Measuring the Activity of Arylamine N‐Acetyltransferase (NAT)

Charlene A. McQueen1

1 University of Arizona, Tucson, Arizona
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 4.6
DOI:  10.1002/0471140856.tx0406s10
Online Posting Date:  February, 2002
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Abstract

This unit describes methods for measuring the activity of arylamine N‐acetyltransferases (NAT). Genetic polymorphisms in NAT 1 and NAT 2 have been associated with susceptibility to aromatic amines carcinogens and effects of therapeutic drugs. Evaluation of the activities associated with substrates of NATs is helpful in elucidating the contribution of these enzymes to the pharmacologic and toxicologic effects of arylamines and hydrazines.

     
 
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Table of Contents

  • Basic Protocol 1: HPLC Assay for NAT Activity Using the Acetyl CoA Recycling System
  • Alternate Protocol 1: HPLC Assay for NAT Activity Using a Fixed Concentration of Acetyl CoA
  • Basic Protocol 2: The Bratton‐Marshall Assay for NAT Activity
  • Support Protocol 1: Preparation of S‐9 from Tissue
  • Support Protocol 2: Preparation of Cytosol
  • Support Protocol 3: Preparing a Standard Curve for HPLC Assay of NAT Activity
  • Support Protocol 4: Preparing a Standard Curve for Colorimetric Assay of NAT Activity
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: HPLC Assay for NAT Activity Using the Acetyl CoA Recycling System

  Materials
  • Cytosolic or S‐9 fraction (see protocol 4Support Protocol 1 or protocol 52)
  • Pierce protein assay kit
  • Homogenization buffer (see recipe)
  • 10× acetyl CoA solution 1 (22.7 mM; see recipe)
  • Recycling mix (RCM; see recipe)
  • 10−2 M arylamine substrate (see recipe and Table 4.6.1)
  • ∼80 U/mg carnitine acetyltransferase (CAT; from pigeon breast muscle; Sigma; keep refrigerated but do not freeze, put on ice, or keep at room temperature >5 min)
  • Methanol, ice‐cold
  • Solvents for mobile phase (see Tables 4.6.2, 4.6.3, and 4.6.4)
  • 1.5‐ml microcentrifuge tubes
  • HPLC with UV detector and column (see Tables 4.6.2, 4.6.3, and 4.6.4)
    Table 4.6.1   Materials   Commonly Used Selective Substrates   Commonly Used Selective SubstratesHPLC Conditions for Sulfamethazine (SMZ)   HPLC Conditions for p‐Aminobenzoic Acid (PABA) d   HPLC Conditions for p‐Aminobenzoic Acid (PABA)HPLC Conditions for 2‐Aminofluorene (2‐AF)

    Species NAT1 NAT2
    Human PABA SMZ
    Mouse INH PABA
    Rat PABA
    Hamster PABA
    Method 1 c Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Adsorbosphere C18 (4.6 × 250–mm, 5 µm)
    Mobile phase 20 mM ammonium acetate (A) and methanol (B) 18% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Linear: 2% to 100% B Isocratic
    Flow rate 1 ml/min 1 ml/min
    Retention (min)
     SMZ 12.0 10.8
     acetyl SMZ 14.0 8.4
    Absorbance wavelength (nm) 254 254
    Method 1 Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Adsorbosphere C18 (4.6 × 250–mm, 5 µm)
    Mobile phase 90% 50 mM acetic acid and 10% acetonitrile 13% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Isocratic Isocratic
    Flow rate 1 ml/min 1 ml/min
    Retention (min)
     PABA 7.0 6.5
     acetyl PABA 13.0 8.8
    Absorbance wavelength (nm) 270 270
    Method 1 e Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Versapack C18 (4.1 × 300–mm, 10 µm)
    Mobile phase 20 mM ammonium acetate (A) and methanol (B) 40% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Linear 35% to 100% B Isocratic
    Flow rate 1 ml/min 2 ml/min
    Retention (min)
     2‐AF 20.4 2.6
     2‐AAF f 22.4 6.8
    Absorbance wavelength (nm) 245 245

     aGrant et al., ; Doll and Hein, ; Ferguson et al., ; Estrada‐Rogers et al., .
     bAbbreviations: INH, isoniazid; PABA, p‐aminobenzoic acid; SMZ, sulfamethazine.
    Table 4.6.2   Materials   Commonly Used Selective Substrates   Commonly Used Selective SubstratesHPLC Conditions for Sulfamethazine (SMZ)   HPLC Conditions for p‐Aminobenzoic Acid (PABA) d   HPLC Conditions for p‐Aminobenzoic Acid (PABA)HPLC Conditions for 2‐Aminofluorene (2‐AF)

