Induction of gene mutations and gene conversions by vinyl chloride metabolites in yeast.

Chloroethylene oxide and 2-chloroacetaldehyde, two metabolites of vinyl chloride, and 2-chloroethanol, a putative metabolic intermediate, were assayed for their genetic activity in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Chloroethylene oxide was found to be the most effective in inducing forward mutations in Sch. pombe and gene conversions in S. cerevisiae, increasing the mutation and conversion frequencies 340 and 50 times, respectively, over those of the controls. In either the presence or the absence of mouse liver microsomes, 2-chloroacetaldehyde showed only feeble genetic activity, and 2-chloroethanol was completely inactive in both yeast strains. In contrast to vinyl chloride, 2-chloroacetaldehyde did not induce forward mutations in Sch. pombe inthe host-mediated assay in mice. The results strongly support the hypothesis that chloroethylene oxide is one of the principal mutagenic agents formed from vinyl chloride in the presence of mouse liver enzymes.

Data selection and treatment of chemicals tested for genotoxicity and carcinogenicity.

A database containing qualitative and quantitative results of experimental studies in the fields of genotoxicity and carcinogenicity has been developed. By analyzing results of the studies performed by the U.S. National Toxicology Program, or by a similar program developed in Japan, or reported in the scientific literature, as well performed by private organizations, information has been collected relating to 3389 chemicals, identified by their CAS number. The studies considered for the database include three genotoxicity/mutagenicity short-term test (STTs), namely, two in vitro (Salmonella, gene mutation assay, and mammalian cells/human lymphocytes chromosome aberration assay) and one in vivo, the rodent bone marrow micronucleus assay. To investigate the possible predictive value of these STT assays for carcinogenicity, the results of animal long-term bioassays have also been collected. We have re-evaluated all the genotoxicity studies and the majority of those cases studied in different laboratories with contrasting results has been resolved; a small proportion of questionable cases is, however, still present in the database. In total, 2898 (85.5%) of the chemicals have been tested in the Salmonella assay; 1399 (41.3%) have been tested in the in vitro chromosome aberration assay; 319 (9.4%) have been tested in the in vivo rodent bone marrow cell micronucleus assay; 716 (21.2%) of the chemicals have been tested in the in vivo animal long-term bioassay. For 1118 chemicals tested in the Salmonella assay, 30,650 quantitative studies have been included in the database, thus allowing a possible classification of mutagenic chemicals according to their mutagenic potency.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenicity of airborne particles from a nonindustrial town in Italy.

The mutagenic activity of airborne particulate matter collected in Pisa, a small nonindustrial town located in Italy, has been monitored over 1 year using the Ames Salmonella Test. Airborne particulate was collected on fibreglass filters using a Hi-Vol sampler and extracted by sonication and Soxhlet acetone extraction in sequence. TA 98 and TA 100 salmonella strains gave positive results with the great majority of samples. The mutagenicity trend fits with a harmonic regression with a peak during December/January and inversely correlates with the temperature. No correlations were observed with other meteorological conditions such as wind, cloud, rainfall, atmospheric pressure, and humidity. The ratio between mutagenicity/microgram of particulate matter with S9 and that without S9 remains more or less constant regardless of seasonal fluctuations, suggesting that during cold months quantitative increases of mutagens onto particulate matter have probably occurred. The comparison of air mutagenicity in different sites suggests that motor vehicle exhaust fumes are the major source of air pollution. Finally, because of high-traffic volume, air mutagenicity at street level is comparable to that observed in several metropolitan areas all over the world.

Bone marrow cell chromosomal aberrations and styrene biotransformation in mice given styrene on a repeated oral schedule.

