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Proc Natl Acad Sci U S A. 1997 September 16; 94(19): 10367–10372. | PMCID: PMC23369 |
Copyright © 1997, The National Academy of Sciences of the USA Medical Sciences Broccoli sprouts: An exceptionally rich source of inducers of
enzymes that protect against  chemical carcinogens Jed W. Fahey, Yuesheng Zhang, and Paul Talalay* Brassica Chemoprotection Laboratory and Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205 Accepted July 3, 1997. Induction of phase 2 detoxication enzymes [e.g., glutathione
transferases, epoxide hydrolase, NAD(P)H: quinone reductase, and
glucuronosyltransferases] is a powerful strategy for achieving
protection against carcinogenesis, mutagenesis, and other forms of
toxicity of electrophiles and reactive forms of oxygen. Since
consumption of large quantities of fruit and vegetables is associated
with a striking reduction in the risk of developing a variety of
malignancies, it is of interest that a number of edible plants contain
substantial quantities of compounds that regulate mammalian enzymes of
xenobiotic metabolism. Thus, edible plants belonging to the family
Cruciferae and genus Brassica (e.g.,
broccoli and cauliflower) contain substantial quantities of
isothiocyanates (mostly in the form of their glucosinolate precursors)
some of which (e.g., sulforaphane or 4-methylsulfinylbutyl
isothiocyanate) are very potent inducers of phase 2 enzymes.
Unexpectedly, 3-day-old sprouts of cultivars of certain crucifers
including broccoli and cauliflower contain 10–100 times higher levels
of glucoraphanin (the glucosinolate of sulforaphane) than do the
corresponding mature plants. Glucosinolates and isothiocyanates can be
efficiently extracted from plants, without hydrolysis of glucosinolates
by myrosinase, by homogenization in a mixture of equal volumes of
dimethyl sulfoxide, dimethylformamide, and acetonitrile at −50°C.
Extracts of 3-day-old broccoli sprouts (containing either glucoraphanin
or sulforaphane as the principal enzyme inducer) were highly effective
in reducing the incidence, multiplicity, and rate of development of
mammary tumors in dimethylbenz(a)anthracene-treated
rats. Notably, sprouts of many broccoli cultivars contain negligible
quantities of indole glucosinolates, which predominate in the mature
vegetable and may give rise to degradation products (e.g.,
indole-3-carbinol) that can enhance tumorigenesis. Hence, small
quantities of crucifer sprouts may protect against the risk of cancer
as effectively as much larger quantities of mature vegetables of the
same variety. Keywords: chemoprotection, glucosinolates, isothiocyanates, sulforaphane, glucoraphanin Many types of chemoprotectors against cancer evoke large
inductions of phase 2 enzymes of xenobiotic metabolism and increase
glutathione levels in animal tissues ( 1). These cellular responses
accelerate the detoxication of electrophiles and reactive forms of
oxygen, and thereby protect cells against mutagenesis and neoplasia.
Substantial evidence suggests that induction of these detoxication
enzymes provides a major strategy for achieving protection against
malignancy ( 1). The chemical specificity of the inducers and the
molecular mechanisms of regulation of phase 2 enzymes are under active
investigation in several laboratories ( 2– 6). Edible plants contain a
wide variety of minor metabolites, some of which are phase 2 enzyme
inducers. Since extensive epidemiological evidence, backed by animal
experiments, shows that diets rich in fruits and vegetables are
associated with large and dose-related reductions in the risk of
developing cancer ( 7– 10), it is likely that these metabolites are at
least partially responsible for protection. Crucifers (e.g., broccoli,
cauliflower, kale, and Brussels sprouts), which are rich in phase 2
enzyme inducers ( 11), may play a special role in affording such
protection ( 12, 13). A simple cell culture system, developed to detect and quantitate the
potency of phase 2 enzyme inducers, measures the elevation of
NAD(P)H:quinone reductase (QR; a typical phase 2 enzyme) in murine
hepatoma cells grown in 96-well microtiter plates ( 11, 14). This assay
was critical for the isolation of the isothiocyanate sulforaphane as
the principal and exceedingly potent monofunctional enzyme inducer in
