2. Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids MI USA
3. Department of Genetics, Michigan State University, East Lansing MI USA
Juglone (5-hydroxy-1,4-naphthalenedione) is a superoxide-generating compound that has been used in acute assays of oxidative stress. Juglone is thought to generate superoxide through redox cycling (Inbaraj and Chignell, 2004 PMID: 14727919). In working with juglone, we noticed that the toxicity of juglone-containing plates decreases rapidly with time. To demonstrate this, we made plates containing 300 µM juglone and transferred superoxide dismutase quintuple mutant worms (sod-12345) to these plates between 1 and 8 hours after the plates were poured. We chose to use sod-12345 worms because they have markedly increased sensitivity to oxidative stress (Van Raamsdonk and Hekimi, 2012 PMID: 22451939) thereby increasing our ability of detecting juglone toxicity. When we tested worm survival one hour after transferring worms to the juglone plates, we found that 100% of worms were dead if the worms were transferred within the first 5 hours. At 6, 7 and 8 hours we observed increased survival, indicating that the juglone plates had lost toxicity over time such that by 8 hours almost all of the sod-12345 worms survived.
As an additional approach to testing the toxicity of juglone-containing plates over time, we used a mitochondrial unfolded protein response (mitoUPR) reporter strain, Phsp-6::GFP, which we have previously shown is activated by oxidative stress (Schaar et al., 2015 PMID: 25671321). Exposing Phsp-6::GFP worms to freshly prepared juglone plates resulted in activation of the mitoUPR reporter. In contrast, exposing these worms to juglone plates 8 hours later, failed to increased GFP expression. This provided additional evidence that juglone plates lose toxicity with time. This may be due to the rapid degradation of juglone, which is unstable in near neutral or alkaline environments (Wright et al., 2007 PMID: 17970515).
Based on these results it is critical to consider timing when carrying out an assay involving juglone. We recommend that (1) juglone plates be used as rapidly as possible, and (2) that the time window that worms are plated to the juglone plates be kept consistent within each study, and between studies.
For those who have not used this assay before, we provide the details and timing of our standard protocol. Juglone plates are made by preparing NGM media and adding 12 mM juglone solution just before pouring. The juglone solution is made by mixing 0.05 g of juglone in 23.926 ml of ethanol. The solution requires extensive stirring to dissolve. We normally begin stirring as soon as we put the media in the autoclave so it will stir for ~1 hour. Since juglone is light sensitive, the container is covered with aluminum foil while stirring. Depending on the strains we are testing, we generally make plates at 180-300 µM juglone and will often test strains at two concentrations (180 µM = 1.5 ml juglone/100 ml NGM, 240 µM = 2 ml juglone/100 ml NGM, 300 µM = 2.5 ml juglone/100 ml NGM). We hand pour 60 mm plates with 10 ml of media using a pipet and spread the plates as a single layer on the bench to accelerate cooling and solidification. As soon as the plates have solidified (~30 mins), we add 40 µl of OP50 bacteria and spread to a thin layer by tilting the plate in order to facilitate drying. We leave plates with lids off next to a flame to dry. Once the bacteria is dry (~30 minutes), we start the assay immediately and check survival every hour. Also, note that the toxicity of juglone varies by batch and thus it is important to always include controls in every assay.