QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wallig, M. A. Articles by Gossman, T. Search for Related Content PubMed PubMed Citation Articles by Wallig, M. A. Articles by Gossman, T.

---

Supplement: International Conference on Diet, Nutrition, and Cancer

Synergy among Phytochemicals within Crucifers: Does It Translate into Chemoprotection?^1,2,3

Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61802

⁴To whom correspondence should be addressed. E-mail: mawallig{at}uiuc.edu.

ABSTRACT

The association between cruciferous vegetables and cancer prevention has been linked to glucosinolate derivatives. These phytochemicals enhance endogenous detoxification, leading to inactivation of potential carcinogens before initiation occurs. Two derivatives, indole-3-carbinol (I3C) and 1-cyano-2-hydroxy-3-butene (crambene) were shown in rats to induce a synergistic enhancement of detoxification enzyme activity. To follow up on these findings, a short-term carcinogenicity study using aflatoxin B1 (AFB₁) was performed in which male F344 rats were fed diets supplemented with these 2 compounds alone or in combination. Groups included a negative control group (no AFB₁, crambene, or I3C), a crambene group (diet 0.150% crambene), an I3C group (diet 0.165% I3C), a high-dose group (diet 0.150% crambene, 0.165% I3C) a low-dose group (diet 0.030% crambene, 0.033% I3C), and a positive control group (AFB₁ treatment only). AFB₁ was administered after 2 wk of dietary pretreatment. Liver sections were scored for lesions including karyomegaly, apoptosis, and biliary hyperplasia and evaluated for expression of the preneoplastic marker glutathione S-transferase- (GSTP). I3C and crambene groups were protected against AFB₁ toxicity whereas the low-dose group was not. The high-dose group had scores close to those of the negative controls. For log₁₀ transformed 2- and 3-dimensional GSTP data, the high-dose group demonstrated synergistic reduction in GSTP-positive area and an additive reduction in GSTP-positive volume compared with the crambene and I3C groups. The low-dose group had no effect. In conclusion, high combination dietary doses of I3C and crambene demonstrated enhanced protection from AFB₁. Low combination doses, as might be realistically in the diet, were not effective.

KEY WORDS: • synergy • phytochemicals • indole-3-carbinol • crambene • aflatoxin B₁ • glutathione S-transferase- • chemoprotection

Diets rich in cruciferous vegetables such as broccoli, Brussels sprouts, cabbage, and kale have been associated with a reduction of the incidence of several types of cancer (1,2). Many of the phytochemicals in these vegetables, most notably those derived from glucosinolates common to all crucifers, induce detoxification enzymes. This induction has been postulated to play a key role in the protection afforded by these vegetables against a variety of cancers (3,4).

Previous research at the University of Illinois identified 2 glucosinolate breakdown products, 1-cyano-2-hydroxy-3-butene (crambene) and indole-3-carbinol (I3C),⁵ as powerful inducers of detoxification enzymes (5–7). Progoitrin, a glucosinolate within the matrix of certain varieties of Brussels sprouts, broccoli, and related cruciferous vegetables (8), is hydrolyzed by an endogenous enzyme, myrosinase, during mastication or cooking (10) to form crambene, whereas I3C is formed similarly from the glucosinolate glucobrassicin (9).

I3C was found to be protective in rat and mouse models against various chemical carcinogens, including experimentally induced aflatoxin B₁ (AFB₁) hepatic carcinogenesis (10), and I3C has generated much interest as a potential cancer chemotherapeutic agent (11). I3C induces both phase I and phase II enzymes and hence is termed a bifunctional inducer. Bifunctional inducers such as I3C (and its major metabolite, diindolylmethane) work via the aryl hydrocarbon receptor pathway that ultimately interacts with the xenobiotic response element (XRE). XRE is present not only in the regulatory regions of the genes for many phase II enzymes, such as the glutathione S-transferases (GSTs) and quinone reductase, but also in the regulatory regions of certain cytochrome P450 (CYP) genes, such as the gene encoding CYP1A, a CYP frequently involved in bioactivation of a number of carcinogens, including AFB₁ (10). Hence, I3C ingestion to prevent carcinogenesis can be a two-edged sword, actually activating certain precarcinogens (12,13).

