Comparison of rat and guinea pig as sources of the S9 fraction in the salmonella/mammalian microsome mutagenicity test

A highly significant enhancement of mutagenicity occurs with 11 polycyclic aromatic hydrocarbons when 3-methylcholanthrene-induced guinea pig liver S9 is substituted for Aroclor-induced rat liver S9 in the Ames test. The use of MC-induced guinea pig liver S9 is particularly valuable for detecting the weak mutagenicity of benz[c]acridine, which is barely positive in a standard Ames assay. However, anthracene and phenanthrene, which are generally considered not to be carcinogens, remain non-mutagenic for strain TA100. This enhancement of mutagenicity does not correlate with arylhydrocarbon hydroxylase activities of the various liver preparations and does not apply to certain other non-PAH mutagens, including β-naphthylamine, aflatoxin B₁ and 4-dimethylaminoazobenzene.     

Photophosphorylation Associated with Photosystem II: II. Effects of Electron Donors, Catalyst Oxidation, and Electron Transport Inhibitors on Photosystem II Cyclic Photophosphorylation

Incubation of KCN-Hg-NH₂OH-inhibited spinach (Spinacia oleracea L.) chloroplasts with p-phenylenediamine for 10 minutes in the dark prior to illumination produced rates of photosystem II cyclic photophosphorylation up to 2-fold greater than the rates obtained without incubation. Partial oxidation of p-phenylenediaine with ferricyanide produced a similar stimulation of ATP synthesis; addition of dithiothreitol suppressed the stimulation observed with incubation. Addition of ferricyanide in amounts sufficient to oxidize completely p-phenylenediamine failed to inhibit completely photosystem II cyclic activity. This is due at least in part to the fact that the ferrocyanide produced by oxidation of p-phenylenediamine is itself a catalyst of photosystem II cyclic photophosphorylation. N,N,N′N′-Tetramethyl-p-phenylenediamine catalyzes photosystem II cyclic photophosphorylation at rates approaching those observed with p-phenylenediamine. The activities of both proton/electron and electron donor catalysts of the photosystem II cycle are inhibited by dibromothyoquinone and antimycin A. These findings are interpreted to indicate that photosystem II cyclic photophosphorylation requires the operation of endogenous membrane-bound electron carriers for optimal coupling of ATP synthesis to electron transport.     

Genotoxicity testing of five compounds in three Drosophila short-term somatic assays

To provide further background data for the somatic mutation and/or recombination tests in Drosophila melanogaster, we have evaluated the responses in 3 assyas (zeste-white, white-ivory and wing spot) of 5 chemicals classified by the U.S. National Toxicology Program (NTP) as genotoxic non-carcinogens (or ambiguous). The selected compounds were 2-chloromethylpyridine, 1-nitronaphthalene, 4-nitro-o-phenylenediamine, 3-nitropropionic acid and p-phenylenediamine. Our results show that all the compounds tested produce significant increases in the frequency of mutant clones, in at least one of the assays, p-phenylenediamine being the compound which present a clearer mutagenic activity, and the wing spot test, the assay the detects more genotoxic compounds (4/5).     

The mutagenicity analysis of imidapril hydrochloride and its degradant,diketopiperazine derivative,nitrosation mixtures by in vitro Ames test with two strains of Salmonella typhimurium

AimThe evaluation of mutagenic properties of imidapril hydrochloride (IMD) and its degradation impurity, diketopiperazine derivative (DKP), nitrosation mixtures was conducted in order to analyze the carcinogenic risk of IMD long-term treatment in patients. In this study an in vitro Ames test with Salmonella enterica serovar Typhimurium TA 98 and TA 100 strains was used.BackgroundIMD and DKP contain nitrogen atoms, which makes them theoretically vulnerable to in vivo nitrosation with the production of N-nitroso compounds (NOC). NOC, in turn, are known animal mutagens indicating that their endogenous production from nitrosable drugs constitutes a carcinogenic hazard.Materials and methodsPure IMD sample was exposed to forced degradation conditions of increased temperature and dry air in order to achieve a DKP sample. Both samples were then treated with a nitrosating agent and the obtained nitrosation mixtures were subjected to mutagenicity analysis by the Ames test with S. typhimurium TA 98 and TA 100 strains in the presence and absence of metabolic activation system (S9 mix) using a commercial Ames MPF 98/100 microplate format mutagenicity assay kit.ResultsNone of the six concentrations of the investigated nitrosation mixtures exhibited any mutagenic potential in both S. typhimurium strains. The addition of S9 mix did not alter the non-mutagenic properties of the studied compounds.ConclusionsThe nitrite treatment of both studied compounds has no impact on their mutagenic properties under the conditions of the present studies. Hence, IMD and DKP nitrosation mixtures are classified as non-mutagens in this test.     

