Transnitrosation of non-mutagenic N-nitrosoproline forms mutagenic N-nitroso-N-methylurea.

N-Nitroso-N-methylurea (NMU) is a potent carcinogen and suspected as a cause of human cancer. In this study, mutagenic NMU was detected by HPLC after the transnitrosation of non-mutagenic N-nitrosoproline (NP) to N-methylurea in the presence of thiourea (TU) under acidic conditions. The structure of NMU was confirmed by comparing (1)H NMR and IR spectra with that of authentic NMU after fractionation by column chromatography. Furthermore, a fraction containing NMU formed by transnitrosation was mutagenic in Salmonella typhimurium TA1535. NMU was formed in the reaction of NP and N-methylurea in the presence of 1,1,3,3-tetramethylthiourea (TTU) or 1,3-dimethylthiourea in place of TU as an accelerator. The reaction rate constants (k) for NMU formation were correlated with their nucleophilicity of sulfur atom in thioureas. The N-methylurea concentration did not affect the NMU formation, whereas the rate of NMU formation correlated linearly with concentrations of NP, TTU and oxonium ion. The observed kinetics suggests a mechanism by which the nitroso group was transferred directly from the protonated NP to the thiourea then to N-methylurea to form NMU. The rate-determining step was the formation of the complex with the protonated NP and thiourea.

Effect of alkyl group on transnitrosation of N-nitrosothiazolidine thiocarboxamides.

S-Nitrosoglutathione (GSNO) relaxes vascular smooth muscles, prevents platelet aggregation, and acts as a potential in vivo nitric oxide donor. 3-Nitroso-1,3-thiazolidine-4-thiocarboxamide (1), a N-nitrosothio-proline analogue, exhibited a high GSNO formation activity. In this study, two compounds (2 and 3) based on compound 1 were newly synthesized by introducing either one or two methyl groups onto a nitrogen atom on the thioamide substituent in 1. The pseudo-first-order rate constants (kobs) for the GSNO formation for the reaction between the compound and glutathione followed the order 1>2≒3. Thus, the introduction of a methyl group(s) onto the thioamide group led to a decrease in the transnitrosation activity. On the basis of density functional theoretical calculations, the transnitrosation for the N-nitrosothiazolidine thiocarboxamides was proposed to proceed via a bridged intermediate pathway. Specifically, the protonated compound 1 forms a bridged structure between the nitrogen atom in the nitroso group and two sulfur atoms-one in the ring and the other in the substituent. The bridged intermediate gives rise to a second intermediate in which the nitroso group is bonded to the sulfur atom in the thioamide group. Finally, the nitroso group is transferred to GSH to form GSNO.

Transnitrosation of alicyclic N-nitrosamines containing a sulfur atom.

Aromatic and aliphatic nitrosamines are known to transfer a nitrosonium ion to another amine. The transnitrosation of alicyclic N-nitroso compounds generates S-nitrosothiols, which are potential nitric oxide donors in vivo. In this study, certain alicyclic N-nitroso compounds based on non-mutagenic N-nitrosoproline or N-nitrosothioproline were synthesised, and the formation of S-nitrosoglutathione (GSNO) was quantified under acidic conditions. We then investigated the effect of a sulfur atom as the substituent and as a ring component on the GSNO formation. In the presence of thiourea under acidic conditions, GSNO was formed from N-nitrosoproline and glutathione, and an N-nitroso compound containing a sulfur atom and glutathione produced GSNO without thiourea. The quantity of GSNO derived from the reaction of the N-nitrosamines containing a sulfur atom and glutathione was higher than that from the N-nitrosoproline and glutathione plus thiourea. Among the analogues that contained a sulfur atom either in the ring or as a substituent, the thiazolidines produced a slightly higher quantity of GSNO than the analogue with a thioamide group. A compound containing sulfur atoms both in the ring and as a substituent exhibited the highest activity for GSNO formation among the alicyclic N-nitrosamines tested. The results indicate that the intramolecular sulfur atom plays an important role in the transnitrosation via alicyclic N-nitroso compounds to form GSNO.

