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

Article abstract of DOI:10.1002/(SICI)1099-1263(199803/04)18:2<117::AID-JAT486>3.0.CO;2-D

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 concentrat

Full text of DOI:10.1002/(SICI)1099-1263(199803/04)18:2<117::AID-JAT486>3.0.CO;2-D

JOURNAL OF APPLIED TOXICOLOGY
J. Appl. Toxicol. 18, 117–123 (1998)
Rhein Affects Arylamine N-Acetyltransferase
Activity in Helicobacter pylori from Peptic
Ulcer Patients
1
2
2
3
3
Jing G. Chung, * Mei F. Tsou, Hwang H. Wang, Hsueh H. Lo, Sue E. Hsieh,
4
1
1
1
5
Yee S. Yen, Lii T. Wu, Shih H. Chang, Chin C. Ho and Chi F. Hung
1
Department of Medicine, China Medical College, Taichung, Taiwan, Republic of China
2
China Medical College Hospital, Taichung, Taiwan, Republic of China
3
Department of Medical Technology, Chungtai Junior College, Taichung, Taiwan, Republic of China
Department of Medical Technology, China Medical College, Taichung, Taiwan, Republic China
Department of Surgery, Jen-AI Hospital, Taichung, Taiwan, Republic of China
4
5
Key words: 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 rhein 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 rhein in H. pylori cytosols. Inhibition of
growth studies from H. pylori demonstrated that rhein elicited dose-dependent bacteriostatic activity in
H. pylori cultures: i.e. the greater the concentration of rhein, the greater the inhibition of growth to
H. pylori. For the cytosol and intact bacteria examination, the apparent values of Km and Vmax were
decreased after co-treatment with 40 ␮M rhein. This report is the first demonstration of rhein inhibition
of arylamine N-acetyltransferase activity and rhein inhibition of growth in the bacterium H. pylori.

1998 John Wiley & Sons, Ltd.
1
7
correlation of H. pylori with active chronic gastritis.
INTRODUCTION
Other investigators have also demonstrated that
H. pylori is a possible causative factor in patients
with gastric cancer. It has been reported that chronic
atrophic gastritis is a precursor lesion of gastric cancer,
and chronic gastritis might be inducd or exacerbated
because gastritis, peptic ulcer and
gastric cancer are related to the presence of this bac-
1
8
Exposure to environmental and occupational chemicals
is thought to be an important cause of chemical car-
cinogenesis. Arylamine carcinogens require metabolic
activation by host enzymes in order to initiate carcino-
1
8–20
by H. pylori
1,2
genesis in specific target organs or tissues. N-Acety-
lation, a major metabolic pathway for these arylamine
carcinogens, is catalysed by cytosolic arylamine N-
acetyltransferase (NAT) using acetyl co-enzyme A as
2
1
terium.
Rhein
(4,5-dihydroxy-2-anthraquinone-carboxylic
acid) is the active metabolite of diacetylrhein and has
been reported to exert several effects both in vivo and
in vitro. Rhein has been shown to exert anti-inflamma-
3
an acetyl donor. Arylamine carcinogens, such as 2-
aminofluorene (AF) are N-acetylated to the 2-acetylam-
inofluorene (AAF), which can undergo further acti-
vation or detoxification reactions. N-Acetyltransferase,
an enzyme involved in several steps of both arylamine
2
2–24
tory and antirheumatic effects in humans.
However,
a specific antineoplastic activity to Ehlrich ascites
tumour cells and human glioma cells has been
4
25,26
activation and detoxification, is found in many tissues
reported.
Other investigators also demonstrated that
5–8
of animals and humans. Humans and other mammals
exhibit a genetic polymorphism in NAT activity
resulting in rapid, slow and some intermediate acety-
rhein interferes with electron transport, inhibiting mito-
chondrial oxidation of NAD- and FAD-linked sub-
26
strates at the dehydrogenase–co-enzyme level. Fur-
thermore, it has been reported that rhein affects energy
metabolism by inducing a decrease in ATP production,
ultimately leading to an impairment of protein syn-
7
,9,10
lation phenotypes.
In fact, human epidemiological
studies suggested an association between rapid acetyl-
11,12
ator phenotype in colorectal cancer,
between slow acetylator phenotype in bladder cancer.
The present authors have also found NAT in the
as well as
13
27
thesis. It has been shown that rhein inhibits glucose
2
8
26
uptake, as well as oxidative phosphorylation, and
14
15
8
27
nematode, the freshwater shrimp and certain fish.
drastically lowers the adenylate energy charge. It has
Warren and Marshall first demonstrated that Helicob-
also been demonstrated that rhein inhibits superoxide
1
6
29,30
acter pylori was present in patients’ stomachs. It
production and the activity of lysosomal enzyme.
has also been demonstrated that there is a etiological
Therefore, other investigators have pointed out that
rhein may exact direct action on plasma and intracellu-
3
1
lar membranes.
*
Correspondence to: Jing G. Chung, Department of Medicine, China
Medical College, Taichung 400, Taiwan, Republic of China.
In previous studies, the present authors found that:
emodin induced cytotoxicity and DNA damage in
CCC 0260–437X/98/020117–07$17.50
Received 11 May 1997
Accepted 17 November 1997

