Vol.
1, /153-1163,
October
Identification
Saliva
Clinical
/995
of a Class
and
Increased
S-Transferases,
Subjects
of Coffee
Norman
E. Sladek2
Department
of Pharmacology,
Minneapolis,
3 abdehyde
a,
and
Matthew
was tested
present
Each
in this fluid.
be abundantly
vegetables
that
the salivary
and
enzymes
these
in the
content
activities
of interest
of these
enzymes
human
diet,
upon
of coffee
rapidly
levels
reflect
to
environmental/industrial/dietary
these
enzymes
(e.g.
of
enzyme
when
bevels
DT-diaphorase
are
potential
bevels,
and
coordinately
of
pothat
quantification
duction
of
viewed
with
to detoxify
enzymes
human
are
diet,
7-9).
and
schedule
in Phase
chemopreventive
tional
diets
selection
several
enzymes;
are
selected
and
or drugs
and
trials
for any
of interest;
use of chemotherapeutic
inactivated,
alternatively,
first
I clinical
and a few are
the antineoplastic
then
a diet
that
putatively
and (c) the raagents, since
activated,
agent
enables
the
in-
interest
monofunctional
hydnoquinone,
bifunctional
inducems4
and
because
and
(e.g.,
(1-3).
In-
DT-diaphorase
these
carcinogens
enzymes
can be
(4).
is
enzymes
Inducers
in certain
components
the Cruciferae
and
are
of these
of the
Liliaceae
Vegetables,
fruits,
and
chemicals
that
induce
these
en-
zymes
prevent
chemopreventive
experimental
cancinogenesis,
i.e., they effect
a
action,
and the former
is thought
to be caus-
ative,
in part,
at beast
sirabbe
dose
both
3,4-benzpymene)
S-tnansfenases
certain
Chemoprevention
potential
the optimal
that
families
of vegetables
(5, 6). Certain
food
additives
(e.g., 2,3tert-butyl-4-hydmoxyanisobe)
and pharmaceuticals
(e.g.,
oltipraz) also act as inducens
of these enzymes
(reviewed
in Refs.
in: (a) preliminarily
the chemopreventive
by
as catechob,
and
abundantly
present
e.g. , members
of
idea
(b) establishing
and
known
in that
assessing
increased
particular
oltipnaz
drugs;
as an
agents
such
glutathione
attractive
and
be useful
is suspected.
,
of the salivary
content
of one or more of these enzymes,
a
noninvasive
and relatively
easy undertaking,
could be useful
diets
also
Celbuban expression
of ALDH-33
and certain
other
glutathione
S-transfenases
and DT-diaphorase)
(e.g.,
markedly
in cer-
as the cytotoxic
and assuming
tissue
toxic
3-methybcholanthnene
the carcinogenic
their
wise
to
to basal
may
to carcinogenic/teratogenic/other-
duce
are known
The
as well
agents,
measurements
be
or broccobi.
potential
would be selected
based
enzyme
activity
would
be favorable
exposure
to
found
especially
and
Such
when
phenolic
antioxidants
2,6-di-tert-butyl-4-hydnoxytobuene)
consumption
S-transferases,
some cancer-producing
agents
tentiab
of some
antineoplastic
of various
of
INTRODUCTION
includantineo-
daily
returned
to pbay in determining
salivary
of cytosobic
were
removed
from
the diet. Given
the
that cytosolic
class
3 abdehyde
dehydroge-
the glutathione
their
was
regard.
indicator
that are known
enzymes
increased
amounts
these
substances
important
role
in that
Further
investigation
revealed
of these enzymes
rapidly,
coordi-
fruits.
markedly
large
thought
Saliva
Quantities
S-transferases
of many xenobiotics,
and some
that are
Inducers
and
relatively
nase,
of
present
tam
nately,
in Human
achieve
its full therapeutic
on whether
high or low
and
Medical
gbutathione
catalyze
the biotransformation
ing some that are carcinogens
agents.
of Minnesota
for the presence
dehydrogenase,
pbastic
Hedge,
55455
and DT-diaphorase,
IT,
Large
1153
Glutathione
in the
Ingest
W.
University
Minnesota
saliva
cbass
,i,
DT-Diaphorase
Continually
Sreerama,
ABSTRACT
Human
Enzyme,
Research
or Broccoli’
Lakshmaiah
School,
Dehydrogenase
of This
Levels
and
Who
3 Aldehyde
Cancer
in such
induction.
One
relevant
cytes,
for
obvious
regard
has
way
tissue
batten
(reviewed
in this
and
been
this
activities
in
serum,
biopsies
(13-15).
is an
7-9).
immenseby
clinical
evaluation
initiated
(10-12).
is an estimate
of doing
in Refs.
manner
reasons,
already
investigations
enzyme
on
of the
mediated
might
of relevant
be
of
Deenzyme
to quantify
peripheral
blood
Limitations
of
the
bymphothese
ap-
by these
could
be
agent
to
The abbreviations
used are: ALDH-3,
human
cytosolic
class 3 aldehyde dehydrogenase;
mIU, milli-International
Unit of enzyme
activity
[nmol
NAD(P)H
formed/mm
in the case of aldehyde
dehydrogenase
activity,
nmol of the conjugate
of 1-chloro-2,4-dinitrobenzene
and glutathione
formed/mm
in the case of glutathione
S-transferase
activity,
nmol
2,6-dichborophenol-indophenol
reduced/mm
in the case of DTdiaphorase
activity,
and nmol p-nitrophenol
formed/mm
in the case of
esterase
activity];
PVDF,
polyvinylidene
difluoride.
4 Monofunctional
inducers
are defined
herein
as agents
that induce
3
Received
1/20/95;
revised
5/15/95;
accepted
5/18/95.
1 Supported
by USPHS
Grant
CA 21737,
Bristol-Myers
Squibb
Company Grant
I00-R220,
and Department
of Defense
Grant
DAMD
1794-J-4057.
Descriptions
of parts of this investigation
have appeared
in
abstract
form (L. Sreerama,
M. Hedge,
and N. E. Sladek.
Proc. Am.
Assoc.
Cancer
Res., 35: 84, 1994).
2
To whom
Pharmacology,
aware
Street
requests
for reprints
should
be addressed,
University
of Minnesota,
3-249
SE.,
Minneapolis,
MN 55455.
Millard
at Department
Hall,
435
of
Del-
ALDH-3,
glutathione
S-transferases,
quinone
oxidoreductase;
NQO1]
but
1A2; bifunctional
inducers
are defined
these enzymes
(Ref. 28 and references
and
DT-diaphorase
not cytochrome
herein
as agents
cited therein).
[NAD(P)H:
P450s
IA1 and
that induce
all of
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1995 American Association for Cancer
Research.
