Comparison of Antioxidant Activity Between Aromatic Indolinonic Nitroxides and Natural and Synthetic Antioxidants

In view of the possible employment of nitroxide compounds in various fields, it is important to know how they compare with other synthetic antioxidant compounds currently used in several industries and with naturally occurring antioxidants. To address this issue, the antioxidant activity of two aromatic indolinonic nitroxides synthesized by us was compared with both commercial phenolic antioxidants (BHT and BHA) and with natural phenolic antioxidants (α-hydroxytyrosol, tyrosol, caffeic acid, α-tocopherol). DPPH radical scavenging ability and the inhibition of both lipid and protein oxidation induced by the peroxyl-radical generator, AAPH, were evaluated. The results obtained show that overall: (i) the reduced forms of the nitroxide compounds are better scavengers of DPPH radical than butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) but less efficient than the natural compounds; (ii) the nitroxides inhibit both linolenic acid micelles and bovine serum albumin (BSA) oxidation to similar extents as most of the other compounds in a concentration-dependent fashion. Since the aromatic nitroxides tested in this study are less toxic than BHT, these compounds may be regarded as potential, alternative sources for several applications. The mechanisms underlying the antioxidant activity of nitroxides were further confirmed by UV–Vis absorption spectroscopy experiments and macroscale reactions in the presence of radicals generated by thermolabile azo-compounds. Distribution coefficients in octanol/buffer of the nitroxides and the other compounds were also determined as a measure of lipophilicity.     

Comparison of antioxidant activity between aromatic indolinonic nitroxides and natural and synthetic antioxidants

In view of the possible employment of nitroxide compounds in various fields, it is important to know how they compare with other synthetic antioxidant compounds currently used in several industries and with naturally occurring antioxidants. To address this issue, the antioxidant activity of two aromatic indolinonic nitroxides synthesized by us was compared with both commercial phenolic antioxidants (BHT and BHA) and with natural phenolic antioxidants (alpha-hydroxytyrosol, tyrosol, caffeic acid, alpha-tocopherol). DPPH radical scavenging ability and the inhibition of both lipid and protein oxidation induced by the peroxyl-radical generator, AAPH, were evaluated. The results obtained show that overall: (i) the reduced forms of the nitroxide compounds are better scavengers of DPPH radical than butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BLT) but less efficient than the natural compounds; (ii) the nitroxides inhibit both linolenic acid micelles and bovine serum albumin (BSA) oxidation to similar extents as most of the other compounds in a concentration-dependent fashion. Since the aromatic nitroxides tested in this study are less toxic than BHT, these compounds may be regarded as potential, alternative sources for several applications. The mechanisms underlying the antioxidant activity of nitroxides were further confirmed by UV-Vis absorption spectroscopy experiments and macroscale reactions in the presence of radicals generated by thermolabile azo-compounds. Distribution coefficients in octanol/buffer of the nitroxides and the other compounds were also determined as a measure of lipophilicity.     

The effects of nitroxide radicals on oxidative DNA damage

The indolinonic and quinolinic aromatic nitroxides synthesized by us are a novel class of biological antioxidants, which afford a good degree of protection against free radical-induced oxidation in different lipid and protein systems. To further our understanding of their antioxidant behavior, we thought it essential to have more information on their effects on DNA exposed to free radicals. Here, we report on the results obtained after exposure of plasmid DNA and calf thymus DNA to peroxyl radicals generated by the water-soluble radical initiator, 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the protective effects of the aromatic nitroxides and their hydroxylamines, using a simple in vitro assay for DNA damage. In addition, we also tested for the potential of these nitroxides to inhibit hydroxyl radical-mediated DNA damage inflicted by Fenton-type reactions using copper and iron ions. The commercial aliphatic nitroxides 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and bis(2,2, 6,6-tetramethyl-1-oxyl-piperidin-4-yl)sebacate (TINUVIN 770) were included for comparison. The results show that the majority of compounds tested protect: (i) both plasmid DNA and calf thymus DNA against AAPH-mediated oxidative damage in a concentration-dependent fashion (1-0.1 mM), (ii) both Fe(II) and Cu(I) induced DNA oxidative damage. However, all compounds failed to protect DNA against damage inflicted by the presence of the transition metals in combination with H(2)O(2). The differences in protection between the compounds are discussed in relation to their molecular structure and chemical reactivity.     

