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Cancer Prevention Research Program [J. W. L., S. L., J. P., M. T. G., K. V. B., Z. F., J. D. P.] and Program in Epidemiology [C. C., D. A. D.], Fred Hutchinson Cancer Research Center , Seattle, Washington 98109, and Bastyr University, Kenmore, Washington 98011 [D. E. M.]
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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concentration (GST-) and GST activity increase with
vegetable consumption and that this effect is GSTM1
genotype dependent. Twenty-one men (10 GSTM1-null and 11
GSTM1+) and 22 women (15 GSTM1-null and 7
GSTM1+), nonsmokers, 20–40 years of age and not on
medications, ate four 6-day controlled diets: basal (vegetable-free),
and basal supplemented with three botanically defined groups of
vegetables (i.e., brassica, allium, and apiaceous).
Fasting blood samples, collected on the last 2 days of each feeding
period, were analyzed for GST-, serum GST activity [against
1-chloro-2,4-dinitrobenzene (CDNB) and
7-chloro-4-nitrobenzo-2- oxa-1,3-diazole (NBD-Cl)] and
peripheral-lymphocyte GST-µ activity (against
trans-stilbene oxide). The brassica, but not allium or
apiaceous, vegetable diets (relative to the basal diet) increased
GST- by 26% (P = 0.005) and GST (NBD-Cl)
activity by 7% (P = 0.02) in the
GSTM1-null individuals, particularly the women.
Apiaceous vegetable supplementation decreased GST- in the
GSTM1+ men (P = 0.03). Among the
GSTM1+ women, both brassica and the allium diets
increased GST-µ activity by 18% (P = 0.02) and
26% (P = 0.001), respectively. The vegetable diets
had no effect on GST (CDNB) activity, irrespective of
GSTM1 genotype or sex. These results demonstrate that
GSTM1 genotype has a significant effect on GST responses
to diet and that brassica vegetables are most effective at inducing
GST-, whereas both brassica and allium vegetables induce GST-µ.
GST responses were more pronounced in women than men, but it is not
clear from this study whether this is a dose-per-body-weight or a
sex-specific effect. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and GST-µ (2)
. A homozygous deletion of the
GSTM1 gene results in no GSTM1 activity in 
50% of
Caucasians; however, because of their broad substrate specificity,
other GST isozymes (e.g., GST-) may be able to compensate
for the lack of GSTM1 activity. GSTM1 also metabolizes constituents of
plant foods, e.g., isothiocyanates (3)
.
GSTM1-null individuals may experience greater exposure to
dietary chemoprotective agents that are typically deactivated by GSTM1,
such that the interaction of GSTM1 genotype and dietary
patterns may be more important than either factor alone
(4)
.
GST- protein and GST activity can be measured in serum; in healthy
individuals, it most probably reflects enzyme release during normal
hepatic cell turnover (5, 6)
. GST-µ protein and activity
can be measured in peripheral lymphocytes (7, 8)
. Thus,
these measures provide noninvasive approaches to monitoring the effect
of interventions on GST protein levels and activities in healthy
individuals (5, 9)
.
The primary aims of our study were: (a) to test the effect
of consumption of three botanically defined vegetable diets, compared
with a vegetable-free diet, on serum GST- concentration and GST
activity in serum and peripheral lymphocytes; and (b) to
test whether the effect depends on GSTM1 genotype or sex.
The secondary aims were to examine the day-to-day variability in these
measures and the relationships between them under controlled dietary
conditions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our intention was to recruit 40 participants, including an equal number of GSTM1+ and GSTM1-null individuals and an equal number of men and women. In all, we randomized 57 individuals (12 GSTM1+ men, 11 GSTM1+ women, 16 GSTM1-null men, and 18 GSTM1-null women). Of these, 6 failed to appear for their first feeding period, 3 dropped out after 1–2 days on the diet, and 5 dropped out after one feeding period. Their reasons for quitting included: difficulty with protocol adherence (time commitment, coming to the center, the study food, and others), illness, wanting to get pregnant (women were advised not to plan a pregnancy during the study), and travel. As these individuals dropped out, new recruits were selected and randomized into the appropriate treatment orders to maintain the blocks. Approximately twice as many GSTM1-null, relative to GSTM1+, women volunteered; the overall prevalence of GSTM1-null genotypes in our recruitment population was 56%.
