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Understanding intramammary estrogen homeostasis constitutes the basis of understanding the role of lifestyle factors in breast cancer etiology. Thus, the aim of the present study was to identify variables influencing levels of the estrogens present in normal breast glandular and adipose tissues (GLT and ADT, i.e., 17β-estradiol, estrone, estrone-3-sulfate, and 2-methoxy-estrone) by multiple linear regression models. Explanatory variables (exVARs) considered were (a) levels of metabolic precursors as well as levels of transcripts encoding proteins involved in estrogen (biotrans)formation, (b) data on breast cancer risk factors (i.e., body mass index, BMI, intake of estrogen-active drugs, and smoking) collected by questionnaire, and (c) tissue characteristics (i.e., mass percentage of oil, oil%, and lobule type of the GLT). Levels of estrogens in GLT and ADT were influenced by both extramammary production (menopausal status, intake of estrogen-active drugs, and BMI) thus showing that variables known to affect levels of circulating estrogens influence estrogen levels in breast tissues as well for the first time. Moreover, intratissue (biotrans)formation (by aromatase, hydroxysteroid-17beta-dehydrogenase 2, and beta-glucuronidase) influenced intratissue estrogen levels, as well. Distinct differences were observed between the exVARs exhibiting significant influence on (a) levels of specific estrogens and (b) the same dependent variables in GLT and ADT. Since oil% and lobule type of GLT influenced levels of some estrogens, these variables may be included in tissue characterization to prevent sample bias. In conclusion, evidence for the intracrine activity of the human breast supports biotransformation-based strategies for breast cancer prevention. The susceptibility of estrogen homeostasis to systemic and tissue-specific modulation renders both beneficial and adverse effects of further variables associated with lifestyle and the environment possible. Electronic supplementary material The online version of this article (10.1007/s00204-020-02807-1) contains supplementary material, which is available to authorized users.

In epidemiological studies, blood levels of 17β-estradiol (E2) are associated with hormone-dependent diseases. The lack of specific methods impedes studies on the role of E2 metabolites and their conjugates in the etiology of hormone-dependent diseases. Stable-isotope dilution tandem mass spectrometry methods (coupled to gas chromatography and liquid chromatography systems) for the analysis of 22 endogenous estrogens, including both oxidative metabolites, as well as sulfates and glucuronides, was validated and the method applied to plasma of women with no breast cancer. No changes in estrogen profile during sample cleanup were observed and values for limit of detection (7fmol/ml - 2 pmol/ml), accuracies (80-122%) as well as intra- and inter-day precision (below 28%) at levels near the limit of quantification were acceptable. In human plasma only seven estrogens were detected and estrone conjugates contributed most to the estrogen profile.

Breast cancer etiology is associated with both proliferation and DNA damage induced by estrogens. Breast cancer risk factors (BCRF) such as body mass index (BMI), smoking, and intake of estrogen-active drugs were recently shown to influence intratissue estrogen levels. Thus, the aim of the present study was to investigate the influence of BCRF on estrogen-induced proliferation and DNA damage in 41 well-characterized breast glandular tissues derived from women without breast cancer. Influence of intramammary estrogen levels and BCRF on estrogen receptor (ESR) activation, ESR-related proliferation (indicated by levels of marker transcripts), oxidative stress (indicated by levels of GCLC transcript and oxidative derivatives of cholesterol), and levels of transcripts encoding enzymes involved in estrogen biotransformation was identified by multiple linear regression models. Metabolic fluxes to adducts of estrogens with DNA (E-DNA) were assessed by a metabolic network model (MNM) which was validated by comparison of calculated fluxes with data on methoxylated and glucuronidated estrogens determined by GC– and UHPLC–MS/MS. Intratissue estrogen levels significantly influenced ESR activation and fluxes to E-DNA within the MNM. Likewise, all BCRF directly and/or indirectly influenced ESR activation, proliferation, and key flux constraints influencing E-DNA (i.e., levels of estrogens, CYP1B1, SULT1A1, SULT1A2, and GSTP1). However, no unambiguous total effect of BCRF on proliferation became apparent. Furthermore, BMI was the only BCRF to indeed influence fluxes to E-DNA (via congruent adverse influence on levels of estrogens, CYP1B1 and SULT1A2).

Because of its assumed role in breast cancer etiology, estrogen biotransformation (and interaction of compounds therewith) has been investigated in human biospecimens for decades. However, little attention has been paid to the well-known fact that large inter-individual variations exist in the proportion of breast glandular (GLT) and adipose (ADT) tissues and less to adequate tissue characterization. To assess the relevance of this, the present study compares estrogen biotransformation in GLT and ADT. GLT and ADT were isolated from 47 reduction mammoplasty specimens derived from women without breast cancer and were characterized histologically and by their percentages of oil. Levels of 12 unconjugated and five conjugated estrogens were analyzed by GC- and UHPLC-MS/MS, respectively, and levels of 27 transcripts encoding proteins involved in estrogen biotransformation by Taqman® probe-based PCR. Unexpectedly, one-third of specimens provided neat GLT only after cryosection. Whereas 17β-estradiol, estrone, and estrone-3-sulfate were detected in both tissues, estrone-3-glucuronide and 2-methoxy-estrone were detected predominately in GLT and ADT, respectively. Estrogen levels as well as ratios 17β-estradiol/estrone and estrone-3-sulfate/estrone differed significantly between GLT and ADT, yet less than between individuals. Furthermore, estrogen levels in GLT and ADT correlated significantly with each other. In contrast, levels of most transcripts encoding enzymes involved in biotransformation differed more than between individuals and did not correlate between ADT and GLT. Thus, mixed breast tissues (and plasma) will not provide meaningful information on local estrogen biotransformation (and interaction of compounds therewith) whereas relative changes in 17β-estradiol levels may be investigated in the more abundant ADT.

