UV-induced skin cancer in a hairless mouse model

Ultraviolet (UV) radiation is a very common carcinogen in our environment, but epidemiological data on the relationship between skin cancers and ambient solar UV radiation are very restricted. In hairless mice the process of UV carcinogenesis can be studied in depth. Experiments with this animal model have yielded quantitative data on how tumor development depends on dose, time and wavelength of the UV radiation. In combination with epidemiological data, these experimental results can be transposed to humans. Comparative studies on molecular, cellular and physiological changes in mouse and man can further our fundamental understanding of UV carcinogenesis in man. This is likely to improve risk assessments such as those related to a stratospheric ozone depletion, and to yield well-targeted intervention schemes, e.g. prescribing a specific drug or diet, for high-risk individuals.     

Evaluation and re-evaluation of genetic radiation hazards in man III. Other relevant data and risk assessment

Some of the advances in mammalian radiation genetics, human genetics and cytogenetics that were made during the last 2–3 years and that have either a direct bearing on, or that may be potentially useful in, the evaluation of genetic radiation hazards in man have been examined. Among these are (1) the new data on the incidence of genetic diseases in man; (2) the latest results of the study of mortality rates among children born to survivors of the atomic bombings of Hiroshima and Nagasaki; (3) new data on the radition-induction of reciprocal translocations in human spermatogonia; (4) new results from radiation studies with mice on skeletal mutations, autosomal recessive lethals, sex-chromosome losses, translocation induction and recovery etc., and (5) a re-analysis of the earlier data on dose-rate effects for the induction of specific locus mutations in mouse spermatogonia. Using the pertinent new information as a basis, quantitative estimates are presented employing both a direct method of expressing risks in terms of effects per unit dose of irradiation and the indirect doubling-dose method of expressing these as increments over the load of genetic disorders occuring spontaneously in man.     

Epidemiological studies on radiation carcinogenesis in human populations following acute exposure: nuclear explosions and medical radiation.

The present review provides an understanding of our current knowledge of the carcinogenic effect of low-dose radiation in man, and surveys the epidemiological studies of human populations exposed to nuclear explosions and medical radiation. Discussion centers on the contributions of quantitative epidemiology to present knowledge, the reliability of the dose-incidence data, and those relevant epidemiological studies that provide the most useful information for risk estimation of cancer induction in man. Reference is made to dose-incidence relationships from laboratory animal experiments where they may obtain, for problems and difficulties in extrapolation from data obtained at high doses to low doses, and from animal data to the human situation. The paper describes the methods of application of such epidemiological data for estimation of excess risk of radiation-induced cancer in exposed human populations and discusses the strengths and limitations of epidemiology in guiding radiation protection philosophy and public health policy.     

Quantitative carbon-14 autoradiography at the cellular level: Principles and application for cell kinetic studies

Summary Amounts of radio-labelled substances as low as 10^–18 moles incorporated into individual cells can be measured by utilizing techniques of quantitative autoradiography. For this purpose, radioactive standard sources are processed with the labelled cells smeared to slides. Carbon-14 is a favourable isotope with regard to minimal loss of -disintegrations due to self-absorption, and to limited cross-fire effects complicating the attribution of silver grains to individual cells. Silver grain densities can be counted by automated microphotometry allowing on-line data processing by an interfaced computer.Rate measurements of¹⁴C-thymidine incorporation into individual cells yield values of the DNA synthesis rate provided that the endogenous pathway of thymidine-phosphate formation has been previously blocked. From the rate values of individual cells the DNA synthesis time of a cell compartment is derived. This is an essential time parameter for the evaluation of kinetic events in proliferating cell populations. This method is applicable to human cells without radiation hazard to man, and provides an optimal source of detailed information on the kinetics of normal and diseased human haematopoiesis. Examples of application consist of thalassaemia, malaria infection, iron deficiency anaemia and acute myelogenous leukaemia.     

