Frequency of chromosomal aberrations in Prague mothers and their newborns.

The capital city of Prague is one of the most polluted areas of the Czech Republic. The impact of air pollution on the level of chromosomal aberrations was systematically studied: analyses were performed using fluorescence in situ hybridization (FISH) with whole-chromosome painting for chromosomes #1 and #4. In the present study, we analyzed the levels of stable (one-way and two-way translocations) and unstable (acentric fragments) chromosomal aberrations in 42 mothers living in Prague and in their newborns. The average age of the mothers was 29 years (range, 20-40 years). Blood samples were collected from October 2007 to February 2008. The average levels of carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) and benzo[a]pyrene (B[a]P) in respirable particles (PM2.5), as determined by stationary monitoring, were 21.0+/-12.3 ng/m(3) and 2.9+/-1.8 ng/m(3), respectively. We did not observe any effect of either c-PAH or B[a]P exposure on the genomic frequency of translocations (per 100 cells, F(G)/100) in either group due to their similar exposure during the winter months. The mean values of F(G)/100 representing stable aberrations were 0.09+/-0.13 vs 0.80+/-0.79 (p<0.001) for newborns vs mothers, indicating a significant increase of F(G)/100 with age. On the other hand, the frequency of unstable aberrations did not differ between the two groups. Our results demonstrate how the patterns of different types of aberration differed between newborns and mothers: we observed 64.3% unstable aberrations and 35.7% stable aberrations in newborns vs 19.7% and 80.3% in mothers, respectively. Our results indicate that after birth the frequencies of aberrations are very low and that the aberrations are represented mainly by acentric fragments. The changes observed in mothers show a shift to stable aberrations represented mainly by two-way translocations. The mother's age affected the level of aberrations in newborns: the group of children born to older mothers (31-40 years) had significantly increased F(G)/100 levels.

The impact of air pollution on the levels of micronuclei measured by automated image analysis.

The measurement of micronuclei (MN) in human peripheral blood lymphocytes is frequently used in molecular epidemiology as one of the preferred methods for assessing chromosomal damage resulting from environmental mutagen exposure. In the present study, we evaluated the effect of exposure to carcinogenic polycyclic aromatic hydrocarbons (c-PAHs), volatile organic compounds (VOC) and smoking on the frequency of MN in a group of 56 city policemen living and working in Prague. The average age of the participants was 34+/-6 years. The study was conducted on the same subjects in February and May 2007. The concentrations of air pollutants were obtained from personal and stationary monitoring. A statistically significant decrease in the levels of pollutants was observed in May when compared with February, with the exception of toluene levels measured by stationary monitoring. The frequency of MN was determined by the automatic image scoring (MetaSystems Metafer 4, version 3.2.1) of DAPI-stained slides. The results of the image analysis indicated a significant difference in the frequency of MN (mean levels 7.32+/-3.42 and 4.67+/-2.92, for February and May, respectively). Our study suggests that automatic image analysis of MN is a highly sensitive method for evaluating the effect of c-PAHs and confirms that there are no differences between smokers and nonsmokers. These results demonstrate the ability of c-PAHs to increase MN frequency, even if the exposure to c-PAHs occurred up to 60 days before the collection of biological material. Our work is the first human biomonitoring study focused on the measurement of MN by automated image analysis for assessing chromosomal damage as a result of environmental mutagen exposure.

Biomarkers of exposure to tobacco smoke and environmental pollutants in mothers and their transplacental transfer to the foetus. Part II. Oxidative damage.

