The cellular and molecular progression of mitochondrial dysfunction induced by 2,4-dinitrophenol in developing zebrafish embryos

Abstract

The etiology of mitochondrial disease is poorly understood. Furthermore, treatment options are limited, and diagnostic methods often lack the sensitivity to detect disease in its early stages. Disrupted oxidative phosphorylation (OXPHOS) that inhibits ATP production is a common phenotype of mitochondrial disorders that can be induced in zebrafish by exposure to 2,4-dinitrophenol (DNP), a FDA-banned weight-loss agent and EPA-regulated environmental toxicant, traditionally used in research labs as an uncoupler of OXPHOS. Despite the DNP-induced OXPHOS inhibition we observed using in vivo respirometry, the development of the DNP-treated and control zebrafish were largely similar during the first half of embryogenesis. During this period, DNP-treated embryos induced gene expression of mitochondrial and nuclear genes that stimulated the production of new mitochondria and increased glycolysis to yield normal levels of ATP. DNP-treated embryos were incapable of sustaining this mitochondrial biogenic response past mid-embryogenesis, as shown by significantly lowered ATP production and ATP levels, decreased gene expression, and the onset of developmental defects. Examining neural tissues commonly affected by mitochondrial disease, we found that DNP exposure also inhibited motor neuron axon arbor outgrowth and the proper formation of the retina. We observed and quantified the molecular and physiological progression of mitochondrial dysfunction during development with this new model of OXPHOS dysfunction, which has great potential for use in diagnostics and therapies for mitochondrial disease.

Triclosan is a mitochondrial uncoupler in live zebrafish

“…As in a previous study, two embryos per well were necessary at the 7–30 hpf stages and three embryos per well were required at the 3 hpf stages, in order to achieve sufficient OCR levels. With 3‐h exposure to 0.5 μM DNP, beginning at 3hpf, basal OCR levels were significantly increased in zebrafish embryos (Bestman et al , ). In comparison, we found that basal respiration of 24 hpf embryos is also increased due to TCS exposure for 1 h at 15 and 30 μM (Fig.…”

“…They also validated the XF e 24 measurements with the Clark electrode method (Stackley et al , ). Another XF‐24 study analyzed the bioenergetics effects of the known mitochondrial uncoupler 2,4‐dinitrophenol (DNP) on 3–72 hpf zebrafish (Bestman et al , ). However, these published studies have utilized relatively low‐throughput 24‐well plates, time‐consuming placement of islet capture screens and multiple fish per well (required to attain suitable signal‐to‐noise ratios).…”

Mitochondrial fusion, fission, and mitochondrial toxicity

“…Finally, even apparently adaptive responses may not preclude longer-term toxicity. For example, acute exposure of developing Danio rerio to the mitochondrial uncoupler 2,4-dinitrophenol resulted in an increase in gene expression of opa1 and peo1 (mtDNA helicase and twinkle homolog, required for mtDNA replication) immediately following the exposure, as the mitochondria fused and replicated as an initial stress response (Bestman et al 2015). However, at increasing time intervals following the initial exposure, opa1, peo1, and drp1 expression decreased, followed by a decrease in overall mitochondrial respiratory capacity, as well as motor neuron and retinal developmental defects.…”

Clinical effects of chemical exposures on mitochondrial function

“…In recent years, the deleterious effect of environmental toxins on mitochondrial function has been studied extensively in humans (Eldakroory et al, 2016, Cremonese et al, 2017, Hongsibsong et al, 2017, Sittitoon et al, 2017) and model organisms such as rodents (O’Brien and Wallace, 2004, Suzuki et al, 2008, Butenhoff et al, 2009, Butenhoff et al, 2012), fish (Ge et al, 2017), zebrafish (Bestman et al, 2015, Liu et al, 2015, Jia et al, 2016, Chen et al, 2016, Raftery et al, 2017), Caenorhabditis elegans ( C. elegans ) (Zhou et al, 2013, Liu et al, 2015, Wyatt et al, 2017), and cellular models (Zieminska et al, 2016, D’Mello et al, 2017, Yang et al, 2017). A large number of environmental factors including l-methyl-4phenyl-l, 2, 3, 6-tetra-hydropyridine (MPTP), and pesticides such as rotenone and paraquat are now widely-recognized mitochondrial toxins (Backer and Weinstein, 1980, Harmon and Sanborn, 1982, Nicklas et al, 1987, Youngster et al, 1987) and specifically, neurotoxins.…”