Search

The presence of N7-alkylguanine adducts in DNA was analysed in a group of 46 patients with larynx tumours. All patients were subjected to laryngectomy and the tissues accessible for analysis by (32)P-post-labelling assay were larynx tumour, larynx non-tumour and peripheral blood leucocytes. N7-Alkylguanine adducts were detected in all the studied DNA samples. The average level of N7-alkylguanines was 26.2/10(7) nucleotides in tumour cells, 22.7/10(7) in non-tumour cells and 13.1/10(7) in blood leucocytes. There was significantly higher level of N7-alkylguanines in the larynx tissues in males than in females. The effect of tobacco smoking on DNA adduct levels was shown by an increase in the average levels of N7-alkylguanines in the subject groups classified according to their smoking habits. A moderate age-related increase in levels of N7-alkylguanine was demonstrated in larynx tumour tissue. The levels of N7-alkylguanine adducts in larynx cells were compared with that of aromatic DNA adducts. Pearson correlation coefficients (0.28 for tumour tissue and 0.30 for non-tumour tissue) indicate independent formation and removal of N7-alkylguanine and aromatic DNA adducts resulting from tobacco smoke exposure.

32 P-Postlabelling was applied to study the distribution of adducts in white blood cells of foundry workers exposed to polycylic aromatic hydrocarbons. The distribution of the adducts among 63 workers followed an apparently trimodal pattern, which could relate to polymorphism in PAH metabolism. A modified postlabelling method is described and some parameters were tested for optimal labelling. The total volume of the polynucleotide kinase reaction is 2 μl, which decreases exposure to radioactivity and costs of isotopes.

1, N 6 -ethenodeoxyadenosine-, 1, N 2 -ethenodeoxyguanosine- and 3, N 4 -ethenodeoxycytidine-3′-monophosphates were labeled by [γ- 32 P] ATP using T4 polynucleotide kinase in conditions commonly used for the 32 P-postlabeling assay. Kinetic studies showed that the reaction is fast reaching a plateau after 15–30 min. The efficiency of phosphorylation, as studied by substrate-product concentration dependency, was between 50–100% at the lower substrate concentrations. The adducts are labeled efficiently at sub-femtomole levels. All the adducts were sensitive to the 3′-dephosphorylation by P1 nuclease although the guanine derivative appeared to be more resistant than the two other adducts.

Polycyclic aromatic hydrocarbons (PAH) are a well-characterized group of mutagens and carcinogens. Benzo(a)pyrene [B(a)P], the best known compound in the group, exerts its genotoxic activity following metabolic activation, when it acquires the properties of an electrophilic reagent that is capable of interacting with DNA. Reactive oxygen species (ROS) can remerge during the PAH metabolic activation. Because of their antioxidant activity, vitamins C and E are thought to act as antimutagenic agents. We designed an in vitro protocol to study the potential protective effect of vitamins C and E toward B(a)P-induced DNA damage. In this study, we examined peripheral blood lymphocytes obtained from healthy nonsmoking female volunteers, 22 to 25 years of age. The cells were exposed in vitro to 1 microM B(a)P in the presence of 40 microM or 100 microM of vitamin C or, alternatively, to 30 microM or 100 microM of vitamin E. The B(a)P-induced DNA damage and repair were estimated as the generation and removal of single-strand DNA breaks measured by the alkaline version of the single-cell gel electrophoresis (comet) assay. The protective effect of vitamins C and E was demonstrated when the vitamins were applied simultaneously with or after the B(a)P. The background level of DNA damage in the presence of vitamins C and E was lower than in the system without the vitamins. The experiments were conducted according to various protocol schemes of the vitamin treatment and the results offer additional evidence of the antigenotoxic activity of vitamins C and E. The vitamin activity does not appear to be connected with the steps in metabolic activation or DNA repair. It seems that both vitamins act as competitors of DNA molecule in reaction with the reactive oxygen species.

Arylamines are known bladder carcinogens deriving from tobacco smoke and environmental pollution. Arylamines are metabolised by NAT1 and NAT2 polymorphic enzymes in reactions of carcinogen activation and detoxification. We analysed genetic polymorphisms in both NAT1 and NAT2 genes in 56 bladder cancer patients and 320 healthy patients. Peripheral blood lymphocytes were collected from each subject and genotyped for NAT1 (six alleles) and NAT2 (four alleles) by PCR-RFLP. A weak association between NAT1 and NAT2 genotypes and bladder cancer risk was found when the genotypes were estimated separately (odds ratio OR 1.2, 95%CI 0.7-2.0, and OR 1.3, 95%CI 0.7-1.9, respectively). Almost all NAT1 genotypes possessing at least one "risk" *10 allele were more frequent in the bladder cancer group than in the control group. There was also an increased frequency of "risk" genotypes along with increased cigarette smoking in bladder cancer patients. The coincidence of NAT1-fast/NAT2-slow appears as a potential risk factor for urinary bladder cancer (OR 1.5, 0.8-3.0), as compared with the other genotype combinations.

