# Patent

## Title
Genetic markers for 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 or for genetic screening to identify pigs on the basis of their 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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims benefit from U.S. application Ser. No. 10/206,118 filed Jul. 29 2002, which is a divisional of U.S. application Ser. No. 09/672,039, filed Sep. 29, 2000 now U.S. Pat. No. 6,448,028 which is a continuation of U.S. provisional application No. 60/156,935, filed Sep. 30, 1999 all of which are incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to novel metabolites of skatole and the identification of novel enzymes involved in the metabolism of skatole. The invention has utility in developing methods to identify and reduce boar taint.
BACKGROUND OF THE INVENTION
Male pigs that are raised for meat production are usually castrated shortly after birth to prevent the development of off-odors and off flavors (boar taint) in the carcass. Boar taint is primarily due to high levels of either the 16-androstene steroids (especially 5α(-androst-16-en-3-one)) or skatole in the fat. Recent results of the EU research program AIR 3-PL94-2482 suggest that skatole contributes more to boar taint than androstenone (Bonneau, M., 1997).
Skatole is produced by bacteria in the hindgut which degrade tryptophan that is available from undigested feed or from the turnover of cells lining the gut of the pig (Jensen and Jensen, 1995). Skatole is absorbed from the gut and metabolized primarily in the liver (Jensen and Jensen, 1995). High levels of skatole can accumulate in the fat, particularly in male pig, and the presence of a recessive gene Ska.sup.1, which results in decreased metabolism and clearance of skatole has been proposed (Lundström et al., 1994; Friis, 1995). Skatole metabolism has been studied extensively in ruminants (Smith, et al., 1993), where it can be produced in large amounts by ruminal bacteria and results in toxic effects on the lungs (reviewed in Yost, 1989). The metabolic pathways involving skatole have not been well described in pigs. In particular, the reasons why only some intact male pigs have high concentrations of skatole in the fat are not clear. Environmental and dietary factors are important (Kjeldsen, 1993; Hansen et al., 1995) but do not sufficiently explain the reasons for the variation in fat skatole concentrations in pigs. Claus et al. (1994) proposed high fat skatole concentrations are a result of an increased intestinal skatole production due to the action of androgens and glucocorticoids. Lundström et al. (1994) reported a genetic influence on the concentrations of skatole in the fat, which may be due to the genetic control of the enzymatic clearance of skatole. The liver is the primary site of metabolism of skatole and liver enzymatic activities could be the controlling factor of skatole deposition in the fat. B.ae butted.k et al. (1995) described several liver metabolites of skatole found in blood and urine with the major being MII and MIII. MII, which is a sulfate conjugate of 6-hydroxyskatole (pro-MII), was only found in high concentrations in plasma of pigs which were able to rapidly clear skatole from the body, whereas high MIII concentrations were related to slow clearance of skatole. Thus the capability of synthesis of MII could be a major step in a rapid metabolic clearance of skatole resulting in low concentrations of skatole in fat and consequently low levels of boar taint.
In view of the foregoing, further work is needed to fully understand the metabolism of skatole in pig liver and to identify the key enzymes involved. Understanding the biochemical events involved in skatole metabolism can lead to novel strategies for treating, reducing or preventing boar taint. In addition, polymorphisms in these candidate genes may be useful as possible markers for low boar taint pigs.
SUMMARY OF THE INVENTION
The present inventors have identified novel metabolites resulting from the phase I metabolism of skatole (3-methylindole, 3MI) by porcine liver microsomes. The metabolites identified 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 hence its susceptibility to boar taint.
The present inventors have also determined that one of the metabolites of skatole, HMI is metabolized to 3-hydroxy-3-methyloxindole (HMOI) by aldehyde oxidase. As a result, enhancing the activity of the aldehyde oxidase may be useful in enhancing skatole metabolism and reducing boar taint. Accordingly, the present invention provides a method for enhancing the metabolism of 3-methylindole and thereby reducing boar taint comprising enhancing the activity of aldehyde oxidase in a pig. The activity of aldehyde oxidase can be enhanced by using substances which (a) increase the activity of aldehyde oxidase; or (b) induce or increase the expression of the aldehyde oxidase gene.
The present inventors have further determined that the cytochrome P450 enzyme, CYP2A6, is also involved in the metabolism of skatole by porcine liver microsomes. As a result, enhancing the activity of the CYP2A6 may be useful in enhancing skatole metabolism and reducing boar taint. Accordingly, the present invention provides a method for enhancing the metabolism of 3-methylindole and thereby reducing boar taint comprising enhancing the activity of CYP2A6 in a pig. The activity of CYP2A6 can be enhanced by using substances which (a) increase the activity of CYP2A6; or (b) induce or increase the expression of the CYP2A6 gene.
The identification of enzymes involved in the metabolism of skatole allows the development of screening assays for substances that interact with these enzymes in skatole metabolism. The screening assays can be used to identify substances that can be used to reduce or treat boar taint.
The present invention also includes a method for producing pigs that have a lower incidence of boar taint by selecting pigs that have high levels of aldehyde oxidase and/or CYP2A6 and breeding the selected pigs.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.



BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which:
FIG. 1 is a chromatographic profile of the main five metabolites produced by pig liver microsomes as detected by UV absorption at 250 nm. Retention times correspond as follows: 9.16 min, UV-1; 11.24 min, 3-hydroxy-3-methyloxindole; 14.42 min, indole-3-carbinol; 17.51 min, 3-methyloxindole; 19.43 min, 2-aminoacetophenone; 22.84 min, parent compound (3-methylindole). (A) Standard mixture containing 2 μg/ml of each metabolite. (B) Incubation mixture.
FIG. 2 is a UV spectra of (A) UV-1 metabolite [λmax (nm): 204, 238]; (B) 3-methyloxindole[λmax (nm): 205, 252]; and (C) 3-hydroxy-3-methyloxindole[HMOI: [λmax (nm): 208, 253].
FIG. 3A is an LC-MS spectrum of metabolite UV-1.
FIG. 3B is an MS/MS spectrum of daughter ion of m/z 148.
FIG. 4 is an 1H-NMR spectrum of metabolite UV-1.
FIG. 5 shows chemical structures and percentages of 3MI metabolites produced by pig liver microsomes.
FIG. 6 shows the oxidative conversion of 3-hydroxy-3-methylindolenine into 3-hydroxy-3-methyloxindole catalyzed by aldehyde oxidase.
FIG. 7 shows the formation of 3-hydroxy-3-methyloxindole (HMOI) from 3-hydroxy-3-methylindolenine, catalyzed by porcine cytosol. Each data point represents the mean of duplicate assays performed for three pigs.
FIG. 8 shows the menadione-induced inhibition of the formation of 3-hydroxy-3-methyloxindole (HMOI) from 3-hydroxy-3-methylindolenine. Each data point represents the mean of duplicate assays performed for three pigs.
FIG. 9 shows the quinacrine-induced inhibition of the formation of 3-hydroxy-3-methyloxindole (HMOI) from 3-hydroxy-3-methylindolenine. Each data point represents the mean of duplicate assays performed for three pigs.
FIG. 10 shows the plot of back fat 3-methylindole content versus hepatic aldehyde oxidase activity in pigs (n=30). Aldehyde oxidase activity measured as nmol of 3-hydroxy-3-methyloxindole (HMOI) formed per mg of cytosolic protein per min.
FIG. 11 shows the sequence alignment of the CYP2A6 gene (SEQ ID NO:3) and the mutation (SEQ ID NO:1), at nt position 1220, indicated in bold.
FIG. 12 shows the nucleotides sequence and deduced amino acid sequences for the pig cytochrome P450 2A6 cDNA from the liver. CYP2A6 was isolated from a pig cDNA library. (SEQ ID NOS: 18 and 19) The nucleotide sequence has been registered in the GenBank (accession number, AY091516). The deduced amino acid sequence is indicated below the corresponding nucleotide sequence. Three active sites for CYP2A6 are underlined. The numbers of nucleotides and amino acids are indicated in the right.
FIG. 13 shows the alignment of amino acid sequence of human CYP2A6 (SEQ ID NO: 20), CYP2A3 (SEQ ID NO: 21) and pig 2A6 (SEQ ID NO: 22). Gln104, Phe209 and His477 are reported to be active site for human CYP2A6 coumarin 7-hydroxylase activity, oxidative metabolism of nicotine and cotinine. The numbers of amino acids are indicated in the right. Asterisk indicated identical for these active site between human and pig.
FIG. 14 shows the results of Northern blot analysis of the CYP2A6′ expression in different porcine tissues. Total RNAs were extracted from spleen, thymus, liver, lung, muscle, ovary, kidney, small intestine heart and testis, respectively. 20 μg of Total RNA (per lane) were electrophoresed on a 1.0% agarose gel containing 2.0 M formaldehyde. The RNAs were transferred to a nylon membrane and then hybridized with dig-labeled porcine CYP2A6 cDNA.
FIG. 15 shows the Genetic polymorphism, sequencing, western blotting analysis, and micosomal enzyme activity and skatole level in fat for CYP2A6 in pig liver. A, PCR-SSCP analysis of CYP2A6 cDNA. M: deletion mutant; W: wild type. B, Sequencing analysis of CYP2A6 for deletion mutant and wild type. M: sequencing data for deletion mutant; W: sequencing data for wild type. C, total proteins from microsome were separated in 12% SDS-PAGE electrophoresis, immunoblotted with mouse anti-human monoclonal 2A6-antibody. Duplicated and 40 μg of total protein from liver microsome was loaded in each lane. M: total protein from individual that has the deletion mutant for CYP2A6; W: total protein from wild type pig liver. D, micosomal CYP2A6 activity and skatole level in fat for both deletion mutant and wild type.



