Effects of the garlic compounds diallyl sulphide and diallyl disulphide on arylamine N-acetyltransferase activity in Klebsiella pneumoniae

Article abstract of DOI:10.1002/(SICI)1099-1263(199903/04)19:2<75::AID-JAT552>3.0.CO;2-P

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) were determined in the bacterium Klebsiella pneumoniae. Cytosols or suspensions of K. pneumoniae with or without specific concentrations of diallyl sulphide (DAS) or diallyl disulphide (DADS) as co-treatment showed different percentages of 2-AF acetylation. The data indicated that there was decreased NAT activity associated with increased levels of DAS or DADS in K. pneumoniae. In growth studies on K. pneumoniae it was demonstrated that DAS or DADS elicited a dose-dependent bacteriocide effect on K. pneumoniae. For the cytosol examinations, the apparent values of K(m) and V(max) were 0.96 ± 0.09 mM and 7.87 ± 0.79 nmol min^-1 mg^-1 protein, respectively, for 2-AF. However, when DAS or DADS was added to the reaction mixtures, the apparent values of K(m) and V(max) were 0.16 ± 0.04 mM and 0.99 ± 0.16 nmol min^-1 mg^-1 protein with DAS, respectively, and 0.14 ± 0.18 mM and 0.85 ± 0.10 nmol min^-1 mg^-1 protein with DADS, respectively, for 2-AF. For the intact bacteria examination, the apparent values of K(m) and V(max) were 0.57 ± 0.06 mM and 2.00 ± 0.14 nmol min^-1 per 10 x 10¹⁰ CFU, respectively, for 2-AF. However, when DAS or DADS was added to the reaction mixtures, the apparent of values of K(m) and V(max) were 0.41 ± 0.04 mM and 1.30 ± 0.10 nmol min^-1 per 10 x 10¹⁰ CFU with DAS, respectively, and 0.34 ± 0.04 mM and 1.08 ± 0.08 nmol min^-1 per 10 x 10¹⁰ CFU with DADS, respectively, for 2-AF. This report is the first demonstration to show that the garlic components DAS and DADS would affect K. pneumoniae growth and NAT activity.

Full text of DOI:10.1002/(SICI)1099-1263(199903/04)19:2<75::AID-JAT552>3.0.CO;2-P

JOURNAL OF APPLIED TOXICOLOGY
J. Appl. Toxicol. 19, 75–81 (1999)
Effects of the Garlic Compounds Diallyl
Sulphide and Diallyl Disulphide on
Arylamine N-Acetyltransferase Activity in
Klebsiella pneumoniae
Guang-Wei Chen,1,4 Jing-Gung Chung,2* Heng-Chien Ho3 and Juang-Geng Lin4
1Department of Surgery, China Medical College Hospital, Taichung 400, Taiwan, Republic of China
2Department of Microbiology, China Medical College, Taichung 400, Taiwan, Republic of China
3Department of Biochemistry, China Medical College, Taichung 400, Taiwan, Republic of China
4Institude of Chinese Medical Science, China Medical College, Taichung 400, Taiwan, Republic of China
Key words: N-acetyltransferase; 2-aminofluorene; N-acetyl-2-aminofluorene; Klebsiella pneumoniae.
Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) were determined in the
bacterium Klebsiella pneumoniae. Cytosols or suspensions of K. pneumoniae with or without specific
concentrations of diallyl sulphide (DAS) or diallyl disulphide (DADS) as co-treatment showed different
percentages of 2-AF acetylation. The data indicated that there was decreased NAT activity associated
with increased levels of DAS or DADS in K. pneumoniae. In growth studies on K. pneumoniae it was
demonstrated that DAS or DADS elicited a dose-dependent bacteriocide effect on K. pneumoniae. For
the cytosol examinations, the apparent values of Km and Vmax were 0.96 ؎ 0.09 mM and 7.87 ؎ 0.79 nmol
min؊1 mg؊1 protein, respectively, for 2-AF. However, when DAS or DADS was added to the reaction
mixtures, the apparent values of Km and Vmax were 0.16 ؎ 0.04 mM and 0.99 ؎ 0.16 nmol min؊1 mg؊1
protein with DAS, respectively, and 0.14 ؎ 0.18 mM and 0.85 ؎ 0.10 nmol min؊1 mg؊1 protein with
DADS, respectively, for 2-AF. For the intact bacteria examination, the apparent values of Km and Vmax
were 0.57 ؎ 0.06 mM and 2.00 ؎ 0.14 nmol min؊1 per 10 ؋ 1010 CFU, respectively, for 2-AF. However,
when DAS or DADS was added to the reaction mixtures, the apparent of values of Km and Vmax were
0.41 ؎ 0.04 mM and 1.30 ؎ 0.10 nmol min؊1 per 10 ؋ 1010 CFU with DAS, respectively, and
0.34 ؎ 0.04 mM and 1.08 ؎ 0.08 nmol min؊1 per 10 ؋ 1010 CFU with DADS, respectively, for 2-AF. This
report is the first demonstration to show that the garlic components DAS and DADS would affect K.
