ICANCER RESEARCH 56. 2683-2687. June 15. 1996|

Advances in Brief

Af-Acetyltransferase Expression and Metabolic Activation of the Food-derived

Heterocyclic Amines in the Human Mammary Gland

Nakissa Sad rieh,' Cindy D. Davis, and Elizabeth G. Snyderwine

Laboratory of Experimental Carcinogenesis. National Cancer Institute, Bethesda. Maryland 20892-4255

Abstract

The heterocyclic amines (HCAs) found in cooked meat are procar-cinogens that are metabolic alh activated by jV-hydroxylation followedby O-acetylation by the /V-acetyltransferases NATI and NAT2. Despitethe importance of metabolic activation in HCA carcinogenicity and thefinding that several HCAs are rodent mammary gland carcinogens,nothing was known about O-acetylation activity in the human mammary gland. The current study examines the expression and catalyticactivity of NAT toward the N-hydroxy-HCAs 2-hydroxyamino-l-meth-yl-6-phenylimidazo[4,5-6]pyridine (¿V-hydroxy-PhIP)and 2-hydroxy-amino-3-methylimidazo[4,5-/]quinoline W-hydroxy-IQ) in the humanmammary gland. Mammary gland cytosol from 10 women and lysatesfrom a primary culture of human mammary epithelial cells metaboli-cally activated 2-hydroxyamino-l-methyl-6-phenylimidazo[4,5-A]pyri-dine and 2-hydroxyamino-3-methylimidazo[4,5-/]quinoline by NAT-mediated 0-acetyltransferase, as measured by the acetyl CoA-enhanced binding of the A'-hydroxylamines to calf thymus DNA invitro. yV-acetylation of p-aminosalicylic, an activity specific to NATI,but not ¿V-acetylationof sulfamethazine, an activity specific to NAT2,was detected in the mammary gland cytosols and human mammaryepithelial cell lysates. Immunohistochemical analysis of human mammary gland sections showed positive staining for NATI protein in theepithelial cells lining the mammary gland ducts. Reverse transcription-PCR analysis showed that mRNA transcripts for both NATI and NAT2were present in human mammary gland; however, no NAT2 catalyticactivity was detectable. Our data demonstrate for the first time that thehuman mammary gland is catalytically active toward the metabolicactivation of HCA food mutagens, and that this activity is most likelycontributed by NATI expressed in the ductular epithelial cells of themammary gland.

Introduction

The etiology of human breast cancer is largely unknown; however,environmental factors, especially diet, appear to influence this disease(1). Humans are exposed to highly mutagenic and carcinogenicHCAs2 from the consumption of cooked meats (2). The finding that

several HCAs, including PhIP and the quinolines IQ and 2-amino-3,4-dimethylimidazo[4,5-/lquinoline, are mammary gland carcino

gens in rats raises the possibility that these compounds may play a rolein human mammary gland cancer (reviewed in Ref. 3; Ref. 4). TheHCAs, like many chemical carcinogens, require metabolic activationfor DNA adduci formation and the initiation of carcinogenesis (3, 5).The first step in the metabolic activation of these compounds occurs

Received 3/22/96: accepted 4/30/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1To whom requests for reprints should be addressed, at Laboratory of Experimental

Carcinogenesis. National Cancer Institute. Building 37. Room 3C28. 37 Convent Drive.MSC4255, Bethesda. MD 20892-4255.

2 The abbreviations used are: HCA. heterocyclic amine; NAT. /V-acetyltransferase;SMZ, sulfamethazine: HMEC. human mammary epithelial cell: PAS. p-aminosalicylicacid; AcCoA. acetyl-CoA; PhIP. 2-amino-l-methyl-6-phenylimidazol4.5-fc]pyridine; IQ.2-amino-3-methylimidazo[4.5-/]quinoline; RFLP. restriction fragment length polymorphism; RAL. relative adduci labeling; RT-PCR, reverse transcription-PCR; HPLC. high-

performance liquid chromatography.

