Integrated multi-omics analysis reveals the underlying molecular mechanism for developmental neurotoxicity of perfluorooctanesulfonic acid in zebrafish
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ABSTRACT: Limited studies on multi-omics have been conducted to comprehensively investigate the molecular mechanism underlying the developmental neurotoxicity of perfluorooctanesulfonic acid (PFOS). In this study, the locomotor behavior of zebrafish larvae was assessed under the exposure to 0.1–20 μM PFOS based on its reported neurobehavioral effect. After the number of zebrafish larvae was optimized for proteomics and metabolomics studies, three kinds of omics (i.e., transcriptomics, proteomics, and metabolomics) were carried out with zebrafish larvae exposed to 0.1, 1, 5, and 10 μM PFOS. More importantly, a data-driven integration of multi-omics was performed to elucidate the toxicity mechanism involved in developmental neurotoxicity. In a concentration-dependent manner, exposure to PFOS provoked hyperactivity and hypoactivity under light and dark conditions, respectively. Individual omics revealed that PFOS exposure caused perturbations in the pathways of neurological function, oxidative stress, and energy metabolism. Integrated omics implied that there were decisive pathways for axonal deformation, neuroinflammatory stimulation, and dysregulation of calcium ion signaling, which are more clearly specified for neurotoxicity. Overall, our findings broaden the molecular understanding of the developmental neurotoxicity of PFOS, for which multi-omics and integrated omics analyses are efficient for discovering the significant molecular pathways related to developmental neurotoxicity in zebrafish.
Project description:The molecular mechanism of perfluorobutanesulfonic acid (PFBS), an alternative to legacy perfluorooctanesulfonic acid (PFOS), remains to be understood. Therefore, we conducted a developmental toxicity evaluation on zebrafish embryos exposed to PFBS and PFOS and assessed neurobehavioral changes at concentrations below each point of departure (POD) determined by embryonic mortality. Using transcriptomics, proteomics, and metabolomics, biomolecular perturbations in response to PFBS were profiled and then integrated for comparison with those for PFOS. Although PFBS (7525.47 M POD) was approximately 700 times less toxic than PFOS (11.42 M POD), similar alteration in behavioral assessment and neurochemical analysis was observed at the corresponding ratios of low and high concentrations. Multi-omics analysis revealed that the PFBS neurotoxicity mechanism was associated with oxidative stress, lipid metabolism, and glycolysis/glucogenesis. The commonalities in developmental neurotoxicity-related mechanisms between PFBS and PFOS interconnected by knowledge-based integration of multi-omics included the calcium signaling pathway, lipid homeostasis, and primary bile acid biosynthesis. Despite being less toxic than PFOS, PFBS exhibited similar dysregulated molecular mechanisms, suggesting that chain length differences do not affect the intrinsic toxicity mechanism. Overall, carefully managing potential toxicity of PFBS can secure its status as an alternative to PFOS.
Project description:Persistent organic pollutants (POPs) are widespread in the environment and bioaccumulate in organisms. Previously we observed hyperactivity of zebrafish larvae exposed to a mixture of POPs based on average blood levels from the Scandinavian population and identified perfluorooctanesulfonic acid (PFOS) as the driving agent for the behavioral changes. We exposed zebrafish larvae from 6 to 96 h post fertilization to the same mixture of POPs in two concentrations or a single PFOS exposure (0.3 and 2.06mg/L) and performed behavioral tests and transcriptomics analysis. Results confirmed previous observations that exposure to POPs and PFOS causes hyperactivity and higher anxiety levels. Transcriptomic analysis showed upregulation of transcripts related to muscle contraction. Muscle contraction is highly regulated by the availability of calcium in the sarcoplasmic reticulum. Ingenuity pathway analysis showed that one of the affected pathways in larvae exposed to the POPs mixture and PFOS was calcium signalling via the activation of the ryanodine receptors. We also found effect on lipid metabolism in those larvae exposed to the lower concentration of PFOS. By using omics technology, we observed that the altered behavioral pattern in exposed zebrafish larvae might be controlled directly by mechanisms affecting muscle function rather than from mechanisms connected to neurotoxicity.
Project description:Condition specific zebrafish metabolic models generated using the COBRA MetaboTools framework. The Wang et al., (2021) zebrafish genome-scale metabolic model (GEM) was constrained with experimental data from 5 days post fertilized (dpf) zebrafish to generate a 'base-model'.
In turn this 5 dpf zebrafish base-model was constrained with experimental (transcriptomics and metabolomics) data from 5 dpf zebrafish exposed to the environmental pollutant perfluorooctane sulfonate (PFOS), at three levels - Low (0.06 uM), Medium (0.6 uM), and High (2 uM) PFOS.