DETAILED DESCRIPTION OF THE INVENTION
I. Skatole Metabolites
The present inventors have identified novel metabolites resulting from the phase I metabolism of skatole (3-methyl indole, 3MI) by porcine liver microsomes. The metabolites identified 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. Accordingly, the present invention provides a method of assessing a pig's ability to metabolize 3-methyl indole comprising testing a sample from the pig for 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.
Since skatole metabolites also undergo Phase II sulfation and glucuronidation reactions, the assay may include measuring the sulfation or glucuronidation products of the metabolites. The sample can be any biological sample from the pig, preferably liver, plasma or fat. Measuring levels of particular metabolites can be used to classify pigs as either good or poor skatole metabolizers. Poor skatole metabolism may be causative of boar taint and therefore the assay may be useful in identifying pigs with boar taint or at risk for developing poor taint. Pigs that have a reduced risk for boar taint (i.e., good metabolizers) may be further selected and bred to produce low boar taint pigs.
II. Enzymes
a) Aldehyde Oxidase
The present inventors have determined that one of the metabolites of skatole, HMI is metabolized to 3-hydroxy-3-methyloxindole (HMOI) by aldehyde oxidase, a cytosolic metalloflavoprotein. The inventors have also determined that aldehyde oxidase plays an important role in the metabolism of skatole (or 3MI) and that its catalytic activity is related to adequate 3MI clearance. As a result, enhancing the activity of the aldehyde oxidase may be useful in enhancing skatole metabolism and reducing boar taint. Accordingly, the present invention provides a method for enhancing the metabolism of 3-methylindole comprising enhancing the activity of aldehyde oxidase in a pig. The activity of aldehyde oxidase can be enhanced by using substances which (a) increase the activity of aldehyde oxidase; or (b) induce or increase the expression of the aldehyde oxidase gene. The activity of aldehyde oxidase may also be enhanced using gene therapy whereby a nucleic acid sequence encoding an dehyde oxidase enzyme in introduced into a pig either ex-vivo or in-vivo. A nucleic acid sequence encoding aldehyde oxidase may be obtained by cloning the pig gene using the information available from the human, bovine and rabbit genes.
As mentioned above, aldehyde oxidase activity is related to 3MI clearance. As a result, testing the enzymatic activity of aldehyde oxidase in a pig can be used to determine a pig's susceptibility to boar taint. Pigs with high aldehyde oxidase activity would be at a lower risk for boar taint than pigs with a low aldehyde oxidase activity. Pigs with high aldehyde oxidase activity may be selected and bred to produce low boar taint pigs. Accordingly, the present invention provides a method of determining a pig's susceptibility to boar taint comprising determining the activity of aldehyde oxidase in a sample from a pig. Methods for determining aldehyde oxidase activity are detailed in Example 2.
b) CYP2A6
The present inventors have further determined that the cytochrome P450 enzyme, CYP2A6, is also involved in the metabolism of skatole by porcine liver microsomes. As a result, enhancing the activity of CYP2A6 may be useful in enhancing skatole metabolism and reducing boar taint. Accordingly, the present invention provides a method for enhancing the metabolism of 3-methylindole comprising enhancing the activity of CYP2A6 in a pig. The activity of CYP2A6 can be enhanced by using substances which (a) increase the activity of CYP2A6; or (b) induce or increase the expression of the CYP2A6 gene. The activity of CYP2A6 may also be enhanced using gene therapy whereby a nucleic acid sequence encoding a CYP2A6 enzyme in introduced into a pig either ex-vivo or in-vivo. A nucleic acid sequence encoding CYP2A6 may be obtained by cloning the pig gene using the information available from the human gene.
Testing the enzymatic activity of CYP2A6 in a pig can be used to determine a pig's susceptibility to boar taint. Pigs with high CYP2A6 activity would be at a lower risk for boar taint than pigs with a low CYP2A6 activity. Pigs with high CYP2A6 activity may be selected and bred to produce low boar taint pigs. Accordingly, the present invention provides a method of determining a pig's susceptibility to boar taint comprising determining the activity of CYP2A6 in a sample from a pig.
c) Screening Assays
The identification of enzymes involved in the metabolism of skatole allows the development of screening assays for substances that interact with these enzymes and thereby modulate skatole metabolism.
In one aspect, the present invention provides a method of screening for a substance that enhances the activity of aldehyde oxidase or CYP2A6.
In one embodiment of the invention, a method is provided 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. Substrates of aldehyde oxidase which may be used in the method of the invention include HMI which is metabolized to HMOI.
The induction of aldehyde oxidase activity can be measured using a variety of techniques including measuring the levels of the aldehyde oxidase protein or mRNA or by testing for aldehyde oxidase activity. Aldehyde oxidase activity can be measured using various assays including the assay described in Example 2 and those described by Rajagopalan et al., 1966.
In another embodiment of the invention, a method is provided 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.
Substrates of CYP2A6 which may be used in the method of the invention for example include skatole and coumarin.
The induction of CYP2A6 activity can be measured using a variety of techniques including measuring the levels of the CYP2A6 protein or mRNA or by testing for CYP2A6 activity as described in Aitio, 1978.
The aldehyde oxidase and CYP2A6 enzymes used in the method of the invention may be obtained from natural, recombinant, or commercial sources. Cells or liver microsomes expressing the enzymes may also be used in the method.
Conditions which permit the formation of a reaction product may be selected having regard to factors such as the nature and amounts of the test substance and the substrate.
The reaction product, unreacted substrate, or unreacted enzyme; may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
To facilitate the assay of the reaction product, unreacted substrate, or unreacted enzyme; antibody against the reaction product or the substance, or a labeled enzyme or substrate, or a labeled substance may be utilized. Antibodies, enzyme, substrate, or the substance may be labeled with a detectable marker such as a radioactive label, antigens that are recognized by a specific labeled antibody, fluorescent compounds, enzymes, antibodies specific for a labeled antigen, and chemiluminescent compounds.
The substrate used in the method of the invention may be insolubilized. For example, it may be bound to a suitable carrier. Examples of suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc. The insolubilized enzyme, substrate, or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
In another aspect, the present invention includes a method for screening for a substance that enhances skatole metabolism by modulating the transcription or translation of an enzyme involved in skatole metabolism.
In one embodiment of the invention, a method is provided 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.
In another embodiment of the invention, a method is provided 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.
A host cell for use in the method of the invention may be prepared by transfecting a suitable host with a nucleic acid molecule comprising a nucleic acid sequence encoding the appropriate enzyme. Suitable transcription and translation elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate transcription and translation elements is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art. Examples of such elements include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other genetic elements, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary transcription and translation elements may be supplied by the native gene of the enzyme and/or its flanking sequences.
Examples of reporter genes are genes encoding a protein such as green fluorescence protein, β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, or an immunoglobulin or portion thereof such as the Fc portion of an immunoglobulin, preferably IgG. Transcription of the reporter gene is monitored by changes in the concentration of the reporter protein such as β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. This makes it possible to visualize and assay for expression of the enzyme and in particular to determine the effect of a substance on expression of enzyme.
Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells, including bacterial, mammalian, yeast or other fungi, viral, plant, or insect cells. Protocols for the transfection of host cells are well known in the art (see, Sambrook et al. Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, which is incorporated herein by reference). Host cells which are commercially available may also be used in the method of the invention. For example, the h2A3 and h2B6 cell lines available from Gentest Corporation are suitable for the screening methods of the invention.
Substances which enhance skatole metabolism by enhancing aldehyde oxidase or CYP2A6 activity (including the substances isolated by the above screening methods) may be used to reduce or treat boar taint or to prepare medicaments to reduce or treat boar taint.
d) Compositions
Substances which enhance skatole metabolism (including substances identified using the methods of the invention which selectively enhance aldehyde oxidase or CYP2A6 activity) may be incorporated into pharmaceutical compositions. Therefore, the invention provides a pharmaceutical composition for use in reducing boar taint comprising an effective amount of one or more substances which enhance skatole metabolism and a pharmaceutically acceptable carrier, diluent, or excipient. The term “effective amount” as used herein means an amount effective, at dosages and for periods of time necessary to achieve the desired result.
In one embodiment, the present invention provides a pharmaceutical composition 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.
The substances for the present invention can be administered for oral, topical, rectal, parenteral, local, inhalant or intracerebral use. Preferably, the active substances are administered orally (in the food or drink) or as an injectable formulation.
In the methods of the present invention, the substances described in detail herein and identified using the method of the invention form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients, or carriers suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, consistent with conventional veterinary practices.
For example, for oral administration the active ingredients may be prepared in the form of a tablet or capsule for inclusion in the food or drink. In such a case, the active substances can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like; for oral administration in liquid form, the oral active substances can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Suitable binders, lubricants, disintegrating agents, and coloring agents can also be incorporated into the dosage form if desired or necessary. Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Suitable lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Examples of disintegrators include starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
Gelatin capsules may contain the active substance and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar carriers and diluents may be used to make compressed tablets. Tablets and capsules can be manufactured as sustained release products to provide for continuous release of active ingredients over a period of time. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration may contain coloring and flavoring agents to increase acceptance.
Water, a suitable oil, saline, aqueous dextrose, and related sugar solutions and glycols such as propylene glycol or polyethylene glycols, may be used as carriers for parenteral solutions. Such solutions also preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Suitable stabilizing agents include antioxidizing agents such as sodium bisulfate, sodium sulfite, or ascorbic acid, either alone or combined, citric acid and its salts and sodium EDTA. Parenteral solutions may also contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.
The substances described in detail herein and identified using the methods of the invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
Substances described in detail herein and identified using the methods of the invention may also be coupled with soluble polymers which are targetable drug carriers. Examples of such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamidephenol, polyhydroxyethyl-aspartamidephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. The substances may also be coupled to biodegradable polymers useful in achieving controlled release of a drug. Suitable polymers include polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
Suitable pharmaceutical carriers and methods of preparing pharmaceutical dosage forms are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.
More than one substance described in detail herein or identified using the methods of the invention may be used to enhance metabolism of skatole. In such cases the substances can be administered by any conventional means available for the use in conjunction with pharmaceuticals, either as individual separate dosage units administered simultaneously or concurrently, or in a physical combination of each component therapeutic agent in a single or combined dosage unit. The active agents can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice as described herein.
e) Genetic Screening
The present invention further includes the identification of polymorphisms in genes encoding the enzymes responsible for skatole metabolism in a pig including aldehyde oxidase and CYP2A6 as described in detail hereinabove. The identification of genes that encode these enzymes from pigs that are high skatole metabolizers (and hence have a low incidence of low boar taint) can be used to develop lines of pigs that have a low incidence of boar taint. In addition, the identification of these genes can be used as markers for identifying pigs that are predisposed to having a low incidence of boar taint.
Accordingly, the present invention provides 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.
Transgenic pigs may also be prepared which produce high levels of aldehyde oxidase and/or CYP2A6. The transgenic pigs may be prepared using conventional techniques. For example, a recombinant molecule may be used to introduce (a) a gene encoding aldehyde oxidase or (b) a gene encoding a CYP2A6. Such recombinant constructs may be introduced into cells such as embryonic stem cells, by a technique such as transfection, electroporation, injection, etc. Cells which show high levels of aldehyde oxidase and/or CYP2A6 may be identified for example by Southern Blotting, Northern Blotting, or by other methods known in the art. Such cells may then be fused to embryonic stem cells to generate transgenic animals. Germline transmission of the mutation may be achieved by, for example, aggregating the embryonic stem cells with early stage embryos, such as eight cell embryos, transferring the resulting blastocysts into recipient females in vitro, and generating germline transmission of the resulting aggregation chimeras. Such a transgenic pig may be mated with pigs having a similar phenotype i.e. producing high levels of aldehyde oxidase and/or CYP2A6 to produce animals having a low incidence of boar taint.
The following non-limiting examples are illustrative of the present invention:
EXAMPLES
Example 1
Identification of Skatole Metabolites
Materials and Methods
Chemicals. 3-Methylindole (3MI), indole-3-carbinol (I3C), indole-3-aldehyde, indole-3-carboxylic acid, 2-aminoacetophenone and sulfatase type H-2 from Helix pomatia were purchased from Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). The oxindoles, 3-methyloxindole (3MOI) and 3-hydroxy-3-methyloxindole (HMOI) were synthesized by the methods of Kende and Hodges (1982) and Skiles et al. (1989), respectively. Authentic 5-OH-3-methylindole and 6-OH-3-methylindole (in the form of 6-sulfatoxyskatole) were donated by Jens Hansen-Møller (Danish Meat Research Institute, Roskilde, Denmark). In order to obtain 6-OH-3-methylindole from 6-sulfatoxyskatole, the compound was hydrolyzed in a total volume of 0.5 ml acetate buffer pH 5.0 containing 90 units/ml of type H-2 sulfatase. Hydrolysis was conducted for 4 hours in a shaking water bath at 40° C. and then 0.5 ml of ice-cold acetonitrile were added both to stop the reaction and precipitate the protein. After centrifugation at 7,500 rpm for 15 min, 50 μl of clear supernatant were injected into the chromatograph, using the conditions described below under “Analytical chromatography”.
Preparation of microsomes. Liver samples were taken from 30 intact male pigs obtained by back-crossing F3 European Wild Pig.times.Swedish Yorkshire boars with Swedish Yorkshire sows (Squires and Lundström, 1997). Liver samples were frozen in liquid nitrogen and stored at −80° C. For the preparation of microsomes, partially thawed liver samples were finely minced and homogenized with 4 volumes of 0.05 M Tris-HCl buffer pH 7.4 (containing 0.15 M KCl, 1 mM EDTA, and 0.25 M sucrose) using a Ultra-Turax homogenizer (Janke and Kunkel, GDR). The homogenate was centrifuged at 10,000 g for 20 min and the resulting supernatant was centrifuged again at 100,000 g for 60 min order to obtain the microsomal pellet. The pellets were suspended in a 0.05 M Tris-HCl buffer, pH 7.4, containing 20% glycerol, 1 mM EDTA, and 0.25 M sucrose to a final concentration of 20 mg protein/ml and stored at −80° C. before analysis. Protein concentrations were determined by the method of Smith et al. (1985) using bicinchoninic acid protein assay reagents purchased from Pierce Chemical Co. (Rockford, Ill., USA) and bovine serum albumin as standard.
Microsomal incubations. Two mg microsomal protein was incubated with 0.4 mM 3MI and 4 mM NADPH in 0.05M sodium phosphate buffer (pH 7.4) containing 5 mM MgCl2 and 1 mM EDTA for 30 min at 37.degree. C. (production of metabolites was determined to be linear over a range of 10 to 40 min). Incubation volumes were 0.5 ml. Reactions were started by the addition of NADPH after 3-minute preincubation periods at 37° C., and stopped with 0.5 ml of ice-cold acetonitrile. Incubations of all 30 samples were run in duplicate and for control incubations NADPH was omitted. After the addition of acetonitrile the mixture was vortexed and centrifuged at 5000 rpm for 20 min. A 50 μl aliquot of the clear supernatant was analyzed by high-performance liquid chromatography (HPLC).
Analytical chromatography. Analytical HPLC was done using a Spectra-Physics system (Spectra-Physics, San Jose, Calif., USA) consisting of a SP8800 gradient pump, a SP8880 autosampler with a 50 μl injection loop, a SP Spectra 100 UV detector, and a Spectra System FL-2000 fluorescent detector. The HPLC method is a modification of a previously reported binary gradient system method (Baek et al., 1995). 3MI and its metabolites were separated using a reverse-phase Prodigy ODS, 5 μm, 250×4.6 mm column (Phenomenex, Torrance, Calif., USA). The mobile phase consisted of two solvents, A (0.01M potassium dihydrogen phosphate buffer pH 3.9) and B (acetonitrile), with the following gradients: 0 min—90% A, 6 min—80% A; 12 min—70% A; 18 min—30% A; 25 min 10% A; 26 min 90% A; 35 min—90% A. All gradients were linear and the flow rate was set at 1.2 ml/min. Absorbance was monitored at 250 nm; fluorescence was monitored at excitation and emission wavelengths of 286 and 350 nm, respectively. HPLC analysis for 3MI metabolites was conducted immediately after the incubations. Metabolites were identified by comparison of retention times, and co-injection of standards (spiking the metabolite mixture with authentic standards).
Isolation and purification of metabolites by preparative HPLC. In order to obtain a sufficient amount of metabolites to conduct UV spectral analysis, a large scale incubation (final volume of 4 ml) was performed, using the same concentrations of reactants as described above. Preparative HPLC was done using a Spectra-Physics SP8800 gradient pump (Spectra-Physics, San Jose, Calif., USA), a manual Rheodyne 7125 injector fitted with a 500 μl injection loop (Rheodyne, Cotati, Calif., USA), and a SP Spectra 100 UV detector. The 3MI metabolites were separated using a reverse-phase Waters preparative HPLC C 18, 10 μm, 300.times.7.6 mm column (Waters Associates, Division of Millipore Corp., Milford, Mass., USA). The mobile phase was the same as above except that the flow rate was set at 3.0 ml/min. The peaks corresponding to the metabolites identified on the basis of their retention times as HMOI, I3C, 3MOI and 2-aminoacetophenone were collected in enough amounts to determine their UV spectra. Purity of the collected fractions was verified by HPLC using the procedure described before under “Analytical chromatography”. One of the metabolites produced by pig liver microsomes could not be identified on the basis of comparison of retention times; this metabolite was named UV-1 due to its absorption in the far UV spectrum and the fact that it was the first metabolite that eluted from the column (Babol et al., 1998a). The peak corresponding to this metabolite, which eluted between 9.1 and 10.1 min, was collected after several 500 μl injections and subjected to HPLC-MS, .sup.1 H-NMR and UV spectra analysis.
Ultraviolet Spectroscopy. UV spectra (200–300 nm) were recorded for the HPLC metabolites UV-1, HMOI, I3C, 3MOI and 2-aminoacetophenone. UV spectra of available authentic standards were also recorded and compared with those of the isolated metabolites. Spectra were recorded on a model 4054 LKB Biochrom UV-Visible spectrophotometer (Pharmacia LKB Biochrom Ltd. Cambridge, UK). Due to their low levels of production, it was not possible to isolate the hydroxyskatoles in enough quantities to determine their UV spectra.
LC/MS of metabolite UV-1. Metabolite UV-1 was analyzed by LC-MS using the following conditions: the HPLC was performed using a Prodigy 5 ODS-2, 5 μm, 150.times.3.2 mm colunm (Phenomenex, Torrance, Calif., USA) and water:acetonitrile (50:50) as mobile phase. The mobile phase was delivered by binary LC pumps (Hewlett Packard 1090 Series II/L, Palo Alto, Calif., USA). The eluent passed through a sample injection valve Rheodyne 7010 (Rheodyne, Cotati, Calif., USA), to an atmospheric pressure chemical ionization (APCI) source configured with a corona discharge pin, at a flow rate of 0.7 ml/min. A sample volume of 20 μl was injected by an autosampler (Hewlett Packard 1090 Series II/L, Palo Alto, Calif., USA). Mass spectrometry (MS) detection was achieved using a VG Quattro II triple quadrupole mass spectrometer (Fisons UK Ltd., Altrincham, UK). Instrument control, data acquisition and data processing were carried out using the MassLynx software package. Liquid nitrogen was used as a drying and sheath gas, at flow rates of 200 and 50 liter/hr, respectively. The instrument was operated in the positive ion mode with an ion source temperature of 150° C., a corona discharge pin potential of +3.75 kV, and a cone voltage of 15V. The total ion chromatogram of LC/MS was obtained by scanning the first quadrupole from m/z 125–700 at a rate of 400 amu/sec in full scan mode with inter-scan delay of 0.10 sec. Data was acquired in continuum mode. The production scan was performed by tandem mass spectrometry (MS/MS) by transmitting the protonated molecular ion ([M+H].sup.+) through the first quadrupole into the second quadrupole containing ultrapure argon. The production chromatogram was recorded by scanning the third quadrupole from m/z 50 to 450 in 1.0 sec. The collision energy was varied between −20 to −50 eV to optimize fragmentation of the selected protonated molecular ion.
NMR spectroscopy of metabolite UV-1. UV-1 metabolite was isolated for NMR analysis using incubation conditions essentially as described above. However, these incubations contained 1 nmol cytochrome P450 content rather than 2 mg of total protein. UV-1 was separated from other microsomal 3MI metabolites by the HPLC conditions described above using a system consisting of an LDC Analytical Constametric 4100 solvent delivery module (ThermoQuest, Riviera Beach, Fla., USA), a Hewlett Packard 1040A diode array detector and a Hewlett Packard 9000 series HPLC workstation (Hewlett Packard Company, Willington, Del., USA). UV-1 was purified by HPLC and pooled from two identical incubations followed by concentration in a Savant Speed-Vac (Savant Instruments, Farmingdale, N.Y., USA). Concentration to dryness was not possible, due to polymerization and degradation of unstable UV-1. Therefore, the sample was evaporated to a volume of 200 L and re-injected on the HPLC for additional purification. In this case however, the aqueous mobile phase consisted of 0.01 M dibasic potassium phosphate buffer, pH 9.0, in 99.9 atom % deuterium oxide. Due to the instability of UV-1 when it was evaporated to dryness, it was necessary to perform the final purification step in the NMR solvent, deuterium oxide. UV-1 was again collected and evaporated to a final volume of 250 L and directly added to the Shigemi NMR tube. The .sup.1 H-NMR spectrum was obtained in deuterium oxide using a Varian Unity Inova 600 MHz NMR (Varian Associates Inc., Palo Alto, Calif., USA).
