Cadmium exposure during pregnancy and lactation: materno-fetal and newborn repercussions of Cd(ii), and Cd-metallothionein complexes.

Cadmium (Cd) is a non-physiological heavy metal that can be harmful at low concentrations. Increasing anthropogenic activities are incrementing the risk of accumulation of this heavy metal in different organs and tissues of the body. In the case of pregnant women, the threat is more serious due to the implications affecting not only their own health but also fetal development as well. Metallothioneins (MTs), small cysteine-rich proteins, are involved in zinc (Zn) and copper homeostasis in mammals but can, however, also bind with Cd if present. The accumulation of Cd in maternal tissues (e.g. placenta, maternal blood, and mammary glands) induces the synthesis of MTs, preferably MT2, in an attempt to sequester the metal to avoid toxicity. The formed Cd-MT complexes will avoid the Cd transport from the placenta to the fetus and end up accumulating in the maternal kidneys. At the same time, high concentrations of MTs will increase the formation of Zn-MT complexes, therefore decreasing the amount of Zn ions available to be transported to the fetus by means of Zn transporters such as ZnT2, ZIP14 and DMT1. Although MTs cannot transport Cd from the mother to the fetus, the divalent DMT1 transporter is suggested to carry the metal to the fetus. As a consequence, the low levels of Zn(ii) in the fetus, together with the presence of Cd(ii) coming from the mother either via the placenta and cord blood or via breast milk induce changes in the fetal development including fetal growth retardation, and low weight or height of the newborn. Likewise, the concentrations of Cd(ii) in the newborn can cause alterations such as cognitive disabilities. In summary, the presence of Cd(ii) in the maternal tissues will induce MT synthesis in an attempt to detoxify these tissues and reduce the possible toxicity of Cd in fetal and newborn tissues.

Bio-monitoring of metal impact on metallothioneins levels in the gastropod Phorcus turbinatus (Born, 1778) in the northeastern and the eastern coasts of Tunisia.

This work is an evaluation of metal contamination degree in Phorcus turbinatus and a monitoring of metals impact on metallothioneins functioning. The gastropod was sampled from six stations along the northeastern and eastern coasts of Tunisia during four seasons (2014-2015). Our results suggested that sedimentological metallic contents (Copper, Zinc and Cadmium) vary significantly depending on stations and not significantly according to seasons except for copper. Nevertheless, differences were no significant between stations and significant between seasons as for ambient factors, metallic intrinsic contents and metallothioneins rates. The monodonta tissue seems to be enriched in the four analyzed metals (Cu, Zn, Cd and Hg) and this species seems to have an ability to accumulate metals. Metal effect on the protein induction may be linked to physicochemical factors (temperature, O2 and Cu contents in sediment). In fact, metallothioneins levels were positively correlated to the dissolved oxygen rates and negatively linked to temperature.

Distribution of exogenous metallothionein following intraperitoneal and intramuscular injection of metallothionein-deficient mice.

Metallothionein-I/II (MT-I/II) is a small metal-binding protein with antioxidant and neuroprotective properties, which has been used experimentally as a neurotherapeutic agent in multiple conditions. Therefore it is important to determine whether exogenous MT-I/II is retained in specific organs or expelled from the body following intramuscular and intraperitoneal injection. The distribution of exogenous MT-IIA (the major human MT-I/II isoform) was examined in MT-I/II-deficient mice, by immunohistochemistry of tissue samples and western blotting of urine samples. MT-IIA was detected within epithelial cells of the kidney cortical and medullary tubules within 1 hour of either intramuscular or intraperitoneal injection. Additionally, MT-IIA was detected within the urine at 1 hour after injection, indicating rapid absorbance into the circulation and filtration through the kidney glomerulus. A portion of the intramuscularly-injected MT-IIA remained within the muscle for at least 24 hours after injection. No MT-IIA was observed within the liver or the brain after either a single injection or a series of MT-IIA injections. These results are consistent with earlier reports that exogenously administered MT-IIA does not cross the intact blood-brain barrier, although a receptor for MT-I/II (megalin) is present in the choroid plexus. We postulate that due to losses through the urine, circulating MT-IIA levels drop rapidly after injection and do not permit transport across the choroid plexus. Peptide analogues of MT-I/II with similar neuroactive properties (emtins) may be more suited for CNS delivery.

