Cruciferous Vegetables

and Cancer Prevention

 Ⅰ. Introduction
• As 30% of all cancers are considered to have a dietary
component.

• Epidemiological studies have identified that a diet rich

in fruits and vegetables is associated with a decreased
risk for a number of different cancers.

• Studies specifically evaluating the effect of cruciferous

vegetables have shown an inverse relationship
between intake of cruciferous vegetables and cancer
incidence.
 Ⅱ. CANCER PREVENTION BY
CRUCIFEROUS VEGETABLES
A. Epidemiological Studies
• “There is sufficient epidemiological evidence to suggest that
consumption of cruciferous vegetables is associated with a reduction
in the incidence of cancer at several sites in humans.”

• Review of 87 case-control studies indicated that 67% described an

inverse association between crucifers and all cancers, with cabbage
intake being associated with the greatest number of studies showing
this effect.

• More recently a study that evaluated the effect of many individual

fruit and vegetables on incidence of bladder cancer among 47,909
men revealed that intake of crucifers, and no other vegetable type
examined, was inversely related to risk for bladder cancer.

• Epidemiological data strongly suggest that a diet rich in broccoli,

cabbage, or a mixture of cruciferous vegetables is able to decrease
one’s risk for cancer.
B. Laboratory Animal Studies
• The results of studies support the epidemiological data, suggesting
that cruciferous vegetables do protect against carcinogenesis.

• For example,
1. When broccoli or cabbage was incorporated into the diets of rats
that had previously received dimethylbenzanthracene(DMBA),
mammary tumor formation was inhibited.
2. Brussels sprouts(20%) given in the diet before, during, and for 2
weeks after DMBA administration also inhibited mammary tumor
formation in rats.

• These, and many other laboratory studies, support the findings of

the epidemiological studies.
• Something distinctive about cruciferous vegetables permits them to
decrease the risk for cancer significantly.
 Ⅲ. CHEMICAL PROFILE OF
CRUCIFEROUS VEGETABLES
• Evidence from epidemiological studies indicating that crucifers are
better able to protect against cancer than many other fruits or
vegetables leads to the logical proposal that chemoprevention by
cruciferous vegetables is associated with some unique aspect of
their biochemistry.

• Cruciferous vegetables contain a series of relatively unique

secondary metabolites of amino acids, termed glucosinolates.

• While glucosinolates are not considered directly bioactive, many

glucosinolate hydrolysis products, particularly the isothiocyanates,
appear to have anticarcinogenic activity.
• The hydrolysis products are glucose, sulfate ions, and an unstable
thiono compound.
• The unstable thiono intermediate can rearrange to form an
isothiocyanate, a nitrile, or a thiocyanate, depending upon such
conditions as pH, temperature, presence of iron, and extent of
hydration.
• The major glucosinolates and their bioactive hydrolysis products
from the common Brassica vegetables are shown in Table.

• A wealth of information is available on the chemistry and

bioactivity of glucosinolate hydrolysis products.
Ⅳ. MECHANISMS OF CHEMOPREVENTION
• In seminal studies planned to determine the mechanism of cancer
prevention by isothiocyanates, Wattenberg treated rats with a
single dose of benzyl isothiocyanate, either 24, 4, or 2h before, or
4h after, giving the carcinogen DMBA.

• When he administered benzyl isothiocyanate in the diet starting 1

week after DMBA treatment, fewer breast tumors developed.

• It has become increasingly clear that the anticarcinogenic effects of

isothiocyanates are unlikely to be due to a single mechanism, or
even limited to a single stage of ccarcinogenesis.
A. Induction Of Detoxification
• The major mechanism of chemoprevention by cruciferous vegetables
is thought to be through upregulation of detoxification enzymes,
resulting in decreased initiation of chemical-induced carcinogenesis.

• 500g of broccoli/day for 12days

-> increased cytochrome P450(CYP)1A2-dependent caffeine metabolism,
it did not alter CYP2E1-dependent chlorzoxazone metabolism.
: This data indicate that dietary crucifers can increase detoxification
enzymes in humans.

• Glucosinolate hydrolysis products upregulate through at least two

response elements in the genes of detoxification enzymes
: (1) bifunctional inducers interact with the xenobiotic response element
(XRE), also termed the aryl hydrocarbon response element(AhRE),
(2) monofunctional inducers interact with the antioxidant response
element(ARE).
• The XRE is found in the DNA regulatory region of the genes for a
number of proteins, including CYP1A/1/2, quinone reductase, and
glutathione transferase Ya2.

