The secretory pathways of rat serum glycoproteins and albumin. Localization of newly formed proteins within the endoplasmic reticulum.

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein. N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membrane-attached polysomes and not into proteins from free polysomes. Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes. Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae. Albumin was mostly recovered (98%) in the cisternae, with negligible amounts in the membranes. To determine whether the radio-active sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the (14)C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins. The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae.

Metal complexes of dietary flavonoids: evaluation of radical scavenger properties and protective activity against oxidative stress in vivo.

Interaction of certain flavonoids with transition metals increases their water solubility and leads to the formation of flavonoid-metal complexes, which may act as superoxide dismutase mimics with high scavenger potencies toward superoxide. Effect of serum albumin on stability of flavonoid-metal complexes was studied and complex of rutin with iron (II) was found to be the most stable. The ability of flavonoid metal complexes to catalyze homolytic cleavage of hydrogen peroxide was also studied and rutin iron (II) complex was found to be relatively poor Fenton catalyst. The potential therapeutic benefits of this new antioxidant agent were studied using experimental model of pathological states associated with oxidative stress in vivo. Acute hepatic injury in MT-I/MT-II null transgenic mice induced by injections of thioacetamide was used for this purpose. It was found that pretreatment with rutin- iron complex protected against thioacetamide induced hepatotoxicity as observed by a significant reduction in the elevated levels of serum enzymes and partial normalization of GSH/GSSG ratio, glutathione peroxidase II and glutathione reductase activity in mice liver. The results demonstrate that flavonoid-metal complexes possess effective free radical scavenger ability and have potent therapeutic benefits for the treatment of oxidative stress-related diseases and dysfunction.

Modulation of resistance to anticancer drugs by inhibition of metallothionein synthesis.

The expression of metallothionein (MT) in certain tumor cells has been associated with resistance to anticancer drugs. In the present study, we examined the effects of inhibition of MT synthesis on resistance to anticancer drugs of human bladder tumor which were inoculated in nude mice. The results show that pretreatment of tumor-bearing mice with zinc salts increased MT content, both in normal and tumor tissues, with a marked reduction in the antitumor activity of cisplatin, Adriamycin, and melphalan. Injection of propargylglycine, an inhibitor of cystathionase, decreased MT induction by zinc in the tumor and diminished the resistance to these drugs. These results suggest a role for MT in drug resistance in tumors, and injection of propargylglycine may provide a potential means to overcome drug resistance caused by elevation of MT levels in certain tumors.

Modulation of both cisplatin nephrotoxicity and drug resistance in murine bladder tumor by controlling metallothionein synthesis.

The role of metallothionein (MT) in cisplatin (cis-DDP) resistance and renal toxicity was investigated in C3H mice inoculated with mouse bladder tumor (MBT-2). C3H mice were inoculated s.c. with 1 x 10(6) MBT-2 cells/mouse on day 0. Mice were given injections of proparglyglycine (PPG) (500 mumol/kg s.c.) once a day for 3 days from day 7 to day 9 and with ZnSO4 (200 mumol/kg s.c.) once a day for 2 days from day 8 to day 9. cis-DDP (50 mumol/kg i.p.) was administered 10 days after MBT-2 cell inoculation. Since MT contents in the tumor and kidneys were significantly increased by administration of ZnSO4, both the antitumor activity of cis-DDP and its renal toxicity were reduced. However, coadministration of PPG reduced MT induction in tumor without affecting the level of renal MT. As a result, PPG could clearly overcome the MT-mediated cis-DDP resistance of tumors without compromising the protective effect exerted by renal MT on nephrotoxicity of the drug. It was suggested, therefore, that PPG may be a promising adjunct in cancer chemotherapy to overcome the drug resistance of tumors caused by the elevated level of MT.

Basal and copper-induced expression of metallothionein isoform 1,2 and 3 genes in epithelial cancer cells: The role of tumor suppressor p53.

