The paramagnetic properties of malaria pigment, hemozoin, yield clues to a low-cost system for its trapping and determination.

The binding of malaria pigment, hemozoin, by a gradient magnetic field has been investigated in a manual trapping column system. Two types of magnetic filling have been tested to produce field gradients: nickel-plated steel wires, wrapped around a steel core, and superparamagnetic microbeads. The latter system allows an efficient trapping (> 80%) of ß-hematin (a synthetic pigment with physical and paramagnetic properties analogous to those of hemozoin). Tests with a Plasmodium falciparum 3D7 culture indicate that hemozoin is similarly trapped. Off-line optical spectroscopy measurements present limited sensitivity as the hemozoin we detected from in vitro cultured parasites would correspond to only a theoretical 0.02% parasitemia (1000 parasites/µL). Further work needs to be undertaken to reduce this threshold to a practical detectability level. Based on these data, a magneto-chromatographic on-line system with reduced dead volumes is proposed as a possible low-cost instrument to be tested as a malaria diagnosis system.

Validation of an automatic comet assay analysis system integrating the curve fitting of combined comet intensity profiles.

In recent years, the single-cell gel electrophoresis (comet) assay has become a reference technique for the assessment of DNA fragmentation both in vitro and in vivo at the cellular level. In order to improve the throughput of genotoxicity screening, development of fully automated systems is clearly a must. This would allow us to increase processing time and to avoid subjectivity brought about by frequent manual settings required for the 'classical' analysis systems. To validate a fully automatic system developed in our laboratory, different experiments were conducted in vitro on murine P388D1 cells with increasing doses of ethyl methanesulfonate (up to 5 mM), thus covering a large range of DNA damage (up to 80% of DNA in the tail). The present study (1) validates our 'in house' fully automatic system versus a widely used semi-automatic commercial system for the image-analysis step, and versus the human eye for the image acquisition step, (2) shows that computing tail DNA a posteriori on the basis of a curve fitting concept that combines intensity profiles [G. Dehon, P. Bogaerts, P. Duez, L. Catoire, J. Dubois, Curve fitting of combined comet intensity profiles: a new global concept to quantify DNA damage by the comet assay, Chemom. Intell. Lab. Syst. 73 (2004) 235-243] gives results not significantly different from the 'classical' approach but is much more accurate and easy to undertake and (3) demonstrates that, with these increased performances, the number of comets to be scored can be reduced to a minimum of 20 comets per slide without sacrificing statistical reliability.

Direct and indirect antimicrobial effects and antioxidant activity of Cordia gilletii De Wild (Boraginaceae).

Cordia gilletii De Wild (Boraginaceae) root bark is traditionally used in Democratic Republic of Congo (DRC) for the treatment of various disorders, including malaria, diarrhea, wounds and skin diseases; part of these activities may rely on antimicrobial and antioxidant properties. Successive extracts of root barks powder with n-hexane, dichloromethane, ethyl acetate, methanol and water were tested for antimicrobial activity, both direct and indirect (antibiotic resistance reversal), against 10 strains of bacteria and 1 strain of fungi by broth microdilution and agar diffusion methods. The eventual synergy between plant extracts and antibiotics was investigated by the determination of the fractional inhibitory concentration index (FIC index). The methanol extract showed direct antimicrobial activity against all tested microorganisms with minimum inhibitory concentrations (MIC) ranging between 125 and 1000 microg/ml, whereas the ethyl acetate and the dichloromethane extracts showed activity on four and three strains, respectively. 200 microg/ml of n-hexane and dichloromethane extracts decreased the MICs of penicillin and streptomycin 4-64-fold for methicillin-resistant Staphylococcus aureus. A synergistic effect was found between the methanol extract and tetracycline, whereas additive effects were observed for the other combinations tested. The methanol and dichloromethane extracts showed the greater antioxidant activity by scavenging the free radical DPPH with IC(50) values of 3.2 and 8.1 microg/ml, respectively. These results support the use of the plant in the treatment of infectious diseases and wounds; they warrant further studies as to the nature of active compounds.

Alkaloids and amides from Triclisia sacleuxii.

