Behavioral changes in metallothionein-null mice after the cessation of long-term, low-level exposure to mercury vapor

The neurobehavioral changes in wild-type and metallothionein (MT)-null mice after the cessation of long-term, low-level exposure to Hg0 were investigated. MT-null and wild-type females were continuously (24 h/day) exposed to mercury vapor (Hg0) at 0.055 mg/m3 (range: 0.043-0.073 mg/m3), which was similar to the current threshold limit value (TLV), for 29 weeks. The effects on behavior, such as locomotor activity in the open field (OPF), learning ability in the passive avoidance response (PA) and spatial learning ability in the Morris water maze (MM) were examined immediately and 12 weeks after the cessation of exposure. Immediately after the exposure had ceased, total locomotor activity in OPF was decreased in the both strain of mice, although the MT-null mice appeared to show more distinct effect. In the PA test, the exposed animals of both strains showed learning impairment as compared to un-exposed mice. Twelve weeks after the cessation of exposure, the locomotor activity in OPF was elevated in the exposed mice of both strains, while the learning ability in the PA test appeared normal in both strains. Spatial learning ability was not affected at all. Immediately after the exposure had ceased, the brain mercury concentration of the exposed wild-type mice was 1.75 microg/g, twofold of that in the MT-null mice. In 12 weeks, brain mercury levels decreased to approximately 1/20 of those in immediately after the exposure in both of the strains. These results for the first time indicated that long-term, low-level exposure to Hg0 could exert neurobehavioral effects, which were not reversible even after a long exposure-free period. Whereas the effects on learning ability were presumably transient, the effects on spontaneous behavior as evaluated in OPF were persistent. Finally, the MT-null mice seemed more susceptible to Hg0-induced neurotoxicity than the wild-type mice, confirming our previous results.     

Neurobehavioral changes in metallothionein-null mice prenatally exposed to mercury vapor

We studied the neurobehavioral effects of prenatal exposure of MT-null and wild-type mice to elemental mercury vapor (Hg0). Pregnant mice of both strains were repeatedly exposed to Hg0 vapor at 0.50 and 0.56 mg/m3 for 6 h/day until the 18th day of gestation. The behavioral effects were evaluated with locomotor activity in the open field, learning ability in the passive avoidance response and spatial learning ability in the Morris water maze at 12 weeks of age. Hg0-exposed MT-null mice showed a significant decrease in total locomotor activity in males, and a learning disability in the passive avoidance response and a retarded acquisition in the Morris water maze in females as compared with the control. In contrast, Hg0-exposed wild-type mice did not differ from controls in the three behavioral measurements. The results indicate that MT-null mice would be more susceptible than wild-type mice to the behavioral neurotoxicity of prenatal Hg0 exposure. Mercury concentrations in the brain of both strains were slightly higher in the exposed group than in the control group, indicating the retention of residual mercury even 12 weeks after the cessation of the exposure. Brain concentrations of mercury were also significantly higher in the exposed-females than exposed-males in either strain. From these results, we suggest that the increased susceptibility of MT-null females to behavioral changes caused by prenatal Hg0 exposure is due to a greater retention of mercury and lack of MT-I,-II in the brain.     

Neurobehavioral changes and alteration of gene expression in the brains of metallothionein-I/II null mice exposed to low levels of mercury vapor during postnatal development

This study examined the neurobehavioral changes and alteration in gene expression in the brains of metallothionein (MT)-I/II null mice exposed to low-levels of mercury vapor (Hg(0)) during postnatal development. MT-I/II null and wild-type mice were repeatedly exposed to Hg(0) at 0.030 mg/m(3) (range: 0.023-0.043 mg/m(3)), which was similar to the current threshold value (TLV), for 6 hr per day until the 20th day postpartum. The behavioral effects were evaluated with locomotor activity in the open field (OPF), learning ability in the passive avoidance response (PA) and spatial learning ability in the Morris water maze (MM) at 12 weeks of age. Hg(0)-exposed MT-I/II null mice showed a significant decrease in total locomotor activity in females, though learning ability and spatial learning ability were not affected. Immediately after Hg(0) exposure, mercury concentrations in the brain did not exceed 0.5 μg/g in any animals. Hg(0) exposure resulted in significant alterations in gene expression in the brains of both strains using DNA microarray analysis. The number of altered genes in MT-I/II null mice was higher than that in wild-type mice and calcium-calmodulin kinase II (Camk2a) involved in learning and memory in down-regulated genes was detected. These results provide useful information to elucidate the development of behavioral toxicity following low-level exposure to Hg(0).     

