Chemico-Biological Interactions Volume 103 Issue 2 1997 [Doi 10.1016%2Fs0009-2797%2896%2903745-3] Dorette T.H. Verhoeven; Hans Verhagen; R.alexandra Goldbohm; Pie -- A Review of Mechanisms

7/25/2019 Chemico-Biological Interactions Volume 103 Issue 2 1997 [Doi 10.1016%2Fs0009-2797%2896%2903745-3] Dorett

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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


2/51

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


3/51

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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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


6/51

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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



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Documents

Chemico-Biological Interactions Volume 103 Issue 2 1997 [Doi 10.1016%2Fs0009-2797%2896%2903745-3] Dorette T.H. Verhoeven; Hans Verhagen; R.alexandra Goldbohm; Pie -- A Review of Mechanisms