Hepatic Metallothionein Levels Research Articles

Induction of metallothionein by exposure to normobaric 100% oxygen atmosphere in rats with different zinc supply

The objective of this study was to determine the effects of an oxygen enriched environment on the induction of the metalloprotein metallothionein (MT) and its relation to zinc metabolism in rats supplied with different levels of dietary zinc. Male albino rats were fed purified diets based on maize starch, egg white, saccharose and soybean oil differing in the concentration of zinc (1; 20; 100; 500 mg Zn/kg diet). At a dietary zinc supply of 1 mg/kg, the rats developed a zinc deficiency indicated by visual and biochemical parameters. At the end of the 37-day feeding period, half of the rats were exposed to 100% oxygen for 12 h.The oxygen treatment significantly reduced plasma zinc in the zinc supplemented rats and reduced it in tendency in the zinc deficient rats. The MT concentration was increased in the zinc supplemented groups in the liver, kidney and lung. The oxygen treatment elevated the metallothionein concentration in the two high zinc supplemented groups (100 and 500 mg Zn/kg diet) in the liver. The response of the zinc concentration in plasma and of hepatic metallothionein levels to oxygen exposure indicates a role of metallothionein in zinc distribution or interactions with other trace elements to support antioxidant capacity, rather than an impact on direct scavenging activity of free radicals.

HEPATOPROTECTIVE EFFECTS OF CYPROTERONE ACETATE IN MICE; MINIMAL ROLE OF METALLOTHIONEIN

Previously, we reported that the antiandrogen cyproterone acetate (CA) modifies cadmium (Cd) distribution and metallothionein (MT) induction in mice given a non-toxic dose of Cd (10 w mol Cd/kg, sc). We have also observed that CA pretreatment will reduce Cd toxicity in in vitro systems. Thus, since the liver is a primary target of acute Cd toxicity, the effects of CA pretreatment on the acute hepatotoxicity of Cd in vivo were studied in male C57 mice. Mice were pretreated with CA (10 mg/kg, sc, at - 48, - 24, and 0 h) prior to Cd (35 or 45 w mol Cd/kg, at 0 h). Cd alone (35 or 45 w mol Cd/kg) induced significant increases in serum alanine aminotransferase (ALT) activity, indicative of hepatotoxicity, 24 h after injection. However, CA pretreatment either prevented (at 35 w mol Cd/kg) or significantly reduced (at 45 w mol Cd/kg) Cd-induced increases in serum ALT. Based on serum creatinine levels, Cd alone was not acutely nephrotoxic. Thus, CA pretreatment substantially reduced the hepatotoxic effects of Cd. CA pretreatment had no effect on hepatic Cd levels 24 h after Cd exposure. The hepatic levels of zinc, a metal known to antagonize Cd toxicity, were modestly altered by CA pretreatment, but not in a consistent fashion. Cd alone markedly increased hepatic MT, a metal-binding protein often associated with tolerance to Cd, 24 h after exposure. CA pretreatment alone did not alter hepatic MT levels and pretreatment with CA before Cd did not alter hepatic levels of MT compared to Cd alone 24 h after injection of the metal. To further examine the tolerance to Cd induced by CA, the effects of CA pretreatment on hepatic Cd levels at early stages were investigated. Hepatic Cd levels were significantly decreased by CA pretreatment 8 h after Cd treatment and returned to control levels by 16 h. These results indicate that CA can substantially reduce the hepatotoxic effects of Cd in C57 mice without activating MT synthesis. The early decreases in liver Cd content may be critical to decreasing the adverse effect of Cd in the liver. Thus, it appears CA induces tolerance to Cd through altered biokinetics and in a manner not involving the MT system.