1 154
Induction
of Salivary
Chemopreventive
proaches
are
that
intensive,
and
the enzyme
(Ref.
be
15 and
to
after
exposure
to be even
It occurred
is present
more
to find
investigation
nase,
one
apparently
was
and
appear
namely,
(17,
isoebectnic
salivary
that,
from
in
DT-diaphonase
were
enzymes
direct
these
proportion
mace,
ethnicity,
had
contributed
between
demonstrated
( 19).
Further
sumption
in
markedly
increased
and
and that
levels
of
in approximately
habits
of the
who
pointed
,
subjects
consumption.
that
the continuous
of coffee
ALDH-3,
such
when
enzyme
fled.
They
con-
did indeed
result
glutathione
S-trans-
were
and/on
abdehyde
analysis
AND
4-Hydroperoxycycbophosphamide
(Asta
Medica
AG, Frankfurt,
Pohb
glutathione
polycbonal
S-transferases
a,
antibodies
specific
were
provided
istry,
Bowman
sity,
MO).
Food
Gray
was
ji, and
for each
School
NC).
purchased
of
(Department
Medicine,
Antinabbit
from
and affinity-purified
of these isozymes
(21)
‘ir
Wake
IgG
Sigma
of BiochemForest
alkaline
Chemical
Univer-
phosphatase
Co.
(St.
Minneapolis,
market,
washed,
chemicals,
sources
Human
reagents,
MN).
and
cooked
was
before
and supplies
or prepared
saliva and
and female
Broccoli
were
purchased
from
CL-6B
concentrated
as described
analysis
consumption.
purchased
All
from
as described
previously
(2, 22).
blood were obtained
from healthy
volunteers
ranging
from
20 to 55 years
other
commen-
(15-20
tinely,
the
activities.
of age.
above
when
plasma
thus
obtained
In those
cases
where
chromatographic
from 1.5 ml saliva)
on reactive
blue
(22)
oxidation
of benzabdehyde
successive
DEAE-Sephaceb
Sephanose
CL-6B
2-Sepharose
to eliminate
was
attempted.
in heparinized
was
syringes
and
4#{176}C
for 15 mm.
Rou-
then
assayed
for
enzyme
SDS-PAGE/immunobbot
after
analysis
which
plasma samples
blue 2-Sephanose
the
resultant
was 41 mIU/mb
exchange
fraction
above.
in the saliva
(NADPtdependent
anion
then
SDS-PAGE/immunobbot
X g and
chromatography,
individual
and
Desalted
preparation
was concentrated,
all as described
abdehyde
dehydrogenase
present
a single
(Ami-
by low-speed
SDS-PAGE
for the presence
of ALDH-3
was to be performed,
were first desabted
and subjected
to reactive
affinity
dehydnoge-
MA)
was
collected
In
(Sephadex
abdehyde
previously
resultant
at 2000
attempted.
concentrators
of ALDH-3
centrifuged
focusof the
S-tnansfenases).
the
was
ml)
obtained
enzyme-catalyzed
saliva)
was purified
chromatography,
cation
exchange
chromatography,
CL-6B
affinity
chromatography
by
CM-
and
reactive
as previously
described
(22),
except
that elution
of aldehyde
dehydrogenase
the affinity column
was with 1, rather than 5, mst NAD.
Ebectrotnansfer
and immunobbot
analysis
of ALDH-3
glutathione
(2,
21,
22);
S-transfenases
were essentially
antibody
dilutions
were 1:500
of ALDH-3
and glutathione
procedures
concentrations
were
strate
linear
ned
out
statistical
using
regression
the
program
for
used
the double-reciprocal
initial
to estimate
in duplicate
concentrations
weighted
used
of
to generate
analysis
plot
(24)
and
All other
previously
rates
(2,
versus
all Km values.
each
off
as described
before
and 1:1000
in the case
respectively.
as described
plots
were
determined
S-transferases,
were
Double-reciprocal
Computer-assisted
adult
and
to be sub-
of PD-10
or column
as described
for the presence
Blood
were
dehydrogenase;
of glutathione
proteins,
Louis,
a local
of
as
to be quanti-
to be
aid
4#{176}C
used
isoebectnic
Danvens,
focusing
nonspecific
experimental
Coffee
was purchased
from the University
of Minnesota
Services
Cafeteria
(University
of Minnesota
Medical
School,
male
was supplied
by Dr. J.
Germany).
Purified
human
were
was
the
& Co.,
aldehyde
2-Sepharose
blue
by Dr. A. J. Townsend
Winston-Salem,
conjugate
cial
METHODS
were
g supemnatant
fractions
(obtained
subjected
to affinity
chromatography
cancinogenesis
MATERIALS
X g and
x
9000
were
experimental
7, 9, and
at 9000
purification
focusing
(isoelectnic
immunobbot
prevent
in Refs.
with
into
obtained
in Centnicon-lO
W. R. Grace
of
placed
they
therein
desabted
(3-5
concentration
analysis,
present
purification
from
(reviewed
when
was
samples
(final
chromatographic
(isoelectnic
centrifugation
in inducens
20).
processed
were
hyaburonidase
therein
immunoblot
concentrated
Division,
CL-6B
(b)
present
column
they
and/or
to (a) be rich
6) and
further
columns
nase)
known
(5,
activities
thus
(22).
essentially
and were
added
fractions
was
saliva
centrifuged
supemnatant
dehydnogenase
of interest
The
enzymes
were
that
1 1 am
was
Pumu-
previously
Briefly,
9 and
Hu-
OH).
antistomach
of saliva
except
(23),
saliva.
at 4#{176}C.
DT1’
when
cases,
fenase,
and DT-diaphorase
levels
as did the continuous
consumption
of relatively
large quantities
of broccoli,
a vegetable
of these
described
processing
et al.
to SDS-PAGE,
immediately
revealed
that coffee
concatechob,
of these enzymes
as
the samples
The
ing,
prepared
between
kept
Columbus,
chicken
to the collected
and
to a direct
and coffee
amounts
salivary
be
strongly
revealed
barge
indeed
age,
activities
experimentation
of the
of the gender,
of the literature
of relatively
initiated
by Takase
by the Cooperative
Division,
and
further
collected
mM),
provided
ALDH-3
were
and
for 15 mm.
con
we ascertained
but
IgY
beakers
G-25)
one,
S-tmansferases
A review
e.g.
to
were
those
indeed
we
it
in the saliva
other
inducens,
3
sa-
reported
did
ml)
mitted
and
identity
investigations,
samples
the
Thus,
found
widely,
and
of
the
glutathione
other.
