Dissertations / Theses on the topic 'Aromatic Heterocyclic Amines'

Heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs) formed during meat processing may pose health risk to the public. This project aimed to investigate the occurrence of HCAs and PAHs in highly consumed cooked meat products including ready-to-eat (RTE) meat, patties and meatballs, and their health risk was also assessed according to the dietary pattern. Different strategies including replacing fat with vegetable oils and adding spices were applied in order to reduce the formation of HCAs and PAHs in final meat products. In addition, inhibitory mechanism of antioxidants in oil and spices on the formation of HCAs and PAHs in meat system were also discussed. In this work, HCAs and PAHs were extracted by solid-phase extraction and analysed by HPLC- Diode array UV/ Fluorescence detector. For RTE meat in UK, chargrilled chicken had the highest level of HCAs (37.45±4.89ng/g) and PAHs (3.11±0.49ng/g), followed by roasted bacon (HCAs 15.24±1.31ng/g, PAHs 1.75±0.17ng/g) in selected RTE meat products. Increase intake of chargrilled chicken and ham could increase breast cancer and colorectal adenoma risk, but other types of meat had relatively lower health risk. Replacing pork back fat with vegetable oils including sunflower oil, olive oil and grape seed oil could not only improve fatty acids profile in cooked meat products, but also reduce HCAs, which could be attributed to the existence of tocopherols and polyphenol compounds in the vegetable oils. However, antioxidants in the oils could not reduce the total amount of PAHs effectively, while the complexity of oil decomposition and antioxidants performance at high temperature could partially explain the case. All 6 spices powder including garlic, onion, red chilli, paprika, black pepper and ginger reduced the formation of total HCAs, while ginger powder achieved the highest inhibition efficiency compared with all other spices. Antioxidant capacity of spices determined their efficiency in prohibiting formation of HCAs and PAHs in great extent, while meat type only affected the formation of HCAs (p < 0.05), but not PAHs (p > 0.05). Regression model suggested that both diallyl disulfide and gallic acid contributed similar inhibitory efficiency on the formation of HCAs and PAHs. Synergistic effect between diallyl disulfide and gallic acid was observed on reducing HCAs (p < 0.05), but not on PAHs (p > 0.05).

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.

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<br>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

Bakgrund: Över 20 mutagena ämnen har detekterats i tillagade livsmedel. Till dessa räknas heterocykliska aminer. Studier har visat att dessa aminer kan orsaka mutationer och därmed öka risken för att utveckla cancer. Heterocykliska aminer bildas under tillagning av kött i höga temperaturer genom Maillardreaktionen. Syfte: Syftet med denna litteraturstudie var att se om marinering med öl, vin eller örtkryddor kan minska bildningen av heterocykliska aminer vid tillagning av kött. Metod: Denna studie är en litteraturstudie med ett urval av artiklar från databaserna PubMed och Web of Science. Totalt inkluderades 6 artiklar varav 3 artiklar studerade marinering med öl/vin och 3 studerade marinering med örtkryddor och örtextrakt. Resultat: Samtliga studier som undersöktes visade att marinering har en reducerande effekt på mängden heterocykliska aminer som bildas vid tillagning. Den mest troliga hypotesen om mekanismen är att effekten beror på den antioxidativa förmågan hos marinaderna. Ett exempel är en marinad med kombinationen gurkmeja och citrongräs som reducerade koncentrationen heterocykliska aminer med 94,8%. Slutsats: Marinering med öl, vin eller örtkryddor visades effektivt reducera mängden heterocykliska aminer. Stora reducerande effekter detekterades i marinader med gurkmeja, citrongräs, ingefära och mörk lager. Mer forskning behövs för att fastställa om reduktionen är kopplad till den antioxidativa effekten hos marinaderna<br>Background: Over 20 different mutagenic substances has been detected in cooked food. These include heterocyclic amines. Studies have shown that these amines can create mutations and increase the risk of developing cancer. Heterocyclic amines are formed in meat during the Maillard reaction which occours at high temperature cooking. Aim: The aim of this study was to investigate if the effect of marinating with beer, wine and herbs/spices can reduce the formation of heterocyclic amines found in cooked meat. Method: This study is a literature study with a selection of articles from databases PubMed and Web of Science. Six articles were included in this study. 3 articles involved marinade with beer/wine and 3 articles involved marinade with herbs/spices and extract. Results: All studies examined showed that marinating has a reducing effect on the concentration of heterocyclic amines formed during cooking. The most credible hypothesis of the mechanism is that the effect depends on the antioxidativ capacity of the marinades. For example one marinade with the combination of turmeric and lemon grass reduced the concentration of heterocyclic amines by 94,8%. Conclusion: Marinades containing beer, wine or herbs/spices was shown to effectively reduce the amount of heterocyclic amines. Great reducing effects were found using turmeric, lemon grass, ginger and black beer. More scientific research is needed to determine if the reduction is linked to the antioxidant effect in marinades.

