Arylamine N-acetyltransferase Activity Research Articles

Expression of arylamine N-acetyltransferase 2 activity in immortalized human bronchial epithelial cells

Lung cancer is the leading cause of cancer deaths in the United States with high incidence in tobacco smokers. Arylamine N-acetyltransferase 2 (NAT2) is a xenobiotic enzyme that catalyzes both N- and O-acetylation of carcinogens present in tobacco smoke and contributes towards the genotoxicity of these carcinogens. NAT2 allelic variants result in slow, intermediate, and rapid acetylation phenotypes. A recent meta-analysis reported NAT2 non-rapid (slow and intermediate) phenotypes had a significantly increased risk of lung cancer. NAT2 activity in humans is thought to be restricted to liver and gastrointestinal tract, and no studies to our knowledge have reported the expression of NAT2 activity in immortalized human lung epithelial cells. Given the importance of NAT2 in cancer and inhalation of various carcinogens directly into the lungs, we investigated NAT2 activity in human lung epithelial cells. Both NAT1 and NAT2 protein were detected by “in-cell” Western. Arylamine N-acetyltransferase activity was determined with selective substrates for NAT1 (p-aminobenzoic acid; PABA) and NAT2 (sulfamethazine; SMZ) in the presence and absence of a selective NAT1 inhibitor. PABA N-acetylation (NAT1 activity) in cell protein lysates was abolished in the presence of 25 μM of NAT1 inhibitor whereas SMZ N-acetylation (NAT2) was unaffected. Incubation with the NAT1 inhibitor partially reduced the N-acetylation of β-naphthylamine and the O-acetylation of N-hydroxy-4-aminobiphenyl consistent with catalysis by both NAT1 and NAT2. Immortalized human lung epithelial cells exhibited dose-dependent N-acetylation of 4-ABP with an apparent KM of 24.4 ± 5.1 μM. These data establish that NAT2 is expressed and functional in immortalized human lung epithelial cells and will help us further our understanding of NAT2 in lung cancer.

The effect of α-solanine on the activity, gene expression, and kinetics of arylamine N-acetyltransferase in HepG2 cells.

α-solanine is one of the major components of Solanumnigrum Linn., the fruits of which are used in China for food. In the present study, α-solanine was selected to assess the inhibition of arylamine N-acetyltransferase (NAT) activity and mRNA expression of NAT and kinetics in HepG2 cells. NAT activity was examined by HPLC. The double-reciprocal plot was contrived to yield a regression equation to calculate Km and Vmax. NAT mRNA expression was determined by PCR. The results revealed that α-solanine could significantly decrease NAT activity in intact HepG2 cells or the cytoplasm. Km did not differ either for intact HepG2 cells or for the cytoplasm, however Vmax was significantly different. α-solanine could decrease the expression of NAT1 mRNA and NAT2 mRNA. In summary, α-solanine was a non-competitive inhibitor of NAT in HepG2 cells. It decreased NAT activity through non-competitive inhibition of NAT activity and by decreasing the expression of NAT1 mRNA and NAT2 mRNA.

Congenic rats with higher arylamine N-acetyltransferase 2 activity exhibit greater carcinogen-induced mammary tumor susceptibility independent of carcinogen metabolism

