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.

Dosimetry of human exposure to furan and 2-methylfuran by monitoring urinary biomarkers.

Furan and 2-methylfuran (2-MF) can form during food processing and accumulate in foods at various concentrations depending on processing technology and beverage/meal preparation methods applied prior to consumption. Here, we report a controlled dosimetry study with 20 volunteers (10 male, 10 female) to monitor dietary furan/2-MF exposure. The volunteers followed an eleven-day furan/2-MF-restricted diet in which they consumed freshly prepared coffee brew containing known amounts of furan and 2-MF on two separate occasions (250 mL and 500 mL on days 4 and 8, respectively). Urine was collected over the whole study period and analyzed for key metabolites derived from the primary oxidative furan metabolite cis-2-butene-1,4-dial (BDA) (i.e., Lys-BDA, AcLys-BDA and cyclic GSH-BDA) and the primary 2-MF metabolite acetylacrolein (AcA, 4-oxo-pent-2-enal) (i.e., Lys-AcA and AcLys-AcA). A previously established stable isotope dilution analysis (SIDA) method was utilized. Excretion kinetics revealed two peaks (at 0-2 and 24-36 h) for AcLys-BDA, Lys-BDA, AcLysAcA and LysAcA, whereas GSH-BDA showed a single peak. Notably, women on average excreted the metabolite GSH-BDA slightly faster than men, indicating gender differences. Overall, the study provided further insights into the spectrum of possible biomarkers of furan and 2-methyfuran metabolites occurring in the urine of volunteers after coffee consumption.

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.

[Scientific policy advice on food safety provided by the Senate Commission on Food Safety]. / Politikberatung zur Lebensmittelsicherheit durch die Senatskommission zur gesundheitlichen Bewertung von Lebensmitteln (SKLM).

The Senate Commission on Food Safety (SKLM, Senatskommission zur gesundheitlichen Bewertung von Lebensmitteln) of the German Research Foundation (DFG, Deutschen Forschungsgemeinschaft) is a transdisciplinary expert committee, providing advice on food safety to the government, parliament, and authorities. Consultation is based on a scientific assessment with the aim to give expert advice to authorities, so that they can make appropriate decisions. The SKLM is independent in its scientific deliberations and under no directive in the selection of issues to work on. Topics considered may result from requests of the Federal Ministry of Food, Agriculture, and Consumer Protection (BMELV, Bundesministeriums für Ernährung, Landwirtschaft und Verbraucherschutz). Other issues may be raised by the SKLM, if they are regarded to be of particular importance for consumer health protection. Issues encompass the scientific assessment of safety and nutritional benefit of food ingredients and additives, of novel and functional food, as well as of novel food technologies. The SKLM identifies gaps in knowledge, research needs, and need for action.

Risk assessment of substances that are both genotoxic and carcinogenic report of an International Conference organized by EFSA and WHO with support of ILSI Europe.

The European Food Safety Authority (EFSA) and the World Health Organization (WHO), with the support of the International Life Sciences Institute, European Branch (ILSI Europe), organized an international conference on 16-18 November 2005 to discuss how regulatory and advisory bodies evaluate the potential risks of the presence in food of substances that are both genotoxic and carcinogenic. The objectives of the conference were to discuss the possible approaches for risk assessment of such substances, how the approaches may be interpreted and whether they meet the needs of risk managers. ALARA (as low as reasonably achievable) provides advice based solely on hazard identification and does not take into account either potency or human exposure. The use of quantitative low-dose extrapolation of dose-response data from an animal bioassay raises numerous scientific uncertainties related to the selection of mathematical models and extrapolation down to levels of human exposure. There was consensus that the margin of exposure (MOE) was the preferred approach because it is based on the available animal dose-response data, without extrapolation, and on human exposures. The MOE can be used for prioritisation of risk management actions but the conference recognised that it is difficult to interpret it in terms of health risk.

Inhibitor binding to active and inactive CDK2: the crystal structure of CDK2-cyclin A/indirubin-5-sulphonate.