Results
HPLC. None of the metabolites produced by pig liver microsomes co-eluted with indole-3-carboxaldehyde or indole-3-carboxylic acid. However, metabolites that coeluted with HMOI, 3MOI, I3C, 2-aminoacetophenone, and the two hydroxyskatoles (5- and 6-OH-3-methylindole) were measured by UV and/or fluorescence detection. The oxindole metabolites (HMOI and 3MOI) and the pyrrole ring opened metabolite (2-aminoacetophenone) were detected and quantitated by UV absorption because they do not fluoresce; I3C and the hydroxyskatoles were detected and quantitated by fluorescence detection. When microsomal incubations were spiked, all metabolites identified on the basis of their retention times, co-chromatographed with their corresponding authentic standards. The chromatographic profile of a microsomal incubation and a standard mixture monitored by UV absorption at 250 nm is shown in FIG. 1.
UV Spectroscopy. The UV spectrum of the metabolites identified on the basis of their retention times on HPLC (HMOI, 3MOI, I3C, and 2-aminoacetophenone) were identical to those of authentic standards. Spectra of metabolites were recorded using water as solvent, and the wavelengths of maximal absorption were as follows: HMOI: λmax (nm): 208, 253; 3MOI: λmax (nm): 205, 252; I3C: λmax (nm): 221, 278; 2-aminoacetophenone: λmax (nm): 228, 257. The UV spectrum of 3-methylindole was: λmax (nm): 224, 281. The UV spectrum of UV-1 metabolite was: λmax (nm): 204, 238. The UV spectra of UV-1 was similar to the spectra of the oxindole metabolites 3MOI and HMOI as shown in FIG. 2. Changing the pH from 3 to 11 did not change the spectrum of UV-1; this lack of a bathochromic shift indicated that the unknown metabolite had no free phenolic group. Isolated UV-1 was kept in acetonitrile:water solution at room temperature and the solution was analyzed by HPLC at 7-day intervals for 6 weeks. After 6 weeks only about 25% of the original compound remained and it was observed that UV-1 was converted into 3MOI. The slopes of the linear regressions of 3MOI and UV-1 over time indicated that the molar response factor for UV-1 on HPLC-UV analysis was 2.95 times that of 3MOI.
Metabolite UV-1 structural data. The mass spectrometry of isolated UV-1 produced a molecular ion at m/z 148 [M+H].sup.+ with major fragments at m/z 133 [M—CH3]+, 104 [M—H3 C—C—OH]+, and 77 (protonated phenyl ring) (FIG. 3). The 1H-NMR spectrum of metabolite UV-1 is shown in FIG. 4. Assignments of the proton signals are provided, listed as chemical shift (multiplicity, integration and assignment): 1.4 (s, 3H, —CH3); 6.8 (d, 2H, H-5 and H-6); 7.2 (d, 2H, H-4 and H-7); 8.4 (s, 1H, H-2). The singlet at 8.4 has been assigned to the proton at C-2 of 3-hydroxy-3-methylindolenine. This proton is attached to the sp.sup.2 hybridized C-2 which is also a deshielded by the adjacent nitrogen. Therefore, this proton is highly deshielded and appears downfield from all other protons in the proposed structure. At 2.0 is a singlet corresponding to the methyl protons of contaminating acetonitrile. Due to the way in which the sample was purified, it was extremely difficult to remove all of the acetonitrile present in the HPLC organic phase.
In summary, seven metabolites of 3MI were found to be produced by pig liver microsomes: 3MOI, HMOI, 6-OH-3-methylindole (6-OH-3MI), I3C, 2-aminoacetophenone, 5-OH-3-methylindole (5-OH-3MI), and the metabolite that was named UV-1. When UV-1 was quantitated assuming a molar absorptivity 2.95 times greater than that of 3MOI, the total amount of nanomoles produced accounted for an average of 96.0% (range of 86.5–105.0%) of the 3MI molecules metabolized during the microsomal incubations. The rates of production of the seven metabolites identified in pig liver microsomal incubations are shown in Table 1. UV-1 metabolite was produced at the highest rate (750.7 pmol/mg protein/min), while 5-OH-3MI was produced at the lowest rate (5.1 pmol/mg protein/min). Large inter-individual differences were noted for the production rates of the same metabolite. For instance, UV-1 metabolite was produced at a rate of 1556.3 pmol/mg protein/min by the microsomes of one pig, while other microsomes produced this compound at a rate of 180.5 pmol/mg/protein/min (Table 1). The metabolite that was produced in larger amounts was UV-1 which, on average, accounted for 45.1% of all metabolites produced. The combined oxindoles accounted for 46.4% of the total metabolites: an average of 27.9% of the metabolites produced corresponded to 3MOI whereas 18.5% corresponded to HMOI. The other metabolites were produced in much lesser amounts. 6-OH-3MI accounted for 4.9% of the metabolites, I3C accounted for 2.7% and 2-aminoacetophenone and 5-OH-3MI accounted for only 0.5% and 0.3% of the metabolites, respectively. The chemical structures and percentages of production of these metabolites are shown in FIG. 5.
Discussion
Only three Phase I metabolites of 3MI had been identified previously in pigs: HMOI, and the hydroxyskatoles, 5-OH-3MI and 6-OH-3MI. HMOI had been found in pig plasma and urine (Baek et al., 1997), and pig liver microsomal incubations (Babol et al., 1998a); 6-OH-3MI had been detected both in pig serum (Baek et al., 1997) and pig liver microsomal incubations (Babol et al., 1998a), while 5-OH-3MI had only been reported to be present in pig serum (Baek et al., 1997). In the present study, all three metabolites were detected in the microsomal incubations and the production of four new metabolites is reported.
One of the pathways of 3MI biotransformation identified in species such as goats, mice and rats is the formation of oxindole derivatives: 3MOI and HMOI (Frydman et al., 1972; Smith et al., 1993). On average, 46.4% of the metabolites produced by pig liver microsomes in the present study corresponded to these two oxindole derivatives; this finding indicates that the oxidole pathway is quantitatively very important in the pig. 3MOI had been identified in rat liver microsomal incubations (Frydman et al., 1972), goat lung and liver microsomal incubations (Huijzer et al., 1987), and in the urine of goats (Hammond et al., 1979). One of the metabolites observed in pig microsomal incubations by Babol et al. (1998a) was named “UV-3” and the results of the present study indicate this metabolite corresponds to 3MOI. The other oxindole derivative of 3MI, HMOI, had already been isolated from the urine of pigs dosed with 3MI (Baek et al., 1997) and was also reported to be produced by pig liver microsomes (Babol et al., 1998a); HMOI is also a major urinary metabolite produced by mice dosed with radiolabeled 3MI (Skiles et al., 1989), additionally it has been found in the urine of humans (Albrecht et al., 1989), and goats (Smith et al., 1993). Interestingly, in the present study, pig liver microsomes produced large amounts of both oxidole derivatives 3MOI and HMOI. In other species studied, one of these metabolites predominates. In goats, production of 3MOI predominates (Hammond et al., 1979), whereas in mice it is HMOI that predominates (Smith et al., 1993).
The 3 methyl group of 3MI may be oxidized to the alcohol, aldehyde and carboxylic acid functions (Hammond et al., 1979). In the present study, only the alcohol function of the 3 methyl group (indole-3-carbinol) was found to be produced by pig liver microsomes. This metabolite exhibits strong fluorescence and also absorbs in the UV and even though it had been previously reported to be produced by pig microsomes (named F-1 by Babol et al., 1998a), its structure was unknown. It is important to note that further metabolism of the alcohol function of indole-3-carbinol could possibly be catalyzed by alcohol dehydrogenase; if this is true, then the product of this reaction, indole-3-carboxaldehyde, would not be produced in microsomal incubations.
Hydroxylation of the aromatic ring of 3MI can occur at any of the carbons 4, 5, 6 or 7; however, the experimental evidence indicates that hydroxylation at positions 5 and 6 predominate. In 1962, Jepson and co-workers showed that rabbit liver microsomes hydroxylate tryptamine, indole acetic acid and related indoles to their corresponding 6-hydroxy derivatives. The microsomal system required NADPH and oxygen and did not form 5- or 7-hydroxyindoles (Jepson et al., 1962). Mahon and Mattok (1967) analyzed the urine of ten normal human subjects and found that all samples contained 6-hydroxyskatole and nine had the 5-isomer, although its excretion rate was approximately 50% of the 6-isomer; 7-hydroxyskatole was detected in three of the samples but its excretion rate was only 5% of the 6-isomer. None of the subjects excreted 4-hydroxyskatole (Mahon and Mattok, 1967). Baek et al. (1995) found conjugates of both 5-OH-3MI and 6-OH-3MI in pig serum. In the present study, the average rate of production of 6-OH-3MI was approximately eleven times greater than the production of the 5 isomer, indicating that hydroxylation at position C6 predominates.
Frydman et al. (1972) found two pyrrole ring opened metabolites produced after incubation of 3-MI with rat liver microsomes. The two compounds were identified as 2-formamidoacetophenone and 2-aminoacetophenone; a total of 33% of the metabolites formed corresponded to 2-formamidoacetophenone, 12% to 2-aminoacetophenone, and 5% to 3-MOI. In the present study, 2-aminoacetophenone was found to be produced by all liver samples analyzed at an average percentage of 0.5%, which is much lower than the percentage reported for rats by Frydman et al. (1972). No previous reports of 2-aminoacetophenone production from 3MI metabolism by pigs were found in the literature.
The 1H-NMR, LC-MS and UV-spectral characteristics of metabolite UV-1 indicate that this compound corresponds to 3-hydroxy-3-methylindolenine. UV-1 was found to be an unstable compound, intermediate between 3MI and 3MOI. The fact that UV-1 was converted into 3MOI suggested that this compound could be a precursor of 3MOI, possibly 2,3-epoxy-3-methylindolenine, the structure of which was postulated by Smith et al. (1993) or, most likely, its ring-opened product, 3-hydroxy-3-methylindolenine (Skordos et al., 1998a, 1998b). The molecular weight of the compound (147) and its fragmentation pattern were compatible with the epoxyde or the imine (FIG. 3), but the UV spectrum, with a λmax at 238 nm (FIG. 2) was more consistent with the imine structure. The molecular weight of 147 could also correspond to an aromatic phenolic metabolite of 3MI; however, when the UV spectrum of isolated UV-1 was taken under different pHs, it did not show the typical bathochromic shift observed in phenolic indoles. Furthermore, the fact that the UV spectrum of metabolite UV-1 was very similar to that of 3MOI and HMOI (FIG. 2) indicated that metabolite UV-1 could be structurally related to any of the two oxindoles; these metabolites, in which the pyrrol ring is oxidized at the 2-carbon position, show very different spectra than 3MI, or other metabolites such as I3C, 2-aminoacetophenone or the hydroxyskatoles. Finally, the 1H-NMR spectrum of UV-1 (FIG. 4) was consistent with the assignment of this metabolite to 3-hydroxy-3-methylindolenine.
The results of the present study indicate that seven major metabolites of 3MI are produced by pig liver microsomes in vitro. In quantitative terms, the main pathway of Phase I biotransformation of 3MI by pig liver microsomes appears to be the formation of oxindole derivatives and the formation of 3-hydroxy-3-methylindolenine. Differences in the metabolic fate of 3MI among species could explain the difference in species susceptibility to 3MI-induced lung toxicity. The extensive metabolism of 3MI to oxindole derivatives may explain the lack of pneumotoxicity showed by pigs and reported by Carlson and Yost (1989). The electrophilic metabolite 3-methylene-indolenine, which is the putative reactive metabolite of 3MI produced by cytochrome P-450 enzymes, is a precursor of I3C in lung microsomal incubations and susceptible species form I3C in appreciable amounts (Skiles and Yost, 1996). In the present in vitro study, less than 3% of the metabolites produced by pig liver microsomes corresponded to I3C, which may also explain the lack of susceptibility of pigs to suffer from 3MI-induced lung lesions. Large inter-individual differences in the rate of production of metabolites were observed. These differences in Phase I metabolism could be due to individual differences in cytochrome P450 enzymes and this issue should be further investigated. It was previously reported that CYP2E1 plays a role in the metabolism of 3MI in the pig (Squires and Lundström, 1997; Babol et al., 1998a), but the role of other isoenzymes remains to be determined. Babol et al. (1998b) reported sulfation and glucuronidation of some 3MI metabolites produced by pig liver microsomes. However, more studies are needed in order to determine the complete Phase II metabolism of the different metabolites of 3MI identified in the present study.
Example 2 
Aldehyde Oxidase
Materials And Methods
Chemicals. Menadione, quinacrine and allopurinol were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). Authentic HMOI was graciously provided by Dr. G. S. Yost, Department of Pharmacology and Toxicology, University of Utah. HMI was produced using porcine liver microsomes and it was isolated and purified using preparative HPLC as described before (Diaz et al., 1999). Isolated HMI was freeze-dried and kept in a dessicator at −20° C. until used.
Preparation of porcine liver cytosol. Liver samples were taken from 30 intact male pigs obtained by back-crossing F3 European Wild Pig×Swedish Yorkshire boars with Swedish Yorkshire sows (Squires and Lundström, 1997). Liver samples were frozen in liquid nitrogen and stored at −80° C. For the preparation of the cytosolic fraction, partially thawed liver samples were finely minced and homogenized with 4 volumes of 0.05 M Tris-HCl buffer pH 7.4 (containing 0.15 M KCl, 1 mM EDTA, and 0.25 M sucrose) using a Ultra-Turax homogenizer (Janke and Kunkel, GDR). The homogenate was centrifuged at 10,000×g for 20 minutes and the resulting supernatant was centrifuged again at 100,000×g for 60 minutes in order to obtain the cytosolic fraction and the microsomal pellet. Cytosolic fractions were stored at −80° C. before analysis. Protein concentrations were determined by the method of Smith et al. (1985) using bicinchoninic acid protein assay reagents purchased from Pierce Chemical Co. (Rockford, Ill., USA) and bovine serum albumin as standard.
Enzyme assays. In order to investigate the role of AO in the conversion of HMI to HMOI, incubations containing HMI, porcine liver cytosol and different concentrations of the selected AO inhibitors menadione and quinacrine were conducted. Each incubation was run in duplicate, and were performed for three randomly selected cytosol porcine samples. HIMOI formation was detected and quantitated by HPLC as described under “Chromatographic analysis”. AO activity was measured as the formation of HMOI per minute per mg of cytosolic protein. Assay mixtures contained 0.05M sodium phosphate buffer (pH 7.4) with 5 mM MgCl2 and 1 mM EDTA, 1 mg cytosolic protein and 1 μg HMI in a final assay volume of 250 μl. For the inhibition experiments, different final concentrations of menadione (0, 2, 5, 10, 25, 50 and 100 μM) or quinacrine (0, 0.05, 0.1, 0.25, 0.5 and 1.0 mM) were tested in the assay mixture. Menadione was dissolved in ethanol (final assay concentration 4%, v/v), which had no effect on activity in controls without inhibitor; quinacrine was dissolved in buffer. Incubations were carried out for 10 min at 37° C. in a shaking water bath; the reaction was stopped with 250 μl ice-cold acetonitrile. After the addition of acetonitrile, the mixture was vortexed and centrifuged at 7,500 rpm for 15 min. A 400 μl aliquot of the clear supernatant was diluted with 400 μl water and 100 μl of the mixture were analyzed immediately by high-performance liquid chromatography (HPLC). Dilution with water was necessary in order to avoid leading of the chromatographic peaks. HMOI production was quantitated by using an external standard. Controls included incubations using boiled cytosol and incubations carried out without the addition of cytosol. Incubations run under the same conditions described above were conducted using 0.1, 0.5 and 1.0 mM allopurinol in order to investigate the role of XO on the enzymatic conversion of HMI into HMOI.
Chromatographic analysis. HPLC was conducted using a Spectra-Physics system (Spectra-Physics, San Jose, Calif., USA) consisting of a SP8800 gradient pump, a SP8880 autosampler with a 100 μl injection loop, and a SP Spectra 100 UV detector. The HPLC method is a modification of a previously reported binary gradient system method (Baek et al., 1997). HMOI and HMI were separated using a reverse-phase Prodigy ODS, 5 μm, 250×4.6 mm column (Phenomenex, Torrance, Calif., USA). The mobile phase consisted of two solvents, A (0.01 M potassium dihydrogen phosphate buffer pH 3.9) and B (acetonitrile), with the following gradients: 0 min—90% A, 6 min—80% A; 12 min—70% A; 18 min—30% A; 25 min 10% A; 26 min 90% A; 35 min—90% A. All gradients were linear and the flow rate was set at 1.2 ml/min. Absorbance was monitored at 250 nm. HPLC analysis was conducted immediately after the incubations.
Measurement of 3MI fat content. For the quantitation of the 3MI fat content, a sample of backfat was taken from each pig and its 3MI content measured with a colorimetric assay (Mortensen and Sorensen, 1984). All analysis were done in duplicate.
Statistical analysis. Pearson correlation coefficients, linear regression analysis and one-way ANOVA were computed using the Statistical Analysis System (SAS, 1995).
Results
Porcine cytosol catalyzed the conversion of HMI to HMOI (FIG. 6) in a time-dependent manner (FIG. 7). Under these assay conditions, the formation of HMOI was found to be linear (r2=0.995) up to 10 min (FIG. 7). No HMOI was formed when cytosol was boiled before the incubation or when no cytosol was added to the assay mixture. The addition of the aldehyde-oxidase inhibitors menadione or quinacrine to the incubation mixtures containing HMI and cytosolic protein decreased the formation of HMOI in a dose-dependent manner. When no inhibitor was added, the total amount of HMOI produced was considered as 100%. At a concentration of 10 μM menadione, only 33.3% of the HMOI formed in the absence of menadione was detected whereas at a concentration of 100 μM menadione, no HMOI was produced (FIG. 8). At a concentration of 50 μM quinacrine, 75.5% of the control HMOI production was observed and at 1 mM 43.4% of the control HMOI was found (FIG. 9). Menadione was a more potent inhibitor of the reaction since even a concentration of quinacrine 10 times higher than that of menadione (1 mM vs 100 μM) was not enough to completely abolish the conversion of HMI to HMOI. The addition of up to 1.0 mM allopurinol to the assay mixture did not affect the conversion of HMI to HMOI (data not shown).
The AO activity, estimated as nmol of HMOI produced per minute per mg cytosolic protein, versus the 3MI fat content of the 30 pigs used in this study are shown in FIG. 10. The Pearson correlation coefficient between these two variables was found to be −0.70 (P<0.001), whereas the determination coefficient was r2=0.49. The linear regression model to explain the 3MI fat content as a function of AO activity was found to be: 3MI in fat=0.22—AO activity 0.042763. This model was found to be highly significant (P<0.001).
The 3MI fat content in all samples ranged from 0.07 to 0.3 mg/kg and had mean value of 0.15 mg/kg, whereas the AO activity ranged from 0.25 to 3.53 nmol HMOI/mg protein/min and had a mean value of 1.27 nmol HMOI/mg protein/min. The results were grouped in three categories according to the 3MI fat content of each pig as follows: large 3MI accumulators (0.2 mg/kg 3MI or more), moderate 3MI accumulators (0.11 to 0.19 mg/kg 3MI) and low accumulators (0.1 mg/kg 3MI or less). Lundström and Bonneau (1996) have suggested that levels of 3MI of 0.2–0.25 mg/kg or greater cause unacceptable taint by sensory analysis. The mean values for 3MI fat content and AO activity for these three categories of pigs are shown in Table 2.
Discussion
Menadione is a well documented inhibitor of AO (Johns, 1967; Krenitzky et al., 1974; Rodrigues, 1994) and biochemical reactions sensitive to inhibition by menadione are attributed to AO (Beedham et al., 1995; Rashidi et al., 1997). Rodrigues (1994) found that at a concentration of 10 μM, menadione completely abolished the oxidation of N1-methylnicotinamide, the model substrate for AO. In the present experiment, a concentration of 10 μM menadione decreased the formation of HMOI by 56.7%, and at 100 μM menadione, no HMOI was formed, indicating a complete inhibition of the enzymatic activity. The inverse dose-response relationship observed between HMOI production and menadione concentration strongly suggests that AO is the enzyme responsible for the biotransformation of HMI into HMOI in porcine cytosol. Quinacrine has been reported as being a competitive inhibitor (Ki=1.5.times.10−6 M) of aldehyde oxidase against all substrates (Rajagopalan and Handler, 1964). In the present trial, quinacrine was less potent than menadione in inhibiting the conversion of HMI into HMOI but it also inhibited the reaction to a large extent. The inhibition of HMOI formation caused by quinacrine also suggests that the production of HMOI from HMI is catalyzed by AO. On the other hand, the lack of inhibition observed when allopurinol was added to the reaction mixture indicates that XO is not involved in the oxidative metabolism of HMI into HMOI.
N-heterocyclic cations constitute a major group of substrates for AO (Beedham, 1985). Quaternization of a ring nitrogen atom activates the heterocycle to nucleophilic substitution and enhances the reactivity of the compound toward enzyme-catalyzed attack (Beedham, 1985). HMI is a recently identified N-heterocyclic quaternized metabolite produced by porcine microsomal enzymes (Diaz et al., 1999) and therefore it constitutes a suitable substrate for AO-catalyzed oxidation. The results of the present study strongly suggest that AO activity present in the cytosol of pigs is responsible for the oxidation of HMI to form a more polar and stable metabolite, HMOI.
When hepatic AO activity (measured as the formation of HMOI) was plotted against the 3MI fat content, a clear inverse relationship was observed (FIG. 9). This finding suggests that hepatic AO activity is related to 3MI clearance. The relatively high determination coefficient (r2 =0.49) indicates that almost 50% of the variation in 3MI fat content is explained by the hepatic enzymatic activity of AO. The results shown on Table 2 also indicate that AO activity may be very significant in the adequate clearance of 3MI in the pig. High 3MI fat levels were associated with low enzymatic activity (mean values of 0.24 mg/kg 3MI and 0.80 nmol HMOI/mg protein/min, respectively), whereas low 3MI levels were associated with high enzymatic activity (mean values of 0.09 mg/kg 3MI and 2.73 nmol HMOI/mg protein/min, respectively). Pigs classified as high 3MI accumulators had a hepatic mean AO activity 3.4 times lower than those pigs classified as low accumulators; this difference was found to be significant (P<0.05).
The results of the present study suggest that AO plays an important role in the metabolism of 3MI in the pig and that its catalytic activity is related to an adequate 3MI clearance. The enzymatic activity of AO in the pig might be used as a potential marker in order to identify pigs containing low levels of 3MI in the fat, which will eventually help to control “boar taint”.
Menadione is customarily used as a source of vitamin K in swine diets (National Research Council, 1987). ecommended levels of inclusion are 2.5 mg/kg for grower diets and 2.0 mg/kg for finisher diets (Patience et al., 1995). Since menadione is a potent inhibitor of AO and the enzyme appears to be important in the metabolism of 3MI, care should be exercised so that excessive levels of menadione are not present in swine diets. It is possible that some of the sporadic episodes of “boar taint” could had been caused by high levels of menadione in the diet resulting in high levels of 3MI in the fat of pigs. Studies are needed in order to determine whether the levels of menadione commonly used in practical pig diets are capable of inhibiting AO activity. Additionally, it has been observed that high levels of dietary copper lead to molybdenum deficiency and thus to low AO activity because molybdenum is a cofactor for this enzyme (Beedham, 1985). It is important to avoid excess copper levels in pig diets in order to avoid a decrease in the activity of AO and the potential occurrence of “boar taint” episodes.
Example 3 
The Role of CYP2A6 in 3-Methylindole Metabolism by Porcine Liver Microsomes
The role of different cytochrome P450 enzymes on the metabolism of 3-methylindole (3MI) was investigated using selective chemical inhibitors. Eight chemical inhibitors of P450 enzymes were screened for their inhibitory specificity towards 3MI metabolism in porcine microsomes: alpha-naphthoflavone (CYP1A2), 8-methoxypsoralen (CYP2A6), menthofuran (CYP2A6), sulphaphenazole (CYP2C9), quinidine (CYP2D6), 4-methylpyrazole (CYP2E1), diethyldithiocarbamate (CYP2E1, CYP2A6), and troleandomycin (CYP3A4). The production of the different 3MI metabolites was only affected by the presence of inhibitors of CYP2E1 and CYP2A6 in the microsomal incubations. In a second experiment, a set of porcine microsomes (n=30) was screened for CYP2A6 content by Western blot analysis and also for their 7-hydroxylation activity (CYP2A6 activity). Protein content and enzymatic activity were found to be correlated with 3MI fat content. The results of the present study indicate that measurement of CYP2A6 levels and/or activity is a useful marker for 3MI-induced boar taint.







