Vitotox Test Research Articles

Mutagenic and antimutagenic evaluation of Asparagus laricinus Burch., Senecio asperulus DC., and Gunnera perpensa L. to hepatic cells

Introduction. The use of traditional medicinal plant concoctions to cure or treat different diseases daily in African folk medicine. However, the effects of most medicinal plants on human health or genetic material remain unknown. This study thus aimed to evaluate the mutagenic and antimutagenic potentials of Asparagus laricinus Burch. cladodes, Senecio asperulus DC., and Gunnera perpensa L. roots extract in vitro. Methods. Neutral red uptake assay, alkaline comet assay, and the VITOTOX test was used with plant extract dilutions of 4, 20, 50, and 100 µg/ml, respectively, on hepatic (C3A) cells and Salmonella Typhimurium TA104 strains. Ethyl methane-sulfonate and 4-nitroquinoline oxide were used as positive controls for the comet and VITOTOX assays, respectively. Results. In vitro cytotoxicity and genotoxicity were not observed from all tested extracts, except for the two dichloromethane (DCM) extracts of S. asperulus and G. perpensa, which appeared to be cytotoxic with S9 metabolic activation, but not genotoxic or mutagenic. From the VITOTOX test results, none of the extracts appeared to have antimutagenic properties after treating S. Typhimurium strains with a known mutagen. Conclusions. These results confirm that previously reported anticarcinogenic properties of A. laricinus, S. asperulus, and G. perpensa did not result from the protective mechanism against genotoxicity but from other ones. Moreover, the negative mutagenic and cytotoxic activities of the tested plants highlighted the safe use of these medicinal plants in vitro. Therefore, S. asperulus and G. perpensa DCM extracts require further investigation for their possible in vivo cytotoxic effects on humans.

Phytochemical screening, antioxidant, anticoagulant and in vitro toxic and genotoxic properties of aerial parts extracts of Fumaria officinalis L. growing in Tunisia

Fumaria is an important plant species with many applications in herbal medicine. In this paper we report on the investigation of the phytochemical composition, antioxidant, anticoagulant and in vitro genotoxic properties of Fumaria officinalis L. which is amongst others widely used in Tunisia. Petroleum ether, ethyl acetate, chloroform and methanol extracts were made and investigated with respect to their total phenolics and flavonoids contents using standard methods. Antioxidant activity was evaluated by DPPH and ABTS methods. The anticoagulant activity was evaluated by prothrombin time (PT) and activated partial thromboplastin time (aPTT) tests. In vitro toxic and genotoxic properties with respectively the Neutral red uptake method and the bacterial Vitotox test and comet assay in human C3A cells. This study showed that methanolic extract had the highest total phenolic and flavonoids contents. The same extract showed surprising anticoagulant and antioxidant activities. The Vitotox test revealed absence of genotoxicity for all extracts in the bacterial Vitotox test. This was confirmed in the comet assay which was conducted on human C3A cells, except for the methanolic extract where DNA damage appeared to be significantly increased at nontoxic concentrations. Thus, showing the potential of the plant as a new source of bioactive molecules for therapeutic purposes, with particular emphasis on the treatment of cardiovascular diseases.

Phytochemical analysis, antioxidant, anticoagulant and in vitro toxicity and genotoxicity testing of methanolic and juice extracts of Beta vulgaris L.

Beet root (Beta vulgaris L.) is well known in cuisine and is also used in traditional medicine to prevent many diseases. We investigated the methanolic extract and juice with respect to the phenolic and flavonoid contents and evaluated their antioxidant, anticoagulant and genotoxic activities. Genotoxicity was studied with the bacterial Vitotox test and the alkaline comet assay involving human C3A liver cells. The methanolic extract had the highest phenolic and flavonoid contents with values of 39.75 mg GAE/g and 20.73 mg CE/g extract, respectively. Methanolic extract and juice showed surprising anticoagulant and antioxidant activities. They were devoid of any genotoxicity or cytotoxic effects. The results show the potential of the plant, especially the juice, as a new source of bioactive molecules for therapeutic purposes, possibly in the treatment of cardiovascular diseases.

Antimutagenic constituents from Monanthotaxis caffra (Sond.) Verdc.