Crambene, on the other hand, is a monofunctional inducer, having minimal effect on phase I enzymes while remaining a potent inducer of hepatic phase II enzymes. It is also an effective long-term inducer of the antioxidant glutathione (14,15). Crambene affects detoxification enzymes via the antioxidant response element (ARE) (16), present in the regulatory regions of many phase II enzymes in rats, including - and -GSTs, quinone reductase, and the glutamate cysteine ligase, the rate-limiting enzyme in glutathione synthesis (17).

Rats treated with I3C and crambene have synergistic rather than additive induction of phase II enzymes, specifically the GSTs and quinone reductases (5,6). This synergy occurs at the gene regulatory level (18), suggesting that this effect may translate into a synergistic effect in the live animal, allowing for lower doses of either compound to be used for chemoprotection. It has been hypothesized that such synergy may explain why higher doses of each compound individually are required to achieve protection experimentally than are typically present in the vegetable plant itself (6). The concept of synergy among phytochemicals as the mechanism of action behind chemoprevention and chemoprotection against cancer afforded by many fruits and vegetables has become increasingly accepted (19). However, the few in vivo studies specifically investigating how individual phytochemicals might interact have been limited to only a few phytochemicals, such as selenium, -tocopherol, soy phytoestrogens, and retinoids (20–23).

The purpose of this study was to examine whether the synergy between crambene and I3C observed with induction of phase II detoxification enzymes would translate into actual chemoprotection, that is, whether the two compounds combined in the diet would interact synergistically to reduce the number of preneoplastic nodules expressing the biomarker enzyme, GST- (GSTP), in the liver after exposure to the carcinogen, AFB₁. The aflatoxin model of hepatocarcinogenesis was selected because it is known to be a naturally occurring human carcinogen, is metabolized in the rat by enzymes affected specifically by both I3C and crambene, and can be used in short-term studies using GSTP expression as a biomarker (24–26).

Materials and methods

    Chemicals. Crambene was isolated and purified from the seeds of Crambe abyssinica as previously described (27). I3C and other chemicals were purchased from Sigma Chemical Company.

    Animals, study design, treatment, and diet. Sixty weanling, male CDF 344 (crl/BR) rats (Charles River Laboratories), weighing 75 g, were randomly assigned to 6 groups of 10 rats each. Included were a negative control group (no AFB₁, crambene, or I3C), a crambene group (diet 0.15% crambene), an I3C group (diet 0.165% I3C), a high-dose combination group (diet 0.15% crambene, 0.165% I3C,) a low-dose combination group (diet 0.030% crambene, 0.033% I3C), and a positive control group fed neither supplement (AFB₁ treatment only) (Table 1). The doses of crambene and I3C were calculated based on short-term pilot feeding studies (data not shown) such as to provide each rat 50 and 56 mg · kg body wt^–1 · d^–1 doses of crambene and I3C, respectively. Both the high and low combination doses as well as the individual doses were shown previously to induce the synergistic response (6). All groups but the first were treated with AFB₁ after 2 wk of dietary pretreatment. Crambene and I3C were supplied in a semipurified antioxidant-free powdered diet (AIN 93G; ICN Biomedicals) (28) for 14 d before AFB₁ treatment (AFB₁ in dimethylformamide via esophageal intubation) on d 14 and 15. After this time, the animals were fed an antioxidant-free semipurified pelleted diet (AIN 93G) for the duration of the experiment. Dietary intake and body weights were monitored daily through d 15 and weekly after that time. The animal protocol and all procedures were approved by the University of Illinois Institutional Animal Care and Use Committee before the experiment began. For evaluation of body weight and feed intake data, 2-way ANOVA was used to determine differences among groups. For terminal body weight data and for liver-body weight ratios, 1-way ANOVA followed by least significant difference for multiple comparisons was performed using statistical analysis software (SAS; SAS Institute).

