Chapter 2 , and

Although and ing phenolics, vitamins and other sul- Major dietary sources of compounds derived from crucif- fur-containing compounds. specific isothiocyanates erous appear to be struc- Isothiocyanates are familiar to turally unrelated, they are both derived many of us as they are largely respon- and indoles from glucosinolates. These -con- sible for the characteristic hot, pun- The concentrations and forms of glu- taining are found in the gent flavours of salad vegetables such cosinolates in commonly eaten crucif- order Capparales, which includes the as , cress, leaves and erous vegetables are summarized in large family . The glu- , and contribute to the Table 6. Several surveys of variations cosinolate molecule comprises a com- flavour of cooked cruciferous vegeta- in content between mon glucone moiety and a variable bles. Indoles do not contribute to the Brass/ca have been reported, aglucone side-chain, derived from one flavour of crucifers. Many of the bio- for example for B. rapa (Carlson et al., of seven amino acids. Isothiocyanates logical and chemico-physical proper- 1987a; Hill et al., 1987) and B. oler- are formed by the degradation of glu- ties of these compounds (e.g. volatility acea (Carlson et al., 1987b; Kushad et cosinolates that have either aliphatic and lipophilicity) are determined by al., 1999), and these are also summa- or aromatic side-chains (Figures 4 & the chemical structure of the isothio- rized in Table 6. While over 90 different 5), derived (mainly) from cyanate side-chain (Figure 5; Appen- isothiocyanate (ITC) glucosinolates and , respectively, dix 1), which, in turn, is determined by have been described, only about six whereas indoles are derived from glu- the structure of the parent glucosino- occur frequently in the diet (Figure 5; cosinolates with indolyl side-chains late molecule. The bioactivity of these Table 6). (4-methyl- derived from (Figures 6 & compounds to humans is thus influ- sulfinylbutyl-ITC) is obtained predomi- 7) (Rosa et al., 1997). Cruciferous enced by a combination of the chemi- nantly from but may also be vegetables also contain other phyto- cal structure of the isothiocyanates obtained from rocket (Eruca sat/va) chemicals that have been associated and their concentration. and some cultivars of and with potential health benefits, includ- Brussels sprouts. 2-Propenyl (allyl')-,

S— SH R-C R—C" R—NCS N N Isothiocyanate os03 os03

Glucosinolate Unstable intermediate

Figure 4 General scheme of the hydrolyss of glucosinolates to isothiocyarates

13 IARC Handbooks of Prevention Volume 9: , isothiocyanates and ind&es

3-butenyl- and 3-methylsulfinyipropyl- ITC (iberin') are derived from con- sumption of certain cultivars of B. 0/er- R NCS acea. Propenyl-ITC can also be obtained from leafy mustard vegeta- bles. 3-Butenyl- and 4-pentenyl-TC R Chemical name: are obtained from leafy B. rapa crops CH3 —S —CH 2 CH2 CH2 3 Methylthiopropyl: and particularly . Phenyl-ethyl-ITC is obtained from watercress and to a lesser extent from 2 —CH2 -- CH3 —S OH2 CH2 CH 4-Methyithiobutyl: rockets root crops such as and . 6-Methylsulfinyihexyl- ITC is derived from the Japanese OH3 S -0H2 —CH2 CH2 3-Mothylsulfinylpropyl ('iberin): broccoli, il some Brussels sprouts and cabbages (Wasabia japonica). Benzyl- o ITC is relatively rare in the diet, as it is obtained from Lepidium (cress) 0113 —S—CH2 CH2 CH2 OH2 - 4-Methy sulfinylbutyl ('su foraphane'): species. In addition to cruciferous veg- broccoli 0 etables, isothiocyanates may also be derived from mustard condiments, CH3 S —CH2 -- 6-Methylsu finylbexyl: wassbi which are eaten mainly in western o countries. Hydroxy-benzyl-ITC is found in 'English' mustard and is obtained OH3 -S—CH3]7 7 Metbylsulfiriylheptyl: watercress il from the seeds of white mustard, L) Sinapis alba. Allyl- (2-propenyl-) and 3- butenyl-ITCs are found in French and CH, S tCH2s - 8-Methylsulfinylocty watercress American mustards and are derived 0 from the seeds of brown (or Indian) 2-Properly ('ally '): mustards, cabbages, mustard, B. juncea. Allyl-ITC is also Ci —CH CH2 - some Brussels sprouts derived from the grated roots of horse- radish (Armoracia rusticana), which is

