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Data from epidemiological studies suggest that consumption of red and processed meat is a factor contributing to colorectal carcinogenesis. Red meat contains high amounts of heme, which in turn can be converted to its nitrosylated form, NO-heme, when adding nitrite-containing curing salt to meat. NO-heme might contribute to colorectal cancer formation by causing gene mutations and could thereby be responsible for the association of (processed) red meat consumption with intestinal cancer. Up to now, neither in vitro nor in vivo studies characterizing the mutagenic and cell transforming potential of NO-heme have been published due to the fact that the pure compound is not readily available. Therefore, in the present study, an already existing synthesis protocol was modified to yield, for the first time, purified NO-heme. Thereafter, newly synthesized NO-heme was chemically characterized and used in various in vitro approaches at dietary concentrations to determine whether it can lead to DNA damage and malignant cell transformation. While NO-heme led to a significant dose-dependent increase in the number of DNA strand breaks in the comet assay and was mutagenic in the HPRT assay, this compound tested negative in the Ames test and failed to induce malignant cell transformation in the BALB/c 3T3 cell transformation assay. Interestingly, the non-nitrosylated heme control showed similar effects, but was additionally able to induce malignant transformation in BALB/c 3T3 murine fibroblasts. Taken together, these results suggest that it is the heme molecule rather than the NO moiety which is involved in driving red meat-associated carcinogenesis. © 2020, The Author(s).

Background: Rodent feeding trials are frequently applied to assess the safety of genetically modified plants. Even though their added value is controversially discussed within the scientific community, the Implementing Regulation (EU) No 503/2013 requests their mandatory conduct during the approval process of genetically modified plants for being placed on the European market. This systematic review aims at providing a comprehensive synthesis and critical appraisal of results from rodent feeding trials with whole genetically modified food/feed being performed to determine their potential to cause general signs of toxicity and/or carcinogenicity in the test animals. Methods: This study follows the systematic review approach to identify, select, appraise and synthesis data from included studies. The selection and critical appraisal process will be done by two reviewers independently from each other, whereas data will be extracted by one reviewer and rechecked by a second one. This review aims at providing a quantitative synthesis of results, whereas in any case a narrative synthesis will be provided. The review protocol was evaluated by external stakeholders with expertise in toxicology and systematic review methodology and their comments were considered during its finalization. In order to increase the efficiency of the review process, the online tool CADIMA will be used during the evidence synthesis process. Discussion: This systematic review aims at contributing to the controversial scientific debate being associated with the safety assessment of genetically modified plants and may support the clarification of potential uncertainties being accompanied with the conduct of rodent feeding trials, using whole genetically modified food/feed as test material.

Highlights • Co-cultures of liver and immune cells can be used to detect iDILI compounds. • Pro-inflammatory factors are involved in the development of iDILI. • The co-exposure of a drug candidate with TNF might be sufficient to predict iDILI., Interactions between hepatocytes and immune cells as well as inflammatory episodes are frequently discussed to play a critical role in the alteration of the individual susceptibility to idiosyncratic drug-induced liver injury (iDILI). To evaluate this hypothesis and to face the urgent need for predictive in vitro models, we established two co-culture systems based on two human cell lines in presence or absence of pro-inflammatory factors (LPS, TNF), i.e. hepatoma HepG2 cells co-cultured with monocytic or macrophage-like THP-1 cells. HepG2 monocultures served as control scenario. Mono- or co-cultures were treated with iDILI reference substances (Troglitazone [TGZ], Trovafloxacin [TVX], Diclofenac [DcL], Ketoconazole [KC]) or their non-iDILI partner compounds (Rosiglitazone, Levofloxacin, Acetylsalicylic Acid, Fluconazole). The liver cell viability was subsequently determined via WST-Assay. An enhanced cytotoxicity (synergy) or a hormetic response compared to the drug effect in the HepG2 monoculture was considered as iDILI positive. TGZ synergized in co-cultures with monocytes without an additional pro-inflammatory stimulus, while DcL and KC showed a hormetic response. All iDILI drugs synergized with TNF in the simple HepG2 monoculture, indicating its relevance as an initiator of iDILI. KC showed a synergy when co-exposed to both, monocytes and LPS, while TVX and DcL showed a synergy under the same conditions with macrophages. All described iDILI responses were not observed with the corresponding non-iDILI partner compounds. Our first results confirm that an inflammatory environment increases the sensitivity of liver cells towards iDILI compounds and point to an involvement of pro-inflammatory factors, especially TNF, in the development of iDILI.

