CERVICAL TRANSFORMATION IN ALCOHOL ABUSE PATIENTS.

Patients with alcohol consumption have unclear risk of developing cervical cancer. The purpose of our work was detection of cervical neoplastic transformation in women with alcohol abuse. We investigated 13 cases of cervical neoplastic transformation (5 cases of invasive carcinoma and 8 case of cervical intraepithelial neoplasia (CIN)) which where detected occasionally during autopsy of alcohol abuse women. Microscopic investigation with immunohistochemistry (IHC) was performed indirect to Ki-67, p16. There are some peculiarities in development of cervical cancer in women with alcohol consumption. Level of cellular proliferation is significantly higher with positive staining Ki67 ranged from 29.1% to 89.7% in alcohol abuse group despite ranged from 27.41% to 75.3% in comparison group depend of transformation stage. Simultaneously, positive staining p16 was ranged from 26.2% to 94.8 % in alcohol abuse group despite ranged from 16.1% to 93.7% in comparison group. Diffuse staining with p16, specific gravity of cells with positive IHC reaction with this protein and high reaction intensity can be used as a specific and sensitive method for detecting CIN III and invasive carcinoma in alcohol abuse.

PECULIARITIES OF PROLIFERATIVE ACTIVITY OF CERVICAL SQUAMOUS CANCER IN HIV INFECTION.

Patients with human immunodeficiency virus (HIV) infection have a statistically significant increased risk of developing cervical cancer. The expression of the human Ki-67 protein is strictly associated with cell proliferation. The purpose of our work was detection of proliferative activity in cervical squamous cancer in women with HIV infection. We investigated 24 cases (12 patients with HIV and 12 patients without HIV infection) of cervical carcinoma, where biopsy had been performed before the treatment. According to histopathological diagnoses, well-differentiated, moderately and poorly differentiated squamous cell carcinoma (7, 13 and 4 cases respectively) was determined. Mean age of women in the group with HIV infection was 32.7 years, and 38.2 years in the group without HIV infection. Detection of protein Ki-67 expression was performed with nuclear staining in the intermediate and superficial cells. The results of this work show that proliferative activity of cervical squamous cancer in women with HIV infection is characterized by a higher level of Ki-67 with averaging level for all histological types of squamous cell carcinoma 62.5±5.6% that is one and half times higher than in group without HIV infection. Depending on a histological type, expression of Ki-67 has increased from 4.7±3.8% in well-differentiated squamous cell carcinoma up to 89.2±5.1% in poorly differentiated squamous cell carcinoma for group with HIV, and from 21.3±2.4% to 79.4±3.7 in group without HIV.

METABOLIC PROFILE OF SERUM FATTY ACIDS IN PATIENTS WITH COMORBIDITY OF CHRO-NIC OBSTRUCTIVE PULMONARY DISEASE AND CHRONIC PANCREATITIS.

Objective - study of blood serum fatty acid metabolic profile in patients with comorbidity of COPD and chronic pancreatitis. 238 patients were examined, of them 131 with combined COPD and chronic pancreatitis and 107 with isolated COPD run. In study of fatty acid spectrum biological materials were prepared and gas chromatography analysis of blood serum lipid fatty acid spectrum performed according to methodology by L.V.Sazonenko et al. The study showed that COPD exacerbation was accompanied with by changes in contents of particular fatty acids both in patients with isolated COPD and in patients with comorbidity in comparison with almost healthy patients. In comparison of values for isolated COPD patients and comorbidity of COPD and chronic pancreatitis patients unidirectional deviations in concentrations of basic FAs were observed in both groups. Although concentrations of basic FAs mostly did not have probable discrepancies, nevertheless, concentration changes of stearic and linoleic acids were significantly more expressed, which caused saturation level reduction and increased unsaturation of fatty acids. The presence of concomitant chronic pancreatitis in patients with COPD substantially strengthened the expression of FAs spectrum deviations due to increase of unsaturation level and the sum of PSFAs which is indicative of lipid peroxidation processes intensification, and this may be treated as one of additional factors of pathology progressing.

[Stimulation of DNA molecules association with amphiphilic derivatives of 1,3-diazaadamantane containing hydrophobic side chanins].

