RELIABILITY OF URINE AS SOURCE FOR BIOLOGICAL INFORMATION FOR RISK ESTIMATION FOR PROSTATE MALIGNANCY

Micromeria pulegium (Rochel) Benth. is an endemic species of Lamiacea family that includes frequently used plants in culinary and folk medicine. As cytotoxic potential of some species of Micromeria genus has been confirmed, this study aimed to test unknown antiproliferative and genotoxic potential of M. pulegium, endemic bh species, aqueous leaf extract in normal (human lymphocytes) and cancer (human melanoma GR-M) cells in order to protect small populations of native M. pulegium populations or promote its controlled micropropagation or cultivation. Cytokinesis-block micronucleus cytome assay was applied for human lymphocyte cultures, while trypan blue exclusion assay was used for evaluation of cytotoxicity in human GR-M melanoma cells. Results demonstrate no genotoxic effects up to concentration of 0.2 mg/ml in human lymphocyte in vitro but significant reduction of cell viability in human GR-M melanoma cell line cultures treated with 0.3 mg/ml of Micromeria extract.

Conventional screening and diagnostic procedures in prostate complaints rely on PSA (Prostate Specific Antigen) concentration which is not specific for prostate cancer and frequently leads to unnecessary invasive procedures in order to exclude malignant disease. It is estimated that approximately 50% of persons who underwent tissue biopsy did so based on false positive PSA value. Therefore a proper and timely differential diagnosis of malignant disease using non-invasive techniques remains one of the biggest challenges in medicine. Urine is the invaluable source of biological information contained in small molecules i.e. RNA that is easily accessible and detectable using molecular genetics techniques. We describe economical and fast method for relative expression analysis applicable to any target gene using urine as a sample. Efficient non-invasive method for identification of malignant or high risk cases prove useful in reduction of patient distress during the diagnostic procedure and significantly reduce healthcare costs.

Tartrazine (E 102) is widely used yellow food colorant. It is used in nonalcoholic and sports drinks, spicy chips, jams, jelly and chewing gum and also found in many non-food products like soaps, cosmetics, shampoo, vitamins and some drugs. Tartrazine belongs to the most important and diverse group of synthetic dyes – azo dyes. Their use often creates controversies in the public since some of them are toxic, carcinogenic, mutagenic and cause different disorders or allergic reactions. In this study we aimed to evaluate genotoxic potential of tartrazine in human lymphocytes culture and its cytotoxic potential in human lymphocytes and melanoma GR-M cell line. For testing of its genotoxic and cytotoxic potential in human lymphocyte culture, we used chromosome aberration analysis and cytokinesis-block micronucleus cytome assay. For the analysis of its cytotoxic potential in human melanoma cell culture, we applied trypan blue exclusion assay.

Genotoxic effects of inorganic molecule dipotassium-trioxohydroxytetrafluorotriborate, K2(B3O3F4OH), a promising new therapeutic for the epidermal changes treatment, have been evaluated. In vitro analysis included evaluation of genotoxic and cytotoxic potential of K2(B3O3F4OH) in concentrations of 0.01, 0.02, 0.05 and 0.06 mg/mL applying cytokinesis-block micronucleus cytome assay in human lymphocyte culture. With the increase of concentration the frequency of micronuclei elevated but the differences were not significant. Also, there were no significant differences among the frequencies of nuclear buds and nucleoplasmic bridges between controls and treated cultures. Nuclear division index and nuclear division cytotoxycity index values did not reveal significant cytotoxic effect of K2(B3O3F4OH). In vivo genotoxic effects were analyzed on BALB/c mice applying reticulocytes micronucleus assay. K2(B3O3F4OH) was administrated intraperitoneally in final concentrations of 10, 20, 50 and 55 mg/kg. Significant decrease of reticulocytes ratio and increase of micronuclei frequencies against pre-treatments were found for both sampling periods of 48 and 72 hours of the highest applied concentration. This study confirmed that K2(B3O3F4OH) is not genotoxic in tested concentrations in vitro as well as in concentrations lower than 55 mg/kg in vivo. This study presents a reliable basis for further pre-clinical and potential clinical investigations.

Introduction: Bioflavonoids delphinidin (2-(3,4,5-Trihydroxyphenyl)chromenylium-3,5,7-triol) and luteolin (2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4-chromenone) have been recognized as promising antioxidants and anticancer substances. Due to their extensive use, the goal of the research was to determine whether they have any genotoxic potential in vitro.Methods: Analysis of genotoxic potential was performed applying chromosome aberrations test in human lymphocyte culture, as this kind of research was not conducted abundantly for these two bioflavonoids. Delphinidin and luteolin were dissolved in DMSO and added to cultures in final concentrations of 25, 50 and 100 μM.Results: In human lymphocytes cultures Delphinidin induced PCDs in all treatments, potentially affecting the cell cycle and topoisomerase II activity. In concentration of 50 μM luteolin showed strong genotoxic effects and caused significant reduction of cell proliferation.Conclusion: Luteolin exhibited certain genotoxic and cytostatic potential. Delphinidin was not considered genotoxic, however its impact on mitosis, especially topoisomerase II activity, was revealed.

Abstract The genotoxicity of halogenated boroxine [K2(B3O3F4OH)], a novel compound with the potential for prevention and/or treatment of various skin changes, has been confirmed in human lymphocytes. The potential of luteolin and delphinidin in inhibition of the genotoxic and cytotoxic effects of halogenated boroxine in vitro was analyzed applying the chromosome aberration analysis and the cytokinesis-block micronucleus cytome assay in human lymphocyte cultures. The in vitro treatments included addition of boroxine and flavonoids independently, and combined treatments of boroxine with luteolin or delphinidin. In the concentration of 50 μM, luteolin significantly decreased the frequency of micronuclei and nuclear buds. Delphinidin suppressed the occurrence of aberrant cells in the presence of the halogenated boroxine, but also affected their induction in respective delphinidin treatment. Detection of endoreduplications in combined treatments indicated that these flavonoids are potential inhibitors of cell cycle or topoisomerase II activity. The obtained results have confirmed antigenotoxic activity of selected bioflavonoids in vitro. The side effects of potential therapeutic applications of halogenated boroxine may be inhibited in the presence of bioflavonoids in appropriate dosage.

