[Restoration of DNA supercoiling in fibroblasts in the rat subjected to gamma ray or fast neutrons]. / Restauration du surenroulement de l'ADN de fibroblastes de rat soumis aux rayons gamma ou aux neutrons rapides.

Nucleoid sedimentation analysis was applied to the study of DNA supercoiling repair in cultured FR 3T3 fibroblasts exposed to low doses of fast neutrons or gamma-rays. Supercoiling was fully restored in both instances upon post-irradiation at 37 degrees C, but the rate of repair of neutron-induced lesions was lower than that for gamma-rays. Non-repairable breaks were not evidenced at the neutral pH used. We suggest that the non-repairable alkali-labile sites evidenced by others in neutron-irradiated DNA do not prevent strand break rejoining and subsequent recovery of the tertiary DNA structure.

Unusual alkaline elution pattern induced in mammalian cell DNA by fast neutrons p(34) + Be.

Syrian hamster fibroblasts (cell line BHK 21/13) were exposed to p(34) + Be fast neutron irradiation and their DNA analysed by the alkaline elution technique. The elution profiles showed an unusual tailing off, characteristic of neutron-irradiated samples, suggesting the presence of a modification in DNA induced by the neutrons. This was not seen with 60Co gamma-irradiation. In neutron-irradiated samples the alteration of DNA appeared to persist even after 2 h of post-treatment incubation (37 degrees C) indicating the absence of repair. The modification of DNA induced by neutrons provides a possible explanation for the reduction of the shoulders in survival curves obtained with neutrons, and the high RBE of neutrons.

Study of several factors in RNA-protein cross-link formation induced by ionizing radiations within 70S ribosomes of E. coli MRE 600.

The induction of RNA-protein crosslinks in E. coli 70S ribosomes by gamma-irradiation was studied by measuring the dependence of cross-link formation on ribosome concentration. The inverse dependence of cross-link percentage upon concentration up to at least 20 A260 nm units ml-1 indicate that indirect effects seem to play a more major part than direct effects for these ribosome concentrations. The effect of various gases and free radical scavengers was used to determine the roles of the radicals H., CO2-., OH. and e-aq and to estimate their relative efficiencies for cross-links. They were found to be: 7.2(H.), 6(CO2-.), 2(OH.) and 1(e-aq). The extent of RNA-protein cross-link production in 70S ribosomes induced by gamma-rays and neutrons in the presence and absence of oxygen was also investigated. Cross-link formation was estimated by separation of linked and unlinked material on nitrocellulose filters or after separation by SDS-sucrose gradient centrifugation under dissociating conditions. Oxygen inhibited cross-link formation by both neutrons and gamma-rays. However, very few cross-links were formed in de-aerated solutions by exposure to neutrons, compared to those produced by gamma-rays under the same conditions. This suggests that molecular oxygen generated along the secondary particle track can reduce the formation of RNA-protein cross-links.

[Comparison of chain breaks produced in DNA in vivo by gamma rays and neutrons; hypothesis of a new DNA radiolesion]. / Comparaison des ruptures de chaîne produites dans l'ADN in vivo par les rayons gamma et les neutrons; hypothèse d'une nouvelle radiolésion de l'ADN.

Using the method of alkaline elution for the treatment of cell DNA in chinese hamster fibroblasts irradiated with low doses of either cobalt-60 gamma rays or p (34 MeV) Be neutrons, we determined the kinetics of radio-induced strand breaks. The comparison gamma rays-neutrons reveals important discrepancies which suggest that neutrons induce a so for unknown reaction in DNA simultaneously with single and double strand breakage. This observation could contribute to explain the high RBE value of high LET particles.

The metabolic activation of dibenzo[a,e]fluoranthene in vitro. Evidence that its bay-region and pseudo-bay-region diol-epoxides react preferentially with guanosine.