OH2 OH -CH2 —CH2 3 Buteryl: Brussels sprouts, Chinese used as a condiment in western cabbages, pak-chol, greens Europe and North America. Cruciferous vegetables may also be CH2 —CH CH2 0H2 —CH2-- 4-Penterryl: Chinese cabbages pak cho: processed by pickling or fermenting, as in sauerkraut in western Europe and in SH—CH2 OH2 CH2 -0H2 ---- 4-Mercaptobutyl: rockets some forms of kimchi in the Korean peninsula. Consumption of these prod- ucts is an additional source of isothio- cyanates in the diet. C H2 Berizyl: Lepid:um cress Six different indolyl glucosinolates have been identified, but only four have been found in cruciferous crops (Figure 7), and, of these, only two,

2-Phenethyl. watercress, , turnips indolylmethyl ('') and 1- ICH2 CH2 methoxy-3-indolylmethyl ('neogluco- brassicin'), are found frequently. The types of indole glucosinolates vary from species to species (Table 6), and there is wide variation in the concen- isothiocyanates and side-chain structures (R) found in commonly Figure 5 Structures of trations of specific indole glucosino- eaten cruciferous vegetables lates. In broccoli, the concentration of

14 Glucosinolates, isothiocyanates and indoles

Table 6. Main glucosinolate side-chains of isothiocyanates occurring in cruciferous vegetables

Cruciferous vegetable Glucosinolate side-chain Content (!LmolIlOO g fresh weight)

Broccoli (B, oleracea var. ita/ica)a 3-Meihylsulfinylpropy 0-330 4-Methylsulfinylbutyl 29-190 Indole-3-methyl 42-100 1 -Methoxyindole-3-methyl 2-18

Cabbage (B. oleracea var. capitata)L 2-Propeny! 4-160 3 Mathylsulfinylpropyl 5-280 Indolylmethyl 9-200

Brussels sprouts (B. oleracea var. gemmifera)G 2-Propenyl 4-390 3-Methylsulfinylpropyl 0-150 3-Buterryl 0-220 4-Methylsulfinylbutyl 0-23 2-1--Iydroxy-3-bulenyl 1-300 lndcle-3-methyl 45-470 1 -Methoxyindole-3-methyl 2-34

Cauliflower (B. oleracea var. botrytis) 2-Propenyl 1-160 3-MethylsuJfinylpropyl 0-330 4-Methylsulfiny butyl 2-190 lndole-3-methyl 14-160 1 -Methoxyindole-3-methyl 1-32

Kale (B. oleracea var. acephafa) 2-Propenyl 62-200 3-Methylsulfinylpropyl 0-50 3-But eryl 6-38 2-Hydroxy-3-butenyl 17 130 I ndole-3-methyl 67-160

Rape (B. rapa, including Chinese cabbage 3-Butenyl 38-290 and turnip tops)d 4-Fertenyl 20-150

All the crops can also contain low concentrations of other glucosinolates (Carlson et al., 1987a,b; Hill et al., 1987; Rosa et aL, 1997; Kushad et al., 1999). The concentrations must be interpreted with care, as different methods of analyses were used in the different stud- ies, and the concentrations are affected by a wide range of environmental factors. In addition, generalizations cannot be made about the glucosinolate content of cabbages and Brussels sprouts as there are large genetic differences between cultivars. The values quoted ndicate the greatest range of values in the reviews referred to above. a All broccoli cultivars produce predominantly 4-netbylsulfinylbutyl glucosinolate, the precursor of sulforaphane. b Some cabbage varieties produce the elongated glucosinolates, 3-butenyl and 4-methylsulfirylbutyl; some also produce their precur- sors, 3-meihylthiopropy and 4-nethylthiobutyl. Surpr singly, the glucosinolate content of Brussels sprouts vanes: some cultivars produce high concentrations of 2-propenyl, while others produce high concentrations of 3-butenyl and 2-bydroxy-3-buteny . Recently developed cu tivars either have low concentrations of these glucosinolates or moderate concentrations of 3-methy sulfinyipropyl or 4-methyl sultinylbutyl. Cultivars tend to have both 3-butenyl and 4 pentenyl (and hydroxylated forms) or just 3-butenyl

