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Carcinogenesis vol.18 no.2 pp.377–381, 1997

Enhancement by indole-3-carbinol of liver and thyroid gland neoplastic development in a rat medium-term multiorgan carcinogenesis model

Dae Joong Kim1,2,5, Beom Seok Han2, Byeongwoo Ahn2, Ryohei Hasegawa3, Tomoyuki Shirai3, Nobuyuki Ito4 and Hiroyuki Tsuda1 1Chemotherapy

Division, National Cancer Centre Research Institute, 5–1–1 Tsukiji, Chuo-ku, Tokyo 104, Japan; 2Department of Pathology, National Institute of Safety Research, Nokbun-dong, Eunpyung-ku, Seoul 122–020, Korea; 3First Department of Pathology, Nagoya City University Medical School, and 4Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467, Japan 5To

whom correspondence should be addressed

The modification potential of indole-3-carbinol (I3C), a naturally occurring compound found in cruciferous vegetables, on neoplastic development was assessed using a rat medium-term multiorgan carcinogenesis model. Onehundred male Sprague–Dawley (SD) rats were randomly divided into three groups and sequentially treated with diethylnitrosamine (DEN; 100 mg/kg b.w., a single i.p.), N-methyl-N-nitrosourea (MNU; 20 mg/kg b.w., four times i.p., at days 5, 8, 11 and 14), and dihydroxy-di-N-propylnitrosamine (DHPN; 0.1% in the drinking water during weeks 1 and 3) (DMD treatment; groups 1 and 2) or the vehicles alone (group 3) in the first 3-week initiation period.

Animals of groups 1 and 3 were then given diet containing 0.25% I3C from week 4 until week 24, followed by a return to basal diet for 28 weeks, and subgroups were killed at weeks 24 and 52. I3C caused significant increases in both number (no./cm2) and area (mm2/cm2) of glutathione Stransferase placental form (GST-P)-positive liver cell foci assessed at week 24 of the experiment (P,0.01, 0.001). The incidence of hepatocellular adenomas in the DMD and I3C group at week 52 showed a tendency for elevation as compared to the DMD alone group, but this was not statistically significant. The thyroid gland tumour incidences in the DMD and I3C groups were significantly increased compared with the DMD alone group values at week 52 (P,0.01). In conclusion, I3C enhanced liver and thyroid gland neoplastic development when given during the promotion stage in the present rat medium-term multiorgan carcinogenesis model.

Introduction Human foodstuffs contain many compounds that inhibit the carcinogenic process in experimental animals (1–3). Recently, *Abbreviations: I3C, Indole-3-carbinol; AFB1, aflatoxin B1; DEN, diethylnitrosamine; DMH, 1,2-dimethylhydrazine; BOP, N-nitrosobis(2-oxopropyl)amine; MNU, N-methyl-N-nitrosourea; DHPN, dihydroxy-di-Npropylnitrosamine; GST-P, glutathione S-transferase placental form; Pg 1, pepsinogen 1; DMD, DEN-MNU-DHPN treatment; PAPG, pepsinogen 1 altered pyloric glands; PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine; AHH, aryl hydrocarbon hydroxylase; GST, glutathione Stransferase; GT, glucuronyl transferase; DTD, DT-diaphorase; PB, phenobarbital; CYP, cytochrome P450; DBN, N,N-dibutylnitrosamine; BITC, benzyl isothiocyanate; BTC, benzyl thiocyanate; UDS, unscheduled DNA synthesis; RDS, replicative DNA synthesis; TSH, thyroid stimulating hormone. © Oxford University Press

many investigations have focused on the chemopreventive effects of naturally occurring compounds. Indole-3-carbinol (I3C*), a major indole metabolite in cruciferous vegetables (cabbages, broccoli, Brussels sprouts and cauliflowers) (2,4,5), has thus been found to inhibit the development of tumours in forestomach (2,6), glandular stomach (7), mammary gland (2,8,9), uterus (10), tongue (11), and liver (12,13) of rodents, as well as in the trout liver (14,15), when administered prior to or during carcinogen exposure by gavage or in the diet.

However, dietary ‘anticarcinogens’ may exhibit adverse promoting activity in certain test protocols or in other organs.

