Mutagenic evaluation of 10 long-term stored medicinal plants commonly used in South Africa

The use of medicinal plants is an increasing phenomenon among the majority of people in many developing countries. Some of the harvested medicinal plants are often stored for shorter or longer periods prior to usage. Evidence from recent studies has demonstrated the pharmacological efficacy of short and long-term stored plant materials when compared to freshly-harvested ones. In an attempt to evaluate the effect of long-term storage on the safety of some commonly used medicinal plants, the Ames test which involved the use of three Salmonella typhimurium tester strains (TA98, TA100 and TA1535) were conducted. Current findings indicate the absence of any mutagenic effects resulting from the storage of medicinal plant materials for as long as 16 years. Although freshly collected Acokanthera oppositifolia extract demonstrated a mutagenic effect against TA1535 strain at the highest concentration tested, no such effect was observed in the stored material. Further studies involving metabolic activation systems and in vivo conditions may further elucidate the effect of long-term storage on the safety of medicinal plants.     

Mutagenicity of styrene and styrene oxide

Incubation of S. typhimurium strain TA 1535 with styrene increased the number of his⁺ revertants/plate in presence of a fortified S9 rat-liver fraction. Styrene was also highly cytotoxic for Salmonella cells. Styrene oxide, the presumed first metabolite, had a mutagenic effect towards strains TA 1535 and TA 100 both with and without metabolic activation. Styrene is probably mutagenic because it is metabolized to styrene oxide.     

Inhibitory effect of Teucrium ramosissimum extracts on aflatoxin B1, benzo[a]pyrene, 4-nitro-o-phenylenediamine and sodium azide induced mutagenicity: Correlation with antioxidant activity

The mutagenic potential of total oligomers flavonoids (TOF), ethyl acetate (EA) and petroleum ether (PE) extracts from aerial parts of Teucrium ramosissimum was assessed using Ames Salmonella tester strains TA98, TA100 and TA1535 with and without metabolic activation (S9). None of the different extracts produced a mutagenic effect. Likewise, the antimutagenicity of the same extracts was tested using the “Ames test”. Our results showed that T. ramosissimum extracts possess antimutagenic activity against all the tested genotoxicants (aflatoxin B₁, benzo[a]pyrene, 4-nitro-o-phenylenediamine and sodium azide) in the Salmonella assay systems used in this study. In addition, all extracts showed important free radical scavenging activity toward the radicals DPPH and ABTS except the PE extract.     

Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs

Quercetin, rhamnetin, isorhamnetin, apigenin and luteolin were isolated from medicinal herbs: Erigeron canadensis L., Anthyllis vulneraria L. and Pyrola chloranta L. The mutagenicity of these naturally occurring flavonoids was tested by the Ames method with S. typhimurium strains TA1535, TA1538, TA97, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Of the above flavonoids only quercetin and rhamnetin revealed mutagenic activity in the Ames test. Quercetin induced point mutations in strains TA97, TA98, TA100 and TA102 of S. typhimurium. The presence of S9 rat liver microsome fraction markedly enhanced the mutagenic activity of quercetin in these strains. Rhamnetin appeared to be a much weaker mutagen in the Ames test. The compound induced mutations in strains TA97, TA98 and TA100 of S. typhimurium but only in the presence of metabolic activation.Comparison of the structure of the studied flavonoids with their mutagenic activity indicates that the mutagenicity of flavonoids is dependent on the presence of hydroxyl groups in the 3′ and 4′ positions of the B ring, and that the presence of a free hydroxy or methoxy group in the 7 position of the A ring also probably contributes to the appearance of mutagenic activity of flavonoids in the Ames test. It also appeared that the presence of methoxy groups, particularly in the B ring of the flavonoid molecule, markedly decreases the mutagenic activity of the compound.     

