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Botryodiplodin induces DNA-protein cross-links (DPC) in mammalian cells in culture. Removal of the mycotoxin followed by incubation of cells in fresh medium leads to the gradual disappearance of DPC. The present experiments attempted to determine to what extent the repair mechanism acts through a system involving transient formation of single-strand breaks (SSB). The data reported here show that no SSB were detected in DNA of various strains of cells, even in the presence of inhibitors (hydroxyurea and 1-beta-D-arabinofuranosyl cytosine). These data demonstrate that neither the base excision, nor the excision repair process was involved in the repair of DPC or of any other lesions induced by botryodiplodin in mammalian cells.

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A newly synthesized fluorescent TPA derivative, O-(N-dansylamino-3-tetradecanoyl)-12,O-acetyl-13-phorbol (Dansyl-TPA), inhibited metabolic cooperation of Chinese hamster V79 cells in culture and also the specific binding of [3H]phorbol dibutyrate to V79 cells. Dansyl-TPA should be a valuable tool for studying the cellular targets of TPA.

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Repair of DNA--protein cross-links (DPC) induced by the mycotoxin botryodiplodin in three cell strains (LF, VA and XP) was studied after removal of the drug and postincubation in fresh medium. Immediately after botryodiplodin was removed, DPC progressively disappeared with an efficiency which depended upon the dose of mycotoxin applied and the cell strain considered. Experiments using [14C]botryodiplodin suggested that DPC repair involved at least one splitting step, located between the DNA and the drug. Botryodiplodin also inhibited the replication process. After incubation of cells in fresh medium, DPC repair was not associated with concomitant resumption of DNA replication ability. Immediately after botryodiplodin removal, inhibition of DNA synthesis was strengthened for a few hours before DNA replication recovered. Possible mechanisms involved in the DPC repair process are discussed.

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Transcriptional activity in nuclei isolated from the liver of rats during the acute inflammatory reaction induced by a 24-hour treatment with turpentine was evaluated by biochemical and ultrastructural techniques. This experimental model was chosen because it is known to induce an increase in hepatic synthesis of several plasma proteins called acute phase reactants. Using an in vitro system for estimating the activity of RNA polymerases A and B as an approach to evaluate RNA synthesis, we have shown that the activity of enzyme A (responsible for pre-rRNA synthesis) was significantly enhanced, whereas the activity of enzyme B (responsible for giant heterogenous RNA synthesis) remained unchanged in treated rats compared with control rats. At the electron microscopic level, using the Miller technique to spread the nuclear contents, the non-nucleolar transcription units were similar in treated and control rats. In contrast to control rats, more nucleolar transcription units were vizualized in treated rats, although the ribonucleoprotein fibrils were less numerous; in addition, in the latter group 10 to 15 times more aggregates constituted of ribonucleoprotein fibrils and particles were observed, the morphology of which was in agreement with a nucleolar origin. These results suggest that, during the acute inflammatory reaction, there was no overall increase in giant heterogenous RNA synthesis, whereas an increase in rRNA synthesis might be observed.

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Adult rat hepatocytes in primary culture were used to study the effect of tryptophan pyrolysis products on the transcriptional process. Hepatocytes were treated with 1, 5 and 10 micrograms/ml of 3-amino-1,4-dimethyl-5H-pyrido-[4,3-b]-indole (Trp-P-1) or 3-amino-1-methyl-5H-pyrido-[4,3-b]-indole (Trp-P-2) for 2 and 4 h. The ultrastructural study revealed the appearance of nucleolar microsegregation accompanied by a reduction in peri- and interchromatin fibrils and granules in hepatocytes exposed to 10 micrograms/ml of each pyrolysate for 1 or 2 h. Biochemical investigation showed that the incorporation of [3H]uridine into nuclear RNA of treated hepatocytes was strongly decreased. Time- and concentration-related inhibition have been established; however, the inhibitory effect of Trp-P-1 was always superior to that of Trp-P-2. The determination of Mg2+-dependent RNA polymerase activity in an in vitro system functioning with isolated rat liver nuclei incubated in the presence of Trp-P-1 or Trp-P-2 showed a 40% inhibition of this activity. After a 1-h exposure of hepatocytes to 5 and 10 micrograms/ml of Trp-P-1, the recovery of RNA synthesis capacity was complete by 2 h and that of normal ultrastructural aspect was achieved within 4 h. All these results indicated that Trp-P-1 and Try-P-2 acted at the nucleolar level by a blockade of pre-rRNA synthesis and at the extranucleolar by decreasing the ultrastructural RNP responsible for hnRNA synthesis.