Assessing the effect of N-oxidation on the mutagenicity of 1-pyrazolines using the Ames assay.

N-Nitrosamines are well known as environmental carcinogens. We have reported that N-nitroso-N-methylbutylamine was oxidized by Fe2+-Cu2+-H2O2 to 5-methyl-5-nitro-1-pyrazoline, a direct-acting N-oxide. 1-Pyrazolines have not been reported to exhibit genotoxicity. In this study, we investigated the effect of N-oxidation on the mutagenicity of 1-pyrazolines using the Ames assay. The mutagenicity of 5-alkyl-5-nitro-1-pyrazoline 1-oxide (1a; methyl, 1b; ethyl), the N-oxide isomer (3-alkyl-3-nitro-1-pyrazoline 1-oxide; 2a; methyl, 2b; ethyl), and the corresponding nonoxides (3-alkyl-3-nitro-1-pyrazoline; 3a; methyl, 3b; ethyl) was assayed in Salmonella typhimurium TA1535 and Escherichia coli WP2uvrA. The ratios of mutagenic potency in S. typhimurium TA1535 versus E. coli WP2uvrA were compared with those of N-alkylnitrosoureas. To predict the reaction site on the pyrazolines with nucleophiles, the electron density of the pyrazolines was obtained by theoretical calculations. The pyrazolines were mutagenic in S. typhimurium TA1535 and E. coli WP2uvrA. The ratio of S. typhimurium TA1535 to E. coli WP2uvrA 1a (87:13) or 1b (90:10) was similar to that of N-ethyl-N-nitrosourea (70:30). In contrast, the mutagenic ratio of 2a (22:78) or 2b (52:48) was similar to that of N-propyl-N-nitrosourea (48:52) or N-butyl-N-nitrosourea (14:86). The ratio of 3a (53:47) or 3b (54:46) was similar to that of N-propyl-N-nitrosourea or N-butyl-N-nitrosourea. The pyrazolines exhibit genotoxicity, and the mutagenic potency of the 1-pyrazolines is influenced by N-oxidation. We estimated that the mutagenicity of 1a or 1b was caused by DNA ethylation, and the isomers or the nonoxides were mutagenic via formation of alkylated DNA, which contains an alkyl chain longer than the propyl.

Chlorine atom substitution influences radical scavenging activity of 6-chromanol.

Synthetic 6-chromanol derivatives were prepared with several chlorine substitutions, which conferred both electron-withdrawing inductive effects and electron-donating resonance effects. A trichlorinated compound (2), a dichlorinated compound (3), and three monochlorinated compounds (4, 5, and 6) were synthesized; compounds 2, 3, and 6 were novel. The antioxidant activities of the compounds, evaluated in terms of their capacities to scavenge galvinoxyl radical, were associated with the number and positioning of chlorine atoms in the aromatic ring of 6-chromanol. The activity of compound 1 (2,2-dimethyl-6-chromanol) was slightly higher than the activities of compounds 2 (2,2-dimethyl-5,7-dichloro-6-chromanol) or 3 (2,2-dimethyl-5,7,8-trichloro-6-chromanol), in which the chlorine atoms were ortho to the phenolic hydroxyl group of 6-chromanol. The scavenging activity of compound 3 was slightly higher than that of 2, which contained an additional chlorine substituted in the 8 position. The activities of polychlorinated compounds 2 and 3 were higher than the activities of any of the monochlorinated compounds (4-6). Compound 6, in which a chlorine was substituted in the 8 position, exhibited the lowest activity. Substitution of a chlorine atom meta to the hydroxyl group of 6-chromanol (compounds 2 and 6) decreased galvinoxyl radical scavenging activity, owing to the electron-withdrawing inductive effect of chlorine. Positioning the chloro group ortho to the hydroxyl group (compounds 4 and 5) retained antioxidant activity because the intermediate radical was stabilized by the electron-donating resonance effect of chlorine in spite of the electron-withdrawing inductive effect of chlorine. Antioxidant activities of the synthesized compounds were evaluated for correlations with the O-H bond dissociation energies (BDEs) and the ionization potentials. The BDEs correlated with the second-order rate constants (k) in the reaction between galvinoxyl radical and the chlorinated 6-chromanol derivatives in acetonitrile. This indicated that the antioxidant mechanism of the synthesized compounds consisted of a one-step hydrogen atom transfer from the phenolic OH group rather than an electron transfer followed by a proton transfer. The synthesized compounds also exhibited hydroxyl radical scavenging capacities in aqueous solution.