Study Design.
We used a four-period cross-over design with different experimental
diets in each period. Eligible participants were grouped according to
sex and GSTM1 genotype. Within each group, each participant
was assigned to receive the four diets in an order drawn randomly from
a four-sequence Latin Square. This approach minimizes the impact of
possible carry-over effects (10)
. Each diet was consumed
for 6 days, and we used a 1-day diet rotation (i.e., the
same food daily) to minimize day-to-day variability. The feeding
periods began with the dinner meal on a Saturday evening and ended with
blood sample collection on the following Friday (day 6) and Saturday
(day 7) mornings. There was at least a 2-week washout period between
each diet period, a sufficient length of time according to data
published previously (11)
.
Study Diets.
During the four feeding periods, participants consumed four different
diets: a basal diet devoid of fruits and vegetables (12)
;
and the basal diet supplemented with brassica, allium, or apiaceous
vegetables. The brassica supplement comprised: 16 g (1/2 cup
United States household measure) of fresh radish sprouts, 150 g (1
cup) of frozen cauliflower (FSA Signature), 200 g (2 cup) of
frozen broccoli (FSA Signature), and 70 g (1 cup) of fresh
shredded cabbage. The allium supplement comprised: 10 g (3
tablespoons) of fresh chopped chives, 100 g (1 1/3 cup) of fresh
chopped leeks, 5 g (1 teaspoon) of fresh minced garlic, and
75 g (1/2 cup) of fresh chopped onion. The apiaceous supplement
comprised: 0.50 g (1 teaspoon) of fresh dill weed, 50 g
(1/2cup) of fresh celery, 5 g (3 tablespoons) of fresh chopped
parsley, 100 g (1 1/4 cup) of fresh grated parsnips, and 110 g (3/4 cup) of frozen carrot coins (FSA Signature). All prepackaged
foods were purchased in case lots, and fresh foods were purchased from
the same vendor. The Food Services Department at Fred Hutchinson Cancer
Research Center prepared the study diets. Nutrient content of the diets
was determined using the Nutrition Data System software program (Food
Database version 12A, release date November 1996; Nutrient Database
version 27, release date November 1996), developed by the Nutrition
Coordinating Center, University of Minnesota, Minneapolis, MN.
Components of the basal diet were adjusted to accommodate the added
vegetables, such that all diets provided a similar percentage of energy
from carbohydrate, protein, and fat: 60, 12, and 29%, respectively.
All four study diets were designed to provide 2000 kcal. Participants
whose daily energy needs exceeded 2000 kcal were provided with
"unit" foods, a combination of foods that were already part of the
basal diet and had the same macronutrient composition as the basal diet
(12)
.
Participants were instructed to consume only the foods and beverages provided to them during the diet periods. The dinner meal was served at the study center each evening, and food for the following days’ breakfast, lunch, and snacks was distributed at that time. The major portion of the vegetables was provided as part of the dinner meal, and study staff monitored participants’ intakes at that meal. Participants were encouraged to report any deviations from the study diets. Overall compliance with the study diet was assessed using a daily checklist. Each checklist listed all foods on the study diets as well as space to record any additional (nonstudy) foods consumed.
GSTM1 and GSTT1 Genotyping:
Identification of Homozygous Null (GSTM1*0/0).
GSTM1 genotyping on all potential study participants was
conducted prior to randomization into the feeding trial. DNA from
participants was extracted from peripheral lymphocytes for
determination of GSTM1 and GSTT1 genotype by PCR.
The primers used for identifying GSTM1 genotypes were 5'-GAA
CTC CCT GAA AAG CTA AAG C-3' and 5'-GTT GGG CTC AAA TAT ACG GTG G-3'
(13)
. Those for the identification of GSTT1
were 5'-TTC CTT ACT GGT CCT CAC ATC TC-3' and 5'-TCA CCG GAT CAT GGC
CAG CA-3' (14)
. ß-globin was coamplified to ensure that
the GSTM1-null and GSTT1-null were attributable
to the deletion of the GSTM1 or GSTT1 alleles and
not to failure of the PCR. PCR results, generated from the DNA samples
of each participant, show a 215-bp band for individuals that are
homozygous or heterozygous for GSTM1 phenotypes
(15)
. GSTM1-null individuals do not show this
band. A 480-bp band is detected for individuals who carry at least one
GSTT1 allele.
