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Background. Metallothioneins (MT) are endogenous metalloproteins involved in the homeostasis of essential metals and detoxification of toxic metals. Some recent experimental studies suggested tumor resistance to cis-diamminedichloroplatin may be associated with overexpression of MT in the tumor. Methods. The presence of MT in 33 primary testicular germ cell tumor specimens was assessed immunohistochemically using a rabbit polyclonal rat liver MT antibody that cross-reacted with human MT. The data were correlated with the patients' clinical course. Results. Seminomas stained weakly or not at all for MT, regardless of the clinical stage. Most nonseminomas stained heavily for MT. The more advanced staged nonseminomas tended to stain more heavily for MT. Conclusions. In view of the considerable experimental evidence as well as some inferential clinical data involving MT in cis-diamminedichloroplatin resistance, there appears to be a role for MT in cis-diamminedichloroplatin resistance in germ cell tumors. Further studies to elucidate the role of MT in germ cell tumor chemore-sistance are warranted.

Hepatocytes in adults are in a nonproliferative state but they have high capacity to regenerate within few hours after an Injury. After partial hepatectomy or chemical injury, hepatocytes undergo a synchronized multistep process consisting of prlming/initiation, proliferation, and termination. These distinct steps are essential for restoring the structure and functions of liver. The mechanisms involved in each of these steps of regeneration are well documented from various laboratories and are described in several reviews. We briefly describe these steps and the Involvement of various cytokines and growth factors for cell regeneration in this short review. Liver cell regeneration may also involve stem cell proliferation. The regenerating cells require large amounts of zinc within a short time, and this requirement is met by induction of a zinc and copper binding protein, metallothionein (MT), during the priming step, soon after an injury. There are several reports on the transfer of zinc from MT to various metalloenzymes and transcription factors. Genetically modified mouse models have been used to study the Involvement of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in cell regeneration. The use of an MT-knockout mouse has enabled us to Investigate the specific role of MT in liver regeneration after partial hepatectomy, chemical injury, and fibrosis. Several studies have suggested a defective liver regeneration after an Injury in MT-knockout mice. There is cumulative evidence that indicates an essential role for MT in liver cell regeneration.

Although the translocation of metallothionein (MT) from cytoplasm to nucleus has been demonstrated in liver during times of high requirement for zinc (fetal development and the neonatal period), the role of MT in cellular growth is not well understood. In this study, a potential role of MT in liver regeneration was investigated in wild type (WT) and MT-I and MT-II gene knockout (MT-null) mice after 35% partial hepatectomy (PH) or sham laparotomy. Hepatic MT levels and proliferation index were measured at 0, 5, 15, 24, 36, 48, and 60 hrs after PH and 48 hrs after sham laparotomy (control). MT levels were increased in WT mice (peak at 24 hrs after PH) and declined to normal levels by 60 hrs after PH. Immunohistochemical staining for MT in WT mice indicated the presence of MT in both nucleus and cytoplasm of hepatocytes at 24 hrs after PH, whereas MT was present mainly in the cytoplasm at 36–60 hrs after PH and 48 hrs after sham laparotomy. Hepatic proliferation Index in both WT and MT-null mice, as determined by argyrophilic nucleolar organizing region staining and proliferating cell nuclear antigen immunohistochemical staining, reached a peak at 48 hrs and declined by 60 hrs after PH. Cell proliferation was significantly less in MT-null mice as compared to WT mice during liver regeneration after PH. These results suggest that MT may play a positive role in hepatic regeneration after PH.

