Screening of medicinal plants used in South African traditional medicine for genotoxic effects

Dichloromethane and 90% methanol extracts from 51 South African medicinal plants were evaluated for potential genotoxic effects using the bacterial Ames and VITOTOX tests with and without metabolic activation. Dichloromethane extracts from bulbs of Crinum macowanii showed mutagenicity in strain TA98 with and without metabolic activation, whereas extracts from leaves of Chaetacme aristata and foliage of Plumbago auriculata showed mutagenicity and/or toxicity. Extracts from the leaves of Catharanthus roseus and twigs of Combretum mkhzense were mutagenic with metabolic activation only. The only 90% methanol extracts that were mutagenic in strain TA98 were from the leaves of C. roseus and Ziziphus mucronata in the presence of metabolic activation. No genotoxic effects were found in strain TA100 or in the VITOTOX test.     

Assessment of toxicity and mutagenicity in air particulate matter from an urban industrial area in the coast of the Rio de la Plata

Chemical characterization and effects assessment of semivolatile organic compounds in organic extracts from air particulate matter from the region of the greater La Plata area was undertaken. Effects covered the study of mutagenicity with the Ames test (Salmonella typhimurium TA100 and TA98 strains with metabolic activation by S9) and cytotoxicity using the Tetrahymena pyriformis test system (growth rate, cell volume, and cell respiration). Chemical analysis of organic extracts was done using gas chromatography. Results demonstrate the presence of polycyclic aromatic hydrocarbons (PAH) in the matrix, high mutagenicity, and cytotoxicity. A higher mutagenic activity detected with TA98 and TA100 strains is associated with an increment of total PAH and total five or six ring PAH content, respectively. Linked with it, a PAH dependent toxicity on Tetrahymena pyriformis has been observed. This cell system proved to be very sensitive. From the results obtained with the cell respiration assay with T. pyriformis it appears that uncoupling agents are present in the samples. The results of this study indicate that air particulate matter from the Rio de La Plata area contains significant genotoxic and cytotoxic activity probably due to the presence of PAHs.     

An evaluation of acetone extracts from six plants in the AMES mutagenicity test

Acetone extracts from six plants were evaluated for mutagenic activity with the Salmonella/mammalian-microsome mutagenicity test (Ames) utilizing tester strains TA98 and TA100 and in the presence and absence of induced rat liver microsomes. Extracts from alfalfa (Medicago sativa), lettuce (Lactuca sativa) and thread-leaf groundsel (Senecio longilobus) produced only negative responses. Comfrey (Symphytum officinale) and tansy ragwort (Senecio jacobaea) extracts produced toxic responses that were abolished in the presence of the microsomal bioactivation system. Bracken (Pteridium aquilinum) and tansy ragwort extracts produced positive responses following bioactivation with the liver microsomal system. The results suggest that the Ames mutagenicity test may be of some value in initial evaluations for potential toxic effects of plants consumed by animals and man.     

Mutagenic activity of the feces of rats following oral administration of tartrazine

After oral administration of the azo dye tartrazine, bile and feces of treated rats were investigated for mutagenicity using the Ames test withSalmonella typhimurium strains TA 98 and TA 100 with and without metabolic activation. In the presence of S 9-mix fecal extracts developed a weak but reproducible dose-related response in strain TA 100. In bile no metabolites exerting mutagenic activity were found.     

Mutagenic activity promoted by amentoflavone and methanolic extract of Byrsonima crassa Niedenzu

Byrsonima crassa is a plant pertaining to the Brazilian central savannah-like belt of vegetation and popularly used for the treatment of gastric dysfunctions and diarrhoea. The methanol extract contains catechin, tannins, terpenes and flavonoids; both mutagenic potential and antioxidant properties have been ascribed to flavonoids. The mutagenicity of some flavonoids is believed to be associated with the formation of reactive oxygen species and seems to depend on the number and position of hydroxyl groups. In the present study the mutagenic activity of the methanol, chloroform and 80% aqueous methanol extracts, as well as acetate and aqueous sub-fractions, of this medicinal plant were evaluated by Salmonella typhimurium assay, using strains TA100, TA98, TA102 and TA97a, and in mouse reticulocytes. The results showed mutagenic activity of the methanolic extract in the TA98 strain without S9, but no mutagenicity to mouse cells in any of the extracts. The acetate fraction showed strong signs of mutagenicity without S9, suggesting that in this enriched fraction were concentrated the compounds that induced mutagenic activity. The aqueous fraction showed no mutagenic activity. The TLC and HSCCC analyses of the acetate fraction with some standard compounds permitted the isolation of the quercetin-3-O-beta-D-galactopyranoside, quercetin-3-O-alpha-L-arabinopyranoside, amentoflavone, methyl gallate and (+)-catechin, of which only the amentoflavone exhibited positive mutagenicity to TA98 (+S9, -S9).     

