2-Amino-N6-hydroxyadenine induces gene/point mutations and multiple-locus mutations, but not multilocus deletion mutations, in the ad-3 region of a two-component heterokaryon of Neurospora crassa.

The mutagenicity of 2-amino-N6-hydroxyadenine (AHA) has been studied in Neurospora crassa by treating a two-component heterokaryon (H-12) and recovering specific-locus mutations induced in the ad-3 region. This assay system permits the identification of ad-3A and/or ad-3B mutants resulting from gene/point mutations, multilocus deletion mutations, and multiple-locus mutations of various genotypes, involving one or both loci. Genetic characterization of the ad-3 mutants recovered from experiments with AHA in H-12 shows that 98.9% (270/273) of the ad-3 mutants are gene/point mutations (ad-3R), 1.1% (3/270) are unknowns, and none is a multilocus deletion mutation (ad-3IR). Among the gene/point mutations, 3.3% (9/273) are multiple-locus mutations (gene/point mutations with a closely-linked recessive lethal mutation [ad-3R + RLCL]). Another 25.3% (69/273) are multiple-locus mutations with a recessive lethal mutation located elsewhere in the genome [ad-3R + RL]. Heterokaryon tests for allelic complementation among the ad-3BR mutants showed that 90.8% (139/153) of the mutants were complementing, and 20.3% (31/153) were leaky. In addition, 32.5% (38/117) of the ad-3AR mutants were leaky. These data are consistent with the hypothesis that AHA produces specific-locus mutations in the ad-3 region of N. crassa by base-pair substitution. The data from the present experiments are compared with the data for 2-aminopurine (2AP)-induced ad-3 mutants in H-12 (de Serres and Brockman, 1991). Whereas, 2AP is a weak mutagen in H-12, AHA is extremely potent (Brockman et al., 1987). In contrast with 2AP, AHA induces ad-3 mutants exclusively by gene/point mutation in H-12. We conclude that whereas AHA induces ad-3 mutants predominantly by AT to GC base-pair transitions, 2AP induces ad-3 mutants by a wide variety of mechanisms including: (1) AT to GC and GC to AT base-pair transitions, (2) frameshift mutations, (3) other, as yet unidentified, intragenic alterations, (4) small multilocus deletion mutations, and (5) multiple-locus ad-3R mutations with closely linked recessive lethal mutations.     

Mutagenic activity and specificity of N-nitrosomethylaniline and N-nitrosodiphenylamine in Salmonella

The carcinogenic nitrosamines, N-nitrosomethylaniline (NMA) and N-nitrosodiphenylamine (NDphA), which have been previously reported negative or very weakly mutagenic in the Salmonella/microsome assay, were found to be mutagenic in the hisG428 Salmonella strain, TA104. NMA was moderately potent and NDphA was about 10% as potent. Mutagenesis by both compounds was dependent on the uvrB mutation and enhanced in strains harboring the plasmid, pKM101. The mutational specificities of NMA and NDphA for base-pair substitutions were determined by assaying their activities in several mutants which are reverted by a limited number, or a single type of base-pair substitution mutation, and additionally by subclassification of revertants. NMA induced predominantly AT----CG transversions and NDphA induced AT----TA transversions. The specificity of NMA and NDphA for mutagenesis at AT base pairs and the lack of sensitivity of the previously employed hisG46 strains for these base changes may be the reason for the previous reports on the lack of mutagenic activity of these compounds. This specificity is quite unusual for nitrosamines and is consistent with the hypothesis that NMA and NDphA lead to DNA damage of different nature than that produced by other nitrosamines.     

A review of the genetic effects of ethyl methanesulfonate

Ethyl methanesulfonate (EMS) is a monofunctional ethylating agent that has been found to be mutagenic in a wide variety of genetic test systems from viruses to mammals. It has also been shown to be carcinogenic in mammals. Alkylation of cellular, nucleophilic sites by EMS occurs via a mixed SN1/SN2 reaction mechanism. While ethylation of DNA occurs principally at nitrogen positions in the bases, because of the partial SN1 character of the reaction, EMS is also able to produce significant levels of alkylation at oxygens such as the O6 of guanine and in the DNA phosphate groups. Genetic data obtained using microorganisms suggest that EMS may produce both GC to AT and AT to GC transition mutations. There is also some evidence that EMS can cause base-pair insertions or deletions as well as more extensive intragenic deletions. In higher organisms, there is clear-cut evidence that EMS is able to break chromosomes, although the mechanisms involved are not well understood. An often cited hypothesis is that DNA bases ethylated by EMS (mostly the N-7 position of guanine) gradually hydrolyze from the deoxyribose on the DNA backbone leaving behind an apurinic (or possibly an apyrimidinic) site that is unstable and can lead to single-strand breakage of the DNA. Data also exist that suggest that ethylation of some chromosomal proteins in mouse spermatids by EMS may be an important factor in causing chromosome breakage.     

