Effects of the UV filter benzophenone-3 (oxybenzone) at low concentrations in zebrafish (Danio rerio).

Organic UV filters including benzophenone-3 (BP-3) are widely used to protect humans and materials from damage by UV irradiation. Despite the environmental occurrence of BP-3 in the aquatic environment, little is known about its effects and modes of action. In the present study we assess molecular and physiological effects of BP-3 in adult male zebrafish (Danio rerio) and in eleuthero-embryos by a targeted gene expression approach focusing on the sex hormone system. Fish and embryos are exposed for 14 days and 120 hours post fertilization, respectively, to 2.4-312 µg/L and 8.2-438 µg/L BP-3. Chemical analysis of water and fish demonstrates that BP-3 is partly transformed to benzophenone-1 (BP-1) and both compounds are accumulated in adult fish. Biotransformation to BP-1 is absent in eleuthero-embryos. BP-3 exposure leads to similar alterations of gene expression in both adult fish and eleuthero-embryos. In the brain of adult males esr1, ar and cyp19b are down-regulated at 84 µg/L BP-3. There is no induction of vitellogenin expression by BP-3, both at the transcriptional and protein level. An overall down-regulation of the hsd3b, hsd17b3, hsd11b2 and cyp11b2 transcripts is observed in the testes, suggesting an antiandrogenic activity. No histological changes were observed in the testes after BP-3 treatment. The study leads to the conclusion that low concentrations of BP-3 exhibit similar multiple hormonal activities at the transcription level in two different life stages of zebrafish. Forthcoming studies should show whether this translates to additional physiological effects.

Protein and metal cluster structure of the wheat metallothionein domain γ-E(c)-1: the second part of the puzzle.

Metallothioneins (MTs) are small cysteine-rich proteins coordinating various transition metal ions, including Zn(II), Cd(II), and Cu(I). MTs are ubiquitously present in all phyla, indicating a successful molecular concept for metal ion binding in all organisms. The plant MT E(c)-1 from Triticum aestivum, common bread wheat, is a Zn(II)-binding protein that comprises two domains and binds up to six metal ions. The structure of the C-terminal four metal ion binding ß(E) domain was recently described. Here we present the structure of the N-terminal second domain, γ-E(c)-1, determined by NMR spectroscopy. The γ-E(c)-1 domain enfolds an M (2) (II) Cys(6) cluster and was characterized as part of the full-length Zn(6)E(c)-1 protein as well as in the form of the separately expressed domain, both in the Zn(II)-containing isoform and the Cd(II)-containing isoform. Extended X-ray absorption fine structure analysis of Zn(2)γ-E(c)-1 clearly shows the presence of a ZnS(4) coordination sphere with average Zn-S distances of 2.33 Å. (113)Cd NMR experiments were used to identify the M(II)-Cys connectivity pattern, and revealed two putative metal cluster conformations. In addition, the general metal ion coordination abilities of γ-E(c)-1 were probed with Cd(II) binding experiments as well as by pH titrations of the Zn(II) and Cd(II) forms, the latter suggesting an interaction of the γ domain and the ß(E) domain within the full-length protein.

The UV-absorber benzophenone-4 alters transcripts of genes involved in hormonal pathways in zebrafish (Danio rerio) eleuthero-embryos and adult males.

Benzophenone-4 (BP-4) is frequently used as UV-absorber in cosmetics and materials protection. Despite its frequent detection in the aquatic environment potential effects on aquatic life are unknown. In this study, we evaluate the effects of BP-4 in eleuthero-embryos and in the liver, testis and brain of adult male fish on the transcriptional level by focusing on target genes involved in hormonal pathways to provide a more complete toxicological profile of this important UV-absorber. Eleuthero-embryos and males of zebrafish were exposed up to 3 days after hatching and for 14 days, respectively, to BP-4 concentrations between 30 and 3000 µg/L. In eleuthero-embryos transcripts of vtg1, vtg3, esr1, esr2b, hsd17ß3, cyp19b cyp19a, hhex and pax8 were induced at 3000 µg/L BP-4, which points to a low estrogenic activity and interference with early thyroid development, respectively. In adult males BP-4 displayed multiple effects on gene expression in different tissues. In the liver vtg1, vtg3, esr1 and esr2b were down-regulated, while in the brain, vtg1, vtg3 and cyp19b transcripts were up-regulated. In conclusion, the transcription profile revealed that BP-4 interferes with the expression of genes involved in hormonal pathways and steroidogenesis. The effects of BP-4 differ in life stages and adult tissues and point to an estrogenic activity in eleuthero-embryos and adult brain, and an antiestrogenic activity in the liver. The results indicate that BP-4 interferes with the sex hormone system of fish, which is important for the risk assessment of this UV-absorber.

The missing piece: sediment records in remote Mountain lakes confirm glaciers being secondary sources of persistent organic pollutants.

