In vivo and ex vivo displacement of zinc from metallothionein by cadmium and by mercury

Divalent cadmium and mercury ions are capable in vitro of displacement of zinc from metallothionein. This process has now been studied in vivo and ex vivo, using the isolated perfused rat liver system, in order to determine if this process can occur in the intact cell. Rats with normal and elevated (via preinduction with zinc) levels of hepatic zinc thionein were studied. Cd(II) completely displaces zinc from normal levels of metallothionein and on a one-to-one basis from elevated levels of metallothionein, both in vivo and ex vivo. Hg(II) displaces zinc from metallothionein (normal or elevated) rather poorly, as compared with Cd(II), in vivo, probably due to the kidneys preference for absorbing this metal. Ex vivo Hg(II) displaces zinc from metallothionein (normal or elevated) on a one-to-one basis, with considerably more mercury being incorporated into the protein than in vivo. The results of double-label ex vivo experiments using metal and [35S]cysteine (+/- cycloheximide) were consistent with the above experiments, indicating that de novo thionein synthesis was not required for short term incorporation of cadmium and mercury into metallothionein. These data are supportive of the hypothesis that cadmium and mercury incorporation into rat hepatic metallothionein during the first few hours after exposure to these metals can occur primarily by displacement of zinc from preexisting zinc thionein by a process which does not require new protein synthesis.     

Tissue-specific expression of two metallothionein genes in common carp during cadmium exposure and temperature shock

Two metallothionein cDNA isoforms (MT-1 and MT-2) were isolated from carp (Cyprinus carpio) by RT-PCR. Sequence analysis of the cDNAs revealed two amino acid differences between the coding regions and markedly different 3'-untranslated ends. Gene-specific primers were selected and used in RT-PCR reactions to measure the basal MT-1 and MT-2 mRNA levels and to follow the inducer-specific expression of MT genes in different tissues during in vivo studies. In the brain and muscle, the uninduced levels of the two MT mRNAs were similar. In the kidney and liver, the MT-1 gene product predominated, while in the heart the relative expression levels of the two genes were opposite. Both the MT-1 and MT-2 mRNA levels increased with Cd concentration in a time- and dose-dependent manner. The expression of MT-2, however, was more responsive to a high Cd concentration. In parallel with the induction of the MTs by Cd, we followed the accumulation of this metal in the kidney and liver. Although the Cd level was always higher in the kidney during treatment, the rate of accumulation was higher in the liver. Cold stress resulted in a significantly higher induction of MT-1 than of MT-2, while heat shock had no effect on the expression of either gene.     

Alterations in metal toxicity and metal-induced metallothionein gene expression elicited by growth medium calcium concentration

The calcium content of the growth medium has been shown to influence the growth and differentiation of primary epithelial cells in culture. The goal of the present study was to determine if growth medium calcium concentration could influence the susceptibility to metal toxicity and metallothionein gene expression of an immortalized human prostate-derived epithelial cell line (RWPE-1). The RWPE-1 cell line was grown in medium containing either 0.1 or 1.4 mM calcium. Confluent cells were exposed to either Zn⁺² (50, 100, or 150 μM) or Cd⁺² (3, 6, or 12 μM) for 13 days, and cell toxicity and MT gene expression were determined along the time course of exposure. It was demonstrated that the calcium content of the growth medium had a marked influence on Zn⁺² toxicity and a lesser but significant effect on Cd⁺² toxicity to the RWPE-1 cells. Calcium concentration of the growth medium was also shown to alter the accumulation of MT-1/2 protein and MT-1E, MT-1X, and MT-2A mRNAs. It was shown that MT-1/2 protein was markedly increased for metal-exposed cells grown in medium containing 0.1 mM calcium; however, the increased expression did not cause an increase in the resistance of the cells to Zn⁺² or Cd⁺² exposure. These observations show that growth medium calcium concentration can influence metal toxicity and the pattern of expression of the MT mRNAs and protein for RWPE-1 cells. The results suggest that caution should be exercised when comparing toxicological responses between cell lines that may be grown in growth formulations differing in calcium concentration.     