Dibenzo[a,e]fluoranthene ( DBF ), a non- alternant carcinogenic polycyclic aromatic hydrocarbon (PAH), binds covalently to DNA. The main adducts were characterized as covalent additions of its bay-region and pseudo-bay-region diol-epoxides. The structure of these 2 adducts was analyzed by mass spectrometry using their persilyl derivatives. 3,4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydro- DBF (3,4-diol-1,2-epoxy- DBF ) and 12,13-dihydroxy-10,11-epoxy-10,11,12,13-tetrahydro- DBF (12, 13-diol-10,11-epoxy- DBF ) obtained by synthesis were allowed to react in vitro with calf thymus DNA or with poly(G). The comigration of DNA and poly(G) adducts isolated after acid hydrolysis of DNA and poly(G) was in good agreement with mass spectroscopic results: both bay-region and pseudo-bay-region DBF diol-epoxides reacted with guanine residues.

DNA-protein crosslinks induced by exposure of cultured mouse fibroblasts to dibenzo[a,e]fluoranthene and its bay- and pseudo-bay region dihydrodiols.

The presence of DNA-protein crosslinks was shown by the alkaline elution technique in cultured mouse fibroblasts treated with dibenzo[a,e]fluoranthene (DBF), a non-alternating carcinogenic PAH. The crosslinks appeared to be between DNA and protein, since the effect disappeared with proteinase treatment. The crosslinking effect increased with time of exposure to DBF. Two major metabolites of DBF were tested under similar conditions. The pseudo-bay region dihydrodiol of DBF induced similar effects. Its isomer, the bay-region dihydrodiol, was inactive. The possible intervention of a bifunctional metabolite of DBF in the DNA-protein crosslinking process is discussed.

Ribosomal RNA-protein cross-links, induced by gamma-irradiation of 40S and 60S ribosomal subunits of L cells.

The 40S and 60S ribosomal subunits of L cells are gamma-irradiated in the absence of oxygen at low radiation doses to keep the integrity of the ribosomal structure. We show that under these experimental conditions, specific cross-links are induced in situ between rRNA and ribosomal proteins due to close contact between their reactive groups. We found that about 15 proteins are cross-linked to the 28S RNA. Most of them belong to the core proteins of the 60S ribosomal subunits. A few high-molecular mass proteins which might be factors are also bound to 28S RNA. Between 8 and 11 proteins are covalently linked to 18S RNA; 8 of these have been previously described as transferable proteins from one subunit to the other. Only 3 are core proteins of the small subunit. The contribution of these results to the study of the three-dimensional ribosomal structure is also discussed.

Binding to DNA of bay region and pseudo bay region diol-epoxides of dibenzo[a,e]fluoranthene and comparison with adducts obtained with dibenzo[a,e]fluoranthene or its dihydrodiols in the presence of microsomes.

When dibenzo[a,e]fluoranthene (DBF) is incubated in vitro with mouse liver microsomes in the presence of calf thymus DNA, several metabolites bind covalently to DNA. The metabolite-nucleoside adducts were separated by h.p.l.c. after enzymatic hydrolysis. The elution profile of this chromatogram exhibits six main peaks, labeled from A to F in order of decreasing polarity. It was compared to those obtained by direct reaction of DNA with 3,4-dihydroxy-1,2-epoxy 1,2,3,4-tetrahydro DBF (the bay region diol-epoxide) or 12,13-dihydroxy 10,11-epoxy 10,11,12,13-tetrahydro DBF (the pseudo bay region diol-epoxide). In both cases the retention period of the peak of the adduct was identical to that of the main peak E. The fluorescence spectra of these two adducts were similar to those of the corresponding tetrols. When DNA is reacted in the presence of microsomes with 3,4-dihydrodihydroxy DBF, the elution profile of the adducts indicates that vicinal epoxidation of the dihydrodiol and direct reaction is dominant. The metabolic reaction with 12,13-dihydrodihydroxy DBF appears more complex as revealed by the observed number of adducts which correspond to vicinal epoxidation of dihydrodiol as well as further oxidation at other sites.

