Carrie L. Barton

Vitamin C Deficiency Activates the Purine Nucleotide Cycle in Zebrafish

Background: We investigated the effects of vitamin C status on the metabolome of adult zebrafish. Results: Levels of inosine monophosphate (IMP) and AMP deaminase (AMPD) activity were enhanced in vitamin C-deficient zebrafish. Conclusion: Vitamin C deficiency activates the purine nucleotide cycle in zebrafish. Significance: The link between vitamin C deficiency and elevated AMPD activity is relevant to metabolic diseases. Vitamin C (ascorbic acid, AA) is a cofactor for many important enzymatic reactions and a powerful antioxidant. AA provides protection against oxidative stress by acting as a scavenger of reactive oxygen species, either directly or indirectly by recycling of the lipid-soluble antioxidant, α-tocopherol (vitamin E). Only a few species, including humans, guinea pigs, and zebrafish, cannot synthesize AA. Using an untargeted metabolomics approach, we examined the effects of α-tocopherol and AA deficiency on the metabolic profiles of adult zebrafish. We found that AA deficiency, compared with subsequent AA repletion, led to oxidative stress (using malondialdehyde production as an index) and to major increases in the metabolites of the purine nucleotide cycle (PNC): IMP, adenylosuccinate, and AMP. The PNC acts as a temporary purine nucleotide reservoir to keep AMP levels low during times of high ATP utilization or impaired oxidative phosphorylation. The PNC promotes ATP regeneration by converting excess AMP into IMP, thereby driving forward the myokinase reaction (2ADP → AMP + ATP). On the basis of this finding, we investigated the activity of AMP deaminase, the enzyme that irreversibly deaminates AMP to form IMP. We found a 47% increase in AMP deaminase activity in the AA-deficient zebrafish, complementary to the 44-fold increase in IMP concentration. These results suggest that vitamin C is crucial for the maintenance of cellular energy metabolism.

Vitamin E Deficiency Decreases Long-Chain PUFA in Zebrafish (Danio rerio)

α-Tocopherol is a required, lipid-soluble antioxidant that protects PUFA. We hypothesized that α-tocopherol deficiency in zebrafish compromises PUFA status. Zebrafish were fed for 1 y either an α-tocopherol-sufficient (E+; 500 mg α-tocopherol/kg) or -deficient (E-; 1.1 mg α-tocopherol/kg) diet containing α-linolenic (ALA) and linoleic (LA) acids but without arachidonic acid (ARA), EPA, or DHA. Vitamin E deficiency in zebrafish decreased by ~20% (n-6) (P < 0.05) and (n-3) (P < 0.05) PUFA and increased the (n-6):(n-3) PUFA ratio (P < 0.05). In E- compared to E+ females, long chain-PUFA status was impaired, as assessed by a ~60% lower DHA:ALA ratio (P < 0.05) and a ~50% lower ARA:LA ratio (P < 0.05). fads2 (P < 0.05) and elovl2 (P < 0.05) mRNA expression was doubled in E- compared to E+ fish. Thus, inadequate vitamin E status led to a depletion of PUFA that may be a result of either or both increased lipid peroxidation and an impaired ability to synthesize sufficient PUFA, especially (n-3) PUFA.

Triclosan Exposure Is Associated with Rapid Restructuring of the Microbiome in Adult Zebrafish

Growing evidence indicates that disrupting the microbial community that comprises the intestinal tract, known as the gut microbiome, can contribute to the development or severity of disease. As a result, it is important to discern the agents responsible for microbiome disruption. While animals are frequently exposed to a diverse array of environmental chemicals, little is known about their effects on gut microbiome stability and structure. Here, we demonstrate how zebrafish can be used to glean insight into the effects of environmental chemical exposure on the structure and ecological dynamics of the gut microbiome. Specifically, we exposed forty-five adult zebrafish to triclosan-laden food for four or seven days or a control diet, and analyzed their microbial communities using 16S rRNA amplicon sequencing. Triclosan exposure was associated with rapid shifts in microbiome structure and diversity. We find evidence that several operational taxonomic units (OTUs) associated with the family Enterobacteriaceae appear to be susceptible to triclosan exposure, while OTUs associated with the genus Pseudomonas appeared to be more resilient and resistant to exposure. We also found that triclosan exposure is associated with topological alterations to microbial interaction networks and results in an overall increase in the number of negative interactions per microbe in these networks. Together these data indicate that triclosan exposure results in altered composition and ecological dynamics of microbial communities in the gut. Our work demonstrates that because zebrafish afford rapid and inexpensive interrogation of a large number of individuals, it is a useful experimental system for the discovery of the gut microbiome’s interaction with environmental chemicals.

