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Effect of Ascorbigen on 7,12-Dimethylbenzanthracene-induced Mammary Tumor in Female Sprague-Dawley Rats
7.6 Effect of Ascorbigen on 7,12Dimethylbenzanthracene-induced Mammary Tumor in Female Sprague-Dawley Rats Xiaokang Ge,' Gad Rennert2 and Shmuel Yannai'* DEPARTMENT O F FOOD ENGINEERING AND BIOTECHNOLOGY, TECHNION - ISRAEL INSTITUTE O F TECHNOLOGY, HAIFA 32000, ISRAEL 'DEPARTMENT OF COMMUNITY MEDICINE AND EPIDEMIOLOGY, NATIONAL CANCER CONTROL CENTER, THE LADY DAVIS CARMEL MEDICAL CENTER, HAIFA, ISRAEL
1 Abstract Epidemiological studies have suggested that consumption of cruciferous vegetables may significantly decrease the risk of various cancers in humans. The anticarcinogenic activity of crucifers may result from the presence of active compounds in the vegetables. Ascorbigen is one of the indole derivatives formed from cruciferous vegetables. In this study, the effect of ascorbigen on the formation of 7,12-dimethyl[a]benzanthracene (DMBA)-induced mammary tumor was studied in female Sprague-Dawley rats. Rats were divided into four groups which received: (1) a single intubation of 15 mg DMBA without ascorbigen; (2) 15 mg DMBA and two daily intubations of 16 mg ascorbigen; (3) 15 mg DMBA and two intubations of 32.5 mg ascorbigen day-'; (4) 15 mg DMBA and two intubations of 65 mgday-' of ascorbigen. The first tumor appeared in rats treated only with DMBA was found 30 days later; while in all ascorbigen-treated groups it appeared after 40 days. After 90 days, 63% of rats receiving only DMBA showed mammary tumors. Rats receiving ascorbigen at dose levels of 32.5 mg day-' and 65 mg day-' had 33% and 39% incidence of mammary tumors, respectively, while rats receiving 16 mg day-' of ascorbigen did not show decreased tumor incidence. The results of this study suggests that ascorbigen may be a promising chemopreventive agent in DMBA-induced mammary carcinogenesis in rats. Correspondenceauthor: Fax: 00972-4-8320742
366
X . Ge, G.Rennert and S. Yannai
367
2 Introduction It has been shown that frequent consumption of vegetables decreases the risk for human cancer.' Identifying dietary photochemicals which have the ability to inhibit carcinogenesis has been a recent focus. Ascorbigen is a natural indole derivative from cruciferous vegetables such as cabbage, cauliflower, Brussels sprouts, broccoli. It is formed in damaged plants and in food processing after myrosinase is liberated. High-pressure liquid chromatography analysis found that the concentration of this compound in stomach juice is much higher than that of indole-3-carbinol, another major indole derivative formed in cruciferous vegetables, when the vegetable is consumed. Recent studies have suggested that ascorbigen has the ability to induce the hepatic microsomal cytochrome P450 enzymes and elevate the activity of C2 hydroxylation of estradiol.* The increase of the C2 hydroxylation of estrogens has been considered to have the potential to decrease the incidence of mammary tumors in animal^.^ However, the anticarcinogenic activity of this compound has not been studied yet. The purpose of this study was to investigate whether ascorbigen was effective in the inhibition of the formation of DMBA-induced mammary tumor in rats.
3 Materials and Methods 3.1 Chemicals Ascorbigen was synthesized according to the method of Kiss and N e ~ k o m . ~ 7,12-Dimethyl[a]anthracene (DMBA) and indole-3-carbinol were purchased from Sigma.
3.2 Animals Virgin female Sprague-Dawley rats were used in this study. The animals were housed in polycarbonate cages (two to five rats/cage), and were kept in a room lighted 12 h day-' and maintained at a temperature of 22 & 1 "C. Rodent diet (Koffolk 1949, purchased from Koffolk, Inc., Tel Aviv, Israel) and tap water were given ad libitum.Ascorbigen and the carcinogen, DMBA, were dissolved in dimethyl sulfoxide (DMSO), either alone or mixed together according to the different experimental protocols described below. The animals used here were approved by the 'Technion Committee for Care and Use of Laboratory Animals'.
