Thesis (Ph. D.)--University of Washington, 2008. / Vita. Includes bibliographical references (leaves 186-207).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

No description available.

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)

Thesis (MEng)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: The expansion of the global fuels industry has caused an increase in the quantity of hydrocarbons produced as a by-product of refinery gas-to-liquid processes. Conversion of hydrocarbons to higher value products is possible using bioprocesses, which are sustainable and environmentally benign. Due to the deficiency of oxygen in the alkane molecule, the supply of sufficient oxygen through aeration is a major obstacle for the optimization of hydrocarbon bioprocesses. While the oxygen solubility is increased in the presence of hydrocarbons, under certain process conditions, the enhanced solubility is outweighed by an increase in viscosity, causing a depression in overall volumetric oxygen transfer coefficient (KLa). The rate at which oxygen is transferred is defined in terms of a concentration driving force (oxygen solubility) and the overall volumetric oxygen transfer coefficient (KLa). The KLa term comprises an oxygen transfer coefficient (KL) and the gas-liquid interfacial area (a), which are dependent on the uid properties and system hydrodynamics. This behaviour is not well understood for hydrocarbon bioprocesses and in a bubble column reactor (BCR). To provide an understanding of oxygen transfer behaviour, a model hydrocarbon bioprocess was developed using a BCR with a porous sparger. To evaluate the interfacial area, the Sauter mean bubble diameter (D32) was measured using an image analysis algorithm and gas holdup (ϵG) was measured by the change in liquid height in the column. Together the D32 and ϵG were used in the calculation of interfacial area in the column. The KLa was evaluated with incorporation of the probe response lag, allowing more accurate representation of the KLa behaviour. The probe response lag was measured at all experimental conditions to ensure accuracy and reliability of data. The model hydrocarbon bioprocess employed C14-20 alkane-aqueous dispersions (2.5 - 20 vol% hydrocarbon) with suspended solids (0.5 - 6 g/l) at discrete super ficial gas velocity (uG) (1 - 3 cm/s). For systems with inert solids (corn our, dp = 13.36 m), the interfacial area and KLa were measured and the behaviour of KLa was described by separation of the in uences of interfacial area and oxygen transfer coefficient (KL). To further the understanding of oxygen transfer behaviour, non-viable yeast cells (dp = 5.059 m) were used as the dispersed solid phase and interfacial area behaviour was determined. This interfacial area behaviour was compared with the behaviour of systems with inert solids to understand the differences with change in solids type. In systems using inert solids, a linear relationship was found between G and uG. An empirical correlation fo rthe prediction of this behaviour showed an accuracy of 83.34% across the experimental range. The interfacial area showed a similar relationship with uG and the empirical correlation provided an accuracy of 78.8% for prediction across the experimental range. In inert solids dispersions, the KLa increased with uG as the result of an increase in interfacial area as well as increases in KL. An increase in solids loading indicated an initial increase in KLa, due to the in uence of liquid-film penetration on KL, followed by a decrease in KL at solids loading greater than 2.5 g/l, due to diffusion blocking effects. In systems with yeast dispersions, the presence of surfactant molecules in the media inhibited coalescence up to a yeast loading of about 3.5 g/l, and resulted in a decrease in D32. Above this yeast loading, the fine yeast particles increased the apparent viscosity of the dispersion sufficiently to overcome the in uence of surfactant and increase the D32. The behaviour of G in yeast dispersions was similar to that found with inert solids and demonstrated a linear increase with uG. However, in yeast dispersions, the interaction between alkane concentration and yeast loading caused a slight increase in dispersion viscosity and therefore G. An empirical