Cellular ATP rescue by different quinones in the presence of rotenone is dependent on their logD value.

<p>(A) Cellular ATP rescue capacity was correlated to the rate of reduction by recombinant NQO1 enzyme in a cell-free assay. Clear outliers (boxed compounds, red circles) are either mitochondrially targeted (#69, 70) or have logD values <2. (Compare to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone-0036153-g003" target="_blank">Figure 3B</a>). Similarly, some compounds demonstrated good ATP rescue activity while being poorly reduced by NQO1 in vitro (boxed compounds, blue circles). Of all compounds tested, about 14% demonstrated very good (>80%) ATP rescue activity (green circles). (B) ATP rescue ability was correlated to the logD value of each compound. Data represent values for rat myoblasts (L6). Similar results were obtained for human immortalized hepatic cells (HepG2) and human myoblasts (9Te) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s002" target="_blank">Figure S2</a>). The data represent one typical experiment out of three experiments, which yielded similar results. The values are means ± SD, n = 3 replicate wells. Color coding of results is analogous to (A). For reasons of clarity, error bars were omitted from graphs but standard deviation values can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s005" target="_blank">Table S2</a>, which lists all results.</p

Reduction of quinones by NQO1 in a cell free assay system is dependent on their logD values.

<p><i>In vitro</i> reduction of 70 quinone derivatives by recombinant NQO1 was correlated to their calculated logD values. Each data point represents the mean from 2 independent experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s004" target="_blank">Table S1</a> for details about the quinones used). For reasons of clarity, error bars were omitted from graphs but standard deviation values can be found in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s005" target="_blank">Table S2</a> which lists all results.</p

Schematic representation of the requirements for cytoplasmic-mitochondrial electron transfer and ATP rescue.

<p>Only quinones with a logD<7 are reduced by cytoplasmic NQO1 or other reductases. Once reduced, and depending on permissive lipophilicity characteristics (27), they can shuttle electrons into the mitochondrial respiratory chain by reducing complex III or integrate into lipid membranes to prevent lipid peroxidation. However, these effects manifest only, if the compounds upon oxidation can return to the cytoplasm to be reduced again, which allows them to act in a catalytical manner. (N: no, Y: yes).</p

Effect of quinones on basal lipid peroxidation is dependent on their logD value.

<p>(A) Cellular levels of basal lipid peroxidation were measured using BODIPY-C₁₁ dye and correlated with the respective logD value. Response to DMSO was set to 100%. Compounds #69 and 70 represent two mitochondrially targeted idebenone molecules. The values are means ± SD, n = 3 replicate wells. (B) Lipid peroxidation values were correlated to ATP rescue efficiency of each quinone. Data represent values for L6 cells. Similar results were obtained for human immortalized hepatic cells (HepG2) and human myoblasts (9Te) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s003" target="_blank">Figure S3</a>). For reasons of clarity, the highly prooxidative (>200%) compounds (# 36,44,52,63 64) and error bars were omitted from graphs but standard deviation values can be found in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s005" target="_blank">Table S2</a>, which lists all results.</p

Efficacy of ATP rescue in the presence of rotenone by different quinones.

<p>(A) ATP rescue by 4 different quinones was determined in rotenone-treated rat myoblasts (L6). ATP rescue is defined as the percentage of quinone-induced increase in ATP levels in the presence of rotenone, relative to the ATP reduction by rotenone alone. Bars represent the mean of 3 wells, error bars represent standard deviation. (B) ATP rescue by different quinones is not cell type- or species-dependent. ATP rescue by 70 quinones was determined in primary human myoblasts (9Te), rat myoblasts (L6) and human immortalized hepatic cells (HepG2). Correlation of values derived from rat myoblasts (L6) and human myoblasts (9Te), are shown (R² = 0.8434). Similar results were obtained for rat myoblasts (L6) vs. human immortalized hepatic cells (HepG2) as well as human myoblasts (9Te) vs. human immortalized hepatic cells (HepG2) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s001" target="_blank">Figure S1</a>). For reasons of clarity, error bars were omitted from (B) but standard deviation (SD) values can be found in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036153#pone.0036153.s005" target="_blank">Table S2</a> which lists all results.</p