Reversions of the two missense and one nonsense trp-mutations induced by UV-light or methyl methanesulfonate in E. coli wild type and its polAi and uvrE502 mutants.

Two missense mutations, trpA58 and trpA78, and one nonsense mutation-trp-ochre, were used to determine the types of base-pair substitution caused by ultra, violet irradiation and methyl methanesulfonate (MMS) in Escherichia coli. UV irradiation of the wild-type bacteria led to the formation of revertants mainly arising as a result of GC yields AT transitions (suppressor revertants of the trpA58 mutant). True revertants of the trp- mutant (arising via transitions of AT pairs) and 5-methyl tryptophan-sensitive (MT-s) Trp+ of the trpA78 mutant (arising via unidentified transversions) occurred at a lower frequency. The polAI mutation did not change the frequency of the UV-induced transitions GC yields AT or that of the substitutions of the AT pairs. The uvrE502 mutation significantly increased the frequency of the UV-induced revertants arising via the transition GC yields AT. Treatment of the wild-type bacteria with MMS resulted in the formation of revertants mainly due to the GC yields AT substitution, and with a lower frequency to the AT yields GC transitions. MMS also induced, with a low frequency, some transversions. The frequency of the MMS-induced GC yields AT transitions was enhanced in the uvrE502 mutant. On the other hand, the uvrE502 mutation eliminated or significantly lowered MMS-induced revertants arising as a result of AT yields GC transitions or transversions.     

Mutagenic specificity of N4-hydroxycytidine

The mutagenic specificity of (oh)⁴Cyd was examined with T4rII phage mutants which allows for discrimination between AT→GC and GC→AT base transitions. AT→GC transitions were induced with a frequency 1–2 orders of magnitude higher than GC→AT transitions. The mechanism of this preferential transition pathway is discussed in the light of base-analogue mutagenesis.     

Mutation spectra in Salmonella of analogues of MX: implications of chemical structure for mutational mechanisms

We determined the mutation spectra in Salmonella of four chlorinated butenoic acid analogues (BA-1 through BA-4) of the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and compared the results with those generated previously by us for MX and a related compound, MCF. We then considered relationships between the properties of mutagenic potency and mutational specificity for these six chlorinated butenoic acid analogues. In TA98, the three most potent mutagens, BA-3, BA-4, MX, and the organic extract, all induced large percentages of complex frameshifts (33-67%), which distinguish these agents from any other class of compound studied previously. In TA100, which has only GC sites for mutation recovery, >71% of the mutations induced by all of the agents were GC-->TA transversions. The availability of both GC and TA sites for mutation in TA104 resulted in greater distinctions in mutational specificity than in TA100. MX targeted GC sites almost exclusively (98%); the structurally similar BA-4 and BA-2 produced mutations at similar frequencies at both GC and AT sites; and the structurally similar BA-3 and BA-1 induced most mutations at AT sites (69%). Thus, large variations in structural properties influencing relative mutagenic potency appeared to be distinct from the more localized similar structural features influencing mutagenic specificity in TA104. Among a set of physicochemical properties examined for the six butenoic acids, a significant correlation was found between pK(a) and mutagenic potency in TA100, even when the unionized fraction of the activity dose was considered. In addition, a correlation in CLOGP for BA-1 to BA-4 suggested a role for bioavailability in determining mutagenic potency. These results illustrate the potential value of structural analyses for exploring the relationship between chemical structure and mutational mechanisms. To our knowledge, this is the first study in which such analyses have been applied to structural analogues for which both mutagenic potency and mutation spectra date were available.     

