Structure-activity relationships of the azide metabolite, azidoalanine, in S. typhimurium

Azide is metabolized to the proximate mutagen, L-azidoalanine in bacterial systems. While this novel mutagenic metabolite plays a key role in azide mutagenesis, the biochemistry of this role is unknown. The chemical synthesis of authentic racemic azidoalanine and several derivatives thereof allowed the exploration of structure-activity relationships with this unique mutagen. We found that whereas azide, azidoalanine and azidoalanine tert.-butyl ester were of comparable mutagenic potency, derivatives which lack the free amino group, such as azidopropionic acid and amino-blocked azidoalanine, were orders of magnitude less active. These findings demonstrate that the free amino group is essential for significant activity, while the carboxyl group may be less important. This conclusion together with the finding that DL-azidoalanine is a less potent mutagen than azide itself, suggests that the metabolite, while necessary for azide mutagenicity, may not be the ultimate mutagenic species. Instead, the data are consistent with the hypothesis that azidoalanine requires further bioactivation.     

Mutagenicity of sodium azide and its metabolite azidoalanine in Drosophila melanogaster

The mutagenic and toxic activities of sodium azide (NaN(3) ) and its organic metabolite L-azidoalanine [N(3)-CH(2)-CH(NH)(2)-COOH] were examined in the different stages of spermatogenesis in Drosophila melanogaster. Both azide and azidoalanine were toxic to the injected males, but azidoalanine was significantly less toxic than sodium azide. Following the injection with 0.2 microl of these compounds in the hemocoel of young adult wild-type males, the minimum concentrations of these compounds with complete toxic effects (zero survival) were 40 mM sodium azide and 160 mM azidoalanine. Sex-linked recessive lethals were scored by the Muller-5 method in three successive broods, representing sperms (brood A), spermatids (brood B), and a compiled group of meiotic and premeiotic germ cell stages (brood C). The results provide strong experimental evidence that azidoalanine is significantly (p<0.01) mutagenic to all stages of spermatogenesis in Drosophila melanogaster. Sodium azide, however, was not significantly (p>0.05) mutagenic and did not increase the rate of sex-linked recessive lethals over those produced by the control group injected with 0.45% NaCl. These results indicate the requirement of metabolic activation of azide in Drosophila as a prerequisite for its mutagenic effects.     

Azidoalanine mutagenicity in Salmonella: effect of homologation and alpha-methyl substitution

Azide mutagenicity in susceptible non-mammalian systems involves the requisite formation of L-azidoalanine, a novel mutagenic amino acid. The biochemical mechanism(s) of azidoalanine-induced mutagenesis, however, is not known. Previous studies of the structural requirements for azidoalanine mutagenicity suggested the importance of free L-amino acid character, and that bioactivation of azidoalanine to the ultimate mutagenic species is required. To gain more insight into possible enzymatic processing, the alpha-methyl analogue, alpha-methyl-azidoalanine, and the homologue, 2-amino-4-azidobutanoic acid, were synthesized and tested for mutagenic potency in Salmonella typhimurium strain TA1530. In addition, azidoacetic acid, a possible azidoalanine metabolite, was prepared and tested. The results show that alpha-methyl substitution effectively blocks the mutagenic effects of azidoalanine with alpha-methyl-azidoalanine being nearly devoid of mutagenic activity. In contrast, homologation of azidoalanine to yield 2-amino-4-azidobutanoic acid produces a marked increase in molar mutagenic potency. As with azidoalanine, the mutagenic activity of this homologue is associated with the L-isomer. Azidoacetic acid, however, was only very weakly mutagenic when tested as either the free acid or ethyl ester. This low mutagenic potency may indicate that bioactivation does not involve the entry of azide-containing azidoalanine catabolite into the Kreb's cycle. The high potency of 2-amino-4-azidobutanoic acid may be indicative of more efficient bioactivation and/or greater intrinsic activity. Importantly, the latter finding clearly shows that potent azido-amino acid mutagenicity is not limited to azidoalanine alone.     

