Hepatic Metallothionein Concentrations Research Articles

Metallothionein content increased in the liver of mice exposed to magnetic fields.

Although recent studies have shown that various stress can induce metallothionein (MT) synthesis in animal tissues, the induction of MT synthesis by exposure to static magnetic fields (SMF) has not been reported. We measured MT levels in the liver, kidney and brain of mice exposed to SMF and also evaluated the effect of SMF exposure on the induction of hepatic MT by treatment with carbon tetrachloride (CCl4). The MT content in the liver was significantly increased by exposure to 4.7 T of SMF for 6, 24, or 48 h, whereas that in the kidney or brain was not changed compared to the control. The combination of CCl4 injection and SMF exposure induced elevation of the hepatic MT content exceeding that induced by either treatment alone. These results indicate that exposure to the strong SMF induces MT synthesis in the liver in mice and enhances the hepatic MT synthesis induced by CCl4 administration.

Metallothionein synthesis induced by interferon alpha/beta in mice of various zinc status.

We studied the ability of interferon alpha/beta (IFN) to induce metallothionein (MT) synthesis in mice. Male mice were injected intraperitoneally with mouse IFN (5 x 10(5) IU/mouse). Plasma Zn levels were reduced at 4 hr after injection, reached a minimum value at 6 hr, and then returned to the control level at 8 hr. Hepatic MT concentrations began to increase at 4 hr and reached maximum values at 6 hr. Induction of MT gene expression and protein synthesis was confirmed by Northern blot analysis and radioimmunoassay, respectively. The induction of MT synthesis in the liver by IFN was dose-dependent. The data suggest that induction of MT-mRNA and the protein in the liver by IFN occurs rapidly but is rather transient. Furthermore, MT synthesis was not induced by IFN in the liver of mice given a Zn-deficient diet, whereas IFN induced increases in the activity of 2',5'-oligoadenylate synthetase in the spleen were unaffected by Zn status. Thus, induction of hepatic MT synthesis by IFN is influenced by Zn status.

Induction of metallothionein synthesis by glutathione depletion after trans- and cis-stilbene oxide administration in rats

To investigate the relationship between glutathione (GSH) depletion and metallothionein (MT) synthesis, the effects of substrates and an inhibitor of GSH S-transferases on concentrations of hepatic GSH, zinc (Zn) and MT were studied in rats. Trans-stilbene oxide (TSO) is an inducer of drug metabolizing enzymes and also a substrate of GSH S-transferase, whereby it covalently reacts with and depletes GSH. The hepatic GSH level was decreased to 25% of the control 2 h after injection of TSO, and returned to the control level by 24 h. TSO significantly increased hepatic concentrations of Zn and MT in a dose-dependent manner. Two isoforms of MT (MT-I and MT-II) were increased by TSO; MT-II was the dominant form. Pretreatment with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, enhanced MT synthesis itself as well as that induced by TSO and cis-stilbene oxide (CSO). On the contrary, injection into rats of perfluorodecanoic acid (PFDA), an inhibitor of GSH S-transferase, resulted in a decrease in basal levels of Zn, and prevented the increase in MT synthesis by TSO and CSO. These results suggest that the decrease of GSH concentration in the liver which causes oxidative stress conditions may be related to MT induction.

Species variation in hepatic metallothionein

Metallothionein (MT) is a low-molecular-weight protein involved in the homeostasis of endogenous metals and in the detoxication of heavy metals. In humans, the levels of hepatic MT have been shown to be up to 100 times the levels found in rat and mouse liver. In order to further investigate this species difference in hepatic MT levels, hepatic MT was quantified in 15 species (human, monkey, dog, cat, cow, pig, sheep, goat, rabbit, chicken, hamster, rat, mice, guinea pig, and frog). Fresh liver was obtained from each species and MT was quantified by 2 different metal-saturation assays. Results from the Cd-heme and Ag-heme assays showed that human, dog, cat, pig, and goat had the highest hepatic MT levels (400–700 μg/g liver). Monkey, cow, and sheep had moderate hepatic MT levels (about 200 μg/g liver), while rodents (mouse, rat, hamster, guinea pig, and rabbit) had low hepatic MT levels (2–10 μg/g liver). Hepatic MT levels in non-mammals (chick and frog) were slightly higher than rodents (about 20 μg/g liver). Sephadex G-75 column elution volumes ranged from 1.7 to 1.8, which implies that MT from all species had approximately the same molecular weight and similar structure. Copper and zinc concentration in the cytosols were measured by atomic absorption spectrophotometry. Dog and cat had the highest levels of Cu (86 and 50 μg/g liver, respectively), and pig and hamster were lowest (about 10 μg/g liver). Human, dog, cat, and goat had the highest levels of zinc (approximately 40–50 μg/g liver) while hamster and guinea pig were lowest (approximately 15 μg/g liver). The results show that there is a marked species difference in hepatic MT concentrations with dog, cat, and human having the highest levels.

