Methods and diagnostic kits utilizing mammalian stress promoters to determine toxicity of a compound

Abstract

This invention provides methods and diagnostic kits for identifying and characterizing toxic compounds. These methods and diagnostic kits measure transcription or translation levels fr4om genes linked to native eukaryotic stress promoters, especially those of mammals. The kits and methods of this invention utilize at least one stress promoter from each of the following groups: redox stress; DNA stress; protein stress and energy/ionic stress. The invention also provides methods and diagnostic kits for identifying and characterizing compounds that are toxic to specific organs, such as skin and the eye, as well as for each of the individual stresses indicated above. The methods and diagnostic kits of this invention yield information concerning the action of a compound on a subcellular level. This information may be utilized to design antitoxins to compounds found to be toxic and in active drug design.

I claim:  
     
         1 . A diagnostic kit for identifying and characterizing a toxic compound comprising: 
 (a) an eukaryotic cell characterized by: 
 (i) at least one promoter which responds to redox stress;  
 (ii) at least one promoter which responds to DNA stress;  
 (iii) at least one promoter which responds to protein stress; and  
 (iv) at least one promoter which responds to energy/ionic stress, each of said promoters being operatively linked to a different gene which encodes a detectable product; and  
   (b) at least four different nucleic acid sequences, each of said nucleic acid sequences being capable of hybridizing to the mRNA transcript of a different one of the genes that is operatively linked to said promoters or to a single stranded cDNA prepared from said mRNA transcript.    
     
     
         2 . A diagnostic kit for identifying and characterizing a toxic compound comprising: 
 (a) an eukaryotic cell comprising at least 4 different promoters which respond to redox stress, each of said promoters being operatively linked to a gene which encodes a different detectable product; and    (b) at least four different nucleic acid sequences, each of said nucleic acid sequences being capable of hybridizing to the mRNA transcript of a different one of the genes that is operatively linked to said promoters or to a single stranded cDNA prepared from said mRNA transcript.    
     
     
         3 . A diagnostic kit for identifying and characterizing a toxic compound comprising: 
 (a) an eukaryotic cell comprising at least 4 different promoters which respond to DNA stress, each of said promoters being operatively linked to a different gene which encodes a detectable product; and    (b) at least four different nucleic acid sequences, each of said nucleic acid sequences being capable of hybridizing to the mRNA transcript of a different one of the genes that is operatively linked to said promoters or to a single stranded cDNA prepared from said mRNA transcript.    
     
     
         4 . A diagnostic kit for identifying and characterizing a toxic compound comprising: 
 (a) an eukaryotic cell comprising at least 4 different promoters which respond to protein stress, each of said promoters being operatively linked to a different gene which encodes a detectable product; and    (b) at least four different nucleic acid sequences, each of said nucleic acid sequences being capable of hybridizing to the mRNA transcript of a different one of the genes that is operatively linked to said promoters or to a single stranded cDNA prepared from said mRNA transcript.    
     
     
         5 . A diagnostic kit for identifying and characterizing a toxic compound comprising: 
 (a) an eukaryotic cell comprising at least 4 different promoters which respond to energy/ionic stress, each of said promoters being operatively linked to a different gene which encodes a detectable product; and    (b) at least four different nucleic acid sequences, each of said nucleic acid sequences being capable of hybridizing to the mRNA transcript of a different one of the genes that is operatively linked to said promoters or to a single stranded cDNA prepared from said mRNA transcript.    
     
     
         6 . A diagnostic kit for identifying and characterizing a toxic compound comprising: 
 (a) a mammalian cell comprising at least 3 different promoters which respond to cell surface receptor stress, each of said promoters being operatively linked to a different gene which encodes a detectable product; and    (b) at least three different nucleic acid sequences, each of said nucleic acid sequences being capable of hybridizing to the mRNA transcript of a different one of the genes that is operatively linked to said promoters or to a single stranded cDNA prepared from said mRNA transcript.    
     
     
         7 . The diagnostic kit according to  claim 1 , wherein said eukaryotic cell is a mammalian cell which additionally comprises a promoter which responds to cell surface receptor-mediated stress, said promoter being operatively linked to a gene which encodes a detectable product, said kit additionally comprising at least one nucleic acid sequence which is capable of hybridizing to either the mRNA transcript of said gene which is operatively linked to said cell surface receptor-mediated promoter or a single stranded cDNA reverse transcribed from the mRNA transcript of each of said genes.  
     
