Histological analysis of the effect of hyperthermia on normal rabbit hepatic vasculature.

The effects of hyperthermia on rabbit hepatic vasculature were studied histologically. To investigate heat-induced vascular damage in the central veins, portal veins, and hepatic arterioles, the left lobes of rabbit liver were heated locally for 30 min in the range of 40-46 degrees C. Hyperthermia was induced by an 8-MHz radiofrequency current heating device using a needle type interstitial applicator. This device allowed application of heat to a central area of 10 x 10 mm no more than 1 degree C below the preset temperature. Within the area of 1 cm2, the percentage of damaged (ruptured or thrombosed) vessels was estimated for each type of hepatic vasculature. Vascular damage following hyperthermia continued up to 24 h after heating for the three types of hepatic vasculature. Central veins were the most thermosensitive followed by portal veins, whereas hepatic arterioles were the most thermoresistant. The temperature causing 50% vascular damage 24 h after heating was 41.5-42.5 degrees C, 42.5-43.5 degrees C, and 44-45 degrees C for central veins, portal veins, and arterioles, respectively. This differential thermal responsiveness of hepatic vasculature may be attributed to the histological structure of the vessels.

Effect of recombinant human granulocyte colony-stimulating factor on granulocytopenia in mice induced by irradiation.

We report the effect of human granulocyte colony-stimulating factor (hG-CSF) on the recovery from granulocytopenia induced by irradiation. Female 9-week old C3H/He mice were used. The irradiation schedule was as follows: Group 1 and 2 received whole-body irradiation of 1 Gy and 5 Gy, respectively, on day 0; Group 3 and 4 received whole-body irradiation of 0.5 and 1.0 Gy, respectively, for 5 consecutive days; Group 5 received upper hemibody irradiation of 3 Gy for 5 consecutive days. Daily subcutaneous injections of G-CSF (3 x 10(5) Unit/mouse) or 0.3 ml of saline to each group were started from the day after the first irradiation and continued for 18 days. Mice were sampled randomly from each group, and the total number of leukocytes, erythrocytes of peripheral blood, nucleated cells in femur, and spleen weight were counted and measured, respectively, on day 0, 3, 5, 7, 9, 12, and 18. The leukocyte counts decreased with an increase in radiation doses. In Group 1 and 2 mice, G-CSF enhanced the leukocyte count more than saline. In Group 3 mice, the recovery of leukocytopenia was facilitated by G-CSF, but in Group 4 mice, G-CSF had no effect on the leukocyte count decrease or on leukocytopenia recovery. In Group 5 mice, G-CSF greatly affected leukocytopenia recovery. Increase in spleen weight paralleled the peripheral leukocyte count. Daily administration of recombinant hG-CSF accelerated the granulocytopenia recovery which was induced by irradiation, and it may be a useful therapeutic agent for treating myelosuppressive cases.

In vivo radiosensitizing activity of a new fluorinated hypoxic cell radiosensitizer, KU-2285, in combination with radiation dose fractionation.

Since most clinical radiotherapy is given as multiple small irradiation fractions, the present study was undertaken to test the in vivo radiosensitizing activity of a new hypoxic cell radiosensitizer, KU-2285, in combination with radiation dose fractionation. Radiosensitizing activity was measured by a growth delay assay using a transplanted mammary tumor in C3H/He mice, and by an in vivo-in vitro assay using the SCC VII tumor. KU-2285 was injected intraperitoneally 30 min before irradiation in all experiments. The in vivo-in vitro assay using SCC VII tumors showed that 12.5 micrograms/g of KU-2285 sensitized the tumors to irradiation (5 Gy/fr x 5 fr/48 hr or 6 Gy/fr x 3 fr/48 hr). KU-2285 also sensitized the transplanted mammary tumors to fractionated irradiation. We concluded that KU-2285 was able to sensitize two different murine tumors when given in combination with radiation dose fractionation.

Effect of recombinant human granulocyte colony-stimulating factor on efficacy of radiation therapy in tumor-bearing rats.

The effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on radiation-induced neutropenia and on growth of transplanted tumors treated by irradiation was investigated using tumor-bearing rats as a model for radiation therapy. In a preliminary study using normal rats, neutropenia induced by upper hemi-body irradiation at 3 Gy/day 5 times a week for 3 weeks was prevented by consecutive subcutaneous injections of rhG-CSF at 100 micrograms/kg/day. Rats bearing Walker-256, a mammary tumor, were scheduled to receive upper hemibody irradiation at 3 Gy/day for 15 times in 3 weeks if white blood cell (WBC) counts were maintained above 3,000/microliters. In control tumor-bearing rats not receiving rhG-CSF, irradiation was often withheld because of the decrease in WBC counts below 3,000/microliters. In contrast, a decrease in WBC counts below 3,000/microliters was rarely found in tumor-bearing rats injected daily with rhG-CSF. The average number of radiation treatments in control rats and rats treated with rhG-CSF was about 8 and 14, respectively, out of the scheduled 15 treatments in 3 weeks. Treatment with rhG-CSF made it possible to complete the radiation therapy regimen and thus inhibit the growth of the transplanted tumor more effectively. These results suggest that rhG-CSF may be useful to ensure radiation therapy on schedule in cancer patients.

Rejoining of DNA single- and double-strand breaks in human white blood cells exposed to ionizing radiation.

PURPOSE: To characterize inter- and intra-individual differences in X-ray-induced DNA strand break rejoining kinetics in human peripheral white blood cells (WBC) obtained from 10 healthy volunteers. MATERIALS AND METHODS: The alkaline and neutral versions of the comet assay were used to measure the rate of rejoining of predominantly single-strand breaks (ssb) following exposure to 8 Gy and double-strand breaks (dsb) following 75 Gy. RESULTS: All cells within a population responded in a similar fashion to induction of ssb and dsb; however, a subset of the WBC appeared to rejoin ssb more rapidly. For the 10 individuals examined, the percentage of ssb rejoined by the rapid component(s) was 47 +/- 16% and the rejoining half-time for the slow component was 1.3 +/- 0.4 h. By 24 h after 8 Gy, 4.9 +/- 3.8% of the initial ssb remained. For dsb rejoining, 58 +/- 11% of the initial damage was still present 4h after 75 Gy and by 24 h 32% of the initial level of damage was still detected. Heavily damaged cells present 24 h after 75 Gy varied from 4% to 50% and were excluded from the analysis of repair rates. CONCLUSIONS: Inter-individual variability exceeded intra-individual variability for 2 of 4 endpoints examined for ssb repair, but not for dsb repair. It was concluded that DNA damage measured using the comet assay could identify a range in the X-ray repair responses of WBC from different normal individuals. Whether these differences correlate with differences in cell killing by radiation remains to be determined.

Use of the micronucleus assay for the selective detection of radiosensitivity in BUdR-unincorporated cells after pulse-labelling of exponentially growing tumour cells.

To determine the radiosensitivity of non-S-phase tumour cells in vitro, survival curves of SCC VII tumour cells were obtained after a short block with hydroxyurea. Dose-response curves of micronucleus (MN) frequency appearing in non-S-phase cells were also determined by excluding S-phase cells with immunofluorescence staining to 5-bromo-2'-deoxyuridine (BUdR). Both the dose-response curves of MN frequency and survival curves were analysed by a linear-quadratic model (surviving fraction = exp (-alpha D-beta D2), MN frequency = aD+bD2+c). A good correlation between the alpha/beta and alpha/beta ratios was observed. In both BUdR-unincorporated and asynchronous cell cultures, the regression lines between the surviving fraction and micronucleus frequency were statistically identical. Therefore, it was shown that cell survival curves, which cannot be obtained directly by the routine colony-formation technique, can be calculated using the micronucleus frequency and the regression line in asynchronous cell cultures. Therefore, it should be possible to detect the response to irradiation of quiescent cells in tumours using the immunofluorescence staining to BUdR and the MN frequency assay.

The scid mutation does not affect slowly repairing potentially lethal damage that is sensitive to 0.23 M NaCl.

