Effect of zinc supplementation on metallothionein,copper, and zinc concentration in various tissues of copper-loaded rats

The present study was designed to investigate the effects of Zn administration on metallothionein concentrations in the liver, kidney, and intestine of copper-loaded rats. Male CD rats were fed a diet containing 12 mg Cu and 67 mg Zn/kg body wt. They were divided into either acute or chronic experimental protocols. Rats undergoing acute experiments received daily ip injections of either Cu (3 mg/kg body wt) or Zn (10 mg/kg body wt) for 3 d. Chronic experiments were carried out on rats receiving Cu ip injections on d 1, 2, 3, 10, 17, and 24, Cu injections plus a Zn-supplemented diet containing 5 g Zn/kg solid diet, or a Zn-supplemented diet alone. Rats injected Zn or Cu had increased MT concentrations in liver and kidney. Zn produced the most important effects and the liver was the most responsive organ. Rats fed a Zn-supplemented diet had significantly higher MT concentrations in liver and intestine with respect to controls. Increased MT synthesis in the liver may contribute to copper detoxification; the hypothesis of copper entrapment in enterocytes cannot be confirmed.     

Unusual accumulation of copper related to induction of metallothionein in the liver of LEC rats.

Copper (Cu), iron (Fe), zinc (Zn) and manganese (Mn) levels in organs of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis, were determined by instrumental neutron activation analysis method. Unusual accumulations of Cu in the liver of LEC rats were found, depending on the age of the animals, the metal concentration being more than approximately 20-40 times those of normal LEA rats (Long-Evans rats with an agouti coat color). Fe and Zn were also accumulated, in addition to Cu, significantly in the LEC rats. The unusual Cu accumulations in the liver of LEC rats were associated with the induction of metallothionein, estimated by radioimmunoassay method, in the liver of LEC rats, rather than that of superoxide dismutase, estimated by electron spin resonance -spin trapping method. These findings suggest that the unusual Cu accumulation in LEC rats is involved in the development of jaundice, hepatic injury and hepatocellular carcinoma.     

Roles of metallothionein in copper homeostasis: responses to Cu-deficient diets in mice.

Metallothionein (MT) protects the body from both harmful non-essential and excessive essential metals. Copper (Cu) is an essential metal, and its concentration in the body is regulated at a constant level between excess and deficient ones. Cu accumulating in the livers of Wilson disease patients and its animal model, Long-Evans rats with a cinnamon-like coat color (LEC) rats, is in the form of Cu,Zn-MT, MT being an antioxidant. Contrary to the efficient production of MT in response to excessive accumulation of Cu in LEC rats, Cu-binding to MT only occurs marginally under normal conditions. However, the present study revealed that Cu binds to MT more with a severe Cu-deficiency. Namely, male C57BL/6J mice were fed a Cu-deficient diet (0.037 mg Cu/g) and deionized water containing trientine, and then the concentration and distribution of Cu were determined. It was suggested that the cessation of biliary excretion and limitation of the Cu supply to ceruloplasmin are the first responses on feeding of a Cu-deficient diet, followed by an increase in Cu-MT with maintenance of the Cu concentration in the liver. These results suggest that MT causes the recruitment of Cu in a Cu-deficient environment by sequestering Cu from degraded Cu-enzymes and delivering it to Cu chaperones.     

Copper-metallothionein induction in the liver of LEC rats.

Recently, copper (Cu) was found to be unusually accumulated, suggesting the induction of metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis. Thus, the direct relationship between the unusual Cu accumulation and the induction of Cu-MT was investigated by giving LEC rats Cu-overloaded or Cu-deficient diets. Results based on the determinations of Cu and MT levels in several organs, as well as the gel-filtration profiles of the cytosols of liver homogenates, showed that dietary Cu induced Cu-MT and development of hepatic injury associated with jaundice.     

