The effect of evening primrose oil on the radiation response and blood flow of mouse normal and tumour tissue.

PURPOSE: To investigate the effect of the oral administration of evening primrose oil on the radiation response and the blood flow of normal tissue and a tumour in BALB/c mice. METHODS AND MATERIALS: Aliquots of evening primrose oil were fed to BALB/c mice daily and the radiation response of the skin was assessed by the determination of ED50 values for the incidence of moist desquamation, using probit analysis. Tumour radiosensitivity was investigated by determining the growth delay caused by irradiation of a transplantable rhabdomyosarcoma. The 86RbCl uptake technique was used to determine the blood flow in normal foot and tumour tissue. The fatty-acid content of red blood cells, plasma and tumour tissue was measured using gas chromatography. RESULTS: Daily evening primrose oil dietary supplementation reduced the sensitivity of skin to radiation-induced moist desquamation and prevented the radiation-associated increase in blood flow that was observed in this tissue. No modification of tumour blood flow or of tumour sensitivity to radiation resulted from evening primrose oil supplementation of mice. Evening primrose oil supplementation resulted in changes in plasma levels of linoleic acid (LA), gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA) and arachidonic acid (AA). These changes were contingent on whether the mice had been irradiated or not. In red blood cells evening primrose oil supplementation increased the GLA level of unirradiated mice and the LA level at 20 days after irradiation. There were no changes in tumour fatty-acid levels as a result of evening primrose oil treatment. CONCLUSIONS: Daily evening primrose oil supplementation reduced the sensitivity of skin to radiation-induced moist desquamation but did not alter tumour sensitivity to radiation.

Effects of low dose irradiation on TK6 and U937 cells: induction of p53 and its role in cell-cycle delay and the adaptive response.

PURPOSE: To investigate the effect of small doses of radiation on the cell-cycle and related processes, and to determine the capacity of small doses of radiation to induce an adaptive response. MATERIALS AND METHODS: TK6, a lymphoblast cell line with wild-type p53, and U937, a monocytic leukaemia cell line with mutant, inactive, p53 were exposed to gamma ray doses ranging from 0.1 Gy to 3 Gy. Cell-cycle distributions and cyclin B1 were assessed by flow cytometry, and p53 and p21 protein levels were measured by Western blotting. Apoptosis was determined by fluorescence microscopy after staining with Hoechst 33342, and by measurement of the pre-G1 cell population by flow cytometry. Micronuclei were determined in cytokinesis-blocked cells by fluorescence microscopy. RESULTS: In TK6 cells, radiation exposure induced elevated p53 and p21 levels and delayed expression of cyclin B1. No changes in these parameters were found in U937 cells. Although both cell lines arrested in G2/M after larger doses of radiation, G2/M-arrest occurred after 0.1 Gy and 0.3 Gy in TK6 cells only. An apoptotic adaptive response was induced in both cell lines by a 0.1 Gy priming dose but an adaptive response with respect to micronuclei was observed only in U937 cells. CONCLUSIONS: The radiation adaptive response can occur in the absence of wild-type p53. A small dose of radiation may not protect cells against both apoptosis and cytogenetic damage caused by a subsequent larger dose of radiation.

The effects of 2-deoxyglucose and amino-oxyacetic acid on the radiation response of mammalian cells in vitro.

Cells use substrates such as glucose and glutamine to provide energy for repair of radiation damage. Glutaminolysis and glycolysis were inhibited by aminooxyacetic acid (AOAA) and 2-deoxyglucose (2DG), respectively, to inhibit metabolism of these substrates in order to determine the effect on radiation response of CHO-K1 cells in vitro. Exposure to treatments which inhibit energy metabolism resulted in alterations in radiosensitivity and, in general, a reduction in cellular recovery rate after y-irradiation but varied with regard to the extent of recovery. The greatest inhibition of recovery relative to that in normal culture medium was found with medium which lacked glucose and glutamine and contained 2DG and AOAA. In contrast, medium lacking glucose and glutamine without the addition of inhibitors resulted in an increase in recovery. It is proposed that the efficiency of energy pathways such as glycolysis and glutaminolysis and their interaction are determinants of both radiosensitivity and recovery.

Assessment of the micronucleus assay as a biological dosimeter using cytokinesis-blocked lymphocytes from cancer patients receiving fractionated partial body-radiotherapy.