Role of megalin and the soluble form of its ligand RAP in Cd-metallothionein endocytosis and Cd-metallothionein-induced nephrotoxicity in vivo.

Orally administered Cd is predominantly distributed to the intestine, and the majority of this mucosal Cd is bound to metallothionein (MT). MT attenuates heavy metal-induced cytotoxicity by sequestering these metals and lowering their intracellular concentrations. In addition, MT acts as an extracellular transporter of orally administered Cd to the kidney. Because of its low molecular weight, the Cd-MT complex is freely filtered at the glomerulus, and the filtered Cd-MT is then incorporated into renal proximal tubular cells. Megalin, a multiligand endocytic receptor (also known as low-density lipoprotein receptor-related protein 2 or Lrp2), acts as the receptor for Cd-MT in a renal proximal tubular cell model. Here, we used the soluble form of 39-kDa receptor-associated protein (sRAP; also known as Lrpap1), a ligand of megalin, to inhibit megalin function, and then analyzed the effect of megalin loss on Cd-MT distribution and Cd-MT-induced nephrotoxicity in an animal model. Administration of sRAP to mice caused acute loss of megalin function by removing megalin in the brush border membrane. The pre-injection of sRAP decreased renal Cd content and decreased Cd-MT-induced kidney damage. Our results demonstrate that sRAP reduces Cd-MT-induced kidney toxicity in vivo.

Redefining the role of metallothionein within the injured brain: extracellular metallothioneins play an important role in the astrocyte-neuron response to injury.

A number of intracellular proteins that are protective after brain injury are classically thought to exert their effect within the expressing cell. The astrocytic metallothioneins (MT) are one example and are thought to act via intracellular free radical scavenging and heavy metal regulation, and in particular zinc. Indeed, we have previously established that astrocytic MTs are required for successful brain healing. Here we provide evidence for a fundamentally different mode of action relying upon intercellular transfer from astrocytes to neurons, which in turn leads to uptake-dependent axonal regeneration. First, we show that MT can be detected within the extracellular fluid of the injured brain, and that cultured astrocytes are capable of actively secreting MT in a regulatable manner. Second, we identify a receptor, megalin, that mediates MT transport into neurons. Third, we directly demonstrate for the first time the transfer of MT from astrocytes to neurons over a specific time course in vitro. Finally, we show that MT is rapidly internalized via the cell bodies of retinal ganglion cells in vivo and is a powerful promoter of axonal regeneration through the inhibitory environment of the completely severed mature optic nerve. Our work suggests that the protective functions of MT in the central nervous system should be widened from a purely astrocytic focus to include extracellular and intra-neuronal roles. This unsuspected action of MT represents a novel paradigm of astrocyte-neuronal interaction after injury and may have implications for the development of MT-based therapeutic agents.

Subcellular distribution of trace elements in kidney of a mother-fetus pair of Dall's porpoises (Phocoenoides dalli).

Total and subcellular renal Zn, Cu, Se, Mn, V, Hg, Cd and Ag were determined by a mother-fetus pair of Dall's porpoises (Phocoenoides dalli). All element concentrations in the maternal individual were higher than those in the fetal individual. Most of total renal elements studied were present in the cytosol of both animals. In maternal cytosol, Mn, Hg and Ag were present in high molecular weight substances (HMW); Se was in low molecular weight substances (LMW); Zn, Cu, and Cd were in metallothionein (MT), mostly; and the distribution of V in percentage among the three renal cytosolic fractions was similar. In fetal cytosol, Zn, Mn, Hg, V, Cd, and Ag were present in HMW, Cu was present in MT, mostly. In contrast, Se was observed mostly in both LMW and HMW. MT isoforms were characterized. Three obvious peaks in retention time were found in either the maternal or fetal MT. The highest elemental ion intensities were in the 7.8min peak for the mother, and in the 4.3min peak for the fetus, respectively, implying that different MT isoforms may be closely associated with elemental accumulation between maternal and fetal renal cytosols.