    GSTP immunohistochemistry. For identification of GSTP-positive foci, sections were baked at 45°C for 30 min before incubation for 30 min with 1% H₂O₂. An avidin-biotin system (VectastainR; Vector Laboratories) was used. Sections were treated with blocking serum (goat) in buffer followed by 1° rabbit antiserum with specificity against GSTP generated from purified rat GSTP and confirmed for specificity in our laboratory (30). Secondary biotinylated antirabbit goat antibody and diaminobenzidine with Meyer’s hematoxylin as a counterstain were used for color development. The area of hepatic tissues in each section was measured as well as the number and mean percent area of GSTP-positive foci within each section, using a Leitz Orthoplan microscope with an attached Leitz Videoplan Sony CCD/RGB video camera. Images of the sections were captured on a computer running Windows 98, then analyzed using NIH Image/J shareware (a public domain image-processing and -analysis program developed at the NIH Research Services Branch) (31). The area data collected for each liver were transformed into 3-dimensional data (% vol foci/cm³ liver) (32). The mean percent area and volume data were log₁₀ transformed because standard errors depended on mean values and consistently increased as mean values increased. One-way ANOVA and least significant difference comparisons using statistical analysis software (SAS) were used to assess for significant variation among and between mean values. Values of P <0.05 were considered significant.

Results

Although diets were formulated to provide 50 and 56 mg · kg body wt^–1 · d^–1 doses of crambene and I3C, respectively, actual doses of each were 90 mg · kg body wt^–1 · d^–1 (data not shown), higher than predicted because of a greater consumption of diet than anticipated by all groups. Accordingly, the high-dose combination group ingested 90 mg · kg body wt^–1 · d^–1 of crambene and 90 mg · kg body wt^–1 · d^–1 of I3C, whereas the low-dose combination group ingested 20% of that, or 18 mg · kg body wt^–1 · d^–1 of both compounds. However, at the time of AFB₁ treatment and at the conclusion of the study, there were no significant differences in body weight or liver–body-weight ratio among the groups (data not shown).

Histologically, there was evidence that high dietary doses of both crambene and I3C afforded protection from AFB₁-mediated chronic injury. Livers from the negative control group (group 1) were generally within normal limits (Fig. 1A) although occasional mild biliary hyperplasia and karyomegaly were observed (Fig. 2). By contrast, the positive control group (group 6) had the severest lesions (Fig. 1B), typified by a high level of karyomegaly, biliary hyperplasia, portal hepatitis, and portal fibrosis. The crambene alone, I3C alone, and high-dose combination groups had virtually no lesions and did not differ from the negative control group; these treatments had significant protective effects against both karyomegaly and biliary hyperplasia, in contrast to the low-dose combination, which was nonprotective for all measures assessed. In addition, the degree of protection against biliary hyperplasia afforded by the high-dose combination treatment was also greater than that afforded by either crambene or I3C alone, but the difference was not significant.

View larger version (126K): [in this window] [in a new window]   FIGURE 1 (A) Normal liver from a negative control group rat (fed neither crambene nor I3C in the diet and not treated with AFB1). A normal portal triad, containing portal veins, hepatic arteriole, and small cholangiole, is featured (circle). (B) Severely affected liver from a positive control group rat (fed neither crambene nor I3C in the diet but treated with AFB1 via esophageal intubation). The liver was harvested 13 wk after AFB1treatment. Features include a hepatocyte with karyomegaly (small arrow), apoptotic bodies (large arrow), and hyperplastic cholangioles (hematoxylin and eosin; circles)

View larger version (29K): [in this window] [in a new window]   FIGURE 2 Effects of 2 wk treatment with dietary crambene alone, I3C alone, high-dose combination of crambene and I3C, and low-dose combination of crambene and I3C on the generation of liver lesions induced by AFB1. Negative control group rats were not exposed to dietary pretreatment or AFB1treatment. Positive control group rats were not exposed to dietary pretreatment but were treated with AFB1. Bars represent mean scores for each lesion assessed (karyomegaly, apoptosis, and biliary hyperplasia) in 3 standard hematoxylin- and eosin-stained liver sections from each rat (n = 10 rats/group). Error bars represent SEM. Bars for a particular lesion without a letter in common differ, P < 0.05.