The repair of slowly repairing potentially lethal damage (PLD) in radiosensitive cells from the severe combined immunodeficient (scid) mouse was compared with that in Balb/c 3T3 cells with "wild-type" radiosensitivity and that in RD13B2 cells derived from scid cells whose sensitivity is normal because of the presence of fragments of human chromosome 8. Treatment with 0.23 M NaCl was used for fixation of slowly repairing PLD. The scid cells repaired PLD sensitive to 0.23 M NaCl to a great extent whin 3-4 h, similarly to Balb/c 3T3 and RD13B2 cells. This indicates that the scid mutation hardly affects the repair of PLD sensitive to 0.23 M NaCl. On the other hand, as reported previously, the rapidly repairing PLD that is sensitive to 0.5 M NaCl was repaired only slowly (3-4 h) in scid cells, in contrast to the rapid repair (within 1 h) seen with Balb/c 3T3 and RD13B2. This suggests that scid mutation is responsible for this repair at reduced rate. To confirm the independence of repair of 0.23 M NaCl-sensitive PLD from that of 0.5 M NaCl-sensitive PLD, both treatments with 0.23 M NaCl and 0.5 M NaCl were combined in each line. It is found that the repair of either PLD was not affected by the other treatment. The scid mutation impaired only the repair of 0.5 M NaCl-sensitive PLD.

System size dependence of the diffusion coefficient in a simple liquid.

An equation to estimate the system size dependence of the self-diffusion coefficient of a tagged particle moving in a simple fluid is given using linear-response theory and linearized hydrodynamics. Estimates made by the equation are compared with the results of the molecular dynamics simulation for a hard-sphere fluid at two densities, rhosigma(3) approximately 0.88 and 0.47, where sigma is the hard-sphere diameter. Good agreement between theory and simulation is obtained at the higher density. At the lower density, the agreement becomes poorer, but it is improved by taking into account the diffusion effect of the tagged particle. The equation gives the same diffusion coefficient for the infinite system as that obtained by taking into account the long-time tail contribution of the velocity autocorrelation function [B. J. Alder, D. M. Gass, and T. E. Wainwright, J. Chem. Phys. 53, 3813 (1970)]. When the tagged particle has a larger mass than the fluid particles, the equation presented here gives the better estimates. It is confirmed by the molecular dynamics calculation.

Effects of losartan on the collagen degradative enzymes in hypertrophic and congestive types of cardiomyopathic hamsters.

The present study was undertaken to determine the effects of AT1 receptor blockade which occurred in response to losartan, on the extracellular matrix (ECM) degradation process in the Bio 14.6 (n = 12) and Bio 53.58 (n = 12) strains which are referred as models of hypertrophic and dilated cardiomyopathy, respectively. The administration of losartan (30 mg/kg/day) in hamsters from 10-20 weeks of age reduced the accumulation of the left ventricular collagen matrix in both of the Bio 14.6 and the Bio 53.58 strains. According to the RT-PCR, the levels of mRNA for matrix metalloproteinase (MMP) and the tissue inhibitor of MMP (TIMP) were examined. MMP-1, -2, -3, and -9 were more enhanced in both myopathic strains than in the control F1beta, strains. With losartan, the levels of MMP-1, -2, -9, TIMP-1 and -2 decreased in the both strains but those for MMP-3 did not in Bio 14.6 strains. TIMP-3 and -4 mRNA levels did not change in any of the experimental hamsters, whether treated or untreated with losartan. The Western blots also showed similar observations in the both strains as seen in mRNA expressions although MMP-2 in the Bio 53.58 strains did not differ between treated and untreated with losartan. Although losartan has an inhibitory effect on collagen accumulation in the development of cardiomyopathy, MMPs (-1, -2, -9) and TIMPs (-1, -2) seem to be susceptible to responding to losartan in Bio cardiomyopathic hamsters.

Electrostatic interactions in protein solution--a comparison between Poisson-Boltzmann and Monte Carlo calculations.

