Anticarcinogenic Activities of Organic Isothiocyanates: Chemistry and Mechanisms1

Home , Benzyl isothiocyanate, Ellagic acid, Glucobrassicin, Glucotropaeolin

(CANCER RESEARCH (SUPPL.) 54. 1976s-198ls. April 1. 1 Anticarcinogenic Activities of Organic Isothiocyanates: Chemistry and Mechanisms1

Yuesheng Zhang and Paul Talalay Dt'fHirtmi'tti itf I'hurrncicologv und Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Abstract the hydrolysis of glucosinolates by myrosinase. The quantities and nature of these products are variable and appear to be controlled by Organic isothioc.vanates block the production of tumors induced in the chemical nature of the glucosinolate, the pH, and the origin and rodents by diverse carcinogens (polve-velie aromatic hydrocarbons, Â¡i/o multiplicity of the myrosinases. A full discussion of the formation of dyes, ethionine, jV-2-fluorenylacetamide, and nitrosamines). Protection is afforded by a-naphthyl-, ÃŸ-naphthyl-, phenyl-, benzyl-, phenethyl-, and these products is provided by Fenwick et al. (2). Our interest in isothiocyanates stemmed from the observations that other arylalkyl isothioc.vanates against tumor development in liver, lung, mammary gland, forestomach, and esophagus. Many isothiocyanates and several members of this family of substances could block the toxic their glucosinolate precursors (ÃŸ-thioglucoside, A'-hydroxysulfate) occur and neoplastic effects of a wide variety of chemical carcinogens (6). naturally and sometimes abundantly in plants consumed by humans, e.g., Furthermore, many isothiocyanates are monofunctional inducers of cruciferous vegetables. Nevertheless, the possible contributions of isothio Phase 2 enzymes (7, 8), a property that is associated with protection cyanates and glucosinolates to the well recognized protective effects against chemical carcinogenesis (9). Recently, a very potent isothio against cancer of high consumptions of vegetables are unclear. The anti- cyanate inducer (sulforaphane) has been isolated from broccoli (10). carcinogenic effects of isothiocyanates appear to be mediated by tandem These observations point to the potential importance of isothiocya and cooperating mechanisms: (a} suppression of carcinogen activation by nates as chemoprotectors against cancer in humans. cytochromes P-450, probably by a combination of down-regulation of This review focuses on the metabolism of Â¡sothiocyanates and on enzyme levels and direct inhibition of their catalytic activities, which thereby lower the levels of ultimate carcinogens formed; and (In induction the mechanisms of their anticarcinogenic effects. We discuss only of Phase 2 enzymes such as glutathione transferases and NAD(P)H: experiments in which pure isothiocyanates have been used and omit quinone reducÃase, which detoxify any residual electrophilic metabolites corroborating information obtained with plants and their extracts, generated by Phase 1 enzymes and thereby destroy their ability to damage because of the presence of other potentially confounding substances. DNA. Since isothiocyanates block carcinogenesis by dual mechanisms and are already present in substantial quantities in human diets, these agents Chemical Properties and Metabolism of Isothiocyanates are ideal candidates for the development of effective chemoprotection of Chemical Reactivity and Spectroscopic Properties. The highly humans against cancer. electrophilic central carbon atom of the â€”N=C=S group reacts rapidly, and under mild conditions with oxygen-, sulfur-, or nitrogen- Introduction centered nucleophiles to give rise to carbamates, thiocarbamates, or Organic isothiocyanates (Râ€”N=C=S), also known as mustard oils, arc widely distributed in plants, many of which are consumed by R'-OH R'-SH humans. They are responsible for the pungent and acrid flavor and odor of condiments such as mustard and horseradish and the familiar biting taste that develops when some cruciferous vegetables are eaten. The designation "mustard oil" originates from the flavor of mustard R - N = C = S seeds which is largely due to the presence of abundant quantities of allyl isothiocyanate (CH,=CHâ€”CH2â€”NCS). In plants, isothiocya R - NH - C = S R - NH -l C = S R - NH - C = S nates are invariably accompanied by usually much larger quantities of O -R' S - R1 NH - R1 their cognate glucosinolates (ÃŸ-thioglucoside, A'-hydroxysulfate). In addition to their characteristic flavors and odors, isothiocyanates have a variety of other pharmacological and toxic activities. These include: thiourea derivatives, respectively. goitrogenic activity; antibacterial, antifungal, and antiprotozoal ac These reaction products have been useful for the spectroscopic tions; the ability to attract or repel insects; cytotoxicity; the induction identification and characterization of isolated isothiocyanates. The of chromosome abnormalities and neoplasia; and the blocking of â€”NCS group of isothiocyanates absorbs UV light with low intensity chemical carcinogenesis. The interesting early history of the chemis near 240 nm (anl about 1000 M"' cm~'). Reactions of isothiocyanates try and biology of these compounds has been reviewed by Challenger with monothiols at pH 7-9 yield thiocarbamates (carbamate thio- (1). Several more recent encyclopedic and scholarly reviews of the esters) that show markedly enhanced UV absorption intensity (am chemistry, distribution in plants, biosynthesis, and biological proper 10,000 M"' cm~') and a characteristic maximum at 270 nm with a ties of glucosinolates and isothiocyanates are available (2^1). broad shoulder near 250 nm (11-13). Zhang et al. (13) have reported Isothiocyanates arise in plants as a result of enzymatic cleavage of recently that isothiocyanates react with vicinal dithiols, such as 2,3- glucosinolates by myrosinase (thioglucoside glucohydrolase, EC dimercaptopropanol or ethane-1,2-dithiol, to give rise initially to the 3.2.3.1), which is released when plant cells are injured. Myrosinase promotes the hydrolysis of glucosinolates and intramolecular (Lossen) expected monothiocarbamates. However, upon further incubation rearrangement of intermediates to yield isothiocyanates, hydrogen these primary products undergo attack by the free thiol group on the sulfate, and glucose as the major products (Fig. 1). electrophilic carbon, resulting in a cyclization reaction with release of the free amine (13, 14). Thus the reaction of any R-NCS with As shown in Fig. 1, a number of other products may also arise from ethane-1,2-dithiol, 2,3-dimercaptopropanol, and 1,2-benzenedithiol 1 Presented at the 4th International Conference on Anlicarcinogenesis & Radiation gives rise to ethylene trithiocarbonate (l,3-dithiolane-2-thione, Amax Protection. April 1S-23, 1^3, Baltimore. MD. Studies from the laboratory of the authors 316 nm; a,,, 16,500 M~' crrT1), 4-hydroxymethyl-l,3-dithiolane-2- were supported by a grant from the National Cancer Institute. Department of Health and thione (Anl.,x316 nm; am 16,400 ivT1cm"1), and l,3-benzodithiole-2- Human Services (FOI C'A 44530). Y. Z. is a Fellow of the Cancer Research Foundation thione (Amax363 nm; am 22,500 M~' cm"1), respectively. Based on the of America. 1976s