Metallothioneins (MTs) are a ubiquitous low-molecular weight, cysteine rich proteins with a high affinity for metal ions. The expression and induction of MTs have been associated with protection against DNA damage, oxidative stress, and apoptosis. Our past research had shown that p53 is an important factor in metal regulation of MTs. The present study was undertaken to explore further the interrelationship between p53 and MTs. We investigated whether silencing of p53 could affect expression pattern of basal and copper induced metallothioneins. The silencing of wild-type p53 (wt-p53) in epithelial breast cancer MCF7 cells affected the basal level of MT-2A RNA, whereas the levels of MT-1A and MT-1X RNA remained largely unchanged. The expression of MT-3 was undetectable in MCF7 with either functional or silenced p53. MCF7 cells with silenced wt-p53 failed to upregulate MT-2A in response to copper and showed a reduced sensitivity toward copper induced cell apoptotic death. Similarly in MCF7-E6 and MDA-MB-231 cells, the presence of inactive/mutated p53 halted MT-1A and MT-2A gene expression in response to copper. Constitutive expression of MT-3 RNA was detectable in the presence of mutated p53 (mtp53). Transient transfection of MDA-MB-231 cells with wt-p53 enabled copper induced upregulation of both MT-1A and MT-2A but not basal level of MT-2A, MT-1E, MT-1X and MT-3. Inactivation of p53 in HepG2 cells amplified the basal expression of studied MT isoforms, including MT-3, as well as copper-induced mRNA expression of MTs except MT-1H and MT-3. Presented data demonstrate a direct relation between p53 and MT-1A and MT-2A and they also indicate that wt-p53 might be a negative regulator of MT-3 in epithelial cancer cells.

Binding of manganese in human and rat plasma.

Albumin, transferrin and 'transmanganin' have all been proposed as the major Mn-binding ligand in plasma. The present investigations were initiated in order to resolve these discrepancies. Compared to other metals tested (109 Cd2+, 65Zn2+, 59Fe3+), 54Mn2+ bound poorly to purified albumin. The addition of exogenous albumin to plasma did not result in an increased 54Mn radioactivity associated with this protein. Also, incubation of 65Zn-albumin in the presence of excess Mn2+ (1 mM) did not result in the displacement of Zn from albumin or Mn binding. In contrast to these results, 54Mn was bound to purified transferrin, not as readily as Fe3+, but better than Zn2+ or Cd2+. Saturation of transferrin with Fe3+ (1.6 micrograms Fe/mg) prevented the binding of 54Mn indicating that Mn probably binds to Fe-binding sites on the protein. Polyacrylamide gel electrophoresis further demonstrated the association of 54Mn with transferrin rather than with albumin in both human and rat plasma. The amount of 54Mn radioactivity recovered with transferrin increased as incubation time was increased, probably due to oxidation of Mn2+ to Mn3+. Mn binding to transferrin reached a maximum within 5 and 12 h of incubation. About 50% of 54Mn migrated with transferrin, whereas only 5% was associated with albumin. A significant portion (20-55%) of the 54Mn radioactivity migrated with electrophoretically slow plasma components whose identity was not determined. Possibilities include alpha 2-macroglobulin, heavy gamma-globulins and/or heavy lipoproteins.

A spectroscopic study of rat liver and Scylla serrata crab metallothioneins.

