Compositions and methods for assessing toxicity using dynamic bh3 profiling

Abstract

The present invention provides methods of assessing toxicity using Dynamic BH3 profiling.

1 - 60 . (canceled) 
     
     
         61 . A method of predicting toxicity of an agent, the method comprising:
 contacting a non-human animal with a sublethal dose of an agent;   sacrificing the animal;   providing one or more cell samples, wherein each cell sample is obtained from a tissue from the animal;   providing a test aliquot of each of the one or more cell samples and a control aliquot of a cell sample not contacted with the agent;   permeabilizing the cells in each aliquot;   contacting the permeabilized cells in the test aliquot with a BH3 domain peptide;   contacting the permeabilized cells in the control aliquot with the BH3 domain peptide; and   measuring an amount of BH3 domain peptide-induced mitochondrial outer membrane permeabilization (MOMP) in the cells in each aliquot,   wherein an increase in the amount of BH3 domain peptide-induced MOMP measured in the cells in the test aliquot compared to the amount measured in the cells in the control aliquot indicates that the agent is toxic to the tissue of the animal from which the cells were obtained.   
     
     
         62 . The method of  claim 61 , wherein the cells in the control aliquot comprise cells from a non-human animal that has not been contacted with the agent. 
     
     
         63 . The method of  claim 61 , wherein the animal is a mammal. 
     
     
         64 . The method of  claim 63 , wherein the mammal is a non-human primate or a rodent. 
     
     
         65 . The method of  claim 61 , wherein the animal is contacted with the agent by injection, inhalation, topical administration, or oral administration. 
     
     
         66 . The method of  claim 61 , wherein the permeabilized cells are contacted with the BH3 domain peptide for 3 hours or less. 
     
     
         67 . The method of  claim 61  further comprising:
 determining a value for percent BH3 domain peptide-induced MOMP in the cells in the test aliquot; 
 determining a value for percent BH3 domain peptide-induced MOMP in the cells in the control aliquot; and 
 determining a value for delta percent priming for the test aliquot, wherein delta percent priming is the difference between the value for percent MOMP determined in the test aliquot and the value for percent MOMP determined in the control aliquot, 
 wherein a delta percent priming of greater than 20 percent indicates that the agent is toxic to the tissue of the animal from which the cells were obtained. 
 
     
     
         68 . The method of  claim 61 , wherein the cells are permeabilized with digitonin or saponin. 
     
     
         69 . The method of  claim 61 , wherein the amount of BH3 domain peptide-induced MOMP is measured by one or more of:
 i) contacting the cells with a potentiometric dye and measuring the emission of said potentiometric dye;   ii) measuring the release of a molecule from the mitochondrial intermembrane space; and   iii) measuring the retention of a molecule from the mitochondrial intermembrane space.   
     
     
         70 . The method of  claim 69 , wherein the potentiometric dye is 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), dihydrorhodamine 123, tetramethylrhodamine methyl ester (TMRM), or tetramethylrhodamine ethyl ester (TMRE). 
     
     
         71 . The method of  claim 69 , wherein the molecule from the mitochondrial intermembrane space is cytochrome c, SMAC/Diablo, Omi, adenylate kinase-2, or apoptosis-inducing factor. 
     
     
         72 . The method of  claim 61 , wherein the BH3 domain peptide is derived from the BH3 domain of a BID, a BIM, a BAD, a NOXA, a PUMA, a BMF, or an HRK polypeptide. 
     
     
         73 . The method of  claim 61 , wherein the BH3 domain peptide is selected from the group consisting of SEQ ID NOs: 1-16. 
     
     
         74 . The method of  claim 61 , wherein the agent comprises one or more compounds. 
     
     
         75 . The method of  claim 61 , wherein the agent is an anticancer agent. 
     
     
         76 . The method of  claim 61 , wherein the agent is a chemotherapeutic agent. 
     
     
         77 . The method of  claim 61 , wherein the agent is a small organic molecule, small inorganic molecule, peptide, protein, protein analog, enzyme, nucleic acid, nucleic acid analog, antibody, antigen, hormone, lipid, polysaccharide, growth factor, virus, cell, bioactive agent, pharmaceutical agent, or a combination or prodrug thereof. 
     
     
         78 . The method of  claim 61 , wherein the agent is gas, fine particles, radiation, electromagnetic radiation, or aerosol. 
     
     
         79 . A method of determining toxicity of an environment, the method comprising:
 exposing a test aliquot of a sample of cells to an environment comprising one or more agents selected from a gas, fine particles, radiation, and an aerosol;   providing a control aliquot of the sample of cells, wherein the control aliquot has not been exposed to the environment;   permeabilizing the cells in each aliquot;   contacting the permeabilized cells in the test aliquot with a BH3 domain peptide;   contacting the permeabilized cells in the control aliquot with the BH3 domain peptide;   measuring an amount of BH3 domain peptide-induced mitochondrial outer membrane permeabilization (MOMP) in the cells in the test aliquot and in the control aliquot,   wherein an increase in the amount of BH3 domain peptide-induced MOMP measured in the cells in the test aliquot compared to the amount measured in the cells in the control aliquot indicates that the one or more agents of the environment to which the cells in the test aliquot were exposed are toxic to cells of the sample.   
     
     
         80 . A method of detecting the presence of a toxic agent in an environment, the method comprising:
 exposing a test aliquot of a sample of cells to an environment;   providing a control aliquot of the sample of cells, wherein the control aliquot has not been exposed to the environment;   permeabilizing the cells in each aliquot;   contacting the permeabilized cells in the test aliquot with a BH3 domain peptide;   contacting the permeabilized cells in the control aliquot with the BH3 domain peptide;   measuring an amount of BH3 domain peptide-induced mitochondrial outer membrane permeabilization (MOMP) in the cells in the test aliquot and in the control aliquot,   wherein an increase in the amount of BH3 domain peptide-induced MOMP measured in the cells in the test aliquot compared to the amount measured in the cells in the control aliquot indicates that the environment to which the cells in the test aliquot were exposed comprises one or more toxic agents selected from a gas, fine particles, radiation, and an aerosol.