[The role of oxidative stress and iron in pathophysiology of rosacea]. / Uloga oksidacijskog stresa i zeljeza u patofiziologiji rozaceje.

Rosacea is a common skin disease of unknown etiology. The aim of the present paper is to explain the role of oxidative stress triggered by UV light and iron metabolism in the pathophysiology of rosacea. It was recently described that the number of ferritin positive cells was significantly higher in skin samples of rosacea patients compared to controls of healthy skin samples. The presence of ferritin was significantly higher in patients with the severe stage of disease. In addition, serum peroxide levels were significantly higher and serum total antioxidative potential levels were significantly lower in rosacea patients than in healthy controls. These results support the role of oxidative stress and affected metabolism of iron in etiology of rosacea. The higher presence of ferritin in skin cells of rosacea patients explains the exacerbation of symptoms by exposure to UV light, that releases ferritin free iron, which is fundamental in the generation of oxidative stress.

Growth modulation of human cells in vitro by mild oxidative stress and 1,4-dihydropyridine derivative antioxidants.

Reactive oxygen species and lipid peroxidation products are not only cytotoxic but may also modulate signal transduction in cells. Accordingly, antioxidants may be considered as modifiers of cellular redox signaling. Therefore, the effects of two novel synthetic antioxidants, analogues of 1,4-dihydropyridine derivatives, cerebrocrast and Z41-74 were analysed in vitro on human osteosarcoma cell line HOS, the growth of which can be modulated by lipid peroxidation. The cells were pretreated with either cerebrocrast or Z41-74 and afterwards exposed to mild, copper induced lipid peroxidation or to 4-hydroxynonenal (HNE), the end product of lipid peroxidation. The results obtained have shown that both antioxidants exert growth modulating effects interfering with the lipid peroxidation. Namely, cells treated with antioxidants showed increased metabolic rate and cell growth, thereby attenuating the effects of lipid peroxidation. Such biomodulating effects of cerebrocrast and Z41-74 resembled growth modulating effects of HNE, suggesting that the antioxidants could eventually promote cellular adaptation to oxidative stress interacting with redox signaling and hydroxynonenal HNE-signal transduction pathways. This may be of particular relevance for better understanding the beneficial role of hydroxynonenal HNE in cell growth control. Therefore, cerebrocrast and Z41-74 could be convenient to study further oxidative homeostasis involving lipid peroxidation.

Effect of ferric ions on reactive oxygen species formation, cervical cancer cell lines growth and E6/E7 oncogene expression.

As iron ions may participate in the pathogenesis of cancer and viral infections, the aim of this study was to monitor their influence on proliferation, E6 and E7 oncogene expression and reactive oxygen species (ROS) production in two human papilloma virus (HPV) positive cervical carcinoma cell lines (HeLa and SiHa) and one HPV negative vulvar cell line (A431). The anti-anaemic drug, ferric-sorbitol-citric acid complex (FSC) as a source of Fe(III) ions was used. Cells were treated with FSC at the concentrations between 0.001 and 1 mM Fe(III) for different time periods. Fe(III) ions inhibited the viability of HeLa and A431 cells while it had no influence on SiHa cells. Furthermore, Fe(III) treatment showed a time-dependent and a higher stimulatory effect on E6/E7 expression in SiHa cells than in HeLa cells. Fe(III) ion treatment with concentrations lower than 0.1mM showed a time and a concentration dependent intracellular ROS production in all tested cell lines, while the treatment with 1mM concentration decreased ROS production in all tested cell lines. In conclusion, Fe(III) ion treatment apart from having an anti-tumour effect, as we previously described, enhances survival of HPV 16-positive cells and might be associated with HPV oncogenesis.

Granulocytes as effective anticancer agent in experimental solid tumor models.