Monanthotaxis caffra (Sond.) Verdc. (Annonaceae) has been reported to possess antitumoural properties. Preliminary screening showed that the crude methanolic leaf extract had strong antimutagenic effects against aflatoxin B1 -induced mutagenicity. The aim of this study was to isolate and evaluate the antimutagenic properties of the active constituents from M. caffra. Different chromatographic, spectroscopic and spectrometric techniques were used for the isolation and identification of the antimutagenic constituents. The antimutagenic effect of the extract and compounds was evaluated using Ames, Vitotox and Comet assays. Bioassay-guided fractionation of the methanolic leaf extract yielded two antimutagenic compounds identified as (+)-crotepoxide and 5,6-diacetoxy1-benzoyloxymethyl-1,3-cyclohexadiene. Crotepoxide had strong antimutagenicity in the Vitotox assay with an IC50 value of 131 μg/ml. 5,6-Diacetoxy-1-benzoyloxymethyl-1,3-cyclohexadiene showed strong antimutagenic activity in the Ames assay with an IC50 value of 348.9 μg/plate and no antimutagenic activity in the Vitotox test. Furthermore, the compound was able to inhibit, block or prevent biotransformation of aflatoxin B1 by repressing the proteins involved in transcription. Crotepoxide and 5,6-diacetoxy-1-benzoyloxymethyl-1,3-cyclohexadiene have the potential to mitigate the risks arising from consumption of aflatoxin B1 -contaminated food and feed.

UPLC-MS/MS analysis of antibiotics in pharmaceutical effluent in Tunisia: ecotoxicological impact and multi-resistant bacteria dissemination.

The UPLC MS/MS analysis showed the presence of the two antibiotics in the pharmaceutical industry discharges during 3 months; norfloxacin and spiramycin which were quantified with the mean concentrations of 226.7 and 84.2ngmL-1, respectively. Sixteen resistant isolates were obtained from the pharmaceutical effluent and identified by sequencing. These isolates belong to different genera, namely Citrobacter, Acinetobacter, Pseudomonas, Delftia, Shewanella, and Rheinheimera. The antibiotic resistance phenotypes of these isolates were determined (27 tested antibiotics-discs). All the studied isolates were found resistant to amoxicillin and gentamicin, and 83.33% of isolates were resistant to ciprofloxacin. Multiple antibiotic resistances were revealed against β-lactams, quinolones, and aminoglycosides families. Our overall results suggest that the obtained bacterial isolates may constitute potential candidates for bioremediation and can be useful for biotechnological applications. Genotoxic effects were assessed by a battery of biotests; the pharmaceutical wastewater was genotoxic according to the bacterial Vitotox test and micronuclei test. Genotoxicity was also evaluated by the comet test; the tail DNA damages reached 38 and 22% for concentrated sample (10×) and non-concentrated sample (1×), respectively. However, the histological sections of kidney and liver's mice treated by pharmaceutical effluent showed normal histology and no visible structural effects or alterations as cytolysis, edema, or ulcerative necrosis were observed. Residual antibiotics can reach water environment through wastewater and provoke dissemination of the antibiotics resistance and induce genotoxic effects.

In vitro toxicity and genotoxic activity of aqueous leaf and fruit extracts of Ruscus hypophyllum L.

Ruscus hypophyllum L. is a rare Mediterranean plant which is used in the traditional medicine. We studied its phenolic content and in vitro toxicity and genotoxicity using the neutral red uptake (NRU) test, the bacterial Vitotox test, and the comet assay in human C3A hepatic cells. Aqueous leaf and fruit extracts were investigated. Antigenotoxicity against 4-nitroquinoline-oxide (4NQO, 0.4 µg/mL) and Benzo(α)pyrene (BaP, 800 µg/mL) was also investigated with the Vitotox test. The extracts appeared to be genotoxic only at high exposure levels in the comet assay. There was no indication of a genotoxic activity in the Vitotox test and also no indication of antigenotoxicity. The moderate polyphenol content may provide an explanation for the absence of antigenotoxicity.

In vitro Toxicity and Genotoxic Activity of Aqueous Leaf Extracts From Four Varieties of Olea europea (L).

