Identification of p53 and its isoforms in human breast carcinoma cells.

In breast carcinoma, disruption of the p53 pathway is one of the most common genetic alterations. The observation that the p53 can express multiple protein isoforms adds a novel level of complexity to the outcome of p53 mutations. p53 expression was analysed by Western immunoblotting and immunohistochemistry using monoclonal antibodies DO-7, Pab240, and polyclonal antiserum CM-1. The more frequently p53-positive nuclear staining has been found in the invasive breast tumors. One of the most intriguing findings is that mutant p53 appears as discrete dot-shaped regions within the nucleus of breast cancer cells. In many malignant cells, the nucleolar sequestration of p53 is evident. These observations support the view that the nucleolus is involved directly in the mediation of p53 function or indirectly by the control of the localization of p53 interplayers. p53 expressed in the nuclear fraction of breast cancer cells revealed a wide spectrum of isoforms. p53 isoforms ΔNp53 (47 kDa) and Δ133p53 ß (35 kDa), known as dominant-negative repressors of p53 function, were detected as the most predominant variants in nuclei of invasive breast carcinoma cells. The isoforms expressed also varied between individual tumors, indicating potential roles of these p53 variants in human breast cancer.

Mutant p53 protein expression and antioxidant status deficiency in breast cancer.

It is well recognized that cancers develop and grow as a result of disordered function of tumor suppressor genes and oncogenes, which may be exploited for screening purposes. Extensive evidence indicated tumor suppressor protein p53 as candidate marker for mutation identification. We have investigated mutant p53 protein expression in human breast tumors in relation to antioxidant status deficiency. The study included 100 breast cancer patients. p53 protein expression was evaluated by Western blot assay and immunostaining using a CM-1, DO-7 and Pab240 antibodies. Antioxidant parameters and lipid peroxidation were estimated by biochemical analyses. Western blotting with epitopespecific monoclonal antibody Pab240 strongly suggests that nuclear extracts from breast cancer cells express mutant forms of p53. It is of interest that the mutant forms of p53 overexpression in conjunction with the appearance of nuclear bodies are observed in highly aggressive carcinomas. Expression of isoform Δp53 (45 kDa) and isoform of ~ 29 kDa were more common in cases with LN metastasis. These studies point out the molecular consequences of oxidative stress (lipid peroxides, LP, p<0.001) and antioxidant status deficiency (copper, zinc superoxid dismutase, SOD, p<0.001; catalase, CAT, p<0.01; glutathione reductase, GR, p<0.001; glutathione, GSH, p<0.05) and indicate the importance of p53 mutation as the commonest genetic alteration detected in breast cancer cells. The expression of mutant p53 is correlated to increased lipid peroxides (0.346, p<0.05 ) and lowered antioxidant activity of CAT (- 0.437, p<0.01) in the breast cancer patients.

Treadmill exercise does not change gene expression of adrenal catecholamine biosynthetic enzymes in chronically stressed rats.

Chronic isolation of adult animals represents a form of psychological stress that produces sympatho-adrenomedullar activation. Exercise training acts as an important modulator of sympatho-adrenomedullary system. This study aimed to investigate physical exercise-related changes in gene expression of catecholamine biosynthetic enzymes (tyrosine hydroxylase, dopamine-ß-hydroxylase and phenylethanolamine N-methyltransferase) and cyclic adenosine monophosphate response element-binding (CREB) in the adrenal medulla, concentrations of catecholamines and corticosterone (CORT) in the plasma and the weight of adrenal glands of chronically psychosocially stressed adult rats exposed daily to 20 min treadmill running for 12 weeks. Also, we examined how additional acute immobilization stress changes the mentioned parameters. Treadmill running did not result in modulation of gene expression of catecholamine synthesizing enzymes and it decreased the level of CREB mRNA in the adrenal medulla of chronically psychosocially stressed adult rats. The potentially negative physiological adaptations after treadmill running were recorded as increased concentrations of catecholamines and decreased morning CORT concentration in the plasma, as well as the adrenal gland hypertrophy of chronically psychosocially stressed rats. The additional acute immobilization stress increases gene expression of catecholamine biosynthetic enzymes in the adrenal medulla, as well as catecholamines and CORT levels in the plasma. Treadmill exercise does not change the activity of sympatho-adrenomedullary system of chronically psychosocially stressed rats.

