4-Methylsulfanyl-3-butenyl isothiocyanate derived from glucoraphasatin is a potent inducer of rat hepatic phase II enzymes and a potential chemopreventive agent

TOXICOKINETICS AND METABOLISM

4-Methylsulfanyl-3-butenyl isothiocyanate derivedfrom glucoraphasatin is a potent inducer of rat hepaticphase II enzymes and a potential chemopreventive agent

Ahmed Faizal Abdull Razis • Gina Rosalinda De Nicola •

Eleonora Pagnotta • Renato Iori • Costas Ioannides

Received: 7 July 2011 / Accepted: 14 September 2011 / Published online: 30 September 2011

� Springer-Verlag 2011

Abstract The objective of this study was to establish

whether the phytochemical glucoraphasatin, a glucosinolate

present in cruciferous vegetables, and its corresponding

isothiocyanate, 4-methylsulfanyl-3-butenyl isothiocyanate,

up-regulate enzymes involved in the detoxification of car-

cinogens and are thus potential chemopreventive agents.

Glucoraphasatin and myrosinase were isolated and purified

from Daikon sprouts and Sinapis alba L., respectively.

Glucoraphasatin (0–10 lM) was incubated for 24 h with

precision-cut rat liver slices in the presence and absence of

myrosinase, the enzyme that converts the glucosinolate to

the isothiocyanate. The intact glucosinolate failed to

influence the O-dealkylations of methoxy- and ethoxyres-

orufin or the apoprotein expression of CYP1 enzymes.

Supplementation with myrosinase led to an increase in the

dealkylation of methoxyresorufin, but only at the highest

concentration of the glucosinolate, and CYP1A2 expres-

sion. In the absence of myrosinase, glucoraphasatin caused

a marked increase in epoxide hydrolase activity at con-

centrations as low as 1 lM paralleled by a rise in the

enzyme protein expression; at the highest concentration

only, a rise was also observed in glucuronosyl transferase

activity, but other phase II enzyme systems were unaf-

fected. Addition of myrosinase to the glucoraphasatin

incubation maintained the rise in epoxide hydrolase and

glucuronosyl transferase activities, further elevated quinone

reductase and glutathione S-transferase activities, and

increased total glutathione concentrations. It is concluded

that at low concentrations, glucoraphasatin, either intact and/

or through the formation of 4-methylsulfanyl-3-butenyl

isothiocyanate, is a potent inducer of hepatic enzymes

involved in the detoxification of chemical carcinogens and

merits further investigation for chemopreventive activity.

Keywords Glucoraphasatin � Isothiocyanates �Glucosinolates � Cruciferous vegetables � Chemoprevention

Introduction

Strong epidemiology has linked the consumption of cru-

ciferous vegetables, such as broccoli, cauliflower, brussel

sprouts and watercress, with low cancer incidence at a

number of sites including lung, bladder, colon, prostate and

breast (Ambrosone et al. 2004; Joseph et al. 2004; Zhao

et al. 2007; Lam et al. 2009; Bhattacharya et al. 2010). This

chemopreventive effect has been attributed to glucosino-

lates that are present at substantial concentrations in these

vegetables (Hayes et al. 2008; Verkerk et al. 2009). When

the vegetable is disturbed, e.g. during harvesting or

chewing, the enzyme myrosinase (b-thioglucoside gluco-

hydrolase) comes into contact with the glucosinolates

converting them to isothiocyanates, which are believed to

mediate the chemopreventive effect of glucosinolates.

Even if myrosinase is deactivated by the cooking process,

microbial myrosinase can release the isothiocyanate from

the glucosinolate in the intestine (Verkerk et al. 2009). In

laboratory studies, isothiocyanates could antagonise the

carcinogenicity of chemical carcinogens of human rele-

vance, including nitrosocompounds and polycyclic aro-

matic hydrocarbons (Zhang 2004).

