Favipiravir induces HuNoV viral mutagenesis and infectivity loss with clinical improvement in immunocompromised patients.

Chronic human norovirus (HuNoV) infections in immunocompromised patients result in severe disease, yet approved antivirals are lacking. RNA-dependent RNA polymerase (RdRp) inhibitors inducing viral mutagenesis display broad-spectrum in vitro antiviral activity, but clinical efficacy in HuNoV infections is anecdotal and the potential emergence of drug-resistant variants is concerning. Upon favipiravir (and nitazoxanide) treatment of four immunocompromised patients with life-threatening HuNoV infections, viral whole-genome sequencing showed accumulation of favipiravir-induced mutations which coincided with clinical improvement although treatment failed to clear HuNoV. Infection of zebrafish larvae demonstrated drug-associated loss of viral infectivity and favipiravir treatment showed efficacy despite occurrence of RdRp variants potentially causing favipiravir resistance. This indicates that within-host resistance evolution did not reverse loss of viral fitness caused by genome-wide accumulation of sequence changes. This off-label approach supports the use of mutagenic antivirals for treating prolonged RNA viral infections and further informs the debate surrounding their impact on virus evolution.

Knockout of lws1 in zebrafish (Danio rerio) reveals its role in regulating feeding and vision-guided behavior.

Long-wave sensitive (LWS) is a G protein-coupled receptor expressed in the retina, and zebrafish is a better model organism for studying vision, but the role of LWS1 in vision-guided behavior of larvae fish has rarely been reported. In this study, we found that zebrafish lws1 and lws2 are tandemly replicated genes, both with six exons, with lws1 being more evolutionarily conserved. The presence of Y277F in the amino acid sequence of lws2 may have contributed to the shift of λmax to green light. We established a lws1 knockout zebrafish model using CRISPR/Cas9 technology. Lws1-/- larvae showed significantly higher levels of feeding and appetite gene (agrp) expression than WT, and significantly lower levels of anorexia gene (pomc, cart) expression. In addition, green light gene compensation was observed in lws1-/- larvae with significantly increased expression levels of rh2-1. The light-dark movement test showed that lws1-/- larvae were more active under light-dark transitions or vibrational stimuli, and the expression of phototransduction-related genes was significantly up-regulated. This study reveals the important role of lws1 gene in the regulation of vision-guided behavior in larvae.

Application of a capillary electrophoresis-mass spectrometry metabolomics workflow in zebrafish larvae reveals new effects of cortisol.

In contemporary biomedical research, the zebrafish (Danio rerio) is increasingly considered a model system, as zebrafish embryos and larvae can (potentially) fill the gap between cultured cells and mammalian animal models, because they can be obtained in large numbers, are small and can easily be manipulated genetically. Given that capillary electrophoresis-mass spectrometry (CE-MS) is a useful analytical separation technique for the analysis of polar ionogenic metabolites in biomass-limited samples, the aim of this study was to develop and assess a CE-MS-based analytical workflow for the profiling of (endogenous) metabolites in extracts from individual zebrafish larvae and pools of small numbers of larvae. The developed CE-MS workflow was used to profile metabolites in extracts from pools of 1, 2, 4, 8, 12, 16, 20, and 40 zebrafish larvae. For six selected endogenous metabolites, a linear response (R2  > 0.98) for peak areas was obtained in extracts from these pools. The repeatability was satisfactory, with inter-day relative standard deviation values for peak area of 9.4%-17.7% for biological replicates (n = 3 over 3 days). Furthermore, the method allowed the analysis of over 70 endogenous metabolites in a pool of 12 zebrafish larvae, and 29 endogenous metabolites in an extract from only 1 zebrafish larva. Finally, we applied the optimized CE-MS workflow to identify potential novel targets of the mineralocorticoid receptor in mediating the effects of cortisol.

Poly(I:C) induces anti-inflammatory response against secondary LPS challenge in zebrafish larvae.