<strong>Introduction</strong> The introduction describes the importance of arylamine compounds to society and provides a brief overview of the methods available for their synthesis. The application of metathesis catalysis to the de novo synthesis of heteroaromatic compounds is also described. <strong>Results and discussion</strong> The first section describes efforts towards the de novo synthesis of arylamines using a cross metathesis/oxidation protocol to form a 1,5-unsaturated dicarbonyl followed by an amine mediated cyclisation. The scope with respect to the 1,5-unsaturated dicarbonyl and amine is covered as well as the utility of some of the products. The section concludes with a modification of the Bohlmann Rahtz pyridine synthesis to furnish arylamines. The next section describes the applications of our methodology to the synthesis of naphthylamines, specifically using the palladium catalysed &alpha;-arylation reaction. A discussion of the α-arylation reaction is included as well as our efforts to explore the scope of the reaction. The third section follows our efforts to apply this methodologyy to the synthesis of five benzo[c]phenanthridine alkaloids including the first reported synthesis of maclekarpine B and C. The final section concludes with a discussion of our efforts towards the de novo synthesis of furans bearing a benzylic stereocentre.

Eine besondere Rolle im Fremdstoffmetabolismus hat die SULT1A1 beim Menschen aufgrund der hohen Expression und breiten Gewebeverteilung. Während die humane SULT1A1 in sehr vielen Geweben exprimiert wird, wurde die murine SULT1A1 vor allem in der Leber, Lunge und Colon gefunden. Neben der Gewebeverteilung spielt auch der Polymorphismus im humanen SULT1A1-Gen eine bedeutende Rolle. Der häufigste Polymorphismus in diesem Gen führt zu einer Aminosäuresubstitution von Arginin zu Histidin an Position 213. Die Genvariante mit Histidin (auch als SULT1A1*2 bezeichnet) codiert für ein Protein mit einer geringen Enzymaktivität und einer reduzierten Enzymmenge in Thrombocyten. Über den Einfluss dieser allelischen Varianten in anderen Geweben ist bislang wenig bekannt. In vorausgegangenen epidemiologischen Studien wurden mögliche Korrelationen zwischen den Genvarianten und der Krebsentstehung in verschiedenen Geweben untersucht. Diese Daten liefern jedoch widersprüchliche Ergebnisse zum Krebsrisiko. Aufgrund der strittigen epidemiologischen Daten sollten Tiermodelle generiert werden, um die häufigsten SULT1A1-Allele hinsichtlich der Empfindlichkeit gegenüber Nahrungs- und Umweltkanzerogenen zu untersuchen. Zur Erzeugung transgener (tg) Mauslinien wurde mittels Mikroinjektion der codierenden Genbereich und große flankierende Humansequenzen stromaufwärts und stromabwärts in das Mausgenom integriert. Es wurden mehrere Mauslinien hergestellt. Zwei davon, die Mauslinie 31 mit dem SULT1A1*1-Allel und die Mauslinie 28 mit dem SULT1A1*2-Allel, wurden eingehend analysiert. In beiden Linien wurde eine identische Kopienzahl des Transgens ermittelt. Proteinbiochemische Charakterisierungen zeigten eine weitgehend dem Menschen entsprechende Gewebeverteilung und zelluläre und subzelluläre Lokalisation der humanen SULT1A1 in der Linie (Li) 28. In Li 31 wurden Unterschiede zu Li 28 sowohl in der Gewebeverteilung als auch in der zellulären Lokalisation des exprimierten humanen Proteins ermittelt. Dabei war die Expression auf Proteinebene in der SULT1A1*2-tg Linie generell stärker als in der SULT1A1*1-Linie. Dieses Ergebnis war überraschend, denn in humanen Thrombocyten führt das SULT1A1*1-Allel zu einem höheren Gehalt an SULT1A1-Protein als das SULT1A1*2-Allel. Zur Analyse der unterschiedlichen Proteinexpressionen in den tg Mauslinien wurde die cDNA und der 5´-flankierende Bereich des SULT1A1-Gens sequenziert. In beiden tg Linien entsprach die Sequenz der cDNA der Referenzsequenz aus der Gendatenbank (Pubmed). In der 5´-flankierenden Region wurden bekannte Polymorphismen analysiert und unterschiedliche Haplotypen in den tg Linien an den Positionen -624 und -396 ermittelt. Dabei wurde in der Li 31 der Haplotyp detektiert, der in der Literatur mit einer höheren SULT1A1-Enzymaktivität beschrieben wird. Der mögliche Zusammenhang zwischen Transkriptionsrate und Proteinexpression wurde in RNA-Expressionsanalysen im codierenden und 5´-nicht codierenden Bereich (mit den alternativen Exons 1B und 1A) untersucht. Im codierenden Bereich und im Exon 1B konnte in den untersuchten Organen eine höhere RNA-Expression in der Li 28 im Vergleich zur Li 31 ermittelt werden. Außer in der Lunge wurde für Exon 1B eine identische RNA-Expression detektiert. RNA, die Exon 1A enthielt, wurde in allen untersuchten Organen der Li 28, aber nur in der Lunge bei der Li 31 gefunden. In beiden tg Linien konnten mit den Exon 1A-Primern jedoch auch größere PCR-Produkte ermittelt werden. Dieser Unterschied im Exon 1A und mögliche Spleißvarianten könnten damit für die unterschiedliche Proteinexpression des humanen SULT1A1-Proteins in den beiden tg Mauslinien sein. Die in dieser Arbeit generierten und charakterisierten tg Mausmodelle wurden in einer toxikologischen Studie eingesetzt. Es wurde das heterozyklische aromatische Amin 2-Amino-1-methyl-6-phenylimidazo-[4,5-b]pyridin (PhIP) verwendet. PhIP wird beim Erhitzen und Braten von Fleisch und