The rat lung lavage (RLL) model is often used to test the effectiveness of new lung surfactant formulations. In this secondary surfactant deficiency model, repetitive lung lavages are performed in anesthetized, tracheostomized and pressure-controlled ventilated animals. This procedure leads to dramatically reduced gas exchange with similarities to acute respiratory distress syndrome (ARDS). After the administration of aerosolized surfactant, the restoration of lung function is indicated by the recovery of oxygenation. For the refinement and reduction of the RLL animal tests, the establishment of an ex vivo model such as the isolated perfused rat lung (IPL) is desirable. We tested the effect of saline lavage procedures using IPL. Sprague-Dawley rats were ventilated under negative conditions (end-inspiratory pressure (Pinsp) / end-expiratory pressure of -15 / -3 cm H2O), an inspiration : expiration ratio of 1 : 1 and 100% oxygen at a respiratory rate of 80 breaths / min. Every 5 min one deep breath with a Pinsp of 20 cm H2O was performed to reduce atelectasis formation. Up to 6 lavages with 0,9 % NaCl-solution caused a decreasing O2 level of at least 200 mm Hg. LDH and total protein were measured in single lavages from IPL and RLL trials. Both biochemical parameters did not differ significantly between lavages. Furthermore, LDH and total protein in lavages from IPL and RLL experiments were in equal ranges. Our data show that a high Pinsp of 15 cm H2O, combined with repeated saline lavages, does not severely damage lung tissue in the IPL model. With this procedure we are able to imitate the oxygenation status of moderate ARDS (100 mm Hg < PaO2 / FiO2 = 200 mm Hg) in the IPL. In addition, lavaged lung tissue in the IPL model is equally affected as lungs in RLL trials. In the future, the development of a new test system for surfactant formulations shall replace the in vivo surfactant formulation testing.

Since the discovery of the excellent features of MWCNT, there has been an increase in potential applications, despite the fact that they have been discussed to have a toxic potential depending on their length and fiber-like shape. For this reason, potential adverse biological effects of MWCNT have been investigated in vivo (rat) in a project funded by the German BMBF (contract No. 03X0109A). Tailor-made MWCNT with different lengths and diameters were produced, suspended in artificial lung medium and injected intraperitoneally in rats in two dose groups (low: 1x109 WHO fibers; high: 5x109 WHO fibers) in a BrdU screening test (MWCNT 1, 2, 3) and a carcinogenicity study (MWCNT 1, 2, 3, 3a). Long amosite asbestos (0.1x109 WHO fibers) served as positive control, ground MWCNT and Printex 90 (5 mg/rat) as negative control. Suspension and length/ diameter distribution were measured in SEM. Proliferation of cells in the diaphragmatic peritoneum was investigated as a shorttime screening test 3 and 6 months after i.p. injection of fibers in rats, using the BrdU method and measurement of peritoneal thickness. Furthermore, animal mortality and tumor development were monitored over two years in a carcinogenicity study. There was a time-independent significant increase in cell proliferation after injection of MWCNT 1(high) (L=7.9 mm; D=0.037 mm), MWCNT 2(low/high) (L=10.24 mm; D=0.04 mm), and MWCNT 3(low/high) (L=8.57 mm; D=0.085 mm), like after exposure to long amosite asbestos (L=13.95 mm; D=0.39 mm) as positive control. MWCNT 2(high) and 3(low/high) induced significant dose-dependent thickening of the peritoneum after i.p. injection, independently of time. In the carcinogenicity study, MWCNT 2, 3 and 3a (L=9.3 mm; D=0.062 mm) showed a higher mesothelioma incidence than MWCNT 1. In conclusion, some MWCNT (MWCNT 2, 3) mediate enhanced proliferation of peritoneal cells in the diaphragm in rats, which may result in mesothelioma development.

