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Biomedical subjects

A Gentil

Publications and source records attributed to A Gentil.

At least 37 records · Page 2Linked to original sources
PubMed1985

Molecular analysis of mutagenesis in mammalian cells.

Mammalian cells are constantly facing various types of mutagens. However, due to the high complexity of the cell genome, the molecular analysis of mutagenesis has not yet been possible. Therefore, we have used simian virus 40 (SV40) as a biological and molecular probe to characterize mutagenesis at the nucleotide level. By using a reversion assay from a temperature-sensitive phenotype towards a wild-type phenotype, we have analysed mutagenesis induced by u.v.-light and by apurinic sites (Ap sites). We report here experiments allowing us to quantify and to compare the mutagenic efficiency of various DNA lesions measured on the SV40 genome. The Ap sites are very mutagenic in this type of assay. The molecular analysis of u.v.-induced mutagenesis reveals that mutations correspond to single base-pair substitutions always located opposite Py-Py lesions. The mutations are almost equally distributed between transition and transversion types, and between the 5' and the 3' side of the Py-Py targets. These results demonstrate for the first time in animal cells the existence of targeted mutations induced by u.v.-light. We propose therefore, the use of SV40 as an efficient biological and molecular probe for assaying mutagenic pathways in mammalian cells.

Acetoxyacetylaminofluorene↗
PubMed1984

Apurinic sites cause mutations in simian virus 40.

SV40 has been used as a molecular probe to study the mutagenicity of apurinic sites (Ap) in mammalian cells. Untreated or UV-irradiated monkey kidney cells were transfected with depurinated DNA from the temperature-sensitive tsB201 SV40 late mutant which grows normally at the permissive temperature of 33 degrees C but which is unable to grow at 41 degrees C. Phenotypic revertants were screened at 41 degrees C for their ability to grow at the restrictive temperature and the mutation frequency was calculated in the viral progeny. Ap sites were introduced into DNA by heating at 70 degrees C under acid conditions (pH 4.8). This treatment induces one Ap site per SV40 genome per 15 min of heating as measured by alkaline denaturation or by treatment with the T4-encoded UV-specific endonuclease which possesses Ap-endonuclease activity. The experiments reported here show that Ap sites strongly decrease virus survival with a lethal hit corresponding roughly to 3 Ap lesions per SV40 genome, and indicate for the first time that apurinic sites produced by heating are highly mutagenic in animal cells. UV irradiation of the host cells 24 h prior to transfection with depurinated DNA did not modify the mutation frequency in the virus progeny.

Animals↗
PubMed1982

Kinetics of induction of sister-chromatid exchanges by X-rays through two cell cycles.

V79 Chinese hamster cells were exposed to X-rays at various times through the two cell cycles required to obtain harlequin-stained chromosomes. A two-fold SCE enhancement was found between the first and the second G1 phase when BrdUrd was incorporated during the first S phase only. This BrdUrd effect was not found when MNNG was used. Furthermore, the kinetics of SCE and aberrations were different, suggesting two separate mechanisms for their formation: SCE activity takes place when DNA damage occurs before the DNA replication, and aberration activity when the DNA damage occurs chiefly after the DNA replication.

Animals↗
PubMed1981

Simian virus 40 as a probe for studying inducible repair functions in mammalian cells.

We describe the use of Simian Virus 40 (SV40) as a molecular probe for studying the cellular functions induced in cultured monkey kidney cells in response to DNA damaging agents. (a) Ultraviolet (UV) irradiation of SV40-infected cells inhibits viral DNA replication. Replication forks are blocked by the first pyrimidine dimer encountered. In some cases, a single-strand break seems to occur at the level of the dimer inhibiting the fork of replication. This break, which can be visualized by electron microscopy studies, might be the first step in an excision repair pathway. (b) Treatment of monkey kidney cells with acetoxy-acetyl-aminofluorene or UV light before infection with UV-irradiated SV40 induces a mutagenic replication mode, as shown by an increase of the mutation frequency of thermosensitive SV40 mutants. (c) A possible recombination assay using various SV40 mutants infecting the same cell is proposed and discussed.

Animals↗
PubMed1980

The effect of the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) on UV- and MNNG-induced sister chromatid exchanges in mammalian cells.

