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In 1989 along the coast of Emilia Romagna…

In 1989 along the coast of Emilia Romagna…. DSP TOXINS. Mytilus galloprovincialis. Mouse Bioassay for DSP Toxins. Intraperitoneal (IP) injection of lipophylic extract of shell-fish digestive glands into 18-20 g mouse

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In 1989 along the coast of Emilia Romagna…

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  1. In 1989 along the coast of Emilia Romagna… DSP TOXINS

  2. Mytilus galloprovincialis

  3. Mouse Bioassay for DSP Toxins • Intraperitoneal (IP) injection of lipophylic extract of shell-fish digestive glands into 18-20 g mouse • Measurement time to death; one mouse unit = amount that kills mouse in 24 hours • Regulatory level: 0.05 MU per g of edible tissue (equivalent to 200 ng/g of okadaic acid) • A validated method accepted by several countries • Rapid and inexpensive • Poor reproducibility and long assay time • False positives mostly due to the presence of high levels of fatty acids • No information on toxins profile

  4. DSP Toxins: Chemistry and Toxicology DL50= 192mg/Kg Diarrhea, nausea, vomiting, tumor promoting activity Okadaic acid DL50= 260mg/Kg Diarrhea, gastrointestinal distress, epatotoxicity Pectenotoxins DL50= 100mg/Kg Cardiotoxicity, ? Yessotoxins

  5. ~200 Kg of mussels extraction hundreds g of extract chromatografic separation Fats Sterols Contaminants Toxins (mg)

  6. NMR spectrum of okadaic acid… Can you spot it??? It was 1989!!! E. Fattorusso, P. Ciminiello, V. Costantino, S. Magno, A. Mangoni, A.Milandri, R. Poletti, M. Pompei, R. Viviani, Mar. Poll. Bull., 24 ,234 (1992).

  7. Adriatic toxin profile: chronicle 1989:for the very first time okadaic acid was spotted in the Adriatic Sea From 1989 on:Okadaic Acid has been the main Adriatic toxin

  8. Let’s now bounce into 1995!!!

  9. Protoceratium reticulatum YESSOTOXIN 1995: In the Northern Adriatic Sea A much better spectrum, isn’t it? P. Ciminiello, E. Fattorusso, M. Forino, S. Magno, R. Poletti, M. Satake, R. Viviani and T. Yasumoto, Toxicon, 35, 177 (1997)

  10. Known Yessotoxin analogues TOXIC AREA 1995:Yessotoxin 1997:Yessotoxin homoYessotoxin 45-hydroxyYessotoxin 45-hydroxy-homoYessotoxin

  11. New Yessotoxin analogues n = 1 Carboxy YTX (1997) n = 2 CarboxyhomoYTX (1998) Adriatoxin (1997) n = 1 OxoYTX (2001) n = 2 OxohomoYTX (1998) P.Ciminiello, E.Fattorusso, M.Forino, S. Magno, R.Poletti, R.Viviani. Tetrahedron Lett. 39, 8897 (1998). P.Ciminiello, E.Fattorusso, M.Forino, R.Poletti and R.Viviani. Eur. J. Org. Chem. 291-294 (2000). P.Ciminiello, E.Fattorusso, M.Forino, R.Poletti and R.Viviani. Chem. Res. Toxicol. 13, 770-774 (2000). P.Ciminiello, E.Fattorusso, M.Forino e R.Poletti Chem. Res. Toxicol. 14, 596-599 (2001).

  12. CYTOTOXIC DERIVATIVES FROM MUSSELS OF ADRIATIC SEA *IC50 16.5 mg/ml WEHI 164 (murine fibrosarcoma) cell lines IC50 10.9 mg/ml on J774 (murine monocyte/macrophage) cell lines P.Ciminiello, C. Dell’Aversano, E.Fattorusso, M. Forino, S. Magno, A. Ianaro, M. Di Rosa Eur.J.Org.Chem. 49-53 (2001).

  13. FURTHER OXAZININES FROM ADRIATIC MUSSELS Ciminiello P., Dell’Aversano C., Fattorusso E., Forino M., Magno S., Santelia F.U., Couladouros E.A., Pitsinos E.N., Moutsos V.I., Tetrahedron, in corso di stampa

  14. CHLOROSULFOLIPID FROM MUSSELS OF ADRIATIC SEA IC50 (mg/ml) P. Ciminiello, M. Di Rosa, E. Fattorusso, M. Forino, A. Ianaro, and R. Poletti. J. Org. Chem. 66(2), 578-582 (2001).