correlation to predict G behaviour with increased uG was developed with an accuracy of 72.55% for the experimental range considered. Comparison of yeast and inert solids dispersions indicated a 37.5% lower G in yeast dispersions compared to inert solids as a result of the apparent viscosity introduced by finer solid particles. This G and D32 data resulted in a linear increase in interfacial area with uG with no significant in uence of alkane concentration and yeast loading. This interfacial area was on average 6.7% lower than interfacial area found in inert solid dispersions as a likely consequence of the apparent viscosity with finer particles. This study provides a fundamental understanding of the parameters which underpin oxygen transfer in a model hydrocarbon bioprocess BCR under discrete hydrodynamic conditions. This fundamental understanding provides a basis for further investigation of hydrocarbon bioprocesses and the prediction of KLa behaviour in these systems. / AFRIKAANSE OPSOMMING: Die uitbreiding van die internasionale brandstofbedryf het 'n toename veroorsaak in die hoeveelheid koolwaterstowwe geproduseer as 'n deur-produk van raffinadery gas-tot-vloeistof prosesse. Omskakeling van koolwaterstowwe na hoër waarde produkte is moontlik met behulp van bioprosesse, wat volhoubaar en omgewingsvriendelik is. As gevolg van die tekort aan suurstof in die alkaan molekule, is die verskaffing van voldoende suurstof deur deurlugting 'n groot uitdaging vir die optimalisering van koolwaterstof bioprosesse. Terwyl die suurstof oplosbaarheid verhoog in die teenwoordigheid van koolwaterstowwe, onder sekere proses voorwaardes is die verhoogde oplosbaarheid oortref deur 'n toename in viskositeit, wat 'n depressive veroorsaak in die algehele volumetriese suurstofoordragkoëffisiënt (KLa). Die suurstof oordrag tempo word gedefinieer in terme van 'n konsentrasie dryfkrag (suurstof oplosbaarheid) en KLa. Die KLa term behels 'n suurstofoordragkoëffisiënt (KL) en die gas-vloeistof oppervlakarea (a), wat afhanklik is van die vloeistof eienskappe en stelsel hidrodinamika. Hierdie gedrag is nie goed verstaan vir koolwaterstof bioprosesse nie, asook in kolom reaktors (BCR). Om 'n begrip van suurstof oordrag gedrag te voorsien, is 'n model koolwaterstof bioproses ontwikkel met 'n BCR met 'n poreuse besproeier. Om die oppervlakarea te evalueer, is die gemiddelde Sauter deursnit (D32) gemeet deur 'n foto-analise algoritme en gas vasvanging ( G) is gemeet deur die verandering in vloeibare hoogte in die kolom. Saam is die D32 en G gebruik in die berekening van die oppervlakarea in die kolom. Die KLa is geëvalueer met insluiting van die meter se reaksie sloering, om n meer akkurate voorstelling van die KLa gedrag te bereken. Die meter reaksie sloering was gemeet op alle eksperimentele toestande om die akkuraatheid en betroubaarheid van data te verseker. Die model koolwaterstof bioproses gebruik n-C14-20 alkaan-water dispersies (2.5 - 20 vol% koolwaterstof) solide partikels (0.5 - 6 g/l) op diskrete oppervlakkige gas snelhede (1 - 3 cm/s). Vir stelsels met inerte solides (koring meel, dp = 13.36 m), is die oppervlakarea en KLa gemeet en die gedrag van KLa beskryf deur skeiding van die invloede van oppervlakarea en KL. Om die begrip van suurstof oordrag se gedrag te bevorder, is nie-lewensvatbare gisselle (dp = 5.059 m) gebruik as die verspreide solide fase en oppervlakarea is bepaal. Hierdie oppervlakarea gedrag is vergelyk met die van stelsels met inerte solides om die verskille met verandering in solide tipes te verstaan. In stelsels met inerte solides, is 'n line^ere verwantskap gevind tussen G en uG. 'n Empiriese korrelasie vir die voorspelling van hierdie gedrag is opgestel met 'n akkuraatheid van 83.34% in die eksperimentele reeks. Die oppervlakarea het 'n soortgelyke verhouding met uG en die empiriese korrelasie verskaf 'n akkuraatheid van 78,8% vir die voorspelling van oppervlakarea oor die eksperimentele reeks. In inerte solide dispersies, het die KLa toegeneem met uG as die gevolg van 'n toename in grens oppervlak asook stygings in KL. 