Luteolin inhibits the growth and arylamine N-acetyl-transferase activity in Neisseria gonorrhoeae

Growth inhibition and arylamine N-acetyltransferase (NAT) activity in Neisseria gonorrhoeae were inhibited by luteolin, a drug which originated from herbs. The growth inhibition was based on changes in optical density (OD) using a spectrophotometer, and arylamine NAT activity with 2-aminofluorene (2-AF) was determined using high pressure liquid chromatography. The inhibition of growth in N. gonorrhoeae demonstrated that luteolin elicited a dose-dependent growth inhibition in the N. gonorrhoeae cultures. Suspensions of N. gonorrhoeae with or without specific concentrations of luteolin cotreatment showed different percentages of 2-AF acetylation. The data indicated that there was reduced NAT activity associated with increased levels of luteolin in N. gonorrhoeae suspensions. Time-course experiments showed that NAT activity measured from intact N. gonorrhoeae cells was inhibited by luteolin for at least 4 h. Using standard steady-state kinetic analysis, it was demonstrated that luteolin was a possible uncompetitive inhibitor to NAT activity in N. gonorrhoeae. This report is the first to show that luteolin can inhibit N. gonorrhoeae NAT activity.     

Drug metabolising N-acetyltransferase activity in human cell lines

Many arylamine and hydrazine drugs and xenobiotics are acetylated by N-acetyltransferase (NAT), a cytosolic enzymic activity which has a wide tissue distribution. Humans can be classified as either fast or slow acetylators on the basis of their ability to metabolise isoniazid or sulphamethazine. These are termed polymorphic substrates. The acetylation of other compounds does not vary amongst individuals, e.g., p-aminobenzoic acid, and are termed monomorphic substrates. NAT from human hepatic and non-hepatic tissues, viz., (i) liver, (ii) the hepatoma cell line HepG2, (iii) tonsil lymphocytes and (iv) the monocytic cell line U937 have been compared with respect to substrate specificity towards polymorphic and monomorphic substrates. The chromatographic and centrifugation behaviour of NAT from these sources has also been investigated. NAT from liver shows 2-fold greater activity towards sulphamethazine than towards p-aminobenzoic acid as substrate. All other cell types tested show at least 70-fold greater activity with p-aminobenzoic as substrate compared to sulphamethazine. NAT from HepG2 cells, U937 cells and tonsil lymphocytes migrates as a single peak during ion-exchange chromatography, whereas the liver NAT activity is separated into two peaks. NAT in HepG2 cells resembles extra-hepatic tissue NAT rather than NAT in liver. HepG2 cells do not therefore represent a good in vitro model for investigation of human metabolism of arylamines or hydrazines. The molecular weight of NAT from U937 cells has been determined by a combination of sucrose density gradient centrifugation and gel filtration to be 31,600 +/- 1200 daltons.     

Effects of luteolin on arylamine N-acetyltransferase activity in rat blood and liver.

In this study we investigated inhibition of Arylamine N-acetyltransferase (NAT) activity in rat blood and liver tissue cytosols by luteolin. Using high-performance liquid chromatography, NAT activity for acetylation of 2-aminofluorene and remaining unacetylated 2-aminofluorene were examined. The NAT activity in rat blood and liver tissue was inhibited by luteolin in a dose-dependent manner: higher concentrations of luteolin in the reaction resulted in greater inhibition of NAT activities in both examined tissues. The data also indicated that luteolin decreased apparent Km and Vmax of NAT enzymes from rat blood and liver tissue cytosols. This report is the first demonstration that luteolin can affect rat blood and liver tissue NAT activity.     

Paeonol promoted 2-aminofluorene and p-aminobenzoic acid acetylations by mononuclear leucocytes from Sprague-Dawley rats

Following exposure of rats to the arylamine carcinogen 2-aminofluorene, DNA-carcinogen adducts were found in the target tissues of the liver and bladder, and also in circulating leucocytes. This work investigated how paeonol affects arylamine (2-aminofluorene and p-aminobenzoic acid) acetylations in rat leucocytes. Evidence is presented showing that rat mononuclear leucocytes are capable of acetylating 2-aminofluorene and p-aminobenzoic acid. Paeonol promoted 2-aminofluorene and p-aminobenzoic acid acetylation. Cultured lymphocytes produced about twice as much N-acetyl-2-aminofluorene from 2-aminofluorene and 2.2-fold as much N-acetyl-p-aminobenzoic acid from p-aminobenzoic acid as monocytes. After cotreatment with paeonol, the lymphocyte and monocyte cultures indicated that paeonol did increase 2-aminofluorene and p-aminobenzoic acid acetylations.     