generally regarded as a detoxication mechanism, recent attention has .S - C,H,,O5 Thioglucosidase pH CSH,,0, been directed to the role of GSH and cysteine conjugates (thiocar R-C H,O bamates) as a means of transporting isothiocyanates to peripheral N - 0 - SO,- (Myrosinase) D-GLUCOSE organs where they can undergo cleavage and contribute to toxicity

GLUCOSINOLATES THIOHYDROXAMATE- (22, 23) or induce enzymes that protect against toxicity (see below). O-SULFONATE Isothiocyanates as Protectors against Chemical Carcinogenesis HSO," The capacity of organic isothiocyanates to block chemical carcino- \ | t genesis was recognized 30 years ago (24, 25). Interest in naphthyl- R-N=C=S R-S-CÂ»N R-CsN+S OXAZOLIDINE-2-THIONE ISOTHIOCYANATE THIOCYANATE MITRILE AMINES NCS arose from its ability to produce profound proliferative damage CYANO-EPITHIO-ALKANES in the liver. In long-term feeding experiments a-naphthyl-NCS sig Fig. 1. Hydrolysis of glucosinolates by myrosinasc and formation of isothiocyanales nificantly reduced (in a dose-dependent manner) the formation of liver and olher producÃs[from Pessina ci al. (5)]. tumors by 3'-methyl-4-dimethylaminoazobenzene. ethionine, and jV-2-fluorenylacetamide in male Wistar rats. Furthermore, animals fed favorable spcctroscopic properties of l,3-benzodithiole-2-thione, a a-naphthyl-NCS did not develop ear duct carcinomas and leukemia, general method for the sensitive, specific, and quantitative analysis of unlike rats receiving the jV-2-fluorenylacetamide alone (24, 25). isothiocyunates was developed (13). Lacassagne et al. (26) confirmed and extended these findings by Metabolism of Isothiocyanates in Vivo. Whereas the nonenzy- demonstrating that not only dietary a-naphthyl-NCS but also ÃŸ-naph- matic reaction of GSH2 with benzyl-NCS is rapid, this reaction is also thyl-NCS (which does not cause marked proliferative changes in the catalyzed by rat liver cytosols, presumably promoted by the glutathi- liver) blocked hepatic tumor formation in rats fed 4-dimethylamino- one transferases present in these preparations (15). The principal azobenzene. Both isothiocyanates produced profound effects on he metabolic products of isothiocyanates administered p.o. to mammals patic enzymes that metabolize xenobiotics (27, 28). are the corresponding thiocarbamates. When six male volunteers were These findings laid the groundwork for many subsequent studies on fed about 100 p,mol of benzyl-NCS, 53.7% of the dose administered the tumor blocking activities of isothiocyanates, which were admin was excreted in the urine as /V-acetyl-S-(iV-benzylthiocarbamoyl)-L- istered usually for only short time periods (Table 1). cysteine, a mercapturic acid, in the first 12 h. No other metabolites Mammary Tumors. In the single dose 9,l()-dimethyl-l,2-bcn- were detected, and the excretion appeared to be complete in this zanthracenc (DMBA)-induced mammary tumor model in female period (16). Similar results were observed in two men and two women Sprague-Dawley rats, single doses of phenyl-NCS, phenethyl-NCS, who were fed watercress, a rich source of phcnethyl-NCS and its and benzyl-NCS markedly reduced the incidence and multiplicity of glucosinolate (gluconasturtiin), which is hydrolyzed by the myrosi- mammary tumors. In a detailed experiment Wattenberg (29) showed nase present in this plant (17). When 30- or 57-g portions of fresh that the timing of administration of the anticarcinogen with respect to watercress (containing 0.72 mg of gluconasturtiin/g) were consumed the DMBA was very important. Thus, when benzyl-NCS was adminis by each individual. 