In utero and neonatal sensitivity of ApcMin/+ mice to radiation-induced intestinal neoplasia

PURPOSE: To assess the sensitivity of ApcMin/+ mice (adenomatous polyposis coli Apc, multiple intestinal neoplasia, Min) to the development of intestinal adenomas after x-irradiation in utero, as neonates, or as young adults. MATERIALS AND METHODS: CHB6 ApcMin/+ mice were exposed to an acute dose of 2 Gy x-rays either in utero on day 7 or 14 post-conception, as 2-day or 10-day neonates or as 35-day young adults. Tumour identification and counting was performed 200-214 days later. RESULTS: Irradiation as 10-day-old neonates resulted in a significantly greater overall tumour incidence (average of about 130 tumours per animal) than irradiation as 35-day-old young adults (about 70 tumours). Irradiation as 2-day-old neonates resulted in an intermediate incidence (about 85 tumours). In contrast, the greatest tumour incidence observed after in utero irradiation of ApcMin/+ mice, of about 44 tumours per animal after 2 Gy irradiation at 14 days post-conception, was significantly lower than the incidence in irradiated adults. Tumour incidences after irradiation as 7-day embryos was not significantly raised above numbers in unirradiated controls (about 30 tumours). These tumour numbers include cystic crypts, largely radiation-induced, which were classed as early stage microadenomas on the basis of loss of wild-type Apc+ and expression of beta-catenin. CONCLUSIONS: The sensitivity of ApcMin/+ mice to the induction of intestinal tumours by radiation was shown to be in the order: 10 d neonates>2 d neonates>35 d young adults>14 d fetus>7 d embryo.     

Rapid rise in FDG uptake in an irradiated human tumour xenograft

In order to investigate early changes in the glucose metabolism of irradiated tumours, tumour uptake of 2-[¹⁸F]fluoro-2-deoxy-d-glucose (¹⁸FDG) was studied in human tumour xenografts. Three human tumour lines [ependymoblastoma (NNE), small cell lung cancer (GLS), and glioblastoma (KYG)] showing different radiosensitivities and incidences of radiation-induced apoptosis were subcutaneously transplanted into nude mice, and were irradiated at a single dose of 10 Gy. Then 0.5 mCi of¹⁸FDG was intravenously administered 1 h before sacrifice. The animals were sacrificed at 2, 4 and 6 h following irradiation, and¹⁸FDG accumulation in the tumours was examined. Before irradiation, GLS and KYG tumours showed significantly higher rates of¹⁸FDG accumulation compared with NNE tumours (P <0.004 andP <0.001, respectively). NNE (the most radiosensitive tumour with the highest incidence of radiation-induced apoptosis), however, displayed a 2.3-fold higher rate of¹⁸FDG accumulation at 2 h following irradiation compared with a non-irradiated group (P <0.01), and thereafter showed a plateau up to 6 h. The accumulation did not increase significantly in the other tumours with lower radiosensitivity and much less radiation-induced apoptosis. The rapidity of the increase in¹⁸FDG accumulation in the most radiosensitive tumour line, occurring as early as 2 h following irradiation, suggests that the increase was independent of recovery phenomena following radiation damage.     

Protection against prenatal irradiation‐induced genomic instability and its consequences in adult mice by Ocimum flavonoids,orientin and vicenin

Purpose: To study the protective effect of orientin and vicenin against early genomic effects of foetal irradiation and their late consequences in mice.

Materials and methods: Fourteen‐day pregnant mice were exposed to 1?Gy ⁶⁰Co gamma‐radiation 30?min after an intraperitoneal injection of orientin or vicenin (50?µg?kg^?1 body weight). Chromosomal aberrations were studied in foetal liver cells and their spleen colonies (three passages, colony‐forming units‐spleen CFU‐S1, CFU‐S2, CFU‐S3) and 1–12 months post‐partum bone marrow. Peripheral blood counts and solid tumours were recorded to 12 and 20 months, respectively.

