The effects of vitamin E on arylamine N-Acetyltransferase activity in strains of Helicobacter pylori from peptic ulcer patients

Article abstract of DOI:10.1016/S0278-6915(99)00047-2

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) and p-aminobenzoic acid (PABA) were determined in the bacterium Helicobacter pylori. Cytosols or suspensions of H. pylori, with or without specific concentrations of vitamin E co-treatment, showed different percentages of 2-AF acetylation. The data indicated that there was increased NAT activity associated with increased levels of vitamin E in H. pylori cytosols and intact bacteria. For the cytosol and intact bacteria examinations, the apparent values of K(m) and V(max) were increased when vitamin E was added to the reaction mixtures for 2-AF and PABA acetylation, respectively. This report is the first demonstration to show that antioxidant agents (vitamin E) can promote H. pylori N-acetyltransferase activity. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0278-6915(99)00047-2

Food and Chemical Toxicology 37 (1999) 655±661
Research Section
The E€ects of Vitamin E on Arylamine N-
Acetyltransferase Activity in Strains of
Helicobacter pylori From Peptic Ulcer
Patients
J. G. CHUNG
Department of Medicine, China Medical College, Taichung 400, Taiwan, Republic of China
(Accepted 10 October 1998)
AbstractÐArylamine N-acetyltransferase (NAT) activities with 2-amino¯uorene (2-AF) and p-amino-
benzoic acid (PABA) were determined in the bacterium Helicobacter pylori. Cytosols or suspensions of
H. pylori, with or without speci®c concentrations of vitamin E co-treatment, showed di€erent percen-
tages of 2-AF acetylation. The data indicated that there was increased NAT activity associated with
increased levels of vitamin E in H. pylori cytosols and intact bacteria. For the cytosol and intact bac-
teria examinations, the apparent values of Km and Vmax were increased when vitamin E was added to
the reaction mixtures for 2-AF and PABA acetylation, respectively. This report is the ®rst demon-
stration to show that antioxidant agents (vitamin E) can promote H. pylori N-acetyltransferase activity.
# 1999 Elsevier Science Ltd. All rights reserved
Keywords: vitamin E; 2-amino¯uorene; N-acetyl-2-amino¯uorene; p-aminobenzoic acid; N-acetyl-p-
aminobenzoic acid; N-acetyltransferase.
Abbreviations: 2-AAF = 2-acetylamino¯uorene; 2-AF = 2-amino¯uorene; EDTA = ethylenediamine-
tetraacetic acid; BSA = bovine serum albimin; DMSO = dimethyl sulfoxide; DTT = dithiothreitol;
CFU = colony-forming
units;
OD = optical
density;
PMSF = phenylmethylsulfonyl¯uoride;
PABA = p-aminobenzoic acid; N-Ac-PABA = acetyl-p-aminobenzoic acid; NAT = N-acetyltransfer-
ase.
INTRODUCTION
1982). Individual genetic variations in humans for
the regulation of the NAT loci have been implicated
Exposure to environmental and occupational chemi-
as important factors in determining susceptibility to
cals is recognized as an important cause of chemical
environmental and occupational arylamine carcino-
carcinogenesis. N-acetylation is thought to play an
important role in arylamine metabolism. Two N-
gens (Lang et al., 1987; Weber and Hein, 1985).
Recently, other investigators have demonstrated
that intestinal micro¯ora plays an important role in
the formation of N-acetyl derivatives from aryla-
mines in dogs (Okumu et al., 1995). Thus, it is sus-
pected that normal ¯ora in the human digestive
system may be involved in the metabolism of aryla-
mines.
Colonization of the human gastric mucosa by
Helicobacter pylori was discovered in 1983 and, in
addition to HCl secretion, is a major contributor to
the development of peptic ulcer disease (Goodwin
and Carrick, 1991; Marshall, 1983; Graham, 1989).
H. pylori has already been shown to survive and
grow in the stomach of humans. Recently, there has
acetyltransferase (NAT) enzymes have been ident-
i®ed and isolated from mammalian tissues, desig-
nated NAT1 and NAT2, catalyse N-acetylation of
various arylamines (Hein et al., 1991; Minchin et al.,
1992; Weber, 1987). NAT2, and most recently
NAT1, has been shown to be
a polymorphic
enzyme that segregates individuals into rapid and
slow acetylator phenotypes (Vatsis and Weber,
1993; Weber and Hein, 1985). For humans, there
are statistical and epidemiological studies linking
rapid acetylation to increased risks of colorectal
cancer (Ilett et al., 1987; Lang et al., 1987; Roberts-
Thomson et al., 1996) and slow acetylation to
increased risks of bladder cancer (Cartwright et al., been increasing evidence that persistent infections
0278-6915/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. Printed in Great Britain
PII S0278-6915(99)00047-2

656
J. G. Chung
with H. pylori can be a risk factor for the develop- density (OD) of the intact bacterial cell suspensions
ment of gastric adenocarcinoma (Blaser and was determined in triplicate by using a Beckman
Parsonnet, 1994; Correa, 1991). Other investigators Spectrophotometer DU 6401. The colony-forming
have demonstrated that chronic atrophic gastritis is units (CFU) were derived from a standard curve
a precursor lesion of gastric cancer, and chronic correlating OD. with plate counts.