Isolation and structural identification of a direct-acting mutagen derived from N-nitroso-N-methylpentylamine and Fenton's reagent with copper ion.

N-Nitrosodialkylamines show their mutagenicity by forming α-hydroxynitrosamines in the presence of rat S9 mix in the Ames assay. The hydroxyl radical derived from Fe(2+)-H(2)O(2) (Fenton's reagent) with Cu(2+) activates N-nitrosamines, with an alkyl chain longer than a propyl constituent, to a direct-acting mutagen. The reactivity of Fe(2+)-Cu(2+)-H(2)O(2) on nitrosamines in relation to their metabolic activation is not fully characterized. Here, we report the identification of the direct-acting mutagen derived from N-nitroso-N-methylpentylamine (NMPe) in the presence of Fe(2+), Cu(2+), H(2)O(2) and nitric oxide (NO), which is a product of nitrosamine metabolism. A dichloromethane extract of the NMPe reaction mixtures was fractionated by silica gel column chromatography several times and by a preparative high performance liquid chromatography (HPLC); we obtained white crystals as a product. The direct-acting mutagen that was isolated was provisionally identified as 5-ethyl-5-nitro-1-pyrazoline 1-oxide by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy, infrared (IR) spectroscopy and X-ray crystallography. To confirm the structure of the mutagen, the authentic compound was synthesized from 2-nitrobutene and diazomethane, followed by N-oxidation with m-chloroperoxybenzoic acid. The (1)H NMR spectral data from the direct-acting mutagen that was synthesized was identical to the data from the isolated mutagen. Furthermore, the authentic 5-ethyl-5-nitro-1-pyrazoline 1-oxide was mutagenic in Salmonella typhimurium TA1535. The results showed that 5-ethyl-5-nitro-1-pyrazoline 1-oxide was a direct-acting mutagen derived from the reaction of NMPe and Fe(2+)-Cu(2+)-H(2)O(2)-NO.

7-azabicyclo[2.2.1]heptane as a structural motif to block mutagenicity of nitrosamines.

Nitrosamines are potent carcinogens and toxicants in the rat and potential genotoxins in humans. They are metabolically activated by hydroxylation at an α-carbon atom with respect to the nitrosoamino group, catalyzed by cytochrome P450. However, there has been little systematic investigation of the structure-mutagenic activity relationship of N-nitrosamines. Herein, we evaluated the mutagenicity of a series of 7-azabicyclo[2.2.1]heptane N-nitrosamines and related monocyclic nitrosamines by using the Ames assay. Our results show that the N-nitrosamine functionality embedded in the bicyclic 7-azabicylo[2.2.1]heptane structure lacks mutagenicity, that is, it is inert to α-hydroxylation, which is the trigger of mutagenic events. Further, the calculated α-C-H bond dissociation energies of the bicyclic nitrosamines are larger in magnitude than those of the corresponding monocyclic nitrosamines and N-nitrosodimethylamine by as much as 20-30 kcal/mol. These results are consistent with lower α-C-H bond reactivity of the bicyclic nitrosamines. Thus, the 7-azabicyclo[2.2.1]heptane structural motif may be useful for the design of nongenotoxic nitrosamine compounds with potential biological/medicinal applications.

Activation mechanism for N-nitroso-N-methylbutylamine mutagenicity by radical species.