1998 John Wiley & Sons, Ltd.

1
18
JING G. CHUNG ET AL.
3
2
33
H. pylori; NAT is present in H. pylori; and emodin
were incubated at 37°C for 10 min and stopped with
50 ␮l of 20% trichloroacetic acid for PABA reactions
and with 100 ␮l of acetonitrile for AF reactions. All
the reactions of the experiments and controls were run
in triplicate. The amounts of acetylated product and
34
affects the NAT activity of H. pylori. Therefore, the
purpose of the present study was to elucidate the
possible effects of rhein on the NAT activity in
H. pylori. Our initial choice of AF and p-aminobenzoic
acid (PABA) as test substrates was based on previous
studies in mice and nematodes and our interest in
comparing the metabolism of a carcinogen (AF) and
to a non-carcinogen (PABA). The results demonstrated
that by using AF and PABA as substrates for NAT
activity determination, rhein did decrease H. pylori
NAT activity in cytosol and in intact bacteria.
remaining non-acetylated substrate were determined by
6
14
6,31
HPLC.
An aliquot of the NAT incubation was
injected onto a C18 reversed-phase column (Spherisorb
4.6 × 250 nm) of a Beckman HPLC (pump 168 and
−1
detector 126) and eluted at a flow rate of 1.2 ml min .
For PABA and N-Ac-PABA the solvent system was
50 mM acetic acid–CH CN (86:14) with detection at
3
2
66 nm. The retention time of PABA was 4 min and
that of N-Ac-PABA was 6.5 min. For AF and AAF,
the solvent system was 20 mM KH PO (pH 4.5)–
2
4
EXPERIMENTAL
CH CN (53:47) with detection at 280 nm. The retention
3
time was 6.5 min for AAF and 9 min for AF. All the
compounds were quantitated by comparison of the
integrated area of the elution peak with that of known
amounts of standards. The activity of NAT is expressed
Chemicals and reagents
Rhein was obtained from Fluka Chemika Co. (Buchs,
Switzerland). Ethylenediaminetetraacetic acid (EDTA),
p-aminobenzoic acid (PABA), acetyl-p-aminobenzoic
acid (N-Ac-PABA),2-aminofluorene (AF), 2-acetyl-ami-
nofluorene (AAF), bovine serum albumin (BSA), phe-
nylmethylsulphonylfluoride (PMSF), TRIS, leupeptin,
acetyl carnitine, dithiothreitol (DTT), carnitine acetyl-
transferase and acetyl co-enzyme A (AcCoA) were
obtained from Sigma Chemical Co. (St Louis, MO).
Acetic acid, acetonitrile, dimethyl sulfoxide (DMSO),
and potassium phosphates were obtained from Merck
Co. (Darmstadt, Germany). All chemicals used were
reagent grade.
−1
−1
as nmol acetylated substrate min mg
cytosolic
protein.
Protein concentrations in the cytosols from H. pylori
36
were determined by the method of Bradford, with
BSA as the standard. All the samples were assayed
in triplicate.
Effect of various concentrations of rhein on
growth of H. pylori
Twenty-two strains of H. pylori, cultured individually
in Brucella anaerobic culture plates in an anaerobic ja8r
for 5 days to obtain growth at the levels of 10
bacteria, were placed in individual tubes containing
brain heart infusion media with or without different
concentrations of rhein (0.04, 0.4, 4, 40 and 400 ␮M).
The culture tubes were incubated at 37°C in a micro-
Helicobacter pylori
Helicobacter pylori bacteria were clinically isolated
from patients who visited our department (China Medi-
cal College Hospital) for endoscopy as described in
aerobic atmosphere (5% O , 10% CO and 85% N )
2 2 2
33,34
previous reports.
and checked for growth after 5 days. This bacterium
grows slowly and usually needs 4–5 days. The determi-
nation of the effects of rhein on the H. pylori was
based on the measurement of the absorbance by an
Preparation of bacteria cytosols
3
4
10
optical density method (OD at 650 nm). The control
groups were prepared under the same conditions as the
rhein-treated groups except without the rhein.
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 centrifuged for 30 min at 10 000 g. The supernatant
was kept on ice until assayed for NAT activity.
Effect of various concentrations of rhein on NAT
activity of H. pylori
Rhein dissolved in DMSO with specific concentrations
ranging from 0.04 to 400 ␮M were prepared. The
reaction mixtures consisted of 50 ␮l of cytosols diluted
as required, 20 ␮l of recycling mixture containing AF
or PABA at specific concentrations as substrates and
N-Acetyltransferase activity determination
The determination of AcCoA-dependent N-acetylation
of PABA and AF was performed as described by
1
0 ␮l of rhein (at a specific concentration). The reac-
35
5
Andres et al. and modified by Mattano and Weber.
tions were started by the addition of AcCoA. The
control reactions had 20 ␮l of distilled water instead
of AcCoA. Following the NAT activity determination,
the procedure was performed to evaluate the effect of
rhein on H. pylori NAT activity.
Incubation mixtures in the assay system consisted of
a total volume of 90 ␮l: tissue cytosol, diluted as
required, in 50 ␮l of lysis buffer (20 mM TRIS-HCl
at pH 7.5, 1 mM DTT, 1 mM EDTA, 1 mM
acetylcarnitine) and AF or PABA at specific concen-
trations for the substrate. The reactions were started
by the addition of 20 ␮l of AcCoA. Control reactions
had 20 ␮l of distilled water instead of AcCoA. The
final concentration of PABA or AF was 0.1 mM, and
that of AcCoA was 0.5 mM. The reaction mixtures
Effect of rhein on kinetic constants of NAT from
H. pylori
Cytosols of H. pylori were co-treated with or without
specific concentrations of rhein (400, 40, 4, 0.4 and
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1998 John Wiley & Sons, Ltd.
J. Appl. Toxicol. Vol. 18, 117–123 (1998)

