Carla Fenoglio

Mechanisms of changes to the liver pigmentary component during the annual cycle (activity and hibernation) of Rana esculenta L.

The present study was performed to elucidate the mechanisms responsible for the changes of melanin content/distribution we had previously discovered in the liver parenchyma of Rana esculenta during natural hibernation. Melanomacrophagic component response was analysed using morphocytochemical methods. The results demonstrated that during the prehibernation period (October–November) the melanomacrophages reach the highest proliferative activity (BrdU, PCNA labelling) which is accompanied by an evident melanosynthesis (dopa‐oxidase activity). In contrast, after hibernation, the decrease of liver pigmentation was the consequence of a partial cell loss by apoptotic mechanisms (TUNEL labelling, pyknosis‐karyorhexis) accompanied by a decrease of melanosome content by autophagy and low melanosynthetic activity. On the basis of these findings, there is evidence that liver melanomacrophages represent a metabolically (melanin synthesis/degradation) and cytokinetically (proliferation/death) active cell population during the annual cycle of the frog. The results are also discussed in relation to the functional synergism between hepatocytes and pigment cells in the adaptation to environmental changes.

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-β1 overexpression by activated type II pneumocytes, leading to fibrotic lesions.

STYRENE-INDUCED ALTERATIONS IN THE RESPIRATORY TRACT OF RATS TREATED BY INHALATION OR INTRAPERITONEALLY

Although exposure to styrene occurs primarily via inhalation, the action of this agent on the respiratory tract has scarcely been investigated. This article describes morphological and biochemical changes occurring in the respiratory tract of rats after either inhalation of styrene vapors (300 ppm, 6 h/d, 5 d/wk, for 2 wk) or systemic (ip) treatment with 40 or 400 mg/kg styrene for 3 consecutive days. Electron microscopy analysis showed diffuse cell damage involving the tracheal, bronchiolar, and alveolar epithelium. In the tracheal epithelium, several cell types were affected. Ciliated cells presented vacuolation, detachment of cilia, blebbing of the apical cytoplasm, and compound cilia. Most secretory cells showed scant secretory granules and blebbings. Dense bodies and fibrillary inclusions were seen in intermediate and basal cells. Styrene also caused alterations of cytoplasmic components in type II pneumocytes and bronchiolar cells as well as thickness of the alveolar wall. These abnormalities were accompanied by depletion of glutathione (GSH) in the lung tissue. Pneumotoxic effects of systemic administration of styrene were dose dependent and tended to be more severe than those seen in the animals exposed for longer periods to styrene by inhalation. Metabolic activation of styrene and subsequent cell damage induced by the reactive metabolite styrene oxide may be involved in the sequence of events culminating in the toxic insult to the respiratory tract.

Frog hepatocyte modifications induced by seasonal variations: A morphological and cytochemical study

A correlated morphological and cytochemical approach was employed to study frog hepatocytes in different periods of their annual cycle, including the natural hibernating period. There were considerable changes in the distribution and organization of hepatic glycogen in different phases of the annual cycle, and distribution of organelles as well. The most striking findings were glycogen storage during the prehibernation and hibernation phases, followed by drastic glycogen depletion. Cytochemical staining of a number of enzymes (succinate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, paranitrophenyl phosphatase, acid phosphatase, and glucose-6-phosphatase) involved in a variety of metabolic pathways, showed various cytoplasmic localizations and differences in intensity of the reaction products as a function of seasonality. Morphological and cytochemical data were interpreted as evidencing different functional requirements during seasonal changes in the frog.

