Long-term effects after treatment with platinum compounds, cisplatin and [Pt(O,O'-acac)(γ-acac)(DMS)]: Autophagy activation in rat B50 neuroblastoma cells.

Cisplatin (cisPt), among the best known components of multi-drug front-line therapies used for the treatments of solid tumors, such as the childhood neuroblastoma, acts through DNA linking. Nevertheless, the cisPt effectiveness is compromised by the onset of severe side effects, including neurotoxicity that results in neurodegeneration, cell death, and drug-resistance. In the field of experimental oncology, aimed at overcoming cytotoxicity and chemoresistance, great efforts are devoted to the synthesis of new platinum-based drugs, such as [Pt(O,O'-acac)(γ-acac)(DMS)] (PtAcacDMS), which shows a specific reactivity with sulfur residues of enzymes involved in apoptosis. Autophagy, an evolutionary conserved degradation pathway for recycling of cytoplasmic components, represents one of the mechanisms adopted by cancer cells which contribute to drug-resistance. In the present study, standard acute (48 h-exposure) and long-term effects (7 day-recovery after treatment or 7 day-recovery followed by reseeding and 96 h-growth), of cisPt and PtAcacDMS (40 and 10 µM, respectively) were investigated in vitro employing rat B50 neuroblastoma as a cancer model. Using fluorescence and electron microscopy, as well as biochemical techniques, our data highlight a key role of the autophagic process in B50 cells. Specifically, long-term effects caused by cisPt lead to inhibition of the apoptotic process and paralleled by the activation of autophagy, thus evidencing that autophagy has a protective role after cisPt exposure, allowing cells to survive. Whereas, long-term effects produced by PtAcacDMS lead toward both apoptosis and autophagy activation. In conclusion, autophagy may represents an alternative cell death pathway, circumventing drug-resistance strategies employed by cancer cells to survive chemoterapy.

Single Silver Nanoparticle Instillation Induced Early and Persisting Moderate Cortical Damage in Rat Kidneys.

The potential toxic effects of silver nanoparticles (AgNPs), administered by a single intratracheal instillation (i.t), was assessed in a rat model using commercial physico-chemical characterized nanosilver. Histopathological changes, overall toxic response and oxidative stress (kidney and plasma protein carbonylation), paralleled by ultrastructural observations (TEM), were evaluated to examine renal responses 7 and 28 days after i.t. application of a low AgNP dose (50 µg/rat), compared to an equivalent dose of ionic silver (7 µg AgNO3/rat). The AgNPs caused moderate renal histopathological and ultrastructural alteration, in a region-specific manner, being the cortex the most affected area. Notably, the bulk AgNO3, caused similar adverse effects with a slightly more marked extent, also triggering apoptotic phenomena. Specifically, 7 days after exposure to both AgNPs and AgNO3, dilatation of the intercapillary and peripheral Bowman's space was observed, together with glomerular shrinkage. At day 28, these effects still persisted after both treatments, accompanied by an additional injury involving the vascular component of the mesangium, with interstitial micro-hemorrhages. Neither AgNPs nor AgNO3 induced oxidative stress effects in kidneys and plasma, at either time point. The AgNP-induced moderate renal effects indicate that, despite their benefits, novel AgNPs employed in consumer products need exhaustive investigation to ensure public health safety.

[Pt(O,O'-acac)(γ-acac)(DMS)] versus cisplatin: apoptotic effects in B50 neuroblastoma cells.

Cisplatin is one of the most active chemotherapeutic agents used in the treatment of childhood and adult malignancies. Cisplatin induces cell death through different pathways. Despite its effectiveness, the continued clinical use of cisplatin is limited by onset of severe side effects (nephrotoxicity, ototoxicity and neurotoxicity) and drug resistance. Therefore, one of the main experimental oncology purpose is related to the search for new platinum-based drugs to create different types of adducts or more specific and effective subcellular targets. Thus, [Pt(O,O'-acac)(γ-acac)(DMS)], which reacts preferentially with protein thiols or thioether, was synthesized. In our research, different approaches were used to compare cisplatin and [Pt(O,O'-acac)(γ-acac)(DMS)] effects in B50 rat neuroblastoma cells. Our results, using immunocytochemical, cytometric and morphological techniques, showed that these compounds exert a cytostatic action and activate apoptosis with different pathways. Long-term effects demonstrated that [Pt(O,O'-acac)(γ-acac)(DMS)] exerts cytotoxic effects in neuronal B50 cell line not inducing drug resistance. Analysis was performed both to compare the ability of these platinum compounds to induce cell death and to investigate the intracellular mechanisms at the basis of their cytotoxicity.