by cytochrome P450-catalyzed AAhydroxylation (5). The resultingA/-hydroxy derivatives are generally poorly reactive with DNA butcan be further metabolized by A/-acetyltransferase-mediated O-acetyl-ation to esters that readily form DNA adducts (5-7). In humans, twoA/-acetyltransferases, designated NATI and NAT2, catalyze A/- andO-acetylation of various arylamines, including the O-acetylation ofW-hydroxy-HCAs (8-10). NAT2, and most recently NATI, have been

shown to be polymorphic enzymes that segregate individuals intorapid and slow acetylator phenotypes (10, 11). Genetic polymorphisms in these enzymes influence the balance of metabolic activationand detoxification of environmental arylamine carcinogens and maymodulate the risk of certain human cancers such as those of thecolorectum and bladder (12-15). Notably, recent studies have indi

cated that NATI activity in the human colon and bladder is animportant determinant of cancer risk and in situ metabolic activationof arylamines (12-14). In light of these findings, it is possible that the

susceptibility of the human mammary gland to arylamine carcinogens,such as the dietary HCAs, may also be influenced by intramammaryNATI or NAT2 activity. However, nothing is known about the NATenzymes in the human mammary gland. In this study, we examine theexpression and activity of NATI and NAT2 in the human mammarygland with the objective of determining if the human mammary glandhas the capacity to carry out the metabolic activation of theA/-hydroxy-HCAs.

Materials and Methods

Chemicals and Antibodies. W-Hydroxy-IQ and /V-hydroxy-PMP were

synthesized as described previously (6). All other reagents were purchasedfrom Sigma Chemical Co. (St. Louis, MO). /V-Acetylated SMZ and a poly-

clonal antibody raised in rabbit against a recombinant human NATI proteinexpressed in Escherichia coli were provided by Dr. Denis Grant (Hospital forSick Children. Toronto. Ontario, Canada).

Animals. Female Sprague-Dawley rats were obtained from NIH AnimalSupply (Frederick, MD) at 60-70 days of age and maintained on NIH Lab

Chow and water ad libidum. Mammary gland and liver cytosol were preparedas described below for human samples.

Preparation of Human Mammary Gland Cytosol. Snap-frozen humanmammary gland samples from patients aged 15-69 years old, undergoing

reduction mammoplasty, were obtained from The Cooperative Human TissueNetwork and stored at -80°C until use. Cytosolic fractions were isolated by

differential ultracentrifugation (16). Five to 10 g of tissue was homogenizedwith a Polytron in 10 ml of TEDK buffer ( 10 mM triethanolamine-HCl, 1 mM

EDTA, 1 mM DTT, and 50 mM KC1, pH 7.0) containing 0.02 mM leupeptin, 0.1mM PMSF, 0.05 mM butylated hydroxytoluene, and 1 /^M pepstatin A. Theprotein concentration of the cytosols was determined colorimetrically by theBCA protein assay (Pierce, Rockford, IL). Due to the lability of the NATenzyme, all NAT enzyme assays were run immediately following cytosol

isolation.Cell Culture and Preparation of Cell Lysates. A primary culture of

HMECs, derived from a 50-year old healthy woman who had undergone

reduction mammoplasty, was purchased from Clonetics (San Diego, CA). Atpassage number 9 or 10, cells from nine confluent T75 flasks were trypsinized,and three cell pellets, constituting a pool of three T75 flasks each, werecollected by centrifugation at 4°C.Subsequently, the cell pellets were washed

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NAT EXPRESSION AND KOOD-DERIVED HETEROCYCLIC AMINES

and resuspended in 1 ml ice-cold TEDK but't'er. The cells were sonicated, and

the cell debris was removed by brief centrifugation. The protein concentrationwas determined, and (he lysate was used immediately for the NAT enzymeassays described below.

iV-Acetyltransferase Assay. SMZ W-acetylation, a measure of NAT2 activity, and PAS N-acelylation, a measure of NATI activity, were determined

spectrophotomeirically essentially as described previously (17). The incubations contained 0.25 HIMAcCoA, 20 n\ of an AcCoA regenerating system (5.4mg acetyl-l)l--carnitine and 1 unit carniline acetyltransferasel. 200 ¡Msubstrate