Mutagenicity testing of condensates of smoke from titanium dioxide/hexachloroethane and zinc/hexachloroethane pyrotechnic mixtures

Condensates of smoke from titanium dioxide/hexachloroethane and zinc/hexachloroethane pyrotechnic mixtures were investigated for their potential to produce genetic damage in the tester strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimurium and in the mouse bone marrow micronucleus assay. Both smoke condensates contained several chlorinated hydrocarbons among which tetrachloroethylene, hexachloroethane, hexachlorobutadiene and hexachlorobenzene were identified by GC/MS. Condensate of smoke from titanium dioxide/hexachloroethane showed a dose-related positive response in the Salmonella assay with strains TA98 and TA100 in the absence of metabolic activation from rat liver S9 fraction. Both smoke condensates were negative in the micronucleus assay but produced a small but significant depression of erythropoietic activity. The results indicate that smoke condensate from titanium dioxide/hexachloroethane mixtures contains unidentified compound(s) that may be considered mutagenic in the Salmonella assay.     

Evaluation of the mutagenic potential of mycotoxins using Salmonella typhimurium and Saccharomyces cerevisiae.

The mutagenic effects of fiteen mycotoxins on Salmonella typhimurium strains TA1535, TA1537 and TA1538 and Saccharomyces cerevisiae strain D-3 were tested. Only aflatoxin B1 and sterigmatocystin were mutagenic. Both were active against S. typhimurium strain TA1538 and S. cerevisiae strain D-3; however, both required activation by the hepatic S-9 enzyme preparation. A positive correlation between the other mycotoxins reported to be carcinogenic and the two in vitro test systems employed was not demonstrated in our hands.     

Evaluation of mutagenic and antimutagenic activities of alpha-bisabolol in the Salmonella/microsome assay

alpha-Bisabolol (BISA) is a sesquiterpene alcohol found in the oils of chamomile (Matricaria chamomilla) and other plants. BISA has been widely used in dermatological and cosmetic formulations. This study was undertaken to investigate the mutagenicity and antimutagenicity of BISA in the Salmonella/microsome assay. Mutagenicity of BISA was evaluated with TA100, TA98, TA97a and TA1535 Salmonella typhimurium strains, without and with addition of S9 mixture. No increase in the number of his+ revertant colonies over the negative (solvent) control values was observed with any of the four tester strains. In the antimutagenicity assays, BISA was tested up to the highest nontoxic dose (i.e. 50 and 150 microg/plate, with and without S9 mix, respectively) against direct-acting (sodium azide, SA; 4-nitroquinoline-N-oxide, 4-NQNO; 2-nitrofluorene, 2-NF; and nitro-o-phenylenediamine, NPD) as well as indirect-acting (cyclophosphamide, CP; benzo[a]pyrene, B[a]P; aflatoxin B1, AFB1; 2-aminoanthracene, 2-AA; and 2-aminofluorene, 2-AF) mutagens. BISA did not alter mutagenic activity of SA and of NPD, and showed only a weak inhibitory effect on the mutagenicity induced by 4-NQNO and 2-NF. The mutagenic effects of AFB1, CP, B[a]P, 2-AA and 2-AF, on the other hand, were all markedly and dose-dependently reduced by BISA. It was also found that BISA inhibited pentoxyresorufin-o-depentylase (PROD, IC50 2.76 microM) and ethoxyresorufin-o-deethylase (EROD, 33.67 microM), which are markers for cytochromes CYP2B1 and 1A1 in rat liver microsomes. Since CYP2B1 converts AFB1 and CP into mutagenic metabolites, and CYP1A1 activates B[a]P, 2-AA and 2-AF, results suggest that BISA-induced antimutagenicity could be mediated by an inhibitory effect on the metabolic activation of these promutagens.     