'n Toename in solides belading het n aanvanklike styging in KLa aangedui, as gevolg van die invloed van die vloeistof-film penetrasie op KL, gevolg deur 'n afname in KL op vastestowwe ladings groter as 2.5 g/l, te danke aan diffusie blokkeer effekte. In stelsels met gis dispersies, het die teenwoordigheid van benattings molekules in die media samesmelting geïnhibeer tot 'n gis lading van ongeveer 3.5 g/l, en het gelei tot 'n afname in D32. Bo hierdie gis lading, het die fyn gis partikels die skynbare viskositeit van die verspreiding verhoog genoegsaam om die invloed van benattings molekules te oorkom en die D32 te verhoog. Die gedrag van G in gis dispersies was soortgelyk aan die van inerte solides en dui op 'n lineêre toename met uG. Maar in gis dispersies, het die interaksie tussen alkaan konsentrasie en gis lading 'n effense toename veroorsaak in die verstrooiing viskositeit en dus in G. 'n Empiriese korrelasie is ontwikkel om G gedrag te voorspel en het 'n akkuraatheid van 72,55% vir die eksperimentele verskeidenheid beskou. Vergelyking van gis en inerte patrikel dispersies wys 'n 37.5% laer G in gis dispersies in vergelyking met inerte vaste stowwe as 'n gevolg van die skynbare viskositeit bekendgestel deur fyner vastestowwe partikels. Hierdie G en D32 data het gelei tot 'n linere toename in grens oppervlak met uG met geen beduidende invloed van alkaan konsentrasie en gis lading nie. Die oppervlakarea was gemiddeld 6.7% laer as oppervlakarea gevind in inerte partikel dispersies as 'n waarskynlike gevolg van die skynbare viskositeit met fyner partikels. Hierdie studie bied 'n fundamentele begrip van die veranderlikes wat die suurstof oordrag definieer in 'n model koolwaterstof bioproses BCR onder diskrete hidrodinamiese voorwaardes. Hierdie fundamentele begrip bied n basis vir verdere ondersoek van koolwaterstof bioprosesse en en die voorspelling van KLa gedrag in hierdie stelsels.

Humans are continuously exposed to mixtures of environmental pollutants. Polycyclic aromatic hydrocarbons (PAHs), such as 2-naphthol, and heavy metals, such as lead, are some of these pollutants. Results from epidemiological studies show associations between exposure to 2-naphthol, exposure to lead, and obesity. However, the individual and combined effects of 2-naphthol and lead on fat cell development (adipogenesis) have not been directly characterized in a biological system. In this study, we evaluated the effects of 2-naphthol and/or lead on adipogenesis using mouse 3T3-L1 cells. Cells were exposed to different doses of 2-naphthol and/or lead. Induced terminal differentiation was evaluated by cell morphology, lipid production, and mRNA expression of marker genes characteristic of either early adipocyte differentiation: CCAAT-enhancer-binding protein β (C/EBPβ), insulin receptor substrate 2 (IRS2), and sterol responsive element binding protein 1 c (SREBP1c); or terminal differentiation: C/EBPα, peroxisome proliferator-activated receptor-γ (PPARγ), and fatty acid binding protein 4 (aP2). Production of antimicrobial peptide cathelicidin (Camp), which is produced by differentiating adipocytes and modulates inflammation and immunity, was also evaluated. Cell morphology changes and increased lipid accumulation indicated that, individually, 2-naphthol and lead induced 3T3-L1 differentiation; however, the highest dose of lead (10 μM) showed the lowest induction level. During terminal differentiation, 2-naphthol and low doses of lead increased C/EBPα, PPARγ, and aP2 expression, whereas 10 μM lead suppressed PPARγ and aP2. During early differentiation, 2-naphthol stimulated C/EBPβ, IRS2, and SREBP1c expression, while lead upregulated C/EBPα and aP2. The 2-naphthol/10 μM lead mixture induced a counterbalancing effect on 3T3-L1 adipogenesis, where 10 μM lead suppressed 2-naphthol-induced adipogenesis. Moreover, 2-naphthol elevated Camp expression in a dose-dependent manner, whereas lead slightly increased Camp at lower doses but suppressed it at 10 μM. The 2-naphthol/10 μM lead mixture showed no effect on Camp expression. In conclusion, 2-naphthol and low lead doses accelerate adipocyte differentiation and Camp production in 3T3-L1 cells; however, high doses of lead attenuate the induction. This effect of lead at high dose counterbalances the upregulation of adipocyte differentiation and Camp production by 2-naphthol. Together, these findings indicate that 2-naphthol and lead play potential roles in the development of inflammation and obesity.