Dehydromenthofurolactone (tonkafuranone) and other bicyclic lactones are common flavour compounds and can be found in a variety of plants. Herein, we present an improved synthesis of the natural product and a series of close analogues, in combination with efficient purification methods for simple preparation from cheap and easily accessible chemicals. A two‐step, one‐pot, reaction procedure, consisting of an aldol condensation step with concomitant lactonization, yields simplified analogues of the natural parent compound (R)‐tonkafuranone and its likewise natural (S)‐enantiomer, lacking one or two of its methyl groups, respectively. In the case of regioisomeric products, these were resolved by preparative high‐performance liquid chromatography, which, in the case of chiral, yet racemic products, was followed by preparative enantio‐resolution on a chiral phase, permitting the isolation of all four pure isomers from one synthetic step. Using the stereo‐analytical method developed, the enantiomeric purity of natural tonkafuranone occurring in Bursera graveolens was analysed and found to be ‘100:0’ in favour of the S‐enantiomer. The chiral, enantiomerically pure products were configurationally assigned by co‐elution, semi‐synthesis, and/or chiroptical methods. With the pure isomers obtained, specific cytotoxicities were tested in two cell lines as a measure for their reactivities towards cellular nucleophiles. Although the synthesized compounds differed in their cytotoxic potential in V79 cells, they exhibited a much smaller cytotoxic potential than the mutagenic α,β,γ,δ‐unsaturated lactone patulin. No toxicity towards murine hippocampal HT‐22 cells was observed at 50 μM. Thus, the molecular mechanism contributing to the high reactivity of patulin towards nucleophiles may not apply for tonkafuranone and structurally related compounds.

Polycyclic aromatic hydrocarbons (PAHs) are products of incomplete combustion of organic compounds, abundant in exhaust fumes and cigarette smoke. They act by binding to the aryl hydrocarbon receptor (AHR) which induces expression of phase 1 and phase 2 enzymes in the liver. PAH induced AHR activation may also lead to adverse effects by modulating other pathways, for example estrogen receptor (ER) signaling in the female reproductive tract. We have investigated the effects of the PAH 3-methylcholanthrene (3-MC) on 17β-estradiol (E2) dependent signaling in the uterus of ovariectomized rats to characterize the cross talk between AHR and ER on an mRNA transcriptome wide scale. A standard three day uterotrophic assay was performed in young adult Lewis rats. Treatment induced effects were analyzed using histology, immunohistochemistry and gene expression analysis by microarray and qPCR. 3-MC shows broad E2 antagonistic effects on uterine mRNA transcription of the vast majority of E2 regulated genes, significantly altering prostaglandin biosynthesis, complement activation, coagulation pathways and other inflammatory response pathways. The regulation of ER expression in the uterus, but not the regulation of E2 metabolism in the liver, was identified as a potentially important factor in mediating this general antiestrogenic effect. The regulation of prostaglandin biosynthesis by E2 is important for inflammation-like events during pregnancy including the initiation of birth. Our results suggest that adverse effects of PAHs on prostaglandin related pathways are likely caused by the interference with E2 signaling, specifically by inhibiting the E2 mediated downregulation of PGF2α. Characterization of the generalized antagonistic effect of 3-MC on E2 dependent signaling in the rat uterus thus contributes to a better understanding of molecular mechanisms of the toxicity of PAHs in female reproductive organs.

Isoflavone (IF) sind Inhaltsstoffe von Soja und anderen Leguminosen mit schwacher estrogener Wirkung. Sie werden in angereicherter Form als Nahrungserganzungsmittel (NEM) angeboten, haufig mit Auslobung einer Wirkung gegen Wechseljahresbeschwerden. Der Beitrag gibt eine Ubersicht uber Vorkommen und Verstoffwechselung, beschreibt die Marktlage und fasst die Datenlage zur Wirksamkeit und Sicherheit von IF bzw. IF-haltigen NEM (IF-NEM) zusammen. Die biologische Wirkung von IF wird v. a. der Aktivierung der Estrogenrezeptoren zugeschrieben. Studien zum Einfluss auf endogene Estrogenspiegel in Frauen zeigen keine einheitlichen Ergebnisse. Die Europaische Behorde fur Lebensmittelsicherheit (EFSA) hat bisher alle beantragten Health Claims fur IF abgelehnt, da wissenschaftlich gesicherte Belege fur die postulierten Wirkungen fehlen. Laut aktueller Risikobewertung der EFSA ergaben die ausgewerteten Humanstudien keine Hinweise auf adverse Effekte durch IF-NEM in den Zielorganen Brustdruse, Uterus und Schilddruse bei gesunden postmenopausalen Frauen. Die Bewertung erlaubt jedoch keine allgemeingultigen Aussagen zur Sicherheit von IF-NEM. Untersuchungen in Tiermodellen sind aufgrund von Unterschieden in der Verstoffwechselung von IF, im biologischen Entwicklungsstadium zum Zeitpunkt der Exposition und in der zeitlich begrenzten Aufnahme von IF in bestimmten Lebensphasen nur eingeschrankt auf den Menschen ubertragbar.

