Oncogenicity Evaluations of Chemopreventive Soy Components in p53 (+/–) (p53 knockout) Mice

Abstract

Epidemiologic data suggest that soy consumption may protect against cancer induction in several tissues in humans. Although the soy components responsible for this activity remain unidentified, isoflavones (e.g., genistein) and protease inhibitors (e.g., Bowman-Birk inhibitor complex [BBIC]) demonstrate chemopreventive activity in several animal cancer models. As part of their preclinical development for cancer prevention, PTI G-2535 (a soy isoflavone mixture containing 45% genistein, 23% daidzein, and 4% glycitein) and BBIC were evaluated for oncogenicity in p53^(+/–) mice. In separate studies, groups of 25 p53^(+/–) mice/sex received daily gavage exposure to PTI G-2535 (0, 250, 1000, or 2500 mg/kg/day) or BBIC (0, 500, 1000, or 2000 mg/kg/day) for 6 months. The high doses of both PTI G-2535 and BBIC were limited by viscosity. p-Cresidine (400 mg/kg/day) served as a positive-control article in both studies. PTI G-2535 induced no gross toxicity in any animal, but did induce a dose-related suppression of body weight gain in male mice. Modest hematologic alterations and increased liver and spleen weights were seen in both sexes exposed to the isoflavone mixture. BBIC had no significant effect on body weight, food consumption, clinical pathology, or organ weights in either sex. Histopathologic evaluations demonstrated no increases in the incidence of either benign or malignant tumors in any group of p53^(+/–) mice exposed to PTI G-2535 or to BBIC. By contrast, the positive-control article, p-cresidine, induced urinary bladder cancers in both studies. Neither PTI G-2535 nor BBIC demonstrates any evidence of oncogenicity in the p53^(+/–) mouse model.

A substantial body of evidence suggests that consumption of soybeans or soy-containing foods confers protection against carcinogenesis in animals, and may protect against cancer induction in humans (reviewed in Messina et al. 1994; Kennedy 1995). Several soy components have biological activities that are consistent with efficacy in cancer chemoprevention: soy contains genistein and other isoflavones that are biologically active (Messina et al. 1994), and also contains protease inhibitors, such as Bowman-Birk inhibitor (BBI; Troll and Wiesner 1983; Kennedy 1995). On the basis of the underlying epidemiologic data linking decreases in human cancer risk to soy consumption, and the activity of different soy components as inhibitors of carcinogenesis in experimental model systems, soy isoflavone mixtures and BBI complex (BBIC) are in preclinical development as cancer chemopreventive agents.

Isoflavones as Chemopreventive Agents

Soy isoflavones are effective inhibitors of mammary carcinogenesis in several experimental model systems (reviewed in Barnes 1997). In addition to activity in breast cancer prevention, isoflavones have been reported to inhibit cancer induction in the prostate (Pollard and Luckert 1997; Bosland et al. 2002), stomach (Tatsuta et al. 1999), intestinal tract (Sorensen et al. 1998; Guo et al. 2004), skin (Wei et al. 1998), and urinary bladder (Zhou et al. 1998) of experimental animals. In vitro, soy isoflavones demonstrate antiproliferative and proapoptotic activity in many cancer cell lines; other activities of isoflavones that are consistent with activity in cancer chemoprevention include alterations in tissue differentiation (LaMartiniere et al. 1998), tyrosine kinase inhibition, antioxidant activity, induction of detoxification enzymes, and regulation of the host immune response (Birt et al. 2001).

It should be noted, however, that although genistein demonstrates a wide range of anticarcinogenic activity in various animal models, Rao and colleagues (1997) have reported that dietary administration of genistein stimulates neoplastic development in the rat colon. Similarly, Allred et al. (2004) report that genistein enhanced the growth of carcinogen-induced mammary cancers in ovariectomized rats.

Estrogenicity appears to underlie the activity of genistein and other isoflavones in modulating tissue differentiation in the mammary gland (LaMartiniere et al. 1998) and may be causally linked to the chemopreventive activity of this class of compounds. However, the estrogenic activity of soy isoflavones also suggests that chronic toxicities and/or oncogenicity could result from prolonged estrogen stimulation in individuals receiving long-term isoflavone exposure.

