Analysis of toxic and mutagenic activities of antiherpesvirus nucleosides against HeLa cells and herpes simplex virus type 1.

The toxic and mutagenic activities of five antiherpesvirus agents to HeLa cells and herpes simplex virus type 1 (HSV-1) were investigated. 5-Iodo-2'-deoxyuridine (IDU) and 9-beta-D-arabinofuranosyl-adenine (araA) showed very potent inhibitory effects on cell growth and the cloning efficiency of HeLa cells, whereas 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU), E-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 9-(2-hydroxyethoxymethyl)guanine (ACV) showed less inhibitory effect. 50% inhibitory doses of BV-araU and BVDU for cell growth were 657 and 253 micrograms/ml, respectively. Although the growth inhibitory activity of BVDU was very weak, as above, the mutagenic activity of this drug to the cells, estimated by induction of colchicine-resistant mutants, was observed to be 4 micrograms/ml, which was a markedly smaller dose than the inhibitory dose for cell growth, and the highest frequency of mutation of the cells was shown at 100 micrograms/ml of BVDU. This activity was more potent than that of IDU. No mutagenic activity of BV-araU, araA and ACV to cells was observed within the concentration range of 1-800 micrograms/ml. IDU showed high mutagenic activity to HSV-1 growing in human embryo lung fibroblasts, and IDU-resistant mutants were induced at a high frequency. BVDU also induced a small amount of BVDU-resistant mutant virus, although this drug induced many mutant cells. No mutagenic activity of BV-araU, araA and ACV to HSV-1 was observed.     

The mutagenic potency of 4 agents at the thymidine kinase locus in mouse lymphoma L5178Y cells in vitro: effects of exposure time

Mutagenic potency at the thymidine kinase (TK) locus in mouse lymphoma L5178Y cells (expressed as induced trifluorothymidine (TFT)-resistant mutants/total dose) was assessed for 4 agents (ethyl methanesulphonate (EMS), benzidine, 1,8-dinitropyrene (1,8-DNP) and ICRF 159) using short (3-4 h) and long (21-24 h) exposure times. The mutagenic potency of EMS was found to be essentially independent of concentration and exposure time when tested over a cytotoxic range consistent with routine testing procedures. Similar results were obtained with benzidine but for both 1,8-DNP and ICRF 159 mutagenic potency was found to be highly dependent on the concentration and exposure time. 1,8-DNP failed to induce any significant increases in mutant frequency when tested at concentrations up to 5 micrograms/ml using short exposure times, whereas the compound was active at concentrations as low as 0.1 microgram/ml when the exposure period was extended to 21 h. Under the latter conditions, however, the molar potency of 1,8-DNP was found to be inversely related to concentration over a range extending from 0.1 to 5 micrograms/ml. ICRF 159 induced increases in the frequency of TFT-resistant mutants using short or long exposure times. When a short exposure time was used, however, the mutagenic potency of the antitumour agent decreased with increasing concentration between 1 and 500 micrograms/ml. Although possible explanations can be offered to account for these observations the results illustrate potential problems which may arise in this system when comparing mutagenic potency values for a range of compounds with a view to assessing relative risk.     

Cytotoxicity and genotoxicity testing of sodium fluoride on Chinese hamster V79 cells and human EUE cells.

The cytotoxic effects of sodium fluoride (NaF) on hamster V79 cells and human EUE cells were studied by measuring the cloning efficiency and DNA, RNA and protein synthesis in cells cultured in the presence of NaF. Potential mutagenicity of NaF was followed on the basis of induced 6-thioguanine-resistant mutants in treated Chinese hamster V79 cells. The results showed that the addition of 10-150 micrograms of NaF per ml of culture medium induced 10-75% cytotoxic effect on hamster V79 cells but had no toxic effect on human EUE cells. NaF was cytotoxic to human EUE cells at considerably higher concentrations (200-600 micrograms/ml). Growth of both cell types with 100 and 200 micrograms of NaF per ml caused inhibition of 14C-thymidine, 14C-uridine and 14C-L-leucine incorporation. This means that NaF inhibits macromolecular synthesis whereby damaging effects were less drastic in human EUE cells. The results of detailed mutagenicity testing on hamster V79 cells showed that NaF did not show any mutagenic effect after long-term (24-h) incubation of hamster cells in the presence of 10-400 micrograms of NaF per ml of culture medium.     