30 to 67% of the phenethyl-NCS was excreted as tered by gavage 2 h prior to the single dose of carcinogen, the average iV-acetylcysteine derivative in the urine in 24 h, and the majority of number of rats bearing tumors was reduced from 77% to 8%. and the number this conjugate was recovered within the initial 8 h (17). This experi of tumors per animal dropped from 1.6 to 0.08. Administration of the ment does not provide unequivocal evidence for the hydrolysis of the isothiocyanate 4 h prior to the carcinogen was somewhat less effective: but if glucosinolate to isothiocyanate in the body, since no information on the isothiocyanate was given 24 h before or 4 h after the DMBA, the tumor the content of free phenethyl-NCS in the watercress was provided. blocking effect was largely abolished. In other experiments with the same rat Furthermore, it is not clear whether breakdown of the glucosinolate to tumor model, Wattenberg (30) observed that dietary benzyl isothiocyanate isothiocyanate resulted from the myrosinase activity of the watercress fed for the entire experimental period, beginning 1 week after the DMBA or from enzymes present in the test subjects (either in the host tissues dose, was also highly effective in reducing mammary tumor incidence and or the microbial flora of the gastrointestinal tract). We are not aware multiplicity. of any experimental information on the ability of human subjects to Forestomach and Lung Tumors. Administration of benzyl-NCS hydrolyze glucosinolatcs to isothiocyanates. (1 mg) to female A/J mice by gavage 15 min prior to each exposure Rats, mice, guinea pigs, and rabbits likewise converted isothiocya to dimcthylnitrosaminc (20 mg/kg; once weekly for 8 weeks) reduced nates efficiently to N-acetylcysteine conjugates, but other metabolites, forestomach tumor incidence and multiplicity but had no effect on including cyclic mercaptopyruvate derivatives (not detected in hu pulmonary adenoma formation. In contrast, administration of benzyl- mans) were also formed (17-20). However, when phenyl-, a-naph- NCS (1 or 2.5 mg, 15 min before carcinogen) reduced, in a dose- thyl-, ÃŸ-naphthyl,or /erf-butyl isothiocyanate were given to rats, no dependent manner, both pulmonary adenomas and forestomach tu cysteine conjugates were detected (15, 16, 21). mors evoked by benzo(Â«)pyrene (3 doses of 2 mg each, given p.o. at The conversion of isothiocyanates to their iV-acctylcysteinc deriv 2-weck intervals) (31). Benzyl-NCS also suppressed the formation of atives (mercapturic acids) proceeds by the conventional route of forestomach tumors in ICR/Ha mice (38). conjugation with glutathione presumably promoted by glutathione The effects of isothiocyanates on lung tumors resulting from administra transfcrases. The resulting conjugates are then hydrolyzed to the tion of the potent tobacco-derived nitrosamine carcinogen NNK have been cysteine derivatives and acetylated. Although this pathway has been studied intensively and reviewed recently by Chung (39). In male F344 rats, phenethyl-NCS was fed for 1 week prior to and during the course of 2 The abbreviations and definitions used are: GSH. glutathione: OR. quinonc reducÃase treatment with NNK (1.76 mg/kg s.c., 3 times weekly for 21 weeks). At the [NAD(P)H:(quinone acceptor) oxidoreductase: EC l.h.99.2]; GST, glutathione trans ferases (EC' 2.5.1.18); Phase I enzymes (principally cytochromes P-450) functionalizc termination of the experiment (104 weeks), the pulmonary tumor incidence xenobiotics largely by oxidative or reductive reactions; Phase 2 enzymes conjugate (adenomas and carcinomas) was reduced from 80% to 43% by the isothio functinnali/cd compounds with endogenous ligands (C.K., GSH. glucuronic acid). OR is cyanate treatment, which also inhibited the methylation and pyridyloxobu- classified as a Phase 2 cn/yme. Monofunctional inducers elevate Phase 2 enzymes without tylation of lung DNA (32). intervention of the Ah (Arvl hydrocarbon) receptor and do not significantly affect Phase 1 en/yme activities; DMBA. ^.lO-dimethyl-l^-ben/anlhraccne; NNK. 4-(methylnitro- In shorter term experiments, female A/J mice received 5 ju.mol of samino)-l-(3-pyridyl)-l-butanone; i.g.. intragastrically. various phenyl-(CH2),,-NCS (n = 0-6) daily by gavage for 4 days. 1977s

