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Breast cancer resistance protein (BCRP/ABCG2) is known to actively transport various anticancer drugs and to restrict the uptake of the food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine from the gut lumen. The present study reveals that BCRP is involved in the transport of phase-2 metabolites of the carcinogen benzo[a]pyrene (BP) in the human intestinal cell line Caco-2. Treatment with the selective BCRP inhibitor Ko 143 (5 microM) inhibited the apical transport of BP-3-sulfate (BP3S) to 83% of control levels in TC7 cells and to 64% of control levels in Caco-2 cells. The apical transport of BP-3-glucuronide was inhibited by Ko 143 to 76% of control levels in TC7 cells. Furthermore, the expression of BCRP is most likely aryl hydrocarbon receptor (AhR) dependent, as treatment of Caco-2 cells with known AhR agonists including 2,3,7,8-tetrachlorodibenzo-p-dioxin, BP, indolo[3,2-b]carbazole and benzo[k]fluoranthene increased both mRNA and protein levels of BCRP. Induced BCRP protein was found to be functionally active, since pre-treatment of TC7 cells with oltipraz, indolo[3,2-b]carbazole or benzo[k]fluoranthene increased the amount of apically transported BP3S to as much as 180% of that in the controls. The induction of BCRP (mRNA and protein expression) by indolo[3,2-b]carbazole was inhibited in Caco-2 cells by co-incubation with the AhR antagonist PD98059 (2'-amino-3'-methoxyflavone). In summary, this study provides strong evidence that BCRP is an important part of the intestinal barrier protecting the body from food-associated contaminants such as the carcinogen BP.

It has been suggested that the teratogenic effects of the antiepileptic drug valproic acid (VPA) is reflected in vitro by the differentiation of F9 cells, activation of peroxisome proliferator-activated receptor delta (PPARdelta), and inhibition of histone deacetylases (HDACs). The aim of this study was to identify genes involved in the differentiation of F9 cells induced by VPA, teratogenic VPA derivatives, or the HDAC inhibitor trichostatin A (TSA) and to characterize the role of PPARdelta. Treatment of the cells with teratogenic VPA derivatives or TSA induced differentiation of F9 cells, mRNA, and protein expression of the neural cell adhesion molecule (NCAM) as well as activated the 5'-flanking region of the NCAM promoter, whereas nonteratogenic VPA derivatives had no effect at all. The polysialyltransferases [ST8SiaIV (PST1) and ST8SiaII] are responsible for the addition of polysialic acid (PSA) to NCAM. The mRNA expression of PST1 was highly induced by only teratogenic VPA derivatives and TSA. As shown by fluorescence-activated cell sorting analysis the level of PSA was higher after treatment of F9 cells with teratogenic VPA derivatives. It is interesting that overexpression of the PPARdelta but not PPARalpha or PPARgamma in F9 cells resulted in higher induction of NCAM mRNA and protein expression and of PST1 mRNA expression (and a higher PSA level) than in mock-transfected F9 cells. Furthermore, repression of PPARdelta activity in F9 cells inhibited these effects. We conclude that NCAM and PST1 are molecular markers in F9 cell differentiation caused by treatment with teratogenic VPA compounds or TSA and suggest that in addition to HDAC inhibition PPARdelta is involved in the signaling pathway.

The small intestine is well equipped with various phase-1 and phase-2 xenobiotic metabolizing enzymes (XME), which contribute to the detoxification process of the body. Many XME are regulated via aryl hydrocarbon receptor (AhR)-dependent pathways, and numerous naturally occurring AhR agonists (e.g. flavonoids, dietary indoles) have been identified to date. In the present study we show that pretreatment of Caco-2 cells with food-associated compounds (flavone and indole-3-carbinol) and with the anticancer chemopreventive agent oltipraz enhances the formation of the major metabolites of the procarcinogen benzo[a]pyrene (BP) formed by intestinal Caco-2 cells, namely BP-1-sulfate and BP-3-sulfate, and their transport to the apical compartment of a Transwell chamber. Oltipraz treatment was most effective in this regard followed by flavone and indole-3-carbinol. The effect observed here after pretreatment with oltipraz, flavone and I3C was the result of the induction of both CYP1A1 and CYP1B1, as was confirmed by analysis of CYP1A1 (protein and mRNA) and CYP1B1 (mRNA) expression. In summary, our study shows that the induction of both CYP1A1 and CYP1B1 resulted in an accelerated metabolism and an enhanced clearance of the potent procarcinogen BP, indicating that flavone, indole-3-carbinol and oltipraz have an impact on the biochemical barrier against BP in intestinal cells.