Multiwall carbon nanotubes (MWCNT) are discussed to exhibit an asbestos-like toxic potential depending on their length and fiber-like shape. For this reason, potential adverse biological effects of MWCNT were investigated in vitro in a German BMBF funded project (contract No. 03X0109A). In this project tailor made MWCNT with different length and diameter were produced to be able to correlate effects with MWCNT characteristics. The present in vitro experiments were performed with the human peritoneal mesothelial cell line LP9/TERT-1 as a model system. Cells were exposed for 24 h to 5 g/cm2 of 6 different MWCNT and data were compared to long amosite asbestos as positive control and more particle-like MWCNT (tangled MWCNT) and particles (milled MWCNT, Printex 90) as negative controls. Pre-experiments demonstrated that incubation of cells at 5 g/cm2 for 24 h differentiated best between the various treatments. For in vitro experiments MWCNT were directly suspended in cell culture medium by using a sonotrode (HD2070; VS70T) twice for 5 minutes at 90 % duty cycle and 100 % amplitude. Suspension, size, and distribution of MWCNT were monitored by scanning electron microscope (SEM). The cytotoxic potential of MWCNT was estimated by means of "relative increase in cell counts" (RICC) and lactate dehydrogenase (LDH)-release. An enzyme (hOGG1)-modified comet assay was used to determine the direct genotoxic potential. In the present study, only CNT3 (length: 8.57 m; diameter: 0,085 m) mediated a significant increase in LDH-release and thus disturbances of membrane integrity, whereas all other MWCNT and reference materials demonstrated no marked effect, compared to the medium control. In the hOGG1-modified comet assay, which detects DNA strand breaks and oxidative DNA-base lesion, no significant direct DNA-damaging effect of the investigated MWCNT was obvious, independent of length and diameter. In contrast, using RICC as an endpoint strong reduction in cell number/proliferation could be demonstrated for CNT1 (length: 7.91 m; diameter: 0.037 m), CNT2 (length: 10.24 m; diameter: 0.04), CNT3, CNT3a (length: 9.3 m; diameter: 0.061 m), and long amosite asbestos (length: 13.95 m; diameter: 0.39 m), as compared to the particulate reference items and the medium control. In conclusion, certain MWCNT exhibit a marked cytotoxic/anti-proliferative activity in human mesothelial cells (LP9/TERT-1) in vitro, without indications for a direct DNA-damaging potential.

Heterocyclic aromatic amines (HCAs) are formed in meat cooked at high temperatures for a long time or over an open flame. In this context 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant HCA in cooked meat, has been suggested to be involved in colon and prostate carcinogenesis. In the latter case it has been reported that: (1) roughly 50% of Fischer F344 male rats treated with PhIP develop carcinomas in the ventral prostate lobe at 1 year of age; (2) inflammation precedes prostatic intraepithelial neoplasia in PhIP-fed rats; (3) inflammation specifically occurs in the ventral prostate lobe of PhIP-fed rats. To test whether PhIP by itself leads to inflammation in the colon and whether a human-relevant concentration of PhIP is able to induce preneoplastic lesions in the colon, male F344 rats were fed 0.1 or 100 ppm PhIP for up to 10 months and thereafter the colon tissue was analyzed histochemically. In none of the experimental groups signs of acute or chronic colonic inflammation were observed. 0.1 ppm PhIP leads to the development of hyperplastic and dysplastic lesions in the colon of single animals, but the incidence of these lesions does not reach a statistical significance. In contrast, in rats fed 100 ppm PhIP for 10 months hyperplastic and dysplastic colonic lesions were induced in a statistically significant number of animals. It is concluded that: (1) the induction of preneoplastic lesions in rat colon by PhIP is not preceded or accompanied by an inflammatory process; (2) a human-relevant concentration of PhIP alone is not sufficient to initiate colon carcinogenesis in rats.

The health promoting effects of a grapevine-shoot extract named Vineatrol (R) 30, which contains resveratrol (Resv) as well as considerable amounts of Resv oligomers, have recently been investigated. In the present study, we analyzed the free radical scavenging capacity, the ability to inhibit lipid peroxidation, and the capacity to enhance the human glutathione peroxidase 1 (GPx) and the human superoxide dismutase 1 (SOD) gene promoter activities of Vineatrol (R) 30. Vineatrol (R) 30 was able to scavenge the 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical cation and led to concentration-dependent inhibition of lipid peroxidation, Vineatrol (R) 30 not being superior to Resv alone in both cases. Vineatrol (R) 30 also enhanced the gene promoter activities of human GPx and SOD expressed in V79 cells, whereas this effect could not be demonstrated for Resv. In summary, the results presented in this study show that the Vineatrol (R) 30 grapevine-shoot extract is a free radical scavenger and potent antioxidant at non- eytotoxic concentrations.