A. F. Abdull Razis � C. Ioannides (&)

Molecular Toxicology Group, Faculty of Health and Medical

Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK

e-mail: [email protected]

G. R. De Nicola � E. Pagnotta � R. Iori

Agricultural Research Council Industrial Crop Research Center

(CRA-CIN), Via di Corticella, 133, 40129 Bologna, Italy

123

Arch Toxicol (2012) 86:183–194

DOI 10.1007/s00204-011-0750-x

One of the principal mechanisms of action of isothiocya-

nates is to perturb the metabolism of carcinogenic com-

pounds, so as to favour their detoxification, thus limiting the

levels of genotoxic intermediates. Indeed, isothiocyanates

prevent the formation of DNA adducts following exposure to

carcinogens (Dingley et al. 2003; Singletary and MacDonald

2000; Bacon et al. 2003). This may be achieved by sup-

pressing the generation of genotoxic intermediates by inhib-

iting cytochrome P450 enzymes in both the liver, the most

active tissue in xenobiotic metabolism, as well as the lung,

one of the target tissues of isothiocyanates, and/or stimulate

the phase II enzymes that are responsible for their detoxifi-

cation. For example, exposure of rats to low doses of sulfo-

raphane, erucin and phenethyl isothiocyanate, simulating

levels of dietary intake, altered hepatic and pulmonary

activities of enzyme systems involved in the metabolism of

chemical carcinogens (Yoxall et al. 2005; Hanlon et al. 2008a;

Konsue and Ioannides 2008). Moreover, in in vitro studies

utilising precision-cut liver and lung slices, isothiocyanates

such as erucin, R,S-sulforaphane and phenethyl isothiocya-

nate, modulated carcinogen-metabolising enzymes at con-

centrations as low as 1 lM (Konsue and Ioannides 2010a, b;

Hanlon et al. 2008b, 2009; Abdull Razis et al. 2011). Mod-

ulation of the same enzymes was also noted when human

precision-cut liver slices were exposed to the same isothio-

cyanates (Konsue and Ioannides 2010a, b; Hanlon et al.

2008b). Recent work emanating from our own laboratory

indicated that the intact glucosinolates have also the potential

to modulate carcinogen-metabolising enzymes in both lung

and liver slices (Abdull Razis et al. 2010a, b, 2011).

4-Methylsulfanyl-3-butenyl glucosinolate (CAS number

28463-23-2, Fig. 1), also referred to as glucoraphasatin,

dehydroerucin or glucodehydroerucin, is a glucosinolate

the most important source of which is Raphanus sativus

(Kaiware Daikon), a white radish that is very extensively

consumed in Japan and increasingly in Europe and North

America (Talalay and Fahey 2001); indeed, radishes are the

only significant sources of glucoraphasatin. The antioxi-

dant activity of glucoraphasatin has already been estab-

lished (Barillari et al. 2006; Papi et al. 2008), and its

potential to enhance quinone reductase activity in HepG2

cells has been reported (Hanlon et al. 2007). As the

potential of isothiocyanates to up-regulate phase II detox-

ification enzyme systems is influenced by the substituent,

and bearing in mind the extensive consumption of this

vegetable, glucoraphasatin has been isolated from Japanese

Daikon sprouts, and a comprehensive study has been

conducted in precision-cut rat liver slices to evaluate the

potential of intact glucoraphasatin and of its corresponding

isothiocyanate, namely 4-methylsulfanyl-3-butenyl isothi-

ocyanate, on cytochromes P450 and phase II detoxification

enzyme systems.

Materials and methods

Ethoxyresorufin, methoxyresorufin, resorufin, NADPH,

1-chloro-2,4-dinitrobenzene (CDNB), cytochrome c perox-

idase-linked anti-rabbit, anti-mouse and anti-goat antibodies

(Sigma Co. Ltd, Poole, Dorset, UK), benzo[a]pyrene 4,5-

epoxide and benzo[a]pyrene 4,5-diol (MidWest Research

Institute, Kansas, USA) and 1-naphthol (Sigma Co. Ltd,

Poole, Dorset, UK), anti-CYP1A1 (AMS Biotechnology,

Abingdon, UK), anti-CYP1A2 (Chemicon International Inc,

Hampshire, UK) and anti-CYP1B1 (BD Biochemicals,

Oxford, UK) were all purchased. Antibodies to human qui-

none reductase, lactate dehydrogenase and b-actin were

obtained from abcam (Cambridge UK); antibody to GST awas from Calbiochem (Lutterworth, UK), and antibodies to

epoxide hydrolase and glucuronosyl transferase (UGT1A6)

as well as donkey anti-goat and goat anti-rabbit antibodies

were from Santa Cruz Biotechnology (California, USA).

