<p>The nasal passages are constantly exposed to both air- and bloodborne foreign compounds. In particular, the olfactory mucosa is demonstrated to be susceptible to a variety of drugs and chemicals. In this thesis, mechanisms involved in tissue-selective toxicity in the olfactory mucosa of rodents have been investigated using the olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO₂) as a model compound. Comparative studies were performed with the non-toxic 2,5-dichlorophenyl methylsulphone (2,5-diClPh-MeSO₂) and the reasons for the strikingly different toxicity were investigated. </p><p>A strong bioactivation and protein adduction of 2,6-diClPh-MeSO₂ in olfactory microsomes and S9-fractions of rodents was demonstrated. In contrast, no significant metabolic activation of 2,5-diClPh-MeSO₂ was observed and the bioactivation in the liver for both chlorinated isomers was negligible. <i>In vitro</i> studies with recombinant yeast cell microsomes expressing mouse cytochrome P450 2A5 (CYP2A5) demonstrated a metabolic activation of 2,6-diClPh-MeSO₂. The 2,6-diClPh-MeSO₂-induced lesions and CYP2A5 expression preferentially occurred in Bowman’s glands and sustentacular cells of the olfactory mucosa. A significant depletion of glutathione (GSH) in the olfactory mucosa was demonstrated <i>in vivo</i>, while no changes were observed in the liver. There was a rapid induction of the endoplasmic reticulum (ER)-specific chaperone Grp78, activation of the ER-specific caspase-12 and the downstream caspase-3 in the Bowman’s glands. Electron microscopy revealed swelling of ER and mitochondria and a lost integrity of the Bowman’s glands. </p><p>Based on these results, the proposed mechanism for 2,6-diClPh-MeSO₂-induced toxicity in the olfactory mucosa is bioactivation by CYP2A5 into a reactive intermediate causing protein adduction and GSH-depletion. This is initiating a sequence of downstream events of ER-stress, changes in ion homeostasis, ultrastructural organelle disruption and apoptotic signalling. In spite of the initial apoptotic signals, the terminal phase of apoptosis seemed to be blocked and necrotic features occurred. The predominant expression of CYP2A5 in the olfactory mucosa is proposed to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO₂.</p>

The nasal passages are constantly exposed to both air- and bloodborne foreign compounds. In particular, the olfactory mucosa is demonstrated to be susceptible to a variety of drugs and chemicals. In this thesis, mechanisms involved in tissue-selective toxicity in the olfactory mucosa of rodents have been investigated using the olfactory toxicant 2,6-dichlorophenyl methylsulphone (2,6-diClPh-MeSO2) as a model compound. Comparative studies were performed with the non-toxic 2,5-dichlorophenyl methylsulphone (2,5-diClPh-MeSO2) and the reasons for the strikingly different toxicity were investigated. A strong bioactivation and protein adduction of 2,6-diClPh-MeSO2 in olfactory microsomes and S9-fractions of rodents was demonstrated. In contrast, no significant metabolic activation of 2,5-diClPh-MeSO2 was observed and the bioactivation in the liver for both chlorinated isomers was negligible. In vitro studies with recombinant yeast cell microsomes expressing mouse cytochrome P450 2A5 (CYP2A5) demonstrated a metabolic activation of 2,6-diClPh-MeSO2. The 2,6-diClPh-MeSO2-induced lesions and CYP2A5 expression preferentially occurred in Bowman’s glands and sustentacular cells of the olfactory mucosa. A significant depletion of glutathione (GSH) in the olfactory mucosa was demonstrated in vivo, while no changes were observed in the liver. There was a rapid induction of the endoplasmic reticulum (ER)-specific chaperone Grp78, activation of the ER-specific caspase-12 and the downstream caspase-3 in the Bowman’s glands. Electron microscopy revealed swelling of ER and mitochondria and a lost integrity of the Bowman’s glands. Based on these results, the proposed mechanism for 2,6-diClPh-MeSO2-induced toxicity in the olfactory mucosa is bioactivation by CYP2A5 into a reactive intermediate causing protein adduction and GSH-depletion. This is initiating a sequence of downstream events of ER-stress, changes in ion homeostasis, ultrastructural organelle disruption and apoptotic signalling. In spite of the initial apoptotic signals, the terminal phase of apoptosis seemed to be blocked and necrotic features occurred. The predominant expression of CYP2A5 in the olfactory mucosa is proposed to play a key role for the tissue- and cell-specific toxicity induced by 2,6-diClPh-MeSO2.