    Species NAT1 NAT2
    Human PABA SMZ
    Mouse INH PABA
    Rat PABA
    Hamster PABA
    Method 1 c Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Adsorbosphere C18 (4.6 × 250–mm, 5 µm)
    Mobile phase 20 mM ammonium acetate (A) and methanol (B) 18% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Linear: 2% to 100% B Isocratic
    Flow rate 1 ml/min 1 ml/min
    Retention (min)
     SMZ 12.0 10.8
     acetyl SMZ 14.0 8.4
    Absorbance wavelength (nm) 254 254
    Method 1 Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Adsorbosphere C18 (4.6 × 250–mm, 5 µm)
    Mobile phase 90% 50 mM acetic acid and 10% acetonitrile 13% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Isocratic Isocratic
    Flow rate 1 ml/min 1 ml/min
    Retention (min)
     PABA 7.0 6.5
     acetyl PABA 13.0 8.8
    Absorbance wavelength (nm) 270 270
    Method 1 e Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Versapack C18 (4.1 × 300–mm, 10 µm)
    Mobile phase 20 mM ammonium acetate (A) and methanol (B) 40% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Linear 35% to 100% B Isocratic
    Flow rate 1 ml/min 2 ml/min
    Retention (min)
     2‐AF 20.4 2.6
     2‐AAF f 22.4 6.8
    Absorbance wavelength (nm) 245 245

     cStevens et al., .
    Table 4.6.3   Materials   Commonly Used Selective Substrates   Commonly Used Selective SubstratesHPLC Conditions for Sulfamethazine (SMZ)   HPLC Conditions for p‐Aminobenzoic Acid (PABA) d   HPLC Conditions for p‐Aminobenzoic Acid (PABA)HPLC Conditions for 2‐Aminofluorene (2‐AF)

    Species NAT1 NAT2
    Human PABA SMZ
    Mouse INH PABA
    Rat PABA
    Hamster PABA
    Method 1 c Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Adsorbosphere C18 (4.6 × 250–mm, 5 µm)
    Mobile phase 20 mM ammonium acetate (A) and methanol (B) 18% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Linear: 2% to 100% B Isocratic
    Flow rate 1 ml/min 1 ml/min
    Retention (min)
     SMZ 12.0 10.8
     acetyl SMZ 14.0 8.4
    Absorbance wavelength (nm) 254 254
    Method 1 Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Adsorbosphere C18 (4.6 × 250–mm, 5 µm)
    Mobile phase 90% 50 mM acetic acid and 10% acetonitrile 13% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Isocratic Isocratic
    Flow rate 1 ml/min 1 ml/min
    Retention (min)
     PABA 7.0 6.5
     acetyl PABA 13.0 8.8
    Absorbance wavelength (nm) 270 270
    Method 1 e Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Versapack C18 (4.1 × 300–mm, 10 µm)
    Mobile phase 20 mM ammonium acetate (A) and methanol (B) 40% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Linear 35% to 100% B Isocratic
    Flow rate 1 ml/min 2 ml/min
    Retention (min)
     2‐AF 20.4 2.6
     2‐AAF f 22.4 6.8
    Absorbance wavelength (nm) 245 245

     dStevens et al., .
    Table 4.6.4   Materials   Commonly Used Selective Substrates   Commonly Used Selective SubstratesHPLC Conditions for Sulfamethazine (SMZ)   HPLC Conditions for p‐Aminobenzoic Acid (PABA) d   HPLC Conditions for p‐Aminobenzoic Acid (PABA)HPLC Conditions for 2‐Aminofluorene (2‐AF)

