Polychlorinated biphenyls (PCBs) are persistent, man-made toxicants that are linked to adverse health effects and diseases such as endocrine disruption, diabetes, obesity, cardiovascular effects, and cancer. Since their manufacturing began in 1929 for industrial use, and was banned in 1979, they have bioaccumulated in water, sediment, food, animals, humans and more. PCBs are also found in indoor air of older buildings and as inadvertent byproducts in the manufacture of paints and pigments. The lower chlorinated PCBs, those with fewer than 5 chlorine atoms, are readily metabolized to form hydroxylated PCBs (OH-PCBs) that are further converted to PCB-sulfates in reactions catalyzed by cytosolic sulfotransferases (SULTs). Steroid sulfotransferases SULT1E1 and SULT2A1 participate in regulating the homeostasis of estrogens and androgens, respectively, through the deactivation of active hormones. The estrogen sulfotransferase (SULT1E1) is also a potential key player in adipogenesis. Recent literature has shown that downregulating expression of SULT1E1 in cells derived from humans and mice caused opposite effects, where adipogenesis was inhibited or stimulated, respectively. Adipogenesis is the maturation of preadipocytes into mature adipocytes, which is regulated by peroxisome proliferating-activator γ (PPARγ). Adipocytes are a main component of adipose tissue, which is important for energy homeostasis, organ protection, and thermoregulation. Adipose tissue also secretes various cytokines such as adiponectin. Adipose tissue dysfunction can result from adipocyte dysfunction, which can be caused by alterations in cell signaling. The objective of this dissertation research was to determine if OH-PCBs and PCB-sulfates are inhibitors of SULT1E1 and SULT2A1 and if inhibition of SULT1E1 by OH-PCBs could potentially affect adipogenesis. We hypothesized that PCB metabolites would inhibit SULT1E1 and SULT2A1 and potentially affect adipogenesis in both human and murine cell models. Using purified recombinant human SULT1E1 and SULT2A1, I found that 4’-OH-PCB 3, 4-OH-PCB 8, 4-OH-PCB 11, 4’-OH-PCB 25, and 4-OH-PCB 52 were potent inhibitors of the sulfation of representative substrates (7.0 nM estradiol for SULT1E1 and 1.0 µM dehydroepiandrosterone for SULT2A1, Figures 3-3 and 3-4, respectively). Moreover, 4-OH-PCB 11 and 4-OH-PCB 52 were the most potent inhibitors of SULT1E1 and SULT2A1 with IC50 values of 7.2 nM and 1.5 μM, tables 3-1 and 3-2, respectively. The least potent inhibitor of SULT1E1 was 4’-OH-PCB 3, with an IC50 of 1300 nM. The PCB-sulfates were not potent inhibitors for either enzyme. 4-OH-PCB 11 inhibited the sulfation of estradiol in the cytosol of both pre-adipocytes and fully differentiated adipocytes (Figure 4-9). Immortalized human adipocytes were treated with 10 µM of triclosan (a known inhibitor of SULT1E1), 4’-OH-PCB 3 and 4-OH-PCB 11. Experiments included exposure to these toxicants for 1) 72 hours to preadipocytes, 2) 72 hours to preadipocytes followed by 11-day differentiation, 3) to differentiating adipocytes and for 48 hours post-differentiation. The lipid accumulation levels remained unaffected, as determined by microscopic imaging and quantification using AdipoRed. The mRNA expression levels of prominent adipogenic markers SULT1E1, PPARγ, and AdipoQ were measured using RT-Q-PCR. Changes in SULT1E1 and PPARγ expression were unaffected upon treatment before, during and after adipogenesis when compared to controls. However, the increase in AdipoQ expression was reduced upon treatment with 4-OH-PCB 11 in differentiated adipocytes and in preadipocytes exposed for 72 hours followed by 11-day differentiation (Figure 4-14). This could be an indicator of adipocyte dysfunction that was not manifested by a change in lipid accumulation. Murine 3T3-L1 cells were also treated with 10 µM of triclosan, 4’-OH-PCB 3 and 4-OH-PCB 11 for 48 hours to preadipocytes, during 8-day differentiation and for 48 hours after differentiation. The mRNA expression levels of prominent markers of cardiovascular and adipogenesis functions, ACE2, PPARγ, FABP4, and AdipoQ were measured using RT-PCR. Compared to controls, the increase in