Survival and infectivity of Toxoplasma gondii and Cryptosporidium parvum oocysts bioaccumulated by Dreissena polymorpha.

AIMS: The study was aimed to understand the depuration process of Cryptosporidium parvum and Toxoplasma gondii oocysts by zebra mussel (Dreissena polymorpha), to consider the use of the zebra mussel as a bioremediation tool. MATERIALS AND METHODS: Two experiments were performed: (i) individual exposure of mussel to investigate oocyst transfers between bivalves and water and (ii) in vivo exposure to assess the ability of the zebra mussel to degrade oocysts. RESULTS: (i) Our results highlighted a transfer of oocysts from the mussels to the water after 3 and 7 days of depuration; however, some oocysts were still bioaccumulated in mussel tissue. (ii) Between 7 days of exposure at 1000 or 10 000 oocysts/mussel/day and 7 days of depuration, the number of bioaccumulated oocysts did not vary but the number of infectious oocysts decreased. CONCLUSION: Results show that D. polymorpha can release oocysts in water via (pseudo)faeces in depuration period. Oocysts remain bioaccumulated and infectious oocyst number decreases during the depuration period in zebra mussel tissues. Results suggest a degradation of bioaccumulated C. parvum and T. gondii oocysts. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the potential use of D. polymorpha as a bioremediation tool to mitigate of protozoan contamination in water resources.

Cytotoxic and genotoxic evaluation of different synthetic amorphous silica nanomaterials in the V79 cell line.

The nature of occupational risks and hazards in industries that produce or use synthetic amorphous silica (SAS) nanoparticles is still under discussion. Manufactured SAS occur in amorphous form and can be divided into two main types according to the production process, namely, pyrogenic silica (powder) and precipitated silica (powder, gel or colloid). The physical and chemical properties of SAS may vary in terms of particle size, surface area, agglomeration state or purity, and differences in their toxicity potential might therefore be expected. The aim of this study was to compare the cytotoxicity and genotoxicity of representative manufactured SAS samples in Chinese hamster lung fibroblasts (V79 cells). Five samples from industrial SAS producers were evaluated, that is, two pyrogenic SAS powders (with primary particle sizes of 20 nm and 25/70 nm), one precipitated SAS powder (20 nm) and two precipitated SAS colloids (15 and 40/80 nm). V79 cell cultures were treated with different concentrations of SAS pre-dispersed in bovine serum albumin -water medium. Pyr (pyrogenic) 20, Pre (precipitated) 20 and Col (colloid) 15 significantly decreased the cell viability after 24 h of exposure, whilst Pyr 25/70 and Col 40/80 had negligible effects. The cytotoxicity of Pyr 20, Pre 20 and Col 15 was revealed by the induction of apoptosis, and Pyr 20 and Col 15 also produced DNA damage. However, none of the SAS samples generated intracellular reactive oxidative species, micronuclei or genomic mutations in V79 cells after 24 h of exposure. Overall, the results of this study show that pyrogenic, precipitated and colloidal manufactured SAS of around 20 nm primary particle size can produce significant cytotoxic and genotoxic effects in V79 cells. In contrast, the coarser-grained pyrogenic and colloid SAS (approximately 50 nm) yielded negligible toxicity, despite having been manufactured by same processes as their finer-grained equivalents. To explain these differences, the influence of particle agglomeration and oxidative species formation is discussed.

Induction of sulfadiazine resistance in vitro in Toxoplasma gondii.

We induced sulfadiazine resistance in two sulfadiazine sensitive strains of Toxoplasma gondii, RH (Type I) and ME-49 (Type II) in vitro by using drug pressure. At first, sulfadiazine susceptibility of the two sensitive strains and two naturally resistant strains of T. gondii was evaluated on Vero cells using an enzyme-linked immunosorbent assay (ELISA). The IC(50) values of sulfadiazine were 77 µg/mL for RH, 51 µg/mL for ME-49 and higher than 1000 µg/mL for the two natural resistant strains. Secondly, induced resistance of the strains by gradually increase sulfadiazine concentration was verified by this test, which resulted IC(50) values at higher than 1000 µg/mL. In conclusion we developed in vitro two sulfadiazine resistant strains called RH-R(SDZ) and ME-49-R(SDZ). These strains resistant to sulfadiazine would be useful to characterize resistance mechanisms to sulfadiazine.

