ICR-induced frameshift mutations in the histidine operon of Salmonella

Both the acridine half-mustard, ICR191, and the nonalkylating azaacridine derivative, ICR364-OH, induce three classes of frameshift mutations in the histidine operon of Salmonella typhimurium. (i) One class is completely stable in reversion tests and is presumed to represent deletion of one or a few critical nucleotide pairs or two nearby frameshifts. One extended deletion was found out of 11 stable mutations. (ii) Of two spontaneously reverting classes which also are considered to predominantly involve base deletions, one is unaffected in reversion with ICR191, nitrosoguanidine, and diethylsulfate, and the other is induced to revert with ICR191. (iii) A third class, considered to predominantly involve base additions, responds in reversion tests with ICR191 as well as with nitrosoguanidine and diethylsulfate. Other investigators have shown that one mutant of this class is a "plus" frameshift and that nitrosoguanidine acts in reversion to delete a guanine plus cytosine base pair. Although such plus frameshifts are found with high frequency among mutations selected from acridine-treated bacteria or when strong selection pressure is applied for their detection in reversion tests, data from this laboratory indicate that this class of plus frameshifts is rare among mutations derived spontaneously or after treatment with a variety of other mutagens. Finally, we demonstrate that the alkylating ICR191 and the nonalkylating ICR364-OH preferentially cause mutations in different chromosome regions and that their spectra of activity only partially overlap that found for spontaneous frameshift mutations.     

Mutagenicity of some intercalating agents in Chinese hamster V79 cells

ICR 170 and ICR 191, but not 9-aminoacridine or chloroquine, induced both 6-thioguanine- and, to a smaller extent, ouabain-resistance in Chinese hamster V79 cells. These results indicate that covalent binding to DNA is necessary for intercalating agents to induce mutation in this cell line, and that this assay can distinguish potential carcinogens from non-carcinogenic analogues of this chemical type. The induction of ouabain-resistance by both ICR 170 and ICR 191 indicates that these frameshift mutagens induce base-pair substitution to some extent in V79 cells.     

Quantitative and molecular analyses of ethyl methanesulfonate- and ICR 191-induced mutation in AS52 cells

A pSV2gpt-transformed Chinese hamster ovary (CHO) cell line has been used to study mutation at the molecular level. This cell line, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line, and has been previously shown to contain a single, functional copy of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. In this study, conditions for its use in the study of mammalian cell mutagenesis have been stringently defined. The spontaneous mutation rate (2 X 10(-6)/cell division) and phenotypic expression time (7 days) of the gpt locus compare favorably with those of the hprt locus in wild-type CHO-K1-BH4 cells. While both cell lines exhibit similar cytotoxic responses to ethyl methanesulfonate (EMSO and ICR 191, significant differences in mutation induction were observed. Ratios of XPRT to HPRT mutants induced per unit dose of EMS and ICR 191 are 0.70 and 1.6, respectively. Southern blot hybridization analyses revealed that most XPRT mutant cell lines which arose following treatment with EMS (20/22) or ICR 191 (20/24) exhibited no alterations of the gpt locus detectable by this technique. Similar observations were made for the hprt locus in EMS-(21/21) and ICR 191-induced (22/22) HPRT mutants. In contrast, most spontaneous gpt mutants (14/23) contained deletions, while most spontaneous hprt mutants (18/23) exhibited no detectable alterations. Results of this study indicate that the AS52 cell line promises to be useful for future study of mutation in mammalian cells at the DNA sequence level.     

In vitro genotoxic evaluation of conventional bleached and biobleached softwood pulp mill effluents

The effluents of pulp and paper mills contain about 300 different chemical compounds; many of them are mutagens and clastogens. Genotoxic studies have shown that chlorination stage liquors are significantly more genotoxic, in the Ames Salmonella assay, than the other process of lignin extraction, and that lyophilized effluents are genotoxic in cultured mammalian cells. Since these effluents from conventional bleaching stages are genotoxic, Chilean industries are interested in changing this process to a less toxic one, such as biobleaching using enzymes. In this study, we tested the in vitro genotoxicity of two types of effluents: an effluent obtained from a conventional radiata pine kraft-bleaching process (effluent D) and one derived from a biobleaching process with hemicellulase (effluent B). Both effluents were tested without any concentration or purification steps in the Ames Salmonella assay (TA100) and in the micronuclei (MN) and sister chromatid exchange (SCE) tests in CHO cells. The results showed that neither effluent induced base pair substitution mutations in the Ames Salmonella assay, and neither increased the micronucleus frequency in CHO cells. But, both increased the SCE frequencies in CHO cells, showing that this assay is more sensitive than the other ones, and that the two effluents contained chemical compounds in amounts enough to induce in vitro genotoxicity measured by the SCE induction.     

