Cylindrospermopsin Genotoxicity and Cytotoxicity: Role Of Cytochrome P-450 and Oxidative Stress

Abstract

Cylindrospermopsin (CYN) is a cyanobacterial toxin found in drinking-water sources world wide. It was the likely cause of human poisonings in Australia and possibly Brazil. Although CYN itself is a potent protein synthesis inhibitor, its acute toxicity appears to be mediated by cytochrome p-450 (CYP450)-generated metabolites. CYN also induces genotoxic effects both in vitro and in vivo, and preliminary evidence suggests that tumors are generated by oral exposure to CYN. To understand the role of CYP450-activated CYN metabolites on in vitro genotoxicity, this study quantified the process in primary mouse hepatocytes using the COMET assay in both the presence and absence of CYP450 inhibitors known to block acute CYN cytotoxicity. CYN was cytotoxic at concentrations above 0.1 microM (EC50 = 0.5 microM) but produced significant increases in Comet tail length, area, and tail moment at 0.05 microM and above; hence genotoxicity is unlikely to be secondary to metabolic disruption due to toxicity. The CYP450 inhibitors omeprazole (100 microM) and SKF525A (50 microM) completely inhibited the genotoxicity induced by CYN. The toxin also inhibits production of glutathione (GSH), a finding confirmed in this study. This could potentiate cytotoxicity, and by implication genotoxicity, via reduced reactive oxygen species (ROS) quenching. The lipid peroxidation marker, malondialdehyde (MDA) was quantified in CYN-treated cells, and the effect of the reduced glutathione (GSSG) reductase (GSSG-rd.) inhibitor 1,3-bis(chloroethyl)-l-nitrosourea (BCNU) on both MDA production and lactate dehydrogenase (LDH) leakage was examined. MDA levels were not elevated by CYN treatment, and block of GSH regeneration by BCNU did not affect lipid peroxidation or cytotoxicity. It therefore seems likely that CYP450-derived metabolites are responsible for both the acute cytotoxicity and genotoxicity induced by CYN.

Experimental immunology Assessment of the potential genotoxic and proapoptotic impact of selected cyanotoxins on fish leukocytes

“…Most studies concerning the cytotoxic/genotoxic influence of CYN was conducted with liver cells or hepatoma cell lines as CYN is known to be a hepatotoxin. These studies confirmed that the toxin is able to induce genotoxic damage, such as DNA strand breaks, the increase in the micronuclei number, nuclear buds and nuclear bridges [15,[33][34][35]. Similar effects were also observed in other metabolically active cells, e.g.…”

“…Similarly to MCs, the main target for CYN is the liver. The studies on CYN genotoxic potential revealed that the toxin is also responsible for DNA damage in hepatocytes [15]. Recently, the toxin-induced formation of DNA single-strand breaks, increase in the frequency of micronuclei and nuclear buds in human peripheral blood lymphocytes exposed to CYN were reported [16].…”

An examination of the antibiotic effects of cylindrospermopsin on common gram‐positive and gram‐negative bacteria and the protozoan Naegleria lovaniensis

“…Extraction and purification of pure cylindrospermopsin was undertaken as previously described by Humpage et al (2005).…”

Análise genômica de isolados brasileiros de Cylindrospermopsis raciborskii com enfoque em cilindrospermopsina e fixação biológica de N2

“…Esta técnica permite providenciar uma informação quantitativa e qualitativa, contudo, para tal análise são necessários padrões da toxina purificada, os quais por vezes são difíceis de serem obtidos, além de necessitar de grandes volumes de amostra quando a toxina se encontra em baixas concentrações, e de passos para purificação das amostras (RIVASSEAU et al, 1999;RAPALA et al, 2002;MATHYS;SURHOLT, 2004;SANGOLKAR;MASKE;CHAKRABARTI, 2006;. A cromatografia líquida acoplada à espectrometria de massas (LC-MS) e a LC-MS/MS também são métodos muito difundidos que fornecem resultados mais precisos sobre a presença desta toxina, bem como disponibilizam dados referentes aos picos gerados pela ionização e fragmentação da molécula (CHISWELL et al, 1999;FALCONER et al, 1999;LI et al, 2001a;LI et al, 2001b;FASTNER et al, 2003;HUMPAGE et al, 2005;FROSCIO et al, 2008;RASMUSSEN et al, 2008;YLMAZ et al, 2008;BLÁHOVÁ et al, 2009;GALLO et al, 2009;PREUβEL et al, 2009;MAZMOUZ et al, 2010;MEREL et al, 2010;ANTAL et al, 2011;BALLOT et al, 2011;EVERSON et al, 2011;MAZMOUZ et al, 2011;Mc GREGOR et al, 2011). IV) Utilizar os dados gerados no sequenciamento gênomico das duas linhagens para mapeamentos e buscas por genes relacionados à síntese de metabólitos secundários, com foco principal na CYN e genes relacionados à fixação biológica do N 2 e diferenciação dos heterócitos.…”