Kinetics and magnitude of metallothionein induction by Cd, Cu, Hg and Zn in European flounder — calibration for environmental monitoring

The inductive response of metallothionein (MT) synthesis to heavy-metal exposure in many Phyla is well documented; however, before this response can be used and interpreted as an environmental biomarker of exposure, the kinetics and dose responses must be known. We have carried out a series of experiments where we have studied these parameters of MT mRNA and MT protein induction in different tissues (liver, kidney, gills) of European flounder after intraperitoneal (ip) injection of Cd (1–320 μg/kg), Cu (25–200 μg/kg), Hg (235–100 μg/kg) and Zn (1–20 mg/kg). Basal hepatic levels of 320 μg MT/g wet tissue for the wild caught flounder used were five times higher than in fish from a pristine area and 10–20 times higher than in plaice or turbot obtained from pristine areas. Transient increases in renal MT mRNA levels were only observed 4–10 days post injection (p.i.) of Cu or Hg and there were no significant increases in MT protein levels in any tissue. Doses of >5 mg/kg Zn induced MT mRNA levels three- to four-fold in all tissues 2–4 days p.i. but only produced a transient elevation in MT levels at 4 days p.i. There was no measurable response to low doses of Cd, doses of >80 μg/kg Cd had no significant effect on hepatic MT levels but caused a significant elevation in gill and kidney levels 10 and 21 days p.i. MT mRNA levels in gill and kidney were induced four- to five-fold 24 h p.i. of Cd (and remained elevated for >10 days) whilst a response was only observed in liver (two-fold) 4 and 10 days p.i. These laboratory results, obtained with a wild population exhibiting moderately elevated basal MT levels indicate that the response to metal exposure is too small and too highly dependent upon the metal and exposure time to provide a robust biomarker of contaminant effect. This supports our previous observation of analyses of flounders from the southern North Sea and Dutch estuaries and would indicate that caging studies with ‘ultra-clean’ animals would be required for any monitoring studies.

Ecotoxicological assessment of persistent organic and heavy metal contamination in Hong Kong coastal sediment.

The aim of the present project is to determine the feasibility of measuring hepatic cytochrome P4501A1 (CYP1A1) and metallothionein (MT) mRNA in fish as an integrative measurement of persistent organic pollutants (POPs) and heavy metal contamination in sediment arising in Hong Kong. Sediment samples were collected from different sites, including Victoria Harbour (VS6), Yim Tin Tsai (YTT) at Tolo Harbour, Mai Po marshes (MPM) at Deep Bay, and Southern Waters (SS6) of coastal waters. The samples were analyzed for total and extractable concentrations of Cd, Cu, Ni, Zn, and Pb, as well as PCBs and PAHs. In addition, biomarker responses were studied in tilapia exposed experimentally to coastal sediment for 7 days. Using RT-PCR technique, hepatic CYP1A1 and MT mRNA were measured. Three control groups were used, including one negative control group maintained in sea water only; the second and third positive control groups were in sea water but were intraperitoneally injected with either beta-naphthoflavone (40 microg/g body weight) or cadmium chloride (10 microg/g body weight), respectively. The chemical data showed that VS6, YTT, and MPM were classified as Class C sediment according to the sediment quality criteria defined by the Hong Kong Environmental Protection Department, indicating the sites were heavily polluted. The exposure of tilapia to the sediment induced hepatic CYP1A1 (VS6 > YTT > MPM > SS6) and MT (VS6 > MPM > YTT > SS6) levels. The induction patterns were comparable to the levels of POPs and metal contamination in the sediment, indicating that the biomarker responses could be used to differentiate low to high levels of contamination among sediment.

Metallothionein-Null Mice Express Altered Genes during Development

Metallothionein (MT) can modulate transcriptional activity in vitro. We examined whether the absence of MT affects gene expression in vivo. We compared the hepatic RNA profiles of wild-type and MT-null neonatal mice using improved differential display. The hepatic MT level was maximal during neonatal development. We identified five cDNA fragments that were expressed in MT-null mice at different levels from those in wild-type mice. Two were fragments of MT-I and mutant MT-I cDNA. The sequences of the other cDNA fragments were identical to those of contrapsin, transketolase, and vanin-3. The latter two were up-regulated, whereas contrapsin was down-regulated in neonatal MT-null mice. These mRNA levels were remarkably different between the two strains of neonatal mice. Further characterization of the regulated mRNA identified here will determine whether or not they are primary or secondary effects of an MT deficiency.

Effect of surgically induced cholestasis on the levels of hepatic zinc and metallothionein in rat liver.

