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LS VI R
Chemica-B~oiogical Interactions 103 1997) 7 129
Mini review
A review of mechanisms underlying
anticarcin~genicity by brassica vegetables
Dorette T.H. Verhoeven**, Hans Verhagen,
R. Alexandra Goldbohm, Piet A. van den Brandtb,
Geert van Poppel
TN Nutrition and Food Research institute, P.O.Bas 360, 3700 AJ Zeist, The Netherkmds
hUniuersity of Limburg, ~@partm~nt of Epjdem~alag~, Maastricht. The
Ne~~~rlunds
Abstract
The mechanisms by which brassica vegetables might decrease the risk of cancer are
reviewed in this paper. Brassicas, including all types of cabbages, broccoli, caulitlower and
Brussels sprouts, may be protective against cancer due to their relatively high glucosinoiate
content. Glucosinolates are usually broken down through hydrolysis catalyzed by myrosi-
nase, an enzyme that is released from damaged plant cells. Some of the hydrolysis products,
viz. indoies and isothiocyanates, are able to influence phase
I
and phase 2 biotransfor~tion
enzyme activities, thereby possibly influencing several processes related to chemical carcino-
genesis, e.g. the metabolism, DNA-binding and mutagenic activity of promutagens. A
reducing effect on tumor formation has been shown in rats and mice. The anticarcinogenic
action of isothiocyanates and indoles depends upon many factors, such as the test system, the
target tissue, the type of carcinogen challenge and the anti~rcinogeni~ compound, their
dosage, as well as the timing of the treatment. Most evidence concerning anti~arcinogenic
effects of glucosinolate hydrolysis products and bras&a vegetables has come from studies in
animals. Animal studies are invaluable in identifying and testing potential anticarcinogens. In
addition, studies carried out in humans using high but still realistic human consumption
levels of indoles and brassica vegetables have shown putative positive effects on health. 6
1997 Elsevier Science Ireland Ltd.
Ktywords:
Anticarcinogenesis; Brassica vegetables; Glucosinolates; Indoles: Isothiocyanates
* Corresponding author. Tel.: +
31 30 6944755: fax: -i-
31
30 6957952.
000%2797/97/ 17.00 Q 1997 Elsevier Science Ireland Ltd. All rights reserved
PlI SOOO9-2797(96)03745-3
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80 D.T.H . Verhoeven et al . 1 Chemi co-Bi ol ogical I nt eracti ons 103 1997) 79-129
1 Introduction
The hypothesis that certain components of plant foods are protective against
cancers at many sites has gained interest in the past two decades and is supported
in many studies [l-3]. A large number of potentially anticarcinogenic agents have
been suggested in fruits and vegetables so far, e.g. fiber, vitamins C and E,
carotenoids, flavonoids, phenols, phytoestrogens, diallylsulfides, limonene and hy-
drolysis products of glucosinolates. The mechanisms by which these agents may act
include dilution and binding of carcinogens in the digestive tract (fiber), antioxidant
effects, inhibition of nitrosamine formation, inhibition of activation of promuta-
gens/procarcinogens, induction of detoxification enzymes, alteration of hormone
metabolism, and others [4-61.
One group of vegetables that has been widely regarded as potentially cancer
protective are vegetables of the Cruciferae family. Cruciferous vegetables are the
major source of glucosinolates in the diet which distinguishes them from other
vegetables. Brassica vegetables, including all cabbage-like vegetables, are a genus of
the family Cruciferae and contribute most to our intake of glucosinolates [7]. In the
1960s interest emerged in the possibility that certain aromatic and indolic glucosi-
nolate hydrolysis products might influence carcinogenesis. In in vivo experiments
animals were fed glucosinolate hydrolysis products together with a carcinogen and
it was found that fewer animals developed tumors in comparison with control
animals not receiving the glucosinolate hydrolysis products [8,9]. From that time on
many studies have been carried out to examine the possible anticarcinogenic effect
of brassica vegetables and glucosinolate hydrolysis products. This paper now
reviews these experimental studies investigating the effects of brassica vegetables
and glucosinolate hydrolysis products on carcinogenesis and cancer risk.
Searches for papers on brassica vegetables, glucosinolates or hydrolysis products
of glucosinolates were carried out with MEDLINE on CD-ROM (1983-1996),
with Current Contents (1995- 1996), and by checking references to find earlier
reports.