Styrene's capacity to induce chromosomal aberrations was studied in bone marrow cells of CD1 male mice. No mutagenic effect could be detected after either a 4-day treatment course with daily oral doses of 500 mg/kg or a 70-day course with daily oral doses of 200 mg/kg. Urinary elimination of styrene metabolites related to styrene-7,8-oxide formation (i.e. phenylethylene glycol, mandelic acid, benzoic acid, phenylglyoxylic acid and total mercapturic acids) was quantitatively evaluated in the group of mice given the 200 mg/kg dose. In parallel, kinetic studies were made on styrene and styrene-7,8-oxide blood concentrations in the same group of animals. These determinations were carried out on days 1 and 70 of treatment by spectrophotometric, gas chromatographic and mass fragmentographic procedures. Not even nanograms of styrene-7,8-oxide were found in the blood of styrene-treated mice. This suggests that the metabolite does not migrate from the cellular compartment where it is formed being immediately metabolized or irreversibly bound to cellular structures. This observation could well explain the lack of mutagenic effects observed.

Kinetics of chromosomal aberrations and first mitosis division in human lymphocytes exposed to mitomycin C.

In order to understand the relationship between the chromosomal damage detectable at the first mitosis after mutagen treatment and the induced mitotic delay we studied the time pattern of both mitotic indices and chromosomal aberration frequencies in human lymphocytes treated in G1 with mitomycin C (2.5 microM) and cultured in vitro in the presence of 5-bromo-2'-deoxyuridine. Mitotic delay was observed in treated cells cultured for 81 h. At this point an increase in the frequency of chromosomal aberrations is evident and a higher proportion of abnormal cells enters mitosis, the long delay being due to the extensiveness of DNA damage. The importance of cell cycle progression for the detection of the maximal amount of induced chromosomal damage is discussed.

Lack of genotoxic properties of the hair-dye component N-methyl-amino-2-nitro-4-N',N'-bis-(2-hydroxyethyl)-aminobenzene, in mammalian cells in vitro, and in yeasts.

N-Methyl-amino-2-nitro-4-N',N'-bis-(2-hydroxyethyl)-aminobenzene is a hair-dye ingredient. Its potential ability to induce gene mutations, in the yeast S. pombe and in cultured mammalian CH-V79 cells, mitotic gene conversion in the yeast S. cerevisiae, and unscheduled DNA synthesis in cultured human HeLa cells was evaluated. The chemical proved unable to induce detectable genotoxic effects according to these tests. The present data, together with others that show that the chemical is not mutagenic in Salmonella typhimurium or Drosophila, and is not clastogenic in mammalian cytogenetic assays (in vitro or in vivo), strongly support the non-genotoxicity of the chemical.

Mutations induced by X-rays and UV radiation during the nuclear cell cycle in the yeast Schizosaccharomyces pombe.

The availability of a cell-division-cycle (cdc) mutant in the fission yeast S. pombe, wee 1-50, has made possible the production of a large population of G1 nuclear-stage synchronized cells. During their development, yeast cells from the G1 into the G2 nuclear stages were treated with X-rays and UV radiation at various doses. The DNA pre-replicative and replicative phases were the most sensitive to both cell lethality and mutant induction with either X-rays or UV radiation. The trends of induced biological effects that were observed suggest that the induction of mutations is dependent on the number of unrepaired DNA lesions that reach the replicating fork or of those that occur at that time. The X-ray-induced mutations were earlier saturated, possibly because of the higher number of lethal lesions so induced.

A mutagenicity methodology for assessing the formation of N-dimethylnitrosamine in vivo.

The mutagenicity test methodology in vitro has been extensively used during recent years in the identification of potential carcinogenic agents. Mutagenic analyses have been applied to the study of chemical reaction products for the demonstration of the formation of mutagenic agents. Recent studies have indicated that secondary and tertiary amines, when reacted with nitrite in acidic conditions, yield N-nitroso compounds, including the potent carcinogen N-dimethylnitrosamine (NDMA). This finding raises the problem of risk evaluation of several food components of human diets for the presence of potential carcinogenic compounds. By combining a mutagenicity test procedure with yeast cells inoculated into the blood system of mice and incubated in the liver for various times (minutes or hours) we have devised a model methodology which allows the detection of the formation of N-dimethylnitrosamine (NDMA) at a level lower than 1 mg/kg. The methodology has been examined for its use in the study of activators of the nitrosation, such as thiocyanate, and of inhibitors of the nitrosation, such as ascorbic acid and tannic acid. Other food components of the human diet, such as red wine, have also been investigated by this methodology.

