Influence of dietary iron deficiency on acute metal intoxication

The influence of dietary iron deficiency on acute nickel, lead or cadmium toxicity as reflected by the induction of hepatic, renal and intestinal metallothionein (MT), disposition of the metals, and alterations in hematological parameters was investigated in rats. The administration of cadmium induced the hepatic, renal and intestinal MT while that of nickel or lead induced hepatic MT only. However, dietary iron deficiency did not influence the cadmium induced tissue MT but enhanced the ability of nickel or lead to restore the normal synthesis of renal and intestinal MT lowered under the influence of reduced body iron status. The accumulation of lead in liver and kidney and that of cadmium enhanced in liver only, while tissue deposition of nickel remained unaffected by iron deficiency. The induction of hepatic MT by three metals appears related to the concomitant rise in the hepatic zinc, calcium and iron levels in normal rats. However, dietary iron deficiency increased the hepatic zinc in response to nickel or cadmium and that of heptic calcium in response to lead.     

Renal metallothionein responds rapidly and site specifically to zinc repletion in growing rats

Metallothionein (MT) is important for heavy metals and free radical protection in the kidney. MT is responsive to zinc and primarily localized within the renal cortex. However, site-specific renal responses to dietary zinc repletion are understudied. The objective of this study was to examine the effects of dietary zinc deficiency and repletion on renal MT concentration and immunolocalization in rats. Weanling male Sprague Dawley rats were randomly assigned to either a zinc-deficient, zinc control, or pair-fed to zinc-deficient group. Half of the zinc-deficient and pair-fed rats were repleted with the control diet ad libitum for an additional 24 h. Renal tissue samples were assessed for total zinc, MT concentrations and MT immunostaining. Dietary zinc deficiency reduced renal zinc and MT concentrations, and attenuated intensity and localization of MT. Dietary zinc repletion for 24 h restored renal zinc and MT concentrations, the latter primarily in the proximal convoluted tubules of the cortex. Concentrations of renal MT, but not zinc, were elevated by diet restriction and MT (μg/mg protein) and partially normalized by 24 h diet repletion. In conclusion, renal MT modification due to zinc deficiency or diet restriction can be rapidly normalized in a site-specific manner with normal dietary zinc intake. The results support a role for MT in kidney homeostasis, in particular at the level of the proximal tubules in the cortex. The speed of MT repletion may have clinical implications for dietary zinc in the treatment of acute and chronic renal pathology due to toxins and free radicals.     

Vitamin D Supplementation Modulates Blood and Tissue Zinc, Liver Glutathione and Blood Biochemical Parameters in Diabetic Rats on a Zinc-Deficient Diet

Previous studies suggest a protective effect of vitamin D3 on zinc deficiency-induced insulin secretion and on pancreas β-cell function. The aim of this study was to investigate the effect of vitamin D on blood biochemical parameters, tissue zinc and liver glutathione in diabetic rats fed a zinc-deficient diet. For that purpose, Alloxan-induced diabetic rats were divided into four groups. The first group was fed a zinc-sufficient diet while the second group was fed a zinc-deficient diet. The third and fourth groups received zinc-sufficient or zinc-deficient diets plus oral vitamin D3 for 27 days. At the end of the experiment, blood, femur, pancreas, kidney and liver samples were taken from all rats. The serum, femur, pancreas, kidney and liver zinc concentrations, liver glutathione, serum alkaline phosphatase activity, daily body weight gain and food intake were lower in the zinc-deficient rats in comparison with those receiving adequate amounts of zinc. These values were increased in the zinc-deficient group that was supplemented with vitamin D3. The serum total cholesterol, triglycerides, total protein, urea, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase and blood glucose values were higher in rats fed a zinc adequate diet, but their concentrations were decreased by vitamin D3 supplementation. The serum total protein levels were not changed by zinc deficiency and vitamin D3 supplementation. These results suggest that vitamin D3 modulates tissue zinc, liver glutathione and blood biochemical values in diabetic rats fed a zinc-deficient diet.     