Early effects of experimental cholestasis on the homeostasis of zinc (Zn) and metallothionein (MT) were studied in rats which had undergone bile duct ligation for 0, 3, 6, 9, 12, 16, 20, and 24 h. Transient increases in hepatic Zn levels were observed at 9 h but returned to control values at 12 h. Serum Zn levels increased at 24 h. Cholestasis was confirmed by increased serum alkaline phosphatase (AP) activity. MT increased at 3 h and reached a maximum level at 12 h and remained elevated even at 24 h after the onset of experimental cholestasis. No hepatocellular damage was detected according to the results of alanine aminotransferase (ALT) activities in serum. These results shown that the increases in Zn observed in liver are related to bile stagnation rather than to a hepatocellular damage and that increased MT occurs concurrently with increased hepatic Zn. These observations suggest that the cellular levels of Zn in cholestasis is regulated by homeostatic mechanisms, of which one could be mediated by MT.

Effects of N-nitrosodimethylamine (NDMA) on the oxidative status of rat liver.

To investigate oxidative effects of N-nitrosodimethylamine (NDMA) on the liver, rats were challenged by the reagent with a dose range of 10 to 40 mg/kg. With lower dose levels, protective responses were prominent, such as elevation of the hepatic glutathione and metallothionein (MT) levels. Increased activities were also evident of gamma-glutamylcysteine synthetase, glucose-6-phosphate dehydrogenase (G6PD), and malic enzyme. In the high dose range, however, toxic responses, such as increases in lipid peroxide levels in liver and serum, and glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and ketone bodies in serum became marked. Some of the protective responses became less marked at the highest dose. Catalase and glutathione peroxidase activities in the liver were also inhibited by NDMA treatment. On the other hand, when NDMA was injected as a series of doses (10 mg/kg on four separate occasions), the effects were less marked, and the hepatic levels of MT and lipid peroxide remained unchanged even after the 4th injection. Only the increase in G6PD activity was more marked after four times repeated injection than after a single injection. These results suggest that oxidative and hepatotoxic effects of NDMA are more moderate when given in repeated doses than in a single dose. In contrast to the liver, elevation of MT levels was the only detectable change in the kidney.

Alterations in Physiological Parameters of Rainbow Trout (Oncorhynchus mykiss) with Exposure to Copper and Copper/Zinc Mixtures

Rainbow trout (Oncorhynchus mykiss) were exposed to sublethal concentrations of copper (Cu, 14 μg/liter) and zinc (Zn, 57 and 81 μg/liter) for a 21-day period. The four treatments included a control, a Cu control, a Cu and low-Zn treatment and a Cu and high-Zn treatment. Selected parameters [e.g., hemoglobin (Hb), hematocrit (Hct), plasma glucose, lactate and cortisol, differential leukocyte count, respiratory burst, tissue metal concentrations, hepatic metallothionein (MT), brain acetylcholinesterase (AChE)] were evaluated at 2, 7, 14, and 21 days of exposure. Whole blood and plasma parameters were not altered by exposure to metals. The percentage of lymphocytes was consistently decreased in the three metal treatments, while percentages of neutrophils and monocytes were increased. Respiratory burst activity was elevated in all metal treatments. Gill Zn concentration was highly variable, with no significant alterations occurring. Gill Cu concentration was elevated above control levels in all metal treatments. Gill Cu concentration in the two Cu/Zn treatments was also elevated above levels in the Cu control. Hepatic metal concentrations and MT levels were not altered from control values. Measurements of brain AChE indicated an elevation in this parameter across metal treatments. In general, alterations in physiological parameters appeared to be due to Cu, with Zn having no interactive effect.

Metallothionein Induction in Rat Brain after Intrastriatal Injection of Zinc and Cadmium Salts(PROCEEDINGS OF 24TH SYMPOSIUM ON TOXICOLOGY AND ENVIRONMENTAL HEALTH)