Fisch gebildet und könnte mit der erhöhten Krebsentstehung im Colon in der westlichen Welt im Zusammenhang stehen. Mittels 32P-Postlabelling sollte der Einfluss der zusätzlichen Expression der humanen SULT-Proteine auf die PhIP-DNA-Adduktbildung analysiert werden. Dabei wurden mehr DNA-Addukte in den tg Tieren als in den Wildtyp-Mäusen ermittelt. Die Konzentration der gebildeten DNA-Addukte korrelierte mit der Expressionsstärke des humanen SULT1A1-Proteins in den tg Mäusen. An den in dieser Arbeit generierten tg Mauslinien mit den häufigsten allelischen Varianten des SULT1A1-Gens konnten Unterschiede auf RNA- und Protein-Ebene ermittelt werden. Zudem konnte gezeigt werden, dass die Expression der humanen SULT1A1 eine Auswirkung sowohl auf die Stärke als auch das Zielgewebe der DNA-Adduktbildung in vivo hat.<br>In humans, SULT1A1 and its polymorphic variants play an important role in xenobiotic metabolism and display a broad tissue distribution and high expression level. This enzyme is expressed in almost every human organ whereas in mice SULT1A1 can only be detected in liver, lung and colon. The most common polymorphism of this gene leads to an amino acid substitution from arginine to histidine at the position 213. In platelets, the allele encoding histidine (also designated as SULT1A1*2) is associated with both low activity and low thermal stability of the SULT protein. However, so far only little is known about the significance of these allelic variants in the other tissues with hSULT1A1 expression. Previous epidemiological studies have made attempts to correlate SULT1A1 allelic variants and cancer development, their data, however, have been contradictory for an appropriate cancer risk assessment. In this thesis, we addressed the effect of the hSULT1A1 genetic variability on the susceptibility to nutritional and environmental carcinogens using transgenic (tg) mouse models. We generated tg mice carrying the most common allelic variants of the human SULT1A1 gene. The coding region and large flanking human sequences upstream and downstream of the hSULT1A1 gene were integrated randomly into the mouse genome by microinjection. Several tg mouse lines were generated. Two of them, line (li) 31 with the SULT1A1*1 allele and li 28 with the SULT1A1*2 allele, were analysed in detail. At first, an identical transgene copy number was detected in both lines. Furthermore, biochemical characterization of li 28 showed that the tissue distribution, the cellular and subcellular localisation of the protein were very similar to those in humans. In contrast, li 31 exhibited differences in tissue distribution and cellular localisation of the human protein compared to li 28. The protein expression level in the tg line with SULT1A1*2 (li 28) was generally higher than in SULT1A1*1 (li 31) mice. These results were surprising since the SULT1A1*1 allele in human platelets usually leads to a higher amount of SULT1A1 protein compared to the SULT1A1*2 allele. To investigate these differences, we sequenced the cDNA and 5´-flanking region of the SULT1A1 gene. In both tg mouse lines, the cDNA sequence was identical to the reference sequence from the gene databank (Pubmed). We subsequently analysed the common polymorphisms of the 5´-flanking region, and determined different haplotypes at position -624 and -396 in the tg mouse lines. According to the literature, the haplotype associated with a higher SULT1A1 enzyme activity, we detected in li 31. We analyzed the possible correlation between gene transcription and protein expression by measuring RNA expression levels of the coding and the non-coding region (with alternative exons 1B and 1A). We detected a higher RNA expression level of the coding region and exon 1B in li 28 compared to li 31, whereas RNA for exon 1A was only found in li 28 in all investigated tissues, but only in lung in li 31. Furthermore we detected with exon 1A-primers larger RNA in both lines. These differences in exon 1A expression accompanied by potential splicing variants could be responsible for the different expression and activity of the human SULT1A1 protein in both tg mouse lines. In order to validate our generated and characterized tg mouse models as toxicological in vivo models, we used them for the evaluation of the heterocyclic aromatic amine 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP). PhIP is typically generated during heating and roasting of meat and fish and is suggested to be associated with an increased colon cancer incidence in the western world. We measured the impact of the additionally expressed human SULT proteins on the PhIP-DNA adduct level by 32P-postlabelling. We detected significantly higher DNA adduct levels in tg compared to wildtype mice, which correlated positively with the expression pattern of the human SULT1A1 protein in the tg mice. In conclusion, in this thesis, we have successfully generated and validated the transgenic mouse lines carrying the most common allelic variants of the human SULT1A1 gene. Interestingly, these lines exhibited differences in both the SULT1A1 RNA and protein levels. Using these transgenic mouse models as in vivo toxicological tools we have shown that the expression of human SULT1A1 in mice has a decisive impact on the strength and the target tissue of DNA adducts.

