Bo Lambert

Clonal chromosome aberrations and genomic instability in X-irradiated human T-lymphocyte cultures

To study the effects of X-irradiation on clone forming ability and karyotypic abnormalities in human peripheral blood lymphocytes, cells were exposed to 3 Gy of X-rays in vitro and either individual T-cell clones or long-term T-cell cultures were established. The karyotypes were analyzed in G-banded chromosome preparations after proliferation for 9-34 days in vitro. T-cell clonal karyotype abnormalities were found in 24 of 37 (65%) irradiated and in two of 43 (5%) control clones. Balanced reciprocal translocations and deletions were the predominating types of clonal aberrations. Complex aberrations and unstable karyotypes were found in about half of the irradiated clones. Some of the T-cell clones demonstrated sequential change from normal to aberrant karyotype. Other clones seemed to develop multiple, heterogeneous chromosomal aberrations during growth in vitro. X-Irradiated T-cells grown in long-term T-cell culture displayed karyotype abnormalities in 60-80% of the cells, and the types of aberrations were similar to those found in the individual, irradiated T-cell clones. An increasing number of cells with the same abnormal karyotype was observed when the cultivation time was extended, indicating clonal proliferation. These results demonstrate that a surprisingly high proportion of T-cells with stable and often complex irradiation-induced chromosome aberrations are able to proliferate and form expanding cell clones in vitro. Furthermore, the results indicate that X-irradiation induces latent chromosome damage and genomic instability in human T-lymphocytes.

Cancer predictive value of cytogenetic markers used in occupational health surveillance programs: a report from an ongoing study by the European Study Group on Cytogenetic Biomarkers and Health

The cytogenetic endpoints in peripheral blood lymphocytes: chromosomal aberrations (CA), sister chromatid exchange (SCE) and micronuclei (MN) are established biomarkers of exposure for mutagens or carcinogens in the work environment. However, it is not clear whether these biomarkers also may serve as biomarkers for genotoxic effects which will result in an enhanced cancer risk. In order to assess this problem, Nordic and Italian cohorts were established, and preliminary results from these two studies indicated a predictive value of CA frequency for cancer risk, whereas no such associations were observed for SCE or MN. A collaborative study between the Nordic and Italian research groups, will enable a more thorough evaluation of the cancer predictivity of the cytogenetic endpoints. We here report on the establishment of a joint data base comprising 5271 subjects, examined 1965-1988 for at least one cytogenetic biomarker. Totally, 3540 subjects had been examined for CA, 2702 for SCE and 1496 for MN. These cohorts have been followed-up with respect to subsequent cancer mortality or cancer incidence, and the expected values have been calculated from rates derived from the general populations in each country. Stratified cohort analyses will be performed with respect to the levels of the cytogenetic biomarkers. The importance of potential effect modifiers such as gender, age at test, and time since test, will be evaluated using Poisson regression models. The remaining two potential effect modifiers, occupational exposures and smoking, will be assessed in a case-referent study within the study base.

A Nordic data base on somatic chromosome damage in humans

Analyses of cytogenetic damage--chromosome aberrations (CA), micronuclei (MN), and sister-chromatid exchange (SCE)--are used to assess genotoxic exposure, on the supposition that higher levels of chromosome damage in peripheral lymphocytes reflect increased cancer risk. We attempt to test this hypothesis prospectively, by relating levels of cytogenetic damage to subsequent cancer morbidity in a cohort comprising 3190 subjects. All these subjects have been analyzed previously (1970-1988) for CA, MN, and/or SCE in studies of occupational and environmental exposure. The present paper describes the data base and assesses how the potential confounders smoking habits, sex, and age influence CA, MN, and SCE levels. Ten Nordic laboratories contributed data. In the present analyses, these data were treated separately to avoid the effects of interlaboratory differences. Point estimates from multiple regression analyses indicate that smoking may increase CA frequencies by up to 10-20% and SCE means by 5-8%, but that it has no effect on MN frequencies. Women had higher CA, MN, and SCE levels than men, but the sex effect was generally smaller than the effect of smoking. Age was positively associated with cytogenetic damage. Compared to the sex effect, the effect of a 10-year age increase was similar on CA, but less, 1-3%, on SCE. The amount of variation explained by the potential confounders taken together was generally low, often less than 20%. Thus, other still unknown factors must be the major sources of CA, MN, and SCE variability.

