4-Aminobiphenyl is an aromatic amine compound that is present in cigarette smoke, diesel exhaust, cooking oil fumes and dye intermediates. It is a well-known human bladder carcinogen and liver carcinogen in experimental animals that is metabolically activated by liver CYP1A1/2. We have used the “Accelerated Cytotoxicity Mechanism Screening” (ACMS) techniques to analyze the molecular cytotoxic mechanisms of 4-aminobiphenyl. Hepatocyte exposure to an inflammatory system significantly increased hepatocyte susceptibility to 4-aminobiphenyl. 4-Aminobiphenyl- induced cytotoxicity and lipid peroxidation were both prevented by altering cellular redox status and with the addition of antioxidants. Toxicity was increased with the depletion of hepatocyte GSH levels and by inhibiting N-acetyltransferase. These results will provide more insight into the cytotoxic and genotoxic mechanisms of 4-aminobiphenyl and also suggest that inflammation may be responsible for an increase in arylamine carcinogenesis.

4-Aminobiphenyl is an aromatic amine compound that is present in cigarette smoke, diesel exhaust, cooking oil fumes and dye intermediates. It is a well-known human bladder carcinogen and liver carcinogen in experimental animals that is metabolically activated by liver CYP1A1/2. We have used the “Accelerated Cytotoxicity Mechanism Screening” (ACMS) techniques to analyze the molecular cytotoxic mechanisms of 4-aminobiphenyl. Hepatocyte exposure to an inflammatory system significantly increased hepatocyte susceptibility to 4-aminobiphenyl. 4-Aminobiphenyl- induced cytotoxicity and lipid peroxidation were both prevented by altering cellular redox status and with the addition of antioxidants. Toxicity was increased with the depletion of hepatocyte GSH levels and by inhibiting N-acetyltransferase. These results will provide more insight into the cytotoxic and genotoxic mechanisms of 4-aminobiphenyl and also suggest that inflammation may be responsible for an increase in arylamine carcinogenesis.

This thesis is concerned with the synthesis and evaluation of various tellurium-based chain-breaking antioxidants. The purpose is to find novel regenerable compounds with improved radical-trapping capacity. In the first part of this work, we explore the possibilities to incorporate tellurium into tocopherols and aromatic amines. Overall, tocopherols carrying alkyltelluro groups are better radical-trapping agents than the corresponding sulfur- and selenium analogues. Among them, 7-octyltelluro δ-tocopherol showed a ca. 17-fold higher reactivity than recorded for α-tocopherol and much better regenerability. Even longer inhibition times were recorded for the corresponding bis(tocopheryl) tellurides. In the aromatic amine series, diphenyl amines carrying alkyltelluro groups were shown to function as efficient radical-quenchers capable of inhibiting peroxidation for 460 min in the presence of N-acetylcysteine. Thiol-consumption experiments suggested that the long inhibition times are due to efficient quenching of in-situ formed alkoxyl radicals in a solvent cage. In the second part of the thesis, we study how the antioxidant properties are affected by variations in the electron density at tellurium and the number of alkyltelluro substituents in the molecule. Evaluation of a series of aryltelluro phenols carrying electron donating and electron withdrawing groups in the para-position of the aryl moiety suggested that a high electron density at the heteroatom prolonged the inhibition time. Among alkyltelluro phenols, alkyltelluro resorcinols and bis(alkyltelluro) phenols, phenols carrying alkyltelluro groups in both ortho positions were superior when it comes to radical-trapping activity and regenerability.

A series of para-substituted phenylethylamine analogues bound to the enzyme aromatic amine dehydrogenase have been simulated using quantum mechanical electronic structure calculations and molecular mechanical molecular dynamics simulations. Trends have been verified connecting bond dissociation energy (and thus driving force) to observed rate constants and activation enthalpy. Trends have been identified in connecting statistics drawn from molecular dynamics simulations and the temperature dependence of the kinetic isotope effect, notably that as the temperature dependence of the kinetic isotope effect increases the flexibility of the promoting vibration decreases. This is explained as being more effected by thermal energy put into the system, and therefore more affected by temperature.

In this study, polymerization mechanisms of aromatic (C6H5 or C6F5) and alkyl (CH3, C2H5, C3H7, C6H13, C12H25 or C18H37) amine based naphthoxazine monomers (15-Na, 15Na-C1, 15Na-C2, 15Na-C3, 15Na-C6, 15Na-C12 and 15Na-C18) and thermal degradation mechanisms of polynaphthoxazines synthesized by curing the naphthoxazine monomers (P-15-Na, P-15NaF, P-15Na-C1, P-15Na-C2, P-15Na-C3, P-15Na-C6, P-15Na-C12 and P-15Na-C18) were studied by direct pyrolysis mass spectrometry. During the curing process, the evolutions of monomer and low mass aromatic or alkyl amines were detected below the curing temperature. The mass spectrometry findings indicated that the monomers were subjected to evaporation and degradation to a certain extent but also to polymerization during the curing process. It has been determined that the polymerization of aromatic amine based naphthoxazine monomer proceeded through the aniline units, either by coupling of the radicals produced by the ring opening of the side chains or by substitution to the benzene ring of aniline. However, even if no direct evidence to confirm polymerization by attack of &ndash / NCH2 groups to naphthalene ring was detected, it is not possible totally eliminate its existence. The evolution of aniline, the main thermal degradation product for this polymer was observed at relatively low temperatures indicating that thermal decomposition started by elimination of aromatic amine involving linkages. Coupling of &ndash / CH2 groups generated by loss of aniline, yielded naphthoxazines with unsaturated linkages that can recombine and form a crosslinked structure with higher thermal stability. Similarly, the polymerization of naphthoxazines based on alkyl amines followed opposing paths. The mass spectral data indicated that the coupling of alkyl amine radicals was the main polymerization pathway. Again, thermal decomposition of alkyl amine based naphthoxazines was started by loss of alkyl amines and diamines. The crosslinked structures produced by coupling of the radicals generated by lose of alkyl amines decomposed at relatively high temperatures.

No description available.

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. / Thesis (Ph.D, Community Health & Epidemiology) -- Queen's University, 2014-04-25 11:32:30.392

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. / 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.

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. / 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.

Recent studies from our laboratory on the aromatic amine carcinogen, 4-aminobiphenyl (ABP) have shown a significantly lower prevalence of ABP-induced liver tumors in male mice lacking the N-acetyltransferases, and a dramatically lower prevalence in females than in males, but no association of tumor prevalence with strain or sex differences in levels of acute ABP-induced DNA damage. This thesis aimed to investigate the possible involvement of acute cytotoxic effects of ABP in the development of a tumor-promoting inflammatory environment. We found that wild-type male mice showed higher acute hepatotoxicity to ABP, as well as, a possible trend towards higher serum levels of the pro-inflammatory cytokine interleukin 6. This correspondence between acute ABP cytotoxicity and inflammatory response with ultimate tumor growth is consistent with a model whereby ABP not only initiates cells by damaging DNA but also promotes tumor growth in a gender-selective fashion that may be governed by gonadal hormone influences.