Humans are exposed to a variety of chemicals including environmental pollutants, cosmetics, food preservatives and drugs. Some of these substances might be harmful to the human body. Traditional toxicological and behavioural investigations performed in animal models are not suitable for the screening of a large number of compounds for potential toxic effects. There is a need for simple and robust in vitro cellular models that allow high-throughput toxicity testing of chemicals, as well as investigation of specific mechanisms of cytotoxicity. The overall aim of the thesis has been to evaluate neuronally differentiated mouse embryonal carcinoma P19 cells (P19 neurons) as a model for such testing. The model has been compared to other cellular models used for neurotoxicity assessment: retinoic acid-differentiated human neuroblastoma SH-SY5Y cells and nerve growth factor-treated rat pheochromocytoma PC12 cells. The chemicals assessed in the studies included the neurotoxicants methylmercury, okadaic acid and acrylamide, the drug of abuse MDMA (“ecstasy”) and a group of piperazine derivatives known as “party pills”. Effects of the chemicals on cell survival, neurite outgrowth and mitochondrial function have been assessed. In Paper I, we describe a fluorescence-based microplate method to detect chemical-induced effects on neurite outgrowth in P19 neurons immunostained against the neuron-specific cytoskeletal protein βIII-tubulin. In Paper II, we show that P19 neurons are more sensitive than differentiated SH-SY5Y and PC12 cells for detection of cytotoxic effects of methylmercury, okadaic acid and acrylamide. Additionally, in P19 neurons and differentiated SH-SY5Y cells, we could demonstrate that toxicity of methylmercury was attenuated by the antioxidant glutathione. In Paper III, we show a time- and temperature-dependent toxicity produced by MDMA in P19 neurons. The mechanisms of MDMA toxicity did not involve inhibition of the serotonin re-uptake transporter or monoamine oxidase, stimulation of 5-HT2A receptors, oxidative stress or loss of mitochondrial membrane potential. In Paper IV, the piperazine derivatives are evaluated for cytotoxicity in P19 neurons and differentiated SH-SY5Y cells. The most toxic compound in both cell models was TFMPP. In P19 neurons, the mechanism of action of TFMPP included loss of mitochondrial membrane potential. In conclusion, P19 neurons are a robust cellular model that may be useful in conjunction with other models for the assessment of chemical-induced neurotoxicity.

Trypanosoma brucei is a causative agent of African sleeping sickness. It is an extracellular parasite which circulates in the blood, lymph and eventually invades the central nervous system. There is a great need for new medicines against the disease and specific properties of nucleoside kinases in the pathogen can be exploited as targets for chemotherapy.  T. brucei contains a gene where two thymidine kinase sequences are fused into a single open reading frame. These types of tandem thymidine kinases were found only in different types of parasites, which made us to believe that it might be beneficial for them. Each thymidine kinase sequence in these tandem enzymes are here referred to as a domain. By cloning and expressing each domain from T. brucei separately, we found that domain 1 was inactive and domain 2 was as active as the full-length enzyme. T. brucei thymidine kinase phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine and to some extent purine nucleosides like deoxyinosine and deoxyguanosine. Human thymidine kinase increases the affinity to its substrates when it forms oligomers. Similarly, the T. brucei two thymidine kinase sequences, which can be viewed as a pseudodimer, had a higher affinity to its substrates than domain 2 alone.  T. brucei lacks de novo purine biosynthesis and it is therefore dependent on salvaging the required purine nucleotides for RNA and DNA synthesis from the host. Purine salvage is considered as a target for drug development. It has been shown that in the presence of deoxyadenosine in the growth medium, the parasites accumulate high levels of dATP and the extensive phosphorylation of deoxyadenosine leads to depleted ATP pools. Initially, we wondered if deoxyadenosine could be used as a drug against T. brucei. However, we found that T. brucei is partially protected against deoxyadenosine because it was cleaved by the enzyme methylthioadenosine phosphorylase (MTAP) to adenine and ribose-1-phosphate. At higher concentration of deoxyadenosine, 3 the formed adenine was not efficiently salvaged into ATP and started to inhibit MTAP instead. The deoxyadenosine was then instead phosphorylated by adenosine kinase leading to accumulation of dATP. The MTAP reaction makes deoxyadenosine itself useless as a drug and instead we focused on finding analogues of deoxyadenosine or adenosine that were cleavage-resistant and at the same time good substrates of T. brucei adenosine kinase. Our best hit was then 9-(2-deoxy-2-fluoro-ß-D-arabinofuranosyl) adenine (FANA-A). An additional advantage of FANA-A as a drug was that it was taken up by the P1 nucleoside transporter family, which makes it useful also against multidrug resistant parasites that often have lost the P2 transporter function and take up their purines solely by the P1 transporter. In parallel with our study of nucleoside metabolism in T. brucei, we also have a collaboration project where we screen essential oils from plants which are used in traditional medicine. If the essential oils are active against the trypanosomes, we further analyze the different components in the oils to identify new drugs against African sleeping sickness. One such compound identified from the plant Smyrnium olusatrum is isofuranodiene, which inhibited T. brucei proliferation with an IC50 value of 3 μM.