Effects of an acridine half-mustard (ICR 191) on growth and ploidy of frog cells in culture

The effects of an acridine half-mustard, ICR 191, on the growth rate and ploidy of four haploid and two diploid lines of Rana pipiens cells in culture were studied. Growth curves indicate that the haploid and diploid cell lines were equally resistant to a 4-hour exposure of this drug (0.1 micrometer to 10 micrometer. ICR 191 treatment induced the haploid cell cultures to become diploid. The proportion of diploid cells increased progressively with respect to time after the 4-hour exposure period. The greater the concentration of ICR 191 applied, the more rapid the rate of conversion. Autoradiographic determinations of percent labelled nuclei indicate that DNA synthesis was not inhibited in haploid or in diploid cells. Therefore, the increased proportion of diploid cells did not originate from the small percentage of diploid cells in the initial population. Instead the haploid cells were converted to diploid cells. Time lapse cinematography indicated that the conversion mechanism was other than cell fusion. Conversion to higher ploidy did not occur when diploid cell cultures were exposed to ICR 191.     

Clastogenicity of lead chromate particles in hamster and human cells.

Several insoluble compounds of chromium, such as lead chromate, are respiratory carcinogens in experimental animals and suspected to be so in humans. Lead chromate induces neoplastic transformation in cultured cells but the mechanism of genotoxicity is unknown. We examined the effect of lead chromate on the integrity of chromosomes of Chinese hamster ovary (CHO) and human foreskin fibroblasts (HFF) after a 24-h exposure. At 0.4 microgram/cm2, 0.8 microgram/cm2, 2 microgram/cm2 and 8 microgram/cm2 lead chromate particles reduced survival of CHO cells to 86%, 62%, 2% and less than 1% respectively. These concentrations induced a dose-dependent 4-19-fold increase in the percent metaphases with damage. The HFF cells exhibited higher sensitivity in both cytotoxicity and clastogenicity. The spectrum of damage observed for both cell types was primarily achromatic lesions affecting one or both chromatids. To test for particle dissolution effects, CHO cells were treated for 24 h with either clarified medium that had been incubated for 24 h with lead chromate particles, or clarified medium that had been pre-conditioned by CHO cells treated with lead chromate particles for 24 h. No damage was detected in these cells, indicating that extracellular dissolution into ionic lead and chromate did not contribute to the genotoxicity. This is consistent with a previous study in which scanning electron micrographs illustrated internalization of the particles. These results suggest that clastogenesis may be a mechanism for lead chromate induced carcinogenesis.     

Isolation and characterization of novel phenotype CHO cell mutants defective in peroxisome assembly, using ICR191 as a potent mutagenic agent

To elucidate molecular and cellular mechanisms of peroxisome biogenesis, we have isolated Chinese hamster ovary (CHO) cell mutants defective in peroxisome biogenesis by making use of enhanced green fluorescent protein (EGFP) and a frameshift-inducing mutagen ICR191. CHO-TKa cells stably expressing Pex2p were transformed with a cDNA encoding EGFP fused with peroxisomal targeting signal type 2 (PTS2-EGFP), termed Tka/EG2. TKa/EG2 cells were mutagenized with ICR191 and cultured in the presence of P9OH (9-(1'-pyrene) nonanol) followed by an exposure to UV. P9OH/UV-resistant and morphologically peroxisome-deficient mutant cells were isolated by directly observing cytosolic localization of EGFP, without cell staining. By a combination of cell-fusion and PEX transfection, we determined complementation groups (CGs) of 16 cell mutants isolated here. The mutants were classified into five CGs, including pex2, pex3, pex5, pex6, and pex7 cell mutants. In contrast to typical pex6 mutants with the impaired import of both PTS1- and PTS2-proteins, two clones, ZPEG236 and ZPEG244, showed a distinct, novel phenotype where PTS1-protein import was normal despite the abrogated PTS2 import. Dysfunction of Pex3p in pex3 ZPEG 238 was due to one base (G) insertion in the codon for Asn7 resulting in a frameshift, thereby inducing a distinct 31 amino-acid sequence and a termination. pex2 ZPEG239 showed a mutation in codon GAG for Glu(201) to a nonsense mutation, TAG. Thus, the method developed here using ICR191 could be useful for isolation of further novel cell mutants impaired in peroxisome biogenesis.     