    Species NAT1 NAT2
    Human PABA SMZ
    Mouse INH PABA
    Rat PABA
    Hamster PABA
    Method 1 c Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Adsorbosphere C18 (4.6 × 250–mm, 5 µm)
    Mobile phase 20 mM ammonium acetate (A) and methanol (B) 18% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Linear: 2% to 100% B Isocratic
    Flow rate 1 ml/min 1 ml/min
    Retention (min)
     SMZ 12.0 10.8
     acetyl SMZ 14.0 8.4
    Absorbance wavelength (nm) 254 254
    Method 1 Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Adsorbosphere C18 (4.6 × 250–mm, 5 µm)
    Mobile phase 90% 50 mM acetic acid and 10% acetonitrile 13% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Isocratic Isocratic
    Flow rate 1 ml/min 1 ml/min
    Retention (min)
     PABA 7.0 6.5
     acetyl PABA 13.0 8.8
    Absorbance wavelength (nm) 270 270
    Method 1 e Method 2
    Column Microsorb‐MV C18 (4.6 × 250–mm, 5 µm) Versapack C18 (4.1 × 300–mm, 10 µm)
    Mobile phase 20 mM ammonium acetate (A) and methanol (B) 40% acetonitrile in H 2O with 0.5% acetic acid
    Gradient Linear 35% to 100% B Isocratic
    Flow rate 1 ml/min 2 ml/min
    Retention (min)
     2‐AF 20.4 2.6
     2‐AAF f 22.4 6.8
    Absorbance wavelength (nm) 245 245

     eStevens et al., .
     f2‐acetylaminofluorene.

Alternate Protocol 1: HPLC Assay for NAT Activity Using a Fixed Concentration of Acetyl CoA

  • 1× acetyl CoA solution 2 (10−2 M; see recipe)
  • 2 × 10−4 M arylamine substrate (see recipe and Table 4.6.1)

Basic Protocol 2: The Bratton‐Marshall Assay for NAT Activity

  Materials
  • Cytosolic or S‐9 fraction (see protocol 4Support Protocol 1 or protocol 52)
  • Pierce protein assay kit
  • Homogenization buffer (see recipe)
  • 1× acetyl CoA solution 2 (10−2 M; see recipe)
  • 2 × 10−4 M arylamine substrate (see recipe and Table 4.6.1)
  • 10% (w/v) trichloracetic acid (TCA; store up to 1 month at room temperature)
  • 0.1% (w/v) sodium nitrite (prepared fresh)
  • 0.5% (w/v) ammonium sulfamate (store up to 1 month at room temperature)
  • 0.05% (w/v) N‐1‐(naphthyl)ethylene‐diamine dihydrochloride (NED; Sigma; store up to 1 month at room temperature)
  • UV spectrophotometer

Support Protocol 1: Preparation of S‐9 from Tissue

  Materials
  • Mammalian liver or cultured cells
  • Homogenization buffer (see recipe), ice‐cold
  • Tissue homogenizer: Polytron (Kinematica) or equivalent
  • Cryovials

Support Protocol 2: Preparation of Cytosol

  Materials
  • Mammalian liver
  • Homogenization buffer (see recipe), ice‐cold
  • Tissue homogenizer: Polytron (Kinematica) or equivalent
  • Ultracentrifuge
  • Cryovials

Support Protocol 3: Preparing a Standard Curve for HPLC Assay of NAT Activity

  Materials
  • Acetyl PABA or other acetylated arylamine of interest
  • DMSO (for acetyl PABA) or other appropriate solvent for acetylated arylamine
  • Mobile phase (see Tables 4.6.2, 4.6.3, and 4.6.4)

Support Protocol 4: Preparing a Standard Curve for Colorimetric Assay of NAT Activity

  Materials
  • 5 × 10−3 M sulfamethazine (SMZ; see recipe) or other arylamine substrate of interest
  • 10% (w/v) trichloracetic acid (TCA; store up to 1 month at room temperature)
  • 0.1% (w/v) sodium nitrate (prepared fresh)
  • 0.5% (w/v) ammonium sulfamate (store up to 1 month at room temperature)
  • 0.05% (w/v) N‐1‐(naphthyl)ethylenediamine dihydrochloride (NED; Sigma; store up to 1 month at room temperature)
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Figures

