Microplastics and BPA Together Weaken Fish Antiviral Defenses Through Oxidative Stress

Freshwater ecosystems rarely confront fish with a single pollutant at a time. Plastic fragments and the industrial chemicals that cling to them drift together through rivers, lakes, and aquaculture ponds, and a new study suggests that this everyday combination can do something neither contaminant achieves alone: it can leave fish measurably more vulnerable to viral infection. Working with juvenile largemouth bass (Micropterus salmoides), one of the most economically important farmed fish in China, researchers found that co-exposure to microplastics and bisphenol A (BPA) suppressed a central antioxidant signaling pathway, damaged liver and brain tissue, and dramatically increased replication of nervous necrosis virus (NNV) after the fish were challenged with the pathogen.

The study, published in Advanced Biotechnology by a team at Sun Yat-sen University led by Jie Gao, Junzhe Zhang, and Junfeng Xie, was designed around environmentally realistic exposure levels. After a preliminary 14-day dose-finding trial, the researchers selected 10 micrograms per liter of green fluorescent polyethylene microplastic particles, roughly 5 micrometers in diameter, and 100 nanograms per liter of BPA. These concentrations caused negligible mortality but sit within ranges documented in freshwater systems worldwide. Microplastic abundances in natural waters span from tens of particles per cubic meter in remote regions such as the Tibetan Plateau to nearly 20,000 particles per cubic meter in urban rivers like the Pearl River, while BPA concentrations in surface waters across Asia range from a few nanograms to several hundred nanograms per liter.

Because BPA is hydrophobic and binds strongly to polymer surfaces, microplastics and BPA frequently co-occur, and the researchers suspected the particles might act as vectors that enhance BPA delivery into tissues. To test this, they exposed juvenile bass for 14 days in five groups: a blank control, a solvent control, microplastics alone, BPA alone, and the combined treatment. Fluorescent imaging confirmed that the particles accumulated progressively in liver tissue over the exposure period, and crucially, the co-exposed fish accumulated significantly more microplastics in their livers than fish receiving particles alone, supporting the vector hypothesis and hinting that the mixture was producing a toxicity greater than the sum of its parts.

The molecular story that emerged centers on NRF2, a transcription factor often described as the master regulator of antioxidant defense. Under normal conditions, NRF2 switches on genes encoding superoxide dismutase (SOD1), catalase (CAT), and glutathione peroxidase (GPx1), enzymes that neutralize reactive oxygen species before they damage cells. In the co-exposed fish, this system collapsed. Enzyme activity assays and quantitative PCR both showed that SOD1, CAT, and GPx activities and transcripts fell most sharply in the combined group, while NRF2 mRNA was downregulated and its cytoplasmic repressor Keap1 was upregulated, a pattern consistent with NRF2 being retained for degradation rather than allowed to activate antioxidant genes.

The downstream consequences were visible at every level the team examined. Malondialdehyde, a standard biomarker of lipid peroxidation, rose to its highest levels in co-exposed livers, and DCFH-DA fluorescence staining revealed intense reactive oxygen species accumulation in the same tissues. Histological sections stained with hematoxylin and eosin showed progressively worsening damage, from mild vacuolization in microplastic-treated fish to disorganized hepatic cords under BPA alone, and finally extensive vacuolization, hepatocellular necrosis, and nuclear pyknosis in the combined group. Liver function markers ALT and AST climbed in parallel, peaking under co-exposure, and the co-exposed livers were visibly hypertrophied.

Energy metabolism told an equally striking story. Hepatic ATP declined sharply in all treated fish, but the synergistic drop under co-exposure suggested that the mixture disrupted mitochondrial bioenergetics more severely than either contaminant individually. This energetic failure was accompanied by activation of the intrinsic apoptotic pathway: the pro-apoptotic genes Bax and Caspase-3 were upregulated, the anti-apoptotic gene Bcl-2 was downregulated, caspase-3 enzyme activity increased, and TUNEL staining revealed abundant apoptotic nuclei in co-exposed liver sections. Transmission electron microscopy extended the damage to the nervous system, showing swollen brain mitochondria with fragmented cristae and vacuolar degeneration in co-exposed fish, while control animals retained intact mitochondrial membranes and densely packed cristae.

