Branislav Rovcanin

Lactate dehydrogenase, Catalase, and Superoxide dismutase in Tumor Tissue of Breast Cancer Patients in Respect to Mammographic Findings

Lactate dehydrogenase (LDH), marker of anaerobic metabolism, is associated with highly invasive and metastatic breast cancer. Novel studies show that increased anaerobic metabolism (LDH), as well as activity of antioxidative enzymes (superoxide dismutase (SOD) and catalase (CAT)), is correlated with higher mammographic density, as known predictor of breast cancer risk. In this study, we measured LDH, MDH, and SOD activity in tumor and adjacent tissues of breast cancer patients by spectrophotometric assay. Mammograms were evaluated according to the American College of Radiology Breast Imaging Reporting and Data system. Mammographically dense breast tissue is associated with higher activity of LDH in tumor tissue of breast cancer patients. Moreover, patients with masses have significantly higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Patients with spiculated mass margin had higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Activity of LDH in patients significantly increases, while activity of CAT significantly decreases with the increase of BIRADS category. These results suggest that the association of activity of LDH and CAT in tumor tissue with mammographic characteristics could help in defining aggressive breast cancers.

Evaluation of Novel Biomarkers of Acute Kidney Injury: The Possibilities and Limitations.

Despite the recent findings concerning pathogenesis and novel therapeutic strategies, the mortality rate in patients with acute kidney injury (AKI) remains very high. Early detection of patients with impaired renal function may help to ensure more aggresive treatment and to improve clinical outcome. Serum creatinine is still gold standard of kidney injury, although it is well known as an insensitive and unreliable biomarker (for example, its concentration does not increase significantly until about half of the kidney function is lost). Considering these data, researches and clinicians are making great efforts in the past decade in order to discover and validate novel AKI biomarkers. Kidney injury molecule-1 (KIM-1), Neutrophil gelatinase-associated lipocalin (NGAL), Interleukin-18 (IL-18), Cystatin C (Cys-C) are some of new, promising markers of kidney damage which are currently in the focus of preclinical and clinical studies. Recent data suggest that some of these new biomarkers represent important parametars of acute tubular necrosis (ATN) and reliable predictors of development and prognosis of AKI. Beside that, monitoring of these markers could have significant importance for early diagnosis and clinical course, not only in patients with various forms of AKI and other renal diseases, but also in patients with cardiorenal syndrome, heart failure, cardiopulmonary bypass, cardiothoracical surgical interventions, in the pediatric emergency setting etc. The aim of this review is to summarize the literature data concerning some new biomarkers, evaluate their role as well as their limitations in the early diagnosis and predict clinical outcome of some renal diseases.

Activity of Superoxide Dismutase, Catalase, Glutathione Peroxidase, and Glutathione Reductase in Different Stages of Colorectal Carcinoma

BackgroundReactive oxygen species are involved in the pathogenesis of colorectal carcinoma. Clarification of oxidative/antioxidant specificities of different stages of colorectal carcinoma is of special importance.AimTo determine oxidative/antioxidant status in plasma of patients with different stages of colorectal carcinoma using malondialdehyde concentration, superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities and distribution of superoxide dismutase isoforms.MethodsLipid peroxidation and antioxidant enzymes activity were estimated using spectrophotometric methods. Reverse zymography was applied for characterization of superoxide dismutase isoforms.ResultsLipid peroxidation is increased in all groups compared to the control, but without differences between different stages of colorectal carcinoma. Total superoxide dismutase activity is lower in all colorectal carcinoma groups than in control, and there is a significant increase in tumor stage IV when compared with tumor stage II. Manganese superoxide dismutase isoform is dominant in all groups and its relative activities are significantly higher than activities of a copper/zinc isoform. Total peroxidase potential reflected in catalase and glutathione peroxidase activity is increased when compared to the control, but without any significant differences between colorectal carcinoma groups. Glutathione reductase activity is lower in all colorectal carcinoma groups than in control, and a significant decrease in glutathione reductase activity was obtained between patients in tumor stage II and III compared to tumor stage IV.ConclusionsColorectal carcinoma is characterized by increased oxidative stress and antioxidant disbalance. Progression of disease is followed by an increase in redox disbalance.