Table I Prulecliim againx! chemical curcinogenesis by aryl ami arylalkyl ixolltiocyanalex in ruis and mice

Carcinogen3'-Me-DAB"Ethioninc(sex/strain)RaI(

S-D) el a!. (24) AAFDABDMBADMBAHI-DENHl>NNKNNKNBMASpeciesRat(cJ,Wistar)Rat(d, Liver,ductLiverMammary leukemia, ear cr-Naphthyl-tt- Sidransky(24)Lacassagne el al.

Wistar)Rat andÃŸ-naphthyl-Phenyl-, (26)Wallenbergel al.

( 9 ,S-D)Mouse glandLung, phcnethyl-Benzyl-,benzyl-, 30)WatlL-nbcrg(29,

(9,ICR/Ha)Mouse forestomachForcstomachForestomachLung, phenethyl-Benzyl-Benzyl-Benzyl-Phenelhyl-Phenyl-(29)Wallenberg

( 9 .ICR/Ha)Mouse (29)Wallenberg

(9,A/J)Mouse (31)Wallenberg

(9,A/I)Rat forestomachLungLungEsophagusProtective (31)Morse

(a,F344)Mouse (32)Morseel al.

(9,A/J)Rat(Â¿. 0-6)Phenethyl-Ref.Sasaki(CH2),,-(n = (33-35)Stonerel al.

F344)Tumor el al. (36, 37) " 3'-Me-DAB, 3'-methyl-4-dimelhylaminoazobenzene: DAB. 4-dimethylaminoazobenzene; AAF, iV-2-fluorenylacctamide: BP. benzo(Â«)pyrene: DEN. diethylnitrosamine: NBMA. A'-nilrosoben/ylmcthy lamine.