Turnover and vectorial properties of cytochrome c oxidase in reconstituted vesicles

1. Proteoliposomes containing cytochrome c oxidase and phospholipid have been made by sonication and by the cholate dialysis procedure. In both methods of preparation, only about 50% of the enzyme molecules are oriented in the membrane with their cytochrome c reaction sites exposed to the outside of the vesicle.2. The activity of cytochrome c oxidase in the reconstituted vesicles is not increased by incubation in 1% Tween 80. Experiments on reconstituted vesicles containing internal (entrapped) cytochrome c indicate that turnover of enzyme oxidising entrapped cytochrome c in the presence of N,N,N′,N′-tetramethyl-p-phenylenediamine or 2,3,5,6-tetramethyl-p-phenylenediamine is at a very much lower rate than enzyme oxidising external ferrocytochrome c.3. Oxidation of ascorbate by externally added cytochrome c results in an electrogenic production of OH^? inside the vesicles, which can be monitored using entrapped phenol red. Polylysine inhibits, but does not abolish, the internal alkalinity change in reconstituted vesicles oxidising internal (entrapped) cytochrome c using externally added ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine. When 2,3,5,6-tetramethyl-p-phenylenediamine is used as the permeable redox mediator, an increase in internal acidity can be monitored under the same conditions.     

Mutagenicity in a Molecule: Identification of Core Structural Features of Mutagenicity Using a Scaffold Analysis

With advances in the development and application of Ames mutagenicity in silico prediction tools, the International Conference on Harmonisation (ICH) has amended its M7 guideline to reflect the use of such prediction models for the detection of mutagenic activity in early drug safety evaluation processes. Since current Ames mutagenicity prediction tools only focus on functional group alerts or side chain modifications of an analog series, these tools are unable to identify mutagenicity derived from core structures or specific scaffolds of a compound. In this study, a large collection of 6512 compounds are used to perform scaffold tree analysis. By relating different scaffolds on constructed scaffold trees with Ames mutagenicity, four major and one minor novel mutagenic groups of scaffold are identified. The recognized mutagenic groups of scaffold can serve as a guide for medicinal chemists to prevent the development of potentially mutagenic therapeutic agents in early drug design or development phases, by modifying the core structures of mutagenic compounds to form non-mutagenic compounds. In addition, five series of substructures are provided as recommendations, for direct modification of potentially mutagenic scaffolds to decrease associated mutagenic activities.     

Spontaneous generation of adriamycin semiquinone radicals at physiologic pH.

Adriamycin semiquinone radicals are spontaneously generated by adriamycin solutions at physiologic pH. Rate of radical formation and equilibrium-state radical yield increase with increasing pH from 7.4 to 8.85. The radicals are oxygen sensitive, but the mechanism of radical formation is oxygen independent and associated with proton removal from the dihydroquinone of adriamycin. The less cardiotoxic and non-mutagenic (Ames test) anthracycline 5-iminodaunorubicin does not form semiquinone radicals spontaneously at physiologic pH.     

Differential responses of N-nitrosoamines and aromatic amines in the plant cell/microbe coincubation assay

The plant cell/microbe coincubation assay is based on employing living tobacco cells in suspension culture as the activating system for promutagens and the Ames/Salmonella cells as the genetic indicator system. In contrast to aromatic amines(e.g. 2-aminofluorene andm-phenylenediamine) that were previously reported to be activated to products mutagenic in theS. typhimurium strains TA98 or YG1024 by tobacco cells, promutagenic N-nitrosoamines (N-nitrosodimethylamine, N-nitroso-morpholine, N-nitrosopiperidine, N-nitrosomethyl-2-hydroxypropylamine) were not activated to product(s) mutagenic inS. typhimurium TA 100.     