BackgroundRecent investigations suggest role(s) of human arylamine N-acetyltransferase 1 (NAT1) in breast cancer. Rat NAT2 is orthologous to human NAT1 and the gene products are functional homologs. We conducted in vivo studies using F344.WKY-Nat2rapid/slow rats, congenic at rat Nat2 for high (rapid) and low (slow) arylamine N-acetyltransferase activity, to assess a possible role for rat NAT2 in mammary tumor susceptibility.MethodsMammary carcinogens, methylnitrosourea (MNU) and 7,12-dimethylbenzanthracene (DMBA) neither of which is metabolized by N-acetyltransferase, were administered to assess mammary tumors. MNU was administered at 3 or 8 weeks of age. DMBA was administered at 8 weeks of age. NAT2 enzymatic activity and endogenous acetyl-coenzyme A (AcCoA) levels were measured in tissue samples and embryonic fibroblasts isolated from the congenic rats.ResultsTumor latency was shorter in rapid NAT2 rats compared to slow NAT2 rats, with statistical significance for MNU administered at 3 and 8 weeks of age (p = 0.009 and 0.050, respectively). Tumor multiplicity and incidence were higher in rapid NAT2 rats compared to slow NAT2 rats administered MNU or DMBA at 8 weeks of age (MNU, p = 0.050 and 0.035; DMBA, p = 0.004 and 0.027, respectively). Recombinant rat rapid-NAT2, as well as tissue samples and embryonic fibroblasts derived from rapid NAT2 rats, catalyzed p-aminobenzoic acid N-acetyl transfer and folate-dependent acetyl-coenzyme A (AcCoA) hydrolysis at higher rates than those derived from rat slow-NAT2. Embryonic fibroblasts isolated from rapid NAT2 rats displayed lower levels of cellular AcCoA than slow NAT2 rats (p < 0.01).ConclusionsA novel role for rat NAT2 in mammary cancer was discovered unrelated to carcinogen metabolism, suggesting a role for human NAT1 in breast cancer.

Treatment of Rats with Apocynin Has Considerable Inhibitory Effects on Arylamine N-Acetyltransferase Activity in the Liver

The effect of apocynin on the activity of arylamine N-acetyltransferases (NATs) in excised liver samples was examined using eighteen Sprague-Dawley rats. Three groups of six animals each were fed a normal diet alone or a treatment of 50 or 100 mg/kg/day of apocynin via gavages for eight (8) weeks. Chronic in vivo administration of apocynin led to significant (p < 0.001) reduction of in vitro liver NAT activity up to 93% as compared with untreated rats (18.80 ± 2.10 μmols p-anisidine/min/μg liver protein). In vitro exposure of untreated liver homogenates to apocynin led to a dose-dependent inhibition of NAT activity with IC50 = 0.69 ± 0.02 mM. In silico modelling of apocynin tautomers and radical species into human NAT crystal structures supported the hypothesis that thiol functionalities in NAT enzymes may be crucial in apocynin binding. The involvement of human NAT enzymes in different pathological conditions, such as cancer, has encouraged the research for selective NAT inhibitors in both humans and animal models with possible chemopreventive properties.

A long-lived luminescent probe to sensitively detect arylamine N-acetyltransferase (NAT) activity of cells

Arylamine N-acetyltransferase (NAT) is an important phase II metabolizing enzyme that influences drug efficacy and adverse effects. Here, we report a long-lived luminescent lanthanide complex as a probe for NAT, employing an intraligand photoinduced electron transfer strategy. The probe shows approximately 100-fold increase of luminescence upon N-acetylation catalyzed by NAT, with relatively high specificity for NAT2 over NAT1. It is the first NAT probe that is suitable for sensitive, homogeneous, and rapid detection of NAT activity of recombinant enzyme or cell lysate, and is expected to be useful for drug discovery and clinical diagnosis.

The role of para‐aminophenol in acetaminophen‐induced methemoglobinemia in dogs and cats

Acetaminophen (APAP) overdose in most species is associated with hepatotoxicity because of the metabolite N-acetyl-p-benzoquinoneimine (NAPQI). In dogs and cats, APAP overdose primarily causes methemoglobinemia and hemolysis. Although NAPQI has been proposed as the responsible intermediate in dogs and cats, it lacks chemical or pharmacokinetic characteristics that favor methemoglobin formation. We hypothesized that para-aminophenol (PAP) rather than NAPQI induces methemoglobinemia and that deficient arylamine N-acetyltransferase (NAT) activity in dogs and cats contributes to this species-dependent methemoglobinemia. Erythrocytes from dogs, cats, mice, and rats were exposed in vitro to APAP, NAPQI, and PAP. Only PAP induced methemoglobin and it induced more methemoglobin formation in dog and cat than rat and mouse erythrocytes. PAP also induced more methemoglobin in erythrocytes from Nat1/Nat2 knockout mice than wildtype (WT) mouse erythrocytes (P < 0.05), but less than in dog and cat erythrocytes (P < 0.01). APAP and PAP toxicity were compared in vivo in WT and Nat1/Nat2 knockout mice. APAP caused no hematotoxicity while PAP induced more methemoglobin in NAT1/NAT2 knockout mice than in WT mice (P < 0.05). These results support the hypothesis that PAP is the metabolite responsible for APAP-induced methemoglobinemia and that deficient NAT activity in dogs and cats contributes to this species-dependent toxicity.