RHEIN AFFECTS BACTERIAL N-ACETYLTRANSFERASE ACTIVITY
119
0
.04 ␮M) and the NAT activity was determined as
a
Table 1. Effect of rhein on the growth of H. pylori
Concentration of rhein (␮M)
described in a previous section. All the reactions were
run in triplicate. For the intact bacteria studies, 3 × 10
bacterial cells in brain heart infusion broth were incu-
bated with arylamine substrate (AF) with or without
specific concentrations of rhein (400, 40, 4, 0.4 and
9
Strain
0
0.04
0.4
4
40
400
0
.04 ␮M) for 96 h at 37°C in a micro-aerobic atmos-
Percentage inhibition
phere (5% O , 10% CO and 85% N ). At the con-
2
2
2
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
0
0
3
2
0
0
0
4
0
0
0
0
3
0
0
4
0
0
0
0
10
10
14
11
14
12
16
10
14
18
10
14
18
10
13
14
16
10
12
16
8
70
61
70
68
64
70
80
60
62
70
64
62
76
60
62
60
70
74
66
70
56
47
44
50
81
74
80
77
76
79
87
69
70
79
72
78
84
74
76
71
84
83
76
83
72
69
61
60
90
82
91
86
88
90
90
84
82
88
86
89
96
84
88
84
94
91
88
96
87
84
72
74
clusion of incubation, the cells and media were
removed and centrifuged. For the experiments with
AF, the supernatant was immediately extracted with
ethylacetate–methanol (95:5), the solvent was evapor-
ated and the residue was redissolved in methanol and
assayed by HPLC. All the samples were run in tripli-
cate. The kinetic constants were calculated with the
Cleland HYPER program, which performs linear
regression using a least-squares method. The amounts
of acetylated product and remaining non-acetylated
substrates were assayed by HPLC as described in the
NAT activity determination. The velocity (1/V) versus
substrate (1/S) data were linearized by plotting 1/S
versus 1/V.
35
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
RESULTS
6
11
10
Effect of various concentrations of rhein on the
growth of H. pylori
b
c
The rationale for the authors’ initial studies was based
on two observations from other reports. First, cytotoxic
effects on cultured cells and bacteria have been associa-
a
Helicobacter pylori was incubated in the presence of various
concentrations of rhein (0.04, 0.4, 4, 40 and 400 ␮M) as
described in the Materials and Methods. The percentage
inhibition was determ ined under a spectrophotom eter and
then calculated. All experim ents and controls were run in
triplicate.
32,33
ted with emodin.
Second, emodin and rhein are
components of Dahung (a Chinese herb medicine).
Helicobacter pylori has been reported to contain NAT
33
activity, therefore an examination of the effect of
rhein on the growth of H. pylori is given in Table 1.
Helicobacter pylori was inhibited by rhein: i.e. the
higher the concentration of rhein in the H. pylori cul-
ture, the higher the inhibition of H. pylori. When the
concentration of rhein reached 400 ␮M, the inhibition
reached to 90%. The data from two other bacteria, a
Gram-negative Escherichia coli and a Gram-positive
Staphylococcus aureus, indicated that the percentage
inhibition was 75% under the same concentrations of
rhein as used on H. pylori. The most significant effect
in both treatment groups was a decrease in the percent-
age of bacterial cells after rhein compared to control
groups (Table 1).
b
E. coli.
S. aureus.
c
Table 2. Effects of rhein on H. pylori N-acetyltransferase
activity in vitro
a
Concentration AAF
of rhein
N-Acetyl-PABA
(nm ol m in m g protein)
−1
−1
−1
−1
(nm ol m in m g
protein)
Control
0.04 ␮M
0
4
40 ␮M
400 ␮M
0.86 ± 0.12
0.83 ± 0.14
0.76 ± 0.11
0.36 ± 0.08
0.24 ± 0.04
0.64 ± 0.08
0.62 ± 0.12
0.55 ± 0.09
0.29 ± 0.06
0.11 ± 0.04
b
b
.4 ␮M
␮M
c
c
d
d
Effect of various concentrations of rhein on the
activity of H. pylori NAT
e
e
0.12 ± 0.04
0.06 ± 0.02
The possible effects of rhein on the NAT activity in
H. pylori in cytosol and in intact bacteria were exam-
ined by HPLC, assessing the percentage acetylation of
AF and PABA. Cytosols of H. pylori with or without
specific concentrations of rhein co-treatment showed
different percentages of AF and PABA acetylation. A
comparison of the relative cytosolic NAT activity with
or without specific concentrations of rhein is presented
in Table 2. Percentage acetylations of AF and PABA
by H. pylori with or without specific concentrations of
rhein co-treatment in intact bacteria are given in Fig. 1.
a
Helicobacter pylori cytosol was incubated in the presence of
various concentrations of rhein (0.04, 0.4, 4, 40 and 400 ␮M)
as described in the Materials and Methods. All experim ents
and control were run in triplicate. Values are m eans ± SD
(
n = 3).
b
Differs between 0.4 ␮M rhein and control; P Ͻ 0.05.
Differs between 4 ␮M rhein and control; P Ͻ 0.02.
Differs between 40 ␮M rhein and control; P Ͻ 0.005.
Differs between 400 ␮M rhein and control; P‘ Ͻ 0.001.
c
d
e
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1998 John Wiley & Sons, Ltd.
J. Appl. Toxicol. Vol. 18, 117–123 (1998)