TABLE 1







Rate of production of 3MI metabolites by pig liver microsomes


(pmol/mg microsomal protein/min) (n = 30)











Rate of Production
Minimum
Maximum



(pmol/mg prot./min)
(pmol/mg
(pmol/mg


Metabolite
±SD
prot./min)
prot./min)













UV-1
750.7 ± 414.5
180.5
1556.3


3-methyloxindole
420.9 ± 118.1
234.4
700.8


3-hydroxy-3-methyloxin-
272.4 ± 91.6 
118.9
516.5


dole


6-OH-3 -methylindole
58.4 ± 47.2
n.d.*
213.7


Indole-3-carbinol
37.1 ± 15.8
12.1
85.7


2-aminoacetophenone
7.8 ± 2.4
3.4
12.7


5-OH-3-methylindole
5.1 ± 5.8
0.7
27.3





*n.d. = not detected

















TABLE 2









Mean (SD





Mean (SD)
aldehyde oxidase





3-methylindole
activity (nmol



3-Methylindole

content
HMOI/mg


Category
fat content
n
(mg/kg)
prot./min)





















High
0.2 mg/kg or
7
0.24
0.4a
0.80
0.61b


accumulator
more


Moderate
0.11–0.19
15
0.15
0.03b
1.40
0.90b


accumulator
mg/kg


Low
0.1 mg/kg or
8
0.09
0.01c
2.73
0.45a


accumulator
less





a–cWithin a column, means lacking a common superscript differ significantly (P < 0.05).






