Toxins in Botanical Drugs and Plant-derived Food and Feed - from Science to Regulation: A Workshop Review.

In September 2022, the 3rd International Workshop on pyrrolizidine alkaloids (PAs) and related phytotoxins was held on-line, entitled 'Toxins in botanical drugs and plant-derived food and feed - from science to regulation'. The workshop focused on new findings about the occurrence, exposure, toxicity, and risk assessment of PAs. In addition, new scientific results related to the risk assessment of alkenylbenzenes, a distinct class of herbal constituents, were presented. The presence of PAs and alkenylbenzenes in plant-derived food, feed, and herbal medicines has raised health concerns with respect to their acute and chronic toxicity but mainly related to the genotoxic and carcinogenic properties of several congeners. The compounds are natural constituents of a variety of plant families and species widely used in medicinal, food, and feed products. Their individual occurrence, levels, and toxic properties, together with the broad range of congeners present in nature, represent a striking challenge to modern toxicology. This review tries to provide an overview of the current knowledge on these compounds and indicates needs and perspectives for future research.

OCT1-dependent uptake of structurally diverse pyrrolizidine alkaloids in human liver cells is crucial for their genotoxic and cytotoxic effects.

Pyrrolizidine alkaloids (PAs) are important plant hepatotoxins, which occur as contaminants in plant-based foods, feeds and phytomedicines. Numerous studies demonstrated that the genotoxicity and cytotoxicity of PAs depend on their chemical structure, allowing for potency ranking and grouping. Organic cation transporter-1 (OCT1) was previously shown to be involved in the cellular uptake of the cyclic PA diesters monocrotaline, retrorsine and senescionine. However, little is known about the structure-dependent transport of PAs. Therefore, we investigated the impact of OCT1 on the uptake and toxicity of three structurally diverse PAs (heliotrine, lasiocarpine and riddelliine) differing in their degree and type of esterification in metabolically competent human liver cell models and hamster fibroblasts. Human HepG2-CYP3A4 liver cells were exposed to the respective PA in the presence or absence of the OCT1-inhibitors D-THP and quinidine, revealing a strongly attenuated cytotoxicity upon OCT1 inhibition. The same experiments were repeated in V79-CYP3A4 hamster fibroblasts, confirming that OCT1 inhibition prevents the cytotoxic effects of all tested PAs. Interestingly, OCT1 protein levels were much lower in V79-CYP3A4 than in HepG2-CYP3A4 cells, which correlated with their lower susceptibility to PA-induced cytotoxicity. The cytoprotective effect of OCT1 inhibiton was also demonstrated in primary human hepatocytes following PA exposure. Our experiments further showed that the genotoxic effects triggered by the three PAs are blocked by OCT1 inhibition as evidenced by strongly reduced γH2AX and p53 levels. Consistently, inhibition of OCT1-mediated uptake suppressed the activation of the DNA damage response (DDR) as revealed by decreased phosphorylation of checkpoint kinases upon PA treatment. In conclusion, we demonstrated that PAs, independent of their degree of esterification, are substrates for OCT1-mediated uptake into human liver cells. We further provided evidence that OCT1 inhibition prevents PA-triggered genotoxicity, DDR activation and subsequent cytotoxicity. These findings highlight the crucial role of OCT1 together with CYP3A4-dependent metabolic activation for PA toxicity.

Novel Insights into Pyrrolizidine Alkaloid Toxicity and Implications for Risk Assessment: Occurrence, Genotoxicity, Toxicokinetics, Risk Assessment-A Workshop Report.

This paper reports on the major contributions and results of the 2nd International Workshop of Pyrrolizidine Alkaloids held in September 2020 in Kaiserslautern, Germany. Pyrrolizidine alkaloids are among the most relevant plant toxins contaminating food, feed, and medicinal products of plant origin. Hundreds of PA congeners with widespread occurrence are known, and thousands of plants are assumed to contain PAs. Due to certain PAs' pronounced liver toxicity and carcinogenicity, their occurrence in food, feed, and phytomedicines has raised serious human health concerns. This is particularly true for herbal teas, certain food supplements, honey, and certain phytomedicinal drugs. Due to the limited availability of animal data, broader use of in vitro data appears warranted to improve the risk assessment of a large number of relevant, 1,2-unsaturated PAs. This is true, for example, for the derivation of both toxicokinetic and toxicodynamic data. These efforts aim to understand better the modes of action, uptake, metabolism, elimination, toxicity, and genotoxicity of PAs to enable a detailed dose-response analysis and ultimately quantify differing toxic potencies between relevant PAs. Accordingly, risk-limiting measures comprising production, marketing, and regulation of food, feed, and medicinal products are discussed.