Radiochemical cross-linking between proteins and RNA within 70 S ribosomal particles from E. coli MRE600.

In the absence of oxygen, gamma-irradiation produces covalent links between some ribosomal proteins and 16 S RNA to 23 S RNA, within 70 S ribosomes from E. coli MRE600. Under optimal conditions minimizing the structural modifications induced by radiations, in situ formed cross-links appear specific and reflect close RNA-protein contacts. In view of these results, the spatial organization of the 30 S, 50 S subunit interfaces is discussed. In addition, the gamma-irradiation technique reveals that subunit association induces modifications of some protein--RNA interactions.

Apparent stimulation of dibenzo[a,e]fluoranthene in vitro metabolism in the presence of norharman.

The effects of norharman (NH) on the metabolism of dibenzo[a,e]-fluoranthene (DBF) and on its fixation on DNA, RNA and proteins have been studied in vitro by incubation with S-9 and microsomes from rats and mice. NH causes a decrease of the activity of microsome monooxygenases proportionally to its concentration but has no effect on the activity of NADPH P-450 reductase nor on that of epoxide hydrolase. Paradoxically, the amount of DBF hydrophobic metabolites and especially that of diols and phenols, increases in the incubation mixture in the presence of NH; this increase is independent of the presence of conjugation enzymes of cytosol. NH does not modify the covalent binding of DBF on the microsome RNA, conversely it decreases the binding of DBF on the DNA and on the proteins of the incubation mixture. This could partly explain the increase of DBF diols and phenols by an accumulative effect. Two higher homologs of NH: benzo[g]-beta carboline and benzo[i]-beta carboline, tested under the same conditions, proved inhibitory.

Heterocyclic polycyclic aromatic hydrocarbon carcinogenesis: 7H-dibenzo[c,g]carbazole metabolism by microsomal enzymes from mouse and rat liver.

The metabolism of dibenzo[c,g]carbazole (DBC), was studied in vitro using microsomal fractions of mouse and rat liver from animals, which were treated with 3-methylcholanthrene (MC). The separation of extractable metabolites by high pressure liquid chromatography (HPLC) and thin-layer chromatography (TLC) as well as identification of most of them by nuclear magnetic resonance, mass spectrometry and comparison with synthetically obtained products are described. The microsomes of both species produced the same twelve compounds of which the following have been identified: five monohydroxylated derivatives (phenols), the product of further oxidation of one of them, and a dihydrodiol. The 5-OH-DBC (60% including its spontaneously-formed dimer) and the 3-OH-DBC (14%) are the main metabolites. Three minor metabolites cochromatographed with synthetically prepared 2-OH-DBC, 4-OH-DBC and 6-OH-DBC. The dihydrodiol detectable in small quantity (4-6%) was tentatively identified as 3,4-dihydroxy-3,4-dihydro-DBC by the sensitivity of its formation to very low concentrations of the inhibitor of microsomal epoxide hydrolase, 1,1,1-trichloropropene oxide, by its molecular ion and major fragment in mass spectrometry and by its dehydration product 3-OH-DBC. No other dihydrodiols were detected. The qualitative and quantitative effects of various modulators of metabolism (enzyme inhibitors, apparently homogeneous epoxide hydrolase, glutathione, supernatant fraction) were investigated. The results are discussed with respect to possible ultimate carcinogens.

Radiochemical cross-linking of proteins to RNA within ribosomal subunits from E. colil MRE 600.

Irradiation in vitro of 30S and 50S ribosomal subunits from E. coli MRE 600 by gamma rays from cobalt 60, in the absence of oxygen, results in the formation of covalent links between the RNAs and some ribosomal proteins. At low radiation doses, just sufficient to keep the integrity of the ribosome structure, the phenomenon appears highly specific. In the 30S irradiated particle, protein S1 attaches to 16S RNA; in the 50S irradiated particle, the proteins L3, L13, L19, L21, L22 and L24 are linked to 23S RNA. The mechanism of formation of these cross-links and their contribution to the study of the tridimensional ribosome structure are also discussed.

