Satellite Meeting to 29th Annual Meeting of EEMS

and

16th Annual Meeting of NordEMS

 

 

ENVIRONMENTAL GENOTOXICOLOGY:

Field and Human Studies in the Nordic/Baltic Region

30 June - 3 July 1999, Vilnius, Lithuania

 

 

ABSTRACTS

 

USE AND MISUSE OF HUMAN GENETICS

Anton Brřgger

Department of Genetics, Institute for Cancer Research, The Norwegian Radium Hospital, N- 0310 Oslo, Norway

Genetics as a science started in 1865 with Gregor Mendel’s identification of hereditary factors. In the same year Francis Galton published two papers on "Hereditary Talent and Character", in which he stressed the importance of heredity for intellectual development and laid the foundations for the use of statistical methods in the analysis of the inheritance of quantitative characters. Galton claimed that natural selection, where the unfit loose in the struggle for life, no longer functioned because medical and social welfare helped the unfit to survive and reproduce. He was concerned that the British population might degenerate and called for social measures to counteract the decline: eugenics (from Greek meaning "well born"). Positive eugenics implied to encourage the gifted, most fit and healthy individuals to have many children. Negative eugenics meant to inhibit reproduction among the unfit, by means of sexual segregation and sterilisation. Eugenics was assumed to improve the genetic constitution of the population.

Eugenics considerations became increasingly popular in Western societies in the beginning of this century. Several countries passed on sterilisation laws aimed at inhibiting reproduction among the persons considered to be unfit. In 1931 more that 30 states in the U.S.A. had such laws. Denmark got their law in 1929, Germany in 1933, Sweden and Norway in 1934, Finland in 1935, and Iceland in 1938. In some of the Nordic countries these laws were not only applied to mentally retarded, but were used against the reproduction among gypsies and "tatere" in a way resembling genocide. Still worse became the development in Germany when Nazis came to power: from sterilisation to euthanasia, from newborn to small children to adults, from the mentally retarded to gypsies, homosexual and epileptic persons ending with Jews and others in the Holocaust. Although sterilisations were still carried out in the Nordic countries until early seventies, the eugenic arguments were fading leaving lack of responsibility and ability to care for children as the most important argument in favor of sterilisation.

In the meantime important advances had been made in medical genetics, and a new concept of application of genetic knowledge was defined: genetic counseling. The measures were now aimed at the single individual rather than the population. Genetic counseling deals with the occurrence or risk of occurrence of a specific genetic disorder in a family. The genetic counselor shall help the family to choose the course of action, which seems appropriate to them in view of their risk. This may be to refrain from having children. In an increasingly number of cases it is possible to obtain a prenatal diagnosis, and the parents may decide to abort the fetus in the case of a severe genetic disorder. In cancer care the identification of genetically disposed high-risk persons may lead to early diagnosis and a more successful treatment. By means of gene technology an increasing number of conditions may be diagnosed. Current research is also aimed at gene therapy, and the future will show to which extent this may cure genetic disease.

 

The Sources, Fate, and Hazards of Mutagens in Surface Waters

Paul A. White

NRC Research Associate, Atlantic Ecology Division, US Environmental Protection Agency, 27 Tarzwell Drive, Narragansett, Rhode Island 02882, USA

A variety of industrial, domestic and agricultural wastes are discharged into rivers, lakes, streams, and estuaries. In some instances, these wastes contain substances that are capable of permanently altering the genetic material of exposed organisms (i.e., they are mutagens). Although the presence of mutagens in aquatic systems has been known for some time, their precise origins, physical-chemical properties, environmental fate, and effects on indigenous biota are poorly understood. Despite the noteworthy potency of mutagens in some industrial wastewaters (e.g., organic chemical production facilities), the greatest potential hazard is posed by facilities having high volumetric discharge rates (e.g., metal foundries, pulp & paper mills, domestic waste treatment plants). A regional analysis of sources in the St. Lawrence River at Montreal (Canada) showed most (~85%) of the surface water mutagenicity is actually non-industrial in origin. More recent analyses conducted in the Providence River system (Rhode Island, USA) confirmed the genotoxic hazards of municipal wastewaters and indicated that the putative mutagens are aromatic amines. Investigations of post-emission behaviour in the St. Lawrence system indicated that direct acting mutagens are rapidly degraded, while promutagenic substances persist in sedimented particulate matter. Investigations of indigenous biota indicated that aquatic organisms can accumulate genotoxic substances, and achieve tissue concentrations 100 to 1000 times higher than water concentrations. Although several organisms were found to accumulate genotoxic substances (e.g., fish, mollusks, crustaceans), tissue concentrations diminished with increasing trophic level. Although effects in St. Lawrence biota were not directly investigated, the Comet assay is currently being employed to examine genotoxic effects in the Providence River system.

 

SAMPLING TECHNIQUES FOR BIOAVAILABLE HYDROPHOBIC POLLUTANTS IN THE ENVIRONMENT: TRENDS AND DEVELOPMENT

Anders Södergren

Lund University, Department of Ecology, Ecology Building, S- 223 62 Lund, Sweden

To predict the distribution and effects of organic pollutants in the environment, long-term monitoring of both abiotic and biotic part of the ecosystem is required. Pollutants with properties such as persistency and lipophilicity tend to end up in organisms and, therefore, the accumulation of these residues will indicate the magnitude of exposure. However, before the pollutants are taken up by organisms, the abiotic part of the ecosystem (water, sediment, soil, air, etc.) may be sampled in order to predict exposure, estimate risks and also to manage the problem. In order to concentrate certain persistent pollutants already during sampling in the field, their lipophilic properties may be utilized. The principles behind, the use of and development of passive samplers to monitor pollutants in the air, in air-borne fallout, in aquatic surface microlayers and in the water will be discussed and their significance as substitutes in biomonitoring will be evaluated.

 

TOXICITY AND GENOTOXICITY OF AQUATIC HYDROPHOBIC POLLUTANTS SAMPLED WITH SEMIPERMEABLE MEMBRANE DEVICES

Darius Sabaliunas1,2, Juozas R. Lazutka2, Inesa Sabaliuniene2

1Department of Ecology, Lund University, S 223 62 Lund, Sweden, 2Faculty of Nature Sciences, Vilnius University, Ciurlionio 21, 2009 Vilnius, Lithuania

Triolein-filled semipermeable membrane devices (SPMDs) were deployed for four weeks in polluted water sources in Lithuania. The SPMD samples were subjected to fractionation using size-exclusion chromatography (SEC). The fraction containing average molecular weight compounds such as PAHs and organochlorine pesticides was screened by gas chromatography and mass-spectrometry. The whole (non-fractionated) samples and their SEC fractions were tested in bioassays such as  MicrotoxTM, MutatoxTM,Daphnia pulex immobilization assay and sister chromatid exchange (SCE) in human lymphocytes in vitro test.

The MicrotoxTM test was most sensitive with the estimated EC50 values in the range of mg or even m g per ml based on the amount of the SPMD triolein. Part of the observed toxicity was caused by elemental sulfur co-sampled by SPMDs from sediments. The sum of toxicity equivalents of the SEC fractions was smaller than the relative toxicity of the whole samples indicating the presence of synergistic interactions in the complex mixtures of chemical pollutants. The toxic or genotoxic response induced by the SPMD samples and their fractions was smaller the D. pulex, MutatoxTM and SCE tests. In MutatoxTM, a positive response was only detected without the S9 metabolic activation which indicates the presence of mainly direct-acting mutagens in the samples. Interpretation of the MutatoxTM data was difficult due to the complexity of dose-response and time-response relationships.

The study has demonstrated the potential as well as some limitations of SPMDs in the monitoring of biological effects of bioavailable organic pollutants in the aquatic environment.

 

A Mass Balance of Surface Water Genotoxicity in the Providence River (Rhode Island, USA)

Paul A. White1, Kay T. Ho1, Takeshi Ohe2, David M. DeMarini3, Christian Blaise4

1Atlantic Ecology Div., U.S. EPA, Narragansett, RI, USA, 2Kyoto Women’s University, Kyoto, Japan. 3Environmental Carcinogenesis Division, U.S. EPA, Research Triangle Park, NC, USA, 4St. Lawrence Center, Environment Canada, Montreal, Canada.

White and Rasmussen (Mutation Res. 410:223-236) used a mass balance approach to demonstrate that over 85% of the total genotoxic loading to the St. Lawrence River at Montreal is non-industrial. To validate the mass balance approach and investigate the sources of genotoxins in surface waters, we conducted a more thorough study of the Providence River. Samples were collected from selected river sites and WWTFs (wastewater treatment facilities). Organics in filtered samples were concentrated via XAD-2 sorption (acetone elution), or direct partitioning into DCM (dichloromethane). pH adjustment provided initial fractionation into base/neutral and acid extracts. Genotoxicity was measured using the SOS Chromotest and the Salmonella mutagenicity test (TA100, TA98, YG1042, YG1041). Initial analyses revealed potent SOS genotoxicity in the base/neutral surface water extracts, with XAD samples yielding potency values 8-10 times greater than matching DCM extracts. Subsequent Salmonella testing revealed activity in the frameshift strains TA98 and YG1041. An 8 to 10-fold increase in YG1041 potency upon S9 addition suggests the presence of aromatic amines. Overall, the results indicate that most (>50%) of the Providence River genotoxic load enters the system via the Seekonk River. Moreover, discharges from the WWTFs can account for local increases in surface water genotoxicity.

METHODS OF SURFACE WATER TREATMENT AND TOTAL MUTAGENIC ACTIVITY (TMA) OF DRINKING WATER

V.S. Zhurkov, L.V. Akhaltseva, T.E. Mozhaeva, N.E. Lukmanova

Research Institute of Human Ecology and Environmental Hygiene, Russian Academy of Medical Sciences, 119833, Pogodinskaya 10, Moscow, Russia

TMA (the mutagenic potential of organic chemicals water pollution) is the integral index for control of mutagenic and carcinogenic activity of drinking water chemicals. It is important to assess the influence of different disinfectants and their combinations on TMA of drinking water from surface water-sources.

Experiments were carried out on pilot drinking water plant with Volga water-source. The treatment by various doses of chlorine and ozone separately and in various combinations were used. Water samples (160-200 l) were passed through column with polymer sorbent Separon SE. Chemicals were extracted by acetone. The eluate was evaporated to dry residue, which was dissolved in 1,5 ml DMSO. Mutagenic activity was assayed by Ames test using S. typhimurium TA 100 and TA 98 without and with S9 fraction of Sowol treated rats liver. The tested doses were equivalent 1/15, 1/75 and 1/375 of water volume passed through the column.

The samples of surface water were not mutagenic. Chlorination (2 and 4 mg/l) induced TMA in all strains/variants, especially with TA 100, S9-. Mutagenic effect was increased with increasing chlorine dose. Ozone (0.5-4.0 mg/l) under ozonation or ozonflotation did not induced TMA of water. Ozone in doses 0.5 and 3.8 mg/l significantly decreased induced by chlorination TMA of water. Pretreatment of water by ozone (1-3 mg/l) before chlorination also decreased TMA of water. The influence of other disinfectants (monochloramine, chlorine dioxide, hypochlorite, hydrogen peroxide and iodine) on surface water TMA were also studied.

 

ECOTOXICITY TESTING: COMMUNITY STRUCTURE APPROACH IN POLLUTED NORTH LITHUANIA RIVERS

Liutauras Stoškus1, Virginija Pliuraite2

1Faculty of Natural Sciences, Vilnius University, Ciurlionio 21, LT-2600, Vilnius, Lithuania

2Institute of Ecology, Akademijos 2, LT-2600, Vilnius, Lithuania

Benthic macroinvertebrates communities provide powerful tool for assessing aquatic environments. The sensitiveness of benthic communities to most forms of human disturbance (chemical pollution and riverbeds physical deterioration) is especially significant attribute for assessment programmes. Benthic communities can be used as indicators over wide temporal and spatial ranges. Macroinvertebrates have been used in many different roles for assessing river health and monitoring responses to remedial management. The ultimate goal of ecotoxicological testing is to predict ecological effects of chemicals. Interpretation of these effects is reasonable accurate and, in other cases, misleading then we collide with physical disturbance of river ecosystems. Integration of existing chemical and physical measures of water quality with biological studies is necessary to understand causes of effects (ecotoxic, physical or natural origin) for suitable river quality management. Results of two years investigation period of macroinvertebrates communities of polluted North Lithuanian rivers indicate how ecotoxicological effects can be separated from responses of physical alteration using Diversity Indices approach.

 

DNA DAMAGE IN ADULTS AND EMBRYOS OF FISH (LIMANDA LIMANDA) FROM THE NORTH SEA

A. Herbert1,2, D. Warnecke1, H.C. da Silva de Assis1,3, A. Sturm1, V. Dethlefsen4,

H. von Westernhagen5, P.- D. Hansen1

1Berlin University of Technology, Institute for Ecology and Biology, Department of Ecotoxicology, Keplerstraße 4- 6, D- 10589, Berlin, Germany, 2Beratungsbüro für Ökotoxikologie und Umweltschutz, An den Buchen 14 B, D- 14979 Grossbeeren, Germany (present address), 3Universidade Federal do Parana, Departamento de Farmacologia, Cx. P. 19031, 81531- 990 Curitiba, Parana, Brasil, 4Bundesforschungsanstalt für Fischerei, Institut für Fischerökologie, Außenstelle Cuxhaven, Deichstraße 12, D- 27472 Cuxhaven, Germany, 5Biologische Anstalt Helgoland, Notkestraße 31, D- 22607 Hamburg, Germany

Genotoxic potentials of pollution interfere with key functions of life, since they affect the primary biological information matrix DNA. To get an impression of the extent of genotoxic impact on marine ecosystem, adult and embryonic fish (dab, Limanda limanda) from the North Sea were examined regarding to DNA damage, other biochemical markers of pollution impact (MFO induction, acetylcholinesterase inhibition), malformations and diseases.

DNA damage was investigated during several sea cruises between 1993 and 1996 at different sampling sites. A semi-authomatized microplate reader technology of the alkaline DNA unwinding assay was used on board of the research ship FFS Walther Herwig. Thus, 2 sampling stations could be investigated per day with 10 individual adult fish each, or twenty stations with pooled samples of fish embryos, allowing sampling in tight spatial and temporal patterns.

Both, adult fish and fish embryos were affected by genotoxic exposure in statistically significant spatial patterns (analysis of variance, P<0.01), with problem areas at the former German offshore waste incineration site, north of the island of Helgoland, at the mouth of the river Rhine, at the Firth of Forth, and at the Danish coast at Esbjerg. Lowest genotoxic impacts were observed in the German Bight and at the Dogger Bank.

Liver DNA from female fish was affected significantly more than DNA from male fish, since DNA damage exhibited co-variation with liver weight (analysis of co-variance, P<0.05), and is possibly due to higher fat contents.

DNA damage in embryos has been much higher that in adult dab liver. As a possible cause, enriched pollutants in embryonic fat deposits must be considered, thus reflecting maternal exposure. The spatial distribution of DNA damage was closely similar in early and in late developmental stages (stages I/II and III/IV, pooled).

Using the observed data, which indicated highly increased somatic mutation rates, model calculations can be made to estimate the pollution-related loss of genetic fitness in fish populations from the North Sea. The calculated estimate would correspond to a profit loss for the German fishery industries of about 50,000,000,- DM per year.

 

DNA DAMAGE IN MUSSELS (DREISSENA POLYMORPHA) FROM THE RIVER ELBE

A. Herbert1,2, H.C. da Silva de Assis1,3, H. Krumbeck1, E. Wittekindt3,

T. Gaumert4, P.- D. Hansen1

1Berlin University of Technology, Institute for Ecology and Biology, Department of Ecotoxicology, Keplerstraße 4- 6, D- 10589, Berlin, Germany, 2Beratungsbüro für Ökotoxikologie und Umweltschutz, An den Buchen 14 B, D- 14979 Grossbeeren, Germany (present address), 3Universidade Federal do Parana, Departamento de Farmacologia, Cx. P. 19031, 81531- 990 Curitiba, Parana, Brasil, 4Arbeitsgemeinschaft für die Reinhaltung der Elbe (ARGE), Wassergütestelle, Neßdeich 120- 121, D- 21129 Hamburg, Germany

The Elbe is considered one of the most polluted rivers in Germany. Mussels (Dreissena polymorpha) were repeatedly exposed along the river at different sites and in different seasons. DNA damage was investigated with the alkaline DNA unwinding assay. In addition, inhibition of acetylcholinesterase was investigated as another biomarker, known to be sensitive to organophosphate and carbamate pesticides. The effects were compared to the soft body contents of heavy metals and arsenic, HCB, HCH isomers, DDT and relatives, and PCBs. Mussels kept in the laboratory and from a clean lake as a reference site were investigated as controls.

Mussels from all sites of the river Elbe exhibited increased body concentrations of almost all investigated pollutants, very high degrees of DNA damage - i.e. increased tenfold and more over backgrounds, and strong inhibition of acetylcholinesterase. There were significant variations of pollution effects between winter and summer on average.

Multivariate statistical analysis revealed that body concentrations of zinc had the closest association to DNA damage in the investigated mussels. This may be seen, however, in the context, that it was the contaminant with by far the highest body concentrations. Among the other investigated elements, lead, cadmium, and copper showed correlations, but not mercury and chromium, and arsenic only slightly.