Absence of mutagenic and antimutagenic activities of fluoride in Ames salmonella assays

The mutagenicity of fluoride (as sodium fluoride, NaF) was investigated with Ames Salmonella/microsome assays in strains of TA97a, TA98, TA100, TA102 and TA1535. The concentrations of NaF tested ranged from 0.44 to 4421 micrograms/plate (0.1 to 1000 ppm F), both with and without microsome activation. In addition, the suggested antimutagenic effect of fluoride was evaluated with known mutagens at various concentrations of NaF (0.44-442.2 micrograms/plate, 0.1-100 ppm F). The data showed that NaF, in amounts from 0.44 to 442.2 micrograms/plate (0.1-100 ppm F), failed to significantly increase the number of the revertants over the number observed in the solvent (distilled deionized water) controls. Increases of NaF to, and beyond, 1100 micrograms/plate (250 ppm F) resulted in a toxic effect and a reduction of the revertants to various degrees among the strains. NaF in the presence of known mutagens did not significantly decrease the number of the revertants. The results of this study indicate that NaF does not have mutagenic or antimutagenic effects in the strains tested with Ames Salmonella assays.     

Cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat tracheal epithelial cells.

The cytotoxicity, genotoxicity and transforming activity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were studied by the assays of colony-forming efficiency (CFE), micronucleus formation (MN), and cell transformation in rat tracheal epithelial (RTE) cells both in vitro and in vivo. Liver S9, primary hepatocytes and RTE cells from normal and Aroclor-1254 induced rats were compared for bioactivation of NNK using Salmonella mutagenesis as the endpoint. Results from the in vitro experiments indicated that low concentrations of NNK (0.01-25 micrograms/ml) caused from 15% to greater than 100% increases in CFE of RTE cells. At high concentrations (100-200 micrograms/ml), NNK was significantly toxic to RTE cells. NNK treatment in vitro (50-200 micrograms/ml) increased MN frequency as much as 3-fold above background and significantly increased the transformation frequency (TF) in 4/5 (50 micrograms/ml) and 6/8 (100 micrograms/ml) experiments. The in vivo exposure of rats to NNK (150-450 mg/kg, given i.p.) resulted in a 60-85% reduction in CFE and a 3-5-fold increase in MN formation in RTE cells. In vivo treatment with cumulative doses of 150 and 300 mg/kg of NNK produced significant increases in TF of tracheal cells from 3/3 and 2/3 rats, respectively. Without activation, NNK was not mutagenic in Salmonella TA1535. The bioactivation of NNK to a mutagenic metabolite was achieved by incubation of NNK with liver S9 fraction from Aroclor-1254 induced rats or primary hepatocytes from both untreated and Aroclor-1254 pretreated rats. RTE cells did not produce sufficient quantities of mutagenic NNK metabolites to be detected by the Salmonella assay.     

Cytogenetic effects of pesticides. IV. Cytogenetic effects of the insecticides Gardona and Dursban.

