Diphenyleneiodonium Chloride, an Inhibitor of Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase, Suppresses Light-Dependent Induction of Clock and DNA Repair Genes in Zebrafish

Abstract

In most species, solar light is both a DNA-damaging agent and the key entraining stimulus for the endogenous circadian clock. The zebrafish is an attractive vertebrate system in which to study the influence of light on gene expression because the DNA repair proteins and circadian oscillators in this species are light-responsive. At the molecular level, light treatment of zebrafish cells induces the production of reactive oxygen species (ROS). ROS both alters the reduction-oxidation (redox) state of these cells and stimulates intracellular extracellular signal-regulated kinase (ERK)/mitogen activated protein kinase (MAPK) cascades that transduce photic signals activating the transcription of particular light-responsive genes, including some clock genes and some DNA repair genes involved in photoreactivation. To date, however, the phototransducing molecules responsible for light-dependent ROS production have not been identified. Flavin-containing oxidases, such as reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, are versatile flavoenzymes that catalyze molecular oxidation in numerous metabolic pathways. Importantly, light induces the photoreduction of the flavin adenine dinucleotide (FAD) moiety in these oxidases, leading to ROS production. Here, we show in cultured zebrafish cells that diphenyleneiodonium chloride (DPI), an inhibitor of NADPH oxidase, both suppresses ERK/MAPK activation and efficiently reduces light-dependent expression of clock and photoreactivation genes. Our results suggest that flavin-containing oxidases may be responsible for light-dependent ROS production and thus light-dependent gene expression in zebrafish. Our findings also support the existence of a regulatory link between photoreactivation and the circadian clock in this species.

Evolution shapes the responsiveness of the D-box enhancer element to light and reactive oxygen species in vertebrates

“…Our findings that ROS serves as a key circadian clock regulator, are consistent with several previous reports from a range of different model systems indicating close links between clocks and oxidative stress 30,47–51 . Our findings that predominantly blue light triggers ROS accumulation in fish cell lines, is also consistent with the ability of violet/blue light to initiate the photoreduction of flavin which can then activate flavin-containing oxidases 35,52 . We reveal a general role for NADPH-flavin-containing oxidases (NOXes) in the regulation of light inducible gene expression using DPI, a general Flavin inhibitor and VAS2870, a well validated NOX inhibitor (non isoform-specific) 36 .…”

Abscisic Acid-Induced Reactive Oxygen Species Are Modulated by Flavonols to Control Stomata Aperture

“…To directly test whether RBOH-produced ROS drives stomatal closure, we blocked RBOH activity with the inhibitor diphenyleneiodonium chloride (DPI; Osaki et al, 2011), which reduces ROS levels and guard cell closure in Arabidopsis (Alvarez et al, 1998;Pei et al, 2000;Singh et al, 2017). Excised leaves were placed in stomatal opening solution with and without 100 mM DPI under white light for 3 h, with samples imaged directly or after the addition of ABA for 45 min.…”

Modulation of NADPH oxidase activity by known uraemic retention solutes

“…For the determination of NADPH oxidase activity, the lysed mononuclear leucocytes were diluted with PBS (pH 7Á4) at a ratio of 1:3, and 50 lL of the lysed cells was transferred into a 96-well plate and incubated for 120 min at 37°C with 150 lL of NADH (100 mM) in the absence or presence of uraemic retention solute or plasma sample (50 ll) and 50 ll of diphenyleneiodonium chloride (DPI) (4Á8 mM), an inhibitor of NADPH oxidase [30]. The change in the absorbance of NADH at 340 nm within 120 minutes was quantified in sextuplicate (n = 6).…”