Oxidative damage to macromolecules may have numerous negative health consequences. We measured oxidative damage to DNA, proteins and lipids in 80 newborns and 79 mothers, analyzed the effect of mother's tobacco smoke exposure on oxidative stress, and assessed correlations between oxidative stress markers and bulky and PAH (polycyclic aromatic hydrocarbons)-specific DNA adducts. Mean levels (+/-S.D.) of 8-oxodeoxyguanosine (8-oxodG) per 10(5) dG in the placenta were 2.85+/-0.78; we did not see a difference between 8-oxodG levels in newborns born to mothers exposed and unexposed to tobacco smoke. Protein carbonyl levels, a marker of protein oxidation, were comparable in the umbilical cord and in maternal venous blood plasma (17.4+/-3.2 and 17.6+/-4.2nmol/ml plasma in newborns and mothers, respectively, p=0.66). Lipid peroxidation measured as levels of 15-F(2t)-isoprostane (15-F(2t)-IsoP) in plasma was significantly higher in newborns than in mothers (362+/-129 and 252+/-130pg/ml in newborns and mothers, respectively, p<0.001). We did not find any effect of tobacco smoke exposure on either biomarker in any group. Levels of both protein carbonyls and 15-F(2t)-IsoP in cord blood significantly correlated with those in maternal plasma (p<0.001). 8-oxodG levels positively correlated with plasma carbonyls in cord plasma, as well as with cotinine levels (marker of tobacco smoke exposure) in maternal plasma. 8-oxodG levels also correlated with bulky DNA adducts in lymphocyte DNA of newborns and mothers and with PAH-DNA adducts in the placenta. Our results showed higher lipid peroxidation in newborns than in mothers, close correlation of analyzed oxidative stress markers between newborns and mothers, and a relationship between oxidative stress and induction of DNA adducts.

Biomarkers of exposure to tobacco smoke and environmental pollutants in mothers and their transplacental transfer to the foetus. Part I: bulky DNA adducts.

(32)P-postlabelling and PAH-ELISA using the antiserum #29 were employed to analyze DNA adducts in venous and umbilical cord blood and the placenta of 79 mothers giving birth to 80 living babies in Prague (Czech Republic). Ambient air exposure was measured by stationary measurements of basic air pollutants (PM2.5, c-PAHs) during the entire pregnancy. Tobacco smoke exposure was assessed by questionnaire data and by plasma cotinine levels. The total DNA adduct levels in the lymphocytes of mothers and newborns were elevated by 30-40% (p<0.001) compared with the placenta. B[a]P-like DNA adduct (adduct with the identical chromatographic mobility on TLC as major BPDE derived DNA adduct) levels were elevated in the blood of mothers compared with the placenta and the blood of newborns (p<0.05 and p<0.01). In tobacco smoke-exposed mothers, higher DNA adduct levels in the blood of mothers and newborns compared with the placenta were found (p<0.001), whereas the total and B[a]P-like adduct levels were comparable in the blood of mothers and newborns. B[a]P-like adducts were elevated in the blood of mothers unexposed to tobacco smoke compared with that of corresponding newborns and the placenta (p<0.01). Total and B[a]P-like DNA adducts were increased in the placenta of tobacco smoke-exposed compared with unexposed mothers (p<0.001 and p<0.01). In lymphocytes of tobacco smoke-exposed mothers, the comparison of total adduct levels (1.18+/-0.67 vs. 0.92+/-0.28) and B[a]P-like DNA adducts (0.22+/-0.12 adducts/10(8) nucleotides vs. 0.15+/-0.06 adducts/10(8) nucleotides) with newborns indicated a 30-40% increase of adducts in mothers. Almost equal PAH-DNA adduct levels were detected by anti-BPDE-DNA ELISA in the placenta of tobacco smoke-exposed and -unexposed mothers. Our results suggest a protective effect of the placental barrier against the genotoxic effect of some tobacco smoke components between the circulation of mother and child. We found a correlation between adduct levels in the blood of mothers and newborns.

Nano-TiO2 stability in medium and size as important factors of toxicity in macrophage-like cells.

