Using the MitoB method to assess levels of reactive oxygen species in ecological studies of oxidative stress

Karine Salin,Andrew G Cairns,Graeme J Anderson,William Mullen,Eugenia M Villasevil,Richard C Hartley,Neil B Metcalfe,Sonya K Auer

doi:10.1038/srep41228

Abstract

In recent years evolutionary ecologists have become increasingly interested in the effects of reactive oxygen species (ROS) on the life-histories of animals. ROS levels have mostly been inferred indirectly due to the limitations of estimating ROS from in vitro methods. However, measuring ROS (hydrogen peroxide, H2O2) content in vivo is now possible using the MitoB probe. Here, we extend and refine the MitoB method to make it suitable for ecological studies of oxidative stress using the brown trout Salmo trutta as model. The MitoB method allows an evaluation of H2O2 levels in living organisms over a timescale from hours to days. The method is flexible with regard to the duration of exposure and initial concentration of the MitoB probe, and there is no transfer of the MitoB probe between fish. H2O2 levels were consistent across subsamples of the same liver but differed between muscle subsamples and between tissues of the same animal. The MitoB method provides a convenient method for measuring ROS levels in living animals over a significant period of time. Given its wide range of possible applications, it opens the opportunity to study the role of ROS in mediating life history trade-offs in ecological settings.

Highlights

Extraction and quantification of the compounds MitoB and MitoP
An important step is to measure the time-course of the MitoB and MitoP compounds in each new organism model to determine the appropriate exposure duration of MitoB: a sufficient time has elapsed when detectable amounts of MitoP have accumulated yet sufficient MitoB still remains in the animal[21]
We showed that the quantification of each of the compounds and their isotopic spikes was very consistent, so that the very high repeatability in the calculated MitoP/MitoB ratio from duplicated quantification of individual extracts (ICC r = 0.878, n = 40, P < 0.001) is unlikely to be a source of variation in the estimation of the MitoP/MitoB ratio

Summary

Introduction

Extraction and quantification of the compounds MitoB and MitoP. The mitochondrial H2O2 level is related to the proportion of MitoB that has been converted into MitoP, expressed as the MitoP/MitoB ratio (Fig. 1). The conversion of MitoB to MitoP by H2O2 is about ten million times slower than the catabolism of H2O2 by the main mitochondrial peroxidase, so that MitoB does not alter physiological levels of H2O219 To date, this method has only been employed in a biomedical context using cultured cells and model organisms (fly Drosophila, worm Caenorhabditis elegans and laboratory mouse Mus musculus18,19,21,22), with the sole exception of our recent study using a species of fish, the brown trout Salmo trutta[23]. The aim of this article is to provide a detailed description of how to develop the MitoB protocol in organisms for which the method has never been applied before, with a general focus on issues that are likely to face ecologists and evolutionary biologists working on oxidative stress We illustrate this by demonstrating how the protocols established for Drosophila by Cochemé, et al.[21] had to be validated and extended for a new model, in our case brown trout[23]. We establish that the method is suitable for aquatic organisms and for use in ecological studies of oxidative stress

Objectives

Methods

Results

Conclusion