Hydrogen abstraction ability of different aromatic nitroxides

Indolinonic aromatic nitroxides have been shown to efficiently inhibit free radical mediated oxidation reactions in biological systems. Since all antioxidants also possess pro-oxidant activity, possibly through a hydrogen abstraction process from suitable substrates, the relative hydrogen abstraction abilities of these compounds were evaluated. Different hydrogen donors were reacted with an indolinic and two indolinonic nitroxides and the rates of hydrogen abstraction were determined using UV-Vis spectroscopy. From the data obtained, a structure-activity relationship was found. In addition, the hydrogen abstraction ability of these compounds was found to be much greater than that of the aliphatic nitroxide TEMPO, despite existing reports indicating that these two classes of compounds show similar antioxidant activities in biological systems.     

Butylated hydroxyanisole, butylated hydroxytoluene and tert.-butylhydroquinone are not mutagenic in the Salmonella/microsome assay using new tester strains

The phenolic antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert.-butylhydroquinone (TBHQ) were reassessed for mutagenic activity using the recently developed Salmonella tester strains TA97, TA102 and TA104, and in addition TA100. None of the phenolic antioxidants showed mutagenic activity, either with or without metabolic activation. At doses of 100 micrograms/plate and higher all 3 phenolic antioxidants exhibited toxic effects. A modification of the assay using the preincubation procedure with strain TA104 did not affect mutation frequencies. Combinations of BHA and BHT, tested to detect possible synergistic effects, did not exert mutagenic activity.     

Comparative mutagenicity of aliphatic epoxides in Salmonella

37 aliphatic epoxides comprising 6 subclasses (unsubstituted aliphatic epoxides, halogenated aliphatic epoxides, glycidyl esters, glycidates, glycidyl ethers and diglycidyl ethers) were tested, under code, for mutagenicity in Salmonella strains TA98, TA100, TA1535 and TA1537 and/or TA97 with and without metabolic activation using a standardized protocol. The 4 halogenated aliphatic epoxides and the 4 diglycidyl ethers were all mutagenic. The 2 glycidates were negative in all strain/activation systems used while all 5 glycidyl esters were mutagenic. 3 of the 8 unsubstituted aliphatic epoxides and 11 of the 12 glycidyl ethers were mutagenic. Glycidol also was mutagenic whereas 9,10-epoxyoctadecanoic acid, 2-ethylhexyl ester was not mutagenic. Of the 28 mutagenic compounds, all but neodecanoic acid, 2,3-epoxypropyl ester and 2-ethylhexyl glycidyl ether were detected in TA100 without activation. The latter two were detected only with activation in TA100 and TA1535. The majority of the other 26 chemicals were also mutagenic in TA1535 without activation. Good intra- and interlaboratory reproducibility was seen in the results of each of the 4 chemicals tested in more than one set of experiments. The current results confirm and extend the observations of other investigators regarding structural effects on the mutagenicity of members of the aliphatic epoxide class of chemicals.     

Evaluation of the mutagenicity of sesquiterpenic compounds and their influence on the susceptibility towards antibiotics of two clinically relevant bacterial strains

Sesquiterpenic compounds are natural chemicals present in organisms from different Phylae or Divisions, which have proved to be important bioactive products, namely in potentiating the action of antibiotics. In the first step, the mutagenicity of nine sesquiterpenic compounds (hydrocarbons and alcohols) was screened in a Salmonella typhimurium his(-)-reversion test with strains TA98 and TA100, in the presence or absence of in vitro metabolic activation. Under the test conditions, none of the compounds showed mutagenicity up to a concentration of 222μg/plate. trans-Farnesol, nerolidol, and α-bisabolol displayed cytotoxicity when tested at concentrations ranging from 14 to 222μg/plate. Then, the combined effect of antibiotic-sesquiterpenic compounds was evaluated on two clinically relevant pathogens, Escherichia coli and Staphylococcus aureus, with well-defined resistance-sensitive profiles. The agar-disc diffusion assay revealed that all the combinations of antibiotic-sesquiterpenic compounds increased the antibacterial activity of the antibiotics tested against S. aureus. For E. coli, an antagonistic effect was observed for various combinations on the growth of this bacterium.     

Mutagenicity of some commercially available nitro compounds for Salmonella typhimurium.