Ellagic acid inhibited 2-aminofluorene and p-aminobenzoic acid acetylation by mononuclear leucocytes from Sprague-Dawley rats

Following exposure of rats to the arylamine carcinogen 2-aminofluorene, DNA-carcinogen adducts were found in the liver and bladder target tissues, and also in circulating leucocytes. This work investigated the effect of ellagic acid on arylamine (2-aminofluorene and p-aminobenzoic acid) acetylations in rat leucocytes. Evidence is presented that rat mononuclear leucocytes are capable of acetylating 2-aminofluorene and p-aminobenzoic acid. Both lymphocytes and monocytes were able to acetylate arylamines during 18 h of culture. Cultured lymphocytes produced about twice as much N-acetyl-2-aminofluorene from 2-aminofluorene and 2.2-fold as much N-acetyl-p-aminobenzoic acid from p-aminobenzoic acid as monocytes. After cotreatment with ellagic acid the lymphocyte and monocyte cultures indicated that ellagic acid reduced 2-aminofluorene acetylation.     

Effects of Carmustine and Lomustine on Arylamine N-Acetyltransferase Activity and 2-Aminofluorene-DNA Adducts in Rat Glial Tumor Cells

Carmustine and lomustine are nitrosourea antitumor chemotherapeutic agents which were used to determine whether or not they could affect arylamine N-acetyltransferase (NAT) activity and DNA-2-aminofluorene adducts in rat glial tumor cell line (C6 glioma). The NAT activity was measured by high preformance liquid chromatography (HPLC) assaying for the amounts of N-acetyl-2-aminofluorene (AAF) and N-acetyl-p-aminobenzoic acid (N-Ac-PABA) and remaining 2-aminofluorene (AF) and p-aminobenzoic acid (PABA). The results indicate that NAT activity in glial tumor cell cytosols and intact tumor cells were decreased by carmustine and lomustine in a dose-dependent manner. The apparent values of Km and Vmax of NAT from rat glial tumor cell also decreased after co-treatment of carmustine and lomustine in both examined cytosols and intact cells. Following exposure of glial tumor cells to the various concentrations of AF with or without co-treatment with carmustine and lomustine, DNA-AF adducts were determined by using -[³²p]-dATP and HPLC. The DNA-AF adducts in rat glial tumor cells were decreased by co-treatment with carmustine and lomustine. This report is the first demonstration to show carmustine and lomustine did inhibit rat glial tumor cells NAT activity and DNA-AF adduct formation.     

Purification and characterization of an arylamine N-acetyltransferase in the nematode Enterobius vermicularis.

N-acetyltransferase (NAT) activities were determined by incubation of Enterobius vermicularis cytosols with 2-aminofluorene (2-AF) as the substrate followed by high pressure liquid chromatography assays. The NAT activity from E. vermicularis was found to be 0.41 +/- 0.08 nmol/min/mg protein for 2-AF. The apparent K(m) and Vmax values obtained were 0.81 +/- 0.11 mM and 2.25 +/- 0.22 nmol/min/mg protein respectively, for 2-AF. The optimal pH value for the enzyme activity was 7.5 for 2-AF. The optimal temperature for enzyme activity was 37 degrees C for the 2-AF substrate. The molecular weight of NAT from E. vermicularis was 44.9 kD. Among a series of divalent cations and salts, Zn2+, Ca2+, and Fe2+ were the most potent inhibitors. Of the protease inhibitors, only ethylenediaminetetraacetic acid significantly protected the NAT. Iodoacetate, in contrast to other agents, markedly inhibited NAT activity. This report is the first demonstration of acetyl coenzyme A-dependent arylamine NAT activity in E. vermicularis and extends the number of phyla in which this activity has been found.     