BACKGROUND: Cyclin-dependent kinase 2 (CDK2) is an important target for structure-based design of antitumor agents. Monomeric CDK2 is inactive. Activation requires rearrangements to key structural elements of the enzyme's active site, which accompany cyclin binding and phosphorylation. To assess the validity of using monomeric CDK2 as a model for the active kinase in structure-based drug design, we have solved the structure of the inhibitor indirubin-5-sulphonate (E226) complexed with phospho-CDK2-cyclin A and compared it with the structure of E226 bound to inactive, monomeric CDK2. RESULTS: Activation of monomeric CDK2 leads to a rotation of its N-terminal domain relative to the C-terminal lobe. The accompanying change in position of E226 follows that of the N-terminal domain, and its interactions with residues forming part of the adenine binding pocket are conserved. The environment of the ATP-ribose site, not explored by E226, is significantly different in the binary complex compared to the monomeric complex due to movement of the glycine loop. Conformational changes also result in subtle differences in hydrogen bonding and electrostatic interactions between E226's sulphonate and CDK2's phosphate binding site. Affinities calculated by LUDI for the interaction of E226 with active or inactive CDK2 differ by a factor of approximately ten. CONCLUSIONS: The accuracy of monomeric CDK2 as an inhibitor design template is restricted to the adenine binding site. The general flexibility observed for the glycine loop and subtle changes to the phosphate binding site suggest a need to study interactions between inhibitors and active CDK2 in structure-based drug design programs.

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.

DNA damage and repair in the bone marrow of rats treated with four chloroethylnitrosoureas.

DNA is considered to be an important target for the antitumor and toxic properties of the chloroethylnitrosoureas. Since the main target for their dose-limiting toxicity and the antileukemic efficacy is believed to be the bone marrow, we have compared the formation and subsequent removal of DNA-DNA interstrand cross-links in the bone marrow of rats which had received a single i.p. injection (100 mumol/kg) of four chloroethylnitrosoureas. The kinetics of cross-link removal was identical for chlorozotocin, which is known to have low chemical carbamoylating activity, to that of 1,3-bis(2-chloroethyl)-1-nitrosourea, a drug with a relatively high carbamoylating capacity. The differential bone marrow toxicity exhibited by these two agents could not, therefore, be explained by a carbamoylation-mediated difference in the rate and extent of DNA-DNA interstrand cross-link removal. The peak level and overall magnitude of cross-links were, however, found to vary considerably with the chemical structure of the analogues. Both 1-(2-chloroethyl)-1-nitroso-3-(methylene-carboxamido)urea and 1-(2-hydroxyethyl)-3-(2-chloroethyl)-3-nitrosourea, were much more effective in inducing interstrand cross-links than 1,3-bis(2-chloroethyl)-1-nitrosourea or chlorozotocin. This differential cross-linking did not, however, parallel the single-dose acute toxicity of these agents but reflected to a greater extent differences in their antileukemic activity. Considering the widely differing biological properties of this class of compounds, the measurement of DNA-DNA interstrand cross-linking in vivo might prove relevant in the evaluation of novel nitrosoureas.

3',5'-Cyclic nucleotide phosphodiesterase in tumor cells as potential target for tumor growth inhibition.

Isoenzymes of 3',5'-cyclic nucleotide phosphodiesterase (PDE) have been characterized in B16 murine melanoma cells and MCF-7 human mammary carcinoma cells. Separation of soluble phosphodiesterase activity by fast protein liquid chromatography on a Mono-Q column resolved three isoenzymes, MCF-7 cells contained a cyclic GMP-specific isoenzyme (PDE-V), a cyclic GMP-activable isoenzyme (PDE-II), and a cyclic AMP-specific isoenzyme (PDE-IV). B16 cells contained a cyclic GMP-specific isoenzyme (PDE-V), a Ca2+/calmodulin-activated isoenzyme (PDE-I), and a cyclic AMP-specific isoenzyme (PDE-IV). A series of PDE inhibitors was tested for their activity spectrum on PDE isoenzymes. Inhibition of PDE activity in B16 cells by the new compound DC-TA-46, was found to result specifically from PDE-IV inhibition [50% inhibition (IC50) = 0.03 microM]. Much lower inhibitory activity was observed for DC-TA-46 toward PDE-I (IC50 = 5 microM) and PDE-V (IC50 = 14 microM). DC-TA-46 was found to inhibit growth of B16 melanoma and MCF-7 mammary carcinoma cells dose dependently (B16: IC50 = 1.7 microM, MCF-7: IC50 = 2 microM). At 2 microM concentration, growth inhibition of B16 melanoma cells was 60%, concomitant with a decrease in PDE activity of 63% and an increase in cAMP level of 59%. In contrast, incubation with inhibitors specific for PDE-I and PDE-V resulted only in marginal or undetectable growth inhibition. The results suggest a correlation between PDE-IV inhibition and growth inhibition. PDE-IV thus appears to be a potential new target for antiproliferative treatment.

