Differences in metallothionein gene expression in primary cultures of rainbow trout hepatocytes and the RTH-149 cell line

Primary cultures of rainbow trout, Salmo gairdneri, hepatocytes were used to study the expression of metallothionein (MT) genes in response to steroid hormone treatment. The expression pattern was compared to that of an immortal cell line (RTH-149). MT mRNA accumulated in both cell cultures after exposure to zinc while 17 beta-oestradiol had no effect in either system. Treatment with cortisol and corticosterone resulted in a 2-fold increase of metallothionein mRNA levels in the primary cultures but had no effect in the RTH-149 cell culture. Primary cultures that were exposed to zinc or cortisol showed a high temporal correlation (r = 0.974) between MT mRNA and MT protein levels. The basal level expression was 3-4-fold higher in primary cultures than in RTH-149 cells. The present study demonstrates the inducibility of rainbow trout MT genes in response to glucocorticoids. It further indicates that primary cultures are to be preferred to immortal cell lines when investigating the inducibility of MT mRNA.     

Glutathione level of Desulfovibrio desulfuricans IMV K-6 under the influence of heavy metal salts

Glutathione is the metal stress protector and changes of its level in the sulfate-reducing bacteria cells under the influence of heavy metal salts have not been studied yet. CdCl2, Pb(NO3)2, CuCl2, and ZnCl2 influence on the total glutathione level in cell-free extracts of sulfate-reducing bacteria Desulfovibrio desulfuricans IMV K-6 was studied. The research has been carried out using Ellman, Lowry methods, statistical processing of the results. It was shown that the glutathione level depends on the heavy metal salts concentration in the medium. The total glutathione level was the highest under the influence of Pb(NO3)2. Other salts were also toxic to bacteria because glutathione level increased in bacterial cells after addition of these salts to the medium. On the basis of the results of our work the range of heavy metal salts influence on D. desulfuricans IMV K-6 cells glutathione level has been formed for the first time: Pb(NO3)2 > CuCl2 > CdCl2 > ZnCl2.     

Impact of deltamethrin exposure on mRNA expression levels of metallothionein A, B and cytochrome P450 1A in rainbow trout muscles

Metallothioneins (MT) are widely utilized to identify specific responses to heavy metal pollution. In addition, there is evidence demonstrating that in vertebrates MT synthesis is stimulated by different endogenous and exogenous agents in particular compounds leading to production of ROS. Also, cytochrome P450 1A can enhance the generation of ROS. On this basis, MT and CYP 1A induction can be considered as biomarkers of oxidative stress. In the current study, we examined the influences of pesticide administration on the expression of MT-A, MT-B and CYP 1A. For this purpose, we produced muscle metallothionein-A, metallothionein-B and cytochrome P450 1A cDNAs and used quantitative RT-PCR to assay mRNAs in rainbow trout exposed to acute and long-term deltamethrin administration. We observed that deltamethrin exposure significantly (p<0.05) increased the expression levels of Cyp1A, MT-A and MT-B in a time dependent manner. Results of our study contributes to the identification of inducers of such biomarkers in addition to well known agents.     

Developmental regulation of metallothionein mRNA, zinc and copper levels in rainbow trout, Salmo gairdneri

The metallothionein (MT) gene expression profile was followed in rainbow trout during early embryo development and in liver and gonads during the period of sexual maturation. The hepatic MT mRNA levels increase at the end of sexual maturation in both male and female rainbow trout. Although both isoforms of MT mRNA accumulate in the liver, there is a preferential increase in MT-A in the female liver. Concomitantly with this increase in MT there is a redistribution of zinc and copper to MT. In the juvenile female there is an abundance of MT mRNA in the ovaries. This is correlated to high levels of zinc in the MT fraction upon Sephadex G-75 chromatography. During ovary development the MT mRNA levels and the MT-bound zinc levels drop, with an increase in zinc being bound to high-molecular-mass proteins. At ovulation most of the zinc is found in the membrane portion upon centrifugation. In contrast to the ovaries, there are no apparent changes in either trace metal distribution or MT mRNA levels during testis development. In the developing embryo there is an increase in MT-bound copper at gastrulation. This is accompanied by an increase in both isoforms of MT mRNA. At hatch both the copper and zinc levels increase in the MT fraction, with a concomitant increase in mainly MT-A mRNA. These findings indicate that the variations in MT mRNA levels during development are closely associated with metal regulation.     