Die Enzyme der Sulfotransferase-Gensuperfamilie (SULT) konjugieren nukleophile Gruppen von kleinen endogenen Verbindungen und Fremdstoffen mit der negativ geladenen Sulfo-Gruppe. Dadurch wird die Polarität dieser Verbindungen erhöht, ihre passive Permeation von Zellmembranen verhindert und somit ihre Ausscheidung erleichtert. Jedoch stellt die Sulfo-Gruppe in bestimmten chemischen Verbindungen eine gute Abgangsgruppen dar. Aus der Spaltung resultierende Carbenium- oder Nitreniumionen können mit DNA oder anderen zellulären Nukleophilen reagieren. In Testsystemen für Mutagenität wurden zahlreiche Verbindungen, darunter Nahrungsinhaltsstoffe und Umweltkontaminanten, durch SULT zu Mutagenen aktiviert. Dabei zeigten sich zum einen eine ausgeprägte Substratspezifität selbst orthologer SULT-Formen unterschiedlicher Spezies und zum anderen Interspezies-Unterschiede in der SULT-Gewebeverteilung. Daher könnten sich die Zielgewebe einer SULT-induzierten Krebsentstehung bei Mensch und Nager unterscheiden. Um die Beteiligung von humanen SULT an der Bioaktivierung von Fremdstoffen im Tiermodell untersuchen zu können, wurden transgene Mauslinien für den Cluster der humanen SULT1A1- und -1A2-Gene sowie für die humane SULT1B1 generiert. Zur Herstellung der transgenen Linien wurden große genomische Konstrukte verwendet, die die SULT-Gene sowie – zum Erreichen einer der Humansituation entsprechenden Gewebeverteilung der Proteinexpression – deren potentielle regulatorische Sequenzen enthielten. Es wurden je drei transgene Linien für hSULT1A1/hSULT1A2 und drei transgene Linien für hSULT1B1 etabliert. Die Expression der humanen Proteine konnte in allen Linien gezeigt werden und fünf der sechs Linien konnten zur Homozygotie bezüglich der Transgene gezüchtet werden. In der molekularbiologischen Charakterisierung der transgenen Linien wurde der chromosomale Integrationsort der Konstrukte bestimmt und die Kopienzahl pro Genom untersucht. Mit Ausnahme einer hSULT1A1/hSULT1A2-transgenen Linie, bei der Kopien des Konstrukts in zwei unterschiedliche Chromosomen integriert vorliegen, wiesen alle Linien nur einen Transgen-Integrationsort auf. Die Untersuchung der Transgen-Kopienzahl ergab, dass die Mauslinien zwischen einer und etwa 20 Kopien des Transgen-Konstrukts pro Genom trugen. In der proteinbiochemischen Charakterisierung wurde gezeigt, dass die transgenen Linien die humanen Proteine mit einer weitgehend der des Menschen entsprechenden Gewebeverteilung exprimieren. Die Intensität der im Immunblot nachgewiesenen Expression korrelierte mit der Kopienzahl der Transgene. Die zelluläre und subzelluläre Verteilung der Transgen-Expression wurden bei einer der hSULT1A1/hSULT1A2-transgenen Linien in Leber, Niere, Lunge, Pankreas, Dünndarm und Kolon und bei einer der hSULT1B1-transgenen Linien im Kolon untersucht. Sie stimmte ebenfalls mit der Verteilung der entsprechenden SULT-Formen im Menschen überein. Da sich die erzeugten transgenen Linien aufgrund ihrer mit dem Menschen vergleichbaren Gewebeverteilung der SULT-Expression als Modellsystem zur Untersuchung der menschlichen SULT-vermittelten metabolischen Aktivierung eigneten, wurde eine der hSULT1A1/hSULT1A2-transgenen Linien für zwei erste toxikologische Untersuchungen eingesetzt. Den Mäusen wurden chemische Verbindungen verabreicht, für die in in-vitro-Versuchen eine hSULT1A1/hSULT1A2-vermittelte Bioaktivierung zu Mutagenen gezeigt worden war. In beiden Untersuchungen wurde die Gewebeverteilung der entstandenen DNA-Addukte als Endpunkt einer gewebespezifischen genotoxischen Wirkung ermittelt. In der ersten Untersuchung wurden 90 mg/kg Körpergewicht 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridin – ein in gebratenem Fleisch gebildetes heterozyklisches aromatisches Amin – transgenen sowie Wildtyp-Mäusen oral verabreicht. Acht Stunden nach Applikation wiesen die transgenen Mäuse signifikant höhere Adduktniveaus als die Wildtyp-Mäuse in Leber, Lunge, Niere, Milz und Kolon auf. In der Leber der transgen Mäuse war das Adduktniveau 17fach höher als in der Leber der Wildtyp-Mäuse. Die Leber war bei den transgenen Tieren das Organ mit dem höchsten, bei den Wildtyp-Tieren hingegen mit dem niedrigsten DNA-Adduktniveau. In der zweiten Untersuchung (Pilotstudie mit geringer Tierzahl) wurde transgenen und Wildtyp-Mäusen 19 mg/kg Körpergewicht des polyzyklischen aromatischen Kohlenwasserstoffs 1-Hydroxymethylpyren – ein Metabolit der Nahrungs- und Umweltkontaminante 1-Methylpyren – intraperitoneal verabreicht. Nach 30 Minuten wurden, verglichen mit den Wildtyp-Mäusen, bis zu 25fach erhöhte Adduktniveaus bei den transgenen Mäusen in Leber, Niere, Lunge und Jejunum nachgewiesen. Somit konnte anhand einer in dieser Arbeit generierten transgenen Mauslinie erstmals gezeigt werden, dass die Expression der humanen SULT1A1/hSULT1A2 tatsächlich sowohl auf die Stärke als auch die Zielgewebe der DNA-Adduktbildung in vivo eine Auswirkung hat.