Metallothioneins (MT) are a group of low-molecular weight, cysteine rich intracellular proteins, which are encoded by a family of genes containing at least 10 functional isoforms in human. The expression and induction of these proteins have been associated with protection against DNA damage, oxidative stress and apoptosis. Moreover, MT may potentially activate certain transcriptional factors by donating zinc. Although MT is a cytosolic protein in resting cells, it can be translocated transiently to the cell nucleus during cell proliferation and differentiation. A number of studies have shown an increased expression of MT in various human tumors of the breast, colon, kidney, liver, lung, nasopharynx, ovary, prostate, salivary gland, testes, thyroid and urinary bladder. However, MT is down-regulated in certain tumors such as hepatocellular carcinoma and liver adenocarcinoma. Hence, the expression of MT is not universal to all human tumors, but may depend on the differentiation status and proliferative index of tumors, along with other tissue factors and gene mutations. In certain tumors such as germ cell carcinoma, the expression of MT is closely related to the tumor grade and proliferative activity. Increased expression of MT has also been observed in less differentiated tumors. Thus, expression of MT may be a potential prognostic marker for certain tumors. There are few reports on the expression of the different isoforms of MT which have been analyzed by specific gene probes. They reveal that certain isoforms are expressed in specific cell types. The factors which can influence MT induction in human tumors are not yet understood. Down-regulation of MT synthesis in hepatic tumors may be related to hypermethylation of the MT-promoter or mutation of other genes such as the p53 tumor suppressor gene. In vitro studies using human cancer cells suggest a possible role for p53 and the estrogen-receptor on the expression and induction of MT in epithelial neoplastic cells. Some of the evidence supporting a role for MT in both intrinsic and acquired drug resistance is critically evaluated in this review. Since chemoresistance in human tumors is a multifactorial phenomenon, it is difficult to conclude that MT is a more crucial factor than others. Therefore, additional experimental data on MT and its isoforms in human tumors, are needed to elucidate the biological functions of MT during tumor progression, along with other tumor markers.

Susceptibility to lead toxicity in MT-null mice and cells, lacking the major forms of the metallothionein (MT) gene, was compared to wild-type (WT) mice or cells. Male MT-null and WT mice received lead in the drinking water (0 to 4000 ppm) for 10 to 20 weeks. Lead did not alter body weight in any group. Unlike WT mice, lead-treated MT-null mice showed dose-related nephromegaly. In addition, after lead exposure renal function was significantly diminished in MT-null mice in comparison to WT mice. MT-null mice accumulated less renal lead than WT mice and did not form lead inclusion bodies, which were present in the kidneys of WT mice. In gene array analysis, renal glutathione S-transferases were up-regulated after lead in MT-null mice only. In vitrostudies on fibroblast cell lines derived from MT-null and WT mice showed that MT-null cells were much more sensitive to lead cytotoxicity. MT-null cells accumulated less lead and formed no inclusion bodies. The MT-null phenotype seems to preclude lead-induced inclusion body formation and increases lead toxicity at the organ and cellular level despite reducing lead accumulation. This study reveals important roles for MT in chronic lead toxicity, lead accumulation, and inclusion body formation.

The transient nuclear localization of metallothionein during cell growth and differentiation may be related to the increased requirement of zinc for DNA synthesis, activation of metalloenzymes, and transcription factors. Treatment of 3T3-L1 fibroblasts with aphidicolin, an inhibitor of nuclear DNA synthesis, caused a cell-cycle block at G1/S phase and a delay in the onset of M phase. This also resulted in the accumulation of both zinc and metallothionein in the nucleus. After removal of aphidicolin, the cells rapidly reentered S phase, and during the G2/M phase of cell cycle both zinc and metallothionein began to relocate to the cytoplasm. Delaying the onset of M phase in 3T3-L1 cells could prevent the cytoplasmic relocation of metallothionein. The nuclear translocation of both zinc and metallothionein during the cell cycle can be considered as a normal process and this may be a general mechanism in response to mitogenic signals. J. Cell. Physiol. 183:247–253, 2000. © 2000 Wiley-Liss, Inc.

Background: Three isoforms of metallothionein (MT‐I, ‐II, and ‐III) have been detected in mice brain but little is known on their inducibility. Therefore, changes in MT levels in brain were investigated in MT‐I overexpressing transgenic mice (MT‐I*) and wild type mice of the same strain (C57BL/6J) after oral administration of ethanol.

