Organic extract of tire debris causes localized damage in the plasma membrane of human lung epithelial cells.

The potential toxicity of tire debris organic extracts on human alveolar epithelial cells (A549) was investigated. We analysed time- and dose dependent modifications produced on plasma membrane molecular composition and on lipid microdomains expression (caveolae and lipid rafts) that represent specific signalling platforms. Cells were exposed to increasing organic extract concentrations (10, 60 and 75mug/ml) for 24, 48 and 72h. An up to three fold dose and time dependent increase in specific protein markers of lipid microdomains was found, suggesting a corresponding increase in signalling platforms. Since the total pool of these plasma membrane markers was unchanged, we supposed that these proteins were translocated within the plasma membrane as to assemble the newly formed lipid microdomains. Despite no major modifications in lipid bilayer composition, a time- and dose dependent toxic effect was documented at 48h of exposure by an increase of cells positive to Trypan Blue assay. After 48h a dose dependent increase in the cell medium of the cytosolic enzyme lactate dehydrogenase was also observed, indicating greater damage of the plasma membrane as prenecrotic sign. The overall ultrastructural morphology of the plasma membrane of treated cells was not greatly modified, suggesting that organic extracts from tire debris cause focalized discontinuities on cell surfaces.

Graphite particles induce ROS formation in cell free systems and human cells.

It is commonly accepted that the toxicity of carbonaceous particulate matter (PM) is due to the production of reactive oxygen species (ROS) which induce biological damage in the exposed cells. It is also known that PM produced during the combustion processes consists of a carbonaceous core "dressed" with other organic and/or inorganic materials. In spite of this knowledge, the role of these materials in the production of ROS has not yet been clear. This work aims at understanding whether "naked" carbonaceous particles are capable of forming ROS either in cell-free or in-cell systems. The problem has been treated based on the data collected from pure graphite samples of different sizes obtained by ball-milling pure graphite for various lengths of time. The experimental approach considered Raman, ESR (spin trapping), cell viability and fluorescence spectroscopy measurements. These techniques allowed us to carry out measurements both in cell and cell-free systems and the results consistently indicate that also pure naked carbonaceous particles can catalyze the electron transfer that produces superoxide ions. The process depends on the particle size and enlightens the role of the edges of the graphitic platelets. Evidence has been collected that even "naked" graphitic nanoparticles are capable of producing ROS and decreasing the cell viability thus representing a potential danger to human health.

Histopathological effects induced by paraquat during Xenopus laevis primary myogenesis.

The oxidative agent paraquat induced tail abnormalities during Xenopus laevis development. Specimens exposed from blastula to the tadpole stage revealed pear-shaped myocytes and irregular intersomitic boundaries. The histological feature of the axial musculature was evaluated in embryos sampled at significant stages of the primary myogenesis. During the somitogenesis PQ-treated embryos showed normal appearing myotomes, but reduced PAS activity in the post-rotating myotomal cells, and myoblasts with slight vacuolations. Once etched from the vitelline envelope, embryos showed severely altered myoblasts with irregular cellular apexes, heavy sarcoplasmic vacuolations, pyknotic nuclei and disorganizing intersomitic boundaries. Myotomes with many necrotic myocytes containing disorganized contractile material and heavily malformed intersomitic boundaries characterized the late myogenic stages. Our results evidence the heaviest PQ histopathological effects to affect myogenesis of post-etched embryos, suggesting a possible linkage between the swimming activity and the oxidative damage to muscle tissue.

Immunocytochemical localization of calmodulin in intact and acrosome-reacted boar sperm.

Localization of calmodulin on intact and acrosome-reacted ionophore A23187 induced boar sperm has been performed with indirect immunofluorescence and immunoelectron microscopy. The results obtained with immunofluorescence are in agreement with previous reports. Immunoelectron microscopy was performed with the methods of preembedding immunostaining and on-grid post-embedding immunostaining of Lowicryl K4M sections with the protein A-colloidal gold. The presence of calmodulin is demonstrated in the acrosomal content and in the equatorial and postacrosomal regions of intact sperm. Apparently, calmodulin is released in association with the plasma membrane and the outer acrosomal membrane in acrosome-reacted sperm and it is maintained in the equatorial and postacrosomal regions. These results provide further evidence that also in mammalian sperm multiple classes of calmodulin-binding proteins may be present.

Identification of actin in boar spermatids and spermatozoa by immunoelectron microscopy.

