In Vitro, In Vivo, and In Silico Analysis of Pyraclostrobin and Cyprodinil and Their Mixture Reveal New Targets and Signaling Mechanisms.

Pyraclostrobin and cyprodinil are broad-spectrum fungicides that are used in crops to control diseases. However, they are excessively used and, as a result, end up in the environment and threaten human health and ecosystems. Hence, knowledge of their mechanisms of action is critical to revealing their environmental fate and negative effects and regulating their use. In the present study, we conducted a comprehensive study to show the adverse effects of pyraclostrobin, cyprodinil, and their mixture using zebrafish larvae and different cell lines. Several end points were investigated, including mortality, development, gene expression, reporter assays, and molecular docking simulations. We found that both compounds and their mixture caused developmental delays and mortality in zebrafish, with a higher effect displayed by pyraclostrobin. Both compounds altered the expression of genes involved in several signaling pathways, including oxidative stress and mitochondrial function, lipid and drug metabolisms, the cell cycle, DNA damage, apoptosis, and inflammation. A noteworthy result of this study is that cyprodinil and the mixture group acted as NFκB activators, while pyraclostrobin demonstrated antagonist activity. The AHR activity was also upregulated by cyprodinil and the mixture group; however, pyraclostrobin did not show any effect. For the first time, we also demonstrated that pyraclostrobin had androgen receptor antagonist activity.

Zebrafish in understanding molecular pathophysiology, disease modeling, and developing effective treatments for Rett syndrome.

Rett syndrome (RTT) is a rare but dreadful X-linked genetic disease that mainly affects young girls. It is a neurological disease that affects nerve cell development and function, resulting in severe motor and intellectual disabilities. To date, no cure is available for treating this disease. In 90% of the cases, RTT is caused by a mutation in methyl-CpG-binding protein 2 (MECP2), a transcription factor involved in the repression and activation of transcription. MECP2 is known to regulate several target genes and is involved in different physiological functions. Mouse models exhibit a broad range of phenotypes in recapitulating human RTT symptoms; however, understanding the disease mechanisms remains incomplete, and many potential RTT treatments developed in mouse models have not shown translational effectiveness in human trials. Recent data hint that the zebrafish model emulates similar disrupted neurological functions following mutation of the mecp2 gene. This suggests that zebrafish can be used to understand the onset and progression of RTT pathophysiology and develop a possible cure. In this review, we elaborate on the molecular basis of RTT pathophysiology in humans and model organisms, including rodents and zebrafish, focusing on the zebrafish model to understand the molecular pathophysiology and the development of therapeutic strategies for RTT. Finally, we propose a rational treatment strategy, including antisense oligonucleotides, small interfering RNA technology and induced pluripotent stem cell-derived cell therapy.

Comparative analysis of diisononyl phthalate and di(isononyl)cyclohexane-1,2 dicarboxylate plasticizers in regulation of lipid metabolism in 3T3-L1 cells.

Diisononyl phthalate (DINP) and di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) are plasticizers introduced to replace previously used phthalate plasticizers in polymeric products. Exposure to DINP and DINCH has been shown to impact lipid metabolism. However, there are limited studies that address the mechanisms of toxicity of these two plasticizers. Here, a comparative toxicity analysis has been performed to evaluate the impacts of DINP and DINCH on 3T3-L1 cells. The preadipocyte 3T3-L1 cells were exposed to 1, 10, and 100 µM of DINP or DINCH for 10 days and assessed for lipid accumulation, gene expression, and protein analysis. Lipid staining showed that higher concentrations of DINP and DINCH can induce adipogenesis. The gene expression analysis demonstrated that both DINP and DINCH could alter the expression of lipid-related genes involved in adipogenesis. DINP and DINCH upregulated Pparγ, Pparα, C/EBPα Fabp4, and Fabp5, while both compounds significantly downregulated Fasn and Gata2. Protein analysis showed that both DINP and DINCH repressed the expression of FASN. Additionally, we analyzed an independent transcriptome dataset encompassing temporal data on lipid differentiation within 3T3-L1 cells. Subsequently, we derived a gene set that accurately portrays significant pathways involved in lipid differentiation, which we subsequently subjected to experimental validation through quantitative polymerase chain reaction. In addition, we extended our analysis to encompass a thorough assessment of the expression profiles of this identical gene set across 40 discrete transcriptome datasets that have linked to diverse pathological conditions to foreseen any potential association with DINP and DINCH exposure. Comparative analysis indicated that DINP could be more effective in regulating lipid metabolism.