Tissue-specific cadmium accumulation and metallothionein-like protein levels during acclimation process in the Chinese crab Eriocheir sinensis

Aquatic organisms chronically exposed to cadmium can increase their resistance to a subsequent elevated exposure. In order to investigate mechanisms involved in acclimation process in the Chinese crab Eriocheir sinensis, we compared Cd level as well as metallothionein-like protein (MTLP) content in different tissues after direct acute exposure (i.e. 500 microg Cd L(-1) for 3 days), and after acute following chronic (i.e. 10 or 50 microg Cd L(-1) for 30 days) exposure. Cadmium accumulation occurred in the following order: anterior gill>hepatopancreas>posterior gill>carapace>hemolymph>muscle. As high concentrations as 188 microg Cd g(-1) w.w. were reported in anterior gills and seem to reach a saturation level. In these gills, the highest MTLP induction was observed after a direct acute exposure, for which a correlation with Cd content occurred. However, the Cd-binding potential by MTLPs was exceeded for any exposure condition. In hepatopancreas, the highest Cd level was reported for crabs acclimated during 30 days to 50 microg Cd L(-1) before challenging with an acute exposure. Moreover, we showed that MTLPs were induced during the acclimation process. In this organ, MTLPs are theoretically sufficient to bind all Cd. These results suggest that during a chronic exposure to 50 microg Cd L(-1), Chinese crabs acquire the capacity to hold more cadmium in hepatopancreas where it can be sequestrated by MTLPs. On the contrary, MTLP induction seems to be a rapid response to acute exposure in anterior gill, but is not sufficient to sequester all Cd. Other sequestration and/or detoxification mechanisms must take place in anterior gill to cope with high Cd levels.     

The distribution kinetics of waterborne silver-110m in juvenile rainbow trout

Juvenile rainbow trout (Oncorhynchus mykiss) were subjected to a 2-day radioactive pulse of 110mAg at 11.9 microg/l (as AgNO3), followed by a 19-day post-tracer exposure to non-radioactive Ag(I) (3.8 microg/l). The distribution of 110mAg in the gills, liver, intestine, kidney, brain and remaining carcass was investigated over a 19-day post-tracer period. Initially, the intestine contained the highest proportion of the 110mAg burden (34%), however, by day 8, less than 5% of the total radioactivity remained in this tissue. The majority of the 110mAg eliminated from the intestine appeared to distribute to the liver. Eventually, the 110mAg content in the liver accounted for as much as 65% of the total radioactivity in the fish. Apart from the liver and intestine, only the gills and carcass contained any appreciable amount (>5%) of the total body 110mAg content. Liver and gill samples were fractionated using differential centrifugation techniques to discern the subcellular distribution of 110mAg in these tissues. In the liver, the 110mAg levels in the cytosolic fraction increased from 35% to 72% of the total cellular burden between days 8 and 19, respectively. The radioactive pulse in the gills was predominantly found in a membrane compartment termed the nuclear fraction ( approximately 60% of the total). Little change was observed over time (day 8 to day 19) to the subcellular distribution of Ag in the gills. Using size-exclusion chromatography, most ( approximately 70%) of the 110mAg content in the liver cytosol eluted at a molecular weight characteristic of metallothionein. The cytosolic distribution of 110mAg in gills was quite diffuse, occurring primarily in the heavy molecular weight fractions.     

Protective effect of zinc against cadmium hepatotoxicity depends on this bioelement intake and level of cadmium exposure: a study in a rat model