For example, exposure to I3C or cabbage during the postinitiation (promotion) stage was found to strongly enhance aflatoxin B1 (AFB1)-induced liver tumourigenesis in the rainbow trout (15–17), diethylnitrosamine (DEN)-induced liver tumourigenesis in newborn or young rats (13), 1,2-dimethylhydrazine (DMH)-induced colon tumourigenesis in rats (18) and mice (19), and N-nitrosobis(2-oxopropyl)amine (BOP)induced pancreas tumourigenesis in hamsters (20). It is, in fact, well established that a chemical may act as a tumour inhibitor in one organ and as a promoter in others.

While the rat medium-term liver bioassay model of 8 weeks duration can detect both promotion and inhibition potential (21,22), in order to determine the spectrum of modifying effects rat medium-term multiorgan carcinogenesis models of 20–36 weeks duration are more applicable (23–28).

To elucidate the influence of I3C in various organs, we therefore conducted a post-initiation study at the whole-body level using one of these models (23–28).

Materials and methods Animals and chemicals One-hundred 6-week-old male Sprague–Dawley (SD) rats were supplied by the National Institute of Safety Research, Seoul, Korea, and were housed in polycarbonate cages with hard wood chips in an air conditioned room (23 6 2°C, 55 6 10% RH) with a 12 h light/dark cycle. Diet (Jeil Sugar Co., Korea) and drinking water were available ad libitum. All animals were fasted for 24 h prior to death. DEN (CAS No. 55–18–5, N-0756), N-methylN-nitrosourea (MNU, CAS No. 684–93–5, Sigma N-4766), and I3C (CAS No. 700–06–1, I-7256) were purchased from Sigma Chemical Co. Ltd., USA. Dihydroxy-di-N-propylnitrosamine (DHPN; CAS No. 53609–64–6) was purchased from Nakarai Tesque, Inc., Japan. Anti-rat-glutathione S-transferase placental form (GST-P) IgG was a generous gift from the late Professor Kiyomi Sato of The Second of Department of Biochemistry, Hirosaki University School of Medicine, Japan. Anti-rat-pepsinogen 1 (Pg 1) IgG was a generous gift from Dr Chie Furihata of The Department of Molecular Oncology, The Institute of Medical Science, The University of Tokyo, Japan.

Treatments One-hundred male SD rats were randomly divided into three groups (Figure 1).

Animals of groups 1 and 2 were sequentially treated with DEN (100 mg/kg b.w., a single i.p. injection, at the commencement of the experiment), MNU (20 mg/kg b.w., i.p., in citrate-buffered solution pH 6.0, four times at days 5, 8, 11 and 14), and DHPN (0.1% in the drinking water for 2 weeks during weeks 1 and 3) (DMD treatment). Non-initiation controls (group 3) were given vehicle alone (i.p. or in the drinking water). After this DMD treatment, the rats were maintained without any treatment for 1 week, and then animals of groups 1 and 3 were given 0.25% I3C in the diet for 20 weeks from week 4, subgroups being killed under ether anaesthesia at weeks 24 and 52 of the

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D.J.Kim et al. treated with diluted normal goat serum, rabbit anti-rat Pg 1 IgG (1:10000), biotin-labelled goat anti-rabbit IgG (1:400) and avidin-biotin-peroxidase complex (Vectastain Elite ABC kit, PK-6101, Vector Lab. Inc., USA). Diaminobenzidine was used as a chromogen to demonstrate the sites of peroxidase binding. The sections were counterstained with haematoxylin for microscopic examination. As a negative control for the specificity of anti-rat-Pg 1 antibody, normal rabbit serum was used instead of anti-rat-Pg 1 IgG. The percentages of pyloric glands with a low Pg 1 content were calculated by counting over 1000 pyloric glands.

Fig. 1. Experimental protocol for the multiorgan carcinogenesis bioassay (DMD model). Animals were sequentially treated with DEN (100 mg/kg b. wt., i.p., single dose), MNU (20 mg/kg b. wt., i.p., four times, on days 5, 8, 11 and 14) and DHPN (0.1% in the drinking water, during weeks 1 and 3). Animals of groups 1 and 3 were given diet containing 0.25% I3C (shaded areas) for 20 weeks after DMD treatment and were given basal diet (clear areas) for 28 weeks. Survivors were killed at weeks 24 and 52.

Quantitative analysis The numbers and areas of GST-P-positive liver cell foci .0.1 mm in diameter were measured with an IBAS automatic image analysis system (Kontron Co. Ltd., Germany). Quantitative data for these were compared for statistical significance using Student’s t-test after ANOVA for GST-P and pepsinogen 1 altered pyloric glands (PAPG) and the Fisher’s exact probability test was applied for tumour incidences.

