Effects of ibuprofen on arylamine N-acetyltransferase activity in human colon tumor cells

Article abstract of DOI:10.1002/(SICI)1099-1263(199901/02)19:1<1::AID-JAT527>3.0.CO;2-3

The inhibition of arylamine N-acetyltransferase (NAT) activity by ibuprofen was determined in a human colon tumour (adenocarcinoma) cell line. Two assay systems were employed, one with cellular cytosols (9000 g supernatant) and the other with intact colon tumour cell suspensions. The NAT activity in a human colon tumour cell line was inhibited by ibuprofen in a dose-dependent manner in both systems, i.e. the greater the concentration of ibuprofen in the reaction, the greater the inhibition of NAT activities In both systems. The data also indicated that ibuprofen decreases the apparent K(m) and V(max) of NAT enzyme from human colon tumour cells in both systems examined. This report is the first demonstration to show that ibuprofen affects human colon tumour cell NAT activity.

Full text of DOI:10.1002/(SICI)1099-1263(199901/02)19:1<1::AID-JAT527>3.0.CO;2-3

JOURNAL OF APPLIED TOXICOLOGY
J. Appl. Toxicol. 19, 1–6 (1999)
Effects of Ibuprofen on Arylamine
N-Acetyltransferase Activity in Human Colon
Tumor Cells
Jing-Gung Chung1,*, Huei-Ling Chang2, Wen-Chuan Lin2, Feng-Tsgh Yeh3 and Chi-Fu
Hung4
1Department of Microbiology, China Medical College, Taichung 400, Taiwan, Republic of China
2Department of Pharmacology, China Medical College, Taichung 400, Taiwan, Republic of China
3Graduate Institute of Chinese Pharmaceutical Sciences, China Medical College, Taichung 400, Taiwan, Republic of
China
4Department of Surgery, Jen-Ai Hospital, 483 Tong-Rong Road, Tali, Taichung 400, Taiwan, Republic of China
Key words: ibuprofen; N-acetyltransferase; 2-aminofluorene; N-acetyl-2-aminofluorene; p-aminobenzoic acid; N-acetyl-p-
aminobenzoic acid.
The inhibition of arylamine N-acetyltransferase (NAT) activity by ibuprofen was determined in a human
colon tumour (adenocarcinoma) cell line. Two assay systems were employed, one with cellular cytosols
(9000 g supernatant) and the other with intact colon tumour cell suspensions. The NAT activity in a
human colon tumour cell line was inhibited by ibuprofen in a dose-dependent manner in both systems,
i.e. the greater the concentration of ibuprofen in the reaction, the greater the inhibition of NAT
activities in both systems. The data also indicated that ibuprofen decreases the apparent Km and Vmax
of NAT enzyme from human colon tumour cells in both systems examined. This report is the first
demonstration to show that ibuprofen affects human colon tumour cell NAT activity.
Wiley & Sons, Ltd.
1998 John
reported in tissues of human colon and bladder.11 Thus,
the genetically mediated variation in acetyltransferase
activities within target organs for arylamine-induced
neoplasm may indicate different risks among the
human population.
INTRODUCTION
Exposure to environmental and occupational carcino-
gens is thought to be responsible for a large proportion
of human cancers. Arylamine carcinogens are well-
known potent carcinogens1 that require metabolic acti-
vation by host enzymes as a prerequisite to the
initiation of carcinogenesis in target organs and
tissues.2 The major site of arylamine metabolism in the
body is usually the liver, however N-acetyltransferase
(NAT), an enzyme mainly in the liver involved in
several steps of both arylamine activation and detox-
ification, is also found in many types of tissues and in
humans.3–6 In fact, in mice the potential N-acetylation
capacity of extrahepatic tissue exceeds that of the
liver.5 N-Acetyltransferase localizes in the cytosol frac-
tion of the liver and other organs or tissues and requires
Ibuprofen, derived from propionic acid, is a non-
steroidal anti-inflammatory analgesic that is relatively
safe and effective for the treatment of pain, dys-
menorrhoea, inflammation and fever, acting as a
cyclooxygenase inhibitor.12,13 Numerous studies have
indicated that ibuprofen may have anti-cancer proper-
ties. Ibuprofen inhibits the growth of some tumour
cells,14 reduces tobacco-specific carcinogen 4-
(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced
pulmonary and gastric tumorigenesis in A/J mice,15
exhibits anti-proliferative effects against human colon
cancer cells,16,17 suppresses aberrant crypt formation or
the progression to foci of multiple aberrant crypts in
the rat colon,18 affects the in vitro invasiveness of a
human transitional bladder cell carcinoma,19 and has
chemopreventive potential against the development of
breast cancer.20
Studies in our laboratory indicate that ibuprofen
also inhhibits NAT activity in Klebsiella pneumoniae.21
There are no reports that address whether ibuprofen
alters the acetylation of arylamine carcinogens in
human tumour cells, therefore the purpose of the
present study was to elucidate the possible effects of
ibuprofen on NAT activity in colon tumour cells. Our
initial choice of 2-aminofluorene (AF) and p-aminoben-
zoic acid (PABA) as test substrates was based on
previous studies in mice5 and current interest in com-
paring the metabolism of a carcinogen (AF) to a non-
acetyl-coenzyme
A (acetyl-CoA) for its activity.
