Katharine P. Hoyes

Transgenerational Effects of Preconception Paternal Contamination with 55Fe

Abstract Hoyes, K. P., Lord, B. I., McCann, C., Hendry, J. H. and Morris I. D. Transgenerational Effects of Preconception Paternal Contamination with 55Fe. Radiat. Res. 156, 488–494 (2001). The conjecture that germline mutations induced by radiation exposure before conception may predispose subsequent offspring to cancer remains contentious. Previous experimental studies have shown that preconception paternal irradiation with 239Pu induces perturbations in the hemopoietic systems of offspring and influences sensitivity to a secondary carcinogen. In the present study, male DBA2 mice were injected intravenously with the Auger electron emitter 55Fe (4 kBq g–1) 18 or 84 days before mating with normal females. Comet analysis showed an increased incidence of DNA strand breaks in sperm from contaminated animals after 84 days, but not after 18 days, indicating spermatogonial rather than spermatid damage. Offspring were either assayed for changes in bone marrow stem cells and committed progenitors or challenged with the chemical carcinogen methyl nitrosourea (MNU, 50 mg/kg) at 10 weeks of age and monitored for the onset of malignancy. Offspring from irradiated fathers had normal peripheral blood profiles, although the stem cell population was amplified in offspring arising from those exposed to 55Fe at 84 days before conception. Exposure to MNU significantly increased the incidence of lympho-hemopoietic malignancies in offspring from the 84-day group, but not in those from the 18-day group. These findings support the hypothesis that aberrations that are potentially leukemogenic may be transmitted to offspring after radiation damage to the paternal germline.

Spermatogenic and Mutagenic Damage after Paternal Exposure to Systemic Indium-114m

The cytotoxic and mutagenic consequences of systemic administration of 114mIn have been examined. Adult male rats were dosed intraperitoneally with 14.8 or 3.7 MBq/kg 114mIn. Approximately 0.25% of the injected radioactivity was localized within the testis by 24 h and was retained with an effective half-life of 49.5 days. Breeding studies were started 3 days after injection, males being housed with two females for seven consecutive mating trials of 19 days, separated by 2 days. Indium-114m caused a reduction in litter size and an increase in the incidence of pre- and postimplantation losses and dominant lethal mutations. These effects became evident from 24 days but were most marked between 87-126 days after treatment and persisted up to 147 days. When animals were mated 200 days after treatment, no significant changes were observed. In a parallel study, administration of 14.8 MBq/kg 114mIn resulted in decreased testis and epididymal weight and sperm reserves. Maximal reduction occurred between 87-108 days after injection followed by recovery toward control values, but neither organ had reached normal levels at 200 days. A single dose of 3.7 MBq/kg, however, had no effect on reproductive organ weight or sperm content. Male F1 progeny from the 14.8 MBq/kg group of the second mating period (commencing at 24 days) displayed decreased testis weights and sperm content and provoked a higher incidence of dominant lethal mutations. This effect was not observed in male progeny from any other time or the alternative dose level.

Testicular toxicity of the transferrin binding radionuclide 114mIn in adult and neonatal rats

Adult (70 d) and neonatal (7 d) male rats were dosed (i.p.) with 37 MBq/kg (1 mCi/kg; approximately 1 microgram elemental indium/kg) 114mIn, a transferrin-binding radionuclide. In adults, approximately 0.25% of the injected activity localised within the testis by 48 h postinjection and remained constant for up to 63 d. In neonates, 0.06% of the activity was in the testis by 48 h, and this declined such that by 63 d only 0.03% remained. At 63 d, treated rats had reduced sperm head counts and abnormal testicular histology that was more marked in animals dosed as adults than as neonates. In vitro, uptake of 114mIn into seminiferous tubules isolated from 7-, 20-, or 70-d-old rats was compared with that of 125I. Both radionuclides were readily accumulated by the tubules. Whilst 114In uptake into 20- and 70-d tubules was inhibited by excess transferrin, uptake into 7-d tubules was unchanged. 125I uptake was not affected by excess transferrin. These data support the contention that some radionuclides may cross the blood-testis barrier by utilisation of the physiologic iron-transferrin pathway, which may lead to greater testicular damage in adult compared to neonatal animals.