Actin was localized in testicular spermatids and in ionophore-treated ejaculated sperm of boar by use of a monoclonal anti-actin antibody labeled with colloidal gold. With the on-grid postembedding immunostaining of Lowicryl K4M sections, actin was identified in the subacrosomal region of differentiating spermatids, in the microfilaments of the surrounding Sertoli cells, and in the myoid cells of the tubular wall. Ejaculated sperm, labeled with the preembedding method, showed actin between the plasma membrane and the outer acrosomal membrane of the equatorial segment. Indirect immunofluorescence was positive in the equatorial segment and in the acrosomal cap of intact sperm, whereas reacted sperm at the anterior head region retained fluorescence only in the inner acrosomal membrane. Rhodamine-phalloidin failed to stain intact and reacted sperm. The distribution of actin in sperm head membranes (inner acrosomal membrane, membranes of the equatorial segment), which are retained after the acrosome reaction, is discussed.

Immunocytochemical identification of actin in rabbit spermiogenesis and spermatozoa.

Actin was localized in testicular spermatids and in spermatozoa of rabbit by using a monoclonal anti-actin antibody and a specific antiserum against actin, labeled with colloidal gold. The antibody reactivity with sperm homogenates was determined by immunoblotting of one-dimensional gels. With on-grid postembedding immunostaining of Lowicryl K4M sections, actin was identified in the subacrosomal region of differentiating spermatids, and in four bulges situated between the inner acrosomal membrane and the nuclear envelope and in the anterior part of the postacrosomal region of ejaculated spermatozoa. Sperm actin was identified on two-dimensional gels as two spots in the isoelectric point and molecular weight corresponding to gamma and beta-isoforms of actin. Immunoblots stained with specific antibodies demonstrated that rabbit spermatozoa express gamma and beta-actin isoforms.

The effects of anticalmodulin drugs on the ultrastructure of boar spermatozoa.

Trifluoperazine, N-6-aminohexyl-5-chloro-1-naphthalene sulfonamide (W7), and calmidazolium are known to be calmodulin inhibitors and cell membrane soluble substances. In mammalian spermatozoa, calmodulin is present and is retained to mediate several sperm processes, such as sperm activation, sperm-egg fusion, microtubule disassembly, etc. We examined the effects of anticalmodulin drugs on the ultrastructure of freshly ejaculated boar spermatozoa. Whereas all the drugs, at the low concentrations tested, appear to prevent acrosomal alterations, at higher concentrations, they induced these alterations. Unexpectedly, the outer acrosomal membrane appeared to be more sensitive to the drugs than the plasma membrane; vesicles formed within the acrosome from the outer acrosomal membrane even when plasma membrane maintained its structural integrity. These findings were confirmed by the analysis carried out by fluorescent light microscopy by utilizing fluoresceinated Ricinus communis agglutinins to specifically stain the acrosomes.

The molecular architecture of an insect midgut brush border cytoskeleton.

The cytoskeletal apparatus of the vertebrate intestinal brush border (BB) has served as a model system for the actin-based cytoskeleton of nonmuscle cells. In this study, we examine the structural organization and molecular architecture of the BB cytoskeleton expressed in the midgut of lepidopteran larvae, Manduca sexta. Electron microscopy of the midgut of the 5th instar larvae revealed enterocytes with an apical BB surface comparable to that in the vertebrate intestine, with both microvillar (MV) and terminal web (TW) domains, the latter defined by a zone of organelle exclusion directly beneath the MV. As reported previously for the larval dragon fly, the MV contain a bundle of actin filaments, as determined by staining with rhodamine phalloidin (Kukulies, J., et al., Protoplasma 121, 157-162 (1984)) and heavy meromyosin decoration (Komnick, H., J. Kukulies, Zoomorphology 107, 241-253 (1987)). Two-dimensional gel analysis revealed the presence of multiple isoelectric variants of actin with the major isoform corresponding to the non-muscle actin isoform II, expressed in Drosophila. Like the vertebrate BB, the Manduca BB can be isolated intact from enterocytes by mechanical shear. Immunochemical analysis of isolated BB fractions or whole homogenates of midgut revealed proteins of appropriate molecular weight immunoreactive with antibodies to the MV core proteins: BB myosin I, villin and fimbrin, and the TW components: spectrin, myosin II and tropomyosin. Immunocytochemical localization of a subset of these proteins at the light microscopic (spectrin) and electron microscopic (actin, villin, spectrin, myosin II, and tropomyosin) level reveals that the molecular architecture of the Manduca BB cytoskeleton is homologous to that found in vertebrates.

Optical scattering (TAOS) by tire debris particles: preliminary results.

Tire debris particles from low severity laboratory wear tests have been investigated by the TAOS optical scattering facility at Yale University. The incident wavelength is 532 nm. After the TAOS event some particle samples have been imaged by a scanning electron microscope and microanalyzed. The TAOS intensity patterns recorded within a solid angle in the backward sector have been processed by cluster analysis and compared with the patterns computed by a T-matrix code. Preliminary agreement has been found between TAOS data and the particle models (size, shape, refractive index). The purpose of the investigation is to obtain signatures of the material, based on its TAOS pattern.

