Espen Mariussen

The effect of brominated flame retardants on neurotransmitter uptake into rat brain synaptosomes and vesicles.

The environmental levels of brominated flame retardants (BFRs) are increasing, but little is known about their toxic effects. In this paper, we show that some of the most important BFRs in commercial use today, have a neurotoxicological potential. Hexabromocyclododecane (HBCD) and tetrabromobisphenol-A (TBBPA) inhibit plasma membrane uptake of the neurotransmitters dopamine, glutamate and gamma-amino-n-butyric acid (GABA) at a concentration level similar to what previously found for polychlorinated biphenyls (PCBs) and even for ecstasy. The IC(50) value for HBCD on dopamine uptake was 4 microM, and the IC(50) values for TBBPA were 9, 6 and 16 microM for dopamine, glutamate and GABA, respectively. HBCD also inhibited glutamate uptake at low concentrations, but never achieved more than 50% inhibition. The inhibition was primarily due to their effect on the membrane potential, measured by the membrane potential marker tetraphenylphosphonium bromide (TPP(+)). Other brominated flame retardants such as octaBDE and decaBDE did not have any effects on uptake. TBBPA, HBCD and even the pentabrominated diphenylether mixture (pentaBDE, DE-71, Great Lakes) also inhibited the vesicular uptake of dopamine with an IC(50) value of 3, 3 and 8 microM, respectively. The neurotoxicological consequences of these findings for environmental contaminants such as BFRs and PCBs are discussed.

Molecular Mechanisms Involved in the Toxic Effects of Polychlorinated Biphenyls (PCBs) and Brominated Flame Retardants (BFRs)

Exposure to polychlorinated biphenyls (PCBs) and brominated flame-retardants (BFRs) in human, primates, and rodents is accompanied by neurobehavioral changes. These involve adverse effects on both memory and learning and motor activity. There are also adverse effects observed on the endocrine and immune system. This review is restricted to our laboratorys recent findings of effects of these compounds on the nervous system and some molecular effects on the immune system. In the nervous system, data showed that PCBs and BFRs produce an effect on neurotransmitter transport mechanisms, in particular the neurotransmitter dopamine. It was demonstrated that this might explain the loss of dopamine in the brain seen after exposure to PCB. Further, it may explain the behavior of dopamine in preparations in vitro from brain tissue after exposure to PCB. Recently it was also reported that PCB and some BFRs induce formation of reactive oxygen species (ROS) in neurons. ROS act as messengers in the nervous system and may also be involved in cell death. In the case of PCB exposure, a correlation between ROS formation and death of neurons was found. In the immune system it was shown that PCBs and some of the BFRs induce formation of ROS in neutrophils (granulocytes). This takes place primarily through phosphorylation and subsequent activation of the NADPH oxidase. This production of ROS may have an adverse effect on the immune system.

Absence of synapsin I and II is accompanied by decreases in vesicular transport of specific neurotransmitters.

Studies of synapsin‐deficient mice have shown decreases in the number of synaptic vesicles but knowledge about the consequences of this decrease, and which classes of vesicles are being affected, has been lacking. In this study, glutamatergic, GABAergic and dopaminergic transport has been analysed in animals where the genes encoding synapsin I and II were inactivated. The levels of the vesicular glutamate transporter (VGLUT) 1, VGLUT2 and the vesicular GABA transporter (VGAT) were decreased by approximately 40% in adult forebrain from mice devoid of synapsin I and II, while vesicular monoamine transporter (VMAT) 2 and VGLUT3 were present in unchanged amounts compared with wild‐type mice. Functional studies on synaptic vesicles showed that the vesicular uptake of glutamate and GABA was decreased by 41 and 23%, respectively, while uptake of dopamine was unaffected by the lack of synapsin I and II. Double‐labelling studies showed that VGLUT1 and VGLUT2 colocalized fully with synapsin I and/or II in the hippocampus and neostriatum, respectively. VGAT showed partial colocalization, while VGLUT3 and VMAT2 did not colocalize with either synapsin I or II in the brain areas studied. In conclusion, distinct vesicular transporters show a variable degree of colocalization with synapsin proteins and, hence, distinct sensitivities to inactivation of the genes encoding synapsin I and II.