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Literature Cited

Literature Cited
   Andres, H.H., Klem, A.J., Szabo, S., and Weber, W.W. 1985. New spectrophotometric and radiochemical assays for acetyl‐CoA: Arylamine N‐acetyltransferase applicable to a variety of arylamine. Anal. Biochem. 145:367‐375.
   Andres, H.H., Klem, A.J., Schopfer, L.M., Harrison, J.K., and Weber, W.W. 1988. On the active site of liver acetyl CoA: Arylamine N‐acetyltransferase from rapid acetylator rabbits (III/J). J. Biol. Chem. 263:7521‐7527.
   Blum, M., Grant, D., McBride, W., Heim, M., and Meyer, U.A. 1990. Human N‐arylamine acetyltransferase genes: Isolation, chromosomal localization and functional expression. DNA Cell Biol. 9:193‐203.
   Blum, M., Demierre, A., Grant, D.M., Heim, M., and Meyer, U.A. 1991. Molecular mechanism of slow acetylation of drugs and carcinogens in humans. Proc. Natl. Acad. Sci. U.S.A. 88:5237‐5241.
   Bratton, A.C. and Marshall, E.K. 1939. A new coupling component for sulfanilamide determination. J. Biol. Chem. 218:537‐550.
   Delclos, K.B. and Kadlubar, F.F. 1997. Carcinogenic aromatic amines and amides. In Comprehensive Toxicology Vol. 12 (I.G. Sipes, C.A. McQueen, and A.J. Gandolfi, eds.) pp. 141‐170. Elsevier Science, Oxford.
   Doll, M.A. and Hein, D.W. 1995. Cloning, sequencing and expression of NAT1 and NAT2 encoding genes from rapid and slow acetylator inbred rats. Pharmacogenetics 5:247‐251.
   Dupret, J.M. and Grant, D.M. 1992. Site‐directed mutagenesis of recombinant human arylamine N‐acetyltransferase expressed in E. coli. J. Biol. Chem. 267:7381‐7385.
   Estrada‐Rogers, L., Levy, G.N., and Weber, W.W. 1998. Substrate selectivity of mouse N‐acetyltransferases 1, 2 and 3 expressed in COS‐1 cells. Drug Metab. Dispos. 26:502‐505.
   Ferguson, R.J., Doll, M.A., Rustan, T.D., and Hein, D.W. 1996. Cloning, expression and functional characterization of rapid and slow acetylator polymorphic N‐acetyltransferase genes of the Syrian hamster. Pharmacogenetics 6:55‐66.
   Fretland, A.J., Doll, M.A., Gray, K., Feng, Y., and Hein, D.W. 1997. Cloning, sequencing and recombinant expression of NAT1, NAT2 and NAT3 derived from C3H/HeJ (rapid) and A/HeJ (slow) acetylator inbred mice: Functional characterization of the activation and detoxification of aromatic amine carcinogens. Toxicol. Appl. Pharmacol. 142:360‐366.
   Glowinski, I.B., Weber, W.W., Fysh, J.M., Vaught, J.B., and King, C.M. 1980. Evidence that arylhydroxamic acid NO‐acetyltransferase are properties of the same enzyme in rabbit liver. J. Biol. Chem. 255:7883‐7890.
   Grant, D.M., Blum, M., Beer, M., and Meyer, U.A. 1991. Monomorphic and polymorphic human arylamine N‐acetyltransferases: A comparison of liver isozymes and expressed products of two cloned genes. Mol. Pharmacol. 59:184‐191.
   Hearse, D.J. and Weber, W.W. 1973. Multiple N‐acetyltransferase and drug metabolism. Biochem. J. 132:519‐526.
   Hein, D.W., Doll, M.A., Fretland, A.J., Leff, M.A., Webb, S.J., Xiao, G.H., Devanaboyina, U.S., Nangju, N.A., and Feng, Y. 2000a. Molecular genetics and cancer epidemiology of the NAT1 and NAT2 acetylation polymorphisms. Cancer Epidemiol. Biomark. Prev. 9:29‐42.
