[CANCER RESEARCH 58, 46.12-4639. October 15, 1998]

Mechanism of Differential Potencies of Isothiocyanates as Inducers ofAnticarcinogenic Phase 2 Enzymes1

Yuesheng Zhang and Paul Talalay2

Depiirtmt'iil of Pharmacolog\ tinti Molecular Sciences. The Johns Hopkins Universit\ School of Medicine. Baltimore. Maryland 21205

ABSTRACT

Isothiocyanates occur in many edible plants and are consumed insubstantial quantities by humans. A number of isothiocyanates blockchemical carcinogenesis in a variety of animal models by inhibiting Phase1 enzymes involved in carcinogen activation and by inducing Phase 2enzymes that accelerate the inactivation of carcinogens. There are largebut unexplained potency differences among individual isothiocyanates.When murine hepatoma (Hepa Iclc?) and several other cell lines wereexposed to low concentrations (1-5 JIM) of certain isothiocyanates, the

intracellular isothiocyanate/dithiocarbamate concentrations (measured bycyclocondensalion with 1,2-benzenedithiol) rose rapidly (30 min at 37°C)

to very high levels (e.g., 800-900 /nn. The intracellular accumulation ofisothiocyanates/dithiocarbamates was temperature, structure, and gluta-

thione dependent and could not be saturated under experimentally achievable conditions. When murine hepatoma cells were exposed to nine isothiocyanates (5 /i\i for 24 h at 37°C) that differed considerably in

structure and Phase 2 enzyme inducer potencies, the intracellular concentrations (area under curve) correlated closely and linearly with theirpotencies as inducers of the Phase 2 enzymes: NAD(P)H:quinone reducíaseand glutathione S-transferases. Isothiocyanates that did not accumu

late to high levels were not inducers. These observations suggest stronglythat induction of Phase 2 enzymes depends on intracellular levels ofisothiocyanates/dithiocarbamates. Depletion of glutathione by treatmentof Hepa cells with buthionine sulfoximine increased the inducer potenciesof several isothiocyanates but could not be directly related to changes inintracellular isothiocyanate/dithiocarbamate concentrations, suggestingthat glutathione may play several roles in the induction process.

INTRODUCTION

Isothiocyanates have engendered widespread interest in a numberof laboratories during the last three decades because of their ability toinhibit tumor formation in several animal models (1, 2). A range ofisothiocyanates with quite different structures are chemoprotectorsagainst tumors evoked by a number of chemical carcinogens in avariety of animal organs, suggesting that there is a common mechanism underlying these effects. It is generally accepted that inductionof cancer-protective Phase 2 enzymes (e.g.. glutathione transferases,

quinone reducíase,and glucuronosyltransferases) and/or inhibition ofcarcinogen-activating Phase 1 enzymes play a major role in protection(3-7). Recently, several isothiocyanates have also been shown to

induce apoptosis in several cell lines, including HeLa cells (8, 9).Because isothiocyanates are present in edible plants, particularly inthe family Cruciferae, and are widely consumed by humans in considerable quantities (up to 100 mg daily; Refs. 10-12), they offer

considerable promise as chemoprotectors against cancer in humans. A

Received 4/8/98; accepted 8/18/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1These studies were supported by Program-Project Grant POI CA 44530 from the

National Cancer Institute. Department of Health and Human Services and by grants fromthe American Institute for Cancer Research and the Cancer Research Foundation ofAmerica. A preliminary account of this work was presented at the eighty-ninth annualmeeting of the American Association for Cancer Research. March 28-April 1, 1998. New

Orleans. LA (Proc. Am. Assoc. Cancer Res.. 39: 267, 1998).2 To whom requests for reprints should be addressed, at Pharmacology and Molecular

Sciences. Johns Hopkins School of Medicine. 725 North Wolfe Street. WBSB. Room 406.Baltimore. MD21205.

better understanding of the absorption, distribution, metabolism, andmechanism of action of this class of compounds is essential to deviserational strategies for chemoprotection.

Exposure of cultured cells and animal tissues to isothiocyanatesleads to coordinate induction of several Phase 2 enzymes, includingglutathione transferases, quinone reducíase, and glucuronosyltransferases (13-15). From our initial studies, it was clear that there were

considerable differences in the potencies of various isothiocyanates ininducing Phase 2 enzymes. Because structure/activity analyses failedto explain the large differences in inducer potencies of various isothiocyanates (13, 14), we undertook a more detailed analysis of themetabolic disposition of these compounds. Our studies were initiallyhampered by lack of a simple and rapid method to measure isothiocyanates and their metabolites in cells and tissues. We thereforedeveloped a method to measure unlabeled isothiocyanates, which hasplayed a crucial role in analyzing the effects of different compoundsof this class. In this report we provide evidence that: (a) certainisothiocyanates are accumulated to very high levels in several celllines; (b) the degree of accumulation correlates with their ability toinduce Phase 2 enzymes; and (c1)cellular GSH3 levels influence their

potencies as inducers of Phase 2 enzymes.

