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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.

<p>Neurological disorders are a major public health concern due to prevalence, severity of symptoms, and impact on caregivers and economic losses. While genetic susceptibility likely has a role in most cases, exposure to toxicants can lead to neurotoxicity, including potentially developmental origins of adult disease or increased risk of disease onset. These exposures are not necessarily large, acute exposures, but could accumulate, with a chronic low-dose exposure, causing toxicity. This research focuses on the potential neurotoxicity of two classes of dietary toxins/toxicants, heterocyclic aromatic amines (HAAs) and per- and polyfluoroalkyl substances (PFAS). HAAs, such as PhIP, harmane, and harmine, are formed in charred or overcooked meat, coffee, tobacco, and other foods. PFAS are largely used in making household materials, but are found in small amounts in eggs and dairy products and largely in contaminated water. While these two classes are diverse in terms of structure, common neurotoxic targets and mechanisms often exist. Therefore, we tested the effects of these chemicals on cell viability and neurotoxicity. In the first aim, we aimed to elucidate the mechanism of toxicity of harmane and harmine, focusing on their ability to cause mitochondrial dysfunction. The second aim was to determine the effects of either harmane or PhIP on the nigrostriatal motor systems and motor function of rats and mice, respectively. The third aim determined the effects of PFAS on neurodevelopment of Northern leopard frogs, focusing on changes in neurotransmitter levels and accumulation in the brain. Harmane did not cause motor dysfunction, but potentially affected the nigro-striatal motor system in an age- or sex-dependent manner. PhIP had differential effects on dopamine levels over time and caused motor dysfunction after subchronic exposure in mice. Perfluorooctane sulfonate (PFOS) accumulated in the brains of frogs and PFAS caused changes in neurotransmitter levels that were dose- and time-dependent. Overall, this research shows that toxins/toxicants humans are exposed to over their whole lives through their diet and contaminated water can cause neurotoxicity, potentially leading to or increasing risk of disease states. </p>

Die Zahl der Kolonkarzinome in den westlichen Industrieländern steigt in den letzten Jahren stetig an. Zu den Verbindungen, die mit der Zubereitung der Nahrung entstehen, mit ihr aufgenommen werden und die Kolonkanzerogenese möglicherweise begünstigen, gehört das heterozyklische aromatische PhIP, das bei der Erhitzung proteinreicher Nahrungsmittel entsteht. Neben zahlreichen Fütterungsversuchen an Nagern existieren auch Zellkulturmodelle zur Untersuchung der molekularen Mechanismen der PhIP-induzierten Kolonkanzerogenese. Die chemische Transformation von Zellen sollte durch wiederholte Exposition gegenüber dem hydroxylierten Metaboliten des Kanzerogens (N2-OH-PhIP) erzielt werden. Es wurden IEC-18-Zellen der Ratte und HCEC-Zellen des Menschen zur Untersuchung verwendet. Die Behandlung der IEC-18-Zellen führt nach 25 Behandlungszyklen mit Konzentrationen von 5 bis 20 µM nicht zur Transformation der Zellen. Die Anwesenheit von N2-OH-PhIP führt zu einer zehnfach erhöhten Induktion der GST-Aktivität, insbesondere der Untereinheiten GST-A1, -A3, -Pi und -T2, die für die effiziente Detoxifizierung des N-Acetoxy-Metaboliten vom N2-OH-PhIP verantwortlich sind. Bereits nach drei Behandlungen mit 1,5 µM N2-OH-PhIP konnte eine maligne Transformation der HCEC-Zellen erzielt werden. Die Zellen zeigten die charakteristischen Zeichen der Transformation: veränderte Wachstumseigenschaften wie klonales dreidimensionales Zellwachstum („pilling up“), Hemmung der Zell-Zell-Kontaktinhibierung, verkürzte Populationsverdopplungszeiten und tumorigene und metastasierende Eigenschaften. Außerdem exprimierten die N2-OH-PhIP-exponierten humanen Kolonzellen mit steigender Anzahl der Behandlungen größere Mengen des trunkierten APC-Proteins. Die bekannten PhIP-spezifischen Mutationen im APC-Gen resultieren in der Expression eines trunkierten Proteinproduktes und werden als frühe Ereignisse in der Kolonkanzerogenese betrachtet. Die zusammenfassende Betrachtung aller Ergebnisse zeigt, dass die IEC-18-Zelllinie zur chemischen Transformation durch N2-OH-PhIP ungeeignet ist. Dagegen wurde erstmalig eine vollständige chemische Transformation von Humandickdarmepithelzellen in vitro durch Exposition der humanen Kolonepithelzelllinie HCEC gegenüber dem Kolonkarzinogen N2-OH-PhIP erzielt. / In the last few years a strong increase in the incidence of colorectal cancer has been observed. As to the specific components in processed food responsible for the induction of colon cancerogenesis / it has been suggested that heterocyclic aromatic amines (HAA), e.g. the most abundant HAA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is formed in protein rich food, when it is cooked at high temperatures or over an open flame, might be involved in this process. Whereas a number of in vivo-models to study PhIP-mediated colon carcinogenesis are known, only a limited number of cell culture systems to study the HAA-mediated transformation of intestinal epithelial cells do in fact exist. In the present study IEC-18 cells (rat intestinal epithelial cells) and HCEC cells (human colon epithelial cells) were incubated with N2-OH-PhIP, the N-hydroxylated metabolite of PhIP. The IEC-18 cells could not be transformed despite 25 treatment cycles with 5 to 20 µM N2-OH-PhIP. This might be due to the fact that GST activity as well as the expression of the GST -A1, -A3, -Pi and -T2 units, which are responsible for the detoxication of the N-acetoxy derivative of PhIP were strongly induced by N2-OH-PhIP. In contrast, HCEC cells were malignantly transformed when exposed three times to 1.5 µM N2-OH-PhIP. The chemically-treated cells showed a reduced population doubling time, they lost cell-cell contact inhibition and started pilling up. Furthermore, if HCEC cells were injected subcutaneously into SCID mice tumors developed at the site of injection in all animals tested. The transformed HCEC cells also express high amounts of truncated APC protein, which in vivo appears at an early stage of colon cancerogenesis. Taken together, it has been shown that IEC-18 cells are not suitable for chemical transformation studies with the HAA metabolite N2-OH-PhIP. For the first time it has been shown that the HAA metabolite N2-OH-PhIP is indeed able to malignantly transform human colon epithelial cells in vitro.