In vitro cytotoxicity and transforming potential of industrial carbon dust (fibers and particles) in syrian hamster embryo (SHE) cells.

Carbon fibers have many applications, mainly in high-tech industries such as the aviation industry. Eleven carbon samples (fibers and particles) coming from an aeronautic group were tested for their cytotoxicity and carcinogenic potential using in vitro short-term assays in Syrian hamster embryo cells. These samples were taken during each important step of the process, i.e. from the initial heating of polyacrylonitrile fibers to pure carbon fibers. They were compared to an asbestos fiber, an amorphous silica, and two commercial graphite powders. Their physical-chemical characteristics and their capacity to release reactive oxygen species (ROS) were determined. This study showed that none of the carbon samples was able to generate ROS as measured by Electron Paramagnetic Resonance analysis, and in our biological assays, they demonstrated no morphological transformation potential and low cytotoxicity compared to positive control (chrysotile asbestos).

Characteristics of microtubules at the different stages of neuronal differentiation and maturation.

The developing nervous system has proved to be a very powerful tool to analyze how MT are involved in basic biological processes such as cell proliferation, cell migration, cell shaping, and transport. A better knowledge of the basic events occurring during neurogenesis also affords us the possibility of establishing the basis of experiments and trying to solve unanswered and important questions. Despite the considerable value of cell culture, we need to use more discrete regions of the developing brain in situ in order to analyze the MT and their modifications into cells developing their "natural" environment. One major problem remains the question of the mode of assembly and disassembly, that is, the behavior of MT in living cells. Dynamic instability and/or treadmilling are accurate interpretations of the dynamics of MT at least in vitro or in cell culture, but we do need more information on what happens in situ and in vitro. One of the main tasks of cell biologists is to devise satisfactory tests to approach this fundamental question. In this view, pharmacological manipulation of embryos treated in whole-embryo culture systems might be a possible way. Microtubules are ubiquitous cell components. However, the extensive heterogeneity of MAP and tubulin in the CNS confers on the neurons a wide range of capabilities of assembly of these proteins and suggests that the neuron has a unique potential of a relation between MT composition and cell function. We have seen that each major event during neurogenesis is related to a specific series of modifications of the MT components. It remains to be determined if there is a causal or just a correlative relationship between the appearance of specific isotypes and the occurrence of specific events and/or functions. We have also to determine the exact spatial and temporal relations among the different isotypes of MT proteins, tubulin, and MAP. Is there a close correspondence between a tubulin and a MAP isotype? Can the appearance of one isotype of tubulin influence the appearance and the assembly of a specific MAP, or vice versa? Recent results obtained with the Tyr- and Glu-MT shed light on these questions and suggest a whole series of possibilities for cells to modulate the structure, behavior, and function of MT in specific domains of the neuron or in specific regions of the brain, by only a minute modification of the molecule of tubulin. Microtubule protein heterogeneity raises also a number of questions.(ABSTRACT TRUNCATED AT 400 WORDS)

Differential gene expression in mesothelioma.

To investigate the molecular events controlling malignant transformation of human pleural cells, we compared constitutive gene expression of mesothelioma cells to that of pleural cells. Using cDNA microarray and high-density filter array, we assessed expression levels of > 6500 genes. Most of the highly expressed transcripts were common to both cell lines and included genes associated with stress response and DNA repair, outcomes consistent with the radio- and chemo-resistance of mesothelioma. Interestingly, of the fewer than 300 genes that differed between cell lines, most functioned in (i) macromolecule stability, (ii) cell adhesion and recognition, (iii) cell migration (invasiveness), and (iv) extended cell division. Expression levels of several of these genes were confirmed by RT-PCR and could be useful as diagnostic markers of human mesothelioma.

Modifications of microtubule proteins in ALS nerve precede detectable histologic and ultrastructural changes.

