A Calcagnile

Istituto Superiore di Sanità, Roma, Latium, Italy

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Publications (40)172.82 Total impact

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    ABSTRACT: The aldehyde reagent methoxyamine is able to interact with apurinic/apyrimidinic sites formed in vivo within cells and displays both an anti-cytotoxic and an antimutagenic activity on N-ethyl-N'-nitro-N-nitrosoguanidine-induced DNA damage in Chinese hamster ovary cells. To clarify the underlying mechanism we have examined the mutational spectra induced by N-ethyl-N'-nitro-N-nitrosoguanidine alone and in the presence of methoxyamine in the hypoxanthine-guanine phosphoribosyltransferase gene of Chinese hamster ovary cells. In both cases all mutations were base pair substitutions, and their distribution among various classes did not differ significantly. Almost 60% were transitions, predominantly GC to AT, and the remaining 40% were transversions, mainly at AT base pairs. The analysis of the proportion of the different types of mutations showed that in the presence of methoxyamine, GC to AT transitions decreased by a factor of 1.8, and AT to CG transversions were reduced by a factor of 13. These data indicate that in mammalian cells the fixation of ethylation damage into mutations occurs by both (a) direct mutagenesis likely driven by O6-ethylguanine adducts and to a minor extent by O4-ethylthymine and (b) apurinic/apyrimidinic site-mediated mutagenesis. These apurinic/apyrimidinic sites are formed during the processing of ethylation at critical sites and are likely to involve O6-ethylguanine and O2-ethylthymine adducts.
    Cancer Research 04/1993; 53(5):1149-55. · 9.28 Impact Factor
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    ABSTRACT: In a previous study we showed that the formation of O6-ethylguanine (O6-EtGua) in the DNA of CHO cells in culture correlated with mutations induced by ethylnitrosourea (ENU) and diethylsulfate (DES) at the hypoxanthine-guanine-phosphoribosyltransferase (hprt) locus but not at the Na, K-ATPase locus. This study was extended to another ethylating agent, ethyl methanesulfonate (EMS). DNA adduct formation and induction of mutation at the two gene loci were determined simultaneously in CHO cells after EMS exposure. The extent of ethylation at the N7 and O6 positions of guanine and at the N3 site of adenine were measured and the possible correlations with 6-thioguanine resistance (6-TGr) and ouabain resistance (ouar) mutations were investigated. A good correlation between the levels of ethylation at O6 guanine and mutation frequency at hprt gene by all three ethylating agents was observed. In the case of the ouar locus, the frequency of O6-EtGua adducts correlated with mutation induction by EMS and ENU but not by DES. Although both EMS and DES have similar reaction mechanisms, these results highlight differences in their mutational specificity. The comparison of this type of analysis with mutational spectra revealed that correlation studies may be inadequate to analyse multicomponent phenomena like mutation formation.
    Environmental and Molecular Mutagenesis 01/1993; 21(2):154-9. DOI:10.1002/em.2850210209 · 2.55 Impact Factor
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    ABSTRACT: In this study we addressed the question as to whether the mutagenesis by methylating agents is affected by the transcriptional activity of the damaged gene. An Epstein-Barr virus (EBV)-derived shuttle vector system was developed where the genetic target for mutation analysis, the bacterial gpt gene, is under the control of an eukaryotic inducible promoter in plasmid pF1-EBV and lacks the eukaryotic promoter in plasmid pF2-EBV. Two human cell lines that episomically maintain these shuttle vectors were established. In clone 6NT cells, which contain pF1-EBV plasmid, the gpt gene is actively transcribed and the transcription rate is regulated by zinc ions. In clone 3 cells, which harbor pF2-EBV plasmid, the gpt gene is not transcribed. Following treatment of both cell lines with the potent alkylating carcinogen N-methyl-N-nitrosourea (MNU), G · C to A · T transitions were the major mutagenic event, consistent with the miscoding potential of O6-methylguanine. The mutations were predominantly generated in the non-transcribed DNA strand of the active gpt gene. The same strand-bias was observed when the gpt gene was transcriptionally inactive, indicating that MNU-induced strand-specific formation of mutations is not due to transcription. Our data identify as major determinants of this phenomenon the sequence-specificity of MNU mutagenesis and the conformational properties of the target protein. Differences in mutation distribution were observed between the transcriptionally active and inactive gpt gene. This finding suggests that the organization of active genes in chromatin might modulate DNA alkylation and/or DNA repair.