(SMZ or PAS), and 50 fil of mammary gland cytosol or epithelial cell lysate(0.25-0.5 mg protein) in a total volume of 100 /il. Concomitantly, controlincubations were run that did not contain AcCoA. Following a 20-minuteincubation at 37°C.the reactions were stopped by the addition of 50 /¿Iof 20%

trichloroacetic acid and centrifugation. One ml of 2.5% dimethylaminobenz-

aldehyde in acetonitrile was added to the supernatants. After IO min. theabsorbance at 450 nm was measured. The level of acetylation. measured as adecrease in the absorbance at 450 nm, was determined using a standard curvefor each substrate. The assays were run under conditions of linearity forincubation lime and cytosolic protein concentration. The limit of detection ofproduct was O.I nmol/min/mg protein of acetylated product in the totalincubation mixtures.

SMZ /V-acetylation in human mammary gland cytosol was also measured by

HPLC. Incubations were carried out as described above and terminated by theaddition of 10 /j.1 of 15% perchloric acid and centrifugation. Fifty ¿ilofsupernatant were analyzed by HPLC. essentially as described previously (18).HPLC was carried out with a Beckman Ultrasphere reversed-phase (?,„ODS

column (5 /¿m;4.6 x 150 mm) using a Gilson model 715 system equippedwith a Croton photodiode array detector. The solvent condition was initially10% acetonitrile in 20 HIMsodium perchlorate buffer (pH 2.5) and followed alinear gradient to 20% acetonitrile over an 8-min period. The flow rate was 1.5nil throughout ihe run. The retention time of the A/-acetylated SMZ wasconfirmed using synthetic standards. A positive control for SMZ A/-acetylation

activity was run using rat and mouse hepatic cytosolic fractions. The limit ofdetection was 0.05 nmol/min/mg protein.

0-Acetyltransferase Assay. NAT-mediated O-acetylation of /V-hydroxy-PhlP and N-hydroxy-IQ was assayed in mammary gland cytosols and inHMEC lysates by the AcCoA-enhanced binding of the hydroxylamines to calf

thymus DNA, as described previously (7). DNA was isolated by phenolextraction, and IQ- and PhlP-DNA adduci levels were quantitated by the<2P-postlabeling method, as described previously (6). DNA adduci values wereexpressed as RAL X IO7.

RT-PCR and RFLP Analysis of NATI and NAT2. The RT-PCR analysiswas carried out using the NATI and NAT2-specific primers reported previ

ously by Kloth et al. (19). Total RNA was isolated from frozen humanmammary glands, reverse transcribed and PCR amplified as described previously (20). The authenticity of the amplified alíeleswas confirmed by RFLPanalysis using Hinc\\ (which digests NAT2 to produce 659- and 248-bpfragments) and ///millI (which digests NATI to produce 786- and 75-bp

fragments) (19).Immunohistochemic'ul Localization of NATI in the Human Mammary

(.lain!. Normal human mammary gland samples, fixed in I09r formalin andembedded in paraffin, were obtained from the Cooperative Human TissueNetwork and cut into 5-/xm sections. Following deparatfinization. the sections

were blocked with a dilution of goat serum for I h and subsequently incubatedovernight with a polyclonal antibody prepared against recombinant humanNATI. The NATI protein was localized using the Vectastain ABC kit (VectorLaboratories. Burlingame. CA). To exclude the possibility of nonspecificbinding of the secondary antibody to human mammary gland sections, controlsections were run that lacked the primary antibody. In addition, other controlsections were run using rabbit serum (1:50 dilution) instead of the NAT1-specific antibody. All the slides were subsequently counterstained with hema-

toxylin.