The cytogenetic effects of the insecticides Gardona and Dursban were investigated. The toxicity and ability of both insecticides to induce chromosome aberrations and sister-chromatid exchange in vitro was tested in a primary culture of mouse spleen cells, in order to assess the potential mutagenicity of both insecticides. The concentrations 10(-7)-10(-3) M were used for testing the toxic effects of the insecticides. Both Gardona and Dursban were toxic to spleen cell cultures and the percentage of viable cells decreased as the concentration of the insecticide was increased. It reached 76.8% and 77.8% of control after treatment with the highest concentration tested (10(-3) M) of Gardona and Dursban respectively. Gardona at 0.25, 0.50, 1.0 and 2.0 micrograms/ml, and Dursban at 0.50, 1.0, 2.0 and 4.0 micrograms/ml were tested for the induction of chromosome aberrations and sister-chromatid exchanges. All of the tested concentrations of both insecticides induced a high percentage of metaphases with chromosomal aberrations in cultured mouse spleen cells after 4-h treatment. The frequency of SCEs/cell increased with increasing concentration of the insecticides. It reached 11.92 +/- 0.14/cell and 13.40 +/- 0.20/cell after treatment with Gardona (2 micrograms/ml) and Dursban (4 micrograms/ml), respectively, compared with 8.2 +/- 0.19/cell and 7.6 +/- 0.15/cell in the solvent control. The presented results indicate that both Gardona and Dursban in the tested concentrations are mutagenic in mouse spleen cell cultures.     

Evaluation of mutagenic and cytotoxic effects of sodium fluoride on mammalian cells influenced by an acid environment

The mutagenic activity of sodium fluoride at reduced pH was studied in the V79/HGPRT system. Statistical analysis of the results of mutagenicity testing suggests that, despite its high toxicity, sodium fluoride has no mutagenic effects at reduced pH on hamster V79 cells. Short-term treatment of cells with sodium fluoride at reduced pH inhibits growth activity of cells as well as synthesis of pulse-labeled nascent DNA and cumulative RNA synthesis and proteosynthesis. From the results of this study we suggest that an acid environment which supports formation of hydrogen fluoride increases toxic but not mutagenic potencies of sodium fluoride.     

Salt-dependent and protein-concentration-dependent changes in the solution structure of the DNA-binding histone-like protein, HBsu, from Bacillus subtilis.

The solution structure of the histone-like DNA-binding protein, HBsu, from Bacillus subtilis in 2 mM sodium cacodylate, pH 7.5, is sensitive to the ionic strength of the buffer. This was shown by circular dichroism measurements at different concentrations of sodium chloride and potassium fluoride. The stability of HBsu is also influenced; at HBsu concentrations of about 0.1 mg.ml-1, melting temperatures of 32 degrees C and 55 degrees C were found in the absence of potassium fluoride and in the presence of 0.5 M potassium fluoride, respectively, exhibiting drastic ionic-strength-dependent differences in the temperature-induced unfolding of HBsu. Furthermore, at low ionic strength, circular dichroism spectra vary markedly depending on the HBsu concentration in the approximate range 0.2-3 mg.ml-1. Such protein-concentration-dependent differences in the spectra were not observed in the presence of 0.5 M potassium fluoride. Very similar circular dichroism spectra of HBsu and the histone-like DNA-binding protein of Bacillus stearothermophilus (HBst) at high ionic strength, indicate comparable structures of both proteins under these conditions. Estimation of the secondary structure content from the circular dichroism spectra yields data which are in satisfactory agreement with the values obtained from the crystal structure of HBst. Transition temperatures of 45 degrees C and 61 degrees C were found in differential scanning calorimetric measurements performed with HBsu in potassium-fluoride-free buffer and in the presence of 0.5 M potassium fluoride, respectively. The thermodynamic data point to the melting of native HBsu dimers into two denatured monomers.     

Transforming activities of sodium fluoride in cultured Syrian hamster embryo and BALB/3T3 cells

The transforming activity of sodium fluoride was studied in the SHE and the BALBl3T3 cell culture systems. Initiating and promoting activities were then investigated by means of the orthogonal methodology. Sodium fluoride was found to induce morphological transformation of SHE cells seeded on a feeder layer of X-irradiated cells at high concentrations (75–125 g/ ml). When the cells were seeded in the absence of a feeder-layer, the transformation frequencies increased in a dose-dependent manner with the concentrations of sodium fluoride ranging from 0 to the highly toxic concentration of 200 g/ml. In the BALBl3T3 cell system, sodium fluoride was negative in the standard Kakunaga procedure, while through the experiment designed by table L₈ (2⁷) of the orthogonal method, an initiating-like effect and a weak promoting activity were detected within the concentrations ranging from a 25 g/ ml to a 50 g/ ml concentration which is highly toxic for BALBl3T3 cells. From these results, it is suggested that, besides a genetic mode of action, sodium fluoride could possibly act through a non-genotoxic mechanism.Abbreviations CE cloning efficiency - NaF sodium fluoride - SHE Syrian hamster embryo - TF transformation frequency     