Genotoxicity and DNA adduct formation of incense smoke condensates: comparison with environmental tobacco smoke condensates

Indoor air pollution has now been recognized as a potentially important problem for public health, since people spend most of their day in closed environments. Incense burning is possibly associated with elevated risks of leukemia and brain tumor in children from the epidemiological studies. Thus, evaluation of the genotoxicity of smoke condensates from incense burning is needed. We examined the genotoxicity of incense smoke condensates (ISC) using the Ames test in S. typhimurium strains with different mutagenic specificity and level of metabolic enzyme, the SOS chromotest in E. coli PQ37, and sister chromatid exchange assay in Chinese hamster ovary cells (SCE/CHO). The genotoxicity of environmental tobacco smoke condensates (TSC) was also evaluated by the three assays to compare with the genotoxicity of ISC, ISC showed a positive response in TA98, but not in TA100. It suggested that ISC only contained frame shift mutagens. The mutagenicity of ISC in both strains of TA98NR with deficient nitroreductase and TA98/1,8-DNP₆ with deficient O-acetyl-transferase was markedly decreased compared to that in TA98 strain. However, the mutagenicity was enhanced in YG1024 with overexpression of O-acetyltransferase activity. Thus, nitroarenes seemed to be responsible in part for the mutagenicity of ISC. Interestingly, all of the four ISC and two TSC samples showed a dose-dependent genotoxic response in the SOS chromotest with E. coli PQ37 but a low SCE induction of those samples were observed in CHO cells. When the genotoxicity was analyzed based on the condensates per one gram of original samples, the genotoxicity of two TSC condensates in prokaryotic cells was higher than that of four ISC samples except for the genotoxicity of TSC-2 in TA98 strain. However, the genotoxicity of certain ISC in eukaryotic cells based on the SCE/CHO assay was higher than that of TSC. To compare the covalent binding of DNA reactive intermediates of ISC and TSC to S. typhimurium TA98, the DNA adducts were evaluated by the ³²P-postlabeling method with butanol extraction version. Similar diagonal radioactive zone (DRZ) was observed between ISC and CSC. However, DNA adduct levels induced by TSC were much greater than that of ISC.     

Genotoxicity assessment of ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN)

Ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN) are relatively insensitive explosive compounds that are being explored as safe alternatives to other more sensitive compounds. When used in combination with other high explosives they are an improvement and may provide additional safety during storage and use. The genetic toxicity of these compounds was evaluated to predict the potential adverse human health effects from exposure by using a standard genetic toxicity test battery which included: a gene mutation test in bacteria (Ames), an in vitro Chinese Hamster Ovary (CHO) cell chromosome aberration test and an in vivo mouse micronucleus test. The results of the Ames test showed that EDDN increased the mean number of revertants per plate with strain TA100, without activation, at 5000μg/plate compared to the solvent control, which indicated a positive result. No positive results were observed with the other tester strains with or without activation in Salmonella typhimurium strains TA98, TA1535, TA1537, and Escherichia coli strain WP2 uvrA. DETN was negative for all Salmonella tester strains and E. coli up to 5000μg/plate both with and without metabolic activation. The CHO cell chromosome aberration assay was performed using EDDN and DETN at concentrations up to 5000μg/mL. The results indicate that these compounds did not induce structural chromosomal aberrations at all tested concentrations in CHO cells, with or without metabolic activation. EDDN and DETN, when tested in vivo in the CD-1 mouse at doses up to 2000mg/kg, did not induce any significant increase in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate that EDDN is mutagenic in one strain of Salmonella (TA100) but was negative in other strains, for in vitro induction of chromosomal aberrations in CHO cells, and for micronuclei in the in vivo mouse micronucleus assay. DETN was not genotoxic in all in vitro and in vivo tests. These results show the in vitro and in vivo genotoxicity potential of these chemicals.     