gastritis might be induced or exacerbated by H.
Preparation of bacterial cytosols
pylori (Dixon, 1989; Kato et al., 1992; Marshall,
1986). Evidence has also shown that gastritis, peptic
ulcer, and gastric cancer are related to the presence
of this bacterium (Marshall et al., 1988).
About 10 Â1010 bacterial colony forming units
(CFU) were washed twice in cold phosphate buf-
fered saline then placed immediately in 1 ml lysis
bu€er [20 mM Tris±HCl, pH 7.5 (at 48C), 1 mM
DTT, 1 mM EDTA, 50 mM PMSF and 10 mM leu-
peptin]. Then the cell suspensions were disrupted in
a sonicator following by centrifugation for 30 min
at 10,000 g. The supernatant was kept on ice until
assayed for NAT activity.
Vitamin E (a-tocopherol) is an antioxidant and
acts against some cancers, including inhibition of
chemical carcinogenesis in some animal studies
(Byers and Perry, 1992; Dorgan and Schatzkin,
1991; Odukoya et al., 1984). The possible role of
vitamin E in the enhancement or inhibition of
colon tumours in mice has also been reported
(Dorgan and Schatzkin, 1991). In addition to its
antioxidant property, vitamin E in¯uences cellular
responses to oxidative stress through modulation of
signal transduction pathways (Azzi et al., 1992).
Vitamin E has been found to inhibit in vitro brain
protein kinase C activity (Mathonery and Azzi,
1989). Vitamin E protected human skin ®broblasts
against the cytotoxic e€ects of UV in tissue culture
(Kondo et al., 1990). But other reports have
demonstrated that rats or mice with intestinal
tumours induced by repeated doses of 1,2-dimethyl-
hydrazine showed higher tumour incidence when
fed a diet with added vitamin E as compared with a
vitamin E-de®cient diet (McIntosh, 1992; Temple
and El-Khatib, 1987). There is no available infor-
mation about vitamin E a€ecting bacterial NAT ac-
tivity. Thus, the present study was focused on the
e€ects of vitamin E on NAT activity in H. pylori.
NAT activity determination
The determination of Ac-CoA-dependent N-
acetylation of 2-AF were as described by Chung
et al. (1997). Incubation mixtures in the assay sys-
tem consisted of a total volume of 90 ml: tissue cyto-
sol, diluted as required in 50 ml lysis bu€er (20 mM
Tris±HCl, pH 7.5, 1 mM DTT, 1 mM EDTA, 1 mM
acetyl carnitine) and 2-AF or PABA at known con-
centrations for substrate. The reactions were started
by addition of 20 ml AcCoA. The controls had 20 ml
water added in place of AcCoA. The ®nal concen-
tration of PABA or 2-AF was 0.1 mM, and that of
AcCoA was 0.5 mM. The reaction mixtures were
incubated at 378C for 10 min and stopped with
50 ml 20% trichloroacetic acid for PABA reactions
and 100 ml acetonitrile for 2-AF reactions. All ex-
perimental reactions and controls were run in tripli-
cate. The amounts of acetylated product and
remaining non-acetylated substrate were determined
by HPLC (Chung et al., 1997). An aliquot of the
NAT reaction mixtures was injected onto a C18
reversed-phase column (Spherisorb 4.6Â 250 nm) of
a Beckman HPLC (pump 168 and detector 126),
and eluted at a ¯ow rate of 1.2 ml/min. 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 of PABA was 4.0 min
and that of N-Ac-PABA was 6.5 min. For 2-AF
and 2-AAF, the solvent system was 20 mM
KH2PO4, pH 4.5/CH3CN (53:47) with detection at
280 nm. The retention time was 6.5 min for AAF
and 9 min for AF. All compounds were quantitated
by comparison of the integrated area of the elution
peak with that of known amounts of standards.
NAT activity is expressed as nmol acetylated per
min per mg of cytosolic protein.