Vitamin A and retinoids are essential nutrients for the differentiation of epithelia. Vitamin A deficiency is accompanied by an impairment in intestinal integrity. We investigated whether retinoids influence the differentiation and permeability of Caco-2 cells under serum-free culture conditions as a model for the intestinal epithelium. Treatment of the Caco-2 cells with retinoic acids (RA) resulted in an increased specific activity, enhanced mRNA expression, and induction of the 5'-flanking promoter activity of the marker enzyme for the differentiation intestinal alkaline phosphatase. Surprisingly, permeability of the Caco-2 monolayer, as measured by transepithelial electric resistance and [3H]-mannitol flux, was found to be enhanced by RA. Treatment with RA had only a slight effect on the mRNA expression of the tight junction-associated proteins occludin, ZO-1, claudin-1, -3, and -4, but enhanced the expression of claudin-2, which was recently suggested to form a paracellular ion channel. The role of retinoids as potent inducers of epithelial differentiation was confirmed for the Caco-2 cells under serum-free culture conditions and it was concluded that IAP is a target gene of RA. The inverse regulation of the permeability by RA under these serum-free conditions showed that other mechanisms, which are essential to regulate intestinal epithelial integrity with respect to decreased permeability, have to be identified.

Owing to their antimicrobial properties, silver nanoparticles are the most commonly used nanoparticles. The constantly increasing use of silver nanoparticles in products related to food and food contact materials is the reason why oral administration of nanoparticles is considered as an important uptake route and should be studied. Therefore, the tissue distribution and excretion of coated silver nanoparticles was investigated in rats after single oral administration including a 7-days post treatment observation time. The animals received silver nanoparticles at a dose of 20 mg/kg body weight and an equimolar dose of silver ions by gavage. The silver content was measured in different organs after days 1, 2, 3 and 7. To assess the effect of the intestinal passage on systemic bioavailability an additional group of rats was intravenously administered with 4 mg/kg body weight silver nanoparticles and a group was treated with an equimolar dose of silver ions. The accumulation of silver was compared after 24 h single intravenous administration. So far, the accumulation of silver was observed in different organs. As expected, most of the nanosilver was excreted via feces and minor amounts were detected in intestine and colon. Orally administered ionic silver was detected in feces and additionally in the urine. Remarkably, also after intravenous injection of nanosilver silver was found in feces, which suggests biliary excretion of silver nanoparticles. It is concluded that the oral resorption of nanosilver results in an accumulation of silver in different organs and feces and the intravenous application suggests a biliary excretion.

Background: The acute toxicity of cyanide after its enzymatic release from cyanogenic glycosides in plants (e.g. bitter almonds) is triggered by the peak level of cyanide in the body. In this regard, not only the content of bound cyanide in foods is relevant, but also the velocity of the release after chewing of the food caused by the β-glucosidase and the following surge of free cyanide in the body. In a human cross-over study (n = 12) with consumption of persipan, linseed, bitter apricot kernels and cassava (dose of bound cyanide: 6.8 mg each), mean peak levels of cyanide in blood were found to be 1.3, 5.7, 14.3 and 15.4 μM, respectively. Additional in-vitro investigations were performed to confirm the velocity of the enzymatic release of cyanide (by plant β-glucosidase) as important factor determining the toxicity of cyanogenic foods. Methods: Food samples containing 1 mg of bound cyanide were homogenized and suspended in 100 ml PBS buffer (pH 4 and 7). Free cyanide was determined at different times in samples taken from the suspension gently stirred at 37 °C (GC–MS method, internal standard: K13C15N). Results: Mean proportion of the total amount released within 10 min after tissue destruction was 0% for persipan, 15% (pH 7) for linseed, 62% (pH 4) for bitter apricot kernels and 78% (pH 7) for cassava. Conclusion: The toxicity of cyanogenic foods is determined not only by its content of bound cyanide, but also by the activity of the β-glucosidase as a plant-specific property.

