Leane Lehmann

Mutagenicity of the mycotoxin patulin in cultured Chinese hamster V79 cells, and its modulation by intracellular glutathione

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.

Hormonal and genotoxic activity of resveratrol

Resveratrol (RES) is a natural polyphenol present in red wines and various human food items. The estrogenic activity of RES was demonstrated in two in vitro assay systems, i.e. binding to human estrogen receptor alpha and stimulation of MCF-7 cell proliferation. To investigate the inhibition of cell proliferation observed at high concentrations of RES, we analyzed the compound for genotoxic potential. RES induced cellular toxicity, micronuclei, and metaphase chromosome displacement in L5178Y mouse lymphoma cells. Likewise, the induction of micronuclei was observed in Chinese hamster V79 cells. Determination of kinetochore signals in micronuclei and cell cycle analysis suggested that RES did not cause a direct disturbance of mitosis. In support of this notion, cell-free tubulin polymerization studies indicated no direct effect of RES on microtubule assembly. According to an estimation of daily intake and bioavailability, concentrations that were found genotoxic in vitro might be reached in human exposure. On the other side, the estrogenic acitivity might be beneficial. Therefore, further investigations of mechanisms, possibly including animal models, would be desirable to clarifiy a potential risk for humans.

Soy isoflavones decrease the catechol-O-methyltransferase-mediated inactivation of 4-hydroxyestradiol in cultured MCF-7 cells

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.

Dose-dependent effects of isoflavone exposure during early lifetime on the rat mammary gland: Studies on estrogen sensitivity, isoflavone metabolism, and DNA methylation

SCOPE 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. METHODS AND RESULTS 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. CONCLUSION 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.

A primaquine-chloroquine hybrid with dual activity against Plasmodium liver and blood stages.

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 parasites 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.

Mode-of-Action Studies of the Novel Bisquaternary Bisnaphthalimide MT02 against Staphylococcus aureus

ABSTRACT 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.

Phytoestrogens Modulate the Expression of 17α-Estradiol Metabolizing Enzymes in Cultured MCF-7 Cells

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.

Gene Expression of 17β-Estradiol-metabolizing Isozymes: Comparison of Normal Human Mammary Gland to Normal Human Liver and to Cultured Human Breast Adenocarcinoma Cells

Metabolic activation of 17beta-estradiol (E2) to catechols and quinones together with lack of deactivation constitute risk factors in human breast carcinogenesis. E2-catchols are generated by cytochrome P450-dependent monooxygenases (CYPs). Deactivation of E2, E2-catechols, and E2-quinones is mediated by UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), catechol-O-methyltransferase (COMT), glutathione-S-transferase (GST), and NADPH-quinone-oxidoreductase (QR) isozymes, respectively. The aim of the present study was to quantify mRNA levels of E2-metabolizing isozymes expressed in MCF-7 cells cultured in the presence/absence of steroids by reverse transcription/competitive PCR in relation to the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase and compare them with expression levels in normal human mammary gland (MG) and liver tissue. CYP1A1, 1B1, SULT1A1, 1A2, membrane-bound and soluble COMT, GSTT1, QR1, and UGT2B7 were detected in both tissues and MCF-7 cells; however, most enzymes were expressed at least tenfold higher in liver. Yet, CYP1B1 was expressed as high in breast as in liver and UGTs were not detected in MCF-7 cells cultured with steroids. MCF-7 cells cultured steroid-free additionally expressed CYP1A2 as well as UGT1A4, 1A8, and 1A9. Normal human liver but not MG expressed CYP1A2, 3A4, UGT1A1, 1A3, 1A4, 1A9, and SULT2A1. UGT1A8 was only detected in MCF7 cells but was not found in human liver. Thus, our study provides a comprehensive overview of expression levels of E2-metabolizing enzymes in a popular in vitro model and in human tissues, which will contribute to the interpretation of in vitro studies concerning the activation/deactivation of E2.

Modelling antibiotic and cytotoxic isoquinoline effects in Staphylococcus aureus, Staphylococcus epidermidis and mammalian cells

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.

Influence of decreased intracellular glutathione level on the mutagenicity of patulin in cultured mouse lymphoma cells

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.