Glyphosate disrupts redox status and up-regulates metallothionein I and II genes expression in the liver of adult rats. Alleviation by quercetin.

The present work evaluated the possible protective effects of quercetin against glyphosate-induced hepatotoxicity in adult rats. Rats were randomly divided into three groups: a control group (C), a glyphosate-treated group (Gly) and a group treated with both glyphosate and quercetin (Gly+QE). During the experimental period (15 days), glyphosate (50 mg/kg b.w.) was administered every two days by intraperitoneal way while quercetin (20 mg/kg b.w./day) was administered daily by gavage. Glyphosate-induced hepatic oxidative stress was evidenced by the increased levels of malondialdehyde, hydrogen peroxide, advanced oxidation protein products and protein carbonyls with a significant decrease in enzymatic (superoxide dismutase, catalase, glutathione peroxidase) and non-enzymatic (non-protein thiols, glutathione, vitamin C) antioxidants. Plasma biomarkers of hepatotoxicity (AST, ALT, ALP, γ-GT, albumin) were also altered. Moreover, glyphosate induced DNA damage, up-regulated metallothionein (MT I and MT II) genes expression and provoked histopathological changes in rats' liver. Quercetin supplementation to glyphosate-treated rats markedly ameliorated all the parameters indicated above as well as the liver histoarchitecture. Therefore, quercetin might have beneficial effects against glyphosate-induced hepatotoxicity in rats.

Zinc chloride for odontogenesis of dental pulp stem cells via metallothionein up-regulation.

INTRODUCTION: Previous studies have shown that zinc chloride (ZnCl(2)) can induce metallthionein (MT) in the liver and kidney to protect tissues against toxicants and shows a better corneal wound healing than conventional drugs do. We hypothesized that ZnCl(2) can promote odontogenesis of dental pulp stem cells (DPSCs) via MT. The purpose of this study was to investigate the effects of ZnCl(2) on human DPSCs and the expression of MT. METHODS: DPSCs were isolated by flow cytometry with selective surface marker CD146 and STRO-1. After they grew into confluence, DPSCs were induced into odontoblasts with or without ZnCl(2) supplemented in the culture medium for 21 days. The effect of ZnCl(2) on DPSCs differentiation was examined followed by alkaline phosphatase staining/activity and quantitative real-time polymerase chain reaction analysis. RESULTS: By treating DPSCs with ZnCl(2), the duration of mineralization was shortened and expressions of differentiation markers into odontoblasts were more significant than those without ZnCl(2) stimulation. Besides, the MT gene expression was increased with the increasing expressions of odontoblasts' markers after treated with ZnCl(2). CONCLUSION: This was the first report that ZnCl(2) could promote odontoblastic differentiation of DPSCs through the up-regulation of gene MT.

Response of metallothionein gene-1 to laboratory exposure to heavy metals and thermal stress in the freshwater prawn Macrobrachium rosenbergii.

Metallothioneins, metal-inducible proteins, are being characterized from different organisms and shown as potential biomarkers of exposure to pollution by certain heavy metals. Here we report the identification of a new metallothionein cDNA (433bp) from the shrimp Macrobrachium rosenbergii, putatively encoding a 61 residue polypeptide. Tissue specific analysis indicated that Mar-MT-I (M. rosenbergii Metallothionein Gene-1) is expressed with the highest levels in the hepatopancreas and lowest in the thoracic ganglia, and none in the gills or muscles. In addition, our data showed that Mar-MT-I is differentially regulated in the hepatopancreas by certain heavy metals and thermal stress: Cd and Cu produce somewhat similar expression profile patterns, Zn has a reductional effect and thermal stress alone entirely stops its expression. These results show that Mar-MT-I mRNA levels can potentially be used as biomarkers for Cd, Cu or Zn pollution individually. However, in the case of combined metal treatment, different combinations of these metals have quite different effect on Mar-MT-I expression. Therefore, factors of such differential behaviors should be kept as a priority for further biomonitoring studies.

L-arginine reduces mercury accumulation in thymus of mercury-exposed mice: role of nitric oxide synthase activity and metallothioneins.