MATERIALS AND METHODS

Materials

Sulforaphane [I-isothiocyanato-4-(methylsulfinyl)butane], erysolin [1-iso-thiocyanato-4-(methylsulfonyl (butane). e.ro-Z-acetyl-í-.ro-ó-norbornyl-NCS,and t'rtí/o-l-acetyl-e.TO-ó-norbornyl-NCS were gifts of Professor G. H. Posner(Department of Chemistry, The Johns Hopkins University). [uC]Phenethyl-

NCS was a gift of Dr. F-L. Chung (American Health Foundation, Valhalla,

NY). All other isothiocyanates were obtained commercially from Aldrich(Milwaukee. WI) or Trans World Chemicals (Rockville, MD), and many weredistilled before use. 1,2-Benzenedithiol and buthionine (/?,S)-sulfoximine werealso from Aldrich. and the former was distilled before use. 'H2O and [I4C-

carboxylated]inulin were obtained from Amersham (Arlington Heights, IL).Dibutyl phthalate, diisononyl phthalate, and ethyl glutathione were purchasedfrom Sigma Chemical Co. (St. Louis, MO).

Cell Culture

All cells were cultured in 5% CO2 (unless otherwise stated) at 37°Cin a

humidified incubator. Media and special growth conditions were as follows:for Hepa Iclc? cells, a-MEM plus 10% fetal bovine serum (Life Technologies, Inc.) treated previously with 1% charcoal at 55°Cfor 90 min and then

filtered (16); for LNCaP cells, a human prostate cell line, RPMI 1640 mediumcontaining 1 mM L-glutamine and 5% fetal bovine serum (17); for MSI cells,

an endothelial cell line from mouse pancreas, DMEM plus 5% fetal bovineserum (18); for 3T3-L1 cells, a fibroblast cell line from mouse, DMEM plus

10% fetal bovine serum in a 10% CO2 atmosphere (19); and for L6 cells, a ratskeletal muscle myoblast line, DMEM with 4 mM L-glutamine, 1.5 g/liter of

sodium bicarbonate, 4.5 g/liter of glucose, 1 mM sodium pyruvate, and 10%fetal bovine serum (20).

3 The abbreviations used are: GSH, glutathione; AUC. area under the curve; QR,NAD(P)H:quinone reducíase; BSO. buthionine-(R.5)-sulfoximine; GST, glutathione S-

transferase; CD. concentration of an inducer required under specified conditions to doublethe QR activity in murine hepatoma Hepa Iclc7 cells grown in 96-well microtiter plates:R-NCS, generic designation of isothiocyanates with differing R group.

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POTENCIES OF ISOTHIOCYANATES AS F.NZYMI: INW'OiKS

Measurement of Intracellular Accumulation of Isothiocyanates

Cell Exposure to Isothiocyanates and Preparation of Cell Lysates. Forthese studies. 7X10' cells were plated in a 10-cm Petri dish with 10 ml ofmedium. After 3 days, the cell number reached 4-5 X 10* cells. This cell

density was selected because intracellular accumulations of isothiocyanates didnot significantly deplete the isothiocyanate concentration in the medium, yetthe accumulated compounds could be accurately measured by the cyclocondensation assay on cells obtained from a single plate. The cell monolayers werethen either directly exposed to specified concentrations of isothiocyanates in10 ml of medium, or suspensions were prepared by treating the cells with 1ml/plate of 0.05% trypsin and then mixing with 9 ml of medium beforeexposure. For monolayers. at the end of exposure to the isothiocyanates. thecells in each plate were harvested by scraping with a razor blade into —¿�0.5ml

of medium and rapidly transferred to a 1.5-ml Eppendorf tube containing 0.8ml of a water-immiscible oil mixture (1 volume ot'diisononyl phthalate and 2

volumes of dibutyl phthalate: Ref. 21). For all cell suspension experimentscarried out at 0—t°C.the entire suspension (10 mil was transferred to a 15-ml

tube containing 2 ml of the same oils but mixed at a 1:1 ratio. The density ofthe oil mixture was such that only cells moved through the oil layer uponcentrifugaron at 4°Cfor 15 s at 14,000 rpm for Eppendorf tubes or for 2 min

at 3000 rpm for 15-ml tubes. After centrifugation. the medium and oil were

aspirated, and the tube tip containing the cell pellet was cut off with a razorblade. The entire pellet was resuspended in 160 fil of H,O and mixed with 20¡JL\of 10 mM ß-mercaptoethanol and 20 ¡j.\of \ck aqueous AMauryl sarcosine.The mixture was then incubated at 37°Cfor l h with occasional Vortex mixing

to ensure complete cell lysis.Quantitation of Isothiocyanates and Dithiocarbamates in Cell Lysates

by the Cyclocondensation Assay. As shown in Fig. 1. isothiocyanates anddithiocarbamates react quantitatively with 1.2-benzenedithiol by a cyclocondensation reaction to give rise to 1.3-benzodithiole-2-thione. which can bequantified spectroscopically after simple isocratic reverse-phase high-perfor

mance liquid chromatography (22. 23). Dithiocarbamates are the conjugationproducts of isothiocyanates with thiol compounds, and intracellular isothiocyanates may exist partly as dithiocarbamates (e.g., GSH conjugates). Theassay was modified for very small quantities as follows. Five or 10 /¿I°'thecell lysate were added to give a 200-fil final volume of reaction solution

containing: 50% methanol (v/v). 25 mM potassium phosphate (pH 8.5). 20 mM1.2-benzenedithiol dissolved in methanol. in a 250-¿¿Iglass insert housed in a4-ml glass vial with compression spring. H-style vial cap. and self-sealingseptum (Waters. Milford. MA). The vial was heated to 65°Cfor 2 h. After the

incubation, the solution was centrifuged at low speed to sediment the celldebris, and the supernatant fraction was injected into a reverse phase high-performance liquid chromatography column for quantitation of 1.3-benzodi-thiole-2-thione (23). This assay can detect as little as a few pmol of isothio

cyanates or dithiocarbamates.Quantitation of Cell Volume. The measured isothiocyanate content of

each cell lysate was converted to intracellular concentration based on theintracellular fluid volume, which was determined from a calibration curverelating intracellular volume to the protein content of the cell lysate. Theprotein content of each cell lysate was measured by the bicinchoninic acidassay (24). For volume determination. 2-10 X IO6 Hepa Iclc? cells were