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.

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.

Current issues and perspectives in food safety and risk assessment.

In this review, current issues and opportunities in food safety assessment are discussed. Food safety is considered an essential element inherent in global food security. Hazard characterization is pivotal within the continuum of risk assessment, but it may be conceived only within a very limited frame as a true alternative to risk assessment. Elucidation of the mode of action underlying a given hazard is vital to create a plausible basis for human toxicology evaluation. Risk assessment, to convey meaningful risk communication, must be based on appropriate and reliable consideration of both exposure and mode of action. New perspectives, provided by monitoring human exogenous and endogenous exposure biomarkers, are considered of great promise to support classical risk extrapolation from animal toxicology.

Assessment of mechanisms driving non-linear dose-response relationships in genotoxicity testing.

In genetic toxicology, risk assessment has traditionally adopted linear dose-responses for any compound that causes genotoxic effects. Increasing evidence of non-linear dose-responses, however, suggests potential cellular tolerance to low levels of many genotoxicants with diverse modes of action. Such putative non-linear dose-responses need to be substantiated by strong mechanistic data that identifies the mechanisms responsible for the tolerance to low doses. This can be achieved by experimental demonstration of cytoprotective mechanisms and by providing experimental support for the existence of tolerance mechanisms against low dose effects. By highlighting key experiments into low dose mechanisms, this review aims to clarify which mechanistic data are required to support the use of non-linear dose-response models in risk assessment. Such key experiments are presented and discussed for alkylating agents, oxidants, particulate matter, nucleoside analogues, topoisomerase inhibitors and aneugens and exemplify the use of gene knockout models or transgenic models as well as chemical modulators of key effectors of relevant pathways and their impact on dose-response relationships. In vitro studies are particularly valuable to elucidate mechanisms of low-dose protection or lack thereof, while in vivo experiments are most appropriate for deriving a safe dose. In order to evaluate the existence of non-linear dose-response relationships for genotoxicants, we suggest that careful attention should be given to the mode of genotoxic action, relevant biomarkers of exposure, as well as to the existence and impact of potential cytoprotective mechanisms like detoxifying metabolism and DNA repair.

An oestradiol-linked nitrosourea and site-directed chemotherapy in mammary carcinoma.

The half-life, peak concentration, peak accumulation and tissue availability of the DNA-crosslinking nitrosourea 1-(2-chloroethyl)-1-nitrosocarbamoyl-L-alanine (CNC-alanine) and its oestradiol-linked derivate (CNC-alanine-oestradiol-17-ester) were studied in liver, lung, spleen, uterus and mammary carcinomas in female Sprague-Dawley rats with chemically induced mammary carcinomas. Compared with CNC-alanine, the ester had a longer half-life, higher peak concentration, increased peak accumulation and enhanced tissue availability in all tissues. In oestradiol receptor positive mammary carcinomas, the oestradiol-linked drug showed a 2 times higher peak concentration, a 5 times longer half-life, a 10 times increased peak accumulation and a 20 times greater tissue availability compared with CNC-alanine. Oestradiol-linked nitrosoureas may offer new perspectives for site-directed chemotherapy of oestradiol receptor positive breast cancer.

Preclinical activity of 17 beta-[N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-L-alanyl]-5 alpha-dihydrotestosterone (E91) against tumour colony forming units and haematopoietic progenitor cells.