The α-Tocopherol Transfer Protein Is Essential for Vertebrate Embryogenesis

The hepatic α-tocopherol transfer protein (TTP) is required for optimal α-tocopherol bioavailability in humans; mutations in the human TTPA gene result in the heritable disorder ataxia with vitamin E deficiency (AVED, OMIM #277460). TTP is also expressed in mammalian uterine and placental cells and in the human embryonic yolk-sac, underscoring TTP’s significance during fetal development. TTP and vitamin E are essential for productive pregnancy in rodents, but their precise physiological role in embryogenesis is unknown. We hypothesize that TTP is required to regulate delivery of α-tocopherol to critical target sites in the developing embryo. We tested to find if TTP is essential for proper vertebrate development, utilizing the zebrafish as a non-placental model. We verify that TTP is expressed in the adult zebrafish and its amino acid sequence is homologous to the human ortholog. We show that embryonic transcription of TTP mRNA increases >7-fold during the first 24 hours following fertilization. In situ hybridization demonstrates that Ttpa transcripts are localized in the developing brain, eyes and tail bud at 1-day post fertilization. Inhibiting TTP expression using oligonucleotide morpholinos results in severe malformations of the head and eyes in nearly all morpholino-injected embryos (88% compared with 5.6% in those injected with control morpholinos or 1.7% in non-injected embryos). We conclude that TTP is essential for early development of the vertebrate central nervous system.

Novel liquid chromatography–mass spectrometry method shows that vitamin E deficiency depletes arachidonic and docosahexaenoic acids in zebrafish (Danio rerio) embryos

To test the hypothesis that embryogenesis depends upon α-tocopherol (E) to protect embryo polyunsaturated fatty acids (PUFAs) from lipid peroxidation, new methodologies were applied to measure α-tocopherol and fatty acids in extracts from saponified zebrafish embryos. A solid phase extraction method was developed to separate the analyte classes, using a mixed mode cartridge (reverse phase, π–π bonding, strong anion exchange), then α-tocopherol and cholesterol were measured using standard techniques, while the fatty acids were quantitated using a novel, reverse phase liquid chromatography–mass spectrometry (LC–MS) approach. We also determined if α-tocopherol status alters embryonic lipid peroxidation products by analyzing 24 different oxidized products of arachidonic or docosahexaenoic (DHA) acids in embryos using LC with hybrid quadrupole-time of flight MS. Adult zebrafish were fed E− or E+ diets for 4 months, and then were spawned to obtain E− and E+ embryos. Between 24 and 72 hours post-fertilization (hpf), arachidonic acid decreased 3-times faster in E− (21 pg/h) compared with E+ embryos (7 pg/h, P<0.0001), while both α-tocopherol and DHA concentrations decreased only in E− embryos. At 36 hpf, E− embryos contained double the 5-hydroxy-eicosatetraenoic acids and 7-hydroxy-DHA concentrations, while other hydroxy-lipids remained unchanged. Vitamin E deficiency during embryogenesis depleted DHA and arachidonic acid, and increased hydroxy-fatty acids derived from these PUFA, suggesting that α-tocopherol is necessary to protect these critical fatty acids.

Toxicity of chlorine to zebrafish embryos

Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce extraovum pathogens that may be released from brood fish during spawning, and this is routinely used in zebrafish Danio rerio research laboratories. Most laboratories use approximately 25 to 50 ppm unbuffered chlorine solution for 5 to 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, but is less effective against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbuffered and buffered chlorine solutions to embryos exposed at 6 or 24 h post-fertilization (hpf) to determine whether higher concentrations can be used for treating zebrafish embryos. Most of our experiments entailed using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6 hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of chlorine. Chlorine is more toxic and germicidal at lower pH, and chlorine causes elevated pH. Consistent with this, we found that unbuffered chlorine solutions (pH ca. 8-9) were less toxic at corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we recommend treating 6 hpf embryos for 10 min and 24 hpf embryos for 5 min with unbuffered chlorine solution at 100 ppm.

The influences of parental diet and vitamin E intake on the embryonic zebrafish transcriptome

The composition of the typical commercial diet fed to zebrafish can dramatically vary. By utilizing defined diets we sought to answer two questions: 1) How does the embryonic zebrafish transcriptome change when the parental adults are fed a commercial lab diet compared with a sufficient, defined diet (E+)? 2) Does a vitamin E-deficient parental diet (E-) further change the embryonic transcriptome? We conducted a global gene expression study using embryos from zebrafish fed a commercial (Lab), an E+ or an E- diet. To capture differentially expressed transcripts prior to onset of overt malformations observed in E- embryos at 48h post-fertilization (hpf), embryos were collected from each group at 36hpf. Lab embryos differentially expressed (p<0.01) 946 transcripts compared with the E+ embryos, and 2656 transcripts compared with the E- embryos. The differences in protein, fat and micronutrient intakes in zebrafish fed the Lab compared with the E+ diet demonstrate that despite overt morphologic consistency, significant differences in gene expression occurred. Moreover, functional analysis of the significant transcripts in the E- embryos suggested perturbed energy metabolism, leading to overt malformations and mortality. Thus, these findings demonstrate that parental zebrafish diet has a direct impact on the embryonic transcriptome.