Two h after the final treatment with isothiocyanates, a single dose of cyanate hydrolysis products. The most clear-cut results have been NNK (10 p,mol hy i.p. injection) was administered and pulmonary obtained with the DMBA-induced mammary tumors of the Sprague- tumors were quantitated 16 weeks later. Phenyl-NCS and benzyl-NCS Dawley rat. Administration of large single doses of glucobrassicin were inactive, but phenethyl-NCS (n = 2) and isothiocyanates with (indolylmethyl glucosinolate) or of glucotropaeolin (benzyl glucosino- n = 3-6 produced marked reductions in lung tumor formation; the late) 4 h prior to the carcinogen substantially reduced both the incidence inhibition was progressively more pronounced as the mÃ©thylÃ¨nechain (from 75% to 25-38%) and the multiplicity (from 1.25 to 0.50-0.69 was lengthened (33, 34). Thus phenylhexyl-NCS (5 /xmol daily for 4 tumor/rat) of mammary tumors. Administration of these glucosinolates or days) reduced pulmonary tumor multiplicity from 7.9 to essentially zero. of glucosinalbin (4-hydroxybenzyl glucosinolate) produced some reduc DNA methylation, as measured by f/'-methylguanine formation, was tion in the multiplicity, but usually not the incidence, of forestomach likewise blocked by these arylalkyl isothiocyanates, again more markedly tumors and pulmonary adenomas of mice treated with benzo(a)pyrene as the mÃ©thylÃ¨nechainof the isothiocyanates was made longer (34). (40). The interpretation of these experiments is complicated. It is not If the administration of benzyl-NCS or phenethyl-NCS (1-3 known to what extent these glucosinolates are degraded in the rodent /xmol/g of diet) was delayed for 1 week after treatment with NNK and bodies or to what extent the glucosinolates or their degradation products then continued to the end of the experiment at 16 weeks, no effect on are responsible for the tumor-blocking effects. If the glucosinolates are pulmonary tumor formation was observed at nontoxic doses of the hydrolyzcd, it is not known whether endogenous or microbial enzymes isothiocyanates (35). are responsible and what products are formed. These issues are especially Tumors of the Esophagus. Stoner et al. (36) have reviewed their complicated for glucobrassicin, since three known degradation products studies on the inhibition by phenethyl-NCS of esophageal tumor of this glucosinolate (indole-3-acetonitrile, 3.3'-diindolylmethane, and production in the rat by the asymmetrical nitrosamine, jV-nitrosoben- indole-3-carbinol) are anticarcinogens and inducers of cytochrome P-450 zylmethylamine. The importance of these studies derives from the (41, 42). A further potentially confounding effect is the conversion of belief that /V-nitroso compounds and their precursors are probably indole-3-carbinol under mildly acid conditions (such as prevail in the causative factors for esophageal cancers in some high incidence stomach) to cyclic derivatives that bind at very low concentrations to the regions (37). Male F344 rats treated with /V-nitrosobenzylmethyl- Ah receptor and thereby become very potent inducers of cytochrome amine, (0.5 mg/kg s.c. once per week for 15 weeks) developed 100% P450IA1 (43). esophageal tumors at the end of the 25-week assay period, and the tumor multiplicity was 11.5/animal. In experimental groups also fed Mechanisms: Effects of Isothiocyanates on Carcinogen phenethyl-NCS (3 /Â¿mol/gof diet), tumor incidence was only 13% and Metabolism average tumor multiplicity was negligible (0.1 Â±0.3/animal). At a higher dose of phenethyl-NC"S(6 fimol/g of diet) no tumors were observed (37). Understanding of the mechanisms of the chemoprotective effects of Phenethyl-NCS treatment blocked the formation of both preneoplastic and isothiocyanates is of great importance not only because these sub neoplastic lesions in the esophagus; even at the lower dose of the isothio stances block the formation of a wide variety of carcinogen-induced cyanate, there was complete inhibition of the appearance of the more ad tumors in rodents, but also because isothiocyanates and their glucosi vanced (papilloma and carcinoma) lesions (37). nolate precursors are widespread Â¡nhuman dietary plants and are Parallel measurements of the effects of phenethyl-NCS on the consumed in substantial quantities. To what extent these substances metabolism of [â€¢iH]nitrosobenzylmethylamine in rat esophageal ex- contribute to the well-recognized protective effects of vegetables plants demonstrated concentration-dependent inhibition of both me against cancer is unclear (44). The only plausible mechanisms pro tabolism of the carcinogen and methylation of DNA, as indicated by posed for the anticarcinogenic effects of isothiocyanates implicate decreased formation of /V7-methyIguanine and O"-methylguanine. modulation of carcinogen metabolism, both depression of activation Tumor-blocking Effects of (Ã¬lucosinolates. A few experiments of carcinogens and acceleration of their disposal. Evidence for these have examined the chemoprotective effects of glucosinolates, which are conclusions is based on measurements of: (a) carcinogen-DNA ad- often present in plants in much higher concentrations than their isothio- duct formation and the accompanying nucleotide modifications; (b) 1978s

