Ibuprofen affects arylamine N-acetyltransferase activity in Helicobacter pylori from peptic ulcer patients

Article abstract of DOI:10.1002/(SICI)1099-1263(199805/06)18:3<179::AID-JAT494>3.0.CO;2-W

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene and p-aminobenzoic acid were determined in the bacterium Helicobacter pylori collected from peptic ulcer patients. Cytosols or suspensions of H. pylori with or without specific concentrations of ibuprofen co-treatment showed different percentages of 2-aminofluorene and p-aminobenzoic acid acetylation. The data indicate that there was decreased NAT activity associated with increased levels of ibuprofen in H. pylori cytosols. Inhibition of growth studies on H. pylori demonstrated that ibuprofen elicited a dose-dependent bactericide effect in H. pylori cultures, i.e. the greater the concentration of ibuprofen, the greater the inhibition of growth to H. pylori. For the cytosol and intact bacteria examinations, the apparent values of K(m) and V(max) were decreased after co-treatment with 40 μM ibuprofen. This report is the first demonstration of ibuprofen inhibition of arylamine N- acetyltransferase activity and ibuprofen inhibition of growth in the bacterium H. pylori.

Full text of DOI:10.1002/(SICI)1099-1263(199805/06)18:3<179::AID-JAT494>3.0.CO;2-W

JOURNAL OF APPLIED TOXICOLOGY
J. Appl. Toxicol. 18, 179–185 (1998)
Ibuprofen Affects Arylamine
N-Acetyltransferase Activity in Helicobacter
pylori from Peptic Ulcer Patients
1
,
1
2
2
2
Shih H. Chang, † Jing G. Chung, Li J. Huang, Sheng C. Chen, and Sheng C. Kuo
1
Department of Medicine, China Medical College, Taichung, Taiwan, Republic of China
Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan, Republic of China
2
Key words: ibuprofen; N-acetyltransferase; 2-aminofluorene; N-acetyl-2-aminofluorene; p-aminobenzoic acid; N-acetyl-p-
aminobenzoic acid.
Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene and p-aminobenzoic acid were
determined in the bacterium Helicobacter pylori collected from peptic ulcer patients. Cytosols or
suspensions of H. pylori with or without specific concentrations of ibuprofen co-treatment showed
different percentages of 2-aminofluorene and p-aminobenzoic acid acetylation. The data indicate that
there was decreased NAT activity associated with increased levels of ibuprofen in H. pylori cytosols.
Inhibition of growth studies on H. pylori demonstrated that ibuprofen elicited a dose-dependent
bactericide effect in H. pylori cultures, i.e. the greater the concentration of ibuprofen, the greater the
inhibition of growth to H. pylori. For the cytosol and intact bacteria examinations, the apparent values
of Km and Vmax were decreased after co-treatment with 40 ␮M ibuprofen. This report is the first
demonstration of ibuprofen inhibition of arylamine N-acetyltransferase activity and ibuprofen inhibition
of growth in the bacterium H. pylori.  1998 John Wiley & Sons, Ltd.
been linked to increased susceptibility to occupational
INTRODUCTION
10
bladder cancer.
Helicobacter pylori has been reported to be present
1
1
Exposure to environmental and occupational carcino-
gens is thought to be responsible for a large portion
of human cancers. Arylamine carcinogens are one of
the most potent carcinogens. Once in the body, arylam-
ine carcinogens are metabolized either to detoxification
products, which are excreted, or they may be further
activated to induce toxicities in target organs and
tissues. The major site of arylamine metabolism in the
body is usually the liver. However, N-acetyltransferase
in the stomachs of patients. It has also been reported
that there is an aetiological correlation of H. pylori
1
2
with active chronic gastritis. Helicobacter pylori is a
possible causative factor in patients with gastric can-
1
3
cer. Based on reports, it has been indicated that
chronic atrophic gastritis is a precursor lesion of gastric
cancer, and chronic gastritis might be induced or
1
4,15
exacerbated by H. pylori
because gastritis, peptic
ulcer and gastric cancer are related to the presence of
this bacterium. The present author’s previous studies
1
6
(NAT), an enzyme mainly found in the liver involved
in several steps of both arylamine activation and detox-
have already demonstrated that H. pylori contains NAT
1
17
ification, is also found in many types of tissues and in
activity in cytosols and in intact bacteria. Therefore,
2–5
humans. In fact, in mice the potential N-acetylation
capacity of extrahepatic tissues exceeds that of the
the investigation of the relationship between H. pylori
and 2-aminofluorene (2-AF) metabolism was under-
taken.
2
liver. Attenuation of NAT activity in the liver has
been associated with several disease processes, such as
Ibuprofen, derived from propionic acid, is one of
the non-steroidal anti-inflammatory analgesics acting as
1,6
bladder and breast cancer. Thus, there is much inter-
est in the role of NAT in chemical carcinogenesis.
Importantly, NAT in the liver of humans and other
mammals shows a genetic polymorphism resulting in
rapid, slow and some intermediate acetylation pheno-
1
8
a cyclooxygenase inhibitor. It has been reported that
ibuprofen is safe and effective for the treatment of
1
9
pain, dysmenorrhoea, inflammation and fever. Several
lines of evidence suggest that ibuprofen may be effec-
tive in preventing cancer:
4,5,7
types.
In humans, the effect and toxicity of a num-
ber of drugs and carcinogens have been reported to
differ markedly among individuals and to depend on
(
i) ibuprofen can reduce tobacco-specific carcinogen
4
-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-
1,6
the N-acetylation polymorphism. For humans, epide-
miological studies suggest that the rapid acetylator
phenotype has been linked to increased risks of col-
induced pulmonary and gastric tumorigenesis in
20
A/J mice,
(ii) ibuprofen exhibits anti-proliferative effects against
8,9
orectal cancer and the slow acetylator phenotype has
21
human colon cancer cells,
(iii) ibuprofen is effective in suppressing aberrant
crypt formation or the progression to foci of
†
Correspondence to: S. H. Chang, Department of Medicine, China
Medical College, Taichung 400, Taiwan, Republic of China.
22
multiple aberrant crypts in the rat colon;
CCC 0260–437X/98/030179–07$17.50
Received 10 July 1997
Accepted 15 December 1997

