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Developmental and reproductive toxicity of natural mixtures of POPs in Zebrafish

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Developmental and reproductive toxicity of natural mixtures of POPs in Zebrafish

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  1. Figure 4. Effect of exposure on body weight. All the exposure groups had significant higher weight compared to controls (P < 0.05; Two-way Anova analysis). Oestrogen (EE2) exposed zebrafish (water exposure of ethinylestradiol; at 10 ng/L). Groups not connected by the same letter are significant different. Figure 1. Concentrations (ng/g lipid weight) of PBDEs in the feed (artemia) and in whole fish. In addition to PBDEs relative high levels of PCBs and DDTs were also detected.The levels of other OPs including HCBs, HCHs and Chlordanes were below the detection limit. Figure 2. Mortality rates during the first generation. Early-life mortality was significantly increased in the exposure groups LOSNA (mixture from low polluted source) and MJØSA HIGH (mixture from high polluted source, high exposure level). Statistical test: Survival analysis (P < 0.05). Figure 3. Sexual maturity as determined by day for first egg laying. The exposure group which received the highest dose of the Lake Mjøsa mixture (MH) had significant advanced sexual maturation (20 days; P < 0.05; Student's t-test). LOSNA= mixture from Lake Losna, low polluted; M= mixture from lake MJØSA, high polluted; H= high exposure level; L= low exposure level; M= medium exposure level. Figure 5. Effect of exposure on sex ratio. There was a clear trend for a changed sex ratio in male direction in exposed groups. Only the group which received the Lake Losna mixture (low polluted source) was significantly different from controls (P < 0.05; Chi Square test). Figure 6. The EROD activity was measured in gills dissected from the fish. The EROD activity/CYP 1A1 induction was significantly increased in all the exposure groups compared to controls (P < 0.05; Student's t-test). Developmental and reproductive toxicity of natural mixtures of POPs in Zebrafish Rasoul Nourizadeh-Lillabadi1*, Jan L. Lyche1, Camilla Almaas1,2, Benedicte Stavik1,2, Elianne Egge2, Jannicke Moe2, Kjetill S. Jackobsen2, Tom Andersen4, Mona Aleksandersen1, Ragnhild Fuglestveit1, Vidar Berg1, Elisabeth Lie1, Kristian Ingebrigtsen1, Nils Chr. Stenseth2, Janneche Utne Skåre1,3, Peter Alestrøm1, Erik Ropstad1. 1) Norweigan School of Veterinary Science, P.BOX. 8143, 0033 Oslo, Norway. 2)Centre for Ecological and Evolutionary Synthesis (CEES), Department of Biology, University of Oslo, P.O. Box 1066 Blindern, N-0316 Oslo, Norway. 3) National Veterinary Institute, P.BOX. 8153, 0033 Oslo, Norway.4)Department of Biology, University of Oslo, P.O. Box 1066 Blindern, N-0316 Oslo, Norway.*Corresponding author: Rasoul.Nouri@veths.no, Introduction Persistent organic pollutants (POPs) occur as mixtures in the environment. There are gaps in the knowledge on which agents occur in mixtures, at which levels they exert toxicity and about their possible mechanisms of action. Polybrominated diphenyl ethers (PBDEs) have become widely distributed as environmental contaminants similar to other POPs such as dioxins, polychlorinated biphenyls (PCBs) and organochlorine pesticides (OP). In contrast to other POPs which have been phased out of production, vast amounts of PBDEs are still produced and used. As a consequence, the levels in humans and wildlife of PCBs and OPs are declining whereas the levels of PBDEs are still rising. The increasing levels of PBDEs emphasize the need for a better understanding of PBDE toxicokinetics and toxicity. Furthermore, data on the combined effects of environmental exposure to mixtures of PBDEs and other POPs is poor. The aim of this project was to study reproductive and developmental endpoints of lifelong exposure to a natural mixture of POPs containing PBDEs, PCBs and DDTs extracted from a freshwater fish (burbot) captured in two lakes with different histories of pollution. Zebrafish (Danio rerio) is widely used as model organism due to important advantages such as easy to breed, short generation interval, well characterized early development together with a sequenced genome.In the present project, zebrafish was used to study the effects on developmental and reproductive toxicology and toxicogenomics after exposure to natural mixtures of POPs. The mortality rates was monitored throughout the experiment. Materials and Methods Zebrafish was exposed via the feed (artemia) to 2 different complex mixtures of POPs (PCBs, DDTs, PBDEs), extracted from burbot (Lota lota) liver oil. One group was exposed to a mixture from Lake Losna (low level of pollution), three groups were given increasing doses (low, medium, high) of a mixture from Lake Mjøsa (high level of pollution) and the controls received fodder containing only solvent (acetone). The exposure of the F0 generation lasted from 6 days post-hatching until sexual maturation. Results Preliminary results show that larvae mortality during the first 20 days post fertilization was significantly higher in the exposed groups compared to controls. The male/female sex ratio and the male weight gain were significantly higher in the fish exposed to both mixtures. Induction of CYP1A1 was significantly elevated in all exposure groups. Significant increased vitellogenin levels and histological changes were found in liver and gonads of fish exposed to the mixture from Lake Mjøsa. Genome-wide RNA profiling of the same tissues successfully detected gene expression effects correlated with the exposure to POPs. Well known oestrogen responsive genes such as vitellogenin and aromatase were differently regulated in the exposed groups compared to controls, suggesting possible oestrogenic effects. The concentrations of POPs in the zebrafish were at a similar level as in wild fish from Lake Mjøsa suggesting that environmental relevant levels of real-life mixtures may produce developmental and reproductive effects. Table 1. Significantly differently expressed genes (+3.5 to -4.9 fold) in the Lake Mjøsa and the Lake Losna groups as compared to the control group. The genes with known (annotated) and unknown function are indicated. Functional Annotation Tool, DAVID Bioinformatics Resources 2007, NIAID/NIH. Table 2. Genes with up-regulated (red) and down-regulated (green) expression in the liver from the Lake Losna group. The genes were clustered according to endocrine functions. Table 3.This table shows genes which are up-regulated (red) and down-regulated (green) in the livers and ovaries from the Lake Mjøsa group. The genes were clustered according to endocrine functions. Acknowledgement: The ZebPOP project was funded by The Research Council of Norway, Grant No. 172017/V10

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