AdipoQ expression was reduced following treatment of preadipocytes with triclosan and 4-OH-PCB 11 and subsequent differentiation (Figure 5-11). The increase in PPARγ expression remained either unchanged from controls or slightly stimulated in differentiating and differentiated adipocytes (Figures 5-11 and 5-13). Angiotensin-converting enzyme 2 (ACE2) expression was decreased compared to control values, upon treatment with 4’-OH-PCB 3 (Figure 5-12), while fatty acid binding protein 4 (FABP4) expression was stimulated to the same extent across all treatment groups in differentiating adipocytes (Figure 5-12). The results, overall, show that these OH-PCBs did not affect lipid accumulation in human adipocytes, but they may affect other signaling pathways in adipogenesis. 4-OH-PCB 11 decreased adiponectin expression compared to the increase that was seen in unexposed differentiating human and mouse adipocytes. Adiponectin is secreted from adipose tissue, and this decrease could indicate a form of dysfunction. This finding is consistent with the results of the purified SULT1E1 study, where 4-OH-PCB 11 potently inhibited SULT1E1, but 4’-OH-PCB 3 did not (Figure 3-3 and Table 3-1). Thus, there is a potential for OH-PCBs to disrupt the expression of adiponectin and perhaps other vital adipokines and this could negatively affect adipose tissue function. Future studies will be needed to determine if these effects are indeed mediated by intracellular estradiol and SULT1E1. Moreover, the potential for in vivo disruption of circulating adiponectin by OH-PCBs and other toxicants that inhibit SULTs remains to be studied.

<p>Heparan sulfate (HS), a linear negatively charged polysaccharide located at the cell surface and in the extracellular matrix, interacts with, and thereby regulates the functions of numerous proteins. HS-protein interactions depend on the fine structure of HS, especially its sulfation pattern. This thesis aimed to understand how differently sulfated domains in HS are generated. Specifically, the substrate specificities of HS hexuronic acid 2-<i>O</i>-sulfotransferase (2OST) and HS glucosaminyl 6-<i>O</i>-sulfotransferases (6OSTs) were investigated. </p><p>Three different 6OSTs (6OST1-3) have been cloned and characterized. To study the mechanisms controlling 6-<i>O</i>-sulfation we incubated the recombinant purified 6-OST isoforms with different 6-<i>O</i>-desulfated poly- and oligosaccharide substrates and the active sulfate donor 3'-phosphoadenosine 5'-phospho[³⁵S]sulfate (³⁵S-labeled PAPS). All three enzymes catalyzed 6-<i>O</i>-sulfation of both <i>N</i>-acetylated (GlcNAc) as well as <i>N</i>-sulfated (GlcNS) glucosamines next to a nonreducing iduronic acid (IdoA) or glucuronic acid (GlcA). Similar specificities were demonstrated, although some differences in substrate preferences were noted.</p><p>To understand how pre-existing 2-<i>O</i>-sulfates affects 6-<i>O</i>-sulfation, 6OST2 and 6OST3 were incubated with pair-wise mixed octasaccharide substrates with different contents of 2-<i>O</i>-sulfates. The specificities for substrates with two or three 2-<i>O</i>-sulfates were higher compared to octasaccharides with no or one 2-<i>O</i>-sulfate indicating that 2-<i>O</i>-sulfate groups substantially promote the subsequent 6-<i>O</i>-sulfation. </p><p>Overexpression of the 6OSTs in a mammalian cell line resulted in increased 6-<i>O</i>-sulfation of -GlcA-GlcNS- and -GlcA-GlcNAc- sequences. The results were not isoform specific, but affected by the overexpression level. </p><p>The 2OST catalyzes 2-<i>O</i>-sulfation of both IdoA and GlcA residues, with high preference for IdoA units. To study how 2-<i>O</i>-sulfation of GlcA and IdoA is regulated, we incubated the enzyme with different substrates and ³⁵S-labeled PAPS. Our findings revealed that the 2OST almost exclusively sulfated IdoA also with a ratio of GlcA to IdoA of 99:1, suggesting that 2-<i>O</i>-sulfation of GlcA occurs before IdoA is formed.</p>

