M (1989) Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology 46:318–322CrossRefPubMedGoogle Scholar
  14. 14.
    Li C-N, Hsu H-L, Wu T-L, Tsao K-C, Sun C-F, Wu JT (2003) Cell-free DNA is released from tumor cells upon cell death: a study of tissue cultures of tumor cell lines. J Clin Lab Anal 17:103–107CrossRefPubMedGoogle Scholar
  15. 15.
    Giacona MB, Reuben GC, Iczkowski KA, Roos TB, Porter DM, Sorenson GD (1998) Cell-free DNA in human blood plasma: length measurements in patients with pancreatic cancer and healthy controls. Pancreas 17:89–97CrossRefPubMedGoogle Scholar
  16. 16.
    Fournié GJ, Courtin J-P, Laval F, Chalé J-J, Pourrat JP, Pujazon M-C, Lauque D, Carles P (1995) Plasma DNA as a marker of cancerous cell death. Investigations in patients suffering from lung cancer and in nude mice bearing human tumours. Cancer Lett 91:221–227CrossRefPubMedGoogle Scholar
  17. 17.
    Stroun M, Anker P, Lyautey J, Lederrey C, Maurice PA (1987) Isolation and characterization of DNA from the plasma of cancer patients. Eur J Cancer Clin Oncol 23:707–712CrossRefPubMedGoogle Scholar
  18. 18.
    El-Hefnawy T, Raja S, Kelly L, Bigbee WL, Kirkwood JM, Luketich JD, Godfrey TE (2004) Characterization of amplifiable circulating RNA in plasma and its potential as a tool for cancer diagnostics. Clin Chem 50:564–573CrossRefPubMedGoogle Scholar
  19. 19.
    Laktionov PP, Tamkovich SN, Rykova EY, Bryzgunova OE, Starikov AV, Kuznetsova NP, Sumarokov SV, Kolomiets SA, Sevostianova NV, Vlassov VV (2004) Extracellular circulating nucleic acids in human plasma in health and disease. Nucleosides Nucleotides Nucleic Acids 23:879–883CrossRefPubMedGoogle Scholar
  20. 20.
    Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO (2007) Exosome-mediated transfer of nRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 9:654–659CrossRefPubMedGoogle Scholar
  21. 21.
    Noerholm M, Balaj L, Limperg T, Salehi A, Zhu LD, Hochberg FH, Breakefield XO, Carter BS, Skog J (2012) RNA expression patterns in serum microvesicles from patients with glioblastoma multiform and controls. BMC Cancer 12:22.  https://doi.org/10.1186/1471-2407-12-22 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Li M, Zeringer E, Barta T, Schageman J, Cheng A, Vlassov AV (2014) Analysis of RNA content of the exosomes derived from blood serum and urine and its potential as biomarkers. Phil Trans R Soc B 369(1652):20130502.  https://doi.org/10.1098/rstb.2013.0502 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Schageman J, Zeringer E, Li M, Barta T, Lea K, Gu J, Magdaleno S, Setterquist R, Vlassov AV (2013) The complete exosome workflow solution: from isolation to characterization of RNA cargo. Biomed Res Int 2013:253957.  https://doi.org/10.1155/2013/253957 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Ling J, Liu WY, Wang TP (1994) Radioassay for RNA N-glycosidase with tritium-labeled sodium-borohydride or amino acid. Bioorg Chem 22:395–404CrossRefGoogle Scholar
  25. 25.
    Palermo M, Alves-Rosa F, Rubel C, Fernandez GC, Fernández-Alonso G, Alberto F, Rivas M, Isturiz M (2000) Pretreatment of mice with lipopolysaccharide (LPS) or IL-1β exerts dose-dependent opposite effects on Shiga toxin-2 lethality. Clin Exp Immunol 119:77–83CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159CrossRefPubMedGoogle Scholar
  27. 27.
    He X, McMahon S, Henderson TD II, Griffey SM, Cheng LW (2010) Ricin toxicokinetics and its sensitive detection in mouse sera or feces using immune-PCR. Plos One 5:e12858.  https://doi.org/10.1371/journal.pone.0012858 Google Scholar
  28. 28.
    Blakey DC, Skilleter DN, Price RJ, Watson GJ, Hart LI, Newell DR, Thorpe PE (1988) Comparison of the pharmacokinetics and hepatotoxic effects of saporin and ricin A-chain immunotoxins on murine liver parenchymal cells. Cancer Res 48:7072–7078PubMedGoogle Scholar
  29. 29.
    Raposo G, Stoorvogel W (2013) Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol 4:373–383CrossRefGoogle Scholar
  30. 30.
    Van Boeckel SR, MacPherson H, Davidson DJ, Norman JE, Stock SJ (2017) Evaluating the role of cell-free DNA in inflammation and spontaneous preterm birth. BioRxiv.  https://doi.org/10.1101/191528 Google Scholar
