Immunohistochemical study of macrophage migration inhibitory factor in rat liver fibrosis induced by thioacetamide.

Macrophage migration inhibitory factor (MIF) is a molecule known to regulate macrophage accumulation at sites of inflammation. To elucidate the role of MIF in progression of liver fibrosis, the immunohistochemical localization of MIF and macrophages in the liver were examined. Male Wistar rats received thioacetamide (TA) injections (200 mg/kg, i.p.) for 1 or 6 weeks. In biochemical and histological tests, it was confirmed that liver fibrosis was induced. In immunohistochemical analyses, the expression of MIF protein was seen in hepatocytes in the areas extending out from the central veins to the portal tracts. In particular, at 6 weeks, immunoreactivity was detected in degenerated hepatocytes adjacent to the fibrotic areas but hardly observed in the fibrotic areas. On the other hand, a number of exudate macrophages stained by antibody ED1 were seen in the areas from the central veins to the portal tracts at 1 week and in the fibrotic areas at 6 weeks. Macrophages also showed a significant increase in number as compared with controls. These results revealed that there was a close relationship between the appearance of MIF expression and ED1-positive exudate macrophages in degenerated hepatocytes during the progression of TA-induced liver fibrosis.

2,4,5-Trichlorophenoxyacetic acid inhibits apoptosis in PC12 cells.

2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) is an endocrine disrupter that exerts cytotoxic effects on organisms. In this study, the influence of 2,4,5-T at low concentrations on apoptosis in PC 12 cells was investigated. Although no apoptotic features were observed in PC12 cells treated with 2,4,5-T, it inhibited the DNA fragmentation induced by serum deprivation. In addition, the cell viability of PC12 cells increased after treatment with 2,4,5-T. In conclusion, 2,4,5-T suppressed the apoptosis of the cultured cells. Since apoptosis is a morphological and biochemical description of a physiological mechanism of cell death that is commonly associated with programmed events necessary for development of individuals and organs, the inhibitory effect of 2,4,5-T on apoptosis might cause serious damage to cell homeostasis and differentiation.

Combined effects of working environmental conditions in VDT work.

The combined effects of city noise and luminance of the computer display were evaluated from the changes in lymphocytes and mental activities of participants. Healthy male students were tested under the following four experimental conditions: (1) a calculating task on a video display terminal (VDT) with luminance of 90 cd m(-2) without city noise; (2) a calculating task on a VDT with luminance of 20 cd m(-2) without city noise; (3) a calculating task on a VDT with luminance of 90 cd m(-2) with city noise of 70 dB(A); and (4) a calculating task on a VDT with luminance of 20 cd m(-2) with city noise of 70 dB(A). A visual reaction test (VRT) was performed, and critical flicker fusion frequency (CFF), heart rate (HR), numbers of circulating white blood cells (WBCs), lymphocyte subsets and subjective symptoms of fatigue were measured (1) before; (2) just after; and (3) 30 min after each 60 min test. Subjective symptoms of fatigue significantly increased just after experiments conducted under the two noisy conditions. VRT and CFF showed significant changes in the case of the high-luminance display with noise. WBCs and neutrophils showed significant increases in the two quiet conditions. These results suggested that high luminance with noise had the most effect on subjective fatigue and mental activities.

Effect of trichloroethylene on spatiotemporal pattern of LTP in mouse hippocampal slices.

The effect of trichloroethylene (TCE) on long-term potentiation (LTP) was studied using both electrical and optical recording. The hippocampi from mice injected with 300 mg/kg or 1000 mg/kg TCE were sliced 24 h after administration. The field potential from the CAI was recorded. After the application of tetanus, population spikes (PS) were potentiated in all groups, but the post-per-pre ratio of PS was smaller in TCE groups than in the control. Optical recording was also carried out in 1000 mg/kg TCE-injected mice and a new analytical method using a high speed camera was employed. After the induction of tetanus, the optical signal was potentiated in both TCE and control groups. However, the post-per-pre ratio of the optical signals and response area were smaller in the TCE groups than in the control. It was suggested that the impairment of LTP is one of the mechanisms of the impairment of immediate memory after acute exposure to TCE in humans.

Deficiencies of hippocampal Zn and ZnT3 accelerate brain aging of Rat.