Radiation protection of the environment: anthropocentric and eco-centric principles

The second half of the XX century was dominated in the field of radiation protection of the environment by the anthropocentric concept stated by the International Commission on Radiological Protection (ICRP). According to this concept "if man is adequately protected by radiological standards then biota are also adequately protected". At the end of the XX--beginning of the XXI centuries in the area of area of radiation protection of nature an ecocentric strategy is beginning to develop where emphasis has swung to the protection of biota in their environment. Inadequacy of ICRP's anthroposentric concept is reported. Issues are discussed such as ecological dosimetry, nonequidosal irradiation of man and biota, criteria for estimating radiation induced changes in biota and man, as well as the need to harmonize permissible exposure doses to man and biota. An urgent need is stressed to develop a single (synthetic) concept of radiation protection which simultaneously ensures protection of human health and biota well-being in their environment. This concept is to be based on the recognition of the integrity of socio-natural ecosystems where man and biota are considered as a unity.     

The construction of computer tomographic phantoms and their application in radiology and radiation protection

Summary In order to assess human organ doses for risk estimates under natural and man made radiation exposure conditions, human phantoms have to be used. As an improvement to the mathematical anthropomorphic phantoms, a new family of phantoms is proposed, constructed from computer tomographic (CT) data. A technique is developed which allows any physical phantom to be converted into computer files to be used for several applications. The new human phantoms present advantages towards the location and shape of the organs, in particular the hard bone and bone marrow. The CT phantoms were used to construct three dimensional images of high resolution; some examples are given and their potential is discussed. The use of CT phantoms is also demonstrated to assess accurately the proportion of bone marrow in the skeleton. Finally, the use of CT phantoms for Monte Carlo (MC) calculations of doses resulting from various photon exposures in radiology and radiation protection is discussed.Dedicated to Prof. W. Jacobi on the occasion of his 60th birthday     

Absence of correlation between the chromosomal radiosensitivity of peripheral blood lymphocytes and stem-cell spermatogonia in mammals

To evaluate the reliability of quantitative extrapolation of radiation-induced chromosomal damage from somatic cells to germ cells, data on the effects of several biological and physical factors on the chromosomal radiosensitivity of blood lymphocytes and stem-cell spermatogonia have been collected from the literature. The results show that most of the factors considered, such as chromosomal constitution, age, genetic constitution, species, sampling time and dose fractionation, had differential effects on the induction of chromosomal aberrations in both systems. These differential effects can easily be explained in terms of the biological differences between in-vitro-stimulated peripheral blood lymphocytes and stem-cell spermatogonia. It is concluded that only direct experiments on germ cells of higher primates and man can be used for a quantitative estimation of human genetic radiation risks arising from structural chromosomal aberrations.     

Human cell transformation in the study of sunlight-induced cancers in the skin of man

Human cell transformation provides a powerful approach to understanding--at the cellular and molecular levels--induction of cancers in the skin of man. A principal approach to this problem is the direct transformation of human skin cells by exposure to ultraviolet and/or near-UV radiation. The frequency of human cells transformed to anchorage independence increases with radiation exposure; the relative transforming efficiencies of different wavelengths implies that direct absorption by nucleic acids is a primary initial event. Partial reversal of potential transforming lesions by photoreactivation suggests that pyrimidine dimers, as well as other lesions, are important in UV transformation of human cells. Human cells can also be transformed by transfection with cloned oncogenes, or with DNAs from tumors or tumor cell lines. Cells treated by the transfection procedure (but without DNA) or cells transfected with DNAs from normal mammalian cells or tissues show only background levels of transformation. Human cells can be transformed to anchorage-independent growth by DNAs ineffective in transformation of NIH 3T3 cells (including most human skin cancers), permitting the analysis of oncogenic molecular changes even in tumor DNAs difficult or impossible to analyze in rodent cell systems.     

Metabolic and functional studies on post-mortem human brain

The evidence that samples of human brain tissue obtained at autopsy may be used as starting material for the isolation of cellular and subcellular preparations which exhibit metabolic and functional activity when incubated in vitro has been reviewed. Supporting evidence has been found in data from model experiments which used animal brain as the source material. Active preparations have been obtained after considerable (up to 24 h) post mortem delays. Such findings are less surprising when the post mortem stability of key tissue components (enzymes, receptors, nucleic acids) and the retention of cellular integrity are examined. The data from these fields have been reviewed and their relevance to functional studies assessed. Studies which use human autopsy material must consider many additional sources of variation not found in experiments with animal brain and the major problems are briefly discussed. It is argued that functional experiments present few, if any, difficulties not already inherent in static analyses of autopsy material and some procedures which help to minimise these difficulties are outlined. Experimentation in this area is greatly aided by the finding that metabolically and functionally active preparations may be obtained from frozen tissue pieces. Dynamic studies provide a new approach for testing hypotheses of the mechanisms underlying human brain disorders and for studying the actions of neuroactive drugs in man.     

