Mutation Research, 291(1993) 217-222 0 1993 Elsevier Science Publishers B.V. All rights
First Annual Meeting of the Italian Section of the European Environmental Mutagen Society (SIMA) Silvio De Flora Institute of Hygiene and Preuentive Medicine, Universiry of Genoa, Genoa, Italy (Received (Accepted
of the European
The First Annual Meeting of the Italian Section of EEMS (SIMA, Societa Italiana di Mutagenesi Ambientale) was held in Pisa on 7-8 October 1992 at the ‘Palazzo dei Congress?. SIMA was founded on 5 October 1991 in AIghero, where Nicola Loprieno had been elected President, Angelo Carere Vice-President, Angelo Abbondandolo, Roberto Barale, Giorgio Cantelli-Forti, Luigi De Carli, Silvio De Flora and Angelo G. Levis members of the Directive Council, and Francesca Pacchierotti Secretary-Treasurer. One of the first tasks of the Directive Committee, which had been scheduled to remain in charge until the First Annual Meeting, was to make a review of the applications for membership, to be updated at each annual meeting. Based on rather selective criteria of the curricula submitted, especially concerning the scientific production in internationally reputable journals, 123 members were accepted as members of SIMA at the end of the starting year of activity. In his presidential address to the General Assembly in Pisa, Nicola Loprieno (University of Piss) stressed that the foundation of SIMA was a
Correspondence: Prof. e Medicina Preventiva, I-16132 Genoa, Italy.
29 January 29 January
Dr. S. De Flora, Istituto di Igiene Universita di Genova, Via Pastore 1,
formal act, aimed at officializing in a national section the contribution given by Italian investigators in the framework of EEMS and IAEMS during the past two decades. The scientific program of the meeting was introduced by Angelo Abbondandolo (IST and University of Genoa) and by Giorgio Bronzetti (CNR, Piss), who successfully acted as the chairman of the local Organizing Committee and took care of the preparation of the proceedings including the abstracts of all oral and poster presentations. Mechanisms mutations
of genie, chromosomic
The first day of the meeting was entirely devoted to a symposium on the general theme of mechanisms of mutation. The morning session, which was chaired by Stefania Bonatti (CNR, Pisa) and Angelo Abbondandolo, included five invited lectures. David M. DeMarini (U.S. Environmental Protection Agency, Research Triangle Park, NC, USA), a scientist of Italian origin, gave a quite interesting presentation dealing with the mutation spectra and molecular mechanisms of mutations induced by urban air and other complex
mixtures at the hisD3052 allele of Salmonella typhimurium. Using colony probe hybridization to detect a common hotspot 2-base GC deletion, followed by PCR and DNA sequencing, nearly 200 different types of mutations were detected in the 75-base target for reversion. Ten mutation spectra were distinctively induced, probably as a consequence of the dominance of particular chemical classes, by three types of complex mixtures, i.e., urban air, cigarette smoke condensate, and municipal waste incinerator emissions. In addition to the hotspot mutations, these mixtures induced a small deletion and a base substitution, suggesting a mechanism involving misinsertion of a base opposite a DNA adduct, followed by a slippage/ mismatch. The assessment of exposure to low doses of mutagens, which represents one of the most classic issues of radiobiology and in general of mutagenesis applied to the human population, was discussed by Gregorio Olivieri (University of Rome). Several uncertainties still exist in this domain, since the direct analysis of the action of low doses in experimental systems is difficult and expensive, and extrapolation of effects from high doses is controversial and questionable. Particular emphasis was given to the sensitive induction of a network of enzymatic systems resulting from an altered expression of several genes in the organism exposed to low doses of mutagens. Angelo Abbondandolo (IST and University of Genoa) highlighted the mechanisms of action of alkylating agents, which have been of invaluable help in mutation research for half a century. While their role in the induction of gene mutations is well delineated, it is still unclear how alkylating agents can cause aneuploidy. He mainly concentrated on several lines of evidence supporting the importance of targets other than DNA in mammalian cells. In particular, the possible involvement of purine nucleotide pools in the aneugenic activity of alkylating agents was analyzed, as assessed both by literature data and by personal experimental work. After a general introduction on the role of QSAR models in risk assessment, Romualdo Benigni (Istituto Superiore di Sanita, Rome) discussed the relationships between chemical structure and aneugenic activity of 35 chlorinated