GSTM1*1/1 Genotyping: Homozygosity Determination by
Long PCR Amplification of GSTM1.
We determined post hoc the homozygosity
(GSTM1*1/1) of GSTM1+ individuals by performing
long-range PCR using the Expand long template PCR system (Boehringer
Mannheim) and primers M2F10, 5'-AAG ACA GAG GAA GGG TGC ATT TGA TA-3'
and M5R16, 5'-ACA GAC ATT CAT TCC CAA AGC GAC CA-3' (16)
.
A 9-kb human tissue plasminogen activator gene fragment was coamplified
using primers 5'-GGA AGT ACA GCT CAG AGT TCT GCA GCA CCC CTG-3' and
5'-CAA AGT CAT GCG GCC ATC GTT CAG ACA CAC C-3', to serve as an
internal control. Fifty µl of reaction mixture contained 500 ng of
genomic DNA, 5 pmol of GSTM1 primers, 10 pmol of tissue
plasminogen activator primers, 500 µM of
deoxynucleotide triphosphates, 1x PCR reaction buffer containing 2.25
mM MgCl2, and 2.5 units of
Expand long template enzyme mixture. The thermal cycling conditions
were: denaturation at 94°C for 2 min; 5 cycles of 93°C for 10 s and 68°C for 8 min; 5 cycles of 93°C for 10 s, 64°C for
30 s, and 68°C for 8 min; 25 cycles of 93°C for 10 s, 64°C for 30 s, and 68°C for 8 min, plus elongation of
15 s for each cycle; 1 cycle at 68°C for 12 min; and cooling at
4°C. The lack of the 4748-bp amplicon is indicative of homozygosity
(1/1) of GSTM1.
Serum GST-.
The concentration of GST- in serum was measured using a commercially
available, enzyme-linked immunoassay kit (High sensitivity Hepkit;
Biotrin International, Dublin, Ireland). Serum was diluted 1:5 using
the assay buffer provided; 100 µl of diluted sample and control were
added in duplicate to a microtiter plate coated with antibody to
GST- and incubated at room temperature for 1 h with shaking.
After washing to remove the unbound fraction, 100 µl of anti-GST IgG
conjugated to horseradish peroxidase were added to each well, incubated
for 60 min at room temperature and washed, and 100 µl of
tetramethylbenzidine substrate were added. After a final 15-min
incubation at room temperature, the reaction was stopped by adding 100
µl of 1 N
H2SO4. Absorbances were
read at 450 nm with a 630-nm reference filter on a Dynatech MR5000
MicroPlate Reader (Dynex Corp., Chantilly, VA). Standards of known
concentrations were included in every run, and the concentrations of
the samples and controls were calculated from a standard curve. The
assay was linear from 200 to 2000 pg/ml. Intra-assay CVs were 6.8 and
3.3% at 262 and 1618 pg/ml, respectively; inter-assay CVs were 13.1
and 9.4% at 433 and 1502 pg/ml, respectively.
Peripheral Lymphocyte GST-µ Activity.
Frozen buffy-coat fractions were thawed on ice and resuspended in PBS
(pH 7.3). While on ice and in a cold room (4–8°C), the cells were
sonicated twice with a Fisher Model 50 Sonic Dismembrator (Fisher
Scientific, Pittsburg, PA) for 10 s each at a setting of 2 W. The
disrupted cells were centrifuged in an Eppendorf 5402C centrifuge at
3800 x g for 30 min at 2–8°C, and the supernatant
was separated into three aliquots. Protein concentration of the cytosol
was measured using the Bradford Coomassie Blue method. Cytosol
containing 100 µg of protein was incubated in the presence of 100
µM 3
[H]TSO (specific
activity, 15 Ci/mmol; American Radiolabeled Chemicals, St.