A systems-based approach to the environmental risk assessment of multiple stressors in honey bees.

The European Parliament requested EFSA to develop a holistic risk assessment of multiple stressors in honey bees. To this end, a systems-based approach that is composed of two core components: a monitoring system and a modelling system are put forward with honey bees taken as a showcase. Key developments in the current scientific opinion (including systematic data collection from sentinel beehives and an agent-based simulation) have the potential to substantially contribute to future development of environmental risk assessments of multiple stressors at larger spatial and temporal scales. For the monitoring, sentinel hives would be placed across representative climatic zones and landscapes in the EU and connected to a platform for data storage and analysis. Data on bee health status, chemical residues and the immediate or broader landscape around the hives would be collected in a harmonised and standardised manner, and would be used to inform stakeholders, and the modelling system, ApisRAM, which simulates as accurately as possible a honey bee colony. ApisRAM would be calibrated and continuously updated with incoming monitoring data and emerging scientific knowledge from research. It will be a supportive tool for beekeeping, farming, research, risk assessment and risk management, and it will benefit the wider society. A societal outlook on the proposed approach is included and this was conducted with targeted social science research with 64 beekeepers from eight EU Member States and with members of the EU Bee Partnership. Gaps and opportunities are identified to further implement the approach. Conclusions and recommendations are made on a way forward, both for the application of the approach and its use in a broader context.

Metabolic Pattern of Hepatotoxic Pyrrolizidine Alkaloids in Liver Cells.

Contamination with 1,2-unsaturated pyrrolizidine alkaloids (PAs) is a serious problem for certain phytomedicines, foods, and animal feeds. Several of these PAs are genotoxic and carcinogenic, primarily in the liver, upon cytochrome P450 (CYP)-catalyzed activation into reactive (pyrrolic and pyrrole-like) metabolites. Here we investigated the metabolism of selected PAs (echimidine, europine, lasiocarpine, lycopsamine, retrorsine, and senecionine) in rat hepatocytes in primary culture and in human CYP3A4-transfected HepG2 cells. The open-chained diesters echimidine and lasiocarpine and the cyclic diester senecionine were extensively metabolized in rat hepatocytes into a broad spectrum of products released into the medium. A large portion of unidentified, possibly irreversibly bound, products remained in the cells while detectable amounts of reactive and other metabolites were found in the incubation media. In HepG2-CYP3A4 cells, lasiocarpine was more extensively metabolized than echimidine and senecionine which also gave rise to the release of pyrrolic metabolites. In human cells, no pyrrolic metabolites were detected in retrorsine or lycopsamine incubations, while no such metabolites were detected from europine in both cell types. Other types of metabolic changes comprised modifications such as side chain demethylation or oxygenation reactions like the formation of N-oxides. The latter, considered as a detoxification step, was a major pathway with cyclic diesters, was less distinctive for echimidine and lycopsamine and almost negligible for lasiocarpine and europine. Our data are in agreement with previously published cyto- and genotoxicity findings and suggests that the metabolic pattern may contribute substantially to the specific toxic potency of a certain congener. In addition, marked differences were found for certain congeners between rat hepatocytes and transfected human HepG2 cells, whereby a high level of bioactivation was found for lasiocarpine, whereas a very low level of bioactivation was observed for monoesters, in particular in human cells.

The mutagenic potency of onion juice vs. its contents of quercetin and rutin.

In spite of its considerable value as a predictor of in vivo genotoxicity and even for carcinogenicity, false positive cases were reported for the Ames test, e.g., with a number of natural food constituents. Here we analyzed the effects of juice of Allium cepa, the common onion, a staple food and traditional remedy used in many civilizations, in the Ames fluctuation assay. We could find mild mutagenicity with an onion juice extract in Salmonella typhimurium strains TA98 and TA100, the latter being less sensitive towards the extract. Mutagenicity was not influenced markedly by the presence of rat liver S9 mix. Onion juice also exerted some toxicity to the bacteria in the same concentration range. Comparative studies with quercetin and rutin, major flavonoid glycosides in onions, revealed a mutagenic potency of quercetin with an EC50-value of 4 µM in TA98. The contents of quercetin and rutin in onion juice were determined as 0.71 ± 0.20, and 0.71 ± 0.21 mg/kg. Calculations of quercetin and rutin concentrations in mutagenic dilutions revealed that both compounds are highly unlikely to cause the mutagenic effects of onion juice and that other yet undefined constituents must be responsible for these effects.