Results: Irradiation significantly increased the percent aberrant cells and aberrations/cell in foetal liver and CFU‐S1. These effects decreased in later passages of CFU‐S and were not seen at 1–6 months post‐partum, but increased significantly from 9 months. Total blood counts showed significant reduction from 6 months, while neutrophils increased from 3 months post‐partum. Solid tumour incidence in adults increased significantly, with a decrease in age at detection. Orientin/vicenin significantly reduced the chromosomal anomalies in foetal and adult haemopoietic cells, restored blood counts to the normal range, and significantly reduced tumour incidence and delayed tumour development to control age.

Conclusions: Orientin and vicenin protect against foetal irradiation‐induced genomic damage and instability, thereby reducing the delayed chromosomal abnormalities and tumorigenesis in adult.     

Dose estimation in B16 tumour bearing mice for future irradiation in the thermal column of the TRIGA reactor after B/Gd/LDL adduct infusion

To test the efficacy of a new ¹⁰B-vector compound, the B/Gd/LDL adduct synthesised at Torino University, in vivo irradiations of murine tumours are in progress at the TRIGA Mark II reactor of the Pavia University. A localised B16 melanoma tumour is generated in C57BL/6 mice and subsequently infused with the adduct. During the irradiation, the mouse will be put in a shield to protect the whole body except the tumour in the back-neck area. To optimise the treatment set-up, MCNP simulations were performed. A very simplified mouse model was built using MCNP geometry capabilities, as well as the geometry of the shield made of 99% ¹⁰B enriched boric acid. A hole in the shield is foreseen in correspondence of the back-neck region. Many configurations of the shield were tested in terms of neutron flux, dose distribution and mean induced activity in the tumour region and in the radiosensitive organs of the mouse. In the final set-up, up to five mice can be treated simultaneously in the reactor thermal column and the neutron fluence in the tumour region for 10 min of irradiation is of about 5×10¹² cm⁻².     

Is sensitization with nicotinamide and carbogen dependent on nicotinamide concentration at the time of irradiation?

Purpose: To determine whether tumour radiosensitization and the therapeutic benefit of administering carbogen with nicotinamide depend upon irradiating at the time of peak drug concentration.

Materials and methods: Local tumour control of CaNT tumours in CBA mice and acute skin reactions in albino WHT mice were assessed after treatment with 10 X‐ray fractions in air, carbogen alone or combined with 0.1, 0.2 or 0.5?mg?g^?1 nicotinamide, injected 15, 30 or 60?min before irradiation. Plasma and tumour drug pharmacokinetics were performed.

Results: Nicotinamide was rapidly taken up into tumours; a six‐ and threefold higher concentration was obtained with 0.5?mg?g^?1 compared with 0.1 and 0.2?mg?g^?1, respectively. Tumour, but not skin, radiosensitization increased as the dose of nicotinamide increased (p=0.03), but at each dose level there was no significant difference in radiosensitivity when irradiations were done at or after the time of peak concentration. An almost eightfold increase in plasma levels increased tumour enhancement ratios from 1.74 to 1.92 (p<0.0001). In tumours all schedules gave significant enhancement relative to carbogen alone (p≤0.04).

Conclusions: Tumour and skin radiosensitivity was independent of time of nicotinamide administration. Higher drug concentrations were not mirrored by proportionally higher enhancement ratios. Lower plasma levels than previously suggested significantly enhanced tumour radiosensitivity relative to carbogen alone. The clinical implications of these findings are discussed.     

Selective inhibition of the epidermal growth factor receptor tyrosine kinase by BIBX1382BS and the improvement of growth delay,but not local control,after fractionated irradiation in human FaDu squamous cell carcinoma in the nude mouse

Purpose: To investigate the effect of BIBX1382BS, an inhibitor of the epidermal growth factor receptor tyrosine kinase, on proliferation and clonogenic cell survival of FaDu human squamous cell carcinoma in vitro, and on tumour growth and local tumour control after fractionated irradiation over 6 weeks in nude mice. FaDu human squamous cell carcinoma is epidermal growth factor receptor positive and significant repopulation during fractionated irradiation was demonstrated in previous experiments.

