BACKGROUND:We have previously demonstrated that ethanol teratogenicity in mice is related to the maternal expression of metallothionein (MT), a zinc (Zn)-binding protein. Ethanol induces maternal liver MT, which causes plasma Zn concentrations to decrease as Zn moves into the liver. During pregnancy it is suggested that this change decreases fetal Zn supply and contributes to abnormal development. Here we investigated whether maternal Zn supplementation at the time of ethanol exposure reduces teratogenicity.METHODS:Mice were injected with 25% ethanol (0.015 ml/g intraperitoneally at 0 and 4 hr) and ZnSO4 (2.5 microg Zn/g subcutaneously at 0 hr) and were killed over 16 hr to ascertain changes in plasma Zn. Plasma Zn concentrations peaked at 2 hr, where levels were 5-fold normal and then returned toward normal over 14 hr. Pregnant mice were treated in a similar manner on gestation day 8 with saline, saline + Zn, ethanol + Zn, or ethanol alone, and fetal abnormalities were assessed on gestation day 18.RESULTS:External abnormalities were most prevalent in offspring from dams treated with ethanol. Zn treatment at the time of ethanol exposure reduced the incidence of fetal abnormalities to basal levels. Litters from dams treated with ethanol + Zn contained more fetuses and fewer fetal resorption sites compared with those from ethanol-treated dams.CONCLUSIONS:These findings demonstrate that Zn supplementation at the time of ethanol exposure significantly negates the deleterious effects of ethanol on the fetus.

BACKGROUND During pregnancy, exposure to lipopolysaccharide (LPS) can lead to abortion, preterm delivery, and teratogenicity. The mechanisms underlying these effects are unclear. Both LPS and ethanol are potent inducers of liver metallothionein (MT), a key Zn binding protein. The teratogenic effects of ethanol have been linked to MT-induced changes in maternal-fetal Zn homeostasis, leading tofetal deficiency. This study was designed to assess whether the teratogenic effects of LPS are also related to MT induction and changes in Zn homeostasis. METHODS Non-pregnant normal (MT +/+) and MT-null (MT -/-) mice were injected subcutaneously with 0.5 microg/gm LPS and killed over 48 hr. In MT +/+ mice, liver MT concentrations were elevated from 6 hr, and were maximal at 24 hr (30-fold basal), whereas liver Zn levels were also increased from 6 hr. Plasma Zn concentrations decreased by 80% at 6 hr, and were below normal between 6 and 24 hr. In MT -/- mice, plasma Zn levels were increased from basal between 6 and 16 hr. Dams were injected with LPS, saline, or LPS and ZnSO4 (2 microg/gm, MT +/+ only) on Day 8 of gestation (GDS), killed on GD18, and the fetuses examined for malformations. RESULTS External abnormalities were most prevalent in fetuses from MT +/+ dams exposed to LPS, where 34% of fetuses in each litterwere affected. MT +/+ dams treated with LPS and ZnSO4, and MT -/- dams treated with LPS had litters in which 5.4 and 4.8% of fetuses were abnormal respectively. CONCLUSIONS The findings of this study strongly support the hypothesis that LPS teratogenicity is mediated at least in part by MT-induced changes in maternal Zn homeostasis,which compromises fetal Zn supply.

