NAD+ precursors and bile acid sequestration treat preclinical refractory environmental enteric dysfunction.

Environmental enteric dysfunction (EED) is a diffuse small bowel disorder associated with poor growth, inadequate responses to oral vaccines, and nutrient malabsorption in millions of children worldwide. We identify loss of the small intestinal Paneth and goblet cells that are critical for innate immunity, reduced villous height, increased bile acids, and dysregulated nicotinamide adenine dinucleotide (NAD+) synthesis signaling as potential mechanisms underlying EED and which also correlated with diminished length-for-age z score. Isocaloric low-protein diet (LPD) consumption in mice recapitulated EED histopathology and transcriptomic changes in a microbiota-independent manner, as well as increases in serum and fecal bile acids. Children with refractory EED harbor single-nucleotide polymorphisms in key enzymes involved in NAD+ synthesis. In mice, deletion of Nampt, the gene encoding the rate-limiting enzyme in the NAD+ salvage pathway, from intestinal epithelium also reduced Paneth cell function, a deficiency that was further aggravated by LPD. Separate supplementation with NAD+ precursors or bile acid sequestrant partially restored LPD-associated Paneth cell defects and, when combined, fully restored all histopathology defects in LPD-fed mice. Therapeutic regimens that increase protein and NAD+ contents while reducing excessive bile acids may benefit children with refractory EED.

Infection and inflammation stimulate expansion of a CD74+ Paneth cell subset to regulate disease progression.

Paneth cells (PCs), a specialized secretory cell type in the small intestine, are increasingly recognized as having an essential role in host responses to microbiome and environmental stresses. Whether and how commensal and pathogenic microbes modify PC composition to modulate inflammation remain unclear. Using newly developed PC-reporter mice under conventional and gnotobiotic conditions, we determined PC transcriptomic heterogeneity in response to commensal and invasive microbes at single cell level. Infection expands the pool of CD74+ PCs, whose number correlates with auto or allogeneic inflammatory disease progressions in mice. Similar correlation was found in human inflammatory disease tissues. Infection-stimulated cytokines increase production of reactive oxygen species (ROS) and expression of a PC-specific mucosal pentraxin (Mptx2) in activated PCs. A PC-specific ablation of MyD88 reduced CD74+ PC population, thus ameliorating pathogen-induced systemic disease. A similar phenotype was also observed in mice lacking Mptx2. Thus, infection stimulates expansion of a PC subset that influences disease progression.

High dissolved oxygen exacerbates ammonia toxicity with sex-dependent manner in zebrafish.

Ammonia nitrogen is one of the important environmental factors, and causes negative effects for fish health in ecosystem and aquaculture. The toxic effects and mechanisms of ammonia in fish deserve further investigation. In the present study, we exposed female and male zebrafish (Danio rerio) to ammonia (50 mg/L NH4Cl) with oxygenated (7.5-7.8 mg/L) or non­oxygenated (3.8-4.5 mg/L) water, to identify the combined effects of dissolved oxygen and ammonia on fish with gender difference. The results showed that oxygenated ammonia exposure increased fish mortality, gill secondary lamellas damage and gill tissue spaces, gene expressions of proinflammatory interleukin 1 beta (il-1ß) and apoptotic caspase8 as compared with non­oxygenated ammonia. Besides, oxygenated ammonia elevated plasma ammonia contents, and decreased the discharge of body ammonia through gills by depressing the enzyme activity of Na+/K+-ATPase. Notably, when zebrafish were subjected to ammonia stress, more severe mortality, gill damage and tissue inflammatory response were observed in males than females. This is the first study to clarify the gender-dependent impacts of ammonia toxicity, and the adverse effects of oxygenation on ammonia resistance in zebrafish.

Vitellogenin 1 is essential for fish reproduction by transporting DHA-containing phosphatidylcholine from liver to ovary.