Despite being widely used to investigate 17β-estradiol (E2)-induced mammary gland (MG) carcinogenesis and prevention thereof, estrogen homeostasis and its significance in the female August Copenhagen Irish (ACI) rat model is unknown. Thus, levels of 12 estrogens including metabolites and conjugates were determined mass spectrometrically in 38 plasmas and 52 tissues exhibiting phenotypes ranging from normal to palpable tumor derived from a representative ACI study using two different diets. In tissues, 40 transcripts encoding proteins involved in estrogen (biotrans)formation, ESR1-mediated signaling, proliferation and oxidative stress were analyzed (TaqMan PCR). Influence of histo(patho)logic phenotypes and diet on estrogen and transcript levels was analyzed by 2-way ANOVA and explanatory variables influencing levels and bioactivity of estrogens in tissues were identified by multiple linear regression models. Estrogen profiles in tissue and plasma and the influence of Hsd17b1 levels on intra-tissue levels of E2 and E1 conclusively indicated intra-mammary formation of E2 in ACI tumors by HSD17B1-mediated conversion of E1. Proliferation in ACI tumors was influenced by Egfr, Igf1r, Hgf and Met levels. 2-MeO-E1, the only oxidative estrogen metabolite detected above 28–42 fmol/g, was predominately observed in hyperplastic tissues and intra-tissue conversion of E1 seemed to contribute to its levels. The association of the occurrence of 2-MeO-E1 with higher levels of oxidative stress observed in hyperplastic and tumor tissues remained equivocal. Thus, the present study provides mechanistic explanation for previous and future results observed in the ACI model.

Human African trypanosomiasis (HAT) is a major tropical disease for which few drugs for treatment are available, driving the need for novel active compounds. Recently, morpholino-substituted benzyl amides of the fluoroquinolone-type antibiotics were identified to be compounds highly active against Trypanosoma brucei brucei . Since the lead compound GHQ168 was challenged by poor water solubility in previous trials, the aim of this study was to introduce structural variations to GHQ168 as well as to formulate GHQ168 with the ultimate goal to increase its aqueous solubility while maintaining its in vitro antitrypanosomal activity. The pharmacokinetic parameters of spray-dried GHQ168 and the newly synthesized compounds GHQ242 and GHQ243 in mice were characterized by elimination half-lives ranging from 1.5 to 3.5 h after intraperitoneal administration (4 mice/compound), moderate to strong human serum albumin binding for GHQ168 (80%) and GHQ243 (45%), and very high human serum albumin binding (>99%) for GHQ242. For the lead compound, GHQ168, the apparent clearance was 112 ml/h and the apparent volume of distribution was 14 liters/kg of body weight (BW). Mice infected with T. b. rhodesiense (STIB900) were treated in a stringent study scheme (2 daily applications between days 3 and 6 postinfection). Exposure to spray-dried GHQ168 in contrast to the control treatment resulted in mean survival durations of 17 versus 9 days, respectively, a difference that was statistically significant. Results that were statistically insignificantly different were obtained between the control and the GHQ242 and GHQ243 treatments. Therefore, GHQ168 was further profiled in an early-treatment scheme (2 daily applications at days 1 to 4 postinfection), and the results were compared with those obtained with a control treatment. The result was statistically significant mean survival times exceeding 32 days (end of the observation period) versus 7 days for the GHQ168 and control treatments, respectively. Spray-dried GHQ168 demonstrated exciting antitrypanosomal efficacy.

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. Electronic supplementary material The online version of this article (10.1007/s00204-018-2279-8) contains supplementary material, which is available to authorized users.

AIMS Sodium intake has been linked to left ventricular hypertrophy independently of blood pressure, but the underlying mechanisms remain unclear. Primary hyperaldosteronism (PHA), a condition characterized by tissue sodium overload due to aldosterone excess, causes accelerated left ventricular hypertrophy compared to blood pressure matched patients with essential hypertension. We therefore hypothesized that the myocardium constitutes a novel site capable of sodium storage explaining the missing link between sodium and left ventricular hypertrophy. METHODS AND RESULTS Using 23Na magnetic resonance imaging, we investigated relative sodium signal intensities (rSSI) in the heart, calf muscle, and skin in 8 PHA patients (6 male, median age 55 years) and 12 normotensive healthy controls (HC) (8 male, median age 61 years). PHA patients had a higher mean systolic 24 h ambulatory blood pressure [152 (140; 163) vs. 125 (122; 130) mmHg, P