It was estimated, in a rat model of moderate and relatively high chronic human exposure to cadmium (Cd), whether enhanced zinc (Zn) consumption may prevent Cd-induced liver injury and if the possible protective effect of this bioelement depends on its intake. For this purpose, the structure and function of the liver of the rats that received Zn (30 and 60 mg/l) or/and Cd (5 and 50 mg/l) for 6 months were evaluated. The treatment with Cd led to, dependent on the exposure level, pathological changes in the liver, including enhanced apoptosis and induction of inflammatory and necrotic processes. Moreover, the serum activities of hepatic marker enzymes (alanine transaminase and aspartate transaminase) and the concentration of proinflammatory cytokine – tumor necrosis factor α were increased. The supplementation with 30 and 60 mg Zn/l (enhancing daily Zn intake by 79% and 151%, respectively) partially or totally prevented from some of the Cd-induced changes in the liver structure and function; however, it provided no protection from necrosis, and the administration of 60 mg Zn/l during the higher Cd exposure even intensified this process. At both levels of Cd treatment, the use of 30 mg Zn/l was more effective in preventing liver injury than that of 60 mg Zn/l. The hepatoprotective impact of Zn may be explained, at least partly, by its antioxidative, antiapoptotic and anti-inflammatory action, ability to stimulate regenerative processes in the liver tissue, and indirect action resulting in a decrease in the liver pool of the non-metallothionein-bound Cd²⁺ ions able to exert toxic action. The results provide strong evidence that enhanced Zn consumption may be beneficial in protection from Cd hepatotoxicity; however, its excessive intake at relatively high exposure to Cd may intensify liver injury.     

Changes in zinc,copper and metallothionein contents during oocyte growth and early development of the teleost Danio rerio (zebrafish)

In the present report, we investigated zinc, copper and metallothionein (MT) contents in zebrafish oocytes and embryos. Our results demonstrate that the metal content increases during oocytes maturation. Zinc increases from 30 ng/oocyte (stage-1 oocytes) to 100 ng/oocyte (stage-3 oocytes); copper varied from 1 ng/oocyte (stage-1 oocytes) to 3.5 ng/oocyte (stage-3 oocytes). During embryogenesis, zinc and copper contents dramatically increase after fertilisation around the 512-cells stage, then slowly decrease until the mid-gastrula stage. During oocyte growth, the changes in the MT level are proportional to metal content, whereas during embryogenesis the pattern of MT accumulation does not parallel that of the two metals. Indeed, the maternal pool of MT decreases steadily during the early stages of the development until the gastrula stage. We have examined the effect of cadmium on the expression of MT during zebrafish development. After cadmium exposure, MT content increases in embryos at the blastula stage, whereas no induction occurs in embryos at the gastrula stage. However, pre-treatment of embryos at the gastrula stage with 5-aza-2'-deoxycytidine induces MT synthesis following exposure to cadmium. These observations show that changes in metal levels are not correlated to MT content in the embryo, whereas DNA methylation is one of the factors regulating MT expression.     

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 inhibition of cystathionase activity on glutathione and metallothionein levels in the adult rat

The effects of alterations in sulfur metabolism on hepatic and renal metallothionein and glutathione metabolism were studied in the adult rat using inhibition of two enzymes of these pathways, hepatic cystathionase and renal gamma-glutamyl transpeptidase. Rats were fed a diet containing both methionine (0.66%) and cystine (0.20%) for 1 week before receiving three consecutive daily intraperitoneal injections of propargylglycine, a selective cystathionase inhibitor, at various doses (2.5–375 μmol/kg). When hepatic cystathionase was inhibited greater than 90% (≥50 μmol propargylglycine/kg), renal and hepatic metallothionein and hepatic glutathione were unaltered except at the highest dose. On the other hand, renal glutathione was increased twofold with a concomitant decrease in renal gamma-glutamyl transpeptidase activity (50% of control). In another experiment, when renal gamma-glutamyl transpeptidase was inhibited greater than 90% with three consecutive daily injections of acivicin, a selective gamma-glutamyl transpeptidase inhibitor (10 mg/kg IP), renal glutathione content was unaltered while hepatic glutathione was decreased. Renal and hepatic metallothionein were not changed. Thus, the cysteine pools for metallothionein and glutathione appear unrelated under the present experimental conditions. In addition, following either propargylglycine or acivicin injections, renal and hepatic glutathione pools appear to be altered differently. These results suggest that renal glutathione may be preferentially maintained even when hepatic glutathione is decreased.     