BACKGROUND:Helicobacter pylori a primary cause of gastritis and peptic ulcer disease, is associated with increased production of reactive oxygen species within the gastric mucosa. Metallothionein (MT), a low-molecular-weight, cysteine-rich, metal-binding ligand, has been shown to sequester reactive oxygen species and reduce tissue damage. This study investigates the role of MT in H. pylori-induced gastritis in mice.MATERIALS AND METHODS:Control (MT+/+) and MT-null (MT-/-) mice were inoculated with either 1 x 108H. pylori or H. felis, and were infected for 4, 8 and 16 weeks or 8 weeks, respectively. H. pylori load was determined by culture. Myloperoxidase activity and MT levels were also determined.RESULTS:The stomachs of H. felis-infected mice were more severely inflamed than those of H. pylori-infected mice. H. felis-induced gastritis was more severe (p =.003) in MT-/- than in MT+/+ mice. MT-/- mice also had higher (60%; p <.05) H. pylori loads than MT+/+ mice 4 weeks after infection but not 8 or 16 weeks after infection. Myloperoxidase activity with H. pylori was similar between MT+/+ and MT-/- mice. Thirty-three per cent greater (p <.05) myloperoxidase activity was observed in MT-/- than in MT+/+ mice infected with H. felis. In MT+/+ mice infected with H. pylori, liver MT was increased by 33 and 39% (p <.05) at 8 and 16 weeks, respectively, whereas gastric MT increased by 46% (p <.05) at 4 weeks and declined to baseline levels at 8 and 16 weeks.CONCLUSIONS:Mice lacking MT are more susceptible to H. pylori colonization and gastric inflammation, indicating that MT may be protective against H. pylori-induced gastritis.

Metallothioneins (MTs) are intracellular, low molecular, low molecular weight, cysteine-rich proteins. Ubiquitous in eukaryotes, MTs have unique structural characteristics to give potent metal-binding and redox capabilities. A primary role has not been identified, and remains elusive, as further functions continue to be discovered. The most widely expressed isoforms in mammals, MT-1 and MT-2, are rapidly induced in the liver by a wide range of metals, drugs and inflammatory mediators. In teh gut and pancreas, MT responds mainly to Zn status. A brain isoform, MT-3, has a specific neuronal growth inhibitory activity, while MT-1 and MT-2 have more diverse functions related to their thiolate cluster structure. These include involvement in Zn homeostasis, protection against heavy metal (especially Cd) and oxidant damage, and metabolic regulation via Zn donation, sequestration and/or redox control. Use of mice with altered gene expression has enhance our understanding of the multifaceted role of MT, emphasised in this review.

The potential of the metal-binding protein, metallothionein, in assessing the progression of normal oesophagus through Barrett's to adenocarcinoma was investigated. Metallothionein was quantitatively determined in resected tissues from patients undergoing oesophagectomy for high grade dysplasia/adenocarcinoma and in biopsies from patients with Barrett's syndrome. In 10 cancer patients, metallothionein concentrations in adenocarcinoma were not significantly different from normal oesophagus, although six had elevated metallothionein concentrations in the metaplastic tissue bordering the adenocarcinoma. In 17 out of 20 non-cancer patients with Barrett's epithelium, metallothionein was significantly increased by 108% (P < 0.004). There was no association between the metallothionein levels in Barrett's epithelium and the presence of inflammatory cells, metaplasia or dysplasia. Metallothionein is a marker of progression from normal to Barrett's epithelium but is not increased in oesophageal adenocarcinoma.

Zn may have an important protective role in the respiratory epithelium and Zn deficiency may enhance airway inflammation and epithelial damage. The effects of mild nutritional Zn deficiency on airway hyperresponsiveness (AHR) and airway inflammation in mice sensitized and challenged with ovalbumin (OVA) to induce an allergic response were investigated. Balb/c mice were given Zn normal (ZN, 50 mg/kg Zn) or Zn limited diets (ZL, 14 mg/kg Zn) before and during induction of allergic airway inflammation, with appropriate controls (saline-treated, SAL). ZL mice had greater levels of AHR than ZN mice, regardless of presence or absence of allergic inflammation. These mice also had increased eosinophilia and mucus cell hyperplasia compared with ZN mice. Second, ZN and ZL OVA-treated mice had significant decreases in airway epithelial Zinquin fluorescence, indicating a lowered availability of Zn compared with their SAL-treated counterparts. In contrast, the pro-apoptotic protein caspase-3, which was co-localized with Zn in the apical epithelium, was significantly increased in both ZN and ZL OVA-treated mice. Immunologically active caspase-3 and apoptosis were increased in OVA-treated mice, especially the ZL group. These findings provide the first data for adverse effects of Zn deficiency on the respiratory epithelium and support a role for altered Zn homeostasis and caspase upregulation in asthma.

