Halina Milnerowicz

The role of metallothionein interactions with other proteins

Metallothionein (MT) is a protein involved in numerous key processes, and the most important include zinc ion homeostasis, detoxification of heavy metals, and protection against oxidative stress. MT by interaction with other proteins fulfills its function, resulting in different effects in the body. Interaction of MT with ferritin, which causes a redox reaction, resulting in the reduction of Fe3+ stored in ferritin and a release of harmful Fe2+, was observed. Referring to the redox function of MT, it has been shown that the pair of GSH/GSSG modulates transfer of Zn between MT and Zn‐binding proteins. Furthermore, it was shown that GSSG, in the presence of GSH, interacts directly with MT. Apothionein‐MT can retrieve Zn from the transcription factors or Zn‐containing enzymes. Apothionein‐MT by taking Zn can deactivate metal‐dependent enzymes while Zn‐MT has the opposite effect. As the effect of MT interaction with low‐density lipoprotein receptors—megalin and lipoprotein receptor related protein 1, the uptake of Cd‐MT occurs and results in the disruption of many functions of proximal tubules. MT is involved in numerous processes and many of them are regulated by protein‐protein interactions. Possibly in the future MT will become a therapeutic agent, which will result in a breakthrough in the field of pharmacy and medicine.

Pro-inflammatory effects of metals in persons and animals exposed to tobacco smoke.

Metals present in tobacco smoke have the ability to cause a pro-oxidant/antioxidant imbalance through the direct generation of free radicals in accordance with the Fenton or Haber-Weiss reaction and redox properties. Metals can also interact with antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and small molecular antioxidants (glutathione) through binding to SH groups or by replacement of metals ions in the catalytic center of enzymes. Excessive free radicals production can induce an inflammatory response. The aim of this study was to review the information on the induction of inflammation by metals present in tobacco smoke such as lead (Pb), cadmium (Cd), arsenic (As), aluminum (Al), nickel (Ni) and mercury (Hg). In cellular immune response, it was demonstrated that radicals induced by metals can disrupt the transcription signaling pathway mediated by the mitogen-activated protein kinase (induced by Pb), NLRP3-ASC-caspase 1 (induced by Ni), tyrosine kinase Src (induced by As) and the nuclear factor κB (induced by Pb, Ni, Hg). The result of this is a gene transcription for early inflammatory cytokines, such as Interleukine 1β, Interleukine 6, and Tumor necrosis factor α). These cytokines can cause leukocytes recruitment and secretions of other pro-inflammatory cytokines and chemokines, which intensifies the inflammatory response. Some metals, such as cadmium (Cd), can activate an inflammatory response through tissue damage induction mediated by free radicals, which also results in leukocytes recruitment and cytokines secretions. Inflammation generated by metals can be reduced by metallothionein, which has the ability to scavenge free radicals and bind toxic metals through the release of Zn and oxidation of SH groups.

Influence of smoking on metallothionein level and other proteins binding essential metals in human milk

Aim: To assess the influence of smoking on the level of total protein, metallothionein (MT), albumin and lactoferrin in human breast milk. Methods: Samples of whole milk and supernatants of milk after centrifugation at 10 000×g and 105 000×g were analysed. Cadmium was determined by graphite furnace atomic absorption spectrometry (GFAAS). The concentration measurements employed the following methods: total protein by Lowry, albumin by colorimetry, cotinine and lactoferrin by ELISA tests, metallothionein by 109Cd/haemoglobin assay. Results: The assessment of tobacco smoke exposure was based on concentrations of cotinine in breast milk (197±98 ng/ml in smokers and 23±11 ng/ml in non‐smokers; p0.001) and serum (179±87 ng/ml and 32±19 ng/ml, respectively; p0.001). The level of cadmium was four times higher in the milk of smoking women than in non‐smokers. The concentration of total protein was lower in smoking (37.3±10.6 mg/ml) than in non‐smoking mothers (51.8±13.8 mg/ml; p0.02). No significant differences between albumin and lactoferrin concentrations were observed. The level of metallothionein was over twice as low in smokers (5.1±1.9 μg/ml) than in non‐smokers (13.4±3.0 μg/ml; p0.001), and an inverse correlation between MT level and cadmium concentration (r=−0.86; p=0.001) was noticed.