Brain parenchyma is protected against excess metals by the barrier system in the brain. To evaluate the expression of metallothionein (MT), a protective protein against heavy metals, in the brain parenchyma, zinc sulfate (0.2 or 2μmol) or cadmium chloride (2 or 20nmol) was injected into the left striatum of rats. Seventy-two h later, the MT level in the ipsilateral striatum injected with 0.2μmol of zinc sulfate was not significantly higher than that after injection with vehicle. When the striatum was injected with 2μmol of zinc sulfate, on the other hand, the MT level in the ipsilateral striatum, showing apparent degeneration, was significantly higher than that after injection with vehicle : the former was approximately 1.5 times the latter. In the case of injection with cadmium chloride at doses of 2 and 20nmol, the MT levels in the ipsilateral striatum was approximately twice that after injection with vehicle. The MT level in the contralateral striatum and other brain regions were not affected by injection with either metal salt at any dose. When zinc sulfate of 100μmol/kg body weight, corresponding to the lower dose tested(0.1μmol/g brain), was subcutaneously injected, the hepatic MT level was approximately four times higher than the normal hepatic level. These results suggest that the changes of MT level in the brain were small compared to those observed in the liver.

Genetic component in the inflammatory response induced by bacterial lipopolysaccharide.

Multiple organ dysfunction syndrome (MODS) appears to be the result of a complex program influenced by multiple factors, including environmental, physiological, and immunological conditions. Thus, an uncontrolled inflammatory response following a stochastic event, the initial injury, is believed to be the cause for the development of this syndrome. Several lines of evidence suggest that a genetic component could contribute to the regulation of the inflammatory response, as well, but no direct evidence demonstrates a heritable predisposition to MODS. In the present study, a genetic contribution was demonstrated for the inflammatory response induced by the administration of bacterial lipopolysaccharide (LPS) in different, genetically distinct strains of inbred mice. A survey of five inbred strains showed that mortality following administration of Escherichia coli LPS (20 mg/kg) was highest in C57BL/6J (B6) mice, while A/J mice were the most resistant. Accordingly, B6 and A/J mice were examined further for differences in the inflammatory response elicited by LPS. B6 mice showed higher levels of circulating interleukin-1beta and interleukin-6, as well as higher mRNA levels of hepatic beta-fibrinogen (an acute-phase gene) and metallothionein. Surprisingly, the circulating levels of tumor necrosis factor-alpha were significantly higher in A/J than in B6 mice after LPS administration. Since B6 and A/J mice were bred and raised in identical environments and received the same LPS challenge, the contrasting inflammatory response that was observed is largely attributable to genetic differences between these two strains. These data illustrate that the response to injury could be modulated by the genetic background of the individual. This information may be pertinent for the care of critically ill patients.

Developmental changes in hepatic metallothionein, zinc, and copper levels in genetically altered mice

Using mice that either overexpress metallothionein 1 (MT-1*) or do not express metallothionein 1 and 2 (MT-null) and a control strain (C57BL/6), the essential metal storage function of hepatic metallothionein and its subcellular localization were investigated during development. Hepatic metallothionein, zinc, and copper levels were measured in all groups from gestational day 20 to 60 days of age. Hepatic metallothionein levels were maximal during the perinatal period in both MT-1* and C57BL/6 mice with levels approximately three times higher in MT-1* mice. MT-null mice had no detectable hepatic metallothionein throughout development. Hepatic zinc levels were highest in the neonatal period of MT-1* and C57BL/6 mice and declined to adult levels by 30 days of age, while hepatic zinc levels in MT-null mice did not vary markedly throughout development. Hepatic copper profiles were very similar in MT-1* and MT-null mice as compared with the C57BL/6 mice. Correlation analysis showed a strong positive correlation between hepatic metallothionein and zinc levels in MT-1* mice, moderate correlation between hepatic metallothionein and metals in C57BL/6 mice, but only a very weak correlation between hepatic metallothionein and copper levels in MT-1* mice. Immunohistochemical localization showed specific nuclear staining in both MT-1* and C57BL/6 mice during the neonatal period with a gradual shift to the cytoplasm. The results show that hepatic metallothionein is a major determinant of zinc but not copper levels during murine development. Additionally, hepatic metallothionein levels and localization are regulated in a similar manner in MT-1* and C57BL/6 mice. The MT-null mice maintain a basal level of zinc sufficient for development, which was found to be 15.9 mug/g. This value was similar to the levels of hepatic zinc that was not bound to metallothionein in MT-1* and C57BL/6 mice during development.Key words: metallothionein, zinc, copper, development, immunohistochemistry.

