Membrane Radiosensitivity of Fatty Acid Supplemented Fibroblasts as Assayed by the Loss of Intracellular Potassium

Summary

Leakage of potassium from mouse fibroblast LM cells, X-irradiated at 0°C with doses up to 400 Gy is shown to be related to plasma membrane lipid composition. Fatty acid supplemented cells, containing about 40 per cent polyunsaturated fatty acids (PUFA) in their membranes were much more sensitive to radiation, as measured by increased permeability, than normal cells, which contained 7 per cent PUFA. The damage observed after irradiation at 0°C was partially repaired during a post-irradiation incubation at 22°C. The o.e.r. for potassium leakage was about 4 for normal fibroblasts and 8 for the PUFA-supplemented cells. No oxygen-dependent radiation damage could be observed in cells treated with high amounts of vitamin E. Depletion of glutathione in PUFA cells sensitized oxic cells to radiation damage, resulting in an increase of the o.e.r. from 8 to 17. No lipid peroxidation (malondialdehyde production and disappearance of fatty acyl chains) could be demonstrated. While PUFA, normal and vitamin E grown cells showed a differential sensitivity in radiation-induced potassium leakage and trypan blue uptake (high doses, interphase death), no difference in radiation-induced clonogenic ability (reproductive death) could be observed after the different cell treatments. The experiments reported are supportive of a role of membranes in the mechanism of radiation-induced interphase death and show that increased damage may be expected when high amounts of polyunsaturated membrane lipids are present under conditions of low amounts of appropriate antioxidants.     

Effect of Hyperthermia on DNA Loop-size in HeLa S3 Cells

Summary

Nuclear matrices of heated and non-heated HeLa S3 cells were isolated and average DNA loop-sizes were compared. Heat treatment (30 min at 45°C) resulted in an ultimate survival level of the cells of about 10 per cent. The loop-size determinations were done on nuclear material isolated from the cells directly after heat treatment. In the nuclear matrices isolated from the heated cells about 1·8 times more protein was bound as compared to the matrices from control cells. Enzymatic analysis using DNase I digestion, followed by centrifugation on neutral sucrose gradients, was performed. Also, halo visualization was combined with autoradiography. Both methods revealed no gross alterations in DNA loop-sizes. The possible function of DNA loop organization in the effect of hyperthermic interference with DNA-related processes is discussed.     

Contrasting Dose-rate Effects of γ-irradiation on Rat Salivary Gland Function

The aim of this study was to investigate the effects of ⁶⁰Co irradiation delivered at high (HDR) and low (LDR) dose-rates on rat salivary gland function. Total-body irradiation (TBI; total doses 7·5, 10 and 12·5 Gy) was applied from a ⁶⁰Co source at dose-rates of 1 cGy/min (LDR) and 40 cGy/min (HDR) followed by syngeneic bone marrow rescue. Four days before and 1–30 days after TBI, submandibular and parotid saliva samples were collected in male albino Wistar rats using Lashley cups. Lag phase and flow rate were recorded, and [Na⁺] and [K⁺] were measured. The severity of salivary gland dysfunction for each dose-rate was dependent on total TBI dose in all parameters. LDR irradiation significantly enhanced the increase of lag phase, while it tended to further decrease flow rate during days 0–3. At later times the reverse effect was seen with significant LDR sparing in most cases. The changes in [Na⁺] and [K⁺] showed similar trends; LDR had an enhancing effect for early damage, while beyond day 3 it consistently produced less damage. From this dose-rate study it is concluded that the early postirradiation changes in salivary gland function are probably predominantly caused by irradiation damage to membrane structures and are less the result of reproductive failure. The later changes in salivary gland function are probably mainly dependent on repopulation of surviving stem cells.     