Materials and methods: Receptor status, receptor phosphorylation, cell cycle distribution, cell proliferation and clonogenic cell survival after irradiation were assayed with and without BIBX1382BS (5?µM) in vitro. Tumour volume doubling time, BrdUrd and Ki67 labelling indices and apoptosis were investigated in unirradiated tumours growing in NMRI nude mice treated daily with BIBX1382BS (50?mg?kg^?1 body weight orally) or carrier. Tumour growth delay and dose–response curves for local tumour control were determined after irradiation with 30 fractions within 6 weeks.

Results: BIBX1382BS blocked radiation‐induced phosphorylation of the epidermal growth factor receptor and reduced the doubling time of FaDu cells growing in vitro by a factor of 4.9 (p=0.008). Radiosensitivity in vitro remained unchanged after incubation with BIBX1382BS for 3 days and decreased moderately after 6 days (p=0.001). BIBX1382BS significantly reduced the volume doubling time of established FaDu tumours in nude mice by factors of 2.6 when given over 15 days (p<0.001) and 3.7 when applied over 6 weeks (p<0.001). When given simultaneously to fractionated irradiation, growth delay was significantly prolonged by an average of 33 days (p=0.003). Local tumour control was not improved by BIBX1382BS. The radiation doses necessary to control 50% of the tumours locally were 63.6?Gy (95% confidence interval 55; 73) for irradiation alone and 67.8?Gy (60; 77) for the combined treatment (p=0.5).

Conclusions: Despite clear antiproliferative activity in rapidly repopulating FaDu human squamous cell carcinoma and significantly increased tumour growth delay when combined with fractionated irradiation, local tumour control was not improved by BIBX1382BS. The results do not disprove that epidermal growth factor receptor inhibition might enhance the results of radiotherapy. However, the results imply that further preclinical investigations using relevant treatment schedules and appropriate endpoints are necessary to explore the mechanisms of action and efficacy of such combinations.     

Comparison of [18F]FDG uptake and distribution with hypoxia and proliferation in FaDu human squamous cell carcinoma (hSCC) xenografts after single dose irradiation

Purpose: This study investigated the uptake of [¹⁸F]2-fluoro-2-deoxy-glucose ([¹⁸F]FDG) in the human tumour xenograft FaDu at early time points after single dose irradiation with Positron-Emission-Tomography (PET), autoradiography and functional histology.

Materials and methods: [¹⁸F]FDG-PET of FaDu hSCC xenografts on nude mice was performed before 25 Gy or 35 Gy single dose irradiation and one, seven or 11 days post irradiation (p.irr.). Before the second PET, mice were injected with pimonidazole (pimo) and bromodeoxyuridine (BrdU). After the PET tumours were excised, sliced and subjected to autoradiography and functional histology staining (pimo, BrdU, Ki67). [¹⁸F]FDG tumour uptake was quantified in the PET scans by maximal standard uptake value (SUV_max) and in the autoradiography after co-registration to the histology slices.

Results: No differences in the overall [¹⁸F]FDG uptake between the two dose groups and time points were found with PET or autoradiography. Comparing autoradiography and histology, the [¹⁸F]FDG uptake was constant in tumour necrosis over time, while it decreased in vital tumour areas and particularly in hypoxic regions. No differences in the [¹⁸F]FDG uptake between positive and negative areas of Ki67 and BrdU were found.

Conclusions: The decline of [¹⁸F]FDG uptake in vital tumour and in pimopositive areas as seen in autoradiography, was not reflected by evaluation of SUV_max determined by PET. These findings suggest that the SUV_max does not necessarily reflect changes in tumour biology after irradiation.     

