Snjezana Ramic - Academia.edu (original) (raw)

Papers by Snjezana Ramic

Biological & Pharmaceutical Bulletin, 2008

The radioprotective effects of water-soluble derivate of propolis (WSDP) collected in Croatia, an... more The radioprotective effects of water-soluble derivate of propolis (WSDP) collected in Croatia, and single flavonoids, caffeic acid, chrysin and naringin in the whole-body irradiated CBA mice were investigated. Irradiation was performed using a gamma-ray source ((60)Co), and absorbed doses were 4 and 9 Gy. The efficiency of test components was evaluated when given intraperitoneally (i.p.) at dose of 100 mg kg(-1) for 3 consecutive days before and/or after irradiation. Moreover, possible genotoxic effects of all test components were assessed on non-irradiated animals. The higher efficiency of test components was observed when given preventively. The results suggest that propolis and related flavonoids given to mice before irradiation protected mice from lethal effects of whole-body irradiation and diminish primary DNA damage in their white blood cells as detected by the alkaline comet assay.

Phytotherapy Research, 2009

Collegium antropologicum

Early stage testicular seminoma is a radiosensitive tumor. Its incidence has significantly increa... more Early stage testicular seminoma is a radiosensitive tumor. Its incidence has significantly increased during the last decade especially in the young population. Although the therapy for testicular seminoma gives very satisfying results, the evaluation of genome damage caused by the therapy is of a great importance in order to recognize possible related health risks. The present study was performed on ten patients diagnosed with seminoma stage I; pT1/2N0M0S0, treated with adjuvant radiotherapy (a radiation dose of 25 Gy divided in 16 fractions) after orchidectomy. To assess the possible existence of an increased baseline DNA/chromosome damage in patients we also selected the appropriate control group often healthy men. The levels of primary DNA/chromosome damage in peripheral blood lymphocytes, as well as the dynamics of their repair were studied using the alkaline comet assay, chromosome aberration and cytokinesis-block micronucleus assay. Altogether four blood samples per patient we...

Archives of Industrial Hygiene and Toxicology, 2000

Tryptophol is an aromatic alcohol and secondary metabolite of the opportunistic fungus Candida al... more Tryptophol is an aromatic alcohol and secondary metabolite of the opportunistic fungus Candida albicans. Although its toxicity profi le at cell level has been poorly investigated, recent data point to cytotoxic, cytostatic, and genotoxic effects in lymphocytes and the induction of apoptosis in leukaemic blood monocytes. In this pilot study we evaluated the genotoxicity of tryptophol in vitro on four permanent cell lines of animal and human origin: ovary cells, alveolar epithelium, liver cells, and blood monocytes using the alkaline comet assay. We selected cells that might be principal targets of tryptophol and other low-molecular geno(toxins) secreted by Candida albicans during host invasion. Our results suggest that tryptophol applied in vitro at 2 mmol L -1 for 24 h damages DNA in HepG2, A549 and THP-1 cells, obviously due to bioactivation and/or decomposition of the parent compound, which results in the formation of more genotoxic compound(s) and production of reactive species that additionally damage DNA. On the other hand, notably lower levels of primary DNA damage were recorded in CHO cells, which lack metabolic activity. Future studies with tryptophol should look further into mechanisms involved in its toxic action and should focus on other cell types prone to infection with Candida spp. such as vaginal epithelial cells or keratinocytes of human origin.

