Angelo Abbondandolo

Are chromosome aberrations in circulating lymphocytes predictive of future cancer onset in humans? Preliminary results of an Italian cohort study

To investigate the existence of an association between the frequency of chromosome aberrations (CA) in non-target tissues and cancer risk, a historical cohort study was carried out in a group of 1455 subjects screened for CA over the last 20 years in Italy. Statistically significant increases in standardized mortality ratio (SMR) for all cancers were found in subjects with medium and high levels of CA in peripheral blood lymphocytes (SMR = 178.5 and SMR = 182.0, respectively) and in subjects with high levels of CA for respiratory tract cancers (SMR = 250.8) and lymphatic and hematopoietic tissue neoplasms (SMR = 548.8). Significant trends in the SMRs were observed for these latter causes of death.

STROMAL DAMAGE AS CONSEQUENCE OF HIGH-DOSE CHEMO/RADIOTHERAPY IN BONE MARROW TRANSPLANT RECIPIENTS

Bone marrow transplant (BMT) relies on the engraftment of donor hemopoietic precursors in the host marrow space. Colony forming units-fibroblasts (CFU-f), the precursor compartment for the osteogenic lineage, are essential to hemopoietic stem cell survival, proliferation and differentiation. We have studied CFU-f in donors (aged 5 months to 62 years) and in patients who had received allogeneic BMT (aged 2 months to 63 years). In donor marrows we found an inverse correlation between CFU-f frequency and age. In BMT recipients CFU-f frequencies were reduced by 60%-90% (p < 0.05) and the numbers did not recover up to 12 years after transplant. Stromal reconstitution to normal levels was found only in patients < 5 years old. In all patients studied CFU-f post-BMT were of host origin. Patients with low CFU-f levels displayed also a decreased bone mineral density (p < 0.05) and significantly reduced levels of long-term culture-initiating cells (LTC-IC) (p < 0.05). Our study demonstrates that the marrow stromal microenvironment is seriously and irreversibly damaged after BMT. Donor cells do not contribute to reconstitute the marrow microenvironment, whose residual CFU-fs remain of host origin.

p53 mutants can often transactivate promoters containing a p21 but not Bax or PIG3 responsive elements.

The human p53 protein acts mainly as a stress inducible transcription factor transactivating several genes involved in cell cycle arrest (e.g. p21) or apoptosis (e.g. Bax, PIG3). Roughly half of all human tumours contains p53 missense mutations. Virtually all tumour-derived p53 mutants are unable to activate Bax transcription but some retain the ability to activate p21 transcription. Identification of these mutants may have valuable clinical implications. We have determined the transactivation ability of 77 p53 mutants using reporter yeast strains containing a p53-regulated ADE2 gene whose promoter is regulated by p53 responsive elements derived from the regulatory region of the p21, Bax and PIG3 genes. We also assessed the influence of temperature on transactivation. Our results indicate that a significant proportion of mutants [16/77 (21%); 10/64 (16%) considering only tumour-derived mutants] are transcriptionally active, especially with the p21 promoter. Discriminant mutants preferentially affect less conserved (P<0.04, Fishers exact test), more rarely mutated (P<0.006, Fishers exact test) amino acids. Temperature sensitivity is frequently observed, but is more common among discriminant than non-discriminant mutants (P<0.003, Fishers exact test). Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours that surprisingly were indistinguishable from wild type in standard transcription, growth suppression and apoptosis assays in human cells, but showed gain of function in transformation assays. The incidence of transcriptionally active mutations among this group was significantly higher than in the panel of mutants studied previously (P<0.001, Fishers exact test). Since it is not possible to predict the behaviour of a mutant from first principles, we propose that the yeast assay be used to compile a functional p53 database and fill the gap between the biophysical, pharmacological and clinical fields.