broccoli ( 15). Sulforaphane induces several phase 2 enzymes in both
cultured cells and mouse tissues ( 15), blocks
7,12-dimethylbenz( a)anthracene (DMBA)-initiated mammary
tumor formation in rats ( 16), and inhibits neoplastic nodule formation
in cultured mouse mammary glands ( 17). Isothiocyanates, including sulforaphane, are synthesized and stored in
plants as relatively stable precursors, known as glucosinolates
(β-thioglucoside N-hydroxysulfates), which are hydrolyzed
to isothiocyanates by myrosinase (β-thioglucoside glucohydrolase; EC
3.2.3.1). Myrosinase (see ref. 18) is normally segregated from
glucosinolates and is released when plant cells are injured. Myrosinase
catalyzes the following reaction:
Determination of the isothiocyanate and glucosinolate contents of
plants has been complicated by the difficulty of quantitatively
extracting both types of compounds while avoiding the hydrolysis of
glucosinolates by myrosinase. We have overcome this problem by a novel
method that provides extracts suitable for nondestructive glucosinolate
analysis by paired-ion chromatography ( 19) and for spectroscopic
measurement of isothiocyanates and glucosinolates (after myrosinase
treatment) by a cyclocondensation reaction with 1,2-benzenedithiol ( 20,
21). As part of our long-term goals of identifying and developing edible
plants for chemoprotection, we describe the presence of extremely high
concentrations of phase 2 enzyme inducer activity in young sprouts of
cruciferous plants (e.g., broccoli), relate this inducer activity to
the presence of specific glucosinolates, and demonstrate the high
potency of extracts of these plants as chemoprotectors against
experimental mammary tumors. Plant Source and Cultivation. Seeds not treated with
pesticides were obtained commercially. Sprouts were produced from seeds
surface-sterilized by a 1-min rinse in 70% ethanol, a 15-min exposure
to 1.3% sodium hypochlorite containing 0.001% Alconox detergent, and
exhaustive rinsing with sterile distilled water. The broccoli
(Brassica oleracea var. italica) cultivar used
was SAGA, unless stated otherwise. Sprouts were grown without added
nutrients either aseptically on 0.7% agar or in inclined perforated
trays (35 × 40 cm) watered with four 15-s gentle spray cycles per
h. All sprouts were grown with a 16-h light and 8-h dark photoperiod
and a corresponding 25/20°C cycle for agar-grown sprouts or a
constant 25°C for tray-grown sprouts. Sprouts were rapidly and gently
collected from the surface of the agar or spray tray immediately before
extraction to minimize hydrolysis of glucosinolates by
endogenous myrosinase. Mature and frozen vegetables were
obtained from local supermarkets. Plants not extracted on the day of
collection were stored at −80°C. Extraction of Phase 2 Enzyme Inducers from Plants. Vegetables
were homogenized with 10 vol of a mixture of equal volumes of dimethyl
sulfoxide, dimethylformamide, and acetonitrile (triple solvent)
maintained at about −50°C in a dry-ice/ethanol bath. Samples were
homogenized, depending on sample size, in a glass homogenizer, a
Brinkmann Polytron homogenizer, or a Waring Blendor. Other extractive
solvents were boiling methanol, boiling water, ice-cold water, and
acetonitrile. In all cases homogenates were centrifuged to remove
remaining particulates and stored at −20°C until analyzed. Myrosinase Purification. The enzyme was purified from
8-day-old Raphanus sativus (daikon) seedlings by sequential
chromatography procedures to be published separately. The dimeric
120-kDa ascorbic acid-requiring enzyme was purified approximately
230-fold to a specific activity of 184 μmol of sinigrin hydrolyzed
per min per mg of protein and was used to hydrolyze glucosinolates for
chemical and inducer activity assays. Bioassay of Inducer Potency. Induction of quinone reductase
was measured in Hepa 1c1c7 murine hepatoma cells grown in 96-well
microtiter plates ( 11, 14). Usually, 15 μl of the solution to be
assayed (in water, methanol, acetonitrile, or triple solvent) was
diluted to 3.0 ml with medium and serial dilutions were used for the
microtiter plates. Excess purified myrosinase and 500 μM ascorbate
were added to each well to achieve complete glucosinolate hydrolysis.
The final concentration of organic solvent was ![[eq-or-less, slanted]](/corehtml/pmc/pmcents/x22DC.gif) 0.5% by volume.