TiO2 along with nano-TiO2 are commonly found in consumer products. In vivo studies have observed an accumulation of nano-TiO2 in macrophages. However, characteristics of nano-TiO2 determining toxicity remain unclear. In our study, the cytotoxic effects of 14 diverse nano-TiO2 on THP-1 macrophage-like cells were measured by 3 cytotoxicity assays (MTS, WST-1 and LDH). Total averaged cytotoxicity was calculated using principal component analysis. Characteristics of all 14 nano-TiO2 included hydrodynamic diameter, zeta potential, shape, polydispersity index (PDI) and concentration; moreover, crystal form, specific surface area and crystallite size were measured for 10 nano-TiO2.The variables affecting cytotoxicity were chosen using LASSO (least absolute shrinkage and selection operator). Except for concentration, PDI in media measured within 1 h after preparation of the nanomaterial dispersion was selected as a variable affecting cytotoxicity: stable dispersion resulted in higher cytotoxic effects. Crystallite size has been shown to have nonlinear effects (particles of sizes between 20 and 60 nm were cytotoxic while smaller and larger ones were not) and thus it has been excluded from LASSO. The shape (particles/fibre) and crystal form did not affect the cytotoxicity. PDI and the nonlinear effect of size could be an explanation for the inconsistencies of the cytotoxicity of nano-TiO2 in various studies.

Chromosomal aberrations and SCEs as biomarkers of cancer risk.

Previous studies have suggested that the frequency of chromosomal aberrations (CAs), but not of sister chromatid exchanges (SCEs), predicts cancer risk. We have further examined this relationship in European cohorts comprising altogether almost 22,000 subjects, in the framework of a European collaborative project (CancerRiskBiomarkers). The present paper gives an overview of some of the results of the project, especially as regards CAs and SCEs. The results confirm that a high level of CAs is associated with an increased risk of cancer and indicate that this association does not depend on the time between CA analysis and cancer detection, i.e., is obviously not explained by undetected cancer. The present evidence indicates that both chromatid-type and chromosome-type CAs predict cancer, even though some data suggest that chromosome-type CAs may have a more pronounced predictive value than chromatid-type CAs. CA frequency appears to predict cancers at various sites, although there seems to be a particular association with gastrointestinal cancers. SCE frequency does not appear to have cancer predictive value, at least partly due to uncontrollable technical variation. A number of genetic polymorphisms of xenobiotic metabolism, DNA repair, and folate metabolism affect the level of CAs and might collectively contribute to the cancer predictivity of CAs. Other factors that may influence the association between CAs and cancer include, e.g., exposure to genotoxic carcinogens and internal generation of genotoxic species. Although the association between CA level and cancer is seen at the group level, an association probably also exists for the individual, although it is not known if an individual approach could be feasible. However, group level evidence should be enough to support the use of CA analysis as a tool in screening programs and prevention policies in occupational and environmental health.

Depletion of CLL-associated patrolling monocytes and macrophages controls disease development and repairs immune dysfunction in vivo.

Chronic lymphocytic leukemia (CLL) is characterized by apoptosis resistance and a dysfunctional immune system. Previous reports suggested a potential role of myeloid cells in mediating these defects. However, the composition and function of CLL-associated myeloid cells have not been thoroughly investigated in vivo. Using the Eµ-TCL1 mouse model, we observed severe skewing of myeloid cell populations with CLL development. Monocytes and M2-like macrophages infiltrated the peritoneal cavity of leukemic mice. Monocytes also accumulated in the spleen in a CCR2-dependent manner, and were severely skewed toward Ly6C(low) patrolling or nonclassical phenotype. In addition, the percentage of MHC-II(hi) dendritic cells and macrophages significantly dropped in the spleen. Gene expression profiling of CLL-associated monocytes revealed aberrantly high PD-L1 expression and secretion of multiple inflammatory and immunosuppressive cytokines like interleukin-10, tumor necrosis factor-α and CXCL9. In vivo myeloid cell depletion using liposomal Clodronate resulted in a significant control of CLL development accompanied by a pronounced repair of innate immune cell phenotypes and a partial resolution of systemic inflammation. In addition, CLL-associated skewing of T cells toward antigen-experienced phenotypes was repaired. The presented data suggest that targeting nonmalignant myeloid cells might serve as a novel immunotherapeutical strategy for CLL.

Effect of the combined action of selenium and arsenic on suspension culture of mice fibroblasts.