Metallothionein turnover, cytosolic distribution and the uptake of Cd by the green mussel Perna viridis.

We examined the relationship between Cd kinetics (uptake from solution and diet, and efflux), metallothionein turnover, and changes in the cytosolic distribution of accumulated Cd between protein fractions in the green mussel Perna viridis. We pre-exposed the mussels to 5, 20, 50 and 200 microg l(-1) of Cd for 1 week and determined the biokinetics of Cd uptake and efflux in the mussels. The dietary assimilation efficiency of Cd increased by 2 times following exposure to 20-200 microg l(-1) Cd, but the dissolved uptake rate was unchanged by pre-exposure to any Cd concentrations. The efflux rate of Cd was also similar among control and Cd pre-exposed mussels. The cytosolic distribution of Cd in the mussels that had been exposed to dissolved Cd, showed that besides metallothionein (7000 - 20,000 Da), high molecular weight proteins (>20,000 Da) were important for Cd binding and depuration. In general, the Cd pre-exposed mussels had higher metallothionein turnover with a higher metallothionein synthesis rate, but similar metallothionein breakdown rates as the control mussels. Metallothionein synthesis rate was correlated to the dietary assimilation of Cd, whereas metallothionein breakdown and Cd efflux rate were independent of each other. This study provides important new information for the role of metallothionein turnover on Cd kinetics in an aquatic invertebrate.

Kinetic model of cadmium accumulation and elimination and metallothionein response in Ruditapes decussatus.

The aim of the present study was to determine the response of metallothionein (MT) during Cd accumulation and elimination in different tissues of the estuarine bivalve Ruditapes decussatus exposed to two nominal Cd concentrations (4 and 40 microg/L) for 40 d, followed by a depuration period of 50 d. Cadmium was accumulated in all tissues of R. decussatus at both exposure concentrations, and the accumulation was tissue dependent. Use of the kinetic model showed that in the gills and remaining tissues, Cd was assimilated faster at the beginning of the exposure and decreased with time, possibly limited by the diffusion rate of this metal within the cell. In the digestive gland, however, the Cd was continuously accumulated. This could reflect that the Cd uptake rate is considerably higher than the loss rate and, therefore, that this tissue has a higher capacity to accumulate Cd compared to the other two tissues. Moreover, the application of this kinetic model in the different subcellular fractions showed that the bioconcentration factor was significantly higher in the low-molecular-weight fraction (where MT is found), suggesting that this fraction binds Cd faster, with a high uptake rate (K(u) = 32/d), and eliminates this metal more slowly (K(1) = 0.005/d). During the depuration phase, MT decreased simultaneously with Cd elimination in all tissues, although with a shorter half-life. In conclusion, the MT response prevented Cd in the tissues of R. decussatus from interfering in the normal clam metabolism; therefore, MT acts as a detoxification mechanism of Cd.

The role of zinc and metallothionein in the dextran sulfate sodium-induced colitis mouse model.

Zinc (Zn) and its binding protein metallothionein (MT) have been proposed to suppress the disease activity in ulcerative colitis. To determine the role of Zn and MT in the dextran sulfate sodium (DSS)-induced model of colitis in mice, a DSS dose-response study was conducted in male C57BL/6 wild-type (MT+/+) and MT-null (MT-/-) mice by supplementing 2%, 3%, and 4% DSS in the drinking water for 6 days. In the intervention study, colitis was induced with 2% DSS, Zn (24 mg/ml as ZnO) was gavaged (0.1 ml) daily, concurrent with DSS administration, and the disease activity index (DAI) was scored daily. Histology, MT levels, and myeloperoxidase (MPO) activity were determined. DAI was increased (P<0.05) by 16% and 21% with 3% and 4% concentrations of DSS, respectively, compared to 2%, evident after 5 days of DSS administration. MPO activity was increased in MT+/+ compared to MT-/- mice and those receiving DSS. Zn administration had a 50% (P<0.05) lower DAI compared to DSS alone. Zn partially prevented the distal colon of MT+/+ by 47% from DSS-induced damage compared to MT-/- mice. MT did not prevent DSS-induced colitis and Zn was partially effective in amelioration of DSS-induced colitis.