Electrophoretic characterization of nucleolar RNA from Chironomus tentans salivary gland cells

Abstract RNA has been extracted from micro-isolated nucleoli from salivary gland cells of Chironomus tentans and analysed by electrophoresis on agarose-acrylamide composite gel. The glands were labelled with tritiated nucleosides in vitro or in vivo. After a short period of labelling, heterodisperse RNA fractions of nucleolar origin were observed between 20 and 30 s. With increased incorporation time a monodisperse 38 s RNA component appeared, which was subsequently converted to one 30 and 23 s component. The fractions designated 38, 30 and 23 s incorporated methyl groups, indicating a relationship with ribosomal RNA. Any further transformation of 30 and 23 s did not take place within the nucleolus, during in vitro or in vivo incorporation. The 30 and 23 s RNA fractions are likely to be precursors to 28 and 18 s ribosomal cytoplasmic RNA. The pattern of formation of RNA compounds was closely similar in the two nucleolar organizers.

Relation Between Sister Chromatid Exchange, Cell Proliferation and Proportion of B and T Cells in Human Lymphocyte Cultures

SummaryHuman B and T lymphocytes differ in the rate of cell proliferation and frequency of sister chromatid exchange (SCE) when cultured separately in short-term cultures. This difference could theoretically be responsible for part of the variation in the SCE-frequency previously observed among healthy subjects since there is individual variation in the proportion of B and T cells in the peripheral blood. We have therefore studied cell proliferation and SCE-frequency in conventional short-term cultures of lymphocytes from 28 healthy subjects with different proportions of B and T cells. The percentage, of B or T lymphocytes did not correlate with the SCE-frequency, nor with the rate of cell proliferation in culture. However, a significantly higher SCE-frequency was found in slowly proliferating cultures than in cultures with a high rate of turn over. Thus, the rate of cell proliferation appears to be an important determinant of the SCE-frequency in conventional lymphocyte cultures. Although the data do not exclude attribution of the difference in SCE-frequency between rapidly and slowly growing cultures to differences in subpopulations of lymphocytes, it appears less likely that B and T cells constitute these tentative subpopulations.

Biomarkers of genotoxicity of urban air pollution: Overview and descriptive data from a molecular epidemiology study on populations exposed to moderate-to-low levels of polycyclic aromatic hydrocarbons: the AULIS project

Epidemiologic studies indicate that prolonged exposure to high pollution levels is associated with increased risk of cancer, especially lung cancer. However, under conditions of moderate or low air pollution, epidemiologic evidence does not permit reliable conclusions. Biomarker-based population studies may serve as complementary tools providing a better understanding of the relative contribution of ambient atmospheric pollution to the overall genotoxic burden suffered by city dwellers. However, past efforts to apply biomarkers to studies of low levels exposure to urban air pollution have given inconclusive results, partly because of the absence of adequate data on personal exposure, covering a time-window which is appropriate for the biomarkers being examined, as well as a battery of biomarkers reflecting different stages of the carcinogenic process. In the present paper, the potential of biomarker-based population studies to aid the assessment of the genotoxic and carcinogenic effects of urban air pollution is reviewed by reference to the achievements and limitations of earlier reported studies. The design and methodology adopted in a recently completed large-scale population study, carried out in the context of the European Union Environment and Climate Programme, known by the short name of AULIS project, is discussed and descriptive statistics of the main findings of the project are presented. These findings indicate that for cohorts suffering moderate-to-low exposures to airborne particulate-bound polycyclic aromatic hydrocarbons (PAHs), no simple correlation with biomarkers of genotoxicity existed and suggest that additional factors made a significant contribution to the overall genotoxic burden.

An evaluation of styrene genotoxicity using several biomarkers in a 3-year follow-up study of hand-lamination workers.

A study employing several biomarkers of styrene exposure and genotoxicity was carried out in a group of lamination (reinforced plastic) workers and controls, who had been repeatedly sampled during a 3-year period. Special attention will be paid to the last sampling (S.VI), reported here for the first time. Styrene concentration in the breathing zone, monitored by personal dosimeters, and urinary mandelic acid (MA) were measured as indicators of external exposure. Blood samples were assayed for styrene-specific O6-guanine adducts in DNA, N-terminal valine adducts of styrene in haemoglobin, DNA single-strand breaks (SSB), determined by use of the single cell gel electrophoresis (Comet) assay), and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutant frequencies (MF) in T-lymphocytes. O6-styrene guanine adduct levels were significantly higher in the exposed group (5.9 +/- 4.9 adducts/10(8) dNp) as compared to laboratory controls (0.7 +/- 0.8 adducts/10(8) dNp; P = 0.001). DNA adduct levels significantly correlated with haemoglobin adducts, SSB parameters and years of employment. Styrene-induced N-terminal valine adducts were detected in the lamination workers (1.7 +/- 1.1 pmol/g globin), but not in the control group (detection limit 0.1 pmol/g globin). N-terminal valine adducts correlated strongly with external exposure indicators, DNA adducts and HPRT MF. No significant correlation was found with SSB parameters. A statistically significant difference in HPRT MF was observed between the laminators (22.3 +/- 10.6/10(6)) and laboratory controls (14.2 +/- 6.5/10(6), P = 0.039). HPRT MF in the laminators significantly correlated with styrene concentration in air, MA and haemoglobin adducts, as well as with years of employment and age of the employees. No significant difference (P = 0.450) in MF between the laminators and the factory controls was observed. Surprisingly, we detected differences in MF between sexes. When data from all measurements were combined, women showed higher MF (geometric mean 15.4 vs. 11.2 in men, P = 0.020). The styrene-exposed group exhibited significantly higher SSB parameters (tail moment (TM), tail length (TL) and the percentage of DNA in the tail (TP)) than the control group (P < 0.001). SSB parameters correlated with indicators of external exposure and with O6-styrene guanine adducts. No significant correlation was found between SSB parameters and haemoglobin adducts or HPRT MF. The data encompassing biomarkers from repeated measurements of the same population over a 3-year period are discussed with respect to the mechanisms of genotoxic effects of styrene and the interrelationship of individual biomarkers.

