Genotoxic and clastogenic activity of saponins extracted from Nauclea bark as assessed by the micronucleus and the comet assays in Chinese Hamster Ovary cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Bark extracts of Nauclea latifolia, Nauclea diderrichii, Nauclea pobeguinii and Nauclea vandergutchii are used in traditional medicine in West and South Africa for the treatment of fevers, diarrhea and malaria. AIM OF THE STUDY: To estimate the possible long-term toxicity and genotoxicity of plant extracts (dichloromethane, methanol, water/methanol, water) and saponins. MATERIALS AND METHODS: The clastogenicity of plant extracts and saponins was assessed by the micronucleus assay performed on Chinese Hamster Ovary cells. The DNA-damaging activity of saponin mixture was assessed by the comet assay on Chinese Hamster ovary cells. RESULTS: Hydromethanolic extracts from Nauclea latifolia, Nauclea diderrichii and Nauclea pobeguinii exhibited a significant clastogenic/aneugenic activity without S9 mix. The hydromethanolic extract from Nauclea diderrichii was the most clastogenic/aneugenic fraction with a Minimal Active Concentration (MAC) of 23.1 µgm L(-1). It was submitted to a separation step leading to six main saponins identified as quinovic acid glycosides (saponins A, D, E, G, J, K). None of the isolated saponins exerted a significant clastogenic/aneugenic activity by the micronucleus assay, however a mixture made with equal quantities of each of the six saponins exhibited a direct genotoxic/clastogenic activity as assessed by both the micronucleus assay and the comet assay on Chinese Hamster Ovary cells. CONCLUSION: Saponins present in the hydromethanolic extracts of Nauclea induced synergistic in vitro DNA-damage and chromosome mutations in mammalian cells. This genotoxic activity was probably due to the capacity of Nauclea saponins to reduce cell defense against oxidative stress through the inhibition of glutathione-S-transferase activity.

Evaluation of the mutagenicity and antimutagenicity of forty-two 3-substituted flavones in the Ames test.

The mutagenic and antimutagenic activities of forty-two synthetic flavones were assessed by the Ames test. The tested flavones included twenty-three 3-nitroflavones, eighteen 3-aminoflavones and the 3-chloroflavone. The mutagenicity was evaluated with Salmonella typhimurium TA100 and YG1042 (an overproducing nitroreductase and O-acetyltransferase TA100 strain) with and without metabolic activation (S9 mix). The antimutagenicity of the non mutagenic derivatives was evaluated against 11 known reference mutagens. A total of 39 synthetic flavones were mutagenic. The mutagenic activities ranged from 0.1 rev/nmole (4'-chloro-6-methoxy-3-nitroflavone) to 6240 rev/nmole (4'-methoxy-3, 3'-diaminoflavone). Two differences were found between the 3-amino and the 3-nitroflavones: (i) the mutagenicity of the 3-aminoflavones required the presence of the metabolic activation; (ii) the 3-amino derivatives were more mutagenic than their 3-nitro counterparts. Increased mutagenicity, as assessed with strain YG1042, was limited to 17/39 derivatives. The mutagenic activity was induced by the presence of the double bond at the 2,3-position for conjugation of the lone-pair electron with the carbonyl group on the 'C' ring. This mutagenicity was modulated by substituents at the 2'-position. Additional mutagenicity was brought by the aminoaromatic and nitroaromatic group reduction by bacterial nitroreductases and by the S9 mix; it was modulated by different substituents on the aromatic rings of the flavones. Three flavones: 3-chloroflavone (1C), 4'-hydroxy-3-nitroflavone (23N) and 2',3-diaminoflavone (2A) showed antimutagenic properties. Compound 1C was efficient against benzo(a)pyrene (BaP), 2-aminofluorene (2AF), 2-aminoanthracene (2AA), 4-nitroquinoline-1-oxide (4NQO) and 1-methyl-3'-nitro-1-nitrosoguanidine (MNNG). Compound 23N inhibited the mutagenicity of BaP and MNNG. The antimutagenic activity of 2A was limited to MNNG.

Evaluation of the genotoxic activity of metronidazole and dimetridazole in human lymphocytes by the comet assay.