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The current/traditional human health risk assessment paradigm is challenged by recent scientific and technical advances, and ethical demands. The current approach is considered too resource intensive, is not always reliable, can raise issues of reproducibility, is mostly animal based and does not necessarily provide an understanding of the underlying mechanisms of toxicity. From an ethical and scientific viewpoint, a paradigm shift is required to deliver testing strategies that enable reliable, animal‐free hazard and risk assessments, which are based on a mechanistic understanding of chemical toxicity and make use of exposure science and epidemiological data. This shift will require a new philosophy, new data, multidisciplinary expertise and more flexible regulations. Re‐engineering of available data is also deemed necessary as data should be accessible, readable, interpretable and usable. Dedicated training to build the capacity in terms of expertise is necessary, together with practical resources allocated to education. The dialogue between risk assessors, risk managers, academia and stakeholders should be promoted further to understand scientific and societal needs. Genuine interest in taking risk assessment forward should drive the change and should be supported by flexible funding. This publication builds upon presentations made and discussions held during the break‐out session ‘Advancing risk assessment science – Human health’ at EFSA's third Scientific Conference ‘Science, Food and Society’ (Parma, Italy, 18–21 September 2018)., EFSA Journal, 17 (S1), ISSN:1831-4732, Proceedings of the Third EFSA Scientific Conference: Science, Food and Society

The diverse community of microbes present in the human gut has emerged as an important factor for cancer risk, potentially by altering exposure to chemical carcinogens. In the present study, human gut bacteria were tested for their capacity to transform the carcinogenic heterocyclic amine 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx). Eubacterium hallii, Lactobacillus reuteri, and Lactobacillus rossiae were able to convert MelQx to a new microbial metabolite characterized on the basis of high-resolution mass spectrometry and NMR as 9-hydroxyl-2,7-dimethyl-7,9,10,11-tetrahydropyrimido[2′,1′:2,3]imidazo[4,5-f]quinoxaline (MelQx-M1), resulting from conjugation with activated glycerol. Acrolein derived from the decomposition of 3-hydroxypropionaldehyde, which is the product of bacterial glycerol/diol dehydratase activity, was identified as the active compound responsible for the formation of MelQx-M1. A complex human gut microbial community obtained from invitro continuous intestinal fermentation was found to also transform MelQx to MelQx-M1. MelQx-M1 had slightly reduced cytotoxic potency toward human colon epithelial cells invitro, and diminished mutagenic potential toward bacteria after metabolic activation. As bacterially derived acrolein also transformed 2 other HCAs, namely 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3-methylimidazo[4,5-f]quinoline, these results generalize the capacity of gut microbiota to detoxify HCAs in the gut, potentially modulating cancer risk., Toxicological Sciences, 159 (1), ISSN:1096-6080, ISSN:1096-0929

Human are constantly exposed to potentially toxic chemicals from the environment, diet, and therapeutic interventions. However, the gut harbors a diverse community of microorganisms, which may alter the exposure and toxicity of these chemicals. Heterocyclic amines (HCAs) are mutagens presented in meat cooked at high temperature, and they are strongly associated with the increased risk of colorectal cancer. Bacterial transformation of HCAs may alter their structures and thus their toxicity, but little is known regarding the influence of gut microbiota on the risk associated with these chemicals due to the existing knowledge gap regarding the microbiota-chemical interactions. The work described in this thesis concerns the chemical and biochemical mechanisms of commensal gut bacterial transformation of HCAs and their