Antioxidant enzymes in women with endometrial polyps: relation with sex hormones.

OBJECTIVE: To investigate whether antioxidant enzyme activities (superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase) and lipid hydroperoxide levels in patients with endometrial polyps are influenced by the changes in sex hormones (estradiol, progesterone, FSH, and LH) during the menstrual cycle and in postmenopause. STUDY DESIGN: The material consisted of blood and endometrial tissue specimens from women diagnosed with endometrial polyps. Patients were divided into groups depending on the phase of the menstrual cycle--follicular or luteal--and the postmenopause. The activities of antioxidant enzymes and the lipid hydroperoxide levels were compared among the phases and a linear regression model was used to evaluate the associations between hormones and antioxidant/oxidant variables. RESULTS: In the blood of examined women, a significant difference in superoxide dismutase activity and lipid hydroperoxide levels was recorded among the phases. There was also a positive correlation between the estradiol concentration and superoxide dismutase. In polyp tissue, we recorded a phase-related difference in superoxide dismutase and glutathione peroxidase activities as well as in the lipid hydroperoxide levels. A negative correlation was observed between FSH/LH and glutathione peroxidase, and between LH and superoxide dismutase. CONCLUSION: Antioxidant enzymes and lipid hydroperoxide levels in patients with endometrial polyps are influenced by the changes in sex hormones during the menstrual cycle and after the menopause, pointing to a role of the observed relationship in polyp etiology.

Forced exercise changes catecholamine synthesis in the spleen of adult rats.

Treadmill training produces modulation of neuro-endocrine and immune functions. This study examined the effects of chronic forced running (CFR) on the plasma concentration of catecholamines and the expression of splenic catecholamine biosynthetic enzymes in rats by using real-time RT-PCR and Western blot analyses. We found that CFR increases the plasma catecholamine levels, decreases splenic tyrosine hydroxylase (TH), dopamine-ß-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT) mRNA levels and increases splenic PNMT protein levels. This shows that CFR is a very strong stressor which activates the sympatho-adrenomedullary system and increases synthesis of splenic PNMT by 20%, which both can modulate the immune function.

Antioxidant protection against curative and palliative doses of ionizing irradiation in human blood decreases with aging.

Reactive oxygen species (ROS) are independently recognized to play a significant role in radiation-induced damage on healthy tissue and in aging process. However, an age-related alteration of antioxidant (AO) system in radiation response in humans is poorly investigated. The aim of this paper was to evaluate the irradiation effects on the activities and expression of AO system in the blood of healthy women during aging. Blood samples were irradiated with curative and palliative doses of 2 Gy or 9 Gy γ-rays. AO capacity for detoxification of O(2)•(-) and H(2)O(2) in response to 2 Gy γ-irradiation decreases in women above 58 years, while in response to 9 Gy shows signs of weakening after 45 years of age. Due to reduction of AO capacity during aging, cytotoxic effects of curative and palliative doses of irradiation, mediated by ROS, may significantly increase in older subjects, while removal of H(2)O(2) excess could reduce them.

Glutathione redox cycle in small intestinal mucosa and peripheral blood of pediatric celiac disease patients.

The celiac disease is an autoimmune gastrointestinal disorder caused by gluten from wheat, rye or barley. In genetically predisposed persons, gluten induces the immune-mediated inflammation of small intestinal mucosa. Histological lesions include intraepithelial lymphocytosis, crypt hypertrophy and villous atrophy, resulting in malabsorption of micro- and macronutrients. The only treatment for celiac patients is a permanent gluten-free diet (GFD). Reactive oxygen species (ROS) and oxidative stress are strongly associated with the celiac disease. Glutathione (GSH) is a main detoxifier of endogenous and exogenous ROS in the intestine. In order to explain the role of glutathione redox cycle in celiac patients, we examined the activities of GSH-related antioxidant (AO) enzymes glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the concentration of GSH in small intestinal biopsies and peripheral blood of children affected by the celiac disease. The concentration of lipid hydroperoxides (LOOH) as markers of oxidative damage was measured in the same samples. The results clearly demonstrate a significant malfunction of GSH redox cycle with a concomitant decrease in the capacity to regenerate GSH and detoxify LOOH in celiac patients, even after several years of GFD. The oral administration of GSH and a diet rich in natural antioxidants, as well as appropriate dietary supplements, could be of great benefit to the patients.