Heparan sulfate (HS), a linear negatively charged polysaccharide located at the cell surface and in the extracellular matrix, interacts with, and thereby regulates the functions of numerous proteins. HS-protein interactions depend on the fine structure of HS, especially its sulfation pattern. This thesis aimed to understand how differently sulfated domains in HS are generated. Specifically, the substrate specificities of HS hexuronic acid 2-O-sulfotransferase (2OST) and HS glucosaminyl 6-O-sulfotransferases (6OSTs) were investigated. Three different 6OSTs (6OST1-3) have been cloned and characterized. To study the mechanisms controlling 6-O-sulfation we incubated the recombinant purified 6-OST isoforms with different 6-O-desulfated poly- and oligosaccharide substrates and the active sulfate donor 3'-phosphoadenosine 5'-phospho[35S]sulfate (35S-labeled PAPS). All three enzymes catalyzed 6-O-sulfation of both N-acetylated (GlcNAc) as well as N-sulfated (GlcNS) glucosamines next to a nonreducing iduronic acid (IdoA) or glucuronic acid (GlcA). Similar specificities were demonstrated, although some differences in substrate preferences were noted. To understand how pre-existing 2-O-sulfates affects 6-O-sulfation, 6OST2 and 6OST3 were incubated with pair-wise mixed octasaccharide substrates with different contents of 2-O-sulfates. The specificities for substrates with two or three 2-O-sulfates were higher compared to octasaccharides with no or one 2-O-sulfate indicating that 2-O-sulfate groups substantially promote the subsequent 6-O-sulfation. Overexpression of the 6OSTs in a mammalian cell line resulted in increased 6-O-sulfation of -GlcA-GlcNS- and -GlcA-GlcNAc- sequences. The results were not isoform specific, but affected by the overexpression level. The 2OST catalyzes 2-O-sulfation of both IdoA and GlcA residues, with high preference for IdoA units. To study how 2-O-sulfation of GlcA and IdoA is regulated, we incubated the enzyme with different substrates and 35S-labeled PAPS. Our findings revealed that the 2OST almost exclusively sulfated IdoA also with a ratio of GlcA to IdoA of 99:1, suggesting that 2-O-sulfation of GlcA occurs before IdoA is formed.

No description available.

<p>Polybrominated diphenyl ethers (PBDEs) are a class of brominated flame retardants (BFRs) that have been heavily used in consumer products such as furniture foams, plastics, and textiles since the mid-1970’s. BFRs are added to products in order to meet state flammability standards intended to increase indoor safety in the event of a fire. The three commercial PBDE mixtures, Penta-, Octa-, and DecaBDE, have all been banned in the United States, however, limited use of DecaBDE is still permitted. PBDEs were phased out of production and added to the Stockholm Convention due to concerns over their environmental persistence and toxicity. Human exposure to PBDEs occurs primarily through the inadvertent ingestion of contaminated house dust, as well as though dietary sources. Despite the phase-out and discontinued use of PBDEs, human exposure to this class of chemicals is likely to continue for decades due to the continued use of treated products and existing environmental reservoirs of PBDEs. Extensive research over the years has shown that PBDEs disrupt thyroid hormone (TH) levels and neurodevelopmental endpoints in rodent and fish models. Additionally, there is growing epidemiological evidence linking PBDE exposure in humans to altered TH homeostasis and neurodevelopmental impairments in children. Due to the importance of THs throughout gestation, there is a great need to understand the effects of BFRs on the developing fetus. Specifically, the placenta plays a critical role in the transport, metabolism, and delivery of THs to the fetal compartment during pregnancy and is a likely target for BFR bioaccumulation and endocrine disruption. The central hypothesis of this dissertation research is that BFRs disrupt the activity of TH sulfotransferase (SULT) enzymes, thereby altering TH concentrations in the placenta.</p><p>In the first aim of this dissertation research, the concentrations of PBDEs and 2,4,6-TBP were measured in a cohort of 102 placenta tissue samples from an ongoing pregnancy cohort in Durham, NC. Methods were developed for the extraction and analysis of the BFR analytes. It was found that 2,4,6-TBP was significantly correlated with all PBDE analytes, indicating that 2,4,6-TBP may share common product applications with PBDEs or that 2,4,6-TBP is a metabolite of PBDE compounds. Additionally, this was the first study to measure 2,4,6-TBP in human placenta tissues.