N-Hexane-Induced Synthesis of Hepatic Metallothionein Is Mediated by IL-6 in Mouse

The mechanism of metallothionein (MT) synthesis in the liver by n-hexane (HX) was examined. The increased synthesis of MT in the liver by HX was inhibited by dexamethasone pretreatment. Serum IL-6 was increased soon after HX injection, reaching a maximum at 8-16 hr, and then decreased, but neither IL-1 nor TNF was increased. The hepatic MT concentration reached a maximum later than did the serum IL-6 concentration, at 2 days after administration. When the MT synthesis induced by HX was inhibited by dexamethasone pretreatment, the concentration of IL-6 in the serum was suppressed to a very low level. Furthermore, the increase in concentration of hepatic MT and plasma fibrinogen was significantly decreased by the anti-mouse IL-6 monoclonal antibody. The concentration of hepatic MT was higher when the concentration of HX in the olive oil of the solution for injection was higher, even when the amount of HX administered was the same. It is suggested that the cytokine(s) is produced by the macrophage and fibroblast through injury and inflammation at the site of administration. These findings suggest that MT synthesis resulting from HX is induced indirectly through cytokine(s) production, especially IL-6.

Altered Zn status by α-hederin in the pregnant rat and its relationship to adverse developmental outcome

The hypothesis that an acute-phase reaction in the pregnant animal causes a systemic redistribution of Zn, resulting in a transient but developmentally adverse Zn deficiency in the embryo, was tested by treating pregnant rats during organogenesis with α-hederin, an agent reported to induce substantial metallothionein (MT) synthesis in rat liver, and determining hepatic MT concentration, hepatic and plasma Zn concentration, and systemic distribution of a pulse of 65Zn after treatment. Developmental toxicity was assessed by evaluating morphologic development in term fetuses. A single dose of α-hederin, injected sc at dosages of 3 to 300 μmol/kg, caused an acute phase response, indicated by decreased Fe and Zn, and increases Cu, α 1-acid glycoprotein, and ceruloplasmin concentration in plasma, along with a dosage-related increase in maternal hepatic MT concentration. The maximum induction of MT was 11 to 15-fold greater than control and occurred at dosages of 30 μmol/kg and higher, and MT concentration reached its peak 12 to 24 h after treatment. Zn concentration in liver and liver cytosol increased along with MT, reaching a maximum level at dosages of 30 μmol/kg and higher. Plasma Zn concentration decreased after α-hederin treatment to a level approximately 75% of control at a dosage of 30 μmol/kg and 50% of control at 300 μmol/kg. Therefore, hepatic MT induction was associated with most, but not all, of the decrease in plasma Zn concentration. Zn distribution was evaluated by giving an oral pulse of 65Zn 8 h after treatment with 0, 30, or 300 μmol/kg α-hederin on gestation day 11, and measuring 65Zn levels 18 h after treatment. The fraction of 65Zn distributed to the liver of treated rats (either dosage) was twice that of control, but distribution of 65Zn to other maternal tissues was decreased. 65Zn accumulation by conceptuses was significantly decreased, attributable to decreased accumulation in decidua, but not in visceral yolk sacs or embryos; however, at this stage of development the decidua accounts for a greater quantity of Zn than either of the other products of conception and may serve as the Zn-storing tissue for the conceptus. Both 30 and 300 μmol/kg increased resorption incidence, and 300 μmol/kg also decreased fetal weight and increased the incidence of abnormal fetuses. Serum collected from rats two hours after α-hederin treatment (i.e., before the onset of MT synthesis) supported rat embryo development in vitro, whereas serum collected 18 h after treatment did not. Adding Zn to this serum restored normal embryonic development. These data are consistent with the hypothesis that systemic changes in Zn status, brought about by a hepatic acute phase response, including a substantial induction of hepatic MT, may be a mechanism for maternally mediated abnormal development.