Zebrafish gonad mutant models reveal neuroendocrine mechanisms of brain sexual dimorphism and male mating behaviors of different brain regions.

BACKGROUND: Sexually dimorphic mating behaviors differ between sexes and involve gonadal hormones and possibly sexually dimorphic gene expression in the brain. However, the associations among the brain, gonad, and sexual behavior in teleosts are still unclear. Here, we utilized germ cells-free tdrd12 knockout (KO) zebrafish, and steroid synthesis enzyme cyp17a1-deficient zebrafish to investigate the differences and interplays in the brain-gonad-behavior axis, and the molecular control of brain dimorphism and male mating behaviors. METHODS: Tdrd12+/-; cyp17a1+/- double heterozygous parents were crossed to obtain tdrd12-/-; cyp17a1+/+ (tdrd12 KO), tdrd12+/+; cyp17a1-/- (cyp17a1 KO), and tdrd12-/-; cyp17a1-/- (double KO) homozygous progenies. Comparative analysis of mating behaviors were evaluated using Viewpoint zebrafish tracking software and sexual traits were thoroughly characterized based on anatomical and histological experiments in these KOs and wild types. The steroid hormone levels (testosterone, 11-ketotestosterone and 17ß-estradiol) in the brains, gonads, and serum were measured using ELISA kits. To achieve a higher resolution view of the differences in region-specific expression patterns of the brain, the brains of these KOs, and control male and female fish were dissected into three regions: the forebrain, midbrain, and hindbrain for transcriptomic analysis. RESULTS: Qualitative analysis of mating behaviors demonstrated that tdrd12-/- fish behaved in the same manner as wild-type males to trigger oviposition behavior, while cyp17a1-/- and double knockout (KO) fish did not exhibit these behaviors. Based on the observation of sex characteristics, mating behaviors and hormone levels in these mutants, we found that the maintenance of secondary sex characteristics and male mating behavior did not depend on the presence of germ cells; rather, they depended mainly on the 11-ketotestosterone and testosterone levels secreted into the brain-gonad regulatory axis. RNA-seq analysis of different brain regions revealed that the brain transcript profile of tdrd12-/- fish was similar to that of wild-type males, especially in the forebrain and midbrain. However, the brain transcript profiles of cyp17a1-/- and double KO fish were distinct from those of wild-type males and were partially biased towards the expression pattern of the female brain. Our results revealed important candidate genes and signaling pathways, such as synaptic signaling/neurotransmission, MAPK signaling, and steroid hormone pathways, that shape brain dimorphism and modulate male mating behavior in zebrafish. CONCLUSIONS: Our results provide comprehensive analyses and new insights regarding the endogenous interactions in the brain-gonad-behavior axis. Moreover, this study revealed the crucial candidate genes and neural signaling pathways of different brain regions that are involved in modulating brain dimorphism and male mating behavior in zebrafish, which would significantly light up the understanding the neuroendocrine and molecular mechanisms modulating brain dimorphism and male mating behavior in zebrafish and other teleost fish.

Differential effects of familial Alzheimer's disease-causing mutations on amyloid precursor protein (APP) trafficking, proteolytic conversion, and synaptogenic activity.

