Paola Irato

Altered Plasma and Mucosal Concentrations of Trace Elements and Antioxidants in Active Ulcerative Colitis

BACKGROUND The production of free radicals is increased in inflammatory bowel disease, and trace elements are crucial components of several antioxidants. Trace elements deficiency may therefore compromise the defense against oxidative damage. The aims of this study were to measure plasma and tissue concentration of trace elements and antioxidants and to relate this to disease activity. METHODS A 10-ml blood sample and six colonic biopsy specimens were obtained from 24 patients with either active ulcerative colitis or in remission and 10 patients with irritable bowel syndrome for measurement of trace elements and trace element-dependent enzymes. RESULTS Patients with moderately active disease had significantly lower plasma iron, selenium, and glutathione peroxidase levels than patients in remission and controls, whereas no significant differences were found between the zinc and copper values of patients and controls. Mucosal concentrations of zinc and metallothionein were reduced, whereas iron and glutathione peroxidase concentrations were increased in patients with endoscopically active disease as compared with controls and patients in remission. CONCLUSIONS Patients with ulcerative colitis have altered plasma and tissue levels of trace elements and antioxidant-related enzymes. The resulting reduced protection against free radicals may contribute to the inflammatory process.

Diagnosis and management of Wilson's disease: results of a single center experience.

Aims To report on the diagnostic features, management, and clinical outcome after different treatments of Wilsons disease patients followed over a mean period of 15 years. Patients Thirty-five patients with Wilsons disease referred to the University of Padovas Department of Gastroenterology for diagnosis or treatment were observed for a mean 15 years. The diagnosis was based on clinical symptoms, laboratory tests (ceruloplasmin, urinary, and hepatic copper concentrations), and uptake of the radiostable isotope 65Cu into the plasma protein pool. Hepatic Cu content was measured by regular follow-up biopsies. Neurologic outcome after therapy was assessed using a newly developed scoring system. Results Twenty-three (65.7%) patients presented with liver disease; 12 (34.3%) had mixed neurologic and hepatic involvement. All patients had been initially treated with either penicillamine (23) or zinc sulfate (12). The neurologic symptoms became worse or remained stationary in 75% of those treated with penicillamine, whereas zinc treatment improved these symptoms in 90% of treated cases. Both treatments were effective in improving the hepatic symptoms. No differences in hepatic Cu content emerged between follow-up biopsies in either treatment group. Six patients (26%) had to abandon the penicillamine treatment due to side effects. In all, 4 patients underwent liver transplantation, which was successful in 3, with a mean survival after transplantation of 4.6 years; the fourth, who had a severe neurologic impairment, died of central pontine myelinolysis. Conclusions Penicillamine and zinc can effectively treat Wilsons disease, though the side effects of penicillamine may be severe enough to prompt its suspension. Liver transplantation remains the treatment of choice for end-stage liver disease.

Zinc Therapy Increases Duodenal Concentrations of Metallothionein and Iron in Wilson's Disease Patients

Objective:Wilsons disease is effectively treated by zinc administration which, in vitro, increases metallothionein concentrations. To ascertain whether the latter also occurs in humans we measured metallothionein and trace element concentrations in the duodenal mucosa of 15 Wilsons disease patients: 12 treated with zinc sulphate, two treated with penicillamine, and one not yet on treatment. The control group consisted of 17 patients with dyspepsia, who underwent the same study protocol.Methods:Metallothionein and trace element concentrations were measured in duodenal mucosa biopsies according to the silver-saturation hemolysate method and atomic absorption spectrophotometry.Results:Metallothionein concentrations increased by 1500% after zinc and 150% after penicillamine in Wilsons disease patients, with respect to controls who had negative endoscopy and Wilsons disease patients who were not treated. A significant correlation was found between metallothionein and duodenal zinc concentrations. Mucosal iron concentration increased in Wilsons disease patients whether they were treated with zinc or penicillamine. Duodenum with duodenitis also had significantly increased iron levels compared with normal duodenum.Conclusions:Zinc administration increases intestinal metallothionein in Wilsons disease patients. The blockade of copper absorption and its elimination in the stools on desquamation of the intestinal cells probably explains one of the mechanisms underlying the effect of zinc treatment. Despite normal endoscopy, Wilsons disease patients present increased mucosal iron concentrations similar to those in controls with duodenitis. Metallothionein may therefore prevent oxidative damage caused by metal toxicity.

