Spectroscopic imaging to assess biochemical alterations in liver carcinoma cells exposed to transition metals.

Despite the invaluable role of transition metals in every living organism, it should be remembered that failure to maintain the proper balance and exceed the appropriate dose may have the opposite effect. In the era of such a popular and propagated need for supplementation in the media, one should bear in mind the harmful effects that may become the result of improper and excessive intake of transition metals. This article establishes the feasibility of Raman (RS) and Fourier-transform infrared (FT-IR) spectroscopic imaging at the single-cell level to investigate the cellular response to various transition metals. These two non-destructive and perfectly complementary methods allow for in-depth monitoring of changes taking place within the cell under the influence of the agent used. HepG2 liver carcinoma cells were exposed to chromium, iron, cobalt, molybdenum, and nickel at 1 and 2 mM concentrations. Spectroscopic results were further supported by biological evaluation of selected caspases concentration. The caspase- 3, 6, 8, 9, and 12 concentrations were determined with the use of the enzyme-linked immunosorbent assay (ELISA) method. This study shows the induction of apoptosis in the intrinsic pathway by all studied transition metals. Cellular metabolism alterations are induced by mitochondrial metabolism changes and endoplasmic reticulum (ER) metabolism variations. Moreover, nickel induces not only the intrinsic pathway of apoptosis but also the extrinsic pathway of this process.

Concentration of Apoptotic Factors in Bronchoalveolar Lavage Fluid, as Potential Brain-Lung Oxygen Relationship, Correspond to the Severity of Brain Injury.

BACKGROUND: The mechanism of acute brain injury initiates a cascade of consequences which can directly cause lung damage, and this can contribute to poor neurological outcomes. The aim of this study was to evaluate concentration of different apoptotic molecules in the bronchoalveolar lavage fluid (BALF) in patients after severe brain injury and to correlate them with selected clinical variables and mortality. METHODS: Patients with brain injury receiving BALF operation were included in the study. BALF samples were collected within the first 6-8 hours after traumatic brain injury (A) and at days 3 (B) and 7 (C) after admission to the intensive care unit (ICU). Changes in the BALF nuclear-encoded protein (Bax), apoptotic regulatory protein (Bcl-2), pro-apoptotic protein (p53) and its upregulated modulator (PUMA), apoptotic protease factor 1 (APAF-1), Bcl-2 associated agonist of cell death (BAD) and caspase-activated DNase (CAD) were analysed. These values were correlated with the selected oxygenation parameters, Rotterdam computed tomography (CT) score, the Glasgow Coma Score and 28-day mortality. RESULTS: We found a significant increase in the concentration of selected apoptotic factors at admission (A), at day 3 (B) and day 7 (C) after severe brain damage contrasted with baseline level A (p < 0.001, separately). That concentration of selected apoptotic factors was significantly correlated with the severity of the injury and mortality. CONCLUSIONS: Activation of different apoptotic pathways seems to be an important process occurring in the lungs of patients in the early phases after severe brain trauma. Levels of apoptotic factors in the BALF correlates with the severity of brain injury.

Nano-Ag Particles Embedded in C-Matrix: Preparation, Properties and Application in Cell Metabolism.

The application of nano-Ag grains as antiviral and antibacterial materials is widely known since ancient times. The problem is the toxicity of the bulk or big-size grain materials. It is known that nano-sized silver grains affect human and animal cells in some medical treatments. The aim of this study is to investigate the influence of nano-Ag grains embedded in a carbonaceous matrix on cytotoxicity, genotoxicity in fibroblasts, and mutagenicity. The nanocomposite film is composed of silver nanograins embedded in a carbonaceous matrix and it was obtained via the PVD method by deposition from two separated sources of fullerenes and silver acetate powders. This method allows for the preparation of material in the form of a film or powder, in which Ag nanograins are stabilized by a carbon network. The structure and morphology of this material were studied using SEM/EDX, XRD, and Raman spectroscopy. The toxicology studies were performed for various types of the material differing in the size of Ag nanograins. Furthermore, it was found that these properties, such as cell viability, genotoxicity, and mutagenicity, depend on Ag grain size.

