Michael Habermeyer

TDP1 Overexpression in Human Cells Counteracts DNA Damage Mediated by Topoisomerases I and II

Tyrosyl DNA phosphodiesterase 1 (TDP1) is a repair enzyme that removes adducts, e.g. of topoisomerase I from the 3′-phosphate of DNA breaks. When expressed in human cells as biofluorescent chimera, TDP1 appeared more mobile than topoisomerase I, less accumulated in nucleoli, and not chromosome-bound at early mitosis. Upon exposure to camptothecin both proteins were cleared from nucleoli and rendered less mobile in the nucleoplasm. However, with TDP1 this happened much more slowly reflecting most likely the redistribution of nucleolar structures upon inhibition of rDNA transcription. Thus, a steady association of TDP1 with topoisomerase I seems unlikely, whereas its integration into repair complexes assembled subsequently to the stabilization of DNA·topoisomerase I intermediates is supported. Cells expressing GFP-tagged TDP1 > 100-fold in excess of endogenous TDP1 exhibited a significant reduction of DNA damage induced by the topoisomerase I poison camptothecin and could be selected by that drug. Surprisingly, DNA damage induced by the topoisomerase II poison VP-16 was also diminished to a similar extent, whereas DNA damage independent of topoisomerase I or II was not affected. Overexpression of the inactive mutant GFP-TDP1H263A at similar levels did not reduce DNA damage by camptothecin or VP-16. These observations confirm a requirement of active TDP1 for the repair of topoisomerase I-mediated DNA damage. Our data also suggest a role of TDP1 in the repair of DNA damage mediated by topoisomerase II, which is less clear. Since overexpression of TDP1 did not compromise cell proliferation, it could be a pleiotropic resistance mechanism in cancer therapy.

Nitrate and nitrite in the diet: How to assess their benefit and risk for human health

Nitrate is a natural constituent of the human diet and an approved food additive. It can be partially converted to nitrogen monoxide, which induces vasodilation and thereby decreases blood pressure. This effect is associated with a reduced risk regarding cardiovascular disease, myocardial infarction, and stroke. Moreover, dietary nitrate has been associated with beneficial effects in patients with gastric ulcer, renal failure, or metabolic syndrome. Recent studies indicate that such beneficial health effects due to dietary nitrate may be achievable at intake levels resulting from the daily consumption of nitrate-rich vegetables. N-nitroso compounds are endogenously formed in humans. However, their relevance for human health has not been adequately explored up to now. Nitrate and nitrite are per se not carcinogenic, but under conditions that result in endogenous nitrosation, it cannot be excluded that ingested nitrate and nitrite may lead to an increased cancer risk and may probably be carcinogenic to humans. In this review, the known beneficial and detrimental health effects related to dietary nitrate/nitrite intake are described and the identified gaps in knowledge as well as the research needs required to perform a reliable benefit/risk assessment in terms of long-term human health consequences due to dietary nitrate/nitrite intake are presented.

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.

Synthesis, topoisomerase-targeting activity and growth inhibition of lycobetaine analogs

The plant alkaloid lycobetaine has potent topoisomerase-targeting properties and shows anticancer activity. Based on these findings, several lycobetaine analogs were synthesized mainly differing in their substituents at 2, 8 and 9 position and their biological activities were evaluated. The topoisomerase-targeting properties and cytotoxicity of these structural analogs were assessed in the human gastric carcinoma cell line GXF251L. Performing a plasmid relaxation assay, an increased inhibition of topoisomerase I was found with N-methylphenanthridinium chlorides bearing a 8,9-methylenedioxy moiety or a methoxy group in 2-position. Furthermore, quaternized phenanthridinium derivatives bearing either a 2-methoxy or a 8,9-methylenedioxy moiety in conjunction with a 2-hydroxy or 2-methoxy group display potent topoisomerase II inhibition as shown by decatenation of kinetoplast DNA. In general, the N-methylphenanthridinium chlorides possess more potency in inhibiting topoisomerase I than topoisomerase II. All quaternized derivatives also exhibited potent inhibition of tumor cell growth in the low micromolar concentration range. Hence, N-methylphenanthridinium compounds were found to represent a promising class of compounds, potently inhibiting both, topoisomerases I and II, and may be further developed into clinically useful topoisomerase inhibitors.

Studies on the inhibition of tumor cell growth and microtubule assembly by 3-hydroxy-4-[(E)-(2-furyl)methylidene]methyl-3-cyclopentene-1,2-dione, an intensively coloured Maillard reaction product

Very recently, 3-hydroxy-4-[(E)-(2-furyl)methylidene]methyl-3-cyclopentene-1,2-dione (1) has been successfully identified as an intensively coloured Maillard product formed from glucose and L-proline upon thermal food processing. Using a biomimetic synthetic strategy, reference material of compound 1 was prepared and purified, and then used to study its effect on the growth of human tumor cells. Compound 1 was found to potently inhibit the growth of human tumor cells in vitro. Using a reporter gene assay we could show that in growth inhibitory concentrations compound 1 effectively inhibits the phosphorylation of the transcription factor Elk-1. In addition, 1 was found to affect the microtubule skeleton. The human mammary carcinoma cell line MCF-7 exhibits a decrease of the microtubule organisation when treated for 24 h with 1 (> or =20 microM). At concentrations of 30 microM and above a loss of microtubule integrity is observed after 1 h incubation. In vitro studies demonstrated that the polymerisation and, to a minor extent, also the depolymerisation of tubulin, isolated and purified from bovine brain, is inhibited in a dose-dependent manner at concentrations of 30 microM and above. This is the first time that a non-enzymatically formed browning compound of known structure was reported to effectively inhibit tumor cell growth and microtubule assembly.