15 IARC Handbooks of Cancer Prevention Volume 9 Cruciferous vegetables, isothiocyanates and indoles

/S —Glucose OH2 N N2 0s03- R

Irdolyl glucosinolate R = H or 00H3

Myrosinase

C

R H20 pH 3-4 Ascorbic acid j Y

H1OHO f CH2-CN CH2 0H (F 0H 0H CR2 +s R R +SCN N

lndolyl-3-carbinOl Ascorbigen lrdolyI3-acetonitriIe

Figure 6 Degradation pathways of indole gluco&nolates

neoglucobrassicin was found in some indole glucosinolate is glucobrassicin, side-chain and partially determines studies to be even higher than that of and 4- met hoxy-3-Lndolylrnethyl glu- the overall amount; glucobrassicin (yang et at., 2001; cosinolate is present at half the con- abiotic and biotic environmental Vallejo et al., 2003a). In Brussels centration (Ciska et al., 2000). factors, which can influence the sprouts, neoglucobrassicin occurs at a Four factors interact to determine overall amount of isothiocyanates much lower concentration than gluco- the exposure of the gastrointestinal produced by the ; brassicin (Kushad et al., 1999; Ciska tract to isothiocyanates and indoles: post-harvest storage, processing et al., 2000), while 4-hydroxy-3- the biochemical genetics of the and ; and indolylmethyl glucosinolate and gluco- biosynthesis of glucosinolates and the thioglucosidase activity of the brassicin are found at similar concen- isothiocyanates within the crop intestinal microbial flora. trations in (Kushad et al., plant, which determines the chemi- 1999). In white cabbage, the main cal structure of the glucosinolate

16 Glucosinolates, isothiocyanates and indoles

and phenylaanine, about 50% CH2 3-Indolylmethyl (ylucobrassicin) of all known glucosinolates, only from the Brassicaceae and the Cappara- H ceac, are synthesized from elongated forms of methionine. Methionine can be elongated by the addition of one to nine methyl groups, but most taxa CH2 1-Mthoxy-3-indolyImethyI (neoglucobrassicin) within the Brassicaceae have only a restricted chain length, which can be N divided into three classes: short 00H3 chains', from the addition of one, two, three or (rarely) four methyl groups to methionine, such as found in crops; long chains', from the addition OH of five or six methyl groups; and 'very long chains'from the addition of seven, CH2 4-Hydroxy-3-indolylmethyl eight or nine methyl groups. Biochemical studies in which [14Clacetate and 14C-labelled amino H acids were administered, with subse- quent analysis of the labelled glucosi- nolates, suggest that the

00H3 elongation is similar to that which occurs during synthesis of 4-Methoxy-3-indolyrmethyl from 2-keto-3-methylbutanoic acid and acetyl coenzyme A (Strassman & Ceci, 1963; Graser et al., 2000). The amino H acid is transaminated to produce an a-keto acid, followed by condensation Figure 7 Structures of the four indolyl glucosinolates found in cruciferous crops with acetyl coenzyme A, isomerization involving a shift in the hydroxyl group, and oxidative decarboxylation to result Biochemical genetics of Biosynthesis of these glucosino- in an elongated keto acid which is glucosinolate biosynthesis lates and their resultant isothio- transaminated to form the elongated cyanates or indole derivatives can be amino acid. It is likely that the elon- separated into four parts: amino acid gated keto acid can undergo further Side-chain structures chain elongation, core glucosinolate condensation with acetyl coenzyme A Glucosinolates with more than 115 synthesis, chain modification and pro- to result in multiple chain elongations. side-chain structures derived from one duction of isothiocyanates and indoles Molecular genetics studies in of eight amino acids (, valine, (Figure 8). The side-chain structure of Ara bidopsis thallana resulted in identi- leucine, , phenylalanine, glucosinolates in any one genotype is fication of a small gene family, desig- methionine, and tryptophan) under strict genetic control, whereas nated MAM (methylthionlky/malate), have been described. These glucosi- the overall level is determined by inter- which catalyse condensation of the nolates all form isothiocyanates upon actions between genotype and envi- keto acids with acetyl coenzyme A hydrolysis, except for those derived ronment. Only an overview is provided (Kroymann etal., 2001). Differences in from tryptophane, which release here; for further details, see Halkier expression and allelic variants of the indoles. The glucosinolate contents of and Du (1997) and Mithen (2001). MAM genes appear to explain the vari- wild and cultivated have been ety of chain lengths observed, surveyed by IJaxenbichler et at. (1991) Amino acid elongation although the details remain to be clari- and Fahey et al. (2001). While some glucosinolates are synthe- fied (Kroymann et al., 2003). Thus, sized from chain-elongated forms of differences in the chain length of