One-month persistence of inflammation and alteration of fibrotic marker and cytoskeletal proteins in rat kidney after Cd-doped silica nanoparticle instillation.

In vivo effects of model cadmium-containing silica nanoparticles (SiNPs-Cd, 1 mg/rat) were investigated by i.t. instillation in rats to evaluate potential effects on secondary target organ, e.g., kidney. Specific endpoints and pathological outcomes were focused to assess inflammation and fibrosis in renal tissue, 7 and 30 days after exposure to SiNPs-Cd, as well as to equivalent amount of CdCl2 or SiNPs. Immunohistochemistry was employed to investigate the presence/distribution of selected markers, i.e., (i) TGF-ß1, (ii) IL-6 (iii) collagen (type I), (iv) fibronectin, and (v) vimentin. An ongoing inflammatory process was demonstrated, together with a general overexpression of extracellular matrix components and alteration of cytoskeletal proteins, mainly in cortex and medulla, 7 days after SiNPs-Cd, lasting until 30th day. Less pronounced effects were observed after CdCl2, while SiNPs did not cause any insult except for IL-6 expression increase. Briefly, engineered SiNPs-Cd cause long-lasting injury in rat kidney, following a single pulmonary exposure. Renal response may be due to (i) translocation, i.e., nanoparticles migration from lung to systemic circulation, or (ii) secondary organ changes, caused by circulating inflammatory factors (e.g., IL-6, TGF-ß1) released from lung following local insult, or (iii) direct renal action of cadmium ions released from the absorbed SiNPs-Cd.

Biological effects of the aqueous extract of Bridelia grandis stem bark on normal and neoplastic human cells: an in vitro preliminary evaluation.

The aim of the work is to investigate the effects of Bridelia grandis (Pierre ex Hutch) stem bark water extract on human HeLa cancer cells and normal monocytes treated in vitro, evaluating the morphological modifications with light and electron microscopy. The phytocomplex obtained from B. grandis caused a significant decrease in the mitotic index of both HeLa cancer cells and normal monocytes. In addition, a reduction of the typical aneuploid-polyploid pattern has been observed in HeLa cells after treatment. Various alterations at fine structural level, both in neoplastic (HeLa cells) and normal (monocytes) cells have been observed. In particular, electron-dense cells containing condensed mitochondria, autophagic vacuoles and dense spherical cytoplasmic inclusions have been observed. The results show that B. grandis water extracts have an antiproliferative effect on human cells, with a different effect on neoplastic and normal cells. The antiproliferative effect is accompanied by the appearance of various subcellular alterations. The morphological alterations observed are likely to represent the condition of 'dark cell' as a possible preliminary phase towards the autophagic and/or apoptotic cell death.

Apoptosis induction and histological changes in rat kidney following Cd-doped silica nanoparticle exposure: evidence of persisting effects.

Abstract Histological and immunocytochemical methods were used to examine rat's renal responses to intratracheal (i.t.) instillation of model cadmium-containing silica nanoparticles (Cd-SiNPs) and also exploring whether these potential modifications would be associated with toxicogenomic changes. Renal effects of Cd-SiNPs (1 mg/rat), CdCl2 (400 µg/rat), SiNPs (600 µg/rat) or 0.1 ml saline (control), assessed 7 and 30 d post-i.t., included (i) induction of apoptosis, (ii) cell proliferation and (iii) the overall toxic response evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, proliferating cell nuclear antigen (PCNA) immunohistochemistry as well as Periodic acid Schiff and Hematoxylin & Eosin, respectively. Area-specific apoptosis was observed in all treatment groups, the cortex and inner medulla being the most affected regions: the apoptotic changes were apparent seven days post-exposure in both areas and were still observable in inner medulla at day 30. Apoptotic frequency increase was more pronounced in Cd-SiNP-treated animals compared to either CdCl2 or SiNPs groups. At day 7, the observed parallel increased number of PCNA immunopositive cells may be associated with an enhanced cell proliferation aimed at replacing the damaged cells. Histopathological findings demonstrated comparable morphological changes of the renal structure (at glomerular and tubular levels) occurring after all treatments at both time-points and more markedly 30 d after instillation. Both morphological and toxicogenomic evaluations confirmed long-lasting renal effects of Cd-SiNPs on apoptosis and regulatory processes. Bare SiNPs i.t. administration caused morphological and apoptotic changes but did not modify gene expression profile in kidney. These findings substantiate the notion that multiple assays and an integrated testing strategy should be recommended to characterize toxicological responses to nanoparticles in mammalian systems.

