The structural basis of the mutagenicity of chemicals in Salmonella typhimurium: the National Toxicology Program Data Base

A portion of the U.S. National Toxicology Program (NTP) Salmonella typhimurium mutagenicity data base was analyzed by CASE, an artificial intelligence SAR system. CASE identified 13 structural determinants which, with a high probability (p less than or equal to 0.05) predicted the likelihood of mutagenicity of the 243 chemicals in the data base (sensitivity = 0.989; specificity = 0.950) as well as of chemicals not included in the data base. CASE also identified an additional set of structures which were highly predictive of mutagenic potency (sensitivity = 0.949; specificity = 1.00). Even though there is little overlap among the chemicals included in the NTP and Gene-Tox Salmonella data bases, CASE found significant similarities between the structural determinants of the mutagenicity in the two data bases, thereby validating the analyses and indicating a commonality in the structural basis of mutagenicity.     

Frameshift mutagenicity in Salmonella typhimurium of furocoumarins in the dark

The dark mutagenicity of 4,5',8-trimethylpsoralen (4,5',8-TMP), 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), 3-carbethoxypsoralen (3-CPs) and two new pyridopsoralens (PyPs and MePyPs) was tested using the Ames Salmonella plating assay in the absence of metabolic activation. 4,5',8-TMP, 8-MOP and the two pyridopsoralens were found to be weak frameshift mutagens in strain TA1537 whereas 5-MOP and 3-CPs did not demonstrate any significant mutagenic activity. These findings support the notion that the genetic risks of these psoralens in the dark may be considered to be negligible.     

Conditions affecting the mutagenicity of sodium bisulfite in Salmonella typhimurium

Sodium bisulfite is a weak mutagen at pH 5 and 6 in S. typhimurium strains carrying the hisG46 and hisD6610 mutations, but is not mutagenic in strains with the hisC3076 or hisD3052 mutations. The bisulfite-induced base-pair substitution mutations were slightly enhanced by the presence of the plasmid, pKM101, but inhibited by the presence of the uvrB and rfa mutations. The hisO1242 mutation which causes constitutive expression of the histidine operon, produced a slight enhancement of frameshift (hisD6610), but not base-pair substitution (hisG46) mutations. Bisulfite-induced mutations appear to be the result of two different mechanisms which may be a function of the repair capacity of the strains. The data suggest that the deamination of cytosine may not be responsible for frameshift mutations, but may be responsible for base-pair substitution mutagenesis. Because the rate of bisulfite autooxidation appears to play a role in the mutagenic process, we are suggesting that the deamination of cytosine may be the result of oxidative damage rather than through the direct formation of a cytosine-bisulfite adduct. This is further supported by the much lower concentrations of bisulfite needed to cause mutagenicity than the 1 M concentrations cited to produce cytosine-bisulfite adducts.     

Synthesis and enantioselective mutagenicity of azidoalanine in Salmonella typhimurium

Azide mutagenicity involves the requisite formation of the putative novel aminoacid metabolite, beta-azidoalanine. The role of this metabolite, however, is unclear. In order to confirm the identity of this metabolite and provide additional information on possible stereochemical requirements for mutagenicity, authentic racemic and L-azidoalanine were synthesized by an unambiguous route and tested for mutagenicity in Salmonella typhimurium TA100, TA1535, hisG46 and Escherichia coli WP2-. A marked antipodal potency ratio was observed in strains TA100 and TA1535 when racemic and L-azidoalanine were compared. The mutagenic activity resided primarily in the L-isomer. The molar potency of L-azidoalanine in TA100 and TA1535 was nearly identical to that of azide. The lack of mutagenic response for racemic or L-azidoalanine in hisG46 and E. coli WP2- was like that reported for azide and is consistent with similar modes of action for these agents.     

Stereochemical effect in the mutagenicity of the aflatoxicols toward Salmonella typhimurium

The mutagenicities of [1R] and [1S] aflatoxicol were measured using the Salmonella microsome test. In strain TA100 the [1R] form (unnatural aflatoxicol, aflatoxicol B) had a mutagenic potency approximately four times that of the [1S] epimer (natural aflatoxicol, aflatoxicol A, Ro) in the presence of S-9 liver microsomal fraction. The order in mutagenic potency compared to some other toxicologically important aflatoxins was as follows: B1 greater than [1R] approximately equal to G1 much greater than [1S] much much greater than B2. Thus, the trans relationship between the vinyl ether and hydroxyl groups leads to greater mutagenicity than the cis relationship. This may be important in the elucidation of stereochemical structure-activity relationships for the aflatoxins.     