Benzoyl chloride and 53 commercially available aromatic heterocyclic and aliphatic nitro compounds were tested for mutagenicity in Salmonella typhimurium TA98 and TA100. 34 of 53 nitro compounds (64%) were mutagenic, 4 in TA100 only, 15 in TA98 only, and 15 in both strains. 13 of the heterocyclic derivatives of pyridine, indole, indazole, quinoline, and benzimidazole were mutagenic. 21 of 34 mutagenic nitro compounds were bactericidal. Nitromethane was the only aliphatic tested and was not mutagenic. Benzoyl chloride, a human carcinogen, was mutagenic for TA98.     

Experimental databases on inhibition of the bacterial mutagenicity of 4-nitroquinoline 1-oxide and cigarette smoke

Two antimutagenicity databases were prepared by applying a co-treatment procedure to the Salmonella reversion assay. Ninety compounds belonging to various chemical classes were quantitatively tested for antimutagenicity towards the direct-acting mutagen 4-nitroquinoline 1-oxide (4NQO) in strain TA100 of S. typhimurium and 63 of them were additionally tested for antimutagenicity towards unfractionated mainstream cigarette smoke (CS) in strain TA98, in the presence of S9 mix. Twelve compounds (13.3%) inhibited 4NQO mutagenicity by at least 50%, with a MID₅₀ (dose inhibiting 50% of mutagenicity) varying over a 1226-fold range. Twenty-six compounds (41.3%) inhibited CS mutagenicity, with a MID₅₀ varying over a 520-fold range. Three compounds only, i.e., bilirubin, curcumin and myricetin, were capable of inhibiting the mutagenicities of both 4NQO and CS. However, myricetin and the other flavonoid rutin were at the same time mutagenic by inducing frameshift mutations following metabolic activation. There was a rather rigorous selectivity of antimutagenicity data depending on the chemical class of inhibitors and it was possible to discriminate protective effects within several pairs or series of structurally related compounds. For instance, all eight thiols and aminothiols inhibited 4NQO mutagenicity, which contrasted with the inactivity of the remaining 17 sulfur compounds tested, all of them lacking a free sulfhydryl group. The mutagenicity of CS was consistently inhibited by the majority of phenols (eight out of 10 tested) and by all two isothiocyanates, two dithiocarbamates, three indole derivatives, three tetrapyrrole compounds and three flavonoids tested. Although the results obtained cannot be extrapolated to other mutagens or test systems, they may provide a useful source of information for research in the area of antimutagenesis and for the development of chemopreventive agents.     

Further mutagenicity studies on pesticides in bacterial reversion assay systems

A total of 228 pesticides (88 insecticides, 60 fungicides, 62 herbicides, 12 plant-growth regulators, 3 metabolites and 3 other compounds) was tested for mutagenicity in bacterial reversion-assay systems with 5 strains (TA100, TA98, TA1535, TA1537 and TA1538) of Salmonella typhimurium and a strain (WP2 hcr) of Escherichia coli. 50 pesticides (25 insecticides, 20 fungicides, 3 herbicides, 1 plant-growth regulator and 1 other compound) were found to be mutagenic. 5 of them required metabolic activation (S9 mix) for their activities. Among various chemical groups, organic phosphates, halogenated alkanes and dithiocarbamates showed higher ratios of mutagens. Although 22 of the pesticides tested have been reported to be carcinogenic, 7 of them, i.e., captain, DBCP, EDB, EDC, ETU, HEH and nitrofen, were detected as mutagens in the present assay. Most of the other 15 non-mutagenic carcinogens were organochlorine pesticides such as alpha-BHC, chlorobenzilate, p,p'-DDT, dieldrin and quintozene.     

Toxicity of Antioxidants to Tetrahymena pyriformis*

SYNOPSIS. The cytotoxicity of 97 antioxidants of various structural types was determined for Tetrahymena pyriformis grown in a peptone medium. Cytotoxicity fo Tetrahymena, generally, was positively associated with acute oral tomicoiciity to rats but not with antioxidant pdency as measured by the Tetrahymena photodynamic assay for antioxidants. Some commonly used hindered-phenol, lipidsoluble anttioxidants, e.g., 2,6-di-tert.-butyl-P-cresol (BHT), had low toxicity (to Tetrahymena but poor antioxidant activity by the photodynamic test. Nmdihydroguahetic acid, with low toxicity to the rat and high Coxicity (to Tetrahymena, had very high activity in the photodynamic test.     

