Stefania Bonatti

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.

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.

The use of organic solvents in mutagenicity testing

13 organic substances (dimethylsulfoxide, methanol, ethanol, n-propyl alcohol, sec-butyl alcohol, tert-butyl alcohol, dl-sec-amyl alcohol, ethylene glycol, ethylene glycol monomethyl ether, 1,4-diethylene dioxide, acetone, methyl acetate and formamide) were considered from the standpoint of their use as solvents for water-insoluble chemicals to be tested for mutagenicity. First, the effect of these solvents on cell survival was studied in the yeast Schizosaccharomyces pombe and in V79 Chinese hamster cells. 8 solvents showing relatively low toxicity on either cell system (dimethylsulfoxide, ethanol, ethylene glycol, ethylene glycol monomethyl ether, 1,4-diethylene dioxide, acetone, methyl acetate and formamide) were tested for their effect on aminopyrine demethylase. 4 solvents (ethanol, 1,4-diethylene dioxide, methyl acetate and formamide) showed a more or less pronounced adverse effect on the microsomal enzymic activity. The remaining 4 and methanol (whose effect on aminopyrine demethylase was not testable) were assayed for mutagenicity in S. pombe. They all gave negative results both with and without the post-mitochondrial fraction from mouse liver.

Scintillometric determination of DNA repair in human cell lines: A critical appraisal

Abstract The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with [3H]thymidine, the radioactivity incorporated in to DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (CHU, THU). The ratios THU/CHU and THU/T:CHU/C, indicating absolute and relative increases of DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation, whereas, no stimulation in inferred when both of them are ⩽1. The scintillometric estimate of DNA repair was always in agreement with the autoradiographic observations, so that the procedure adopted can be used as a rapid test for screening investigations. Agents giving a relative but no an absolute increase of DNA radioactivity are generally not inducers of repair synthesis as estimated by autoradiography. However, the same scintillometric results are also occasionally observed with DNA repair inducers, such as methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), owing to alterations of thymidine pool radioactivity. These chemicals, besides affecting the levels of labelled precursors in the intracellular pool in the T series, differently modified the increase of pool radioactivity which is a regular effect of HU. With such chemicals, DNA repair synthesis can be detected only after normalization of th DNA radioactivity on the basis of pool alterations. The quantitative value of the autoradiographic estimate of DNA repair is also affected by the changes in the radioactivity of the thymidine pool although autoradiography retains its qualitative value. Dimethylnitrosamine, mitomycin C and potassium dichromate, described by other authors as inducers of DNA repair, also gave negative results by the scintillometric procedure after normalization of DNA radioactivities. However, in our hands, these agents were unable to stimulated repair synthesis, according to the results of autoradiography and isopynic centrifugation. The proposed scintillometric procedure is effective in indicating false negative inducers of DNA repair, not giving rise to false positives.

Sister chromatid exchange induction in peripheral blood lymphocytes of traffic police workers.

Traffic police workers, as a population exposed to urban atmosphere, were compared with a control population exposed to indoor air pollution levels. Sister chromatid exchanges (SCEs) as a biomarker of effect were measured in peripheral blood lymphocytes of 54 exposed subjects and 35 controls, and environmental concentration of polynuclear aromatic hydrocarbon (PAH) tracer compounds was detected by personal air samplers. The mean exposure level to benzo[a]pyrene in our group of traffic policemen (3.4 mg/m3) was in the range that has been estimated in urban areas in Europe during the last 10 years. No difference in SCE levels was found between exposed workers (7.36, SD 1.35) and controls (7.47, SD 1.28). No correlation was observed between SCE/cell and airborne PAH concentration in the traffic worker population. A positive regression of SCE on exposure estimate was found only in the non-smoking group of police workers. Our findings suggest that exposure to urban air pollution does not induce relevant cytogenetic effects.

Study on aneuploidy and p53 mutations in astrocytonias

To determine whether a correlation exists between aneuploidy and p53 status in astrocytic tumors we analyzed 48 astrocytomas with different grades of malignancy for the presence of p53 mutations and aneuploidy of chromosomes 10 and 17 (Ch10, Ch17), known to be particularly involved with this type of tumor. We used polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis on exons 5-8 of the p53 gene, and fluorescence in situ hybridization (FISH) analysis on interphase nuclei using chromosome specific pericentromeric probes, respectively. Our results showed that Ch10/Ch17 aneuploidy is a common early event in astrocytomas (90% of low grade tumors are aneuploid). p53 mutations and Ch17 aneuploidy are early events, but their incidence is not dependent on tumor grade. Loss of Ch10 is the only alteration that significantly correlates with tumor progression. No significant correlation between the presence of Ch10/Ch17 aneuploidy and p53 mutations was found. However, the coexistence of p53 mutations and aneuploidy, was observed in a subset of cases. The presence of p53 mutations appeared to be a significant predictor of a poor prognosis. In conclusion, genomic instability may or may not be associated with p53 mutations in astrocytomas, thus suggesting that other cellular determinants can also be responsible for the aneuploidy observed.

Analysis of the mosaicism induced by hydroxylamine and nitrous acid in Schizosaccharomyces pombe

Abstract The mutagenic action of hydroxylamine and nitrous acid on the ad-6 and ad-7 loci of Schizosaccharomyces pombe was studied by analyzing the relative frequencies of complete and mosaic mutant colonies (ad-, purple phenotype) and the sizes of the purple sectors in the mutant mosaics. With both hydroxylamine and nitrous acid, the delay in the phenotypic expression of ad- clones was found to be related to the survival level.

The mutagenic activity of N-nitroso-N-methylurethane and N-nitroso-N-ethylurethane in Schizosaccharomyces pombe

Abstract An investigation on the comparative mutagenic activity of N-nitroso-N-methylurethane (NMU) and N-nitroso-N-ethylurethane (NEU) in Schizosaccharomyces pombe was carried out. NMU, NEU and four related compounds were tested for reversion to methionine independence on teh met4-, D 19, h- strain, NMU was found to be much more mutagenic and toxic than NEU. A genetic analysis of the revertants induced by NMU and NEU did not reveal any difference between the two mutagens as to proportion of reversions due to suppressor mutations. For both NMU and NEU, mutagenic activity and breakdown of the compound increased with increasing pH. Urethane, N-methylurethane and N-ethylurethane, tested in the same conditions as NMU and NEU, were not mutagenic; the mutagenic activity of nitrous acid was much lower than that of the nitroso compounds. The results confirm the hypothesis that the mutagenic activity of nitroso compounds is due to the production in the cell of diazoalkanes, whose formation in vitro is known to be favoured by alkali conditions.