The MetaboTools framework was used to construct three condition-sepcific models: Low, Medium, and High PFOS.
Key simulation predictions of effects on the carnitine shuttle and lipid metabolism were confirmed in wild-caught fish and dolphins (stranded animals) sampled from the northern Gulf of Mexico - published in Nolen et al., (2024) https://doi.org/10.1016/j.cbpc.2023.109817
Project description:Acute exposure to acrylamide (ACR), a type-2 alkene, may lead to a ataxia, skeletal muscles weakness and numbness of the extremities in exposed human and laboratory animals. Recently, a zebrafish model for ACR neurotoxicity mimicking most of the pathophysiological processes described in mammalian models, was generated in 8 days post-fertilization larvae. In order to better understand the predictive value of the zebrafish larvae model of acute ACR neurotoxicity, in the present manuscript the ACR acute neurotoxicity has been characterized in the brain of adult zebrafish, and the results compared with those obtained with the whole-larvae. Although qualitative and quantitative analysis of the data shows important differences in the ACR effects between the adult brain and the whole-larvae, the overall effects of ACR in adult zebrafish, including a significant decrease in locomotor activity, altered expression of transcriptional markers of proteins involved in synaptic vesicle cycle, presence of ACR-adducts on cysteine residues of some synaptic proteins, and changes in the profile of some neurotransmitter systems, are similar to those described in the larvae. Thus, these results support the suitability of the zebrafish ACR acute neurotoxicity recently developed in larvae for screening of molecules with therapeutic value to treat this toxic neuropathy.
Project description:Complex mixtures of persistent organic pollutants (POPs) are regularly detected in the environment and animal tissues. Often these chemicals are associated with latent effects following early-life exposures, following the developmental origin of health and disease paradigm. We investigated the long-term effects of a human relevant mixture of 29 POPs on adult zebrafish following a developmental exposure, in addition to a single PFOS exposure for comparison, as it was the compound with the highest concentration within the mixture. Zebrafish embryos were exposed from 6 to 96 hours post fertilization to x10 and x70 the level of POP mixture or PFOS found in human blood before being transferred to clean water. We measured growth, swimming performance, and reproductive output at different life stages. In addition, we assessed anxiety behavior of the adults and their offspring, as well as performing a transcriptomic analysis on the adult zebrafish brain, as the POP mixture and PFOS concentrations used are known to affect larval behavior. Exposure to POP mixture and PFOS reduced swimming performance and increased length and weight, compared to controls. No effect of developmental exposure was observed on reproductive output or anxiety behavior. Additionally, RNA-seq did not reveal pathways related to anxiety although pathways related to synapse biology were affected at the x10 PFOS level. Furthermore, pathway analysis of the brain transcriptome of adults exposed as larvae to the low concentration of PFOS revealed enrichment in pathways such as calcium, MAPK, and GABA signaling, all of which are important for learning and memory. Based on our results we can conclude that some effects on the endpoints measured were apparent, but if these effects lead to adversities at population levels remains elusive.
Project description:Recently it was discovered that the perfluorooctane sulfonate (PFOS) detected in wildlife, such as fish-eating birds, had a greater proportion of linear PFOS (L-PFOS) than the manufactured technical product (T-PFOS), which contains linear and branched isomers. This suggests toxicological studies based on T-PFOS data may inaccurately assess exposure risk to wildlife. To determine if PFOS effects were influenced by isomer content we compared the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either L-PFOS or T-PFOS using Agilent microarrays. At equal concentrations (10 μM), T-PFOS altered the expression of more transcripts (340, >1.5 fold change, p<0.05) compared to L-PFOS (130 transcripts). Higher concentrations of L-PFOS (40 μM) were also less transcriptionally disruptive (217 transcripts) than T-PFOS at 10 μM. Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, hepatic system development and cellular growth and proliferation. Pathway and interactome analysis suggested that genes may be affected through the RXR receptor, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling and PPARγ and SREBP receptors. In all functional categories and pathways examined, the response elicited by T-PFOS was greater than L-PFOS. These data show that T-PFOS elicits a greater transcriptional response in CEH than L-PFOS alone and demonstrates the importance of considering the isomer-specific toxicological properties of PFOS when assessing exposure risk. Reference Design. Reference = pool of equal parts of all control and treated samples. Control groups and 5 treatment groups. Control samples were CEH exposed DMSO only (vehicle solvent). Treatments were: CEH exposed to 10 uM L-PFOS, 40 uM L-PFOS, 10 uM T-PFOS, 0.03 nM TCDD and 1 nM TCDD.
