Genotoxicity of iron compounds in Salmonella typhimurium and L5178Y mouse lymphoma cells

The mutagenic activity of elemental and salt forms of iron (Fe), including compounds currently being used in dietary supplements and for food fortification, were evaluated for mutagenicity in Salmonella typhimurium and L5178Y mouse lymphoma cells. Except for the weak response obtained with ferrous fumarate, none of the compounds induced a mutagenic response in Salmonella. In the mouse lymphoma assay, responses were related to the Fe compound and/or reduction of ferric (Fe+3) to ferrous (Fe+2). Responses with the elemental forms of Fe were divergent. Electrolytic Fe with a relatively larger particle size and irregular shape was negative. The smaller-sized carbonyl Fe, which after 4 hr attached to and was taken up by the cells, induced mutagenic responses both with and without S9. With ferric chloride (FeCl3) and ferric phosphate (FePO4), there was an increase in mutant frequency only with S9. With the Fe+2 compounds, ferrous sulfate (FeSO4) and ferrous fumarate (FeC4H2O4), positive responses were observed without S9. The Fe chelate, sodium Fe(III)EDTA was positive in both the presence and absence of S9. The lowest effective doses (LED) for induction of mutagenicity were identified for these compounds and an LED ratio calculated. The LED ratio ranges from 1 for FeSO4 to 30 for carbonyl Fe, which are similar to oral LD50 values obtained in animal studies.     

A screening method for isolating DNA repair-deficient mutants of CHO cells

A simple procedure for isolating mutagen-sensitive clones of CHO cells was developed and applied in mutant hunts in which colonies were screened for hypersensitivity to killing by ultraviolet radiation (UV), ethyl methanesulfonate (EMS), or mitomycin C (MMC). Each of two UV-sensitive clones studied in detail had a D₃₇ dose of 1.0 J/m² compared to 7.0 J/m² for the wild-type cells, and each was shown to have no detectable repair replication following exposure to UV doses of up to 26 J/m². Although these mutants resemble xeroderma pigmentosum human mutants with respect to their repair defect and cross-sensitivity to the carcinogen 4-nitroquinoline-1-oxide, one of two clones (UV-20) is characterized by extreme hypersensitivity to MMC (80-fold as compared to the wild type). Clones having hypersensitivity to alkylating agents, but not UV, were obtained using MMC and EMS. In the latter case the two clones had significantly increased sensitivity to the killing action of⁶⁰Co -rays.Notice: This report was prepared as an account of work sponsored by the United States Government. Neither the United States nor the United States Department of Energy, nor any of their employees, nor any of their contractors, subcontractors, or their employees, makes any warranty, express or implied, or assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product or process disclosed, or represents that its use would not infringe privately owned rights.     

Development and characterization of CHO repair-proficient cell lines for comparative mutagenicity and metabolism of heterocyclic amines from cooked food

In order to understand the role of repair and metabolism in the mutagenicity of heterocyclic amines from cooked foods, we previously developed the nucleotide excision repair-deficient CHO 5P3NAT2 cell line engineered to coexpress the mouse CYP1A2 and human N-acetyltransferase genes. In the present study, we have made a matched repair-competent cell line by mutagenizing 5P3NAT2 cells with ethyl methanesulfonate and selecting for resistance to cytotoxicity by 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). In the differential cytotoxicity (DC) assay, 4 out of 15 clones showed no cytotoxic effect with IQ at the highest dose (30 microg/ml) tested, in contrast to repair-deficient 5P3NAT2 cells, which showed approximately 100% cytotoxicity at 0.3 microg/ml. Subsequently, these IQ-resistant clones were examined for resistance to killing by UV irradiation. All four IQ-resistant clones, which show resistance to UV similar to that of repair-proficient AA8 cells, still express both the CYP1A2 and N-acetyltransferase genes. Sequence analysis of CXPD cDNA from the 5P3NAT2R9 clone revealed an A:T-->G:C reversion event at the site of the UV5 mutation. This base change results in reversion of the codon 116 tyrosine in UV5 cells back to the original cysteine in AA8 cells, thereby restoring wild-type CXPD activity and repair function. In contrast to 5P3NAT2 cells, the repair-proficient 5P3NAT2R9 revertant cell line shows little IQ-induced cell killing, and dramatically lower levels of induced mutation at the adenine phosphoribosyltransferase (Aprt) gene locus over the range of 2-40 microg/ml IQ. This matched pair of repair-proficient/deficient cell lines can provide insight not only into the genotoxicity of heterocyclic amine dietary carcinogens such as IQ and PhIP, but also into the effects of nucleotide excision repair on the ultimate mutagenicity of these compounds.     