17 IARC Handbooks of Cancer Prevention Volume 9: Cruciferous vegetables, isothiocyanates and indoles

by cytochrome P450 (GYP) monooxy- genases of the CYP79 family. The best Amino acid characterized system is conversion of tyrosine and phenylalanine to their cor- Chain elongation (MAM genes) responding (Du et al., 1995; Bak et al., 1999) as precursors of ben- Elongated amino acid zyl and hydroxybenzyl glucosinolates. In Arabidopsis, oximes from chain- CYP79 elongated methionine homologues are formed by the action of the GYP 79Ff and GYP 79F2 genes. The first of these Oximes can catalyse many homologues of chain-elongated forms of methionine, 1 CYP83, glycosylation, whereas the latter can catalyse only sulfotransferase long-chain methionine homologues. In Arabidopsis, conversion of the G I ucosi nol ate to the thiohydroximate is proba- bly catalysed by another cytochome P450, CYP83131 (Hansen et al., 2001). Oxidation, desatura tien, hydroxylation etc. As thiohydroximates would be toxic to (2-0x0 glutarate-dependent dioxygenases) + plant tissue, they are effectively detox- ified by glycosylation by a soluble Chain modification U D PG :thiohydroximate S-gl ucosyl- transferase to produce a desulfoglu- I Degradation (plant , epithiospeci- fier protein and microbial thioglucosidases) cosinolate (Reed et al. 1993; Groot- wassink et al., 1994: Guo & Poulton, 1994). This is sulfated by a soluble 3'- Degradation products, phosphoadenosine 5'-phosphosulfate: isothiocyanates, etc. desulfoglucosinolate sultotransferase (Jain et al., 1990), to produce the glu- Mammalian metabolism of isothiocyanates I (glutathione-S-transferases and enzymes cosinolate. T of mercapturic acid pathway) Chain modification N-Acetylcysti ne—isoth iocyanate The chain structure of glucosinolates conjugates derived from any amino acid can undergo modification. As for other aspects of glucosinolates, most atten- tion has been paid to methionine- derived compounds. Excretion After the biosynthesis of methylth- ioalkyl glucosinolates from methionine, the side-chain can undergo various Figure 8 Biosynthesis and metabolism of glucosinolates and isothiocyanates modifications. Initial oxidation results in Indole glucosiralate degradation products are not metabolized via the nrercapturic acid pathway methylsulfinylalkyl, which accumulates in broccoli. Additional oxidation results methionine-derived glucosinolates in Core g I ucosi nolate biosynthesis in methylsulfonylalkyl glucosinolates, cruciferous crops is probably due to The first step in glucosinolate biosyn- which accumulate in certain non-culti- allelic differences in MAM genes that thesis is conversion of the amino acid vated cruciferous vegetables. Remo- result in different specificities of their to an oxime. Recent studies, largely val of the methylsulfinyl group and enzymic products for elongated keto with A. thaliana, suggest that all amino desaturation result in alkenyl glucosi- acids. acid—oxime conversions are catalysed nolates. Alternatively, hydroxylation

