The sole purpose of radio-contrast agents is to increase the absorption of X rays in blood vessels and soft tissues. Current diagnostic agents take advantage of the properties of iodine with its acceptably low clinical toxicity and moderately high atomic number (53), the latter yielding a relatively large absorption coefficient for photons in the diagnostic X-ray energy range, owing to the photoelectric effect. One consequence of this, not often appreciated, is that cells in the vicinity of an iodine-rich solution will receive a higher radiation dose than would be the case if iodine were not present. This is partly responsible for the reported cytogenetic changes in lymphocytes in vitro and in vivo (Adams et al, 1977;Norman et al, 1978;Cochran et al, 1980;Cochran & Norman, 1982;Hadaghy et al, 1982). Though these data clearly signal an undesirable, if unavoidable, phenomenon in diagnostic radiology, such a cytotoxic effect may be capable of exploitation in radiotherapy.To examine this latter possibility, cells in vitro were treated with various concentrations of the contrast agent meglumine ioglycamide (Biligram, Schering AG) and subjected to orthovoltage X rays. Marked radiation enhancement was obtained at an iodine concentration of 50mg ml" 1 , a concentration that is well within the range achievable in the bloodstream. METHODChinese hamster ovary (CHO) cells were grown in monolayer culture in Hepes-buffered minimum essential medium (MEM) supplemented with 15% fetal calf serum, nonessential amino acids and L-glutamine. Tissue culture flasks (25 cm 2 ; Nunclon) were seeded with 3.5 x 10 5 cells 24 h before irradiation, so that cells were in the exponential phase of growth during treatment.Biligram was obtained as a 17% aqueous solution (85 mg ml" 1 iodine) and diluted with complete medium to the required concentration. Biligram cannot pass through the cell membrane and a 10% aqueous solution was not cytotoxic towards a cell monolayer for 1 h at 37°C. Monolayers of CHO cells were given 5 or 10 ml of fresh complete medium (depth of medium 2 or 4 mm above cell monolayer), containing the necessary quantity of Biligram, 5 min before irradiation.Cell monolayers growing on the bottom of flat plastic flasks were irradiated from above with X rays generated at 250 kVp (half value layer 0.83 mm Cu) and at a dose-rate of 1.60 cGy min ~1. The incident beam passed through 4 mm of iodine-rich medium before reaching the cell layer.In a further experiment, to examine the importance of the direction of the incident beam in relation to the iodine-rich medium and cell layer, the monolayer was irradiated from below, i.e. the X-ray beam passed through the bottom of the dish and the cells before traversing the overlying layer of medium (2 mm deep). The dose-rate for this irradiation was 1.81 cGy min" 1 .The experiment was repeated with photons from a cobalt-60 source (1.25 MeV) to confirm that there is little enhancement at this energy where photoelectric absorption is minimal. In this case, cells were irradiated from above at a dose-r...

Most tumours grow more slowly when implanted into pre-irradiated sites--the tumour bed effect (TBE). The TBE is usually assayed by measuring the delay for tumours growing in irradiated sites compared with that for tumours growing in mock-irradiated sites to reach a certain arbitrary, externally-measured volume. The resulting X-ray dose-response curves for the TBE are generally S-shaped, with little effect up to doses of 5 Gy, a dose-dependent effect between 5 and 20 Gy and a plateau at higher doses. In this study such a dose-dependent TBE was demonstrated for two contrasting transplantable tumours (a rapidly growing mammary adeno-carcinoma and the RIF-1 fibrosarcoma) growing in the flank skin of C3H/He mice. Cell kinetic and histological methods were used to investigate the mechanism of the reduced tumour growth rate in irradiated sites. By combining information from tumour growth curves and metaphase-arrest lines, tumour cell birth and cell loss rates were estimated. In addition the necrotic and viable fractions of tissue were measured by means of Chalkley point counting. In both tumours, marked increases in cell loss rate and degree of necrosis were found to be dependent on the dose of X rays previously given to the stroma. Surprisingly, cell birth rate and mitotic index were significantly increased in mammary tumours growing in irradiated sites. The estimation of viable, as opposed to external, volume of tumours growing in sites which had received between 0 and 60 Gy X rays suggested that the conventional TBE assay method may underestimate the extent of the TBE and may distort the shape of the TBE dose response curve.

The radiation response of mammary tumours transplanted into syngeneic C3H mice has been measured with the animals breathing air or 100% oxygen at 290 kPa (HPO), either with or without ketamine plus diazepam anaesthesia. The single doses needed to cure 37% of tumours within 40 days (TCD37/40) for mice anaesthetised with ketamine plus diazepam and for unanaesthetised mice irradiated in air were not significantly different, 66.5 Gy and 68.8 Gy respectively. When animals were irradiated in HPO, the TCD37 value was significantly reduced from 60 Gy with no anaesthetic to 41 Gy with ketamine plus diazepam anaesthesia; an enhancement ratio (ER) of 1.5. The total ER from no anaesthetic in air to anaesthetic in HPO was 1.7 (68.8/41). There was less CNS toxicity for ketamine plus diazepam than for sodium pentobarbitone anaesthesia in mice treated in HPO. The combination of ketamine and diazepam is an unusual anaesthetic in that it maintains blood pressure, cardiac output and respiration in man. Vascular effects and lowered body and tumour temperatures may also have influenced tumour oxygenation and radiation response.

The reduction in tumour growth rate seen when tumours are implanted into preirradiated sites, the tumour bed effect (TBE), is believed to be due to radiation damage to vascular stroma, leading to defective angiogenesis in the tumour. The present work examined whether or not the functional inadequacy of irradiated stroma was accompanied by an increased hypoxic fraction in tumours growing in irradiated beds. Mouse flank skin was given 0 or 20 Gy X-rays and RIF-1 fibrosarcoma cells were implanted i.d. into the centre of the treatment field one week later. Tumours of 200 mm3 were irradiated under clamped or unclamped conditions and the hypoxic fraction measured from the displacement of the corresponding survival curves, assayed in vitro. Results indicated a small increase in the hypoxic fraction. Averaging values from three independent experiments, the percentage of hypoxic cells increased from 2.5 per cent for cells in tumours growing in unirradiated beds to 4.6 per cent for those from tumours in beds given 20 Gy. Thus an irradiated vascular bed is still to some extent able to maintain the proportion of oxic: hypoxic tumour cells found in tumours growing in unirradiated beds, despite manifest changes in tumour necrosis and growth rate.

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