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

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

Evaluation of MT expression and detection of apoptotic cells in LEC rat kidneys

To confirm our previous observations on the effectiveness of long term treatment with Zn on Long-Evans Cinnamon (LEC) rats, we extended these studies determining the effects of Zn on trace elements, metallothionein (MT) concentrations and immunolocalization, and on the levels of both MT-1 and MT-2 mRNAs in the LEC rat kidneys. We also localized the renal cells that had chromatin condensation and nuclear fragmentation typical of apoptosis. The results demonstrate that the amount of Zn increased in the treated rats with respect to both untreated and basal rats. In the treated rats the amount of Cu and Fe was similar to that of the basal rats. MT concentrations did not change either with or without Zn treatment, but were higher than the basal group. However, if we consider the percentage of oxidized MT (MTox), we note that Zn treatment is very effective in reducing this value. MTox is not able to bind metals, so it does not perform a "scavenger" function. Moreover, quantification of mRNA indicates that the MT-1 isoform was significantly higher than the MT-2 isoform following Zn treatment. Untreated group sections showed a confocal fluorescent signal that highlighted the irregular nuclei and small apoptotic bodies. The intensity and quantity of fluorescence decreased in the treated group sections. These findings suggest that, in LEC rats, Zn may contribute to cytoprotection through the regulation of MT expression which may provide a cellular defence strategy in response to DNA damage.     

Interactions between Zn and Cu in LEC rats, an animal model of Wilson's disease

The effect of oral Zn treatment was studied in the liver and kidneys of 26 male Long-Evans Cinnamon (LEC) rats (mutant animals, 5 weeks old) in relation to both the interaction between Zn and Cu and the localisation and concentration of metallothionein (MT). Rats receiving 80 mg zinc acetate daily by gavage and control rats receiving no treatment were killed after 1 or 2 weeks. By immunohistochemical and analytical chemical techniques we revealed that treated rats had higher levels of MT in the hepatic and renal cells compared to untreated ones. Tissue Zn concentrations were significantly higher in treated rats compared to untreated whereas Cu concentrations decreased in the liver and kidneys as indicated by analytical chemical analyses. MT levels also decreased with treatment period. A histochemical procedure, obtained using autofluorescence of Cu-metallothioneins, confirms these findings: after 2 weeks, the signal decreased in both the liver and kidney sections. This gives a greater understanding of the mechanism of Cu metabolism in the two tissues considered. These results suggest that Zn acts both to compete for absorption on the luminal side of the intestinal epithelium and to induce the synthesis of MT.     

Metabolic fate of the insoluble copper/tetrathiomolybdate complex formed in the liver of LEC rats with excess tetrathiomolybdate

Copper (Cu) accumulating in a form bound to metallothionein (MT) in the liver of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, can be removed from the MT with tetrathiomolybdate (TTM). However, the insoluble Cu/TTM complex formed with excess TTM is known to be deposited in the liver. The metabolic fate of the insoluble Cu/TTM complex was investigated in the present study. LEC rats were injected with TTM at the dose of 10 mg/kg body weight for 8 consecutive days and were fed with a standard or low Cu diet for 14 days after the last injection. About 95% of the Cu in the liver became insoluble together with Mo. The concentration of Cu in the liver supernatants of rats fed with the standard diet increased significantly compared with that in rats dissected 24 h after the last injection (control rats), while the concentration in rats fed with the low Cu diet remained at a comparable level to that in the controls. The rate of Cu accumulation in the livers of rats fed with the standard diet did not differ before and after the treatment, suggesting that there was no rebound effect by treatment with TTM. These results suggest that the insoluble Cu/TTM complex is resolubilized in the liver, and that the solubilized complex is excreted into the bile and blood, i.e., the insoluble Cu/TTM complex is not the source of Cu re-accumulation in the form bound to MT in the liver after TTM treatment. It was concluded that, once Cu is complexed with TTM, the metal is excreted either immediately in the soluble form or slowly in the insoluble form into the bile and blood.     

Effect and possible role of Zn treatment in LEC rats,an animal model of Wilson's disease

The effect of oral zinc (Zn) treatment was studied in the liver, kidneys and intestine of Long-Evans Cinnamon (LEC) rats in relation to metals interaction and concentration of metallothionein (MT) and glutathione (GSH). We also investigated the change in the activity of antioxidant enzymes and determined the biochemical profile in the blood and metal levels in urine. We showed that the Zn-treated group had higher levels of MT in the hepatic and intestinal cells compared to both untreated and basal groups. Tissue Zn concentrations were significantly higher in the Zn-treated group compared to those untreated and basal, whereas Cu and Fe concentrations decreased. The antioxidant enzyme activities in the Zn-treated group did not change significantly with respect to those in the basal group, except for hepatic glutathione peroxidase activity. Moreover, the biochemical data in the blood of Zn-treated group clearly ascertain no liver damage. These observations suggest an important role for Zn in relation not only to its ability to compete with other metals at the level of absorption in the gastrointestinal tract producing a decrease in the hepatic and renal Cu and Fe deposits, but also to MT induction as free radical scavenger.     

