Toxicity and genotoxicity of antimalarial alkaloid rich extracts derived from Mitragyna inermis O. Kuntze and Nauclea latifolia.

The toxicity and the genotoxicity of antimalarial alkaloid rich extracts derived from two plants used in traditional medicine in Mali (Mitragyna inermis (Willd.) O. Kuntze Rubiaceae and Nauclea latifolia (Sm.) Rubiaceae) were evaluated on in vitro and in vivo systems. The results demonstrated that an alkaloid rich extract derived from M. inermis induced a strong inhibition of protein synthesis in mammalian cells but did not exhibit mutagenic or genotoxic activity. An alkaloid rich extract derived from N. latifolia could interact in vitro with DNA of bacteria and mammalian cells, leading to G2-M cell cycle arrest and heritable DNA-damage, as well as inducing in vivo single-strand breaks in liver, kidney and blood cells.

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.

Optimization of the Salmonella/mammalian microsome assay for urine mutagenesis by experimental designs.

Assessing urine mutagenicity with the Salmonella mutagenicity test is often limited by the volumes of the samples. Optimization of the assay was performed with factorial and Doehlert designs. Two fractional factorial designs 2(3-1) (3 factors, 4 experiments) were used to estimate the main effects of the percent S9 in the mix, the time of liquid incubation, the inoculum size and the growth conditions. A Doehlert design (3 factors, 13 experiments) was used to study the main effects and the interactions of the NADP, G6P and S9 in the mix. The positive markers were benzo[a]pyrene (BaP, 0.3 microgram/plate) and a pool of smokers' urine (SU, 1.25 ml equivalent/plate). The response was limited to the induction factor (IF, number of induced revertants/number of spontaneous revertants) with Salmonella typhimurium TA98. The optimal conditions for BaP were: a 60 min period of liquid incubation and a volume of 0.1 ml (approx. 10(8) cells/plate) of an overnight culture grown in 50 ml of Nutrient Broth No. 2 from a 250 ml flask. The S9 mix (0.1 ml, final volume) included 1.5% of S9, 1.0 mM NADP and 4.4 mM G6P. The maximal IF was 15.79. The optimal conditions for SU were: a 60 min period of liquid incubation and a volume of 0.1 ml (approx. 10(8) cells/plate) of an overnight culture grown in 7 ml of Nutrient Broth No. 2 from a 20 x 180 mm tube. The S9 mix (0.1 ml, final volume) included: 4% S9, 4.2 mM NADP and 5.2 mM G6P. The maximal IF was 10.95. These optimal conditions did not modify the spontaneous frequencies of the tester strains: TA97a, TA98, TA100 and TA102. The dose-response curves of mutagenic urine samples were found to be non-linear. This micromethod required 8-fold less urine sample and 12.5-fold less liver homogenate as compared to the standard plate incorporation assay and was from 6.2- to 11.8-fold more sensitive to evaluate urine mutagenicity. The sensitivity of this technique was found to be limited to individuals smoking more than approx. 5 cigarettes/day by the standard extraction-concentration procedure.

Monitoring exposure of hospital personnel handling cytostatic drugs and contaminated materials.

A study was undertaken to evaluate the urine mutagenicity of 63 individuals working in four hospital departments. The exposed group included 38 subjects who were exposed to various cytostatic drugs and/or contaminated material from treated patients. The control group included 25 individuals of the hospital personnel. Urine mutagenicity was monitored by the Ames test using tester strains TA 98 + S9 Mix and TA 102-S9 Mix. Urine samples were collected before and after the working periods. A total of 29/116 (25%) urine samples were mutagenic for either strain. Among the mutagenic samples, 24/29 were mutagenic for tester strain TA 98 exclusively. No significant correlation could be found between occupational exposure to cytostatic drugs and urine mutagenicity evaluated by the strain TA 98 + S9 Mix. Smoking was the main environmental factor that modulated urine mutagenicity with TA 98. Three subjects in the exposed group had mutagenic urine samples at the end of the working period with strain TA 102-S9 Mix. This mutagenicity was related to occupational exposure to cisplatin. In the control group, one individual had mutagenic samples before and after the working period. Assessing occupational exposure to cytostatic drugs with strain TA 102 requires additional studies to determine environmental mutagens which can be detected by this strain.

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.

Flow cytometry to evaluate the parasitemia of Plasmodium falciparum.

The resistance of plasmodiums to the current antimalarial agents has spurred the search for new active molecules of vegetal origin or chemical synthesis. The screening of antimalarial molecules "in vitro" was done by simple but tedious techniques such as quantification of parasitemia through optical microscopy or by using radioactive markers. We have developed a new method to evaluate parasitemia by using the ODAM ATC 3000 flow cytometer and biological cell sorter. We selected the ethidium bromide for labelling the nucleic acids of Plasmodium falciparum, and we optimised the method by using a mathematical model: the design of Hadamar. This simple technique presents the advantage of being an objective and rapid count of large number of red cells (10(6) x 10(7)). This method is rapid, reliable, reproducible, devoid of subjectivity and provides more precise results than those of optical microscopy. The good correlation between paraitemia measured by optical microscopy and fluorescence obtained by flow cytometry allows us to recommend this technic for the screening of new antimalarial molecules.

[Homogeneous suspensions of mycobacteria: application to the evaluation of bactericidal activity (author's transl)]. / Préparation de suspensions homogénes de mycobactéries: application à la détermination de l'activité mycobactéricide.

This work describes a technique of emulsification of mycobacterial cells (including tubercle bacilli) in order to obtain homogeneous suspensions containing up to 10(8) cells/ml. The suspensions are used in a modified AFNOR method for the evaluation of bacterial activity: within five min virulent strains are more resistant than are avirulent ones but, within 30 min and in the presence of proteins, the behaviour of all strains is quite similar.