Isolation of glucoraphasatin and myrosinase

Glucoraphasatin was isolated from 7-day-old freeze-dried

Japanese Daikon sprouts. The glucoraphasatin content of

Daikon sprouts is 75 lmol/g dry weight (Barillari et al.

2005). The sample was treated with boiling 70% ethanol in

order to quickly deactivate the endogenous myrosinase. The

sprouts were homogenised using an Ultraturrax homogeniser

at medium speed for 15 min, and the resulting homogenate

was centrifuged at 17,7009g for 30 min. The isolation of

glucoraphasatin from the extract was carried out by one-step

anion exchange chromatography, as previously described

(Barillari et al. 2005). The purity of glucoraphasatin was

further improved by gel filtration performed using a XK

26/100 column packed with Sephadex G10 chromatography

media (GE Healthcare, UK), connected to an AKTApurifier

(GE Healthcare, UK). Individual fractions were analysed by

O

OH

HOHO

OH

S

N

S

-O3SO

Glucoraphasatin 4-methylsulfanyl-3-butenyl isothiocyanate

SNCS

Myrosinase PBS, pH 7.4

Glucose + HSO4

-

Fig. 1 Formation of

4-methylsulfanyl-3-butenyl

isothiocyanate from

glucoraphasatin by catalysed

enzymatic hydrolysis reaction at

pH 7.4

184 Arch Toxicol (2012) 86:183–194

123

HPLC, and those containing pure glucosinolate were pooled

and freeze-dried. Glucoraphasatin was characterised by 1H

and 13C NMR spectroscopy, and the absolute purity, esti-

mated by HPLC analysis of the desulpho-derivative,

according to the ISO 9167-1 method (EEC Regulation No

1861/90, 1990), was close to 95% including the minor peak

corresponding to the Z stereoisomer (Montaut et al. 2010).

The enzyme myrosinase (b-thioglucoside glucohydrolase,

EC.3.2.1.147) was isolated from seeds of Sinapis alba L., as

described by Pessina et al. (1990) with some modification.

The specific activity of the stock solution used in the present

study was about 60 U/mg of soluble protein. The enzymatic

activity was 30 U/ml, and the solution was stored at 4�C in

sterile distilled water until use. One myrosinase unit was

defined as the amount of enzyme able to hydrolyse 1 lmol

sinigrin per minute at pH 6.5 and 37�C.

Preparation and culture of precision-cut liver slices

Male Wistar albino rats (200–250 g) were obtained from

Charles River UK Ltd (Margate, Kent, UK). The animals

were housed at 22 ± 2�C, 30–40% relative humidity, in an

alternating 12-h light/dark cycle with light onset at 07.00 h.

Rat liver slices (200–300 lm) were prepared from 8-mm

cylindrical cores using a Krumdieck tissue slicer (Alabama

Research and Development Corporation, Munsford, AL,

USA) as previously described (Hashemi et al. 1999). The

multiwell plate procedure, using 12-well culture plates,

was used to culture the slices in the presence of glucosin-

olate (0–10 lM), or glucosinolate (0–10 lM) plus myro-

sinase (0.018 U). One slice was placed in each well, in

1.5 ml of culture medium. Slices were incubated under

sterile conditions for 24 h on a reciprocating plate shaker

housed in a humidified incubator, at a temperature of 37�C

and under an atmosphere of 95% air/5% CO2. The slices

were initially pre-incubated for 30 min in order to slough

off any dead cells due to slicing. Three different slice

pools, each comprising 10 rat liver slices, were used per

concentration.

Enzyme assays

Following incubation, slices were removed from the cul-

ture medium, homogenised and post-mitochondrial super-

natants prepared and stored at -80�C. When required,

microsomes were isolated by centrifugation (105,000g 9

1 h). The following assays were carried out on isolated

microsomes: the dealkylations of methoxy- (Burke and

Mayer 1983) and ethoxyresorufin (Burke and Mayer 1974),

glucuronosyl transferase (UDP-GT) using 1-naphthol as

substrate (Bock and White 1974) and epoxide hydrolase

(EH) using benzo[a]pyrene 4,5-epoxide (Dansette et al.

