Identification of matrix metalloproteinase-12 as a candidate molecule for prevention and treatment of cardiometabolic disease.

Obesity is strongly associated with metabolic syndrome, a combination of risk factors that predispose to the development of the cardiometabolic diseases: atherosclerotic cardiovascular disease and type 2 diabetes mellitus. Prevention of metabolic syndrome requires novel interventions to address this health challenge. The objective of this study was the identification of candidate molecules for the prevention and treatment of insulin resistance and atherosclerosis, conditions that underlie type 2 diabetes mellitus and cardiovascular disease, respectively. We used an unbiased bioinformatics approach to identify molecules that are upregulated in both conditions by combining murine and human data from a microarray experiment and meta-analyses. We obtained a pool of eight genes that were upregulated in all the databases analysed. This included well known and novel molecules involved in the pathophysiology of type 2 diabetes mellitus and cardiovascular disease. Notably, matrix metalloproteinase 12 (MMP12) was highly ranked in all analyses and was therefore chosen for further investigation. Analyses of visceral and subcutaneous white adipose tissue from obese compared to lean mice and humans convincingly confirmed the up-regulation of MMP12 in obesity at mRNA, protein and activity levels. In conclusion, using this unbiased approach an interesting pool of candidate molecules was identified, all of which have potential as targets in the treatment and prevention of cardiometabolic diseases.

Common dysregulated pathways in obese adipose tissue and atherosclerosis.

BACKGROUND: The metabolic syndrome is becoming increasingly prevalent in the general population that is at simultaneous risk for both type 2 diabetes and cardiovascular disease. The critical pathogenic mechanisms underlying these diseases are obesity-driven insulin resistance and atherosclerosis, respectively. To obtain a better understanding of molecular mechanisms involved in pathogenesis of the metabolic syndrome as a basis for future treatment strategies, studies considering both inherent risks, namely metabolic and cardiovascular, are needed. Hence, the aim of this study was to identify pathways commonly dysregulated in obese adipose tissue and atherosclerotic plaques. METHODS: We carried out a gene set enrichment analysis utilizing data from two microarray experiments with obese white adipose tissue and atherosclerotic aortae as well as respective controls using a combined insulin resistance-atherosclerosis mouse model. RESULTS: We identified 22 dysregulated pathways common to both tissues with p values below 0.05, and selected inflammatory response and oxidative phosphorylation pathways from the Hallmark gene set to conduct a deeper evaluation at the single gene level. This analysis provided evidence of a vast overlap in gene expression alterations in obese adipose tissue and atherosclerosis with Il7r, C3ar1, Tlr1, Rgs1 and Semad4d being the highest ranked genes for the inflammatory response pathway and Maob, Bckdha, Aldh6a1, Echs1 and Cox8a for the oxidative phosphorylation pathway. CONCLUSIONS: In conclusion, this study provides extensive evidence for common pathogenic pathways underlying obesity-driven insulin resistance and atherogenesis which could provide a basis for the development of novel strategies to simultaneously prevent type 2 diabetes and cardiovascular disease in patients with metabolic syndrome.

Fulvestrant induces resistance by modulating GPER and CDK6 expression: implication of methyltransferases, deacetylases and the hSWI/SNF chromatin remodelling complex.