HER-2/neu oncogene is amplified/overexpressed in 15-30% cases of invasive human breast carcinomas... more HER-2/neu oncogene is amplified/overexpressed in 15-30% cases of invasive human breast carcinomas (BC), resulting with a high risk of relapse and poor survival. Due to reduction in BC recurrence and markedly improved survival, treatment of HER-2/neu overexpressing patients with humanized monoclonal antibody trastuzumab (Herceptin®) has become a worldwide treatment of choice. Unfortunately, in some patients responses to trastuzumab vary in magnitude and can be short-lived, with clinical limitations resulting from acquisition of trastuzumab resistance. We evaluated the expression of Tyr-1248 phosphorylated form of HER2 receptor (pHER2) among 83 patients diagnosed with ductal invasive BC. Immunohistochemically detected expression of pHER2 was correlated with known prognostic markers for BC. 42 selected patients were HER2 negative while 41 showed HER2 overexpression and were scheduled for trastuzumab therapy. Overexpressed HER2 was mainly in its phosphorylated/active form (p<0.001). ...

Journal of Environmental Science and Health Part A

Present study aimed at an integral assessment of sister chromatid exchange (SCE) frequencies in t... more Present study aimed at an integral assessment of sister chromatid exchange (SCE) frequencies in the health care workers occupationally exposed to cytostatics. The results of 500 individual analyses were evaluated. Drug handling practice was investigated in parallel and the results showed that cytostatics are mostly prepared outside hospital pharmacy (98%) and mainly handled by nurses (96%). Mean frequency of SCE was 5.63 +/- 2.28, while HFC represented 9.65% of the cells analysed. Both values were higher compared to previously established control values for Croatian population. The duration of exposure, profession, age, gender, smoking habit, medical exposures, and simultaneous exposure to other occupational mutagens significantly contributed to SCE and HFC values. The usefulness both biomarkers in the assessment of cytogenetic damage is confirmed. Since current practice in Croatian hospitals does not include regular monitoring of workplaces, to ensure maximal occupational safety, a...

Collegium antropologicum, 2014

Endometrial cancer is the most common gynecological malignancy in Croatia. The aim of this study ... more Endometrial cancer is the most common gynecological malignancy in Croatia. The aim of this study was to determine the immunohistochemical expression of estrogen receptors (ER) and progesterone receptors (PGR) in cancer cells and in the myometrium and to correlate it with prognostic factors of endometrial carcinoma. ER positivity in carcinoma cell nuclei was found in 42 cases (73.7%) and PGR positivity was found in 39 cases (68.4%). Loss of ER in carcinoma cell nuclei correlated with larger tumor size (p = 0.041), poor carcinoma differentiation (p = 0.012), a more aggressive histological type (p < 0.001), lymphovascular space invasion (p = 0.002) and a higher surgical stage (p = 0.037). Loss of PGR in carcinoma cell nuclei correlated with an increased age in patients (p = 0.009), poor tumor differentiation (p = 0.002), a more aggressive histological type (p < 0.001), lymphovascular space invasion (p = 0.002) and a higher surgical stage (p < 0.001). The lower expression of bo...

Anticancer research, 2013

To determine the correlation of phosphorylated human epidermal growth factor receptor-2 (pHER2) w... more To determine the correlation of phosphorylated human epidermal growth factor receptor-2 (pHER2) with clinicopathological characteristics of breast cancer (BC) and patients' response to trastuzumab-based therapy. pHER2 was determinated immuno-histochemically in 88 cases of HER2-positive and 50 cases of HER2-negative BC. All patients with HER2-positive BC received trastuzumab-based therapy and 16 of them (18.2%) had disease progression during therapy treatment (i.e. trastuzumab-resistant). pHER2 was predominantly expressed in HER2-positive BC, with 55 cases (62.5%) of tumours expressing pHER2. Six cases of HER2-negative cancer (12.5%) displayed positive expression of pHER2. Expression of pHER2 correlated with younger age of patients and negative oestrogen receptor status. Acquisition of resistance to trastuzumab correlated with negativity for pHER2 (p=0.028). Positive expression of pHER2 may yield additional information regarding the poor prognosis of BC and could be used for pre-...