View larger version (116K): [in this window] [in a new window]   FIGURE 3 (A) Normal liver from a negative control group rat (fed neither crambene nor I3C in the diet and not treated with AFB1). Cholangioles (arrows), which are the only cell type that stains positively for GSTP in normal liver, are featured. (B) Liver from a positive control group rat (fed neither crambene nor I3C in the diet but treated with AFB1 via esophageal intubation). The liver was harvested 13 wk after AFB1treatment. A nodule containing enlarged hepatocytes expressing GSTP is featured at the center of the image (avidin-biotin complex, hematoxylin counterstain).

View larger version (30K): [in this window] [in a new window]   FIGURE 4 Effects of 2 wk treatment with dietary crambene alone, I3C alone, high-dose combination of crambene and I3C, and low-dose combination of crambene and I3C on the generation of GSTP-positive preneoplastic foci and nodules induced in the liver by AFB1. Negative control group rats were not exposed to dietary pretreatment or AFB1 treatment. Positive control group rats were not exposed to dietary pretreatment but were treated with AFB1. Bars represent log10 mean area (cm2) and log10 mean estimated volume (cm3) of foci and nodules in 3 standard liver sections from each rat (n = 19 rats/group). Error bars represent SEM. Bars for a particular lesion without a letter in common differ, P < 0.05.

These data are the first in vivo evidence that crambene is chemoprotective not only against toxicity but also against initiation of carcinogenesis, supporting previous biochemical and molecular evidence that it is a phytochemical with cancer-protective potential. Although a weak inducer of detoxification enzymes in in vitro systems, crambene is equipotent with sulforaphane in vivo (33). Like sulforaphane, crambene exerts its effect on detoxification enzymes via the Nrf2/keap1 pathway that ultimately interacts with the ARE (16). In addition, the precursor glucosinolate for crambene, progoitrin, is in the same synthetic pathway as sulforaphane (34), being a derivative of methionine, and varieties of broccoli that are low in glucoraphanin, the precursor for sulforaphane, tend to have higher levels of progoitrin (35). Hence, greater dietary exposure to crambene than sulforaphane is possible in some situations.

With regard to crambene and I3C acting synergistically, the present study demonstrated that although dietary crambene and I3C were indeed individually protective against AFB₁-induced degenerative and/or inflammatory lesions and preneoplastic lesions, high combination doses of I3C and crambene were associated with enhanced protection from AFB₁ hepatic injury and carcinogenesis. This enhancement approached synergy in the suppression of the area of GSTP induction and achieved additivity in suppression of the volume of GSTP-positive hepatocytes within the liver. However, because of a high degree of variability in response among individual animals within groups, the apparent additivity and synergy was not significant at the P < 0.05 level. Nevertheless, a clear trend toward enhanced protection by a combination dose of both compounds was present, but the doses producing the effect were supraphysiologic, being far higher than could be realistically encountered even in an exclusively cruciferous diet, where a dose of 10 mg/kg body wt is on the outer realm of possibility (6).

Although the results of this study are somewhat equivocal with regard to synergy, they provide preliminary evidence that crambene and I3C can interact to produce an enhanced protective effect against the initiation stages of carcinogenesis. A longer study (e.g., 24 or 30 wk) may have allowed additional foci and nodules to form in the positive control group and fewer foci and nodules in the treatment groups. This would have enhanced the differences in response and made them stronger statistically. To definitely determine whether synergy can occur between crambene and I3C, a full-blown 2-y carcinogenicity study will have to be undertaken to more clearly define tumor incidence, multiplicity, and growth in animals treated with these compounds alone and in combination.

The low-dose combination group, which nevertheless received doses of crambene and I3C that would be somewhat unrealistic even in a diet very high in crucifers, was not protected, suggesting that synergy does not function at the low doses that might be encountered in the diet. The inability of the low-dose combination treatment to protect is disappointing and does not support the idea that synergistic interactions among phytochemicals that act via XRE and ARE account for the disparity between the high doses of purified phytochemicals needed to provide chemoprotection experimentally and the substantially lower doses of these same phytochemicals present in fruits and vegetables. Rather, the results seem to support a threshold phenomenon that occurs only at relatively high doses of compounds interacting via XRE or ARE. Further dose-response studies are needed to determine whether the threshold occurs and, if it does, at what level it occurs.