Isoflavones (IFs) from soy and other legumes have weak estrogenic properties. Isolated IFs are available as dietary supplements and advertised to alleviate symptoms of menopause. The present chapter provides an overview of the occurrence, the chemical structure of IFs and their metabolites, the market situation and reviews the current evidence on the efficacy and safety of IF-containing dietary supplements.The biological effectiveness of IFs is attributable to the activation of the estrogen receptor (ER). Studies on the influence of IFs on endogenous estrogen levels in women show inconsistent results. So far, the European Food Safety Authority (EFSA) has rejected all submitted health claims for IFs due to insufficient scientific evidence for any of the postulated health effects. Based on the results of their recent risk assessment, the EFSA concluded that the available human studies did not support the hypothesis of adverse effects of isolated IFs on the human mammary gland, uterus or thyroid in healthy postmenopausal women. However, the assessment does not allow a general statement on the safety of IF-containing dietary supplements. Studies in animal models are often not comparable with the complex interactions in humans due to differences in the metabolism of IFs, in the developmental stage at time of consumption and in the temporarily restricted uptake of IFs during certain stages of life.So far, for none of the advertised functions is unequivocal scientific evidence available. On the basis of available data, potential unwanted side effects cannot be fully excluded. This holds particularly true for women with undiagnosed diseases, especially for those with undetected precancerous lesions in the mammary gland.

The α,β-unsaturated carbonyl group is recognized as alert for mutagenicity, attributed to (1) its direct reaction with DNA, counteractable by glutathione (GSH), and (2) oxidative stress caused indirectly by GSH depletion. Accordingly, the α,β,γ,δ-unsaturated lactone patulin (PAT), a mycotoxin detected in fruits and products derived thereof, is known to induce gene, chromosome, and genome mutations in vitro, its mutagenicity correlating inversely with intracellular GSH levels. Thus, the reactivity of PAT against DNA bases and nucleosides in the absence and presence of GSH and glutathione S-transferases (GSTs) was investigated under cell-free conditions using HPLC mass spectrometry techniques for identification of reaction products. Adduct formation with all four nucleobases as well as with purine base nucleosides occurred even in the presence of GSH, revealing several adducts of PAT, mono- and disubstituted with nucleobases/nucleosides as well as novel GSH-PAT adducts. In addition, novel mixed GSH-PAT-nucleobase adducts were observed. These adducts exhibited a ketohexanoic acid-type structure of the PAT molecule, C6 substituted with GSH and linking C1 of PAT with nitrogens of nucleobases/nucleosides via an amide bond. Formation of GSH-PAT-adenine adducts was not prevented by GSTs, and excess of GSH needed to reduce their formation was higher than for PAT-adenine adducts. The formation of mixed GSH-DNA base adducts has not been described for PAT or any other α,β-unsaturated carbonyl before, although the reaction mechanism seems to be applicable to a variety of α,β-unsaturated carbonyls occurring in food and in the environment.

We present a new class of hybrid molecules consisting of the established antiplasmodial drugs primaquine and chloroquine. No drug is known to date that acts comparably against all stages of Plasmodium in its life cycle. Starting from available precursors, we designed and synthesized a new-generation compound consisting of both primaquine and chloroquine components, with the intent to produce agents that exhibit bioactivity against different stages of the parasite's life cycle. In vitro, the hybrid molecule 3 displays activity against both asexual and sexual P. falciparum blood stages as well as P. berghei sporozoites and liver stages. In vivo, the hybrid elicits activity against P. berghei liver and blood stages. Our results successfully validate the concept of utilizing one compound to combine different modes of action that attack different Plasmodium stages in the mammalian host. It is our hope that the novel design of such compounds will outwit the pathogen in the spread of drug resistance. Based on the optimized synthetic pathway, the compound is accessible in a smooth and versatile way and open for potential further molecular modification.

A recent intervention study demonstrated the occurrence of irilone as second most abundant isoflavone next to daidzein in human plasma after consumption of a red clover-based dietary supplement (RCDS) containing predominately formononetin ≫ biochanin A > irilone (12 % of these isoflavones). To elucidate the relevance of this finding, in the present study (1) the representativeness of the isoflavone composition of the RCDS and (2) the estrogenic activity of irilone were investigated. Thus, major isoflavones were quantified in eight commercially available RCDS. Furthermore, the estrogenic activities of irilone and other isoflavones were determined by marker gene expression in Ishikawa and cell proliferation in MCF-7 cells. Irilone amounted to 1.8–10.9 mg/g capsule content and 5–18 % of the three major isoflavones, respectively, demonstrating the general occurrence of irilone in RCDS. Moreover, irilone significantly induced the activity of alkaline phosphatase (AlP) as well as AlP, progesterone receptor, and androgen receptor mRNA levels in Ishikawa cells. Furthermore, irilone significantly induced MCF-7 cell proliferation. Neither 17β-estradiol (E2)-induced AlP activity nor E2-induced MCF-7 cell proliferation was affected by irilone. ICI182,780 antagonized IRI-induced effects on both AlP activity and cell proliferation, suggesting an estrogen receptor agonistic mode of action. Taking into account the estrogenic activity of red clover isoflavones (formononetin, biochanin A, prunetin, glycitein) and their biotransformation products (daidzein, genistein, ethylphenol) as well as published plasma levels of isoflavones after consumption of RCDS, irilone could contribute approximately 50 % of the E2 equivalents estimated for daidzein.