Fast axonal transport is altered in nerves from amyotrophic lateral sclerosis (ALS) patients. Microtubules are involved in axonal transport. We analyzed the possibility of an involvement of microtubule modifications underlying the alterations in transport using biochemical and morphologic analysis of intercostal nerves from ALS and control patients. In intercostal nerves displaying no morphologic signs of acute neuronal degeneration, two-dimensional gels showed modifications of the group of beta tubulins and abnormal spots of proteins, some appearing to be closely related to tau proteins. These results suggest that microtubule proteins are modified in ALS before ultrastructural axonal degeneration, but the significance of these abnormalities remains hypothetical.

In situ analysis of the action of Navelbine on various types of microtubules using immunofluorescence.

Preliminary clinical studies demonstrated that 5' nor-anhydro-vinblastine, Navelbine (NVB) has a broader antitumor activity and fewer neurotoxic effects than vinblastine or vincristine. The tectal plate anlage of mouse embryos at the earliest stages of neuronal differentiation were used to analyze and compare the effect of NVB, vincristine and vinblastine on axonal and mitotic microtubules after culture of post-implantation embryos in a medium containing the agent. All drugs are active on mitotic microtubules at the same concentration (0.1 mumol/L), inducing a depolymerization of microtubules and a blockade of cells at metaphase. At higher concentrations. NVB is the only one of the three drugs that induces a blockade of the cells at prophase. A depolymerization of axonal microtubules occurs at higher concentrations with NVB than with the two other vinca alkaloids. These results demonstrate that NVB is as active on mitotic microtubules and less active on axonal microtubules than vincristine and vinblastine. These findings can be related to the potent antitumor effect of the drug with minor neurotoxicity.

Biochemical effects of Navelbine on tubulin and associated proteins.

Navelbine (NVB) or 5' nor-anhydro-vinblastine was shown to present a broader antitumor activity and to induce fewer side effects than vinblastine (VBL) or vincristine (VCR). The possible mechanisms of these differences were analyzed with in vitro methods. At substoichiometric concentrations, the three drugs inhibit microtubule assembly. NVB, in comparison with VCR and VBL, is shown to have a lower inhibitory effect. At stoichiometric concentrations, the three drugs are able to induce tubulin aggregation into spirals and paracrystals. This process involves a microtubule-associated protein (MAPs) family referred to as Tau and is inhibited by another MAPs family referred to as MAP2. However, dramatic quantitative and qualitative differences are observed between NVB and VLB or VCR in TAU-induced aggregation of tubulin. The rate and extent of NVB-induced tubulin aggregation is much lower. With NVB, only certain TAU isoforms are able to induce paracrystals, while all TAU isoforms may contribute to VCR-induced or VBL-induced paracrystals. The TAU isoforms that are not able to induce crystallization with NVB, at least in a certain range of concentrations, are probably involved in mitotic microtubules--the hypothetical antitumoral target of vinca alkaloids (VAS). The present work shows for the first time that an anticancer drug is able to discriminate between the various types of microtubules. A next step will be to investigate whether this property is limited to a modulating effect of the various TAU isoforms on the affinity of VAS for tubulin. These biochemical investigations will be extended to tubulins extracted from tumor cell lines in order to further discriminate NVB from the other VAS.

Inhaled crocidolite mutagenicity in lung DNA.

We used transgenic mice carrying the lacI reporter gene to study the mutagenesis potential of asbestos crocidolite. The animals were exposed by nose-only inhalation to an aerosol containing 5.75 mg/m(3) crocidolite dust for 6 hr/day and 5 consecutive days. After 1, 4, and 12 weeks, we examined four end points: the cytology of bronchoalveolar lavage, the lung load of crocidolite, the hydrophobic DNA adducts, and the mutations in the lacI reporter gene. Twelve weeks after exposure, nearly 10% of the inhaled fibers remained in the lung (227 +/- 103 ng/mg lung). There was evidence of a typical inflammatory response consisting of multinucleate macrophages at weeks 4 and 12, whereas immediately after the exposure, we observed numerous polymorphonuclear neutrophils. The mutant frequency significatively increased during the fourth week after the exposure: 13.5 [time] 10(-5) in the exposed group versus 6. 9 10(-5) in the control group. The induction factor, defined by the ratio of checked mutants of exposed mice to checked mutants of control mice, was 1.96. The mutation spectrum of control lung DNA and exposed lung DNA was similar, suggesting the possible involvement of a DNA repair decrease in crocidolite-treated animals. We used the (32)P-postlabeling method and did not detect any increase of either 5 mC or bulky adduct in treated mice. This is the first study that demonstrates asbestos mutagenicity in vivo after a nose-only inhalation.

