The influence of automobile exhausts on mutagenicity of soils: contamination with, fractionation, separation, and preliminary identification of mutagens in the Salmonella/reversion assay and effects of solvent fractions on the sister-chromatid exchanges in human lymphocyte cultures and in the in vivo mouse bone marrow micronucleus assay

To test the assumption that automobile exhausts contribute to soil mutagenicity, two soils with low levels of mutagenic activities were exposed to traffic exhausts at a heavily charged junction of German motorways (Autobahnen) for 3, 7, 10, 13, 17, 21, and 26 weeks. Indeed, in the presence of a metabolic activation system from rat liver (S9), an average increase of 8 and 9 (4 and 12) revertants per gram per week was found in Salmonella typhimurium TA 98 (TA 100). In the absence of S9, meaningful measurements were impossible on account of a concurrent dose dependent increase of toxicity. No correlation between the increase of mutagenicity and the contents of polycyclic aromatic hydrocarbons (PAH) could be detected. In another series, soils sampled at the roadside and at distances of 10 and 50 m of five roads near Mainz expressed 10–20-fold higher mutagenicity (revertants per gram) under identical test conditions as compared with the average of agricultural soils. Toxic effects, however, again confounded the results and no correlation between the distance from roads and the levels of mutagenicity could be demonstrated. Subsequently, Soxhlet-extraction with the solvent sequence dichloromethane, acetone, and toluene/diethylketone was found to be an optimum procedure for soils at roadsides. The mass balance of solvent fractionation of such soils revealed that <2% each belonged to organic acids and bases, 4% to fractions designed polar neutrals, 8% to polar aromatics, 7% to dichloromethane solubles, and 79% to cylohexane solubles, among them 63% acetone soluble compounds. The major part of mutagenicity (55–65%) was present in the fraction of polar aromatics, followed by polar neutrals and the acetone subfraction of cyclohexane solubles (10% each) summarizing the results obtained with S. typhimurium TA 98, TA 98NR, YG 1021, YG 1024, TA 100, YG 1026, and YG 1029 with and without addition of S9. The modified tester strains, either deficient in nitroreductase (TA 98NR) or overproducing nitroreductase (YG 1021, 1026) or O-acetyl-transferase (YG 1024, 1026), indicated a major contribution of nitroarenes to soil mutagenicity. With respect to mutagenic PAH, high pressure liquid chromatography (HPLC) revealed that >90% of dibenz[a,h]anthracene (4.18 mg/kg soil), benzo[a]pyrene (1.96 mg), benzofluoranthenes (0.14 mg), and benz[a]anthracene (0.18 mg) were present in the acetone subfraction of cyclohexane solubles. Concentrations and mutagenic activities, however, did not correlate. Additional preparative and analytical HPLC of the solvent fractions of polar neutrals and polar aromatics, resulted in the tentative identification of 2-nitrofluorene. Analysis of the vertical profile of soil revealed an increase of mutagenicity per gram from the surface to a maximum at 5–15 cm depth and a subsequent decrease with very little activity remaining deeper than 35 cm. In human lymphocyte cultures, the fraction of polar aromatics, 0.01–0.3 μg/ml, induced 11.27±4.76–20.70±6.19 sister-chromatid exchanges (SCE) per cell in the absence of S9 (solvent control: 10.16±4.83 SCE per cell) and 12.77±6.53–17.87±4.93 SCE per cell in the presence of S9 (solvent control: 8.37±3.92 SCE per cell). However, no activities could be detected in the fractions of polar neutrals and non-polar neutrals. Again, negative results were obtained in the in vivo mouse bone marrow micronucleus assay at 2000 mg/kg p.o. with all fractions.     

Induction of chromosome damage and sister-chromatid exchanges in human lymphocyte cultures by the antitumour antibiotic Neocarzinostatin

Neocarzinostatin (NCS), a chemotherapeutic antibiotic, was investigated for the ability to induce chromosomal aberrations and sister-chromatid exchanges (SCEs) in human lymphocyte cultures. It was observed that the antibiotic causes a cell-cycle delay and reduces the mitotic index. Analysis of the induced chromosomal abnormalities showed that they are mainly chromosome and chromatid breaks; while the frequency of SCEs was increased, the magnitude indicates that NCS cannot be considered a potent inducer of SCEs.     