Photolysis primes biodegradation of benzo[a]pyrene

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.     

Photolysis primes biodegradation of benzo[a]pyrene.

14C-labeled benzo[a]pyrene (BaP) was used as a model-compound for polycyclic aromatic hydrocarbons (PAH) in order to assess the effect of photolytic pretreatment on the subsequent fate of BaP in sewage sludge and soil test systems. Photolysis was performed in methanolic solution with or without 0.1 M H2O2, under either UV light (300 nm) or natural sunlight. The presence of H2O2 greatly enhanced the rate of photolysis both with UV and with natural sunlight. Intact BaP resisted biodegradation in both test systems. Photolysis transformed BaP to polar materials that were subject to increased mineralization and binding in both biological test systems. As shown by the Ames assay, photolysis decreased the mutagenicity of BaP to test strains TA98 and TA104 only moderately. The photolysate had an increased acute toxicity and lost its need for activation by S-9 enzymes. However, during subsequent incubation in soil or sewage sludge, mutagenicity decreased rapidly by one to two orders of magnitude and acute toxicity disappeared due to the mineralization and binding of photoproducts to humic materials. Photolysis of BaP and similar PAH compounds represents a useful treatment option that could be applied to certain PAH-containing petroleum refinery sludge and to coal tar residues in order to facilitate their detoxification and environmentally safe disposal.     

Subsequent products after antioxidant actions of beta-carotene and alpha-tocopherol have no Salmonella mutagenicity

Beta-carotene and alpha-tocopherol are important antioxidants biologically, but whether their oxidized products are toxic or not remains to be discovered. Here, we chromatographically separated 5 pure products or isomeric mixtures from reaction mixtures of beta-carotene and reactive oxygens, and 17 lipid-radical scavenging products of alpha-tocopherol. The products were tested for mutagenicity using Salmonella typhimurium TA98, TA100, TA102, and TA104, in the presence and absence of S9. None showed mutagenicity against any of the four strains, or cytotoxicity that influenced the survival of the bacteria. Lipid-peroxides have been known to increase the formation of mutagens from dietary procarcinogens such as heterocyclic amines. So, we also measured the activity to increase 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) mutagenicity. The products from beta-carotene and alpha-tocopherol did not increase, but rather several of them suppressed, the mutagenicity of Trp-P-2. Thus, the products of beta-carotene and alpha-tocopherol formed after the antioxidant actions were not genotoxic.     

Mutagenicity of aliphatic epoxides.

The mutagenicity of 17 aliphatic epoxides was determined using the specially constructed mutants of Salmonella typhimurium developed by Ames. The activity of these epoxides together with those reported in the literature as mutagens in strains TA100 and TA1535 depended on the degree of substitution around the oxirane ring. Monosubstituted oxiranes were the most potent mutagens in both strains. 1,1-Disubstitution resulted in the complete loss or reduction of mutagenicity, trans-1,2-Disubstituted, and tetrasubstituted oxiranes all lacked mutagenicity, while the cis-1,2-disubstituted oxiranes tested were weakly mutagenic in strain TA100 only. For the monosubstituted compounds the presence of electron-withdrawing substituents increased mutagenicity.     

Fluorescence study on rat epithelial cells and liposomes exposed to aromatic nitroxides

This study was performed to evaluate the effects, if any, of aromatic nitroxides, namely, indolinic nitroxides, on membrane fluidity of rat epithelial cells using steady-state fluorescence. These nitroxides are being increasingly considered as new and versatile compounds to reduce oxidative stress in biological systems. Hence, the results obtained in this study will give more insights on the interaction of these compounds with biological structures which at present is lacking, especially in view of their possible application as antioxidant therapeutic agents. The probes DPH and Laurdan which give information on the hydrophobic and hydrophilic-hydrophobic regions of the membrane bilayer, respectively, showed that nitroxide 1 (1,2-dihydro-2-methyl-3H-indole-3-one-1-oxyl) significantly increases membrane fluidity, whereas the corresponding phenylimino nitroxide derivative 2 (1,2-dihydro-2-methyl-3H-indole-3-phenylimino-1-oxyl) leads to membrane rigidification. The aliphatic nitroxide TEMPO included in this study for comparison produced no modifications. Consequently, it appears that the structure of the heterocyclic rings (aromatic or aliphatic) and the substituents may affect membrane fluidity differently.     