Bowman-Birk Inhibitor as a Chemopreventive Agent

Epidemiologic data demonstrate an inverse association between human consumption of diets rich in protease inhibitors and cancer incidences in the breast, colon, and prostate (reviewed in Troll and Wiesner 1983; Kennedy 1995, 1998). The epidemiology literature suggesting that protease inhibitors may protect against cancer is supported by a growing database demonstrating that natural or synthetic protease inhibitors can inhibit carcinogenesis in laboratory animals.

Kennedy and colleagues have demonstrated that BBI, a soy-derived serine protease inhibitor, has significant cancer chemopreventive activity in both in vivo and in vitro bioassay systems (reviewed in Kennedy 1998). In vivo, BBI inhibits carcinogenesis in the colon (Weed, McGandy, and Kennedy 1985; Kennedy et al. 1996), esophagus (von Hofe, Newberne, and Kennedy 1992), liver (St. Clair et al. 1990), lung (Witschi and Kennedy 1989), oral mucosa (Messadi et al. 1986; Kennedy et al. 1993), and prostate (Bosland et al. 2002). BBI also demonstrates desirable activity against established neoplastic lesions, as it inhibits the growth of human prostate cancer xenografts in nude mice (Wan et al. 1999), and prevents the formation of pulmonary metastases in mice after subcutaneous injections of tumor cells (Kobayashi et al. 2004).

In vitro studies have shown that BBI can inhibit malignant transformation of C3H/10T 1/2 cells by x-rays (Yavelow et al. 1985) or 3-methylcholanthrene (St. Clair 1991). Similarly, BBI suppresses the enhancement of radiation-induced transformation by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (Su, Toscano, and Kennedy 1991), and the induction of premalignant lesions induced in the mouse mammary gland by 7,12-dimethylbenz[a]anthracene (Du, Beloussow, and Shen 2001). BBI can also suppress the in vitro growth of human small cell lung cancer cell lines (Clark et al. 1993) and a number of human prostate cancer cell lines (Kennedy and Wan 2002).

Development of Soy Components as Chemopreventive Agents

The broad range of anticarcinogenic activity of soy isoflavones and BBIC in experimental model systems suggests that both agents have considerable potential utility in human cancer chemoprevention. Initial clinical trials have been conducted with soy isoflavones (Ullman et al. 2005) and with BBIC (Armstrong et al. 2000a, 2000b; Malkowicz et al. 2001), and both agents appear to be well tolerated in humans receiving short-term exposure. However, evaluating isoflavones or BBIC for activity in cancer chemoprevention will require the conduct of randomized clinical trials involving chronic administration protocols. Prior to the conduct of such trials, it is essential to demonstrate that soy isoflavones and BBIC are not, in themselves, carcinogenic.

The present studies were performed to evaluate the possible oncogenic activity of PTI G-2535, a characterized mixture of soy isoflavones, and BBIC in the TSG-p53^(+/–) mouse (p53 knockout mouse). The p53 knockout mouse is accepted by several regulatory agencies around the world as an alternative model for oncogenicity bioassays (McDonald et al. 2004), and demonstrates a > 85% concordance with the results of chronic rodent oncogenicity bioassays (Storer et al. 2001). These studies address a key regulatory requirement for the entry of soy isoflavones and BBI into long-term clinical trials for cancer prevention, and also provide data relevant to an issue that could limit the use of soy isoflavones for cancer prevention in humans.

MATERIALS AND METHODS

Animal Welfare

Prior to the initiation of experimentation, all study protocols were reviewed and approved by the IIT Research Institute Animal Care and Use Committee. All work involving experimental animals was performed in full compliance with National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

Animals and Animal Husbandry

C57BL/6 and TSG-p53^(+/–) (heterozygous p53 knockout) mice were purchased from Taconic, Germantown, New York. Mice were received at 6 to 8 weeks of age and were quarantined for 2 weeks prior to the administration of test or control articles. Mice were housed individually in suspended stainless steel cages in a windowless, climate-controlled room that was maintained on a 12-h light/12-h dark cycle. At all times during the quarantine and dosing periods, animals were permitted free access to certified Purina Laboratory Chow 5002 (PMI Feeds, Richmond, Indiana) and coarse-filtered City of Chicago water (delivered via an automatic watering system).

