Novel enzymes and metabolites involved in skatole metabolism

Abstract

Novel metabolites and enzymes involved in skatole metabolism are disclosed. The novel metabolites are 3-OH-3-methylindolenine (HMI); 3-methyloxindole (3MOI); indole-3-carbinol (I-3C); and 2-aminoacetophenone (2-AM). Measuring levels of these metabolites in a pig may be useful in identifying the pig's ability to metabolize skatole and its susceptibility to boar taint. The novel enzymes involved in skatole metabolism are aldehyde oxidase and CYP2A6. Enhancing the activity of these enzymes may be useful in enhancing skatole metabolism and reducing boar taint. The identification of the enzyme also allows the development of screening assays for substances that interact with these enzymes and skatole metabolism. Pigs having high levels of these enzymes may be selected and bred to produce pigs that have a lower incidence of boar taint.

We claim:  
     
         1 . A method of assessing a pig's ability to metabolize 3-methyl indole comprising testing a sample from the pig for (a) one or more metabolites selected from the group consisting of 3-OH-3-methylindolenine (HMI); 3-methyloxindole (3MOI); indole-3-carbinol (I-3C); and 2-aminoacetophenone; (b) aldehyde oxidase activity; and/or (c) CYP2A6 activity wherein (a) the presence of one or more metabolites; (b) high aldehyde oxidase activity and/or (c) high CYP2A6 activity indicates that the pig can metabolize 3-methylindole.  
     
     
         2 . A method according to  claim 1  wherein the sample is from liver, plasma or fat.  
     
     
         3 . A method according to  claim 1  for determining a pig's susceptibility to boar taint wherein the ability to metabolize 3-methylindole indicates that the pig is at a reduced risk for boar taint.  
     
     
         4 . A method for enhancing the metabolism of 3-methylindole in a pig comprising enhancing the activity of aldehyde oxidase in a pig.  
     
     
         5 . A method according to  claim 4  wherein the activity of the aldehyde oxidase is enhanced by using substances which (a) increase the activity of aldehyde oxidase; or (b) induce or increase the expression of the aldehyde oxidase gene.  
     
     
         6 . A method according to  claim 4  wherein the method reduces boar taint in the pig.  
     
     
         7 . A method for enhancing the metabolism of 3-methylindole comprising enhancing the activity of CYP2A6 in a pig.  
     
     
         8 . A method according to  claim 6  wherein the activity of the CYP2A6 is enhanced by using substances which (a) increase the activity of CYP2A6; or (b) induce or increase the expression of the CYP2A6 gene.  
     
     
         9 . A method according to  claim 7  wherein the method reduces boar taint in the pig.  
     
     
         10 . A method for screening for a substance that enhances skatole metabolism in a pig by enhancing aldehyde oxidase activity comprising the steps of: 
 (a) reacting a substrate of aldehyde oxidase and aldehyde oxidase, in the presence of a test substance, under conditions such that aldehyde oxidase is capable of converting the substrate into a reaction product;    (b) assaying for reaction product, unreacted substrate or unreacted aldehyde oxidase;    (c) comparing to controls to determine if the test substance selectively enhances aldehyde oxidase activity and thereby is capable of enhancing skatole metabolism in a pig.    
     
     
         11 . A method for screening for a substance that enhances skatole metabolism in a pig by enhancing CYP2A6 activity comprising the steps of: 
 (a) reacting a substrate of CYP2A6 and CYP2A6, in the presence of a test substance, under conditions such that CYP2A6 is capable of converting the substrate into a reaction product;    (b) assaying for reaction product, unreacted substrate or unreacted CYP2A6;    (c) comparing to controls to determine if the test substance selectively enhances CYP2A6 activity and thereby is capable of enhancing skatole metabolism in a pig.    
     
     
         12 . A method for screening for a substance that enhances skatole metabolism by enhancing transcription and/or translation of the gene encoding aldehyde oxidase comprising the steps of: 
 (a) culturing a host cell comprising a nucleic acid molecule containing a nucleic acid sequence encoding aldehyde oxidase and the necessary elements for the transcription or translation of the nucleic acid sequence, and optionally a reporter gene, in the presence of a test substance; and    (b) comparing the level of expression of aldehyde oxidase, or the expression of the protein encoded by the reporter gene with a control cell transfected with a nucleic acid molecule in the absence of the test substance.    
     
     
         13 . A method for screening for a substance that enhances skatole metabolism by enhancing transcription and/or translation of the gene encoding CYP2A6 comprising the steps of: 
 (a) culturing a host cell comprising a nucleic acid molecule containing a nucleic acid sequence encoding CYP2A6 and the necessary elements for the transcription or translation of the nucleic acid sequence, and optionally a reporter gene, in the presence of a test substance; and    (b) comparing the level of expression of CYP2A6, or the expression of the protein encoded by the reporter gene with a control cell transfected with a nucleic acid molecule in the absence of the test substance.    
     
     
         14 . A use of substance identified according to any one of claims  10 - 13  to prepare a medicament to reduce boar taint in a pig.  
     
     
         15 . A pharmaceutical composition for use in reducing boar taint comprising an effective amount of a substance which is selected from the group consisting of: 
 (a) a substance that increases the activity of an aldehyde oxidase enzyme;    (b) a substance that induces or increases the expression of an aldehyde oxidase gene;    (c) a substance that increases the activity of an CYP2A6 enzyme; and    (d) a substance that induces or increases the expression of an CYP2A6 gene.    
     
     
         16 . A method for producing pigs which have a lower incidence of boar taint comprising selecting pigs that express high levels of aldehyde oxidase and/or CYP2A6; and breeding the selected pigs.