Pulmonary toxicity of instilled cadmium-doped silica nanoparticles during acute and subacute stages in rats.

Potential risk associated with new nanomaterial exposure needs to be assessed. This in vivo study investigated pulmonary effects of engineered cadmium-containing silica nanoparticles Cd/SiNPs (1 mg/rat), silica SiNPs (600 µg/rat) and CdCl2 (400 µg/rat) 1, 7 and 30 days after intratracheal instillation. Comprehensive histopathological and immunocytochemical characterization of lung damage in terms of apoptosis, cell proliferation, inflammation, fibrosis and metabolism were obtained. After exposure to all treatments, lung parenchyma showed injury patterns characterized by collapsed alveoli, inflammation, granuloma formation, thickened alveolar septa and bronchiolar epithelium exfoliation. Type II pneumocytes, containing scarcely surfactant-lamellated bodies, were also observed. Apoptotic phenomena enhanced as following, Cd/SiNPs>CdCl2> SiNPs. In parallel with these findings, a significant increase of PCNA-immunoreactive cells was detected together with high mitotic activity. Cellular localization and distribution of IL-6, IP-10 and TGF-ß1 revealed an increased expression of these cytokines as evidence of an enhanced cellular inflammatory response. CYP450-immunoreactivity was also enhanced, at bronchiolar (e.g. Clara cells) and alveolar (e.g. macrophages) level after both Cd/SiNPs and CdCl2. These overall effects were observed acutely and lasted until the 30th day, with Cd/SiNPs producing the most marked effects. Collagen-immunolabelling changed particularly 7 and 30 days after Cd/SiNPs, when a strong stromal fibrogenic reaction occurred. The present findings suggest that Cd/SiNPs produce significantly greater pulmonary alterations than either SiNPs or CdCl2 under the present experimental conditions.

Mitochondrial fusion: a mechanism of cisplatin-induced resistance in neuroblastoma cells?

Cisplatin induces apoptosis through different pathways. The intrinsic apoptotic pathway is mediated by mitochondria, which, as a result of cisplatin treatment, undergo morphological alterations. The aim of this study was to investigate cisplatin-induced mitochondrial functional and morphological long-term effects in neuroblastoma B50 rat cells. To this purpose, we followed evaluated different several apoptotic markers by means of flow cytometry, confocal and electron microscopy and western blotting techniques. We applied different treatment protocols based on the incubation of the neuroblastoma B50 rat cells with 40 µM cisplatin: (i) for 48 h and harvesting of the cells at the end of the treatment; (ii) further recovery in drug-free medium for 7 days post-treatment; (iii) conditions as in (ii) followed by re-seeding in normal medium and growth for a further 4 days. We observed apoptosis induction after the first treatment and after the recovery from cell death after long-term culture in drug-free medium. Interestingly, the latter phenomenon was characterized by mitochondrial elongation and mitochondrial protein rearrangement. In recovered and re-seeded cells, mitochondrial equilibrium moved toward fusion, possibly protecting cells from apoptosis.

Long-lasting oxidative pulmonary insult in rat after intratracheal instillation of silica nanoparticles doped with cadmium.