Role of placental metallothionein in maternal to fetal transfer of cadmium in genetically altered mice

The role of placental metallothionein (MT) as a barrier for maternal to fetal transfer of cadmium (Cd) was investigated using mice which overexpressed the MT-1 isoform (MT-1*), mice which did not express the MT-1 and 2 isoforms (MT-null) and control mice (C57BL/6). In addition, immunohistochemical localization of MT in the placenta was determined in these mice. Two days prior to parturition, the mice were injected with radioactive 109Cd chloride (4 μCi, 0.6 ng Cd/mouse) and killed 24 h later. Organs and fetuses were collected and radioactivity, MT and metal levels were measured. Cd accumulated mainly in the liver and kidney (80% of administered dose) with very low levels (0.1–0.3%) detected in fetuses. When analyzed on a per organ or per gram basis, MT-null fetuses accumulated significantly more Cd (3–10-fold) than the control fetuses and there was no significant difference in fetal Cd accumulation in the MT-1* and control fetuses. As expected, MT and zinc levels were higher in MT-1* than C57BL/6 mice and no MT was detected in MT-null mice. Most striking was the high hepatic MT levels in MT-1* dams (4 mg/g). Immunohistochemical analysis showed MT staining in spongiotrophoblasts, glycogen cells, visceral yolk sac, trophoblast giant cells and maternal decidual cells with the MT-1* placenta staining much more intensely as compared to control placenta. The results suggest that placental MT reduces maternal to fetal Cd transfer, however the low doses of Cd administered in the present experiment resulted in high levels of Cd accumulation in liver and kidney in all groups of mice with a low concentration of Cd reaching the placenta. Thus, the role of placental MT as a barrier for Cd is inconclusive.

Developmental changes in hepatic metallothionein, zinc, and copper levels in genetically altered mice.

Using mice that either overexpress metallothionein 1 (MT-1*) or do not express metallothionein 1 and 2 (MT-null) and a control strain (C57BL/6), the essential metal storage function of hepatic metallothionein and its subcellular localization were investigated during development. Hepatic metallothionein, zinc, and copper levels were measured in all groups from gestational day 20 to 60 days of age. Hepatic metallothionein levels were maximal during the perinatal period in both MT-1* and C57BL/6 mice with levels approximately three times higher in MT-1* mice. MT-null mice had no detectable hepatic metallothionein throughout development. Hepatic zinc levels were highest in the neonatal period of MT-1* and C57BL/6 mice and declined to adult levels by 30 days of age, while hepatic zinc levels in MT-null mice did not vary markedly throughout development. Hepatic copper profiles were very similar in MT-1* and MT-null mice as compared with the C57BL/6 mice. Correlation analysis showed a strong positive correlation between hepatic metallothionein and zinc levels in MT-1* mice, moderate correlation between hepatic metallothionein and metals in C57BL/6 mice, but only a very weak correlation between hepatic metallothionein and copper levels in MT-1* mice. Immunohistochemical localization showed specific nuclear staining in both MT-1* and C57BL/6 mice during the neonatal period with a gradual shift to the cytoplasm. The results show that hepatic metallothionein is a major determinant of zinc but not copper levels during murine development. Additionally, hepatic metallothionein levels and localization are regulated in a similar manner in MT-1* and C57BL/6 mice. The MT-null mice maintain a basel level of zinc sufficient for development, which was found to be 15.9 micrograms/g. This value was similar to the levels of hepatic zinc that was not bound to metallothionein in MT-1* and C57BL/6 mice during development.

