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.

Modulation of 3',5'-cyclic AMP homeostasis in human platelets by coffee and individual coffee constituents.

3',5'-Cyclic AMP (cAMP) is one of the most important second messengers in mammalian cells, mediating a multitude of diverse cellular signalling responses. Its homeostasis is primarily regulated by adenylate cyclases and phosphodiesterases (PDE), the activities of which are partially dependent on the downstream events of adenosine receptor signalling. The present study was conducted to determine whether coffee constituents other than caffeine can influence the homeostasis of intracellular cAMP in vitro and in vivo by evaluating the effects of selected constituents present in coffee, coffee brews and coffee extracts on platelet PDE activity. In addition, to evaluate the potential effects of these constituents on platelet cAMP concentrations and PDE activity in humans, a 7-week pilot intervention study with eight subjects was conducted. The subjects consumed a regular commercial coffee and a low-caffeine coffee at a rate of 750 ml/d for 2 weeks each. The in vivo results revealed a highly significant inhibition of PDE activity (P< 0·001) after coffee intervention that was not directly dependent on the caffeine content of coffee. Although our in vitro and in vivo findings suggest that caffeine plays some role in the modulation of platelet cAMP status, other natural and roasting-associated compounds such as pyrazines and other currently unidentified species also appear to contribute significantly. In conclusion, moderate consumption of coffee can modulate platelet PDE activity and cAMP concentrations in humans, which may contribute to the putative beneficial health effects of coffee. Further detailed mechanistic investigations will be required to substantiate these beneficial effects and to elucidate the underlying mechanisms.

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,ß-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,ß-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.

Toxicokinetics of acrylamide in primary rat hepatocytes: coupling to glutathione is faster than conversion to glycidamide.

Acrylamide (AA), classified as class 2A carcinogen (probably carcinogenic to humans) by the International Agency for Research on Cancer (IARC), is formed during heating of food from reducing carbohydrates and asparagine by Maillard reaction chemistry. After dietary uptake, AA is in part metabolically converted into the proximate genotoxic phase I metabolite glycidamide (GA). GA reacts with nucleophilic base positions in DNA, primarily forming N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) adducts. In a competing phase II biotransformation pathway AA, as well as its phase I metabolite GA, is coupled to glutathione (GSH). The GSH coupling products are further biotransformed and excreted via urine as mercapturic acids (MA), N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA). In the present study, hepatic biotransformation pathways and DNA adduct formation were studied in primary rat hepatocytes, incubated with AA (0.2-2,000 µM) for up to 24 h. Contents of AA-GSH, GA, AAMA, and GAMA were measured in the cell culture medium after solid phase extraction (SPE). N7-GA-Gua adducts in DNA of hepatocytes were determined by HPLC-ESI-MS/MS after lysis of the cells and neutral thermal hydrolysis. Formation of AA-GSH was linear with AA concentration and incubation time and became detectable already at 0.2 µM (4 h). In contrast to AA, GA was not detected before 16 h incubation at 10-fold higher AA concentration (2 µM). In summary, the rate of AA-GSH formation was found to be about 1.5-3 times higher than that of GA formation. N7-GA-Gua adducts were found only at the highest AA concentration tested (2,000 µM).

Opinion on the use of plasma processes for treatment of foods.

The working group "Food technology and safety" of the DFG Senate Commission on Food Safety (SKLM) advises on new technologies concerning food processing. Treatment with plasma is a newly developed process, which is currently used only on a pilot scale in Europe. The novel plasma treatment technology is experimentally applied to consumer goods. There are also potential applications in the food sector, e.g. to inactivate microorganisms on food surfaces. There is still insufficient information on concomitant physical and chemical processes and changes induced in the food. On May 25th 2012, the SKLM issued a first statement on plasma treatment of foods in German. The English version was agreed on December 14th 2012.

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.

The role of the concept of "history of safe use" in the safety assessment of novel foods and novel food ingredients. Opinion of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG).