BACKGROUND: Breast cancer is the leading cause of cancer death in women living in the western hemisphere. Despite major advances in first-line endocrine therapy of advanced oestrogen receptor (ER)-positive breast cancer, the frequent recurrence of resistant cancer cells represents a serious obstacle to successful treatment. Understanding the mechanisms leading to acquired resistance, therefore, could pave the way to the development of second-line therapeutics. To this end, we generated an ER-positive breast cancer cell line (MCF-7) with resistance to the therapeutic anti-oestrogen fulvestrant (FUL) and studied the molecular changes involved in resistance. METHODS: Naive MCF-7 cells were treated with increasing FUL concentrations and the gene expression profile of the resulting FUL-resistant strain (FR.MCF-7) was compared with that of naive cells using GeneChip arrays. After validation by real-time PCR and/or western blotting, selected resistance-associated genes were functionally studied by siRNA-mediated silencing or pharmacological inhibition. Furthermore, general mechanisms causing aberrant gene expression were investigated. RESULTS: Fulvestrant resistance was associated with repression of GPER and the overexpression of CDK6, whereas ERBB2, ABCG2, ER and ER-related genes (GREB1, RERG) or genes expressed in resistant breast cancer (BCAR1, BCAR3) did not contribute to resistance. Aberrant GPER and CDK6 expression was most likely caused by modification of DNA methylation and histone acetylation, respectively. Therefore, part of the resistance mechanism was loss of RB1 control. The hSWI/SNF (human SWItch/Sucrose NonFermentable) chromatin remodelling complex, which is tightly linked to nucleosome acetylation and repositioning, was also affected, because as a stress response to FUL treatment-naive cells altered the expression of five subunits within a few hours (BRG1, BAF250A, BAF170, BAF155, BAF47). The aberrant constitutive expression of BAF250A, BAF170 and BAF155 and a deviant stress response of BRG1, BAF170 and BAF47 in FR.MCF-7 cells to FUL treatment accompanied acquired FUL resistance. The regular and aberrant expression profiles of BAF155 correlated directly with that of CDK6 in naive and in FR.MCF-7 cells corroborating the finding that CDK6 overexpression was due to nucleosome alterations. CONCLUSION: The study revealed that FUL resistance is associated with the dysregulation of GPER and CDK6. A mechanism leading to aberrant gene expression was most likely unscheduled chromatin remodelling by hSWI/SNF. Hence, three targets should be conceptually addressed in a second-line adjuvant therapy: the catalytic centre of SWI/SNF (BRG1) to delay the development of FUL resistance, GPER to increase sensitivity to FUL and the reconstitution of the RB1 pathway to overcome resistance.

Oncostatin M is a FIP1L1/PDGFRA-dependent mediator of cytokine production in chronic eosinophilic leukemia.

BACKGROUND: Chronic eosinophilic leukemia (CEL) is a myeloproliferative neoplasm characterized by expansion of neoplastic eosinophils, tissue infiltration, and organ damage. In a subset of these patients, the FIP1L1/PDGFRA (F/P) oncoprotein is detectable. F/P exhibits constitutive tyrosine kinase activity and activates a number of signaling pathways. So far, however, little is known about the role of F/P-dependent proteins in the pathogenesis of CEL. METHODS: A screen for F/P-dependent cytokines was performed in growth factor-dependent human cell lines lentivirally transduced with F/P. Signal transduction pathways were characterized in Ba/F3 cells with doxycycline-inducible expression of F/P and in EOL-1 cells. Cytokine expression was confirmed in patients' material by immunohistochemistry, immunofluorescence, and confocal microscopy. Gene expression analysis, proliferation assays, and chemotaxis assays were used to elucidate paracrine interactions between neoplastic eosinophils and stromal cells. RESULTS: We show that F/P upregulates expression of oncostatin M (OSM) in various cell line models in a STAT5-dependent manner. Correspondingly, neoplastic eosinophils in the bone marrow were found to overexpress OSM. OSM derived from F/P + cells stimulated proliferation of stromal cells. Moreover, OSM-containing supernatants from F/P + cells were found to upregulate production of stromal cell-derived factor-1 (SDF-1)/CXCL12 in human fibroblasts. SDF-1, in turn, induced migration of EOL-1 cells in a dose-dependent manner. CONCLUSIONS: We have identified a F/P-driven paracrine interaction between neoplastic eosinophils and stromal cells that may contribute to tissue fibrosis and accumulation of neoplastic eosinophils in CEL.

Heme oxygenase-1 end-products carbon monoxide and biliverdin ameliorate murine collagen induced arthritis.

