Evaluation of the genotoxicity induced by the fungicide fenarimol in mammalian and plant cells by use of the single-cell gel electrophoresis assay.

Fenarimol, a systemic pyrimidine carbinol fungicide, is considered to be not genotoxic or weakly genotoxic, although the available toxicological data are controversial and incomplete. Our results obtained in vitro with leukocytes of two different rodent species (rat and mouse) show that fenarimol affects DNA, as detected by the single-cell gel electrophoresis (SCGE, Comet) assay. This fungicide is able to induce DNA damage in a dose-related manner, with significant effectiveness at 36 nM, but without significant interspecies differences. Simultaneous exposure of rat leukocytes to fenarimol (36-290 nM) and a model genotoxic compound (50 microg/ml bleomycin) produced a supra-additive cytotoxic and genotoxic effect. This supports previous findings suggesting possible co-toxic, co-mutagenic, cancer-promoting and co-carcinogenic potential of fenarimol, and modification of the effects of other xenobiotics found to be influenced by this agrotoxic chemical, with consequent different toxicological events. The potential for DNA strand breaks to act as a biomarker of genetic toxicity in plants in vivo was also considered, in view of the fact that higher plants represent reliable sensors in an ecosystem. Significant DNA breakage was observed in the nuclei of Impatiens balsamina leaves after in vivo treatment with fenarimol (145 nM, 1h). More than 50% of the cells showed such DNA damage.

Evaluation of the migration of mutagens/carcinogens from PET bottles into mineral water by Tradescantia/micronuclei test, Comet assay on leukocytes and GC/MS.

This study monitored the release of mutagenic/carcinogenic compounds into mineral water (natural and carbonated) from polyethylene terephthalate (PET) bottles, using a plant mutagenicity test which reveals micronuclei formation in Tradescantia pollen cells (Trad/MCN test), a DNA damage assay (Comet assay) on human leukocytes and gas chromatography/mass spectrometry (GC/MS) for the characterisation of migrants. The water samples were collected at a bottling plant and stored in PET bottles for a period ranging from 1 to 12 months. Every month some samples were randomly collected and lyophilised, the residual powders were extracted with organic solvents and then analysed by GC/MS and tested for DNA damage in human leukocytes, or reconstituted with distilled water to obtain concentrates for the exposure of Tradescantia inflorescences. Micronuclei increase in pollen was found only in natural mineral water stored for 2 months. DNA-damaging activity was found in many of the natural and carbonated water samples. Spring water was negative in the plant micronuclei test and the Comet assay, whereas distributed spring water showed DNA-damaging effects, suggesting a possible introduction of genotoxins through the distribution pipelines. GC/MS analysis showed the presence in mineral water of di(2-ethylhexyl)phthalate, a nongenotoxic hepatocarcinogenic plasticizer, after 9 months of storage in PET bottles.

Amifostine (WR-2721) selective protection against melphalan genotoxicity.

Amifostine (WR-2721) is an aminothiol compound dephosphorylated at the tissue site by alkaline phosphatase to the active metabolite, which is able to inactivate electrophilic substances and scavenge free radicals. Amifostine effects against melphalan-induced DNA strand breaks were studied in normal human white blood cells (WBC) and K562 leukemic cells using the single cell gel electrophoresis (SCGE) or Comet assay, a reported method for measuring DNA damage in individual cells. Prior to treatment (1 h, 37 degrees C) with increasing doses of melphalan, with or without S9, the cells were treated (15 min, 37 degrees C) with a control medium or amifostine (3 mg/ml). Treatment of normal and leukemic cells with melphalan induced a dose-dependent 'comet formation'. Melphalan-induced DNA damage follows a normal distribution in WBC. On the other hand, in K562, a significant proportion of undamaged cells remains even with doses at which mean DNA damage is serious. Pretreatment with WR-2721 protects WBC, but not K562, against the genotoxic effect of melphalan. Amifostine might even strengthen the action of the antiblastic drug against K562 cells. S9 addition appears to enhance melphalan effectiveness. SCGE appears as a suitable primary screening method for in vitro and in vivo studies on drug-DNA interactions and their modulations by endogenous/exogenous factors.

DNA damage by tobacco smoke and some antiblastic drugs evaluated using the Comet assay.