1979). The following determinations were carried out in

the cytosolic fraction: quinone reductase (NQO1) using

menadione as substrate (Prohaska and Santamaria 1988),

glutathione S-transferase activity (Habig et al. 1974)

monitored using CDNB as accepting substrate, and total

glutathione levels (Akerboom and Sies 1981). Protein

concentration was determined in both cellular subfractions

using bovine serum albumin as standard (Bradford 1976).

Finally, in order to monitor changes in enzyme protein

expression, Western blot analysis was performed. Hepatic

microsomal or cytosolic proteins from pooled slices were

loaded on to 10% (w/v) SDS–PAGE and then transferred

electrophoretically to Hybond-P polyvinylidene difluoride

membrane. The immunoblot analysis of rat proteins was

carried out by exposure to the primary antibodies followed

by the appropriate peroxidase-labelled secondary antibody.

b-Actin and lactate dehydrogenase were used as the

housekeeping protein to normalise protein loading.

Immunoblots were quantitated by densitometry using the

GeneTool software (Syngene Corporation, Cambridge,

UK), with the control band designated as 100%.

Evaluation of cellular toxicity

LDH release from liver slices into the incubation medium

was used as an index of cytotoxicity and was determined

employing a cytotoxicity detection kit plus (Roche Diag-

nostics, Mannheim, Germany). On completion of a 24-h

incubation, the culture medium was aspirated and the tissue

slices were each homogenised in 1.5 ml of phosphate-

buffered saline (PBS), pH 7.4. The media and homogenates

were centrifuged at 2,000g 9 5 min at 4�C using a bench

centrifuge. Duplicate aliquots (0.1 ml) of each triplicate

incubation were used for analysis according to the manu-

facturer’s instructions.

Statistical evaluation

Results are presented as mean ± standard deviation of 3

pools, each comprising 10 slices. Statistical evaluation was

carried out by one-way ANOVA followed by the Dunnett’s

test.

Results

In order to choose a range of concentrations of the gluco-

sinolate/isothiocyanate that do not compromise the viabil-

ity of rat liver slices, an initial study was conducted, where

slices were exposed for 24 h to glucoraphasatin

(0–25 lM), in the presence and absence of myrosinase, and

toxicity was assessed using the leakage of lactate dehy-

drogenase as biomarker. Glucoraphasatin displayed no

toxicity at the concentrations studied, but in contrast, when

Arch Toxicol (2012) 86:183–194 185

123

myrosinase was added to generate 4-methylsulfanyl-3-

butenyl isothiocyanate, toxicity was evident at the highest

concentrations (Fig. 2).

Incubation of precision-cut rat liver slices with gluco-

raphasatin (0–10 lM), in the absence of myrosinase, failed

to influence the dealkylations of methoxy- and ethoxyres-

orufin (Fig. 3). Similarly, the intact glucosinolate did not

modulate the protein expression of the three enzymes

belonging to the CYP1 family, namely CYP1A1, CYP1A2

and CYP1B1. When, however, the incubation medium was

supplemented with myrosinase, a clear rise in the expres-

sion of CYP1B1/A2 was evident (Fig. 3). Similarly, a

statistically significant increase in the O-demethylation of

methoxyresorufin was observed, but only at the highest

concentration.

Microsomal glucuronosyl transferase activity was ele-

vated only at the highest concentration of glucoraphasatin,

but addition of myrosinase led to significant increases in

activity at lower concentrations, and in both cases, the rise

in activity was paralleled by increased expression of the

protein determined immunologically (Fig. 4). Both the

glucosinolate and, in particular, the generated isothiocya-

nate markedly induced the epoxidation of benzo[a]pyrene

4,5-epoxide; protein expression was similarly enhanced

(Fig. 4).

Exposure of the rat liver slices to glucoraphasatin sup-

pressed quinone reductase activity at the highest concen-

trations studied, but addition of myrosinase to the incubation

medium led to more than doubling of the activity; compa-

rable changes were observed at the protein level (Fig. 5).

When glutathione S-transferase activity was monitored, as in

the case of quinone reductase, activity was impaired at the

higher concentrations of the glucosinolate, but doubled fol-

lowing supplementation of the incubation medium with

myrosinase; a similar picture emerged at the protein level

when GST a levels were determined (Fig. 5). Finally, total

glutathione levels were not altered by the exposure of the

slices to glucoraphasatin, but following addition of myrosi-

nase to the incubation mixture, a significant increase was

observed.