We examined the link of hippocampal Zn to the functional impairments with aging using senescence-accelerated mouse prone 10 (SAMP10) with deficits in learning and memory. Zn in hippocampal mossy fiber pathway was less distributed in aged SAMP10 than that in the age-matched control. Furthermore, expression of Zn transporter 3, ZnT3, which plays to accumulate Zn in synaptic vesicles in the mossy fiber pathway, was markedly reduced in the hippocampal region even in young SAMP10. Moreover, excessive presynaptic release of glutamate as well as glycine and expression of glial fibrillary acidic protein, a marker of neuronal cell injury, were observed in the hippocampus of aged SAMP10 compared to the control. The present results suggest that age-dependent deficiencies of Zn in synaptic vesicles of the mossy fiber pathway induced by low expression of ZnT3 cause glutamatergic excitotoxicity in the hippocampal neurons and the deterioration of learning and memory in SAMP10.

Protective role of metallothionein on DNA damage in rat kidney caused by cis-diamminedichloroplatinum.

Cis-diamminedichloroplatinum (cis-DDP) has been used as an anticancer agent but it also causes nephrotoxicity. To study the cis-DDP metabolism and its effects, the induction of metallothioneins and DNA damage caused by cis-DDP were observed. Cis-DDP or trans-DDP was administered to seven-week-old Wistar male rats as three daily injections of 8.0 mg/kg body wt., intraperitoneally. Using the obtained kidneys, gel filtration assay, metal analysis, immunohistochemistry and terminal deoxy transferase-mediated deoxy uracil triphosphate nick end labeling (TUNEL) method were carried out to examine localization of metallothioneins and DNA damage caused by cis-DDP. Platinum (Pt) contents, 26.05+/-12.01 microg/g body wt. and 51.29+/-4.59 microg/g body wt. (average+/-S.E.) were detected in the kidney of rats injected with both cis- and trans-DDP, respectively. Metallothionein was detected in the cortex of the kidney in rats administrated cis-DDP or trans-DDP. The mRNA was also detected in the same region. On the other hand cis-DDP showed induction of DNA damage on the cells in the outer stripe of the outer medulla but trans-DDP did not show any damage. The region-induced DNA damage differed from that induced by metallothioneins. Cis-DDP is suggested to be mainly trapped at the proximal tubules by metallothioneins, and the rest of cis-DDP induces DNA damage at the outer stripe of the outer medulla. Metallothioneins are considered to contribute to the protection against cis-DDP in the rat cortex.

Diverse effect of tributyltin on apoptosis in PC12 cells.

It has not been fully elucidated how endocrine-disrupting chemicals disrupt hormone functions or how strong their effects are compared with natural hormones. There is little information concerning the effects of tributyltin (TBT), one of the endocrine disrupters on living organisms. Although TBT at high concentration induced apoptosis in PC12 cells, TBT at low concentration inhibited the DNA fragmentation in the cells cultured in serum-free medium or in medium containing 6-hydroxydopamine. The cell viability grown in both medium conditions increased after treatment with TBT. These findings suggest that TBT exerted a apoptosis-inducing and -inhibiting effect. These diverse effect of TBT on apoptosis would cause serious damages on cell differentiation.

Histochemical characterization of silver-induced metallothionein in rat kidney.

Histochemical characterizations of Ag-induced metallothionein (MT) in the kidney of the rat have been reported. Ag, Cu and Zn contents increased in kidney and liver after Ag injection. In particular, the Cu content in kidneys increased dramatically after three injections of Ag. Sephadex G-75 elution profiles of the renal cytosol of rats injected with Ag revealed that the accumulated Cu in the kidney was bound to MT as were Ag and Zn. In addition, localization of Cu- and Ag-MT in the kidney was studied using autofluorescent signals, which are dependent on Cu- or Ag-thiol clusters, and immunohistochemistry. Although the MT induced by Ag was predominantly observed in the cortex of the kidney, some MT signals were also detected in the outer stripe of the outer medulla, as well as in the kidneys of LEC rats, an animal model of Wilson disease (a hereditary disorder of Cu metabolism). In these LEC rats, the Cu-MT also accumulated in the outer stripe of the outer medulla of the kidney. From these results, one possibility could explain that the Cu-MT detected in the outer stripe of the outer medulla in the kidney of Ag-injected rat was associated with the Cu transporter affected by Ag.

Distribution of zinc-binding metallothionein in cirrhotic liver of rats administered zinc.