A method for studying the mutagenicity of some gaseous compounds in Salmonella typhimurium

A dynamic flow-through exposure system was designed for mutagenicity studies of gaseous compounds in Salmonella. Salmonella typhimurium strain TA100 was the primary tester strain. The dose ranges were 0.5-20% of vinyl chloride, ethene, propene, and 1,3-butadiene, 1-200 ppm of ethylene oxide, 0.5-20 ppm of nitrogen dioxide, and 0.1-3.5 ppm of ozone. The gas flow rate was 250, 500, or 1,000 ml/min, and the exposure time was 6 or 7 hours. Of the tested gases, vinyl chloride, ethylene oxide, and nitrogen dioxide were mutagenic. Ethene, propene, and 1,3-butadiene were not mutagenic in this system. Ozone is bacteriotoxic, and no mutagenic effect could be demonstrated in the nontoxic dose range. The exposure system was considered suitable for studies on gaseous chemicals.     

1,8-Dinitropyrene: comparative mutagenicity in Chinese hamster V79 and CHO cells

1,8-Dinitropyrene (1,8-DNP) was clearly mutagenic at the hprt locus in CHO cells, but not detectably mutagenic in V79 cells, following a 3-h treatment period. Preliminary data indicate that CHO, but not V79, cells have measurable levels of N-acetyltransferase activity, and this may contribute to the differential sensitivity of the two cell lines to the mutagenicity of 1,8-DNP.     

Prediction of Salmonella mutagenicity

The ability of a number of prediction systems was examined todetermine how well they could predict Salmonella mutagenicity.Theprediction systems included two computer-based systems (CASE⁰and TOPKAT⁰), the measurement of a physiochemical parameter(k_e) and the use of structural alerts by an expert chemist.The computer based systems operators and the chemist were suppliedwith the structures of 100 chemicals that had been tested formutagenicity in the Salmonella test; the actual chemicals wereneeded for the physiochemical measurement. None of the participantswas provided with the chemical names or Salmonella test resultsprior to submitting their predictions. The three systems thatpredicted the mutagenicity from the structure of the chemicalsproduced equivalent results (71–76% concordance with theSalmonella results); the physiochemical system produced a lower(60–61 %) concordance. ⁷To whom correspondence should be addressed at: WC-05, NIEHS, PO BOX 12233, Research Triangle Park, NC 27709, USA     

LUMO energies and hydrophobicity as determinants of mutagenicity by nitroaromatic compounds in Salmonella typhimurium

Quantitative structure-activity relationships have been derived for the mutagenic activity of 47 nitroaromatic compounds acting on Salmonella typhimurium (TA100) and 66 acting on TA98. The mutagenicity is linearly dependent on the energy of the lowest occupied molecular orbital and bilinearly dependent on the hydrophobicity (octanol/water log P) of the mutagens. The mechanism of action is considered in the light of these findings.     

Genotoxicity of vanadium compounds in yeast and cultured mammalian cells.

The ability of vanadium compounds to induce genetic activity was investigated in D7 and D61M strains of Saccharomyces cerevisiae and in Chinese hamster V79 cell line. In our previous work, ammonium metavanadate (pentavalent form, V5) induced mitotic gene conversion and point reverse mutation in the D7 strain of yeast. The genotoxicity was reduced by the presence of S9 fraction, which probably reduced pentavalent vanadium to the tetravalent form. In the present study, vanadyl sulfate (tetravalent form, V4) induced no convertants and revertants in yeast cells harvested from stationary growth phase. With yeast cells from logarithmic growth phase, which contain high levels of cytochrome P-450, a significant increase in genetic effects was observed. Further experiments, performed by treating cells harvested from logarithmic growth phase in the presence of cytochrome P-450 inhibitors, indicated that the monooxygenase system influenced the genotoxicity of metavanadate while the genetic activity of vanadyl remained unaffected. Aneuploidy effect in the D61M strain of Saccharomyces cerevisiae was induced by either V5 or V4, confirming that vanadium compounds are potentially antitubulin agents in eukaryotic cells. Although these compounds are very toxic in V79 cells, no mutagenic effect was observed in the presence or in the absence of S9 fraction.     

