The induction of synthesis of the "female" yolk precursor protein vitellogenin (VTG) in male fish by estrogenic chemicals in the environment has been demonstrated in many recent reports. However, little is known about the organismal and biological significance of this phenomenon. To examine the relationship between VTG production in male fish and reproductive impairment, adult male medaka were exposed to 4-tert-octylphenol (OP), a known environmental estrogen, in concentrations ranging ftom 20 to 230 ppb for 21 days, under flow-through conditions. Following exposure, male fish were mated, in the absence of OP, with unexposed females.Breeding groups composed of exposed males and control females produced about 50% fewer eggs than control groups. VTG levels in serum of male fish increased with increasing OP exposure concentration and decreased after OP exposure was discontinued. Nevertheless, significant correlations (pO.0l) were observed between VTG levds in exposed male fish and 1) OP exposure concentrations, 2) percent of fertilized eggs, and 3) survival of embryos. OP-induced VTG synthesis and reproductive impairment appear to be closely linked phenomena. Histological exmination indicated spermatogenesis in OP-exposed fish was inhibited, and some exposed fish had oocytes in their testes. Finally, OP caused a significant increase in the number ofabnormally developing embryos, suggesting that .OP may be genotoxic as well as estrogenic. Environmental estrogens, or xenoestrogens, chemicals with bioactivity similar to the endogenous female hormone estrogen, are known to affect development and sexual maturation of (in)vertebrates. Xenoestrogens can exert their action by binding to the cell's estrogen receptor (ER), but they can also act through ER-independent mechanisms (5). Reported adverse effects in humans include increased incidences of breast cancer and reduced sperm counts, whereas wildlife populations affected by xenoestrogens display a variety of reproductive alterations such as cryptorchidism in the Florida panther, small baculum in young male otters, small penises in alligators, sex reversal in fish, and egg-shell thinning and altered social behavior in birds (3,4,6-8).Alkylphenol polyethoxylates (APEs) are nonionic surfactants widely used in the manufacturing of cleaning agents, plastics, paper, cosmetics, and food products (9). APEs are discharged from industrial wastewater as nontoxic, hydrophilic compounds. However, bacteria metabolize APEs into hydrophobic, estrogenic by-products, including p-nonylphenol and 4-tertoctylphenol (OP), that bioaccumulate in aquatic wildlife and may affect reproductive ability (10,11). These metabolites bind to the ER of fish and mammals (12)(13)(14), induce transcriptional activation of estrogen-responsive genes (15), and induce production of the yolk protein vitellogenin (VTG) in fish hepatocyte cell culture and in male rainbow trout (16)(17)(18)(19)(20). Of the alkylphenols examined, OP appears to be the most biologically active (9).VTG is normally synthesized in the liv...

Metallothionein is a cysteine-rich metal-binding protein whose biosynthesis is closely regulated by the level of exposure of an organism to zinc, copper, cadmium, and other metal salts. The metallothionein from Callinectes sapidus is known to bind six divalent metal ions in two separate metal-binding clusters. Heteronuclear 1H-113Cd and homonuclear 1H-1H NMR correlation experiments have been used to establish that the two clusters reside in two distinct protein domains. The three-dimensional solution structure of the metallothionein has been determined using the distance and angle constraints derived from these two-dimensional NMR data sets and a distance geometry/simulated annealing protocol. There are no interdomain short distance (< or = 4.5 A) constraints observed in this protein, enabling the calculation of structures for the N-terminal, beta domain and the C-terminal, alpha domain separately. A total of 18 structures were obtained for each domain. The structures are based on a total of 364 experimental NMR restraints consisting of 277 approximate interproton distance restraints, 12 chi 1 and 51 phi angular restraints, and 24 metal-to-cysteine connectivities obtained from 1H-113Cd correlation experiments. The only element of regular secondary structure in either of the two domains is a short segment of helix in the C-terminal alpha domain between Lys42 and Thr48. The folding of the polypeptide backbone chain in each domain, however, gives rise to several type I beta turns. There are no type II beta turns.

The blue crab, Callinectes sapidus, which uses the copper-dependent protein haemocyanin for oxygen transport, lacks the ubiquitous cytosolic copper-dependent enzyme copper/zinc superoxide dismutase (Cu,ZnSOD) as evidenced by undetectable levels of Cu,ZnSOD activity, protein and mRNA in the hepatopancreas (the site of haemocyanin synthesis) and gills. Instead, the crab has an unusual cytosolic manganese SOD (cytMnSOD), which is retained in the cytosol, because it lacks a mitochondrial transit peptide. A second familiar MnSOD is present in the mitochondria (mtMnSOD). This unique phenomenon occurs in all Crustacea that use haemocyanin for oxygen transport. Molecular phylogeny analysis suggests the MnSOD gene duplication is as old as the origin of the arthropod phylum. cytMnSOD activity in the hepatopancreas changes during the moulting cycle of the crab. Activity is high in intermoult crabs and non-detectable in postmoult papershell crabs. mtMnSOD is present in all stages of the moulting cycle. Despite the lack of cytCu,ZnSOD, crabs have an extracellular Cu,ZnSOD (ecCu,ZnSOD) that is produced by haemocytes, and is part of a large, approx. 160 kDa, covalently-linked protein complex. ecCu,ZnSOD is absent from the hepatopancreas of intermoult crabs, but appears in this tissue at premoult. However, no ecCu,ZnSOD mRNA can be detected, suggesting that the protein is recruited from the haemolymph. Screening of different taxa of the arthropod phylum for Cu,ZnSOD activity shows that those crustaceans that use haemoglobin for oxygen transport have retained cytCu,ZnSOD. It appears, therefore, that the replacement of cytCu,ZnSOD with cytMnSOD is part of an adaptive response to the dynamic, haemocyanin-linked, fluctuations in copper metabolism that occur during the moulting cycle of the crab.

No abstract