Effect of zinc supplementation from different sources on growth, nutrient digestibility, blood metabolic profile, and immune response of male guinea pigs

Forty weaned male guinea pigs of 208.20±6.62 g mean body weight were divided into 4 groups of 10 animals in a randomized block design. All of the guinea pigs were fed a basal diet [25% ground maize hay, 30% ground maize grain, 22% ground chickpea (Cicer arietinum L.), 9.5% deoiled rice bran, 6% soybean meal, 6% fish meal, 1.45% mineral supplement (without Zn) and 0.05% ascorbic acid] and available green fodder. Group I served as the control (no Zn supplementation), whereas 20 ppm Zn was added in the diet in groups II, III, and IV either as zinc sulfate (ZnSO₄), zinc amino acid complex (ZAAC), and ZnSO₄ ⁺ ZAAC in equal parts, respectively. Experimental feeding lasted for 70 d, including a 3-d digestibility trial. Blood was collected through cardiac puncture from four animals in each group at d 0 and subsequently at the end of experimental feeding. After 40 d of experimental feeding, four animals from each group were injected with 0.4 mL of Brucella abortus cotton strain-19 vaccine to assess the humoral immune response of the animals. After 10 wk of study, four animals from each group were sacrificed to study the concentration of Zn, Cu, Co, Fe, and Mn in the liver, pancreas and spleen. Results revealed no significant difference in the feed intake, body weight gain, and digestibility of the nutrients, except for crude protein (CP) digestibility, which was significantly (p<0.05) lower in group IV. Although concentrations of serum glucose, Ca, and P and the albumin:globulin (A:G) ratio were similar in the different groups, the total protein, albumin, and serum alkaline phosphatase activity were higher in all of the Zn-supplemented groups on d 70. The serum Zn levels at the end of experimental feeding were significantly higher in groups II and III, whereas serum Mn levels were found to be significantly (p<0.05) higher in groups III and IV. The organ weights (as percentage of body weights) did not show any differences among the treatment groups. Although the Mn concentration was significantly (p<0.05) higher in the pancreas, the Cu concentration was significantly (p<0.05) reduced in the spleen in all of the Zn-supplemented groups. The humoral immune response (antibody titer values) on d 14 of vaccination was significantly (p<0.05) higher in all of the Zn-supplemented groups. It was concluded that the 20-ppm level of Zn in the diet might be adequate for growth and nutrient utilization in guinea pigs, but supplementation of 20-ppm zinc significantly improved the immune response and impact was more prominent with the ZAAC (organic source) compared to ZnSO₄ (inorganic source).     

Iron depletion prevents adenine nucleotide decomposition and an increase of xanthine oxidase activity in the liver of the Long Evans Cinnamon (LEC) rat, an animal model of Wilson's disease.

The Long Evans Cinnamon (LEC) rat, which accumulates excess Cu in the liver as in patients with Wilson's disease, is a mutant strain displaying spontaneous hepatitis. It was reported that Fe, like Cu, increases in the liver and that the severity of hepatitis is modified by Fe in the diet. In this experiment, oxidative stress increased by Fe was investigated before the onset of hepatitis. To examine the effect of Fe on the progress into hepatitis, LEC female rats were fed an Fe-regular (Fe 214 microg/g; Fe(+) group) or an Fe-restricted (Fe 14 microg/g; Fe(-) group) diet from 53 days of age for 35 days. Fischer rats were also fed as control animals. Adenine nucleotide decomposition was determined as an index of oxidative stress based on xanthine oxidase activity. The size of the hepatic pool of adenine nucleotides (ATP+ADP+AMP) was significantly smaller in LEC rats than Fischer rats. The energy charge (ATP+0.5ADP)/(ATP+ADP+AMP) was smaller in Fe(+) groups than in Fe(-) groups. In the LEC rat liver, the Fe concentration in the Fe(+) group was 160% of that in Fe(-) group and the correlation coefficient between the hepatic Fe concentration and the energy charge was significant. In this strain, an increase of xanthine oxidase activity resulted in an increase of xanthine, an oxidized metabolite of hypoxanthine in the liver. The results suggest the involvement of the Fe in the progression into hepatitis in the LEC rat, even if the dietary Fe concentration is similar to that of commercial diet.     