The induction of sister chromatid exchanges (SCE) by the tumour promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) has been studied in V79 Chinese hamster cells comparing control untreated cells with either UV-irradiation or MNNG-treated cells. In untreated cells TPA induced SCE at a very low concentration. An increase of approximately 50% over the level of spontaneous SCE formation was observed. In UV-irradiated cells, TPA, when present after the UV-irradiation, significantly enhanced UV-induced SCE formation, more SCE being induced than the expected sum of SCE formed by the separate treatments. TPA also enhanced MNNG-induced SCE but only when it was present for 28 h before, and not after, the MNNG-treatment. The protease inhibitor antipain fully inhibited TPA-induced SCE but did not inhibit UV-induced SCE. When UV-irradiated cells were treated with both TPA and antipain the enhanced UV-induced SCE induction was only partly inhibited by antipain, showing that TPA is responsible for two independent effects, both leading to SCE formation. These results are discussed in relation to the hypothesis that tumour promotion by TPA may depend on the enhancement of mitotic recombination leading to the segregation of a mutational event.

Animals↗
PubMed1980

DNA excision repair in human cells treated with ultraviolet radiation and 7,12-dimethylbenz[a]anthracene 5,6-oxide.

Excision repair was measured in normal human and xeroderma pigmentosum group C cells treated with 7,12-dimethylbenz[a]-anthracene 5,6-oxide and with ultraviolet radiation by the techniques of unscheduled DNA synthesis, repair replication, a modification of bromodeoxyuridine photolysis employing the dye Hoechst 33258 and 365 nm radiation, and endonuclease-sensitive sites assay. Radioautography and repair replication showed that in normal cells the magnitude of repair after a saturation dose of epoxide (approx. 10 microM) to be 0.1-0.2 that after a saturating ultraviolet dose (20 J/m2 at 254), though survival data showed that both doses gave nearly similar killings. Repair was of the long-patch type and repair kinetics after the epoxide treatment were similar to ultraviolet. After a combined treatment with both agents, unscheduled synthesis in normal cells was more than additive, although, considering the experimental errors, these data and those of repair replication are consistent with additivity. The epoxide did not inhibit loss of sites sensitive to the ultraviolet endonuclease. However, after a combined treatment to xeroderma pigmentosum cells there was appreciably less unscheduled synthesis than for the sum of both treatments and the epoxide inhibited the loss of nuclease-sensitive sites. We interpret the data to indicate that there are different rate-limiting steps in the removal of the ultraviolet and the epoxide damages, and that the residual repair activity in xeroderma pigmentosum cells is accomplished by different, not just fewer, enzymes than in normal cells.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1979

The initiation of tumours on mouse skin by dihydrodiols derived from 7,12-dimethylbenz(a)anthracene and 3-methylcholanthrene.

The cis-2a,3-diol and the trans-4,5-, trans-7,8-, trans-9,10- and trans-11,12-dihydrodiols of 3-methylcholanthrene and the trans-3,4, trans-5,6-, trans-8,9. and trans-10,11- dihydrodiols of 7,12-dimethylbenz[a]anthrancene have been tested, in comparison with the parent hydrocarbons, for their abilities to initiate skin tumours in female CDI mice. Groups of mice received a single topical application (25 micrograms) of a diol or of a hydrocarbon, and 1 week later repeated topical applications (1 microgram) of 12-0-tetradecanoylphorbol-13-acetate were commenced. The results show that the diol of 3-methylcholanthrene and the 3,4-diol of 7,12-dimethylbenz[a]anthrancene were active as initiating agents but that they were no more active than their parent hydrocarbon. The K-region 5,6-diol of 7,12-dimethylbenz[a]anthrancene, which cannot be converted directly into a vicinal diol-epoxide, was also active as a tumour-initiating agent when applied to mouse skin.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1979

Biological activities of dihydrodiols derived from two polycyclic hydrocarbons in rodent test systems.

Comparisons have been made between (a) the initiation of tumours in mouse skin, (b) the induction of hyperplasia and the suppression of sebaceous glands in mouse skin and (c) the induction of s.c. tumours in rats, by either benzo[a]pyrene or 7-methylbenz[a]anthracene and their related K-region and non-K-region dihydrodiols. Whilst the 3,4-dihydrodiol derived from 7-methylbenz[a]anthracene is more active than the hydrocarbon in initiating tumours in mouse skin (subsequently promoted by a phorbol ester) the 7,8-dihydrodiol of benzo[a]pyrene is very much less active than benzo[a]pyrene itself in the induction of hyperplasia or the suppression of sebaceous glands in mouse skin or in the induction of s.c. sarcomas in rats. Since much other evidence suggests that the 3,4-dihydrodiol of 7-methylbenz[a]anthracene and the 7,8-dihydrodiol of benzo[a]pyrene are the dihydrodiols involved, via the related vicinal diol-epoxides, in the metabolic activation of these hydrocarbons, mouse skin initiation-promotion experiments may be more useful for the identification of such diols than the other two in vivo tests for biological activity used here.

Animals↗
PubMed1977

Two-stage carcinogenesis with rat embryo cells in tissue culture.