</p><p>In the second aim of this dissertation research, the placenta tissue concentrations of THs, as well as the endogenous activity of deiodinase (DI) and TH SULT enzymes were quantified using the same cohort of 102 placenta tissue samples. Enzyme activity was detected in all samples and this was the first study to measure TH DI and SULT activity in human placenta tissues. Enzyme activities and TH concentrations were compared with BFR concentrations measured in Aim 1. There were few statistically significant associations observed for the combined data, however, upon stratifying the data set based on infant sex, additional significant associations were observed. For example, among males, those with the highest concentrations of BDE-99 in placenta had T3 levels 0.80 times those with the lowest concentration of BDE-99 (95% confidence interval (CI): 0.59, 1.07). Whereas females with the highest concentrations of BDE-99 in placenta had T3 levels 1.50 times those with the lowest concentration of BDE-99 (95% CI: 1.10, 2.04). Additionally, all BFR analyte concentrations were higher in the placenta of males versus females and they were significantly higher for 2,4,6-TBP and BDE-209. 3,3’-T2 SULT activity was significantly higher in female placenta tissues, while type 3 DI activity was significantly higher in male placenta tissues. This research is the first to show sex-specific differences in the bioaccumulation of BFRs in human placenta tissue, as well as differences in TH concentrations and endogenous DI and SULT activity. The underlying mechanisms of these observed sex differences warrant further investigation. </p><p>In the third aim of this dissertation research, the effects of BFRs were examined in a human choriocarcinoma placenta cell line, BeWo. Michaelis-Menten parameters and inhibition curves were calculated for 2,4,6-TBP, 3-OH BDE-47, and 6-OH BDE-47. 2,4,6-TBP was shown to be the most potent inhibitor of 3,3’-T2 SULT activity with a calculated IC50 value of 11.6 nM. It was also shown that 2,4,6-TBP and 3-OH BDE-47 exhibit mixed inhibition of 3,3’-T2 sulfation in BeWo cell homogenates. Next, a series of cell culture exposure experiments were performed using 1, 6, 12, and 24 hour exposure durations. Once again, 2,4,6-TBP was shown to be the most potent inhibitor of basal 3,3’-T2 SULT activity by significantly decreasing activity at the high and medium dose (1 M and 0.5 M, respectively) at all measured time points. Interestingly, BDE-99 was also shown to inhibit basal 3,3’-T2 SULT activity in BeWo cells following the 24 hour exposure, despite exhibiting no inhibitory effects in the BeWo cell homogenate experiments. This indicates that BDE-99 must act through a pathway other than direct enzyme inhibition. Following exposures, the TH concentrations in the cell culture growth media and mRNA expression of TH-related genes were also examined. There was no observed effect of BFR treatment on these endpoints. Future work should focus on determining the downstream biological effects of TH SULT disruption in placental cells, as well as the underlying mechanisms of action responsible for reductions in basal TH SULT activity following BFR exposure. </p><p>This was one of the first studies to measure BFRs in a cohort of placenta tissue samples from the United States and the first study to measure THs, DI activity, and SULT activity in human placenta tissues. This research provides a novel contribution to our growing understanding of the effects of BFRs on TH homeostasis within the human placenta, and provides further evidence for sex-specific differences within this important organ. Future research should continue to investigate the effects of environmental contaminants on TH homeostasis within the placenta, as this represents the most critical and vulnerable stage of human development.</p> / Dissertation

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. Includes bibliographical references.