The genotoxicity of metronidazole (MZ) and dimetridazole (DZ) has been evaluated in human lymphocytes using the comet assay. The test has been performed using 3 doses (58.4, 175.2 and 292.1 microM for MZ; and 70.9, 212.6 and 354.3 microM for DZ) under 3 experimental protocols: aerobiosis, anaerobiosis (90% N2, 10% CO2) and with the presence of the microsomal fraction S9 mix. The effects of 4 antioxidants (8-hydroxyquinoline (8HQ), vitamin C (VitC), catalase (CAT) and superoxide dismutase (SOD), have been investigated on DNA damage generated by fixed concentrations of MZ (292.1 microM) and DZ (354.4 microM). In aerobic conditions, MZ and DZ produced significant dose-response relationships. The dose-related effects of both drugs decreased or were abolished in anaerobic conditions or in presence of S9 mix. 8HQ, VitC, CAT and SOD induced dose-related protective responses against DNA damage due to MZ and DZ. These findings suggest that MZ and DZ induce DNA damage in human lymphocytes through the futile cycle. The one-electron reduction of the drugs leads to the production of nitro radical anions. In the presence of oxygen, these radicals are reoxidized and generate oxygen-activated species.

The Salmonella sulA-test: a new in vitro system to detect genotoxins.

The Salmonella sulA-test is a newly developed colorimetric assay to detect genotoxins. This technique is based on the ability of DNA-damaging agents to induce the sulA gene, one of the SOS response genes. A constructed plasmid, pEM1968, carrying a fused sulA'::'lacZ was introduced into Salmonella typhimurium TA1538. Monitoring sulA gene expression was performed by assaying the beta-galactosidase activity in the transformed strain S. typhimurium TA1538/pEM1968. A simple, fast and sensitive liquid incubation procedure has been developed after optimization of the S9 mix composition and beta-galactosidase assay. The SOS-inducing potency (SOSIP, microM-1) was defined as the slopes of the non-linear dose-response relationships. Twenty-one chemicals with different modes of action were examined for a preliminary evaluation of the test. Nineteen chemicals were genotoxic in the Salmonella sulA-test. The SOSIP ranged from 1.2 x 10(-4) microM-1 (ethyl methanesulfonate) to 419.9 microM-1 (bleomycin). Sodium azide and 5-fluorouracil were not genotoxic. Frameshift, base-pair and oxidative genotoxins were detected by the tester strain. The calculated SOSIP and the minimum concentrations detected (MCD) in the Salmonella sulA-test were compared to the reported values obtained with two similar assays: the SOS Chromotest and umu-test. The SOSIP values of 12 compounds were the highest in this new assay. Five chemicals tested in the Salmonella sulA-test gave similar SOSIP values with those of one of the two other tests. ICR-191 had the highest SOSIP with the SOS Chromotest and 3-methylchloranthrene showed the highest SOSIP with the umu-test. Similarly, the lowest MCD values were found for 12 compounds in the Salmonella sulA-test. Four compounds had close MCD values in this assay and one of the two other techniques. The SOS Chromotest remained the most sensitive assay for cisplatin and ICR 191. The umu-test was the technique of choice for 3-methylchloranthrene.

[The Ames test: application to smokers' urine]. / Le test d'Ames: application aux urines de fumeurs.

The Ames test is a rapid and sensitive in vitro technique for detecting mutagens by using Salmonella typhimurium hypersensibilized strains. We applied this assay to the study of smoker's urine mutagenicity. Eighteen smokers and 18 non-smokers were investigated. Statistical analysis showed a significant increase of the smoker's urine mutagenicity. The Ames test could be used to determine the mutagenicity level of smokers. Moreover, the results suggest the applicability of the Ames test to urines of ex-smokers and passive smokers.

[A new mode of expression for the assessment of capacities of DNA repair by flow cytometry]. / Nouveau mode d'expression pour l'évaluation des capacités de réparation de l'ADN par cytométrie en flux.

