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京都大学 / 新制・課程博士 / 博士(工学) / 甲第23927号 / 工博第5014号 / 新制||工||1783(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 松田 建児, 教授 杉野目 道紀, 教授 生越 友樹 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Cross-conjugation in molecules has been seen in nature for many years but was not pursued due to the difficulty of their synthesis and their lack of stability. Recently, it has become more interesting due to the rise of molecular electronics. Linear conjugation serves well as the wires to conduct electrons, but molecular electronics are made up of more than just wires. Molecules are needed that possess an on/off switch that can allow or deter conduction. Cross-conjugated systems show promise in their ability to be turned on or off from external stimuli. Pentacene quinone is a well-known cross-conjugated molecule that already shows promise in the field of molecular semiconductors. By synthetically fusing the pentacene quinone to the β, β' positions of a porphyrin, it has been shown that both the solubility and stability have been greatly improved. This has allowed us to pursue functionalization of the quinone moiety. Several new cross-conjugated pentacene quinone fused porphyrin systems were synthesized and studied. It was found that cross-conjugated platinum porphyrins show enhanced fluorescence, and phosphorescence that shifts toward the Near IR. Additionally, strong electron withdrawing groups show potential in charge transfer, and a lower HOMO to LUMO gap, while mildly withdrawing groups have a higher HOMO to LUMO gap. Furthermore, a new method to introduce halogenated methine bridges at the pentacene quinone core was developed, thus opening the doors to new polycyclic aromatic hydrocarbons to be synthesized and studied.

In recent years the nitrated polycyclic aromatic hydrocarbons (nitroPAH's) have been recognized as powerful mutagens in the Ames Salmonella test. Most nitroPAH’s are direct-acting mutagens in the Ames test i.e. they induce mutation in the absence of S9, and appear to be activated through nitroreduction by bacterial enzymes. Others, however, such as 3-nitroperylene, are indirect-acting mutagens and show maximum activity only when S9 is present. Studies using the Ames test have indicated that the cytochrome P-450-dependent mixed function oxidase system of S9 is responsible for the activation of 3-nitroperylene to mutagenic species. However, the pattern of P-450 isozymes involved in this process appears to be different from that involved in the conversion of most PAH's, such as the standard indirect-acting mutagen benzo(a)pyrene (B(a)P), to proximate mutagens. 6-NitroB(a)P, in contrast, behaves in an analogous manner to its parent hydrocarbon. Using appropriate Salmonella mutants, the activation of 3-nitroperylene was found to require bacterial involvement, although the nature of the bacterial contribution has yet to be determined. Studies with other mutants have indicated that nitroreduction, at least as a primary activation step, does not appear to be important. Incubation of 3-nitroperylene with high concentrations of S9 led to the formation of a number of metabolites, of which phenolic derivatives were prominent. In addition, S9-derived microsomes were able to catalyse the conversion of 3-nitroperylene to species which were able to bind to protein and DNA. Under the conditions employed in these binding studies, 3-nitroperylene appears to be acting like a simple PAH, and such experiments with very high concentrations of liver protein may be unrepresentative of the processes responsible for the mutagenesis of the compound. / Thesis / Master of Science (MSc)

Background: The International Agency for Research on Cancer has classified diesel exhaust as a carcinogen, and specific polycyclic aromatic hydrocarbons (PAHs) as probable carcinogens. Urban air pollution is one source of PAH exposure. These facts provided motivation to pursue three thesis objectives: 1) to critically review environmental inequity research in Canada and methods used in previous studies; 2) to determine associations between socio-demographic factors and residential traffic exposure; and, 3) to assess correlations between two PAH biomarkers and their relationship with a newer geographic information system (GIS) method (a proxy of PAH exposure measurement), and explore determinants of these two PAH biomarkers. Methods: The first objective was achieved