SOS induction in Escherichia coli and Salmonella mutagenicity: a comparison using 330 compounds

To examine the concordance of two microbial genotoxicity short-termassays, 330 experimental results for the SOS chromotest usingtester strain Escherichia coli PQ37 were compared with the resultsof the Salmonella/mammalian microsome mutagenicity assay withSalmonella typhimurium TA97, TA98, TA100, TA102, TA104, TA1535,TA1537 and/or TA1538. With respect to qualitative features,the concordance between SOS chromotest and Salmonella mutagenicitytest results was 86.4% (sensitivity, 78.6%; specificity, 100%;     

Genotoxicity of two arsenic compounds in germ cells and somatic cells of Drosophila melanogaster

Two arsenic compounds, sodium arsenite (NaAsO₂) and sodium arsenate (Na₂HAsO₄), were tested for their possible genotoxicity in germinal and somatic cells of Drosophila melanogaster. For germinal cells, the sex-linked recessive lethal test (SLRLT) and the sex chromosome loss test (SCLT) were used. In both tests, a brood scheme of 2–3–3 days was employed. Two routes of administration were used for the SLRLT: adult male injection (0.38, 0.77 mM for sodium arsenite; and 0.54, 1.08 mM for sodium arsenate) and larval feeding (0.008, 0.01, 0.02 mM for sodium arsenite; and 0.01, 0.02 mM for sodium arsenate). For the SCLT the compounds were injected into males. Controls were treated with a solution of 5% sucrose which was employed as solvent. The somatic mutation and recombination test (SMART) was run in the w⁺/w eye assay as well as in the mwh +/+ flr³ wing test, employing the standard and insecticide-resistant strains. In both tests, third instar larvae were treated for 6 hr with sodium arsenite (0.38, 0.77, 1.15 mM), and sodium arsenate (0.54, 1.34, 2.69 mM). In the SLRLT, both compounds were positive, but they were negative in the SCLT. The genotoxicity of both compounds was localized mainly in somatic cells, in agreement with reports on the carcinogenic potential of arsenical compounds. Sodium arsenite was an order of magnitude more toxic and mutagenic than sodium arsenate. This study confirms the reliability of the Drosophila in vivo system to test the genotoxicity of environmental compounds. © 1995 Wiley-Liss, Inc.     

A test of the mutagenicity of cooked meats in vivo

There is a correlation between intestinal cancer and diets high in meat, so fried beef, chicken, lamb, pork and fish were tested for their ability to induce mutations in the small intestine of mice. The mice were bred to be heterozygous at the Dlb-1 locus so that loss of the dominant Dlb-1 b allele by mutation could be detected. Mice were fed the AIN-76A diet (which contains 50% of the calories in the form of sucrose) or an isocaloric diet in which the sucrose was replaced by meat or fish, for 5 or 9 weeks. Manifestation of mutants requires approximately 1 week in this system, so this corresponds to an effective exposure of 4 and 8 weeks, respectively. There was no significant difference in the weights of animals on the different diets, and no difference in mutant frequency. Several food mutagens were present, but at low levels. These results, when considered in the light of tests of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine and amino(alpha)carboline at much higher doses (Zhang,X.-B., Tao,K.S., Urlando,C., Shaver-Walker,P. and Heddle,J.A. (1996) MUTAGENESIS:, 11, 43-48), indicate that there is no highly mutagenic compound missed by previous testing with bacterial assays and that mixtures of heterocyclic amines at low levels do not show great synergy.     