  15. CHLOROSULFOLIPID FROM MUSSELS OF ADRIATIC SEA 6.2 mg IC50 (mg/ml) RAW 274.7: 20 mg/ml WEHI 164: 13.5 mg/ml P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, S. Magno and Poletti J. Am. Chem. Soc. 124, 13114 (2002)

  16. A FURTHER CHLOROSULFOLIPID FROM MUSSELS OF ADRIATIC SEA 2.1 mg IC50 (mg/ml) 214= 16384 J774: 20 mg/ml WEHI 164: 10.4 mg/ml P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, S. Magno, M. Di Rosa, A. Ianaro, and R.Poletti, Tetrahedron, 60, 7093 (2004) .

  17. High variation in toxins composition • New Biotoxins • New Cytotoxins

  18. A NEW TARGET • Monitoring program • qualitative analysis • quantitative analysis

  19. Mytilus galloprovincialis hepatopancreas Acetone extraction ripartizione AcOEt H2O ripartizione n-e,hexane MeOH 80% ripartizione MeOH 40% CH2Cl2 LiChroprep RP-8 MeOH 100% Yasumoto test Cytoxicity tests MeOH 90% MeOH 60% MeOH 80% Toyopearl HW-40 MeOH 50 ml RP-18 H2O/MeOH 3:7 H2O/MeOH/CH3CN 4:4:2 Luna RP-18 OMOYTX CARBOXY-OMOYTX 45OH-OMOYTX

  20. Analysis Methods: LC-MS • Molecular weight & elemental composition (HRMS) • Structural information (MS/MS) • Wide range of analytes possible • Universal AND selective • High sensitivity • Excellent quantitation • Automated analysis • Legally-acceptable confirmation • Discovery of new toxins, analogs and metabolites • Multiple toxin determinations

  21. Components of a Tandem Mass Spectrometer Sanple Inlet System Syringe, CE,HPLC Ion Detector Ionization Source Mass Analyzer 1 Collision Cell Mass Analyzer 2 High Vacuum (10-5 to 10-6 torr) EI, CI, FAB LD, FD,ESI Data Acquisition Sector, Quadrupole, TOF,Ion Trap

  22. COMPONENTS OF TANDEM MASS SPECTROMETER DATA Inlet system HPLC DETECTOR S O U R C E ANALYZER ESI Ion trap

  23. C TIC MS D B TIC A XIC m/z 1173.5 B XIC ION TRAP • Drawback • Non linear • calibration • curves • Advantage: • High sensitivity • in Full Scan SELECTIVITY

  24. Temp= 150°C Voltage = -10V Tube Lens off set = -60V Spray Voltage = 4,5KV YTX DL68pg FSMS YTX [M-2Na+H]- 14.1mg/ml 4,7mg/ml 1,6mg/ml 0,52mg/ml 0,17mg/ml 0,057mg/ml YTX [M-Na]- Hypersil C-8 BDS

  25. HPLC-ESI-ion trap-MS Method for Direct Detection of Yessotoxins DL 70 pg FINNIGAN LCQ Mass Spectrometer Hypersil 3mm C8 BDS, 50 x 2.0 mm Gradient H2O/ACN 2mM AF/50mM FA P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, S. Magno, and R. Poletti J. Chromat. A,968, 61 (2002).

  26. LC-MS 8.65 min 8.60 min 9.23 min 8.30 min FSMS FSMS 8.25 min FSMS XIC 1187.5 1173.5 FSMS XIC TIC MS Mytilus galloprovincialis Cesenatico coasts in July 2001 1141.5 1171.5 1157.5 YTX XIC XIC 45-OHYTX 45-OHhomoYTX 1047.5 CarboxyYTX CarboxyhomoYTX New YTX P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, S. Magno, and R. Poletti Chem. Res. Toxicol. 15, 979 (2002)

  27. Protoceratium reticulatum as the producing organism of… LC-multiple tandem MS Ultrafree (6000 rpm, 10 min) SPE Load Wash Eluate Extract (500 mL) YTX CarboxyYTX + MeOH (125 mL x 3) NoroxoYTX HomoYTX 45-OHYTX

  28. Conclusions Some YTX analogues present in large quantities in contaminated shellfish – 45-hydroxyYTX, carboxyYTX, noroxoYTX - could to derive from oxidative modification of the absorbedYTX within the mollusk .