Sister-chromatid exchange and chromosome aberrations for 4 aliphatic epoxides in mice

Sister-chromatid exchange (SCE) and chromosome aberrations (CA) in bone marrow cells were analyzed after in vivo exposure in mice to 4 aliphatic epoxides, namely 1-naphthyl glycidyl ether (NGE), 1-naphthyl propylene oxide (NPO), 4-nitrophenyl glycidyl ether (NPGE) and trichloropropylene oxide (TCPO). These compounds were selected as being among the most mutagenic aliphatic epoxides in our previous structure-mutagenicity studies with the Ames test. There were significant dose-related increases in SCE and CA results for all 4 epoxides. The order of genotoxicity as established through SCE was NGE greater than NPO greater than NPGE approximately equal to TCPO greater than solvent control. It is of interest that Ames Salmonella results are consistent with in vivo genotoxicity for these compounds. However, only the plate test version of the Ames procedure is consistent with this order of in vivo genotoxicity and neither preincubation Ames testing results nor chemical alkylation rates would have predicted this order.     

Genotoxicity and PAC analysis of particulate and vapour phases of environmental tobacco smoke

Samples of indoor air were collected from an office room (88 m3) both before smoking and during experimental smoking of 96 cigarettes by 10 persons within 6 h. The particulates were collected on glass-fibre filters and the vapour-phase compounds on XAD-2 resin. The samples were extracted with acetone and analysed quantitatively for polycyclic aromatic compounds and qualitatively with GC-MS. The extracts of filters and XAD-2 resins were fractionated into neutral/acidic and 2 basic (strong and weak bases) fractions; all these fractions were tested with the sister-chromatid exchange (SCE) assay in Chinese hamster ovary (CHO) cells and with the Salmonella/microsome test (strain TA98). Total concentrations of PAC were 205 ng/m3 in the background sample and 1207 ng/m3 after contamination by cigarette smoking. The total PAC concentrations were 4-6 times higher in the vapour phase than in the particulate phase. The fractions of the particulate samples collected before smoking showed mainly marginal genotoxic activity, whereas after smoking their genotoxicity increased dramatically. The fractions of the vapour phase samples were not genotoxic before smoking, but after smoking the neutral/acidic and strong basic fractions induced responses in both assays. The SCE assay was more sensitive towards the vapour-phase mutagens of environmental tobacco smoke (ETS). The relative responses of the two basic fractions, whereas the fraction containing neutral and acidic compounds was the most potent in the SCE assay. In the Salmonella test, the mutagenic activity was mainly detected with metabolic activation, while the induction of SCE in CHO cells was also seen without an exogenous metabolic activation system.     

Frameshift mutagenesis by 9-aminoacridine and ICR191 in Escherichia coli: effects of uvrB, recA and lexA mutations and of plasmid pKM101

We have studied the effects of different repair capacities on reversion of two Escherichia coli strains (lacZ19124 and lacZ19136) by 9-aminoacridine (9AA) and the acridine half-mustard ICR191. Introduction of a uvrB mutation into these strains led to enhanced ICR191-induced reversion of lacZ19136 and reduced ICR191-induced reversion of lacZ19124. 9AA-induced reversion of lacZ19124 was essentially unchanged while reversion of lacZ19136 was reduced. Plasmid pKM101 reduced reversion of the two markers by each of the mutagens, except in the case of ICR191-induced reversion of the lacZ19124 marker where mutagenesis was slightly enhanced. Mutations in the recA and lexA genes had minimal effects on ICR191- and on 9AA-induced reversion of the lacZ markers; although 9AA-induced reversion of the lacZ19124 marker was somewhat reduced, most of the other results indicated that mutation yields were if anything higher in the recA or lexA backgrounds. Mutagenesis by 9AA and ICR191 would therefore appear to occur independently of the inducible error-prone repair process commonly referred to as SOS repair.     

Genotoxic effects of allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC)

