Role of radioadaptation on radiation-induced thymic lymphoma in mice

Thymic lymphoma (TL) was observed in different stages of development in 46% of male mice (23/50) following exposure to an acute challenge dose of 2 Gy ⁶⁰Co γ-rays. With an adapting dose of 1 cGy 24 h prior to the challenge dose of 2 Gy, similar growth of TL was seen in 42.5% of mice (17/40). TL was not found in unirradiated control mice (0/50) or in the group treated with 1 cGy (0/50). Multiple adapting doses for 5 or 10 consecutive days induced TL in 8/50 and 9/50 mice, respectively (17% in average). When multiple adapting doses were followed by the challenge dose, the yield of TL was much lower, 16% (8/50) and 30% (15/30), respectively. By 15, 30, 60, 90, and 120 days after exposure with 3 Gy of ⁶⁰Co γ-rays, TL developed in 30, 70, 70, 80 and 85% of the female mice, respectively. When mice were conditioned with an adapting dose of 1 cGy 24 h prior to the challenge dose, TL was not found 15 days post-irradiation, while about a 25% reduction in the occurrence of TL was noticed at all other intervals. The results suggested that an adapting dose could play a role in bringing about a change in terms of delay and inhibition of the acute effects of radiation, i.e., the onset of TL in mice.     

Chromosomal responses to ionizing radiation reminiscent of and adaptive response in cultured Chinese hamster cells

When Chinese hamster V79 cells were internally exposed to low level chronic β-rays from incorporated tritiated thymidine (³H-dThd), the showed an “adaptive” response to the induction of chromosomal damage by subsequent higher acute doses of γ-rays.

The yield of sister-chromatid exchanges (SCEs) in the ³H-dThd pretreated cells was less than the yield induced by γ-rays alone (protective effects), and the micronucleus frequency was less than the sum of the induced frequencies by ³H-dThd and γ-rays separately (below-additivity effects). No adaptation to the micronucleus induction by γ-rays was observed after the ³H-adapted cells had divided once and when 3-aminobenzamide (3AB) was given before the challenge doses. The cross-resistance study revealed that the ³H-adapted cells were resistant to SCE induction but not to the micronucleus inductions by the challenge doses of reactor radiations. The results suggest that the SCE adaptation and the micronucleus adaptation or clastogenic adaptation are probably caused by different, inducible adaptive repair pathways.     

The influence of anoxia or oxygenation on the induction of chromosome aberrations in human lymphocytes by 15-MeV neutrons

Dicentric and total aberration yields induced in human lymphocytes by 15-MeV neutrons under conditions of oxygenation and of anoxia have been fitted to a dose-response curve using the function Y = D + βD². An oxygen-enhancement ratio (OER) ranging from 3.7 at low yields to 1.6 at high yields was calculated from the co-efficients of the dicentric yield curves and evidence is presented which suggests that oxygen does not act as a dose-modifying agent in this system. High dose RBE values of 1.2 and 2.1 with respect to 250 kVp X-rays for oxygenated and anoxic conditions were also obtained. Coefficients for total aberration yield gave similar values to dicentrics for both OER and RBE     

Effect of gamma radiation and alpha particles on gene recombination in Chlamydomonas reinhardi

Low doses of ⁶⁰Co γ radiation, which kill no more than about 5% of the zygospores, change gene recombination at only 2 short stages during the course of meiosis in Chlamydomonas reinhardi, but higher doses, which kill more than 10% of the spores, depress recombination at all stages up to pachytene. Irradiation with particles having a mean linear energy transfer (LET) of about 1300 and 1600 MeV g⁻¹ cm² changes recombination in a manner which appears to combine the effects characteristic of both low and high doses of γ-radiation simultaneously. The “γ high-dose” type of response has a relative biological effectiveness (RBE) of between 20 and 35, and the “γ low-dose” RBE is greater than 1 although precise evaluation is impossible due to the complexity of the response. The RBE for survival was 16.5 at the low dose levels studied.     