Collegium antropologicum, 2007

The aim of this study was to ascertain which histological types of melanoma can clinically and mo... more The aim of this study was to ascertain which histological types of melanoma can clinically and morphologically appear as polypoid melanomas. In 645 cases of primary cutaneous melanoma we have analyzed criteria for diagnosis of polypoid cutaneous melanoma and afterwards we have analyzed growth phase in each polypoid melanoma, histological type of atypical melanocytes, the number of epidermal ridges which are occupied by atypical melanocytes, and distribution according to age, sex and location, as well as the disease free survival. According to the criteria for polypoid melanomas we have found 147 (22.8%) polypoid cutaneous melanomas. Analyzing the growth phases, histological types of atypical melanocytes and the number of affected epidermal ridges in the group of polypoid melanomas we have ascertained 2 (1.4%) ALMs, 4 (2.8%) LMMs, 42 (28.6%) SSMs and 99 (67.2%) NMs. Our conclusion is that polypoid cutaneous melanomas are morphological forms of various histological melanoma types (ALM...

ABSTRACT Breast cancer (BC) is the most prevailing malignancy among worldwide female population, ... more ABSTRACT Breast cancer (BC) is the most prevailing malignancy among worldwide female population, with constantly increasing incidence and serious consequences on morbidity and mortality of affected patients. Genotypically and phenotypically BCs represent very heterogeneous group of tumours, diverse in their pathohistological features, expression of molecular markers, malignant/metastatic potential, clinical presentation, prognosis, and response to therapy. The purpose of this preliminary study was to determine the expression pattern of pannexin family (Panx 1/2/3) of gap junction proteins in invasive ductal breast carcinoma. A total of 27 female patients with diagnosed invasive ductal BC were included in the study. All cases were graded according to the modified criteria of Scarff Bloom-Richardson, as described by Elston and Ellis. Tumour tissue samples surgically removed from each patient (primary tumor and positive axillary lymph nodes) were embedded in paraffin blocks for immunohistological staining or snap-frozen in liquid N2 for RNA isolation. Immunohistochemical staining was performed with rabbit polyclonal anti-human pannexin-1, 2 and 3 antibody (AbCam ab78380, ab55917, ab98093) using DAB substrate and HRP reaction. RNA isolation, in vitro transcription and qRT-PCR were performed using TRI Reagent and RNeasy Mini kit, gene specific Quanti Tect Primer Assay (Qiagen) and 7500 qRT-PCR system (Applied Biosystem). Obtained results showed positive (protein, mRNA) expression pattern ranging from diffuse (Panx2) to granular (Panx1, Panx3) cytoplasmatic staining with slight or no membrane positivity in tumor cells of all analyzed samples. However, the significance, if any, of their expression for tumorigenesis of invasive ductal breast carcinoma has still to be elucidated.

ABSTRACT AIMS: The epidermal growth factor receptor family (EGFR) consisting of four tyrosine kin... more ABSTRACT AIMS: The epidermal growth factor receptor family (EGFR) consisting of four tyrosine kinas receptors, EGFR1/c-erbB1, c-erbB2, cerbB3 and cerbB4. They are involved in tumor cell proliferation, differentiation, survival and angiogenesis. Co-expressed in the same neoplastic cell source with accompanying hetero-dimerization they further enhance pathologically important signaling transduction pathways. In the presented study, we analyzed the expression and cellular distribution of EGFR family members in sporadic cardiac myxoma lesions of human heart. METHODS AND RESULTS: The study included 36 patients, who were treated with surgical excision of the lesion. Expression of EGFR1/c-erbB1, c-erbB2, cerbB3 and cerbB4 was studied by immunohistochemical staining and QRT-PCR. The patient population was comprised of 27 (75%) women and 9 (25%) men. The mean age of the patients were 63 years, with an age range of 34 to 87 years. All study cases were sporadic myxomas. All lesions were located in the left atrium. Majority of examined cardiac myxoma cases demonstrate moderateto- strong and overlapping expression of EGFR family protein members in all commonly present histological types of neoplastic myxoma cells except the c-erbb2 which was immunohistochemically negative in all cases. c-erbB4 showed the highest and statistically significant decrease in expression on mRNA level compared to normal left atrial tissue. Compared relative quantities of EGFR protein members with each other that c-erbB3 and crbB2 are the only ones exhibiting statistically significant (r=0.721, p=0.01304), and positive correlation. CONCLUSION: EGFR1/c-erbB1, cerbB3 and cerbB4 are co-expressed in cardiac myxoma lesions and may play a distinct role in their pathogenesis.

