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The ever-increasing production and use of nanocrystaline semiconductors (Quantum dots; QDs) will inevitably result in increased appearance of these nanomaterials in the aquatic environment. However, the behavior and potential toxicity of heavy metal constituted nanoparticulates in aquatic invertebrates is largely unknown, especially with regard to molecular responses. The freshwater crustacean Daphnia pulex is a well-suited toxicological and ecological model to study molecular responses to environmental stressors. In this study, D. pulex were exposed for 48 h to sublethal doses of QDs (25% and 50% of LC50) with differing spectral properties (CdTe and CdSe/ZnS QDs) and Cd and Zn salts. Our data suggest that acute exposure to both CdSO4 and Cd-based QDs leads to Cd uptake in vivo, which was biologically supported by the observation of increased expression of metallothionein (MT-1). Furthermore, Cd, Zn, and CdSe/ZnS QDs induced different patterns of gene expression regarding stress defense and DNA repair, which furthers our knowledge regarding which response pathways are affected by nanoparticulate forms of metals versus ionic forms in aquatic crustaceans., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Female squirrelfish (Fam. Holocentridae) can accumulate and temporarily sequester copious amounts of zinc (Zn) in their livers. There, it is initially compartmentalized before a subsequent, estrogen-triggered redistribution to the ovaries. Here we show that cellular uptake of Zn is also influenced by estrogen signaling, and that estrogen increases concentrations of the plasma Zn-binding protein vitellogenin (VTG). However, estrogen-mediated increases in VTG are not sufficient to accommodate the magnitude of hepato-ovarian Zn transfer in female squirrelfish (Holocentrus adscensionis). These findings suggest that holocentrids have acquired the ability to use hormonal cues to drive hepatic uptake and storage of Zn, signal for its physiological redistribution, and influence the capacity for systemic transport of Zn beyond the mediation of increased plasma VTG concentrations. Such specific adaptations suggest an advantage for the oocyte, which is corroborated in further studies where we determined that oocyte Zn concentrations are positively correlated with egg viability in captive-spawned squirrelfish. The novel nature of these findings underlies the importance of Zn in squirrelfish reproductive biology.

Zinc is a vital micronutrient, yet as an environmental toxicant it can be deleterious to aquatic organisms such as fish. Consequently, the study of zinc uptake mechanisms is essential for understanding nutrition, toxicity, and metabolism of this metal. Intestinal zinc uptake was studied in two marine teleosts, using both in vitro (in vitro perfusion and intestinal sacs) and in vivo techniques (in situ bolus). Female squirrelfish (Holocentrus adscensionis) exhibited significantly increased epithelial zinc uptake associated with enhanced hepatic zinc accumulation. This confirms this zinc-hyperaccumulating teleost as a potential model of zinc absorption. Intestinal zinc uptake in the gulf toadfish (Opsanus beta) was biphasic with respect to zinc concentration (0.3-500 microM), exhibiting both saturable and passive uptake components. In both species, the passage of zinc into the postintestinal compartment was highly dependent on technique. Decreased proportions of postintestinal zinc in vivo, coupled with concentration-dependent distribution of zinc accumulation, suggested mechanisms may act to control the movement of zinc into the circulation. In addition, the results of this study were used to reinterpret previous findings of zinc uptake in freshwater fish and allowed a critique of techniques used to study intestinal metal uptake.

Females of the squirrelfish family (Holocentridae) accumulate higher levels of hepatic zinc than any other studied animal. This accumulation is accompanied by high expression of the zinc-binding protein, metallothionein (MT), and is strongly correlated to the onset of sexual maturity. In an attempt to further characterize the timeframe of this accumulation, and to possibly discern any potential mediators, we examined the physiology and endocrinology of the yearly reproductive cycle of mature female squirrelfish. There are two separate reproductive events during the year in December-January and again in March-April, as evidenced by peaks in ovarian growth, VTG production, steroid levels, zinc accumulation and redistribution. Increased hepatic zinc seems to be preceded by a necessary increase in MT, but this was not clearly correlated to plasma 17beta-estradiol, testosterone, or progesterone levels. The plasma zinc protein vitellogenin (VTG) is one, but probably not the predominant, vehicle for the transport of hepatic zinc to the ovary.