liver metabolites, as well as toxicological and physiological relevance of the microbial transformations. The knowledge obtained provides mechanistic insights on how gut microbiota alter chemical toxicity and supports gut microbiota as a factor in the risk assessment of toxic chemicals. Chapter 1 introduces the background information concerning the microbial metabolism of glycerol in the human gut and its relevance to HCA transformation. In addition, an overview of the potential targets of gut bacteria-derived acrolein is given. In Chapter 2, we aimed to address the generality of bacterial conjugation of HCAs with acrolein. MeIQx is an imidazoquinoxaline mutagen ten times more mutagenic than PhIP toward bacterial DNA in vitro assays. E. hallii, Lactobacillus reuteri, and Lactobacillus rossiae were found to convert MeIQx to a new microbial metabolite characterized on the basis of HRMS and NMR as 9-hydroxyl-2,7-dimethyl-7,9,10,11-tetrahydropyrimido-[2′,1′:2,3]imidazo[4,5-f]quinoxaline (MeIQx-M1). Acrolein derived from the decomposition of 3-HPA, which is a product of glycerol reduction mediated by GDH activity, was identified as the active compound responsible for the formation of MeIQx-M1. MeIQx-M1 appears to have slightly reduced cytotoxic potency toward human colon epithelial cells, and diminished mutagenic potential toward bacteria after metabolic activation. In Chapter 3, the physiological and toxicological relevance of the microbial transformation of MeIQx to MeIQx-M1 was characterized. To address whether the microbial transformation influences the intestinal transport of MeIQx, the intestinal uptake of MeIQx and its metabolite MeIQx-M1 was quantified with ex vivo rat intestinal segments, however, only negligible amounts of both MeIQx and MeIQx-M1 were transported. In addition, neither MeIQx nor MeIQx-M1 were cytotoxic towards liver HepaRG cells at dietary levels or higher concentrations. Physiologically based pharmacokinetic modeling suggests that increased microbial transformation of MeIQx can reduce plasma levels of MeIQx, potentially contributing to reduced systemic exposure of MeIQx in human. In Chapter 4, the impact of commensal gut microbes on the transformation of HCA liver metabolites, especially glucuronide conjugates was investigated. The aim was to gain knowledge regarding how gut microbes coordinate to transform HCA-glucuronides to release HCAs and further convert them to HCA-M1s. The beta-glucuronidase (GUSB) produced by species belong to Eubacterium and Faecalibacterium catalyzed the re-activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine N2-β-D-glucuronide (PhIP-G), a liver metabolite of PhIP, to form PhIP. GDH of Flavonifractor plautii, Blautia obeum, E. hallii, and L. reuteri contributed the detoxification of PhIP to form PhIP-M1. Co-culture of GUSB+ strain and GDH+ strain led to the conversion of PhIP-G to PhIP and PhIP-M1. These results for the first time demonstrate the gut microbial transformation of PhIP-G to PhIP-M1, providing a mechanistic model for reducing PhIP exposure from enterohepatic circulation by linking the modification of a carcinogen with the metabolic activity of predominant gut microbes. In Chapter 5, the chemical scope of the reaction of acrolein with HCAs was expanded. The aim of this study was to gain knowledge on the reactivity of heterocyclic guanines (HCGs) with α,β-unsaturated aldehydes (UAs). Results suggest that HCGs react with acrolein to form products bearing a hydroxyltetrahydropyrimidine ring. Substitution on both α and β positions of acrolein dramatically reduced reactivity and acrolein had the highest reactivity among the tested UAs. Moreover, two drugs bearing a guanidine moiety can readily react with acrolein with 491-721 times faster than that of DNA base with acrolein. These findings suggest the guanidine conjugation with acrolein is a general reaction of HCGs mediated by bacterial acrolein and HCGs are scavengers of acrolein. Chapter 6 summarizes the findings and gives an outlook. A critical discussion of achievements and limitations is provided and ongoing future directions are presented.