Cytoskeletal elements in mammalian spermiogenesis and spermatozoa.

Identification of the cytoskeletal elements and their role in the formation as well as the maintenance of head membrane compartmentalization is a much debated issue in mammalian spermatozoa. Data which have emerged during the last ten years are summarized. Those which have converged in a common opinion, such as the distribution of actin in mammalian spermiogenesis, are distinguished from those which have to be confirmed, such as the role of actin related proteins and actin in mature spermatozoa.

N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces cytoskeletal alterations on 'Swiss 3T3' mouse fibroblasts.

The effect of the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) on Swiss 3T3 cells was investigated. Cell morphology alterations were observed when 3T3 cell cultures were exposed for 6 h to 1.5 mM MPTP. Using indirect immunofluorescence technique, cytoskeletal elements' organization of microfilaments and microtubules, has been analysed. MPTP modified both actin and tubulin networks, stress fibers appeared less sharp and microtubules were disorganized. The effect of MPTP was completely reversible and cell viability was unaffected. These results suggest that changes in cytoskeletal organization may be the first visible effect related to biochemical alterations induced by MPTP.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) affects the actin cytoskeleton and calcium level of Swiss 3T3 mouse fibroblasts.

Organization of the actin cytoskeleton, the cytosolic free-calcium concentrations and ATP levels were analyzed in 3T3 mouse fibroblasts treated with 0.75 or 1.5 mM MPTP. In the presence of the drug actin filaments were time- and dose-dependently disorganized, ATP level was unaffected and intracellular calcium increased within 5 s. The correlation between MPTP cytotoxicity and [Ca2+]i level emerging from these results, suggests that the primary effect of the molecule itself is on the plasma membrane's integrity for calcium ion regulation.

Benomyl affects the microtubule cytoskeleton and the glutathione level of mammalian primary cultured hepatocytes.

Rat primary hepatocyte cultures have been used to study the effect of Benomyl alone or in combination with Pirimiphos-methyl. The results presented demonstrate that Benomyl alone is responsible for the microtubular disorganization in both a time- and dose-dependent manner, that the effect is reversible after the agent is removed, and that Benomyl is a potent glutathione-depleting agent. Pirimiphos-methyl, alone or combined with Benomyl had no effect on microtubule organization, but reinforced the decrease in glutathione.

Overexpression of HSP70 is induced by ionizing radiation in C3H 10T1/2 cells and protects from DNA damage.

Various kinds of stress such as heat, UV, gamma-rays and chemicals that cause DNA damage induce heat shock proteins (Hsps), and in particular Hsp70. The Hsps cytoprotective function is not fully understood, although these proteins act as molecular chaperones or modulators of intracellular levels of reactive oxygen species (ROS). Recently, Hsps have been proposed to play a significant role in DNA repair after UV or gamma-ray irradiation. Ionizing radiation targets DNA molecules either via direct interaction or via production of free radicals and ROS. When exposed to gamma-rays C3H 10T1/2 cells are radiosensitive, therefore we decided to use them to investigate Hsp induction after ionizing radiation and their protective role against DNA damage. Here we demonstrate the induction of Hsps by gamma-rays, and investigate the kinetics of expression after irradiation at different doses. We also show that Hsp70 overexpression acts as a radioprotective mechanism towards the first event of DNA damage and increases long term viability. A preliminary investigation on the cell cycle does not evidence a significant protective action of inducible Hsp70 on it.

Copper and zinc uptake and hsp70 expression in HepG2 cells.

The aim of this work is to study the accumulation in HepG2 cells of two essential metals with toxic potency and to analyse the induction of the heat shock protein 70 kDa (hsp70) consequent to metal exposure. Cu and Zn were the metals considered and were analysed both as single compounds and in combination in order to evidence synergic effects of the mixture. The use of HepG2 cells provided an in vitro system that retains morphological and metabolic properties and the expression of specific genes typical of liver parenchymal cells. Moreover, the hepatic cells represent a suitable model for their susceptibility to metal toxicity since liver, gastrointestinal tract and renal tubular cells are involved in the uptake, transport, detoxification and secretion of these compounds. The uptake of Cu and Zn followed a time-dependent accumulation when they were used separate. The combination of the two metals produced a higher accumulation of Zn. The stress protein hsp70 was expressed before the metals accumulated within the cells, as shown by the measures obtained with the ICP-AES technique. Moreover, the accumulation of hsp70 by a sublethal shock provided a protective mechanism against metal cytotoxicity.