In Eastern Asia, coronary heart disease as well as breast and prostate cancer are less frequent than in Western countries, which is considered to be due to the Asian diet. Because Asians consume much more soy-based food products, putative beneficial health effects of soy and soy isoflavones have attracted much attention in the past two decades. Yet, although a plethora of cell-free studies, studies in vitro and in vivo, and studies in humans have investigated a multitude of putatively beneficial biological effects, no health claim directly related to isoflavones has been approved up to now. Likewise, despite ongoing safety evaluation of isoflavones by scientific expert panels, no conclusion is expected to be released in the near future. The present chapter summarizes the current knowledge on toxicity and efficacy of isoflavones, focusing on biokinetic properties of isoflavones necessary to understand the heterogeneity of study results and other difficulties in evaluating safety and efficacy of isoflavones.

Screening of various bisquaternary bisnaphthalimides against a variety of human pathogens revealed one compound, designated MT02, with strong inhibitory effects against Gram-positive bacteria. The MICs ranged from 0.31 μg/ml against community-acquired methicillin-resistant Staphylococcus aureus (MRSA) lineage USA300 to 20 μg/ml against Streptococcus pneumoniae . Radioactive whole-cell labeling experiments indicated a strong impact of MT02 on bacterial DNA replication. DNA microarray studies generated a transcriptional signature characterized by stronger expression of genes involved in DNA metabolism, DNA replication, SOS response, and transport of positively charged compounds. Furthermore, surface plasmon resonance and gel retardation experiments demonstrated direct binding of MT02 to DNA in a concentration-dependent, reversible, and non-sequence-specific manner. The data presented suggest that the bisquaternary bisnaphthalimide MT02 exerts anti-Gram-positive activity by binding to DNA and thereby preventing appropriate DNA replication.

The tissue concentrations of the female sex hormone 17beta-estradiol (E2) and its reactive catechol metabolites such as 4-hydroxyestradiol (4-HO-E2) play important roles in hormonal carcinogenesis. They are influenced by the activity of local enzymes involved in the metabolic activation and inactivation of E2. In the mammary gland, catechol estrogens are predominately inactivated by catechol-O-methyltransferase (COMT). Food supplements containing the soy isoflavones genistein and daidzein are consumed because they are believed to protect from breast cancer; however, this proposed benefit is controversial. The aim of the present study was to investigate the influence of soy isoflavones on the gene expression and activity of COMT in cultured human mammary adenocarcinoma MCF-7 cells. Levels of COMT messenger RNA (mRNA) were determined by reverse transcription/competitive polymerase chain reaction and COMT activity was determined by high-performance liquid chromatography analysis of the methylation products of both the model substrate quercetin and the physiological relevant substrate 4-HO-E2. Our study demonstrates for the first time that soy isoflavones at hormonally active concentrations cause a significant reduction of both COMT mRNA levels and COMT activity as well as of the methylation of 4-HO-E2. Experiments using the estrogen receptor (ER) antagonist ICI 182,780 support a role of the ER in the isoflavone-induced down-regulation of COMT expression. Thus, this study not only demonstrates that hormonally active concentrations of soy isoflavones inhibit the detoxification of catechols in this human breast cancer cell line but also implies that diet might influence COMT activity to a greater extent than heretofore recognized.

Phytosterols are constituents of plant membranes and are thus contained in low concentrations in vegetable products as well as at high concentrations in functional food designed to reduce serum cholesterol levels. Similar to ChOL, phytosterols are oxidized chemically in food and by biotransformation in vivo. Although oxyphytosterols have been detected in the serum of healthy human subjects, little is known of their biological activity. Therefore, the estrogenic and antiestrogenic activities of a mixture of six oxidation products of stigmasterol (oxy-StOL) were determined at the following endpoints: (i) the affinity to isolated human estrogen receptors (ER), (ii) the basal and 17beta-estradiol (E2)-induced expression of the alkaline phosphatase (AlP) in human endometrial adenocarcinoma (Ishikawa) cells, and (iii) the basal and E2-induced proliferation of human breast adenocarcinoma (MCF-7) cells. Oxy-StOL was able to replace E2 from human ERalpha and ERbeta and induced a weak estrogenic response in MCF-7 cells. Moreover, the E2-induced activity of the AlP in Ishikawa cells as well as the E2-induced proliferation of MCF-7 cells were decreased at noncytotoxic concentrations (up to 10 microM), indicating that at least one component of oxy-StOL represents an estrogen-active compound which might interfere with endogenous estrogens.

There is an ongoing debate whether the intake of soy-derived isoflavones (sISO) mediates beneficial or adverse effects with regard to breast cancer risk. Therefore, we investigated whether nutritional exposure to a sISO-enriched diet from conception until adulthood impacts on 17β-estradiol (E2)-induced carcinogenesis in the rat mammary gland (MG). August-Copenhagen-Irish (ACI) rats were exposed to dietary sISO from conception until postnatal day 285. Silastic tubes containing E2 were used to induce MG tumorigenesis. Body weight, food intake, and tumor growth were recorded weekly. At necropsy, the number, position, size, and weight of each tumor were determined. Plasma samples underwent sISO analysis, and the morphology of MG was analyzed. Tumor incidence and multiplicity were reduced by 20 and 56 %, respectively, in the sISO-exposed rats compared to the control rats. Time-to-tumor onset was shortened from 25 to 20 weeks, and larger tumors developed in the sISO-exposed rats. The histological phenotype of the MG tumors was independent of the sISO diet received, and it included both comedo and cribriform phenotypes. Morphological analyses of the whole-mounted MGs also showed no diet-dependent differences. Lifelong exposure to sISO reduced the overall incidence of MG carcinomas in ACI rats, although the time-to-tumor was significantly shortened.