Endocrine disrupting chemicals, or EDCs, are some of the most prevalent toxic chemicals found in the environment because of human activity and they have a variety of adverse effects on both humans and wildlife. A proposed mechanism through which EDCs can negatively affect an organism is via an epigenetic mechanism known as DNA methylation, which can affect the development of the organism with negative outcomes later in life. The aim of this project was to investigate the effect of a prenatal exposure to mixture of EDCs, called Mixture N1, and its adverse effects. Mixture N1 has in a previous study been detected in the first semester of mothers, and linked to language delay in the offspring in the SELMA cohort study. We investigated relationships with DNA methylation pattern changes in key genes with exposure, gene expression and behaviour in the adult brains of the exposed mice.  Our results showed correlative as well as linear relationships between methylation and different behavioural outcomes for target genes. One gene in particular - Nr3c1 - stood out among the results, having links to both stress and sociability. Specifically, DNA methylation of this gene correlates to active stress coping behaviours as well as sociability, but with no mediation component in these relationships. These results are promising for the use of methylation analysis as a biomarker of EDC mixture exposure, but more so as a predictor of negative behavioural outcomes later in life. More research could strengthen this use, and uncover the mechanism through which methylation alone might affect changes in behaviour.

This study has examined how cooking affects the levels of the toxicantsaflatoxin, arsenic, lead, dioxins, cadmium, mercury, perfluorinatedcompounds and polybrominated diphenyl ethers in rice, potatoes and fish.Not every toxicant was examined for all three types of food, they were onlyexamined for those types of food where current levels of the toxicant in thattype of food are relevant from a risk assessment perspective. To determineif there is a danger of negative effects due to the exposure of thesetoxicants from food, articles from the Swedish National Food Agency andother scientific articles have been compiled. A minor experimentalsubstudy was also performed where rice bought in Uppsala was rinsed andboiled in different ways to see if that affected the residue levels of arsenic.The results of this study indicate that the levels of some toxic substancescan be lowered on a dry weight basis by cooking, and that this reduction isdependent on the cooking method used, the properties of the food andtoxin. The levels of cadmium and mercury were generally unchanged bycooking whereas the results for lead and perfluorinated compounds wereconflicting. Cooking can lover the levels of aflatoxin, arsenic, dioxins andpolybrominated diphenyl ethers on a dry weight basis. The effects ofcooking on toxicant levels should be considered when performing riskassessments, but further studies are needed to achieve a better basis for decision-making.

The persistent and toxic perfluoroalkyl acids (PFAAs) are ubiquitously present in the environment and reach humans predominantly via food and drinking water. The aim of the present study was to investigate the effect of low-grade (&lt;10 ng L-1 of single PFAAs) contaminated PFAAs drinking water on serum PFAA concentrations in a representative Swedish adolescent population, and to examine the influence of potential determinants on the variation of the PFAAs serum concentrations. This was done by using multivariate regression analysis on the possible determinants of blood serum PFAA concentrations in 479 Swedish adolescents, 10 to 21 years of age, who had left complete dietary and life style information in 2016-17 in the nation-wide food consumption survey Riksmaten Ungdom. Raw and drinking water samples (DW) from water treatment plants (WTPs) that delivered DW to participants schools were sampled in 2018, analysed for PFAAs, and used for assessing the participants DW PFAA exposure. Maternal education level and maternal birth country, consumption of fish, as well as age and sex were significantly associated with the participants PFAAs serum concentrations. DW concentrations as low as &lt;1 ng L for PFOA and PFHxS, &lt;0.45 ng L-1 for PFNA and &lt;4 ng L-1 for PFOS were significantly associated with increased adolescent serum concentrations of the PFAAs in question, which suggests that low-grade contaminated drinking water is an important exposure route for Swedish adolescents. For risk assessment purposes, it was investigated whether parts of the adolescent population exceeded the serum PFOS and PFOA concentrations corresponding to the current health-based reference intakes as assessed by the European Food Safety Authority (EFSA) and the U.S. Agency for Toxic Substances and Disease Registry (ATSDR). Around 1.7% and 2.7% of participants had PFOS serum concentrations exceeding serum levels used to derive the tolerable daily intake (TDI) (EFSA) and the minimum risk level (MRL) (ATSDR), respectively and a cause for concern was consequently identified. However, the high serum concentrations of participants exceeding the TDI and MRL serum concentrations belong to participants suspected to have been previously exposed to highly contaminated drinking water and not from consuming foods and beverages containing background concentrations of PFAAs.

