[The Lafarge affair: a judgment in which toxicology didn't win fame!]. / L'affaire Lafarge: un dossier contesté où la toxicologie ne s'est guère illustrée!

The famous Lafarge affair which took place in Tulle (Corrèze, France) in 1840, has never ceased to interest the general public because some doubts have remained even after the pronunciation of the judgment. Another hypothesis than arsenicum poisoning has been formulated in 1980 by Professor P. Lepine who believes in a paratyphoid fever occuring after food poisoning. This assumption and the discovery of exceptional family documents in 2004 by Edouard de Lamaze, a distant cousin of Emma Pontier, the confidante of Marie Lafarge, sheds a new light on this mysterious affair.

Selectivity assessment of chlorfenvinphos reevaluated by including physiological and behavioral effects on an important beneficial insect.

Selectivity is an important factor in identifying candidate pesticides to be used in crop protection since it characterizes chemicals that, while being effective against target pests, exert an acceptable impact on the other components of the environment. Extrapolated to an integrated pest management (IPM) context, selectivity implies that candidate pesticides may preserve the ability of beneficial insects to significantly control target pest populations. In the present study, we assess the physiological selectivity of the organophosphate chlorfenvinphos, used to protect cruciferous crops against the cabbage root fly, Delia radicum (Diptera: Anthomyiidae), by investigating both the lethal and sublethal effects exerted on its main parasitoid Trybliographa rapae (Hymenoptera: Figitidae). The comparison of the median lethal doses showed that T. rapae was at least seven times less sensitive than D. radicum to chlorfenvinphos. However, longevity of parasitoids surviving a sublethal dose was reduced by half. The potential fecundity of females was decreased by 9.6 to 22.8%. Chlorfenvinphos also induced important behavioral changes in both sexes and reduced the chances for parasitoids to mate by more than 70%. While most behavioral changes were reversible, effects on mating and on fecundity were not, thereby suggesting long-term effects on the reproduction of the parasitoid. These cumulative effects of chlorfenvinphos would have dramatic consequences on the efficacy of parasitoids contacting such doses of chlorfenvinphos in the field and therefore there is question about the intrinsic selectivity of this insecticide.

Measurement of insecticide uptake and effective fraction in a beneficial insect using solid-phase microextraction.

The determination of insecticide uptake in beneficial insects is important for quantifying the doses that are responsible for the toxicological effects and to compare them with the doses that insects may absorb in treated fields. Because of the small size of some beneficial species, the amount of insecticide absorbed may be very low. Herein, we present a method that relies on the sensitivity and specificity of SPME (solid-phase microextraction) as a sampling technique that can be used to measure very small amounts of an organophosphorus insecticide in small insects. In our study, the method was applied to quantify the internal dose and free dissolved fraction of chlorfenvinphos in beneficial parasitoids exposed through a topical application. Up to 0.5 ng of the insecticide could be quantified in these fractions, that is, 10 times less than when using solvent extraction techniques. The penetration and elimination rates of the insecticide in the insect were also determined. The method proved to be suitable to quantify internal doses in parasitoids collected in a treated field.

Resistance of human multidrug resistance-associated protein 1-overexpressing lung tumor cells to the anticancer drug arsenic trioxide.

The human multidrug-resistance protein (MRP1) confers resistance to some heavy metals such as arsenic and antimony, mainly through mediating an increased cellular efflux of metal. However, it was recently suggested that arsenic, used under its trioxide derivative form as anticancer drug, is not handled by MRP1. The aim of the present study was to test this hypothesis in MRP1-overexpressing human lung tumor GLC4/Sb30 cells. Using the cytotoxicity MTT assay, GLC4/Sb30 cells were found to be 10.8-fold more resistant to arsenic trioxide (As2O3) than parental GLC4 cells. MK571, a potent inhibitor of MRP1 activity, almost totally reversed resistance of GLC4/Sb30 cells, but did not alter the sensitivity of GLC4 cells. Moreover, As2O3-loaded GLC4/Sb30 cells poorly accumulated arsenic through an increased MK571-sensitive efflux of metal. Finally, depletion of cellular glutathione levels in buthionine sulfoximine-treated GLC4/Sb30 cells was found to result in increased accumulation and reduced efflux of arsenic in cells exposed to As2O3, outlining the glutathione-dependence of MRP1-mediated transport of the metal. These results indicate that MRP1 overexpression in human tumor cells can confer resistance to As2O3, which may limit the clinical use of this anticancer drug for treatment of MRP1-positive tumors.

