Localization and role of metallothioneins in the olfactory pathway after exposure to mercury vapor.

We have investigated the localization and physiological roles of metallothioneins (MTs) in the olfactory pathway after exposure to mercury (Hg0) vapor. Male MT-null and wild-type mice were examined for the distribution of mercury, MT immunoreactivity and MT-III mRNA expression. There were no signs of histological changes in MT-null or wild-type mice. Light and electron microscopy of the samples stained with autometallography demonstrated chronological transfer of exposed mercury granules to the olfactory bulb by way of the olfactory tract. Basal expression of MT-I and -II immunoreactivity was observed in supporting cells, basal cells and acinar cells of the Bowman's gland of the olfactory mucosa in wild-type mice even without mercury exposure. In situ hybridization showed that signals for MT-III mRNA dominated in the olfactory cells of the olfactory mucosa, neurons in the olfactory bulb and those of brain in MT-null and wild-type mice. No difference in these findings was observed between samples taken at any interval after mercury exposure.

Induction of metallothionein in the livers of female Sprague-Dawley rats treated with 2,3,7 ,8-tetrachlorodibenzo-p-dioxin.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects against metal toxicity and external stimuli including ionizing or ultraviolet B irradiation. Since 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to cause an exaggerated oxidative stress response in animals and in different organs, we have studied possible involvement of MT in the oxidative responses induced by TCDD. Female Sprague-Dawley (SD) rats (6-week old) were administered a single oral dose of TCDD that varied from 1.0 to 4.0 microg/kg body weight. The serum and tissues were collected 7 days after dosing. Indicators of oxidative damage were assessed. Significant increases in serum 8-hydroxydeoxyguanosine (8-OHdG) levels were observed in the rats dosed with 2.0 and 4.0 microg TCDD/kg bw. Only 4.0 microg TCDD/kg bw produced a decrease in reduced glutathione concentration in the liver. Immunohistochemical staining revealed a TCDD-induced increase in heme oxygenase-1 (HO-1) expression in the hepatic macrophages (Kupffer cells). Under these conditions, MT protein as well as the mRNAs of MT-I and MT-II, were dose-dependently induced in the liver by TCDD doses from 1.0 microg/kg bw. TCDD-induced MT was found to localize in the parenchymal cells of the liver. Serum concentrations of cytokines (TNF-alpha, IL-1beta and IL-6) were not affected by TCDD. The hepatic concentrations of Cu, Zn and Fe were all increased significantly by TCDD administration. Our results suggest that MT levels are increased in the liver upon exposure to TCDD, perhaps by TCDD-generated reactive oxygen species, and that it may play a protective role in TCDD-induced oxidative stress responses as an antioxidant.

Isolation and identification of metallothionein isoforms (MT-1 and MT-2) in the rat testis.

It has been a long-lasting controversial issue as to whether or not the male genital organs, such as the testis and prostate, contain metallothioneins (MTs), a group of cysteine-rich heavy-metal-binding proteins that play a role in detoxifying heavy metals such as cadmium (Cd). Earlier studies reported that the rodent testis lacks MTs and concluded that this is why the testis is very susceptible to Cd, although other indirect experimental evidence suggests that MTs are present in this organ. A deficiency of MTs in the testis was originally suspected on the basis of amino acid composition analysis, since MT-like proteins isolated as Cd-binding proteins did not have a characteristic MT structure. In the present study, we demonstrate that the rat testis indeed expresses Cd-binding proteins with sequences identical to those previously described for MT-1 and MT-2, the major isoforms. To confirm that MT-1 and MT-2 are present in the rat testis, we purified and isolated Cd-binding proteins by homogenization using Cd-containing buffer, followed by sequential purification using Sephadex G-75 gel filtration chromatography and anion HPLC column chromatography, which yielded Cd-binding protein-1 (Cd-BP-1) and -2 (Cd-BP-2). After pyridylethylation, Cd-BP-1 and Cd-BP-2 were subjected to specific protein fragmentation by acids and endopeptidases, which revealed that these Cd-binding proteins have the same primary structures as MT-1 and MT-2 respectively. Thus we believe that the present results clearly resolve the long-standing debate about the presence of MTs in the testis, at least in the rodent.