Table 2 Induction of Phase 2 enzymes by IsOtUuCyanaU* in rodent tissues

of enzyme specific activities EnzymeGSTGSTGSTGSTGSTGSTQRORORORORR-NCSBenzyl-NCSBenzyl-NCSAllyl-NCSPhenethyl-NCSPhenylbulyl-NOSPhcnylhcxyl-NCSSulforaphaneErucinErysolinBenzyl-NCSPhenethyl-NCSPhcnylhulyl-NCSPhenylhexyl-NCSa-Naphlhyl-NCSÃŸ-Naphthyl-NCSSulforaphaneErueinErysolinSpecies(sex/strain)Mouse (treated/control)2.08-2.591463.204.38-9.363.43-^.293.0-3.51.63-1.731.35-1.375.27-8.311.251.26-2.253.07-3.931.31.351.371.08-3.002.572.052.822.274.781.802.415.1~1i1.441.632.8-4.42.231.05-3.10Ref.Sparnins ICR/Ha)Mouse(female; 49,50)Bensonel al. (38,

bowelForestomachLiverLungKidneySmall

ICR/Ha)Ral(female; (51)Bensonand Barretlo (52)Bogaardset al.

bowelColonLiverSmall

(male;F344)Rat (53)Guo et al. bowelLiverLiverLiverForestomachGlandular (male;F344)Ral et al.(47)Guo

(male;F344)Mouse el al.(57)Zhang

CD-I)Mouse(female; (10)Bensonet al. stomachLiverSmall

bowelColonForestomachLiverLungKidneySmall

CD-I)Ral(female; (51)Bensonand Barretto

(52)Guo et al.

bowelColonBladderLiverLungNasal

(male;F344)Ral et al.(47)Guo

mucosaLiverLiverLiverLiverForestomachGlandular

(male;F344)Rat et al.(57)Leonard

(male;F344)Mouse 28)Zhanget al. (27,

(female; CD-I)TissueEsophagusForestomachLiverSmall el al. (10) stomachLiverSmall

bowelColonRatio

rates of activation of carcinogens and levels of Phase 1 enzymes; and ageal tumors by /V-nitrosobenzylmethylamine in rats also paralleled (c) activities of Phase 2 enzymes and levels of GSH. inhibition of the binding of the carcinogen to DNA and the formation Reactions of Carcinogens with DNA. There is a striking parallel of A^-methylguanine and O''-methylguanine. between the inhibitory effects of various arylalkyl isothiocyanates on Regulation of Phase 1 Enzymes by Isothiocyanates. Administra lung tumor formation by NNK, the potent carcinogenic nitrosamine of tion of isothiocyanates to rodents produced either increases or de tobacco, and the ability of these isothiocyanates to block O''-methyl- creases of microsomal cytochrome P-450 content and the activities of guanine formation in lungs of rats and mice. The very systematic several cytochrome P-450-dependent monooxygenases. The effects studies by Chung, Hecht and their colleagues should be consulted for appear to depend on experimental conditions: the nature of the iso- details [Refs. 32, 39, and 45; reviewed by Chung (46)]. These workers thiocyanate; the treatment regimen; the target tissue examined; and and others (46) have also studied the metabolic activation of NNK by the specific monooxygenase measured. lung tissue and microsomes and have concluded that the chcmopro- The most dramatic increases have been reported with indole-3-carbinol and 3,3'-diindolylmethane and to a lesser degree with indole-3- tective isothiocyanates act principally on the enzymes involved in the metabolic activation of NNK. By the use of inhibitory antibodies acetonitrile. These Ã•ndolesarehydrolytic rearrangement products of glu- specific for cytochromes P-450, Guo el al. (46) identified cyto- cobrassicin (indolylmethyl glucosinolate). Single i.g. administrations of chromes P450IIB1 and 2 but not P450IA1 or P450IIE1 as important 10-KK) /Â¿molofthese Ã•ndolestofemale Sprague-Dawley rats raised the participants in the activation process. aryl hydrocarbon hydroxylase activities of homogenates of liver and Similar experiments by Stoner et al. (36, 37) have established that small intestine as much as 25-fold. Clearly these effects cannot be directly the inhibitory effects of phenethyl-NCS on the production of esoph- attributed to the actions of isothiocyanates (41). As already mentioned. 1479s