This study was undertaken in order to investigate the effect of zinc (Zn) administration on induction of Zn-binding metallothionein in rat liver with thioacetamide-induced cirrhosis, and the localization of metallothionein in the liver. Normal and cirrhotic rats received intraperitoneal injections with or without Zn. Subsequently, metal analyses, purification of metallothionein by gel filtration and immunohistochemical assessments of metallothionein were carried out. Although in Zn-injected cirrhotic rats, the Zn contents in the liver and plasma increased significantly depending upon the dose of Zn, the Zn contents in the liver and plasma of the cirrhotic rats were lower than those of normal rats after the same dose of Zn. The results of gel filtration also showed that the levels of Zn-metallothionein in the cirrhotic liver were reduced in comparison with those of the normal liver. By the immunohistochemical method, the presence of metallothionein in the parenchymal areas but not in the fibrotic areas of the cirrhotic liver was confirmed. These results suggested that the induced metallothionein was only located in the parenchymal areas. The metallothionein induced in the parenchymal areas was considered to play a role in protecting the parenchymal cells against the progression of fibrosis, because metallothionein has been thought to be involved in the cellular defense against oxidative stress.

Role of metallothionein on Ag accumulation in hepatic and renal cytosol after Ag injection to rats.

To examine the role of metallothionein on Ag accumulation in liver or kidney of rat after Ag injection, the relative Ag-binding capacity of Ag-induced metallothionein in hepatic or renal cytosol of rat after Ag injection was determined. The greater part of Ag increment in hepatic cytosol was attributable to a low molecular weight protein, while the main part of Ag increment in renal cytosol was ascribed to high molecular weight proteins. The low molecular weight, metal-binding protein was identified as metallothionein using ELISA. The maximal levels of hepatic and renal metallothionein mRNA induced by Ag occurred at 7 hr after Ag injection. There was a close relationship between Ag contents in the hepatic or renal cytosol and metallothionein after Ag injection. In dose-response and time-course studies, approximately 60-70% of the Ag increments in hepatic cytosol and approximately 30% of the Ag increments in renal cytosol were bound to metallothionein. These results suggest that the role of metallothionein on Ag accumulation in the liver after Ag injection is different from that of metallothionein on Ag accumulation in the kidney.

The effect of heavy metal-induced metallothionein on Zn, Cu and Cd accumulation in rat kidney.

To examine the role of metallothionein on heavy metal accumulation in kidneys of rats after Zn, Cd or Cu injection, the relative Zn, Cd or Cu-binding capacity of heavy metal-induced metallothionein in renal cytosol of rats after Zn, Cd or Cu injection was determined. The Zn or Cu increment in renal cytosol after Zn or Cu injection was attributable to low and high molecular weight proteins, while most of the Cd increment was attributable to a low molecular weight protein. The low molecular weight, metal-binding protein was identified as metallothionein using a competitive ELISA. There was a close relationship between heavy metal contents in the renal cytosol and metallothionein of all heavy metal-injected rats. In dose-response and time-course studies, approximately 45, 40 and 85% of the Zn, Cu and Cd increments in renal cytosol were bound to metallothionein after Zn, Cu and Cd injection, respectively. Therefore the order of relative binding capacities of Zn, Cu and Cd-induced metallothionein in kidney was determined to be Cd>Zn approximately Cu. These results suggest that the role of metallothionein in Zn or Cu accumulation in the kidney after Zn or Cu injection is different from that of metallothionein in Cd accumulation in the kidney after Cd injection.

Immunohistochemical determination of the Wilson Copper-transporting P-type ATPase in the brain tissues of the rat.

Immunohistochemical localization of Copper-transporting P-type ATPase (ATP7B), a gene product responsible for Wilson disease, was visualized in the brain tissues of the Long-Evans agouti rat in detail using tissue-blotting technique and confocal laser microscopy. The ATP7B was intensely detected in neuronal cells of the hippocampal formation, olfactory bulbs, cerebellum, cerebral cortex and nuclei in the brainstem in which high amounts of copper and cuproenzymes, dopamine beta hydroxylase and Cu,Zn-superoxide dismutase (Cu,Zn-SOD) were detected. The present results suggest that ATP7B plays key roles in neurotransmissions of catecholamine pathway and preventing brain tissues from injury by superoxide radicals to regulate the cellular Cu concentration and/or activities of cuproenzymes related to neurotransmissions and a free radical metabolism. Furthermore, it is reasonable to assume that neurotoxicity due to abnormal copper accumulation or irregular regulation of cuproenzymes in the critical brain regions by mutation of the ATP7B gene leads to neurological failures of Wilson disease.

Histochemical localization of superoxide dismutase activity in rat brain.