Louis, MO) and 5 mM GSH in a final volume of 100
µl. The enzymatic reaction was initiated by the addition of GSH, and
the reaction continued for 30 min at 37°C in a water bath, after
which time it was stopped by the addition of 200 µl of hexanol. After
vortexing, the phases were allowed to separate overnight and then were
centrifuged in an Eppendorf 5402C centrifuge at 3800 x
g for 10 min. Ten µl of the aqueous phase was added to 4
ml of Sigmafluor and counted in a Wallac 1409 scintillation counter for
1 min. Samples were assayed in duplicate. A sample control consisting
of cytosol that was heat-inactivated for 15 min at 100°C was included
as a blank. Activity of the blank was subtracted from the corresponding
sample result to correct for nonenzymatic conjugation. Results were
expressed as pmol product formed/mg protein/min. The intra- and
inter-assay CVs were 6 and 10%, respectively.
Serum GST (CDNB) Activity.
Serum GST activity using CDNB as a substrate was determined according
to a modification of the method of Habig et al.
(17)
on a Roche Cobas Mira Plus (Branchburg, NJ)
centrifugal analyzer. Serum and controls were incubated at 37°C in
the presence of 1.22 µM CDNB in 0.1
M potassium phosphate buffer (pH 6.25) and 10.85
mM GSH in a final volume of 365 µl. The rate of
formation of a colored product was measured at 340 nm. Enzyme activity
(units/l) was calculated based on the extinction coefficient for the
substrate CDNB of 9.2 mM-1
cm-1, with a unit defined as that amount of
enzyme that catalyzes formation of 1 µmol of product/min. Aliquots of
control serum, frozen at -80°C, were thawed and run at the beginning
and end of each batch of 60 samples. Intra- and inter-assay CVs were
7.7 and 11.8%, respectively (mean activity, 12.55 units/l).
Serum GST (NBD-Cl) Activity.
Serum GST activity using NBD-Cl as a substrate was determined according
to a modification of the method of Ricci et al.
(18)
on a Roche Cobas Mira Plus (Branchburg, NJ)
centrifugal analyzer. Serum and controls were incubated at 37°C in
the presence of 200 µM NBD-Cl in 0.1
M acetate buffer (pH 5.0) and 500
µM GSH in a final volume of 360 µl. The rate
of formation of a colored product was measured at 340 nm. Enzyme
activity (units/l) was calculated based on the extinction coefficient
for the substrate NBD-Cl of 14.5
mM-1
cm-1. Aliquots of control serum, frozen at
-80°C, were thawed and run at the beginning and end of each batch of
60 samples. Intra- and inter-assay CVs were 5.2 and 7.6%, respectively
(mean activity, 61.32 units/l).
Statistical Analysis.
GST measures were made on blood samples collected on the last 2
consecutive days of each feeding period. GST (CDNB) and GST-µ
activities were approximately normally distributed, although the
distribution of GST-µ was bimodal. Serum GST- concentration and
GST activity (NBD-Cl), as well as the macronutrient intake data, were
skewed and were log-transformed (natural log) prior to analysis.
This study was a randomized cross-over trial, a type of longitudinal
study in which participants receive different treatments at different
times. For the purpose of analysis, this design can be thought of as a
randomized block design with subjects as blocks; sex, GSTM1
genotype, and their interaction as between-block factors; and feeding
periods, diet treatments, carry-over, sampling day (day 6 or 7), and
their interactions with between-block factors as within-block factors.
We analyzed the data using a linear mixed model (Ref. 19
;
PROC MIXED in SAS, Release 6.12), with study participants as a random
effect and all other factors as fixed effects. For comparisons between
vegetable-supplemented and basal diets, stratified by GSTM1
genotype and sex, the corresponding contrasts were used. For GST-µ
and GST (CDNB) activities, we present LS-means and standard errors for
the difference between the vegetable diets and the basal diet by
genotype and sex, adjusted for other effects in the model. Because the
statistical analyses for GST- and GST (NBD-Cl) activity were done on
the log-transformed variables, the difference of the log-transformed
means after back-transformation is presented as the ratio of the
LS-means on the original scale. Pearson correlation was used to
evaluate the correlations between GST values on days 6 and 7 and
between different GST measures.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Only two individuals (a GSTM1-null female and a
GSTM1+ male) were GSTT1-null. Energy and
macronutrient intakes on the different diets are presented in Table 2
. A similar carbohydrate, protein, and fat distribution was maintained
across the four diets; however, we did not attempt to adjust for the
additional dietary fiber in the vegetables. Participant compliance to
the study diets was excellent. On the basis of the participants’
self-reported deviation from the study diets, only 1.6% (19 of 1204)
"person feeding days" included consumption of nonstudy foods. The
majority of these were attributable to participants having to make food
substitutions (about which they consulted the study staff), because a
snow storm prevented travel to the feeding center.