Occupational cancer in France: epidemiology, toxicology, prevention, and compensation.

This article is a description of the current situation in France with regard to occupational cancer: research, prevention, and occupation. Toxicologic experiments are carried out using (italic)in vitro(/italic) and (italic)in vivo(/italic) tests, particularly using transgenic mice. Several epidemiologic studies have been conducted over the last decades: population-based case-control studies; mortality studies and cancer incidence studies carried out in historical cohorts of workers employed in the industry; and case-control studies nested in occupational cohorts. French ethical aspects of toxicologic and epidemiologic studies are described. The results thus obtained are used to establish regulations for the prevention and the compensation of cancers attributable to occupational exposure. This French regulation for prevention of occupational cancer involves several partners: (italic)a(/italic)) the states authorities, including labor inspectors, responsible for preparing and implementing the labor legislation and for supervising its application, particularly in the fields of occupational health and safety and working conditions; (italic)b(/italic)) the Social Security Organisation for the analysis of present or potential occupational risks based on tests, visits in plants, complaints or requests from various sources, and statistics. These activities are performed within the framework of the general French policy for the prevention of occupational cancer. This organization includes the National Institute for Research and Safety, particularly involved in research in the various fields of occupational risks--animal toxicology, biologic monitoring, exposure measurements epidemiology, psychology, ergonomy, electronic systems and machineries, exposure to chemicals, noise, heat, vibration, and lighting; and (italic)c(/italic)) companies where the regulation defines the role of the plant manager, the occupational physician, and the Health, Safety and Working Conditions Committee (comprising the manager, employees' representatives, the occupational physician, and the safety department) in dealing with any problem regarding safety, occupational hygiene, and working conditions. These organizations along with medical practitioners are involved with the compensation of occupational cancers. The regulation for compensation includes the tables of occupational cancer, the possibility of recognition of a cancer case when the requirements of the tables are not met, and the postprofessional follow-up of workers exposed to a carcinogenic agent.

Regional specificity of tubulin heterogeneity in adult mouse brain.

Eight alpha and twelve beta isoforms of tubulin were isolated from discrete regions of the mouse brain using high-resolution isoelectric focusing and identified by two-dimensional gel electrophoresis and immunoreactivity. In the different regions, the number of isoforms was identical, but their relative proportion varied except for alpha 2, 3, 4, 5, 6 and beta 1 bands. The patterns of the inferior and superior colliculi were nearly similar. The cerebellum, compared with the inferior and superior colliculi is characterized by a decrease of alpha band 7 and beta bands 3 and 7 and an increase of alpha band 8 and beta bands 2, 4, 10, 11, 12. The forebrain displays an intermediate pattern between the cerebellum and the colliculi. These results suggest that in functionally different regions of the brain the number of isotypes of tubulin is identical but their relative proportion differs with an apparent correlation between the function of the neuronal subpopulations and the pattern of isotubulins.

Modifications of tubulin heterogeneity during axonal growth in the embryonic nervous system.

Tectal plates of mouse embryos were used to analyze the heterogeneity of tubulin with high resolution isoelectric focusing, at time of appearance of the young neurons and axons. In the cold-labile pool of tubulin, isotypes 6 and 7 appear, and isotypes 1, 3, 11, 13 increase their relative quantities. The increase of the prominence of the alpha-group with regard to the beta-group in the cold-stable pool of tubulin raises the question as to the exact role of tubulin in the cold stability. A role of other specific microtubule components also appears probable.

Biochemical basis of microtubule cold stability in the peripheral and central nervous systems.