Genotoxic activity of potassium permanganate in acidic solutions

Potassium permanganate (KMnO4) combined with sulfuric acid is a strongly oxidizing mixture which has been recommended for the destruction and the decontamination of various mutagens/carcinogens in the publication series of the International Agency for Research on Cancer. Evaluation of the genotoxicity of 4 potassium permanganate solutions was performed using a microtechnique of the Ames test with the tester strains TA97, TA98, TA100 and TA102 with and without metabolic activation. Presence of direct-acting mutagens was detected in all the samples with the tester strain TA102 without S9 mix (163-357 revertants/microliters of the solutions). Three samples containing either acetone or ethanol as an organic solvent also induced a mutagenic response on tester strain TA100 without S9 mix (167-337 revertants/microliters). In addition, DNA damage in human peripheral blood lymphocytes was also measured for one of the mixtures by a new technique: the single-cell gel assay (SCGA). A sample with no organic solvent induced DNA damage in human lymphocytes with a dose-response relationship as determined by SCGA. The major mutagenic agent generated by the permanganate solutions was found to be manganese ion (Mn2+). Both manganese sulfate (MnSO4) and manganese chloride (MnCl2) gave mutagenic dose-response relationships on tester strain TA102 without S9 mix. The mutagenic potencies were 2.8 and 2.4 revertant/nmole for MnSO4 and MnCl2 respectively. MnCl2 also induced DNA damage in human lymphocytes as determined by the SCGA. The genotoxic effects of KMnO4 in acidic conditions were probably mediated by the conversion of MnO4- to Mn2+. KMnO4 in alkaline solutions did not produce mutagenic species and may offer an alternative for the degradation of genotoxic compounds.     

Absence of mutagenic activity of benorylate,paracetamol and aspirin in the Salmonella//mammalian microsome test

Benorylate and its two major hydroyssis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.     

Mutagenicity of N-acetyl and N,N'-diacetyl derivatives of 3 aromatic amines used as epoxy-resin hardeners

3 epoxy-resin hardeners, 4,4'-diaminodiphenyl ether (DDE), 4,4'-diaminodiphenylmethane (DDM), and 4,4'-diaminodiphenylsulfone (DDS), and their N-acetyl and N,N'-diacetyl derivatives were examined for their mutagenicity using Salmonella typhimurium TA98 and TA100 as the tester stains and an S9 mix containing a rat-liver 9000 X g supernatant fraction as the metabolic activation system. DDE and DDM were mutagenic towards TA98 and TA100 in the presence of S9 mix while DDS exhibited no significant mutagenic activity towards these tester strains. These epoxy-resin hardeners were metabolized in vivo and their N-acetyl and N,N'-diacetyl metabolites were found in the urine. Among these acetyl metabolites, only N-acetyl-DDE was found to be mutagenic towards TA98 and TA100 in the presence of S9 mix. None of these acetyl metabolites exhibited significant mutagenic activity towards these tester strains in the absence of S9 mix.     

Potential cytotoxic and mutagenic effect of Pinus wallichiana,Daphne oleiodes and Bidens chinensis

The Pinus wallichiana, Daphne oleiodes and Bidens chinensis have a long history of being used traditionally for treatment of various types of disorders. Most of the uses have been without any scientific evidence and toxicological assessment. We evaluated the mutagenic and cytotoxic capabilities of various parts of P. wallichiana, D. oleoides and B. chinensis. Ames Salmonella mutagenicity assay determined the mutagenicity activity against TA 98 and TA 100 bacterial strains of Salmonella typhimurium without metabolic activator S9 system. The number of mutant colonies in negative control was considered as limit to determine the mutagenicity effects of every extract. Brine shrimps lethality bioassay was used to determine the cytotoxic capabilities of the selected plants. The P. wallichiana, D. oleiodes and B. chinensis did not showed any mutagenic activity both for frameshift mutation (TA98) and base-pair substitution (TA100) without S9 mixture. The crude methanolic extract of P. wallichiana stem showed moderate cytotoxicity (53.33%) at 1000 μg/ml with LD₅₀ value 599.634. The D. oleoides fruit showed a toxicity of 60% at 1000 μg/ml with LD₅₀ value 367.730. The B. chinensis (whole plant) showed lethality of 63.3% at 1000 μg/ml, with LD₅₀ 204.833. The absence of any mutagenic activity of crude extract of the tested plants in both bacteria strains, TA 98 and TA 100 without the S9 mix confirms the safety of these plants to the consumers.     

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.     