Results Body and liver weights At week 24 of the experiment, the body weights in the DMD plus I3C group was significantly decreased as compared with the DMD alone group value (P,0.05) and the relative liver weight was significantly increased (P,0.001). However, no intergroup variation in the body or relative liver weights was evident at week 52 of the experiment after the return to basal diet (Table I).

Fig. 2. Number and area of GST-P-positive liver cell foci in SD rats treated with DMD with or without subsequent I3C administration. Values are for weeks 24 and 52. DMD and I3C group (shaded areas); DMD alone group (clear areas). **,***Significantly different from the DMD alone group at P,0.01 and P,0.001, respectively. experiment. The body weights and relative liver weights were measured at death.

Histological examination The major organs, including the liver, thyroid gland, lungs, heart, kidneys, oesophagus, stomach, intestine, adrenal glands and testes were taken, fixed in 10% neutral phosphate-buffered formalin, and embedded in paraffin for routine processing and examination of H&E stained sections. Liver tissue and glandular stomach were also processed for immunohistochemical staining of GST-P and Pg 1.

Immunohistochemical staining of GST-P From the three major lobes of the liver 4–5-mm thick slices were cut with a razor blade, and fixed in ice cold acetone, embedded in paraffin and sectioned at 4 µm for subsequent immunohistochemical staining of GST-P, as described previously (13,22,29). Endogenous peroxidase activity was blocked by treatment with methanolic hydrogen peroxide. After being passed through xylene and a graded alcohol series the sections were sequentially treated with diluted normal goat serum, rabbit anti-rat-GST-P (1:10,000) IgG, affinity-purified biotin-labelled goat anti-rabbit IgG, and avidin-biotin-peroxidase complex (Vectastain Elite ABC kit, PK-6101, Vector Lab. Inc., USA). Diaminobenzidine was used as a chromogen to demonstrate the sites of peroxidase binding. The sections were counterstained with haematoxylin for microscopic examination.

As a negative control for the specificity of anti-rat-GST-P IgG, normal rabbit serum was substituted for antiserum.

Immunohistochemical staining of Pg 1 The glandular stomachs of animals killed at the end of week 52 were fixed in sublimated formalin and cut into about eight strips, which were embedded in paraffin for subsequent immunohistochemical staining of Pg 1, as detailed earlier (7,29,30). The ABC method was used to determine the location of Pg 1 in the pyloric mucosa. Endogenous peroxidase activity was blocked by treatment with methanolic hydrogen peroxide. Sections were sequentially

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Quantitative values for GST-P-positive liver cell foci Quantitative values for GST-P-positive liver cell foci per cm2 are shown in Figure 2. The average number (no./cm2) and area (mm2/cm2) of GST-P-positive liver cell foci in the DMD plus I3C group as well as the Dmax of GST-P-positive liver cell foci were significantly increased as compared to the respective DMD alone group values at week 24 of the experiment (P,0.01, 0.001 and 0.05, respectively). At week 52 of the experiment, these DMD plus I3C group values for GST-P-positive liver cell foci still showed tendencies for increase, but these values were not significant.

PAPG in pyloric mucosa The numbers of PAPG in pyloric mucosa in each group showed tendencies for decrease, but these PAPG in the pyloric mucosa did not significantly different between groups at week 52 of the experiment (data not shown).

Incidences of tumours Incidences of neoplastic lesions are shown in Table II. The combined incidence of hepatocellular adenomas or carcinomas in the DMD plus I3C group at week 52 was significantly higher than in the same group at week 24 of the experiment (38 vs. 5%, P,0.05) with a tendency for increase as compared with DMD alone group (38 vs. 18%). However this was not statistically significant. At week 52 the combined incidence of follicular cell adenomas and adenocarcinomas of the thyroid gland was also significantly increased in the DMD plus I3C group with a preponderance of solid and poorly differentiated follicular cell adenocarcinomas (P,0.01) (Table II). Colloid follicles of the thyroid gland appeared normal. The numbers of neoplastic lesions of other organs in each group did not significantly differ. Rats in both the DMD treated groups developed alveolar hyperplasias and adenomas and/or adenocarcinomas of the lung but no effect of I3C was evident.

Pathological changes were also observed in the adrenal gland (an adenocarcinoma) and kidney (renal cell adenomas and a nephroblastoma).

