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Polybrominated diphenyl ethers (PBDEs) are environmental contaminants found in human and animal tissues worldwide. Neonatal exposure to the flame retardant 2,2', 4,4',5-pentabromodiphenyl ether (PBDE-99) disrupts normal brain development in mice, and results in disturbed spontaneous behavior in the adult. The mechanisms underlying the late effects of early exposure are not clear. To gain insight into the initial neurodevelopmental damage inflicted by PBDE-99, we investigated the short-term effects of PBDE-99 on protein expression in the developing cerebral cortex of neonatal mice, and the cytotoxic and apoptotic effects of PBDE-99 in primary cultures of fetal rat cortical cells. We used two-dimensional difference gel electrophoresis (2D-DIGE) to analyze protein samples isolated from the cortex of NMRI mice 24h after exposure to a single oral dose of 12 mg/kg PBDE-99 on post-natal day 10. Protein resolution was enhanced by sample pre-fractionation. In the cell model, we determined cell viability using the trypan blue exclusion assay, and apoptosis using immunocytochemical detection of cleaved caspase-3. We determined the identity of 111 differentially expressed proteins, 32 (29%) of which are known to be cytoskeleton-related. Similar to previous findings in the striatum, we found elevated levels of the neuron growth-associated protein Gap43 in the cortex. In cultured cortical cells, a high concentration of PBDE-99 (30 microM) induced cell death without any apparent increase in caspase-3 activity. These results indicate that the permanent neurological damage induced by PBDE-99 during the brain growth spurt involve detrimental effects on cytoskeletal regulation and neuronal maturation in the developing cerebral cortex.

Exposure to the brominated flame retardant 2,2 ,4,4 ,5-pentabromodiphenyl ether (PBDE-99) during the brain growth spurt disrupts normal brain development in mice and results in disturbed spontaneous behavior in adulthood. The neurodevelopmental toxicity of PBDE-99 has been reported to affect the cholinergic and catecholaminergic systems. In this study we use a proteomics approach to study the early effect of PBDE-99 in two distinct regions of the neonatal mouse brain, the striatum and the hippocampus. A single oral dose of PBDE-99 (12 mg/kg body weight) or vehicle was administered to male NMRI mice on neonatal day 10, and the striatum and the hippocampus were isolated. Using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), we found 40 and 56 protein spots with significantly (p < 0.01) altered levels in the striatum and the hippocampus, respectively. We used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-ToF-MS) to determine the protein identity of 11 spots from the striatum and 10 from the hippocampus. We found that the levels of proteins involved in neurodegeneration and neuroplasticity (e.g., Gap-43/neuromodulin, stathmin) were typically altered in the striatum, and proteins involved in metabolism and energy production [e.g., alpha-enolase; gamma-enolase; ATP synthase, H+ transporting, mitochondrial F1 complex, beta subunit (Atp5b); and alpha-synuclein] were typically altered in the hippocampus. Interestingly, many of the identified proteins have been linked to protein kinase C signaling. In conclusion, we identify responses to early exposure to PBDE-99 that could contribute to persistent neurotoxic effects. This study also shows the usefulness of proteomics to identify potential biomarkers of developmental neurotoxicity of organohalogen compounds.

Bakgrund: År 1978 introduceras ett klassificeringssystem i FASS (Farmaceutiska specialiteter i Sverige) för att underlätta bedömningen vid förskrivning av läkemedel till gravida och ammande kvinnor. Detta gäller bedömningen av ett läkemedels risk för bieffekter under graviditet och amning utifrån vilken kategori det placeras i. När Sverige 1995 går med i EU beslutar den medicinska expertgruppen att inte längre granska FASS- texter utan lämna över uppgiften till själva läkemedelsföretagen. Syfte: I detta projektarbete jämförs FASS-texter under rubrikerna Graviditet och Amning med läkemedelskategorin som de är placerade i. Metod och Material: Läkemedelsprodukter som granskas i arbetet är totalt 422 produkter med indikationsområden för andningsorgan (ATC- grupp R) samt ögon och öron (ATC-grupp S). Läkemedelsgrupperna är intressanta eftersom de innehåller läkemedel som rekommenderas under graviditet samt amning som till exempel inhalationspreparat mot astma eller ögondroppar mot glaukom. De texter som granskas har hämtats från FASS hemsida www.fass.se. Ytterligare information såsom namn, form, styrka, beredningsform och tillverkare för läkemedlen har hämtats från NPL (Nationellt Produktregister för Läkemedel). Resultat: Studien visar att ca 5 % respektive 13 % av läkemedel inom indikationsområden Andningsorgan (ATC-grupp R) samt Ögon och Öron (ATC-grupp S) är felklassificerade i förhållande till kriterierna för en FASS-text. Procenten inkluderar även läkemedel med saknad klassificering. Diskussion: Eftersom granskning av FASS-texter inte sköts av någon myndighet längre är läkemedelsföretagen själva ansvariga för informationen som står i FASS idag. Detta har lett till problem i form av saknad klassificering eller fel klassificering av läkemedel i FASS-texter något som är viktigt ur graviditets- och amningsperspektiv. Problemet bör uppmärksammas av ansvariga läkemedelsföretag och kontrollmyndigheter.

