Search

Mycotoxins are secondary fungal metabolites which exhibit toxic effects in low concentrations. Several mycotoxins are described as carcinogenic or immunosuppressive, but their underlying modes of action especially on molecular level have not yet been entirely elucidated. Metabolic profiling as part of the omics methods is a powerful tool to study the toxicity and the mode of action of xenobiotics. The use of hydrophilic interaction chromatography in combination with targeted mass spectrometric detection enables the selective and sensitive analysis of more than 100 polar and ionic metabolites and allows the evaluation of metabolic alterations caused by xenobiotics such as mycotoxins. For metabolic profiling, the hepato-cellular carcinoma cell line HepG2 was treated with sub-cytotoxic concentrations of 20 mycotoxins. Moniliformin and citrinin significantly affected target elements of the citric acid cycle, but also influenced glycolytic pathways and energy metabolism. Penitrem A, zearalenone, and T2 toxin mainly interfered with the urea cycle and the amino acid homeostasis. The formation of reactive oxygen species seemed to be influenced by T2 toxin and gliotoxin. Glycolysis was altered by ochratoxin A and DNA synthesis was affected by several mycotoxins. The observed effects were not limited to these metabolic reactions as the metabolic pathways are closely interrelated. In general, metabolic profiling proved to be a highly sensitive tool for hazard identification in comparison to single-target cytotoxicity assays as metabolic alterations were already observed at sub-toxic concentrations. Metabolic profiling could therefore be a powerful tool for the overall evaluation of the toxic properties of xenobiotics.

A family of Pt(II) complexes bearing monoanionic C^N^N ligands as luminophoric units as well as a set of monodentate ligands derived from allenylidene and carbene species were synthesized and characterized in terms of structure and photophysical properties. In addition, we present the extraordinary molecular structure of a phosphorescent complex carrying an allenylidene ligand. Depending on the co-ligand, an effect can be observed in the photoluminescence lifetimes and quantum yields as well as in the radiative and radiation less deactivation rate constants. Their correlation with the substitution pattern was analyzed by comparing the photoluminescence in fluid solution at room temperature and in frozen glassy matrices at 77 K. Moreover, in order to gain a deeper understanding of the electronic states responsible for the optical properties, density functional theory calculations were performed. Finally, the cytotoxicity of the complexes was evaluated

The specific energy/energy density of state-of-the-art (SOTA) Li ion batteries can be increased by raising the upper charge voltage. However, instability of SOTA cathodes, i.e. LiNiyCoxMnyO2 (x + y + z = 1; NCM) triggers electrode crosstalk via enhanced transition metal (TM) dissolution, and contributes to severe capacity fade, in worst case, to a sudden death ("roll-over failure"). Lithium difluorophosphate (LiDFP) as electrolyte additive is able to boost high voltage performance via scavenging of dissolved TMs. However, LiDFP is chemically unstable and rapidly decomposes to toxic (oligo-) organofluorophosphates (OFPs) at elevated temperature; a process which can be precisely analyzed by means of high-performance liquid chromatography - high resolution mass spectroscopy. The toxicity of LiDFP can be proven via the well-known acetylcholinesterase inhibition test. Interestingly, while fluoroethylene carbonate (FEC) is inappropriate for high voltage applications as single electrolyte additive due to rollover failure, it is able to suppress formation of toxic OFPs. Based on this, a synergistic LiDFP/FEC dual-additive approach is suggested in this work, showing characteristic benefits of both individual additives, i.e. good capacity retention at high voltage in the presence of LiDFP and decreased OFP formation/toxicity induced by FEC.

In recent years, it has been shown that food contact materials can be a potential source of microplastics (MP). Recently, it was reported that more than 16 million polypropylene (PP) particles L-1 may be released from infant feeding bottles (IFBs) made of PP. In the present study seven different IFBs were investigated by the same method used in the aforementioned publication. In our tests, however, only one IFB showed a level of MP above the limit of detection. More importantly, the MP detected were not of the same material as the bottle and are more likely the result of contamination. In addition, there was a notable difference in released MP particles when the water simulant was filtered for µ-Raman spectroscopy at hot temperature (70°C) instead of filtering it after cooling down to room temperature. Thermal desorption gas chromatography mass spectrometry showed that these differences may be the result of migration and precipitation of additives such as fatty acid esters, often used as release agents in bottle production. These observations, that migrating additives could result in false positive errors for MP, indicate the need for critical consideration when polymers have been subjected to heat.