A new alkaloid, named gasabiimine (1), together with four known bisbenzyltetrahydroisoquinoline alkaloids (phaeanthine, 1,2-dehydroapateline, N-methylapateline, O-methylcocsoline) has been isolated from the roots of Triclisia sacleuxii. The structure of the new alkaloid was elucidated by spectroscopic data analysis.

Chromatographic determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA: a validation study.

Although a series of biomarkers are widely used for the estimation of oxidative damage to biomolecules, validations of the analytical methods have seldom been presented. Formal validation, that is the study of the analytical performances of a method, is however recognized as the best safeguard against the generation and publication of data with low reliability. Classical validation parameters were investigated for the determination of an oxidative stress biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in cellular DNA, by high-performance liquid chromatography coupled to amperometric detection (HPLC-EC); this modified base is increasingly considered as a marker of oxidative damage to DNA, but many questions are still raised on the analytical methods in use. Upon a rigorous statistical evaluation of the quality criteria currently required for assays in biological media, including selectivity, linearity, accuracy, repeatability, sensitivity, limits of detection and quantification, ruggedness and storage at different stop points in the procedure, the HPLC-EC assay method is found mostly reliable. The present validation attempt demonstrates that (i) the HPLC-EC assay of 8-oxo-dG provides consistent data allowing to reliably detect an increase of this biomarker in cellular DNA; (ii) a harsh oxidative stress does not hinder the enzymatic digestion of DNA by nuclease P1; and (iii) the analytical results must be expressed relative to the internal standard dG which significantly improves both repeatability and sensitivity. Whereas the described assay minimizes the artifactual production of the analyte from processing and storage, this cannot be totally ruled out; the true 8-oxo-dG base levels still lack a definitive assay method, which remains a considerable analytical challenge and the object of controversy.

Use of an Amoeba proteus model for in vitro cytotoxicity testing in phytochemical research. Application to Euphorbia hirta extracts.

Amoeba proteus is proposed as a low-cost multi-purpose biochemical tool for screening and standardizing cytotoxic plant extracts with possible application in the laboratories of developing countries. Advantages and limitations of this test are examined and different mathematical treatments (probit analysis versus curve fitting to Von Bertalanffy and Hill functions) are investigated. Known anti-cancer (doxorubicin, daunorubicin, dacarbazine, 5-fluorouracil) and antiparasitic (emetine, dehydroemetine, metronidazole, cucurbitine, chloroquine) drugs were tested using this method and only metronidazole appeared inactive. Application of this model to Euphorbia hirta established that a 100 degrees C aqueous extraction of fresh aerial parts allows efficient extraction of active constituents and that drying the plant material before extraction considerably reduces activity.

Cytotoxic constituents from Plumbago zeylanica.

The bioassay-guided fractionation of the dichloromethane extract of aerial parts of Plumbago zeylanica led to the isolation of beta-sitosterol, beta-sitosteryl-3beta-glucopyranoside, beta-sitosteryl-3beta-glucopyranoside-6'-O-palmitate (1), lupenone, lupeol acetate, plumbagin and trilinolein. Compound 1 showed cytotoxic activity against MCF7 and Bowes cancer cell lines (IC50 113 microM and 152 microM, respectively), beta-sitosterol inhibited Bowes cell growth (IC50 36.5 microM) and plumbagin was cytotoxic against MCF7 and Bowes cells (IC50 1.28 microM and 1.39 microM, respectively).

[Cannabis: experts agree more than they admit]. / Cannabis: les scientifiques sont d'accord plus qu'ils ne l'admettent.