The regional brain distribution of metallothionein (MT), zinc, and copper in the brain was determined in nine anatomical regions (olfactory bulb, cortex, corpus striatum, hippocampus, thalamus plus hypothalamus, pons plus medulla oblongata, cerebellum, midbrain, and white matter) and was compared between two different strains of rat (Sprague-Dawley [SD] and Lewis). No significant difference was observed in the whole-brain MT level between the two strains (17.8 ± 3.4 μg/g in SD rats and 20.3 ± 2.3 μg/g in Lewis rats). In SD rats, however, MT was more highly expressed in the white matter than in the other regions studied. In contrast, MT concentration was highest in the cortex and lowest in the olfactory bulb in Lewis rats. The MT levels in the cortex, corpus striatum, hippocampus, and thalamus plus hypothalamus were significantly lower in SD rats than in Lewis rats. In both strains, the olfactory bulb contained markedly higher levels of both zinc and copper than the other regions (27.9 ±6.8 μg/g zinc in SD rats and 27.6 ± 6.9 μg/g zinc in Lewis rats, and 5.2 ± 1.5 μg/g copper in SD rats and 11.1 ± 4.8 μg/g copper in Lewis rats). The next high-est zinc levels were seen in the hippocampus, whereas the next highest copper levels were in the corpus striatum in both SD and Lewis rats. The high levels of zinc and copper in the olfactory bulb were not accompanied by concomitant high MT concentrations. These results indicate that the strain of rat as well as the anatomical brain region should be taken into account in MT and metal distribution studies. However, the highest concentrations of zinc and copper in olfactory bulb were common to both SD and Lewis rats. The discrepancy between MT and the metal levels in olfactory bulb suggests a role for other proteins in addition to MT in the homeostatic control of zinc and copper.

The presence of high levels of metallothionein (MT) in developing mammalian cells is well documented. It has been suggested that the developmental profile and gene expression of MT is similar to that of the so-called oncodevelopmental gene products such as a-fetoprotein. In this study tissue sections of nine human embryonal carcinomas of the testis were tested by means of the avidin-biotin peroxidase complex for the presence of MT. The antigen was localized in variable amounts in the cytoplasm and nucleus in tumour cells in all cases. There was evidence that immunoreactivity was related to the histological growth pattern of tumour cells. These findings suggest that MT may be considered an oncodevelopmental product which could be useful as a tumour marker. In addition, the histology of these tumours might predict MT expression; this may prove of value in testing the hypothesis of MT-related emergence of drug-resistant cell lines in the course of treatment of tumours with metal-containing chemotherapeutic agents.

Summary: Markers of chemoresistance have been rarely investigated in human ovarian cancer. This study evaluates the clinical value in ovarian cancer of metallothionein (MT), heat-shock protein-27 (HSP-27) and glutathione-S-transferase × and α (GSTπ, GSTα), recognized for their relation with drug resistance in vitro. The expression of these markers was evaluated by immunohistochemistry on paraffin-embedded tumor specimens from 86 patients with ovarian carcinomas diagnosed between 1977 and 1990 who received chemotherapy. Response to chemotherapy was evaluated using well-defined criteria. Marker expression was evaluated on a section of the primary tumor (81 cases) and, when available on a section of tumor following chemotherapy (48 cases). MT was expressed in 38.3 of primary tumors unexposed to chemotherapy, HSP-27 in 50.6, GST × in 37, and GST α in 50.6. The expression of all four markers did not help to predict chemoresistance. The concordance between marker expression by the tumor before and after chemotherapy was weak (concordance, 51.2-70.7). Immunostaining was not associated (p > 0.1) with any prognostic factor such as stage, residual tumor after surgery and grade. Ovarian cancer is a highly heterogeneous neoplasm and the expression of markers of chemoresistance reflects this heterogeneity. Our data suggest that chemoresistance is more likely multifactorial and confirms the complexity of the in vivo model.

Pregnancy-associated changes in metallothionein (MT) concentrations in blood plasma were examined using a competitive enzyme-linked immunosorbent assay with a rabbit polyclonal antibody to rat liver MT. Plasma MT of pregnant rats significantly increased after 8 days of gestation and remained high during pregnancy and for 7 days after delivery. Gel filtration showed that both Cu and Zn were associated with the plasma MT of pregnant rats. These results suggest that plasma MT may play a role in the transport of essential metals such as Cu and Zn to fetus during pregnancy, but the source of plasma MT is unknown. Injections of cadmium chloride and cadmium-metallothionein to pregnant rats further increased the plasma MT concentrations. After injection of CdCl2, both MT and Cd concentrations in the liver of the pregnant rats were significantly lower than those of the nonpregnant rats, whereas renal Cd and MT levels were higher in pregnant rats. This increased accumulation of Cd in the kidney of pregnant rats may be related to the increase of plasma MT during pregnancy. About 5% of 6 pg of in vitroadded Cd (20 pg/ml) was bound to the MT in the plasma. Therefore, Cd may be transported by the circulating MT to the kidney, leading to an increase in renal accumulation of Cd in pregnant rats.