7-Benzylamino-6-chloro-2-piperazino-4-pyrrolidino-pteridine, a potent inhibitor of cAMP-specific phosphodiesterase, enhancing nuclear protein binding to the CRE consensus sequence in human tumour cells.

The cAMP-specific phosphodiesterase isoenzyme family PDE4 represents the highest cAMP-hydrolysing activity in many human cancer cell lines including the human large cell lung carcinoma cell line LXFL529L. Treatment of LXFL529L cells with the potent PDE4 inhibitor 7-benzylamino-6-chloro-2-piperazino-4-pyrrolidino-pteridine (DC-TA-46) induces dose-dependent growth inhibition. Cells are arrested in the G(1)-phase of the cell cycle and the induction of apoptosis is observed. In this study, we investigated the effect of DC-TA-46 on downstream elements of the cAMP-pathway. DC-TA-46 mediated inhibition of PDE4 activity in LXFL529L cells resulted in an increase of the intracellular cAMP level and significant induction of the activity of protein kinase A (PKA). The regulatory PKA subunit RIalpha was predominantly expressed in LXFL529L cells. In contrast to effects induced by cAMP analogues like 8-Cl-cAMP, the expression of the regulatory subunits of PKA remained unaffected by DC-TA-46. Treatment of LXFL529L cells with DC-TA-46 enhanced the binding of nuclear proteins to the cAMP-responsive element (CRE) consensus sequence TGACGTCA in a time- and dose-dependent manner, indicating the activation of transcription factors by PKA phosphorylation.

Microbial food cultures – opinion of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG)

In the EU, there are no specific legal regulations regarding microbial food cultures. However, at European and national level, there are regulations that require microbial cultures to be checked in terms of their compliance with legal requirements. Due to the lack of definitions for microbial food cultures with various applications, there are uncertainties regarding how they are to be assessed. The increased elaboration of microbial ecology and modern taxonomy has allowed the description of numerous new species that are attractive for use in food cultures or are already in use, on which, however, only limited experience is available. In view of these developments, the SKLM has prepared this statement, focusing on definitions, gaps in knowledge and further research needs. It aims to support the producers and users of microbial cultures as well as authorities responsible for consumer health protection with respect to safety assessment and to contribute to consumer information. The scientific status concerning these cultures in food technology, the traditional roots of their application and their potential for sustaining and/or furthering food variety and quality have not been adequately described up to now. This is the subject of the present SKLM statement. In addition, definitions are proposed for cultures used in food technology that may also be useful for the assessment in a legal context. The opinion was released in German on 29 March 2010, the English version was agreed on 15 November 2010.

Thermally induced process-related contaminants: the example of acrolein and the comparison with acrylamide: opinion of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG).

α,β-Unsaturated aliphatic carbonyl compounds are naturally widespread in food, but are also formed during the thermal treatment of food. This applies, for example, to the genotoxic carcinogen acrylamide (AA), but also to acrolein (AC), the simplest α,β-unsaturated aldehyde. First observations indicate that human exposure to AC may be higher than the exposure to AA. The DFG Senate Commission on Food Safety therefore compared data on AC and AA available in the scientific literature, evaluating current knowledge on formation, occurrence, exposure, metabolism, biological effects, toxicity, and carcinogenicity and defined knowledge gaps as well as research needs in an opinion on November 19, 2012, in German. The English version was agreed on April 17, 2013.

Update of the toxicological assessment of furanocoumarins in foodstuffs (Update of the SKLM statement of 23/24 September 2004)--Opinion of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG).

The DFG Senate Commission on Food Safety (SKLM) has discussed the toxicological assessment of furanocoumarins in foodstuffs and adopted an opinion on 23/24 September 2004 [SKLM, English version: Toxicological assessment of furanocumarins in foodstuffs, 2006; Mol. Nutr. Food Res. 2007, 51, 367-373]. At that time, no analytical data were available on the occurrence and content of furanocoumarins in citrus oils, especially in lime oil and the foodstuffs produced from it. According to the SKLM, the highest levels were likely to be found in products containing lime or bergamot oil. Distilled and cold pressed oils differ in their levels of furanocoumarins; in distilled oils, no furanocoumarins were found. The original estimate of the average daily intake of furanocoumarins in Germany made by the SKLM is based on the assumption that flavoured foods contain cold-pressed citrus oils exclusively (worst case scenario). Recent data, however, indicate that distilled citrus oils are mainly used in flavoured soft drinks. The SKLM has therefore decided to update the assessment of the average intake of furanocoumarins from flavoured food. The following opinion was released in German on 25 January 2010, the English version was agreed on 27/28 September 2010.

N-Nitroso Compounds in Foods

N-nitroso compounds (NOC) are a class of potent environmental carcinogens and some NOC have been classified as “probably” or “possibly” carcinogenic to humans. NOC are chemically rather simple compounds, encompassing N-nitrosamines and N-nitrosamides, and compounds carrying further functional groups, such as certain N-nitrosated amino acids. Chemical and biological properties are primarily governed by the respective NOC structure. NOC are generated by reaction of precursor molecules with nitrosating agents. The basic reaction mechanisms and formation pathways, including in-vivo formation, are being delineated. In addition, the occurrence of NOCs in major food classes is reviewed in connection with estimates of resulting human exposure. The toxicological properties and the toxicokinetics are summarized within the frame of current concepts and approaches for risk assessment.