Silica/cadmium containing nanomaterials are now produced on industrial scale due to their potential for a variety of technological applications. Nevertheless, information on toxicity, exposure and health impact of these nanomaterials is still limited. In this study, in vivo effects of silica nanoparticles (SiNPs) doped with Cd (SiNPs-Cd, 1mg/rat), soluble CdCl(2) (400 µg/rat), or SiNPs (600 µg/rat) have been investigated by evaluating F(2)-isoprostanes (F(2)-IsoPs), superoxide dismutase (SOD1), inducible nitric oxide synthase (iNOS) and cyclooxygenase type 2 (COX-2) enzymes, as markers of oxidative stress, 24h, 7 and 30 days after intra-tracheal (i.t.) instillation to rats. Free and esterified F(2)-IsoPs were evaluated in lung and plasma samples by GC/NICI-MS/MS analysis, and SOD1, iNOS and COX-2 expression in pulmonary tissue by immunocytochemistry. Thirty days after exposure, pulmonary total F(2)-IsoPs were increased by 56% and 43% in CdCl(2) and SiNPs-Cd groups, respectively, compared to controls (32.8 ± 7.8 ng/g). Parallel elevation of free F(2)-IsoPs was observed in plasma samples (by 113% and 95% in CdCl(2) and SiNPs-Cd groups, respectively), compared to controls (28 ± 8 pg/ml). These effects were already detectable at day 7 and lasted until day 30 post-exposure. Pulmonary SOD1-, iNOS-, and COX-2-immunoreactivity was significantly enhanced in a time-dependent manner (7 days <30 days) after both CdCl(2) and SiNPs-Cd treatments. SiNPs did not influence any of the evaluated endpoints. The results indicate the capacity of engineered SiNPs-Cd to cause long-lasting oxidative tissue injury following pulmonary exposure in rat.

Effects of Cisplatin in neuroblastoma rat cells: damage to cellular organelles.

Cisplatin (cisPt) is a chemotherapy agent used as a treatment for several types of cancer. The main cytotoxic effect of cisplatin is generally accepted to be DNA damage. Recently, the mechanism by which cisPt generates the cascade of events involved in the apoptotic process has been demonstrated. In particular it has been shown that some organelles are cisPt target and are involved in cell death. This paper aims to describe the morphological and functional changes of the Golgi apparatus and lysosomes during apoptosis induced in neuronal rat cells (B50) by cisplatin. The results obtained show that the cellular organelles are the target of cisPt, so their damage can induce cell death.

Cell proliferation and death in the brain of active and hibernating frogs.

'Binomial' cell proliferation and cell death have been studied in only a few non-mammalian vertebrates, such as fish. We thought it of interest to map cell proliferation/apoptosis in the brain of the frog (Rana esculenta L.) as this animal species undergoes, during the annual cycle, physiological events that could be associated with central nervous system damage. Therefore, we compared the active period and the deep underground hibernation of the frog. Using western blot analysis for proliferating cell nuclear antigen (PCNA), we revealed a positive 36 kDa band in all samples and found higher optical density values in the hibernating frogs than in active frogs. In both active and hibernating frogs, we found regional differences in PCNA-immunoreactive cells and terminal transferase dUTP nick-end labelling apoptotic cells in the ventricular zones and parenchyma areas of the main encephalon subdivisions. During the active period of the frogs, the highest concentration of PCNA-immunoreactive cells was found in the ventricle dorsal zone of the cerebral hemispheres but only some of the cells were apoptotic. By contrast, the tectal and cerebellar ventricular zones had a small or medium amount of PCNA-immunoreactive cells, respectively, and a higher number of apoptotic cells. During hibernation, an increased PCNA-immunoreactive cell number was observed in both the brain ventricles and parenchyma compared with active frogs. This increase was primarily evident in the lateral ventricles, a region known to be a proliferation 'hot spot'. Although differences existed among the brain areas, a general increase of apoptotic cell death was found in hibernating frogs, with the highest number of apoptotic cells being detected in the parenchyma of the cerebral hemispheres and optic tectum. In particular, the increased number of apoptotic cells in the hibernating frogs compared with active frogs in the parenchyma of these brain areas occurred when cell proliferation was higher in the corresponding ventricular zones. We suggest that the high number of dying cells found in the parenchymal regions of hibernating frogs might provide the stimulus for the ventricular zones to proliferate. Hibernating frogs could utilize an increased cell proliferation in the brain areas as a neuroprotective strategy to face cell death and the onset of neurological damages. Therefore, the hibernator promises to be a valuable model for studying the mechanisms naturally carried out by the central nervous system in order to adapt itself or survive adverse conditions.

Exposure to heptachlor: evaluation of the effects on the larval and adult epidermis of Rana kl. esculenta.