Example 4
According to the invention, the association of alternate forms of cytochrome P450 enzymes such as the CYP2A6 may be used to identify and select pigs with differences in boar taint. For example, according to the invention, a deletion mutant of the CYP2A6 gene has been identified that results in a frame shift and loss of function mutation, which resulted in higher skatole levels in the pig.
We have cloned the pig isoforms of CYP2A6. We found a deletion mutation that results in a frame shift and premature stop. This animal has zero enzyme activity for CYP2A6 (coumarin 7-hydroxylase) in the liver and high skatole levels in fat. Another polymorphism was identified which resulted in a t to c transition at nt number 124 and a change from Phe to Leu at amino acid number 42 of SEQ ID NO:3 (wild type).
Further according to the invention, other polymorphisms in genes related to skatole metabolism (other cytochrome P450 related genes) in the pig may be identified to genetically identify and select pigs based upon their proclivity to boar taint. Once an association between a gene or gene product and a particular trait is made, genes encoding these proteins may be screened for polymorphism or markers which may be used to indicate differences in these animals with respect to the correlated trait. These polymorphisms with these genes enables genetic markers to be identified for specific breeds or genetic lines or animals, boar taint potential early in the animal's life.
An alternate form of CYP2A6 has been identified according to the invention which results in a frameshift causing a premature stop codon and loss of function resulting in higher skatole levels in the pig. Tests for the presence of this alternate form may be developed using the novel sequence for CYP2A6 as disclosed herein, SEQ ID NO: 18 or 3 supra and the mutations disclosed herein in SEQ ID NO:1 (both 124 nt and 422 deletion), SEQ ID NO:5 (124nt only) and SEQ ID NO:7 (422 deletion only). These tests include but are not limited to PCR, SSCP, and the like.
The invention thus relates to genetic markers for economically valuable traits in animals. The markers represent alleles or alternate gene forms that are associated boar taint, based upon the findings that the aldehyde oxidase pathway and CYP2A6 are associated with skatole production.
Thus, the invention relates to genetic markers and methods of identifying those markers in an animal of a particular animal, breed, strain, population, or group, whereby the animal is has increased, decreased or otherwise altered skatole metabolism, and thus boar taint.
Any method of identifying the presence or absence of these markers may be used, including, for example, single-strand conformation polymorphism (SSCP) analysis, base excision sequence scanning (BESS), RFLP analysis, heteroduplex analysis, denaturing gradient gel electrophoresis, and temperature gradient electrophoresis, allelic PCR, ligase chain reaction direct sequencing, mini sequencing, nucleic acid hybridization, micro-array-type detection of genes encoding enzymes involved in skatole metabolism. Also within the scope of the invention includes assaying for protein conformational or sequences changes which occur in the presence of this polymorphism. The polymorphism may or may not be the causative mutation but will be indicative of the presence of this change and one may assay for the genetic or protein bases for the phenotypic difference.
The following is a general overview of techniques which can be used to assay for the genetic marker of the invention.
In the present invention, a sample of genetic material is obtained from an animal. Samples can be obtained from blood, tissue, semen, etc. Generally, peripheral blood cells are used as the source, and the genetic material is DNA. A sufficient amount of cells are obtained to provide a sufficient amount of DNA for analysis. This amount will be known or readily determinable by those skilled in the art. The DNA is isolated from the blood cells by techniques known to those skilled in the art.
Isolation and Amplification of Nucleic Acid
Samples of genomic DNA are isolated from any convenient source including saliva, buccal cells, hair roots, blood, cord blood, amniotic fluid, interstitial fluid, peritoneal fluid, chorionic villus, and any other suitable cell or tissue sample with intact interphase nuclei or metaphase cells. The cells can be obtained from solid tissue as from a fresh or preserved organ or from a tissue sample or biopsy. The sample can contain compounds which are not naturally intermixed with the biological material such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
Methods for isolation of genomic DNA from these various sources are described in, for example, Kirby, DNA Fingerprinting, An Introduction, W. H. Freeman & Co. New York (1992). Genomic DNA can also be isolated from cultured primary or secondary cell cultures or from transformed cell lines derived from any of the aforementioned tissue samples.
Samples of animal RNA can also be used. RNA can be isolated from tissues expressing the gene as described in Sambrook et al., supra. RNA can be total cellular RNA, mRNA, poly A+ RNA, or any combination thereof. For best results, the RNA is purified, but can also be unpurified cytoplasmic RNA. RNA can be reverse transcribed to form DNA which is then used as the amplification template, such that the PCR indirectly amplifies a specific population of RNA transcripts. See, e.g., Sambrook, supra, Kawasaki et al., Chapter 8 in PCR Technology, (1992) supra, and Berg et al., Hum. Genet. 85:655–658 (1990).
PCR Amplification
The most common means for amplification is polymerase chain reaction (PCR), as described in U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188 each of which is hereby incorporated by reference. If PCR is used to amplify the target regions in blood cells, heparinized whole blood should be drawn in a sealed vacuum tube kept separated from other samples and handled with clean gloves. For best results, blood should be processed immediately after collection; if this is impossible, it should be kept in a sealed container at 4° C. until use. Cells in other physiological fluids may also be assayed. When using any of these fluids, the cells in the fluid should be separated from the fluid component by centrifugation.
Tissues should be roughly minced using a sterile, disposable scalpel and a sterile needle (or two scalpels) in a 5 mm Petri dish. Procedures for removing paraffin from tissue sections are described in a variety of specialized handbooks well known to those skilled in the art.
To amplify a target nucleic acid sequence in a sample by PCR, the sequence must be accessible to the components of the amplification system. One method of isolating target DNA is crude extraction which is useful for relatively large samples. Briefly, mononuclear cells from samples of blood, amniocytes from amniotic fluid, cultured chorionic villus cells, or the like are isolated by layering on a sterile Ficoll-Hypaque gradient by standard procedures. Interphase cells are collected and washed three times in sterile phosphate buffered saline before DNA extraction. If testing DNA from peripheral blood lymphocytes, an osmotic shock (treatment of the pellet for 10 sec with distilled water) is suggested, followed by two additional washings if residual red blood cells are visible following the initial washes. This will prevent the inhibitory effect of the heme group carried by hemoglobin on the PCR reaction. If PCR testing is not performed immediately after sample collection, aliquots of 106 cells can be pelleted in sterile Eppendorf tubes and the dry pellet frozen at −20° C. until use.
The cells are resuspended (106 nucleated cells per 100 μl) in a buffer of 50 mM Tris-HCl (pH 8.3), 50 mM KCl 1.5 mM MgCl2, 0.5% Tween 20, and 0.5% NP40 supplemented with 100 μg/ml of proteinase K. After incubating at 56° C. for 2 hr. the cells are heated to 95° C. for 10 min to inactivate the proteinase K and immediately moved to wet ice (snap-cool). If gross aggregates are present, another cycle of digestion in the same buffer should be undertaken. Ten μl of this extract is used for amplification.
When extracting DNA from tissues, e.g., chorionic villus cells or confluent cultured cells, the amount of the above mentioned buffer with proteinase K may vary according to the size of the tissue sample. The extract is incubated for 4–10 hrs at 50°-60° C. and then at 95° C. for 10 minutes to inactivate the proteinase. During longer incubations, fresh proteinase K should be added after about 4 hr at the original concentration.
When the sample contains a small number of cells, extraction may be accomplished by methods as described in Higuchi, “Simple and Rapid Preparation of Samples for PCR”, in PCR Technology, Ehrlich, H. A. (ed.), Stockton Press, New York, which is incorporated herein by reference. PCR can be employed to amplify target regions in very small numbers of cells (1000–5000) derived from individual colonies from bone marrow and peripheral blood cultures. The cells in the sample are suspended in 20 μl of PCR lysis buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.45% NP40, 0.45% Tween 20) and frozen until use. When PCR is to be performed, 0.6 μl of proteinase K (2 mg/ml) is added to the cells in the PCR lysis buffer. The sample is then heated to about 60° C. and incubated for 1 hr. Digestion is stopped through inactivation of the proteinase K by heating the samples to 95° C. for 10 min and then cooling on ice.
A relatively easy procedure for extracting DNA for PCR is a salting out procedure adapted from the method described by Miller et al., Nucleic Acids Res. 16:1215 (1988), which is incorporated herein by reference. Mononuclear cells are separated on a Ficoll-Hypaque gradient. The cells are resuspended in 3 ml of lysis buffer (10 mM Tris-HCl, 400 mM NaCl, 2 mM Na2 EDTA, pH 8.2). Fifty μl of a 20 mg/ml solution of proteinase K and 150 μl of a 20% SDS solution are added to the cells and then incubated at 37° C. overnight. Rocking the tubes during incubation will improve the digestion of the sample. If the proteinase K digestion is incomplete after overnight incubation (fragments are still visible), an additional 50 μl of the 20 mg/ml proteinase K solution is mixed in the solution and incubated for another night at 37° C. on a gently rocking or rotating platform. Following adequate digestion, one ml of a 6M NaCl solution is added to the sample and vigorously mixed. The resulting solution is centrifuged for 15 minutes at 3000 rpm. The pellet contains the precipitated cellular proteins, while the supernatant contains the DNA. The supernatant is removed to a 15 ml tube that contains 4 ml of isopropanol. The contents of the tube are mixed gently until the water and the alcohol phases have mixed and a white DNA precipitate has formed. The DNA precipitate is removed and dipped in a solution of 70% ethanol and gently mixed. The DNA precipitate is removed from the ethanol and air-dried. The precipitate is placed in distilled water and dissolved.
Kits for the extraction of high-molecular weight DNA for PCR include a Genomic Isolation Kit A.S.A.P. (Boehringer Mannheim, Indianapolis, Ind.), Genomic DNA Isolation System (GIBCO BRL, Gaithersburg, Md.), Elu-Quik DNA Purification Kit (Schleicher & Schuell, Keene, N.H.), DNA Extraction Kit (Stratagene, Lajolla, Calif.), TurboGen Isolation Kit (Invitrogen, San Diego, Calif.), and the like. Use of these kits according to the manufacturer's instructions is generally acceptable for purification of DNA prior to practicing the methods of the present invention.
The concentration and purity of the extracted DNA can be determined by spectrophotometric analysis of the absorbance of a diluted aliquot at 260 nm and 280 nm. After extraction of the DNA, PCR amplification may proceed. The first step of each cycle of the PCR involves the separation of the nucleic acid duplex formed by the primer extension. Once the strands are separated, the next step in PCR involves hybridizing the separated strands with primers that flank the target sequence. The primers are then extended to form complementary copies of the target strands. For successful PCR amplification, the primers are designed so that the position at which each primer hybridizes along a duplex sequence is such that an extension product synthesized from one primer, when separated from the template (complement), serves as a template for the extension of the other primer. The cycle of denaturation, hybridization, and extension is repeated as many times as necessary to obtain the desired amount of amplified nucleic acid.
In a particularly useful embodiment of PCR amplification, strand separation is achieved by heating the reaction to a sufficiently high temperature for a sufficient time to cause the denaturation of the duplex but not to cause an irreversible denaturation of the polymerase (see U.S. Pat. No. 4,965,188, incorporated herein by reference). Typical heat denaturation involves temperatures ranging from about 80° C. to 105° C. for times ranging from seconds to minutes. Strand separation, however, can be accomplished by any suitable denaturing method including physical, chemical, or enzymatic means. Strand separation may be induced by a helicase, for example, or an enzyme capable of exhibiting helicase activity. For example, the enzyme RecA has helicase activity in the presence of ATP. The reaction conditions suitable for strand separation by helicases are known in the art (see Kuhn Hoffman-Berling, 1978, CSH-Quantitative Biology, 43:63–67; and Radding, 1982, Ann. Rev. Genetics 16:405–436, each of which is incorporated herein by reference).
Template-dependent extension of primers in PCR is catalyzed by a polymerizing agent in the presence of adequate amounts of four deoxyribonucleotide triphosphates (typically dATP, dGTP, dCTP, and dTTP) in a reaction medium comprised of the appropriate salts, metal cations, and pH buffering systems. Suitable polymerizing agents are enzymes known to catalyze template-dependent DNA synthesis. In some cases, the target regions may encode at least a portion of a protein expressed by the cell. In this instance, MRNA may be used for amplification of the target region. Alternatively, PCR can be used to generate a cDNA library from RNA for further amplification, the initial template for primer extension is RNA. Polymerizing agents suitable for synthesizing a complementary, copy-DNA (cDNA) sequence from the RNA template are reverse transcriptase (RT), such as avian myeloblastosis virus RT, Moloney murine leukemia virus RT, or Thermus thermophilus (Tth) DNA polymerase, a thermostable DNA polymerase with reverse transcriptase activity marketed by Perkin Elmer Cetus, Inc. Typically, the genomic RNA template is heat degraded during the first denaturation step after the initial reverse transcription step leaving only DNA template. Suitable polymerases for use with a DNA template include, for example, E. coli DNA polymerase I or its Klenow fragment, T4 DNA polymerase, Tth polymerase, and Taq polymerase, a heat-stable DNA polymerase isolated from Thermus aquaticus and commercially available from Perkin Elmer Cetus, Inc. The latter enzyme is widely used in the amplification and sequencing of nucleic acids. The reaction conditions for using Taq polymerase are known in the art and are described in Gelfand, 1989, PCR Technology, supra.
Allele Specific PCR
Allele-specific PCR differentiates between target regions differing in the presence of absence of a variation or polymorphism. PCR amplification primers are chosen which bind only to certain alleles of the target sequence. This method is described by Gibbs, Nucleic Acid Res. 17:12427–2448 (1989).
Allele Specific Oligonucleotide Screening Methods
Further diagnostic screening methods employ the allele-specific oligonucleotide (ASO) screening methods, as described by Saiki et al., Nature 324:163–166 (1986). Oligonucleotides with one or more base pair mismatches are generated for any particular allele. ASO screening methods detect mismatches between variant target genomic or PCR amplified DNA and non-mutant oligonucleotides, showing decreased binding of the oligonucleotide relative to a mutant oligonucleotide. Oligonucleotide probes can be designed so that under low stringency, they will bind to both polymorphic forms of the allele, but at high stringency, bind to the allele to which they correspond. Alternatively, stringency conditions can be devised in which an essentially binary response is obtained, i.e., an ASO corresponding to a variant form of the target gene will hybridize to that allele, and not to the wild-type allele.
Ligase Mediated Allele Detection Method
Target regions of a test subject's DNA can be compared with target regions in unaffected and affected family members by ligase-mediated allele detection. See Landegren et al., Science 241:107–1080 (1988). Ligase may also be used to detect point mutations in the ligation amplification reaction described in Wu et al., Genomics 4:560–569 (1989). The ligation amplification reaction (LAR) utilizes amplification of specific DNA sequence using sequential rounds of template dependent ligation as described in Wu, supra, and Barany, Proc. Nat. Acad. Sci. 88:189–193 (1990).
Denaturing Gradient Gel Electrophoresis
Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of DNA in solution. DNA molecules melt in segments, termed melting domains, under conditions of increased temperature or denaturation. Each melting domain melts cooperatively at a distinct, base-specific melting temperature (Tm). Melting domains are at least 20 base pairs in length, and may be up to several hundred base pairs in length.
Differentiation between alleles based on sequence specific melting domain differences can be assessed using polyacrylamide gel electrophoresis, as described in Chapter 7 of Erlich, ed., PCR Technology, “Principles and Applications for DNA Amplification”, W. H. Freeman and Co., New York (1992), the contents of which are hereby incorporated by reference.
Generally, a target region to be analyzed by denaturing gradient gel electrophoresis is amplified using PCR primers flanking the target region. The amplified PCR product is applied to a polyacrylamide gel with a linear denaturing gradient as described in Myers et al., Meth. Enzymol. 155:501–527 (1986), and Myers et al., in Genomic Analysis, A Practical Approach, K. Davies Ed. IRL Press Limited, Oxford, pp. 95–139 (1988), the contents of which are hereby incorporated by reference. The electrophoresis system is maintained at a temperature slightly below the Tm of the melting domains of the target sequences.
In an alternative method of denaturing gradient gel electrophoresis, the target sequences may be initially attached to a stretch of GC nucleotides, termed a GC clamp, as described in Chapter 7 of Erlich, supra. Preferably, at least 80% of the nucleotides in the GC clamp are either guanine or cytosine. Preferably, the GC clamp is at least 30 bases long. This method is particularly suited to target sequences with high Tm's.
Generally, the target region is amplified by the polymerase chain reaction as described above. One of the oligonucleotide PCR primers carries at its 5′ end, the GC clamp region, at least 30 bases of the GC rich sequence, which is incorporated into the 5′ end of the target region during amplification. The resulting amplified target region is run on an electrophoresis gel under denaturing gradient conditions as described above. DNA fragments differing by a single base change will migrate through the gel to different positions, which may be visualized by ethidium bromide staining.
Temperature Gradient Gel Electrophoresis
Temperature gradient gel electrophoresis (TGGE) is based on the same underlying principles as denaturing gradient gel electrophoresis, except the denaturing gradient is produced by differences in temperature instead of differences in the concentration of a chemical denaturant. Standard TGGE utilizes an electrophoresis apparatus with a temperature gradient running along the electrophoresis path. As samples migrate through a gel with a uniform concentration of a chemical denaturant, they encounter increasing temperatures. An alternative method of TGGE, temporal temperature gradient gel electrophoresis (TTGE or tTGGE) uses a steadily increasing temperature of the entire electrophoresis gel to achieve the same result. As the samples migrate through the gel the temperature of the entire gel increases, leading the samples to encounter increasing temperature as they migrate through the gel. Preparation of samples, including PCR amplification with incorporation of a GC clamp, and visualization of products are the same as for denaturing gradient gel electrophoresis.
Single-Strand Conformation Polymorphism Analysis
Target sequences or alleles at the chosen boar taint loci can be differentiated using single-strand conformation polymorphism analysis, which identifies base differences by alteration in electrophoretic migration of single-stranded PCR products, as described in Orita et al., Proc. Nat. Acad. Sci. 85:2766–2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single-stranded amplification products. Single-stranded nucleic acids may refold or form secondary structures which are partially dependent on the base sequence. Thus, electrophoretic mobility of single-stranded amplification products can detect base-sequence difference between alleles or target sequences.
Chemical or Enzymatic Cleavage of Mismatches
Differences between target sequences can also be detected by differential chemical cleavage of mismatched base pairs, as described in Grompe et al., Am. J. Hum. Genet. 48:212–222 (1991). In another method, differences between target sequences can be detected by enzymatic cleavage of mismatched base pairs, as described in Nelson et al., Nature Genetics 4:11–18 (1993). Briefly, genetic material from an animal and an affected family member may be used to generate mismatch free heterohybrid DNA duplexes. As used herein, “heterohybrid” means a DNA duplex strand comprising one strand of DNA from one animal, and a second DNA strand from another animal, usually an animal differing in the phenotype for the trait of interest. Positive selection for heterohybrids free of mismatches allows determination of small insertions, deletions or other polymorphisms that may be associated with polymorphisms.
Non-Gel Systems