Changes in pro/antioxidant balance in smoking and non-smoking pregnant women with intrauterine growth restriction

The aim of this study was to investigate the influence of cigarette smoking on the pro/antioxidant balance in pregnant women with intrauterine growth restriction (IUGR). The studies have shown a 2-fold increase of Cd concentration in blood of women with IUGR in labour and a 10-fold increase in smoking pregnant women with IUGR. The increase of malondialdehyde concentration in plasma and 8-hydroxydeoxyguanosine in serum and Cu/Zn superoxide dismutase activity in erythrocyte lysate of pregnants with IUGR, reinforced by smoking, was revealed. We observed a decrease in the concentration of glutathione in blood and glutathione peroxidase activity in plasma and in erythrocyte lysate. A 4-fold higher metallothionein concentration in the plasma of women with IUGR in labour suggests that metallothionein may be one of the IUGR markers. Metallothionein concentration was intensified by smoking up to 7-fold in comparison to the controls. The pro/antioxidant balance during pregnancy is significantly affected by smoking.

Change of zinc, copper, and metallothionein concentrations and the copper-zinc superoxide dismutase activity in patients with pancreatitis.

Objectives: The aims of the present studies were to measure the concentrations of zinc (Zn), copper (Cu), and metallothionein and the Cu/Zn superoxide dismutase activity as elements engaged in an essential manner in the prooxidative and antioxidative balance of organism and to demonstrate the degree to which metallothionein and Cu/Zn superoxide dismutase are involved in the inflammatory processes occurring in the pancreas. Methods: The concentration of metallothionein was measured by immunoenzymatic method. Serum Cu/Zn superoxide dismutase activity was determined using a commercial test. The measurements of Zn and Cu concentrations in serum were assessed with the use of flame atomic absorption spectrometry. Results: Lowered serum Zn concentration and higher Cu level were observed in the serum of patients with chronic exacerbated pancreatitis and chronic pancreatitis. The significant increase of metallothionein concentration and Cu/Zn superoxide dismutase activity was observed in the blood of patients with chronic exacerbated pancreatitis and chronic pancreatitis. In slices of the pancreas during pancreatitis, we observed in immunohistochemical reaction the variable involvement of Cu/Zn superoxide dismutase and metallothionein. Conclusions: The results presented in these studies indicate an essential and variable involvement of antioxidants such Cu/Zn superoxide dismutase and metallothionein and disordered Cu and Zn homeostasis depending on the progression of inflammatory processes in patients with pancreatitis.

The Influence of the Occupational Exposure to Heavy Metals and Tobacco Smoke on the Selected Oxidative Stress Markers in Smelters

The aim of the study was to verify if there is any association between exposure to Cu, Zn, Cd, Pb, As and the formation of malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), advanced oxidation protein products (AOPP), and whether in this process cigarette smoking plays a role. The investigations were performed in the 352 smelters occupationally exposed to heavy metals and 73 persons of control group. Metals concentration was determined by atomic absorption spectrometry. MDA and AOPP concentrations were determined by spectrophotometric methods. The concentration of 8-OHdG was determined by ELISA method. It was demonstrated an increased Cu concentration in smoking smelters compared to non-smoking control group. It was noted no differences in Zn and Mg concentrations between the examined groups. Pb concentration was more than sixfold higher in the group of smoking smelters and about fivefold higher in the group of non-smoking smelters compared to the control groups (smokers and non-smokers). It was shown that Cd concentration in the blood was nearly fivefold higher in the smoking control group compared to the non-smoking control group and more than threefold higher in the group of smoking smelters compared to non-smoking. It was shown an increased As concentration (more than fourfold) and decreased Ca concentration in both groups of smelters compared to control groups. In groups of smelters (smokers and non-smokers), twofold higher MDA and AOPP concentrations, and AOPP/albumin index compared to control groups (smokers and non-smokers) were shown. Tobacco smoke is the major source of Cd in the blood of smelters. Occupational exposure causes accumulation of Pb in the blood. Occupational exposure to heavy metals causes raise of MDA concentration and causes greater increase in AOPP concentration than tobacco smoke.

Dysfunction of the pancreas in healthy smoking persons and patients with chronic pancreatitis.