Cold-stable, cold-labile and unpolymerized tubulins extracted from thalamic nuclei (soma-enriched fraction) and various nerves (both central and peripheral: axon-enriched fractions) appear different when analyzed by high-resolution isoelectric focusing. Cold-labile tubulin appears identical to unpolymerized tubulin. The axonal fractions contain fewer tubulin isotypes than the soma-enriched fraction; the peripheral axonal fraction has fewer isotypes than the central fraction. Cold-stable tubulin exhibits a specific pattern characterized by the abundance of two isotypes of alpha-tubulin, 7 and 8, and one beta-tubulin, isotype 9, with slightly different patterns of the axon-enriched fractions from the central and peripheral nervous systems. Our results suggest that the cold stability of microtubules is based on biochemical properties of tubulin, and confirm the domain specificity of the heterogeneity of tubulin.

In situ appearance of the cold-stable microtubules in the growing axons of the tectal plate of mouse investigated immunocytochemically after polyethyleneglycol (PEG) embedding.

Tubulin immunostaining of semi-thin sections after polyethylene glycol embedding was used in the tectal plate of the embryonic mouse at 10 days postmating to analyze the effects of cold treatment on the microtubules of the different cell types seen at this stage. Three sets of microtubules are observed. In the radially oriented bipolar columnar cells, dense bundles of microtubules are present in the ventricular processes between the cell nucleus and the ventricular surface. In the mitotic cells, located just at the surface of the ventricle, microtubules are among condensed chromosomal figures. In the apical region, the intermediate zone, tangentially oriented axonal profiles contain dense bundles of microtubules among tangentially oriented young neurons. Cold treatment does not modify the organization of the cells. However, it depolymerizes whole cytoplasmic and mitotic microtubules of the bipolar cells and a large number of microtubules in the growing axons. In the axonal profiles, the cold-stable fraction of microtubules displays the appearance of short fragments. Some of these are regularly organized, suggesting that they could be the remnants of the same individual microtubule. These fragments are approximately 1 micron long and seem to represent nearly 10% of the total microtubules in the axons. These cold-stable fragments might fulfill a function in the axon analogous to the microtubule organizing centers in the perikaryon and their presence can explain some properties of the growing axons suggested by previous studies on the guidance of neurites and growth cones as well as on the growth of isolated axons.

Modifications of tubulin heterogeneity during embryonic and postnatal stages in a specific region of mouse brain.

An auditory nucleus (the inferior colliculus of the mouse) was used to study the modifications of the heterogeneity of tubulin which occur at various stages of development and maturation. Both the cold-stable (CS) and cold-labile (CL) fractions of tubulin were analyzed by high-resolution isoelectric focusing. Our results suggest that tubulin heterogeneity is modified at critical stages of development and maturation, with specific variations of the two fractions. Stage E10 corresponding to the appearance of the first young neurons and axonal profiles is marked by the modification of the CS fraction with the emergence of isotypes alpha 5 to alpha 8 and beta 12 and beta 17. Stage E12 is characterized by the modifications of the CL fraction, particularly the beta-group; at this stage the first dendrites become visible. At birth, all isotypes increase in both the CL and CS fractions. At stages P7-P10 transient modifications of a group of both CS and CL fractions and of the beta group of CS fraction occur. These are associated with the emergence of isotypes beta 17 to beta 20 in the CL fraction. This period precludes the initial period of functional maturation of the auditory system which occurs from P10 to P20. During this period, the CL fraction (alpha and beta group) remains unmodified, whereas all isotypes of the CS fraction, except 3-4, display complex variations with an initial decrease until P12, an increase until P17, and a final decrease until P20.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneity of cold-stable and cold-labile tubulin in axon- and soma-enriched portions of the adult mouse brain.

