Effekter av persistente organiske miljøgifter på steroidogenesen i primære neonatale Leydigceller fra gris

After World War II the production of manmade chemicals has increased immensely. They are made to give desired practical properties and functions. Some are added to consumer products to provide conductivity, increase flame resistance, as plasticizer and some act as both grease and water repellants. Others are widely used as pesticides and fungicides. A number of these compounds are spread via water, air, soil leading to exposure of living organisms, including humans. Of particular concern are so-called Persistent Organic Pollutants (POPs), which break down slowly, bio-accumulate in the food web and spread though water and air which make these compounds reach places far from where they have been used or produced. Since the late 1960s exposures to POPs has been associated with disturbance of the reproductive function of birds and fish affecting the hormone systems. Thinning of bird eggshell due to exposure of the pesticide dichlorodiphenyltrichloroethane (DDT) was one of the earliest published reports to get much publicity. Not long after, DDT was banned as a pesticide in Europe and northern America and comities were formed to protect the environment against POPs with harmful effects. Exposures of POPs occur as complex mixtures of POPs in the environment. One POP alone can have different effect than as part of a mixture of many POPs. Further, POPs can break down to metabolites with more and different effects than the mother substances, which also is shown with DDT and its metabolites. In Paper I of this thesis, POPs mixtures extracted from fish oil were exposed to unstimulated and LH-stimulated primary neonatal porcine Leydig cells. The endpoints studied were toxicity, hormone secretion of testosterone (T) and estradiol 17β (E2) in addition to gene expression of selected steroidogenic genes. The three other papers investigated the same endpoints in the same primary cells exposure to the DDT-metabolites; o,p’-DDD, 3-MeSO2-DDE and 3,3’-(bis)MeSO2-DDE. The exception was Paper III, where proteomic analysis was used instead of gene expression. In this article a broad range of proteins were investigated in unstimulated and LH-stimulated cells exposed to 3-MeSO2-DDE. With this we wanted to get closer to an explanation of the effects found in Paper II, and especially why we get increased dose related secretion of hormones in unstimulated cells while reduced secretion was found in LH-stimulated cells. The target for all of our studies was to investigate the effects on hormone secretion at concentrations of POPs or DDT-metabolites not related to cell toxicity. Of the DDTvii metabolites, o,p’-DDD showed toxicity at concentrations ≥10 μM (Paper IV). Some toxicity was also observed in LH-stimulated cells exposed to 3-MeSO2-DDE at concentrations ≥20 μM (Paper II). This thesis contain studies in which differential or partly differential effects with regard to hormone secretion were observed with DDT metabolites and POPs mixtures dependent on whether they were stimulated with LH or not. All POPs mixtures from fish oil and DDT metabolites caused reduced secretion of E2 and T in LH-stimulated Leydig cells while increased secretion of hormones was found in unstimulated Leydig cells (Paper I-IV). The expression of selected genes relevant to the steroidogenesis were mainly downregulated in LH-stimulated and unstimulated Leydig cells exposed to the POP mixtures and DDT metabolites (Paper I, II, IV). The genes STAR, CYP11A, HSD3B, CYP17A1 and CYP19A1 were most often affected. In the proteomic study (Paper III) exposing 3-MeSO2-DDE to Leydig cells 145 proteins in LH-stimulated cells and 86 proteins in unstimulated cells were regulated by 3-MeSO2-DDE. Eleven of these proteins were common for each culture condition. This study indicated that multiple pathways were affected by exposure, including mitochondrial function, oxidative phosphorylation, eukaryotic initiation factor 2 (EIF2) signaling, glutathione detoxification. These results can give a base for new mechanistic studies. Our studies showed that POPs mixtures extracted from fish and the DDT metabolites; o,p’-DDD, 3-MeSO2-DDE and 3,3’-(bis) MeSO2-DDE alters secretion of the hormones E2 and T differently dependent on the presence of absence of LH-stimulation. The Proteomic study (Paper III) indicated that environmental pollutants can affect important cell functions that may indirectly affect hormone secretion of the cell. These results can give grounds for future mechanistic studies.Etter andre verdenskrig har produksjonen av menneskeskapte kjemikalier økt kraftig. De er skapt for å gi ulike produkter ønskede egenskaper, eksempelvis, strømledende, flammeavstøtende, plastifiserende, fett- og vannavstøtende. I tillegg finnes plantevernmidler som er laget for å hemme eller forebygge angrep av skadedyr, sopp og ugras som skader planter. Ulempen med mange av disse forbindelsene er at de brytes ned sakte og akkumuleres i næringskjeder. Disse kjemiske forbindelsene kalles gjerne persistente organiske forurensningsstoffer og benevnes gjerne som POPs (engelsk: Persistent Organic Pollutants). Spredning gjennom vann og luft gjør at disse stoffene kan ende opp langt fra steder de er benyttet eller produsert. Sent på 1960 tallet ble det oppdaget at eksponering for POPs kunne være forbundet med skade på forplantningsevnen hos fugl og fisk. Tynning av fugleskall på grunn av eksponering for plantevernmiddelet dichlorodiphenyltrichloroethane (DDT) var et av de første funnene som fikk mye publisitet. Ikke lenge etter ble DDT forbudt som plantevernmiddel i Europa og NordAmerika, og det ble iverksatt tiltak for å redusere utslipp av POPs med skadelige effekter. POPs forekommer som komplekse blandinger i miljøet. Effekter av disse kan ha andre effekter enn stoffene enkeltvis. POPs blir nedbrutt i varierende grad til metabolitter som kan gi andre effekter enn morsubstansen. Dette er også vist med DDT og dets metabolitter.acceptedVersio