through an extensive and critical literature review. The second and third objectives were achieved through conducting a cross-sectional study in Montreal where 107 female and 93 male volunteers completed a questionnaire and provided a urine sample for measurement of 1-hydroxypyrene (1-OHP) and 1-hydroxypyrene glucuronide (1-OHPG). GIS-based distance-weighted traffic density (DWTD) at participants’ residences and time- and distance-weighted traffic density (TDWTD) for all participants’ locations in the 48 hours before urine collection were calculated. Results: Participants with lower household income and unemployment/student status were more likely to be exposed to higher traffic density at their residence. DWTD was related to self-reported living within 100 meters of highway/major roads. Detection rates for the two biomarkers were over 95%, and females have higher 1-OHP and 1-OHPG levels (exp β: 1.56, 95% CI: 1.17 to 2.09; exp β: 1.49, 95% CI: 1.05 to 2.11, respectively) than males. Smoking in the 48-hour period before urine collection significantly predicted levels of biomarkers, and among non-smokers barbecued/grilled meat consumption was implicated in increases in 1-OHP. Conclusions: Those with lower household income and unemployment/student status experienced increased traffic exposure, while education, marital status and ethnicity were not associated with traffic exposure. While higher levels among females and an interaction with sex needs further study, PAH biomarkers are useful in capturing recent PAH exposure from smoking, and barbecued/grilled meat consumption. PAH biomarkers can be easily used in epidemiologic studies to assess general population exposures. / Thesis (Ph.D, Community Health & Epidemiology) -- Queen's University, 2013-07-30 10:41:50.321

Contamination of soil with Polycyclic Aromatic Hydrocarbons (PAHs) is a serious environmental issue because some PAHs are toxic, carcinogenic and mutagenic. Bioremediation is a promising option to completely remove PAHs from the environment or convert them to less harmful compounds. One of the main challenges in bioremediation of PAHs in a conventional roller bioreactor is the limitation on mass transfer due to the strong hydrophobicity and low water solubility of these compounds. To address this challenge, a novel bead mill bioreactor (BMB) was developed by Riess et al. (2005) which demonstrated a significant improvement in the rates of mass transfer and biodegradation of PAHs. <p> In this study, to further improve mass transfer rates, baffles have been installed in both the conventional and bead mill bioreactors. Mass transfer rates of 1000 mg L-1 suspended naphthalene, 2-methylnaphthalene and 1,5-dimethylnaphthalene, three model compounds of PAHs, have been investigated in four bioreactors: conventional (control), baffled, BMB and baffled bead mill bioreactors. The baffled bioreactor provided mass transfer coefficients (KLa) that were up to 7 times higher than those of the control bioreactor. <p> Bioremediation of suspended naphthalene or 2-methylnaphthalene as a single substrate and their mixtures was studied using the bacterium <i>Pseudomonas putida </i>ATCC 17484. Both baffled and bead mill bioreactors provided maximum bioremediation rates that were 2 times higher than the control bioreactor. The maximum bioremediation rates of 2-methylnaphthalene were further increased in the presence of naphthalene by a factor of 1.5 to 2 compared to the single substrate. <p> Another rate-limiting step for bioremediation of PAH-contaminated soil is the strong sorption between the contaminant and soil. To find out the effect of sorption on the bioavailability of naphthalene, the appropriate sorption isotherms for three types of soils (sand, silt and clay) have been determined. It was observed that the sorption capacity of soils for naphthalene was proportional to the organic carbon content of the soils. The mass transfer of soil-bound naphthalene from the artificially prepared contaminated soils with short contamination history to the aqueous phase was studied in both the control and bead mill bioreactors. It was observed that the mass transfer was unexpectedly fast due to the increased interfacial surface area of naphthalene particles and the weak sorption between naphthalene and soils. It was concluded that artificially, naphthalene contaminated soils would likely not be any more difficult to bioremediate than pure naphthalene particles.

