Post-initiation effects of chlorophyllin and indole-3-carbinol in rats given 1,2-dimethylhydrazine or 2-amino-3-methyl- imidazo[4,5-f]quinoline
Meirong Xu1,
Gayle A. Orner1,2,
George S. Bailey2,
Gary D. Stoner3,
David T. Horio4 and
Roderick H. Dashwood1,2,5
1 Linus Pauling Institute and
2 Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331,
3 School of Public Health, Ohio State University, Columbus, OH 43210 and
4 Department of Pathology, St Francis Medical Center, Honolulu, HI 96817, USA
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Abstract
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Chlorophyllin (CHL) is a water-soluble derivative of chlorophyll, the ubiquitous pigment in green and leafy vegetables, whereas indole-3-carbinol (I3C) is present in cruciferous vegetables such as cabbage, broccoli and cauliflower. In rats initiated with 1,2-dimethylhydrazine (DMH), CHL and I3C reportedly promoted or enhanced the incidence of colon tumors when they were administered after, or during and after the carcinogen exposure, respectively. The same compounds given post-initiation inhibited the formation of colonic aberrant crypts induced by heterocyclic amines, such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), but tumor suppression was not examined in the latter studies. In the present investigation, male F344 rats were treated with IQ or DMH during the first 5 weeks of a 1 year study; IQ was given in the diet (0.03%), whereas DMH was administered once a week by s.c. injection (20 mg/kg body wt). Beginning 1 week after the last dose of IQ or DMH until sacrifice, rats received 0.001, 0.01 or 0.1% (w/v) CHL in the drinking water or 0.001, 0.01 or 0.1% I3C in the diet. Compared with controls given carcinogen alone, 0.1% I3C treatment suppressed the multiplicity of IQ-induced colon tumors, and CHL inhibited in a dose-related manner the incidence of IQ-induced liver tumors. However, 0.001% CHL increased significantly the multiplicity of DMH-induced colon tumors while having no effect on the colon tumors induced by IQ. These results indicate that both the choice of carcinogen as well as the dose of the tumor modulator can be important determinants of the events that occur during post-initiation exposure to CHL or I3C. Based on the present findings and data in the literature, it is possible for CHL and I3C to act as tumor promoters or anticarcinogens, depending upon the test species, initiating agent and exposure protocol.
Abbreviations: CHL, chlorophyllin; I3C, indole-3-carbinol; DMH, 1,2-dimethylhydrazine; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; PhlP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
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Introduction
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It is well established that the human diet contains both carcinogens and cancer chemopreventive agents (14). Dietary anticarcinogens and antimutagens have been identified using a range of end points, from a reduction in the frequency or multiplicity of tumors and intermediate biomarkers of cancer in vivo to inhibitory activity in short-term genotoxicity assays in vitro. The list of cancer chemopreventive agents includes both natural and synthetic derivatives of phytochemicals that occur in fruits and vegetables (14). Two that have received considerable attention during the past decade are chlorophyllin and indole-3-carbinol.
Chlorophyllin (CHL), a water-soluble salt of chlorophyll, acts as an anticarcinogen during the initiation phase of carcinogenesis (5) and is being evaluated by one of us (G.S.B.), in collaboration with Dr Tom Kensler and colleagues of Johns Hopkins University, against aflatoxin exposure in a clinical intervention biomarker trial in Daxin, China. During the post-initiation phase, 0.1% CHL (w/v) in the drinking water inhibited the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP)- and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced colonic aberrant crypts in the rat (6; R.H.Dashwood, unpublished results), but promoted 1,2-dimethylhydrazine (DMH)-induced colon tumor formation in the same species (7).
Indole-3-carbinol (I3C), a natural constituent of brassica vegetables that has been used in studies of estrogen metabolism in women (8), has both anticarcinogenic and tumor promoting activities in animal models depending upon the initiator, exposure protocol and species (see ref. 9 for a review). Inhibition of the formation of colonic aberrant crypt foci, which are putative preneoplastic lesions and intermediate biomarkers of colon cancer (10,11), was observed in rats given 0.1% I3C in the diet before, during and after exposure to IQ (12). Potent inhibition of PhlP-induced colonic aberrant crypts also was observed in the rat when 0.1% dietary I3C was administered during the initiation phase, post-initiation phase or continuously throughout the entire study (6). However, 0.1% dietary I3C given before, during and after DMH treatment reportedly enhanced the formation of colon tumors in the rat (13). Collectively, these results suggested that the initiating agent, namely DMH or heterocyclic amine, might influence the response to a tumor modulator (CHL or I3C) administered for a prolonged period of time during the post-initiation phase. However, the doseresponse relationships have not been adequately addressed, and the molecular mechanisms have yet to be studied in any detail.
