Biomedical subjects

R K Sachs

Publications and source records attributed to R K Sachs.

68 records · Page 4Linked to original sources

PubMed1993

DNA damage in non-proliferating cells subjected to ionizing irradiation at high or low dose rates.

Ionizing radiation damage to the genome of a non-cycling mammalian cell is analyzed using continuous time Markov chains. Immediate damage induced by the radiation is modeled as a batch Poisson arrival process of DNA double strand breaks (DSBs). Different kinds of radiation, for example gamma rays or alpha particles, have different batch probabilities. Enzymatic modulation of the immediate damage is modeled as a Markov process similar to the processes described by the master equation of stochastic chemical kinetics. An illustrative example is the restitution/complete exchange model, which postulates that radiation induced DSBs can subsequently either undergo enzymatically mediated repair (restitution) or can participate pairwise in chromosome exchanges, some of which make irremediable lesions such as dicentric chromosome aberrations. One may have rapid irradiation followed by enzymatic DSB processing or have prolonged irradiation with both DSB arrival and enzymatic DSB processing continuing throughout the irradiation period. A complete solution of the Markov chain is known for the case that the exchange rate constant is negligible so that no irremediable chromosome lesions are produced and DSBs are the only damage to the genome. Using PDEs for generating functions, a perturbation calculation is made assuming the exchange rate constant is small compared to the repair rate constant. Some non-perturbative results applicable to very prolonged irradiation are also obtained using matrix methods: Perron-Frobenius theory, variational methods and numerical approximations of eigenvalues. Applications to experimental results on expected values, variances and statistical distributions of DNA lesions are briefly outlined. Continuous time Markov chain models are the most systematic of those current radiation damage models which treat DSB-DSB interactions within the cell nucleus as homogeneous (e.g. ignore diffusion limitations). They contain most other homogeneous models as special cases, limiting cases or approximations. However, applying the continuous time Markov chain models to studying spatial dependence of DSB interactions, which is generally believed to be very important in some situations, presents difficulties.

Animals↗

PubMed1993

Effect of LET on chromosomal aberration yields. I. Do long-lived, exchange-prone double strand breaks play a role?

Dicentric chromosomal aberrations produced by ionizing radiation probably result from pairwise interaction of DNA double strand breaks (dsbs). It has been suggested that high LET radiation may preferentially produce a subclass of 'severe' dsbs that are long-lived and/or exchange-prone, and that it is the production of these severe dsbs which account for the increased biological effectiveness of high-LET radiation. We present a quantitative formalism to describe the induction of these severe dsbs, and the subsequent production of exchange-type chromosomal aberrations. Using a Markov model and microdosimetric methods, we conclude that dicentric production by such severe dsbs has properties similar to those observed at high LET. Specifically, at high doses, the yield is nearly linear with dose even if dsbs from different tracks interact. The model is applied to published data on dicentric aberrations produced by irradiation of human lymphocytes in vitro. Corrections for the effects of interphase death are estimated. From comparisons with the experiments we conclude that interaction of severe dsbs could make a significant contribution to the observed dicentric production at high LET and also perhaps for low doses (though not high doses) at low LET. Proximity explanations of high-LET effects continue to offer the main prospect for obtaining a unified picture of chromosomal aberration formation by all ionizing radiation types, but a hybrid model in which severe dsbs contribute to the high-LET aberration yield cannot be ruled out. If all or part of high-LET radiation damage is qualitatively different from low-LET radiation damage, as this severe dsb model may suggest, there could be far-reaching implications for the field of radiation protection.

Animals↗

PubMed1993

Ratios of radiation-produced chromosome aberrations as indicators of large-scale DNA geometry during interphase.

Chromosome aberrations produced by ionizing radiation are assumed to develop from DNA double-strand breaks (DSBs) which interact pairwise. Stable chromosome aberrations that exemplify inter- and intra-chromosomal exchanges are, respectively, translocations and pericentric inversions. By comparing the number of these for each chromosome one can infer results on the randomness of DSB induction or exchange formation and on large-scale chromosome geometry. We analyze frequencies of translocations and pericentric inversions in lymphocytes from 38 A-bomb survivors, using G-banding. A total of 636 translocations and 102 pericentric inversions were found. The 636/102 ratio of translocations to pericentric inversions is approximately 14 times smaller than predicted by a random model, in general agreement with earlier results and results on the ratio of dicentrics to centric rings for in vitro irradiation. Presumably the excess of intra-chromosomal exchanges is due to a spatial proximity effect, implying a localization of chromosomes within the cell nucleus during and shortly after irradiation. The distribution of the pericentric inversions among different chromosomes indicates this proximity effect is roughly the same for all chromosomes, regardless of DNA content; i.e., the ratio of pericentric inversions for two different chromosomes approximately equals the ratio given by a model which takes into account chromosome lengths and centromere locations but otherwise assumes randomness. Possible exceptions are chromosomes 7 and 12, which show some excess of pericentric inversions. The percentage of translocations involving each chromosome corresponds roughly to the percentage expected assuming randomness, except that for chromosome 1 there is a significant excess.