pneumoniae growth and NAT activity. Copyright 1999 John Wiley & Sons, Ltd.
loci have been implicated as important factors in
INTRODUCTION
determining susceptibility to environmental and occu-
pational arylamine carcinogens.5,9 In microorganisms,
Salmonella typhimurium and Helicobacter pylori have
been shown to contain NAT activity.10,11 Recently,
other investigators have demonstrated that the intestinal
microflora play an important role in the formation of
N-acetyl derivatives from arylamines in dogs.12 Thus,
it is suspected that the normal flora in the human
digestive system may be involved in the metabolism
of arylamines.
Genetic and environmental factors interact in chemical
carcinogenesis. Arylamine carcinogens require meta-
bolic activation by host enzymes in order to initiate
carcinogenesis in specific target organs or tissues.1 N-
Acetylation, a major metabolic pathway of arylamines
and hydrazines, is catalysed by cytosolic N-acetyl-
transferase (NAT) using acetyl coenzyme A as an
acetyl donor.2 In humans, two NATs, which were
identified and isolated from mammalian tissue, were
designated NAT1 and NAT2 and they catalysed the
N-acetylation of various arylamines.3–5 Both NAT2 and,
more recently, NAT1 have been shown to be polymor-
phic enzymes that segregate individuals into rapid and
slow acetylator phenotypes.5,6 Genetic polymorphism
influences the metabolic activation and detoxification
of environmental arylamine carcinogens and could
modulate the risks of human cancers, such as those
of the bladder and colorectum.7,8 Individual genetic
variations in humans for the regulation of the NAT
Garlic (Allium sativum) has been demonstrated to
possess anti-helmentic, anti-thrombotic, antibiotic,
fungicidal and anti-neoplastic cell properties.13–18 It has
been reported that garlic may be effective in the pre-
vention or treatment of several chronic diseases, such
as cardiovascular disease, hypertension and cancer.19–21 It
has also been reported that the components of garlic,
such as diallyl sulphide (DAS) and diallyl disulphide
(DADS), are effective inhibitors of the growth of neo-
plastic CMT-13 cells.18 Both DAS and DADS have
also been shown to be effective inhibitors of the pro-
motion phase of dimethylbene[a]anthracene- induced
skin tumours in mice.17,22 In the present authors’ lab-
oratory, it has been found that Klebsiella pneumoniae,
one member of the normal flora in the human intestine,
* Correspondence to: J.-G. Chung, Department of Medicine, China
Medical College, 91 Hsueh-Shih Road, Tiachung 400, Republic
of China.
CCC 0260–437X/99/020075–07$17.50
Copyright 1999 John Wiley & Sons, Ltd.
Received 6 May 1997
Accepted 12 April 1998

76
G.-W. CHEN ET AL
expressed higher NAT activity than other enteric bac-
teria examined. Other investigators have already dem-
onstrated that attenuation of liver NAT activity is
associated with bladder and breast disease processes.2,5
Therefore, the present study was focused on the effects
of DAS or DADS on the growth and NAT activity of
K. pneumoniae.
Arylamine N-acetyltransferase activity
determination
The determination of AcCoA-dependent N-acetylation
of 2-AF was performed as described by Chung et al.11
Protein concentrations
Protein concentrations in the cytosols from K. pneu-
moniae were determined by the method of Bradford23
with BSA as the standard. All of the samples were
assayed in triplicate.