Poly(I:C) is known as an agonist of the TLR3 receptor which could prime inflammation and elicit the host immune response, which is widely applied as adjuvant or antivirus treatment. However, the negative effects of poly(I:C) on regulating immune response to protect the host from inflammatory diseases remain largely unknown. Here, we establish an in vivo model to pre-treat zebrafish larvae with poly(I:C) at 2 dpf, then challenge them with LPS at 6 dpf, and find that poly(I:C) training could significantly alleviate the LPS challenge-induced septic shock and inflammatory phenotypes. Moreover, the poly(I:C)-trained larvae exhibit decreased number of macrophages, but not neutrophils, after secondary LPS challenge. Furthermore, training the larvae with poly(I:C) could elevate the transcripts of mTOR signaling and heighten the H3K4me3-mediated epigenetic modifications. And interestingly, we find that inhibiting the H3K4me3 modification, rather than mTOR signaling, could recover the number of macrophages in poly(I:C)-trained larvae, which is consistent with the observations of inflammatory phenotypes. Taken together, these results suggest that poly(I:C) training could induce epigenetic rewiring to mediate the anti-inflammatory response against secondary LPS challenge-induced septic shock through decreasing macrophages' number in vivo, which might expand our understanding of poly(I:C) in regulating fish immune response.

Effect of Ishige okamurae Extract on Osteoclastogenesis In Vitro and In Vivo.

We demonstrated the effect of Ishige okamurae extract (IOE) on the receptor activator of nuclear factor-κB ligand (RANKL)-promoted osteoclastogenesis in RAW 264.7 cells and confirmed that IOE inhibited RANKL-induced tartrate-resistant acid phosphatase (TRAP) activity and osteoclast differentiation. IOE inhibited protein expression of TRAP, metallopeptidase-9 (MMP-9), the calcitonin receptor (CTR), and cathepsin K (CTK). IOE treatment suppressed the expression of activated T cell cytoplasmic 1 and activator protein-1, thus controlling the expression of osteoclast-related factors. Moreover, IOE significantly reduced RANKL-phosphorylated extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK). It also reduced the RANKL-induced phosphorylation of NF-κB and nuclear translocation of p65. IOE inhibited Dex-induced bone loss and osteoclast-related gene expression in zebrafish larvae. HPLC analysis shows that IOE consists of 3.13% and 3.42% DPHC and IPA, respectively. Our results show that IOE has inhibitory effects on osteoclastogenesis in vitro and in vivo and is a potential therapeutic for osteoporosis.

Caffeine Decreases Topiramate Levels in Zebrafish Larvae in a Pentylenetetrazol-Induced Seizure Model.

Epilepsy ranks as the second-most prevalent neurological disease, and is characterized by seizures resulting in neurobiological and behavioral impairment. Naturally occurring in coffee beans or tea leaves, the alkaloid caffeine (CAF) is the most prevalent global stimulant. Caffeine has been observed to influence epileptic seizures and the efficacy of antiepileptic medications, with a notable impact on topiramate (TPM). This study aimed to explore the influence of CAF on TPM's anticonvulsant effects in zebrafish larvae within a PTZ-induced seizure model, concurrently determining TPM concentrations through a sophisticated analytical approach based on ultrahigh-performance liquid chromatography and subsequent mass spectrometric detection. Zebrafish larvae four days post-fertilization were incubated for 18 h with varying doses of TPM or combinations of CAF + TPM, and locomotor activity was then assessed. Seizures were induced by introducing a PTZ solution to achieve a final concentration of 20 mM. Utilizing liquid chromatography-mass spectrometry (LC-MS/MS), TPM levels in the larvae were quantified. CAF co-administration (especially in higher doses) with TPM caused a decrease in the average locomotor activity in the larvae compared to TPM alone. Moreover, CAF decreased TPM levels in the larvae at all investigated doses. In conclusion, these findings offer a novel perspective on the interplay between CAF and TPM, shedding light on previously unexplored facets. The potential impact of CAF consumption in assisting with epileptic seizure control, unless proven otherwise, suggests a noteworthy consideration for future research and clinical practices.