OBJECTIVES: Heme oxygenase-1 (HO-1) which degrades Heme to free iron, biliverdin and carbon monoxide (CO) plays an important role in inflammation. There are, however, conflicting data concerning the role of HO-1 in rheumatoid arthritis (RA) and the therapeutic potential of individual heme degradation products remains to be determined. We therefore investigated the effect of CO and biliverdin upon therapeutic administration in the murine collagen induced arthritis (CIA) model of RA. METHODS: CIA was induced in DBA/1 mice. Anti-CII antibody levels were determined by ELISA. Mice were scored for paw swelling and grip strength. After the first clinical signs of arthritis one group of animals was treated with biliverdin, the second group was treated with CO. After 60 days all animals were sacrificed and analysed for histomorphological signs of arthritis. RESULTS: All animals immunised with CII developed serum anti-CII antibodies. Antibody levels were decreased in the CO-treated group. Both, Biliverdin and the CO-treated animals, showed an improvement in clinical disease activity. Histological analysis revealed significantly less inflammation, erosion and reduced numbers of osteoclasts in CO-treated animals only, whereas cartilage degradation was prevented in both biliverdin and CO-treated animals. CONCLUSIONS: Our data demonstrate a beneficial effect of CO, in particular, and biliverdin, on inflammation and bone destruction in the CIA mouse model.

Gene expression signature of chronic lymphocytic leukaemia with Trisomy 12.

BACKGROUND: The prognosis of chronic lymphocytic leukaemia (CLL) patients is largely determined by the karyotype of the malignant clone. We have investigated the gene expression profile associated with trisomy 12 (+12). DESIGN: Initially, unselected peripheral blood mononuclear cells of four patients with +12 were compared with 16 CLL controls using microarray analysis. RESULTS: were validated by quantitative real-time PCR with RNA from 61 patients (29 with +12, 32 CLL controls). Results Seven genes showing the strongest correlation with +12 in microarray analysis were selected for real-time PCR: HIP1R, MYF6, SLC2A6, CD9 (overexpressed); CD200, P2RY14, RASGRP3 (underexpressed). Four genes were significantly associated with +12: HIP1R (P<0.0001), MYF6 (P=0.007), P2RY14 (P=0.014), CD200 (P=0.028). Receiver Operating Characteristic curve analysis revealed that HIP1R expression was a highly sensitive and specific marker for +12 in CLL patients. MYF6 was exclusively expressed in normal or malignant B cells in peripheral blood but was poorly predictive for +12. As expected, a number of overexpressed genes are located on chromosome 12 (HIP1R, MYF6). Interestingly, both significantly underexpressed genes (P2RY14, CD200) reside on the long arm of chromosome 3 pointing to trans-repression in this region. CONCLUSIONS: Analysis of the molecular signature of trisomy 12 in CLL resulted in: (i) identification of a surrogate marker for PCR (HIP1R); (ii) observation of a gene dosage effect; and (iii) detection of specific underexpression of genes located on chromosome 3. These results should help to improve diagnosis and treatment decisions for patients with CLL and trisomy 12.

Reduced TGF-beta1 expression and its target genes in human insulinomas.

Aiming to identify signalling pathways relevant for ss-cell growth we performed an explorative micro-array analysis comparing the gene expression profiles of three human insulinomas and one normal pancreatic islet preparation. This revealed an insulinoma-associated down-regulation of the transforming growth factor beta 1 (TGF-beta1) and its target genes. Comparative quantitative real-time PCR (qRT-PCR) including an expanded sample number of both insulinomas (n=9) and pancreatic islet preparations (n=4) confirmed the decreased TGF-beta1 expression and its target molecules (TGFBI, NNMT, RPN2) in insulinomas. Similarly, TGF-beta1 immunofluorescence analysis revealed reduced expression in insulinomas when compared to pancreatic islets. In contrast, TGFBR2 (transforming growth factor beta receptor II) was found up-regulated. However, the consistent down-regulation of the TGF-beta1 targets TGFBI (transforming growth factor, beta-induced), NNMT (nicotinamide N-methyltransferase), RPN2 (ribophorin II) indicates that the parallel up-regulation of TGFBR2 does not compensate for the only marginal TGF-beta1 expression levels in insulinomas. TGFBR2 expression was confirmed at the protein level in insulinomas. SMAD2/3 protein expression was found at higher levels in human pancreatic islets when compared with insulinomas by dual colour confocal microscopy. TGF-beta1 signalling is known to be involved in cell replication and is abrogated in ductal pancreatic tumours. The down-regulation of TGF-beta1 expression and its target molecules in insulinomas is a new aspect of this cytokine. Our data underline parallels in endocrine and exocrine pancreatic tumour development, which may implicate common progenitor cells.