Cannabis is evermore present in society, whether within the general public or as a subject for scientific debate. Mass consumption of cannabis, for example, is stabilising around 22 percent of 18-year old who admit to having used it at least once during the previous month; however, this consumption rate falls off as they enter later adulthood. This article describes the emerging scientific consensus about the effects of this drug. The psychotropic effects of cannabis--the result of cannabinoids contained in its resin that activate specific receptors--include general euphoria, a mild release from inhibitions and, in certain cases, some distortion of sensory perception. Some patients also experience drowsiness, a stimulated appetite and anxiolysis, while others anticipate a more intense experience such as an altered state of consciousness. The toxicity of smoked cannabis and its acute, chronic secondary effects are described, as well as the problematic relationship between cannabis consumption and psychosis. The damages and toxic effects attributed to such consumption are presented via three, related themes: the growth of dependency, negative somatic consequences (including cognitive impairment and its consequences for driving an automobile, and damaging psychosocial effects. "Escalation theory" is criticized. In their conclusion, the authors cast doubt on the scientific grounds for penalisation of cannabis consumption, and recommend a "de-demonisation" of the drug. An analysis and discussion of the current penalties applied in Belgium are presented.

Antiplasmodial and antitrypanosomal activity of Triclisia sacleuxii (Pierre) Diels.

The antiplasmodial and antitrypanosomal activities of Triclisia sacleuxii (Pierre) Diels were investigated on three Plasmodium falciparum strains [FcB1, 3D7 (chloroquine-sensitive) and W2 (chloroquine-resistant) strains] and on Trypanosoma brucei Tbsf 221. Roots, stems and leaves ethanolic extracts as well as crude tertiary and quaternary alkaloids fractions were considered. Whereas the ethanolic extracts and quaternary crude alkaloids fractions exhibited no significant activity, the roots and stems tertiary alkaloid fractions revealed interesting growth inhibition against the Plasmodium FcB1 and Trypanosoma Tbsf 221 strains. The IC(50) were 1.04 and 0.89 microg/ml for roots, 2.50 and 0.91 microg/ml for stems. The leaves tertiary alkaloids fraction also showed a promising antitrypanosomal activity (IC(50): 1.85 microg/ml). Phytochemical analysis of the roots tertiary alkaloids fraction yielded four major compounds, phaeanthine, N-methylapateline, 1,2-dehydroapateline and 1,2-dehydrotelobine, which were identified on the basis of their spectroscopic data. The four compounds displayed (in vitro) antitrypanosomal activity with IC(50) of 2.68, 1.19, 1.06 and 1.11 microM, respectively. They also demonstrated antiplasmodial activity on Plasmodium falciparum 3D7, with IC(50) of 1.72, 0.93, 1.39 and 12.4 microM respectively and on the chloroquine-resistant W2 with IC(50) of 0.35, 1.10, 1.63 and 1.52 microM.

A validation protocol for the HPTLC standardization of herbal products: application to the determination of acteoside in leaves of Plantago palmata Hook. f.s.

Formal validation, that is the study of the analytical performances of a method, is recognized as the best safeguard against the generation and publication of data with low reliability. Although the topic of HPTLC validations has been largely investigated, there is still a need for a general validation method applicable whenever a blank matrix cannot be reconstituted, notably herbs and their extracts. This work proposes two validation schemes aiming at generate linearity, accuracy and precision data in a minimal number of HPTLC plates, taking the standardization of Plantago palmata as an example with both UV and visible (post-chromatographic derivatization with a sulphuric acid-vanillin reagent) detections. A major problem associated with HPTLC determinations is underlined, namely the low range of linearity which makes spiking studies quite difficult as care must be taken to avoid overloading, whereas keeping the analyte detectable in blank extracts and avoiding spikes too close to endogenous levels. A second problem is the use of general post-chromatographic derivatization reagents that compromise the selectivity of the method by reacting with compounds that may not be resolved from the compound of interest. The use of such reagents is clearly not without danger, especially given the relatively low resolution of planar chromatography. In conclusion, the retained validation protocol effectively yields the main validation data whereas allowing to pinpoint major analytical drawbacks. It was not possible to simultaneously validate aucubin and acteoside assays as both analytes are present at too different levels/detectabilities.

Gas chromatographic profiling and determination of urinary acylcarnitines.