Aim:Despite its therapeutic value almost nothing is known about potential adverse health effects of Olea europea L. We therefore investigated the in vitro toxicity and genotoxicity of leaf extracts of this plant.Material and Methods:Extracts from olive tree leaves were obtained from four different regions in Tunisia. We investigated the in vitro toxicity, genotoxicity and antigenotoxicity of their aqueous extracts using the neutral red (NR) uptake, Vitotox and alkaline comet assays.Results:None of the extracts were found to be toxic and none of them were genotoxic, although some doubt exists for the extract obtained at Meski (North of Tunisia). On the basis of the Vitotox test only, none of the extracts appeared to have antigenotoxic (or cogenotoxic) properties.Discussion:The negative genotoxicity underline the safe use of the leaves, for example, as hypoglycemic and antidiabetic preparations. Lack of antigenotoxicity may indicate that the previously reported anticancer effects do not result from protection against genotoxicity.HIGHLIGHTS We investigated the in vitro toxicity and genotoxicity of aqueous extracts of olivesThe neutral Red Uptake test, Vitotox and alkaline comet assay were usedLeaf extracts from 4 different origins were investigatedNone of them showed in vitro toxicity or genotoxicityThe extracts also didn’t have antigenotoxic properties Abbreviation list: BaP: benzo(α)pyrene, EMS: ethyl methane sulfonate, LMP: low melting point, NI50: 50% inhibition of NRU, NR: neutral red, NRU: neutral red uptake, OD: optical density, PBS: phosphate buffer saline, SDS: sodium dodecyl sulphate, S/N: signal to noise ratio, 4NQO: 4-nitroquinoline oxide

Phytochemical and in vitro antimicrobial and genotoxic activity in the brown algae Dictyopteris membranacea

The emergence and increase of microbial resistance towards many conventional therapeutics necessitate the investigation and discovery of new natural sources of antimicrobial agents. In this study the Mediterranean brown alga, Dictyopteris membranacea, was investigated. The total phenolic, flavonoid and tannin contents were quantified and antibacterial, antifungal and antitubercular activities of the crude extract and semi-purified ethanol, acetone and methanol/CH2Cl2 fractions were determined. The antimicrobial activity was determined by the agar diffusion method against six strains of Gram-positive, two strains of Gram-negative bacteria and one yeast strain. The MIC and MBC or MFC values served to identify the bactericidal and bacteriostatic effects of all prepared fractions against the different strains. The acetone fraction presented a source of phenolic (112mgGAE/g of sample) and tannin (130mgCAE/g sample) compounds whereas the ethanol fraction showed the highest concentration of flavonoids (120mgQE/g). Ethanol and acetone fractions exhibited considerable antibacterial, antifungal and antitubercular activities with a good correlation between the phytochemical composition of all prepared fractions and the different biological activities.This study on the phytochemical and in vitro antimicrobial activity of this brown alga was supplemented by an in vitro investigation of the potential genotoxicity of the extract and fractions using the bacterial Vitotox test and micronucleus test on human C3A cells. The results show that all prepared fractions were not genotoxic in the presence or absence of S9.Overall, the results are therefore encouraging and justify further investigations on the therapeutic and other values of this alga.

Co-exposure of ELF-magnetic fields and chemical mutagens: An investigation of genotoxicity with the SOS-based VITOTOX test in Salmonella typhimurium

It is believed that extreme low frequency magnetic fields (ELF-MF) are not mutagenic, at least at exposure levels below 100μT. Synergistic or co-operative effects with environmental mutagens remain possible yet. We therefore investigated the effects of ELF-MF in conjunction with 4 different well known chemical mutagens having different modes of action. For this purpose the bacterial Vitotox test was used. Our study confirmed previous results which showed that a 100μT magnetic field (50Hz) does not damage DNA and hence is not mutagenic in this assay and that there was also no influence on the DNA damaging capacity of the used mutagens.