Benzene and the genotoxicity of its metabolites. I. Transplacental activity in mouse fetuses and in their dams.

Benzene and some of its metabolites (hydroquinone, phenol, catechol, 1,2,4-benzenetriol, p-benzoquinone, o,o'-biphenol, p,p'-biphenol) have been tested for their capability to induce micronuclei in bone marrow cells of pregnant mice and, transplacentally, in fetal liver cells. Dams are scarcely sensitive to the genotoxic activity of benzene and its metabolites while the latter are able to produce only evident toxic effects. Benzene and hydroquinone transplacentally induce micronuclei in fetal liver cells while all other metabolites show weak or negative genotoxicity, although they produce severe cellular toxicity.

Benzene and the genotoxicity of its metabolites. II. The effect of the route of administration on the micronuclei and bone marrow depression in mouse bone marrow cells.

Benzene (880 mg/kg) and 4 of its metabolites, i.e., phenol (265 mg/kg), hydroquinone (80 mg/kg), catechol (40 mg/kg), and p-benzoquinone (5-20 mg/kg) have been tested for their capability to induce micronuclei in bone marrow cells of male mice after oral administration or intraperitoneal injection. Oral administration of benzene shows more activity than intraperitoneal injection, whereas the metabolites show more activity if administered by the latter method. The respective genotoxic strengths of the benzene metabolites are the following: hydroquinone much greater than phenol greater than catechol = p-benzoquinone. This last is active when administered orally.

The human lymphocyte micronucleus assay: a comparison between whole-blood and separated-lymphocyte cultures.

The induction of micronuclei (MN) by vincristine, mitomycin C and cyclophosphamide was compared in purified lymphocytes and in whole-blood cultures. With both assays, cytokinesis was blocked by cytochalasin B and MN were only scored in binucleate cells. The data suggest that whole-blood cultures may be considered a better experimental condition for the detection of MN induced by chemicals in vitro.

Induction of chromosomal aberrations and SCE by chloramphenicol.

The induction of chromosomal aberrations and sister-chromatid exchanges (SCE) was studied in human lymphocyte cultures treated with chloramphenicol (CAP), an antimicrobial agent acting by inhibiting protein synthesis. Moreover chromosomal aberrations and sister-chromatid exchanges were studied in bone marrow cells of treated mice and in Chinese hamster cell cultures (V79) respectively. While no aberrations were induced by short treatments in human lymphocytes exposed in G1 and G2 phases, high frequencies of aberrations, exclusively of the chromatid type, were induced when the drug was administered during a whole cell cycle. Aberrant metaphases were detected only at the end and a few hours after the end of treatment; at later times aberrant cells reached control values. Doses producing aberrations only slightly increased SCE both in human lymphocytes and in V79 cells. In mouse bone marrow cells CAP induced a high mitotic delay and few structural aberrations; intrachromosomal vacuoles were observed.

Mutagenic activity of some coal-derived humic compounds evaluated by the Ames test.

Two coal-derived humic substances (Sulcis and South Africa, Eniricerche, Italy) have been evaluated for their mutagenic activity on TA98 and TA100 Salmonella typhimurium strains, either in presence or in absence of metabolic activation (S9). Both compounds showed no effect on the two strains, as observed with natural humic acid (Fluka). After chlorination, coal-derived humic acids induced a strong dose-related increase in the number of revertants on TA100 without S9, whose extent was directly proportional to the chlorination ratios. Such effect was completely suppressed when a sodium thiosulfate solution (10%) was added at the end of the chlorination period (about 90 h). The analogies with natural humic acid mutagenicity are discussed.