Acute irradiation effects on morphology and function of rat submandibular glands

In this study the morphologic and functional changes were compared after irradiation (single dose, 15 Gy) of rat submandibular salivary glands. Before and 1-10 days after local irradiation of the gland region, samples of submandibular saliva were collected after stimulation by pilocarpine. At the same time-points and also 3 h postirradiation submandibular glands were carefully extirpated and prepared for histocytologic examination (LM, TEM). Maximal increase of the lag phase and decrease of the flow rate were observed 3 days after irradiation, while [K+] and [Na+] increased and decreased, respectively, from days 1 and 3 after irradiation. Morphologic changes were observed from the third hour after irradiation, were maximal 3 days after irradiation and had partially recovered by day 10. Three hours and 1 day after irradiation degranulation of convoluted granulated tubes (CGT) was observed. Three days after irradiation the most striking morphologic changes in serous and mucous cells were distension of the cisternae of the RER, degeneration of mitochondria and vacuolization of the cytoplasm. Fibril-like condensations of electron dense material in the mucous granules were observed 3 h, 1 and 6 days after irradiation. Regranulation of CGT cells was observed from day 6. From this study it is concluded that changes in salivary gland function can be observed before major morphologic changes occur. Functional changes persist after the morphologic changes seem to have virtually returned to normal.     

Reduced levels of glutathione in liver and tumour tissue of the mouse after administration of eupatoriopicrin

In this study the effect of the cytostatic acting sesquiterpene lactone eupatoriopicrin, isolated from Eupatorium cannabinum L., on gluthathione (GSH) levels in liver and tumour tissue of the mouse is described. C57Bl mice, bearing a solidly growing Lewis Lung carcinoma or a FIO 26 fibrosarcoma, with a volume between 500–1000 μL, were injected with eupatoriopicrin 20 or 40 mg/kg, intraperitoneally (i.p.) or intravenously (i.v.). At different time points, between 0–48 h after administration, the GSH content in liver and tumour tissue was determined. A dose dependent reduction of the GSH content was found. No difference in reduction was seen between i.p. and i.v. administration, but the route of administration appeared to be of great importance regarding acute toxicity: 40 mg/kg i.p. was lethal within 48 h, whereas mice receiving the same dose i.v. survived for over three months.     

Cisplatin sensitivity and thermochemosensitisation in thermotolerant cDDP-sensitive and -resistant cell lines.

Development of thermotolerance is an important phenomenon that must be considered when thermochemotherapy with multiple heat treatments is used clinically. To study the effect of thermotolerance on cellular cisplatin (cDDP) sensitivity at 37 degrees C and 43 degrees C in cell lines with different cDDP sensitivities, two Ehrlich ascites tumour cell lines (one with high cDDP sensitivity and one with in vitro acquired cDDP resistance) were used. The results indicate that in both cell lines the state of thermotolerance per se did not affect the cDDP sensitivity at 37 degrees C. Thus, general elevations in ''all'' heat shock protein levels as found in thermotolerant cells apparently do not influence cDDP sensitivity to a considerable extent. The sensitising effect of a (second) heat treatment given simultaneously with a cDDP treatment was less in thermotolerant cells. Thermal enhancement ratios (TERs) at the 10% survival level for heat doses of 43 degrees C for 30 min or 43 degrees C for 60 min were reduced by a factor of 1.6 and 2.1 in cDDP-resistant and -sensitive thermotolerant cells respectively, as compared with control cells. Thus, protection against heat damage in thermotolerant cells seems to be paralleled by diminished thermal chemosensitisation. Although the effect of thermotolerance on the cDDP-sensitising effect was less pronounced in the resistant cells, a modifying effect on the resistance factor was not achieved.     