Spatial diastereomer patterns of hexabromocyclododecane (HBCD) in a Norwegian fjord

Hexabromocyclododecane (HBCD) is the third most used brominated flame retardant globally, and has been found widely distributed in the environment. The present study reports concentrations and spatial patterns of alpha, beta and gamma-HBCD in a contaminated Norwegian fjord. Intertidal surface sediment and selected species from the marine food web were sampled at five locations in increasing distance from a known point source of HBCD. All sediment and biota samples were analyzed for the three HBCD diastereomers by liquid chromatography and mass spectrometry (LC/MS). The results demonstrated a HBCD gradient with decreasing concentrations at increasing distance from the point source in sediment and sedentary species, but less so in the species with large feeding ranges. Mean concentrations of Sigma HBCD at the closest/most remote locations relative to the point source were 9000/300 ng g(-1) TOC in sediment and 150/90 ng g(-1) lw in the species with largest feeding range (great black-backed gull). The HBCD diastereomer patterns were similar for each of the matrices (sediment, organisms) independent of distance from the source, indicating no difference in environmental partitioning between the diastereomers. However, the concentration ratio of diastereomers in each matrix ranged from 3:1:10 (alpha:beta:gamma) in the sediments to 55:1 (alpha:gamma) in the highest trophic level species, suggesting diastereomer-specific bioaccumulation in the organisms.

Spatial patterns of polybrominated diphenyl ethers (PBDEs) in mosses, herbivores and a carnivore from the Norwegian terrestrial biota

The widespread occurrence of polybrominated diphenyl ethers (PBDEs) in the environment has attracted considerable attention, leading to concerns about the extent and magnitude of wildlife and human exposure. In this work, we focus on the occurrence and fate of PBDEs in a Norwegian air-plant-herbivore-carnivore system. Specifically, we have analysed for PBDEs in moss, livers from various terrestrial herbivores (moose, grouse, and European roe deer) and, for the first time, livers from the top predator lynx. The samples were collected from different sites and time periods (1990-2004) to identify possible spatial and temporal trends in contaminant levels and patterns. The general finding was that PBDEs were found in all (biotic) samples, although at lower concentrations than previously observed in mammals from the marine environment. The PBDE levels in the herbivores ranged from less than 0.5 ng/g lipid weight to 9.4 ng/g lipid weight as the highest. The median PBDE concentration in lynx was approximately one order of magnitude higher than in the herbivores. In the lynx samples there was a predominance of BDE-153 whereas BDE-47 and 99 dominated in the herbivores. This probably reflects different bioaccumulation properties or metabolic transformation processes of the BDE-congeners, and food choice. Levels of PBDEs in both moss and herbivores showed a general decline towards the northern parts of Norway. No clear temporal trends were observed. The PBDE levels observed in this study were low and are probably of limited toxicological significance.

Episodic discharge of lead, copper and antimony from a Norwegian small arm shooting range

Small arm shooting ranges are major deposits of lead (Pb), copper (Cu), antimony (Sb), and zinc (Zn) from use of ammunition. Metals and metalloids from the ammunition residues may leach into the soil and surrounding watercourses and may pose a threat to exposed wildlife and humans. Discharge of elements is dependent on several factors such as soil properties, hydrological conditions, precipitation and time. In order to identify strategies to limit spread of ammunition residues from shooting ranges it is of importance to gain knowledge of the mobility of the elements from the spent bullets. Here we have monitored the levels of Pb, Cu and Sb in a small drainage stream from a Norwegian military small arm shooting range in 2001 and 2006. The first campaign in 2001 was initiated ahead of the snow-melting period in the spring and continued two months in order to quantify discharge and discharge patterns of the selected elements in the drainage stream. Two shorter campaigns were performed in autumn 2001 during an excavation, and in summer 2006. Mean levels of Pb, Cu and Sb in the stream during the first monitoring period in 2001 were 14 microg/l, 39 microg/l and 9 microg/l respectively. High flow, following precipitation, led to an approximately fourfold increase in the concentration of Pb and threefold increase in the level of Cu and Sb compared to low discharge concentrations. An estimation of discharge of the metals showed that the snow melting period and precipitation events constituted to a large proportion of the total release. A sudden increase in the levels can induce more stress and reduce survival of exposed aquatic animals due to the short time available for adaptation.

Antimony (Sb) and lead (Pb) in contaminated shooting range soils: Sb and Pb mobility and immobilization by iron based sorbents, a field study