EXPERIMENTAL
Chemicals and reagents
Preparation of specific concentrations of DAS or
DADS to measure the growth of K. pneumoniae
Diallyl sulphide (DAS) and diallyl disulphide (DADS)
were obtained from Fluka Chemika (Bucha,
Switzerland). Ethylenediaminetetraacetic acid (EDTA),
2-aminofluorene (2-AF), 2-acetylaminofluorene (2-
AAF), bovine serum albumin (BSA), phenylmethylsul-
phonylfluoride (PMSF), TRIS, leupeptin, acetyl carnit-
ine, dithiothreitol (DTT), carnitine acetyltransferase and
acetyl coenzyme A (AcCoA) were obtained from
Sigma Chemical Co. (St. Louis. MO). Acetic acid,
acetonitrile, dimethyl sulphoxide (DMSO) and potass-
ium phosphates were obtained from Merck (Darmstadt,
Germany). All of the chemicals used were reagent
grade. The VITEK GNI card was obtained from
bioMerieux Vitek, Inc. (Hazelwood, MO). The Bio-
Rad protein assay kit was obtained from Bio-Rad
Laboratories (Hercules, CA).
Either DAS or DADS was dissolved in DMSO with
specific concentrations ranging from 0.004 to 400 ␮M.
About 108 bacteria were placed in individual tubes
containing trypticase soy broth with or without specific
concentrations of DAS or DADS (0.004, 0.04, 0.4, 4,
40, 400 ␮M). The culture tubes were incubated at 35°C
in an aerobic atmosphere (5% O2, 10% CO2 and 85%
N2) for 24 h and checked for growth. The control
groups (treated with DMSO) were prepared under the
same conditions as the DAS- or DADS-treated groups
except without DAS or DADS.
Preparation of reaction mixture
The reaction mixtures consisted of 50 ␮l of the cytosols
diluted as follows: 20 ␮l of recycling mixture contain-
ing 2-AF at a specific concentration as substrate and
10 ␮l of DAS or DADS (at specific concentrations).
The reactions were started by the addition of 20 ␮l of
AcCoA. The control reactions had 20 ␮l of distilled
water in place of AcCoA.
Preparation of bacterial cytosols and intact
bacteria
Klebsiella pneumoniae, strain N3, was isolated from
patients at the China Medical College Hospital and
identified by using the VITEK GNI kit in conjugation
with the VITEK System. Klebsiella pneumoniae was
incubated in 100 ml of trypticase soy broth at 35°C
overnight and then centrifugated at 3500 g and 4°C for
20 min. The pellet was washed twice in cold phos-
phate-buffered saline and then used for the following
two procedures: (i) for cytosol NAT assays, the bac-
terial pellet (ca. 1 ml) was placed immediately in 2 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; (ii) the pellet was
resuspended in 2 ml of trypticase soy broth for all
intact cell assay systems. The absorbance of the intact
bacterial cell suspension was determined in triplicate
by using a Beckman Spectrophotometer DU 6401.
The colony-forming units (CFU) were derived from a
standard curve correlating absorbance with plate counts.
The determination of the affects of DAS or DADS
on the growth of K. pneumoniae was based on the
measurement of the absorbance as described above.
Growth inhibition (%) was determined using the fol-
lowing equation:
Time-course study measuring the effects of DAS
or DADS on NAT activity in K. pneumoniae
The reaction mixtures (containing 0.087 mM 2-AF) in
the presence or absence of DAS or DADS (40 ␮M)
were incubated at 37°C for 0.25, 1, 2 and 4 h, respect-
ively. The NAT activity was assayed as described pre-
viously.