Differential DNA strand breaking abilities of *OH and ROS generating radiomimetic chemicals and gamma-rays: study of plasmid DNA, pMTa4, in vitro

Reactive oxygen species (ROS) interact with components of a living cell. Among them ^⋅OH is known to cause major oxidative damage to living cells and is proposed to be involved in pathogenesis including carcinogenesis. Proper understanding of consequences of such damage is, therefore, medically relevant. In this report, aqueous solution of plasmid DNA, pMTa4, has been exposed to Fenton oxidant and Haber-Weiss oxidant mediated free radical generating chemical systems, and ⁶⁰Co γ-rays in vitro either alone or in combination to study their strand breaking abilities. The exposed pMTa4 was analyzed by agarose gel electrophoresis. The results show qualitative differences in the induction of strand breaks on the plasmid pMTa4 molecule by the iron (Fe²⁺), copper (Cu²⁺) or γ-rays mediated ^⋅OH and other ROS.     

Calibration of the comet assay for the measurement of DNA damage in mammalian cells

We used X-rays from a linear accelerator and from a low energy therapeutic source to calibrate the single cell gel electrophoresis (comet assay), a widely used method to measure DNA damage. γ-Rays from ⁶⁰Co, with known efficiency in inducing DNA breakage, were used as reference. Human lymphocytes and one murine tumour cell line, F10-M3 cells, were irradiated under different experimental conditions. A similar relationship between radiation dose and induced DNA damage was obtained with γ- and X-rays. A calibration curve was constructed to convert the comet assay raw data into break frequency. The median levels of DNA breaks and oxidative damage in circulating lymphocytes from healthy volunteers were calculated to be 0.76 and 0.80 breaks/10⁹ Da, respectively, (0.50 and 0.52 breaks/10⁶ bp). The values of oxidative DNA damage were in the same order of magnitude as those found by others with HPLC methods.     

Comparative study of gamma-ray and high-energy carbon ion irradiation on negative gravitaxis inEuglena gracilisZ

The effects of ⁶⁰Co gamma-ray and 290 MeV/amu carbon ion irradiation on negative gravitaxis was studied in the photosynthetic flagellate Euglena gracilis strain Z in a dose-response dependent manner. Cells were exposed to the doses (0-200 Gy for water). The negative gravitaxis was quantified by the r-value observed in a recently developed biomonitoring system. The present results demonstrate the inhibitory effects of gamma-rays and 290 MeV/amu carbon ions on negative gravitaxis of the Euglena gracilis strain Z. The 290 MeV/amu carbon ions had a greater impact at a low dose (<40 Gy) than the ⁶⁰Co gamma-rays.     

Toxicity and mutagenicity of X-rays and [125I]dUrd or [3H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.

Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.     

Chromosome aberrations in workers of nuclear-power plants

The frequencies of chromosome aberrations in 135 workers from nuclear-power plants were compared with those in 135 age-matched controls. A total of 135,000 cells was scored. The frequencies of dicentric chromosome were 1.67 × 10⁻³ in the exposed group and 0.49 × 10⁻³ in the control group and those of chromosome-type deletion were 3.33 × 10⁻³ and 1.10 × 10⁻³, respectively. The frequencies of all types of chromosome aberrations in the exposed subjects were higher than those in the control group, but no significant trend of dose-dependent increase was observed when only the exposed group were considered. Poisson regression analysis, with both exposed and control included, showed that there was a significant association of chromosome aberration with radiation dose and the duration of work, but not with age, smoking habit and alcohol intake. It was also found that recent exposure to radiation, within the last 5 years, had contributed more to the observed chromosome aberration than earlier exposure.     

The effect of hydroxyl radicals on the rheological performance of hylan and hyaluronan

Shear flow, dynamic oscillation and extensional viscosity measurements were used to compare the rheological performance of several hylan samples (M_v 1.6, 3.2, 3.7, 4.7 and 5.6×10⁶) and hyaluronan (M_v 1.4 and 1.8×10⁶) before and after hydroxyl radicals (√OH) induced degradation. It was found that the higher molecular weight cross-linked structure of hylan was more resistant to degradation than hyaluronan and that this superior stability was reflected in various rheological parameters. The √OH degradation of the initial hylan and hyaluronan samples produced a range of polysaccharides based on hylan and hyaluronan with molecular weight covering a range from 0.5–5.6×10⁶. The rheological parameters associated with the polysaccharides could then also be studied. Zero shear values of the complex viscosity (η*), dynamic viscosity (η′) and shear viscosity (η) were calculated using the method of Morris¹ and shown to approach the same value at zero shear or frequency. An adaptation of the method of Gibbs et al.² gave a ‘master curve’ for the storage and loss modulus of hyaluronan and hylan, which encompasses a 10-fold molecular weight and a 5-fold concentration variation. In all instances for hylan, the storage modulus predominates over the loss modulus, whereas for hyaluronan, the reverse is true, demonstrating the greater elasticity of hylan throughout the whole experimental range of molecular weights and concentrations.     

The regulation of mortality and fecundity in Schistosoma mattheei following a single experimental infection in sheep.

The regulation of mortality and fecundity of Schistosoma mattheei in sheep was examined using a series of mathematical models applied to data culled from the literature. Parasite mortality (μ) was found to be an increasing linear function of the magnitude of the initial infection over the ranges of doses examined (200–91, 000 cercariae) where μ = 9.78 × 10⁻³ + 3.476 × 10^−7* infection dose. Parasite fecundity (λ) was found to be inversely related to the duration of the infection. The best fit model for parasite fecundity was one in which fecundity decreased exponentially with time since initial infection, λ = λ₀e^−δ1−r. There was no evidence for density-dependent regulation of fecundity.     

Interaction of sialosyl cholesterol with the cell surface of rat astrocytes and its biological activities

The interaction between sialosyl cholesterol (- or neuraminyl cholesterol, - or β-SC) and the plasma membrane of astrocytes was investigated by the use of ¹⁴C-labeled - or β-SC. Both - and β-SC were dose-dependently and time-dependently bound to rat astrocytes. The Scatchard plot analyses showed that rat astrocytes bound apparently 9.69 × 10⁹ molecules of both -SC/cell (apparent K_d = 2.29 × 10⁻⁵ M) and β-SC/cell (apparent K_d = 5.39 × 10⁻⁵ M) at 37°C. Both the binding of -SC to astrocytes and the subsequent inhibition of DNA synthesis were decreased at the low temperature (4°C), and also suppressed by serum proteins including albumin. One molecule of bovine serum albumin (BSA) bound 2.3 molecules of -SC with the slightly lower K_d-value (8.03 × 10⁻⁶ M) than that for the binding site on astrocytes. BSA not only suppressed the -SC-binding to astrocytes but also increased its release from the cells to the culture media. Gangliosides such as GM1 and GM3 unaffected the -SC-binding, promoted the small release of -SC from the cell surface, and inhibited the morphological changes of astrocytes induced by -SC. The mechanism of -SC-binding to cultured astrocytes with reference to the effects of serum or gangliosides is discussed.     

DNA polymerase delta mediates increase in exchange production by X-radiation in human lymphocytes moving from G0 to G1

Earlier work of several laboratories established that the yields of radiation-induced ring and dicentric chromosomes are greater when human peripheral blood lymphocytes are irradiated in GH₁ some hours after phytohemagglutinin stimulation than if they are irradiated in G₀ before stimulation. Post-treatment of lymphocytes irradiated in G₀ with the DNA polymerase inhibitor aphidicolin, which is effective against both pol and pol δ, produces a similar increase in ring and dicentric yield. We found that aphidicolin post-treatment was much less effective in increasing ring and dicentric yield increases in cells irradiated in G₁ four to five hours after stimulation. Because we had earlier found specific inhibitors of DNA pol ineffective in producing increased yields in either G₀ or G₁ lymphocytes, we conclude that much of the G₀ to G₁ increase in yields is mediated by pol δ.     

Human fibroblast culture on a crosslinked dermal porcine collagen matrix

The use of a novel porcine-derived collagen biomaterial as a dermal tissue engineering matrix was examined. The matrix is derived from porcine dermis, and is processed to retain the native collagen (Type 1) and elastin structure. Human primary fibroblasts were cultured on the matrix to examine its potential for creating a dermal replacement. Attachment of fibroblasts on the collagen was compared to tissue culture plastic and PET membranes. Cell proliferation was assessed using the MTT assay and DAPI staining. For seeding densities of 5×10⁴ and 1×10⁵ cells cm⁻², PET and plastic demonstrated >95% attachment of seeded numbers after 3 h. The collagen matrix reached levels >80% after 3–4 h with no influence of the seeding density. Matrix samples with perforating pores of 40 μm diameter were also studied. After 216 h culture in static culture, with media replacement every 3 days, the final cell numbers reached 2.1×10⁵ (perforated) and 2.0×10⁵ cells cm⁻² (unperforated). In comparison fibroblast culture in a perfusion bioreactor, with continuous media replacement, reached 2.3×10⁵ (unperforated) and 2.5×10⁵ cells cm⁻² (perforated) after 216 h.     

Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.     

Antigenic complexes of steroid hormones formed by coupling to protein through position 7: Preparation from Δ -3-oxosteroids and characterization of antibodies to testosterone and androstenedione

A general method for rendering Δ³-3-oxosteroids antigenic by coupling to a macromolecule through position 7 is described. It involves nucleophilic attack on the 6, 7-dehydroderivatives of the steroids by ambidentate reagents to form 7-thioether alkanoic acids. These were covalently attached to bovine serum albumin (BSA) by use of the carbodiimide reagent. Addition products with mercapoacetic acid and β-mercaptopropionic acid and their BSA-conjugates, were thus obtained from testosterone androst-4-ene-3, 17-dione, progesterone and 17-hydroxyprogesterone through the respective 4, 6-dienes.

Immunization of rabbits with testosterone-7-carboxymethyl-thioether-BSA and the homologous testosterone-7-carboxyethyl-thioether-BSA gave rise to antisera of high affinity for testosterone (Ka=9. 4×10⁹ 1/mol) that showed little cross-reaction with androstenedione (< 1%) and with a variety of 17-oxoandrostane compounds ( 0.5%). Conversely, immunization with androstenedione-7 -carboxyethyl-thioether-BSA yielded an antiserum with high affinity for androstenedione (Ka = 1. 04 × 10¹⁰ I/mol) but minimal cross-reaction with testosterone (< 0.5%) and 17β-hydroxy-androstane compounds ( 1%). The reaction of anti-testosterone and anti-androstenedione sera with their homologous haptens was not significantly inhibited by the closely related steroids 17- estosterone and dehydroepiandrosterone, or by 11-deoxycorticosterone, progesterone, 17-hydroxyprogesterone, estrone and estradiol-17β. However, anti-testosterone sera cross-reacted with 5-dihydrotestosterone (40–50%) and to a lesser extent with 5β-dihydrotestosterone (5%). Analogously, the anti-androstenedione sera cross-reacted with 5-dihydroandrostenedione (71%) and to a minor extent with 5β-dihydroandrostenedione (8%).

A radioimmunoassay procedure for the determination of testosterone in plasma is described, which makes use of the new anti-testosterone serum. Preliminary results suggest that it can be applied to ether extracts from human sera without Chromatographic purification.     

Reactivity study of cyclopentadienyl osmium(II) bisphosphine azido complexes with activated alkynes and nitriles: Isolation of osmium triazolato and tetrazolato complexes by 1,3-dipolar addition

The cyclopentadienyl osmium(II) complexes [(η⁵-C₅H₅)Os(PPh₃)₂X] [X = Br (1), CH₃CN (2)] reacts with sodium azide (NaN₃) to yield the corresponding azido complex [(η⁵-C₅H₅)Os(PPh₃)₂N₃] (3). This undergoes [3+2] dipolar cycloaddition reaction with activated alkynes like dimethyl and diethyl acetylenedicarboxylate to yield triazolato complexes [(η⁵-C₅H₅)Os(PPh₃)₂{N₃C₂(CO₂R)₂}] [R = –CH₂CH₃ (4) and –CH₃ (5)]. The complex 3 also reacts with nitriles such as tetracyanoethylene (TCE), fumaronitrile and p-nitrobenzonitrile to yield complexes of the type [(η⁵-C₅H₅)Os(PPh₃)₂{N₄C₂(CN)C(CN)₂}] (6), [(η⁵-C₅H₅)Os(PPh₃)₂{N₃C₂HCN}] (7) and [(η⁵-C₅H₅)Os(PPh₃)₂{N₄C(C₆H₄–p-NO₂)}] (8). These complexes were fully characterized on the basis of microanalyses, FT-IR and NMR spectroscopic data. The molecular structure of the representative complex [(η⁵-C₅H₅)Os(PPh₃)₂{N₃C₂(CO₂CH₂CH₃)₂}] (4) was determined by single crystal X-ray analysis.     

Hydrodynamic characteristics and equilibrium rigidity of pullulan molecules

The hydrodynamic characteristics of the polysaccharide pullulan (polymaltotriose) in water have been investigated and its molecular characteristics have been determined. Experimental values varied over the following ranges: velocity sedimentation coefficient (S): 0.9 < S < 11.2, translational diffusion coefficient (10⁷ cm² s⁻¹): 1.1 < D < 14.7 and intrinsic viscosity (cm³ g⁻¹): 6.7 < [η] < 164, which corresponds to a change in molecular weight (× 10³) in the range 3.9 < M_SD < 644. On the basis of analysis of the literature and our experimental data, excluded volume effects have been shown to have a prevailing influence on the chain length of these polysaccharides. The equilibrium rigidity and hydrodynamic chain diameter of pullulan were evaluated on the basis of the theory of hydrodynamic properties of a wormlike necklace, taking into account excluded volume effects. At low M (< 30 × 10³) the translation friction data (in contrast to viscometric data) cannot be described in the framework of the theory of linear molecules.     

Δ-THC and 11-OH-Δ-THC: Behavioral effects and relationship to plasma and brain levels

The time course of effects following i.p. injections of 0.3–10.0 mg/kg Δ⁹-tetrahydrocannabinol (δ⁹-THC) and 0.1–3.0 mg/kg 11-OH-Δ⁹-THC were determined in rats during 6 hour sessions under a fixed-interval 90-second schedule of food reinforcement. In addition, the acute and chronic effects of these two drugs were tested in different rats trained on a differential reinforcement of low rate 15-second schedule. The onset of activity of 11-OH-Δ⁹-THC was faster, and usually abruptly suppressed all responding, while Δ⁹-THC's onset was slower and often resulted in a decreased and steady pattern of responding. 11-OH-Δ⁹-THC was about 3 times more potent, had a shorter duration of effect and when responding resumed, it returned to control rates within a shorter time than for Δ⁹-THC. Tolerance and cross-tolerance developed at the same rate to equipotent doses of the two drugs. The time course for plasma and brain levels of radioactivity were studied in other rats after i.p. administration of H³-Δ⁹-THC and H³-11-OH-Δ⁹-THC. 11-OH-Δ⁹-THC was absorbed more quickly than Δ⁹-THC and reached peak plasma and brain levels earlier. In addition, higher plasma and brain levels, and larger brain to plasma ratio of radioactivity were attained after 11-OH-Δ⁹-THC. Therefore, differences in behavioral effects produced by Δ⁹-THC and it's 11-hydroxy metabolite were accompanied by differences in absorption and disposition.     

Luminescent yeast cells entrapped in hydrogels for estrogenic endocrine disrupting chemical biodetection

In the construction of luminescent yeast cell based fibre-optic biosensors, we demonstrate a novel approach for estrogenic endocrine disrupting chemical (EDC) biodetection by entrapping genetically modified Saccharomyces cerevisiae cells, containing the estrogen receptor alpha-mediated expression of the luc reporter gene, in hydrogel matrices based on calcium alginate or PVA. In order to insure a significant signal, an optimal immobilization ratio of 1:2 alginate 3% (w/v): 5 × 10⁶ [cells/ml], respectively, was used with the highest 17-β-estradiol (β-E2) induction factor after 2.5 h of incubation with 10 [nM] β-E2. It was shown that biocompatible alginate beads, 4.27–4.55 × 10⁵ [CFU/bead], which were characterized by a detection limit of 0.08 [μg l⁻¹] and an EC50 of 0.64 [μg l⁻¹] for β-E2, retained their viability for luminescence measurements after 1 month of storage at −80 °C slow freeze condition, and thus repeated cell cultivations were not required. The assay reproducibility for each tested EDC, represented by the coefficients of variation (CV), ranged from 4.35 to 18.47%. An alternative immobilization method, based on a room temperature partial drying of polyvinyl alcohol (PVA) solution (LentiKat^® Liquid) and cell suspension mix, was investigated with only a slightly lower detection limit for β-E2 than that reported with alginate beads. Alginate yeast based hydrogels may also be applicable to the analysis of environmental water samples since the trend of detected estrogenic activities with alginate beads roughly correlated with LC–MS–MS analytical results.