16

12o_3O> 8

TO3

U2

0.5 1 1.5 2.5

Protein in Cell Lysates (mg)

Fig. 2. Standard curve relating the protein content in cell lysates to the intrucelluluraqueous volume. The cell volume was determined by ^H^O uptake hy cells and correctedfor trapped extracellular water hy means of ¡nulin['4C]carbonytìc acid measurement, asdescribed in "Materials and Methods."

incubated in 5 ml of medium containing both "H,O and | '4C]inulin for 5 min

at 37°C.which was sufficient to establish uptake equilibrium. The specificactivities of the incubation medium were 6.7 X H)4 cpm/ml for 'H:O and3.1 x H)4 cpin/ml for |'4C]inulin (1.75 ftg/ml). The cells were centrifuged

through a layer of the oil mixture, the cell pellet was retrieved as describedabove, and lysed in 200 /¿Iof 0.1 % N-lauryl sarcosine. and the radioactivity ofboth 'H and 14C and the protein content in the lysates were determined. Inulincrosses the cell membrane extremely slowly, and the amount of I4C measured

in the lysates was assumed to arise from inulin trapped in the water of theextracellular space. Assuming that the extracellular contamination of inulinwas the same as that of water, the intracellular volume was calculated as thetotal 'H contained in the lysate minus the total [4C contained in the lysate. The

contribution of |'4C]inulin to the total radioactivity in the lysate was —¿�8%.A

linear relation (r = 0.99) between cell volume and protein content of the lysate

was thus established (Fig. 2).Calculation of the AUC of Intracellular Isothiocyanate Concentrations.

Intracellular isothiocyanate concentrations were measured at multiple timepoints, and the data were processed by an interactive computer program todetermine the AUC. The computer program (LACRAN method) was developed by Rocci and Jusko (25) and further modified by Ediss and Tarn (26).

Induction of Phase 2 Enzymes

Phase 2 enzyme induction was measured in Hepa Iclc7 cells that weregrown either in 96-well microtiter plates or 10-cm plastic Petri dishes. The

microtiter plate assay was performed as described ( 16. 27); only induction ofQR was measured by this method. Briefly. lO.(XX)cells were cultured in eachmicrotiter well with 200 /M!of medium for 24 h and then exposed to a seriesof concentrations of the inducer in 150 n\ of medium/well for 24 or 48 h. Theinducers were all dissolved in acetonitrile (final concentration of acetonitrile.0.1% by volume). When BSO was used to deplete cellular GSH. the cells weregrown for 24 h in the microtiter plate, treated with 1(X)J^MBSO in 150 /il of

Isothiocyanate

Fig. I. The cyelocondensation reaction of isothiocyanates (above) and dithiocarhamales (below) wiih 1.2-

benzenedithiol.

R-N-C=S H

S

y>1.2-Benzenedithiol

R-NH-C

Dithiocarbamate

4633

R-NH2

R-NH2 + Rf-SH

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POTENCIES OF ISOTHIOCYANATES AS ENZYME INDUCERS

5,000

3,000

2,000

o _^

itc

§" 1,000o ,

II 500

li 3°°S* 200

100

Measured with C

iMeasured with thecyclocondensationassay

40 100

Extracellular Phenethyl-NCS (uM)

Fig. 3. Intracellular accumulation of [l4C]phenethyl-NCS measured by radioactivityand the cyclocondensation assay. For each assay, 4-5 X IO6 Hepa Iclc? cells suspendedin 10 ml of medium were exposed to specified concentrations of [l4C]phenethyl-NCS at37°Cfor 30 min. The cells were then quickly separated from the medium by centrifugation

through oil and lysed; the content of phenethyl-NCS (or its derivatives) was measured by

both radioactivity and the cyclocondensation assay and expressed as intracellular concentrations, based on volume.

medium/well for 24 h, and finally exposed to the test inducer in fresh medium(150 ^I/well) for another 24 h in the presence of 100 fj.MBSO.

The 10-cm Petri dish assay was performed as follows. Cells (5 X 10*) were

grown in each plate with 10 ml of medium for 48 h and then exposed to the testinducer in 10 ml of fresh medium/plate for another 24 h. After exposure, thecells were washed with 10 ml of Dulbecco's buffer, harvested by scraping, and

centrifuged at 2000 rpm for 5 min at 4°C.The buffer was removed, the cell

pellet was resuspended in 0.25 ml of fresh buffer and homogenized in a glasshomogenizer (Kontes, Vineland. NJ). and (he homogenates were centrifuged at10.(XK)rpm for 30 min at 4°C.The resultant supernatant fraction was assayed

tor both QR and GST activities, and the amount of protein was determined(28). QR and GST activities were measured as described (13, 14).