aliphatic hydrocarbons, as inferred from a database in Aspergillus nidulans presented by Riccardo Crebelli from the same institution. Steric factors, described by molar refractivity, were mainly responsible for the toxic effects of these compounds, whereas electronic factors were related to aneuploidy. The specific QSAR equations predicted the activity of six additional congeneric chemicals consistently with new experimental data. The morning session was concluded by Italo Barrai (University of Ferrara), who covered aspects of population genetics and in particular illustrated models evaluating the genetic load in humans, defined as the pool of recessive damaging events, resulting from either mutational or segregational mechanisms, which affect the common genetic background. He also provided examples of epidemiologic models, aimed at distinguishing the relative contribution of genetic and environmental factors to the frequency of biological phenomena. Oral communications
The afternoon session was chaired by Silvana Castellino (Farmitalia Carlo Erba, Milan) and Roberto Barale (University of Ferrara). It included an intensive sequence of 18 oral communications, where not only experienced speakers but also younger scientists had the opportunity to present new stimulating data on the general subject of mutation mechanisms. Paola Fortini (Istituto Superiore di Sanita, Rome) reported the results of a collaborative study with the University of Leiden (The Nederlands) on the mutagenic processing of ethylation damage in mammalian cells, involving in particular the use of methoxyamine to study the apurinic/ apyrimidinic sites formed during the processing of DNA ethylation by ENNG in the hprt gene of CHO cells. Gilbert0 Fronza (IST, Genoa) analyzed the mutation spectrum induced by acetoxy-4-aminoquinoline l-oxide, the ultimate metabolite of 4NQ0, in the DNA of a single-stranded shuttle vector (plasmid pZ1891 transfected into CVl monkey cells. As assessed in collaboration with CNRS (Villejuif, France), the lesions on ssDNA were found to be identical in eukaryotes and prokaryotes. Two collaborative studies between the Isti-
tuto Superiore di Sanitl (Rome) and the Imperial Cancer Research Fund (South Mimms, UK) were presented by Gabriele Aquilina and Margherita Bignami. In the first one, evidence was provided that resistance to the alkylating agents MNU and MNNG is associated with a defect in a DNA mismatch binding protein and the mutator phenotype. The second study dealt with an SV40based assay for in vitro replication by HeLa cellfree extracts, showing that 06-methylguanine inhibited DNA replication. Tamara Basic-Zaninovic (Istituto Superiore di Sanita, Rome, and University of Zagreb, Croatia) investigated the fidelity of replication of the leading and lagging DNA strands opposite MNU-induced DNA damage, using human cells harbouring an EBV-derived shuttle vector. The general role of poly(ADPribose) polymerase activity as a physiological mechanism of response to DNA damage was elucidated by Linda Scarabelli (University of Genoa), who also discussed the relationships between induction of DNA strand breaks and endogenous levels of pADPRP activity in the liver of rats treated with the pesticides Zineb and Ziram. Paola Vagnarelli (University of Pavia) developed a mutation assay in V79 cells treated with nitrosoguanidine, based on the direct analysis of DNA by minisatellite probes. Studies on the antimutagenic activity of cinnamaldehyde in strain D7 of Saccharomyces cerevisiue were described by Clara Della Croce (CNR, Pisa); a significant decrease of UV-induced mutations was observed in mutants defective in excision repair, which may result in a longer persistence of the dimer in the DNA helix. After a short break, which was felt to be necessary by all attendants in order to improve their resistance to mutation mechanisms, the second set of oral presentations was started by Franca Majone (University of Padua). She reported collaborative studies with NIH (Bethesda, MD, USA), showing the role of the HTLV-1 transactivating protein Tax in the induction of micronuclei, resulting from both clastogenic and aneuploidizing effects in COS cells. Cecilia Betti (University of Pisa) demonstrated the ability of methylmercury chloride to induce structural and numerical chromosomal aberrations in human lymphocytes, even after liquid holding in phase