Structure-dependent genotoxic potencies of selected pyrrolizidine alkaloids in metabolically competent HepG2 cells.

1,2-unsaturated pyrrolizidine alkaloids (PAs) are natural plant constituents comprising more than 600 different structures. A major source of human exposure is thought to be cross-contamination of food, feed and phytomedicines with PA plants. In humans, laboratory and farm animals, certain PAs exert pronounced liver toxicity and can induce malignant liver tumors in rodents. Here, we investigated the cytotoxicity and genotoxicity of eleven PAs belonging to different structural classes. Although all PAs were negative in the fluctuation Ames test in Salmonella, they were cytotoxic and induced micronuclei in human HepG2 hepatoblastoma cells over-expressing human cytochrome P450 3A4. Lasiocarpine and cyclic diesters except monocrotaline were the most potent congeners both in cytotoxicity and micronucleus assays with concentrations below 3 µM inducing a doubling in micronuclei counts. Other open di-esters and all monoesters exhibited weaker or much weaker geno- and cytotoxicity. The findings were in agreement with recently suggested interim Relative Potency (iREP) factors with the exceptions of europine and monocrotaline. A more detailed micronuclei analysis at low concentrations of lasiocarpine, retrorsine or senecionine indicated that pronounced hypolinearity of the concentration-response curves was evident for retrorsine and senecionine but not for lasiocarpine. Our findings show that the genotoxic and cytotoxic potencies of PAs in a human hepatic cell line vary in a structure-dependent manner. Both the low potency of monoesters and the shape of prototype concentration-response relationships warrant a substance- and structure-specific approach in the risk assessment of PAs.

Estragole: DNA adduct formation in primary rat hepatocytes and genotoxic potential in HepG2-CYP1A2 cells.

Estragole is a natural constituent in herbs and spices and in products thereof such as essential oils or herbal teas. After cytochrome P450-catalyzed hydroxylation and subsequent sulfation, estragole acts as a genotoxic hepatocarcinogen forming DNA adducts in rodent liver. Because of the genotoxic mode of action and the widespread occurrence in food and phytomedicines a refined risk assessment for estragole is needed. We analyzed the time- and concentration-dependent levels of the DNA adducts N2-(isoestragole-3'-yl)-2'-desoxyguanosine (E3'N2dG) and N6-(isoestragole-3'-yl)-desoxyadenosine (E3'N6dA), reported to be the major adducts formed in rat liver, in rat hepatocytes (pRH) in primary culture after incubation with estragole. DNA adduct levels were measured via UHPLC-ESI-MS/MS using stable isotope dilution analysis. Both adducts were formed in pRH and could already be quantified after an incubation time of 1 h (E3'N6dA at 10 µM, E3'N2dG at 1µM estragole). E3'N2dG, the main adduct at all incubation times and concentrations, could be detected at estragole concentrations < 0.1 µM after 24 h and < 0.5 µM after 48 h. Adduct levels were highest after 6 h and showed a downward trend at later time-points, possibly due to DNA repair and/or apoptosis. While the concentration-response characteristics of adduct formation were apparently linear over the whole concentration range, strong indication for marked hypo-linearity was obtained when the modeling was based on concentrations < 1 µM only. In the micronucleus assay no mutagenic potential of estragole was found in HepG2 cells whereas in HepG2-CYP1A2 cells 1 µM estragole led to a 3.2 fold and 300 µM to a 7.1 fold increase in micronuclei counts. Our findings suggest the existence of a 'practical threshold' dose for DNA adduct formation as an initiating key event of the carcinogenicity of estragole indicating that the default assumption of concentration-response-linearity is questionable, at least for the two major adducts studied here.

Pyrrolizidine alkaloids in food and phytomedicine: Occurrence, exposure, toxicity, mechanisms, and risk assessment - A review.