Antifouling paints are applied on vessels to prevent growth of fouling organisms such hasbarnacles. Presently, there are a number of different paints available on the Swedish marketwith different strategies and active substances. The paints might work by either continuouslyreleasing biocides or physically by peeling off or provide an easily cleansed surface whereorganisms cannot attach. The physically working paints do not need to register an activesubstance since its purpose is not to affect living organisms by a chemical or biological modeof action. In this study, two commercially available paints, the copper-based Fabi 3959(International Paint Ltd) and physically eroding, biocide-free labelled Mille Light (HempelFärg AB) were compared to Hard Racing superior, containing copper and the forbiddensubstance Tributyltin. Fabi International is only allowed to be used on the Swedish west coastdue to 6% added as active substance while the biocide-free Mille Light is eligible for eastcoast usage. The toxic effect from respective paint was investigated by assembling a growthinhibition test with the red macro alga Ceramium tenuicorne. The results show that all thestudied paints had a negative effect on growth and therefore leaked substances inconcentrations high enough to be harmful to the alga. The toxic response differed with theeffect on growth being in the following order, Hard racing superior>Fabi >Mille Light.Implications regarding the current legalization involving biocide-free labelled antifoulingpaints are discussed.

The dietary intake of polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD) have been estimated for Swedish children. A dietary survey performed in 2003, including 4, 8-9 and 11-12 year olds, and concentrations in individual food items were combined. The food included in the study was mainly of animal origin, consisting of fish and shellfish, dairy products, meat products, eggs, animal and vegetable fats and fats from miscellaneous food products. The medium-bound intake of PBDEs (9 congeners) were estimated to 23.0 ng/day, 30.9 ng/day and 27.7 ng/day for 4, 8-9 and 11-12 years olds respectively. The corresponding estimations for HBCD were 7.94 ng/day 10.7 ng/day and 9.46 ng/day for 4, 8-9 and 11-12 years olds respectively. These results show a higher daily intake for 8-9 year olds compared with the other age groups. However, when estimating the daily intake per kg bw, the intake decreases with age. BDE-47 contributed the most to the total intake of PBDEs, with approximately 40%. The food group contributing the most to the intake of PBDEs and HBCD was fish and shellfish, of which non-Baltic fatty fish was the largest contributor. There were no considerable differences between boys and girls in any of the aspects examined. The result from this study show a lower intake of PBDEs and HBCD in Swedish children compared with children in other studies made in Europe and the United States. / Bromerade flamskyddsmedel används för att skydda brännbara material från att fatta eld, till exempel skyddas textilier och plaster i bland annat elektronik, fordon och möbler. Två typer av bromerade flamskyddsmedel är polybromerade difenyletrar (PBDE) och hexabromocyklododekan (HBCD). Dessa är additiva flamskyddsmedel och blandas i materialet som ska skyddas men binder inte in i produkten och kan därför lätt läcka ut i miljön, vilket också har skett. Halter har påträffats i miljön och i biota långt från plaster där ämnena produceras eller används. PBDE och HBCD har visats ha hormonstörande och neurotoxiska effekter i studier på råtta och mus. Thyroxinnivåerna sjunker vid exponering av PBDE och HBCD, vilket skulle kunna leda till sköldkörtelproblem och störd utveckling av bland annat hjärnan om exponering sker perinatalt. De neurotoxiska effekterna inkluderar inlärnings- och minnessvårigheter och ett förändrat beteende med hyper- och hypoaktivitet som följd. Human exponering för PBDE och HBCD sker främst via födan och speciellt via animaliska produkter då dessa ämnen är lipofila, bioackumulerande