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V79 379A cells were irradiated and then exposed to anisotonic PBS for 20 min. This enhanced the radiation effect resulting from the fixation of potentially lethal damage. The induction of DNA single- and double-strand breaks is not increased by this treatment. Anisotonic treatment delayed the onset of repair of DNA damage. However when cells were returned to normal medium, they repaired the damage to a similar extent as cells not exposed to the anisotonic treatment. We suggest that the fixation of damage by post-irradiation anisotonic treatment is mediated through an increased probability of misrepair of DNA damage due to the delay in the onset of repair. This is supported by the observation that there is a reduced effect of post-irradiation anisotonic treatment on cells that have a markedly reduced ability to repair double-strand breaks.

The induction and repair of DNA double-strand breaks (DSB) following exposure to 238Pu α-particles was examined in V79-379A cells. The technique of neutral filter elution was used in these investigations at both pH 9·6 and pH 7·2. The initial dsb yield was found to be similar to that seen after 250 kVp X-ray or 2·3 MeV neutron exposure. However, the pattern of dsb rejoining after α-particle irradiation did not follow that seen after X-rays or neutrons. A very fast initial component, complete within 2 min of incubation following irradiation, removed 70 per cent of the dsb seen after 40 Gy α-particles; very little slow rejoining was seen. This contrasts sharply with the dsb rejoining seen after X-ray or neutron exposure, and presumably reflects the differences in the nature of the dsb induced and the way they are repaired.

The ability to determine the proportion of hypoxic cells within a tumour during treatment could lead to a more rational assessment of benefit when planning and executing radiotherapy. Until recently the only method available to determine the hypoxic fraction has been an indirect radiobiological assessment. This is applicable to experimental tumour systems but not to human patients. We have therefore sought a more direct and rapid quantitative method for detecting hypoxic cells which could eventually be applicable in the clinic. The method is based on the production of fluorescence in hypoxic cells which can be detected by microscopy or by using a flow cytometer.The rationale for the present work was based on the observations that:—(1) with certain nitroaromatic compounds the quantum efficiencies of fluorescence for the nitroreduction products (amines) are at least four orders of magnitude greater than that of the parent compound (McGlynn et al, 1969); (2) the amine is only produced under hypoxic conditions by cellular enzymes, because the initial reduction step to the nitro radical-anion, an obligate intermediate, is rapidly reversed in the presence of oxygen (Mason, 1982); (3) the reduction products formed from the bioreductive metabolism of misonidazole, metronidazole and nitrofurazone bind intracellularly in hypoxic cells.Radiolabelling of misonidazole or its analogues, followed by measurement of bound label using scintillation counting (Verghese et al, 1967), autoradiography (Chapman et al, 1981), or (potentially) external gamma scanning (Rasey etal, 1982) have been proposed as the basis for clinical detection and imaging of hypoxic cells in tumours.

Two compounds, nitroakridin 3582 (NA) and a 3-nitronaphthalimide (DM113), have been tested as potential fluorescent markers for hypoxic cells. Cellular fluorescence in three cell lines (V79–379A, WHF1B, EMT6) was measured by flow cytometry and high-performance liquid chromatography (HPLC) after incubating the cells with the drugs for various times in air or hypoxia. In all three cell lines, both drugs showed greater fluorescence in hypoxic than in oxic cells. There were, however, differences between the cell lines in respect of the magnitudes of hypoxic and oxic cell fluorescence and in the ratio of hypoxic to oxic fluorescence. Differences in hypoxic cell fluorescence were due to differences in the rate and extent of nitroreduction. Drug uptake and DNA content per cell were relatively unimportant factors in determining the magnitude of fluorescence. There was not a good correlation between cytotoxicity of the drugs and hypoxic fluorescence. Nitroakridin was more toxic to hypoxic than to aerated cells but the reverse was true for DM113. The dependence of fluorescence and radiosensitivity on oxygen concentration were compared for the three cell lines and only small differences between the “K”-curves for the two end points were found.Two problems with the present compounds which should be addressed in designing future fluorogenic compounds as hypoxic markers were oxic cell fluorescence and leakage of fluorescent products from hypoxic cells.