The results concerning examination of DNA adducts in oral (23 patients), pharyngeal (23 patients) and larynx cancer (10 patients) subjects are presented. DNA adduct levels were compared in respect to anatomical structure (primary tumour location), number of cigarettes smoked, TNM stage, and age of patients. DNA was isolated from removed tissue (tumour and non-tumour surrounding tissue) using detergent/phenol extraction. 32P-postlabelling assay including nuclease P1-enhancement modification was applied. Aromatic DNA adducts were found in all studied tissues. Total DNA adduct levels (tumour and non-tumour tissues) was lowest in larynx cancer, higher in oral cancer and highest in pharyngeal cancer. There were no influence of age into formation of DNA adducts. The higher level of DNA adducts was found in tumour tissue of oral cancer in the group of smokers with metastasis into lymph nodes.

The examples of so far application of taxol in head and neck cancer are not completely clear. This is the first representative of a totally new group of cytostatics known as taxones. Presently, paclitaxel (TAXOL) is the anti-cancer drug commonly used in ovary cancer patients, resistant to platinum analogues. Taxones have the different functioning than the other anti-cancer drugs. They are responsible for originating microtubules and their further stabilisation. Moreover, paclitaxel (TAXOL) is a chemical compound sensitizing tumor cells for ionizing radiation. The authors are trying to specify the role of taxones nowadays and for the future use.

The bleomycin test was established in the laboratory of Hsu et al. (M.D. Anderson Cancer Center, Houston, Texas) for quantitative estimation of hidden chromosome instability. The article presents a concept, method and applicability of the test. Chromosome sensitivity initially studied in respect to estimate a general genetic risk to develop cancer was found as a technique of an extended potential to study a genetic risk of familial cancer, young adultsneck cancer and other associations.

The aim of this study was to identify subjects with 35delG mutation of GJB2 gene as the most frequent genetic cause of deafness. Deaf patients receiving cochlear implantation at the ENT Clinic at University of Medical Sciences in Poznań and their family members were recruited to the study. Peripheral blood lymphocytes DNA was amplified in allele-specific PCR and analysed for single strand conformation polymorphism (SSCP) to detect mutation at DFNB1 locus. 35delG mutation at both alleles was found at 42.9% of deaf patients and 29.4% of health relatives were found to be carrier of the mutation at one allele. The study is thought to be a first step in analysis of typical mutations in Polish deaf population.

A classical cytogenetic analysis was applied to analyse karyotypes of 9 cell lines derived from laryngeal cancer. The most frequent aberration was a deletion of the whole sex chromosome Y. An abundance of somatic chromosome alterations was further analysed to find correlation with tumour staging and grading. A conventional cytogenetic analysis seems to be not sufficient to recognize chromosome alterations specific for a given tumor stage. On the other hand, an analysis in respect to histologic grading has indicated for an association between rearrangement of 9 chromosome and a high tumor aggressiveness. It seems that a combination of conventional cytogenetics with molecular methods (FISH, CGH) would be helpful in diagnosing of laryngeal cancer.

An exposure to tobacco smoke carcinogens is followed by an interaction of chemical carcinogens with DNA molecule resulted by the formation of carcinogen: DNA adducts. The study subjects were 40 oral and pharyngeal cancer patients with primary tumours diagnosed as squamous cell carcinoma. The biological samples purchased for analysis included tumour biopsy, the surrounding tissue (histopathologically recognised as non-malignant) and peripheral blood leucocytes. For DNA adducts analysis first DNA was isolated using phenol detergent extraction followed by 32P-postlabelling assay including P1 nuclease enhancement. Aromatic DNA adducts were found in all studied tissues. The average levels of DNA adducts in tumour and non-tumour tissues were found higher than in leucocytes. Biopsies from pharynx contained higher levels of DNA adducts than from oral cavities. Confounding effect of tobacco smoking and alcohol consumption on DNA adducts level was observed but it has not reached statistical significance. Formation of DNA adducts was not depended on such factors as patients age, sex, pulmonary tuberculosis, cancer progression (TNM), environmental pollution and mechanical irritation.