<br>The enzymes of the sulfotransferase gene superfamily (SULT) conjugate nucleophilic groups of small endogenous compounds and xenobiotics with the negatively charged sulfo group. Thus, the polarity of the compounds is increased, their passive permeation of cell membranes is hindered and their excretion facilitated. The sulfate groups, however, form a good leaving group in certain chemical linkages due to their electron-withdrawing characteristics. Carbenium or nitrenium ions resulting from a spontaneous cleavage may react with DNA and other cellular nucleophiles. In test systems for mutagenicity, a large amount of compounds including ingredients of nutrition and environmental contaminants were activated to mutagens by SULT. A pronounced substrate specificity even of orthologous SULT forms of different species was evidenced. Also, the tissue distribution of SULT exhibited pronounced interspecies differences. The target tissues of a SULT induced carcinogenesis might thus be different in humans and rodents. To investigate the involvement of human SULT in the bioactivation of xenobiotics in an animal model, transgenic mouse lines for the human SULT1A1- and -1A2 gene cluster as well as for human SULT1B1 were generated. For the construction of the transgenic lines, large genomic constructs were used, containing the SULT genes plus their potential regulatory sequences to cause a tissue distribution of protein expression corresponding to the situation in humans. Three transgenic lines for hSULT1A1/hSULT1A2 and three transgenic lines for hSULT1B1 were established. The expression of the human proteins could be shown for all lines and except for one line, all could be bred to transgene homozygosity. By molecular biological characterization of the transgenic lines, the chromosomal integration locus of the constructs was identified and the copy number per genome was investigated. With the exception of one hSULT1A1/hSULT1A2 transgenic line, where the construct had integrated into two different chromosomes, all lines exhibited just one transgene integration locus. By investigating the transgene copy number it was deduced that the mouse lines carry between one and 20 copies of the transgene construct per genome. The protein biochemical characterization showed that the transgenic mouse lines express the human proteins with a tissue distribution largely similar to the distribution in humans. The intensity of the proteins detected by immunoblotting correlated with the copy number of the transgenes. The cellular and subcellular distribution of the transgene expression was investigated for one of the hSULT1A1/1A2 transgenic lines in liver, kidney, lung, pancreas, small intestine and colon and for one of the hSULT1B1 transgenic lines in colon. It also accorded with the distribution of the respective SULT in humans. Owing to the similarity of transgene expression to the corresponding human tissue distribution, the transgenic lines were considered suitable as model systems for the investigation of the human SULT-mediated metabolic activation. One of the hSULT1A1/hSULT1A2 transgenic lines was used in two first toxicological investigations with chemical compounds for which in vitro experiments had demonstrated a hSULT1A1/hSULT1A2 mediated bioactivation. In both investigations, the tissue distribution of the resulting DNA adducts was determined as an end point for a tissue-specific genotoxic effect. For the first investigation, 90 mg/kg bodyweight of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine – a heterocyclic amine formed in cooked meat – were orally administered to transgenic and wild type mice. Eight hours after application, the transgenic mice exhibited significantly higher adduct levels than the wild type controls in liver, lung, kidney, spleen and colon. The adduct level in the liver of the transgenic mice exceeded that in the wild type liver by a factor of 17. Furthermore, the liver was the organ with the highest adduct level in the transgenic mice and with the lowest adduct level in the wild type mice. For the second investigation (a pilot study with few animals), 19 mg/kg bodyweight of the polycyclic aromatic hydrocarbon 1-hydroxymethylpyrene – a metabolite of the nutritional and environmental contaminant 1-methylpyrene – were administered intraperitoneally to transgenic and wild type mice. After 30 minutes, up to 25 fold higher adduct levels compared to the wild type were detected in liver, kidney, lung and jejunum of the transgenic mice. Thus, by means of one of the transgenic mouse line generated in this thesis, it could be shown for the first time that the expression of human SULT1A1/SULT1A2 has in fact an impact on the strength as well as on the target tissue of DNA-adduct generation in vivo.