Deregulated expression of fat and muscle genes in B-cell chronic lymphocytic leukemia with high lipoprotein lipase expression.

Lipoprotein lipase (LPL) is a prognostic marker in B-cell chronic lymphocytic leukemia (B-CLL) related to immunoglobulin V(H) gene (IgV(H))mutational status. We determined gene expression profiles using Affymetrix U133A GeneChips in two groups of B-CLLs selected for either high ('LPL+', n=10) or low ('LPL-', n=10) LPL mRNA expression. Selected genes were verified by real-time PCR in an extended patient cohort (n=42). A total of 111 genes discriminated LPL+ from LPL- B-CLLs. Of these, the top three genes associated with time to first treatment were Septin10, DMD and Gravin (P

Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.

Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.

Presence of alcohol in suicide victims.

A number of studies have established a strong connection between acute inebriation, alcohol addiction and suicides, as the last act of alcoholism or an act of desperation in an alcoholic's family, an act of escape from restraints in state of depression or as a way of self-destruction. In recent years in average 600 people per year committed suicide. Slovenia is a country with extremely high and variable suicide tendencies and harmful alcohol use levels, as well as a high level of alcohol-related troubles. The aim of our research was to ascertain some typical features, especially those connected to the inebriation of suicide victims from a wider Ljubljana region. Autopsies were carried out on the victims in the period between 1995 and 1999. There were 508 (31.2%) suicides among all the analyzed violent deaths; 73.2% of them were men. The average age of the victims was 46.5 years. Most suicides were committed at home (50.0%). 25.4% were completely sober in the moment of the act, while in all other cases inebriation was established, the average value being 9.57 g/kg. Men were drunk in 87.1% of cases, women only in 12.9% and the given alcohol levels were substantially higher with men (0.65:0.26 g/kg). The share of inebriated persons decreases with age-reaching its peak in the 35-54 age group. Regarding the method, the predominant ones are intoxication and the use of firearms, which is a typical way of committing suicide among men, while women rather choose jumping from great heights and drowning. Alcohol was present in as many as 55.7% of suicides with intoxication and in 68.8% of all suicides committed by using firearms, while the highest alcohol levels were found in those who died from cutting their veins (2.01 g/kg). Based on this and on other research, more effort should be focused on alcohol abuse prevention, making all people aware of the consequences of alcohol abuse, the possibilities of treatment and their availability as well as possible co-morbid depressions. Simultaneously, due to an established link, the national alcohol policy and strategy for prevention of suicides should be professionally harmonized.

Harmful alcohol use of those who died a violent death (the extended region of Ljubljana 1995-1999).

Consumption of alcohol increases the risk of dying a violent death. We wanted to establish a connection between harmful alcohol use and dying a violent death. We analyzed all such victims in the extended region of Ljubljana. The research included 1630 deceased, who were autopsied at the Forensic Institute of the Ljubljana, Faculty of Medicine in the period from 1995 to 1999. Presence of alcohol was established in 76.3% of the cases. From all included in the research, 38.2% of all work accident victims, 28.8% of all murder victims, 25.4% of suicides, 24.6% of victims involved in traffic accidents and 19.3% of those who died in accidents at home. 23.2% of all violent death victims had a concentration of alcohol above 1.5 g/kg; among those, victims of traffic accidents, suicides and accidents at home represent the largest part. The lowest values of alcohol in blood were found in those who died because of accidents at work. The highest values were found in males aged 35-44. The research confirmed that consumption of alcohol in Slovenia was strongly connected to violent deaths. The blood levels of alcohol of the victims are distinctively higher where there are practically no limitations of alcohol consumption and lower in the environment or activities where legal restrictions prohibit or at least explicitly limit harmful use of alcohol (working environment).