Cadmium was preferentially bound to metaliothionein In tissues 24 h after CdCl2 injection. Of a number of chelating agents examined, only 2,3-dimercapto-1-propanol (BAL) was effective in mobilizing Cd from metaliothionein into bile. Structurally similar dithiols such as 1,3-dimercaptopropanol and 2,3-dimercapto-1-propanesulfonic acid were not effective. Diethylenetriamine pentaacetic acid increased only the urinary excretion of Cd. Biliary excretion of Cd increased with increasing dose of BAL, and there was a concurrent decrease in hepatic Cd levels without any increase in renal concentration. BAL was effective even 14 d after Cd injection. The form of Cd excreted in the bile after BAL injection in chronic exposure has not yet been characterized. Initial studies suggested that it was not metaliothionein but was a low-molecular-weight Cd complex, probably with BAL.

The effects of several mono-, di-, and trithiols (400 μmol/kg) in mobilizing Cd from metallothionein were studied in rats 24 h after a single injection of 109CdCI2 (1 mg kg/Cd). BAL (2,3-dimercaptopropanol) and propanetrithiol (1,2,3-trimercaptopropane) were the most effective mercaptans in increasing the biliary excretion of Cd (3.1 and 5.5% of administered dose, respectively, compared to 0.04% in control injected with propylene glycol) in in situ experiments with a significant decrease in hepatic Cd. Propane-1-thiol was inactive and propanetrithiol was the most toxic of the compounds studied. A number of propane dithiols having sulfhydryl groups at different carbon positions with and without substituents (OH or SO-3) and dimercaptoethane were also tested for effectiveness in removing Cd. All the lipophilic compounds with two adjacent sulhydryl groups were effective in increasing the biliary excretion of Cd, but were less effective than BAL and propanetrithiol. The major form of Cd in liver cytosols of rats pretreated with CdCI2 and injected with mercaptans was metallothionein. A small amount of Cd in liver cytosol and a major portion of biliary Cd in propanetrithiol-injected rats were bound to high-molecular-weight proteins when fractionated on Sephadex G-75 columns. On the other hand, after injection of BAL, most of the Cd in the bile was associated with a fraction of molecular weight 10,000. Even though Cd was present mainly as metallothionein in livers of Cd-pretreated rats, the biliary forms of Cd after injection of BAL and propanetrithiol were different. Similar results were obtained when Cd was added in vitro to bile samples collected from control rats that were injected with these chelating agents alone. However, the Sephadex G-75 elution profile of Cd-BAL and Cd-propanetrithiol after direct addition to control rat bile showed Cd complexes of identical molecular weight (<6000). These results suggested that the Cd-binding ligands present in bile after injection of BAL and propanetrithiol were different from and had a higher molecular weight than the complexes in vitro with Cd and the respective chelating agents.