An efficient Fmoc strategy for the rapid synthesis of peptide para-nitroanilides

A new strategy has been developed for the rapid synthesis ofpeptide para-nitroanilides (pNA). The method involves derivatization of commercially available tritylchloride resin(TCP-resin) with 1,4-phenylenediamine, subsequent coupling withdesired amino acids by the standard Fmoc protocol, and oxidationof the intermediate para-aminoanilides (pAA) with Oxone®. This procedure allows easy assembly of the desired para-aminoanilides (pAA) on standard resin and efficient oxidation and purification of the corresponding para-nitroanilides (pNA). The method allows easy access to any desired peptide para-nitroanilides, which are useful substrates for the characterization and study of proteolytic enzymes.     

Development of a toxicity test to be coupled to the Ames Salmonella assay and the method of construction of the required strains

A ‘toxicity’ test protocol is described here to be used for determining the bactericidal effect of the chemicals which are tested for their mutagenic activity by the Ames method. Two sets of strains, isogenic with the Ames tester strains except for their his character, are constructed.One set is the his⁺ derivatives of the tester strains which are used for measuring the survival of the inocolum cells after exposure to the chemical. The other set is the stable his⁻ derivatives of the tester strains which are used for simulating the background growth in the Ames mutagenicity plate test. The per cent survival of the his⁺ cells in the incculum in the presence of the ‘filler cells’ is used as a measure of the toxic effect of the chemical.     

Measurement of drug-metabolizing systems in Salmonella typhimurium strains G46, TA1535, TA100, TA1538 and TA98

Salmonella typhimurium strains which are commonly used in the Ames test for screening potential carcinogens were examined for a number of drug-metabolizing systems. Neither cytochrome P-450 itself nor two activities catalyzed by the cytochrome P-450 system in mammalian cells, i.e., benzpyrene monooxygenase and ethoxycoumarin O-deethylation, could be detected. Nor do these bacterial strains demonstrate any ability to detoxify epoxides by hydrating them or to conjugate p-nitrophenol with glucuronic acid.On the other hand, S. typhimurium strains G46, TA1535, TA100, TA1538 and TA98 contain considerable amounts of acid-soluble thiols, approx. 5–10% of which is glutathione. These bacteria can also enzymatically conjugate glutathione with 1-chloro-2,4-dinitrobenzene (CDNB) and can reduce oxidized glutathione using NADPH as cofactor.Thus, enzymatic and non-enzymatic reaction of immediate carcinogens with thiol groups in S. typhimurium may have a significant effect on the outcome of the Ames test in certain cases.     

The effect of p-phenylenediamine and dibromothymoquinone on the postosynthetic electron transport and proton pump activities of isolated barley chloroplats

The effect of p-phenylenediamine and dibromothymoquinone (DBMIB) on photosynthetic electron transport was studied using thylakoids from barley chloroplasts. p-Phenylenediamine (0.1 mm) converted the light saturation curve for electron transport between water and ferricyanide from a hyperbolic one saturating at low light intensity into a linear one which was not saturated at any light intensity studied. DBMIB at a concentration of 1 μm had little inhibitory effect on the basal electron transport activity of coupled thylakoids in either the presence or the absence of 0.1 mmp-phenylenediamine. However, activity was inhibited by DBMIB after uncoupling with 60 mm methylamine. These results may indicate that there is a rate-limiting step in electron transport at or after plastoquinone which is circumvented by phenylenediamine. Its appearance or elaboration could explain the conversion of a linear light saturation curve to a hyperbolic one during chloroplast development. The movement of protons into thylakoids upon illumination was reduced about 50% by 1 μm DBMIB, a concentration which maximally inhibits ferricyanide-Hill activity after uncoupling. Fifty percent inhibition occurred irrespective of whether or not phenylenediamine was included in the reaction mixture. Most of the residual DBMIB-insensitive pH change was inhibited by 3 μm 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), suggesting that photosystem II was required for the latter activity. In the presence of both DBMIB and DCMU, proton pump activity could be reestablished by including isoascorbate and p-phenylenediamine in the reaction mixture.     