Chromosomal aberrations, sister-chromatid exchanges, cell-cycle kinetics and satellite associations in human lymphocyte cultures exposed to vanadium pentoxide

Treatment of human lymphocytes with vanadium pentoxide (V2O5) was not found to increase the frequency of structural chromosomal aberrations (CA) and sister-chromatid exchanges (SCE). However, V2O5 significantly increased polyploid cell frequency; also, the mitotic index (MI) was significantly decreased, and the average generation time (AGT) was significantly increased. Finally, the frequency of cells with satellite associations (SA), the frequency of SA per cell, and the frequency of chromosomes associated increased in treated cultures.     

Erythrocytes modulate the baseline frequency of sister-chromatid exchanges and the kinetics of lymphocyte division in culture

The baseline sister-chromatid exchange (SCE) frequencies of human plasma lymphocyte cultures (PLC), but not pig PLC, were nearly twice as high as those of whole-blood cultures (WBC). Addition of human red blood cells (RBCs) to human PLC decreased the SCE frequency in proportion to the RBC-leukocyte co-incubation interval. When the period of RBC-leukocyte co-incubation was equivalent to the total length of the culture period (72 h), the SCE frequency was similar to that observed in WBC. Shorter co-incubation periods yielded SCE frequencies intermediate between those of PLC and WBC. Regardless of the species, cell proliferation was slower in PLC than in WBC. Experiments where RBCs were added to PLC showed that the time sequence of RBC incorporation also affects the cell-cycle progression of human and pig lymphocytes. When either human or pig RBCs were added immediately after PLC stimulation, the cell-cycle kinetics was similar to that of WBC. Shorter co-incubation periods made cell-cycle progression intermediate between PLC and WBC values. Thus, PBCs modulate the baseline frequency of SCEs in human PLC and the cell-cycle progression of both human and pig lymphocytes in a time-dependent manner. Two possible hypotheses for the heightened frequency of SCEs of human lymphocytes in RBC-free cultures were assessed. The loss of RBC-to-lymphocyte cellular contact in PLC did not influence the SCE frequencies of lymphocytes. Finally, the increase of SCEs in human PLC could not be related to differences in the generation time of lymphocytes in culture.     

Induction of sister-chromatid exchanges (SCE), polyploidy, and micronuclei by plant flavonoids in human lymphocyte cultures. A comparative study of 19 flavonoids

Nineteen naturally occurring flavonoids were studied with regard to their SCE-inducing potency and their capability of inducing polyploidy and micronuclei in human lymphocyte cultures. The cells were treated for a period of 48 h. The flavone C-glycosides, vitexin and orientin, exhibited a moderate SCE-inducing activity, whereas the other compounds displayed only weak effects or were inactive. Polyploidy was induced by procyanidins consisting of 3 or 4 flavanol units and to a lesser extent by flavone, flavonol, and anthocyanidin aglycones. The aglycones as well as the C-glycosides and the O-glycosides, spiraeoside and luteolin-7-glucoside, were more or less active in inducing micronuclei in the lymphocytes. The flavonol O-glycosides, rutin and hyperoside, and the monomeric and dimeric flavanols failed to produce any genotoxic effects. The results are discussed with respect to a possible structure-activity relationship.     

Comparative study of sister-chromatid exchanges and chromosomal aberrations induced by airborne particulates from an urban and a highly industrialized location in human lymphocyte cultures

Cytogenetic effects induced by extracts of airborne particulates in human lymphocyte cultures were studied with regard to local and seasonal variations. Samples of airborne particulates were collected from an urban and a highly industrialized area in March and October, respectively. All extracts of particulates induced a significant increase of sister-chromatid exchanges (SCE) in a dose-dependent manner. Referring to the induction of sister-chromatid exchanges, local and seasonal differences were observed. Samples from the industrialized area revealed the highest activities. In addition, SCE rates found for March samples were always higher than those for October for both locations. Furthermore, a remarkable, significant induction of chromosomal aberrations occurred with all samples from both locations and sampling periods. Aspects of health risk evaluation for exposed human populations are discussed with respect to the observed cytogenetic effects of airborne particulates in human lymphocyte cultures.     