1998 John Wiley & Sons, Ltd.

1
80
S. H. CHANG ET AL.
(
(
(
iv) ibuprofen inhibits the growth of some tumour
sol, diluted as required in 50 ␮l of lysis buffer (20 mM
TRIS·HCl, pH 7.5, 1 mM DTT, 1 mM EDTA and
1 mM acetylcarnitine), and 2-AF or PABA at a specific
concentration for the substrate. The reactions were
started by addition of 20 ␮l of Ac-CoA. The control
reactions had 20 ␮l of distilled water in place of Ac-
CoA. The final concentration of PABA or 2-AF was
0.1 mM, and that for Ac-CoA was 0.5 mM. The reac-
tion mixtures were incubated at 37°C for 10 min and
stopped with 50 ␮l of 20% trichloroacetic acid for
PABA reactions and 100 ␮l of acetonitrile for 2-AF
reactions. All of the reactions of the experiments and
controls were run in triplicate. The amounts of acetyl-
ated product and remaining non-acetylated substrate
23
cells;
v) ibuprofen affects the in vitro invasiveness of a
24
human transitional bladder cell carcinoma;
vi) ibuprofen has chemopreventive potential against
25
the development of breast cancer and is con-
sidered to be a potential cancer chemopreven-
26
tive agent.
There are no reports available that address whether
ibuprofen would alter the acetylation of arylamine car-
cinogens. Therefore, the purpose of the present study
was to elucidate the possible effects of ibuprofen on
NAT activity and growth in the bacterium H. pylori.
The initial choice of 2-AF and p-aminobenzoic acid
6
,19
were determined by HPLC.
An aliquot of the NAT
(
PABA) as test substrates was based on previous stud-
incubation mixture was injected onto the C18 reversed-
phase column (Spherisorb 4.6 × 250 mm) of a Beckman
HPLC (pump 168 and detector 126) and eluted at a
6 19
ies in mice, bacteria and the author’s interest in
comparing the metabolism of a carcinogen (2-AF) to
a non-carcinogen (PABA). The results presented in this
report demonstrated that by using 2-AF and PABA as
substrates for NAT activity determinations, ibuprofen
did decrease H. pylori NAT activity in cytosols and
in intact bacteria.
−1
flow rate of 1.2 ml min . For PABA and N-Ac-PABA,
the solvent system was 50 mM acetic acid/CH CN
3
(
86:14), with detection at 266 nm. The retention time
for PABA was 4.0 min and that of N-Ac-PABA was
.5 min. For 2-AF and 2-AAF, the solvent system
was 20 mM KH PO (pH 4.5)/CH CN (53:47), with
6
2
4
3
detection at 280 nm. The retention time was 6.5 min
for 2-AAF and 9 min for 2-AF. All of the compounds
were quantitated by comparison of the integrated area
of the elution peak with that of known amounts of
standards. The NAT activity is expressed as nmol
EXPERIMENTAL
Chemicals and reagents
−1
−1
Ibuprofen, ethylenediaminetetraacetic acid (EDTA), p-
aminobenzoic acid (PABA), N-acetyl-p-aminobenzoic
acid (N-Ac-PABA), 2-aminofluorene (2-AF), 2-acetyl-
aminofluorene (2-AAF), bovine serum albumin (BSA),
phenylmethylsulphonylfluoride (PMSF), TRIS·HCl, leu-
peptin, acetyl carnitine, dithiothreitol (DTT), carnitine
acetyltransferase and acetyl-coenzyme A (Ac-CoA)
were obtained from Sigma Chemical Co. (St. Louis,
MO). Acetic acid, acetonitrile, dimethyl sulphoxide
acetylated substrate min mg cytosolic protein.
Protein concentration
Protein concentration in the cytosols from H. pylori
27
were determined by the method of Bradford, with
BSA as the standard. All of the samples were assayed
in triplicate.
(DMSO) and potassium phosphates were obtained from
Merck Co. (Darmstadt, Germany). All of the chemicals
used were reagent grade.
Effects of various concentrations of ibuprofen on
growth of H. pylori
Helicobacter pylori
Twenty-two strains of H. pylori, cultured individually
in Brucella anaerobic culture plates in an anaerobic jar
Helicobacter pylori bacteria were clinically isolated
from patients who visited the Department (China Medi-
cal College Hospital) for endoscopy as described in
8
for 5 days to obtain growth at the level of 10 bacteria,
18
were placed in individual tubes containing brain heart
infusion media with or without different concentrations
of ibuprofen (4, 8, 16, 32 and 64 mM). The culture
tubes were incubated at 37°C in a microaerobic atmos-
previous reports.
Preparation of bacterial cytosols
10
phere (5% O , 10% CO and 85% N ) and checked
About 10 × 10 colony-forming units (CFU) were
washed twice in cold phosphate-buffered saline, placed
immediately in 1 ml of 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), disrupted by a sonicator
and then centrifuged for 30 min at 10 000 g. The super-
natant was kept on ice until assayed for NAT activity.
2
2
2
for growth after 5 days. This bacterium grows slow
and usually needs 4–5 days of incubation. The determi-
nation of the effects of ibuprofen on the growth of H.
pylori was based on measurements of absorbance by
an optical density method (OD at 650 nm). The control
groups were prepared under the same conditions as the
ibuprofen-treated groups except without the ibuprofen.
Growth inhibition (%) was determined using the fol-
lowing equation:
NAT activity determination
The determination of Ac-CoA-dependent N-acetylation
of PABA and 2-AF was performed as described by
Chung et al. Incubation mixtures in the assay system
consisted of a total volume of 90 ␮l of bacterial cyto-
17
Original OD−Final OD(+ ibuprofen)
1
−
× 100
ͩ
ͪ
Original OD

1998 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 18, 179–185 (1998)