Phytochemical Composition and Potential Genotoxic effects of Important Egyptian Medicinal Plants

Introduction: We performed a phytochemical analysis and investigated the potential genotoxic properties of ten different methanol extracts of plants that are frequently used in Egypt for medicinal purposes. Such studies are important because these plants were never thoroughly investigated with respect to their possible potential sideeffects and risks for humans. Materials and Methods: In this study we used the bacterial Vitotox test and alkaline comet assay in human C3A cells to estimate their genotoxic potential. The Vitotox test is an interesting screening test which correlates very well with the well-known Ames assay but has the advantage of being rapid, sensitive and requiring only small amounts of a test compound. The test was conducted in the presence and absence of a metabolizing S9-enzyme fraction. The comet assay is now a widely used and validated genotoxicity test which can be applied in all DNA-containing cells. In this study it was conducted in human C3A cells which conserved phase I and II biotransformation capabilities. The in vitro NRU assay was used to investigate toxicity and utilized as a dose-finding test. Results: None of the plants have shown genotoxic properties although one of them, Derris rubusta, showed borderline genotoxicity in both tests. This plant also contains alkaloids and coumarins, besides flavonoids, carbohydrates, tannins and triterpenes that were also found in the other plants. Conclusion: So far all 10 investigated medicinal plants appeared not genotoxic but due to borderline effects, Derris robusta deserves further complementary investigations.

Toxic and mutagenic properties of extracts from Tunisian traditional medicinal plants investigated by the neutral red uptake, VITOTOX and alkaline comet assays

We investigated the genotoxic properties of a number of extracts from Tunisian traditional medicinal plants with the bacterial VITOTOX test in Salmonella typhimurium and the alkaline comet assay in human C3A cells. Ethyl acetate and methanol extracts from Marrubium alysson L. and Retama raetam (Forsk.) Webb and methanol extracts from Peganum harmala L. were investigated. Toxicity was furthermore studied with the neutral red uptake test that served for dose-finding. All extracts showed antigenotoxic properties against 4-nitroquinoline-oxide (4-NQO) and benzo(α)pyrene in the VITOTOX test, except the methanol extracts from R. raetam where antigenotoxicity was not found against the mutagen 4-NQO (in the absence of S9). The ethyl acetate extract from R. raetam was found mutagenic with the VITOTOX test in the absence of S9, whereas both ethylacetate and methanol extracts of M. alysson L. induced DNA damage according to the alkaline comet assay in C3A cells.

Genotoxicity investigation of ELF-magnetic fields in Salmonella typhimurium with the sensitive SOS-based VITOTOX test

We performed a genotoxicity investigation of extremely low-frequency (ELF) magnetic fields (MFs, 50 Hz, 100 and 500 µT, 1 and 2 h exposure) alone and in combination with known chemical mutagens using the VITOTOX test. This test is a very sensitive reporter assay of Salmonella typhimurium bacteria based on the SOS response. Our study showed that ELF-MFs do not induce SOS-based mutagenicity in S. typhimurium bacteria and do not show any synergetic effect when combined with chemical mutagens.

Assessing the cytotoxic and mutagenic effects of secondary metabolites produced by several fungal biological control agents with the Ames assay and the VITOTOX® test

The potential genotoxic effects of several pure secondary metabolites produced by fungi used as biological control agents (BCAs) were studied with the Ames Salmonella/microsome mutagenicity assay and the Vitotox test, with and without metabolic activation. A complete set of Salmonella tester strains was used to avoid false negative results. To detect possible mutagenic and/or cytotoxic effects of fungal secondary metabolites due to synergistic action, crude extracts and fungal cell extracts of the BCAs were also examined. Although the sensitivity of the methods varied depending on the metabolite used, clearly no genotoxicity was observed in all cases. The results of the two assays are discussed in the light of being used in a complementary fashion for a convincing risk-assessment evaluation of fungal BCAs and their secondary metabolites.

Evaluation of Genotoxicity of Medicinal Plant Extracts by the Comet and VITOTOX Tests

We report the results of our genotoxic evaluation of extracts from three medicinal plants Acacia nilotica, Juglans regia, and Terminalia chebula and the herbal drug Triphala employing the VITOTOX and comet tests.These tests detect DNA damage in prokaryotic and eukaryotic test systems, respectively. In the VITOTOX test, none of the extracts were identified as genotoxic. In the comet assay, extracts of Acacia nilotica showed statistically significant DNA damage only in a concentration of 2500 ppm (highest tested dose), whereas extracts from Juglans regia showed significant damage in concentrations above 250 ppm and more. Extracts from Terminalia chebula and Tripahala significantly increased DNA damage in a concentration above 500 ppm. This is not considered contradictory, because DNA damage in the alkaline comet assay may not be permanent and hence may not necessarily result in mutations. All the extracts were previously found in the Ames assay to have potent antimutagenic effects against the direct acting mutagens NPD, sodium azide, and the S9-dependent mutagen 2-AF. The results of the previous study using the Ames assay are in conformity with those of the VITOTOX test. It was found that the extracts were safe in concentrations of up to 1000 microg/0.1 mL and 2500 microg/0.1 mL. A literature survey also showed that plant extracts can be mutagenic as well as antimutagenic depending on the test system used. This indicates that a battery of assays is needed before any conclusion can be reached.