Defining signal thresholds in DNA microarrays: exemplary application for invasive cancer.

BACKGROUND: Genome-wide or application-targeted microarrays containing a subset of genes of interest have become widely used as a research tool with the prospect of diagnostic application. Intrinsic variability of microarray measurements poses a major problem in defining signal thresholds for absent/present or differentially expressed genes. Most strategies have used fold-change threshold values, but variability at low signal intensities may invalidate this approach and it does not provide information about false-positives and false negatives. RESULTS: We introduce a method to filter false-positives and false-negatives from DNA microarray experiments. This is achieved by evaluating a set of positive and negative controls by receiver operating characteristic (ROC) analysis. As an advantage of this approach, users may define thresholds on the basis of sensitivity and specificity considerations. The area under the ROC curve allows quality control of microarray hybridizations. This method has been applied to custom made microarrays developed for the analysis of invasive melanoma derived tumor cells. It demonstrated that ROC analysis yields a threshold with reduced missclassified genes in microarray experiments. CONCLUSIONS: Provided that a set of appropriate positive and negative controls is included on the microarray, ROC analysis obviates the inherent problem of arbitrarily selecting threshold levels in microarray experiments. The proposed method is applicable to both custom made and commercially available DNA microarrays and will help to improve the reliability of predictions from DNA microarray experiments.

DNA microarrays: a novel approach to investigate genomics in trophoblast invasion--a review.

The events that regulate trophoblast invasion need to be characterized at the transcriptional level. Several types of gene products may be involved in various stages oftrophoblast infiltration, including integrins, matrix metalloproteases (MMPs) and extracellular matrix (ECM) proteins. Autocrine or paracrine regulators of cytotrophoblast proliferation or differentiation in vitro (e.g. growth factors and cytokines, as well as oxygen tension) could be characterized mechanistically at the transcriptional level. Large-scale gene expression profiling of trophoblasts of distinct invasive stages could be carried out on fixed tissue obtained by laser-directed microdissection. This information may shed light on physiological implantation and placentation, as well as on the interpretation of pathological processes such as pre-eclampsia. The applications of DNA microarrays are ideal for studies of genomic structure (e.g. mutation and polymorphism analyses) and monitoring of gene expression. The ultimate goal is to understand the critical events underlying growth, development, homeostasis, 'behaviour and the onset of disease at a genomic level. Microarrays detect gene expression levels in parallel by measuring the hybridization of labelled, single-stranded DNA to many thousands of partial or whole gene sequences immobilized on a glass surface (the 'chip'). Microarrays are available both commercially and can be manufactured in house.

Genotoxic effects of radiotherapy and chemotherapy on circulating lymphocytes in patients with Hodgkin's disease.

PURPOSE: The aim of this study was to find out the structural chromosomal changes in somatic cells after chemotherapy (CT) with or without radiotherapy (RT). METHODS AND MATERIALS: This prospective study included 30 Hodgkin's disease (HD) patients. The patients of Group I(1) had only MOPP/ABV CT. The patients of Group II(2) also had irradiation. Group III(3) (control group) consisted of healthy subjects without any reported malignant disease. Mutagenetic testing was performed at the time of diagnosis and was repeated immediately after treatment and again 6 months later. The following tests were applied: structural chromosomal aberrations (CA), sister chromatid exchange (SCE) and micronucleus (MN) tests. RESULTS: Prior to treatment, the chromosome damage in our patients was not higher than that in the control group. Immediately after the complete treatment, we observed a strong inhibition of the mitotic activity of lymphocytes as well as a significant increase in the frequency of CA, MN and SCE in the Groups I and II. In patients treated by RT, we found statistically significant differences between the Groups I and II in MN (P<0.005) and CA frequencies (P<0.005), and an increased number of dicentrics (P=0.021). Six months after the complete treatment, the mitotic activity was found to be nearly normal, but chromosome damage occurred. CA and SCE values did not differ much from the values measured immediately after treatment, whereas MN values decreased without returning to the baseline levels. The chromosome damage persisted even 6 months after combined RT and CT. The damage in the genome of individual cells was in some cases even greater than immediately after treatment. The possible risk of neoplastic transformation posed by these heavily damaged cells, if viable, due to the changes in the expression of oncogenes or tumour suppresser genes, is discussed.

