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E Dogliotti

Publications and source records attributed to E Dogliotti.

71 records · Page 4Linked to original sources
PubMed1989

The origin of DNA single strand breaks induced by ethylating agents in mammalian cells.

Chinese hamster ovary (CHO) cells were treated with two ethylating agents, N-ethyl-N-nitrosourea (ENU) and diethylsulfate (DES), and the kinetics of DNA single strand break (ssb) induction and rejoining were determined in parallel with DNA adduct formation and removal. In the case of DES, DNA ssb as determined by alkaline elution (AE) were repaired very slowly with more than 50% of the lesions still present on DNA 3 h after treatment. In contrast, 45% of ENU-induced ssb were repaired within 10 min. From the relative concentration of the different ethylated products and their repair rates as measured by high performance liquid chromatography (HPLC) analysis of the ethylated DNA, a theoretical function was constructed that describes the number of ssb expected at each time point after exposure to the mutagen. DES-induced ssb are explained by excision repair processes active on the ethylated purines, mainly 3-ethyladenine (3-EtAde) and 7-ethylguanine (7-EtGua). On the same basis, the rapidly repaired ENU-induced ssb remain unexplained. These results are also discussed in relation to the sensitivity of the two techniques, AE and HPLC, for detecting DNA damage.

Alkylating Agents↗
PubMed1988

Relationship between specific alkylated bases and mutations at two gene loci induced by ethylnitrosourea and diethyl sulfate in CHO cells.

DNA adduct formation and induction of mutations at 2 gene loci, hypoxanthine-guanine-phosphoribosyltransferase (HPRT) and Na,K-ATPase, were determined simultaneously in Chinese hamster ovary (CHO) cells after treatment with 2 ethylating agents, ethylnitrosourea (ENU) or diethyl sulfate (DES). Doses of DES and ENU, which resulted in equal levels of O6-ethylguanine (O6-EtGua) and O4-ethylthymine (O4-EtThy) in the DNA, were found to induce very similar frequencies of 6-thioguanine-resistant (6-TGr) mutants. Formation of these DNA adducts might therefore be correlated with mutations induced at the HPRT locus. When, however, the same analysis was applied to ouabain-resistant (ouar) mutants, it was found that, at similar levels of O6-EtGua and O4-EtThy, DES induced many more ouar mutants than ENU. This result supports the notion that primary DNA lesions other than O6-EtGua and O4-EtThy are involved in the fixation of ENU- and DES-induced mutations at the Na,K-ATPase gene locus.

Alkylating Agents↗
PubMed1988

Isolation of clones displaying enhanced resistance to methylating agents in O6-methylguanine-DNA methyltransferase-proficient CHO cells.

O6-Methylguanine-DNA methyltransferase (MT)-proficient Chinese hamster ovary cells were grown in the presence of low, gradually increasing levels of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with the aim of selecting MNNG-resistant cell lines. Six resistant clones with two levels of resistance were isolated. A 3-fold increase in survival was observed in clones 13, 14 and 15 and a greater than 10-fold increase in clones A, B and C. Cross resistance to N-methyl-N-nitrosourea but not to mitomycin C was observed. By comparison with the parental MT-proficient cells, MT activity was doubled in two resistant clones (13 and B) irrespective of their resistance levels. DNA glycosylase activity responsible for the removal of 7-methylguanine and 3-methyladenine showed similar levels in resistant clones 13 and B, in the MT-proficient cells and in the original MT-deficient cells. Alkylation-induced DNA damage, as measured by alkaline elution at the same MNNG dose, was higher in clones 13 and B than in the parental cells. The induction of sister chromatid exchanges by MNNG was inversely related to the resistance levels, thus paralleling the induction of cytotoxicity. These results suggest the existence in Chinese hamster ovary cells of at least two independent functions which control resistance to methylating agents, one possibly being the capacity to repair O6-methylguanine.

Alkylating Agents↗
PubMed1987

Effects of buffer composition on water stability of alkylating agents. The example of N-ethyl-N-nitrosourea.