Sub-cellular partitioning of Cd, Cu and Zn in tissues of indigenous unionid bivalves living along a metal exposure gradient and links to metal-induced effects.

We studied organ and sub-cellular distributions of several trace metals in a freshwater bivalve that has been proposed for use as a metal biomonitor. Specimens of Pyganodon grandis were collected from nine lakes located along a Cd, Cu and Zn concentration gradient (Rouyn-Noranda area, Quebec). Gills and digestive gland were isolated, homogenized and six sub-cellular fractions were separated by differential centrifugation and analysed for their Cd, Cu and Zn content. Metallothionein was quantified independently. Gill tissues contained abundant calcium concretions that accounted for over 60% of the total gill burden of each metal. Cadmium and Zn concentrations in this granule fraction reflected ambient metal concentrations. Metal concentrations in the digestive gland also responded to the metal contamination gradient, but to a lesser extent than the gills, reflecting the lower abundance of granules in the digestive gland. Metals (Cd, Cu) in this organ were present largely in the "heat-stable proteins" fraction, and metal concentrations in this fraction were strongly correlated with those of both metallothionein and, to a lesser extent, the "lysosomes+microsomes" and "mitochondria" fractions. In both organs, Cd concentrations in the "heat-denaturable protein" fraction remained low and constant, suggesting reasonably effective metal detoxification. Some evidence for oxidative stress was noted in the gills but not in the digestive gland. Overall, we conclude that in nature metals in P. grandis are bound differently in the gills and in the digestive gland and that metal detoxification in the former organ may be less effective than in the latter.

Is the digestive gland of Mytilus galloprovincialis a tissue of choice for estimating cadmium exposure by means of metallothioneins?

A study performed over 12 months with caged mussels Mytilus galloprovincialis in the coastal marine zone, which is under urban pressure, reveals a temporal variation of digestive gland mass, which causes "biological dilution" of cytosolic metallothionein (MT) and trace metal (Cd, Cu, Zn, Fe, Mn) concentrations. The dilution effect was corrected by expressing the cytosolic MT and metal concentrations as the tissue content. Consequently, the changes of the average digestive gland mass coincide with the changes of MT and trace metal contents. From February to June, MT contents are nearly twice and trace metal contents nearly three times higher than those of the other months. The period of increased average digestive gland mass, of MT and trace metal contents probably overlaps with the sexual maturation of mussels (gametogenesis) and enhanced food availability. Since natural factors contribute more to the MT content than the sublethal levels of Cd, the digestive gland of M. galloprovincialis is not considered as a tissue of choice for estimating Cd exposure by means of MTs.

Metallothionein concentrations in natural populations of gudgeon (Gobio gobio): relationship with metal concentrations in tissues and environment.

A field monitoring campaign investigating the suitability of (Cd, Zn)-metallothionein concentrations (MTs) in different tissues of the gudgeon as a biomarker for metal contamination in the aquatic environment was conducted. Gudgeons were captured at 10 sampling sites on a river system in Flanders (Belgium). Nine sampling sites were situated along a Cd and Zn gradient with a nearby tributary as the reference site. Cadmium, Cu, and Zn concentrations were measured in the water and sediments. Concentrations of (Cd, Zn)-MT were measured in different organs (gill, liver, kidney) of gudgeon (Gobio gobio). The hepatic and gill Cd and Zn concentrations, as well as the hepatic (Cd, Zn)-MT concentrations, reflected the polymetallic contamination gradient. Moreover, the hepatic Cd and Zn concentrations could describe 72% of the variance in the (Cd, Zn)-MT concentrations in the liver, illustrating the possible use of hepatic MT concentrations as a biomarker for environmental metal contamination. In this way a dose-response relationship could be established under natural conditions. However, a poor negative relation between the Cd and Zn concentrations in the gills and the corresponding (Cd, Zn)-MT concentrations was found. No relation between the Cd and Zn concentrations in the kidney tissue and the corresponding (Cd, Zn)-MT concentrations could be established. These results clearly illustrate the tissue-specificity of the MT concentrations, thus for monitoring purposes MT concentrations should be measured in liver tissues, rather than in kidney or gill tissues.