Introduction: Occurrence and emission of chemical pollutants such as organic compounds, heavy metals and pharmaceuticals are high in the aquatic environment and a concern worldwide due to the risk of affecting organisms negatively. Biomarkers can indicate the magnitude of organism response to environmental pollutants and provide the causal link between the presence of a chemical and ecological effect. These pollution indicators can be used in effect-based environmental monitoring. Cytochrome p450 1A (CYP1A), metallothionein (MT) and cytochrome p450 3A (CYP3A) are examples which can be used as biomarkers for identification of exposure of pollutants.

The aims of this project were to find functioning primers for MT, CYP1A and CYP3A, and examine whether mRNA levels of these genes are relevant to use as biomarkers to demonstrate exposure of metals, dioxin-like chemicals and ethinyl estradiol (EE2).

Methods: The tadpoles were exposed to Cadmium chloride (CdCl₂), β-naphthoflavone (BNF) and EE2. RNA of the liver tissue samples was isolated, and concentration and integrity were measured. The primers were designed based on different strategies, A, existing gene mRNA sequences, B, Short Read Archive (SRA) database of NBCI and C, extended RNA-sequencing. qRTPCR was used for RNA-primer-testing and mRNA expression level measurement.

The results show significantly differences between sites and individuals, expression level of CYP1A was significantly induced but no induction of CYP3A and MT was achieved.

Conclusions: Applying CYP1A gene as biomarker on wild tadpoles looks promising. For the use of CYP3A and MT as biomarkers, further efforts are needed.

Occurrence of pharmaceutical residues in surface and ground waters, used as a source for drinking water supply has been studied over the years. Wastewater treatment plants are generally not efficient enough to remove pharmaceuticals from wastewater and sewage sludge. As a result, they are released into the environment mainly via effluent discharges to surface water bodies, the reuse of biosolids in agriculture as soil amendment, and the disposal of biosolids to landfill areas.

The aim of this study was to investigate the leaching behavior of pharmaceuticals from several therapeutic groups (i.e. atorvastatin, bicalutamide, carbamazepine, furosemide, hydrochlorothiazide, losartan, oxazepam, valsartan, and venlafaxine) using undisturbed soil columns (loamy sand, loam, and clay) and disturbed soil columns (till mixed with peat) amended with sewage sludge at laboratory-scale. The experiment was performed under controlled conditions to gain a better understanding of the leaching behavior of pharmaceuticals in the natural environment. Another objective was to single out the pharmaceuticals with the highest leaching potential (i.e. carbamazepine, hydrochlorothiazide, oxazepam, and valsartan) in order to analyze their effects on the environment based on earlier studies on the subject. Oxazepam, for example, has shown to have effects on fish such as increased feeding rate and activity.

Leachate analysis show that, in general, pharmaceuticals leach slower in loamy sand compared to loam and clay which might be due to macro pores and funnel flow in the loamy and clayish soil which enhance the leaching rate. The accumulated mass (µg) in the leachate from clay (13) were also higher than in loam (5.7), loamy sand (2.6), and the till and peat (0) soil.  This indicates that the slow leaching of pharmaceuticals in loamy sand, and till and peat might enhance the sorption or degradation of pharmaceuticals compared to clay and loam.