Determination of Cellular GSH

Cellular GSH was measured by a fluorometric assay (29). Briefly, celllysates were diluted with 0.1 M potassium phosphate buffer, pH 8.0, andproteins were precipitated with trichloroacetic acid. A 100-/J.1sample was thenmixed with I(X)¡j.\of o-phthaldialdehyde (10 mg in 10ml of methanol) and 1.8

ml of 0.1 M potassium phosphate buffer (pH 8.0) containing 5 mM EDTA. Thesolution was incubated at room temperature for 10 min, and formation ofGSH-phthaldialdehyde conjugate was then measured by a luminescence spectrometer (Perkin-Elmer Model LS50). The conjugate was excited at a wave

length of 350 nm, and the fluorescence emission was measured at 420 nm.

Statistics

To measure intracellular concentrations of isothiocyanates or GSH, twoseparate experiments, each of which was assayed in duplicate, were carriedout. To measure cellular enzyme activities (QR and GST), three separateexperiments, each of which was assayed in duplicate or quadruplicate, werecarried out. Each value represents the mean of these analyses. Replicateanalyses routinely had SEs of <10% of the mean.

RESULTS

Validation of the Cyclocondensation Assay for Measurement ofIntracellular Isothiocyanate and Dithiocarbamate Concentrations. The cyclocondensation assay is the only available method forthe accurate and highly sensitive quantitation of isothiocyanates thatdoes not require their radiolabeling. The procedure for measuringcellular accumulation of isothiocyanates is described under "Materialsand Methods." Because we did not know whether isothiocyanates

might be metabolized in cells to products that were not detected by thecyclocondensation assay, we measured the cellular uptake of twoisothiocyanates that could be quantitated by other means: radioactive

phenethyl-NCS (C6H5-CH,-14CH2-NCS) and fluorescein-NCS. Insuspensions of Hepa Iclc? cells exposed to [14C]phenethyl-NCS in

concentrations up to 100 /AMin the medium, radioactivity accumulated up to the equivalent of more than 3 mM in just 30 min at 37°C.

As shown in Fig. 3, 80-90% of phenethyl-NCS accumulated in cells

(measured by radioactivity) was also measured by the cyclocondensation assay, and this percentage remained constant as the intracellularconcentration increased from 200 /UMto more than 3 mM. Hence, themajority of cellular phenethyl-NCS is measurable by the cyclocondensation assay. Fluorescein-NCS accumulated to much lesser extent

in Hepa Iclc7 cells, but nearly all of the isothiocyanates in the cells(measured by fluorescence) were also detected by the cyclocondensation assay. Moreover, when several other isothiocyanates wereincubated with Hepa IclcV cell lysates for 1 h at 37°C,nearly all of

the isothiocyanate added was detected by the cyclocondensation assay(data not shown). Therefore, these experiments established that thecyclocondensation assay provides a valid measurement of intracellular isothiocyanates or their dithiocarbamate metabolites. Isothiocyanates are metabolized to dithiocarbamates, i.e., GSH derivatives. Thecyclocondensation assay cannot distinguish between these two classesof compounds but measures both species quantitatively (23). However, for reasons of simplicity, we refer to the total amounts ofisothiocyanates/dithiocarbamates in cells measured by the cyclocondensation assay as intracellular concentrations of "isothiocyanates," as

indicated in Figs. 3-10.

Isothiocyanates Accumulate to Very Different Concentrationsin Hepa Iclc? Cells. The accumulation of phenethyl-NCS in Hepa

Iclc7 cells in the above experiment (Fig. 3) was surprisingly high.Therefore, we used the cyclocondensation assay to examine the accumulation of other isothiocyanates. Hepa Iclc7 cells in monolayerswere exposed to 5 /XMconcentrations of sulforaphane, benzyl-NCS,phenethyl-NCS, phenyl-NCS, or a-naphthyl-NCS. As shown in Fig.4, sulforaphane and benzyl-NCS accumulated very rapidly to very

high concentrations in Hepa Iclc? cells, reaching levels of 280 and370 /LIM,respectively, in just 30 min, whereas the highest intracellularlevels of of phenethyl-NCS attained were more modest. Although thecellular concentration of benzyl-NCS began to decrease rapidly after

30 min of incubation, that of sulforaphane continued to accumulate for6 h, reaching 350 /H.M,and the subsequent decrease was relativelyslow. In contrast, very little phenyl-NCS or a-naphthyl-NCS accumulated, with maximal intracellular concentrations rising <5-fold,

and none were detected after 3 h of incubation. Thus, it became clearthat, whereas some isothiocyanates accumulated rapidly to very highconcentrations in Hepa Iclc7 cells, the degree of the maximal accumulation apparently depended on the specific isothiocyanate. Our

400

(min) Exposure Time

Fig. 4. Time course of accumulation of five isothiocyanates in Hepa Iclc? cells. Foreach assay, 4-5 X IO6 cells in monolayers in 10-cm dishes were exposed to 5 JIM

benzyl-NCS (•),sulforaphane (D). phenethyl-NCS (•),a-naphthyl-NCS (O), or phenyl-NCS (X) in 10 ml of medium at 37°Cfor specified times. At the end of exposure, cells

were quickly harvested, separated from medium, and lysed. and the content of isothiocyanates (or their derivatives) in the lysate was measured by the cyclocondensation assay.