2,
by ALDH-3,
not.
and
enzyme
review
tains
was
present
dietary
1,
enzyme
exhibited
an
in human
most
evaluate
varied
salivary
A subsequent
those
of these
also
class
this
were
dehydroge-
ALDH-V,
of
values,
to each
and
relationship
known
the
across
aldehyde
Although
to ALDH-3,
each
of
the
an
added
5
to look
We
not
was
(Midwest
mucosa
ALDH-3
small
enzymes
in saliva.
stomach
as described
event,
documenting
mucosa
Network
Collection
approach
in any
we did come
of
dehydnogenase
addition
this
stomach
Tissue
cosa
e.g.,
during,
tissue/fluid
enzymes
to further
In the course
ALDH-3.
ideal
dubbed
18).
point
aldehyde
the
properties
designed
rifled
of the relevant
for reports
from
physical
experiments
and,
However,
and
to be different
(16);
presence
different
demonstrated
catalytic
be
of these
dehydnogenases
it would
before,
inducer
Human
man
parameters,
specific,
labor
intensive.
a report.
the
are
bow on nib
of doing
substrate
if one or more
it might
in which
abdehyde
as
the literature
such
way
test
to be
labor
saliva,
of at least
unable
liva
likeby
we searched
presence
Another
two
are very
phammacokinetic
to us that
in the
at. Thus,
study).
to the suspected
not
latter
in serum
of an appropriate
invasive,
likely
the
levels
various
half-life,
is also
is invasive,
the present
determine
plasma
and
each
Enzymes
of the
each
was
22).
substrate
Initial
mates
six to eight
value.
used
sub-
Wilkinson
to fit lines
to
values.
unweighted
STATView
to generate
regression
(Brainpower,
all other
analysis
Inc.,
linear
was
Calabas,
functions.
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1995 American Association for Cancer
Research.
carCA)
Clinical
Table
I
Aldehyde
dehydro
genase
purified
activity
saliva”
Saliva
Stomach
Mucosa”
31,000
33,000
Measurement
Specific
fro m human
(mIU/mg
protein)
Yield (%)
Fold purification
51
Research
1155
123
!1#{231}
7.35
60
1,340
Cancer
6.85
423
Enzyme purification
was as described
in ‘ ‘ Materials
and Methods.’ ‘ Benzaldehyde
(4 mM) and NAD
(1 mM) were
used as substrate
and cofactor, respectively,
to monitor aldehyde dehydrogenase
activity.
Aldehyde
dehydrogenase
activity in the starting material (9000 X g
“
supernatant
fraction
of saliva)
was 41 mIU/ml.
F, Values
for purified
stomach
mucosa
ALDH-3
publication
(22); they are included
for comparative
are from
purposes.
6.55
#{149}1
:1
5.85
a previous
5.20
RESULTS
Preliminary
studies
revealed
dependent
enzyme
that catalyzed
to benzoic
this
acid
enzyme
in human
activity
independent
lected,
of
in
the
approximately
either
from
genase
was
followed
NAD
aldehyde
just
similar,
one
if not
and
that
kinetic
properties
of
The
hibited
apparently
pure
a specific
activity
in Refs.
point
22 and
exhibited
pattern
of ALDH-3
both
the denatured
mucosa
to inexplicably
in Refs.
linear
I 10,000
gradient
(Fig.
enzyme,
2)
not shown)
the relative
as well
subunit
and
2).
It was
concentration
catalytic
was
incubated
aldehyde
partially
of disulfiram
activity
it was
only
at 56#{176}C
(data
exhibited
estenolytic
physical
ticab
to those
the
activity
and catalytic
exhibited
to NADP
inhibited
(50
lost
as
(<30%)
and
jiM)
completely
dehydnogenases,
these
NAD
was
in less
heat
than
not presented).
purified
(8390
mIU/mg
characteristics
by stomach
mucosa
Like
been,
a high
i.e.,
when
all known
also
Each
of
iden(22).
This
fan,
mucosa
to
at,
distin-
and saliva
in the two human
booked
ALDH-3
abdophosphamide
characteristic
in stomach
individuals,
salivary
(Fig. 3). Lange intenindividual
S-transfenase
and DT-diaphorase
3).
Indicative
of
be directly
protein).
thus
3).
the salivary
of
tumor
namely,
and colon
C carcinoma,
the reaction
at measurably
by
are essentially
ALDH-3
have
adenocarcinoma
latter catalyze
a cofacton
enzyme
present
that are present
expression/secretion
relative
amounts
labile,
(Table
the ALDH-3
Among
as
salivary
to acet-
10 mm
salivary
that
ALDH-3,
oxidation
from
celb lines
MCF-7
breast
in that those in the
greaten rates (2, 3, 22,
26-28).
molec-
aldehyde
the
carboxyphosphamide
mucosa
As judged
by Km values,
the
much preferred
benzabdehyde
as a substrate,
molec-
mucosa
catalyzed
the ALDH-3s
with
native
Antistomach
the stomach
poorly
guishes
banding
somewhat
its relative
(data
and
1). While
exact
vary
to
and 25). As judged
by
PAGE
and gel permeation
not shown).
the native
identical
(Fig.
the
Like
only
22
5-200,
(data
not exactly
ALDH-3
exI),
(reviewed
ular weight
was 54,500.
aldehyde
dehydmogenase
(Table
by
dehydrogenase
protein
(Table
of 5.7-6.4,
seems
recognized
IgY
were
mucosa
p1 mange
on Sephacryl
was
ALDH-3
stomach
of ALDH-3
(reviewed
nondenatuming
chromatography
they
stomach
in the
of origin
ular weight
purified
characteristic
although
by
fabling
tissue
exhibited
Thus, the salivary
of its physical
and
salivary
abdehyde
of 31,000
mIU/mg
values
25),
always
the
as
of a
made.
and isoebectnic
those
those
ob-
an electro-
to that
not shown).
comparison
with
present
presence
I
Isoelectric
focusing
of the aldehyde
dehydrogenase
purified
from human
saliva.
Purification
and isoelectric
focusing
of the aldehyde
dehydrogenase
present
in human
saliva were as described
in “Materials
and Methods.”
Subjected
to isoelectric
focusing
were isoelectnic
point
standards
(Lane
/), and amounts
of purified
stomach
mucosa
ALDH-3
(Lane
2) and the aldehyde
dehydrogenase
purified from human saliva
(Lane
3) sufficient
to generate
approximately
10-15
nmol NADH/min
(as determined
by spectrophotometric
assay)
when
benzaldehyde
(4
mM)
and NAD
(I mM) were
used as substrate
and cofactor,
respectively.
Lane
/, stained
with Coomassie
brilliant
blue R-250
for the
presence
of proteins.
Lanes 2 and 3, stained
for aldehyde
dehydrogenase
activity
as described
in “Materials
and Methods”;
benzaldehyde
(4 mM)
and NAD
(4 mM) were used as the substrate
and cofactor,
respectively.
