Poul Bjerregaard

The aim of this study was to compare results obtained by eight different short-term assays of estrogenlike actions of chemicals conducted in 10 different laboratories in five countries. Twenty chemicals were selected to represent direct-acting estrogens, compounds with estrogenic metabolites, estrogenic antagonists, and a known cytotoxic agent. Also included in the test panel were 17beta++-estradiol as a positive control and ethanol as solvent control. The test compounds were coded before distribution. Test methods included direct binding to the estrogen receptor (ER), proliferation of MCF-7 cells, transient reporter gene expression in MCF-7 cells, reporter gene expression in yeast strains stably transfected with the human ER and an estrogen-responsive reporter gene, and vitellogenin production in juvenile rainbow trout. 17beta-Estradiol, 17alpha-ethynyl estradiol, and diethylstilbestrol induced a strong estrogenic response in all test systems. Colchicine caused cytotoxicity only. Bisphenol A induced an estrogenic response in all assays. The results obtained for the remaining test compounds--tamoxifen, ICI 182.780, testosterone, bisphenol A dimethacrylate, 4-n-octylphenol, 4-n-nonylphenol, nonylphenol dodecylethoxylate, butylbenzylphthalate, dibutylphthalate, methoxychlor, o,p'-DDT, p,p'-DDE, endosulfan, chlomequat chloride, and ethanol--varied among the assays. The results demonstrate that careful standardization is necessary to obtain a reasonable degree of reproducibility. Also, similar methods vary in their sensitivity to estrogenic compounds. Thus, short-term tests are useful for screening purposes, but the methods must be further validated by additional interlaboratory and interassay comparisons to document the reliability of the methods.

Author(s): Bergman, Åke; Heindel, Jerrold J; Kasten, Tim; Kidd, Karen A; Jobling, Susan; Neira, Maria; Zoeller, R Thomas; Becher, Georg; Bjerregaard, Poul; Bornman, Riana; Brandt, Ingvar; Kortenkamp, Andreas; Muir, Derek; Drisse, Marie-Noël Brune; Ochieng, Roseline; Skakkebaek, Niels E; Byléhn, Agneta Sundén; Iguchi, Taisen; Toppari, Jorma; Woodruff, Tracey J

The abilities of seven clones of marine phytoplankton, belonging to six different algal classes, to accumulate transuranic elements were evaluated in laboratory culture experiments. Plutonium, americium, and californium were rapidly concentrated by all species, resulting in volume/volume concentration factors generally >10 5 for all species and all isotopes. Two natural assemblages from the coastal Mediterranean behaved like the algal cultures. Isotopes associated with cells by a passive adsorption to cell surfaces, with equilibrium between cells and water reached in 3–4 days. Uptake of isotope was directly proportional to the number of suspended particles and the isotope concentration in the culture. Equilibrium isotope concentrations differed between species, reflecting different numbers of transuranic binding sites on the cell surfaces. Generally, the green and blue‐green cells had less reactive surfaces than the diatoms. Once accumulated, Am was lost more rapidly by green algae than by diatoms. Elimination proceeded in two phases, a rapid initial loss and then a slower release. The biological half‐life for Am turnover in the slowly exchanging compartment in the diatom was 10–12 days. No substantial differences in uptake were noted between cultures receiving Pu in the III–IV and V–VI oxidation states. In contrast to the other elements, neptunium showed no detectable accumulation by any of the cells. The results suggest that Pu, Cf, and Am would associate with marine particles which could transport them vertically, transfer them into the marine food web, or both, while Np would behave essentially conservatively in seawater.

BACKGROUND: The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation. RESULTS: In the present study, we have used quantitative real-time PCR to investigate the expression of ar, sox9a, dmrt1, fig alpha, cyp19a1a and cyp19a1b during the expected sex determination and gonadal sex differentiation period. The expression of the genes expected to be high in males (ar, sox9a and dmrt1a) and high in females (fig alpha and cyp19a1a) was segregated in two groups with more than 10 times difference in expression levels. All of the investigated genes showed peaks in expression levels during the time of sex determination and gonadal sex differentiation. Expression of all genes was investigated on cDNA from the same fish allowing comparison of the high and low expressers of genes that are expected to be highest expressed in either males or females. There were 78% high or low expressers of all three "male" genes (ar, sox9a and dmrt1) in the investigated period and 81% were high or low expressers of both "female" genes (fig alpha and cyp19a1a). When comparing all five genes with expected sex related expression 56% show expression expected for either male or female. Furthermore, the expression of all genes was investigated in different tissue of adult male and female zebrafish. CONCLUSION: In zebrafish, the first significant peak in gene expression during the investigated period (2-40 dph) was dmrt1 at 10 dph which indicates involvement of this gene in the early gonadal sex differentiation of males.