The protective influence of arsenic against the toxic action of selenium has been tested on suspension cultures of mice fibroblasts LA 115. The growth of the cells was observed after the isolated and combined action of sodium arsenite NaAsO2 and sodium selenite Na2SeO3. The concentration range of both substances in cultivation medium MEm (USOL) was 10(-5) -10(-11)M. The growth of treated cultures was analyzed daily during 5 days of exposure. From the results obtained, growth curves of the cell cultures were constructed and analyzed. The results of every determination were evaluated in relation to the corresponding control culture. The results obtained demonstrate that decreasing concentrations of arsenic enhanced its protective effect in the range of the concentrations used. In contrast, a low protective effect of selenium against arsenic was noted in the concentrations employed. The cell cultures have proved to be very suitable for toxicological studies of the combined effects of different substances.

Monitoring genotoxic exposure in uranium mines.

Recent data from deep uranium mines in Czechoslovakia indicated that mines are exposed to other mutagenic factors in addition to radon daughter products. Mycotoxins were identified as a possible source of mutagens in these mines. Mycotoxins were examined in 38 samples from mines and in throat swabs taken from 116 miners and 78 controls. The following mycotoxins were identified from mines samples: aflatoxins B1 and G1, citrinin, citreoviridin, mycophenolic acid, and sterigmatocystin. Some mold strains isolated from mines and throat swabs were investigated for mutagenic activity by the SOS chromotest and Salmonella assay with strains TA100 and TA98. Mutagenicity was observed, especially with metabolic activation in vitro. These data suggest that mycotoxins produced by molds in uranium mines are a new genotoxic factor for uranium miners.

Risk of cancer in an occupationally exposed cohort with increased level of chromosomal aberrations.

We used cytogenetic analysis to carry out a cohort study in which the major objective was to test the association between frequency of chromosomal aberrations and subsequent risk of cancer. In spite of the extensive use of the cytogenetic analysis of human peripheral blood lymphocytes in biomonitoring of exposure to various mutagens and carcinogens on an ecologic level, the long-term effects of an increased frequency of chromosomal aberrations in individuals are still uncertain. Few epidemiologic studies have addressed this issue, and a moderate risk of cancer in individuals with an elevated frequency of chromosomal aberrations has been observed. In the present study, we analyzed data on 8,962 cytogenetic tests and 3,973 subjects. We found a significant and strong association between the frequency of chromosomal aberrations and cancer incidence in a group of miners exposed to radon, where a 1% increase in frequency of chromosomal aberrations was followed by a 64% increase in risk of cancer (p < 0.000). In contrast, the collected data are inadequate for a critical evaluation of the association with exposure to other chemicals.

Biomarker studies in northern Bohemia.

Studies were conducted in northern Bohemia to simultaneously evaluate personal exposures to air pollution in the form of respirable particles containing polycyclic aromatic hydrocarbons (PAHs) and biomarkers of exposure, biological effective dose, genetic effects, and metabolic susceptibility. The series of biomarkers included PAH metabolites in urine, urine mutagenicity, PAH-DNA adducts in white blood cells determined by 32P-postlabeling, PAH-albumin adducts determined by enzyme-linked immunosorbent assay (ELISA), DNA damage in lymphocytes detected by comet assay, chromosomal aberrations, sister chromatid exchanges, and glutathione S-transferase M1 (GSTM1) genotypes. For these studies, a group of women who work outdoors about 30% of their daily time was selected. In a pilot study, a group of women from a polluted area of the Teplice district (northern Bohemia) was compared with a group of women from a control district of southern Bohemia (Prachatice). In a follow-up repeated-measures study, a group of nonsmoking women from Teplice was sampled repeatedly during the winter season of 1993 to 1994. Personal exposure monitoring for respirable particles (< 2.5 microns) was conducted for the 24-hr period before collection of blood and urine. Particle extracts were analyzed for carcinogenic PAHs. In the pilot study and in the follow-up study, a highly significant correlation between individual personal exposures to PAHs and DNA adducts was found (r = 0.54, p = 0.016; r = 0.710, p < 0.001, respectively). The comet parameter (percentage DNA in tail; %T) correlated with exposures to respirable particles (r = 0.304, p = 0.015). The GSTM1 genotype had a significant effect on urinary PAH metabolites, urine mutagenicity, and comet parameters (% T and tail moment) when the GSTM1 genotype was considered as a single factor affecting these biomarkers. Multifactor analysis o variance considering exposure and adjusting the data for GSTM1, age, and diet showed that the effect of personal exposures to PAHs on the variability of biomarkers (DNA adducts, comet parameters, urine mutagenicity) might be higher than the effect of the GSTM1 genotype. These results show the importance of considering all potential factors that may affect the biomarkers being analyzed.

