X-ray-induced translocations in spermatoginia II. Fractionation responses in mice

The dose-response curve for reciprocal translocations induced by X-rays in spermatognial stem cells, and observed in primary spermatocytes of mice, is “hymp-shaped”, with a maximum yield at about 600 R. To test the hypothesis that the decrease in yield with increasing dose above 600 R is a consequence of the different sensitivities of cells in different stages of the cell cycle to both cell killing and chromosome aberration induction, several fractionation experiments were carried out.A total dose of 2800 R was given in repeated doses of 400 R, separated by 8-week intervals. The yield of translocations is that expected for additivity; for example, the yield at 1600 R is approximately equal to that for four separate 400-R doses.When the total dose (500 R) which gives a translocation yield on the ascending part of the dose-response curve is given as two equal fractions separated by intervals of 30, 90, or 150 min, the translocation yield decreases with increasing interval. However, when a total dose (1000 R) which would give a translocation yield on the descending part of the dose-response curve is given in two equal fractions separated by intervals of from 30 min to 6 weeks, the response is different; the translocation yield increases with intervals up to 18 h, then decreases with intervals up to 4 weeks, and finally increases again to a yield equal to additivity with an interval of 6 weeks. These changes in translocation yield with changes in interval between the two doses are explained in terms of the differential sensitivity of cells to killing and aberration induction in the different phases of the cell cycle, and by assuming that the cells surviving the first dose and repopulating the testis different cycle characteristics from normal cells.     

A comparative study of the frequencies of radiation-induced chromosome aberrations in somatic and germ cells of the rhesus monkey (Macaca mulatta)

Frequencies of radiation-induced chromosome aberrations in spermatogonia, peripheral blood lymphocytes and bone-marrow cells of the rhesus monkey (Macaca mulatta) and in human blood lymphocytes, were determined at different exposures of X-rays. The dose-response curve for the induction of reciprocal translocations in spermatogonia suggested a “hump” at about 200 rad. The absolute frequencies of chromosome aberrations in somatic and germ cells of the rhesus monkey were low in comparison with most other mammalian species and the ratio between aberrations in the two tissues was 25 to 1 at the 100 rad level. Although the numbers of “effective chromosome arms” in man and rhesus monkey are similar (81 vs. 83), the rhesus monkey showed a lower rate of induction of dicentrics in blood lymphocytes than man at all doses, reaching statistical significance at the 300 rad level.     

X-Ray-Induced Chromosome Aberrations in Mouse Dictyate Oocytes. II. Fractionation and Dose Rate Effects

Split-dose experiments were done on maturing dictyate oocytes to determine if the magnitude of the first dose influenced the "rejoining time" of radiation-induced chromosomal lesions. A total dose of 400r was split into various combinations with varying fractionation intervals. The data derived from analyzing interchanges indicate that there is no difference in the rejoining time whether the first dose was 100, 200, or 300r. It thus appears that the radiation dose in the ranges studied does not significantly alter the rate of repair of the chromosomal lesions. This conclusion is contrary to that which has been propounded to explain the nonlinear dose curves obtained for specific locus mutations.

Chronic ⁶⁰Co γ-ray exposures were given to female mice over an 8-day period. The exposures were delivered during the period of peak sensitivity, i.e., 8–16 days prior to ovulation. The doses given were 117, 240, 348, and 483r. The aberration yields observed were dramatically lower than for comparable doses of acute X rays even when the RBE of γ rays compared with X rays is taken into account. The large drop in yields at the low dose rates is interpreted as resulting from a large two-track component in the acute curve, and as being independent of effects on repair systems.

     

Activation of T cells by glutaraldehyde-fixed erythrocyte antigens: radiosensitivity of cells mediating delayed-type hypersensitivity reactions in the mouse.