  31. 31.
    Barrett JS, Wagner JG, Fisher SJ, Wahl RL (1991) Effect of intraperitoneal injection volume and antibody protein dose on the pharmacokinetics of intraperitoneally administered IgG2ak murine monoclonal antibody in the rat. Cancer Res 51:3434–3444PubMedGoogle Scholar
  32. 32.
    Wahl RL, Barrett J, Geatti O, Liebert M, Wilson BS, Fisher S, Wagner JG (1988) The intraperitoneal delivery of radiolabeled monoclonal antibodies: studies on the regional delivery advantage. Cancer Immunol Immunother 26:187–201CrossRefPubMedGoogle Scholar
  33. 33.
    Roy CJ, Song K, Sivasubramani SK, Gardner DJ, Pincus SH (2012) Animal models of ricin toxicosis. Curr Top Microbiol Immunol 357:243–257PubMedPubMedCentralGoogle Scholar
  34. 34.
    Schep LJ, Temple WA, Butt GA, Beasley MD (2009) Ricin as a weapon of mass terror—separating fact from fiction. Environ Int 35:1267–1271CrossRefPubMedGoogle Scholar
  35. 35.
    Lim H, Kim HJ, Cho YS (2009) A case of ricin poisoning following ingestion of Korean castor bean. Emerg Med J 26:301–302CrossRefPubMedGoogle Scholar
  36. 36.
    Sekine I, Kawase Y, Nishimori I, Mitarai M, Harada H, Ishiguro M, Kikutani M (1986) Pathological study on mucosal changes in small intestine of rat by oral administration of ricin: I. Microscopical observation. Acta Pathol Jpn 36:1205–1212PubMedGoogle Scholar
  37. 37.
    Ishiguro M, Tanabe S, Matori Y, Sakakibara R (1992) Biochemical studies on oral toxicity of ricin. IV. A fate of orally administered ricin in rats. J Pharmacobiodyn 15:147–156CrossRefPubMedGoogle Scholar
  38. 38.
    Gal Y, Mazor O, Alcalay R, Seliger N, Aftalion M, Sapoznikov A, Falach R, Kronman C, Sabo T (2014) Antibody/doxycycline combined therapy for pulmonary ricinosis: attenuation of inflammation improves survival of ricin-intoxicated mice. Toxicol Rep 1:496–504CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Gal Y, Sapoznikov A, Falach R, Ehrlich S, Aftalion M, Sabo T, Kronman C (2016) Potent antiedematous and protective effects of ciprofloxacin in pulmonary ricinosis. Antimicrob Agents Chemother 60:7153–7158PubMedPubMedCentralGoogle Scholar
  40. 40.
    Sabo T, Gal Y, Elhanany E, Sapoznikov A, Falach R, Mazor O, Kronman C (2015) Antibody treatment against pulmonary exposure to abrin confers significantly higher levels of protection than treatment against ricin intoxication. Toxicol Lett 237:72–78CrossRefPubMedGoogle Scholar
  41. 41.
    Griffiths GD, Rice P, Allenby AC, Bailey SC, Upshall DG (1995) Inhalation toxicology and histopathology of ricin and abrin toxins. Inhal Toxicol 7:269–288CrossRefGoogle Scholar
  42. 42.
    Wilhelmsen C (2000) Inhaled ricin dose ranging and pathology in inbred strains of mice. USAMRIID Technical Report, USAMRIID, FrederickGoogle Scholar
  43. 43.
    Falach R, Sapoznikov A, Gal Y, Israeli O, Leitner M, Seliger N, Erlich S, Kronman C, Sabo T (2016) Quantitative profiling of the in vivo enzymatic activity of ricin reveals disparate depurination of different pulmonary cell types. Toxicol Lett 258:11–19CrossRefPubMedGoogle Scholar
  44. 44.
    Zhao YQ, Song J, Wang HL, Xu B, Liu F, He K, Wang N (2016) Rapid detection of ricin in serum based on Cu-chelated magnetic beads using mass spectrometry. J Am Soc Mass Spectrom 27:748–751CrossRefPubMedGoogle Scholar
  45. 45.
    Wu J, Wang Y, Jia P, Wang C, Zhao Y, Peng H, Wei W, Li H (2011) Immunochromatography detection of ricin in environmental and biological sample. Nano Biomed Eng 3:169–173.  https://doi.org/10.5101/nbe.v3i3.p169-173 CrossRefGoogle Scholar

Copyright information

© Japanese Association of Forensic Toxicology and Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Reut Falach
    • 1
  • Ofir Israeli
    • 1
  • Yoav Gal
    • 1
  • Anita Sapoznikov
    • 1
  • Ohad Shifman
    • 1
  • Sharon Ehrlich
    • 1
  • Moshe Aftalion
    • 1
  • Adi Beth-Din
    • 1
  • Tamar Sabo
    • 1
  • Chanoch Kronman
    • 1
  1. 1.Department of Biochemistry and Molecular GeneticsIsrael Institute for Biological ResearchNess-ZionaIsrael

Personalised recommendations