There were strong positive correlations to HCHs, but not HCB. Another statistical influences on DNA damage were DDT and DDD, and DDE to a lower extent. There was no correlation to the investigated PCB congener concentrations.

It must be clearly pointed out, that the found statistical influences of the investigated pollutants on DNA damage may only be explained in part by their genotoxic potential, but reflect also strongly the environmental concentrations, bioavailability and toxicokinetics.

The study is not only an illustrative example of the high genotoxic impact of pollutants on aquatic ecosystems, but gives in addition an idea, which pollutants could contribute to these effects mostly.

 

ALLOZYME ANALYSIS OF FRESHWATER MOLLUSCS IN THE LITHUANIAN INDUSTRIAL AND NON-INDUSTRIAL REGIONS

Vaida Deveniene1, Algimantas Paulauskas1,2

1Laboratory of Immunogenetics, Institute of Ecology, Vilnius, Lithuania, 2Vytautas Magnus University, Faculty of Environmental Sciences, Daukanto 28, Kaunas, Lithuania

Freshwater molluscs Limnaea stagnalis and Planobarbius corneus are widespread in the aquatic ecosystems in Lithuania. They are perfect bioindicators of bioaccumulation of pollutants due to they sedimentary way of life, ability to concentrate chemicals up to very high concentration. These molluscs can be used as bioindicators of environmental variability.

For genetic variability studies, molluscs were collected from non-industrial (Šakiai district, Simnas and Kroku water meadow) and industrial regions (Elektrenai, Šiauliai and Kedainiai) of Lithuania in 1997-1998.

Using polyacrilamide gel electrophoresis six isoenzyme systems - esterase (EST), malate dehydrogenase (MDH), lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6PDH), malic enzyme (ME), and 6-phosphogluconate dehydrogenase (6-PGDH) - were identified. Thre of these isoenzyme systems (G-6PDH, MDH, and EST) were polymorphic.

These sampled populations have been investigated and described by the frequency of gene and of genotype, the proportion of polymorphic loci (P), the total gene diversity (H), observed (Ho) and expected (He) mean heterozygosity, the mean number of alleles at all loci (A).

Relative genetic distance and similarity between these populations were quantified according to Nei and Roger.

The analysis among populations showed genetic differences between industrial and non-industrial regions.

 

THE COMET ASSAY IN ENVIRONMENTAL BIOMONITORING

L. Verschaeve, U. Van Gorp, G. Koppen

VITO, Environmental Toxicology, Boeretang 200, B- 2400 Mol, Belgium

The single cell gel electrophoresis assay or comet test is since several years successfully applied to evaluate the genotoxicity of chemicals. It is also used for environmental biomonitoring and may also be considered a very important tool for environmental studies. Indeed, so far environmental monitoring was essentially performed using analytical techniques characterizing the levels of particular (known) pollutants. These methods are of course useful, but they do not provide insight into bioavailability of pollutants and the biological hazards associated with complex mixtures. With the comet assay an easy and sensitive method is available for the (geno)toxicological evaluation of environmental samples. Although so far essentially mammalian cells were investigated, cells from other organisms are now investigated as well (e.g., fish, molluscs, …).

In our laboratory we are interested in, among others, soil and water pollution and therefore adapted the comet assay for investigation of earthworm coelomocytes, plant root and leaf cells and oyster gill cells.

Earthworms are breeded in the laboratory and exposed to polluted soil samples. The water content of pH of the samples as well as the exposure time were shown to be important parameters that influence the outcome of a comet assay. Yet, earthworms proved to be good sentinel organisms for soil pollution studies, although we failed to demonstrate dose-effect relationship (e.g., PAH’s contaminated soils), probably because such samples contain complex mixtures of chemicals rather than only one pollutant. Furthermore a dose-range (e.g., dilution with standard black earth) should be carefully established as highly polluted samples sometime give less DNA damage than less polluted samples due to toxicity and consequently loss of highly damaged cells.

Also plant cells (we usually investigated root tips from Vicia faba) proved to be useful tool for environmental biomonitoring, although most of the drawbacks encountered with earthworms also holds true here. Furthermore, it should be noted that when leaves are investigated, the DNA damage is, even in control plants, increasing with the age of the leaves. This may be one of the reasons why the plant comet test should be conducted on root tips or plantlets in the laboratory rather than on plants found in the (polluted) area of interest.

Since a short period of time we also apply the comet assay in oysters as part of a aquaculture project in the sluice dock in Ostend (Belgium). In this project we demonstrated that the comet assay in the gills of oysters perfectly reflects the degree of pollution of the different sites that were investigated. There was much less inter-, and intra-individual variation in gill cells compared to blood cells.

In conclusion, our investigation show that the comet assay can be successfully used for environmental biomonitoring of soils, as well as fresh and marine waters. However, one should be very attentive to the sample characteristics (e.g., pH) and other conditions that may influence the results. Standardised exposure of the test organisms to the samples is an absolute prerequisite to obtain reproducible results.

ENVIRONMENTAL MONITORING OF DNA DAMAGES IN CELLS FROM THE BLUE MUSSEL, MYTILUS EDULIS, USING THE COMET ASSAY

Jette Rank

Department of Environment, Technology and Social Studies, Roskilde University, DK-4000 Roskilde, Denmark

Wastewater discharged to marine waters from households and industries in Denmark as well as in other countries have shown genotoxic effects. Therefore, it would be of interest to know if genotoxic wastewater can harm marine organisms. As mussels are among the most used organisms for biomonitoring in coastal waters, they were chosen in the present study as indicator organisms for genotoxic effects. Blue mussels, Mytilus edulis, were sampled during four summer month from coastal waters in Křge Bay at four stations with various distance from wastewater discharge. Haemolymph cells were examined for DNA breaks in the single cell gel electrophoresis (the comet) assay. The results showed variation among the four stations as well as seasonal variations. The tail moments were in the range 1.56- 5.38. Samples from other coastal waters were found to be in the same range (0.97- 4.54). The results were inconclusive as no pattern could be seen with regard to increased tail moments at the contaminated sites. Neither was there any consistent seasonal variation during the four month of observation. Further studies using gill cells are ongoing. It is obvious that biomonitoring of DNA damages in mussels or other aquatic species will need more comprehensive knowledge of the background levels of DNA damages in wild populations. It is concluded that bio-monitoring of DNA damages in marine organisms seems to be very difficult and the concept "environmental monitoring of DNA damages" needs to be developed further.

CYTOGENETIC ANALYSIS OF AQUATIC ORGANISMS AS A TOOL FOR MONITORING OF ENVIRONMENTAL EXPOSURE

Janina Baršiene

Institute of Ecology, Akademijos 2, 2600 Vilnius, Lithuania

Urban sewage, specific industrial and agricultural effluents as well as naturally produced substances can affect aquatic organisms by genotoxic and cancerogenic compounds. However, sometimes even high concentrations of pollutants may be tolerated by organisms due to mechanisms of elimination or detoxification, which could evolve the course of evolution. In this case the correlation between concentrations of pollutants in water, sediments, organisms and the biological effects might be absent. Moreover, due to composition of genotoxic pollutants in aquatic systems, bioaccumulation and biotransformation processes, very specific ecological conditions may be created. Main aims of genetic toxicology are to investigate the extent to which ecosystems are polluted with genotoxins and to determine adverse effects as reduced fitness, changes in gene frequencies and therefore impact on genetic diversity in populations, communities, associated with genotoxic exposure (Depledge, 1994). However the ecological effects of many genotoxic compounds remain poorly understood.

The cytogenetic techniques used in this study proved to be a good tool in the evaluation of peculiarities of genotoxic damage induced by heavy metals PCBs, radionuclides and other dangerous environmental agents. As a biomarker of environmental influence the cytogenetic disturbances – aneuploidy, polyploidy, meiotic injures, chromosomal aberrations, centromere dissociation and fragmented polyploid nuclei were evaluated in somatic and gonadal cells of molluscs, fish, helminthes and crustaceans. Wide distribution, abundance, relative sensitivity to environmental contamination, sedentary, filter or detritus feeding style of the life, different life cycles were the main criteria for selection of indicator species.

The environmental genotoxicity in vivo assessments were performed mainly in molluscs inhabiting highly contaminated sites of rise and orange-tree fields, Jugar River (Cofrentes NPP), mountain and geothermal springs in eastern Spain, in different water bodies of Lithuania, Switzerland, and Poland. The highest level of cytogenetic disturbances was found in molluscs from the vicinities of Cofrentes NPP, from geothermal spring and canals in orange-tree fields (eastern Spain). Comparatively high frequency of aneugenic and clastogenic effects were determined in Murten Lake (Switzerland, in the vicinities of which two NPPs are located), Nialk Lake (Poland, contamination by sewage effluents), in Klaipeda Port area, Nemunas near Birštonas and Neris below Vilnius (Lithuania). The comparison of genotoxic effects in the tissues of fish, molluscs and helminthes inhabiting the same localities showed that bivalve molluscs were most sensitive, and helminthes were most resistant organisms to environmental exposure. The evaluation of cytogenetic effects of pollution in Klaipeda Port by using passive monitoring showed that after the sediment dredging genotoxic influence in 1995-98 period decreased considerably and in 1998 fragmented polyploid (cancer or apoptotic) cells were not observed at all. Active biomonitoring experiments indicated that genotoxic carcinogenic compounds are discharged into the Klaipeda Port area from Smelte and Dane Rivers. High aneugenic and clastogenic effects of pollutants were determined in estuary of Vilhelmo Channel near the waterworks, which is potentially dangerous not only for aquatic organisms but also for humans. The frequency of aneugenic and clastogenic effects in molluscs inhabiting highly contaminated sites was 3-10 times higher than in molluscs from the reference sites. The role of pollutants inducing cytogenetical injures in aquatic organisms will be discussed.

 

CHICK EMBRYO IN OVO – A PERSPECTIVE ALTERNATIVE SYSTEM FOR EVALUATION OF SHORT- AND LONG-TERM CONSEQUENCES OF EXPOSURE TO XENOBIOTICS DURING ORGANOGENESIS: THE EFFECTS OF 1,2-DIBROMOETHANE

Bozena Novotná1, Zdena Zemanová2, Zdík Dušek1, Iva Holejšovská1,2,

Jolana Vanková1

1Institute of Experimental Medicine and 2Institute of Physiology, Czech Academy of Sciences, Vídenská 1083, 14220 Praha 4, Czech Republic

Exposure to genotoxic compounds during organogenesis may result in pregnancy outcome, associated frequently with dysmorphogenesis. Survivals with external malformations are usually detected at the birth while the manifestation of other consequences, including carcinogenesis, occurs later during postnatal life. Dibromoethane (DBE), a human carcinogen still used as a solvent in industry, exhibits in adult organisms strong nefrotoxicity which is usually related to further bioactivation of hepatic DBE-glutathione conjugates by renal enzymes. It is assumed that embryonic tissues may exert even the higher sensitivity to genotoxic action. The data about embryotoxic effects of DBE in experimental animals are, however, confounding and some sporadic information has been obtained from experiments with rat embryos in vitro.

Three-day-old chick embryos were treated intraamniotically with DBE with the aim to investigate genotoxicity and embryotoxicity of the compound, with the special concern for hematopoiesis and kidney development. Although the most pronounced effect seemed to be an early death of embryos (within 24 h) resulting the most probably from extraembryonic vessel bleeding into amniotic cavity, survivals exhibited on incubation day 10 a significant increase of kidney malformations (namely cystic dilatations of tubules). External morphology was affected only in low frequency and in the spectrum of detected malformations prevailed brain defects (exencephaly and hydrocephaly) followed by malformations of great vessels.

Cytogenetic analysis did not show in blood cells of exposed embryos any changes in the frequency of chromosomal aberrations when compared with untreated group. Only mitotic activity exceeded the control level 24 h after the treatment in consequence of accumulation of mitoses following the initial inhibition of cell division. In contrast, comet assay revealed there dose-dependent increase of DNA fragmentation, detected already 3 h after the injection of DBE. Exposed groups did not differ from controls 6 h later suggesting probably the repair of induced damage. Nevertheless, during the following intervals new wave of DNA fragmentation has been detected, persisting up to incubation day 10. Also kidney tissue exhibited an increased level of DNA breaks 24 h after the treatment with DBE, further intervals have not been analyzed yet.

In conclusion, chick embryo represents a perspective tool for evaluation of long-term consequences of genotoxic exposure in vivo. Identification of DNA fragmentation origin in relation to cell differentiation and cell death is the subject of further research.

The work was supported by grant from Grant Agency of the Czech Republic No 304/98/1613 and by NATO grant ENVIR.LG 960330

 

IN OVO KINETICS OF ENVIRONMENTAL XENOBIOTICS;

APPLICABILITY IN THE ESTIMATION OF BIOLOGICALY EFFECTIVE DOSES

L.Vodicková1, E.Frantík1, M.Peterka2, B.Novotná2, Z.Dušek2, P.Vodicka2

1Natl. Inst. Publ. Health, Šrobárova 48, 10042 Prague 10, Czech Republic

2Inst. Exper. Med., Czech Acad. Sci., Vídenská 1083, 14220 Prague 4, Czech Republic

 

Most studies on embryotoxic, teratogenic and genotoxic effects of xenobiotics using chick embryo system and different routes of administration lack data on the kinetics and distribution of the xenobiotic and do not permit to assess the real dose received by the embryo (internal exposure).

Kinetics of three model compounds, important contaminants exhibiting extremely different physical-chemical properties, were compared at different routes of exposure: acetone (polar), 1,2–dibromoethane (DBE, less polar) and styrene (ST, non-polar). Simultaneously, the principal metabolite of styrene, styrene-7,8-oxide was investigated. The chick embryo in ovo was treated either by intraamnial injection or by exposure of the egg to vapours. Tissue concentrations of above chemicals were analysed by head–space GC.

After 3-hr exposure to saturated vapours DBE reached much higher tissue concentration than ST: the concentration was comparable to maximum concentration found after intraamnial injection of 300 micrograms of DBE in 6% ethanol. At both routes, only about 3% of the administered dose were found in the embryo; a major part of the amount was in the yolk and white in the case of exposure to vapours, and at injection – in the amnial fluid. The concentration decrease was exponential, with the half time significantly shorter for DBE than for ST.

Our results indicate that at exposure to vapours, the dose received by the embryo is flexibly controlled by the vapours concentration and duration of exposure, and depends critically on the properties of the compound. Shell window can modify the distribution and concentration gradients in the egg. At intraamnial injection, most of the applied dose remains in the amnion, the absorption is extremely variable, depends on the properties of the substance and in addition on the vehicle used; a considerable part of the dose leaks out and is found in the paraffin barrier.

This study is a part of the project: "1,2 DBE – the significance of its genotoxic effects in the developing organism." and will be supplemented by the determination of biomarkers of the genotoxic effect (single strand breaks in DNA, specific DNA adducts). The parameters of internal exposure are intended to enhance the quantitative information on the potency of chemicals to induce embryonal damage (embryotoxicity, teratogenicity, genotoxicity).

This study was supported by grants GACR 304/98/1613 and 313/99/1460

 

Micronuclei in reindeer lymphocytes – a field study

Ingvild Svorkmo Espelien

Norwegian Institute for Nature Research, Tungasletta 2, N-7485 Trondheim, Norway

As a part of an ongoing larger project, micronucleus analyses were carried out on peripheral blood lymphocytes from wild and semi-domesticated reindeer by the method of Cytochalasin-Bcytokinesis-blocking. The methodology was adapted for field conditions in extreme winter conditions for reindeer blood and blood of reindeer foetuses. Reindeer from 4 different populations were sampled for micronucleus preparation, totally 25 animals including 15 adults and 10 fetuses. Chemical and physical analyses of metals and radioactivity in tissue samples from the reindeer were compared with the frequency of micronuclei in the different populations. The number of animals sampled were, however, low. The application of the micronucleus assay as a biomarker in large wild living animals will be discussed.

 

 

GENOTOXIC ACTION OF THE Co(NO3)2 ON THE FIELD BEANS CULTIVAR ‘AUŠRA’

D. Barysas, L. Balciuniene

Botanical Garden of Vilnius University, Kairenu 43, LT 2040 Vilnius, Lithuania

Field beans (Vicia faba L.) are very suitable for evaluation of the frequency of morphoses. On the young lower leaves various agents such as ionising radiation, alkylating and chelating substances, and ageing are capable of inducing the leave-spots. Extent of expression of morphoses may also be estimated. Salts of Co2+ may induce the phenocopies of the chlorophyll mutations. This type of morphoses was examined in the present work.

Seed material of the field bean c. 'Aušra' was obtained from the Lithuanian Institute of Agriculture. The seeds were treated with solution of various concentrations of Co(NO3)2: 0 (without), 0.25, 0.5 and 1×10-2M. Seeds were soaked in solutions for 15 h. Then they were washed with distilled water and planted in experimental field of the Botanical garden. All 'steadfast' (unshelled) seeds were removed. Types of morphoses were determined one month after the sowing. Degree of morphosis was expressed by number in the strengthening order. The leaf color was evaluated from 0 (normal green plant) to 5 (plant with yellow leaves).