Fig.
dehydno-
exhibited
mucosa
ALDH-3
(data
was purified
and a direct
was
(10-15
h) eating.
exceeded
acetabdehyde
the
identical,
cob-
fractions
suggested
stomach
enzyme
ALDH-3
before
(15-16
volume
for aldehyde
octanal,
as cofactor)
was
of the proteins
by staining
3.50
was
sample
X g supernatant
dehydnogenase,
mobility
collected
of
individual
the
mm), on long after
when
the sample
(benzabdehyde,
and
(concentration)
given
focusing
4.55
of an NA(D)Pt
of benzaldehyde
level
any
at which
x g on 105,000
activity
phonetic
day
the sample
saliva
substrates
single
of
10 ml. Isoebectnic
9000
tamed
The
saliva
of
after (10-15
it did decrease
the presence
the oxidation
saliva.
the
time
on whether
mm), just
However,
in
:1
some
sort
ALDH-3
variations
levels
of
rebated
alcohol
consumption,
animal
products.
(Fig.
coordinated
regulation
salivary
gender,
the use of tobacco,
since
on
substantially
this
widely
of
it did appear
the salivary
beverage
the
the
to
4).
However,
consumption
consumed
varied
of these
enzymes
was the fact that
of each in any given saliva sample
appeared
Intenindividuab
variability
in the
enzymes
could
not be attributed
to
average,
bevels
in salivary
glutathione
were also observed
(Fig.
higher
(at least
bevels
levels
race,
of these
ethnicity,
or the consumption
to be rebated
of all three
in individuals
150 ml daily)
enzymes
who
when
of
to coffee
were,
regularly
compared
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1995 American Association for Cancer
Research.
1 156 Induction
of Salivary
Chemopreventive
Enzymes
A
B
12
S
.:wr
97.4
66.0
54.5
I’
45.0
-.
kDa
31.0
21.5
14.3
Fig. 2
Subunit
molecular
weight
of the aldehyde
dehydrogenase
purified
from human
saliva
as determined
by SDS-PAGE
and recognition
of the
denatured
enzyme
by antistomach
mucosa
ALDH-3
IgY. Purification
and SDS-PAGE
of the aldehyde
dehydrogenase
present
in human
saliva were
as described
in “Materials
and Methods.”
A, subjected
to SDS-PAGE
were molecular
weight
markers
(Lane
1) and 3 jig each of purified
stomach
mucosa
ALDH-3
(Lane 2) and the aldehyde
dehydrogenase
purified
from human
saliva (Lane
3). Molecular
weight markers were lysozyme
(14.3
kDa), trypsin
inhibitor
(21.5 kDa), carbonic
anhydrase
(31 kDa), ovalbumin
(45 kDa),
BSA monomer
(66 kDa),
and phosphorylase
b (97.4 kDa).
Proteins
in each lane were visualized
by staining
with Coomassie
brilliant
blue R-250.
A plot of log Mr versus mobility
was used to estimate
subunit
molecular
weights.
B, purified
stomach
mucosa
ALDH-3
and the aldehyde
dehydrogenase
purified
from human
saliva
(3 jig each) were submitted
to SDS-PAGE
and then electrotransferred
onto an Immobibon-PVDF
transfer
membrane.
The membrane
was then probed
with antistomach
mucosa
ALDH-3 IgY as described in “Materials
and Methods” to visualize stomach mucosa ALDH-3 (Lane I) and the aldehyde dehydnogenase
purified from
human
saliva
(Lane
2).
Table
2
Substrate
and
dehydrogenase
cofactor
purified
preferences
from human
Table
of the aldehyde
saliva
Km
3
Catalysis
by the aldehyde
of aldophosphamide
and benzabdehyde
dehydrogenase
purified
from human
relative rates”
(i.M)
nmol Aldophosphamide
Aldehyde
(mM)
Cofactor
Benzaldehyde
(0.05-4)
Acetaldehyde
(25-200)
Benzaldehyde
(4)
NADP
(1)
(4)
NAD
(1)
NADP
465
463
NADP
Source
Saliva
Stomach
505
486
85,000
oxidized/mm/mg
Benzaldehyde
(1000)
oxidized/mm/mg
0.32
0.29
mucosa”
dehydrogenase
activity
was quantified
as described
in
“Materials
and Methods”;
aldophosphamide
(160 i.M) or benzaldehyde
(4 mM) was the substrate,
and NAD
(1 mM) was the cofactor.
b The
value for purified
stomach
mucosa
ALDH-3
is from a previous publication
(27); it is included
here for comparative
purposes.
81,000
40
1,250
nmol
a Aldehyde
80,000
85,000
(4)
(0.01-1)
(0.1-4)
NAD
Stomach
Muscosa”
Saliva”
(mM)
NAD
oxidation
saliva:
54
1,000
Each value is the mean of three determinations.
Values
for purified
stomach
mucosa
ALDH-3
are from a previous
publication
(22); they are included
here for comparative
purposes.
“
I’
salivary
ALDH-3,
activities
that
tiveby
to the average
who
ALDH-3
uabs
was
who
revealed
and
Roasted
(19).
known
Thus,
indeed
regularly
that
‘n were
other
salivary
bevels
did not drink coffee
Immunobbot
analysis
relatively
drank
salivary
each
coffee
inducens,
elevated
relatively
levels
relatively
it seemed
of these
enzymes
at all (Figs. 3 and
(Fig. 5) confirmed
of
barge
elevated
beans
e.g.
likely
are
,
that
that
the
level
source
the relatively
of
of individ-
of coffee,
S-tmansfemases
in such
a rich
ably
saliva
amounts
hydroquinone,
brought
4).
in the
glutathione
in individuals
and
a,
ji,
of catechob
elevated
about
induced
and
enzymes
levels
of
coffee
by an agent(s)
individuals
on
volunteer
who
of
erage
for
(Fig.
alternately
and
several
the
7)
confirmed
days.
The
that
the
mela-
was,
in fact,
basis
that
these
without
relatively
(Fig.
DT-diaphorase
consumed
in the coffee,
salivary
activities
went
drinking
notion
a daily
hydroquinone,
expression/secretion
days
and
who
present
and/or
To further
test this notion,
S-tnansferase,
and DT-diaphonase
supported
of
in
catechob
the
S-tmansfenase,
observed
amounts
largely
several
individuals.
of these
large
glutathione
we
large
results
of
6).
Again,
bevels
of
most
prob-
coordinately
enzymes.
ALDH-3,
glutathione
were quantified
in a
drinking
amounts
the
coffee
experiment
immunobbot
ALDH-3
for
of the bev-
were
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1995 American Association for Cancer
Research.
fully
analysis
relatively
Clinical
A
.
Cancer
Research
B
.
S
E
.
80
S
E
60
E
E
I>
I.-
E
S
S
I
S
z
30
LU
o
20
LU
20
S
S
S
S
S
S
N
z
C,
S
S
a’
(I)
S
S
N
I-
S
LU
0
40
4
0
$
LU
S
I>
I-
>
I-
S
a’
S
a’
0
a,
I
-
0
=-
GST
DT-D
I
30
ACTIVITY,
r=0.88
40
mIU/mI
E
005
0
ALDH-3
I
20
ALDH-3
0
S
05
S
a’
005
a’
a’
0
S
S
S
0
0
§xaS
I
10
0
S
S
0
a’
8
$
.