Chromosome Aberrations↗

PubMed1992

Evolution of DNA damage in irradiated cells.

Ionizing radiation damage to a mammalian genome is modeled using continuous time Markov chains. Models are given for the initial infliction of DNA double strand breaks by radiation and for the enzymatic processing of this initial damage. Damage processing pathways include DNA double strand break repair and chromosome exchanges. Linear, saturable, or inducible repair is considered, competing kinetically with pairwise interactions of the DNA double strand breaks. As endpoints, both chromosome aberrations and the inability of cells to form clones are analyzed. For the post-irradiation behavior, using the discrete time Markov chain embedded at transitions gives the ultimate distribution of damage more simply than does integrating the Kolmogorov forward equations. In a representative special case explicit expressions for the probability distribution of damage at large times are given in the form used for numerical computations and comparisons with experiments on human lymphocytes. A principle of branching ratios, that late assays can only measure appropriate ratios of repair and interaction functions, not the functions themselves, is derived and discussed.

Cell Survival↗

PubMed1992

DNA damage caused by ionizing radiation.

A survey is given of continuous-time Markov chain models for ionizing radiation damage to the genome of mammalian cells. In such models, immediate damage induced by the radiation is regarded as a batch-Poisson arrival process of DNA double-strand breaks (DSBs). Enzymatic modification of the immediate damage is modeled as a Markov process similar to those described by the master equation of stochastic chemical kinetics. An illustrative example is the restitution/complete-exchange model. The model postulates that, after being induced by radiation, DSBs subsequently either undergo enzymatically mediated restitution (repair) or participate pairwise in chromosome exchanges. Some of the exchanges make irremediable lesions such as dicentric chromosome aberrations. One may have rapid irradiation followed by enzymatic DSB processing or have prolonged irradiation with both DSB arrival and enzymatic DSB processing continuing throughout the irradiation period. Methods for analyzing the Markov chains include using an approximate model for expected values, the discrete-time Markov chain embedded at transitions, partial differential equations for generating functions, normal perturbation theory, singular perturbation theory with scaling, numerical computations, and certain matrix methods that combine Perron-Frobenius theory with variational estimates. Applications to experimental results on expected values, variances, and statistical distributions of DNA lesions are briefly outlined. Continuous-time Markov chains are the most systematic of those radiation damage models that treat DSB-DSB interactions within the cell nucleus as homogeneous (e.g., ignore diffusion limitations). They contain virtually all other relevant homogeneous models and semiempirical summaries as special cases, limiting cases, or approximations. However, the Markov models do not seem to be well suited for studying spatial dependence of DSB interactions, which is known to be important in some situations.

Animals↗

PubMed1992

Modelling the formation of polycentric chromosome aberrations.

Exchange-type chromosome aberrations produced by ionizing radiation or restriction enzymes are believed to result from pairwise interaction of DNA double-strand breaks (dsb). In addition to dicentrics, such aberrations may include higher-order polycentrics (tricentrics, tetracentrics, etc.). We have developed computer programs that calculate the probability of the various polycentrics for a given average number of pairwise interactions. Two models are used. Model I incorporates kinetic competition between restitution, complete exchanges (illegitimate recombination events), and incomplete exchanges. Model II allows unrestituted breaks even if there is no recombination. The models were applied to experimental observations of aberrations produced in G1 Chinese hamster ovary cells after electroporation with the restriction enzyme PvuII, which produces blunt-end dsb. We found, experimentally and theoretically, that there was a maximum in the number and multiplicity of polycentrics per cell: beyond a certain PvuII concentration no additional or higher-order polycentrics were produced. Computer-generated relationships, which were remarkably similar for both models and for all values of the adjustable parameters, were found between dicentrics per cell and higher-order polycentrics per cell. Excellent agreement was found between the experimental observations and the consensus theoretical curve relating tricentrics per cell to dicentrics per cell. The observed number of higher polycentrics per cell for a given number of dicentrics per cell was somewhat larger than the consensus theoretical prediction. The observed number of centric rings per cell was markedly larger than the consensus theoretical value, presumably owing to intrachromosomal localization ('proximity effects'). The computer models also provided estimates for the adjustable parameters; for example, in model I the fraction of incomplete exchanges was found to be about 35%.

Animals↗

PubMed1992

The ratio of dicentrics to centric rings produced in human lymphocytes by acute low-LET radiation.