Another aspect of synergy not addressed by this study is the blocking versus suppressing properties of crambene and I3C (36). The study investigated the potential for I3C and crambene to block initiation of carcinogenesis rather than to suppress initiated cells from proceeding down the path toward neoplasia. I3C and its major metabolite diindolylmethane, for example, affect the cell cycle and the apoptotic pathways as well (10), pathways that act almost exclusively in the promotional stages of carcinogenesis. Crambene has as yet unspecified effects on the cell cycle (33,37) but is known to induce long-term elevations in pancreas and liver of the antioxidant glutathione as well as induce apoptosis in pancreatic acinar cells (15). At this point, however, it is not known whether the apoptotic effect can be induced in initiated cells or cells of nonpancreatic origin. Important synergistic interactions can and do occur in the promotional stages of carcinogenesis, and these interactions may be of equal or greater importance than interactions that prevent initiation. Additional in vivo investigations using well-defined models of carcinogenesis are needed in which potentially synergistic compounds such as crambene and I3C are provided in the diet after initiation.

In any discussion of synergy, however, many complicating factors must be acknowledged, factors affecting any interpretation of results and, more important, their potential applications to humans. As summarized in a recent review on the topic, there are "inadequate chemical identification of compounds, lack of relevant endpoints and inconsistencies in mechanistic hypotheses and experimental methodologies [that] leave many critical gaps in our understanding of the benefits of these compounds" (4). One of these factors is precisely defining the compounds and interactions among these compounds that produce the putative synergistic effect. It is becoming increasingly apparent that it may take >2 bioactive compounds and >1 biochemical pathway to produce chemoprotection (presumably via synergy) by cruciferous vegetables and fruits and vegetables in general (19). I3C and crambene are not the only bioactive phytochemicals in cruciferous vegetables. Sulforaphane, phenylethyl isothiocyanate, goitrin, 1,2-dithiol-2-thiones, and various polyphenols, to name just a few (38–41), are abundant in cruciferous vegetables. In most cases, their interactions with each other as well as with other chemoprotective nutritive compounds, such as vitamins, fiber, and minerals, are unknown or as yet undefined.

A case in point is selenium, which can be readily taken up by cruciferous vegetables (42). Selenium and its metabolites interact synergistically with other chemoprotective compounds, including retinoids and vitamin E, to inhibit carcinogenesis in experimental situations (20,21). However, enrichment of broccoli with selenium recently was shown to substantially decrease sulforaphane and polyphenol content in the enriched broccoli, potentially dampening the chemoprotective contribution of these compounds (43). Interactions of selenium with either I3C or crambene in the process of cancer prevention have yet to be defined, although selenium has been shown to interact with crambene in vitro to synergistically inhibit the growth of and to kill MCF-7 canine mammary cells at doses that leave normal canine mammary epithelium unaffected (44).

The data presented here are preliminary and represent only an early step in the process of truly demonstrating the occurrence of synergy in in vivo carcinogenesis models and then relating this to the underlying biochemical and molecular mechanisms that have resulted from the numerous in vitro studies performed to date. The in vivo studies necessary to identify and prove that the synergistic mechanisms identified in vitro do indeed translate into anticarcinogenesis will be expensive, time consuming, and difficult to perform, but they will be necessary to provide firm and definitive answers to the question of synergy among phytochemicals.

ACKNOWLEDGMENTS

The authors thank Anna Peters, Elisabeth Peters, Szymon Perkowski, and William Love for their excellent technical assistance.

FOOTNOTES

¹ Published in a supplement to The Journal of Nutrition. Presented as part of the International Research Conference on Food, Nutrition, and Cancer held in Washington, DC, July 14–15, 2005. This conference was organized by the American Institute for Cancer Research and the World Cancer Research Fund International and sponsored by (in alphabetical order) California Avocado Commission; California Walnut Commission; Campbell Soup Company; The Cranberry Institute; Danisco USA, Inc.; The Hormel Institute; National Fisheries Institute; The Solae Company; and United Soybean Board. Guest editors for this symposium were Vay Liang W. Go, Ritva R. Butrum, and Helen A. Norman. Guest Editor Disclosure: R. R. Butrum and H. Norman are employed by conference sponsor American Institute for Cancer Research; V.L.W. Go, no relationships to disclose.

