Caffeine release of radiation induced S and G2 phase arrest in V79 hamster cells: increase of histone messenger RNA levels and p34cdc2 activation.

The serine/threonine protein kinase p34cdc2 activity in V79 hamster cells 4 h after treatment with 7-Gy X-rays is similar to that of unirradiated cells. Nevertheless, the irradiated cells are arrested in the S and G2 phases of the cell cycle. The mRNA concentrations of histones H1 and H4 are reduced by a factor of about 2 in irradiated cells compared to unirradiated cells, as opposed to the mRNAs of high-mobility group I(Y) and 17 proteins which appear unchanged. Both the p34cdc2 activity and the mRNA concentrations of the histones rise within 30 min after the release of the radiation induced cell cycle block by caffeine. During this time span the p34cdc2 activity increases about 4-fold and the histone mRNA levels recover approximately to those of an exponentially growing cell population. Regulatory pathways influenced in irradiated and in subsequently caffeine treated cells apparently interact with basic cell cycle control mechanisms.

Calorimetric measurements of the transition enthalpy of DNA in aqueous urea solutions.

The helix-coil equilibrium of DNA is delicately affected by the nature of the solvent. In this investigation the helical secondary structure was destabilized by an increasing concentration of urea. We found a linear dependence of the transition enthalpy deltaH on the urea concentration for calf thymus DNA as well as for salmon sperm DNA.

Fluorescent antibody localization of Microciona prolifera aggregation factor and its baseplate component.

Specific rabbit antisera were prepared against purified aggregation factor and its membrane-associated receptor, baseplate, derived from the marine sponge. Microciona prolifera. They were utilized in conjunction with fluorescent-labeled goat anti-rabbit IgG in an assay to demonstrate the surface localizations of both components. The specificity of antibody preparations for AF and BP was demonstrated through inhibition of the rotation-mediated assay by homotypic antibody. This study confirms the presence of aggregation factor on the surface of disaggregated sponge cells maintained in the presence of the divalent cations, Ca++ and Mg++, and its absence when cells are maintained in Ca++ and Mg++-free seawater. The location of BP could also be demonstrated on the cell surface. Aggregation factors and baseplate appear to be heavily distributed on archeocytes and choanocytes, but are localized less intensely on gray cells. Gray cells are typified by yellowish autofluorescence of their intracellular granules in stained and control preparations. The reaction of anti-Microciona aggregation factor with its homotypic factor appeared to be species specificity judged by immunofluorescence assays and by inhibition of rotation-mediated assay by anti-homotypic AF since antibodies prepared against heterotypic AF preparations were unreactive.

Resolution of DNA damage at the single-cell level shows largely different actions of X-rays and bleomycin.

Both X-rays and the radiomimetic agent bleomycin (BLM) induce DNA strand breaks, predominantly via reactive radicals. To compare the induction of breaks with the two agents in Chinese hamster (CHO-K1) cells, two different alkaline unwinding methods, a 3H tracer-based analysis of large cell populations and an optical adaption allowing measurement of single cells, were applied. Radiation and BLM show qualitatively similar dose responses when the average number of DNA strand breaks is measured in a large cell population. However, the breakage pattern at the single-cell level indicates large discrepancies between the actions of the two agents. Irradiated cells show a uniform distribution of DNA strand breaks over the cell population. Effects of treatment with 30 micrograms x ml-1 BLM for 2 hr vary from practically zero in some cells to high levels of DNA strand breakage in others. Unlike the repair of radiation-induced DNA breaks, the repair efficiency of BLM-induced DNA strand breaks, as measured at the single-cell level, varies strongly among cells of the same population. Such heterogeneity at the cellular level potentially reduces BLM's usefulness for tumor therapy because the appearance of BLM-resistant subpopulations may critically impair treatment outcome.

Down syndrome clusters in Germany after the Chernobyl accident.