ABSTRACT BACKGROUND: Cardiac myxoma represents the most prevalent type of primary cardiac tumors ... more ABSTRACT BACKGROUND: Cardiac myxoma represents the most prevalent type of primary cardiac tumors in adults with very serious consequences for morbidity and mortality of affected patients. Clinically useful biomarkers and patophysiological mechanisms responsible for their etiology and progression are still vastly unknown. The tripartite pannexin family is the most recently discovered member of gap junction proteins. The functional properties and tissue distribution of known pannexin isoforms in the settings of normal and diseased human heart is still incompletely understood. In this study we examined the expression of pannexin family members in sporadic cardiac myxoma tissue in order to gain a better understanding of this neoplastic disease and expand the currently limited repertoire of its biomarkers. METHODS: The study included 39 patients with cardiac myxoma treated with surgical excision of the lesion. All studied cases were sporadic cardiac myxomas. All lesions were located in the left atrium. Expression of Panx1, Panx2 and Panx3 was studied by immunohistochemical staining and qRT-PCR. RESULTS: All three pannexin family members were immunohistochemically visualized in majority of CM samples (66.7%). Comparison of relative mRNA quantities of pannexin members (qRT-PCR positive in all tissue samples) revealed statistically significant and positive correlation (p=0.0014 ; r=0.732) between Panx3 and Panx2. Panx3 also showed the statistically significant (p= 0.009) increase in mRNA expression compared to the control tissue. CONCLUSION: All three pannexin family members are expressed in neoplastic tissue of sporadic cardiac myxoma and may play distinct role in their pathogenesis

The epidermal growth factor receptor family (HER) of tyrosine kinase receptors includes HER-1, HE... more The epidermal growth factor receptor family (HER) of tyrosine kinase receptors includes HER-1, HER-2, HER-3 and HER-4 with the ability to dimerize after activation. HER-2 is over-expressed in 15%-25% of breast cancers with worse prognosis. HER-2 interaction with other receptors can influence the response of breast cancer targeted therapy. Other HER receptors are not routinely tested. We determined immunohistochemical expression of HER-1, HER-3 and HER-4 on 40 HER-2 positive and 42 HER-2 negative breast cancer biopsies. There was no statistically significant correlation of HER-1 and HER-2 ; 77.5% of HER-2 positive tumors had strong co-expression of HER-3 compared with 7.1% of HER-2 negative cases. HER-3 over-expression is correlated with poor prognostic factors such as negative estrogen and progesterone receptor and positive nodal status. HER-3 receptor was active in proliferating tumor cells with negative HER-2. HER-4 receptor showed controversial results. HER-2 negative tumors had ...