Claviceps purpurea is an ergot fungus known for its neurotropic alkaloids, which have been identified as the main cause of ergotism, a livestock and human disease triggered by ergot consumption. Tetrahydroxanthone dimers, the so-called ergopigments, presumably also contribute to this toxic effect. Overexpression of the cluster-specific transcription factor responsible for the formation of these pigments in C. purpurea led to the isolation of three new metabolites (8-10). The new pigments were characterized utilizing HRMS, NMR techniques, and CD spectroscopy and shown to be xanthone dimers. Secalonic acid A and its 2,4'- and 4,4'-linked isomers were also isolated, and their absolute configuration was investigated. The contribution of secalonic acid A, its isomers, and new metabolites to the toxicity of C. purpurea was investigated in HepG2 and CCF-STTG1 cells. Along with cytotoxic properties, secalonic acid A was found to inhibit topoisomerase I and II activity.

Aronia melanocarpa (MICHX.) ELLIOTT, which belongs to the Rosaceae family, has increasingly come into focus of research due to the high content of polyphenols. In addition to antioxidative properties, further health-promoting effects of these polyphenols are still of interest. Especially, the proanthocyanidins offer thereby huge opportunities due to their high structural heterogeneity. Therefore, the present study focuses on the topoisomerase inhibiting effects of oligomeric proanthocyanidins (PACs), which are potentially depended on their degree of polymerization. The investigated PACs isolated from Aronia berries were characterized by chromatographic techniques and liquid chromatography-high-resolution mass spectrometry. Four PAC enriched fractions were obtained from Aronia pomace containing 47 PACs with a degree of polymerization from three to six. Due to the low yield of hexamers, the potential inhibiting effects against human topoisomerase were investigated for the trimer to pentamer fractions. The relaxation and decatenation assays were performed to examine the inhibiting effect on topoisomerases under cell-free conditions. Moreover, rapid isolation of topoisomerase cleavage complexes in human colon carcinoma HT29 cells was performed to evaluate the effect on topoisomerases in a cell-based system. The fractions demonstrated inhibitory potential on topoisomerases I and II. In sum, an increasing effect strength depending on the degree of polymerization was shown.

Dietary polyphenols like epigallocatechin-3-gallate (EGCG)—which represents the most abundant flavan-3-ol in green tea—are subject of several studies regarding their bioactivity and health-related properties. On many occasions, cell culture or in vitro experiments form the basis of published data. Although the stability of these compounds is observed to be low, many reported effects are directly related to the parent compounds whereas the impact of EGCG degradation and autoxidation products is not yet understood and merely studied. EGCG autoxidation products like its dimers theasinensin A and D, “P2” and oolongtheanin are yet to be characterized in the same extent as their parental polyphenol. However, to investigate the bioactivity of autoxidation products—which would minimize the discrepancy between in vitro and in vivo data—isolation and structure elucidation techniques are urgently needed. In this study, a new protocol to acquire the dimers theasinensin A and D as well as oolongtheanin is depicted, including a variety of spectroscopic and quadrupole time-of-flight high-resolution mass spectrometric (qTOF-HRMS) data to characterize and assign these isolates. Through nuclear magnetic resonance (NMR) spectroscopy, polarimetry, and especially circular dichroism (CD) spectroscopy after enzymatic hydrolysis the complementary atropisomeric stereochemistry of the isolated theasinensins is illuminated and elucidated. Lastly, a direct comparison between the isolated EGCG autoxidation products and the monomer itself is carried out regarding their antioxidant properties featuring Trolox equivalent antioxidant capacity (TEAC) values. These findings help to characterize these products regarding their cellular effects and—which is of special interest in the flavonoid group—their redox properties.

α-Asarone and β-asarone are reported as bioactive constituents of Acorus calamus. Phase I metabolism of asarone isomers results in a multiple spectrum of genotoxic metabolites. Thus, the question arises whether structural analogues of the known phase I metabolites also naturally occur in A. calamus-based food products. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for three product classes, herbal infusions, alcoholic beverages, and food supplements. High asarone contents were detected in herbal infusions (total mean 9.13 mg/kg, n = 8) and food supplements (total mean 14.52 mg/kg, n = 6); hence, these food products can highly contribute to human exposure to genotoxic asarone derivatives. Also, the occurrence of asarone oxidation products found in food and food supplements has to be taken under consideration because data on toxicity is limited so far.

Asarone isomers are naturally occurring in Acorus calamus Linné, Guatteria gaumeri Greenman, and Aniba hostmanniana Nees. These secondary plant metabolites belong to the class of phenylpropenes (phenylpropanoids or alkenylbenzenes). They are further chemically classified into the propenylic trans- and cis-isomers α-asarone and β-asarone and the allylic γ-asarone. Flavoring, as well as potentially pharmacologically useful properties, enables the application of asarone isomers in fragrances, food, and traditional phytomedicine not only since their isolation in the 1950s. However, efficacy and safety in humans are still not known. Preclinical evidence has not been systematically studied, and several pharmacological effects have been reported for extracts of Acorus calamus and propenylic asarone isomers. Toxicological data are rare and not critically evaluated altogether in the 21st century yet. Therefore, within this review, available toxicological data of asarone isomers were assessed in detail. This assessment revealed that cardiotoxicity, hepatotoxicity, reproductive toxicity, and mutagenicity as well as carcinogenicity were described for propenylic asarone isomers with varying levels of reliability. The toxicodynamic profile of γ-asarone is unknown except for mutagenicity. Based on the estimated daily exposure and reported adverse effects, officials restricted or published recommendations for the use of β-asarone and preparations of Acorus calamus. In contrast, α-asarone and γ-asarone were not directly addressed due to a limited data situation.