The half-life of N-ethyl-N-nitrosourea (ENU) in water solution was measured at pH 6.1, 6.8 and 7.3 using buffer systems characterized by differences in quality and quantity of the component species. The decay of the concentration of ENU as a function of time was detected both indirectly, by measuring its residual capability of inducing alkali-labile sites in CHO DNA, and directly, by measuring its residual concentration by means of 1H-nmr spectroscopy. Under controlled pH, temperature, and buffering conditions, the rate of hydrolytic loss of ENU measured by the biological and chemical experiments compared quite well, whereas at constant pH and temperature, it was substantially dependent upon the concentration of the buffer. This was attributed to the occurrence of general base catalysis promoted by the nucleophilic anions present in the buffer systems. This conclusion, largely supported by analysis of previously reported data concerning the kinetics of ENU in water, suggests that buffering conditions may play a role in determining the actual levels of reactive substrates in in vitro assays and, therefore, that different buffering conditions may lead to poorly reproducible results.

Alkylating Agents↗
PubMed1987

Induction kinetics of mutations at two genetic loci, DNA damage and repair in CHO cells after different exposure times to N-ethyl-N-nitrosourea.

Ouabain resistance (ouar) and 6-thioguanine resistance (6-TGr) mutation frequencies were measured in Chinese hamster ovary cells after treatment with N-ethyl-N-nitrosourea (ENU) for varying periods of time. Maximal mutation frequency at the Na+/K+ ATPase gene locus (ouar mutations) was attained within 5 min of exposure, whereas the mutation frequency at the hypoxanthine guanine phosphoribosyltransferase locus (6-TGr mutations) continued to increase up to 60 min, following the theoretical curve for exponential decay of ENU with time. Detection of DNA single strand breaks (ssb) by alkaline elution showed that maximal levels were attained within 5 min of treatment with ENU. Fast repair of DNA ssb occurred early after exposure (greater than 50% repair within 10 min). Analysis of DNA ethylation products by h.p.l.c. showed initially rapid removal of O2-ethylcytosine (25% in the first hour), slow removal of 7-ethylguanine, 3-ethyladenine and 3-ethylguanine and no removal at all of O6-ethylguanine, O4-ethylthymine and ethylphosphotriesters. These time-course studies reveal different target gene responses in the fixation of DNA damage into mutations.

Animals↗
PubMed1987

Cytotoxicity, mutations and SCEs induced by methylating agents are reduced in CHO cells expressing an active mammalian O6-methylguanine-DNA methyltransferase gene.

Alkylation at the O6 position of guanine leading to miscoding during DNA replication has been shown to correlate with mutagenesis both in bacteria and mammalian cells. The widely used Chinese hamster ovary cells (CHO) are unable to remove O6-methylguanine (O6-meG) due to the absence of O6-meG DNA methyltransferase (MT) activity. Recently Ding et al. [Mol. Cell. Biol. (1985) 5, 3293-3296] transfected CHO cells with human liver DNA obtaining a line provided with a function for the repair of O6-meG. We confirmed the presence of MT activity in this particular clone (14,300 molecules/cell). We used this MT-proficient cell line as compared with the original MT-deficient CHO cell line to analyse the relevance of repair of this lesion on cell killing, ouabain resistance (ouar) mutations and sister chromatid exchanges (SCEs) induced by methylating agents. MT-proficient cells were more resistant than MT-deficient ones to the cytotoxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU). Furthermore a lower number of MNNG-induced SCEs were found in MT-proficient CHO than in MT-deficient cells. Similar ouar mutation frequencies were recorded in the two cell lines after 4-nitroquinoline-1-oxide (4NQO) treatment showing that the differences in cytotoxicity and mutagenesis are restricted to treatment with alkylating agents.

4-Nitroquinoline-1-oxide↗
PubMed1984

Temporal dissociation in the exposure times required for maximal induction of cytotoxicity, mutation, and transformation by N-methyl-N'-nitro-N-nitrosoguanidine in the BALB/3T3 ClA31-1-1 cell line.