Monitoring genotoxic exposure in uranium miners.

Recent data from deep uranium mines in Czechoslovakia indicated that in addition to radon daughter products, miners are also exposed to chemical mutagens. Mycotoxins were identified as a possible source of mutagenicity present in the mines. Various methods of biomonitoring were used to examine three groups of miners from different uranium mines. Cytogenetic analysis of peripheral lymphocytes, unscheduled DNA synthesis (UDS) in lymphocytes, and lipid peroxidation (LPO) in both plasma and lymphocytes were studied on 66 exposed miners and 56 controls. Throat swabs were taken from 116 miners and 78 controls. Significantly increased numbers of aberrant cells were found in all groups of miners, as well as decreased UDS values in lymphocytes and increased LPO plasma levels in comparison to controls. Molds were detected in throat swabs from 27% of miners, and 58% of these molds were embryotoxic. Only 5% of the control samples contained molds and none of them was embryotoxic. The following mycotoxins were isolated from miners' throat swab samples: rugulosin, sterigmatocystin, mycophenolic acid, brevianamid A, citreoviridin, citrinin, penicilic acid, and secalonic acid. These data suggest that mycotoxins are a genotoxic factor affecting uranium miners.

Tumour vaccines expressing IL-2, CD80, and IL-2 plus CD80 gene.

Experiments were designed to investigate immunogenicity and therapeutic efficacy of tumour vaccines constructed by transfection of poorly immunogenic murine sarcoma Mc12 with synergistic CD80 and IL-2 genes. Immunization/challenge experiments demonstrated that both, IL-2(+) and IL-2(+) plus CD80(+) live cell vaccines can exert an immunizing stimulus, the IL-2(+) plus CD80(+) vaccine being superior to the IL-2(+) vaccine. Live CD80(+) Mc12 cell vaccine could not be tested, since the vaccine was highly tumorigenic in the doses required for immunization. Preimmunization with IL-2(+) and IL-2(+) plus CD80(+) vaccines induced regressions of a proportion of the parental Mc12 challenge inocula after their temporary growth. Areas of necrosis and extensive infiltration with Mac1(+) and CD4(+) leukocytes have been observed in the regressing sarcomas. When the therapeutic efficacy of the irradiated CD80(+), IL-2(+), and mixed CD80(+) plus IL-2(+) vaccines was compared, it was found that the insertion of the IL-2, but not CD80 gene alone was efficient. The mixed IL-2(+) plus CD80(+) tumour vaccine was able to protect and prolong survival of a higher proportion of mice than the IL-2(+) tumour vaccine.

Genetically modified tumour vaccines.

Two genes, the gene coding for IL-2 and the gene encoding the CD80 molecule, were inserted into murine sarcoma MC12 cells. Tumorigenicity of a variety of cell clones with different expression of the inserted genes was assessed. Most of the genetically manipulated MC12 cell clones were less tumorigenic than the parental MC12 cell population. Tumorigenicity of the clones declined with increasing production of IL-2 as well as with the increasing expression of the CD80 molecule. When the tumorigenicity of the clones carrying an inserted IL-2 gene was compared with that of the clones carrying an inserted CD80 gene, it was found that the insertion of the IL-2 gene suppresses tumorigenicity more efficiently than insertion of the CD80 gene. Admixture of the IL-2-producing MC12 clones to the tumorigenic CD80(+) MC12 cell doses could completely inhibit the tumorigenicity of the CD80(+) cells. Insertion of the CD80 gene into sarcoma cells substantially enhanced the adhesive interaction between the MC12 sarcoma and syngeneic T lymphocytes.

Immunogenicity, immunosensitivity and cell surface adhesiveness of tumour vaccines carrying an inserted CD80 gene.