• The ARE is found in the regulatory regions of the genes for

quinone reductase and glutathione transferase Ya2, but not
CYP1A1/2.

• Thus,
gene sequences for these phase Ⅱ enzymes containregulatory
regions with both an XRE and an ARE, while gene sequences for
CYP1A/2 lack the ARE and lack the ability to be stimulated by
monofunctional inducers.
B. Inhibition of Activation
• A large number of precarcinogens are bioactibated to the ultimate
carcinogen by CYP-dependent oxidation.
• It has been proposed that glucosinolate breakdown products may
protect against initiation of cancer not only by induction of phase Ⅱ
detoxification enzymes, but also by inhibiting CYP-dependent
activation of precarciongens.

• Very recently benzyl isothiocyanate was shown to cause the

destruction of CYP2E1 during metabolism.

• In a temporal study using rats, PEITC was found to cause a

significant loss of CYP1A1/2 and CYP3A activity, in addition to the
sever loss of CYP2E1 activity.
• In contrast to the in vitro study that had reported acute inhibition of
CYP2B enzyme activity, here PEITC was found to induce CYP2B
enzymes.
C. Inhibition of Cell Proliferation and Apoptosis

• In a separate study using the human undifferentiated colon cancer

cell line HT28, the parent glucosinolate, glucobrassicin, was shown
to have no effect, while the hydrolysis products diindolyl methane
and sulforaphane decreased cell viability by approximately 50%.

• Studies have also implicated cell cycle arrest. A study in HeLa cells
reported that allyl isothiocyanate, benzyl isothiocyanate, and PEITC
all caused cell cycle arrest at G2/M.

• Isothiocyanates have been found to cause apoptosis in vitro.

• c-Jun N-terminal kinase(JNK), which lies upstream of caspase 3 in

the apoptotic cascade, was also actibated by isothiocyanates.
 Ⅴ. DEVELOPMENT OF CANCER PREVENTATIVE
AGENTS FROM CRUCIFEROUS VEGETABLES

• The cancer preventative activity of isothiocyanates derived from

hydrolysis of glucosinolates.

• The majority of the work has focused on just a few, including

indole-3-carbinol, PEITC, sulforaphane, and benzyl isothiocyanate.

• Scattered studies have shown protection from other indolyl

compounds, such as brassinin, which gas been shown to protect
against mammary carcinoma and skin tumors in mice given
DMBA, and allyl isothiocyanate, effective in combating colon
carcinogenesis.
A. Phenylethyl Isothiocyanate(PEITC)
• Watercress is a particularly good source of gluconasturtiin, the
parent glucosinolate to PEITC.

• In an evaluation of the effect of PEITC on NNK bioactivation in

rats, it was found that PEITC(1 mmol/kg rat) had an inhibitory
effect on CYC2E1, effectively blocking the bioactivation of NNK.
Also, PEITC induced a number of phase Ⅱ enzymes which could
then clear any bioactivated products that were formed.

• One study reports that, when PEITC and benzyl isothiocyanate

were given postinitiation to rats that had received the urinary
bladder carcinogen diethylnitrosamine, there was a significant
increase in papillary hyperplasia and carcinoma compared with
rats receiving the carcinogen alone.
B. Indole-3-Carbinol
• A breast cancer prevention trial is being conducted at Strang Cancer
Center, based on mechanistic studies showing that in three breast
cancer cell lines, indole-3-carbinol caused a>50% inhibition in growth,
with an increase in the quiescent cell fraction, and a doubling of the
apoptotic rate.
• Indole-3-carbinol condensation products have the ability to bind to the
SRE and cause induction of CYP1A1/2, which is involved in the
bioactivation of many precarcinogens.

• When indole-3-carbinol was administered to mice prior to the

pulmonary carcinogen NNK, pulmonary NNK-DNA adduct formation
was decreased, but hepatic NNK-DNA adduct formation was increased.

• Indole-3-carbinol and at least some of these condensation products

have an interactive effect with estrogen, affecting estrogen metabolism
and estrogen binding to the estrogen response element.

• A number of studies in rodents and humans show that indole-3-

carbinol causes an increase in 2-hydroxylation of estrogen, increasing
the ratio of 2:16 hydroxylated products.
C. Sulforaphane and Sulforaphane Analogues

• Sulforaphane decreases mammary tumor incidence when

administered to rats before and during administration of DMBA,
and inhibits neoplastic nodule formation in mouse mammary
gland cultures.