1
20
JING G. CHUNG ET AL.
Figure 1. Percentage acetylation of 2-am inofluorene and p-
am inobenzoic acid by H. pylori N-acetyltransferase with or
without specific concentrations of rhein. The cytosols were
prepared as described in Materials and Methods. The AcCoA
concentration was 0.1 m M. Values are the m ean ±SD of 1
−1
−1
nm ol m in m g protein; n = 3 as described in the text.
The data indicate that there was decreased NAT
activity associated with increased rhein levels in
H. pylori cytosols: i.e. the higher the concentration of
rhein in the reaction mixtures, the higher the inhibition
of NAT activity. Because 400 ␮M rhein showed inhi-
bition of NAT activity both in cytosol and in intact
bacteria (inhibition ratios were 90% and 93%, respect-
ively, for AF and PABA in cytosol and 40% and 37%,
respectively, for both substrates in intact bacteria), the
inhibition of rhein to the acetylation of PABA was
slightly higher than that seen for AF.
Effect of various concentrations of rhein on the
kinetic constants of H. pylori NAT
The kinetic constants determined for H. pylori NAT
using AF and PABA as substrates with or without
4
0 ␮M rhein are shown in Figs 2 and 3. For the
cytosol examination, the apparent values of Km and
Vmax were 3.25 ± 0.66 mM and 17.26 ± 4.86 nmol
−1
−1
min mg protein, respectively, for 2-AF (Table 3)
−1
−1
and 2.60 ± 0.44 mM and 11.90 ± 2.28 nmol min mg
protein, respectively, for PABA (Table 4). However,
when rhein was added to the reaction mixtures, the
Figure 2. Lineweaver–Burk double reciprocal plot of H. pylori
N-acetyltransferase activity as a function of 2-am inofluorene
concentration in cytosol (A) and in intact bacteria (B). The
cytosols and suspensions were prepared as described in
Materials and Methods. The AcCoA concentration was 0.1 m M.
apparent values of Km and V
were 2.54 ± 0.5 mM
max
1 −1
−
and 10.48 ± 3.06 nmol min mg protein, respectively,
for 2-AF (Table 3) and 2.26 ± 0.36 mM and 6.72 ±
−1
−1
Values are the m ean ± SD of 1 nm ol m in m g protein; n = 3
as described in the text.
−
1
−1
0
.70 nmol min mg protein, respectively, for PABA
Table 4). For the intact bacteria examination, the
apparent values of K and V were 3.18 ± 0.76 mM
(
m
max
−1
10
and 16.02 ± 3.48 nmol min (10 × 10 colony forming
ture, the apparent values of Km and V
were
max
10
−
1
−1
units (CFU)) , respectively, for 2-AF (Table 3) and
2.67 ± 0.18 mM and 9.76 ± 0.92 nmol min (10 × 10
−1
−1
3
.26
10 × 10 CFU) , respectively, for PABA (Table 4).
However, when rhein was added to the reaction mix-
±
0.64 mM and 18.10
±
4.18 nmol min
CFU) , respectively, for AF (Table 3) and
10
−1
−1
10
(
2.16 ± 0.18 mM and 6.72 ± 0.70 nmol min (10 × 10
−1
CFU) , respectively, for PABA (Table 4).
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1998 John Wiley & Sons, Ltd.
J. Appl. Toxicol. Vol. 18, 117–123 (1998)