Mercury, an occupational and environmental contaminant, is a well-recognized health hazard. The thymus is a target for inorganic mercury (Hg2+); thymic function is impaired in Hg2+ intoxication and is partially restored by simultaneous L-arginine supplementation. The nitric oxide (NO)-nitric oxide synthase (NOS) pathway and metallothioneins (MTs) were studied to investigate the role of L-arginine in thymic function restoration after mercury exposure. Mice received a higher and a lower dose of inorganic mercury, with and without L-arginine supplementation. Saline-treated mice were used as controls. Thymus weight and thymulin were measured as indices of thymic function. Mice treated with Hg2+ alone displayed an accumulation of metal in the thymus, reduced NOS activity, a lower plasma nitrite plus nitrate concentration and an increased MTs expression compared with control mice. L-arginine supplementation was associated with lower Hg2+ concentrations in the organ and partial preservation of other measures. Reduced accumulation of Hg2+ in mice dosed with L-arginine was probably related to greater NO production and NO-MTs interactions.

Effect of zinc supplementation on plasma IL-6 and MCP-1 production and NK cell function in healthy elderly: interactive influence of +647 MT1a and -174 IL-6 polymorphic alleles.

Pro-inflammatory cytokine response and NK activity are controlled by the availability of zinc ion, whose intra-cellular transport is regulated by metallothioneins. In order to closely examine the importance of circulating zinc in the modulation of immune response during ageing, in the balance of Th2/Th1 equilibrium and finally in the reversibility of systemic low grade inflammation, we evaluated the changes occurring in plasma IL-6 and MCP-1 concentrations and NK lytic activity in a healthy low grade inflamed elderly population, following zinc-aspartate supplementation. In addition, we aimed to highlight the potential interaction among circulating zinc increments, changes in immunological parameters and +647 MT1a and -174 IL-6 polymorphic alleles. Thirty-nine healthy individuals (60-83 years) from the ZINCAGE cohort (previously typed for +647 MT1a and -174 IL-6 polymorphisms) were supplied with zinc-aspartate. Blood samples collected before and after supplementation underwent basal laboratory determinations (circulating zinc, albumin and C-reactive protein) and immunological studies (plasma IL-6 and MCP-1 and NK lytic activity). Zinc supplementation in subjects with low or borderline-normal circulating zinc increased the concentration of this ion and modulated plasmatic IL-6 and MCP-1 as well as NK lytic activity. An interactive effect of polymorphic alleles of MT1a and IL-6 genes on zinc, IL-6, MCP-1 and NK activity was evidenced following supplementation, indicating the genetic background as one of the determinants for identifying groups of subjects that can take advantage of therapeutic intervention.

Systemic imbalance of essential metals and cardiac gene expression in rats following acute pulmonary zinc exposure.

It was recently demonstrated that particulate matter (PM) containing water-soluble zinc produces cardiac injury following pulmonary exposure. To investigate whether pulmonary zinc exposure produces systemic metal imbalance and direct cardiac effects, male Wistar Kyoto (WKY) rats (12-14 wk age) were intratracheally (IT) instilled with saline or 2 micromol/kg zinc sulfate. Temporal analysis was performed for systemic levels of essential metals (zinc, copper, and selenium), and induction of zinc transporter-2 (ZT-2) and metallothionein-1 (MT-1) mRNA in the lung, heart, and liver. Additionally, cardiac gene expression profile was evaluated using Affymetrix GeneChips (rat 230A) arrays to identify zinc-specific effects. Pulmonary zinc instillation produced an increase in plasma zinc to approximately 20% at 1 and 4 h postexposure with concomitant decline in the lung levels. At 24 and 48 h postexposure, zinc levels rose significantly (approximately 35%) in the liver. At these time points, plasma and liver levels of copper and selenium also increased significantly, suggesting systemic disturbance in essential metals. Zinc exposure was associated with marked induction of MT-1 and ZT-2 mRNA in lung, heart, and liver, suggesting systemic metal sequestration response. Given the functional role of zinc in hundreds of proteins, the gene expression profiles demonstrated changes that are expected based on its physiological role. Zinc exposure produced an increase in expression of kinases and inhibition of expression of phosphatases; up- or downregulation of genes involved in mitochondrial function; changes in calcium regulatory proteins suggestive of elevated intracellular free calcium and increases in sulfotransferases; upregulation of potassium channel genes; and changes in free radical-sensitive proteins. Some of these expression changes are reflective of a direct effect of zinc on myocardium following pulmonary exposure, which may result in impaired mitochondrial respiration, stimulated cell signaling, altered Ca2+ homeostasis, and increased transcription of sulfotransferases. Cardiotoxicity may be an outcome of acute zinc toxicosis and occupational exposures to metal fumes containing soluble zinc. Imbalance of systemic metal homeostasis as a result of pulmonary zinc exposure may underlie the cause of extrapulmonary effects.