On-line solid-phase extraction–capillary electrophoresis for enhanced detection sensitivity and selectivity: application to the analysis of metallothionein isoforms in sheep fetal liver

A simple and rapid capillary electrophoresis (CE) method for analysis of metallothionein (MT) isoforms is described. A modified two-step solvent extraction procedure was used in combination with CE and an on-line solid-phase pre-concentration device for sensitive and reproducible detection of MT isoforms in sheep fetal liver. Preparation of twenty samples was practicable within a working day with subsequent automated overnight analysis by solid-phase extraction (SPE)–CE. A commercially available divinylbenzene-based reversed-phase resin was found to be most suitable for the SPE device because it is resistant to extremes of pH and can be adequately regenerated between analyses. Each SPE device was readily constructed from commonly available materials and was used for the reproducible separation of over 100 biological samples before replacement. Precision of analysis within or between sample batches was <10% and usually <5% while detection limits were at least 28 ng/ml for standards and 272 ng/ml for biological samples. This would indicate a detection limit of about 0.5 μg/g wet weight of tissue. Recovery of MT from tissue cytosols by solvent extraction was measured using radiolabeled MT and was found to be just over 50% increasing to almost 70% by addition of NaCl to the homogenisation buffer. The combined solvent extraction and SPE–CE methodology was applied to the analysis of MT in sheep fetal liver and the results compared favorably with those obtained by high-resolution chromatography. MT-1 levels were 2 to 4-times higher than those of MT-2 and both isoforms decreased from day 89 to day 136 of gestation. These results were compared with MT levels in fetal liver from sheep embryos that had been perturbed by temporary transfer to an advanced uterine environment. Hepatic MT levels at day 136 of gestation were 3 to 8-times higher than in normal fetal liver and significant differences were observed with both isoforms.

Metallothionein-independent hepatoprotection by zinc and sakuraso-saponin

Hepatoprotective activities of zinc and sakuraso-saponin against toxicity of carbon tetrachloride were investigated in metallothionein (MT)-deficient mice. Pretreatment of control 129/Sv mice with zinc or sakuraso-saponin blocked carbon tetrachloride-induced elevation of plasma transaminase activities. Quantitatively equivalent protection against carbon tetrachloride-induced hepatic damage was also observed in MT-deficient mice. Zinc and sakuraso-saponin caused elevation of hepatic MT levels in control 129/Sv mice, whereas hepatic MT was undetectable in MT-deficient mice. To examine the possibility that sakuraso-saponin-induced hepatoprotection is mediated by endogenous zinc, the hepatic concentration of zinc was analyzed. Hepatic zinc concentration in MT-deficient mice was not changed by the treatment of sakuraso-saponin. Injection of sakuraso-saponin caused a decrease of activity of aniline hydroxylation. The suppression of cytochrome P450 appears to be a mechanism by which sakuraso-saponin protects mice from the hepatotoxic effects of carbon tetrachloride. These findings indicate that the hepatoprotective activity of zinc or sakuraso-saponin is not dependent on their MT-inducing activity.

Repeated administration of alpha-hederin results in alterations in maternal zinc status and adverse developmental outcome in the rat.

The administration of alpha-hederin, an inducer of metallothionein, results in a secondary zinc deficiency that may be an important maternally mediated mechanism of developmental toxicity. Previous studies have shown adverse developmental outcome with a single administration of alpha-hederin to rats on gestation day (GD) 8 or 11. The objective of this study was to determine whether dosing of alpha-hederin throughout organogenesis would result in a sustained elevation of maternal hepatic metallothionein and subsequent developmental abnormalities. Rats were administered dosage levels of 0 (vehicle only), 20, or 30 mumol/kg from GD 6-15. Maternal hepatic metallothionein levels were 10-fold higher on GD 16 in the treatment groups than the controls. Consequently, liver zinc concentrations increased 60% and 54%, whereas plasma levels decreased 23% and 33% in the 20 and 30 mumol/kg treatment groups, respectively. At GD 20, mean fetal weights of the treatment litters were 11% less than control litters. The administration of alpha-hederin resulted in a threefold increase in the number of offspring that exhibited developmental abnormalities, including visceral and skeletal malformations. Following an oral pulse of 65Zn subsequent to treatment with 0 or 20 mumol/kg of alpha-hederin, the distribution of 65Zn to the liver of treated dams was twice that of controls, whereas the radiolabeled zinc apportioned to the decidua and uterus decreased by 44%. Furthermore, the 65Zn detected in the embryos from treated dams was 70% lower than in embryos from control dams. In conclusion, low doses of a metallothionein inducer administered to the dam from GD 6-15 resulted in a sustained elevation of hepatic metallothionein and a subsequent redistribution of zinc leading to a decrease in the zinc available to the embryo and ultimately to adverse development of the offspring. Repeated dosing throughout organogenesis, as required in regulated safety assessment testing, increased the severity of the effects previously observed with single large dosages of the toxicant administered during midgestation.