Mice immunized with glutaraldehyde-fixed sheep red blood cells (G-SRBC) show delayed-type hypersensitivity (DTH) reactions to G-SRBC or SRBC. The specificity of the DTH reaction of mice sensitized with glutaraldehyde-fixed antigens is similar to that found after sensitization with unfixed antigens. The dose-response curve for sensitization by glutaraldehyde-fixed SRBC was very different from the curve for normal SRBC. At low doses, both antigens were effective in sensitizing to show DTH but neither induced an antibody response. However, at high antigen doses, only the glutaraldehyde-fixed antigen was efficient in sensitizing to show DTH and it failed to raise an antibody titer. Spleen cells of mice sensitized with fixed RBC can transfer DTH locally but if the donor cells are irradiated (500 R), the transfer is abrogated. In contrast, the transfer of DTH by spleen cells of mice immunized with unfixed antigen is not affected by 500 R. The transfer of DTH by spleen cells of mice immunized with fixed antigen can be blocked by “in vitro desensitization” while the transfer of DTH by spleen cells from mice primed with normal antigen is resistant to “in vitro desensitization.” These results suggest that immunization of mice with different physical states of the same antigen can result in the activation of antigen-specific T cells which exhibit markedly different properties.     

Fourier-transform,infrared difference-spectrometry for structural analysis of dextrans

The Fourier-transform (F.t.), infrared (i.r.) spectra of a series of branched dextrans were examined. The dextrans studied were those from the N R R L collection designated Leuconostoc mesenteroides B-1142, B-1191, B-1299 fraction S, B-1355 fraction S, B-1402, and B-1422, and Streptobacterium dextranicum B-1254 fractions S[L] and L[S]. The spectrum of a levan, N R R L L. mesenteroides B-523 fraction M, was also examined, for comparison with the spectra of the dextrans. Meaningful results were obtained by “weight-normalizing” the spectral absorbance to that of the dextran of very low degree of branching (dextran B-1254 fraction L[S]), and then subtracting this spectrum of linear dextran from each of the other polysaccharide spectra. The resulting i.r.-absorbance difference-spectra were plotted, at uniform scale-expansion across the 1800-400-cm^?1 region, resulting in difference-absorbance features at ≈ 1100 and ≈ 800 cm^?1 for all branched dextrans. These absorbance differences could be correlated to the type and degree of dextran branching, which had previously been established by permethylation analysis. It was concluded that such F.t.-i.r. difference-spectra have general application for the structural analysis of polysaccharides.     

Fucose-containing polysaccharides in the brown algae Ascophyllum nodosum and Fucus vesiculosus

The fucose-containing, sulfated polysaccharides from Ascophyllum nodosum and Fucus vesiculosus were isolated by extraction with water adjusted to pH 2. Pure fractions were carefully separated by fractional precipitation with ethanol from aqueous solutions containing magnesium or calcium chloride. Progress in the fractionation efforts and purity of the fractions isolated were established by free-boundary and cellulose acetate clectrophoresis. Ascophyllan, two “complexes”, and a galactofucan were isolated from A. nodosum. An ascophyllan-like fraction, and a “complex” were isolated from F. vesiculosus. Mild, acid hydrolysis (0.02m hydrochloric acid, 1 h, 80°) converted each of the “complexes” into an electrophoretically faster-moving and a slower-moving component. The “complex” from F. vesiculosus comprised a greater proportion of the extract than did the two “complexes” from A. nodosum. In addition, the Fucus “complex” was richer in fucose*. However, the data suggest that neither species contains a pure fucan sulfate in the native state.     

Relative biological effectiveness of x-rays and fast neutrons in inducing translocations in mouse spermatogonia

The dose-response relationships for inducing translocations in the spermatogonia of mice were studied, and they were compared for 200 kVp X-rays and 2 MeV fast neutrons. The dose response for fast neutrons was markedly convex; more precisely, the response obtained was linear in the dose range from 24 to 94 rad with a regression coefficient of 11.36·10^?4, but decreased for a further increase in dose up to 267 rad. On the other hand, that for X-rays showed a linear dose-response relationship from 48 to 672 rad with a regression coefficient of 2.69·10^?4. The relative biological effectiveness for inducing translocations in the spermatogonia of mice was compared for the linear parts of the dose response in both types of radiation, and the relative biological effectiveness (RBE) value was 4.22.     

