Study of Tissue-Specific Reactive Oxygen Species Formation by Cell Membrane Microarrays for the Characterization of Bioactive Compounds

Abstract

The production of reactive oxygen species (ROS) increases considerably in situations of cellular stress, inducing lipid peroxidation and multiple alterations in proteins and nucleic acids. However, sensitivity to oxidative damage varies between organs and tissues depending on the triggering process. Certain drugs used in the treatment of diverse diseases such as malaria have side effects similar to those produced by oxidative damage, although no specific study has been conducted. For this purpose, cell membrane microarrays were developed and the superoxide production evoked by the mitochondrial activity was assayed in the presence of specific inhibitors: rotenone, antimycin A and azide. Once the protocol was set up on cell membrane isolated from rat brain areas, the effect of six antimalarial drugs (atovaquone, quinidine, doxycycline, mefloquine, artemisinin, and tafenoquine) and two essential oils (Rosmarinus officinalis and Origanum majoricum) were evaluated in multiple human samples. The basal activity was different depending on the type of tissue, the liver, jejunum and adrenal gland being the ones with the highest amount of superoxide. The antimalarial drugs studied showed specific behavior according to the type of human tissue analyzed, with atovaquone and quinidine producing the highest percentage of superoxide formation, and doxycycline the lowest. In conclusion, the analysis of superoxide production evaluated in cell membranes of a collection of human tissues allowed for the characterization of the safety profile of these antimalarial drugs against toxicity mediated by oxidative stress.

Protective Actions of α-Tocopherol on Cell Membrane Lipids of Paraquat-Stressed Human Astrocytes Using Microarray Technology, MALDI-MS and Lipidomic Analysis

“…CMMAs have previously been used in different lipidomic studies to analyze the lipid fingerprint of nerve and peripheral tissue in animal models [ 35 , 40 ]. An additional advantage of this technology is that protein expression or activity assays can be performed, as the protein-lipid relation and protein functionality is maintained [ 35 , 39 , 40 , 41 , 42 ]. Thus, the combination of membrane microarray technology with mass spectrometry results in a powerful technique to determine the effects of different compounds over lipid composition.…”

“…The crude homogenate was subjected to a 1500-rpm centrifugation (AllegraTM X 22R centrifuge, Beckman Coulter, CA, USA) for 5 min at 4 °C, and the resulting supernatant was collected and centrifuged at 18,000× g (Microfuge ® 22R centrifuge, Beckman Coulter, CA, USA) for 15 min (4 °C). With this protocol, a fraction enriched in plasma membrane and membranes from internal organelles, including mitochondria, was obtained, as it has been demonstrated by detection of GLUT-4 transporter and Insulin Receptor β subunit [ 36 , 37 ], and in previous studies by cytochrome C detection [ 36 ], mETC enzymes and acetylcholinesterase activity [ 18 , 38 , 39 ]. The tubes were finally decanted, and the pellets were frozen at −80 °C, with the exception of one aliquot, which was used to determine the protein concentration.…”

“…The CMMAs were stored at −20 °C until usage. The CMMAs were validated before usage by different methods, including Bradford staining, for protein determination, enzyme activity assays (NADH oxidoreductase, succinate dehydrogenase, and cytochrome c oxidase), and radioligand binding assays [ 18 , 38 , 39 , 40 ].…”

Microarrays, Enzymatic Assays, and MALDI-MS for Determining Specific Alterations to Mitochondrial Electron Transport Chain Activity, ROS Formation, and Lipid Composition in a Monkey Model of Parkinson’s Disease

“…Printing was carried out under controlled humidity (relative humidity 60%) at a controlled temperature of 4 °C. CMMAs were stored at −20 °C until usage [ 65 ]. CMMAs were validated before usage via different methods, including Bradford staining for protein determination, and enzyme activity assays (succinate dehydrogenase and cytochrome c oxidase) [ 60 , 65 , 66 ].…”

Analysis of Mitochondrial Function in Cell Membranes as Indicator of Tissue Vulnerability to Drugs in Humans

“…In this sense, the aim of this study was to analyze the superoxide formation evoked by drugs and compounds with antipsychotic, anticholinergic, narcotic, and analgesic properties in isolated bovine heart membranes and on human cell membrane microarrays (CMMAs). These CMMAs (Figure 1) consisted of a collection of membranes isolated from 10 human tissues, which maintain the membrane environment and protein functionality, enabling their use in superoxide assays [54].…”