This paper is one of several prepared under the project "Food Safety In Europe: Risk Assessment of Chemicals in Food and Diet" (FOSIE), a European Commission Concerted Action Programme, organised by the International Life Sciences Institute, Europe (ILSI). The aim of the FOSIE project is to review the current state of the science of risk assessment of chemicals in food and diet, by consideration of the four stages of risk assessment, that is, hazard identification, hazard characterisation, exposure assessment and risk characterisation. The contribution of animal-based methods in toxicology to hazard identification of chemicals in food and diet is discussed. The importance of first applying existing technical and chemical knowledge to the design of safety testing programs for food chemicals is emphasised. There is consideration of the presently available and commonly used toxicity testing approaches and methodologies, including acute and repeated dose toxicity, reproductive and developmental toxicity, neurotoxicity, genotoxicity, carcinogenicity, immunotoxicity and food allergy. They are considered from the perspective of whether they are appropriate for assessing food chemicals and whether they are adequate to detect currently known or anticipated hazards from food. Gaps in knowledge and future research needs are identified; research on these could lead to improvements in the methods of hazard identification for food chemicals. The potential impact of some emerging techniques and toxicological issues on hazard identification for food chemicals, such as new measurement techniques, the use of transgenic animals, assessment of hormone balance and the possibilities for conducting studies in which common human diseases have been modelled, is also considered.

Folate sind als Überträger von C1-Fragmenten verschiedener Oxidationsstufen direkt an der Nukleinsäuresynthese beteiligt und spielen somit eine entscheidende Rolle im Zellstoffwechsel. So kann ein suboptimaler Folatstatus zur Ausprägung einer klinischen Symptomatik führen, die sich primär in Zellen mit hoher Teilungsrate manifestiert. Dabei gilt die megaloblastäre Anämie als Leitsymptom für einen akuten Folatmangel. Herausragende Bedeutung besitzen Folate in der Schwangerschaft, wo ihnen bei der Entwicklung des fetalen Nervensystems eine besondere Rolle zukommt. Bei einem zu niedrigen Serumfolatspiegel der Mutter steigt das Risiko für embryonale Missbildungen wie Neuralrohrdefekte stark an. Die weitere medizinische Relevanz der Folate steht momentan im Fokus der Forschung, wie z. B. der protektive Effekt in Bezug auf bestimmte Krebsformen, die Risikoreduktion für verschiedene kardiovaskuläre Erkrankungen sowie Zusammenhänge zwischen dem Folatstatus und neurologischen Störungen (Alzheimer, Depressionen). Allerdings kann Folat als essentielles Vitamin von Säugetieren nicht selbst synthetisiert werden, sondern muss aus externen Quellen über den Verdauungstrakt aufgenommen werden. Die Resorption aus dem Darm erfolgt nach aktuellem Kenntnisstand insbesondere über den Reduced Folate Carrier (RFC, SLC19A1) und den Proton-Coupled Folate Transporter (PCFT, SLC46A1). Trotz der Existenz dieser spezifischen Transporter ist der Folatmangel des Menschen der häufigste Vitaminmangel in Mitteleuropa. Er lässt sich durch eine ausschließlich ernährungsphysiologische Problematik nur unzureichend erklären. Interessanterweise besitzen sowohl das PCFT-Gen als auch das RFC-Gen in ihrer Promoterregion verschiedene Regulationselemente, unter anderem auch funktionell aktive DREs (Dioxin Response Element), die als Bindungsstelle für einen ligandenaktivierten Transkriptionsfaktor, den nukleären Arylhydrocarbon-Rezeptor (AhR), dienen. DREs wurden bisher hauptsächlich bei fremdstoffmetabolisierenden Enzymen wie z. B. Cytochrom P450-Isoenzymen gefunden und vermitteln bekanntermaßen die toxischen und karzinogenen Effekte von AhR-Liganden. Von besonderem Interesse sind in diesem Zusammenhang ubiquitär verbreitete Umweltkontaminanten wie die polyzyklischen und die halogenierten aromatischen Kohlenwasserstoffe (PAK, HAK), da