och ofta biomagnifierande vilket gör att de påträffas i högre koncentrationer högre upp i trofinivåerna. Studier från bland annat Sverige och Finland visar att fisk och skaldjur är den största källan till intag av PBDE. De flesta intagsberäkningar av PBDE och HBCD baseras på livsmedelskonsumtionen hos vuxna och visar följaktligen endast hur intaget ser ut för den delen av populationen. För barn, som är en av de känsligaste grupperna i populationen, finns inte många studier att tillgå, varken från Sverige eller andra delar av världen. I den här studien har därför intaget av PBDE (summan av 9 kongener) och HBCD beräknats för barn i Sverige. I en rikstäckande kostundersökning utförd 2003 deltog barn i åldrarna 4, 8-9 och 11-12 år. De fick i en matdagbok ange sin konsumtion under fyra på varandra följande dagar. Data från denna undersökning kombinerades sedan med haltdata från olika livsmedel för att räkna ut intaget av PBDE och HBCD på individbasis. Undersökningen innefattade främst animaliska livsmedel och innehöll därför fisk och skaldjur, mejeriprodukter, köttprodukter, ägg, animaliskt och vegetabiliskt fett och fett från övriga livsmedel. Resultaten visar att födointaget av PBDE var 23,0 ng/dag, 30,9 ng/dag och 27,7 ng/dag för 4, 8-9 respektive 11-12 åringar. Intaget av HBCD beräknades till 7,94 ng/dag, 10,7 ng/dag och 9,46 ng/dag för 4, 8-9 respektive 11-12 åringar. Detta visar att 8-9 åringar har det högsta dagliga intaget av PBDE och HBCD. När intaget beräknas på kroppsvikt däremot, har de yngsta barnen det högsta intaget som sedan sjunker med åldern. Fisk och skaldjur var den största källan till intaget av PBDE och HBCD, trots att konsumtionen av dessa livsmedel var relativt lågt. Det fanns ingen större skillnad mellan pojkar och flickor, varken i intag av PBDE eller av HBCD. Jämfört med de få studier som gjorts i andra länder, är det tydligt att svenska barn har ett lägre intag av PBDE och HBCD. Undersökningen tyder också på att intaget av PBDE och HBCD hos svenska barn, utifrån de kunskaper vi har idag, inte utgör någon risk med avseende på de effekter av PBDE och HBCD som påträffats i toxikologiska studier. Däremot är barn i ett känsligt skede i livet och upprepad exponering samt exponering för flera miljögifter samtidigt skulle kunna påverka deras utveckling negativt.

Antibiotics are essential drugs in modern medicine. After consumption antibiotics are excreted in unmetabolized form in the urine and reach our sewage treatment plants (STP). STP are not able to degrade all antibiotics leading to release of antibiotics into the aquatic environment. Aquatic animals are thus continuously exposed to antibiotics. This study involved assessment of the toxicity of eight antibiotics previously detected in the river Fyrisån, Uppsala, Sweden, in developing zebrafish (Danio rerio) embryos up to day six of age. The experiments included assessment of embryonal toxicity for the individual antibiotics as well as mixtures of all antibiotics.  The mixtures were based on previously measured concentrations in river Fyrisån and tested in increasing concentrations up to 1000-times higher concentrations. In the toxicity assessment different lethal and sublethal endpoints were observed, such as early movements, heart rate, hatching time and length. These experiments were followed by behaviour study observing the swimming activity during alternating dark-light alternations. Lastly, a bioaccumulation study was performed on mixtures of antibiotics to determine if these antibiotics were bioaccumulative in zebrafish embryos. The results showed that these eight antibiotics, individually and as a mixture did not affect any of the endpoints. As for bioaccumulation, none of the eight antibiotics were bioaccumulating in zebrafish embryos. These antibiotics seem to be non-toxic during fish embryonal development. However, the results cannot determine the long-term effects of antibiotic exposure and thus further studies are needed to assess the potential toxicity of environmentally present antibiotics to fish.