The protective role of metallothionein (MT) in Cd-mediated hepatotoxicity was investigated in vivo and in vitro. Following injection of Cd (2 mg/kg, intraperitoneal or subcutaneous) hepatoxicity was significantly greater at 20 h in metallothionein-null (MT-/-) mice, compared with controls (MT+/+). The decrease in the blood and liver glucose concentrations correlated with the extent of hepatotoxicity, with blood glucose 43% lower in MT-/- mice. Zinc (50 microM) and/or Dex (1 microM) were used in hepatocyte cultures to raise MT 2-5-fold. When Cd at 10 microM was co-treated with Zn and/or Dex, lactate dehydrogenase (LD) leakage in the MT+/+ and MT-/- hepatocytes was reduced only when Zn was present. Cellular glutathione (GSH) was the same in control MT+/+ and MT-/- cultures and was uninfluenced by Zn and Dex. After treatment with 5 and 10 microM Cd, GSH levels were lower in MT-/- than MT+/+ hepatocytes in the control and Dex groups. Higher GSH concentrations were maintained in Zn co-treated cultures from both genotypes, indicating that the superior protective effect of Zn may in part derive from its influence on cellular GSH. Pre-treatment with Zn and/or Dex provided no further protection than co-treatment. Tolerance to brief (15 min) Cd exposure was also investigated in the presence of MT inducers including progesterone (100 microM). Zn, Dex and progesterone treated hepatocytes had less LD leakage than controls with Zn giving the greatest protection (LD leakage 18% of controls at 100 microM Cd). Zn pre-treated cells had higher cytosolic/particulate ratios of Cd. These findings demonstrate that MT protects primary cultures of mouse hepatocytes from short-term exposure to Cd. Zn enhances the protection through MT and non-MT mechanisms.

Background: Ethanol causes significant teratogenicity in normal (MT+/+) but not metallothionein-null (MT−/−) fetuses. Impaired maternal fetal zinc (Zn) transfer is indicated, because ethanol significantly reduces plasma Zn concentrations in MT+/+ dams while increasing concentrations in MT−/− dams. In this study we examined maternal-fetal Zn homeostasis in response to ethanol in MT+/+ and MT−/− mice and the origins of the increase in plasma Zn in MT−/− mice. Methods and Results: Mice were treated with saline or ethanol (0.015 ml/g intraperitoneally at 0 and 4 hr) on day 12 of gestation. An additional subcutaneous injection of 65 Zn tracer was administered after the second ethanol injection before mice were killed 3 hr later. Maternal liver MT levels were not different between ethanol and saline MT+/+ mice. Both liver Zn and 65 Zn levels were higher in MT+/+ mice. Plasma Zn concentrations were higher in MT−/− mice, with MT−/− ethanol-treated mice having levels greater than those of MT−/− saline-treated controls. MT+/+ ethanol-treated fetuses exhibited lower 65 Zn transfer and whole Zn concentrations compared with MT+/+ and MT−/− saline and MT−/− ethanol fetuses. So we could examine changes in plasma Zn after ethanol treatment, MT+/+ and MT−/− mice were injected with 65 Zn 3 days before they received ethanol treatment. Muscle and skin showed a decrease in 65 Zn retention in both genotypes over 3 hr. There was a trend toward greater 65 Zn release from skin and muscle at an earlier time in MT−/− mice: 24% vs. 2% decrease (MT−/− vs. MT+/+) for muscle and 28% vs. 15% decrease (MT−/− vs. MT+/+) for skin at 2 hr. Conclusions: The results show (a) that ethanol interferes with the transfer of Zn to the fetus, and that this is MT dependent, and (b) that the increase in plasma Zn seen in MT−/− mice after ethanol administration is a result of Zn release from the skin and muscle, in the absence of hepatic Zn sequestration.

