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Since epidemiological studies have firmly implicated co-exposure to iron oxides and polycyclic aromatic hydrocarbons as a potential etiological factor involved in the excess mortality due to lung cancer in miners, experimental studies have been performed to investigate the role of iron particles in benzo[a]pyrene (B[a]P)-induced lung pathogenesis. In a previous study using the Comet assay in vivo in the rat, we demonstrated that iron particles enhanced B[a]P genotoxicity. To determine whether co-exposure (B[a]P/iron oxides) induces a real genotoxic activity or is only due to inhibition of DNA repair, the unscheduled DNA synthesis (UDS) assay was implemented in vivo in the rat. The UDS assay was used to measure DNA repair in two cell types (lung cells and hepatocytes) of OFA Sprague-Dawley rats, 24 h after endotracheal administration of a single dose of an iron oxide (hematite, Fe2O3) (0.75 mg), of B[a]P (0.75 mg) or of B[a]P (0.75 mg) coated on hematite particles (0.75 mg). No difference in UDS was observed in the two organs investigated in rats treated with iron oxide alone compared with control animals, while a significant increase in UDS was observed in lungs and liver of rats treated with B[a]P alone compared with control animals. The main finding was a significant increase in UDS observed in both lung and liver cells of rats treated with B[a]P coated on hematite when compared with those treated with B[a]P alone. The current study demonstrates (i) that iron particles did not inhibit UDS in lung cells and hepatocytes of OFA Sprague-Dawley treated rats with B[a]P coated on hematite and (ii) a potent genotoxic activity of co-exposure to B[a]P coated on hematite. Therefore, our data may contribute to explaining the excess mortality due to lung cancer in epidemiological studies and overall why exposure to B[a]P coated on Fe2O3 particles resulted in a higher tumor incidence in rodents compared with exposure to B[a]P alone. [ABSTRACT FROM AUTHOR]

Since epidemiological studies have firmly implicated co-exposure to iron oxides and polycyclic aromatic hydrocarbons as a potential etiological factor involved in the excess mortality due to lung cancer in miners, experimental studies have been performed to investigate the role of iron particles in benzo[a]pyrene (B[a]P)-induced lung pathogenesis. In a previous study using the Comet assay in vivo in the rat, we demonstrated that iron particles enhanced B[a]P genotoxicity. To determine whether co-exposure (B[a]P/iron oxides) induces a real genotoxic activity or is only due to inhibition of DNA repair, the unscheduled DNA synthesis (UDS) assay was implemented in vivo in the rat. The UDS assay was used to measure DNA repair in two cell types (lung cells and hepatocytes) of OFA Sprague-Dawley rats, 24 h after endotracheal administration of a single dose of an iron oxide (hematite, Fe2O3) (0.75 mg), of B[a]P (0.75 mg) or of B[a]P (0.75 mg) coated on hematite particles (0.75 mg). No difference in UDS was observed in the two organs investigated in rats treated with iron oxide alone compared with control animals, while a significant increase in UDS was observed in lungs and liver of rats treated with B[a]P alone compared with control animals. The main finding was a significant increase in UDS observed in both lung and liver cells of rats treated with B[a]P coated on hematite when compared with those treated with B[a]P alone. The current study demonstrates (i) that iron particles did not inhibit UDS in lung cells and hepatocytes of OFA Sprague-Dawley treated rats with B[a]P coated on hematite and (ii) a potent genotoxic activity of co-exposure to B[a]P coated on hematite. Therefore, our data may contribute to explaining the excess mortality due to lung cancer in epidemiological studies and overall why exposure to B[a]P coated on Fe2O3 particles resulted in a higher tumor incidence in rodents compared with exposure to B[a]P alone.