A review of the genetic toxicology of chlorinated dibenzo-p-dioxins.

Information from both published and unpublished sources considered relevant to the understanding of the genetic toxicology of chlorinated dibenzo-p-dioxins is summarized in this review. Interest in writing this paper was stimulated by the fact that this class of compounds, particularly 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has gained notoriety as an extreme environmental and industrial hazard. The potential for human exposure occurs in the work place when dioxins are formed during the synthesis of a number of commercially important compounds such as 2,4,5-trichlorophenoxyacetic acid, hexachlorophene, and pentachlorophenol. Environmental contamination may result from manufacturing processes and from dioxin contaminants in marketed products. Research on dioxins as potential mutagens was initiated because of their structural similarity to acridines, a class of known intercalating agents. To date, only 4 dioxin compounds have been evaluated for mutagenicity: the di-, tetra-, and octa-chlorinated derivatives and the unsubstituted dibenzo-p-dioxin. Since only a few of the many possible structural forms of dioxins have been tested, no definite conclusions can be made about their potential mutagenicity. Furthermore, the positive mutagenicity and cytological effects reported thus far with the few dioxin isomers examined seems to depend on the position of chlorine substitution. The most active form of the molecule is the 2,3,7,8-derivative (TCDD). Data available for assessing the mutagenic potential of TCDD are conflicting and scarce. Differences in testing results reported in these studies could be attributed to solubility problems with the test chemical, treatment protocols, purity of test samples, or toxicity. Because there are conflicting data, additional experiments are needed before the mutagenic potential of TCDD and other dioxins can be determined. Studies exploring the promoting effect of dioxins on the mutagenicity of other compounds are also recommended because experiments have shown TCDD to be an extremely active liver enzyme inducing agent that enhances the mutagenicity of certain polycyclic hydrocarbons such as 3-methylcholanthrene in vitro. The importance of discerning the hazards to human health from dioxin compounds became apparent after an accidental release of TCDD from a chemical plant contaminated the Seveso, Italy area in July 1976. This accident revealed that insufficient data were available to properly evaluate the long-term health risks posed by dioxin compounds. Several research projects were therefore initiated after the Seveso incident; it is hoped that many of the questions concerning the mutagenicity of TCDD and possibly of other dioxin congeners will be answered as a result of this work.

Mutagenic action of structurally related alkene oxides on Schizosaccharomyces pombe: the influence, 'in vitro', of mouse-liver metabolizing system.

A series of aliphatic epoxides were tested for their ability to induce forward mutations in the yeast Schizosaccharomyces pombe. For all the compounds under study, a linear dose-response relationship was found, and the ranking of the relative specific activity was: epichlorohydrin greater than ethylene oxide greater than glycidol greater than 1,2-epoxybutane greater than 1,1,1-trichloropropylene oxide greater than propylene oxide greater than 2,3-epoxybutane. The influence of the metabolic conversion of the epoxides by the mouse-liver S9 fraction was also investigated. In such conditions, except for the 2,3-epoxybutane, the genetic activity of epoxides seems to be reduced.

The intragastric host-mediated assay for the assessment of the formation of direct mutagens in vivo.

The intragastric host-mediated assay (h.m.a.) was devised and carried out with a view to assessing the formation of direct mutagens in the gastrointestinal tract of mammals. The h.m.a. consists in the injection of nitrosable compounds, NaNO2 and cells of the yeast S. pombe, by gavage into the animals' stomachs and in the recovery of the target cells from the faeces for mutation-induction analysis. Methylurea was chosen as a model nitrosable compound, and the effects of nitrosation modulators such as ascorbic acid and thiocyanate were studied. Cimetidine, a drug nitrosable in vitro, was tested with the system. Positive results were obtained only at very large doses and in artificially produced low pH. The new host-mediated assay seems to be efficient in revealing the formation, in vivo, of direct, short-living mutagens.

