Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells

Author(s)

Gonzalez-Blanco, Carlos ^{[1, 2, 3]} ; Dorr, Felipe Augusto ^[1] ; Albuquerque, Renata ^[1] ; Onuki, Janice ^[2] ; Pinto, Ernani ^{[4, 1]}

Affiliation

^[1] Univ Sao Paulo, Sch Pharmaceut Sci, Dept Clin & Toxicol Analyses, BR-05508900 Sao Paulo, SP - Brazil

^[2] Butantan Inst, Lab Dev & Innovat, Ave Vital Brasil 1500, BR-05503900 Sao Paulo, SP - Brazil

^[3] Organismo Invest Judicial, Dept Ciencias Forenses, Secc Toxicol, Heredia 40801 - Costa Rica

^[4] Univ Sao Paulo, Ctr Nucl Energy Agr, BR-13416000 Piracicaba, SP - Brazil

• Document type: Journal article
• Source: Molecules; v. 25, n. 13 JUL 2020.
• Web of Science Citations: 2

Abstract

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(2)method, which takes advantage of the UV absorption of the uracil group (lambda 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS(1)data confirms the presence of the protonated alkaloids, and the MS(2)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. (AU)

• FAPESP's process: 14/50420-9 - Secondary metabolites from aquatic microrganisms and their impact on seafood and freshwater fish aquaculture
• Grantee: Ernani Pinto Junior
• Support Opportunities: Regular Research Grants