Mutagenicity evaluation of HC Blue No. 1 and HC Blue No. 2. III. Effects in the Salmonella typhimurium/Escherichia coli reversion assay and the mouse lymphoma L5178Y TK+/- forward mutation assay

The hair-dye ingredients, HC Blue No. 1 (HCB1) and HC Blue No. 2 (HCB2), were tested for the induction of bacterial mutation using Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100; and Escherichia coli strains WP2uvrA-. In addition, both dyes were evaluated in the mouse lymphoma L5178Y TK+/- assay (MLA) for the potential to induce forward mutation. A liver homogenate (S9) prepared from Aroclor 1254-induced male Fischer 344 rats was used to provide a means for metabolic activation. HCB1 was not mutagenic in the Ames assay, but was weakly mutagenic in the MLA only in the presence of metabolic activation. In contrast, HCB2 was a strong mutagen in the Ames assay in tester strain TA98 both in the presence and absence of metabolic activation. A positive response was also noted with HCB2 in the MLA, both in the presence and absence of metabolic activation. Negative findings from the Ames assay of this study agree with other published results where an identical lot of HCB1 was used. Using the same lot, a weak positive result was observed in the MLA, however, the activation requirements and magnitude of the response were different from that of a lot evaluated by the NTP. In contrast, HCB2 appears to be both a bacterial and mammalian cell mutagen independent of lot variability.     

A comparison of aflatoxin B1-induced cytotoxicity,mutagenicity and prophage induction in Salmonella typhimurium mutagen tester strains TA1535, TA1538, TA98 and TA100

Treatment of Ames mutagen tester strains with aflatoxin B1 (AFB1) and S9 mix results not only in the production of a poten mutagen, but induces a pathway that leads to the induction of prophages present in all Ames tester strains.Characterization of the prophage induction and mutagenic response following AFB1 treatment showed that plasmid pKM101 dramatically enhances mutagenesis, but suppressed prophage induction. Spontaneous release of phage by TA98 and TA100 was also lower than in TA1535 and TA1538.In addition to mutagenesis and prophage induction, survival of all 4 tester strains was quantitated after AFB1 treatment. The data show that the frameshift tester strains (TA1538 and TA98) are more sensitive to the bactericidal action of AFB1 than the base-pair tester strains (TA1535 and TA100), survival being significantly affected above 100 ng. One of several hypotheses examined was the difference in the number and types of prophages present in base-pair tester strains that are not detectable in the frame-shift tester strains.These data suggest that prophage induction can detect DNA damage that is non-mutagenic; and that it is important to characterize the lysogenic nature of the Ames strains since it may influence the observed histidine revertant rate and the survival of the tester strain.     

Absence of mutagenic activity of benorylate, paracetamol and aspirin in the Salmonella/mammalian microsome test

Benorylate and its two major hydrolysis products, paracetamol and aspirin were examined for mutagenicity in the Salmonella/mammalian microsome screening test. The compounds were tested in 6 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100, TA97 and TA98) in the presence and absence of a rat-liver microsome activation system. Benorylate did not show evidence of mutagenic activity in the 6 strains tested with or without metabolic activation at concentrations ranging from 0.006 to 3 mg per plate. Paracetamol and aspirin likewise did not show any evidence of mutagenic activity at concentrations ranging from 0.1 to 50 mg per plate for the former and 0.01 to 50 mg per plate for the latter.     

Mutagenic effect of furocoumarin monoadducts and cross-links on bacteriophage lambda

The mutagenicities of 17 closely related oxiranes were determined in 4 tester strains (Salmonella typhimurium TA98, TA100, TA1535, TA1537). The test compounds comprised all possible oxides of benzene and its partially hydrogenated congeners. In TA100 and TA1535, 12 of the tested oxiranes were weak to moderate mutagens. 4 of these were also active in TA98. No mutagenicity was observed with the remaining 5 compounds in any of the 4 strains.The presence of a double bond in formal conjugation with the epoxide ring increased the mutagenicity relative to that of the saturated oxirane. Interestingly, additional epoxide rings within the same molecule did not markedly increase the mutagenic activity, and for the oxiranes that are not activated by a double bond, the relationship between mutagenic activity and the number of epoxide rings in the molecule was even inverse.The influence of bromo and hydroxyl substitution on oxirane mutagenicity is discussed. Most notably, a compound having a 4-hydroxyl group in syn position to a 1,2-epoxide ring fused to the cyclohexane ring, a structure which has been suggested to increase the electrophilic reactivity of dihydrodiol epoxides through hydrogen bonding, was almost inactive.     