Chromium picolinate (CrPic) is a synthetic nutritional supplement primarily used for weight loss and muscle building. Recent studies have indicated that CrPic might be genotoxic and these findings together with the wide-spread consumer use, have increased the concern about its safety. In the present study we investigated the potential genotoxicity of CrPic in mice given a single intraperitoneal injection (up to 3 mg/kg b.wt.) by evaluating the frequency of micronucleated polychromatic erythrocytes (fMNPCE) in peripheral blood, and DNA damage in lymphocytes and hepatocytes. The fMNPCE was evaluated after 42 h and DNA damage after 16 h. Using the Comet assay DNA damage was also monitored in extended-term cultures of human lymphocytes and in L5178Y mouse lymphoma cells that had been exposed for 3 h to 500 μM CrPic under different exposure conditions.

A slight, but significant CrPic-induced increase in DNA damage (P < 0.001) was observed in the human lymphocytes, but only when these cells were exposed in the absence of serum. In all other experiments CrPic was found to be without genotoxic effects, both in vivo and in vitro. Taken together, our results suggest that a high concentration of CrPic might be DNA damaging, but only under non-physiological conditions.

It has been suggested that extended-term cultures of human lymphocytes could be used as a complement to cell lines based on transformed cells when testing the genotoxicity of chemicals. To investigate whether the pattern of induced DNA damage and its subsequent repair differs significantly between cultures based on different blood donors, hydrogen peroxide (H₂O₂)-induced DNA damage was measured in cultures from four different subjects using the comet assay. The DNA damage was significantly increased in all cultures after 10 min exposure to 0.25 mmol/L H₂O₂, and there was a significant decrease in the H₂O₂-induced DNA damage in all cultures after 30 min of DNA repair. The level of damage varied between the different donors, especially after the repair. Using PCR and DNA sequencing, exon 5 of the p53 gene was sequenced in the lymphocytes from the donors with the lowest and highest residual damage. No such mutation was found. Mouse lymphoma L5178Y cells carrying the p53 mutation in exon 5 were included as a reference. These cells were found to be less sensitive toward the H₂O₂-induced DNA damage, and they were also found to have a rather low DNA repair capacity. The demonstrated variation in H₂O₂-induced DNA damage and DNA repair capacity between the cultures from the different subjects may be important from a risk assessment perspective, but is obviously not of decisive importance when it comes to the development of a routine assay for genotoxicity.

The xenobiotic-metabolizing activity of avian heart was investigated in chicken and Eider duck embryos exposed to aryl hydrocarbon (Ah) receptor agonists in ovo. Both beta-naphthoflavone (BNF) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) induced 7-ethoxyresorufin O-deethylase (EROD) activities in chicken embryo hearts whereas Eider duck embryos only responded to BNF. The differential responses of chicken and Eider duck embryos were used to examine the involvement of Ah receptor-mediated enzyme induction in the activation of the environmental and food mutagen 3-amino- 1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1). As determined by light microscopic autoradiography, there was a highly selective binding of non-extractable 3H-Trp-P-1-derived radioactivity in endothelial cells of large vessels and capillaries in hearts of BNF- and PCB 126-treated chicken embryos. No binding occurred at these sites in vehicle-treated controls. There was also a strong endothelial binding of 3H-Trp-P-1 in hearts of BNF-treated Eider duck embryos whereas no binding occurred in hearts of PCB 126-treated Eider duck embryos. A positive correlation between induction of EROD activity and covalent binding of 3H-Trp-P-1 to protein in heart homogenates from BNF- and PCB 126-treated chicken and Eider duck embryos was also observed. The results suggest a cytochrome P450 1A (CYP1A)-mediated activation of Trp-P-1 in avian heart endothelial cells although involvement of other Ah receptor-regulated enzymes is also possible. We propose that heart endothelial cells may be targets for bioactivation and toxicity of environmental contaminants in birds exposed to Ah receptor agonists.

The chorioallantoic membrane (CAM) is a highly vascularized tissue that takes part in the respiratory exchange of gases through the eggshell. Although the CAM may be exposed to environmental contaminants, its response to pollutants has not been studied. We examined the cytochrome P4501A (CYP1A)-catalyzed deethylation of 7-ethoxyresorufin (EROD) in the CAM during chicken embryo development. EROD was constitutively present and was inducible by the aryl hydrocarbon (Ah) receptor agonist 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126). Our results suggest the CAM as a first line of defence of the avian embryo against toxic compounds, but also as a target for CYP1A-activated chemicals.