1,2-Unsaturated pyrrolizidine alkaloids (PA) belong to the most toxic compounds. These substances are found in several plants such as Asteraceae and Boraginaceae families. Acute PA poisoning by food contamination causes severe damage to liver; long-term, sub-lethal doses may cause cumulative damage or cancer. A previous whole genome μ-array analysis of PA in primary human hepatocytes revealed potential interactions of PA with certain nuclear receptors acting or working as transcription factors. Employing reporter gene assays it was analyzed whether PA can activate or inhibit nuclear receptors such as RARα, RXRα, LXRα, FXR, PPARα, PPARγ, PPARδ, PXR, ERα and ERβ. As PXR and VDR mediate CYP3A4 promoter activity a potential induction or inhibition of CYP3A4 promoter activity was additionally investigated. To cover the most frequently occurring PA structures (retronecine, heliotridine and otonecine type as well as monoester, diester and cyclic diester) the four PA senecionine, heliotrine, echimidine and senkirkine were selected as representative PA. Heliotrine was found to inhibit the transactivation of LXRα, RXRα, PPARα, PPARγ, PPARδ and PXR. Senecionine activated ERα and β. Echimidine was the only PA that activated PXR. Consequently, it also exclusively induced CYP3A4 promoter activity. Neither interactions nor transactivation could be observed for senkirkine. In conclusion, the prediction from the μ-array data suggesting an interaction of PA with different nuclear receptors could be confirmed. However, the interactions were found to be heterogeneous between the structurally different PA. This suggests a specific structure–activity relationship of PA concerning their interaction with nuclear receptors.

Marine biotoxins are produced by microalgae and may concentrate in filter-feeding bivalves. Intoxication can occur after consumption of contaminated shellfish containing several marine biotoxins. Okadaic acid (OA) and yessotoxin (YTX) are two important representatives coexisting in nature in different quantities. The aim of this study was to evaluate combined hepatotoxic effects caused by OA and YTX in HepG2 cells. Additionally, the induction of toxicological relevant transcription factors was investigated by using transactivation assays in regard to mono- vs. co-exposure. Pure OA and YTX were used to treat human liver HepG2 cells including treatments in a proportion of 1:3, 1:1 and 3:1. In addition human CYP enzymes (S9) were included as external metabolic activation system to analyze metabolism dependent effects. Cell viability was measured using neutral red assay. Molecular effects were investigated in mono- and co-exposure on nuclear receptors by using a transactivation assay in HEK-293T- cells. The study clearly showed that combined effects of OA and YTX occur in regard to cytotoxicity in HepG2 cells. The viability decreased with higher YTX than OA concentrations. Such strong effects were not observed for mono-exposure of YTX. Metabolic activation enhanced toxicity after both, mono- and co-exposure. Additionally, co-exposure differently affects the activation of transcription factors compared to mono-exposure. In conclusion, co-exposure of OA and YTX lead to stronger effects than mono-exposure. The proportion of YTX to OA may play a key role in regard to the final resulting toxicity if both toxins are simultaneously present.