Antioxidant status and lipid peroxidation in the blood of breast cancer patients of different ages after chemotherapy with 5-fluorouracil, doxorubicin and cyclophosphamide.

OBJECTIVES: Breast carcinoma is related to the increase of lipid peroxidation in plasma with concomitant decrease of antioxidant (AO) defense capacity in blood cells, which becomes more pronounced during aging of the patients. This work evaluated the potential age-related effect of chemotherapy with 5-fluorouracil, doxorubicin and cyclophosphamide (FAC) on the level of lipid hydroperoxides (LP), glutathione (GSH), AO enzyme activities of copper, zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) in breast cancer patients. The level of CuZnSOD protein was assessed after the FAC therapy and radiotherapy of breast cancer. DESIGN AND METHODS: AO parameters were measured in the blood of 58 breast cancer patients and 60 healthy age-matched healthy subjects by biochemical and Western blot analyses. RESULTS: Increased oxidative stress (LP: p<0.05) and decreased AO enzyme activities (CuZnSOD: p<0.01, GPx: p<0.05, GR: p<0.01) and GSH level (p<0.01) in the blood of breast cancer patients in response to FAC chemotherapy seem not to be age-dependent. CuZnSOD enzyme expression decreased after the FAC chemotherapy (p<0.05), while it increased after the radiotherapy of breast cancer (p<0.05). CONCLUSION: FAC chemotherapy and radiotherapy promote further oxidative shift, which potentiate already existing chronic oxidative stress linked to breast cancer. In these effects, impaired capacity for H(2)O(2) detoxification (CAT, GPX and GSH) seems to have major contribution.

Antioxidant enzymes and lipid peroxidation in endometrium of patients with polyps, myoma, hyperplasia and adenocarcinoma.

BACKGROUND: Oxidative stress and impaired antioxidant system have been proposed as a potential factors involved in the pathophysiology of diverse disease states, including carcinogenesis. In this study, we explored the lipid peroxidation levels and antioxidant enzyme activities in women diagnosed with different forms of gynecological diseases in order to evaluate the antioxidant status in endometrium of such patients. METHODS: Endometrial tissues of gynecological patients with different diagnoses were collected and subjected to assays for superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and lipid hydroperoxides. RESULTS: Superoxide dismutase activity was significantly decreased (50% in average) in hyperplastic and adenocarcinoma patients. Activities of both glutathione peroxidase and glutathione reductase were increased 60% and 100% on average, in hyperplastic patients, while in adenocarcinoma patients only glutathione reductase activity was elevated 100%. Catalase activity was significantly decreased in adenocarcinoma patients (47%). Lipid hydroperoxides level was negatively correlated to superoxide dismutase and catalase activities, and positively correlated to glutathione peroxidase and glutathione reductase activities. CONCLUSIONS: This study provided the first comparison of antioxidant status and lipid peroxidation in endometrial tissues of patients with polyps, myoma, hyperplasia and adenocarcinoma. The results showed that patients with premalignant (hyperplastic) and malignant (adenocarcinoma) lesions had enhanced lipid peroxidation and altered uterine antioxidant enzyme activities than patients with benign uterine diseases, polyps and myoma, although the extent of disturbance varied with the diagnosis. Further investigation is needed to clarify the mechanisms responsible for the observed alterations and whether lipid hydroperoxide levels and antioxidant enzyme activities in uterus of gynecological patients might be used as additional parameter in clinical evaluation of gynecological disorders.

