Thais Braga Gomes Araujo^1,2, Gabriel de Farias Araujo², Lorena Oliveira Souza Soares^1,2, Guilherme Conceição Concas³, Mariana Gisbert Jardim dos Santos³, Natália Reis de Almeida⁴, Brena Rodrigues Mota Ikehara⁴, Tommaso Del Rosso³, Frederico Garcia Pinto⁴, Fabio Verissimo Correia^1,5, Enrico Mendes Saggioro^1,2

Introduction
Functional nanoparticles have gained attention for their ability to modulate biological processes and potential therapeutic applications. Among them, carbon monoxide-rich gold nanoparticles (COR-AuNPs), developed via pulsed laser ablation, have been investigated for controlled carbon monoxide (CO) delivery, a molecule with anti-inflammatory, antioxidant, and pro-angiogenic properties. However, their behavior in aquatic organisms and the resulting metabolic effects remain poorly elucidated, limiting both their biomedical potential and understanding of possible environmental impacts. This study aimed to characterize the metabolomic profile of zebrafish (Danio rerio) following sublethal exposure to COR-AuNPs.

Materials and Methods
COR-AuNPs were synthesized by pulsed laser ablation at the Nanolaser Laboratory (PUC-Rio). Adult zebrafish (n = 70) were acclimated for 7 days in a 150 L aquarium under controlled conditions and exposed to sublethal concentrations of AuNPs (5, 10, 20, 35, and 75 µg L⁻¹) for 96 hours in a semi-static system with 50% water renewal every 48 hours. After exposure, fish were euthanized by hypothermia, and brain and liver tissues were collected (pools of n = 15) and stored at -80°C. Metabolites were extracted using methanol, chloroform, and water, derivatized with methoxyamine and BSTFA, and analyzed by gas chromatography mass spectrometry (GC-MS). Metabolic pathways were mapped using MetaboAnalyst. The study was approved by the Animal Ethics Committee of IOC/FIOCRUZ (CEUA No. 035/2022).

Results and Discussion
The analyses revealed clear, concentration-dependent alterations in the metabolic profiles of zebrafish brain and liver. In the brain, PCA showed distinct separation between control and exposed groups. At 5 and 10 µg L⁻¹, changes predominantly affected amino acid and carbohydrate metabolism, along with modulation of neurotransmitter precursors. At 20 and 35 µg L⁻¹, significant impacts were observed on antioxidant pathways (notably glutathione metabolism), lipid metabolism, and energy pathways, suggesting adaptive responses to nanoparticle interaction. At 75 µg L⁻¹, alterations in lipid biosynthesis, energy metabolism, and secondary metabolites became prominent. In the liver, the most marked effects occurred at 10 and 35 µg L⁻¹, affecting carbohydrate metabolism (glucose, fructose, lactate), amino acids, antioxidant pathways, and lipids involved in membrane dynamics. The alterations suggest metabolic reprogramming in response to nanoparticle exposure, with organ-specific patterns. These findings suggest cellular and metabolic interactions with COR-AuNPs, highlighting the need for further studies to clarify their biomedical potential and possible long-term environmental effects

Conclusion
COR-AuNPs modulated key metabolic pathways in zebrafish, evidencing their bioactivity even in the absence of overt toxicity. These results position this nanomaterial as a promising tool for biomedical research, while highlighting the importance of comprehensive studies on its long-term biological and environmental effects.

Agradecimentos: The authors thank the Laboratory for Environmental Health Assessment and Promotion (IOC/Fiocruz) and the Nanolaser Laboratory (PUC-Rio) for infrastructure and technical support. The authors also express their gratitude to Dr. Renata Jurema Medeiros and her team for providing the facilities for animal experimentation at the National Institute for Quality Control in Health (INCQS) of the Oswaldo Cruz Foundation (FIOCRUZ). This study was supported by CAPES, FAPERJ, CNPq, and FIOCRUZ.