PURPOSE: To assess the suitability of the cytokinesis block micronucleus assay as a biological dosimeter following in-vivo radiation using cancer patients undergoing radiotherapy. METHODS: Blood from 4 healthy donors was irradiated in vitro with gamma-rays and the dose response of induced micronuclei in binucleate lymphocytes following cytokinesis block was determined. Micronucleus frequency was ascertained before and at intervals during radiotherapy treatment in 6 patients with various tumors in the pelvic region. Equivalent whole body doses (physical doses) at these times were calculated from radiation treatment plans and cumulative dose volume histograms. RESULTS: Linear dose response relationships were found for induced micronucleus frequency in lymphocytes resulting from both in-vitro and in-vivo irradiation. Doses resulting from in-vivo irradiation (biological doses) were estimated by substitution of micronucleus frequency observed in radiotherapy patients into the dose response curve from in-vitro irradiation of blood. The relationship between the biologically estimated dose (BD) and the calculated equivalent whole body dose (PD) was BD = 0.868 (+/- 0.043)PD + 0.117 (+/- 0.075). CONCLUSION: The micronucleus assay appears to offer a reliable and consistent method for equivalent whole body radiation dose estimation, although our findings should be confirmed using lymphocytes from radiotherapy patients with tumors at anatomical sites other than the pelvis. Except at doses lower than about ).4 Gy, the method yields dose estimates acceptably close to "true" physically determined doses. The assay can be performed relatively rapidly and can be used as a "first line" biological dosimeter in situations where accidental exposure to relatively high radiation doses has occurred.

Tumor blood flow measurements using coincidence counting on patients treated with neutrons.

PURPOSE: The purpose of this work was to measure blood flow in tumors using a coincidence counting technique on patients undergoing treatment with neutrons. METHODS AND MATERIALS: The half-time, Tw, for the washout of 15O from neutron-activated tumors was measured with two 10 cm NaI(Tl) crystals coupled to a PC-based coincidence counting system. Blood flow measurements were made in 33 patients, 19 of whom had cancers of the head and neck region, 6 had breast cancer, 5 had sarcomas, and 3 patients had mesotheliomas. RESULTS: Blood flow as indicated by Tw of mobile 15O formed by neutron activation could be readily determined in tumors of patients undergoing neutron radiotherapy. The general reduction in the value for Tw was noted towards the end of treatment and did not seem to be dependent on the initial tumor volume. There was a tendency for larger lesions to be associated with longer half-times of 15O washout. CONCLUSION: It appears possible to obtain a reasonable estimate of tumor blood flow using a simple coincidence counting technique. In view of the large variation in blood flow between tumors, it did not appear to be possible to identify potentially hypoxic tumors that would respond to neutron therapy.

The response of mouse skin to fractionated doses of fast neutrons (66 MeVp-Be) with variable interfraction and overall treatment times.

PURPOSE: Beam availability for neutron therapy at the National Accelerator Centre at Faure, South Africa is such that treatment fractions are given at irregularly spaced intervals. Such treatment scheduling may not be optimal. METHODS: Investigations were made using the acute skin reaction of the mouse foot to determine the effect of different numbers of regularly and irregularly spaced fractions of p(66)/Be neutrons. Assessment of results was both by average skin reaction and by ED50 values for the incidence of moist desquamation as established by probit analysis. RESULTS: Different numbers of fractions (between 6 and 11) and different times between fractions did not appear to affect the mouse foot response significantly when fractionation was completed within 11 days, i.e. before repopulation began to have an effect. When overall treatment times were longer than 11 days, the mouse foot responses to 6 and 9 fractions with variable interfraction times were similar, provided the overall treatment times were the same and the fractions were at least 24 h apart. The alpha/beta ratio was 87 +/- 27 (SE) Gy for the early response of the BALB/c mouse foot skin to p(66)/Be neutrons. CONCLUSIONS: The response of mouse skin to fractionated p(66)/Be neutrons was independent of fraction number or interfraction time, provided that the overall treatment time was the same.

RBE and OER measurements on the p(66) + Be neutron beam at Faure, South Africa.

Results reported are for single dose exposures and refer to 60Co-gamma-irradiation. The RBE determined by V79 cell survival and based on the Do ratio was found to be 1.70 +/- 0.4 ranging from 1.5 to 1.8. In the case of the regeneration of mouse jejunal crypts the RBE was calculated at ten cell survival and was found to be 1.68. The maximum acute mouse skin reaction at a skin score of 2.0 was found to be 2.1 while the average skin reaction was 1.7. Growth retardation of Vicia faba bean roots measured at the level of 50% indicated an average RBE of 3.0 and a range of 2.7 to 3.7. The OER obtained for V79 cell survival was found to be 1.7 to 1.8. Comparison is made with the RBE and OER measurements for the neutron facilities at Clatterbridge, Fermilab and Louvain-la-Neuve which produce neutrons by the same nuclear reaction and whose physical specifications closely resemble those of the Faure neutrons. This comparison indicates that the Faure beam shows no unusual biological features and that its biological effectiveness is in line with that expected from its physical characteristics.