Modifications of the published procedure ( 11) included ( i)
use of fetal calf serum treated with charcoal (1 g/100 ml) for 90 min
at 55°C and ( ii) assessment of cytotoxicity by measurement
of protein concentration as follows: a 20-μl aliquot of the digitonin
cell lysate was transferred to a replica 96-well microtiter plate and
300 μl of bicinchoninic acid reagent was added ( 22). One unit of
inducer activity is the amount that doubles the QR activity in a
microtiter well containing 150 μl of medium. Hence, a compound with a
CD value (the concentration of a compound required to double the QR
specific activity in Hepa 1c1c7 murine hepatoma cells) of 1.0 μM has
6,667 units of inducer activity per μmol. We express the inducer
potency of plant extracts as units/g fresh weight (fr. wt.) or dry
weight. Measurement of Isothiocyanates and Glucosinolates. Isothiocyanate concentrations of plant extracts were determined
spectroscopically by cyclocondensation with 1,2-benzenedithiol to
produce 1,3-benzodithiole-2-thione ( ![[var epsilon]](/corehtml/pmc/pmcents/epsiv.gif) of 23,000
M −1![[center dot]](/corehtml/pmc/pmcents/middot.gif) cm −1 at 365 nm) ( 20, 21). Aqueous
extracts were used directly; organic solvent extracts were first
evaporated to dryness and then redissolved in water. Glucosinolates in
these extracts were quantitatively converted to isothiocyanates by
treatment with purified myrosinase for 2 h at 37°C and then
subjected to cyclocondensation. Indole glucosinolates cannot be
measured in this fashion because their isothiocyanates are unstable
(see below). Paired-Ion Chromatography of Glucosinolates. Plant extracts
were chromatographed in acetonitrile/water (1:1, vol/vol)
containing 5 mM tetradecylammonium bromide at a flow rate of 3
ml/min, on reverse-phase columns (Whatman Partisil 10 ODS-2; 250
× 4 mm) on a Waters HPLC system equipped with a photodiode array
detector ( 19). Sinigrin (allyl glucosinolate) was used as a standard.
The relative integrated absorbance areas at 235 nm for
alkylthioglucosinolates such as glucoraphanin, glucobrassicin, and
neoglucobrassicin were 1.00, 1.22, and 2.70 times, respectively, those
of an equimolar quantity of sinigrin. Inhibition of Mammary Tumor Development in Rats. Mammary
tumors were produced in female Sprague–Dawley rats by feeding single
10-mg doses of DMBA by gavage at age 50 days ( 16). Glucosinolate or
isothiocyanate preparations obtained from broccoli sprouts and vehicle
control (all in 1.0 ml of equal volumes of Emulphor 620P and water)
were administered by daily gavage on days 47–51 (2 h before the DMBA
dose on day 50). Plant Preparation. Three-day-old broccoli sprouts were rapidly
plunged into 5 vol of boiling water and boiling was continued for 3
min. The mixture was then cooled, filtered, and lyophilized to provide
a dry powder. To prepare glucosinolates, the dried powder was dissolved in water and
analyzed for inducer activity and for glucosinolate and isothiocyanate
content. Excess purified myrosinase was added to both analyses. The
preparation was then diluted with water and mixed with an equal volume
of Emulphor 620P, so that 1.0 ml contained 25 or 100 μmol of
glucosinolates (Table 1).
| Table 1Analyses of 3-day-old broccoli sprout preparations used
for rat mammary tumor
inhibitionstudies |
To prepare isothiocyanates, the above-described powder was mixed with
2% (wt/wt) (based on the original fr. wt. of broccoli sprouts) of
9-day-old daikon ( Raphanus sativus) sprouts as an abundant
source of crude myrosinase. The mixture was homogenized and incubated
for 3 h at 37°C, which resulted in quantitative conversion of
glucosinolates to isothiocyanates. It was filtered, lyophilized, and
assayed for inducer activity and for isothiocyanate content. The added
daikon accounted for less than 1% of the isothiocyanate content and of
the total inducer activity. The preparation was then diluted with water
and mixed with an equal volume of Emulphor 620P so that 1.0 ml
contained 25, 50, or 100 μmol of isothiocyanates (Table 1). Progression of tumor development was monitored by palpation at weekly
intervals. Kaplan–Meier analyses followed by log-rank comparisons were
used to test for differences in tumor incidence. Animals were
necropsied at 167 days of age, and mammary tumors were excised,
counted, and weighed. All animal experiments were in compliance with
National Institutes of Health Guidelines and were approved by a Johns
Hopkins University Animal Care and Use Committee. Extraction and Quantitation of Glucosinolates and Isothiocyanates
from Crucifers. The isothiocyanate sulforaphane was isolated from
acetonitrile extracts of lyophilized aqueous homogenates of broccoli
and was identified as the principal and very potent phase 2 enzyme
inducer of this crucifer ( 15). This finding, and the subsequent
demonstration of the tumor blocking activity of sulforaphane ( 16),
focused our attention on the potential of plant-derived isothiocyanates
as chemoprotectors and emphasized the long-recognized chemoprotective
properties of this class of compounds ( 23). However, it is now apparent
that the isolated sulforaphane was derived largely from its
glucosinolate (glucoraphanin) through the action of
endogenous myrosinase during the isolation procedure.