Multiple biomarker response in rainbow trout during exposure to hexavalent chromium

This research investigated hexavalent chromium toxicity in rainbow trout using a panel of biomarkers at different levels of biological organization. A time-course experiment in which rainbow trout were exposed in hard water (63.5 mg/L CaCO3) to a sublethal concentration of hexavalent chromium (10 mg/L) for a period of 28 days was conducted. The responses of multiple biomarkers were measured in gill and liver tissues at varying time points. Significant differences in metallothionein induction, superoxide dismutase activity, lipid peroxidation, cellular morphology, and growth were observed. Results indicated that gill tissues were more sensitive than hepatic tissues to chromium toxicity, yet hepatic tissues appeared to play a larger role in the organism's adaptive response to chromium compared to gill tissues. This study highlights the importance of using a set of integrated biomarkers to assess contaminant exposure and effects.     

IGF and GH mRNA levels are suppressed upon exposure to micromolar concentrations of cobalt and zinc in rainbow trout white muscle

The objective of this study was to assess the effects of cobalt and zinc exposure of rainbow trout (Oncorhynchus mykiss) on insulin like growth factors (IGF) and growth hormone (GH). Mature rainbow trouts were exposed to 0.42, 2.1, 4.2, 21 and 42μmol/L Co(2+) (added as CoCl(2)·6H(2)O) and 0.34, 1.7, 3.4, 17 and 34μmol/L Zn(2+) (added as ZnSO(4)i·7H(2)O). After 6, 12, 24 and 48h of treatment, expressions of white muscle IGF-I, IGF-II and GH mRNAs were measured by means of quantitative Real Time PCR. During the exposure experiments, no mortalities occurred. The most effective metal concentrations, which caused significant alterations, were determined to be 42μmol/L Co(2+) (10mg CoCl(2)·6H(2)O/L) and 3.4μmol/L Zn(+2) (1mg ZnSO(4)·7H(2)O/L). The following results were obtained for these concentrations. Expression of IGF-I did not change at 6h in zinc treatment while the decrease (p<0.05) was observed at 12h and 24h, and this decrease became stronger at 48h. Cobalt exposure caused a decrease in IGF-I mRNA level at 6h, 12h, 24h and 48h (p<0.05). Both zinc and cobalt exposure resulted in significant decreases in GH expression at 6h. Exposure of trout to Zn resulted in a decrease in expression of IGF-II starting from 6h whereas the significant decrease started at 6h in cobalt exposure and this decrease elevated at 24h. The results indicate that micromolar cobalt and zinc exposure causes significant attenuation in the expressions of these three genes' time dependently. Our findings show that IGF-I is the most resistant and GH is the most sensitive component against cobalt and zinc exposure. We conclude that IGF/GH axis might be strongly affected by the short term exposure to low micromolar concentrations of zinc and cobalt due to alterations of these genes.     

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.     

The effect of orally administered melatonin on tissue accumulation and toxicity of cadmium in mice

The aim of this study was to determine whether an oral administration of melatonin, a known antioxidant, free radical scavenger and metal chelator, influences tissue accumulation and toxicity of cadmium (Cd) in mice exposed subchronically to the metal. The animals received drinking water containing 50 μg Cd/mL only or with additional 2, 4 or 6 μg/mL melatonin for 8 weeks. Melatonin co-treatment brought about a dose-dependent decrease in the renal, hepatic and intestinal Cd concentrations, and the renal and hepatic metallothionein levels followed a pattern similar to that of the Cd accumulation. Histopathological changes occurred only in the kidneys (glomerular swelling and focal tubular degeneration) in all mice from the Cd alone group. In mice co-treated with melatonin, only slight (2 μg/mL melatonin) or no damage (4 and 6 μg/mL melatonin) was seen. The Cd and melatonin treatments did not affect renal lipid peroxidation and iron concentration. These data indicate that orally administered melatonin together with Cd reduces tissue accumulation of this metal; in particular, the reduction of renal Cd accumulation by melatonin is probably responsible for the prevention of Cd-induced injury in this organ.     