Earlier, a new class of compounds--amphiphilic derivatives of 1,3-diazaadamantanes, capable of facilitating the strand exchange in the system of short oligonucleotides was revealed. Longer hydrophobic side chains of 1,3-diazaadamantanes promoted stronger acceleration of the reaction. In this study, interaction with DNA of two 1,3-diazaadamantane derivatives containing different side chains was investigated by use of optical methods. Concentration of the investigated 1,3-diazaadamantans micelles formation were determined by the means of monitoring fluorescence intensity enhancement of 1-anilinonaphtalene-8-sulphonate probe; as well as the ranges of concentrations where the compounds/water mixtures existed as true solutions. 1,3-diazaadamantanes affinity to DNA was determined with Fluorescent Intercalator Displacement (FID) approach. Significant increase in hydrodynamic volume of short DNA hairpins in the complexes with 1,3-diazaadamantanes was revealed by estimation of the fluorescence polarization of ethidium bromide probe bound to the hairpins. Intermolecular association of DNA hairpins upon binding with 1,3-diazaadamantans was confirmed by Förster resonance energy transfer in system of an equimolar mixture of fluorescently labeled with Cy-3 and Cy-5 hairpins. In this study, the number of positive charges at 1,3-diazaadamantane derivatives containing side chains of different lengths was demonstrated to affect their affinity to DNA, whereas longer length of the hydrophobic side chains ensured more efficient interaction between the DNA duplexes that may facilitate, in particular, DNA strand exchange.

Balkan endemic nephropathy and associated urinary tract tumours: a review on aetiological causes and the potential role of mycotoxins.

A series of publications in the 1950s described a kidney disease in Bulgaria, the former Yugoslavia and Romania that became known as Balkan endemic nephropathy (BEN). The disease was qualified by World Health Organisation (WHO) experts as 'progressive and very gradually developing renal failure with insidious onset.... The last stage shows marked fibrosis...'. BEN is characterized by tubular degeneration, interstitial fibrosis and hyalinization of glomeruli accompanied by enzymuria and impaired renal function without nephrotic syndrome. Later, an association between BEN and tumours of the kidney pelvis and ureter was recognized, so that the problem of BEN became not only nephrological, but also oncological. There may also be an association with increased urinary bladder cancer incidence, although many confounding factors may interfere in the analysis of data for this organ. In view of the very intimate association between BEN and the urinary tract tumours (UTT), the term 'endemic uropathy' has been proposed. Several hypothesis concerning the aetiology of these diseases has been investigated, which include: predisposing genes factors, environmental factors (heavy metals, minerals, bacteria, leptospira, viruses, fungal toxins and, most recently, pliocene lignites). This paper reviews the different hypotheses about the aetiology of endemic uropathy and pays particular attention to the role of fungal toxins.

Experimental mycotoxic nephropathy in pigs provoked by a diet containing ochratoxin A and penicillic acid.

Mycotoxic nephropathy was induced in 18 young pigs by diets contaminated with strains of Aspergillus ochraceus containing ochratoxin A (OTA) and penicillic acid (PA) at levels corresponding to those naturally encountered in animal feeds in Bulgaria. Haematological and biochemical parameters, as well as the morphological and ultrastructural changes in various internal organs, and especially in the kidneys, were examined at different stages of development of the disease. A mottled surface of the kidneys was only seen in pigs exposed to a mouldy diet containing 180 ppb OTA for 3 months, but microscopic lesions, as well as changes in various haematological and biochemical parameters, were observed in all groups exposed to the same mouldy diet containing only 90 or 180 ppb OTA. Histological examination showed two types of change: degenerative changes affecting the epithelial cells of the proximal tubules, which predominated at the initial stage, and proliferative changes in the interstitium, which predominated at the later stage of the disease. Telangiectasis and lymph stasis were also seen, as well as degenerative changes in the capillary endothelium. The characteristic renal lesions were similar to those observed in spontaneous cases of mycotoxic porcine nephropathy in Bulgaria, but they were a little different from the classic Danish porcine nephropathy. The enhanced toxicity of OTA in our study may be due to a synergistic effect between OTA and PA or to some other unknown metabolites produced by the same ochratoxinogenic strains of A. ochraceus.

Binding, annealing and strand exchange between oligonucleotides in different sites of the RecA protein filament.

The efficiency of single-stranded (ss) oligonucleotides binding at the secondary site of the RecA protein filament is demonstrated to depend on the strandedness of DNA bound at the primary site. When the primary site is occupied by a ss-oligonucleotide, the binding of another ss-oligonucleotide at the secondary site is characterized by higher affinity and a lower rate of dissociation than is the case when the primary site is occupied by a double-stranded oligonucleotide. In contrast to a DNA strand exchange reaction suppressed by a heterologous oligonucleotide bound at the secondary site of the RecA filament, the occupation of the secondary site by a heterologous oligonucleotide does not prevent renaturation between the oligonucleotides bound at the primary site and complementary oligonucleotides from solution demonstrating that the binding of a DNA strand in the secondary site is not a necessary intermediate step in RecA-promoted DNA renaturation.

