Effects of steroid hormones on reproduction- and detoxification-related gene expression in adult male mosquitofish, Gambusia affinis

The molecular mechanisms that mediate fish reproduction and detoxification in response to steroid hormones were studied by using adult male western mosquitofish (Gambusia affinis) as sentinel species. The expression patterns of three vitellogenins (VtgA, VtgB and VtgC), two estrogen receptors (ERα and ERβ), two androgen receptors (ARα and ARβ), metallothionein (MT) and cytochrome P450 1A (CYP1A) in the liver and testis of adult male mosquitofish were assessed through exposure treatments with progesterone (P), testosterone (T) and 17β-estradiol (E2), alone and in combination for eight days. The results showed that expression patterns of Vtg subtype, ER subtype, AR subtype, MT and CYP1A genes in male mosquitofish varied according to tissue and specific hormone stress. Vtg subtype mRNA expression was induced in the liver in E2-added treatments, and an up-regulation of ERα mRNA expression was also observed. In addition, hormone treatments increased three Vtg subtype mRNA expression levels in the testis, at least to some extent. All hormone treatments significantly inhibited ERα, ERβ and ARβ mRNA expression in the testis. Some of hormone treatments could affect MT and CYP1A gene expression in mosquitofish. In general, multiple hormone treatments showed different effects on target gene expression compared with corresponding hormone alone. The results from the present study provided valuable information on the toxicological effects of steroid hormones in mosquitofish.     

Topographical analysis of As-induced folding of α-MT1a

Metallothionein binds multiple metals into two clustered domains. While the structure of the fully metalated protein is well known for the Cd- and Zn-containing protein, there is little known about the structures of the metal-free protein (apo-metallothionein) and even less about the partially metalated forms. However, the partially-metalated species are vitally important intermediates in the passage of the protein from translational synthesis to its homeostatic buffer or metal chaperone roles. Because multiple metals bind to metallothioneins, the partially-metalated species span a wide range depending on the metal bound. Up to 3 As³⁺ bind stepwise to the α-domain fragment in a manner that allows measurement of each of the 4 species simultaneously with the number of free cysteines diminishing by 3 for every As³⁺ bound: apo- (11 Cys), As₁- (8 Cys), As₂- (5 Cys) and As₃-α-MT (2 Cys). The cysteine modifier benzoquinone (Bq), was used to determine the relative accessibility of the free cysteines in the α-MT fragment as a function of the number of As³⁺ bound. The effect of each As³⁺ was to induce folding in the protein. The ESI-MS results show that the whole protein folds significantly even when just one of the three As³⁺ has bound. The profile of the Bq reacting with the unbound cysteines shows effects of steric hindrance in slowing down the reaction. By freezing the reaction midway to the endpoint, the mass spectral data show the ‘mid-flight’ concentrations of all the key species, 27 in all. Analysis of this mid-flight reaction profile gives insight into the topology of the partially metalated MT from the differential access to the unbound cysteinyl thiols by the Bq. Significantly, the metal-free, apo-α-MT also adopts a folded structure in the presence of the As³⁺ even though there is no As³⁺ bound. This can only happen if the apo-protein wraps around other metalated proteins in solution via protein–protein interactions.     

Temporal variations of metallothionein and metal concentrations in the digestive gland of oysters (Crassostrea gigas) from a clean and a metal-rich site

The concentrations of metallothionein (MT) in bivalves, a potential biomarker of metal pollution, are variable according to specific organs, the highest concentrations being encountered in the digestive glands of oysters. Thus, the present study has been focussed on this organ with a view to validate the use of MT as a biomarker in the field, the temporal changes of metal and metallothionein concentrations have been examined from March to October 1997 in the digestive gland of resident oysters from a clean site (Bay of Bourgneuf, France) and a metal-rich site, the Gironde estuary which has been shown as the most Cd-contaminated marine area in France but is also enriched with Cu and Zn. Moreover, oysters from the clean site have been translocated to the Gironde estuary over the same period. Taking into account all the samples collected over the 7 months of the study, MT concentrations in the digestive gland were positively correlated with weight whereas metal levels were negatively correlated with weight. However, considering monthly samples including specimens from both sites (resident or translocated oysters), a positive correlation was shown between MT and metal concentrations in autumn (September and October) but not in spring and summer. These findings limit the interest of using the digestive gland of oysters as the preferred tissue for the determination of MT concentration as a biomarker.The alternative use of gills should be considered.     