Different induction of metallothioneins and Hsp70 and presence of the membrane transporter ZnT-1 in HepG2 cells exposed to copper and zinc.

Eukaryotic cells respond to stressful environmental stimuli, such as toxic concentrations of heavy metals, by rapidly synthesising defence proteins: the metallothioneins (MT) and the heat shock protein 70 (Hsp70). In this study we have analysed how the human hepatoblastoma cell line HepG2 responds to exposure to excess copper (30 microg/ml) and zinc (50 microg/ml) for long exposure times (48 and 72 h). Accumulation of the two metals, as measured by ICP-AES, was time-dependent reaching a plateau after 72 h. HepG2 cells responded by dramatically increasing levels of MT during stress, mostly during zinc exposure. A time lag in Hsp70 induction was observed as the levels of this protein increased only after removal of the stress from culture medium (recovery) for 24 h, thus suggesting that the two defence mechanisms are not coordinated in a metal-induced stress response. Moreover in HepG2 cells, immunochemical and fluorescence techniques showed the presence and the localisation of the zinc membrane exporter ZnT-1 as a further mechanism of defence/homeostasis against zinc toxicity.

Evidence that a Ca2+ chelator and a calmodulin blocker interfere with the structure of inter-Sertoli junctions.

Ca2+ dependence of tight junction structure has been well documented in cultured epithelial tissues, and regulatory mechanisms have been identified. To analyse the possible control exerted on inter-Sertoli junctions, we exposed guinea-pig seminiferous tubules to the presence of a Ca2+ chelator (EGTA) and to a calmodulin blocker (Trifluoperazine, TFP) in vitro, for times ranging from 30 to 120 min. We observed the morphology of junctional complexes and the basal cytoplasmic regions in sections and replicas. Sertoli cell response to Ca2+ depletion involved several events: retraction of cells toward the base of the tubule and a consequent stretching of the points of fusion, augmented density of the cytoplasm, and destabilization of the array of intramembrane particles. Exposure of tubules to TFP resulted in disruption of the interactions between actin filaments and membrane junctional specialization, as well as a disorganization of other cytoskeletal elements. Thus, in vitro, junction integrity appears to be related to Ca2+ level, and Ca2+ depletion apparently interferes with Ca2+ distribution inside the cell and on microfilaments involved in junction regulation. Our results do not provide direct evidence for any particular mechanism of action of TFP, but a multiple effect is evident. TFP, which affects Ca2+ regulation and membrane fluidity, probably acts indirectly on junction-associated filaments. Both the experimental conditions tested suggest a Ca2+-mediated regulatory role of microfilaments of this complex junction.

Immunoelectron microscopic localization of actin in migrating cells during planarian wound healing.

Wound repair in planarians is mainly characterized by two cell-migratory events involving the epidermis adjacent to the wound and its basement membrane. The first event is the migration of epidermal cells to cover the wound surface; the second one is the migration of newly differentiating replacement epidermal cells from the parenchyma to the epidermis. In addition to these events, migration of fixed parenchymal cells is observed during wound healing. All migrating cells were characterized by the presence of actin, as shown by the results obtained by means of indirect immunolocalization with fluorescent and electron microscopy. Migrating cells were heavily labeled with gold particles, which clustered at the level of cell-matrix and cell-cell contacts.

Actin localization at the tight junctions of invertebrate ciliated epithelia.

Indirect immunofluorescencc, rhodamine-phalloidin staining and immunoelectron microscopy performed with the on-grid postembedding immunostaining of Lowicryl K4M sections, were used to identify actin in the branchial epithelium of the lower chordate ascidians. The ciliated cells of these invertebrates present two distinct junctional patterns. One consists only of an extended tight junction whereas in the other the tight junction is accompanied by a prominent zonula adhaerens. Evidence is given of the localization of actin at the tight junction. The absence of reaction in the zonula adhaerens suggests that the definition of this junction in the model here presented must be reconsidered.

Immunoelectron microscopic localization of a fibronectin-like molecule in Dugesia lugubris s.l.

Fibronectin (FN)-like protein has been localized by immunoelectron microscopy in the extracellular matrix (ECM) of planaria Dugesia lugubris s.l. The immunolabeling was present in both intercellular spaces of epidermal cells and the basement membrane, however the amount and distribution of gold particles seemed to be substantially different. FN-like material increased markedly during the passage of migrating cells through the basement membrane from the parenchyma to the epidermis. Gold particles were often found at cell-matrix contacts. Our result suggest that FN-like molecules detected in planarian ECM may be involved not only in cell adhesion but also in promoting cell migration and in regulating the epidermal cell turnover.