Histochemical localization of superoxide anion (O2.-) scavenging activity in rat brain was visualized by the tissue-blotting technique. The activity was thought to mainly depend on Cu/Zn-SOD, because the localization of the activity was identical with the immunohistochemistry of Cu/Zn-SOD and the localization of its mRNA in the brain. Moreover, the activity was dramatically decreased after treatment of Cu (I) chelater. The activity was detected in pyramidal cells of the cortex, granular, and mitral cells of the olfactory bulbs, pyramidal cell layer CA1 to CA3, and dentate gyrus of hippocampus formation and granular cells of the cerebellum. Moreover, the activity was detected in the pontine nuclei of brain stem. Olfactory bulbs, hippocampus, and cerebellum were believed to be bestowed high brain functions, i.e., long-term potentiation and long-term depression. A part of the function was regulated by a retrograde neurotransmitter, nitric oxide (.NO). Our findings suggest that the SOD is colocalized with NO synthase in olfactory bulbs, hippocampus, and cerebellum, where .NO plays the important roles. In contrast, low SOD activity was observed in the axonal neurofiber bundles, although the regions contain a lot of membrane lipids, which was thought to be peroxidized by O2.- and related radicals such as .OH in the regions. From these findings, it was suggested that the SOD did not only play a role in protecting the neurons from endogenously formed O2.-, but also play a role in preservation of beneficial natures of .NO in the brain.

Copper metabolism in the kidney of rats administered copper and copper-metallothionein.

To gain a greater understanding of the mechanism of Cu metabolism in kidneys of rats, using autofluorescence of Cu-metallothioneins (Cu-MTs) we revealed the behavior of Cu-MT in the kidneys of rats administered Cu-MT. Yellow and orange fluorescent signals of Cu-MT were observed in the cortex. By microscopic studies, Cu-MT was dominant in the proximal convolute tubular cells of the cortex. A high concentration of Cu-MT presented in the lysosome-like organelles of the proximal convolute tubular adjacent to the glomeruli. During the time course after the injection, the orange signal in lysosome-like organelles gradually converted to a yellow signal, indicating that the Cu-MT was involved in a degradation process in lysosomes by oxidation, and the MT mRNA increased in the cortex, although the immunoreactivity of MT was almost constant in the same region. These results suggested that Cu bound to the injected MT was released in lysosomes and became a new inducer of MT biosynthesis in the cortex. In conclusion, the biosynthesis and degradation of Cu-MT occur repeatedly in the proximal convolute tubular cells.

Copper-metallothionein in the kidney of macular mice: a model for Menkes disease.

Menkes disease is an X-linked disorder of copper metabolism. Excess amounts of copper in the kidney of Macular mice, a model for this disease, were found as copper-metallothionein (Cu-MT) from kidney of the mice. Histochemical studies of Cu-MT based on its autofluorescent emission properties showed that the protein was predominant in the proximal convoluted tubule (PCT) cells of the cortex. PCT cells are known to be the primary site of the nephrotoxicity caused by heavy metals. MT mRNA was also observed in the cortex, indicating that the protein was biosynthesized in this region. On the basis of these results, we suggest that biosynthesis and degradation of Cu-MT occur repeatedly in the PCT cells of the cortex. We also compared the histochemical localization of Cu-MT in Macular mice and Long-Evans cinnamon rats, a model for Wilson's disease. The significance of this comparison is discussed.

Purification and characterization of guinea-pig liver microsomal deacetylase involved in the deacetylation of the O-glucoside of N-hydroxyacetanilide.

A microsomal deacetylase that catalyses the deacetylation of the O-glucoside of N-hydroxyacetanilide (GHA) was purified from guinea-pig liver. The activity was located exclusively in the microsomes and not detected in the cytosol. The purified GHA deacetylase was a trimeric protein with a molecular mass of 160+/-10 (S.D.) kDa composed of subunits of 53+/-2 kDa; its pI was 4.7. The N-terminal amino acid sequence of GHA deacetylase was similar to those reported for guinea-pig and rat liver microsomal carboxylesterases. The GHA deacetylase showed a comparable hydrolytic activity towards p-nitrophenyl acetate (PNPA), although the activities towards N-hydroxyacetanilide, acetanilide and some endogenous acylated compounds were very low or not detectable. The deacetylase activity towards GHA was inhibited by organophosphates but not by p-chloromercuribenzoate, suggesting that GHA deacetylase can be classified as a B-esterase. The enzyme exhibited a positive homotropic co-operativity towards GHA. The values of the Hill coefficient, the half-saturating concentration ([S]0.5) for GHA, and Vmax were 1.59+/-0.03, 5.51+/-0.07 mM and 32.5+/-1.4 micromol/min per mg respectively, at the optimum pH of 8.5. The bell-shaped pH dependence of the Vmax/[S]0.5 profile indicated pKa values attributed to histidine and lysine residues. The study of stoichiometric inhibition by di-isopropyl fluorophosphate and kinetic analysis with the Monod-Wyman-Changeux model suggests that GHA deacetylase has six substrate binding sites and three catalytically essential serine residues per enzyme molecule.