Contamination of soil with Polycyclic Aromatic Hydrocarbons (PAHs) is a serious environmental issue because some PAHs are toxic, carcinogenic and mutagenic. Bioremediation is a promising option to completely remove PAHs from the environment or convert them to less harmful compounds. One of the main challenges in bioremediation of PAHs in a conventional roller bioreactor is the limitation on mass transfer due to the strong hydrophobicity and low water solubility of these compounds. To address this challenge, a novel bead mill bioreactor (BMB) was developed by Riess et al. (2005) which demonstrated a significant improvement in the rates of mass transfer and biodegradation of PAHs. <p> In this study, to further improve mass transfer rates, baffles have been installed in both the conventional and bead mill bioreactors. Mass transfer rates of 1000 mg L-1 suspended naphthalene, 2-methylnaphthalene and 1,5-dimethylnaphthalene, three model compounds of PAHs, have been investigated in four bioreactors: conventional (control), baffled, BMB and baffled bead mill bioreactors. The baffled bioreactor provided mass transfer coefficients (KLa) that were up to 7 times higher than those of the control bioreactor. <p> Bioremediation of suspended naphthalene or 2-methylnaphthalene as a single substrate and their mixtures was studied using the bacterium <i>Pseudomonas putida </i>ATCC 17484. Both baffled and bead mill bioreactors provided maximum bioremediation rates that were 2 times higher than the control bioreactor. The maximum bioremediation rates of 2-methylnaphthalene were further increased in the presence of naphthalene by a factor of 1.5 to 2 compared to the single substrate. <p> Another rate-limiting step for bioremediation of PAH-contaminated soil is the strong sorption between the contaminant and soil. To find out the effect of sorption on the bioavailability of naphthalene, the appropriate sorption isotherms for three types of soils (sand, silt and clay) have been determined. It was observed that the sorption capacity of soils for naphthalene was proportional to the organic carbon content of the soils. The mass transfer of soil-bound naphthalene from the artificially prepared contaminated soils with short contamination history to the aqueous phase was studied in both the control and bead mill bioreactors. It was observed that the mass transfer was unexpectedly fast due to the increased interfacial surface area of naphthalene particles and the weak sorption between naphthalene and soils. It was concluded that artificially, naphthalene contaminated soils would likely not be any more difficult to bioremediate than pure naphthalene particles.

<p>Polycyclic aromatic hydrocarbons (PAHs) are important contaminants that are found in increasing amounts in aquatic ecosystems. One of the sites that that is contaminated by extremely high levels of PAHs is the Atlantic Wood Industries Superfund Site on the Elizabeth River, VA. The Atlantic killifish (<italic>Fundulus heteroclitus</italic>) from this site exhibit increased levels of antioxidants, increased sensitivity to hypoxia, and increased expression of enzymes involved in glycolytic metabolism, suggesting that exposure to PAHs in the environment may induce changes in mitochondrial function and energy metabolism. Normal mitochondrial activity is crucial to an organism's survival. Therefore, gaining a better understanding of how mitochondria are affected by environmental contaminants such as PAHs is a pressing research objective. As a first step in understanding changes in cellular bioenergetics of aquatic organisms in response to PAHs, this research focused on the effect of benzo[a]pyrene (BaP), a representative PAH, on mitochondria the killifish model and on comparison of the mitochondria of the PAH-adapted killifish from the Elizabeth River Superfund Site to reference site fish. In order to assess the extent of mitochondrial DNA damage in the killifish, a PCR-based assay (LA-QPCR) for nuclear and mitochondrial DNA (nDNA, mtDNA) damage was adapted to this model and validated in with UV exposure and BaP exposure studies, as well as with <italic>ex situ</italic> study examining DNA damage in killifish inhabiting the Elizabeth River Superfund site. With the newly adapted LA-QPCR, mtDNA and nDNA damage in the killifish from the Elizabeth River Superfund site and from a reference site (King's Creek, VA) that were treated with BaP were examined. Similar increases in mitochondrial and nuclear DNA damage were observed in King's Creek fish treated with BaP. Killifish from the Elizabeth River showed high levels of basal nDNA and mtDNA damage compared to fish from the reference site, but the level of damage induced due to BaP treatment was much lower in Elizabeth River killifish. Laboratory-reared offspring from both populations showed