Kinetic study on the reaction of cisplatin with metallothionein.

The binding of cisplatin to metallothionein (MT) was investigated at 37 degrees C in 10 mM Tris-NO3 (pH approximately 7.4) and 4.62 mM NaCl. The conditions were chosen to mimic passage of clinical concentrations of cisplatin through the cytosol. The reactions were monitored by high-performance liquid chromatography (HPLC), atomic absorption spectroscopy, and ultraviolet (UV) absorption spectroscopy. The UV data showed that several reactions occur, the first of which does not affect the absorbance (no Pt-sulfur bond formation). They also suggested that if [cisplatin] is large compared with [MT], the rate of subsequent reaction is between first and second order in [cisplatin] and between zeroth and first order in [MT]. HPLC eluates with 24 < retention time (tR) < 27 min contained undialyzable Pt, which increased with reaction time and corresponded to Pt-thionein product. Eluates with 3 < tR < 7 min corresponded to unbound cisplatin and allowed determination of second-order rate constants (k), using the second-order rate equation. The k value for cisplatin reacting with apo-MT was approximately 0.14 M-1 s-1, Cd/Zn-MT approximately 0.75 M-1 s-1, Cd7-MT approximately 0.53 M-1 s-1, and Zn7-MT approximately 0.65 M-1 s-1. Thus, cisplatin displaced Cd and Zn equally well. Leukocyte MT concentration was approximately 1.0 mM, so that the kinetics of cisplatin binding to cellular MT is pseudo-first order (pseudo-first-order rate constant, approximately 0.63 x 10-3 s-1; half-life, approximately 18 min). With [cisplatin] = 10 microM, the rate of cisplatin reaction with MT is approximately 6.3 micromol s-1 cm-3. We conclude that cellular MT can trap significant amounts of cisplatin and may efficiently contribute to cisplatin resistance.

Accumulation, distribution and toxicology of dietary nickel in lake whitefish (Coregonus clupeaformis) and lake trout (Salvelinus namaycush).

An 18-day experiment was conducted to investigate the uptake and sublethal toxicity of dietary Ni in adult lake whitefish (LWF, Coregonus clupeaformis) and lake trout (LT, Salvelinus namaycush) fed diets containing 0, 1000 and 10000 microg Ni/g, prepared with and without brine shrimp. The results of this experiment were used to design an experiment of longer duration in which one of the fish species was selected and exposed to lower dietary Ni doses. In the present study feed refusal was observed in LT and LWF fed 10000 microg Ni/g, after three and 4-5 feedings, respectively. LT fed Ni-contaminated diets exhibited different patterns of Ni accumulation than LWF. Increased Ni concentrations in all LWF tissues, except the intestine, were associated with increased doses of Ni. Copper and Zn concentrations in kidney and liver of LWF were altered. Metallothionein concentrations in kidneys of LT fed 1000 microg Ni/g and 10000 microg Ni/g and LWF fed 10000 microg Ni/g and in livers of LWF fed 10000 microg Ni/g (diet without shrimp only) increased significantly. Increased lipid peroxide production in the plasma of LT fed 10000 microg Ni/g was observed. Blood glucose and electrolytes were affected by Ni exposure. Histopathological alterations were observed in kidneys of LWF fed low and high dose diets, livers of whitefish fed high dose diets, and intestines of LWF fed high dose diets and LT fed low and high dose diets. LT fed high dose diets exhibited significant decreases in weight.

Cadmium in caribou (Rangifer tarandus) kidneys: speciation, effects of preparation and toxicokinetics.

