M. S.

N,N'-Dimethyldiaminodiphenylmethanes and N,N'-dialkyldiaminobenzophenones were successfully prepared from the respective primary amines via alkylation of their methanesulfonamide derivatives. Initially N,N'-diaminodiphenylmethanes and N,N'-diaminobenzophenones were treated 0 with 2.2 equivalents of methanesulfonyl chloride in pyridine at 80° to afford the corresponding bis methanesulfonamide derivatives in 79-91% yields. Alkylation was accomplished by reacting the sulfonamides with 2.2 equivalents of dry potassium hydroxide in hexamethylphosphoramide at 75°, followed by addition of 2.2 equivalents of alkylating agent (methyl iodide, ethyl iodide, and benzyl chloride) to afford 70-84% of the alkylated sulfonamides. Subsequently the N,N'-dialkylsulfonamides were cleanly cleaved using a mixture of concentrated sulfuric/acetic acids (40-60%, vol/vol) to afford N,N'-dimethyldiaminodiphenylmethanes and N,N'-dialkyldiaminobenzophenones in 68-87% and 64-85% yields, respectively. β-Hydroxy sulfonamides were prepared by treating N-methyl methanesulfonanilide with a 10% excess of n-butyllithium in tetrahydrofuran at -78°, followed by addition of benzophenone or acetone to afford the corresponding β-hydroxy sulfonanilides in 76% yield. Upon heating the sulfonanilide-benzophenone adduct in refluxing toluene, water was eliminated to form the corresponding unsaturated sulfonanilide in 89% yield. The attempted preparation of β-hydroxy sulfinamides involved initially the synthesis of N-methanesulfin-p-toluidide following the procedure of Corey and Durst. This compound was subsequently alkylated with sodium hydride and methyl iodide in 1.2-dimethoxyethane in 76% yield. Treatment of the resulting methylated sulfinamide with lithium 0 diisopropylamide at - 78° followed by addition of water, resulted in elimination of the methanesulfine group to afford N-methyl-p-toluidine as the major product. / M.S.

Amino polyaromatic hydrocarbons (amino PAHs) and nitro polyaromatic hydrocarbons (nitro PAHs), two highly mutagenic classes of compounds, are proposed to be metabolized to an electrophilic aryl nitrenium ion which attacks DNA and forms DNA adducts. A structure-activity relationship was investigated between aryl nitrenium ion stability and mutagenic activity. Arylnitrenium stability for seventeen amino PAHs was measured using electron ionization mass spectrometry. The ratio of [M-1]⁺/M⁺ intensities was used as an indicator of nitrenium ion stability. These values were then compared to mutagenicity data from the Ames Salmonella assay. Physical descriptors of isomer classification (based on ring size and shape) and isomer position of substitution were used in the statistical analysis. A strong correlation between mutagenicity, isomer classification, and stability was found (r²= 0.899 and p<0.001). This finding agrees with reported literature that the compound's physical features of ring size and position of substitution affect mutagenicity. A high correlation of r² = 0.955 was found between mutagenicity, isomer classification, and stability for three sets of isomers: 1- and 2-aminoanthracene, 1- and 2-aminonaphthalene, and 1- and 2-aminofluorene. The nitrenium ion formed from the 2 position was found to be less stable and more mutagenic than the 1 position. This finding agrees with correlations found between mutagenicity and LUMO (Lowest Unoccupied Molecular Orbital) calculations of the electrophilicity of the nitrenium ion. These results indicate that the less stable the nitrenium ion within an isomer pair, the more mutagenic the amino PAH. / Master of Science

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.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第24100号 / 工博第5022号 / 新制||工||1748(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 米田 稔, 教授 清水 芳久, 教授 松井 康人 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

The author was desirous of investigating the properties of "quinolineins" as compared with those of corresponding phthaleins. As there was no quinolinic acid available in the laboratory, an attempt was made to prepare it. It was found that most methods gave very small yields, with the exception of one. / Thesis / Master of Arts (MA)