The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of native rat liver and crab (Scylla serrata) Cd,Zn-metallothionein have been measured and the data are compared. The MCD data indicate that there are close similarities in the geometries of the cadmium-binding sites in both of these proteins; however, the CD spectra are quite different for the rat liver and crab proteins. The CD spectrum for the crab metallothionein is unlike any previously reported for a cadmium-containing metallothionein. This suggests that the CD spectrum is sensitive to the different bridging pattern used in the binding sites in the crab compared with the rat-liver metallothionein. Cadmium binding to the metal-free metallothionein is demonstrated for both proteins and it is shown that there are only minor structural differences between the native and remetallated proteins. The structural changes that occur near to the cadmium-binding sites during cadmium loading to the native proteins have been followed using absorption and CD spectroscopy. Marked changes are observed in the CD spectrum which can be associated with a two-phase reaction: initially Zn2+ is displaced by the Cd2+, then at higher concentrations of Cd2+ the tetrahedral geometry of the Cd2+-binding sites is lost as more Cd2+ is bound using the same thiolate groups. While this latter reaction results in considerable change to the CD spectrum, only minor changes are observed in the absorption spectrum. A significant red shift is observed in the S leads to Cd charge transfer transition region of the MCD spectrum (230-270 nm) following both cadmium loading of native rat liver, Cd,Zn-metallothionein and the metallation of metal-free metallothionein with cadmium. There are two contributions to this effect in Cd,Zn-metallothionein: (i) there is a S leads to Zn band underlying the S leads to Cd band; and (ii) the occupation of zinc sites by cadmium changes the energy of the S leads to Cd transition.

Effects of heavy metal cations, sulfhydryl reagents and other chemical agents on striatal D2 dopamine receptors.

To investigate aspects of the biochemical nature of the membrane-bound D2 dopamine receptor, rat striatal homogenates were pretreated with heavy metal cations and a variety of other chemical agents, and their effects on D2 receptor density were subsequently determined using a standard [3H]spiperone binding assay. Preincubation of striatal membranes in the presence of 3 mM Mn2+, Fe2+, Co2+, EDTA, or ascorbate enhanced subsequently measured stereospecific binding of [3H]spiperone compared to control (e.g. control: Bmax = 140 fmoles/mg protein, KD = 0.21 nM; Mn2+-treated: Bmax = 253 fmoles/mg protein, KD = 0.20 nM). Another group of metal cations, that is Zn2+, Cd2+, Cu2+, Hg2+ and CH3Hg+, all of which have significant -SH reactivity, as well as the -SH alkylating agent N-ethylmaleimide (NEM), caused a decrease in specific binding sites. Pretreatment with 3 mM Cd2+ or Cu2+ resulted in a 40-60% reduction in the subsequently measured stereospecific binding of [3H]spiperone, whereas 1 mM Hg2+ or 3 mM NEM completely abolished specific [3H]spiperone binding. The effect of Hg2+ could not be reversed by washing the membranes, nor by further incubation of the membranes in the presence of excess EDTA or 2,3-dimercapto-1-propanesulfonic acid (DMPS). Further incubation in the presence of 3 mM dithioerythritol (DTE) resulted in the regeneration of about 40% of lost sites. Agents which enhanced receptor density, such as Mn2+ or EDTA, could not antagonize the effect of Hg2+, nor could the mercury-chelating agent DMPS, when added to crude homogenates prior to Hg2+. Ascorbate protected 25-35% of specific binding sites by virtue of its ability to reduce Hg2+ to insoluble Hg+. Only 3 mM DTE afforded complete protection against 1 mM Hg2+. Prior formation of the spiperone/receptor complex also demonstrated considerable ability to protect receptors from destruction by Hg2+. Preincubation of striatal membranes in the presence of 0.5 mM spiperone protected about 80% of sites from the subsequent addition of 1 mM Hg2+. A major conclusion of these studies is that one or more free -SH groups on or adjacent to the active site may be a requirement for specific antagonist binding to the membrane-bound D2 receptor. Occlusion of these -SH groups by sulfhydryl reagents results in partial to complete abolition of subsequently measured specific 3H-antagonist binding. Only agents which can regenerate free -SH groups, such as DTE, are able to induce any recovery in specific binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular localization of metallothionein in human term placenta.