The Council of the European Union has proposed a revision on the EU regulation on novel foods and novel food ingredients concerning safety assessment of traditional foods from non-EU countries and their introduction onto the EU market. The proposal stipulates that such foods may be placed on the EU market if their history of safe use in the country of origin is appropriately documented. The present statement of the SKLM gives an overview on current discussions on practical implementation of the "history of safe use" concept as well as examples of its application. The SKLM, in principle, agrees with these concepts, underscores, however, in connection with convincing evidence for a "history of safe use" the need for a range of additional information to achieve a comprehensive risk assessment. In the opinion of the SKLM such information must comprise compositional data as well as experience on adverse effects. A list of questions considered essential is presented. The following opinion was adopted on December 23rd 2010.

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.

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.

Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity.

In the present study, we investigated the effect of anthocyanidins on human topoisomerases I and II and its relevance for DNA integrity within human cells. Anthocyanidins bearing vicinal hydroxy groups at the B-ring (delphinidin, DEL; cyanidin, CY) were found to potently inhibit the catalytic activity of human topoisomerases I and II, without discriminating between the IIalpha and the IIbeta isoforms. However, in contrast to topoisomerase poisons, DEL and CY did not stabilize the covalent DNA-topoisomerase intermediates (cleavable complex) of topoisomerase I or II. Using recombinant topoisomerase I, the presence of CY or DEL (> or = 1 microM) effectively prohibited the stabilization of the cleavable complex by the topoisomerase I poison camptothecin. We furthermore investigated whether the potential protective effect vs topoisomerase I poisons is reflected also on the cellular level, affecting the DNA damaging properties of camptothecin. Indeed, in HT29 cells, low micromolar concentrations of DEL (1-10 microM) significantly diminished the DNA strand breaking effect of camptothecin (100 microM). However, at concentrations > or = 50 microM, all anthocyanidins tested (delphinidin, cyanidin, malvidin, pelargonidin, and paeonidin), including those not interfering with topoisomerases, were found to induce DNA strand breaks in the comet assay. All of these analogues were able to compete with ethidium bromide for the intercalation into calf thymus DNA and to replace the minor groove binder Hoechst 33258. These data indicate substantial affinity to double-stranded DNA, which might contribute at least to the DNA strand breaking effect of anthocyanidins at higher concentrations (> or = 50 microM).

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-TDP1(H263A) 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.

Maillard reaction products modulating the growth of human tumor cells in vitro.

We investigated the effect of a series of Maillard reaction products formed from carbohydrates under household heating conditions on the growth of human tumor cells in vitro. 4-Hydroxy-5-methyl-3-(2H)-furanone (1) was found to potently enhance the proliferation of human tumor cells. In contrast, the Maillard-type chromophores 2-(2-furyl)methylidene-4-hydroxy-5-methyl-2H-furan-3-one (2), 4-(2-furyl)-7-[(2-furyl)methylidene]-2-hydroxy-2H,7H,8aH-pyrano[2,3-b]- pyran-3-one (6), and 3-hydroxy-4[(E)-(2-furyl)methylidene]methyl-3-cyclopentene-1,2 dione (13) inhibited the growth of human tumor cells in vitro in the low micromolar range. GXF251L cells (gastric carcinoma), synchronized by serum deprivation, were retained in the G1-phase of the cell cycle after treatment with 2, 6, or 13 for 24 h. Concomitantly, a distinct sub-G1 peak was observed, indicative for apoptosis induction. DNA fragmentation was further investigated by ELISA using antibodies raised against histones and DNA. 2 induced a significant increase of fragmented DNA at concentrations > or = 30 microM. After treatment with compound 6, DNA fragmentation was observed at a higher concentration range (> or = 50 microM), whereas incubation with 13 resulted in a marked DNA fragmentation already at 20 microM. On the protein level, the activation of caspase 3, as an early marker for apoptosis induction, was determined. The results were almost identical to those obtained in the DNA fragmentation ELISA. In summary, Maillard reaction products potently modulating the growth of human tumor cells were identified. The Maillard-type chromophores 2, 6, and 13 were found to interfere with the proliferation of gastric carcinoma cells, causing cell cycle arrest and apoptosis induction.

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.