Cytogenetic biomonitoring of an Italian population exposed to pesticides : chromosome aberration and sister-chromatid exchange analysis in peripheral blood lymphocytes

Chromosome aberrations (CA) and sister-chromatid exchanges (SCE) were measured in lymphocytes of (A) 32 healthy individuals working in the flower industry and exposed to pesticides, (B) 32 individuals exposed as above and hospitalized for bladder cancer, and (C) 31 controls. Compounds to which floriculturists were exposed included 18 nitro-organic herbicides and fungicides, 9 nitro-organic fungicides, 12 organophosphate and organothiophosphate insecticides, 4 hydrocarbon derivative herbicides and 5 inorganic fungicides and insecticides. 150 and 70 metaphases per individual were scored for CA and SCE, respectively. A significant increase in the incidence of CA and SCE was observed in both exposed groups. Cancer patients showed the presence of rare rearrangements (dicentrics, rings and quadriradials) that were not observed in controls and were present at a lower frequency in healthy exposed people. Hyperdiploid and polyploid metaphases were also significantly increased in the 2 exposed groups compared to controls. Stratifying for age or smoking habits, although affecting the significance of individual data, did not change the substance of the results.

Mutagenicity of industrial compounds styrene and its possible metabolite styrene oxide

Styrene and its presumed metabolite, styrene oxide, were tested for their mutagenic effect on a forward mutation system of yeast and of Chinese hamster cells, and on a gene-conversion system of yeast. Experiments with liver microsomal preparations and host-mediated assay with yeast were also carried out. Styrene oxide was mutagenic in all test systems. Styrene was mutagenic only in the host-mediated assay.

Cytogenetic biomonitoring in traffic police workers: micronucleus test in peripheral blood lymphocytes.

Atmospheric pollution represents a relevant environmental hazard which has been associated with considerable excess mortality, morbidity, and increased rates of respiratory diseases in humans. To date, more than 3,000 environmental chemical compounds have been identified in the ambient atmosphere, including a variety of mutagenic and/or carcinogenic agents, such as polycyclic aromatic hydrocarbons (PAHs), aromatic amines, and heterocyclic compounds. Positive associations between cytogenetic markers and airborne levels of PAHs have been reported by experimental and human studies. Traffic has been implicated as the major determinant for the concentration of PAHs and, therefore, for the genotoxic activity of urban air. A biomonitoring study has been conducted in 82 Italian traffic police workers exposed to air pollutants and 34 control subjects (matched by age, gender, and smoking habits) not exposed to traffic pollutants. The aim of this study was to assess the cytogenetic effects, such as micronucleus frequency in peripheral blood lymphocytes, and to estimate the association with individual exposure to PAH. Statistical analysis of the frequency of micronuclei in binucleated cells showed higher mean levels in referent subjects (4.03%) than in traffic police officers (3.73%). Smoking showed no effect on the frequency of micronuclei. The study failed to detect any association between micronucleus frequency and individual level of benzo(a)pyrene, considered a marker of exposure to PAHs. These findings indicate that exposure to urban air pollutants does not result in increased levels of micronuclei in peripheral white blood cells. Environ. Mol. Mutagen. 30:396–402, 1997

Tumour p53 mutations exhibit promoter selective dominance over wild type p53.

The tumour suppressor gene p53 is frequently mutated in human cancer. Tumour derived p53 mutants are usually transcriptionally inactive, but some mutants retain the ability to transactivate a subset of p53 target genes. In addition to simple loss of function, some p53 mutants may be carcinogenic through a dominant negative mechanism. Aiming at a more general classification of p53 mutants into predictive functional categories it is important to determine (i) which p53 mutants are dominant, (ii) what features characterize dominant mutants and (iii) whether dominance is target gene specific. The ability of 71 p53 mutants to inhibit wild type p53 was determined using a simple yeast transcriptional assay. Approximately 30% of the mutants were dominant. They preferentially affect highly conserved amino acids (P<0.005), which are frequently mutated in tumours (P<0.005), and usually located near the DNA binding surface of the protein (P<0.001). Different tumour-derived amino acid substitutions at the same codon usually have the same dominance phenotype. To determine whether the ability of p53 mutants to inhibit wild type p53 is target gene specific, the dominance towards p21, bax, and PIG3 binding sites was examined. Approximately 40% of the 45 mutants examined were dominant for the p21 (17/45) or PIG3 (20/45) responsive elements and 71% (32/45) were dominant for the bax responsive element. These differences are statistically significant (p21 vs bax, P<0.003; bax vs PIG3, P<0.02, Fishers exact test) and defined a hierarchy of dominance. Finally, we extended the analysis to a group of mutants isolated in BRCA-associated tumours, some of which retained wild type level of transcription in yeast as well as in human cells, but show gain of function in transformation assays. Since transformation assays require transdominant inhibition of the endogenous wild type allele, one possible explanation for the behaviour of the BRCA-associated mutants is that they adopt conformations able to bind DNA alone but not in mixed tetramers with wild type p53. The yeast data do not support this explanation, because all BRCA-associated mutants that behaved as wild type in transcription assay were recessive in dominance assays.