Salmonella/human S9 mutagenicity test: a collaborative study with 58 compounds

A large and extensive body of data on the use of human liver S9 fractions in the Salmonella mutagenicity test (Ames test) is presented; the data were obtained from a collaborative study by JEMS/BMS (Bacterial Mutagenicity Test Study Group) members and the Human and Animal Bridging Research Organization (HAB). In this study, the mutagenicity of 58 chemicals, many of which were judged to be human carcinogens by the IARC, was determined by the Ames test (the pre-incubation method at 37 degrees C for 20 min) in the presence of a selected human liver S9 fraction with a high drug-metabolic activity or a pooled human liver S9 fraction with a moderate drug-metabolic activity. For reference, mutagenicity was also examined in the presence of a phenobarbital/5,6-benzoflavone-pretreated rat liver S9 fraction, which is normally used in mutagenicity testing systems. The bacterial test strains consisted of Salmonella typhimurium TA100, TA98 or YG7108. The data indicated that the mutagenicity of chemicals in the rat and human liver S9 fractions varied considerably, depending on the chemicals in question. In addition, a large inter-individual diversity in the mutagenic response to mutagens, depending on the chemical structures of the mutagens, was also demonstrated using two selected human S9 fractions. Most of the mutagens tested in this study (75%; 36 out of 48 compounds that were judged to be mutagenic in at least one S9 fraction) were less mutagenic in the presence of the two human S9 fractions than in the presence of the rat S9 fraction. On the other hand, the other compounds (25%), including some aromatic amines and nitrosamines, showed a more potent mutagenicity in the presence of either one of the two human S9 fractions than in the presence of the rat S9 fraction. These data strongly suggest that the use of human liver S9 fraction in mutagenicity testing systems may be useful for a better understanding of the mutagenic effects of chemicals on humans.     

Comparison of mutagenicity results for nine compounds evaluated at the hgprt locus in the standard and suspension CHO assays.

The Chinese hamster ovary (CHO) assay, which measures newly induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus, has been widely used for mutagenesis testing. The insensitivity of the standard assay to some genotoxic agents has been speculated to be due to the relatively small number of cells used in the assay. In the present study, we have compared the standard monolayer assay with a suspension adapted CHO assay that uses cell numbers comparable to that of the L5178Y mouse lymphoma assay. Nine compounds, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), 2-methoxy-6-chloro-9-[3-(ethyl-2-chloroethyl)-aminopropylamino]-acridine 2HCl (ICR 170), methyl acrylate, ethyl acrylate, tetraethylene glycol diacrylate, trimethylolpropane triacrylate, 2-ethylhexyl acrylate and dicyclopentenyloxyethyl methacrylate were evaluated in the monolayer and suspension assays. Both assays gave the same overall qualitative results for the test compounds. There were some quantitative differences in the mutant frequency for the three compounds found to be mutagenic (EMS, MMS and ICR 170). The acrylates (many of which appear to exert their genotoxic effect through a clastogenic mechanism) were negative in both test systems. The use of the suspension assay did not improve the ability of the hgprt locus to detect the genotoxicity of the acrylates. Thus, increasing the number of cells does not improve the ability of the CHO/HGPRT assay to detect compounds that act primarily by a clastogenic mechanism.     

Conditions affecting the mutagenicity of trichloroethylene in Salmonella

Trichloroethylene (TCE) is a high production volume chemical frequently stabilized with oxiranes. These oxiranes may be responsible for the mutagenic activity of TCE in Salmonella, which has been occasionally, but not consistently, reported. High purity and oxirane-stabilized TCE samples were tested for their mutagenic potential in Salmonella typhimurium strains TA 1535, TA 98, and TA 100. Stabilized TCE was tested using a preincubation protocol up to a dose level of 10,000 micrograms per plate, but no mutagenic response was observed in either the presence or absence of a supplementary metabolic activation system (S9 mix) derived from Aroclor 1254-induced male rat liver. TCE without oxirane stabilizers also was nonmutagenic when tested in a vapor delivery system at nominal concentrations of up to 20% and using S9 mix derived from either rat or hamster. TCE containing 0.5-0.6% 1,2-epoxybutane did induce mutagenic responses from strains TA 1535 and TA 100 in the presence and absence of S9 mix. The lowest effective dose was about 0.63% in TA 1535 in the absence of S9 mix. Vapor-phase tests with 1,2-epoxybutane showed that an atmospheric concentration of 0.009% could induce 12-fold and 3-fold increases, respectively, in strains TA 1535 and TA 100. These increases would account for the mutagenic activity of the stabilized TCE sample. Epichlorohydrin (another commonly used stabilizer) induced similar increases in mutant numbers at an atmospheric concentration of 0.0009%. The absence of a significant response caused by unstabilized TCE in the presence of S9 mix is probably due to a lack of assay sensitivity, since chloral, a metabolite of TCE, is a mutagen in TA 100 [Haworth et al.: Environ Mutagen [Supplement 1] 5:3-142, 1983].     