MATERIALS AND METHODS
Chemicals and reagents
Vitamin E (a-tocopherol), ethylenediaminetetra-
acetic acid (EDTA), 2-amino¯uorene (2-AF), 2-
acetylamino¯uorene (2-AAF), p-aminobenzoic acid
(PABA), acetyl-p-aminobenzoic acid (N-Ac-PABA),
carnitine acetyltransferase, Tris, leupeptin, bovine
serum albumin (BSA), phenylmethylsulfonyl¯uoride
(PMSF), acetyl carnitine, dithiothreitol (DTT) and
acetyl-coenzyme
A were obtained from Sigma
Chemical Co. (St Louis. MO, USA). Acetonitrile,
dimethyl sulfoxide (DMSO) and potassium phos-
phates were obtained from Merck Co (Darmastadt,
Germany). All of the chemicals used were reagent
grade. The Bio-Rad protein assay kit was obtained
from Bio-Rad Laboratories (Hercules, CA, USA).
Helicobacter pylori
Protein determination
H. pylori was clinically isolated from patients
Protein concentrations in the cytosols of H. pylori
who visited the department of medicine (China were determined by the method of Bradford (1976)
Medical College) for endoscopy, as described in with BSA as the standard. All of the samples were
previous studies Chung et al. (1997). The optical assayed in triplicate.

E€ects of vitamin E on bacterial NAT activity
657
Table 1. E€ects of vitamin E on H. pylori NAT activity in cytosols
Vitamin E concn
AAF (nmol/min/mg protein
N-Acetyl-PABA (nmol/min/mg protein)
Control
0.04 mM
0.4 mM
4 mM
40 mM
400 mM
0.9120.12
0.9620.12
a1.1820.16
b1.4620.28
c1.6420.22
d1.7020.25
0.7520.08
0.8020.10
a0.9220.11
b1.1820.16
c1.2920.24
d1.3220.28
H. pylori cytosol was incubated in the presence of various concentrations of vitamin E (0.04, 0.4, 4, 40 and 400 mM). The NAT activity
was determined as described in Materials and Methods. Values are means2SD of three individual experiments. aDi€ers between
0.4 mM vitamin E and control; P < 0.05. bDi€ers between 4 mM vitamin E and control; P < 0.01. cDi€ers between 40 mM vitamin
E and control; P < 0.005. dDi€ers between 400 mM vitamin E and control; P < 0.001.
Table 2. E€ect of vitamin E on H. pylori NAT activity in intact bacteria
Vitamin E concn
AAF (nmol/min/mg protein)
N-Acetyl-PABA (nmol/min/mg protein)
Control
0.04 mM
0.4 mM
4 mM
40 mM
400 mM
0.7520.14
0.8020.12
a0.8820.11
b0.9620.20
c1.0720.23
d1.1220.27
0.6820.10
0.7120.12
a0.8020.09
b0.8920.14
c1.0120.22
d1.0720.24
H. pylori was incubated in the presence of various concentrations of vitamin E (0.04, 0.4, 4, 40 and 400 mM). The NAT activity was deter-
mined as described in Materials and Methods. Values are means2SD of three individual experiments. aDi€ers between 0.4 mM vita-
min E and control; P < 0.05. bDi€ers between 4 mM vitamin E and control; P < 0.01. cDi€ers between 40 mM vitamin E and
control; P < 0.005. dDi€ers between 400 mM vitamin E and control; P < 0.001.
Time-course study measuring the e€ects of vitamin E speci®c concentration of vitamin E (40 mM) for
on NAT activity in H. pylori
96 hr at 378C in an aerobic atmosphere ( 5% O2,
10% CO2, 85% N2). At the conclusion of incu-
bation, the cells and media were removed and cen-
trifuged. For the experiments with 2-AF, the
supernatant was immediately extracted with ethyla-
cetate±methanol (95:5), the solvent evaporated, and
the residue redissolved in methanol, and assayed by
HPLC. For the experiments with PABA, the super-
natant was direct assayed by HPLC (Chung et al.,
1997). All of the samples were run in triplicate.
The reaction mixtures (containing 0.087 mM 2-
AF) contained 3 Â109 cells in the presence or
absence of vitamin E (40 mM, which is a normal
concentration in humans) were incubated at 378C
for 0.25, 1, 2 and 4 hr, respectively. The NAT ac-
tivity was assayed as described above.