Cell surface adhesiveness, immunogenicity and immunosensitivity of tumour vaccines modified by the CD80 gene transfection was examined and compared to that of the parental MC12 murine sarcoma. Insertion of the CD80 gene substantially enhanced the adhesiveness of the genetically modified tumour cells to nylon wool non-adherent (T) but not to nylon wool adherent (B) lymphocytes. The increased adhesive interaction could be inhibited by anti-CD80 monoclonal antibody. CD80+ transfectants were more sensitive to the cytotolytic effect of MC12-immune splenocytes and IL-2-activated spleen cells than the parental MC12 sarcoma. Similarly, spleen cells from syngeneic mice immunized with CD80+ transfectants displayed a higher cytolytic activity when allowed to react with MC12 cells than splenocytes from mice immunized with the parental MC12 cells. These results suggest that a positive correlation exists among the expression of the CD80 molecules, T cell adhesion to the genetically modified cells, immunosensitivity of the CD80+ transfectants and the capacity of the transfectants to activate cytolytic, tumour-reactive effector cells in vivo. This correlation provides a rationale for gene therapy based on the construction of CD80- modified tumour vaccines.

Tumour inhibitory effects of plasmid DNA.

The experiments were designed to examine whether direct in vivo transfer of plasmid DNA not carrying any 'therapeutic' genes ('empty' plasmid DNA) can influence tumour growth. Murine MC-induced sarcoma Mc12 was transplanted i.m. in syngeneic recipients and the tumour-inoculated mice were treated either with repeated peritumoral (i.m.) injections of the naked plasmid DNA or with a single peritumoral (i.m.) injection of plasmid DNA incorporated in liposomes. Two plasmid DNA preparations, BCMGNeo and pON1 were utilized. The direct in vivo transfer of both 'empty' plasmid DNA preparations inhibited tumour growth and significantly prolonged survival of tumour-bearing mice. Our results emphasize the importance of plasmid DNA controls for evaluation of gene therapy of cancer based on the transfer of 'therapeutic' genes in tumour-bearing individuals.

CD80 and IL-2 signals cooperate in the regression of tumors transplanted in congenitally athymic mice.

Interleukin-2 and CD80 transfectants of a methylcholanthrene-induced murine sarcoma Mc12 (Mc12-IL-2 and Mc12-CD80 cells) with similar tumorigenicity in euthymic mice were utilized for experiments designed to investigate a co-stimulatory role of the CD80 molecule in allogeneic, congenitally athymic (nu/nu) mice. The CD80-transfected cells were as tumorigenic in nu/nu mice as the parental Mc12 sarcoma. The IL-2-transfected cells grew only transiently and regressed in all nu/nu recipients during four weeks after challenge with doses up to 5x10(7) cells. The 1:1 mixture of parental Mc12 with Mc12-CD80 cells grew progressively in all inoculated nu/nu mice; in a 1:1 mixture with parental Mc12 cells, Mc12-IL-2 cells were able to cause regressions in approximately 50% of nu/nu mice; the 1:1 mixture of Mc12-IL-2 and Mc12-CD80 transfectants showed only transient growth and regressed during four weeks in all inoculated nu/nu mice. Adoptive transfer of cell-mediated immunity revealed that spleen cells from tumor regressors were capable of transferring the resistance to Mc12 tumor in nu/nu mice. The spleen cells from tumor regressors were not cytolytic when allowed to react in vitro with Mc12, Mc12-IL-2, or Mc12-CD80 target cells. However, when grown in IL-2-containing medium, splenocytes from tumor regressors, but not the splenocytes from tumor progressors, could develop cytolytic activity directed against Mc12 target cells that was comparable to that of the splenocytes from tumor-free controls. These results suggest that the rejection of tumors in nu/nu mice was mediated by IL-2-dependent mechanisms in which the CD80 molecule played a co-stimulatory role; the results also indicate that the ability to be activated by IL-2 and to give rise to cytolytic activity of nu/nu splenocytes from tumor progressors is decreased.

Biological effect monitoring in industrial workers from the Czech Republic exposed to low levels of butadiene.