Present work confirmed results about a strong morphosis-inducing effect of the cobalt salts. Phenocopies of the chlorophyll mutations were observed as well.

The morphosis-inducing effect of Co(NO3)2 directly depended on concentration: the number of normal plants decreased from 100 % among untreated plants to 27.0 % after the treatment with 1.0×10-2 M of Co(NO3)2. The main group of morphoses was presented by plants with signs of the strongest affections (poor plants with yellow leaves), and the proportion of these plants depended to the dose of Co(NO3)2. Total frequency of this group of morphoses and its part in the general frequency of morphoses proportionally arose with increasing dose of Co(NO3)2.

It was proved that alterations of colour of chlorophyll are uninherited in M2. So, they are really phenocopies of chlorophyll mutations.

Connection between degree of the defeat of the plants in M1 and mutability of their progenies was not statistically significant. But without any doubt the treatment of field beans with Co(NO3)2 increased variability of plants in M2: frequency of the altered plants was about 3-3.5 times higher when compared to the progenies of untreated plants.

The nature of these altered plants must be verified in M3.

 

DETECTION OF GENOMIC DAMAGE BY ALKALINE COMET ASSAY AND VARIABILITY OF SUPEROXIDE DISMUTASE (SOD) SPECTRUM IN BARLEY

K. Zukas, V. Kleizaite, S. Šulcaite

Department of Botany and Genetics, Vilnius University, M.K. Ciurlionio 21, 2009 Vilnius, Lithuania

The response of barley single cells to methyl methanesulfonate (MMS) and UV radiation (UVC) has been studied. The level of MMS- and UVC-induced DNA single strand breaks (SSBs) was evaluated by single-cell alkaline gel electrophoresis (comet assay) followed by visual scoring. Direct correlation between the SSBs induction and MMS concentration was detected while SSBs induction did not depend on dose of UVC. Both studied mutagens modify the isozyme spectrum of superoxide dismutase (SOD). Relationship between the induction of oxidative damage and SOD spectrum will be discussed.

 

 

 

THE ROLE OF SCREENING AND PRIORITIZATION IN THE ASSESSMENT AND MANAGEMENT OF CHEMICALS

C.E. Cowan-Ellsberry1, D. Sabaliunas2, Tom Feijtel3

1The Procter & Gamble Company, 5299 Spring Grove Ave, Cincinnati, OH 45242, 2The Procter & Gamble Company, Egham, UK, and 3The Procter & Gamble Company, Strombeek-Bever, Belgium

As the scientific basis for conducting human health and environmental risk assessments to determine the likelihood of significant adverse human health and/or environmental effects (i.e., risk) from chemicals has improved, the time and effort require to conduct these assessments has increased dramatically. At the same time, there is a general recognition that much of this effort is not required as many substances do not result in significant exposures and that the potential to exert adverse effects is unlikely to occur. Thus recently, there has been an increasing focus on how to streamline the risk assessment process and thus focus resources toward conducting assessments on those substances that are most likely to result in exposure and adverse effects. It will be these substances which will require in-depth review and potentially management. This work has focused on developing and applying screening and risk prioritization approaches and on determining when these approaches can be appropriately used in the overall process of management of chemicals. There are several screening and prioritization projects underway in Europe, Canada and the United States. These projects primarily emphasize how information on potential adverse effects from exposure to the substance and physical, chemical and degradation properties of the substance is used in these screening and prioritization approaches. Other discussions have focused on whether these properties should be used individually or in some combination, how to estimate these properties when data are limited or missing, and the appropriate criteria for these properties which indicate that there is a need for in-depth review and risk assessment of the substance. In this presentation, the appropriate role of screening and prioritization in the overall chemical management process and especially in streamlining and focusing resources on substances most likely to be of concern is discussed. In addition, the basic elements of these screening and prioritization approaches will be described including the criteria used and processes to estimate substance properties. This presentation will also discuss the future direction of these screening and prioritization activities with recommendations on how these processes can be improved.

THE ESTIMATION OF MUTATION RATES FROM DNA DAMAGE DATA IN ENVIRONMENTAL STUDIES AND GENETIC RISK ASSESSMENT

A. Herbert1,2, P.- D. Hansen1

1Berlin University of Technology, Institute for Ecology and Biology, Department of Ecotoxicology, Keplerstraße 4- 6, D- 10589, Berlin, Germany, and 2Beratungsüro für Ökotoxikologie und Umweltschutz, An den Buchen 14 B, D- 14979 Grossbeeren, Germany (present address)

Decision making in environmental management is highly influenced by cost-benefit analysis, and this again depends highly on environmental risk assessment. The method of choice is the use of models, which simplify the view of complex ecosystems, but are exact enough to fulfill the desired purposes at a satisfying degree. At the moment, cybernetic models with strong deterministic elements are favored in ecology, but stochastic models have proven to be useful tools in toxicology. In genotoxicology, linear and quasi-linear stochastic models for dose-response relationships provide in many cases a good basis for risk assessment in a first approximation. This appears to be also true for the rate of de novo-formation of DNA damage and mutation rates. De novo-formation of DNA damage can be estimated from the steady-state levels of DNA damage and DNA repair rates. Thus, exogeneously induced increases of steady-state levels of DNA damage can be used to estimate the relative induction of spontaneous mutations rates.

Animals of various crustacean, mussel and fish species have been treated with genotoxic model agents (MNNG, NQO, BaP) in laboratory experiments, and steady-state levels of DNA damage were determined by the alkaline DNA unwinding assay. Results were used to estimate relative somatic mutation rates (in percent).

The results indicate that in static exposure experiments high concentrations of model agents are necessary to induce the mutation rates considerably. Thus, treatment of trout (Oncorhynchus mykiss) with 20 m g/l of BaP for 96 h induced the estimated mutation rate only about tenfold (1000 % ). Compared to these results, field studies on mussels and fish reveal quite frequently results in the same order of magnitude. These highly induced somatic mutation rates surprised in the first moment, but review of literature soon confirmed these results by similar observations involving different methodological approaches, e.g. chromosomal or pigmentation aberrations (Even a recent investigation of heritable germ-line mutations in environmentally exposed herring gulls reports an increase of 400% of the background mutation rate).

Reasons for these highly induced mutation rates in pollution impacted aquatic ecosystems may be seen in toxicokinetics related phenomena like bioconcentration or metabolism, but additive and synergistic effects of possibly hundreds or thousands of different genotoxic pollutants may explain another part of the effects.

From studies on fish in the North Sea a rough loss of genetic fitness of 10 percent due to genotoxic pollution impacts can be estimated, equivalent to approximately 50,000,000, - DM loss in fishery profits per year in Germany. Alone this aspect points out the necessity for better environmental management concerning genotoxic pollution. Cost-effective DNA damage assays for aquatic organisms appear to be useful tools to accomplish this without high additional costs.

 

GENETIC EFFECTS OF HIGH AND LOW EXPOSURE TO HEAVY METALS IN LAPLAND NATURE RESERVE

Ĺse Krřkje, Chris Bingham

Department of Botany, Norwegian University of Science and Technology, 7491 Trondheim, Norway.

The project aims at investigating the genotoxic effects of heavy metals on plants and small mammals in the Lapland Biospheric Reserve, in the Kola Peninsula, Russia. The area has a high level of pollution. The study area in the reserve is about 5 000 km2, and stretches from the part where the ecosystem is completely destroyed and into the area, which seems to be untouched by pollutants. In this area the Severonickel Smelter Complex is the only local source of atmospheric industrial pollution. Emissions from the Severonickel Smelter complex include 4 000 tons nickel and 4 000 tons copper annually. Plant material has been collected over a period of five years and small mammals were collected in two years. Levels of metals have been measured in plant species and tissues from small mammals. The frequency of chromosome aberrations was examined in lymphocytes from Clethrionomys rufocanus (greyside mouse) and in root-tips from Picea abies (spruce) to study the effect of pollution on cell division. The results from the study of small mammals are consistent with a concentration/effect relationship between chromosome aberrations in lymphocytes and distance from the smelter stacks. The results from the spruce, so far, do not show the same trend.

 

EVALUATION OF HEALTH RISKS CAUSED BY NITRO MUSKS

Sebastian Kevekordes1, Hartmut Dunkelberg1, Volker Mersch-Sundermann2

1Medical Institute of General Hygiene and Environmental Health, University of Göttingen, Windausweg 2, 37073 Göttingen, Germany, and 2Department of Medical Microbiology and Hygiene, Faculty of Clinical Medicine Mannheim, University of Heidelberg, P.O. Box 100023, 68135 Mannheim Germany

Nitro musks, i.e. musk ambrette, musk ketone, musk xylene, musk tibetene and musk moskene, are synthetic scents. According to the International Fragrance Association (IFRA) musk ketone and musk xylene are widely used in cosmetics and detergents. Additionally, relevant amounts of nitro musks are used in Indian joss sticks. In the European Community (EC) musk tibetene and musk moskene are produced only in small quantities. Because of its allergic potency musk ambrette is forbidden in EC.

Several studies indicated that lipophilic nitro musks persist in the environment. Additionally, it has been shown that synthetic nitro musks particularly musk xylene and musk ketone accumulate in human fat tissue and breast milk.

 

Breast milk

Human fat tissues

Human blood

Musk Xylene

10-1200 µg/kg milk fat

6-600 µg/kg fat

0,1-1,1 µg/l plasma

Musk Ketone

10-90 µg/kg milk fat

10-50 µg/kg fat

no data available

Musk Ambrette

Musk Tibetene

Musk Moskene

not detected

not detected

no data available

 

The five nitro musk compounds were examined for mutagenicity in the Salmonella/mammalian microsome assay (TA97, TA98, TA100, TA102), and SOS repair inducing potency in the E.coli PQ37 genotoxicity assay (SOS chromotest) in the presence and absence of an exogenous metabolizing system containing microsomal enzyme fractions of livers of Aroclor 1254 pretreated rats (S9 mix). Additionally, the fragrances were examined for genotoxicity using the sister chromatid exchange test (SCE) with human lymphocytes and the micronucleus test (MNT) with human hepatoma cells (Hep-G2) and human lymphocytes.

Musk ambrette, a well-known photoallergen, exhibited mutagenic effects in the Ames test. Except musk ambrette none of the nitro containing musks exhibited direct mutagenicity or genotoxicity in the assays used.

Comparable to PCB musk xylene and musk ketone were identified as enzyme inducers and co-genotoxicants.

 

GENOTOXIC ACTIVITIES OF DILL (ANETHUM GRAVEOLENS) ESSENTIAL OIL ON HUMAN LYMPHOCYTES IN VITRO AND DROSOPHILA MELANOGASTER SOMATIC CELLS IN VIVO

J. Mierauskiene, G. Slapšyte, V. Dedonyte, J. R. Lazutka

Department of Botany and Genetics, Vilnius University, M. K. Ciurlionio 21, 2009 Vilnius, Lithuania

Essential oils extracted from plants are widely used as fragrances in cosmetics, flavouring additives of food and beverages, scenting agents in a variety of household products (e.g., detergents, soaps, room-air-fresheners, and insect repellents), constituents of some old over-the-counter drugs, and intermediates in the synthesis of perfume chemicals. Some plant extracts, essential oils and their derivatives have been previously shown to have genotoxic properties.

The present study was undertaken to evaluate the genotoxic activity of the essential oil isolated from the plant dill (Anethum graveolens) in chromosome aberration (CA) and sister chromatid exchange (SCE) tests in human lymphocytes in vitro and in Drosophila melanogaster somatic mutation and recombination test (SMART) in vivo.

Dill essential oil appeared to be very strong clastogen: even lowest concentration (0.01 m l/ml) induced CA, and at a concentration of 0.25 m l/ml as many as 32% of cells contained CA. Dependence of the number of aberrant cells on the concentration of dill essential oil may be described by exponential equation Y=1.825e10.72X, where Y is the number of aberrant cells, and X is the concentration (m l/ml) of dill essential oil in culture. This dose- response dependence is statistically significant (r2 = 0.9484). Dill essential oil was capable of inducing SCE in human lymphocytes as well. Dose- response dependence is linear and may be described by the equation Y = 8.63 + 50.79X (r2=0.9962), where Y is number of SCE per cell, and X is concentration (m l/ml) of dill essential oil in the culture.

The rather strong insecticidal activity of the dill essential oil was observed in a preliminary experiment with adult flies. The average survival of adult flies after exposure to 0.75% (v/v) of dill essential oil was about 60% , and all adult flies were dead after the treatment with 1% and higher concentrations. The slight but statistically significant increase in the frequency of single spots was observed (P<0.05).

It may be concluded that dill essential oil is mutagenic for human lymphocytes in vitro and D. melanogaster somatic cells in vivo. The chemical constituents responsible for mutagenicity of dill essential oil remain to be determined

 

ESTIMATION OF MUTAGENICITY OF THE CHLORINATION PRODUCTS OF CYCLOHEXENE

L.P.Sycheva, Z.I.Zholdakova, E.E.Poliakova, N.E.Lukmanova, L.V.Akhaltseva,V.S.Zhurkov

Research Institute of Human Ecology and Environmental Hygiene, Russian Academy of Medical Sciences, 119833, Pogodinskaya 10, Moscow, Russia

Ames test on strains TA98 and TA 100 (S9+/S9- ) as well as colon and bone marrow micronuclei test in mice were used to estimate mutagenicity of chlorination products of cyclohexene. Cyclohexene was chlorinated in the dark at 25° C in distilled water with addition of phospfate buffer (0.02M, pH 6.8-7.0). Two liters of solution was used for micronuclei test and 250 ml for Ames test. The reaction mixture contained cyclohexene (32.8 mg/l) and active chlorine (0.56 g/l). Reactive time was 24 h. HCl was added later to obtain pH 2. Chlorination products were extracted with methylene dichloride. The extracts were dried over anhydrous sodium sulfate, concentrated in Claisen flask up to 1 ml and later totally dried at room temperature. For the experiments with mice the dry matter was dissolved in 10 ml of sunflower oil. The obtained suspension as well as two and four times diluted solutions were administrated by gavage daily during 4 days (0.1 ml per 10 g of weight). The colon and bone marrow were isolated at 24 h after the last injection. The colon was fixed with formaline. Suspended samples were prepared by 50% KOH dissociation of cells and stained by acetoorcein. In Ames test the dried matter was dissolved in 4 ml of DMSO and 0.1 ml of the solution as well as of 5 and 25 times diluted solution were introduced per each plate.

The two maximal doses of the samples statistically significant increased of the rate of micronucleated cells in colon. The mutagenic effect of the chlorination by-products was not detected in the cells of bone marrow. Mutagenicity of cyclohexene was not revealed in Ames test. Otherwise mutagenic effect of its chlorination by-products was noted for the TA 100 (S9- ). The observed mutagenic effects are typical for the transformation products of organic compounds in natural water during chlorination. The induction of mutation in mice colon agrees with the observations of epidemiologists concerning increased the rate of cancer due to water chlorination.

 

GENOTOXICITY OF RECOMBINANT HUMAN CYTOKINES

S. Rudaitiene, J.R. Lazutka

Department of Botany and Genetics, Vilnius University, 21 Ciurlionis St., 2009 Vilnius, Lithuania

Recombinant human proteins have got widespread clinical application during the last decade. Patients are frequently exposed to the high doses of recombinant proteins during the long period of time. Some of human recombinant proteins such as growth hormone, steroid hormones, few cytokines are known to possess genotoxic properties in cultured human lymphocytes. The main aim of this study was to evaluate genotoxicity of recombinant human cytokines from different groups, such as interferons, interleukins, colony-stimulating factors, and tumor necrosis factors. For this purposes peripheral blood lymphocytes from healthy donors were grown for 72h with different concentrations of cytokines (from 10 to 10 000 U/ml). Cytokines - interferon a and g (IFNa , IFNg ), interleukin-2 (IL-2), granulocyte colony-stimulating factor (G-CSF) and tumor necrosis factor a (TNFa ) - were present in culture from the 0 or 24h of cell growth. Genotoxicity of cytokines was evaluated by the analysis of SCEs in second-division cells. Lymphocytes from two different donors were used for each cytokine.

Two human recombinant interferons IFNa and IFNg were investigated in our study. It was established that they possess opposite cytogenetical activities in cultured human lymphocytes. IFNg increased SCE frequency in dose-independent manner. The rate of SCE induced by IFNg reached 18% . Meanwhile two concentrations (100 and 500 U/ml) of IFNa statistically significantly (P<0.05) decreased SCE rate in cultured human lymphocytes. Two forms of recombinant T cell growth factor IL-2, known as efficacious anticancer drug, were investigated in our study. The first form is prepared for laboratory studies, the other - for clinical use. The first form of IL-2 increased SCE rate in dose-independent mane. SCE rate induced by IL-2 varied from 4 to 17% . The therapeutic form of IL-2 statistically significantly decreased SCE value. The percentage of reduction varied from 18 to 26. The haematopoietic growth factor G-CSF, widely used in medicine as immunostimulatory drug, have no effect on SCE rate in cultured human lymphocytes. In the same experimental system pro-inflammatory cytokine TNFa considerably increased SCE frequency (from 35 to 48% ). The highest genotoxicity of TNFa was observed at concentrations from 10 to 100 U/ml, with a peak response at 50 U/ml.