>.
S
S
.
60
40
E
0
1157
ALDH-3
GST
E
80
DT-D
>-
Fig.
ALDH-3,
3
in human
of protein
“Materials
were
glutathione
saliva.
Collection
concentrations
and Methods.”
used as substrate
S-transferase,
and DT-diaphorase
levels
and processing
of saliva and determinations
and enzyme
activities
were as described
in
Benzaldehyde
(4 mM) and NADP
(4 mM)
and cofactor,
respectively,
to quantify
ALDH-3
activity.
Reduced
glutathione
(5 mM) and 1-chloro-2,4-dinitrobenzene
(I mM) were used to quantify
glutathione
S-transferase
(GS7)
activity.
2,6-Dichlorophenol-indophenol
(40 p.M), NADH (160 jiM),
and dicumarol (10 jiM) were used as substrate,
cofactor,
and inhibitor,
respectively,
to quantify
DT-diaphorase
(DT-D) activity.
Points
are the mean
of duplicate
determinations
made on single samples (3-5 ml) collected
from each donor. Enzyme activity was normalized
for salivary
volume
(A) and salivary
protein
(B). Overall
mean
± SD values were 9 ± 9
mIU/ml saliva and 5 ± 5 mIU/mg
salivary
9000 X g supernatant
protein
for ALDH-3,
36 ± 21 mIU/ml saliva and 20 ± 12 mIU/mg salivary
9000 X g supernatant
protein
for glutathione
S-transferase,
and 21 ± 19
mIU/ml saliva and 1 1 ± 11 mIU/mg
salivary 9000 X g supernatant
protein
for DT-diaphorase.
#{149},
subjects
who consumed
at least 150 ml of
coffee
daily; mean
± SD values
were 15 ± 12 mlU/ml
saliva
and 8 ±
6 mIU/mg salivary 9000 X g supernatant
protein
for ALDH-3,
51 ± 21
mIU/ml saliva and 28 ± 12 mIU/mg salivary 9000 X g supernatant
protein
for glutathione
S-transferase,
and 31 ± 22 mIU/ml
saliva and
17 ± 12 mIU/mg
salivary
9000 X g supernatant
protein
for DTdiaphorase.
0, subjects
who did not drink
coffee
at all; mean
± SD
values
were 5 ± 2 mIU/ml
saliva and 3 ± 1 mIU/mg
salivary
9000
X
g supernatant
protein
for ALDH-3,
23 ± 7 mIU/ml
saliva and 12 ± 3
mIU/mg
salivary
9000
X g supernatant
protein
for glutathione
Stransferase,
and 1 1 ± 8 mIU/ml saliva and 6 ± 4 mIU/mg
salivary
900()
x g supernatant
protein
for DT-diaphorase.
In all cases, salivary
enzyme
activities
of coffee-drinking
volunteers
were significantly
(P
0.005)
greater
than were those of volunteers
who did not drink coffee.
I>
IU
4
LU
C”
4
60
40
0
0.
20
4
l.
10
20
ALDH-3
30
ACTIVITY,
40
mIU/mI
E
E
80
>-
I>
IU
4
60
LU
C”
4
40
0
a.
20
4
I-
elevated
in salivary
samples
coffee
consumption,
and
thione
S-tnansfenases
a,
during
these
Numerous
known
serum
subjects
vegetables,
activities
levels
who
during
periods
of high
salivary
bevels
each relatively
the
of glutaelevated
periods.
to coordinately
diaphonase
taken
revealed
that
ji, and ‘rr were
induce
e.g.,
glutathione
in various
of glutathione
had consumed
crucifers
models
such
as broccoli,
S-transferase
(5,
6).
S-transferase
a were
relatively
barge amounts
are
and DTFurthermore,
elevated
in
of brussels
20
40
GST
60
ACTIVITY,
80
mIU/mI
Fig. 4
ALDH-3,
glutathione
S-transferase
(GST),
and DT-diaphorase
levels
in human
saliva.
In the investigation
presented
in Fig. 3, one or
more of the three enzyme
activities
of interest
were quantified
in single
samples
obtained
from each of 33 subjects;
all three were quantified
in
25 subjects.
Correlations
between
in the latter are shown
here.
of coffee
daily. 0, subjects
the magnitudes
#{149},
subjects
who
did
of enzyme
who consumed
not drink coffee
at least
at all.
activities
150
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Research.
ml
1 158 Induction
of Salivary
Chemopreventive
Enzymes
1
2
34
5
6
7
1234567
__5
GST-a
p
ALDH-3
GST-ji
S
-w
L5
GST-it
Fig.
5
Immunoblot
analysis
of six
human
saliva
samples
for the physical
presence
____
--tI_,
‘
of ALDH-3
and
glutathione
S-transferases
a,
p., and
ir.
Three
IJ.g of purified
stomach
mucosa
ALDH-3
(left panel,
Lane 1), 1 jig each of purified
human
glutathione
S-transferases
(GST) a, p., and i (right
panels,
Lane 1), and equal amounts
of processed
saliva
samples
[left panel,
relevant
protein
pools (15-20
jig) recovered
from 9000 X g saliva
supernatant
fractions
(1.5 ml) subjected
to reactive
blue 2-Sepharose
CL-6B
affinity
chromatography;
right panels,
aliquots
(200 i.g protein)
of 9000 X g saliva
supernatant
fractions]
obtained
from each of three subjects
who did not drink coffee
(Lanes
2-4)
and three subjects
who consumed
at least 600 ml
of coffee
daily (Lanes
5-7) were first subjected
to SDS-PAGE.
Proteins
thus resolved
were then electrotransferred
onto Immobilon-PVDF
transfer
membranes,
after which
the membranes
were probed
with antibodies
against
stomach
mucosa
ALDH-3
and human
glutathione
S-transferases
a, p.,
and ‘rr as described
in “Materials
and Methods.”
Aldehyde
dehydrogenase
activities
(4 mM benzaldehyde,
4 mM NADP)
in the original
saliva samples
were 2, 3, 7, 27, 32, and 42 mlU/ml
(left panel,
Lanes
2-7, respectively).