Global transcriptional profiles ofStaphylococcus aureustreated with berberine chloride

In this study, we show that berberine chloride (BBR) has antimicrobial activities against all 43 tested strains of Staphylococcus aureus, an important human and animal pathogen. However, the response mechanisms of S. aureus to BBR are still poorly understood. Affymetrix GeneChips were used to determine the global transcription of S. aureus triggered by treatment with subinhibitory concentrations of BBR. 468 genes were up-regulated and 262 genes were down-regulated upon exposure to BBR. There was elevated transcription of various transporter genes, including genes involved in multidrug resistance, members of the multidrug and toxin extrusion family, the ferrous iron transporter, the amino acid transporter, the Na(+)/H(+) antiporter, and the potassium cation transporter. Measurements of active transport were used to demonstrate a phenotypic correlation between efflux transporter overexpression and inhibition of BBR uptake. Furthermore, BBR induced the expression of urease genes, sortase enzyme, and iron-regulated surface determinant genes, but repressed transcription of a gene encoding arylamine N-acetyltransferase activity (N315-SA2490). To our knowledge, this is the first analysis of a genome-wide transcription profile of S. aureus cells in response to BBR treatment. These results will pave the way to exploring the mechanisms of BBR against S. aureus.

Bacillus cereus strain 10-L-2 produces two arylamine N-acetyltransferases that transform 4-phenylenediamine into 4-aminoacetanilide

A bacterium, strain 10-L-2, that was isolated from soil and identified as Bacillus cereus grew well on medium containing 4-phenylenediamine and Polypepton. Strain 10-L-2 converted a wide variety of anilines, including 4-phenylenediamine, to their corresponding acetanilides. Growing cells acetylated a single amino group of 4-phenylenediamine to form 4-aminoacetanilide with a 97% molar yield, as shown by mass spectrometry and HPLC. Cell extracts exhibited arylamine N-acetyltransferase (NAT) activity toward 4-phenylenediamine. Two NATs, namely, NAT-a and NAT-b, were separated by DE52 column chromatography and were further purified and characterized. The subunit molecular masses of NAT-a and NAT-b were 31.0 and 27.5 kDa, respectively, as determined by SDS-PAGE analysis. The two enzymes had similar pH- and thermo-stabilities and were similarly affected by pH, temperature, and several reagents. The enzymes showed peak activity toward 5-aminosalicylic acid of the substrates tested, but they differed in substrate specificity. Only NAT-a had activity toward sulfamethazine. Although other wild-type bacterial cultures also synthesize NAT, the ability of strain 10-L-2 to convert and detoxify 4-phenylenediamine is much higher. This report provides the first evidence of two NATs in a eubacterium.

Microbial transformation of aniline derivatives: Regioselective biotransformation and detoxification of 2-phenylenediamine by Bacillus cereus strain PDa-1

A bacterial isolate, strain PDa-1, grew well on basal medium supplemented with 2-phenylenediamine, sucrose, and ammonium nitrate and completely transformed 2-phenylenediamine. The isolate was identified as Bacillus cereus. The product formed from 2-phenylenediamine was identified by EI-MS and NMR as 2-aminoacetanilide; whole cells converted 2-phenylenediamine to the product with a 76% molar yield. Whole cells also showed a broad substrate specificity toward 20 of 26 tested arylamines with substituent groups of various size and positions. Especially 2-aminobenzoic acid, 4-aminosalicylic acid, 5-aminosalicylic acid, and 2-aminofluorene were converted completely to the corresponding product with an aminoacetyl group. Cell extracts of strain PDa-1 had a high arylamine N-acetyltransferase activity. The partially purified enzyme converted 2-phenylenediamine to 2-aminoacetanilide. Strain PDa-1 constitutively expressed the enzyme in the absence of 2-phenylenediamine. Effects of 2-phenylenediamine and 2-aminoacetanilide on growth indicated that this enzyme probably plays a role in the detoxification of toxic arylamines in this strain.