• When sulforaphane and compound 30were administered to rats

given DMBA, they were both effective as anticarcinogens.
 Ⅵ. BIOACTIVE COMPONENTS
OTHER THAN ISOTHIOCYANATES
A. Crambene
• In evaluation of glucosinolate hydrolysis products as
anticarcinogens, almost all the research has focused on
isothiocyanates.
• Studies have revealed that crambene is a
monofunctional inducer, causing upregulation of
quinone reductase and glutathione transferase while
having no effect on CYP1A1/2 levels.
• When rats were given both crambene and indole-3-
carbinol together, total upregulation of quinone
reductase was significantly greater than expected when
adding together their individual effects.
B. S-Methyl Cysteine Sulfoxide(SMCSO)
• Crucifers contain high levels of SMCSO.
C. Dithiolethione
• The natural dithiolethione present in cruciferous vegetables.

• Both dithiolethione and oltipraz(antischistosomal drug) have been

shown to upregulate phase Ⅱ enzymes via the ARE, making them
classic monofunctionall inducers.

• Healthy adults three groups

1group : given either a placebo
2group : given 500mg oltipraz once weekly
3group : given 125mg oltipraz daily
↓ after 1 month
urinary analysis revealed that the intermittent high dose inhibited phaseⅠ
Bioactivation of aflatoxin, while daily low-dose oltipraz increased phase Ⅱ
detoxification of bioactivated aflatoxin.
 Ⅶ. SAFETY OF CRUCIFEROUS VEGETABLES
• Genotoxicity of vegetable extracts
Brussels sprouts > cauliflower > cabbage, kohlrabi, broccoli > Turnip
(This ranking was not strictly related to total isothiocyanate content, but the authors suggested that this
might only be because individual isothiocyanates differ in their genotoxic potency.)

• Isothiocyanates were both promoters and complete carcinogens,

initiating cancer and promoting mitosis of mutant cells.
• Cruciferous vegetables gave been a part of the diet for many
centuries. Any compound exhibiting bioactivity can be expected to
exert adverse, or toxic, effects at sufficiently high doses.
• The meal from industrial oilseeds is high in glucosinolates, often
containing 3 to 7% glucosinolates by weight. In contrast, cruciferous
vegetables contain 0.02% to 0.4% glucosinolate by weight, although
levels in cruciferous vegetable seeds are typically tenfold higher than
in the plant.
• Therefore, intake of cruciferous vegetable seeds as a significant
percentage of the diet could produce adverse effects.
Ⅷ. IMPACTING THE AMERICAN DIET
• When glucosinolates isolated from Brussels sprouts were fed to rats,
they had no measurable effect on clearance of the drug antipyrine
unless they were first hydrolyzed by adding myrosinase. When the
hydrolyzed products were fed to rats, antipyrine clearance was
increased by 66%.

• Information of this type has been used to suggest that glucosinolate

breakdown products are the active molecules in upregulation of
detoxification enzymes. This is important as one considers whether it is
better to cook cruciferous vegetables or eat them raw to obtain their
health benefits.

• Most animal studies have been carried out using freeze-dried raw
vegetables. While there are relatively few human studies, most have
employed cooked vegetables. Feeding cooked cabbage(200g/day)and
Brussels sprouts (300g/day)to humans caused an increase in
detoxification enzymes, and feeding cooked brussels sprouts(300g/day)
decreased oxidative DNA damage.
• New broccoli products, including broccoflower and broccolini are
on the market, but glucosinolate levels in these varieties are not
yet reported.

• Tablets containing broccoli and other cruciferous vegetables are

becoming available in health food stores.

• Crucifers offer a valuable health benefit to the diet that may not
be obtained through other functional foods.
• Only time remains before there are cruciferous cultivars
commercially available that are nutritious, palatable, and
sufficiently potent when integrated into the diet on a regular
basis, to offer protection from the risks of cancer.
 Ⅸ. SUMMARY
• A diet rich in cruciferous vegetables, such as broccoli and cabbage,
is associated with a decreased risk for a number of cancers.
• Crucifers contain a family of secondary plant metabolites known as
glucosinolates.
• Isothiocyanates have also been shown to increase or upregulate
several detoxification enzymes.
• Crucifers may act through inhibition of cytochrome P450-dependent
bioactivation of carcinogens resulting in decreased initiation of
cancer and, through cell cycle arrest and apoptosis, resulting in
decreased progression of tumor growth.