Vasculogenesis: a crucial player in the resistance of solid tumours to radiotherapy

Tumours have two main ways to develop a vasculature: by angiogenesis, the sprouting of endothelial cells from nearby blood vessels, and vasculogenesis, the formation of blood vessels from circulating cells. Because tumour irradiation abrogates local angiogenesis, the tumour must rely on the vasculogenesis pathway for regrowth after irradiation. Tumour irradiation produces a marked influx of CD11b⁺ myeloid cells (macrophages) into the tumours, and these are crucial to the formation of blood vessels in the tumours after irradiation and for the recurrence of the tumours. This process is driven by increased tumour hypoxia, which increases levels of HIF-1 (hypoxia-inducible factor 1), which in turn upregulates SDF-1 (stromal cell-derived factor 1 or CXCL12), the main driver of the vasculogenesis pathway. Inhibition of HIF-1 or of its downstream target SDF-1 prevents the radiation-induced influx of the CD11b⁺ myeloid cells and delays or prevents the tumours from recurring following irradiation. Others and we have shown that with a variety of tumours in both mice and rats, the inhibition of the SDF-1/CXCR4 pathway delays or prevents the recurrence of implanted or autochthonous tumours following irradiation or following treatment with vascular disrupting agents or some chemotherapeutic drugs such as paclitaxel. In addition to the recruited macrophages, endothelial progenitor cells (EPCs) are also recruited to the irradiated tumours, a process also driven by SDF-1. Together, the recruited proangiogenic macrophages and the EPCs reform the tumour vasculature and allow the tumour to regrow following irradiation. This is a new paradigm with major implications for the treatment of solid tumours by radiotherapy.     

Abscopal effect of radiation therapy: Interplay between radiation dose and p53 status

Abstract

Purpose: This study investigates whether the abscopal effect induced by radiation-therapy (RT) is able to sterilize non-irradiated tumour cells through bystander signals.

Material and methods: Wild-type (wt)-p53 or p53-null HCT116 human colon cancer cells were xenografted into both flanks of athymic female nude mice. When tumours reached a volume of 0.2 cm³, irradiation was performed, under strict dose monitoring, with a dedicated mobile accelerator designed for intra-Operative-RT (IORT). A dose of 10 or 20 Gy (IR groups), delivered by a 10 MeV electron beam, was delivered to a tumour established in one side flank, leaving the other non-irradiated (NIR groups). A subset of mice were sacrificed early on to carry out short-term molecular analyses.

Results: All directly-irradiated tumours, showed a dose-dependent delayed and reduced regrowth, independent of the p53 status. Importantly, a significant effect on tumour-growth inhibition was also demonstrated in NIR wt-p53 tumours in the 20 Gy-irradiation group, with a moderate effect also evident after 10 Gy-irradiation. In contrast, no significant difference was observed in the NIR p53-null tumours, independent of the dose delivered. Molecular analyses indicate that p53-dependent signals might be responsible for the abscopal effect in our model system, via a pro-apoptotic pathway.

Conclusions: We suggest that the interplay between delivered dose and p53 status might help to sterilize out-of-field tumour cells.     

Time benefit in the assessment of recurrences following fractionated radiotherapy in an experimental tumour system using positron‐emission tomography with 18F‐fluorodeoxyglucose

Purpose: To determine the sensitivity and specificity of ¹⁸F‐fluorodeoxyglucose‐positron‐emission tomography (FDG‐PET) in the diagnosis of R1H tumours after fractionated radiotherapy, and the dependency of sensitivity and specificity on time after therapy. In addition, the time benefit of FDG‐PET concerning early recognition of recurrences after fractionated radiotherapy was assessed.

Material and methods: Subcutaneously growing rat rhabdomyosarcoma R1H tumours were irradiated by applying total doses of 80 or 85?Gy after reaching a start volume of 0.8?cm³. Twenty animals were treated. Tumour volume was determined twice a week. FDG‐PET was performed weekly before, during and for 6 months after therapy using a conventional full‐ring whole‐body PET scanner. In total, 600 PET results were evaluated qualitatively using a six‐scale score. PET results and actual tumour volumes were compared. The sensitivity and specificity of tumour detection by PET was calculated for different times after the onset of therapy. The optimal score for tumour detection and the influence of time after therapy on the quality of PET (time benefit) was evaluated using receiver‐operating characteristics.

