Much interest exists in the studies that would help to understand whether there is a causal association between cancer and various types of molecular or cytogenetic damage detected in human cells. It is unlikely that a single biological marker for any particular genotoxic effect will be found, because of the dynamics and adaptability of biological systems, and the multiplicity of the reactions and mechanisms involved in genotoxic end-points affecting either structure or function. This is of particular interest in relation to whether any of the damage detected could be used as early biomarkers of diseases. Among the several critical markers appropriate for biological monitoring is the inherent susceptibility associated with genetic traits. One major process of oncogenesis is activation of proto-oncogenes by point mutations or chromosomal translocation. There is substantial evidence that indicates that loss of heterozygosity of certain chromosomes is involved in human carcinogenesis. Such an event can be caused by loss of a whole chromosome, partial deletion, or somatic recombination. In general, biological monitoring is aimed at identifying one or several critical changes in the host resulting in an association with a specific pathology. Autosomal dominant polycystic kidney disease (ADPKD) is one of the most frequent genetically related diseases of the anatomy of kidneys. The main feature of this disease is the development of a thin-walled cystic formation that leads to dysfunction and progressive degeneration of the kidneys. In 90% of autosomally dominant inherited disease, the presence of 16PKD gene in the short arm of chromosome 16 (polycystic kidney disease 1) has been described. ADPKD is most often established between the ages of 30 and 50 years in 1% of the population in general. There are no reports explaining whether ADPKD patients are vulnerable to any other diseases.

Materials and Methods

Three members of a family with ADPKD were randomly chosen for studies; the young girl at the age of 19, her brother of 9 years of age, and their maternal aunt at the age of 41 years. Results of the analysis performed on the blood samples of the ADPKD patients were compared with average values measured in the control group from the epidemiological survey. The control group contained 24 persons, healthy donors who were chosen from two areas, an industrial and a countryside region of southern Poland (the same region from which patients in the study originated). The industrial area controls were administrative staff of the petroleum plant close to a highly polluted region, and the countryside regions were two neighboring villages with a low level of pollution and a low level of total cancer cases. The average age in the control group was 40 years. Among the control group, 36% were smokers and 64% were nonsmokers, and 44% were females. Individuals in the healthy control group were of similar socio-economic status and were represented by farmers and forest workers, skilled state workers, teachers or clerks with a liberal or technical education and low medium income. Interviews were performed with a questionnaire considering health, lifestyles and habits, job conditions, and possibilities of hazardous exposure leading to increased genotoxic risk. The questionnaire was in good agreement with recommendation for a biological monitoring type of study. The whole blood samples for cytogenetic procedures were collected by venipuncture and divided into two parts. The first part was collected in heparinized tubes and was transported to the DREB Laboratory at the Institute of Nuclear Physics, where appropriate lymphocyte isolation for DNA analysis and culturing of samples were processed as soon as possible. From the second part, blood plasma was extracted, frozen at ?70°C, and transported in dry ice to the laboratory of the BIBRA Institute (Carshalton, UK) for testing p21ras protein levels. Results from all of the analyses performed were compared to the average levels observed in the control group.

Blood culturing and cytogenetic screening

The samples of heparinized whole blood were incubated at 37°C using Eagle’s medium supplemented with 20% of fetal calf serum and antibiotics. Lymphocytes were stimulated with LF-7, a Polish substitute of PHA and cultured with an addition of a proper amount of BrdU, in the case of chromosomal aberrations for 48 h, and in the case of SCE cultures, for 72 h. Two hours before the end of culturing, colcemid was added (0.01 ml/ml) to each sample. Part of cultured samples was taken for special staining (banding technique) to analyze karyotypes. In the rest of the cultured samples, fixation and staining were performed by standard cytological procedures for either CA or SCE analysis. Chromosome and chromatid type aberrations were scored in the cells in the first mitosis and expressed as total aberration frequency including gaps (TAbF), and excluding gaps (AbF). Sister chromatid exchanges (SCE) were screened in the second mitosis, and breaks per each cell containing at least 44 chromosomes were counted. Percent of cells displaying a higher number of exchanges per cell than the value of SCE characteristic for 95% at cells in the reference group (HFC high frequency cells) was evaluated on the basis of SCE distribution in the cells. Proliferative rate index was estimated from the ratio of cells entering various mitosis according to the following formula:

Results

The main characteristics of donors and number of lymphocytes analyzed in comparison with the control group.

The analysis of p21ras protein levels with an application of the marker from Oncology Science, with the 10 ng sensitivity level, was performed in the Laboratory of BIBRA Institute (Carshalton, UK), and did not show positive results (Dr. Diana Anderson, personal communication 1997). Neither analysis of karyotype revealed any significantly visible difference between each other or from the normal karyotype.

The number of metaphases accepted for analysis in the first mitosis (M₁) and the number of metaphases accepted for analysis in the second mitosis (M₂) Results of the cytogenetic analysis in each acceptable good spread metaphase and mean values for various biomarkers levels and DNA analysis..

The analysis performed in the first mitosis for presence of chromosomal damage revealed, in all samples, a statistically significant increase in the level of aberration frequencies including gaps (TAbF). Aberration frequencies excluding gaps (AbF) were significantly higher in blood samples of probands with codes 1 and 2 in comparison to the control group results. Percents of aberrant cells (AbC) in all samples were also visibly higher than an average level observed in the control group.presents the distribution of SCE detected in the blood of lymphocytes under study. Sister chromatid exchanges (SCE) and percent of cells with elevated number of exchanges were higher in the lymphocytes of donors with code 1 and 3 when compared to the mean values observed in the control group in environmental survey. Differences in SCE values of samples with codes 1:2, and 2:3 are significant with values for p <0.0000 and p = 0.0043, respectively. The distribution of cells with sister chromatid exchanges presented in shows in cases of codes 1 and 3 the more frequent presence of cells outside the distributions. That is in good agreement with the significantly higher level of HFC detected in the blood of those donors.

DNA damage induced by in vitro irradiation with a 0.5 Gy dose of gamma radiation. show the distribution of cells with various levels of DNA damage expressed as Tail Moment and DNA tail, respectively. There were no statistically significant differences detected in sensitivities to radiation of lymphocytes, all of them were in the range of average DNA Tail Moment (TM) for control sample ± 2SD (0.49–3.69). However, higher responses were observed in samples with codes 1 and 2 and lower response in the lymphocytes from the sample with the code 3. The lymphocytes of proband with the code 3 also showed the lowest level of chromosomal aberration induced in vivo), and this might explain a lower susceptibility to this environmental agent (i.e., ionizing radiation).