Protective properties of five newly synthesized cyclic compounds against sodium azide and N -methyl- N ′-nitro- N -nitrosoguanidine genotoxicity

Abstract

The current study aims to determine the antimutagenic potential of five newly synthesized cyclic compounds against the genotoxic agents sodium azide (NaN₃) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The mutant bacterial tester strains were NaN₃-sensitive Salmonella typhimurium TA1535 and MNNG-sensitive Escherichia coli WP2uvrA. According to the results, all the test compounds showed significant antimutagenic activity. The inhibition rates ranged from 26.05% (Compound 4—1 µg/plate) to 68.54% (Compound 5—0.01 µg/plate) for NaN₃ and from 32.44% (Compound 3—1 µg/plate) to 60.77% (Compound 5—1 µg/plate) for MNNG genotoxicity. Moreover, the mutagenic potential of the test compounds was investigated using the same strains. The results showed that all the test compounds do not have mutagenic potential on the bacterial strains at the tested concentrations. Thus, the findings of the present study give valuable information about chemical prevention from NaN₃ and MNNG genotoxicity.

Keywords

Synthetic cyclic compounds antimutagenicity mutagenicity bacterial reverse mutation test MNNG sodium azide

Introduction

Sodium azide (NaN₃) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) are two well-known genotoxic agents, widely affecting many organisms. The NaN₃ is known as a powerful mutagen in several organisms including bacteria, plants and animals (Sadiq and Owais, 2000). Previous studies showed clearly that the mutagenicity of NaN₃ is mediated through the production of an organic metabolite of azide called l-azidoalanine (Ciesla et al., 1980; Owais and Kleinhofs, 1988; Raicu and Mixich, 1982; Sadiq and Owais, 2000). On the other hand, MNNG is a well-known carcinogen and is known to exert its mutagenic and lethal effects by methylation of DNA (Kumaresan et al., 1995). Recent studies showed that O⁶-methylguanine is one of its important products, which appears to be responsible for the mutagenic action (Eadie et al., 1984; Loveless 1969).

To date, there have been a lot of studies focused on NaN₃ and MNNG genotoxicity in order to determine their effect mechanisms and prevention perspectives, because of their wide-ranging hazardous potential (Ciesla et al., 1980; Eadie et al., 1984; Kumaresan et al., 1995; Loveless 1969; Owais and Kleinhofs, 1988; Raicu and Mixich, 1982; Sadiq and Owais, 2000). In this manner, combination of natural or synthetic heterocyclic compounds and effective genotoxicity test systems has a great importance and potential to develop preservative agents against mutagens and their negative effects (Gulluce et al., 2010, 2011; Guvenalp et al., 2010).

Thus, the present study was designed to evaluate the antimutagenic potential of five newly synthesized nitrogen- and oxygen-containing heterocyclic compounds (Figure 1 ) using Ames/Salmonella and Escherichia coli WP2 bacterial reverse mutation assay systems.

Materials and Methods

Synthesis of test compounds

Synthesis method for β-aminoketones (Compounds 1 and 2)

To a solution of Bi(OTf)₃.4H₂O (0.05 mmol) in 1 mL of water the aldehyde (1 mmol; thiophene-2-carbaldehyde and 4-nitrobenzaldehyde), aniline (1 mmol) and cyclohexanone (2 mmol) were added. The reaction mixture was stirred vigorously with a magnetic stirrer during the mentioned time at room temperature and then stopped by the addition of EtOAc. The aqueous phase was extracted with EtOAc (3 × 10 mL). The organic phases were combined and washed with water (2 mL) and brine (2 mL) consecutively, dried over MgSO₄, and filtered. The solvents were removed under reduced pressure. The crude mixture was purified by column chromatography (hexane/EtOAc; Ollevier et al., 2006).

Synthesis method for oxazine (Compound 3)

Aldehyde (2 mmol; naphthalene-2-carbaldehyde) and 25% methanolic ammonia solution (0.5 mL) were added to a solution of 2-naphthol (1 mmol) in absolute methanol (MeOH; 0.5 mL). The mixture was left to stand in cooler for 2 days, and then the crystalline product formed was separated out. The crude crystals were filtered off, washed with cold MeOH (2 × 2 mL) and purified by column chromatography, eluting with the indicated solvents (Lázár and Fülöp, 2005).