Berberine inhibits arylamine N-acetyltransferase activity and gene expression in mouse leukemia L 1210 cells

N-acetyltransferases (NATs) are recognized to play a key role in the primary step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylators, which are being thought to involve cancer risk related to environmental exposure. Berberine has been shown to induce apoptosis and affect NAT activity in human leukemia cells. The purpose of this study is to examine whether or not berberine could affect arylamine NAT activity and gene expression (NAT mRNA) and the levels of NAT protein in mouse leukemia cells (L 1210). N-acetylated and non-N-acetylated AF were determined and quantited by using high performance liquid chromatography. NAT mRNA was determined and quantited by using RT-PCR. The levels of NAT protein were examined by western blotting and determined by using flow cytometry. Berberine displayed a dose-dependent inhibition to cytosolic NAT activity and intact mice leukemia cells. Time-course experiments indicated that N-acetylation of AF measured from intact mice leukemia cells were inhibited by berberine for up to 24 h. The NAT1 mRNA and NAT proteins in mouse leukemia cells were also inhibited by berberine. This report is the first demonstration, which showed berberine affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and levels of NAT protein.     

Molecular and genetic analyses of arylamine N-acetyltransferase polymorphism of rabbit liver

A cDNA clone encoding the full coding region of polymorphic arylamine N-acetyltransferase was isolated from rabbit liver and expressed in Chinese hamster ovary cells. The expressed enzyme acetylated 2-aminofluorene, procainamide, sulfamethazine, and p-aminobenzoic acid at equivalent rates. N-Acetyltransferase activity was measured in 17 rabbits from an inbred colony which were classified into rapid, intermediate, and slow acetylators. The livers of the rapid and intermediate acetylators efficiently acetylated all four substrates, while the liver from the slow acetylator showed a low but significant activity with p-aminobenzoic acid. Immunoblot and Northern blot analyses of rabbit livers indicated that the differences in N-acetyltransferase activity were due to differences in N-acetyltransferase protein and mRNA content. Genomic clones of N-acetyltransferase were isolated from the rapid and slow acetylator rabbits. The nucleotide sequence of the gene from rapid acetylator rabbit was identical to that of the cDNA, while the sequence of the gene from slow acetylator rabbit was homologous, but not identical, to the cDNA sequence. Genomic Southern blot and polymerase chain reaction analyses of the genomic DNAs and cDNAs from the three types of acetylator indicated that the gene for polymorphic arylamine N-acetyltransferase is totally deleted in the slow acetylator rabbit, while the gene from slow acetylator rabbit is expressed in all rabbits and might encode another N-acetyltransferase. Thus the genetic mechanism of N-acetyltransferase polymorphism in rabbit liver is essentially different from that of human liver as demonstrated in this laboratory (Ohsako, S., and Deguchi, T. (1990) J. Biol. Chem. 265, 4630-4634; Deguchi, T., Mashimo, M., and Suzuki, T. (1990) J. Biol. Chem. 265, 12757-12760).     

Acetylation of Serotonin in the Rabbit Pineal Gland: An N-Acetyltransferase with Properties Distinct from NAT1 and NAT2 Is Responsible

Two rabbit arylamine N-acetyltransferases (NAT1 and NAT2, EC 2.3.1.5) have been cloned and characterized recently in this laboratory. They catalyze the acetylation of primary arylamine and hydrazine drugs and other substrates in the liver, including sulfamethazine, p-aminosalicylic acid, and p-aminobenzoic acid. In the pineal gland, serotonin is metabolized to N-acetylserotonin by an unknown N-acetyl-transferase. Similarity of the liver enzymes and the pineal gland arylalkylamine N-acetyltransferase (AA-NAT) has been suggested, because pineal gland homogenates were shown to metabolize arylamine substrates as p-phenetidine, aniline, or phenylethylamine, and liver homogenates or partially purified liver enzyme preparations catalyzed the N-acetylation of serotonin. The present study was undertaken to elucidate the possible role of NAT1 or NAT2 in serotonin acetylation in the pineal gland. We transiently expressed rNAT1 and rNAT2 genes in COS cells, studied the kinetics of the enzymes produced with various substrates, and compared these data with activities of rabbit pineal glands and livers. These enzymatic studies were complemented with western blot analysis with antibodies against NAT1 and NAT2. Cross-hybridization of rNAT1 or rNAT2 to the gene for the pineal gland AA-NAT was tested by Southern blot studies of genomic rabbit DNA. Our results indicate that although NAT1 is expressed in the pineal gland, it is not involved in the physiologically important step of N-acetylation of serotonin.     