Location and incidence of chromosome and chromatid breaks in patients with Hodgkin's disease or testicular tumors.

In 90 patients aged 17 to 35 who suffered from Hodgkin's disease (HD) or had testicular tumors (TT), the location of chromosome and chromatid breaks on individual chromosome segments was reviewed using an adapted Funes-Cravioto scheme, in addition to examining the percentage of structural chromosomal aberrations. On the basis of an analysis of 1121 breaks in patients with HD or TT, the results were presented graphically as multiples of the expected number of breaks for the normal population. Before the beginning of treatment, the number of structural chromosomal aberrations (SCA) in patients with TT or HD was equal to that in a control group of subjects with malignant diseases. This, however, does not apply to the location of chromosome and chromatid breaks. In patients with HD, the dominant unstable sites are located on group A2 chromosomes, segments 2 and 5, and on group B chromosomes, segment 5. In patients with TT, the number of chromosome and chromatid breaks is also increased on group A2 chromosomes, segments 2 and 5, and in addition, also on group B chromosomes, segment 4.

Incidence of chromosomal aberrations and micronuclei in cave tour guides.

An analysis of structural chromosomal aberrations (SCA) and micronucleus tests (MN) were performed in 38 subjects, cave tour guides and in appropriate control group. The dominant type of chromosomal aberrations in tourist guides were chromosomal breaks (0.013 per cell) and acentric fragments (0.011 per cell). In the control group, these aberrations were present up to 0.008 on cells. Considering the analysed cells of the guides in total (33,556), the incidence of dicentric and rings range is below 0.0008 on cells, even though three dicentric and ring chromosoms were found already in the first 1000 in vitro metaphases of some guides. Only 0.0003 dicentrics and neither other translocations were found in control group (ambiental exposure). The incidence of micronuclei in cytokinesis blocked lymphocytes ranged from 12-32 per 500 CB cells in the cave tour guides and from 4-11 per 500 CB cells in control group. Measurements of radon and its daughters were performed at different locations in the cave. Annual doses from 40-60 mSv were estimated per 2000 work hours for cave guides. The changes found in the genome of somatic cells may be related to the exposure doses of radon and its daughters, although smoking should not be ignored.

Chromosome aberrations study of pupils in high radon level elementary school.

The ICRP Publication 65 recommends 200-600 Bq x m(-3) as the indoor radon action level for the general public. In Slovenia, a value of 400 Bq x m(-3) has been proposed but not yet approved. In a nation-wide radon project financed by the Health Inspectorate of Slovenia, it was discovered that the elementary school named "S3" belongs to a group of schools with elevated winter indoor radon concentrations up to 7,000 Bq x m(-3). Opening windows and doors during classes substantially decreased radon concentrations, but very seldom below 1,000 Bq x m(-3). Yearly effective doses for pupils, estimated according to ICRP 65, ranged from 7 to 11 mSv. Because the pupils have been subjected to the elevated radon concentrations, special preventive health checks have been performed. The examination protocol included mutagenetic tests, one for structural chromosomal aberrations and the other a micronucleus test. Altogether 85 pupils (37 girls and 48 boys) from the first four grades between the ages of 9 and 12 y were examined. An increase in cytogenetic damage was found for these pupils, compared to the control group, composed of pupils of the same age from another area with indoor radon concentrations in their school of below 400 Bq x m(-3). The incidence of structural chromosomal aberrations reached 2.0% (0.5-4) and micronucleus test was 6.52 per 500 cells with a maximum of 15 in some cases. In the control group structural chromosomal aberrations varied from 0.5 to 2.5%, while the maximum incidence of micronucleus was 9 micronucleus per 500 CB cells. The results obtained are preliminary and suggest a need to expand the study. A long-term radon survey, at least over a year, of the homes and wider residential environment of the pupils would be necessary to assess the correlation between radon exposure and both structural chromosomal aberrations and micronucleus findings.