Effects of interaction between65Zn,cadmium, and copper in rats

Distribution and retention of zinc in the presence of cadmium and copper was studied in rats exposed repeatedly to these metals. The experiment was performed on white rats of the Wistar strain. The animals were divided into four groups/five rats each: 1)⁶⁵ZnCl₂; 2)⁶⁵ZnCl₂+CdCl₂; 3)⁶⁵ZnCl₂+CuCl₂; and 4) control group. Rats were administered sc every other day for two weeks:⁶⁵ZnCl₂−5 mg Zn/kg; CdCl₂−0,3 Cd/kg; and CuCl₂−2 mg Cu/kg. The zinc content was measured in rat tissues by γ-counting. Effect of Cd and Cu on subcellular distribution of zinc in the kidney and liver and on the level of metallothionein were also examined. Whole body retention of zinc under the influence of cadmium was lower than that observed in animals treated with zinc alone. However, copper increased twofold the whole body retention of zinc. Cadmium elevated the accumulation of zinc only in the kidneys nuclear fraction and liver soluble fraction. In the kidneys and liver, copper elevated the accumulation of zinc, in the nuclear, mitochondrial, and soluble fractions. The level of metallothionein-like proteins (MT) in the kidneys after a combined supply of zinc and copper was significantly increased with respect to the group of animals treated with zinc alone. These results indicated complex interactions between cadmium, copper, and zinc that can affect the metabolism of each of the metals.     

Low iron diet and parenteral cadmium exposure in pregnant rats: the effects on trace elements and fetal viability

The effects of latent iron deficiency combined with parenteral subchronic or acute cadmium exposure during pregnancy on maternal and fetal tissue distribution of cadmium, iron and zinc, and on fetal viability were evaluated. Timed-pregnant Sprague-Dawley rats were fed on semisynthetic test diets with either high iron (240 mg kg) or low iron (10 mg kg), and concomitantly exposed to 0, 3 or 5 mg cadmium (as anhydrous CdCl₂) per kilogram body weight. Animals were exposed to cadmium from gestation day 1 through 19 by subcutaneously implanted mini pumps (Subchronic exposure) or on gestation day 15 by a single subcutaneous injection (Acute exposure). All rats were killed on gestation day 19. Blood samples, selected organs and fetuses were removed and prepared for element analyses by atomic absorption spectrometry. Low iron diet caused decreases in maternal body weight, maternal and fetal liver weights, placental weights and tissue iron concentrations. By cadmium exposure, both subchronic and acute, tissue cadmium concentrations were increased and the increase was dose-related, maternal liver and kidney zinc concentrations were increased, and fetal zinc concentration was decreased. Cadmium concentration in maternal liver was additionally increased by low iron diet. Acute cadmium exposure caused lower maternal body and organ weights, high fetal mortality, and decreased fetal weights of survivors. In conclusion, parenteral cadmium exposure during pregnancy causes perturbations in essential elements in maternal and fetal compartments. Acute cadmium exposure in the last trimester of gestation poses a risk for fetal viability especially when combined with low iron in maternal diet.     

Rat liver metabollothionein interactions of silver,zinc, and cadmium

Weanling rats were fed diets either alone or with combinations of silver, elevated zinc, or elevated cadmium for 7 weeks. The rats were then killed, and the silver, zinc, and cadmium proteins isolated from the livers by gel filtration and ion exchange chromatography. When silver was fed alone in diet, low levels of this metal were eluted as two peaks from the ion exchange column. In contrast, when silver was fed with cadmium or elevated zinc, three metal-containing peaks were eluted from this ion exchange column. Amino acid analysis revealed that the major proteins binding these metals are metallothioneins, as judged by high cysteine content.     

Zinc deficiency and the desaturation of linoleic acid in rats force-fed fat-free diets

Recent studies with rats force-fed zinc-deficient diets containing various types of fat failed to demonstrate a role of zinc in desaturation of linoleic acid. The present study was conducted to investigate the effect of zinc deficiency on desaturation of linoleic acid in rats that were initially force-fed fat-free diets to stimulate activity of desaturases. Therefore, rats were fed zinc-adequate and zinc-deficient fat-free diets for 6 d. After that period, the groups were divided and half of the rats continued feeding the fat-free diet for another 3.5 d whereas the other half was switched to a fat diet by supplementing the fat-free diet with 5% safflower oil. In order to assess desaturation of linoleic acid, fatty acid compositions of liver phosphatidylcholine, ethanolamine, and-serine were considered, particularly levels of individual (n-6) polyunsaturated fatty acids (PUFA). Levels of total and individual (n-6) PUFA were similar in zinc-adequate and zinc-deficient rats fed the fat-free diet throughout the experiment. Addition of 5% safflower oil increased levels of total and individual (n-6) PUFA in both zinc-adequate and zinc-deficient rats. However, total (n-6) PUFA in all types of phospholipids were higher in zinc-adequate rats than in zinc-deficient rats. Additionally, in zinc-deficient rats there were changes of (n-6) PUFA levels typical for impaired Δ5 and Δ6 desaturation: linoleic acid and dihomo-γ-linolenic acid were elevated; arachidonic acid, docosatetraenoic acid, and docosapentaenoic were lowered by zinc deficiency. Therefore, the study shows that zinc deficiency impairs desaturation of linoleic acid in rats force-fed fat-free diets and therefore supports results from former convential zinc deficiency experiments suggesting a role of zinc for desaturation of linoleic acid.     