Results

Mammary gland cytosol from 10 women carried out the NAT-cata-lyzed AcCoA-mediated binding of N-hydroxy-PhIP to calf thymus DNA(Fig. 1/1). The binding of N-hydroxy-PhIP to calf thymus DNA waslinear, with substrate up to 100 /LIMper incubation (Fig. Iß).The adduc-

O

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2.50

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1 23456789 10

Individuai cytosol

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so 100 150 200 250

N-OH-PhIP (jjM)Fig. 1. AcCoA-dependcnt NAT-medialed O-acetylation of /V-hydroxy-PhlP in human

mammary gland cylosol. A. AcCoA-depcndent binding of N-hydroxy-PhlP to calf thymusDNA in mammary gland cytosols from 10 women shown individually. The values are themean PhlP-DNA adduci levels observed in duplicate cytosolic incubations with N-hvdroxy-PhlP(2 JIM) in the absence (0) or presence (•)of AcCoA. B. AcCoA-depcndentbinding of W-hydroxy-PhIP to calf thymus DNA at différentconcentrations of W-hydroxy-PhlP. . incubations in the presence of AcCoA; -—,incubations in the absence ofAcCoA. The values are the mean PhlP-DNA adduci levels observed in two cytosolic

preparations run in duplicate: bars, SD. All cytosolic incubations were conducted asdescribed previously with PhlP-DNA adduci levels measured by ^-P-postlabeling (7). The

average W-hydroxy-PhIP adduci levels found in call thymus DNA incubated with AcCoAhut without cytosol was 0.24 (RAL X IO7).

tion of N-hydroxy-PhIP to DNA in the presence of human mammarygland cytosol was enhanced at least 50-fold by the addition of the NAT

cofactor AcCoA (Fig. 1). The NAT activation in human mammary glandvaried from 0.44 to 2.26 PhIP adducts (RAL X IO7),with an average of

0.94 ±0.5 (mean ±SD) in the 10 individuals (Table 1).Mammary gland cytosols also catalyzed the O-acetylation of N-

hydroxy-IQ as measured by the AcCoA-enhanced binding of N-hydroxy-IQ to calf thymus DNA in vitro (Table 1). O-Acetylationactivity toward /V-hydroxy-PhlP and A'-hydroxy-IQ was also observedin HMEC lysates (Table 1). /V-Acetylation of PAS. an activity specific

to NATI, was detected in all human mammary gland samples examined and in lysates of HMECs (Table 1). Female Sprague-Dawley ratmammary gland cytosol showed comparable PAS N-acetylation(641.22 ±310.7 pmol/min/mg protein, mean ±SD, n = 3) to that

seen with human mammary gland cytosol and with HMEC lysates. Incontrast, SMZ acetylation, an activity specific to NAT2, was not

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NAT EXPRESSION AND FOOD-DERIVED HKTEROCYCLIC AMINES

Table 1 N-Act'tyltranxferaxe activity in hnmtin mammary gland cytosol and in human

mammary epithelial cells

SubstrateN-OH-IQ

N-OH-PhIPPASSMZMammary

glandcytosol"nr

Mean ±SD10

2.34 ±1.56'10 0.94 ±0.50'

8 395 ±12/8 ND*HMEC

lysates*Mean

±SDJ7.01

±2.51'1.64 ±0.52'

840 ±18/ND

" Human mammary gland cytosol was prepared as described in "Materials and Methods."

' A primary culture of human mammary epithelial cells was lysed by sonicalion. and

the cell lysates were used.' Number of individual human mammary gland cytosol samples that were assayed in

duplicate.'' Mean of three to four separate experiments.'Relative adduci level (RAL) X IO7 nucleoiides/mg of cytosolic protein/20-min

incubation.•¿�'pmoles of W-acetylatedproduct formed/minute/mg of cytosolic protein.f ND. not detected.

detected with any of the human or rut mammary gland samples or inHMEC lysates (Table 1). SMZ /V-acetylation was, however, found in

rat liver cytosol (167.33 ±73.13 pmol/min/mg protein, mean ±SD,n = 4). In addition to the standard colorimetrie assay, /V-acetyl-SMZ

could not be detected in incubations with human mammary glandcytosol or HMEC lysate, using an HPLC method. Pentachlorphenol

(250 /*M), an inhibitor of NAT activity, inhibited PAS JV-acetylationand iV-hydroxy-PhlP O-acetylation reactions by an average of 60% in

both human mammary gland cytosolic incubations and in HMEC celllysate incubations (data not shown).

