Early phosphoproteomic changes for adverse outcome pathway development in the fathead minnow (Pimephales promelas) brain

Abstract

Adverse outcome pathways (AOPs) are conceptual frameworks that organize and link contaminant-induced mechanistic molecular changes to adverse biological responses at the individual and population level. AOPs leverage molecular and high content mechanistic information for regulatory decision-making, but most current AOPs for hormonally active agents (HAAs) focus on nuclear receptor-mediated effects only despite the overwhelming evidence that HAAs also activate membrane receptors. Activation of membrane receptors triggers non-genomic signaling cascades often transduced by protein phosphorylation leading to phenotypic changes. We utilized label-free LC-MS/MS to identify proteins differentially phosphorylated in the brain of fathead minnows (Pimephales promelas) aqueously exposed for 30 minutes to two HAAs, 17α-ethinylestradiol (EE2), a strong estrogenic substance, and levonorgestrel (LNG), a progestin, both components of the birth control pill. EE2 promoted differential phosphorylation of proteins involved in neuronal processes such as nervous system development, synaptic transmission, and neuroprotection, while LNG induced differential phosphorylation of proteins involved in axon cargo transport and calcium ion homeostasis. EE2 and LNG caused similar enrichment of synaptic plasticity and neurogenesis. This study is the first to identify molecular changes in vivo in fish after short-term exposure and highlights transduction of rapid signaling mechanisms as targets of HAAs, in addition to nuclear receptor-mediated pathways.

Differential Hepatic Gene Expression Profile of Male Fathead Minnows Exposed to Daily Varying Dose of Environmental Contaminants Individually and in Mixture

“…Advances in biological science and the applications of Omics technologies along with advances in computational analytical tools may provide valuable information on mechanistic pathways that link molecular events to adverse outcomes at the ecological level. Transcriptomics combined with computational pathway analysis could provide some useful information on the key events at the cellular or organ level which facilitates predictive outcome effects in organisms or population (59, 68, 115–117). The Omics approach could characterize the biochemical pathways targeted by the contaminants and the common regulators perturbed by the chemical exposure which can be subsequently applied to ecotoxicology research (66).…”

Tissue-Based Mapping of the Fathead Minnow (Pimephales promelas) Transcriptome and Proteome

“…Interestingly, in the gut and liver, the majority of the upstream regulatory targets were nuclear transcription factors (48% gut, 47% liver, 31% brain); however, in the brain a higher proportion of the upstream regulatory targets were extracellular proteins and ligands (18% for gut and liver and 27% for brain), or membrane receptors (26% gut, 22% liver, 38% brain). These data are intriguing given the growing appreciation for the importance of membrane receptors and endocrine ligands and their signaling mechanisms in the brain, particularly for neuroendocrine functions and responses to endocrine modulators such as ethinylestradiol or levonorgestrel ( 28 , 52 ).…”

“…The relatively recent interest in contaminants that act as endocrine disruptors has focused on effects on the endocrine system of fish, since these organisms are present in contaminated environments. Studies analyzing effects on reproduction ( 21 – 23 ), thyroid function ( 24 , 25 ), neuroendocrine control ( 26 – 28 ) or effects on sex differentiation during sensitive periods of development ( 29 – 32 ) require good molecular tools for data interpretation. Thus it is important to develop well-annoted sequence databases to have a more comprehensive evaluation of the effects of endocrine disruptors on fathead minnows using functional genomic approaches.…”

Low Electric Treatment activates Rho GTPase via Heat Shock Protein 90 and Protein Kinase C for Intracellular Delivery of siRNA

“…To understand the biological processes, molecular functions and cellular components associated with the phosphorylated proteins that were up-regulated by LET, a GO analysis was carried out at Filgen, Inc. (Nagoya, Japan) using the MicroArray Data Analysis Tool Ver 3.2 (Filgen, Inc). GO slim analysis is a useful tool for understanding the functional role of changed proteins group by statistically addressing the frequency of GO term-attached proteins 54 . To determine interactions among the 15 proteins showing the largest upregulation in phosphorylation following LET, STRING analysis was performed by using STRING ver 10.5 (ELIXIR Core Data Resources).…”