Vitellogenins (Vtgs) are essential for female reproduction in oviparous animals, yet the exact roles and mechanisms remain unknown. In the present study, we knocked out vtg1, which is the most abundant Vtg in zebrafish, Danio rerio via the CRISPR/Cas 9 technology. We aimed to identify the roles of Vtg1 and related mechanisms in reproduction and development. We found that, the Vtg1-deficient female zebrafish reduced gonadosomatic index, egg production, yolk granules and mature follicles in ovary compared to the wide type (WT). Moreover, the Vtg1-deficient zebrafish diminished hatching rates, cumulative survival rate, swimming capacity and food intake, but increased malformation rate, and delayed swim bladder development during embryo and early-larval phases. The Vtg1-deficiency in female broodstock inhibited docosahexaenoic acid-enriched phosphatidylcholine (DHA-PC) transportation from liver to ovary, which lowered DHA-PC content in ovary and offspring during larval stage. However, the Vtg1-deficient zebrafish increased gradually the total DHA-PC content via exogeneous food intake, and the differences in swimming capacity and food intake returned to normal as they matured. Furthermore, supplementing Vtg1-deficient zebrafish with dietary PC and DHA partly ameliorated the impaired female reproductive capacity and larval development during early phases. This study indicates that, DHA and PC carried by Vtg1 are crucial for female fecundity, and affect embryo and larval development through maternal-nutrition effects. This is the first study elucidating the nutrient and physiological functions of Vtg1 and the underlying biochemical mechanisms in fish reproduction and development.

Environmental estrogen exposure converts lipid metabolism in male fish to a female pattern mediated by AMPK and mTOR signaling pathways.

Environmental estrogens, including bisphenol A (BPA) and 17ß-estradiol (E2), which are widely used in industries and medicine, pose a severe ecological threat to fish due to feminization induction. However, the related metabolic basis for reproductive feminization in male fish has not been well addressed. We first found that female zebrafish exhibited higher lipid accumulation and lipogenesis activity than males. Next, we exposed male and female zebrafish to E2 (200 ng/L) or BPA (100 µg/L) for six weeks, and observed an early-phase reproductive feminization in males, accompanied with reduced spermatids, significant fat deposition and lipogenic gene expressions that mimicked female patterns. Cellular signaling assays revealed that, E2 or BPA modulated lipid metabolism in males mainly through lowering 5' AMP-activated protein kinase (AMPK) and upregulating the lipogenic mechanistic target of rapamycin (mTOR) pathways. For the first time, we show that environmental estrogens could alter lipid metabolism in male fish to a female pattern (metabolic feminization) prior to gonad feminization in male fish, to allows males to accumulate efficiently lipids to harmonize with the feminized gonads. This study suggests that negative effects of environmental estrogens, as hazardous materials, on vertebrate health are more complicated than originally thought.

Concentration-dependent effects of 17ß-estradiol and bisphenol A on lipid deposition, inflammation and antioxidant response in male zebrafish (Danio rerio).

Environmental estrogenic compounds are important pollutants, which are widely distributed in natural water bodies. They produce various adverse effects on fish, but their concentration-dependent toxicities in fish metabolism and health are not fully understood. This study investigated the effects of 17ß-estradiol (E2) and bisphenol A (BPA) at low and high concentrations on lipid deposition, inflammation and antioxidant response in male zebrafish. We measured fish growth parameters, gonad development, lipid contents and the activities of inflammatory and antioxidant enzymes, as well as their mRNA expressions. All E2 and BPA concentrations used increased body weight, damaged gonad structure and induced feminization in male zebrafish. The exposure of zebrafish to E2 and BPA promoted lipid accumulation by increasing total fat, liver triglycerides and free fatty acid contents, and also upregulated lipogenic genes expression, although they decreased total cholesterol content. Notably, zebrafish exposed to low concentrations of E2 (200 ng/L) and BPA (100 µg/L) had higher lipid synthesis and deposition compared to high concentrations (2000 ng/L and 2000 µg/L, respectively). However, the high concentrations of E2 and BPA increased inflammation and antioxidant response. Furthermore, BPA caused greater damage to fish gonad development and more severe lipid peroxidation compared to E2. Overall, the results suggest that the toxic effects of E2 and BPA on zebrafish are concentration-dependent such that, the relative low concentrations used induced lipid deposition, whereas the high ones caused adverse effects on inflammation and antioxidant response.

Reduced oxidative stress increases acute cold stress tolerance in zebrafish.

Cold stress is a major threat to fish in both nature and aquaculture, and can induce oxidative stress in various fish. While the exact role of oxidative stress in cold-caused mortality is still unknown. The purpose of the present study was to evaluate the effects of oxidative stress on cold tolerance in fish and verify whether changing oxidative status could affect cold tolerance. We firstly demonstrated that acute cold exposure induced high oxidative stress in zebrafish liver, which may lead to mortality. Then we performed in vivo and in vitro experiments to determine the effects of the altered oxidative status on cold tolerance in zebrafish and zebrafish liver cell line (ZFL), respectively. In the in vivo study, the zebrafish which were fed with α-lipoic acid or reduced glutathione had lower cold-caused oxidative stress and tissues damage, and showed higher cold tolerance. In the experiment using zebrafish cells, increasing oxidative stress by H2O2 decreased the cellular cold tolerance, and the cold tolerance was partly recovered when oxidative stress was reduced by the addition of Vitamin C (VC). Taken together, we conclude that the reduction of oxidative stress increases cold tolerance in fish.