Chemotactic attraction of Crassostrea virginica hemolymph cells to Staphylococcus lactus.

The nuclear polyhedrosis virus (NPV) of Porthetria dispar was isolated and purified through a two-step zonal centrifugation procedure. The LD₅₀ of the purified NPV was determined by a dose-response assay. Quantitative analyses were made of whole polyhedra and of separated fractions of polyhedral protein, virus rods, and denatured material, i.e., the pellet obtained from low speed centrifugation of dissolved polyhedra, to determine the protein, DNA, and “RNA” (orcinol-positive material) present in this NPV. Approximately one-half the “RNA” was present in the denatured material. Trace elements were also determined, and four, Fe, Mg, Cu, and Zn, of the ten assayed were present in the polyhedral protein fraction, while only Mg and Zn were in the virus rod fraction.     

More Accurate Dose-Response Estimation Using Monte-Carlo Uncertainty Analysis: The Data Cube Approach

The traditional q₁ ^* methodology for constructing upper confidence limits (UCLs) for the low-dose slopes of quantal dose-response functions has two limitations: (i) it is based on an asymptotic statistical result that has been shown via Monte Carlo simulation not to hold in practice for small, real bioassay experiments (Portier and Hoel, 1983); and (ii) it assumes that the multistage model (which represents cumulative hazard as a polynomial function of dose) is correct. This paper presents an uncertainty analysis approach for fitting dose-response functions to data that does not require specific parametric assumptions or depend on asymptotic results. It has the advantage that the resulting estimates of the dose-response function (and uncertainties about it) no longer depend on the validity of an assumed parametric family nor on the accuracy of the asymptotic approximation. The method derives posterior densities for the true response rates in the dose groups, rather than deriving posterior densities for model parameters, as in other Bayesian approaches (Sielken, 1991), or resampling the observed data points, as in the bootstrap and other resampling methods. It does so by conditioning constrained maximum-entropy priors on the observed data. Monte Carlo sampling of the posterior (constrained, conditioned) probability distributions generate values of response probabilities that might be observed if the experiment were repeated with very large sample sizes. A dose-response curve is fit to each such simulated dataset. If no parametric model has been specified, then a generalized representation (e.g., a power-series or orthonormal polynomial expansion) of the unknown dose-response function is fit to each simulated dataset using “model-free” methods. The simulation-based frequency distribution of all the dose-response curves fit to the simulated datasets yields a posterior distribution function for the low-dose slope of the dose-response curve. An upper confidence limit on the low-dose slope is obtained directly from this posterior distribution. This “Data Cube” procedure is illustrated with a real dataset for benzene, and is seen to produce more policy-relevant insights than does the traditional q₁ ^* methodology. For example, it shows how far apart are the 90%, 95%, and 99% limits and reveals how uncertainty about total and incremental risk vary with dose level (typically being dominated at low doses by uncertainty about the response of the control group, and being dominated at high doses by sampling variability). Strengths and limitations of the Data Cube approach are summarized, and potential decision-analytic applications to making better informed risk management decisions are briefly discussed.     

Translocations in mouse spermatogonia after exposure to unequally fractionated doses of x-rays

The influence of various X-ray fractionation regimes on translocation induction in mouse spermatogonia was studied. Splitting a single exposure of 600 R into two fractions of 300 R separated by 24 h did not influence the translocation yield (7.58% vs. 8.35%). When the dose was given in the unequal fractions,100 R+500 R and 500 R+100 R, with the same 24 h between the fractions, the translocation frequency was significantly altered (10,19 and 4.94%, respectively). As a possible explanation of these findings it is assumed that the relatively resistant cells surviving the first fraction of the dose are sensitized towards translocation induction when receiving the second fraction of the dose.     