Test and Control Articles

PTI G-2535 (a mixture of soy isoflavones that contains 45% genistein, 23% daidzein, and 4% glycitein) and BBIC were obtained from the Chemopreventive Agent Repository maintained by the Division of Cancer Prevention, National Cancer Institute (Bethesda, Maryland). PTI G-2535 was administered as a suspension in a vehicle of 0.5% aqueous carboxymethylcellulose; BBIC was administered as a suspension in distilled water. Mice in the vehicle-control groups received daily gavage administration of 0.5% aqueous carboxymethylcellulose only (isoflavone study) or distilled water only (BBIC study). Mice in the positive control group in each study received daily oral (gavage) exposure to p-cresidine (Sigma Chemical, St. Louis, Missouri). p-Cresidine induces urinary bladder cancers in TSG-p53^(+/–) mice, and has been used previously as a positive control article for oncogenicity bioassays in this animal model (Storer et al. 2001).

Range-Finding Study Designs

Four-week range-finding studies were conducted in C57BL/6 mice to support dose selection for the definitive oncogenicity evaluations; maximum doses evaluated in these studies were 5000 mg/kg/day for PTI G-2535 and 3000 mg/kg/day for BBIC. In these range-finding studies, it was determined that the maximum dose levels of PTI G-2535 and BBIC that can be administered via gavage to mice in a repeat-dose protocol are limited by the viscosity of the dosing formulations, rather than by any evidence of limiting toxicity (data not shown). On the basis of dosing formulation viscosity, the maximum dose levels selected for use in the oncogenicity studies were 2500 mg/kg/day for PTI G-2535 and 2000 mg/kg/day for BBIC.

Oncogenicity Study Designs

In the definitive oncogenicity study of PTI G-2535, groups of 25 TSG-53^(+/–) mice per sex received daily oral (gavage) exposure to the soy isoflavone mixture at doses of 250, 1000, or 2500 mg/kg body weight/day for 6 months. In the definitive oncogenicity study of BBIC, groups of 25 TSG-53^(+/–) mice per sex received daily oral (gavage) exposure to BBIC at doses of 500, 1000, or 2000 mg/kg body weight/day for 6 months. In both studies, a dosing volume of 10 ml/kg/day was used; the maximum dose of each agent that could be administered was limited by viscosity. Mice in the negative control group (25 per sex) in each study received daily gavage exposure to an identical volume of either 0.5% aqueous carboxymethylcellulose (isoflavone study) or distilled water (BBIC study) for 6 months. Mice in the positive control group (15 per sex in the isoflavone study; 25 per sex in the BBIC study) received daily gavage exposure to p-cresidine (400 mg/kg body weight/day) for the same period.

In both studies, mice were observed twice daily throughout the quarantine and dosing periods for mortality or evidence of toxicity. Body weights and food consumption were measured individually for each animal once per week, and each animal underwent a detailed, hand-held clinical and physical observation on a weekly basis. Hematology and clinical chemistry assays were performed on blood samples collected from the first ten animals per sex per group immediately prior to the terminal necropsy. Clinical chemistry assays were performed using an automated clinical chemistry analyzer (Synchron LX-20; Beckman-Coulter, Brea, California) and hematology assays were performed using an automated hematology analyzer (Advia 120; Bayer Healthcare, Tarrytown, New York).

All mice underwent a complete necropsy with tissue collection. Intercurrent deaths were necropsied immediately after their discovery. At the end of the 6-month dosing period, surviving animals were euthanized in random order by CO₂ asphyxiation; necropsy was initiated within 5 min of euthanasia. At the terminal necropsy, weights of the adrenals, brain, heart, kidneys, liver, spleen, testes/ovaries, and thymus were collected individually for each animal. Histopathologic evaluation of tissues from the vehicle control group and all isoflavone or BBIC-treated groups included all gross lesions identified at necropsy and approximately 45 tissues per animal. In the positive-control group, histopathology was limited to the kidney and urinary bladder, known target tissues for the oncogenicity of p-cresidine (Storer et al. 2001).