3.3 Treatment of Animals Female Sprague-Dawley rats, at 48 days of age were divided into four groups randomized by weight. 0
Group 1. At 48 days of age, animals were fed by oesophageal intubation with 0.5 ml DMSO, once a day, for two days. On the third day (age, 50
368
0
0
0
Effect of Ascorbigen on 7,12-Dimethylbenranthracene-inducedMammary Tumor
days), animals were fed by one intubation of 15 mg DMBA in 0.5 ml DMSO. Group 2. At 48 days of age, animals were fed by intubation with 16 mg ascorbigen in 0.5 ml DMSO, once a day, for two days. On the third day (age, 50 days), animals were fed by intubation with both 16 mg ascorbigen and 15 mg DMBA in 0.5 ml DMSO. Group 3. At 48 days of age, animals were fed by intubation with 32.5 mg ascorbigen in 0.5 ml DMSO, once a day, for two days. On the third day (age, 50 days), animals were fed by intubation with both 32.5 mg ascorbigen and 15 mg DMBA in 0.5 ml DMSO. Group 4. At 48 days of age, animals were fed by intubation with 65 mg ascorbigen in 0.5 ml DMSO, once a day, for two days. On the third day (age, 50 days), animals were fed by intubation with both 65 mg ascorbigen and 15 mg DMBA in 0.5 ml DMSO.
The animals were weighed every one or two weeks. Rats were palpated for mammary tumors every one or two weeks starting 6 weeks after the day of administration of DMBA. The experiments were finished 90 days after the day of administration of DMBA. At the end of experiment, all the animals were sacrificed by COz asphyxiation.
4 Results The development of palpable mammary tumors in rats treated with DMBA only or DMBA with different doses of ascorbigen is shown in Figure 1 and Table 1. At 60 days after the day of given DMBA, tumor incidence was decreased in all ascorbigen treated groups. Table 1 shows that at 90 days after the day of given DMBA, 63% of the rats receiving DMBA only (group 1) showed mammary tumors. Rats receiving dose levels of 32.5 mg day-' (group 3) and 65 mg day-' (group 4) had a decreased incidence of mammary tumors, Table 1 Effect of ascorbigen on DMBA-initiated mammary tumors Number Group of rats Carcinogena Treatmentb 1 2 3
4
21 20 18 18
DMBA DMBA DMBA DMBA
Vehicle Ascorbigen, 16 mg day-' Ascorbigen, 32.5 mg day-' Ascorbigen, 65 mg day-'
Mammary tumor Average incidence (%)' no./rar 63 60 33d 39"
1.32 1.20 0.53' 0.12
~~~~
a
DMBA (15 mg/rat) was fed by intubation to female Sprague-Dawley rats at 50 days of age.
* Ascorbigen was given by intubation when rats were at 48 days of age for continuous 3 days; vehicle,
0.5 ml DMSO (dimethyl sulfoxide). 'Tumor incidence (%) at 90 days after the day of administration of DMBA. 'Different from group 1 . X2-test,p < 0.05. 'Different from group I . &test,p c 0.05.
369
X . Ge, G . Rennert and S . Yannai ~
65 mg/rat
AGE (days)
II
ASG+DMSO ASG+DMSO i)MBA+ASG+DMSO
I
1 ,
I 0
I
,
2/18 animals with tumor
6/18 animals with tumor
4/20 animals with tumor
12/20 animals with tumor
I
111
40 49 50
Control
7/18 animals
with tumor
32.5 mg/rat 16 mg/rat
4/18 animals
110
t
t
1,;;A+DMs0
13/27 animals with tumor
DMSO
with tumor
1
t
140
17/27 animals with tumor
Figure 1 Scheme for the study of the effect of ascorbigen on the formation of DMBAinitiated mammary tumors. AGE, the age of the anaimals. DMSO, dimethyl sulfoxide. DMBA, dimethylbenz[a]anthracene. ASG, ascorbigen
'
while rats receiving 16 mg day- dose level (group 2) did not show the decreased incidence (Table 1). Table 1 also shows that at the dose of 65 mgday-' the tumor incidence has not been further decreased compared to that occurred in 32.5 mg day-'. Figure 2 shows that the latency period of the mammary tumors occurring in rats that received 32.5 and 65 mg kg-' ascorbigen was also increased. The appearance of first tumor in the group receiving DMBA only was 30 days after the day of administration of DMBA, while in all the ascorbigen-treated groups the first tumor was found after 40 days.