Distribution of 1AL.1RS and 1BL.1RS wheat-rye translocations in Triticum aestivum using specific PCR

Chromosome arm 1RS of rye (Secale cereale) is a valuable resource for wheat (Triticum aestivum) improvement. 1AL.1RS and 1BL.1RS translocations play an important role in wheat breeding, since wheat carrying these chromosomal translocations has higher tolerance to biotic and abiotic stress. In this study, the presence of 1RS and the distribution of 1AL.1RS and 1BL.1RS wheat-rye translocations were examined in 66 Iranian cultivars and 70 regional foreign accessions of bread wheat, using three rye-specific primers (“RYER3/F3”, “O-SEC5′-A/O-SEC3′-R”, “PAWS5/S6”). Based on “RyeR3/F3”, the presence of 1RS was verified in 15 (23%) Iranian cultivars and in two (3%) foreign accessions. Further, “O-SEC5′-A/O-SEC3′-R” and “PAWS5/S6” were used to distinguish 1AL.1RS and 1BL.1RS translocations. According to results from these primers, 1BL.1RS was identified in 14 (21%) Iranian cultivars and two (3%) foreign accessions. The results confirm that “Sholeh” is the only cultivar (1.5%), among all cultivars and accessions, that carries 1AL.1RS. This study provides a useful tool in marker-assisted selection of materials containing 1RS, and in the creation of new Iranian common wheat cultivars with a larger genetic diversity in wheat breeding programs.     

The induction of thymidine- and IUdR-resistant variants in P388 mouse lymphoma cells by x-rays, UV and mono- and bi-functional alkylating agents

Dose-response curves for “mutation” to resistance to 5-iodo-2-deoxyuridine (IUdR) and excess thymidine (TdR) in P388 mouse lymphoma cells have been established after exposure of these cells to six chemical mutagens, UV |and| ionising radiations. The dose-response curves for all mutagens in both selective system show considerable similarities when induced mutation frequencies are plotted against survival. Expression time for both types of variants, IUdR^r and TdR^r, are similar, i.e. maximum frequencies are reached by 48 h and there is no fall in variant frequency at late expression times up to 144 h. Over the range of survival levels studied there appears to be little or no dependence of expression time on dose of mutagen. Some loss of mutants after high doses (i.e. at low survival levels) was observed due to the fact that a significant proportion of both TdR^r and IUdR^r clones were more sensitive to the mutagens than the wild-type population. The similarities in induced dose-response curves for different mutagens suggest that the mutants have a common origin, probably an error in repair, but it seems unlikely that errors in “cut and patch” repair are responsible. A comparison of spontaneous frequencies of IUdR^r and TdR^r variants suggests that IUdR is mutagenic in P388 cells.     

Studies on the induction of translocations in mouse spermatogonia. IV. Effects of acute gamma-irradiation

The rate of induction of reciprocal translocations by 56–816 R exposures of mouse spermatogonia to acute γ-irradiation (95 R/min) was determined by cytological examination of descendant spermatocytes. The dose-response relationship did not differ significantly from linearity and had a regression coefficient of 1.8·10⁻⁴ per R with respect to translocations per spermatocyte. Further analysis at exposures below 816 R (considered less likely to produce distortion) showed that the quadratic regression of best fit had too small a square-law component to account for the very low frequency of translocations obtained after chronic γ-exposures in a previous experiment. The possibility is discussed that there is some extra factor, besides the diminution of the square-law component, which operates to reduce the yield after protracted exposures.     