Familial hypercholesterolemia is a genetic disease affecting about 10 million people around the world. Those who carry the disease have a very high risk of developing cardiovascular diseases and commonly encounter myocardial infarction at the early age of 40. Therefore, a diagnosis and immediate treatment are very important for these patients. Despite many combinations of available drugs, there are many patients who still cannot reach the desired cholesterol levels. Mipomersen is a new lipid-lowering drug which inhibits the synthesis of apolipoprotein B, a common component of lipoproteins such as low-density lipoprotein. Inhibition of this protein leads to reduced production of these lipoproteins and reduces the risk of cardiovascular diseases. The drug is currently only indicated for treating patients with homozygous familial hypercholesterolemia.  Unfortunately, there have been many reports of adverse events in patients using mipomersen which has proven problematic.         The aim of this thesis is to analyze the efficacy and safety of mipomersen when treating patients with familial hypercholesterolemia. This has been done by searching for five clinical trials in the database Web of Science. The studies were required to include patients with familial hypercholesterolemia, use mipomersen as the study drug and analyze its effect and safety.   The studies showed that mipomersen has a very good effect in decreasing low-density lipoproteins as well as other lipoproteins in comparison to placebo. Many of the patients who were treated with mipomersen displayed several adverse events and the most common were injection-site reaction and influenza-like symptoms. Elevated levels of aminotransaminase and increased fat deposit in the liver were also common. Based on the five clinical trials analyzed in this thesis, mipomersen is an effective lipid-lowering drug which reduces low density lipoprotein cholesterol, apolipoprotein B and lipoprotein (a) in patients with familial hypercholesterolemia. Elevations in alanine aminotransferase and aspartate aminotransferase are common in patients treated with mipomersen. This could indicate a negative impact on the liver. To be more certain of its safety profile, more research could be needed. There are however, new treatments that combines statins and a proprotein convertase subtilisin/kexin 9 inhibitor, which could be the future of lipid-lowering treatments and mipomersen would then likely be substituted.

<p>Radiotherapy is one of the most common therapeutic methods for treatment of many types of cancer. Despite many decades of development and experience there is much to improve, both in efficacy of treatment and to decrease the incidences of adverse healthy tissue reactions. Around 20 % of the radiotherapy patients show a broad range in the severity of normal tissue reactions to radiotherapy, and dose limits are governed by severe reactions in the most radiosensitive patients (< 5 %). Identification of patients with low, moderate or high clinical radiosensitivity before commencing of radiotherapy would allow individual adaptation of the maximum dose with an overall increase in the cure rate. Characterization of factors that may modify the biological effects of ionizing radiation has been a subject of intense research efforts. Still, there is no assay currently available that can reliably predict the clinical radiosensitivity. The aim of this work has been to investigate the role of oxidative stress in individual radiosensitivity and evaluate novel markers of radiation response, which could be adapted for clinical use.</p><p>8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a general marker of oxidative stress, is one of the major products of interaction of ionizing radiation with DNA and the nucleotide pool of the cell. As 8-oxo-dG is highly mutagenic due to incorrect base pairing with deoxyadenosine, various repair mechanisms recognize and remove 8-oxo-dG. The repaired lesions are released from cells to the extracellular milieu (serum, urine and cell culture medium) where they can be detected as markers for free radical reactions with the nucleic acids.</p><p>Significant variations in background levels as well as in radiation induced levels of 8-oxo-dG in urine have been demonstrated in breast cancer patients (paper 1). Two major patterns were observed: high background and no therapy-related increase vs. low background and significant increase during radiotherapy for the radiosensitive and non radiosensitive patients respectively.</p><p>Studies in paper 2 indicated major contribution of the nucleotide pool to the extracellular 8-oxo-dG levels. The results also implicated induction of prolonged endogenous oxidative stress in the irradiated cells. RNA “knock-down” experiments on the nucleotide pool sanitization enzyme hMTH1 in paper 3 lend further experimental evidence to this assumption.</p><p>The applicability of 8-oxo-dG as a diagnostic marker of oxidative stress was demonstrated in paper 4. Studies on dialysis patients revealed a good correlation between inflammatory responses (known to be associated with persistent oxidative stress) and extracellular 8-oxo-dG.</p><p>In summary, our results confirm that extracellular 8-oxo-dG is a sensitive <i>in vivo</i> biomarker of oxidative stress, primarily formed by oxidative damage of dGTP in the nucleotide pool with a potential to become a clinical tool for prediction of individual responses to radiotherapy.</p>

<p>The toxicity of perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) was measured. When Caco-2 cells from human adenocarcinoma are cultivated on a filter a monolayer is formed with properties similar to human duodenum epithelium. The Caco-2 cells grown on filter were exposed to the environmental contaminants PFOS and PFOA. The effects on the Caco-2 epithelium were examined by four different methods: trans-epithelial resistance (TEER), leakage of the intracellular protein lactate dehydrogenase (LDH), 14C-mannitol passage through the epithelium and protein content of the epithelium. TEER and C-mannitol passage show the Caco-2 cellmonolayer integrity, LDH leakage gives information of cytotoxicity and protein content of the epithelium shows cell adhension to the filter.</p><p>In the first study TEER decreased at the highest concentrations of PFOS and PFOA (1Mm). The 14C-mannitol passage increased at the highest PFOS concentration. No cytotoxicity was shown and protein-loss was not observed. The second study with PFOS doses of 0, 1, 10, 100 and 500µM and 1 and 10mM showed that the effect of PFOS on TEER was dose-dependent. The 14C-mannitol passage was very high at the highest PFOS-concentration (10mM) and a dose-response was indicated. No cytotoxicity was demonstrated and protein-quantity was not affected. In the third study it was demonstrated that the toxicity of PFOS did not depend on the different concentrations of the oil-emulsion used to dissolve PFOS and PFOA.</p>