A mutagenic metabolite synthesized by Salmonella typhimurium grown in the presence of azide is azidoalanine

A mutagenic azide metabolite was purified from the medium in which Salmonella typhimurium cells were grown in the presence of azide. This metabolite was identified to be azidoalanine based on infrared and mass spectroscopy and elemental analysis. This compound appeared to be identical to the mutagenic compound synthesized in vitro from azide and O-acetylserine by partially purified O-acetylserine sulfhydrylase. The metabolite (azidoalanine) mutagenic efficiency and spectrum in S. typhimurium was similar to that of inorganic azide. The compounds 2-azidoethylamine, 2-bromoethylamine, 3-bromopropionic acid and N-(azidomethyl) phthalimide were also mutagenic with a similar spectrum to azide and azidoalanine, but with lower efficiency. The compounds 3-azidopropylamine, 4-azidobutylamine, 3-chloroalanine and ethylamine were only weakly or nonmutagenic. Numerous other chloro, bromo and azido phthalimide derivatives tested were nonmutagenic. It is suggested that the lack of azide mutagenicity (and perhaps carcinogenicity) in mammalian cells may be due to their inability to convert azide to azidoalanine.     

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.     

Azidoalanine mutagenicity in Salmonella: Effect of homologation and α-methyl substitution

Azide mutagenicity in susceptible non-mammalian systems involves the requisite formation of l-azidoalanine, a novel mutagenic amino acid. The biochemical mechanism(s) of azidoalanine-induced mutagenesis, however, is not known. Previous studies of the structural requirements for azidoalanine mutagenicity suggested the importance of free l-amino acid character, and that bioactivation of azidoalanine to the ultimate mutagenic species is required. To gain more insight into possible enzymatic processing, the α-methyl analogue, α-methylazidialanine, and the homologue, 2-amino-4-azidobutonoic acid, were synthesized and tested for mutagenic potency in Salmonella typhimurium strain TA1530. In addition, azidoacetic acid, a possible azidoalanine metabolite, was prepared and tested. The results show that α-methyl substitution effectively blocks the mutagenic effects of azidoalanine with α-methyl-azidoalanine being nearly devoid of mutagenic activity. In contrast, homologation of azidoalanine to yield 2-amino-4-azidobutanoic acid produces a marked increase in molar mutagenic potency. As with azidoalanine, the mutagenic activity of this homologue is associated with the l-isomer. Azidoacetic acid, however, was only very weakly mutagenic when tested as either the free acid or ethyl ester. This low mutagenic potency may indicate that bioactivation does not involve the entry of azide-containing azidoalanine catabolite into the Kreb's cycle. The high potency of 2-amino-4-azidobutanoic acid may be indicative of more efficient bioactivation and/or greater intrinsic activity. Importantly, the latter finding clearly shows that potent azido-amino acid mutagenicity is not limited to azidoalanine alone.     

Sodium azide mutagenesis in mammals: inability of mammalian cells to convert azide to a mutagenic intermediate

Sodium azide is unique among mutagens. It is highly mutagenic in many plant and bacterial species but marginally mutagenic in mammalian cells. A possible explanation for this difference in mutagenic efficiency may lie in the inability of mammalian cells to convert azide to the putative ultimate mutagen. Normal human fibroblasts and Chinese hamster cells or cell-free extracts from these cell lines were treated with azide and the sonicates tested for mutagenicity in Salmonella strain TA1530. The data suggest that neither cell line was capable of converting azide to a mutagenic intermediate. In addition, both cell lines expressed the enzyme O-acetylserine(thio)-lyase which is responsible for the conversion of azide to azidoalanine, the putative mutagenic intermediate. Although mammalian cells possess the enzyme responsible for the conversion of azide to azidoalanine, they appear incapable of converting azide into a mutagenic intermediate in appreciable quantities. Further, the data support the conclusion that azide may be further modified in mammalian cells to an intermediate that is not genotoxic.     

Metabolic activation of the mutagen azide in biological systems

Inorganic azide (N3-) mutagenicity is mediated through a metabolically synthesized organic azide, L-azidoalanine (N3-CH2-CH(-NH2)-COOH). L-Azidoalanine appears to be formed by the action of O-acetylserine (thiol)-Lyase (EC 4.2.99.8) using O-acetylserine and azide as substrates. In both plants and bacteria tested, azide substitutes for the natural substrate sulfide (S2-) in this reaction. Azide (L-azidoalanine) mutagenesis is highly attenuated by a deficiency in the excision of UV-like DNA damage (uvr-). Thus a premutation lesion recognizable by the bacterial excision-repair enzymes must be formed. Mutagenesis appears to proceed from this by 'direct mispairing' pathway. Azide (L-azidoalanine) mutagenicity is highly specific and involves a stereoselective process, but the molecular nature of the specificity has not been determined.     

Effect of glutathione L-cystein and L-djenkolic acid in the synthesis and mutagenicity of azide metabolite in Bacillus subtilis ATCC 6633 strain.