The effect of two fractions of radiation, delivered in air or in hyperbaric oxygen, on the Fib/T tumour in WHT mice pretreated with a hypoxic gas mixture.

The response of the Fib/T tumour to equal fractions of radiation, spaced by an interval of 24 hours, was determined in tumour-bearing mice that were pretreated either with 8% oxygen for 48 h or with air. An increase in tumour cell kill occurred in the group of animals that received a low oxygen pretreatment. Tumour cell kill was further and significantly increased if mice were retained in the low oxygen environment for the 24-h interval between radiation fractions. Possible explanations for these findings are proposed and discussed. The effect of a 48 h 8% oxygen exposure in modifying the response of the Fib/T tumour to two fractions of radiation, both delivered in hyperbaric oxygen, was also investigated. The low oxygen pretreatment did not significantly alter the tumour response to radiation given under conditions of hyperbaric oxygenation.

Time modulation effect of diethyldithiocarbamate (DDC) on radiosensitization by superoxide dismutase (SOD) inhibition.

Superoxide dismutase (SOD) is known to protect cells from the lethal effects of ionizing radiation by the dismutation of oxygen radicals. Diethyldithiocarbamate (DDC) is known inhibitor of SOD and may therefore be useful as a radiosensitizer. DDC however, is also a thiol radioprotector due to its ability to scavenge radiation induced free radicals. We have shown that DDC, if administered to tumours 1 hour prior to x-irradiation exerts a protective effect, whereas if administered 4 hours prior to irradiation, it radiosensitizes. This time modulation effect is not apparent after neutron irradiation where DDC protects in both situations. We have also examined the effect of DDC on the LD50/30 in mice after total body irradiation.

The effect of irradiation in air or in hyperbaric oxygen on the Fib/T tumor in WHT mice pretreated with a hypoxic gas mixture.

The effect of exposing WHT mice bearing the Fib/T tumor to a low-oxygen environment (8, 10, and 15% oxygen) for 48 h or 72 h before irradiation was compared, using an in vitro colony-forming excision assay, to the effect obtained when mice were pretreated with air. The response of the Fib/T tumor to radiation delivered in air was improved both by a 48-h and by a 72-h exposure of the animals to 8, 10, and 15% oxygen. However, the greatest tumor sensitization was achieved when mice were kept in 8% oxygen for 48 h before irradiation. These results are interpreted and discussed in relation to increases in the 2,3-DPG concentration, which were shown to occur when mice were exposed to a reduced oxygen environment. The relative importance of two models proposed to explain these findings is assessed. If mice pretreated with air were irradiated in hyperbaric oxygen, a similar tumor response was observed compared to that when mice were exposed to 8% oxygen for 48 h and then irradiated in air.

Chromium-cage complex as contrast agent in MR imaging--biodistribution studies of the [57Co]cobalt analogue.

The synthesis and T1 and T2 relaxivities of the Cr(III)-(NH2)-Sar-cage complex is reported. An outer-sphere relaxation mechanism is postulated for the relaxivity of the complex. Tissue distribution studies in mice using a [57Co]cobalt analogue as a radioactive tracer showed that the complex is excreted rapidly in the urine. Some renal uptake of the complex is seen. Appreciable uptake of labelled cage complex was observed in 3-methylcholanthrene induced murine rhabdomyosarcoma.

Preclinical studies with the Faure high energy neutron facility: response of pig skin to fractionated doses of fast neutrons (66 MeVp----Be).

The early and late responses of pig skin to fractionated doses of both unfiltered and filtered (i.e. hardened) neutrons using the Faure neutron therapy facility (66 MeVp----Be) were determined and compared with those following fractionated doses with 60Co gamma-rays. Dose-effect curves for the quantal responses of moist desquamation (early epithelial response) and dermal necrosis (late response) were fitted by probit analysis and ED50 values obtained. For a neutron fractionation scheme comprised of 12 fractions in 26 days, and using an unfiltered beam, the ED50 values for moist desquamation and dermal necrosis were 18.67 +/- 2.22 and 22.25 +/- 0.48 Gy, respectively, whereas in the case of the filtered beam, the corresponding ED50 values were 24.78 +/- 1.44 and 23.30 +/- 0.47 Gy. In order to provide a comparison, the values for 24 fractions of 60Co gamma-rays given in 39 days (a clinical protocol used in the Groote Schuur Hospital) were 74.02 +/- 2.92 and 66.72 +/- 1.93 Gy for moist desquamation and dermal necrosis, respectively. For the unfiltered beam, values for the comparative biological effectiveness (CBE) were 3.96 and 3.00 for the early and late skin response, respectively. The corresponding CBE values were for the filtered beam 2.99 and 2.86. These results for the Faure neutron therapy facility can be extrapolated to the human situation with a high degree of confidence, so that the neutron dose which would yield acceptable skin damage in patients may be determined using the data presented here.