DNA repair and frequency of x-ray and u.v.-light induced chromosome aberrations in leukocytes from patients with Down's syndrome.

DNA‐repair and the frequency of chromosome aberration after u.v. and X‐ray irradiation was studied on leukocytes from patients with Downs syndrome.

Repeated DNA sequences in a Balbiani ring

Abstract Newly synthesized RNA from a short region of chromosome IV of Chironomus tentans salivary gland cells was studied by RNA-DNA hybridization. The region is dominated by a puff, Balbiani ring 2, likely to be involved in the genetic determination of secretory proteins. Hybridization of filter-bound Chironomus tentans DNA by BR-2 † RNA was studied in a micro-assay for RNA-DNA hybrids. The proportion of DNA hybridized at saturation was 0.17%, corresponding to 200 × 106 daltons of DNA per haploid genome. Cytological hybridization showed that this DNA is mainly located in the BR-2 region. Since the size of BR-2 RNA is about one order of magnitude smaller than the size of BR-2 DNA, several transcription units may be contained in the BR-2 region. The rate of hybrid formation was determined with DNA in excess to measure the number of binding sites for BR-2 RNA. Nucleolar 38 s RNA, known to have about 100 binding sites in the haploid genome, was used for comparison. BR-2 RNA hybridized twice as fast as 38 s RNA, and retained an almost linear reaction rate until 15% of the input RNA had hybridized. This result suggests that the average multiplicity of sequences within BR-2 DNA is about 200, and that a considerable part of BR-2 RNA hybridizes with repeated DNA sequences. Furthermore, the results indicate that BR-2 RNA and its template DNA are internally repeated, because the multiplicity of BR-2 DNA is about one order of magnitude greater than the maximal number of transcription units. Cytological hybridization experiments with RNA from two additional Balbiani rings (BR-1 and BR-3) as well as from BR-2 indicated that the three BRs differ with regard to information content, and that BR-1 like BR-2 contains repeated nucleotide sequences. Thus it is likely that the three BRs code for different secretory proteins.

Delayed Chromosomal Instability in Human T-lymphocyte Clones Exposed to Ionizing Radiation

Recent studies have demonstrated that cells which survive alpha-particle and X-ray exposure may show chromosomal instability, i.e. they continue to develop chromosomal aberrations at an increased frequency for many division cycles after the exposure. To characterize this delayed response, we carried out repeated karyotype analyses of X-irradiated T-lymphocytes during clonal expansion in vitro. Human peripheral blood lymphocytes were obtained from a healthy donor and exposed to 3-Gy X-irradiation. Cell survival, estimated by a cell cloning assay, was 5%. Non-irradiated, control cells were studied in parallel. Monoclonal cell lines were established using the T-cell cloning procedure. G-band karyotype analyses were carried out at several intervals during expansion of the clones for up to 2 months. The irradiated clones did not differ from the control clones with regard to growth rate or cytometric DNA profile. Non-irradiated cell clones showed a normal karyotype, with < 10% of sporadic, non-clonal chromosome and chromatid breaks. In the irradiated clones, the karyotypes showed different (sub)clonal chromosome rearrangements, which developed successively during the cultivation time. In addition to these karyotypic abnormalities, > 20% of the cells in these clones had sporadic, non-clonal chromosome aberrations, and there was a tendency of increasing frequency of such aberrations by length of cultivation. Thus, two types of radiation-induced chromosomal instability were observed; (sub)clonal karyotypic abnormalities and sporadic, non-clonal chromosome aberrations. The frequency and kinetics by which these alterations occur in the progeny of X-irradiated T-cells suggest that they arise through different pathways, and argue against their causation by mutation or persistent DNA damage.