Effect of glutathione depletion on removal of copper from LEC rat livers by tetrathiomolybdate

Tetrathiomolybdate (TTM) is a powerful and selective copper (Cu) chelator that is used as a therapeutic agent for Wilson disease. TTM is the sole agent that can remove Cu bound to metallothionein (MT) in the livers of Long-Evans rats with a cinnamon-like coat color (LEC rats). However, the administration of excess TTM causes the deposition of Cu and molybdenum (Mo) in the liver. In the present study, the effect of hepatic glutathione (GSH) depletion on the removal of Cu from the livers of LEC rats was evaluated to establish an effective therapy by TTM. Pretreatment with l-buthionine sulfoximine (BSO), a depletor of GSH in vivo, reduced the amounts of Cu and Mo excreted into both the bile and the bloodstream, and increased the amounts of Cu and Mo deposited in the livers of LEC rats in the form of an insoluble complex 4 h after the TTM injection. The results suggest that GSH depletion creates an oxidative environment in the livers of LEC rats, and the oxidative environment facilitates the insolubilization of Cu and Mo in the livers of LEC rats after the TTM injection. Therefore, the effect of TTM on the removal of Cu from the liver was reduced in the oxidized condition. Wilson disease patients and LEC rats develop liver injury caused by oxidative damage. From a clinical viewpoint, increasing in the GSH concentration is expected to enhance the effect of TTM.     

Effects of dietary zinc levels on the activities of enzymes, weights of organs, and the concentrations of zinc and copper in growing rats

Zinc (Zn) is an essential nutrient that is required in humans and animals for many physiological functions, including immune and antioxidant function, growth, and reproduction. The present study was performed to investigate the effects of three Zn levels, including Zn adequate (35.94 mg/kg, as a control), Zn deficiency (3.15 mg/kg), and Zn overload (347.50 mg/kg) in growing male rats for 6 wk. This allowed for evaluation of the effects that these Zn levels might have on body weight, organ weight, enzymes activities, and tissues concentrations of Zn and Cu. The results showed that Zn deficiency has negative effects on growth, organ weight, and biological parameters such as alkaline phosphatase (ALP) and Cu−Zn superoxide dismutase (Cu−Zn SOD) activities, whereas Zn overload played an effective role in promoting growth, improving the developments of organs and enhancing immune system. Hepatic metallothionein (MT) concentration showed an identical increase tendency in rats fed both Zn-deficient and Zn-overload diets. The actual mechanism of reduction of Cu concentration of jejunum in rats fed a Zn-overload diet might involve the modulation or inhibition of a Cu transporter protein by Zn and not by the induction of MT.     

Interaction between pesticides and essential metal copper increases the accumulation of copper in the kidneys of rats

Male Sprague-Dawley rats, weighing 175-200 g, six per group were fed AIN 93M diet (CON) or diets containing 500 mg Ca (LCa), 7 mg Zn (LZn), 2 mg Cu (LCu), 60 mg Zn (HZn), or 12 mg Cu (HCu) per kilogram of diet in the following combinations: control (CON), LCa+LZn (LC+Z), LCa+LZn+LCu (LC+Z+C), or HZn+HCu (HZ+C) without or with a pesticides mixture (PM); Endosulfan, Thiram, and Acephate were added to the diets at 25% of LD50/kg. Rats were fed for 2 wk (small intestinal changes) or 4 wk (tissues changes). Plasma Zn was 47% lower than CON in the experimental groups. Plasma Cu and ceruloplasmin concomitantly decreased in the LC+Z+C group and increased with the addition of PM. Kidney Cu was 40% lower in LC+Z+C group, than CON and increased by 31% with PM; in the HZ+C+PM group, kidney Cu was 38% higher than the HZ+C group. Mucosal and small intestines Ca declined by 47% in all experimental groups; PM increased Zn in the LC+Z+C and HZ+C groups; PM further decreased intestinal and mucosal Cu retention in the LC+Z+C and HZ+C groups. Data suggest that low levels of PM in the diet can induce Cu accumulation in the kidney when dietary Zn and Cu are low or high.     