OF MICE AND MOLECULES: POLLUTION AND HUMAN SURVIVAL. by E. Skjei and M. D. Whorton, Dial Press, New York, 1983, 347 pp., US $17.95.CRC HANDBOOK OF NATURALLY OCCURRING FOOD TOXICANTS. Edited by Miloslav Rechcigl, Jr., CRC Press, Boca Raton, FL, 1983, 338 pp., US $63.00.NEPHROTOXIC MECHANISMS OF DRUGS AND ENVIRONMENTAL TOXINS. Edited by G. A. Potter, Plenum Press, New York, 1982.SOCIETAL RISK ASSESSMENT: HOW SAFE IS SAFE ENOUGH? Edited by Richard C. Schwing and Walter A. Albers Jr., Plenum Press, New York, 1980, 374 pp., illus., US $29.50.PEST RESISTANCE TO PESTICIDES. Proceedings of a U.S.-Japan Cooperative Science Program Seminar on Pest Resistance to Pesticides; Challenges and Prospects, Held Dec. 3-7, 1979, in Palm Springs, California. Edited by George Georghiou and Tetsuo Saito, Plenum Press, New York, 1983, 809 pp., US $89.50.BIODEGRADATION TECHNIQUES FOR INDUSTRIAL ORGANIC WASTES. Edited by D. J. De Renzo, Noyes Data Corporation, Park Ridge, NJ, 1980, 358 pp., US$28.00.APPLICATION OF ON-LINE ANALYTICAL INSTRUMENTATION TO PROCESS CONTROL. Proceedings of the First Annual Conference on Activated Sludge Process Control Held in Chicago, November 10-11, 1981. Edited by R. M. Arthur, Ann Arbor Science Publishers, Ann Arbor, Michigan, 1982, 222 pp., US $29.95.ORGAN AND SPECIES SPECIFICITY IN CHEMICAL CARCINOGENESIS. Edited by Robert Langenbach, Stephen Nesnow, and Jerry M. Rice, Plenum Press, New York, 1983, 693 pp., US $79.50.HANDBOOK OF PLASTICS FLAMMABILITY AND COMBUSTION TOXICOLOGY. by Arthur H. Landrock, Noyes Publications, Park Ridge, NJ, 1983, 308 pp., US $36.00.ALCOHOLS TOXICOLOGY Edited by William W. Wimer, John A. Russell, and Harold L. Kaplan, Noyes Data Corporation, Park Ridge, NJ, 1983, 278 pp., US $36.00.INDUSTRIAL WASTE. Proceedings of the Fourteenth Mid-Atlantic Conference Held at the University of Maryland, College Park, Maryland, June 27-29, 1982. Edited by James E. Alleman and Joseph T. Kavanagh, Ann Arbor Science Publishers, Ann Arbor, Michigan, 1982, 612 pp. US$39.95.TRACE ELEMENTS IN HEALTH. Edited by J. Rose, Butterworth Publishers, Woburn, Massachusetts, 1983, 317 pp., US $69.95.EVALUATION AND MANAGEMENT OF HOSPITAL INFECTIONS. Edited by Ralph van Furth, Martinus Nijhoff Publishers, The Hague/Boston/London, 1982, 266 pp. US $39.00.SELENIUM-A POTENTIAL ENVIRONMENTAL POISON AND A NECESSARY FOOD CONSTITUENT. Edited by Charles G. Wilber, Charles C. Thomas, Springfield, Illinois, 1983, 126 pp., US$19.50.

A recent study from our laboratory has shown that cadmium, a toxic heavy metal, is one of the most effective agents known for inducing hepatocytic transdifferentiation of the rat pancreas. With repeated injections of cadmium, the incidence of rats with pancreatic hepatocytic foci can be as high as 93%. Cadmium is also well known as a very potent inducer of metallothionein, a metal-binding protein that appears to be important in the biologic response to several toxic heavy metals in most tissues, including the pancreas. Therefore, the present study sought to determine if metallothionein was associated with cadmium-induced transdifferentiation of pancreatic cells. Expression of metallothionein was studied immunohistochemically by the peroxidase-antiperoxidase method in tissue sections of the pancreas of rats with pancreatic hepatocytes. High levels of metallothionein were localized primarily within the pancreatic hepatocytes. Surrounding normal pancreatic islet and acinar cells were not immunoreactive. Thus, metallothionein is expressed actively in cells transdifferentiated to hepatocytes by cadmium within the pancreas.