The amyloid precursor protein (APP) is a key player in Alzheimer`s disease (AD) and the precursor of the Aß peptide, which is generated by consecutive cleavages of ß- and γ-secretases. Familial Alzheimer's disease (FAD) describes a hereditary subgroup of AD that represents a low percentage of AD cases with an early onset of the disease. Different APP FAD mutations are thought to have qualitatively different effects on its proteolytic conversion. However, few studies have explored the pathogenic and putative physiological differences in more detail. Here, we compared different FAD mutations, located at the ß- (Swedish), α- (Flemish, Arctic, Iowa) or γ-secretase (Iberian) cleavage sites. We examined heterologous expression of APP WT and FAD mutants in non-neuronal cells and their impact on presynaptic differentiation in contacting axons of co-cultured neurons. To decipher the underlying molecular mechanism, we tested the subcellular localization, the endocytosis rate and the proteolytic processing in detail by immunoprecipitation-mass spectrometry. Interestingly, we found that only the Iberian mutation showed altered synaptogenic function. Furthermore, the APP Iowa mutant shows significantly decreased α-secretase processing which is in line with our results that APP carrying the Iowa mutation was significantly increased in early endosomes. However, most interestingly, immunoprecipitation-mass spectrometry analysis revealed that the amino acid substitutions of APP FAD mutants have a decisive impact on their processing reflected in altered Aß profiles. Importantly, N-terminally truncated Aß peptides starting at position 5 were detected preferentially for APP Flemish, Arctic, and Iowa mutants containing amino acid substitutions around the α-secretase cleavage site. The strongest change in the ratio of Aß40/Aß42 was observed for the Iberian mutation while APP Swedish showed a substantial increase in Aß1-17 peptides. Together, our data indicate that familial AD mutations located at the α-, ß-, and γ-secretase cleavage sites show considerable differences in the underlying pathogenic mechanisms.

In silico analysis decodes transthyretin (TTR) binding and thyroid disrupting effects of per- and polyfluoroalkyl substances (PFAS).

Transthyretin (TTR) is a homo-tetramer protein involved in the transport of thyroid hormone (thyroxine; T4) in the plasma and cerebrospinal fluid. Many pollutants have been shown to bind to TTR, which could be alarming as disruption in the thyroid hormone system can lead to several physiological problems. It is also indicated that the monomerization of tetramer and destabilization of monomer can lead to amyloidogenesis. Many compounds are identified that can bind to tetramer and stabilize the tetramer leading to the inhibition of amyloid fibril formation. Other compounds are known to bind tetramer and induce amyloid fibril formation. Among the pollutants, per- and polyfluoroalkyl substances (PFAS) are known to disrupt the thyroid hormone system. The molecular mechanisms of thyroid hormone disruption could be diverse, as some are known to bind with thyroid hormone receptors, and others can bind to membrane transporters. Binding to TTR could also be one of the important pathways to alter thyroid signaling. However, the molecular interactions that drive thyroid-disrupting effects of long-chain and short-chain PFASs are not comprehensively understood at the molecular level. In this study, using a computational approach, we show that carbon chain length and functional group in PFASs are structural determinants, in which longer carbon chains of PFASs and sulfur-containing PFASs favor stronger interactions with TTR than their shorter-chained counterparts. Interestingly, short-chain PFAS also showed strong binding capacity, and the interaction energy for some was as close to the longer-chain PFAS. This suggests that short-chain PFASs are not completely safe, and their use and build-up in the environment should be carefully regulated. Of note, TTR homologs analysis suggests that thyroid-disrupting effects of PFASs could be most likely translated to TTR-like proteins and other species.

Sex-specific differences in zebrafish brains.