E€ects of various concentrations of vitamin E on
NAT activity of H. pylori
Vitamin E was dissolved in DMSO at various
concentrations ranging from 0.04 to 400 mM. The
reaction mixtures consisted of 50 ml cytosols diluted
as required, 20 ml recycling mixture containing 2-
AF or PABA at selected concentration for sub-
strates, and 10 ml vitamin E (at selected concen-
tration). The reactions were started by addition of
20 ml Ac-CoA. The control reactions had 20 ml dis-
tilled water in place of Ac-CoA. Following these
steps, the NAT determination was determined as
described above for the e€ects of vitamin E on
NAT activity in H. pylori (Chung et al., 1997).
Statistical analysis
Statistical analysis of the data was performed
with an unpaired Students t-test. The kinetic con-
stants were calculated with the Cleland HYPER
Program (Cleland, 1967) that performs linear re-
gression using a least-squares method. The velocity
1/V) versus substrate (1/S) data were linearized by
plotting (1/S) v. (1/V).
RESULTS
The possible e€ects of vitamin E on NAT activity
in H. pylori in cytosols and intact bacteria were
examined by HPLC assessing the percentage of
E€ects of vitamin E on the kinetic constants of NAT
from H. pylori
Cytosols of H. pylori were co-treated with or acetylation of 2-AF and PABA. Cytosols or suspen-
without speci®c concentration of vitamin sions of H. pylori, with or without speci®c concen-
a
E
(40 mM) and the NAT activity determined as trations of vitamin E co-treatment, showed di€erent
described above. All of the reactions were run in percentages of 2-AF and PABA acetylation. A com-
triplicate. For the intact bacterial studies, 3 Â109 parison of the relative cytosolic and intact bacterial
bacterial cells in trypticase soy broth were incubated NAT activity, with or without speci®c concen-
with arylamine as the substrate (22, 45, 67.5, 90, trations of vitamin E, are presented in Tables 1 and
112.5, 135 mM 2-AF or PABA) with or without a 2. The data indicated that there was increased NAT

658
J. G. Chung
Fig. 2. Lineweaver±Burk double reciprocal plot of NAT in
H. pylori by cytosol examination. Recycling mixtures con-
taining 0.087, 0.174, 0.384, 0.696 and 1.392 mM 2-AF (A)
and PABA (B) were used. Cytosols were prepared as
described in Materials and Methods. Lineweaver±Burk
double reciprocal plots were measured by using linear re-
gression analysis of reciprocal substrate concentrations
plotted against reciprocal initial velocities. Q= control.
* = vitamin E-treated.
Fig. 1. Time-course e€ects of 40 mM vitamin E on NAT
activity in H. pylori. Reaction mixtures (with or without
40 mM vitamin E) [(A) for cytosol examination and (B) for
intact bacteria examination] were incubated at 378C and
assayed at 0.25, 1, 2 and 4 hr, respectively. Time-course
e€ects were determined as described in Materials and
Methods. Open bar = control; Shaded bar = vitamin E-
treated.
activity associated with increased vitamin E levels
in cytosols and intact bacteria, that is, the higher
the concentration of vitamin E in the reaction mix-
tures, the higher the promotion of NAT activity.
To determine the time-course e€ect of 40 mM
vitamin E on NAT activity both in cytosols and
intact bacteria, reaction mixtures with or without
40 mM vitamin E were incubated at 378C and
assayed at di€erent time intervals. The results are
shown in Fig. 1(a, b). For the cytosol examination,
the amounts of acetylated product increased
6.020.8%, 8.420.9%, 9.620.7%, 12.220.8%
for the 0.25, 1, 2 and 4-hr incubations for 2-AF
acetylation, respectively. For the intact bacteria
examination, the amounts of acetylated product
increased 16.822.2%, 28.426.1%, 19.621.4%
and 24.822.6% for the 0.25, 1, 2 and 4-hr incu-
bations for 2-AF acetylation, respectively. Similar
results are found in PABA, which is used as sub-
strate in cytosol and intact bacteria examinations
(data not shown).
protein, respectively, for 2-AF, and 0.6920.07 mM
and 4.3320.27 nmol/min/mg protein, respectively,
for PABA. However, when vitamin E was added to
the reaction mixture, the apparent values of Km
and Vmax were increased 1.0- and 0.72-fold, respect-
ively, for 2-AF and 0.32- and 0.51-fold, respectively,
for PABA. For the intact bacteria examination, the
apparent values of Km and Vmax were
0.7020.07 mM and 3.1220.26 nmol/min/10 Â 1010
CFU, respectively, for 2-AF, 0.4520.06 mM and
1.1720.09 nmol/min/10 Â 1010 CFU, respectively,
for PABA. However, when vitamin E was added to
the reaction mixture, the apparent values of Km
and Vmax were increased 0.22- and 0.34-fold,
respectively, for 2-AF, and 0.24-and 0.13-fold,
respectively, for PABA.