Mercury accumulation and its distribution to metallothionein in mouse brain after sub-chronic pulse exposure to mercury vapor

Previously we found that exposure to mercury vapor effectively induced metallothionein (MT) biosynthesis in rat brain. Although the induction of not only MT-I/II but also MT-III was evident, the induction rate of the latter was much lower than that of the former. The brain of an MT-null mouse lacks MT-I/II, but has MT-III. Here we examined the effects of sub-chronic pulse exposure to mercury vapor on the brain MT in MT-null mice and their wild type controls. MT-null and wild type mice were preliminarily exposed to mercury vapor for 2 weeks at 0.1 mg Hg/m³ for 1 h/day for 3 days a week, and then exposed for 11 weeks at 4.1 mg Hg/m³ for 30 min/day for 3 days a week. This exposure caused no toxic signs such as abnormal behavior or loss of body weight gain in the mice of either strain throughout the experimental period. Twenty-four hours after the termination of the exposure, mice were sacrificed and brain samples were subjected to mercury analysis, MT assay, and pathological examination. The MT-null mice showed lower accumulation of mercury in the brain than the wild type mice. Mercury exposure resulted in a 70% increase of brain MT in the wild type mice, which was mostly accounted for by the increase in MT-I/II. On the other hand, the brain MT in the MT-null mice increased by 19%, suggesting less reactivity of the MT-III gene to mercury vapor. Although histochemical examination revealed silver-mercury grains in the cytoplasm of nerve cells and glial cells throughout the brains of both strains, no significant difference was observed between the two strains.     

Distribution and retention of mercury in metallothionen-null mice after exposure to mercury vapor

We studied the role of metallothionein (MT) in the distribution and retention of mercury in the brain, lung, liver and kidney of MT-null and wild-type mice after exposure to mercury (Hg0) vapor. Mice were exposed to Hg0 vapor at 5.5-6.7 mg/m3 for 3 h and killed at 1, 24, 72 or 168 h after exposure. One hour after exposure to Hg0 vapor, there were no differences in mercury concentrations in these organs from MT-null and wild-type mice. However, the elimination rate of mercury from the organs, except the brain, were remarkably faster in MT-null mice than in wild-type mice. MT-I and -II levels in the lung and kidney were increased significantly in wild-type mice but not in MT-null mice at 24 h after exposure to Hg0 vapor. At this time point, over 65% of the mercury was retained in the MT fraction of the cytosol of organs from wild-type mice. In contrast, mercury appeared mainly in the high-molecular-weight protein fractions in the cytosol of organs from MT-null mice. In the brain, a large amount of mercury was bound to MT in both strains of mice immediately after exposure. No difference was observed in the elimination rate of mercury from the brain between both strains of mice. Brain MT levels were elevated slightly in wild-type mice at 168 h after exposure but could not be detected in MT-null mice. These data suggest that no detectable MT-I and -II levels were found in the brain of MT-null mice and that mercury was apparently bound to MT-III. Using MT-null mice, we showed also that MT-III may play an important role in the retention of mercury in the brain.     

Maternal-to-fetus transfer of mercury in metallothionein-null pregnant mice after exposure to mercury vapor

This study examined the role of placenta metallothionein (MT) in maternal-to-fetal mercury transfer in MT-null and wild-type mice after exposure to elemental mercury (Hg(0)) vapor. Both strains were exposed to Hg(0) vapor at 5.5-6.7 mg/m(3) for 3 h during late gestation. Twenty-four hours after exposure to Hg(0) vapor, accumulation of mercury in the major organs, except the brain, of MT-null maternal mice was significantly lower than that in organs of wild-type mice. In contrast to mercury levels in maternal organs, fetal mercury levels were significantly higher in MT-null mice than in wild-type mice. In placenta, mercury concentrations were not significantly different between the two strains. Although MT levels in major organs, except the brain, of wild type mice were markedly elevated after the exposure to Hg(0) vapor, the placental MT levels were not elevated. However, endogenous MT level in the placenta is significantly higher than that in other organs, except the liver. Gel filtration profile of the placental cytosol in the wild-type mice revealed that a large amount of placental mercury was associated with MT. In MT-null mice, mercury in placental cytosol appeared mainly in the high-molecular-weight protein fractions. Mercury in the placenta was localized mainly in the yolk sac and decidual cells in the deep layer of the decidua in both mouse strains. The similar localization of MT was found in the placenta of wild type mice. These results suggest that MT in the placenta has a defensive role in preventing maternal-to-fetal mercury transfer.     