Therefore, in the present study, we compared the post-initiation effects of CHL and I3C, administered at three different concentrations within the range of human exposure, in male F344 rats initiated with DMH or IQ. The results indicated that a low concentration of CHL increased the multiplicity of colon tumors in rats given DMH but had no effect on the colon tumors initiated by IQ, and that I3C inhibited the multiplicity and incidence of IQ-induced colon and liver tumors, respectively, but had no effect on the tumors initiated by DMH.
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Materials and methods
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Chemicals
I3C and CHL were purchased from Sigma Chemical Company (St Louis, MO), IQ was from Toronto Research Chemicals (Ontario, Canada) and DMH was from Aldrich Chemical Company (Milwaukee, WI).
Tumor bioassay
Male F344 rats, 45 weeks of age (103 ± 10 g body wt), were purchased from Taconic (Germantown, NY) and divided randomly into groups of 2025 animals (23 rats per cage). Water and AIN-93G diet were provided ad libitum. During the first 5 weeks of a 1 year study, animals were given either DMH or IQ in order to induce tumors in the colon and other target organs (Figure 1
). IQ was administered in the diet at a concentration of 0.03%, whereas DMH was injected once a week (20 mg/kg body wt, s.c.). Starting 1 week after the last carcinogen treatment until the end of the study, rats were given 0.1, 0.01 or 0.001% CHL (w/v, based on total chlorins) in the drinking water, or 0.1, 0.01 or 0.001% I3C in the diet. These concentrations of CHL and I3C were chosen since they represent the lower range of possible human exposure, and they were well below the levels reported to exhibit promotional activity (5,7,13). CHL solutions were prepared fresh every other day, and AIN-93 diet (Dyets, Bethlehem, PA) was mixed with IQ or I3C on a weekly basis, based on the protocol in Figure 1
. Food consumption and animal body weights were monitored weekly. After 1 year, or earlier in the case of animals that became moribund, rats were sacrificed and subjected to a gross necropsy examination of all organs. One portion of each tumor was fixed in 10% formalin and processed for histological examination, as described previously (14,15), and other portion(s) were frozen on liquid nitrogen and stored at -80°C for molecular analyses.

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Fig. 1. Modulation of DMH- and IQ-induced tumorigenesis in the rat following post-initiation treatment with CHL or I3C. Male F344 rats, 2025/group (103 ± 10 g), were given AIN-93G diet containing 0.03% IQ for 5 weeks, or they were given AIN-93G diet alone and injected s.c. with DMH, once a week for 5 weeks (20 mg/kg body wt, in 0.9% NaCl, 0.001M EDTA pH 6.8). Negative controls were given AIN-93G diet and injected once a week with test vehicle containing no carcinogen. Starting 1 week after the last dose of carcinogen until the end of the study, rats were given 0.1, 0.01 or 0.001% CHL (w/v) in the drinking water, or 0.1, 0.01 or 0.001% I3C in the diet. Final data for tumor incidence (%) and multiplicity (mean number of tumors/tumor-bearing tissue) are shown in Tables I and II .
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Statistics
Tumor incidence and multiplicity results were determined for the various groups and statistical comparisons were made, initially using one-way ANOVA, followed by pairwise comparisons using Fisher's LSD model.
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Results
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The results for groups initiated with DMH or IQ are summarized in Tables I and II
, respectively. No significant effects of the CHL or I3C treatments were seen on the body weights of the animal throughout the study, except at the highest concentration of 0.1% I3C in rats initiated with IQ; at the time of sacrifice, average body weights (g) were 531 ± 38.9 and 489 ± 47.5 for IQ and IQ + 0.1%I3C groups, respectively (P < 0.05). None of the animals treated with vehicle and post-treated with CHL or I3C had tumors (not shown).
In rats initiated with DMH, tumors were found in the colon, small intestine and Zymbal's gland, whereas IQ produced tumors in these organs plus the liver and skin, as reported previously (1417). Examples of the tumors induced by IQ are shown in Figure 2
. Tumors induced by DMH and IQ in the colon and small intestine were identified almost exclusively as adenocarcinomas, and the skin tumors and Zymbal's gland tumors were squamous cell carcinomas. Liver tumors induced by IQ were primarily hepatocellular carcinomas, and occasionally adenomas or mixed hepatocellular and ductal carcinomas. The proportion of the latter tumor types was in the order of 10% in all IQ groups, and although the numbers of tumors did not permit statistical comparisons, none of the modulator treatments appeared to alter the overall frequency of carcinomas versus adenomas (data not shown).