RHEIN AFFECTS BACTERIAL N-ACETYLTRANSFERASE ACTIVITY
121
Table 3. Kinetic data for acetylator of 2-aminofluorene
a
in H. pylori
In cytosol
(m M)
In intact bacteria
m
K (m M)
K
m
V
m ax
V
m ax
−1
−1
10
−1
(nm ol m in m g
)
(nm ol (10 × 10 CFU) )
Control 3.25 ± 0.66
Em odin 2.54 ± 0.48
17.26 ± 4.86 3.18 ± 1.76
10.48 ± 2.56 2.67 ± 0.18
16.02 ± 3.48
9.76 ± 0.92
b
c
d
e
a
Values are m eans ± SD (n = 3). The acetyl CoA and rhein
concentrations were 0.1 m M and 40 ␮M, and the kinetic
constants were calculated from the m odified HYPER Program
of Cleland. All experim ents and controls were run in triplicate.
b
Differs between 40 ␮M rhein and control; P Ͻ 0.05.
Differs between 40 ␮M rhein and control; P Ͻ 0.001.
Differs between 40 ␮M rhein and control; P Ͻ 0.005.
Differs between 40 ␮M rhein and control; P Ͻ 0.001.
c
d
e
Table 4. Kinetic data for acetylator of p-aminobenzoic
a
acid in H. pylori
In cytosol
(m M)
In intact bacteria
m
K (m M)
K
m
V
m ax
V
m ax
−1
−1
10
−1
(nm ol m in m g
)
(nm ol (10 × 10 CFU) )
Control 2.60 ± 0.44
Em odin 2.26 ± 0.36
11.90 ± 2.28 3.26 ± 0.66
18.10 ± 4.18
6.72 ± 0.70
b
c
d
e
6.72 ± 0.70
2.16 ± 0.18
a
Values are m eans ± SD (n = 3). The acetyl CoA and rhein
concentrations were 0.1 m M and 40 ␮M, and the kinetic
constants were calculated from the m odified HYPER Program
of Cleland. All experim ents and controls were run in triplicate.
b
Differs between 40 ␮M rhein and control; P Ͻ 0.05.
Differs between 40 ␮M rhein and control; P Ͻ 0.005.
Differs between 40 ␮M rhein and control; P Ͻ 0.005.
Differs between 40 ␮M rhein and control; P Ͻ 0.0001.
c
d
e
(
i) The AcCoA-dependent arylamine NAT enzyme
has been reported to be present in many kinds
3,4
of experimental animals, including humans, and
NAT has been shown to be involved in some
3
8,39
chemical carcinogenesis.
(
ii) Rapid and slow acetylation has been demonstrated
as a predisposing factor for the sensitivity of
individuals to the toxicity during exposure to
1
1,12
many arylamines.
Therefore, the genetically
mediated variations in NAT activities within tar-
get tissues or organs for arylamine-induced neo-
plasm may indicate differential risks among the
human population.
Figure 3. Lineweaver–Burk double reciprocal plot of H. pylori
N-acetyltransferase activity as a function of p-am inobenzoic
acid concentration in cytosol (A) and intact bacteria (B). The
cytosols and suspensions were prepared as described in
Materials and Methods. The AcCoA concentration was 0.1 m M.
(
iii) Some enzymes of enteric bacteria are known to
contribute to the metabolic activation of chemical
4
0,41
carcinogens in animal studies.
−1
−1
Values are the m ean ± SD of 1 nm ol m in m g protein; n = 3
as described in the text.
(iv) The present authors’ previous studies already
showed that emodin induced the inhibition of
growth and DNA damage in H. pylori, and
32,33
emodin decreased NAT activity in H. pylori.
(
v) According to the present authors’ preliminary
studies, many kinds of enteric bacteria such as
Klebsiella pneumoniae, Salmonella group B and
E. coli, exhibit NAT activity (manuscript in
preparation). It was found that H. pylori cytosols
DISCUSSION
There are many events that are likely to be prerequi-
sites for the observed effects of rhein on NAT activity:
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1998 John Wiley & Sons, Ltd.
J. Appl. Toxicol. Vol. 18, 117–123 (1998)