Project description:Polychlorinated biphenyls (PCBs) are persistent and ubiquitously distributed environmental pollutants. Based on their chemical structure, PCBs are classified into non-ortho substituted and ortho-substituted congeners. Non-ortho-substituted PCBs are structurally similar to dioxin or TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) and their mode of action and toxic effects are well established. In contrast, much less is known about the effects of ortho-substituted PCBs. Studies conducted so far have focused on tissue-specific effects but there is limited knowledge about the effects on the whole organism, particularly the sensitive developmental stages in vertebrates. Hence, in this study we investigated the effects of exposure to an environmentally relevant ortho-substituted PCB (2,2’,4,4’,5,5’-hexachlorobiphenyl; PCB153) on zebrafish embryos. We exposed zebrafish embryos to either DMSO (0.1%; solvent control) or three different concentrations of PCB153 (0.1, 1 and 10 μM) from 4 hours post-fertilization (hpf) to 120 hpf. At the end of the exposure, larvae were sampled for determining transcriptional changes (RNA sequencing) and the remaining embryos were maintained in contaminant-free environment. At 7 and 14 days post-fertilization (dpf), zebrafish larvae were assessed for locomotory behavior. We did not observe any overt phenotypes during the exposure period, but observed a spinal phenotype in the 10μM PCB153 treated group starting at 7 dpf. This phenotype was observed in a dose-dependent manner and majority of the embryos with this phenotype died by 14 dpf. RNA sequencing of 5 dpf larvae exposed to PCB153 also revealed dose-dependent changes in gene expression patterns. A total of 633, 2227, and 3378 differentially expressed genes were observed in 0.1, 1 and 10 μM PCB153 treated embryos, respectively. Among these, 301 genes were common to all treatment groups, and KEGG pathway analysis revealed enrichment of genes related to circadian rhythm, FOXO signaling and insulin resistance pathways. We are currently investigating the functions of genes that are uniquely altered by different PCB153 concentrations. Overall, these results suggest that developmental exposure to PCB153, a PCB congener highly prevalent in the environment, targets multiple physiological processes including photoperiod regulation and metabolism. [Funded by NIH P01ES021923 and NSF OCE-1314642].
Project description:Integrated multi-omics analysis reveals the underlying molecular mechanism for developmental neurotoxicity of perfluorooctanesulfonic acid in zebrafish
Project description:Per- and poly-fluoroalkyl substances (PFAS), a class of ubiquitous, persistent, and toxic anthropogenic chemicals that exert harmful effects on environmental and human health, but the mechanisms that drive these effects are not well understood. Perfluorooctanesulfonic acid (PFOS) is a legacy PFAS that elicits numerous adverse effects on the endocrine, immune, nervous, reproductive, and developmental processes. The aim of this study was to identify transcriptomic changes in zebrafish (Danio rerio) exposed to PFOS that provide insight into sensitive target tissues and molecular mechanisms of developmental toxicity. Zebrafish embryos were exposed to DMSO (0.01%) or PFOS (16 µM) from 3–72 hours post fertilization (hpf). Larvae were collected, cells isolated and assessed for viability, and single-cell gene expression profiling was performed. Data analysis was conducted in three different ways: pseudo-bulk analysis, untargeted cluster analysis, and a targeted cluster analysis where unique clusters were defined for liver (fabp10a, and apoa2). Results from the pseudo-bulk analysis showed that 8.63% (2390/ 27698) genes were significantly differentially expressed between DMSO and PFOS groups. The top five pathways impacted by these significant DEGs were neuron development, visual perception, central nervous system development, eye development, and synaptic signaling. Results from untargeted cluster analysis revealed 22 distinct clusters that were annotated to specific tissues. The top five clusters with the highest number of significant DEGs were digestive organs, muscle, otolith, cardiac muscle, and optic lens. Additionally, we looked at the distribution of pathway-specific genes for epigenetic modifications, Nfe2l2 pathway, and PPAR pathway within clusters to identify sensitive tissues—blood-related tissue, retinal rod, and cardiac muscle were more likely be impacted by epigenetic modifications by DNA methylation; while blood-related tissue and periderm are sensitive to be impacted by both Nfe2l2 and PPAR pathway. Another potential mechanism induced by PFOS is beta-oxidation of fatty acids—which was significantly downregulated in blood-related tissue and upregulated in digestive organs. Results of targeted or curated liver cluster showed 220 DEGs in the liver were significant with PFOS exposure with Notch signaling pathway more likely to be impacted with PFOS exposures. The sensitivity pathways for PFOS-induced toxicity in larval zebrafish are PPAR signaling (overall, and digestive organs) and Notch signaling pathway (liver). Overall, scRNA-seq is a powerful tool to identify tissue-specific changes and novel mechanisms by which toxicants like PFOS disrupt embryonic development.