After atmospheric deposition and storage in the ice, glaciers are temporary reservoirs of persistent organic pollutants (POPs). Recently, the hypothesis that melting glaciers represent secondary sources of these pollutants has been introduced by investigations of the historical trend of POPs in a dated sediment core from the proglacial Alpine Lake Oberaar. Here, the hypothesis is further confirmed by the comparison of sediment data gathered from two Alpine lakes with a glaciated and a nonglaciated hydrological catchment. The two lakes (Lake Engstlen and Lake Stein in the Bernese Alps in Switzerland) are situated only 8 km apart at similar altitude and in the same meteorological catchment. In the nonglacial lake sediment of Lake Engstlen, PCBs and DDT (polychlorinated biphenyls and dichlorodiphenyl trichloroethane) levels culminated with the historic usage of these chemicals some 30-50 years ago. In the glacial Lake Stein, this peak was followed by a reincrease in the 1990s, which goes along with the accelerated melting of the adjacent glacier. This study confirms the hypothesis of glaciers being a secondary source of these pollutants and is in accordance with the earlier findings in Lake Oberaar.

Accumulation and effects of the UV-filter octocrylene in adult and embryonic zebrafish (Danio rerio).

Wide application of the UV-filter octocrylene (OC) in cosmetics leads to contamination of the aquatic environment, but effects of OC remain unclear. Here we determine bioaccumulation and molecular effects of OC. Adult male zebrafish were exposed to 22, 209 and 383 µg/L and embryos to 69, 293 and 925 µg/L OC. OC accumulated in fish up to 17 µg/g. Calculated BCF varied between 41 and 136. Microarray analysis in brain and liver following exposure to 383 µg/L OC revealed alteration of 628 and 136 transcripts, respectively. Most prominent GO processes included developmental processes, organ development, hematopoiesis, formation of blood vessels, blood circulation, fat cell differentiation and metabolism. Validation by RT-qPCR in brain and liver of adult fish and embryos included a series of genes. Blood levels of 11-ketotestosterone were not altered. The transcriptomics data suggest that OC mainly affects transcription of genes related to developmental processes in the brain and liver as well as metabolic processes in the liver.

Effects of low concentrations of the antiprogestin mifepristone (RU486) in adults and embryos of zebrafish (Danio rerio): 1. Reproductive and early developmental effects.

Effects of synthetic progestins have recently been reported in fish, but potential effects of the synthetic antiprogestin mifepristone (MIF), also called RU486, have not been studied. The present study provides first insights into reproductive effects of MIF in zebrafish in comparison to the progesterone receptor agonist, progesterone (P4). We carried out a reproductive study using breeding groups of adult zebrafish. After a 14 day pre-exposure, zebrafish were exposed for 21 days to 5, 39, 77 ng/L MIF, 25 ng/L P4 and water and solvent controls. In addition, embryos originating from exposed adult fish were continuously exposed to 3, 15, 26 ng/L MIF, and 254 ng/L P4, respectively, for 96 h post fertilization. We found a significant U-shaped increase in egg production after exposure to 5 and 77 ng/L MIF, but no effects at 25 ng/L P4. Levels of sex steroid hormones in blood plasma of adult males (11-ketotestosterone) and females (17 ß-estradiol) were not altered. In addition to an increase of mature vitellogenic oocytes in ovaries of females exposed to MIF and P4, we observed several histopathological changes in ovaries, including post-ovulatory follicles, atretic follicles and proteinaceous fluid. Male gonads showed no or less alterations and no histopathological effects. Fertility of eggs and hatching success of embryos (F1 generation) was not affected at 3-26 ng/L MIF and 254 ng/L P4, respectively. The data lead to the conclusion that trace quantities of MIF affect reproduction of zebrafish and ovaries of female zebrafish. Effects on transcriptional changes in adult and embryonic zebrafish of this study in comparison to in vitro effects are reported in the associated report (Blüthgen et al., 2013a).

Effects of low concentrations of the antiprogestin mifepristone (RU486) in adults and embryos of zebrafish (Danio rerio): 2. Gene expression analysis and in vitro activity.

Here, we analyzed the transcriptional effects of the antiprogestin mifepristone (MIF, RU486) and progesterone (P4) in zebrafish as well as their in vitro activities in yeast-based reporter gene assays. This study is associated with the reproduction study in adult zebrafish and embryos exposed for 21 days to 5, 39, 77 ng/L MIF, and 25 ng/L P4 (Blüthgen et al., 2013a). The in vitro activities of MIF and P4 were investigated using a series of recombinant yeast-based assays (YES, YAS, YPS) and compared to transcriptional alterations obtained in fish tissues and embryos from the exposure study. MIF elicited antiestrogenic, androgenic and progestogenic activities in recombinant yeast, similar to P4, and no antiprogestogenic activity in vitro. The transcriptional alterations of steroid hormone receptors were similar in adult males and females, and more pronounced in embryos. MIF tended to transcriptionally down-regulate the androgen (ar), progesterone (pgr) and glucocorticoid (gr) receptors in adult fish and embryos. Transcripts of the estrogen receptor (esr1) and vitellogenin (vtg1) were not significantly altered. A trend for down-regulation was observed for transcripts of genes belonging to steroidogenic enzymes including 17ß-hydroxysteroid dehydrogenase type 3 (hsd17b3), 3 ß-hydroxysteroid dehydrogenase (hsd3b), P450 aromatase A (cyp19a) and 11ß-hydroxylase (cyp11b). P4 resulted in similar transcriptional alterations as MIF. The data indicate that gene expression changes (here and later gene expression is taken as synonym to gene transcription) and in vitro activities match only in part including the lack of antiprogestogenic activity of MIF. Additionally, effects on reproduction and gonad histology described in the associated report (Blüthgen et al., 2013a) can only partly be explained by gene expression data presented here.