Toxicology Letters, 2006

Hyperbaric oxygen (HBO) treatment is able to generate reactive oxygen species and induce DNA dama... more Hyperbaric oxygen (HBO) treatment is able to generate reactive oxygen species and induce DNA damage. The aim of this study was to evaluate the sister chromatid exchange (SCE) frequencies in lymphocytes from the patients undergoing HBO therapy (HBOT) and to determine the sensitivity of lymphocytes from these patients to SCE induction by 20 and 40 ng/ml mitomycin C (MMC). Patients were exposed to 10 consecutive daily HBO treatment according to a routine HBOT for diabetic feet. In this study, there were 12 patients at the beginning; however, it was not possible to sample all of the patients at all HBO therapy sessions; thus, towards the end of the therapy, the number of patients gradually decreased. A statistically significant induction in mean SCE/cell (p < 0.05, n = 11) was observed immediately after the first session of HBOT. The mean SCE frequency gradually decreased after 5th and 10th HBOT sessions relative to its frequency after the 1st treatment and reached baseline (pretherapy) levels 1 day after the last treatment in the sampled four patients. The mean MMC-induced SCE frequency was highest in lymphocytes sampled immediately after the first HBOT session and significantly higher than the MMC-induced SCE frequencies in cells sampled before HBOT. MMC-induced SCE frequencies remained high in lymphocytes at later stages of therapy, unlike the case with untreated cells and mean MMC-induced SCE frequencies were significantly higher (p < 0.05, n = 4) in lymphocytes sampled 1 day after the last session of HBOT than in lymphocytes sampled from these patients to the beginning of the therapy. In conclusion, HBOT induces SCE and one day after completing the therapy, lymphocytes retain increased sensitivity to the genotoxicity of MMC.

Journal of Environmental Science and Health, Part A, 2004

Water extracts genotoxicity of the coagulant produced from industrial waste (red mud and waste ba... more Water extracts genotoxicity of the coagulant produced from industrial waste (red mud and waste base) and its waste mud remained after waste water treatment by the coagulation/flocculation process were studied. Tests were conducted in order to confirm nontoxicity of this new product prior to commercial production and usage and also to recommend a safe way for a handling and disposal of the remaining waste material. The toxicity investigation included (i) determining frequency of the cell survival, (ii) the Ames assays, (iii) micronucleus assay, and (iv) cell proliferation kinetics (expressed as mitotic index). These techniques were also employed in toxicity testing of the different concentrations of metal salts, zinc chloride, and lead nitrate in this case since these two elements were present in high concentrations in the waste water intended for the purification with the new coagulant. Mixture of metal salts in the concentrations that represent maximum allowed values for water extracts of technological waste was also tested. Two strains of Salmonella typhimurium, TA98, and TA100 were used for determining cytotoxicity and for the Ames test, while the cytogenetic investigations were performed on human peripheral blood lymphocytes. Water extracts of the coagulant and its waste mud did not induce a significant increase of the micronuclei in human peripheral blood lymphocytes. They also did not disturb lymphocyte proliferation kinetics in vitro. As regards lead nitrate it proved not to be cytotoxic on bacterial strains in the tested concentration range (1-100 mg/L), whereas zinc chloride showed cytotoxic effect for the concentrations above 25 mg/L. The Ames test results for the noncytotoxic concentrations of these metals were negative. Comparing to the negative control a significantly higher number of the micronuclei was determined after the treatment of the whole blood with 100 mg/L of zinc chloride, as well as with 10 and 100 mg/L of lead nitrate. A linear, dose dependent increase was obtained for both salts. Similar results were obtained on the basis of the mitotic index.

Journal of Environmental Science and Health, Part A, 2009

Journal of Environmental Science and Health, Part A, 2010

The primary and residual genome damage and its elimination rate were evaluated in peripheral bloo... more The primary and residual genome damage and its elimination rate were evaluated in peripheral blood lymphocytes of breast cancer patients treated with adjuvant radiotherapy after surgical removal of the tumor by mastectomy or quadrantectomy. The levels of DNA/chromosome damage were estimated before, throughout, as well as after six months, respectively one year after the radiotherapy, using the alkaline comet assay, the chromosome aberration analysis and the cytokinesis-block micronucleus assay. The marked individual differences in the baseline genome damage were observed in patients, which additionally increased until the end of the radiotherapy cycle. The levels of DNA/cytogenetic damage slowly declined during post-irradiation period; although in the majority of subjects they did not return to pre-therapy levels. In addition to the well-established comet parameters, the long-tailed nuclei were also proved as a useful indicator of individual DNA damage and response to radiation. One of the most important observation was that older breast cancer patients, irradiated after mastectomy, had higher values of almost all parameters evaluated. We found positive correlations between the comet assay parameters and the cytogenetic biomarkers that confirmed their complementary value in the assessment of the radiation sensitivity/susceptibility in elderly breast cancer patients. The specific patterns of DNA damage observed in the majority of subjects after a prolonged exposure to ionizing radiation indicate the possibility of adaptive response. Such results may also be linked to the hormesis theory and support previous observations, but the underlying mechanisms should be further investigated on a much larger population.