The presence of amplified regions affects the stability of chromosomes in drug-resistant Chinese hamster cells.

The stability of chromosomes carrying amplified CAD (carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase) or DHFR (dihydrofolate reductase) genes was studied in V79 Chinese hamster cell derivatives resistant to PALA (N-phosphonacetyl-L-aspartate) and MTX (methotrexate), respectively. Cells were maintained in the presence of the selective drugs during the study. In both metaphase chromosomes and interphase nuclei, amplified regions were localized by in situ hybridization. In MTX-resistant cells, the amplification-bearing chromosome moved sluggishly at anaphase and gave rise to bud-shaped formations in interphase nuclei. It is suggested that these buds could eventually separate as micronuclei. In both MTX- and PALA-resistant cells, amplified DNA was observed in micronuclei in interphase and in displaced chromosomes in metaphase. Finally, amplification-bearing dicentric chromosomes were found in both drug-resistant cell lines. Cumulatively, these observations indicate that the presence of the amplified region in a chromosome renders it unstable: chromosomes bearing an amplified region tended to be excluded from cells, and rearrangements were more frequent than in normal chromosomes.

A comparative study of different experimental protocols for mutagenesis assys with the 9-azaguanine resistance system in cultured Chinese hamster cells ☆

Abstract Both spontaneous and EMS-induced mutant frequencies were determined in cultured cells from V79 Chinese hamsters using three different experimental protocols. After optimal expression time was attained, mutation frequencies only remained constant when a protocol was used in which the cell density was maintained below critical values both before and during mutant selection. The identification of such a palteau allows, besides more reliable and reproducible estimated of mutation frequency, reduction in the size of experiments for quantitative evaluation of mutagenicity. Determination of mutation frequencies over a wide range of expression times becomes in fact unnecessary.

Determining mutational fingerprints at the human p53 locus with a yeast functional assay: a new tool for molecular epidemiology

In order to isolate experimentally induced p53 mutations, a yeast expression vector harbouring a human wild-type p53 cDNA was treated in vitro with the antineoplastic drug chloroethyl-cyclohexyl-nitroso-urea (CCNU) and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. p53 mutations were identified in 32 out of 39 plasmids rescued from independent ade- transformants. Ninety-two percent of CCNU induced mutations were GC-targeted single base pair substitutions, and GC>AT transitions represented 73% of all single base pair substitutions. In 70% of the cases the mutated G was preceded 5′ by a purine. The distribution of the mutations along the p53 cDNA was not random: positions 734 and 785 appeared as CCNU mutational hotspots (n=3, P<0.0003) and CCNU induced only GC>AT transitions at those positions. The features of these CCNU-induced mutations are consistent with the hypothesis that O6-alkylguanine is the major causative lesion. One third of the CCNU-induced mutants were absent from a huge collection of 4496 p53 mutations in human tumours and cell lines, thus demonstrating that CCNU has a mutational spectrum which is uniquely different from that of naturally selected mutations. This strategy allows direct comparison of observed natural mutation spectra with experimentally induced mutation spectra and opens the way to a more rigorous approach in the field of molecular epidemiology.