Chromosome aberrations produced by ionizing radiation are assumed to develop from DNA double-strand breaks (DSBs) which interact pairwise, in an exchange event. Dicentrics and centric rings are aberrations that exemplify inter- and intrachromosomal exchanges, respectively. We show from a survey of published data that for acute low-LET irradiation of resting human lymphocytes the observed ratio of dicentrics to centric rings is approximately five times smaller than predicted by a pairwise interaction model which assumes complete randomness. Such a low ratio can be interpreted as evidence for a proximity effect, favoring exchanges of an intrachromosomal type. That is, since DSBs induced close together have an above-average chance of pairwise interaction, the observed excess of centric rings indicates that at the time of irradiation there is some degree of spatial confinement for the two arms of a single chromosome. Assuming the excess of centric rings is indeed due to proximity effects, the data are used to estimate that the volume of a domain, within which any one lymphocyte chromosome is localized at one instant during the G0/G1 phase, is at most approximately 20% of the nuclear volume.

Chromosome Aberrations↗

PubMed1990

Dose-rate dependent stochastic effects in radiation cell-survival models.

When cells are subjected to ionizing radiation the specific energy rate (microscopic analog of dose-rate) varies from cell to cell. Within one cell, this rate fluctuates during the course of time; a crossing of a sensitive cellular site by a high energy charged particle produces many ionizations almost simultaneously, but during the interval between events no ionizations occur. In any cell-survival model one can incorporate the effect of such fluctuations without changing the basic biological assumptions. Using stochastic differential equations and Monte Carlo methods to take into account stochastic effects we calculated the dose-survival relationships in a number of current cell survival models. Some of the models assume quadratic misrepair; others assume saturable repair enzyme systems. It was found that a significant effect of random fluctuations is to decrease the theoretically predicted amount of dose-rate sparing. In the limit of low dose-rates neglecting the stochastic nature of specific energy rates often leads to qualitatively misleading results by overestimating the surviving fraction drastically. In the opposite limit of acute irradiation, analyzing the fluctuations in rates merely amounts to analyzing fluctuations in total specific energy via the usual microdosimetric specific energy distribution function, and neglecting fluctuations usually underestimates the surviving fraction. The MOnte Carlo methods interpolate systematically between the low dose-rate and high dose-rate limits. As in other approaches, the slope of the survival curve at low dose-rates is virtually independent of dose and equals the initial slope of the survival curve for acute radiation.

Cell Survival↗

PubMed1990

Incorporating dose-rate effects in Markov radiation cell survival models.

Markov models for the survival of cells subjected to ionizing radiation take stochastic fluctuations into account more systematically than do non-Markov counterparts. Albright's Markov RMR (repair-misrepair) model (Radiat. Res. 118, 1-20, 1989) and Curtis's Markov LPL (lethal-potentially lethal) model [in Quantitative Mathematical Models in Radiation Biology (J. Kiefer, Ed.), pp. 127-146. Springer, New York, 1989], which assume acute irradiation, are here generalized to finite dose rates. Instead of treating irradiation as an instantaneous event we introduce an irradiation period T and analyze processes during the interval T as well as afterward. Albright's RMR transition matrix is used throughout for computing the time development of repair and misrepair. During irradiation an additional matrix is added to describe the evolving radiation damage. Albright's and Curtis's Markov models are recovered as limiting cases by taking T----0 with total dose fixed; the opposite limit, of low dose rates, is also analyzed. Deviations from Poisson behavior in the statistical distributions of lesions are calculated. Other continuous-time Markov chain models ("compartmental models") are discussed briefly, for example, models which incorporate cell proliferation and saturable repair models. It is found that for low dose rates the Markov RMR and LPL models give lower survivals compared to the original non-Markov versions. For acute irradiation and high doses, the Markov models predict higher survivals. In general, theoretical extrapolations which neglect some random fluctuations have a systematic bias toward overoptimism when damage to irradiated tumors is compared with damage to surrounding tissues.

Cell Survival↗

PubMed1989

Detection of an intrinsic marker in hypoxic cells.

An autoradiographic method is presented for detecting, within a cell population, those cells which have been subjected to chronic hypoxia. No radioisotope is administered; rather the photographic emulsion is chemically reduced by intrinsic constituents of the cells. Hypoxic regions in the sandwich system, a multicellular in vitro tumor model, were detected in this manner. These regions were then compared with hypoxic sandwich regions as demonstrated by [3H]misonidazole labeling. Auxiliary studies, including studies on hypoxic monolayers, were consistent with the sandwich results. In all cases, the intracellular distribution of the chemographic grains was found to be cytosolic. Often the grains were clustered near the nucleus, perhaps in the region of the endoplasmic reticulum and the Golgi. We conclude that cells in a state of hypoxia and nutrient deprivation similar to that found in solid tumors retain a detectably altered biology for a significant period after reoxygenation. Therefore systematic methods of detecting previous hypoxia in histological tumor sections are feasible.