Mutagenic activity in roadside soils

Mutagenicity of soils sampled at median strips, roadsides and a park neighboring arterial roads in Kurume City was determined by Ames test. Organic extracts of soils were mutagenic in strains TA98, TA100, YG1041 and YG1042 with and without S9mix. No sample showed mutagenic responses in strains YG3003 or YG7108. Extracts from soils of median strips and beside intersections showed higher mutagenicity and concentrations of PAHs and heavy metals than others, and mutagenic activity of soils correlated significantly with concentrations of PAHs and heavy metals. However, PAHs accounted for less than 12% of total mutagenicity in strains TA98 and TA100 of soil extracts. These extracts showed much higher mutagenicity in YG strains than in TA strains. The results indicate that these soils may be polluted with nitroarenes and aromatic amines.     

The Salmonella mutagenicity of water and sediments from the Porsuk River in Turkey

In this study, water and sediments from the Porsuk River were investigated for their potential mutagenicity in Salmonella typhimurium TA 98 and TA 100 strains by performing Ames test (plate incorporation assay) without metabolic activation. Different columns of XAD 4 and XAD 16 were used to fractionate the water samples. Positive results in XAD 4 extracts of water samples were obtained for TA 98 in two of these stations. Extracts of the sediment samples were assayed in five different doses of concentrations and mutagenic results were obtained for both of the two strains in different sampling sites. Total metal concentrations in the stream sediment samples were determined by atomic absorption spectrophotometry (AAS) in order to explain some of these mutagenic results. The presence of mutagens causing frameshift and base-pair substitution mutations in water and sediments of the Porsuk River was suggested by the results and this mutagenicity was discussed.     

Mutagenicity and DNA-damaging activity caused by decomposed products of potassium sorbate reacting with ascorbic acid in the presence of Fe salt.

Although potassium sorbate (PS), ascorbic acid and ferric or ferrous salts (Fe-salts) are used widely in combination as food additives, the strong reactivity of PS and oxidative potency of ascorbic acid in the presence of Fe-salts might form toxic compounds in food during its deposit and distribution. In the present paper, the reaction mixture of PS, ascorbic acid and Fe-salts was evaluated for mutagenicity and DNA-damaging activity by means of the Ames test and rec-assay. Effective lethality was observed in the rec-assay. No mutagenicity was induced in either Salmonella typhimurium strains TA98 (with or without S-9 mix) or TA100 (with S-9 mix). In contrast, a dose-dependent mutagenic effect was obtained when applied to strain TA100 without S-9 mix. The mutagenic activity became stronger increasing with the reaction period. Furthermore, the reaction products obtained in a nitrogen atmosphere did not show any mutagenic and DNA-damaging activity. PS, ascorbic acid and Fe-salts were inactive when they were used separately. Omission of one component from the mixture of PS, ascorbic acid and Fe-salt turned the reaction system inactive. These results demonstrate that ascorbic acid and Fe-salt oxidized PS and the oxidative products caused mutagenicity and DNA-damaging activity.     

The mutagenic, DNA-damaging and antioxidative properties of bark and leaf extracts from Coutarea hexandra (Jacq.) K. Schum

Coutarea hexandra is a species commonly known in Brazil as quina, and its bark is used in folk medicine. In this study, we assess the mutagenic and DNA-damaging effects of ethanol extracts from C. hexandra stem bark (SCH) and leaves (LCH) by employing the Ames test on the TA98 and TA100 strains of Salmonella typhimurium in addition to a plasmid treatment test. Furthermore, we performed a phytochemical analysis by TLC and HPLC, a quantification of the phenolic constituents and an assessment of the antioxidative activity. SCH and LCH showed mutagenic action in the Ames test for TA98 strains after metabolic activation. LCH also showed mutagenicity for the TA100 strain after metabolic activation. The findings from the plasmid treatment test did not indicate any DNA-damaging activity for either of the extracts with the tested dosages. SCH showed greater flavonoid content and greater antioxidative potential in relation to LCH. This study suggests that caution is advisable in the use of this plant. However, in vivo studies should be conducted to confirm these data.     