Each year over 260,000 new cases of breast cancer will be diagnosed in the U.S. and approximately 40,000 women will die of metastatic breast cancer. The etiology of breast cancer is poorly understood and only 5 -10% of cases can be attributed to genetic factors. This suggests that the development of breast cancer may involve environmental factors including diet, lifestyle, and exposure to chemicals. Several lines of experimental and epidemiological evidence have highlighted COX-2 as a potential target for breast cancer prevention. The central hypothesis of this proposal is that activation of COX-2 transcription by epigenetic effectors can be prevented by dietary agents that target the activator protein-1 (AP-1) transcription factor and the aromatic hydrocarbon receptor (AhR). The first specific aim was to determine the mechanism through which conjugated linoleic acid (CLA) and rosmarinic acid (RA) inhibit TPA-induced COX-2 trancription. These studies documented that CLA and RA repressed COX-2 transcription by antagonizing the AP-1 transcription factor. The second specific aim was to investigate whether or not the AhR plays a role in TCDD-induced COX-2 transcription and effects of chemopreventive agents. Results indicated that AhR agonists induced the binding of the AhR to COX-2 and was prevented by CLA and the AhR antagonist, resveratrol (RES) and 3-methoxy-4-nitroflavone (3M4NF). The third specific aim was to examine the effects of AhR agonists and dietary selective AhR modulators on chromatin modifications associated with the COX-2 promoter. Chromatin immunoprecipitation (ChIP) assays revealed that the AhR is recruited to the region of the COX-2 promoter containing a xenobiotic response element and accompanied by recruitment of p300 and acetylation of histone H4. Transcriptional regulation of COX-2 by AhR agonists and dietary antagonists may also involve other post-transcriptional modifications of histones, which along with chromatin remodeling factors modulate the structure of chromatin and recruitment of RNA polymerase II. Overall, the results demonstrated that COX-2 transcription can be targeted by a variety of dietary agents that act through different mechanisms. Therefore, inhibition of transcriptional regulation of COX-2 by selected dietary factors may be a breast cancer preventive strategy that bypasses the side effects of drugs that target COX-2.