Genotoxicity of cytokines in cultured human lymphocytes may range from slight effect (IFNg , IL-2) to more evident (TNFa ); also some of cytokines (IFNa and therapeutic form of IL-2) may decrease SCE value. It is obvious that the mechanism by which cytokines exhibit their genotoxic or antigenotoxic effect is indirect and probably related to the changes in cell’s enzyme activities or to the formation of free radicals.

 

THE GENETIC DIVERSITY OF SMALL RODENTS (MAMMALIA: RODENTIA ) IN

ECOLOGICALLY DIFFERENT LOCALITIES

Daina Skiriute1, Algimantas Paulauskas1,2

1Vytautas Magnus University, Faculty of Environmental sciences, Daukanto

28, Kaunas, Lithuania, and 2Laboratory of Immunogenetics, Institute of Ecology, Vilnius, Lithuania

 

Genetic diversity is found in all wild animal and plant populations and plays the most important role in the evolutionary processes and also it takes place as one of the conditions to everyday population survival in different and very changeable, un-constant and dynamic environment.

Ecological plasticity of population, which depends on genetic diversity, is very important in adaptation. In this study genetic diversity is investigated in wild animal populations which are the best indicators of different ecosystems and can be the most objective reflection of different processes in ecosystems and its genotoxic effects on the organisms. Studies of genetic diversity can reflect differences between ecosystems.

The aims of our study are as follows: 1. To establish genetic diversity in the wild animal populations from ecologically different localities. 2. Investigation of enzymes of small rodents. 3. Inter-specific genetic diversity. 4. Genetic diversity inside populations. 5. Correlation between environmental pollution and genetic diversity in local populations;

The main parameters of genetic diversity in our work are the following: Gene frequency, genotype frequency, polymorphism, heterozygosity of loci, main heterozygosity, lack of heterozygosity. These parameters help to decide about the state of wild populations. The best test-object in such studies is small rodent populations that were used in our work. We investigated two families Muridae and Cricetidae widely spread in every ecological locality: Apodemus flavicolis, A. agrarius, A. sylvaticus, Mus musculus, Microtus arvalis, Microtus agrestis, and Clethrionomys glareolus. There were studied the following enzyme systems: ADH, LDH, XDH, Est, G-6-PDG, ME, Alfa-GPD. Method of study: protein electrophoresis in PAAG. Material of study: tissue of liver and kidney. Anthropogenic influence on wild animal populations can be one of the ecological reasons of genetic polymorphism in many species. High extent of genetic polymorphism of organisms can be caused by economic use of the territory. Genetic polymorphism is one of the reasons of individual resistance to unfavorable environmental factors.

ENVIRONMENTAL RISK ASSESSMENT OF MEDICINAL PRODUCTS

Lisbeth Ehlert Knudsen

Danish Medicines Agency, Frederiksundsvej 378, 2700 Brřnshřj, Denmark

Adverse effects on the environment from use and disposal of medicinal products is a matter of concern and article 4.6 of Directive 65/66/EEC requires the applicant to address potential risks presented by the medicinal product for the environment. The identification of potential risks to the environment is normally achieved by an Environmental Risk Assessment (ERA), implying:

- Assessment of human exposure levels

- General processes of release, dispersion and elimination

- Estimation of relevant parameters in the aquatic, soil and atmospheric compartments

- Assessment of ecotoxicity, environmental exposure and environmental risk

- Worst case approaches

Data requirements according to Draft directive, currently negotiated, to set a Predicted Environmental Concentration (PEC) include:

- Amount placed on the market by the applicant, per year

- Use pattern

- Excretion and metabolite pattern in humans (clinical studies)

- Physico-chemical properties: Molecular weight, water solubility, n-octanol/water partition coefficient (Kow, Po/w, Log10 Po/w etc.), dissociation constant, hydrolysis, vapour pressure, other degradation processes e.g. oxidation, photolysis

- Information relating to removal and passage from one environmental compartment into another: Biodegradation, adsorption to sewage sludge, adsorption to soil

In most cases no specific data is available and a first estimate of the for example the predicted environmental concentration in surface waters can be obtained using the formula:

PEC, crude estimate [g/l] = A´ (100- R)/(365´ P´ V´ D´ 100),

where

- A[kg] = predicted amount used per year in the EU country with the maximum use;

- R[%] = removal rate (due to loss by adsorption to sludge particles, by volatization, by hydrolysis, by biodegradation etc); under worst case conditions R can be counted as zero;

- P = number of inhabitants of the country;

- V [m3] = volume of waste water per capita and day (generally 0.15 to 0.30 m3);

- D = factor for dilution of wastewater by surface water flow (average factor 10).

Following estimation of PEC and no effect levels, including safety factors (103) no further actions may or may not be necessary. Currently concentrations in water below 0.001 – 0.01 m g/l do not give rise for concern, unless a special ecotoxic effects is known or expected.

 

constructive materials mutagenicity in Drosophila

T. Tsutsman

Research Institute of Human Ecology & Environmental Hygiene, Russian Academy of Medical Sciences, Pogodinskaya st., 10, Moscow, 119833, Russia

Constructive and interior decorative materials mutagenicity by inhalation and oral exposure was studied in Dominant Lethal Test (DLT) and Somatic Mutation and Recombination Test (SMART) of Drosophila melanogaster.

Experimental chamber with volatile polymeric materials was used for vapour exposure (DLT and SMART). 48 chemicals were identified within chamber (high concentrations of acetaldehyde, carbon sulfide, vinyl acetate, acetophenone, formaldehyde were detected).

Non-volatile constructive materials were used for oral treatment (SMART). Water suspensions of concrete and galvanic production scraps different ratio were analyzed.

DLT. Males of D-32 wild type stock were treated (72 h) with vapours, mated in mass to the same stock virgin females. Females were placed into chambers with replaceable bottom exchanged 4 times every 12 h. For the first time eggs were calculated immediately after exchange, for the second time - after 48 h, 250C incubation. Changed colour eggs were registered as late embryonal lethals and interpreted as male dominant lethal mutations.

SMART. Larvae and eggs obtained from virgin yellow females and white, singed3 males mating (48 h) are treated by inhalation at experimental chamber and by feeding suspension of choice till imago stage. F1 females were analyzed. Head, thorax and scutellum yellow or singed macro- and microhairs and eye white facets are interpreted as somatic mutations or/and recombinations.

Results. Significant increase of male dominant lethal mutations frequency (4,86% of experimental vs. 1,11% of unexposed control) and mutation/recombination frequency (1,94% of experimental vs. 0,74% of unexposed control) from inhalation exposure are revealed. We detected four-day delay of adult flies’ hatch for exposed cultures.

Constructive materials oral exposure reduced females’ survival for 15-25%. Spectrum ratio alteration of mutations/recombinations was shown for cultures exposed to concrete suspension. But total number of these events did not differ from control. Thus no one constructive materials suspensions had mutagenic or/and recombinogenic properties in SMART.

DLT and SMART of Drosophila melanogaster appear to be useful for hygienic studies aimed to forecast of constructive materials genotoxic effects.

 

 

 

DOSE ASSESSMENT OF PAST ACCIDENTAL OR CHRONIC EXPOSURE USING FISH CHROMOSOME PAINTING

S. Salomaa, C. Lindholm

Radiation and Nuclear Safety Authority (STUK), P.O.Box 14, FIN-00881 Helsinki, Finland

FISH chromosome painting for scoring of stable translocations has become a relevant method in biodosimetry of past exposures of both acute and chronic type. The validation of this technique is, however, still in progress. To evaluate the practicality for retrospective biodosimetry, more information on the stability of translocations by time and the control level is needed. In a follow-up study of four accidentally exposed subjects, we have shown that translocation frequency remained relatively stable throughout the first four post-accidental years. A decline in translocation yields was observed in one subject exposed to both high-dose and partial-body irradiation that had lead to severe aplasia. However, the decrease was significant only for terminal (one-way) translocations.

In practice, however, retrospective biodosimetry is mainly applied to chronically exposed populations. By comparing the translocation frequency to a carefully matched control population, we concluded that average dose from the Chernobyl fallout to the residents of a heavily contaminated village in the Bryansk region of Russia was less than 100 mGy during the 7 first years after the accident. No increase in translocation frequencies was observed in Estonian Chernobyl liquidators who had an average recorded of 110 mSv. It was concluded that the recorded dose might overestimate their average bone marrow doses. In a material of 84 persons in which the effect of chronic radon exposure on chromosomal aberration yields was studied, no increase of translocations was obtained with increasing domestic radon concentration. Instead, the results showed a significant age-dependence and wide translocation range within an age group.

The large variability of translocation frequency and the relatively high control level as compared to dicentrics will influence the sensitivity of FISH biodosimetry and thus limit the applicability of the method. The data obtained from our studies supports the use of translocations for retrospective dosimetry of individual with high doses. At low doses and chronic exposures, the method is more applicable at a group level. Large numbers of cells are needed for reliable dose calculations and a careful matching of controls is required, especially with regard to age.

 

Collaborative epidemiological study of Chernobyl cleanup WORKERS FROM THE BALTIC COUNTRIES

Mare Tekkel1, Mati Rahu1, Toomas Veidebaum1, Anssi Auvinen2, Timo Hakulinen2, Peter D. Inskip3, John D.Boice Jr.4

 

1Department of Epidemiology and Biostatistics, Estonian Institute of Experimental and Clinical Medicine, Hiiu 42, 11619 Tallinn, Estonia, 2Finnish Cancer Registry, Liisankatu 21B, FIN-00170 Helsinki, Finland, 3Radiation Epidemiology Branch, National Cancer Institute, 6130 Executive Blvd., Executive Plaza North, Bethesda, MD 20852, USA, 4International Epidemiology Institute, 1550 Research Blvd., Rockville,

MD 20850, U.S.A

The project was to evaluate the feasibility of measuring cancer occurrence among the Chernobyl cleanup workers from the Baltic countries using record-linkage techniques, and to characterize the exposure of workers exposed between 1986-1990 during the cleanup activities of the contaminated facility and surrounding environment. Project started in December 1992 in Estonia and was expanded to Latvia, somewhat later to Lithuania. The cohort of cleanup workers from Estonia numbers 4,832, from Latvia - 6,019 and from Lithuania - 6,330 including persons from Ignalina Nuclear Power Station. Special questionnaires were completed by workers in all three countries to obtain detailed information on work history at Chernobyl, about some cancer risk factors and demographic information. The overall response rate was 81,4% in Estonia. Through 1997, completed questionnaires have been obtained for 59,9% cleanup workers in Latvia. Blood samples have been collected from 3,197 cleanup workers from Estonia, 2,274 from Latvia and 603 from Lithuania for GPA and/or FISH analyses. Biodosimetry results were similar among three counties. During the thyroid screening in Estonia 1,984 men were examined within a two-week period. Fine needle aspiration biopsy revealed two papillary carcinomas and three follicular neoplasms. There was no indication that thyroid nodularity was associated with any measure of radiation exposure. Linkages with the database of the Estonian Cancer Registry revealed no excess of cancer through 1993 (25 cancers observed versus 26 expected). Through 1995, there were still no leukemias or thyroid cancers among Estonian cleanup workers. In Latvia and Lithuania correspondingly 61 and 71 new cancer cases (seven cases of leukemia, three thyroid cancers in Latvia; five and three cases in Lithuania) were registered in the cohort in 1986-1996. Mortality linkages in Estonia as of 1993 revealed strikingly high numbers of death due to accidents, poisonings and suicides. A high proportion of deaths due to external causes also was observed in Latvia.

 

MULTIENDPOINT STUDY OF CYTOGENETIC DAMAGES IN PEOPLE SURVIVED CHERNOBYL ACCIDENT AND APPROACHES TO DOSE ESTIMATION

I.E. Vorobtsova

Central Research Institute of Roentgenology and Radiology, Leningradskaya St., 70/4. Pesochny- 2,

St. Petersburg, Russia

People exposed to ionizing radiation after Chernobyl accident may be at increased risk for cancer and other negative health effects. Due to uncertainty of the physical doses, biodosimetry became the method of choice for determination of exposure levels. Biodosimetry based on various genetic end points (multifactorial biodosimetry) might be more practical for prediction of health effects than physical dosimetry itself.

We studied several cohorts of people suffered from Chernobyl accident: liquidators, their children, people evacuated from radioactive areas and control population using several end points: unstable chromosome aberrations (CA), stable CA detected by FISH, micronuclei, in vitro radiosensivity of peripheral lymphocytes. Long term radiation genotoxicity was manifested as increased number of unstable and stable CA, micronuclei in lymphocytes and erythrocytes (liquidators, evacuated people), and as increased chromosomal radiosensivity of lymphocytes in vitro (children of liquidators, evacuated children). Based on the results obtained for each individual, group of people with the maximal values of end points studied was selected for further biomedical monitoring.

Analysis of CA in human lymphocytes is known to be the most powerful tool for biodosimetry. For this goal an in vitro calibration dose-effect curve is used as a rule. The assumption was made that lymphocytes exposed in vivo respond to irradiation in the same manner as do lymphocytes exposed in vitro. However, more precise dose estimations are possible if in vivo dose-response curve for CA would be available. We studied both in vitro and in vivo dose-effect relationships for chromosomal exchanges in lymphocytes of cancer patients received whole-body irradiation before local radiotherapy. Irradiation was performed at a single dose 10 cGy each day up to total dose of 50 cGy, and blood was sampled after each fraction. Blood sampled before whole-body exposure was irradiated in vitro at the same dose-range. Slides of metaphase lymphocytes prepared using routine protocol were stained with Giemsa or hybridized with DNA probes specific to 1, 4 and 8 (or 12) chromosomes using FISH technique. For translocations in vivo and in vitro dose-response did not differ significantly, but for dicentrics (both Giemsa and FISH) irradiation of lymphocytes in vitro was more effective than irradiation in vivo. Thus, if biodosimetry is performed by dicentrics using their in vitro dose-response as reference, the amount of radiation individual received could be underestimated at least in the case of protracted irradiation. It seems to be more reasonable to estimate absorbed dose by translocations, even in the case of early performed biodosimetry.

An important moment for biodosimetry based on stable CA is the knowledge of their baseline frequency. It was found that in control population stable CA accumulate with age in accordance with square model. Thus, for estimation of individual dose by translocations the age factor should be taken into account.

 

LESSONS FROM CYTOGENETIC STUDIES OF CHERNOBYL ACCIDENT VICTIMS

J.R. Lazutka

Department of Botany and Genetics, Vilnius University, 21 Ciurlionis St., 2009 Vilnius, Lithuania

Cytogenetic analyses of the Chernobyl accident victims usually have two goals: (a) to evaluate radiation dose received by exposed persons, and (b) to make a prognosis of malignant diseases among exposed persons.

There are many reports of increased frequency of chromosome aberrations in persons exposed to Chernobyl radiation. The cases include clean-up workers and residents of contaminated areas. Increased frequency of dicentric chromosomes in lymphocytes from clean-up workers was demonstrated in some very extensive studies, eg. In reports from Obninsk (Sevan’kaev et al., 1995) and Vilnius (Lazutka et al., 1997). As expected, it was demonstrated that the frequency of dicentric chromosomes and acentric fragments declined with increasing time intervals after the exposure. Increased frequency of chromosome-type aberrations was observed in residents of contaminated areas in Ukraine, Belorus, and Russia. It was noted that the frequency of aberratiosn increased with the time spent in contaminated area.

On the basis of cytogenetic findings, some attempts were made to reconstruct the dose received by Chernobyl clean-up workers. These dose reconstructions were in good agreement with available data of physical dosimetry as well as with the resultsobtained by other methods of dosimetry.

Although chromosomal events are often involved in formation of neoplasias, relationship between the rates oc chromosome damage and cancer risk is not very well established. According to fairly precise calculations, by now approximately 4,000 victims of the Chernobyl accident are examined cytogenetically. The most comprehensive cohorts are in Obninsk (Russia) – approx. 2,000 individuals, Kiev (Ukraine) – approx. 1,000 individuals, and in Vilnius (Lithuania) – approx. 500 individuals. Thus, there is quite good background to establish international study similar to that one established in the Nordic countries.

 

GENOTOXICAL EFFECTS OF LOW DOSES OF IONIZING RADIATION IN

CHERNOBYL CLEAN-UP WORKERS WHO WORKED AT THE STATION

DIFFERENT YEARS AFTER THE ACCIDENT

E. Neronova, N. Slozina, T. Kharchenko, A. Nikiforov

All Russian Centre of Emergency and Radiation Medicine, Emercom of Russia

194044 St.Petersburg, Lebedeva 4/2, Russia

359 clean-up workers were cytogenetically investigated in ARCERM EMERCOM of Russia from 1992 till 1998. Unstable chromosomal aberrations (single and double fragments, chromatid exchanges, dicentrics and rings chromosomes, and atypical chromosomes) in peripheral blood lymphocytes were studied. For all subjects questionnaires were filled in. Control group consisted of 51 persons of the same age and health status with no history of radiation exposure.