Use of the cytokinesis-block method for the analysis of micronuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze micronuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for micronucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 micrograms/ml CYB for varying durations (8-48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for micronucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of micronuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125-1.0 micrograms/ml) and cyclophosphamide (2-12 micrograms/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in micronucleus frequency over controls at the highest concentration tested (1.0 micrograms/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in micronucleus frequency over controls at the highest tested concentration (12 micrograms/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze micronucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.     

Mutagenicity of gossypol analyzed by induction of meiotic micronuclei in vitro

Chromosome breakage caused by mutagens in male germ cells can be analyzed by micronucleus induction during meiotic division. This can be followed in vitro by culturing seminiferous tubular segments from stages of the epithelial cycle that contain late pachytene and diakinetic primary spermatocytes. We studied the mutagenic potential of a male contraceptive, gossypol, in this test system using adriamycin (10 ng/ml) as a reference mutagen. A small but significant increase in the frequency of micronuclei was induced with concentrations of 10 and 20 micrograms/ml of gossypol, while cytotoxic effects appeared at concentration of 20 micrograms/ml and were evident at 50 micrograms/ml. Analysis of meiotic micronucleus induction in vitro seems to be a sensitive test system of male germ-cell mutagenesis, but further studies on the possible mutagenic effects of gossypol are needed.     

Evaluation of diallate and triallate herbicides for genotoxic effects in a battery of in vitro and short-term in vivo tests

Commercial-grade preparations of two thiocarbamate herbicides, diallate and triallate, were evaluated for their mutagenic potential in a battery of short-term bioassays. All in vitro bioassays were performed with and without mammalian metabolic activation, and all such tests were repeated after an interval of at least 1 week. Diallate and triallate were tested in the Salmonella/microsome assay over dose ranges of 0.59 to 118.0 micrograms/plate and 6.37 to 1273 micrograms/plate, respectively. Both diallate and triallate gave positive results in S. typhimurium strains TA1535, TA98, and TA100 only in the presence of a rat-liver metabolic activation system. In Saccharomyces cerevisiae strain D7, diallate was tested at concentrations from 1.18 to 29.50 micrograms/ml, and triallate was tested at 0.955 to 9.548 micrograms/ml. Both diallate and triallate gave negative results for mitotic gene conversion, mitotic crossing-over, and reverse mutation. In the mouse lymphoma L5178Y TK+/- assay, diallate was tested at concentrations ranging from 1 to 72 micrograms/ml, and triallate was tested at 0.5 to 60 micrograms/ml. Both herbicides produced mutagenic responses in the mouse lymphoma assay in the presence of metabolic activation. In the Drosophila sex-linked recessive lethal test, flies were exposed to 0.0004% diallate and 0.001% triallate. In this assay, diallate was considered mutagenic, whereas triallate did not produce a detectable mutagenic response.     

A multiple end-point approach to evaluation of cytotoxicity and genotoxicity of erythrosine (FD and C Red No. 3) in a V79 hepatocyte-mediated mutation assay

V79 Chinese hamster lung cells were used to evaluate in vitro the cytotoxicity and genotoxicity of erythrosine (2', 4', 5', 7'-tetraiodofluorescein disodium salt; FD and C Red No. 3), a color additive used widely in foods, drugs and cosmetics. Erythrosine reduced colony size at 200 micrograms/ml and was lethal to 90% or more of the cells at 400 micrograms/ml. At dose levels of 100, 200 and 300 micrograms/ml of medium, erythrosine was non-mutagenic to V79 cells at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) and sodium, potassium ATPase (Na+, K+ -ATPase) gene loci and did not increase the frequency of sister-chromatid exchanges with or without rat hepatocyte-mediated activation. Erythrosine at 300 micrograms/ml, unlike lower dose levels, produced an increase in micronucleus frequency in the absence of hepatocytes. An erythrosine dose-related increase in the mitotic frequency was due to an increase in the number of first mitoses at the expense of later cell divisions. Hepatocytes moderated the effect of erythrosine treatment on micronucleus frequency, mitotic frequency and MII/MI ratio. These results demonstrate the advantage of a multiple end-point approach to the evaluation of cytotoxicity and genotoxicity within a single-assay system.     