Other possible techniques include non-gel systems such as TAQMANTM (Perkin Elmer). In this system, oligonucleotide PCR primers are designed that flank the mutation in question and allow PCR amplification of the region. A third oligonucleotide probe is then designed to hybridize to the region containing the base subject to change between different alleles of the gene. This probe is labeled with fluorescent dyes at both the 5′ and 3′ ends. These dyes are chosen such that while in this proximity to each other the fluorescence of one of them is quenched by the other and cannot be detected. Extension by Taq DNA polymerase from the PCR primer positioned 5′ on the template relative to the probe leads to the cleavage of the dye attached to the 5′ end of the annealed probe through the 5′ nuclease activity of the Taq DNA polymerase. This removes the quenching effect allowing detection of the fluorescence from the dye at the 3′ end of the probe. The discrimination between different DNA sequences arises through the fact that if the hybridization of the probe to the template molecule is not complete, i.e., there is a mismatch of some form, the cleavage of the dye does not take place. Thus, only if the nucleotide sequence of the oligonucleotide probe is completely complimentary to the template molecule to which it is bound will quenching be removed. A reaction mix can contain two different probe sequences each designed against different alleles that might be present thus allowing the detection of both alleles in one reaction.
Yet another technique includes an Invader Assay, which includes isothermic amplification that relies on a catalytic release of fluorescence. See Third Wave Technology at www.twt.com.
Non-PCR Based DNA Diagnostics
The identification of a DNA sequence linked to sequences encoding enzymes involved in skatole metabolism can be made without an amplification step, based on polymorphisms including restriction fragment length polymorphisms in an animal and a family member. Hybridization probes are generally oligonucleotides which bind through complementary base pairing to all or part of a target nucleic acid. Probes typically bind target sequences lacking complete complementarity with the probe sequence depending on the stringency of the hybridization conditions. The probes are preferably labeled directly or indirectly, such that by assaying for the presence or absence of the probe, one can detect the presence or absence of the target sequence. Direct labeling methods include radioisotope labeling, such as with p32 or S35. Indirect labeling methods include fluorescent tags, biotin complexes which may be bound to avidin or streptavidin, or peptide or protein tags. Visual detection methods include photoluminescents, Texas red, rhodamine and its derivatives, red leuco dye and 3,3′,5,5′-tetramethylbenzidine (TMB), fluorescein, and its derivatives, dansyl, umbelliferone and the like or with horse radish peroxidase, alkaline phosphatase and the like.
Hybridization probes include any nucleotide sequence capable of hybridizing to the porcine chromosome where the CYP2A6 gene or other gene involved in skatole metabolism resides, and thus defining a genetic marker linked to the gene, including a restriction fragment length polymorphism, a hypervariable region, repetitive element, or a variable number tandem repeat. Hybridization probes can be any gene or a suitable analog. Further suitable hybridization probes include exon fragments or portions of cDNAs or genes known to map to the relevant region of the chromosome.
Preferred tandem repeat hybridization probes for use according to the present invention are those that recognize a small number of fragments at a specific locus at high stringency hybridization conditions, or that recognize a larger number of fragments at that locus when the stringency conditions are lowered.
One or more additional restriction enzymes and/or probes and/or primers can be used. Additional enzymes, constructed probes, and primers can be determined by routine experimentation by those of ordinary skill in the art and are intended to be within the scope of the invention.
According to the invention, polymorphisms in genes encoding enzymes involved in skatole metabolism have been identified which have an association with boar taint. The presence or absence of the markers, in one embodiment may be assayed by PCR-RFLP analysis using the restriction endonucleases and amplification primers may be designed using analogous human, pig or other sequences due to the high homology in the region surrounding the polymorphisms, or may be designed using known gene sequence data as exemplified in GenBank or even designed from sequences obtained from linkage data from closely surrounding genes based upon the teachings and references herein. The sequences surrounding the polymorphism will facilitate the development of alternate PCR tests in which a primer of about 4–30 contiguous bases taken from the sequence immediately adjacent to the polymorphism is used in connection with a polymerase chain reaction to greatly amplify the region before treatment with the desired restriction enzyme. The primers need not be the exact complement; substantially equivalent sequences are acceptable. The design of primers for amplification by PCR is known to those of skill in the art and is discussed in detail in Ausubel (ed.), Short Protocols in Molecular Biology, 4th Edition, John Wiley and Sons (1999).
The following is a brief description of primer design.
Primer Design Strategy
Increased use of polymerase chain reaction (PCR) methods has stimulated the development of many programs to aid in the design or selection of oligonucleotides used as primers for PCR. Four examples of such programs that are freely available via the Internet are: PRIMER by Mark Daly and Steve Lincoln of the Whitehead Institute (UNIX, VMS, DOS, and Macintosh), Oligonucleotide Selection Program (OSP) by Phil Green and LaDeana Hiller of Washington University in St. Louis (UNIX, VMS, DOS, and Macintosh), PGEN by Yoshi (DOS only), and Amplify by Bill Engels of the University of Wisconsin (Macintosh only). Generally these programs help in the design of PCR primers by searching for bits of known repeated-sequence elements and then optimizing the Tm by analyzing the length and GC content of a putative primer. Commercial software is also available and primer selection procedures are rapidly being included in most general sequence analysis packages.
Sequencing and PCR Primers
Designing oligonucleotides for use as either sequencing or PCR primers requires selection of an appropriate sequence that specifically recognizes the target, and then testing the sequence to eliminate the possibility that the oligonucleotide will have a stable secondary structure. Inverted repeats in the sequence can be identified using a repeat-identification or RNA-folding program such as those described above. If a possible stem structure is observed, the sequence of the primer can be shifted a few nucleotides in either direction to minimize the predicted secondary structure. The sequence of the oligonucleotide should also be compared with the sequences of both strands of the appropriate vector and insert DNA. Obviously, a sequencing primer should only have a single match to the target DNA. It is also advisable to exclude primers that have only a single mismatch with an undesired target DNA sequence. For PCR primers used to amplify genomic DNA, the primer sequence should be compared to the sequences in the GenBank database to determine if any significant matches occur. If the oligonucleotide sequence is present in any known DNA sequence or, more importantly, in any known repetitive elements, the primer sequence should be changed.
The methods and materials of the invention may also be used more generally to evaluate pig DNA, genetically type individual pigs, and detect genetic differences in pigs. In particular, a sample of pig genomic DNA may be evaluated by reference to one or more controls to determine if a polymorphism in the particular gene is present. Preferably, RFLP analysis is performed with respect to the pig gene, and the results are compared with a control. The control is the result of a RFLP analysis of the pig gene of a different pig where the polymorphism(s) of the pig gene is/are known. Similarly, the genotype of a pig may be determined by obtaining a sample of its genomic DNA, conducting RFLP analysis of the gene in the DNA, and comparing the results with a control. Again, the control is the result of RFLP analysis of the gene of a different pig. The results genetically type the pig by specifying the polymorphism(s) in its genes. Finally, genetic differences among pigs can be detected by obtaining samples of the genomic DNA from at least two pigs, identifying the presence or absence of a polymorphism in the gene, and comparing the results.
These assays are useful for identifying the genetic markers relating to boar taint, as discussed above, for identifying other polymorphisms in the genes encoding enzymes involved in skatole metabolism and for the general scientific analysis of pig genotypes and phenotypes.
The examples and methods herein disclose certain gene(s) which has been identified to have a polymorphism(s) which is associated either positively or negatively with a beneficial trait that will have an effect on meat quality, heavy muscling, and/or skeletal muscle cramping disease for animals carrying this polymorphism. The identification of the existence of a polymorphism within a gene is often made by a single base alternative that results in a restriction site in certain allelic forms. A certain allele, however, as demonstrated and discussed herein, may have a number of base changes associated with it that could be assayed for which are indicative of the same polymorphism (allele). Further, other genetic markers or genes may be linked to the polymorphisms disclosed herein so that assays may involve identification of other genes or gene fragments, but which ultimately rely upon genetic characterization of animals for the same polymorphism. Any assay which sorts and identifies animals based upon the allelic differences disclosed herein are intended to be included within the scope of this invention. One of skill in the art, once a polymorphism has been identified and a correlation to a particular trait established will understand that there are many ways to genotype animals for this polymorphism. The design of such alternative tests merely represents optimization of parameters known to those of skill in the art and is intended to be within the scope of this invention as fully described herein.
Example 5
Cloning, Expression and Functional Characterization of Cytochrome P450 2A6 Gene from Pig Liver
Entire male pigs are used for meat production in pig industry, due to a better feed conversion, improved carcass leanness and addressed animal welfare. Therefore, raising male pigs may improve the profitability of pork production by up to 30% (Babol et al. 1995). However, the frequent occurrence of off-odors in cooked pork from uncastrated male pigs, commonly known as “boar taint”, is highly objectionable to consumers. Skatole is one of the major contributors to boar taint (Gonzalo et al. 2000). Skatole is absorbed from the gut and then metabolized primarily in the liver. In pigs, cytochrome P450 enzymes have been found to have significant impact on metabolism of skatole. It has been shown that CYP2A6 is one of major key enzymes in the metabolism of skatole (Gonzalo el al. 2000). In pigs, CYP2A6 has been found to be highly and negatively correlated with skatole accumulation in fat (Babol et al. 1998; Gonzalo et al. 2000). Therefore CYP2A6 plays an important role in the metabolism and clearance of skatole from the body in pigs.
Cytochrome P450 is a superfamily of hemoprotein (Ingelman-Sundberg et al 1999). In human, CYP2A6 is predominantly expressed in the liver (Koskela et al. 1999; Oscarson, 2001). It is a major hepatic member of the family, which metabolizes pharmaceutical (Miles et al. 1990) and many other drugs and environment chemicals (Yamazaki et al 1992). In human, CYP2A6 was first identified as the coumarin-7 hydroxylase (Yamano et al., 1990), and has received a lot of attention since then, due to its principle role in nicotine C-oxidation and possible involvement in smoking behavior and lung cancer susceptibility (Xu et al., 2002; Oscarson, 2001). The knowledge concerning CYP2A6 in human has substantially increased. However, the information about the CYP2A6 gene, its expression and how a genetic variant of CYP2A6 affect skatole level in pigs is remains empty.
In present study, we constructed the cDNA library from pig liver by rapid amplification of cDNA ends (RACE) method and reported the sequence of porcine CYP2A6 cDNA. We examined the expression pattern of the CYP2A6 mRNA species in different tissues in pigs by Northern analysis. Polymerase chain reaction technique combined with single strand conformational polymorphism (PCR-SSCP) was used to scan and identify any genetic polymorphism of CYP2A6 coding region from porcine liver tissues, which may alter the metabolic capacities of the enzyme. Furthermore, functional studies with this genetic polymorphism of CYP2A6 were carried out.
Tissue Samples
Liver tissues were obtained from a male pig for construction of cDNA library. To identify any genetic polymorphism in CYP2A6, sixty-nine pigs from a variety of breeds, including Yorkshire, Duroc, Landrace, and Pietrain, as well as crosses between Landrace and Duroc, Large White and Duroc, and Large White and Pertain, were slaughtered at an average live weight of 144 kg (144 kg±33) at the Department of Animal and Poultry Science abattoir. A sample of liver was taken immediately following exsanguinations, frozen in liquid nitrogen and stored at −70° C. for until use.
Isolation of Total RNA
One hundred milligram of tissue samples were homogenized in 1 ml of Tri-Reagent (Sigma) and incubated for 10 min at room temperature. After incubation, 0.2 ml of chloroform was added and vortexed. The samples were centrifuged at 12,000×g for 10 min at 4° C. and then aqueous phase was transferred in to a sterile tube. The aqueous phase was mixed with 0.5 ml of isopropanol and incubated at room temperature for 10 min to precipitate the RNA. Pellet was obtained by centrifugation (12,000×g for 10 min at 4° C.). The pellet was washed with 75% ethanol and then suspended into 50 μl of DEPC water.
Construction and Screening of a Pig cDNA RACE Library
5′ and 3′ rapid amplification of cDNAs (RACE) were constructed from 1 μg of total RNA from liver separately by use of Smart RACE cDNA Amplification kit (Clontech), and used as templates in the subsequent PCR screening of porcine CYP2A6. The 5′RACE was performed by synthesizing the first strand cDNA with a modified lock-docking oligo (dT) primer and then tailing the product 5′AAG CAG TGG TAT CAA CGC AGA GTA CGC GGG 3′(SEQ ID NO:9) (anchor primer) in 5′ end via terminal transferase. The 3′RACE was performed with oligo (dT) primer but includes the same lock-docking nucleotide positions as in 5′RACE. The first fragment of CYP2A6 was amplified with the primers designed from the conserved region of human 2A6, mouse 2A5, and rat 2A3 cDNA sequence. The forward primer is 5′AGG ACA AAG AGT TCC TGT CAC TG 3′, (SEQ ID NO:10) reverse primer is 5′CAA TCT CCT CAT GGA CCT TGG 3′(SEQ ID NO: 11). To obtain full-length porcine CYP2A6, following primers were used in the subsequent PCR-based screening: 5′ATG AGC AGC AGG AAG CCG TAG 3′(SEQ ID NO:12) and anchor primer with 5′Race as a template; 5′CTA CGG CTT CCT GCT GCT CAT 3′(SEQ ID NO:13) and anchor primer with 3′Race as a template; 5′CAC AAC GAT GCG CTA CGG CT 3′(SEQ ID NO:14) and 5′GCAGGAAGCTCATGGTGTAG 3′(SEQ ID NO:15) with either 3′ or 5′Race as a template. The PCR consisted of 35 cycles of denaturing for 1 minute at 94° C., optimal annealing for 1 minute, and extending for 1 minute, with a final 10 minutes extension step at 72° C. 10 μl of the PCR products were analyzed by electrophoresis on a 1% agarose gel.
Colony Hybridization
When there were multiple bands to be amplified from both 3′ and 5′Race templates, the PCR products were cloned into pGEM-T Easy Vector System (Promega), and then subjected to colonies hybridization to confirm the specificity of amplified fragment prior to DNA sequencing. Colonies were lifted up to positively charged nylon membrane (Roche), then subjected to lysis and fixation in 0.5M NaCl for 5 minutes, rinsing in 5×SSC for 1 minutes, and air dry; Colonies hybridization was performed with ECL nucleotide DNA labeling and detection kit (Amersham Life Science). The probe used in the hybridization was the fragment first amplified by the primers designed from the human 2A6, mouse 2A5, and rat 2A3 cDNA conserved region. After hybridization overnight at 42° C., the membrane was washed with 0.15×SSC for 20 minutes twice and exposed to x-ray film (Kodak). The colony that gives the strongest signal is subjected to be sequencing.
Isolation of Full-Length Porcine CYP2A6 cDNA
To obtain full-length porcine CYP2A6 sequence, forward primer 5′CTC GCA GTG CCA CCA TGC TG 3′(SEQ ID NO:16) and reverse primer 5′GCA GGA AGC TCA TGG TGT AGG TC (SEQ ID NO: 17) 3′ were designed based on the sequence obtained from the 5′ and 3′RACE, and used to amplify the full-length porcine CYP 2A6 either with 5′ or 3′RACE cDNA as a template. PCR profile was 3 min at 94° C., followed by 35 cycles of 1 min at 94° C., 1 min 30 sec at 64° C., 2 min at 72° C. and final extension of 10 min at 72° C. and two drops of mineral oil were added. The PCR fragment was cloned into T-Easy vector (Promega) and subjected to sequence analysis.
Northern Blot Analysis
Total RNAs were isolated from porcine spleen, thymus, liver, lung, muscle, ovary, kidney, small intestine, heart, and testis tissues with Tri-Reagent (Sigma). 20 μg of total RNA from each tissue was subjected to electrophoresis in the 2.0M formaldehyde-containing 1% agarose gel and transferred to nylon membrane (Amersham Pharmacia Biotech) with downward capillary. Full-length of the porcine CYP2A6 (1498 bp) was created from forward primer 5′CTC GCA GTG CCA CCA TGC TG 3′(SEQ ID NO:16) and reverse primer 5′GCA GGA AGC TCA TGG TGT AGG TC 3′(SEQ ID NO:17) from pig liver cDNA library we created. CYP2A6 eDNA was labeled using random primers with digoxigenin-dUTP (Roche Molecular Biochemicals) and hybridized at 50° C. overnight. After prewashing with 2×SSC containing 0.1% SDS, the membrane was washed with 0.2×SSC containing 0.1% SDS for 15 minutes twice at 67°. The hybridized probes are immunodetected with anti-digoxigenin-alkaline phosphatase conjugate, detected with the colorimetric substrates (DIG, Roche), and exposed to Kodak Scientific Imaging film (Kodak) for 1 hour at room temperature.
Sequencing Analysis
The PCR fragments were ligated into pGEM-T Easy Vector System (Promega), and then transformed into competent DH5α cells. DNAs were purified and subject to sequencing using an Applied Biosystems model ABI 377 DNA sequencer.
RT-PCR
To scan any genetic polymorphism in the CYP2A6 from individuals, RT-PCR products that cover its whole coding region were amplified and then subjected to SSCP analysis. First strand cDNA was synthesized from 1 to 5 μg of total RNA from liver samples using SuperScript reverse transcriptase (Invitrogen) and oligo (dT) primer (Sigma). Following the reverse transcription, 2.5 μl of the first strand cDNA was used as the template for PCR. The PCR mixtures (50 ul) contained 1×PCR buffer (100 mM Tris-HCl, pH 8.3; 500 mM KCl, 11 mM MgCl2, 0.1% gelatin), 0.2 mM dNTP, 0.4 mM primers (forward and reverse primer) and 2.5 U of Red Taq polymerase (Sigma). The primer pair (forward primer, 5′CTC GCA GTG CCA CCA TGC TG 3′, (SEQ ID NO:16) reverse sequence, 5′GCA GGA AGC TCA TGG TGT AGG TC 3′) (SEQ ID NO:17) was designed to amplify the entire coding region of porcine CYP2A6, based on our isolated CYP2A6 (GenBank accession number AY091516). The PCR profile was 3 min at 94° C., followed by 35 cycles of 1 min at 94° C., 1 min at 65° C., 1 min at 72° C. and final extension of 10 min at 72° C.
Single-Strand Conformational Polymorphism Analysis
PCR products were first cut into fragments with BstxI enzyme, and then resolved by SSCP analysis. 5 μl of PCR product amplified was digested with BstxI in 20 μl reaction at 37° C. for 3 hours. A total of 7 μl of digested fragments were then diluted with 13 μl of loading buffer (10% of Sucrose, 0.01% of Bromophenol blue and 0.01% of Xylene cyanol FF). Each digestion reaction was denatured at 100° C. for 5 min, chilled on ice and resolved on 10% of polyacrylamide gel. The electrophoresis was carried in a vertical unit (Bio-Rad Laboratories, 130×160×1.0 mm), in 0.6×TBE buffer for 17 hours at 15° C. at 160 V. The gels were then silver stained.
CYP2A6 Activity
CYP2A6 activity is assayed by measurement of coumarin 7-hydroxylase activity on pig liver microsomal samples. 20 μl of microsomal suspension containing 0.4 mg microsomal protein were mixed with 200 μl of coumarin hydroxylase reaction mix (0.05M Tris buffer pH 7.4, 5 mM MgCl2 and 0.2 mM coumarin). The reaction was started by adding 15 μl of 25 mM NADPH. After incubation at 37° C. for 15 minutes, the reaction was stopped by the addition of 50 μl of 20% trichloroacetic acid, followed by cenrifugation at 10,000 g for 2 min. Two hundred microliters of the supernatant fraction was diluted with 2 ml of 0.1 M Tris buffer (pH 9.0), and the fluorescence was determined at wavelengths of 390 nm for excitation at and 440 nm for emission.
Measurement of Skatole Level in Fat
A backfat sample was collected at the midline point of 11th rib and frozen at −20° C. until assayed for skatole. The skatole content was measured with a colorimetric assay, according to the method described by Gonzalo et al. (2000).
Western Analysis
Liver tissue (1 g) was homogenized in 5 ml of sample buffer (1% cholic acid, 0.1% SDS in PBS buffer) and the protein concentrations of homogenates were determined using the BCA kit (Pierce). 40 μg of total protein were subjected to sodium dodecyl sulphate gel electrophoresis using a 12% polyacrylamide gel. The protein was transferred to a nitro-cellulose filter (BioRad), incubated with mouse anti-human monoclonal 2A6-antibody MAB-2A6 (Gentest), and subsequently anti-mouse IGG peroxidase conjugate developed in goat (Sigma). Immunoreactive bands were stained by a chemiluminescence procedure (ECL, Amersham Life Science) and visualized by autoradiography.
The CYP2A6 cDNA Sequence and Sequence Characterization
Pig CYP2A6 cDNA was isolated by PCR screening of the liver cDNA library constructed with RACE. The nucleotide sequence of the CYP2A6 cDNA was 1519 bp long and contained a 1485 bp-long open reading frame (ORF), which encodes 497 amino acids (FIG. 12). Pig CYP2A6 cDNA sequence was submitted to Genbank database under the accession number AY091516.