Effects of acute 17alpha-methyltestosterone,acute 17beta-estradiol,and chronic 17alpha-methyltestosterone on dopamine,norepinephrine and serotonin levels in the pituitary,hypothalamus and telencephalon of rainbow trout (Oncorhynchus mykiss)

This study investigated: (a) the effects of acute 17alpha-methyltestosterone (MT) or 17beta-estradiol (E(2)) administration on norepinephrine (NE), dopamine (DA), serotonin (5-HT), 3,4, dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) contents in the hypothalamus, telencephalon and pituitary of previtellogenic female rainbow trout Oncorhynchus mykiss, and (b) the effects of chronic MT administration on the levels of these neurotransmitters in these brain regions in immature male rainbow trout. The acute administration of MT induced a significant decrease in pituitary levels of DOPAC as well as in the DOPAC/DA ratio. On the other hand, the acute administration of E(2) induced an increase in pituitary 5-HT levels as well as a decrease in the 5-HIAA/5-HT ratio. In a second experiment, 20 mg MT per kilogram body weight was implanted for 10, 20 or 40 days into sexually immature male rainbow trout. Implanted rainbow trout showed increased testosterone and decreased E(2) levels. In the pituitary, MT induced long-term decreases in NE, DA, DOPAC and 5-HT levels, as well as in the DOPAC/DA ratio. Hypothalamic and telencephalic DA, NE and 5-HT levels were not affected by MT implantation. However, 5-HIAA levels and the 5-HIAA/5-HT ratio were reduced by MT implantation in both brain regions. These results show that chronic treatment with MT exerts both long-term and region-specific effects on NE, DA, and 5-HT contents and metabolism, and thus that this androgen could inhibit pituitary catecholamine and 5-HT synthesis. A possible role for testosterone in the control of pituitary dopaminergic activity and gonadotropin II release is also discussed.     

Metallothionein mRNA and protein induction by cadmium in peripheral-blood leucocytes.

To help to evaluate the role of metallothionein (MT) in peripheral-blood leucocytes, we examined MT protein and mRNA levels in these cells before and after exposure to CdCl2 in culture. Protein was assayed by 109Cd2+ binding, and RNA by dot-blot hybridization. MT was induced in both lymphocytes and adherent monocytes about 10-fold with a 12 h exposure to 10 microM-CdCl2, but absolute levels were 3-fold higher in monocytes: 57 x 10(5) (+Cd2+) versus 6 x 10(5) (-Cd2+) molecules/cell for monocytes; 18 x 10(5) (+Cd2+) versus 2 x 10(5) (-Cd2+) for lymphocytes. Polymorphonuclear cells expressed relatively little MT (0.6 x 10(5) molecules/cell), and this did not change with phorbol ester stimulation or exposure to Cd2+, arguing against a direct protective role for MT in activated neutrophils. MT mRNA levels corresponded qualitatively to expression of protein in these cells. Our data provide quantitative comparisons of leucocyte MT expression and regulation in the human population. Variation in both basal and induced MT mRNA levels reflects environmental or experimental (intra-individual) and possibly genetic (inter-individual) differences.     

Cadmium affects the expression of metallothionein (MT) and glutathione peroxidase (GPX) mRNA in goldfish, Carassius auratus

Cadmium (Cd) is a widespread non-essential heavy metal that enters the aquatic environment as a result of natural and human-caused activities, including industrial effluent, mining, and agricultural runoff. In the present study, we investigated time and dose-related effect of CdCl(2) on metallothionein (MT) and glutathione peroxidase (GPX) mRNA levels in a number of goldfish tissues, in vivo. Basal MT and GPX mRNA levels remained unchanged in the tissues tested throughout the experiment. Injection with CdCl(2) significantly increased MT mRNA levels in the brain, liver, kidney and intestine in a dose-dependant manner at all time tested (6, 12, 24 and 36 h). We isolated the full length GPX cDNA from goldfish kidneys, and found it to contain 785 nucleotides, including an open reading frame, predicted to encode a protein of 142 amino acids. In contrast, injection with CdCl(2) significantly decreased GPX mRNA levels in the liver and kidney in a time-, and dose-, dependant, and became undetectable after 12, 24 and 36 h. The findings provide molecular characterization of MT and GPX in goldfish and suggest that exposure to Cd results in significant physiological changes in goldfish.     

Formation of a double salt of phosphatidylcholine and zinc chloride.

