32P-post-labelling analysis of DNA adducts formed in the upper gastrointestinal tissue of mice fed bracken extract or bracken spores

Bracken toxicity to both domestic and laboratory animals is well established and tumours are formed when rodents are treated with either bracken extracts or bracken spores. In this study we have administered bracken spores and extract to mice in order to investigate whether such exposure leads to the formation of DNA adducts. DNA, isolated from the upper gastrointestinal tract and liver, was digested to 3'-nucleotides. Adducts were extracted with butanol, 32P-post-labelled, separated by thin layer chromatography (TLC) and visualised and quantified using storage-phosphor technology. A cluster of adducts was clearly seen in the DNA of the upper gastrointestinal tract, but not liver, 5 and 24 h after treatment with bracken extract or bracken spores. These adducts were not observed in DNA extracted from vehicle-treated animals. Whereas, after 5 h adduct levels in extract and spore-treated animals were similar, after 24 h adduct levels in the extract-treated animals had diminished by > 75%, but levels in spore-treated animals remained similar to those found after 5 h. This suggests that the DNA-reactive compounds were being released slowly from the spores, even though the spores had been sonicated before administration. Adducts were also quantified after the addition of an internal standard (deoxyinosine 3'-monophosphate) by comparing the amount of label incorporated into the adducts with that found in a known amount of the internal standard. Adduct levels using this internal standard approach were similar to those found by direct measurement of radioactivity incorporated into the adduct, indicating that the labelling of adducts was quantitative. We have tried, unsuccessfully, to synthesise ptaquiloside, the principal carcinogenic component present within bracken. However, similar patterns of adducts were observed when two other compounds, (1-(4-chlorophenyl sulphonyl)-l-cyclopropane carbonitrile and 3-cyclopropylindeno [1,2-c] pyrazol-4-(O-methyl)oxime), which both contain a cyclopropyl ring, were administered to mice. The adducts detected in bracken-treated animals may, thus, have arisen from ptaquiloside but, whether these adducts arise directly from the compounds and bracken spores/extract themselves or via an indirect mechanism, remains to be determined. As bracken-induced DNA adducts are detectable in rodent tissues by a 32P-post-labelling procedure commonly employed to investigate DNA damage in human populations, it may prove possible to apply such approaches to determine human exposure.     

Effects of DDT on reproduction in multiple generations of beagle dogs

The effects of chronic oral exposure to 1, 5, and 10 mg of technical DDT/kg/day on: 1) age at puberty, length of gestation, fertility, success of pregnancy, litter size, and lactational ability of dams; 2) viability, survival to weaning, sex distribution and growth of pups; and 3) morbidity, mortality, organ/body weight ratios, gross and histologic abnormalities in all animals were studied through three generations of Beagle dogs. There were a total of 135 adult female and 63 adult male dogs in the project which produced 650 pups. There were no statistically significant differences among control and DDT-treated dogs in any of the reproductive variables, with the exception of age at puberty of the females. DDT-treated females had their first estrous cycles 2 to 3 months earlier (P less than .001) than the control dogs. Selected DDT-treated females, held for a second breeding period, had normal anestrous periods between their first and second estrous cycles. There was no effect of DDT on survival, growth, and sex distribution of pups, nor was there any influence on morbidity, mortality, gross or histologic findings in any of the dogs. All organ/body weight ratios were normal, with the possible exception of an increase in liver/body weight ratio in some DDT-treated animals.