The present study focuses on the association between metabolic capacity and toxicity of the natural occurring flavonoid nevadensin in vitro. Human colon (HT29), liver (HepG2) and bone marrow (KG1) carcinoma cells were used and strong cell line dependent differences in toxic effect strength were found. HepG2 and KG1 cells were more sensitive against nevadensin treatment in comparison to HT29 cells. High resolution mass spectrometry experiments showed that nevadensin is rapidly glucuronidated in HT29 cells, whereas KG1 cells do not metabolize nevadensin, thus glucuronidation was supposed to be a crucial metabolic pathway in vitro. To proof this suggestion, nevadensin glucuronides were isolated from pig liver microsomes und structurally elucidated via NMR spectroscopy. In HepG2 cells a cellular enrichment of nevadensin itself as well as nevadensin-7-O-glucuronide was determined by tandem mass spectrometry. A proteomic screening of uridine 5'-diphospho (UDP)-glucuronosyltransferase (UGT) in HT29 and HepG2 cells provided first hints that the isoforms UGT1A6 and UGT1A1 are responsible for nevadensin glucuronidation. Additionally, nevadensin was found to be a potent SULT inhibitor in HepG2 cells. In sum, the present study clearly illustrates the importance of obtaining detailed information about metabolic competence of cell lines which should be considered in the evaluation of toxic endpoints.

Nevadensin, an abundant polyphenol of basil, is reported to reduce alkenylbenzene DNA adduct formation. Furthermore, it has a wide spectrum of further pharmacological properties. The presented study focuses the impact of nevadensin on topoisomerases (TOPO) in vitro. Considering the DNA-intercalating properties of flavonoids, first, minor groove binding properties (IC50 = 31.63 µM), as well as DNA intercalation (IC50 = 296.91 µM) of nevadensin, was found. To determine potential in vitro effects on TOPO I and TOPO IIα, the relaxation and decatenation assay was performed in a concentration range of 1–500 µM nevadensin. A partial inhibition was detected for TOPO I at concentrations ≥ 100 µM, whereas TOPO IIα activity is only inhibited at concentrations ≥ 250 µM. To clarify the mode of action, the isolating in vivo complex of enzyme assay was carried out using human colon carcinoma HT29 cells. After 1 h of incubation, the amount of TOPO I linked to DNA was significantly increased by nevadensin (500 µM), why nevadensin was characterized as TOPO I poison. However, no effects on TOPO IIα were detected in the cellular test system. As a subsequent cellular response to TOPO I poisoning, a highly significant increase of DNA damage after 2 h and a decrease of cell viability after 48 h at the same concentration range were found. Furthermore, after 24 h of incubation a G2/M arrest was observed at concentrations ≥ 100 µM by flow cytometry. The analysis of cell death revealed that nevadensin induces the intrinsic apoptotic pathway via activation of caspase-9 and caspase-3. The results suggest that cell cycle disruption and apoptotic events play key roles in the cellular response to TOPO I poisoning caused by nevadensin in HT29 cells. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-021-03162-5.

Influencing the redox balance of pancreatic beta cells could be a promising strategy for the treatment of diabetes. Nuclear factor erythroid 2p45-related factor 2 (Nrf2) is present in beta cells and regulates numerous genes involved in antioxidant defense. As reactive oxygen species (ROS) are important for beta cell signaling but induce oxidative stress when present in excess, this study elucidates the influence of Nrf2-activating compounds on different kinds of ROS and correlates changes in redox balance to effects on mitochondrial function, insulin release, and cell viability. Acute glucose stimulation (15 mmol/L) of murine islet cells of C57Bl/6N mice affects ROS and redox status of the cells differently. Those ROS monitored by dihydroethidium, which detects superoxide radical anions, decrease. By contrast, oxidant status, monitored by dichlorodihydrofluorescein, as well as intracellular H2O2, increases. Glucolipotoxicity completely prevents these fast, glucose-mediated alterations and inhibits glucose-induced NAD(P)H production, mitochondrial hyperpolarization, and ATP synthesis. Oltipraz (10 μmol/L) or dimethyl fumarate (DMF, 50 μmol/L) leads to nuclear accumulation of Nrf2, restores mitochondrial activity and glucose-dependent ROS turnover, and antagonizes glucolipotoxicity-induced inhibition of insulin release and apoptosis. Importantly, these beneficial effects only occur when beta cells are challenged and damaged by high lipid and carbohydrate supply. At physiological conditions, insulin release is markedly reduced in response to both Nrf2 activators. This is not associated with severe impairment of glucose-induced mitochondrial hyperpolarization or a rise in apoptosis but coincides with altered ROS handling. In conclusion, Nrf2 activators protect beta cells against glucolipotoxicity by preserving mitochondrial function and redox balance. As our data show that this maintains glucose-stimulated insulin secretion, targeting Nrf2 might be suited to ameliorate progression of type 2 diabetes mellitus. By contrast, nonstressed beta cells do not benefit from Nrf2 activation, thus underlining the importance of physiological shifts in ROS homeostasis for the regulation of beta cell function.

