Effects of isoflavones on breast tissue and the thyroid hormone system in humans: a comprehensive safety evaluation.

Isoflavones are secondary plant constituents of certain foods and feeds such as soy, linseeds, and red clover. Furthermore, isoflavone-containing preparations are marketed as food supplements and so-called dietary food for special medical purposes to alleviate health complaints of peri- and postmenopausal women. Based on the bioactivity of isoflavones, especially their hormonal properties, there is an ongoing discussion regarding their potential adverse effects on human health. This review evaluates and summarises the evidence from interventional and observational studies addressing potential unintended effects of isoflavones on the female breast in healthy women as well as in breast cancer patients and on the thyroid hormone system. In addition, evidence from animal and in vitro studies considered relevant in this context was taken into account along with their strengths and limitations. Key factors influencing the biological effects of isoflavones, e.g., bioavailability, plasma and tissue concentrations, metabolism, temporality (pre- vs. postmenopausal women), and duration of isoflavone exposure, were also addressed. Final conclusions on the safety of isoflavones are guided by the aim of precautionary consumer protection.

Consumption of a dark roast coffee decreases the level of spontaneous DNA strand breaks: a randomized controlled trial.

PURPOSE: Coffee consumption has been reported to decrease oxidative damage in peripheral white blood cells (WBC). However, effects on the level of spontaneous DNA strand breaks, a well established marker of health risk, have not been specifically reported yet. We analyzed the impact of consuming a dark roast coffee blend on the level of spontaneous DNA strand breaks. METHODS: Healthy men (n = 84) were randomized to consume daily for 4 weeks either 750 ml of fresh coffee brew or 750 ml of water, subsequent to a run in washout phase of 4 weeks. The study coffee was a blend providing high amounts of both caffeoylquinic acids (10.18 ± 0.33 mg/g) and the roast product N-methylpyridinium (1.10 ± 0.05 mg/g). Before and after the coffee/water consumption phase, spontaneous strand breaks were determined by comet assay. RESULTS: At baseline, both groups exhibited a similar level of spontaneous DNA strand breaks. In the intervention phase, spontaneous DNA strand breaks slightly increased in the control (water only) group whereas they significantly decreased in the coffee group, leading to a 27% difference within both arms (p = 0.0002). Food frequency questionnaires indicated no differences in the overall diet between groups, and mean body weight during the intervention phases remained stable. The consumption of the study coffee substantially lowered the level of spontaneous DNA strand breaks in WBC. CONCLUSION: We conclude that regular coffee consumption contributes to DNA integrity.

Anti-mitotic properties of indirubin-3'-monoxime, a CDK/GSK-3 inhibitor: induction of endoreplication following prophase arrest.

The bis-indole indirubin is the active ingredient of the Traditional Chinese Medicine recipe Danggui Longhui Wan used against chronic myelocytic leukemia. We have previously shown that indirubins are potent inhibitors of cyclin-dependent kinases and glycogen synthase kinase-3. We here investigated the anti-mitotic properties of this class of compounds using the cell permeable indirubin-3'-monoxime and the HBL-100 cell line. Indirubin-3'-monoxime reversibly arrests asynchronous HBL-100 cells in G2. This arrest is not accompanied by any significant change in expression of the major cell cycle regulators. However indirubin-3'-monoxime inhibits the phosphorylation of consensus CDK phosphorylation sites as well as of nucleolin at a specific CDK1/cyclin B phosphorylation site, suggesting a direct action on the mitotic CDK1/cyclin B. When indirubin-3'-monoxime is added to HBL-100 cells synchronized in M phase by nocodazole, cells undergo an endoreplication leading to an 8n DNA content. As soon as indirubin-3'-monoxime is washed away, these polyploid cells become aneuploid and later die from necrosis. This mechanism of endoreplication followed by cell death may contribute to the anti-tumour properties of indirubins.

Indirubins inhibit glycogen synthase kinase-3 beta and CDK5/p25, two protein kinases involved in abnormal tau phosphorylation in Alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors?