Breast cancer is the second leading cause of cancer-related death in women. Mutations in the tumor suppressor gene BRCA-1 confer a high risk of breast tumor development. However, in sporadic breast cancers, which represent 90-95% of breast cancer cases, BRCA-1 expression is downregulated in the absence of mutations in the BRCA-1 gene. This suggests that epigenetic effectors may contribute to disruption of BRCA-1 expression and the onset of mammary tumors.Prototypical environmental contaminants found in industrial pollution, tobacco smoke, and cooked foods include benzo[a]pyrene (B[a]P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which have been shown to alter mammary gland development, act as endocrine disruptors and tumor promoters. Population studies detected accumulation of TCDD in women's adipose tissue and breast milk. Moreover, sporadic breast tissue exhibited statistically significant higher levels of PAH-DNA adducts. Based on this information, we examined the effect of B[a]P on the tumor suppressor BRCA-1and observed that exposure to B[a]P led to repression of BRCA-1 transcription through a p53-dependent mechanism. We have also demonstrated that 17β-estradiol (E2) stimulated the recruitment of ERα and AP-1 family members to a region of the BRCA-1 promoter flanking an AP-1-like site. However, accumulation of p53 prevented E2-mediated BRCA-1 transcription and recruitment of ERα, potentially providing one mechanism of B[a]P-mediated repression.In addition, the effects of B[a]P and TCDD are mediated through binding of the liganded aromatic hydrocarbon receptor (AhR) to dioxin or xenobiotic-responsive elements (XRE). We have evidence that suggests B[a]P and TCDD may modulate repression of E2-stimulated BRCA-1 expression through 1) binding of the liganded AhR to XREs on the BRCA-1 promoter and 2) preventing promoter occupancy by p300 and SRC-1.Taken together, the data presented here suggest that the transcriptional regulation of BRCA-1 is complex and involves modulation of the recruitment of ERα, AhR, p53, and their cofactors. An important implication of these findings is a greater understanding of the role of ERα, AhR, and p53 in regulation of BRCA-1 which could lead to the development of therapeutic strategies that target these interactions to enhance upregulation of BRCA-1 expression in sporadic breast tumors.

Epigenetic mechanisms may contribute to reduced expression of the tumor suppressor gene BRCA-1 in sporadic breast cancers. Through environmental exposure and diet, humans are exposed to xenobiotics and food compounds that bind the aromatic hydrocarbon receptor (AhR). AhR-ligands include the dioxin-like and tumor promoter 2,3,7,8 tetrachlorobenzo-p-dioxin (TCDD). The activated AhR regulates transcription through binding to xenobiotic response elements (XRE=GCGTG) and interactions with transcription cofactors. Previously, we reported on the presence of several XRE in the proximal BRCA-1 promoter, and that the expression of endogenous AhR was required for silencing of BRCA-1 expression by TCDD. Here, we document that in estrogen receptor-alpha-positive and BRCA-1 wild-type MCF-7 breast cancer cells, the treatment with TCDD attenuated 17-beta estradiol (E2)-dependent stimulation of BRCA-1 protein and induced hypermethylation of a CpG island spanning the BRCA-1 transcriptional start site of exon-1a. Additionally, we found that TCDD enhanced the association of the AhR, DNA methyl transferases (DNMT)1, DNMT3a, and DNMT3b; methyl binding protein (MBD)2; and tri-methylated H3K9 (H3K9me3) with the BRCA-1 promoter. Conversely, the phytoalexin resveratrol, selected as a prototype dietary AhR antagonist, antagonized at physiologically relevant doses the TCDD-induced repression of BRCA-1 protein, BRCA-1 promoter methylation, and the recruitment of the AhR, MBD2, H3K9me3, and DNMTs (1, 3a, and 3b). Taken together, these observations provide evidence for a mechanistic role for AhR-agonists in establishment of BRCA-1 promoter hypermethylation and the basis for the development of prevention strategies based on AhR antagonists.

Cultured human lymphocytes may be assayed for aryl hydrocarbon hydroxylase (AHH) in whole cell preparations. The optimum assay conditions are pH 8.5, and 1.5 mM Mg++. The reaction is linear with time and cell number, and is inhibited by CO. Estradiol may inhibit induction of AHH by 3-methylcholanthrene, but is a poor competitor for the enzyme. A Caucasian population was assayed for AHH activity. The distribution was lognormal; no difference was found in cultured cells from males and females or smokers and nonsmokers. Cells from relatives of lung cancer patients showed higher activity. An American Indian population showed no difference from the Caucasian population in enzyme level. No linkage was found between AHH and 16a-hydroxylase.