Glucosinolates per se are not inducers but are converted
quantitatively by treatment with myrosinase to their cognate
isothiocyanates, which are inducers of phase 2 enzymes (Fig.
Left).
Our search for edible plants with high levels of inducer activity
required development of methods for efficient extraction and
quantitation (preferably in the same extracts) of generally hydrophobic
isothiocyanates and water-soluble glucosinolates, under conditions that
prevented the hydrolysis of glucinolates by myrosinase. It was also
desirable to obtain extracts without use of hot solvents to avoid loss
of volatile and highly reactive isothiocyanates and to use
water-miscible solvents that could be directly assayed for QR inducer
potency ( 11, 14) and for isothiocyanate content by cyclocondensation
with 1,2-benzenedithiol ( 20, 21). Of a range of solvents and solvent combinations examined, a mixture of
equal volumes of dimethyl sulfoxide, dimethylformamide, and
acetonitrile (triple solvent) was particularly effective. Rapid
immersion and homogenization of fresh or frozen plant samples in 10 vol
of triple solvent at −50°C, followed by filtration or
centrifugation, provided soluble extracts that contained both
glucosinolates and isothiocyanates in high yield. We compared the
inducer activities of myrosinase-treated extracts of five freshly
harvested adult heads of broccoli. Several extraction procedures were
used including ice-cold water, boiling water, boiling aqueous 80%
methanol, and triple solvent, as well as acetonitrile extraction of
lyophilized aqueous homogenates. The triple solvent and 80% methanol
extracts had very high inducer activity (38,000–45,000 units/g fr.
wt.), whereas ice-cold and boiling water extracts contained only about
30% and acetonitrile extracts contained about 10% of this activity.
Furthermore, triple solvent extracts showed essentially no inducer
activity without added myrosinase, which is not itself an inducer (Fig.
Right), whereas the smaller amounts of inducer activity
extracted with water were not increased by myrosinase treatment. Cold
triple solvent is a simple, efficient, and reproducible extractant for
both glucosinolates and isothiocyanates and also prevents myrosinase
action. By using this extraction procedure, followed by the addition of
purified myrosinase, we have shown that nearly all the inducer activity
of the cruciferous plants evaluated is derived from glucosinolates. Comparison of Inducer Activity of Fresh and Frozen Mature Stage
Vegetables. By using triple solvent for extracting glucosinolates
and converting them to their bioactive isothiocyanates with myrosinase,
we compared phase 2 enzyme inducer activities from a variety of
cruciferous plants (arugula, broccoli, Brussels sprouts, cabbage,
cauliflower, Chinese cabbage, collards, crambe, daikon, kale, kohlrabi,
mustard, red radish, turnip, and watercress). Although our earlier
study found less than 3,400 units of inducer activity per g fr. wt of a
variety of such vegetables including broccoli (recalculated from ref.
11 to permit dry/fr. wt. comparisons), extraction with triple solvent
provided between 10,000 and 100,000 units/g fr. wt. This finding of
dramatically higher inducer activities is attributable to much more
efficient extraction of the inducers and hydrolysis of glucosinolates
by added myrosinase. Because broccoli extracts were typically the most
potent, we compared the inducer activities of 7 samples of frozen
broccoli (five national brands) with those of 22 randomly collected
fresh broccoli samples (cultivars unknown) obtained from local
supermarkets. Activities of frozen samples ranged from 9,000 to 15,000
units/g fr. wt., whereas the fresh samples had an almost 8-fold range
of potencies from 11,100 to 83,300 units/g fr. wt. (mean =
35,100 units/g fr. wt.; median = 30,800 units/g fr. wt.; Fig.