Glutathione
S-transferase
activities
in the original
saliva samples
were 22,
16, 36, 62, 62, and 88 mIU/ml
(right panels,
Lanes
2-7, respectively).
sprouts
(29).
thione
tively
high
(Fig.
that
during
the
that
salivary
were
each
Once
the
bevels
period
high
of
origin
is the
observation
glands
(30-32).
synthesized
in more
they
ultimately
The
presence
Both
33).
arrive
in
the
samples,
subjects
who
Prochaska
of
even
had
and
human
either
when
such
large
(15)
liver,
and
that
were
also
reported
On
who
(29,
unable
taken
coffee
to
in
from
(Table
to
the
S-tnansfenase
donors
4).
detect
other
any
hand,
activity
regularly
was
consumed
barge amounts
of coffee as well as in those obtained
who did not drink coffee at all. Plasma
glutathione
from donors
S-tnansfenase
levels
reflected
relative
were
levels
higher
of
in
this
the
enzyme
former
and
in the
LU
20
40
60
80
DAYS
activities
were
unable
serum.
>-
system.
were
of
20
Ui
glutathione
DT-diaphonase
samples
glutathione
from
or
amounts
in human
samples
are
and
We
4
z
they
been
(15).
40
U
N
is found
that
and DT-diaphorase
activity
bow-level
is that
circulatory
has
>-
>
I-
S-trans-
enzymes
a
ALDH-3
consumed
in plasma
the
plasma
serum
‘in
into the saliva
Supporting
this
e.g.,
via
60
E
and
ji,
possibility
is
S-transferase
Femnandes
DT-diaphonase
relatively
saliva
80
I-
One
organs/tissues,
the
E
revealed
glutathione
of these
4
9) con-
period.
possibility
in rodent
presence
a,
each
S-tnansferase
present
plasma
present
in
glutathione
are reportedly
detect
distant
activity
S-tnansferases
the
1
Cl)
elevated
and
this
a
4
>
this
after
(Fig.
and then secreted
salivary
glands.
that
by
and
relatively
is not known.
of gbutathione
S-transferase
were
Another
neba-
consumption
before
consumption,
ALDH-3,
gluta-
were
analysis
during
and DT-diaphonase
these
lower
gbutathione
enzymes
are synthesized
one or more of the various
in
broccoli
ALDH-3
of salivary
scenario
high
much
broccoli
elevated
ALDH-3,
activities
immunobbot
of
of
levels
of
were
again,
relatively
The
fenases,
they
salivary
DT-diaphorase
a period
8).
firmed
then,
and
whereas
period
by
unexpectedly
during
volunteer,
these
Not
S-tnansfenase,
roughly
the
Fig.
6
Effect
of drinking
coffee
on the levels
of ALDH-3,
glutathione
S-transferase,
and DT-diaphorase
activities
in human
saliva.
ALDH-3
(#{149}),
glutathione
S-transferase
(Lx),
and DT-diaphorase
(0) activities
in
the saliva
of a healthy
male subject,
age 27 years,
who had not consumed
coffee
for at least 3 weeks
prior to the beginning
of this experiment,
and who partook
of a “normal”
diet and did not partake
of
alcohol,
carbonated
beverages,
tobacco,
or prescription
medications
during
the experiment,
were
quantified
for 9 days to establish
basal
levels of these enzyme
activities.
The subject
then drank
1-1.2
liters of
regular
black
coffee/day
for 12 consecutive
days
(days
9-20),
after
which
he completely
stopped
consuming
coffee
for a period
of 22 days
(days 21-42).
The sequence
of drinking
coffee
(days 43-59)
and then
stopping
(day 60 and beyond)
was then repeated.
Collection
and processing
of saliva,
and quantification
of ALDH-3
(4 mM benzaldehyde,
4
mM NADP),
glutathione
S-transferase,
and DT-diaphorase
activities
were as described
in “Materials
and Methods.”
saliva.
DISCUSSION
Hanada
5
L. Sreerama
and N. E. Sladek,
manuscript
in preparation.
abdehyde
et al.
(17)
dehydrogenase
were
the
first
in human
to report
saliva.
the
Upon
presence
characterizing
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1995 American Association for Cancer
Research.
of an
Clinical
1
I
2345
.
Cancer
Research
1159
2345
.-
GST-a
ALDH-3
.
‘.
i.T,
GST-i
GST-ir
Fig. 7 Immunoblot
analysis of human saliva samples obtained at various time points during the course of the experiment
described
in the legend
to Fig. 6 for the physical
presence
of ALDH-3
and glutathione
S-tnansferases
a, p., and in. Three p.g of purified
stomach
mucosa
ALDH-3
(left panel,
Lane 1), 1 p.g each of purified
human
glutathione
S-transferases
(GST) a, p., and ‘in (right
panels,
Lane 1), and equal amounts
of processed
(exactly
as detailed
in the legend
to Fig. 5) saliva samples
obtained
on days 1 (Lane 2), 17 (Lane 3), 22 (Lane 4), and 42 (Lane 5) of the experiment
described
in the legend
to Fig. 6 were first subjected
to SDS-PAGE.
Proteins
thus resolved
were
then electrotransferred
onto Immobibon-PVDF
transfer
membranes,
after which
the membranes
were probed
with antibodies
against
stomach
mucosa
ALDH-3
and human
glutathione
S-transferases
a, p.,
and ‘in as described
the
enzyme
against
in “Materials
[subunit
the
ALDH-2,
Mr
salivary
and Methods.”
48,000;
enzyme
failure
or ALDH-4
ALDH-3,
of
to recognize
antiserum
raised
authentic
(glutamic
ALDH-1,
y-semiabdehyde
de-
hydrogenase);
a Km of 106 jiM when
acetaldehyde
substrate],
they declared
it to be a unique,
theretofore
was the
unidenti-
fled,
abdehyde
ALDH-V
(18).
Herein,
dehydrogenase
we,
too,
and
report
gave
it the
the presence
name
of an aldehyde
It was
in
this
the only
fluid.
laboratory
aldehyde
The
and
reason
that
for
of Hanada
remote,
possibility
is that
tamed
the salivary
sample(s)
a variant
Unknown
serves.
that we could
discrepancy
et al. (18)
the
find
between
is unclean.
subject(s)
from
happened
our
One,
whom
to be one(s)
albeit
they
ob-
Quit
60
E
E
40
I-
>
I-
U
4
20
UI
>-
N
z
UI
expressing
10
is the
Saliva
is viewed
of
salivary
purpose
that
S-transfemases,
volumes
it are
glands
potentially
enzymes,
37-39)
ALDH-3,
can
be
in
ml/day
the
many
secretion.
of which
present
generated
Large
by
the
S-transferases,
of
and
are substrates
for
in the environment/diet
by
microflora
in
the
oral
cavity
(40, 41). Thus,
it is tempting
to speculate
that these
enzymes
are present
in the saliva,
in cells lining the alimentary
canal,
and
“ports
of entry,”
agents,
although,
is actually
alimentary
lining
(42).
S-tmansferase,
high
in
canal
other
tissues
that
constitute
so-called
8
Effect
of eating
broccoli
on the levels of ALDH-3,
glutathione
S-transfenase,
and DT-diaphorase
activities
in human
saliva.