EILATox-Oregon Workshop: blind study evaluation of Vitotox test with genotoxic and cytotoxic sample library.

In order to assess the robustness, sensitivity and specificity of a recently developed Vitotox test, 17 blind coded chemicals and three environmental water samples were tested at the EILATox-Oregon Workshop using the Thermo Electron Vitotox kit. The Vitotox test is a rapid geno- and cytotoxicity test using standard 96- or 384-well microtitre plates. The genotoxicity test is based on two genetically modified Salmonella typhimurium strains containing bacterial luciferase operon from Vibrio fisheri under the SOS inducible promoter. The SOS system is an inducible network in Escherichia coli that responds to DNA damage and activates DNA repair. The Vitotox genotoxicity test bacteria strain carries bacterial luciferase genes under the control of SOS inducible promoter and therefore any DNA damage inside the cells induces the production of bacterial luciferase. The luciferase expression is then followed with a microtitre plate luminometer for 3 h after mixing different dilutions of sample with the test bacteria. The genotoxicity index is calculated for each dilution and the genotoxicity of the sample is interpreted based on kinetic time curves and genotoxicity vs concentration/dilution curves. Cytotoxicity of the sample is determined simultaneously with another test strain containing the same luciferase operon controlled by the constitutive promoter. This bacterium produces constant bioluminescence and any decrease of the bioluminescence production is used as a marker for cytotoxicity. As a miniaturized microtitre plate assay the Vitotox test requires a very small quantity of the sample material. The samples used in the workshop were diluted 1 : 10 or 1 : 100 before testing. Genotoxicity and cytotoxicity data were collected at dilutions of 1 : 10-1 : 2000. When the samples of the EILATox-Oregon Workshop were tested using the Vitotox test, four coded chemicals out of 17 were determined to be genotoxic. Seven chemicals and one environmental sample were found to be cytotoxic. Three chemical samples were found to be both geno- and cytotoxic.

Genotoxicity of PM 10 and extracted organics collected in an industrial, urban and rural area in Flanders, Belgium

The variation in the genotoxic potency of PM 10 in vitro in relation to the particle source type was investigated. Particles were collected at one urban, one rural, and one industrial site in Flanders. Genotoxicity was assessed using four different in vitro test systems exposed to PM 10 in suspension and to the organic extracts of PM 10. Two of these systems were bacterial assays: the Salmonella mutagenicity test and the Vitotox test. In addition, the Comet assay and Micronucleus test were performed using human blood cells. Results show that exposure to PM 10 and the organic extracts from both urban and industrial areas causes significant genetic damage. The Salmonella mutagenicity test was most suitable for the screening of PM 10 and the organic extracts; the Micronucleus test was most suitable only for the screening of organic extracts, and original particles were toxic for the exposed lymphocytes. Clear dose–response curves were not established in the Comet and Vitotox assay, and organic extracts were apparently toxic in the latter. The total polycyclic aromatic hydrocarbon content of the organic extracts, as measured with GC/MS, ranged between 1 and 6 ng/m 3. Results obtained in this study suggest that PM 10 causes DNA damage and mutations. The use of biological tests for the screening of air samples is useful to complement air quality control by chemical measurements.

Genotoxicity of melanoidin fractions derived from a standard glucose/glycine model.

Genotoxic compounds can act at various levels in the cell (causing gene, chromosome, or genome mutations), necessitating the use of a range of genotoxicity assays designed to detect these different types of mutations. The production of melanoidins during the processing and cooking of foods is associated with changes in their nutritional character, and the discovery of mutagenic substances in pyrolyzed protein and amino acids has raised concern about the safety of these foods. The aim of this work was to test melanoidin fractions in three different in vitro assays (Ames test, Vitotox test, and micronucleus test). These melanoidin fractions were produced from the condensation of glucose with glycine and their separation was conducted by dialysis. The crude reaction mixture (before dialysis) and both the LMW and HMW fractions obtained by dialysis showed no genotoxicity in these assays, despite being tested at concentrations much higher than those naturally found in food products. The LMW fraction, however, showed toxicity at these high concentrations. The volatile fraction produced in this reaction showed genotoxicity only in the Vitotox test, at high concentrations.