Altered maternal zinc metabolism following exposure to diverse developmental toxicants

It has been hypothesized that one mechanism contributing to the developmental toxicity of some xenobiotics is an embryonic/fetal zinc (Zn) deficiency that occurs secondary to toxicant-induced changes in maternal Zn metabolism. We studied the influence of diverse toxicants (urethane, ethanol, melphalan, arsenic, and α-hederin) on maternal-embryonic Zn metabolism and maternal liver metallothionein (MT) induction in Sprague-Dawley rats given a 65Zn-labelled meal by gavage 8 h after toxicant exposure and killed 10 h later on gestation day 12.5. Exposure to the toxicants resulted in increases in maternal hepatic MT concentrations that generally exceeded that which could be accounted for by reductions in food intake. 65Zinc retention was higher in maternal liver and lower in the products of conception in the toxicant-exposed groups. Strong linear relationships were found; as maternal liver MT concentrations increased, 65Zn retention in maternal liver was increased and 65Zn distribution to the conceptuses was decreased. These results support the hypothesis that diverse insults can produce developmental toxicity, in part, by altering maternal and embryonic Zn metabolism.

Cadmium disposition and metallothionein induction in mice: strain-, sex-, age-, and dose-dependent differences

The strain- and sex-related differences in tissue cadmium (Cd) and metallothionein (MT) levels were examined in young adult (4–6 months old) C3H/HeJ, DBA/2J, 129/J, CD-1 and A/J mice, 24 h after a subcutaneous (s.c.) injection of 5–30 μgmmol CdCl 2/kg. In many cases the tissue Cd concentrations were inversely related to the tissue weights. Also, the strain and sex differences were more obvious at 20–30-μmol dose levels. At such doses the hepatosensitive C3H males had as much as 30% higher hepatic Cd concentrations as the resistant DBA males, but similar concentrations to the resistant A/J and CD-1 males. This observation suggests that tissue Cd level is not the only determinant of strain differences in hepatotoxicity in males. Among the females, the A/J had 30–47% higher hepatic Cd concentration than the other strains. Livers of male C3H, CD-1 and 129/J mice had 17–57% higher Cd concentrations than those of the females; the greatest difference was in the C3H strain in which only the males exhibited hepatotoxicity at the 30 μmol dose. In all animals the hepatic MT concentration increased with the increasing Cd concentration. However, the 129/J males and all the females reached a plateau in MT concentration at Cd concentrations of 20–30 μg/g liver. Even at the highest Cd concentration, the C3H males had MT concentrations similar to some of the hepatoresistant males, suggesting that their sensitivity to Cd was not due to compromised MT levels. The renal Cd concentration was similar in males of most strains but not in females. For examples, at the 30-μmol dose 129/J females had a Cd concentration which was 70% higher than in the DBA females. Also, at this dose level the A/J, C3H and 129/J females had 30–50% more Cd than the males. The DBA and C3H males had approximately twice the MT concentration of the A/J and 129/J males at 10 μg Cd/g kidney. At similar Cd concentration, the DBA females also had 1.6–2.0 times the MT concentration of the other females. The renal MT levels in females were 1.4–2.9 times higher than in males. Strains susceptible to the testicular toxicity of Cd had up to three times greater testicular Cd accumulation than the resistant strains. However, there was no increase in testicular MT in any strain. The effect of age on Cd and MT levels was studied in 19-month-old A/J and DBA mice at the 25-μmol dose. The aged mice had 15–42% higher hepatic Cd concentration than the younger animals; however, their MT levels were the same. The renal Cd, but not MT, concentration in the older animals was 25–33% lower than in young animals. Despite the larger body weights the Cd-injected aged males had 22–29% smaller testes, but about the same amount of Cd, as the young males, suggesting that the older DBA males may exhibit testicular toxicity at a lower dose of Cd than the young animals. Thus, it is concluded that not only the dose but also the strain, sex and age of the animals are important factors in determining the disposition of Cd in mice. The differences in the tissue Cd accumulation may relate to variations in hormonal and other intinsic factors affecting the cellular uptake and subcellular distribution of Cd and biosynthesis and degradation of MT.