Metallothionein deficiency promotes mouse skin carcinogenesis induced by 7,12-dimethylbenz[a]anthracene.

To investigate a possible involvement of metallothionein (MT) in chemical carcinogenesis, MT-I and MT-II gene-deficient [MT (-/-)] transgenic mice and wild-type [MT (+/+)] control mice were topically applied at a single dose of 100, 250, 500, or 1000 microg of 7,12-dimethylbenz[a]anthracene (DMBA) on the dorsal skin and thereafter compared. After 14 weeks of DMBA treatment, the skin tumor occurred in MT (-/-) mice only and in a dose-dependent manner, whereas no change was observed in MT (+/+) mouse skin given the same DMBA treatment. The tumor cells showed proliferative activity, as shown by proliferative cell nuclear antigen staining. These results demonstrate that MT acts as an endogenous defensive factor against DMBA-induced skin tumorigenesis.

Gene expression in skin tumors induced in hairless mice by chronic exposure to ultraviolet B irradiation.

We investigated the expressions of c-Ha-ras, c-jun, c-fos, c-myc genes and p53 protein in the development of skin tumors induced by chronic exposure to UVB without a photosensitizer using hairless mice. When mice were exposed to UVB at a dose of 2 kJ/m2 three times a week, increased c-Ha-ras and c-myc transcripts were detected after only 5 weeks of exposure, while no tumor appeared on the exposed skin. The increase in gene expression continued until 25 weeks, when tumors, identified pathologically as mainly squamous cell carcinomas (SCC), developed in the dorsal skin. In these SCC, overexpression of c-fos mRNA was also observed along with the increases in c-Ha-ras and c-myc. A single dose of UVB (2 kJ/m2) applied to the backs of hairless mice transiently induced overexpression of the early event genes c-fos, c-jun and c-myc, but not c-Ha-ras, in the exposed area of skin. Accumulation of p53 protein was determined by Western blotting analysis or immunohistochemistry using monoclonal antibodies PAb 240 or 246, which recognize mutant or wild type, respectively. In the SCC, a mutant p53 protein accumulated in the cytoplasm and nucleus. After single-dose irradiation, the increased wild-type p53 protein was observed in the nuclei of epidermal cells. The present results suggest that overexpression of the c-fos, c-myc and c-Ha-ras genes, and the mutational changes in p53 protein might be associated with skin photocarcinogenesis. Moreover, overexpression of the c-Ha-ras and c-myc genes might be an early event in the development of UVB-induced skin tumors in mice.

Testosterone-dependent induction of metallothionein in genital organs of male rats.

Metallothioneins (MTs) are a group of cysteine-rich heavy-metal-binding proteins. We have investigated MT gene expression in the ventral and dorsolateral lobes of the prostate and coagulating gland of male Wistar rats. In intact rats, both MT mRNA and MT were present in the dorsolateral lobe and coagulating gland but not in the ventral lobe. Orchidectomy caused involution of the above organs, and both MT mRNA and MT were considerably decreased or become undetectable. An injection of testosterone propionate into orchidectomized rats restored not only the size of these organs, but also MT mRNA and MT concentrations, particularly in the dorsolateral lobe and coagulating gland. In the dorsolateral lobe, no selective uptake of Zn2+ preceding the increase in MT was observed, suggesting that Zn2+ ions are not associated with the increased expression of the MT gene. The present result suggests that of the male auxiliary genital organs, the dorsolateral lobe and coagulating gland, but not the ventral lobe, contain MT, the biosynthesis of which is regulated by testosterone.

Difference in cadmium cytotoxicity in two kidney cell lines (LLC-PK(1) and MDCK) with special reference to metallothionein.