Results: After irradiation, 8/20 tumours (40%) were locally controlled, while 12/20 recurred. In this tumour model, evidence of relapse is assured when a volume of 0.1?cm³ is reached. Sensitivity of tumour diagnosis by PET increases with time, i.e. with the volume of recurrent tumours after the onset of therapy, mounting to >0.95 after 100 days. Specificities of 0.95–1.0 were determined after therapy, showing no increase with time. Tumour diagnosis by PET is highly accurate when performed 80 days after the start of treatment. On average, tumours were recognized by PET on 31, 62, 74 and 81 days (median) before approaching volumes of 0.2, 0.5, 0.8 or 1.0?cm³, respectively.

Conclusion: An experimental system was implemented that allows reproducible detection of recurrent R1H tumours after radiotherapy using FDG‐PET. The usefulness of PET as a diagnostic test for R1H tumours is very good and a reliable resolution for PET is demonstrated for volumes<1?cm³. The results indicate that FDG‐PET enables early recognition of recurrences after fractionated radiotherapy.     

Impact of increased cell loss on the repopulation rate during fractionated irradiation in human FaDu squamous cell carcinoma growing in nude mice

Purpose: To determine the impact of increased necrotic cell loss on the repopulation rate of clonogenic cells during fractionated irradiation in human FaDu squamous cell carcinoma in nude mice.

Materials and methods: FaDu tumours were transplanted into pre‐irradiated subcutaneous tissues. This manoeuvre has previously been shown to result in a clear‐cut tumour bed effect, i.e. tumours grow at a slower rate compared with control tumours. This tumour bed effect was caused by an increased necrotic cell loss with a constant cell production rate. After increasing numbers of 3‐Gy fractions (time intervals 24 or 48?h), graded top‐up doses were given to determine the dose required to control 50% of the tumours (TCD₅₀). All irradiations were given under clamp hypoxia.

Results: With increasing numbers of daily fractions, the top‐up TCD₅₀ decreased from 37.9?Gy (95% CI: 31; 45) after single dose irradiation to 14.1?Gy (8; 20) after irradiation with 15 fractions in 15 days. Irradiation with 18 daily 3‐Gy fractions controlled more than 50% of the tumours without a top‐up dose. After irradiation with six fractions every second day, the top‐up TCD₅₀ decreased to 26.9?Gy (22; 32). No further decrease of the TCD₅₀ was observed after 12 and 18 irradiations every second day. Assuming a constant increase of TCD₅₀ with time, the calculated doubling time of the clonogenic tumour cells (T_clon) was 7.8 days (4.4; 11.3). The T_clon calculated for FaDu tumours growing in pre‐irradiated tissues was significantly longer (p=0.0004) than the T_clon of 5.1 days (3.7; 6.5) determined under the same assumptions in a previous study for FaDu tumours growing in normal subcutaneous tissues.

Conclusions: Increased necrotic cell loss by pre‐irradiation of the tumour bed resulted in longer clonogen doubling times during fractionated radiotherapy of human FaDu squamous cell carcinoma. This implies that a decreased necrotic cell loss might be the link between reoxygenation and repopulation demonstrated previously in the same tumour model.     

In vivo measurement of the hypoxia marker EF5 in Shionogi tumours using 19F magnetic resonance spectroscopy

Purpose:?¹⁹F magnetic resonance spectroscopy (MRS) was used to non-invasively detect EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] adducts in the Shionogi tumour model of prostate cancer to evaluate hypoxia.

Material and methods:?¹⁹F MRS signal of EF5 in Shionogi mouse tumours was acquired using a 2 cm diameter solenoid volume coil with a 7.05 T Bruker scanner. MRS signal was observed in mouse tumours longitudinally following intraperitoneal (IP) injection of EF5. Another mouse group was injected intravenously (IV) with EF5, and in vivo MRS signal was obtained two hours after injection. This data was compared with the ex vivo percentage of hypoxic cells present in the corresponding excised tumours, determined by flow cytometry of bound EF5.

Results:?Longitudinal ¹⁹F MRS signal attributable to EF5 began to decline within five hours of EF5 administration. Flow cytometry comparisons yielded an inverse correlation (p-value < 0.006) between the MRS signal and tumour hypoxic cell percentage. The tumours exhibited an average cell viability of 34 ± 26%.

Conclusions:?The results confirmed that MRS of EF5 in mice is an unsuitable technique for the determination of EF5 binding as a measure of tumour hypoxia.     