The isoflavone irilone is found in human plasma after ingestion of red clover-based dietary supplements, but information allowing safety assessment is rare. Here, data in support of the mutagenic potential of irilone in cultured V79 cells [1] are presented. These data include (i) a quantitative assessment of irilone in the culture medium during the cell culture experiments, (ii) changes in the mutation spectrum in cDNA of the hypoxanthine-guanine phosphoribosyltransferase locus of irilone-treated V79 cells, (iii) occurrence of karyorrhexis and apoptosis as well as (iv) number of micronucleated cells containing whole chromosomes or chromosomal fragments. Also exemplary micrographs, used for the fluorescence microscopic assessment of (iii) and (iv) are presented.

The mycotoxin patulin (PAT), which occurs in various foods, induces mutations in cultured mammalian cells. However, the mechanism underlying the mutagenic potential of PAT is far from clear. In order to further clarify the mutagenic mode of action of PAT, we investigated the potential of PAT to induce DNA strand breaks, oxidative DNA modifications, and DNA–DNA cross-links using an alkaline filter elution technique and a fluorescence-based assay. In concentrations known to induce mutations in V79 cells (0.5–2.5 μM), PAT also induced DNA–DNA cross-links. In contrast, no effect on the number of DNA strand breaks (i.e., DNA single and double strand breaks as well as alkali-labile sites) or fpg-sensitive sites (i.e., oxidative DNA modifications) was observed in the non-cytotoxic concentration range. Thus, the present study demonstrates for the first time the direct reactivity of PAT towards DNA in a cellular system, which might have implications for the toxicological evaluation of this widespread mycotoxin.

The mycotoxin alternariol (AOH) is an important contaminant of fruit and cereal products. Concern about exposure to low levels of AOH was raised after the disclosure that contamination of food with the AOH-producing species Alternaria alternata is associated with oesophagal cancer. Previously we have reported that AOH induces kinetochore-negative micronuclei in cultured Chinese hamster V79 cells. The present study investigates the mutagenicity of AOH at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus in V79 cells and at the thymidine kinase (TK) gene locus in mouse lymphoma L5178Y tk +/− cells (MLC). Concentrations of 10 μM AOH and more gave rise to a significant and concentration-dependent induction of HPRT and TK mutations in V79 cells and in MLC, respectively. The mutagenic potency of AOH was about 50-fold lower than that of the established mutagen 4-nitroquinoline- N -oxide in both cell lines. Discrimination between small and large colonies in the TK assay revealed the predominant induction of small colonies, which are indicative for extensive chromosomal deletions and which correlated with the induction of micronuclei in MLC. The mutagenicity of AOH may have a bearing on the carcinogenicity of this mycotoxin.

The glycoprotein Wnt-7a participates in a signaling pathway that transmits information among uterine cell types. Disruption of this pathway by the transplacentally acting carcinogen diethylstilbestrol (DES) is associated with morphological abnormalities of the female reproductive tract (FRT). This raises the question whether estrogens in the diet might also interfere with this pathway. Therefore, this study investigated the influence of the steroid hormone 17beta-estradiol (E2), the mycotoxin zearalenone (ZEN), the soy phytoestrogen genistein (GEN), and DES on the expression of Wnt-7a in an endometrial adenocarcinoma cell line (Ishikawa cells) by reverse transcription/competitive PCR. In addition, the enzymatic activity of alkaline phosphatase (ALP) was determined, which is estrogen receptor (ER)-dependently regulated in Ishikawa cells. After treatment of Ishikawa cells with E2, ZEN, GEN, and DES, a decrease in the gene expression of Wnt-7a was observed. Maximum effect (50% reduction) was observed after treatment with concentrations that induced maximum expression of the ALP. Experiments in the presence of the ER antagonist (ICI 182,780) suggested that the ER is involved in the regulation of Wnt-7a in Ishikawa cells. In conclusion, interference with the expression of Wnt genes in the FRT might be a novel mechanism by which estrogens disrupt the function of the FRT.