Effects of DAS or DADS on the kinetic constants
of NAT from K. pneumoniae
Cytosols of K. pneumoniae were co-treated with or
without 40 ␮M DAS or DADS and the NAT activity
was determined as described above. All of the reactions
were run in triplicate. For the intact bacteria studies,
3 ϫ 108 bacterial cells in trypticase soy broth were
incubated with arylamine as the substrate (22, 45, 67.5,
90, 112.5, 135 ␮M 2-AF) with or without a specific
concentration of DAS or DADS (40 ␮M) for 4 h at
37°C in an aerobic atmosphere (5% O2, 10% CO2 and
85% N2). At the conclusion of incubation, the cells
and media were removed and centrifuged. For the
experiments with 2-AF, the supernatant was immedi-
ately extracted with ethyl acetate–methanol (95:5), the
solvent was evaporated and the residue was redissolved
in methanol and assayed for percentage utilization of
Original absorbance−final absorbance(ϩDAS or DADS)
1Ϫ
ϫ100
ͩ
ͪ
Original absorbance
Copyright
1999 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 19, 75–81 (1999)

NAT ACTIVITY IN K. PNEUMONIAE
77
2-AF by high performance liquid chromatography
(HPLC). All of the samples were run in triplicate.
Table 2. Effect of DAS on K. pneumoniae NAT activity
in cytosol examinationsa
Ϫ1
Concentration of DAS (␮M)
AAF (nmol minϪ1 mg protein)
Data analysis
Control
0.004
0.04
0.4
4
40
0.68 Ϯ 0.09
0.60 Ϯ 0.10
0.54 Ϯ 0.09
0.42 Ϯ 0.08b
0.32 Ϯ 0.07c
0.18 Ϯ 0.05d
0.08 Ϯ 0.02e
The average of three independent experiments was
presented with the standard deviation (mean Ϯ SD).
Statistical analysis of the data was performed with
unpaired Student’s t-test; P Ͻ 0.05 was considered sig-
nificant. The kinetic constants were calculated with
the Cleland HYPER Program,24 which performs linear
regression using a least squares method. The velocity
versus substrate data were linearized by plotting (1/V)
versus (1/S).
400
aValues are the means Ϯ SD of three independent experi-
ments. The AcCoA concentration was 0.1 mM for cytosol
examinations.
bDiffers between 0.4 ␮M DAS and control; P Ͻ 0.05.
cDiffers between 4 ␮M DAS and control; P Ͻ 0.01.
dDiffers between 40 ␮M DAS and control; P Ͻ 0.005.
eDiffers between 400 ␮M DAS and control; P Ͻ 0.001.
RESULTS
Effect of specific concentrations of DAS or DADS
on the growth of K. pneumoniae
are presented in Tables 2 and 3. In the presence of
0.004, 0.04, 0.4, 4, 40 and 400 ␮M DAS or DADS,
the NAT activities were decreased by 11.8 Ϯ 2.2%,
21.6 Ϯ 3.2%, 38.3 Ϯ 25.2%, 53.7 Ϯ 5.9%, 73.6 Ϯ
7.1% and 82.3 Ϯ 6.0% for DAS (Table 2) and by 8.8
Ϯ 0.6%, 25.0 Ϯ 1.3%, 41.2 Ϯ 2.4%, 58.9 Ϯ 3.9%,
70.5 Ϯ 4.4% and 91.2 Ϯ 5.6% for DADS (Table 3).
The NAT activities for the acetylation of 2-AF co-
treated with 40 ␮M DAS or DADS were decreased by
more than 70% and 90%, respectively. The data indi-
cated that there was decreased NAT activity associated
with increased DAS or DADS levels in K. pneumoniae
cytosols, i.e. the higher the concentration of DAS or
DADS in the reaction mixtures, the higher the inhi-
bition of NAT activity.
The effects of DAS or DADS on the growth of K.
pneumoniae are given in Table 1. Klebsiella pneumon-
iae was inhibited by DAS or DADS in a dose-depen-
dent manner, i.e. the higher the concentration of DAS
or DADS in the K. pneumoniae culture, the higher the
inhibition of K. pneumoniae. When the concentration
of DAS or DADS reached 400 ␮M, the inhibition
reached above 80%. The inhibition level of DADS was
slightly higher than that of DAS.