ZnCl2 forms a 1 : 1 double salt with phosphatidylcholine. This compound resembles the long-known double salt of CdCl2 and phosphatidylcholine except that the latter has the composition (CdCl2)3(phosphatidylcholine)2. Treatment of phosphatidylcholine with a mixture of equimolar amounts of ZnCl2 and CdCl2 yields the ZnCl2 double salt. The ZnCl2 double salt can be obtained as an amorphous flocculent precipitate and as small spherules. These results are discussed in relation to the toxic action of cadmium salts on the mammalian testis and to the protection afforded by zinc salts. It is suggested that membrane phospholipids are essential components of specific sites for reversible binding of Zn2+ and Cd2+.     

Analysis of stress-induced gene expression in fish cell lines exposed to heavy meals and heat shock

We have examined the effect of heavy metals on the expression of two major groups of stress-induced proteins in fish cell lines: the 70 kDa heat-shock proteins (hsp70) and metallothioneins (MTs). The rainbow trout hepatoma (RTH) cell line synthesized the hsp70 protein in response to zinc and heat shock, while chinook salmon embryonic (CHSE) cells synthesized this protein in response to these inducers, as well as cadmium. The synthesis of this 70 kDa protein was correlated with the accumulation of hsp70 mRNA as measured by hybridization to a trout hsp70 gene probe. Heavy metals also induced the synthesis of MT in RTH cells. However, heat shock did not result in induction of MT and its mRNA. Unlike RTH cells, CHSE cells did not synthesize MT following exposure to cadmium or zinc. When these cells were treated with 5-azacytidine prior to heavy metal treatment, accumulation of MT mRNA was observed. Northern blot analysis of total RNA from 5-azacytidine treated CHSE cells, using a trout MT (tMT-B) cDNA probe, indicated that the time-course of induction and the maximal level of MT mRNA accumulation in response to cadmium and zinc paralleled that observed in RTH cells. Copper and dexamethasone were ineffective in inducing MT mRNA in 5-azacytidine-treated CHSE cells. These results indicate that MT is specifically induced in response to heavy metal treatment, whereas the synthesis of hsp70 appears to be a general stress response. Furthermore, MT is differentially regulated by heavy metals and dexamethasone in these cell lines and the expression of MT is cell-type-specific.     

The rainbow trout metallothionein-B gene promoter: contributions of distal promoter elements to metal and oxidant regulation

In this report, the contributions of the distal 5'-regulatory sequences of the rainbow trout (Oncorhynchus mykiss) metallothionein (tMT)-B gene promoter (-738 to +5) were studied. Transfection of the -738 promoter fragment in a rainbow trout hepatoma cell line (RTH-149) resulted in 4- to 5-fold greater activity compared to the proximal -137 promoter region. Mutation of the proximal MREa abolishes the basal activity of the -738 fragment indicating that the distal regulatory elements require a cooperative interaction with MREa. However, the fragments containing both distal MREs, c and d (positioning -570 and -680, respectively), or MREc alone could confer basal and metal-induced activity when fused to the TATA box. This suggests that these distal elements are functional and therefore may play a role as basal elements in their natural state. The trout MT genes are also induced by oxidants including H2O2, tBHP and tBHQ. The larger promoter fragment -738 responds to H2O2, while the -137 fragment does not. However, fusion of the isolated MREc fragment (-648 to -533) in its native orientation, upstream of the -137 promoter elicits a response to H2O2, although no response is seen with MREc in reverse. These data suggest that this distal fragment contains functional oxidant responsive elements which have resemblance to the mammalian antioxidant responsive element (AREs).     

Effects of acute iron overload on Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

Distribution of radioiron to various tissues after intraperitoneal injections was examined in Atlantic salmon and rainbow trout. Liver and spleen were found to be the major iron storage tissues. Injections of 1 or 5 mg iron as ferric ammonium citrate led to a fall in hemoglobin levels in both species after 2 d. Hemoglobin levels returned to normal levels in rainbow trout after 8 d, but Atlantic salmon had not recovered, and Hb levels fell below 3 g/100 mL. In both species, the fall in Hb was associated with a raise in iron levels in spleen and liver, suggesting damage to erythrocytes. Atlantic salmon liver ferritin showed a two- to threefold increase, while rainbow trout showed a sixfold increase, and a more rapid response. The toxic effect of iron in fish appears to be different from the effect in other vertebrates.     