Mobilization of Hepatic Cadmium in Pregnant Rats

Mobilization of hepatic cadmium (Cd) in pregnant rats was investigated. Female Sprague-Dawley rats (200-250 g) were injected subcutaneously, daily with 1.0 mg Cd/kg body weight as CdCl2 for 8 days. A group of these rats was made pregnant. Copper (Cu), zinc (Zn), Cd, and metallothionein (MT) concentrations in the liver, kidney, and plasma of the control and Cd-injected, pregnant and nonpregnant rats, were compared. The hepatic Cd concentration of the Cd-injected rats decreased by 40% during pregnancy and became significantly lower than that of the nonpregnant Cd-injected rats. On the other hand, there was a concomitant increase (60%) of Cd concentrations in the kidney of the Cd-injected pregnant rats. MT in the Cd-injected rats also showed a similar pattern of decrease in hepatic concentrations and increase in renal concentrations during pregnancy. Both Cd and MT contents in the placenta of the Cd-injected rats were higher than those of the control and there was a significant increase over the gestational period. Plasma Cd and MT concentrations of the Cd-injected pregnant rats were higher than those of Cd-injected nonpregnant rats. These results suggest that pregnancy can mobilize the hepatic Cd which can be transferred to the kidney and placenta through the blood plasma. Moreover, the blood urea nitrogen levels of the pregnant Cd-injected rats were increased on Gestation Day 21 and 7 days after delivery, indicating signs of Cd nephropathy. The hepatic Cd, Cu, Zn, and MT concentrations of the newborn rats, however, were unaltered by Cd injections. Therefore, it is possible that pregnancy may induce a high risk of Cd nephrotoxicity in women with chronic Cd exposure.

Stress-related induction of hepatic metallothionein synthesis and increase in peripheral polymorphonuclear leukocytes in mice.

This experiment examined whether hepatic metallothionein (MT) synthesis induced by stressful stimuli could reinforce the peripheral leukocyte defense mechanism in mice. A 2 x 2 cm section of dorsal skin was excised from male ICR mice (7 w.o.), then the hepatic MT concentration and superoxide anion production (SOA) in peripheral leukocytes were measured at 6 and 24 hr after the excision. The 6 hr-hepatic MT level was 6 times greater in the skin-excised mice than in the controls. SOA in the skin-excised mice was 2.3 times greater at 6 hr than in the controls, then decreased to the control level by 24 hr. Food deprivation increased the hepatic MT and SOA levels at 24 and 48 hr to a remarkably greater level than in the controls. The increases in SOA, which was measured by chemiluminescence response (CL) were found to be due to an increase in the number of polymorphonuclear leukocytes (PMNs) in the peripheral leukocytes in both the skin excision and food deprivation groups. These results suggest that skin excision causes an inflammatory response in mice that results in an acute increase in the number of PMNs concomitant with the acute activation of hepatic MT synthesis. Food deprivation might result in physiologic stress 24 hr or more after food deprivation and cause "emergency" increases in MT synthesis and PMN defense mechanisms. Thus, some unknown linked mechanisms might exist between hepatic MT synthesis and increased peripheral PMNs.

Prevention of Immunologic Stress Contributes to the Growth-Permitting Ability of Dietary Antibiotics in Chicks ,

The growth-permitting ability of antibiotics fed to broiler chicks was studied as it relates to the state of activation of the immune system. In Experiment 1, chicks were fed two levels of antibiotics (0 or 100 mg streptomycin + 100 mg penicillin/kg diet) and were raised either in an environment with poor sanitation to create a chronic immune stress or in a clean environment. Chicks raised in the unsanitary environment and not fed antibiotics had significantly lower (P < 0.05) rates of weight gain and efficiencies of feed utilization, and higher levels of plasma interleukin-1, compared with chicks raised in the clean environment or chicks raised in the unsanitary environment and fed antibiotics. Adding antibiotics to the diet of birds in the clean environment did not affect any variable. In Experiment 2, chicks were raised in a conventional environment and fed two levels of an antibiotic (0 or 100 mg tetracycline/kg diet). After a 15-d feeding period, half of the chicks were injected with Salmonella typhimurium lipopolysac-charide to create an acute immunologic stress. Feeding antibiotic resulted in improved weight gain, feed consumption and efficiency of feed utilization. Lipopolysac-charide-injected birds developed heavier livers, spleens and intestines relative to body weights and higher rectal temperatures and hepatic metallothionein concentrations, presumably due to an immunologic stress. Omitting antibiotic from the diet resulted in similar changes. These results indicate that feeding antibiotics may permit growth by preventing immunologic stress and associated metabolic changes brought about by monokines including interleukin-1.