Effect of dietary vitamin C on radiation induced damage to the testis

We have investigated the ability of dietary vitamin C (ascorbic acid) to mitigate radiation induced damage to the testis arising from exposure to x-rays or the Auger emitting radionuclide 114mIn (half-life 50 days). Male mice were maintained on a normal or an ascorbate enriched diet (1% by weight) for 5 days then irradiated with 3 Gy 300kVp x-rays or injected intraperitoneally with 114mIn (1.85-14.8 MBq kg-1). Diets were continued for the duration of the experiment. At 35 days post-irradiation animals were killed and testicular damage was assessed using testis weight and spermhead counts as biological endpoints. Acute irradiation with 3 Gy x-rays resulted in severe damage to the seminiferous epithelium manifested as a 50% reduction in testis weight and more then 80% reduction in spermhead count, no differences were observed between control and ascorbate treated animals. Similarly chronic irradiation by tissue-incorporated 114mIn caused a dose-dependent reduction in testis weight and spermhead count and no abrogation of this effect was observed with ascorbate. While the enriched diet did result in elevated testicular ascorbate levels this effect was short-lived and appeared to be subject to diurnal variation. These results suggest that vitamin C does not have a role as a radioprotectant in the testis against medical or environmental radiation exposures.

In Vivo Induction of O6-Alkylguanine-DNA-Alkyltransferase in Response to Indium-114m

The effect of systemic administration of the radionuclide 114mIn on O6-alkylguanine-DNA-alkyltransferase (ATase) activity has been examined in rats. In response to 14.8 MBq/kg 114mIn injected intraperitoneally, hepatic ATase was induced maximally approximately fivefold at 7 days after injection, at which time the cumulative radiation dose to the liver was approximately 2 Gy. At 63 days after injection ATase activity was still approximately twofold elevated and remained so at 126 days after injection. By 200 days after injection ATase activity had returned to control values. The 114mIn content of the liver increased to a maximum of 28.7 kBq/g 48-72 h after injection, after which it began to decrease such that at 126 days only 0.3 kBq/g remained and at 200 days 0.03 kBq/g. In response to 4.44 MBq/kg 114mIn, hepatic ATase was induced twofold by 7 days after injection, when the liver had received a radiation dose of 0.6 Gy, and was still slightly elevated at 63 days. There was no ATase induction after 0.44 MBq/kg 114mIn up to 7 days after injection; however, at 42 days after injection activity was approximately twofold higher. These results suggest that induction of hepatic ATase activity by 114mIn is dependent upon cumulative radiation dose and dose rate; both must be above minimum threshold values for induction to occur. The induction of a DNA repair enzyme by radiation exposure from an internal radionuclide may have important consequences for risk assessments of occupational, medical and environmental exposures.

Cell cycle synchronisation by 3-hydroxypyridin-4-one iron chelators

We describe a protocol for the synchronisation of normal and tumour cells grown in suspension cultures using 3-hydroxypyridin-4-one iron chelators. These compounds inhibit ribonucleotide reductase, one of the rate limiting enzymes in DNA synthesis, and so block the cell cycle in late G1 phase. After removal of the chelator or repletion of cellular iron, cells progress through the cycle and remain synchronised for at least one full cell cycle. Cell viability is unaffected for at least 72 hours post-incubation and chelator treatment has no effect on RNA and protein synthesis. This method of synchronisation has been successful with all cell lines tested including normal and leukaemic human cell lines.

Genotoxic Consequences of Testicular Localization of Indium-114m

The incidence of childhood leukaemia in the offspring of workers at the Sellafield nuclear reprocessing plant has been associated with preconception al paternal radiation exposure (Gardner et al., 1990). One possible causative factor for this effect may be that in addition to external radiation, workers may also be subject to internal radionuclide contamination. Local testicular exposure to radionuclides offers a route for increased potential for transmission of genetic damage via sperm, irradiated during their developmental stages, which could be a contributory factor to childhood leukaemogenesis.