Mitochondrial Toxicity Screening Overview

Mitochondrial toxicity studies are typically initiated as a follow-up to either animal experiments or high content in vitro screening after one or both have suggested mitochondrial toxicity. MitoTox™ assays are organized into two stages. Stage I assays are designed to either confirm or disprove mitotoxic effects, and when results are confirmatory, to guide more detailed mechanistic Stage II studies.

Whenever possible it is preferable to make the screening process highly interactive between the client and MitoSciences. This is due to the fact that the numbers and kinds of measurements that are needed to demonstrate and characterize mitochondrial toxicity depend to a significant extent on the compound class, specific structure, and primary target. Moreover, information about Cmax and target affinity can be useful in designing the most efficient possible screening projects.


Stage I Assays

1. Cell Viability - This assay confirms the cell toxicity of a compound and provides the working range for evaluation of this toxicity.

2. Membrane Potential - This assay provides important insight into whether the compound can act as an uncoupler of oxidative phosporylation, which in turn is relevant to whether the compound can become accumulated into the organelle.

3. Mitochondrial Biogenesis - This assay monitors longer term effects than cell viability and membrane potential, and identifies commonly seen late-stage toxicity not seen in initial screens. It is a particularly important assay to conduct when examining antivirals and antibacterials due to the similarity between the mitochondrion's replication and protein synthesis machinery and that of prokaryotes.

4. OXPHOS Activity - This assay identifies any inhibitory (or activating) reaction of a compound with the key enzymes of energy metabolism.

Each of these assays can be done in 96-well format spectrophotometrically and IC₅₀s determined for the effect of the compound on each parameter. For the cell viability, membrane potential, and biogenesis assays, the IC₅₀ measurements can be compared to help distinguish primary from secondary events. Taken together the results provide useful information to guide go / no-go decisions for a compound with respect to the potential for subsequent in vivo studies, and provide important comparative data for a set of closely similar compounds to determine which is the least mito-toxic.

Careful evaluation of the data obtained in Stage I is necessary to see if strategic decisions can be made, and wherever possible access to existing data sets on comparator drugs that have been assayed identically are provided. When further work is required to more definitively characterize the mechanism(s) of mitochondrial toxicity of any compound or drug class, a set of Stage II investigational assays is available.


Stage II Assays

1. Protein Expression - Cellular metabolism involves a highly interconnected and tightly controlled set of pathways for catabolizing sugars, fats and amino acids, plus the corresponding pathways for synthesizing these cellular components. Perturbation of cellular homeostasis by adverse effects of compounds inevitably leads to alterations in the transcriptional and post-transcriptional regulation, which is reflected in altered levels of various metabolic enzymes. We assay such changes by ELISA of a set of key proteins in the various metabolic pathways, including glycolysis, oxidative phosphorylation, fatty acid oxidation, Krebs cycle, urea cycle, gluceonogenesis etc. Altered patterns of protein levels provide important information about transcriptional networks that are being affected by the compound(s) under study.

2. Oxidative Stress - This assay measures the end-point of oxidative stress, which is oxidative and nitrative damage to proteins. Briefly, a set of key mitochondrial and cytosolic proteins known to be sensitive to oxidative stress are arrayed by immunocapture and the relative levels of post-translational modification of these are measured. For many of these proteins, and particularly those involved in Oxidative Phosphorylation, activity measurements can be obtained simultaneously to better understand the consequences of the observed oxidative damage. Evaluation of oxidative stress is particularly important to do when altered Oxidative Phosphorylation has been observed, because sub-threshold reduction of energy production can lead to chronic increase in oxidative stress that over time becomes pathogenic.

3. Apoptosis - Mitochondria are often called the central executioner in the apoptotic pathway. Many signals of cellular disfunction, and in particular reduction in energy production, lead to mitochondrially induced apoptosis. Our assay monitors the release of cytochrome c to the cytosol that triggers most apoptotic events and allows measurement of caspase independent apoptosis triggered by release of AIF from mitochondria and its uptake into the nucleus.

4. Phosphorylation - An important aspect of cellular signaling is the direct control of metabolism through inhibition/activation of key enzymes in various pathways. Regulation of metabolism also occurs via altered transcription caused by phosphorylation/dephosphorylation of transcription factors and their activators in the cell. There are many kinases and phosphatases in the cell involved in these regulatory processes. Examination of altered phosphorylation can be multiplexed with the transcription assay above. This assay should be a key part of any evaluation of non-specific and adverse effects of compounds designed to act as kinase inhibitors for the treatment of cancer, diabetes and other diseases.

All of the Stage II assays are conducted on cells in culture and all of the measurements can be done on one sample of cells after their growth in the presence of the compound of interest. IC₅₀ measurements can be obtained that can be referred back to the levels that alter cell viability and other properties analyzed in the Stage I assays.

More details of each assay for application with cell culture material are described. Our preferred cell line at present is HepG2 cells because these are a standard for hepatotoxicity studies and we have collected considerable comparator data for these. However other human cells lines can be used. Moreover several of the assays can be conducted on tissues from animals that have been dosed with the compound of interest. A review of the use of our assays for in vivo drug analysis is provided elsewhere.