Among the tests used to determine the mutagenic potential of chemicals, the chromosomal aberrations and micronucleus assays play an important role. These tests score either chromosomal structural aberrations at metaphase or micronuclei at interphase. One of the hallmarks of apoptosis is DNA fragmentation into 50-300 kpb leading to oligonucleosomal fragmentation that can interfere with the results of clastogenic assays. In this case, apoptosis may be a confusing factor in the evaluation of the mutagenic potential of molecules and lead to false positive results. For these reasons we have developed a cell line able to demonstrate the interference of apoptosis in two mutagenicity tests: the in vitro micronucleus test and metaphase analysis in vitro. We used a murine cytotoxic T cell line, CTLL-2 Bcl2, in which a stably transfected bcl2 gene is known to protect these cells from apoptosis induced by various stimuli. A comparison between results obtained in parental CTLL-2 cells and in CTLL-2 Bcl2 cells treated with non-genotoxic apoptosis inducers, such as dexamethasone or gliotoxin, leads us to conclude that apoptosis could give false positive results due to DNA fragmentation. Moreover, with etoposide, a clastogen that also induces apoptosis, we observed that the percentages of aberrant cells and numbers of micronuclei were significantly increased in CTLL-2 cells compared with CTLL-2 Bcl2 cells. This observation suggests that apoptosis leads to an overestimation of the genotoxic potential of chemicals. Finally, with nocodazole, an aneugen, we confirm that this model can also detect agents that have only genotoxic potential and thus allows a better estimation of the genotoxic threshold in studies with aneugens, thus avoiding overestimation of the mutagenic risk of such a compound.

Chlorohydroxyfuranones (CHFs) are mutagenic disinfection by-products found in chlorine-treated drinking water. In the current study, the genotoxicity of four CHFs, 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA), 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), was determined. Two in vitro assays, the microscale micronucleus assay on L5178Y mouse lymphoma cells and the unscheduled DNA synthesis assay on a hepatocyte primary culture from Fisher F344 rats, were carried out. All four CHFs demonstrated genotoxic effects in both assays. In the two systems used, CMCF was the most genotoxic compound, followed by MCA, MX and MCF, respectively. This work was the first study of the DNA damaging properties of all four CHFs in two in vitro genotoxicity tests. These new data expand the range of genetic damages induced by this group of compounds.

A micromethod for the in vitro micronucleus assay was developed using L5178Y cells to enable the rapid screening of a large number of molecules. The method is quick, simple to perform and needs very small amounts of compound, i.e. <10 mg. In this methodology, three types of treatment were carried out in parallel, enabling an optimal detection of both aneugenic and clastogenic compounds: two treatments without metabolic activation with or without a recovery period after a 24 h continuous treatment and one treatment with metabolic activation by Aroclor 1254-induced rat or hamster liver S9 mix. Seventeen known genotoxins (12 clastogens and five aneugens) and seven known non-genotoxins were tested. The in vitro micronucleus micromethod using L5178Y cells exhibited good sensitivity (16 positive/17 known genotoxins tested) and specificity (7 negative/7 known non-genotoxins tested) for the 24 test compounds studied with or without metabolic activation. Furthermore, this test showed a good correlation with other in vitro micronucleus tests performed using macromethods with various mammalian cell cultures. We conclude that the in vitro micronucleus micromethod with L5178Y cells could be used in the earliest stages of development of new molecules as a preliminary short-term screening assay before starting regulatory tests.

Three short-term assays (SOS chromotest, Ames fluctuation test and newt micronucleus test) were performed to detect the genotoxic activity of organohalides, compounds likely to be found in chlorinated and/or ozonated drinking water: monochloro-, dichloro- and trichloroacetic acids and monobromo-, dibromo- and tribromoacetic acids. With the SOS chromotest, only three of the chemicals studied (dichloroacetic acid, dibromo- and tribromoacetic acids) were found to induce primary DNA damage in Escherichia coli PQ 37. In the Ames fluctuation test, all the compounds except monochloroacetic acid showed mutagenic activity in Salmonella typhimurium strain TA100. In these two in vitro tests, a good correlation between increasing number of substituents and decreasing mutagenicity was observed. Namely, the toxicity of brominated and chlorinated acetic acids decreased when the number of substituents increased. The newt micronucleus test detected a weak clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae for trichloroacetic acid only.

Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of four trihalomethanes (chloroform, bromodichloromethane, chlorodibromomethane and bromoform). With the SOS chromotest, all the chemicals studied except chloroform were found to induce primary DNA damage in Escherichia coli PQ37. In the Ames-fluctuation test, only bromoform showed mutagenic activity on Salmonella typhimurium strain TA100. The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae for bromodichloromethane and bromoform. It appeared that the presence of bromine substituent(s) generally led to significant genotoxic activity. Moreover, the use of the metabolic system significantly increased the genotoxicity of the brominated trihalomethanes in the SOS chromotest. Unlike previous investigations in which the SOS chromotest was always the least interesting assay, this study exhibited the good efficiency of this in vitro test on E.coli for the detection of trihalomethanes with bromine substituents.

The genotoxic activity of alpha-Ni3S2 was assessed on human lymphocytes from nickel-hypersensitized (SSL) and nickel-unsensitized (USL) subjects. Three genotoxicity tests were performed: the sister chromatid exchange (SCE) test, the metaphase analysis test and the micronucleus test. (i) The SCE test (3-100 micrograms/ml) showed a weak but statistically significant increase in the number of SCE in both lymphocyte types with respect to controls, USL presenting a slightly higher SCE incidence but only at one concentration. (ii) The metaphase analysis test demonstrated a high dose-dependent clastogenic activity of alpha-Ni3S2 in both lymphocyte types. The frequency of chromosomal anomalies was significantly higher in USL than in SSL for all concentrations applied. (iii) The micronucleus test confirmed the dose-dependent clastogenic activity of alpha-Ni3S2 and the differences already observed between USL and SSL, i.e. the number of cells with micronuclei was statistically higher in USL. Finally, the incorporation study with alpha-63Ni3S2 showed a higher uptake of its solubilized fraction by USL. This allows an explanation of the different genotoxic action of nickel on the two cell types. In this study we demonstrated that hypersensitivity has an influence on the incorporation of alpha-Ni3S2 and subsequently on the different induction of chromosomal aberrations in human lymphocytes.

Four mutagenicity tests of alpha-Ni3S2 were performed: the Ames test on five Salmonella typhimurium strains, HPRT test on V79 cells, in vitro chromosomal aberrations on human lymphocytes and the in vivo micronucleus test in mice. The in vitro tests were carried out without metabolic activation. (i) The Ames test (5-1500 micrograms/plate) demonstrated no mutagenic activity, thus confirming previous observations by other authors. (ii) The HPRT test was carried out under standard conditions (3 h exposure) with alpha-Ni3S2 concentrations from 30 to 1000 micrograms/ml. No significant difference was observed with the control cells. Ultrastructural examination revealed alpha-Ni3S2 binding to the cell membrane. Very few particles were found in the cytoplasm but not in the nucleus. (iii) In vitro metaphase analysis in human lymphocytes were performed after exposure to total alpha-Ni3S2 suspension and to the soluble fraction in culture medium with or without fetal calf serum (FCS) (3-100 micrograms/ml, 24 h exposure). Nickel concentrations in the soluble fraction were determined by electrothermal atomic absorption spectrometry. A clastogenic effect of alpha-Ni3S2 became evident under all experimental conditions with a significant additional increase of chromosomal aberrations in 20% FCS complemented medium. No difference was observed between total suspension and soluble fraction. (iv) The micronucleus test confirmed the clastogenic effect of alpha-Ni3S2 in vivo after administration of 250 mg/kg (i.p.). This test revealed a clear increase of micronuclei frequency in polychromatic erythrocytes 24, 48 and 72 h after the treatment. In addition, we observed a statistically significant decrease in the number (%) of polychromatic erythrocytes after 24 and 48 h exposure. The soluble, i.e. cell-entering, fraction seems to play a great part in the clastogenic effect, as has also been shown with other test methods by other authors.