Immunohistochemical analysis of human mammary gland samplesshowed positive staining using an NATl-specific antibody (Fig. 2).

The staining was limited to the ductular epithelial cells of the mammary gland. Notably, no staining was observed in control slides wherethe primary antibody was omitted (Fig. 2, B and F} or in samplesincubated with rabbit serum (Fig. 2D).

RNA was isolated from frozen human mammary gland samples andreverse transcribed using NATI- and A/A72-specific primers, as de

scribed previously (19). When the total RNA from 12 individualwomen was isolated and analyzed, all showed the presence of NATIand NAT2 transcripts (Fig. 3A). The authenticity of the products wasconfirmed by RFPL analysis using Hindi, which digests the NAT2product (907 bp) into two fragments (659 and 248 bp), and ///«dill,which cleaves the NATI product (861 bp) into a 786-bp fragment and75-bp fragment (Fig. 3ß).

Discussion

The metabolic activation of the HCAs at particular tissue sites maybe one important determinant of the target organ specificity tor HCA

Fig. 2. Immunohistochemical localization of NATI inrepresentative human mammary gland sections. A. humanmammary gland stained with anti-NATI (X 1(K)).B. controlstaining without primary antibody (XIOO). C. human mammary gland stained with anli-NATl (X200). D. controlstaining with rabbit IgG (X200I. £.human mammary glandstained with anli-NATl (X400). F. control staining withoutprimary antibody (X4(X)). Anti-NATI antibody is a poly-clonal antibody raised in rabbit against recomhinant E. coli-expressed human NATI. Slides were counterstained withhematoxylin.

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NAT EXPRESSION AND FOOD-DERIVED HETEROCYCLIC AMINES

Sample

NATI

NAT 2

123456 789 10

~ - ~

L l 2 3B

861786

•¿�659

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Fig. 3. Expression of NATI and NAT2 in human mammary gland as determined byRT-PCR analysis. A. RT-PCR analysis of human mammary gland RNA from 10 womenusing NATI and NAT2-specific primers. B, RFLP patterns of NAT] (Limes 1-3) andNAT2 (Lanes 4-6) from the PCR amplification of mammary gland RNA was digested

with Hindi (Lanes 2 and 5) or Hindlll (Lanes 3 and 6), or without enzymes (Lanes I and4). Arrows, NATI and NAT2 PCR products and restriction fragment sizes. RFLP is shownfor one representative individual. L. ladder.

carcinogenesis. With regard to the mammary gland, studies in ratshave suggested that the in situ O-acetylation capacity of the rat

mammary gland may be associated with the susceptibility of the glandto PhlP-induced carcinogenesis (21). With the present study, we

demonstrate, for the first time, that NATI is expressed in the humanmammary gland ductular epithelial cells and that it is catalyticallyactive toward the N-acelylation of PAS and the O-acetylation ofA/-hydroxylamines. Human mammary gland cytosols catalyzed theAcCoA-dependent O-acetyltransferase-mediated binding of N-hy-droxy-PhlP and W-hydroxy-IQ to DNA in vitro. This activity was also

observed in lysate of a primary culture of HMECs. Comparison of theO-acetylation activity observed in human mammary gland cytosol

with that reported previously for rat mammary gland cytosol (21)indicates that O-acetylation of A/-hydroxy-PhIP and A/-hydroxy-IQ is

exceptionally high in the rat, despite the similar activity of PASiV-acetylation (reported herein). This finding suggests, in part, that theiV-hydroxy-HCA substrate specificity is different between the rat and

the human mammary gland NATs. Although the limited in vitro datasuggest that the human may be less susceptible than the rat toHCA-induced mammary carcinogenesis, it may be hasty to speculate

on the relative susceptibility of the human mammary gland to theHCAs. Nonetheless, the finding that human mammary gland NAT iscatalytically active toward /V-hydroxy-HCA is consistent with the

notion that the human mammary gland may be susceptible to thecarcinogenic effects of these compounds.