Neurobehavioral effects of combined prenatal exposure to low-level mercury vapor and methylmercury

We evaluated the effects of prenatal exposure to low-level mercury (Hg(0)) or methylmercury (MeHg) as well as combined exposure (Hg(0) + MeHg exposure) on the neurobehavioral function of mice. The Hg(0) exposure group was exposed to Hg(0) at a mean concentration of 0.030 mg/m(3) for 6 hr/day during gestation period. The MeHg exposure was supplied with food containing 5 ppm of MeHg from gestational day 1 to postnatal day 10. The combined exposure group was exposed to both Hg(0) vapor and MeHg according to above described procedure. After delivery, when their offspring reached the age of 8 weeks, behavioral analysis was performed. Open field (OPF) tests of the offspring showed an increase and decrease in voluntary activity in male and female mice, respectively, in the MeHg exposure group. In addition, the rate of central entries was significantly higher in this group than in the control group. The results of OPF tests in the Hg(0) + MeHg exposure group were similar to those in the MeHg exposure group in both males and females. The results in the Hg(0) exposure group did not significantly differ from those in the control group in males or females. Passive avoidance response (PA) tests revealed no significant differences in avoidance latency in the retention trial between the Hg(0), MeHg, or Hg(0) + MeHg exposure group and the control group in males or females. Morris water maze tests showed a delay in the latency to reach the platform in the MeHg and Hg(0) + MeHg exposure groups compared with the control group in males but no significant differences between the Hg(0), MeHg, or Hg(0) + MeHg exposure group and the control group in females. The results of OPF tests revealed only slight effects of prenatal low-level Hg(0) exposure (0.03 mg/m(3)), close to the no-observable-effect level (NOEL) stated by the WHO (0.025 mg/m(3)), on the subsequent neurobehavioral function. However, prenatal exposure to 5 ppm of MeHg affected exploratory activity in the OPF test, and, in particular, male mice were highly sensitive to MeHg. The MeHg and Hg(0) + MeHg exposure groups showed similar neurobehavioral effects. Concerning the effects of prenatal mercury exposure under the conditions of this study, the effects of MeHg exposure may be more marked than those of Hg(0) exposure.     

Increased radiation-induced apoptosis in mouse thymus in the absence of metallothionein.

Metallothionein (MT) has been shown to protect cells from free radical induced DNA damage after exposure to copper, hydrogen peroxide and also radiation. In order to study the role of MT in radiation induced apoptosis, age-matched male control mice (C57BL/6J), MT-I overexpressing (MT-I*) and MT-null transgenic mice were exposed to whole body cobalt 60 gamma-irradiation at 0, 5, or 10 Gy, and their thymus were removed 24 h later. The basal levels of MT and zinc concentrations in the thymus were measured by 109Cadmium-heme assay and atomic absorption spectrophotometry, respectively. The MT expression after radiation was determined by immunohistochemical staining using a polyclonal antibody to MT. The extent of apoptosis in thymocytes was determined by histology (H&E stain). DNA was isolated from the thymus, and DNA fragmentation was determined by agarose gel electrophoresis. The results showed that the basal level of MT protein in MT-I* thymus was 2.4-fold higher than control mice, and that MT was inducible in both MT-I* and control C57BL6 thymus after radiation exposure. Minimal MT protein was detected in MT-null mice thymus before or after radiation, while, a significantly higher number of apoptotic cells and DNA fragmentation were found in MT-null thymus after whole body irradiation. These data demonstrated a protective role for MT in radiation-induced apoptosis in mouse thymus.     

Metallothionein-I/II null mice are more sensitive than wild-type mice to the hepatotoxic and nephrotoxic effects of chronic oral or injected inorganic arsenicals.