Differential mutagenic activity of IQ (2-amino-3-methylimidazo[4,5-f]quinoline) in Salmonella typhimurium strains in vitro and in vivo, in Drosophila, and in mice

IQ, a heterocyclic aromatic amine which is formed during the frying of meat, was prepared by chemical synthesis. Its genotoxic potential was studied in bacteria, Drosophila and in mice. A mutagenic effect of IQ (frameshift induction) was detected in Salmonella typhimurium in experiments without metabolic activation; this effect was several orders of magnitude lower than that observed in the presence of an activation system. Ames tests with liver-homogenate S9 fraction from PCB-induced mice and rats confirmed the high mutagenic potency of IQ metabolites (Kasai et al., 1980a). Comparative studies on diagnostic Salmonella strains revealed that the high frameshift-inducing activity is independent of the plasmid pkM101; it is, however, greatly reduced by an intact excision-repair system for DNA lesions. The mutagenic activity of the metabolite(s) formed in vitro by S9 mix has a half-life of ca. 14 min. In the fruit fly, Drosophila melanogaster, IQ induced when used at sublethal concentrations, X-chromosomal recessive lethal mutations in male germ cells in a dose-dependent manner. In mice, tests were performed to detect somatic mutations: chromosomal anomalies (micronuclei) in bone marrow, and gene mutations (affecting coat pigmentation) in mice exposed to IQ in utero. No genotoxic effects were observed in these assays. However, the formation of mutagenic metabolites in the liver of IQ-treated mice was unequivocally demonstrated in host-mediated assays using Salmonella as mutagen probes in mice. The data demonstrate genotoxic activity of IQ in prokaryotic and eukaryotic organisms. The possible reasons for the different response of mammalian systems in vivo and the Salmonella system are discussed.     

First characterization of the mutagenic specificity of 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000) in Salmonella typhimurium

In order to investigate the mutational events induced by the very potent genotoxic agent 7-methoxy-2-nitronaphtho[2,1-b]furan (R7000), we evaluated its mutagenic potency in a battery of his- mutants of Salmonella typhimurium designed to study mutagenic specificity (Levin and Ames, 1986). Using this system we could show that R7000 is able to induce base-pair substitutions, mainly GC----TA transversions and, perhaps to a lower extent, AT----TA transversions. In addition, our results suggest that this chemical may be a very efficient inducer of base-pair deletions/insertions resulting in frameshifts. Whereas the induction of base-pair substitutions by R7000 is dependent on the presence of plasmid pKM101 carrying the mucA,B operon, the induction of base-pair deletions/insertions is independent of the presence of the plasmid.     

Mutagenic potency and specificity of procarbazine in the ad-3 forward-mutation test in growing cultures of heterokaryon 12 of Neurospora crassa.

Procarbazine (Natulan) was tested for its mutagenic potency and specificity in the ad-3 forward-mutation test in heterokaryon 12 (H-12) of Neurospora crassa. In these experiments, procarbazine was a weak mutagen when present in growing cultures but nonmutagenic when conidial suspensions (nongrowing conidia) were treated. A total of 208 ad-3 mutants recovered after exposure of growing cultures of H-12 to 1 mg of procarbazine/ml, and 2 ad-3 mutants of spontaneous origin, were characterized genetically. These tests distinguish among gene/point mutations (ad-3R) at the ad-3A or ad-3B locus, multilocus deletion mutations ([ad-3]IR) covering one or more loci in the ad-3 and immediately adjacent regions, and 3 different classes of multiple-locus mutations: gene/point ad-3 mutations with a recessive lethal mutation elsewhere in the genome (ad-3R + RL), gene/point mutations with a closely linked recessive lethal mutation (ad-3R + RLCL), and multilocus deletion mutations with a closely linked recessive lethal mutation ([ad-3]IR + RLCL). All of the procarbazine-induced ad-3 mutants resulted from gene/point mutations; 92.2% (200/217) resulted from gene/point mutations at the ad-3A or ad-3B locus, and 3.7% (8/217) resulted from gene/point mutations with a recessive lethal mutation elsewhere in the genome. Identical percentages (15.4% [20/130] and 15.4% [12/78]) of the sigma ad-3BR and sigma ad-3AR mutants were leaky, and a high percentage (71.5% [93/130]) of the sigma ad-3BR mutants had nonpolarized complementation patterns. These results indicate that procarbazine-induced ad-3 mutants of Neurospora crassa are composed solely of gene/point mutations (ad-3R) that resulted, predominantly or exclusively, from base-pair substitutions. The Neurospora specific-locus data on procarbazine-induced ad-3 mutants are compared with data from similar experiments with the mouse using the morphological specific-locus assay; marked similarities were found between the mutagenic effects of procarbazine in the 2 specific-locus assays.     