La maladie d’Alzheimer (MA), la forme la plus répandue de démence, est caractérisée par une accumulation cérébrale de plaques amyloïdes formées de peptide beta-amyloïde, et d’enchevêtrements neurofibrillaires (NFT) de protéine tau anormalement phosphorylée (P-tau). Depuis plusieurs années, l’évidence d’une implication majeure d’altérations synaptiques dans la pathologie a émergée. De plus, il a été observé dans les cerveaux MA que les héparanes sulfates (HS), normalement extracellulaires, accumulent à l’intérieur des neurones, où ils co-localisent avec tau. Le laboratoire CRRET a mis en évidence que la 3-sulfotransferase 2 (HS3ST2), enzyme prédominante dans le cerveau où elle génère des HS 3-O-sulfatés (3S-HS) de rôle inconnu, est impliquée dans les mécanismes à l’origine de la tauopathie. Puisque la HS3ST2 et les 3S-HS n’ont jamais été caractérisés à la synapse où ils pourraient participer au développement de la tauopathie, les objectifs de ce travail sont : 1) déterminer si la HS3ST2 et les 3S-HS sont présents à la synapse et étudier des possibles rôles physiologiques ; 2) déterminer si les 3S-HS accumulent au niveau intracellulaire dans des cellules neuronales et/ou dans de synaptosomes issus d’un modèle murin de tauopathie ; et 3) examiner si les 3S-HS intracellulaires produits par la HS3ST2 sont impliqués dans le développement ou évolution de la tauopathie au niveau synaptique.Dans ce travail, nous avons montré la présence des 3S-HS et de la HS3ST2 à la synapse de cellules hippocampiques et accumulé des preuves de leur implication dans la stabilité et l’activité synaptique, toutes deux altérées par des peptides se liant aux 3S-HS ont pu bloquer cette activité. Nous avons implémenté et caractérisé le modèle murin de tauopathie rTg4510 et mise en place les cultures primaires de leur neurones hippocampiques. Dans ces cellules, nous avons montré l’accumulation intracellulaire des 3S-HS et une surexpression de la HS3ST2 corrélant avec l’accumulation de P-tau. La digestion enzymatique des HS dans les synaptosomes a résulté dans l’inhibition de la tauopathie.Ce travail révèle pour la première fois un rôle fondamental de la 3-O-sulfatation des chaines d’HS à la synapse, aussi bien dans des conditions physiologiques que pathologiques. Pour la première fois, l’enzyme HS3ST2 est décrite à la synapse. De plus, ce travail donne la preuve d’un lien fort entre l’expression d’HS3ST2, l’accumulation de 3S-HS et la tauopathie au niveau synaptique, ouvrant de nouvelles opportunités pour mieux comprendre la MA. / Alzheimer’s disease (AD), the main form of dementia in the world, is characterized by brain accumulation of amyloid plaques formed of amyloid beta, and neurofibrillary tangles (NFT) made of tau protein in an abnormally hyperphosphorylated form (P-tau). Strong evidences show that synaptic changes are central to the disease process. Moreover, previous observations in AD have shown that heparan sulfates (HS), typically present outside the cell , accumulate inside neurons of AD in where they interact with tau. Recently, the CRRET laboratory demonstrated that the neural 3-O-sulfotransferase 2 (HS3ST2), which generates 3-O-sulfated HS (3S-HS) of still unrevealed physiological roles, is involved in the mechanisms leading to tauopathy. Since it was unknown whether HS3ST2 and 3S-HS are expressed at the synapse and if there they participate to tauopathy development and/or evolution, the objectives of this work were: 1) to determine if HS3ST2 and 3S-HS are present at the synapse and to get insights on their physiological role; 2) to investigate whether 3S-HS accumulate intracellularly in hippocampal cells and/or in synaptosomes from a mice model of tauopathy; and 3) to investigate whether intracellular 3S-HS made by HS3ST2 are involved in tauopathy development and/or evolution at the synaptic level.We described here the presence of 3S-HS and HS3ST2 at the synapse and the role that may play 3S-HS in maintaining synaptic transmission and stability in primary cell culture from mice. These roles are the results of potential multiple implications of 3S-HS in various processes. Secondly, we implement and characterized the rTg4510 mice model of AD-related tauopathy and set primary cultures of hippocampal cells from these mice. In the tauopathic cells, we showed the intracellular accumulation of 3S-HS and HS3ST2 overexpression. Finally, we cleaned P-tau in synaptosomes from the rTg4510 mice aged of 2 months by digesting HS.The present work reveals, for the first time, the presence and a possible fundamental role of HS3ST2 and 3S-HS at the synapse. We give evidences of an interplay between 3S-HS, produced by HS3ST2, and tau and the synaptic level, leading to its abnormal phosphorylation. The results of these work open a new way to understand the phenomenon leading to synaptic impairment in AD patients and could reveal new targets to elaborate protection strategies against the AD pathological lesions.