The human CYP2A6, mouse CYP2A5, rat CYP2A3 were identified as the coumarin-7 hydroxylase. We compared pig CYP2A6 ORF to above genes, it showed 87% homology to human CYP2A6, 85% to mouse CYP2A5, and 86% to rat 2A3. The deduced amino acid sequence for pig CYP2A6 showed 87% homology to human 2A6, 90% to mouse 2A5, and 89% to rat 2A3 (FIG. 13). In human CYP2A6, Gln104, Phe209 and His477 were reported to be active sites for CYP2A6 coumarin 7-hydroxylase activity, oxidative metabolism of nicotine and cotinine (Lewis et al. 1999). R128 was represents one of key binding residues for human CYP2A6 (Kiragawa et al, 2001; Lewis et al, 1999). All above active sites are conserved in the putative pig CYP2A6.
Expression of CYP2A6 mRNA Species in Various Tissues
The expression patterns of CYP2A6 mRNA in various tissues, including spleen, thymus, liver, lung, muscle, ovary, kidney, small intestine heart and testis from pigs, were investigated by Northern blotting by using pig CYP2A6 cDNA as a probe. The result showed that CYP2A6 are only expressed in liver and kidney tissue (FIG. 14). A much higher level of CYP2A6 mRNA was observed in the liver, and a lower level of CYP2A6 mRNA was expressed in the kidney. The result showed the CYP2A6 is predominantly expressed in pig liver tissue. It indicated the liver is the major tissue that plays important role in CYP2A6 metabolism in pigs.
CYP2A6 Genetic Polymorphism
In order to identify any genetic polymorphism of CYP2A6, which may alter the metabolic capacities of the enzyme, polymerase chain reaction technique combined with single strand conformational polymorphism (PCR-SSCP) was used to scan CYP2A6 coding region from porcine liver tissues. In pig, CYP2A6 full-length cDNA was amplified by PCR with primer pair: forward primer 5′CTC GCA GTG CCA CCA TGC TG 3′(SEQ ID NO:16) and reverse primer 5′GCA GGA AGC TCA TGG TGT AGG TC 3′(SEQ ID NO:17) from liver tissues. The resulting PCR products were about 1500 bp in size. Digested PCR products with BstxI were subjected to SSCP analysis using our optimized system. We found that there are several different polymorphisms existing in CYP2A6 coding region (data not shown). Of which, one of deletion that resulted in coding region frame shifting received our most attention. Due to one G missing, the length of ORF region of CYP2A6 changes from 1485 bp to 612 bp. This also causes the length of its encoded gene product change from 495 amino acid to 204 amino acid. It is suggested that the deletion might also result in inactivation of CYP2A6 activity for the individual that contains such deletion. It has been shown that CYP2A6 is one of major key enzymes in the metabolism of skatole (Gonzalo et al. 2000). CYP2A6 is negatively correlated with skatole accumulation in fat (Babol et al. 1998). Therefore, we infer that CYP2A6 activity for the sample that exists such deletion would be zero for its comarin 7-hydroxylase activity due to coding region frame shifting of CYP2A6 gene, and that skatole level should be higher due to losing this enzyme activity to clear skatole from the body.
To evaluate above hypothesis and investigate the association of this genetic polymorphism of CYP2A6 with skatole level, the phenotyping using skatole level measurement, coumarin 7-hydroxylase activity assay and immunoblotted with monoclonal anti-human CYP2A6 anti-body (Gentest) for the samples showed different genotype, were further carried out. The results showed that the skatole level is much higher for the sample with deleted mutation than that in wild type samples. Coumarin 7-hydroxylase assay and immunoblotting analysis also told us zero for coumarin 7-hydroxylase activity and negative immunoreactive band for the sample that has deleted mutation, while remaining lower skatole level, higher activity and detectable immunoreactive bands for wild type samples (FIG. 15). The results are strongly supporting our suggestion that the CYP2A6 deletion caused a complete lack of enzymatic activity, and hence caused higher level skatole level in pig.
In human, CPY2A6 gene has been extensively studied; however, the information about the CYP2A6 gene, its expression and how a genetic variant of CYP2A6 affect skatole level in pigs is remains empty. In this study, we reported the molecular cloning, functional characterization of CYP2A6 gene in pig. We designed the primers based on conserved region of human 2A6, mouse 2A5 and rat 2A3 cDNA sequence. Coumarin 7-hydroxylation is catalysed by a high-affinity CYP2A6 and CYP2A5 enzyme in human and mouse (Miles et al., 1990; Donato et al., 2000), and that by CYP2A3 in rat. The formation of 7-hydroxycoumarin has been used as an in vivo and invitro probe for CYP2A6 in human, CYP2A5 in mouse, and 2A3 in rat (Rodrigues et al., 1994; Rautio et al., 1992; Fernandez-Salguero et al, 1995). Therefore, by using the designed primers, we screened out the first fragment, subsequently the whole sequence of pig CYP2A6 cDNA.
The CYP2A6 in human, CYP2A5 in mouse, and CYP2A3 was sequenced (Access number: U22027 for human, BC046605 for mouse, and M33190 for rat), and has been mapped to chromosome 19q13.2 (b: Fernandez-Salguero et al., 1995) chromosome 7 (Kent et al.,1987) and chromosome 1 (STS: D1Mgh28), respectively. As indicated in the results, when comparing pig CYP 2A6 sequence to its orthologous genes, sequence of human CYP2A6, mouse 2A5 and rat CYP2A3, we found that it has high homology to its orthologs both in cDNA sequence and amino acid sequence. And all the important active sites of amino acid sequence in human CYP2A6 are also conserved in our putative pig CYP2A6 sequence. Furthermore, we searched against human, mouse and rat genomic database with pig CYP2A6 cDNA sequence, we found that pig CYP2A6 only hit a human genomic clone (NT—011109) from chromosome 19q13.2, mouse genomic clone (NT—039410) from chromosome 7, and rat genomic clone (NW—043361) from chromosome 1q21, respectively. The hit scores showed that pig CYP2A6 cDNA sequence has highest identity with human CYP2A6 genomic clone (91%) at human chromosome 19q13.2, with mouse 2A5 genomic clone (89%) at mouse chromosome 7, and with rat CYP2A3 genomic clone (88%) at rat chromosome 1q21. All these findings taken together thus led us to conclude that the putative CYP2A6 is indeed pig CYP2A6.
In this study, we performed northern blot analysis for pig CYP2A6 mRNA distribution in different tissues, the results showed that CYP2A6 is expressed predominantly in liver and at a much higher level in liver, lower level in kidney. This indicated liver is the most important tissue for clearance of skatole from body in pig. In spite of high similarities of pig CYP2A6 with its orthologous genes, these enzymes differ in tissue distribution. It has been reported that mRNA expression is observed mainly in liver for human 2A6 (Koskela et al. 1999; Oscarson, 2001), in liver, kidney and small intestine for mouse 2A5 (Su et al., 1998), and in olfactory mucosa and lung for rat 2A3 (Su, et al., 1996; Kimura et al., 1989). In our study, we found that CYP2A6 is not expressed in small intestine and lung in pig. The difference of expression for CYP2A6 mRNA and its orthologous genes in various tissues suggest there might be difference in their promoter region, this difference may be useful for study regulation of tissue-specific gene expression.
In human, CYP2A6 has been one of most important enzyme in nicotine C-oxisation, due to the important of CYP2A6 in nicotine metabolism, and possible involvement in smoking behavior and lung cancer susceptibility (Xu et al., 2002; Oscarson, 2001). Polymorphism in the human CYP2A6 gene may thus impact on both smoking behavior and lung cancer susceptibility. Therefore, substantial efforts have been focused on detecting genetic polymorphism and its consequences (Paschke, et al., 2001; Kamataki, et al., 1999; Oscarson, et al., 2001; Kitagawa, et al., 2001). In human, large interindividual differences has been seen in the levels of CYP2A6 enzyme, due to the genetic variants mainly located in the open reading frame (Nakajima et al., 2002). A number of genetic polymorphisms have been detected for the CYP2A6 in human, including SNPs in the coding region that lead to inactivation, such as Gly479Val (Oscarson et al., 2001) and Arg128Gln (Kitagawa et al., 2001). The progress in such researches will facilitate molecular study to clarify how critical the CYP2A6 polymorphism in causing genetic difference and its subsequent consequence.
The role of cytochrome P450 enzyme including CYP2A6 in the metabolism of skatole has been investigated in human, mouse, and rabbit (Thornton-Manning et al., 1996). In pigs, It has been reported that CYP2A6 is one of key enzymes in the hepatic metabolism of skatole (Gonzalo et al. 2000) and CYP2A6 is negatively correlated with skatole accumulation in fat (Babol et al. 1998). Therefore, pigs with high levels of these enzyme incuding CYP2A6 have low levels of skatole in the fat, since skatole is rapidly metabolised and celared from the body, pigs with low levels of these enzyme can have high levels of skatole in the fat. Therefore, CYP2A6 could be use as an genetic marker to select against skatole, once CYP2A6 genetic variant and its consequence on skatole has been investigated. Because there is no information on CYP2A6 gene, we first isolated pig CYP2A6 from liver tissue using RACE method, then performed PCR-SSCP analysis to scan pig CYP2A6 coding region based on our optimized genotyping system. In this study, we focus our efforts on evaluation of CYP2A6 functional region and its genetic polymorphism. We have identified one genetic polymorphism, resulting in a frame shifting in the coding region and inactivation of the enzyme activity. Due to deletion of CYP2A6, coumarin 7-hydroxylation and CYP2A6 gene product are not detectable. It is not known at which age the unregulation of CYP2A6 occurs. In our CYP2A6 phenotyping studies using coumarin, western analysis with mouse anti-human monoclonal 2A6-antibody, and skatole measurement in pig, we also found that there are the existence of additional alleles outside of coding region that modulate or inactive CYP2A6 activity (data not shown). Therefore, it would be helpful to investigate the promoter region of CYP2A6, in combination with phenotype individuals with either coumarin, immunodectected band as indicators of in vivo and invitro CYP2A6 activity in future study, since there may be other CYP2A6 alleles that have not yet known.
In this study, we isolated pig CYP2A6 cDNA from liver and found the CYP2A6 deletion in ORF region, which resulted in a complete lack of the enzymatic activity. There has been no published study that investigates the impact of genetic polymorphism in CYP2A6 on its clearance of skatole from body in pig. The data presented in this study suggest that the CYP2A6 gene deletion might play an important role in the development of genetic marker for skatole.
REFRENCES
Diaz, G. J. and Squires, E. J. (2000). Metabolism of 3-Methylindole by Porcine Liver Microsomes: Responsible Cytochrome P450 Enzyme. Toxicological Science 55, 284–292.
Babol, J., Squires, E. J. and Lundström, K. (1998) Relationship between Oxidation and Conjugation Metabolism of Skatole in Pig Liver and Concentrations of Skatole in Fat. Journal of Animal Science 76, 829–838.
Donato, M. T., Viitala, P., Rodriguez-Antona, C., Lindfors, A., Castell, J. V., Raunio, H., Gómez-Lechón, M. J. and Pelkonen, O. (2000) CYP2A5/CYP2A6 expression in mouse and human hepatocytes treated with various in vivo induces. Drug Metabolism and Disposion 28, 1321–1326
Fernandez-Salguero, P. and Gonzalez, F. J. (1995) The CYP2A gene subfamily: species differences, regulation, catalytic activities and role in chemical carcinogenesis. Pharmacogenetics 5, 123–128
Fernandez-Salguero, P., Hoffman, S. M., Cholerton, S., Mohrenweiser, H., Raunio, H., Rautio, A., Pelkonen, J. D., Humang, W. E., Eeans, J. R. and Idle. (1995) A genetic polymorphism in coumarin 7-hydroxylation: sequence of the human CYP2A genes and identification of variant CYP2A6 alleles. The American Journal of Human Genetics 57, 651–660
Ingelman-Sundberg, M., Oscarson, M. and Mclellan, R. A (1999) Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment. Trends in Pharmacological Science 20, 342–349
Kamataki, T., Nunoya, K. I., Sakai, Y., Kushida, H. and Fujita, K. I. (1999) Genetic polymorphism of CYP2A6 in relation to cancer. Mutation Research 428, 125–130 Kent, R. B., Fallows, D. A., Geissler, E., Glaser, T., Emanuel, J. R., Lalley, P. A., Levenson, R. and Housman, D. E. (1987) Genes encoding alpha and beta subunits of Na, K-ATPase are located on three different chromosomes in the mouse. Proceedings of the National Academy of Science, USA 84, 5369–5373
Kimura, S., Kozak, C. A. and Gonzalez, F. J. (1989) Identification of a novel P450 expression in rat lung: cDNA cloning and sequence, chromosome mapping, and induction by 3-methylcholanthrene. Biochemistry 28, 3798–3803
Kitagawa, K., Kunugita, N., Kitagawa, M. and Kawamoto, T. (2001) CYP2A6*6, a novel polymorphism in cytochrome P450 2A6, has a single amino acid substitution (R128Q) that inactivates enzymes. The Journal of Biological Chemistry 276, 17830–17835
Koskela, S., Hakkola, J., Hukkanen, J., Pelkonen, O., Sorri, M. and Saranen, A. (1999) Expression of CYP2A gene in human liver and extrahepatic tissue. Biochemical Pharmacology 57, 1407–1413
Lewis, D. F. V., Dickins, M., Lake, B. G., Eddershaw, P. J., Tarbit, M. H. and Goldfarb, P. S. (1999) Molecular modeling of the human cytochrome P450 isoform CYP2A6 and investigations of CYP2A substrate selectivity. Toxicology 133, 1–33
Miles, J. S., Mclaren, S. W, Forrester, L. M., Glancey, M. J., Lang, M. A. and Wolf, C. R. (1990) Identification of the human liver cytochrome P-450 responsible for coumarin 7-hydroxylase activity. Biochemistry Journal 267, 365–371
Nakajima, M., Kuroiwa, Y. and Yokoi, T. (2002) Interindividual differences in nicotine metabolism and genetic polymorphism of human 2A6. Drug Metabolism Reviews 34, 865–877
Oscarson, M. (2001) Genetic polymorphisms in cytochrome P450 2A6 (CYP2A6) gene: implications for interindividual differences in nicotine metabolism. Drug Metabolism and Disposition 29, 91–95
Paschke, T., Riefler, M., Schuler-Metz, Annette., Wolz, Lucie., Scherer, Gerhard., Mcbride. M. and Bepler, G. (2001) Comparison of cytochrome P450 2A6 polymorphism frequencies in Caucasians and African-Americans using a new one-step PCR-RFLP geneotyping method. Toxicology 168, 259–268
Rautio, A., Kraul, H., Kojo, A., Salmela, E., Pelkonen, O. (1992) Interindividual variability of coumarin 7-hydroxylation in health individuals. Pharmacogenetics 2, 227–233
Rodrigues, A. D. (1994) Use of in vitro human metabolism studies in drug development: an industrial perspective. Biochemical Pharmacology 48, 2147–2156
Su, T., He, W. L., Lipinskas, T. W. and Ding, X. (1998) Differential xenobiotic induction of CYP2A5 in mouse liver, kidney, lung, and olfactory mucosa. Drug Metabolism and Disposition 26, 822–824
Su, T., Sheng, J. J, Lipinskas, T. W. and Ding, X. (1996) Expression of CYP2A6 genes in rodent and human nasal mucosa. Drug Metabolism and Disposition 24, 884–890
Thornton-Manning, J., Appleton, M. L., Gonzalez, F. J., and Yost, G. S. (1996) Metabolism of 3-methylindole by vaccinia-expressed P450 enzymes: correlation of 3-methyleneindolenine formation and protein-binding. The Journal of Pharmacology and Experimental Therapeutics 276, 21–29
Xu, C., Goodz, S., Sellers, E. M. and Tyndale, R. F. (2002) CYP2A6 genetic variation and potential consequences. Advanced Drug Delivery Review 54, 1245–1256
Yamazaki, H., Inui, Y., Yun, C. H., Guengerich, F. P. and Shimada, T. (1992) Cytochrome P450 2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-nitrosodialkylamines and tobacco-related nitrosamines in human liver microsomes. Carcinogenesis 13, 1789–1794.
Yamano, S., Tatsuno, J. and Gonzalez, F. J (1990) The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human. Biochemistry 29, 1322–1329
Aitio, A. (1978) A simple and sensitive assay of 7-ethoxycoumarin deethylation. Anal. Biochem. 85, 488–491.
Albrecht C F, Chorn D J and Wessels P (1989) Detection of 3-hydroxy-3-methyloxindole in human urine. Life Sci 45:1119–1126.
Babol J, Squires E J and Lundström K (1998a) Hepatic metabolism of skatole in pigs by cytochrome P4502E1. J Anim Sci 76:822–828.
Babol J, Squires E J and Lundström K (1998b) Relationship between oxidation and conjugation metabolism of skatole in pig liver and concentrations of skatole in fat. J Anim Sci 76:829–838.
Baek C E, Hansen-Mller J, Friis C and Hansen S H (1995) Identification and quantification of selected metabolites of skatole—possibilities for metabolic profiling of pigs. Proc. EAAP Working Group Production and Utilisation of Meat from Entire Male Pigs, Milton Keynes, INRA and MLC.
Baek CE, Hansen-Mller J, Friis C, Cornett C and Hansen S H (1997) Identification of selected metabolites of skatole in plasma and urine from pigs. J Agric Food Chem 45:2332–2340.
Beedham, C. (1985) Molybdenum hydroxylases as drug-metabolizing enzymes. Drug Metab. Rev., 16, 119–156.
Beedham, C.; Peet, C. F.; Panoutsopoulos, G. I.; Carter, H.; Smith, J. A. (1995) Role of aldehyde oxidase in biogenic amine metabolism. Prog. Brain Res., 106, 345–353.
Bonneau, M. 1997. Proc. EAAP Working Group on the Production and Utilization of Meat from Entire Male Pigs, Stockholm.
Carlson J R and Yost G S (1989) 3-Methylindole-induced acute lung injury resulting from ruminal fermentation of tryptophan, in Toxicants of Plant Origin. Volume III. Protein and Amino Acids (Cheeke P R ed) pp 107–123, CRC Press, Boca Raton.
Claus, R., U. Weiler, and A. Herzog. 1994. Physiological aspects of androstenone and skatole formation in the boar—a review with experimental data. Meat Sci. 38:289–305.
Diaz, G. J.; Skordos, K.; Yost, G. S; Squires, E. J. (1999, in press) Identification of Phase I metabolites of 3-methylindole produced by pig liver microsomes. Drug Metab. Dispos. 
Friis, C. 1993. Distribution, metabolic fate and elimination of skatole in the pig. In: M. Bonneau (Ed.) Measurement and prevention of boar taint in intact male pigs. p 113–115. INRA Edition, Paris.
Frydman R B, Tomaro M L and Frydman B (1972) Pyrrolooxygenases: isolation, properties, and products formed. Biochim Biophys Acta 284:63–79.
Hammond A C, Carlson J R and Willett J D (1979) The metabolism and disposition of 3-methylindole in goats. Life Sci 25:1301–1306.
Hansen L L, Larsen A E and Hansen-Møller J (1995) Influence of keeping pigs heavily fouled with faeces plus urine on skatole and indole concentration (boar taint) in subcutaneous fat. Acta Agric Scand 45:178–185.
Huijzer J C, Adams J D and Yost G S (1987) Decreased pneumotoxicity of deuterated 3-methylindole: bioactivation requires methyl C—H bond breakage. Toxicol Appl Pharmacol 90:60–68.
Jensen M T, Cox R P and Jensen B B (1995) Microbial production of skatole in the hind gut of pigs given different diets and its relation to skatole deposition in backfat. Anim Sci 61:293–304.
Jepson J B, Zaltzman P and Udenfriend S (1962) Microsomal hydroxylation of tryptamine, indole acetic acid and related compounds, to 6-hydroxy derivatives. Biochim Biophys Acta 62:91–102.
Johns, D. G. (1967) Human liver aldehyde oxidase: differential inhibition of oxidation of charged and uncharged substrates. J. Clin. Invest., 46, 1492–1505.
Kende A S and Hodges J C (1982) Regioselective C-3 alkylations of oxindole dianion. Synth Commun 12:1–10.
Kjeldsen, N. 1993. Practical experience with production and slaughter of intact male pigs. In: M. Bonneau (Ed.) Measurement and prevention of boar taint in intact male pigs. p 137-144. INRA Edition, Paris.
Krenitsky, T. A.; Tuttle, J. V.; Cattau, E. L. Jr.; Wang, P. (1974) A comparison of the distribution and electron acceptor specificities of xanthine oxidase and aldehyde oxidase. Comp. Biochem. Physiol., 49B, 687–703.
Lundström, K.; Bonneau, M. (1996) Off-flavour in meat with particular emphasis on boar taint. In Meat Quality and Meat Packaging; Taylor, S., Raimundo A., Severini, M.; Smulders, F. J. M., Eds., ECCEAMST, Utrecht.
Lundström, K., B. Malmfors, S. Stern, L. Rydhmer, L. Eliasson-Selling, A. B. Mortensen, and H. P. Mortensen. 1994. Skatole levels in pigs selected for high lean tissue growth rate on different protein levels. Livest. Prod. Sci. 38:125–132.
Mahon M E and Mattok G L (1967) The differential determination of conjugated hydroxyskatoles in human urine. Can J Biochem 45:1317–1322.
Mortensen, A. B.; S.o slashed.rensen, S. E. (1984) Relationship between boar taint and skatole determination with a new analysis method. Proc. 30.sup.th Eur. Mtg. Res. Workers, Bristol. Paper 8–11, p. 395.
National Research Council. (1987) Vitamin Tolerance of Animals. National Academy Press, Washington.
Patience, J. F.; Thacker, P. A.; de Lange C. F. M. (1995) Swine Nutrition Guide. 2nd Ed. Prairie Swine Centre Inc., Saskatoon.
Rajagopalan, K. V.; Handler, P. (1964) Hepatic aldehyde oxidase. III. The substrate binding site. J. Biol. Chem., 239, 2027–2035.
Rajagopalan, K. V.; Handler, P. (1966) P. Aldehyde oxidase. Methods Enzymol. 9, 364–368.
Rashidi, M. R.; Smith, J. A.; Clarke, S. E.; Beedham, C. (1997) In vitro oxidation of famciclovir and 6-deoxypenciclovir by aldehyde oxidase from human, guinea pig, rabbit, and rat liver. Drug Metab. Dispos., 25, 805–813.
Rodrigues, A. D. (1994) Comparison of levels of aldehyde oxidase with cytochrome P450 activities in human liver in vitro. Biochem. Pharmacol., 48,197–200.
Ruangyuttikarn W, Appleton M L and Yost G S (1991) Metabolism of 3-methylindole in human tissues. Drug Metab Dispos 19:977–984.
SAS. (1995) SAS System for Windows (Release 6.11). SAS Institute Inc., Cary, N.C.
Sambrook et al. (1989) Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press
Skiles G L and Yost G S (1996) Mechanistic studies on the cytochrome P450-catalyzed dehydrogenation of 3-methylindole. Chem Res Toxicol 9:291–297.
Skiles G L, Adams J D and Yost G S (1989) Isolation and identification of 3-hydroxy-3-methyloxindole, the major murine metabolite of 3-methylindole. Chem Res Toxicol 2:254–259.
Skordos K W, Skiles G L, Laycock J D, Lanza D L and Yost G S (1998a) Evidence supporting the formation of 2,3-epoxy-3-methylindoline: a reactive intermediate of the pneumotoxin 3-methylindole. Chem Res Toxicol 11:741–749.
Skordos K W, Laycock J D and Yost G S (1998b) Thioether adducts of a new imine reactive intermediate of the pneumotoxin 3-methylindole. Chem Res Toxicol 11: 1326–1231.
Smith P K, Krohn R I, Hermanson G T, Mallia A K, Gartner F H, Provenzano M D, Fujimoto E K, Goeke N M, Olson B J and Klenk D C (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85.
Smith D J, Skiles G L, Appleton M L, Carlson J R and Yost G S (1993) Identification of goat and mouse urinary metabolites of the pneumotoxin, 3 methylindole. Xenobiotica 23:1025–1044.
Squires E J and Lundström K (1997) Relationship between cytochrome P450IIE1 in liver and levels of skatole and its metabolites in intact male pigs. J Anim Sci 75:2506–2511.
While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