Time course of matrix metalloproteases and tissue inhibitors in bleomycin-induced pulmonary fibrosis

To investigate simultaneously localization and relative activity of MMPs during extracellular matrix (ECM) remodeling in bleomycin-induced pulmonary fibrosis in rat, we analyzed the time course of the expression, activity and/or concentration of gelatinases MMP-2 and MMP-9, collagenase MMP-1, matrylisin MMP-7, TIMP-1 and TIMP-2, both in alveolar space (cellular and extracellular compartments) and in lung tissue. MMP and TIMP expression was detected (immunohistochemistry) in lung tissue. MMP activity (zymography) and TIMP concentration (ELISA) were evaluated in lung tissue homogenate (LTH), BAL supernatant (BALs) and BAL cell pellet (BALp) 3, 7, 14, and 28 days after bleomycin intratracheal instillation. Immunohistochemistry showed an extensive MMP and TIMP expression from day 7 in a wide range of structural and inflammatory cells in treated rats. MMP-2 was present mainly in epithelia, MMP-9 in inflammatory cells. MMP-2 and MMP-9 activity was increased respectively in BAL fluid and BAL cells, with a peak at day 7. TIMP-1 and TIMP-2 concentration (ELISA) enhancement was delayed at day 14. In conclusion gelatinases and their inhibitors are significantly activated during bleomycin-induced pulmonary fibrosis. Marked changes in gelatinases activity are observed early in the alveolar compartment, with a prevailing extracellular activity of MMP-2 and a predominant intracellular distribution of MMP-9, while enzyme activity changes in lung parenchyma were less evident. In the repairing phase the reduction of gelatinases activity is synchronous with a peak of alveolar concentration of their inhibitors.

Nitric oxide‐containing neurons in the nervous ganglia of Helix aspersa during rest and activity: Immunocytochemical and enzyme histochemical detection

Nitric oxide synthase (NOS) immunoreactivity and staining for nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐diaphorase) activity are two cytochemical markers for nitric oxide (NO)‐containing neurons. The authors examined the changes in the distribution of NOS immunolabeling and NADPH‐diaphorase reactivity in the cerebral and buccal ganglia of the terrestrial snail Helix aspersa during resting and active phases. During inactivity and after 1 day of activity, in the mesocerebrum and metacerebrum of the snails, there were several reactive neurons for both markers; after 7 days of activity, the number of reactive neurons was lower. Opposite results were obtained in the buccal ganglia, in which increased staining and numbers of reactive neurons were present in the active snails (after 1 day and 7 days of activity). Although the staining patterns for the two reactions were similar, colocalization was not always observed. The comparison between inactive and active animals provided a more precise survey of NOS‐containing neurons in the snail cerebral ganglia than previously described. Moreover, it suggested that not only is NO involved in distinct nervous circuits, but, as a ubiquitous molecule, it also plays a role in neuroprotection and neuropeptide release. J. Comp. Neurol. 409:274–284, 1999.

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.

Protective effect of procaine hydrochloride on cisplatin-induced alterations in rat kidney

Efforts have been made to reduce the undesirable side effects of cisplatin, mainly nephro- and neurotoxicity, but their reduction is usually accompanied by a concomitant inhibition of antitumor activity. The local anesthetic procaine hydrochloride (P.HCl) improves the therapeutic index of cisplatin not only by the reduction of its nephro- and hemotoxicity, but also by an increase of its antitumor activity. We therefore investigated the effects of a combined treatment of cisplatin and P.HCl on rat kidneys and compared this to kidneys from rats treated with a toxic dose of cisplatin or P.HCl alone. Treatment with a saline solution was used as control. Dehydrogenase activities [succinate dehydrogenase (SDH) and NADPH diaphorase reaction demonstrating nitric oxide synthase (NOS/NADPHd)] and phosphatase activities [K+ p-nitrophenyl phosphatase (K+ pNPPase), alkaline phosphatase (AlPase) and acid phosphatase (AcPase)] were studied on cryostatic sections of kidneys from controls and treated rats. Evidence of heavy morphological damage and altered AlPase and AcPase activities induced by cisplatin were observed in the S3 segment of the proximal tubules. In addition, SDH and K+ pNPPase activities showed some changes in the distal tubule cells. The NOS/NADPHd activity in macula densa was drastically reduced. Combined treatment of cisplatin and P.HCl greatly attenuated morphological alterations of the rat kidney and reduced the changes in enzyme activities, except for NOS/NADPHd activity, compared to the cisplatin-treated group of animals. The study indicates that, in cisplatin-induced nephrotoxicity, a significant role is played by enzyme activities, in particular K+ pNPPase and NOS/NADPHd, and that P.HCl can mitigate the nephrotoxicity of cisplatin, possibly by influencing some enzyme activities involved in important renal metabolic pathways.