Effects of specific concentrations of DAS or DADS
on the NAT activity in K. pneumoniae
The possible effects of DAS or DADS on the NAT
activity in K. pneumoniae in cytosols and intact bac-
teria were examined by HPLC, assessing the percentage
of acetylation of 2-AF. Cytosols or suspensions of K.
pneumoniae with or without specific concentrations
of DAS or DADS as co-treatment showed different
percentages of 2-AF acetylation. Comparisons of the
relative cytosolic and intact bacteria NAT activity with
or without specific concentrations of DAS or DADS
Time-course effect of DAS or DADS on NAT
activity in K. pneumoniae
To determine the time-course effect of 40 ␮M DAS or
DADS on NAT activity both in cytosols and in intact
bacteria, reaction mixtures with or without 40 ␮M DAS
or DADS were incubated at 37°C and assayed at
different time intervals. The results are shown in Figs
Table 3. Effect of DADS on K. pneumoniae NAT activity
Table 1. Effect of DAS or DADS on the growth of K.
in cytosol examinations.a
pneumoniaea
Concentration of DADS (␮M) AAF (nmol minϪ1 mgϪ1 protein)
Concentration (␮M)
Control
0.004
0.04
0.4
4
40
0.68 Ϯ 0.09
0.62 Ϯ 0.12
0.51 Ϯ 0.10
0.40 Ϯ 0.09b
0.28 Ϯ 0.08c
0.14 Ϯ 0.05d
0.06 Ϯ 0.02e
0
0.004
0.04 0.4
% Inhibition
4
40
400
Control
DAS
DADS
0
0
0
0
12
14
0
28
31
0
48
53
0
56
62
0
67
72
0
82
89
400
aKlebsiella pneumoniae was incubated in the presence of spe-
cific concentrations of DAS or DADS (0.0004, 0.04, 0.4, 4, 40
and 400 ␮M) as described in Materials and Methods. The
percentage inhibition was determined under a spectrophoto-
meter and then calculated as described in Materials and
Methods.
aValues are means Ϯ SD of three independent experiments.
The AcCoA concentration was 0.1 mM for cytosol examination.
bDiffers between 0.4 ␮M DADS and control; P Ͻ 0.05.
cDiffers between 4 ␮M DADS and control; PϽ0.005.
dDiffers between 40 ␮M DADS and control; PϽ0.001.
eDiffers between 400 ␮M DADS and control; PϽ0.0001.
Copyright
1999 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 19, 75–81 (1999)

78
G.-W. CHEN ET AL
acetylated product decreased by 44.3%, 23.5%, 36.8%
and 34.5% for the 0.25-, 1-, 2- and 4-h incubations,
respectively. In the presence of DADS, the total
amounts of acetylated product decreased by 52.0%,
40%, 43.3% and 40.6% for the 0.25-, 1-, 2- and 4-h
incubations, respectively. For the intact bacteria
examination, the total amounts of acetylated product
were 4.0 Ϯ 0.4, 9.6 Ϯ 0.8, 15.8 Ϯ 1.3 and 23.7 Ϯ
1.6 nmol 1010 CFU after 0.25-, 1-, 2-, and 4-h incu-
bations, respectively. In the presence of DAS, the
total amounts of acetylated product decreased by 20%,
18.8%, 14% and 11.6% for the 0.25-, 1-, 2- and 4-h
incubations, respectively. In the presence of DADS,
the total amounts of acetylated product decreased by
30%, 27%, 23% and 22% for the 0.25-, 1-, 2-, and 4-
h incubations, respectively.
Effects of DAS or DADS on the kinetic Constants
of NAT in K. pneumoniae
The kinetic constants determined for K. pneumoniae
NAT using 2-AF as the substrate with or without
40 ␮M DAS or DADS are shown in Figs 3 and 4 and
given in Table 4. For the cytosol examination, the
apparent values of Km and Vmax were 0.96 Ϯ 0.09 mM
and 7.87 Ϯ 0.79 nmol minϪ1 mgϪ1 protein, respect-
ively. When DAS was added to the reaction mixtures,
the apparent values of Km and Vmax decreased by 83.4%
and 82.5%, respectively. When DADS was added to
the reaction mixtures, the apparent values of Km and
Vmax decreased by 85.5% and 90.2%, respectively. For
Figure 1. Time-course effects of 40 ␮M DAS or DADS on NAT
activity in K. pneumoniae by cytosol examination. Reaction
mixtures (with or without DAS or DADS) were incubated at
37°C and assayed at 0.25, 1, 2 and 4 h, respectively. Time-
course effects were determined as described in the
Experimental. White bars = control; light bars = DAS; black
bars = DADS.