Mutagenic activity of nine N,N-disubstituted hydrazines in the Salmonella/mammalian microsome assay.

The mutagenic activity of N,N-dimethyl-, N,N-diethyl-, N,N-dibutyl-, N,N-diisobutyl-, N,N-di(p-tolyl)-, N-ethyl-N-phenyl-, N,N-dibenzyl-, N,N-diphenyl- and N,N-diisopropylhydrazine was examined in the Salmonella/mammalian microsome assay using the strains TA1535, TA1537, TA97, TA98, TA100, TA102 and TA1530. All nine hydrazines were mutagenic in at least one tester strain, although of borderline significance for some of the compounds. The mutagenic potencies of the hydrazines varied 2-3 orders of magnitude, from very weak to moderate mutagenic activity. In general, the addition of S9 resulted in a lowering of the mutagenic activity and a lowering of the toxic properties of the hydrazines. The test results were relatively difficult to evaluate due to toxic effects of many of the test compounds on the test bacteria which may have resulted in an underestimation of the mutagenic potencies of some of the compounds. The pattern of mutagenic activity of the hydrazines in the different tester strains indicates that more than one mechanism of action may be involved in the mutagenicity.     

Study of promutagen biotransformation in the Ames test. III. The role of conjugation with glucuronic acid and sulfate in the modification of mutagenic effect of nitrosomorpholine, diethylnitrosamine and cyclophosphamide

The role of reactions of conjugation with uridine diphosphoglucuronic acid (UDPGA) and with 3-phosphoadenosine-5-phosphosulfate (PAPS) in modification of the mutagenic effect of diethyl nitrosamine (DENA), nitrosomorpholine (NM) and cyclophosphane (CP) was studied by the Ames test. It was shown that adding UDPGA to the activating mixture significantly decreased the level of the mutagenic effect of DENA, NM and CP on bacteria Salmonella typhimurium TA 1950, when S9 and microsomal fractions of rat liver homogenate were used. Adding PAPS to the activating mixture when S9 and cytosole fractions were used, did not affect mutagenic action of DENA on S. typhimurium TA 1950 and TA 1535, enhancing the mutagenic effect of CP on TA 1535, with no such influence on TA 1950. Introduction of PAPS into the activating mixture elevated the mutagenic effect of NM on both bacterial strains using S9 fraction but not cytosole fraction.     

Structural determination of a directly mutagenic amino-nitrobiphenyl as the S9 metabolite of 2,4,2′,4′-tetranitrobiphenyl in Salmonella typhimuriumTA98

In order to elucidate the mechanisms of mutagenic activation of nitrobiphenyls by mammalian activation systems, 2,4,2′,4′-tetranitrobiphenyl was incubated with S9 and its mutagenic metabolites were separated by SiO₂ and Al₂O₃ column chromatography. The most mutagenic diamino-dinitrobiphenyl was isolated from the reaction mixture of 2,4,2′ ,4′-tetranitrobiphenyl with S9 mix at 37°C for 48 h, and its mutability was 4646 revertants/50 ng in Salmonella typhimurium TA98 without S9 mix. The deamination product of this most mutagenic metabolite was identical to 2,4′-dinitrobiphenyl by gas chromatography-mass spectrometry. Therefore, the structure of the metabolite was determined as 2,4′-diamino-2′,4-dinitrobiphenyl by its chemical and physico-chemical properties.     

Genotoxicity of 6 oxime compounds in the Salmonella/mammalian-microsome assay and mouse lymphoma TK+/− assay

To aid in the selection of chemical candidates for in vivo tests, the mutagenicity of 6 oxime compounds was evaluated in the Salmonella plate incorporation assay and mouse lymphoma L5178Y TK^+/− assay. All of the oximes were mutagenic in the mouse lymphoma assay in the absence of exogenous metabolic activation. Acetaldehyde oxime was also mutagenic in the presence of S9 activation. In contrast to these results, a positive response was noted only for 2-(hydroxyimino)-N-phenyl-acetamide oxime in strain TA1535 in the absence of activation in the Salmonella/microsome test.