  29. Adriatic toxin profile: chronicle In 1995: Yessotoxin was detected for the first time In the Adriatic Sea 1989:for the very first time okadaic acid was spotted in the Adriatic Sea From 1995 on:Yessotoxinshave become the main Adriatic toxins While Okadaic acid has slowly subsided From 1989 on:Okadaic Acid has been the main Adriatic toxin At the beginning of the new millennium: Okadaic Acid had virtually disappeared

  30. Anomalous toxicity of Adriatic mussels cultivated in November 2004

  31. ALLOWANCE LEVELS SUGGESTED BY EU TOXIN 100g SHELLFISH OKADAIC ACID 16 mg YESSOTOXIN 100 mg

  32. Mytilus galloprovincialis hepatopancreas Acetone extraction AcOEt H2O CH2Cl2 H2O MeOH 80% n-hexane H2O BuOH MeOH 40% CH2Cl2 YESSOTOXINS OKADAIC ACID Yasumoto test OKADAIC ACID YESSOTOXINS NEW PROCEDURE OLD PROCEDURE Yasumoto test

  33. In summer 2004 CH2Cl2 phase deriving From Adriatic mussels Was found TOXIC!!!

  34. Isolation of the new toxins CH2Cl2 . ODS Acetone extract HW-40 RP-8 H2O HPLC RP-18 H2O/MeOH/CH3CN 4:4:2 MeOH/H2O 8:2 MeOH/H2O 6:4 NEW TOXIN B NEW TOXIN A

  35. H O O C 1 0 0 8 4 4 M e 9 4 0 H O H 4 0 O 8 6 9 H H H2-1 O H H-4 H-5 7 2 7 M e O H O H H 3.0 2.0 1.0 5.0 4.0 H O O H H M e H H H H H M e O O O 2 R O 1 3 7 E A B C D 2 a O M e 6 M e 4 R ' O O O H H H H H 1 New Toxin- A:Carboxyhomo-1-desulfoYTX

  36. Toxin A Spin system C-1/C-2a

  37. H O O C 1 0 0 8 4 4 M e 9 4 0 H O H 4 0 O 8 6 9 H H H2-1 O H H-4 H-5 7 2 7 M e O H O H H 3.0 2.0 1.0 5.0 4.0 H O O H H M e H H H H H M e O O O 2 R O 1 3 7 E A B C D 2 a O M e 6 M e 4 R ' O O O H H H H H 4 New Toxin- B :Carboxyhomo-4-desulfoYTX

  38. Toxin B: spin system C-4/C-8

  39. Two new toxins in Adriatic mussels: Desulfohomoyessotoxins P. Ciminiello, C. Dell'Aversano, E. Fattorusso, M. Forino, L. Grauso, S. Magno, R. Poletti, L. Tartaglione Chem. Res. in Toxicol.,20, 95 (2007)

  40. What issues do CarboxyhomodesulfoYTXs Raise? Mytilus galloprovincialis hepatopancreas Acetone extraction NEW PROCEDURE CH2Cl2 H2O H2O BuOH EU official protocol for extracting Marine Biotoxins needs to be updated CarboxyhomodesulfoYTX toxicology should be investigated Who is responsible for the lack of the sulfate ester in carboxyhomodesulfoYTXs? YESSOTOXINS OKADAIC ACID

  41. In November 2003… Alexandrium ostenfeldii massively bloomed in the Adriatic Sea

  42. spirolides

  43. HPLC Rp-8 Three major Spirolides @ m/z: 678,5692,5694,5 SPE MeOH: H2O 8 : 2 MPLC C18 H2O:CH3CN 90% A to 100% B H2O:CH3CN Extraction of Alexandrium ostenfeldii culture

  44. Spirolide @ 678 13,19-didesmethylspirolideC

  45. Spirolide @ 692 13-desmethylspirolideC

  46. Spirolide @ 694 P. Ciminiello, C. Dell’Aversano, E. Fattorusso, M. Forino, L. Grauso, S. G. Magno, R. Poletti, L. Tartaglione. Chem. Res. Toxicol. 20, 95-98, 2007.

  47. Mytilus galloprovincialis Cesenatico and Trieste ACN/H2O 8:2 +0,1% formic acid Filtrate Residue partition n-hexane ACN/H2O partition ACN/H2O 2:1 CH2Cl2 dead live

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