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Prevention of Cyclophosphamide-induced Micronucleus Formation in Mouse Bone Marrow by Indole-3-carbinol1
Food and Chemical Toxicology 36 (1998) 975±977
Prevention of Cyclophosphamide-induced Micronucleus Formation in Mouse Bone Marrow by Indole-3-carbinol* R. C. AGRAWAL{ and S. KUMAR Environmental Carcinogenesis Laboratory, Industrial Toxicology Research Centre, Post Box No. 80, Mahatma Gandhi Marg, Lucknow-226 001, India (Accepted 17 February 1998) AbstractÐIndole-3-carbinol (I3C) is a glucobrassicin derivative isolated from cruciferous vegetables. In this study, the protective eect of I3C is reported against cyclophosphamide (CP)-induced micronuclei formation in mouse bone marrow cells. The three test doses, namely 500, 250 and 125 mg/kg body weight of I3C provided protection when given 48 hr prior to the single ip administration of cyclophosphamide (50 mg/kg). The ecacy of the test doses of I3C was also evaluated using a lower dose of CP (25 mg/kg body weight). A signi®cant inhibition in micronuclei formation was noticed with I3C at 250 and 125 mg/kg body weight dose. I3C could not induce micronuclei formation at the test doses 500 and 250 mg/kg body weight. I3C, therefore seems to have a preventive potential against CP-induced micronuclei formation in Swiss mouse bone marrow cells. # 1998 Elsevier Science Ltd. All rights reserved Keywords: mutagenicity; indole-3-carbinol; cyclophosphamide; bone marrow; micronucleus. Abbreviations: CP=cyclophosphamide; I3C=indole-3-carbinol.
Introduction Epidemiological data indicate that consumption of cruciferous vegetables is associated with a decreased incidence of cancer in human population (Graham, 1983; Hirayama, 1986). The anticarcinogenic properties of cruciferous vegetables and isolated compounds have been studied in several investigations where animals were ®rst fed diet rich with cruciferous vegetables and then exposed to various carcinogens (Boyd et al., 1982; Stoewsand et al., 1978; Wattenberg, 1983). The compounds involved in inhibition of carcinogenesis were found to be I3C and 3-3 dienolymethane (Loub et al., 1975). These compounds inhibit the benzo[a]pyrene-induced forestomach tumorigenesis. The naturally occurring indoles also reduce the benzo[a]pyrene-induced sister chromatid exchange (SCE) frequencies in vitro in primary chick embryo hepatocytes (Jongen et al., 1989). Conversely, there are reports that I3C enhanced liver and thyroid gland neoplastic development when given during the promotional stage of the rat multi-organ carcinogenesis test system. (Dashwood et al., 1991; Kim et al., 1997). I3C induced ornithine dicarboxylase activity in mouse *ITRC communication no. 1967. {Author for correspondence.
epidermis and thus presented potential for enhancement of tumour promotion (Birt et al., 1986). Studies reporting preventive role of I3C against the mutagenic changes and chemical carcinogenesis are limited and are of considerable importance with respect to nutrition and cancer. Therefore, we undertook the evaluation of the preventive eect of I3C using the micronucleus assay. The bone marrow micronucleus assay has being carried out in this laboratory for several years (Agrawal et al., 1996 and 1997; Agrawal and Mehrotra, 1997a,b).
Materials and Methods I3C and CP were purchased from Sigma Chemical Co. (St Louis, MO, USA). Other Reagent grade chemicals were procured locally. Male Swiss albino mice, 6±8-wk old and weighing 15±20 g, were obtained from the animal colony of the Industrial Toxicology Research Centre, Lucknow. Animals were housed in plastic cages and provided standard pellet diet and water ad lib. For the micronucleus assay, the requisite dose of I3C was dissolved in 5 ml dimethyl sulfoxide (DMSO) and administered as single ip dose, 0.2 ml/ mouse 48 hr prior to the CP administration, to six animals. Control mice were injected with an equal
0278-6915/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(98)00032-5
976
R. C. Agrawal and S. Kumar
volume of vehicle alone. The positive control group also received a single ip injection of 50 and 25 mg/ kg CP in 0.9% saline. The animals were killed 24 hr after the CP administration by cervical dislocation, and slides of bone marrow were prepared essentially as described by Schmid (1975) and modi®ed by Aron et al. (1989). After staining with May± Gruenwald and Giemsa, a total of 2000 cells were scored at a magni®cation of 1000 (100 10 ) for each animal. The data are expressed as the average number of micronucleated cells/thousand (polychromatid erythrocytes (PCE) cells/animal (2SE) for a group of six animals. The results were compared with the vehicle control group using Student's onetailed t-test with signi®cance determined at P < 0.05.