The spectra of base substitutions induced by the impCAB,mucAB and umuDC error-prone DNA repair operons differ following exposure to methyl methanesulfonate

We have used the lacZ reversion assay to study the mutation spectra induced by the Escherichia coli chromosomal umuDC operon and of its two plasmid-borne analogues impCAB and mucAB following exposure of cells to UV light and methyl methane-sulfonate (MMS). We have shown that the impCAB, mucAB and umuDC operons all produce a similar response to UV light which results almost exclusively in AT → GC transitions. However, we found that the three operons produced different responses to alkylating agents. We found that with MMS the chromosomal umuDC operon produced almost exclusively AT → GC transitions, whilst both mucAB and impCAB produced predominantly transversions. In the case of the impCAB operon the mutation spectrum contained more AT → TA than GC → TA transversions; this balance was reversed with mucAB. The effect of the copy number of the error-prone DNA repair operons upon the mutagenic spectra was also studied. The results obtained suggest that the copy number of the imp operon does not greatly affect the specificity of base substitutions observed. However, an increase in the copy number of the umuDC operon greatly affected the specificity of base substitution, such that virtually no transitions were produced and the spectrum was dominated by GC/AT → TA transversions. It appears that the three error-prone DNA repair operons impCAB, mucAB and umuDC, despite showing strong structural and functional homologies, can display major differences in the spectrum of base changes induced during mutagenesis. We propose that the type of misincorporation/chain extension which DNA polymerase III is allowed to synthesize on a damaged DNA template is extremely sensitive to both the amount and type of error-prone repair proteins present. The modulation of these events by the different proteins can result in widely different mutagenic changes in the repaired DNA.     

The genetic activity of N6-hydroxyadenine and 2-amino-N6-hydroxyadenine in Escherichia coli, Salmonella typhimurium and Saccharomyces cerevisiae

The genetic activity of 2-amino-N6-hydroxyadenine or 2-amino-N-hydroxylaminopurine (AHA) and N6-hydroxyadenine or 6-N-hydroxylaminopurine (HAP) was studied in S. typhimurium, E. coli and Saccharomyces cerevisiae strains. AHA was a more potent mutagen for bacteria and a less potent mutagen for yeast than HAP. The mutagenic activity of analogs was not influenced by excision, mutagenic or double-strand DNA repair mutations. On the other hand, the uvrBdel mutation has a drastic effect on the mutagenicity and toxicity of both analogs in the Salmonella strains studied. HAP was a very potent mutagen in yeast with a low capability of inducing mitotic recombination contrary to common mutagens, possessed unique intergenic specificity and was able to induce mutations in diploids at rather high frequency.     

Two N-hydroxylaminopurines are highly mutagenic in the ad-3 forward-mutation test in growing cultures of heterokaryon 12 of Neurospora crassa

3 purine analogs were tested for their mutagenic activities in the ad-3 forward-mutation test in heterokaryon 12 (H-12) of Neurospora crassa. In growing cultures of H-12, the N-hydroxylaminopurines 2-amino-6-N-hydroxylaminopurine (AHA) and 6-N-hydroxylaminopurine (HAP) are potent and strong mutagens, respectively, whereas 2-aminopurine (AP) is a weak mutagen. AHA and HAP are about equally mutagenic at low doses, but AHA is more mutagenic than HAP at high doses. Despite their potent mutagenicity in growing cultures, AHA and HAP are not mutagenic in nongrowing conidia under the conditions of our experiments. AHA is the most potent mutagen tested in the ad-3 forward-mutation test in N. crassa. At the highest dose tested (30 micrograms/ml), it gave an ad-3 mutant frequency of 0.7 X 10(-2), about a 12,000-fold increase over the average spontaneous ad-3 mutant frequency. The potent mutagenicity of AHA may make it (and possibly HAP) especially useful for obtaining specific-locus mutations in other organisms.     