The aim of the study was to elucidate the effects of murine granulocytes on the growth of solid murine tumors when administrated in the vicinity of W256 carcinoma growing in Sprague Dawley rats, and in the vicinity of Ehrlich ascites tumor (EAT) growing in BALBc mice. The administration of granulocytes significantly improved the survival of W256-bearing rats, and increased the tumor regression incidence from 17% up to 75%. Rats with regressing tumors had 2.5 times increased levels of granulocytes in peripheral blood, which were also cytotoxic in vitro for W256 carcinoma cells. However, blood levels of cytokine-induced neutrophil chemoattractant-2, tumor necrosis factor α and interleukin 6 were similar between rats with regressing tumors and control healthy rats, suggesting that the observed regression of W256 carcinoma was caused by specific anticancer effects of the applied granulocytes. Anticancer effects of granulocytes were also found in BALBc mice bearing solid form of EAT, resulting in a 20% increase of survival in EAT-bearing mice. Therefore, the administration of granulocytes, isolated from healthy animals and applied at the site of solid tumors in rats and in mice, reduced experimental tumor growth, and extended the survival of tumor-bearing animals, while in some rats it even caused a W256 regression.

Altered iron metabolism, inflammation, transferrin receptors, and ferritin expression in non-small-cell lung cancer.

The involvement of iron and inflammation parameters on overall survival in non-small-cell lung cancer (NSCLC) patients was studied. Furthermore, transferrin receptors 1 (TfR1) and ferritin expression in tumor tissue, tumor stroma, and normal lung tissue were analyzed. Iron metabolism and inflammation parameters were determined by automated laboratory measurements at the time of diagnosis. TfR1 and ferritin expression were determined by immuno-histochemical methods. About 50% of patients survived 12 months only. At the time of diagnosis more than half of the patients had anemia and significantly elevated serum ferritin. Iron content of serum ferritin (ICF) was below the reference values in 90% of patients. Furthermore, ICF showed positive correlation with iron metabolic parameters and survival but negative correlation with serum ferritin and ESR. The expression of TfR1 and ferritin in tumor cells was observed in 88% or 62% of patients, respectively. Tumor stroma was TfR1 negative and sporadically ferritin positive. Tumor tissue ferritin expression showed negative correlation with serum iron and hematokrit (Ht), and positive correlation with ferritin, erythrocyte sedimentation rate (ESR), alpha-1 globulin, and alpha-2 globulin. Positive correlation was found between TfR1 expression in tumor tissue and alpha-globulin. The correlation between TfR1/ferritin expression in tumor tissue and ICF or survival was not observed. Therefore, we conclude that elevated serum ferritin in sera of NSCLC patients is the result of inflammation and oxidative stress rather than body iron overload. Higher expression of ferritin in tumor tissue may be the consequence of iron deficiency or local toxicity induced by environmental factors.

Specific thermographic changes during Walker 256 carcinoma development: differential infrared imaging of tumour, inflammation and haematoma.

BACKGROUND: Infrared imaging measures spatial variations in the skin temperature aiming to determine pathological processes; hence possible use of this non-invasive analytical method in cancer detection is emerging. METHODS: Infrared thermal imaging was used to detect changes in rat skin surface temperature associated with experimental cancer development (Walker 256 carcinoma), inflammation (upon s.c. Sephadex injection) and haematoma (provoked by s.c. blood coagulate injection). Infrared camera with a geometric resolution of 76,800 pixels, spectral range of 8-14 microm and the minimal detectable temperature resolution of 0.07 degrees C with spatial resolution of 0.48 mm at measuring distance of 30 cm was used to obtain computerised thermal scans. Genuine ThermoWEB software developed for remote internet control as open source software was used. RESULTS: The raise of peripheral temperature was observed after induction of local inflammation or haematoma. Opposite to that, transient decrease of the skin surface temperature was observed after tumour transplantation. Progressive growth of tumour was associated with the raise of the skin surface temperature from the 10th day after tumour inoculation, when the tumours developed supportive neoangiogenic blood supply, as verified by histology. CONCLUSION: While the raise of peripheral temperature in advanced tumour was caused by neoangiogenesis, the reduction in skin surface temperature in an early period after tumour cell inoculation indicated a decay of transplanted tumour cells due to the immune response and the lack of blood supply. Thus, infrared thermal imaging may have considerable value in evaluation of the tumour development and discrimination of cancer from inflammation and haematoma.

Acute immunomodulatory effects of iron polyisomaltosate in rats.