DISCUSSION
There are many events which are likely to be fac-
tors for the observed e€ects of vitamin E on NAT
activity in H. pylori. First, the NAT enzyme exists
in many kinds of experimental animals including
humans, and NAT has been shown to be involved
The kinetic constants determined for H. pylori
NAT using 2-AF and PABA as substrates with or
without 40 mM vitamin E are shown in Figs 2 and
3 and given in Tables 3 and 4. For the cytosol
examination, the apparent values of Km and Vmax in metabolism of arylamine carcinogens (Grant
were 0.9020.09 mM and 5.1820.28 nmol/min/mg et al., 1992; Minchin et al., 1992; Weber, 1987).

E€ects of vitamin E on bacterial NAT activity
659
available reports that address vitamin E e€ects on
NAT activity in H. pylori. Thus, the present studies
were focused on the e€ects of vitamin E on NAT
activity in H. pylori cytosols and intact bacteria.
According to other reports, the micro¯ora may
be involved in metabolism of chemical compounds
in human or animal intestine. The intestinal micro-
¯ora in mice have been reported to play an import-
ant role on absorption and metabolic activation of
1-nitropyrene (Kinouchi et al., 1993). But in
humans, 1-nitropyrene can be metabolized to 1-ami-
nopyrene and N-formyl-1-aminopyrene by human
micro¯ora (Manning, 1986), and the acetyltransfer-
ase enzyme of human enteric bacteria may contrib-
ute to the metabolic activation of 2-amino-3-
methylimidazo[4,5-f]quinoline (Watanabe et al.,
1992). Other investigators have demonstrated that
some anaerobic and facultative bacteria normally
associated with the mammalian intestine could per-
form N-acetylation, as well as N-formation, of aryl-
amine (Okumu et al., 1995). The nature of the
bacterial enzyme responsible for N-formation
remains unknown. Thus, H. pylori may be involved
in the metabolic activation or detoxi®cation of the
arylamine carcinogen in humans, because the pre-
Fig. 3. Lineweaver±Burk double reciprocal plot of NAT in sent author's earlier study demonstrated that H.
H. pylori by intact bacteria examination. Recycling mix-
pylori could acetylate 2-AF and PABA in vitro and
tures containing 0.087, 0.174, 0.384, 0.696 and 1.392 mM
intact bacteria cells (Chung et al., 1997).
Vitamin E has been widely studied for its e€ects
2-AF (A) and PABA (B) were used. Intact bacteria were
prepared as described in Materials and Methods.
on biological functions. The data presented here
clearly demonstrated that vitamin E did a€ect H.
pylori NAT activity, in concentrations from 0.004
to 400 mM in cytosols and in intact bacteria exam-
inations. Vitamin E did increase the formation of
Lineweaver±Burk double reciprocal plots were measured
by using linear regression analysis of reciprocal substrate
concentrations plotted against reciprocal initial velocities.
Q= control. * = vitamin E-treated.
Secondly, some enzymes of enteric bacteria are acetylated products of 2-AF and PABA by H.
known to contribute to the metabolic activation of pylori. The results show that vitamin E had a dose-
chemical carcinogens in animal studies (Kinouchi dependent e€ect on the NAT activity in H. pylori,
et al., 1993; Larsen, 1988). Thirdly, it has been that is, the higher the concentration of vitamin E,
reported that several species of dog intestinal bac- the higher the promotion of NAT activity. The pro-
teria exhibit N-acetylation activities towards aryl- motion of vitamin E to the acetylation of 2-AF is
amines in varing degrees (Okumura et al., 1995). slightly higher than with PABA. Substrate speci®ci-
Fourthly, earlier studies demonstrated that H. ties for NATs are di€erent, but overlapping. For
pylori contains NAT activity (Chung et al., 1997). example, in humans, NAT1 acetylates PABA and
Recently, the present author's data also showed p-aminosalicyclic acid, whereas, NAT2 does not.
that vitamin C promotes NAT activity in Klebsiella NAT2 acetylates sulfamethazine, whereas, NAT1
pneumoniae. Fifthly, other investigators have shows negligible activity towards this substrate.
already demonstrated that attenuation of NAT ac- However, both enzymes show signi®cant activity
tivity is associated with several disease processes with carcinogenic arylamines, such as 2-AF.