The accuracy of the Poisson-Boltzmann (PB) approximation and its linearized version is investigated by comparison to results obtained from Monte Carlo simulations. The dependence of the calcium binding constant of the protein calbindin as a function of salt concentration and mutation is used as a test case. The protein is modeled as a collection of charged and neutral spheres immersed in the electrolyte solution. The PB equation is solved using a finite difference technique on a grid in a spherical polar coordinate system, which is the preferred choice for a globular protein like calbindin. Both MC and PB give quantitative agreement with experimental results. The linearized PB equation is almost as accurate, but it becomes less reliable in systems with divalent ions. However, the linearized PB equation fails to describe the concentration profiles for cations and anions outside the protein even in a 1:1 salt solution.

[Promotive effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on recovery from neutropenia induced by fractionated irradiation in mice].

The effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on the recovery from neutropenia induced by fractionated whole-body irradiation was investigated in mice. Male 7-week old C3H/HeN mice received a total of ten exposures of 0.25 Gy/day from day 1 to 5 and from day 8 to 12. Peripheral neutropenia with a nadir on day 17 was caused by the fractionated irradiation. Daily subcutaneous injections of rhG-CSF at 0.25 and 2.5 micrograms/body/day from day 1 to 21 promoted the recovery of neutrophils in a dose-dependent manner. The kinetics of morphologically identifiable bone marrow cells were studied to clarify the mechanism behind the promotive effect of this factor. A slight decrease in mitotic immature granulocytes, such as myeloblasts, promyelocytes and myelocytes on day 5, and a drastic decrease in metamyelocytes and marrow neutrophils on days 5, 9, and 17 were seen in the femur of irradiated mice. Treatment using rhG-CSF caused an increase in immature granulocytes of all differential stages in the femur. Microscopic findings of the femurs and spleens also revealed an increase in immature granulocytes in these organs in mice injected with rhG-CSF. These results indicate that rhG-CSF accelerates granulopoiesis in the femur and spleen, thereby promoting recovery from neutropenia induced by fractionated irradiation.

Combined effects of radiation and IUdR on experimental liver tumors and hepatocytes.

IUdR, a thymidine analogue, increased the radiosensitivity of SCC VII tumor cells. A decrease in Dq value of cell survival curve was more prominent compared with that in D0 value. However, in experimental liver tumors made by transplanting SCC VII tumors, the decrease in D0 value was apparent. Proportion of IUdR incorporated tumor cells in the liver increased with an increase in number of IUdR injections, and reached 52% after five administrations of the drug at an interval of twelve hours. However, few hepatocytes incorporated IUdR. The response of hepatocytes to radiation was evaluated by micronucleus appearance frequency. To accelerate the appearance of micronucleus in hepatocytes, partial hepatectomy was performed immediately after irradiations. No differences in micronucleus frequency were observed between the treated and untreated with IUdR.

Gentamicin nephrotoxicity: application of multivariate analysis.

A retrospective study was conducted to identify patient risk factors associated with gentamicin nephrotoxicity using multivariate analysis. Patients were evaluated for nephrotoxicity using an algorithm that considers serum creatinine, previous general experience with the drug, alternative etiologic candidates, timing of events, drug concentrations or evidence of overdosage, dechallenge, and rechallenge. There were 220 patients who met the criteria for inclusion in the study (103 men and 117 women). Variables found not to be significant discriminators between toxic and nontoxic patients included height, weight, sex, dose, estimated peak gentamicin concentration, age, and initial renal function. Variables that were found to enter into the discriminant analysis included presence of complicating factors with nephrotoxic potential, number of concurrent potentially nephrotoxic drugs, intensity index or estimated trough gentamicin concentration, and the total dose or treatment duration. Two models were developed; the probability of correctly classifying a patient as toxic was 63.5% and 65.4%, and as nontoxic was 72.1% and 68.8% for the two models, respectively. Although the discriminant models developed were statistically significant, the discrimination between toxic and nontoxic patients was not satisfactory to allow development of a generalized predictive model. Multivariate techniques were found to be essential in allowing for the differentiation of variables causing gentamicin nephrotoxicity.