Blood samples were collected twice (in 1993 and 1994) from 19 workers exposed to 1,3-butadiene and 19 matched controls. Three exposed and three control subjects were the same in 1993 and 1994. Personal passive dosimetry was performed in 1993 and twice in 1994 on the day preceding blood sampling. Mean exposure level in 1994 was 1.76 +/- 4.20 ppm (S.D.) and individual exposure levels ranged between 0.012 ppm (detection limit) and 19.77 ppm. Using the clonal assay, geometric mean of hprt mutant frequencies adjusted for cloning efficiency, age and smoking were, respectively, 7.85 (+/- 7.09) x 10(-6) and 10.14 (+/- 9.16) x 10(-6) in pooled (1993 plus 1994) exposed and control subjects. The difference was not statistically significant indicating that 1,3-butadiene did not induce a detectable increase in mutations at the hprt locus. A similar result was obtained for the 1994 subjects alone. There was no difference between adjusted geometric mean mutant frequencies of exposed and unexposed non-smokers or between exposed and unexposed smokers. Analysis of chromosomal aberrations in lymphocytes from 1994 subjects indicated that the percentage of aberrant cells was significantly enhanced in exposed subjects. In 1993 (data not shown), it was impossible to demonstrate a significant increase of aberrant cells in subjects exposed to 1,3-butadiene. Frequencies of micronuclei in cytochalasin-B blocked binucleate lymphocytes in exposed and unexposed 1994 subjects were not significantly different. This was also the case for earlier samples analyzed in the same plant. Using the comet assay for 1994 subjects, no statistically significant difference was found between the whole group of exposed and unexposed subjects. This was true for both the comet tail length and the percentage of DNA in the tail. In exposed smokers, however, the comet tail length was significantly longer than in unexposed smokers. Unexpectedly, in unexposed smokers the tail length was significantly shorter than in unexposed non-smokers. It was also unexpected that the percentage of DNA in the comet tail was significantly lower in exposed non-smokers than in unexposed non-smokers.

The use of YG bacterial tester strains for the monitoring of drinking water mutagenicity.

The organic extract from the 50 drinking water specimens taken in four cities were tested for mutagenicity in the Ames test (plate incorporation assay) using the parent TA98 and TA100 strains, and derived YG1041 and YG1042 strains. Four dose levels of extractable organic matter (EOM) with duplicate plate per dose were used. Slopes (revertants/mg EOM) were calculated by the Bernstein linear regression rejection model using GeneTox Manager software. The mutagenicity observed in the conventional strains TA98 and TA100 did not reach the significant increase in all tested samples with the higher mutagenic response found in TA100-S9. With the YG1041 and YG1042 tester strains, the results obtained demonstrated the clear-cut direct dose-related mutagenicity response in all tested drinking water extracts. Compared with TA98 and TA100 strains, the numbers of YG induced revertants were approximately 20 times higher. The high sensitivity of the YG tester strains could facilitate the mutagenicity monitoring in drinking water extracts, and help reduce the volume of sample required. However, to identify the chemical contaminants in drinking water responsible for the mutagenicity further studies are required.

Chromosomal aberrations in peripheral lymphocytes of children as biomarkers of environmental exposure and life style.

The original purpose of our study was to determine if the detection of chromosomal aberrations in peripheral lymphocytes of children might be used as a biomarker of environmental pollution and life style. We compared the results of cytogenetic analyses performed in children and adolescents in the periods 1984-1993 and 1994-1999, in a total of 3402 subjects. The frequency of aberrant cells (AB.C.) markedly decreased in the period 1994-1999 compared with the period 1984-1993. The decreases in AB.C. were significant in the age groups 7-15 and 16-19 years: 1.63% AB.C. versus 1.14% AB.C. and 2.02% AB.C. versus 1.08% AB.C., respectively (P<0.01). No difference in the frequency of AB.C. was observed in newborns. Based on our experience, we believe that monitoring the spontaneous level of chromosomal aberrations in children over 5 year periods may be used to examine the general changes in environmental pollution in larger geographic areas.