(Weber, 1987; Weber and Hein, 1985). There are no Although several intestinal bacteria show di€erent
Table 3. Kinetic data for acetylator of 2-AF in H. pylori
Cytosol
Vmax(nmol/min/mg)
Intact bacteria
Km (mM)
Km(mM)
Vmax(nmol/10 Â1010CFU)
Control
Vitamin E
0.9020.09
a1.8220.27
5.1820.28
b8.6920.35
0.7020.07
b0.8520.08
3.1220.26
d4.1620.28
Values are means2SD of three independent experiments. The acetyl CoA and vitamin E concentrations were 0.1 mM and 40 mM, respect-
ively. The kinetic constants were calculated from the modi®ed HYPER Program of Cleland (1967). aDi€ers between 40 mM vitamin
E and control; P < 0.001. bDi€ers between 40 mM vitamin E and control; P < 0.05.

660
J. G. Chung
Table 4. Kinetic data for acetylator of PABA in H. pylori
Cytosol
Intact bacteria
Km(mM)
Vmax(nmol/min/mg)
Km(mM)
Vmax(nmol/10 Â1010CFU)
Control
Vitamin E
0.6920.07
a0.9020.08
4.3320.27
b6.5320.22
0.4520.06
b0.5620.08
1.1720.09
d1.3320.12
Values are means2SD of three independent experiments. The acetyl CoA and vitamin E concentrations were 0.1 mM and 40 mM, respect-
ively. The kinetic constants were calculated from the modi®ed HYPER Program of Cleland (1967).aDi€ers between 40 mM vitamin E
and control; P < 0.01.bDi€ers between 40 mM vitamin E and control; P < 0.05
N-acetylation activities for 2-AF and PABA, most et al., 1991). Other studies have demonstrated that
of these bacterial species show higher activity for attenuation of NAT activity is associated with sev-
eral disease processes (Weber, 1987; Weber and
Hein, 1985). This needs further investigation. Four
patients were o€ered orally vitamin E 400 (IU/soft
gells twice per day for 4 days), followed by the iso-
lation of the bacteria then H. pylori were examined
in vitro for NAT activity. The results demonstrated
that vitamin E did increase NAT activity in two
patients (P < 0.05) (data not shown), but two
strains isolated from another two patients did not
show signi®cant di€erences between the vitamin E
treated group and the group that was not treated
with vitamin E. Humans may take vitamin E as a
dietary supplement and may be exposed simul-
taneously to arylamine carcinogens through food
consumption (Wakabayashi et al., 1992); further in-
vestigations are needed to determine the enhancing
e€ect of vitamin E on H. pylori NAT activity.
Vitamin E could promote the metabolism of aryl-
amine carcinogens in humans. Therefore, this ®nd-
ing is very important and o€ers information about
the role of an antioxidant agent (vitamin E) which
may or may not protect 2-AF-induced carcinogen-
esis.
acetylation of 2-AF than for PABA. The di€erence
between these two types of activity may be due to
bacterial species variations for NAT activity.
Currently, there are no available reports that
demonstrate whether bacterial NAT belongs to
NAT1 or NAT2, and no bacterial NAT sequences
have been reported, although several reports have
demonstrated that bacteria contain NAT activity
using arylamine as the substrate.
The results in the time-course study showed that
the promotion rates of vitamin E slightly increases
as the incubation time continues. There are two
reasons for choosing 40 mM vitamin E for the
reported examinations. First, NAT activity in H.
pylori was promoted about 50% after co-treated
with vitamin E. Secondly, the amounts of vitamin E
(40 mM) was close to the normal concentration (12±
35 mM) in human (Stuart, 1982). As vitamin E did
promote NAT activity in H. pylori, the kinetic con-
stants were also a€ected. For the cytosol examin-
ations, co-treatment with vitamin E indicated that
the apparent values of Km and Vmax increased 0.22-
and 0.54-fold for 2-AF, and 0.26- and 0.58-fold for
acetylation of PABA. For the intact bacteria exam-
ination, co-treatment with vitamin E indicated that
the apparent values of Km and Vmax increased 0.10-
and 0.44- fold for acetylation of 2-AF, 0.12- and
0.49-fold for acetylation of PABA. The results from
kinetic studies showed that vitamin E increased the
apparent Km and Vmax values in both assay sys-
tems. These results suggest that vitamin E promotes
the N-acetylation of both substrates at high ex-
posure levels.
REFERENCES
Azzi A., Boscobonik D. and Hensey C. (1992) The protein
kinase C family. European Journal of Biochemistry 208,
547±557.
Blaser M. J. and Parsonnet J. (1994) Parasitism by the
``slow'' bacterium Helicobacter pylori leads to altered
gastric homeostasia and neoplasia. Journal of Clinical
Investigation 94, 4±8.