Les amines hétérocycliques aromatiques (AHA) sont des contaminants de l'environnement et de l'alimentation, majoritairement formés lors de la cuisson de viande et poisson ainsi que dans la fumée de cigarette et les gaz d'échappements. Les AHA sont mutagènes chez la bactérie, cancérogènes multi-sites chez le rongeur et sont classées comme cancérogènes possibles ou probables chez l'Homme par l'IARC. Il est aujourd'hui indispensable de caractériser des biomarqueurs d'exposition dérivés des AHA (adduits à l'ADN et métabolites) pour améliorer l'estimation du risque chez l'Homme. Des résultats de l'équipe ont démontré que le 2-amino-9H-pyrido[2,3-b]indole (AαC) forme des niveaux d'adduits à l'ADN élevés dans les hépatocytes humains. Ces niveaux sont plus élevés que ceux formés par les autres AHA. L'objectif de cette thèse est de mieux comprendre le potentiel génotoxique d'AαC chez l'Homme. Nos travaux ont démontré que les adduits à l'ADN dérivés d'AαC sont persistants dans les hépatocytes humains et formés à des doses aussi faibles que 1nM. De plus, le CYP1A2 a été confirmé comme enzyme majoritaire dans la bioactivation d'AαC dans le foie humain. Nous avons également caractérisé les métabolites majeurs dérivés d'AαC dans les hépatocytes humains. Cette étude a permis d'établir pour la première fois une corrélation entre l'activité catalytique du CYP1A2, la formation d'AαC-HN2-O-Gl et la formation des adduits à l'ADN dérivés d'AαC. Le métabolite AαC-HN2-O-Gl étant réactif vis-à-vis de l'ADN in vitro, nos travaux confortent l'hypothèse que la voie des UDP-Glucuronosyltransférases (UGTs) est une nouvelle voie de bioactivation d'AαC dans le foie humain. De plus, nous avons montré que les adduits à l'ADN dérivés des AHA sont formés dans les lymphocytes T humains activés et en particulier les adduits en position C8 de la guanine dérivés d'AαC. Au total, ces travaux ont permis l'identification de métabolites stables et des adduits à l'ADN, potentiels biomarqueurs d'exposition à AαC, qui sont indispensables pour une meilleure estimation du risque génotoxique d'AαC chez l'Homme. / Heterocyclic aromatic amines (HAA) are environmental and food contaminants, mainly formed during meat and fish cooking, but also in cigarette smoke and exhaust gaz. HAA are mutagenic in bacteria, carcinogenic in rodents and are classified as possible or probable human carcinogens by IARC. Today it is essential to characterize exposure biomarkers i.e. DNA adducts and metabolites, to assess the human risk associated with HAA. The research team has previously demonstrated that 2-amino-9H-pyrido[2,3-b]indole (AαC) form high levels of DNA adducts in human hepatocytes. These levels are greater that those derived from other HAAs. Thus, the aim of this thesis was to better understand the genotoxic potential of AαC in human. We demonstrated that in human hepatocytes, DNA adducts derived from AαC are persistent and formed at doses as low as 1nM. Moreover, we confirmed that CYP1A2 is the major enzyme implicated in the bioactivation of AαC in human liver. We have also characterized the major metabolites derived from AαC formed in human hepatocytes. This study allows, for the first time, the establishment of a correlation between the catalytic activity of CYP1A2, AαC-HN2-O-Gl formation and AαC derived DNA adducts formation. AαC-HN2-O-Gl being reactive toward DNA in vitro, our work reinforces the hypothesis that the UDP-glucuronosyltransferase (UGTs) pathway is a new bioactivation pathway for AαC in human liver. Moreover, we demonstrated the formation of HAA derived DNA adducts, especially those derived from AαC at position C8 of guanine, in activated human T lymphocytes. Taken together, our data lead to the identification of stable metabolites as well as DNA adducts which are potentials AαC exposure biomarkers in human. These biomarkers are essential for a better assessment of the genotoxic risk of AαC in human.