In this systematic review, we highlight the differences between the male and female zebrafish brains to understand their differentiation and their use in studying sex-specific neurological diseases. Male and female brains display subtle differences at the cellular level which may be important in driving sex-specific signaling. Sex differences in the brain have been observed in humans as well as in non-human species. However, the molecular mechanisms of brain sex differentiation remain unclear. The classical model of brain sex differentiation suggests that the steroid hormones derived from the gonads are the primary determinants in establishing male and female neural networks. Recent studies indicate that the developing brain shows sex-specific differences in gene expression prior to gonadal hormone action. Hence, genetic differences may also be responsible for differentiating the brain into male and female types. Understanding the signaling mechanisms involved in brain sex differentiation could help further elucidate the sex-specific incidences of certain neurological diseases. The zebrafish model could be appropriate for enhancing our understanding of brain sex differentiation and the signaling involved in neurological diseases. Zebrafish brains show sex-specific differences at the hormonal level, and recent advances in RNA sequencing have highlighted critical sex-specific differences at the transcript level. The differences are also evident at the cellular and metabolite levels, which could be important in organizing sex-specific neuronal signaling. Furthermore, in addition to having one ortholog for 70% of the human gene, zebrafish also shares brain structural similarities with other higher eukaryotes, including mammals. Hence, deciphering brain sex differentiation in zebrafish will help further enhance the diagnostic and pharmacological intervention of neurological diseases.

Plasticizers: negative impacts on the thyroid hormone system.

This review aims to understand the impacts of plasticizers on the thyroid system of animals and humans. The thyroid gland is one of the earliest endocrine glands that appear during embryogenesis. The thyroid gland synthesizes thyroid hormones (TH), triiodothyronine (T3), and thyroxine (T4) that are important in the regulation of body homeostasis. TH plays critical roles in regulating different physiological functions, including metabolism, cell growth, circadian rhythm, and nervous system development. Alteration in thyroid function can lead to different medical problems. In recent years, thyroid-related medical problems have increased and this could be due to rising environmental pollutants. Plasticizers are one such group of a pollutant that impacts thyroid function. Plasticizers are man-made chemicals used in a wide range of products, such as children's toys, food packaging items, building materials, medical devices, cosmetics, and ink. The increased use of plasticizers has resulted in their detection in the environment, animals, and humans. Studies indicated that plasticizers could alter thyroid function in both animals and humans at different levels. Several studies demonstrated a positive and/or negative correlation between plasticizers and serum T4 and T3 levels. Plasticizers could also change the expression of various TH-related genes and proteins, including thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), and transporters. Histological analyses demonstrated thyroid follicular cell hypertrophy and hyperplasia in response to several plasticizers. In conclusion, plasticizers could disrupt TH homeostasis and the mechanisms of toxicity could be diverse.

Di(isononyl) cyclohexane-1,2-dicarboxylate (DINCH) alters transcriptional profiles, lipid metabolism and behavior in zebrafish larvae.

Plasticizers are commonly used in different consumer goods and personal care products to provide flexibility, durability and elasticity to polymers. Due to their reported toxicity, the use of several plasticizers, including phthalates has been regulated and/or banned from the market. Di(isononyl) cyclohexane-1,2-dicarboxylate (DINCH) is an alternative plasticizer that was introduced to replace toxic plasticizers. Increasing global demand and lack of toxicity data and safety assessment of DINCH have raised the concern to human and animal health. Hence, in the present study, we investigated the adverse effects of DINCH (at concentrations ranging from 0.01 to 10 µM) in early developmental stages of zebrafish using different endpoints such as hatching rate, developmental abnormalities, lipid content, behavior analysis and gene expression. We found that DINCH caused hatching delay in a dose-dependent manner and altered the expression of genes involved in stress response. Lipid staining using Oil Red O stain showed a slight lipid accumulation around the yolk, brain, eye and neck with increasing concentration. Genes associated with lipid transport such as fatty acid synthesis, ß-oxidation, elongation, lipid transport were significantly altered by DINCH. Genes involved in cholesterol biosynthesis and homeostasis were also affected by DINCH indicating possible developmental neurotoxicity. Behavioral analysis of larvae demonstrated a distinct locomotor activity upon exposure to DINCH. The present data shows that DINCH could induce physiological and metabolic toxicity to aquatic organisms. Hence, further analyses and environmental monitoring of DINCH should be conducted to determine its safety and toxicity levels.