    Journal of Molecular Biology 09/1992; 226(3):909. DOI:10.1016/0022-2836(92)90974-O · 3.96 Impact Factor
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    ABSTRACT: The biological effects of the interaction of methoxyamine (MX) with apurinic/apyrimidinic (AP) sites produced in CHO cells by treatment with alkylating agents were examined. A decrease in cytotoxicity was observed after a 10 min treatment with the SN1 alkylating agents ethylnitrosourea (ENU), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and N-methyl-nitrosourea when MX was present in the culture medium. Furthermore MX reduced the number of mutations to 6-thioguanine resistance induced by ENU and ENNG and the number of sister chromatid exchanges induced by ENU. In contrast, no protective effect of MX on survival was observed after a 10 min treatment with the SN2 alkylating agents diethylsulfate (DES), ethyl methane sulfonate and methyl methane sulfonate. A 3 h exposure to MX abolished the protective effect of MX on ENU-induced cytotoxicity and increased the cytotoxicity of DES. In vitro studies with synthetic oligonucleotides containing a single AP site opposite a normal guanine or O6-methylguanine showed that MX inhibits the cleavage of AP sites by the CHO AP endonuclease(s). A model is proposed in which different DNA lesions are involved in AP site formation after treatment with SN2 or SN2 alkylating agents. The involvement of specific alkylation products in cytotoxicity and mutagenesis is also discussed.
    Carcinogenesis 02/1992; 13(1):87-93. DOI:10.1093/carcin/13.1.87 · 5.27 Impact Factor
  • Mutation Research/Environmental Mutagenesis and Related Subjects 04/1991; 252(2):208. DOI:10.1016/0165-1161(91)90100-M
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    ABSTRACT: The induction of unscheduled DNA synthesis (UDS) and the alteration of semiconservative DNA replication by the structurally related intercalating agents proflavine and 9-aminoacridine were studied in MRC-5 human fibroblasts in culture. Autoradiographic determinations of both parameters were carried out simultaneously in the same culture specimens. Proflavine affected DNA synthesis, but did not elicit any UDS. 9-Aminoacridine inhibited DNA synthesis only at the highest concentration and caused UDS to a low but significant extent. These results suggest that the ability to induce UDS is not a general property of the intercalating agents and that the alterations of the DNA structure, typical of the "pure" intercalative process, are not handled by pathways involving unscheduled synthesis.
    Journal of Toxicology and Environmental Health 11/1990; 31(2):117-24. DOI:10.1080/15287399009531441 · 1.81 Impact Factor
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    ABSTRACT: 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.
    Annali dell'Istituto superiore di sanita 02/1989; 25(1):51-5. · 0.77 Impact Factor
  • F Palombo, A Calcagnile, E Dogliotti
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    ABSTRACT: The use of shuttle vectors has been applied in recent years to develop a better understanding of the molecular mechanisms of mutagenesis in mammalian cells. These recombinant DNA molecules replicate together with the host eukaryotic cells and can be retrieved in bacteria for rapid detection and analysis of mutation. Two approaches based on the use of shuttle vectors for studying the mutagenic effects of DNA lesions induced by alkylating agents are presented.
    Annali dell'Istituto superiore di sanita 02/1989; 25(4):557-61. · 0.77 Impact Factor
  • Mutation Research/Environmental Mutagenesis and Related Subjects 06/1988; 203(3):218. DOI:10.1016/0165-1161(88)90141-0
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    ABSTRACT: 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.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 02/1988; 193(1):43-51. DOI:10.1016/0167-8817(88)90006-5 · 4.44 Impact Factor
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    ABSTRACT: 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.