In two independent studies using different approaches and covering West Berlin and Bavaria, respectively, highly significant temporal clusters of Down syndrome were found. Both sharp increases occurred in areas receiving relatively low Chernobyl fallout and concomitant radiation exposures. Only for the Berlin cluster was fallout present at the time of the affected meioses, whereas the Nuremberg cluster preceded the radioactive contamination by 1 month. Hypotheses on possible causal relationships are compared. Radiation from the Chernobyl accident is an unlikely factor, because the associated cumulative dose was so low in comparison with natural background. Microdosimetric considerations would indicate that fewer than 1 in 200 oocyte nuclei would have experienced an ionizing event from Chernobyl radioactivity. Given the lack of understanding of what causes Down syndrome, other than factors associated with increased maternal age, additional research into environmental and infectious risk factors is warranted.

Enhanced neoplastic transformation in an inhomogeneous radiation field: an effect of the presence of heavily damaged cells.

In the inhomogeneous radiation field surrounding small beta-particle sources, nonlethally and heavily damaged cells are in proximity, permitting interaction via extracellular signals. This situation is typical of hot particles such as those released during the accident at Chernobyl. Beta-particle-emitting yttrium-90 wires (average energy 934 keV) were employed to investigate radiation-induced neoplastic transformation under these conditions. Integrated 24-h doses ranging from 0 to 750 Gy across the exposure field were applied. At equal levels of toxicity a 10-fold enhancement of neoplastic transformation frequency in C3H 10T1/2 cells was observed in the presence of heavily damaged cells. Homogeneous fields of low-dose-rate beta-particle radiation produced neoplastic transformation frequencies typical for comparable photon exposures reported in the literature.

Staurosporine- and radiation-induced G2-phase cell cycle blocks are equally released by caffeine.

We show here that the arrests of cells in G2 phase of the cell cycle induced by either staurosporine or ionizing radiation are closely related phenomena governed by a common kinase signaling pathway. The protein kinase inhibitor staurosporine induces a complete G2-phase arrest in exponentially growing TK6 human lymphoblastoid and V79 Chinese hamster fibroblast cells. Both cell types are equally sensitive to the kinase inhibitor and the arrest is dependent on its continued presence. Caffeine completely abrogates this arrest at concentrations comparable to those which abrogate radiation-induced G2-phase arrest. The kinetics of caffeine-induced release of both kinds of arrest are essentially identical. The activity of p34cdc2 kinase was also found to increase in a parallel fashion after caffeine-induced release of both kinds of arrest. As opposed to those transformed cell types which arrest only in G2 phase in response to staurosporine, immortalized C3H 10T1/2 fibroblasts and Muntjak skin fibroblasts display both G1- and G2-phase arrests. The results suggest that staurosporine and radiation interact with regulatory pathways in the cell cycle, and specifically with a caffeine-sensitive signal transduction pathway which recognizes DNA damage, regulates the G2/M-phase transition, and attenuates the biological consequences of radiation exposure.

The German uranium miners cohort study: feasibility and first results.

In Germany, the largest single cohort study on uranium miners to date is being conducted. The cohort includes about 64,000 workers of the former Wismut company in eastern Germany. Inclusion criteria were: a date of employment between 1946 and 1989, a minimum period of employment of 180 days, and complete information on working history. Due to poor working conditions in the late 1940s and early 1950s, miners were exposed to high levels of radiation, while later radiation exposure was significantly reduced. The aim of the cohort study is to evaluate the risk of lung cancer and other cancers associated with several indicators of exposure to radon and its progeny, with particular attention to low levels of radiation. Radon exposure will be estimated by a detailed job- exposure matrix. Some information about smoking, dust and arsenic is already available. About 49,000 miners are defined as exposed (underground or processing), while the internal control group (surface only) consists of 15,000 workers. A total of 1,436 lung cancer deaths among cohort members have been reported. The first mortality follow-up will be finished early in 2002, and a total of about 3,000 lung cancer deaths are expected by then.

Choline acetyltransferase induction in cultured neurons: dissociated spinal cord cells are dependent on muscle cells, organotypic explants are not.

The dependence of the induction of choline acetyltransferase (CAT) on the type of tissue culture, monolayer or piece explants, of spinal cord (SC) and muscle (M) was studied. CAT activities of dissociated SC cells were low but were increased by M monolayers. In piece explants of SC, high CAT activities were achieved independently of muscle cells (chick, mouse). It is therefore likely that in tntact SC tissue factors other than the trophic influence of muscle are able to induce cholinergic maturation. M piece explants on SC monolayers did not induce CAT in, and their outgrowth was inhibited by, SC monolayers.

