Oxidative effects of the biotoxin domoic acid on Phaeodactylum tricornutum

CABRERA J.; GONZÁLEZ P.M.; PUNTARULO S.

Mar del Plata

Congreso; VIII Congreso Argentino de la Sociedad de Toxicología y Química Ambiental, SETAC Capítulo Argentino; 2022

Domoic acid (DA) is a marine biotoxin produced duringharmful algal blooms (HAB) of diatoms, such as Pseudo-nitzchia sp, Amphoracoffeaeformis and Nitzschianavis-varingica. The hypothesis ofthis work was that the DA triggers the generation of reactive oxygen species (ROS)in the intracellular environment that could reach the marine non-toxic diatom Pheodactylumtricornutum, modifying its cellular redox balance and its antioxidantcapacity. It wasevaluated in P. tricornutum the generation of active species usingthe dye 2′,7′ dichlorofluorescein diacetate (DCFH-DA, measured fluorometrically), the content of reduced glutathione(GSH, determined by reverse phase HPLC), and the activity of the antioxidantenzyme glutathione peroxidase (GPX, measured spectrophotometrically). The oxidativedamage was tested measuring spectrophotometrically protein carbonyls and thiobarbituric acid reactive substances (TBARs) contents. In all measurements, cells of P. tricornutum were concentrated by centrifugation at 9,000gfor 10 min at 4°C, and were supplemented with DA to a final concentration of 64µM during 12 min. After exposure, cells were washed with F/2 medium and werecentrifuged three times at 9,000g for10 min at 4°C. All determinations were performed in latent (LAG, 4 days ofgrowth), exponential (EXP, 7 days of growth) and early stationary (STA, 12 daysof growth) phases of cellular development. In the EXP phase, the oxidation rateof DCFH-DA was increased by 7-fold, as compared to cells incubated in theabsence of DA (control). STA and LAG phases showed a lower enhancement of theoxidation rate of DCFH-DA as compared to EXP phase (3-fold and 2-fold,respectively). In the three growth phases, after exposure to DA significantincreases in the activity of the antioxidant enzyme GPx were measured. GSHcontent was significantly decreased after exposure to DA by 35, 36 and 18% ascompared to controls, in LAG, EXP and STA phases of cells growth, respectively.Protein carbonyls and TBARs contents showed an increase in the three growth phasesafter 12 min of exposure to DA, as compared to control cells. The resultssuggested that the exposure of P.tricornutum to DA causes oxidative stress with effects on the antioxidantcellular capacity. In spite of the increase in the enzymatic activity of GPx toavoid cellular oxidative damage, the biomarkers of the deleterious effects wereobserved.