Fatal aluminum phosphide poisoning.

A 39-year-old man committed suicide by ingestion of aluminum phosphide, a potent mole pesticide, which was available at the victim's workplace. The judicial authority ordered an autopsy, which ruled out any other cause of death. The victim was discovered 10 days after the ingestion of the pesticide. When aluminum phosphide comes into contact with humidity, it releases large quantities of hydrogen phosphine (PH3), a very toxic gas. Macroscopic examination during the autopsy revealed a very important asphyxia syndrome with major visceral congestion. Blood, urine, liver, kidney, adrenal, and heart samples were analyzed. Phosphine gas was absent in the blood and urine but present in the brain (94 mL/g), the liver (24 mL/g), and the kidneys (41 mL/g). High levels of phosphorus were found in the blood (76.3 mg/L) and liver (8.22 mg/g). Aluminum concentrations were very high in the blood (1.54 mg/L), brain (36 microg/g), and liver (75 microg/g) compared to the usual published values. Microscopic examination revealed congestion of all the organs studied and obvious asphyxia lesions in the pulmonary parenchyma. All these results confirmed a diagnosis of poisoning by aluminum phosphide. This report points out that this type of poisoning is rare and that hydrogen phosphine is very toxic. The phosphorus and aluminum concentrations observed and their distribution in the different viscera are discussed in relation to data in the literature.

Differential sensitivities of MRP1-overexpressing lung tumor cells to cytotoxic metals.

The human multidrug-resistance protein (MRP1), known to mediate cellular efflux of a wide range of xenobiotics, including anticancer drugs, has also been shown to transport antimony, thereby conferring resistance to this heavy metal. The aim of the present study was to investigate whether other cytotoxic metals could be handled by MRPI using MRP1-overexpressing lung tumor GLC4/Sb30 cells. Such cells were found to be 3.4-, 12.7- and 16.3-fold more resistant than parental GLC4 cells to mercuric ion, arsenite and arsenate, respectively, whereas they remained sensitive to other cytotoxic metals tested such as copper, chromium, cobalt or aluminium. MK571, a potent inhibitor of MRP1 activity, almost totally reversed resistance of GLC4/Sb30 cells to mercuric ions and arsenic while it did not significantly alter sensitivity of GLC4 cells to metals. Arsenate-treated GLC4/Sb30 cells were found to poorly accumulate arsenic through increased MK571-inhibitable efflux of the metal. Arsenate, however, failed to alter MRP1-mediated transport of known MRP1 substrates such as calcein and vincristine. In conclusion, these findings demonstrated that MRP1 likely handled some, but not all, cytotoxic metals such as arsenic and mercuric ions in addition to antimony, therefore resulting in reduced toxicity of these compounds towards MRP1-overexpressing cells.

Overexpression of the multidrug resistance-associated protein (MRP1) in human heavy metal-selected tumor cells.