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1994 American Association for Cancer Research. ANTICARCINOGENIC ACTIVITIES OF ORGANIC ISOTHIOCYANATES recent experiments (43) have shown that indole-3-carbinol is readily Tahle 3 Effects of benzyl isotltiocyanate on glutathitmt' levels in female ICR/Ha mouse converted both in vitro and in vivo, under mildly acid conditions to tissues polymeric Ã•ndolesthathind with very high affinity to the Ah receptor, and of GSH concentrations thereby enhance transcription of cytochrome P450IA1. TissueEsophagus (treated/control)1.63-1.750.77 Single i.g. doses (1(K) /j.mol) of phenethyl-NCS to male F344 rats et ai (49) caused marked increases of liver microsomal pentoxyresorufin O-de- Forestomach Sparnins and Wallenberg (3K) alkylase activity (10-fold) and the content of cytochrome P4501IB1 Small bowelRatio 1.64-1.66Ref.Sparnins Sparnins et al. (49) protein (6.6-fold) (47). In contrast, cytochrome P-450 content, ethoxyresorufin O-dealkylase, and erythromycin /V-demethylase activi Table 4 Indiiclio/i of (Â¡Â¡linonereducÃasein Hepa Iclc7 murine hcpaioina cells by ties all fell moderately during the first 12-18 h and then recovered by the isolhiocyanatesa end of the experiment at 48 h (47). Although these effects appear to be Concentration required somewhat contradictory, the above-described treatment with phenethyl- to double QR NCS depressed the formation of reactive metabolites of NNK by lung Isolhiocyanate (and to a lesser degree by liver) microsomes. The principal effect of Sulforaphane |CH,SO(CHÃ¯)4NCS) 0.2 phenethyl-NCS appears to be depression of the a-hydroxylation of NNK iV-Hexyl-NCS 15 Benzyl-NCS 1.9 which is critical for DNA adduct formation. Furthermore, the direct Cyclooctyl-NCS 10 addition of a number of isothiocyanates to microsomes of rat and mouse Cyclohexyl-NCS 50 " From PresterÃ et al. (55). lungs and rat nasal mucosa potently depressed the metabolic activation reactions of NNK. Thus, 50% inhibition was achieved by phenylethyl- NCS at 120-200 nM, by phenylbutyl-NCS at 30-75 nM, and by phenyl- sented by elevations of GST and QR in various tissues, appears to be hexyl-NCS at 15-90 nM depending on the specific metabolites. The KÂ¡ a constant property of a variety of isothiocyanates. Benzyl-NCS also values for the inhibition of NNK metabolism by phenylhexyl-NCS were raised GSH levels in esophagus and small bowel (but not forestom very low (10.9-16.8 nM) for various reactions (57). ach) of mice by 63-75% (Table 3; Ref. 38). Straightforward results were obtained in experiments in which In common with many chemically unrelated chemoprotective com 100-300 ^mol daily i.g. doses of allyl-NCS were given to male pounds, the administration of isothiocyanates to rodents evokes a gener outbred rats for 3 days. The hepatic aminopyrine yV-demethylase, alized "electrophile counterattack" response, characterized by the induc /j-nitroanisole O-demethylase, and aniline /j-hydroxylase activities tion of Phase 2 enzymes and increases in tissue GSH levels (54, 55). were all decreased in a dose-dependent manner to as low as 31-46% Much has been learned about the chemical structure and mecha of control values (48). nism of induction of chemoprotective compounds by the measurement Although both a- and ÃŸ-naphthyl-NCS block neoplasia in the liver of OR activity in Hepa Iclc7 murine hepatoma cells in culture (7), (see above), dietary administration of these compounds to male F344 and especially in cells grown in 96-well microtiter plates (56). Ele rats for 2-4 weeks had opposite effects on cytochrome P-450 content vation of QR in this cell line accurately predicts induction of both QR and monooxygenase activities of liver microsomes. Whereas the and GST in animal tissues. This system played a critical role in the a-naphthyl-NCS depressed total P-450 content, as