Anti-anaemic drug ferric-sorbitol citrate showed immunomodulatory effects, activating NF-kappaB in peritoneal macrophages, which consequently secrete tumour necrosis factor-alpha (TNF-alpha). TNF-alpha activates NF-kappaB in spleen cells. The aim of this study was to investigate the effect of iron polyisomaltosate, an iron (Fe(3+)) compound, on serum iron, interleukin-6 (IL-6) and serotonin (5-HT) concentration, neutrophil activity, and NF-kappaB activation in peritoneal macrophages and spleen cells in rats. Female Wistar rats were injected i.p. with 7.5mg iron/kg of iron polyisomaltosate 1.5, 3, 6, 24 and 48h before sacrifice. Serum iron, 5-HT and IL-6 concentration was determined by colorimetric, spectrofluorimetric and ELISA methods, neutrophil activity by a chemiluminescence assay, and NF-kappaB expression/activation by a Dot-Blot method. Iron polyisomaltosate significantly increased serum iron and 5-HT concentrations during the first 6h, IL-6 levels 3 and 6h, and diminished respiratory burst of granulocytes 1.5h after the injection. Iron polyisomaltosate stimulated activation of p65, p50 and RelB subunits of NF-kappaB in the peritoneal macrophages after 6h, and RelB subunit was additionally increased after 24 and 48h. In the spleen cells iron polyisomaltosate stimulated p65 subunit after 48h and RelB subunit after 24h. The results showed time-dependent immunomodulatory effects of iron polyisomaltosate. These effects might be achieved via induction of the intracellular signalling for NF-kappaB activation in peritoneal macrophages and later in spleen cells, together with increase of serum 5-HT, and IL-6 but with diminished respiratory burst of granulocytes. Iron polyisomaltosate presumably activated reactive oxygen species resulting in the stimulation of the acute phase reactants in the liver.

Oxidative stress and ferritin expression in the skin of patients with rosacea.

BACKGROUND: Rosacea is a common chronic light-sensitive inflammatory skin disease of unknown origin. The purpose of this work was to determine the parameters of oxidative stress, antioxidative capacity, and the pathophysiologic role of ferritin expression in skin cells of patients with rosacea. OBJECTIVES: The investigation consisted of measurements of serum peroxide levels, serum total antioxidative potential levels, and immunohistochemical analyses of ferritin in skin tissue samples. RESULTS: Serum peroxide levels were significantly higher and serum total antioxidative potential levels were significantly lower in patients with rosacea than in healthy control subjects (P < .05). Compared with control subjects, the number of ferritin-positive cells was significantly higher (P < .001) in skin samples from patients with rosacea, especially those with severe disease. LIMITATIONS: Patients with rosacea in the study were aged 30 to 70 years (average age was 56 years). Younger patients with flushing only were not included according to the request of the ethics committee, limiting the use of diagnostic biopsies only to the necessary cases. CONCLUSION: The statistically significant differences in the expression of ferritin, higher peroxide levels, and lower antioxidative potential support the onset of systemic oxidative stress in patients with rosacea.

Comparative study on the antioxidant and biological activities of carvacrol, thymol, and eugenol derivatives.

Four derivatives of thymol, carvacrol, and eugenol were synthesized: 4-(hydroxymethyl)-5-isopropyl-2-methylphenol, 4,4'-methylenebis(5-isopropyl-2-methyl)phenol, 4-allyl-6-(hydroxymethyl)-2-methoxyphenol, and 4-(hydroxymethyl)-2-isopropyl-5-methylphenol. The obtained derivatives showed remarkably better antioxidative properties according to 1,1-diphenyl-2-picrylhydrazyl assay (50% inhibitory concentrations = 4-156 microg/mL) and Rancimat assay (protection factors = 1.55-5.84) when compared with parent compounds and values similar to or better than those of butylated hydroxytoluene and vitamin C. At concentrations of 10 mM carvacrol derivatives had no toxic effect on viability of Escherichia coli K-12 (determined by minimum inhibitory concentrations). Other phenol derivatives showed reduced cytotoxic effect on E. coli K-12 at concentrations of 2-5 mM on the basis of 50% lethal dose measurements. In comparison with the parent compounds, phenol derivatives showed reduced cytotoxic effect for Saccharomyces cerevisiae cells (determined by yeast colony reduction). On the other hand, the majority of synthesized compounds had dose-dependent antiproliferative effects on human uterine carcinoma cells (HeLa), which makes them potentially interesting for the adjuvant experimental cancer treatments. The 4,4'-methylenebis(5-isopropyl-2-methyl)phenol derivative of carvacrol showed lower inhibiting capacity also for the HeLa cells, which makes this particular derivative attractive as an efficient antioxidant with negligible cytotoxic effects.