N-Nitrosodialkylamines are known to be potent indirect-acting mutagens/carcinogens, which are activated by cytochrome P450. The reaction product of N-nitroso-N-methylbutylamine (NMB) with modified Fenton's reagent supplemented with copper salt (Fe²(+)-Cu²(+)-H2O2) was reported to be mutagenic in Salmonella typhimurium TA1535 without S9 mix. In this study, the NMB activation mechanism was investigated by ESR spectroscopy with radical trapping agents to detect radical species and also by observing changes in mutagenic potency with a Salmonella strain in the Ames assay in the presence of radical trapping agents. In ESR spectroscopy experiments, the hydroxyl radical generated from the modified Fenton's reagent was detected using the hydroxyl radical trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO). Since the amount of the DMPO-OH adduct decreased with the addition of NMB, hydroxyl radical was presumed to react with NMB followed by the generation of nitric oxide (NO), which was detected as CarboxyPTI through reaction with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (CarboxyPTIO). The mutagenicity of the reaction extract decreased following the addition of DMPO or CarboxyPTIO. Furthermore, the mutagenicity of the reaction product in the presence of DMPO was enhanced by the addition of NO. The reaction product from NMB with Fe²(+)-Cu²(+)-NO in the absence of H2O2 was mutagenic, and this activity increased with the introduction of additional NO. These findings suggest that hydroxyl radical takes part in the generation of NO from NMB and that NO plays an important role in NMB activation in the presence of Fe²(+) and Cu²(+).

Post-operative Rehabilitation of Atypical Femoral Fracture in a Single Center.

OBJECTIVE: Increasing numbers of reports have described atypical femoral fracture (AFF) in patients being treated with oral bone resorption inhibitors, such as bisphosphonates. Most AFF patients undergo surgical treatment. However, there is little information about post-operative rehabilitation and patient activity levels after surgery for such fractures. Here we report the outcome of surgical treatment and postoperative rehabilitation for AFF at a single center in Japan. METHODS: We retrospectively reviewed 13 patients (14 AFFs) who underwent surgery at Nagano Matsushiro General Hospital between January 2013 and December 2016. The clinical backgrounds of the patients were evaluated. RESULTS: The patients comprised 1 man (1 AFF) and 12 women (13 AFFs). The mean age at surgery was 77.7±7.1 years (mean±SD). Before AFF occurred, 12 of the 13 patients had used bisphosphonates for osteoporosis. An intramedullary nail was inserted in all patients. Partial weight bearing was started on average 2 weeks after surgery, and full weight-bearing gait was permitted on average 3 weeks after surgery. The average time to bone union was 9.9±6.1 months, ranging from 3 to 23 months. None of the patients required additional surgical procedures, including revision surgery for pseudoarthrosis (nonunion) or delayed union. Before AFF, 12 patients walked independently, and 1 patient walked with a single cane. At the final follow-up (mean duration: 34.5±15.7 months), 8 patients could walk independently and 5 patients walked with a single cane. CONCLUSIONS: We recognized that rigid fixation for AFF supported early weight-bearing gait after surgery.

Unique behavior of 2,6-bis(bromomethyl)naphthalene as a highly active organic DNA crosslinking molecule.

Among 14 bis-halomethylated naphthalenes and quinolines, 2,6-bis(bromomethyl)naphthalene was found to have highly active crosslinking activity on DNA. The unique behavior of high microbial mutagenicity, even though it had a low propensity to form double-strands in linearized plasmid DNA, suggested that it would offer a new seed, capable of forming intrastrand crosslinks similar to cisplatin. The electron withdrawal extent of the halogen atoms, the substitution patterns of two halomethyl groups, and the introduction of a nitrogen atom into the aromatic nucleus had remarkable effects on the activity of the molecule.

Antimutagenic components in Spatholobus suberectus Dunn against N-methyl-N-nitrosourea.