Acorus calamus is a swamp herb, which is widely spread in northern hemisphere. It is used in infusions and in bitters but also in food supplements and in traditional herbal medicine. However, the main A. calamus ingredients, propenylic 2,4,5-trimethoxyphenylpropene isomers, termed alpha- (trans) and beta- (cis) asarone, are known carcinogens in rodents. Genotoxic and mutagenic properties are proposed. The presented in vitro cytotoxicity study focused on time-dependent and combinatory exposure scenarios. All experiments performed in HepG2 cells show moderate (in middle micromolar range) cytotoxicity with a time-dependent increase in effectiveness. The combination of the two asarone isomers in short time experiments (1 h) did not show any effect, whereas asarone isomer interaction changes from synergistic to antagonistic with an extended duration of exposure up to 72 h. The antagonism occurred predominantly in the naturally occurring trans/cis-asarone ratio of approximately 1:10. Combinatory cytotoxicity of asarones and selected, dietary relevant flavonoids in constant ratios was mainly attributed to flavonoid toxicity.

α-Asarone and β-asarone are reported as bioactive constituents of

In the course of gaining new insights into the secondary metabolite profile of various Stachybotrys strains, in particular concerning triprenyl phenol-like compounds, so far, unknown metabolites with analogous structural features were discovered. Three novel meroterpenoids containing a chromene ring moiety, namely stachybotrychromenes A–C, were isolated from solid culture of the filamentous fungus Stachybotrys chartarum DSMZ 12880 (chemotype S). Their structures were elucidated by means of comprehensive spectroscopic analysis (1D and 2D NMR, ESI-HRMS, and CD) as well as by comparison with spectroscopic data of structural analogues described in literature. Stachybotrychromenes A and B exhibited moderate cytotoxic effects on HepG2 cells after 24 h with corresponding IC50 values of 73.7 and 28.2 μM, respectively. Stachybotrychromene C showed no significant cytotoxic activity up to 100 μM. Moreover, it is noteworthy that stachybotrychromenes A–C are produced not only by S. chartarum chemotype S but also S. chartarum chemotype A and Stachybotrys chlorohalonata. Electronic supplementary material The online version of this article (10.1007/s12550-018-0312-7) contains supplementary material, which is available to authorized users.

Previously the food carcinogen methyleugenol was found to be cytotoxic and genotoxic in multiple cell lines and in primary hepatocytes. In this study, the question addressed was whether methyleugenol and the selected oxidative metabolites, 1′-hydroxymethyleugenol, methyleugenol-2′,3′-epoxide and 3′-oxomethylisoeugenol trigger a DNA damage response in the human colon carcinoma HT29 cell line. Most notably investigations by flow cytometry revealed that the metabolites induce an accumulation of HT29 cells in the G 2 phase of the cell cycle. DNA damage response is characterised by a time-delayed phosphorylation of ATM (ataxia-telangiectasia, mutated)/ATR (ATM- and Rad3-related) kinases and checkpoint kinase 1 after 2 h of incubation, and the tumour suppressor protein p53 only after 24 h of incubation. The test compounds induced apoptotic cell death indicated by cleavage of caspase 3 and poly-(ADP-ribose)-polymerase after a prolonged incubation time up to 72 h. In addition, activation of ATM/ATR-signalling cascade might contribute to apoptosis induction to a certain extent. However, clarification of this relationship awaits experimental confirmation.

The present study focused on genotoxic properties of the carcinogenic phenylpropanoids α-asarone and β-asarone, which are found in several herbs and spices, such as Acorus calamus or Acorus gramineus. Cytotoxic and genotoxic effects were determined in human liver carinoma HepG2 cells, in hamster lung fibroblast V79 cells and in human cytochrome P450 1A2 and human sulfotransferase 1C2 transfected V79 cells (tV79). The Alamar blue assay was used to measure cytotoxicity of both isomers prior to the identification of DNA damaging properties by single cell gel electrophoresis (comet assay). Furthermore, the phosphorylation status of the histone H2AX, as a response of DNA double strand breaks, was investigated in HepG2 cells by Western blot analysis and visualized by immunofluorescence microscopy. After 24 h of incubation a significant reduction of cell viability was found. Moreover, both asarone isomers induced DNA strand breaks in V79 cells after 1 h of incubation. In tV79 cells even more pronounced DNA damaging properties were exhibited, whereas in HepG2 cells the compounds were found to be less effective. Furthermore, in tV79 cells a significant increase of formamidopyrimidine-DNA-glycosylase-sensitive sites was observed. DNA strand breaks, induced by aA, were to some extent characterized as DNA double strand breaks. In summary, asarone-induced cytotoxicity and genotoxicity is strongly influenced by the cellular metabolic enzyme status and therefore, a contribution of their respective metabolites to in vitro toxicity can be suggested.