Evaluation of the Vitotox test as a high-throughput genotoxicity assay

The VitotoxTM test is a high-throughput bacterial genotoxicity test based on the SOS DNA-repair system induced by genotoxic compounds. Two genetically engineered Salmonella typhimurium strains are used in this system, TA104recN2-4 (Genox strain), that contains the bacterial luciferase (lux) operon (luxCDABE) under transcriptional control of recN promoter, and TA104 pr1 (Cytox strain), that constitutively expresses lux operon.The performance of the VitotoxTM test was evaluated with 33 known Ames positive chemicals, 26 known Ames negatives and 18 drug candidates developed at Mitsubishi Pharma Corporation. Ten compounds had inconclusive results because they caused SOS-independent enhancement of light emission. Among 49 known chemicals with conclusive results, 89% of the Ames positive compounds were detected as positive (genotoxic) with the VitotoxTM test, and all of the Ames negative compounds were detected as negative. There was a 94% concordance between the Ames test results and the VitotoxTM test results.In a practical validation study using 18 drug candidates developed at Mitsubishi Pharma Corporation, 7 of 8 Ames positive compounds were detected as genotoxic and all of the Ames negative compounds gave negative results with the VitotoxTM test. The concordance between the VitotoxTM test results and the Ames test results for 18 drug candidates was 94% (17/18). Moreover, the VitotoxTM test required a smaller sample quantity than the Ames test to detect genotoxicity.The present results indicate that the VitotoxTM test is useful for rapid screening of large numbers of chemicals when only a small quantity of a chemical is available.

VITOTOX� bacterial genotoxicity and toxicity test for the rapid screening of chemicals

The VITOTOX test is a new bacterial genotoxicity test that was previously shown to be very rapid and sensitive. Initially only one Salmonella typhimurium strain (TA104 recN2-4) was used in the test. In this paper we introduce a second strain (TA104pr1) that can be used as an internal control to further enhance the reliability of the test. We demonstrate the usefulness of this pr1 strain in genotoxicity and toxicity testing. We also report on the results of a study where the VITOTOX test was performed on newly synthesized pharmaceutical compounds, or intermediate products in the synthesis of drug candidates. We demonstrate that the test gives identical results when performed independently in two different laboratories and that it correlates well with either the Ames test or SOS chromotest.

The VITOTOX® test, an SOS bioluminescence Salmonella typhimurium test to measure genotoxicity kinetics

A new test to detect genotoxicity, that we refer to as the VITOTOX® test, was developed. Four gene fusions that are based on the Escherichia coli recN promoter were constructed and evaluated for their SOS response-dependent induction. The wild-type recN promoter, a derivative mutated in the second LexA binding site, a derivative with a mutated −35 region, and a derivative from which both the second LexA binding site and the −35 region were mutated, were cloned upstream of the promoterless Vibrio fischeri luxCDABE operon of pMOL877, in such a way that lux became under transcriptional control of the recN promoter derivatives. The inducibility by the SOS response of the promoter constructs was tested in both E. coli and in the Ames test Salmonella typhimurium strains TA98, TA100 and TA104. In all strains, the highest sensitivity and induction was observed with the plasmids pMOL1067 and pMOL1068, that contain the lux operon under control of the recN promoter mutated in the second LexA binding site, or a recN promoter with a mutated −35 region, respectively. Therefore, strains containing pMOL1067 or pMOL1068 were further used for genotoxicity testing. With the VITOTOX test, genotoxicity was detected within 1–4 h. The VITOTOX test is very sensitive: for most products tested, the minimal detectable concentration (MDC) values were considerably lower (5 to >100 times) than those described for the Ames test and the SOS chromotest. A good correlation was observed with the results from the Ames tests, but certain PAHs that are not mutagenic in the Ames test were genotoxic in the VITOTOX test. With the VITOTOX strains, the kinetics of SOS induction can be determined. This feature made it possible to distinguish between compounds in mixtures of genotoxic products so long as they had different induction kinetics.