Chemopreventive effect of vanadium in a rodent model of chemical hepatocarcinogenesis: reflections in oxidative DNA damage, energy-dispersive X-ray fluorescence profile and metallothionein expression.

In the present study, we investigated the antitumour efficacy of vanadium in a defined rodent model of experimental hepatocarcinogenesis. Hepatic preneoplasia was induced in male Sprague-Dawley rats with a single, necrogenic, intraperitoneal injection of diethylnitrosamine (DEN) (200 mg/kg body weight) followed by promotion with phenobarbital (PB). The levels of modified DNA bases 8-hydroxy-2'-deoxyguanosine (8-OHdG), a potential marker involved in the initiation of carcinogenesis, were measured by high-performance liquid chromatography, whereas tissue trace element status and expression of metallothionein (MT), a Cu-Zn metalloprotein associated with neoplastic cell growth and subsequent development of premalignant phenotype of the cell, were studied by energy-dispersive X-ray fluorescence spectrometry and enzyme-coupled immunohistochemistry, respectively. There was a significant and steady elevation of modified bases (8-OHdG) along with substantial increase in MT immunoexpression and disturbance in trace element homeostasis following DEN exposure. Supplementation of vanadium at a dose of 0.5 ppm for four consecutive weeks strictly abated the formation of 8-OHdG (P < 0.0001; 81.28%) in preneoplastic rat liver. In a long-term DEN plus PB regimen, vanadium was able to limit in situ MT expression with a concomitant decrease in MT immunoreactivity (P < 0.05). Furthermore, vanadium treatment throughout the study restored hepatic levels of essential trace elements and decreased nodular incidence (58.34%) and nodule multiplicity (P < 0.001; 66.89%) in rats treated with DEN plus PB. Taken together, the study provides evidence in support of the chemopreventive potential of vanadium in limiting neoplastic transformation during the preneoplastic stages of hepatocarcinogenesis in rats.

A Kampo formula Juzen-taiho-to induces expression of metallothioneins in mice.

Juzen-taiho-to (JTX), one of the commonly prescribed traditional Japanese herbal medicines (Kampo), is indicated for adjunctive treatment of cancers and autoimmune diseases. To understand the mechanisms underlying the clinical effects of JTX, the effects of orally administered JTX on the expression of metallothioneins (MTs) were examined in the liver, spleen, small and large intestines of mice. In addition, the expression of MTs in specific pathogen free (SPF) mice was examined to understand the participation of intestinal bacteria in the expression of MTs. JTX enhanced expression of MT-I and -II significantly in the liver of SPF mice. Induction of MT-II expression was observed also in the small intestine. Intestinal bacteria appeared to have no effect on MTs expression. Neither expression of MT-III nor its induction was observed in any tissue. These findings strongly suggest that MTs should mediate at least some effects of JTX in mice.

Involvement of cytokines in the hepatic metallothionein expression by alpha-hederin.