Vitamin C Inhibited DNA Adduct Formation and Arylamine N-Acetyltransferase Activity and Gene Expression in Rat Glial Tumor Cells

Studies have been demonstrated that vitamin C (ascorbic acid) exhibit the protective role of vin in certain types of cancer. Rat glial tumor cells also have been shown have N-acetyltransferase activity. In this study, we reported the effects of vitamin C on arylamine N-acetyltransferase (NAT) activity and DNA adduct formation in rat glial tumor cell line (C6 glioma). The activity of NAT was measured by high performance liquid chromatography assaying for the amounts of acetylated 2-aminofluorene and p-aminobenzoic acid and nonacetylated 2-aminofluorene and p-amonibenzoic acid. Rat C6 glioma cells were used for examining NAT activity and gene expression and 2-aminofluorene-DNA adduct formation. The results demonstrated that NAT activity and 2-aminofluorene-DNA adduct formation in C6 glioma cells were inhibited and decreased by vitamin C in a dose-dependent manner. But vitamin C did not affect NAT gene expression in examined cells. The apparent kinetic parameters (apparent values of Km and Vmax) from C6 glioma cells were also determined with or without vitamin C cotreatment. The data also indicated that vitamin C decreased the apparent values of Km and Vmax from C6 glioma cells.     

Effects of caffeic acid, chlorogenic acid and ferulic acid on growth and arylamine N-acetyltransferase activity in Shigella sonnei (group D)

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) as substrates were determined in Shigella sonnei (group D) collected from patients with diarrhoeal disease. The NAT activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. Inhibition of growth studies from S. sonnei (group D) demonstrated that caffeic acid (CA), chlorogenic acid (CGA) and ferulic acid (FA) elicited a dose-dependent bactericidal effect in S. sonnei (group D) cultures, i.e. the greater the concentration of CA, CGA and FA, the greater the inhibition of growth of S. sonnei (group D). Cytosols or suspensions of S. sonnei (group D) with and without selected concentrations of CA, CGA and FA co-treatment showed different percentages of 2-AF acetylation. The data indicated that there was reduced NAT activity associated with increased CA, CGA and FA in Shigella dysenteriae (group D) cytosols and intact cells. For the cytosol and intact bacteria examinations, the apparent values of K(m) and Vmax decreased after being co-treated with 400 microM CA, CGA and FA. This report is the first demonstration of plant phenolic inhibition (CA, CGA and FA) of arylamine NAT activity and growth in the bacterium S. sonnei (group D).     

Effects of the butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the arylamines N-acetyltransferase activity in rat white blood cells

Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used to determine any effects on the N-acetyltransferase (NAT) activity in rat whole blood and white blood cells as measured by high performance liquid chromatography assay for the amounts of N-acetyl-2-aminofluorene (AAF) and 2-aminofluorene (AF). Two assay systems were performed, one with cellular cytosols, the other with intact white blood cells. The NAT activity in the whole blood and white blood cell cytosols was suppressed by BHA and BHT in a dose-dependent manner, i.e. the higher the concentrations of BHA and BHT, the higher the inhibition of NAT activity. Time-course experiments showed that NAT activity measured from the intact white blood cells was inhibited by BHA and BHT up to 24 h. The results suggest that BHA and BHT suppressed AF acetylation in rat blood with intact white blood cells.     

Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and functional expression

N-Acetylation by hepatic arylamine N-acetyltransferase (NAT, EC 2.3.1.5) is a major route in the metabolism and detoxification of numerous drugs and foreign chemicals. NAT is the target of a common genetic polymorphism of clinical relevance in human populations. We have used our recently isolated rabbit cDNA rnat to clone three human NAT genes from human leukocyte DNA. None of the three genomic coding sequences was interrupted by introns. Two genes, designated NAT1 and NAT2, each possessed open reading frames of 870 bp. Both genes have been assigned to human chromosome 8, pter-q11. Following transfection they were transiently expressed in monkey kidney COS-1 cells. NAT1 and NAT2 gave rise to functional NAT proteins, as judged by their NAT enzyme activity with the arylamine substrate sulfamethazine. Western blots with NAT-specific antisera detected proteins of apparent molecular weight of 33 and 31 kD in NAT1- and NAT2-transfected cultures, respectively. The product of NAT2 had an identical apparent molecular weight as that of NAT detected in human liver cytosol. The deduced amino acid sequence of NAT2 also contained 6 peptide sequences which had previously been determined from tryptic peptides of the polymorphic NAT purified from human liver. These data suggest that NAT2 encodes the polymorphic NAT protein. The third gene, NATP, had multiple deleterious mutations and did not encode a functional NAT protein; it most likely represents a pseudogene.     

Berberine Inhibited Arylamine N-Acetyltransferase Activity and Gene Expression and DNA Adduct Formation in Human Malignant Astrocytoma (G9T/VGH) and Brain Glioblastoma Multiforms (GBM 8401) Cells

Studies have demonstrated that berberine exhibits the antineoplastic action in rat model. Rat glial tumor cells also have been shown to have N-acetyltransferase activity. In this study, we reported the effects of berberine on arylamine N-acetyltransferase (NAT) activity, gene expression, and DNA adduct formation in human brain tumor cell lines (G95/VGH and GBM 8401). The activity of NAT (N-acetylation of substrate) was measured and determined by high-performance liquid chromatography (HPLC) assaying for the amounts of acetylated 2-aminofluorene (AF) and nonacetylated AF. Human brain tumor cells (G9T/VGH and GBM 8401) were used for examining NAT activity and gene expression and AF-DNA adduct formation. NAT gene expression was determined by polymerase chain reaction (PCR) for the levels of mRNA NAT in both examined cells lines. The amounts of AF-DNA adducts were also determined and quantities by HPLC. The results demonstrated that NAT activity, levels of mRNA NAT1 and AF-DNA adduct formation in both examined cell were inhibited and decreased by berberine in a dose-dependent manner. The apparent values of Km and Vmax from NAT of both examined cells were also determined with or without berberine cotreatment. The data also indicated that berberine decreased the apparent values of Km and Vmax. These effects also indicate that berberine is a uncompetitive inhibitor.     

Quercetin glucuronides inhibited 2-aminofluorene acetylation in human acute myeloid HL-60 leukemia cells

Our earlier study has demonstrated that following the exposure of rat to the arylamine carcinogen 2-aminofluorene, DNA-2-aminofluorene adducts were found in the target tissues liver, bladder, colon, lung and also in circulating leukocytes (lymphocytes and monocytes). The result also demonstrated that orally treated antioxidants decreased N-acetylation of 2-aminofluorene in target tissues and leukocytes. Therefore, this study investigated whether quercetin glucuronides could affect N-acetylation of 2-aminofluorene in human acute myeloid leukemia HL-60 cells. Evidence is presented here that human leukemia cells are capable of acetylating 2-aminofluorene. Quercetin glucuronides did inhibit 2-aminofluorene acetylation in intact cells. The results also indicated that quercetin glucuronides induced cytotoxicity in dose-dependent manner in the examined human acute myeloid leukemia HL-60 cells.     

Ibuprofen Inhibits Arylamine N-Acetyltransferase Activity in the Bacteria Klebsiella pneumoniae

Ibuprofen, one of the nonsteroidal anti-inflammatory drugs, inhibited arylamine N-acetyltransferase activity of Klebsiella pneumoniae both in vitro and in vivo. The NAT activities of Klebsiella pneumoniae were inhibited by ibuprofen in a dose-dependent manner both in vitro and in vivo. In vitro, the NAT activity was 0.675 ± 0.028 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.506 ± 0.002 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In vivo, the NAT activity was 0.279 ± 0.016 nmol/min/10¹⁰ colony forming units (CFU) for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was 0.228 ± 0.008 nmol/min/10¹⁰ CFU for the acetylation of 2-aminofluorene. The inhibition of NAT activity by ibuprofen was shown to persist for at least 4 h. For in vitro examination, the values of apparent K _m and V _max were 1.08 ± 0.05 mM and 9.17 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the reaction mixtures, the values of apparent K _m and V _max were 1.19 ± 0.01 mM and 6.67 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. For in vivo examination, the values of apparent K _m and V _max were 1.24 ± 0.48 mM and 4.18 ± 1.06 nmol/min/10 × 10¹⁰ CFU, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the culture, the values of apparent K _m and V _max were 0.95 ± 0.29 mM and 2.77 ± 0.37 nmol/min/mg protein, respectively, for 2-aminofluorene, respectively. This report is the first finding of ibuprofen inhibition of arylamine N-acetyltransferase activity in a strain of Klebsiella pneumoniae. Received: 28 January 1997 / Accepted: 12 February 1997     