In fish, the synthesis of metallothionein (MT) is increased by a number of heavy metals. The rainbow trout MT-A gene promoter region contains six known metal responsive elements (MREs), that mediate promoter activation by metals. In the present study, two fish cell lines differing in their ability to produce MT, RTG-2 (produce MT protein) and CHSE-214 (produce no detectable MT protein), were used to help elucidate the roles of Zn, Ag and MT in the activation of the MT promoter. The hypothesis tested was that Ag activates the MT-A promoter indirectly by displacing Zn from pre-existing Zn-MT and that this liberated Zn subsequently induces MT synthesis. Both cell lines were transfected with a luciferase reporter gene construct containing the rainbow trout MT-A promoter, exposed to various concentrations of Zn or Ag, and assayed for luciferase activity. CHSE-214 cells showed five times greater production of luciferase than RTG-2 cells when exposed to identical concentrations of Ag. Thus, Ag can likely induce MT transcription without displacing Zn from pre-existing Zn-MT. Furthermore, Ag activated the MT promoter at concentrations 100-fold lower than those required for Zn to initiate transcription, suggesting that zinc displaced from other sites by such low concentrations of Ag would not be sufficient to initiate MT transcription. This interpretation was further supported by radiotracer studies indicating that Ag did not cause a redistribution of 65Zn within either of the two cell types. These combined results indicate that Ag may be a direct inducer of MT.

Female squirrelfish (Holocentridae) accumulate higher concentrations of hepatic zinc than any other known organism. In the liver cells, up to 70% of zinc is bound to metallothionein (MT), which is expressed at extremely high levels. These attributes are related to reproduction in ways that have not been fully characterized. In the present study, we have demonstrated that female-specific zinc and MT accumulation and distribution are strongly correlated to the onset of sexual maturity in Holocentrus adsenscionis. Sexual maturation not only resulted in increased concentrations of zinc in the liver and plasma, but also increased levels of hepatic MT mRNA. Furthermore, mature female squirrelfish exhibited greater proportions of MT protein in the nuclear liver cell fractions. To characterize the physiology further, we have examined the influence of the female sex hormone 17 beta-estradiol (E(2)). E(2) was not sufficient to elicit an increase in hepatic zinc concentrations or MT mRNA levels. E(2) administration did, however, result in increased levels of MT in the nuclear fraction as well as overall hepatic MT protein. E(2) also increased concentrations of zinc in the plasma. The changes in zinc concentration in the bloodstream followed the same time course as vitellogenin (VTG) transport from the liver. However, the high ratio of molar concentrations of zinc to VTG in the bloodstream suggest that VTG may not be the primary vehicle for hepato-ovarian zinc transport in squirrelfish.

Females of the squirrelfish family (Holocentridae) accumulate higher levels of zinc in the liver than any other known animal. This zinc accumulation is made possible by high expression of the zinc-binding protein, metallothionein (MT). In the present study, the squirrelfish (Holocentrus ascensionis) MT cDNA was cloned and sequenced. The deduced amino acid sequence was very similar to other teleost MT. The role of estrogens on zinc metabolism was investigated by injecting male and immature female squirrelfish with 17 beta-estradiol (E(2)). E(2) treatment triggered transient increases in plasma zinc and vitellogenin (VTG) levels, and both of these variables showed very similar time courses. These results suggest that E(2) is responsible for the large hepatoovarian translocation of zinc observed in female squirrelfish and that VTG might be a vehicle for zinc. E(2) did not directly alter the levels of zinc or MT mRNA in the liver. However, the hepatic MT protein concentration increased differentially in the nuclear fraction. Thus E(2) is probably responsible for the association of MT with the nuclear fraction previously observed in untreated mature female squirrelfish.