18 Glucosinolates, isothiocyanates and indoles results in 3-hydroxypropyl or 4-hydroxy- butenyl glucosinolate (') neously rearranges to several prod- butyl glucosinolate (Mithen et al., owing to the goitrogenic achvity of its ucts. Most frequently, it undergoes a 1995; Giamoustaris & Mithen, 1996; major hydrolysis product, 5-vinyloxazo- Lessen rearrangement to produce an Hall et al., 2001). There is considerable lidine-2-thione (), when incorpo- isothiocyanate. If the glucosinolate scope for variation in this part of the rated into animal feed. Thus, glucosi- side-chain contains a double bond, pathway. For example, 4-methyl- nolate concentrations have been and in the presence of an epithiospec- sulfinylbutenyl glucosinolate, found reduced from greater than 80 ftmol/g ifier protein (see below), the isothio- exclusively in Raphanus, probably to less that 5 pmol/g in spring rape and cyanate may rearrange to produce an results from desaturation of the corre- to less than 15 imol/g in winter rape. epithionitrile (Figure 9a). If the glucosi- sponding methylsulfinylbutyl glucosi- This was shown to be a complex nolate lacks a double bond, the sulfur nolate but without associated methyl- genetic trait determined by alleles at may be lost and a formed. sulfinyltransferase activity. The alkenyl four or five quantitative trait loci (Figure 9b). A few glucosinolates have glucosinolates 3-butenyl and 4-pen- (Toroser et al., 1995; Howell et al., been shown to produce , tenyl can undergo 3-hydroxylation, with 2003). Studies in broccoli have although the mechanism by which this important consequences for the nature focused on increasing the concentra- occurs is unknown. Aglucones from of the hydrolytic products. While the tion of methylsulfinylalkyl glucosino- glucosinolates which contain j3-hydroxy- details of the biochemical processes lates. Surveys of glucosinolates in lated side-chains, such as progoitrin and the genes that determine these existing broccoli cultivars showed that found in the seeds of oilseed rape and modifications are not fully understood, the concentration of 4-methylsu Ifinyl- in the edible parts of Chinese cabbage studies with Arabidopsis suggest that butyl glucosinolates varied from 0.8 and Brussels sprouts, spontaneously many of the processes are due to the mmollg dry weight to 21.7 mmol!g cyclize to form the corresponding activity of 2-oxogluturate-dependent (Kushad et al., 1999). In an effort to -2-thiones (Figure Oc). dioxygenases (Hall et al., 2001). As for obtain higher concentrations, Faulkner side-chain elongation, genetic control et al. (1998) used B. viiosa and other Glucosinolate hydrolysis and of the side-chain structure is very wild members of the nine B. oleracea formation of indoles strict. Thus, a specific genotype always species complex. Hybrids between The formation of indoles from indole produces the same chain structure, these accessions and broccoli con- glucosinolates has been reviewed by regardless of the environment in which tained concentrations in excess of 80 yang and Dragsted (1996). After tissue it is grown. imol/g dry weight. After several back- disruption, myrosinase cleaves the ndole glucosinolates derived from crosses, isothiocyanate-enriched broc- thioglucose bond, like other glucosino- tryptophan are also modified by addi- coli was developed from these initial lates, but the resulting isothiocyanate tion of hydroxyl or methoxy groups hybrids (Mithen et al., 2003). Some of is unstable and degrades to the corre- (Figure 7), but the biochemical mecha- the quantitative trait loci important in sponding alcohol (Figure 5). The alco- nisms and genetic basis of these mod- enhancing the glucosinolate concen- hol can condense to 3,3'-diindoyl- ifications are unknown. Although mod- tration of broccoli are in positions in the methane. As with methionine-derived ification of the side-chains of isothio- genome similar to those that deter- glucosinolates, indolyl-3-acetonitrile cyanates, producing glucosinolates, is mine reduction of glucosinolate levels can be formed instead of an unstable under strict genetic control, environ- in rape. Identification of the genes isothiocyanate. The factors that deter- mental factors can affect modification involved in these quantitative trait loci mine the degradation pathway are of indole side-chains. might facilitate a genetic approach to largely unknown. Moreover, in the enhancing glucosinolates in horticul- acidic conditions of the stomach, Genetic basis of glucosinolate tural crops. indole-3-carbinol (and related products accumulation from other indolyl glucosinolates) can While several genes that determine Glucosinolate hydrolysis and for- undergo several condensation reac- side-chain structure have now been mation of isothiocyanates tions, to produce at least 15 different cloned, the genetic control of total glu- When tissue is disrupted, an endoge- oligomeric products (Anderton et al., cosinolate accumulation is still far from nous plant thioglucosidase ('myrosi- 2003). lndole-3-carbinol can also react understood. Most studies have been nase', see below) causes cleavage of with ascorbic acid to form ascorbigen conducted on spring or winter rape (B. the thio—glucose bond to give rise to (Piironen & Virtanen, 1962; Hrncirik et riapus), in which breeders have sought unstable thiohydroximate O-sulfonate al., 2001). As for isothiocyanate-pro- to reduce the content of 2-hydroxy-3- (Figure 4). This aglycone sponta- ducing glucosinolates, if cooking IARC Handbooks of Cancer Prevention Volume 9: Cruciferous vegetables, isothiocyanates and indoles

Epithiospecifier CH2—CH—CH2--CN protein (a) /SGIuc0 CH2 =CH—CH2 C Myrosinase N 0S0 3- CH2=CH—CH2 N=C=S

2-Propenenyl glucosinolate 2-Propenyl (ally!') isothiocyanate (')

E pithiospecifier O (b) protein? 6 —{CH2]4-- CN /SGIu cose .- - il /!ethylsulfinylbutyl nitrile C S [C H 214 Myrosinase N O 0S03- il S [C H2 ]4 N C =S