A method is described for the routine profiling and determination in urine of most of the acylcarnitines clinically relevant for the diagnosis of organic acidurias. The procedure, which does not require expensive apparatus, involves extraction of the acylcarnitines on strong cation-exchange disposable columns, mild alkaline hydrolysis and gas chromatography of the liberated monocarboxylic acids. The different steps were optimized in order to increase the analytical performance. No significant interferences were encountered, the limit of detection (signal-to-noise ratio = 3:1) ranged from 0.1 to 4 mg/l and the between-day coefficient of variation from 3.6 to 17.7%, depending on the acyl species. The rapidity of the method results from the application of a single solid-phase extraction on disposable columns. The acyl moieties are chromatographed underivatized in order to permit the identification of short-, medium- and long-chain acylcarnitines. The method was assessed by analysing fourteen urine specimens from patients presenting an organic aciduria.

Photodynamic DNA damage mediated by delta-aminolevulinic acid-induced porphyrins.

The mutagenic properties of UVA are thought to be predominantly radical-mediated, which supposes endogenous sensitizers. In order to investigate a possible role of porphyrins, their synthesis was induced in a murine leukemia P388D1 cell model by treatment with delta-aminolevulinic acid (delta-ala). Intra-cellular protoporphyrin IX reached a plateau after about 2 h, whereas soluble porphyrins, probably the photostable uro- and/or coproporphyrins, were excreted. Irradiation of treated cells by UVA (tanning lamp) but also by visible light was found to generate in DNA a significant increase of 8-oxo-7,8-dihydro-2'-deoxyguanosine, a mutagenic marker of oxidative damage. The different parameters involved in this photodynamic effect are reported, namely delta-ala concentration and loading time, light dosage and the influence of intracellular and medium-excreted porphyrins. These results point to an implication of porphyrins in solar-induced carcinogenicity but also in possible adverse effects of the medical applications of photodynamic therapy and diagnosis.

GC-MS profiling of urinary organic acids evaluated as a quantitative method.

We assessed the quantitative performances of a classical method for profiling urinary organic acids: ethyl acetate extraction/oxime-trimethylsilyl derivatization/GC-MS. Twenty-seven acids were quantified on the basis of specific ions in both scan and selected-ion monitoring modes. We found that the tuning of the mass detector severely affects the calibration factors, being critical to achieve quantitative results, and we propose a practical procedure for reproducible tuning. Of seven compounds tested, tropic acid was retained as the internal standard suitable for most of the acids of clinical interest; a second internal standard, 2-ketocaproic acid, was used in quantifying keto-acids. The within-day and total relative standard deviations (CVs), estimated from scan-mode analyses of urine, ranged from 2.6% to 12.7% and from 4.2% to 11.8%, respectively. Curvilinear relationships between analytical response and concentration were observed for most of the acids investigated.

Validation of raw data measurements in the comet assay.

General guidance recently proposed for the comet assay concluded that "the method should be adjusted scientifically at each laboratory to obtain valid and reproducible results". However, the comet widely used metrics, Tail DNA and Tail moment, are actually based on a ratio of fluorescence signals, a relative and semi-quantitative measurement, and are quite difficult to validate according to classical criteria. As the validation of analytical methods increasingly becomes an absolute requirement in many fields, this paper investigates a scheme to study the variability of raw data measurements for computer-assisted comet measurement, including the between-operators reproducibility. In the overall analysis process, we show that the image acquisition step gives the highest variability, notably for the Tail length parameter that negatively influences the Olive tail moment. However, when the operator interacts with the system to correct obviously mistaken measurements, the reproducibility is sensibly improved. For the metrics Tail DNA and Olive tail moment, the total variability in measurements for a panel of comets quantified by different operators in real conditions is about 4%. The proposed validation scheme allows to assess the measurement process and to verify if there are any major difference between trained operators, an essential requirement for long-term investigations.

Statistics of the Comet assay: a key to discriminate between genotoxic effects.