The mycotoxin alternariol (AOH) is found in food and beverages infected by Alternaria alternata. Because consumption of foodstuffs contaminated with A. alternata has been implicated in an elevated incidence of esophageal carcinogenesis, we have investigated the estrogenic potential, the effect on cell proliferation, and the genotoxic effect of AOH in cultured mammalian cells. AOH replaced E2 from isolated human estrogen receptors alpha and beta and increased the level of alkaline phosphatase (ALP) mRNA and the enzymatic activity of ALP in a human endometrial adenocarcinoma cell line (Ishikawa cells). The estrogenicity of AOH was about 0.01% of that of E2. The effects in Ishikawa cells were reversed by the ER antagonist ICI 182,780. Analysis of cell proliferation by flow cytometry and microscopy of Ishikawa and Chinese hamster V79 cells revealed that AOH inhibited cell proliferation by interference with the cell cycle. The genotoxic potential was assessed by the micronucleus (MN) assay and immunochemical differentiation between MN containing whole chromosomes (kinetochore-positive) and DNA fragments (kinetochore-negative) in Ishikawa and V79 cells. AOH induced kinetochore-negative MN in both cell lines. This is the first report on the estrogenic potential, inhibition of cell proliferation and clastogenicity of AOH in Ishikawa and V79 cells in vitro.

Because the ability of the mycotoxin patulin (PAT) to cause gene mutations in mammalian cells is still ambiguous, we have studied the mutagenicity of PAT at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus in cultured Chinese hamster V79 cells with normal, depleted, and elevated glutathione (GSH) levels. PAT was more toxic to GSH-depleted cells than to normal cells and caused an increase of the intracellular GSH level in normal and GSH-depleted cells. It also caused synchronization of the cell cycle due to a temporary accumulation of cells in the G2/M phase; this G2/M arrest was more persistent in GSH-depleted than in normal cells. PAT gave rise to a clear and concentration-dependent induction of HPRT mutations at non-cytotoxic concentrations in V79 cells with normal GSH level; the lowest PAT concentration causing a significant number of mutant cells was 0.3 micromolar, and the mutagenic potency of PAT equaled that of the established mutagen 4-nitroquinoline-N-oxide. The mutagenicity of PAT was again more pronounced, by a factor of about three, in GSH-depleted V79 cells. Elevated GSH levels abolished all observed effects of PAT. These data support the notion that PAT is a mutagenic mycotoxin, in particular in cells with low GSH concentration. The ability of PAT to cause gene mutations in mammalian cells might have a bearing on its carcinogenicity.

Bisphenol A (BPA), a monomer of polycarbonate plastics and epoxy resins, has previously been reported to induce micronuclei containing whole chromosomes in Chinese hamster V79 cells. In the present study, the aneuploidogenic potential of BPA was investigated in cultured human AG01522C fibroblasts. In contrast to the known aneugens diethylstilbestrol (DES) and 17beta-estradiol, which caused mitotic arrest and the induction of kinetochore-positive micronuclei, BPA did not induce micronuclei and inhibited the proliferation of AG01522C cells in G2 phase and probably also in G1 phase. Fluorescence microscopy of the BPA-treated cells after immunofluorescent staining of microtubules revealed structural abnormalities of the cytoplasmic microtubule complex (CMTC): densely stained rings and loops of tubulin were observed, which increased in number with increasing BPA concentration and were more stable against low temperature than normal microtubules. The mechanisms of the growth inhibition and the interference with microtubules elicited by BPA in AG01522C cells are presently unknown. The formation of rings and loops in the CMTC of AG01522C cells was also observed with two congeners of BPA carrying one and two, respectively, additional methyl groups in ortho-position to the phenolic hydroxyl group at each aromatic ring. However, in contrast to BPA itself, these congeners of BPA behaved "DES-like" by inducing mitotic arrest and kinetochore-positive micronuclei in AG01522C cells.

The soy isoflavones daidzein, genistein and glycitein are extensively metabolized by rat liver microsomes to a variety of catechol metabolites. Hydroxylated metabolites of daidzein and genistein have also been demonstrated in incubations with human hepatic microsomes and in the urine of humans after ingestion of soy food. Although the microsomal metabolism of formononetin and biochanin A is dominated by demethylation to daidzein and genistein, respectively, catechols of the parent isoflavones and of the demethylation products are also formed. Thus, oxidative metabolism appears to be common among isoflavones and may have implications for their biological activities. As genistein but not daidzein exhibits clastogenic activity in cultured mammalian cells, the role of oxidative metabolism for the genotoxicity of isoflavones is of particular interest.

Isoflavone (ISO) exposure during adolescence modulates 17β-estradiol (E2) sensitivity of the adult mammary gland. The present study investigated the dose dependency of these effects focusing on proliferation, estrogen receptor dependent and independent gene expression, as well as DNA methylation and ISO metabolism.Female Wistar rats were lifelong exposed to an ISO-depleted diet or to diets enriched with a soy ISO extract (ISO-rich diet (IRD)) causing plasma concentrations as observed minimally (IRDlow) and maximally (IRDhigh) in Asian women. The extract was characterized by both phytochemical analysis and E-Screen. Rats were ovariectomized at postnatal day (PND) 80 and treated with E2 from PND94 to 97. In contrast to uterine response, body weight and visceral fat mass were affected by ISO. In the mammary gland, both E2-induced proliferation (proliferating cell nuclear antigen staining) and estrogen receptor activation (progesterone receptor staining) were significantly reduced by IRDhigh but not by IRDlow, which however attenuated Gdf15 mRNA expression. DNA methylation analysis revealed significant differences in the promoter regions of Aldhl1, Extl1, and WAP between IRDhigh and ISO-depleted diet.Lifelong exposure to ISO results in dose-dependent differential effects on proliferation, gene expression, and DNA methylation in rat mammary glands. Yet, a decrease in estrogen responsiveness was only achieved by IRDhigh.

