Gene expression, Antioxidant Responses, and metabolomic Profiling Reveal Chlorella vulgaris Mediated Protection against acetaminophen and microplastic Co-toxicity in Oreochromis niloticus

Abstract Emerging water pollutants such as pharmaceutical (PhACs) and microplastics (MPs) are progressively detected in aquatic environments, raising alarms about their mutual toxicological effects on non-target biota, particularly fish. Although the individual effects of these pollutants have been investigated, there is little data available on their interactions and potential strategies to reduce toxicity. This study was designed to explore the synergistic effects of acetaminophen (APAP) and MPs on inflammatory, antioxidant, and metabolic responses in O. niloticus for 14 days, and to evaluate the protective potential of dietary Chlorella supplementation. Fish subjected to APAP alone or coupled with MPs exhibited marked upregulation of inflammatory (TNF-α, IL-6, and IL-1β) and antioxidant (SOD, CAT, and GPx) genes, increased oxidative/nitrosative stress biomarkers (XO, NO, and MDA), and significant alterations in antioxidant enzyme activities. Co-exposure revealed noticeable metabolic fluctuations in amino acids, lysophosphatidylcholines (LPCs), and fatty acids levels and their related pathways. In contrast, Chlorella -supplemented treatments exhibited significant restoration of antioxidant defences, reduction of inflammatory responses, and normalization of the altered metabolites along with their enriched pathways, particularly proline, arginine, and glycerophospholipid metabolism, along with biosynthesis of unsaturated fatty acids. The present findings displayed a clear relationship between the transcriptional upregulation of antioxidant defense genes and the corresponding physiological and metabolic responses across the experimental groups of O. niloticus . Moreover, the study provides evidence that Chlorella supplementation can mitigate the combined toxicity of pharmaceuticals and microplastics in fish, thereby offering a sustainable dietary strategy to enhance fish resilience in contaminated aquatic environments.