Metabolic conjugation reduces in vitro toxicity of the flavonoid nevadensin.

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.

Topoisomerase poisoning by the flavonoid nevadensin triggers DNA damage and apoptosis in human colon carcinoma HT29 cells.

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.

α-Asarone, ß-asarone, and γ-asarone: Current status of toxicological evaluation.

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 ß-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 ß-asarone and preparations of Acorus calamus. In contrast, α-asarone and γ-asarone were not directly addressed due to a limited data situation.

DNA double strand break repair as cellular response to genotoxic asarone isomers considering phase I metabolism.

The phenylpropenes α-asarone and ß-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.

In vitro combinatory cytotoxicity of hepatocarcinogenic asarone isomers and flavonoids.

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.

Three challenges to the complementarity of the logic and the pragmatics of science.

The bipartite metatheory thesis attributes to Rudolf Carnap, Philipp Frank and Otto Neurath a conception of the nature of post-metaphysical philosophy of science that sees the purely formal-logical analyses of the logic of science as complemented by empirical inquiries into the psychology, sociology and history of science. Three challenges to this thesis are considered in this paper: that Carnap did not share this conception of the nature of philosophy of science even on a programmatic level, that Carnap's detailed analysis of the language of science is incompatible with one developed by Neurath for the pursuit of empirical studies of science, and, finally, that Neurath himself was confused about the programme of which the bipartite metatheory thesis makes him a representative. I argue that all three challenges can be met and refuted.