There are currently over 430 known SLC transporters, over 30% of which have an unknown function. Compared to other transporter gene families, the SLC genes are relatively understudied with many orphan genes. SLC transporters have a high disease relevance and can be associated with many different diseases like gout, type 2 diabetes and different forms of cancer. SLC transporters also appear to be very druggable, thus offering a rare opportunity of an underexplored gene family, that can be linked to many diseases and seem to have a general druggability with small organic molecules. This thesis is evaluating three specific SLC transporters of the SLC38 family to discover their different roles and purposes. In this project CRISPR-Cas9 is used to knockout three SLC38 transporters, called SNAT6, SNAT8 and SNAT10. The cell-line used is HEK293 cells, as they are easy to transfect and are thought to express the three genes, however it is not certain that they do express the three SNAT genes. The project aims to optimize the method for best possible transfection by trying different protocols. A literature study is done on what the future experiments of the knocked-out cells could be, including; ensuring the HEK293 cells express the three genes, controlling the effectiveness of the transfection and analyzing the result of such a transfection. To confirm that the HEK293 cells do express the three SNATs a western blot assay could be performed. RT-qPCR is found to be useful in evaluating whether the knockouts are successful, by measuring if the three SNAT transporter proteins are present or not in the knocked-out cells. A metabolic analysis study to determine the result of the knockouts is also described as a future experiment. The experimental finding was a CRISPR-Cas9 transfection method that yielded enough RNA, enabling future experiments such as RT-qPCR.

Food contact materials are materials that are intended to come into contact with food, and we are exposed to different types of chemicals that exist in the packages on a daily basis. In this study, a battery of effect‑based in vitro cellular bioassays was used to evaluate the presence of bioactive compounds in commonly used food contact materials made of paper and cardboard, retrieved from the Swedish market. Sample extracts were tested at concentrations 0.3, 1, 3 and 10 mg food contact material/mL cell culture medium. The use of effect-based bioassays allowed for screening of multiple health-relevant endpoints in a non-targeted approach. Hence, taking unknown substances and mixtures into consideration when addressing potential toxicity of the materials. In essence, detection of bioactivity could be considered as moderate to high in assays of positive effects. Antiandrogenic and antiestrogenic effects were found in 72% of the samples, followed by 47% bioactivity in the Nrf2 assay. No androgenic effect was detected. Usage of effect-based bioassays allows for high sensitivity and low detection limits, and these can be used as a first approach to evaluate package materials to ensure the safety of consumers. / Livsmedelsförpackningar är material som är avsedda att komma i kontakt med livsmedel. Vi exponeras för olika typer av kemikalier som existerar i dessa förpackningar varje dag. I denna studie användes ett batteri av effektbaserade in vitro bioanalytiska metoder för att undersöka förekomst av bioaktiva ämnen i vanligen använda livsmedelsförpackningar tillverkade av papper och kartong, insamlade från den svenska marknaden. Provextrakten testades i koncentrationerna 0.3, 1, 3 och 10 mg livsmedelsförpackning/mL cellmedium. Att använda effektbaserade bioanalyser möjliggör undersökning av flertalet hälsorelevanta effekter genom en icke-riktad strategi. På så sätt tas okända substanser och komplexa blandningar i beaktande. Andelen bioaktiva prover kan anses måttlig till hög för positiva analyser. Antiandrogena och antiöstrogena effekter detekterades i 72% av proverna, följt av 47% bioaktivitet i Nrf2 analysen. Ingen agonistisk androgen effekt observerades. Att använda effektbaserade bioanalyser möjliggör hög sensitivitet och detektion vid låga koncentrationer, därför kan dessa användas som ett första steg för att evaluera förpackningsmaterial för att säkra konsumenternas hälsa.