sie in der Umwelt sehr persistent sind und sich dadurch in der Lebensmittelkette anreichern. Infolgedessen wurde im Rahmen dieser Studie der mögliche Einfluss von AhR-Liganden wie TCDD (2,3,7,8-tetrachlordibenzo-p-dioxin) und B[a]P (Benzo[a]pyren) auf die carriervermittelte intestinale Folatresorption beim Menschen untersucht sowie die Regulation der Transportproteine RFC und PCFT auf transkriptioneller Ebene experimentell überprüft. Als adäquates in vitro Modell diente dabei die humane Kolonzelllinie LS180, für die zunächst eine Charakterisierung erfolgte. Mittels RT-PCR wurde der Nachweis erbracht, dass alle am Folattransport beteiligten Import-und Exportcarrier auf mRNA-Ebene exprimiert werden. Für die Transportproteine RFC und PCFT erfolgte über den Western Blot auch der Nachweis auf Proteinebene. Die Funktionalität der AhR-Signalkaskade, die über methylcholanthrenartige Induktoren wie TCDD zur Induktion von Cytochrom P450 führt, konnte im Folgenden mittels Ethoxyresorufin-O-deethylase-Assay (EROD) überprüft werden. Es zeigte sich nach Induktion mit TCDD (0,01 - 10 nM) oder B[a]P (0,01 - 1 μM) über 12 – 96 h ein hochsignifikanter, dosis- und zeitabhängiger Effekt auf die Cyp1A1 vermittelte Enzymaktivität der Ethoxyresorufin-O-deethylase in den LS180-Zellen. Dabei konnten ligandenabhängige Unterschiede im Induktionsmuster ermittelt werden. Im Anschluss wurde im intestinalen Zellmodell die initiale konzentrations- und zeitabhängige Folataufnahme bei pH 5.5 über funktionelle Aufnahmeversuche mit Tritium-markierter Folsäure untersucht und charakterisiert. Sie stellte sich als aktiver, sättigbarer Prozess dar, wobei in den unbehandelten LS180-Zellen ca. eine Verdreifachung der intrazellulären Radioaktivität über einen Zeitraum von 2,5 min beobachtet werden konnte. Als kinetische Parameter wurden ein Km-Wert von 27,91 μM sowie ein Vmax-Wert von 281,2 pmol/min berechnet. Nachfolgende Untersuchungen mit den spezifischen Inhibitoren Raltitrexed (RTX) und Pemetrexed (PMX) konnten zeigen, dass sowohl PCFT als auch RFC an der funktionellen Folsäureaufnahme bei einem pH-Wert von 5.5 in dem gewählten Versuchsaufbau beteiligt sind. Der RFC scheint jedoch einen etwas höheren Anteil an der Gesamtaufnahme zu haben als der PCFT. In LS180-Zellen, die vorher mit den Modellsubstanzen TCDD (1 bzw. 10 nM) oder B[a]P (0,1 bzw. 1 μM) über 24 - 120 h inkubiert wurden, konnte eine zeit- und dosisabhängige, statistisch signifikante Reduktion der carriervermittelten Folatresorption beobachtet werden, wobei die maximale Verminderung der Aufnahmerate ca. 75 % betrug. Da sich dieser Effekt durch die AhR-Antagonisten Salicylamid (SAL) und CH-223191 (CH) dosisabhängig umkehren ließ, erfolgt die Regulation vermutlich über den AhR-Signalweg. Um den Mechanismus der Regulation zu klären, wurde mit Hilfe der quantitativen qRT-PCR unter Verwendung von TaqMan-Sonden die Genexpression von RFC und PCFT nach Vorbehandlung mit 1 - 10 nM TCDD oder 0,1 – 1 μM B[a]P über 12 - 120 h untersucht. Analog zu den Aufnahmeversuchen konnte hier eine zeit- und dosisabhängige Reduktion beider Transporter auf transkriptioneller Ebene beobachtet werden. Auch führte eine Vorbehandlung mit CH und SAL wiederum zu einer Umkehr des Effektes, wobei CH wahrscheinlich einen ligandenselektiven AhR-Antagonisten darstellt. Zusammenfassend konnte durch die vorliegende Arbeit nachgewiesen werden, dass Cytochrom P450-Induktoren über die AhR-Signalkaskade die carriervermittelte Folatresorption in humanen Kolonzellen herabregulieren. Es erscheint anhand der gewonnen Erkenntnisse möglich, dass die Folathomoöstase durch verschiedene Umweltkontaminanten wie z. B. TCDD negativ beeinflusst werden kann und als Folge daraus auch eventuell der Folatmangel der Bevölkerung in Industrienationen zumindest teilweise erklärbar wird. Es ist darüber hinaus denkbar, dass auch andere Fremdstoffe die Folataufnahme beeinflussen, da große planare Strukturen häufig über den AhR-Weg wirken. Weiterführende Studien sind notwendig, um das Verständnis für den Einfluss von Umweltkontaminanten auf die intestinale Folataufnahme zu verbessern.