In vivo imaging of herpes simplex virus type 1 thymidine kinase gene expression: early kinetics of radiolabelled FIAU

Previous studies have shown that the herpes simplex virus type 1 thymidine kinase gene (HSV1-tk), in combination with appropriate radiolabelled substrates (e.g. [I*]-2’-fluoro-2’-deoxy-5-iodo-1-β-d-arabinofuranosyluracil; I*-FIAU, where the asterisk indicates that any of the various radioactive iodine isotopes can be used), can be used as a reporter gene forin vivo monitoring of gene transfer and expression. The aim of our study was to examine the early kinetics of I*-FIAU and the possibility of utilising iodine-123-labelled FIAU for imaging of gene expression. CMS-5 fibrosarcoma cells were transduced in vitrowith the retroviral vector STK containing the HSV1-tk gene. BALB/c mice were inoculated subcutaneously with HSV1-tk(+) and tk(-) cells into both flanks. FAU (2’-fluoro-2’-deoxy-1-β-d-arabinofuranosyluracil was radioiodinated (¹²³I, ¹²⁵I) using the iodogen method. High-performance liquid chromatography purification resulted in high specific activity and radiochemical purity for both tracers ([¹²³I]FIAU and [¹²⁵I]FIAU). Biodistribution studies and gamma camera imaging were performed at 0.5, 1, 2 and 4 h p.i. In addition, the genomic DNA of the tumours was isolated for measurement of the activity accumulation resulting from the [¹²⁵I]FIAU incorporation. Biodistribution studies 0.5 h p.i. showed tumour/blood and tumour/muscle ratios of 3.8 and 7.2, respectively, for the HSV1-tk(+) tumours, and 0.6 and 1.2, respectively, for negative control tumours. Fast renal elimination of the tracer from the body resulted in rapidly increasing tumour/blood and tumour/muscle ratios which reached values of 32 and 88 at 4 h p.i., respectively. Tracer clearance from blood was bi-exponential, with an initial half-life of 0.6 h followed by a half-life of 4.6 h. The tracer half-life in herpes simplex viral thymidine kinase-expressing tumours was 35.7 h. The highest activity accumulation (20.3%±5.7% ID/g) in HSV1-tk(+) tumours was observed 1 h p.i. At that time, about 46% of the total activity found in HSV1-tk(+) tumours was incorporated into genomic DNA. Planar gamma camera imaging showed a distinct tracer accumulation as early as 0.5 h p.i., with an increase in contrast over time. These results suggest that sufficient tumour/background ratios for in vivo imaging of HSV1-tk expression with [¹²³I]FIAU are reached as early as 1 h p.i. Received 17 July and in revised form 12 November 1999     

Protracted Treatment of C57B1 Mice with Zn-DTPA after 241Am Injection Reduces the Long-term Radiation Effects

Summary

Repeated intraperitoneal injections of ZnNa₃-diethylenetriaminepenta-acetate (ZnDTPA), once a week, during 8 successive weeks, and starting 4 days after injection of 58 and 373 kBq ²⁴¹Am/kg to C57B1 mice, were an effective protection against long-term radiation damage. At both dose levels of ²⁴¹Am, Zn-DTPA reduced the ²⁴¹Am concentration in bones by between 33% and 45%, and in the liver by 97%. Mean survival with ²⁴¹Am was shortened in Zn-DTPA-treated mice, by 17% at the lower dose level and by 70% at the higher dose level. After treatment with DTPA at the lower ²⁴¹Am level, survival became equal to that of control mice without ²⁴¹Am, while at the higher level life span was still shortened. After the lower ²⁴¹Am dose the incidence of bone tumours, liver carcinomas and the total number of all malignant tumours were significantly reduced by chelation therapy. The decrease in bone tumour incidence was proportional to the decrease in ²⁴¹Am concentration and reduction in cumulative radiation dose in bone after chelation therapy. The incidence of liver carcinomas was reduced to that in non-²⁴¹Am-injected mice and the reduction was thus proportional to the 97% reduction in ²⁴¹Am concentration of the liver at the end of chelation therapy. After the higher ²⁴¹Am dose no tumours showed up in sham-treated mice, probably due to the overkill effect on the cells at risk. In the corresponding Zn-DTPA-treated mice, bone tumours and a few other malignant tumours were observed.     