Discussion

A principal mechanism through which a chemical agent

can exert its chemopreventive effect is by preventing the

interaction of chemical carcinogens with DNA. The most

effective means for achieving this is by facilitating the

detoxification of the reactive genotoxic metabolites of

carcinogenic compounds following the up-regulation of the

relevant phase II detoxification enzyme systems by the

chemopreventive agent. Indeed, numerous studies have

established that chemopreventive glucosinolates and their

degradation products, the isothiocyanates, are capable of

elevating the activity of such enzymes in both liver and

lung of rats treated with isothiocyanates, or in vitro in rat

and human slices (Hanlon et al. 2008a, b, 2009; Konsue

and Ioannides 2008; Abdull Razis et al. 2010a, b, 2011).

An alternative mechanism is to impair the activity of

cytochrome P450 enzymes that contribute to the bioacti-

vation of chemical carcinogens to their reactive interme-

diates; however, bearing in mind the role of the

cytochrome P450 enzymes in the metabolism of endoge-

nous substrates, such as hormones, and in the detoxification

of medicinal drugs, such an approach would be more

problematic. In the present study, we have evaluated the

potential of glucoraphasatin, the major glucosinolate in

Daikon, to modulate the activity of both cytochromes P450

Fig. 2 Leakage of LDH following incubation of precision-cut rat

liver slices with glucoraphasatin in the absence and presence of

myrosinase. LDH leakage was measured in rat liver slices incubated

with glucoraphasatin alone (0–25 lM) and in the presence of

myrosinase (0.018 U) for 24 h. Results are presented as mean ± SD

of three slices, each analysed in duplicate. *P \ 0.05; **P \ 0.01;

***P \ 0.001

186 Arch Toxicol (2012) 86:183–194

123

and phase II enzyme systems in rat liver slices and con-

sequently function as a potential dietary chemopreventive

phytochemical. Glucoraphasatin was isolated from Daikon

sprouts, a major source of the glucosinolate. The use of

precision-cut tissue slices allows us to distinguish between

the effects of glucoraphasatin per se from that of the iso-

thiocyanate, generated by the addition of myrosinase.

Intact glucoraphasatin had no significant effect on the

dealkylation of ethoxy- and methoxyresorufin, markers of

the CYP1 enzymes, which is the cytochrome P450 family

most closely linked to the bioactivation of chemical car-

cinogens (Ioannides and Lewis 2004; Zhou et al. 2009). In

concordance, apoprotein levels of CYP1A1, CYP1A2

and CYP1B1 were not altered by the treatment with

glucoraphasatin. These observations contradict our previ-

ous findings, where the glucosinolates, glucoraphanin and

glucoerucin, elevated the activity and apoprotein expres-

sion of these enzymes, in both rat and lung liver slices

(Abdull Razis et al. 2010a, b, 2011), indicating that

individual glucosinolates differ in their potential to perturb

cytochrome P450 enzymes. Supplementation of the

glucoraphasatin incubation system with myrosinase to form

4-methylsulfanyl-3-butenyl isothiocyanate did not impact

on the deethylation of ethoxyresorufin, whereas the

demethylation of methoxyresorufin increased by some

30%, but only at the highest concentration employed; in

order to ascertain whether the increase in activity reflects

increased enzyme availability, immunoblot analyses were

performed on pooled slices, which showed that the

CYP1A2 and CYP1B1 apoprotein levels also rose. It is

pertinent to point out that exposure of glucoraphasatin to

myrosinase at pH 7.4 leads to its complete conversion to

4-methylsulfanyl-3-butenyl isothiocyanate (Papi et al.

2008).

Epoxides have been implicated in the carcinogenicity of

numerous chemical carcinogens including polycyclic

aromatic hydrocarbons, mycotoxins such as aflatoxin

B1, and halogenated aliphatic compounds, such as vinyl

Fig. 3 Effect of glucoraphasatin on CYP1 activities, and expression

in precision-cut rat liver slices. Precision-cut rat liver slices were

incubated with glucoraphasatin (0–10 lM) or glucoraphasatin

(0–10 lM) plus myrosinase (0.018 U) for 24 h; slices were pooled

and microsomes isolated, and the O-dealkylations of methoxy- and

ethoxyresorufin determined. Results are presented as mean ± SD for

three pools of slices, each comprising ten slices. The immunoblot

analysis was carried out by exposure to mouse anti-rat CYP1A1,

mouse anti-rat CYP1A2 or rabbit anti-rat CYP1B1 primary antibodies

followed by the appropriate peroxidase-labelled secondary antibody.