BACKGROUND: An extract from Spatholobus suberectus (S. suberectus) Dunn has been reported to show potent antimutagenic effects against N-alkyl-N-nitrosoureas in umu screening. The aim of this study was to identify the antimutagenic components from extracts of S. suberectus against N-methyl-N-nitrosourea (MNU) in the Ames assay with Salmonella typhimurium strain TA1535 and to elucidate the antimutagenic mechanism of the flavonoids. RESULTS: From the ethyl acetate fraction obtained from fractionation of the methanol extract of S. suberectus Dunn, medicarpin, formononetin and isoliquiritigenin were successfully isolated through a combination of normal- and reversed-phase chromatography. Genistein and naringenin, which were already reported to be contained in S. suberectus Dunn, were also tested for their antimutagenicity towards MNU, along with formononetin, isoliquiritigenin and medicarpin. Our results demonstrated that genistein, isoliquiritigenin, medicarpin and naringenin were antimutagenic against MNU without showing cytotoxicity. MNU is reported to cause not only DNA alkylation but also induce reactive oxygen species. The hydroxyl radical scavenging capacity of the flavonoids was correlated with the antimutagenic capacity, indicating that the hydroxyl radical scavenging activity was involved in their antimutagenicity towards MNU. CONCLUSIONS: It is important to prevent DNA damage by N-nitrosamines for cancer chemoprevention. Genistein, isoliquiritigenin, medicarpin and naringenin were demonstrated to possess an antigenotoxic effects against carcinogenic MNU due to their radical scavenging activity.

Oxidative transformation of 2-acetylaminofluorene by a chemical model for cytochrome P450: A water-insoluble porphyrin and tert-butyl hydroperoxide.

Oxidation of 2-acetylaminofluorene (AAF), a carcinogen, by a chemical model for cytochrome P450 was investigated to identify an active mutagen and elucidate the oxidation pathway. The oxidation system consisted of a water-insoluble tetrakis(pentafluorophenyl)porphyrinatoiron(III) chloride and tert-butyl hydroperoxide. The mutagen derived from AAF by the chemical model was 2-nitro-9-fluorenone (NO(2)=FO), which was mutagenic in Salmonella typhimurium TA1538. AAF was oxidized initially at position 9 of the fluorene carbon by the chemical model forming 2-acetylamino-9-fluorenol (AAF-OH), and then oxidized further to 2-acetylamino-9-fluorenone (AAF=O) as a major product. Initial oxidation of the nitrogen formed 2-nitrofluorene (NO(2)F), and further oxidation yielded 2-nitro-9-fluorenol (NO(2)F-OH) as a minor product. These products, AAF-OH, AAF=O, NO(2)F, and NO(2)F-OH, and their presumable common intermediate, N-hydroxy-2-acetylaminofluorene, were oxidized by the chemical model, and the formation of NO(2)F=O was determined. These results showed that NO(2)F=O was the mutagen derived from AAF in the presence of the chemical model and was formed via oxidation of N-OH-AAF, NO(2)F, and NO(2)F-OH. These results may lead to a new metabolic pathway of AAF.

On-line liquid chromatography and circular dichroism detection of stereo-isomers of alpha-tocopherol derivatives generated by an electrochemical reaction.

The electrochemical oxidation of (+/-)-alpha-tocopherol on a porous graphite electrode was performed in the presence of methanol, and successive separation and detection of the products were performed by an on-line liquid chromatography/mass spectrometry system. Three products were identified, one of which was determined to be alpha-tocopheryl quinone, because its m/z was 469 [M+Na](+). The other two products showed identical mass and UV spectra, and were suspected to be diastereomers of 9-methoxy-alpha-tocopheron, because their molecular weights were m/z 483 [M+Na](+), and also because it is known that the chemical oxidation of alpha-tocopherol by benzoyl peroxide or N-bromosuccinimide in the presence of methanol should provide 9-methoxy-alpha-tocopheron. To confirm that these two compounds were diastereomers, a circular dichroism detector was used. The signs of both peaks detected by the circular dichroism detector at 230 nm were opposite. In addition to observations of identical mass and ultraviolet spectra, these results indicated that the two products were diastereomers of 9-methoxy-alpha-tocopheron, whose stereochemistry is different at the newly generated chiral center of the 9-position. The on-line use of a circular dichroism detector with an electrochemical cell/liquid chromatography system may expand the utility of the system to study the metabolism of a chiral drug.