Bradford M. M. (1976) A rapid and sensitive method for
the quantitation of microgram quantities of protein uti-
lizing the principle of protein-dye binding. Analytical
Biochemistry 72, 248±254.
Byers T. and Perry G. (1992) Dietary carotene, vitamin C,
and vitamin E as protective antioxidants in human can-
cer. Annual Review of Nutrition 12, 139±159.
Cartwright R. A., Glasham R. W., Rogers H. J., Ahmed
R. A., Barham-Hall D., Higgens E. and Kahn
M. A. (1982) Role of N-acetyltransferase phenotype in
bladder carcinogenesis: A pharmacogenetic epidemiolo-
gical approach to bladder cancer. Lancet ii, 842±846.
Chung J. G., Wang H. H., Tsou M. F., Hsien S. E., Lo
H. H., Yen Y. S., Chang S. S., Wu L. T., Lee J. H. and
Hung C. F. (1997) Evidence for arylamine N-acetyl-
transferase in the bacteria Helicobacter pylori.
Toxicology Letters 91, 63±71.
Vitamin E has been demonstrated to inhibit
chemical carcinogenesis in some but not all animal
studies. Both inhibition and enhancement of colon
tumours in mice have been reported (Linder, 1985).
Vitamin E increases the rate of substrate acetylation
by NAT, and it may or may not increase the risk of
tumour formation. It may depend on whether the
host was exposed to the arylamine carcinogens. If
the host was exposed to the arylamine carcinogen
then the increased NAT activity may lead to the
increased risk of tumour formation. Other reports
have demonstrated that elevated levels of NAT ac-
tivity may be associated with increased sensitivity to
the mutagenic e€ects of many arylamines (Einisto
Cleland W. W. (1967) The statistical analysis of enzyme
kinetics data. Advances in Enzymology 29, 1±30.

E€ects of vitamin E on bacterial NAT activity
661
Correa P. (1991) Is gastric carcinoma an infectious dis-
ease? New England Journal of Mededicine 325, 1170±
1171.
Dixon M. F. (1989) Campylobacter pylori and chronic gas-
tritis. In Campylobacter pylori and Gastroduodenal
Disease, pp. 106±116. Blackwell, Oxford.
Dorgan J. F. and Schatzkin A. (1991) Antioxidant micro-
nutrients in cancer prevention. Hematology and
Oncology Clinics of North America 5, 43±68.
Einisto P., Watanabe P., Ishidate M. and Nohmi T. (1991)
Mutagenicity of 30 chemicals in S. typhimurium posses-
sing di€erent nitroreductase or O-acetyltransferase ac-
tivities. Mutation Research 259, 95±102.
Manning B. W. (1986) Biotransformation of 1-nitropyrene
to 1-aminopyrene and N-formyl-1-aminopyrene by
human intestinal microbiota. Journal of Toxicology and
Environmental Health 18, 339±346.
Marshall B. (1983) Unidenti®ed curved bacilli on gastric
epithelium in active chronic gastritis. Lancet i, 1273±
1275.
Marshall B. (1986) Campylobacter pylori and gastritis.
Journal of Infectious Diseases 153, 650±657.
Marshall B., Goodwin C. S. and Warren J. R. (1988)
Prospective double-blind trial of duodenal ulcer relapse
after eradication of H. pylori. Cancer Research 2, 1437±
1447.
Mathonery C. M. and Azzi A. (1989) Vitamin E inhibits
protein kinase C activity. Biochemical and Biophysical
Research Communications 154, 694±697.
Minchin R. F., Reeves P. T., Teitel C. H., McManus
M. E., Mojarrabi T., Ilett K. F. and Kadlubar
F. F. (1992) N-and O-acetylation of aromatic and het-
erocyclic amine carcinogens by human monomorphic
and polymorphic acetyltransferase expressed in COS-1
Goodwin C. S. and Carrick J. (1991) Peptic ulcer disease
and Helicobacter pylori infection. Current Opinions in
Gastroenterology 7, 1008±1115.
Graham D. Y. (1989) Campylobacter pylori and peptic
ulcer disease. Gastroenterology 96, 615±625.
Grant D. M., Josephy P. D. and Lord H. I. (1992)
Salmonella typhimurium strains expressing human aryla-
mine N-acetyltransferase: metabolism and mutagenic ac-
tivation of aromatic amines. Cancer Research 52, 3961±
3964.
Hein D. W., Rustan J. D., Bucher K. D., Furman E. J. and
Martin W. J. (1991) Extrahepatic expression of the N-
acetylation polymorphism toward arylamine carcinogens
in tumor target organs of an inbred rat model. Journal
Pharmacology Experimental Therapy 258, 232±236.