Localization of renal Cu-binding metallothionein induced by Au injection into rats.

The localization of Au-induced metallothionein (MT) in kidneys is reported. Au, Cu and Zn contents in kidneys and liver increased after Au injection. Especially the Cu content in the kidney increased in comparison with the Zn content. The yellow-orange autofluorescent signals which are a marker of Cu-MT were observed predominantly in the outer stripe of the outer medulla with a ring shape in the kidneys of Au-injected rats. MT mRNA was also located in only the outer stripe of the outer medulla. Neither autofluorescence nor MT mRNA was found in the kidneys of control rats. These results indicate that MT was biosynthesized in only the outer stripe of the outer medulla and the biosynthesized MT was bound to Cu. Representative Sephadex G-75 elution profiles of the renal cytosol of rats injected with Au showed that Au, Cu and Zn contents in MT fractions increased after Au injection. Interestingly, Cu in MT fractions dramatically increased in comparison with Zn in the MT fractions in spite of Au injection into rats. Only the Cu-containing MT fractions emitted a yellow-orange autofluorescence. The accumulated Cu in the kidneys of Au-injected rat was thought to be associated with renal toxicity.

Effects of amino acid replacements on cadmium binding of metallothionein alpha-fragment.

To evaluate whether only 20 cysteine residues at invariant positions are needed to bind and coordinate the metals in metallothioneins (MTs), and whether changing the positions of cysteine residues in the sequence affects the metal-binding capacity and the coordination of MTs, we examined the cadmium-binding affinities of seven mutant MT alpha s using an Escherichia coli expression system. Five mutant MT alpha s in which the constitutive amino acid residues other than cysteines of the alpha-fragment were replaced with glycine residues, and the remaining two mutant MT alpha s in which the invariant positions of the cysteine residues of the alpha-fragment were shifted, were analysed for their ability to be expressed as cadmium-binding forms and for their biochemical properties. The results showed that extreme alteration of the constitutive amino acid residues other than cysteines in the MT alpha-fragment leads to disruption of their cadmium-binding abilities and of their structure. However, mutant MT alpha s containing changes of the invariant positions of the cysteine residues were expressed in a cadmium-binding form in Escherichia coli, although the invariant positions of 20 cysteine residues in the MTs are thought to be important for their metal-binding abilities. These results suggest that the position of cysteine residues and the chemical nature of the other amino acids in the amino acid sequence of MTs are less critical than expected.

Restriction of cadmium transfer to eggs from laying hens exposed to cadmium.

The transfer of Cd to eggs of white Leghorn laying hens has been shown to be restricted. After Cd was injected ip into laying hens, the Cd concentrations in the blood, livers, ovaries, and eggs were measured. Although the Cd concentrations in the maternal blood and livers increased remarkably at certain levels of administrations, the Cd concentration in the yolks of eggs was not significantly increased, and was less than 0.04 microgram/g wet weight. After egg production stopped in the highest injected group (7.5 mg Cd/kg), Cd in the yolks of eggs had an accumulated range of 0.02-0.03 microgram/g wet weight. This was despite the high Cd accumulation in the liver. Furthermore, the Cd concentration in the follicle walls of the ovary increased and was 13- to 52-fold higher than in the follicle yolks. An additional experiment was conducted in order to estimate whether hatching success is affected by the Cd in the laid eggs of Cd-injected laying hens. The ratio of hatching success in the 0.3 or 1.2 micrograms Cd/egg-injected groups was similar to that in the saline-injected group, indicating that a small amount of Cd in the eggs might exert no marked influence on the hatching success. In conclusion, Cd transfer from laying hen to eggs was restricted after the maternal bird was exposed to Cd. Furthermore, Cd accumulates in the follicle walls of ovary. These results suggest that the follicle walls might play a role in protecting the follicle yolks against Cd toxicity.