Ames test-negative carcinogen, ortho-phenyl phenol, binds tubulin and causes aneuploidy in budding yeast.

Ortho-phenyl phenol (OPP) is broad-spectrum of fungicides and antibacterial agents. OPP tested negative in an Ames system and positive with respect to the formation of tumors in the urinary bladder in rats when administered in diet, showing attributes of an Ames test-negative carcinogen. It has also been demonstrated that OPP does not bind or cleave DNA in vivo or in vitro, rather dose-dependent protein binding in OPP-treated rats was observed. OPP, however, generates chromosomal aberrations including aneuploidy. Thus, the steps by which Ames test-negative carcinogens exert their effects need to be elucidated. Here, we used an assay of loss of heterozygosity (LOH) in Saccharomyces cerevisiae to determine the biological effects of OPP and its hepatic metabolite phenyl hydroquinone (PHQ). LOH was found to be induced by OPP and PHQ because of a functional chromosome loss: aneuploidy. PHQ bound to and interfered with the depolymerization of tubulin in vitro and arrested the cell-cycle at M and G1. These results indicate that OPP and PHQ damaged tubulin to cause mis-segregation of chromosome by delaying cell-cycle progression through mitosis, and as a consequence caused aneuploidy.

Effects of melophlins on colony formation of Chinese hamster V79 cells and IL-8 production in PMA-stimulated HL-60 cells.

We have recently isolated four new melophlins P (1), Q (2), R (3), and S (4) together with seven known melophlins A (5), D (6), E (7), G (8), H (9), I (10), and O (11) from two marine sponges of the genus Melophlus collected in Palau. In this study, the influence of these compounds on the colony formation of Chinese hamster V79 cells and the production of IL-8 in PMA-stimulated HL-60 cells were examined. These 11 compounds did not show any effect on IL-8 production. The EC50 values of compounds 2, 3, 4, 5, 7, 9, 10, and 11 against V79 cells were 44.0, 13.3, 16.7, 27.2, 19.8, 8.5, 23.1, and 9.6 microM, respectively. The linear-chain-type compounds (1, 6, and 8) were not active against V79 cells at 50 microM. Although the growth inhibitory activity of these melophlins was not remarkable, some structure-activity relationships of these compounds against V79 and murine leukemia L1210 cells were observed.

A sesquiterpene quinone, 5-Epi-smenospongine, promotes TNF-α production in LPS-stimulated RAW 264.7 Cells.

Eight sesquiterpene quinones: ilimaquinone (1), smenospongidine (3), smenospongiarine (5), smenospongine (7), and their corresponding 5-epimers 2, 4, 6, and 8, isolated from the Palauan marine sponge Hippospongia sp., were examined regarding their effects on TNF-α production in LPS-stimulated RAW 264.7 cells. 5-Epi-smenospongine (8) promoted the production of TNF-α to a level three times greater than the control at 10 micron, but compounds 1-7 did not show apparent activity. The results suggest that the cis-decaline ring and a primary amine in the benzoquinone ring are necessary for activity. This is the first study to report the modulation of TNF-α production by a sesquiterpene quinone.

DMPO-OH radical formation from 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in hot water.

When an aqueous solution of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was heated at 70 degrees C for 30 min, formation of DMPO-OH was observed by ESR. This DMPO-OH radical formation was suppressed under an argon atmosphere. When water was replaced with ultra-pure water for ICP-MS experiments, DMPO-OH radical formation was also diminished. Under an argon atmosphere in ultra-pure water, the intensity of the DMPO-OH signal decreased to about 1/20 of that observed under aerobic conditions with regular purified water. The addition of hydroxyl radical scavengers such as mannitol did not affect the formation of DMPO-OH, but the signal turned faint in the presence of EDTA. We suggest that DMPO reacted with dissolved oxygen to form DMPO-OH.