In silico and in vitro assessment of androgen receptor antagonists.

There is a growing concern for male reproductive health as studies suggest that there is a sharp increase in prostate cancer and other fertility related problems. Apart from lifestyle, pollutants are also known to negatively affect the reproductive system. In addition to many other compounds that have been shown to alter androgen signaling, several environmental pollutants are known to disrupt androgen signaling via binding to androgen receptor (AR) or indirectly affecting the androgen synthesis. We analyzed here the molecular mechanism of the interaction between the human AR Ligand Binding Domain (hAR-LBD) and two environmental pollutants, linuron (a herbicide) and procymidone (a pesticide), and compared with the steroid agonist dihydrotestosterone (DHT) and well-known hAR antagonists bicalutamide and enzalutamide. Using molecular docking and dynamics simulations, we showed that the co-activator interaction site of the hAR-LBD is disrupted in different ways by different ligands. Binding free energies of the ligands were also ordered in increasing order as follows: linuron, procymidone, DHT, bicalutamide, and enzalutamide. These data were confirmed by in vitro assays. Reporter assay with MDA-kb2 cells showed that linuron, procymidone, bicalutamide and enzalutamide can inhibit androgen mediated activation of luciferase activity. Gene expression analysis further showed that these compounds can inhibit the expression of prostate specific antigen (PSA) and microseminoprotein beta (MSMB) in prostate cell line LNCaP. Comparative analysis showed that procymidone is more potent than linuron in inhibiting AR activity. Furthermore, procymidone at 10 µM dose showed equivalent and higher activity to AR inhibitor enzalutamide and bicalutamide respectively.

The brominated flame retardants TBECH and DPTE alter prostate growth, histology and gene expression patterns in the mouse.

The brominated flame retardants (BFRs), 1,2-dibromo-4-(1,2 dibromoethyl)cyclohexane (TBECH) and 2,3-dibromopropyl-2,4,6-tribromophenyl ether (DPTE) bind to the androgen receptor (AR). in vitro bioassays have shown that TBECH is a potent androgen agonist while DPTE is a potent AR antagonist. Both TBECH and DPTE alter gene expression associated with AR regulation. However, it remains to be determined if TBECH and DPTE can affect the prostate. For this reason, we exposed CD1 mice to a 1:1 mixture of TBECH diastereomers α and ß, a 1:1 mixture of γ and δ, and to DPTE, and tested their effects on prostate growth, histology and gene expression profiles. Castrated mice were used to study the androgenic effects of TBECHαß and TBECHγδ while the antagonistic effects of DPTE were studied in non-castrated mice. We observed that testosterone and TBECHγδ increased body and prostate weights while TBECHαß affected neither of them; and that DPTE had no effect on body weight but reduced prostate weight drastically. Histomorphometric analysis of the prostate revealed epithelial and glandular alterations in the TBECHγδ group comparable to those in testosterone group while alterations in the TBECHαß group were less pronounced. DPTE displayed androgen antagonist activity reminiscent of castration. The transcription profile of the prostate was altered by castration and exposure to testosterone and to TBECHγδ reversed several of these changes. Testosterone and TBECHγδ also regulated the expression of several androgen responsive genes implicated in prostate growth and cancer. While DPTE resulted in a drastic reduction in prostate weight, it only affected a small number of genes. The results indicate that TBECHγδ and DPTE are of high human health concern as they may contribute to changes in prostate growth, histology and function.

Thyroid hormone: sex-dependent role in nervous system regulation and disease.