Retinoids are essential for growth and cell differentiation of epithelial tissues. The effects of the food compounds phytol, the phytol metabolite phytanic acid, and the fatty acid docosahexaenoic acid (DHA) on the retinoid signaling pathway in intestinal cells were studied. Phytol inhibited the formation of all-trans-retinoic acid (RA) from dietary retinol in intestinal cells. Phytanic acid, a known retinoic X receptor (RXRalpha) and peroxisome proliferator activating receptor (PPARalpha) activator, also activated PPARdelta, and to a lesser degree PPARgamma, in a transactivation assay. Phytanic acid had no effect on intestinal RA hydroxylase CYP26 (also named P450RAI) gene expression and metabolism of all-trans-RA in intestinal Caco-2 cells. However, in combination with retinoic acid receptor (RAR)-ligands (all-trans-RA or synthetic Am580) phytanic acid enhanced the induction of CYP26 and RA-metabolism in comparison to treatments with all-trans-RA or Am580 alone. Also treatment with DHA did not affect CYP26 gene expression and RA-metabolism but cotreatment of the cells with DHA and all-trans-RA or Am580 enhanced the induction of CYP26, in comparison to the induction caused by all-trans-RA or Am580 alone. This study indicates that food compounds such as phytanic acid and DHA that are RXR-agonists and have an impact on intestinal CYP26 gene expression and metabolism of all-trans-RA in intestinal cells.

Therapy with the antiepileptic drug valproic acid (2-propylpentanoic acid, VPA) during early pregnancy can cause teratogenic effects (neural tube defects) in humans and in mice. VPA and a teratogenic derivative specifically induce differentiation of F9 teratocarcinoma cells and activate PPARdelta. We have now studied structure-activity relationships of 11 VPA-related compounds by quantitatively comparing their teratogenic potency with their effects in the two novel in vitro systems. Based on the induction of a Rous sarcoma virus (RSV) promoter-driven reporter gene, which is associated with the differentiation of F9 cells, a system suitable for high-throughput and quantitative screening was established. Structure-activity investigations showed that only teratogenic derivatives of VPA induced the response in F9 cells as well as activated the PPARdelta-dependent reporter system in Chinese hamster ovary (CHO) cells. Increases in the length of the side chain in the VPA-related 2-alkyl-pentynoic acid generate more potent inducers in the cell-culture-based assays, which also show higher teratogenicity and embryonic lethality rates. Activation of PPARdelta correlated well with the effects in the F9 cell assay and with teratogenic potency in vivo (p < 0.007). Evaluation of the effects of the presented set of compounds allows the conclusion that the in vitro systems faithfully reflect teratogenicity of VPA-related compounds. Whether the activation of PPARdelta is causally related to the disruption of proper embryonic development or whether it reflects other yet unknown VPA-induced events remains to be established.

1. The macrolide tacrolimus (FK506), used as an immunosuppressant, is a cytochrome P450 (CYP) 3A substrate in the liver. The metabolism of tacrolimus and the transport of its metabolites in the pig gut was studied in the Ussing chamber. Tacrolimus and its metabolites were quantified by h.p.l.c./mass spectrometry. 2. In the Ussing chamber, demethyl, didemethyl, hydroxy and hydroxy-demethyl tacrolimus were generated. Their formation was concentration- and time-dependent. The metabolite pattern was not different from that after incubation of tacrolimus with human small intestinal microsomes. 3. The metabolite formation was highest in the duodenum and declined in the order duodenum > jejunum > ileum > colon > stomach. 4. Since tacrolimus metabolism was inhibited by the specific CYP3A inhibitors, troleandomycin and ketoconazole, we concluded that these enzymes are involved in intestinal metabolism of tacrolimus. 5. Tacrolimus metabolites re-entered the mucosa chamber (> 90%) and passed through the small intestinal preparation into the serosa chamber. 6. It is concluded that tacrolimus is metabolized in the intestine, that the metabolites are able to re-enter the gut lumen and also enter into the portal vein and that small intestinal metabolism and transport is at least in part responsible for the low oral bioavailability of tacrolimus.