Evaluation of the mutagenicity and antimutagenicity of Ziziphus joazeiro Mart. bark in the micronucleus assay

The aim of this study was to evaluate the mutagenicity (clastogenicity/aneugenicity) of a glycolic extract of Ziziphus joazeiro bark (GEZJ) by the micronucleus assay in mice bone marrow. Antimutagenic activity was also assessed using treatments associated with GEZJ and doxorubicin (DXR). Mice were evaluated 24–48 h after exposure to positive (N-nitroso-N-ethylurea, NEU - 50 mg.kg⁻¹ and DXR - 5 mg.kg⁻¹) and negative (150 mM NaCl) controls, as well as treatment with GEZJ (0.5–2 g.kg⁻¹), GEZJ (2 g.kg⁻¹) + NEU and GEZJ (2 g.kg⁻¹) + DXR. There were no significant differences in the frequencies of micronucleated polychromatic erythrocytes in mice treated with GEJZ and GEJZ + DXR compared to the negative controls, indicating that GEZJ was not mutagenic. Analysis of the polychromatic:normochromatic erythrocyte ratio revealed significant differences in the responses to doses of 0.5 g.kg⁻¹ and 1–2 g.kg⁻¹ and the positive control (NEU). These results indicated no systemic toxicity and moderate toxicity at lower and higher doses of GEZJ. The lack of mutagenicity and systemic toxicity in the antimutagenic assays, especially for treatment with GEZJ + DXR, suggested that phytochemical compounds in Z. joazeiro bark attenuated DXR-induced mutagenicity and the moderate systemic toxicity of a high dose of Z. joazeiro bark (2 g.kg⁻¹). Further studies on the genotoxicity of Z. joazeiro extracts are necessary to establish the possible health risk in humans and to determine the potential as a chemopreventive agent for therapeutic use.     

Inhibition of mitomycin C-induced sister-chromatid exchanges in mouse bone marrow cells by the immunopotentiators Krestin and Lentinan

We have carried out an investigation to determine whether or not the sister-chromatid exchange frequencies (SCEs) observed in bone marrow cells in mice treated with mitomycin C (MMC) are inhibited by the immunopotentiators Krestin and Lentinan. We found that mitomycin C (2 mg/kg, i.v.)-induced SCEs were inhibited in 27% of the mice treated with Krestin (300 mg/kg, i.p.) and in 23% of the mice treated with Lentinan (1 mg/kg, i.p.). The effects of Krestin were found to be dose-dependent in inhibition of MMC-induced SCEs while those of Lentinan were not. Our findings therefore suggest that Krestin and Lentinan are not only useful for cancer treatment as immunopotentiators in combination with anticancer drugs but may also prevent the increase of chromosomal damage induced by anticancer drugs.     

Activation of mutagens by hepatocytes and liver 9000 X g supernatant from human origin in the Salmonella typhimurium mutagenicity assay. Comparison with rat liver preparations

The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.     

Effects of pH on weak and positive control mutagens in the Ames Salmonella plate assay

The effects of pH on the mutagenic activity of several chemicals were evaluated in the standard Ames Salmonella typhimurium plate-incorporation assay. The pH of the base agar was varied between 6.0 and 8.0. The positive control compounds routinely used in this laboratory, 2-aminoanthracene, 4-nitro-o-phenylenediamine, sodium azide and nitrofurantoin, showed increasing mutagenic activity as the pH was decreased to 6.0. However, the activity of two weakly mutagenic cosmetic ingredients, 2,2',4,4'-tetrahydroxybenzophenone and trans-4-phenyl-3-buten-2-one, was completely eliminated at pH levels near 6.0. It is concluded that plates poured with agar with pH levels below 7.0 can result in strong responses for the positive control chemicals but give negative results for some mutagens.     

Comparison of the induction by cigarette smoke condensates of sister-chromatid exchanges in Chinese hamster cells and of mutations in Salmonella typhimurium

Three cigarette smoke condensates were tested for the induction of sister-chromatid exchanges in ovary cells of the Chinese hamster and for mutations in Salmonella typhimuriumIn the sister-chromatid exchange test an effect was obtained that was not enhanced by the inclusion of a system for metabolic activation. In the Salmonella test, an effect was only obtained by including rat-liver homogenates derived from rats treated with inducers of the enzyme systems necessary for metabolic activation.It appears that the SCE test and the Salmonella test are sensitive to different components of cigarette smoke condensates.     

Mutagenic and genotoxic effects of theophylline and theobromine in Salmonella assay and in vivo sister chromatid exchanges in bone marrow cells of mice.