The bis-indole indirubin is an active ingredient of Danggui Longhui Wan, a traditional Chinese medicine recipe used in the treatment of chronic diseases such as leukemias. The antitumoral properties of indirubin appear to correlate with their antimitotic effects. Indirubins were recently described as potent (IC(50): 50-100 nm) inhibitors of cyclin-dependent kinases (CDKs). We report here that indirubins are also powerful inhibitors (IC(50): 5-50 nm) of an evolutionarily related kinase, glycogen synthase kinase-3beta (GSK-3 beta). Testing of a series of indoles and bis-indoles against GSK-3 beta, CDK1/cyclin B, and CDK5/p25 shows that only indirubins inhibit these kinases. The structure-activity relationship study also suggests that indirubins bind to GSK-3 beta's ATP binding pocket in a way similar to their binding to CDKs, the details of which were recently revealed by crystallographic analysis. GSK-3 beta, along with CDK5, is responsible for most of the abnormal hyperphosphorylation of the microtubule-binding protein tau observed in Alzheimer's disease. Indirubin-3'-monoxime inhibits tau phosphorylation in vitro and in vivo at Alzheimer's disease-specific sites. Indirubins may thus have important implications in the study and treatment of neurodegenerative disorders. Indirubin-3'-monoxime also inhibits the in vivo phosphorylation of DARPP-32 by CDK5 on Thr-75, thereby mimicking one of the effects of dopamine in the striatum. Finally, we show that many, but not all, reported CDK inhibitors are powerful inhibitors of GSK-3 beta. To which extent these GSK-3 beta effects of CDK inhibitors actually contribute to their antimitotic and antitumoral properties remains to be determined. Indirubins constitute the first family of low nanomolar inhibitors of GSK-3 beta to be described.

Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases.

Indirubin is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine to treat chronic diseases. Here we identify indirubin and its analogues as potent inhibitors of cyclin-dependent kinases (CDKs). The crystal structure of CDK2 in complex with indirubin derivatives shows that indirubin interacts with the kinase's ATP-binding site through van der Waals interactions and three hydrogen bonds. Indirubin-3'-monoxime inhibits the proliferation of a large range of cells, mainly through arresting the cells in the G2/M phase of the cell cycle. These results have implications for therapeutic optimization of indigoids.

Novel steroid-linked conjugates of 17 beta-[N-[N'-(2-chloroethyl)-N'-nitroso]carbamoyl]amino acids and their antineoplastic activity against Noble Nb prostate carcinoma model in rats.

The novel steroid conjugates 17 beta-[N-[N'-(2-chloroethyl)-N'-nitroso] carbamoyl]-glycyl-19-nortestosterone (1) and 17 beta-[N-[N'-(2-chloroethyl)-N'-nitroso]carbamoyl]-L-alyanyl-19- nortestosterone (2) were synthesized and characterized with respect to affinity for steroid receptors and for androgenic efficacy. At an i.p. dosage of 50 mg/kg, conjugates 1 and 2 induced strong tumor inhibition of Nobel Nb prostate carcinoma in rats, but also a marked loss of body weight. In two further experiments, treatment with conjugate 2 at a dosage of 25 mg/kg demonstrated high antitumor activity without indication of toxicity. Conjugate 2 achieved the same tumor growth inhibition as a nearly twofold molar dose of cyclophosphamide. The results indicate reproducibly high antitumor activity of 2 in the Noble Nb model at a well tolerated dosage. A low dose equimolar mixture of unlinked N-(2-chloroethyl)-N-nitrosocarbamoyl (CNC)-alanine and 19-nortestosterone was significantly more toxic than conjugate 2, showing about the same adverse effect on the body weight as the conjugate at high dosage. CNC-L-alanine at equimolar dosage was highly toxic, causing early death of all animals.

Biological activity of hydroxylated chloroethylnitrosoureas.

1-Nitroso-1-(2-hydroxyethyl)-3-(2-chloroethyl)urea (Compound I) and 1-nitroso-1-(2-hydroxypropyl)-3-(2-chloroethyl)urea (Compound II) display significantly reduced antitumor activity compared to the corresponding isomeric derivatives 1-nitroso-1-(2-chloroethyl)-3-(2-hydroxyethyl) urea (Compound III) and 1-nitroso-1-(2-chloroethyl)-3-(2-hydroxypropyl) urea (Compound IV). Their low therapeutic activity is paralleled by low toxicity while mutagenicity and carcinogenicity are high. A comparative investigation of the genotoxicity of Compounds I and III using primary cultures of fetal hamster lung cells revealed an about 14-fold higher rate of DNA single-strand breaks following exposure (100 microM, 1 h) to Compound I as compared to Compound III. The rate of DNA interstrand cross-links, on the other hand, was 11-fold higher following Compound III as compared to Compound I. The results underline that the therapeutic activity of chloroethylnitrosoureas is mainly attributable to their cross-linking potential while induction of DNA single-strand breaks plays a decisive role for mutagenicity and carcinogenicity but appears not to be relevant for antineoplastic effectiveness.