    Carcinogenesis 11/1987; 8(10):1417-21. · 5.27 Impact Factor
  • Carcinogenesis 10/1987; 8(10):1417-1421. DOI:10.1093/carcin/8.10.1417 · 5.27 Impact Factor
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    ABSTRACT: 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.
    Carcinogenesis 02/1987; 8(1):25-31. DOI:10.1093/carcin/8.1.25 · 5.27 Impact Factor
  • Mutation Research/Environmental Mutagenesis and Related Subjects 10/1985; 147(5):286. DOI:10.1016/0165-1161(85)90139-6
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    ABSTRACT: DNA-repair ability was estimated in a group of vulcanizers by measuring in vitro UV-induced unscheduled. DNA synthesis (UDS) in peripheral lymphocytes, and compared with that of an adequate control group. A considerable interindividual variability was shown by the UDS responses of the subjects studied both in the control and exposed population. Significantly (P = 0.0158) decreased UDS values were observed among the vulcanizers as compared to the referents. Neither age nor cigarette-smoking was observed to affect the UDS response, thus suggesting an association between the industrial exposure and decrease in the DNA-repair rate.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 11/1984; 138(1):105-11. DOI:10.1016/0165-1218(84)90092-2 · 4.44 Impact Factor
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    ABSTRACT: Two structurally related organophosphorous insecticides, dichlorvos and trichlorfon, and their main metabolite dichloroacetaldehyde, were assayed for their ability to induce DNA repair, detected as unscheduled DNA synthesis in human epithelial-like cell (EUE) cultures in vitro. A dose-response relationship was found for treatment with the two pesticides, but negative results were obtained with dichloroacetaldehyde. Tests for induction of gene mutation, as ouabain resistance in Chinese hamster cells (V79), failed to show any mutagenic activity by these compounds.
    Pesticide Science 10/1984; 15(5):439 - 442. DOI:10.1002/ps.2780150504
  • Mutation Research/Environmental Mutagenesis and Related Subjects 06/1984; 130(3):246-246. DOI:10.1016/0165-1161(84)90277-2
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    ABSTRACT: 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.
    Teratogenesis Carcinogenesis and Mutagenesis 02/1983; 3(6):481-90. DOI:10.1002/1520-6866(1990)3:63.0.CO;2-9
  • Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 04/1982; 103(3-6):385-90. DOI:10.1016/0165-7992(82)90072-0 · 4.44 Impact Factor
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    ABSTRACT: The aldehyde reagent methoxyamine is able to interact with apurinic/ apyrimidinic sites formed in vivo within cells and displays both an anti- cytotoxic and an antimutagenic activity on ,V-e(hyl-/\"-nitro-,V-ni- trosoguanidine-induced DNA damage in Chinese hamster ovary cells. To clarify the underlying mechanism we have examined the mutational spec tra induced by A'-ethyl-W-nitro-A'-nitrosoguanidine alone and in the pres ence of methoxyamine in the hypoxanthine-guanine phosphoribosyltrans- ferase gene of Chinese hamster ovary cells. In both cases all mutations were base pair substitutions, and their distribution among various classes did not differ significantly. Almost 60% were transitions, predominantly GC to AT, and the remaining 40% were transversions, mainly at AT base pairs. The analysis of the proportion of the different types of mutations showed that in the presence of methoxyamine, GC to AT transitions de creased by a factor of 1.8, and AT to CG transversions were reduced by a factor of 13. These data indicate that in mammalian cells the fixation of ethylation damage into mutations occurs by both (a) direct mutagenesis likely driven by 06-ethylguanine adducts and to a minor extent by (>4- ethylthymine and il»apurinic/apyrimidinic site-mediated mutagenesis. These apurinic/apyrimidinic sites are formed during the processing of ethylation at critical sites and are likely to involve 06-ethylguanine and O2-ethylthymine adducts.

Publication Stats

732 Citations
172.82 Total Impact Points

Institutions

  • 1982–2012
    • Istituto Superiore di Sanità
      • Department of Environment and Primary Prevention
      Roma, Latium, Italy
  • 2004
    • Tuscia University
      Viterbo, Latium, Italy
  • 1996
    • Istituto Dermopatico dell'Immacolata
      Roma, Latium, Italy
  • 1988
    • TNO
      Delft, South Holland, Netherlands