5 Discussions In this study, ascorbigen has been investigated for its capacity to suppress carcinogen dimethylbenzanthracene (DMBA)-induced mammary tumor formation. The experiment described here demonstrates that ascorbigen has an activity against DMBA-initiated mammary tumor formation in rats. The carcinogen-initiated rat mammary tumor model has been extensively used to investigate the development of rat mammary tumors using diverse carcinogens and chemicals which modulate tumor formation (reviewed by Welsch5). Ascorbigen inhibited DMBA-induced tumor incidence at doses 32.5 mg/rat and 65 mg/rat. In this study, ascorbigen was given two days before DMBA was given. That means it was given before and during the tumor initiation stage. Compounds which decrease enzyme activity responsible for carcinogen activa-
370
Effect of Ascorbigen on 7,12-Dimethylbenzanthracene-inducedMammary Tumor
30
40
50
60
70
80
90
100
Days after DMBA Figure 2 Effect of ascorbigen on the time of appearance of DMBA-initiated mammary tumors. Different doses of ascorbigen and vehicle were intubated to rats at 48 days of agefor three days. A t SO days of age, all the rats were intubated a single dose of 15 mglrat of DMBA. The groups were: A Group I, DMBA; Group 2, DMBA + ascorbigen 16 mg day-' for 3 days; 0 Group 3, DMBA + ascorbigen 32.5 mg day-' for 3 days; Q Group 4 . DMBA +- ascorbigen 65 mg day-' for 3 days
tion, increase enzyme activity responsible for carcinogen detoxification, and scavenge amount of reactive ultimate carcinogen can lead to the inhibition of tumor initiation. Ascorbigen is a dietary ingredient and present in cruciferous vegetables. Mukhanov and colleagues have found that some ascorbigen derivatives inhibited the growth of transplanted adenocarcinoma of large intestine in athymic BALB/c mice. The stable and unstable transformation products of ascorbigen could determine the biological properties of the original compound. In gastric juice ascorbigen can be transformed to 5 1 1 -dihydroindole[3,2-b]carbazole,a compound with an affinity for the aromatic hydrocarbon.' It has recently been reported that when 2000 ppm ascorbigen was fed to female Sprague-Dawley rats, the activity of 2-hydroxylation of estradiol (cytochrome P450 1Al dependent) was induced in liver microsomes.* It has been reported that increased 2-hydroxylation of estradiol may be a result of the decreased mammary tumor incidence.' The alteration in cytochrome P450 1Al activity by ascorbigen may be responsible for its detoxification of carcinogens. Moreover, ascorbigen may also have an ability to sequester the reactive carcinogen. Our in vitro study has shown that ascorbigen may also act as antioxidant.* In conclusion, this study shows that ascorbigen may be an anticarcinogenic agent in rats. Although the exact mechanisms by which ascorbigen inhibited the DMBA-initiated mammary tumor formation are not completely understood, the beneficial effects of dietary ascorbigen in cancer prevention are being established.
X . Ge. G. Rennert and S. Yannai
371
6 References 1 Freudenheim JL, Marshall JR, Vena JE, Laughlin R, Brasure JR, Swanson MK, Nemoto T and Graham S. 1996. Premenopausal breast cancer risk and intake of vegetable, fruit and related nutrients. J Natl Cancer Inst 88,340-348. 2 Sepkovic DW, Bradlow HL, Michnovicz J, Murtezani S, Levy I and Osborne MP. 1994. Catechol estrogen production in rat microsomes after treatment with indole-3carbinol, ascorbigen, or p-naphthaflavone: A comparision of stable isotope dilution gas chromatography-mass spectrometry and radiometric methods. Steroids 59, 3 18323. 3 Bradlow HL, Michnovicz JJ, Telang T and Osborne M. 1991. Effects of dietary indole3-carbinol on estradiol metabolism and spontaneous mammary tumors in mice. Carcinogenesis 12, 171-1574. 4 Kiss G and Neukom H. 1966. Uber die Struktur des Ascorbigens. Hefv Chim Acta 49, 989-992. 5 Welsch CW. 1985. Host factors affecting the growth of carcinogen-induced rat mammary carcinogens: a review and tribute to Charles Brenton Huggins. Cancer Res 45,3415-3443. 6 Mukanov VI. 1984. Study of ascorbigen and its derivatives. Bioorg. Khim 10,544-559. 7 Preobrazhenskaya MN, Bukhman VM, Korolev AM and Efimov SA. 1993. Ascorbigen and other indole-derived compounds from Brassica vegetables and their analogs as anticarcinogenic and immunomodulating agents. Pharm Therapeutics 60,301-3 13. 8 Ge X, Karmansky I and Yannai S. 1999. Indole derivatives, 3,3’-diindolymethane and ascorbigen, inhibit oxidation of human low-density lipoproteins in vitro. Pharmacy Pharmacol Commun in press.