Acrylamide (AA), a genotoxic carcinogen (IARC class 2A), is a food contaminant formed by thermal treatment of food. The margin of exposure (MOE) of AA, representing the distance between the bench mark dose associated with 10% tumor incidence in rats and the estimated average human exposure, is considered to be low and of human health concern. After uptake, AA is partly converted by CYP450 2E1 into the genotoxic epoxide glycidamide (GA). GA forms DNA adducts, primarily at N7 of guanine (N7-GA-Gua). AA and its genotoxic metabolite GA are conjugated to glutathione and excreted via urine as mercapturic acids (MA), namely N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA), and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)-cysteine (GAMA) which are used as biomarkers for exposure. Female Sprague Dawley (SD) rats were kept on an AA free diet. AA was given by gavage in a broad dosage range, covering single doses of 0.1-10,000 µg/kg bw. Formation of urinary MAs and of N7-GA-Gua DNA adducts in liver, kidney and lung was measured 16 h after application. The lowest dosage of 0.1 µg AA/kg bw did not result in significantly enhanced MA excretion, as compared to untreated controls, found to excrete about 0.8 nmol MA. Since the animals may have ingested at best 0.4 nmol AA/d with the diet, this findings suggests a background level of endogenously formed AA. At the lowest dosage (0.1 µg AA/kg bw), N7-GA-Gua adducts above the limit of detection (LOD, 0.2 adducts/10exp8 nucleotides) were not detected in any organ tested. At 1 µg/kg bw, enhanced adduct levels were found in kidney (around 1 adduct/10exp8 nucleotides) and lung (< 1 adduct/10exp8 nucleotides), but not in liver. At 10 and 100 µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at 1 µg AA/kg bw (about 1-2 adducts/10exp8 nucleotides). Taken together, the results of this in vivo study allow to conclude that exposure to single doses of AA in the range of human dietary exposure leads to N7-GA-Gua adduct levels in tissues of SD rats at the low end of steady state background DNA lesions of various origin in human and rat tissues. This study was supported by ISIC, the Institute for Scientific Information on Coffee. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5732. doi:1538-7445.AM2012-5732

Purpose: Engineered nanomaterials may exhibit properties differing significantly from those observed in the bulk materials, because of their small dimensions and large surface-to-volume ratio. Due to their unique qualities silver nanoparticles are used in a wide range of consumer products. Even the use of nanoscaled silver hydrosol for nutritional purposes has been requested. Despite the wide applications of silver nanoparticles, there is still lack of information concerning the impact on human health after oral application. Methods: To investigate the effects of silver nanoparticles on human intestinal cells, we monitored the reaction of Caco-2 cells, as a model for the first intestinal barrier of the human body, and AgPure silver nanoparticles. AgPure has already been used in consumer products. The radius of gyration, hydrodynamic radius and size distribution were determined by SAXS and DLS. We studied the effects of AgPure on cell viability and proliferation by CellTiter-Blue assay, DAPI staining and xCELLigence impedance measurement. Additionally, we tested for membrane damage with LDH assay, for apoptotic effects with Annexin-V/7AAD staining and for reactive oxygen species with dichlorofluorescein assay.Results: AgPure are spherical with metal core radii of 7.4 nm and hydrodynamic radii of 22 nm with stabiliser. The size is Gaussian-distributed with a polydispersity of 17 %. When proliferating Caco-2 cells are exposed to AgPure, morphological abnormally adherence and particle dose- and time-dependant cytotoxicity was observed. However, apoptosis or membrane damage did not occur, but results of the dichlorofluorescein assay suggested the formation of reactive oxygen species as possible mechanism of cytotoxicity.