Mutagenicity of N-nitrosopyrrolidine derivatives in Salmonella (Ames) and Escherichia coli K-12 (343/113) assays

The mutagenicity of nitrosopyrrolidine (NPYR) and its derivatives was determined by use of the Ames Salmonella assay. A clear specificity to revert the missense stain of TA1535 and a requirement for the phenobarbital-induced rat-liver activation system (S9 mix) were noted. 3,4-Dichloronitrosopyrrolidine was more mutagenic than NPYR, whereas 3-hydroxynitrosopyrrolidine was weakly mutagenic. The carcinogenic nitroso-3-pyrrolidine was not mutagenic under the test conditions. The noncarcinogenic derivatives (2,5-dimethylnitrosopyrrolidine, nitrosoproline and 4-hydroxynitrosoproline) were not mutagenic. Liquid preincubation assays were not any more effective than the pour-plate assays. Selected derivatives of NPYR were tested in the Escherichia coli K-12 (343/113) assay A specificity to revert the missense mutation at the arg locus and a dependence on phenobarbital-induced rat-liver S9 mix were noted with NPYR and its derivatives. 3,4-Dibromonitrosopyrrolidine, which was not mutagenic in Salmonella, was effective in E. coli, and the weakly carcinogenic NPRL was a weak mutagen resulting in a 2-fold enhancement in the E. coli arginine reversion assay.     

Toxic genetic effects of flunitrazepam

The mutagenic activity of Flunitrazepam, the active ingredient of the drug Rohypnol, has been investigated by using the Salmonella/microsome mutagenicity test. A dose-related mutagenic effect was observed on Salmonella typhimurium strain TA 100 either in the absence or in the presence of a rat liver microsomal fraction (S9) as in vitro metabolic activation system. By adopting a modification of the Salmonella test, the mutagenicity of urines from rats or patients treated with the drug was evaluated. In these cases mutagenic activity was detected toward the Salmonella strains TA 98 and TA 100 both in presence and in absence of the metabolic activation system. The data indicate that Flunitrazepam and/or its urinary metabolites can induce both base-pair substitutions or frame-shift point mutations.     

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

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

In vitro and in vivo analysis of somatic and germline mutability of 2-amino-N6-hydroxyadenine in Drosophila melanogaster

Two complementary assays were employed to examine the mutagenicity of 2-amino-N6-hydroxyadenine (AHA) in Drosophila melanogaster. A lambda phage-based shuttle vector system, utilizing the supF transfer RNA gene of Escherichia coli, questioned the mutagenicity of AHA in established cell cultures derived from somatic tissue while the standard sex-linked recessive lethal assay measured mutational events in vivo. Consistent with studies in other systems, AHA appears strongly mutagenic when cells are exposed directly. Conversely, in vivo studies suggest that AHA is not a strong mutagen. Further studies will determine if AHA is weakly or not mutagenic in vivo and, using the supF system, what the nature of the mutational events at the molecular level is.     

Bacterial mutagenicity of aceanthrylene: a novel cyclopenta-fused polycyclic aromatic hydrocarbon of low molecular weight

Aceanthrylene, a non-alternant cyclopenta-fused hydrocarbon, was shown to be weakly mutagenic without S9 and strongly mutagenic with S9 in the Ames Salmonella plate incorporation assay. The compound was most active in strain TA100 (35 revertants/nmole in the presence of 0.3 mg of S9 protein), and less active in strains TA98, TA1537 and TA1538 (20, 10 and 3.1 rev/nmole respectively, + S9). Strain TA1535 was unresponsive, suggesting that this compound induces frameshift mutations rather than base-pair substitutions. The mutagenic potency of aceanthrylene is consistent with predictions of its activity based on the relatively large delocalization energy (delta E deloc/beta = 0.931) of the carbonium ion which would result from oxirane ring opening of the 1,2-epoxide, a potential active metabolite.     