Alpha-hederin, a triterpenoid saponin, has been reported to induce hepatic metallothionein (MT). However, the mechanism underlying its effects is unknown. This study investigated the effects of alpha-hederin on the regulation of MT expression in an in vitro model using the murine hepatoma cell line, Hepa-1c1c7, and the murine macrophage cell line, RAW 264.7. Alpha-hederin that was added directly to Hepa-1c1c7 cells had no effect on MT induction. However, MT and its mRNA levels increased markedly when the Hepa-1c1c7 cells were cultured with the alpha-hederin-treated, conditioned medium from the RAW 264.7 cells. Co-treating the RAW 264.7 cells with alpha-hederin and pentoxifylline, a TNF-alpha synthesis inhibitor, resulted in decreased effects of alpha-hederin on MT induction. In the alpha-hederin-exposed RAW 264.7 cell cultures, production and mRNA levels of TNF-alpha and IL-6 were increased. Accordingly, it was found that the MT induction activity was inhibited when antibodies to TNF-alpha and/or IL-6 were added to the alpha-hederin-treated, conditioned medium from the RAW 264.7 cells. These results suggest that the upregulation of MT expression by alpha-hederin is mediated by TNF-alpha and IL-6.

Relationship between metallothionine and zinc in the protection against DNA damage in zinc-treated Long-Evans Cinnamon rat liver.

The aims of the work presented here were to determine the effect of long term treatment with zinc (Zn) on both total metallothionine (MT) and, in particular, oxidized MT (MTox) concentrations in Long-Evans Cinnamon (LEC) rat liver. We also evaluated semi-quantitatively the cell death index using TUNEL assay as it is a useful method to localize the nuclear fragmentation occurring in oxidative stress conditions. The results demonstrate there were no statistically different MT concentrations between Zn-treated and untreated rats, whereas the Zn treatment was very effective in reducing the percentage of oxidized MT (MTox). MTox is not able to bind metals, so it does not perform its "scavenger" action against copper (Cu) accumulation in LEC rats. The intensity and quantity of fluorescent staining observed in untreated rat sections decreased compared to the treated ones. These findings suggest that in LEC rats one of zinc's roles is to protect from oxidative stress, however, its mode of action remains partially unknown: a hypothesis is competition for Cu binding sites. A new insight is that Zn induced MT can protect efficiently against DNA damage by free radicals.

Estrogen response element-independent regulation of gene expression by genistein in intestinal cells.

Isoflavone, genistein, was shown to have antioxidant and antitumor activities. We have reported the stimulatory effect of genistein on the expression of antioxidant and metal-binding protein, metallothionein IIA (MTIIA), in human intestinal Caco-2 cells. Genistein has been shown to up-regulate the expression of other genes through estrogen response element (ERE) but the ERE sequence is not in the MTIIA promoter. In this paper, we investigated the ERE-independent mechanism that mediates the stimulatory effect of genistein. Genistein enhanced the expression of human MTIIA promoter (up to -426)-containing reporter genes, thus supporting a promoter-specific transcriptional regulation. A shorter MTIIA promoter (-83 to +27) was found to be able to mediate the full reporter gene response to genistein in a dose- and time-dependent fashion. Further deletion and mutation analysis revealed that the GC-rich Sp1 binding sequence was the target of the stimulation. Genistein was known to bind to estrogen receptors (ER). When cells were cotransfected with ER beta, the stimulatory effect of genistein on the reporter gene containing the GC-rich promoter sequence increased further and a similar result was observed for breast cancer MCF-7 cells. Inhibitors of protein kinase A could block the response to genistein but the phosphorylation of Sp1 protein per se was not affected by the genistein treatment. Our observation could help to evaluate the biological significance of genistein, which is used widely as a supplement.

Nephrotoxic actions of low-dose mercury in mice: protection by zinc.

The authors conducted this study to determine if very-low-dose (i.e., 4 ppm) mercury is nephrotoxic and, if so, whether the nephrotoxic actions of mercury in mice could be prevented by zinc intake. Animals were administered 4 ppm mercuric chloride and/or 800 ppm zinc chloride in their drinking water for 12 wk. The animals were sacrificed at the end of the exposure period, and their kidneys were excised, weighed, and processed for histological study. Both metals reduced significantly (p < .05) the absolute and relative kidney weights of the animals. Zinc-treated animals showed normal kidney histology that was comparable with that of the control. Mercury treatment produced necrosis and widening of the glomeruli, whereas a combination of both metals resulted in protection from the toxic effects, with most nephrons resembling the control. The results indicate that low-dose mercury exposure in mice kidney induces some degenerative effects, which are prevented by zinc.