Flow cytometry technic was used to study DNA synthesis of Hep G2 cells following mitomycin C and adriblastine treatments during 24 hours. DNA synthesis was expressed by 2 methods: the new expression global DNA synthesis (S+G2)/G1 that considered the cells during scheduled and unscheduled DNA syntheses of S and G2 phases and the cell cycle (Fox program) that evaluated the cells during scheduled DNA synthesis by the terms G1 = 2n, S = 2n+x and G2 = 4n which excluded unscheduled DNA synthesis. The experimental data treated with this new expression led to the determination of threshold concentrations for the two tested compounds where the DNA repair mechanisms were overloaded, leading to cell death. This term was shown to be more accurate to describe the genotoxic action of compounds. Furthermore, these threshold concentrations of DNA damages was found to be linked with significant increase of micronuclei in the micronucleus test.

The micronucleus assay in human lymphocytes: screening for inter-individual variability and application to biomonitoring.

Micronuclei levels were assessed in cytokinesis-blocked lymphocytes of 200 male and female healthy donors not occupationally exposed to genotoxic risks and of 33 male industrial painters handling genotoxic substances. Frequency of micronucleated cells was 9.87 +/- 3.1 per 1000 in the control population and was shown to have a large inter-individual variability. The study of factors contributing to this variability showed that only smoking could affect micronucleated cell rate, inducing an increase of 25%, whereas age and sex had no effect. Among the industrial painters, frequency of micronucleated cells averaged 18.30 +/- 7.39 per 1000: the difference between the two populations studied was shown to be statistically significant by the Mann-Whitney rank sum test (one-sided U test) and indicated that exposed painters need preventive measures.

The single cell gel electrophoresis assay (comet assay): a European review.

The single cell gel electrophoresis (SCGE) assay is a rapid, simple, visual and sensitive technique for measuring DNA breakage in individual mammalian cells. Here we review the development of the SCGE assay (with particular reference to the alkaline version), existing protocols for the detection and analysis of comets, the relevant underlying principles determining the behaviour of DNA, and the potential applications of the technique.

[Assay of micronuclei in binucleated T-lymphocytes. Analysis of the distribution and variation factors in a population of 100 subjects]. / Le test de numération des micronoyaux dans les lymphocytes T binucléés. Analyse de la répartition et des facteurs de variation sur une population de 100 sujets.

The authors analysed micronuclei levels distribution in lymphocytes of 100 non occupationally exposed subjects and studied the effect of age, sex and smoking of donors on the distribution. Results showed that micronucleated cells were distributed according to a normal distribution (average = 9.5 +/- 4 micronucleated cells in 1,000 binucleated lymphocytes). Age and sex of donors had no effect on the distribution but, concerning smoking, the results showed that micronuclei levels were correlated to the number of cigarettes daily smoked.

[The mode of action of a nonpolyenic antifungal (desertomycin) produced by a strain of Streptomyces spectabilis]. / Recherches sur le mode d'action d'un antifongique non polyénique (désertomycine) produit par une souche de Streptomyces spectabilis.

A metabolite with antifungal activity, of non polyenic macrolide structure, was extracted and purified from the culture supernatant of a soil-isolated Streptomyces spectabilis strain, BT 352. This product was found to be related to (or being) desertomycin. Six yeast and five filamentous fungus strains were used to determine minimum concentration of the metabolite that inhibits growth by 80% (IMC); it was established at 50 micrograms/mL for the fungi and at 100 micrograms/mL or more for the yeasts tested. Short-term genotoxicity tests showed no antifungal effect on the bacterial genome, and desertomycin at concentration levels of 100 micrograms/mL or more affected protein synthesis. The antifungal metabolite had no immediate inhibiting effect upon yeast respiration, even at high concentrations; however, the respiration activity of cells grown in the presence of subinhibiting doses and collected during their growth phase was reduced by as much as 40%. Saccharomyces uvarum spheroplast regeneration in a liquid medium containing desertomycin was inhibited at doses fivefold weaker than the IMC determined with intact cells. Contrary to amphotericin B, desertomycin subinhibiting doses do not modify, and if so lightly, the yeast latent phase or the spheroplast wall regeneration phase, thus indicating a fungicidal action. Moreover, following a 30-min contact with desertomycin subinhibiting and inhibiting doses, yeasts liberated potassium in large amounts, indicating that plasma membranes were affected.

Applicability of the SOS Chromotest to detect urinary mutagenicity caused by smoking.