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POTENCIES OF ISOTHIOCYANATES AS ENZYME INDl'CKRS

1,000

Hepa1c1c7

LNCaP MS1 3T3-L1

Fig. 5. Accumulation of sulforaphane and benzyl-NCS in five cell lines. Suspensionsof Hepa Iclc?, LNCaP. MSI, 3T3-L1, and L6 cells were each incuhaled with 5 »IMsulforaphane (•)or henzyl-NCS (D) for 30 min at 37°C,and the content of isothiocya-

nales (or their derivatives) in the lysate was measured by the cyclocondensation assay.

preliminary experiments suggested that isothiocyanate accumulationand removal are likely to be a continuous process, and cellularconcentration of an isothiocyanate at a given time during the exposureperiod may depend on its degradation rate in the medium (data notshown).

Isothiocyanates Accumulate in Various Types of Cells. We nextexamined whether the rapid and concentrative uptake of isothiocyanates was unique to Hepa Iclc? cells. We focused on four cell lines:mouse pancreatic endothelial cells (MSI); mouse fibroblast cells(3T3-L1); rat skeletal muscle myoblast cells (L6); and human prostatecancer cells (LNCaP). Both sulforaphane and benzyl-NCS rapidly

accumulated in all four cell lines, although the degree of accumulationdiffered. As shown in Fig. 5, when these cells were exposed insuspensions to either 5 JJ.Msulforaphane or 5 ¿IMbenzyl-NCS in themedium at 37°Cfor 30 min, the intracellular concentrations attained

were 40-180 times higher than the initial extracellular isothiocyanate

concentration. Hence, these experiments established that cellular accumulation of isothiocyanates is likely to be a common property ofmany cell types from a variety of species. Interestingly, in all cell linestested, the maximal accumulation of benzyl-NCS was always higher

than that of sulforaphane.The Phase 2 Enzyme Inducer Potencies of Isothiocyanates Cor

relate with Their Levels of Accumulation in Cells. The aboveexperiments not only showed that various isothiocyanates accumulated to very different levels in Hepa Iclc? cells but also suggestedthat the accumulations are correlated with the inducer potencies ofthese compounds. Thus, the rank order of their potencies in inducingQR (induced for 48 h in the microtiter assay), i.e., sulforaphane (CD,0.2 ¿IM)> benzyl-NCS (CD, 1.5 JAM)> phenethyl-NCS (CD, 4.5UM), whereas phenyl-NCS and a-naphthyl-NCS were not inducers,

matched the rank order of their cellular accumulations. Furthermore,other isothiocyanates, including fluorescein-NCS and p-tolyl-NCS,

which did not accumulate to high levels in Hepa Iclc7 cells, also didnot induce QR. To clarify to what degree the inducer potencies ofisothiocyanates depended on their cellular accumulations, we examined accumulation of nine isothiocyanates with very different structures and inducer potencies (Fig. 6A). Hepa Iclc7 cells in monolayerswere exposed to each compound at 5 /XMin the medium for 24 h. Thecells were then harvested, and the activities of QR and GST wereassayed. Another set of cells were exposed to the same isothiocyanates at the same concentration but for different time periods. Thecells were harvested at 1, 3, 6, 12, or 24 h, at which times theintracellular concentration of each isothiocyanate was determined bythe cyclocondensation assay, and the area under the concentrationwith respect to time curve (AUC) of the intracellular isothiocyanateswas computed. As shown in Fig. 6B, the maximal accumulation and

the time course of accumulation varied widely among these isothio-cyanates. Moreover, both QR and GST were induced by these iso-

thiocyanates but also to very different levels. For example, whereas 5juM sulforaphane raised QR activity —¿�10-fold,no induction wasobserved upon exposure to 5 JUMphenyl-NCS. Although the level of

GST induction by isothiocyanates was uniformly much lower thanthat of QR, there was a very close correlation between the inductionof the two enzymes, i.e., an isothiocyanate that was a more potentinducer of QR was also a more potent inducer of GST. When thespecific activities of both QR and GST of cells treated with isothiocyanates were divided by the values of control cells and comparedwith the AUC of the intracellular concentrations of these isothiocyanates, a remarkable correlation was found (Fig. 6C); cells that accumulated higher total concentrations of isothiocyanates also induced

(a)CH3—s—(CH2)„—NCS

(b) CH3-S-<CH2)4-NCS

o

H2—NCS

B

I OL

l °U

sC

400

300

200

100

01 3 6 12 18Exposure Time (h)

24

1 12

è 8

¡i•¿�53-

oO

coir

QR

2000 4000 6000AUC of Intracellular R-NCS Within 24 h Exposure

(h x \jM)

Fig. 6. Relationship of intracellular concentration of ¡solhiocyanates and inducerpotency of Phase 2 enzymes. For measuring accumulation of nine isothiocyanates in HepaIclc? cell monolayers, cells were exposed to 5 /J.Mconcentrations of each of the followingisothiocyanates at 37°Cfor the times specified, and isothiocyanates in the cell lysates weremeasured by the cyclocondensation assay: sulforaphane (a. •¿�);erysolin (h. D); endo-2-acetyl-e.ra-6-norbornyl-NCS (c, •¿�);ejr«-2-acetyl-f.vo-6-norbomyl-NCS (d, O); benzyl-NCS (e. A); phenethyl-NCS tf. A); «o-norbornyl-NCS (jc. T); cyclooctyl-NCS (h. V); orphenyl-NCS (/. +). The AUC was then calculated for each isothiocyanate for the 24-h

period. For measuring enzyme activity, monolayers were similarly exposed to the isothiocyanates for 24 h, after which the cells were harvested, and the specific activities ofQR and GST were measured as described in "Materials and Methods." A, the chemical

structures of nine isothiocyanates: R. intracellular accumulation of the nine isothiocyanates: C, relation of isothiocyanale accumulation (AUC) at 24 h and QR and GSTactivities (ratio of specific activities of treated cells over control cells).