The influence of gender on traffic accidents--the case of the Republic of Slovenia.

Modern road traffic (RT) requires great efforts from all drivers and the highest use of all their physical and mental capacities. In addition, drivers must possess a certain character to ensure safe driving. Only a few decades ago, the driving of motor vehicles was a rarity and women behind the steering wheel were even scarcer. With the development of motorisation, their share of RT has been on a constant increase, as has, logically, their share of road traffic accidents (RTA). This research presents the increasingly important role of women in RT and attempts to show the special attributes of female drivers, namely that above all, they cause fewer accidents, and notably, those they cause are less likely to be fatal.

Road traffic accidents caused by elderly drivers.

Numerous factors have contributed to an increase in the number of elderly people in developed countries. This has caused an increase in the share of elderly drivers, drivers with all diseases and troubles problems brought about by old age. In 1993, less than 12% of the population of Slovenia was older than 65 years and owned slightly over 6% of personal automobiles, or were holders of slightly more than 5% of driver's licences. On average, these drivers caused about 6% of road traffic accidents (RTAs). Among fatalities who caused RTAs, about 19% were older than 65 years. Overall, the share of fatalities over 65 years of age was about 26% of all fatalities in RTAs; among pedestrians, their share was almost 42%. For the elderly, driving is an important means of maintaining freedom and independence. Their quality of life is certainly lower without their own transport. It is therefore necessary to protect the right of individuals to drive motor vehicles, but only if they possess the necessary capabilities.

Alcoholic drinkers and road safety in the Republic of Slovenia.

In this study we were therefore interested in the percentage of road traffic offenses (RTO) and road traffic accidents (RTA) involving inebriated drivers one year before and one year after the passing of the new Law on Road Traffic Safety (LRTS) as well as measures (referrals, punishments and final decisions on the revoking of driver's licenses due to drunk driving). One year before the passing of the new LRTS, there were 40,702 RTA-s in the Republic of Slovenia (12.2% caused by drunk drivers). The average alcohol concentration in exhaled air for those analysed was 1.19 g/kg. One year after the passing of this law there were 36,479 RTA-s (8.6% caused by drunk drivers). The average alcohol concentration in exhaled air for those analysed was 1.32 g/kg (the differences were statistically significant). In 13.8% cases the reason for performing a measurement of the alcohol concentration in exhaled aier was an RTA with an average alcohol concentration in exhaled air of 1.22 g/kg and in 86.2% of cases an RTO with an average alcohol concentration in exhaled air of 1.25 g/kg (the differences were statistically significant). We found it interesting that the number of events minvolving lower concentrations decreased, but the percentage involving higher alcohol concentrations even increased. The results of this study indicate without a doubt that the law was not successful enough with its repressive and preventative measures in the field of drunk drivers. Experts on alcohol believe that punishment cannot make alcoholics and other drivers abandon their behavioural patterns and stop driving under the influence of alcohol. This can be achieved only by treatment, and the present practice (police--misdenveanour counts--repeat general medical check-up) has been ineffective as prevention among alcoholic drivers. We therefore believe that supplements to the LRTS should be adopted urgently, that would contribute, through better medical selection, to a reduction in the number of drunk drivers behind the wheel, both those who are alcohol dependent (and should be referred to treatment).