     
         8 . A diagnostic kit for identifying and characterizing a toxic compound comprising: 
 (a) an eukaryotic cell which harbors at least one promoter or response element that responds to redox stress;    (b) an eukaryotic cell which harbors at least one promoter or response element that responds to DNA stress;    (c) an eukaryotic cell which harbors at least one promoter or response element that responds to protein stress; and    (d) an eukaryotic cell which harbors at least one promoter or response element that responds to energy/ionic stress; wherein, each of said promoters or response elements is operatively linked to a heterologous gene encoding a detectable product.    
     
     
         9 . A diagnostic kit for identifying and characterizing a toxic compound comprising at least 4 different eukaryotic cells, each of which harbors a different promoter or response element that responds to redox stress and is operatively linked to a heterologous gene encoding a detectable product.  
     
     
         10 . A diagnostic kit for identifying and characterizing a toxic compound comprising at least 4 different eukaryotic cells, each of which harbors a different promoter or response element that responds to DNA stress and is operatively linked to a heterologous gene encoding a detectable product.  
     
     
         11 . A diagnostic kit for identifying and characterizing a toxic compound comprising at least 4 different eukaryotic cells, each of which harbors a different promoter or response element that responds to protein stress and is operatively linked to a heterologous gene encoding a detectable product.  
     
     
         12 . A diagnostic kit for identifying and characterizing a toxic compound comprising at least 4 different eukaryotic cells, each of which harbors a different promoter or response element that responds to energy/ionic stress and is operatively linked to a heterologous gene encoding a detectable product.  
     
     
         13 . A diagnostic kit for identifying and characterizing a toxic compound comprising at least 3 different eukaryotic cells, each of which harbors a different promoter or response element that responds to cell surface receptor-mediated stress and is operatively linked to a heterologous gene encoding a detectable product.  
     
     
         14 . The diagnostic kit according to  claim 8 , additionally comprising a mammalian cell harboring a promoter which responds to cell surface receptor-mediated stress, said promoter being operatively linked to a heterologous gene encoding a detectable product.  
     
     
         15 . The diagnostic kit according to any one of claims  1 - 5  or  8 - 12 , wherein said eukaryotic cell is a mammalian cell.  
     
     
         16 . The diagnostic kit according to  claim 15 , wherein said mammalian cell is a HepG2 cell.  
     
     
         17 . The diagnostic kit according to any one of claims  6 - 7  or  13 - 14 , wherein said mammalian cell is a cell derived from the skin or from the eye.  
     
     
         18 . The diagnostic kit according to any one of claims  1 ,  2 ,  7 ,  8 ,  9  or  14  wherein said promoter or response element that responds to redox stress is selected from CYP1A1, GST Ya, JUN, XRE, NFkBRE, RARE, ThRE, PPRE, ERE, NMO1, ALDH1, ALDH2, HMO, MnSOD, UGT, CYP1B2, Cu.ZnSOD, ADPRT, GP, FAOxase, PBE, PPAR, EH, CYP2B2, CYP2E1, CYP3A3, P450b, P450d, PPa, PKC, GST2, GAPDH, NQO or ARE.  
     
     
         19 . The diagnostic kit according to any one of claims  1 ,  3 ,  7 ,  8 ,  10  or  14 , wherein said promoter or response element which responds to DNA stress is selected from GST Ya, GADD45, JUN, FOS, XHF, GADD153, TRE, p53RE, HMO, DRA, MnSOD, MDR-1, EGR-1, GAS 2,3, MGMT, DNA Pol, beta-pol, DHFR, TK, PCNA, PGHS, LOX, ISG15, 2′-5′ AS, EH, CYP2E1, TPO1, TPO2, PCNA or PPa.  
     
     
         20 . The diagnostic kit according to any one of claims  1 ,  4 ,  7 ,  8 ,  11  or  14 , wherein said promoter or response element which responds to protein stress is selected from GRP78, JUN, FOS, HSP70, MT 1A, MT IIA, MT III or GP.  
     
     
         21 . The diagnostic kit according to any one of claims  1 ,  5 ,  7 ,  8 ,  12  or  14 , wherein said promoter which responds to energy/ionic stress is selected from GRP78, FOS, CRE, CYP11B2, 2′-5′ AS, TH, DBH, ODC, CYP2E1, G6PD, PKC or PVALB.  
     