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Fig. 2. Tumors induced in rats given IQ during the first 5 weeks of a 1 year study. (A) Gross view of a colon tumor; (B) detail of a carcinoma of the colon (100x magnification); (C) a liver adenoma (200x); (D) gross view of tumor in the small intestine; (E) squamous carcinoma of the Zymbal's gland (100x); (F) squamous carcinoma in a skin tumor (200x). The sections (B), (C), (E) and (F) were stained with hematoxylin and eosin.
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In the present study, 87% of the rats given DMH alone had colon tumors, and each rat had on average 2.7 tumors per colon (Table I
). Fifty-two percent and 17% of the DMH-induced animals also had tumors in the small intestine and Zymbal's gland, respectively. The high incidence of colon tumors in this investigation, compared with only 10% reported before, might be due to the fact that a 20-week time point was used in the earlier study with DMH and CHL (7). In addition, the previous study used Purina chow diet (7), and we have reported recently that feeding chow versus AIN-76A or AIN-93G diets plays a significant role in determining the number of colonic aberrant crypts induced by IQ in the rat (18).
In the groups given DMH and post-treated with CHL or I3C, there was no obvious doseresponse relationship for suppression or promotion of tumor incidence or multiplicity in the colon, small intestine or Zymbal's gland (Table I
). However, the lowest concentration of 0.001% CHL significantly increased the multiplicity of colon tumors, from a value of 2.70 ± 1.05 for DMH alone to 4.89 ± 2.3 tumors/tumor-bearing colon for DMH plus CHL (P < 0.05, mean ± SD). The finding that promotion occurred at the lowest concentration of CHL only is discussed further below.
In all of the groups given IQ, except the group administered the highest concentration of 0.1% I3C, the overall incidence of colon tumors was around 910% and the average multiplicity was 1.01.5 tumors/tumor-bearing colon (Table II
). Thus, in contrast with the results with DMH, no tumor promotion was seen by CHL or I3C in the colon of rats induced with the heterocyclic amine. Indeed, in the group given IQ plus 0.1% I3C there was a complete absence of colon tumors, and the reduced multiplicity of colon tumors proved to be statistically significant compared with the group given IQ alone (1.5 ± 0.71 versus 0.0 ± 0.0 tumors/tumor-bearing colon, P < 0.05). No effect was seen for CHL or I3C on the incidence or multiplicity of IQ-induced tumors in the small intestine, Zymbal's gland or skin (Table II
). However, in the liver, which was the major target organ for IQ-induced tumorigenesis, the highest concentration of 0.1% CHL suppressed the incidence of tumors compared with the group given IQ alone (P < 0.05). Moreover, the doseresponse for suppression by CHL in the liver was statistically significant: tumor incidence results were 73, 60, 50 and 36% for groups given IQ alone, IQ + 0.001% CHL, IQ + 0.01% CHL and IQ + 0.1% CHL, respectively (P < 0.05 for the trend). No significant effects were detected on liver tumor multiplicity in these groups.
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Discussion
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To our knowledge, this is the most detailed doseresponse analysis to date for CHL and I3C in the rat using a post-initiation exposure protocol and comparing two different initiating agents. In previous studies, short-term treatment of rats with PhlP, followed by post-initiation exposure to a high-fat diet, caused the rapid induction of colonic aberrant crypts or mammary tumors (19,20); however, this is the first paper to describe the induction of tumors in the rat using a short, 5-week dietary exposure protocol for IQ. Based on the original carcinogenicity bioassay that defined IQ as a carcinogen in the F344 rat, in which IQ was given continuously for over 1 year at a dietary level of 0.03% (16), the present exposure protocol was successful at inducing tumors in all of the major target organs. The 73% liver tumor incidence in this study (Table II
) compares favorably with the 68% liver tumor incidence reported previously for IQ (16,21). However, the incidence of tumors in other target organs was lower than expected, including tumors of the colon, the target organ of principle interest at the outset of this investigation. Whereas the original report found a colon tumor incidence of 63% (16), the present study detected only a 9% incidence of IQ-induced colon tumors.
In previous studies of IQ- and PhlP-induced colonic aberrant crypts (6,12), CHL and I3C were shown to be effective inhibitors in the rat colon when given at the time of carcinogen exposure, and they were more or less equally potent when tested as suppressing agents during the post-initiation phase. In the present investigation, rats given IQ and post-treated with CHL showed no inhibition in the colon, but, unexpectedly, there was evidence for dose-related suppression of liver tumorigenesis. CHL has been shown to complex with IQ during the initiation phase and to reduce hepatic IQDNA adducts and overall carcinogen bioavailability in vivo (2225). However, the 1-week period between the last dose of IQ and the first treatment with CHL (Figure 1
) should have allowed for most of the carcinogen to be metabolized and excreted via the urine and feces (2628). The complexing of CHL with residual amounts of IQ or its metabolites cannot be completely ruled out, but this is unlikely to represent a major inhibitory mechanism during the post-initiation phase. Although it is generally assumed that an oral dose of CHL is largely retained within the GI tract of the rat (29), suppression of IQ-induced liver tumorigenesis in the present study points to systemic mechanisms, and suggests at least some degree of CHL uptake. There is currently no information available on the mechanisms by which CHL might protect in the liver when administered post-initiation, but the results are noteworthy in view of the intervention trail currently under way in Daxin, China (see Introduction). Assuming that uptake also occurs in people ingesting an oral dose, CHL might operate as a suppressing agent in individuals exposed unavoidably to hepatocarcinogens in the diet (30,31), as well as protecting via molecular complex formation or other blocking mechanisms (5).