Tracking pollutants in dietary fish oil: From ocean to table.

Dietary fish oil used in aquafeed transfers marine pollutants to farmed fish. However, the entire transfer route of marine pollutants in dietary fish oil from ocean to table fish has not been tracked quantitatively. To track the entire transfer route of marine pollutants from wild fish to farmed fish through dietary fish oil and evaluate the related human health risks, we obtained crude and refined fish oils originating from the same batch of wild ocean anchovy and prepared fish oil-containing purified aquafeeds to feed omnivorous lean Nile tilapia and carnivorous fatty yellow catfish for eight weeks. The potential human health risk of consumption of these fish was evaluated. Marine persistent organic pollutants (POPs) were concentrated in fish oil, but were largely removed by the refining process, particularly dioxins and polychlorinated biphenyls (PCBs). The differences in the POP concentrations between crude and refined fish oils were retained in the fillets of the farmed fish. Fillets fat content and fish growth were positively and negatively correlated to the final POPs deposition in fillets, respectively. The retention rates of marine POPs in the final fillets through fish oil-contained aquafeeds were 1.3%-5.2%, and were correlated with the POPs concentrations in feeds and fillets, feed utilization and carcass ratios. The dietary crude fish oil-contained aquafeeds are a higher hazard ratio to consumers. Prohibiting the use of crude fish oil in aquafeed and improving growth and feed efficiency in farmed fish are promising strategies to reduce health risks originating from marine POPs.

Chronic exposure to low environmental concentrations and legal aquaculture doses of antibiotics cause systemic adverse effects in Nile tilapia and provoke differential human health risk.

BACKGROUND: Antibiotics used globally to treat human and animal diseases exist ubiquitously in the environment at low doses because of misuse, overdose and poor absorption after ingestion, coupled with their high-water solubility and degradation resistance. However, the systemic chronic effects of exposure to low environmental concentrations of antibiotics (LECAs) and legal aquaculture doses of antibiotics (LADAs) in fish and their human health risk are currently unknown. OBJECTIVE: To investigate the in vivo chronic effects of exposure to LECAs and LADAs using oxytetracycline (OTC) and sulfamethoxazole (SMZ) in Nile tilapia (Oreochromis niloticus) and their human health risk. METHODS: Twenty O. niloticus weighing 27.73 ±â€¯0.81 g were exposed to water containing LECAs (OTC at 420 ng/L and SMZ at 260 ng/L) and diets supplemented with LADAs (OTC 80 mg/kg/day and SMZ 100 mg/kg/day) for twelve weeks. General physiological functions, metabolic activities, intestinal and hepatic health were systemically evaluated. The possible human health risks of the consumption of the experimental Nile tilapia fillets in adults and children were assessed by using risk quotient. RESULTS: After exposure, we observed retarded growth performance accompanied by reduced nutrients digestibility, feed efficiency, organ indices, and lipid body composition in treated fish. Antibiotics distorted intestinal morphological features subsequently induced microbiota dysbiosis and suppressed intestinal tight junction proteins. Exposure of fish to LECAs and LADAs induced oxidative stress, suppressed innate immunity, stimulated inflammatory and detoxification responses, concomitantly inhibited antioxidant capacity and caused lipid peroxidation in intestine and liver organs. Both LECAs and LADAs enhanced gluconeogenesis, inhibited lipogenesis and fatty acid beta oxidation in intestine and liver organs. The exposure of fish to LECAs and LADAs induced anaerobic glycolytic pathway and affected intestinal fat catabolism in intestine while halted aerobic glycolysis, increased hepatic fat catabolism, and induced DNA damage in liver. The hazard risk quotient in children for fish treated with OTCD was >1 indicating human health risk. CONCLUSION: Overall, both LECAs and LADAs impair general physiological functions, nutritional metabolism, and compromise fish immune system. Consumption of fish fed with legal OTC provokes health risk in children. Global stringent prohibition policy for use of antibiotics in aquaculture production and strategies to limit their release into the environment are urgently required to protect human health.