     
         22 . The diagnostic kit according to any one of claims  6 ,  7 ,  13  or  14 , wherein said promoter which responds to cell surface receptor-mediated stress is selected from IL-1 alpha, G-CSF, GM-CSF, TNF alpha, IL-3, IL-8, IL-6, ICAM-, IL-10 and M-CSF.  
     
     
         23 . The diagnostic kit according to  claim 1 , wherein: said promoters comprise ALDH1, CYP1A1, FOS, GADD153, HMO, HSP70, JUN and MTIIA; and said eukaryotic cell is a HepG2 cell.  
     
     
         24 . The diagnostic kit according to  claim 8 , wherein: said promoters or response elements comprise CY1A1, GST Ya, GADD45, FOS, XHF, HSP70, MT IIA, GADD153, CRE, XRE, NFkBRE, RARE and p53RE; and 
 said eukaryotic cells are HepG2 cells.    
     
     
         25 . The diagnostic kit according to  claim 24 , wherein said gene encoding a detectable product is the CAT gene.  
     
     
         26 . A method for determining the toxicity of a compound comprising the steps of: 
 (a) culturing: 
 (i) a eukaryotic cell comprising at least one promoter or response element which responds to redox stress;  
 (ii) a eukaryotic cell comprising at least one promoter or response element which responds to DNA stress;  
 (iii) a eukaryotic cell comprising at least one promoter or response element which responds to protein stress; and  
 (iv) a eukaryotic cell comprising at least one promoter or response element which responds to energy/ionic stress; wherein each of said promoters or response elements is operatively linked to a gene encoding a detectable product;  
   (b) exposing said cells to said compound; and    (c) detecting the product of each of said genes.    
     
     
         27 . A method for determining the toxicity of a compound comprising the steps of: 
 (a) culturing: 
 (i) a eukaryotic cell harboring a first redox stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (ii) a eukaryotic cell harboring a second redox stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (iii) a eukaryotic cell harboring a third redox stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (iv) a eukaryotic cell harboring a fourth redox stress promoter or response element operatively linked to a gene encoding a detectable product;  
   (b) exposing said cells to said compound; and    (c) detecting the product of each of said genes.    
     
     
         28 . A method for determining the toxicity of a compound comprising the steps of: 
 (a) culturing: 
 (i) a eukaryotic cell harboring a first DNA stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (ii) a eukaryotic cell harboring a second DNA stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (iii) a eukaryotic cell harboring a third DNA stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (iv) a eukaryotic cell harboring a fourth DNA stress promoter or response element operatively linked to a gene encoding a detectable product;  
   (b) exposing said cells to said compound; and    (c) detecting the product of each of said genes.    
     
     
         29 . A method for determining the toxicity of a compound comprising the steps of: 
 (a) culturing: 
 (i) a eukaryotic cell harboring a first protein stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (ii) a eukaryotic cell harboring a second protein stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (iii) a eukaryotic cell harboring a third protein stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (iv) a eukaryotic cell harboring a fourth protein stress promoter or response element operatively linked to a gene encoding a detectable product;  
   (b) exposing said cells to said compound; and    (c) detecting the product of each of said genes.    
     
     
         30 . A method for determining the toxicity of a compound comprising the steps of: 
 (a) culturing: 
 (i) a eukaryotic cell harboring a first energy/ionic stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (ii) a eukaryotic cell harboring a second energy/ionic stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (i) a eukaryotic cell harboring a third energy/ionic stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (i) a eukaryotic cell harboring a fourth energy/ionic stress promoter or response element operatively linked to a gene encoding a detectable product;  
   (b) exposing said cells to said compound; and    (c) detecting the product of each of said genes.    
     
     
         31 . A method for determining the toxicity of a compound comprising the steps of: 
 (a) culturing: 
 (i) a mammalian cell harboring a first cell surface receptor-mediated stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (ii) a mammalian cell harboring a second cell surface receptor-mediated stress promoter or response element operatively linked to a gene encoding a detectable product;  
 (iii) a mammalian cell harboring a third cell surface receptor-mediated stress promoter or response element operatively linked to a gene encoding a detectable product;  
   (b) exposing said cells to said compound; and    (c) detecting the product of each of said genes.    
     
     
         32 . The method according to  claim 26 , comprising the additional step of culturing a mammalian cell comprising at least one promoter or response element which responds to cell surface receptor-mediated stress.  
     
     
         33 . The method according to any one of claims  26 - 32 , wherein: 
 (a) each of said promoters or response elements is operatively linked to the same gene encoding a detectable product, said gene being heterologous to each of said promoters or response elements; and    (b) each of said cells harbors a different promoter or response element operatively linked to said gene and is cultured separately prior to exposure to said compound.    
     