Cytotoxicity and mutagenicity of coal oils in the CHO/HGPRT assay

We used the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl-transferase (CHO/HGPRT) assay to determine the cytotoxicity and mutagenicity of a crude coal oil, the neutral fraction of this crude, and the following three subfractions of the neutral fraction: aliphatic, neutral polar, and a subfraction composed of polycyclic aromatic hydrocarbons plus neutral nitrogen heterocyclics. We also studied the cytotoxicity and mutagenicity of a blend of light and heavy coal-derived fuel oils before and after hydrogenation. All seven mixtures were highly cytotoxic to CHO cells, but the addition of S9 reduced the cytotoxicity. Also, hydrogenation reduced the cytotoxicity of the blend of coal-derived fuel oils. Although highly cytotoxic, none of the seven mixtures induced a clear mutagenic response in the CHO/HGPRT assay. However, previous work has shown that all of the mixtures except the aliphatic subfraction and the blend after hydrogenation are mutagenic in the histidine-reversion assay in Salmonella typhimurium. Based on chemical analyses of the mixtures, the differential sensitivity of Salmonella and CHO cells to nonmutagenic cytotoxins, and studies of the neutral fraction to which additional benzo[a]pyrene had been added, we conclude that the disparity between the results in Salmonella and those obtained in the CHO/HGPRT assay is probably due to the much greater sensitivity of CHO cells (relative to Salmonella) to the cytotoxins in these coal oils. This sensitivity, coupled with the low concentrations of mutagens relative to nonmutagenic cytotoxins in the coal oils, prevents exposure of the cells to concentrations of the mutagens in the mixtures that are high enough to be quantified in the CHO/HGPRT assay.     

The mutagenicity of 45 nitrosamines in the Salmonella typhimurium

The correlation between mutagenicity in the rat liver microsome--mediated Salmonella Mutagenicity Assay of Ames and carcinogenicity in rats was examined with three groups of nitrosamines. Qualitatively the correlation was good, but there was poor correlation between mutagenic potency and carcinogenic potency. Of 23 cyclic nitrosamines, 19 were carcinogenic and mutagenic, and two were carcinogenic but not mutagenic, and the carcinogenicity studies of the remaining two are not complete. Of six symmetrical aliphatic nitrosamines, five were carcinogenic and mutagenic while only one carcinogen was not mutagenic. The greatest discrepancy occurred among 16 asymmetric nitrosamines, where 11 were both carcinogenic and mutagenic, four were carcinogenic but nonmutagenic, and one carcinogenicity study is incomplete.     

Genotoxicity studies of methyl isocyanate in Salmonella, Drosophila, and cultured Chinese hamster ovary cells

The genotoxic effects of methyl isocyanate (MIC) were investigated using four short-term tests: the Salmonella reversion assay (Ames test), the Drosophila sex-linked recessive lethal assay, and the sister chromatid exchange (SCE) and chromosomal aberration assays in cultured Chinese hamster ovary (CHO) cells. No evidence was found for the induction of mutations in either Salmonella or Drosophila. MIC did, however, induce SCEs and chromosomal aberrations in CHO cells both in the presence and absence of Aroclor-induced rat liver S-9.     

Cytotoxicity and mutagenicity of dimethylnitrosamine in mammalian cells (CHO/HGPRT system): Enhancement by calcium phosphate

The cytotoxicity and mutagenicity of dimethylnitrosamine (DMN) was determined in the CHO/HGPRT system. Metabolic activation of the promutagen was achieved by use of a liver homogenate supernatant (S9) prepared from Aroclor 1254-induced Sprague-Dawley rats. The cytotoxic and mutagenic effects of DMN were enhanced by the inclusion of calcium chloride in the incubation mix, and this enhancement was dependent on the presence of sodium phosphate. Under conditions that yielded maximal activity (10 mM calcium chloride, 10 mM magnesium chloride, 50 mM sodium phosphate), an apparent calcium phospate precipitate was observed. DMN activity increased with increasing amounts of S9 protein over the range 0.3–3.0 mg/ml in the S9 mix and appeared to plateau at higher concentrations. The mutagenicity of DMN can be described as 110 mutants/10⁶ cells per mM DMN per mg/ml S9 protein per hour.     

Cytotoxicity and mutagenicity of the fungicides captan and folpet in cultured mammalian cells (CHO/HGPRT system)

The cytotoxicity and mutagenicity of the fungicides captan and folpet were determined in the CHO/HGPRT system which utilizes Chinese hamster ovary cells and resistance to 6-thioguanine to estimate mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus. Treatment of cultures with each compound for 5 hr in serum-free medium resulted in reproducible, significant, concentration-dependent increases in the frequency of 6-thioguanine-resistant mutants.