4-Methyl (sulfinylbutyl glucosinolate 4-Methylsulfinylbutyl isothiocyanate () (sulforaphane)

(c) S —Glucose CH2=CH —HC ----CH 2 CH2 =CH—CHCH — --C ______OH N Myrosinase H 0503

2-Hydroxy-3-butenyl 5-Vinyloxazolidine-2-thione goitrin) glucosinolate (progoitrin')

Figure 9 (a) Production of 2-propenyl- or 1-cyano -2,3-epithiopropane by hydrolysis of 2-propenyl glucosinolates, which are a result of expression of the epithiospecifier protein. (b) Production of 4-methylsulfinylbutyl isothiocyanate or nitrile by hydrolysis of the corre- sponding glucosinolate, as a result of expression of e pith iospecifier-li ke protein. (c) Production of 54nyioxazolidine-2-thione (goitrin) from 2-hydroxy-3-butenyl glucosinolate

denatures myrosinase activitiy, the Myrosinases glucosinolate-containing plants, and intact indole glucosinolate can be Bones and Rossiter (1996) and Rask myrosinase (i.e. thiogl ucosidase) degraded in the colon, in which the et aI. (2000) have reviewed the data on activity has also been detected in contrasting pH may result in production myrosinases, and only a brief sum- insects, fungi and bacteria. In plants, of different degradation products and mary is provided here. Many myrosi- the expression of isozymes varies both their absorption into the bloodstream. nase isozymes have been detected in between species and within the organs

20 Glucosinolates, isothiocyanates and indoles

of the same individual (Lenman et al., cosinolates, such as those with methyl- glucosinolates and larger alterations in 1993). In general, isozymes with sulfinylalkyl side-chains found in broc- the ratio of indolyl:methionine-derived activity towards the glucosinolate chain coli and watercress. In this case, the glucosinolates. In contrast, sulfur fertil- structure appear to have little substrate sulfur from the glucone is lost, as it ization alone had either a small effect specificity, although a myrosinase cannot be re-incorporated into the or no effect at all on the glucosinolate highly specific to epiprogoitrin was degradation product since it lacks a content of broccoli (Vallejo of al., described in Crambe (Bernardi of al., terminal double bond. 2003a,b). It is likely that any effect of 2003). Molecular studies with A. The ratio of isoth iocyanates: n itri les sulfur depends strongly on the thaliana, Brass/ca and Sinapis have varies according to genotype supply. Soil fertilization (combined N shown that myrosinases comprise a (Matusheski et aI., 2003; Mithen et al., and S) was reported to have a signifi- gene family (Xue et al., 1992) with 2003), and this may be related to cant effect on glucosinolates in broc- three subclasses, denoted MA, MS expression of the epithiospecifier pro- coli (Mithen et al., 2003). This study and MC. Members of each of these tein. Glucosinolates in ecotypes of A. also showed that certain genotypes subfamilies occur in A. thaliana (Xue et f/ia/lana vary with respect to the have a greater response than others. al., 1995). As would be expected, relative ratio of isothiocyanate to Nitrogen fertilizer in a hydroponic many more copies occur in the B. epithionitrile or to nitrile they form after system enhanced the concentration of napus genome, because of genome tissue disruption, due partly to allelic glucosinolates in pak-chol (Shattuck & replication. All myrosinases are glyco- variation at a quantitative trait locus Wang, 1994). Water stress induced sylated, and the extent of associated with gene coding for glucosinolates in and rape glycosylation varies between sub- epithiospecifier protein (Lambrix of al., (Bouchereau et al., 1996; Jensen et classes. It is likely that the subdivision 2001). Likewise, while in standard al.•. 1996) and is likely to have a similar of myrosinases will be revised as new cultivars of broccoli hydrolysis results effect in horticultural cruciferous data on sequences become available. in about a 20:80% ratio of isothio- vegetables. The effect of temperature MB and MC myrosinases are linked cyanates:nitriles, isothiocyanate en- has not been studied in detail, and, like with myrosinase-binding proteins and riched broccoli produces about 95% other environmental factors, affects myrosinase-associated proteins, res- isothiocyanates after hydrolysis general plant growth parameters. pectively (Lenman et al., 1990). The (Mithen et al., 2003). Pereira et al. (2002) reported that tem- roles of these proteins are not under- perature affected the glucosinolate con- stood, but they seem to have no signif- centrations in seedlings ('sprouts') of icance for the generation of glucosino- Factors that affect gluco- broccoli, those grown at higher temper- late degradation products after con- sinolate concentrations atures having more glucosinolates than sumption of cruciferous vegetables. those grown at lower temperatures. Not only abiotic factors but also Epithiospecifier protein Abiotic and biotic environmental insects and pathogens induce glucosi- The epithiospecifier protein was first factors nolate accumulation, although the described by Tookey (1973) and was In general terms, while the ratio of indi- effects are mainly on indolyl glucosino- purified from B. napus (Bernardi et al., vidual glucosinolates within a particu- lates as opposed to methionine- 2000; Fee et al., 2000). This protein lar class (e.g. those derived from derived (isothiocyanate-producing) appears to have no inherent enzymatic methionine) is relatively constant and glucosinolates (Birch et al., 1992; activity; it does not interact with glu- is unaffected by environmental factors, Shattuck & Wang, 1994). Jasmonic cosinolates but only with the unstable the total concentration is affected by acid, a signalling molecule involved in thiohyd roximate O-sulfonate after several such factors. Our understand- several plant—insect or —pathogen myrosinase activity. Fee et al. (2000) ing of these is relatively poor, and it is interactions, also induced glucosino- suggested that the mode of action of likely that there are often significant lates (Bodnaryk, 1994). epithiospecifier protein is similar to that genotype—environment interactions. of a cytochrome F450, such as in iron- Soil fertility is probably a major factor. Post-harvest storage, processing dependent epoxidation reactions, Zhao et al. (1994) reported that the and cooking While this protein has been considered sulfur and nitrogen supply affected the Few studies have been conducted on only in the context of the generation of glucosinolate content of rapeseed and the effects of post-harvest events on epithionitriles, it might also be involved described minor alterations in the the concentration of glucosinolates in in the production of nitriles from glu- ratios of individual methioninederived horticultural cruciferous vegetables.

