Increased CO2 Concentration Mitigates the Impact of Nitrite on Zebrafish (Danio rerio) Liver and Gills

Nitrite and carbon dioxide (CO2) are common environmental substances in intensive aquaculture ponds. However, the effects and mechanisms of their combined exposure on aquatic animals remain unclear. In this study, we investigated the toxic effects of 2.5, 5, and 10 mg/L CO2 in the presence of 2 mg/L nitrite on hematological, blood gas parameters, and liver physiological and pathological changes in zebrafish (Danio rerio) over 14 days and 28 days. Our results demonstrated a reduced nitrite uptake and accumulation in the gills and liver of zebrafish exposed to nitrite and varying levels of CO2. Increased CO2 levels also led to a decrease in the expression of gill ae1, whereas the transcriptional levels of nhe1 and nhe3b, nkcc and nbc1 were notably upregulated. Moreover, there was a decrease in Cl− and Na+ concentrations, along with an increase in K+ concentrations. These changes suggested that zebrafish responded to increased CO2 stress by reducing their absorption of chloride-dependent nitrite, excreting H+ and maintaining their internal pH. Exposure to higher CO2 levels in the presence of nitrite resulted in lower blood MetHb levels and liver oxidative stress compared to the nitrite single-exposure treatment. Furthermore, co-treatment with CO2 and nitrite attenuated the nitrite-induced damage to genes related to mitochondrial respiratory chain function (ndufs1, mtnd5, mtycb, atp5f1b, mtatp8), leading to elevated ATP levels. Exposure to nitrite alone increased the expression of lipolytic genes (hsla, cpt1aa, atgl) and decreased the expression of lipid synthesis genes (fasn, acaca), resulting in a decrease in TG and TC content in zebrafish liver. However, co-treatment with CO2 and nitrite prevented the nitrite-induced disruption of lipid metabolism, as evidenced by the improvement in TG and TC levels, as well as transcriptional levels of lipid metabolism-related genes. In conclusion, our study suggests that in the presence of 2 mg/L nitrite, increased CO2 (2.5–10 mg/L) may modulate ion transporter genes to reduce the chloride-dependent nitrite uptake and maintain pH homeostasis, concurrently alleviating oxidative stress, restoring mitochondrial respiratory function, and improving lipid metabolism in a dose-dependent manner. These changes may be related to the increase in the concentration of bicarbonate ions in the water as the CO2 level rises. These findings shed light on the potential protective effects of CO2 in mitigating the harmful effects of nitrite exposure in aquatic animals.