The mutagenicity of urine obtained from five cigarette smokers was investigated using two bacterial assays: the Ames test and the SOS Chromotest. Urinary mutagens were extracted on Amberlite XAD-2 resin. Four urine samples showed activity towards Salmonella typhimurium tester strain TA98 with S9 mix while no SOS-inducing activity could be measured with Escherichia coli strain PQ37 in the SOS Chromotest. Using factorial design and a positive control benzo[a]pyrene (BaP), the concentration of S9, nicotinamide adenine dinucleotide phosphate (NADP) and glucose-6-phosphate (G6P) were optimized (2%, 0.5 mM and 10 mM respectively) for the SOS Chromotest. The SOS-inducing power of BaP was 1.42/nM with the standard S9 mix and 3.26/nM with the optimized S9 mix. B buffer and the age of L-broth were found to decrease the sensitivity of beta-galactosidase assays in the SOS Chromotest. A 4000-fold urine concentrate from a smoker was finally tested using the Ames test and the modified SOS Chromotest. Mutagenic and toxic activities were found toward tester strain TA98 (+S9 mix) showing that the SOS Chromotest is not at present suitable for assaying urinary mutagens in the presence of an in vitro metabolic activating mixture.

Handling of cytostatic drugs and urine mutagenesis.

As part of a French national epidemiologic study on human reproduction among hospital personnel, we investigated urine mutagenicity of nurses and personnel from oncology units exposed to cytostatic drugs. During a first series of experiments, urine mutagenicity of 47 subjects working in six oncology units was investigated in the Marseille regional's hospital. A control group of 37 individuals working in one cardiology clinic was also included. Urinary mutagens were extracted on XAD-2 resin and tested by two bacterial mutagenicity tests: the Ames test with tester strains Salmonella typhimurium TA 97, TA 98, TA 100 and TA 102 with or without metabolic activation (S9 MiX) and the SOS Chromotest with tester strain Escherichia coli PQ 37-S9 MIX. Bactericidal activity towards the tester strains was found in 40% of the urine samples (36/90). During a second series of experiments, urine mutagenicity of 17 office clerks was also investigated. Toxicity was found in six of the 21 urine samples. No significant difference of toxicity distribution and no relationship between toxicity and cigarette smoking were found. Qualitative analysis of the data showed no significant difference among the exposed groups and the control group (Chi 2 = 0.529, df = 2) with tester strain TA 98 + S9 MIX. Cigarette smoking was found to be the main factor of increased urinary mutagenicity (Chi 2 = 0.529, df = 1). Quantitative analysis of the data showed that mutagenic potencies varied from 0.332 +/- 0.539 revertants/mg creatinine to 7.226 +/- 6.743 revertants/mg creatinine with TA 98 + S9 MIX.(ABSTRACT TRUNCATED AT 250 WORDS)

Urine mutagenicity of steel workers exposed to coke oven emissions.

Urine mutagenicity of 19 individuals was investigated at a steel mill. All the subjects worked on the coal processing unit. Urine samples were collected at the end of a working day. Urine samples of two exposed workers were collected at the end of two periods of rest and two periods of working. Mutagens were extracted on XAD-2 resin and tested by the Salmonella microsomal assay and the SOS spot test. Mutagenic potencies of exposed smokers and exposed non-smokers were 8.62 +/- 6.56 and 1.1 +/- 0.48 revertants/mg creatinine respectively with Salmonella typhimurium strain TA98 + S9. Both values were significantly higher than those of unexposed smokers and non-smokers (5.07 +/- 3.33 and 0.47 +/- 0.72 revertants/mg creatinine respectively). The urinary mutagenic potency of the two exposed individuals increased at the end of periods of working (15.97 +/- 2.57 revertants/mg creatinine) and decreased at the end of periods of rest (12.31 +/- 2.45 revertants/mg creatinine). Urinary mutagens were detected with S. typhimurium strain TA100 + S9 to a lesser extent. No direct-acting mutagens were detected by the SOS spot test. Atmospheric benzo[a]pyrene (BaP) were also measured by h.p.l.c. on the coke battery. BaP concentrations ranged between 0.01 and 0.6 microgram/m3 air at the different working sites. Biological monitoring with short-term tests is discussed.