Early stress exposure on zebrafish development: effects on survival, malformations and molecular alterations.

The effects of stress during early vertebrate development can be especially harmful. Avoiding stressors in fish larvae is essential to ensure the health of adult fish and their reproductive performance and overall production. We examined the consequences of direct exposure to successive acute stressors during early development, including their effects on miR-29a and its targets, survival, hatching and malformation rates, larval behaviour and cartilage and eye development. Our aim was to shed light on the pleiotropic effects of early-induced stress in this vertebrate model species. Our results showed that direct exposure to successive acute stressors during early development significantly upregulated miR-29a and downregulated essential collagen transcripts col2a1a, col6a2 and col11a1a, decreased survival and increased malformation rates (swim bladder, otoliths, cardiac oedema and ocular malformations), promoting higher rates of immobility in larvae. Our results revealed that stress in early stages can induce different eye tissular architecture and cranioencephalic cartilage development alterations. Our research contributes to the understanding of the impact of stressful conditions during the early stages of zebrafish development, serving as a valuable model for vertebrate research. This holds paramount significance in the fields of developmental biology and aquaculture and also highlights miR-29a as a potential molecular marker for assessing novel larval rearing programmes in teleost species.

BPA induces hepatotoxicity in zebrafish through oxidative stress and apoptosis pathways.

BPA is so ubiquitous that 27 million tons of BPA-containing plastic, including mineral water bottles and baby bottles, is produced worldwide each year. The potential toxicity of BPA to humans and aquatic organisms has been the subject of intense research. In this study, a zebrafish model system was used to assess BPA-mediated hepatotoxicity. Zebrafish larvae at 72-144 hpf were exposed to BPA at different concentrations (0,1, 3 and 5mg/L). For example, BPA-treated zebrafish larvae showed increased mortality, delayed uptake of nutrients in yolk sac, shortened body length, smaller liver area, abnormal expression of genes related to liver development, and pathological changes in the liver tissue. Mechanistically, BPA exposure induced excessive oxidative stress in the liver of zebrafish and increased the level of hepatocyte apoptosis in zebrafish larvae, and the antioxidant astaxanthin could rescue the BPA-mediated liver toxicity.

Cell-Derived Vesicles for Antibiotic Delivery-Understanding the Challenges of a Biogenic Carrier System.

Recently, extracellular vesicles (EVs) sparked substantial therapeutic interest, particularly due to their ability to mediate targeted transport between tissues and cells. Yet, EVs' technological translation as therapeutics strongly depends on better biocompatibility assessments in more complex models and elementary in vitro-in vivo correlation, and comparison of mammalian versus bacterial vesicles. With this in mind, two new types of EVs derived from human B-lymphoid cells with low immunogenicity and from non-pathogenic myxobacteria SBSr073 are introduced here. A large-scale isolation protocol to reduce plastic waste and cultivation space toward sustainable EV research is established. The biocompatibility of mammalian and bacterial EVs is comprehensively evaluated using cytokine release and endotoxin assays in vitro, and an in vivo zebrafish larvae model is applied. A complex three-dimensional human cell culture model is used to understand the spatial distribution of vesicles in epithelial and immune cells and again used zebrafish larvae to study the biodistribution in vivo. Finally, vesicles are successfully loaded with the fluoroquinolone ciprofloxacin (CPX) and showed lower toxicity in zebrafish larvae than free CPX. The loaded vesicles are then tested effectively on enteropathogenic Shigella, whose infections are currently showing increasing resistance against available antibiotics.

Valbenazine promotes body growth via growth hormone signaling during zebrafish embryonic development.