Normal metallothionein [(MT)+/+] and MT-null (MT-/-) mice were used to examine the influence of MT on Zn retention and the metabolic consequences of 2 d food deprivation, with and without inflammation induced by intraperitoneal injection of bacterial endotoxin lipopolysaccharide (LPS). LPS reduced fecal Zn concentration in MT+/+ mice from 5.9 +/- 0.2 micromol/g on d 1 to 2.2 +/- 0.2 micromol/g on d 2, but not in MT-/- mice, 5.9 +/- 0.2 and 5.7 +/- 0. 5 micromol/g, respectively. MT+/+ mice fed an 8 mg Zn/kg diet and injected with LPS excreted 40% less Zn over 2 d than their MT-/- counterparts. Starvation for 2 d did not lower fecal Zn concentration in either genotype, although in MT+/+ mice, urinary Zn excretion was reduced from 12.7 +/- 1.3 nmol on d 1 to 5.9 +/- 1.8 nmol on d 2 and plasma Zn concentration was lowered to 9.8 +/- 0.4 micromol/L. Zn was not reduced in urine or plasma of MT-/- mice, with respective values of 10.8 +/- 2.0 nmol on d 1, 9.3 +/- 2.9 nmol on d 2 and 13.0 +/- 1.0 micromol/L. LPS injection resulted in much higher total liver Zn (677 +/- 27 nmol) and MT (106 +/- 2 nmol Cd bound/g) than starvation (Zn = 405 +/- 21, MT = 9 +/- 3) in MT+/+ mice after 2 d, but did not further reduce urinary Zn. LPS-injected MT-/- mice had no rise in liver Zn or fall in plasma and urine Zn. MT-/- mice fed a Zn-deficient (0.8 mg Zn/kg) diet lost 10% of body weight over 25 d compared with no loss in MT+/+ mice. Despite this, MT-/- mice excreted no more Zn via the gut than did MT+/+ mice. In summary, MT inhibits intestinal Zn loss when highly expressed. When uninduced, typically during Zn deficiency, MT appears to conserve Zn and body mass by reducing only urinary and other nonintestinal Zn losses.

The influence of hepatic metallothionein (MT) and zinc (Zn) on glycolysis was investigated in primary cultures of mouse hepatocytes prepared from MT-normal (+/+) and MT-null (−/−) mice. In MT +/+ mice, a close relationship was observed between the Zn concentration in the incubation medium (10–150 µ M ), increased MT levels in the cells, and increased glycolysis (accumulation of lactate + pyruvate) over 24 h, with significant effects seen at physiological levels of Zn (10–25 µ M ). Hepatocytes from MT −/− mice had significantly lower basal rates of glycolysis and demonstrated increased glycolysis only at Zn concentrations of 50 µ M or greater. The lactate: pyruvate ratio was higher in the MT +/+ hepatocytes. The oxidation of endogenous fatty acid (accumulation of the ketone bodies, 3-hydroxybutyrate and acetoacetate) was initially greater in the MT +/+ hepatocytes, although only MT −/− hepatocytes showed increased ketone body production in response to Zn. The 3-hydroxybutyrate: acetoacetate ratio was higher in the MT +/+ hepatocytes and increased with increasing Zn concentrations. Intracellular Zn accumulation was 60% greater in the MT +/+ hepatocytes, with approximately 80% of the extra Zn associated with MT. The results implicate MT-associated Zn rather than increased intracellular Zn per se in the regulation of hepatic carbohydrate metabolism.

Metallothionein (MT) has been assigned a role in intestinal Zn absorption and secretion. The influence of MT was investigated in isolated segments of the small intestine from mice lacking the expression of MT I and II genes (MT−/−).