Cellular localization of metallothionein (MT) in placenta may provide information on its function as a metal binding protein. Rabbit antibodies to rat liver MT cross-reacted with human MT and were used to localize MT in human term placenta by avidin-biotin peroxidase technique. Serial sections (5 microns) were cut from paraffin-embedded placentae obtained at term from five normal women and incubated with rabbit antibodies to MT. Normal rabbit serum was used as a negative control. The slides were incubated with biotinylated swine anti-rabbit IgG (linking antibody) then with avidin-biotin horseradish peroxidase complex and developed with diaminobenzidine in hydrogen peroxide (0.03 per cent) substrate. The optimum staining of MT was obtained at a 1:800 antibody dilution. MT was identified in fetal amniotic cells, syncytial trophoblasts and villous interstitial cells, and in maternal decidual cells. The presence of MT at specific cellular sites suggests that it may regulate the transplacental transport of metals such as zinc, copper and cadmium. Since the level of cadmium is lower and that of zinc and copper higher in fetal than in maternal blood, this may suggest that placental MT may restrict cadmium while enhancing zinc and copper transport.

Chelation of cadmium without increased renal cadmium deposition.

Cadmium (Cd) is mainly accumulated in liver and kidney bound to metallothionein (MT) and excreted very slowly from the body. In chronic exposure, Cd is gradually transported from liver to kidney; the renal toxic effects appear when renal Cd concentration exceeds the critical concentration. In order to prevent the Cd-induced renal disease, it is important to control the movement of Cd to the kidney and its renal deposition. However, the chelation of Cd from liver is difficult because of the high affinity of intracellular MT for Cd. A number of chelating agents containing both carboxyl and thiol groups were able to mobilize and excrete Cd more easily in a short time (1/2 hr) after Cd exposure than longer times (24 hr), after MT synthesis. The renal deposition of Cd increased on BAL (2,3-dimercaptopropanol) treatment a short time (1/2 hr) after Cd exposure. However, it was observed that if BAL was administered 24 hr after Cd exposure, it could mobilize Cd from hepatic MT and increase the biliary excretion of Cd without any increase in renal Cd concentration. Studies using a number of structurally related thiols (mono-, di- and trithiols) showed that the major structural requirement for in vivo chelation of Cd from intracellular MT were the vicinal thiol groups on an aliphatic chain, and lipophilicity. BAL was the most effective of all the compounds studied and it did not mobilize Cd to the kidney, when most of the intracellular Cd was bound to MT. Furthermore, a delayed treatment with BAL or DTPA (diethylenetriamine pentaacetic acid) after synthesis of MT resulted in an increase in fecal or urinary excretion of Cd in rat model experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of orally administered cadmium-metallothionein in mice.

The main source of cadmium in the diet is cereal or meat, especially in liver and kidney. Since the cadmium in both liver and kidney is bound to metallothionein, a heat-stable protein, the gastrointestinal absorption and metabolism of cadmium metallothionein (CdMt) was studied in detail. The selective renal cadmium deposition after oral CdMt was analogous to the studies on injected CdMt. Metallothionein with 109Cd or 35S-cysteine radioactive label was isolated from rat liver and administered orally (60 microgram Cd) through a gastric tube to mice (C57 BL/6J). After 4 hr, a major portion of the ingested CdMt was isolated intact from intestinal mucosal cells. However, only a small amount of cadmium bound metallothionein was present in the kidney supernatant. The protein moeity was also degraded completely in kidney. The absorption and tissue distribution of cadmium from oral-cysteine and cadmium-glutathione complexes were similar to that after oral CdCl2 in mice. These results that oral CdMt may be absorbed intact from the gastrointestinal tract and the protein is degraded during renal deposition.

The significance of the nuclear and cytoplasmic localization of metallothionein in human liver and tumor cells.

