Autofluorescence microscopy: A non-destructive tool to monitor mitochondrial toxicity

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Sep 30, 2011
Rodrigues R. M., Macko P., Palosaari T., Whelan M. P.
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JRC-IHCP scientists on a novel non-invasive approach to explore chemical induced toxicity

The aim of this study, published by scientists of the Joint Research Centre, Institute for Health and Consumer Protection, is to demonstrate that autofluorescence imaging of mitochondria in living eukaryotic cells provides structural and morphological information that can be used to assess mitochondrial health.

Mitochondria, the small organelles that are called 'the powerhouse of the cell', convert food we eat into cellular energy through a process dependent on the presence of oxygen. Mitochondria can be poisoned by several substances, which can induce excess production of reactive oxygen species (ROS), cause the oxidation and damage of the mitochondria and therefore reduce their ability to produce energy. This oxidation is a common factor in a variety of diseases. The assessment of mitochondrial health (which is closely correlated with overall cellular health) is performed by several methods in genomic toxicology, which evaluate mitochondrial toxicity of different substances and chemicals.

In this study, mitochondria were analysed with the technique of fluorescence microscopy, which makes it possible to distinguish them inside living cells, allowing structure analysis of these organelles in a non-invasive way. This is obtained through visualization of NADH, a derivative of nicotinamide adenine dinucleotide (NAD+), an important coenzyme in cell metabolism and energy production.

Mitochondrial morphology is determined by the occurrence of mitochondrial fission and fusion. During normal cell function mitochondria appear as elongated tubular structures. However, cellular malfunction induces mitochondria to fragment into punctiform, vesicular structures. This change in morphology is associated with the generation of reactive oxygen species (ROS) and early apoptosis (programmed cell death).

The authors firstly established the illumination conditions that do not affect mitochondrial structure and calculated the maximum safe light dose to which the cells can be exposed. Subsequently, they performed sequential recording of mitochondrial fluorescence and monitored changes in mitochondrial morphology in a continuous non-destructive way. This approach was then used to assess mitochondrial toxicity induced by potential toxicants exposed to mammalian cells. Both mouse and human cells were used to evaluate mitochondrial toxicity of different compounds with different toxicities.

This technique constitutes a novel and promising approach to explore chemical induced toxicity because of its reliability to monitor mitochondrial morphology changes and corresponding toxicity in a non-invasive way. It also represents a solid basis for complementary toxicological tests, including for instance the testing of a broad range of chemical concentrations, which would provide full toxicological profiles of the chemical compounds used. Quantification of the morphological changes of mitochondria by image analysis represents the next challenge of further development of this technique.

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R. M. Rodrigues*, P. Macko*, T. Palosaari* , M. P. Whelan*, Autofluorescence microscopy: A non-destructive tool to monitor mitochondrial toxicity.
Toxicol Lett. 2011 Oct 30; 206(3):281-8. Epub 2011 Jul 20. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

*European Commission Joint Research Centre, Institute for Health and Customer Protection, Via E. Fermi 2749, 21027 Ispra (Varese), Italy.

Photo: Fluorescence microscopy image: NADH in mitochondria. Source: JRC-IHCP research.