[Quantification of metallothionein induced with selenium by using competitive ELISA].

Metallothionein induced by selenium was assayed with competitive ELISA. It was found that in control mice, the liver contained (2.47+/-0.90) microgram MT/g wet weight tissue, while the mice administered with ZnSO(4) contained (8.15+/-2.20) microgram MT/g wet weight liver tissue and the mice given with selenium malt contained (12.80+/-1.44) microgram MT/g wet weight liver. A significant difference was found between the control and the latter two groups (P<0.05, P<0.05). The MT content of selenium group was significantly higher than the zinc group. It is suggested that the selenium was more potent in induction of MT and was a stronger inducer than ZnSO(4) in its effective dose range, therefore selenium may be developed into a novel and safe MT inducer.

Catalytic oxidation of zinc/sulfur coordination sites in proteins by selenium compounds.

Zinc/thiolate (cysteine) coordination occurs in a very large number of proteins. These coordination sites are thermodynamically quite stable. Yet the redox chemistry of thiolate ligands confers extraordinary reactivities on these sites. The significance of such ligand-centered reactions is that they affect the binding and release of zinc, thus helping to distribute zinc, and perhaps controlling zinc-dependent cellular events. One new aspect focuses on the thiolate ligands of zinc as targets for the redox action of selenium compounds. A distinctive feature of this chemistry is the capacity of selenols to catalyze the oxidation of zinc/thiolate sites. We here use a chromophoric compound, 2-nitrophenylselenocyanate, to investigate its reaction mechanism with the zinc/thiolate clusters of metallothionein, a protein that is a cellular reservoir for zinc and together with its apoprotein, thionein, is involved in zinc distribution as a zinc donor/acceptor pair. The reaction is particularly revealing as it occurs in two steps. A selenenylsulfide intermediate is formed in the fast oxidative step, followed by the generation of 2-nitrophenylselenol that initiates the second, catalytic step. The findings demonstrate the high reactivity of selenium compounds with zinc/thiolate coordination sites and the potent catalytic roles that selenoproteins and selenium redox drugs may have in affecting gene expression via modulation of the zinc content of zinc finger proteins.

Metallothionein mRNA in monocytes and peripheral blood mononuclear cells and in cells from dried blood spots increases after zinc supplementation of men.

A specific, sensitive and reliable index for assessment of human zinc status has not been developed, and continues to present a considerable challenge for nutritionists in the trace element field. We have focused on metallothionein (MT) expression as a potential index. A protocol involving 16 men and a 10-d supplementation period plus a 4-d postsupplementation period was used to examine the relative response of MT expression in erythrocytes, monocytes, peripheral blood mononuclear cells (PBMC) and cells from a dried blood spot (DBS). Zinc was supplemented at the current adult male recommended dietary allowance (RDA) of 15 mg. Erythrocyte MT protein, as measured by ELISA, increased gradually to about twofold over the placebo group during zinc supplementation and remained elevated for 4 d postsupplementation. Competitive reverse transcriptase-polymerase chain reaction showed that MT mRNA levels in both monocytes and PBMC increased (up to 4.7- and 2.7-fold, respectively) after 2 d of supplementation, with greater expression in monocytes compared with PBMC. Total RNA extracted from dried blood spots, representing cells from 50 microL of blood, showed a comparable change in MT mRNA upon zinc supplementation. In each leukocyte population isolated, when zinc supplementation was withdrawn, MT mRNA levels decreased. Collectively, these experiments show that, in men, MT gene expression increases during supplementation at the RDA, and that the DBS sampling method will be of value in measuring MT expression in a variety of clinical and survey situations.

Effect of arsenic trioxide on metallothionein and its conversion to different arsenic metabolites in hen liver.