Microtubules from the optic nerve (axonal tubulin) and lateral geniculate nucleus (cell tubulin) were separated by cold treatment and the cold-soluble fraction was purified in the presence of Taxol. Isoforms of cold-stable and cold-soluble tubulin were resolved by the use of high-resolution isoelectric focusing. The cold-soluble fraction of axonal tubulin has only 14 of the 20 isotypes seen in the same fraction of cell tubulin. The two cold-stable fractions have 20 isotypes but axonal tubulin has a specific pattern of isotypes 1, 2 and 5. Cold-stable fractions of both axonal and cell tubulin display the existence of an intensely stained alpha-isotype, isotype 7, which seems associated with the property of cold stability. Our results highly favor the hypothesis of a physiological role of the heterogeneity of tubulin in neuronal microtubules.

Quantitative analysis of monoclonal immunoglobulins in serum of patients with amyotrophic lateral sclerosis.

In a prospective study, we analysed the presence of monoclonal immunoglobulin (moIg) in the serum from 30 patients with amyotrophic lateral sclerosis (ALS) and 30 matched controls using a sensitive Western blot technique. The incidence of serum moIg was 60% in the ALS group and 13.3% in the control group. Most ALS sera contained 2 or 3 monoclonal components. They were IgG (72.7%) and IgM (27.3%). These results corroborate the concept of a probable association between ALS and serum moIg.

Effects of nickel sulfate on pulmonary natural immunity in Wistar rats.

This study was designed to investigate the in vivo effect of nickel sulfate on the pulmonary non-specific immune defences. Groups of four male Wistar rats were treated with a single intratracheal instillation of NiSO(4) at different doses: 1, 2, 4 and 8 micromol of NiSO(4) per rat. Control rats received a corresponding instillation of the saline vehicle. The effect of NiSO(4) on the cytotoxic activity of the pulmonary natural killer (NK) cells and alveolar macrophages (AM), as well as the pulmonary production of cytokines such as alpha-tumor necrosis factor (TNF-alpha) and gamma-interferon (IFN-gamma), were examined 1, 2 and 7 days later. Spontaneous NK-cytotoxicity towards mouse-derived tumor cell line, Yac-1 was suppressed 1 day after treatment at doses of 2 micromol/rat and above with only one result significant (P<0.05); 2 days after treatment the suppression was increased with all results significant at the same doses; 1 week after treatment NK activity restoration was observed except for the highest dose, 8 micromol/rat. AM-mediated cytotoxicity towards mouse-derived tumor cell line, 3T12, did not show any significant difference in treated and untreated animals. In contrast, whereas moderate levels of TNF-alpha were detected in the broncho-alveolar lavage (BAL) fluid supernatants of controls, the NiSO(4) treatment highly suppressed TNF-alpha production with a maximum observed after 2 days. TNF-alpha suppression was found to be transient, at least with the lowest NiSO(4) dose, with levels returning to normal after 7 days. A non-significant increase in IFN-gamma was observed in the BAL fluids of treated animals at each time of examination. Taken together, these results indicate that NK cell activity and TNF-alpha secretion are sensitive targets for instilled NiSO(4) in Wistar rats.

Histopathological study in B6C3F1 mice chronically exposed by inhalation to glutaraldehyde.

Glutaraldehyde vapors are irritating for the skin, eyes, nose and lungs; respiratory symptoms and headaches have been described among workers exposed to low concentrations of glutaraldehyde far below to 190 ppb. This study was initiated to determine the chronic effects in mice of inhaled glutaraldehyde vapors. B6C3F1 mice were exposed using whole-body inhalation chambers, 6 h/day, 5 days/week, for 52 and 78 weeks to 100 ppb, or to filtered air (controls). In nasal passages at the level of the vestibule, hyperplasia of the squamous epithelium lining of the dorsal wall and lateral aspect of the atrioturbinate was observed in a greater number of exposed females than in controls. Epidermal erosion and ulceration as well as squamous and inflammatory exfoliation were also seen in the nasal lumens. All these changes were dependent on the length of glutaraldehyde exposure. The present data suggest that glutaraldehyde long term exposure only led to changes in nasal passages of female mice but did not induce mortality and/or tumors in nasal passages, in all mice. These results, along with the previous subchronic inhalation study of Gross et al., 1994, demonstrates that in a long term study, chronic glutaraldehyde exposure close to the current threshold limit values induced lesions at the more anterior part of the nasal passages in mice and that they likely result from an irritation mechanism (antero-posterior gradient).