Binding of dibenzo(a,e)fluoranthene, a carcinogenic, polycyclic hydrocarbon without K-region, to nucleic acids in a subcellular microsomal system.

Dibenzo(a,e)fluoranthene (DBF), a highly carcinogenic polycyclic hydrocarbon without an apparent K-region, binds covalently to DNA, transfer RNA, and polyribonucleotides when incubated with hepatic microsomal fractions under standard conditions. Optimal binding conditions for [3H]DBF were established. Methylcholanthrene-pretreated mouse liver microsomes induced a higher level of binding of [3H]DBF to DNA than did similarly induced rat liver microsomes. 7,8-Benzoflavone strongly inhibited the binding of this polycyclic aromatic hydrocarbon to DNA, while cyclohexene oxide and trichloropropene oxide had an enhancing effect when used in the presence of rat liver microsomes. An unexpected inhibitory effect was observed with cyclohexene oxide in mouse liver microsome-enriched medium. [3H]DBF bound twice as much to denatured as to native DNA. Incubation of [3H]DBF in the presence of liver microsomes and polyribonucleotides (polyadenylate, polyuridylate, polyguanylate, and polyinosinate) indicated that binding occurs mainly with guanine. Binding of [3H]DBF to DNA of various origins was found to be directly proportional to the amount of GC pairs. Preliminary results indicate a covalent bond between DBF and nucleic acids.

Biochemical analysis of damage induced in yeast by formaldehyde. II. Induction of cross-links between DNA and protein.

Exposure of Saccharomyces cerevisiae cells to formaldehyde-induced cross-links between DNA and proteins. This damage was demonstrated by three different techniques. Ultraviolet irradiation also produced cross-links between DNA and proteins in yeast.

Biochemical analysis of damage induced in yeast by formaldehyde. I. Induction of single-strand breaks in DNA and their repair.

Analysis of sedimentation profiles in alkaline sucrose gradients showed that, through a metabolic process, formaldehyde (FA) produced single-strand breaks in DNA of exponential phase cells of haploid wild-type Saccharomyces cerevisiae. The production of this type of lesion was dose-dependent. Strains defective in excision-repair of pyrimidine dimers induced by ultraviolet (UV) irradiation showed a reduced capacity to undergo single-stand breaks after treatment with FA. This indicates that the repair pathways of damage induced by UV and FA share a common step. Post-treatment incubation of wild-type cells in growth medium indicate a lag in cell division during which a slow recovery of DNA with a normal size was observed.

[Formation of covalent linkages between the nucleic acid and protein constituents of E. coli MRE 600 ribosomes during gamma irradiation]. / Formation de liaisons covalentes entre les constituants nucléiques et protéiques des ribosomes d'E. coli MRE 600 irradiés par des rayons gamma

Gamma-irrdiation of E. coli MRE 600 ribosomes in aqueous suspensions leads to covalent linkages between the RNA and some ribosomal proteins. The presence of oxygen during the irradiation strongly inhibits this phenomenon. It appears clearly that only a few proteins are able to participate in these cross-linking reactions, which occur simultaneously in the two sub-units. The radiochemical yield was determined at several concentrations and was relatively low.

[Formation, by gamma irradiation, of RNA-protein cross-links in E. coli ribosomes]. / Production, par les rayons gamma, de liaisons ARN-protéines dans les ribosomes d'E. coli

Gamma irradiation in desaerated conditions of E. coli MRE 600 ribosomes, labelled with C14 uracil, leads to a decrease of extractibility of C14 RNA by lithium chloride 4 M-urea 8 M. On the other hand, the radioactivity of the protein fraction increases with irradiation. These results strongly suggest that RNA-protein cross links are formed in irradiated ribosomes.