Metallothioneins are a group of low-molecular-weight intracellular proteins present in high levels in fetal mammalian livers, bound to zinc and copper. They are also present in two major isoforms in low basal levels in various organs of adults in several species. Although a number of functions have been proposed for metallothioneins, their major biological roles may be in the storage of zinc and copper during rapid growth and development, and also in the detoxification of certain toxic metals. In adult liver, metallothionein is mainly localized in the cytoplasm, it is localized also in the hepatocyte nuclei in human fetal liver and fetal and neonatal rat liver, as determined by immunohistochemical staining with a specific metallothionein antibody. Because of its high expression in fetal development, the potential role of metallothioneins in human tumors was investigated. The cellular localization of metallothionein was demonstrated in various human tumors such as thyroid tumors, testicular germ cell carcinoma, bladder transitional cell carcinomas, and salivary gland tumors. In most of these tumor tissues, metallothioneins were found in high levels in nucleus and cytoplasm in both benign and malignant tumors, although the proliferating edge of the malignant tumors showed most intense metallothionein staining. The expression of metallothionein is not universal to all tumor growth; its presence may depend on various factors, such as the type of tumor, cellular origin, morphological heterogeneity, or stage of growth. Human testicular seminomas, which are well differentiated, showed little expression of metallothionein irrespective of the staging, as compared to less well-differentiated embryonal carcinomas.(ABSTRACT TRUNCATED AT 250 WORDS)

Physicochemical and metabolic properties of modified metallothioneins.

Attempts to crosslink metallothionein through lysine residues have resulted in trapping of octameric structures. Two such polymers have been characterized. They are of a critical size for glomerular filtration, and unique tissue distributions of cadmium arise when they are injected into rats.

Effects of cadmium and zinc on tissue levels of metallothionein.

Although the induced synthesis of metallothionein (MT) after exposure to certain metals has been known for some time, there is little information on the quantitation of MT in various tissues. In this study, tissue MT concentrations were measured by a modified Cd-saturation method in tissues of adult male rats after injection of different metal salts. There were differences in tissue levels of MT, depending on the injected metals. Of all the metals studied, Cd2+ was the most effective element in increasing MT concentrations in liver, kidney, pancreas and small intestine. The highest increase in tissue MT concentration after CdCl2 injection was found in the liver, while the pancreas contained the highest MT level after ZnSO4 injection. Co and Ni salts increased MT levels in both liver and kidney, while Mn and Ca increased MT levels only in liver. A direct correlation between tissue MT levels and Cd or Zn concentration was observed in most of the tissues after injection of CdCl2 or ZnSO4. Although there was no positive relationship between tissue levels of MT and tissue Mn and Ni concentrations, the increase in hepatic Zn after injection of these metals was related to hepatic MT levels. The tissue distribution of injected Cd2+ in control adult rats and Zn-deficient rats was similar. However, there was no increase in pancreatic MT levels in Zn-deficient rats after injection of CdCl2. The high concentration of MT in pancreas after ZnSO4 injection in adult rats and the inability of the pancreas to synthesize MT in Zn-deficient rats suggest that the induction of pancreatic MT synthesis is sensitive to Zn status.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between cadmium-induced pulmonary carcinogenicity, metallothionein expression, and inflammatory processes: a species comparison.

There is sufficient evidence for pulmonary carcinogenicity of inhaled Cd compounds in rats whereas no such evidence was found in mice and hamsters; the evidence in humans has been termed limited, indicating significant species differences in pulmonary response to inhaled Cd. We hypothesized that expression of metallothionein (MT) protein in the lung after inhalation of Cd differs between species thereby providing different degrees of sequestration of Cd and protection from its effects. Rats and mice were exposed to 100 micrograms CdCl2 aerosols/m3 for 4 weeks, and the presence of MT was determined in lung and free lung cell homogenates as well as by immunocytochemistry in lung sections up to 28 days postexposure. In addition, pulmonary inflammatory, and cell proliferative responses were determined. Cd exposure significantly increased MT in homogenates of total lung in both species; however, no significant increase of MT in rat lung tissue after removal of free lung cells by lavage was found whereas MT was still significantly increased in lavaged mouse lung tissue throughout the postexposure time. In contrast, exposed rats showed significant increases in MT in the lavageable lung cells and mice did not. Histochemical analysis of lung sections revealed that mainly the epithelial cells of the bronchi, bronchioli, and alveoli of Cd-exposed mice expressed MT. Mice also exhibited a marked and sustained pulmonary inflammatory and cell proliferative response upon CdCl2 exposure which was not observed in rats. The retained Cd dose per gram lung was about 2-fold greater in mice, which is consistent with a greater deposition efficiency of inhaled Cd-aerosols in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization studies on the cadmium-binding proteins from two species of New Zealand oysters.