1 Abstract Epidemiological studies have suggested that consumption of cruciferous vegetables may significantly decrease the risk of various cancers in humans. The anticarcinogenic activity of crucifers may result from the presence of active compounds in the vegetables. Ascorbigen is one of the indole derivatives formed from cruciferous vegetables. In this study, the effect of ascorbigen on the formation of 7,12-dimethyl[a]benzanthracene (DMBA)-induced mammary tumor was studied in female Sprague-Dawley rats. Rats were divided into four groups which received: (1) a single intubation of 15 mg DMBA without ascorbigen; (2) 15 mg DMBA and two daily intubations of 16 mg ascorbigen; (3) 15 mg DMBA and two intubations of 32.5 mg ascorbigen day-'; (4) 15 mg DMBA and two intubations of 65 mgday-' of ascorbigen. The first tumor appeared in rats treated only with DMBA was found 30 days later; while in all ascorbigen-treated groups it appeared after 40 days. After 90 days, 63% of rats receiving only DMBA showed mammary tumors. Rats receiving ascorbigen at dose levels of 32.5 mg day-' and 65 mg day-' had 33% and 39% incidence of mammary tumors, respectively, while rats receiving 16 mg day-' of ascorbigen did not show decreased tumor incidence. The results of this study suggests that ascorbigen may be a promising chemopreventive agent in DMBA-induced mammary carcinogenesis in rats. Correspondenceauthor: Fax: 00972-4-8320742
366
X . Ge, G.Rennert and S. Yannai
367
2 Introduction It has been shown that frequent consumption of vegetables decreases the risk for human cancer.' Identifying dietary photochemicals which have the ability to inhibit carcinogenesis has been a recent focus. Ascorbigen is a natural indole derivative from cruciferous vegetables such as cabbage, cauliflower, Brussels sprouts, broccoli. It is formed in damaged plants and in food processing after myrosinase is liberated. High-pressure liquid chromatography analysis found that the concentration of this compound in stomach juice is much higher than that of indole-3-carbinol, another major indole derivative formed in cruciferous vegetables, when the vegetable is consumed. Recent studies have suggested that ascorbigen has the ability to induce the hepatic microsomal cytochrome P450 enzymes and elevate the activity of C2 hydroxylation of estradiol.* The increase of the C2 hydroxylation of estrogens has been considered to have the potential to decrease the incidence of mammary tumors in animal^.^ However, the anticarcinogenic activity of this compound has not been studied yet. The purpose of this study was to investigate whether ascorbigen was effective in the inhibition of the formation of DMBA-induced mammary tumor in rats.
3 Materials and Methods 3.1 Chemicals Ascorbigen was synthesized according to the method of Kiss and N e ~ k o m . ~ 7,12-Dimethyl[a]anthracene (DMBA) and indole-3-carbinol were purchased from Sigma.
3.2 Animals Virgin female Sprague-Dawley rats were used in this study. The animals were housed in polycarbonate cages (two to five rats/cage), and were kept in a room lighted 12 h day-' and maintained at a temperature of 22 & 1 "C. Rodent diet (Koffolk 1949, purchased from Koffolk, Inc., Tel Aviv, Israel) and tap water were given ad libitum.Ascorbigen and the carcinogen, DMBA, were dissolved in dimethyl sulfoxide (DMSO), either alone or mixed together according to the different experimental protocols described below. The animals used here were approved by the 'Technion Committee for Care and Use of Laboratory Animals'.