   Hein, D.W., Grank, D.M., and Sim, E. 2000b. Update on consensus arylamine N‐acetyltransferase gene nomenclature. Pharmacogenetics 10:291‐292.
   Hickman, D.A., Risch, A., Buckle, V., Spurr, N., Jeremiah, S.J., McCarthy, A., and Sim, E. 1994. Chromosomal localization of human genes for arylamine N‐acetyltransferases. Biochem. J. 297:441‐445.
   Lemke, L.E. and McQueen, C.A. 1995. Acetylation and its role in the mutagenicity of the antihypertensive agent hydralazine. Drug Metab. Dispos. 23:559‐565.
   Mattano, S.S. and Weber, W.W. 1987. Kinetics of arylamine N‐acetyltransferase in tissues from rapid and slow acetylator mice. Carcinogenesis 8:133‐137.
   Minchin, R.F. 1995. Acetylation of p‐aminobenzoylglutamate, a folic acid catabolite, by recombinant human arylamine N‐acetyltransferase and U937 cells. Biochem. J. 307:1‐3.
   Stevens, G.J., Payton, M., Sim, E., and McQueen, C.A. 1999. N‐acetylation of the heterocyclic amine batracylin by human liver. Drug Metab. Dispos. 27:966‐971.
   Stevens, G.J., Burkey, J.L., and McQueen, C.A. 2000. Toxicity of the heterocyclic amine batracylin: Investigation of rodent N‐acetyltransferase activity and potential contribution of cytochrome P450 3A. Cell Biol. Toxicol. 16:31‐39.
   Vatsis, K.P. and Weber, W.W. 1997. Acetyltransferases. In Comprehensive Toxicology, Vol. 3, (I.G. Sipes, C.A. McQueen, and A.J. Gandolfi, eds.) pp. 385‐499. Elsevier Science, Oxford.
   Vatsis, K.P., Martell, K.J., and Weber, W.W. 1991. Diverse point mutations in the human gene for polymorphic N‐acetyltransferase. Proc. Natl. Acad. Sci U.S.A. 88:6333‐6337.
   Vatsis, K.P., Weber, W.W., Bell, D.A., Dupret, J.M., Price‐Evans, D.A., Grant, D.M., Hein, D.W., Lin, H.J., Meyer, U.A., Relling, M.V., Sim, E., Suzuki, T., and Yamazoe, Y. 1995. Nomenclature for N‐acetyltransferases. Pharmacogenetics 5:1‐17.
   Ward, A., Summers, M.J., and Sim, E. 1995. Purification of recombinant human N‐acetyltransferase type 1 (NAT 1) expressed in E. coli and characterization of its potential role in folate metabolism. Biochem. Pharmacol. 49:1759‐1767.
   Weber, W.W. and Cohen, S.N. 1968. The mechanism of isoniazid acetylation by human N‐acetyltransferase. Biochem. Biophys. Acta 151:276‐278.
   Windmill, K.F., Gaedigk Hall, P.D., Samaratunga, H., Grant, D.M., and McManus, M.E. 2000. Localization of N‐acetyltransferases NAT1 and NAT2 in human tissues. Toxicol. Sci. 54:19‐29.
Key References
   Andres et al., 1985. See .
  Describes the recycling system for acetyl CoA.
   Hearse and Weber, 1973. See .
  Describes the Bratton‐Marshall method.
   Hein et al., 2000a. See .
  Recent review of the molecular genetics and epidemiology of human NATs.
   Stevens et al., 1999. See .
  Describes HPLC conditions for PABA and SMZ.
   Stevens et al., 2000. See .
  Describes HPLC conditions for 2‐AF.
   Vatsis and Weber, 1997. See .
  Recent review of NATs.
Internet Resources
   http://www.louisville.edu/medschool/pharmacology/NAT.html
  Web site for NAT alleles and nomenclature.
   http://www.gene.uc.ac.uk/nomenclature
  Web site for Human Gene Nomenclature Committee.
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