Regulation of the ontogeny of rat liver metallothionein mRNA by zinc

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in livers of fetal and newborn rats from dams which were fed with a control or zinc-deficient or copper-deficient or iron-deficient diet from day 12 of gestation. The liver samples were analyzed for MT-mRNA levels using a mouse MT-I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT-I mRNA were significantly decreased only in pups born from zinc-deficient dams. Injection of the zinc-deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT-I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT-I mRNA levels were observed to increase significantly in control fetal rat liver on days 17-21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc-deficient dams during the late gestational period. The MT-I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc-deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc-deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT-I mRNA but also in the localization of MT in newborn rat liver.     

Zinc deficiency and the activities of lipoprotein lipase in plasma and tissues of rats force-fed diets with coconut oil or fish oil

The present study was performed to investigate the effect of zinc deficiency on the activities of lipoprotein lipase in postheparin serum and tissues of rats fed diets containing either coconut oil or fish oil as dietary fat, using a bifactorial experimental design. To ensure an adequate food intake, all the rats were force-fed by gastric tube. Experimental diets contained either 0.8 mg zinc/kg (zinc-deficient diets) or 40 mg zinc/kg (zinc-adequate diets). The effects of zinc deficiency on the activities of lipoprotein lipase in postheparin serum and postprandial triglyceride concentrations and distribution of apolipoproteins in serum lipoproteins depended on the type of dietary fat. Zinc-deficient rats fed the coconut oil diet exhibited a reduced activity of lipoprotein lipase in postheparin serum and adipose tissue, markedly increased concentrations of triglycerides in serum, and a markedly reduced content of apolipoprotein C in triglyceride-rich lipoproteins and high density lipoproteins compared with zinc-adequate rats fed coconut oil. By contrast, zinc-deficient rats fed the fish oil diet did not exhibit reduced activities of lipoprotein lipase in postheparin serum and adipose tissue and increased concentrations of serum lipids compared with zinc-adequate rats fed the fish oil diet. This study suggests that a reduced activity of lipoprotein lipase might contribute to increased postprandial concentrations of serum triglycerides observed in zinc-deficient animals. However, it also demonstrates that the effects of zinc deficiency on lipoprotein metabolism are influenced by dietary fatty acids.     

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.     

The mobility and reactivity of maleimide-binding proteins in the rat erythrocyte membrane. Effects of dietary zinc deficiency and incubation with zinc in vitro

Erythrocyte ghosts, prepared from rats fed zinc-deficient diets, were analyzed for the mobility of membrane proteins by electron spin resonance spectroscopy of the sulfhydryl-binding spin probe, 4-maleimido-2,2,6,6-tetramethylpiperidine-N-oxyl. Compared with erythrocyte membranes from rats fed zinc-adequate diets ad libitum or pair-fed, erythrocyte membranes from zinc-deficient rats had a significantly increased ratio of weakly immobilized to strongly immobilized probe-binding proteins. This suggests that dietary zinc deficiency causes a conformational change in erythrocyte membrane proteins. Dietary zinc deficiency did not significantly affect N-ethylmaleimide (NEM)-induced thermal sensitivity or NEM-induced mechanical fragility in rat erythrocytes; however, the addition of zinc in vitro to red cells significantly inhibits NEM-induced mechanical fragility.     

Zinc depletion suppresses tumor growth in mice

The hypothesis that in tumor-bearing animals an increase of host hepatic zinc metallothionein (Zn-MT) causes a restriction of zinc in the tumor tissue was studied. Three types of tumors were induced in laboratory mice by cell transplant. Tumor growth appears to be inhibited under zinc-deficient conditions, even in cases where zinc deficiency was started after tumor cell transplant. The survival times of tumor-bearing mice were prolonged by administration of cadmium chloride, which induces the synthesis of a combined zinc-cadmium metallothionein derivative in the host liver, but not in the tumor tissue, leading to an increase of hepatic zinc in the treated animals. The uptake of⁶⁵Zn by the liver of Cd-treated, tumor bearing mice was significantly higher than that of controls whereas uptake of⁶⁵Zn by tumor cells was significantly higher in controls than in the treated animals. These results suggest that restriction of zinc intake suppresses tumor growth.     