Autometallography and metallothionein immunohistochemistry in hepatocytes of turbot (Scophthalmus maximus L.) after exposure to cadmium and depuration treatment

In this study, autometallography and immunohistochemistry were used to localize and quantify cadmium and metallothionein (MT) levels, respectively, in cellular compartments of turbot liver on exposure to cadmium for 7 days and further depuration treatment for 14 days. Metals weakly bound to proteins (i.e. MTs) in hepatocyte lysosomes were visualized as black silver deposits (BSDs) using a light microscope. With the aid of a newly developed immunohistochemical procedure, MTs were localized and semi-quantified in both the cytosolic and the lysosomal compartments of hepatocytes. The BSD extent in the lysosomes of hepatocytes increased significantly as a result of cadmium exposure. This response was evidenced after 1h. Further, a progressive increase in the volume density of BSDs occurred up to the seventh day. Total MT immunohistochemical levels increased at a lower rate, starting after 1 day of cadmium exposure. BSD extent values recovered after depuration, whilst MT levels remain unchanged. It is possible that the detoxification rate of metals via lysosomes was diminished, whilst MT levels remained unchanged, at least after 14 days of depuration. It can be concluded that autometallography and MT immunohistochemistry are good tools for clarifying metal and metal-MT trafficking routes in hepatocytes, and also that BSD extent and MT immunohistochemical levels in the lysosomes and cytosol of fish hepatocytes can be considered to be useful biomarkers of metal exposure.     

Autometallography and metallothionein immunohistochemistry in hepatocytes of turbot (Scophthalmus maximus L.) after exposure to cadmium and depuration treatment

In this study, autometallography and immunohistochemistry were used to localize and quantify cadmium and metallothionein (MT) levels, respectively, in cellular compartments of turbot liver on exposure to cadmium for 7 days and further depuration treatment for 14 days. Metals weakly bound to proteins (i.e. MTs) in hepatocyte lysosomes were visualized as black silver deposits (BSDs) using a light microscope. With the aid of a newly developed immunohistochemical procedure, MTs were localized and semi-quantified in both the cytosolic and the lysosomal compartments of hepatocytes. The BSD extent in the lysosomes of hepatocytes increased significantly as a result of cadmium exposure. This response was evidenced after 1h. Further, a progressive increase in the volume density of BSDs occurred up to the seventh day. Total MT immunohistochemical levels increased at a lower rate, starting after 1 day of cadmium exposure. BSD extent values recovered after depuration, whilst MT levels remain unchanged. It is possible that the detoxification rate of metals via lysosomes was diminished, whilst MT levels remained unchanged, at least after 14 days of depuration. It can be concluded that autometallography and MT immunohistochemistry are good tools for clarifying metal and metal-MT trafficking routes in hepatocytes, and also that BSD extent and MT immunohistochemical levels in the lysosomes and cytosol of fish hepatocytes can be considered to be useful biomarkers of metal exposure.     

Uptake and accumulation of zinc in juvenile rainbow trout,Salmo gairdneri

Synopsis The toxicity of zinc to rainbow trout was determined and the 72 h median lethal concentration was found to be 2.00 mg l^–1 in freshwater, hardness 7.50 mg l^–1 as calcium. An insignificant increase in zinc concentration of internal tissues occurred in fish exposed to 1.52 mg l^–1 in freshwater for 72 h. However, there was a significant uptake of zinc by gills and the body surface. Fish exposed to 10 mg l^–1 zinc for 72 h in two-thirds sea water showed significant zinc uptake by liver, rectum and muscle, when compared to control fish. Drinking rate decreased from 1.43 to 0.26 ml kg^–1 h^–1 when zinc sulphate was added to freshwater. Trout adapted to two-thirds sea water showed no decrease in drinking, about 7 ml kg^–1 h^–1 when zinc was added to the water.     