). The inducer activities of the
fresh broccoli samples were unrelated to their physical appearance or
whether grown under conventional or organic conditions. In all 7
samples of frozen broccoli and in 21 of 22 triple solvent extracts of
fresh mature broccoli, no inducer activity was detectable before
addition of myrosinase. Therefore, we conclude that the contribution of
phytochemicals other than glucosinolates to the inducer activity of
these extracts is negligible. The generally lower potencies of frozen
broccoli samples may have been due to unfavorable storage conditions or
to removal of glucosinolates during the blanching process. Both
cultivar and many environmental factors have significant effects on
glucosinolate content and, consequently, on inducer potency.
Effects of Plant Age on Inducer Potencies of Cruciferous
Vegetables. Preliminary experiments indicated that inducer
potencies (expressed per g of plant) of extracts of young sprouts of
arugula, bok choy, broccoli, Brussels sprouts, cabbage, cauliflower,
Chinese cabbage, collards, cress, daikon, kale, kohlrabi, mustard,
turnip, and watercress ranged from 10 to 100 times those of mature
field-grown plants. Similarly, in sprouts of eight broccoli cultivars,
grown without exogenous nutrients, the inducer activity (nearly all of
which arose from glucosinolates) per unit plant weight declined
initially in an exponential manner from a maximum in the seed (Fig.
) and continued to decline
thereafter, approaching the values in mature broccoli heads after about
15 days (data not shown), whereas the total inducer activity per plant
remained constant. The inducer activity fell from 1.8 million units/g
of seeds to 180,000 units/g fr. wt. at 9 days, largely due to an
increase in plant weight from seeds (3.3 mg) to 9-day-old sprouts (60
mg). Apparently no significant net synthesis of glucosinolates occurred
under these conditions.
When broccoli sprout extracts were tested with mutant murine hepatoma
cells defective in cytochrome P450 activity, they were fully active as
inducers (data not shown), demonstrating that they contain inducers
that are monofunctional ( 24). This is to be expected since most of the
inducer activity of sprouts arises from sulforaphane, which is a
monofunctional inducer ( 15). In contrast bifunctional inducers raise
both phase 2 enzymes and certain phase 1 enzymes (cytochromes P450) via
an aryl hydrocarbon receptor-dependent mechanisms. Whereas inducer activity was high for sprouts of all crucifers
examined, it was consistently highest for broccoli and cauliflower
cultivars. Three-day-old sprouts of random commercial cultivars of
broccoli (n = 26) and cauliflower (n =
28) produced inducer potencies ranging from 92,500 to 769,000 units/g
fr. wt. (mean = 293,000 units/g fr. wt.) and from 50,000 to
560,000 units/g fr. wt. (mean = 251,000 units/g fr. wt.),
respectively. Repetitive kilogram-scale harvests of 3-day-old broccoli
(cultivar SAGA) sprouts yielded an average of 511,000 units/g fr. wt.
(n = 14). Glucosinolate Profiles of Sprouts and Mature Broccoli. The
dramatic quantitative differences between the inducer potencies of
young sprouts and mature crucifers grown from the same seed lots are
associated with equally striking qualitative and quantitative
differences in the glucosinolate profiles of these plants. Paired-ion
chromatography confirmed that the major glucosinolates in mature
broccoli are typically indoles: glucobrassicin, neoglucobrassicin ( 18,
25, 26), and smaller quantities of 4-hydroxyglucobrassicin (for
example, see Fig. ). Although
the indole glucosinolate composition varied considerably among
cultivars and even among different samples of the same cultivar, indole
glucosinolates accounted, on average, for 67% of the total
glucosinolates in extracts prepared from 7 broccoli cultivars (DeCicco,
Emperor, Everest, Excelsior, Green Comet, Green Valiant, and SAGA). In
contrast, in most 3-day-old broccoli and cauliflower sprouts and seeds
(>70 cultivars examined), less than 10% of total glucosinolates were
indoles, and in many cultivars none could be detected (Fig. ). These
sprouts contained primarily alkylthioglucosinolates: typically
glucoraphanin with smaller quantities of glucoerucin and glucoiberin
(3-methylsulfinylpropyl glucosinolate). The total glucosinolate
contents are 22.7 and 3.37 μmol/g fr. wt. for the sprouts and
mature broccoli, respectively. Indole glucosinolates, however,
accounted for only 3% of the total glucosinolates in these sprouts,
compared with 68% of those in the mature plant. There are 20 times
more methylsulfinylalkyl glucosinolates (glucoraphanin and glucoerucin)
in the sprouts compared with the mature broccoli. A 100-g serving of
mature broccoli would, therefore, provide 108 μmol of
methylsulfinylalkyl glucosinolates and 229 μmol of indole
glucosinolates, whereas consumption of an equivalent quantity of
methylsulfinylalkyl glucosinolates via a much smaller serving of
sprouts (5 g) would result in the consumption of only 11.2 μmol of
indole glucosinolates.