Dietary zinc attenuates renal lead deposition but metallothionein is not directly involved

Chronic lead exposure irreversibly damages the kidneys and may be associated with hypertension and renal insufficiency at sub-clinically toxic levels. Zinc supplementation reduces lead absorption and tissue retention in rodent models but the mechanisms are unknown. Metallothionein (MT) may function in lead detoxification. Our objective was to investigate the effects of marginal zinc (MZ) and supplemental zinc (SZ) intakes on renal lead and zinc accumulation, renal MT immunolocalization and levels. Weanling Sprague Dawley rats were assigned to MZ (8 mg Zn/kg diet), zinc-adequate control (CT; 30 mg Zn/kg), zinc-adequate diet-restricted (DR; 30 mg Zn/kg) or SZ (300 mg Zn/kg) groups, with and without lead acetate-containing drinking water (200 mg Pb/L) for 3 weeks. Kidneys were analyzed for lead and zinc by inductively coupled plasma spectroscopy and MT by immunolocalization and Western blotting. MZ had higher renal lead and lower renal zinc concentrations than CT. SZ was more protective than CT against renal lead accumulation. Renal MT levels reflected dietary intake (SZ ≥ DR ≥ CT ≥ MZ) but lead had no effect on MT staining intensity, distribution, or relative protein amounts. In summary, while SZ lowered renal lead concentration, MT did not appear to function in renal lead accumulation. Future studies should explore alternate mechanisms of renal lead detoxification.     

STUDIES ON A CADMIUM-INDUCED METALLOTHIONEIN IN HOUSEFLY LARVAE, MUSCA DOMESTICA

Abstract  The induction of metallothionein (MT) was studied in the housefly larvae ( Musca domestica ). Upon dietary exposure to Cd, two Cd-binding proteins were isolated from the whole body homogenates, using gel filtration and ion exchange chromatography. Mass spectrometric measurement revealed that they have high purity and the molecular weight are 9045.9Da and 11560.2Da, respectively. Amino acid analysis showed that the content of cysteine is the highest, attaining to 18.2%. However, aromatic amino acid residues such as tyrosine (2.5%) and phenylalanine (3.1%) were also detected. In addition, MT from housefly larvae has strong heat stability. All these results suggest that the properties of MT isolated from housefly are very similar to that of mammalian MT, but some differences still exist.     

Evaluation of cadmium,lead and metallothionein contents in the tissues of mussels (Mytilus galloprovincialis) from the Campania coast (Italy): Levels and seasonal trends

The biological effect of seasonality on cadmium, lead and metallothionein contents was assessed in mussels Mytilus galloprovincialis from natural banks located along the coastline of the Gulf of Naples (Campania, Italy). Heavy metals and metallothionein concentrations were measured in digestive and reproductive glands. The results showed a clear correlation between metallothionein content and the reproductive gland status determined during the seasons; on the contrary, no correlation was found between metallothionein and metal contents. Data allow us to hypothesize that metallothionein functions go beyond metal detoxification, thus opening new scenarios for these proteins in invertebrates. The effect of seasons on metals concentration in mussel tissues showed similar seasonal patterns between the sites, regardless of their anthropogenic impacts. Cadmium content was not strictly related to seasonal periods, whereas lead content was significantly lower in summer. The results also indicate that the metal contents in mussels from the Gulf of Naples do not represent a risk to human health, even in the period of their maximum accumulation, and that the relaying of mussels before marketing could improve the animal stress conditions, but having a slight effect on metal excretion.     

Incorporation of a glycine within the conserved TCPCP motif of human neuronal growth inhibitory factor significantly reduces its bioactivity