ALDH-3
(#{149}),
glutathione
S-transferase
(A), and DT-diaphorase
(0) activities
in
the saliva of a healthy
female
subject,
age 28 years, who quit consuming
Fig.
coffee
the
lung
(reviewed
Consistent
and
and
with
DT-diaphonase
in
in Refs.
the
tissues
this
notion,
that
1, 43, and 44).
constitute
However,
the
experiment,
of coffee,
described
salivary
are mel-
this
diet and did not partake
beginning
of
and
who
alcohol,
partook
carbonated
of
a
bever-
ages, tobacco,
or prescription
medications
during
the experiment,
were
quantified
for 8 days to establish
basal levels of these enzyme
activities.
The subject
then consumed
300 g of microwave
oven-cooked
(power
output
of 650 W for 2 mm) broccoli/day
for 12 days (days 8-20),
after
which
she completely
stopped
consuming
broccoli
(day 21 and thereafter).
Collection
and
processing
of saliva,
and
quantification
of
ALDH-3,
glutathione
S-transferase,
and DT-diaphorase
activities were
as
ALDH-3,
activities
at
“normal”
e.g., lungs, for the purpose
of detoxifying
such
in the case of certain
xenobiotics,
toxification
effected
glutathione
atively
in cells
DAYS
saliva
in humans)
glutathione
20
the
the
to catalyze
the detoxification
in Refs. 4, 35, and 36). Toxic,
are abundantly
and/or
of ALDH-3,
as a digestive
(600
xenobiotics,
presence
DT-diaphonase
primarily
are known
(reviewed
toxic,
the
and
secreted
(34).
and DT-diaphonase
various
xenobiotics
(4,
the
4
Cl)
ALDH-3.
glutathione
these
dehydrogenase
Quit
Broccoli
-I
dehy-
drogenase
in human
salivary
samples.
However,
the enzyme
that we found exhibited
physical
and catalytic
properties
essentialby identical
to those exhibited
by stomach
mucosa
ALDH-3
(22).
4
>
in “Materials
aspect
the
at
fenases,
notion
and NAD(P)
NAD(P)H,
the
of this
and Methods.”
concentrations
DT-diaphomase,
is problematic.
would
have
to
enable
sufficient
and
ALDH-3,
First,
to be present
the
glutathione
respectively,
glutathione,
in the saliva
S-transto catalyze
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1995 American Association for Cancer
Research.
1 160 Induction
of Salivary
Chemopreventive
Enzymes
I
I
2345
2345
GST..cc
ALDH-3
,.
.
.
V.
GST-1L
GST-ic
Fig.
Immunobbot
9
analysis
of human
saliva
samples
obtained
at various
time points
-
during
the course
of the experiment
described
in the legend
to Fig. 8 for the physical
presence
of ALDH-3
and glutathione
S-transferases
a, p., and Pin. Three
p.g of purified
stomach
mucosa
ALDH-3
(left panel,
Lane 1), 1 p.g each of purified
human
glutathione
S-transferases
(GST) a, p., and ‘in (right panels,
Lane I), and equal amounts
of processed
(exactly
as detailed
in the legend
to Fig. 5) saliva samples
obtained
on days 7 (Lane 2), 13 (Lane 3), 21 (Lane 4), and 28 (Lane 5) of the experiment
described
in the legend
to Fig. 8 were first subjected
to SDS-PAGE.
Proteins
thus resolved
were then electrotransferred
onto Immobilon-PVDF
transfer
membranes,
after which
the membranes
were probed
with antibodies
against
stomach
mucosa
ALDH-3
and human
glutathione
S-transferases
a, p.,
and ‘in as described
in “Materials
and Methods.”
Table
4
ALDH-3,
in matching
activities
glutathione
plasma
human
S-transferase,
and
saliva
NADH,
and DT-diaphorase
samples
obtained
from
male volunteers”
Enzyme
activit y (mIU/ml)
swallowed
catalytically
Saliva
Plasma
Glutathione
Glutathione
in
Subject”
S-transferase
ALDH-3
I
2.6
16.7
14.0
6.4
2
3
4
S
3.5
5.0
17.0
35.5
28.0
16.0
71.0
76.0
4.6
8.0
36.0
39.6
7.2
9.0
10.5
11.0
the
is
detoxifying
the
lyzed
estenolytic
tathione
cofacton
reactions
would
not
at
be required
detoxification/digestion.
are reportedly
1-3
jiM
(45);
meaningful
for
enzyme-catalyzed
contents,6
and
none
(c)
are catalytically
rates,
active
in the contents
unclear
at
this
of the upper
point
as
to
small
which
nase,
but
theme
constituents
are
cafestol
pabmitate,
transferase
activity
coffee beans
(19), agents
inducems)
human
is known
DT-diaphonase
P450s
and
mouse
IA1
glutathione
and
1A2,
of NAD,
bevels
e.g.
,
liven,
that
hope,
tumor
cells
(6)
S-transfenase,
(thus,
in cultured
prevent
The
colon
stomach,
certain
and
experimental
even
again
munine
dietary
expectation,
L. Sreerama
and
N. E. Sladek,
unpublished
to
not
by defhepatoma
intestine
(6).
constituents
cancinogenesis
is that
al-
a
(3).
and
but
of glu-
NADP,
and
and hydroquinone
ALDH-3,
glutathi-
in broccoli
activities
inducer)
chemicals
7-9).
and
and
tissues,
in Refs.
certain
palmitate
but not cytochnome
P450
definition,
monofunctionab
present
webb-established
man-made
Thus,
kahweol
in catechol
to induce
breast
to be
a monofunctionab
It is now
candidates.
e.g.,
of
increased
salivary
and DT-diapho-
have been shown
to induce
glutathione
Sin mouse liver and intestine
(46), and roasted
in cultured
cytochnome
cells
likely
beans,
and DT-diaphonase,
(thus,
they are, by
Sulforaphane
inition,
very
coffee
are known
to be rich
that have been shown
one S-transferase,
IA1
activities
induce
some
of green
intestine.
component(s)
ALDH-3-cata-
Salivary
those
Second,
coffee/broccoli
is(ane)
responsible
for the
bevels of ALDH-3,
glutathione
S-transferases,
(reviewed
respective
unknown.
in a matter of seconds,
(b) none of these enzymes
are
active at the bow pH (--2.0)
that ordinarily
prevails
prevails
It
and
though
are
in the oral cavity would
have to be effected
very
(a) any food that is placed
therein
is ordinarily
stomach
naniby
“ Collection
of blood
and saliva
from human
donors,
further
processing
of these
fluids,
and determination
of ALDH-3,
glutathione
S-transferase,
and DT-diaphorase
(DT-D)
activities
were as described
in
‘ ‘ Materials
and Methods.
‘ ‘ Each
value is the mean of duplicate
determinations
made on each sample.
