Exposure to nitrate and nitrite disrupts cardiovascular development through estrogen receptor in zebrafish embryos and larvae.

Increasing nitrate concentration on surface and groundwater due to anthropogenic activities is an environmental concern. In this study, Tg(fli1: EGFP) zebrafish embryos were exposed to nitrate (NO3-) and nitrite (NO2-), and their cardiovascular development were investigated. Exposure to 10 mg/L NO3-N and 1 and 10 mg/L NO2-N decreased heart rate at 48-96-h post-fertilization (hpf), ventricular volume, and red blood cell flow rate at 96 hpf. Similar concentrations increased the number of embryos and larvae with pericardial edema and missing intersegmental and parachordal vessels in the caudal region at 48-96 hpf. Addition of ICI 182,720 (ICI) reversed the effects of nitrate and nitrite, suggesting estrogen receptors (ER) are involved. 10 mg/L NO3-N and 1 mg/L NO2-N decreased cardiovascular-related genes, gata4,5,6, hand2, nkx2.5, nkx2.7, tbx2a, tbx2b, and fgf1a. Gene expressions of ovarian aromatase and brain aromatase (cyp19a1a and cyp19a1b, respectively) decreased in the exposed groups, whereas ERs (esr1, esr2a, and esr2b) and nitric oxide synthase 2a (nos2a) increased. The effects on gene expression were also reversed by addition of ICI. Taken together, nitrate and nitrite disrupt cardiovascular system through ER in developing zebrafish, implying that environmental nitrate and nitrite contamination may be harmful to aquatic organisms.

Dietary administration of Bacillus amyloliquefaciens R8 reduces hepatic oxidative stress and enhances nutrient metabolism and immunity against Aeromonas hydrophila and Streptococcus agalactiae in zebrafish (Danio rerio).

Zebrafish (Danio rerio) are an excellent model for assessing the beneficial effects of probiotics before applying them in aquaculture. This study evaluated the effects on zebrafish of dietary supplementation with the probiotic Bacillus amyloliquefaciens R8, which heterologously expresses xylanase from rumen fungi. Nutrient metabolism, hepatic oxidative stress, and innate immunity against pathogen infections were investigated. Treated zebrafish received feed supplemented with B. amyloliquefaciens R8 for 30 days and then were compared to zebrafish that were fed a control diet. The treated fish showed significant increases in xylanase activity in the intestines. The livers of the treated fish showed increased mRNA expressions of glycolysis-related genes of hexokinase, glucokinase, glucose-6-phosphatase, and pyruvate kinase; and higher enzyme activities of 3-hydroxyacyl-coenzyme A dehydrogenase and citrate synthase which are associated with fatty acid ß-oxidation and mitochondrial integrity. The livers of treated fish also showed decreased mRNA expressions of oxidative stress-related genes (SOD, Gpx, NOS2, and Hsp70) and an apoptotic gene (tp53), as well as increased expression of an anti-apoptotic gene (bcl-2). The probiotics-treated fish had increased expression of innate immune-related genes (IL-1ß, IL-6, IL-21, TNF-α, and TLR-1, -3, and -4). Following challenge with Aeromonas hydrophila and Streptococcus agalactiae, treated fish showed increased a higher survival rate than control fish. Overall, results showed that the administration of xylanase-expressing B. amyloliquefaciens R8 can potentially improve nutrient metabolism and hepatic stress tolerance, and enhance immunity and disease resistance against A. hydrophila and S. agalactiae in zebrafish.

Dietary supplementation with xylanase-expressing B. amyloliquefaciens R8 improves growth performance and enhances immunity against Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus).