Use of four short-term tests to evaluate the mutagenicity of municipal water

Some ways in which four short-term tests may be used to evaluate the mutagenicity of drinking water were explored by testing raw and treated water from Lake Bloomington, which serves the town of Bloomington, Illinois (population, 44,000). The water was collected from February 1976 to October 1977 and was concentrated by evaporation or by use of XAD-2 resin. The water was tested for the ability to induce reverse mutation in a prokaryote, Salmonella typhimurium; forward mutation in a mold, Neurospora crassa; mitotic gene conversion in a yeast, Saccharomyces cerevisiae; and reverse mutation in maize, Zea mays. Because of the large number of water samples (54) and the limited amounts of the samples, it was not possible to test all samples in all four tests by all the protocols. Thus, the sensitivities of the four tests to potential mutagens in the water samples could not be rigorously compared. However, the results do show that lake and tap water samples collected during 1976 were toxic but not mutagenic in N. crassa and neither toxic nor genotoxic in S. cerevisiae; lake water collected during 1977 was mutagenic in one line of Z. mays and slightly mutagenic in S. typhimurium strain TA1536 in the presence of rat liver S9. The results suggest that tests that detect a variety of genetic end points should be used when testing complex mixtures such as drinking water. The advantages and disadvantages of the tests and protocols are discussed in terms of their applicability to the study of the mutagenicity of drinking water.     

A comparative study, with 40 chemicals, of the efficiency of the Salmonella assay and the SOS chromotest (kit procedure)

A comparison was made, for 40 compounds belonging to different chemical classes, of the mutagenicity as measured by the Salmonella assay and of the SOS-inducing potency as measured by the SOS chromotest kit procedure. It was found that most (78%) of the chemicals described as mutagens/carcinogens (14 compounds) were detected with a simplified Ames test procedure, using 3 strains (TA 97, TA 98, TA 100) and 3 concentrations of the tested material. The SOS chromotest, carried out following the recommendations of the commercially available kits, revealed that only 4 Ames test-positive compounds were mutagenic towards E. coli strain PQ 37.     

Mutagenicity and toxicity studies of p-phenylenediamine and its derivatives

The mutagenicity of p-phenylenediamine and its derivatives was tested using Ames Salmonella strains TA98 and TA100. p-Phenylenediamine was weakly mutagenic to TA98 with metabolic activation. 2-Nitro-p-phenylenediamine was directly mutagenic to both strains, while 2-methyl-p-phenylenediamine required S9 mix. All the test compounds induced a dose-related increase in chromosomal aberrations m Chinese hamster ovary (CHO) cells in the absence of the S9 mix. The mutagenicity and toxicity of these compounds did not correlate with their oxidation potentials, or any other tested physicochemical properties including the energy difference between the lowest unoccupied and the highest occupied molecular orbital, ionization potential, and dipole moment.     

Tofisopam--evaluation of mutagenic and genotoxic properties

The mutagenic properties of tofisopam, the member of the 2,3-benzodiazepine family, were evaluated on the basis of Ames test with Salmonella typhimurium TA1537, TA97, TA98, TA100 and TA102 strains. The genotoxic properties of tofisopam were estimated on L929 cell line with the cytokinesis-block technique. Under the experimental conditions, no mutagenic activity of tofisopam in tester bacteria strains was found, and no genotoxic activity was observed.     

Contribution of coffee aroma constituents to the mutagenicity of coffee

About 40 coffee aroma constituents belonging to the classes of dicarbonyls, sulphur-containing compounds, furfuryls, N-heterocyclics and others were systematically evaluated in three Ames tester strains. Only aliphatic dicarbonyl compounds showed notable direct mutagenic activity, which mainly affected 'base-pair substitution' in Ames tester strains TA100 and TA102. Very weak effects were also seen with some N-heterocyclics, mainly affecting frameshift tester strain TA98 upon metabolic activation. However, it was shown that these N-heterocyclics do not contribute substantially to the mutagenicity in coffee. The hydrogen peroxide and methylglyoxal contents of coffee were determined up to 26 hr after preparation. Their concentrations tended to decrease whereas mutagenic activity decreased significantly with time in tester strains TA100 and TA102. It is concluded that several highly labile coffee constituents contribute to the bacterial mutagenicity and also that the synergism between hydrogen peroxide and methylglyoxal is not the main factor. The absence of coffee mutagenicity/carcinogenicity in rodents with these highly reactive coffee aroma compounds can be explained in part by detoxification of microsomal enzyme systems.     