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.

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.

Serotonin metabolism in rat skin: characterization by liquid chromatography–mass spectrometry

We have recently uncovered the full expression of novel cutaneous serotoninergic and melatoninergic systems in the human and hamster skin. In this work, we have characterized serotonin metabolism in the rat skin using liquid chromatography–mass spectrometry and found that serotonin undergoes acetylation in the presence of acetyl coenzyme A. Inhibition of serotonin acetylation with Cole bisubstrate inhibitor shows that rat skin expresses both arylalkylamine and arylamine N-acetyltransferase activities. The serotonin degradation product—5-hydroxyindole acetic acid is also detected and pargyline (monoaminooxidase inhibitor) suppresses almost completely 5-hydroxyindole acetic acid accumulation. Together with previous data, the present study clearly demonstrates that biotransformation of serotonin in mammalian skin follows two alternate pathways. In the first pathway, serotonin is acetylated by arylalkylamine and arylamine N-acetyltransferases to generate the precursor of melatonin. Alternately, serotonin may undergo oxidative deamination by monoaminooxidase followed by enzymatic degradation by aldehyde dehydrogenase into 5-hydroxyindole acetic acid, which is presumably devoid of biological activity. Thus, the current methodological development of a liquid chromatography–mass spectrometry-based assay allows rapid resolution of the cutaneous metabolism of serotonin.

Inhibition of N-acetyltransferase activity and gene expression in human colon cancer cell lines by diallyl sulfide

Diallyl sulfide (DAS) is one of the major components of garlic ( Allium sativum) and is widely used in the world for food. In this study, DAS was selected for testing the inhibition of arylamine N-acetyltransferase (NAT) activity ( N-acetylation of 2-aminofluorene) and gene expresseion (mRNA NAT) in human colon cancer cell lines (colo 205, colo 320 DM and colo 320 HSR). The NAT activity was examined by high preformance liquid chromatography and indicated that a 24 h DAS treatment decreases N-acetylation of 2-aminofluorene in three colon (colo 205, 320 DM and colo 320 HSR) cancer cell lines. The NAT enzymes (protein) were analyzed by western blotting and flow cytometry and it indicated that DAS decreased the levels of NAT in three colon (colo 205, 320 DM and colo 320 HSR) cancer cell lines. The gene expression of NAT (mRNAT NAT) was determined by polymerase chain reaction (PCR), it was shown that DAS affect mRNA NAT expression in examined human colon cancer cell lines. This report is the first to demonstrate that DAS does inhibit human colon cancer cell NAT activity and gene expression.

Shikonin Inhibited Growth and N-acetylation of 2-aminofluorene in Bacteria Strains Staphylococcus aureus and Klebsiella pneumoniae

Objectives. N-acetyltransferase (NAT) is the first step in the metabolism (N-acetylation) of arylamine carcinogens (2-aminofluorene) and drugs (sulfamethazine). Angiogenesis is critical for tumor growth and inflammation, and shikonin has been reported to inhibit angiogenesis and induce apoptosis in vivo and in vitro. Therefore, our objective was to investigate the effect shikonin has on growth and N-acetylation of 2-aminofluorene in bacteria. Methods. In this study, growth inhibition of S. aureus and K. pneumoniae was determined by measuring absorbance by an optical density method (OD at 650 nm) using a Beckman Spectrophotometer (DU 6401). We examined arylamine N-acetyltransferase (NAT) activity in the bacteria S. aureus and K. pneu1I1oniae collected from patients and examined the levels of Nacetylation of 2-aminofluorene by high performance liquid chromatography. Results. Shikonin elicited dose-dependent bacteriostatic activity in both examined bacteria cultures. Cytosols and suspensions of S. aureus and K. pneumoniae with and without specific concentrations of shikonin co-treatment showed different percentages of 2-aminofluorene acetylation. The data indicated that the decrease in N-acetylation of 2-aminofluorene was associated with increased levels of shikonin in both examined bacteria cytosols and intact cells. The apparent values of Km and Vmax decreased after co-treatment with 4 μM shikonin. Conclusions. Shikonin induces inhibition of growth and inhibition of arylamine NAT activity (N-acetylation of 2-aminofluorene) in S. aureus and K. pneumoniae.