Cytotoxicity, alkali-labile DNA lesions, ouabain resistance mutations, and neoplastic transformation were analyzed concurrently in the BALB/3T3 ClA31 -1-1 cell line treated with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for different exposure times (15, 30, 60, 90, 120, and 240 min; 24, 48, and 72 hr). The half-life of MNNG in complete medium was approximately 70 min, both without cells and with cell numbers as used in the assays for cytotoxicity (2 X 10(2) cells/60-mm dish), transformation (1 X 10(4) cells/dish), and mutation (1 X 10(5) cells/dish). The cytotoxic effect of MNNG (0.5 or 2 micrograms/ml) appeared to be completed after an exposure time between 100 and 200 min. Maximal frequency of ouabain resistance mutations, however, was reached after a much shorter treatment time (30 to 60 min). Detection of DNA damage by alkaline elution analysis showed maximal increase in single-strand breaks already after treatment for 30 min. Exposures for 30 min followed by posttreatment incubation for 30 or 90 min showed active repair of single-strand breaks during these periods, indicative of an even balance between the additional MNNG-induced damage and its repair. Morphological transformation assays, at the same treatment times and concentrations used in the mutation assays, yielded frequency curves that reached their maxima 1 to 3 hr later than did the mutation frequencies. The ratio of transformation to ouabain resistance mutation frequencies was 3.7 for short treatment times (30 to 60 min), while it increased to more than 20 for exposure times of 240 min or longer. The temporal dissociation in the exposure times for maximal induction of mutation and transformation, observed with MNNG in this cell line, supports the hypothesis that a single gene mutational event is not sufficient to account for the full expression of neoplastic transformation.

Animals↗
PubMed1983

DNA repair induction by cytostatic drugs in proliferating and quiescent MRC-5 cells.

The repair of DNA damage is closely related to cell cycle, in terms of both modulation of repairing ability throughout the cell cycle and perturbations in replication. We have studied the induction of Unscheduled DNA Synthesis (UDS) in human fibroblasts (MRC-5) by some chemotherapeutic agents, selected for their different mechanisms of action. To take into account the interaction between repair and replication systems, the experiments were performed in both proliferating and quiescent cultures; semiconservative DNA synthesis inhibition was examined by microphotometric measurements in the same autoradiographic preparations used for UDS analysis. Vincristine, methotrexate and 6-thioguanine induced no UDS. Adriamycin, actinomycin D, and, to a lesser extent, cyclophosphamide and thiotepa gave positive results only in proliferating cultures, whereas bleomycin was an effective inducer of UDS in quiescent cells, with weak UDS levels in proliferating ones. In all these cases, the combined analysis of UDS and semiconservative DNA synthesis inhibition proved to be of value in the assessment of interaction between drugs and DNA in proliferating cultures. The results of our experiments emphasize the importance of the cycling conditions in the cellular response to DNA damage.

Antineoplastic Agents↗
PubMed1982

Molecular dosimetry of DNA damage caused by alkylation. I. Single-strand breaks induced by ethylating agents in cultured mammalian cells in relation to survival.

Cultured Chinese hamster ovary cells were treated with ethylating agents. DNA lesions giving rise to single-strand breaks (ssb) or alkali-labile sites were measured by centrifugation in alkaline sucrose gradients after lysis in alkali. 4 agents with different tendencies to ethylate preferentially either at N or O atoms were compared, namely N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), N-ethyl-N-nitrosourea (ENU), ethyl methanesulphonate (EMS) and diethyl sulphonate (DES). The compounds differed greatly in their potency to induce the lesions measured when compared on a molar basis, but comparison at equicytotoxic doses showed relatively small differences. Upon prolonged incubation of the DNA in alkali, the number of ssb increased considerably. DNA from untreated cells showed biphasic kinetics: slow ssb formation for about 10 h, then the rate increased and remained constant for up to 40 h. Treated cells showed an accelerated, dose-dependent linear generation of ssb for 10 h, followed by a short plateau; then ssb were formed again at a constant rate, somewhat higher than that in controls. Ssb formed in the initial phase are ascribed to phosphotriester hydrolysis, those after the plateau to unidentified causes. Zero intercepts appeared to be a measure of apurinic sites generated intracellularly. A 24-h repair period preceding lysis reduced the ENNG intercept, but not that of DES. Rapid degradation of DES during the 1-h treatment occurred, so most "apurinic-site lesions" were induced in the beginning of exposure and possibly were already repaired at the end. The types of lesion distinguished (reparable and non-reparable apurinic sites, phosphotriesters) appeared of little consequence for cell survival.

Alkylating Agents↗
PubMed1979

Mutational studies with diquat and paraquat in vitro.

Diquat and paraquat were assayed in the following tests. (1) Ames test in Salmonella typhimurium (strains TA1535, TA1537, TA1538, TA98 and TA100) with and without rat-liver microsomal fractions. (2) Resistance to 8-azaguanine in Salmonella typhimurium (strain hisG46, TA92 and TA1535. (3) Repair test in Salmonella typhimurium (strains TA1538 and TA1978). (4) Gene mutations in Aspergillus nidulans: 8-AG resistance and methionine suppression (meth A1 locus). (5) Lethal recessive damage in Aspergillus nidulans. (6) Unscheduled DNA synthesis (UDS) in human epithelial-like cells (EUE). Diquat and paraquat were positive in S. typhimurium (in the repair test and the 8-AG resistance system), in A. nidulans (for gene mutations and lethal recessive damage induction) and in EUE cells (UDS induction).