Enhancement of liver and thyroid gland neoplasia by I3C

Table I. Body and relative liver weights for SD rats sequentially treated with DMD followed by I3C at weeks 24 and 52 Treatments

DMDa→I3Cb DMD alone I3C

No. of rats

Body wt (g)

Relative liver wt (g%)

24 weeks

52 weeks

0 weeks

24 weeks

52 weeks

24 weeks

52 weeks

20 19 10

14 16 10

159.6 6 11.6 158.8 6 8.85 160.0 6 9.96

439.5 6 57.1* 471.7 6 38.4 484.9 6 32.1

635.8 6 85.8 622.1 6 70.1 677.0 6 74.0

3.54 6 0.27*** 2.58 6 0.28 2.88 6 0.22

2.56 6 0.34 2.39 6 0.21 2.53 6 0.31

DMD alone (n 5 17)b

I3C (n 510)

aDMD

represents ‘DEN1MNU1DHPN’ treatment.bI3C was given at 0.25% in the diet for 20 weeks. *, ***Significantly different from the DMD alone group at P,0.05 and 0.001, respectively.

Table II. Histological findings at weeks 24 and 52 of the experiment in rats treated with DMD and / or I3C Organ/lesions

Treatment 24 weeks DMD→I3C (n 5 20)

Liver Hepatocellular adenoma (HA) Hepatocellular carcinoma (HCC) HA/ HCC Thyroid gland Follicular cell hyperplasia Follicular cell adenoma Follicular cell adenocarcinoma Adenoma/ adenocarcinoma Lung Alveolar hyperplasia Adenoma Adenocarcinoma Adenoma/ adenocarcinoma Adrenal gland Adenocarcinoma Kidney Renal cell adenoma Nephroblastoma Testis Interstitial cell adenoma

52 weeks DMD alone (n 5 19)

I3C (n 5 10)

DMD→I3C (n 5 16)a 6 (38)†† 0 6 (38)†

0 1 (5)c 1 (5)

0 0 0

0 0 0

1 (5) 1 (5) 0 1 (5)

1 (5) 1 (5) 0 1 (5)

0 0 0 0

12 8 4 12

(75)*††† (50)*† (29)† (75)**†††

16 (84) 2 (10) 0 2 (10)

0 0 0 0

16 6 1 7

(100) (38)† (6) (44)†

13 1 1 2

(65) (5) (5) (10)

2 (12) 1 (6) 3 (18)

0 0 0

4 2 2 4

(29) (12) (12) (29)

0 0 0 0

17 8 1 9

(100) (47)† (6) (53)†

0 0 0 0

0

0

0

1 (6)

0

0

0 0

0 0

0 0

2 (14) 1 (6)

1 (6) 0

0 0

0

0

0

3 (19)a

3 (18)

0

aTwo moribund cPercentages in

animals in the DMD1I3C group are included.bOne moribund animal in the DMD alone group is included. parentheses. *, **: Significantly different from the DMD alone group at P,0.05 and 0.01, respectively. †,††,†††Significantly different from those of same group at week 24 at P,0.05, 0.01 and 0.001, respectively.

Discussion The results of the present study demonstrate clearly enhancing effects of I3C on liver and thyroid gland neoplastic development in the post-initiation (promotion) stage of the present rat medium-term multiorgan carcinogenesis model (DMD treatment). The liver results are in line with our previous findings in a rat medium-term bioassay (13).

Biphasic modifying effects of I3C on hepatocarcinogenesis were earlier demonstrated in terms of AFB1-induced liver tumours in the rainbow trout (14–17). Exposure during the initiation stage reduced the yield of hepatocellular carcinomas, while post-initiation administration caused a significant increase (14,15). Dietary exposure to I3C or cabbage of rodents also enhanced the development of colon tumours after DMH treatment (18,19) although inhibition of aberrant crypt formation by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was observed for both initiation and promotion stages (31). The mechanisms responsible for the observed promotion effects of I3C (13,15–19) remain to be clarified.

However, in most of the studies, high levels of I3C were

administered daily over long periods and this would be expected to result in induction of aryl hydrocarbon hydroxylase (AHH) activity (4,5), as well as glutathione S-transferase (GST) (32–35), glucuronyl transferase (GT) (33), and DTdiaphorase (DTD) (33). Although this property of I3C might normally be expected to inhibit carcinogen action (2,4,35), the same type of pleiotropic response has been observed for phenobarbital (PB)-type enzyme inducers resulting in promotion/enhancement depending on the period of administration of I3C on the liver (36–38). I3C has been shown to induce hepatic cytochrome P450 (CYP) 2B1/2, as well as CYP 1A1 and 1A2 (33).