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POTENCIES OF ISOTHKX'YANATKS AS EN/YME INWCERS

1,200

10 20Exposure Time (min)

30

Fig. 7. Effect of temperature on uptake of sulforaphane hy Hepa Iclc? cells. Cellsuspensions were exposed to 5 ¡IMsulforaphane at 4°Cor 26°Cfor the indicated time, and

the accumulated sulloraphanc was measured in lysates by the cyclocondensation assayand expressed as intracellular concentration, based on cell volume.

120 150

Initial Extracellular Sulforaphane (nM)

Fig. 8. Concentration-dependent uptake of sulforaphane hy Hepa Iclc7 cells at 4°C.Cell suspensions (4-5 X IO6 cells) were exposed to 0.8.4. 20. 80, or 150 JIM sulforaphaneal 4°C for 1, 3, 6, 9, and 12 min. After exposure, the content of sulforaphane (or

derivatives) in the lysale was measured hy Ihe cyclocondensation assay, and the initialrales of uptake of sulforaphane by the cells were calculated.

higher levels of QR and GST, regardless of the structural differencesof these compounds. A similar correlation was also observed whencells were exposed to these isothiocyanates for only 12 h, but theinduced levels of both enzymes were lower than those after 24 h oftreatment (data not shown). Therefore, we conclude that inducerpotencies of isothiocyanates in Hepa IclcV cells depend largely, if notentirely, on their intracellular accumulation.

Initial Velocity of Uptake of Isothiocyanates into Hepa Iclc?.The rates of accumulation of the active isothiocyanates were extremely rapid at 37°C,effectively precluding determination of the

initial uptake kinetics. When suspensions of Hepa Iclc? cells wereexposed to 5 /J.M sulforaphane, the initial rate of accumulation ofsulforaphane was reduced by at least 4-fold when the temperature waslowered from 26°Cto 4°C(Fig. 7). Similar temperature effects were

also observed for several other isothiocyanates tested (data notshown). At 4°C,the initial uptake of several isothiocyanates in Hepa

Iclc7 cells was linear with time for at least the first 12 min and wasdirectly proportional to extracellular concentration. For sulforaphane,the uptake rate was concentration-dependent up to more than 150 ¡JLM

but could not be accurately determined at higher concentrations (Fig.8). The initial rate of sulforaphane accumulation in these cells (2.7/xM/min for each 1 piM extracellular concentration of sulforaphane)was not saturable under experimentally attainable conditions. Moreover, the initial rates of uptake of allyl- and benzyl-NCS (at l /J.Minthe medium) over the first 12 min at 4°Cwere also linear and were

considerably faster than those of sulforaphane. Allyl-NCS accumulated 9 times (22 /iM/min) and benzyl-NCS 18 times (46 ju,M/min).

respectively, more rapidly than sulforaphane (results not shown),emphasizing that uptake rates are also dependent on structure. Notably, the initial uptake rate of sulforaphane was the slowest of the threecompounds, yet the ultimate accumulation of sulforaphane was thehighest (Fig. 4).

Isothiocyanate Accumulation in Cells Is Influenced by CellularGSH Levels. How the cell achieves such rapid and high cellularaccumulation of isothiocyanates is not yet clear, but the accumulationis apparently influenced by cellular GSH concentrations. There aretwo reasons for examining the relation between intracellular GSHlevels and the uptake of isothiocyanates into cells: (a) isothiocyanatesreact rapidly with GSH to form dithiocarbamates both nonenzymati-cally and enzymatically (30-32); and (b) GSH levels have beenimplicated in the regulation of Phase 2 enzymes (33-36). Hepa Iclc7

cells normally contain 5 HIMGSH, but this level is reduced to 0.8 mMwhen the cells are treated for 24 h with 100 ¿J.MBSO, which is widelyused to deplete GSH because it inhibits -y-glutamylcysteine synthetase

(37). When cells, with or without BSO treatment, were exposed insuspension for 30 min to either sulforaphane or benzyl-NCS at 0.9,9.1, or 91 fjiMin the medium at 37°C,the intracellular accumulation

of both compounds was drastically reduced in the BSO-treated cells

(Fig. 9). Without BSO treatment, cellular sulforaphane concentrationsreached 0.2, 1.2, or 6 mM, respectively, and with treatment the cellularconcentrations reached only 0.07. 0.3, or 1.5 mM, respectively, a3-4-fold decrease. Cellular accumulation of benzyl-NCS was simi

larly affected. Thus, depletion of cellular GSH reduced the uptake ofisothiocyanates. Surprisingly, over longer periods of exposure (1-24

h) to these two isothiocyanates, although the cellular accumulation ofsulforaphane was continuously depressed in BSO-treated cells, thecellular accumulation of benzyl-NCS was unexpectedly higher inBSO-treated cells than that in untreated cells (Fig. 10). Two additionalisothiocyanates, allyl-NCS and phenethyl-NCS were also examined.