A reactive metabolite of furan, cis-2-butene-1,4-dial, is mutagenic in the Ames assay

Furan is classified as a nongenotoxic hepatocarcinogen. It is thought to be activated to a toxic metabolite, cis-2-butene-1,4-dial, which is acutely toxic to liver cells. The resulting cytotoxicity is followed by compensatory cell proliferation, increasing the likelihood of tumor production. We examined the genotoxic activity of cis-2-butene-1,4-dial in several strains of Salmonella typhimurium commonly used in the Ames assay. This reactive compound tested positive in TA104, a strain that is sensitive to aldehydes. Mutagenic activity was concentration-dependent (1000 +/- 180 revertants/micromol). Incubation of cis-2-butene-1,4-dial with glutathione prior to addition of bacteria inhibited both the acute toxic and genotoxic activity of this compound. No evidence of mutagenic activity was seen at nontoxic concentrations in TA97, TA98, TA100, and TA102. Our findings are consistent with the hypothesis that cis-2-butene-1,4-dial reacts with DNA to form mutagenic adducts. Our data suggest that cis-2-butene-1,4-dial may be an important genotoxic as well as toxic intermediate in furan-induced tumorigenesis.     

Assessment of mutagenic activity in drinking water from São Paulo City,Brazil

During the last decade, studies carried out in several countries throughout the world have disclosed the presence of halogenated hydrocarbons and other potentially carcinogenic compounds in drinking water, causing public concern with respect to the quality of drinking water. Considering this problem, chlorinated drinking water from seven water treatment plants in São Paulo and from its water sources were tested for mutagenicity in the Ames/Salmonella assay. Different volumes of raw and treated waters were concentrated by XAD-2 resin, and eluted in methanol and methylene chloride. Organic extracts ressuspended in DMSO were assayed for dose response in Salmonella typhimrium strains TA98 and TA100, with and without metabolic activation, using the plate incorporation technique. Results showed higher incidence of mutagenic activity in water samples after chlorination than in raw water. The majority of positive responses were decreased by addition of metabolic activation, confirming the presence of direct-acting mutagens, produced during water chlorination, although significant mutagenicity was also observed in the presence of S9. Mutagenic levels ranged from 104 to 569 revertants/L in raw water, and in chlorinated drinking water, between 10 and 418 revertants/L. Based on the results obtained in this study, it is essential to study and identify mutagenic compounds evolving from the chlorination processes, and to determine, for water treatment plants with high incidence of mutagenic compounds, the procedures to avoid or eliminate this problem, as well as to establish an effective monitoring program.     

Genotoxicity assessment through the Ames test of medicinal plants commonly used in Brazil

Ten medicinal herbs commonly used as popular medicine in Brazil—Bauhinia forficata L., Bauhinia variegata L., Cymbopogon citratus D.C. Stapf, Echinodorus macrophyllum (Kunth) Micheli, Hidrocotyle asiatica L, Matricaria chamomila L., Pfaffia iresinoides (Kunth) Sprengel, Plaffia paniculata (Martius) Kuntze, Phyllanthus tenellus Roxb, and Solidago microglossa D.C.—were tested for mutagenicity by the Ames test using Salmonella typhimurium TA 97a, TA 98, TA 100, and TA 104 strains, with and without metabolic activation. The genotoxicity assessment of these medicinal plants was performed in aqueous extracts 1:5. Seventy percent of these herbs presented mutagenic effects with at least one of the Ames strains used in this study. Bauhinia variegata L., E. macrophyllum K., and M. chamomilla L. showed no mutagenic activity. The mutagenic effects were detected mainly with the strains TA 98 related to frameshift mutations. The higher mutagenicity ratio was obtained with S. microglossa D.C. (known as arnica-do-Brazil) when TA 98 strain was used with metabolic activation (MR = 6.55) and with TA 97a strain with and without the addition of S9. Medicinal plants are now used by all the segments of the population, more intensively in the last years. These results indicate the need to establish rules to assess the safety of the use of medicinal herbs. © 1994 by John Wiley & Sons, Inc..     

Safety evaluation of water extracts of Toona sinensis Roemor leaf.