Caribou kidney is a major source of cadmium (Cd) in the traditional diets of many aboriginal communities in the Arctic. In order to characterize the risk of Cd exposure, we studied the speciation of Cd in caribou kidneys and how, it can be affected by food preparation. Cd in caribou kidneys was bound to metallothionein (MT) (40%) high molecular weight proteins (50-500 kDa) (30%) and existed as free ions (10%). There was no change in Cd concentrations after the samples were baked at 350 degrees C for 30 min but the MT level was significantly decreased. Food preparation also denatured the high molecular weight proteins, resulting in a significant increase of free Cd. The toxicokinetics of Cd in caribou kidney was studied using a rodent model. Four groups (n = 6) of adult Sprague-Dawley rats were fed with rat chow with the addition of one of the following: 10% veal kidney with 0 microg/g Cd, 10% veal kidney spiked with CdCl2 to make up to 66 microg Cd; 10% caribou kidney containing 20 microg/g Cd and 10% caribou kidney containing 66 microg/g Cd. After 14 days, Cd accumulation increased with dose but less than 1% of the dosed Cd accumulated in the liver and kidney of rats. The percentage of Cd accumulated in the liver was higher in the group fed with caribou kidney than that dosed with spiked veal kidney. Our results suggest that caribou kidney is a major source of Cd exposure among the Arctic populations but moderate consumption may be considered unavoidable.

Renal cortical mitochondrial dysfunction upon cadmium metallothionein administration to Sprague-Dawley rats.

A bolus dose of cadmium metallothionein (CdMT) produces renal proximal tubular dysfunction because it accumulates in the tubular epithelial cells and undergoes rapid degradation, releasing Cd. Morphologically, mitochondria appear to be the target organelle. The present study examined changes in renal cortical mitochondrial function following CdMT administration and investigated whether some of these effects could be ascribed to Cd2+ accumulation in the mitochondria. Sprague-Dawley rats were injected ip with 0.3 mg Cd as CdMT/kg and the animals were sacrificed after 6, 8, or 12 h. Two- to threefold increases in urinary protein excretion and LDH activity were evident at 8 h, with marked elevations (11- and 29-fold) thereafter. Renal cortical mitochondria were swollen and rounded at 12 h. The mitochondrial Cd level was 399 pmol/mg protein at 6 h and did not change significantly during the next 6 h; however, mitochondrial respiratory function declined with time. At 12 h, state 3 oxygen consumption, respiratory control ratio (RCR), and ADP:O (P/O) ratio were 48, 49, and 76% of control values, respectively, indicating inhibition of electron transfer and oxidative phosphorylation. The direct effect of Cd on mitochondrial function was examined by incubating mitochondria from untreated rats with 0.1-2 microM CdCl2. Rapid uptake of Cd resulted in concentration-dependent effects on respiration. After 1 min of incubation with 2 microM Cd, the mitochondria contained 262 microgCd/mg protein and state 3 respiration and RCR values were 75 and 33% of control levels, respectively. Thus, renal proximal tubular cell damage following a bolus dose of CdMT involves perturbations in mitochondrial respiration, brought on by the accumulation of Cd.

Dynamics of (Cd,Zn)-metallothioneins in gills, liver and kidney of common carp Cyprinus carpio during cadmium exposure.