Paclitaxel (taxol) inhibits the arylamine N-acetyltransferase activity and gene expression (mRNA NAT1) and 2-aminofluorene-DNA adduct formation in human bladder carcinoma cells (T24 and TSGH 8301).

Acetylator polymorphism in man results from differential expression of human liver N-acetyltransferase. N-Acetyltransferase enzyme activity has been demonstrated to be involved in some types of chemical carcinogenesis. Paclitaxel (taxol) had been shown to affect N-acetyltransferase activity of human lung cancer cells. In this study, paclitaxel was chosen to investigate the effects of arylamine N-acetyltransferase activity (N-acetylation of substrate), gene expression and 2-aminofluorene-DNA adduct formation in human bladder carcinoma cell lines (T24 and TSGH 8301). The N-acetyltransferase activity (N-acetylation of substrates) was determined by high performance liquid chromatography assaying for the amounts of acetylated 2-aminofluorene and p-aminobenzoic acid and nonacetylated 2-aminofluorene and p-aminobenzoic acid. Intact human bladder carcinoma T24 and TSGH 8301 cells were used for examining N-acetyltransferase activity, gene expression and 2-aminofluorene-DNA adduct formation. The results demonstrated that the N-acetyltransferase activity, gene expression (NAT1 mRNA) and 2-aminofluorene-DNA adduct formation in intact human bladder carcinoma cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The effects of paclitaxel on the apparent values of Km and Vmax of N-acetyltransferase enzyme from intact human bladder carcinoma cells were also determined in these cell lines. A marked influence of paclitaxel was observed on the decreasing apparent values of Km and Vmax from intact human bladder carcinoma cells (T24 and TSGH 8301). Thus, paclitaxel is an uncompetitive inhibitor to the NAT enzyme.

Aloe-emodin inhibited N-acetylation and DNA adduct of 2-aminofluorene and arylamine N-acetyltransferase gene expression in mouse leukemia L 1210 cells

N-Acetyltransferases (NATs) plays an important role in the first step of arylamine compounds metabolism. Polymorphic NAT is coded for rapid or slow acetylatoion phenotypes, which are recognized to affect cancer risk related to environmental exposure. Aloe-emodin has been shown to exit anticancer activity. The purpose of this study is to examine whether or not aloe-emodin could affect arylamine N-acetyltransferase (NAT) activity and gene expression (NAT mRNA) and DNA–2-aminofluorene (DNA–AF) adduct formation in mouse leukemia cells (L 1210). By using high performance liquid chromatography, N-acetylation and non- N-acetylation of AF were determined and quantitated. By using reverse transcriptase-polymerase chain reaction (RT-PCR) and PCR, NAT mRNA was determined and quantitated. Aloe-emodin 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 aloe-emodin for up to 24 h. Using standard steady-state kinetic analysis, it was demonstrated that aloe-emodin was a possible uncompetitive inhibitor to NAT activity in cytosols. The DNA–AF adduct formation in mouse leukemia cells were inhibited by aloe-emodin. The NAT1 mRNA in mouse leukemia cells were also inhibited by aloe-emodin. This report is the first demonstration which showed aloe-emodin affect mice leukemia cells NAT activity, gene expression (NAT1 mRNA) and DNA–AF on adduct formation.