The mutagenic and genotoxic effects of two methylxanthines, theophylline (TH) and theobromine (TB), were assessed in the Ames mutagenicity assay (in strains TA97a, TA100, TA102 and TA104) and in vivo sister chromatid exchanges (SCEs) in bone marrow cells of mice. These are the two most commonly used nervous system stimulators throughout the world. TH is used in the long-term treatment of asthma. Bacterial mutagenicity assay showed very weak mutagenic effects of both drugs in Salmonella strains TA102 and TA104 only in certain concentrations when S9 was added to it. No mutagenic effects were observed in any other strains used in this assay either with or without metabolic activation. But results of in vivo SCE assay indicate that these two drugs can induce significant SCE in bone marrow cells of mice.     

Comparison of the induction by cigarette smoke condensates of sister-chromatid exchanges in Chinese hamster cells and of mutations in Salmonella typhimurium.

Three cigarette smoke condensates were tested for the induction of sister-chromatid exchanges in ovary cells of the Chinese hamster and for mutations in Salmonella typhimurium. In the sister-chromatid exchange test an effect was obtained that was not enhanced by the inclusion of a system for metabolic activation. In the Salmonella test, an effect was only obtained by including rat-liver homogenates derived from rats treated with inducers of the enzyme systems necessary for metabolic activation. It appears that the SCE test and the Salmonella test are sensitive to different components of cigarette smoke condensates.     

Comparison of urethane-induced sister-chromatid exchanges in various murine strains, and the effect of enzyme inducers

The induction of sister-chromatid exchanges (SCEs) by urethane, 150 and 300 mg/kg administered i.p., was examined in bone-marrow cells of AKR, BALB/c, C3Hf, C57BL/6J and DBA/2 male mice. In all strains, the base-line level of SCE/cell was similar, ranging from 4.3 to 8.7, and the response increased with the dose of urethane. DBA/2 mice were the most susceptible to urethane at both dose levels, with 30.6 SCE/cell after treatment with 300 mg/kg, whereas the response of the other strains was from 17.4 to 21.5 SCE/cell at the same urethane dose. Pretreatment of C57BL/6J and DBA/2 mice with phenobarbital decreased the SCE frequencies induced by urethane, 300 mg/kg, to 70%, whereas a prior administration of beta-naphthoflavone reduced SCE levels in C57BL/6J but not in DBA/2 mice.     

Inhibition of dinitropyrene mutagenicity in vitro and in vivo using Salmonella typhimurium and the intrasanguinous host-mediated assay

Dinitropyrenes (DNP), present in polluted air, are potent direct-acting mutagens in Salmonella typhimurium TA98. This mutagenicity is markedly reduced in the presence of rat-liver S9 or microsomes. This has now been confirmed using mouse hepatic fractions. Since most in vitro test systems do not adequately simulate conditions encountered in the intact animal, we have investigated dinitropyrene mutagenicity to Salmonella in the host-mediated assay. 1,8-Dinitropyrene (1,8-DNP) given p.o. to BALB/c mice induced a weak mutagenic effect in S. typhimurium TA98 recovered from the liver 1 h after i.v. administration (optimum time). Over the entire dose range tested no toxicity to bacterial cells was detected. Mutation induction in vivo was dose-related with maximum response at 1 mg DNP/kg body weight. This optimum dose, however, was non-mutagenic to strains TA98/1,8-DNP6 (O-transacetylase-deficient) or TA98NR/1,8-DNP6 (nitroreductase- and O-transacetylase-deficient). 1,3-Dinitropyrene and 1,6-dinitropyrene were weakly mutagenic to TA98 at doses similar to 1,8-DNP. Studies with [14C]1,8-DNP showed that 1 h after oral dosing (1 mg/kg), over 100 ng of 1,8-DNP equivalents were present in the liver (= 0.73% dose). However, only about 5.5 ng were present in the bacterial pellet, suggesting that hepatic components in vivo, as in vitro, bind to DNP, thus interfering with its interaction with Salmonella.     

Evaluation of a multi-endpoint assay in rats, combining the bone-marrow micronucleus test, the Comet assay and the flow-cytometric peripheral blood micronucleus test