Widely used in the past against termites and soil insects, the chlorinated insecticide heptachlor (H) is a toxic contaminant which represents a risk for both terrestrial and aquatic organisms. Like many organochlorine pesticides, heptachlor and heptachlor epoxide (HE), with oxidation products synthesized by many plant and animal species, degrade slowly since many of the derived compounds are persistent. This increases the status of heptachlor as a hazardous pollutant. In the present experimental study we exposed specimens of Rana kl. esculenta, from the tadpole stage through to their complete metamorphosis, to three different concentrations of heptachlor (4, 40 and 400 ppb). Mortality and HE bioaccumulation were evaluated on all the experimental groups. Since amphibian integument directly interacts with the environmental constituents (water, air and soil), we investigated the toxic effects on the ventral epidermis of both tadpole and adult samples by employing such histo-cytopathological biomarkers as ultrastructural morphology, certain enzyme activities (acid and alkaline phosphatases, AcPase, and AlkPase; succinic dehydrogenase, SDH; alpha-naphtyl butyrate esterase, ANBE; nitric oxide synthase/NADPH diaphorase, NOS/NADPHd). Also, the levels of reactive oxygen species (ROS) in the different conditions were evaluated. The results obtained were of ecological relevance, in particular as regards the effects of this environmental toxicant on the samples of tadpole epidermis. Severe morphological alterations were observed in the larval epidermal cells (apical and skein cells), whereas the cell epidermis (keratinocytes and mitochondria-rich cells) of the adult survivors showed changes in enzyme activities, particularly those involved in the protective response to xenobiotic injury. In general, morpho-histochemical studies, analysis of HE bioaccumulation and mortality showed a relation to the H doses employed.

Specific rearrangement of the microtubular network during the cell cycle phases and correlation with micronucleation and death induced by anticancer drugs in NIH/3T3 subcultures.

The changes of the microtubular network induced by microtubule destabilizing (Vinblastine, VBL) and stabilizing (Taxol, TAX) agents were studied in NIH/3T3 fibroblastic cells in conventional culture conditions (NIHb) and in a subpopulation (NIHs), obtained after serum deprivation and expressing different morphofunctional features and higher cytokinetic activity. In this cell model, we analyzed VBL and TAX effects on cell cycle and microtubular network, in relation to cell death. In NIHb cells, VBL induced higher microtubule depolymerization, prevalence of tubulin paracrystals and micronucleation, while, in NIHs cells, lower depolymerization and appearance of tubulin spiral-like structures, with lower micronucleation, increase of apoptosis and disappearance of high polyploid cells. DNA static cytofluorometry of cells showing paracrystals or spirals permitted correlation of the appearance of these tubulin aggregation forms with the cell cycle phases. In NIHb cultures, the DNA content curves, in cells with paracrystals or spirals, showed a similar trend, with a higher frequency of the two anomalies in the G2/M phase. In NIHs cultures, paracrystals and spirals are found in G2/M cells, while G1 cells showed prevailingly paracrystals. TAX induced the appearance of microtubule bundles in the two cultures. The prevalence of circular bundles was found in NIHb cells, while a higher number of linear bundles was shown in NIHs cells. In NIHb cells, circular bundles were related to higher apoptosis and micronucleation. DNA cytofluorometry, in cells with linear or circular bundles, showed that the latter was present with high frequency in NIHb cells in all the cell cycle phases; in NIHs cells, they appeared, with lower frequency, prevailing in the S-G2/M phase. Furthermore, in NIHs cells, the appearance of linear bundles in G1 cells was related to a lower micronucleation. These finding showed that microtubule reorganization in different cell cycle phases could play a role in the progression of nuclear fragmentation/micronucleation relating to cell death.

Correlation between the liver temperature employed during machine perfusion and reperfusion damage: role of Ca2+.

This study compares the effects of machine perfusion (MP) at different temperatures with simple cold storage. In addition, the role of Ca(2+) levels in the MP medium was evaluated. For MP, rat livers were perfused for 6 hours with Krebs-Henseleit (KH) solution (with 1.25 or 2.5 mM CaCl(2)) at 4 degrees C, 10 degrees C, 20 degrees C, 25 degrees C, 30 degrees C, or 37 degrees C. For cold storage, livers were perfused in situ and preserved with Celsior solution at 4 degrees C for 6 hours. The reperfusion period (2 hours at 37 degrees C) was performed under the same conditions used for MP-preserved and cold storage-preserved livers. Hepatic enzyme release, bile production, adenosine triphosphate (ATP) levels, and morphology were evaluated during MP and reperfusion. MP at 37 degrees C caused marked enzyme release; the same findings were obtained during reperfusion. By contrast, MP temperature lowering induced a significant decrease in liver damage. High levels of biliary gamma-glutamyltransferase and lactate dehydrogenase were found with MP at 4 degrees C and 10 degrees C but not with MP at 20 degrees C. When a KH-1.25 mM CaCl(2) solution was used during MP at 20 degrees C, very low enzyme release was observed and significantly lower hepatic damage was present at the end of the reperfusion period in comparison with cold storage. The same results were obtained when ruthenium red, a calcium uniporter blocker, was added to KH-2.5 mM CaCl(2). ATP levels were higher and morphology was better in liver preserved with KH-1.25 mM CaCl(2). MP at 20 degrees C with KH-1.25 mM CaCl(2) resulted in better quality liver preservation, improving hepatocyte and endothelial biliary cell survival, in comparison with cold storage. This raises the need to reconsider the temperature and calcium levels to be used during liver MP.