For the estimation of genotoxical effects of low doses of ionizing radiation we analyzed obtained results according to the year clean-up workers worked at the Chernobyl station. So all clean-up workers were divided into 4 groups: I (252 persons) – 1986, II (73) – 1987, III (29) - 1988 and IV (5) - 1989-90. Performed investigations revealed that the frequency of single and double fragments varied in different groups of clean-up workers and did not exceed control data some years. But despite of these variations the yield of cytogenetical markers - dicentrics and ring chromosomes was statistically higher in all groups of clean-up workers than in controls. The cytogenetical data were compared with the official doses picked out from the clean- up workers questionnaires. The doses were ranged: 20.4± 1.08 cGy in I group, 13.5± 1.73 cGy in II group, 4.87± 0.82 cGy in III and 8.4± 4.2 cGy in IV group. It is known that the official doses could not correspond to real physical doses, nevertheless performed analysis of correlation showed statistical dependence between official doses and the yield of dicentrics and ring chromosomes.

Therefore our results showed that different types of unstable chromosomal aberrations were found in Chernobyl clean-up workers. Some of aberrations like single fragments, chromatid exchanges can testify the action of different environmental factors or life-style factors. But dicentrics and rings suggested about genotoxical effect of ionizing radiation. This effect was found in all groups of clean-up workers who worked at the station different years after the accident and who suffered from wide range low doses of radiation and this effect still exists more than 10 years after exposure.

 

MICRONUCLEUS TEST IN PERIPHERAL BLOOD ERYTHROCYTES FOR GENOTOXICITY TESTING OF CHERNOBYL CLEAN-UP WORKERS AND CANCER PATIENTS

Zanda Auce

Faculty of Biology, University of Latvia, Kronvalda bulv. 4, LV-11842, Riga, Latvia

The micronucleus test for genotoxic damages has been used in many monitoring studies. Micronucleus in peripheral blood erythrocytes provides a simple and rapid method for detection of chromosomal damage by chemical and physical agents.

Our particular interest is in determining mutational changes in Chernobyl clean-up workers and cancer patients, and we consider the micronucleus assay to be potentially suitable for this purpose. We suggest that more comprehensive picture of morphofunctional changes in erythron system can be obtained by erythrocyte refractive index properties at different pH values together with scoring micronucleus index in peripheral blood erythrocytes.

From March 1998 to March 1999 blood samples from Chernobyl cleaning-up workers, cancer patients and control group were obtained. At the time of blood drawing for micronucleus test, a short personal history questionnaire was administrated. The questions dealt with factors thought to affect micronucleus level such as working site at the Chernobyl reactor, age, sex, smoking habits, medication, type of cancer, phase of therapy and work history.

Whole blood samples were obtained by venipuncture in preservative-free heparin. The blood was smeared on a clean glass slide, fixed with 96% ethanol for 5 min, air dried, hydrolysed in 1N Hl at 60° C in a water bath for 8 min and stained with basic fuchsin in acid alcohol for 30 min by a simplified alternative to the Schiff method (Horobin and Kevill-Davies, 1971). Unreacted stain was removed by rinsing with tap water.

Preparations were coded and observed blindly. The slides were screened at a magnification of x100 for regions of suitable technical quality, where the cells were well spread and undamaged. Total 2000 and 20 000 cells by two observers were counted at x100 magnification for each individual.

Preliminary work indicated that counts of 2000 cells per individual were too low due to the rarity of micronucleated erythrocytes, also erythrocytes are not homogeneously distributed on slides. We suggested that counting of 20 000 erythrocytes per individual provided more suitable statistical power.

Microscopically, micronuclei in peripheral blood erythrocytes were round in shape and had a strong red colour. Since they are the only coloured bodies in the uncoloured erythrocytes, they were easily seen.

The preliminary date obtained seems to be significant and will appear in near future.

OXIDATIVE STRESS AS POSSIBLE COMMON MECHANISM OF CHRONIC INJURY BY POLYCHLORINATED DIBENZO-p-DIOXINS/FURANS AND RADIATION

L.V.Khripach, J.A.Revazova, B.A.Revich

Research Institute of Human Ecology and Environmental Hygiene, Russian Academy of Medical Sciences, 119833, Pogodinskaya 10, Moscow, Russia

Literature and our own data permit to suppose that expressive signs of oxidative stress can be common features for chronic radiation and PCDD/PCDF damage. During recent observation of 45 exposed women tight positive correlation (up to r 0.75, p Ł 0.002 ) was revealed between induced by H2O2 blood plasma luminol-dependent chemiluminescence (CL) and different PCDD/PCDF congeners in plasma lipids. Even slightly more tight but negative correlation was obtained for saliva CL, which can be a sign of local neuthrophils depression. Levels of chromosomal aberrations (CA) in whole blood cultures of these persons were determined in Medico-Genetical Center by V.Platonova and L.Katosova. Statistical analysis of these data have revealed lack of direct correlation between CA and PCDD/PCDF blood burdens; in the row "relative control – colony near plant – herbicide plant" all CA types have increased insignificantly except for significant increasing of "fresh" chromosomal exchanges (Zhurkov et al., 1999, in press). Nevertheless sufficiently close relations can be revealed between cytogenetic damage and CL intensity. Total CA levels have increased proportionally to CL intensity for moderately exposed persons, while sharp fall of the curve was observed for highly exposed plant workers. Shape of CA distribution for the sub-cohort of plant workers looked like mirror variant of closed to chi-square distribution for relative controls; besides additive group of outliers with highest CL signals have appeared on its left flank - the result which can be also treated as an evidence that in this sub-cohort DNA oxidative damage appoints to its maximum and begins to drop. Plasma CL had negative correlation with predominant single plus pair fragments (r= - 0.41, pŁ 0.02), but positive one with chromosomal exchanges (r= 0.46, pŁ 0.009). Transient rise with subsequent decreasing of cytogenetic effect was described both for PCDD/PCDF (Umnova N.V. et al., 1997) and radiation exposure (Gevorkyan A.L., 1998). Delayed but inevitable macrophagal reaction can be a reason of such dynamics, since elevated flux of reactive oxygen species can damage all cell macromolecules in parallel, outer polysaccharide pattern including. Theoretically this scheme means that mutagenic factors with sufficiently high oxidative components in their mechanisms are in vivo mutagenic and auto – antimutagenic in the same time, giving possibility to elect cells having not only DNA breaks, but breaks+adducts also, and all this is the mirror of high evolutionary adaptation of aerobic organisms to permanent endogenic oxidative damage. Practically it can embarrass the revealing of corresponding cytogenetic gradients during biomonitoring, since all investigators strive for including in observation the most exposed persons, which have already falling signs of cytogenetic damage. Another side of this hypothesis concludes in prediction of similar CA patterns for radiation and for any other mutagenic factor with high oxidative component, since more oxidatively unstable blood cells with unrepaired breaks will be eliminated but minor more stable fraction (possibly monocytes) can accumulate, having erroneously repaired breaks as exchanges.

 

DOSE-RESPONSE OF STABLE CHROMOSOME ABERRATIONS DETECTED BY FISH IN HUMAN LYMPHOCYTES IRRADIATED IN VIVO AND IN VITRO

A. Kanayeva1, I. Vorobtsova1, N. Timofeyeva1, A. Semyonov1, F. Darroudi2,

A.T. Natarajan2

1Central Research Institute of Roentgenology and radiology, Leningradskaya St., 70/4. Pesochni-2, 189646 St. Petersburg, Russia, and 2MGC Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, Wassenaarseweg 72, P.O. Box 9503, 2300 RA Leiden, The Netherlands

To define the exposure and evaluate the radiation dose by chromosome exchanges, their in vitro dose-response curve is used as reference. Several studies are available on an in vivo dose-response for unstable chromosome aberrations (dicentrics). As what about stable chromosome aberrations detected by "chromosome painting", their in vivo dose response has not been studied up till now. For the retrospective biological dosimetry such studies are extraordinary important. Analysis of translocations is useful for determination of previous chronic exposure, because in contrast to dicentrics they do not eliminate from blood in time.

The study was performed on the 4 cancer patients. Whole body irradiation of patients (in vivo irradiation of lymphocytes) was carried out with Co60 gamma-rays unit at a single dose 10 cGy each day up to total dose of 50 cGy. In vitro irradiation of blood collected before whole body exposure was performed with the same gamma-rays source at doses of 0, 8, 16, 24, 32, and 40 cGy. Slides from lymphocyte cultures were prepared using routine methods. FISH was carried out using 1, 4 and 8 chromosome specific DNA probes as well as pancentromeric probe. For some patients all chromosomes were labeled with FITC, pancentromeric probe – with Texas-Red. For other patients chromosome 1 was biotinilated and FITC labeled, chromosome 4 – biotinilated and labeled with Texas-Red, chromosome 8 and pancentromeric probe – FITC labeled. For each donor, about 1500-4500 first mitotic – division cells were scored per dose point on coded slides. All types of chromosome aberrations were registered, but the data are presented for dicentrics and translocations, both terminal and reciprocal. There is an essential inter-individual variability in the base level of translocations. The yield of dicentrics significantly faster increases with dose in lymphocytes irradiated in vitro as compared to those irradiated in vivo. No difference between in vivo and in vitro dose-response curves is observed for translocations. The frequency of translocations grows significantly more rapid then the frequency of dicentrics. Since for dose reconstruction the individual frequency of dicentrics is usually referred to the in vitro calibration dose-response curve, the real amount of absorbed radiation in case of protracted exposure is likely to be underestimated. As our data show, for more correct estimation of radiation dose the individual frequency of dicentrics should be referred to the in vivo calibration dose-response curve. In contrast, translocations being independent on the regimen of irradiation seem to be more adequate end point for both early and retrospective biodosimetry using the in vitro dose-response calibration curve. Inconsistent results have been previously reported regarding the ratio of translocations to dicentrics measured using chromosome painting. Our data provide evidence on the higher frequency of translocations as compared to dicentrics at the dose-range studied.

 

RADIOADAPTIVE RESPONSE IN HUMAN AND IT'S MODIFICATION

L.V Tskhovrebova

Institute of General Genetics, Russian Academy of Sciences, 3 Gubkin St., Moscow, Russia

Presented work is the effort to research and explain some aspects of radioadaptive response in human. In this study, we performed assays in five different cultures of cells derived from healthy donors, patients with homocystinuria, Marfan syndrome, Ehlers-Danlos syndrome and schizophrenia. Given disorders were described earlier as deficient in DNA repair (Zasukhina et al, 1982; Sinelschikova et al, 1987). Present study was performed to achieve a few practical goals:

- To analyze the frequency and spectrum of structural chromosome mutations induced by gamma-quants and 8-MOP in lymphocytes of healthy donors and patients with different hereditary disorders;

- To induce the radioadaptive response in all named cells by applying consequently low and high doses of gamma-radiation followed by analysis of chromosome aberrations;

- To analyze the effect of interferon pretreatment on chromosome aberration induction by both given mutagens;

- To perform comparative analysis of radioadaptive response and protective effect of interferon using chromosome aberration criterion;

- To study features of radioadaptive response in persons living in the areas of higher radioactivity - Chernobyl's catastrophe contamination areas.

As a result of these assays we have found:

1. There is no correlation between DNA repair efficiency and presence or absence of radioadaptive response. Seemingly, radioadaptive response depends on other processes than DNA repair. It could be very well connected with the process of formation of primary DNA lesions, or some processes that precede primary DNA damage.

2. Comparison of radioadaptive response and protective effects of interferon has shown that in some part the "machinery" of these two could be the same: on the one hand, it was demonstrated earlier that interferon has non-reparative component in its ability to defend cells against 8-MOP, mechanism of this defense consists in prevention of transformation monoadducts into crosslinks (Yakubovskaya et al, 1993); on the other hand, the radioadaptivity induced by low-dose radiation has defensive effect similar to that one induced by interferon when cells were treated with 8-MOP instead of high dose of radiation.

3. In this work, for the first time, the features of radioadaptive response were investigated in the cells of patients with hereditary disorders mentioned above. We have found that radioadaptive response has been repressed completely in the cells of patients with homocystinuria.

 

STUDIES OF LITHUANIAN CHERNOBYL CLEAN-UP WORKERS

G.Rimdeika1, J.R. Lazutka2

1Lithuanian Chernobyl Medical Center, Sapiega Hospital, Antakalnio St. 17, 2055 Vilnius, Lithuania

2Department of Botany and Genetics, Vilnius University, 21 Ciurlionis St., 2009 Vilnius, Lithuania

The cohort of Chernobyl clean-up workers from Lithuania consists of 7152 Lithuanian residents. 5446 persons have been registered at the Chernobyl Medical Center by April 1996. 5211 clean-up workers are on active follow-up. At arrival, the most common age group of clean-up workers was 30-34 years, the average dose received estimated to be 114 mSv. The radiation dose is recorded only for 69% of clean-up workers. The highest doses were received in 1986 (mean 200 mSv).

Several genetical studies were carried out at Lithuanian Chernobyl Medical Center in collaboration with Vilnius University and some Lithuanian and foreign research institutions. Five hundred and nine Chernobyl clean-up workers were examined cytogenetically between 1991 and 1994. The main conclusion of the study was that even up to 8 years after the irradiation, cytogenetic effects in lymphocytes of Chernobyl clean-up workers were still observable. However, increased frequencies of chromosomal damage were characteristic for 35% of clean-up workers only. The rest part of examined clean-up workers had frequency of chromosomal aberrations not different from the frequency in control individuals. It must be noted, however, that both groups of clean-up workers had almost identical distribution of recorded doses of exposure to ionizing radiation.

Lithuanian Chernobyl Medical Center also participated in collaborative epidemiological study of Chernobyl clean-up workers from the Baltic countries. In the frame of this epidemiological study, retrospective dose assessment using GPA mutations analysis or stable chromosome aberration analysis by means of FISH were performed on blood samples from 603 Lithuanian Chernobyl clean-up workers. The main conclusion of this study was that Chernobyl clean-up workers received the dose of ionizing radiation that do not significantly differ from the records of their estimated doses.

MOLECULAR EPIDEMIOLOGY - NEW DEVELOPMENTS AND OLD RESTRAINTS

Harri Vainio

Karolinska Institutet, P.O.Box 210, S-1717 77 Stockholm, Sweden

Molecular epidemiology has emerged as a natural outgrowth of the attempts to apply information derived from the explosion in molecular biology to disease in human populations. The incorporation of biomarkers into classical epidemiological designs holds the promise of unraveling mechanisms, elucidating gene-environment interactions, and dissecting the heterogeneity of disease. The primary interest of molecular epidemiology is in the identification of factors in the physical and social environment that affect risk for disease and which are amenable to preventive intervention. The explosion in molecular technology has not, so far, resulted in widespread improvements in epidemiological results, and that has led to a sense of frustration in the public health community. As experience accumulates, there is a new appreciation that attention to study design, infrastructure, and biomarker validation can improve the results. Increased knowledge on molecular genetics can help us better define the molecular mechanisms underlying human health and disease, and subdivide diseases that are clinically indistinguishable into molecularly-distinct forms thereby improving the physician’s ability to choose rational treatments or preventative measures. The advances in genomics research will help us identify genotypic or other molecular markers that predict therapeutic response; identify new therapeutic targets; reveal pathogenic organisms; and fractionate the population into individuals with more or less risk of specific disease, allowing preventative measures to be used judiciously. With the advances in the development of tools to identify susceptible subgroups of people, however, comes the responsibility of devising strategies to anticipate the ethical and legal implications of these tools. Occupational and environmental health can reap the benefits of advances in molecular biology and genetics, but it will take a concerted and cautious effort.

 

P53 mutations as a biomarker in molecular epidemiology studies

Kirsti Husgafvel-Pursiainen

Finnish Institute of Occupational Health, FIN-00250 Helsinki, Finland

Molecular biomarkers used in research on environmental cancer are multiple. These include markers of exposure, susceptibility and effect. For example, molecular dose of a certain carcinogen (or a group of carcinogens) can be estimated by measuring e.g. DNA–adducts. Similarly, frequency and type of mutations in genes controlling cell growth and differentiation are being used as molecular ‘fingerprints’ of different exposures.

Mutations of the tumor suppressor gene p53, which encodes a transcription factor controlling cellular response to DNA damage, represent a potentially useful biomarker in search for etiology, molecular mechanisms, and, hopefully, prevention of environmental cancers. P53 mutations are particularly common in cancers related to exposure to genotoxic agents, such as UV-induced skin cancer, hepatocellular carcinoma associated with dietary exposure to the mycotoxin aflatoxin B1, and lung cancer in smokers. In human lung cancer, mutations of the p53 gene occur in about 50% of tumours, and they appear to exhibit typical features. In addition, many studies suggest that p53 mutations may be associated with poor prognosis, especially in non-small cell lung cancer. Not only smoking but also exposure of non-smokers to environmental tobacco smoke (ETS) constitutes a health risk; epidemiological studies have suggested a causal association between lung cancer risk and exposure to environmental tobacco smoke in lifetime non-smokers. Extensive data on the biological effects of tobacco smoking have allowed use of biomarkers also in studies on ETS exposure. P53 mutations as a biomarker of tobacco-related lung carcinogenesis in smokers and non-smokers will be discussed.

 

Correlation between cytogenetic and molecular markers of individual susceptibility to chemical mutagens.