Effects of persistent organic pollutants (POPs) on the steroidogenesis of primary neonatal porcine Leydig cells

After World War II the production of manmade chemicals has increased immensely. They are made to give desired practical properties and functions. Some are added to consumer products to provide conductivity, increase flame resistance, as plasticizer and some act as both grease and water repellants. Others are widely used as pesticides and fungicides. A number of these compounds are spread via water, air, soil leading to exposure of living organisms, including humans. Of particular concern are so-called Persistent Organic Pollutants (POPs), which break down slowly, bio-accumulate in the food web and spread though water and air which make these compounds reach places far from where they have been used or produced. Since the late 1960s exposures to POPs has been associated with disturbance of the reproductive function of birds and fish affecting the hormone systems. Thinning of bird eggshell due to exposure of the pesticide dichlorodiphenyltrichloroethane (DDT) was one of the earliest published reports to get much publicity. Not long after, DDT was banned as a pesticide in Europe and northern America and comities were formed to protect the environment against POPs with harmful effects. Exposures of POPs occur as complex mixtures of POPs in the environment. One POP alone can have different effect than as part of a mixture of many POPs. Further, POPs can break down to metabolites with more and different effects than the mother substances, which also is shown with DDT and its metabolites. In Paper I of this thesis, POPs mixtures extracted from fish oil were exposed to unstimulated and LH-stimulated primary neonatal porcine Leydig cells. The endpoints studied were toxicity, hormone secretion of testosterone (T) and estradiol 17β (E2) in addition to gene expression of selected steroidogenic genes. The three other papers investigated the same endpoints in the same primary cells exposure to the DDT-metabolites; o,p’-DDD, 3-MeSO2-DDE and 3,3’-(bis)MeSO2-DDE. The exception was Paper III, where proteomic analysis was used instead of gene expression. In this article a broad range of proteins were investigated in unstimulated and LH-stimulated cells exposed to 3-MeSO2-DDE. With this we wanted to get closer to an explanation of the effects found in Paper II, and especially why we get increased dose related secretion of hormones in unstimulated cells while reduced secretion was found in LH-stimulated cells. The target for all of our studies was to investigate the effects on hormone secretion at concentrations of POPs or DDT-metabolites not related to cell toxicity. Of the DDTvii metabolites, o,p’-DDD showed toxicity at concentrations ≥10 μM (Paper IV). Some toxicity was also observed in LH-stimulated cells exposed to 3-MeSO2-DDE at concentrations ≥20 μM (Paper II). This thesis contain studies in which differential or partly differential effects with regard to hormone secretion were observed with DDT metabolites and POPs mixtures dependent on whether they were stimulated with LH or not. All POPs mixtures from fish oil and DDT metabolites caused reduced secretion of E2 and T in LH-stimulated Leydig cells while increased secretion of hormones was found in unstimulated Leydig cells (Paper I-IV). The expression of selected genes relevant to the steroidogenesis were mainly downregulated in LH-stimulated and unstimulated Leydig cells exposed to the POP mixtures and DDT metabolites (Paper I, II, IV). The genes STAR, CYP11A, HSD3B, CYP17A1 and CYP19A1 were most often affected. In the proteomic study (Paper III) exposing 3-MeSO2-DDE to Leydig cells 145 proteins in LH-stimulated cells and 86 proteins in unstimulated cells were regulated by 3-MeSO2-DDE. Eleven of these proteins were common for each culture condition. This study indicated that multiple pathways were affected by exposure, including mitochondrial function, oxidative phosphorylation, eukaryotic initiation factor 2 (EIF2) signaling, glutathione detoxification. These results can give a base for new mechanistic studies. Our studies showed that POPs mixtures extracted from fish and the DDT metabolites; o,p’-DDD, 3-MeSO2-DDE and 3,3’-(bis) MeSO2-DDE alters secretion of the hormones E2 and T differently dependent on the presence of absence of LH-stimulation. The Proteomic study (Paper III) indicated that environmental pollutants can affect important cell functions that may indirectly affect hormone secretion of the cell. These results can give grounds for future mechanistic studies