Results I3C administered ip at 500, 250 and 125 mg/kg body weight was found to inhibit the micronuclei formation induced by CP given ip at 25 and 50 mg/ kg body weight. A lower dose of I3C (62.5 mg/kg body weight) remained ineective (Table 1). A dose-dependent response was remarkable and statistically signi®cant. In the positive control group CP induced micronuclei formation at both 25 and 50 mg/kg body weight. It was noteworthy that dierent doses of I3C and CP used in the present experiments were not cytotoxic for PCE/NCE (normochromatid erythrocytes) ratio in I3C-treated and positive controls as compared to the solvent control group, which remained unchanged. I3C was ineective at 62.5 mg/kg dose level even with the 25 mg/kg body weight dose of CP. I3C failed to induce micronuclei formation at all the test dose levels (Table 1). Table 1. Modulation of mutagenicity by I3C induced by CP in bone marrow of Swiss mice Treatment (ip) CP (50 mg/kg) I3Ca+CP a. 500 mg/kg + 50 mg/kg b. 250 mg/kg + 50 mg/kg c. 125 mg/kg + 50 mg/kg d. 62.5 mg/kg + 50 mg/kg CP (25 mg/kg) I3Ca+CP a. 250 mg/kg + 25 mg/kg b. 125 mg/kg + 25 mg/kg c. 62.5 mg/kg + 25 mg/kg I3Ca a. 500 mg/kg b. 250 mg/kg Solvent control (DMSO)
MN PCE 2SE
PCE/NCE ratio + SE
3.32 0.56
0.455 2 0.219
1.02 0.40* 0.92 0.41* 0.52 0.30* 2.22 0.30 2.02 0.25
0.301 2 0.123 0.280 2 0.148 0.317 2 0.157 0.260 2 0.140 0.415 2 0.075
0.25 2 0.04* 0.752 0.05* 1.58 2 0.64
0.202 2 0.087 0.141 2 0.040 0.301 2 0.102
0.62 0.03 0.52 0.07 0.452 0.20
0.38 20.080 0.36 20.20 0.60 20.200
*Denotes statistically signi®cant in Student's t-test at P < 0.050, when compared with respective positive control group. Each groups consists of six animals. a I3C was administered 48 hr prior to the CP or vehicle administration. PCE/NCE denotes polychromatid erythrocytes/normochromatid erythrocytes).
Discussion In our study, a single ip administration of I3C resulted in a dose-dependent inhibition of micronuclei formation induced by CP in mouse bone marrow cells. Its eectivity was observed at two clastogenic dose levels of CP. I3C, when tested for mutagenic eect at various test dose levels, failed to induce micronucleus formation. The non-mutagenic eect of I3C has been observed also in Salmonella typhimurium and in Chinese hamster ovary test systems (Kuo et al., 1992; Takahashi et al., 1995). We have also found an antimutagenic eect of I3C during the assessment of mutagenic potential of propoxur and its modulation by I3C (Agrawal et al., 1997). The anticarcinogenic eect of I3C is also reported in polycyclic aromatic hydrocarbon (PAH)-induced tumorigenesis in rats (Wattenberg and Loub, 1978). All these reports indicate the protective eect of the glucobrassicin derivative. The mechanism is unknown; however, it may be linked to the observations that I3C induces phase I and phase II biotransformation reactions, namely cytochrome P-450 and glutathione S-transferase activities (Sparnin et al., 1986). Few more reports on such an eect of I3C on biotransformation reactions are available (Baldwin et al., 1992; Shertzer and Sainsbury, 1991; Wortelboer et al., 1992). Induction of these enzymes which constitute Phase I and II activity of the mixed-function oxidase function for metabolism and disposition of xenobiotics, can be understood to play a signi®cant role in the observed antimutagenic eects of I3C. The amount of I3C present in cruciferous vegetables