DNA Strand Breaks from Tritium Decay: A Local Effect for Cytosine-6-3H

Summary

Twelve-week-old female (C3H × 101)F₁ mice were injected intravenously with an ultrafiltered solution of ²³⁹Pu in 1 per cent trisodium citrate, and mated to uninjected PCT males. The plutonium content was examined radiochemically and autoradiographically in placentae and foetuses on the 12th and 18th days of gestation, and in neonates during the 24 hours after birth and also at 18 days post-natally.

Plutonium was distributed in most tissues of the late foetus and the suckling as it is in adult mice. However, on both the 12th and 18th days of gestation the concentration in the yolk-sac splanchnopleure was much higher than in any other foetal tissue. The amount of ²³⁹Pu in 18-day-old sucklings was between two and seven times as great as in 1-day-old neonates because of ingestion of milk from the lactating dams. In the first litter following administration of the radionuclide to the dam, about 0·02 per cent of the plutonium injected was transferred to an individual offspring by the time of birth, and a further 0·08 per cent by the time of weaning.     

Exacerbation of autoimmune thyroiditis by a single low dose of whole-body irradiation in non-obese diabetic-H2h4 mice

Purpose: To evaluate how irradiation affects thyroid autoimmunity in mouse models of Hashimoto's thyroiditis and Graves' hyperthyroidism.

Materials and methods: Non-obese diabetic (NOD)-H2^h4 mice spontaneously develop anti-thyroglobulin (Tg) antibodies and thyroiditis when supplied with sodium iodine (NaI) in the drinking water. BALB/c mice develop anti-thyrotropin receptor (TSHR) antibodies and hyperthyroidism following immunization with adenovirus expressing TSHR (Ad-TSHR). Mice were irradiated as follows: A single whole-body irradiation with 0.05, 0.5 or 3 Gy one week before or after the beginning of NaI or immunization with Ad-TSHR, fractionated whole-body irradiations with 0.05 Gy twice a week or 0.5 Gy once a week from one week before NaI or Ad-TSHR immunization, or a single regional irradiation to the thyroid gland with 0.5 Gy one week before NaI. The effect of a single irradiation with 0.05, 0.5 or 3 Gy on splenocytes was also evaluated.

Results: A single whole-body irradiation with 0.5 Gy one week before NaI exacerbated thyroiditis and increased anti-Tg antibody titers in NOD-H2^h4 mice. In contrast, any irradiation protocols employed did not affect incidence of hyperthyroidism or anti-TSHR antibody titers in BALB/c mice. High-dose irradiation increased the relative ratios of effector T cells to regulatory T cells (an indication of enhanced immune status) but kills most of T cells.

Conclusions: These results indicate that a single whole-body low-dose irradiation with 0.5 Gy exacerbates thyroiditis in NOD-H2^h4 mice, data consistent with some clinical evidence for increased incidence of thyroid autoimmunity by environmental irradiation.     

Dose and Dose-splitting Effects of X-rays on Lung Tumour Induction in Mice

Summary

This paper reports lung-tumour induction 12 months after single or split doses of X-rays in C3H/He male mice. The early proliferative response of lung cells after doses which induced lung tumours was also examined after single X-irradiation. The lung-tumour incidence tended to increase with increasing dose after a single irradiation and peaked at 5 Gy. At more than 10 Gy it decreased sharply to the control level. The mean tumour diameters tended to increase with doses up to 7·5 Gy and then decreased beyond 10 Gy. These results suggested that suppression of tumour growth reduced the tumour incidence at doses of over 10 Gy. The lung-tumour incidence decreased with increasing intervals between two equal doses of 2·5 or 5 Gy. The decrease was thought to be caused by the repair of the tumorigenic injury. However, the tumour incidence after two 2·5 Gy irradiations at 1 day intervals or two 5 Gy irradiations at 6 h intervals was higher than that observed after a single dose. This phenomenon was regarded as a progression of the tumorigenic injury. The labelling indices of the lung cells, determined using tritiated thymidine after a single irradiation, were higher than those of non-irradiated control cells. This response was delayed as the dose increased. The responses versus days after irradiation could be classified into three patterns on the basis of their peaks. The possibility that the larger delay after higher doses had some relation to the reduction of target cells for tumour incidence is suggested.     