Effects of low copper and high zinc intakes and related changes in Cu,Zn-superoxide dismutase activity on DMBA-induced mammary tumorigenesis

The effect of low copper and high zinc intakes on Cu,Zn-superoxide dismutase (Cu,Zn-SOD) activity and mammary tumorigenesis induced by 9,10-dimethyl-1,2-benzanthracene (DMBA) was investigated. Groups of 40 weanling female Sprague-Dawley rats were fed a modified AIN-76 diet containing the following (/kg diet): 1 mg Cu (0.016 mmol) and 30 mg Zn (0.459 mmol); 6 mg Cu (0.094 mmol) and 30 mg Zn (0.459 mmol) (control); or 6 mg Cu (0.094 mmol) and 150 mg Zn (2.295 mmol) for 21 wk. At 5 wk, 30 rats/group were given 4 mg (15.6 mumol) DMBA in corn oil intragastrically, and controls (10/group) received corn oil alone. Erythrocyte Cu,Zn-SOD activity was measured at 3, 5 (just before DMBA), 9, 13, 17, and 21 wk. The group fed the high-Zn diet had a slightly lower weight gain and food consumption. DMBA treatment had no effect on these parameters. Plasma and liver Cu concentration decreased in the low-Cu group. Femur zinc was significantly elevated in the high-Zn group. Erythrocyte Cu,Zn-SOD activity was decreased in the low-Cu group from 3 to 21 wk and was significantly elevated in the high-Zn group at 3 and 5 wk. In the low-Cu group, there were 5 nonmalignant adenomas and 3 malignant adenocarcinomas; in the control group, there were 4 adenomas and 3 adenocarcinomas; in the high-Zn group, there were 5 adenomas and 3 adenocarcinomas. No relationship between Cu,Zn-SOD activity and the presence of tumors could be found.     

Effect of high dietary zinc on plasma ceruloplasmin and erythrocyte superoxide dismutase activities in Copper-depleted and repleted rats

The effect of moderately high dietary zinc (Zn) on the activities of plasma (PL) ceruloplasmin (CP), and PL and erythrocyte (RBC) copper (Cu), Zn superoxide dismutase (SOD) was determined in weanling rats fed Cu-deficient (DEF; <1 mg Cu/kg), marginal (MAR; 2 mg Cu/kg), or control (CON; 5 mg Cu/kg) copper diets containing normal or high Zn (HZn; 60 mg/kg) for 4 wk and supplemented with oral Cu (CuS; 5 mg/L) in drinking water for 0, 1, 3, or 7 d. PL Cu decreased (67% compared to CON;p≤0.05) in the DEF and increased to control level after 3 d of CuS; increased in the MAR group after 1 d of CuS. HZn reduced overall PL Cu by 27% in all groups, but did not alter the linear increase in PL Cu between 0 and 3 d of Cu S. PL CP activity altered concomitantly with PL Cu levels: The time course of increase in CP activity after 0–3 d of CuS was not influenced by HZn in the diet and CP declined in the DEF group by 92%. There was no correlation between dietary Cu level and PL CP. PL SOD activity decreased by 46% (p≤.05) in the DEF group, increased to control activity after 1 d of CuS and declined slighty after 7 d; MAR diet did not alter PL SOD. HZn diet increased PL SOD activity in all groups by 150%, reduced activity in the DEF and MAR groups by 65 and 37% and delayed the recovery of PL SOD after CuS. RBC SOD declined in the DEF and MAR groups by 56 and 33% (p≤0.05) and did not respond to CuS; HZn diet did not influence RBC SOD activity. These data indicate that moderately high Zn in the diet reduces PL Cu, but not PL CP activity or the recovery of PL Cu or CP activity after oral CuS of Cu-deficient rats, modifies the response of PL SOD to dietary Cu, but does not influence RBC SOD activity.     