is reportedly in the range of 410 to 1090 mg/kg fruit weight (Fenwick and Heany, 1983). It is expected that a person on vegetarian diet consumes about 100 g cruciferous vegetables/day and the daily intake of I3C may fall with in the range of 41 to 109 mg/person. Therefore, the dose used is although about 20 times higher than the actual consumption in humans. It is noteworthy that the employed dose was eective against a noted mutagen. These results are important with respect to preventive aspects of diet and nutrition in chemical carcinogenesis. AcknowledgementÐThe authors are grateful to Dr P.K. Seth, Director of ITRC, for his encouragement and providing facilities to carry out this work. REFERENCES
Agrawal R. C. and Mehrotra N. K. (1997a) Eect of diuron on the germ cells of mice using dominant lethal test. Indian Journal of Experimental Biology 35, 1256± 1257. Agrawal R. C. and Mehrotra N. K. (1997b) Assessment of mutagenic potential of propoxur and its modulation by indole-3-carbinol. Food and Chemical Toxicology 35, 1081±1984. Agrawal R. C., Kumar S. and Mehrotra N. K. (1996) Micronucleus induction by diuron in mouse bone marrow. Toxicology Letters 86, 1±4.
Prevention of mutagenicity by indole-3-carbinol Agrawal R. C., Shukla Y. and Mehrotra N. K. (1997) Assessment of mutagenic potential of thiram. Food and Chemical Toxicology 35, 523±525. Aron C. S., Sorg R. and Zimmer D. (1989) The mouse bone marrow micronucleus test. Evaluation of 21 drug candidates. Mutation Research 223, 129±140. Baldwin W. S. and Leblanc G. A. (1992) The anticarcinogenic plant compound indole-3-carbinol dierentially modulate P450 mediated steroid hydroxylase activities in mice. Chemico±Biological Interaction 83, 155±169. Birt D. F., Walker B., Tibbels M. G. and Bresnick E. (1986) Antimutagenesis and antipromotion by apigenin, robenetin and indole-3-carbinol. Carcinogenesis 7, 959±963. Boyd J. N., Babish J. G. and Stoewsand G. S. (1982) Modi®cation by beet and cabbage diet of a¯atoxin B1 induced rat plasma a-foetoprotein elevation, hepatic tumorigenesis and mutagenicity of urine. Food and Chemical Toxicology 20, 47±52. Dashwood R. H., Fong A. T., Williams D. E., Hendricks J. D. and Bailey G. S. (1991) Promotion of a¯atoxin B1 carcinogenesis by the natural tumour modulator indole3-carbinol: in¯uence of dose, duration and intermittent exposure on indole-3-carbinol promotional potency. Cancer Research 51, 2362±2365. Fenwick G. R. and Heany R. K. (1983) Glucosinolates and their breakdown products in cruciferous crops, foods and feedingstu. Food Chemistry 11, 249±271. Graham S. (1983) Results of case control studies of diet and cancer in Bualo, New York. Cancer Research 43, 2409. Hirayama T. (1986) Diet and cancer: feasibility and importance of prospective cohort study. In Diet and human carcinogenesis, ed. J. V. Jossens, M. J. Hill and J. Geboers, 191. Proceedings of the Second ECP Symposium. Aarhus, Denmark. pp. 191. Excerpta Medica, Amsterdam. Jongen W. M. F., Topp R. J., Wienk K. J. H. and Homan E. C. (1989) Modulating eects of naturally occurring indoles on SCE induction depend largely on the type of mutagen. Mutation Research 222, 263±269. Kim D. J., Han B. S., Ahn B., Hasegawa R., Shirai T., Ito N. and Tsuda N. (1997) Enhancement by indole-3carbinol of liver and thyroid gland neoplastic develop-
977