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and 4 . There was no carry-over effect for any of the GST measures. Overall,
for the four controlled diets, serum GST- concentrations did not
differ between the GSTM1-null and GSTM1+
genotypes (3283 ± 415 versus 4124 ± 617;
P = 0.3) but were higher in men than women (4634 ± 628 versus 2922 ± 414; P = 0.02).
GST- concentrations increased significantly with consumption of the
brassica diet relative to the basal, vegetable-free diet (13%;
P = 0.05; Table 3
). This increase was exclusively among
the GSTM1-null individuals (26%; P =
0.005), particularly the GSTM1-null women (38%;
P = 0.002). Among GSTM1+ men, GST-
concentrations decreased by 22% with the addition of apiaceous
vegetables (P = 0.03).
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concentration. Among GSTM1-null women, there was a small
(8%; P = 0.04) increase in serum GST (NBD-Cl) activity
with addition of brassica vegetables.
As expected, LS-mean peripheral lymphocyte GST-µ activity was
significantly higher in GSTM1+ than in the
GSTM1-null individuals (257 ± 14 versus
17 ± 12; P = 0.0001) for the four experimental
diets. GST-µ activity was higher in GSTM1+ men than
GSTM1+ women (290 ± 18 versus 224 ±
22; P = 0.03). Among the GSTM1+ women,
GST-µ activity increased by 52 ± 16 (P = 0.001)
and 36 ± 16 pmol mg-1
min-1 (P = 0.02), with the
addition of allium and brassica vegetables, respectively (Table 4)
. Of
the GSTM1+ individuals, 12 (5 women and 7 men) were
GSTM1*1/0 and 6 (2 women and 4 men) were
GSTM1*1/1. Further evaluation of GST-µ activity in
GSTM1*1/0 and GSTM1*1/1 participants showed no
significant (P = 0.8) effect of the interaction of
homozygosity-by-diet on GST-µ activity, and on each diet, mean
GST-µ activity was similar in both groups (Fig. 1
). With allium vegetable supplementation specifically, GST-µ activity
increased 19% among the GSTM1 heterozygotes
(P = 0.02), whereas it increased only 5% among the
homozygotes (P = 0.7). The study size was not large
enough to examine the combined effect of GSTM1+ homozygosity
and sex.
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7 g of brassica vegetables per kg
body weight, whereas the men received a significantly
(P = 0.001) lower dose of 6 g per kg). Among the
GSTM1-null individuals (i.e., the group in which
GST- responded to the brassica diet), women who weighed 
62 kg
(median weight for women) had a similar GST- response as did women
who weighed >62 kg (an increase of 1162 ± 876 and 1329 ±
776 pg/ml, respectively). On average, men who weighed 70 kg (median
weight for men), relative to those who weighed >70 kg, had a greater
but statistically nonsignificant (P = 0.6) increase in
serum GST- (1199 ± 1533 and 234 ± 267 pg/ml,
respectively).
Correlations between GST measures on days 6 and 7 were 0.87, 0.93,
0.47, and 0.66 for GST- concentration and GST-µ, GST (CDNB), and
GST (NBD-Cl) activities, respectively; however, the scatter for the
serum GST activities was large. Mean GST- concentrations and GST-µ
activities, but not serum GST activities, on day 7 were statistically
significantly higher than on day 6 (P < 0.004); day 7
values were 9 and 11% higher for GST- concentrations and GST-µ
activities, respectively. Overall, the correlations between serum
GST- concentration and the serum GST activities were not strong;
GST- concentration was weakly correlated with GST (NBD-Cl) activity
(r = 0.18; P = 0.02) but not GST (CDNB)
activity (r = 0.048; P = 0.54). The
correlation between GST (CDNB) and GST (NBD-Cl) activity was 0.23
(P = 0.0024).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We had postulated that lack of GSTM1 enzyme activity would result in a
greater effect of vegetable diets on GST-, potentially as a result
of decreased inactivation of biologically active dietary constituents.