I’ Subjects
1-3 did not drink
coffee
at all; subjects
4 and 5 consumed
at least 500 ml of coffee
daily.
C Measurable
levels of ALDH-3
and DT-diaphorase
activities
were not
found in any of the plasma samples. The minimum amounts
of ALDH-3,
glutathione
S-transferase,
and DT-diaphorase
activities
detectable
by the
spectrophotometric
assay used herein
were 1, 1, and 2 mIU/ml
saliva or
plasma,
respectively.
In a further attempt
to detect the presence
of ALDH-3
in the plasma,
8-10-ml
blood samples
were taken from the same individuals and processed
as before.
The plasma
samples
thus obtained
were
subjected
to reactive
blue 2-Sepharose
CL-6B
affinity chromatography,
and
the resultant
preparations
were concentrated
and submitted
to SDS-PAGE/
immunoblot
analysis
as well as spectrophotometric
assay
for catalytic
activity;
no ALDH-3
was found.
their
NADPH
after a brief exposure
(30 mm) to a pH of 2.0,6 i.e., they would
not be active
upon reaching
the slightly
alkaline
pH that ordi-
S-transferase
DT-D
and
detoxification
quickly
since
observations.
Downloaded from clincancerres.aacrjournals.org on June 17, 2017. © 1995 American Association for Cancer
Research.
they
Clinical
will
do
so
in
humans.
support
this
clinical
trials
Many
of the
agents
in experimental
(3,
substances
known
the
lectively
are known
(1-3,
human
Unknown
one S-transferases,
insight
in
studied
e.g.
26-28,
where
a
when
some
aromatic
tive
also
that
be used
potential
of various
diets
brought
by such
drugs,
Coordinated
and
could
not be ascertained
was
tionab
effected
by one
inducems,
e.g.,
of the
salivary
be important
enzymes
cytochnome
not
of
is
inducers
given
diet
activity,
if chemoprevention
only
P450s
effect-
induction
enzyme
induced
IA1
and
by
bifunc-
IA2.
epidemiobogical
broccoli
predict
that continuous
in fact, a chemopreventive
studies
already
published
ingestion
action.
A
suggests
of either
review
of
that
C and
2 and
reflect
given
levels
of them,
glutathione
S-transferase,
be of value
in the optimization
protocols
(choice
induction
of these
benefit
of these
first
introducing
levels
rapidly,
that
2 and
a drug
and,
may
with
3 and
( effective)
a few
sensitivity
(resistance)
the
present
deliberate
a few
to basal
is
It is possible
at one
may
weeks
after
the inducer
study).
drug
in-
days
return
after
in diet,
be of
maximum
(ineffective)
the
1ev-
could
Finally,
levels
on even
to
of
salivary
Moreover,
of days
change
months
bevels
DT-diaphorase
enzyme
be effective
a relevant
salivary
only
a matter
of
(discussed
monitoring
rapidly,
and
within
on bifunc-
to chemotherapy
occurs
E09
coordinated
of chemotherapeutic
scenarios.
the inducer,
(Refs.
time,
in certain
again
the
that
as webb.
prior
anti-
effectiveness
and/or
dose)
enzymes
that
Thus,
enzymes
Con-
of certain
that of others
ebs could
of drug,
55).
monofunctional
and increase
3).
chbonam-
Ref.
the therapeutic
tissue
the
the indoboquinone
then,
by either
decrease
e.g.,
in
activation
ALDH-3,
point
in
be ineffective
later,
would
i.e.
,
(appear
tumor
to)
be
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would
DT-diaphomase
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by measuring
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They
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42).
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22).
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in the case of any
but the distinction
in
I clinical
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in subsequent
enzymes
and
action
in Ref.
risk
48-50),
the detoxification
mitomycin
e.g.,
suggest
the
of cycbophosphamide
22, 26-28,
chemothemapeutic
DT-diaphonase
(reviewed
these
(1-3,
and
of
occur-
(52-54).
catalyze
other
drugs,
seem likely.
of salivary
carcinogens,
and
mebphalan,
cancer
reduces
the detoxification
S-transfenases
risk
published
humans
and
the
prostate
1161
occur-
beukemia,
change
already
by
occurrence
catalyzes
neoplastic
some
as 3-methylchobanthnene
to preliminarily
glutathione
ALDH-3,
such
bucil,
cancer
not
Research
cancer
cancer,
and
studies
of broccoli
lung
of colon
does
pancreatic,
oxazaphosphorines
alkybating
glutathie.g. , colon
environmental/industnial/di-
these
other
induction
by inducing
the exprescould also be useful
are known
induce
ing a chemoprotective
be identified
to
of which
hydrocarbons
3,4-benzpynene,
cob-
cycbophosphamide
chemopreventive
exposure
indicator
could
agents
and
and
kidney,
and
vemseby,
(only one would have to
induced
by both mono-
to be used
suspected
cobonectal
the risk
of endometniab
Epidemiobogical
glutathione
but it is known
but that would
the measurement
schedules
(19).
reduces
risk
occurrence,
consumption
tionab
as
cyclic
induces
these
of potential
(51).
of these enzymes,
to be the case,
cancer
ALDH-3
been
Oxazaphosphonines
and bifunctional
inducers)
in Phase
a relatively
easy and noninvasive
agents,
it has
is whether
salivary
levels of ALDH-3,
and DT-diaphomase
reflect
tissue,
cases
are
the
bladder,
of known
Thus,
,
48-50).
chemoprevention
Such measurements
an
DT-diaphorase
antineoplastic
thought
to bring about
sion of these enzymes.
etary
that
in that regard,
of the
22,
as to the dosage
those
nence
activi-
a substance
detoxification
levels of one on more of these enzymes
be measured
since they are coordinately
functional
would
be
induce
of coffee
increases
breast,
coordinately
35).
ingestion
rence,
ovarian
enzymes
is likely to be the causal
(reviewed
in Refs. 4, 7-9, and
carcinogens
epithebium,
levels
Should
that prove
I
(10-12).
of a number
4 and
Phase
DT-diaphonase
and
as oxazaphosphonines,
ifosfamide
and
chemopreventive
also
detoxification
detoxification
known
initiated
and
in Refs.
has not been
the
been
S-tnansfenases
by the induced
of chemoprevention
to catalyze
47),
as so-called
carcinogenesis
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ALDH-3
and
act
uous
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10 and
already
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and is chemopreventive,
carcinogens
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35).
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(reviewed
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enzymes
have
S-transferase,
to catalyze
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6, 15).
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drug
Identification of a class 3 aldehyde dehydrogenase in human
saliva and increased levels of this enzyme, glutathione
S-transferases, and DT-diaphorase in the saliva of subjects who
continually ingest large quantities of coffee or broccoli.
L Sreerama, M W Hedge and N E Sladek
Clin Cancer Res 1995;1:1153-1163.
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