Cellular and molecular mechanisms involved in the resistance to cytotoxic heavy metals remain largely to be characterized in mammalian cells. To this end, we have analyzed a metal-resistant variant of the human lung cancer GLC4 cell line that we have selected by a step-wise procedure in potassium antimony tartrate. Antimony-selected cells, termed GLC4/Sb30 cells, poorly accumulated antimony through an enhanced cellular efflux of metal, thus suggesting up-regulation of a membrane export system in these cells. Indeed, GLC4/Sb30 cells were found to display a functional overexpression of the multidrug resistance-associated protein MRP1, a drug export pump, as demonstrated by Western blotting, reverse transcriptase-polymerase chain reaction and calcein accumulation assays. Moreover, MK571, a potent inhibitor of MRP1 activity, was found to markedly down-modulate resistance of GLC4/Sb30 cells to antimony and to decrease cellular export of the metal. Taken together, our data support the conclusion that overexpression of functional MRP1 likely represents one major mechanism by which human cells can escape the cytotoxic effects of heavy metals.

Iron-induced oxidative DNA damage and its repair in primary rat hepatocyte culture.

Iron-overload diseases frequently develop hepatocellular carcinoma. The genotoxic mechanism whereby iron is involved in hepatocarcinogenesis might involve an oxidative process via the intermediate production of reactive oxygen species. This was presently investigated by examining kinetics of formation and repair of DNA base lesions in primary rat hepatocyte cultures supplemented with the iron chelate, ferric nitrilotriacetate Fe-NTA (10 and 100 microM). Seven DNA base oxidation products have been identified in DNA extracts by gas chromatography-mass spectrometry, which showed a predominance of oxidized-purines (8-oxo-guanine, xanthine, fapy-adenine, 2-oxo-adenine) above oxidized pyrimidines (5-OHMe-uracil, 5-OH-uracil, 5-OH-cytosine) in control cultures. All these DNA oxidation products revealed a significant dose-dependent increase at 4 to 48 h after Fe-NTA supplementation, among which fapy-adenine showed the highest increase and 5-OH-cytosine was the least prominent. Involvement of iron in this oxidative process was established by a correlation between extent in DNA oxidation and intracellular level of toxic low molecular weight iron. DNA excision-repair activity was estimated by release of DNA oxidation products in culture medium. All the seven DNA oxidation products were detected in the medium of control cultures and showed basal repair activity. This DNA repair activity was increased in a time- and dose-dependent fashion with Fe-NTA. Oxidized-pyrimidines, among which was 5-OHMe-Uracil, were preferentially repaired, which explains the low levels detected in oxidized DNA. Since oxidized bases substantially differed from one another in terms of excision rates from cellular DNA, specific excision-repair enzymes might be involved. Our findings, however, demonstrate that even though DNA repair pathways were activated in iron-loaded hepatocyte cultures, these processes were not stimulated enough to prevent an accumulation of highly mutagenic DNA oxidative products in genomic DNA. The resulting genotoxic effect of Fe-NTA might be relevant in understanding the hepatocarcinogenic evolution of iron-overload diseases.

Influence of fatty acid ethanolamides and delta9-tetrahydrocannabinol on cytokine and arachidonate release by mononuclear cells.

The effects of arachidonic acid ethanolamide (anandamide), palmitoylethanolamide and delta9-tetrahydrocannabinol on the production of tumor necrosis factor-alpha (TNF-alpha), interleukin-4, interleukin-6, interleukin-8, interleukin-10, interferon-gamma, p55 and p75 TNF-alpha soluble receptors by stimulated human peripheral blood mononuclear cells as well as [3H]arachidonic acid release by non-stimulated and N-formyl-Met-Leu-Phe (fMLP)-stimulated human monocytes were investigated. Anandamide was shown to diminish interleukin-6 and interleukin-8 production at low nanomolar concentrations (3-30 nM) but inhibited the production of TNF-alpha, interferon-gamma, interleukin-4 and p75 TNF-alpha soluble receptors at higher concentrations (0.3-3 microM). Palmitoylethanolamide inhibited interleukin-4, interleukin-6, interleukin-8 synthesis and the production of p75 TNF-alpha soluble receptors at concentrations similar to those of anandamide but failed to influence TNF-alpha and interferon-gamma production. The effect of both compounds on interleukin-6 and interleukin-8 production disappeared with an increase in the concentration used. Neither anandamide nor palmitoylethanolamide influenced interleukin-10 synthesis. delta9-Tetrahydrocannabinol exerted a biphasic action on pro-inflammatory cytokine production. TNF-alpha, interleukin-6 and interleukin-8 synthesis was maximally inhibited by 3 nM delta9-tetrahydrocannabinol but stimulated by 3 microM delta9-tetrahydrocannabinol, as was interleukin-8 and interferon-gamma synthesis. The level of interleukin-4, interleukin-10 and p75 TNF-alpha soluble receptors was diminished by 3 microM delta9-tetrahydrocannabinol. [3H]Arachidonate release was stimulated only by high delta9-tetrahydrocannabinol and anandamide concentrations (30 microM). These results suggest that the inhibitory properties of anandamide, palmitoylethanolamide and delta9-tetrahydrocannabinol are determined by the activation of the peripheral-type cannabinoid receptors, and that various endogenous fatty acid ethanolamides may participate in the regulation of the immune response.