Attenuation of NAT activity in the liver has been
associated with several disease processes.7,8 N-Acetyl-
transferase in the liver of humans and other mammals
shows a genetic polymorphism resulting in rapid, slow
and some intermediate acetylation phenotypes.6,9–10 In
humans, the effect and toxicity of a number of drugs
and carcinogens have been reported to differ markedly
among individuals and to depend on the N-acetylation
polymorphism. Extrahepatic expression of acetylator-
genotype-dependent NAT activity has also been
* Correspondence to: Dr Jing-Gung Chung, Department of Micro-
biology, China Medical College, Taichung 400, Taiwan, Republic
of China
Received 26 March 1998
Revised 16 June 1998
Accepted 21 July 1998
CCC 0260–437X/99/010001–06$17.50
1998 John Wiley & Sons, Ltd.

2
J.-G. CHUNG ET AL.
carcinogen (PABA). The results presented in this report
demonstrate that by using AF and PABA as substrates
for NAT activity determinations, ibuprofen did decrease
colon tumour cell NAT activity in cytosols and in
intact cells.
measurements, the final concentrations were Ͻ 0.1 mM
for AF or PABA and 0.5 mM for acetyl-CoA. The
reaction mixtures with or without specific concen-
trations of ibuprofen were incubated at 37°C for 10 min
and stopped with 50 ␮l of 20% trichloroacetic acid for
the PABA reactions and with 100 ␮l of acetonitrile for
the AF reactions. All the reactions (experiments and
controls) were run in triplicate. The amounts of acetyl-
ated product and remaining non-acetylated substrate
were determined by HPLC.21,22 An aliquot of the reac-
tion mixture was injected onto a C18 reversed-phase
column (Spherisorb, 4.6 × 250 mm) of a Beckman
HPLC (pump 168 and detector 126) and eluted at a
flow rate of 1.2 ml min−1. For PABA and N-Ac-PABA,
the solvent system was 50 mM acetic acid/CH3CN
(86:14) with detection at 266 nm. The retention time
for PABA was 8.0 min and that for N-Ac-PABA was
11.0 min. For AF and AAF, the solvent system was
20 mM KH2PO4 (pH 4.5)/CH3CN (53:47) with detec-
tion at 280 nm. The retention time for AAF was
6.5 min and that for AF was 9.0 min. All the com-
pounds were quantitated by comparison of the inte-
grated area of the elution peak with that of known
amounts of standards. The NAT activity was expressed
as nmol acetylated substrate min−1 mg−1 cytosolic pro-
tein.
EXPERIMENTAL
Chemicals and reagents
Ibuprofen, ethylenediaminetetraacetic acid (EDTA), p-
aminobenzoic acid (PABA), N-acetyl-p-aminobenzoic
acid (N-Ac-PABA), acetylcarnitine, leupeptin, bovine
serum albumin (BSA), phenylmethylsulphonylfluoride
(PMSF). TRIS, dimethyl sulphoxide (DMSO), dithi-
othreitol (DTT) and carnitine acetyltransferase were
obtained from Sigma Chemical Co. (St. Louis, MO).
Acetyl-coenzyme A (acetyl-CoA) was obtained from
P-L Biochemicals Inc. (Milwaukee, WI). 2-Aminofluo-
rene (AF) and N-acetyl-2-aminofluorene (AAF) were
obtained from K and K Laboratories (Plainview, NY).
All the chemicals used were reagent grade.
Human colon tumour cell line
Human colon adenocarcinoma cell line (Colo 205 from
a male 70-year-old Caucasian) were obtained from the
Taipei Veterinary Hospital (Taipei, Taiwan). The cells
were placed into 75-cm2 tissue culture flasks and grown
at 37°C under a humidified 5% CO2 atmosphere in
RPMI 1640 medium (Sigma Chemical Company,
St. Louis, MO) supplemented with 10% fetal bovine
serum (Gibco BRL, Grand Island, NY) and 2% penicil-
lin–streptomycin (10,000 U ml−1 penicillin and
10 mg ml−1 streptomycin).