Effect of pH shifts on the mutant frequency at the thymidine kinase locus in mouse lymphoma L5178Y TK+/- cells

Evidence has been accumulating that conditions of nonphysiological pH may affect the results of in vitro genetic tests by mechanisms unrelated to the chemical being tested. Medium was pH-adjusted with HCl, NaOH or with organic buffers (Good's zwitterions). In the absence of S9 mix, no changes in mutant frequency were observed over a pH range of 6.4-9.2; a small, 1.9-fold increase was observed for a moderately toxic treatment (24% relative growth) at pH 6.3. However, in the presence of S9 mix, the mutant frequency increased sharply for pH values below 6.8. At pH 6.4, a 4-fold increase was induced, and pH 6.0 resulted in a 10-fold increase in mutant frequency. Basic pH shifts in the presence of S9 mix caused no changes in mutant frequency up to pH 8.0; treatment with pH 8.8 was highly toxic (5.3% relative growth) and caused a 3-fold increase in mutant frequency. Thirteen mutant clones induced at pH 6.0 with S9 mix were challenged with trifluorothymidine after their expansion in nonselective medium and all retained their resistance; another 14 clones were tested for thymidine utilization and all incorporated only 0.1-5.5% of the 14C-labeled thymidine used by the parental line. The induced mutants were primarily of the small-colony phenotype, which indicated clastogenic activity. This was confirmed with chromosome studies which showed a large increase in cells with aberrations consisting of chromatid breaks and complex rearrangements. The results show that the combination of weak acidity (pH 6-6.8) and S9 mix is mutagenic and clastogenic to L5178Y TK+/- cells.     

Platinum-induced mutations to 8-azaguanine resistance in Chinese hamster ovary cells.

6 platinum (Pt) compounds were compared in suspension cultured Chinese hamster ovary (CHO-S) cells with respect to their inhibition of growth, their reduction of cloning efficiency, and their induction of mutants resistant to 200 microM (30 micrograms/ml) 8-azaguanine (8-AG) and 3 mM ouabain (OUA), respectively. The toxicity of these compounds can be ranked by the medium concentrations which decrease suspension growth/or cloning efficiency by 50%: cis-Pt(NH3)2-Cl2 (0.9/1.5 microM) greater than Pt(SO4)2 + methylcobalamin (MeB-12) methylation product (20/10 microM) greater than K2PtCl4 (32/50 microM) = K2PtCl6 (34/50 microM) = MePtCl2-3 (60/50 microM) greater than Pt(SO4)2 (66/105 microM). Following 20 h exposures to concentrations which resulted in relative survivals of 80-2%, none of the foregoing compounds increased consistently the frequency of OUA(R) mutants above the spontaneous frequency (6.0 x 10(-6)). Parallel treatments with 800 microM (100 micrograms/ml) ethyl methanesulfonate (EMS) increased the OUA(R) mutant frequency 10--12-fold. Using 8-AG for mutant selection, dose-dependent increases of 5--7-fold above the spontaneous frequency (3--8 x 10(-5) were obtained with cis-Pt(NH3)2Cl2, Pt(S04)2, and the product from Pt(SO4)2 + MeB-12. Identical 20 h exposures to varying amounts of K2PtCl4, K2PtCl6, and MePtCl2-3 did not induce 8-AG(R) mutants. Optimal detection of Pt-induced 8-AG(R) mutants required 7 post-treatments, expression doublings in suspension culture. Under our selection conditions 8/8 spontaneous and 24/24 Pt-induced 8-AG(R) variants contained reduced hypoxanthine-guanine phosphoribosyl transferase (HGPRT) specific activities (means ranging from 3 to 11% of the parental CHO-S cells). When compared from linear plots of the 8-Ag(r) frequency against the initial medium concentration, cis-Pt(NH3)2Cl2 is 134 times and Pt(SO4)2 si 3.5 times more mutagenic than EMS. However, on a cell-survival basis EMS is 8--10-fold more mutagenic than these two Pt-compounds. 6-Thioguanine (10 microM) can be substituted for 8-AG to assay mutant induction by cis-Pt(NH3)2Cl2 and Pt(SO4)2 in CHO-S cells. The sensitivity of the CHO-S HGPRT locus for detecting mutagenesis by Pt complexes can be increased several fold by continuous subculture in the presence of these agents for 10--25 population doublings. By this procedure K2PtCl6 is seen to be weakly mutagenic and 20 microM Pt(SO4)2 produces 8-AG(R) mutants at frequencies requiring 7--8-fold higher concentrations when a fixed 20 h exposure is used.     