Maria Sasiadek, Kamila Schlade-Bartusiak, Leszek Noga

Genetic Department, Wroclaw Medical University, Marcinkowskiego 1, 50-368 Wroclaw, Poland

Although the whole population is exposed to environmental mutagens, only some individuals within it develop cancer. Variations in individual sensitivity to genotoxic effects of carcinogenic compounds justify this fact. Susceptibility to such chemicals can be associated with individual differences in carcinogen metabolism and/or the ability to repair DNA lesions caused by a particular agent. Biotransformation, involving metabolic activation and detoxification pathways, is considered to be essentially responsible for variations in susceptibility to xenobiotics. Genetically conditioned polymorphic variants of the enzymes playing crucial functions in the above mentioned pathways may reduce or increase the efficiency of carcinogen metabolism. Then the individual sensitivity to particular chemical mutagens can be established by studying the influence of genetic polymorphisms on genotoxic response of human cells in in vitro experiments. The most useful assays in analysing genotoxic response are cytogenetic tests because they can be applied to any mutagen known to produce chromosomal alterations.

The aim of our study was to analyse the correlation between cytogenetic and molecular markers of individual sensitivity to diepoxybutane (DEB). The study referred to correlation between the results of cytogenetic tests for chromosomal aberrations (CA) – measured by classical and molecular cytogenetics (FISH), sister chromatid exchanges (SCEs), and molecular markers: polymorphisms of epoxide-detoxifying enzymes – glutathione S-transferases M and T (GSTM1 and GSTT1). The results revealed that GSTT1 null genotype correlated with a significant increase in SCE frequency. The GSTM1 null genotype did not influence the SCE frequency and therefore could not be applied as a marker of individual sensitivity to DEB. There was also neither any correlation between the individual sensitivity to DEB characterised by high SCE frequency and CA induction, nor between the individual level of CA induction and GSTT1 genotypes.

 

GENOTYPE CORRELATED TO CYTOGENETIC DAMAGE IN TWO CASE-CONTROL STUDIES OF OCCUPATIONALLY EXPOSED WORKERS.

Marit Thorsen, Elin H. Kure, Inger-Lise Hansteen

Department of Occupational and Environmental Medicine, Telemark Central Hospital, 3710 Skien, Norway.

Ten maintenance workers in an ethylene plant monitored for benzene and 1,3-butadiene exposure were compared to maintenance workers from a chloralkali production plant matched for sex, age and smoking. In the other study, 30 oil exposed workers from a cable manufacturing company exposed to naphtenic oils of both low and high viscosity were compared to male office workers.

Cytogenetic damage was studied in conventional whole blood cultures, in cultures inhibited for DNA synthesis and repair the last three hours in culture, and as SCE's.

Susceptibility genes studied were GSTm 1, GSTq 1, GSTp 1, mEH (exon 3 and 4), CYP1A1 (m1 and m2), and CYP2E1( Rsa I and Dra I polymorphism).

The genotype distribution of polymorphisms for the 80 persons studied, and for the two groups viewed separately, is in accordance with frequencies observed in the general population, as expected. The mean number of chromatid breaks and of cells with aberrations (CA) scored in the conventional cultures, was related to genotype. No statistical differences could be observed between the genotypes for either parameter. The results of CA were trichotomized on the basis of the absolute values as "low" (1-33 percentile), "medium" (34-66 percentile) and "high" (67-100 percentile). A "high" level of CA is associated with cancer development. Combining this high frequency with susceptibility genotypes, a trend with higher percentage of persons with the polymorphic GSTp 1 genotype was observed even if it was not statistically significant due to small numbers.

For the 20 maintenance workers the inhibited cultures showed significant increases in chromatid breaks and in CA (p<0.05) for the GSTp 1 heterozygotes compared to homozygote wildtype individuals. For mEH exon 3 significant increases for the same cytogenetic endpoints were found for homozygote wildtype individuals compared to the heterozygotes. For smokers a non-significant increase in CA for the GSTm 1 null-genotypes was observed. This trend was not seen for non-smokers. For mEH exon 3 homozygote wildtype smokers had an increase in CA which was not observed for non-smokers.

SCE was studied for the 60 oil exposed workers and controls. No difference in SCE frequency could be found for any polymorphisms of the studied genotypes.

 

ANALYSIS OF MICRONUCLEI IN BLOOD LYMPHOCYTES OF PESTICIDE-EXPOSED GREENHOUSE WORKERS WITH THE ANTIBROMODEOXYURIDINE METHOD

Ghita C.-M. Falck1, Ari Hirvonen1, Roberto Scarpato2, Sirkku T. Saarikoski1,

Lucia Migliore2, Hannu Norppa1

1Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, Topeliuksenkatu 41 a A, FIN-00250, Helsinki, Finland, 2Dipartimento di Scienze dell’Ambiente e del Territorio, Universitŕ di Pisa, Via San Giuseppe 22, 56100 Pisa, Italy

The frequency of micronuclei (MN) in cultured peripheral lymphocytes was used as a biomarker of genotoxic effects in 34 pesticide-exposed greenhouse workers (14 women, 20 men) and 33 unexposed referents (16 women, 17 men) matched with the exposed workers for age and smoking habits. To restrict the analysis primarily to cells that have divided once in vitro, MN were scored only in cells showing label after a 42-h incubation with bromodeoxyuridine (BrdU), as detected by immunofluorescence (anti-BrdU technique). Two different concentrations of BrdU (0.5 and 1 m g/ml) were compared. Individual frequencies of micronucleated cells (MNCs) obtained with the two concentrations of BrdU significantly correlated with each other (r=0.55, P<0.001). Higher mean MNCs frequencies (per 1000 cells) were detected among exposed smokers (9.0 at 0.5 m g/ml BrdU and 7.8 at 1 m g/ml BrdU) than in smoking referents (6.3 and 5.9, respectively). In multiple regression analysis controlling for age, sex, smoking, and genotypes, a significant elevation of MNC frequency (P=0.004 at 1 m g/ml BrdU; P=0.052 at 0.5 m g/ml BrdU) was observed in greenhouse workers with a work history of extensive pesticide spraying (n=17). In the present study the possible influence of the genetic polymorphisms of xenobiotic metabolizing enzymes glutathione S-transferase M1 (GSTM1), T1 (GSTT1), and N-acetyltransferase 2 (NAT2) was also evaluated. Increased MNC frequencies were associated with ageing at 0.5 m g/ml BrdU, with the GSTM1-positive genotype at both 1 (P=0.028) and 0.5 (P=0.056) m g/ml BrdU in all subjects, and with the NAT2 fast acetylator genotype in smokers at 0.5 m g/ml BrdU (P=0.043). The results indicate that MN rates are increased in greenhouse workers, especially in those involved in pesticide spraying. The GSTM1 positive and NAT2 fast genotypes appear to be associated with elevated MNC frequencies, which contradicts with earlier results on elevated chromosomal aberration rates in GSTM1 null smokers and NAT2 slow subjects.

 

FLUORESCENCE IN SITU HYBRIDIZATION (FISH) IN GENETIC TOXICOLOGY

A. T. Natarajan

Department of Genetics, Radiation Genetics and Chemical Mutagenesis, Medical Genetics Centre South-West Netherlands – MGC, Leiden University, Wassenaarseweg 72, P.O. Box 9503, 2300 RA Leiden, The Netherlands

Fluorescence in situ hybridization (FISH) using DNA libraries has revolutionized the field of cytogenetics. Whole chromosome specific DNA libraries, chromosome arm specific probes, centromeric, telomeric as well as other region specific probes have become available, which increase the resolution of this technique. FISH technique has extensively been used in assessing radiation induced chromosome aberrations in human and rodents. New insights into the mechanisms of formation of aberrations have emerged, among which the observed heterogeneity between chromosomes and chromosome regions. Centromere specific probes have been employed to study aneuploidy induced by chemical agents with success. In combination with cytokinesis-block method, not only the nature of induced micronuclei (arising from fragments or whole chromosomes), but also mal-segregation of specific chromosomes can be monitored. The technique has been extended to male germ cells to study chemically induced aneuploidy in mouse, rat and human FISH technique has also been used in biomonitoring studies of human populations exposed to chemicals and ionizing radiation. One of the important achievements is the use of FISH technique in detecting stable translocations in radiation accident victims, the frequencies of which can be used for retrospective dosimetry of absorbed radiation dose.

 

MOLECULAR ANALYSIS OF SOMATIC IN VIVO MUTATIONS IN HUMANS: MUTATIONAL SPECTRA AT THE HPRT LOCUS

Bo Lambert, Andrej Podlutsky, Sai-Mei Hou, Fredrik Nyberg, Göran Pershagen

Karolinska Institutet, Departments of Biosciences at Novum and Institute of Environmental Medicine, SE-141 57 Huddinge, Sweden

Somatic mutagenesis is a critical step in carcinogenesis, and is likely to be involved in the biology of aging. Several partly overlapping processes are known to contribute to different types of somatic mutation, e.g. spontaneous degradation of DNA, random mistakes of DNA replication and repair enzymes, and DNA damage induced by environmental exposures. Since the knowledge about factors contributing to somatic in vivo mutation in human cells is very limited, we have used the T-cell clonal assay to study the mutation frequency (MF) and molecular spectrum of mutation at the hypoxanthine guanine phosphoribosyltransferase (hprt) locus in peripheral blood samples of healthy adults. Two major factors are associated with an increase of the hprt MF, donor age and smoking. In adult populations, the hprt MF increases with about 1- 3 % from one year of age to the next, and in smokers (S), the hprt MF is approximately 50% higher than in non-smokers (NS). The age-related increase of hprt MF seems to be steeper among S than among NS. In an attempt to elucidate the mechanisms involved in age- and smoking related hprt mutagenesis, we have studied the mutational spectra in S and NS, and in young and old individuals. Types and frequencies of in vivo mutation in the hypoxanthine phosphoribosyltransferase (hprt) gene was studied in 142 T-cell mutants from 78 healthy non-smoking and smoking adults with a mean of 65 years, and hprt MF of 18.7±12.0x10-6 and 26.6±18.5x10-6 (P<0.001), respectively. GC>AT transitions were the predominant type of mutation, with 50% of all single base pair substitutions (SBS). The mutations showed a non-random distribution along the coding sequence, with three significant hotspots. There was no difference between S and NS with regard to the distribution of mutations at these hotspots. However, 85% of the mutations at GC base pairs and 88% of the mutations at AT base pairs in S occurred at sites with guanine or thymine respectively, in the non-transcribed DNA strand. Moreover, S had a higher frequency of transversions and lower frequency of transitions than NS. Particularly GC>TA transversions were increased in S (11%) compared to NS (2%), which suggests that tobacco-smoke induced adducts at guanine bases in the non-transcribed DNA strand contributes to the increase of hprt mutation in S. Overall, these results were very similar to the mutational spectra in a younger study populations reported previously (Podlutsky et al., Carcinogenesis 19, 1998, 557). With the possible exception of an increase of mutations at hotspot position 143, and a decrease of 5-methylcytosine deamination mediated transitions at CpG-sites in the older individuals, there were no differences between the mutational spectra of old and young adults. In conclusion, both smoking and aging seem to have minor influences on the spectrum of hprt mutation in T-cells.

 

SPECTRUM OF DNA ADDUCTS INDUCED BY STYRENE: PROSPECTS IN HUMAN BIOMONITORING

Pavel Vodicka1, Mikko Koskinen2, Kari Hemminki2

1Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Vídenská 1083, 14220 Prague 4, Czech Republic

2Department of Biosciences at NOVUM, Karolinska Institute, Hälsovägen 7, 14157 Huddinge, Sweden

Styrene, high volume and important industrial chemical, is metabolized by monooxigenase system to styrene oxide (SO). SO attacks nucleophilic centers of biological macromolecules, including proteins and DNA.

Alkylation products arising from the treatment of DNA with SO have been characterized. 93% of the total alkylation takes place at the N-7 position guanine residues, either through a or b carbon of SO. As a novel finding was the detection of N-3 adenine adduct, accounting for 4% of the total alkylation. Both 7-alkylguanine and N-3 adenine adducts are labile, being prone to spontaneous depurination. The most abundant stable adduct is N2-guanine (1.5%). Interestingly, we have also identified N-1 adenine adduct, although the relative proportion is not feasible due to the tendencies of this adduct to either deamination or Dimroth rearrangement to corresponding N6-adenine adducts. The relative proportion of N6-adenine adducts (formed directly or via Dimroth rearrangement) was approximately 1%, an additional product of the N-1 adenine conversion, N-1 hypoxanthine, accounts for 0.1%.

DNA adducts are believed to play important role in the cascade of genotoxic effects. In cell culture system the close association of SO-7-alkylguanine adducts to single strand breaks in DNA has been proven. In cultured human lymphocytes SO induces HPRT mutations at A-T nucleotide pairs (GC-TA, AT-TA base substitutions). These base substitutions could not be related to the O6-guanine adducts, previously detected in styrene-exposed population. It may be that very labile N-7 guanine and N-3 adenine adducts are depurinated and adenine is inserted opposite to apurinic site in the course of replicative by-pass.

Our in vitro studies suggest that N-7 guanine, N-3 adenine, N6-adenine and N2-guanine adducts should be the target for in vivo experiments. These adducts are hot candidates in human biomonitoring.

The role of styrene-induced DNA adducts and their relationship with other parameters of genotoxic effects are discussed. The design of human biomonitoring study is proposed with the aims to understand the complex mechanism of genotoxicity and individual susceptibility.

[This study was supported by the grant GACR 313/99/1460 and by Swedish Work-Environment Fund]

 

OXIDATIVE DNA BASE MODIFICATIONS IN LARYNX CANCER

Pawel Jaloszynski1, Pawel Jaruga2, Ryszard Olinski2, Krzysztof Szyfter1

1Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60-479 Poznan, Poland

2Department of Clinical Biochemistry, Medical School, Karlowicza 24, 85-094 Bydgoszcz, Poland

Tobacco smoking is a major causative factor for larynx cancer. Taking into account a relatively high content of reactive oxygen species in tobacco smoke one may expect, that mutagenesis associated with oxidative DNA base modifications plays a role in development of tobacco-related cancers. Quantitative characterisation of oxidised DNA bases in cancerous and non-cancerous larynx tissues is a step on the way of understanding of larynx cancer biology.

Using gas chromatography/mass spectrometry with selected-ion monitoring (GC/MS-SIM), DNA isolated from larynx tissues of larynx cancer patients was analysed. The levels of oxidised bases were compared in cancerous and non-cancerous, adjacent tissue. Samples of tissue were obtained from patients who underwent total or partial laryngectomy. All tissue samples were subjected to histopathological analysis, which allowed excluding samples with heavy inflammation. Finally, 10 samples of tumor tissue and 10 samples of relevant adjacent non-cancer larynx epithelium were selected. All tissue samples were obtained from males 36-75 years old (mean 60,3), smoking 20-30 cigarettes per day, tumor stages were T3N0M0 (4 patients), T3N1M0 (2), T3N2M0 (3), T4N1M0 (1).

DNA was isolated by proteinase K digestion under argon and four subsequent extractions with chloroform/isoamyl alk. mixture. After hydrolysis with formic acid DNA samples were derivatized with bis(trimethylsilyl)trifrluoroacetamide at room temperature. Stable isotope (13C, 15N) labeled bases were used as internal standards. Using GC/MS-SIM the following base oxidative modifications were analysed:

8-oxoguanine (8-OH-Gua),

8-oxoadenine (8-OH-Ade),

5-hydroxyuracyl (5-OH-Ura).

Considerable differences in the level of all modified bases in cancerous versus normal tissue were observed. The most distinct differences were present for purines modifications - elevated level of 8-OH-Ade in tumor samples was observed in all 10 cases, the level of 8-OH-Gua was elevated in 9 cases. The level of pyrimidine modification, 5-OH-Ura, was higher in tumor samples in 6 cases per 10.

The above results are preliminary data of the project that was designed in order to obtain a detailed information on the role of oxidative DNA damage in the biology of larynx cancer, in particular on the effect of tobacco smoke on the level of oxidative DNA base modifications in larynx tissues.

BASE AND NUCLEOTIDE EXCISION REPAIR IN HUMAN AND RAT GERM CELLS

Gunnar Brunborg, Ann-Karin Olsen

Department of Environmental Medicine, National Institute of Public Health, P.O. Box 4404 Torshov, N-0403 Oslo, Norway

In all cells, most probably also in germ cells, normal metabolic activities introduce a high number DNA lesions such as abasic (AP) sites, oxidative DNA lesions and alkylation damage. Such damage is mainly processed through the base excision repair (BER) mechanism correcting small base modifications and AP-sites. Environmental factors (ionising and UV radiation, pesticides, pollutants, food and food contaminants) are responsible for other lesions mostly repaired by the nucleotide excision repair system (NER).

We have investigated BER and NER processes in germ cells from humans and rats, with emphasis on both the species differences and differences between germ and somatic cells. BER related DNA repair proteins were assayed in vitro by measuring the enzyme activities of cell extracts towards DNA substrates with defined lesions.