The response of a murine tumour to single doses of X rays has been measured using three different assays—animal cure, cell survival in vitroafter irradiation in vivo, and tumour growth delay. The dose to cure 50% of the animals, the TCD50, was 79.0 Gy. This was not affected by clamping the tumours to render all the cells hypoxic at the time of irradiation, implying that most of the cells in the tumour were hypoxic already. The enhancement ratio for the hypoxic cell sensitizer Ro-07-0582 was 2.1. The cell survival assay gave an enhancement ratio of 1.6 and an hypoxic fraction of 5%. The discrepancy in the estimates of the hypoxic fraction can be explained by the ability of the naturally hypoxic cells, but not the oxic ones, to recover from potentially lethal damage in vivo. Neither the cell survival assay nor the growth delay assay agreed with the TCD50assay as to the effect of the hypoxic cell sensitizer, even allowing for recovery from potentially lethal damage. It is doubtful whether the measured survival curve would predict the measured TCD50.

The Secretariat, UKCCCR, The Medical Research Council, 20 Park Crescent, London W1N 4AL, United Kingdom

A comparison has been made of the effects of the 2-nitroimidazole Ro-07-0582 on tumour growth delay after irradiation and tumour cell survival in vitroafter irradiation in vivo. This compound has previously been shown to be a specific sensitizer of hypoxic cells. A dose of 1 mg/g body weight gave an enhancement ratio of 2-2 for both growth delay and cell survival in a system where high pressure oxygen has been shown to have no effect. However, while the hypoxic fraction in the tumour was estimated to be less then 10% from the growth delay curves, the survival curves gave a value in excess of 50%. This discrepancy probably reflects differences in the response of cells left in situ or removed and assayed in vitro.

Misonidazole (MISO) has been shown to affect the pharmacokinetics of both cyclophosphamide (CY) and melphalan (MEL) in WHT mice resulting in increased plasma levels of the cytotoxic drugs. The effect is not solely due to the reduction in body temperature observed with large single doses of MISO, as a change in MEL pharmacokinetics was still observed when the mice were maintained at 37 degrees C. Inhibition of cytotoxic drug metabolism may also be a possible mechanism. Such a pharmacokinetic effect could account for part of the potentiation of MEL and CY action observed in tumours with large single doses of MISO. However, a chronic low dosing schedule of MISO did not affect the plasma half-life of either cytotoxic drug, although a significant potentiation of each drug in combination with a chronic MISO dose has been obtained in some tumours. These results suggest that potentiation of chemotherapeutic drug action by MISO in the clinical situation is unlikely to be due to changes in drug pharmacokinetics.

Misonidazole (MISO) potentiates the action of cyclophosphamide (CY) and melphalan in the WHFIB culture-adapted fibrosarcoma, whether assayed by cell survival or tumour-growth delay. In the case of CY, MISO also inhibited recovery from potentially lethal drug damage. The optimum effect was seen when MISO was given 1 h before CY, though it was also effective when given 6 h before or 1 h after the drug. Other radiosensitizers also potentiated the action of CY. There was only a small effect of MISO on the LD50 of CY and no effect on CY toxicity as assayed by changes in blood counts or damage to bladder epithelium. However, mice bearing multiple lung tumours were less able to cope with the combined treatment than those bearing s.c. tumours.