The influence of metallothionein (MT)(2) on Zn absorption was investigated in MT-null (MT-/-) and normal (MT+/+) mice fed Zn-depleted (ZnD) diets for 7 d and compared with those fed Zn-replete (ZnR) diets in a previous study. Mice were starved for 20 h, then administered an oral gavage of aqueous (65)ZnSO(4) solution at doses of 154, 770 or 1540 nmol of Zn, and the amount transferred into nongut tissues was determined 4 h later. (65)Zn transfer did not differ between genotypes in ZnR mice. However ZnD MT+/+ mice had a 30-40% greater transfer from the 154 and 770 Zn doses compared to ZnR MT+/+ mice. This was not observed in MT-/- mice. In MT+/+ mice, Zn depletion enhanced the induction of MT by Zn in the intestine and pancreas. (65)Zn uptakes in the liver and pancreas were greater in MT+/+ than MT-/- mice, and this was greater (50%) at the 154 and 770 doses in mice fed ZnD diets. Plasma Zn concentrations were raised to a similar extent in ZnR and ZnD MT-/- mice. ZnR MT+/+ mice had significantly lower plasma Zn levels than MT-/-mice; this difference was less marked in the ZnD mice. We conclude that a MT-facilitated enhancement in Zn absorption occurs in response to dietary Zn deficiency.

BACKGROUND:Ethanol profoundly affects fetal development, and this is proposed to be due primarily to a transient fetal zinc (Zn) deficiency that arises from the binding of Zn by metallothionein (MT) in the maternal liver. Zn homeostasis and fetal outcome were investigated in normal (MT+/+) and metallothionein-null (MT-/-) mice in response to ethanol exposure.METHODS/RESULTS:Mice were treated with saline or ethanol (0.015 m/g intraperitoneally at 0 and 4 hr) on day 8 of gestation (Gd8), and the degree of fetal dysmorphology was assessed on Gd18. The incidence of external abnormalities was significantly increased in offspring from MT+/+ dams exposed to ethanol, where 27.4% of fetuses were affected. MT-/- ethanol-, MT+/+ saline-, and MT-/- saline-treated dams had fetuses in which the frequencies of abnormalities were 2.2, 6.4, and 6.9%, respectively. To investigate Zn homeostasis, nonpregnant mice were killed at intervals over 16 hr after ethanol injection. Liver MT concentrations in MT+/+ mice were increased 20-fold by 16 hr, with a significant elevation evident by 4 hr, whereas liver Zn levels were also significantly increased by 2 hr and maintained for 16 hr. In parallel with these changes, plasma Zn concentrations in MT+/+ mice decreased by 65%, with minimum levels of 4.5+/-0.3 micromol/liter at 8 hr. Conversely, MT-/- mice exhibited increased plasma Zn concentrations, with peak values of 20.8+/-0.3 observed at 4 hr.CONCLUSION:These findings link the teratogenic effect of ethanol to the induction of maternal MT and the limitation of fetal Zn supply from the plasma.

BACKGROUNDZinc (Zn) is protective and enhances epithelial repair in gut diseases. In this study we investigate the localization and distribution of Zn and its binding protein, metallothionein (MT), in the gut of rats fed diets varying in Zn content.METHODSMale-Sprague Dawley rats were fed low, normal, high, or excess Zn in their diets (10, 100, 400, or 1000 mg Zn/kg, respectively) and killed 7 days later. Blood, liver, and gut tissues were collected. Tissue Zn was determined with atomic absorption spectrophotometery and MT with a Cd/haem affinity assay. Zn and MT were immunohistochemically localized in the small-intestinal wall with zinquin and an anti-MT antibody.RESULTSMost Zn in the intestinal wall was present in the mucosal scrapings, with 94% membrane-bound and 6% cytosolic, irrespective of dietary Zn. MT levels increased in all gut regions at dietary Zn levels above 100 mg Zn/kg. MT was 40% higher in the ileum than in other gut regions in rats fed low- and normal-Zn diets. The Zn content of the ileum was also 20% higher than that of other gut regions in rats fed low-, normal-, or high-Zn diets. Zn and MT were colocalized in the base of the intestinal crypts, most visibly in the ileum.CONCLUSIONMucosal cytosolic Zn and MT concentrations are increased only at high or excessive Zn intakes in all gut regions except the ileum, which can respond to a lower Zn intake. As the cytosolic Zn pool most likely influences mucosal protection and repair mechanisms, it is proposed that an increased MT may indicate the adequacy of oral Zn therapy in gut disease.