The cerebral neurons of Helix aspersa during hibernation. Changes in the cytochemical detection of calmodulin, cytoskeletal components and phosphatases.

Some markers of the intracellular systems that regulate neuronal activity and morphology were analyzed in the cerebral ganglion of hibernating snails (Helix aspersa), in comparison with active animals. The immunocytochemical expression of a calcium-binding protein, i.e. calmodulin, and some cytoskeletal components, i.e. 200 kDa phosphorylated neurofilament protein (pNFH), microtubule associated protein 2 (MAP2) and alpha-tubulin were analyzed by the use of a panel of antibodies raised against mammal antigens. Moreover, by enzymatic reactions the Ca(2+)-ATPase and alkaline phosphatase (AIPase) activities were demonstrated. In comparison with the active phase, the hibernation induced an increase in the immunopositivity for calmodulin in all the neurons. The increase may be linked to unmasking of immunoreactive epitopes due to conformational changes of the protein, which in turn may be a consequence of a reduction or absence of binding with calcium ions or of a real increase in the amount of calmodulin in the somata of neurons. In any event, both the hypotheses indicate that neurons have decreased or suppressed the Ca(2+)-dependent mechanisms as also shown by the lower Ca(2+)-ATPase activity. Nevertheless, the AIPase activity, which was localized in the epineural sheat, was not significantly changed during hibernation and this supports that some metabolic activities are preserved in the hibernated animals. Changes in the immunopositivity for cytoskeletal components were found. There was an increase in the epitopes recognized by the mammalian pNF antibody, that concerned both the positivity of the entire cytoplasm of some clusters of metacerebral neurons and the intensity of the reaction. This would be aimed to improve the stability of the somata and primary neurites. Moreover, the decrease of alpha-tubulin and MAP2 immunopositivity, suggests that a disassembly of microtubules have occurred. The findings indicate that the transport of vesicles in the axons is slowed down during hibernation. In fact, research in progress show that the patterns of neurotransmission and neuromodulation are also deeply modified.

Naphthylnitrobutadienes as pharmacologically active molecules: evaluation of the in vivo antitumour activity

SummaryOn the basis of our previous interesting results in vitro on the antiproliferative activity of (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1-Naph-DNB) we have designed and synthesized the new molecule methyl (2Z,4E)-2-methylsulphanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate (1-Naph-NMCB) characterized by the same naphthylnitrobutadiene array but with a different functional group at one end of the diene system. This new molecule showed an in vitro antiproliferative activity more significant than that found for the original 1-Naph-DNB.In order to verify in vivo our in vitro results we have tested the antitumour activity of 1-Naph-DNB and 1-Naph-NMCB in several murine tumour models, namely the myelomonocytic P388 and the Lewis lung carcinoma 3LL in BDF1 mice, the melanoma B16 in C57Bl mice, the fibrosarcoma WEHI 164 in nude mice and, finally, the C51 colon cancer in Balb/c mice. In the case of 1-Naph-NMCB the analysis of the antitumour activity has been preceded by toxicological experiments on CD-1 mice, in order to determine the lethal (LD) and the maximal tolerated (MTD) doses together with the spectrum of histological alterations caused by its iv administration.The results obtained show that the modification of the original structure of 1-Naph-DNB according to the molecular-simplification strategy has led to an asymmetric nitrobutadiene array, i.e. that of 1-Naph-NMCB, endowed with an antitumour activity which is in some cases even better than that showed by the parental compound itself, together with differences in tumour selectivity and negligible histological toxic effects.A promising, versatile route to new, more active and/or safe nitrobutadiene derivatives has thus been positively tested.