Although their accumulations in Hepa Iclc7 cells were similarlyaffected by GSH levels in short-term exposure (<1 h), their accumu

lations were not significantly affected by cellular GSH level when thecells were exposed for longer periods of time ( 1-24 h; Fig. 10). Thus,

the effect of GSH on cellular accumulation is isothiocyanate specific.Although most isothiocyanates are known to react readily with GSH,to what degree dithiocarbamate formation is directly responsible foraccumulation of these compounds is not known.

| 6

u u

Extracellular R-NCS Concentration (uM)

Fig. 9. Effect of cellular GSH on accumulation of sulforaphane and ben/.yl-NCS inHepa Iclc7 cells. Monolayers (2-3 X 10* cells) were exposed to medium with (D) orwithout (•) 100 JIM BSO for 24 h at 37°C. The cells were then trypsini/ed, and

suspensions were exposed to the ¡sothiocyanates for an additional 30 min, again with orwithout 100 /AM BSO. and cell lysates were analy/ed for isothiocyanates accumulated hythe cyclocondensation assay. The GSH conteníof cells treated with or without BSO forthe first 24 h was also determined. Untreated cells. 5 mM: BSO-treated cells. 0.8 mM.

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l'Oli NOES OF ISOTHIOCYANATES AS ENZYME INDUCFRS

Fig. 10. Time course of isothiocyanate accumulation in Hepa Iclc?cells with or without GSH depletion by BSO. The experiment wassimilar to that in Fig. 9, except that cells were exposed in monolayers toextracellular concentrations of 5 fiM isothiocyanates at 37°Cfor 1, 3. 6,

12. or 24 h: at the end of each incubation, cells were harvested, and thecellular contents of isothiocyanates (or derivatives) were determined bythe cyclocondensalion assay. Cells treated with BSO (•)and withoutBSO (D) are shown.

300

O

1Ifu ¿ 200

3

100

Sulforaphane

Benzyl-NCS Allyl-NCS Phenethyl-NCS

1 3 6 12 24 1 3 6 12 24 1 3 6 12

Incubation Time (h)

24 1 3 6 12 24

Cellular GSH Levels Modulate Induction of Phase 2 Enzymesby Isothiocyanates. The observation that cellular GSH level affectedaccumulation of several isothiocyanates in Hepa Iclc7 cells led us toexamine whether the potency of these compounds as inducers ofPhase 2 enzymes was similarly affected. Hepa Iclc7 cells grown inmicrotiter plate wells were treated with 100 ¿IMBSO for 24 h and thenexposed in fresh medium to an isothiocyanate for an additional 24 hin the presence of 100 p.MBSO. Parallel controls were not treated withBSO. Four isothiocyanates, sulforaphane. benzyl-NCS, allyl-NCS,and phenethyl-NCS, were examined because their cellular concentrations had been measured in both BSO-treated and control cells.Surprisingly, in BSO-treated cells, all four isothiocyanates showedhigher potencies in inducing QR and were from 1.3-fold (sulforaphane) to as much as 3.8-fold (allyl-NCS) more potent than they were

in control cells (Table 1). As discussed above, depletion of GSH byBSO in Hepa Iclc? cells results in uniformly lower accumulation ofall four isothiocyanates after short-term exposure (30 min). Overlonger periods of exposure (1-24 h), sulforaphane accumulation inBSO-treated cells was continuously depressed, whereas allyl-NCSand phenethyl-NCS accumulations were similar to those in untreatedcells. Benzyl-NCS accumulation was —¿�40%higher than that in un

treated cells. Clearly, the enhanced inducer potency of these fourisothiocyanates in GSH-depleted cells cannot be attributed to changes

in their cellular accumulations. Changes in GSH levels alone did notappear to affect the enzyme levels, because neither treatment of cellswith 100 JAMBSO for 48 h nor treatment with 5 mM ethyl-GSH for24 h resulted in any significant elevation of QR activity. Ethyl-GSH

is transported intact into cells and raises GSH levels (38). Thus, ourexperiments clearly suggest that the normally high cellular GSH

Table I Effect of cellular GSH depletion hy BSO on intlucer potenciesof isotliioi-yanates

QR inducer activity (CD.^IM)"IsothiocyanateSulforaphane

Bcn/yl-NCSAllyl-NCSPhenethyl-NCSWith

BSO0.46

0.882.200.93Without

BSO0.58

2.598.302.40

" Hepa Iclc7 cells were grown in microtiter plate wells for 24 h. treated with 100 U.M

BSO for 24 h. and then exposed to serial dilutions of the indicated isothiocyanates in newmedium for an additional 24 h in the presence of 100 fj.M BSO. QR activity was thenassayed, and the CD values were calculated. Three types of controls were included: (ti)cells were treated with 100 /¿MBSO only during the last 48 h of growth (change ofmedium at 24 h); (h) cells were treated with 5 imi ethyl-GSH only in the last 24 h of theexperiment: and (r) cells received no additions of BSO. ethyl-GSH. or isothiocyanates.BSO and ethyl-GSH produced no significant (<30%) increases in QR-specific activity.

levels appear to lower the inducer potencies of these isothiocyanates.Although the exact reasons are unknown, it is possible that freeisothiocyanates are the important signals for enzyme induction, andthe concentration of free isothiocyanates in cells depends inversely oncellular GSH concentrations because isothiocyanates are readily andreversibly converted to dithiocarbamates.