Metal interaction and regulation of Tetrahymena pigmentosa metallothionein genes.

The patterns of expression of two metallothionein (MT) genes, MT-1 and MT-2, previously identified as Cd-MT and Cu-MT, were analysed in Tetrahymena pigmentosa in response to metal inducers cadmium, copper and zinc and to a mixture of copper and cadmium at appropriate concentrations. Co-treatment induces synergistic accumulation of both metals and higher expression of MT-mRNAs in the first few hours. mRNA levels were observed not to completely correlate with MT-protein levels, suggesting that post-transcriptional regulation may be involved in MT induction. MT-1 is induced to higher levels than MT-2. Zinc does not induce any MT expression. The lowest level of mRNA was observed for MT-2, induced only by copper. Cadmium is a powerful inducer of the MT-1 gene, although a very low transcription rate by copper occurs in the first hour.

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.

Cadmium-thionein in Tetrahymena thermophila and Tetrahymena pyriformis.

The treatment of Tetrahymena thermophila with cadmium causes a reduction in growth rate according to dose; almost all the metal is accumulated in the cytosol where the Zn content is also increased threefold. Bio-Gel and Water 160 (HPLC) column chromatography show that Cd and Zn are bound to a protein with an ultraviolet (UV) spectrum that appears to be similar to that of Cd-metallothioneins isolated by higher organisms, but its molecular weight is greater: about 28 000 D, comparable to that of metallothionein isolated from Tetrahymena pyriformis. Further purification of these proteins by ion exchange chromatography revealed the presence of two peaks, considered as two isoforms of the metallothioneins present in both T. thermophila and T. pyriformis (MT 1 and MT 2). Their amino acid analyses confirmed that they are different isometallothioneins, MT 1 and MT 2, with about 30% cysteine, and aspartic acid, glycine and lysine as major amino acids. From our analyses we may conclude that Tetrahymena pyriformis MTs are similar to those present in invertebrates and vertebrates, while Tetrahymena thermophila MTs are peculiar in that they have cyclic amino acid histidine in both MT 1 and MT 2; furthermore, aromatic amino acid phenylalanine is also present in MT 2.

Identification, cloning and characterisation of a novel copper-metallothionein in tetrahymena pigmentosa. Sequencing of cDNA and expression.

The protist Tetrahymena pigmentosa accumulates large amounts of metal ions, particularly cadmium and copper. This capability is linked to the induction of metallothioneins (MTs), cysteine-rich metal-binding proteins found in protists, plants and animals. The present study focuses on a novel inducible MT-isoform isolated from Tetrahymena after exposure to a non-toxic dose of copper. The cDNA sequence was determined utilising the partial peptide sequence of purified protein. The Cu-MT cDNA encodes 96 amino acids containing 28 cysteine residues (29%) arranged in motifs characteristic of the metal-binding regions of vertebrate and invertebrate MTs. Both the amino acid and nucleotide sequences differ, not only from other animal MTs, but also from the previously characterised Tetrahymena Cd-MT. Both MTs contain the structural pattern GTXXXCKCXXCKC, which may be proposed as a conservative sequence of Tetrahymena MTs. Cu-dependent regulation of MT expression was also investigated by measuring MT-mRNA and MT levels. MT synthesis occurs very quickly and MT contents increase with Cu accumulation. The induction of Cu-MT mRNA is very rapid, with no observable lag period, and is characterised by transient fluctuation, similar to that described for Cd-MT mRNA. The data reported here indicate that, also in the unicellular organism Tetrahymena, two very different MT isoforms, which perform different biological functions, are expressed according to the inducing metal, Cu or Cd.