1
22
JING G. CHUNG ET AL.
contained NAT activity. Therefore, the present
studies were focused on the effect of rhein on
the NAT activity of H. pylori.
Because rhein does inhibit the NAT activity of
H. pylori, the kinetic constants were also affected. For
cytosol examination, the apparent values of Km and
Vmax decreased by 0.23- and 0.30-fold for acetylation
of AF and by 0.58- and 0.16-fold for acetylation of
PABA. For the intact bacteria examination, the
apparent values of Km and Vmax decreased by 0.39-
and 0.33-fold for acetylation of AF and by 0.36- and
Thus, the data presented in this report clearly demon-
strated that rhein did affect H. pylori NAT activity and
growth. The results clearly indicated that rhein, in
concentrations of 0.4–400 ␮m for cytosol tests,
decreased the acetylated product of AF and PABA by
H. pylori. The results also show that when rhein
decreased the NAT activity in H. pylori, it was a dose-
dependent effect: i.e. the higher the concentration of
rhein, the higher the inhibition of NAT activity. The
data presented from the intact bacteria tests also
showed that rhein decreased the percentage of acetyl-
ated products of AF and PABA. Rhein also induced a
dose-dependent effect on the NAT activity in H. pylori.
The data also demonstrated that rhein induced inhi-
bition of the growth effect on the H. pylori culture:
i.e. the higher the concentration of rhein, the higher
the inhibition of H. pylori growth. In other words,
rhein can be used as a bactericide to H. pylori. This
is the first report to show that rhein could act as an
antimicrobial agent.
0
.54-fold for acetylation of PABA. Based on the kinetic
constant decreases, it was suggested that rhein may
act as an uncompetitive inhibitor. This needs further
investigation. The cytosol and intact bacteria data
showed different degrees of rhein inhibition on the
NAT enzyme. Therefore, this finding is very important
to 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-
4
2
genic effects of many arylamines and attenuation of
NAT activity is associated with several disease pro-
3
,4
cess.
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