Insensitive munition compounds (IMC) are an emerging class of explosives that are less susceptible to accidental explosions compared to the conventional explosives they will be replacing. An IMC that has been incorporated in several explosives formulations is 2,4-dinitroanisole (DNAN). As the manufacture, storage, and use of these compounds increases, the expected releases in natural and engineered systems might pose an environmental hazard to public health and ecosystems. To date there is little information on the environmental fate and toxicology of DNAN. However, nitroaromatic compounds are known to be toxic, mutagenic and difficult to completely biodegrade. In order to study the fate and (bio)transformation of DNAN, microcosm studies with soils and anaerobic wastewater sludge were performed to determine (bio)transformation pathways and key factors influencing (bio)conversion. Transformation was enhanced in anaerobic conditions, in particular when exogenous electron donor was added. Abiotic transformation (in heat-killed soil) was also significant and dominated transformation reactions in soils that were not amended with exogenous electron donor. The organic carbon content of soils was a key factor that correlated to the anaerobic biotransformation rate. Having identified (bio)transformation products using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry, an overall pathway of (bio)transformation was devised and consistent with nitro-group reduction to form aromatic amines. During the nitro-group reduction, reactive products (e.g. nitroso-intermediates) coupled with amines to form azo-dimers and oligomers. Subsequent transformation pathways included N-alkylation, N-acetylation, and stepwise demethoxylation of these oligomers. The assessment of the toxicity of DNAN and its (bio)transformation products was performed utilizing microbial toxicity assays and ecotoxicity evaluation with zebrafish (Danio rerio) embryos. Overall DNAN severely inhibited methanogens (IC₅₀ = 41 μM ), the bioluminescent marine bacterium Aliivibrio fischeri utilized in the Microtox test (IC₅₀ = 57 μM), and nitrifiers (IC₅₀ = 49 μM). Reduced aromatic amine products in general were less toxic than DNAN with the exception of 2-methoxy-5-nitroaniline and 3-nitro-4-methoxyaniline, which were similar in toxicity to some of the test organisms as DNAN. Azo-oligomer surrogates were as toxic or more toxic than DNAN, although at trace levels they significantly stimulated activity. N-acetylated amines were found to have by far the lowest toxicity to microorganisms. In zebrafish embryos, the (bio)transformation product or surrogates 3-nitro-4-methoxyaniline and 2,2'-dimethoxy-4,4'-azodianiline caused developmental abnormalities (each with lowest observable effect level of 6.4 μM). An integrated approach which monitored (bio)transformation product mixture profile in parallel with their toxicity to microbial and zebrafish toxicity was used to characterize toxicity during the time course of the anaerobic (bio)transformation of DNAN. Enhanced inhibition of methanogenic activity and zebrafish mortality were associated with the onset of dimer formation indicating they were being mostly impacted by reactive intermediates formed early in the biotransformation of DNAN. Further accumulation of oligomers was associated with a decrease toxicity. On the other hand, A. fischeri bioluminescence became more and more inhibited as the oligomers formed, indicating different responses depending on target organism. Taken globally, the results indicate that DNAN can be readily transformed in soils and wastewater sludge forming both highly toxic (e.g. azo-oligomers) and non-toxic intermediates (e.g. N-acetylated 2,4-diaminoanisole). Depending on target organism, the prolonged formation of oligomer mixtures either resulted in detoxification or recovery of activity.