The effect of paclitaxel on 2-aminofluorene–DNA adducts formation and arylamine N-acetyltransferase activity and gene expression in human lung tumor cells (A549)

In this study, paclitaxel was used to determine inhibition of arylamine N-acetyltransferase (NAT) activity, gene expresssion and 2-aminofluorene–DNA adduct formation in a human lung tumor cell line (A549). The activity of NAT was measured by HPLC assaying for the amounts of N-acetyl-2-aminofluorene (2-AAF) and remaining 2-aminofluorene (2-AF). Human lung tumor cell cytosols and intact cells were used for examining NAT activity and carcinogen–DNA adduct formation. The results demonstrated that NAT activity, gene expression (NAT1 mRNA) and 2-AF–DNA adduct formation in human lung tumor cells were inhibited and decreased by paclitaxel in a dose-dependent manner. The effects of paclitaxel on the values of the apparent K m and V max of NAT from human lung tumor cells were also determined in both examined systems. The result also indicated that paclitaxel decreased the apparent values of K m and V max from human lung tumor cells in both cytosol and intact cells. Thus, paclitaxel is an uncompetitive inhibitor to NAT enzyme.

The effect of paclitaxel on gene expression and activity of arylamine N-acetyltransferase and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cells

N-Acetylation is recognized as the first step in arylamine metabolism. The enzyme responsible for N-acetylation is called arylamine N-acetyltransferase (NAT),which uses acetyl coenzyme A as the acetyl group donor. Paclitaxel has been shown to exhibit antineoplastic and anticancer activity. In this study, paclitaxel was selected to determine the inhibition of arylamine N-acetyltransferase activity, gene expression (NAT mRNA) and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cell line. Paclitaxel (0.01–l μ m) did decrease the level of NAT mRNA in a dose-dependent manner. The results demonstrated that paclitaxel inhibited NAT activity and DNA-2-aminofluorene adduct formation in human leukemia HL-60 cells in a dose-dependent manner. Using standard steady-state kinetic analysis, it was demonstrated that paclitaxel was a possible uncompetitive inhibitor to NAT activity in cytosols based on the decrease in apparent values of K m and V max. This report is the first demonstration that paclitaxel affected human leukemia HL-60 cells NAT activity and DNA–2-aminofluorene adduct formation.

Effects of garlic components diallyl sulfide and diallyl disulfide on arylamine N-acetyltransferase activity and 2-aminofluorene-DNA adducts in human promyelocytic leukemia cells.

Two components of garlic, diallyl sulfide (DAS) and diallyl disulfide (DADS), inhibited arylamine N-acetyltransferase (NAT) activity and 2-aminofluorene-DNA adduct in human promyelocytic leukemia cells (HL-60). The NAT activity was measured by high performance liquid chromatography assaying for amounts of N-acetyl-2-aminofluorene (2-AAF) and remaining 2-aminofluorene (2-AF). Cellular cytosols and intact cell suspensions were assayed. The inhibition of NAT activity and 2-AF-DNA adduct formation in human leukemia cells by DAS and DADS were dose-dependent and were directly proportional. The data also indicated that DAS and DADS decrease the apparent values of Km and Vmax from human leukemia cells in both assays. This is the first report of garlic components affecting human leukemia cell NAT activity and 2-AF-DNA adduct formation.

Effects of butylated hydroxyanisole and butylated hydroxytoluene on DNA adduct formation and arylamine N-acetyltransferase activity in human bladder tumour cells.

In this study, butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used to determine the inhibition of arylamine N-acetyltransferase (NAT) activity and DNA adduct formation in human bladder tumour cell line T-24. The activity of NAT was measured by high-performance liquid chromatography, assaying for the amounts of N-acetyl-2-aminofluorene and N-acetyl-p-aminobenzoic acid and remaining 2-aminofluorene and p-aminobenzoic acid. Human bladder tumour cell line T-24 cytosols and intact cells were used for examining NAT activity and carcinogen-DNA adduct formation. The results demonstrated that NAT activity and 2-aminofluorene-DNA adduct formation in human bladder tumour cells were inhibited and decreased by BHA and BHT in a dose-dependent manner. The effects of BHA and BHT on the values of the apparent K(m) and V(max) also were determined in both systems examined. The results indicated that BHA and BHT decreased the apparent values of K(m) and V(max) from human bladder tumour cells in both cytosol and intact cells.