The Comet assay was used in human leukocytes to detect, in vivo, DNA strand-breaks induced by smoking habit to evaluate the test sensitivity to an environmental factor, and by several antiblastic drugs to note their effectiveness at single cell level. Differences related to smoking habit, gender and age are evident. Melphalan shows the widest DNA damage. The damage induced by etoposide can be ascribed to the balancing between the production of strand-breaks and cross-links which limit the migration of DNA fragments. Interferon, fludarabine, prednisone, and oncocarbide appear to induce unexpected strand-breaks. Single cell gel electrophoresis (SCGE) is highly effective in revealing the association between DNA damage and environmental, genetic, and acquired factors, providing further data on the possible applicability of this assay in genotoxic human surveillance in addition to established tests. Moreover, the ability to point out cell subpopulations varying in mean damage levels could allow detection of potentially emerging drug-resistant populations.

Bleomycin genotoxicity alteration by glutathione and cytochrome P-450 cellular content in respiratory proficient and deficient strains of Saccharomyces cerevisiae.

The genotoxic effects of the antiblastic drug bleomycin were studied in the D7 strain of Saccharomyces cerevisiae and on its derivative mitochondrial mutant rho degree at different cellular concentrations of two drug metabolizing systems, glutathione (GSH) and cytochrome P-450. Bleomycin mutagenic activity was evaluated as frequencies of mitotic gene conversion, reversion and total aberrations under different physiological conditions. In the D7 strain, petite mutant induction was also detected. This is important due to the role of the mitochondrial genome in cancer induction, ageing and degenerative diseases. Both strains showed higher convertant than revertant induction. At high cytochrome P-450 levels, bleomycin-induced gene conversion was enhanced in both strains although mitochondrial functionality showed a detoxicant role while cellular GSH content decreased the induction of convertants only in the respiratory proficient strain. Cell metabolic conditions, such as cell cycle, aerobic/hypoxic conditions of the cell and content of drug metabolizing enzymes, appeared to interact with the genotoxic effectiveness of bleomycin. Moreover, the usefulness of S.cerevisiae as a model organism for drug assessment for mutagenicity was emphasized.

Modulation of mitomycin C mutagenicity on Saccharomyces cerevisiae by glutathione, cytochrome P-450, and mitochondria interactions.

It is well established that most anticancer drugs also have mutagenic effects and require metabolic activation before exerting their mutagenic/antiblastic activity. Antitumoral compound effects strongly depend on the biochemical/physiological conditions of the tumoral cells, and especially on the activation of specific drugs metabolizing enzymes and on respiration. We examined the mitomycin C-induced mutagenic effects on the D7 strain of Saccharomyces cerevisiae and on its derivative mitochondrial mutant p degrees at different contents of glutathione and cytochrome P-450, molecules able to activate/detoxicate xenobiotics. The mutagenic activity of the drug was evaluated as frequency of mitotic gene conversion and reversion in different physiological conditions. The highest frequencies of reversion and especially of gene conversion were observed at the highest cytochrome P-450 contents in the D7 strain with a further increase at high glutathione level. In the respiratory-deficient strain, the highest frequency of convertants was shown at low glutathione level and lack of cytochrome P-450. These results suggest the relevance of mitochondrial functionality for the expression of genotoxic activity of this anticancer drug.

Occupational genotoxicity assessment by mutagenicity assays.

Mutagenic activity measured by Ames test and by gene conversion, point mutation and mitochondrial mutability in Saccharomyces cerevisiae D7 strain was determined in the indoor environment of a glass factory. The results suggest that the increase in mutagenicity of air sample collected near the machinery is due to the thermal decomposition of oils. Modified assays were therefore compared for their ability to detect mutagens contained in urinary concentrates of exposed workers. The bacterial tests were performed by microsuspension assay in TA98, TA100 strains and in YG1024, YG1029 strains which overproduce O-acetyltransferase. Significant differences are evidenced both in the eukaryotic and prokaryotic systems.

Urban air pollution: use of different mutagenicity assays to evaluate environmental genetic hazard.

The genotoxic activities associated with airborne particulate matter collected in Parma (northern Italy) have been determined. The airborne particle extracts were tested for mutagenicity using Salmonella frameshift (TA98) and base-substitution (TA100) tester strains with and without S9 microsomal activation and Saccharomyces cerevisiae strain D7 in order to determine the frequency of mitotic gene conversion and ilv1-92 mutant reversion in cells harvested at stationary and logarithmic growth phase. The relationship between mitochondrial DNA mutations and ageing, degenerative diseases and cancer prompted us to take into account the mitochondrial informational target, i.e., the respiratory-deficient (RD) mutants. The results obtained show a variability in the response for the different test systems during different months. The Salmonella mutagenicity trend was directly correlated with carbon monoxide, nitrogen oxides (NOx) and Pb concentration in airborne particulates and inversely correlated with temperature, whereas the mitochondrial genotoxic effect was higher during spring and late summer. These data suggest that the genotoxic risk assessment is a time-dependent value strictly correlated with the evaluation system being tested.