G,. The frequency of chromosomal aberrations was evaluated by Cecilia Tiveron (ENEA, Rome) at the first-cleavage metaphase of mouse eggs fertilized in vivo with sperm of chlorambuciltreated males; the results confirmed the highly specific responsiveness of early spermatids to the induction of chromosomal lesions by this compound. As reported by Lucia Migliore (University of Pisa), none of 14 hydrogenated isoindolonic compounds compared favorably to the structurally related cytochalasin B in blocking cytodieresis in cultured human lymphocytes, when assayed according to the micronucleus test procedure. Giorgio Bronzetti (CNR, Pisa) provided evidence that 5-methoxypsoralen (5-MOP) is metabolized by yeast cells of Succharomyces cerevisiue, and that its metabolites, purified by HPLC, are less genotoxic than the parent compound. In addition, 5-MOP was found to induce cytochrome P450 at the transcriptional level in S. cerevisiue D7 cells grown in logarithmic phase. Pasquale Mossesso (University of Viterbo) investigated the interindividual variability in the induction of chromatid aberrations in human lymphocytes treated in phase G, with camptothecin, an inhibitor of topoisomerase I. The preliminary results of a population monitoring program, discussed by Stefano Landi (University of Pisa), showed a considerable interindividual variability in the in vitro induction of SCE by diepoxybutane in human lymphocytes, according to a procedure which is commonly utilized in the diagnosis of Fanconi anemia. A study presented by Emanuela Dorigo (University of Padua) led to the conclusion that preclastogenic lesions induced by mitomycin C and benzo[a]pyrene, as assessed by the micronucleus test in peripheral blood reticulocytes of mice, can persist after several cell divisions. Luigina Renzi (University of Padua, in collaboration with the Finnish Center for Radiation and Nuclear Safety and the Institute of Occupational Health, Helsinki, Finland) applied both in situ hybridization with DNA probes and antikinetochore antibody staining for the detection of the kinetochore region. Carla Gambina (CNR, Pisa) dealt with gene targeting techniques exploiting homolog recombination mechanisms in order to introduce gene-specific mutations into mammalian cells.
Perspectives in environmental mutagenesis The second day of the meeting was opened, under the chairmanships of Angelo G. Levis (University of Padua), by a lecture covering mutagenesis in somatic cells, chronic degenerative diseases and their prevention, which was given by Silvio De Flora (University of Genoa). After having analyzed their epidemiologic impact, he commented on some common features and mechanisms of degenerative diseases, such as their multiplicity, multifactorial origin and multistep pathogenesis, the influence of cell proliferation, the length of the latency period and the exponentially increasing prevalence with age. Mutations in the DNA of somatic cells, either nuclear or mitochondrial, may play a fundamental role not only in carcinogenesis but also in the pathogenesis of cardiovascular diseases, dismetabolic conditions, neurologic disorders, and aging phenomena. Biomonitoring techniques provide evidence that environmental genotoxins can produce DNA damage in different organs, bearing a distinctive pathological meaning, and that chemopreventive agents can trigger a broad spectrum of protection. Studies are in progress on the involvement of molecular and biochemical changes in cardiomyopathies, arteriosclerosis and diabetes in both experimental animals and humans. This lecture was followed by a visit to the posters, which covered a variety of areas related to environmental mutagenesis. Poster presentations were divided into three general areas, and each of them was highlighted by one or two coordinators with the contribution of the authors and discussants from the audience. For the sake of conformity with the abstract book, each presentation will be identified with the first author of the abstract, although in some cases this did not coincide with the main discussant of the group. Poster session on mechanisms and methods in eukaryotes This session was coordinated by Miria Stefanini (CNR, Pavia), according to three main tracks. A series of posters dealt with the evaluation of genotoxic effects. In cultured human lym-