Identification of the zinc-binding protein specifically present in male rat liver as carbonic anhydrase III

A zinc (Zn)-binding protein that is present specifically in the livers of male adult rats was detected by HPLC with in-line detection by mass spectrometry (ICP MS). The Zn-binding protein was purified on Sephadex G-75 and G3000SW HPLC columns. and was identified as carbonic anhydrase III (CAIII) based on the amino acid sequence of a peptide obtained on lysyl endopeptidase digestion. CAIII is expressed as one of the major Zn-binding proteins in the livers of male rats in an age-dependent manner, a comparable amount of Zn to that of copper, Zn-superoxide dismutase (Cu,Zn-SOD) being bound to CAIII at 8 weeks of age. Castration at 4 or 8 weeks of age was shown to reduce Zn bound to CAIII to 47.5% of the sham-operated control level, suggesting that the sex-dependent expression of CAIII is partly regulated by a sex hormone, androgen. The concentration of CAIII in the livers of Long-Evans rats with a cinnamon-like coat color (LEC rats), an animal model of Wilson disease, was also estimated as Zn bound to CAIII and shown to be lower than that in Wistar rats before the onset of hepatitis. The concentration of CAIII was decreased specifically by repeated injections of cupric ions without the Cu,Zn-SOD concentration being affected.     

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.     

Metallothionein-1 and metallothionein-2 gene expression and localisation of apoptotic cells in Zn-treated LEC rat liver

The aims of the present work were to determine the effect of long-term treatment with zinc (Zn) on metallothionein (MT) concentrations and to study the levels of both MT-1 and MT-2 mRNAs in Long-Evans Cinnamon (LEC) rat liver. We also identified apoptotic cells comparing two cytochemical techniques. Thirteen rats received 50 mg zinc acetate daily by gavage, 13 rats received no treatment, and both groups were killed after 60 days. Finally four rats were killed 35 days after birth (T(0)). The results demonstrate that the Zn-treated group had higher levels of MT than both the untreated and basal ones. Quantification of mRNA indicates that the level of the Zn-treated group was significantly higher than the untreated group. Confocal fluorescent staining with monoclonal antibody (Mab) against single-strand DNA localised the hepatic cells that had chromatin condensation and nuclear fragmentation typical of apoptosis, especially in the untreated group sections. The intensity and quantity of fluorescence decreased in both the treated and basal groups. The higher sensitivity of Mab staining compared to TUNEL, which revealed both apoptotic and necrotic cells, reflects the different action mechanism of the two techniques. These findings confirm, in LEC rats, the important role of Zn in cellular protection in relation to MT expression and apoptotic processes as cellular responses to DNA damage by free radicals.     

Dietary pharmacological or excess zinc and phytase effects on tissue mineral concentrations, metallothionein, and apparent mineral retention in the newly weaned pig

Feeding pharmacological zinc (Zn) to weaned pigs improves growth, and dietary phytase improves P and Zn availability. Metallothionein (MT) increases in the duodenum, kidney, and liver of pigs fed 1000 mg Zn/kg with phytase or 2000 mg Zn/kg with or without phytase when fed for 14 d postweaning. The goal of this study was to determine the effects of feeding pharmacological Zn and phytase on tissue minerals, MT, mineral excretion, and apparent retention. Twenty-four newly weaned pigs (20 d; 7.2 kg) were individually fed twice daily, a basal diet supplemented with 0, 1000, or 4000 mg Zn/kg as Zn oxide, without or with phytase (500 phytase units [FTU]/kg) for 14 d, followed by a basal diet (100 mg Zn/kg) without phytase for 7 d. Pigs fed 4000 mg Zn/kg without phytase had higher (p=0.01) plasma, hepatic, renal Zn, renal Cu, and hepatic, renal, and jejunal MT than pigs fed the basal diet or 1000 mg Zn/kg. Duodenal MT was higher (p=0.0001) in pigs fed 1000 and 4000 mg Zn/kg than in pigs fed the basal diet. In pigs fed 1000 and 4000 mg Zn/kg, Zn loading occurred during the first 11 d of supplementation; by d 14, excess Zn was being excreted in the feces.