Unincorporated iron pool is linked to oxidative stress and iron levels in Caenorhabditis elegans.

Free radicals or reactive oxygen species (ROS) are relatively short-lived and are difficult to measure directly; so indirect methods have been explored for measuring these transient species. One technique that has been developed using Escherichia coli and Saccharomyces cerevisiae systems, relies on a connection between elevated superoxide levels and the build-up of a high-spin form of iron (Fe(III)) that is detectable by electron paramagnetic resonance (EPR) spectroscopy at g = 4.3. This form of iron is referred to as "free" iron. EPR signals at g = 4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S = 5/2) Fe(III) ions in sites of low symmetry. Unincorporated iron in this study refers to this high-spin Fe(III) that is captured by desferrioxamine which is detected by EPR at g value of 4.3. Previously, we published an adaptation of Fe(III) EPR methodology that was developed for Caenorhabditis elegans, a multi-cellular organism. In the current study, we have systematically characterized various factors that modulate this unincorporated iron pool. Our results demonstrate that the unincorporated iron as monitored by Fe(III) EPR at g = 4.3 increased under conditions that were known to elevate steady-state ROS levels in vivo, including: paraquat treatment, hydrogen peroxide exposure, heat shock treatment, or exposure to higher growth temperature. Besides the exogenous inducers of oxidative stress, physiological aging, which is associated with elevated ROS and ROS-mediated macromolecular damage, also caused a build-up of this iron. In addition, increased iron availability increased the unincorporated iron pool as well as generalized oxidative stress. Overall, unincorporated iron increased under conditions of oxidative stress with no change in total iron levels. However, when total iron levels increased in vivo, an increase in both the pool of unincorporated iron and oxidative stress was observed suggesting that the status of the unincorporated iron pool is linked to oxidative stress and iron levels.

Measuring "free" iron levels in Caenorhabditis elegans using low-temperature Fe(III) electron paramagnetic resonance spectroscopy.

Oxidative stress, caused by free radicals within the body, has been associated with the process of aging and many human diseases. Because free radicals, in particular superoxide, are difficult to measure, an alternative indirect method for measuring oxidative stress levels has been used successfully in Escherichia coli and yeast. This method is based on a proposed connection between elevated superoxide levels and release of iron from solvent-exposed [4Fe-4S] enzyme clusters that eventually leads to an increase in hydroxyl radical production. In past studies using bacteria and yeast, a positive correlation was found between superoxide production or oxidative stress due to superoxide within the organism and electron paramagnetic resonance (EPR) detectable "free" iron levels. In the current study, we have developed a reliable and efficient method for measuring "free" iron levels in Caenorhabditis elegans using low-temperature Fe(III) EPR at g=4.3. This method uses synchronized worm cultures grown on plates that are homogenized and treated with desferrioxamine, an Fe(III) chelator, prior to packing the EPR tube. Homogenization was found not to alter "free" iron levels, whereas desferrioxamine treatment significantly raised these levels, indicating the presence of both Fe(II) and Fe(III) in the "free" iron pool. The correlation between free radical levels and the observed "free" iron levels was examined by using heat stress and paraquat treatment. The intensity of the Fe(III) EPR signal, and thus the concentration of the "free" iron pool, varied with the treatments that altered radical levels without changing the total iron levels. This study provides the groundwork needed to uncover the correlation among oxidative stress, "free" iron levels, and longevity in C. elegans.