Lack of correlation between the inducibility of metallothionein mRNA and metallothionein protein in cadmium-exposed rodents

Cadmium (Cd) is carcinogenic in humans and laboratory animals. Depending on the duration and route of exposure, Cd can also induce damage in the liver, kidneys and lungs. In certain tissues, metallothionein (MT) proteins are induced by Cd exposure and associated with native and acquired tolerance to the metal. Rats are generally more sensitive than mice to Cd carcinogenicity; however, sensitivity can vary markedly between different strains of the same rodent species. To further define the role of MT in Cd toxicity and Carcinogenesis, adult male Wistar rats and adult male C57 and DBA mice were treated with CdCl 2 and liver, kidney, and lung were analyzed for Cd, MT mRNA, and MT protein 24 h later. Dose-related increases in Cd were detected in the livers and kidneys of all animals tested; however, increases in pulmonary Cd were observed only in C57 mice, and only at the highest CdCl 2 dose. While hepatic Cd concentrations were similar in the rats and mice, renal Cd concentrations were similar in the rats and DBA mice but were nearly 2-fold higher in C57 mice at the highest CdCl 2 dose. Dose-related increases in MT mRNA occurred in the livers and lungs of all animals tested. Hepatic MT mRNA concentrations were highest in the rats, and C57 mice exhibited the greatest magnitude of hepatic MT mRNA induction. Dose-related increases in renal MT mRNA were also detected in both strains of mice, but between the two strains, C57 mice exhibited substantially higher levels of renal MT mRNA induction. Basal levels of renal MT mRNA were higher in the rats than in the mice, and transcription of the MT gene was not inducible in the rat kidney at any of the CdCl 2 doses used. In comparison, basal levels of pulmonary MT mRNA were similar in the rats and DBA mice, were substantially lower in C57 mice, and increases in pulmonary MT mRNA were most pronounced in the rats. Analysis of MT protein revealed dose-related increases in the livers and kidneys of all animals tested. C57 mice had the lowest basal and induced levels of hepatic MT, and basal levels of renal MT were much higher in the rats than in mice of either strain. Although dose-related increases in pulmonary MT were similar in both strains of mice, pulmonary MT levels were much lower and not inducible in the rats. Overall our experiments revealed complex profiles of Cd distribution and MT expression that varied between tissues, species and strains, and often did not directly correlate with sensitivity to damage. The results suggest that Cd distribution, inducibility of the MT gene, and levels of MT protein, must all be considered when predicting susceptibility to Cd toxicity and carcinogenicity at particular target sites.

Sequence of exposure to cadmium and arsenic determines the extent of toxic effects in male Fischer rats

Arsenic and cadmium are both priority hazardous substances and human carcinogens. Although there is the potential for simultaneous exposure to both metals, the interactions of cadmium and arsenic are not well defined. We examined the toxicity of these metals when given alone or in alternating sequence to adult male Fischer rats. In the first study, a non-toxic dose of arsenic (22.5 μmol NaAsO2/kg s.c.) was given 24 h before cadmium (10, 20, or 30 μmol CdCl2/kg, s.c.) and toxicity was assessed 24 h later. Arsenic pretreatment markedly reduced mortality in rats given the high dose of cadmium (9 survivors/10 treated) compared to rats given cadmium alone (210). Arsenic pretreatment also reduced cadmium-induced hepatotoxicity, as indicated by serum glutamic oxalacetic transaminase (SGOT) activity, and markedly reduced cadmium-induced testicular hemorrhagic necrosis. Arsenic pretreatment produced an 8-fold increase in hepatic levels of metallothionein (MT), a metal-binding protein often associated with cadmium tolerance. In the second study, a non-toxic dose of cadmium (3 μmol CdCl2/kg s.c.) was given 24 h before arsenic (68, 79, 84, or 90 μmol NaAsO2/kg s.c.) and toxicity was assessed 24 h later. Cadmium pretreatment did not alter the lethality of the high dose of arsenic and had no effect on arsenic-induced hepatotoxicity. Although cadmium pretreatment had no effect on arsenic toxicity, it produced large increases in hepatic MT (26-fold) before the arsenic challenge and greatly enhanced MT induction after the challenge. Thus, even though both arsenic and cadmium induce MT synthesis, only arsenic pretreatment protects against cadmium intoxication, and cadmium pretreatment does not effect arsenic toxicity. Thus, toxic interactions of arsenic and cadmium appear to depend on the sequence of exposure.

