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.

Particle size, chemical composition, seasons of the year and urban, rural or remote site origins as determinants of biological effects of particulate matter on pulmonary cells.

Particulate matter (PM), a complex mix of chemical compounds, results to be associated with various health effects. However there is still lack of information on the impact of its different components. PM2.5 and PM1 samples, collected during the different seasons at an urban, rural and remote site, were chemically characterized and the biological effects induced on A549 cells were assessed. A Partial Least Square Discriminant Analysis has been performed to relate PM chemical composition to the toxic effects observed. Results show that PM-induced biological effects changed with the seasons and sites, and such variations may be explained by chemical constituents of PM, derived both from primary and secondary sources. The first-time here reported biological responses induced by PM from a remote site at high altitude were associated with the high concentrations of metals and secondary species typical of the free tropospheric aerosol, influenced by long range transports and aging.

Season linked responses to fine and quasi-ultrafine Milan PM in cultured cells.

Exposure to urbane airborne particulate matter (PM) is related to the onset and exacerbation of cardiovascular and respiratory diseases. The fine (PM1), and quasi-ultrafine (PM0.4) Milan particles collected during different seasons have been characterised and the biological effects on human epithelial lung A549, monocytes THP-1 cells and their co-culture, evaluated and compared with the results obtained on the PM10 and PM2.5 fractions. Chemical composition and transmission electron microscopy (TEM) analysis of PM0.4 showed that this fraction was very similar to PM1 for biological responses and dimension. All the winter fractions increased within 1h the level of reactive oxygen species (ROS), while only summer PM2.5 had this effect on A549 cells. The phosphorylation of H2AX (γH2AX), a marker of double strand DNA breaks (DSBs), was increased by all the winter fractions on A549 and THP-1 cells while summer PM samples did not induced this effect. PM0.4 and PM1 biological effects are partly similar and related to the season of sampling, with effects on ROS and DNA damage induced only by winter PM fractions. The winter PM damaging effect on DNA correlates with the presence of organic compounds.

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.

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.

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.

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.

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells.

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of 137CS gamma-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation.

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.

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.

Cellular and Molecular Targets of Benzo[a]pyrene and Metal Toxicity in Xenopus laevis Embryos and in Hep G2 Cells.

This paper describes the use of two in vitro systems (stage 35 Xenopus laevis embryos and the human hepatoblastoma cell line, Hep G2) to study effects of some environmental contaminants (benzo[a]pyrene, copper and zinc), which are representative of chemicals with different cell targets and mechanisms of action. The ability to activate benzo[a[pyrene and to metabolise it with the cytochrome P4501A isozyme were demonstrated in both in vitro systems by assessing the formation of water-soluble and protein-bound benzo[a]pyrene metabolites and by immunochemical analysis. In X. laevis embryos, the formation of DNA adducts demonstrated the ability to produce benzo[a]pyrene reactive metabolites. Moreover, in Hep G2 cells, the cytoskeletal protein, tubulin, and the reduced form of glutathione proved to be the cellular targets of copper and zinc toxicity. In response to metal-induced stress in Hep G2 cells, there was a cytoplasmic reorganisation of heat shock protein, Hsp 70. In conclusion, the in vitro systems provide for a rapid evaluation of heterogeneous compounds such as benzo[a]pyrene and heavy metals that differ in toxic potency and mechanisms of action. They could also be used to study the mechanisms of toxic action and to identify specific cellular and molecular targets.

Physical-chemical and ecotoxicological evaluation of water based drilling fluids used in Italian off-shore.

In order to evaluate the effects on the marine ecosystem caused by an eventual discharge into sea of water based drilling fluids, as current legislation allows, chemical and ecotoxicological analyses were performed on the most common drilling muds and products used in Italian off-shore activities. The chemical analysis on drilling fluids involved the leaching test and the measurement of total content of heavy metals, whereas biodegradation tests were performed on the products used in mud's formulations. As for ecotoxicological evaluation, two marine organisms, the crustacean Artemia salina and the diatom Phaeodactylum tricornutum, were selected to determine the LC50 and the EC50 respectively.

Molecular approaches to evaluate pollutants.

Many organisms are in use to test pollutants and their extensive variability clearly emerges from reviews since researchers in the world are involved in continuous effort to set up new assays and to improve those already in use. In the present paper we focus the attention on the mixed function oxidase system and the DNA adduct formation which are two biomarkers widely used and extensively studied in mammals and fish by different Authors. We compare their results with the ones we obtained in amphibians, which result to be a good model. Moreover we present some significative results obtained by the use of cultured cell lines to test the herbicide MCPA. The results obtained demonstrate that the amphibian Xenopus is a suitable indicator for induction of cytochrome P-450 by B[a]P as well as for production of DNA adducts. Cultured cells evidenced that cytoskeletal array and thiol proteins are molecular targets of the herbicide used, demonstrating that risk assessment can be properly analysed in in vitro systems.

Human-derived cell lines to study xenobiotic metabolism.

In vitro systems make for rapid identification of xenobiotic effects and can be used to study cellular and subcellular toxicity mechanisms. In this report the metabolic competence of two human-derived cell lines, a hepatic (Hep G2) and a pulmonary one (A549) was tested. In the two cell systems the capability to activate Benzo[a]Pyrene through the cytochrome P450 enzyme system and to form reactive metabolites was analysed. 3H-BaP and the scintillation counting analysis were used to show the differences of the metabolic activity in Hep G2 and A549. A similar time course of 3H-BaP uptake was observed in the cell systems. Nevertheless, in the two cell lines the distribution of radioactive metabolites seemed to reflect a specific tissue response to toxicity.

Identification of a multixenobiotic resistance mechanism in Xenopus laevis embryos.

In the '90's a membrane-associated transport protein, discovered in aquatic organisms, was considered to be expressed in response to environmental xenobiotics. Like the multidrug resistance protein found in mammalian tumor cell lines, this protein confers resistance in organisms in polluted areas by binding xenobiotics and transporting them out of the cells in an energy-dependent manner. This study investigates the expression and the activity of a P-glycoprotein (Pgp) involved in a multixenobiotic resistance mechanism (MXRM) during the early developmental stages and in tissues of adult Xenopus laevis.

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.

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.

Lethality, teratogenicity and growth inhibition of heptanol in Xenopus assayed by a modified frog embryo teratogenesis assay-Xenopus (FETAX) procedure.

The frog embryo teratogenesis assay-Xenopus (FETAX), a powerful test for the presence of developmental toxicants, has been modified mainly by performing an in vitro fertilization and increasing the exposure time to 112 h. The modified assay (modFETAX) that presents several advantages over the original FETAX methodology has been validated by the use of ZnSO4, a standard teratogen for FETAX. The modFETAX has been applied to evaluate the 1-heptanol effects on mortality, malformation and growth inhibition. The results indicate that heptanol causes a significant growth inhibition of Xenopus tadpoles and that LC50 and TC50 at 120 h are, respectively, 1.49 and 0.37 mM; the resulting teratogenic index (TI50) of 4.03 suggests that heptanol is a strong teratogen.