IBUPROFEN AFFECTS BACTERIAL N-ACETYLTRANSFERASE ACTIVITY
181
Effects of various concentrations of ibuprofen on
NAT activity in H. pylori
of H. pylori are given in Table 1. Helicobacter pylori
was inhibited by ibuprofen, i.e. the higher the concen-
tration of ibuprofen in the H. pylori culture, the higher
the inhibition of H. pylori. When the concentration
of ibuprofen reached 64 mM, the inhibition reached
about 60%.
Ibuprofen dissolved in DMSO at specific concentrations
ranging from 0.04 to 400 mM were prepared. The
reaction mixtures consisted of 50 ␮l of cytosol, diluted
as required, 20 ␮l of recycling mixture containing 2-
AF or PABA at specific concentrations as substrates
and 10 ␮l of ibuprofen (at a specific concentration).
The reactions were started by the addition of Ac-CoA.
The control reactions had 20 ␮l of distilled water in
place of Ac-CoA. Following the NAT activity, determi-
nations were made to assess the effect of ibuprofen on
NAT activity in H. pylori.
Effects of various concentrations of ibuprofen on
the NAT activity in H. pylori
The possible effects of ibuprofen on the NAT activity
in H. pylori in cytosols and in intact bacteria were
examined by HPLP assessment of the percentage acety-
lation of 2-AF and PABA. Cytosols of H. pylori with
or without specific concentrations of ibuprofen co-
treatment showed different percentages of 2-AF and
PABA acetylation. A comparison of the relative cyto-
solic NAT activity with or without specific concen-
trations of ibuprofen is presented in Table 2. Percentage
acetylation of 2-AF and PABA by H. pylori with or
without specific concentrations of ibuprofen co-treat-
ment in intact cells is shown in Fig. 1. The data
indicate that there was decreased NAT activity associa-
ted with increased ibuprofen levels in H. pylori in intact
bacteria, i.e the higher the concentration of ibuprofen in
the reaction mixtures, the higher the inhibition of NAT
activity. Because 32 mM of ibuprofen showed inhi-
bition of the NAT activity both in cytosol and in
intact bacteria (inhibition ratios were 51% and 53%
Effects of ibuprofen on kinetic constants of NAT
from H. pylori
Cytosols of H. pylori were co-treated with or without
specific concentrations of ibuprofen (4, 8, 16, 32 and
6
4 mM) and the NAT activity was determined as
described above. All of the reactions were run in
9
triplicate. For the intact bacteria studies, 3 × 10
bacterial cells in brain heart infusion broth were incu-
bated with arylamine substrates (2-AF or PABA), with
or without specific concentrations of ibuprofen, for
9
1
6 h at 37°C in a microaerobic atmosphere (5% O2,
0% CO and 85% N ). At the conclusion of incu-
2
2
bation, the cells and media were removed and centri-