Statistical Analysis

Statistical analysis of continuous data (body weight, food consumption, clinical pathology) was performed using analysis of variance, with post hoc comparisons made using Dunnett’s test. Incidence data (survival, clinical observations, histopathology) were compared using χ² analysis. A minimum significance level of p < .05 was used for all comparisons.

RESULTS

Oncogenicity Study of PTI G-2535

Daily gavage administration of PTI G-2535 at dose levels of up to 2500 mg/kg/day had no significant effect on animal survival, and induced no evidence of gross toxicity in any treated animal. At study termination, survival in all study groups ranged from 92% to 100%.

Weekly clinical observations failed to identify any evidence of toxicity in groups exposed to either PTI G-2535 or to p-cresidine; clinical signs were unremarkable in all mice treated with PTI G-2535, regardless of dose. By contrast, however, PTI G-2535 did induce a dose-related suppression of body weight gain in male mice (Figure 1), but not in female mice (Figure 2). The finding that body weight gain is suppressed only in male mice suggests that this effect may be associated with the weak estrogenicity of the soy isoflavone mixture. Body weight gain was significantly suppressed in both sexes of p53^(+/–) mice exposed to the positive control article, p-cresidine (Figures 1 and 2).

Necropsy findings were unremarkable in the negative-control group and in all groups exposed to PTI G-2535. No treatment-associated pattern of gross lesions or other evidence of toxicity was identified in any animal exposed to the soy isoflavone mixture. By contrast to the lack of gross pathology in isoflavone-treated mice, renal lesions consistent with nephropathy were a common finding at necropsy in both sexes of mice receiving oral exposure to p-cresidine. Organ weight data collected at the terminal necropsy demonstrated statistically significant increases in liver and spleen weights in both sexes of mice receiving chronic isoflavone exposure (Table 1).

Clinical pathology evaluations performed on samples collected at the terminal necropsy demonstrated modest, dose-related decreases in red blood cell (RBC) counts in both sexes of mice exposed to PTI G-2535; these were accompanied by compensatory increases in RBC volume and mean corpuscular hemoglobin. Female mice also demonstrated increased plasma cholesterol and triglycerides. No other differences from negative controls were observed in mice exposed to soy isoflavones. Summaries of statistically significant hematology and clinical chemistry findings in isoflavone-treated mice are presented in Tables 2 and 3.

Histopathologic evaluation of tissues provided no evidence of oncogenicity of PTI G-2535 in p53^(+/–) mice. The total incidence of neoplasms in the vehicle control and isoflavone-treated groups was low, and was comparable in all groups. All observed neoplasms were interpreted as incidental findings. A summary of neoplastic and preneoplastic lesions identified in study animals is provided in Table 4.

By contrast to the lack of oncogenicity of PTI G-2535, the positive-control article (p-cresidine) induced both neoplastic and preneoplastic lesions in the urinary bladder of p53^(+/–) mice. In the positive-control group, 4/15 male mice demonstrated transitional cell carcinoma of the urinary bladder, and 1/15 male mice developed a urinary bladder sarcoma. One of 15 female mice developed a transitional cell carcinoma of the urinary bladder. Essentially all mice in the positive-control group demonstrated preneoplastic lesions in the urinary bladder. In males, 4/15 mice developed transitional cell papillomas, 5/15 male mice demonstrated squamous metaplasia, and 15/15 male mice demonstrated urothelial hyperplasia. In female mice in the positive-control group, 2/15 females developed transitional cell papillomas, 11/15 females demonstrated squamous metaplasia, and 14/15 female mice developed urothelial hyperplasia.