These differences in glucosinolate profiles between young sprouts and
mature broccoli are of considerable interest and potential importance
in devising chemoprotective strategies in humans. The
methylsulfinylalkyl glucosinolates contained in high concentrations in
sprouts are monofunctional inducers (see above, and ref. 15). Moreover,
sulforaphane does not appear to be significantly genotoxic in that it
stimulates neither unscheduled DNA synthesis in hepatocytes nor the
formation of histidine revertants in the Salmonella
typhimurium test ( 27). In contrast, hydrolysis of indole
glucosinolates by myrosinase gives rise to bifunctional inducers, such
as indole-3-carbinol and indole-3-nitrile, and to condensation
products, such as 3,3′-diindolylmethane and indole-3-carbazole, which
bind to the aryl hydrocarbon receptor ( 28). Indole-3-carbinol is both
an inhibitor and an enhancer of tumor formation in animals, depending
upon the experimental system and the timing of administration in
relation to exposure to carcinogen (see references in ref. 29).
Consequently, there are potential limitations to the use of indole
glucosinolates as chemoprotectors in humans because they ( i)
are weak inducers of phase 2 enzymes, ( ii) are bifunctional
inducers that activate phase 1 enzymes, ( iii) may have
estrogen receptor binding activity ( 30), and ( iv) are
potential tumor promoters. Inhibition of DMBA-Elicited Mammary Tumor Development in Rats by
Broccoli Sprout Extracts. Sulforaphane and several synthetic
acetylnorbornyl isothiocyanate analogues, which are potent phase 2
enzyme inducers, reduced the incidence, multiplicity, and weight of
mammary tumors and retarded tumor development in female Sprague–Dawley
rats treated with a single dose of DMBA ( 16). We therefore reasoned
that extracts of cruciferous sprouts containing high levels of
glucoraphanin and associated high inducer activity should likewise have
antitumor activity. Accordingly, boiling water extracts of 3-day-old
broccoli sprouts rich in glucoraphanin were evaluated as inhibitors in
the DMBA rat mammary tumor system. Although triple solvent is a more
efficient solvent, it was not used to make these preparations because
solvent residues could confound interpretation of feeding experiments.
Since the inducer activity of 3-day-old broccoli sprout extracts is
derived nearly exclusively from two glucosinolates (usually about 75%
glucoraphanin and 25% glucoerucin), we examined the extracts before
and after myrosinase treatment to determine the effects of
glucosinolates and isothiocyanates, respectively, on tumor inhibition.
These sprout preparations were standardized according to their inducer
activities in the murine hepatoma cell assay, by assuming as an
approximation that the entire inducer activity was derived from
glucoraphanin or sulforaphane. Daily doses of glucosinolates (25 and
100 μmol) and isothiocyanates (25, 50, and 100 μmol) were
administered on days 47–51 to groups of 20 rats that also received 10
mg of DMBA at 50 days of age, 2 h after administration of the
sprout extract, according to a standard protocol ( 16). Mammary tumor development, monitored by palpation at weekly intervals,
was significantly retarded compared with controls, with respect to
tumor multiplicity (tumors per animal), and incidence (fraction of
tumor-bearing animals) (Fig. ).