21 IARC Handbooks of Cancer Prevention Volume 9; Cruciferous vegetables, isothiocyanates and indoles

Vallejo et al. (2003c) reported a loss of intact glucosinolates will be consumed Indole glucosinolates are likely to be 70-80% of the total glucosinolate con- which may be degraded to isothio- degraded in the gastrointestinal tract. tent alter a period of simulated cold cyanates or other compounds by the When they were fed to rats, similar bio- storage during transport and storage intestinal microflora, as discussed below. logical effects were seen as when before sale. Storage of cut cabbage Glucobrassicin is chemically and degradation products were fed, sug- reduced methionine-derived glucosi- thermally stable, as no degradation was gesting microbial degradation of intact nolates, as expected, but enhanced observed after 2 h in aqueous media glucosinolates (Bonnesen et ai., 1999). the concentration of indolyl glucosino- with pH values ranging from 2 to 11. lates (Verkerk et at, 2001). Storage of Moreover, glucobrassicin was weakly broccoli under various conditions also degraded by heat treatment (10% after Estimates of dietary intake enhanced indole glucosinolates 1 h) (Chevolleau et al., 1997). of isothiocyanates and (Hansen et al., 1995). indoles As described above, myrosinase- Intestinal microbial flora mediated glucosinolate degradation Cooking glucosinolate-containing veg- results in initial formation of an unsta- etables for more than 2 min inactivates Estimates of dietary intake of isothio- ble intermediate and then in the forma- myrosinase, as described above. cyanates and indoles are clearly tion of an isothiocyanate or a nitrile Several studies have shown that isoth- needed. The existing databases on (Figure 9a and b), the latter due to the iocyanate metabolites can be detected consumption of crucifers (section 1) presence of an epithionitrile specifier in blood and urine after cooked glu- and glucosinolate content (Table 6) or an opithiospecifier nitrile-like protein. cosinolate-containing are eaten, might be considered adequate to pro- When raw Brassica vegetables are although to a lesser extent than when vide a basis for such estimates, but the macerated, about 80% of the methion- raw glucosinolate-containing foods are wide variation in glucosinolate content ne-derived glucosinolates can be con- consumed (Getahun & Chung, 1999; due to the factors described above and verted to nitriles, as opposed to isoth- Conaway et al., 2000). A likely source the difficulty in estimating the conver- iocyanates, the precise ratio of isothio- of isothiocyanates is microbial degra- sion of glucosinolates to isothio- cyanates:nitriles depending on geno- dation of glucosinolates by the intesti- cyanates and indoles during consump- type (Mithen et at., 2003). Both myrosi- nal microflora. Thus, isothiocyanates tion reduce the confidence with which nase and ethiospecific protein can be can be generated by incubation of such estimates can be made. degraded by cooking. Mild cooking, for cooked (i.e. myrosinase denatured) example steaming broccoli florets for watercress with human faeces under Measurement of isothiocyanates less than 3 mm, denatured the epi- anerobic conditions (Getahun & in vegetables before consumption thionitrile specifier protein while leav- Chung, 1999). Rouzaud et al. (2003) Jiao et aL (1998) in Singapore and ing at least some of the endogenous compared urinary isothiocyanate Shapiro et al. (1998) in the USA myrosinase intact, enhancing the metabolites in gnotobiotic rats harbour- reported the isothiocyanate concentra- isothiocyanate content. Further cook- ing whole human faecal flora and in tiens found in vegetables. In each ing, e.g. heating broccoli for 10 or 20 germ-tree rats and found, as expected, case, the vegetables were analysed by min at 50 C, subsequently denatured that after feeding with glucosino-late- condensation with 1 ,2-benzenedithiol myrosinase, preventing any immediate containing food the greatest excretion (see section 3), which results in quan- isothiocyanate production. Thus, the of isothiocyanates was observed when tification of total isothiocyanates. degree of cooking can have significant the plant myrosinase was intact. When Endogenous plant myrosinase was effects on the delivery of isothio- the plant myrosinase was denatured by denatured by cooking before treatment cyanate to the gastrointestinal tract: heat treatment, the amount of isothio- with exogenous myrosinase (Table 7). consumption of raw vegetables may cyanate produced was estimated to be Thus, the estimate of isothiocyanate result in exposure to a significant reduced by > 85% in the gnotobiotic production is probably higher than that amount of nitriles; mild cooking may rats. Some isothiocyanates also which occurs within the gastrointesti- result in consumption of large amounts appeared to be produced in the germ- nal tract, on the assumption that of isothiocyanates, with topical expo- free rats, and some evidence was myrosinase activity is a limiting factor. sure of the upper gastrointestinal tract obtained that the presence of human to biologically significant concentra- faecal flora actually reduced the Intake of indole glucosinolates tions; while more extensive cooking pre- amount of isothiocyanate available for Estimates of the average daily intake vents isothiocyanate formation, and absorption. of indole glucosinolates are based on