Tese de doutoramento em Ciências do Consumo Alimentar e Nutrição apresentada à Faculdade de Ciências da Nutrição e Alimentação da Universidade do Porto, sob orientação de Professora Doutora Olívia Maria de Castro Pinho (Faculdade de Ciências da Nutrição e Alimentação da Universidade do Porto) e coorientação de Professora Dr.ª Isabel Maria Pinto Leite Viegas Oliveira Ferreira (Faculdade de Farmácia Universidade do Porto).<br>Resumo da tese: As aminas aromáticas heterocíclicas (HAs) são consideradas um fator de risco para o desenvolvimento de cancro. A capacidade de formação das HAs em carnes e pescado cozinhados pelos métodos culinários habituais (fritos, assados, grelhados na frigideira, no carvão ou em dispositivo elétrico), mesmo em quantidades muito pequenas (ppb), implicam uma frequente exposição pela população em geral. Nos últimos 30 anos, realizaram-se inúmeros estudos visando a minimização do risco para a saúde associado às HAs. Contudo, as três principais áreas de estudo das Has ainda são um desafio: a formação em alimentos, as estratégias de inibição da formação, e a procura de compostos quimiopreventivos. Além disso, vários investigadores têm realçado a necessidade do estudo de outros carcinogénios que ocorram em simultâneo com as HAs, como os hidrocarbonetos policíclicos aromáticos (PAHs). A presença de HAs e PAHs foi avaliada em carnes (bife e frango) e pescado (salmão e sardinha) cozinhados de diferentes formas, refletindo as práticas portuguesas. O conteúdo em HAs nas amostras foi determinado usando um método de referência, que consiste em extração e purificação por extração em fase sólida (SPE) e separação e deteção por cromatografia líquida de alta eficiência acoplada a sistemas de deteção por díodos e fluorescência (HPLC-DAD/FLD). Os PAHs foram analisados através de uma metodologia determinada na presente dissertação, que consiste também em extração e purificação por SPE e separação e deteção por HPLC-FLD. Relativamente à formação de HAs nas amostras analisadas, em geral, a HA mais abundante foi o PhlP (1,45-33,8 ng/g), seguido de AªC (1-19 ng/g) e MelQx (não detectado-9,07ng/g). A formação das HAs IQ, 4,8-DiMelQx, MeAªC, Trp-P-1, Trp-P-2 e Glu-P-1 verificou-se em algumas amostras em função do tipo de músculo ou método culinário.(...)/ Thesis abstract: Heterocyclic aromatic amines (HAs) are considered a dietary risk factor for human cancer. Their capability of formation on muscle foods during ordinary cooking practices (grilling, broiling, barbecuing, roasting, frying, pan-frying), even at low parts-per-billion (ppb), implies frequent exposure by the general public. Over the past 30 years, numerous studies have been stimulated aiming to alleviate human health risk associated with HAs. The three main areas are still a challenge: their occurrence in foods, the strategies to inhibit their formation, and the search for chemopreventive agents. Furthermore, several researchers highlighted an urgent need of studying HAs and other concomitant mutagens at the same tíme, as polycyclic aromatic hydrocarbons (PAHs). The occurrence of HAs and PAHs in different cooked muscle foods (beef, salmon, and sardines) and different cooking procedure (barbecuing, grilling and pan-frying) on Portuguese household cooking procedures were evaluated. The samples were analyzed for HAs contents usíng a reference method, which consists in solid-phase extraction (SPE) and high-performance liquid chromatography-diode array detection/ fluorescente detection (HPLC-DAD/FLD). To PAHs a similar methodology was ascertained, which also consists in SPE and separation and detection by HPLC-FLD. Concerming the HAs formation im analyzed samples, in general the most abundant HA was PhIP (1,45-33,8 ng/g) followed by AaC (1-19 ng/g) and MelQx (not detected-9,07ng/g). The HAs, IQ, 4,8-DiMelQx, MeAaC, Trp-P-1, Trp-p-2 ND glu-p-1 were also formed in some muscle foods and cooking procedures with relative significance.(...)