Intact cell NAT activity determination
Human colon tumour cells (in 1 ml of RPMI 1640
medium with glutamine and 10% fetal calf serum)
were incubated with arylamine substrate at 1 × 106
cells ml−1 in individual wells of 24-well cell culture
plates with or without ibuprofen co-treatment for the
time indicated at 37°C in 95% air/5% CO2. At the
end of incubation, the cells and media were removed
and centrifuged. For experiments with AF, the
supernatant was immediately extracted with ethyl
acetate/methanol (95:5), the solvent was evaporated
and the residue was redissolved in methanol and
assayed for AAF as described above. For experiments
with PABA, aliquots of the supernatant were assayed
directly for N-Ac-PABA.
Preparation of human colon tumour cell cytosols
Approximately 5 × 107 cells were placed in 2 ml of
the lysis buffer (20 mM TRIS·HCl, pH 7.5, at 4°C,
1 mM DTT, 1 mM EDTA, 50 ␮M PMSF and 10 ␮M
leupeptin) as described previously.4 The suspensions
were centrifuged at 9000 g for 1 min in a Model 3200
Eppendorf/Brinkman centrifuge and the supernatant
fraction was subsequently centrifuged at 10,000 g for
60 min. The supernatant was kept on ice for NAT
activity and protein determinations.
Protein determination
Protein concentrations in the human colon tumour cell
cytosols were determined by the method of Bradford23
with bovine serum albumin as the standard. All the
samples were assayed in triplicate.
N-Acetyltransferase activity determinations
Statistical treatment of data
The determination of acetyl-CoA-dependent N-acety-
lation of PABA and AF was performed as described
by Chung et al.5 Incubation mixtures in the assay
system consisted of a total volume of 90 ␮l: cell
cytosol in 50 ␮l of lysis buffer (20 mM TRIS·HCl, pH
7.5, 1 mM DTT and 1 mM EDTA); 20 ␮l of an acetyl-
CoA recycling mixture of 50 mM TRIS·HCl (pH 7.5),
0.2 mM EDTA, 2 mM DTT, 15 mM acetylcarnitine,
and 2 U ml−1 carnitine acetyltransferase and AF or
PABA at the specified concentrations, and the reactions
were started by the addition of 20 ␮l of acetyl-CoA.
The control reactions had 20 ␮l of distilled water
instead of acetyl-CoA. For the single-point activity
Statistical analysis of the data was performed with an
unpaired student’s t-test. The kinetic constants were
calculated with the Cleland HYPER Program,24 which
performs linear regression using a least-squares method.
RESULTS
Effects of various concentrations of ibuprofen on
human colon tumour cell NAT activity in cytosol
The possible effects of ibuprofen on NAT activity in
human colon tumour cells, both in cytosols and in
1998 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 19, 1–6 (1999)

IBUPROFEN EFFECTS ON ARYLAMINE N-ACETYLTRANSFERASE ACTIVITY
3
intact cells, were examined by HPLC, assessing the
percentage of acetylation of AF and PABA. The means
± SD of NAT activity co-treated with or without
ibuprofen with both substrates are given in Table 1.
The data indicate that there is decreased NAT activity
associated with increased ibuprofen both in cytosols
and in intact cells. For the cytosol examinations in the
presence of 0.02, 0.2, 2 and 20 mM ibuprofen, the
NAT activities were inhibited by 6.2, 34.3, 53.1 and
67.1% for AF and by 6.6, 28.3, 53.8 and 71.7% for
PABA, respectively. For the intact cell examinations
in the presence of 0.02, 0.2, 2 and 20 mM ibuprofen,
the NAT activities were inhibited by 4.2, 31.6, 54.7
and 76.1% for AF and by 4.1, 25.0, 52.0 and 80.2%
for PABA, respectively.
Effects of 2 mM ibuprofen on human colon
tumour cell NAT activity in intact cells
The NAT activities for the acetylation of AF (15,
30, 60 and 100 ␮M) measured from intact cells were
1.68± 0.08, 3.10 ± 0.34, 6.48 ± 0.69 and 5.89 ± 0.48
nmol 10−6 cells for acetylation of AF without co-
treatment with ibuprofen. In the presence of 2 mM
ibuprofen, the NAT activities were decreased by about
43–55% for AF (Fig. 1a). The NAT activities for the
acetylation of PABA (15, 30, 60 and 100 ␮M)
measured from intact cells were 1.18 ± 0.07,
2.46 ± 0.30, 5.12 ± 0.43 and 5.06 ± 0.46 nmol 10−6
cells, respectively, for acetylation of PABA without
co-treatment with ibuprofen. In the presence of 2 mM
ibuprofen, the NAT activities were decreased by about
48–59% (Fig. 1b).
