Cylindrospermopsin (CYN) and microcystin-LR (MC-LR) are cyanotoxins of significant concern due to their widespread occurrence and toxic potential. Among exposure routes, ingestion through contaminated water and food is particularly relevant. Although CYN and MC-LR are primarily classified as cytotoxin and hepatotoxin, respectively, both cyanotoxins have shown potential neurotoxic effects. In addition, a recent study has reported CYN metabolites in the brain of orally exposed rats. However, to our knowledge, the combined impact of CYN and MC-LR on the brain remains unexplored. This study aimed to identify CYN, MC-LR, and their potential metabolites in the brain of Wistar rats after oral exposure to dose combinations of pure CYN and MC-LR (7.5+75, 23.7+237, and 75+750 µg/kg body weight) by ultra-high performance liquid chromatography coupled to a tandem mass spectrometry system (UHPLC-MS/MS). Furthermore, several oxidative stress biomarkers were assessed, including lipid peroxidation (LPO) levels and superoxide dismutase (SOD) and catalase (CAT) activities. Although CYN and MC-LR were not detected in brain tissue, up to nine potential CYN metabolites and eight MC-LR metabolites produced by different metabolic pathways were identified. Moreover, sex-dependent differences were observed in oxidative stress biomarkers. In males, exposure to the intermediate dose (23.7 + 237 µg/kg) significantly increased LPO levels (1.3-fold) and SOD (1.3-fold) and CAT (1.6-fold) activities compared to controls, while in females only changes in LPO levels were observed, where a significant increase was observed at all doses tested. These findings suggest that oral exposure to CYN+MC-LR mixtures may cause neurotoxic effects in rats and highlight the importance of considering sex as a biological variable in cyanotoxins toxicological assessment.

Acknowledgement - Project PID2023-147444OB-I00 funded by MICIU/AEI /10.13039/501100011033 and FEDER, UE. Plata-Calzado thanks the Consejería de Transformación Económica, Industria, Conocimiento y Universidades de la Junta de Andalucía for awarding the predoctoral contract (PREDOC_00447) in the Predoctores 2021 grant.