Synthesis method for dihydropyridines (Compounds 4 and 5)

To a stirred mixture of 1,3-dione compound (1 mmol), ethyl acetoacetate (1 mmol) and Yb(OTf)₃ (5 mol%) in ethanol (5 mL), aldehyde (3-ethoxy-4-hydroxybenzaldehyde and m-phenoxybenzaldehyde; 1 mmol) and ammonium acetate (1 mmol) were added at room temperature. The reaction mixture was stirred for 6 h (thin-layer chromatography (TLC)) at room temperature then the resulting solid product was filtered, washed with water and dried in vacuum to derive the crude product. A pure product was obtained by further recrystallization using ethanol as a solvent. The filtrate containing the catalyst could be evaporated under reduced pressure to give a white solid. After completion of the reaction (monitored by TLC), the reaction mass was filtered in hot condition to separate the catalyst and poured on ice water. The obtained solid condensation product was further purified by recrystallization in ethanol. The recovered catalyst was washed with ethyl acetate, then dried at 70°C and activated at 120°C prior to use for next run in model reaction. And it was found that the recovered catalyst shows good yield with three successive reactions (Wang et al., 2005).

Chemicals

Direct acting mutagens NaN₃ and MNNG were obtained from Sigma-Aldrich (St. Louis, MO, USA) and ABCR GmbH & Co. KG (Karlsruhe, Germany), respectively. Other solvents and pure chemicals including magnesium sulfate (MgSO₄), sodium ammonium phosphate (Na₂NH₂PO₄), d-glucose, d-biotin, sodium chloride (NaCl), l-histidine HCl, l-tryptophane, sodium phosphate dibasic (Na₂HPO₄), crystal violet, citric acid monohydrate, potassium phosphate dibasic (K₂HPO₄) and sodium phosphate monobasic (NaH₂PO₄) were also obtained from Difo (New Jersey, USA), Fluka (Steinheim, Germany), Merck (Darmstadt, Germany) and Sigma (St. Louis, MO, USA).

Bacterial Strains

Salmonella typhimurium TA1535 (ATCC^® Number: 29629) strain was provided by The American Type Culture Collection—Bacteria Department of Georgetown University, Washington, USA, and E. coli WP2uvrA (ATCC^® Number: 49979) strain was provided from LGC Standards, Middlesex, UK. All strains were stored at –80°C. Working cultures were prepared by inoculating nutrient broth with the frozen cultures, followed by an overnight incubation at 37°C with gentle agitation (Oh et al., 2008).

Viability Assays and Determination of Test Concentrations

The toxicity of chemicals toward S. typhimurium TA1535 and E. coli WP2uvrA strains was determined as described in detail elsewhere (Santana-Rios et al., 2001; Yu et al., 2001). These tests confirmed that there was normal growth of the background lawn, spontaneous colony numbers within the regular range and no significant reduction in cell survival. Thus, for the concentrations and conditions reported here, no toxicity or other adverse effects were observed.

Bacterial Reverse Mutation Assay

The bacterial mutagenicity and antimutagenicity assays were performed according to the details described elsewhere (Mortelmans and Riccio, 2000; Mortelmans and Zeiger, 2000). The known mutagens NaN₃ (in distilled water—1 µg/plate) for S. typhimurium TA1535 and MNNG (in 10% dimethylsulfoxide (DMSO)—1 µg/plate) for E. coli WP2uvrA were used as positive controls and 10% DMSO was used as negative control in these studies.

In the mutagenicity test performed with TA1535 strain of S. typhimurium, 100 µL of the overnight bacterial culture, 50 µL test compounds at different concentrations (0.01, 0.1 and 1 µg/plate in 10% DMSO) and 500 µL buffer solution were added to 2 mL of the top agar containing 0.5 mM histidine/biotin. The mixture was poured onto minimal glucose agar plates. Histidine-independent revertant colonies and viable cells were scored on plates after incubation at 37°C for 48 h.

In the antimutagenicity test performed with the same strain, 100 µL of the overnight bacterial culture, 50 µL mutagen, 50 µL test compounds at different concentrations (0.01, 0.1, 1 µg/plate in 10% DMSO) and 500 µL buffer were added to 2 mL of the top agar containing 0.5 mM histidine/biotin. The mixture was poured onto minimal glucose agar plates. Histidine-independent revertant colonies and viable cells were scored on plates after incubation at 37°C for 48 h.

