Capillary Electrophoresis Monitors Enhancement in Subcellular Reactive Oxygen Species Production upon Treatment with Doxorubicin

Abstract

This study investigated the role of doxorubicin (DOX) accumulation in the ROS production detected in individually electrophoresed organelles, including mitochondria, acidic organelles, and peroxisomes. While bulk measurements of ROS production in cells and organelles are not capable of discriminating between the effects of preparative procedures on measured ROS production, capillary electrophoresis with dual laser-induced detection of individual organelles demonstrated a difference in the measured ROS production as a result of various preparative procedures. Using this technique, the three different types of detected organelles: (i) produce ROS and do not have detectable levels of DOX, (ii) contain detectable DOX but do not produce ROS, or (iii) produce ROS and accumulate DOX. The third type displays two sub-populations of organelles, one of which demonstrated a direct relationship between DOX uptake and subsequent ROS production, corresponding most likely to mitochondria, and a second one with low DOX uptake but large variation in ROS production, corresponding most likely to acidic organelles.

Continuous intact cell detection and viability determination by CE with dual‐wavelength detection

“…Using these methods, cell viability can be determined by analyzing populations of cells, but not based on the state of individual cells. In recent years, Arriaga and his colleagues used CE for separation and detection of individual particles including nuclei [5], mitochondria [6-9, 23, 24], acidic organelles [10][11][12][13][14], liposomes [25], microspheres [26], and latex beads [27]. In all these reported experiments, CZE mode was used to analyze individual particles.…”

Recent advances in the analysis of biological particles by capillary electrophoresis

“…A general review covering the effort to develop techniques for the single-cell analysis of individual organelles has appeared [38]. The CE analysis of subcellular particles has included the analysis of new types of subcellular particles [7,39,40], separation conditions [41], improved detection arrangements [42,43], new applications [44][45][46] and new sampling and labeling schemes [47][48][49].…”

“…Recent reports have included the use of CE-LIF for the analysis of acidic organelles [40], drug content [39,46], mitochondrial DNA content [45] and superoxide production [44]. It is hypothesized that acidic organelles, including endosomes and lysosomes, play an important role in the cellular metabolism of chemotherapeutic drugs in certain forms of multidrug resistance.…”

“…3). A similar technique for monitoring the effect of DOX on ROS production in individual mitochondria using a different fluorogenic dye, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), has been published by Eder et al [46].…”

Analysis of the bioactivity of magnetically immunoisolated peroxisomes

“…[13] Capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF) has been used to analyze both DOX and its metabolites in plasma,[14, 15] cells,[16] subcellular fractions,[17, 18] and individual organelles. [19, 20] The requirement of small volume (i.e., nanoliters) of samples by CE and the high sensitivity of LIF detection make it possible to analyze fluorescent analytes in limited sample volumes such as those obtained from immunoisolated organelles.…”