Misonidazole (MISO) given as a large single dose enhanced the action of cyclophosphamide (Cy) and melphalan (L-PAM) in two mouse tumours. Below a dose of about 500 mg kg-1 it had no chemosensitizing effect. When MISO was given as a series of small doses by repeat injection over an 8 h period, in order to stimulate human pharmacokinetics, it significantly enhanced the action of Cy in the SA F tumour. It also enhanced the action of Cy and L-PAM in the WHFIB tumour as assayed by tumour cell survival in vitro following treatment in vivo but not when the assay was tumour growth delay. There was no enhancement by MISO of the leukopenia due to Cy or L-PAM. The results suggest that, in some tumours there may be benefit from the combination of clinically relevant MISO doses with alkylating agents. The leucopenia induced by these agents should not be enhanced by the MISO.

Two mouse sarcomas have been used to assess the sensitivity to Adriamycin of tumour cells in vivo and in vitro. Both tumours were tissue-culture adapted so that cell survival could be assayed in vitro after treatment either in vivo or in vitro. For both tumours (WHFIB and CBSAF) cells were highly sensitive when treated in vitro yet very resistant to treatment in vivo, whether assayed by cell survival or regrowth delay. Cells from both tumours treated in vitro with Adriamycin immediately after excision were slightly more resistant than the cells maintained in vitro. However, this was not adequate to explain the marked discrepancy between in vivo resistance and in vitro sensitivity. The discrepancy was not due to a failure of drug delivery. Phase of cell growth was the factor was most significantly affecting chemosensitivity in vitro, plateau-phase cells being much more resistant than log-phase cells. Hypoxia was also an important factor leading to reduced chemosensitivity. Tumour diameter, in the range 2-8 mm, did not appear to be important.

The causes of failure after radiotherapy or chemotherapy may be related to physical or biological factors. The physical factors will include geographic miss in radiotherapy. The biological reasons may be inadequate drug delivery in chemotherapy or resistance of nutritionally deprived or hypoxic cells, a capacity to repair damage or rapid proliferation of the tumour cells so that repopulation may occur during treatment. There is much current interest in repopulation and it would be of value to identify those tumours which have rapid cell proliferation for, in these cases, a change in drug scheduling or shortening the overall treatment time with radiotherapy may lead to increased success by overcoming the effect of repopulation (Fowler, 1985).We have previously described a method for labelling proliferating cells in human tumours by measuring the uptake of bromodeoxyuridine (BrdUrd) into cells synthesizing DNA, following an intravenous injection, and the potential applications of this procedure to the measurement of proliferation rates in human tumours (Begg et al, 1985; Wilson et al, 1985). In this preliminary communication we report on three human tumours where kinetic studies have been performed and demonstrate the potential advantages of the technique.

We have compared the effects of mixed fractionation schedules with X rays and neutrons on growth delay of a murine tumour and skin reactions in mice. The schedules were five daily fractions of X rays, neutrons or mixtures (NNXXX, XXXNN or NXXXN). For clamped tumours or skin all three mixed schedules had the same effect. In contrast, for undamped tumours giving the neutrons first (NNXXX) was more effective than the other two mixed schedules. This represented a true therapeutic gain and implies that if neutrons are used clinically as only part of a course of fractionated radiotherapy, they should be given at the beginning rather than at the end of treatment.

Bromodeoxyuridine (BrdU) incorporation and flow cytometry have been used to measure the kinetics of V79 cells growing at different temperatures in vitroand cells of the murine Sa F tumour growing in vivo. By simultaneously measuring total DNA content and BrdU incorporation in individual cells at different times after pulse labelling with BrdU, it is a simple procedure to quantify the movement of cells through the cell cycle. The method has the advantage of speed (2 × 104or more cells are analysed within a day or so of the experiment) and the ability to analyse the results in different ways. A G2 block in tumour cell progression in vivois readily detected after a dose as low as 2 Gy. The neutron relative biological effectiveness for this G2 block is probably larger than that for tumour growth delay.