Metallothionein, a high cysteine-containing protein, can bind with both essential and nonessential metals and thus play an important role as a metal storage protein and also in the detoxification of toxic metals. Although in the human fetus, levels of trace minerals and metallothionein are very high, their postnatal changes are not well documented. The purpose of the present investigation, therefore, was to quantify the accumulation of metallothionein in premature and full-term infants during the first year of life and to identify factors affecting its accumulation. From 47 postmortem samples, it was determined that hepatic metallothionein levels were highest in newborn premature and full-term infants falling to levels found in older children by 4.4 months of age. Hepatic zinc levels were also highest in the youngest infants, falling with increasing postnatal age. There was a significant positive correlation between zinc and metallothionein at all ages. However, there was a negative correlation between hepatic metallothionein levels and cystathionase activity. Hepatic copper and metallothionein levels were unrelated. The renal concentration of metallothionein, zinc, and copper were significantly lower than corresponding hepatic levels. The fall in hepatic levels of zinc and metallothionein during the first months of life correspond to a period of negative zinc balance and low endogenous cysteine production in the newborn. Thus metallothionein may play an important role as a storage depot for these two essential nutrients during this critical period of active growth. (Pediatr Res24: 326–329, 1988)

Body retention and tissue distribution of Cd from oral 109 CdCl2 and 109 Cd-metallothionein were studied in C57BL/6J mice given identical doses (60 μg Cd and 2 μCi 109 Cd). Measurement of109 Cd radioactivity in mice showed identical absorption but differences in tissue distribution of Cd from the two forms. A significantly greater deposition of Cd was observed in the kidney of mice fed Cd-metallothionein than in the CdCl2 group where a major portion of Cd was deposited in the liver. Most of the radioactive Cd in the renal and hepatic tissues and in the duodenum was bound to metaltothionein 24 h after oral Cd-metallothionein, whereas only 30 and 38% of the cadmium in liver and kidney supernatants, respectively, were bound to metallothionein after oral CdCI2. These results suggest that the dietary form of Cd may influence the renal deposition of Cd.

Metallothioneins (MT) are low molecular weight, metal-binding proteins. The induction of MT synthesis by cytokines, hormones and other cytotoxic agents indicates its role in cellular proliferation and differentiation as well as in cellular defense mechanisms. Previous studies have detected expression of MT in various human tumors by immunohistochemical staining. In certain cases the presence of MT in a tumor may be associated with its resistance against radiation and chemotherapeutic agents.

We describe a competitive, solid-phase radioimmunoassay for metallothionein, which employs a rabbit antiserum directed against rat MT-2 to detect metallothionein (MT) from several different species (rabbit, mouse, rat, Chinese hamster, and human). The lower limit of detection of the assay for rat MT-2 was 0.7 ng; for rabbit MT-2 it was 2 ng. The method is capable of measuring both isoforms of MT (MT-1 and MT-2). When MT levels in rat and mouse tissues were estimated with this RIA and the silver-saturation method, both assays gave the same pattern of MT induction in control and cadmium-treated animals. Both methods measured high levels of MT in human liver samples. Chinese hamster ovary cells induced with cadmium also showed elevated MT expression. The detectability of MTs from a broad range of species is facilitated by the use of solid-phase MT, which has an avidity for the antiserum similar to that of the MT in the tested sample.

To investigate the role of metallothionein (MT) in the sequestration and storage of zinc in newborn rat livers, a cross-fostering experiment was performed in which zinc-deficient (Zn-D) pups were suckled from Zn-sufficient (Zn-S) dams and vice versa. At consecutive days during lactation, groups of pups were killed. The experiment was continued for 22 d. Zinc concentrations in various tissues and MT concentrations in livers and kidneys were analyzed. The retention of injected 65Zn, as well as body weight, was also studied in both groups. Higher whole-body retention of 65Zn in the zinc-depleted (Zn-Dp) rats indicates a decreased zinc turnover. These rats also showed markedly reduced growth. The observation that the zinc-repleted (Zn-Rp) pups, although showing 65Zn retention similar to that of controls, grew less than controls suggests that Zn-Rp may not compensate for gestational zinc deficiency. Zn-Rp pups showed an increased accumulation of zinc into hepatic MT until d 10, whereas Zn-Dp pups showed a more accelerated degradation of MT than controls. These data indicate that hepatic MT levels fluctuate directly in response to dietary zinc status in newborn rats. Various tissues such as spleen, heart, lung and intestine showed no difference in zinc concentration among all groups at d 22 postpartum. Thus the rapid degradation of hepatic MT in zinc deficiency that may occur to maintain the required zinc levels in other tissues supports the role of MT as a zinc storage protein in newborn rats.