E91 (17 beta-[N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-L-alanyl]-5 alpha-dihydrotestosterone) (CNC-ala-DHT) is a newly synthesised alkylating compound consisting of N-[N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-L-alanine (CNC-ala) as the alkylating moiety and of 5 alpha-dihydrotestosterone (DHT) as a steroid carrier molecule. We studied the antitumour activity of E91 (final concentrations: 0.1, 1, 10 and 30 mumol/l) against freshly explanted human tumours, using an in vitro soft agar cloning system. A total of 54 tumour samples was evaluated using 1 h-exposure and 51 tumour specimens were studied using a continuous exposure for 21-28 days. In addition, the compound's activity was compared with other clinically used anticancer agents. After short-term exposure, 49 of 53 evaluable specimens (92%) had adequate colony formation, as compared with 49 of 50 (98%) after long-term exposure. After short-term exposure, E91 exhibited only marginal antitumour activity. However, in long-term exposure experiments, E91 had marked and concentration-dependent antitumour activity (P < 0.001). At concentrations of > 10 mumol/l, E91 was as active as the other clinically used antineoplastic agents and at 30 mumol/l, E91 was significantly more active than 5-fluorouracil (P = 0.041). E91 showed activity against a wide spectrum of tumour types. The highest activity was observed against colorectal carcinomas (3/4 tumour specimens inhibited at 30 mumol/l). Sensitivity was also high remarkable in breast cancer specimens with 3/6 specimens inhibited at 30 mumol/l. In vitro myelotoxicity was less than that of doxorubicin. At 30 mumol/l, E91 induced a reduction of colony forming units-granulocyte macrophage (CFU-GM) to only 53% of control and of CFU-GEMM to 20% of control. We conclude that because of broad activity and reduced myelotoxicity further clinical development of E91 appears warranted.

Synthesis of 7-benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine and novel derivatives free of positional isomers. Potent inhibitors of cAMP-specific phosphodiesterase and of malignant tumor cell growth.

7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine (7a) is a potent inhibitor of the cAMP-specific phosphodiesterase isoenzyme family PDE4 and induces growth inhibition in a panel of tumor cell lines. In this study, we describe a synthesis that yields 7a and novel derivatives free of positional isomers. The synthesis of alkylamino substituted pteridines is based on the successive nucleophilic aromatic substitution of the chlorine atoms of 2,4,6, 7-tetrachloropteridine. For the reaction with secondary amines, the positional order of reactivity was found to be C4 > C7 > C2 > C6. Final structural proof is given by X-ray crystallography. To unravel structural elements of 7a crucial for the interaction with the target enzyme, the compound was modified systematically. The impact of the modifications on activity was tested by evaluating the ability of the compounds to inhibit cAMP hydrolysis by cAMP-specific phosphodiesterase (PDE4) purified from the solid human large cell lung tumor xenograft LXFL529. Growth inhibitory properties were determined by in vitro treatment of the respective cell line LXFL529L using the sulforhodamine B assay (SRB). The results show that for high activity, the heterocyclic substituent in position 2 of the pteridine ring system requires the presence of a basic nitrogen in 4'-position, as represented by piperazine.

Carcinogenicity of N-nitrosoephedrine in rats.

N-nitrosoephedrine was administered orally to 32 male Srague--Dawley rats at doses of 120 mg/kg body wt. twice weekly. Of the treated animals, 50% died with preneoplastic and malignant lesions mainly in the liver, lung and forestomach. The median time of death of tumor bearing animals was 522 days after the beginning of the experiment. The observation of hyperkeratosis, papillomas, and 1 squamous cell carcinoma of the forestomach suggests that the compound not only exhibits systemic effects but is probably also a weak local carcinogen.

Induction of DNA strand breaks by nitrosocimetidine.

The DNA of a transformed epithelial mouse cell line was studied by means of the alkaline filter elution test. Addition of N-nitrosocimetidine (NC) to the cells in vitro resulted in a concentration-dependent increase in DNA damage. In contrast cimetidine itself had no effect. The capacity of NC to generate DNA strand breaks was found to be smaller by comparison with the potent gastric carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

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).