Vesicular monoamine transporter 2 (VMAT-2) functions by uptake of cytoplasmic monoamines into vesicles for storage. Valbenazine (VBZ) is a newly FDA-approved oral VMAT-2 inhibitor used for the treatment of movement disorders such as tardive dyskinesia (TD), and Tourette syndrome (TS). Clinical data shows that VBZ is a relatively safe drug with no cardiotoxicity or hepatotoxicity. However, the effect of VBZ on embryonic development remains unknown. Here, we use zebrafish larvae as an animal model to demonstrate that VBZ exposure causes premature hatching and increased body size and hyperactivity-like behaviors in zebrafish larvae. In addition, VBZ exposure leads to increased dopamine (DA) and Glutamate (Glu) levels. Moreover, an increase of growth hormone (gh) and enriched PI3K/AKT signaling were found in VBZ-exposed zebrafish larvae, which may explain their accelerated development. In summary, VBZ exposure may be developmentally toxic in zebrafish larvae.

Neural System Impairment and Involved Microglia-Neuron Regulation of Broflanilide in Zebrafish Larvae.

Broflanilide is widely used to control pests and has attracted attention due to its adverse effects on aquatic organisms. Our previous study showed that broflanilide has a negative impact on the central nervous system (CNS) at lethal dosages; however, its neural effects under practical situations and the underlying mechanisms remain unknown. To elucidate how broflanilide affects the CNS, we exposed zebrafish larvae to broflanilide at 16.9 and 88.0 µg/L (the environmentally relevant concentrations) for 120 h. Zebrafish locomotion was significantly disturbed at 88.0 µg/L, with a decreased moving distance and velocity accompanied by an inhibited neurotransmitter level. In vivo neuroimaging analysis indicated that the nerves of zebrafish larvae, including the axons, myelin sheaths, and neurons, were impaired. The number of neurons was significantly reduced after exposure, with an impaired morphological structure. These changes were accompanied by the abnormal transcription of genes involved in early CNS development. In addition, an increased total number of microglia and an elevated proportion of amoeboid microglia were observed after 88.0 µg/L broflanilide exposure, pointing out to an upstream role of microglia activation in mediating broflanilide neurotoxicity. Meanwhile, increased inflammatory cytokine levels and brain neutrophil numbers were observed, implicating significant inflammatory response and immune toxicity. Our findings indicate that broflanilide interferes with microglia-neuron regulation and induces neurodevelopmental disorders.

A refined toxicokinetic model for quantifying the interaction between Cd and Cu in zebrafish larvae.

The interaction between metals is ubiquitous, but there is still a lack of quantitative models considering the interaction between metals, which leads to the deviations in predicting the joint toxicity of metals. The present study estimated the uptake rate constants (kin) and elimination rate constants (kout) and elucidated how the presence of one metal (Cu or Cd) affects the absorption and excretion of another metal (Cd or Cu) in zebrafish larvae. The results showed that Cd and Cu inhibited each other in the process of absorption and excretion by comparing separately kin and kout of Cd or Cu with the other metal Cu or Cd mixed concentrations increased, thereby affecting the Cd and Cu bioaccumulation in the zebrafish larvae. Then the interactions between Cd and Cu in the uptake and elimination processes were quantified to obtain a refined toxicokinetic model. Verification with independent experiment data showed that the refined toxicokinetic model could significantly improve the prediction of the Cd or Cu bioaccumulation in the zebrafish larvae. This study contributes to understand the toxicokinetic process of the Cd-Cu mixture in the zebrafish larvae, and the developed model could be used to predict the toxicity of the metal mixtures.

Developmental and behavioral toxicity assessment of opicapone in zebrafish embryos.