Antioxidant status and lipid peroxidation in small intestinal mucosa of children with celiac disease.

OBJECTIVE: To explain the role of oxidative stress in the pathology of celiac disease. DESIGN AND METHODS: The activities of antioxidant enzymes and the levels of glutathione and lipid hydroperoxides were measured in the samples of small intestinal biopsies from 39 children with different forms of the disease and in 19 control subjects. RESULTS: The activities of analyzed enzymes varied significantly between the examined groups. An increase in the activities of superoxide dismutase was observed in patients with active and silent celiac disease, while the activities of glutathione peroxidase and glutathione reductase and the glutathione content were significantly reduced. The level of lipid hydroperoxides was significantly elevated in these groups. CONCLUSIONS: Oxidative stress is an important factor in the pathogenesis of celiac disease. The antioxidant capacity of celiac patients is significantly reduced, mostly by a depletion of glutathione. Natural antioxidants and appropriate dietary supplements could be important complements to the classic therapy of celiac disease.

Antioxidant status and lipid peroxidation in the blood of breast cancer patients of different ages.

Oxidative stress is considered to be implicated in the pathophysiology of breast cancers. In this study we investigated the level of oxidative stress and antioxidant (AO) status in the blood of breast cancer patients of different ages. The level of lipid hydroperoxides (LP) was measured in blood plasma and the activities of copper, zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) enzymes, as well as the level of total glutathione (GSH) and CuZnSOD protein were measured in blood cells of breast cancer patients and age-matched healthy subjects. Our results showed that breast carcinoma is related to increase of lipid peroxidation in plasma with concomitant decrease of AO defense capacity in blood cells, which becomes more pronounced during aging of the patients. Suppression of CuZnSOD activity related to breast cancer is most likely caused by decreased de novo synthesis of this enzyme. Similar patterns of suppression in CuZnSOD and CAT activities related to aging were recorded both in controls and patients. Age-related decrease in CuZnSOD activity seems not to be caused by altered protein levels of this enzyme. Suppression of AO enzymes associated with breast cancer and aging is most likely the cause of increased levels of reactive oxygen species (ROS). Our results indicate significant role of oxidative-induced injury in the breast carcinogenesis, particularly during the later stages of aging. Overall, our data support the importance of endogenous AOs in the etiology of breast cancer across all levels of predicted risk.

Antioxidant enzymes, glutathione and lipid peroxidation in peripheral blood of children affected by coeliac disease.

BACKGROUND: Oxidative stress has been implicated in the pathogenesis of coeliac disease. The aim of this study was to examine the modulation of the biochemical response to oxidative stress in untreated and treated coeliac disease. METHODS: The study involved peripheral blood samples from 39 paediatric patients (18 with active, 11 with silent form of the disease, 10 on gluten-free diet [GFD]) and 30 control subjects. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), as well as the concentrations of total glutathione (GSH) and lipid hydroperoxides (LOOH) were determined in patients and controls. RESULTS: In comparison to the controls, a significant increase in SOD activity was found in the active group (P<0.05), while CAT activity was elevated in GFD group (P<0.05). GPx activity was lower in patients than in controls (active and silent, P<0.001; GFD, P<0.01). GSH contents were significantly reduced in all patient groups (P<0.001) as well, while the concentration of LOOH was elevated in active and silent group (P<0.001). The concentration of LOOH correlated negatively with the activity of GPx (r = -0.32, P<0.01) and the concentration of GSH (r = -0.70, P<0.001). A significant positive correlation was found between the concentration of GSH and the activity of GPx (r = 0.57, P<0.001). CONCLUSIONS: The results show evidence of increased oxidative stress in untreated coeliac disease. Although LOOH were not significantly elevated in the GFD group, changes in antioxidant enzyme activities and GSH content demonstrate that oxidative stress persists even in treated patients.

Lipid peroxidation and antioxidant status in blood of patients with uterine myoma, endometrial polypus, hyperplastic and malignant endometrium.