The unusual effect of pKM101 on the mutagenicity of acetaldehyde oxime in Salmonella typhimurium

Acetaldehyde oxime was found to induce more revertants in Salmonella typhimurium strain TA1535 than in TA100 in the absence of S9 metabolic activation. TA100 was originally constructed from TA1535 by the addition of the plasmid pKM101, carrying mucAB which generally enhances sensitivity to the mutagenic effects of chemicals. The role of pKM101 in lowering the sensitivity to acetaldehyde oxime was explored by: (1) increasing the incubation time of the selective agar plates from 2 to 3 days; (2) using a new strain, isogenic to TA100, constructed by introducing pKM101 into the TA1535 isolate used in these experiments; (3) by testing a strain constructed by inserting into TA1535 a plasmid carrying mucAB but otherwise unrelated to pKM101. Each of these alterations increased the number of revertants per plate in the presence of acetaldehyde oxime, indicating that the apparent nonmutagenicity of this chemical in TA100 is due to multiple factors.     

New structural features of the flagellar base in Salmonella typhimurium revealed by rapid-freeze electron microscopy.

The structure of the flagellar base in Salmonella typhimurium has been studied by rapid-freeze techniques. Freeze-substituted thin sections and freeze-etched replicas of cell envelope preparations have provided complementary information about the flagellar base. The flagellar base has a bell-shaped extension reaching as far as 50 nm into the bacterial cytoplasm. This structure can be recognized in intact bacteria but was studied in detail in cell envelopes, where some flagella lacking parts of the bell were helpful in understanding its substructure. Structural relationships may be inferred between this cytoplasmic component of the flagellum and the recently described flagellar intramembrane particle rings as well as the structures associated with the basal body in isolated, chemically fixed flagella.     

Studies on mutagenicity of irradiated sugar solutions in Salmonella typhimurium.

Irradiated sugar solutions are mutagenic towards Salmonella typhimurium, the effect being dose-dependent up to 2.0 Mrad. At all doses, ribose solution exhibited greater mutagenicity than did sucrose solution. The mutagenic effect was observed only in dividing cells and appears to be directly related to the growth rate. A larger proportion of revertants was observed after incubation with irradiated sugar solution for a period of 4 h than for 24 h. Irradiation of the sugar solutions in the frozen conditions was effective in completely preventing the development of mutagenic potential. Post-irradiation storage of the sugar solutions for a prolonged period (25 weeks) also minimized their mutagenic effect. The irradiated sugar solutions gave rise to both missense and frame-shift (additon as well as deletion) types of mutation; ribose was more effective in inducing the latter type. The irradiated sugar solutions failed to show a mutagenic response in the host-mediated assay with mice as the mammalian host.     

Toxicity and mutagenicity of hexavalent chromium on Salmonella typhimurium.

Four hexavalent and two trivalent chromium compounds were tested for toxicity and mutagenicity by means of the Salmonella typhimurium/mammalian-microsome test. All hexavalent compounds yielded a complete inhibition of bacterial growth at doses of 400 to 800 mug/plate, a significant increase of his(+) revertant colonies at doses ranging from 10 to 200 mug, and no effect at doses of less than 10 mug. The distinctive sensitivity of the four Salmonella strains tested (TA1535, TA1537, TA98, and TA100) suggested that hexavalent chromium directly interacts with bacterial deoxyribonucleic acid by causing both frameshift mutations and basepair substitutions. The latter mutations, which are prevalent, are amplified by an error-prone recombinational repair of the damaged deoxyribonucleic acid. On the average, 1 mumol of hexavalent chromium yielded approximately 500 revertants of the TA100 strain, irrespective of the compound tested (sodium dichromate, calcium chromate, potassium chromate, or chromic acid). The mutagenic potency of the hexavalent metal was not enhanced by adding the microsomal fraction of rat hepatocytes, induced either with sodium barbital or with Aroclor 1254. The two trivalent compounds (chromium potassium sulfate and chromic chloride), with or without the microsomal fraction, were neither toxic nor mutagenic for the bacterial tester strains.     

Collaborative study of mutagenicity with Salmonella typhimurium TA102.

Thirty compounds of various chemical classes were investigated for mutagenicity in a collaborative study (3 laboratories) using Salmonella typhimurium TA102. With 5 compounds, namely hydrazine sulfate, phenylhydrazine, hydralazine, glutardialdehyde and glyoxal, mutagenicity was detected by all laboratories. Formaldehyde was assessed as weakly mutagenic in only 1 of 3 laboratories. The remaining 24 agents were uniformly described as non-genotoxic in TA102. In spite of the overall good qualitative agreement in the mutagenicity results between the 3 laboratories some quantitative discrepancies occurred in the dose response of the mutagenic compounds. Varying inter- and intra-laboratory differences in the spontaneous rate of revertants were obtained. The usefulness of the tester strain TA102 in routine mutagenicity testing is discussed.     