The mutagenicity of the mycotoxin patulin was assessed by the thymidine kinase mutation assay, which is based on point mutations and deletions. Patulin was mutagenic in cultured mouse lymphoma cells and the mutagenicity was significantly increased in cells pretreated with buthionine sulfoximine, which reduces intracellular glutathione levels.

Isoquinolines (IQs) are natural substances with an antibiotic potential we aim to optimize. Specifically, IQ-238 is a synthetic analog of the novel-type N,C-coupled naphthylisoquinoline (NIQ) alkaloid ancisheynine. Recently, we developed and tested other IQs such as IQ-143. By utilizing genome-wide gene expression data, metabolic network modelling and Voronoi tessalation based data analysis - as well as cytotoxicity measurements, chemical properties calculations and principal component analysis of the NIQs - we show that IQ-238 has strong antibiotic potential for staphylococci and low cytotoxicity against murine or human cells. Compared to IQ-143, systemic effects are less pronounced. Most enzyme activity changes due to IQ-238 are located in the carbohydrate metabolism. Validation includes metabolite measurements on biological replicates. IQ-238 delineates key properties and a chemical space for a good therapeutic window. The combination of analysis methods allows suggestions for further lead development and yields an in-depth look at staphylococcal adaptation and network changes after antibiosis. Results are compared to eukaryotic host cells.

Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants with myriad biological effects, including carcinogenicity. We present data showing gender-specific genotoxicity in Fischer 344 transgenic BigBlue rodents exposed to 4-chlorobiphenyl (PCB3), a hydroxylated metabolite, and the positive control 3-methylcholanthrene (3-MC) where female rats are more resistant to the genotoxic effects of the test compounds compared to their male counterparts. This difference is further highlighted through our examination of gene expression, organ-specific weight changes, and tissue morphology. The purpose of the present study was to explore the complex and multifaceted issues of lower molecular weight PCBs as initiators of carcinogenesis, by examining the mutagenicity of PCB3, a hydroxylated metabolite (4′-OH-PCB3), and 3-methylcholanthrene (3-MC, positive control) in a transgenic rodent model. Previous findings indicated that PCB3 is mutagenic in the liver of male BigBlue transgenic rats under identical exposure conditions. We expected that female rats would be equally, if not more sensitive than male rats, since a 2-year carcinogenesis bioassay with Sprague–Dawley rats and commercial PCB mixtures reported much higher liver cancer rates in female than in male rats. The current study, however, revealed a similar trend in the mutation frequencies across all four treatment groups in females as reported previously in males, but increased variability among animals within each group and a lower overall effect, led to non significant differences in mutation frequencies. A closer analysis of the possible reasons for this negative result using microarray, organ weight and histology data comparisons shows that female Fischer 344 rats 1) had a higher baseline mutation frequency in the corn oil control group and greater variability than male rats; 2) responded with robust gene expression changes, which may also play a role in our observation of 3) highly increased liver, spleen, and lung weight in 3-MC and PCB3-treated female rats and thus changed distribution and kinetics of the test compounds. Our analysis indicates that female transgenic BigBlue Fischer 344 rats are more resistant to PCB3 and 3-MC genotoxicity compared to their male counterparts. Keywords: Polychlorinated biphenyl, PCB3, 3-methylcholanthrene, 3-MC, Gender difference, Genotoxicity, Biotransformation, Carcinogenicity

The activation of 17beta-estradiol (E2) to 2-hydroxyestradiol (2-HO-E2), the more genotoxic 4-hydroxyestradiol (4-HO-E2), and the oxidation to the respective quinones constitutes a risk factor in hormonal carcinogenesis. 2-HO-E2 is formed by cytochrome P450 CYP1A1, and 4-HO-E2 is formed by CYP1B1. Both are detoxified by catechol-O-methyltransferase (COMT), whereas their quinones are inactivated by NADPH-quinone-oxidoreductase (QR). Since the soy isoflavones genistein (GEN) and daidzein (DAI) are widely consumed due to their putative protective function in breast carcinogenesis, we examined the influence of E2, GEN, and DAI on CYP1A1/1B1, COMT, and QR expression in MCF-7 cells by reverse transcription/competitive PCR. CYP1A1 and COMT enzyme activity were determined using ethoxyresorufin and quercetin as substrates. Furthermore, estrogen receptor (ER)-regulated cell proliferation was determined by E-screen. E2, GEN, and DAI inhibited the expression of CYP1A1, COMT, and QR. The maximum effect (reduction by 40-80%, depending on the gene product and compound) was obtained at 100 pM E2, 1 microM GEN, and 10 microM DAI, which also induced the most pronounced cell proliferation in the E-screen. In contrast, expression of CYP1B1 was only slightly affected. CYP1A1 and COMT mRNA levels correlated with enzyme activities. The ER antagonist ICI 182,780 reversed the E2- and isoflavone-mediated effects. Thus, GEN and DAI at estrogen-active concentrations stimulate the formation of the more E2 genotoxic metabolites and inhibit the detoxification of catechol and quinone estrogens in estrogen-responsive tumor cells.