Alterations of tissue glutathione levels and metallothionein mRNA in rainbow trout during single and combined exposure to cadmium and zinc

The objective of this study was to assess the effects of Cd and Zn exposure of rainbow trout (Oncorhynchus mykiss) on (a) hepatic glutathione (GSH) levels; and (b) hepatic and branchial metallothionein (MT) mRNA expression. Juvenile rainbow trout were exposed to waterborne Cd (nominal concentrations: 1.5 or 10 microg Cd l(-1)), Zn (150 or 1000 microg Zn l(-1)) or Cd/Zn mixtures (1.5 microg Cd l(-1) with 200 microg Zn l(-1) or 10 microg Cd l(-1) with 1000 microg Zn l(-1)). After 14 and 28 days of treatment, hepatic concentrations of total glutathione, oxidized glutathione (GSSG) and cysteine were determined by means of fluorometric high performance liquid chromatography (HPLC). Branchial and hepatic expression of MT mRNA was measured by means of semi-quantitative RT-PCR. Exposure of trout to Zn did not result in significantly elevated tissue levels of Zn, whereas Cd accumulation factors changed significantly with time and concentration. Despite of the absence of Zn accumulation, hepatic GSH but not MT mRNA levels were significantly altered in Zn-exposed fish. Cd, on the contrary, affected mainly the MT response but not GSH. Also tissue specific differences in the regulation of the two thiol pools were expressed. The thiol response after exposure to metal mixtures could not be explained by simple addition of the effects of the individual metals. The results indicate that cellular thiol pools show different reaction patterns with respect to specific metals and metal mixtures. Under conditions of long-term, low dose metal exposure, the function of GSH appears to go beyond that of a transitory, first line defense.     

Effect of chronic non-lethal doses of non-metals and metals on hepatic metallothionein in Channa punctatus (Bloch).

Compared to non-metal toxicants (ammonia, 1.56 ppm; and phenol, 10 ppm), the metals (CdCl2, 30 ppm; HgCl2, 16.7 ppb; and ZnCl2, 6 ppm) significantly induced hepatic metallothionein (MT) concentrations in C. punctatus, exposed independently to non-lethal doses of these toxicants for 28 days. It is suggested that hepatic MT serves as a metal-sequestering protein and is involved in the detoxication of metals only and ensures protection from toxic chemicals in ambience.     

Effects of cadmium and zinc on solar-simulated light-irradiated cells: potential role of zinc-metallothionein in zinc-induced genoprotection

Zinc is an essential oligoelement for cell growth and cell survival and has been demonstrated to protect cells from oxidative stress induced by UVA or from genotoxic stress due to UVB. In a recent work we demonstrated that the antioxidant role of zinc could be related to its ability to induce metallothioneins (MTs). In this study we identified the mechanism of zinc protection against solar-simulated light (SSL) injury. Cultured human keratinocytes (HaCaT) were used to examine MTs expression and localization in response to solar-simulated radiation. We found translocation to the nucleus, with overexpression of MTs in irradiated cells, a novel observation. The genoprotective effect of zinc was dependent on time and protein synthesis. DNA damage was significantly decreased after 48 h of ZnCl(2) (100 microM) treatment and is inhibited by actinomycin D. ZnCl(2) treatment (100 microM) led to an intense induction, redistribution, and accumulation of MT in the nucleus of irradiated cells. MT expression correlated with the time period of ZnCl(2) treatment. CdCl(2), a potent MT inducer, did not show any genoprotection, although the MTs were expressed in the nucleus. Overall our findings demonstrate that MTs could be a good candidate for explaining the genoprotection mediated by zinc on irradiated cells.     

Involvement of differential metallothionein expression in free radical sensitivity of RTG-2 and CHSE-214 cells

The role of metallothionein (MT) in free radical regulation and scavenging was investigated using two fish cell lines, the rainbow trout gonadal (RTG-2) cell line and the chinook salmon embryonic (CHSE-214) cell line. Exposure of RTG-2 cells to H(2)O(2) resulted in upregulation of both MT mRNA and MT protein and was also demonstrated by immunocytochemistry, confirming that MT was regulated by free radicals. We then compared the H(2)O(2) resistance in RTG-2 and CHSE-214 cells following metal treatment with Zn or Cd to induce MT. Comparison of survival of control cells and metal-exposed cells showed that metal treatment, which induced MT, significantly raised the H(2)O(2) tolerance in a dose-dependent manner in RTG-2 cells, while no increased H(2)O(2) resistance was observed in CHSE-214 cells. Transient over-expression of MT in CHSE-214: 59 cells also resulted in a dose-dependent increase in resistance to H(2)O(2) exposure. The raised resistance against H(2)O(2) in metal treated RTG-2 cells as well as transfected CHSE-214: 59 cells strongly demonstrate that MT is involved in the protection against H(2)O(2) and suggest a physiologically important function for MT when cells or whole organisms are exposed to oxidative stress.