Citrinin in the diet of young and healthy persons living in balkan endemic nephropathy areas.

The dietary citrinin (CT) intake of 19 persons living in highrisk "Balkan Endemic Nephropathy" areas in Bulgaria was studied. Over 4 weeks, volunteers collected aliquots of their daily meals. Weekly samples were homogenized and analysed for CT by enzyme immunoassay (detection limit: 1ng/g). CT was found at least once in the weekly diet of 11 persons, maximum levels were at 6 ng/g. Considering the total amount of food consumed, the weekly CT intake of several persons exceeded 10 microgram. The data suggest that people living in high-risk nephropathy areas are exposed to dietary CT at considerable levels.

The properties of human DNA fingerprints produced by polymeric monocore probes (PMC probes).

The properties of human DNA fingerprints detected by multilocus polymeric monocore probes (PMC probes) have been investigated. The PMC probes were produced by the polymerase chain reaction with two partly complementary oligonucleotides homologous to various minisatellite or microsatellite core sequences (Ijdo J, Wells RA, Baldini A, Reeders ST. Nucleic Acids Res 1991;19:4780). It has been shown that these probes possess increased sensitivity, they detect considerably more hypervariable fragments in genomic DNA thus exhibiting advantages over the corresponding oligonucleotides and natural polycore minisatellite probes. Variation in the DNA fingerprints of different individuals produced by these probes indicates that the probability of accidental identity is very low (< 10(-12)). According to the data of cross-hybridization with PMC probes of various specificity, several distinct families can be distinguished among G-rich hypervariable sequences of the human genome.

Formation of DNA adducts in tissues of mouse progeny through transplacental contamination and/or lactation after administration of a single dose of ochratoxin A to the pregnant mother.

Ochratoxin A (OTA) is a mycotoxin which has been detected in foods of plant origin, in edible animal tissues, and in human sera, urine, and milk in many countries. OTA is nephrotoxic and carcinogenic in mice and rats and is suspected to play a key role in the etiology of Balkan endemic nephropathy and/or associated urinary tract tumors. In the present study, some early signs of genetic impairment, including the presence of DNA adducts in target tissues from the progeny of mice after administration of a single OTA dose during late pregnancy, have been investigated. By the 32P-postlabeling method, several characteristic DNA adducts with the same Rf values were detected in kidney and liver of both the OTA-treated mice and their progeny the fetus and the offspring. No adduct was found in tissues from control animals. Different adducts were most important in kidney and liver DNA and some were organ-specific. High levels of DNA adducts were detected in the kidneys of male progeny, whereas in the female progeny and the mothers they were detected almost exclusively in the liver. This result correlates well with the carcinogenicity in mice: the target organ for males is the kidney, while for females it is the liver. High levels of DNA adducts were also found in fetuses. These results provide evidence for a direct genotoxic action of OTA in the progeny through transplacental contamination, which constitutes a new serious health hazard of exposure to this toxin.

Periodicity in recA protein-DNA complexes.

The reaction of guanine residues with dimethylsulfate was studied for complexes of recA protein with fluorescent dye tagged double stranded oligonucleotides. The patterns of dimethylsulfate modification obtained demonstrate a similarity of DNA states in the complexes with recA protein formed as a result of recA promoted strand exchange and renaturation reactions. The guanine modification efficiency varies periodically as a function of the base position along the oligonucleotide axis, with a period of 3 nucleotides. This effect suggests that the arrangement of recA monomers along the oligonucleotide is strictly ordered, and the dimethylsulfate reactivity of a guanine residue depends on the site of its binding in a recA monomer.

Efficient interaction of recA protein with fluorescent dye-labeled oligonucleotides.

Some fluorescein derivatives attached to the 5'-end of oligonucleotides stimulate recA protein-oligonucleotide binding. The complex formation at near stoichiometric DNA/protein ratios is demonstrated for 18-bases-long oligonucleotides. The complexes with dye-labeled oligonucleotides are shown to be active in the reaction of homologous strand exchange. The strand exchange reaction in the presence of adenosine-5'-O-(3-thiotriphosphate) proceeds with the formation of a stable complex of recA protein with the double stranded oligonucleotide, which is a product of the strand exchange. The displaced single-stranded oligonucleotide is shown to be bound weakly.

Ochratoxin A in human blood in relation to Balkan endemic nephropathy and urinary tract tumours in Bulgaria.