Excessive accumulation of hepatic copper in LEC rats aged 80 days without hepatitis and 130 days with hepatitis.

The Cu concentration was about 40 and 60 times higher in the liver in Long-Evans with a cinnamon-like coat color (LEC) rats aged 80 days (without hepatitis) and 130 days (with hepatitis), respectively than in the liver in Fischer rats. Most hepatic Cu was recovered in the cytosol fraction. Furthermore, about 96% and 84% of the cytosolic Cu was found in the metallothionein region on a Sephadex G-75 column in LEC rats aged 80 and 130 days, respectively. The hepatic metallothionein concentration was about 130 to 140 times higher in LEC rats than in Fischer rats when the concentration was expressed as metallothionein-bound Cu. Three forms of Cu-metallothionein were isolated by DEAE-cartridge. Although the concentration of hepatic Cu-metallothionein and its composition of polymorphic form were not changed greatly in hepatitis phase (in the 130-day-old LEC rats), activities of serum enzymes, aspartate aminotransferase (GOT) and alanine aminotransferase (GPT) were increased significantly. The LEC rat showed a significantly low concentration of biliary Cu and markedly low activity of ceruloplasmin (as ferroxidase). Serum Cu showed a low concentration in the 80-day-old LEC rats, but recovered to the control level in the 130-day-old LEC rats. The abnormal accumulation of Cu may be due to the inherent reduction of excretion of Cu into the bile and blood. Such deposition may be a trigger for the onset of the spontaneous hepatitis occurring at 90-120 days after birth and for the onset of hepatoma later.

Changes in plasma zinc, copper, iron, and hepatic metallothionein in adjuvant-induced arthritis treated with cyclosporin

The early changes in hepatic metallothionein (MT) and plasma zinc (Zn), copper (Cu), and iron (Fe) were investigated during the induction of adjuvant (AJ) arthritis in rats in conjunction with cyclosporin (CsA) treatment. Plasma Zn decreased after AJ injection (60% of control values at 8 h), and this was associated with a 4.5-fold increase in hepatic MT at 8 h. Plasma Zn was lowest at 16 h (40% of control), whereas hepatic MT concentrations increased to a maximum of 20-fold at 16 h. Changes in plasma Fe paralleled those of Zn, whereas plasma Cu levels were increased. Plasma metal and hepatic MT concentrations returned toward normal from d 1-7. At d 14, when marked paw swelling was apparent, hepatic MT and plasma Cu were again increased and plasma Zn decreased. Administration of CsA decreased MT induction in rats injected with AJ and also caused a marked recovery in plasma Zn and Fe levels. These changes were small but significant even in the early stages (up to 24 h) after AJ injection and were followed by a sustained improvement in all parameters, corresponding to the nonappearance of clinical arthropathy in CsA-treated rats. TNF-alpha and IL-6 production by peritoneal macrophages isolated from AJ-injected rats was significantly decreased by CsA treatment at d 7 and 14. The inhibition of hepatic MT induction during acute and chronic inflammation by cyclosporin emphasizes the role of the immune system in altered metal homeostasis in inflammation.

Induction of metallothionein by diethyl maleate

Metallothionein (MT) is a sulfhydryl-rich protein whose levels are increased by administration of a variety of agents including metals, cytokines, and oxidative stress agents. Recent studies have suggested that MT is involved in protecting against various forms of oxidative stress, but little is known about the induction of MT by oxidative stress agents. Diethyl maleate (DEM) causes oxidative stress by depleting glutathione levels and is quite effective at increasing hepatic concentrations of MT. The purpose of the current study was to learn more about the relationship between induction of MT and oxidative stress by characterizing this increase in hepatic MT levels produced by DEM. Administration of DEM (3 to 9 mmol/kg, sc) increased hepatic MT concentration in mice as much as 37-fold to 213 μg MT/g liver, which is similar to the hepatic MT level seen after administration of other effective MT inducers, such as Cd. The maximal increase of hepatic MT took place 12 to 24 hr after administration of 5 mmol DEM/kg. This rise in MT was preceded by a 60% depletion of hepatic glutathione 3 hr after DEM and increases in both MT-I and MT-II mRNA, which reached a peak 6 to 9 hr after DEM. Administration of DEM (3–5 mmol/kg, sc) also increased MT levels in Sprague-Dawley rats. Pretreatment with DEM protected against Cd-induced hepatotoxicity in a fashion which suggested that a functional MT was being synthesized. In summary, DEM is a highly effective inducer of MT which increases MT at the mRNA level.