Mutagenicity assessment of Salacia chinensis by bacterial reverse mutation assay using histidine dependent Salmonella typhimurium tester strains

Background and objectiveGenotoxicity analysis is one of the most important non-clinical environmental safety investigations required for pharmaceutical and agrochemical product registration. Any medicinal product must undergo a risk evaluation to determine its mutagenicity and carcinogenicity.Materials and methodsThe Ames test is a commonly used in vitro test for determining a test chemical's mutagenic activity. Histidine-dependent Salmonella typhimurium strains with a defective gene that causes the bacteria to synthesis the necessary amino acid histidine for life were tested for mutagenic potential. In order to reveal pro-mutagens and mutagens, the mutagenic potential of both plate integration and pre-incubation techniques was examined in the presence and absence of metabolizing system. Salacia chinensis has been widely used in ayurveda to treat various ailments. However, the information of mutagenicity of Salacia chinensis is scarce as per available literature.ResultsThe mutagenicity of a Salacia chinensis root extract was investigated utilizing the Ames assay with plate incorporation and pre-incubation protocols using the appropriate Salmonella typhimurium tester strains: TA98, TA100, TA1537, TA1535, and TA102 in the presence and absence of S9. The concentrations used were 0.3123, 0.625, 1.25, 2.5 and 5 mg/plate. The extract of Salacia chinensis root did not show any mutagenic effect in any of the Salmonella typhimurium strains at the concentrations tested in the absence or presence of metabolic activation.ConclusionThe root of Salacia chinensis was hence confirmed to be non-mutagenic and at least according to the results of this genotoxicity evaluation can be regarded as being safe for human use.     

Coloured and low conductive fabrics by in situ laccase-catalysed polymerization

Coloured and conductive fabrics were obtained through “in situ” laccase polymerization of catechol and p-phenylenediamine under high-pressure homogenization. Both monomers, catechol and p-phenylenediamine, were polymerized by different laccase forms, namely native, PEGylated and Epoxy-PEGylated. All the catalysts were placed inside a textile fabric bag which served simultaneously as enzyme support and as substrate for coating with the newly produced polymers. The PEGylated laccase forms gave rise to a higher amount of oligomers/polymers and higher colouration level of polyethylene terephthalate (PET), cotton and wool fabrics compared to native laccase. Both functional polymers were able to confer conductivity to the substrates however in a different extent. Fabrics coated with poly(p-phenylenediamine) present higher conductivity, rather due to its polymerized structure than to the amount of polymer produced by enzyme catalysis. Herein a green approach was presented to produce polyphenols with increased fixation onto different textile substrates. These substrates reach high levels of colouration and good fastness behaviour after washing.     

Chemically-induced sister-chromatid exchange in vivo in bone marrow of Chinese hamsters An evaluation of 24 compounds

Chemically-induced sister-chromatid exchange (SCE) was measured in vivo in bone marrow of Chinese hamsters. Chemicals were administered either intraperitoneally or orally and increased SCE frequencies were noted with 6 of 6 direct-acting genotoxins and with 9 of 14 activation-dependent genotoxins. Metronidazole, O-toluidine, 4-nitro-O-phenylenediamine and 2-nitro-p-phenylenediamine, compounds which have shown either mutagenic or carcinogenic activity, did not induce SCE in vivo. 4 non-genotoxins and 4 different control treatments did not induce SCE. The results show that the in vivo SCE method may be useful for the identification of genotoxins and that the outcome of the test is, for certain chemicals, dependent upon the route of exposure.     

Rate-limiting step in oxidation of physiological and artificial reductants by Azotobacter vinelandii membrane vesicles.

The respiration of Azotobacter vinelandii membrane vesicles was investigated in order to determine the partial rates of electron fluxes at each segment of its branched respiratory chain. It is concluded that under physiological conditions only 20 to 30% of the total flux is carried through the c₄, c₅ → a₁,o chain. Steady state analysis indicates that the limited capacity of the chain is due to the slow rate of oxidation of the cytochromes c by the a₁,o oxidases. This rate-limiting step is bypassed by the artificial electron donors, ascorbate-2,6-dichlorophenol indophenol and ascorbate-N,N,N′,N′-tetramethyl-p-phenylenediamine, which directly reduce the highly active a₁,o oxidases. During the oxidation of ascorbate-N,N,N′,N′-tetramethyl-p-phenylenediamine by the membrane vesicles, an accumulation of oxidized N,N,N′,N′-tetramethyl-p-phenylenediamine occurs. Such accumulation of positively charged molecules should lead to a generation of a membrane potential. This fact and previous data concerning coupling site III of A. vinelandii are discussed.     

Test paper for detection of lipoxygenase.