Tese de doutoramento em Ciências do Consumo Alimentar e Nutrição apresentada à Faculdade de Ciências da Nutrição e Alimentação da Universidade do Porto, sob orientação de Professora Doutora Olívia Maria de Castro Pinho (Faculdade de Ciências da Nutrição e Alimentação da Universidade do Porto) e coorientação de Professora Dr.ª Isabel Maria Pinto Leite Viegas Oliveira Ferreira (Faculdade de Farmácia Universidade do Porto).<br>Resumo da tese: As aminas aromáticas heterocíclicas (HAs) são consideradas um fator de risco para o desenvolvimento de cancro. A capacidade de formação das HAs em carnes e pescado cozinhados pelos métodos culinários habituais (fritos, assados, grelhados na frigideira, no carvão ou em dispositivo elétrico), mesmo em quantidades muito pequenas (ppb), implicam uma frequente exposição pela população em geral. Nos últimos 30 anos, realizaram-se inúmeros estudos visando a minimização do risco para a saúde associado às HAs. Contudo, as três principais áreas de estudo das Has ainda são um desafio: a formação em alimentos, as estratégias de inibição da formação, e a procura de compostos quimiopreventivos. Além disso, vários investigadores têm realçado a necessidade do estudo de outros carcinogénios que ocorram em simultâneo com as HAs, como os hidrocarbonetos policíclicos aromáticos (PAHs). A presença de HAs e PAHs foi avaliada em carnes (bife e frango) e pescado (salmão e sardinha) cozinhados de diferentes formas, refletindo as práticas portuguesas. O conteúdo em HAs nas amostras foi determinado usando um método de referência, que consiste em extração e purificação por extração em fase sólida (SPE) e separação e deteção por cromatografia líquida de alta eficiência acoplada a sistemas de deteção por díodos e fluorescência (HPLC-DAD/FLD). Os PAHs foram analisados através de uma metodologia determinada na presente dissertação, que consiste também em extração e purificação por SPE e separação e deteção por HPLC-FLD. Relativamente à formação de HAs nas amostras analisadas, em geral, a HA mais abundante foi o PhlP (1,45-33,8 ng/g), seguido de AªC (1-19 ng/g) e MelQx (não detectado-9,07ng/g). A formação das HAs IQ, 4,8-DiMelQx, MeAªC, Trp-P-1, Trp-P-2 e Glu-P-1 verificou-se em algumas amostras em função do tipo de músculo ou método culinário.(...)/ Thesis abstract: Heterocyclic aromatic amines (HAs) are considered a dietary risk factor for human cancer. Their capability of formation on muscle foods during ordinary cooking practices (grilling, broiling, barbecuing, roasting, frying, pan-frying), even at low parts-per-billion (ppb), implies frequent exposure by the general public. Over the past 30 years, numerous studies have been stimulated aiming to alleviate human health risk associated with HAs. The three main areas are still a challenge: their occurrence in foods, the strategies to inhibit their formation, and the search for chemopreventive agents. Furthermore, several researchers highlighted an urgent need of studying HAs and other concomitant mutagens at the same tíme, as polycyclic aromatic hydrocarbons (PAHs). The occurrence of HAs and PAHs in different cooked muscle foods (beef, salmon, and sardines) and different cooking procedure (barbecuing, grilling and pan-frying) on Portuguese household cooking procedures were evaluated. The samples were analyzed for HAs contents usíng a reference method, which consists in solid-phase extraction (SPE) and high-performance liquid chromatography-diode array detection/ fluorescente detection (HPLC-DAD/FLD). To PAHs a similar methodology was ascertained, which also consists in SPE and separation and detection by HPLC-FLD. Concerming the HAs formation im analyzed samples, in general the most abundant HA was PhIP (1,45-33,8 ng/g) followed by AaC (1-19 ng/g) and MelQx (not detected-9,07ng/g). The HAs, IQ, 4,8-DiMelQx, MeAaC, Trp-P-1, Trp-p-2 ND glu-p-1 were also formed in some muscle foods and cooking procedures with relative significance.(...)

碩士<br>國立臺灣師範大學<br>化學系<br>105<br>Heterocyclic aromatic amines (HCAs) comprise of a class of > 25 compounds, which primarily generated unintended hazardous substances by heating or processing of meats from cooking of proteinaceous foods at temperatures above 150℃. In addition, several of them have been found to be carcinogenic in animals, causing tumors in diverse organs across multiple species. The International Agency for Research on Cancer (IARC) has classified three HCAs (MeIQ, MeIQx, and PhIP) as being possible human carcinogens (Group 2B) and one HCA (IQ) to be a probable human carcinogen (Group 2A). In this study, two sample preparation strategies, liquid-liquid extraction (LLE) with solid-phase extraction (SPE) and quick, easy, cheap, effective, rugged, and safe extractions (QuEChERS) method, were investigated for the determination of 11 types of HCAs in meat products by LC-MS/MS. HCAs in sample were first extracted with acetonitrile by LLE, and followed by SPE using Oasis® MCX cartridges. In QuEChERS extraction, acetonitrile is used as LLME solvent, and PSA, C18EC and MgSO4 are served as dSPE sorbent. Both methods showed good performances on precision (RSD < 15.15%), accuracy (79.80-117.64%), recovery (52.39-116.88%), limit of quantitation for the spiked meat extract (0.01-10 ppb) and correlation coefficients (>0.993). QuEChERS extraction strategy gives better linear dynamic range and superior sensitivity in comparison with LLE-SPE approach. Eventually, HCAs were successfully quantified in real samples by two proposed approaches on LC-MS/MS system.