Gene expression profiles of normal human fibroblasts after exposure to ionizing radiation: a comparative study of low and high doses

Several types of cellular responses to ionizing radiation, such as the adaptive response or the bystander effect, suggest that low-dose radiation may possess characteristics that distinguish it from its high-dose counterpart. Accumulated evidence also implies that the biological effects of low-dose and high-dose ionizing radiation are not linearly distributed. We have investigated, for the first time, global gene expression changes induced by ionizing radiation at doses as low as 2 cGy and have compared this to expression changes at 4 Gy. We applied cDNA microarray analyses to G1-arrested normal human skin fibroblasts subjected to X irradiation. Our data suggest that both qualitative and quantitative differences exist between gene expression profiles induced by 2 cGy and 4 Gy. The predominant functional groups responding to low-dose radiation are those involved in cell-cell signaling, signal transduction, development and DNA damage responses. At high dose, the responding genes are involved in apoptosis and cell proliferation. Interestingly, several genes, such as cytoskeleton components ANLN and KRT15 and cell-cell signaling genes GRAP2 and GPR51, were found to respond to low-dose radiation but not to high-dose radiation. Pathways that are specifically activated by low-dose radiation were also evident. These quantitative and qualitative differences in gene expression changes may help explain the non-linear correlation of biological effects of ionizing radiation from low dose to high dose.     

Models and assumptions underlying genetic risk assessment

Various methods employed for estimating the genetic risks of radiation are reviewed. With the doubling-dose method, genetic damage is expressed as an increase in cases of known genetic disease. The actual doubling dose is based on figures obtained with the mouse. There have been no recent data on induced mutation frequencies. Recent results suggest that the prevalence figure for multifactorial disease may be at least one order of magnitude higher than before. Various assumptions underlying the doubling-dose concept are discussed in the light of recent findings on: (1) spontaneous mutations resulting from insertion elements, and (2) the comparability between spontaneous and induced mutations. The so-called direct method makes use of figures for induction of dominant mutations affecting the skeleton and the lens of the eye in the mouse, and of translocation induction in monkeys. Induction rates are converted to overall rates of induced dominant effects in man by applying certain assumptions. The proportionality between dose and effect is the basis for all genetic risk assessments. The possible significance of data on human lymphocytes indicating a threshold below 4 rad and the induction of repair enzymes by low radiation doses is discussed. The parallelogram approach is based on the principle that estimates can be obtained on the amount of genetic damage that cannot always be assessed directly. Thus mutations in mouse germ cells can be predicted by using mutation frequencies in cultured mammalian cells and O6-ethylguanine adducts. Measurement of haemoglobin mutations in human and mouse erythrocytes, and of HPRT-deficient mutations in lymphocytes of man and mouse should make more precise estimates of mutation frequencies in human germ cells possible. The development of a database on mutations in somatic cells of the mouse, their induction frequencies and molecular nature are considered an important priority. Used in combination with mouse germ-cell mutation frequencies, they should enable more precise risk estimates on the basis of mutations in somatic cells of man.     

Proteome alterations in gamma-irradiated human T-lymphocyte leukemia cells

Analyses of the protein expression profiles of irradiated cells may be beneficial for identification of new biomolecules of radiation-induced cell damage. Therefore, in this study we exploited the proteomic approach to identify proteins whose expression is significantly altered in gamma-irradiated human T-lymphocyte leukemia cells. MOLT-4 cells were irradiated with 7.5 Gy and the cell lysates were collected at different times after irradiation (2, 5 and 12 h). The proteins were separated by two-dimensional electrophoresis and quantified using an image evaluation system. Proteins exhibiting significant radiation-induced alterations in abundance were identified by peptide mass fingerprinting. We identified 14 proteins that were either up- or down-regulated. Cellular levels of four of the proteins (Rho GDP dissociation inhibitor 1 and 2, Ran binding protein 1, serine/threonine protein kinase PAK2) were further analyzed by two-dimensional immunoblotting to confirm the data obtained from proteome analysis. All identified proteins were classified according to their cellular function, including their participation in biochemical and signaling pathways. Taken together, our results suggest the feasibility of the proteome method for monitoring of cellular radiation responses.     