It has been reported that the ⁶CPCP⁹ motif near the N-terminus is pivotal to the inhibitory activity of human neuronal growth inhibitory factor (hGIF). In order to better understand the biological significance of this region on the structure, property and function of hGIF, we introduced a highly flexible residue, Gly, either in front of the ⁶CPCP⁹ motif (the IG6 mutant, TGCPCP) or in the middle of it (the IG8 mutant, TCPGCP) and investigated their structural and metal binding properties in detail. The results showed that the overall structure and the stability of the metal-thiolate clusters of the two mutants were comparable to that of hGIF. However, the bioassay results showed that the bioactivity of the IG6 mutant decreased significantly, while the bioactivity of the IG8 mutant was almost abolished. Molecular dynamics simulation results showed that the backbone of the IG6 mutant exhibited high similarity to that of hGIF, and the two prolines could still induce structural constraints on the ⁶CPCP⁹ tetrapeptide and form a similar conformation with that of hGIF, however, the conformation of the first five amino acid residues in the N-terminus was quite different. In hGIF, the five residues are twisted and form a restricted conformation, while in the IG6 mutant this peptide extends more naturally and smoothly, which is similar to that of MT2. As to the IG8 mutant, the Gly insertion broke the ⁶CPCP⁹ motif, thus probably abolishing the interactions with other molecules and eliminating its inhibitory activity. Based on these results, we suggested that although the structure adopted by the ⁶CPCP⁹ motif is the determinant factor of the inhibitory bioactivity of hGIF, other residues within the N-terminal fragment (residue 1-13) may also influence the peptide conformation and contribute to the protein’s bioactivity.     

Renal metallothionein responds rapidly and site specifically to zinc repletion in growing rats

Metallothionein (MT) is important for heavy metals and free radical protection in the kidney. MT is responsive to zinc and primarily localized within the renal cortex. However, site-specific renal responses to dietary zinc repletion are understudied. The objective of this study was to examine the effects of dietary zinc deficiency and repletion on renal MT concentration and immunolocalization in rats. Weanling male Sprague Dawley rats were randomly assigned to either a zinc-deficient, zinc control, or pair-fed to zinc-deficient group. Half of the zinc-deficient and pair-fed rats were repleted with the control diet ad libitum for an additional 24 h. Renal tissue samples were assessed for total zinc, MT concentrations and MT immunostaining. Dietary zinc deficiency reduced renal zinc and MT concentrations, and attenuated intensity and localization of MT. Dietary zinc repletion for 24 h restored renal zinc and MT concentrations, the latter primarily in the proximal convoluted tubules of the cortex. Concentrations of renal MT, but not zinc, were elevated by diet restriction and MT (μg/mg protein) and partially normalized by 24 h diet repletion. In conclusion, renal MT modification due to zinc deficiency or diet restriction can be rapidly normalized in a site-specific manner with normal dietary zinc intake. The results support a role for MT in kidney homeostasis, in particular at the level of the proximal tubules in the cortex. The speed of MT repletion may have clinical implications for dietary zinc in the treatment of acute and chronic renal pathology due to toxins and free radicals.     

The cadmium binding domains in the metallothionein isoform Cd7-MT10 from Mytilus galloprovincialis revealed by NMR spectroscopy

The metal–thiolate connectivity of recombinant Cd₇-MT10 metallothionein from the sea mussel Mytilus galloprovincialis has been investigated for the first time by means of multinuclear, multidimensional NMR spectroscopy. The internal backbone dynamics of the protein have been assessed by the analysis of ¹⁵N T ₁ and T ₂ relaxation times and steady state {¹H}–¹⁵N heteronuclear NOEs. The ¹¹³Cd NMR spectrum of mussel MT10 shows unique features, with a remarkably wide dispersion (210 ppm) of ¹¹³Cd NMR signals. The complete assignment of cysteine Hα and Hβ proton resonances and the analysis of 2D ¹¹³Cd–¹¹³Cd COSY and ¹H–¹¹³Cd HMQC type spectra allowed us to identify a four metal–thiolate cluster (α-domain) and a three metal–thiolate cluster (β-domain), located at the N-terminal and the C-terminal, respectively. With respect to vertebrate MTs, the mussel MT10 displays an inversion of the α and β domains inside the chain, similar to what observed in the echinoderm MT-A. Moreover, unlike the MTs characterized so far, the α-domain of mussel Cd₇-MT10 is of the form M₄S₁₂ instead of M₄S₁₁, and has a novel topology. The β-domain has a metal–thiolate binding pattern similar to other vertebrate MTs, but it is conformationally more rigid. This feature is quite unusual for MTs, in which the β-domain displays a more disordered conformation than the α-domain. It is concluded that in mussel Cd₇-MT10, the spacing of cysteine residues and the plasticity of the protein backbone (due to the high number of glycine residues) increase the adaptability of the protein backbone towards enfolding around the metal–thiolate clusters, resulting in minimal alterations of the ideal tetrahedral geometry around the metal centres.