A quick, simple, and specific test for lipoxygenase was developed. Filter paper impregnated with linoleic acid and N,N′-dimethyl-p-phenylenediamine gave a blue color as a positive test. The specificity of the test was established by: use of inhibitors of lipoxygenase and heme-catalyzed peroxidation, use of nonsubstrates, use of inactivated enzymes, proportionality of color intensity to amount of enzyme applied, and use of nonoptimum pH. Heattreated tomato samples showed decreasing color intensities with increasing temperatures. The technique should prove useful as a quality control tool in the quick detection of lipoxygenase during processing and especially in in situ localization of the enzyme in fruits and vegetables.     

Peroxidative oxidation of leuco-dichlorofluorescein by prostaglandin H synthase in prostaglandin biosynthesis from polyunsaturated fatty acids

Prostaglandin H synthase can oxidize arachidonic acid with leuco-dichlorofluorescein as reducing cosubstrate. Addition of 0.5 mM phenol increases the oxidation of leuco-dichlorofluorescein 5-fold, probably by acting as a cyclic intermediate in the oxidation. Tetramethyl-p-phenylenediamine is also oxidized as cosubstrate. Its oxidation is not influenced by phenol. A stoichiometry of close to one mole of tetramethyl-p-phenylenediamine or leuco-dichlorofluorescein consumed per mole of arachidonic acid was found in the initial phase of the reaction. In the presence of phenol + leuco-dichlorofluorescein, the oxidation rate of arachidonic acid is about 40% lower than with phenol alone as cosubstrate. Since dichlorofluorescein has a molar extinction coefficient of 91 · 10³ at 502 nm, the oxidation of less than 1 μM leuco-dichlorofluorescein can be detected spectrophotometrically. The rate of extinction change with leuco-dichlorofluorescein (at 502 nm) is about 4-fold more rapid than with tetramethyl-p-phenylenediamine (at 611 nm). With this spectrophotometric assay we have confirmed that arachidonic acid, linolenic acid, adrenic acid, γ-linolenic acid, eicosapentaenoic acid, are substrates for prostaglandin H synthase with decreasing reaction rates in the mentioned order. The same order of reaction rates were found when oxygen consumption was measured. The assay also shows that docosahexaenoic acid is substrate for the enzyme. The reaction rate of the enzyme evidently is decreased both by a n − 3 double bond and by deviation from a 20 carbon chain length of the fatty acid substrate.     

Kinetic study of the inhibition of rat liver ornithine decarboxylase by diamines; considerations on the mechanism of interaction between enzyme and inhibitor

• 1.1. Partially purified rat liver ornithine decarboxylase is inhibited by several diamines including putrescine, 1,3-diaminopropane, cadaverine and p-phenylenediamine.
• 2.2. The inhibition is dependent on pH, being strong at pH above 8 and negligible below pH 6.5.
• 3.3. The kinetic study of the inhibition showed that while the aromatic diamine behaved as a simple competitive inhibitor, the aliphatic diamines presented a more complex pattern of inhibition in which two molecules of inhibitor might bind to the enzyme active site.
• 4.4. The K_I values for the different inhibitors were calculated and the degree of affinity for the enzyme was p-phenylenediamine > putrescine > cadaverine > 1,3-diaminopropane.
• 5.5. A molecular mechanism explaining how one or two molecules of inhibitor can bind to the enzyme is proposed.

     

Structure—activity relationships in the mutagenicity of quinone methides of 7-hydroxyflavylium salts for Salmonella typhimurium

Several synthetic 7-hydroxyflavylium salts related to apigeninidin, a natural 3-deoxyanthocyanidin, have been studied in the Ames mutagenicity test using strain TA1537 of Salmonella typhimurium. Under the neutral pH conditions of the test, these flavylium salts are deprotonated through ionization of the C7-OH (pK_a′ = 4.2–4.4) to form quinone methides. Only the quinone methides of 4-methyl-7-hydroxyflavylium chloride and 4′-methoxy-4-methyl-7-hydroxy-flavylium chloride showed mutagenicity. Responses of 4–8 times the background were observed at the higher doses (1000 μg/plate), both with and without metabolic activation. It was concluded that the induction of frameshift mutagenicity by this group of compounds is caused by those quinone methides that have non-ionic, stable polycyclic structures at neutral pH.