Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells
The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are...
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MDPI AG
2020-07-01
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| Series: | Molecules |
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| author | Carlos González-Blanco Felipe Augusto Dörr Renata Albuquerque Janice Onuki Ernani Pinto |
| author_facet | Carlos González-Blanco Felipe Augusto Dörr Renata Albuquerque Janice Onuki Ernani Pinto |
| author_sort | Carlos González-Blanco |
| collection | DOAJ |
| description | The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS<sup>2</sup> method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS<sup>1</sup> data confirms the presence of the protonated alkaloids, and the MS<sup>2</sup> fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN’s lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue. |
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| language | English |
| last_indexed | 2024-03-10T18:43:38Z |
| publishDate | 2020-07-01 |
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| series | Molecules |
| spelling | doaj.art-7f004bd1d68647feb4be461cf3cf89be2023-11-20T05:40:43ZengMDPI AGMolecules1420-30492020-07-012513302710.3390/molecules25133027Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 CellsCarlos González-Blanco0Felipe Augusto Dörr1Renata Albuquerque2Janice Onuki3Ernani Pinto4Department of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo 05508-900, SP, BrazilDepartment of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo 05508-900, SP, BrazilDepartment of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo 05508-900, SP, BrazilLaboratory of Development and Innovation, Butantan Institute, Av. Vital Brasil, 1500, São Paulo 05503-900, SP, BrazilDepartment of Clinical and Toxicological Analyses, School of Pharmaceutical Sciences, University of São Paulo, São Paulo 05508-900, SP, BrazilThe term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS<sup>2</sup> method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS<sup>1</sup> data confirms the presence of the protonated alkaloids, and the MS<sup>2</sup> fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN’s lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue.https://www.mdpi.com/1420-3049/25/13/3027LC-MS<sup>2</sup>LC-DADcylindrospermopsin7-deoxy-cylindrospermopsin7-deoxy-desulfo-cylindrospermopsincyanotoxins |
| spellingShingle | Carlos González-Blanco Felipe Augusto Dörr Renata Albuquerque Janice Onuki Ernani Pinto Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells Molecules LC-MS<sup>2</sup> LC-DAD cylindrospermopsin 7-deoxy-cylindrospermopsin 7-deoxy-desulfo-cylindrospermopsin cyanotoxins |
| title | Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells |
| title_full | Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells |
| title_fullStr | Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells |
| title_full_unstemmed | Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells |
| title_short | Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells |
| title_sort | alternative isolation protocol for desulfo and zwitterionic cylindrospermopsin alkaloids and comparison of their toxicity in hepg2 cells |
| topic | LC-MS<sup>2</sup> LC-DAD cylindrospermopsin 7-deoxy-cylindrospermopsin 7-deoxy-desulfo-cylindrospermopsin cyanotoxins |
| url | https://www.mdpi.com/1420-3049/25/13/3027 |
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