Metallothionein (MT) is a low-molecular-weight, sulfhydryl-rich, metal-binding protein that can protect against the toxicity of cadmium, mercury, and copper. However, the role of MT in arsenic (As)-induced toxicity is less certain. To better define the ability of MT to modify As toxicity, MT-I/II knockout (MT-null) mice and the corresponding wild-type mice (WT) were exposed to arsenite [As(III)] or arsenate [As(V)] either through the drinking water for 48 weeks, or through repeated sc injections (5 days/week) for 15 weeks. Chronic As exposure increased tissue MT concentrations (2-5-fold) in the WT but not in MT-null mice. Arsenic by both routes produced damage to the liver (fatty infiltration, inflammation, and focal necrosis) and kidney (tubular cell vacuolization, inflammatory cell infiltration, and interstitial fibrosis) in both MT-null and WT mice. However, in MT-null mice, the pathological lesions were more frequent and severe when compared to WT mice. This was confirmed biochemically, in that, at the higher oral doses of As, blood urea nitrogen (BUN) levels were increased more in MT-null mice (60%) than in WT mice (30%). Chronic As exposures produced 2-10 fold elevation of serum interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha levels, with greater increases seen by repeated injections than by oral exposure, and again, MT-null mice had higher serum cytokines than WT mice after As exposure. Repeated As injections also decreased hepatic glutathione (GSH) by 35%, but GSH-peroxidase and GSH-reductase were minimally affected. MT-null mice were more sensitive than WT mice to the effect of GSH depletion by As(V). Hepatic caspase-3 activity was increased (2-3-fold) in both WT and MT-null mice, indicative of apoptotic cell death. In summary, chronic inorganic As exposure produced injuries to multiple organs, and MT-null mice are generally more susceptible than WT mice to As-induced toxicity regardless of route of exposure, suggesting that MT could be a cellular factor in protecting against chronic As toxicity.     

Early life inorganic lead exposure induces testicular teratoma and renal and urinary bladder preneoplasia in adult metallothionein-knockout mice but not in wild type mice

Inorganic lead compounds are carcinogenic in animals and have carcinogenic potential in humans. In mice, lead (Pb) is a transplacental carcinogen in the kidney. Metallothionein (MT) is a metal-binding protein that can reduce the toxicity of various metals, including Pb, either by direct sequestration or as an antioxidant for metals that generate reactive oxygen species. Although MT appears to reduce Pb carcinogenicity in adult mice it is unknown how MT deficiency may affect Pb carcinogenicity from early life exposure. Thus, groups (n = 10) of pregnant MT-I/II double knockout (MT-null) or 129/SVJ MT wild type (WT) mice were exposed to Pb acetate in the drinking water (0, 2000, 4000 ppm Pb) from gestation day 8 through birth and during lactation. Maternal drinking water Pb exposure continued to wean at 4 weeks of age and the male offspring were then directly exposed to Pb until 8 weeks of age and observed until 2 years old. High dose (4000 ppm) but not low dose (2000 ppm) Pb reduced survival in the latter part of the study in both MT-null and WT mice. In MT-null mice, but not WT, early life Pb exposure caused a dose-related increase in testicular teratomas, to a maximum incidence of 28% compared to control (4%). Pb-induced renal cystic hyperplasia, considered preneoplastic, was a prominent occurrence in MT-null mice but nearly absent in WT mice. Pb dose-related increases in renal cystic hyperplasia occurred in adult MT-null with early life exposure with maximal incidence of 52%. Pb-treated MT-null mice also showed dose-related increases in urinary bladder hyperplasia with occasional papilloma that were absent in WT mice. Thus, MT deficiency made mice more sensitive to early life Pb exposure with regard to testes tumors, and renal and urinary bladder preneoplastic lesions.     

Metallothionein-null mice are more susceptible than wild-type mice to chronic CdCl(2)-induced bone injury.

Cadmium (Cd) is an environmental pollutant and is toxic to a number of organs. Chronic exposure to Cd causes loss of bone mass and increased incidence of bone fractures, as seen in Itai-itai patients and laboratory animals. Metallothionein (MT), a low-molecular weight, cysteine-rich, metal-binding protein, has been shown to play an important role in the detoxication of Cd. Thus, this study was designed to test the hypothesis that MT protects against Cd-induced bone injury. Wild-type and MT-I/II knockout (MT-null) mice were given repeated sc injections of CdCl(2) over a wide range of doses for 10 weeks, and Cd-induced bone injury was examined. Cd produced dose- and time-dependent increases in bone Cd content. However, the concentration of Cd in bone was much lower than that found in the liver and kidney (11 vs 400 and 120 microg/g, respectively) of the same mice. There was no difference in bone Cd content between wild-type and MT-null mice. Repeated Cd injections produced a dose-dependent loss of bone mass (up to 25%), as shown by analysis of the femur, tibia, and lumbar vertebrae. The loss of bone mass was more marked in MT-null mice than in wild-type mice, as shown by dry bone weight, defatted bone weight, bone ash weight, and total calcium content. X-ray photography showed decreasing bone density along the entire bone length with increasing dose and time of Cd exposure. The decrease in bone density was more marked in MT-null mice than in wild-type mice at the same dose and time points. Histopathology showed dilatation of haversian canals with increased osteoid seams, rounded osteocytes with expanded pericellular space, and expansion of hyperplastic bone marrow into metaphyseal cortical bone. Again, these lesions were more marked in MT-null mice. In conclusion, this study demonstrates that deficiency in MT renders animals more susceptible to Cd-induced bone mass loss and bone injury, and thus indicates that MT plays a protective role in Cd-induced toxicity in bone, as it does in other tissues.     