Induction of Hypoxia in the KHT Sarcoma by Tumour Necrosis Factor and Flavone Acetic Acid

Summary

The interaction of FAA or TNF with radiation was studied in the murine KHT sarcoma. When used alone both agents showed a dose- and time-dependent toxicity towards the tumour cells and significantly reduced tumour blood flow within 1 h of treatment. When used in combination with radiation, both TNF and FAA caused an increase in the fraction of hypoxic cells in the KHT tumour. This was assessed by an in vivo/in vitro clonogenic assay and by a comparison with the radioprotection provided by clamping tumours prior to and during irradiation. When TNF was given at a dose of 2·5 × 10⁵ U/kg an increase in tumour hypoxia was seen after 30 min. Close to 100% radiobiological hypoxia was reached by 1 h after treatment, lasting for up to 16 h. Doses of TNF below 0·25 × 10⁵ U/kg did not induce levels of hypoxia comparable to clamping when administered 3 h prior to irradiation. Similarly, FAA produced a rapid increase in tumour hypoxia: a dose of 200 mg/kg induced close to 100% radiobiological hypoxia when give 1 h prior to irradiation. Complete tumour hypoxia was still apparent 18 h after treatment with FAA. Administered doses of FAA below 100 mg/kg did not produce close to 100% radiobiological hypoxia when administered 3 h prior to irradiation.     

p-[123I]iodo-l-phenylalanine for detection of pancreatic cancer: basic investigations of the uptake characteristics in primary human pancreatic tumour cells and evaluation in in vivo models of human pancreatic adenocarcinoma

Pancreatic cancer is associated with the worst 5-year survival rate of any human cancer. This high mortality is due, in part, to difficulties in establishing early and accurate diagnosis. Because most tumours share the ability to accumulate amino acids more effectively than normal tissues and any other pathology, assessment of amino acid transport in tumour cells using radiolabelled amino acids has become one of the most promising tools for tumour imaging. This study investigated the potential of p-[¹²³I]iodo-l-phenylalanine (IPA) for detection of pancreatic cancer by single-photon emission tomography. IPA affinity for pancreatic tumour was investigated in human pancreatic adenocarcinoma PaCa44 and PanC1 cells, followed by analysis of the underlying mechanisms of tracer accumulation in neoplastic cells. Thereafter, IPA was evaluated for targeting of pancreatic tumours using SCID mice engrafted with primary human pancreatic adenocarcinoma cells, as well as in acute inflammation models in immunocompetent mice and rats. IPA accumulated intensively in human pancreatic tumour cells. Radioactivity accumulation in tumour cells following a 30-min incubation at 37°C/pH 7.4 varied from 41% to 58% of the total loaded activity per 10⁶ cells. The cellular uptake was temperature and pH dependent and predominantly mediated by specific carriers for neutral amino acids, namely the sodium-independent and l-leucine-preferring (L-system) transporter and the alanine-, serine- and cysteine-preferring (ASC-system) transporter. Protein incorporation was less than 8%. Biodistribution studies showed rapid localization of the tracer to tumours, reaching 10%±2.5% to 15%±3% of the injected dose per gram (I.D./g) in heterotopic tumours compared with 17%±3.5% to 22%±4.3% I.D./g in the orthotopic tumours, at 60 and 240 min post injection of IPA, respectively. In contrast, IPA uptake in the gastrointestinal tract and areas of inflammation remained moderate and decreased with time. Excellent tumour detection was obtained by gamma camera imaging. The specific and high-level targeting of IPA to tumour and the negligible uptake in the gastrointestinal tract and areas of inflammation indicate that p-[¹²³I]iodo-l-phenylalanine is a promising tracer for differential diagnosis of pancreatic cancer.