The distribution and excretion of endogenous Zn, including the role of the pancreas, were examined in fasted MT+/+ and MT-/- mice. At 3 and 6 h after receiving 65Zn tracer by subcutaneous injection, 65Zn levels were compared in tissues of MT+/+ and MT-/- mice. 65Zn levels were significantly higher in the liver and pancreas of the MT+/+ mice, whereas in the MT-/- mice, 65Zn levels were significantly higher in muscle, skin, and most of the gastrointestinal tract other than the stomach and upper small intestine. In MT-/- mice, 3% of the injected 65Zn was recovered in the luminal contents of the small intestine over 3-6 h, compared with <1.5% in the MT+/+ mice. A loading dose of Zn (150 microg, s.c.) sufficient to raise the plasma Zn concentration by fourfold to fivefold in both MT+/+ and MT-/- mice resulted in similar increases in pancreatic Zn levels in each genotype, although more Zn appeared in the lower small intestine of MT-/- mice. Pancreatectomy decreased the level of 65Zn in the small intestine of MT-/- but not MT+/+ mice. Longer-term studies over 4 days demonstrated few differences in tissue 65Zn between MT+/+ and MT-/- mice, with the exception of the pancreas, where 65Zn retention after fasting in MT-/- mice was half that of MT+/+ mice. MT-/- mice also had significantly lower Zn concentrations in the pancreas. Fecal excretion of 65Zn in MT-/- mice was greater than that of MT+/+ mice in the first 24 h (24.7 vs. 18.2% of injected dose; p < 0.05). Besides metallothionein (MT), there were no significant differences in the molecular weight distribution of Zn binding ligands in the lumen of the small intestine between MT+/+ and MT-/- mice. Mice lacking MT I and II lose more endogenous Zn into the gut because of a relative failure of the pancreas to retain Zn. However, increased Zn secretion via the small intestinal mucosa may also contribute to intestinal Zn loss in MT-/- mice.

The influence of metallothionein (MT) on Zn transfer into non-gut tissues was investigated in MT-null (MT−/−) and normal (MT+/+) mice 4 h after oral gavage of aqueous 65ZnSO4solution at doses of 154, 385, 770 and 1540 nmol Zn per mouse. Zn transfer was not significantly different between MT+/+ and MT−/− mice and was directly proportional to the oral dose (slope = 0.127, r = 0.991; 0.146, r = 0.994, respectively). Blood 65Zn and plasma Zn concentrations increased progressively in MT−/− mice at doses >154 nmol Zn, reaching levels of 2.4% of oral dose and 60 μmol/L, respectively, at the 1540 nmol Zn dose. The corresponding values for MT+/+ mice were approximately half, 1.0% and 29 μmol/L. Intergenotypic differences were found in tissue distribution of 65Zn within the body; MT−/− mice had higher 65Zn levels in muscle, skin, heart and brain, whereas MT+/+ mice retained progressively more Zn in the liver, in conjunction with a linear increase in hepatic MT up to the highest Zn dose. MT induction in the small intestine reached its maximum at an oral dose of 385 nmol Zn and did not differ at higher doses. Absorption of a 770 nmol 65Zn dose from a solid egg-white diet was only one fourth (MT+/+) and one eighth (MT−/−) of the Zn absorption from the same dose of 65Zn in aqueous solution. MT+/+ mice had greater (P < 0.05) Zn absorption from the egg-white diet than did MT−/− mice, indicating that gut MT confers an absorptive advantage, but only when Zn is incorporated into solid food.