The metabolism of arsenic, its affinity to metallothionein (MT), its influence on selenium levels, and its biotransformation to different metabolites in the liver tissue of laying hens exposed to arsenic trioxide (As2O3) was investigated. The experiment was performed with two groups of hens fed for 19 d with either a standard diet or with the same diet enriched in arsenic (30 microg/g). The major findings were as follows: 1. After 19 d exposure, about 65% of the total liver As was found in the water-soluble phase (100,000g centrifuged supernatant). In liver supernatant, As binding was found mostly in the range of very low-molecular-weight proteins (Mr < 10,000). Although after exposure the amount of MT-like proteins increased, the As bound to it was only in trace amounts. The protein was identified by convential procedures as Zn,Cu-thionein with traces of selenium and arsenic. 2. Arsenic exposure resulted in almost unchanged Se levels regarding its tissue concentrations and distribution between supernatant and pellet, where about 10% of total Se was found in the supernatant. On the contrary, As exposure did affect Cd levels. Tissue Cd concentration was slightly diminished, but the percentage of tissue Cd found in the water-soluble phase was increased from 20% to 40%. 3. In methanol extracts of tissue and supernatant of the As-exposed group, only two arsenic compounds were detected, As(III) and dimethylarsinic acid (DMA), the latter prevailing.

Effect of metal ions on calcifying growth plate cartilage chondrocytes.

The effects of the trace metals zinc (Zn), manganese (Mn), and cadmium (Cd) on the metabolism of growth plate chondrocytes was examined using a mineralizing culture system. Supplementation of serum-free primary cultures of growth plate chondrocytes with 10-100 mu m Zn resulted in an increase in cell protein and greatly increased alkaline phosphatase (AP) activity; however, above 25 mu m Zn mineralization of the cultures was reduced. The effects of Zn on cellular protein and AP activity were enhanced by the addition of the albumin to the culture media. Removal of Zn from basal culture media resulted in recoverable reductions in cellular protein and AP activities. Cadmium was acutely toxic to chondrocyte cell cultures at concentrations above 5 mu m. Even at very low concentrations (0.25 mu m) Cd caused significant reductions in DNA, cellular protein, and matrix protein synthesis. In contrast, Cd had negligible effects on AP activity or culture mineralization. Manganese treatment (50 mu m) resulted in reduced levels of proteoglycan, cell protein, DNA synthesis, and collagen synthesis, although AP specific activity did not change. At 10 mu m, Mn significantly reduced mineralization but had only minor influence on other culture parameters. Both Zn (200 mu m) and Cd (0.1 mu m), but not Mn, induced the synthesis of metallothionein. The physiological and biochemical effects of specific metal ions is largely dependent on their physicochemical properties, especially their ligand affinities. Knowledge of these properties allows predictions to be made regarding whether the organic or the mineral phase are most likely to be affected in a mineralized tissue.

Competitive reverse transcriptase-polymerase chain reaction shows that dietary zinc supplementation in humans increases monocyte metallothionein mRNA levels.

Zinc status is difficult to evaluate in humans. Metallothionein gene expression is transcriptionally regulated by dietary zinc and thus could serve as an assessment parameter based on zinc-dependent function. We used semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to establish that MT mRNA is increased in a human monocytic cell line by addition of zinc to the medium. To examine this response in human subjects, a dietary supplement of 50 mg zinc gluconate/d was given for 15 d. Monocytes were purified from venous blood using NycoPrep 1.068. Monocyte purity was determined by flow cytometry using fluorescent anti-human monocyte CD14 antibodies. Total monocyte RNA was extracted and converted to cDNA by reverse transcription. Competitive RT-PCR was used to analyze differences between cDNA levels that are proportional to MT mRNA levels in monocytes from zinc-supplemented and control subjects. RT-PCR oligonucleotide primers were designed to amplify both a 201 bp segment of the human MT cDNA and a 180 bp competitor cDNA template. The 180 bp competitor cDNA template was used for MT cDNA quantitation. The RT-PCR data show that there was a significant increase in monocyte MT mRNA in subjects within 6 d of zinc supplementation, which remained elevated at d 15 of supplementation. In contrast, plasma zinc was greater at d 6 of zinc supplementation, but by d 15 of supplementation, while still elevated, was close to control levels. These data suggest that monocyte MT mRNA levels respond to zinc supplementation and that the response could serve as a more useful assessment variable than plasma zinc for the measurement of zinc status in humans.