The Connection Between Selected Caspases Levels in Bronchoalveolar Lavage Fluid and Severity After Brain Injury.

Objective: The interaction between the brain and lungs has been the subject of many clinical reports, while the exact impact of brain injury on the physiology of the respiratory system is still subject to numerous experimental studies. The purpose of this study was to investigate the activation of selected caspases levels in bronchoalveolar lavage fluid (mini BALF) of patients after isolated brain injury and their correlation with the severity of the injury. Methods: The analysis was performed on patients who were admitted to the intensive care unit (ICU) for severe isolated brain injury from March 2018 to April 2020. All patients were intubated and mechanically ventilated. Mini BALF was collected within the first 6-8 h after trauma and on days 3 and 7 after admission. The concentrations of selected caspases were determined and correlated with the severity of brain injury evaluated by the Rotterdam CT Score, Glasgow Coma Score, and 28-day mortality. Results: Our results showed significantly elevated levels of selected caspases on days 3 and 7 after brain injury, and revealed apoptosis activation during the first 7 days after brain trauma. We found a significant different correlation between the elevation of selected caspases 3, 6, 8, and 9, and the Glasgow Coma Score, Rotterdam CT scale, and 28-day mortality. Conclusions: The increased levels of selected caspases in the mini BALF in our patients indicate an intensified activation of apoptosis in the lungs, which is related to brain injury itself via various apoptotic pathways and correlates with the severity of brain injury.

The Brain-gut Axis-where are we now and how can we Modulate these Connections?

A traumatic brain injury (TBI) initiates an inflammatory response with molecular cascades triggered by the presence of necrotic debris, including damaged myelin, hemorrhages and injured neuronal cells. Molecular cascades prominent in TBI-induced inflammation include the release of an excess of proinflammatory cytokines and angiogenic factors, the degradation of tight junctions (TJs), cytoskeletal rearrangements and leukocyte and protein extravasation promoted by increased expression of adhesion molecules. The brain-gut axis consists of a complex network involving neuroendocrine and immunological signaling pathways and bi-directional neural mechanisms. Importantly, modifying the gut microbiome alters this axis, and in turn may influence brain injury and neuroinflammatory processes. In recent years it has been demonstrated that the activity and composition of the gastrointestinal (GI) microbiome population influences the brain through all of above-mentioned pathways affecting homeostasis of the central nervous system (CNS). The GI microbiome is involved in the modulation of cellular and molecular processes which are fundamental to the progression of TBI-induced pathologies, including neuroinflammation, abnormal blood brain barrier (BBB) permeability, immune system responses, microglial activation, and mitochondrial dysfunction. It has been postulated that interaction between the brain and gut microbiome occurs mainly via the enteric nervous system and the vagus nerve through neuroactive compounds including serotonin or dopamine and activation by bacterial metabolites including endotoxin, neurotransmitters, neurotrophic factors, and cytokines. In recent years the multifactorial impact of selected immunomodulatory drugs on immune processes occurring in the CNS and involving the brain-gut axis has been under intensive investigation.

Cell cycle and transmembrane mitochondrial potential analysis after treatment with chromium(iii), iron(iii), molybdenum(iii) or nickel(ii) and their mixtures.

The aim of this study was to examine the effect of chromium(iii), iron(iii), molybdenum(iii) and nickel(ii) and their combinations on the cell cycle and mitochondrial transmembrane potential (MTP) in BALB/3T3 and HepG2 cells. A statistically significant dose related decrease of the percentage of cells in the G0/G1 and S phases was observed. However, a statistically significant dose related increase of the percentage of cells in the G2/M phase after exposure to chromium(iii), nickel(ii) or molybdenum(iii) at 200-1000 µM concentrations in both cell lines was observed. Moreover, an increase of the percentage of cells in the subG1 phase was observed. In both cell lines a statistically significant dose related decrease of the percentage of cells in the G2/M phase after exposure to iron(iii) at 200-1000 µM concentrations was observed. However, a statistically significant dose related increase of the percentage of cells in the G0/G1 phase after exposure to iron(iii) at 200-1000 µM concentrations was observed. A concentration dependent statistically significant decrease in the level of the MTP was observed in both cell lines after exposure to chromium(iii), iron(iii), nickel(ii) and molybdenum(iii). The results obtained from both cell lines show that HepG2 cells are more sensitive when compared to BALB/3T3 cells. Additions of Cr(iii) at 200 µM plus Fe(iii) at 1000 µM showed a synergistic effect on the cell cycle and MTP. In the case of Cr(iii) at 200 µM plus Mo(iii) at 1000 µM, an antagonistic effect was observed in both analyses. In the case of Cr(iii) at 1000 µM plus Mo(iii), Ni(ii) and Fe(iii) at 200 µM, no changes in the percentage of cells in all phases were observed in both cell lines in both analyses.