Computer simulations have been made of four radiotherapy fractionation regimes either in current use or proposed for clinical trials of misonidazole. A variety of cell-survival parameters and reoxygenation patterns have been used. The models allow the relative importance of repair capacity, reoxygenation rate, and dose per fraction to be assessed for these four schedules in the presence or absence of misonidazole.Unlike hyperbaric oxygen, the dose of misonidazole and the fractionation scheme to be used are critically interdependent, because the total drug dose is limited to 12 g/m2by its neurotoxicity, regardless of the extent to which it is fractionated.The largest sensitizing effect is always demonstrated with six fractions, each given with 2 g/m2of misonidazole. In the absence of reoxygenation a sensitizer enhancement ratio of 1.7 is predicted, but this falls to 1.1–1.2 if extensive reoxygenation occurs. Less sensitization is observed with 30 fractions, each with 0.4 g/m2of drug.However, for clinical use, the important question is which treatment kills the maximum number of tumour cells. Many of the simulations predict a marked disadvantage of reducing the fraction number for X rays alone. The circumstances in which this disadvantage is offset by the large SER values with a six-fraction schedule are few. The model calculations suggest that many small fractions, each with a low drug dose, are safest unless the clinician has some prior knowledge that a change in fraction number is not disadvantageous.

Recently, Wheldon (1979) and Zeitz and McDonald (1978) have considered the effects of different fractionated irradiation schedules on a model tumour having properties similar to melanoma cells in culture. Using essentially similar assumptions, they came to different conclusions as to which of the four different schedules gave the lowest survival. Zeitz and McDonald (1978) considered the response of a totally oxic population of cells using the cell-survival data of Dewey (1971). They considered three forms of the equation for cell survival and showed that a multi-target model with initial slope gave the best fit to Dewey's data. However, regardless of the exact form of the cell-survival curve chosen, the greatest extent of cell kill was achieved when a few large radiation doses were used (3 × 12.00 Gy), and the least when a large number of small fractions were used (31 × 2.00 Gy). However, they demonstrated that when a large number of small radiation doses are used large differences in the estimates of cell kill can be obtained, depending on the exact form chosen for the cell survival equation. This is because survival is being determined by the shoulder region of the survival curve and that region is the least well described experimentally.

The ability of acutely hypoxic tumour cells to recover from sublethal damage following irradiation in vivoand in vitrohas been measured using a single tumour system. The methods of assay were tumour growth delay, local tumour control and tumour cell survival in vitrofollowing treatment in vivoor in vitro. Tumours in vivoor cells in vitrorendered acutely hypoxic during irradiation were irradiated with either single doses or two doses 24 hours apart. Cells left in situhad a greater capacity for recovery than those treated either in vivoor in vitroand then assayed in vitro. It is suggested that tumours may not show a systematically reduced capacity for recovery relative to normal tissues, unless chronically hypoxic tumour cells have a reduced capacity for recovery and determine the response. However, the results imply that deductions as to the ability of tumour cells to recover from sublethal damage (whether chronically hypoxic or not), which rely on in vitroassays, may underestimate the extent of recovery.

A simple, safe, controllable technique of intravenous anaesthesia employing a mixture of meperidine (Demerol®) and succinylcholine has been described. Our experience with the technique in 160 surgical procedures is discussed.

Ehrlich mouse ascites cells have been used as a biological dosimeter to check physical methods of measurement of central axis depth doses of a beam of fast neutrons. The cells were irradiated in suspension in Perspex tubes, using air or nitrogen bubbling. Irradiations with fast neutrons under aerobic conditions were carried out at four depths in a tissue-equivalent phantom and in the absence of the phantom. The relative intensities of the radiation under these conditions agreed well with measurements using a special ionisation chamber, provided allowance was made for accompanying ? radiation. The oxygen enhancement ratio was found to be 2·78 with 250 kV X rays and 1·80 with the fast neutron beam. No signicant change of OER was observed for irradiations at 8·7 cm deep in the phantom.