Hsieh S. H., Lo H. H., Yen Y. S. and Chung J. G. (1997)
The e€ect of vitamin C on N-acetyltransferase activity
in Klebsiella pneumoniae. Food and Chemical Toxicology
35, 1151±1157.
cells.
Biochemical
and
Communications 185, 839±844.
Biophysical
Research
Odukoya O., Hawach F. and Shklar G. (1984)
Retardation of experimental oral cancer by topical vita-
min E. American Journal of Clinical Nutrition 53, 2835±
2865.
Okumura F., Uedo O., Kitamuru S. and Tatsumi
K. (1995) N-acetylation and N-formation of carcino-
genic arylamines and related compounds in dogs.
Carcinogenesis 16, 71±76.
Roberts-Thomson I. C., Ryan P., Khoo K. K., Hart W. J.,
McMichael A. J. and Butler R. N. (1996) Diet, acetyla-
tor phenotype, and risk of colorectal neoplasia. Lancet
18, 1372±1373.
Stuart M. J. (1982) Vitamin E de®ciency: its e€ect on pla-
telet-vascular interaction in various pathologic states.
Annals of the New York Academy of Sciences 393, 277±
278.
Temple N. J. and El-Khatib S. M. (1987) Cabbage and
vitamin E: Their e€ect on colon tumor formation in
mice. Cancer Letters 35, 71±77.
Vatsis K. P. and Weber W. W. (1993) Structural hetero-
geneity of Caucasian N-acetyltransferase at the NAT1
gene locus. Archives of Biochemistry and Biophysics 301,
71±76.
Wakabayashi K., Nagao M., Esumi H. and Sugimura
T. (1992) Food-derived mutagens and carcinogens.
Cancer Research 52 (Suppl.), 2092s±2098s.
Wang H. H. and Chung J. G. (1997) Emodin induced
growth inhibition and DNA damage in the Helicobacter
pylori. Current Microbiology 35, 262±266.
Ilett K. K., David B. M., Detchon P., Castledon W. M. and
Kwa R. (1987) Acetylator phenotype in colorectal carci-
noma. Cancer Research 47, 1466±1469.
Kato J., Tominaga S. and Ito Y. (1992) A prospective
study of atrophic gastritis and stomach cancer risk.
Japnanese Journal of Cancer Research 83, 1137±1142.
Kinouchi T., Kataoka K., Miyanishi K., Akimoto S. and
Ohnishi Y. (1993) Biological activities of the intestinal
micro¯ora in mice treated with antibiotics or untreated
and the e€ects of the micro¯ora on absorption and
metabolic activation of orally administrated glutathione
conjugates of K-region epoxides of 1-nitropyrene.
Carcinogenesis 14, 869±874.
Kondo S., Mamada A., Yamaguchi J. and Fukuro
S. (1990) Protective e€ect of dl-alpha-tocoperol on the
cytotoxicity of ultraviolet B against human skin ®bro-
blasts in vitro. Photodermatology, Photoimmunology,
Photomedicine 7, 173±177.
Lang N. P., Chu Z. J., Hunter C. F., Kendell D. C.,
Flammang J. J. and Kadlubar F. (1987) Role of aro-
matic amine acetyltransferase in human colorectal can-
cer. Archives of Surgery 121, 1259±1261.
Larsen G. L. (1988) Deconjugation of biliary metabolites
by micro¯oral b-glucronidase, sulphatase and cysteine
conjugate b-lyase and their subsequent enterohepatic
circulation. In Role of Gut Flora in Toxicity and Cancer,
ed. I. Rowland, pp. 79±107. Academic Press, London.
Linder M. (1985) Nutrition and metabolism of vitamins.
In Nutritional Biochemistry and Metabolism with Clinical
Applications, ed. M. Linder, pp. 115±122. Elsevier, New
York.
Watanabe M., Sofuni T. and Nohmi T. (1992)
Involvement of Cys69 residue in the catalytic mechan-
ism of N-hydroxylamine O-acetyltransferase of salmo-
nella typhimurium: sequence similarity at the amino
acid level suggests a common catalytic mechansim of
acetyltransferase for S. typhimurium and higher organ-
isms. Journal of Biological Chemistry 267, 8429±8436.
Weber W. W. and Hein D. W. (1985) N-acetylation phar-
macogenetics. Pharmacological Reviews 37, 25±79.
Weber W. W. (1987) The Acetylator Genes and Drug
Response. Oxford University Press, New York.
McIntosh G. H. (1992) The in¯uence of dietary vitamin E:
Their e€ect on colon tumor formation in mice. Cancer
17, 47±55.