Glucocorticoid inhibition of inflammation-induced metallothionein synthesis in mouse liver

The effects of glucocorticoid treatment on the induction of hepatic metallothionein (MT) during inflammation initiated by turpentine oil (TUR) or endotoxin (LPS) were studied in mice. The administration of TUR increased concentrations of hepatic MT as well as that of plasma fibrinogen. Although hepatic MT was modestly induced by dexamethasone (DEX) alone, pretreatment with DEX (12.5 to 100 mg/kg, sc) inhibited the increases both in hepatic MT and in plasma fibrinogen from a subsequent dose of TUR 6 hr after DEX administration. The concentration of hepatic MT in the DEX-pretreated (25 mg/kg) group was higher than that in the nonpretreated group 4 hr after administration of TUR, but after 24 hr, the MT concentration in the DEX-pretreated group was inhibited to 20% of that in the nonpretreated group. These inhibitory effects were also observed by prednisolone (PRE) but not by salicylic acid. The inhibitory effect of DEX on the induction of MT synthesis during inflammation was observed after administration of the exudate obtained from inflamed tissue. When inflammation was initiated by an injection of LPS as well as TUR, pretreatment of either DEX or PRE inhibited the increase of hepatic MT. Pretreatment of DEX did not affect the induction of hepatic MT synthesis by cadmium. In contrast to inflammation initiated by TUR or LPS, pretreatment of DEX caused an additive increase of hepatic MT concentration after administration of zinc. These results suggest that glucocorticoids, despite being direct inducers of MT, inhibit the induction of MT synthesis during inflammation by the suppression of cytokine production.

Induction of hepatic metallothionein by nonmetallic compounds associated with acute-phase response in inflammation

Induction of hepatic metallothionein (MT) synthesis by several nonmetallic compounds and its relationship to an acute-phase response in inflammation were studied in mice. Subcutaneous injections of menadione, paraquat, carbon tetrachloride (CCl 4), and several organic solvents caused an increase of hepatic MT concentration. This MT contained only zinc. Menadione and n-hexane caused the greatest accumulation of hepatic MT among these nonmetallic compounds (about 13-fold). The concentration of Zn was significantly decreased in plasma in contrast to liver after an injection of these nonmetallic compounds. When 65ZnCl 2 was injected iv after these injections, uptake of 65Zn to the liver was increased. This effect was not observed after treatment with cycloheximide. The association with inflammation of this induction of MT accumulation was examined by determination of acutephase proteins. The concentration of fibrinogen in the plasma was significantly increased following injection of those nonmetallic compounds which caused marked hepatic MT accumulation. An injection of 1 n NaOH, 1 n HCl, turpentine oil, or endotoxin caused a significant increase in the plasma concentration of fibrinogen and in the hepatic MT concentration. Injections of n-hexane as well as turpentine oil significantly increased hepatic MT concentration and plasma concentration of fibrinogen and ceruloplasmin with time. The concentration of fibrinogen was significantly correlated ( r = 0.789) with the concentration of hepatic MT. Neither adrenalectomy nor pretreatment with dexamethasone prevented hepatic MT accumulation caused by these compounds. These results indicate that induction of hepatic MT synthesis by these nonmetallic compounds is associated with an acute-phase response in inflammation and is independent of glucocorticoids.

Heme oxygenase activity and cytochrome P‐450 content associated with induced metallothionein in the liver of rats treated with various metals

Abstract The relationship between heme oxygenase (HO) activity and metallothionein (MT) concentration in rat liver was investigated. The responses to treatments with metals of the ?‐series metals that can bind to MT in vivo (e.g., cadmium, zinc and copper) and the B‐series metals that cannot bind to MT in vivo (e.g., cobalt, nickel and manganese) were compared. A single treatment with any metal in the A‐ or B‐series increases both hepatic HO activity and MT concentration. However, repeated treatments with ?‐series metals decreases HO activities more than a single injection despite appreciable increases in MT concentrations. Repeated treatments with B‐series metals increase both HO activities and MT concentrations. On the other hand, cytochrome P‐450 concentrations are depressed by single or repeated treatments with all metals tested. These results suggest that the binding abilities of metals to MT in vivo, or the replaceable zinc concentration in MT, affect the magnitude of HO activity.