Pro- and antioxidant activity of chromium(III), iron(III), molybdenum(III) or nickel(II) and their mixtures.

The aim of this study was to examine the effect of chromium(III), iron(III), molybdenum(III) and nickel(II) and their combinations on pro- and antioxidant activity in mouse embryo fibroblasts and liver cancer cells. The present study shows that chromium(III), iron(III), nickel(II) and molybdenum(III) induce oxidative stress. In the case of chromium(III), nickel(II) and molybdenum(III) the intracellular ROS were dominant. However, in the case of iron(III) MDA was dominant - the end product of lipid peroxidation. Antioxidant activity of superoxide dismutase and catalase increased in low concentration of chromium(III); however, they decreased in higher concentrations. The same enzymes decreased after iron(III), nickel(II) and molybdenum(III) treatment in dose dependent manner. The activity of glutathione peroxidise decreased in dose dependent manner in all used microelements. Additions of Cr(III) at 200 µM plus Fe(III) at 1000 µM showed synergistic effect in ROS production and in lowering antioxidant activity. The same type of interaction in pairs Cr(III) at 1000 µM plus Fe(III) or Ni(II) or Mo(III) at concentration of 200 µM was observed. The protective effects of Cr(III) in antioxidant activity and in lowering intracellular ROS production in pairs of Cr(III) at 200 µM and Ni(II) or Mo(III) at concentration of 1000 µM were observed.

Influence of Inosine Pranobex on Cell Viability in Normal Fibroblasts and Liver Cancer Cells.

INTRODUCTION: Inosine pranobex (Isoprinosine) stimulates cell-mediated immune responses to viral infections in humans and might have also therapeutic use in animals. The aim of this study was to compare three in vitro cytotoxicity assays on mouse embryo fibroblasts and liver cancer cells and determine their ability to detect early cytotoxic effects for inosine pranobex. MATERIAL AND METHODS: BALB/3T3 clone A31and HepG2 cells were incubated with inosine pranobex at concentrations from 0.1 to 1,000 µg/mL. Cell viability was determined with the MTT reduction, the LHD release, and the NRU tests. RESULTS: A decrease in the cell viability was observed after incubating the BALB/3T3 clone A31and HepG2 cells with inosine pranobex. CONCLUSIONS: Based on the cytotoxicity endpoints measured in these investigations in BALB/3T3 clone A31cells, it can be concluded that the cell membrane may be the first part of the cell to be affected by inosine pranobex. The disintegration of lysosomes and mitochondria follows mitochondria damage. In HepG2 cells likewise, the cell membrane may be the first part of the cell to be affected by inosine pranobex. Also in liver cancer cells, the disintegration of mitochondria (assessed with the MTT reduction assay) and next of lysosomes (assessed with the NRU assay) follows mitochondria damage.

Genotoxicity and Mutagenicity of Inosine Pranobex.