Animals↗

PubMed1989

3H-misonidazole labeling and viability of hypoxic cells in the sandwich system, an in vitro tumor analogue.

3H-misonidazole was used as a marker of hypoxic cells in an in vitro tumor analogue, the sandwich system. MISO binding was assessed in situ, using autoradiography. Binding profiles indicate that there are regions of radiobiological hypoxia surrounding the necrotic center in sandwiches of the V79 cell line and in sandwiches of the 9L cell line. Grains per cell were counted and detailed statistics on the variation of intrinsic binding among cells in the same microenvironment are presented. There is a systematic decrease in the standard deviation of grains per cell as one examines populations of cells further and further from the nutrient and oxygen source. Kinetic studies show that the growth fraction of the cell population also decreases with distance from the nutrient source. These findings taken together suggest that MISO binding is proportional to cell size and cells in the inner noncycling portion of the sandwich are more nearly uniform in size. Sandwich cells which exhibit heavy MISO binding, and are presumably radiobiologically hypoxic, were shown to be still viable if restored to good nutrient and oxygen conditions.

Animals↗

PubMed1989

Comparison of 3H-misonidazole binding between CHO and 9L cells using the sandwich system.

3H-misonidazole binding of 9L cells was compared with that of CHO cells using an in vitro tumor analog, the sandwich system. In sandwiches there is a gradient of microenvironments, with cells adjacent to the necrotic center subjected to low concentrations of oxygen and glucose and to high concentrations of metabolites. Mixed sandwiches, having 9L and CHO cells interspersed, were used along with sandwiches of each individual cell line. MISO binding was assessed in situ, using autoradiography. Grains per cell were counted and detailed statistics were obtained on the variation in MISO binding among cells located in the same microenvironment. In all cases binding in the regions near the necrotic center was more than 50 times the binding found at the sandwich edge and found in control monolayers, indicating radiobiological hypoxia near the necrotic center. 9L cells began to significantly increase binding of MISO metabolites at a somewhat higher oxygen concentration than did CHO cells. At all oxygen tensions, average per cell binding of the 9L cells was 3 times or more that of the CHO cells, a factor greater than can be explained by the ratio of cell volumes alone. Statistical analyses of the variation in binding among cells in a given microenvironment give some evidence that in the mixed CHO/9L sandwiches there are interactions between the cells of the two different lines which affect the growth patterns of the cells. No preferred binding of misonidazole in the nucleus or cytoplasm was noted within the cells.

Animals↗

PubMed1989

Reducing the hypoxic fraction of a tumour model by growth in low glucose.

The question of whether growth under low glucose conditions leads to a reduced amount of cell hypoxia was investigated using an in vitro tumour analogue, the sandwich system. In this multicellular system, the interplay between diffusion and consumption of oxygen and nutrients results in spatial gradients of these environmental factors. Gradients in the environment lead to biological heterogeneity within the cell population. A necrotic centre, surrounded by a viable cell border, subsequently develops. Cells adjacent to the necrotic centre in sandwiches are hypoxic and are in an environment somewhat analogous to that of cells adjacent to necrotic regions in solid tumours. Using sandwiches of the 9L and V79 cell lines, the effects of growth under low glucose conditions on the degree of hypoxia in regions adjacent to the necrotic centre were investigated. Per-cell binding of 3H-misonidazole, assessed by autoradiography, was used as an indicator of oxygen deprivation. It was found that the extent of the hypoxic region and the severity of hypoxia were considerably reduced by growing sandwiches in a glucose concentration of 0.6 mM rather than 6.5 mM. This reduction was found in conjunction with a smaller viable border; it occurred despite the fact that the average per-cell oxygen consumption is higher in the low glucose sandwiches. The data are qualitatively consistent with a joint oxygen-glucose deprivation model for cell necrosis.

Animals↗

PubMed1988

Joint oxygen-glucose deprivation as the cause of necrosis in a tumor analog.

The sandwich system was recently developed as an in vitro tumor analog. Like spheroids, sandwiches are organized, multicellular systems in which the interplay between diffusion and consumption leads to the formation of spatial gradients; a necrotic center and a viable cell border subsequently develop. Using sandwiches of the 9L and V79 cell lines, the effects of oxygen and glucose deprivation on the onset and formation of necrosis were investigated. The data indicate that in sandwiches necrosis is a result of a shortage of both substances. Complementary cell monolayer experiments to determine a number of consumption parameters were performed. On the basis of the data, we propose a joint oxygen-glucose deprivation model for V79 cell necrosis. It is assumed a cell dies when oxygen deprivation in conjunction with glucose deprivation lowers the cell's ATP production rate below a critical value. Interactions of the concentrations and consumptions of oxygen and glucose are analyzed theoretically; concentration profiles are obtained by numerically solving coupled non-linear integral equations arising from the diffusion equation. The predicted viable border widths are in good agreement with the observed values.

Animals↗