phocytes treated with two diastereoisomeric metabolites of benzo[a]pyrene, anti-BPDE was more toxic and mutagenic at the hgprt locus than syn-BPDE (Nicola Zanesi et al., University of Padua); in addition, anti-BPDE produced more DNA adducts and induced a greater DNA repair CUDS), whereas DNA breaks (FADU) were of similar magnitude for the two diastereoisomers (Paola Ferraro et al., University of Padua). In lymphocytes of patients affected by psoriasis, treatment with mineral tar affected neither benzo[a]pyrene metabolism nor formation of BPDE-DNA adducts (Sofia Pavane110 et al., University of Padua). The micronucleus test in bone marrow erythrocytes of mice treated in vivo was negative with the pesticide Ridomil and its components Mancozeb and Matalaxil (Livia Bianchi et al., University of Pavia), whereas the same technique showed a moderate clastogenicity and a spindle-type aneuploidizing activity of three vanadium salts (Rosalba Ciranni et al., Universities of Pisa and Ferrara); the SMART test in Drosophila melanogaster larvae treated with benzo[a]pyrene correlated with the formation of BPDE-DNA adducts, whose chromosomal localization was investigated (Mauro Zordan et al., University of Padua). The optimization of the experimental approach was pursued by studies concerning the micronucleus test coupled with the detection of the kinetochore region either by means of in situ hybridization with DNA probes (Francesca Lo Jacono et al., University of Pisa) or by antikinetochore antibody staining (Corrado Ferri et al., ENEA and University of Rome). Other methodological presentations covered the cytogenetic analysis of peripheral blood human lymphocytes (Paola Prati et al., Universities of Pisa and Ferrara) and the SCE analysis of mutagens requiring metabolic activation in two mouse embryo liver cell lines (Paolo Guidotti et al., Universities of Pisa and Ferrara; CNR, Pisa). Poster presentations investigating fundamental mechanisms provided evidence that the mating type influences DNA repair and mutability in S. cerevisiae (Alvaro Galli et al., CNR, Pisa); DNA topoisomerases do not play a primary role in excision repair in Chinese hamster ovary CHO cells (Guide Frosina and Ottavio Rossi, IST,
Genoa); a significant increase in the frequency of CREST-positive micronuclei and chromosome fragments containing amplified material occurs in hamster cells with amplified CAD or DHFR genes, thereby suggesting that amplification is a destabilizing factor for both segregation and structural integrity of chromosomes (Stefania Bonatti et al., IST and University of Genoa; CNR, Pisa). Poster session on metabolism the genotoxic response
This session, co-chaired by Patrizia Hrelia and Moreno Paolini (University of Bologna), included poster presentations on the following topics: modulation of several enzyme activities in the liver of rats treated with the mutagen 3-chloro4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), isolated from chlorinated water (Silvano Monarca et al., Universities of Brescia and Perugia; U.S. Environmental Protection Agency, Cincinnati, OH, USA); induction of P450IIBI isoforms in rodents treated with Fenarimol, and an antagonistic effect of this fungicide towards the in vivo clastogenicity of trichloroethylene (Francesca Maffei et al., University of Bologna); lack of mutagenicity of the fungicide Zineb in the Ames Salmonella test, and its genotoxicity in logarithmically growing S. cerevisiae D7 cells, an activity which was inhibited by chlorophyllin (Elena Morichetti et al., CNR and University of Pisa); ability of the polyamine spermine to inhibit the mutagenicity of EMS and MMS, whereas diallylsulfide inhibited the genotoxicity of DMNA at the hgprt locus in mammalian cultured cells (Roberto Fiorio et al., CNR, Pisa); selective formation of BPDE-DNA adducts in pulmonary alveolar macrophages of smokers, and prevention by oral N-acetylcysteine of molecular, cytogenetic, histopathologic and metabolic alterations in various tissues of rats exposed to cigarette smoke (Albert0 Izzotti et al., University of Genoa and Institute G. Gaslini, Genoa; Centre of Oncology, Sofia, Bulgaria); increase of the urinary excretion of thioethers and modulation of several liver enzyme activities in rats treated with the pyrethroid insecticide Deltametrin (Giuseppina Scassellati Sforzolini et al., Universities of Peru-
gia and Brescia); interaction between three benzene metabolites, i.e., hydroquinone, phenol and catechol, in the in vivo induction of micronuclei in mouse bone marrow erythrocytes (Antonella Marazzini et al., Universities of Pisa and Ferrara); relationships between induction of the cytochromes P450IA1, IA2, IIEl, IIIA and IVA and formation of superoxide anion, as assessed by EPR spectroscopy in purified mouse liver microsomes and in a continuous line of mouse hepatocytes (Laura Pozzetti et al., University of Bologna). Poster session