Figure 1. Effects of ibuprofen on the production of AAF and
N-Ac-PABA by human colon tumour cells. Tumour cells were
incubated as described for 18 h with AF (a) and PABA (b) and
with 2 mM ibuprofen co-treatment. Both AAF and N-Ac-PABA
were measured by HPLC assay. Each point represents the
mean of triplicate assays of three incubations of cells.
Time course effect of 2 mM ibuprofen on NAT
activity in human colon tumour intact cells
To determine the time course effect of 2 mM ibuprofen
on NAT activity in human colon tumour cells, the
cells were incubated at 37°C and harvested at 6, 12,
18 and 24 h, respectively. Increased time of incubation
led to increased AAF and N-Ac-PABA production up
to 24 h (Fig. 2). Figure 2 illustrates the time course of
AAF and N-Ac-PABA production from human colon
tumour cells at both 30 and 60 ␮M AF and PABA.
Effect of 2 mM ibuprofen on kinetic constants of
NAT activity in human colon tumour cells
In the presence or absence of ibuprofen, specific con-
centrations of AF and PABA (0.373, 0.435, 0.543,
0.745, 1.102 and 2.205 mM, respectively) were added
to the recycling mixtures for determining human colon
Table 1. Effects of ibuprofen on human colon tumour cell NAT activity in cytosol and in intact cellsa
Concentration
of ibuprofen
In cytosol
N-Ac-PABA
In intact cells
N-Ac-PABA
AAF
AAF
Control
0.02 mM
0.2 mM
2 mM
1.28 ± 0.23
1.20 ± 0.18
0.84 ± 0.12*
0.60 ± 0.08**
0.42 ± 0.04***
1.06 ± 0.22
0.99 ± 0.14
0.76 ± 0.10*
0.49 ± 0.08**
0.30 ± 0.04***
1.17 ± 0.26
1.12 ± 0.18
0.80 ± 0.16*
0.53 ± 0.08**
0.28 ± 0.04***
0.96 ± 0.20
0.92 ± 0.12
0.72 ± 0.10*
0.46 ± 0.06**
0.19 ± 0.03***
20 mM
aValues are the mean ± SD of determinations on three experiments. The NAT activity is expressed as nmol/min−1 mg−1 protein
in cytosol and as nmol/min−1 10−6 cells in intact cells. Significant differences between 0.2 mM ibuprofen and control: *PϽ0.05, **
PϽ0.01 and ***PϽ0.001.
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J.-G. CHUNG ET AL.
values were decreased by 54.2% and 49.6% for acety-
lation of AF and by 11.3% and 13.6% for acetylation
of PABA, respectively. For the intact cell examinations,
the apparent Km and Vmax values were 6.99 ± 0.72 mM
and 40.00 ± 7.89 nmol min−1 mg−1 protein for AF and
3.37 ± 0.48 mM and 14.70 ± 2.25 nmol min−1 mg−1
protein for PABA, respectively (Tables 2 and 3). When
2 mM ibuprofen was added to the reaction mixtures,
the apparent Km and Vmax values were decreased by
33.0% and 36.8% for acetylation of AF and by 15.1%
and 24.7% for acetylation of PABA, respectively.
Clearly, the Km and Vmax values for the human colon
tumour cell NAT activity were decreased in the pres-
ence of ibuprofen both in cytosol and in intact cell
examinations.
DISCUSSION
The NAT enzyme has been reported to exist in many
kinds of experimental animals, including humans, and
NAT has been shown to be involved in some chemical
carcinogenesis.25,26 The genetically mediated variation
in NAT activities within target tissues for arylamine-
induced neoplasm may indicate differential risks among
the human population. It was reported that colorectal
adenoma and cancer risks were in relation to meat
intake and acetylator status.27 Arylamine NAT activity
towards AF and PABA was detected in human colon
tumour cells.28 Ibuprofen decreased the NAT activity
of K. pneumoniae in cytosol and in intact bacteria.22 It
is reasonable to hypothesize that ibuprofen may also
alter the acetylation of arylamine carcinogens such as
AF, therefore the present studies were focused on the
ibuprofen effects on NAT activity in human colon
tumour cells.
Ibuprofen, clinically used as an anti-inflammatory
drug, has been considered as a possible chemopreven-
tive drug. We were interested in studying whether or
not ibuprofen would affect the NAT activity of human
colon tumour cells both in cytosol and in intact cells.