Studies on the role of iron in the reversal of cadmium toxicity in chicks

Studies were conducted to determine the effect of dietary iron (Fe) levels ranging from a deficiency to an excess on the toxicity of cadmium (Cd) in chicks. In Fe-deficient animals, cadmium was found to be more toxic than in Fe supplemented animals as measured by growth. The liver Cd burdens were increased significantly in the presence of dietary Fe supplementation, and there was a significant Cd−Fe interaction in the Cd concentration of the kidney, indicating that iron deficiency increased the concentration of Cd in the kidneys of those chicks receiving this element. Cd tended to reduce the Fe concentration in both the liver and kidney. The absorption of Cd as measured by the amount of¹⁰⁹Cd that disappeared from an isolated duodenal segment in one h was not affected by the Fe content of the diet, but the amount of isotope appearing in the liver compared to the amount present in the blood was increased in the Fe supplemented chicks. Separation of the Cd binding ligands by column chromatography revealed that more of the Cd in the liver, but not the kidney, was associated with ligands which eluted in a column volume that contained metallothionein in those chicks receiving Fe than in the livers from Fe deficient animals. The inverse relationship between the amount of Cd bound to the metallothionein containing fraction and toxicity may be related causally. Paper No. 10538 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7601. The use of trade names in this publication does not imply endorsement by the NC Agricultural Research Service of the products named nor criticism of similar ones not mentioned.     

Effects of dietary zinc on free radical generation, lipid peroxidation, and superoxide dismutase in trained mice.

The purpose of this study was to investigate the effects of dietary zinc on free radical generation, lipid peroxidation, and superoxide dismutase (SOD) in exercised mice. In the first part of the study, 48 male weanling mice were randomly divided into three groups. They were fed a zinc-deficient diet containing 1.6 mg/kg zinc or were pair-fed or fed ad libitum a zinc-adequate diet supplemented with 50 mg/kg zinc. Half of each group received an exercise training program that consisted of swimming for 60 min per day in deionized water. The diets and exercise program persisted for 6 weeks. In the second part of the study, 64 mice were fed zinc-deficient diets for 6 weeks, and then one group was fed the zinc-deficient diet for an additional 3 weeks, and the other three groups were fed diets supplemented with 5, 50, and 500 mg/kg zinc, respectively. Half of each group also received the exercise program. Both blood and liver samples were examined. Free radicals in liver were directly detected by electron spin resonance techniques and the extent of lipid peroxidation was indicated by malonic dialdehyde (MDA). Both CuZn-SOD and Mn-SOD were measured. The results showed that exercise training increased the metabolism of zinc, and zinc deficiency induced an increased free radical generation and lipid peroxidation and a decreased hepatic CuZn-SOD activity in exercised mice. Furthermore, although exercise training had no effect on the level of free radicals in zinc-adequate mice, it could increase the hepatic mitochondrial MDA formation further in zinc-deficient animals and zinc deficiency would eliminate the exercise-induced increase in SOD activities which existed in zinc-adequate mice. A total of 50 mg/kg zinc supplemented in the diet was adequate to correct the zinc-deficient status in exercised mice while 5 mg/kg zinc had a satisfactory effect on the recovery of only sedentary zinc-deficient mice. However, 500 mg/kg zinc had a harmful effect on both sedentary and exercised zinc-deficient animals.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

The binding of gold(I) to metallothionein

The binding of gold(I) to metallothionein, MT, has been unambiguously established by the reaction of Na₂AuTM with purified horse kidney MT. Zinc was displaced more readily than cadmium although the latter could be displaced using large Au/Cd ratios. The metal exchange reactions were complete within 2 hr of mixing. Further evidence that such reactions might be physiologically significant were obtained by studying in vitro metal displacements in the liver cytosol of in vivo metal treated rats: When Na₂AuTM was added to the cytosol of rats administered CdCl₂ in vivo, zinc, copper and cadmium were displaced in 2/1/1 ratios from the metallothionein fraction. The zinc and cadmium displacement provide direct evidence that the gold was binding to MT. Addition of Cd⁺² to liver cytosol of gold-treated rats resulted in displacement of copper and zinc, but not gold, from the MT fractions. When liver MT is prepared from rats exposed to Au or Cd, the Cd/protein ratio increased during the preparation, but the Au/protein ratio decreased. The Mt-bound metals account for 95% of the cytosolic Cd but only 15%–30% of the cytosolic gold in these studies. Thus, the nonspecific binding of gold to MT in vivo should be considered as one aspect in its equilibration among protein binding sites, which include, inter alia, metallothionein. Gold was found to coelute with zinc and cadmium in the MT fraction of rat kidney cytosol, when both Cd and Na₂AuTM were administered to the rats. The possible significance of gold binding to MT in the treatment of rheumatoid arthritis-chrysotherapy-is briefly discussed.     