The involvement of granulocytes in spontaneous regression of Walker 256 carcinoma.

We described before that oxidative burst of granulocytes is cytotoxic for melanoma B16F10 and for Walker 256 carcinoma (W256). Therefore, we assumed that granulocytes could also be important mechanism of the host defence against tumour. In current study we report massive granulocyte infiltration at the site of W256 transplanted in the hind limb of Sprague-Dawley associated with spontaneous tumour regression observed for 22/25 rats (87%). Peripheral blood granulocytes of these animals were highly cytotoxic for W256 cells cultured in vitro. After the tumour disappearance the inflammatory oxidative burst of the granulocytes ended. Distraction of granulocytes from the tumour by s.c. Sephadex injection decreased the incidence of the W256 regression to only 7/25 animals (30%). These results suggest that innate immunity based on immune competent granulocytes may be the cause of well known phenomenon of spontaneous regression of W256 carcinoma.

Oxidative burst of neutrophils against melanoma B16-F10.

Intensive oxidative burst was determined by chemiluminescence of peripheral blood neutrophils of mice that were intramuscularly injected with melanoma B16-F10 and/or subcutaneously with Sephadex G-200. The neutrophils from papula developed at the site of Sephadex injection were cytotoxic for the B16-F10 cells in vitro. However, survival of Sephadex injected tumour-bearing mice was lower than of control animals bearing B16-F10, while their tumours grew faster and were less necrotic. Thus, it is likely that injection of Sephadex distracted the neutrophils from the tumour allowing faster progression of the tumour, indicating that neutrophils may have an important role in the host defence against malignant cells in the early stage of tumour development.

Altered iron metabolism, transferrin receptor 1 and ferritin in patients with colon cancer.

In this study, the level and distribution of transferrin receptor 1 (TfR1) and ferritin in colorectal carcinoma and in normal colon epithelium has been determined relative to the tumor stage and iron status of patients using immunohistochemical staining methods. While the majority of carcinoma patients were anemic, no relationship between the level of colon tissue ferritin and TfR1 and the systemic parameters of iron metabolism was evident. Furthermore, no association between ferritin content and the grade of colorectal carcinoma was observed. However, a relationship between the expression of TfR1 and the grade of colorectal carcinoma was observed. In this case high expression of TfR1 was found in colorectal carcinoma samples of Dukes A or B grade, and well differentiated colorectal carcinoma cells. In comparison, weak or no expression of TfR1 was observed in carcinoma samples of Dukes C or D grade with poorly differentiated cells and in carcinoma samples that had lymph node infiltration and distant metastasis.

Uptake of anti-anemic substance ferric-sorbitol-citrate by normal and malignant cells and its effects on expression of transferrin receptor 1 and ferritin.

Iron-containing antianemic drug ferric-sorbitol-citrate (FSC) inhibits the proliferation of various cancer cell lines in vitro and causes a regression of experimental murine tumors in vivo but does not affect the proliferation of nonmalignant cells. Growth modification caused by FSC iron involves a diminished expression of Bcl-2 and an overexpression of p53 proto-oncogene, accompanied by an increased incidence of apoptosis. Aiming to evaluate further the activity principle of the anticancer effects of this antianemic drug, in this study, we analyzed the utilization of iron from FSC and the effects of FSC iron on transferrin receptor 1 (TfR1) and ferritin expression. Without FSC iron, all the cell lines had an equal expression of TfR1, but if cultured in FSC-supplemented medium, human colon SW620 and laryngeal carcinoma Hep cells exhibited a lower expression of TfR1-positive cells than nonmalignant Wi38 fibroblasts and pancreatic carcinoma MiaPaCa2 cells. The most sensitive to FSC iron were colon carcinoma SW620 cells, whereas Wi38 fibroblasts were not sensitive at all. Increased iron uptake by colon carcinoma cells was noticed in the first 3 hours of the incubation with FSC iron, whereas higher FSC iron concentrations and longer incubation also impaired ferritin expression in SW260 colon carcinoma cells. Thus, the anticancer ability of FSC could result from its higher initial utilization of iron and consecutive negative signal for the expression of TfR1 in tumor cells. Tumor cells containing lower amounts of ferritin are probably more sensitive to oxidative stress caused by iron overload, whereas FSC iron itself was proven to be chemically stable and did not induce lipid peroxidation.