Primary sclerosing cholangitis (PSC) is a rare inflammatory chronic liver disease that causes damage to the intra- and or extrahepatic bile ducts leading to cholestasis. As the disease proceeds the development of cirrhosis and eventually liver failure occurs. This study aims to determine the role of IgG subclasses in the prognosis of PSC and its outcome. A retrospective analysis was performed of 183 patients followed at the Department of Upper Abdominal Diseases at the Karolinska University Hospital. Factors that were analysed were sex, age at PSC diagnosis, total IgG values, IgG subclasses values and events of autoimmune hepatitis (AIH), inflammatory bowel disease (IBD), colectomy, cirrhosis, cholangitis, endoscopic retrograde cholangiopancreatography (ERCP), liver transplantation and cholangiocarcinoma. This study showed that high IgG4 levels were associated with a higher incidence of cirrhosis, liver transplantation, cholangitis and ERCP, while low IgG4 levels were associated with a prior IBD diagnosis. In conclusion, elevated IgG4 levels were associated with a higher occurrence of cirrhosis, cholangitis, ERCP and liver transplantation. It seems that IgG4 could be of importance for outcome prediction in PSC.

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.

Internal fertilization usually implies that a spermatozoon, with intact attributes for zygote formation, passes all hurdles during its transport through the female genitalia and reaches the oocyte. During this journey, millions to billions of other spermatozoa perish. Spermatozoa are highly differentiated motile cells without synthetic capabilities. They generate energy via glycolysis and oxidative phosphorylation to sustain motility and to maintain the stability and functionality of their plasma membrane. In vivo, they spend their short lifespan bathing in female genital tract fluids of different origins, or are in vitro exposed to defined media during diverse sperm handling i.e. extension, cryopreservation, in vitro fertilization, etc. Being excitable cells, spermatozoa respond in vivo to various stimuli during pre-fertilization (capacitation, hyperactivation, oocyte location) and fertilization (acrosome reaction, interaction with the oocyte) events, mediated via diverse membrane ion-conducting channels and ligand-gated receptors. The present Thesis has mapped the presence and reactivity (sperm intactness and kinematics) of selected receptors, water and ion channels in ejaculated boar spermatozoa. The final aim was to find a relevant alternative cell type for in vitro bioassays that could ease the early scrutiny of candidate drugs as well as decreasing our needs for experimental animals according to the 3R principles. Spermatozoa are often extended, cooled and thawed to warrant their availability as fertile gametes for breeding or in vitro testing. Such manipulations stress the cells via osmotic variations and hence spermatozoa need to maintain membrane intactness by controlling the exchange of water and the common cryoprotectant glycerol, via aquaporins (AQPs). Both AQPs-7 and -9 were studied for membrane domain changes in cauda- and ejaculated spermatozoa (un-processed, extended, chilled or frozen-thawed). While AQP-9 maintained location through source and handling, thawing of ejaculated spermatozoa clearly relocated the labelling of AQP-7, thus appearing as a relevant marker for non-empirical studies of sperm cryopreservation. Alongside water, spermatozoa interact with calcium (Ca2+) via the main Ca2+ sperm channel CatSper. Increments in intracellular Ca2+ initiate motility hyperactivation and the acrosome reaction. The four subunits of the CatSper channel were present in boar spermatozoa, mediating changes in sperm motility under in vitro capacitation-inducing conditions (increased extracellular Ca2+ availability and bicarbonate) or challenge by the CatSper antagonists mibefradil and NNC 55-0396. Uterine and oviduct fluids are richest in endogenous opioids as β-endorphins during mating and ovulation. Both μ- and δ- opioid receptors were present in boar spermatozoa modulating sperm motility, as in vitro challenge with known agonists (μ: morphine; δ: DPDPE and κ: U 50488) and antagonists (μ: naloxone; δ: naltrindole and κ: nor-binaltrorphimine) showed that the μ-opioid receptor maintained or increased motility while the δ-opioid receptor mediated decreased motility over time. Finally, boar spermatozoa depicted dose-response effects on sperm kinematics and mitochondrial potential following in vitro challenge with 130 pharmacological drugs and toxic compounds as well as with eight known mito-toxic compounds. In conclusion, boar spermatozoa expressing functional water (AQPs-7 and -9) and ion (CatSper 1-4) channels as well as μ- and δ-opioid receptors are able to adapt to stressful environmental variations, capacitation and pharmacological compounds and drug components. Ejaculated sperm suspensions are easily and painlessly obtained from breeding boars, and are suitable biosensors for in vitro drug-induced testing, complying with the 3R principles of reduction and replacement of experimental animals, during early toxicology screening.