Animals↗
PubMedDate not supplied

Construction of a shuttle vector containing a single O6-methylguanine: a probe for mutagenesis in mammalian cells.

A shuttle vector, pKE15, was constructed for investigating the mechanisms by which single carcinogen-DNA adducts induce mutations in mammalian cells. pKE15 contains the SV40 origin of replication, the neomycin resistance gene, SV40 polyadenylation sequences and the pML2 origin of replication. Transfection of pKE15 into CHO cells established the G418-resistant phenotype; the frequency of G418-resistant clones was approximately 10(-4), a value that is similar to those obtained with other SV40-based vectors expressing the neomycin resistance gene. A tetranucleotide containing O6-methylguanine, a DNA adduct formed by carcinogenic alkylating agents, was incorporated into a 4-base gap positioned in the center of a PstI site. The tetranucleotide containing the adduct was physically mapped to a 14-base-pair region of the shuttle vector that included the ligation target, the PstI site. It was incorporated approximately equally into either of the complementary strands of the shuttle vector. The ligation efficiency of the tetranucleotide into the gapped genome was approximately 100% and was independent of the concentration of tetranucleotide used at concentrations ranging over one order of magnitude. The potential applications of the site-specifically modified genome for establishing the mutagenic fate of O6-methylguanine in repair-proficient and -deficient CHO cells are discussed.

Alkylation↗
PubMedDate not supplied

Molecular dosimetry of DNA damage caused by alkylation. II. The induction and repair of different classes of single-strand breaks in cultured mammalian cells treated with ethylating agents.

Cultured Chinese hamster ovary cells were treated with ethylating agents. DNA lesions giving rise to single-strand breaks (SSB) or alkali-labile sites were measured by elution through membrane filters at pH 12.0 and pH 12.6, and by centrifugation in alkaline sucrose gradients after 1 h and 21 h lysis in alkali. Two agents with different tendencies to ethylate preferentially either at N or O atoms were compared, namely N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and diethyl sulphate (DES). The compounds differed greatly in their potency to induce lesions, but the ratios of SSB, measured with different methods after a treatment for 30 min, did not differ significantly. This suggested that the spectrum of lesions induced by the two compounds is very similar. However, when both agents were studied with alkaline elution at pH 12.0 after a short treatment time (5 min) only ENNG was found to induce rapidly-repairable SSB. Most of these were rejoined already within 5 min after treatment. These results suggest that rapidly-repairable lesions occurring in DNA after treatment of mammalian cells with ethylating agents are due mainly to alkylation at O-atoms.

Alkylation↗
PubMedDate not supplied

Split-dose exposure to N-methyl-N'-nitro-N-nitrosoguanidine in BALB/3T3 C1 a31-1-1 cells: evidence of DNA repair by alkaline elution without changes in cell survival, mutation and transformation rates.

Dose fractionation of a direct-acting chemical carcinogen, the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), was studied for its concurrent effects on survival, DNA damage and repair, ouabain resistance (Ouar) mutations and neoplastic transformation, in the mouse embryo cell line BALB/3T3 C1A31-1-1. MNNG doses of 0.5, 1 and 2 micrograms/ml were added to the cells either as a single exposure or in two equal fractions separated by 1, 3 or 5 h intervals. No significant difference in cytotoxicity was found when single and split-dose treatments were compared. No recovery from sublethal damage was therefore found in this cell line by split-dose administration of MNNG, although such an effect was found when the same cell line was treated with single and split doses of X-rays. Repair of DNA damage as measured by alkaline elution was studied up to 24 h after a single MNNG exposure (0.5 micrograms/ml). DNA repair was rapid during the first 5 h after treatment and slow thereafter. DNA damage detected after split doses of MNNG at 1 and 5 h intervals was significantly lower than after a corresponding single dose. With both single and split doses, rejoining of single-strand breaks (ssb) was nearly complete after 24 h of repair time. Ouar mutation and neoplastic transformation frequencies were determined for single and split doses of MNNG with the second treatment being given during (1 h) or after (5 h) the period of rapid DNA repair. No significant differences in either effect were detected for dose splitting at any tested dose.

Animals↗