The treatment of tumors by the induction of anemia and irradiation in hyperbaric oxygen.

Because increased effects have been achieved when murine tumors are irradiated after a period of hypoxia and because of anecdotal clinical experiences of an improved result after irradiation of previously anemic patients in hyperbaric oxygen, the relationship between irradiation and increased survival was investigated in seventy-two patients with advanced head and neck or cervical cancer. Anemia was achieved by means of a two-stage isovolemic venesection maintained for seventy-two hours, hemoglobin was returned to a normal level, and treatment in hyperbaric oxygen was started. Marked tumor shrinkage after the induction of anemia and before radiotherapy was seen and was probably disease, site, and hemoglobin level related. As a result, a possible new approach to cancer therapy is suggested. After completion of therapy, the 1-year disease-free survival for patients with head and neck and cervical cancer was not improved, but the 21-month survival for cervical cancer was improved. Further studies are strongly urged.

In vivo radiosensitization by diethyldithiocarbamate.

Diethyldithiocarbamate (DDC) has been suggested to have both radiosensitizing (due to superoxide dismutase (SOD) inhibition) and radioprotective properties. We have studied the activity of SOD up to 24 h after intratumoral administration of 50, 100, 150, and 300 mg/kg DDC in 3-methylcholanthrene-induced tumors in BALB/c mice. Maximal inhibition of SOD (8% of control) was obtained 1 h after administration of 100 mg/kg DDC. Tumor response to DDC and X irradiation was assessed using a tumor growth-delay assay, after 11 Gy 100-kVp X rays given up to 24 h after DDC administration. Radiation-induced tumor growth delay (7.11 +/- 1.76 days) was enhanced only when tumors were irradiated 2-4 h after 50 mg/kg DDC. When higher doses of DDC were used, tumor cure was noted when DDC was injected 1-6 h before irradiation. We suggest our findings are consistent with radiosensitization being due to SOD inhibition, but that if insufficient time is allowed between DDC injection and irradiation, the sensitization is masked by a radioprotective effect. We believe that further investigations as to the therapeutic potential of DDC in human patients with cancer are warranted.

Interstitial misonidazole: clinical experience in advanced mouth cancer.

In an attempt to obtain full radiosensitisation and avoid dose-limiting neurotoxicity, a needle has been specially developed for the injection of misonidazole pellets into tumours. The methods of production and insertion of the pellets are described. Thirteen advanced, untreated squamous carcinomas of the mouth were injected and then irradiated to tolerance. Twelve out of 13 implanted lesions had completely regressed at the end of treatment but only six remained healed after 1 year.

OER and RBE for 125I and 192Ir at low dose rate on mammalian cells.

The oxygen enhancement ratio (OER) for 125I and 192Ir as well as the relative biological effectiveness (RBE) at low dose rates (40-80 cGy h-1) were determined for B16 melanoma cells in culture. The OER was found to be 2.1 +/- 0.03 for 125I and 2.7 +/- 0.04 for 192Ir. The RBE for 125I relative to 192Ir was determined as 1.8 +/- 0.03 under aerated conditions and as 2.4 +/- 0.03 under hypoxia.

Effect of X irradiation on adenosine triphosphate and glucose-6-phosphate dehydrogenase in the CaNT mouse tumor.

The levels of adenosine triphosphate (ATP) in the transplantable CaNT murine tumor grown in CBA mice at various times following 5, 10, and 15 Gy X rays (100 kVp) were increased within 45 min. Maximal ATP levels occurred at 2.5 h following the 10 Gy dose (3.8 times that of unirradiated controls), returning almost to control levels by 13 h after irradiation. The specific activity of glucose-6-phosphate dehydrogenase (G-6-PDH) after 10 Gy increased about 1.5-fold 1 h after irradiation, returning to control levels by 48 h. It is suggested that the increased ATP following irradiation might play a major role in energy provision when cellular repair processes are able to operate. The increased G-6-PDH activity after irradiation may reflect enhanced metabolism associated with cellular repair mechanisms.