Ammonia and nano-TiO2 are commonly found pollutants in aquatic environments around the world. NH3 has been proved to be absorbed on nano-TiO2 surface, therefore, the biosafety and environmental effects of ammonia and co-occurring nano-TiO2 in aquatic environments has increased considerably in recent years. To explore the potential interactive effects and mechanisms of ammonia and nano-TiO2 on the intestinal immune system, three-month-old female zebrafish were exposed to total ammonia nitrogen (TAN; 0, 3, 30 mg/L) with or without nano-TiO2 (1 mg/L) for 60 d. The results showed that intestinal ammonia levels increased with the increase of TAN exposure concentration in the presence of nano-TiO2. Histopathological analysis demonstrated that both TAN and nano-TiO2 caused cell vacuolation, lymphocyte infiltration and goblet cells hyperplasia in the intestine mucosa. Our study also found that the contents and gene expression levels of lysozyme (lys) and β-defensin (def-β) in the intestine of zebrafish exposed to TAN alone or combined with nano-TiO2 were significantly reduced, suggesting a decline in the intestinal innate immunity of fish. A broad upregulation of TLRs-related genes indicated that TAN and nano-TiO2 could activate TLR4/5-mediated MyD88-dependent pathway, and eventually induce intestinal inflammation. It should be noted that TAN combined with nano-TiO2 had more significant inhibitory effects on the intestinal structure and innate immune responses than TAN alone. Current data suggested that ammonia and nano-TiO2 had a synergistic inhibitory effect on intestinal mucosal immunity, and their associated health risk to aquatic animals and the water ecosystem should not be underestimated.
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