well as ethoxycou- identification of sulforaphane as the major Phase 2 enzyme inducer in marin O-deethylase and benzphetamine O-demethylase activities, the SAGA broccoli (10). Extensive information is now available on the ÃŸisomer had the opposite effects (27, 28). For instance, dietary inducer potency of a wide variety of isothiocyanates in Hepa cells. ÃŸ-naphthyl-NCS (5.4 jamol/g diet for 28 days) raised microsomal Some illustrative inducer concentrations required in the assay system cytochrome P-450 content, ethoxycoumarin O-deethylase, and benz to double the specific activity of QR in Hepa Iclc7 murine hepatoma phetamine /V-demethylase 1.6-, 1.74-, and 2.2-fold, respectively (28). cells are given in Table 4. In Hepa Iclc7 murine hepatoma cells in culture. benzyl-NCS had In an effort to understand the basis for the high inducer potency of no effect on aryl hydrocarbon hydroxylase activity (8). sulforaphane, a large number of bifunctional isothiocyanates were re Although the effects of isothiocyanates on Phase 1 enzymes appear cently synthesized and evaluated as inducers (58). It was found that to vary greatly with experimental conditions and the activity meas among these analogues, the methylsulfinyl group (CH,SOâ€”) of sulfora ured, the conclusion that these compounds depress the activational phane could be replaced by either CH,COâ€” or CH,SO2â€” groups metabolism of two carcinogens, NNK and /V-nitrosobenzylmethyl- without significant effect on inducer potency. For optimal potency, these amine, appears to be firmly established. functional groups had to be separated from the â€”NCS function by 3 or 4 carbon atoms. In some of the analogues, the link separating the two Regulation of Phase 2 Enzymes and Glutathione Levels by functions was flexible whereas in others it was relatively rigid. Certain Isothiocyanates bifunctional norbornyl-NCS analogues were potent inducers of QR in murine hepatoma cells and inducers of OR and GST in mouse tissues. In contrast to the complex effects of isothiocyanates on Phase 1 Since there is much persuasive evidence that induction of Phase 2 enzymes, the effect of these agents on Phase 2 enzymes of rodent enzymes plays a major role in the chemoprotective effects of many tissues is quite straightforward. Table 2 summarizes the changes in different classes of compounds (9), it appears very likely that isothio specific activities of GST and OR in the cytosols of several organs of cyanates also exert at least a part of their protective actions through mice and rats treated with benzyl-NCS, phenethyl-NCS, allyl-NCS, this mechanism. Isothiocyanates are potent electrophiles like most a-naphthyl-NCS, ÃŸ-naphthyl-NCS, or sulforaphane [1-isothiocya- other inducers of Phase 2 enzymes (7, 54, 55). Isothiocyanates resem nato-(4/?)-(methylsulfinyl)butane]. The compounds were given either ble other monofunctional enzyme inducers in that they stimulate in the diet (3-34 fxmol/g diet) for 5-28 days or by i.g. administration transcription of Phase 2 enzymes via a common AP-1-like enhancer (5-100 /j.mol in single or several daily doses). The organs examined element present in the upstream regulatory regions of certain GST and included: liver; esophagus; forestomach; glandular stomach; small QR genes (54, 55). bowel; colon; lung; kidney; and bladder. Inductions of GST and QR, expressed as ratios of specific activities of tissues obtained from References treated and control animals varied from 1.2 to 9.4 and were mostly in 1. Challenger, F. Aspects of the Organic Chemistry of Sulphur, pp. 151-161. London: the 2-4-fold elevation range. Phase 2 enzyme induction, as repre- Butterworths. 1959. HMOs

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1981s

Yuesheng Zhang and Paul Talalay

Cancer Res 1994;54:1976s-1981s.

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