Mutagenic studies on the hair dye 2-(2',4'-diaminophenoxy)ethanol with different genetic systems.

A new hair-dye coupler, 2-(2',4'-diaminophenoxy)ethanol was analyzed for its potential mutagenic activity in different genotoxic assays, namely gene reverse mutations in Salmonella typhimurium, forward mutations in the yeast Schizosaccharomyces pombe, and in the V79 Chinese hamster cell line grown in vitro (HGPRT forward mutation system). Two other genetic test systems, measuring the mitotic gene conversion in Saccharomyces cerevisiae (strain D4) and the unscheduled DNA-repair synthesis in a HeLa cell line grown in vitro, were also used. 2,4-Diaminoanisole, a mutagenic/carcinogenic structurally related hair-dye coupler, and a group of well-known mutagens, namely methyl methanesulfonate, ethyl methanesulfonate, cychlophosphamide, hycanthone and N-nitrosodimethylamine, were used as positive controls. The new aromatic amine, 2-(2',4'-diaminophenoxy)ethanol, was negative in all the assays performed, under the same treatment conditions as in the case of all the positive controls.

In vivo genotoxic interactions among three phenolic benzene metabolites.

Three benzene metabolites, hydroquinone (HQ), cathecol (CAT) and phenol (PHE) were studied to define their possible interaction in inducing micronuclei (Mn) in mouse bone marrow polychromatic erythrocytes (PCEs). HQ and CAT, administered separately, induced Mn while PHE showed no genotoxic effects. Binary and ternary mixtures of two or three metabolites gave different results, causing considerable increase or decrease in Mn induction. HQ and PHE, in binary mixtures, as well as PHE and CAT, increased Mn synergistically, while HQ and CAT interacted negatively. The genotoxicity of ternary mixtures was mainly the consequence of two metabolites: HQ and CAT. The maximal effect obtained is far below the induction of Mn consequent to benzene treatment. These data suggest that toxic and genotoxic effects of benzene alone could be the result of more complex interactions among these and other metabolites.

Microgel electrophoresis assay (comet test) and SCE analysis in human lymphocytes from 100 normal subjects.

Microscopic examination of individual human lymphocytes embedded in agarose, subjected to electrophoresis and stained with a fluorescent DNA-binding dye, provides a novel way of measuring DNA damage as extent of migration of DNA fragments, mainly single-strand breaks. With this relatively simple method, DNA damage arising as a consequence of smoking, age and other factors was examined in peripheral human lymphocytes from 100 healthy individuals living in Pisa (Italy). The extent of DNA migration was found to be significantly increased by smoking. It is noteworthy that the effect of smoking was more significant in men than in women and that DNA migration was similar in the young and in the older people. SCE analysis did not reveal any significant effect of smoking, sex or age in the same population, suggesting a higher responsiveness of the comet test to DNA-damaging agents.

Comparative studies by comet test and SCE analysis in human lymphocytes from 200 healthy subjects.

The comet test (single cell gel electrophoresis, SCGE) appears to be a promising tool to estimate DNA damage at the single cell level and it provides information on the presence of damage among individual cells. Previously, we analyzed the degree of DNA damage in peripheral human lymphocytes from 100 healthy subjects living in Pisa (Italy) taking into account age, gender and smoking habit, and we also reported some results aiming at the assessment of the comet test (Betti el al., 1994). In addition, SCE analysis was carried out in order to compare the two endpoints. Because of the interesting results obtained, the present study was extended to 200 individuals, and data analyzed included information concerning number of cigarettes smoked a day, tar/cigarette and job. Data obtained confirmed that the SCGE is more sensitive than SCE in revealing smoking habit effects but comet induction did not seem to be related to the amount of cigarette tar inhaled. Moreover, sampling time was found to play a greater role in the comet assay as compared to SCE. Job position did not significantly influence SCE mean/subject or comet length mean/subject.