Studies on T cells in newborns. Higher reactivity of umbilical cord blood lymphocytes to PHA as measured by whole blood microtechnique.

The capacity of human foetal lymphocytes to respond to PHA and to form E-rosettes have been compared with data from adult individuals. For this purpose a microculture system that uses whole blood and avoids the problems of lymphocyte separation, has been developed. Foetal lymphocytes reached optimal stimulation with lower dosis of PHA (31,2 microgram/ml) as compared with adult cells (125-252 microgram/ml). However their quantitative response (measured by 14C-thymidine uptake) was equal in both groups. In addition, peripheral T cells (E-rosetting cells) reached values of 36.47 +/- 9% in newborn and 49.6 +/- 10% in normal adult controls. These results are discussed as to the status and development of cellular inmunity in human foetus.     

Human γγ-Enolase: Two-Site Immunoradiometric Assay with a Single Monoclonal Antibody

Abstract: A monoclonal antibody (mAb), termed BBS/NC/VI-H14 (H14), that reacts with the human enzyme γγ-enolase was prepared. It was directed against the γ-subunit and did not cross-react with the α- or β-subunit. The mAb H14 can be used for quantitative determination of γγ-enolase in a two-site immunoradiometric assay (two-site IRMA). It is also suitable for immunostaining formalin-fixed tissues. The specific identification of γγ-enolase provided by the two-site IRMA with H14 is discussed in relation to the cellular distribution of this protein.     

Mapping of 443 porcine EST improves the comparative maps for SSC1 and SSC7 with the human genome

Numerous mapping studies of complex traits in the pig have resulted in quantitative trait loci (QTL) intervals of 10-20 cM. To improve the chances to identify the genes located in such intervals, increased expressed sequence tags (EST)-based marker density, coupled with comparative mapping with species whose genomes have been sequenced such as human and mouse, is the most efficient tool. In this study, we mapped 443 porcine EST with a radiation hybrid (RH) panel (384 had LOD > 6.0) and a somatic cell hybrid panel. Requiring no discrepancy between two-point and multipoint RH data allowed robust assignment of 309 EST, of which most were located on porcine chromosomes (SSC) 1, 4, 7, 8 and X. Moreover, we built framework maps for two chromosomes, SSC1 and SSC7, with mapped QTL in regions with known rearrangement between pig and human genomes. Using the Blast tool, we found orthologies between 407 of the 443 pig cDNA sequences and human genes, or to existing pig genes. Our porcine/human comparative mapping results reveal possible new homologies for SSC1, SSC3, SSC5, SSC6, SSC12 and SSC14 and add markers in synteny breakpoints for chromosome 7.     

The origin of modern humans: A cytogenetic model

A new model of the origin of man is proposed on the basis of recent studies on cytogenetics of chromosomal Q-heterochromatin regions (Q-HRs) in man and other higher primates. This model is based on the following facts: a) chromosomal Q-HRs were found in the genome of only three higher primates (man, the chimpanzee and the gorilla); b) chromosomal Q-HRs in the human genome, unlike those in apes, exhibit considerable quantitative variability; c) the number of human chromosomal Q-HRs in the genome has a selective value in the adaptation of human populations to various environmental conditions. According to this model, the three major morphofunctional distinctions of man—great physiological flexibility, characteristic morphological structure, and conceptual thinking—arose as a result of the capacity of our remote ancestors to broadly change their genome mass owing to features of chromosomal Q-HRs that are only intrinsic to man. We feel that genome-mass variability through chromosomal Q-HRs allowed man to adapt himself to various environments over such a short period of time.     

Predicting cancer rates in astronauts from animal carcinogenesis studies and cellular markers