Thyroid hormone (TH) regulates many functions including metabolism, cell differentiation, and nervous system development. Alteration of thyroid hormone level in the body can lead to nervous system-related problems linked to cognition, visual attention, visual processing, motor skills, language, and memory skills. TH has also been associated with neuropsychiatric disorders including schizophrenia, bipolar disorder, anxiety, and depression. Males and females display sex-specific differences in neuronal signaling. Steroid hormones including testosterone and estrogen are considered to be the prime regulators for programing the neuronal signaling in a male- and female-specific manner. However, other than steroid hormones, TH could also be one of the key signaling molecules to regulate different brain signaling in a male- and female-specific manner. Thyroid-related diseases and neurological diseases show sex-specific incidence; however, the molecular mechanisms behind this are not clear. Hence, it will be very beneficial to understand how TH acts in male and female brains and what are the critical genes and signaling networks. In this review, we have highlighted the role of TH in nervous system regulation and disease outcome and given special emphasis on its sex-specific role in male and female brains. A network model is also presented that provides critical information on TH-regulated genes, signaling, and disease.

Transcriptomic analysis of nonylphenol effect on Saccharomyces cerevisiae.

Nonylphenol (NP) is a bioaccumulative environmental estrogen that is widely used as a nonionic surfactant. We have previously examined short-term effects of NP on yeast cells using microarray technology. In the present study, we investigated the adaptive response of Saccharomyces cerevisiae BY4742 cells to NP exposure by analyzing genome-wide transcriptional profiles using RNA-sequencing. We used 2 mg/L NP concentration for 40 days of exposure. Gene expression analysis showed that a total of 948 genes were differentially expressed. Of these, 834 genes were downregulated, while 114 genes were significantly upregulated. GO enrichment analysis revealed that 369 GO terms were significantly affected by NP exposure. Further analysis showed that many of the differentially expressed genes were associated with oxidative phosphorylation, iron and copper acquisition, autophagy, pleiotropic drug resistance and cell cycle progression related processes such as DNA and mismatch repair, chromosome segregation, spindle checkpoint activity, and kinetochore organization. Overall, these results provide considerable information and a comprehensive understanding of the adaptive response to NP exposure at the gene expression level.

Major Spinal Surgery Between Two Documented COVID-19 Infections in an Elderly Female: A Case Report.

Documented re-infection of COVID-19 is uncommon and doing a major spinal surgery in an elderly patient right after the recovery from the first event is itself a major undertaking. Re-infection after successful surgery points to the possibility of COVID-19 infection being a post-surgical complication. Here, we report a case of a 72-years-old elderly female who had presented to us with features of COVID-19 infection confirmed by reverse transcription polymerase chain reaction assay and unstable spinal fracture who underwent a pedicle screw fixation for the fracture of the third and fourth thoracic vertebrae after two consecutive negative serology assays. A month after discharge from the hospital, she presented with severe symptoms of COVID-19 again confirmed by two consecutive polymerase chain reaction assays. She was managed conservatively and was discharged without significant respiratory and neurological complications. We described this case in detail in addition to reviewing the pertinent literature.

Sex differences in severity and mortality from COVID-19: are males more vulnerable?

Coronavirus disease 2019 (COVID-19) has shown high infection and mortality rates all over the world, and despite the global efforts, there is so far no specific therapy available for COVID-19. Interestingly, while the severity and mortality of COVID-19 are higher in males than in females, the underlying molecular mechanisms are unclear. In this review, we explore sex-related differences that may be contributing factors to the observed male-biased mortality from COVID-19. Males are considered the weaker sex in aspects related to endurance and infection control. Studies show that viral RNA clearance is delayed in males with COVID-19. A recent study has indicated that the testis can harbor coronavirus, and consequently, males show delayed viral clearance. However, the role of testis involvement in COVID-19 severity and mortality needs further research. Males and females show a distinct difference in immune system responses with females eliciting stronger immune responses to pathogens. This difference in immune system responses may be a major contributing factor to viral load, disease severity, and mortality. In addition, differences in sex hormone milieus could also be a determinant of viral infections as estrogen has immunoenhancing effects while testosterone has immunosuppressive effects. The sex-specific severity of COVID-19 infections indicates that further research on understanding the sex differences is needed. Inclusion of both males and females in basic research and clinical trials is required to provide critical information on sex-related differences that may help to better understand disease outcome and therapy.