Ochratoxin A is suspected of being one of the etiological agents responsible for Balkan endemic nephropathy and the associated urinary tract tumours. Contamination of cereals by this mycotoxin has been found to be more frequent in areas of endemic nephropathy than in areas where the disease is absent. As ochratoxin A binds to serum albumin, it should be detectable in biological fluids from exposed populations. A survey was thus conducted to determine the occurrence of ochratoxin A in blood from people living in the endemic area who were either affected or unaffected by the two diseases and in blood from people living in control regions where these diseases do not occur. Blood samples were collected in 1984, 1986, 1989 and 1990. Ochratoxin A was found more frequently and at higher levels in blood from patients with Balkan endemic nephropathy and/or urinary tract tumours than in blood from unaffected people from endemic and control areas. These findings suggest further that ochratoxin A is involved in the etiology of the two diseases.

Concentrations of ochratoxin A in the urine of endemic nephropathy patients and controls in Bulgaria: lack of detection of 4-hydroxyochratoxin A.

Ochratoxin A has been detected more frequently and at higher levels as a contaminant in staple food consumed by subjects affected by Balkan endemic nephropathy or urinary tract tumours in the Vratza district (Bulgaria) than in samples from control populations in and outside the endemic area. Serum from patients with Balkan endemic nephropathy also contained ochratoxin A more frequently and at higher levels than serum from controls. Metabolic phenotyping of subjects in the Vratza district with debrisoquine revealed a preponderance of extensive metabolizers among subjects at high risk for Balkan endemic nephropathy. In rats, ochratoxin A is metabolized to 4-hydroxyochratoxin A, and rat strains shown to be poor or extensive metabolizers of debrisoquine were also poor or extensive metabolizers of ochratoxin A. In order to determine whether the metabolic phenotype for debrisoquine also parallels that of ochratoxin A in humans, a sensitive method was developed for quantifying ochratoxin A and its 4-hydroxy metabolite in human urine. This method was subsequently used to analyse urine from subjects who had previously been phenotyped for debrisoquine. Ochratoxin A was detected more frequently and at higher levels in urine from members of families affected by Balkan endemic nephropathy than in samples taken from subjects in control areas. No 4-hydroxyochratoxin A was found in any of these samples (detection limit, 15 ng/l urine). On the basis of results from human studies and animal models, the role of genetic polymorphism in drug oxidation and disease susceptibility is discussed briefly.

Ochratoxin A and other mycotoxins in cereals from an area of Balkan endemic nephropathy and urinary tract tumours in Bulgaria.

The etiology of Balkan endemic nephropathy and urinary tract tumours in the rural population of the endemic regions remains unknown. As one hypothesis involves mycotoxins, a survey was carried out to investigate the possible involvement of the nephrotoxic mycotoxins ochratoxin A and citrinin. Recently, this survey was extended to screening for the presence of other mycotoxins--aflatoxins, citrinin, sterigmatocystin and zearalenone. A total of 524 samples of home-produced and home-stored beans and maize from the harvests of 1984, 1985, 1986, 1989 and 1990 were analysed. Ochratoxin A was found in samples from both endemic and nonendemic areas, but more of the samples from affected families were contaminated, and at higher levels, than those from unaffected households. Citrinin and aflatoxins B1 and G1 were also found more frequently in samples from endemic areas. These results support the theory that mycotoxins are involved in the etiology of Balkan endemic nephropathy and urinary tract tumours.

Induction of characteristic chromosomal aberrations, particularly X-trisomy, in cultured human lymphocytes treated by ochratoxin A, a mycotoxin implicated in Balkan endemic nephropathy.

Ochratoxin A (OA), a mycotoxin which induces nephropathy and kidney tumours in rats and mice, is a contaminant of food consumed by a population with a high incidence of endemic nephropathy (EN). It was therefore tested in vitro for its ability to induce chromosomal aberrations in human peripheral lymphocytes in a small number of subjects, in the presence or absence of a kidney microsomal metabolic activation system. OA was found to induce aberrations on X chromosomes of similar types to those previously detected in lymphocytes from patients suffering from endemic nephropathy.

Ochratoxin A in human blood in relation to Balkan endemic nephropathy and urinary system tumours in Bulgaria.

In continuing the effort to provide further evidence for the hypothesis that ochratoxin A might be involved in the aetiology of Balkan endemic nephropathy and the associated urinary system tumours, a survey to determine the occurrence of ochratoxin A in human blood was conducted in affected and unaffected areas of Bulgaria, where both diseases are prevalent. Ochratoxin A, positive samples, were present more often in blood from affected patients and the contamination levels were generally higher.