Metallothionein-I/II null mice are sensitive to chronic oral cadmium-induced nephrotoxicity.

Chronic exposure to cadmium (Cd) via food and drinking water is a major human health concern. We have previously shown that metallothionein (MT), a metal-binding protein, plays an important role in protecting against Cd toxicity produced by repeated sc injections. However, it is unclear whether MT protects against Cd-induced nephrotoxicity following chronic oral exposure, a route with obvious human relevance. To clarify this issue, MT-I/II knockout (MT-null) and background-matched wild-type (WT) mice were allowed free access to drinking water containing CdCl(2) (30, 100, and 300 ppm Cd), or feed containing CdCl(2) (100 ppm Cd) for 6 months, and the resultant nephrotoxicity was examined. Chronic oral Cd exposure produced a dose-dependent accumulation of Cd in liver and kidney of WT mice, reaching levels up to 50 microg Cd/g tissue. Immunohistological localization of renal MT indicated that chronic oral Cd exposure in WT mice greatly increased MT in the proximal tubules and the medulla, with cellular localization in both the cytoplasm and nuclei. As expected, no MT was detected in kidneys of MT-null mice. After 6 months of Cd exposure, tissue Cd concentrations in MT-null mice were only about one-fifth of that in WT mice. Even though the renal Cd concentrations were much lower in the MT-null mice, they were more sensitive than WT mice to Cd-induced renal injury, as evidenced by more severe nephropathic lesions, increased urinary excretion of gamma-glutamyl-transferase and glucose, and elevated blood urea nitrogen. Six months of Cd exposure to MT-null animals resulted in greater increases in renal caspase-3 activity, an indicator of apoptosis, than to WT mice. In conclusion, this study demonstrates that lack of MT renders MT-null mice vulnerable to Cd-induced nephrotoxicity after chronic oral exposure, the primary route of human Cd exposure.     

Distribution of mercury in metallothionein-null mice after exposure to mercury vapor: amount of metallothionein isoform does not affect accumulation of mercury in the brain

To examine the contribution of metallothionein (MT) to mercury accumulation in mouse tissues, 129 strain female mice and MT null mice were exposed to metallic mercury vapor at a sub-toxic level, and Hg levels in the brain, kidney and liver were determined on 1, 3 and 7 days after the exposure. After exposure to mercury vapor, significant Hg accumulation was observed in the brains of wild-type and MT-I/II null and MT-III null mice, as well as in the liver and kidneys. No strain difference was observed in the tissue Hg accumulations 24 hr after the exposure except for the kidneys, where the highest accumulation was found in MT-III null mice. Although the brains of MT-III null mice showed slightly higher Hg accumulation than the other two strains, no significant difference was observed except in the cerebrum on Day 7. Gel chromatograms of cerebrum soluble fractions revealed that a significant amount of Hg existed as an MT-bound form in all the mouse strains. On the other hand, MT-bound Hg was found as a minor fraction in soluble fractions of the kidneys and livers in wild-type and MT-III null mice. Despite a significant strain difference in total MT levels in the cerebrum, there was no difference among the three strains in the amount of Hg accumulated in the cerebrum and its distribution rates in MT fractions. The present study demonstrated that brain uptake of Hg⁰ and its accumulation as Hg²⁺ did not depend on the amount of MT isoform in the tissue, at least in the early phase.     