The phenylpropenes α-asarone and β-asarone are widely spread in the marsh plant Acorus calamus. Both isomers are classified as carcinogenic in rodents. However, the respective genotoxic mechanisms are not elucidated so far. The present study gives deeper insights into the genotoxic effects of asarone isomers as well as their known oxidative phase I metabolites, (E)-3′-oxoasarone and asarone epoxide. We show that asarone metabolites highly increase DNA strand breaks after 1 h of incubation, markedly metabolic activation contributes to their carcinogenic mode of action. All test compounds act as aneugens and potently enhance the amounts of micronuclei in binuclear cells. However, a prolonged incubation time of 24 h results in a decrease of DNA damage. This work suggests that asarone metabolites also induce DNA double strand breaks , why we put a strong focus on homologous recombination and non-homologous end joining. The obtained results herein indicate that asarone epoxide-induced DNA strand breaks are repaired via a homologous repair pathway.

This chapter presents and discusses a number of chemicals formed during food processing or storage, which have been in the focus of safety concerns because of their possible adverse health effects. The chemicals discussed here are ethyl carbamate, biogenic amines, trans–fatty acids, and fatty acid peroxides. The processes leading to these compounds include fermentation, acid hydrolysis, oil refining, hydrogenation, and oxidation. They are composed of legal, state-of-the-art, as well as illegal practices such as those having led to the Spanish toxic oil syndrome. This chapter reflects the broad spectrum of pathways that may eventually lead to process-generated contaminants.

Evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. The inhibition of histone deacetylase (HDAC) activity and the disruption of the HDAC complex have been recognized as a potent strategy for cancer therapy and chemoprevention. In the present study, we investigated whether selected plant constituents affect HDAC activity or HDAC1 protein status in the human colon carcinoma cell line HT29. The polyphenols (−)-epigallocatechin-3-gallate (EGCG) and genistein (GEN) as well as two oxidative methyleugenol (ME) metabolites were shown to inhibit HDAC activity in intact HT29 cells. Concomitantly, a significant decrease of the HDAC1 protein level was observed after incubation with EGCG and GEN, whereas the investigated ME metabolites did not affect HDAC1 protein status. In conclusion, dietary compounds were found to possess promising HDAC-inhibitory properties, contributing to epigenetic alterations in colon tumor cells, which should be taken into account in further risk/benefit assessments of polyphenols and alkenylbenzenes.

The use of nanostructured silica (SiO2) particles is no longer restricted to biomedical and (bio-) technological fields but rather finding applications in products of the food industry. Thus, our studies on the toxicological relevance of SiO2 nanoparticles focused on cytotoxic effects, the modulation of the cellular redox status and the impact on DNA integrity in human colon carcinoma cells (HT29). The results indicate that these SiO2 nanoparticles stimulate the proliferation of HT29 cells, depending on the incubation time and the particle size. The cytotoxicity of the investigated SiO2 nanoparticles was found to depend on the concentration, size and on the FCS content of the culture medium. Furthermore, SiO2 seem to interfere with glutathione biosynthesis. The results indicate further that effects of SiO2 NPs are not mediated by oxidative stress but by interference with the MAPK/ERK1/2 as well as the Nrf2/ARE signalling pathways. Additionally, investigations regarding DNA integrity revealed no substantial (oxidative) DNA damage.

Irinotecan is an anticancer agent that stabilizes topoisomerase I/DNA complexes. So far, no test system has been reported for directly determining irinotecan-induced stabilization of topoisomerase I/DNA complexes in organs in vivo. We adapted an 'in vivo complexes of enzyme to DNA' (ICE) bioassay to assess irinotecan activity in the stomach, duodenum, colon and liver of male Wistar rats after a single treatment with irinotecan (100 mg/kg body weight, intraperitoneally). This was compared to the control group receiving 0.9% sodium chloride intraperitoneally. In addition, the DNA strand breaking properties of irinotecan were measured in mucosal cells from the distal colon by single-cell gel electrophoresis (comet assay) to investigate the association of topoisomerase poisoning and DNA damage in vivo. A single dose of irinotecan significantly increased amounts of topoisomerase I covalently bound to DNA in stomach, duodenum, colon and liver. Concomitantly, the irinotecan-treated group showed significantly higher amounts of DNA strand breaks in colon mucosa cells compared to the control group. The ICE bioassay and the comet assay represent two test systems for investigating the impact of topoisomerase I poisons on DNA integrity in colon tissues of Wistar rats.