The mutagenicity of 2-amino-N6-hydroxyadenine to L5178Y tk +/- 3.7.2C mouse lymphoma cells: measurement of mutations to ouabain, 6-thioguanine and trifluorothymidine resistance, and the induction of micronuclei

2-Amino-N6-hydroxyadenine (AHA) was tested in the mouse lymphoma L5178Y tk +/- assay using the microtitre cloning technique over concentrations from 0.005 micrograms/ml-1 (100% viability) to 6 micrograms/ml (10% viability) as measured by cloning efficiency immediately after treatment. At low, non-toxic concentrations (0.005-0.25 micrograms/ml) a dose-related linear increase in the frequency of ouabain-resistant mutants was seen, in addition to an increase in 6-thioguanine- and trifluorothymidine-resistant mutants. No consistent induction of micronucleated cells was observed in this concentration range. Toxic concentrations (20-90% kill) induced a dose-related increase in micronuclei, while the frequency of ouabain-resistant mutants fell (although it was still highly significantly above the control value). These results suggest that the mechanism of action of AHA depends on the concentration, with point mutations being induced at low, non-toxic doses and detectable chromosome breakage occurring only at higher doses. Both large-colony and small-colony trifluorothymidine-resistant mutants were induced at all concentrations. The utility of using multiple genetic end-points in one cell line and the importance of dose range selection for risk assessment and an understanding of the mode of action of test substances is underlined.     

2,4-Dichlorophenoxyacetic acid causes chromatin and chromosome abnormalities in plant cells and mutation in cultured mammalian cells

The cytotoxic and mutagenic effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) on shallot root tip cells and on V79 Chinese hamster fibroblast cells were examined and compared. In shallot root tips 2,4-D caused changes in mitotic activity, as well as changes in chromosome and chromatin structure, and also changes during the cell cycle. 2,4-D also showed mutagenic and cytotoxic effects on V79 cells in culture in concentrations higher than 10 micrograms/ml. The results in both systems (plant and mammalian cells) were in agreement showing mutagenic activity of 2,4-D in the concentration range higher than usually used in establishing plant tissue culture (greater than 5 micrograms/ml).     

Evaluation of genotoxicity of N-nitrosodibenzylamine in Chinese hamster V79 cells and in Salmonella

