Tools to evaluate estrogenic potency of dietary phytoestrogens:A consensus paper from the EU Thematic Network "Phytohealth" (QLKI-2002-2453).

Phytoestrogens are naturally occurring plantderived polyphenols with estrogenic potency. They are ubiquitous in diet and therefore, generally consumed. Among Europeans, the diet is rich in multiple putative phytoestrogens including flavonoids, tannins, stilbenoids, and lignans. These compounds have been suggested to provide beneficial effects on multiple menopause-related conditions as well as on development of hormone-dependent cancers, which has increased the interest in products and foods with high phytoestrogen content. However, phytoestrogens may as well have adverse estrogenicity related effects similar to any estrogen. Therefore, the assessment of estrogenic potency of dietary compounds is of critical importance. Due to the complex nature of estrogenicity, no single comprehensive test approach is available. Instead, several in vitro and in vivo assays are applied to evaluate estrogenic potency. In vitro estrogen receptor (ER) binding assays provide information on the ability of the compound to I) interact with ERs, II) bind to estrogen responsive element on promoter of the target gene as ligand-ER complex, and III) interact between the co-activator and ERs in ligand-dependent manner. In addition, transactivation assays in cells screen for ligand-induced ERmediated gene activation. Biochemical in vitro analysis can be used to test for possible effects on protein activities and E-screen assays to measure (anti)proliferative response in estrogen responsive cells. However, for assessment of estrogenicity in organs and tissues, in vivo approaches are essential. In females, the uterotrophic assay is applicable for testing ERa agonistic and antagonistic dietary compounds in immature or adult ovariectomized animals. In addition, mammary gland targeted estrogenicity can be detected as stimulated ductal elongation and altered formation of terminal end buds in immature or peripubertal animals. In males, Hershberger assay in peri-pubertal castrated rats can be used to detect (anti)androgenic/ (anti)estrogenic responses in accessory sex glands and other hormone regulated tissues. In addition to these short-term assays, sub-acute and chronic reproductive toxicity assays as well as two-generation studies can be applied for phytoestrogens to confirm their safety in long-term use. For reliable assessment of estrogenicity of dietary phytoestrogens in vivo, special emphasis should be focused on selection of the basal diet, route and doses of administration, and possible metabolic differences between the species used and humans. In conclusion, further development and standardization of the estrogenicity test methods are needed for better interpretation of both the potential benefits and risks of increasing consumption of phytoestrogens from diets and supplements.

The acute effects of mono(2-ethylhexyl)phthalate (MEHP) on testes of prepubertal Wistar rats.

A single oral dose of 400 mg/kg body weight of mono(2-ethylhexyl)phthalate (MEHP), the testis toxic metabolite of di(2-ethylhexyl)phthalate, was given to 28-day-old male Wistar rats and the testis toxic effects were investigated 3,6, and 12 h after exposure. Detachment and sloughing of germ cells were observed, and in the Sertoli cells the cytoplasmatic intermediate filament vimentin collapsed. In the immunohistochemical investigation the androgen receptor distribution was unchanged between the control group and treated groups. The expression of the testosterone-repressed-prostatic-message-2 gene in rat testis increased after 3 h, but returned to control levels after 6 and 12 h. Caspase-3 activity increased 3 and 12 h after MEHP exposure. This increase could not be correlated to an increase in DNA fragmentation or increase in apoptotic numbers of germ cells. In conclusion, the effect of MEHP in testis is apparently not involving the androgen receptor. Vimentin localisation in the Sertoli cells, and increased levels of caspase-3 activity appear to be sensitive and early markers of MEHP testis toxicity.

Evaluation of the Emu-pim-1 transgenic mouse model for short-term carcinogenicity testing.

The value of the chronic rodent carcinogenicity assay in adequately predicting cancer risk in humans has become a matter of debate over the past few years. Therefore, more rapid and accurate alternative tests are urgently needed. Transgenic mouse models, those harboring genetic changes that are relevant to the multistage cancer process, may provide such alternative tests. Transgenic Emu-pim-1 mice, developed by Berns and coworkers in 1989, contain the pimn-1 oncogene, which is expressed at elevated levels in their lymphoid compartments. As a result, these mice are predisposed to the development of T-cell lymphomas. Because of the low incidence of spontaneous tumors and the increased sensitivity to N-ethyl-N-nitrosourea-induced carcinogenesis, Emu-pim-1 mice were suggested to be one of the first potential and attractive candidates to be used in short-term carcinogenicity testing. In the present article, the results from 2 recent short-term assays (with mitomycin C and x-rays) are briefly presented, together with a review of all 11 performed bioassays and their corresponding histopathologic and molecular data. The overall results allow the first evaluation of the Emu-pim-1 mouse model with regard to its usefulness in short-term carcinogenicity testing. It has been shown that the model is primarily suitable as a sensitive short-term assay for genotoxic carcinogens that not only induce (at least) gene mutations and/or large deletions and rearrangements but that also sufficiently target the lymphoid system. However, the Emu-pim-1 mice lack sufficient sensitivity to justify their routine use in short-term carcinogenicity testing in general.

The effect of soy isoflavones on the development of intestinal neoplasia in ApcMin mouse.