Chemical carcinogens a review and analysis of the literature of selected chemicals and the establishment of the Gene-Tox carcinogen data base: A report of the U.S. environmental protection agency Gene-Tox program

The literature on 506 selected chemicals has been evaluated for evidence that these chemicals induce tumors in experimental animals and this assessment comprises the Gene-Tox Carcinogen Data Base. Three major sources of information were used to create this evaluated data base: all 185 chemicals determined by the International Agency for Research on Cancer to have Sufficient evidence of carcinogenic activity in experimental animals, 28 selected chemicals bioassayed for carcinogenic activity by the National Toxicology Program/National Cancer Institute and found to induce tumors in mice and rats, and 293 selected chemicals which had been evaluated in genetic toxicology and related bioassays as determined from previous Gene-Tox reports. The literature data on the 239 chemicals were analyzed by the Gene-Tox Carcinogenesis Panel in an organized, rational and consistent manner. Criteria were established to assess individual studies employing single chemicals and 4 categories of response were developed: Positive, Negative, Inconclusive (Equivocal) and Inconclusive. After evaluating each of the individual studies on the 293 chemicals, the Panel placed each of the 506 chemicals in an overall classification category based on the strength of the evidence indicating the presence or absence of carcinogenic effects. An 8-category decision scheme was established using a modified version of the International Agency for Research on Cancer approach. This scheme included two categories of Positive (Sufficient and Limited), two categories of Negative (Sufficient and Limited), a category of Equivocal (the evidence of carcinogenicity from well-conducted and well-reported lifetime studies had uncertain significance and was neither clearly positive nor negative), and three categories of Inadequate (the evidence of carcinogenicity was insufficient to make a decision, however, the data suggested a positive or negative indication). Of the 506 chemicals in the Gene-Tox Carcinogen Data Base, 252 were evaluated as Sufficient Positive, 99 as Limited Positive, 40 as Sufficient Negative, 21 as Limited Negative, 1 as Equivocal, 13 as Inadequate with the data suggesting a positive indication, 32 as Inadequate with the data suggesting a negative indication, and 48 Inadequate with the data not suggesting any indication of activity.This data base was analyzed and examined according to chemical class, using a 29 chemical class scheme. The major chemical classes represented were: acyl, alkyl and aryl halides (38 chemicals); alcohols and phenols (28 chemicals); alkyl and aryl epoxides (20 chemicals); amines, amides and sulfonamides (70 chemicals); aromatic azo, azide, azoxy, diazo, hydrazo and nitrile chemicals (28 chemicals); aziridines, nitrogen and sulfur mustards (25 chemicals); carbamates, dicarboximides, thioureas and ureas (21 chemicals); metals and organometallics (41 chemicals); nitroalkanes, nitroaromatics, nitrofurans, nitroimidazoles and nitroquinolines (23 chemicals); nitrosamines (19 chemicals); and polycyclic aromatic hydrocarbons and dihydrodiol derivatives (57 chemicals). The Gene-Tox Carcinogen Data Base provides a basis for future in-depth analyses of genetic toxicology bioassay systems with regard to their ability to predict the carcinogenic effects of chemicals.     

Diethylstilbestrol and 11 derivatives: a mutagenicity study with Salmonella typhimurium.

Diethylstilbestrol was tested for mutagenicity with his- S. typhimurium strains under 10 different matabolic situations (no exogenous metabolizing system; S9 mix from liver homogenate of rats induced with Aroclor 1254, with or without inhibition of epoxide hydratase; liver and/or kidney S9 mix from control or hamsters treated with Aroclor 1254; horse-radish peroxidase + H2O2). Under none of these conditions did diethylstilbestrol give any indication of a mutagenic effect. Furthermore, 11 metabolites and other derivatives of diethylstilbestrol, 2 of them potent inducers of sister-chromatid exchange in cultured fibroblasts, were not mutagenic with any of the 4 tester strains (S. typhimurium TA100, TA98, TA1537, TA1535) in the presence or absence of S9 mix from liver homogenate of rats induced with Aroclor 1254. Thus, one of the few known human carcinogens is very resistant to detection by the mammalian enzyme-mediated Salmonella typhimurium mutagenicity test (Ames test). This is especially remarkable since the metabolizing systems used included: (1) some of very high metabolic activity (S9 mix from liver homogenate of rats and hamsters induced with Aroclor 1254); (2) metabolizing systems from organs susceptible to the carcinogenic activity of diethylstilbestrol (hamster kidney); as well as (3) a mixture of (1) and (2) in case both activities are required for the carcinogenic effect in the whole animal.     

Antimutagenic effect of p-aminobenzoic acid on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium

p-Aminobenzoic acid (PABA) exhibited antimutagenic activity toward N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)-induced mutagenicity in the Ames assay in Salmonella typhimurium. The antimutagenic effects were associated with an increased rate of decomposition of MNNG in the presence of PABA. The participation of other mechanisms, such as the alteration of cellular processes by PABA, however, cannot be excluded.