Purpose: Cassia cinnamon contains high levels (up to 1%) of coumarin. Heavy consumption of this spice may result in a dose exceeding the tolerable daily intake (TDI) which is derived from toxicological data on isolated coumarin (animal experiments and medicinal drug in humans). In this context, the question was raised whether coumarin in the plant matrix of cinnamon has the same bioavailability as isolated coumarin. Methods: A crossover study was performed, in which the same dose of 12mg coumarin was administered to 24 healthy volunteers. They received four different formulations: isolated coumarin in a capsule (reference), cinnamonin capsules, cinnamon tea prepared from the same cinnamon, and cinnamon in rice pudding as a typical meal. The relative extent of absorption was measured as urinary excretion of the main metabolite 7-hydroxycoumarin (7OHC) within 8 h after application. In order to monitor the velocity of absorption, 7OHC plasma levels were measured for 105 min after administration. Results: Plasma levels of 7OHC revealed a fast absorption of coumarin especially from cinnamon tea leading to the highest peak concentrations. Mean urinary excretion of 7OHC was found to be 62.8% for isolated coumarin in a capsule, 56.0% for cinnamon in capsules, 66.1% for cinnamon tea, and 54.7% for cinnamon in rice pudding. Therefore,the relative extent of absorption of coumarin from powder of cassia cinnamon was only slightly lower than that of isolated coumarin,and the TDI of 0.1 mg/kg bw can be used for risk assessment of coumarin exposure from cinnamon-containing meals.

Purpose: 1,2-Unsaturated pyrrolizidine alkaloids (PA) belong to the most toxic compounds. These substances are found in several plants such as Asteraceae and Boraginaceae families. Acute PA poisoning via food contamination causes severe damage to the liver; long-term, sub-lethal doses may cause cumulative damage or cancer. Methods: In this study, we analyzed the toxicological effects (cytotoxicity, ATP-content, and oxidative stress) of selected PA (retrorsine, senecionine, and seneciphylline) on the hepatocarcinoma cell line HepG2 by using a bioactivating system (S9-fraction). Furthermore, we characterized the uptake of senecionine and its N-oxidated form across the intestinal barrier by using the well-characterized human Caco-2 cell Transwell-model as an in vitro system for the human gut barrier. The PA content was analyzed by LC-MS/MS. Results: The metabolic bioactivation of PA with the S9-fraction of rat liver homogenate resulted in a weak increase of cellular cytotoxicity including a slight reduction of cellular ATP-content, as well as in a weak increase of the apoptosis rate and of oxidative stress. The Caco-2 cell absorption studies revealed a fast passage of senecionine from the apical into the basolateral compartment suggesting that active transport mechanisms are involved in the uptake and excretion of the substance. In contrast, the oxidized metabolite senecionine-N-oxide did not cross the differentiated Caco-2 monolayer from the apical to the basolateral side. In addition, we observed the conversion of senecionine into the oxidated form, as well as the reduction of senecionine-N-oxide. This indicates that Caco-2 cells are able to metabolize this compound.

Purpose: In vitro transport studies in the human intestinal Caco-2 cells revealed that the ATP-binding cassette carrier Breast Cancer Resistance Protein (BCRP) is involved in the active efflux of phase II metabolites of benzo[a]pyrene (BP). Therefore we investigated the metabolism and excretion of BP in vivo using the bcrp-/- knockout mice. Besides BCRP, several xenobiotic metabolizing enzymes are involved in the detoxification of BP, and these can be induced by the aryl hydrocarbon receptor (AhR). The flavonoid quercetin have been identified as AhR agonists suggesting that these compounds can promote detoxification and elimination of dietary toxins such as BP. Methods: Bcrp-/- knockout mice and bcrp+/+ wild-type FVB mice were exposed i.v. and p.o. to BP (1.0mg/kg), including 3µCi of the radiolabeled BP per mouse. Additionally, mice were fed for 3 days with 10 mg/kg quercetin before administration of BP. Blood and bile samples were collected and at the last indicated time point; the organs were removed and homogenized. The levels of radioactivity were quantified by a Wallac 1409 liquid scintillation counter. Results: In BCRP-deficient mice, hepatobiliary excretion of BP metabolites significantly decreased compared to wild-type mice and also intestinal secretion of BP was significantly reduced. Additionally, bcrp-/- knockout mice showed higher plasma and liver levels of BP compared to wild-type mice clearly show the important role of BCRP in BP elimination also in vivo. Additionally, quercetin dramatically enhanced the hepatobiliary, urinary and intestinal excretion of BP and reduced the general tissue concentrations compared with non-quercetin fed mice.