Proximal tubular dysfunction is a main toxic sign of chronic cadmium (Cd) poisoning, but it is not clear why the proximal tubular epithelium is more susceptible than distal tubular cells to Cd toxicity. Kidney cell lines of proximal (LLC-PK(1)) and distal (MDCK) tubular origins have been used to study comparative Cd toxicity. From cell morphology, cell numbers and cellular protein amounts, LLC-PK, cells were found to be more susceptible to Cd than MDCK cells. In LLC-PK(1) cells, cell number was decreased with time at concentrations of 25 mum Cd or more, whereas MDCK cells proliferated even under 50 mum Cd until hr 24. Cd accumulated in both cell types in a time- and dose-dependent manner; the accumulated Cd level was greater in LLC-PK(1) cells than in MDCK cells. The basal metallothionein (MT) amount was low and similar between the two cell lines; MT was induced in a dose- and time-dependent manner by Cd in MDCK cells whereas MT induction in LLC-PK(1) cells was observed only until Cd cytotoxicity overwhelmed cell viability. The vulnerability of LLC-PK(1) cells can be explained by the fact that they accumulate more Cd than MDCK cells, with insufficiently induced MT to sequester Cd ions.

Enhanced synthesis of metallothionein as a possible cause of abnormal copper accumulation in LEC rats.

Long-Evans rats with a cinnamon-like coat color (LEC) is an inbred strain accumulating copper (Cu) in the liver abnormally and showing spontaneous hepatitis and hepatoma. The present study was intended to clarify how Cu accumulates in the LEC rat liver. For this purpose, the distribution profiles of Cu and zinc (Zn) and the inducibility of metallothionein (MT) synthesis were examined in the liver between Cu-loaded Long Evans agouti (LEA, the original strain of LEC) rats and were compared with those in control LEC rats. LEA rats (female, five weeks old) were injected subcutaneously with CuCl2 daily at a dose of 3 mg Cu/kg body weight for 2, 4, 6, and 9 days. The concentration of Cu (124 micrograms/g) accumulated in the LEA rat liver after four injections was comparable to that in control LEC rats. Only 20% of Cu in the liver of LEA rats was recovered in the supernatant fraction in the form of MT, while Cu in the LEC rat liver (113 micrograms/g) was recovered mostly in the supernatant fraction, and was bound to MT. Although the increased concentration of Cu in the LEA rat liver was further elevated after additional injections of Cu, the amount of MT did not increase further. The MT mRNA content in the LEA rat liver remained lower than that of LEC rats even after further injections of Cu. Therefore, the present results suggest that LEC rats can accumulate Cu at a high concentration in the liver because of their extremely high inducibility of MT.

Metallothionein mRNA in the testis and prostate of the rat detected by digoxigenin-labeled riboprobe.

Metallothionein (MT), a cysteine-rich heavy metal-binding protein, has been considered to play a role in the homeostatic control and detoxification of heavy metals, such as zinc, copper, and cadmium. In the present study, we have utilized a digoxigenin-labeled riboprobe to localize MT mRNA only by bright-field optics in the testis and prostate of the rat. In the rat testis, MT mRNA was found predominantly in primary spermatocytes and also in secondary spermatocytes and spermatids, but not in the spermatogonia, Sertoli cells, and Leydig cells. On the other hand, MT protein was present in these spermatogenic cells as well as in spermatozoa and Sertoli cells. In the prostate, MT mRNA was found predominantly in the epithelium of the dorsolateral lobes, but not in the ventral lobe, which is in agreement with the observed localization of MT protein. The utilization of both in situ hybridization and immunohistochemical staining on the same tissue specimens show MT gene expression in specific cell types in the male genital organs.

Selective enhancement of metallothionein mRNA expression by copper in primary cultured liver parenchymal cells of LEC rats.