The alkaline Comet assay is a widely used single cell gel electrophoresis technique for the quantification of DNA strand breaks, crosslinks and alkali-labile sites induced by a series of physical and chemical agents. DNA migration in an electric field, supposed proportional to strand breakage, is a proposed estimation of genotoxicity. Breaks are quantified from geometric and fluorescence measurements by image analysis of comet-shaped DNA, often reported parameters being tail DNA and tail moment. Although a variety of statistical approaches have been used in the literature, most of these do not take into account the distribution patterns of comet data. In order to investigate a methodology for statistically demonstrating a comet effect, two different experiments, a reproducibility study and a trend analysis, were undertaken on a murine lymphoma cell line (P388D1) photodynamically stressed after induction of porphyrins with delta-aminolaevulinic acid. This treatment results in significant heterogeneity of DNA damage, producing values ranging from 0 to 100% tail DNA in the same sample. The comparison of distribution curves for stressed and non-stressed samples shows that none of the application conditions are verified, either for parametric tests (which require normal distributions), or non-parametric tests (which assume essentially similar distributions). Meaningful statistics (median and 75th percentile) were consequently extracted from repeated experiments and found suitable for comparing stress conditions in an ANOVA and in a trend analysis; the 75th percentile is theoretically more sensitive but tends to more rapidly saturate at extensive stress levels. We conclude that a trend analysis of median comet metrics from repeated experiments at different stress levels is certainly an efficient way to statistically demonstrate a genotoxic effect. Whether the considered comet parameter is tail DNA or tail moment had no influence on the conclusions of our experiments, which were carried up to stress levels leading to a median 70% tail DNA.

Cysteine but not glutathione modulates the radiosensitivity of human melanoma cells by affecting both survival and DNA damage.

Glutathione (GSH) and its precursor cysteine (Cys) are both known to react within any cells with oxidative species and thus play an important role in cellular defense mechanisms against oxidative stress. In melanocytes, these are also important precursors of melanogenesis by reacting non-enzymatically with l-dopaquinone to form the sulfur-containing pheomelanin. Our aim was to assess pigment role in the cellular radioprotection mechanism using a human melanoma cell model of mixed-type melanin under GSH depletion to obtain a radiosensitizing effect. The latter has been achieved either by Cys deprivation or GSH specific depletion. We first compared cell survival of Cys-deprived and GSH-depleted cells vs. control cells. Cys deprivation was achieved by decreasing Cys concentration in the culture medium for 24 h. In this condition, no toxicity was observed, Cys and GSH levels decreased, melanogenesis switched to a higher eumelanin synthesis and cells were significantly more resistant to 10-Gy dose of ionizing radiations than untreated cells. Glutathione depletion was achieved with the gamma-glutamylcysteine synthetase inhibitor buthionine-S-sulfoximine (BSO) for 24 h at 50 microM, a concentration yielding no toxicity. In this condition, intracellular GSH level decreased but no change in pigmentation was observed and cells were slightly but significantly more sensitive to radiation than the control. We then compared DNA radio-induced damages by Comet assay in control cells, cells treated as above and cells with stimulated pigmentation by increasing Tyr concentration in the medium. Our results showed that, when intracellular eumelanin content increased, DNA damage decreased. By contrast, DNA damage increased in cells treated with BSO alone. It is concluded that increasing the intracellular eumelanin content by the melanin precursor Tyr or by favoring the Pheo- to Eumelanin switch, compensates for the loss of the two intracellular radioprotectors that are GSH and Cys.

Assessment of an electron-impact GC-MS method for organic acids and glycine conjugates in amniotic fluid.

We assessed the reliability of a method designed for common electron-impact GC-MS systems to determine in a single run most organic acids and glycine conjugates of clinical interest in amniotic fluid. Suitable sensitivity was achieved by dividing the selected-ion chromatogram into 12 time segments during which the monitoring dwelt on specific ions. Twelve metabolites were simultaneously quantified in amniotic fluid, with performances ranging from very good to clinically acceptable. The total coefficient of variation was 2.5-14.1% and the detection limit was well below the lower value of the physiological range. For five other metabolites, the precision was lower and/or the detection limit was near the physiological range. The method was clinically assessed by the prenatal detection of three cases of tyrosinaemia type I and one case of propionic acidaemia. Analytical and clinical evaluation of the method showed that GC-MS with electron-impact fragmentation can be an informative analytical approach for low-level organic acids in physiological fluids. Apart from the case of glycine conjugates, the method shows a fair reliability for amniotic fluid analysis, which might warrant its use for prenatal diagnosis of organic acidurias. However, this method cannot replace procedures using isotopic internal standards, nor GC-MS based on chemical ionization fragmentation, which remain confirmatory analytical methods of choice.