Oxidative burst and anticancer activities of rat neutrophils.

It is assumed that oxidative damage caused by reactive oxygen species (ROS) from activated neutrophil granulocytes may contribute to pathology of tumors. ROS are crucial in neutrophil-mediated tumor cell lysis. The present study is focused on the oxidative burst and antitumorous activities of neutrophils when challenged with Walker carcinoma W256. Survival and tumor growth dynamics were monitored in vivo, while tumor cell proliferation when mixed with neutrophils was studied in vitro together with the generation/release of neutrophil respiratory burst products, primarily 1O2. Neutrophils were collected upon Sephadex injection. The survival of Sephadex injected animals was slightly improved, while their tumors grew less than in controls. The presence of tumor cells in vitro activated neutrophils to produce singlet oxygen similar to phorbol ester. Neutrophils from Sephadex-bearing animals diminished tumor cell proliferation in vitro (measured by 3H-TdR incorporation), while neutrophils from Sephadex and the tumor-bearing animals did not show such activity in vitro. Our results confirm that in the case of rapidly growing tumors such as murine W256 carcinoma neutrophils have antitumorous effects in the early phase of tumor development.

Effect of ferric sorbitol citrate (FSC) administration on NF-kappaB in macrophages and spleen cells of rats.

Interaction of iron metabolism and the immune system is complex and pathological changes in one system affect the other. Ferric sorbitol citrate (FSC), non-toxic compound of ferric ions with sorbitol and citrate. has immunomodulatory effect in treated mice. We investigated an effect of FSC on NF-kappaB expression/activation in peritoneal macrophages and spleen cells of rats. TNF-alpha concentrations in sera of control and FSC intraperitoneal (i.p.) treated Wistar rats were measured by ELISA. Furthermore, peritoneal macrophages (PM) were counted and splenocytes were isolated. PM and splenocytes were lysed and their cytoplasmic and nuclear fractions were separated by centrifugation. The influence of FSC on NF-kappaB expression and/or activity as well as expression of its inhibitor IkappaB-alpha was measured by Western blot. 1.5 and three hours after FSC treatment TNF-alpha level in sera was significantly (p < or = 0.05) increased. Activation of transcription factor NF-kappaB in PM was detected three hours after treatment, followed by significant increment in PM number. In splenocytes NF-kappaB was activated six and 48 hours after FSC application. The results indicate that, after i.p. application, FSC acts as a modulator of the immune system activating NF-kappaB in PM. PM consequently secrete TNF-alpha that activates NF-kappaB in splenocytes.

Differing effects of two iron compounds on experimental arthritis, TNF-alpha levels and immune response in mice.

The effects of ferric-sorbitol-citrate and ferric-citrate on the severity of experimental arthritis, TNF-alpha secretion and the immune status were examined in mice. Arthritis was induced by footpad injection of methylated BSA and intraperitoneal injection of Bordetella pertussis. Joint and footpad swelling were measured weekly by a caliper. TNF-alpha serum levels were measured by ELISA. The immune status was determined by the response of mouse lymphocytes to ConA in vitro and by the antigen-presenting cell assay. Experimental arthritis was aggravated by ferric-citrate, whereas ferric-sorbitol-citrate did not promote it. If applied to normal (non-arthritic) mice three times a week for 4 weeks, ferric-sorbitol-citrate stimulated isolated splenocytes to increase production of TNF-alpha, the function of antigen-presenting cells and lymphocyte proliferation in response to ConA in vitro. TNF-alpha production by cultured splenocytes was also stimulated. In mice with antigen-induced arthritis, iron compounds did not additionally stimulate TNF-alpha production. Thus, we have shown that ferric-sorbitol-citrate stimulated TNF-alpha production, antigen-presenting cell activity and cellular immune response. Development of antigen-induced arthritis and TNF-alpha production in arthritic mice were not stimulated.