Cisplatin treatment of NIH/3T3 cultures induces a form of autophagic death in polyploid cells.

The effects induced by different concentrations (50, 75, 100 microM) of the cytostatic drug cisplatin (cDDP) in NIH/3T3 cells were analyzed. Sub-confluent cultures of this mouse fibroblast line, obtained after serum deprivation, showed the presence of aneuploid/polyploid cells with ploidy values ranging from 4c to 24c. DNA content cytofluorometry demonstrated that 50 and 75 microM cDDP induced a cytostatic effect; 100 microM concentration showed lower antiproliferative action. All treatments caused a partial cell detachment and apoptosis, the incidence of which appeared to be cDDP concentration-dependent. Ultrastructural and fluorescence microscopy integrated analyses of the still adherent cells demonstrated the presence of alternative degeneration patterns, especially in polyploid cells, with extensive modifications at both nuclear and cytoplasmic levels. There were events of micronucleation and phenomena of multilobulation and furrows of the nucleus that preceded the formation of heterogeneous fragments. These events were correlated, at cytoplasmic level, with actin reorganization and the appearance of autophagocytotic processes. In our cell model, the same pharmacological treatment was able to induce different cell death phenomena relating to cell dimension and ploidy. More actively proliferating cells (2c-4c DNA content) die throughout canonical apoptosis, while polyploid cells prevailingly degenerate by mechanisms partly referable to autophagic cell death.

Iron accumulation in mammary tumor suggests a tug of war between tumor and host for the microelement.

Iron is indispensable for the metabolism and proliferation of both normal and malignant cells. Recycling from senescent erythrocytes in the liver and spleen is critical for iron supply to all tissues. In the liver and spleen from MMTV-neu (erbB-2) mice bearing a mammary carcinoma, we noticed the scarcity of hemosiderin pigment and its abundance in the stroma of the tumor. Thus iron (III) was investigated with the Perls' reaction in tissues from normal and MMTV-neu mice. With respect to normal animals, in MMTV-neu mice, staining for iron was almost absent in the liver and scarce in the red pulp of the spleen. By contrast, iron was abundant in stromal and tumor cells in the invasion, angiogenic, necrotic and hemorrhagic regions and also in the interstitial fluid. These observations suggest that the tumor subverts iron recycling to its own advantage, by directly utilizing iron released from erythrocytes and dead tumor cells. Our findings are in keeping with the development of iron chelating drugs as chemotherapic agents.

Expression of cell kinetics and death during monocyte-macrophage differentiation: effects of Actinomycin D and Vinblastine treatments.

The different effects of two cytostatic drugs, Actinomycin D and Vinblastine, during macrophage-like differentiation induced in THP-1 monocytic cell line by phorbol ester phorbol 12-myristate 13-acetate (PMA) (6, 30, and 60 nM), were studied by morpho-cytochemical approaches. In PMA-unstimulated monocytic cells, the cytostatic effects of Actinomycin D (an antimetabolic drug) were characterized by a drastic reduction of the G2/M cells accompanied by dramatic death of the G1 cells; on the contrary, Vinblastine (a microtubule-depolymerizating drug) induced an accumulation of the G2/M cells with the appearance of aneugenic micronuclei and scarce cell death mainly from the G1 cells. After 60 nM PMA stimulation, the culture was mostly composed by macrophagic cells characterized by low proliferation and the appearance of mono-/binucleated polyploid cells; in this condition, the cytotoxicity of the two drugs, more effective for Vinblastine, induced cell death in the different ploidy classes (2c, 4c, 8c). Cell death appeared to be of apoptotic nature, but with some morpho-phenotypic differences due to the action mechanism of the drugs and dependent on cell culture growth and differentiation. As a consequence of the different block-action of the two drugs on the cell cycle phases and in relation to the different subcellular targets, the effects changed during the transition from not-adhering/proliferating monocytes to adhering/low-proliferating differentiated macrophages.

