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Developmental exposure to Pb2+ induces transgenerational changes to zebrafish brain transcriptome.

ABSTRACT: Lead (Pb2+) is a major public health hazard for urban children, with profound and well-characterized developmental and behavioral implications across the lifespan. The ability of early Pb2+ exposure to induce epigenetic changes is well-established, suggesting that Pb2+-induced neurobehavioral deficits may be heritable across generations. Understanding the long-term and multigenerational repercussions of lead exposure is crucial for clarifying both the genotypic alterations behind these behavioral outcomes and the potential mechanism of heritability. To study this, zebrafish (Danio rerio) embryos (<2 h post fertilization; EK strain) were exposed for 24 h to waterborne Pb2+ at a concentration of 10 μM. This exposed F0 generation was raised to adulthood and spawned to produce the F1 generation, which was subsequently spawned to produce the F2 generation. Previous avoidance conditioning studies determined that a 10 μM Pb2+ dose resulted in learning impairments persisting through the F2 generation. RNA was extracted from control- and 10 μM Pb2+-lineage F2 brains, (n = 10 for each group), sequenced, and transcript expression was quantified utilizing Quant-Seq. 648 genes were differentially expressed in the brains of F2 lead-lineage fish versus F2 control-lineage fish. Pathway analysis revealed altered genes in processes including synaptic function and plasticity, neurogenesis, endocrine homeostasis, and epigenetic modification, all of which are implicated in lead-induced neurobehavioral deficits and/or their inheritance. These data will inform future investigations to elucidate the mechanism of adult-onset and transgenerational health effects of developmental lead exposure.

SUBMITTER: Meyer DN

PROVIDER: S-EPMC7015790 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The synthetic estrogen 17α-ethinylestradiol (EE2) is an endocrine disrupting compound of concern due to its persistence and widespread presence in the aquatic environment. Effects of developmental exposure to low concentrations of EE2 in fish on reproduction and behavior not only persisted to adulthood, but have also been observed to be transmitted to several generations of unexposed progeny. To investigate the possible biological mechanisms of the persistent anxiogenic phenotype, we exposed zebrafish embryos for 80 days post fertilization to 0, 3, and 10 ng/L EE2 (measured concentrations 2.14 and 7.34 ng/L). After discontinued exposure, the animals were allowed to recover for 120 days in clean water. Adult males and females were later tested for changes in stress response and shoal cohesion, and whole-brain gene expression was analyzed with RNA sequencing. The results show increased anxiety in the novel tank and scototaxis tests, and increased shoal cohesion in fish exposed during development to EE2. RNA sequencing revealed 34 coding genes differentially expressed in male brains and 62 in female brains as a result of EE2 exposure. Several differences were observed between males and females in differential gene expression, with only one gene, sv2b, coding for a synaptic vesicle protein, that was affected by EE2 in both sexes. Functional analyses showed that in female brains, EE2 had significant effects on pathways connected to the circadian rhythm, cytoskeleton and motor proteins and synaptic proteins. A large number of non-coding sequences including 19 novel miRNAs were also differentially expressed in the female brain. The largest treatment effect in male brains was observed in pathways related to cholesterol biosynthesis and synaptic proteins. Circadian rhythm and cholesterol biosynthesis, previously implicated in anxiety behavior, might represent possible candidate pathways connecting the transcriptome changes to the alterations to behavior. Further the observed alteration in expression of genes involved in synaptogenesis and synaptic function may be important for the developmental modulations resulting in an anxiety phenotype. This study represents an initial survey of the fish brain transcriptome by RNA sequencing after long-term recovery from developmental exposure to an estrogenic compound.

Project description:Lead (Pb) poisoning has been a major public health issue globally and the recent Flint water crisis has drawn nation-wide attention to its effects. To better understand how lead plays a role as a neurotoxin, we utilized the Drosophila melanogaster model to study the genetic effects of lead exposure during development and identified lead-responsive genes. In our previous studies, we have successfully identified hundreds of lead-responsive expression QTLs (eQTLs) by using RNA-seq analysis on heads collected from the Drosophila Synthetic Population Resource. Cis-eQTLs, also known as allele-specific expression (ASE) polymorphisms, are generally single-nucleotide polymorphisms in the promoter regions of genes that affect expression of the gene, such as by inhibiting the binding of transcription factors. Trans-eQTLs are genes that regulate mRNA levels for many genes, and are generally thought to be SNPs in trans-acting transcription or translation factors. In this study, we focused our attention on alternative splicing events that are affected by lead exposure. Splicing QTLs (sQTLs), which can be caused by SNPs that alter splicing or alternative splicing (AS), such as by changing the sequence-specific binding affinity of splicing factors to the pre-mRNA. We applied two methods in search for sQTLs by using RNA-seq data from control and lead-exposed w1118Drosophila heads. First, we used the fraction of reads in a gene that falls in each exon as the phenotype. Second, we directly compared the transcript counts among the various splicing isoforms as the phenotype. Among the 1,236 potential Pb-responsive sQTLs (p < 0.0001, FDR < 0.39), mostly cis-sQTLs, one of the most distinct genes is Dscam1 (Down Syndrome Cell Adhesion Molecule), which has over 30,000 potential alternative splicing isoforms. We have also identified a candidate Pb-responsive trans-sQTL hotspot that appears to regulate 129 genes that are enriched in the "cation channel" gene ontology category, suggesting a model in which alternative splicing of these channels might lead to an increase in the elimination of Pb2+ from the neurons encoding these channels. To our knowledge, this is the first paper that uses sQTL analyses to understand the neurotoxicology of an environmental toxin in any organism, and the first reported discovery of a candidate trans-sQTL hotspot.

Project description:BackgroundAdverse environmental exposure during the prenatal period can lead to diseases in the offspring, including hypertension. Whether or not the hypertensive phenotype can be transgenerationally transmitted is not known.MethodsPregnant Sprague Dawley rats were intraperitoneally injected with lipopolysaccharide (LPS) on gestation days 6, 8, 10, and 12 to generate the prenatal LPS exposure model. Blood pressure was monitored by both telemetry and tail-cuff method. RNA sequencing was performed to analyze transcriptome alteration in the kidney of the third generation. Tempol and spironolactone were used to test the potential preventative and therapeutic effect of targeting reactive oxygen species and mineralocorticoid receptor signaling, respectively. Molecular biological experiments were performed to illustrate the mechanism of epigenetic and transcription regulation.ResultsPrenatal LPS exposure can impair the ability to excrete a salt load and induce hypertension from the first to the third generations, with the fourth and fifth generations, inducing salt-sensitive hypertension. Compared with control pups, the transcriptome in the kidney of the hypertensive third-generation prenatal LPS-exposed offspring have upregulation of the Ras-related C3 botulinum toxin substrate 1 (Rac1) gene and activation of mineralocorticoid receptor signaling. Furthermore, we found that LPS exposure during pregnancy triggered oxidative stress that upregulated KDM3B (histone lysine demethylase 3B) in the oocytes of first-generation female rats, leading to an inheritable low level of H3K9me2 (histone H3 lysine 9 dimethylation), resulting in the transgenerational upregulation of Rac1. Based on these findings, we treated the LPS-exposed pregnant rats with the reactive oxygen species scavenger, tempol, which successfully prevented hypertension in the first-generation offspring and the transgenerational inheritance of hypertension.ConclusionsThese findings show that adverse prenatal exposure induces transgenerational hypertension through an epigenetic-regulated mechanism and identify potentially preventive and therapeutic strategies for hypertension.

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Developmental exposure to Pb2+ induces transgenerational changes to zebrafish brain transcriptome.

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