The use of clinical psychoactive drugs often poses unpredictable threats to fetal development. Catechol-O-methyltransferase (COMT) is a key enzyme that regulates dopamine metabolism and a promising target for modulation of cognitive functions. Opicapone, a newly effective third-generation peripheral COMT inhibitor, is used for the treatment of Parkinson's disease (PD) and possibly to improve other dopamine-related disorders such as alcohol use disorder (AUD) and obsessive-compulsive disorder (OCD). The widespread use of opicapone will inevitably lead to biological exposure and damage to the human body, such as affecting fetal development. However, the effect of opicapone on embryonic development remains unknown. Here, zebrafish larvae were used as an animal model and demonstrated that a high concentration (30 µM) of opicapone exposure was teratogenic and lethal, while a low concentration also caused developmental delay such as a shortened body size, a smaller head, and reduced locomotor behaviors in zebrafish larvae. Meanwhile, opicapone treatment specifically increased the level of dopamine (DA) in zebrafish larvae. The depletion response of the total glutathione level (including oxidized and reduced forms of glutathione) and changed antioxidant enzymes activities in zebrafish larvae suggest oxidative damage caused by opicapone. In addition, enhanced glutathione metabolism and cytokine-cytokine receptor interaction were found in zebrafish larvae treated with opicapone, indicating that opicapone treatment caused an oxidation process and immune responses. Our results provide a new insight into the significant developmental toxicity of opicapone in zebrafish larvae.

Developmental hazards of 2,2',4,4'-tetrabromodiphenyl ether induced endoplasmic reticulum stress on early life stages of zebrafish (Danio rerio).

Polybrominated diphenyl ether flame retardants are known to have adverse effects on the development of organisms. We investigated the molecular mechanisms associated with the developmental hazards of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in zebrafish, as well as the behavioral and morphological alterations involved, focusing on endoplasmic reticulum stress (ERS), oxidative stress, and apoptosis. Our study revealed behavioral alterations in zebrafish exposed to BDE-47, including impaired motor activity, reduced exploration, and abnormal swimming patterns. In addition, we observed malformations in craniofacial regions and other developmental abnormalities that may be associated with ERS-induced cellular dysfunction. BDE-47 exposure showed apparent changes in ERS, oxidative stress, and apoptosis biomarkers at different developmental stages in zebrafish through gene expression analysis and enzyme activity assays. The study indicated that exposure to BDE-47 results in ERS, as supported by the upregulation of ERS-related genes and increased activity of ERS markers. In addition, oxidative stress-related genes showed different expression patterns, suggesting that oxidative stress is involved in the BDE-47 toxic effects. Moreover, an assessment of apoptotic biomarkers revealed an imbalance in the expression levels of pro- and anti-apoptotic genes, suggesting that BDE-47 exposure activated the apoptotic pathway. These results highlight the complex interactions between ERS, oxidative stress, apoptosis, behavioral alterations, and morphological malformations following BDE-47 exposure in zebrafish. Understanding the mechanisms of toxicity of developmental hazards is essential to elucidate the toxicological effects of environmental contaminants. The knowledge can help develop strategies to mitigate their adverse effects on the health of ecosystems and humans.

A New In Vivo Zebrafish Bioassay Evaluating Liver Steatosis Identifies DDE as a Steatogenic Endocrine Disruptor, Partly through SCD1 Regulation.

Non-alcoholic fatty liver disease (NAFLD), which starts with liver steatosis, is a growing worldwide epidemic responsible for chronic liver diseases. Among its risk factors, exposure to environmental contaminants, such as endocrine disrupting compounds (EDC), has been recently emphasized. Given this important public health concern, regulation agencies need novel simple and fast biological tests to evaluate chemical risks. In this context, we developed a new in vivo bioassay called StAZ (Steatogenic Assay on Zebrafish) using an alternative model to animal experimentation, the zebrafish larva, to screen EDCs for their steatogenic properties. Taking advantage of the transparency of zebrafish larvae, we established a method based on fluorescent staining with Nile red to estimate liver lipid content. Following testing of known steatogenic molecules, 10 EDCs suspected to induce metabolic disorders were screened and DDE, the main metabolite of the insecticide DDT, was identified as a potent inducer of steatosis. To confirm this and optimize the assay, we used it in a transgenic zebrafish line expressing a blue fluorescent liver protein reporter. To obtain insight into DDE's effect, the expression of several genes related to steatosis was analyzed; an up-regulation of scd1 expression, probably relying on PXR activation, was found, partly responsible for both membrane remodeling and steatosis.