increased BaP-induced damage in mtDNA, relative to nDNA. Similar to the adult experiment, the Elizabeth River larvae had higher levels of basal DNA damage than those from the reference site, but were less impacted by BaP exposure. Results suggest that BaP exposure can have important energetic consequences and that multi-generational exposure in the wild may lead to adaptation that dampens DNA damage arising from BaP exposure. Since the toxic effects of many PAHs are the result of bioactivation by cytochrome P4501A (CYP1A), the existence of enzymes that can potentially metabolize PAHs in mitochondria was verified. Using Western blot, protein similar in size to microsomal CYP1A was identified with monoclonal antibody against scup CYP1A in the mitochondrial fraction from adult male killifish livers. The size of the protein in the mitochondria was the similar to that of microsomal CYP1A. Fish dosed with BaP had increased EROD activity in the liver mitochondrial fraction compared to controls. In killifish larvae dosed with BaP and benzo[k]fluoranthene (BkF), CYP1A protein levels as well as enzyme activity were elevated. However, fish from the Elizabeth River Superfund site showed recalcitrant mitochondrial CYP1A protein levels and enzyme activity in a similar manner to microsomal CYP1A. Finally, the hypothesis that energy metabolism of BaP-treated fish may be different from the control group and that killifish from the Elizabeth River Superfund site may also have altered energy metabolism compared to reference site fish was tested. Respiration of killifish embryos treated with BaP from both populations was measured. Compared to the King's Creek control fish, all other treatment groups showed decrease in oxygen consumption, indicating lower respiration rate. However, when activities of key enzymes involved in glycolysis (PK) and anaerobic metabolism (LDH) in adult killifish liver and muscle were measured, no differences in the enzyme activities were observed in BaP-treated group compared to the control group. Moreover, metabolomic analysis on BaP treated King's Creek and Elizabeth River killifish showed no difference in the profile in all four treatment groups. The findings in this thesis contribute to the understanding of how BaP, a common environmental pollutant in the aquatic ecosystem, targets the mitochondria in fish model. Nevertheless, deeper examination of how BaP may impact mitochondrial function in killifish and potentially influence adaptation of killifish at a highly contaminated site is necessary. Further studies will elucidate whether such impacts can potentially affect the energy budget and organism level fitness in populations in the wild.</p> / Dissertation

<p>Chronic exposure to toxicant mixtures is a serious threat to environmental and human health. It is especially important to understand the effects of these exposures for contaminants, such as polycyclic aromatic hydrocarbons (PAHs), which are toxic, ubiquitous, and increasingly prevalent. Furthermore, estuarine systems are of particular concern, as they are highly impacted by a wide variety of pollutants; fish there are often exposed to some of the highest levels of contaminants of any vertebrate populations, along with other stressors such as fluctuations in water level, dissolved oxygen, and temperature. A population of <italic>Fundulus heteroclitus</italic> (the Atlantic killifish or mummichog, hereafter referred to as killifish) inhabits a Superfund site heavily contaminated with a mixture of PAHs from former creosote operations; they have developed resistance to the acute toxicity and teratogenic effects caused by the mixture of PAHs in sediment from the site. The primary goal of this dissertation was to better understand the mechanism(s) by which Elizabeth River killifish resist the developmental toxicity of a complex mixture of PAHs and to investigate the tradeoffs associated with this resistance. Because the aryl hydrocarbon receptor (AHR) pathway plays an important role in mediating the effects of PAHs, one major hypothesis of my work was that suppression of the AHR response plays an important role in the resistance of Elizabeth River killifish. For this reason, investigation of the activation of the AHR pathway, as measured by CYP induction, is a unifying thread throughout the work. Another major hypothesis of this work is that adaptation to PAHs has secondary consequences for Elizabeth River killifish, such as altering their response to other xenobiotics. To investigate these hypotheses, a series of experiments were carried out in PAH-adapted killifish from the Elizabeth River and in reference fish. The morpholino gene knockdown technique was modified for use in killifish; we demonstrated that CYP1A knockdown exacerbates PAH-driven cardiac teratogenesis and AHR2 (but not AHR1) knockdown