Isothiocyanates, produced from glucosinolates in cruciferous
vegetables, are conjugated by GST. In vitro studies suggest
that several of the isothiocyanates are good substrates for GSTM1
(3)
, whereas in vivo data (an observational
study) showed that a greater urinary excretion of total isothiocyanates
was associated with only the GSTT1+, but not the
GSTM1+, genotype (22)
. We were unable to assess
the effect of GSTT1 genotype on GST- in this study
population; only 2 individuals (a GSTM1-null woman and a
GSTM1+ man) had the GSTT1-null genotype. However,
our results suggest that, under controlled dietary conditions,
GSTM1 genotype modulates effects of vegetable
supplementation.
Overall, among our participants, LS-mean serum GST- concentrations
and GST (CDNB) activity were higher in men than in women. We observed
similar but nonsignificant differences in GST-µ and GST (NBD-Cl)
activities. Sex differences in serum concentrations of GST-
(23)
and the specific GST- isoform, GSTA1-1
(24)
, but not lymphocyte GST-µ concentration
(8)
or serum GST (CDNB) activity (6)
, have
been reported previously in observational studies.
Differences in vegetable-dose-per-body-weight between men and women may
contribute to the observed sex-dependent response to diet in our study;
however, body weight and sex were confounded, and this effect could not
be tested effectively. In the study of Bogaards et al.
(9)
, heavier men (>80 kg) had a smaller change in GST-
than lighter men (<80 kg) in response to consumption of Brussels
sprouts. Together, these results suggest that body size may influence
response; however, we cannot resolve whether this is specific to men.
There may be a threshold dose-per-body-weight, such that most
individuals, irrespective of sex, respond similarly and only the
heaviest ones have a lower GST- response. On the other hand, in the
study of Nijhoff et al. (5)
, supplementation
with 300 g of Brussels sprouts daily increased GST- in men
(n = 5), but not in women (n = 5).
Furthermore, in our study, addition of apiaceous vegetables decreased
GST- concentrations among GSTM1+ men but not women. Thus,
taking the literature as a whole, it remains unclear whether the
differences in responses to cruciferous feeding in humans are the
result of doseper-body-weight or physiological effects. It is also
unclear whether men or women are better responders.
Peripheral lymphocyte GST-µ activity, as determined by conjugation of TSO, increased with allium and brassica supplementation in GSTM1+ women but not in men. In rodents, both hepatic and intestinal GST-µ have been shown to increase with various dietary and drug treatments (21, 25) ; however, the capacity to induce GST-µ in humans, particularly in circulating cells (which presumably are not exposed to the same concentrations of phytochemicals as are the intestine and liver) has not been reported previously. In humans, Nijhoff et al. (26) found no effect of Brussels sprouts (300 g) on GST-µ protein in duodenum, rectum, or lymphocytes. On the other hand, Pool-Zobel et al. (27) reported increased expression of GSTP1 protein in lymphocytes in a subset of individuals fed carotenoid-rich beverages, indicating that certain GST isozymes in circulating cells may be responsive to dietary changes. On this basis, our results suggest that, over a week, at moderate, readily achievable levels of vegetable intake, GST-µ activity can be induced by vegetables in the Brassicaceae and Alliaceae families. With the present diet design, we cannot compare the difference in magnitude of response among vegetable treatments; each botanical grouping was fed as different gram weights, and the vegetables were provided in different forms (e.g., the allium vegetables were fresh, whereas the majority of the brassica vegetables was frozen). The effect of frozen versus fresh on differences in response across the botanical groupings is unknown and would depend in part on the potency, stability, and metabolism of the various phytochemicals found in each botanical grouping.
Our study does not support the hypothesis that the variation in peripheral lymphocyte GST-µ activity (against TSO) among GSTM1+ individuals is attributable to an allelic dose effect (16) . We found no difference in GST-µ activity between GSTM1*1/0 and GSTM1*1/1 individuals in this selected population, under defined dietary conditions. However, the number of participants was small, especially GSTM1*1/1 individuals (n = 6), and these results may be attributable to chance. This hypothesis needs to be tested further with a larger sample size.