Optimization of the Zebrafish Larvae Pentylenetetrazol-Induced Seizure Model for the Study of Caffeine and Topiramate Interactions.

Epilepsy is a common neurological disorder characterized by seizures that cause neurobiological and behavioral impairment. Caffeine (CAF), which is the most widely consumed stimulant in the world, is reported to influence epileptic seizures and antiepileptic drugs, especially topiramate (TPM). The aim of the study was to optimize the zebrafish larvae pentylenetetrazol-induced seizure model for the study of CAF and TPM interactions, which include the determination of dose space, and the delivery of an analytical method for monitoring CAF, TPM, and CAF metabolite paraxanthine (PAR) in Zebrafish larvae. Methods: The zebrafish larvae, 4 days post-fertilization, were incubated for 18 h with CAF, TPM, or CAF + TPM, with subsequent locomotor activity assessment. Seizures were evoked by adding PTZ solution to obtain a final concentration of 20 mM. Subsequently, the liquid chromatography-mass spectrometry (LC-MS/MS) analytical method was used to simultaneously assess the levels of both CAF and TPM in the larvae. CAF (50 mg/L) and TPM (75 µM) given separately decreased the average larvae locomotor activity compared to the PTZ group but, however, were not able to lower it to the control level. Co-administration of 25 mg/L CAF and 50 µM TPM suppressed the activity to the same level. Adding 25 µM TPM to 50 mg/L CAF decrease the measured CAF level in the larvae. Until proven otherwise, CAF consumption should be regarded as a potential determinant in the modulation of TPM's efficacy in the management of epileptic seizures. The optimized model will contribute to the standardization of studying CAF and TPM interactions and building the understanding of the molecular bases of the interaction.

Optimization of Mass Spectrometry Imaging for Drug Metabolism and Distribution Studies in the Zebrafish Larvae Model: A Case Study with the Opioid Antagonist Naloxone.

Zebrafish (ZF; Danio rerio) larvae have emerged as a promising in vivo model in drug metabolism studies. Here, we set out to ready this model for integrated mass spectrometry imaging (MSI) to comprehensively study the spatial distribution of drugs and their metabolites inside ZF larvae. In our pilot study with the overall goal to improve MSI protocols for ZF larvae, we investigated the metabolism of the opioid antagonist naloxone. We confirmed that the metabolic modification of naloxone is in high accordance with metabolites detected in HepaRG cells, human biosamples, and other in vivo models. In particular, all three major human metabolites were detected at high abundance in the ZF larvae model. Next, the in vivo distribution of naloxone was investigated in three body sections of ZF larvae using LC-HRMS/MS showing that the opioid antagonist is mainly present in the head and body sections, as suspected from published human pharmacological data. Having optimized sample preparation procedures for MSI (i.e., embedding layer composition, cryosectioning, and matrix composition and spraying), we were able to record MS images of naloxone and its metabolites in ZF larvae, providing highly informative distributional images. In conclusion, we demonstrate that all major ADMET (absorption, distribution, metabolism, excretion, and toxicity) parameters, as part of in vivo pharmacokinetic studies, can be assessed in a simple and cost-effective ZF larvae model. Our established protocols for ZF larvae using naloxone are broadly applicable, particularly for MSI sample preparation, to various types of compounds, and they will help to predict and understand human metabolism and pharmacokinetics.

Design, Synthesis, Anti-Tubercular Evaluation and Teratogenicity Studies of Furanyl Pyrazolo[3,4-b] Quinoline-5-Ones.

