ABSTRACT
The purpose of this work was to investigate the administration of very low but repeated doses of a genotoxic carcinogen and an eventual correlation with cellular DNA synthesis. The compound 7H-dibenzo[c,g]carbazole is a genotoxic carcinogen in the mouse liver and was administered topically at the dose of 13.35 microg per animal every 2 days to give a total of 13 applications. Animals were sacrificed 48 hours after every 2 applications until the 10th treatment, then 48 hours after every treatment. Postulated genotoxic effects such as DNA adduct formation were detected by the 32P-post labeling assay. Liver sections were examined for microscopic changes and DNA synthesis. Results showed an increase of the total DNA adduct level in the liver throughout the study with a slowing down in the level after the sixth application of the compound. This change could correspond to the onset of DNA synthesis and to the moderate hepatocellular apoptosis which was observed. The DNA synthesis, which was considered to be secondary to the cytotoxicity induced by the high level of DNA adducts altering normal cellular activity, could also be the opportunity to fix the DNA adducts into heritable mutations. These results raise the question regarding the risk assessment in humans exposed regularly to very low doses of chemicals in the environment: for non-proliferating tissue, the regular accumulation of DNA adducts could remain silent until a "threshold level" is reached from which stimulation of the DNA synthesis may fix the DNA adducts into heritable mutations, eventually leading to tumors.
Subject(s)
Carbazoles/toxicity , Carcinogens/toxicity , DNA Adducts/biosynthesis , DNA Replication/drug effects , Liver/metabolism , Mutagens/toxicity , Administration, Topical , Animals , Apoptosis/drug effects , Carbazoles/administration & dosage , Cell Division/drug effects , DNA Adducts/drug effects , Dose-Response Relationship, Drug , Female , Hepatocytes/drug effects , Hepatocytes/pathology , Liver/drug effects , Mice , Mice, Inbred DBAABSTRACT
Samples of DNA irradiated at 405 and/or 365 nm in the presence of 8-methoxypsoralen (8-MOP) were analysed via a modified postlabelling assay using three hydrolysis enzymes other than those employed previously. These enzymes (deoxyribonucleaseI, venom phosphodiesterase and alkaline phosphatase) liberated 3'-adducted dinucleotide monophosphate instead of the 5'-modified dinucleotide monophosphate normally obtained. The first separation chromatography (D1) of samples irradiated in the presence of 8-MOP showed a single spot above the origin, and the next separation (D2) resolved this spot into two components (spots I and II). Double irradiation experiments in which samples of DNA were first irradiated at 405 nm before being irradiated at 365 nm showed that spot II could be transformed into spot I. The use of 6,4,4'-trimethylangelicin, which induced only photomonoadducts under UVA irradiation, gave only spot II. These two results indicated that spots I and II were respectively due to interstrand cross-links and monoadducts. Dose-effect experiments showed that spots I and II were dose dependent, and low-dose irradiations permitted us to measure one interstrand cross-link and two monoadducts per 10(8) base pairs.
Subject(s)
DNA/analysis , HeLa Cells/chemistry , Methoxsalen/analysis , Autoradiography , Chromatography/methods , DNA/metabolism , DNA/radiation effects , Evaluation Studies as Topic , Furocoumarins/analysis , Furocoumarins/metabolism , Furocoumarins/pharmacology , Humans , Isotope Labeling , Kinetics , Methoxsalen/metabolism , Methoxsalen/pharmacology , PUVA Therapy , Phosphorus RadioisotopesABSTRACT
8-Methoxypsoralen is a bifunctional furocoumarin used in human photochemotherapy. It can form two kinds of DNA adduct, monoadducts and interstrand cross-links. These adducts have been detected by 32P-postlabelling using hydrolysis with DNase1, alkaline phosphatase and snake venom phosphodiesterase, and longer labelling than usual, with more T4 polynucleotide kinase. Using preliminary two-dimensional chromatography (D1, D2) followed by transfer of adducts to separate PEI cellulose sheets for further development (D3, D4), we observed three spots corresponding to the adducts sought. Two experiments (dose-effects and shift of radioactivity) have confirmed the origin of the three spots.
Subject(s)
DNA Adducts , DNA/analysis , Furocoumarins/analysis , Methoxsalen/analysis , Phosphorus Radioisotopes , DNA/drug effects , DNA/radiation effects , DNA Damage , Dose-Response Relationship, Radiation , HeLa Cells , Humans , Hydrolysis , PUVA Therapy/adverse effectsABSTRACT
Endothelin-1 (ET-1) induced a concentration- and time-dependent increase in arachidonic acid release from guinea pig alveolar macrophages, a maximum being reached at 1 nM ET-1. Regarding the time-course study, maximal release of arachidonic acid was observed after 30 s of incubation with ET-1 and then followed by a marked decrease, suggesting a reuptake of free arachidonic acid. ET-1 (0.1-10 nM) induced a concentration- and time-dependent release of TXB2, the stable metabolite of TXA2, evaluated by an ELISA technique. Maximum release was also obtained with 1 nM ET-1 but only after a 1-min incubation period. By contrast, ET-1 (1 pM-1 microM) did not stimulate the release of superoxide anion at any time point of the kinetic study. These results suggests that ET-1 may activate guinea pig alveolar macrophages, probably through a mechanism involving the arachidonic acid pathway.