Adverse effects of parental zinc deficiency on metal homeostasis and embryonic development in a zebrafish model

The high prevalence of zinc deficiency is a global public health concern, and suboptimal maternal zinc consumption has been associated with adverse effects ranging from impaired glucose tolerance to low birthweights. The mechanisms that contribute to altered development and poor health in zinc deficient offspring are not completely understood. To address this gap, we utilized the Danio rerio model and investigated the impact of dietary zinc deficiency on adults and their developing progeny. Zinc deficient adult fish were significantly smaller in size, and had decreases in learning and fitness. We hypothesized that parental zinc deficiency would have an impact on their offsprings mineral homeostasis and embryonic development. Results from mineral analysis showed that parental zinc deficiency caused their progeny to be zinc deficient. Furthermore, parental dietary zinc deficiency had adverse consequences for their offspring including a significant increase in mortality and decreased physical activity. Zinc deficient embryos had altered expression of genes that regulate metal homeostasis including several zinc transporters (ZnT8, ZnT9) and the metal-regulatory transcription factor 1 (MTF-1). Zinc deficiency was also associated with decreased expression of genes related to diabetes and pancreatic development in the embryo (Insa, Pax4, Pdx1). Decreased expression of DNA methyltransferases (Dnmt4, Dnmt6) was also found in zinc deficient offspring, which suggests that zinc deficiency in parents may cause altered epigenetic profiles for their progeny. These data should inform future studies regarding zinc deficiency and pregnancy and suggest that supplementation of zinc deficient mothers prior to pregnancy may be beneficial.

A Novel Zebrafish Model for Assessing In Vivo Delivery of Morpholino Oligomers

Morpholino oligomers have great therapeutic potential for treatment of a broad range of human diseases, including viral, bacterial, age-related, and genetic diseases, but they suffer from poor systemic delivery into cells. Although various approaches have been undertaken to address the delivery problem, it remains as the major barrier of morpholinos to be used as effective therapeutics. This slow development is in part due to the cost of materials and the animal models used for screening the efficacy and safety of those delivery approaches. The need to have an inexpensive vertebrate model for assessing in vivo delivery of morpholinos is evident. Therefore, we have produced a novel transgenic zebrafish model containing a dual reporter cassette for determination of in vivo delivery, bio-distribution, and safety of a morpholino. The levels of morpholino delivered to the cells in various tissues can be determined by changes in reporter gene expressions caused by morpholino-induced exon skipping. This chapter provides a description of the reagents, equipment, and procedure for successful retro-orbital injection of a peptide-conjugated morpholino into the blood stream of the adult zebrafish to cause targeted exon skipping in the heart of the zebrafish.

Combinatorial effects of zinc deficiency and arsenic exposure on zebrafish (Danio rerio) development

Zinc deficiency and chronic low level exposures to inorganic arsenic in drinking water are both significant public health concerns that affect millions of people including pregnant women. These two conditions can co-exist in the human population but little is known about their interaction, and in particular, whether zinc deficiency sensitizes individuals to arsenic exposure and toxicity, especially during critical windows of development. To address this, we utilized the Danio rerio (zebrafish) model to test the hypothesis that parental zinc deficiency sensitizes the developing embryo to low-concentration arsenic toxicity, leading to altered developmental outcomes. Adult zebrafish were fed defined zinc deficient and zinc adequate diets and were spawned resulting in zinc adequate and zinc deficient embryos. The embryos were treated with environmentally relevant concentrations of 0, 50, and 500 ppb arsenic. Arsenic exposure significantly reduced the amount of zinc in the developing embryo by ~7%. The combination of zinc deficiency and low-level arsenic exposures did not sensitize the developing embryo to increased developmental malformations or mortality. The combination did cause a 40% decline in physical activity of the embryos, and this decline was significantly greater than what was observed with zinc deficiency or arsenic exposure alone. Significant changes in RNA expression of genes that regulate zinc homeostasis, response to oxidative stress and insulin production (including zip1, znt7, nrf2, ogg1, pax4, and insa) were found in zinc deficient, or zinc deficiency and arsenic exposed embryos. Overall, the data suggests that the combination of zinc deficiency and arsenic exposure has harmful effects on the developing embryo and may increase the risk for developing chronic diseases like diabetes.