Left renal vein hypertension in patients with left renal bleeding of unknown origin.

The pullback pressure from the left renal vein (LRV) to the inferior vena cava was studied in 16 patients with left renal bleeding of unknown origin (group A), 15 patients with hematuria of miscellaneous laterality (group B), and nine control subjects (group C). The mean pressure gradients were 5.0 +/- 2.0 mm Hg (mean +/- SD) in group A, 1.9 +/- 1.0 mm Hg in group B, and 1.1 +/- 0.9 mm Hg in group C. The mean pressure gradient of group A was significantly higher than that of groups B and C (P less than .001). From the results of control studies, we regarded pressure gradients greater than or equal to 3.0 mm Hg as indicative of LRV hypertension. With this criterion, 88% (14 of 16) of the patients in group A had LRV hypertension, which was considered a cause of hematuria. Analysis of the results of renal angiography in the 31 patients revealed that opacification of collateral pathways of the LRV was a significant angiographic finding in LRV hypertension.

Computed tomography analysis of causes of local failure in radiotherapy for cervical carcinoma.

BACKGROUND: The authors analyzed the radiation dose to the periphery of the cervix and area of the cervix in relation to local failure of radiotherapy for carcinoma of the cervix using computed tomography (CT) images. METHODS: Between 1981-1990, 127 consecutive patients were treated with definitive radiotherapy. Ninety-nine of these patients had CT images taken at the time of intracavitary therapy. Of these 99 patients, 80 were eligible for this analysis. After CT scanning, isodose curves relative to the point A dose were superimposed on the CT images. The minimum percent dose and minimum dose at the periphery of the cervix were estimated. The area of the cervix also was measured. These factors were examined in relation to the local tumor control rate. RESULTS: Histograms of both the minimum percent dose and the cervical area showed significant differences between the local control and local failure groups (P <0.001). The local control rates were related to both the minimum percent dose and the cervical area, and differed significantly over and below the values of 60% and 18 cm2 (P <0.001 each), respectively. The local control patients, over and below the line: Y = -0.220X + 21.2, in which X (gray [Gy]) and Y (Gy) are the whole pelvis dose and the minimum dose, respectively, could be well differentiated with significance (91.7% vs. 25.0%; P <0.001). CONCLUSIONS: Computed tomography analysis indicated that the local tumor control rate was related strongly to the minimum percent dose, the cervical area, and the pair of whole pelvis and minimum dose values. These factors were found to be more useful than the point A dose in predicting local tumor control.

[Hypoxic cell radiosensitizer present status and problems from viewpoint of clinical radiotherapy].

The results of clinical trials of hypoxic cell radiosensitizers were reviewed in order to clarify the present status and problems in this field. Most trials failed to show an effectiveness of the combination, however, some ENT tumors and bladder cancers were improved in local tumor responses and survivals. A selection of the patients suitable for the combination and development of simple method for the selection are indispensable. SERs necessary for clinical radiotherapy are considered to be larger than 1.2 at 2 Gy fraction level. Present hypoxic cell radiosensitizers have no significant effects on alpha values. Since radiation responses at low radiation dose ranges are dominated by alpha values, the chemicals having an effects on alpha values have to be developed.

[Present status of hypoxic cell sensitizers and PLDR inhibitors].

Clinical and basic studies in hypoxic cell radiosensitizers and potentially lethal damage (PLD) repair inhibitors were reviewed. Most clinical trails on misonidazole (MISO) show that the toxicity of MISO is a major reason for failure in the trials and an adequate selection of tumors is needed for the success. Phase III studies on SR-2508 and RO 03-8799, less toxic and more effective sensitizers, are in progress. AK-2123 and RK-28 developed in Japan are drugs with similar clinical applicability of MISO. Thiol depletion of tumors is one of the way to increase the radiosensitization. The measurement of intercapillary distance on histopathological microslide is a valuable method to select the tumors containing much hypoxic cell fractions. Stable and effective chemicals as MISO in hypoxic cell sensitization must be developed to investigate the clinical applicability of PLD repair inhibitors.