We have synthesized novel pyrazolo-quinoline analogues (P1-10) in an effort to create newer antitubercular drugs against the rising bacterial resistance. NMR, IR and ESI-MS spectra were utilized to characterize the synthesised compounds. The antitubercular activity of the target compounds was evaluated against Mycobacterium tuberculosis. Six derivatives (P1-6) displayed very significant activity at 1.6 µg/mL concentration and were found to be more active than pyrazinamide standard. Thus, as per the drug susceptibility results the MIC value could be considered between 1.6 and 0.8 µg/mL. In addition, all the synthesised compounds were subjected to molecular docking studies against specific protein, Enoyl acyl carrier protein reductase (InhA) in complex with N-(4-methylbenzoyl)-4-benzylpiperidine, PDB ID: 2NSD. Among all the compounds the most effective compounds were found an Autodock score of 11.6 and 11.2 against 2NSD, respectively. Further, Zebrafish larvae have been used to test the teratogenicity of the synthesised compounds. There were no indications of abnormalities with (P2), (P4), (P5), (P6), and (P10) at 0.5 µM. Supplementary Information: The online version contains supplementary material available at 10.1134/S1068162023010053.

Transcriptome reveals overview of Ca2+ dose-dependent metabolism disorders in zebrafish larvae after Cd2+ exposure.

Cadmium (Cd), a ubiquitous environmental hazardous heavy metal, poses a significant threat to the health of aquatic organisms, including teleosts. Although the toxic profile of Cd is well recognized, little is known regarding the overall view of toxic responses to varying aquatic environmental parameters (e.g., water hardness) at an individual level. Herein, differences in water hardness were partially mimicked by adjusting Ca2+ levels in E3 medium. As an in vivo model, zebrafish embryos were exposed to variable Ca2+ levels (NV, normal Ca2+; LV, low Ca2+; HV, high Ca2+) alone or combined with 30.7 µg/L Cd2+ (NC, LC, and HC, respectively) until 144 hr post-fertilization. The genome-wide transcriptome revealed differentially expressed genes between groups. Functional enrichment analysis found that biological processes related to metabolism, particularly lipid metabolism, were significantly disrupted in NC and LC treatments, while a remission was observed in the HC group. Biochemical assays confirmed that the decrease in Ca2+ enhanced synthesis, inhibited mobilization and increased the storage of lipids in Cd2+ treatments. This study suggests that the toxic effect of Cd on biological pathways will be influenced by Ca2+, which will improve the toxicological understanding and facilitate accurate assessment of Cd.

Characterization of two novel ammonia transporters, Hiat1a and Hiat1b, in the teleost model system Danio rerio.

Ammonia excretion in fish excretory epithelia is a complex interplay of multiple membrane transport proteins and mechanisms. Using the model system of zebrafish (Danio rerio) larvae, here we identified three paralogues of a novel ammonia transporter, hippocampus-abundant transcript 1 (DrHiat1), also found in most vertebrates. When functionally expressed in Xenopus laevis oocytes, DrHiat1a and DrHiat1b promoted methylamine uptake in a competitive manner with ammonia. In situ hybridization experiments showed that both transporters were expressed as early as the 4-cell stage in zebrafish embryos and could be identified in most tissues 4 days post-fertilization. Larvae experiencing morpholino-mediated knockdown of DrHiat1b exhibited significantly lower whole-body ammonia excretion rates compared with control larvae. Markedly decreased site-specific total ammonia excretion of up to 85% was observed in both the pharyngeal region (site of developing gills) and the yolk sac (region shown to have the highest NH4+ flux). This study is the first to identify DrHiat1b/DrHIAT1 in particular as an important contributor to ammonia excretion in larval zebrafish. Being evolutionarily conserved, these proteins are likely involved in multiple other general ammonia-handling mechanisms, making them worthy candidates for future studies on nitrogen regulation in fishes and across the animal kingdom.