Inflammation in the pleural cavity following injection of multi-walled carbon nanotubes is dependent on their characteristics and the presence of IL-1 genes

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Perfluoroalkyl acids potentiate glutamate excitotoxicity in rat cerebellar granule neurons

Perfluoroalkyl acids (PFAAs) are persistent man-made chemicals, ubiquitous in nature and present in human samples. Although restrictions are being introduced, they are still used in industrial processes as well as in consumer products. PFAAs cross the blood-brain-barrier and have been observed to induce adverse neurobehavioural effects in humans and animals as well as adverse effects in neuronal in vitro studies. The sulfonated PFAA perfluorooctane sulfonic acid (PFOS), has been shown to induce excitotoxicity via the N-methyl-D-aspartate receptor (NMDA-R) in cultures of rat cerebellar granule neurons (CGNs). In the present study the aim was to further characterise PFOS-induced toxicity (1–60 μM) in rat CGNs, by examining interactions between PFOS and elements of glutamatergic signalling and excitotoxicity. Effects of the carboxylated PFAA, perfluorooctanoic acid (PFOA, 300–500 μM) on the same endpoints were also examined. During experiments in immature cultures at days in vitro (DIV) 8, PFOS increased both the potency and efficacy of glutamate, whereas in mature cultures at DIV 14 only increased potency was observed. PFOA also increased potency at DIV 14. PFOS-enhanced glutamate toxicity was further antagonised by the competitive NMDA-R antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) at DIV 8. At DIV 8, PFOS also induced glutamate release (9–13 fold increase vs DMSO control) after 1−3 and 24 h exposure, whereas for PFOA a large (80 fold) increase was observed, but only after 24 h. PFOS and PFOA both also increased alanine and decreased serine levels after 24 h exposure. In conclusion, our results indicate that PFOS at concentrations relevant in an occupational setting, may be inducing excitotoxicity, and potentiation of glutamate signalling, via an allosteric action on the NMDA-R or by actions on other elements regulating glutamate release or NMDA-R function. Our results further support our previous findings that PFOS and PFOA at equipotent concentrations induce toxicity via different mechanisms of action

Perfluoroalkyl acids potentiate glutamate excitotoxicity in rat cerebellar granule neurons