² Author Disclosure: No relationships to disclose.

³ This project was supported in part by Grant 00B016 from the American Institute for Cancer Research.

⁵ Abbreviations used: AFB₁, aflatoxin B₁; ARE, antioxidant response element; CYP, cytochrome P450; GST, glutathione S-transferase; GSTP, glutathione S-transferase-; I3C, indole-3-carbinol; XRE, xenobiotic response element.

LITERATURE CITED

1. Steinmetz KA, Potter JD. Vegetables, fruit and cancer. Cancer Cause Cont. 1991;2:325-357.

2. Verhoeven DT, Goldbohm RA, van Poppel G, Verhagen H, van den Brandt PA. Epidemiological studies on brassica vegetables and cancer risk. Cancer Epidemiol Biomark Prev. 1996;5:733-748.[Abstract/Free Full Text]

3. Keck AS, Finley JW. Cruciferous vegetables: Cancer protective mechanisms of glucosinolate hydrolysis products and selenium. Integr Cancer Ther. 2004;3(1):5-12.[Abstract/Free Full Text]

4. Finley JW. Proposed criteria for assessing the efficacy of cancer reduction by plant foods enriched in carotenoids, glucosinolates, polyphenols and selenocompounds. Ann Bot (Lond). 2005;95(7):1075-1096.[Abstract/Free Full Text]

5. Wallig MA, Kingston S, Staack R, Jeffery EH. Induction of rat pancreatic glutathione S-transferase and quinone reductase activities by a mixture of glucosinolate breakdown derivatives found in Brussels sprouts. Food Chem Toxicol. 1998;36(5):365-373.[Medline]

6. Staack R, Kingston S, Wallig MA, Jeffery EH. A comparison of the individual and collective effects of four glucosinolate breakdown products from Brussels sprouts on induction of detoxification enzymes. Toxicol Appl Pharmacol. 1998;149(1):17-23.[Medline]

7. March TH, Jeffery EH, Wallig MA. The cruciferous nitrile, crambene, induces rat hepatic and pancreatic glutathione S-transferases. Toxicol Sci. 1998;442(4):82-90.

8. Kushad MM, Brown AF, Kurilich AC, Juvik JA, Klein BP, Wallig MA, Jeffery EH. Variation of glucosinolates in vegetable crops of Brassica oleracea. J Agric Food Chem. 1999;47(4):1541-1548.[Medline]

9. Fenwick GR, Heaney RK, Mullin WJ. Glucosinolates and their breakdown products in food and food plants. CRC Crit Rev Food Sci Nutr. 1983;18:123-201.

10. Kim YS, Milner JA. Targets for indole-3-carbinol in cancer prevention. J Nutr Biochem. 2005;16(2):65-73.[Medline]

11. Bradlow HL, Sepkovic DW, Telang NT, Osborne MP. Multifunctional aspects of the action of indole-3-carbinol as an antitumor agent. Ann NY Acad Sci. 1999;889:204-213.[Medline]

12. Bailey GS, Dashwood RH, Fon AT, Williams DE, Scanlan RA, Hendricks JD. Modulation of mycotoxin and nitrosamine carcinogenesis by indole-3-carbinol quantitative analysis of inhibition versus promotion. O’Neil IK Chen J Bartsch H eds. Modulation of mycotoxin and nitrosamine carcinogenesis by indole-3-carbinol quantitative analysis of inhibition versus promotion. Relevance to human cancer of N-nitroso compounds. :275-280 International Agency for Research on Cancer Lyon.