Supercritical fluid reactive deposition was used for the deposition of highly dispersed platinum nanoparticles with controllable metal content and particle size distribution on beta-cyclodextrin. The average particle size and size distribution were steered by the precursor reduction conditions, resulting in particle preparations20,100, and100 nm as characterized by transmission electron microscopy and scanning electron microscopy (SEM). These particle preparations of different size distributions were used to address the question as to whether metallic platinum particles are able to invade cells of the gastrointestinal tract as exemplified for the human colon carcinoma cell line HT29 and thus affect the cellular redox status and DNA integrity. Combined focused ion beam and SEM demonstrated that platinum nanoparticles were taken up into HT29 cells in their particulate form. The chemical composition of the particles within the cells was confirmed by energy-dispersive X-ray spectroscopy. The potential influence of platinum nanoparticles on cellular redoxsystems was determined in the DCF assay, on the translocation of Nrf-2 and by monitoring the intracellular glutathione (GSH) levels. The impact on DNA integrity was investigated by single cell gel electrophoresis (comet assay) including the formation of sites sensitive to formamidopyrimidine-DNA-glycosylase. Platinum nanoparticles were found to decrease the cellular GSH level and to impair DNA integrity with a maximal effect at 1 ng/cm(2). These effects were correlated with the particle size in an inverse manner and were enhanced with increasing incubation time but appeared not to be based on the formation of reactive oxygen species.

The anthocyanidin delphinidin (DEL) has recently been shown to inhibit human topoisomerase I and II, without stabilizing the covalent DNA/topoisomerase intermediate [Habermeyer, M., Fritz, J., Barthelmes, H. U., Christensen, M. O., Larsen, M. K., Boege, F., and Marko, D. (2005) Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity. Chem. Res. Toxicol. 18, 1395-404]. In the present study, we demonstrated that DEL affects the catalytic activity of topoisomerase IIalpha in a redox-independent manner. Furthermore, this potent inhibitory effect is not limited to a cell-free system, but is also of relevance within intact cells. DEL at micromolar concentrations was found to significantly decrease the level of topoisomerase IIalpha/DNA intermediates stabilized by the topoisomerase II poison doxorubicin in the human colon carcinoma cell line (HT29). In addition, DEL diminished the DNA-damaging properties of topoisomerase II poisons in HT29 cells without affecting the level of sites sensitive to formamidopyrimidine-DNA-glycosylase. However, the preventive effect on DNA damage exhibited an apparent maximum at a concentration of 10 microM DEL, followed by a recurrence of DNA damage at higher DEL concentrations. Furthermore, the incubation of HT29 cells with 10 microM DEL resulted in a decrease of etoposide (ETO)-induced DNA strand breaks. However, the level of ETO-stabilized covalent topoisomerase/DNA intermediates did not affect DEL, indicating an additional mechanism of action. An impact of DEL on genes involved in the repair of DNA double-strand breaks and the onset of apoptosis has to be considered. In conclusion, the natural food constituent DEL represents, depending on the concentration range, a protective factor against the DNA-damaging effects of topoisomerase II poisons in vitro. Further studies are needed to clarify whether in vivo a high DEL intake might compromise the therapeutic outcome of these anticancer agents.

In the present study, delphinidin was found to suppress the phosphorylation of the epidermal growth factor receptor (EGFR) within human tumour cells (human colon carcinoma cell line (HT29), human vulva carcinoma cell line (A431)), albeit less effective than the flavonol quercetin. The higher potency of quercetin was also observed downstream on the level of the mitogen-activated protein kinase (MAPK) cascade. In addition, delphinidin, quercetin and (-)-epigallocatechin-3-gallate (EGCG) were found to suppress the phosphorylation of the ErbB2 receptor, with delphinidin exhibiting the strongest inhibitory properties. Their potency to suppress the ErbB2 receptor phosphorylation can be summarised as delphinidin > EGCG > quercetin. The effectiveness of delphinidin against the EGFR and the ErbB2 receptor was comparable, indicating a broader spectrum of activity against receptor tyrosine kinases. At low micromolar concentrations delphinidin showed some preference towards the ErbB2 receptor. In summary, quercetin and delphinidin appear to differ in their activity profile towards the ErbB receptor family members. Whereas quercetin was most effective against the EGFR, delphinidin exhibited some preference towards the ErbB2 receptor.