PURPOSE: There is evidence that drug-induced embryonic arrhythmia initiates phenytoin (PHT) teratogenicity. The arrhythmia, which links to the potential of PHT to inhibit a specific potassium channel (Ikr), may result in episodes of embryonic ischemia and generation of reactive oxygen species (ROS) at reperfusion. This study sought to determine whether the proposed mechanism might be relevant for the teratogenic antiepileptic drug trimethadione (TMO). METHODS: Effects on embryonic heart rhythm during various stages of organogenesis were examined in CD-1 mice after maternal administration (125-1,000 mg/kg) of dimethadione (DMO), the pharmacologically active metabolite of TMO. Palatal development was examined after administration of a teratogenic dose of DMO and after simultaneous treatment with DMO and a ROS-capturing agent (alpha-phenyl-N-tert-butyl-nitrone; PBN). The Ikr blocking potentials of TMO and DMO were investigated in HERG-transfected cells by using voltage patch-clamping tests. RESULTS: DMO caused stage-specific (gestation days 9-13 only) and dose-dependent embryonic bradycardia and arrhythmia at clinically relevant maternal plasma concentrations (3-11 mM). Hemorrhage in the nasopharyngeal part of the embryonic palate (within 24 h) preceded cleft palate in fetuses at term. Simultaneous treatment with PBN significantly reduced the incidence of DMO-induced cleft palate, from 40 to 13%. Voltage patch-clamping studies showed that particularly DMO (70% inhibition), but also TMO, had Ikr blocking potential at clinically relevant concentrations. CONCLUSIONS: TMO teratogenicity, in the same way as previously shown for PHT, was associated with Ikr-mediated episodes of embryonic cardiac arrhythmia and hypoxia/reoxygenation damage.

The blood-brain barrier (BBB) is formed by endothelial cells, connected by tight junction proteins restricting paracellular transport. Transport of substances into the brain may also be limited due to active efflux transporters. Not much is known about the development of tight junction proteins and active transporters in the BBB, and how this affects drug distribution to the brain at different ages. The aim of this study was to analyze possible differences in expression of tight junction proteins and active transporters in the BBB at different postnatal ages in the rat. Brain capillary-rich fractions (BCRF) were collected and gene expression levels were compared at three postnatal ages using microarray analysis. BCRF was also analyzed for the mRNA expression levels of P-gp Abcb1 (P-gp), Abcg2 (Bcrp), Slc22a8 (Oat3) and occludin using real-time PCR at 12 different postnatal ages between postnatal Day 1 and Adult. In the array analysis, 28 genes of interest were found to be differentially expressed. The mRNA levels of Abcg2 decreased to one seventh from postnatal Day 1 to Day 15. The levels of Slc22a8 increased from birth and reached a plateau at Day 3 - 12 followed by a decrease to Day 15. These findings show that active transporters are differentially expressed in the developing BBB, possibly affecting drug distribution to the brain. No change in the mRNA levels for Abcb1 or occludin was observed.

Abstract

Background: Aromatase, encoded by CYP19A1, is an enzyme that converts androgens to estrogens. The enzyme is expressed in various tissues in the human body. The aim of this study is to investigate if there is a tissue-specific effect of 1α,25-dihydroxyvitamin D₃ by analyzing the gene expression of CYP19A1 in cell lines representing the human adrenal cortex, the human ovarian, human osteoblastic cells and human breast glands. The results could be important for the evaluation of the NCI-H295R cell line as a toxicological screening method in which aromatase is an important enzyme and in the development of endocrine cancer therapy with CYP19A1 as a target.Methods: The change in gene expression followed exposure for 1α,25-dihydroxyvitamin D₃ was measured  using qRT-PCR. Primers were specific for the total aromatase gene or promoter-specific regions of the gene.Results: The results show that 1α,25-dihydroxyvitamin D₃ exerts a tissue specific effect on CYP19A1 expression. The gene expression was increased in the ovarian cells, the osteoblastic cells and the adrenocarcinoma cells, while it was decreased in the breast cancer cells. The results also showed an altered gene expression due to a promoter-specific regulation.Conclusion: It has become more clear that 1α,25-dihydroxyvitamin D₃ could be a potential anti-breast cancer agent in the future. However, the difference in aromatase gene expression between the breast cancer cells and the adrenocarcinoma NCI-H295R cells indicates that the NCI-H295R cell line might not be reliable for toxicological screening on sex hormone production on its own, unless there is a way to take into account the tissue-specific regulation of CYP19A1.