Non-neoplastic findings in mice exposed to PTI G-2535 were limited to dose-related increases in the incidence of extramedullary hematopoiesis in the spleen; this effect was seen in both sexes. In comparison to a 4% (1/25) incidence in both male and female mice in the vehicle-control group, incidences of extramedullary hematopoiesis in male mice exposed to the low, middle, and high doses of PTI G-2535 were 4%, 8%, and 48%, respectively. Similarly, the incidences of extramedullary hematopoiesis in female mice exposed to PTI G-2535 were 4%, 96%, and 92% in the low-, middle-, and high-dose groups. Mice in the positive-control group demonstrated a number of degenerative and necrotic changes in the kidneys, with males being affected more severely than females. Microscopic alterations identified in the kidneys of mice in the positive-control group receiving chronic oral exposure to p-cresidine included papillary necrosis and mineralization.

Oncogenicity Study of BBIC

Daily gavage administration of BBIC at dose levels of up to 2000 mg/kg/day had no significant effect on survival, and induced no clinical evidence of toxicity in any treated animal. After 6 months of exposure, survival in groups treated with BBIC ranged from 88% to 100%; this survival compares to survival of 96% and 100% in male and female mice in the vehicle-control group, and 100% and 96% in male and female mice in the positive-control group, respectively.

No evidence of BBIC toxicity was identified during weekly clinical and physical observations; clinical signs were unremarkable in all mice treated with BBIC, regardless of dose. Similarly, no clinical evidence of toxicity was identified in animals in the positive-control group treated with p-cresidine.

Chronic oral administration of BBIC had no effect on group mean body weight in either sex (Figures 3 and 4). As was seen in the isoflavone study, a statistically significant suppression of body weight gain was seen in both sexes in the positive-control group receiving p-cresidine (Figures 3 and 4). Statistically significant reductions in group mean body weight were initially seen in both sexes after 5 weeks of exposure to p-cresidine, and continued throughout the remainder of the study.

Necropsy findings were unremarkable in the negative control group and in groups exposed to all dose levels of BBIC. No treatment-related pattern of gross lesions or other evidence of toxicity was identified in any animal exposed to BBIC. By contrast, a high incidence of gross renal lesions consistent with nephropathy was seen in both sexes of mice exposed to p-cresidine.

BBIC had no statistically significant effect on any clinical chemistry parameter evaluated (data not shown). Similarly, the results of hematology assays failed to identify any toxicity of BBIC. Mean absolute and relative weights of the adrenals, brain, heart, kidneys, liver, spleen, testes/ovaries, and thymus in groups receiving chronic dietary exposure to BBIC were comparable to those in sex-matched vehicle controls.

Histopathologic evaluation of tissues provided no evidence of oncogenicity of BBIC in p53^(+/–) mice (Table 5). In male mice, only two malignant lesions were observed in mice receiving BBIC or vehicle; both were found in the group receiving the low dose of BBIC. In female mice, three malignant lymphomas were seen in the vehicle-control group, and four malignant lymphomas were observed in the group receiving high-dose BBIC. One osteosarcoma was identified in a female mouse in the low-dose BBIC group, and one histocytic sarcoma was present in a female mouse in the high-dose BBIC group.

By contrast to the low incidences of malignancy in vehicle controls and in mice treated with BBIC, 11/25 males and 1/25 females in the positive-control group developed transitional cell carcinoma (TCC) of the urinary bladder. In addition, 1/25 males in the positive-control group developed a renal TCC. Both sexes of mice treated with p-cresidine demonstrated 100% incidences of hyperplastic and metaplastic changes in the urinary bladder epithelium.

Other than occasional incidental lesions that were identified in all groups, no non-neoplastic lesions were seen in any mouse in the vehicle control group. Similarly, no pattern of treatment-related non-neoplastic lesions was identified in any group receiving chronic oral exposure to BBIC. As was seen in the isoflavone study, renal papillary necrosis and renal mineralization were common findings in mice in the positive-control group receiving chronic oral exposure to p-cresidine.

DISCUSSION

Data from a large number of epidemiology studies demonstrate reductions in cancer risk in groups consuming soy and soy products. These epidemiology data, when considered with the expanding data set demonstrating cancer chemoprevention by soy components in animal models, clearly support the development of soy constituents for possible use in human cancer prevention. The present oncogenicity studies in p53^(+/–) mice address critical scientific and regulatory steps in the preclinical development of BBIC and PTI G-2535 for clinical study in cancer chemoprevention.