Effect of a hyperthermic treatment on the pluripotent haemopoietic stem cell in normal and anaemic mice

Up to now, the hyperthermic sensitivity of pluripotent haemopoietic stem cells is unknown, and the few existing data from reports in the literature are conflicting. There are two main drawbacks in the set-up of those studies: (1) only CFU-S day 9 results were presented, whereas it is questionable if this assay gives a true reflection of the pluripotent stem cell, and (2) no attention has been paid to heat effects on the seeding efficiency, i.e. the amount of stem cells which will lodge in the spleen. The present study focused on the procedural differences and compared the results of a hyperthermic treatment (60 min, 42 degrees C) on the stem cells, assayed with the CFU-S day 9 and the CFU-S day 12 method, using the following three stem cell suspensions, all differing in their proliferative activity: bone marrow from normal mice and bone marrow and spleen cells from anaemic mice. Furthermore, we investigated the seeding efficiency before and after heat treatment. Resting stem cells, assayed with the CFU-S day 12 method, turned out to be resistant to hyperthermia as compared with the active cycling stem cells, while with the CFU-S day 9 assay the stem showed the same thermosensitivity in the two bone marrow suspensions. The active cycling stem cells do not significantly differ in thermosensitivity, in CFU-S day 9 and day 12 assays, although there is a difference between bone marrow and spleen. Hyperthermia appears to influence the seeding efficiency for spleen CFU-S; an increase of 1.73 was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Study on the relationship of serum fetuin-A concentration with aortic stiffness in patients on dialysis.

BACKGROUND: An increase in aortic stiffness, as reflected by an increase in pulse wave velocity (PWV) or aortic augmentation index (AI) is an important predictor of cardiovascular mortality in dialysis patients. Dysregulation of calcification inhibitors, such as fetuin-A, is involved in vascular pathology in dialysis patients and fetuin-A is inversely related to mortality in dialysis patients. In this study, the relation between serum fetuin-A concentration and parameters of aortic stiffness was investigated in patients with end-stage renal disease. METHODS: In a cross-sectional study we included 131 dialysis patients, aged 62+/-14 years (33 on peritoneal dialysis and 98 on haemodialysis), and 41 controls, aged 60+/-8 years. Time-averaged pre-dialysis values of serum albumin, Ca, P and intact parathyroid hormone were included in multiregression analysis, as were high-sensitivity C-reactive protein (hsCRP), fetuin-A, age, mean arterial pressure (MAP) and dialysis modality. PWV and AI were measured with the SphygmoCor device. RESULTS: Mean fetuin-A concentration in dialysis patients (0.63+/-0.16 g/l) did not differ from controls (0.63+/-0.11 g/l). Median hsCRP levels in dialysis patients were higher compared with controls (4.0 vs 1.9 mg/l; P<0.0001). PWV but not AI was higher in dialysis patients than in controls (9.9 vs 7.9 m/s; P<0.0001). In univariate analysis in dialysis patients, fetuin-A levels were inversely related to both PWV (r = - 0.25, P = 0.007) and AI (r = - 0.26, P = 0.006), respectively. However, after correction for age, gender, MAP and diabetes mellitus, this relation lost statistical significance. CONCLUSIONS: In a dialysis population with a relatively low level of inflammatory activity, the soluble calcification inhibitor fetuin-A could not be identified as an independent predictor of aortic stiffness as measured with PWV and AI.     

Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1.

Resistance of Lactococcus lactis to cytotoxic compounds shares features with the multidrug resistance phenotype of mammalian tumor cells. Here, we report the gene cloning and functional characterization in Escherichia coli of LmrA, a lactococcal structural and functional homolog of the human multidrug resistance P-glycoprotein MDR1. LmrA is a 590-aa polypeptide that has a putative topology of six alpha-helical transmembrane segments in the N-terminal hydrophobic domain, followed by a hydrophilic domain containing the ATP-binding site. LmrA is similar to each of the two halves of MDR1 and may function as a homodimer. The sequence conservation between LmrA and MDR1 includes particular regions in the transmembrane domains and connecting loops, which, in MDR1 and the MDR1 homologs in other mammalian species, have been implicated as determinants of drug recognition and binding. LmrA and MDR1 extrude a similar spectrum of amphiphilic cationic compounds, and the activity of both systems is reversed by reserpine and verapamil. As LmrA can be functionally expressed in E. coli, it offers a useful prokaryotic model for future studies on the molecular mechanism of MDR1-like multidrug transporters.