rescues PAH-driven cardiac teratogenesis. Using acute toxicity tests of larval killifish, we showed that Elizabeth River killifish are less sensitive than reference larvae to chlorpyrifos, permethrin, and carbaryl. These results demonstrated that the adaptation was able to protect from multiple xenobiotics, not just PAHs. Using the in ovo ethoxyresorufin-o-deethylase (EROD) assay and a subjective cardiac deformity screen, we showed that the adaptation was spread throughout the killifish subpopulations of the Elizabeth River estuary. However, the adaptive response varied greatly among the subpopulations, which showed that AHR pathway suppression was not required for some level of protection from PAH toxicity. Finally, using the quantitative real-time PCR, the EROD assay, and cardiac deformity screening, we demonstrated that the adaptation was heritable for two generations of fish reared in clean laboratory conditions. The findings in this dissertation will help to reveal how mixtures of PAHs exert their toxic action in un-adapted organisms. Furthermore, these studies will hopefully demonstrate how chronic exposure to PAH mixtures can affect organisms at the population and even evolutionary level. Perhaps most importantly, they will help us to better predict the consequences and tradeoffs for organisms and populations persisting in PAH-contaminated environments.</p> / Dissertation

The presence of complex mixtures of compounds in ambient air, many of which are either unknown or uncharacterised makes an assessment of risk associated with these exposures problematic. Bioanalytical methods can provide an integrative assessment of complex mixture potency for specific mechanisms of toxicity within these contexts. The aim of this study was to evaluate the suitability of monitoring ambient air exposures as sampled by (polyurethane foam) PUF passive air samplers (PAS) using effect based techniques (bioanalytical methods). Passive samplers have the advantage of offering a low-tech inexpensive monitoring strategy which can thereby increase sampling capacity across a broader range of scenarios simultaneously. One challenge posed by the application of passive samplers in particular for these assessments has been the expression of potency estimates in relatively non-comparable terms specific to a given dose of the sampler or for a specific deployment period. The project was therefore designed in order to address these aims and previously identified challenges by investigating the applicability of these techniques for: monitoring in both indoor and outdoor air, the determination of seasonal exposure gradients; the determination of exposure gradients in different locations (urban capitals, regional centres, background); and the application of in-situ calibration to provide comparable effect measurements in terms of equivalent reference compound air concentrations. Air sampled using PUF PAS was monitored for its capacity to induce biological responses which are mechanistically relevant to critical health endpoints in these scenarios. The mechanisms assessed included genotoxicity (DNA damage – umuC assay), Aryl hydrocarbon receptor (AhR) activity (CAFLUX assay), and estrogenicity (ESCREEN assay). The findings from this effect based monitoring revealed that the level of biological response measured changes with the exposure scenario (indoor vs. outdoor; summer vs. winter; urban capital cities vs. background locations). Estrogenicity for example assessed as estradiol equivalent air concentrations (E Eq BIO) averaged 54 pg.m-3 (1.5 - 185 pg.m-3) in indoor air, while samples from ambient air were found to be not estrogenic. Total aryl hydrocarbon receptor (AhR) activity assessed as 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalent air concentrations (TCDD Eq BIO) averaged 4.1 pg.m-3 (1.3 – 7.2 pg.m-3) in indoor air while samples from ambient air averaged 15 pg.m-3 (1.5 – 46 pg.m-3)in summer and 53 pg.m-3 (2.2 – 251 pg.m-3) in winter. The relationship for both direct (-S9) and indirect (+S9) acting genotoxicity and AhR activity were found to be relatively consistent with respect to both season (elevated in winter) and location (elevated in urban capital cities). Overall suitable techniques were developed for combining passive sampling with multiple end-point toxicity testing and it was demonstrated that these techniques may be applied across different exposure scenarios. During the course of this method development and interpretation process a range of limitations were identified relating to: the use and application of effect based techniques to monitor environmental samples; the use of passive samplers within this context specifically; and also with the application of in-situ calibration techniques to passive samplers to improve the comparability of these assessments.