13. Johnson F, Huff J. Development of a multi-organ rat model for evaluation of chemopreventive agents: efficacy of indole-3-carbinol—certain health supplements may cause both carcinogenic and anticarcinogenic effects. Carcinogenesis. 2002;23(10):1767-1768.[Free Full Text]

14. Wallig MA, Jeffery EH. Enhancement of pancreatic and hepatic glutathione levels in rats during cyanohydroxybutene intoxication. Fund Appl Toxicol. 1990;14:144-159.[Medline]

15. Wallig MA, Kore AM, Crawshaw J, Jeffery EH. Separation of the toxic and glutathione-enhancing effects of the naturally occurring nitrile, cyanohydroxybutene. Fund Appl Toxicol. 1992;19:598-606.[Medline]

16. Nho CW, Jeffery EH. Crambene, a bioactive nitrile derived from glucosinolate hydrolysis, acts via the antioxidant response element to upregulate quinone reductase alone or synergistically with indole-3-carbinol. Toxicol Appl Pharmacol. 2004;198(1):40-48.[Medline]

17. Nguyen T, Sheratt PJ, Pickett CB. Regulatory mechanism controlling gene expression mediated by the antioxidant response element. Ann Rev Pharmacol Toxicol. 2003;43:233-260.[Medline]

18. Nho CW, Jeffery EH. The synergistic upregulation of phase II detoxification enzymes by glucosinolate breakdown products in cruciferous vegetables. Toxicol Appl Pharmacol. 2001;174:146-152.[Medline]

19. Liu RH. Potential synergy of phytochemicals in cancer prevention: Mechanism of action. J Nutr. 2004;134:3479S-3485S.[Abstract/Free Full Text]

20. Horvath PM, Ip C. Synergistic effect of vitamin E and selenium in the chemoprevention of mammary carcinogenesis in rats. Cancer Res. 1983;43(11):5335-5341.[Abstract/Free Full Text]

21. Curphey TJ, Kulhmann ET, Roebuck BB, Longnecker DS. Inhibition of pancreatic and liver carcinogenesis in rats by retinoid- and selenium-supplemented diets. Pancreas. 1988;3(1):36-40.[Medline]

22. Shklar B, Schwartz J, Trickler D, Cherverie SR. The effect of a mixture of beta-carotene, alpha-tocopherol, glutathione and ascorbic acid for cancer prevention. Nutr Cancer. 1983;20(2):145-151.

23. Zhou JR, You L, Zhong Y, Blackburn GL. Soy phytochemicals and tea bioactive components synergistically inhibit androgen-sensitive human prostate tumors in mice. J Nutr. 2003;133(2):516-521.[Abstract/Free Full Text]

24. Manson MM, Hudson EA, Ball HWJ, Barrett MC, Clark HL, Judah DJ, Verschoyle RD, Neal GE. Chemoprevention of aflatoxin B₁-induced carcinogenesis by indole-3-carbinaol in rat liver—predicting the outcome using early biomarkers. Carcinogenesis. 1998;19(10):1829-1836.[Abstract/Free Full Text]

25. Beutler TM, Bammler TK, Hayer JD, Eaton DL. Oltipraz-mediated changes in aflatoxin B₁ biotransformation in rat liver: Implications for human chemointervention. Cancer Res. 1996;56(10):2306-2313.[Abstract/Free Full Text]

26. Kim DJD, Lee KK, Han BS, Ahn B, Bae JH, Jang JJ. Biphasic modifying effect of indole-3-carbinol on diethylnitrosamine-induced preneoplastic glutathione-S-transferase placental form-positive liver-cell foci in Sprague-Dawley rats. Jpn J Cancer Res. 1994;85:578-583.[Medline]

27. Niedoborski T, Klein BP, Wallig MA. Rapid isolation and purification of 1-cyano-2-hydroxy-3-butene (crambene) from Crambe abyssinica seed meal using immiscible solvent extraction and high-performance liquid chromatography. J Agric Food Chem. 2001;49(8):3594-3599.[Medline]

28. American Institute of Nutrition. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr. 1993;123:1939-1951.[Abstract/Free Full Text]

29. Steele RGD, Torrie JH. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. Principles and procedures of statistics: a biometrical approach. 2nd ed. McGraw-Hill New York.