Two different types of New Zealand oysters--Ostrea lutaria (OL) and Crassostrea glomerata (CG)--contained different concentrations of zinc, copper, and cadmium. OL oysters had 5.3 micrograms Cd/g, 3.4 micrograms Cu/g, 100 micrograms Zn/g; CG oysters had 1.4 micrograms Cd/g and 936 micrograms Zn/g. Both kinds of oysters were shown by gel filtration (G-75) to contain cadmium and zinc in fractions corresponding to a high molecular weight protein (corresponding to the size of albumin or larger) which was heat labile. OL oysters contained cadmium in fractions corresponding to a molecular weight of approximately 6500. The cadmium-binding protein in these fractions was heat-stable. This protein contained no detectable amounts of zinc and was not present in the CG oysters. Further purification by gel filtration (G-50) was performed to obtain a purer protein fraction. Isoelectric focusing of the protein obtained by G-50 filtration showed one main fraction of protein with a pI approximately 5.9 at approximately 13 degrees C. CG oysters contained cadmium and zinc in a polypeptide with low molecular weight (MW 1000). The cadmium-binding oyster proteins are minimally reactive in a competitive binding radioimmunoassay in comparison to the reactivity of a typical vertebrate metallothionein; the proteins may be metallothioneins, but, if so, they do not exhibit the principal determinants characteristic of vertebrate metallothioneins.

Effects of gamma radiation on levels of brain metallothionein and lipid peroxidation in transgenic mice.

The induction of metallothionein (MT) synthesis in the brain was investigated in MT-I isoform-overexpressing transgenic (MT-I*) and control mice after exposure to increasing doses of 2 to 20 Gy of whole-body gamma radiation. Although the MT-I isoform was the major isoform of MT in this transgenic mouse, the other isoforms, MT-II and MT-III, were also present in the brain. The total concentration of MT in the brain was measured by a cadmium-binding assay, while zinc and lipid peroxides were measured by atomic absorption spectrophotometry and by the thiobarbituric acid method, respectively. In MT-I* mice at 24 h after radiation exposure, the level of MT in the brain was increased from a basal level of 44.4 +/- 4.0 microg/g to a maximum level of 91.0 +/- 9.0 microg/g after 5 Gy and remained high after 10 and 20 Gy. In a time-course experiment with 5 Gy, the concentration of MT in the brain of MT-I* mice increased at 3 h and reached a maximum of 175.3 +/- 15.3 microg/g at 6 h. This high level of MT remained unchanged for 48 h after radiation exposure. Metallothionein was not induced markedly in the brains of control mice either at 24 h after exposure to 2-20 Gy gamma radiation or at different times after exposure to 5 Gy gamma radiation. In both strains of mice, the total concentration of zinc in the brain decreased with increasing radiation dose. No differences in lipid peroxide levels were seen in control mice exposed to 5 Gy at 6 and 12 h or after exposure to three other doses (2, 10 and 20 Gy) at 24 h. Slight increases (1.35 and 1.22, respectively) in lipid peroxide levels were observed in control mice at 24 and 48 h after exposure to 5 Gy. Lipid peroxide levels in the brain were not changed in irradiated MT-I* mice. The results show a marked increase in the levels of MT in the brain of transgenic mice after exposure to gamma radiation. The induced synthesis of MT may be only one of several mechanisms that prevent the induction of lipid peroxidation in the brain by gamma radiation.

Sensitivity to radiation treatment and changes in metallothionein synthesis in a transplanted murine tumor.