Although tamoxifen has been successfully utilized in the treatment and prevention of estrogen-dependent breast cancer for decades, its use is limited by its low incidence of endometrial cancer. The carcinogenic effects of tamoxifen are complex and may involve a combination of estrogen receptor-mediated hormonal effects as well as the metabolic activation of tamoxifen to reactive electrophiles that are genotoxic. Moreover, a significant population of patients develop clinical resistance to tamoxifen, which leads to breast cancer recurrence and a decrease in patient survival. Therefore, the goal of the current study was to examine the interactions of major metabolites of tamoxifen with the human cytosolic sulfotransferases hSULT2A1, hSULT1E1, and hSULT1A1*1. Changes in the catalytic activity of hSULT2A1 by tamoxifen metabolites may inhibit the formation of the genotoxic Α-sulfooxy tamoxifen intermediate catalyzed by this enzyme. Moreover, tamoxifen metabolites might interfere in the inactivation of hydroxysteroids catalyzed by hSULT2A1 as a part of the variable responses to tamoxifen therapy. Endoxifen was the most potent inhibitor of the hSULT2A1, which suggests that this metabolite may inhibit the role of hSULT2A1 in the metabolic pathway for genotoxicity that is seen with tamoxifen.N-desmethyltamoxifen (N-desTAM) was a substrate for the hSULT2A1, and the product of this reaction, N-desmethyltamoxifen sulfamate (N-desTAM-S), displayed greater inhibition of the enzyme than its unconjugated precursor. Thus, endoxifen, N-desTAM, and N-desTAM-S might serve protective roles in some tissues as they may inhibit the role of hSULT2A1 in the genotoxicity of tamoxifen. Metabolites of tamoxifen were then examined as inhibitors of hSULT1E1 and hSULT1A1*1 due to the roles of these enzymes in the inactivation of estrogens. Each of the metabolites studied were weak inhibitors of hSULT1E1; thus, endoxifen is not likely to promote increased estrogen signaling in breast tissue when administered as an independent breast cancer therapeutic agent in ongoing clinical trials. However, 4-hydroxytamoxifen (4-OHTAM) was a very potent inhibitor of hSULT1A1*1 when examined with estradiol as substrate. This suggests the potential for 4-OHTAM to interfere in estrogen metabolism in tissues where hSULT1A1*1 is expressed and hSULT1E1 is not. This information will be useful when interpreting the clinical trials of endoxifen and will aid in the design of related molecules

Alkylierte polyzyklische aromatische Kohlenwasserstoffe werden in vielen Matrizes wie Fahrzeugabgasen und Tabakrauch und auch als Kontaminanten in Nahrungsmitteln neben rein aromatischen Kongeneren gefunden. Alkylierte PAK können über die Alkylseitenkette über benzylische Hydroxylierung und nachfolgende Sulfonierung katalysiert über Sulfotransferasen (SULT) zu reaktiven Schwefelsäureestern umgesetzt werden. Die SULT-vermittelte Bioaktivierung zu einem genotoxischen Schwefelsäureester wurde für den benzylischen Alkohol 1-Hydroxymethylpyren des Hepatokanzerogens 1-Methylpyren in früheren Arbeiten gezeigt. In der vorliegenden Arbeit wurde überprüft, ob die benzylischen Alkohole weiterer alkylierter PAK über Sulfonierung zu genotoxischen Schwefelsäureestern umgesetzt werden. Hierzu wurde eine Gruppe von 17 Modellsubstanzen ausgewählt, um die Ableitung von Struktur-Aktivitäts-Beziehungen zu ermöglichen. Das genotoxische Potenzial authentischer benzylischer Schwefelsäureester der Modellsubstanzen wurde zunächst in vitro über DNA-Adduktbildung im zellfreien System und Mutagenität im Salmonella-Rückmutationstest untersucht. Die Sulfate zeigten große Reaktivitätsunterschiede in Abhängigkeit von der Struktur des aromatischen Systems und der Position der Alkylseitenkette, wobei die Endpunkte DNA-Adduktbildung und Mutagenität gut korrelierten. Des Weiteren wurde der Salmonella-Mutagenitätstest