DISCUSSION

Isothiocyanates have been shown to block chemical carcinogenesisagainst a diverse group of carcinogens in many target tissues ofseveral animal species (1, 2). They also inhibit Phase 1 enzymes(cytochromes P-450) involved in carcinogen activation and induce

Phase 2 enzymes that accelerate cellular disposal of activated carcinogens in a variety of cells and animal tissues (1, 2, 15). Blockingcarcinogen activation and/or detoxifying activated carcinogens arerecognized to be effective strategies in reducing cancer risk in animals(3-7). However, the relative importance of inhibition of Phase 1

enzymes and induction of Phase 2 enzymes by isothiocyanates hasbeen difficult to assess. Efforts to relate the structures of isothiocyanates to their potencies have been only partially successful (13, 14,39-42). Nevertheless, the anticarcinogenic and the enzyme-regulat

ing activities of isothiocyanates are clearly concentration dependent.For these reasons, the previously unrecognized rapid and concentra-

tive accumulation of isothiocyanate enzyme inducers in cell lines is ofspecial interest. Studies in Hepa Iclc7 cells clearly indicated that theinitial rates of uptake of isothiocyanates are structure-related and are

influenced by cellular GSH levels. Because isothiocyanates can undergo conjugation with GSH, nonenzymatically and enzymatically, itis possible that GSH conjugation with isothiocyanates is involved intheir concentrative cellular accumulation, and this may be furtherenhanced by GSTs. In fact, the initial uptake rates of benzyl-NCS.allyl-NCS, and sulforaphane in Hepa Iclc7 cells appear to be corre

lated with the second order rate constants for their reaction with GSH(30). However, these observations are in conflict with the fact thatother isothiocyanates. such as phenyl-NCS and a-naphthyl-NCS,

which are rather reactive with GSH were not accumulated. Moreover,the effect of GSH levels on cellular accumulation of isothiocyanatesseems unpredictable when cells were exposed to these compounds forlonger periods of time (> 1 h). For example, although the accumulation of sulforaphane was continuously depressed during the entireexposure period, benzyl-NCS accumulated ultimately to a higher levelthan that in GSH-undepleted cells. These results raised the possibilitythat GSH may be involved in both the uptake and export of isothio-

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POTENCIES OF ISOTHIOCYANATES AS ENZYME INDUCERS

cyanates, and depletion of cellular GSH may decrease the export ofcellular isothiocyanates or their metabolites. In this connection, it iswell known that elimination of many drugs in cells by drug transporters (e.g., multiple resistance pumps) requires GSH (43, 44).

To understand how cellular concentrations of isothiocyanates mightaffect their biological potencies, we focused on the induction of twoPhase 2 enzymes. Undoubtedly, the rapid and concentrative accumulation of isothiocyanates will also increase the inhibition of Phase 1enzymes. The finding that inducer potencies of isothiocyanates inHepa Iclc? cells depend closely on the degree to which they areaccumulated in cells is significant in that it may provide an understanding of the structure-activity relationship and point to ways to

design more effective chemoprotective agents. It is very likely thatstrategies that increase the levels and prolong accumulation of isothiocyanates in cells will also increase their Phase 2 enzyme inducerpotencies. Moreover, because these isothiocyanates differ widely intheir chemical structures, our results also suggest that the specificstructures of isothiocyanates may have little importance in enzymeinduction once these compounds have entered cells.

Our experiments also clearly show that decreasing cellular GSHlevels in Hepu IclcV cells increases the inducer potencies of isothiocyanates. This finding is consistent with several previous observations. For example, depletion of cellular GSH also increased thepotency of compounds such as mercury chloride in inducing QR inHepa IclcV cells (36). In a transient gene expression assay in HepG2cells, lowering cellular GSH levels with BSO stimulated the responseof the electrophile responsive element to terf-butylhydroquinone and

other compounds (33). Moreover, isothiocyanates that reacted morerapidly with GSH were found to be less potent as inhibitors of lungtumor formation by 4-(methylnitrosamino)-l-(3-pyridyl)-l-butane

(41). This finding may help us to control isothiocyanate inducerpotency and to understand the mechanism of enzyme induction byisothiocyanates and possibly other compounds. However, furtherstudy is needed to clarify the inhibitory mechanism of GSH.

In summary, by following cellular accumulation of isothiocyanateswith this cyclocondensation assay, we have discovered that manyisothiocyanates are rapidly accumulated to high concentrations in anumber of cultured mammalian cells and that the magnitude of suchaccumulation is related to the specific isothiocyanates, incubationtemperature, and cellular GSH content. We also found that the Phase2 enzyme inducer potencies of isothiocyanates correlate with theirlevels of accumulation in Hepa Iclc7 cells, thus suggesting a plausible mechanism for the differential potencies of these compounds ininducing Phase 2 enzymes. Moreover, our experiments also indicatedthat cellular GSH may negatively modulate the potencies of isothiocyanates as inducers of Phase 2 enzymes.

ACKNOWLEDGMENTS

We thank Kristina L. Wade. Alhena Dinkova-Kostova, and Katherine K.

Stephenson for much valuable assistance and W. David Holtzclaw and PeterMaloney for helpful discussions.

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1998;58:4632-4639. Cancer Res   Yuesheng Zhang and Paul Talalay  Inducers of Anticarcinogenic Phase 2 EnzymesMechanism of Differential Potencies of Isothiocyanates as

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