ment in a rat medium term multiorgan carcinogenesis model. Carcinogenesis 7, 371±386. Kuo M. L., Lee K. C. and Lin J. K. (1992) Genotoxicities of nitroprens and their modulation by apegenin, tannic acid, ellagic acid and indole-3-carbinol in the Salmonella and CHO systems. Mutation Research 270, 87±95. Loub W. D., Wattenberg L. W. and Davis D. W. (1975) Aryl hydrocarbon hydroxylase induction in rat tissue by naturally occurring indoles of cruciferous plants. Journal of the National Cancer Institute 54, 985. Schmid W. (1975) The micronucleus test. Mutation Research 31, 9±15. Shertzer H. G. and Sainsbury M. C. (1991) Intrinsic acute toxicity and hepatic enzyme inducing properties of the chemoprotectants, indole-3-carbinol and 5,10-dihydroindeno[1,2-b]indole in mice. Food and Chemical Toxicology 29, 237±249. Sparnin V. L., Mott A. W., Barang G. and Wattenberg L. W. (1986) Eect of allyl methyl trisul®de on glutathione-S-transferase activity and B(a)P induced neoplasia in mice. Nutrition and Cancer 8, 211±215. Stoewsand G. S., Babish J. P. and Wimberley H. C. (1978) Inhibition of hepatic toxicities from polybrominated biphenyl and a¯atoxin B1 in rats fed cauli¯ower. Journal of Environmental Pathology and Toxicology 2, 399. Takahashi N., Dashwood R. H., Bjeldanes L. F., Williams D. E. and Bailey G. S. (1995) Mechanisms of indole-3carbinol (I3C) anticarcinogenesis: inhibition of a¯atoxin B1-DNA adduction and mutagenesis by I3C acid condensation products. Food and Chemical Toxicology 33, 851±857. Wattenberg L. W. (1983) Inhibition of neoplasia by minor dietary constituents. Cancer Research 43, 2448. Wattenberg L. W. and Loub W. D. (1978) Inhibition of polycyclic aromatic hydrocarbon induced neoplasia by naturally occurring indoles. Cancer Research 34, 1410± 1413. Wortelboer H. M., van der Linden E. C. M., de Kruif C. A., Noordhoeck J., Blauuboer B. J., van Bladeren P. J. and Falke H. E. (1992) Eects of indole-3-carbinol on biotransformation enzymes in the rat in vivo changes in liver and small intestinal mucosa in comparison with primary hepatocyte cultures. Food and Chemical Toxicology 30, 589±599.
Prevention of Cyclophosphamide-induced Micronucleus Formation in Mouse Bone Marrow by Indole-3-carbinol* R. C. AGRAWAL{ and S. KUMAR Environmental Carcinogenesis Laboratory, Industrial Toxicology Research Centre, Post Box No. 80, Mahatma Gandhi Marg, Lucknow-226 001, India (Accepted 17 February 1998) AbstractÐIndole-3-carbinol (I3C) is a glucobrassicin derivative isolated from cruciferous vegetables. In this study, the protective eect of I3C is reported against cyclophosphamide (CP)-induced micronuclei formation in mouse bone marrow cells. The three test doses, namely 500, 250 and 125 mg/kg body weight of I3C provided protection when given 48 hr prior to the single ip administration of cyclophosphamide (50 mg/kg). The ecacy of the test doses of I3C was also evaluated using a lower dose of CP (25 mg/kg body weight). A signi®cant inhibition in micronuclei formation was noticed with I3C at 250 and 125 mg/kg body weight dose. I3C could not induce micronuclei formation at the test doses 500 and 250 mg/kg body weight. I3C, therefore seems to have a preventive potential against CP-induced micronuclei formation in Swiss mouse bone marrow cells. # 1998 Elsevier Science Ltd. All rights reserved Keywords: mutagenicity; indole-3-carbinol; cyclophosphamide; bone marrow; micronucleus. Abbreviations: CP=cyclophosphamide; I3C=indole-3-carbinol.