In this way DNA-glycosylases and AP-endonucleases were shown to be highly proficient in germ cells compared to somatic cells, in both rats and humans. DNA lesions such as methylated bases, hypoxanthine and 1,N6-ethenoadenine, all processed by the methylpurine DNA glycosylase, were excised more efficiently by germ cells than by somatic cells. The oxidative DNA lesion 8-hydroxyguanine (8oxoG) was not efficiently removed by any of the protein extracts, with germ cells possessing the highest activity. Formamidopyrimidine (FaPyG) is known to be incised in situ by both the human 8-oxoguanine glycosylase 1 (hOGG1) and the endonuclease II homologue 1 (hNTH1). In our assay FaPyG was incised by the germ cell extracts more efficiently than by the somatic cell extracts. Uracil and AP-sites were excised at least as efficiently by germ compared to somatic cell extracts, from both rats and humans. The presence of some of the endogenous enzymes was confirmed by western analysis using specific antibodies.

Although the enzyme activities were present in the cell extracts, their functionality may be questioned in living cells. To address this, we utilised the alkaline single cell gel electrophoresis (Comet) assay, combined with appropriate purified DNA-glycosylases and AP-endonucleases, to study BER in cells exposed to X-rays. The results indicate that BER is proficient in human and rat male germ cells.

In contrast to BER, the NER mechanism did not seem to be functional in male germ cells. The Comet assay was used in combination with the T4 endonuclease V (T4endoV) which incises next to cyclobutane pyrimidine dimers (CPD). CPDs induced by low doses of UV-C in germ cell suspensions, and CPDs induced by UV-B in germ cells within seminiferous tubules, both failed to be incised.

In conclusion, the BER mechanism, correcting DNA lesions induced by normal metabolic activities, is proficient in male (rat and human) germ cells. On the other hand, the NER mechanism, repairing DNA lesions introduced by exogenous agents, appears to be non-functional.

This work was supported by The European Commission (ENV4-CT95-0204).

 

METABOLIC ENZYME POLYMORPHISMS AND CHROMOSOME DAMAGE IN HUMAN LYMPHOCYTES

H. Norppa, H. Järventaus, S. Bernardini, L. Knudsen1, S. Salomaa2, A. Hirvonen

Department of Industrial Hygiene and Toxicology, Finnish Institute of Occupational Health, FIN-00250 Helsinki, Finland; 1Danish National Institute of Occupational Health, DK-2100 Copenhagen, Denmark; 2Finnish Centre for Radiation and Nuclear Safety, FIN-00881 Helsinki, Finland

Recent epidemiological studies have shown that high frequencies of structural chromosomal aberrations (CAs) in peripheral lymphocytes are predictive of increased cancer risk. This association appears to exist regardless of the cause of the high CA level; it is not modified by smoking or occupational exposure to carcinogens and is also found in "unexposed" subjects. Besides genotoxic exposure, elevated CA rates may also indicate increased susceptibility which could derive from acquired factors - such as low levels of antioxidants, induction or inhibition of carcinogen-activating and detoxifying enzymes, and inhibition of DNA repair – or from inherited factors such as genotypes of enzymes involved in DNA repair and in the carcinogen metabolism.

Genetic polymorphisms of xenobiotic-metabolizing enzymes have lately received much interest. Many such polymorphisms are common in human populations and could thus influence the level of genotoxicity biomarkers. The recognition of polymorphisms that significantly affect cytogenetic parameters may facilitate the identification of risk groups and increase the sensitivity of cytogenetic assays. In evaluating genotype effects, it has to be borne in mind that, within an exposed population, there is individual variation also in the extent and nature of the exposure. Unless this variation can adequately be dealt with, it may be difficult to see any genotype effects. Correct identification of various exposure groups thus becomes essential. The GSTM1 gene, coding for the detoxification enzyme glutathione S-transferase M1, is homozygously deleted from about a half of Caucasians. In three independent studies, we have seen that individuals lacking the GSTM1 gene (null genotype) have an increased frequency of chromosomal aberrations in association with smoking or other exposure. A similar kind of deletion polymorphism also affects the GSTT1 gene (glutathione S-transferase T1), but in Caucasians the prevalence of the null genotype is relatively low (10-20%), which makes it often difficult to evaluate the influence of this polymorphism. Nevertheless, the GSTT1 null genotype appears to be associated with an elevated "baseline" rate of sister chromatid exchanges (SCEs) and possibly also chromosomal aberrations. Another important polymorphic phase II enzyme is N-acetyltransferase 2 (NAT2); the various genotypes are associated with either fast or slow acetylation capacity. In our studies, the NAT2 slow genotype was associated with an increased frequency of CAs, apparently in the absence of identified environmental exposure.

In vitro studies in cultures of human lymphocytes can be used to identify the influence of genetic polymorphisms on chromosome damage. In whole-blood lymphocyte cultures, especially erythrocytic GSTT1 activity appears to be important, and several genotoxins, including diepoxybutane, monoepoxybutene, styrene oxide, and styrene, have been shown to induce more SCEs in cultured lymphocytes of GSTT1 null than GSTT1 positive individuals. GSTM1 genotype has been observed to affect SCE induction by monoepoxybutene and trans-stilbene oxide. Genotype differences between cell donors may actually explain some discrepancies in genotoxicity assay results between different laboratories. Cultured human cells are widely used in genotoxicity testing, but it is not well characterized which polymorphisms are expressed in such cultures to the extent that they can affect test results.

In conclusions, genetic polymorphisms of some xenobiotic-metabolizing enzymes appear to influence the level of chromosome damage observed in human lymphocytes after in vivo or in vitro genotoxic exposure. Other polymorphisms seem to affect the "baseline" level of chromosome damage, which may reflect modulation of the effects of unidentified genotoxic exposures or a role in natural metabolism.

 

THE SIMULTANEOUS INVESTIGATION OF ENVIRONMENTAL COMPLEX MUTAGENS AND HUMAN GENETIC HEALTH IN RUSSIA.

J. Revazova

Research Institute of Human Ecology & Environmental Hygiene,

Russian Academy of Medical Sciences, Pogodinskaya st., 10, Moscow, 119833, Russia

Complete and scientifically proved recommendations on estimation of real genetic danger from environmental genotoxicants are absent at the moment. For this reason, we are looking for the relationships between genetic effects in exposed people and total mutagenic activity of environmental pollutants. Biotesting of complex pollutants in air, water, wastes and other samples were carried out by classical genetic tests with Salmonella (Ames-test), Drosophila melanogaster (SMART and DLT) and mammals (chromosomal aberrations (CA) and micronuclei). CA and UDS were estimated in human blood cells from exposed people. In parallel, oxidative status and emotional stress level (as indicators of the individual sensitivity) of the same persons were investigated. Important component of our investigation was detection of accumulation of some genotoxicants (heavy metals, PCDD etc.) in human biosubstrates.

It was shown that: - Drosophila tests are very comfortable for detection of complex mixture mutagenicity in wastes, river water, etc.; - air samples must be prepared for genetic analyze using water and organic solvents; - single investigation of pollutant mutagenicity in environment is not enough for conclusion about presence or absence of genetic hazards. Results of our studies demonstrated the relationship between level of PCDD in human blood serum and aberrations of chromosomal type (dicentrics and ring chromosomes). It was shown that human oxidative status and emotional stress level might be used as indicators of the individual sensitivity to influence of genotoxicants. We have to note, that level of chromosomal aberrations in humans from some Russian regions at present is higher (up to 3- 4%) among control cohorts when compared to the similar data received ten years ago. So, relationship between high concentration of genotoxicants in environment and the level of genetic damage in human cells exists in most cases, particularly in conditions of occupational exposure.

 

CHROMOSOME ABERRATIONS IN TOBACCO SMOKE-ASSOCIATED LARYNGEAL CANCER

K.(Chris) Szyfter1, M. Kujawski1, P. Dabrowski2, M. Jarmuz1, S. Kita2

1Institute of Human Genetics, Polish Academy of Sciences, ul. Strzeszynska 32, 60-479 Poznan, Poland, and 2Clinics of Otolaryngology, K.Marcinkowski Medical University, ul. Przybyszewskiego 49, 60-355 Poznan, Poland

General features of cancer include genomic instability and dynamic changes of chromosome aberrations (CA) specific for a given type of cancer. Hence, understanding of clastogenicity can help to explain genetic predisposition, the process of carcino- and tumorigenicity as well as tumor staging.

The study concerned laryngeal cancer subjects at variable stages of the disease. The post-surgery biopsies were: laryngeal tumor, laryngeal non-tumor mucosa, adjacent lymph nodes with metastasis and peripheral blood lymphocytes (PBL).

PBL were analysed by the bleomycin test for hidden chromosome instability. The index of instability in the group of larynx cancer subjects (74 persons) significantly exceeded that in controls (30 persons). Inter-individual variability of results indicated an occurrence of genuine instability and over-instability only among cancer patient but not in controls. An analysis of results seems to indicate for (i) a lack of influence of tumor progression on chromosome instability in PBL and (ii) a link between chromosome instability and histological aggressiveness.

Supplementation of the bleomycin test with an identification of aberrations has proven a non-random distribution of CA. The most frequent CA found in the group of 14 patients were attributed to chromosomes 2, 6, 9, 12 and 14.

Another series of experiments was addressed to the question of male/female disproportional morbidity for larynx cancer. The presence of Y chromosome was studied in PBL analysed by classical karyotyping and tumour and non-tumor laryngeal biopsies by FISH with the use Y centromere-specific probe. Numerical aberrations of Y chromosome were detected only in tumor tissue.

Clastogenecity in the target tissue was studied was studied by comparative genome hybridisation (CGH). Chromosome imbalances were detected in majority of 38 laryngeal subjects. Gene copy amplification was most frequently found in chromosomes 3q, 8q and 9q, but losses at 18q, 3p and 4. At the stage of metastasis to the adjacent lymph nodes, losses of genetic material exceeded amplifications. The total number of CA in metastatic lymph nodes was higher than that in primary tumors.

The final target that is an application of CA analysis in laryngeal tumor staging requires an extension of the study for a larger group.

 

CHROMOSOME ABERRATIONS AND SISTER-CHROMATID EXCHANGES IN LITHUANIAN POPULATION: EFFECTS OF OCCUPATIONAL AND ENVIRONMENTAL EXPOSURES

J.R. Lazutka, R. Lekevicius, V. Dedonyte, L. Maciuleviciute-Gervers,

J. Mierauskiene, S. Rudaitiene, G. Slapšyte

Laboratory of Ecological Genetics, Department of Botany and Genetics, Vilnius University, 21 Ciurlionis St., 2009 Vilnius, Lithuania

Cytogenetic analysis of chromosome aberrations in 175,229 cells from 1,113 individuals, both unexposed and occupationally or environmentally exposed to heavy metals (mercury and lead), organic (styrene, formaldehyde, phenol and benzo[a]pyrene) and inorganic (sulfur and nitrogen oxides, hydrogen and ammonium fluorides) volatile substances and/or ionizing radiation was performed. In addition, 11,250 cells from 225 individuals were scored for the frequency of sister-chromatid exchanges (SCE).

Increased frequencies of chromosome aberrations were found in all occupationally exposed groups. A principal difference between the exposure to heavy metals and organic substances was found: increase in the chromosome aberration frequency was dependent on duration of exposure to mercury but not dependent on duration of exposure to styrene, formaldehyde and phenol. Higher chromosome aberration incidence was found in lymphocytes of children living in vicinity of a plant manufacturing phosphate fertilizers. This indicates that children are sensitive study group for the assessment of environmental exposure. However, the results of SCE analysis in these children were inconclusive.

Exposure to ionizing radiation was found to cause chromosome breaks and chromatid exchanges in Chernobyl clean-up workers and chromatid breaks, chromatid exchanges, dicentric chromosomes and chromosome translocations in workers from Ignalina Nuclear Power Plant. Increased frequency of chromatid exchanges in individuals exposed to ionizing radiation was quite unexpected. This may be attributed to the action of some unrecognized life-style or occupational factors, or to be a result of radiation-induced genomic instability. Also increased SCE frequency was found in lymphocytes of Chernobyl clean-up workers.

 

BIOLOGICAL AND GENOTOXIC MONITORING OF OCCUPATIONAL EXPOSURE TO COBALT-CONTAINING DUST

M. De Boeck1, S. Lardau2, M. Kirsch-Volders1, D. Lison2

1Vrije Universiteit Brussel, Laboratorium voor Cellulaire Genetica,Pleinlaan 2, 1050 Brussel, Belgium, and 2Université Catholique de Louvain, Unité de Toxicologie Industrielle et Médecine du Travail, Clos Chapelle-aux-Champs 30.54, 1200 Brussel, Belgium.

Occupational exposure to cobalt-containing dust has been associated with an increased risk of lung cancer when exposure was to a mixture of cobalt with tungsten carbide (hard metal industry), but not in cobalt refineries where workers were exposed to cobalt metal alone. In vitro studies have confirmed an increased genotoxic potential of cobalt metal dust when mixed with tungsten carbide particles.

To refine the risk assessment in these industrial settings at the current exposure levels, genotoxicity studies were conducted in the hard metal industry and in cobalt refineries. Different biological and genotoxicity parameters were evaluated in blood and urine of 2 x 40 exposed and 40 matched non-exposed workers : (1) exposure indexes: urinary cobalt, nickel, cotinine corrected for creatinine content, (2) effect parameters: lymphocyte micronucleus frequency, DNA strand breakage and oxidative DNA damage measured by the alkaline comet assay as well as urinary 8-hydroxydeoxyguanosine and (3) candidate susceptibility index: serum vitamin E. In addition, the donors were subjected to an extensive questionnaire on their occupational exposure, medical background, food, smoking and drinking habits.

The preliminary results from the genotoxic analysis conducted in cobalt refineries do not indicate significant genotoxic damage levels in exposed workers.

 

THE MICRONUCLEUS TEST: A SENSITIVE TOOL FOR HUMAN EFFECT MONITORING

Sebastian Kevekordes, Thomas Gebel, Hartmut Dunkelberg

Medical Institute of General Hygiene and Environmental Health, University of Göttingen, Windausweg 2, 37073 Göttingen, Germany

Aim. The aim of the study was to investigate a putatively elevated DNA damage in subjects possibly exposed to higher amounts of antineoplastic agents and to test the usefulness of the cytokinesis-block micronucleus assay concerning for human effect monitoring.

Methods. The level of genetic damage in peripheral mononuclear blood cells was determined using the sister chromatid exchange test, the alkaline elution technique, and the cytokinesis-block micronucleus test.

Results. The putatively elevated exposure of the study subjects was caused by a malfunction of a safety hood resulting in leakage of air during antineoplastic drug infusion preparation. Two months after a new safety hood was installed, the frequencies of micronuclei and sister chromatid exchanges of exposed nurses (n=10) were still significantly elevated when compared to a matched control group (p<0.01 and p<0.05 in the one-sided Wilcoxon-test, respectively). In a second examination seven months later, the frequency of micro-nuclei was significantly decreased to control values (p<0.05, one-sided Wilcoxon-test, n=6). When comparing the study subjects according to smoking status, it was observed that smokers (n=8) had significantly elevated frequencies of micronuclei and sister chromatid exchanges (p<0.01 and p<0.05 in the one-sided U-test, respectively). No differences in the rate of DNA damage could be detected with the alkaline elution technique suggesting the sensitivity of this method to be low.

Conclusions. The cytokinesis-block micronucleus test seems to be a rapid, sensitive and specific tool for the monitoring of biological effects in comparison to the classical and established sister chromatid exchange test and alkaline elution technique.

Control measures on the level of biological effect should be performed regularly to ensure maximum safety precautions for workers potentially exposed to genotoxic agents.

 

CHROMOSOME STUDIES OF PEDIATRIC PATIENTS ON LONG-TERM NITROFURANTOIN OR FURAGIN THERAPY FOR URINARY TRACT INFECTION

G. Slapšyte1, J.Mierauskiene1, A. Jankauskiene2

1Ecological Genetics Laboratory, Vilnius University, Ciurlionio 21, 2009 Vilnius, Lithuania, and 2Vilnius University Children’s Hospital, Vilnius, Lithuania

Nitrofurantoin and furagin are chemically and structurally similar 2-substituted 5-nitrofurans. Both of them possess excellent antibacterial activities and are widely used in the treatment of urinary tract infection (UTI). Earlier findings have indicated certain carcinogenic and genotoxic effects of nitrofurantoin. Little information exists on genetic effects of furagin. As both drugs are rather frequently used for a long-term low-dose antibacterial prophylaxis not only in adult patients but also in children, they have a certain priority for being tested for their genotoxic potential. Objectives of the present study were to estimate the prevalence of chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) in peripheral lymphocytes of pediatric patients on nitrofurantoin or furagin therapy. 89 patients were included into the study. Their ages ranged from 0.2 to 13, with a mean of 5.4 years. The mean blood urea and creatinine levels of all patients were within normal range. Cytogenetic analyses were performed prior to the start of therapy, after 1, 6 and 12 months of treatment. The treatment consisted of oral administration of drugs in doses of 3-5 mg/kg per day for the first 7 days of treatment, and in doses of 1-2 mg/kg per day for the rest days. No increase in the aberration rate over the level in controls (i.e., the same patients prior to the start of therapy plus patients with the same diagnosis) was observed after different steps of therapy in all patients under study. Statistical analysis did not show any significant differences between the mean SCE rates in nitrofurantoin-treated (maximum 7.22 SCE/cell frequency was determined after 6 month treatment) and controls (6.56 SCE/cell), indicating a lack of genotoxic potential of nitrofurantoin within the therapeutic dose range. Significantly increased frequencies of SCEs were determined in patients on furagin therapy, however no correlation was observed between the SCE rates and duration of therapy. Mean frequencies of SCEs after 1, 6 and 12 months of treatment were 8.24, 7.38 and 8.33 SCE/cell. The results of this study advocate cautious use of furagin in the management of UTI. Long-term low-dose nitrofurantoin therapy does not seem to have genotoxic effect as expressed by induction of SCEs and chromosome aberrations.