Transformation of rat embryo fibroblasts in vitro has been investigated using initiation with either benzo(a)pyrene (BaP), 7,12-dimethylbena(a)anthracent (DMBA) or benzo(e)pyrene (BeP) and promotion with either phorbol ester (TPA) or croton oil (Cr.Oil). The criteria used to assess in vitro transformation were (a) the efficiency of cloning in liquid medium, (b) abnormal cellular morphology and (c) the development of malignant tumours following s.c. inoculation of newborn rats. The results show that the cloning efficiency, which remained low in the control cells, was increased to a variable extent in the treated groups. Transformation occurred in all groups, but occurred earliest in cells that were initiated and promoted. Initiation with DMBA or BaP and promotion with TPA or Cr.Oil led to the earliest acquisition of malignancy. Correlations were found between the transformation of cells in vitro and the acquisition of malignant potential, and between the carcinogenic action of the compounds in vitro and their action in vivo, but cloning efficiency was not a reliable indicator of in vitro transformation or of malignancy. In most cases in vitro transformation appeared to precede the acquisition of malignancy, but in two cases it occurred later. The studies also show that BeP, which is a tumour initiator in vivo, also acts in this way in vitro. The conclusion drawn from a discussion of these results and of two-stage carcinogenesis in vivo is that two-stage carcinogenesis can be reproduced in tissue culture; this model may be useful in studies of those mechanisms of chemical carcinogenesis that involve the processes of initiation and promotion.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1977

Metabolism and cytotoxicity of 7,12-dimethylbenz[a]anthracene by hamster, rat and rabbit embryo cell cultures.

1. The metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) by rat, hamster and rabbit fibroblasts has been studied and compared with the metabolism of the hydrocarbon by liver homogenates. The metabolsim of DMBA by cell cultures and by liver homogenates was very similar. The ethyl acetate-soluble metabolites identified included dihydrodiols, phenols and hydroxymethyl derivatives. Other more polar metabolites and unidentified water-soluble metabolites were also formed. 2. High yields of phenols and other more polar metabolites, which may be tetrahydrotetrols, were produced by rabbit fibroblasts. 3. Kinetic studies showed that the metabolic activity of rabbit fibroblasts was high but that the conversion of DMBA into water-soluble metabolites was lower than with hamster and rat fibroblasts. 4. 7,12-Dimethylbenz[a]anthracene-induced cytotoxicity was inversely related to the conversion of metabolized DMBA to water-soluble derivatives.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1976

Tumour-initiating activities on mouse skin of dihydrodiols derived from benzo[a]pyrene.

Three dihydrodiols that are metabolites of benzo[a]pyrene and benzo[a]-pyrene itself have been tested in a comparative experiment for their activities as initiators of tumours in mouse skin. A single application (25 mug) of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene, of 7,8-dihydro-7,8-dihydroxybenzo[a]pyrene, of 9,10-dihydro-9,10-dihydroxybenzo[a]pyrene, or of benzo[a]pyrene was made to the shaved dorsal skin of adult female CDI mice; this was followed 2 weeks later by multiple thrice-or twice-weekly applications (1 mug) of 12-O-tetradecanoyl-phorbol-13-acetate as promoting agent. A control group of 30 mice received the promoting agent alone. The experiments were terminated 52 weeks after initiation. At this stage, all the groups contained mice bearing skin papillomas, some of which had progressed to malignancy. Quantitatively the results show that the 7,8-dihydrodiol is almost as active an initiator of mouse skin tumours as benzo[a]pyrene itself; the 4,5- and 9,10-dihydrodiols were significantly less active. The significance of these results is discussed in relation to the hypothesis that diol-epoxides are important in the metabolic activation of polycyclic hydrocarbons like benzo[a]pyrene.

Animals↗
PubMed1975

[Carcinogenic effect of 7,12-dimethylbenz(a)anthracene 5,6-oxide in mice].

The comparative carcinogenic activities of 7,12-dimethylbenz(a)anthracene 5,6-oxide (DMBA-5,6-oxide) and 7,12-dimethylbenz(a)anthracene (DMBA) to skin and subcutaneous tissue were studied. It was found that DMBA-5,6-oxide was less carcinogenic to both the skin and subcutaneous tissue, than DMBA. Moreover it does not show any significant initiating acitivity. The results are discussed.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1975

Benzo (a) pyrene uptake by normal and by tumour cells in vitro.

A method for determining the quantitative uptake of benzo(a)pyrene/B(a)P/by cells in culture is described. B(a)P uptake is very rapid and increases with concentration. The amount of B(a)P taken up by normal or by tumour cells depends on the cell type, and also, on the species from which they were derived.

Animals↗