The effect of misonidazole (Ro 07–0582, Roche Products Ltd., Welwyn Garden City, Herts.) as a radiosensitizer of hypoxic cells has been reported for several cell lines in vitroand for many tumour lines grown in vivo (Adams et al., 1976a). However, there have been no direct comparisons of the effects of this drug in vivoand in vitrobecause of a lack of data on the intra-tumour concentration of the drug. Recently Flockhart et al.(1977) have developed a method to measure the concentration of unchanged, misonidazole in tumours by gas-liquid chromatography. This article reports measurements of the ability of misonidazole to sensitize hypoxic cells in four different tumours to radiation as a function of its measured concentration in the tumours at the time of irradiation. These results are compared with those previously obtained for cells in culture.

It has been suggested by Brown and Howes (1974) that damage to vascular endothelial cells can contribute significantly to growth delay in tumours after irradiation with doses greater than about 2,000 rads and that this can account for the discrepancy between deductions based on the measurements of cell survival and those based on growth delay measurements, in particular the discrepancy in the estimates of the hypoxic fraction (McNally, 1973). If this is so, one would expect the dose effect curves for the gross response of a tumour to doses less than 2,000 rads would not contain this contribution due to vascular damage. However, this does not remove the discrepancy between the two methods of assay, since the growth delay curves for two related rat fibrosarcomas, RIB5and RIB5C, both indicated that below 2,000 rads the response was being governed by a predominantly well oxygenated component, which would not be the case if, as the measured survival curves suggested, as many as 15 per cent of the cells were hypoxic (McNally, 1973).Alterations in tumour growth after irradiation undoubtedly reflect damage to all the components which may make up the tumour.

Dr Mould has done it again. Repeat prescriptions are usually given because the first dose failed; here it is just the opposite, the first was such a success that the patient is crying out for more of the same! Ambrose Bierce defined a prescription as a physician's guess at what will best prolong the situation with least harm to the patient; in this case, any harm will be due to sore ribs from laughter.

High linear energy transfer (LET) beams for radiotherapy are always contaminated with gamma rays. This will be a few per cent in a typical high-energy neutron beam (Fowler, 1981), increasing with depth in tissue as neutrons are captured (Maughan et al, 1981). For instance, for a typical high-energy cyclotron producing 8–22 MeV (mean energy) therapy neutrons at 10 cm depth or more, the gamma-ray component may exceed 20% (Fowler, 1981). This will decrease the biological effectiveness of the beam by decreasing the effective LET. In addition, there are fast protons which also are effectively low-LET particles.

Twenty-five years ago, if you asked a trainee what he wanted to be when he grew up, as likely as not he would say a vascular radiologist. That was the time when a young radiologist worth his salt was busy catheterising the aorta and, if he was very good, trying his hand at selective renal and mesenteric injections.

Two questions of concern in the clinical application of hypoxic cell sensitizers are (a) will chemical sensitizers increase damage to normal tissues in a manner similar to hyperbaric oxygen and (b) can sensitizers be combined such that their sensitizing abilities are additive, but their toxicities are not? The first question arises because there is evidence from clinical trials of hyperbaric oxygen of a small, but significant, increase in normal tissue damage relative to air (Dische, 1978). Will the same effect be seen with chemical radiosensitizers? The second question arises because the amount of a sensitizer that can be used, and hence its enhancement of radiation damage, is limited by its toxic side-effects. In the case of misonidazole (Ro 07–0582, Roche Products Ltd.), the dose-limiting side effect is neurotoxicity (Dische et al., 1977). It might be possible to design another sensitizer less toxic than misonidazole such that the two could be combined to give significant sensitization with reduced toxicity. How do the radiosensitizing properties of chemical sensitizers add together?

Several authors have demonstrated that the electron affinic radio-sensitizing drugs can also have a very marked cytotoxic effect on hypoxic cells in the absence of radiation. For this cytotoxicity, prolonged contact time of several hours under hypoxic conditions is necessary. We recently summarized the results on mouse tumours, which showed that cytotoxicity is a small or negligible effect compared with direct radiosensitization (Denekamp, 1978). However, Stratford and Adams (1978) have stressed the importance of the biological half-life of the drug in determining its cytoxic action. A half-life of 1–1½ hours in the mouse prevents prolonged exposure to high concentrations of misonidazole, whereas the longer half-life of 12 hours in man does not.