Two isothiocyanates (ITCs) commonly found in human diet, allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC), were tested for genotoxic effects in a battery of assays: Salmonella/microsome assay with TA 98 and TA 100, differential DNA repair assay with E. coli and micronucleus (MN) induction assay with human derived Hep G2 cells. Albeit to a different degree, both ITCs induced genotoxic effects in all test systems. AITC was more genotoxic in bacterial test systems than in Hep G2 cells; in contrast, the effect of PEITC was stronger in Hep G2 cells. In in vivo assays with E. coli indicators in which mice were exposed to relatively high doses of the compounds (90 and 270 mg/kg), AITC induced moderate but significant effects; PEITC failed to induce significant effects in any of the organs. To find out the reason for the weak genotoxicity of AITC and PEITC under in vivo test conditions, we exposed E. coli indicator cells to the test substances in the absence or presence of rat liver homogenate (with and without cofactors), bovine serum albumin (BSA) and human saliva. All of them markedly attenuated the genotoxicity of AITC and PEITC, implying that the test substances are detoxified by direct non-enzymatic binding to proteins. Additional experiments carried out on the mechanistic aspects of AITC and PEITC-induced genotoxicity showed that the compounds induce the formation of thiobarbituric acid reactive substances (TBARS) in Hep G2 cells. Furthermore, in in vitro assays with E. coli, radical scavengers reduced the differential DNA damage induced by AITC and PEITC. The latter two findings give a clue that reactive oxygen species might be involved in the genotoxic effect of the ITCs. Although ITCs have been repeatedly advocated as very promising anticancer agents, the data presented here indicate that the compounds are genotoxic, and probably carcinogenic, in their own right.     

Cyto- and genotoxic effects of novel aromatic nitroxide radicals in vitro

Because of the increasing interest in the use of nitroxide radicals as antioxidants and probes for various applications in biological systems, the question of their toxicity is of paramount importance. Cytotoxicity and mutagenicity studies have been extensively performed with the commercially available aliphatic nitroxides, and the general outcome is that these compounds are nonmutagenic and relatively noncytotoxic. In this study, the cytotoxicity and genotoxicity of a new class of aromatic nitroxides that we have synthesized (i.e., indolinonic and quinolinic nitroxides), whose antioxidant activity has been established in both chemical and biological systems, were evaluated and compared with those of two commercial nitroxides and with that of butylated hydroxytoluene (BHT). The mutagenicity assay was performed using Salmonella typhimurium tester strains TA98, TA100, and TA102, chosen on the basis of their ability to detect various types of mutations and their sensitivity to oxidative damage. None of the compounds tested were found to be mutagenic. The colony-forming assay (CFA) using Chinese hamster ovary (CHO) AS52 cells was employed for determining the cytotoxicity of the test compounds. On comparing the effective dose that inhibits the CFA by 50% (IC(50)), most of the compounds tested on an equal molar concentration basis were less toxic than BHT. Therefore, the overall results obtained correlate well with the data reported in the literature on the toxicity of aliphatic nitroxides and lend support to the possible use of these compounds as therapeutic antioxidants.     

Chromosome aberrations in Chinese hamster and human cells: a comparison using compounds with various genotoxicity profiles

Chromosome aberrations (Cabs) can be induced in vitro by non-DNA damaging compounds, often associated with cytotoxicity and DNA synthesis inhibition, and under conditions that would not be relevant in vivo. Such misleading positive results are reported both in Chinese hamster cell lines and in human peripheral blood lymphocytes (HL). We assessed the response of HL to compounds with varied genetic toxicity profiles, all of which induced Cabs in CHO cells Seven of 10 compounds were negative or equivocal in HL. Results in purified lymphocytes for four verified that the difference was not due to the presence of blood in cultures. Two compounds that were weakly positive in the Ames test and one that induced DNA adducts were negative or equivocal in the HL assay; their overall mutagenic potential in vivo is not clear. Of four Ames-negative compounds, three of which inhibited DNA synthesis in CHO cells, three were negative and one was equivocal in the HL assay. A potent Cab inducer, which also induced micronuclei in vivo (but was negative in the Ames test) was clearly positive in the HL assay. Two compounds were clearly positive in HL only when the mitotic indices (MI) were below 50% of control. These are genotoxic in other assays but our evidence suggests that Cab induction is related more to toxicity than to primary DNA damage. For this limited set of 10 compounds, HL were more likely than CHO cells to give negative or equivocal results. It is likely that more stringent checkpoint controls in human cells prevent damaged cells reaching mitosis, and may also influence the reported greater sensitivity to induction of aneuploidy and polyploidy of normal rodent compared with human cells. In the studies reported here, two strong inducers of polyploidy in CHO cells gave weaker increases in HL. Human lymphocytes have disadvantages as a routine screening assay (finding donors, known individual variability, increased time required and the inadequacy of the MI as a toxicity measure), but may be useful in follow-up testing to assess weight of evidence about genotoxic risk to humans, for compounds that are positive in the Chinese hamster cell Cabs assays.     