Effects of ionizing- and UV B-radiation on proteins controlling cell cycle progression in human cells: comparison of the MCF-7 adenocarcinoma and the SCL-2 squamous cell carcinoma cell line.

MCF-7 and SCL-2 cells were irradiated with UV B-radiation or with 137Cs gamma-radiation, in order to investigate cell cycle checkpoint control mechanisms. Effects of both qualities of radiation were investigated for the two cell lines in regard to p53 protein levels, and alterations in Cdk1 (cyclin dependent kinase 1) and Cdk2 phosphorylation were monitored. SCL-2 cells constitutively overexpressed a form of p53 protein whose abundance remained unchanged after irradiation, whereas MCF-7 cells expressed wild type p53 whose abundance increased after irradiation. Accordingly, MCF-7 cells showed a strong G1 phase arrest, whereas SCL-2 cells were only delayed in S phase (after UV B-irradiation) and arrested in G2 phase (after gamma-irradiation and UV B-irradiation), as monitored by flow cytometry. In MCF-7 cells increased p53 levels were observed for up to 30 h after gamma-irradiation and up to 20 h after UV B-irradiation. Only in SCL-2 cells was there a significant radiation induced inactivation of Cdk1 by hyperphosphorylation. This effect was prevented by culturing cells in the presence of caffeine after irradiation. After UV B-irradiation the inactivation of Cdk1 was less pronounced and only partially diminished in the presence of caffeine. No alteration in Cdk2 phosphorylation was observed after irradiation in either cell line.

UV-B-induced cell cycle perturbations, micronucleus induction, and modulation by caffeine in human keratinocytes.

UV-B-induced perturbations of cell cycle progression in asynchronous human keratinocytes were analysed during two cell cycles with respect to their cell cycle stage at the time of irradiation using BrdUrd/Hoechst flow cytometry. Exponentially growing SCL-2-keratinocytes exposed to UV-B radiation showed a short delay in G1-phase exit and were blocked in the S and G2/M phases of the first cell cycle. UV-A wavelengths did not show any detectable effect on cell cycle progression. In contrast, 137Cs-irradiation of these cells induced a temporary G2 block only. Micronucleus frequency increased in gamma-irradiated cells as soon as the cells started to divide and reached a plateau when most of the cells had divided. Continuous treatment with caffeine starting immediately after 137Cs gamma-irradiation prevented accumulation of cells in G2 phase, but did not influence the frequency of micronuclei. In UV-B-irradiated keratinocytes, however, the damage-induced cell cycle perturbations were merely reduced by caffeine, but not eliminated. Compared with gamma-irradiation a moderate induction of micronuclei was observed in UV-B-irradiated cells. Caffeine, however, potentiated the induction of micronuclei by UV-B. These different effects on cell cycle kinetics and micronucleus induction indicate different mechanisms of DNA damage caused by UV-B- and gamma-irradiation that may be repaired through different pathways.

Resistance and cross-resistance to chromosome damage in human blood lymphocytes adapted to bleomycin.

Human peripheral blood lymphocytes cultured in the presence of low concentrations of bleomycin (BLM), 0.01-0.1 microgram/ml, for 48 h and then treated with a high concentration (1.5 microgram/ml) of the same agent or with 1.5 Gy X-rays, became significantly less sensitive to the induction of chromosomal damage than those which did not receive the pre-treatment with BLM. They responded with lower frequencies of chromatid and isochromatid breaks. These results lend further support to the operation of an adaptive repair system in lymphocytes which offers resistance and cross-resistance to the induction of chromosomal damage by the same or similar DNA-damaging agents.

Health risks from combined exposures: mechanistic considerations on deviations from additivity.