Mechanisms for the abnormal copper (Cu) accumulation in the liver of LEC rats were examined using primary cultured liver parenchymal cells prepared from mutant LEC rats and those from control LEA rats (original strain). The Cu and metallothionein (MT) mRNA levels in the liver of LEC rats were caused to decrease to the same levels as those of LEA rats by removing Cu in vivo selectively with tetrathiomolybdate. Cu was taken up by LEC rat cells to the same extent as LEA rat cells by exposure to low medium Cu and to a higher extent by exposure to high medium Cu, while the MT mRNA level in LEC rat cells increased dose-dependently at a much higher rate than that in LEA rats. MT mRNA levels in both cells were comparable by exposure to cadmium, zinc and dexamethasone. The results indicate that expression of MT mRNA is selectively enhanced by Cu in LEC cells despite the fact that uptake of Cu is comparable with normal cells.

Induction of metallothionein and its localization in the nucleus of rat hepatocytes after partial hepatectomy.

Metallothioneins, a group of cysteine-rich heavy-metal binding proteins, are induced in the regenerating rat liver in response to the stimuli evoked by partial hepatectomy. We have investigated the expression of metallothionein genes and proto-oncogenes (c-fos, c-jun and c-myc), as well as specific localization of metallothionein in the liver cells after partial hepatectomy. Metallothionein mRNA was detected as early as 3 hr and reached a maximal level by 6 hr. Expression of the proto-oncogenes apparently preceded the elevation of metallothionein protein because the latter was maximal 18 hr after partial hepatectomy, followed by a decrease until 70 hr. Hepatocytes of the intact rat liver have metallothionein in the cytoplasm only. Interestingly, metallothionein was localized predominantly in the nucleus as early as 6 hr after partial hepatectomy, and the staining intensity of metallothionein became maximal at 15 hr, followed by detection in both the cytoplasm and nucleus at 24 hr or longer. The use of a confocal laser scanning microscope with both tissue sections and isolated nuclei has clearly shown that metallothionein immunofluorescence exists inside hepatocyte nuclei after partial hepatectomy. Expression of the proto-oncogenes c-fos and c-jun is elevated after partial hepatectomy, and the resultant heterodimer of gene products may contribute to the observed metallothionein gene induction. However, the observation that metallothionein protein levels were elevated until 18 hr after partial hepatectomy suggests that an alternative pathway for the induction of metallothionein gene expression may also be present.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging and extrapyramidal function.

Measurements on human brain samples of some enzymes concerned with neurotransmitter synthesis suggest serious losses with age. The most severe loss found was that in striatal tyrosine hydroxylase activity, the rate-controlling enzyme in the synthesis of dopamine. Cell counts in the substantia nigra where this dopaminergic tract originates suggest that the decrease in enzyme activity is partly due to cell loss, but must largely reflect decreased activity of residual cells. It is possible that this loss may account for some of the difficulties in movement seen in aged individuals and that it might be less if pigment formation in these cells could be inhibited.

Delta 9-Tetrahydrocannabinol, ethanol, and amphetamine as discriminative stimuli-generalization tests with other drugs.

Three groups of rats (A, B, C) were trained in a T-maze discriminate between drug-and control solution-induced internal discriminative stimuli. The drugs used to induce discriminative stimuli were: delta 9-THC, 5.0 mg/kg (Group A); ethanol, 1.2 g/kg (Group B), and amphetamine, 1.0 mg/kg(Group C). After discrimination acquisition several drugs were tested for generalization in each group. Group A was tested with delta 8-THC, CBD, CBN, ethanol, pentobarbital,chlorpromazine, amphetamine, and apomorphine; only delta8-THC and CBN induced delta9-THC-like responses. Group B was tested with delta 9-THC, delta 8-THC, CBD, CBN, pentobarbital, and amphetamine; pentobarbital induced ethanol-like response. Group C was tested with delta 9-THC, apomorphine, and ethanol; delta 9-THC and apomorphine elicited amphetamine-like responses.