With the publication of revised draft ICH guidelines (Draft ICH S2), there is scope and potential to establish a combined multi-end point in vivo assay to alleviate the need for multiple in vivo assays, thereby reducing time, cost and use of animals. Presented here are the results of an evaluation trial in which the bone-marrow and peripheral blood (via MicroFlow(?) flow cytometry) micronucleus tests (looking at potential chromosome breakage and whole chromosome loss) in developing erythrocytes or young reticulocytes were combined with the Comet assay (measuring DNA strand-breakage), in stomach, liver and blood lymphocytes. This allowed a variety of potential target tissues (site of contact, site of metabolism and peripheral distribution) to be assessed for DNA damage. This combination approach was performed with minimal changes to the standard and regulatory recommended sampling times for the stand-alone assays. A series of eight in vivo genotoxins (2-acetylaminofluorene, benzo[a]pyrene, carbendazim, cyclophosphamide, dimethylnitrosamine, ethyl methanesulfonate, ethyl nitrosourea and mitomycin C), which are known to act via different modes of action (direct- and indirect-acting clastogens, alkylating agents, gene mutagens, cross-linking and aneugenic compounds) were tested. Male rats were dosed at 0, 24 and 45 h, and bone marrow and peripheral blood (micronucleus endpoint), liver, whole blood and stomach (Comet endpoint) were sampled at three hours after the last dose. Comet and micronucleus responses were as expected based on available data for conventional (acute) stand-alone assays. All compounds were detected as genotoxic in at least one of the endpoints. The importance of evaluating both endpoints was highlighted by the uniquely positive responses for certain chemicals (benzo[a]pyrene and 2-acetylaminofluorene) with the Comet endpoint and certain other chemicals (carbendazim and mitomycin C) with the micronucleus endpoint. The data generated from these investigations demonstrate the suitability of the multi-endpoint design.     

Activity of a nitroalkene derivative, 1-(5-bromofur-2-il)-2-bromo-2-nitroethene, in the Salmonella/microsome assay and the mouse bone marrow micronucleus test

Mutagenicity of a substituted nitroalkene, 1-(5-bromofur-2-il)-2-bromo-2-nitroethene (BNF) was tested in the Salmonella/microsome assay using the strains TA 98, TA 100 and TA 100NR (nitroreductase deficient). BNF was a direct mutagen in TA 98 and TA 100; the response was lowered when exogenous metabolic activation (S9) was used. A further decrease in mutagenicity was observed in strain TA 100NR, as compared to the parental TA 100, which showed the involvement of nitroreduction in the overall response elicited by BNF. The micronucleus assay was carried out in Swiss male mice which were given a single i.p. dose of 10–20 mg/kg of BNF dissolved in peanut oil, bone marrow being sampled 24 and 48 h later. The micronucleated polychromatic erythrocyte counts (MNPCE) showed a weak response in the dose range of 10–17.5 mg/kg at the second sampling (48 h) and a significant rise for 20 mg/kg at 24 and 48 h.     

Genotoxic effects of styrene-7,8-oxide in human white blood cells: comet assay in relation to the induction of sister-chromatid exchanges and micronuclei

Styrene is used in the production of plastics, resins and rubber. The highest human exposures to styrene take place by inhalation during the production of fiberglass reinforced plastics. Styrene is metabolized mainly in the liver to styrene-7,8-oxide (SO), its principal in vivo mutagenic metabolite. In this study, human peripheral white blood cells were exposed to several SO concentrations (10-200 microM) in order to evaluate its genotoxic properties by means of comet assay, sister-chromatid exchanges (SCE) and cytokinesis-blocked micronucleus (MN) test, in addition to determine its clastogenic or aneugenic properties by combining MN with fluorescence in situ hybridization (FISH) procedures. Our results show that SO induces DNA damage, SCE and MN in human leukocytes in vitro at concentrations above 50 microM, and that there is a strong relationship between DNA damage, as measured by the comet assay, and cytogenetic damage induced by SO at the doses employed. SO shows preferentially a clastogenic activity and produces a cytostatic effect at high doses, reflected by the significant decrease of the calculated proliferation indices. A good dose-effect relationship is obtained in the three tests performed at the concentration range assayed.     

Activity of 1,2-dimethylhydrazine in the mouse bone marrow micronucleus assay using a triple- and a single-dosing protocol

In the present study the ability of 1,2-dimethylhydrazine (DMH) to induce micronuclei in mouse bone marrow erythrocytes was investigated using 2 different dosing regimens. DMH caused an increase in micronuclei in both male and female mice following single administration and sampling after 24 h. The effect was more pronounced in female than in male animals. Triple administration of DMH at concentrations corresponding to 80, 40 and 20% of the median lethal dose (MLD) did not increase the incidence of micronuclei in either sex. Small increases in micronucleus incidence were observed after triple dosing at 100% of the MLD value. These results suggest that a future micronucleus protocol should include animals of both sexes and single and repeated administration of the test substance.