Interaction of benzo[a]pyrene, 2,3,3′,4,4′,5 hexachlorobiphenyl (PCB 156) and cadmium on biomarker responses in flounder (Platichthys flesus L.)

Interactive effects of a mixed pollutant exposure on biomarker responses were studied in European flounder (Platichthys flesus L.). The model chemicals, benzo[a]pyrene (BaP, 2.5 mg kg-1), 2,3,3′,4,4′5 hexachlorobiphenyl (PCB-156, 2.5 mg kg-1), and cadmium (cadmium, 1 mg kg-1), were administered to fish by subcutaneous injections. Biomarker responses were quantified both following administration of single chemicals and sequential combinations of the chemicals in pairs. Significant induction of CYP1A protein levels and corresponding ethoxyresorufin-O-deethylase (EROD) activities was observed in BaP and PCB treated flounder after 2 and 8 days, respectively. The strongest induction (44 fold) was caused by BaP. No further induction was observed after additional treatment with PCB 156. CYP1A induction caused by BaP was inhibited (40% compared with BaP treatment alone) in flounder pre treated with cadmium, whereas induction by PCB 156 appeared to be unaffected by pre treatment with cadmium. Flounder treated with cadmium only had significantly elevated hepatic levels of metallothionein (MT) after 15 days. Pre treatment with BaP and PCB prior to cadmium inhibited the MT induction (30-50%) compared with cadmium alone. Furthermore, significantly higher glutathione S transferase activities were observed in flounder administered cadmium alone, and in flounder treated with BaP or PCB 156 prior to cadmium. GST selenium independent peroxidase activities appeared to be unaffected by any of the treatments in the present study. The results indicate that chemical mixtures may affect biomarker responses differently from compounds administered alone, and that the sensitivity of both CYP1A and MT are influenced by pollutants other than their primary inducers.

Retrorsine in breast milk influences copper handling in suckling rat pups

Aims: To explore the hypothesis that a second xenobiotic agent is required with excess copper to produce Indian Childhood Cirrhosis, this study investigated the effect of the pyrrolizidine alkaloid retrorsine fed to the mother during the suckling period upon the serial changes in neonatal copper status. Methods: Female Wistar rats with new-born litters were fed either a control or a retrorsine (50 mg/kg) diet. At 0, 4, 8, 11, 15, 18 and 21 days, pups from each litter were weighed, sacrificed and their livers removed for copper, DNA and metallothionein analysis. Serum samples were assayed for caeruloplasmin oxidase activity and albumin. Results: 1) Higher than adult level of hepatic copper in normal rats which rose post-natally before declining from day 11 after birth, 2) raised hepatic copper concentrations and total copper in the retrorsine group from day 15; levels were higher than adult at birth, 3) reduced serum caeruloplasmin oxidase activity and albumin levels in retrorsine group, but both groups lower than adult, 4) lower hepatic metallothionein levels in retrorsine group, but both groups higher than adult, and 5) reduced liver DNA in the retrorsine group when expressed as total DNA and per gram of tissue. These changes were not secondary to under-nutrition as a small study on under-nourished rat neonates showed that copper handling is not significantly altered when compared to well-nourished rats. Conclusions: Retrorsine passing to rat neonates via breast milk causes: 1) the accumulation of hepatic copper, 2) impairment of the rise in serum caeruloplasmin, which could indicate a decline in synthesis or failure of copper incorporation into the apo-protein, 3) a decrease in hepatic metallothionein and serum albumin levels, again suggesting diminished protein synthesis, and 4) reduced hepatic DNA indicative of decreased cell number but increased cell size. Accumulation of liver copper but reduction of copper-binding proteins could result in free copper and explain the synergistic hepatotoxicity of copper and retrorsine.