EPR determination of low molecular weight iron content applied to whole rat hepatocytes.

Electron paramagnetic resonance (EPR) has been described as suitable for the evaluation of low molecular weight (LMW) iron in liver homogenates after chelation by desferrioxamine. LMW iron is a highly toxic iron species incriminated in free radical production. The first aim of the study was to evaluate the conditions of EPR application for LMW iron content determination in whole rat hepatocytes. For this purpose, LMW iron was simultaneously quantified by EPR and by atomic absorption spectrometry, EPR determination of LMW iron needed a preincubation of hepatocyte cultures with the iron chelator for at least on hr. Deferiprone as LMW iron chelator was revealed to be more suited than desferrioxamine. Secondly, we showed the applicability of this methods for evaluating the prooxidant status during an oxidative stress. As an example, oxidative stress induced by ethanol in hepatocytes was studied during inflammatory circumstances, well-known to lead to nitric oxide production. In hepatocyte cultures supplemented with ethanol, an evaluation of LMW iron content was observed in cells. But when nitric oxide donors or a supplementation constituted of lipopolysaccharide and gamma-interferon, able to induce nitric oxide synthase, were added, LMW iron content decreased. Thus EPR determination of LMW iron content in whole hepatocytes could give some insight about the mechanism of induction or inhibition of a oxidative stress.

Stimulation of Rb+ influx by bradykinin through Na+/K+/Cl- cotransport and Na+/K(+)-ATPase in NIH-3T3 fibroblasts.

Bradykinin receptor stimulation results in G-protein-coupled phospholipase activation, initiating protein kinase C (PKC) stimulation and cytosolic free Ca2+ concentration ([Ca2+]i) rises as signalling pathways. Using Rb+ as a tracer for K+, we have studied the mechanisms involved in bradykinin-stimulated Rb+ influx in NIH-3T3 fibroblasts. The furosemide-sensitive Na+/K+/Cl- cotransport and the ouabain-sensitive Na+/K(+)-ATPase were both involved in Rb+ influx under resting conditions with a ratio Na+/K+/Cl- cotransport/Na+/K(+)-ATPase (r) = 0.73. Bradykinin stimulated Rb+ influx (+82.6%) through both systems without changing their ratio (r = 0.72). PKC stimulation by a 15-min-treatment with phorbol 12-myristate 13-acetate (PMA) (2x10(-7) M) increased Rb+ influx in resting cells by 75.7% without affecting r (0.75). PKC inhibition by H-7, and PKC down-regulation by 24-h PMA (10(-6) M) treatment decreased the bradykinin-induced stimulation of Rb+ influx (+31% and +14.9% above control, respectively). Both down-regulation and inhibition of PKC dramatically reduced the furosemide-sensitive Na+/K+/Cl- cotransport, as r fell to 0.239 and 0.032 in bradykinin-stimulated cells after H-7 and 24-h PMA treatments, respectively. BAPTA/AM pretreatment (10(-4) M, 60 min), which complexed with [Ca2+]i, not only prevented the bradykinin-induced [Ca2+]i raise, but also partially inhibited bradykinin-induced Rb+ influx stimulation (+39% above control), without modifying r (0.76). We conclude that stimulation of PKC is a major pathway involved in bradykinin stimulation of Rb+ influx in NIH-3T3 fibroblasts, and that rises in [Ca2+]i participate in bradykinin signalling, possibly through PKC activation. Our data also suggest that active PKC is required for basal and bradykinin-stimulated Na+/K+/Cl- cotransport activity in these cells.