Metallothionein-I/II double knockout mice are no more sensitive to the carcinogenic effects of nickel subsulfide than wild-type mice

Metallothionein (MT) is a high-affinity metal-binding protein thought to mitigate the toxicity of various metals. MT may limit the toxicity of a metal by direct binding or through action as an antioxidant for metals that generate reactive oxygen species. Nickel compounds have carcinogenic potential in humans and animals, possibly by production of oxidative stress. The impact of MT deficiency on the carcinogenic effects of nickel is unknown. Thus, groups (n=25) of male MT-I/II double knockout (MT-null) or MT wild-type (WT) mice were exposed to a single treatment of nickel (0.5 or 1.0 mg Ni3S2/site, intramuscularly, [i.m.], into both hind legs), or left untreated (control) and observed over the next 104 weeks. There were no differences in the incidence of spontaneous tumors in MT-null and WT mice. Nickel induced injection site fibrosarcomas in a dose-related fashion to a similar extent in both WT and MT-null mice. Nickel-treatment had no effect on total lung tumor incidence, although some phenotypic-specific differences occurred in the proportion of benign and malignant pulmonary tumors. Overall, MT-null mice appear no more sensitive to the carcinogenic effects of nickel than WT mice. Thus, poor MT production does not appear to be a predisposing factor for nickel carcinogenesis.     

Analysis of toxicity using metallothionein knockout mice

Two research groups produced metallothionein (MT)-I/II knockout mice with null mutation of MT-I and MT-II genes. In 1993, Choo et al. produced MT-I/II knockout mice with a mixed genetic background of 129 Ola and C57BL/6 strains. Palmiter et al. also produced MT-I/II knockout mice with a genetic background of 129/Sv strain in 1994. Subsequently, MT-I/II knockout mice have been used to clarify the biological function and physiological role of MT by many research groups. We were also provided MT-I/II knockout mice from Dr. Choo (Australia). F1 hybrid mice were mated with C57BL/6, and their offspring were back-crossed to C57BL/6 for ten generations. MT-I/II knockout (MT(-/-)) mice and wild-type (MT(+/+)) mice were obtained by mating of those heterozygous (MT(+/-)) mice. We have been investigating the susceptibility of MT-I/II knockout mice to toxicity of harmful factors and some diseases. Our present studies found that MT-I/II knockout mice have an increased sensitivity to harmful metals such as cadmium, mercury, and arsenic, oxidative stress, chemical carcinogenesis and neurodegenerative diseases. These results clearly indicate that MT plays an important role in defense of these toxicities. In this review, we present our findings and summarize recent reports with MT-I/II knockout mice concerning the role of MT as a biological protective factor.     

Tissue accumulation of cadmium following oral administration to metallothionein-null mice

To verify the roles of intestinal metallothionein (MT) as a barrier against ingested cadmium (Cd) and as a transporter of mucosal Cd to the kidneys, the distribution of orally administered Cd was compared between normal and MT-I and -II knock-out (MT-null) mice. Following single administration of a low dose of Cd (0.1 mg/kg), hepatic Cd levels and the sum of total Cd in the liver and kidney (K+L) were significantly less in the controls than in MT-null mice. The ratio of Cd in the kidney to the liver (K/L) was significantly lower in the MT-null mice. On the other hand, at a high Cd dose (2.0 mg/kg), K+L and K/L were not significantly different between the two groups. However, following oral pretreatment with zinc (Zn) to the high dose control mice, K+L significantly decreased and K/L significantly increased. No such effects of Zn pretreatment were observed in MT-null mice. Similar differences in K+L and K/L were also observed between the control and MT-null mice groups following the Zn pretreatment. Repeated administration of Cd for 4 weeks resulted in significantly larger K/L distribution in control mice over null mice. These results suggest that MT in the intestinal mucosa functions both as a protective barrier against Cd absorption and as an extracellular transporter of Cd to the kidney.     

Protection against carbon tetrachloride hepatotoxicity by oleanolic acid is not mediated through metallothionein