mit den benzylischen Alkoholen der untersuchten alkylierten PAK und gentechnisch veränderten S. typhimurium-Stämmen, die SULT-Formen des Menschen heterolog exprimieren, durchgeführt. Bis auf die Alkohole 2- und 4-HMP zeigten alle untersuchten benzylischen Alkohole deutliche mutagene Effekte in einem oder mehreren humane SULT exprimierenden Stämmen. Die durchgeführten in vitro-Versuche zeigten das Potenzial der benzylischen Metabolite alkylierter PAK für genotoxische Wirkungen. Nachfolgend musste geklärt werden, welche Relevanz die beobachteten Effekte für die komplexere in vivo-Situation haben. Nach Verabreichung verschiedener benzylischer Schwefelsäureester und Alkohole an männliche Ratten konnten DNA-Addukte in den untersuchten Organen detektiert werden, was im Fall der Schwefelsäureester deren systemische Bioverfügbarkeit und im Fall der benzylischen Alkohole deren Umsatz durch SULT der männlichen Ratte zeigte. Da im Gegensatz zum Menschen die SULT-Expression in der Ratte auf die Leber fokussiert ist, musste ein Großteil des Umsatzes zu genotoxischen Sulfaten in der Leber stattgefunden haben. DNA-Addukte wurden jedoch auch in extrahepatischen Organen gefunden, was über einen hepatischen Export der gebildeten reaktiven Sulfate und deren Transport über den Blutkreislauf zu diesen Geweben erklärt werden kann. Für die weiterführenden in vivo-Studien wurden die benzylischen Alkohole 1-HMP und 1-HM-8-MP ausgewählt, die trotz großer struktureller Ähnlichkeit toxikodynamische Unterschiede zeigten. Zur Untersuchung der Bedeutung des SULT-vermittelten Toxifizierungsweges als auch konkurrierender detoxifizierender oxidativer Stoffwechselprozesse, wurden für 1-HMP und 1-HM-8-MP in vivo-Inhibitionsstudien mit SULT-Inhibitoren und für 1-HM-8-MP auch mit ADH/ALDH-Inhibitoren durchgeführt. Eine Vorbehandlung mit dem SULT-Hemmstoff Pentachlorphenol führte zu einer Reduktion der DNA-Adduktniveaus in Organen 1-HMP- und 1-HM-8-MP-behandelter Tiere. Die Verabreichung von Quercetin hatte keine Auswirkung auf die DNA-Adduktniveaus. Die Hemmung der DNA-Adduktbildung bei Verabreichung von Pentachlorphenol verdeutlichte jedoch, dass benzylische Alkohole alkylierter PAK in vivo über Sulfonierung bioaktiviert werden. Eine Vorbehandlung mit dem ADH-Inhibitor 4-Methylpyrazol und dem ADH-Substrat Ethanol führte zu erhöhten DNA-Adduktniveaus in Organen 1-HM-8-MP-behandelter Tiere. Den gleichen Effekt, jedoch in geringerem Ausmaß, hatte auch die Vorbehandlung mit dem ALDH-Inhibitor Disulfiram. Dies deutet darauf hin, dass oxidative Modifikationen an der Seitenkette des 1-HM-8-MP einen Detoxifizierungsmechanismus darstellen. Nach Verabreichung benzylischer Metabolite alkylierter PAK wurden oftmals hohe Adduktniveaus in der Niere detektiert. Als mögliche Ursache hierfür wurde eine Transporter-vermittelte renale Sekretion reaktiver Sulfate postuliert, die über Vorbehandlung mit Probenecid vor Verabreichung von 1-HMP und 1-HM-8-MP überprüft wurde. Der Haupteffekt der Probenecid-Behandlung wurde jedoch nicht in der Niere, sondern in der Leber beobachtet, die stark erhöhte Adduktniveaus zeigte. Eine mögliche Erklärung hierfür ist die Hemmung des Exportes in der Leber gebildeter reaktiver Sulfate über Inhibition hepatischer organischer Anionentransporter. / Alkylated polycyclic aromatic hydrocarbons are found besides purely aromatic congeners in numerous matrices like car engine exhausts and tobacco smoke and as contaminants in foods. Alkylated PAH can be converted at the alkyl side chain to reactive sulfuric acid esters via benzylic hydroxylation and subsequent sulfonation catalysed by sulfotransferases (SULT). The SULT-mediated bioactivation to a genotoxic sulfuric acid ester was shown for the benzylic alcohol 1-hydroxymethylpyrene of the hepatocarcinogen 1-methylpyrene in previous studies. In the thesis at hand it was studied if the benzylic