Oxidative stress is considered to be involved in pathogenesis of many disorders of the female genital tract. In this study, we explored the lipid peroxidation levels and antioxidant enzyme activities in women diagnosed with different forms of uterine diseases in order to evaluate the extent of oxidative stress in blood of such patients. Blood samples of healthy subjects and gynecological patients were collected and subjected to assays for superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and lipid hydroperoxides. The results show that alterations of measured parameters vary with the enzyme type and diagnosis. However, both reduction in antioxidants and elevation of lipid peroxidation were observed in general. Lipid hydroperoxides level was negatively correlated to superoxide dismutase and glutathione peroxidase activities, as well as positively correlated to catalase activity. In addition, the lipid hydroperoxides/ glutathione peroxidase ratio was found to be increased, according to the type of uterine disease. The obtained results show that perturbation of antioxidant status is more pronounced in blood of patients with premalignant (hyperplastic) and malignant (adenocarcinoma) lesions, compared to those with benign uterine changes such as polypus and myoma.

Lipid peroxidation and antioxidant status in blood of patients with uterine myoma, endometrial polypus, hyperplastic and malignant endometrium

Oxidative stress is considered to be involved in pathogenesis of many disorders of the female genital tract. In this study, we explored the lipid peroxidation levels and antioxidant enzyme activities in women diagnosed with different forms of uterine diseases in order to evaluate the extent of oxidative stress in blood of such patients. Blood samples of healthy subjects and gynecological patients were collected and subjected to assays for superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and lipid hydroperoxides. The results show that alterations of measured parameters vary with the enzyme type and diagnosis. However, both reduction in antioxidants and elevation of lipid peroxidation were observed in general. Lipid hydroperoxides level was negatively correlated to superoxide dismutase and glutathione peroxidase activities, as well as positively correlated to catalase activity. In addition, the lipid hydroperoxides/ glutathione peroxidase ratio was found to be increased, according to the type of uterine disease. The obtained results show that perturbation of antioxidant status is more pronounced in blood of patients with premalignant (hyperplastic) and malignant (adenocarcinoma) lesions, compared to those with benign uterine changes such as polypus and myoma.

Superoxide dismutase activity in colostrum, transitional and mature human milk.

Colostrum and mature human milk are rich sources of nutrients and contain biologically active molecules that are essential for specific antioxidant functions. The aim of the present study was to determine the activity of copper, zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) activity in different phases of lactation. Specific enzyme activity was determined in colostral milk (3rd-5th days after delivery), and in mature milk in the third week (15-20 days), and the fourth and seventh months of lactation. In the third week of lactation, the activity of CuZnSOD and MnSOD was significantly higher in comparison to the colostral phase. In the fourth month of lactation, the activity of both enzymes was suppressed, while in the seventh month of lactation the MnSOD activity was increased, and the CuZnSOD activity was not significantly changed. These findings show that the activities of superoxide dismutases significantly change during different phases of lactation.

Differences in antioxidative response of rat hippocampus and cortex after exposure to clinical dose of gamma-rays.

Ionizing radiation increases intracellular production of reactive oxygen species, which can damage cell structure and function. The brain is particularly vulnerable to oxidative injury, and in an area-dependent manner. In order to elucidate differences in enzymatic antioxidative response of rat hippocampus and cortex, we measured activities of CuZnSOD, MnSOD, and CAT in those two brain regions, isolated 1 h and 24 h after exposure to 2 Gy of gamma-rays. Our results indicate that lower MnSOD activities and inducibility, found in the hippocampus, are probably some of the main reasons for the particularly great oxidative vulnerability of this brain region.

Antioxidant enzyme activity in rat hippocampus after chronic and acute stress exposure.

Chronic exposure to stress alters the prooxidant-antioxidant balance, which might lead to the development of various human pathological states. In order to explain the role of antioxidant response in stress-induced injury, we examined the effects of two types of acute stress, as well as combined effects of chronic and acute stress on manganese-superoxide dismutase, copper,zinc-superoxide dismutase, and catalase activities in rat brain hippocampus. Our results show that chronic stress induces an increase in oxidative enzyme activities and that adaptation to chronic stress might alter hippocampal antioxidant mechanisms' response to acute stress.