The aim of the article is a review of own cytogenic studies on laryngeal cancer confronted with the literature data. Spontaneous and bleomycin-induced chromosome instability was analysed in peripheral blood lymphocytes in relation to genetic risk of cancer incidence and progression. Comparative genome hybridization (CGH) was applied to demonstrate gains and losses of DNA copy number in tumour and non-tumour laryngeal mucosa. The profiles of imbalances of DNA copy number were shown to differ between metastazing and non-metastazing tumours. Preliminary data indicate a frequent loss of Y chromosome in tumour cells. The loss of heterozygosity at chromosome p53 locus (17p) has been shown to be more frequent than at chromosome locus coding 16 gene (9p). Altogether, the experiments have proven that a dynamics of chromosome aberrations is highest at the stage of metastasis.

Squamous cell carcinomas of the head and neck show frequent and complex chromosome aberrations, but little is known about the changes that occur during the metastatic process. To compare the accumulation of changes in primary and metastatic tumors we analyzed 19 pairs of primary larynx cancer tumors and their metastases. The most frequent changes were found at 3p, 3q, 5p, 9, and 13. Losses at 13, 8p, and 9q were more frequent in metastases than in primary tumors.

Chromosome instability is associated with an increased risk of malignancy. However, the quantitative analysis of chromosome breaks provided by the bleomycin test requires additional analysis aimed for the localisation of chromosome aberrations. For this reason, the metaphasis slides prepared for bleomycin test were stained with fluorochrome DAPI to estimate chromosome breaks in particular chromosomes. The additional staining of chromosomes can be recognised as an extension of the classical bleomycin test addressed for identification of structural aberrations. Preliminary results indicate that the most frequent chromosome breaks were found in chromosomes 1, 2, 3, 7 and 13.

The bleomycin test is a recognised method of evaluation of hidden genetical instability. The concept of the test consists in inducing chromosome aberrations in lymphocytes exposed in vitro to bleomycin and subsequent quantitative analysis of chromosome breaks. The study material was whole venous blood from 61 laryngeal cancer patients and from 30 healthy persons taken as a control. For each patient two parallel cultures where carried out in the standard procedure. Bleomycin was added to one of the cultures to induce chromosome breaks. Then, in microscopic metaphasal plates stained by Giemsa dye, the chromosome instability index estimated as a number of chromosome breaks per cell (b/c) and the percentage of cells with chromosome breaks were calculated. Higher indices of chromosome instability were demonstrated in laryngeal cancer patients in comparison to the controls. The persons with chromosome instability (b/c0.8) or with chromosome oversensitivity to mutagens (b/c1) were identified only among larynx cancer subjects. Furthermore, it was established that an increased chromosome instability is associated with high aggressiveness recognised by histological grading. The latter finding requires confirmation on an enlarged group of subjects.

An inter-individual variability in sensitivity to mutagen-induced genotoxicity was studied in respect to an estimation of larynx cancer risk. Bleomycin induced chromosome breaks were analysed in blood lymphocytes proliferating in vitro in a group of 35 larynx cancer subjects and in 18 healthy controls. A significantly higher index of chromosome breaks was found in larynx cancer subjects as compared with the controls. The distribution of individual results indicates that subjects oversensitive to bleomycine were identified only among laryngeal tumour patients. A potential usage of bleomycin test in tumour prognosis is discussed.

According to epidemiological data concerning laryngeal cancer there is almost ninefold higher morbidity in male population than in females. The aim of this study was to analyse on molecular level the genotoxic effects arising under the same exposure to tobacco smoke in males and females. In biopsies received from laryngectomy material we determined levels of aromatic and alkylated DNA adducts in tumours and in adjacent non-tumour tissue. It was established that levels of DNA adducts found in male DNA samples exceeded those in female DNA samples 1.5-3.8 times, which was recognised as a molecular evidence for epidemiological differences. Next, testosterone and sex hormones binding globulin (SHBG) were determined in male subjects blood serum. The levels of testosterone and SHBG very weakly correlated with DNA adduct levels even when subjects were separated into age groups. It is concluded that levels of testosterone and SHBG are not the proper markers of individual susceptibility to genotoxicity of tobacco smoke carcinogens.