Perfluorinated alkyl substances impede growth, reproduction, lipid metabolism and lifespan in Daphnia magna.

Per- and polyfluorinated alkyl substances (PFASs) are synthetic organofluorine compounds with unique stability accompanied with hydrophobic and lipophobic properties. Perfluorooctane sulfonate (PFOS) and Perfluorooctanoic acid (PFOA) are of high concern due to their wide application in consumer and industrial products, extreme persistence, abundant occurrence in the environment and their toxic effect to humans and animals. However, knowledge on the molecular mechanisms of toxicity and the effects on reproduction output remain scarce. In this study, we analyzed the effects of PFOS and PFOA on Daphnia magna. Acute toxicity, development, reproduction, lipid metabolism (lipid-accumulation) and lifespan was investigated, as well as the expression of genes related to these endpoints. Exposure of PFOS and PFOA at 1, 10 and 25 µM did not cause acute lethality. Hatching was reduced following exposure to both compounds, and lifespan was decreased following exposure to 25 µM PFOS. Body length of Daphnia magna was reduced significantly by 25 µM PFOS following 7 days exposure. Lipid staining revealed that all PFAS exposures increased lipid accumulation. qRT-PCR analysis of genes involved in lipid metabolism suggests that the increase in lipid content could be due to inhibition of genes involved on absorption and catabolism of fatty acids. Exposure to both PFOA and PFOS reduced the fecundity significantly. Downregulation of genes involved in development and reproductive process, including vtg2, vasa, EcRA, EcRB, usp, jhe, HR3, ftz-F1, E74 and E75 were observed. The alterations in developmental and reproductive genes as well as the disturbed lipid metabolism provides mechanistic insight into the possible causes for decreased fecundity and lifespan observed following exposure to both PFOS and PFOA.

Nonsteroidal anti-inflammatory drugs (NSAIDs) cause male-biased sex differentiation in zebrafish.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used pharmaceuticals to treat pain, fever and inflammation. NSAIDs are also known to have many side effects including adverse effects on reproduction in both humans and animals. As NSAIDs usage is not regulated they are frequently detected at high concentrations in the environment. In order to understand the effect of NSAIDs on zebrafish sex differentiation, we used seven different NSAIDs which were either Cox-1 selective, Cox-1 biased, non-selective or COX-2 selective. We show that at higher concentration, NSAIDs are toxic to zebrafish embryo as they lead to mortality and hatching delay. Gene expression analysis following short term exposure of NSAIDs led to downregulation of female specific genes including zp2, vtg2 foxl2 and wnt4. Long term exposure of larvae to environmentally relevant concentrations of Cox-2 selective and non-selective NSAIDs resulted in male-biased sex ratio which confirmed the qRT-PCR analysis. However, the Cox-1 selective acetylsalicylic acid and the Cox-1 biased ketoprofen did not alter sex ratio. The observed male-biased sex ratio could also be due to induction of apoptosis process as the genes including p21 and casp8 were significantly upregulated following exposure to the Cox-2 selective and the non-selective NSAIDs. The present study indicates that NSAIDs alter sex differentiation in zebrafish, primarily through inhibition of Cox-2. This study clearly demonstrates that the use of NSAIDs and their release into the aquatic environment should be carefully monitored to avoid adverse effects to the aquatic organisms.

Zebrafish cyp17a1 knockout reveals that androgen-mediated signaling is important for male brain sex differentiation.