alcohols of further alkylated PAH are converted to genotoxic sulfuric acid esters via sulfonation. For this purpose a group of 17 model substances was chosen to allow for deduction of structure activity relationships. The genotoxic potential of authentic benzylic sulfuric acid esters of the model substances was initially investigated in vitro via DNA adduct formation in a cell free system and mutagenicity in the Salmonella reverse mutation test. The sulfates showed large differences in reactivity depending on the structure of the aromatic system and the position of the alkyl side chain whereupon the endpoints DNA adduct formation and mutagenicity correlated well. Furthermore, the Salmonella mutagenicity test was carried out with the benzylic alcohols of the alkylated PAH studied and S. typhimurium strains genetically engineered for the heterologous expression of human SULT forms. Except for the alcohols 2- and 4-HMP all benzylic alcohols studied showed clear mutagenic effects in one or more SULT-expressing strains. The studies performed in vitro demonstrated the potential of benzylic metabolites of alkylated PAH for genotoxic effects. Consecutively, the relevance of the observed effects for the more complex in vivo situation had to be clarified. After administration of different benzylic sulfuric acid esters and alcohols to male rats DNA adducts were detected in the organs studied, in case of the sulfuric acid esters showing their systemic bioavailability and in case of the benzylic alcohols demonstrating their conversion to the corresponding reactive benzylic sulfuric acid esters by SULT of the male rat. Since in contrast to man SULT expression in the rat is focused on the liver, a large part of the conversion to genotoxic sulfates must have been taken place in the liver. However, DNA adducts were also found in extrahepatic tissues which can be attributed to a hepatic export of the reactive sulfates formed and their transport to these tissues via circulation. For the continuative in vivo studies the benzylic alcohols 1-HMP and 1-HM-8-MP were chosen that demonstrated toxicodynamic differences in spite of their great structural resemblance. To investigate the importance of the SULT-mediated toxification pathway as well as competing detoxifying oxidative metabolic pathways, in vivo inhibition studies with SULT inhibitors were performed for 1-HMP and 1-HM-8-MP and with ADH/ALDH inhibitors also for 1-HM-8-MP. A pretreatment with the SULT inhibitor pentachlorophenol led to a reduction of DNA adduct levels in organs of animals treated with 1-HMP and 1-HM-8-MP. Administration of quercetin had no impact on the DNA adduct levels. However, inhibition of DNA adduct formation at administration of pentachlorophenol demonstrated that benzylic alcohols of alkylated PAH are bioactivated via sulfonation in vivo. A pretreatment with the ADH inhibitor 4-methylpyrazole and the ADH substrate ethanol led to increased DNA adduct levels in organs of animals treated with 1-HM-8-MP. The same effect but to a lesser extent was caused by a pretreatment with the ALDH inhibitor disulfiram. This indicates that oxidative modifications at the side chain of 1-HM-8-MP represent a detoxification mechanism. After administration of benzylic metabolites of alkylated PAH often high DNA adduct levels were detected in kidney. A transporter-mediated renal secretion was postulated as possible cause which was investigated using a pretreatment with probenecid before administration of 1-HMP and 1-HM-8-MP. However, the main effect of the treatment with probenecid was not observed in kidney but in liver that showed strongly increased adduct levels. A possible explanation for this effect is the inhibition of the export of reactive sulfates formed in liver via inhibition of hepatic organic anion transporters.

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.