1 and 2. For the cytosol examination, the total amounts
of acetylated product were 10.4 Ϯ 1.0, 18.5 Ϯ 1.8,
23.2 Ϯ 2.6 and 25.4 Ϯ 1.6 AAF nmol minϪ1 mgϪ1
protein for 0.25-, 1-, 2- and 4- h incubations, respect-
ively. In the presence of DAS, the total amounts of
Figure 3. Lineweaver-Burk double reciprocal plot of NAT
activity in K. pneumoniae by cytosol examination. Recycling
mixtures containing 0.087, 0.174, 0.384, 0.696 and 1.392 mM
2-AF were used. Cytosols were prepared as described in the
Experimental. Lineweaver–Burk double reciprocal plots were
measured by using linear regression analysis of reciprocal
substrate concentrations plotted against reciprocal initial velo-
cities. Circle = control; square = DAS; triangle = DADS.
Figure 2. Time-course effects of 40 ␮M DAS or DADS on NAT
activity in K. pneumoniae by intact bacteria examination.
Reaction mixtures (with or without DAS or DADS) were incu-
bated at 37°C and assayed at 0.25, 1, 2 and 4 h, respectively.
Time-course effects were determined as described in the
Experimental. White bars = control; light bars = DAS; black
bars = DADS.
Copyright
1999 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 19, 75–81 (1999)

NAT ACTIVITY IN K. PNEUMONIAE
79
DISCUSSION
The data presented in this report clearly indicate that
DAS and DADS do affect K. pneumoniae growth and
NAT activity. The effects of DAS or DADS on the
NAT activity in K. pneumoniae were dose-dependent,
i.e. the higher the concentration of DAS or DADS,
the higher the inhibition of NAT activity. For the intact
bacteria examinations, the data also showed that DAS
or DADS decreased the percentage of acetylated pro-
ducts of 2-AF. Both DAS and DADS also induced a
dose-dependent effect on the NAT activity in the intact
bacteria examinations. The data further demonstrated
DAS- or DADS-induced bacteriocidal effects on the
K. pneumoniae culture, i.e. the higher the concentration
of DAS or DADS, the higher the inhibition of K.
pneumoniae growth. This is the first report to show
that DAS or DADS could act as an antimicrobial agent
for K. pneumoniae. This finding is in agreement with
earlier studies from other investigators who have dem-
onstrated that garlic components show antibiotic
activity.15 Some enzymes of enteric bacteria are known
to contribute to the metabolic activation of chemical
carcinogens in animal studies.25–28 Other investigators
have demonstrated that attenuation of liver NAT
activity is associated with breast and bladder cancer
processes.3,5 But the decrease of NAT activity, whether
it could indicate the decrease of arylamine-induced
carcinogenesis or not, still needs further investigations.
The substrate specificities of the NATs are different
but overlapping: for example, in humans, NAT1 acetyl-
ates p-aminobenzoic acid (PABA) and p-aminosal-
icyclic acid whereas NAT2 does not; and NAT2 acetyl-
ates sulphamethazine, whereas NAT1 shows negligible
activity toward this substrate.2,5,29 However, both
enzymes show significant activity with carcinogenic
arylamines, such as 2-AF.29 Although several intestinal
bacteria show different N-acetylation activity for 2-AF
and PABA, most of these bacterial species show higher
activity for acetylation of 2-AF than for PABA.12 The
intestinal microflora in mice have been reported to
play an important role in absorption and metabolic
activation of 1-nitropyrene,27 but in humans 1-nitropy-
ene can be metabolized to 1-aminopyrene and N-
formyl-1-aminopyrene by human microflora29, and the
Figure 4. Lineweaver–Burk double reciprocal plot of NAT
activity in K. pneumoniae by intact bacteria examination.