Metal accumulation and metallothionein in brown trout, Salmo trutta, from two Norwegian rivers differently contaminated with Cd, Cu and Zn

In this work we have studied the accumulation of heavy metals in two brown trout (Salmo trutta) populations in their natural environment and the participation of metal binding to metallothionein (MT) in this process. Cd, Cu and Zn concentrations, total MT (including Cu MT) and Cd/Zn MT were measured in the gills, liver and kidney of trout inhabiting two rivers, one Cu-contaminated and the other Cd/Zn-contaminated, located at Røros, Central Norway. In both populations, high levels of Cu were found in the liver, whereas Cd was accumulated in liver and particularly in the kidney. The proportions of Cd/Zn MT and Cu MT in liver and kidney, but not in gills, reflected the accumulated and the environmental concentrations of these metals. The total Cu MT concentrations in the investigated tissues, however, were highest in trout from the river with the lowest ambient Cu concentration. It is suggested that MTs are of less importance in Cu-acclimated trout. The data also suggest that acclimation to a Cu-rich environment involves reduced Cu accumulation or increased Cu elimination. In trout from the Cd-rich environment, this metal was mainly bound to MT, whereas in trout from the Cu-rich environment Cd was also associated with non-MT proteins. These findings emphasize the importance to determine both Cd/Zn MT and Cu MT levels, when the participation of this protein in metal handling in trout tissues is investigated.     

Cadmium transport in isolated enterocytes of freshwater rainbow trout: interactions with zinc and iron, effects of complexation with cysteine, and an ATPase-coupled efflux

The present study investigated the mechanisms of intestinal cadmium (Cd) uptake and efflux, using isolated enterocytes of freshwater rainbow trout (Oncorhynchus mykiss) as the experimental model. The apical uptake of free Cd(2+) in the enterocytes was a saturable and high-affinity transport process. Both zinc (Zn(2+)) and iron (Fe(2+)) inhibited cellular Cd(2+) uptake through a competitive interaction, suggesting that Cd(2+) enters enterocytes via both Zn(2+) (e.g., ZIP8) and Fe(2+) (e.g., DMT1) transport pathways. Cellular Cd(2+) uptake increased in the presence of HCO(3)(-), which resembled the function of mammalian ZIP8. Cellular Cd(2+) uptake was unaffected by Ca(2+), indicating that Cd(2+) does not compete with Ca(2+) for apical uptake. Interestingly, Cd uptake was influenced by the presence of l-cysteine, and under the exposure condition where Cd(Cys)(+) was the predominant Cd species, cellular Cd uptake rate increased with the increased concentration of Cd(Cys)(+). The kinetic analysis indicated that the uptake of Cd(Cys)(+) occurs through a low capacity transport mechanism relative to that of free Cd(2+). In addition, Cd efflux from the enterocytes decreased in the presence of an ATPase inhibitor (orthovanadate), suggesting the existence of an ATPase-coupled extrusion process. Overall, our findings provide new mechanistic insights into the intestinal Cd transport in freshwater fish.     

Developmental variation in copper,zinc and metallothionein mRNA in brindled mutant and nutritionally copper deficient mice

The concentrations of copper, zinc and metallothionein-I (MT-I) mRNA were determined in the liver, kidney and brain of the brindled mutant mouse from birth until the time of death. Despite accumulation of copper in the kidney of the mutant, MT-I mRNA concentrations were normal. There was no difference between the MT-I mRNA in the brain of mutant and normal in the first 10 days of life, but after day 10 metallothionein mRNA levels were increased in the mutant. The concentration of copper was very low in the liver of the mutant, and on day 6 after birth the metallothionein mRNA was also reduced by about 50%. This reduction was not seen in copper-deficient 6-day-old pups, despite very low hepatic copper levels. This suggests that the lower hepatic MT-I mRNA in the day 6 brindled mouse was not simply due to the reduction in hepatic copper and also that hepatic copper is not regulating metallothionein gene expression the liver of neonatal mice. After day 12 hepatic MT-I mRNA levels were elevated in mutant and in copper deficient mice, both of which die at 14 to 16 days. These increases and the increase in brain MT-I mRNA in older mutant mice are likely to be caused by stress. Overall the results support the conclusions that the brindled mutation does not cause a constitutive activation of the metallothionein genes, and that the differences in metallothionein mRNA between mutant and normal are most probably secondary consequences of the mutation.