The procedures of mutagenicity and antimutagenicity assays described for the Salmonella assay are all applicable to the E. coli WP2 reverse mutation assay. The only procedural difference is the addition of limited tryptophan (0.01 mM) instead of histidine to the top agar (Mortelmans and Riccio, 2000).

The plate incorporation method was used to assess the results of mutagenicity and antimutagenicity assays (Maron and Ames, 1983).

In mutagenicity assays, the mutagenic index was calculated for each concentration, which is the average number of revertants per plate divided by the average number of revertants per plate with the negative (solvent) control. A sample is considered mutagenic when a dose–response relationship was observed and a two-fold increase in the number of mutants with at least one concentration was observed (Gulluce et al., 2010; Santos et al., 2008).

In antimutagenicity assays, the inhibition of mutagenicity was calculated using the following equation (M: number of revertants/plate induced by mutagen alone, S ₀: number of spontaneous revertants, S ₁: number of revertants/plate induced by the test compound plus the mutagen): %Inhibition = 1 – [(M – S ₁) / (M – S ₀)] × 100.

In all, 25–40% inhibition was defined as moderate antimutagenicity; 40% or more inhibition as strong antimutagenicity and 25% or less inhibition as no antimutagenicity (Evandri et al., 2005).

Statistical analysis

The results are presented as the average and standard error of three experiments with triplicate plates/dose experiment. The data were further analyzed for statistical significance using analysis of variance (ANOVA), and the difference among means was compared by high-range statistical domain using Tukey’s test. A level of probability was taken as <0.05, indicating statistical significance (Gulluce et al., 2010).

Results

According to the mutagenicity assay results, no test compounds have mutagenic potential for S. typhimurium TA1535 and E. coli WP2uvrA at tested concentrations (Table 1 and Figure 2(a) and (b) ).

Table 1.

The mutagenicity assay results of the test compounds for S. typhimurium TA1535 and E. coli WP2uvrA strains

			Number of revertants
			S. typhimurium TA1535		E. coli WP2uvrA
Test item concentration (µg/plate)			Mean ± SE	Mutation %	Mean ± SE	Mutation %
Entry	MNNG^a	1			413.00 ± 2.04
	NaN₃ ^a	1	426.00 ± 2.48
	DMSO^b (µL/plate)	100	18.00 ± 0.91		16.00 ± 0.40
1	C₁₈H₂₁NOS	0.01	17.00 ± 1.47	–	13.00 ± 0.81	–
		0.1	19.25 ± 1.88	–	11.00 ± 1.29	–
		1	19.75 ± 1.89	–	10.00 ± 0.82	–
2	C₂₀H₂₂N₂O₃	0.01	15.00 ± 2.19	–	17.00 ± 0.91	–
		0.1	16.50 ± 2.04	–	16.00 ± 0.90	–
		1	14.00 ± 0.91	–	16.75 ± 1.37	–
3	C₃₂H₂₃NO	0.01	15.00 ± 1.47	–	16.00 ± 1.68	–
		0.1	12.00 ± 1.82	–	17.50 ± 0.40	–
		1	15.50 ± 0.91	–	16.50 ± 0.91	–
4	C₂₁H₂₅NO₅	0.01	09.50 ± 0.90	–	08.00 ± 1.68	–
		0.1	16.00 ± 1.82	–	16.00 ± 1.82	–
		1	22.00 ± 0.58	–	11.00 ± 1.30	–
5	C₂₅H₂₅NO₄	0.01	13.50 ± 0.90	–	07.00 ± 1.40	–
		0.1	13.00 ± 0.90	–	14.50 ± 2.05	–
		1	14.00 ± 2.58	–	14.25 ± 0.85	–

MNNG: N-methyl-N’-nitro-N-nitrosoguanidine, NaN₃: sodium azide; S. typhimurium: Salmonella typhimurium; E. coli: Escherichia coli.

^aMNNG (1 µg/plate) and NaN₃ (1 µg/plate) were used as positive controls for E. coli WP2uvrA and S. typhimurium TA1535 strains, respectively.