3.3 Treatment of Animals Female Sprague-Dawley rats, at 48 days of age were divided into four groups randomized by weight. 0
Group 1. At 48 days of age, animals were fed by oesophageal intubation with 0.5 ml DMSO, once a day, for two days. On the third day (age, 50
368
0
0
0
Effect of Ascorbigen on 7,12-Dimethylbenranthracene-inducedMammary Tumor
days), animals were fed by one intubation of 15 mg DMBA in 0.5 ml DMSO. Group 2. At 48 days of age, animals were fed by intubation with 16 mg ascorbigen in 0.5 ml DMSO, once a day, for two days. On the third day (age, 50 days), animals were fed by intubation with both 16 mg ascorbigen and 15 mg DMBA in 0.5 ml DMSO. Group 3. At 48 days of age, animals were fed by intubation with 32.5 mg ascorbigen in 0.5 ml DMSO, once a day, for two days. On the third day (age, 50 days), animals were fed by intubation with both 32.5 mg ascorbigen and 15 mg DMBA in 0.5 ml DMSO. Group 4. At 48 days of age, animals were fed by intubation with 65 mg ascorbigen in 0.5 ml DMSO, once a day, for two days. On the third day (age, 50 days), animals were fed by intubation with both 65 mg ascorbigen and 15 mg DMBA in 0.5 ml DMSO.
The animals were weighed every one or two weeks. Rats were palpated for mammary tumors every one or two weeks starting 6 weeks after the day of administration of DMBA. The experiments were finished 90 days after the day of administration of DMBA. At the end of experiment, all the animals were sacrificed by COz asphyxiation.
4 Results The development of palpable mammary tumors in rats treated with DMBA only or DMBA with different doses of ascorbigen is shown in Figure 1 and Table 1. At 60 days after the day of given DMBA, tumor incidence was decreased in all ascorbigen treated groups. Table 1 shows that at 90 days after the day of given DMBA, 63% of the rats receiving DMBA only (group 1) showed mammary tumors. Rats receiving dose levels of 32.5 mg day-' (group 3) and 65 mg day-' (group 4) had a decreased incidence of mammary tumors, Table 1 Effect of ascorbigen on DMBA-initiated mammary tumors Number Group of rats Carcinogena Treatmentb 1 2 3
4
21 20 18 18
DMBA DMBA DMBA DMBA
Vehicle Ascorbigen, 16 mg day-' Ascorbigen, 32.5 mg day-' Ascorbigen, 65 mg day-'
Mammary tumor Average incidence (%)' no./rar 63 60 33d 39"
1.32 1.20 0.53' 0.12
~~~~
a
DMBA (15 mg/rat) was fed by intubation to female Sprague-Dawley rats at 50 days of age.
* Ascorbigen was given by intubation when rats were at 48 days of age for continuous 3 days; vehicle,
0.5 ml DMSO (dimethyl sulfoxide). 'Tumor incidence (%) at 90 days after the day of administration of DMBA. 'Different from group 1 . X2-test,p < 0.05. 'Different from group I . &test,p c 0.05.
369
X . Ge, G . Rennert and S . Yannai ~
65 mg/rat
AGE (days)
II
ASG+DMSO ASG+DMSO i)MBA+ASG+DMSO
I
1 ,
I 0
I
,
2/18 animals with tumor
6/18 animals with tumor
4/20 animals with tumor
12/20 animals with tumor
I
111
40 49 50
Control
7/18 animals
with tumor
32.5 mg/rat 16 mg/rat
4/18 animals
110
t
t
1,;;A+DMs0
13/27 animals with tumor
DMSO
with tumor
1
t
140
17/27 animals with tumor
Figure 1 Scheme for the study of the effect of ascorbigen on the formation of DMBAinitiated mammary tumors. AGE, the age of the anaimals. DMSO, dimethyl sulfoxide. DMBA, dimethylbenz[a]anthracene. ASG, ascorbigen
'
while rats receiving 16 mg day- dose level (group 2) did not show the decreased incidence (Table 1). Table 1 also shows that at the dose of 65 mgday-' the tumor incidence has not been further decreased compared to that occurred in 32.5 mg day-'. Figure 2 shows that the latency period of the mammary tumors occurring in rats that received 32.5 and 65 mg kg-' ascorbigen was also increased. The appearance of first tumor in the group receiving DMBA only was 30 days after the day of administration of DMBA, while in all the ascorbigen-treated groups the first tumor was found after 40 days.