Comparison of the mutagenic specificity induced by four nitro-group-containing aromatic amines in Salmonella typhimurium his genes

Four nitrated aromatic amines (2-nitro-p-phenylenediamine [2NPD], 3-nitro-o-phenylenediamine [3NPD], 4-nitro-o-phenylenediamine [4NPD] and 4,4'-dinitro-2-biphenylamine [DNBA]) are direct-acting mutagens in Salmonella typhimurium strain TA100. These compounds were tested further using the Xenometrix strains of S. typhimurium: TA7001, TA7002, TA7003, TA7004, TA7005, and TA7006, with and without S9 mix in the plate incorporation assay. The direct-acting mutagenicity of 2NPD, 4NPD, and DNBA was detected with TA7002, TA7004 and TA7005. 2NPD and DNBA showed some activity in TA7006; DNBA also showed some activity in TA7003. Mutagenicity was generally decreased in these strains when S9 was added. 3NPD was mutagenic in TA7004 without S9 and in TA7005 with and without S9. These data suggest that 2NPD, 4NPD and DNBA induced TA-->AT and CG-->AT transversions as well as GC-->AT transitions in the his gene. 3NPD induced CG-->AT transversions and GC-->AT transitions. 2NPD and DNBA also induced a small portion of CG-->GC transversions.     

Mutational specificities of 1′-acetoxysafrole,N-benzoyloxy-N-methyl-4-aminoazobenzene,and ethyl methanesulfonate in human cells

We have used an oriP-tk shuttle vector t determine the types of mutations induced in human cells by ethyl methanesulfonate (EMS), 1'-acetoxysafrole (AcOS), and N-benzoyloxy-N-methyl-4-aminoazobenzene (BzOMAB). Plasmid DNA was treated in vitro with mutagen and electroporated into human lymphoblastoid cells. After replication of the vector in human cells, plasmids were analyzed for mutations in the herpes simplex virus type 1 thymidine kinase gene. Ethyl methanesulfonate induced predominantly GC → AT transition mutations. Treatment of the shuttle vector with AcOS induced 5 of the 6 possible base substitution mutations, including GC → AT (32%) and AT → GC (14%) transition mutations, GC → TA (%), GC → CG (18%), and AT → TA (14%) transversion mutations, as well as a low frequency (9%) of −1 frameshift mutations at GC base pairs. Replication in human cells of DNA modified with BzOMAB yielded a significant increase (17-fold) in the frequency of deletion mutations relative to solvent-treated DNA. A majority (94%) of the point mutations induced by BzOMAB occurred at GC base pairs and were predomianntly GC → AT transitions (33%) and −1 frameshift (22%) mutations, with the remainder consisting mainly of transversions at GC base pairs (28%). The broad spectrum of base substitution mutations observed for AcOS and BzOMAB may indicate the frequent insertion of a variety of bases during replicative bypass of aralkylated bases in human cells.     

The capacity of some nitro- and amino-heterocyclic sulfur compounds to induce base-pair substitutions

The capacity of 27 heterocyclic sulfur compounds to induce base-pair substitutions was investigated with Klebsiella pneumoniae ur- pro- and Salmonella typhimurium TA100 as test organisms. Among the compounds tested, all sulfur compounds with nitro groups and some thiazoles with an amino group were mutagenic. Among the nitrothiazoles, the most potent mutagen was niridazole, followed by 2-acetamido-5-nitrothiazole, 2-bromo-5-nitrothiazole, N-(5-nitrothiazol-2-yl)benzamide, and 2-amino-5-nitrothiazole. Of the nitrothiophenes, 2-nitrothiophene was more mutagenic than 3-nitrothiophene and 2,4-dinitrothiophene. 4-Nitroisothiazole was also mutagenic. Of the aminothiazoles, 2-amino-5-bromothiazole and 2-amino-5-chlorothiazole were mutagenic to both test organisms. With 2-amino-5-(p-nitrophenylsulfonyl)thiazole, a mutagenic action was only found with Salmonella typhimurium TA100, whereas 2-aminothiazole and 2-amino-4-methylthiazole were only mutagenic with Klebsiella pneumoniae. With the other 13 compounds, no mutagenic activity was observed. Of the coccidiostatics, 2-acetamido-5-nitrothiazole was also mutagenic on Escherichia coli K12 and Saccharomyces cerevisiae D4 but non-mutagenic on Salmonella typhimurium TA1530, TA1535, TA1537 and TA98, while 2-amino-5-nitrothiazole was mutagenic on Escherichia coli K12, Salmonella typhimurium TA1530, TA1535 and TA98, and non-mutagenic on strain TA1537 and on Saccharomyces cerevisiae D4.