This paper will give a short overview of the occurrence and intake of glucosino-
lates, and of the chemistry of glucosinolates and their hydrolysis products. This will
be followed by the putative beneficial properties and adverse effects of brassicas,
glucosinolates and their hydrolysis products. Finally, an overall conclusion and
suggestions for further research will be given.
2. Occurrence and intake of glucosinolates
So far, over one hundred glucosinolates have been identified which occur
predominantly, but not exclusively, in vegetables of the family Cruciferae [lo]. In
Table 1 an overview is given of common vegetables of the family Cruciferae.
Vegetables of the genus
Brassica,
in which 15-20 glucosinolates have been found,
contribute most to our intake of glucosinolates and include all kinds of cabbages,
kale, broccoli, cauliflower, Brussels sprouts, kohlrabi, rapeseed, rutabaga and
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turnip. Detailed information on the consumption of brass&as is not readily
available. In Table 2 the ~ons~ption of some brassicas, as determined from food
cons~ption surveys in the UK [I l] and in the Netherlands [121, in 1992 is given.
The glucosinolate content of plants depends upon variety, cultivation conditions.
climate and the agronomic factors associated with plant growth, while the levels in
a particular plant vary between the parts of the plant [7]. Indolglucosinolates are
found in young shoots and growing leaves [17]. The total glucosinolate content of
a variety of brassica vegetables is also shown in Table 2. Very little is known about
the average daily intake of glucosinolates. An intake of 12- 16 mg glucosinolates
per person per day has been suggested by Fenwiek and I-Ieany [IO].
3. Chemistry of gluc~inolates and their hy~olysis products
3.1.
Chemical structure
The chemistry of glucosinolates and their hydrolysis products has been reviewed
extensively by Fenwick and Heany [7,10]. All glucosinolates share a common basic
skeleton containing a j?-r>-thioglucose grouping, a side chain and a sulfonated
oxime moiety, but differ in side chain (R):
S - p - D glucose
/
R-C
\\
N-OSO,--
with R = alkyl, alkenyl, arylalkyl, alkylthioalkyl, a-hydroxyalkyl or indolylmethyl.
Table 1
Common vegetables of the family Cruciferae
Genus
Armoracia
Brassica
Species Common name
Rusticana Horseradish
C~~esr~is Turnip
~kine~s~s
Pak choy
Juncea Brown mustard
_-...- _.__
Napus
Nigra
Oleracea
Pekinensis
Sativum
O~~~ina~e
Sat iw
Aha
Rape, Swede, rutabaga
Black mustard
Cabbage, kale, Brussels sprouts, cauliflower. broccoli. kohlrabi
Chinese cabbage
Garden cress
Watercress
Radish
Mustard
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D.T.H. Verhoeven et al. / Ch~~i~~- ~o~o~i~al nteru~ti~tls IO.? [i997] 19-i-79
83
Glucosinolate hydrolysis products make a significant contribution to the typical
flavour of brassica vegetables. The enzyme myrosinase (thioglucoside glycohydro-
lase EC 3:2:3:1) catalyses the hydrolysis of glucosinolates [lo]. Myrosinase is found
in plant cells in a separate compartment from glucosinolates. When the plant cells
are damaged, e.g. by cutting or chewing, the myrosinase comes in contact with the
glucosinolates and hydrolysis occurs. All kinds of processing lead to a certain
degree of glucosinolate hydrolysis by myrosinase hydrolysis or other chemical
reactions. The effects of processing on glucosinolate levels in vegetables have been
reviewed by De Vos and Bhjleven [18]. When the vegetables are cooked, myrosinase
is inactivated and thermal degradation and wash out occur, leading to a 30 --60(X1
loss of intact glucosinolates.
It should be noted that myrosinase activity has also been found in certain
intestinal microflora. This could be of importance when intact glucosinolates are
digested, although very little is known about this metabolic pathway [lo].