Isolation and characterization of antimutagenic components of Glycyrrhiza aspera against N-methyl-N-nitrosourea.

BACKGROUND: A powdered ethanolic extract of Glycyrrhiza aspera root exhibits antimutagenic activity against N-methyl-N-nitrosourea (MNU) based on the Ames assay with Salmonella typhimurium TA1535. The aim of this study was to identify the antimutagenic components of the powdered ethanolic extract of G. aspera root. RESULTS: The powdered ethanolic extract of G. aspera root was sequentially suspended in n-hexane, carbon tetrachloride, dichloromethane, ethyl acetate, and ethanol, and each solvent soluble fraction and the residue were assayed for antimutagenic activity against MNU in S. typhimurium TA1535. The dichloromethane soluble fraction exhibited the highest antimutagenicity and was fractionated several times by silica gel chromatography. The fraction with the highest antimutagenic activity was further purified using HPLC, and the fractions were assayed for antimutagenicity against MNU in S. typhimurium TA1535. Finally, five components with antimutagenic activity against MNU were identified as glyurallin A, glyasperin B, licoricidin, 1-methoxyphaseollin, and licoisoflavone B. CONCLUSIONS: The five components were demonstrated to possess an antigenotoxic effect against carcinogenic MNU for the first time. It is important to prevent DNA damage by N-nitrosamines for cancer chemoprevention.

The mutagenic mechanism of oxygenated alkylhydrazones occurs through alkyl radicals and alkyldiazonium ions.

Acetone alkylhydrazones have been reported to be mutagenic in Salmonella typhimurium TA1535 after exposure to oxygen, and the corresponding 2-alkylazo-2-propyl hydroperoxides are formed by autoxidation as a result. The aims of this study were to investigate the mutagenic mechanisms of a methyl analogue, 2-methylazo-2-propyl hydroperoxide (MAPH), by comparing the mutagenic potency of specific Salmonella strains, detecting the DNA adducts that cause mutagenicity, and observing the hydroxyl radical and methyl radical with the electron spin resonance (ESR) spin-trapping method. MAPH showed stronger mutagenicity in both Salmonella typhimurium YG3001, a strain sensitive to hydroxyl radicals, and Salmonella typhimurium YG7108, a strain sensitive to alkylating agents, than the original Salmonella typhimurium TA1535 strain. Moreover, MAPH resulted in the formation of 8-hydroxy-2'-deoxyguanosine and O6-methyl-2'-deoxyguanosine in a reaction with DNA. These results showed that the mutagenicity of hydrazones was ascribed to the generation of reactive species by autoxidation, namely that of the alkyldiazonium ion and also the hydroxyl radical.

Antimutagenic components in Glycyrrhiza against N-methyl-N-nitrosourea in the Ames assay.

Antimutagenesis against N-nitroso compounds contribute to prevention of human cancer. We have found that Glycyrrhiza aspera ethanolic extract exhibits antimutagenic activity against N-methyl-N-nitrosourea (MNU) using the Ames assay with Salmonella typhimurium TA1535. In the present study, eight purified components from Glycyrrhiza, namely glabridin, glycyrrhetinic acid, glycyrrhizin, licochalcone A, licoricesaponin H2, licoricesaponin G2, liquiritigenin and liquiritin were evaluated for their antimutagenicity against MNU in the Ames assay with S. typhimurium TA1535. Glycyrrhetinic acid, glycyrrhizin, licoricesaponin G2, licoricesaponin H2 and liquiritin did not show the antimutagenicity against MNU in S. typhimurium TA1535. Glabridin, licochalcone A and liquiritigenin reduced revertant colonies derived from MNU in S. typhimurium TA1535 without showing cytotoxic effects, indicating that these compounds possess antimutagenic activity against MNU. The inhibitory activity of glabridin and licochalcone A was more effective than that of liquiritigenin. Thus, Glycyrrhiza contains antimutagenic components against DNA alkylating, direct-acting carcinogens.