The p53 knockout mouse is a genetically engineered animal strain that is accepted by the United States Food and Drug Administration as an alternative model for oncogenicity bioassays of agents that demonstrate either clear or equivocal genotoxic activity. The model is more broadly accepted in Europe, as the European Committee for Proprietary Medicinal Products accepts oncogenicity studies in this model for all agents, regardless of genotoxicity (McDonald et al. 2004). In previous genotoxicity studies, PTI G-2535 demonstrated a positive result in the L5178Y/tk ^(+/–) (mouse lymphoma) assay, both with and without metabolic activation. The isoflavone mixture was also weakly positive (with metabolic activation only) in one of six tester strains (Salmonella typhimurium strain TA 100) in the Ames test (Misra et al. 2002). BBIC was not genotoxic in a battery of in vitro and in vivo genetic toxicity bioassays (National Cancer Institute Division of Cancer Prevention, unpublished data).

An important issue related to the interface between the epidemiologic and experimental data sets for cancer prevention by soy is the identification of the component (or components) in which chemopreventive activity resides. BBIC and several soy isoflavones demonstrate chemopreventive activity in laboratory animals. The isoflavones present in the highest concentrations in soybeans are daidzen, glycitein, genistein, and their 7-O-glucosides, 7-O-malonylglucosides and 7-O-acetylglucosides (Franke et al. 1998). Chemoprevention studies of isoflavones have generally focused on genistein; however, the literature suggests that several of the more abundant soy isoflavones listed above, as well as other less abundant isoflavones, may also have anticarcinogenic activity. As such, the chemopreventive efficacy of whole soy may result from the activity of several isoflavones, rather than a single compound alone. Such activity would suggest that development of isoflavone mixtures, rather than single pure isoflavone compounds, may provide a superior strategy for cancer chemoprevention. This approach is rational in consideration of the likely possibilities that (a) more than one soy isoflavone has biological activities related to cancer chemoprevention, and (b) complete fractionation and separation of soy to produce chemically pure isoflavone formulations is likely to be both economically unfeasible and biologically undesirable.

An essential part of the preclinical development of any natural product or other potential chemopreventive agent to which humans may receive chronic exposure at supraphysiologic or pharmacologic doses is the demonstration that the agent is not carcinogenic in laboratory animals. The results of the present studies demonstrate that PTI G-2535 and BBIC, two soy components with known anticarcinogenic activity in experimental models, are not oncogenic in TSG-p53^(+/–) (p53 knockout) mice when administered at their maximum deliverable doses for 6 months. The 6-month oncogenicity study in the p53 knockout mouse is accepted by the United States Food and Drug Administration as a suitable preclinical model for carcinogenicity evaluations. As such, lack of oncogenicity in the p53 knockout mouse is generally accepted as adequate evidence of lack of carcinogenicity in murine model systems.

The lack of oncogenicity of the soy isoflavone mixture (PTI G-2535) in the present study is particularly important in consideration of the hypothesis put forth by Ross, Potter, and Robison (1994) and Strick et al. (2000), who suggested that exposure to genistein and other isoflavones may be causally related to the induction of pediatric leukemias. Although their data supporting the proposed link between isoflavones and leukemogenesis did not include assessments of in vivo oncogenicity, Strick and colleagues (2000) suggest that genistein and several other flavonoids may contribute to leukemia through a mechanism involving inhibition of topoisomerase II and cleavage of the MLL gene. Although the mechanistic data presented by Strick et al. (2000) are suggestive, the results of the present bioassay do not support this hypothesis. Indeed, although in utero and pediatric exposures were not included in the design of the present bioassay in p53 knockout mice, the results of the oncogenicity bioassay of PTI G-2535 suggest that chronic isoflavone exposure is unlikely to be an important risk factor for leukemia, even in individuals bearing genetic alterations that predispose them to neoplasia.