The glucosinolate hydrolysis products consist of equimolar amounts of an
aglucon, glucose and sulphate. The aglucones are unstable and undergo further
reactions to form for instance thiocyanates, nitrites or isothiocyanates. The nature
of the hydrolysis products depends primarily on the side chain of the glucosinolate,
besides the conditions of the hydrolysis and the presence of any cofactors. Glucosi-
nolates with an indole side chain form indoles, whereas oxazolidine-2-thiones are
formed from glucosinolates with a ~-hydroxy-alkyl side chain. Table 3, derived
from De Vos and Blijleven [1X], shows some glucosinolates and their hydrolysis
products that occur in cruciferous vegetables,
Of the indole glucosinolates, which are predominant in brassica vegetables,
glucobrassicin is amongst the most prevalent. At neutral pH glucobrassicin forms
an unstable isothiocyanate which degrades to indole-3-carbinol (13C) and a thio-
cyanate ion. I3C may condense to 3,3-diindolylmethane (DIM) or react with
ascorbic acid to form ascorbigen. At a more acidic pH, glucobrassicin forms
indole-3-acetonitrile (13A) and elemental sulphur [7,17]. In many publications it was
suggested that 13C and I3A were the most abundant metabolites, but recently it has
been stated that ascorbigen is the major indole-containiIlg product of gluco-bras-
sicin biotransformation [19].
3.3. Analysis q lucosinoiate composition und content
The analysis methods for determining glucosinolate composition and content
have been reviewed by McGregor et al. [20]. Distinction has been made between
total glucosinolates, individual glucosinolates and hydrolysis products. For determi-
nation of total glucosinolate content, calorimetric measurement of glucose released
by myrosinase hydrolysis seems most appropriate. Both gas-liquid chromatography
(GLC) and high performance liquid chromatography (HPLC) can be used to
determine the content of individual glucosinolates. Because of the thermal instabil-
ity of indolglucosinolates, HPLC is the most suitable method for determination of
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D.T.H. Verhoeuw et al. / ~hemi co-Biol ~gi~ul I~ t er~~ t i~ ~ 103 1997) 79-129 85
these compounds. rsothiocyanates and nitriles can be analyzed by GLC. HPLC
with UV detection may be used for analysis of oxazolidinethiones and indoles.
4.
Putative beneficial properties of glucosinolate hydrolysis products and brassicas
4 1 Postulated mechanism of anticarcinogenic action
Carcinogenesis is a multistage process; cells susceptible to genetic changes
(initiation) and epigenetic changes (promotions may gain a growth advantage and
undergo clonal expansion. Genetic changes are considered to result from interac-
tions between DNA and a carcinogen/mutagen, which can be metabolised into an
electrophilic intermediate and bind to DNA. If repair of the damage does not
occur, replication of DNA can lead to permanent DNA lesion, and in presence of
a tumor promotor to preneoplastic cells, neoplastic cells and finally metastases [21].
At each stage of the carcinogenic process a possibility of intervention exists. One
anticarcinogenic action is the modulation of metabolism of carcinogenic/mutagenic
compounds, thereby preventing the formation of electrophilic intermediates. This
modulation of metabolism comprises inhibition of activation of promutagens/pro-
carcinogens, induction of detoxifying mechanisms, and stimulation of activation
coordinated with detoxification and blocking of reactive metabolites [22]. Involved
in this modulation are phase 1 and phase 2 biotransfo~ation enzymes [23]. Phase
1 involves oxidation, reduction and hydrolysis reactions, thereby making xenobi-
otics more hydrophilic (which can result in inhibition of activation but also in
activation of the compound) as well as susceptible to detoxification. The most
important phase 1 enzymes are the cytochrome P450 enzymes. Phase 2 metabolism,
a detoxifying mechanism, comprises conjugation reactions making phase 1 metabo-
lites more polar and readily excretable. Examples of phase 2 enzymes are
glutathione S-transferases and UDP-glucuronyl transferases. Alteration of bio-
transformation enzyme activities is supposed to be involved, at least partly, in the
alteration of the toxicity, mutagenicity and tumorigenicity of specific chemicals [24].
Isothiocyanates and indoles, both glucosinolate hydrolysis products, are consid-
ered to be able to modulate biotransfo~ation enzyme activities ]17,24-261. Indoles
thereby also affect estradiol metabolism, which is P450 dependent, and may reduce
the risk of estrogen-dependent diseases such as mammary cancer [27]. Besides
modulators of biotransformation enzymes isothiocyanates are seen as suppressing
agents [6,28]. Suppressing agents act during the promotion phase of the neoplastic
process via prevention of the evolution of the neoplastic process in cells.