Small-arm shooting ranges often receive a significant input of lead (Pb), copper (Cu) and antimony (Sb) from ammunition. The goal of the present study was to investigate the mobility, distribution and speciation of Pb and Sb pollution under field conditions in both untreated and sorbent-amended shooting range soil. Elevated Sb (19-349μgL(-1)) and Pb (7-1495μgPbL(-1)) concentrations in the porewater of untreated soil over the four-year test period indicated a long-term Sb and Pb source to the adjacent environment in the absence of remedial measures. Mixing ferric oxyhydroxide powder (CFH-12) (2%) together with limestone (1%) into the soil resulted in an average decrease of Sb and Pb porewater concentrations of 66% and 97%, respectively. A similar reduction was achieved by adding 2% zerovalent iron (Fe°) to the soil. The remediation effect was stable over the four-year experimental period indicating no remobilization. Water- and 1M NH4NO3-extractable levels of Sb and Pb in field soil samples indicated significant immobilization by both treatments (89-90% for Sb and 89-99% for Pb). Results from sequential extraction analysis indicate fixation of Sb and Pb in less accessible fractions like amorphous iron oxides or even more crystalline and residual mineral phases, respectively. This work shows that amendment with Fe-based sorbents can be an effective method to reduce the mobility of metals both in cationic and anionic form in polluted shooting range soil.

Diastereomer-specific bioaccumulation of hexabromocyclododecane (HBCD) in a coastal food web, Western Norway

The present study reports diastereomer-specific accumulation of HBCD from a point source in five marine species representing a typical food web in a Norwegian coastal area. Samples of mussels, polychaetes, crabs and seabird eggs were analyzed for the diastereomers α-, β- and γ-HBCD, as well as lipid content and stable isotopes of nitrogen ((15)N/(14)N) to estimate trophic level. Accumulated HBCD did not correlate well with lipid content for most of the species, thus wet-weight based concentrations were included in an assessment of biomagnification. In contrast to β- and γ-HBCD, the α-diastereomer increased significantly with trophic level, resulting in magnification factors >1 in this coastal marine ecosystem. Data for poikilotherms did not show the same positive correlation between the α-diastereomer and trophic position as homeotherms. The apparent biomagnification of the α-HBCD could be due to bioisomerization or diastereomer-specific elimination that differed between poikilotherms and homeotherms.

Accumulation and disposition of hexabromocyclododecane (HBCD) in juvenile rainbow trout (Oncorhynchus mykiss)

The brominated flame retardant hexabromocyclododecane (HBCD) has been reported in environmental samples worldwide. The three diastereomers, alpha-, beta- and gamma-HBCD, behave differently in aquatic food webs; likely depending on different factors influencing assimilation efficiency and metabolism. In the present study, two oral exposure experiments with rainbow trout were performed to assess the role of selective uptake on diastereomer-specific accumulation and disposition of HBCD to liver, brain and muscle. In both experiments, juvenile rainbow trout (Oncorhynchus mykiss) were administrated a technical HBCD-mixture in commercial feed (10mgkg(-1)), followed by up to 21 days of food deprivation. Already 6h after exposure, the HBCD accumulation was significant, and the concentrations peaked 4-8 days after the exposure. The relative change in HBCD pattern during the accumulation process (0-8 days), suggested that there was a diastereomer-selective uptake of alpha- and beta-HBCD in the rainbow trout. During the initial 48h, considerable amounts of all three diastereomers were distributed to liver, brain and muscle. A 70% reduction in SigmaHBCD levels after 21 days, indicated elimination of HBCD from brain and liver, but no clear elimination from the muscle was observed. Differences in HBCD pattern between organs at the end of the experiment support a proposal of an organ-specific diastereomer accumulation.

Perfluoroalkylated compounds induce cell death and formation of reactive oxygen species in cultured cerebellar granule cells.

The present communication investigates the effects of different perfluoroalkylated compounds (PFCs) on formation of reactive oxygen species (ROS) and cell death in cultured cerebellar granule cells. This allows direct comparison with similar effects found for other environmental contaminants like polychlorinated biphenyls and brominated flame-retardants. The increase in ROS formation and cell death was assayed using the fluorescent probe 2,7-dichlorofluorescin diacetate (DCFH-DA) and the trypan blue exclusion assay. The effects of the PFCs were structure dependent. Cell death was induced at relatively low concentrations by perfluorooctyl sulfonate (PFOS), perfluorooctane sulfonylamide (PFOSA) and the fluorotelomer alcohol 1H, 1H, 2H, 2H-perfluorodecanol (FTOH 8:2) with EC(50)-values of 62 ± 7.6, 13 ± 1.8 and 15 ± 4.2 μM (mean ± SD) respectively. PFOS, perfluorooctanoic acid (PFOA) and PFOSA induced a concentration dependent increase in ROS formation with EC(50)-values of 27 ± 9.0, 25 ± 11 and 57 ± 19μM respectively. Reduced cell viability and ROS formation were observed at concentration level close to what is found in serum of occupationally exposed workers. The effect of PFCs on ROS formation and cell viability was compared with other halogenated compounds and future investigations should emphasize effects of mixtures and how physical chemical properties of the compounds influence their toxicity.