Bathymetric adaptations of reef-building corals at davies reef,great barrier reef,Australia. II. Light saturation curves for photosynthesis and respiration

Light saturation (P-I) curves for oxygen production and consumption were constructed in the laboratory for corals collected from depths of 1 to 45 m on the southeastern (seaward) side of Davies Reef, Great Barrier Reef, Australia. This depth range represents a gradient of from 82.6 to 2.9% of surface light, which was measured as photosynthetic photon flux density (PPFD) between 400 and 700 nm. Adaptative changes in photokinetic parameters were observed over the entire range of light intensities. The compensation intensity (I_c), I_k, the intensity at which photosynthesis was 95% light saturated (I_0.95), and the respiration rate (?R), all decreased with decreasing light intensity. The maximal rate of photosynthesis when normalized on the basis of coral chlorophyll-a content (P_ma^g) tended to decrease with decreasing irradiance but the change was not statistically significant. The $\text{P}\text{R}$ ratio ($\text{P}{}_{\mathrm{m}}{}^{\mathrm{g}}\text{?R}$), the initial slope of a light saturation curve (α), and the maximum rate of photosynthesis when normalized by coral protein content (P_mp^g), all increased with decreasing light intensity. The logarithms of the values for each variable parameter were proportional to the logarithms of the fraction of the surface PPFD (T) transmitted to the depth at which the corals grew. This enabled changes in these parameters to be accurately estimated for corals growing at any depth on the reef. Comparison of experimental data with published values for “saturating” irradiance and P_ma^g, suggests that the endosymbiotic algae within reef-building corals are photosynthetically intermediate between classical “sun” and “shade” plants.     

Interindividual variability in the enantiomeric disposition of ibuprofen following the oral administration of the racemic drug to healthy volunteers

The plasma disposition of the enantiomers of ibuprofen has been investigated following the oral administration of the racemic drug (400 mg) to 24 healthy male volunteers. The plasma elimination of (R)-ibuprofen was found to be more rapid than that of the S-enantiomer [plasma half-life: (R) 2.03 h; (S) 3.05 h; 2P less than 0.001], resulting in a progressive enrichment in the plasma content of this isomer, some 64% of the total area under the plasma concentration time curves (AUC) being due to the pharmacologically active enantiomer. The influence of dose on the pharmacokinetic characteristics of the enantiomers of ibuprofen, over the range 200-800 mg, was investigated in three subjects. Examination of dose-normalized AUC values and oral clearance indicate the dose dependence of (R)-ibuprofen disposition.     

An improved method for preparing rat small intestine microsomal fractions for studying drug metabolism.

Epithelial cells from isolated rat small intestine were harvested by vibration of the everted intestine in 0.14 m NaCl containing 5 mm EDTA. These cells, which were largely free of mucus contamination, were homogenized in hypotonic (74 mm) sucrose using a Potter-Elvehjem homogeniser. After successively sedimenting a “brush border plus nuclei” and a “mitochondrial” fraction, microsomes were prepared from the postmitochondrial supernatant by ultracentrifugation or by precipitation at pH 5.0. The isolation and fractionation procedure was validated by the distribution of marker enzymes and by light microscopy and found to be largely uncontaminated by other subcellular components or by haemoglobin. The usefulness of this preparation was demonstrated by determining drug-metabolising enzyme activity and by substrate- and metabolite-binding spectra to cytochrome P-450. A comparison of precipitated “acid” and “normal” intestinal microsomes indicated similar apparent K_m and V_max values for a number of drug-metabolising enzymes. The content of components of the microsomal electron transport system were also similar in both preparations while the “acid” microsomes contained approximately 50% more protein.     

Bioassay of mosquito iridescent virus of Aedes taeniorhynchus in cell cultures of Aedes aegypti.