The editor, John Lett, in his preface to the 11th volume emphasises the need for Advances in Radiation Biology to include more reviews on new research areas, in particular molecular biology and modern biochemistry: “the classical era is at an end”. This volume represents a good balance between the “classical era” and “modern times”, providing eight reviews ranging from free radical chemistry through DNA repair to radiation risks. Any reader interested in the broad field of radiobiology will find several chapters in this book worth his attention.

General interest in the biological effects of radiation has waxed and waned over the last 20 years and in the recent past the subject has lost its popularity as a scientific discipline. However, the recent resurgence of public concern about nuclear power and nuclear weapons has demonstrated that there will continue to be a need to train scientists in the subject of radiation biology, quite apart from the needs of strictly academic research and clinical oncology. The nature of the subject is such that there are few suitable text-books available.

These two volumes represent the proceedings of the 5th International Symposium on Hyperthermic Oncology held in Kyoto in September 1988. There is an enormous amount of information here, with over 1700 pages and more than 400 presentations from the most basic, through clinical experience to the physics of hyperthermia.Volume 1 contains a vast number of summary papers, up to three pages in length, on all aspects of hyperthermia. Only the really dedicated “hyperthermist” will find this volume of value.

In her review of the first edition of this book, Dr Spittle commented on the absence of sections on cytotoxic chemotherapy and on statistics, but said it should be a standard text for Part 1 of the FRCR examination. In what is now a single author book, Professor Nias has expanded the text on chemotherapy but, correctly in my view, not included a chapter on statistics. The other comments by Dr Spittle are, in general, as valid for this edition as for the first.

What should one look for from the proceedings of a large international conference covering radiation research from basic physics to clinical trials? Perhaps a compendium of the very latest results. But for how long will they be the “latest”? Perhaps a summary of current trends in research, the current “in” subjects.

This book contains 55 papers presented at the Second International Meeting on Progress in Radio-Oncology held under the auspices of the Austrian/International Club for Radio-Oncologists. The subjects covered include particle beam therapy, radiosensitisers, altered fractionation, and hyperthermia. There are introductory papers and a summary. A problem always present in the publication of such proceedings is that the papers may be quite brief and the information may soon be out of date. To a certain extent this book does suffer in this respect.

In 1985 the British Journal of Radiologyintroduced a requirement that authors sign a statement that the use of animals in research studies reported in the Journalwas in conformity with the Biological Council Guidelines on the Use of Living Animals in Scientific Investigations. An editorial outlining these Guidelines was published in the January 1985 issue.On 1st January 1987 the Animals (Scientific Procedures) Act 1986, governing the use of animals in experimentation in the United Kingdom, came into effect. This Act made extensive changes to the law and, as a result, the Biological Council has issued a second edition of their Guidelines taking account of these changes (Biological Council, 1987). Subsequently the United Kingdom Co-ordinating Committee on Cancer Research (UKCCR) representing the main cancer charities and the Medical Research Council of the United Kingdom prepared a set of Guidelines for the Welfare of Animals in Experimental Neoplasia (UKCCR, 1988).

This large volume, the 32nd of the excellent Houston Symposia on Cancer Research, covers almost the whole spectrum of radiobiological research as it relates to the treatment of cancer by radiation. There are 41 papers, grouped into six sections on mechanisms of radiation injury, responses at low doses and low dose rates, variations in radiation response, kinetic changes after irradiation, tissue response to radiation, and potential therapeutic advantages.

This book arose out of a course of lectures aimed at clinical trainees in medical and radiation oncology, undergraduate and graduate students, and postdoctoral fellows. This might seem to be a rather heterogeneous audience in terms of both stage of learning and interest.