[Chemotherapeutic characterization of new nitrosourea compounds]. / Chemotherapeutische Charakterisierung neuer Nitrosoharnstoffe.

The development of new nitrosoureas is described using selected examples. Results obtained with water-soluble analogs and with compounds linked to biomolecules as for instance amino acids, oligopeptides and steroids, are presented. The pronounced antineoplastic effect of some water-soluble analogs is paralleled by an increased rate of DNA-interstrand cross-links and by an increased suppression of hematopoietic stem cells. The suppression of bone marrow stem cells is followed by their rapid regeneration. Water-soluble nitrosoureas induce significant less inhibition of glutathione reductase as compared with established compounds. With regard to long-term toxicity and carcinogenicity water-soluble are superior to established compounds as for instance BCNU. Linking of the nitrosourea moiety to amino acids and oligopeptides led to some analogs with outstanding therapeutic ratio. Out of a group of steroid-linked nitrosoureas, CNC-L-alanine-estradiol-17-ester (CNC-ala-17-E2) is chosen to demonstrate the possibility of reducing bone marrow toxicity despite unchanged or increased therapeutic activity by attachment of the nitrosourea moiety to a steroid. Results of a comparative interspecies in vitro evaluation of CNC-ala-17-E2 in transplanted MXT mammary carcinoma of the mouse, MNU-induced autochthonous rat mammary carcinoma and primary human mammary carcinomas are presented and the question is discussed to what extent in vitro activity of such receptor agents using the tumor stem cell assay reflects their in vivo activity.

Influence of ascorbic acid on the endogenous (intragastral) formation of N-nitroso compounds.

N-Nitroso compounds are not only environmentally occurring carcinogens, but can also be generated in vivo from their precursors. Nitrosation rates depend on the chemical structures of the compounds to be nitrosated, their basicity, the pH value of the reaction medium and the availability of nitrosating agents. Yields of N-nitroso compounds can be enhanced by catalysts or decreased by inhibitors. One of the most potent inhibitors of N-nitrosation is ascorbic acid, which has been shown to block formation of N-nitroso compounds very effectively in vitro and also in animal experiments at high dosages; alpha-tocopherol seems to be of similar effectiveness. In the human situation, ascorbic acid can be applied to prevent nitrosamine formation in the gastrointestinal tract. However, the pharmacokinetics of the nitrosable compounds also play an important role: recirculation of drugs with long half-lives through the bloodstream via the salivary glands would need a continuous intake of ascorbic acid as long as the drug is present in the plasma, since ascorbic acid practically does not follow the blood-saliva circulation process.

Anticancer activity of new nitrosoureas against Walder carcinosarcoma 256 and DMBA-induced mammary cancer of the rat.

Three new nitrosoureas and chlorozotocin were screened for anticancer activity against Walker carcinoma 256 and DMBA-induced mammary cancer of the rat. High single doses (80% of LD50) of water-soluble 1-(2-chloroethyl)-1-nitroso-3-(methylencarboxamido)urea and 2-[3-(2-chloroethyl)-3-nitrosoureido]ethylmethansulfonate effected a similar tumor weight inhibition than BCNU in treatment of subcutaneously implanted Walker 256. In dose-response studies low doses of the new analogs effected a higher tumor weight inhibition than BCNU in the treatment of subcutaneously implanted Walker 256. The therapeutic index calculated as LD50/ED50, was 2.7 and 2.4 for the new compounds in comparison to BCNU with 2.1. Chlorozotocin and 1-(methylenecarboxyethyl)-1-nitroso-3-phenylurea were not active. Against DMBA-induced mammary cancer the new BCNU analogs yielded a greater tumor weight inhibition than adriamycin and BCNU. At a dose-level of approximately LD10, the remission rates of individual tumors, which were at least 0.6 g at the beginning of treatment were 46% (13/28) for therapy with 2-[3-(2-chloroethyl)-3-nitrosoureido]ethylmethanesulfonate, 32% (9/28) for 1-(2-chloroethyl)-1-nitro-3-(methylenecarboxamido)urea, 26% (6/23) for chlorozotocin, 21% (7/33) for adriamycin and 17% for BCNU, respectively. As single agents the 2 new analogs 2[3-(1-chloroethyl)-3-nitrossureido[ethylmethanesulfonate, the water-soluble 1-(2-chloroethyl)-1-nitroso-3-(methylenecarboxamido)urea and chlorozotocin were the most effective compounds against DMBA-induced mammary cancer of the rat.

