PROTECTIVE EFFECTS OF ANTIOXIDANTS TO PREVENT DOXORUBICIN-INDUCED SKELETAL DEFORMITIES IN FISH LARVAE

Introduction

Oxidative stress has been related to various skeletal pathologies altering the activity of osteoclasts and osteoblasts, affecting bone remodelling. Reactive oxygen species are key components to increase oxidative stress, increase bone resorption and inhibit bone formation. A large number of studies suggests the importance of the antioxidant system to reduce the effect of bone pathologies and promote better skeletogenesis in aquaculture produced fish .

 Materials and Methods

Microdiets were prepared manually by mixing squid powder first with water-soluble components, then with fat and lipid-soluble vitamins, and finally with gelation dissolved warm water. Resveratrol (34mg/kg) and Doxorubicin (30mg/kg) were dissolved on polar molecules whereas MitoTempo was dissolved in water . S. aurata larvae  at 30 days were randomly stocked into 18 experimental tanks at a density of 2100 larvae/tank. The larvae were fed experimental microdiets with added antioxidants and pro-oxidants, alone or in combination. The antioxidant microdiets were fed every hour from 8:00 to 20:00, whereas  the pro-oxidant (doxorubicin) diet was fed only once a week and was continued with a respective combination of control or antioxidant diets. Similarly, zebrafish  (5 dpf )  were randomly stocked into 24 experimental tanks at a density of 100 larvae/tank for 30 days. The larvae were fed with experimental microdiets with antioxidant and pro-oxidants alone or in combination . The antioxidant supplemented microdiets were fed  by standardized spatula 15mg/day and increased by 5mg/day every week.  Antioxidant supplemented microdiets were fed 4 times a day,  whereas  the pro- oxidant  diet was fed only once a week. The microdiet combinations were Control, Resveratrol (RES), MitoTempo (MT), Doxorubicin (DOX), Doxorubicin + Resveratrol and Doxorubicin + MitoTempo. The effect of antioxidant and pro-oxidant supplemented diet on skeletal formation, the incidence and distribution of skeletal deformities and the stages of bone mineralization , mineral content and molecular markers of bone  were determined.

Results

At 45 dah , S. aurata  fed microdiets supplemented with pro-oxidant s  increased the incidence of bone deformities , deformities charge and decreased bone mineralization. These effects were significantly reversed while co-feeding with microdiets supplemented with antioxidants. Doxorubicin significantly reduced the total length of larvae which was significantly reversed with the co-treatment with Resveratrol . Deformities charge  was significantly higher on  the  Doxorubicin supplemented group and were significantly reduced on  the groups co-feeding  with antioxidants. Cluster analysis  showed  a  distinct difference between the groups on  the  distribution of skeleton anomalies. The mineralization of the skeleton elements  was  significantly affected by Doxorubicin. However, co-feedi ng with Resveratrol the e ffect was rescued. The differences were also observed on Meristic count between the groups supplemented with antioxidants and pro-oxidants supplemented microdiets. Calcium and Phosphorus content were also decreased on Doxorubicin supplemented groups. Similarly, on  the  zebrafish trial, the effect of microdiets on standard length was compared with the standard Zebrafeed diet (Sparos Lda , Olhão , Portugal). Resveratrol supplementation significantly increased the length as compared to Zebrafeed and control microdiet. S urvival of zebrafish larvae was significantly decreased under doxorubicin supplementation but significantly reversed upon co-feeding with antioxidants . Dox orubicin supplementation also delayed larvae development and significantly decreased total length however, this effect  was significantly reversed by co-treatment with antioxidants.  Calcium, Phosphorus, Sodium, Potassium and Magnesium contents were also decreased on Doxorubicin supplemented groups whereas, while in co-feeding with antioxidants, the loss of mineral content was significantly rescued.

Conclusion

O ur results showed that antioxidant supplements effectively prevent  the incidence of bone anomalies and mineralization defects induced by pro-oxidants on both models. Resveratrol and MitoTempo supplementation can reverse pro-oxidant induced effects on bone anomalies, mineralization and oxidative stress. The use of antioxidants in fish diets can be beneficial for improving the health and quality of aquaculture produced fish.

Keywords: Oxidative stress, Resveratrol, MitoTempo, Doxorubicin, Secondary Osteoporosis

Acknowledge ments: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766347- BioMedAqu, and by the Portuguese Foundation for Science and Technology (FCT) through the project UIDB/04326/2020.