4.2.
Results of studies on anticarcinogenic action
In Tables 4-8 the results of studies on the antimutagenic and anticarcinogenic
properties of isothiocyanates, indoles and brassica vegetables are presented. A
distinction has been made between in vitro and in vivo studies. One of the most
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96 D .T.H. Verhoeven et al . 1 Chemi co-Bi ol ogical I nt eracri ons 103 3997) 79-129
interesting in vitro systems for identifying compounds with potential chemopreven-
tive activity has been developed by Prochaska and colleagues in the laboratory of
Talalay 1291.This resulted e.g. in the identification of sulforaphane as a hydrolysis
product of glucosinolates [95]. Most data originate from experiments using labora-
tory animals. There is a limited number of studies dealing with effects of isothio-
cyanates, indoles and brassicas in man. The results of these studies are presented
separately in Table 8.
Tables 4 and 5 presents the alteration of biotransfo~ation enzyme activities by
isothiocyanates, indoles and brassicas. In various test systems isothiocyanates,
indoles and brassica vegetables induced both phase 1 enzymes and phase 2 enzymes,
although the induction pattern differs among tissues. Isothiocyanates blocked the
activity of some phase 1 enzymes, depending upon the target tissue and the phase
1 enzyme examined.
Table 6 summarizes the modulation of metabolism and mutagenicity of various
agents by isothiocyanates, indoles and brassicas in different test systems. Isothio-
cyanates decreased the activation of mutagenic compounds, mostly nitrosamines. In
addition, isothiocyanates reduced the DNA adduct formation induced by 4-
(methylnitrosamino)- 1-(3-pyridyl)-1 -butanone (NNK) and N-nitrosomethylbenzy-
lamine (NMBA), but not the DNA adduct formation induced by benzo(a)pyrene
(B(a)P) and 2-amino-l-methyl-6-phenylimidazoI4,5-~]pyridine (PhIP). Indoles were
inhibitors of the DNA-binding of aflatoxin B, (AFB,), B(a)P, PhIP and N-ni-
trosodimethylamine (NDMA), increased nitrosamine metabolism, and reduced the
number of sister chromatid exchanges induced by mutagens, depending on the kind
of mutagen. Brassica vegetables reduced AFB,-DNA binding.
The in vivo alteration of carcinogenicity of various agents by isot~ocyanates,
indoles and brassica vegetables is presented in Table 7. Both isothiocyanates,
indoles and brassicas reduced tumor formation in miscellaneous tissues, test systems
and using various carcinogenic compounds. When added in vitro to human
erythroleukemic KS62 cells various glucosinolates, viz. sinigrin, gluconapin, pro-
goitrin, epi-progoitrin, sinalbin, glucotropaeolin, glucoerucin, glucocheirolin and
glucoraphenin, had no effect on tumor cell growth [194]. Their hydrolysis-delved
products, produced using myrosinase, showed an inhibition of human ery-
throleukemic K562 cefl growth, which was most evident for the hydrolysis products,
from sinigrin, glucotropaeolin, glucoerucin and glucocheirolin [1941. The hydroly-
sis-derived products from glucoraphenin decreased the growth of several other
tumor ceils, viz. FL (murine erythroleukemic cells), Jurkat (human T-lymphoid
cells), HeLa (human cervix carcinoma cells), H9 (human T-lymphoid cells) and
H3-TI-1 cells (obtained by transfection of HeLa with a LTR-HIV-l-CAT plasmid)
[194]. 2-Phenethyl isothiocyanate (PEITC) and its mercapturic acid pathway
metabolites inhibited the growth of HL60 (human leukaemia 60) cells in vitro. The
adduct with L-cysteine was the most potent inhibitor [195].
Table 8 shows the effects of indoles and brassica vegetables given to hnmans. I3C
induced estradiol 2-hydroxylase, a phase
I
enzyme. Brassica vegetables, mainly
Brussels sprouts, reduced oxidative DNA damage, increased glutathione S-trans-
ferase levels, and induced also estradiol 2-hydroxylase. When 11 smokers ate 56.8
-
7/25/2019 Chemico-Biological Interactions Volume 103 Issue 2 1997 [Doi 10.1016%2Fs0009-2797%2896%2903745-3] Dorett
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