## Claims

1. A method of genetically typing pigs to determine those with boar taint characteristics, comprising:
obtaining a sample of genetic material from said pig; and
assaying for the presence of a genotype in said pig which is associated with boar taint, said genotype characterized by the following:
a) a polymorphism in the CYP2A6 gene as set forth in SEQ ID NO:3, wherein said polymorphism is a t/c polymorphism at nucleotide position 124 of SEQ ID NO:3 or a deletion of guanine at nucleotide position 422 of SEQ ID NO:3, said polymorphisms being associated with boar taint characteristics.

2. The method of claim 1 wherein said polymorphism of a deletion of guanine at nucleotide position 422 of SEQ ID NO:3 results in a loss of function mutation of CYP2A6.

3. The method of claim 1 wherein said t/c polymorphism at nucleotide position 124 of SEQ ID NO:3 results in a Phe to Leu change at position 42 of SEQ ID NQ:4.

4. The method of claim 1 wherein said polymorphism of a deletion of guanine at nucleotide position 422 of SEQ ID NO:3 results in a truncated CYP2A6 protein of SEQ ID NO:8.

5. The method of claim 1 wherein said step of assaying is selected from the group consisting of: restriction fragment length polymorphism (RFLP) analysis, minisequencing, MALD-TOF, SINE, beteroduplex analysis, one base extension methods, single strand conformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE).

6. A method of genetically typing pigs according to skatole metabolism comprising:
obtaining a sample of genetic material from said pig;
assaying for the presence of an allele characterized by a polymorphism in a CYP2AG gene as set forth in SEQ ID NO:3 present in said sample, wherein said polymorphism results in a deletion of guanine at position 422 of SEQ ID NO:3, or a c/t transition at position 124 of SEQ ID NO:3; and
correlating said allele with skatole metabolism, and concomitant boar taint.

7. The method of claim 6 wherein said step of assaying is selected from the group consisting of: restriction fragment length polymorphism (RFLP) analysis, minisequencing, MALD-TOF, SINE, heteroduplex analysis, one base extension methods, single strand eanformational polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE).