Late functional and biochemical changes in mouse lung after irradiation: differential effects of WR-2721

The radioprotective effect of WR-2721 on late damage after whole thorax irradiation has been studied after split doses of radiation using the standard death and breathing rate assays at monthly intervals between 3 and 15 months after irradiation, as well as two biochemical measurements of injury at 15 months, hydroxyproline (HP), an indicator of tissue fibrosis, and DNA content, an indicator of tissue cellularity. A comparison of HP/lung and breaths per minute (BPM) in each dose group in the WR-2721 and non-WR-2721-treated mice 15 months after irradiation showed that the relationship between these two assays of late lung injury was not the same. There were large dose-related increases in breathing rate corresponding to relatively small changes in HP in the lungs of mice given radiation alone. In contrast, the mice given WR-2721 before irradiation showed large dose-related increases in HP/lung, but BPM remained relatively constant independent of dose. These data suggest then that changes in breathing rate and deaths later than 9 months after whole lung irradiation may not be due to collagen accumulation in the lung. WR-2721 did protect better against late lung functional changes (protection factors (PF) = 1.6) and late deaths (PF = 1.51) than against earlier changes in these same assays (PF = 1.4 and 1.28, respectively). Although the earlier-appearing injury after whole thoracic irradiation is most likely related to lung damage with deaths and increases in breathing rate resulting from pneumonitis, the cause of the late-appearing functional injury in the lung after radiation is not clear. Thus protection of late lung damage measured from either lethality or breathing rate is not related to the prevention of lung fibrosis.     

Characteristics of the common bean, Phaseolus vulgaris, associated with resistance to the leafhopper Empoasca kraemeri

Experiments were conducted using the common bean, Phaseolus vulgaris, and its most important pest in Latin America, Empoasca kraemeri, in order to assess the value of insect counts and various plant characteristics as selection criteria in a plant breeding programme. The combination of insect counts with measurements of damage symptoms should make it possible to distinguish tolerance from the resistance mechanisms of antibiosis and non-preference. By this means, and by a knowledge of the plant characteristics associated with resistance, different forms of resistance may be combined in the progeny of crosses. In a comparison of two contrasting genotypes the more severely damaged genotype was the more heavily infested at early stages of plant growth, but had the lower level of infestation at later stages. The association of high early counts with high subsequent damage was not confirmed in an experiment on six genotypes, but the association of high damage with low late counts was partially confirmed. These results indicate that the level of early infestation of E. kraemeri is not consistently reflected in the subsequent levels of damage which P. vulgaris genotypes display. This suggests that these two criteria represent distinct forms of resistance which can be combined in hybrid progeny. Resistance was also associated with late maturity, indeterminate growth habit, purple flowers and black or beige seeds. However, consumer preferences and the requirements of agricultural systems place constraints upon the use of these relationships in resistance breeding.     

The relationship between 'early' and 'late' radiation-induced skin reactions as seen in two strains of mice that differ in radiosensitivity

The acute skin reaction and time-course of skin contraction following graded single doses of X-rays were studied in CBA and C57B1 mice, in order to elucidate the relationship between 'early' and 'late' skin damage in strains that are known to differ in radiosensitivity. A dose-dependent acute reaction and rapid skin contraction was noted 0-50 days after irradiation in both strains, and both parameters of response were greatest in the C57B1 mice. Following this initial shrinkage there was a slight relaxation, which was again more evident in the C57s. A gradual, continuous, dose-independent contraction from 50-466 days was evident in CBA mice, whereas in C57s there was no evidence of late shrinkage. Histological examination at 466 days post-irradiation revealed a greater epidermal hyperplasia in CBA than in C57 mice. Comparison of these results with data on acute intestinal damage and early and late lung damage shows no general relationship between the incidence of early and late radiation damage in these two strains of mice.     

Experimental habitat fragmentation and invertebrate grazing in a herbaceous grassland species

A field experiment was conducted to examine the effects of habitat fragmentation on herbivore damage to individually tagged leaves of Betonica officinalis rosettes. Fragments of different size and corresponding control plots were established at three study sites in nutrient-poor calcareous grasslands in the northern Swiss Jura mountains. Leaf damage was recorded three times over the growing season (late spring, summer and early autumn). Five years after the initiation of the fragmentation, the density of rosettes did not differ between fragments and control plots. The number of leaves per rosette was higher in fragments than in control plots in summer but not in late spring and early autumn. The extent of leaf damage, expressed as proportion of leaf area removed by invertebrate herbivores, increased over the vegetation period. Leaf damage was greater in fragments than in control plots at two study sites, whereas the opposite (less strongly expressed) was found at the third site. Number of species and density (individuals per m²) of potential herbivores (gastropods and grasshoppers) were recorded in all fragments and control plots. Effects of fragmentation on the number of species and densities depended on plot size and differed between gastropods and grasshoppers. Leaf damage in fragments increased with increasing density of gastropods if the third site, which had lowest leaf damage, was excluded. Such a positive relationship was neither found in control plots nor for grasshopper densities. Thus, movement of gastropods in fragments was probably restricted which resulted in increased feeding pressure at least in two sites. However, even if our fragmentation experiment was well designed and replicated, the interpretation of these experimental results remains difficult because there was large site-to-site and seasonal variation.     

The influence of G2 progression on X-ray sensitivity of two-cell mouse embryos

Naturally synchronous, two-cell mouse embryos were X-irradiated in vitro. In experiment 1, irradiation was either in the early or in the late G2 phase, which lasts about 14 hours. In experiment 2, irradiation of all the embryos was in late G2 but embryos with different intervals between irradiation and the first mitosis after irradiation were separated and investigated independently. After 2 Gy the time interval between irradiation in late G2 and the first mitosis post-irradiation was on the average about 9 hours; after irradiation in the early G2 phase about 13.5 hours. Development (hatching of blastocysts) and cell proliferation (cell number per embryo at the stage of the hatched blastocyst) was most impaired and the frequency of micronuclei (determined in four- or eight-cell embryos) was highest in the case of a short interval between irradiation in G2 and the first mitosis post-irradiation. It is concluded that a longer interval allows a longer period of DNA repair. The results also demonstrate a positive correlation between the extent of chromosomal damage (micronuclei) and the extent of cell death as well as the impairment of the development of the whole biological system.     

X-ray response of Chinese hamster ovary cells during the latter part of G2.

The ability of Chinese hamster ovary (CHO) cells to repair x-radiation damage during the transit from the late G2 to early M cell cycle stages was investigated by conventional dose-fractionation techniques. Despite their relatively high radiation sensitivity, CHO cells positioned in late G2 exhibit increased survival when a given dose of ionizing radiation is administered as two fractions (separated by 40-50 min) instead of as a single fraction. This increased survival apparently represents repair since neither cell cycle progression nor changes in the number of "effective targets" can account for the observed dose-sparing effect.     

Autophagy-Regulated ROS from Xanthine Oxidase Acts as an Early Effector for Triggering Late Mitochondria-Dependent Apoptosis in Cathepsin S-Targeted Tumor Cells

Cathepsin S (CTSS), which is highly expressed in various malignant tumor cells, has been proposed to promote tumor progression, migration, and invasion. CTSS inhibition not only blocks tumor cell invasion and endothelial tube formation but also induces cellular cytotoxicity. In our previous studies, we have observed that CTSS inhibition induces autophagy, which is responsible for up-regulating xanthine oxidase for early ROS generation and consequent cell death. However, whether the autophagy-regulated early ROS triggers apoptosis remains unclear. We conducted a long-term follow-up study to investigate the relationship between early autophagy and late mitochondria-dependent apoptosis. We demonstrated that early ROS generation is critical for mitochondria damage and the activation of intrinsic apoptotic pathway. Attenuating the early ROS level diminished later mitochondrial damage and downstream apoptotic signaling. Collectively, mitochondria-dependent apoptosis is regulated by autophagy-regulated early ROS, which serves as an early effector that triggers mitochondrial signaling for late apoptosis. The data emphasize the essential role of autophagy-regulated early ROS in triggering late apoptotic signaling.     

Oxygen-dependent protection of radiation lung damage in mice by WR 2721

The modification of early and late radiation damage to the mouse lung by oxygen and WR 2721 has been studied by measurement of breathing rate, lethality, pleural fluid and hydroxyproline content. Protection by hypoxia and sensitization by hyperoxia of early radiation pneumonitis were demonstrated. There was a tendency for the protective effect of WR 2721 to decrease as the breathed oxygen concentration was raised above normal levels. WR 2721 protection of the late damage was higher (PF = 1.6-1.65) than was seen for early pneumonitis (PF = 1.3-1.35) when either breathing rate or lethality were used. Protection factors (PF) gained from measurements of pleural fluid at a year after treatment were similar to those for other endpoints of late damage (PF = 1.7). In contrast, the measurement of fibrosis through determination of lung hydroxyproline at 1 year gave a somewhat lower protection factor for WR 2721. In the same experiments the degree of epilation on the dorsal thorax was scored at 6 weeks. One hundred per cent oxygen gave enhancement (dose enhancement factor (DEF) = 1.2), 9 per cent oxygen reduced damage (DEF less than 0.7) and WR 2721 gave PF values in excess of 1.4 at all oxygen concentrations used. This showed that the radiation response of hair follicles was more sensitive to WR 2721 or to changes of oxygen than the lung. The results presented indicate a competitive interaction between WR 2721 and oxygen for the same injury site causing a shift in the oxygen K curve to higher oxygen concentrations. The validity of applying functional or survival measurements to assess the extent of pulmonary fibrosis is discussed.     

Vascular damage after fractionated whole-brain irradiation in rats

Whole-brain irradiation of animals and humans has been reported to lead to late delayed structural (vascular damage, demyelination, white matter necrosis) and functional (cognitive impairment) alterations. However, most of the experimental data on late delayed radiation-induced brain injury have been generated with large single doses or short fractionation schemes that may provide a less accurate indication of the events that occur after clinical whole-brain radiotherapy. The pilot study reported here investigates cerebral vascular pathology in male Fischer 344 rats after whole-brain irradiation with a fractionated total dose of 137Cs gamma rays that is expected to be biologically similar to that given to brain tumor patients. The brains of young adult rats (4 months old) were irradiated with a total dose of 40 Gy, given as eight 5-Gy fractions twice per week for 4 weeks. Brain capillary and arteriole pathology was studied using an alkaline phosphatase enzyme histochemistry method; vessel density and length were quantified using a stereology method with computerized image processing and analysis. Vessel density and length were unchanged 24 h after the last dose, but at 10 weeks postirradiation, both were substantially decreased. After 20 weeks, the rate of decline in the vessel density and length in irradiated rats was similar to that in unirradiated age-matched controls. No gross gliosis or demyelination was observed 12 months postirradiation using conventional histopathology techniques. We suggest that the early (10-week) and persistent vascular damage that occurs after a prolonged whole-brain irradiation fractionation scheme may play an important role in the development of late delayed radiation-induced brain injury.     

The Effects of Increased Illumination and Shading on the Low-Light-Induced Decline in Photosynthesis in Cotton Leaves

An experiment was carried out to study whether low-light-induced damage to the photosynthetic system in leaves of cotton (Gossypium hirsutum cv. Deltapine) which are below the compensation point in the canopy can be arrested and reversed by increased illumination. In addition it was intended to find out whether the photosynthetic system in leaves of shade plants show a greater resistance to low-light-induced damage than leaves of plants from more exposed habitats. The plants were grown at high density, and increased illumination to the shade leaves in the canopy was achieved by thinning the stand. Thinning was carried out at two stages and its effects on the decline in the photosynthetic capacity of the 4th leaf were followed. An early thinning was carried out shortly after the 4th leaf dropped below the compensation point and a late thinning 2 weeks later. Comparison was also made between the low-light-induced damage to the photosynthetic capacity of the 4th leaf in plants grown under two light regimes during the progressive increase in self-shading of the 4th leaf within the canopy. It was observed that both types of thinning arrested the low-light-induced damage to the photosynthetic system in shade leaves. The decline in photosynthetic capacity of the 4th leaf was stopped after both early and late thinning. The dry weight of the shoot system in the early and late thinned plants was not significantly different. It was double that of the control plants. The plants thinned early did not have higher shoot weight than the late thinned plants since there was a rapid shedding of flowers and fruits after early thinning. The 4th leaf in the early thinned plants showed a 30% increase in chlorophyll content and dry weight per unit leaf area. It is suggested that shedding of flowers and fruits, and increases in chlorophyll and dry weight per unit leaf area in the early thinned plants were caused by a change in the hormonal balance of the plants. The photosynthetic system in leaves of shade plants showed a greater resistance to damage by low light intensity than the photosynthetic system in leaves of plants grown at higher light intensities.     

Leaf damage by herbivores affects attractiveness to pollinators in wild radish, Raphanus raphanistrum

 We carried out two experiments to determine the effect of leaf damage on plant attractiveness to pollinators using wild radish, Raphanus raphanistrum (Brassicaceae), a self-incompatible annual herb. Pairs of plants from 36 full-sib families were grown in pots in the greenhouse. One member of each pair was damaged by Pieris rapae larvae that were allowed to remove half of the leaf area of each of the first four rosette leaves. The plants were subsequently taken out for pollinator observations once a week from the beginning of flowering in late June until the end of August. We conducted two experiments to examine how foliar damage affected visitation by pollinators. In the first experiment, numbers of pollinators visiting plants were compared between damaged and control sibling plants. In the second experiment, the number of open flowers during observations was controlled to be the same for both damaged and undamaged sibs. Damage significantly decreased the number and size of flowers during the first observations in late June. Damaged plants received fewer visits by native bees during the first week of observations. Since damage did not affect native bee visits when the number of open flowers was equalized between treatments, flower number was probably the main cue attracting native bees to plants. In the experiment without flower number control, syrphid flies, the other abundant pollinator taxon, spent more time per flower on the undamaged than on the damaged plants. When flower number was controlled, flies probed significantly more flowers during each visit on the undamaged than on the damaged plants and had higher visitation rates to undamaged plants early in the season. Since syrphid flies preferred undamaged plants both with and without flower number control, they apparently used cues apart from flower number for visitation. The difference between undamaged and damaged plants in floral characteristics and pollinator visitation vanished within a few weeks after the start of flowering. This result suggests that early damage may not have a strong fitness effect through reduction in mating success. However, poor weather conditions can cause early mortality of plants in the field, and nutrient depletion and competition decrease fruit set of later flowers. Therefore, conditions exist under which visitation to early flowers may affect plant fitness. Received: 30 July 1996 / Accepted: 10 February 1997     

The expression of early and late damage after thoracic irradiation: a comparison between CBA and C57B1 mice

Lung injury after localized irradiation of the thorax was quantified and compared in CBA and C57B1 mice. Using lethality and breathing rate as end points, two phases of damage separated in time were distinguished in CBA mice as an early pneumonitic phase and a later phase associated with pleural effusions. C57B1 mice failed to show the pneumonitic response over a large dose range extending beyond 20 Gy. In this respect they differ from most other mouse strains so far studied. At the lower doses the extent of the late phase was similar between these two strains. The interstrain comparison presented suggests that damage to separate tissue compartments was responsible for the acute and chronic responses.     

The Role of Wnt/β-Catenin Signaling in Enterocyte Turnover during Methotrexate-Induced Intestinal Mucositis in a Rat

Background/Aims

Intestinal mucositis is a common side-effect in patients who receive aggressive chemotherapy. The Wnt signaling pathway is critical for establishing and maintaining the proliferative compartment of the intestine. In the present study, we tested whether Wnt/β-catenin signaling is involved in methotrexate (MTX)-induced intestinal damage in a rat model.

Methods

Non-pretreated and pretreated with MTX Caco-2 cells were evaluated for cell proliferation and apoptosis using FACS analysis. Adult rats were divided into three experimental groups: Control rats; MTX-2 animals were treated with a single dose of MTX given IP and were sacrificed on day 2, and MTX-4 rats were treated with MTX similar to group B and were sacrificed on day 4. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation, and enterocyte apoptosis were measured at sacrifice. Real Time PCR and Western blot was used to determine the level of Wnt/β-catenin related genes and protein expression.

Results

In the vitro experiment, treatment with MTX resulted in marked decrease in early cell proliferation rates following by a 17-fold increase in late cell proliferation rates compared to early proliferation. Treatment with MTX resulted in a significant increase in early and late apoptosis compared to Caco-2 untreated cells. In the vivo experiment, MTX-2 and MTX-4 rats demonstrated intestinal mucosal hypoplasia. MTX-2 rats demonstrated a significant decrease in FRZ-2, Wnt 3A Wnt 5A, β-catenin, c-myc mRNA expression and a significant decrease in β-catenin and Akt protein levels compared to control animals. Four days following MTX administration, rats demonstrated a trend toward a restoration of Wnt/β-catenin signaling especially in ileum.

Conclusions

Wnt/β-catenin signaling is involved in enterocyte turnover during MTX-induced intestinal mucositis in a rat.     

Gamma-radiation dose-rate effects on DNA damage and toxicity in bacterial cells

In order to investigate the relationship between radiation dose-rate and bacterial DNA damage as well as general cellular toxicity, two recombinant Escherichia coli strains, DPD2794 and GC2 were used. Following gamma-ray irradiation, these bioluminescent bacteria showed quantitative stress responses in terms of DNA damage and general toxicity depending on the dose rates of energy deposition, i.e. dose-rate of radiation. In addition, an inverse relationship was found, at lower dose rates between 0.5 and 1 Gy/h and a parabolic relationship at dose rates between 0.5 and 2.6 Gy/h.     

Functional and histological changes in rat lung after boron neutron capture therapy

The motivation for this work was an unexpected occurrence of lung side effects in two human subjects undergoing cranial boron neutron capture therapy (BNCT). The objectives were to determine experimentally the biological weighting factors in rat lung for the high-LET dose components for a retrospective assessment of the dose to human lung during cranial BNCT. Lung damage after whole-thorax irradiation was assessed by serial measurement of breathing rate and evaluation of terminal lung histology. A positive response was defined as a breathing rate 20% above the control group mean and categorized as occurring either early (<110 days) or late (>110 days). The ED(50) values derived from probit analyses of the early breathing rate dose-response data for X rays and neutrons were 11.4+/-0.4 and 9.2+/-0.6 Gy, respectively, and were similar for the other end points. The ED(50) values for irradiation with neutrons plus p-boronophenylalanine were 8.7+/-1.0 and 6.7+/-0.4 for the early and late breathing rate responses, respectively, and 7.0+/-0.5 Gy for the histological response. The RBEs for thermal neutrons ranged between 2.9+/-0.7 and 3.1+/-1.2 for all end points. The weighting factors for the boron component of the dose differed significantly between the early (1.4+/-0.3) and late (2.3+/-0.3) breathing rate end points. A reassessment of doses in patients during cranial BNCT confirmed that the maximum weighted doses were well below the threshold for the onset of pneumonitis in healthy human lung.     

Review: total doses in fractionated radiotherapy--implications of new radiobiological data

The total dose in radiotherapy has been adjusted in the past for different fractionation schedules by the use of empirical formulae such as NSD, TDF and CRE. It is now appropriate to consider fractionation factors which include more biological insight in their formulation than was possible earlier. It has become clear, from both clinical and experimental animal data, that the total dose in multi-fraction irradiations depends more critically on size of dose-per-fraction for late than for early damage to normal tissues. This difference has been interpreted as due to different shapes of the underlying dose-response curves. The late reactions respond with more curvature in the dose-response curve, i.e. with more repair capability at very low doses per fraction, than the early tissue reactions. A linear-quadratic relationship for the dose-response curves has been found to fit experimental data well, with few exceptions. This paper reviews this interpretation and explores some of its implications for radiotherapy and for radiobiology applied to therapy. Of many repair factors that have been suggested, the ratio alpha/beta (of the linear to the quadratic coefficients) is one that should be independent of the level of damage assayed. Values of alpha/beta of about 10 Gy have been reported for a number of early tissue responses but a range of values from about 1 to 5 or 6 Gy for late responses. It is a current challenge to radiobiology to explain why this difference occurs. Once such values are known for different tissues--and the dangers of premature assumptions are emphasized--calculations are possible which might be useful in radiotherapy as an alternative to NSD, TDF, CRE etc. Some data are presented on the magnitude of differences from these previously used empirical formulae, with a discussion about how easily detected the discrepancies might be in clinical practice. Applications to hypofractionation, hyperfractionation and accelerated fractionation are illustrated.     

X-ray sensitivity and DNA synthesis in synchronous culture of Plasmodium falciparum

The relationship between parasite development and sensitivity to irradiation with X-rays was investigated during a single synchronous cycle of Plasmodium falciparum in culture. The sensitivity of the parasites to irradiation was closely correlated with the phases of DNA synthesis. Their sensitivity was greatest at the ring stage in development, but decreased at the trophozoite stage when DNA synthesis begins. Lowest sensitivity was found when DNA synthesis was most rapid as the parasites were transforming from late trophozoite to schizont forms. These findings suggest that DNA is the target of the lethal radiation damage in the parasites.     

Role of medial prefrontal cortex dopamine in age differences in response to amphetamine in rats: Locomotor activity after intra-mPFC injections of dopaminergic ligands

Changes in medial prefrontal cortex (mPFC) dopamine receptor expression and in mPFC projections to the nucleus accumbens in adolescence suggest that there may be age differences in the regulation of drug‐related behavior by the mPFC. The age‐specific role of prelimbic D1 dopamine receptors on amphetamine‐induced locomotor activity was investigated. In experiment 1, rats aged postnatal day 30 (P30), P45, and P75, corresponding to early and late adolescence and adulthood, were given an injection of D1 and D2 antagonists into the prelimbic mPFC before a systemic injection of 1.5 mg/kg of amphetamine and locomotor activity was recorded. In experiment 2, effects of intra‐prelimbic injections of a D1 agonist and antagonist on locomotor activity produced by a lower dose (0.5 mg/kg) of amphetamine were investigated. D2 receptor antagonist did not alter amphetamine‐induced activity, whereas the D1 receptor antagonist reduced activity produced by 1.5 mg/kg of amphetamine more in P30 than in P45 and P75 rats. In addition, D1 agonist enhanced the locomotor activating effects of 0.5 mg/kg of amphetamine in adolescent rats and decreased activity in adult rats. These results suggest that insufficient activation of mPFC D1 receptors may underlie the reduced activity at the low dose of amphetamine in early adolescent compared to adult rats. © 2011 Wiley Periodicals, Inc. Develop Neurobiol, 2012     

Effect of timing of second prostaglandin F2α administration in a 5-day, progesterone-based CO-Synch protocol on AI pregnancy rates in beef cows

The objective was to compare the timed AI pregnancy rate of Angus-cross beef cows synchronized with a 5-d CO-Synch + CIDR (a progesterone-releasing intravaginal insert) protocol and given two doses of PGF_2α (PGF), with the first dose in conjunction with CIDR withdrawal on Day 5, and the second dose given either early or late relative to the first dose. All cows (N = 1782) at 16 locations received 100 μg of GnRH + CIDR on Day 0. Cows received 25 mg of PGF concurrent with removal of the CIDR on Day 5, and were randomly allocated within locations to receive a second PGF either early (N = 881; from 0.5 to 3.9 h) or late (N = 901; from 4.5 to 8.15 h) relative to the first PGF treatment. On Day 8 (72 h after CIDR removal), all cows were inseminated and concurrently given 100 μg of GnRH. Cows were fitted with a pressure-sensitive mount detection device (Kamar) at CIDR removal. Cows were observed twice daily through Day 7 and at the time of AI on Day 8 for estrus and Kamar status (estrus - red, partial and lost Kamar versus no estrus - white Kamar) was recorded. Accounting for location, season, AI sire, cow observed in estrus or not at or before timed AI, and treatment by cows observed in estrus interaction, timed AI pregnancy rates were greater for the late (6.45 ± 0.03 h) than the early (2.25 ± 0.05 h) interval, 57.2 vs. 52.7%, respectively (P < 0.05). In conclusion, cows that received the second PGF late after the first PGF on the day of CIDR removal in a 5 d CO-Synch + CIDR synchronization protocol had significantly higher timed AI pregnancy rates than those receiving the second PGF early after the first PGF.