Carcinogenic activity in Sprague-Dawley rats of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-gluco-pyranose (chlorozotocin).

Sprague--Dawley rats of both sexes received 2 mg/kg body wt (group 1) or 0.4 mg/kg body wt (group 2) of 2-[3-(2-chloroethyl)-3-nitrosoureido]-D-glucopyranose (chlorozotocin) intraperitoneally in weekly injections for life. A dose-dependent carcinogenic effect of chlorozotocin was found (animals with malignant tumors in group 1: males 88%, females 89%, in group 2: males 83%, females 63%, in controls: males 10%, females 24%). Chlorozotocin reduced the life expectancy of treated animals significantly, compared to solvent controls. Histologically, the chlorozotocin-induced tumors were shown to be mesotheliomas or sarcomas of the peritoneal cavity (undifferentiated type, fibrosarcomas, myosarcomas).

Examination of four newly synthesized 2-chloroethylnitrosoureas in comparison with BCNU, CCNU, MeCCNU, chlorozotocin and hydroxyethyl-CNU in preterminal rat leukemia L 5222.

The newly synthesized nitrosoureas 1-(2-chloroethyl)-1-nitroso-3-(methylenecarboxamido)-urea (Acetamido-CNU), 1-(2-chloroethyl)-1-nitroso-3-(4-morpholino)-urea (Morpholino-CNU), 1-(2-chloroethyl)-1-nitroso-3-(1-piperidino)-urea (Piperidino-CNU), and 2-[3-(2-chloroethyl)-3-nitrosoureido]-ethylmethanesulfonate (Ethylmethanesulfonato-CNU) were compared in their chemotherapeutic activity against preterminal rat leukemia L 5222 with BCNU, CCNU, MeCCNU, Chlorozotocin, and Hydroxyethyl-CNU. With respect to the dose range effecting a median survival time of more than 90 days, MeCCNU was superior to the other substances. Chlorozotocin, on the other hand, was the only substance which achieved no cures in this experimental arrangement. From the four newly introduced substances the water-soluble substances Acetamido-CNU and Morpholino-CNU were approximately comparable to CCNU with regard to the dose range effecting a median survival time of greater than 90 days. Piperidino-CNU and Ethylmethanesulfonato-CNU also effected cures; however, only Piperidino-CNU in one dosage effected a median survival time of greater than 90 days.

Chemotherapeutic activity of new 2-chloroethylnitrosoureas in rat L5222 leukemia: comparison of bifunctional and water-soluble derivatives with 1,3-bis(2-chloroethyl)-1-nitrosourea.

The chemotherapeutic activity of eight nitrosourea derivatives was compared with that of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in transplantable rat leukemia L5222 cells. Bifunctional 1,1'-polymethylenebis-3-(2-chloroethyl)-3-nitrosoureas effected cure rates between 30 and 75% in single equitoxic doses in the therapy of advanced ip implanted L5222 [staging of L5222 leukemia development (hr before median day of death in controls): early = greater than 120; advanced = 120--61; late = 60--25; and preterminal = 24--0]. Of three water-soluble monofunctional alkylating 1-(omega-hydroxyalkyl)-3-nitrosoureas, 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea yielded more cures (90%) than did BCNU (cure rate, 70%) and was also superior to the other substances. Against preterminal ip implanted and late intracerebrally implanted L5222, the hydroxyethyl compound was significantly superior to BCNU.