Radiotherapy is one of the most common therapeutic methods for treatment of many types of cancer. Despite many decades of development and experience there is much to improve, both in efficacy of treatment and to decrease the incidences of adverse healthy tissue reactions. Around 20 % of the radiotherapy patients show a broad range in the severity of normal tissue reactions to radiotherapy, and dose limits are governed by severe reactions in the most radiosensitive patients (&lt; 5 %). Identification of patients with low, moderate or high clinical radiosensitivity before commencing of radiotherapy would allow individual adaptation of the maximum dose with an overall increase in the cure rate. Characterization of factors that may modify the biological effects of ionizing radiation has been a subject of intense research efforts. Still, there is no assay currently available that can reliably predict the clinical radiosensitivity. The aim of this work has been to investigate the role of oxidative stress in individual radiosensitivity and evaluate novel markers of radiation response, which could be adapted for clinical use. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a general marker of oxidative stress, is one of the major products of interaction of ionizing radiation with DNA and the nucleotide pool of the cell. As 8-oxo-dG is highly mutagenic due to incorrect base pairing with deoxyadenosine, various repair mechanisms recognize and remove 8-oxo-dG. The repaired lesions are released from cells to the extracellular milieu (serum, urine and cell culture medium) where they can be detected as markers for free radical reactions with the nucleic acids. Significant variations in background levels as well as in radiation induced levels of 8-oxo-dG in urine have been demonstrated in breast cancer patients (paper 1). Two major patterns were observed: high background and no therapy-related increase vs. low background and significant increase during radiotherapy for the radiosensitive and non radiosensitive patients respectively. Studies in paper 2 indicated major contribution of the nucleotide pool to the extracellular 8-oxo-dG levels. The results also implicated induction of prolonged endogenous oxidative stress in the irradiated cells. RNA “knock-down” experiments on the nucleotide pool sanitization enzyme hMTH1 in paper 3 lend further experimental evidence to this assumption. The applicability of 8-oxo-dG as a diagnostic marker of oxidative stress was demonstrated in paper 4. Studies on dialysis patients revealed a good correlation between inflammatory responses (known to be associated with persistent oxidative stress) and extracellular 8-oxo-dG. In summary, our results confirm that extracellular 8-oxo-dG is a sensitive in vivo biomarker of oxidative stress, primarily formed by oxidative damage of dGTP in the nucleotide pool with a potential to become a clinical tool for prediction of individual responses to radiotherapy.

Exposure to xenoestrogens during development has been shown to impair sexual differentiation in various species. The major aim of this thesis was to elucidate the respective roles of the two estrogen receptors ERα and ERβ in normal and disrupted differentiation of sex organs and copulatory behavior in the Japanese quail (Coturnix japonica). The expression of ERα mRNA was much stronger than that of ERβ mRNA in the gonads and Müllerian ducts (embryonic oviducts) in early embryos. By contrast, ERβ seemed to be predominantly expressed in regions of the embryonic brain that are associated with male sexual behavior. Embryos were exposed to the selective ERα agonists propyl-pyrazole-triol (PPT) and 16α-lactone-estradiol (16α-LE2). The estrogens 17β-estradiol (E2) and 17α-ethynylestradiol (EE2), which activate both ERα and ERβ, were used as positive controls. All substances impaired reproductive organ differentiation. The effects observed included oviductal malformations in females and partial development of oviducts in males. All substances also induced testis feminization (ovotestis) in male embryos. The male copulatory behavior was severely impaired by the positive controls but was unaffected by PPT and 16α-LE2 at doses that disrupted sex organ differentiation. A higher dose of 16α-LE2 significantly suppressed the behavior. However, it is possible that this effect was caused by cross-activation of ERβ. The substances also induced hepatic expression of mRNA encoding the egg-yolk proteins vitellogenin II and very low-density apolipoprotein II, which are commonly used as indicators of estrogen exposure. In conclusion, the results suggest that ERα is important for female reproductive organ differentiation. Excess activation of ERα by xenoestrogens impairs differentiation in both females and males and induces hepatic expression of egg-yolk proteins. The results also indicate that ERα alone cannot mediate demasculinization of male copulatory behavior in quail, although further studies are needed to test this hypothesis.