fuged. For the experiments with 2-AF, the supernatant
was immediately extracted with ethylacetate/methanol
a
Table 1. Effect of ibuprofen on the growth of H. pylori
(95:5), the solvent evaporated and the residue redis-
solved in methanol and assayed by HPLC. All of the
samples were run in triplicate. The kinetic constants
were calculated by the Cleland HYPER Program
which performs linear regression using a least-squares
method. The amounts of acetylated product and remain-
ing non-acetylated substrates were assayed by HPLC
as described above under NAT activity determination.
The velocity (1/V) versus substrate (1/S) data were
linearized by plotting 1/S versus 1/V.
Concentration of ibuprofen (m M)
2
8
0
4
8
16
32
64
Strain
Percentage inhibition
1
2
3
4
5
6
7
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
16 ± 6
14 ± 6
12 ± 4
11 ± 4
15 ± 4
17 ± 6
12 ± 4
14 ± 6
12 ± 4
16 ± 6
9 ± 2
12 ± 4
16 ± 6
10 ± 3
13 ± 4
14 ± 4
9 ± 2
34 ± 9
31 ± 8
28 ± 6
21 ± 4
28 ± 4
32 ± 6
24 ± 6
20 ± 6
21 ± 6
28 ± 6
17 ± 4
24 ± 6
28 ± 6
20 ± 6
23 ± 6
24 ± 6
16 ± 4
19 ± 4
22 ± 4
19 ± 6
21 ± 6
26 ± 4
47 ± 9
41 ± 9
34 ± 6
27 ± 5
34 ± 5
40 ± 8
30 ± 6
31 ± 6
22 ± 6
40 ± 4
24 ± 4
32 ± 5
36 ± 8
31 ± 8
32 ± 6
35 ± 6
26 ± 6
34 ± 6
36 ± 6
31 ± 8
36 ± 6
37 ± 6
56 ± 12 68 ± 14
54 ± 10 62 ± 12
46 ±
40 ±
46 ±
8
8
8
61 ± 12
51 ± 10
58 ± 12
Statistical treatment of data
59 ± 12 70 ± 14
47 ±
47 ±
40 ±
8
8
8
60 ± 12
61 ± 12
52 ± 10
Statistical analysis of the data was performed with an
unpaired Student’s t-test. The mean significant differ-
ence between ibuprofen-treated and control groups
was P Ͻ 0.05.
9
10
11
59 ± 10 68 ± 12
32 ±
48 ±
6
8
46 ±
59 ± 12
8
1
1
1
2
3
4
54 ± 10 66 ± 14
42 ±
46 ±
48 ±
34 ±
43 ±
46 ±
43 ±
45 ±
49 ±
8
8
8
6
8
8
8
8
8
53 ± 10
58 ± 12
64 ± 14
49 ± 10
61 ± 12
58 ± 10
56 ±
57 ±
64 ± 12
RESULTS
15
1
1
1
1
2
2
2
6
7
8
9
0
1
2
12 ± 3
14 ± 4
11 ± 3
12 ± 3
15 ± 4
Effects of various concentrations of ibuprofen on
the growth of H. pylori
8
8
The rationale for the author’s initial studies was based
on two observations from other reports. First, earlier
studies have already demonstrated that H. pylori con-
tains NAT activity in cytosols and in intact bacteria
a
Helicobacter pylori was incubated in the presence of various
concentrations of ibuprofen (4, 8, 16, 32 and 64 m M) as
described in the experim ental section. The percentage inhi-
17
with 2-AF and PABA used as substrates. Second,
bition was determ ined by using
a spectrophotom eter and
ibuprofen has been shown to decrease Klebsiella pneu-
29
then calculating the percentage inhibition. All experim ents and
controls were run in triplicate.
moniae NAT activity. Therefore, the results of an
examination for the effects of ibuprofen on the growth