Genotoxicities of nitropyrenes and their modulation by apigenin, tannic acid, ellagic acid and indole-3-carbinol in the Salmonella and CHO systems

Four naturally occurring compounds, indole-3-carbinol (I3C), apigenin (Api), ellagic acid (EA) and tannic acid (TA), were tested for their inhibitory effects against 1-nitropyrene- (1-NP) or 1,6-dinitropyrene (1,6-DNP)-induced genotoxicity in Salmonella tester strains and Chinese hamster ovary (CHO) cells. Api and TA strongly inhibited the bacterial mutagenesis induced by nitropyrenes, while 13C and EA had little or no effect. For example, in TA98, 0.2 μmole Api resulted in 48% and 56% inhibition of the mutagenicity induced by 4 nmole 1-NP and 0.035 nmole 1,6-DNP, respectively. With an equal dose, expected, a good correlation was observed between the antimutagenicity of nitropyrenes and their inhibitory effect on nitroreductase activity. This indicated that one of the possible antimutagenic mechanisms of Api or TA was to inactivate the metabolism of nitropyrenes. Two biological end-points, cytotoxicity and sister-chromatid exchange (SCEs), were used to screen the antigenotoxic effects of these compounds in CHO cells. At the sub-cytotoxic dose, 13C, Api and TA all protected against the cytotoxicity induced by 1-NP and 1,6-DNP, but only TA and Api gave a significant reduction of the frequency of SCEs. Moreover, this reduction was found to be highly dose-dependent.     

The Salmonella mutagenicity test: evaluation of 29 drug candidates

The Salmonella mutagenicity test (Ames assay) is part of the routine screening battery applied to all new drugs at The Upjohn Company. The purpose of this paper is to report results for 29 compounds. These compounds are very diverse in chemical structure and represent classes of compounds selected because of known biological activity and other reasons. None of the compounds reported here produced an increase in revertant colonies in the Salmonella strains employed (TA98, TA100, TA1535, TA1537 and TA1538) and therefore the Salmonella mutagenicity results with these materials do not suggest potential for mutagenesis or carcinogenesis.     

Azido analogs of acridine: Photoaffinity probes for frameshift mutagenesis in Salmonella typhimurium

In order to identify a photoaffinity probe for 9-aminoacridine frameshift mutagenesis, 20 azido analogs of acridine were synthesized and tested in Ames' Salmonella tester strains, TA1535, TA1537, TA1538 and their corresponding excision-repair-proficient strains TA1975, TA1977, and TA1978, to determine their mutagenicity and toxicity relative to 9-aminoacridine. The substituent-mutagenicity patterns observed for these compounds agree very well with those obtained previously for non-azidoacridines. The results presented here show that the 2-azido-analog of 9-aminoacridine demonstrates biological activity similar to 9-aminoacridine prior to photolytic activation. With light activation, however, the 9-amino-2-azido derivative becomes more effective at producing frameshift mutations characteristics of 9-aminoacridine. Furthemore, this photolytic enhancement of mutagenesis appears to be due to the repairable lesion suggesting that covalent attachment of the drug occurs.     

Bacterial mutagenicity of some methyl 2-cyanoacrylates and methyl 2-cyano-3-phenylacrylates

The mutagenic potential of three alkyl 2-cyanoacrylate adhesives, three commercial alkyl 2-cyanoacrylate adhesives and three methyl 2-cyano-3-phenylacrylates, was assessed using the Salmonella/microsome mutagenicity assay. Compounds were tested with and without Aroclor 1254-induced rat-liver homogenate (S9 mix). The methyl 2-cyanoacrylate adhesives were mutagenic in the standard plate test with S. typhimurium strain TA100 with and without S9 activation. Methyl 2-cyano-3-(2-bromophenyl)acrylate revealed a direct mutagenic action to S. typhimurium strain TA1535. The compounds most toxic towards the bacterium S. typhimurium, were the methyl 2-cyanoacrylate adhesives (greater than 500 micrograms/plate). All alkyl 2-cyanoacrylate adhesives were tested in a modified spot test for volatile compounds with tester strain TA100. Mutagenic and toxic effects were observed with the three methyl 2-cyanoacrylate adhesives. It can be concluded from the results that the bacterial toxicity and mutagenicity of methyl 2-cyanoacrylate adhesives may be due to the methyl 2-cyanoacrylate monomer.