Furosemide-sensitive K+ transport in transformed and nontransformed rat liver epithelial cells: regulation by protein kinase C and involvement in cell growth.

Using Rb+ as a K+ tracer and atomic absorption spectrophotometry for measuring the Rb+ stable isotope, we studied K+ transport systems and their regulation by protein kinase C in nontransformed and spontaneously transformed rat liver epithelial cells. Ouabain-sensitive Na+/K(+)-ATPase and the furosemide-sensitive Na+/K+/Cl- cotransport had comparable activity ratios in both cell types (0.92 and 1 in nontransformed and transformed rat liver epithelial cells, respectively). The protein kinase C activators, dioctanoylglycerol and phorbol myristate acetate, partly inhibited the Na+/K+/Cl- cotransport in both cell types but their effect was stronger in nontransformed cells, suggesting that, in transformed cells, the Na+/K+/Cl- cotransport had partly lost the ability to be inhibited by protein kinase C. In both cell types, phorbol myristate acetate had little and dioctanoylglycerol had no inhibitory effect on Na+/K(+)-ATPase. Furosemide (1 mM) partly inhibited the [3H]thymidine incorporation in both cell types, suggesting an involvement of the Na+/K+/Cl- cotransport in rat liver epithelial cell growth.

[Absolute and relative bioavailability of germanium in the rabbit]. / Biodisponibilités absolue et relative du germanium chez le lapin.

The debated consumption of germanium suggested the authors to compare biopharmaceutical parameters of germanium oxide and germanium sesquioxide. A first evaluation, in rabbit, has been based on Germanium blood levels determined by atomic absorption spectrometry, after cross administration of both products by the I.V. and oral routes. When given orally, the apparent oxide bioavailability is very low (about 10%) but better than that of the sesquioxide. That difference could result from differences of disposition parameters of both products, which have to be studied late.

[Comparative urinary elimination of caffeine in sedentary and sportsmen after administration of Guronsan]. / Elimination urinaire comparée de la caféine chez le sédentaire et le sportif aprés administration de GURONSAN.

The authors present the results of a study on urinary excretion of caffeine, after a single oral intake of 100 mg of caffeine, in two populations of students at rest and during exercise. Whether expressed in mg/l or mg/g creatinine no significant difference in urinary excretion of caffeine was observed between the two populations and it proves to be lower than the limit level authorized by the IOC (12 mg/l).

[Subacute and subchronic oral toxicity of beta-bis carboxyethyl sesquioxide of germanium in the rat]. / Toxicités orales subaiguë et sub chronique du beta-bis carboxyéthyl sesquioxyde de germanium, chez le rat.

After a brief recall of toxicological data about germanium compounds, the authors relate subacute and subchronic oral toxicities of beta bis carboxyethyl-germanium sesquioxide in rats. During 28 days and six months, male and female animals have received 1 mg/kg/day. No particular toxic symptoms, no behaviour trouble except a small decrease of body weight, in male rats, at the end of the 6-month experimentation, were observed. A light decrease of erythropoiesis and a general stimulation of cellular metabolism has been noticed after 28 days. The only marked effect was a moderate renal deficiency characterized by a tubular disease with presence of cylinders, swelling of tubulus cells and floculus amounts after 6 months. Germanium urinary excretion was constant and linked to the received dose. Six months later, no preferential accumulation in organs was observed.