INTRODUCTION: Inosine pranobex (Methisoprinol, ISO, Isoprinosine) is an immuno-modulatory antiviral drug that has been licensed since 1971 in several countries worldwide. In humans, the drug is approved for the treatment of viral infections, and it might also have therapeutic use in animals. The aims of the presented work were to investigate the genotoxicity of inosine pranobex on BALB/3T3 clone A1 and HepG2 cell lines and to elucidate its mutagenicity using the Ames test. MATERIAL AND METHODS: The BALB/3T3 clone A1 and HepG2 cells were incubated with inosine pranobex at concentrations from 0.1 to 1,000 µg/mL. The genotoxicity was determined by comet and micronucleus assays, and the mutagenicity was determined by Ames assay. RESULTS: Inosine pranobex did not induce a significant dose-related increase in the number of comets or micronuclei in BALB/3T3 clone A1 and HepG2 cells. Moreover, based on the results of the Ames test, it was concluded that inosine pranobex is not mutagenic in the Salmonella typhimurium reverse mutation assay. CONCLUSION: Based on the results of a comet assay, micronucleus assay, and Ames test, it was concluded that inosine pranobex is neither genotoxic nor mutagenic.

Interactions between chromium(III) and iron(III), molybdenum(III) or nickel(II): Cytotoxicity, genotoxicity and mutagenicity studies.

The aim of this study was to examine the effect of chromium(III) and iron(III) and molybdenum(III) and nickel(II) and their combinations on cyto-, genotoxicity and mutagenicity in BALB/3T3 and HepG2 cells. The results obtained from cytotoxicity assays indicate that there are differences between BALB/3T3 and HepG2 cell lines in their sensitivity to chromium chloride, iron chloride, molybdenum trioxide and nickel chloride. The statistically significant increase of DNA damage of all used microelements in both cell lines was observed. The micronucleus assay performed with the use of all concentrations shows statistically significant induction of chromosomal aberrations in all tested microelements in both cell lines. Moreover, treated cells display characteristic apoptosis in comparison to control cells. In all tested microelements, the increase of number of reverse mutations was observed with and without metabolic activation. Additions of Cr(III) at 200 µM plus Fe(III) at 1000 µM showed synergistic effect in decrease of cell viability and increase of comets, micronuclei and number of revertants in both cell lines. In case of Cr(III) at 200 µM plus Mo(III) at 1000 µM, a protective effect of chromium against molybdenum at 1000 µM toxicity in both cell lines (assessed by MTT, LDH and NRU, comet, micronucleus and Ames assays) was observed. The protective effect of Cr(III) in decrease of cell viability was observed in pair of Cr(III) at 200 µM and Ni(II) at 1000 µM in BALB/3T3 and HepG2 cell lines assessed by MTT, LDH and NRU, comet, micronucleus and Ames assays.

Cytotoxicity of Iron (III), Molybdenum (III), and their Mixtures in BALB/3T3 and HepG2 Cells.

INTRODUCTION: Iron and molybdenum are essential trace elements for cell metabolism. They are involved in maintaining proper functions of enzymes, cell proliferation, and metabolism of DNA. MATERIAL AND METHODS: BALB/3T3 and HepG2 cells were incubated with iron chloride or molybdenum trioxide at concentrations from 100 to 1,400 µM. The cells were also incubated in mixtures of iron chloride at 200 µM plus molybdenum trioxide at 1,000 µM or iron chloride at 1,000 µM plus molybdenum trioxide at 200 µM. Cell viability was determined with MTT reduction, LHD release, and NRU tests. RESULTS: A decrease in cell viability was observed after incubating both cell lines with iron chloride or molybdenum trioxide. In cells incubated with mixtures of these trace elements, a decrease in cell viability was observed, assessed by all the used assays. CONCLUSION: Iron (III) and molybdenum (III) decrease cell viability in normal and cancer cells. A synergistic effect of the mixture of these elements was observed.

Cell Viability in Normal Fibroblasts and Liver Cancer Cells After Treatment with Iron (III), Nickel (II), and their Mixture.