Among naturally occurring plant constituents, the 1,2-unsaturated pyrrolizidine alkaloids (in the following termed 'PAs') play a distinct role because of the large number of congeners occurring in nature and the pronounced toxicity of some congeners. Several PAs are hepatotoxic in humans, experimental and farm animals and were shown to be potent hepatocarcinogens in laboratory rodents. Although the general mode of action leading to toxicity has been elucidated, i.e., being mediated by metabolic conversion of the parent molecule into a highly reactive electrophile capable of attacking cellular target molecules, major questions related to the risk assessment of PAs remain unresolved. It was the aim of a workshop held in September 2018 to shed more light on the occurrence, exposure, mode of action, toxicokinetics and -dynamics of PAs to improve the scientific basis for an advanced toxicological risk assessment. The contributions in nine chapters describe the scientific progress using advanced analytical methods, studies in subcellular fractions, cell culture, experimental animals and humans and the use of PBPK modeling and structure-activity relationship considerations aiming at a better understanding of PA toxicity and genotoxicity. Since PAs differ considerably in their toxic potencies and substantial species differences in sensitivity towards PA exposure exist, a special emphasis was placed on these issues.

Characterization of the cytotoxicity of selected Chelidonium alkaloids in rat hepatocytes.

Phytomedicinal preparations containing extracts of the plant Chelidonium majus (Greater Celandine) have been used in the therapy of upper abdominal disorders. C. majus alkaloids (CAL) were suspected to be responsible for reported cases of liver symptoms including cases of acute liver failure in patients upon treatment with certain C. majus preparations. Based on these reports, a safe oral daily dose limit of not more than 2.5 mg CAL was established in the EU. However, C. majus extracts and individual CAL were not able to elicit similar adverse effects when given orally to pigs or rats. We found that CAL differ considerably in their cytotoxicity in rat hepatocytes in culture. The cationic congeners chelerythrine, coptisine and sanguinarine were the most toxic ones (EC20 values ≤2 µM) while the neutral congeners chelidonine, dihydrosanguinarine and protopine were less toxic, with a rank order of toxicity of coptisine > chelerythrine > sanguinarine > chelidonine > protopine > dihydrosanguinarine. Calculation of octanol-water partition coefficients revealed that the most cytotoxic CAL in hepatocytes were the cationic polar ones. At cytotoxic concentrations sanguinarine led to a marked decrease in reduced and oxidized intracellular glutathione while the much less cytotoxic dihydrosanguinarine did not. After glutathione depletion with menadione, CAL toxicity was only slightly enhanced. Comparison of the cytotoxic concentrations to reported liver levels in experimental animals suggests that the latter were too low to cause hepatotoxicity, probably due to an extremely low oral availability of certain CAL.

Estimates of Ethanol Exposure in Children from Food not Labeled as Alcohol-Containing.

Ethanol is widely used in herbal medicines, e.g., for children. Furthermore, alcohol is a constituent of fermented food such as bread or yogurt and "non-fermented" food such as fruit juices. At the same time, exposure to very low levels of ethanol in children is discussed as possibly having adverse effects on psychomotoric functions. Here, we have analyzed alcohol levels in different food products from the German market. It was found that orange, apple and grape juice contain substantial amounts of ethanol (up to 0.77 g/L). Furthermore, certain packed bakery products such as burger rolls or sweet milk rolls contained more than 1.2 g ethanol/100 g. We designed a scenario for average ethanol exposure by a 6-year-old child. Consumption data for the "categories" bananas, bread and bakery products and apple juice were derived from US and German surveys. An average daily exposure of 10.3 mg ethanol/kg body weight (b.w.) was estimated. If a high (acute) consumption level was assumed for one of the "categories," exposure rose to 12.5-23.3 mg/kg b.w. This amount is almost 2-fold (average) or up to 4-fold (high) higher than the lowest exposure from herbal medicines (6 mg/kg b.w.) suggested to require warning hints for the use in children.

Interim relative potency factors for the toxicological risk assessment of pyrrolizidine alkaloids in food and herbal medicines.