Introduction Epidemiological data indicate that consumption of cruciferous vegetables is associated with a decreased incidence of cancer in human population (Graham, 1983; Hirayama, 1986). The anticarcinogenic properties of cruciferous vegetables and isolated compounds have been studied in several investigations where animals were ®rst fed diet rich with cruciferous vegetables and then exposed to various carcinogens (Boyd et al., 1982; Stoewsand et al., 1978; Wattenberg, 1983). The compounds involved in inhibition of carcinogenesis were found to be I3C and 3-3 dienolymethane (Loub et al., 1975). These compounds inhibit the benzo[a]pyrene-induced forestomach tumorigenesis. The naturally occurring indoles also reduce the benzo[a]pyrene-induced sister chromatid exchange (SCE) frequencies in vitro in primary chick embryo hepatocytes (Jongen et al., 1989). Conversely, there are reports that I3C enhanced liver and thyroid gland neoplastic development when given during the promotional stage of the rat multi-organ carcinogenesis test system. (Dashwood et al., 1991; Kim et al., 1997). I3C induced ornithine dicarboxylase activity in mouse *ITRC communication no. 1967. {Author for correspondence.
epidermis and thus presented potential for enhancement of tumour promotion (Birt et al., 1986). Studies reporting preventive role of I3C against the mutagenic changes and chemical carcinogenesis are limited and are of considerable importance with respect to nutrition and cancer. Therefore, we undertook the evaluation of the preventive eect of I3C using the micronucleus assay. The bone marrow micronucleus assay has being carried out in this laboratory for several years (Agrawal et al., 1996 and 1997; Agrawal and Mehrotra, 1997a,b).
Materials and Methods I3C and CP were purchased from Sigma Chemical Co. (St Louis, MO, USA). Other Reagent grade chemicals were procured locally. Male Swiss albino mice, 6±8-wk old and weighing 15±20 g, were obtained from the animal colony of the Industrial Toxicology Research Centre, Lucknow. Animals were housed in plastic cages and provided standard pellet diet and water ad lib. For the micronucleus assay, the requisite dose of I3C was dissolved in 5 ml dimethyl sulfoxide (DMSO) and administered as single ip dose, 0.2 ml/ mouse 48 hr prior to the CP administration, to six animals. Control mice were injected with an equal
0278-6915/98/$19.00+0.00 # 1998 Elsevier Science Ltd. All rights reserved. Printed in Great Britain PII S0278-6915(98)00032-5
976
R. C. Agrawal and S. Kumar
volume of vehicle alone. The positive control group also received a single ip injection of 50 and 25 mg/ kg CP in 0.9% saline. The animals were killed 24 hr after the CP administration by cervical dislocation, and slides of bone marrow were prepared essentially as described by Schmid (1975) and modi®ed by Aron et al. (1989). After staining with May± Gruenwald and Giemsa, a total of 2000 cells were scored at a magni®cation of 1000 (100 10 ) for each animal. The data are expressed as the average number of micronucleated cells/thousand (polychromatid erythrocytes (PCE) cells/animal (2SE) for a group of six animals. The results were compared with the vehicle control group using Student's onetailed t-test with signi®cance determined at P < 0.05.
Results I3C administered ip at 500, 250 and 125 mg/kg body weight was found to inhibit the micronuclei formation induced by CP given ip at 25 and 50 mg/ kg body weight. A lower dose of I3C (62.5 mg/kg body weight) remained ineective (Table 1). A dose-dependent response was remarkable and statistically signi®cant. In the positive control group CP induced micronuclei formation at both 25 and 50 mg/kg body weight. It was noteworthy that dierent doses of I3C and CP used in the present experiments were not cytotoxic for PCE/NCE (normochromatid erythrocytes) ratio in I3C-treated and positive controls as compared to the solvent control group, which remained unchanged. I3C was ineective at 62.5 mg/kg dose level even with the 25 mg/kg body weight dose of CP. I3C failed to induce micronuclei formation at all the test dose levels (Table 1). Table 1. Modulation of mutagenicity by I3C induced by CP in bone marrow of Swiss mice Treatment (ip) CP (50 mg/kg) I3Ca+CP a. 500 mg/kg + 50 mg/kg b. 250 mg/kg + 50 mg/kg c. 125 mg/kg + 50 mg/kg d. 62.5 mg/kg + 50 mg/kg CP (25 mg/kg) I3Ca+CP a. 250 mg/kg + 25 mg/kg b. 125 mg/kg + 25 mg/kg c. 62.5 mg/kg + 25 mg/kg I3Ca a. 500 mg/kg b. 250 mg/kg Solvent control (DMSO)
MN PCE 2SE
PCE/NCE ratio + SE
3.32 0.56
0.455 2 0.219
1.02 0.40* 0.92 0.41* 0.52 0.30* 2.22 0.30 2.02 0.25
0.301 2 0.123 0.280 2 0.148 0.317 2 0.157 0.260 2 0.140 0.415 2 0.075
0.25 2 0.04* 0.752 0.05* 1.58 2 0.64
0.202 2 0.087 0.141 2 0.040 0.301 2 0.102
0.62 0.03 0.52 0.07 0.452 0.20
0.38 20.080 0.36 20.20 0.60 20.200
*Denotes statistically signi®cant in Student's t-test at P < 0.050, when compared with respective positive control group. Each groups consists of six animals. a I3C was administered 48 hr prior to the CP or vehicle administration. PCE/NCE denotes polychromatid erythrocytes/normochromatid erythrocytes).