The purposes of this study were to evaluate the safety of water extracts of Toona sinensis Roemor leaf (TSL-1). The mutagenic properties of TSL-1 was investigated using the Ames test, and no mutagenicity was found toward all tester strains (Salmonella typhimurium TA98, TA100, TA102 and TA1535). In the acute oral toxicity study, a single limit dose of 5000 mgTSL-1/kg bw was given to male and female ICR mice, then observed for a 14-day period. In the subacute study, TSL-1 was administered as oral daily dose of 1000 mg/kg bw/day for 28 days. The results showed no acute lethal effect at a maximal tested dose of 5000 mg/kg bw TSL-1 in male and female mice. The subacute toxicity showed the oral administration of 1000 mg/kg bw for consecutive 28 days was safe in male mice. TSL-1 treated female mice showed decreases of food intake and kidney relative weight in acute oral toxicity test, and decreases of body weight gain, food intake and lung relative weight in subacute toxicity trial. However, no remarked toxic effects were found in the biochemical and histopathological parameters of TSL-1 treated female mice. These effects whether related to the major components or other ingredients in TSL-1 need to elucidate in the further studies.     

Effect of pyrolysis temperature on the mutagenicity of tobacco smoke condensate.

Tobacco smoke aerosols with fewer mutagens in the particulate fraction may present reduced risk to the smoker. The objective of this study was to test the hypothesis that the temperature at which tobacco is pyrolyzed or combusted can affect the mutagenicity of the particulate fraction of the smoke aerosol. Tobacco smoke aerosol was generated under precisely controlled temperature conditions from 250 to 550 degrees C by heating compressed tobacco tablets in air. The tobacco aerosols generated had a cigarette smoke-like appearance and aroma. The tobacco smoke aerosol was passed through a Cambridge filter pad to collect the particulate fraction, termed the smoke condensate. Although condensates of tobacco smoke and whole cigarette mainstream smoke share many of the same chemical components, there are physical and chemical differences between the two complex mixtures. The condensates from smoke aerosols prepared at different temperatures were assayed in the Ames Salmonella microsome test with metabolic activation by rat liver S9 using tester strains TA98 and TA100. Tobacco smoke condensates were not detectably mutagenic in strain TA98 when the tobacco smoke aerosol was generated at temperatures below 400 degrees C. Above 400 degrees C, condensates were mutagenic in strain TA98. Similarly, condensates prepared from tobacco smoke aerosols generated at temperatures below 475 degrees C were not detectably mutagenic in strain TA100. In contrast, tobacco tablets heated to temperatures of 475 degrees C or greater generated smoke aerosol that was detectably mutagenic as measured in TA100. Therefore, heating and pyrolyzing tobacco at temperatures below those found in tobacco burning cigarettes reduces the mutagenicity of the smoke condensate. Highly mutagenic heterocyclic amines derived from the pyrolysis of tobacco leaf protein may be important contributors to the high temperature production of tobacco smoke Ames Salmonella mutagens. The relevance of these findings regarding cancer risk in humans is difficult to assess because of the lack of a direct correlation between mutagenicity in the Ames Salmonella test and carcinogenicity.     

Genotoxicity and mutagenicity of melanoidins isolated from a roasted glucose-glycine model in human lymphocyte cultures, intestinal Caco-2 cells and in the Salmonella typhimurium strains TA98 and TA102 applying the AMES test.

Melanoidins are formed during household cooking procedures and are part of our daily diet, but data on their toxicological potential are still scarce. Therefore, the mutagenic, cytotoxic and genotoxic activity of the water soluble total fraction (sol A), the water soluble high molecular weight fraction (HMW; Molecular weight>12,400 Da) and the remaining water soluble low molecular weight fraction (LMW) isolated from a glucose-glycine model system roasted at 125 degrees C was comprehensively studied in human lymphocytes (genetic end point: sister chromatid exchange (SCE)), Caco-2 cells (SCE, cell viability, cell proliferation) and in the Salmonella typhimurium strains TA98 and TA102 (Ames test). Tests were performed in a dose- and time-dependent manner. The results indicate a significant increase in SCE formation in human lymphocytes after the exposure to 0.05% and 0.1% of the melanoidin fractions. In Caco-2 cells, only the exposure to LMW increased the SCE formation as a matter of concentration. Cell's proliferation and viability decreased significantly after exposure to melanoidins. In the AMES test, melanoidins did not show a mutagenic potential, neither using the TA98 nor the TA102 strain. These results show that melanoidins isolated from the glucose-glycine mixture exhibited modest but significant genotoxic effects in human lymphocytes and, in particular the LMW, in Caco-2 cells, but they induce neither in low nor in very high concentrations mutagenicity in bacteria strains.