Recycling mixtures containing 0.087, 0.174, 0.384, 0.696. and
1.392 mM 2-AF were used. Intact bacteria were prepared as
described in the Experimental. Lineweaver–Burk double
reciprocal plots were measured by using linear regression
analysis of reciprocal substrate concentrations plotted against
reciprocal initial velocities. Circle
= control; square = DAS;
triangle = DADS.
the intact bacteria examination, the apparent values of
Km and Vmax were 0.57 Ϯ 0.06 mM and 2.04 Ϯ
0.14 nmol minϪ1 per 10 ϫ 1010 CFU, respectively.
When DAS was added to the reaction mixture, the
apparent values of Km and Vmax were decreased by
28.1% and 35%, respectively. When DADS was added
to the reaction mixtures, the apparent values of Km and
Vmax were decreased by 30.4% and 46%, respectively.
Table 4. Kinetic data for acetylation of 2-aminofluorene in K. pneumoniaea
Cytosol
Intact bacteria
Vmax
Km (mM)
Vmax
Km (mM)
(nmol minϪ1 mg
)
(nmol per 10ϫ1010 CFU)
Ϫ1
Control
DAS
DADS
0.96 Ϯ 0.09
0.16 Ϯ 0.04b
0.14 Ϯ 0.03c
7.87 Ϯ 0.79
0.99 Ϯ 0.16c
0.85 Ϯ 0.10c
0.57 Ϯ 0.06
0.41 Ϯ 0.04d
0.34 Ϯ 0.04e
2.00 Ϯ 0.14
1.30 Ϯ 0.10e
1.08 Ϯ 0.08e
aValues are means Ϯ SD of three independent experiments. The AcCoA and DAS or DADS concentrations were 0.1 mM and
40 ␮M, respectively, and the kinetic constants were calculated from the modified HYPER Program of Cleland.
bDiffers between 40 ␮M DAS and control; P Ͻ 0.0001.
cDiffers between 40 ␮M DADS and control; PϽ 0.0001.
dDiffers between 40 ␮M DAS and control; P Ͻ 0.05.
eDiffers between 40 ␮M DADS and control; P Ͻ 0.05.
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1999 John Wiley & Sons, Ltd.
J. Appl. Toxicol. 19, 75–81 (1999)

80
G.-W. CHEN ET AL
acetyltransferase enzyme of human enteric bacteria may
contribute to the metabolic activation of 2-amino-3-
methylimidazo[4,5-f]quinoline.10 Thus, enteric bacteria
such as K. pneumoniae may be involved in the
metabolic activation or detoxification of arylamine car-
cinogens.
This point needs further investigation. The in vitro
data showed different degrees of DAS or DADS inhi-
bition on the NAT enzyme, therefore, the findings in
the present report are very important for the possibility
of decreasing arylamine carcinogens in induced car-
cinogenesis, because other reports have demonstrated
that elevated levels of NAT activity may be associated
with increased sensitivity to the mutagenic affects of
many arylamines.30 Other reports have demonstrated
that attenuation of liver NAT activity could be associa-
ted with several disease processes.2,5
In conclusion, the present studies showed that the
two main components of garlic, DAS and DADS, are
not equally effective inhibitors of K. pneumoniae
growth and NAT activity and that the oil-soluble
component of garlic, DADS, is more effective in
inhibiting K. pneumoniae growth and its NAT activity.
Because DAS and DADS do inhibit NAT activity
in K. pneumoniae, the kinetic constants were also
affected. We selected 40 ␮M DAS and DADS for
these studies and K. pneumoniae growth was inhibited
by more than 50% after co-treatment with DAS or
DADS. For cytosol examinations, co-treatment with
DAS or DADS indicated that the apparent values of
Km and Vmax decreased by 0.83- and 0.82-fold, respect-
ively, with DAS and by 0.85- and 0.90-fold, respect-
ively, with DADS for acetylation of 2-AF. For the
intact bacteria examinations, co-treatment with DAS or
DADS indicated that the apparent values of Km and
Vmax decreased by 0.28- and 0.35-fold, respectively,
with DAS, and by 0.30- and 0.46-fold, respectively,
with DADS for the acetylation of 2-AF. Based on the
kinetic constant decreases, it was suggested that DAS
or DADS could act as a non-competitive inhibitor.
Acknowledgement
This work was supported by grant CMC-SCIWCM-86 C001 from
the Supervisory Committee for the Integration of Western Chinese
Medicine.
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