30. March TH, Jeffery EH, Wallig MA. Characterization of rat pancreatic glutathione S-transferases by chromatofocusing, reverse phase HPLC and immunohistochemistry. Pancreas. 1998;17(3):217-228.[Medline]

31. Image J [homepage on the Internet]. Bethesda (MD): Research Services Branch, National Institute of Mental Health [cited 2005 Aug 16]. Available from: http://rsb.info.nih.gov/ij/.

32. Lhoste ER, Roebuck BD, Brinck-Johnson T, Longenecker DS. Effect of castration and hormone replacement on azaserine-induced pancreatic carcinogenesis in male and female Fischer rats. Carcinogenesis. 1987;8:699-703.[Abstract/Free Full Text]

33. Keck AS, Staack R, Jeffery EH. The cruciferous nitrile crambene has bioactivity similar to sulforaphane when administered to Fischer 344 rats but is far less potent in cell culture. Nutr Cancer. 2002;42(2):233-240.[Medline]

34. Reichelt M, Brown PD, Schneider B, Oldham NJ, Stauber E, Tokuhisa J, Kliebenstein DJ, Mitchell-Olds T, Gershenzon J. Benzoic acid glucosinolate esters and other glucosinolates from Arabidopsis thaliana. Phytochemistry. 2002;59:663-671.[Medline]

35. Brown AF, Yousef GG, Jeffery EH, Klein BP, Kushad MM, Wallig MA, Juvik JA. Glucosinolate profiles in broccoli (Brassica oleracea L.): stability over environments and implications for cancer chemoprotection. J Hort Sci. 2002;127:807-813.

36. Wattenberg LW. Inhibition of chemical carcinogenesis. J Natl Cancer Inst. 1978;60(1):11-18.[Medline]

37. Jarrell VL, Epps D, Wallig MA, Jeffery EH. Antiproliferative effects of crambene, a nitrile common in cruciferous vegetables [abstract]. FASEB J. 1999;13(5):A918.

38. Guo Z, Smith TJ, Wang E, Sadrieh N, Ma Q, Thomas PE, Yang CS. Effects of phenylethylisothiocyanate, a carcinogenesis inhibitor, on xenobiotic-metabolizing enzymes and nitrosamine metabolism in rats. Carcinogenesis. 1992;13:2205-2210.[Abstract/Free Full Text]

39. Kelley MK, Bjeldanes LF. Modulation of glutathione S-transferase activity and isozyme pattern in liver and small intestine of rats fed goitrin- and T3-supplemented diets. Food Chem Toxicol. 1995;33(2):129-137.[Medline]

40. Kensler TW, Groopman JD, Eaton DL, Curphey TJ, Roebuck BD. Potent inhibition of aflatoxin-induced hepatic tumorigenesis by the monofunctional enzyme inducer 1,2-dithiole-3-thione. Carcinogenesis. 1992;13(1):95-100.[Abstract/Free Full Text]

41. Chu Y-F, Sun J, Wu X, Liu RH. Antioxidants and antiproliferative activities of vegetables. J Agric Food Chem. 2002;50:6910-6916.[Medline]

42. Banuelos GS. Irrigation of broccoli and canola with boron- and selenium-laden effluent. J Environ Qual. 2002;31(6):1802-1808.[Abstract/Free Full Text]

43. Finley JW, Keck AS, Robbins RJ, Hintze KJ. Selenium enrichment of broccoli: Interaction between selenium and secondary plant compounds. J Nutr. 2005;135:1236-1238.[Abstract/Free Full Text]

44. Wallig MA, Kuchan MJ, Milner JA. Differential effects of cyanohydroxybutene and selenium on normal and neoplastic canine mammary cells in vitro. Toxicol Lett. 1993;69:97-105.[Medline]

This article has been cited by other articles:

				G. Pappa, J. Strathmann, M. Lowinger, H. Bartsch, and C. Gerhauser Quantitative combination effects between sulforaphane and 3,3'-diindolylmethane on proliferation of human colon cancer cells in vitro Carcinogenesis, July 1, 2007; 28(7): 1471 - 1477. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wallig, M. A. Articles by Gossman, T. Search for Related Content PubMed PubMed Citation Articles by Wallig, M. A. Articles by Gossman, T.