A protective role for metallothionein (MT) in cellular injury caused by ionizing radiation has been proposed. To elucidate the role of MT in the sensitivity of tumors to radiation, we examined the effectiveness of radiation treatment of tumors with altered synthesis of MT after injection of tumor-bearing mice with zinc and/or propargyl glycine (PPG). The mice were inoculated with Meth-A fibrosarcoma cells and the antitumor activity of X radiation was tested in mice with and without induced synthesis of MT. Exposure to X radiation decreased the tumor weight markedly. In mice pretreated with zinc to induce MT, the tumor weight did not change compared to the controls. However, injection of PPG, an inhibitor of cystathionase, decreased the zinc-induced MT synthesis in the tumors and also decreased the tumor weight after exposure to X radiation. These results suggest that in radiation therapy one of the factors involved in radiosensitivity may be the expression of MT in certain tumors.

Organ-specific metallothionein induction in mice by X irradiation.

Metallothioneins (MTs) are induced in cultured cells and experimental animal tissues by a variety of chemical agents. We report that whole-body X irradiation (1 to 80 Gy) induces MT-1 mRNA transcription and protein expression and accumulation in liver but not in kidney or spleen. The degree of induction was comparable to maximum levels achieved after treatment with metal salts, but the peak of MT-1 mRNA and protein accumulation was approximately 10 h later than with treatment with metal salts and remained high for an extended period. Because of the lack of induction of MT-1 by X rays in cultured cells, and the similarity of the tissue pattern of MT induction between X rays and other agents that also do not induce MT expression in cultured cells, it appears that these agents may act through the mediation of tissue-specific factors. The implications of radiation-induced metallothionein synthesis and organ-specific resistance to cellular damage are discussed.

Repeated cadmium exposures enhance the malignant progression of ensuing tumors in rats.

Prior studies show that a single subcutaneous (sc) exposure to cadmium (Cd) will induce injection site sarcomas (ISS) in rats. These tumors, thought clearly malignant, do not often metastasize or invade subdermal muscle layers because of their location. Recent evidence indicates that when tumorigenic cells chronically exposed to Cd in vitro are inoculated into mice, tumor progression and invasiveness in the mice are enhanced. Thus, we studied the effects of repeated Cd exposures on tumor incidence, progression, and metastatic potential in rats. Wistar (WF) and Fischer (F344) rats (30 per group) were injected sc in the dorsal thoracic midline with CdCl2 once weekly for 18 weeks with doses of 0, 10, 20, or 30 micromol Cd/kg. This resulted in total doses of 0, 180, 360, or 540 micromol/kg. One other group of each strain received a low, loading dose of Cd (3 micromol/kg) prior to 17 weekly injections of 30 micromol/kg (total dose 513 micromol/kg). Rats were observed for 2 years. Many F344 rats (57%) died within one week after the first injection of the highest dose, but WF rats were not affected. The low loading dose prevented acute lethality of the high dose in F344 rats. Surprisingly, latency (time to death by tumor) of ISS was the shortest in the groups given the low loading dose in both strains. ISS in these groups also showed the highest rate of metastasis and subdermal muscle layer invasion. Based on ISS incidence in the groups given the lowest total dose of Cd (180 micromoles/kg), F344 rats were more sensitive to tumor induction, showing an incidence of 37% compared to 3% in WF rats. On the other hand, Cd-induced ISS showed a higher overall metastatic rate in WF rats (18 metastatic ISS/68 total tumors in all treated groups; 27%) compared to F344 rats (6%). Immunohistochemically, the primary ISS showed high levels of metallothionein (MT), a cadmium-binding protein, while metastases were essentially devoid of MT. These results indicate that repeated Cd exposures more rapidly induce ISS. An initial low exposure to Cd further accelerates the appearance and enhances the metastatic potential and invasiveness of these tumors. The primary and metastatic ISS appear to have a differing phenotype, at least with regard to MT production. The association between multiple Cd exposures and enhanced metastatic potential of the ensuing tumors may have important implications in chronic exposures to Cd, or in cases of co-exposure of Cd with organic carcinogens, as in tobacco smoking.