In the general population, food constitutes the major environmental source of cadmium (Cd) in nonsmokers. It is established that leafy vegetables, roots, and grains (wheat or rice) can accumulate relatively high amounts of Cd from the soil. Beef liver and kidney and shellfish are also major dietary sources of Cd. The daily intake of Cd in various parts of the world is different and depends on both the dietary habits and concentration of Cd in foodstuffs. Because of the long biological half-life of Cd in humans and absence of any specific indicators of its toxicity, the environmental exposure of Cd should be monitored in various countries. Although environmental Cd poisoning is rare, there are isolated reports on excessive exposure to Cd in Japan and Shipham, a zinc-mining town in England. The body retention and toxicity of Cd depends on various factors, such as daily intake, the form of Cd in food, its interactions with essential elements, and nutritional status of the population. Since kidney is considered a critical organ in Cd toxicity, the indicators of renal dysfunction have been widely used for evaluation of Cd poisoning in occupationally exposed people. It is unclear whether similar indicators can be used for monitoring environmental Cd exposure.

The objectives of this investigation were to study the role of zinc-thionein in the metabolism of excessive amounts of zinc and the re-utilization of zinc bound to this protein. Rats were injected ip with 5 mg Zn/kg body weight as zinc chloride for 5 days to induce the synthesis of zinc-thionein. Proteins with molecular weight of 10,000 with high affinity for zinc were synthesized in rat liver and intestine. These proteins were heat stable. Large scale isolation of this protein from rat liver was achieved by a simple method consisting of a heating step and sephadex gel fractionation. Intravenous injection of isolated 65Zn-thionein to rats resulted in similar organ distribution of zinc as that after 65ZnCl2injection. Gel filtration experiments with liver supernatant from rats injected with 65Zn-thionein showed that a major portion of 65Zn was transfered to other protein fractions and was re-utilized similar to inorganic salts of zinc. Morphological studies showed no toxic effects after injection of zinc-thionein. These results show that zinc-thionein is metabolically active and non-toxic.

The effects of changes in sulfur-containing intracellular ligands on biliary excretion of cadmium were studied in rats. Injection of zinc or copper salts 24 h before intravenous injection of 109CdCl2 (1 mg/kg Cd) decreased biliary excretion of Cd. Pretreatment with cysteine (25 mg/kg) had a similar effect. Depletion of intracellular thiol by injection of diethylmaleate had little effect. The effect of chelating agents on the pharmacokinetics of Cd depended on time of administration of the agents after exposure to Cd. When chelating agents were administered ½> h after Cd injection (before the synthesis of metallothioneinj, the thiol-containing agents (2,3-dimercapto-1-propanol (BAL), DL-penicillamine, N-acetylpenicillamine, and dithioerythritolj increased the biliary excretion of Cd, while the carboxyl-containing ones (EDTA and nitrilotriacetate) increased the urinary excretion of Cd. BAL was the most effective chelating agent, but there was also an increase in the renal concentration of Cd. However, when these chelating agents were administered 24 h after Cd injection (after the synthesis of metallothionein), only BAL increased the biliary excretion of Cd. Renal and hepatic Cd concentrations decreased concurrently after BAL treatment.

Biliary excretion of cadmium was studied in rats after intravenous injection of different doses of cadmium chloride (0.1-2 mg Cd/kg). The rate of bile flow was not affected by cadmium injection and cadmium was excreted into bile during the first 2 hr after injection. The biliary excretion of cadmium increased with increasing dose of CdCl1. Cumulative biliary excretion of cadmium for 5 hr was 0.065% of the administered dose for groups injected with 0.1 mg Cd/kg as compared to 16.9% of the administered dose for 2 mg Cd/kg. During the 5 hr experimental period, most of the cadmium in liver cytosol was bound to high-molecular-weight proteins and less than 10% was bound to the metallothionein fraction. The biliary cadmium was recovered as a low-molecular-weight compound (less than 4,000) in experiments with various doses of cadmium and no cadmium was attached to high-molecular-weight proteins or metallothionein in the bile. The low-molecular-weight cadmium complex in bile was partially characterized as Cd-glutathione by thin-layer chromatography and amino acid analysis.