     
         34 . The method according to any one of claims  26 - 30 , wherein said eukaryotic cell is a mammalian cell.  
     
     
         35 . The method according to  claim 31  or  32 , wherein said mammalian cell is derived from the skin or the eye.  
     
     
         36 . The method according to  claim 33 , wherein said cell is a mammalian cell.  
     
     
         37 . The method according to  claim 34 , wherein said mammalian cell is HepG2.  
     
     
         38 . The method according to any one of claims  26 ,  28  or  32 , wherein said promoter or response element that responds to redox stress is selected from CY1A1, GST Ya, JUN, XRE, NFkBRE, RARE, ThRE, PPRE, ERE, NMO1, ALDH1, ALDH2, HMO, MnSOD, UGT, CYP11B2, Cu.ZnSOD, ADPRT, GP, FAOxase, PBE, PPAR, EH, CYP2B2, CYP2E1, CYP3A3, P450b, P450d, PPa, PKC, GST2, GAPDH, NQO or ARE.  
     
     
         39 . The method according to any one of claims  26 ,  29  or  32 , wherein said promoter or response element that responds to DNA stress is selected from GST Ya, GADD45, JUN, FOS, XHF, GADD1S3, TRE, p53RE, HMO, DRA, MnSOD, MDR-1, EGR-1, GAS 2,3, MGMT, DNA Pol, beta-pol, DHFR, TK, PCNA, PGHS, LOX, ISG15, 2′-5′ AS, EH, CYP2El, TPO1, TPO2, PCNA or PPa.  
     
     
         40 . The method according to any one of claims  26 ,  30  or  32 , wherein said promoter or response element that responds to protein stress is selected from GRP78, JUN, FOS, HSP70, MT 1A, MT IIA, MT III or GP.  
     
     
         41 . The method according to any one of claims  26 ,  31  or  32 , wherein said promoter or response element that responds to energy/ionic stress is selected from GRP78, FOS, CRE, CYP11B2, 2′-5′ AS, TH, DBH, ODC, CYP2E1, G6PD, PKC or PVALB.  
     
     
         42 . The method according to any one of claims  26 ,  27  or  32 , wherein said promoter or response element that responds to cell surface receptor-mediated stress is selected from IL-1 alpha, G-CSF, GM-CSF, TNF alpha, IL-3, IL-8, IL-6, ICAM-1, IL-10 and M-CSF.  
     
     
         43 . The method according to  claim 26 , wherein the detection of said gene products comprises the steps of: 
 (a) isolating mRNA from said exposed culture;    (b) quantitating the amount of mRNA transcribed from each of said genes that is operatively linked to a stress promoter or response element.    
     
     
         44 . The method according to  claim 36 , wherein said gene encoding a detectable product is the CAT gene.  
     
     
         45 . The method according to any one of claims  26 - 30  or  32 , comprising the additional step of incubating said compound with an S9 liver extract prior to exposing said cells to said compound.  
     
     
         46 . The method according to  claim 44 , wherein: 
 said promoters or response elements comprise CYP1A1, GST Ya, GADD45, FOS, XHF, HSP70, MT IIA, GADD153, CRE, XRE, NFkBRE, RARE and p53RE; and 
 said mammalian cells are HepG2 cells.  
   
     
     
         47 . A method of identifying an antitoxin to a new toxic compound comprising the steps of: 
 (a) determining the types of stresses caused by said new toxic compound by the method according to any one of claims  26 - 46 ;    (b) identifying a known toxic compound which, in the process according to any one of claims  26 - 46 , causes stresses similar to those caused by said toxic compound; and    (c) repeating the method used to determine the types of stresses caused by said new toxic compound according to step (a) with the additional step of treating the eukaryotic cells employed in said method with an antitoxin to said known toxic compound identified in step (b).    
     
     
         48 . A method of decreasing the toxicity of a drug, comprising the steps of: 
 (a) determining the type of stresses caused by said drug using the methods according to any one of  claims 26  to  46 ; and    (b) modifying said drug to alter or eliminate the portion thereof suspected of causing said determined stresses.    
     
     
         49 . The method according to  claim 48 , further comprising, after step (b), the additional step of: 
 (c) repeating the method used to determine the types of stresses caused by said drug according to step (a) using the modified drug according to step (b).    
     
     
         50 . A modified drug produced by the method according to  claim 49  or  50 .