In humans, two Af-acetyltransferases, NATI and NAT2, canO-acetylate the /V-hydroxylamine derivatives of the HCAs (8-9). Theresults from the studies shown here suggest that O-acetylation of(V-hydroxy-PhIP and /V-hydroxy-IQ in the human mammary gland is

largely carried out by NATI. This conclusion is supported by thefinding that SMZ 7V-acetylation, an NAT2 activity, was not detectable

in mammary gland cytosols or HMEC cell lysate, whereas PASW-acelylalion, an NATI activity, was clearly observed in both thecytosols and cell lysates. The results from RT-PCR, although not

quantitative, indicate that NAT2 as well as NATI mRNA is ex

pressed, and thus we cannot rule out the possibility of a very low levelof NAT2 activity in the human mammary gland. Nevertheless, theconclusion that NATI is the predominant NAT responsible for theyV-acetyllransferase activity in the human mammary gland is consist

ent with the finding that other extrahepatic tissues, such as colon andbladder epithelium, show NATI activity but lack detectable NAT2activity (14, 22). In addition, the results from our immunohistochem-ical studies of the human mammary gland using an NATI-specific

antibody further demonstrate the expression of NATI protein in theepithelium lining the ducts of the human mammary gland.

Recent studies have shown that NATI, previously referred to as amonomorphic enzyme, is polymorphic owing to allelic variances thatalter tissue levels of the enzyme and hence NATI activity (11-14). Inthe human bladder, a specific NATI genetic polymorphism (NATI* 10

alíele),which increases NATI expression in the bladder, is associatedwith higher carcinogen-DNA adduci levels (14). Furthermore, studies

in the human colon showed that NATI genetic polymorphisms thatincrease NATI activity also increase the risk of colorectal cancer (12,13). Thus, the evidence to date suggests that NATI genetic polymorphisms that cause variations in in situ metabolic activation may playan important role in target organ susceptibility. Notably, our resultsshow that NATI is expressed in the ductal epithelial cells, which areconsidered to be the cells associated with mammary gland cancer (23).Thus, metabolic activation of the A/-hydroxylamines by O-acetylation

is likely to occur at the critical site for the initiation of mammarygland cancer by the HCAs. In addition, the variation in W-hydroxy-PhlP O-acetylation observed in the mammary gland samples raises the

possibility that the NATI genotype, and hence susceptibility to HCAcarcinogenesis, may be different among the women we examined.

Several epidemiológica! studies have failed to show an associationbetween NAT acetylation phenotype and the incidence of breastcancer in women (24, 25). However, these studies measuredA'-acetylated SMZ in blood samples from women given SMZ and,

therefore, assessed the NAT2 rather than the NATI acetylator phenotype. In light of previous studies supporting the association betweenthe NATI genotype and colorectal cancer and our current studyshowing that NATI is catalytically active and expressed in the humanmammary gland, additional studies are needed to determine if the NATIgenotype is a risk factor for human mammary gland cancer. In addition,to better assess the role of dietary HCAs in human breast cancer, theNATI genotype and human mammary gland cancer incidence amongwomen who regularly eat well-done cooked meats is warranted.

Acknowledgments

We thank Dr. Denis G. Gram (Hospital for Sick Children, Toronto, Ontario,Canada) for providing the polyclonal anti-human NATI antibody and technical

advice. Dr. Herman A. J. Schul (Medical College of Ohio, Toledo, Ohio) forthe 32P-postlabeling analysis, and Dr. Snorri S. Thorgeirsson (National Cancer

Instilute, Bethesda. Maryland) for support and helpful discussions.

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1996;56:2683-2687. Cancer Res   Nakissa Sadrieh, Cindy D. Davis and Elizabeth G. Snyderwine  GlandFood-derived Heterocyclic Amines in the Human Mammary

-Acetyltransferase Expression and Metabolic Activation of theN

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