The radiation space environment includes particles such as protons and multiple species of heavy ions, with much of the exposure to these radiations occurring at extremely low average dose-rates. Limitations in databases needed to predict cancer hazards in human beings from such radiations are significant and currently do not provide confidence that such predictions are acceptably precise or accurate. In this article, we outline the need for animal carcinogenesis data based on a more sophisticated understanding of the dose-response relationship for induction of cancer and correlative cellular endpoints by representative space radiations. We stress the need for a model that can interrelate human and animal carcinogenesis data with cellular mechanisms. Using a broad model for dose-response patterns which we term the "subalpha-alpha-omega (SAO) model", we explore examples in the literature for radiation-induced cancer and for radiation-induced cellular events to illustrate the need for data that define the dose-response patterns more precisely over specific dose ranges, with special attention to low dose, low dose-rate exposure. We present data for multiple endpoints in cells, which vary in their radiosensitivity, that also support the proposed model. We have measured induction of complex chromosome aberrations in multiple cell types by two space radiations, Fe-ions and protons, and compared these to photons delivered at high dose-rate or low dose-rate. Our data demonstrate that at least three factors modulate the relative efficacy of Fe-ions compared to photons: (i) intrinsic radiosensitivity of irradiated cells; (ii) dose-rate; and (iii) another unspecified effect perhaps related to reparability of DNA lesions. These factors can produce respectively up to at least 7-, 6- and 3-fold variability. These data demonstrate the need to understand better the role of intrinsic radiosensitivity and dose-rate effects in mammalian cell response to ionizing radiation. Such understanding is critical in extrapolating databases between cellular response, animal carcinogenesis and human carcinogenesis, and we suggest that the SAO model is a useful tool for such extrapolation.     

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.     

Cellular transformation by radiation: Induction,promotion, and inhibition

Mammalian cell cultures offer powerful tools for evaluating qualitatively and quantitatively the oncogenic potential of radiation over a wide range of doses with particular importance at the low dose range that is relevant to human exposure and risk. Our studies have shown that early events in the process of radiation induced transformation in both rodent and human cells requires initial replication for fixation of transformation as a hereditary property of the cells and further clonal expansion for full expression. Early events (fixation) are inhibited by cell–cell contact and high cell density but can be modified at low temperature where repair processes are slowed. Cell–cell contact and communication in tissue organization may be in part responsible for our findings that radiation oncogenesis induced in utero in hamsters is expressed at a lower frequency than that induced in vitro. Quantitative studies carried out on hamster embryo cells indicate that neutrons are more effective in their carcinogenic potential than x-rays but also more toxic, that splitting the dose of x-rays at low doses leads to enhanced transformation, but that at high doses protracted radiation has a sparing effect. At all dose ranges survival was increased by protracting the radiation dose, thus suggesting that different repair processes must be involved for survival and transformation. Similar observations were seen when the protease inhibitor Antipain was found to enhance transformation in rodent and human cells when present at the time of radiation, but was protective when added after radiation. Survival was not modified under any of those conditions, and Antipain did not affect DNA replication and repair. In our qualitative studies, once cells are transformed by radiation, they exhibit a wide range of structural and functional phenotypic changes, some of which are membrane-associated and are expressed within days after induction. Our current studies on nutritional and hormonal influences on radiation transformation indicate the following: Pyrolysate products from broiled protein foods act in synergism with radiation to produce transformation, whereas vitamin A analogs are powerful, preventive agents. Retinoids inhibit both x-ray-induced transformation and its promotion by TPA: these modifications (enhancement by TPA, inhibition by retinoids) are not reflected in sister chromatid exchanges, but are reflected in the level of membrane associated enzymes Na/K ATPase. Whereas retinoids modify late events (expression, promotion), we find that thyroid hormone plays a crucial role in the early phases of radiation and chemically induced transformation. Under hypothyroid conditions no transformation is observed. The addition of triiodothyronine at physiological levels results in a transformation rate that is dose-related. Our recent success in transforming human skin fibroblasts will enable quantitative and qualitative studies of radiation carcinogenesis in a system relevant to man.     

Should human chondrocytes fly? The impact of electromagnetic irradiation on chondrocyte viability and implications for their use in tissue engineering

A significant logistic factor as to the successful clinical application of the autologous tissue engineering concept is efficient transportation: the donor cells need to be delivered to tissue processing facilities which in most cases requires air transportation. This study was designed to evaluate how human chondrocytes react to X-ray exposure. Primary cell cultures were established, cultured, incubated and exposed to different doses and time periods of radiation. Subsequently, quantitative cell proliferation assays were done and qualitative evaluation of cellular protein production were performed. Our results show that after irradiation of chondrocytes with different doses, no significant differences in terms of cellular viability occurred compared with the control group. These results were obtained when chondrocytes were exposed to luggage transillumination doses as well as exposure to clinically used radiation doses. Any damage affecting cell growth or quality was not observed in our study. However, information about damage of cellular DNA remains incomplete.