Bacillus amyloliquefaciens has attracted attention as a probiotic in aquaculture due to its immunostimulatory activity against pathogenic infection. Xylanases are extensively used in animal feed to degrade plant ingredients, enhancing nutrient utilization and increasing the growth rate of various animals. In the present study, the effects of dietary supplementation with B. amyloliquefaciens and xylanase-expressing B. amyloliquefaciens R8 on the growth of Nile tilapia (Oreochromis niloticus) and immunity against Aeromonas hydrophila were evaluated. The results showed that the xylanase activity in the intestine, weight gain (WG), feed efficiency (FE) and condition factor (CF) of Nile tilapia fed B. amyloliquefaciens R8 for 2 months were significantly increased compared with those of the fish fed the control diet and B. amyloliquefaciens. Moreover, the mRNA expression of growth- and metabolism-related genes, such as insulin-like growth factor-1 (igf-1), glucokinase (GK), glucose-6-phosphate 1-dehydrogenase (G6PD), and glucose-6-phosphatase (G6Pase), was significantly induced in Nile tilapia fed administered B. amyloliquefaciens R8, and this group also exhibited a higher survival rate than the control fish following a challenge with A. hydrophila. The phagocytic activity and respiratory burst activity of head kidney leukocytes as well as the serum lysozyme activity of B. amyloliquefaciens R8-fed Nile tilapia were significantly higher than those of fish fed the control diet for 2 months. Superoxide dismutase (SOD) levels in the head kidney leukocytes of Nile tilapia fed B. amyloliquefaciens R8 differed from those of fish fed the control diet, but this was not significant. These results indicate that dietary supplementation with xylanase-expressing B. amyloliquefaciens R8 improves growth performance and enhances immunity and disease resistance against A. hydrophila in Nile tilapia.

Nitrate and Nitrite Exposure Induces Visual Impairments in Adult Zebrafish.

Nitrate and nitrite have emerged as increasingly common environmental pollutants, posing significant risks to various forms of life within ecosystems. To understand their impact on the visual system of zebrafish, adult zebrafish were exposed to environmentally relevant concentrations of nitrate (10 mg/L) and nitrite (1 mg/L) for 7 days. Visual behaviors were examined using optomotor and avoidance response. The eyeballs of the zebrafish were collected for H&E staining, IHC, and qPCR. Exposure decreased visual behavior and the thickness of most retinal layers. Exposure decreased expression of pax6a, pax6b, gpx1a, and bcl2a. Exposure increased expression of esr1, esr1a, esr2b, cyp19a1b, sod1a, nos2a, casps3, and tp53, and increased retinal brain aromatase expression by IHC. Collectively, our findings demonstrate that nitrate and nitrite exposure negatively impacted the visual system of adult zebrafish, highlighting the potential hazards of these environmental pollutants on aquatic organisms.

Oxidative stress induced by hydrogen peroxide disrupts zebrafish visual development by altering apoptosis, antioxidant and estrogen related genes.

Hydrogen peroxide is considered deleterious molecule that cause cellular damage integrity and function. Its key redox signaling molecule in oxidative stress and exerts toxicity on a wide range of organisms. Thus, to understand whether oxidative stress alters visual development, zebrafish embryos were exposed to H2O2 at concentration of 0.02 to 62.5 mM for 7 days. Eye to body length ratio (EBR) and apoptosis in retina at 48 hpf, and optomotor response (OMR) at 7 dpf were all measured. To investigate whether hydrogen peroxide-induced effects were mediated by oxidative stress, embryos were co-incubated with the antioxidant, glutathione (GSH) at 50 µM. Results revealed that concentrations of H2O2 at or above 0.1 mM induced developmental toxicity, leading to increased mortality and hatching delay. Furthermore, exposure to 0.1 mM H2O2 decreased EBR at 48 hpf and impaired OMR visual behavior at 7 dpf. Additionally, exposure increased the area of apoptotic cells in the retina at 48 hpf. The addition of GSH reversed the effects of H2O2, suggesting the involvement of oxidative stress. H2O2 decreased the expression of eye development-related genes, pax6α and pax6ß. The expression of apoptosis-related genes, tp53, casp3 and bax, significantly increased, while bcl2α expression decreased. Antioxidant-related genes sod1, cat and gpx1a showed decreased expression. Expression levels of estrogen receptors (ERs) (esr1, esr2α, and esr2ß) and ovarian and brain aromatase genes (cyp19a1a and cyp19a1b, respectively) were also significantly reduced. Interestingly, co-incubation of GSH effectivity reversed the impact of H2O2 on most parameters. Overall, these results demonstrate that H2O2 induces adverse effects on visual development via oxidative stress, which leads to alter apoptosis, diminished antioxidant defenses and reduced estrogen production.