Discussion In our study, a single ip administration of I3C resulted in a dose-dependent inhibition of micronuclei formation induced by CP in mouse bone marrow cells. Its eectivity was observed at two clastogenic dose levels of CP. I3C, when tested for mutagenic eect at various test dose levels, failed to induce micronucleus formation. The non-mutagenic eect of I3C has been observed also in Salmonella typhimurium and in Chinese hamster ovary test systems (Kuo et al., 1992; Takahashi et al., 1995). We have also found an antimutagenic eect of I3C during the assessment of mutagenic potential of propoxur and its modulation by I3C (Agrawal et al., 1997). The anticarcinogenic eect of I3C is also reported in polycyclic aromatic hydrocarbon (PAH)-induced tumorigenesis in rats (Wattenberg and Loub, 1978). All these reports indicate the protective eect of the glucobrassicin derivative. The mechanism is unknown; however, it may be linked to the observations that I3C induces phase I and phase II biotransformation reactions, namely cytochrome P-450 and glutathione S-transferase activities (Sparnin et al., 1986). Few more reports on such an eect of I3C on biotransformation reactions are available (Baldwin et al., 1992; Shertzer and Sainsbury, 1991; Wortelboer et al., 1992). Induction of these enzymes which constitute Phase I and II activity of the mixed-function oxidase function for metabolism and disposition of xenobiotics, can be understood to play a signi®cant role in the observed antimutagenic eects of I3C. The amount of I3C present in cruciferous vegetables is reportedly in the range of 410 to 1090 mg/kg fruit weight (Fenwick and Heany, 1983). It is expected that a person on vegetarian diet consumes about 100 g cruciferous vegetables/day and the daily intake of I3C may fall with in the range of 41 to 109 mg/person. Therefore, the dose used is although about 20 times higher than the actual consumption in humans. It is noteworthy that the employed dose was eective against a noted mutagen. These results are important with respect to preventive aspects of diet and nutrition in chemical carcinogenesis. AcknowledgementÐThe authors are grateful to Dr P.K. Seth, Director of ITRC, for his encouragement and providing facilities to carry out this work. REFERENCES
Agrawal R. C. and Mehrotra N. K. (1997a) Eect of diuron on the germ cells of mice using dominant lethal test. Indian Journal of Experimental Biology 35, 1256± 1257. Agrawal R. C. and Mehrotra N. K. (1997b) Assessment of mutagenic potential of propoxur and its modulation by indole-3-carbinol. Food and Chemical Toxicology 35, 1081±1984. Agrawal R. C., Kumar S. and Mehrotra N. K. (1996) Micronucleus induction by diuron in mouse bone marrow. Toxicology Letters 86, 1±4.