Kidney Injury Molecule-1 and Cardiovascular Diseases: From Basic Science to Clinical Practice

Despite the recent findings concerning pathogenesis and novel therapeutic strategies, cardiovascular disease (CVD) still stays the leading cause of morbidity and mortality in patients with renal dysfunction, especially acute kidney injury (AKI). Early detection of patients with impaired renal function with cardiovascular risk may help ensure more aggressive treatment and improve clinical outcome. Kidney injury molecule-1 (KIM-1) is a new, promising marker of kidney damage which is currently the focus of countless studies worldwide. Some recent animal and human studies established KIM-1 as an important marker of acute tubular necrosis (ATN) and reliable predictor of development and prognosis of AKI. Food and Drug Administration (FDA) in USA acclaimed KIM-1 as an AKI biomarker for preclinical drug development. Recent data suggest the importance of monitoring of KIM-1 for early diagnosis and clinical course not only in patients with various forms of AKI and other renal diseases but also in patients with cardiorenal syndrome, heart failure, cardiopulmonary bypass, cardiothoracic surgical interventions in the pediatric emergency setting, and so forth. The aim of this review article is to summarize the literature data concerning KIM-1 as a potential novel marker in the early diagnosis and prediction of clinical outcome of certain cardiovascular diseases.

Acute Pretreatment with Chloroquine Attenuates Renal I/R Injury in Rats

Background Acute kidney injury (AKI) still remains an unresolved problem in pharmacotherapy and renal inflammation is a major factor in its development. Chloroquine, a well-known antimalarial drug, posses pleitropic effects as well: antiinflammatory, anticoagulant and vascular actions. The effects of chloroquine on renal function may involve significant increase in urine flow rate, glomerular filtration rate and sodium excretion, as well as stimulation of nitric oxide synthase. However, its role in experimental models of renal I/R injury is unknown. We aimed to analyze the acute effects of a single-dose intravenous chloroquine administered at three different times in the experimental model of I/R injury in rat. Methods Rats were subjected to bilateral renal ischemia (45 min) followed by reperfusion with saline lasting 4 hours. Chloroquine was administered in doses of 0.3 mg/kg i.v. and 3 mg/kg i.v. 30 min before ischemia, 30 min before reperfusion and 5 min before reperfusion. Selected a hemodynamic, biochemical and morphological parameters were followed in the Sham-operated animals and rats subjected to I/R injury and pretreated with saline or chloroquine. Results Chloroquine (0.3 and 3 mg/kg, i.v.) protected the I/R injured kidney in an U-shaped manner. Both doses were protective regarding biochemical and histological markers of the I/R injury (serum urea, creatinine and fractional excretion of sodium, as well as total histological score, tubular necrosis score and KIM-1 staining score) (P<0.05 vs. corresponding controls, i.e. rats subjected to I/R injury and treated with saline only). The protective effects of the lower dose of chloroquine were more profound. Time-related differences between pretreatments were not observed (P>0.05, all). Conclusion Our study shows for the first time that a single dose of chloroquine (0.3 mg/kg i.v.) could afford significant protection of the injured rat kidney.

Inter-individual variability in the response of human peripheral blood lymphocytes to ionizing radiation: comparison of the dicentric and micronucleus assays

Ionizing radiation can induce a wide range of DNA damage that leads to chromosomal aberrations. Some of those aberrations (dicentrics and micronuclei) are applied in biodosimetry. Biological dosimetry assumes similar radiosensitivity of each donor, but it does not exclude inter-individual variations in radiation susceptibility. Therefore, for biological reasons, it is always challenging to investigate inter-individual variability in response to radiation. For mechanistic reasons, it is also interesting to investigate the correlation between dicentric and micronuclei formation in response to radiation. In this experiment, irradiated blood specimens from 14 healthy male and female donors have been used to evaluate inter-individual variability in response to the genotoxic effects of X-ray radiation, as well as the dose–response relationship and test sensitivity using two endpoints (dicentrics and micronuclei). The results showed similar patterns of cytogenetic biomarker distribution between donors, but differences in the response of some donors at some doses. Data also showed that responses of male donors were better detected using the dicentric test, while for females, micronucleus frequencies were higher in response to the same dose of radiation. No influence of smoking status or age on specific responses was observed. Group variability in response to radiation was evaluated using coefficient of variation for each group of individuals irradiated with the same doses; as the dose increases, group variability becomes substantially lower. Despite sporadic inter-individual variability, trend of radiation-induced changes was similar. Produced calibration curves for both types of damage revealed dicentrics as genetic damage more typical for radiation than micronuclei.

Matrix metalloproteinases and membrane damage markers in sera of patients with acute myocardial infarction

Coronary artery disease is a multifunctional disease and represents one of the leading causes of death worldwide. Oxidative stress appears as an etiological factor for myocardial damage during acute myocardial infarction. Some data suggest that acute coronary syndromes may also be influenced by matrix metalloproteinases through degradation of the fibrous cap of vulnerable atherosclerotic lesions. It has been indicated that gelatinases A and B play a key role in acute myocardial infarction and deoxyribonuclease I has been postulated to be a novel early phase marker of disease. The aim was to study activity of gelatinases A and B in acute myocardial infarction and its association with some membrane damage markers. Seventy-five patients with disease and seventy-five healthy controls were enrolled. Activities of lactate dehydrogenase, malate dehydrogenase, and deoxyribonuclease I were estimated using standard spectrophotometric assay and isoforms of lactate and malate dehydrogenases were determined using direct zymography. Activity of dehydrogenases was significantly higher in patients, while deoxyribonuclease I was lower. Isoform 2 of lactate dehydrogenase was significantly higher in the patient group. Gelatinases A and B were detected only in patients group. The results suggest determination of serum malate dehydrogenase activity to be used as an additional parameter for acute myocardial infarction diagnosis. Those findings suggest important role of gelatinases A and B as biomarkers of early stage of acute myocardial infarction together with membrane damage parameters.