Oleanolic acid is a triterpenoid compound that has been shown to protect against liver injury produced by some hepatotoxicants. This study was designed to characterize the protective effects of oleanolic acid on carbon tetrachloride-induced hepatotoxicity, and the role of metallothionein in the protection. Oleanolic acid pretreatment (100–400 μmol/kg, sc) protected Sprague–Dawley rats and mice from carbon tetrachloride-induced liver injury in a dose- and time-dependent manner, as evidenced by serum alanine aminotransferase and sorbitol dehydrogenase activities, as well as by histopathology. The protection against carbon tetrachloride hepatotoxicity was not evident until animals were pretreated with oleanolic acid 12 h, and lasted for 72 h after a single injection. This suggests that the protection might be due to induction of some adaptive mechanisms. Metallothionein (MT), an acute-phase protein proposed to decrease carbon tetrachloride-induced liver injury, was dramatically induced following oleanolic acid treatment. To examine whether oleanolic acid protection is mediated through MT, MT-I and II knock-out (MT-null) mice were utilized. Oleanolic acid pretreatment increased MT levels in control mice (20-fold), but not in MT-null mice, however, it protected equally against carbon tetrachloride-induced hepatotoxicity in both control and MT-null mice. These data indicate that oleanolic acid is effective in protecting rats and mice from the hepatotoxicity produced by carbon tetrachloride, and the protection is not mediated through induction of MT.     

Urinary excretion of metallothionein-I and its degradation product in rats treated with cadmium, copper, zinc or mercury

The metallothionein-I (MT-I) content of urine following administration of cadmium (Cd), copper (Cu), mercury (Hg) or zinc (Zn) to rats was determined by radioimmunoassay. Urinary excretion of MT-I was increased significantly after injection of each of these metals. Fractionation of urine from Cd-treated rats on Sephadex G-50 showed a single immunoreactive component corresponding to native MT-I, whereas in urine from Cu, Zn or Hg-treated rats 2 immunoreactive components corresponding to MT-I and a possible degradation production were observed. Since a comparable low molecular weight component corresponding to this degradation product was not detected to the same extent on fractionation of plasma from Cu-exposed rat, it seemed to be derived from degradation of MT in the kidney.     

Basal metallothionein-I/II protects against NMDA-mediated oxidative injury in cortical neuron/astrocyte cultures

N-Methyl-D-aspartate (NMDA) receptor overactivation-mediated oxidative stress has been proposed to contribute to brain injury. Metallothionein-I/II (MT-I/II), a member of cysteine-rich metalloproteins, has been found to express in the central nervous system primarily in cortical tissues and be upregulated following brain injury. To address the role of MT-I/II on NMDA-mediated oxidative injury, we established primary cortical neuron/astrocyte cultures from neonatal MT-I/II deficient (MT?/?) and wild type (MT+/+) mice to test whether basal MT-I/II protects cortical cultures against NMDA-mediated injury. We found that MT-I/II expression was increased by NMDA in MT+/+ cultures but was not detectable in MT?/? cultures. NMDA concentration-dependently induced oxidative injury in both MT+/+ and MT?/? cultures as evidenced by decrease of cell viability, increases of lipid peroxidation and DNA damage. However, these toxic effects were greater in MT?/? than MT+/+ cultures. NMDA significantly increased reactive oxygen species (ROS) generation and disrupted mitochondrial membrane potential in neurons in MT+/+ cultures, and these effects were exaggerated in MT?/? cultures. Our findings clearly show that basal MT-I/II provides protection against NMDA-mediated oxidative injury in cortical neuron/astrocyte cultures, and suggest that the protective effects are possibly associated with inhibition of ROS generation and preservation of mitochondrial membrane potential.     

Alterations of metallothionein isomers in Hg<Superscript>0</Superscript>-exposed rat brain

Previously we found that exposure to mercury vapor effectively induced brain metallothionein (MT) in rats. Here, using FPLC-gel chromatography, we examined time-dependent alterations in the MT isomers, MT-I/II and MT-III, following 3 weeks of exposure. Rats were exposed to mercury vapor at 8.3 mg/m3 for 15 h in total over 5 consecutive days. Total MT levels in rat cerebrum and cerebellum increased by 65% and 155%, respectively, 24 h after the final exposure. The increased levels in both tissues remained unchanged for at least 2 weeks after termination of exposure. Interestingly, most MT in control rat cerebrum and cerebellum was accounted for by MT-III, with MT-I/II being less than 10%. Through mercury vapor exposure, MT-I/II was quickly induced to a significant extent in both tissues, reaching a level comparable to that of MT-III. The induction rate of MT-I/II in the cerebellum was somewhat higher than in the cerebrum. Chromatograms showed that the MT-I/II thus induced began to decline at an early stage in both tissues. In the cerebrum, the amount of MT-I/II on day 22 was about 30% of the maximum level on day 1. On the other hand, the induction of MT-III was not that dramatic, but it did become evident, at least in the latter stage, when MT-I/II had begun to decrease. Thus, though the induction rate of MT-III was not as high as MT-I/II, it was sustained throughout the experimental period.