Objectives: The aim of the study was to assess the effect of cigarette smoking on the endocrine pancreatic function by determining the levels of serum glucose and plasma insulin as well as by defining immunohistochemical localization of insulin and glucagon in tissue specimens of the pancreata derived from healthy persons and smoking and nonsmoking patients with diagnosed chronic pancreatitis (CP). Methods: The oxidative method was used to measure fasting glycemia in blood plasma and the method enzyme-linked immunoassay to determine the level of insulin in plasma. Immunohistochemical localization of hormones in paraffin tissue specimens of the pancreas was performed using the LSAB2-HRP visual test with polyclonal insulin and glucagon antibodies. The intensity of immunohistochemical reaction was calculated with digital imaging methodology. Results: The study revealed a substantially higher level of serum glucose in smoking CP patients and in healthy persons compared with nonsmoking patients and healthy persons, whereas insulin concentration in smoking patients was statistically lower than in nonsmokers. Smoking patients showed significantly lower expression of insulin and glucagon in the pancreas compared with nonsmoking patients and healthy persons. Conclusions: Impairment of the endocrine function of &bgr; and &agr; cells in the pancreatic islets is frequently manifested by complications in pancreatitis resulting among others from long-term smoking.

The influence of the intensity of smoking and years of work in the metallurgy on pro-oxidant/antioxidant balance in the blood of smelters.

The aim of this study was to investigate the effect of cigarette smoking and occupational exposure to heavy metals on the degree of pro-oxidant/antioxidant imbalance in smelters. The investigations were performed on the blood and urine of 400 subjects: 300 male copper smelters and 100 nonexposed male subjects. Biological material was divided into three groups: nonsmokers, those who smoked less than 20 cigarettes a day and those who smoked more than 20 cigarettes a day. The results showed a significant increase in the concentration of lead, cadmium and arsenic in the blood and urine of smelters, while smoking more than 20 cigarettes a day caused a further increase in the concentration of these metals. The level of malondialdehyde was approximately twofold higher in the plasma of the smelters compared to the control group. We have observed a disturbance in the level of antioxidants in erythrocyte lysate manifested by an increase in metallothionein and glutathione concentrations as well as superoxide dismutase and glutathione peroxidase activities and the decrease in glutathione S-transferase activity. Cigarette smoking, years of work in metallurgy and age of smelters were additional factors significantly affecting the pro-oxidant/antioxidant balance.

The influence of biological and environmental factors on metallothionein concentration in the blood.

The concentration of metallothionein (MT), a low-molecular-weight protein, is regulated by many factors, primarily metals (zinc, cadmium, copper), cytokines, glucocorticoides and free radicals. These factors are determined by such aspects of human biology as gender, pregnancy and age, as well as by environmental factors including the use of oral contraceptives and cigarette smoking, all which may affect MT levels in the body. The aim of this study was to investigate the influence of these biological and environmental factors on MT concentrations in erythrocyte lysate and in plasma. MT concentrations were determined by a two-step direct enzyme-linked immunosorbent assay. Evaluation of exposure to cigarette smoking was performed by checking cotinine levels in the plasma of subjects. The studies showed higher MT concentrations in both the erythrocyte lysate and plasma of women when compared to men. Furthermore, pregnancy causes an increase of MT concentration in plasma, while oral contraceptives cause an elevated concentration of MT in erythrocyte lysate. Age impacts plasma MT concentrations in men, whereas it does not affect concentrations of MT in erythrocyte lysate.

The Application of Capillary Electrophoresis in the Determination of Glutathione in Healthy Women's Blood

The purpose of this work was to compare glutathione status in the blood of women taking oral contraceptives, female smokers and pregnant women using both the capillary electrophoresis (CE) and alloxan methods. The studies were performed on the whole blood prepared by deproteinization by 25% metaphosphoric acid. The reduced glutathione (GSH) was measured by alloxan and CE methods. Oxidized glutathione (GSSG) was measured by CE and the GSH/GSSG ratio was calculated. In pregnant women (Group A), women taking oral contraceptives (Group B) and female smokers (Group C), lower concentrations of GSH were observed compared with the control group (Group D) as measured by CE and alloxan methods, suggesting the presence of oxidative stress. The level of GSSG in Group C was higher than in Group D, indicating glutathione oxidation. In Groups A and B, reduced levels of GSSG were observed, which indicates that other processes besides oxidation affect glutathione status. In Groups A and C, a lower GSH/GSSG ratio was observed than in Group D, while in Group B no statistical change was observed. In conclusion, the advantage of CE is the possibility of measuring GSSG, which could allow for a more accurate interpretation of the status of GSH in the human body. Oxidation of glutathione in female smokers was indicated, while in pregnant women and women taking oral contraceptives processes other than oxidation can be associated with a decrease in glutathione levels.