5 Discussions In this study, ascorbigen has been investigated for its capacity to suppress carcinogen dimethylbenzanthracene (DMBA)-induced mammary tumor formation. The experiment described here demonstrates that ascorbigen has an activity against DMBA-initiated mammary tumor formation in rats. The carcinogen-initiated rat mammary tumor model has been extensively used to investigate the development of rat mammary tumors using diverse carcinogens and chemicals which modulate tumor formation (reviewed by Welsch5). Ascorbigen inhibited DMBA-induced tumor incidence at doses 32.5 mg/rat and 65 mg/rat. In this study, ascorbigen was given two days before DMBA was given. That means it was given before and during the tumor initiation stage. Compounds which decrease enzyme activity responsible for carcinogen activa-
370
Effect of Ascorbigen on 7,12-Dimethylbenzanthracene-inducedMammary Tumor
30
40
50
60
70
80
90
100
Days after DMBA Figure 2 Effect of ascorbigen on the time of appearance of DMBA-initiated mammary tumors. Different doses of ascorbigen and vehicle were intubated to rats at 48 days of agefor three days. A t SO days of age, all the rats were intubated a single dose of 15 mglrat of DMBA. The groups were: A Group I, DMBA; Group 2, DMBA + ascorbigen 16 mg day-' for 3 days; 0 Group 3, DMBA + ascorbigen 32.5 mg day-' for 3 days; Q Group 4 . DMBA +- ascorbigen 65 mg day-' for 3 days
tion, increase enzyme activity responsible for carcinogen detoxification, and scavenge amount of reactive ultimate carcinogen can lead to the inhibition of tumor initiation. Ascorbigen is a dietary ingredient and present in cruciferous vegetables. Mukhanov and colleagues have found that some ascorbigen derivatives inhibited the growth of transplanted adenocarcinoma of large intestine in athymic BALB/c mice. The stable and unstable transformation products of ascorbigen could determine the biological properties of the original compound. In gastric juice ascorbigen can be transformed to 5 1 1 -dihydroindole[3,2-b]carbazole,a compound with an affinity for the aromatic hydrocarbon.' It has recently been reported that when 2000 ppm ascorbigen was fed to female Sprague-Dawley rats, the activity of 2-hydroxylation of estradiol (cytochrome P450 1Al dependent) was induced in liver microsomes.* It has been reported that increased 2-hydroxylation of estradiol may be a result of the decreased mammary tumor incidence.' The alteration in cytochrome P450 1Al activity by ascorbigen may be responsible for its detoxification of carcinogens. Moreover, ascorbigen may also have an ability to sequester the reactive carcinogen. Our in vitro study has shown that ascorbigen may also act as antioxidant.* In conclusion, this study shows that ascorbigen may be an anticarcinogenic agent in rats. Although the exact mechanisms by which ascorbigen inhibited the DMBA-initiated mammary tumor formation are not completely understood, the beneficial effects of dietary ascorbigen in cancer prevention are being established.
X . Ge. G. Rennert and S. Yannai
371
6 References 1 Freudenheim JL, Marshall JR, Vena JE, Laughlin R, Brasure JR, Swanson MK, Nemoto T and Graham S. 1996. Premenopausal breast cancer risk and intake of vegetable, fruit and related nutrients. J Natl Cancer Inst 88,340-348. 2 Sepkovic DW, Bradlow HL, Michnovicz J, Murtezani S, Levy I and Osborne MP. 1994. Catechol estrogen production in rat microsomes after treatment with indole-3carbinol, ascorbigen, or p-naphthaflavone: A comparision of stable isotope dilution gas chromatography-mass spectrometry and radiometric methods. Steroids 59, 3 18323. 3 Bradlow HL, Michnovicz JJ, Telang T and Osborne M. 1991. Effects of dietary indole3-carbinol on estradiol metabolism and spontaneous mammary tumors in mice. Carcinogenesis 12, 171-1574. 4 Kiss G and Neukom H. 1966. Uber die Struktur des Ascorbigens. Hefv Chim Acta 49, 989-992. 5 Welsch CW. 1985. Host factors affecting the growth of carcinogen-induced rat mammary carcinogens: a review and tribute to Charles Brenton Huggins. Cancer Res 45,3415-3443. 6 Mukanov VI. 1984. Study of ascorbigen and its derivatives. Bioorg. Khim 10,544-559. 7 Preobrazhenskaya MN, Bukhman VM, Korolev AM and Efimov SA. 1993. Ascorbigen and other indole-derived compounds from Brassica vegetables and their analogs as anticarcinogenic and immunomodulating agents. Pharm Therapeutics 60,301-3 13. 8 Ge X, Karmansky I and Yannai S. 1999. Indole derivatives, 3,3’-diindolymethane and ascorbigen, inhibit oxidation of human low-density lipoproteins in vitro. Pharmacy Pharmacol Commun in press.