Evaluation of biomarkers for genotoxic exposure are important for future use of such biomarkers in cancer prevention. We have studied a group of aluminum plant workers for a period of 2.5 years. The level of polycyclic aromatic hydrocarbons (PAHs) has been monitored at the work place (cathode relining). During the study period, urine and blood were sampled up to seven times from the same workers. Mean level of urinary 1-hydroxypyrene varied from 1.08 to 2.44 mumol/mol creatinine in the exposed groups compared with 0.36 and 0.20 mumol/mol creatinine in the two reference groups. For a group of 14 workers the intraindividual variation of urinary 1-hydroxypyrene was analyzed. The relative standard deviation of the values wasor = 0.50 for half of the workers; the relative standard deviation was calculated for individual values divided by mean of each time point. Polycyclic aromatic hydrocarbon-DNA adducts in white blood cells from exposed and nonexposed workers were measured by both immunoassay and 32P-postlabeling. By 32P-postlabeling mean values of 12.0 adducts/10(8) and 10.8 adducts/10(8) nucleotides were found in a PAH-exposed group and a reference group, respectively. Intraindividual variation of PAH-DNA adducts was also analyzed.

Genotoxical agents as PAH, nitrosamines, aromatic amines located in tobacco smoke are responsible for disorders of structure and function of DNA chromosomes, proteins and also initiation of carcinogenesis. DNA adducts are recognized as measure of the biological effective dose of exposure to environmental genotoxicant. So far most studies on DNA adducts after human exposure to tobacco smoke have ben carried out on white blood cells, lungs and oral cavities tissue. The aim of this work was estimation of relationship between state of neoplastic disease (TNM system) and number of aromatic DNA adducts. The subject were 37 patients with primary larynx tumor. In every case histopathological investigation revealed squamous cell carcinoma. There were 33 total and 4 partial laryngectomies performed. From tumour and nontumour larynx tissue DNA was isolated. Analysis of DNA adducts was performed by the 32P-postlabelling method. The results were characterized by individual differentiation. The highest level of DNA adducts was found in larynx tumour cells. In case of strong smokers (30-40 cigarettes per day) the level of aromatic adducts was higher then in non- or ex-smokers. In both tissues (tumour and non-tumour) the highest level of aromatic adducts was in T3 stage, the lowes in cause of T2 stage. In tissues with T4 stage the level of DNA adducts was intermediate. One has been observed decrease tendency in level of DNA adducts which is connected with increase of TNM stage.

Cel pracy Celem pracy jest przedstawienie stanu wiedzy na temat ciągu wydarzen na poziomie molekularnym i subkomorkowym ktore powodują, ze uszkodzenia struktury DNA i chromosomow prowadzą do zmiany genotypu, nieprawidlowej ekspresji genow, a w konsekwencji do zmiany fenotypu na nowotworowy. Z drugiej strony jak ten stan wiedzy jest wykorzystywany w klinice i czy pomaga poprawic wskaźniki przezycia dla tej grupy nowotworow. Material i metodyka Badaniami objeto grupe ponad 200 chorych nanowotwory glowy i szyi. Wykonano badania dotyczące uszkodzenia struktury DNA, struktury i funkcji określonych genow, zaklocenia cyklu zyciowego i struktury komorek oraz uszkodzenia chromosomow. Wyniki Wielokierunkowe badania biologii nowotworow krtani wykazaly akumulacje uszkodzen molekularnych i chromosomowych w przebiegu choroby. Kolejne uszkodzenia DNA prowadzą do wzrostu liczby mutacji. U chorych z nowotworami glowy i szyi w genomowym DNA nastepuje akumulacja 8–11 mutacji. Aberacje chromosomowe z przewagą delecji zmieniają funkcjonowanie genomu. Mnogośc uszkodzen materialu genetycznego prowadzi do niestabilności genetycznej, ktora ulatwia generowanie kolejnych uszkodzen. Oznacza to zmiany na poziomie komorkowym obejmujące kolejno transformacje nowotworową, a w dalszym etapie wzrost agresywności i powstawanie przerzutow regionalnych i odleglych. Wnioski Pomimo tak duzego postepu w naukach podstawowych nadal jest brak podstawowego markera monitorującego przebieg i określającego prognoze nowotworow glowy i szyi. Wydaje sie, ze najblizsza przyszlośc i oczekiwania to patologia molekularna.

Larynx cancer is strongly associated with tobacco smoking. The objective of this work was an analysis of aromatic DNA adducts in tumour and non-tumour larynx cells by means of the 32 P-postlabelling method. Peripheral blood leukocytes were used as a reference tissue. The presence of aromatic DNA adducts was demonstrated in all the studied tissues obtained after surgery of larynx tumours. The highest level of DNA adducts was found in larynx tumour cells, followed by non-tumour larynx cells, which exceeded that found in leukocytes almost 2.5 times. Large interindividual differences were detected between subjects. The adduct level in tumour/non-tumour correlated only moderately. However a high correlation was found between the level of DNA adducts in larynx (tumour and non-tumour) cells and that in leukocytes

Das erste Produkt der Zusammenwirkung von genotoxischen Faktoren mit der DNS sind sog. Addukte. Sie stellen das Mass der effektiven biologischen Dosis des genotoxischen Faktors dar. Dabei werden DNS-Addukte als vorklinisches Stadium des kanzerogenetischen Prozesses angesehen [2, 4].