^bDimethylsulfoxide (DMSO; 100 µL/plate) was used as negative control.

The possible antimutagenic potential of test materials was examined against NaN₃ and MNNG in S. typhimurium TA1535 and E. coli WP2uvrA, respectively. The results were evaluated using standard plate incorporation method and summarized in Table 2 showing the antimutagenic activities of test materials tested at three different concentrations (0.01, 0.1 and 1 µg/plate).

Table 2.

The antimutagenicity assay results of the test compounds for S. typhimurium TA1535 and E. coli WP2uvrA strains

			Number of revertants
			S. typhimurium TA1535		E.coli WP2uvr
Test Items Concentration(µg/plate)			Mean ± SE	Inhibition %	Mean ± SE	Inhibition %
Entry	MNNG^a	1			413.00 ± 2.04
	NaN₃ ^a	1	426.00 ± 2.48
	DMSO^b (µL/plate)	100	18.00 ± 0.91		16.00 ± 0.40
1	C₁₈H₂₁NOS	0.01	299.50 ± 1.68	29.69^c	200.00 ± 4.56	51.57^c
		0.1	305.50 ± 2.19	28.28^c	255.00 ± 2.21	38.25^c
		1	296.00 ± 1.82	30.51^c	253.50 ± 1.47	38.61^c
2	C₂₀H₂₂N₂O₃	0.01	282.00 ± 1.47	33.80^c	276.50 ± 1.82	33.05^c
		0.1	275.00 ± 3.42	33.41^c	238.00 ± 2.19	42.37^c
		1	289.25 ± 1.88	32.10^c	162.00 ± 3.48	58.83^c
3	C₃₂H₂₃NO	0.01	307.50 ± 1.08	27.81^c	214.00 ± 2.94	48.18^c
		0.1	249.50 ± 2.08	41.43^c	241.50 ± 3.70	41.52^c
		1	238.00 ± 2.19	44.13^c	279.00 ± 2.48	32.44^c
4	C₂₁H₂₅NO₅	0.01	246.25 ± 1.50	42.19^c	238.00 ± 2.14	42.37^c
		0.1	315.00 ± 2.38	43.66^c	271.50 ± 4.02	34.26^c
		1	240.00 ± 2.19	26.05^c	250.00 ± 2.38	39.46^c
5	C₂₅H₂₅NO₄	0.01	134.00 ± 2.58	68.54^c	200.00 ± 4.56	51.57^c
		0.1	158.00 ± 0.91	62.91^c	215.00 ± 2.38	47.94^c
		1	180.00 ± 2.58	57.74^c	170.00 ± 4.55	60.77^c

MNNG: N-methyl-N’-nitro-N-nitrosoguanidine, NaN₃: sodium azide; S. typhimurium: Salmonella typhimurium; E. coli: Escherichia coli.

^aMNNG (1 µg/plate), and NaN₃ (1 µg/plate) were used as positive controls for E. coli WP2uvrA and S. typhimurium TA1535 strains, respectively.

^bDimethylsulfoxide (DMSO; 100 µL/plate) was used as negative control.

^c p < 0.05.

In the antimutagenicity assays performed with S. typhimurium TA1535 strain, all the test compounds exhibited significant antimutagenic activity against NaN₃ mutagenicity at all test concentrations (Table 2 and Figure 3(a) ). The inhibition rates ranged from 26.05% (Compound 4—1 µg/plate) to 68.54% (Compound 5—0.01 µg/plate).

Besides, all the test compounds also showed significant antimutagenic activity on E. coli WP2uvrA strain at all tested concentrations (Table 2 and Figure 3(b)). The inhibition rates of these compounds were between 32.44% (Compound 3—1 µg/plate) and 60.77% (Compound 5—1 µg/plate).

Discussion

Heterocyclic compounds represent important biological and medicinal scaffolds. In the current study, the mutagenic and antimutagenic properties of newly synthesized five compounds with three different groups, including mannich bases (Compounds 1 and 2), oxazine (Compound 3) and dihydropyridines (Compounds 4 and 5; Figure 1), have been investigated using AMES-Salmonella and E. coli WP2 bacterial test systems.