Living organisms are exposed every day to a multitude of physical, chemical and biological agents. However, assessments of possible deleterious outcomes from these exposures concentrate on single agents and neglect the potential for combined effects, i.e., synergisms or antagonisms. Biomechanistic considerations based on multistep processes such as carcinogenesis indicate the potential for highly detrimental interactions, if two or more consecutive rate limiting steps are specifically effected by different agents. However, this depends on the specificity of the individual agents for well defined molecular structures or DNA sequences. Low specificity towards molecular structure or DNA-sequence-and therefore exchangeability-of many genotoxic agents indicate little functional specificity of most agents and therefore little vulnerability towards synergism at most occupational and non-occupational exposure situations. In addition, the relative insignificance of combined actions for those common exposure situations where highly non-linear dose effect relationships for non-genotoxic acting agents are involved is also evident. However, an experimental proof of the quantitative assessment of the contribution of synergistic interactions to the total detriment from natural and man-made toxicants is remote. Surprises therefore cannot be excluded.

Adaptive response to 2 low doses of X-rays in human blood lymphocytes.

Human peripheral blood lymphocytes exposed to a single adaptive dose of 1 cGy X-rays or 2 adaptive doses, each of 1 cGy, were found to be equally resistant to the induction of chromosome damage by subsequent challenge with a high dose of 1 Gy X-rays, as compared to cells that were not pre-exposed. They responded with a significantly reduced incidence of chromatid and isochromatid breaks. These results indicate the presence of an inducible chromosomal repair mechanism in human blood lymphocytes and confirm the observations made by earlier investigators. The incidence of chromosome damage was found to be similar in the lymphocytes pre-exposed to a single or 2 adaptive doses, suggesting that, under the conditions tested, the second adaptive dose did not offer any additional protection against the chromosome damage induced by the challenge dose.

Lack of adaptive response to low doses of ionizing radiation in human lymphocytes from five different donors.

Various investigators reported a reduced yield of chromosome and chromatid aberrations in short-term cultures of human lymphocytes if a 'challenge' exposure to ionizing radiation was preceded by an 'adaptive' exposure. In order to examine the cell cycle dependence of the 'adaptive response', chromosome and chromatid aberration yields were estimated after challenge doses in the G1, S or G2 phase of lymphocytes which had been adapted in the early G1 phase. On testing two donors no protective adaptive response was found. Blood samples of four donors were tested for their capability to evoke the adaptive response in a standard experiment with the adaptive dose in the S phase and the challenge dose in the G2 phase. A synergistic response occurred in one out of two similar experiments performed with the same blood sample. The three other blood samples tested did not respond. Apparently these data indicate a high frequency of human lymphocyte cultures that do not display an adaptive response.

Human cytogenetic consequences of the Chernobyl accident.

The frequency of chromosomal aberrations was evaluated in more than 500 liquidators of the Chernobyl accident. The 'sarcophagus' builders and the dosimetrists showed the highest frequency of aberrations per 100 cells: 3.24 +/- 0.25 and 3.11 +/- 0.43. For Chernobyl Atomic Power Station staff members the mean frequencies of aberrations per 100 cells was 2.37 +/- 0.20. The mean yields of aberrations in the other groups was between 1.31 and 1.47 per 100 cells. If the mean frequencies of aberrations are converted into equivalent whole body doses, values between 136 and 414 mGy are obtained. Especially in the group of 'sarcophagus' builders, the yields of aberrations varied interindividually and corresponded to equivalent whole body doses of up to about 2 Gy.

Combined effect of radiation and other agents: is there a synergism trap?

Most assessments of possible deleterious outcomes from environmental and occupational exposures concentrate on single agents and neglect the potential for combined effects--that is, synergisms or antagonisms. Biomechanistic considerations based on multistep processes, such as carcinogenesis, indicate the potential for highly detrimental interactions if two or more consecutive rate-limiting steps are specifically effected by different agents. However, low specificity toward molecular structure or DNA sequence--and, therefore, exchangeability--of many genotoxic agents indicate little functional specificity and, hence, little vulnerability toward synergism in most occupational and environmental exposure situations. It is also evident that a low potential exists for the combined effects of common low-exposure situations wherein nongenotoxic agents with highly nonlinear dose-effect relationships and apparent thresholds are involved. A quantitative assessment of the contribution of synergistic interactions to the total detriment from natural and man-made toxicants based on experimental data is far away. There are important examples of combined exposures shown to lead to health effect risks that differ from those expected from simple addition-for example, the influence of smoking on radon- or asbestos-induced lung cancer and on ethanol-induced esophageal cancer. The existing data on combined effects is rudimentary, mainly descriptive, and rarely covers exposure ranges large enough to make direct inferences to present-day low-dose exposure situations. In view of the multitude of possible interactions among the large number of potentially harmful agents in the human environment, descriptive approaches will have to be supplemented by the use of mechanistic models for critical health endpoints, such as cancer. Finally, considering the shape of dose-effect relationships for ionizing radiation, an important question arises from the unresolved question of whether real or apparent thresholds may be used for any genotoxic agent, separately or one time, for an exposed genome.