In light of the broad spectrum of products containing nanosilver, the harmfulness of nanosilver to human health and the environment was intensively discussed at a conference held in February 2012 at the BfR. The conference agenda covered the aspects of analytics of nanosilver materials, human exposure and toxicology as well as effects on microorganisms and the environment. The discussion recovered major gaps related to commonly agreed guidelines for sample preparation and central analytical techniques. In particular, the characterization of the nanoparticles in complex matrices was regarded as a challenge which might become a pitfall for further innovation and application. Historical and anecdotal records of colloidal silver have been sometimes taken as empirical proof for the general low toxicity of nanosilver. Yet as reported herein, a growing number of animal studies following modern performance standards of toxicity testing have been carried out recently revealing well-characterized adverse effects on different routes of exposure in addition to argyria. Furthermore, recent approaches in exposure assessment were reported. However, consumer exposure scenarios are only starting to be developed and reliable exposure data are still rare. It was further widely agreed on the workshop that the use of silver may lead to the selection of silver resistant bacteria. With respect to its environmental behavior, it was suggested that nanosilver released to wastewater may have negligible ecotoxicological effects. Finally, the presentations and discussion on risk assessment and regulation of nanosilver applications gave insights into different approaches of risk assessment of nanomaterials to be performed under the various regulatory frameworks.

In the past, it was generally accepted as a default assumption that No-Observed-Adverse-Effect Levels (NOAELs) or Lowest-Observed-Adverse-Effect Levels (LOAELs) in long-term toxicity studies are lower than in short-term ones, i.e. the toxic potency increases with prolonged exposure duration. Recent studies on pesticides and industrial chemicals reported that subacute, subchronic or chronic NOAELs/LOAELs are similar when study design factors are appropriately considered. We investigated whether these findings also apply to certain food constituents. After reviewing subchronic and chronic toxicity studies on more than 100 compounds, a total of 32 compounds could be included in the analysis. Geometric mean (GM) values of subchronic vs. chronic NOAEL or LOAEL ratios ranged from 1.0 to 2.0, with a geometric standard deviation from 2.2 to 4.2, which is consistent with data reported in the literature. While for many of the investigated compounds the ratio is around 1 - suggesting that health-based guidance values could appropriately be derived from subchronic toxicity studies - our study also identified some substances with higher ratios leading to a GM of around 2. The EFSA Scientific Committee suggested to apply an uncertainty factor of 2 to extrapolate from subchronic to chronic studies and, as a precautionary approach, we concur with this suggestion. published

The conventional approach for testing the genotoxic potential of chemicals in vitro includes a battery of bacterial and mammalian mutagenicity tests. Toxicogenomics analyses may provide information about DNA-damaging properties of test compounds but are not routinely used for identification of a genotoxic potential. In this study, metabolically active human HepaRG hepatocarcinoma cells were exposed to five food-relevant genotoxic carcinogens. Transcriptomic responses were analyzed using RNA sequencing technology and validated by real-time polymerase chain reaction. Biostatistical approaches revealed a characteristic transcript signature of 37 differentially expressed genes, which were commonly regulated by the test chemicals. Specificity of the transcript signature was confirmed by using non-genotoxic carcinogens as comparators. Pathway analyses showed that the obtained transcript signature was closely related to DNA damage response and p53 activation. In conclusion, we have established a characteristic transcript marker pattern to monitor genotoxicity in human HepaRG cells, and to distinguish genotoxic from non-genotoxic carcinogens. Our analyses underline that a common response related to DNA damages response, cell cycle alterations and cell death is initiated in HepaRG cells upon exposure to genotoxic compounds and allows for the identification of a common transcriptomic signature for genotoxic stress.

Deliverable 14.2 aimed to carry out a wide literature survey in order to create an inventory of available biomarkers of effects for the first set of prioritized chemical families: Bisphenols, Phthalates, Polycyclic Aromatic Hydrocarbons (PAHs), Perfluorinated compounds (PFAS, PFOAs), Organophosphate (OPFR) and Brominated (BFR) Flame retardants, and Cadmium (Cd).