According to the results, none of the test compounds showed mutagenic activity on S. typhimurium TA1535 and E. coli WP2uvrA tester strains. However, all of them have significant antimutagenic activity against NaN₃- and MNNG-induced mutagenesis on the same strains at all tested concentrations.

The first investigated group was mannich bases. Mannich reaction is one of the classical methods for the construction of nitrogenous compounds especially β-amino carbonyl compounds that are versatile intermediates for the synthesis of β-amino alcohols and acids, which have remarkable biological significance (Shailaja et al., 2010). β-aminocarbonyl derivatives are an important class of heterocyclic compounds that occur in various pharmaceuticals, natural products and versatile synthetic intermediates (Tramontini and Angiolini, 1990; Yi et al., 1991). For example; aminoketone derivatives exhibit remarkable antitubercular, antifungal, antimalarial, antidiabetic, anti-inflammatory and antitumor activities (Connolly et al., 2005; Ma et al., 2004; Mhaske and Argade, 2006; Witt and Bergman, 2003).

The second one was oxazine derivatives. Synthesis of 1,3-oxazines have attracted attention because of their potential as antibiotics, antitumor agents, analgesics and anticonvulsants. 1,3-Oxazines have generated great interest as antipsychotic agents and as possible effectors for serotonin and dopamine receptors. Additionally, benzo-1,3-oxazines are known to be biologically active antimalarial, antianginal, antihypertensive and potent antirheumatic agents. Several methods have previously been reported for the preparations of 1,3-oxazine derivatives. The ring-chain tautomeric interconversion of N-unsubstituted 1,3-N-O-heterocycles and the corresponding hydroxyalkylimines can often be exploited advantageously in different areas of organic synthesis and also in physical, medicinal and peptide chemistry. Hence, the synthesis of these derivatives is of considerable interest (Damodiran et al., 2009).

The last organic group is dihydropyridines. Synthetic derivatives of 1,4-dihydropyridine (DHP) possess important biochemical and pharmacological properties. They show modulating activity on cardiovascular and neuronal processes and on corticosteroid regulatory circuits, prevent inflammatory, diabetic processes and some of them show antineoplastic, geroprotective, radioprotective and radiosensitizing effects. Some of the positive effects of 1,4-DHPs are long term; and due to their low toxicity, this group of compounds appears to be a promising one for medicinal applications. Most of the 1,4-DHPs that differed in chemical structure showed antimutagenic activity and significantly reduced spontaneous and alkylation-induced point mutations and chromosome breaks in the germ cells of Drosophila, alkylation-induced micronuclei in mouse bone-marrow cells, and radiation-induced chromosome aberrations and other cytogenetic end points in fish. Previous studies performed with 1-DHP derivatives suggested that the 1-DHPs inhibit spontaneous, chemical or radiation induced mutagenesis by the modulation of DNA repair mechanisms in Drosophila and in vitro human cell lines (Abbas et al., 2010; Antonyraj and Kannan, 2008; Chan et al., 2010; Chikhale et al., 2009; Choi et al., 2010; Fassihi et al., 2009; Filipan-Litvic et al., 2008; Kumar and Maurya, 2007a, 2007b; Kumar et al., 2010; Pandey et al., 2010).

The positive mutagens of the present study were model mutagens, NaN₃ and MNNG, which have hazardous effects on various organisms, from bacteria to human being. The mutagenicity of NaN₃ is mainly explained by the mechanism, which is mediated through the production of an organic metabolite of azide called l-azidoalanine (Ciesla et al., 1980; Owais and Kleinhofs, 1988; Raicu and Mixich, 1982; Sadiq and Owais, 2000). On the other hand, the mutagenicity and carcinogenicity mechanism of MNNG depends on the formation of a metabolite called O⁶-methylguanine (Eadie et al., 1984; Kumaresan et al., 1995; Loveless, 1969). According to the antimutagenicity results, the possible antimutagenicity mechanism of the test materials can be explained by the inhibiton of l-azidoalanine and O⁶-methylguanine production.

The results give preliminary information about protective potential of the test materials, and these findings are valuable for the prevention of and drug-discovering studies against NaN₃ and MNNG genotoxicity.

Footnotes

Funding

This work was partly supported by Yildiz Technical University with the project number BAPK 2010-01-02-DOP01.

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