Compartmentalization in environmental science and the perversion of multiple thresholds.

Nature and living organisms are separated into compartments. The self-assembly of phospholipid micelles was as fundamental to the emergence of life and evolution as the formation of DNA precursors and their self-replication. Also, modern science owes much of its success to the study of single compartments, the dissection of complex structures and event chains into smaller study objects which can be manipulated with a set of more and more sophisticated equipment. However, in environmental science, these insights are obtained at a price: firstly, it is difficult to recognize, let alone to take into account what is lost during fragmentation and dissection; and secondly, artificial compartments such as scientific disciplines become self-sustaining, leading to new and unnecessary boundaries, subtly framing scientific culture and impeding progress in holistic understanding. The long-standing but fruitless quest to define dose-effect relationships and thresholds for single toxic agents in our environment is a central part of the problem. Debating single-agent toxicity in splendid isolation is deeply flawed in view of a modern world where people are exposed to low levels of a multitude of genotoxic and non-genotoxic agents. Its potential danger lies in the unwarranted postulation of separate thresholds for agents with similar action. A unifying concept involving toxicology and radiation biology is needed for a full mechanistic assessment of environmental health risks. The threat of synergism may be less than expected, but this may also hold for the safety margin commonly thought to be a consequence of linear no-threshold dose-effect relationship assumptions.

Main sources of indoor radon in the Swiss Central Alps.

To estimate the amount of radon gas transferred from the water to the indoor atmosphere of dwellings in the Swiss Central Alps, the radon concentrations of tap water of about 50 public water supplies were analysed. The radon levels of water in alpine communities fit a lognormal distribution with a geometric mean of 26.5 Bq/1 and a geometric standard deviation of 3.15. Radon monitoring in typical homes in the Swiss Midland revealed a significant influence of wind velocity and temperature difference indoor/outdoor on the indoor radon-daughter levels during the winter period.

Quantifying health effects from the combined action of low-level radiation and other environmental agents: can new approaches solve the enigma?

Efforts to assess the quantify deleterious effects from toxicants are directed mainly towards single agents, whereas real world environmental and occupational exposures to natural and anthropogenic agents quite often entail the concomitant presence of several toxicants. These combined exposures may lead to health risks that differ from those expected from simple addition of the individual risks. For example, combined exposures to physical and chemical agents such as radon and smoking or asbestos and smoking produce over-additive effects at exposure levels typical for earlier workplaces. In tumour therapy, the modulation of radiation effects by cytotoxic drugs is widely used to enhance the therapeutic gain. Whether interactions occurring at high exposure levels are important at the low exposure levels set for the public and for modern workplaces is difficult to answer. A scientifically sound extrapolation from these high to low-dose levels should be based on dose-effect relationships of the relevant agents alone and in combination. In general this information is not available. The existing data base on combined effects is rudimentary, mainly descriptive and rarely covers exposure ranges large enough to make direct inferences to present day low-dose exposure situations. In view of the multitude of possible interactions between the large number of potentially harmful agents in the human environment, descriptive approaches will have to be supplemented by the use of mechanistic models for critical health endpoints such as cancer. To generalise and predict the outcome of combined exposures, agents will have to be grouped depending on their physical or chemical mode of action on the molecular and cellular level. Such a grouping must be guided by specific mechanistic studies designed to examine the underlying hypothesis regarding how various classes of agents interact.