Comparison of the continuous rat hepatoma cell line 2sFou with primary rat hepatocyte cultures for the induction of DNA repair synthesis by nitrosamines, benzo[a]pyrene and hydroxyurea

We have examined the suitability of the continuous rat hepatoma cell line 2sFou for testing the genotoxicity of chemicals in comparison with that of primary rat hepatocyte cultures (HPC). The capacity of the cells for metabolic activation was assessed by measuring induction of DNA-repair synthesis and inhibition of replicative DNA synthesis by the test compounds dimethylnitrosamine (DMN), diethylnitrosamine (DEN), hydroxyurea (HU) and benzo[a]pyrene (BaP), which are substrates for major hepatic and extrahepatic forms of cytochrome P-450 dependent monooxygenases. The cellular capacity for DNA-repair synthesis was assessed using UV-light as a DNA-damaging agent. Repair-specific incorporation of [3H]deoxycytidine (3H-dCyd) caused by UV-light was higher in 2sFou cells than in HPC. In contrast, background repair incorporation of 3H-dCyd in 2sFou cells was only 1/3 that found in HPC. All the test agents induced DNA repair and inhibited DNA synthesis in both 2sFou cells and HPC. The two nitrosamines were more effective in HPC than in 2sFou cells. HU and BaP affected DNA repair and DNA synthesis in the two cell systems at a similar range of concentrations. In general, DNA repair in the 2sFou cells increased near linearly with the concentrations of the test compounds. The data indicate that 2sFou cells are capable of activating hepatotropic pro-mutagens/carcinogens such as dialkylnitrosamines, and are sensitive indicators of DNA damage. In contrast, BaP, a non-hepatotoxic compound, caused only little DNA repair in these cells. Thus, continuously growing cells, such as 2sFou, show a qualitatively similar response to genotoxic chemicals as HPC and offer a potential alternative to HPC for genotoxicity testing.     

Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.     

Correcting for toxic inhibition in quantification of genotoxic response in the umuC test.

An improved procedure for quantification of results from the umuC tests for genotoxicity is presented. The calculation method better separates toxic growth inhibition (cytotoxicity) from genotoxic effects than currently used methods and therefore, greatly extends the applicability of genotoxicity tests on environmental samples. The basic principle is to normalize the genotoxic response compensating for both decreasing biomass and growth rate reduction that results from cytotoxicity. The improved method and the currently used method was compared for umuC tests on the pure compounds: methylmethanesulfonate (MMS), N-methyl-N'-nitro-N-nitroguanidine (MNNG), sodium azide (NaN3), and 4-nitroquinoline-1-oxide (4-NQO). For compounds with no or low cytotoxicity, the two calculation methods gave practically identical results, while for highly cytotoxic compounds, the traditional method overestimated genotoxicity. umuC tests were also carried out on leachate polluted groundwater sampled downgradient of a landfill (Grindsted, Denmark). All polluted samples showed high cytotoxicity concomitant with high genotoxicity when the results were quantified in the traditional way. The new method showed that these results were in fact false positive, as the apparent genotoxicity was a result of cytotoxicity. Based on the mathematical analysis leading to the improved procedure for correction for cytotoxicity, it is suggested to alter the present test design of the umuC test in order to obtain well-defined exposure concentrations as well as mathematical consistency in the quantification of genotoxicity.     

Comparative genotoxicity of nitrosamine drinking water disinfection byproducts in Salmonella and mammalian cells

Nitrosamine water disinfection byproducts (DBPs) are an emerging class of non-halogenated, nitrogen-containing water contaminants. Five nitrosamine DBPs were analyzed for genotoxicity (N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomorpholine (NMOR) and N-nitrosodiphenylamine (NDPhA). Using Salmonella typhimurium strain YG7108 the descending rank order of mutagenicity was NDMA>NPIP>NMOR>NPYR; NDPhA was not mutagenic. We developed and calibrated an exogenous S9 mix that was highly effective in activating NDMA in Chinese hamster ovary (CHO) cells using the SCGE (Comet) assay. The descending rank order for genotoxicity was NDMA>NPIP>NMOR. NDPhA was genotoxic only at one concentration and NPYR was not genotoxic. The genotoxic potencies in S. typhimurium and CHO cells were highly correlated. Based on their comparative genotoxicity attention should be focused on the generation and occurrence of NDMA, NPIP and NMOR. Current drinking water disinfection processes may need to be modified such that the generation of nitrosamine DBPs is effectively limited in order to protect the environment and the public health.