碩士<br>國立清華大學<br>工程與系統科學系<br>104<br>Huge metals are discharged into the environment for their wide applications in semiconductor, panel, textile dyeing, printing inks, wood preservation, paints, etc. resulting in environmental pollution. Usually, metals exist in metal ions forms in aqueous environment. Therefore, the strategy based on metal ions reduction and consequently precipitation was often used for metal ions removal. In this work, the applicability of polymerized aromatic amines for the removal of copper and palladium from various water samples has been investigated. Besides, the effect of mass transfer, concentrations of reactants and the addition of the initiator on the morphology of polymerized aromatic amines was studied. With using different aniline analogues as the monomers, we could tune the nitrogen/carbon composition. In the meantime, we can get a high capacity adsorbent for metals by increasing the amount of amines, imines and surface area. Key words: polymerized aromatic amines、surface area、amines and imines

Background: Meat consumption is associated with an elevated risk of colorectal cancer (CRC); exposure to heterocyclic aromatic amines (HAAs), carcinogens produced when meat is cooked at high temperatures, is one hypothesized explanation for this relationship. HAAs form adducts with DNA; left unrepaired, DNA adducts can induce mutations which may initiate and/or promote the development of colorectal adenomas, precursors to the vast majority of CRCs. Along this continuum, genetic differences in the ability to biotransform or metabolize HAAs and repair DNA is postulated to modify the HAA-CRC relationship. Methods: This thesis examined the HAA-CRC relationship in two studies (Phase 1 and 2). In a cross-sectional study of 99 healthy volunteers, Phase 1 investigated the relationship between dietary exposure to HAAs and the levels of bulky DNA adducts in blood leukocytes. In Phase 2, a cross-sectional study examined the relationships between dietary exposures to: a) HAAs and; b) meat mutagenicity, and the prevalence of colorectal adenomas among 342 patients undergoing a screening colonoscopy. Both Phase 1 and 2 examined potential gene-diet interactions between dietary HAAs and genetic factors relevant to the biotransformation of HAAs and DNA repair. Results: In Phase 1, an interaction was observed for dietary HAAs and NAT1 polymorphisms where a positive association between HAA intakes and bulky DNA adduct levels was found among those with the NAT1 slow acetylator genotype, hypothesized to confer a lower ability to biotransform HAAs. In Phase 2, polymorphisms in genes involved in the biotransformation of HAAs (CYP1B1 rs10012 and rs1056827) and DNA repair (XPC rs2228001) were found to determine colorectal adenoma risk. As well, gene-diet interactions were observed for dietary HAAs/meat mutagenicity exposures and polymorphisms in CYP1B1 and XPD (rs13181 and rs1799793). Overall, a higher risk of colorectal adenoma was observed with higher HAA and/or meat mutagenicity exposures among those with polymorphisms which confer a greater activity to biotransform HAAs and/or a lower ability to repair DNA. Conclusion: This research supports the contribution of dietary HAAs and genetic susceptibility to the risk of developing colorectal adenomas and highlighted bulky DNA adduct formation as a potential biologic pathway through which HAAs may influence cancer risk.<br>Thesis (Ph.D, Community Health & Epidemiology) -- Queen's University, 2014-04-25 11:32:30.392

The antimutagenic potency of wheat grain and berry extracts was studied in vitro against several heterocyclic amines (HCAs) using the Salmonella mutagenicity assay and the anticarcinogencity of wheat grain was studied in vivo using the rat colonic aberrant crypt focus assay. Wheat bran, which binds HCAs in vitro, as well as refined wheat and unrefined whole wheat, inhibited the mutagenic activities of 2-amino-3- methylimidazo [4, 5-f] quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) when they were co-incubated and the supernatant (minus grain) was added to the Salmonella mutagenicity assay. The water-soluble fraction alone from refined and unrefined wheat, but not bran, also inhibited these mutagens in vitro. In vivo, AIN- 93G diets containing refined wheat or unrefined wheat were examined for their ability to inhibit IQ-induced colonic aberrant crypt foci (ACF) in the F344 rat. A slight increase in the number of aberrant crypts/ACF (AC/ACF) was seen after 16 weeks in rats treated post-initiation with refined wheat (p<0.05), and fewer foci with 2 or 3 aberrant crypts (ACF-2) were found in rats given unrefined whole wheat post-initiation compared with animals treated with the same diet during the initiation phase (p<0.05). There was no significant difference in the profile of IQ urinary metabolites or excretion of promutagens 0-48 hours after carcinogen dosing, and grains had no effect on hepatic cytochrome P450 (CYP) 1A1, CYP1A2, aryl sulfotransferase, or N-acetyltransferase activities; however, a slightly higher UDP-glucuronosyl transferase activity was observed in rats fed unrefined wheat compared with refined wheat diets (p<0.05). Thus, despite their antimutagenic activities in vitro, only marginal effects were seen with refined and unrefined wheat in vivo with respect to induction of hepatic enzyme activities, carcinogen metabolism, or IQ-induced ACF in the rat colon. The fresh juice and extract of crandall black currant (Ribes aureum) were not mutagens in the Salmonella mutagenicity assay. Berry extract or fresh juice at levels to 50 ��l (22 mg berry) in a 500 ��l pre-incubation system significantly inhibited the mutagenicity of IQ, a mutagen from cooked meat, by 32% when rat liver S9 bioactivation system was present. One hundred ��l of crandall black currant extract gave 89% inhibition of IQ mutagenicity (p<0.05). However, the mutagenicity of 2-hydroxyamino-3-methylimidazo[4,5-f] quinoline (N-hydroxy- IQ), a direct-acting metabolite of IQ, was not affected. An in vitro fluorometric assay showed the activity of cytochrome P 450 (CYP) 1A1 and CYP 1A2 was decreased. Inhibition of CYP 1A2 activity may be an important mechanism of antimutagenicity of crandall black currant extract. Similar results were also observed with other berry samples. Key word: cereal grains, black currant, berry, aberrant crypt foci, heterocyclic amines, CYP1A1, CYP1A2, Salmonella mutagenicity assay.<br>Graduation date: 2003

<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>