The tumor incidence at all doses of glucosinolate and isothiocyanate
treatment was lower than in controls (P = 0.0197 and
0.0190, respectively). Analysis of tumor progression by palpation was
terminated at 147 days of age. Retardation of tumor development was
clearly dose-dependent for the isothiocyanates, but the potency of the
25 μmol dose of glucosinolates appeared to be similar to that of the
100-μmol dose, suggesting that the lower dose may have produced a
nearly maximal effect. All treatments markedly reduced the total number
of tumors found at necropsy. There were 34 tumors in the control group
(20 surviving animals) and 11–19 tumors in the treated groups (19–20
surviving animals).
The tumor inhibitory effects of sprout extracts in these experiments
are comparable to those observed with 75 μmol of synthetic
sulforaphane in a similar protocol ( 16). It therefore seems unlikely
that broccoli sprouts contain significantly potent chemoprotectors
other than the glucosinolates/isothiocyanates identified in these
studies. Although the chemoprotective effects of isothiocyanates are
well recognized ( 23), to our knowledge the only antitumor experiments
with glucosinolates are those of Wattenberg et al. ( 31) who
reported that relatively large single doses of benzyl glucosinolate
(147 μmol) and glucobrassicin (134 μmol) significantly decreased
mammary tumor formation in DMBA-treated rats. The possibility that the
chemoprotective effect of glucosinolates may not depend on hydrolysis
to isothiocyanates needs consideration. The high potency of lyophilized hot-water extracts of broccoli sprouts
in suppressing mammary tumor development is in sharp contrast to the
few previous studies on the tumor suppressive effects of feeding dried
mature crucifers to rodents. In the present study (Fig. ) daily
administration of dried sprout extracts ranging from 81 to 424 mg
(i.e., 0.54 to 2.83% of an estimated 15-g daily food intake) provided
a highly significant tumor suppressor effect. In contrast, 5–40%
dried mature crucifers in the diet were required to achieve protection
(see ref. 10 for references). Large quantities of inducers of enzymes that protect against
carcinogens can be delivered in the diet by small quantities of young
crucifer sprouts (e.g., 3-day-old broccoli sprouts) that contain as
much inducer activity as 10–100 times larger quantities of mature
vegetables. Moreover, the inducer activity arises primarily from
glucoraphanin (the glucosinolate of sulforaphane) and such sprouts
contain relatively low quantities of indole glucosinolates, which are
potential tumor promoters. Because little is known of the metabolism of
glucosinolates in humans, we have undertaken studies (to be published
separately) that demonstrate efficient conversion of glucosinolates to
isothiocyanates in humans in the absence of plant myrosinase. Observations reported in this paper are the subject of issued and
pending patents. We thank Katherine K. Stephenson, Kristina L. Wade,
and Mark Wrona for expert technical assistance; W. David Holtzclaw and
Tory Prestera for help in isolation and identification of the
glucosinolates; Mikio Shikita for purification of myrosinase; Patrick
M. Dolan for assistance with the rat mammary tumor experiments; Stephen
J. Gange for statistical analysis; and Thomas W. Kensler and Theresa A.
Shapiro for constructive review of the manuscript. We are grateful to
the following growers for providing mature cruciferous vegetables grown
to our specifications: G. & Y. Johnson, M. Rice, M. Voelkel, M. Heller,
J. Martin, W. & S. Hastings, J. & L. Carty, P. Holloway, and T. & L.
Harding. These studies were supported by generous gifts from Lewis B.
Cullman, Charles B. Benenson, and other Friends of the Brassica
Chemoprotection Laboratory; by a Program-Project Grant (P01 CA 44530)
from the National Cancer Institute, Department of Health and Human
Services; and by grants from the American Institute for Cancer Research
and the Cancer Research Foundation of America. | | QR | quinone reductase
[NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] | | | DMBA | 7,12-dimethylbenz(a)anthracene | | | glucoraphanin | the
glucosinolate of sulforaphane or 4-methylsulfinylbutyl isothiocyanate
[CH3S(O)(CH2)4NCS] | | | glucoerucin | the glucosinolate of erucin or 4-methylthiobutyl isothiocyanate
[CH3S(CH2)4NCS] | | | glucobrassicin | indol-3-ylmethyl glucosinolate | | | neoglucobrassicin | 1-methoxyindol-3-ylmethyl glucosinolate | | | 4-hydroxyglucobrassicin | 4-hydroxyindol-3-ylmethyl glucosinolate | | | fr. wt. | fresh weight |
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