Although PTI G-2535 demonstrated no evidence of oncogenicity in the present study, the soy isoflavone mixture did have a number of non-neoplastic effects. Statistically significant, dose-related increases in absolute and relative liver and spleen weights were present in both sexes (Table 1). Although the mechanism(s) underlying these effects are unknown, the observed hepatomegaly could be related to the effects of genistein and other isoflavones on hepatic cytochrome p450 levels or activity (Shay and Banz 2005; Moon, Wang, and Morris 2006). No microscopic alterations were identified in the liver of mice receiving chronic dietary exposure to PTI G-2535, and splenic alterations were limited to extramedullary hematopoiesis. As such, the increase in liver and spleen weights seen in isoflavone-treated mice appear to be of limited toxicologic significance.

Chronic administration of the soy isoflavone mixture also induced small, but statistically significant and dose-related reductions in RBC counts in both sexes. Compensatory changes that appear to be secondary to isoflavone-induced anemia included increased mean corpuscular volume, increased mean corpuscular hemoglobin, and extramedullary hematopoiesis. These effects had no effect on animal survival, and were not associated with any gross clinical evidence of toxicity.

Small but statistically significant elevations in serum cholesterol and triglycerides were also observed in female mice exposed to PTI G-2535. Although soy isoflavones have been reported to modulate the activity of enzymes involved in cholesterol and fatty acid biosynthesis, most literature reports suggest that isoflavones are hypo- rather than hyperlipidemic (Kirk et al. 1998; Song et al. 2003). Similarly, a recent meta-analysis of 23 clinical intervention trials with soy proteins containing isoflavones demonstrated an overall trend towards decreases in serum cholesterol and triglycerides (Zhan and Ho 2005). As such, the small increases (<30 mg/dl) in cholesterol and triglycerides observed in the present study appear to have limited biological relevance to the possible use of soy isoflavones for cancer prevention in humans.

Although BBI has a broad range of chemopreventive activity in animal carcinogenesis models, the chemopreventive properties of soy protease inhibitors are not universal. For example, Furukawa and colleagues have reported both cancer inhibitory and potential cancer promoting effects of soybean trypsin inhibitor in animal models for pancreatic cancer (Furukawa et al. 1992a, 1992b). In this regard, a key finding in the present study is that chronic oral administration of BBIC had no adverse effects on pancreatic structure or function, as determined by the results of histopathologic evaluation of tissues and clinical pathology assays. By contrast to the lack of pancreatic toxicity of BBIC observed in the present study, a range of toxic effects has been identified in the pancreas of rodents fed raw soy flour or other soy-derived protease inhibitors. These effects include the induction of pancreatic hypertrophy, nodular hyperplasia, acinar cell proliferation, and acinar adenomas in rats or mice fed soy flour or soybean trypsin inhibitors (McGuinness et al. 1980; Liener and Hasdai 1986; Gumbmann et al. 1989), and increases in the incidence of carcinogen-induced preneoplastic and neoplastic pancreatic lesions in rats fed raw soy flour or soy protein isolates (Gumbmann et al. 1986).

The lack of oncogenicity of PTI G-2535 and the overall lack of toxicity of BBIC in the present studies are not related to poor oral bioavailability. PTI G-2535 induced a dose-related suppression of body weight gain in male mice, thus providing clear evidence of systemic distribution of the isoflavone mixture. Although no comparable toxicity was seen when BBIC was administered at its maximum deliverable dose, previous tissue distribution studies of [¹²⁵I]BBI in mice have identified the intact protease inhibitor in several tissues following its oral administration (Billings et al. 1992). Furthermore, data from the range-finding study of BBIC conducted as a component of the present studies demonstrated that mean urinary BBI levels in groups receiving gavage exposure to BBIC exceeded those in vehicle controls by more than 40-fold (data not shown). Mean urinary BBI levels (50 to 80 ng/ml) measured in mice exposed to BBIC in the present range-finding study also substantially exceed the range of BBI concentrations (2 to 21 ng/ml) measured in the urine of humans enrolled in phase I clinical trials of BBIC (Armstrong et al. 2000a).

The results of the present studies provide no evidence that either PTI G-2535 or BBIC are oncogenic in mice lacking one allele of the p53 tumor suppressor gene. In consideration of their demonstrated chemopreventive activity in animal models for cancer in several sites, and their relative lack of toxicity and oncogenicity in predictive animal model systems, PTI G-2535 and BBIC merit consideration for further evaluation in human cancer chemoprevention trials.