In contrast with our present results and the data from our earlier study (13), Jang et al. (39) reported that dietary intake of 0.5% I3C significantly decreased the development of hyperplastic nodules and GST-P-positive liver cell foci after sequential treatment with DEN, MNU, and N, N-dibutylnitrosamine (DBN). The reason for this discrepancy is unclear since the strains of rats, dose, duration and kinds of carcinogens applied were basically the same.

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Dietary supplements of 0.1% I3C and 0.12% sinigrin, major constituents of cruciferous vegetables, were found to exert an inhibitory effect on DEN-induced hepatocarcinogenesis in ACI/N rats when given prior to and during carcinogen exposure (12). Ethanol extracts from Chinese cabbages were also reported to inhibit the development of GST-P-positive liver cell foci in newborn SD rats after initiation with DEN (40).

This, however, could have been due to other components such as sinigrin, benzyl isothiocyanate (BITC) and benzyl thiocyanate (BTC) (41). BITC and BTC are considered effective in the pre-initiation, as well as the post-initiation (promotion) stages (42), with effects on the inactivation or detoxification of the carcinogen. Thus, they both inhibit unscheduled DNA synthesis (UDS) and replicative DNA synthesis (RDS) in rat hepatocytes in response to DEN exposure (43). However, I3C increased liver tumour induction in F344 rats by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone associated with augmented 7-methylguanidine adduct formation in liver DNA while sinigrin only affected the latter (44). Thus, there is no simple explanation for inhibition and promotion of hepatocarcinogenesis by members of the Cruciferae family.

The mechanisms of I3C promotion of thyroid tumourigenesis are also unclear, but might be related to its effects on the liver.

Several PB-type inducers of hepatic microsomal enzymes (including CYP2B1/2) are known to enhance follicular cell tumour development initiated by a variety of carcinogens in rats (45–50). The cumulative effect of PB-type inducers on various drug metabolizing activities and on liver mass leads to increased metabolic clearance of thyroid hormones, resulting in a hyperplasiogenic influence through the thyroid stimulating hormone (TSH) feedback loop (47–51). Dietary exposure of rats to cooked Brussels sprouts for only 2 days can change the metabolic activity of CYP2B1/2, which is the predominant phase I form induced in the small intestine and liver (33). A number of glucosinolate hydrolysis products have been shown to be goitrogenic, the most potent of which, 5-vinyloxazolidine2-thione (derived from progoitrin, IV) is potentially present in large amounts in Brussels sprouts (52–54). Chronic feeding trials of materials containing glucosinolates have shown these to be dose-dependently linked to lesions in the liver, kidney and pancreas, haemorrhage and death (55,56). Pancreatic cancer development in the hamster model was also markedly increased in animals fed cabbages and a high fat diet (20).

We did not observe gross or microscopic lesions in organs other than the liver, thyroid gland, lung, kidney, and adrenal gland in our experiment. It should be borne in mind that I3C reduced the life-span of the rats compared with the DMD alone group, perhaps due to the presence of liver or thyroid gland tumour masses (Table II). From the available results, we conclude that I3C is capable of causing toxicity and enhancing tumourigenesis in the liver and thyroid gland of experimental animals. Therefore, the efficacy and risk potential of cruciferous vegetables deserves further careful attention.

Acknowledgements The authors would like to express their sincere gratitude to the late Professor Kiyomi Sato of the Second Department of Biochemistry, Hirosaki University School of Medicine for generous provision of GST-P antibody and to Dr Chie Furihata of the Department of Molecular Oncology, The Institute of Medical Science, The University of Tokyo, for generous provision of Pg 1 antibody.

This work was supported, in part, by a Grant-in-Aid for the Second Term Comprehensive 10-year Strategy for Cancer Control from the Ministry of Health and Welfare, by a Grant-in-Aid for Cancer Research from the Ministry

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of Health and Welfare in Japan, and Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan. Dr D.J.Kim is the recipient of support from the Korea Science & Engineering Foundation (KOSEF, Daejeon, Korea) and the Foundation for Promotion of Cancer Research in Japan (FPCR, Tokyo, Japan).

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Received on July 10, 1996; revised on September 17, 1996; accepted on November 4, 1996

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