1998 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 18, 179–185 (1998)

1
82
S. H. CHANG ET AL.
(
P Ͻ 0.001), respectively, for 2-AF and PABA in cyto-
Table 2. Effects of ibuprofen on H. pylori N-acetyltransfer-
ase activity in cytosols
sols, and 41% and 46%, respectively, for both sub-
strates in intact bacteria), we chose this concentration
to perform the following studies. The inhibition by
ibuprofen on the acetylation of PABA was slightly
higher than that seen for 2-AF.
9
Concentration
of ibuprofen
AAF
(nm ol m in m g
protein)
N-Acetyl-PABA
(nm ol m in m g
−1
−1
−1
−1
protein)
Control
0.86 ± 0.12
0.73 ± 0.12
0.64 ± 0.08
Effects of various concentrations of ibuprofen on
the kinetic constants of H. pylori NAT
b
a
4
8
1
3
6
m M
m M
6 m M
2 m M
4 m M
0.53 ± 0.11
0.42 ± 0.10
c
b
0.66 ± 0.10
0.53 ± 0.09
0.44 ± 0.08
d
c
0.38 ± 0.08
0.31 ± 0.06
0.26 ± 0.04
The kinetic constants determined for H. pylori NAT
using 2-AF and PABA as substrates with or without
e
d
f
e
0.36 ± 0.06
3
2 mM ibuprofen are shown in Fig. 2 for the cytosol
a
examination and Fig. 3 for the intact bacteria
examination. For the cytosol examinations, the apparent
Helicobacter pylori cytosols were incubated in the presence
of various concentrations of ibuprofen (4, 8, 16, 32 and 64 ␮M)
as described in the experim ental section. All experim ents and
controls were run in triplicate. Values are m eans ± SD (n = 3).
Statistical analysis of the data was perform ed with an unpaired
Student’s t-test.
values of Km and V
were 3.72 ± 0.49 mM and
max
−1
−1
1
6.67 ± 4.30 nmol min mg protein, respectively, for
-AF (Table 3) and 3.71 ± 0.49 mM and 20.46 ±
4.48 nmol min mg protein, respectively, for PABA
(Table 4). However, when ibuprofen was added to the
reaction mixtures, the apparent values of K and V
2
−1
−1
b
Differs between 4 m M ibuprofen and control (P Ͻ 0.05).
Differs between 8 m M ibuprofen and control (P Ͻ 0.02).
Differs between 16 m M ibuprofen and control (P Ͻ 0.01).
Differs between 32 m M ibuprofen and control (P Ͻ 0.005).
c
d
e
m
max
−1
−1
f
were 2.38 ± 0.28 mM and 7.69 ± 1.24 nmol min mg
Differs between 64 m M ibuprofen and control (P Ͻ 0.001).
protein, respectively, for 2-AF (Table 3) and
−1
−1
1
.85 ± 0.28 mM and 5.57 ± 0.45 nmol min mg pro-
tein, respectively, for PABA (Table 4). For the intact
Figure 2. Lineweaver-Burk double-reciprocal plot of H. pylori
N-acetyltransferase activity as a function of 2-am inofluorene
and p-am inobenzoic acid concentrations in cytosols. (●)
norm al; (■) with ibuprofen. The cytosols were prepared as
described in the experim ental section. The Ac-CoA and ibup-
rofen concentrations were 0.1 m M and 32 m M, respectively.
Values are m eans ± SD (n = 3).
Figure 1. Percentage acetylation of 2-am inofluorene and
p-am inobenzoic acid by H. pylori with or without specific
concentrations of ibuprofen. The intact bacteria were prepared
as described in the experim ental section. The Ac-CoA concen-
tration was 0.1 m M. Values are m eans ± SD (n = 3).