 

The sister chromatid exchanges in the last in vitro cell division measured in the third in vitro division cells of lymphocyte cultures Correlate with the level of chromosomal aberrations in persons Exposed to genotoxic pollutants

Tomasz Kretowicz1, Teresa Pawlak2, Janusz Limon2, Andrzej L. Pawlak3

1Department of Genetics, Gdansk University, 2Chair and Department of Human Genetics, Medical University Gdansk, and 3Institute of Human Genetics, Polish Academy of Sciences, Poznan, ul. Strzeszynska 32, 60-479 Poland

In groups of persons differing in the levels of exposure to industrial pollutants the frequencies of SCEs were assessed in peripheral lymphocyte cultures both in the second division metaphases and in the consecutive divisions (1+2-nd and the 3-rd) in the third division metaphases. Parallel tests for the frequency of chromosomal aberrations (CA) were performed in the same persons. Among the SCE-related parameters the SCE frequencies in the third division of the third division cells showed the highest correlation with the level of chromosomal aberrations in the studied persons. This finding draws attention because of the recent observation (Hagmar et al., Cancer Res. 1998, 58: 4117) that the increased frequency of CA is a marker of the higher risk of cancer.

The same SCE-related parameter proved to be sensitive to genotoxic effects of industrial pollutants with the increase in frequency of SCEs roughly equal to that of the SCEs frequency in the standard test in the second division metaphases. The increases in frequency in relation to the control group were by 39% and 42.6%, respectively.

The frequency of SCEs in the 10% of cells showing the highest levels (HFCSCEs) was increased by genotoxic exposure by 29.4% in the second division metaphases, whereas it increased only by 8% in comparison in the third division of the third division cells. Then the sensitivity of the third division in vitro SCEs in the detection of the effects of the high level and long lasting in vivo genotoxic exposure does not extend to the parameter of HFCSCEs. This last finding was related to the high frequency of HFCSCEs in the third division of the third division cells of the cultures of control group.

The number of cell divisions in vitro seems to be as an important determinant of the SCE frequency in cells subjected to SCE inducers in vivo, in particular when the HFCSCEs are considered. The assumed mechanism of the observed influence of the number of cell divisions in vitro on the sensitivity of the SCE-related parameters to the effects of genotoxic exposure in vivo may involve the different nature of DNA lesions involved in generation of the basal (background) vs. induced by genotoxic activity levels of SCEs .

 

A PROTECTIVE EFFECT OF VITAMINS C AND E AGAINST B(a)P-INDUCED GENOTOXICITY IN HUMAN LYMPHOCYTES STUDIED BY COMET ASSAY

Marzena Gajecka1, Maciej Kujawski1, Jan Gawecki2, Krzysztof (Chris) Szyfter1

1Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60-479 Poznan, Poland, 2Department of Human Nutrition and Food Hygiene, The University of Agriculture, Poznan, Poland

Polycyclic aromatic hydrocarbons (PAH) are considerably well characterised group of mutagens and carcinogens. Benzo(a)pyrene, the best known compound of the group, exerts its genotoxic activity after metabolic activation when is getting properties of electrophilic reagent capable to interact with DNA. Concurrently, reactive oxygen species (ROS) can emerge in the course of metabolic activation of PAH. Vitamins C and E because of antioxidant activity were claimed to act as antimutagenic agents. To study a potential protective effect of vitamin C and E (a -tocopherol) towards benzo(a)pyrene-induced DNA damage the in vitro protocol was designed.

The study material were peripheral blood lymphocytes derived from 19 and 4 healthy female volunteers (age: 22-25 years, non-smokers) in the experiments with vitamin C and in the experiments with vitamin E, respectively. Cells were exposed in vitro to 1 m M B(a)P in the presence of vitamin C at concentration 40 m M or 100 m M or, alternatively, 30 m M or 100 m M vitamin E. B(a)P-induced DNA damage and repair were estimated as generation and removal of single strand DNA breaks measured by the alkaline version of the single cell gel electrophoresis (comet assay).

A protective effect of vitamin C and E was demonstrated when vitamins were applied the same time with B(a)P or afterwards. The background level of DNA damage in the presence of vitamins was also lower than in the system without vitamins.

The results obtained in the experiments carried according to various protocols schemes of the vitamins treatment provide another evidence of anti-genotoxic activity of vitamins C and E. The activity of vitamins appears to be not connected with the steps of metabolic activation neither DNA repair. It seems, both vitamins act as competitors of DNA molecule in reactions with the reactive oxygen species.

 

CYTOGENETICAL DISTURBANCES LONG PERIOD AFTER ACCIDENTAL EXPOSURE TO LOW DOSES OF RADIATION.

N. Slozina , E. Neronova, T. Kharchenko, A. Nikiforov

All-Russian Center of Emergency and Radiation Medicine, Emercom of Russia

194044 Lebedeva 4/2, St.Petersburg, Russia

The main task of the laboratory of biodosimetry and clinical cytogenetics of ARCERM EMERCOM of Russia is the investigation of genomic disturbances of people who suffered from the radiation and other accidents in North-West region. The laboratory was organized in 1992. Our database contains information about more than 600 cytogenetically investigated persons. At this moment the most numerous groups are the groups of people who were irradiated some years ago. On the first place among them is the group of the clean-up workers of Chernobyl accident living mainly in North-West region. We performed conventional chromosomal analysis of 359 clean-up workers. During all period of observation the frequency of chromatid-type aberrations fluctuates like in our control group. The rate of dicentrics did not decrease with time: the frequency of dicentrics and rings chromosomes in 1992-93 years was 0.11± 0.06%, in 1994 - 0.24± 0.04%, in 1995 - 0.21± 0.04%, in 1996 - 0.39± 0.08%, in 1997 - 0.53± 0.14%, and in 1998 - 0.39± 0.09% that significantly (p<0.05-0.001) exceeds our control level (0.03± 0.02%). The dicentrics were found in more than 20% of clean-up workers. We compared different parameters reflected in questionnaires (like type and duration of work, life style after the accident and so on) between two groups of clean-up workers- "with dicentrics" and "without dicentrics" and revealed no differences. In most of cytogenetical analyses we analysed 200 metaphases. So we decided to enlarge the quantity of metaphases and in a group of 5 clean-up workers not less than 500 metaphases from every person were analyzed. The dicentrics were found in all clean-up workers. The frequency of dicentrics fluctuated from 0.2% to 1.0% in these persons. According to these data it is possible to suppose that dicentrics could be found in most of clean- up workers if the number of cells analyzed is large enough. Another cytogenetical finding in clean-up workers that seems to be interesting are chromatid exchanges that were not found in our control group. The frequency of this type of aberrations was significantly higher in smoking than in nonsmoking clean-up workers. So it is possible to suppose some specific reaction of clean-up workers to environmental mutagens. The use of micronucleus test with cytochalazin B did not reveal any differences between the clean-up workers and the control group. This method seems to be uninformative long period after radiation exposure. We have also investigated other groups of people who were accidentally irradiated some years ago, such as crewmembers of nuclear submarines and others. These groups were not numerous, but the frequency of dicentrics in all these groups exceeded the control level.

Thus the cytogenetical disturbances were found long period after accidental exposure to low doses of radiation. The rate of dicentrics in different groups of irradiated people was slightly but statistically significantly increased. Possible mechanisms of "stability" of unstable aberrations could be discussed.

 

GENETIC EFFECTS OF THE SKRUNDA RADIO LOCATION STATION

Isaak Rashal1, Zanda Auce2, Valdis Balodis2

1Institute of Biology, University of Latvia, Miera Str. 3, Salaspils, LV-2169, Latvia, and

2Faculty of Biology, University of Latvia, Kronvalda blvd. 4, Riga, LV-1842, Latvia

The Soviet Army operated the Skrunda Radio Location Station (RLS) during 1971-1998 for strategic defence against nuclear attack. The four stations of the Skrunda radar have irradiated natural ecosystems and humans with pulse electromagnetic radiation in a frequency range 156-162 MHz for over 25 years. About 2000 people were lived in front of the radar and were irradiated constantly. The particularity of the Skrunda RLS was a high power of short impulses: peak power of one antenna was 1.25 MW with duration of the peak 0.8 ms, nevertheless that mean power of one radar was only 50 kW. Biological effects of such high power and short impulse radiation were not tested before.

Complex investigations of biological effects of the Skrunda radar emissions were begun in 1990. Two methods were performed to test genetic effects. Metaphase chromosome assay of human lymphocytes was not successful for this purpose. Chromosome aberrations and aneuploid cells were used as indicators of genotoxicity. The frequency of abnormal cells in exposed and unexposed samples did not differ significantly (Nagle et al., 1998). The micronucleus test in peripheral erythrocytes of cows was the second method. In opposite, this assay could indicate genetic effect of the irradiation by the radar: the frequency of micronuclei in exposed group was significantly higher than in the control (Balode, 1996).

Data will be discussed in connection with other observations that indicated the presence of the biological effects of the radar on plants and humans. It was found that radiation exposure of the Skrunda RLS decreased of the incremental growth of pine trees (Balodis et al., 1996). It was shown also increased level of immunoglobulin A and decreased ability to produce interferons in exposed people (Bruvere et al., 1997). School children living in front of the Skrunda RLS had less developed motor and psychological function in comparison with unexposed children (Kolodinski and Kolodynska, 1996).

 

CYTOGENETICAL MONITORING OF DOMESTIC ANIMAL POPULATION UNDER THE CONDITIONS OF ENVIRONMENTAL POLLUTION

I. Miceikiene, K. Janušauskas

Laboratory of Animal Genetics, Lithuanian Veterinary Academy, Kaunas 3026, Lithuania

A large amount of chemical substances such as different fertilizers, industrial wastes emitted by a variety of factories, oil-fired power plants, smelters and industrial boilers contaminate large weather systems, water and soil. Near ecologically dangerous objects we can find not only common waste products but also specific substances that can be contributed to the class of very dangerous hazards having mutagenic and clastogenic effects.

All living organisms, including farm animals, may be exposed to numerous mutagens under such environmental conditions. Industrial effluents and waste products can cause immunopathology, respiratory, digestive diseases, cancer, lead to reproductive failure and malformations, and effect hereditary material - genome.

Different testing methods are used to evaluate the effect of environmental contamination to living organisms.

Our study focuses on the estimation of environmental chemical hazards to the heritable material and reproductive performance of cattle near two ecologically dangerous sources: Thermoelectric power station and Biochemical plant. In total, 260 cows were investigated cytogenetically and 2460 cows on reproductive data. Cows kept in polluted areas had statistically significant (P=0.0010 higher percentages of cells with chromosome aberrations (3.85± 0.67% and 6.25± 2.04%) in comparison with cows from control area (2.40± 0.16%), and higher frequencies of sister chromatid exchanges (7.21± 0.45 SCE/metaphase) as compared to control frequencies (4.50± 0.35 SCE/metaphase). The replication index (index of cytokinetics in cultured cells) is decreased in cows from contaminated environment (1.84± 0.31, P=0.024) in comparison with control (2.11± 0.13). Reproductive records tended to be worse from contaminated farm cows.

 

ETHICAL DIMENSIONS IN GENETIC TESTING

Marja Sorsa

Ministry of Education, Department for Education and Science Policy, Meritullinkatu 10, P.O.Box 293,

FIN-00171 Helsinki, Finland

The rapid expansion of molecular genetic techniques in different areas of biomedical, biotechnological and industrial production and research has brought along strong ethical concerns in different areas of the application, including human genetic testing.

The European Group on Ethics in Science and New Technologies, being a modified successor of the former Group of Advisors on Ethical Implications in Biotechnology is working on the issues as an advisory group nominated by the European Commission. The present group has 12 members representing multidisciplinary independent expertise, and its expanded mandate relates not only to modern biotechnology but also to other areas of science, including information technology. The EGE is presided by Ms. Noelle Lenoir (France). EGE has an inter-institutional position within the EU; in addition to its own initiative requests for its advisory ethical opinion may come from the European Commission, the Council of Ministers or from the European Parliament.

An opinion on Genetic testing/Human genomics banks is on the near future priority list of EGE, as mentioned in the EGE opinion No. 11 on Human Tissue Banking given on 21 July 1998. This future opinion relates also to the earlier (1996) opinion No. 6 on prenatal diagnostics from the group, as well as to the Bioconvention of the Council of Europe (1997), its Recommendations R(90)13 on genetic tests in prenatal diagnostics, R(92)3 on genetic testing and screening and R(97)5 on data protection of medical information. Genetic testing also relates to UNESCO’s Universal Declaration on the Human Genome and Human Rights (1997), the appropriate ILO and WHO recommendations as well as on existing national legislations on genetic testing already available in some countries.

The special features of genetic testing, in relation to other medical testing, are relevant also for the ethical analysis: (1) genetic test data is permanent and does not change during the person’s lifetime; (2) there may be a long latency between the analysis of the genetic test and manifestation of the disease outcome; (3) genetic testing, either population-based genetic screening or individual-based genetic testing, gives some information also of the relatives of the person tested.

The various uses and potential applications of genetic testing entail a variety of ethical questions, many of which focus on the ownership use of the genetic information as part of human integrity. The controversies between paternalism vs. autonomy of decision, protection of confidentiality or anonymity, prevention of discrimination and stigmatization, safety aspects in relation to testing as well as the scientific quality requirements are among the main ethical issues involved with genetic testing. Commercialization of genetic testing poses also specific ethical questions, which may be especially relevant for future possibilities to test employees or insurance applicants. These questions will be addressed in the future opinion of EGE.

 

NEW LEGISLATION ON CENTRALIZED HEALTH DATABASE IN ICELAND

Jorunn Erla Eyfjörd

Molecular and Cell Biology Research Laboratory, Icelandic Cancer Society, Skogarhlid 8, Box 5420, 125 Reykjavik, Iceland

New Icelandic legislation authorizing the establishment of a centralized nationwide health sector databank, with provisions to grant monopoly for its establishment and use to a biotechnology company, has caused a severe controversy and raised many ethical questions.

The original idea behind this came from deCode Genetics, a company founded and incorporated in Delaware USA in 1996. It is generally assumed that deCode will be granted this monopoly. The presentation of the bill to the public has been almost entirely in the hands of deCode, which has acquired the trust of the nation by presenting a very favourable picture of the potential benefits to Iceland and the world.

There has, however, been strong opposition in Iceland, initially from the medical and scientific community but this is now spreading.

The main concerns that have been raised are in the following areas and these will be discussed:

Consent and ethical review. The legislation allows only for people to opt out of the database but does not require any form of consent. No independent ethical or scientific review is required of applications, scientific or commercial, that the licensee may make of the data.

Confidentiality and privacy. Privacy will be protected by means of encryption of the data and by restricting the type of query that a subscriber can make. In the opinion of experts these measures, though useful, do not make the data anonymous.

Monopoly and scientific freedom. The licensee will acquire monopoly control over the data needed for genetics and medical research in Iceland. This will be a threat to the research of independent scientists.

 

ETHICAL ISSUES WITH FOCUS ON INDIVIDUALS PARTICIPATION IN BIOMONITORING STUDIES AND CLINICAL TRIALS

Lisbeth Ehlert Knudsen

Danish Medicines Agency, Frederiksundsvej 378, 2700 Brřnshřj, Denmark

The principles in recruiting study persons for biomonitoring studies and clinical trials rely on voluntariness and informed consent, while some access to health information is part of the medical treatment, pre-employment, occupational and insurance demands to the individuals.

In the review of research projects and clinical trials ethical issues play an important role The rationale of each project is to be considered to see whether the rules set in the Helsinki declaration and by WHO are met: Is new information obtained by the study detailed in the protocol and what are the benefits from this information.

Review of the ‘popular’ information given as background for the informed consent of each participating individual is important to avoid misinformation or lack of important information.

The principles of respect for autonomy, non-maleficence and beneficence should be followed:

- The individuals right to withdraw at any time of the study/trial without any consequences in further treatment.

- The individuals right to be informed – or not to be informed about study results.

- The responsibility of the investigator to prevent disease or disease progress by offering the most appropriate treatment or evoking immediate changes in e.g. hazardous working conditions.

- Confidentiality in handling of data obtained from the study persons and full information about future use and handling of individual data and samples.

As information about heritable characteristics confer special ethical considerations some special sets of rules should apply:

- If genetic testing is part of a study/trial the genes/pre-dispositions under investigation must be specified. No general consent should be requested for testing of e.g. genes predisposing of cancer, cardiovascular diseases or respiratory diseases. This issue is currently under discussion in clinical trials.

- The right of the individual to desist from genetic testing or from knowing the result. This was laid down as one principle in Danish legislation of use of health information. If a justified (validated) test is offered a (potential) employee a 48 hours interval for consideration and consultation of experts should be allowed before consent.

- Confidential handling of study results to secure the individuals right to know who has the information (potential employers, insurance companies, a.o.)