Cadmium concentrations, (Cd,Zn)-metallothionein (MT) concentrations, MT synthesis and the relative amounts of cadmium bound to (Cd,Zn)-MTs were determined in gills, liver and kidney of common carp Cyprinus carpio exposed to 0, 0.5 microM (0.06 mg.l(-1)), 2.5 microM (0.28 mg.l(-1)) and 7 microM (0.79 mg.l(-1)) Cd for up to 29 days. Cadmium accumulation was in the order kidney > liver > gills. Control levels of hepatic (Cd,Zn)-MT were four times higher compared to those of gills and kidney. No increases in (Cd,Zn)-MT concentrations were observed in liver during the exposure period. In comparison with control carp, (Cd,Zn)-MT concentrations increased up to 4.5 times in kidney and two times in gills. In both these organs, (Cd,Zn)-MT concentrations were linearly related with cadmium tissue levels and with the de novo synthesis of MTs. Hepatic cadmium was almost completely bound to (Cd,Zn)-MT, while percentages of non-MT-bound cadmium were at least 40% in gills and 25% in kidney. This corresponded with a total saturation of (Cd,Zn)-MT by cadmium in kidney and a saturation of approximately 50 and 60% in gills and liver, respectively. The final order of non-MT-bound cadmium was kidney > gills > liver. Our results indicate that cadmium exposure causes toxic effects, which cannot be correlated with the accumulated levels of the metal in tissues. Although cadmium clearly leads to the de novo synthesis of MT and higher (Cd,Zn)-MT concentrations, the role of this protein in the detoxification process is clearly organ-specific and its synthesis does not keep track with cadmium accumulation.

Metallothionein I+II expression and their role in experimental autoimmune encephalomyelitis.

We examined the expression and roles of neuroprotective metallothionein-I+II (MT-I+II) in the rat CNS in experimental autoimmune encephalomyelitis (EAE), an animal model for the human autoimmune disease, multiple sclerosis (MS). EAE caused significant macrophage activation, T-lymphocyte infiltration, and astrogliosis in spinal cord, brain stem, and cerebellum, which peaked 14-18 days after immunization. The remission of symptoms and histopathological changes began at days 19-21 and were completed by days 30-40. MT-I+II expression was increased significantly in EAE infiltrates. In order to study the effects of increased MT levels, we administered Zn-MT-II intraperitoneally (i.p.) to rats during EAE. Clinically, Zn-MT-II treatment reduced the severity of EAE symptoms and mortality in a time- and dose-dependent manner. Histopathologically, Zn-MT-II increased reactive astrogliosis and decreased macrophages and T lymphocytes significantly in the CNS. In spleen sections, the number of macrophages both in control and EAE-sensitized rats was reduced by Zn-MT-II, while the number of lymphocytes remained unaltered by Zn-MT-II. Therefore, we suggest that MT-II has peripheral mechanisms of action on macrophages, while T lymphocytes are affected locally in the CNS. During EAE, oxidative stress was decreased by Zn-MT-II, which could contribute to the diminished clinical scores observed. None of the effects caused by Zn-MT-II could be attributable to the zinc content. These results suggest MT-I+II as potentially useful factors for the treatment of EAE/MS.

Nephrotoxicity of cadmium-metallothionein: protection by zinc and role of glutathione.

Chronic cadmium (Cd) exposure can cause renal proximal tubular dysfunction resulting from the release of Cd metallothionein (CdMT) from the liver and its accumulation and degradation in the renal tubular epithelial cells. Pretreatment with zinc (Zn) can protect against acute CdMT nephrotoxicity. While induction of MT by Zn plays a part in Zn protection, other factors, such as glutathione (GSH), may also be involved because protection is offered even in MT-null mice. The present study was designed to investigate the involvement of GSH in Zn protection against acute CdMT nephrotoxicity. The study was carried out in MT-null mice to remove the induction of MT by Zn as a confounding variable. Three approaches were used to modulate renal cortex GSH levels: buthionine sulfoximine (BSO) was administered to inhibit GSH synthesis, and GSH and Zn were administered to increase the GSH levels. Both GSH and Zn were effective in protecting against CdMT nephrotoxicity. Elevation in renal cortex GSH levels, however, was not essential for Zn protection, as a low dose of Zn that caused no significant increase in renal GSH also protected against CdMT. On the other hand, maintenance of normal GSH status was essential for Zn protection, as inhibition of GSH synthesis abolished this protection. Both GSH and Zn reduced the accumulation of Cd as well as MT in the renal cortex, with Zn causing greater reduction in Cd accumulation than that of MT. The relative intracellular distribution of Cd was unaltered. These results suggest that in MT-null mice Zn protects against CdMT nephrotoxicity by possibly displacing some of the Cd from CdMT as well as reducing the uptake of CdMT, and that this protection requires the maintenance of normal GSH status.