The Bacillus subtilis ATCC 6633 strain synthesizes a mutagenic metabolite from sodium azide and O-acetylserine. Mutagenicity of azide was decreased in growth media containing 10(-4) M glutathione, L-cysteine or L-djenkolic acid whereas dithiothritol (DTT) added at the same concentration did not reduce the mutagenicity of azide. Likewise, glutathione, L-cysteine, L-djenkolic acid, and DTT were found to have no effect in reducing the mutagenicity of the in vitro produced metabolite using bacterial cell-free extract. These results suggest that O-acetyl-serine sulfhydrylase catalyzes the reaction of azide and O-acetylserine to form a mutagenic metabolite, which is ninhydrin positive and migrates in TLC to an Rf value similar to that of azidoalanine in both acidic and basic solvent systems.     

Differential metabolism of sodium azide in maize callus and germinating embryos

Sodium azide is a potent mutagen of maize (Zea mays L.) kernels that may have potential as a point mutagen for inducing biochemical mutations in maize tissue cultures. Azide mutagenicity was evaluated in friable, embryogenic maize callus and a nonregenerable maize suspension culture by determining the number of resistant variant cell lines able to grow on media containing inhibitory concentrations of lysine plus threonine (LT). The number of LT-resistant variants selected from either culture type did not increase in response to azide treatment. In addition, there was no increase in somatic mutations in more than 100 plants regenerated from azide treated LT-resistant lines. The levels of mutagenic metabolite of azide (presumably azidoalanine), were determined by bioassay in the two azide-treated maize callus types and compared to levels of mutagenic metabolite in embryos isolated from azide-treated kernels. The two types of maize tissue cultures and isolated embryos contained similar levels of mutagenic metabolite 4 h after azide treatment indicating similar uptake and conversion of azide to mutagenic metabolite in the three tissues. Mutagenic metabolite in azide-treated embryos did not significantly decrease after 40 h. However, mutagenic metabolite levels in both azide-treated tissue cultures decreased to near background levels within 20 h providing evidence for rapid metabolism of the azide mutagenic metabolite. The lack of evidence for azide mutagenicity in maize callus and its known potent mutagenicity in kernels appears to be associated with specific differences in azide metabolism between callus tissues and kernel embryos.     

In vitro synthesis of a mutagenic azide metabolite by cell-free bacterial extracts

Cell-free extracts of Salmonella typhimurium synthesize a mutagenic azide metabolite from sodium azide and O-acetylserine. S. typhimurium mutant DW379 (O-acetylserine sulfhydrylase-deficient) extracts were neither able to carry out this reaction nor produce the mutagenic azide metabolite in vivo. The in vitro reaction was inhibited by sulfide but not by l-cysteine. The catalytic activity responsible for the mutagenic metabolite synthesis was stable to brief heating up to 55°C and had a pH optimum between 7–7.4. These results suggest that the enzyme O-acetylserine sulfhydrylase catalyzes the reaction of azide with O-acetylserine to form a mutagenic azide metabolite.     

The very low mutagenic activity of sodium azide inArabidopsis thaliana

Sodium azide, reported to be a strong mutagen in barley, revealed a very weak mutagenic activity inArabidopsis.     

Effect of sodium azide on sister-chromatid exchanges in human lymphocytes and Chinese hamster cells

Previous reports from this laboratory and others indicate that sodium azide is a unique mutagen. It is highly mutagenic in S. typhimurium TA1530 as well as in barley, rice, peas, yeast and Chinese hamster V79 cells. However, azide apparently does not produce chromosome breaks in barley, Vicia or human lymphocytes. Therefore, a study of the effects of azide on sister-chromatid exchanges (SCE) appeared warranted.Human whole blood and Chinese hamster K1 cell line were exposed for 4 and 2 h resp. to various concentrations of sodium azide ranging from 10⁻³ to 10⁻⁷ M. Cells were harvested and chromosomes stained by the FPG technique. In human lumphocytes, concentrations above 10⁻⁴ induced lethality whereas the K1 cell line was sensitive to concentrations above 10⁻⁵ M. The lower concentrations of azide produced no significant increase in SCE frequency above controls. Concurrent mitomycin C treatments produced significant increases in SCE levels.This apparent lack of induction of SCEs above background combined with previous data demonstrating negative clastogenic but very positive mutagenic activity of azide confirms the uniqueness of this mutagen. It would appear that azide is one of the few known potent mutagens that does not increase SCEs and/or break chromosomes.     