Correlations were strong for GST- concentration and GST-µ activity
measured in blood samples drawn on 2 consecutive days. However, 10%
higher values were obtained on day 7 than on day 6, particularly on the
brassica diet, suggesting that response to diet may not have reached a
steady state with 6 days of feeding. Typically, inducible
biotransformation enzymes respond rapidly to changes in diet. Sreerama
et al. (11)
demonstrated that salivary GST
activity increased and reached a plateau within 5 days in response to
broccoli feeding. Whether rate of adaptation is affected by
individuals’ baseline GST levels under habitual diet conditions could
not be determined here; we did not collect and test blood samples at
the start of each period. Other aspects of dietary adaptation may also
need to be considered. Recent studies have shown that intestinal
bacteria hydrolyze glucosinolates that have not been cleaved by
myrosinase in the cruciferous vegetable (28)
. In our
study, a predominant portion of the brassica diet was fed as cooked
vegetables, in which most of the myrosinase has been inactivated.
Bacterial hydrolysis could have a major influence on exposure to
isothiocyanates in cooked vegetables, and thus, ultimately, GST-
response. The adaptation of intestinal microflora to the diet may
result in an increasing dose of isothiocyanates as bacterial enzymes
are induced or bacterial populations are altered.
The lack of association between GST- and serum GST activities,
measured using CDNB and NBD-Cl as substrates, is not altogether
unexpected. CDNB has been used for decades as a general measure of GST
activity; it is a good substrate for many of the GST isozyme classes
(18)
. Therefore, activity against CDNB in serum most
probably reflects not only GST- and -µ (in GSTM1+
individuals) circulating as a result of hepatic cell turnover
(26)
but also GSTs from other tissues (2)
and
leakage from erythrocytes either in vivo or ex
vivo in the collected blood sample (29)
. NBD-Cl has
been identified as a substrate that is more specific for GST-
(18)
. To our knowledge, this is the first attempt to
quantify serum GST activity against NBD-Cl. The low correlation between
serum GST (NBD-Cl) activity and GST- concentration implies that the
GST (NBD-Cl) assay is not highly specific for GST- in serum;
however, the small, but statistically significant, increase in GST
(NBD-Cl) activity in GSTM1-null women supplemented with
brassica vegetables follows a similar pattern as observed for GST-
concentrations. Overall, serum GST activities in these healthy
individuals were very low and nonenzymatic conjugation in the serum was
high, suggesting that these measures are not sensitive enough to
monitor effectively responses to dietary interventions, even under
controlled conditions.
In summary, brassica, but not allium or apiaceous, vegetable
supplementation under controlled dietary conditions significantly
increased serum GST- concentrations and GST (NBD-Cl) activity in
GSTM1-null individuals, particularly in women. Both brassica
and allium vegetables increased peripheral lymphocyte GST-µ activity
in GSTM1+ women. These results suggest that the capacity of
moderate intakes of cruciferous vegetables to induce GST- is
dependent in part on the lack of GSTM1 activity. Whether sex
differences in GST- in response to diet are a function of a
dose-per-body-weight effect or a genuine difference in physiological
response between men and women remains to be clearly established.
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Acknowledgments |
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Footnotes |
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1 Supported by National Cancer Institute Grant R01
CA70913 (to J. D. P.) and Fred Hutchinson Cancer Research Center. 
2 To whom requests for reprints should be
addressed, at Fred Hutchinson Cancer Research Center, 1100 Fairview
Ave. N., MP-900, Seattle, WA 98109-1024. Phone: (206) 667-6580; Fax:
(206) 667-7850; E-mail: jlampe{at}fhcrc.org
3 The abbreviations used are: GST, glutathione
S-transferase; FSA, Food Services of America; CV,
coefficient of variation; GSH, reduced glutathione; CDNB,
1-chloro-2,4-dinitrobenzene; NBD-Cl, 7-chloro-4-nitrobenz-2-oxa 1,3
diaxole; LS, least squares; TSO, trans-stilbene
oxide.
Received 11/ 2/99; revised 5/10/00; accepted 5/24/00.
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in humans. Carcinogenesis (Lond.), 16: 955-957, 1995.-class glutathione S-transferase levels in human blood plasma. Carcinogenesis (Lond.), 15: 1073-1075, 1994. in serum. Clin. Chem., 40: 184-189, 1994. 1-1: plasma concentrations in controls and in patients with gastrointestinal disorders. Clin. Chem., 42: 416-419, 1996.This article has been cited by other articles:
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