In protecting the body against the noxious effects of dietary cadmium ions, cadmium is bound to metallothionein in the proximal intestine, and subsequently excreted into the lumen with desquamation of the epithelium. The purpose of this study was to determine the extent to which cadmium in the form of intestinal cadmium-thionein is absorbed from the intestinal lumen and to appraise the toxicity of cadmium-thionein on the intestinal mucosa. With open-ended duodenal perfusion, equivalent amounts of cadmium administered as CdCl2 or cadmium-thionein entered the mucosa, but significantly less cadmium from the perfusate of cadmium-thionein passed into the body. Exposure of the mucosa to CdCl2 for 1 hr led to minor abnormalities in the form of broadening of villi with pseudostratification of epithelium, and swelling of mitochondria, whereas cadmium-thionein produced extensive necrosis of absorptive cells. The results suggest that cadmium-thionein may play a paradoxical role, providing protection against the cadmium ion in the intracellular milieu, but promoting cadmium toxicity when it is present in sufficient amounts in the lumen of the intestine.

Intravenous injection of 109Cd-labeled CdCI2 (I mg/kg) to control rats and rats that had been injected with CdCI2 (0.25 mg/kg) 24 hr earlier showed that there was a decrease in the biliary excretion of 109Cd in the latter group. The 24 hr pretreatment with CdCI2 resulted in induced synthesis of metallothionein in rat liver and kidney. The binding of cadmium to liver tissue was increased and the renal accumulation of cadmium was unaltered on cadmium pretreatment.Rat liver metallothionein was isolated from rats injected repeatedly with CdCl2 Intravenous injection of cadmium-bound metallothionein gave a different distribution of cadmium in the tissues and biological fluids as compared to injection of CdCl2. A major percentage of cadmium was deposited in the kidney with urinary excretion after injection of rat cadmium-metallothionein to control rats. The biliary excretion of cadmium after cadmium-metallothionein injection was minimal. About 90% of cadmium in the kidney cortex and urine could be recovered as cadmium-thionein in sephadex gel filtration, 3 hr after cadmium-thionein injection.

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 14C-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.

Metallothionein is a metal binding protein thiol found in high concentrations in the liver and kidney. Recent evidence has linked overexpression of cellular metallothionein with tumor cell resistance to chemotherapeutic drugs, such as alkylating agents and cis-diamminedichloroplatinum (II) (cisplatin). We studied the metallothionein content of 9 human transitional cell carcinomas of the bladder with immunohistochemical methods. All tumors stained positive for metallothionein and the staining was localized almost exclusively to the cytoplasm. Uroepithelium displaying dysplastic changes or carcinoma in situ demonstrated the greatest intensity of staining, while staining in the invasive portions of the tumor was weak and variable. These findings were of interest, since combination chemotherapy of invasive transitional cell carcinoma of the bladder often is ineffective against carcinoma in situ. Normal uroepithelium stained strongly in all 3 patients who experienced disease progression and death, and in only 1 of the 5 who are currently without evidence of disease.

Cadmium is unique among non-essential metals in several of its toxicological effects because of its long biological half life, slow excretion and delayed action on the kidneys1. Although cadmium poisoning in humans is uncommon, there are reports of chronic cadmium poisoning in workmen2–4and also in others in certain polluted areas5,6. At present there are no suitable methods either to measure the body burden of cadmium or to treat cadmium poisoning7. The therapeutic effects of various chelating agents including 2,3-dimercaptopropanol (BAL) and its soluble glycosides have been studied without much success in acute cadmium intoxication8–11. However, those studies were done before anything was known of the specific binding of cadmium to metallothionein, an intracellular low molecular weight protein12. The potential role of this protein in the detoxification and toxicity of metals has been reviewed recently13,14. The induced synthesis of metallothionein has a marked effect on the pharmacokinetics of cadmium and other divalent metals15,16. Thus it is important to consider the intracellular binding of cadmium with metallothionein in developing a suitable chelation therapy for chronic exposure to cadmium17. The in vivo chelation of cadmium with BAL or BAL and diethylenetriamine pentaacetic acid (DTPA) from rats exposed to cadmium is reported here.