In a previous investigation (7), post-initiation treatment with 0.1% CHL in the drinking water for 20 weeks reportedly increased the incidence of DMH-induced colon tumors from 10 to 47%; however, promotional effects of CHL or I3C on tumor incidence may have been masked in the present study since 87% of the rats given DMH alone had colon tumors (Table I
). Nonetheless, the lowest concentration of 0.001% CHL increased significantly the multiplicity of colon tumors induced by DMH, without promoting the colon tumors induced by IQ. These findings with CHL raise two important issues.
First, the results suggest that the choice of initiating agent might, in some cases, influence the response to a modular given post-initiation. In other words, it may be possible for the same modulator to act as a tumor promoter against one initiating agent but to have no effect, or potentially to operate as a suppressing agent, against other carcinogens. This concept is well established for compounds that are effective during the time of initiation, when changes in carcinogen metabolizing enzymes can facilitate the detoxification of one class of carcinogen but augment the metabolic activation of others (32). However, it has received less attention during the post-initiation phase, largely due to the fact that, unlike the case of `blocking agents', the mechanisms are often poorly defined for suppressing agents (2). As an additional level of complication, humans most likely receive a lifetime of exposure to endogenous and exogenous carcinogens, with modulators acting on previous as well as concurrent initiation events. For these reasons, we are cautious about extrapolating the present data with CHL and I3C to the situation of people consuming chlorophyll- or indole-rich foods.
Second, the results reported here and elsewhere (7) suggest that different mechanisms operate in the colon according to the concentration and duration of CHL exposure. Recent studies with butyrate, a short-chain fatty acid that protects against colon cancer in the rat (33), provided evidence for cell cycle arrest in colon cancer cells at low concentrations, but at higher concentrations caused the induction of apoptosis (34). We have reported on the loss of apoptosis during IQ-induced colon tumor formation in the rat (15), as evidenced by increased expression of anti-apoptotic Bcl-2, decreased levels of pro-apoptotic Bax and loss of terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL). Moreover, a recent investigation of TUNEL and bromodeoxyuridine labeling indices in the rat colon showed that cell proliferation was increased following post-initiation treatment with 0.001% CHL, with no corresponding changes in TUNEL, whereas higher CHL concentrations augmented both of these end-points, such that the net balance between cell proliferation and apoptosis was unaffected (35). We have also shown that DMH-induced colon tumors from the present study, in particular those from the group promoted by 0.001% CHL, contained a unique spectrum of ß-catenin mutations compared with the tumors from rats given DMH alone (36). The results strongly implicated the Apc/ß-catenin pathway, and indicated that during the post-initiation phase of carcinogenesis, CHL and I3C might alter the expression of ß-catenin/Tcf/Lef target genes. We are now conducting further studies in this direction, in light of the important `gatekeeping' function of the APC/ß-catenin pathway in human colon cancer (37).
In summary, the present investigation examined the post-initiation effects of CHL and I3C in rats given DMH or IQ. Suppression of IQ-induced colon tumor multiplicity was significant at the highest concentration of 0.1% I3C, which is in general accordance with previous studies using heterocyclic amine-induced colonic aberrant crypts as the end-point (6,12). However, there was no significant effect of I3C on DMH-induced colon tumors, in contrast with a previous study showing enhancement of colon tumorigenesis when I3C was given before, during and after DMH exposure (13). In rats given CHL, significant, dose-related suppression of IQ-induced liver tumorigenesis was observed, but at the lowest concentration tested, this compound increased the multiplicity of DMH-induced colon tumors while having no promotional effect on the colon tumors induced by IQ.
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Notes
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5 To whom correspondence should be addressed E-mail: Rod.Dashwood{at}orst.edu 
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Acknowledgments
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This study was supported in part by NIH grants CA65525, CA80176, CA34732, ES00210 and ES03850. Support for G.A.O. was provided by NIEHS training grant T32 ES07060. We thank Hugh Luk of St Francis Medical Center, Honolulu, for preparing the tissue sections for histopathological evaluation.
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Received August 18, 2000;
revised November 22, 2000;
accepted November 27, 2000.