1998 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 18, 179–185 (1998)

IBUPROFEN AFFECTS BACTERIAL N-ACETYLTRANSFERASE ACTIVITY
183
−
1
0
1
.46 ± 0.18 nM and 4.00 ± 0.48 nmol min (10 ×
1
0 −1
0 ) CFU, respectively, for PABA (Table 4).
DISCUSSION
There are many observations that are likely to be
prerequisites for the observed effects of ibuprofen on
NAT activity:
(
i) the NAT enzyme exists in many kinds of experi-
1,2,4,5
mental animals, including humans,
and NAT
activity has been shown to be involved in some
3
0,31
chemical carcinogenesis,
(
ii) rapid and slow acetylations have been demon-
strated as a predisposing factor for the sensitivity
of individuals to toxicity during exposure to many
8
–10
arylamines;
(iii) some enzymes of enteric bacteria are known to
contribute to the metabolic activation of chemical
3
2,33
carcinogens in animal studies;
(
iv) the present author’s previous studies have already
shown that ibuprofen induces the inhibition of
growth and decreases NAT activity in Klebsi-
2
9
ella pneumoniae
(
v) according to the present author’s preliminary
studies, many kinds of enteric bacteria, such as
K. pneumoniae, Salmonella group B and E. coli,
exhibit NAT activity (manuscript in preparation).
It was found that H. pylori cytosols contained NAT
Figure 3. Lineweaver-Burk double-reciprocal plot of H. pylori
N-acetyltransferase activity as a function of 2-am inofluorene
and p-am inobenzoic acid concentration in intact bacteria: (●)
norm al; (■) with ibuprofen. The suspensions were prepared
as described in the experim ental section. The Ac-CoA and
ibuprofen concentrations were 0.1 m M and 32 m M, respect-
ively. Values are m eans ± SD (n = 3).
1
7
activity. Therefore, the present studies were focused
on the effects of ibuprofen on NAT activity in
H. pylori.
The data presented in this report clearly demonstrate
that ibuprofen does affect H. pylori NAT activity and
growth. The results clearly indicate that ibuprofen, in
concentrations from 4 mM to 64 mM for cytosol tests,
decreases the acetylated product of 2-AF and PABA
by H. pylori. The results also show that when ibuprofen
decreases the NAT activity in H. pylori it is a dose-
dependent affect, i.e. the higher the concentration of
ibuprofen, the higher the inhibition of NAT activity.
The data presented from the intact bacteria tests also
show that ibuprofen decreases the percentage of acetyl-
ated products of 2-AF and PABA. The concentrations
(4–64 mM) of ibuprofen used in this study are reason-
bacteria examination, the apparent values of Km and
−
1
Vmax were 0.46 ± 0.09 mM and 1.63 ± 0.18 nmol min
10 −1
(
10 × 10 ) colony forming units CFU, respectively,
for 2-AF (Table 3) and 0.56 ± 0.11 mM and 6.66 ±
−1
10 −1
0
.69 nmol min (10 × 10 ) CFU, respectively, for
PABA (Table 4). However, when ibuprofen was added
to the reaction mixture, the apparent values of K and
m
−1
Vmax were 0.25 ± 0.06 mM and 1.29 ± 0.11 nmol min
10 −1
(
10 × 10 ) CFU, respectively, for AF (Table 3) and
a
Table 3. Kinetic data for acetylation of 2-aminofluorene in H. pylori
In cytosol
In intact bacteria
m ax (nm ol 10 × 10 )
1
0 −1
K
m
(m M)
V
m ax
K
m
(m M)
V
−1
−1
(
nm ol m in m g
)
CFU)
Control
Ibuprofen
3.72 ± 0.49
2.38 ± 0.28
16.67 ± 4.30
7.69 ± 1.24
0.46 ± 0.09
0.25 ± 0.06
1.63 ± 0.18
1.29 ± 0.11
b
c
d
e
a
Values are m eans ± SD (n = 3). The acetyl-CoA and ibuprofen concentrations were 0.1 m M and 32 m M, and the kinetic constants
were calculated from the m odified HYPER Program of Cleland. All experim ents and controls were run in triplicate. Statistical
b
analysis of the data was perform ed with an unpaired Student’s t-test. Differs between 32 m M ibuprofen and control (P Ͻ 0.05).
c
Differs between 32 m M ibuprofen and control (P Ͻ 0.001).
Differs between 32 m M ibuprofen and control (P Ͻ 0.01).
Differs between 32 m M ibuprofen and control (P Ͻ 0.005).
d
e