Perfluoroalkyl acids (PFAAs) are persistent man-made chemicals, ubiquitous in nature and present in human samples. Although restrictions are being introduced, they are still used in industrial processes as well as in consumer products. PFAAs cross the blood-brain-barrier and have been observed to induce adverse neurobehavioural effects in humans and animals as well as adverse effects in neuronal in vitro studies. The sulfonated PFAA perfluorooctane sulfonic acid (PFOS), has been shown to induce excitotoxicity via the N-methyl-Daspartate receptor (NMDA-R) in cultures of rat cerebellar granule neurons (CGNs). In the present study the aim was to further characterise PFOS-induced toxicity (1–60 μM) in rat CGNs, by examining interactions between PFOS and elements of glutamatergic signalling and excitotoxicity. Effects of the carboxylated PFAA, perfluorooctanoic acid (PFOA, 300–500 μM) on the same endpoints were also examined. During experiments in immature cultures at days in vitro (DIV) 8, PFOS increased both the potency and efficacy of glutamate, whereas in mature cultures at DIV 14 only increased potency was observed. PFOA also increased potency at DIV 14. PFOSenhanced glutamate toxicity was further antagonised by the competitive NMDA-R antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) at DIV 8. At DIV 8, PFOS also induced glutamate release (9–13 fold increase vs DMSO control) after 1− 3 and 24 h exposure, whereas for PFOA a large (80 fold) increase was observed, but only after 24 h. PFOS and PFOA both also increased alanine and decreased serine levels after 24 hexposure. In conclusion, our results indicate that PFOS at concentrations relevant in an occupational setting, may be inducing excitotoxicity, and potentiation of glutamate signalling, via an allosteric action on the NMDA-R or by actions on other elements regulating glutamate release or NMDA-R function. Our results further support our previous findings that PFOS and PFOA at equipotent concentrations induce toxicity via different mechanisms of action

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Effects of a human-based mixture of persistent organic pollutants on the in vivo exposed cerebellum and cerebellar neuronal cultures exposed in vitro

Exposure to persistent organic pollutants (POPs), encompassing chlorinated (Cl), brominated (Br) and perfluoroalkyl acid (PFAA) compounds is associated with adverse neurobehaviour in humans and animals, and is observed to cause adverse effects in nerve cell cultures. Most studies focus on single POPs, whereas studies on effects of complex mixtures are limited. We examined the effects of a mixture of 29 persistent compounds (Cl + Br + PFAA, named Total mixture), as well as 6 sub-mixtures on in vitro exposed rat cerebellar granule neurons (CGNs). Protein expression studies of cerebella from in vivo exposed mice offspring were also conducted. The selection of chemicals for the POP mixture was based on compounds being prominent in food, breast milk or blood from the Scandinavian human population. The Total mixture and sub-mixtures containing PFAAs caused greater toxicity in rat CGNs than the single or combined Cl/Br sub-mixtures, with significant impact on viability from 500x human blood levels. The potencies for these mixtures based on LC50 values were Br + PFAA mixture > Total mixture > Cl + PFAA mixture > PFAA mixture. These mixtures also accelerated induced lipid peroxidation. Protection by the competitive N-methyl-D-aspartate (NMDA) receptor antagonist 3-((R)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) indicated involvement of the NMDA receptor in PFAA and Total mixture-, but not Cl mixture-induced toxicity. Gene-expression studies in rat CGNs using a sub-toxic and marginally toxic concentration ((0.4 nM-5.5 µM) 333x and (1 nM-8.2 µM) 500x human blood levels) of the mixtures, revealed differential expression of genes involved in apoptosis, oxidative stress, neurotransmission and cerebellar development, with more genes affected at the marginally toxic concentration. The two important neurodevelopmental markers Pax6 and Grin2b were downregulated at 500x human blood levels, accompanied by decreases in PAX6 and GluN2B protein levels, in cerebellum of offspring mice from mothers exposed to the Total mixture throughout pregnancy and lactation. In rat CGNs, the glutathione peroxidase gene Prdx6 and the regulatory transmembrane glycoprotein gene Sirpa were highly upregulated at both concentrations. In conclusion, our results support that early-life exposure to mixtures of POPs can cause adverse neurodevelopmental effects