Each lane was loaded with 30 lg of total microsomal protein from

pooled slices. The blots were stripped and re-probed with anti-b-actin

antibody to normalise for differences in protein loading. *P \ 0.05;

**P \ 0.01; ***P \ 0.001

Arch Toxicol (2012) 86:183–194 187

123

Fig. 3 continued

188 Arch Toxicol (2012) 86:183–194

123

chloride (Decker et al. 2009). Two enzymes are effective in

the detoxification of such epoxides, namely the microsomal

epoxide hydrolase and the cytosolic glutathione S-trans-

ferases. Glucoraphasatin failed to increase glutathione

S-transferase activity, but in contrast, it was a potent inducer

of epoxide hydrolase activity, monitored using benzo[a]-

pyrene 4,5-oxide as the model substrate, as well as of pro-

tein expression determined immunologically. Indeed,

activity more than trebled at a concentration of 1 lM of

glucoraphasatin, the lowest concentration employed in the

present studies, the effect being more pronounced than

those elicited by glucoerucin and glucoraphanin under

identical conditions (Abdull Razis et al. 2011). Formation

of 4-methylsulfanyl-3-butenyl isothiocyanate by supple-

mentation of the incubation medium with pure myrosinase

maintained the increase in epoxide hydrolase activity and

expression, indicating that the isothiocyanate, similar to the

parent glucosinolate, up-regulates this enzyme. Moreover,

generation of 4-methylsulfanyl-3-butenyl isothiocyanate

from glucoraphasatin led to a doubling of glutathione

S-transferase, monitored using CDNB as substrate, whose

conjugation with glutathione is catalysed by a number of

glutathione S-transferases isoenzymes (Sherratt and Hayes

2002), and thus provides a general picture of changes in

this activity. An increase in GST a protein levels was

observed in slices exposed to glucoraphasatin plus

myrosinase, indicating that increased enzyme levels

contribute to the rise in activity. Finally, an increase of

50% was also noted in the levels of total glutathione in

the slices exposed to a combination of glucoraphasatin

and myrosinase, similar to the effect of glucoraphanin

and glucoerucin, when incubated under similar condi-

tions in a myrosinase-supplemented medium, thus pre-

venting the levels of the tripeptide from becoming

limiting during glutathione conjugation (Abdull Razis

et al. 2010a, b).

Quinone reductase is an enzyme involved in the

detoxification of quinones by catalysing their two-electron

reduction to hydroquinones, thus preventing them from

undergoing redox cycling generating reactive oxygen spe-

cies. This enzyme was not stimulated by the exposure of rat

liver slices to glucoraphasatin; in fact, activity was

impaired at the highest concentrations of the glucosinolate.

However, supplementation of the glucoraphasatin incuba-

tion system with myrosinase resulted in doubling of the

enzyme activity and a rise in enzyme expression.

Fig. 4 Modulation of UDP-

glucuronosyl transferase and

epoxide hydrolase activities and

expression in precision-cut rat

liver slices by glucoraphasatin.

Precision-cut rat liver slices

were incubated with

glucoraphasatin (0–10 lM) or

glucoraphasatin (0–10 lM) with

myrosinase (0.018 U) for 24 h;

slices were pooled, and

microsomes were isolated, and

epoxide hydrolase and UDP-

glucuronosyl transferase

activities were determined.

Results are presented as

mean ± SD for three pools of

slices, each comprising ten

slices. The immunoblot analysis

was carried out by exposure to

antibodies against epoxide

hydrolase or glucuronosyl

transferase (UGT1A6) followed

by the appropriate peroxidase-

labelled secondary antibody.

Each lane was loaded with

30 lg of total microsomal

protein from pooled slices. The

blots were stripped and

re-probed with anti-b-actin

antibody to normalise for

differences in protein loading.