<P align=justify>Aromatische Amine und Amide (aAA) sind aufgrund ihrer starken Verbreitung in der menschlichen Umwelt und ihres kanzerogenen Potenzials von großer toxikologischer Bedeutung. Die Kanzerogenität der aAA wird durch die Mutagenität hochreaktiver Stoffwechselprodukte vermittelt, die in zwei sequenziellen katalytischen Reaktionen entstehen. Die erste ist meistens eine <I>N</I>-Hydroxylierung, die oft durch Cytochrom P450 1A2 (CYP1A2) katalysiert wird. Daran schließt sich eine <I>O</I>-Konjugation durch Sulfotransferasen (SULT) oder <I>N</I>-Acetyltransferasen (NAT) an. Die Bioaktivierung ist ein kritischer Parameter für die Übertragbarkeit von Ergebnissen aus Tiermodellen auf den Menschen. </P><P align=justify>Rekombinante <I>in vitro</I> Systeme, die fremdstoffmetabolisierende Enzyme verschiedener Spezies exprimieren, ermöglichen die vergleichende Untersuchung der Bioaktivierung im Menschen und in Versuchstieren. Ziel des Projektes war die Aufklärung der Bioaktivierung der aAA durch humane Enzyme. Im Vordergrund stand die Untersuchung der Rolle humaner SULT in diesem Prozess. Es wurden rekombinante <I>in vitro</I> Systeme, konstruiert, die CYP1A2 und SULT des Menschen koexprimieren. SULT-cDNAs wurden in den Säugerzell Expressionsvektor pMPSV kloniert und in Standardindikatorzellen für Mutagenitätsuntersuchungen (V79 Zellen aus dem Chinesischen Hamster) transfiziert. Das Expressionsniveau von CYP1A2 und SULT wurde mittels Immunblotanalyse und radiometrischen Aktivitätsmessungen charakterisiert. In den rekombinanten Zellen wurden vier aAA als Modellsubstanzen (2-Acetylaminofluoren, 2-Aminoanthracen, 3&prime;-Methyl-4-dimethylaminoazobenzol, 2,4-Diaminotoluol) auf ihre Mutagenität am <I>hprt</I>-Locus hin untersucht.</P><P align=justify>Die aAA waren in Zellen, die keine rekombinanten Enzyme oder lediglich CYP1A2 exprimierten, nicht mutagen. In Zellen, die CYP1A2 und SULT der Subfamilie 1A koexprimierten, erzeugten sie bereits in geringen Konzentrationen klare mutagene Effekte (0,3 &#181;M für 2-Acetylaminofluoren <br /> und 3&prime;-Methyl-4-dimethylaminoazobenzol; 0,1 &#181;M für 2-Aminoanthracen; 10 &#181;M für 2,4-Diaminotoluol). Die stärkste Aktivierung von 2-Acetylaminofluoren und 3&prime;-Methyl-4-dimethylaminoazobenzol erfolgte in der Zelllinie, die CYP1A2 und SULT1A2 koexprimierte; die stärkste Aktivierung von 2,4-Diaminotoluol und 2-Aminoanthracen erfolgte in der Zelllinie, die CYP1A2 und SULT1A1 koexprimierte. </P><P align=justify>Sowohl SULT1A1 als auch SULT1A2 sind im Menschen genetisch polymorph. Ein unterschiedlich starkes Aktivierungspotenzial der Alloenzyme könnte eine individuell unterschiedliche Suszeptibilität für die durch aAA ausgelöste Kanzerogenese bedingen. In HPRT-Mutationsuntersuchungen mit rekombinanten Zellen zeigten die allelischen Varianten der SULT1A2 starke Unterschiede in ihrem Aktivierungpotenzial. Nur in der Zelllinie, die das Alloenzym SULT1A2*1 mit CYP1A2 koexprimierte, wurde 2-Acetylaminofluoren zum Mutagen aktiviert. Zur Aktivierung von 3&prime;-Methyl-4-dimethylaminoazobenzol waren jedoch sowohl das Alloenzym SULT1A2*1 als auch das Alloenzym SULT1A2*2 in der Lage. Die Alloenzyme der SULT1A1 zeigten ein ähnlich gutes Aktivierungspotenzial für aAA. </P><P align=justify>In früheren Studien wurde gezeigt, dass die SULT1C1 der Ratte eine wichtige Rolle bei der Aktivierung der aAA in dieser Spezies spielt. Dahingegen war die humane SULT1C1 nicht in der Lage die untersuchten aAA zu aktivieren. Die Kenntnis solcher Spezieunterschiede könnte wichtig sein um unterschiedliche Organotropismen aAA in Menschen und Tiermodellen zu erklären, da SULT mit starker Gewebespezifität exprimiert werden und das Expressionsmuster für die einzelnen SULT-Formen in Menschen und Ratten sich stark unterscheidet.</P><br> / <P align=justify>Aromatic amines and amides (aAA) represent a group of chemicals with great toxicological importance due to their wide distribution in the environment and their carcinogenic potency. The carcinogenicity of aAA is mediated by the mutagenic action of highly reactive metabolites. They are frequently formed by <I>N</I>-hydroxylation of the exocyclic amino group, usually catalysed by cytochrome P450 1A2 (CYP1A2) and subsequent <I>O</I>-conjugation by phase-II enzymes e.g. sulfotransferases (SULT) or <I>N</I>-acetyltransferases. </P> <P align=justify>The bioactivation constitutes a critical parameter for the transfer of results from animal models on man. Recombinant <I>in vitro</I> systems expressing xenobiotic metabolizing enzymes of different species allow the comparative study of the bioactivation in humans and animal models. <BR>The aim of this project was to elucidate the bioactivation of aAA by human xenobiotic enzymes. The investigation focused on the role of SULT in this process. SULT-cDNAs were cloned into the mammalian expression vector pMPSV and transfected in V79 Chinese Hamster cells, which represent standard indicator cells for mutagenicity tests. Selected SULT-cDNAs were also co-expressed with human CYP1A2. These cells were able to catalyse internally both enzymatic reactions that are necessary for the bioactivation of aAA. The expression level of CYP1A2 and SULT in the co-expressing cell clones was characterised by immunoblot analysis and radiometric SULT-activity measurement. The mutagenicity of four aAA model compounds, 2-aminoanthracene, 2-acetylaminofluorene, 3'-methyl-4-dimethylaminoazobenzene and 2,4-diaminotoluene, at the <I>hprt</I> locus of the recombinant cell lines was investigated.</P><br /> <P align=justify>These aAA were not or only marginally mutagenic in wild type cells or in recombinant cells expressing CYP1A2 alone. If CYP1A2 was co-expressed with SULT forms of the 1A subfamily clear mutagenic effects occured in low concentrations of the aAA (0,3 &#181;M for 2-acetylaminofluorene and 3&prime;-methyl-4-dimethylaminoazobenzene; 0,1 &#181;M for 2-aminoanthracene; 10 &#181;M for 2,4-diaminotoluene). The strongest activation of 2-acetylaminofluorene and 3'-methyl-4-dimethylaminoazobenzene was mediated by SULTA2 and of 2-aminoanthracene and 2,4-diaminotoluene by SULT1A1. </P><br /> <P align=justify>SULT1A1 and SULT1A2 are expressed polymorphically in humans. Differences in the activation potency of distinct alloenzymes for aAA may cause divergent individual susceptibilities for cancer induced by aAA. Briefly, the allelic variants of SULT1A2 showed substantial differences regarding their activation potencies for the investigated aAA. Only alloenzyme SULT1A2*1 was able to activate 2-acetylaminofluorene to a mutagen whereas 3&prime;-methyl-4-di-methylaminoazobenzene was activated by alloenzymes SULT1A2*1 and SULT1A2*2. The investigated alloenzymes of SULT1A1 showed equal activation potencies for aAA. </P><br /> <P align=justify>In previous studies it had been shown that the SULT1C1 plays an important role in the activation of aAA in rats. However, the human SULT1C1 was not able to activate the investigated aAA in the study presented here. Such species differences might be important for the elucidation of divergent organotropisms of aAA in humans and animal models, since SULT are expressed with strong tissue specificities and the pattern of expression in humans and rats is severely different.</P><br>