The last poster session, coordinated by Antonio De Marco (CNR, Rome), was devoted to monitoring of environmental agents in a variety of genotoxicity test systems. Among metal compounds, ammonium metavanadate inhibited cytochrome P450 in purified yeast microsomes and decreased the specific mRNA levels (Renata Del Carratore et al., CNR, Pisa), and methylmercury chloride was more efficient than dimethylmercury in inducing DNA damage in cultured human lymphocytes and rat lymphocytes, liver, and gastric mucosa (Cecilia Betti et al., Universities of Pisa and Ferrara; Federal Research Centre for Nutrition, Karlsruhe, Germany). The food dye erythrosine, a suspected rodent carcinogen, failed to enhance the frequencies of SCE in peripheral blood lymphocytes and of micronuclei in reticulocytes of mice (Andrea Zijno et al., Istituto Superiore di SanitB, Rome). Three presentations dealt with humic acids from either natural or anthropogenic sources: those derived from coal manufacture induced base-pair substitutions in S. typhimurium only following chlorination, while a sample inducing chromosomal aberrations in cultured human lymphocytes lost its clastogenicity following chlorination (Francesca Bernacchi et al., Universities of Pisa and Ferrara; Eniricerche, Rome); likewise, chlorinated humic acid was mutagenic in both S. typhimurium and E. coli, and induced SOS response in E. co/i, but under field conditions chlorination of water was devoid of genotoxic effects (Paola Venier et al., University of Padua); humic and fulvic acids in soil protected Kciu fuba
against the induction of micronuclei by the herbicide maleic hydrazide (Antonio De Marco and Claudio De Simone, CNR, Rome, and Istituto Sperimentale Studio e Difesa Suolo, Rieti). Two studies addressed the problem of monitoring the marine environment by using Myrilus guZloprovincialis as a target for genotoxic agents; isolated mytilus gills, used in vitro, were as sensitive to the induction of micronuclei by mitomycin C as following in vivo exposure of the whole organism (Marco Gabriele et al., University of Padua); treatment of mytilus with benzo[a]pyrene resulted in the formation of BPDE-DNA adducts, as assessed by 32P postlabeling (Sofia Pavane110 et al., University of Padua). The mutagenic monitoring of atmospheric particulate was carried out in four studies. Air samplings in four Italian towns (Turin, Rome, Naples and Syracuse) showed mutagenic responses in S. typhimurium TA98 and TA98/1,&DNP,, mainly recovered from the acidic, polar and PAH fractions, and caused an increase of SCE but not of chromosomal aberrations in cultured human lymphocytes (Lorenza Giromini et al., Universities of Pisa and Ferrara); the mutagenicity in S. typhimurium TA98, TA98/1,8-DNP, and TAlOO of atmospheric particulate recovered near a treatment plant was higher than that of urban air from heavy-traffic areas (Roberto Scarpato et al., Universities of Pisa and Ferrara); various extracts of atmospheric particulate from a residential area in Parma were analyzed in S. typhimurium TA98 and TAlOO, and in S. cerevisiue D7 (Carlo Rossi et al., University of Parma; USL4, Parma; Biotecnologika, Felino); the mutagenicity of atmospheric particulate in Pisa was monitored in S. typhimurium TA98 and TAlOO (Rita Vellosi et al., CNR and USL12, Pisa). Finally, the genotoxicity of purification sludges was investigated in S. typhimurium TA98 and TAlOO as well as in cultured human lymphocytes for the induction of chromosomal aberrations, micronuclei and SCE
(Cinzia Sbrana et al., Universities Ferrara).
of Pisa and
Conclusions The program of the meeting was particularly stringent, condensing in two days 58 presentations authored by a total of 190 investigators, most of them Italian, but with an important contribution of scientists from other countries, including Bulgaria, China, Croatia, Finland, France, Germany, The Nederlands, the UK and the USA. In spite of this tour de force, the meeting was well organized, and all attendants agreed in commenting favorably on its scientific success. The meeting was concluded by the general assembly of SIMA, which included in the agenda the reports of the President and of the Secretary-Treasurer, the admission of new members, and the election of the Directive Committee for the biennium 1993-94. All members of the Society expressed their gratitude to Nicola Loprieno, who regretfully declined its nomination as President, according to the statute of the Society demanding this charge for a round only. The former Vice-President, Angelo Carere, was elected President, and Gregorio Olivieri VicePresident. Angelo Abbondandolo, Roberto Barale, Giorgio Bronzetti, Silvio De Flora, Angelo G. Levis and Miria Stefanini were elected members of the Directive Council, and Francesca Pacchierotti was re-confirmed as Secretary-Treasurer. One of the tasks of the new Directive Committee will be the organization of the Second Annual Meeting of SIMA, which is tentatively scheduled for October 1993 in the area of Rome. Acknowledgement I thank all members on the SIMA Directive Committee for revising this report.