Prevention of mutagenicity by indole-3-carbinol Agrawal R. C., Shukla Y. and Mehrotra N. K. (1997) Assessment of mutagenic potential of thiram. Food and Chemical Toxicology 35, 523±525. Aron C. S., Sorg R. and Zimmer D. (1989) The mouse bone marrow micronucleus test. Evaluation of 21 drug candidates. Mutation Research 223, 129±140. Baldwin W. S. and Leblanc G. A. (1992) The anticarcinogenic plant compound indole-3-carbinol dierentially modulate P450 mediated steroid hydroxylase activities in mice. Chemico±Biological Interaction 83, 155±169. Birt D. F., Walker B., Tibbels M. G. and Bresnick E. (1986) Antimutagenesis and antipromotion by apigenin, robenetin and indole-3-carbinol. Carcinogenesis 7, 959±963. Boyd J. N., Babish J. G. and Stoewsand G. S. (1982) Modi®cation by beet and cabbage diet of a¯atoxin B1 induced rat plasma a-foetoprotein elevation, hepatic tumorigenesis and mutagenicity of urine. Food and Chemical Toxicology 20, 47±52. Dashwood R. H., Fong A. T., Williams D. E., Hendricks J. D. and Bailey G. S. (1991) Promotion of a¯atoxin B1 carcinogenesis by the natural tumour modulator indole3-carbinol: in¯uence of dose, duration and intermittent exposure on indole-3-carbinol promotional potency. Cancer Research 51, 2362±2365. Fenwick G. R. and Heany R. K. (1983) Glucosinolates and their breakdown products in cruciferous crops, foods and feedingstu. Food Chemistry 11, 249±271. Graham S. (1983) Results of case control studies of diet and cancer in Bualo, New York. Cancer Research 43, 2409. Hirayama T. (1986) Diet and cancer: feasibility and importance of prospective cohort study. In Diet and human carcinogenesis, ed. J. V. Jossens, M. J. Hill and J. Geboers, 191. Proceedings of the Second ECP Symposium. Aarhus, Denmark. pp. 191. Excerpta Medica, Amsterdam. Jongen W. M. F., Topp R. J., Wienk K. J. H. and Homan E. C. (1989) Modulating eects of naturally occurring indoles on SCE induction depend largely on the type of mutagen. Mutation Research 222, 263±269. Kim D. J., Han B. S., Ahn B., Hasegawa R., Shirai T., Ito N. and Tsuda N. (1997) Enhancement by indole-3carbinol of liver and thyroid gland neoplastic develop-
977
ment in a rat medium term multiorgan carcinogenesis model. Carcinogenesis 7, 371±386. Kuo M. L., Lee K. C. and Lin J. K. (1992) Genotoxicities of nitroprens and their modulation by apegenin, tannic acid, ellagic acid and indole-3-carbinol in the Salmonella and CHO systems. Mutation Research 270, 87±95. Loub W. D., Wattenberg L. W. and Davis D. W. (1975) Aryl hydrocarbon hydroxylase induction in rat tissue by naturally occurring indoles of cruciferous plants. Journal of the National Cancer Institute 54, 985. Schmid W. (1975) The micronucleus test. Mutation Research 31, 9±15. Shertzer H. G. and Sainsbury M. C. (1991) Intrinsic acute toxicity and hepatic enzyme inducing properties of the chemoprotectants, indole-3-carbinol and 5,10-dihydroindeno[1,2-b]indole in mice. Food and Chemical Toxicology 29, 237±249. Sparnin V. L., Mott A. W., Barang G. and Wattenberg L. W. (1986) Eect of allyl methyl trisul®de on glutathione-S-transferase activity and B(a)P induced neoplasia in mice. Nutrition and Cancer 8, 211±215. Stoewsand G. S., Babish J. P. and Wimberley H. C. (1978) Inhibition of hepatic toxicities from polybrominated biphenyl and a¯atoxin B1 in rats fed cauli¯ower. Journal of Environmental Pathology and Toxicology 2, 399. Takahashi N., Dashwood R. H., Bjeldanes L. F., Williams D. E. and Bailey G. S. (1995) Mechanisms of indole-3carbinol (I3C) anticarcinogenesis: inhibition of a¯atoxin B1-DNA adduction and mutagenesis by I3C acid condensation products. Food and Chemical Toxicology 33, 851±857. Wattenberg L. W. (1983) Inhibition of neoplasia by minor dietary constituents. Cancer Research 43, 2448. Wattenberg L. W. and Loub W. D. (1978) Inhibition of polycyclic aromatic hydrocarbon induced neoplasia by naturally occurring indoles. Cancer Research 34, 1410± 1413. Wortelboer H. M., van der Linden E. C. M., de Kruif C. A., Noordhoeck J., Blauuboer B. J., van Bladeren P. J. and Falke H. E. (1992) Eects of indole-3-carbinol on biotransformation enzymes in the rat in vivo changes in liver and small intestinal mucosa in comparison with primary hepatocyte cultures. Food and Chemical Toxicology 30, 589±599.