Jeffrey C. Philcox

Paracetamol hepatotoxicity in metallothionein-null mice.

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.

Metallothionein induction in cultured rat hepatocytes by arthritic rat serum, activated macrophages, interleukin-6, interleukin-11 and leukaemia inhibitory factor

Potential mediators of hepatic metallothionein (MT) synthesis in adjuvant-induced arthritis were investigated in cultured rat hepatocytes. Sera from arthritic rats (14 d post-adjuvant treatment) in the presence of Zn (50 µmol/L) + dexamethasone (Dex; 1 µmol/L) increased metallothionein (MT) accumulation by 34% above that obtained with control rat serum with Zn + Dex. Endogenous IL-6 activity in serum from arthritic rats was 93 ± 49 U/mL and was undetectable in control rat serum. The activities of TNF, IL-1 and corticosterone concentrations were the same in control and arthritic rats. The accumulation of MT in hepatocytes in the presence of Zn (10 µmol/L) + Dex (1 µmol/L) was enhanced 29% and 49% by media from lipopolysaccharide (LPS)-stimulated peritoneal macrophage (PMM) and Kupffer cell cultures (KCM), respectively. The response with PMM and KCM was quantitatively the same as that with interleukin-6 (IL-6). Analysis of PMM and KCM showed activities of 1,000–10,000 U/mL for IL-6, 100–1000 U/mL for TNF and <10,000 U/mL for IL-1, the latter detected only in PMM. LPS alone enhanced the accumulation of MT above Zn + Dex in a dose dependent manner. A significant LPS response was obtained at 5 mg/L with a maximal stimulation above Zn + Dex of 38% at 10 mg/L. This direct stimulation of MT by LPS was not part of the response observed with PMM and KCM where the final LPS concentration in culture was only 0.1 mg/L. Other cytokines capable of synergy with Zn + Dex on MT synthesis were investigated. Interleukin-11 (IL-11) increased the Zn + Dex induction in a dose dependent manner with maximal stimulation at 100 U/mL of 40%. A small stimulation of 12% above Zn + Dex was obtained with leukaemia inhibitory factor (LIF) at concentrations greater than 100 U/mL. No enhancement of the Zn + Dex response was obtained with interleukin-3 (1000 U/mL), interleukin-4 (10 µg/L), platelet activating factor (5 nmol/L) or granulocyte-colony stimulating factor (5 µg/L). Neither IL-11 nor LIF enhanced the response obtained with Zn + Dex + IL-6. The results demonstrate that mediators present in arthritic rat serum and in LPS-stimulated PMM and KCM cause a quantitatively similar response on MT accumulation as IL-6. IL-11 and to a lesser extent LIF, are also potential mediators of MT synthesis in inflammation.

Regional distribution and localization of Zinc and metallothionein in the intestine of rats fed diets differing in zinc content

BACKGROUND Zinc (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. METHODS Male-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. RESULTS Most 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. CONCLUSION Mucosal 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.

Regional Distribution of Metallothionein and Zinc in the Mouse Gut - Comparison With Metallothionien-Null Mice

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.

Helicobacter-induced Gastritis in Mice Not Expressing Metallothionein-I and II*

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.

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-α 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.

Activation of glycolysis by zinc is diminished in hepatocytes from metallothionein-null mice

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 µM), 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 µM). Hepatocytes from MT −/− mice had significantly lower basal rates of glycolysis and demonstrated increased glycolysis only at Zn concentrations of 50 µM 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 induction in freshly isolated rat hepatocytes

The control of metallothionein (MT) synthesis was investigated in freshly prepared rat hepatocytes in experiments of short-term duration. Viability and metabolic function were maintained in incubations of 6-h duration. MT synthesis was measurable in hepatocytes from fed rats at Zn concentrations down to 1 μM. Zn and dexamethasone induced concentration-dependent increases in the synthesis of MT with maximal increases above the 5-h control of 3.2- and 2.5-fold, respectively. Zn induction of MT was first measurable at 2 h and was inhibited by actinomycin C. Although initial (0 h) MT concentrations in hepatocytes from fasted rats were double those from fed rats, after 6-h incubation in the presence of 50 μM Zn, the fasted rat hepatocytes showed only half the MT concentrations of the fed rat hepatocytes. Glucagon and interleukin-6 (IL-6) were less effective inducers and increased MT synthesis by 28 and 17%, respectively. IL-6 (100 U/mL) was found to have an additive effect on MT synthesis above that of Zn alone (1–50 μM) or Zn plus dexamethasone (1 μM). A supernatant from LPS-stimulated macrophages increased MT synthesis by 40%. The basal MT synthesis was not increased by either tumor necrosis factor-α (TNF-α) or interleukin-1 (IL-1).All incubations were carried out in the presence of RPMI 1640 medium with Hepes (20 mM), bicarbonate (24 mM), and fatty acid-free albumin (FAFA; 0.5% w/v). MT synthesis was also seen using Krebs bicarbonate buffer with glucose (10 mM), Hepes (20 mM), and FAFA (0.5% w/v), and although the level of MT synthesis was less than in RPMI, the increases in concentrations of MT at 5 h were 225, 139, 36 and 20% for Zn, dexamethasone, glucagon, and control, respectively.It is concluded that MT synthesis occurs in freshly prepared hepatocytes and that these cells are responsive to some of the established inducers of MT. This system enables the study of MT synthesis in individual rats in various metabolic and pathological states.

Tolerance to cadmium hepatotoxicity by metallothionein and zinc: in vivo and in vitro studies with MT-null mice

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.

Metallothionein and zinc homeostasis during tumor progression : effect of methotrexate treatment

Zinc homeostasis was studied during the induction, growth, and methotrexate (MTX) treatment of Dark Agouti rat mammary adenocarcinomas (DAMA). A progressive fall in plasma Zn concentration (pZn), significant at a tumor burden of less than 1% body weight (bw), was sustained during tumor enlargement to give a 54% reduction in pZn at 16.3% bw (n=6/group). The hypozincemia was attributed to the increasing Zn demand for tumor growth. Zn content of the 16.3% bw tumors equaled that of muscle (normally 60% of total body Zn). Tumor metallothionein (tMT) was sufficient to bind <3% of total tumor Zn, and hepatic MT (hMT) remained at basal concentrations during early tumor growth, doubling only in the presence of significant necrosis in large tumors. Methotrexate (MTX, 0.5 mg/Kg im x 2 d) at respective tumor burdens of 5 and 10% bw (n=9, 10/group) gave 2 therapeutic effects, dependent on tumor size: 1.5% bw tumors in 7 rats remained close to their original size until experiment end when pZn, hMT, and tMT were typical of 5% bw untreated tumors. 2. Tumors in 5 rats given MTX at 10% bw had marked subcapsular necrosis and regression to a size similar to those in group 1; pZn returned toward normal, whereas hMT was 6 times its 5% bw counterpart. Host weight loss was significantly reduced, as were tumor-associated changes in plasma glucose and calcium.In summary, neither tMT nor hMT appears to play a role in the hypozincemia that follows DAMA Zn sequestration and growth. Critically timed MTX can result in tumor regression and return of plasma Zn, Ca, and glucose toward normal. This is associated with an increase in hMT and reduction in host weight loss, suggesting a flow of Zn from the resorbing tumor to the host, enabling the synthesis of hMT and retention of host structural proteins.