Health concerns have arisen due to the formation of N-nitrosodibenzylamine (NDBzA; CAS No. 5336-53-8) in pork processed in a new type of rubber netting. In view of the potent carcinogenicity of related nitrosamines (e.g. N-nitroso-n-dibutylamine and N-nitrosodiethylamine), NDBzA was evaluated for genotoxicity in vitro in both Chinese hamster V79 cells and in Salmonella. In V79 cells, concentrations up to 25 micrograms/ml were tested with and without activation by rat or hamster hepatocytes. Significant elevation of SCE frequency was seen only at 25 micrograms/ml in the presence of uninduced hamster hepatocytes. Mutation to 6-thioguanine resistance was observed at 25 micrograms/ml, in the absence of hepatocytes and in the presence of induced (Aroclor 1254) or uninduced hamster hepatocytes, but not with rat hepatocytes. With uninduced rat hepatocytes, a small but significant (p less than 0.05) increase in the mutation frequency was seen with 10 micrograms/ml NDBzA. In the Salmonella assay, using a pre-incubation protocol and concentrations up to 1000 micrograms/ml, NDBzA was negative in strain TA98, and in TA100 with rat S9, but was positive at the highest dose in TA100 with hamster S9, and more strongly with Aroclor 1254-induced hamster S9. When activated by uninduced rat or hamster hepatocytes, as opposed to S9, NDBzA was negative with all tester strains. Hamster hepatocytes activated more than rat in the V79 studies, and hamster S9 was more strongly activating in the Salmonella assay. These results indicate that NDBzA is weakly mutagenic to both Salmonella and V79 cells.     

Genetic toxicity of the benzene metabolite trans, trans-muconaldehyde in mammalian and bacterial cells

Previous studies in our laboratory identified trans,trans-muconaldehyde (MUC), a six-carbon diene dialdehyde, as a microsomal metabolite of benzene. This ring-opened metabolite of benzene was also shown to be hematotoxic in mice in a manner similar to benzene. To further explore the role of MUC in relation to benzene toxicity, a number of test systems were utilized to determine its genotoxic potential. In B6C3F1 mice, MUC induced a highly significant increase in sister-chromatid exchange (SCE), the lowest effective dose being 3 mg/kg, but failed to induce any micronuclei (MN). In Chinese hamster ovary (CHO) cells, MUC at concentrations up to 0.8 micrograms/ml was negative in the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) assay. Dose-related increases in the percentage of cells with MN were observed in CHO cells treated with 0.4-0.8 micrograms/ml MUC. MUC did not-cause unscheduled DNA synthesis in rat primary hepatocytes. Treatment of Salmonella typhimurium TA97 with MUC induced a low level of mutations at concentrations ranging from 10 to 70 micrograms/ml with or without S9 activation. MUC was inactive in strains TA1535, TA100, TA1538 and TA98. In CHO cells and rat primary hepatocytes, MUC was cytotoxic at 0.4 and 4.0 micrograms/ml, respectively. Concentrations of 100 micrograms/plate MUC were toxic for bacterial cells. The present findings indicate that MUC is nonmutagenic or minimally mutagenic in bacterial and mammalian in vitro systems. In mammalian cells, MUC is highly cytotoxic and genotoxic.     

Combined effects of solutes and food preservatives on rates of inactivation of and colony formation by heated spores and vegetative cells of molds.

The combined and independent effects of sucrose, sodium chloride, potassium sorbate, and sodium benzoate on heat inactivation of conidia of Aspergillus flavus and Penicillium puberulum, ascospores of Byssochlamys nivea, and vegetative cells of Geotrichum candidum were studied. In addition, the effects of solutes and preservatives on colony formation by unheated and heated conidia of A. flavus were evaluated. Increased concentrations of sucrose were accompanied by increased tolerance to heat by A. flavus, B. nivea, and G. candidum. Low concentrations (3 and 6%) of sodium chloride protected A. flavus and G. candidum, whereas up to 12% sodium chloride protected B. nivea, but had little effect on the heat stability of P. puberulum. Potassium sorbate and sodium benzoate acted synergistically with heat to inactivate all four molds. At the same concentration, the two preservatives had varied degrees of effectiveness on molds and were influenced by the type of solute in the heating menstrua. Heated conidia of A. flavus had increased sensitivity to preservatives and reduced water activity, whether achieved by the presence of sucrose or sodium chloride, thus demonstrating heat-induced injury. At the same concentration, potassium sorbate was clearly more inhibitory than was sodium benzoate to colony formation by A. flavus, and the presence of sucrose and sodium chloride enhanced this inhibition.