Tolerance induced by all-trans-retinol to the hepatotoxic effects of cadmium in rats: role of metallothionein expression.

Recently, it has been shown that large doses of all-trans-retinol (vitamin A) can potentiate the hepatotoxicity of several organic chemicals in the rat. Whether retinol pretreatment can alter the acute hepatotoxicity of an inorganic chemical, such as cadmium, is unknown. Therefore, the objective of this study was to determine how retinol might affect the acute toxicity of cadmium chloride (CdCl2) and to elucidate possible mechanisms. Cadmium exposure can induce acute, lethal hepatocellular necrosis in rodents, as well as lesions in the lung, kidney, testis, and gastrointestinal tract. In the present studies, male Sprague-Dawley rats were pretreated with retinol (75 mg/kg/day, po) for 7 consecutive days. One day after the last dose of retinol, animals were given a single injection of CdCl2 (2.5 to 4.0 mg/kg, iv). Cadmium chloride administration to unpretreated control rats caused extensive hepatic, renal, pulmonary, and testicular toxicity at 6, 24, and 48 hr postdosing as evaluated by plasma enzymes and/or histopathology. In retinol-pretreated rats, a significant attenuation of CdCl2-induced tissue injury was observed. Since the inducible cadmium-binding protein metallothionein (MT) is often an essential aspect of cadmium tolerance, its content in tissue was assessed using the cadmium-hemoglobin assay. Interestingly, retinol pretreatment significantly increased MT in the liver by sevenfold, but had no effect on lung, kidney, testicular, or pancreatic MT content. Although this increase in hepatic MT was much less than that induced by CdCl2, it was additive to the induction of CdCl2. Furthermore, the tissue distribution of cadmium was significantly altered by retinol pretreatment. The liver accumulated more cadmium, while less cadmium was found in the lung, kidney, and testis in retinol-pretreated rats than in controls. In monolayers of primary isolated hepatocytes, CdCl2-induced toxicity was significantly reduced in cells isolated from retinol-pretreated rats compared to those isolated from control rats. The dose response was shifted to the right and the in vitro cadmium LC50 was increased by in vivo retinol exposure from 1.1 +/- 0.1 to 2.4 +/- 0.04 microM. From these data it is concluded that the induction of hepatic MT is an essential aspect of retinol-induced tolerance to CdCl2 hepatotoxicity, as well as toxicity in other tissues.

Characterization of calf liver Cu,Zn-metallothionein: naturally variable Cu and Zn stoichiometries.

Cu,Zn-metallothioneins were purified from bovine calf liver in order to examine the stoichiometry of metal binding to the protein. Copper and zinc analyses were carried out by atomic absorption spectrophotometry. Consistent quantitative thiolate analyses were obtained spectrophotometrically with Ellman's reagent and amperometrically with phenylmercuric acetate. These were used to define protein concentration. A complementary method to assess the sum of the thiol and Cu(I) content of metallothionein involved titration of the reducing equivalents of the protein with ferricyanide. The stoichiometry of reaction was consistent with the oxidation of all the sulphydryl groups to disulphides and all of the bound Cu from the cuprous to the cupric oxidation state. Accordingly to these methods, total numbers of zinc plus copper ions bound to metallothionein isolated from a number of calf livers centred on about 7, 10-12, or 15 g-atoms of metal per mol of protein. The reactivity of ferricyanide and 4,7-phenylsulphonyl-2,9-dimethyl-1, 10-phenanthroline (BCS) with Cu,Zn-metallothioneins of various metal ratios was assessed. Zinc metallothionein reacted almost entirely in two slow steps with ferricyanide. As the Cu content of the protein increased, the fraction of reaction occurring in the time of mixing increased in parallel. BCS was able to remove 70-80% of metallothionein-bound Cu as Cu(I). The rest was resistant to reaction.