Berberine Inhibits Arylamine N-Acetyltransferase Activity and Gene Expression in Salmonella Typhi

The effects of berberine on growth, arylamine N-acetyltransferase (NAT) activity, and gene expression in Salmonella Typhi (Typhi) were described. The growth inhibition of Typhi was determined by measuring absorbance by optical density (OD at 650 nm). The NAT activity was determined by measuring the levels of 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF) by high-performance liquid chromatography. The results demonstrated that 24-h berberine treatment decreased bacteria growth and amounts of AAF in Typhi. Western blotting and flow cytometry were used for examining the levels of NAT after bacteria were cotreated with or without various concentrations of berberine, and results indicated that berberine decreased the levels of NAT in Typhi. Polymerase chain reaction was used for examining the gene expression of NAT (mRNA NAT), and results indicated that berberine affects mRNA NAT1 expression in Typhi.     

Effects of Ellagic Acid by Oral Administration on N-Acetylation and Metabolism of 2-Aminofluorene in Rat Brain Tissues

Numerous studies have demonstrated that the Acetyl Coenzyme A-dependent arylamine NAT enzyme exist in many tissues of experimental animals including humans, and that NAT has been shown to be exist in mouse brain tissue. Increased NAT activity levels are associated with increased sensitivity to the mutagenic effects of arylamine carcinogens. Attenuation of liver NAT activity is related to breast and bladder cancer processes. Therefore, the effects of ellagic acid (EA) on the in vitro and in vivo N-acetylation of 2-aminofluorene (AF) were investigated in cerebrum, cerebellum and pineal gland tissues from male Sprague-Dawley rats. For in vitro examination, cytosols with or without EA (0.5–500 M) co-treatment decreased 7–72%, 15–63% and 10–78% of AF acetylation for cerebrum, cerebellum and pineal gland tissues, respectively. For in vivo examination, EA and AF at the same time treated groups with all 3 examined tissues did show significant differences (the changes of total amounts of AF and AF metabolites based on the Anova analysis) when compared to the ones without EA cotreatment rats. The pretreatment of male rats with EA (10 mg/kg) 24 hr prior to the administration of AF (50 mg/kg) (one day of EA administration suffice to induce large changes in phase II enzyme activity) resulted in a 76% decrease in total AF and metabolites in pineal gland but did not show significant differences in cerebrum and cerebellum tissues. This is the first demonstration to show that EA decreases the N-acetylation of carcinogens in rat brain tissues.     

Placental arylamine N-acetyltransferase type 1: potential contributory source of urinary folate catabolite p-acetamidobenzoylglutamate during pregnancy

Human arylamine N-acetyltransferase type 1 (NAT1), better known as a drug-metabolising enzyme, has been proposed to acetylate the folate catabolite p-aminobenzoylglutamate (p-abaglu) to N-acetamidobenzoylglutamate (ap-abaglu) which is a major urinary folate catabolite. Using mass spectroscopic analysis, we demonstrate the formation of ap-abaglu by recombinant human NAT1 and human placental homogenates. Using density gradient centrifugation the placental enzymic activity which acetylates p-aba and the placental enzymic activity acetylating p-abaglu both have an S(20,w) value of 3.25 S. This is the expected value for a monomer of human NAT1 (33 kDa). The specific NAT1 inhibitor 5-iodosalicylate inhibits acetylation of both p-aba and p-abaglu catalysed by either recombinant human NAT1 or placental samples as the source of enzyme. These data demonstrate that NAT1 is the major placental enzyme involved in acetylating p-abaglu.