Evaluation of Rana snk esculenta blood cell response to chemical stressors in the environment during the larval and adult phases.

The assessment of the biological effects on aquatic vertebrate species is frequently employed to monitor water pollution, as it provides significant information on bioavailability and actual concentration levels. In anamniote vertebrates (fish and amphibians), significant correlations have been observed between exposure to contaminants - both natural and experimental - and blood modification. We investigated the changes in some circulating blood cell parameters of green frog (Rana snk esculenta) tadpoles and adults collected at two sample rice fields, one heavily polluted and the other relatively unpolluted. The frequency of eosinophilic leucocytes, mitotic, anucleated and micronucleated erythrocytes was evaluated also regarding the haemopoietic/haemocatheretic and NOS expression of the liver. Haematological indicators in polluted samples were found to be significantly different from controls as regards both larval and adult exposure, and provided information on long-term background pollution of the habitats under investigation. The population of the polluted area showed evident effects of chronic exposure to contaminants, to a degree which could lead to sub-lethal alterations of their health status. The general nature of responses to this kind of stress emphasizes the role of amphibian peripheral blood as a sensitive indicator regarding contamination in aquatic environments.

In situ demonstration of improvement of liver mitochondria function by melatonin after cold ischemia.

In a previous investigation, reperfusion with a melatonin-containing medium was demonstrated to enhance bile production and tissue ATP levels in rat livers, cold-preserved with University of Wisconsin (UW) or Celsior solutions, with respect to melatonin-free reperfusion; lipid peroxidation products in the perfusate were not influenced by the indole. This was ascribed to an increased efficiency of the hepatocyte mitochondria induced by melatonin. Reactive oxygen species (ROS) normally leak from the electron transfer chain in mitochondria and excessive ROS production is presumed to mediate ischemia-reperfusion (I/R) damage. A histochemical reaction was used to demonstrate ROS on the same model. Compared to the lobular zonation of ROS in control livers, the stained area of cold-preserved livers reperfused without melatonin was restricted to a narrow portal region, in keeping with the much lower ATP content. When reperfusion was performed with melatonin, the liver morphology was improved and the ROS reaction in hepatocytes more intense, though not reaching the control liver pattern. Sinusoidal cells were poorly-stained in both cases. In conclusion, with this different approach, melatonin was confirmed to improve mitochondrial performance and to discriminate parenchymal from sinusoidal cell behavior. Our observations confirm that melatonin mitigates I/R injury and support its potential in liver transplantation.

In situ assessment of oxidant and nitrogenic stress in bleomycin pulmonary fibrosis.

Reactive oxygen species (ROS) and nitric oxide (NO) have a role in the development of pulmonary fibrosis after bleomycin administration. The ROS production induces an antioxidant response, involving superoxide dismutases (SODs), catalase, and glutathione peroxidases. We compared in situ oxidative burden and antioxidant enzyme activity in bleomycin-injured rat lungs and normal controls. ROS expression and catalase, glucose-6-phosphate-dehydrogenase (G6PHD), and NOS/NADPH-diaphorase activity were investigated by using histochemical reactions. Nitric oxide synthase (e-NOS and i-NOS) and SOD (MnSOD, Cu/ZnSOD, ECSOD) expression was investigated immunohistochemically. After treatment ROS production was enhanced in both phagocytes and in type II alveolar epithelial cells. Mn, Cu/Zn, and ECSOD were overexpressed in parenchymal cells, whereas interstitium expressed ECSOD. Catalase and G6PHD activity was moderately increased in parenchymal and inflammatory cells. NOS/NADPH-d activity and i-NOS expression increased in alveolar and bronchiolar epithelia and in inflammatory cells. It can be suggested that the concomitant activation of antioxidant enzymes is not adequate to scavenge the oxidant burden induced by bleomycin lung damage. Inflammatory cells and also epithelial cells are responsible of ROS and NO production. This oxidative and nitrosative stress may be a substantial trigger in TGF-beta1 overexpression by activated type II pneumocytes, leading to fibrotic lesions.