There is known a strong association between induction of cancer of respiratory tracts and tobacco smoking. Exposure to polycyclic aromatic hydrocarbons (PAH) present in tobacco smoke can induce DNA lesions including bulky DNA adducts. The studied group consisted of 6 men, aged 46-71 years, all intensive cigarette smokers, without reported occupational exposition to PAH. Treatment did not include radio- and chemotherapy. DNA isolated from surgically removed tumors, surrounding tissue histologically characterized as non-malignant and peripheral blood white cells was analyzed by 32P-postlabelling assay with P1 nuclease enhancement. Both, P1 nuclease digestion and the chromatographic systems used for separation of DNA adducts were established for PAH: DNA analysis. The main adduct was identified as diol-epoxide metabolite of B (a) P. The mean number of adducts in tumor samples, surrounding tissue and white blood cells was calculated, respectively, to 82.3, 59.9 and 41.7 PAH: DNA adducts per 10(8) nucleotides. The only accessible reference was for DNA from white blood cells. The amount of PAH: DNA adducts in white blood cells of larynx cancer patients exceeded that found in healthy blood donors (both: smokers and non-smokers) as well as in workers exposed occupationally to high concentration of PAHs in the ambient air. Hence, it is concluded that carcinogen: DNA adducts as pre-mutagenic lesions can provide a diagnostic information. It concerns the target cells as well as a surrogate tissue. In any case the study has only a preliminary character because of limited number of subjects studied.

Repeated blood samples were obtained from volunteers who were occupationally exposed to polycyclic aromatic hydrocarbons in a Finnish iron foundry. Aromatic adducts were determined in white blood cell deoxyribonucleic acid of the subjects with the 32P-postlabeling technique after nuclease P1 treatment and butanol extraction, which showed no major difference. When repeated samples were analyzed, it appeared that the oldest ones displayed the lowest adduct levels, probably due to the instability of the adducts upon storage at -20 degrees C. The workers tended to maintain their adducts at a uniform level, a finding suggesting the contribution of host factors in the control of adduct levels.

Two isoaccepting t RNA Phe were isolated from yellow lupin seeds by DEAE-cellulose, BD-cellulose and reversed phase chromatography. The products obtained were characterized by aminoacylation and fluorescence. The chromatographic behaviour and some properties of the isolated t RNAs are discussed and compared with the known t RNA Phe from other sources.

Human tonsillar lymphocytes incorporate about 7-10 times more [3H]-deoxy-thymidine than [3H]-deoxycytidine at the same extracellular nucleoside concentration, and both incorporations could be inhibited by 10(-5) M arabinosyl-cytosine to 99%. On the other hand, the crude extract of these cells had about 10 times more deoxycytidine kinase than deoxythymidine kinase specific activity, as it was reported previously as well. Therefore, the differences in the labelling of these cells by [3H]-deoxycytidine and [3H]-thymidine cannot be simple due to the different levels of phosphorylating enzymes in the cells. The deoxycytidine kinase of human tonsillar lymphocytes was purified 38.6-fold by DEAE-Sephadex chromatography, and some kinetic and regulatory properties of the purified enzyme were investigated. Deoxycytidine kinase was eluted as a single peak from DEAE-Sephadex column and also from Sephadex G-100 column indicating a molecular weight of about 60 000; no isoenzymes could be detected. During the purification procedure an increase of the enzyme activity was observed suggesting the inhibition of the enzyme in the cells. An apparent Km of 13 microM for deoxycytidine and Ki of 55 microM for arabinosyl-cytosine were found for the tonsillar deoxycytidine kinase. The enzyme was strongly inhibited by dCTP, but not by the other deoxyribonucleoside triphosphates.

Lymphocytes during culture release a part of their DNA into medium, where its function remains unclear. Released DNA was removed by nuclease digestion. The effect of the removal of released SNA on cellular DNA synthesis was analysed. The culture of human lymphocytes treated with DNase for 6-48 hrs before harvest was characterized by significant lowering of 3H-T incorporation, dependent on time and DNase dose. Analogical experiment with RNase A did not change 3H-T incorporation as compared with standard culture. Lowering of incorporation is not connected with interaction of nucleases with mitogen, neither with cell death. Although not directly proved it is likely that released DNA is involved in regulation of lymphocyte proliferation.