Susceptibility to kainate-induced seizures under dietary zinc deficiency

Zinc homeostasis in the brain is altered by dietary zinc deficiency, and its alteration may be associated with the etiology and manifestation of epileptic seizures. In the present study, susceptibility to kainate-induced seizures was enhanced in mice fed a zinc-deficient diet for 4 weeks. When Timm's stain was performed to estimate zinc concentrations in synaptic vesicles, Timm's stain in the brain was attenuated in the zinc-deficient mice. In rats fed the zinc-deficient diet for 4 weeks, susceptibility to kainate-induced seizures was also enhanced. When the release of zinc and neurotransmitters in the hippocampal extracellular fluid of the zinc-deficient rats was studied using in vivo microdialysis, the zinc concentration in the perfusate was less than 50% of that of the control rats and the increased levels of zinc by treatment with kainate were lower than the basal level in control rats, suggesting that vesicular zinc is responsive to dietary zinc deficiency. The levels of glutamate in the perfusate of the zinc-deficient rats were more increased than in the control rats, whereas the levels of GABA in the perfusate were not at all increased in the zinc-deficient rats, unlike in the control rats. The present results demonstrate an enhanced release of glutamate associated with a decrease in GABA concentrations as a possible mechanism for the increased seizure susceptibility under zinc deficiency.     

Metallothionein mRNA levels are influenced by dietary cyclodextrins in rats

The relationship between metallothionein mRNA levels and the amounts of copper and zinc in liver, kidney and small intestine by feeding dietary cyclodextrin was examined in growing Wistar rats. alpha-, beta- or gamma-cyclodextrin was fed at 50 g/kg of diet for a 7-days period (ad libitum). After feeding, the liver zinc of rats fed beta-cyclodextrin was greater than those of rats fed the other three diets. Copper accumulated in kidney of rats fed alpha- or beta-cyclodextrin. Copper content in the small intestine did not show any alterations among rats fed all kinds of diets. The cyclodextrin-supplemented diets were ineffective in zinc content in every organ. There was the greatest level of copper in serum of rats fed beta-cyclodextrin, whereas the highest level of serum zinc was observed in rats fed gamma-cyclodextrin diet. Northern blot analysis demonstrated that dietary beta- and gamma-cyclodextrins, but not alpha-cyclodextrin markedly increased the metallothionein mRNA in the liver, whereas small intestinal metallothionein mRNA levels were markedly decreased. Kidney metallothionien mRNA levels were raised appreciably by all dietary cyclodextrin intakes. Metallothionein gene expressions in liver, kidney and small intestine were not proportional to liver and serum copper or zinc levels in those tissues. These results suggest that regulation of the metallothionein mRNA levels may at least partly involved with the accumulation of metals as copper in liver and kidney of rats fed cyclodextrins.     

Inducibility and amounts of metallothionein as influenced by cadmium and zinc in the developing rat

Information on the accumulation and/or depletion of Zn in metallothionein (MT) of rat fetus, rat pup, and maternal rat liver at various ages was obtained with pregnant rats fed a basal casein diet or this diet plus either 100 ppm Zn or 50 ppm Cd. Rats fed each of the respective diets were sacrificed on 12, 16, and 20 d of gestation and 0, 7, 14, and 28 d post-partum. No Cd was detected in the placenta or fetal tissue and the Cd did not affect the accumulation of Zn in the fetal MT, but it did increase the Zn content in liver MT of the dams. Very little Zn in MT was found on day 12 of gestation, but Zn rapidly increased in MT to a maximum at time of birth. The accumulation of Zn in MT was independent of the diet for the fetuses, but the Zn accumulation in the dam and pup tissues was diet dependent. In order to study age-dependent difference in the inducibility of MT, newborn, 5-week-old, or 24-week-old rats were injected with zinc at the levels of 0, 3, 6, or 9 mg/kg and 5 h later injected with³⁵S-cystine. In rats sacrificed 1 h later, the amount of radioactivity in liver MT demonstrated that this protein in older animals was more readily induced by Zn than in younger animals.