1998 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 18, 179–185 (1998)

1
84
S. H. CHANG ET AL.
a
Table 4. Kinetic data for acetylation of p-aminobenzoic acid in H. pylori
In cytosol
In intact bacteria
m ax (nm ol 10 × 10 )
−1
−1
10 −1
K
m
(m M)
Vm ax (nm ol m in m g
)
K
m
(m M)
V
CFU)
Control
Ibuprofen
3.71 ± 0.49
1.85 ± 0.28
20.46 ± 4.48
5.57 ± 0.45
0.65 ± 0.11
0.46 ± 0.08
6.66 ± 0.69
4.00 ± 0.48
b
c
d
e
a
Values are m ean ± SD (n = 3). The acetyl-CoA and ibuprofen concentrations were 0.1 m M and 32 m M, and the kinetic constants
were calculated from the m odified HYPER Program of Cleland. All experim ents and controls were run in triplicate. Statistical
analysis of the data was perform ed with an unpaired Student’s t-test.
b
Differs between 32 m M ibuprofen and control (P Ͻ 0.005).
Differs between 32 m M ibuprofen and control (P Ͻ 0.0001).
Differs between 32 m M ibuprofen and control (P Ͻ 0.01).
Differs between 32 m M ibuprofen and control (P Ͻ 0.001).
c
d
e
able because they are based on literature indicating
that the analgesic dose of ibuprofen is 0.97–.3.88 mM
and the antirheumatic dose is 3.88–15.51 mM for
Vmax decreased 0.45- and 0.20-fold for acetylation of
2-AF, and 0.30- and 0.40-fold for acetylation of PABA.
Based on the kinetic constant decreases, it was sug-
gested that ibuprofen may act like a non-competitive
inhibitor, a result that is consistent with that reported
3
4
human daily used. The data also demonstrate that
ibuprofen induces the inhibition of growth in H. pylori
culture, i.e. the higher the concentration of ibuprofen,
the higher the inhibition of H. pylori growth. Based
on the observations, if there is no inhibition of growth,
then there is no decreased NAT activity by ibuprofen.
Because ibuprofen did inhibit the NAT activity of
H. pylori, the kinetic constants were also affected. The
reason for selecting 32 mM of ibuprofen for kinetic
constant studies is that this concentration has already
reduced acetylation by 50%. For the cytosol examin-
ations, the apparent values of Km and Vmax decreased
3
5
for arylamine NAT from pigeon liver. While the
nature of the interaction and the NAT protein domains
involved in this interaction remain unclear, ibuprofen
inhibition may be a useful tool to distinguish between
different aromatic amine NATs. This point needs
further investigation and is very important for the
possibility of decreasing arylamine carcinogens in
induced carcinogenesis, because other reports have
demonstrated that elevated levels of NAT activity may
be associated with increased sensitivity to the muta-
3
6
0
.36- and 0.53-fold for acetylation of 2-AF, and 0.50-
genic affects of many arylamines and attenuation of
NAT activity has been reported to be associated with
and 0.72-fold for acetylation of PABA. For the intact
bacteria examinations, the apparent values of Km and
1
,2,4,5
several disease processes.
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