*P \ 0.05; **P \ 0.01;

***P \ 0.001

Arch Toxicol (2012) 86:183–194 189

123

UDP-glucuronosyl transferases constitute a very impor-

tant phase II microsomal enzyme system participating in the

detoxification of major classes of chemical carcinogens

including polycyclic aromatic hydrocarbons and aromatic

and heterocyclic amines (Bock 2006). Glucoraphasatin

failed to up-regulate this enzyme at the concentrations

studied, commensurate with previous studies using other

glucosinolates (Abdull Razis et al. 2011). The presence of

myrosinase, however, in the incubation mixture led to a

significant increase in activity; a rise in UGT1A6 expres-

sion, the most important isoform in carcinogen metabolism

(Bock 2006), was also noted.

Extrapolation of the present data to the in vivo situa-

tion is hampered by the lack of pharmacokinetic data for

both 4-methylsulfanyl-3-butenyl isothiocyanate and

glucoraphasatin. Pharmacokinetic studies performed in

rats with only single low dietary doses of sulforaphane, a

structurally closely related isothiocyanate, and phenethyl

Fig. 4 continued

190 Arch Toxicol (2012) 86:183–194

123

isothiocyanate revealed that plasma concentrations of

about 0.5–2 lM could be attained, and it is likely that

higher levels can be attained following consumption of

glucosinolate supplements (Hanlon et al. 2008c; Konsue

et al. 2010). However, hepatic intracellular concentrations

may be much higher than those in the plasma; it has been

demonstrated in in vitro studies that peak intracellular

concentrations of isothiocyanates are attained within 3 h

of exposure, and intracellular concentration may be as

much as 200-fold higher than extracellular concentration

(Ye and Zhang 2001; Zhang and Callaway 2002). Con-

sequently, it would not be unreasonable to speculate that

similar plasma/intracellular concentrations of 4-meth-

ylsulfanyl-3-butenyl isothiocyanate may be achieved fol-

lowing intake of dietary doses of glucoraphasatin.

A number of studies have reported that intact glucosino-

lates can be absorbed intact through the intestinal epi-

thelium, and this probably involves carrier-mediated

transport mechanisms (Holst and Williamson 2004).

Indeed, at least in rats and dogs, glucosinolates, such as

glucoraphanin, could be absorbed intact, following oral

intake (Bheemreddy and Jeffery 2007; Cwik et al. 2010),

and could conceivably modulate carcinogen-metabolising

enzyme systems, and thus contribute to the chemopre-

ventive activity associated with the consumption of

cruciferous vegetables.

In conclusion, the present studies have established that

low concentrations of glucoraphasatin, either intact and/or

through its degradation product 4-methylsulfanyl-3-butenyl

isothiocyanate, is a potent inducer of hepatic enzymes

involved in the detoxification of ubiquitous chemical car-

cinogens, such as polycyclic aromatic hydrocarbons, het-

erocyclic amines and mycotoxins; whereas it does not

influence the cytochrome P450 enzymes such as the CYP1

family. It is our view that this glucosinolate merits further

investigation through laboratory studies in animal models

of cancer to evaluate its chemopreventive potential, and

epidemiological studies to establish whether a link exists

Fig. 5 Effect of glucoraphasatin on quinone reductase and glutathi-

one S-transferase activities and expression, and glutathione levels in

precision-cut rat liver slices. Precision-cut rat liver slices were

incubated with glucoraphasatin (0–10 lM) or glucoraphanin

(0–10 lM) plus myrosinase (0.018 U) for 24 h; slices were pooled

and cytosol isolated, and glutathione S-transferase and quinone

transferase activities, as well as glutathione levels, were determined.

Results are presented as mean ± SD for three pools of slices, each

comprising ten slices. The immunoblot analysis was carried out by

exposure to rabbit anti-rat GSTA1-1 or quinone reductase primary

antibodies followed by the appropriate peroxidase-labelled secondary

antibody. Each lane was loaded with 30 lg of total cytosolic protein

from pooled slices. The blots were stripped and re-probed with anti-

LDH antibody to normalise for differences in protein loading.

*P \ 0.05; **P \ 0.01; ***P \ 0.001

Arch Toxicol (2012) 86:183–194 191

123

between this extensively consumed glucosinolate and

cancer incidence.

Acknowledgments The authors thank the Malaysian Government

for funding this work through a PhD award to one of them (AF Abdull

Razis).

Conflict of interest The authors declare that they have no conflict

of interest.

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