INTRODUCTION: Nickel and iron are very commonly occurring metals. Nickel is used in industry, but nowadays it is also used in medical biomaterials. Iron is an element necessary for cell metabolism and is used in diet supplements and biomaterials, whence it may be released along with nickel. MATERIAL AND METHODS: BALB/3T3 and HepG2 cells were incubated with iron chloride or nickel chloride at concentrations ranging from 100 to 1,400 µM. The following mixtures were used: iron chloride 200 µM plus nickel chloride 1,000 µM, or iron chloride 1,000 µM plus nickel chloride 200 µM. The cell viability was determined with MTT, LHD, and NRU tests. RESULTS: A decrease in cell viability was observed after incubating the BALB/3T3 and HepG2 cells with iron chloride or nickel chloride. A synergistic effect was observed after iron chloride 1,000 µM plus nickel chloride 200 µM treatment in all assays. Moreover, the same effect was observed in the pair iron chloride 200 µM plus nickel chloride 1,000 µM in the LDH and NRU assays. CONCLUSIONS: Iron (III) and nickel (II) decrease cell viability. Iron chloride at a concentration of 200 µM protects mitochondria from nickel chloride toxicity.

Chromium(III) and iron(III) inhibits replication of DNA and RNA viruses.

The aim of this study was to examine the effect of treating of chromium(III) and iron(III) and their combinations on Herpes Simplex Virus type 1 (HSV-1) and Bovine Viral Diarrhoea virus (BVDV) replication. The antiviral efficacies of chromium(III) and iron(III) on HSV-1 and BVDV were evaluated using Real Time PCR method. Moreover, the cytotoxicity of these microelements was examined using the MTT reduction assay. The IC50 (50% inhibiotory concentration) for the chromium chloride was 1100 µM for Hep-2 cells and 1400 µM for BT cells. The IC50 for the iron chloride was 1200 µM for Hep-2 cells and more than1400 µM for BT cells. The concentration-dependent antiviral activity of chromium chloride and iron chloride against HSV-1 and BVDV viruses was observed. In cultures simultaneously treated with (1) 200 µM of CrCl3 and 1000 µM of FeCl3, (2) 1000 µM of CrCl3 and 200 µM of FeCl3, (3) 400 µM of CrCl3 and 800 µM of FeCl3, (4) 800 µM of CrCl3 and 400 µM of FeCl3 a decrease in number of DNA or RNA copies was observed compared with control cells and cells incubated with chromium(III) and iron(III) used separately. The synergistic antiviral effects were observed for chromium(III) and iron(III) against HSV-1 and BVDV.

Selected aspects of the action of cobalt ions in the human body.

Cobalt is widespread in the natural environment and can be formed as an effect of anthropogenic activity. This element is used in numerous industrial applications and nuclear power plants. Cobalt is an essential trace element for the human body and can occur in organic and inorganic forms. The organic form is a necessary component of vitamin B12 and plays a very important role in forming amino acids and some proteins in nerve cells, and in creating neurotransmitters that are indispensable for correct functioning of the organism. Its excess or deficiency will influence it unfavourably. Salts of cobalt have been applied in medicine in the treatment of anaemia, as well as in sport as an attractive alternative to traditional blood doping. Inorganic forms of cobalt present in ion form, are toxic to the human body, and the longer they are stored in the body, the more changes they cause in cells. Cobalt gets into the body in several ways: firstly, with food; secondly by the respiratory system; thirdly, by the skin; and finally, as a component of biomaterials. Cobalt and its alloys are fundamental components in orthopaedic implants and have been used for about 40 years. The corrosion of metal is the main problem in the construction of implants. These released metal ions may cause type IV inflammatory and hypersensitivity reactions, and alternations in bone modelling that lead to aseptic loosening and implant failure. The ions of cobalt released from the surface of the implant are absorbed by present macrophages, which are involved in many of the processes associated with phagocytose orthopaedic biomaterials particles and release pro-inflammatory mediators such as interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor α (TNF-α), and prostaglandin.

[The role of chromium in cell biology and medicine]. / Znaczenie chromu w biologii komórki i medycynie.

Chromium (Cr3+) is an essential trace element for animals and humans. Its deficiency in organisms causes e.g. disturbances of carbohydrate and lipid metabolism, hypoglycemia, impaired glucose tolerance, elevated cholesterol and triglycerides in blood, but a decrease in HDL-cholesterol. It plays an important role in insulin-receptor activation. In the literature, antioxidative and anti-diabetic effects of chromium were also described. An excess of trivalent chromium can act as a prooxidant.