To probe the mechanism, the team turned to computational tools. Molecular docking using an AlphaFold2-predicted structure of largemouth bass NRF2 showed that BPA binds stably within a hydrophobic pocket of the protein, with a calculated affinity of minus 6.2 kilocalories per mole. Key residues including Tyr33, Asn34, Gln37, and Arg36 form hydrogen bonds and van der Waals contacts that could destabilize NRF2’s conformation and impair its transcriptional activity. Network toxicology analysis identified 260 overlapping gene targets between BPA and polystyrene exposure, and enrichment analysis pointed to redox-related signaling cascades, including the PI3K-Akt, mTOR, and FoxO pathways, reinforcing the idea that both contaminants converge on NRF2-mediated redox control.

The most consequential experiment came last. After the 14-day exposure, fish were transferred to clean water and challenged with NNV by immersion at 2 times 10 to the 2 TCID50 per milliliter for six hours. NNV is a neurotropic pathogen that infects more than 120 marine and freshwater species, causing viral encephalopathy and retinopathy, with symptoms such as erratic swimming, anorexia, and lethargy, and it is known to exploit redox imbalance to evade immune defense. When the researchers measured viral capsid protein and RNA-dependent RNA polymerase transcripts in brain tissue at 0, 3, and 7 days post-infection, they found elevated viral gene expression in all exposed groups compared with controls, but the highest viral loads appeared exclusively in the co-exposed fish, peaking at both 3 and 7 days post-infection. Single exposures alone did not produce this enhancement, underscoring the synergistic nature of the effect.

The authors frame these findings within an adverse outcome pathway, positioning NRF2 inhibition as the molecular initiating event that cascades through oxidative stress, mitochondrial failure, and apoptosis to the organism-level outcome of heightened viral susceptibility. This mechanistic chain matters because most ecotoxicological risk assessments still evaluate chemicals one at a time, even though aquatic organisms in farmed and natural waters are bathed in complex mixtures. The results suggest that sublethal concentrations of two common contaminants, neither individually capable of compromising antiviral defense, can together push fish past a cellular tolerance threshold, with microplastics potentially amplifying BPA uptake and intracellular delivery.

For aquaculture, the implications are direct. Largemouth bass production in China has exceeded 100,000 tons annually since the species was introduced to Guangdong in the 1980s, but intensive farming has been associated with declining disease resistance, and NNV outbreaks cause high mortality with few treatment options. The researchers propose incorporating mixture-responsive biomarkers, such as NRF2 suppression, ATP depletion, and viral susceptibility, into environmental monitoring, and they call for extending this adverse outcome pathway framework across other species, developmental stages, and pollutant combinations. If the pattern holds broadly, the quiet synergy between plastic debris and its chemical passengers may represent an underappreciated driver of infectious disease in freshwater fish, one that single-compound toxicology has been structurally unable to see.

Subject of Research: Combined microplastic and bisphenol A exposure increases viral susceptibility in largemouth bass via NRF2-mediated oxidative stress

Article Title: Co-exposure to microplastics and bisphenol A increases viral susceptibility in largemouth bass (Micropterus salmoides) via oxidative stress

Article References: Gao, J., Zhang, J., Zheng, R., Jiang, J., Huang, S., Miao, Q., Wu, B., Tang, W., He, J., & Xie, J. (2025). Co-exposure to microplastics and bisphenol A increases viral susceptibility in largemouth bass (Micropterus salmoides) via oxidative stress. Advanced Biotechnology, 3(4), Article 31. https://doi.org/10.1007/s44307-025-00085-5

Image Credits: AI Generated

DOI: 10.1007/s44307-025-00085-5

Keywords: microplastics, bisphenol A, largemouth bass, oxidative stress, NRF2, nervous necrosis virus, aquaculture, ecotoxicology, apoptosis, mitochondrial dysfunction, freshwater pollution, mixture toxicity