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Botryodiplodin, a mycotoxin isolated from a strain of Penicillium roqueforti, induces DNA-protein cross-links in mammalian cells. This action depends upon the presence of the hydroxyl group at the position 2 of the molecule. The mechanism involved in the formation of cross-linkings is discussed.

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The effects of botryodiplodia, a mycotoxin synthesized by Botryodiplodia theobromae and by some strains of Penicillium roqueforti. The toxin inhibited cell multiplication in growing cultures at concentrations which were without toxic effect on cultures nearing or at confluence. In growing cultures the toxin affected DNA, RNA and protein synthesis, the effect on DNA synthesis being the greatest. The removal of mycotoxin from the culture medium brought about partial recovery of the synthetic activities of the cells. The implications of the results with respect of the recovery of DNA synthesis are discussed.

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PR toxin, a mycotoxin from Penicillium roqueforti, induces DNA--protein cross-links in chromatin of both cultured cells and isolated rat-liver nuclei. The presence of the aldehyde group in the PRT molecule is required for the induction of cross-linking; methylene bridges between nucleic acid and protein are presumably involved in the complex formation. The role of other functional groups of PR toxin is discussed.

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Patulin, a carcinogenic mycotoxin, inhibits in vivo and in vitro protein synthesis in rat liver. The in vivo inhibition culminates 5 h after toxin administration and reaches a maximum of 65% with regard to control values; a breakdown of polysomes is associated with the translational blockage. However in in vitro systems, the cellular fractions obtained from patulin-treated rats appear equally as active in protein synthesizing ability and as sensitive to the toxin action as those prepared from control animals. The in vitro inhibition by patulin is dose related. The postmitochondrial system is less sensitive than that functioning with isolated polysomes and pH 5 enzyme. This difference might be due to the presence of soluble factor(s) that counteract patulin action. We propose that the inhibition of protein synthesis by patulin may result from an interaction of the drug with active SH groups at the membrane level (amino acid transport, ion equilibrium) and/or at the cytoplasmic level (enzymes and factors involved in the translational process).

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The action of the ingestion of balanced diets containing pesticides [lindane (125 ppm) or/and zineb (5200 ppm)] on aflatoxin B1-induced toxic effects (inhibition of RNA and protein syntheses in liver) has been determined in two animal species. In the rat, the ingestion of these diets for 4 weeks before aflatoxin B1 treatment (0,7 mg/kg for 3 h), strongly decreased the toxic effects on both transcription and translation. By contrast, in the mouse, the above-mentioned diets did not modify the extent of the inhibition produced by aflatoxin B1 (60 mg/kg for 3 hr) on RNA and protein syntheses. These results were discussed.

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Zineb (zinc ethylene bisdithiocarbamate) inhibits the transcriptional activity of an in vitro system functioning with either RNA polymerases of isolated rat liver nuclei or purified E. coli RNA polymerase. Zineb also inhibits the in vitro aminoacid incorporation mediated by a polysomal standard system, by interacting with some pH 5 enzyme components(s). When rats fed a balanced semi-synthetic diet containing zineb (5 200 mg/kg) for ten weeks, one may observe a decrease in the RNA and protein syntheses in liver. The consequences of the ingestion of small but repeated doses of zineb are discussed.

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PR toxin impairs liver cell metabolism by inhibiting RNA and protein synthesis. In vitro, the drug inhibits the transcription carried out by isolated rat liver nuclei and the translation promoted by polysomes. The action of monovalent cations on the biological activities of PR toxin has been studied. The increased ionic strength due to the presence of salt in the incubation medium, lowers the inhibitory action of PR toxin on in vitro transcription and translation activities; this action is reversible. Besides the overall effect of the ionic strength, ammonium salts possess a specific ability to suppress irreversibly the biological properties of PR toxin (in vivo toxicity and capacity of inhibiting RNA and protein synthesis). The mechanism of this action is discussed.

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The problem of the chemical structure--biological effects relationships has been studied for various eremophilane compounds related to PR toxin (PRT), a mycotoxin synthesized by Penicillium roqueforti. The biological tests were based on in vivo toxicity for male Swiss mice and inhibition of in vitro transcription and tranlation. The results showed a good correlation between the responses obtained for the three tests by the different compounds; thus, the toxic potency and the capacity of inhibiting transcription and translation should be directed by a common chemical structure. The data also indicated that the biological properties are related to the existence of an aldehyde group in position 12. In addition, evidence has been obtained demonstrating the hydrolysis of PRT imine with formation of a PRT-like compound.

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