The effect of zinc and the role of p53 in copper-induced cellular stress responses

Metals can directly or indirectly cause an increase in reactive oxygen species (ROS) accumulation in cells, and this may result in programmed cell death. A number of previous studies have shown that zinc (Zn) modulates mitogenic activity via several signalling pathways, such as AKT, mitogen‐activated protein kinase (MAPK), nuclear factor‐kappa B (NF ‐κB), AP‐1 and p53. The exact role that Zn plays in the regulation of apoptosis remains ambiguous. Intracellular free Zn modulates p53 activity and stability, and excess Zn alters the p53 protein structure and down‐regulates p53s binding to DNA. Copper (Cu) accumulation causes apoptosis that seems to be mediated by DNA damage and subsequent p53 activation. Cu can also displace Zn from its normal binding site on p53, resulting in abnormal protein folding and disruption of p53 function. In spite of the induction of the tumour suppressor p53, hepatic Cu accumulation significantly increases the risk of cancerous neoplasm both in humans and rats, suggesting that p53 function may be impaired in these cells. It is generally understood that imbalances in Cu and Zn levels may lead to a higher prevalence of p53 mutations. An increased number of p53 mutations have been found in liver samples from Wilsons disease (WD) patients. High levels of the p53 mutation most probably contribute to the pathogenesis of cancer in individuals with WD, but the cause and effect are not clear. The protein p53 also plays a crucial role in the transcriptional regulation of metallothionein, which indicates a novel regulatory role for p53. This review discusses the central role of p53 and the redox‐inert metal Zn in the cellular stress responses induced by the redox active biometal Cu. Copyright

Effect of penicillamine and zinc on iron metabolism in Wilson's disease

Objective. The physiology of iron metabolism in Wilsons disease is largely unknown, and there is a paucity of data on the real presence and progression of iron accumulation. The purpose of this study was to assess the iron metabolism parameters, including hepatic iron concentration, in follow-up liver biopsies and serum, and urinary pro-hepcidin. Material and methods. Twenty-three Wilsons disease patients undergoing long-term treatment were enrolled in the study. Results. Hepatic iron content was significantly increased in penicillamine-treated patients compared with zinc-treated patients. Serum and urinary pro-hepcidin concentrations were significantly higher in Wilsons disease patients than in healthy volunteers, despite a normal biochemical pattern of iron metabolism. Conclusions. Long-term penicillamine treatment seems to be responsible for a more marked iron accumulation in the liver. This observation may justify a revision of long-term Wilsons disease treatment modalities with penicillamine. The finding that serum and urinary pro-hepcidin is significantly increased in Wilsons disease patients compared with healthy volunteers suggests a role for hepcidin in iron metabolism in Wilsons disease, but this needs to be confirmed by a study of hepatic hepcidin expression in these patients.

Effect of zinc supplementation on trace elements and intestinal metallothionein concentrations in experimental colitis in the rat

BACKGROUND AND AIM Zinc enhances cell protection against infection and injury and the healing processes themselves. We evaluated the effect of zinc supplementation at different doses on a model of experimental colitis in the rat. METHODS Colitis, induced by intra-rectal instillation of dinitrobenzen-sulphonic acid, was assessed at 1 week by examining: general outcome and macroscopic damage, myeloperoxidase activity, mucosal zinc, iron and metallothionein concentrations. Rats received zinc sulphate, 2 mg/kg or 30 mg/kg, twice a day by gavage for 9 days, starting 3 days before the induction of colitis, or intrarectal instillation of zinc (20 mg/kg) once daily starting 8 hours after the induction of colitis and for 6 days thereafter RESULTS Zinc-treated rats had less diarrhoea, higher body weight and lower colonic weight than untreated rats but no effect was observed on macroscopic inflammation, adhesions, colonic distension and neutrophil infiltration of the colonic mucosa. Zinc supplementation did not affect mucosal iron and zinc concentrations or plasma zinc levels in colitic rats. Metallothionein synthesis was induced in control rats and to a lesser extent in colitic rats. CONCLUSION Zinc administration induces metallothionein synthesis but has little effect on the short-term course of experimental colitis.