Pyrrolizidine alkaloids (PAs) are among the most potent natural toxins occurring in a broad spectrum of plant species from various families. Recently, findings of considerable contamination of teas/herbal infusions prepared from non-PA plants have been reported. These are obviously due to cross-contamination with minor amounts of PA plants and can affect both food and herbal medicines. Another source of human exposure is honey collected from PA plants. These findings illustrate the requirement for a comprehensive risk assessment of PAs, hampered by the enormous number of different PA congeners occurring in nature. Up to now, risk assessment is based on the carcinogenicity of certain PAs after chronic application to rats using the sum of detected PAs as dose metric. Because of the well-documented large structure-dependent differences between sub-groups of PA congeners with respect to their genotoxicity and (cyto)toxicity, however, this procedure is inadequate. Here we provide an overview of recent attempts to assess the risk of PA exposure and the available literature on the toxic effects and potencies of different congeners. Based on these considerations, we have derived interim Relative Potency (REP) factors for a number of abundant PAs suggesting a factor of 1.0 for cyclic di-esters and open-chain di-esters with 7S configuration, of 0.3 for mono-esters with 7S configuration, of 0.1 for open-chain di-esters with 7R configuration and of 0.01 for mono-esters with 7R configuration. For N-oxides we suggest to apply the REP factor of the corresponding PA. We are confident that the use of these values can provide a more scientific basis for PA risk assessment until a more detailed experimental analysis of the potencies of all relevant congeners can be carried out.

Comparative investigation of the mutagenicity of propenylic and allylic asarone isomers in the Ames fluctuation assay.

α-, ß- and γ-asarone are naturally occurring phenylpropenes that occur in different plant families, mainly in Aristolochiaceae, Acoraceae and Lauraceae. Plants containing asarones are used as flavouring ingredients in alcoholic beverages (bitters), traditional phytomedicines and the rhizome of e.g. Acorus calamus is used to prepare tea. Although α- and ß-asarone show a potential in the treatment of several diseases, previous studies have shown carcinogenicity in rodents (duodenum, liver). However, the mechanism of action remained unclear. Studies on the mutagenicity of propenylic α- and ß-asarone are inconsistent and data on carcinogenicity and genotoxicity of allylic γ-asarone are lacking completely. Thus, the present study determined the mutagenicity of the three asarone isomers using the Ames fluctuation assay with and without exogenous metabolic activation (S9 mix) in the standard Salmonella typhimurium strains TA98 and TA100. A concentration dependent increase in mutagenicity could be verified for α- and ß-asarone in strain TA100 in the presence of rat liver homogenate. The side-chain epoxides of α- and ß-asarone, major metabolites formed in liver microsomes, caused mutations in TA100, supporting the hypothesis that epoxidation of the side chain plays a key role in mutagenicity of the propenylic alkenylbenzenes. The allylic γ-asarone, not undergoing detectable side-chain epoxidation in liver microsomes, was supposed to be activated via side-chain hydroxylation and further sulphonation, a typical pathway for other allylic alkenylbenzenes like estragole or methyleugenol. However, neither y-asarone nor 1'-OH-γ-asarone showed any mutagenic effect even in the human SULT-expressing Salmonella strains (TA100-hSULT1A1 and TA100-hSULT1C2), while 1'-OH-methyleugenol used as a positive control was mutagenic under these conditions. These results indicate that the propenylic asarones are genotoxic via metabolic formation of side-chain epoxides while the side-chain hydroxylation/sulphonation pathway is either not operative or does not lead to mutagenicity with the allylic γ-asarone.

Hepatic metabolism of carcinogenic ß-asarone.