INTRODUCTION Precancerous changes of the cervix frequently occur in women in their reproductive age and are associated with sexually transmitted diseases. The evolution of these lesions qualifies them as precursors of malignancy, and their origination is associate with various risk factors, human papillomavirus (HPV) being the most important one. A proper clinical approach to and treatment of these changes depend on histologic diagnosis, which must be both terminologically adequate and apprehensive. PREVIOUS CLASSIFICATIONS AND TERMINOLOGY The continuous change in nomenclature and lack of a uniform terminology has become the source of confusion and misunderstanding between gynecologists and pathologists. The term carcinoma in situ was first introduced in 1930 to denote the lesion, which is a reliable precursor of malignancy. Less intensive epithelial changes of the cervix were classified as dysplasia. Depending on extensive the change was, dysplasias were subclassified into mild, moderate and severe. Carcinoma in situ and various degrees of dysplasias were more precisely defined at the First International Congress of Exfoliative Cytology, which has also enabled the biological differentiation between these entities. The histologic differentiation of these lesions was, however, subjective and quite unreliable. UP-TO-DATE CLASSIFICATION AND TERMINOLOGY: Cellular changes in carcinoma in situ and in severe dysplasias were mutually so similar that pathologists could not make a reproducible difference between these lesions. Therefore, a conclusion was reached that these changes were one and the same process, whereas the differences were merely of quantitative nature. This discovery resulted in a terminological change, i.e. in a unique term--cervical intraepithelial neoplasia (CIN), with its gradation from 1 to 3. The 3rd grade of cervical intraepithelial neoplasia, according to the new terminology, encompassed changes which pathologists could not properly differentiate before. Many other changes with various, mostly descriptive terminology have also been included in the CIN category, thus preventing misunderstanding between pathologists and gynecologists. Besides the CIN classification, which has been most widely used today, there is also a division into only two biologically different categories: low-grade cervical intraepithelial neoplasia (Lo-CIN) and high-grade cervical intraepithelial neoplasia (Hi-CIN). The latter modification is included in the Bethesda system of cytologic diagnoses as low-grade squamous intraepithelia lesion (L-Sil) and high-grade squamous intraepithelial lesions (H-Sil). CONCLUSION The use of a uniform terminology and classification minimizes the problem of diagnosing precancerous cervical lesions and enables adequate clinical treatment of these patients.

INTRODUCTION Human papillomavirus (HPV) infection of the female genital tract is a sexually transmissible disease most frequently manifested by warts on the vulva, anogenital region, vagina and cervix. Precancerous lesions of vulvar intraepithelial neoplasia (VIN) as well as the development of invasive malignant neoplasms are also related to the infections caused by some HPV types. Infections with HPV-6 and HPV-11, the disease is often polycentric, sometimes reaching gigantic dimensions, elicit venereal warts. Amongst several histological criteria for diagnosing this change, the most typical is koilocytosis, with perinuclear halo formation with a thick cytoplasmic border. Precancerous changes of the vulva and anogenital region are displastic changes of the squamous epithelium, characterized by high mitotic activity, disturbance of nucleocytoplasmic relationship and lack of differentiation in the upper epithelial layers. The changes in the epithelium divided into thirds starting from basal membrane is the main criterion in estimating the degree of dysplasia, marked by VIN 1, VIN 2 and VIN 3. According to the nuclear and cytoplasmic characteristics. VINs are subclassified into three types: basaloid, verrucous (condylomatous) and well differentiated. Basaloid and verrucous (condylomatous) VIN types are morphological markers of HPV infection. The squamous cell carcinoma of the vulva and anogenital region is a morphologically heteregenous neoplasm with particular histological entities connected with HPV infection. CASE REPORTS Four cases of patients with changes in the anogenital region in the form of small to gigantic polypoid formations were reported. The histological features corresponded to vulvar intraepithelial neoplasms (VIN lesions), as well as to neoplastic changes characteristic of HPV infection. The evolution of the changes from typical condylomas through VIN lesions to infiltrative neoplasms, taking place over the years, was also verified. DISCUSSION Some histologically typical epithelial changes in the scope of VIN, as well as some histological types of malignant neoplasms, are associated with HPV-16 and HPV-18, and with HPV-31 to a smaller extent. The oncogenic potential of these viruses is established by in vitro cultures, but also by their finding in 50-90% of genital neoplasia in different series analyzed. The oncogenic potential of the virus depends on numerous heteregenous and complex factors denoted as risk factors. A typical, morphologically well-differentiated change is condyloma acuminatum, with the finding of acanthosis, hyperkeratosis, parakeratosis, dyskeratosis, and koilocytosis, which is always an accompanying morphological quality in these changes. In precancerous lesions and infiltrative neoplasms, koilocytosis is not a necessary finding. CONCLUSION The HPV infection of the vulva and anogenital region is reflected in a spectrum of histological changes. Condylomatous verrucous lesions, smaller papular or plaque-like changes with VIN histological features, as well as infiltrative malignant neoplasia with certain histological properties, could be induced by different HPV types. In the absence of data of in situ hybridization and immuno-electron microscopy as the reliable evidence of the presence of a certain HPV genotype, histological changes mostly characteristic of HPV infection could serve as an indirect pathway.