The Importance of Metallothionein for the Accumulation of Copper, Zinc and Cadmium in Environmentally Exposed Perch, Perca fluviatilis*

A field study of the role of metallothionein (MT) in the binding of heavy metals in perch (Perca fluviatilis), exposed to moderate levels of copper, zinc and cadmium, was performed. Perch were sampled at four sites in Sweden in September during two consecutive years. Two sites were located in the vicinity of a brassworks and two outside the emission range. The first year, fish from the two brassworks sites and from one of the uncontaminated sites were collected. The second year, fish from the most contaminated site and from the two uncontaminated sites were caught. The levels of hepatic copper, zinc and cadmium reflected the concentrations of these metals in water and were increased in fish from the two contaminated sites. The level of cadmium in liver was relatively low. MT was induced in liver of perch caught at the most contaminated site. The hepatic MT content in individual livers correlated well to the accumulation of copper (r = 0.85, P less than 0.001) and zinc (r = 0.75, P less than 0.001). There was a low but significant correlation between the levels of MT and cadmium in the liver (r = 0.48, P less than 0.001). The relationship between MT and metals was very similar both years. Subcellular fractionation of the metals in the liver revealed that an induction of MT was followed by an increased amount of copper, zinc and cadmium bound to the protein. The relative fraction of the total hepatic copper and cadmium bound to MT was increased at the most contaminated site, whereas there was no difference in subcellular distribution of zinc between the sites. In perch from the most contaminated site, the portions of hepatic copper, zinc and cadmium found in the cytosolic fraction were 70, 57 and 81%, respectively. Seventy-one % of the copper, 29% of the zinc and 84% of the cadmium found in hepatic cytosol of fish from the same site, eluted together with MT after gel filtration chromatography. The analysis of the subcellular distribution of copper, zinc and cadmium demonstrates that MT is responsible for the binding of a large amount of the total hepatic cellular content of copper and cadmium in perch.

Marginal Zinc Deficiency and Changes in Behavioral Salt Taste Threshold and Salt Preference in Mice.

An animal model of marginal zinc deficiency was tested in mice, and salt taste threshold and salt preference were investigated in the state of marginal zinc deficiency. Two experiments were conducted. In Experiment 1, four-week-old male ICR mice were fed diets of three different levels of zinc content (3.17, 9.27, or 48.13 micrograms Zn/g) for 60 days. Food intake, growth, zinc levels in tissues, hepatic metallothionein (MT) content, and activities of selected enzymes (hepatic and RBC delta-amino-levulinic acid dehydratase (ALAD) and plasma alkaline phosphatase (ALP] did not differ among the groups throughout the experiment, and no overt signs of zinc deficiency were manifested in any group. In Experiment 2, four-week-old male ICR mice were fed a zinc-deficient diet (1.98 micrograms Zn/g) or a zinc-adequate diet (49.14 micrograms Zn/g) for 56 days. Food intake and growth did not differ between the two groups, and no overt signs of zinc deficiency were observed throughout the experiment. Zinc levels in the plasma and femur--but not those in the brain, kidneys, liver, and red blood cell (RBC)--and plasma ALP activities were significantly lower on Day 42 and Day 56 of the experiment in the mice fed the zinc-deficient diet (ZnD group) than in those fed the zinc-adequate diet (ZnA group). Hepatic MT contents were lower in the ZnD group than in the ZnA group on Day 56 only. Salt taste threshold was 0.05% in the ZnA group, while it was 0.1% in the ZnD group, between Day 30 and Day 38, and between Day 44 and Day 52 of the experiment. Preference for 0.9% NaCl solution was no different in the two groups when tested between Day 38 and Day 40 or between Day 52 and Day 54, but that for 1.6% NaCl solution was significantly higher in the ZnD group than in the ZnA group between Day 40 and Day 42, and between Day 54 and Day 56.

Hepatic metallothionein levels in roach ( Rutilus rutilus L.) continuously exposed to water-borne cadmium

1. The molecular weight of roach metallothionein was determined to be 6340 (±838) dallons by SDS-PAGE. 2. Hepatic metallothionein and cadmium concentrations both increased during the period of exposure and were found to be positively correlated. 3. The rate of increase of hepatic metallothionein concentration was quantified and was more rapid than that previously recorded for cadmium sensitive species.