Chemical and metabolomic screens identify novel biomarkers and antidotes for cyanide exposure (original) (raw)
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PloS one, 2018
Cyanide is a potent toxic agent, and the few available antidotes are not amenable to rapid deployment in mass exposures. As a result, there are ongoing efforts to exploit different animal models to identify novel countermeasures. We have created a pipeline that combines high-throughput screening in zebrafish with subsequent validation in two mammalian small animal models as well as a porcine large animal model. We found that zebrafish embryos in the first 3 days post fertilization (dpf) are highly resistant to cyanide, becoming progressively more sensitive thereafter. Unbiased analysis of gene expression in response to several hours of ultimately lethal doses of cyanide in both 1 and 7 dpf zebrafish revealed modest changes in iron-related proteins associated with the age-dependent cyanide resistance. Metabolomics measurements demonstrated significant age-dependent differences in energy metabolism during cyanide exposure which prompted us to test modulators of the tricarboxylic acid ...
Pharmacologic modeling of primary mitochondrial respiratory chain dysfunction in zebrafish
Neurochemistry International, 2017
Mitochondrial respiratory chain (RC) disease is a heterogeneous and highly morbid group of energy deficiency disorders for which no proven effective therapies exist. Robust vertebrate animal models of primary RC dysfunction are needed to explore the effects of variation in RC disease subtypes, tissue-specific manifestations, and major pathogenic factors contributing to each disorder, as well as their pre-clinical response to therapeutic candidates. We have developed a series of zebrafish (Danio rerio) models that inhibit, to variable degrees, distinct aspects of RC function, and enable quantification of animal development, survival, behaviors, and organ-level treatment effects on function as well as mitochondrial biochemistry and physiology. Here, we characterize four pharmacologic inhibitor models of mitochondrial RC dysfunction in early larval zebrafish, including rotenone (complex I inhibitor), azide (complex IV inhibitor), oligomycin (complex V inhibitor), and chloramphenicol (mitochondrial translation inhibitor that leads to multiple RC dysfunction). A range of concentrations and exposure times of each RC inhibitor were systematically evaluated on early larval development, animal survival, integrated behaviors (touch and startle responses), organ physiology (brain death, neurologic tone, heart rate), and fluorescence-based mitochondrial physiology in zebrafish skeletal muscle. Pharmacologic RC inhibitor effects were validated by spectrophotometric analysis of Complex I, II and IV activities, or relative quantitation of ATP levels in larvae. Outcomes were prioritized that utilize in vivo animal imaging and quantitative behavioral assessments, as may optimally inform the translational potential of pre36 clinical drug screens for future clinical study in human mitochondrial disease subjects. The RC complex inhibitors each delayed early embryo development, with short-term exposures of these three agents or chloramphenicol from 5-7 days post fertilization also causing #
Sea lamprey cardiac mitochondrial bioenergetics after exposure to TFM and its metabolites
Aquatic Toxicology, 2020
Population control of invasive sea lamprey relies heavily on lampricide treatment of infested streams. The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is thought to impair mitochondrial ATP production through uncoupling oxidative phosphorylation. However, the effect of TFM on the entire electron transport chain (complexes I to V) in the mitochondria is not clear. In addition, TFM is reduced in phase I metabolism by sea lamprey at higher levels than in other fish species. The effects of these TFM reductive metabolites on mitochondria have not been explored. In this study, we sought to examine the effects of TFM and its reductive metabolite amino-TFM (TFMa) on cardiac mitochondrial oxygen consumption and membrane potential to delineate potential mechanisms for toxicity. To determine if molecules with similar structure also exhibit similar effects on mitochondria, we used 4-nitro-3-methylphenol (NMP) and its reductive metabolites 4-amino-3-methylphenol (NMPa) and 4-nitroso-3-methylphenol (NMPn) for comparisons. We found that mitochondrial bioenergetics was heavily affected with increasing concentrations of TFM, NMP, and NMPa when complexes I and II of the electron transport chain were examined, indicating that the toxic action of these compounds was exerted not only by uncoupling complex V, but also affecting complexes I and II.
and on Oxidative Phosphorylation of Isolated Trout Heart Mitochondria. SINHASENI, P
1982
223. Acclimated normal rainbow trout were exposed to 130 ppb hexachlorocyclopentadiene (HEX) in a flow-through well water circuit which was designed to permit measurements of oxygen consumption by the fish. Compared to preHEX values, HEX increased oxygen con-sumption rates by 186 + 24 % (2 f SEM), with maximum oxygen consumption rates being reached in approximately 84 min after HEX exposure. Oxygen consumption subsequently decreased, and all HEX-exposed fish died within 6.5 hr of exposure. Fish exposed to HEX-free vehicle (acetone) showed no changes of oxygen consumption. When added to normal isolated trout heart mitochondria, HEX appeared to uncouple oxidative phosphorylation, with calculated re-spiratory control ratios being decreased 50 % from control values at a HEX concentration of 0.4 1 FM. We postulate that one important mechanism of HEX intoxication in the intact animal may be due to increased oxygen consumption and impaired oxidative ATP synthesis due to the mitochondrial u...