ß-Asarone (1) belongs to the group of naturally occurring phenylpropenes like eugenol or anethole. Compound 1 is found in several plants, e.g., Acorus calamus or Asarum europaeum. Compound 1-containing plant materials and essential oils thereof are used to flavor foods and alcoholic beverages and as ingredients of many drugs in traditional phytomedicines. Although 1 has been claimed to have several positive pharmacological effects, it was found to be genotoxic and carcinogenic in rodents (liver and small intestine). The mechanism of action of carcinogenic allylic phenylpropenes consists of the metabolic activation via cytochrome P450 enzymes and sulfotransferases. In vivo experiments suggested that this pathway does not play a major role in the carcinogenicity of the propenylic compound 1 as is the case for other propenylic compounds, e.g., anethole. Since the metabolic pathways of 1 have not been investigated and its carcinogenic mode of action is unknown, we investigated the metabolism of 1 in liver microsomes of rats, bovines, porcines, and humans using (1)H NMR, HPLC-DAD, and LC-ESI-MS/MS techniques. We synthesized the majority of identified metabolites which were used as reference compounds for the quantification and final verification of metabolites. Microsomal epoxidation of the side chain of 1 presumably yielded (Z)-asarone-1',2'-epoxide (8a) which instantly was hydrolyzed to the corresponding erythro- and threo-configurated diols (9b, 9a) and the ketone 2,4,5-trimethoxyphenylacetone (13). This was the main metabolic pathway in the metabolism of 1 in all investigated liver microsomes. Hydroxylation of the side chain of 1 led to the formation of three alcohols at total yields of less than 30%: 1'-hydroxyasarone (2), (E)- and (Z)-3'-hydroxyasarone (4 and 6), with 6 being the mainly formed alcohol and 2 being detectable only in liver microsomes of Aroclor 1254-pretreated rats. Small amounts of 4 and 6 were further oxidized to the corresponding carbonyl compounds (E)- and (Z)-3'-oxoasarone (5, 7). 1'-Oxoasarone (3) was probably also formed in incubations with 1 but was not detectable, possibly due to its rapid reaction with nucleophiles. Eventually, three mono-O-demethylated metabolites of 1 were detected in minor concentrations. The time course of metabolite formation and determined kinetic parameters show little species-specific differences in the microsomal metabolism of 1. Furthermore, the kinetic parameters imply a very low dependence of the pattern of metabolite formation from substrate concentration. In human liver microsomes, 71-75% of 1 will be metabolized via epoxidation, 21-15% via hydroxylation (and further oxidation), and 8-10% via demethylation at lower as well as higher concentrations of 1, respectively (relative values). On the basis of our results, we hypothesize that the genotoxic epoxides of 1 are the ultimate carcinogens formed from 1.

Aroma Characterization and Safety Assessment of a Beverage Fermented by Trametes versicolor.

A cereal-based beverage was developed by fermentation of wort with the basidiomycete Trametes versicolor. The beverage possessed a fruity, fresh, and slightly floral aroma. The volatiles of the beverage were isolated by liquid-liquid extraction (LLE) and additionally by headspace solid phase microextraction (HS-SPME). The aroma compounds were analyzed by a gas chromatography system equipped with a tandem mass spectrometer and an olfactory detection port (GC-MS/MS-O) followed by aroma (extract) dilution analysis. Thirty-four different odor impressions were perceived, and 27 corresponding compounds were identified. Fifteen key odorants with flavor dilution (FD) factors ranging from 8 to 128 were quantitated, and their respective odor activity values (OAVs) were calculated. Six key odorants were synthesized de novo by T. versicolor. Furthermore, quantitative changes during the fermentation process were analyzed. To prepare for the market introduction of the beverage, a comprehensive safety assessment was performed.

Evaluation of the cytotoxic and mutagenic potential of three ginkgolic acids.

Ginkgolic acids (GAs) are alkylphenols which can be found in the fruits and leaves of Ginkgo biloba L. (Ginkgoaceae) used in herbal teas, drugs and food supplements. Standardized leaf extracts of G. biloba are widely used in the therapy of cognitive decline including Alzheimer's diseases. However, GAs are known to have cytotoxic and allergenic potential and are suspected to possess genotoxic properties. Therefore, we examined in this study the cytotoxicity and mutagenicity of three major GAs with different alkyl or alkenyl groups (13:0, 15:1, 17:1). Cytotoxicity was assessed in male Chinese hamster lung fibroblasts (V79 cells) using the resazurin reduction assay. The substances showed concentration dependent cytotoxic effects after 24h of incubation at concentrations of 50µM and higher. Mutagenicity was determined by using the Ames fluctuation assay in different Salmonella typhimurium strains (TA97a, TA98, TA100 and TA102) with and without exogenous metabolic activation (S9 mix). Furthermore, we analyzed the mutagenic potency of the three major GAs in V79 cells by performing the hypoxanthine phosphoribosyl transferase (HPRT) assay which detects gene mutations at the HPRT locus. None of the mutagenic assays showed any increase in mutagenicity above background. Therefore, these data provide evidence that the GAs tested have some cytotoxic potency but are not mutagenic. Thus, our findings contribute to the risk assessment of preparations containing plant extracts from G. biloba.