Brain sex differentiation is a complex process, wherein genes and steroid hormones act to induce specific gender brain differentiation. Testosterone (T) derived from the gonads has been linked to neural circuit modeling in a sex-specific manner. Previously, we have shown that cyp17a1 knockout (KO) zebrafish have low plasma androgen levels, and display compromised male-typical mating behaviors. In this study, we demonstrated that treatment of cyp17a1 KO males with T or 11-ketotestosterone (11-KT) is sufficient to rescue mating impairment by restoring the male-typical secondary sex characters (SSCs) and mating behaviors, confirming an essential role of androgen in maintaining SSCs and mating behaviors. Brain steroid hormone analysis revealed that cyp17a1 KO fish have reduced levels of T and 11-KT. We performed RNA sequencing on brain samples of control and cyp17a1 KO male zebrafish to get insights regarding the impact of cyp17a1 KO on gene expression pattern, and to correlate it with the observed disruption of male-typical mating behaviors. Transcriptome analysis of cyp17a1 KO males showed a differential gene expression when compared to control males. In total, 358 genes were differentially regulated between control males and KO males. Important genes including brain aromatase (cyp19a1b), progesterone receptor (pgr), deiodinase (dio2), and insulin-like growth factor 1 (igf1) that are involved in brain functions, as well as androgen response genes including igf1, frem1a, elovl1a, pax3a, mmp13b, hsc70, ogg1 were regulated. RT-qPCR analysis following rescue of cyp17a1 KO with T and 11-KT further suggested that androgen-mediated signaling is disrupted in the cyp17a1 KO fish. Our results indicated that cyp17a1 KO fish have an incomplete masculinization and altered brain gene expression, which could be due to decreased androgen levels.

The food preservative ethoxyquin impairs zebrafish development, behavior and alters gene expression profile.

In the present study, we investigated the detrimental effects of ethoxyquin (EQ) on zebrafish embryonic development using different endpoints including lethality, malformations, locomotion and gene expression. EQ is primarily used as a preservative in animal feed and it has been shown to have negative impacts on different laboratory animals. However, studies on the adverse effects of EQ in aquatic animals are still limited. In this study, zebrafish eggs were exposed to different concentrations of EQ ranging from 1 to 100 µM for six days. In the 100 µM treated groups 95 and 100% mortality was observed at 24 and 48 h, respectively. Delayed development, decreased pigmentation and pericardial edema were observed in larvae. Behavioral analysis of larvae demonstrated a distinct locomotive pattern in response to EQ both in light and dark indicating a possible developmental neurotoxicity and deficits in locomotion. The expression levels of genes involved in several physiological pathways including stress response, cell cycle and DNA damage were altered by EQ. Our results demonstrate that EQ could cause developmental and physiological toxicity to aquatic organisms. Hence, its toxic effect should be further analyzed and its use and levels in the environment must be monitored carefully.

Comparative transcriptional analysis of methylparaben and propylparaben in zebrafish.

Parabens are widely used as preservatives in different commercial items including food, cosmetics and pharmaceuticals, and their wide use has resulted in accumulation in the environment. Parabens have been shown to have negative effects on animals as well as human health. In this study, we carried out a comprehensive study to determine the adverse effects associated with propylparaben (PP) and methylparaben (MP) on early developmental stages of zebrafish. Mortality, hatching, developmental abnormalities and gene expression profiles were investigated in embryos exposed to both compounds. The semi-static exposure conditions showed that both MP (≥100 µM) and PP (≥10 µM) are toxic to the embryos in a concentration-dependent manner and lead to developmental abnormality. Malformations such as spinal defects, pericardial edema, and pigmentation defects were observed following both MP and PP treatments. Hatching delay, mortality and developmental abnormality data indicate that PP is more toxic than MP. For gene expression analysis, 1 and 10 µM doses of MP and PP were analyzed. Genes from physiological pathways including stress response, cell cycle and DNA damage, inflammation, fatty acid metabolism and endocrine functions were affected by MP and PP. The gene expression profiles show that parabens cause toxicity by inducing oxidative stress, DNA double-strand breaks, apoptosis as well as by altering fatty acid metabolism. Altered expression of androgen receptor (ar) and estrogen receptor 2 alpha (esr2a) indicates an antiandrogenic and estrogenic activity of parabens in zebrafish. Overall, the present study provides considerable information on the negative effects of MP and PP using physiological endpoints and motivates further studies to explore the molecular mechanism of the toxicity associated with parabens.