Skin and Sural Nerve Biopsies: Ultrastructural Findings in the First Genetically Confirmed Cases of CADASIL in Serbia

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited vascular disorder caused by Notch3 gene mutations. The main histopathological hallmark is granular osmiophilic material (GOM) deposited in the close vicinity of vascular smooth muscle cells (VSMCs). The authors report the first 7 ultrastructurally and genetically confirmed cases of CADASIL in Serbia. Samples of skin and sural nerve were investigated by transmission electron microscopy. GOM deposits were observed around degenerated VSMCs in all the skin biopsies examined. Sural nerve biopsies revealed severe alterations of nerve fibers, endoneurial blood vessels with GOM deposits, endoneurial fibroblasts, and perineurial myofibroblasts. Total genomic DNA was extracted from peripheral blood leukocytes, and exons 2–6 of the Notch3 gene were amplified by PCR and subsequently sequenced. Four different mutations in exons 2 (Cys65Tyr), 3 (Gly89Cys and Arg90Cys), and 6 (Ala319Cys), which determine the CADASIL disease, were detected among all described patients. A novel missense mutation Gly89Cys involving exon 3 was detected. Due to the difficulties in the determination of the Notch3 mutations, these data suggest that electron microscopic analysis for GOMs in dermal vessel wall provides a rapid and reliable screening method for this disease.

Papillary Thyroid Carcinoma: A Malignant Tumor with Increased Antioxidant Defense Capacity

Papillary thyroid carcinoma (PTC) is the commonest thyroid malignancy worldwide for which the radiation exposure is the most influential risk factor. The levels of oxidative stress in PTC are not well characterized on the tissue level. The objective of this study was to evaluate total oxidant status (TOS) and total antioxidant status (TAS) in PTC and benign goiter (BG) tissues and to examine their association with clinicopathological characteristics. Tumor and normal thyroid tissue samples were collected from 59 PTC patients, and goiter tissues were collected from 50 BG patients. TOS and TAS were quantified in the tissue homogenates by spectrophotometric assays. TOS values in tumor tissues did not differ significantly from normal and goiter tissues; however, PTC tissues have significantly higher TAS values than normal and goiter tissues. TOS values correlated with retrosternal growth in BG patients. The significant correlations were found between TOS and TAS values and thyroid function parameters. In 17 PTC patients with multiple tumor foci (multicentric phenotype), TAS values were significantly lower, compared to 42 patients with unicentric PTC. TAS and TOS are the most useful predictors of thyroid capsular invasion by PTC. The age, sex, body mass index, smoking, familial history of thyroid disease and nodule size did not influence TOS and TAS in PTC or BG patients. In conclusion, we show the profiles of TOS and TAS in PTC and BG tissues. Importantly, PTC tissues possess increased antioxidant capacity. The redox status influences the parameters of the thyroid function and tumors biological behavior.

The influence of redox status on inter-individual variability in the response of human peripheral blood lymphocytes to ionizing radiation

Abstract Purpose: Ionizing radiation (IR) can act on atomic structures, producing damage to biomolecules. Earlier investigations evaluating individual radiosensitivity in vitro were focused on cytogenetic biomarkers (chromosomal aberrations – CA and micronuclei – MN). Since IR can also cause oxidative damage by producing reactive oxygen species, the main goal of this investigation was to establish the influence of redox status on CA and MN frequency in human peripheral blood lymphocytes. Materials and methods: Blood samples from 56 healthy donors were irradiated at doses of 0, 0.75, 1.5 and 3 Gy and then analyzed cytogenetically and biochemically. Results: The results showed inter-individual variability in all analyzed parameters, as well as dose-dependent increases in almost all of them. Correlation analysis indicated no association between CA, MN and oxidative stress parameters. However, findings for overall response (HRR) parameters showed that donors with lower values for parameters of antioxidant status had increased levels of cytogenetic damage and higher responses to irradiation and vice versa. Conclusion: Besides well-established cytogenetic biomarkers of radiation exposure, our results indicated promising future use for biochemical oxidative status parameters in routine radiation protection practice, since together they can provide a complete radiation response profile in cases of continuous low-dose exposure, as well as in a radiation emergency.