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium     

Involvement of the L-cysteine biosynthetic pathway in azide-induced mutagenesis in Salmonella typhimurium

L-Cystine and L-cysteine specifically reverse the mutagenic action of azide in Salmonella typhimurium and Escherichia coli. To establish whether the L-cysteine biosynthetic pathway is involved in azide-induced mutagenesis, several derivatives of a mutagen tester-strain of S. typhimurium bearing mutations in different cys genes were isolated. No mutagenic effect of azide was observed in a strain carrying mutation in the cysE gene, unless the incubation medium was supplemented with exogenous O-acetylserine. Out of 16 cysK mutants 14 were mutagenized by azide very poorly or not at all. These results indicate that the activity of O-acetylserine sulfhydrylase A, and the availability of O-acetylserine, one of the two co-substrates of the enzyme, are essential for the mutagenic action of azide in S. typhimurium     

Structure-function characterization for ethidium photoaffinity labels as mutagens in salmonella

The development of photoaffinity probes to characterize the binding process and subsequent biological activity of a drug has recently been emphasized by the synthesis of two ethidium azide analogs. The initial findings showed that one of the azido analogs, the 8-azido-3-amino derivative, was at least 40-fold more mutagenic and toxic in Salmonella tester strain TA1538 than the other analog, the 3,8-diazido derivative. These observations suggested the need to examine the structural requirements of ethidium photoaffinity labels for frameshift mutagenic activity in Salmonella. Thus, the isomer of the monoazide, the 3-azido-8-amino derivative, and two deaminated monoazide derivatives were synthesized and all of the ethidium analogs were screened in two Salmonella frameshift tester strains, TA1537 and TA1538, and in their excision-repair positive isogenic strains. The results presented in this paper demonstrate that two substituents are needed to produce significant mutagenicity and toxicity by the compound. One substituent, usually the amino group, is required for mutagenic activity, perhaps by orienting the phenanthridinium ring into its mutagenic configuration. The other substituent, the azido group, is required for covalent attachment, a requisite for mutagenic activity.Thus, photoaffinity labeling has provided a means of comparing structure with mutagenic activity for ethidium compounds.     

Production of frameshift mutations in Salmonella by phenanthridinium derivatives: enzymatic activation and photoaffinity labeling

The effect of metabolic activation on the mutagenic potential of some phenanthridinium compounds was examined in Salmonella typhimurium strains TA1538 and TA1978 . All of the compounds tested were mutagenic in TA1538, a DNA excision-repair-deficient strain, when metabolizing enzymes were included in the assay. Reversions were not detected when these compounds were examined under the same conditions in TA1978 , the isogenic strain of TA1538 proficient in DNA repair. The mutagenic activity of an azido analog of propidium iodide was also examined using photoactivation and enzymatic activation, and with both conditions, reversions were observed in TA1538 but not in TA1978 . Furthermore, the ranking of mutagenic activity of propidium azide relative to ethidium azide analogs was comparable for both types of activation. The evidence from several studies suggests that the structural requirements for mutagenic activity for this series of phenanthridinium compounds appear to be the same whether mutagenesis is induced via photoactivation or metabolic activation. The interaction with DNA resulting in covalent alteration of the DNA is implicated as the mutagenic mechanism whether the active species is generated by metabolic- or photo-activation.     

Isolation of an azide mutagenic metabolite in Salmonella typhimurium

A scheme that employs a cation-exchange column and high-pressure liquid chromatography (HPLC) is devised to isolate and process large quantities of azide metabolite produced by S. typhimurium TA1530 strain. The mutagenic metabolite adheres strongly to the cation-exchange column, thus providing a convenient way to separate the metabolite from unreacted azide (N₃⁻). The metabolite is very polar and only sparingly soluble in most organic solvents. Recrystallization in a methanol-carbontetrachloride solvent system gave rise to microcrystalline material that decomposes with charring and gas evolution at 173–176°C. The infrared spectrum indicates the presence of a covalently bound azide moiety.     

In vitro production of azide mutagenic metabolite in Arabidopsis,Drosophila and Neurospora

The ability of Arabidopsis, Drosophila and Neurospora to convert azide to its mutagenic metabolite was investigated. Cultures of these organisms all contained significant levels of O-acetylserine sulfhydrylase activity. Extracts from each organism produced a product from O-acetylserine and azide in vitro which was mutagenic in Salmonella typhimurium TA1530.     

Mutagenic effects of sodium azide in Drosophila melanogaster.

The mutagenic effects of sodium azide (NaN3) were studied at low pH in male Drosophila melanogaster using the sex-linked recessive-lethal test. No significant increase in the mutation frequency was observed after abdominal injection of azide solutions buffered at either pH 3.8 OR 4.6. However, a weak mutagenic effect was noticed in the flies fed for 3 days on 0.1 mM azide (pH 4.6) solution.