International Journal of Hygiene and Environmental Health, 2009

The aim of the present study was to evaluate genome damage induced in peripheral blood lymphocyte... more The aim of the present study was to evaluate genome damage induced in peripheral blood lymphocytes of Croatian health care workers occupationally exposed to cytotoxic drugs. A comprehensive multi-biomarker approach using the alkaline comet assay and cytogenetic endpoints (analysis of structural chromosome aberrations, SCE assay, lymphocyte proliferation kinetics and cytokinesis-block micronucleus assay) was employed. The study included two populations of subjects: 50 health care workers occupationally exposed to cytotoxic drugs and 50 control subjects matched in age, gender and smoking habit. An investigation regarding the handling practice with cytotoxic drugs was conducted in parallel. Results obtained indicate high exposure levels at workplace that should be reduced. The values recorded among the occupationally exposed subjects were as follows: mean comet tail length: 17.4670.08 mm; the incidence of long-tailed nuclei: 54.6873.93%; 4.4870.33 structural chromosome aberrations per 200 cells; 5.8170.04 SCE per 50 cells; 29.2872.21% of high-frequency cells; proliferation rate index: 1.9770.12; and 16.3270.85 micronuclei per 1000 binuclear cells. All these values indicated higher levels of DNA and cytogenetic damage compared to the general population. Obtained results also confirmed that the frequency of long-tailed nuclei in the alkaline comet assay represents a helpful complement to other well-established comet parameters. The age of subjects and smoking habit significantly influenced the values of both comet and cytogenetic endpoints. Overall results of this study confirmed that handling cytotoxic drugs without appropriate safety precautions involves a potential genotoxic risk for exposed subjects. Before a strict monitoring of exposure levels on each workplace becomes a standard practice in Croatian hospitals, cytogenetic surveillance of exposed workers is also recommended, at least in cases of accidental exposure.

International Endodontic Journal, 2009

Brzovic V, Miletic I, Zeljezic D, Mladinic M, Kasuba V, Ramic S, Anic I. In vitro genotoxicity of... more Brzovic V, Miletic I, Zeljezic D, Mladinic M, Kasuba V, Ramic S, Anic I. In vitro genotoxicity of root canal sealers.

Food and Chemical Toxicology, 2007

Mycotoxins can frequently occur in animal feed and human food. T-2 toxin, as the most toxic trich... more Mycotoxins can frequently occur in animal feed and human food. T-2 toxin, as the most toxic trichothecene, has been implicated as the causative agent in a variety of animal diseases and is associated with some human diseases. The comet assay was performed as a test for detection of DNA damage caused by T-2 toxin in peripheral blood cells of chicken. The suitability of the comet assay as a biomarker for genotoxic analysis has been applied in studies using human white blood cells. It can be applied to any tissue from which a single cell suspension can be obtained. The method has already been applied to chicken as a foodstuff for detection of irradiation of food containing DNA. However, application of the method on chicken blood cells has not been set up yet. The aim of this research was to develop a protocol for detection of DNA damage induced by T-2 toxin in chicken blood cells. Chickens were administered orally with T-2 toxin and the samples of whole blood were collected at 24 h post treatment. The DNA damage was determined by an increase in the comet parameters in tested animals. Our results show that T-2 toxin had induced significant DNA damage in treated chicken as compared with control animals, indicating that the assay can be used for the assessment of primary DNA damage caused by mycotoxins.