The ellagitannins castalagin and vescalagin, and the C-glycosides grandinin and roburin E as well as ellagic acid were found to potently inhibit the growth of human colon carcinoma cells (HT29) in vitro. In a cell-free system these compounds were identified as potent inhibitors of the protein tyrosine kinase activity of the epidermal growth factor receptor (EGFR) with IC 50 values in the low nanomolar range. To address the question of whether the interference with the activity of the isolated EGFR also plays a role within intact cells, effects on the phosphorylation status of the EGFR, as a measure for its activity, were determined in HT29 cells. As exemplified for castalagin and grandinin, both the nonglycosylated and the glycosylated ellagitannins effectively suppressed EGFR phosphorylation, but only at concentrationsor =10 microM, thus, in a concentration range where growth inhibition was observed. These results indicate that the suppression of EGFR-mediated signaling might contribute to the growth inhibitory effects of these compounds present in oak-matured wines and spirits such as whiskey. In contrast, despite substantial growth inhibitory properties, ellagic acid did not significantly affect EGFR phosphorylation in HT29 cells up to 100 microM.

Methyleugenol is a substituted alkenylbenzene found in several herbs and spices. It is classified by the European Union's Scientific Committee on Food as a genotoxic carcinogen. We addressed the biological mechanism of the genotoxic properties of methyleugenol and its oxidative metabolites. Methyleugenol and the oxidative metabolites significantly enhanced the DNA damage in human colon carcinoma cells (HT29). Methyleugenol did not affect the protein status of γH2AX, a biomarker of DNA double-strand breaks, whereas its metabolites methyleugenol-2',3'-epoxide and 3'-oxomethylisoeugenol significantly increased the cellular phosphorylated H2AX level. Both of these metabolites also showed a significant induction of micronuclei in HT29 cells. Furthermore, we investigated whether topoisomerase interaction contribute to the observed effect on DNA integrity. Methyleugenol-2',3'-epoxide and 3'-oxomethylisoeugenol inhibited the activity of recombinant topoisomerase I. In HT29 cells, neither methyleugenol nor the metabolites affected the level of topoisomerase protein bound to DNA, excluding a topoisomerase poisoning mode of action. In addition, 3'-oxomethylisoeugenol potently diminished the level of camptothecin-stabilized topoisomerase I/DNA intermediates and camptothecin-induced DNA strand breaks. In conclusion, it could be suggested that 3'-oxomethylisoeugenol may also interact with classical or food-borne topoisomerase I poisons, diminishing their poisoning effectiveness.

Complex polyphenol-rich extracts from apples are known to inhibit the activity of the epidermal growth factor receptor (EGFR) in vitro. The aim of the present study was to identify the bioactive constituents of the apple juice extract which contribute substantially to this potentially chemopreventive effect and to address the question whether the effect is specific to the EGFR or whether other members of the ErbB-receptor family might also be affected. Apple-derived dihydrochalcones and their respective glycosides were found to decrease EGFR activity under cell-free conditions with IC50-values ranging from 0.4 ± 0.1 to 267.0 ± 50.0 μM but showed no activity on human cancer cells. The concentration of quercetin or its glycosides in the extract was too low to contribute substantially to the EGFR-inhibitory properties. In contrast, fractions derived from the apple juice extract comprising ≥86% oligomeric procyanidins (OPCs) suppressed the activity of the EGFR in cell culture with an IC50 ∼ 100 μg mL(-1). In addition, the activity of further members of the ErbB-receptor family was potently inhibited, with ErbB3 receptor activity being most potently decreased (IC50 ∼ 10 μg mL(-1)). From the apple polyphenols identified so far OPCs were found to add the highest contribution to the inhibitory effects towards members of the ErbB-receptor family. Considering the crucial role of the ErbB-receptors in carcinogenesis, these results support the hypothesis that apple-derived OPCs as well as OPC-rich apple preparations might be of interest with respect to chemoprevention.

Prostaglandins are endogenous mediators formed from arachidonic acid by cyclooxygenases and prostaglandin synthases during inflammatory processes. The five-membered ring can be dehydrated, and α,β-unsaturated cyclopentenone PGs (cyPGs) are generated. Recent studies have been focused on their potential pharmacological use against inflammation and cancer. However, little is known so far about possible adverse health effects of cyPGs. We addressed the question whether selected cyPGs at a concentration range of 0.1-10 μM exhibit mutagenic and genotoxic properties in the hamster lung fibroblast V79 cell line and whether these effects are accompanied by a depletion of intracellular glutathione (GSH). The cyPGs 15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2) and prostaglandin A2 (PGA2) significantly induced DNA damage in V79 cells after 1 h of incubation. Furthermore, a more pronounced increase in formamidopyrimidine-DNA glycosylase (FPG) sensitive sites, indicative of oxidative DNA-damage, was observed. The findings on DNA-damaging properties were supported by our results that 15dPGJ(2) acts as an aneugenic agent which induces the amount of kinetochore positive micronuclei associated with an increase of apoptosis. The strong potency of cyPGs to rapidly bind GSH measured in a chemical assay and to significantly reduce the GSH level after only 1 h of incubation may contribute to the observed oxidative DNA strand breaks, whereas directly induced oxidative stress via reactive oxygen species could be excluded. However, after an extended incubation time of 24 h no genotoxicity could be measured, this may contribute to the lack of mutagenicity in the hypoxanthine phosphorybosyltransferase (HPRT) assay. In conclusion, potential in vitro genotoxicity of cyPG and a strong impact on GSH homeostasis have been demonstrated, which may be involved in carcinogenesis mediated by chronic inflammation.