A bioassay of mosquito iridescent virus (MIV) of Aedes taeniorhynchus was developed using cell cultures of Aedes aegypti. The dilution end point technique was based on the occurrence of cytopathic effects which were optimum at 31°C. Peleg's A. aegypti cell line was more sensitive and reliable than Singh's A. aegypti cell line for infectivity titration of the “R” and “T” strains of MIV. The highest tissue culture infectivity dose 50s (TCID₅₀) were elicited by virion:cell ratios of approximately 10. TCID₅₀ titers were significantly reduced by virus neutralization with either homologous or heterologous antiserum to either RMIV or TMIV. The virus propagated in either cell line was not infectious to A. taeniorhynchus larvae, or to the respective cells from which the virus was produced. All plaque assay attempts were unsuccessful.     

Flow and thrombosis at orifices simulating mechanical heart valve leakage regions

BACKGROUND: While it is established that mechanical heart valves (MHVs) damage blood elements during leakage and forward flow, the role in thrombus formation of platelet activation by high shear flow geometries remains unclear. In this study, continuously recalcified blood was used to measure the effects of blood flow through orifices, which model MHVs, on the generation of procoagulant thrombin and the resulting formation of thrombus. The contribution of platelets to this process was also assessed. METHOD OF APPROACH: 200, 400, 800, and 1200 microm orifices simulated the hinge region of bileaflet MHVs, and 200, 400, and 800 microm wide slits modeled the centerline where the two leaflets meet when the MHV is closed. To assess activation of coagulation during blood recirculation, samples were withdrawn over 0-47 min and the plasmas assayed for thrombin-antithrombin-llI (TAT) levels. Model geometries were also inspected visually. RESULTS: The 200 and 400 microm round orifices induced significant TAT generation and thrombosis over the study interval. In contrast, thrombin generation by the slit orifices, and by the 800 and 1200 microm round orifices, was negligible. In additional experiments with nonrecalcified or platelet-depleted blood, TAT levels were markedly reduced versus the studies with fully anticoagulated whole blood (p < 0.05). CONCLUSIONS: Using the present method, a significant increase in TAT concentration was found for 200 and 400 microm orifices, but not 800 and 1200 microm orifices, indicating that these flow geometries exhibit a critical threshold for activation of coagulation and resulting formation of thrombus. Markedly lower TAT levels were produced in studies with platelet-depleted blood, documenting a key role for platelets in the thrombotic process.     

A systems-based mathematical modelling framework for investigating the effect of drugs on solid tumours

Background and Purpose

Most information on the dose-response of radiation-induced cancer is derived from data on the A-bomb survivors. Since, for radiation protection purposes, the dose span of main interest is between zero and one Gy, the analysis of the A-bomb survivors is usually focused on this range. However, estimates of cancer risk for doses larger than one Gy are becoming more important for radiotherapy patients. Therefore in this work, emphasis is placed on doses relevant for radiotherapy with respect to radiation induced solid cancer.

Materials and methods

For various organs and tissues the analysis of cancer induction was extended by an attempted combination of the linear-no-threshold model from the A-bomb survivors in the low dose range and the cancer risk data of patients receiving radiotherapy for Hodgkin's disease in the high dose range. The data were fitted using organ equivalent dose (OED) calculated for a group of different dose-response models including a linear model, a model including fractionation, a bell-shaped model and a plateau-dose-response relationship.

Results

The quality of the applied fits shows that the linear model fits best colon, cervix and skin. All other organs are best fitted by the model including fractionation indicating that the repopulation/repair ability of tissue is neither 0 nor 100% but somewhere in between. Bone and soft tissue sarcoma were fitted well by all the models. In the low dose range beyond 1 Gy sarcoma risk is negligible. For increasing dose, sarcoma risk increases rapidly and reaches a plateau at around 30 Gy.

Conclusions

In this work OED for various organs was calculated for a linear, a bell-shaped, a plateau and a mixture between a bell-shaped and plateau dose-response relationship for typical treatment plans of Hodgkin's disease patients. The model parameters (α and R) were obtained by a fit of the dose-response relationships to these OED data and to the A-bomb survivors. For any three-dimensional inhomogenous dose distribution, cancer risk can be compared by computing OED using the coefficients obtained in this work.