We found that the NAT activities decreased as the
ibuprofen concentration increased, i.e. the higher the
concentration of ibuprofen, the higher the inhibition of
NAT activity. In order to simulate the conditions of
the human large intestine, a pH of 7.5 was selected
for all the experiments. This pH is close to that of the
normal large intestine. The data from the cytosol and
intact human colon tumour cell studies showed that
Figure 2. Effects of incubation time on AAF and N-Ac-PABA
production by human colon tumour cells. Tumour cells were
incubated with AF (a) and PABA (b) at 30 ␮M and 60 ␮M and
with 2 mM ibuprofen co-treatment for the time shown. Both
AAF and N-Ac-PABA were measured by HPLC assay. Each
point represents the mean of triplicate assays of three
incubations of cells.
tumour cell NAT kinetic constants. For the cytosol
examinations, the apparent Km and Vmax values were
7.04 ± 1.26 mM and 28.57 ± 6.18 nmol min−1 mg−1 pro-
tein for AF and 2.21 ± 0.18 mM and 13.16
±
1.99 nmol min−1 mg−1 protein for PABA, respectively
(Tables 2 and 3). When 2 mM ibuprofen was added
to the reaction mixtures, the apparent Km and Vmax
Table 2. Kinetic data for acetylation of 2-AF in human colon tumour cellsa
In cytosol
In intact cells
Vmax(nmol) 10−6 cells)
Km (mM)
Vmax(nmol/min−1 mg−1
protein)
Km(mM)
Control
Ibuprofen
7.04 ± 1.26
3.22 ± 0.32*
28.57 ± 6.18
14.38 ± 2.40***
6.99 ± 0.72
4.68 ± 0.64†
40.00 ± 7.89
25.26 ± 4.92***
aValues are means ± SD; n = 3. The acetyl-CoA and ibuprofen concentrations were 0.1 mM and 2 mM, and the kinetic constants
were calculated from the modified HYPER Program of Cleland.24 Significant differences between 2 mM ibuprofen and control:
PϽ0.05, ***PϽ0.001 and †PϽ0.005.
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IBUPROFEN EFFECTS ON ARYLAMINE N-ACETYLTRANSFERASE ACTIVITY
5
Table 3. Kinetic data for acetylation of PABA in human colon tumour cellsa
In cytosol
In intact cells
Vmax(nmol) 10−6 cells)
Km (mM)
Vmax(nmol/min−1 mg−1
Km(mM)
protein)
Control
Ibuprofen
2.21 ± 0.18
1.96 ± 0.16*
13.16 ± 1.99
11.44 ± 1.52***
3.37 ± 0.48
2.86 ± 0.44†
14.70 ± 2.25
11.06 ± 1.42***
aValues are means ± SD; n = 3. The Acetyl CoA and ibuprofen concentrations were 0.1 mM and 2 mM, and the kinetic constants
were calculated from the modified HYPER Program of Cleland.24 Significant differences between 2 mM ibuprofen and control: *
PϽ0.05, ***PϽ0.001 and †PϽ0.005.
there were significant differences of NAT activity
between the control and ibuprofen treatment groups.
The present studies demonstrate that ibuprofen can
markedly inhibit the NAT activity of human colon
tumour cells in both examined systems and that the
inhibition is dose dependent. The data also indicate
that ibuprofen decreases the NAT kinetic constants in
the cytosols and in intact cells. Although it is not
known whether a decrease of NAT activity would
decrease tumour production or whether ibuprofen could
prevent the development of colon cancer, other investi-
gators have demonstrated that elevated levels of NAT
activity are associated with increased sensitivity to the
mutagenic effects of many arylamines.29 It has reported
that attenuation of liver NAT activity is related to
bladder and breast cancer.7 Further investigation is
needed.
the O-acetylation of the N-hydroxy metabolites to
DNA-reactive metabolites,31,32 which could initiate
large bowel cancer. Recently, it has been demonstrated
that the combination of fast-acetylator phenotypes with
a high consumption of meat increased the risk of
colorectal cancer.27 The present study may offer some
information about ibuprofen effects on NAT activity,
because NATs are expressed in human ileum and colon
and are able to metabolize arylamine compounds.33,34
In conclusion, this study indicates that ibuprofen
inhibits NAT activity in human colon tumour cells in
vitro and in intact cells. This report is also the first
demonstration to show that ibuprofen can inhibit the
NAT activity of a human colon tumour cell line.
Acknowledgement
Flammang and colleagues have found high levels of
acetyltransferase activity in human colonic mucosa.30
They have also found that the mucosa itself catalyses
This work was supported by grant CMC86-M-11 from the Research
Section of China Medical College.
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