Application of the equivalency factor concept to the phototoxicity and -genotoxicity of furocoumarin mixtures.

Furocoumarins (FCs) are natural constituents widely occurring in plants used as food or in phytomedicines, cosmetics, etc. Some FCs exert dermal photo-toxicity and -genotoxicity when combined with UVA irradiation. For a few congeners, skin tumor formation has been described in humans and laboratory animals. Since almost no information is available on the photo-toxic properties of several congeners, we analyzed the photo-cytotoxic, photo-mutagenic, and photo-clastogenic properties in V79 cells for thirteen naturally occurring FCs, and for the coumarin limettin. Furthermore, nine FC mixtures including one mixture based on the FC pattern of an Angelica archangelica extract were tested in the same assays. We found that the concept of relative potency factors for photo-cytotoxic, -mutagenic, and -clastogenicpotencies of FCs, setting the value for 5-methoxypsoralen at 1.00, was applicable to all congeners tested. The concept was used successfully to describe the photo-toxic properties of binary mixtures of 5- and 8-methoxypsoralen. Furthermore, the photo-genotoxic (photo-mutagenic and -clastogenic) properties of complex FC mixtures comprising up to nine different congeners could be predicted. These data suggest that FCs can differ widely in their photo-toxic and photo-genotoxic properties but show relatively strict additivity with respect to their on target-effects when occurring as complex mixtures.

The antioxidant quercetin inhibits cellular proliferation via HIF-1-dependent induction of p21WAF.

Flavonoids are dietary antioxidants that may play a role as adjunct nutritional supplements in cancer or during inflammatory disorders. Hypoxia and the transcription factor hypoxia-inducible factor-1alpha also appear to play a key role in many human cancers. In this study, we investigated the role of quercetin in the hypoxia-dependent HIF-1alpha induction. It was shown that quercetin induced HIF-1alpha expression and HIF-1 activity under normoxia and hypoxia in human HepG2 hepatoma cells. By using actinomycin D and cycloheximide, we showed that quercetin acted post-transcriptionally by prolonging the HIF-1alpha protein half-life. Thereby quercetin interfered with the proline hydroxylation-dependent HIF-1alpha protein destabilization in the N-terminal HIF-1alpha transactivation domain. Experiments with quercetin analogues revealed that a flavonol structure and the presence of hydroxyl groups at position 3' and 4' are a prerequisite for the HIF-1alpha stabilizing effects. Further, quercetin inhibited cell proliferation and induced expression of the cell cycle inhibitor p21WAF and knocking-down HIF-1alpha disrupted these effects. These results provide evidence that quercetin inhibits the cell cycle and that induction of the HIF-system contributes to these effects of quercetin.

Application of the concept of relative photomutagenic potencies to selected furocoumarins in V79 cells.

Furocoumarins are phototoxic and photomutagenic natural plant constituents found in many medicinal plants and food items. Since plants contain mixtures of several furocoumarins, there is a need for a comparative risk assessment of a large number of furocoumarins. Previously, we have introduced the concept of relative Photomutagenicity Equivalency Factors (PMEFs) derived from the slope of the concentration-response curve of photomutagenicity of individual furocoumarins in V79 cells using the HPRT mutation assay in the presence of UVA irradiation at 125mJ/cm(2). Here we have applied this method to the furocoumarins bergamottin, isopimpinellin and psoralen using 5-methoxypsoralen (5-MOP) as a reference compound with a PMEF of 1.0. We found that neither bergamottin nor isopimpinellin, two furocoumarins abundant in plants, food etc., exerted any significant photomutagenicity while psoralen was clearly photomutagenic with a PMEF of 0.36. Similarly, isopimpinellin was not phototoxic in V79 cells, while bergamottin showed some cytotoxicity which, however, was completely independent of UVA irradiation. Only psoralen was photocytotoxic showing a similar concentration-response relationship for photomutagenicity, and for photocytotoxicity (at 72h after irradiation). Data from the micronucleus assay for DNA damage at 20h after irradiation were in complete agreement with the HPRT mutation data. Our findings indicate that individual furocoumarins differ enormously in their photomutagenic potency, and that a specific toxicological risk assessment is required for each furocoumarin instead of an assessment based on the sum of furocoumarins in a given sample.