22 Glucasinolates, isothiocyanates and indoles

Table 7. Isothiocyanate (ITC) content of cruciferous vegetables in Sones et al. (1984) estimated the Singapore and the USA per-capita intake of indole glucosino- lates in the United Kingdom to be 19.4 Vegetable Mean content (pmol/100 g fresh weight mg/day for glucobrassicin and 3.1 (range)) mg/day for neoglucobrassicin. yang Singapore USA (Shapiro and Dragsted (1996) estimated the per (Jiao et ai., 1998) etaL, 1998) capita intake in Denmark to be 5 mg/day for glucobrassicin and 0.5 Broccoli (B. oleracea var. itailca) 38.6 (10.1-62.0) 13.7 mg/day for neoglucobrasscin, and the Cabbage (B. oleracea var. cap/tata) 27.5 (11.9-62.7) 4.4 respective values in Finland to be 2.5 Cauliflower (B. oleracea var. botrytis) 11.6 (2.7-24.0) mg/day and 0.3 mg/day. Broadbent (B. oleracea var. acephala) 18.2 and Broadbent (1998a) in the USA Kai Ian (B, oleracea var. alboglabra) 15.4 (3.1-35.9) estimated the per capita intake of Turnip (B. raps var. rapa) 1.4 glucobrassicin to be 8.1 mg/day. The Bok chol (B, rapa var. chinensis) 4.9 (2.0-7.5) total intake of indole glucosinolates, Choi sum (B. rape var. parachinensis) 11.1(3.5-23.4) including the two major ones and both Watercress (Nasturtium officinale) 81.3 (17.1-144.6) fresh and cooked cruciferous vegeta- Kai choi (B. juncea ver. rugosa) 71.2 (25.6-1384) bles, was about 22.5 mg/day per capita. The more recent estimates indicate wide variations between coun- the intake of specific cruciferous vary considerably depending on grow- tries, which are due to the use of dif- vegetables and their content of the var- ing conditions, the , storage and ferent methods for estimating intake of ious indole glucosinolates. The esti- preparation conditions, as consider- cruciferous vegetables and indole mates are only approximate, as the able amounts of glucosinolates can be glucosinolates. concentrations of indole glucosinolates lost during storage and processing.

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