The role of metallothionein (MT) in protecting the liver against paracetamol (PCT) toxicity was investigated in vivo and in vitro in mice lacking expression of MT-1 and MT-2 genes (MT -/-). In the fed, glycogen replete state, hepatotoxicity (PCT 300 mg/kg i.p.) at 6 h was significantly greater in MT -/- than MT +/+ mice. Plasma lactate dehydrogenase (LD) and alanine aminotransferase (ALT) were 5- and 13-fold greater respectively than in MT +/+ mice. Liver glycogen, glucose and zinc levels were significantly lower in MT -/- mice at this time. In contrast, hepatotoxicity (PCT 135 mg/kg i.p.) at 6 h was similar in both MT +/+ and MT -/- mice fasted 24 h, despite a doubling in liver MT in MT +/+ mice. No differences were found between MT -/- and MT +/+ mice in cytochrome P450 activity. Liver glutathione levels were the same in both groups of mice prior to fasting and were decreased to a similar extent (55-65%) following PCT treatment. Investigation of lower PCT doses (< or = 120 mg/kg) in fasted mice over 24 h demonstrated a greater susceptibility in female MT -/- mice with plasma LD, 2.4-fold and ALT, 7.5-fold greater than in MT +/+ mice at 120 mg/kg PCT. In male MT -/- mice, there was only a trend towards greater susceptibility at 110 mg/kg PCT compared to male MT +/+ mice, and at 120 mg/kg, both male genotypes were equally affected. Investigations with cultured hepatocytes supported the in vivo findings in that there was a trend towards greater toxicity (PCT at 1 and 5 mM for 24 h) in hepatocytes from fed MT -/- mice, with the difference diminished in association with greater hepatotoxicity in hepatocytes from fasted mice. Use of dexamethasone (Dex) to increase MT in the MT +/+ mouse hepatocytes protected from PCT toxicity. Zn alone was not protective. Zn plus Dex offered no protection despite higher MT levels. Generation of apo-MT with Dex may offer more protection than Zn-MT. In conclusion, MT -/- mice were more susceptible than MT +/+ mice to PCT toxicity in the fed state, but the increased susceptibility was much smaller, but still significant, when the effects of glycogen were minimised by fasting.

Gut Zn homeostatic responses to low, replete, and excess dietary Zn (10, 150, and 400 mg Zn/kg, respectively) were compared in mice with (MT+/+) and without (MT-/-) metallothionein (MT) expression. MT concentrations decreased progressively from stomach (12.9 nmol Cd bound/g) to colon (4.6 nmol Cd bound/g). Small intestinal MT was increased in mice fed the 400-mg Zn/kg diet (+130%, duodenum; +56%, jejunum; +29%, terminal ileum), but not in the stomach, cecum and colon. Zn concentrations were much higher in the distal gut at increasing Zn intakes in MT+/+ mice but to a lesser extent in MT-/- mice. On the 10-mg Zn/kg diet, MT-/- mice had 45% more Zn in the jejunum/ileum than MT+/+ mice. In fasted (20 h) mice, Zn concentrations in all gut regions were similar to those of MT+/+ mice fed the 10-mg Zn/kg diet, irrespective of prior Zn intake or genotype. Liver MT quadrupled in mice fasted after the 10-mg Zn/kg diet but only doubled after the 400-mg Zn/kg diet, a trend also present in gut MT. Glucagon administration stimulated gut as well as liver MT, implicating it as a major component of the MT response to fasting. MT-/- mice had five times more variation than MT+/+ mice in plasma Zn over all dietary groups. Together, these findings demonstrate that without MT, there is little modification of regional gut Zn concentrations in response to extremes of dietary Zn and poorer regulation of Zn homeostasis.