This chapter discusses a number of toxicants in foods generated by non-thermal processes, including ethyl carbamate, 3-monochloropropane-1,2-diol, glycidol, biogenic amines, trans fatty acids and fatty acid peroxides. The occurrence and formation of these toxicants are discussed, as well as the toxicity and toxicokinetics. The risk of exposure to humans and the various subsequent effects are outlined, including epidemiological data, carcinogenicity, genotoxicity and reproductive toxicity. A brief case study of Spanish Toxic Oil Syndrome is also summarized.

In the present study, we addressed the question whether cyanidin-3-glucoside (C3G) or complex C3G-rich blackberry extracts affect human topoisomerases with special emphasis on the contribution of the potential degradation products phloroglucinol aldehyde (PGA) and protocatechuic acid (PCA). In HT29 colon carcinoma cells a C3G-rich blackberry extract suppressed camptothecin- (CPT-) or doxorubicin- (DOX-) induced stabilization of the covalent DNA-topoisomerase intermediate, thus antagonizing the effects of these classical topoisomerase poisons on DNA integrity. As a single compound, C3G (100 μM) decreased the DNA-damaging effects of CPT as well, but did not significantly affect those induced by DOX. At the highest applied concentration (100 μM), cyanidin protected DNA from CPT- and DOX-induced damage. Earlier reports on DNA-damaging properties of cyanidin were found to result most likely from the formation of hydrogen peroxide as an artifact in the cell culture medium when the incubation was performed in the absence of catalase. The suppression of hydrogen peroxide accumulation, achieved by the addition of catalase, demonstrated that cyanidin does not exhibit DNA-damaging properties in HT29 cells (up to 100 μM). The observed effects on topoisomerase interference and DNA protection against CPT or DOX were clearly limited to the parent compound and were not observed for the potential cyanidin degradation products PGA and PCA.

Scope: The effect of two anthocyanin-rich berry extracts (A, bilberry; B, red grape) on topoisomerases was investigated in a cell-free system and in human HT29 colon carcinoma cells. In parallel, their impact on DNA integrity was determined. Methods and results: The berry extracts suppressed the activity of topoisomerase I at concentrations ≥50 μg/mL. The activity of the topoisomerase II isoform was preferentially diminished (≥1 μg/mL). Within HT29 cells, the extracts were found to act as catalytic inhibitors without stabilizing the cleavable complex. Although topoisomerase activity was inhibited, none of the extracts induced DNA strand breaks up to 50 μg/mL. Moreover, pre- and coincubation of HT29 cells with A (≥1 μg/mL) significantly suppressed (p-value ≤0.001) the strand-breaking effects of camptothecin, whereas B was found to be less effective (1 μg/mL; p-value ≤0.05). Both extracts were found to significantly diminish doxorubicin-mediated DNA strand breaks at concentrations ≥1 μg/mL (p-value ≤0.001). Consistent with these results, the extracts suppressed doxorubicin-mediated enhancement of levels of topoisomerase II covalently linked to DNA in HT29 cells. Conclusion: These results raise the possibility that high intake of berry extracts may protect DNA and thus counteract the therapeutic effectiveness of orally applied topoisomerase poisons during chemotherapy.

Delphinidin has been found to possess DNA strand-breaking properties in cell culture. In the present study, we demonstrated that the extent of DNA damage by delphinidin is not affected by the expression of tyrosyl-DNA-phosphodiesterase 1, indicating that the induction of DNA strand breaks is not predominantly topoisomerase-mediated. However, the DNA-damaging properties of delphinidin were decreased by the addition of catalase to the cell culture medium, counteracting delphinidin-mediated hydrogen peroxide formation. Under these conditions, delphinidin showed clearly antioxidative properties in HT29 cells, preventing menadione-induced oxidative DNA damage. In contrast, in the absence of catalase, delphinidin lacked antioxidative properties. In conclusion, delphinidin acted as an effective antioxidant within intact cells if the formation of hydrogen peroxide was prevented. In the absence of catalase, the accumulated hydrogen peroxide appears to play a substantial role for the observed DNA-damaging properties of delphinidin and the apparent lack of antioxidative properties of this anthocyanidin.

Nanoparticles might enter the food chain, e.g. as a contaminant on the surface of food plants. However, there is only little knowledge about their uptake into cells of the gastrointestinal tract. Therefore, it is of general interest to study the correlation between particle properties like size, shape, and crystal structure on the one hand and biological responses on the other (see, e.g., [1]).