We previously observed that postnatal exposure of mice to the procarcinogen 4-aminobiphenyl (ABP) produced liver tumors only in wild-type males, while arylamine N-acetyltransferase deficient males and females of either strain were protected. Others have also observed a sex difference in liver tumors in mice using the procarcinogen diethylnitrosamine (DEN). Reasons for these sex and strain differences are unclear, but differences in acute hepatotoxicity and inflammation may be involved. In this thesis we found that neither ABP nor DEN produced overt hepatotoxicity in postnatally exposed mice, and only DEN caused an increase in levels of the pro-inflammatory cytokine interleukin-6 but was not sex-dependent. The lack of sex difference suggests that sex hormone modulation of inflammation following sexual maturation might favour growth of initiated cells in males. However, the lack of detectable inflammation following ABP exposure may be due to localized responses, or that inflammation may be a DEN-specific effect.

We previously observed that postnatal exposure of mice to the procarcinogen 4-aminobiphenyl (ABP) produced liver tumors only in wild-type males, while arylamine N-acetyltransferase deficient males and females of either strain were protected. Others have also observed a sex difference in liver tumors in mice using the procarcinogen diethylnitrosamine (DEN). Reasons for these sex and strain differences are unclear, but differences in acute hepatotoxicity and inflammation may be involved. In this thesis we found that neither ABP nor DEN produced overt hepatotoxicity in postnatally exposed mice, and only DEN caused an increase in levels of the pro-inflammatory cytokine interleukin-6 but was not sex-dependent. The lack of sex difference suggests that sex hormone modulation of inflammation following sexual maturation might favour growth of initiated cells in males. However, the lack of detectable inflammation following ABP exposure may be due to localized responses, or that inflammation may be a DEN-specific effect.