Zebrafish: A Model for the Study of Toxicants Affecting Muscle Development and Function
International Journal of Molecular Sciences, 2016
The rapid progress in medicine, agriculture, and allied sciences has enabled the development of a large amount of potentially useful bioactive compounds, such as drugs and pesticides. However, there is another side of this phenomenon, which includes side effects and environmental pollution. To avoid or minimize the uncontrollable consequences of using the newly developed compounds, researchers seek a quick and effective means of their evaluation. In achieving this goal, the zebrafish (Danio rerio) has proven to be a highly useful tool, mostly because of its fast growth and development, as well as the ability to absorb the molecules diluted in water through its skin and gills. In this review, we focus on the reports concerning the application of zebrafish as a model for assessing the impact of toxicants on skeletal muscles, which share many structural and functional similarities among vertebrates, including zebrafish and humans.
Application of Zebrafish in Mitochondrial Dysfunction
IntechOpen eBooks, 2024
This chapter provides an overview of the zebrafish (Danio rerio) as a model organism for studies of mitochondrial dysfunction. Zebrafish possess a genetic similarity with humans and have conserved mitochondrial genomes, rendering them a valuable research tool for examining the intricate mechanisms that govern mitochondrial processes at diverse developmental stages. The chapter explores several methods for evaluating mitochondrial health and function. Examples include in vitro cell culture and in vivo analysis in embryos, larvae, and adults. The chapter discusses the use of zebrafish models in toxicological research to investigate mitochondrial reactions to environmental stressors and xenobiotics. The importance of implementing standardized protocols, validating marker, integrating different omics data, and using in vivo and in vitro approaches to advance mitochondrial research will be highlighted. In summary, zebrafish are suitable for analyzing both mitochondrial function and dysfunction, as well as their impact on human health.
An Investigation of the Bioactivation Potential and Metabolism Profile of Zebrafish versus Human
Journal of Biomolecular Screening, 2012
The zebrafish model has been increasingly explored as an alternative model for toxicity screening of pharmaceutical drugs. However, little is understood about the bioactivation of drug to reactive metabolite and phase I and II metabolism of chemical in zebrafish as compared with human. The primary aim of our study was to establish the bioactivation potential of zebrafish using acetaminophen as a probe substrate. Our secondary aim was to perform metabolite profiling experiments on testosterone, a CYP3A probe substrate, in zebrafish and compare the metabolite profiles with that of human. The glutathione trapping assay of N-acetyl-p-benzoquinone imine demonstrated that zebrafish generates the same reactive metabolite as humans from the bioactivation of acetaminophen. Zebrafish possesses functional CYP3A4/5like and UDP-glucuronosyltransferase metabolic activities on testosterone. Differential testosterone metabolism was observed among the two species. In silico docking studies suggested that the zebrafish CYP3A65 was responsible for the bioactivation of acetaminophen and phase I hydroxylation of testosterone. Our findings reinforce the need to further characterize the drug metabolism phenotype of zebrafish before the model can fully achieve its potential as an alternative toxicity screening model in drug research.
Transport of cyanide into guinea pig cardiac mitochondria∗
Toxicology Letters, 1991
The transport of cyanide (CN) into cells has been presumed to be by passive diffusion. Recently, there have been reports that CN, in the form of an anion, may enter the cell by active or facilitated transport. To characterize the mechanism(s) and kinetics of CN movement across the cell membrane, we measured the rate of r4CN (Na salt) uptake into guinea-pig mitochondria. Initial velocities of CN movement into mitochondria were determined at time points ranging from 10-100 msec and at CN concentrations ranging from 1 FM-10 mM using a rapid filtration device. A Hofstee plot of the data suggests that an active or facilitated transport predominates at lower CN concentrations (< 10 PM), whereas passive diffusion of CN predominates at higher CN concentrations. The kinetic constants for the active phase transport were J-0.9 pmol/ms and Kt = 14 ,uM. These results suggest that a large portion of CN movement across maxthe cell membrane is due to an active or facilitated transport phenomenon. *The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. In conducting the research described in this report, the investigators'adhered to the 'Guide of the Care and Use of Laboratory Animals' of the