Data from epidemiological studies suggest that isoflavones in soy may have a protective effect on the development of colon cancer in humans. Therefore, we have investigated whether soy isoflavones will inhibit intestinal tumour development in Apc(Min) mice. The mice were fed a Western-type high risk diet (high fat, low fibre and calcium) containing two different isolates of soy protein as a protein source. For the control and test groups this resulted in the administration of about 16 and 475 mg of total isoflavones per kg diet, respectively. As a positive control. a third group of mice was administered a low isoflavone diet supplemented with 300 ppm sulindac. No significant differences in the incidence, multiplicity, size and distribution of intestinal tumours were observed between Min mice fed low and high isoflavone-containing diets. However, a clear reduction in the number of small intestinal tumours was observed for the sulindac diet. Thus, in contrast to epidemiological studies, our results demonstrate that high amounts of soy isoflavones present in a Western-type high risk diet do not protect against intestinal tumour development in a relevant animal model such as the Min mice.

Effects of long-term feeding with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in C57BL/ByA and E mu-pim-1 mice.

The heterocyclic amine PhIP, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, has been shown to be carcinogenic in rats producing colon and mammary carcinomas. There is, so far, only a single long-term study report on PhIP carcinogenicity in mice. However, this study focuses on the lymphoma induction only. Whether PhIP induces tumors in other organs in mice after long-term exposure is yet to be established. To contribute to the knowledge about the long-term effects of PhIP feeding the remaining animals from a 7 months feeding study (0.03% PhIP) were put on standard diet and followed up to 18 months. The results confirmed that PhIP is a potent mouse lymphomagen. In addition, treatment with 0.03% PhIP in the diet for the first 7 months of a life-time study (18 months) gave rise to very few and sporadic tumors in other tissues, indicating weak, if any, carcinogenicity of PhIP to organs other than the lymphatic system under the present study design.

Short-term carcinogenicity testing of a potent murine intestinal mutagen, 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), in Apc1638N transgenic mice.

Transgenic Apc1638N mice, heterozygous for a targeted frameshift mutation at codon 1638 of the endogenous adenomatous polyposis coli (APC) gene, are predisposed to develop multiple adenomas and adenocarcinomas along the intestinal tract and to a number of extra-intestinal lesions including, among others, mammary tumors. We have studied these mice in a short-term carcinogenicity test with 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), a potent murine small intestinal mutagen and lymphomagen. Upon dietary administration of 0.03% PhIP in a short-term (6 months) study, a significantly increased number of small intestinal tumors as well as an increased number of aberrant crypt foci (ACF) were observed in male Apc+/Apc1638N mice compared with untreated transgenic mice. No differences in intestinal and mammary tumor multiplicity were observed between treated and control Apc+/Apc1638N females.

Lymphoma induction by heterocyclic amines in E mu-pim-1 transgenic mice.

The usefulness of transgenic E mu-pim-1 mice bearing in their genome the pim-1 oncogene supplemented with an upstream immunoglobulin enhancer and a downstream murine leukaemia virus long terminal repeat, as sensitive test organisms was studied in two short-term carcinogenicity studies. The mice were fed standard diet Altromin 1314 supplemented either with 0.03% 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) for 7 months or with 0.03% 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) for 6 months. PhIP and IQ are heterocyclic amines formed during cooking of meat and fish and are mutagenic to bacteria and cultured mammalian cells. PhIP is a potent mouse lymphomagen, while IQ is a liver, lung and forestomach carcinogen in mice. We found that transgenic E mu-pim-1 mice are highly susceptible to PhIP induced lymphomagenesis but do not respond to IQ treatment. PhIP feeding of E mu-pim-1 mice not only increased the total number of T-cell lymphomas but also decreased the latency time compared to either transgenic or wild-type controls. The effect was most pronounced in the treated female E mu-pim-1 mice, which showed a higher incidence of PhIP induced T-cell lymphomas than transgenic males and a strongly reduced latency period after PhIP treatment compared to non-transgenic mice. Our results suggest that the transgenic E mu-pim-1 mouse may be a useful model for short-term carcinogenicity screening of potential genotoxic carcinogens having the lymphoid system as target tissue. Carcinogens that do not target this tissue, like IQ, however will not be recognised.

Short-term carcinogenicity testing of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in E(mu)-pim-1 transgenic mice.

The usefulness of transgenic E(mu)-pim-1 mice over-expressing the pim-1 oncogene in lymphoid tissues, as sensitive test organisms was studied in a short-term carcinogenicity study. The mice were fed standard diet Altromin 1314 supplemented either with 0.03% 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) for 7 months or with 0.03% 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) for 6 months. PhIP and IQ are heterocyclic amines formed during cooking of meat and fish and are mutagenic to bacteria and cultured mammalian cells. PhIP is a potent mouse lymphomagen, while IQ is a liver carcinogen and also causes lung tumors and tumors of the forestomach in mice. We found that transgenic E(mu)-pim-1 mice are highly susceptible to PhIP induced lymphomagenesis but do not respond to the IQ treatment. PhIP feeding of E(mu)-pim-1 mice not only increased the total number of T-cell lymphomas but also decreased the latency time compared to either transgenic or wild-type controls. The effect was most pronounced in the treated female E(mu)-pim-1 mice, which showed a higher incidence of PhIP induced T-cell lymphomas than transgenic males and a strongly reduced latency period after PhIP treatment compared to non-transgenic mice. Our results suggest that the transgenic E(mu)-pim-1 mouse may be a useful model for short-term carcinogenicity screening of potential genotoxic carcinogens having the lymphoid system as target tissue. The carcinogen IQ which does not have the lymphoid system as a target was not recognized in this model.