Potentiation of cisplatin-induced antiproliferative and apoptotic activities by the antiarrhythmic drug procainamide hydrochloride.

BACKGROUND: We describe the potentiation of antiproliferative and apoptotic activities triggered by cis-diamminedichloroplatinum(II) (DDP), and obtained in vitro by the co-administration of procainamide hydrochloride (PdHCl) in murine P388, and human A2780 and A549 cells. METHODS: We determined the antiproliferative and apoptotic activities of DDP and PdHCl combinations by different techniques. Moreover, cell cycle analysis, restriction enzyme inhibition followed by agarose gel electrophoresis, and TUNEL analysis of tumour cells in vivo were also used to strengthen our hypothesis. RESULTS: Our results show that PdHCl may significantly increase the inhibition of cell proliferation and apoptosis. Experiments in vivo showed that the co-administration of DDP and PdHCl increased the percentage of apoptotic cells compared to DDP alone treatment, both in subcutaneous (sc) and intraperitoneal (ip) P388 tumours. We finally demonstrated that the co-administration of PdHCl prevents DNA digestion accounting for a restriction enzyme inhibition that in some cases was greater than that obtained by DDP alone. Moreover, when PdHCl was mixed with the reaction products (RP) of DDP (RP-PdHCl) we obtained a restriction enzyme inhibition greater for some enzymes (Bsp1407I, Hin1II, and Psp1406I) than that obtained by the DDP-PdHCl solution. CONCLUSIONS: On the whole our data demonstrate that the class I antiarrhythmic drug PdHCl may increase the antiproliferative activity of DDP by improving its triggering of apoptosis, and that this phenomenon may be likely linked to the formation of a new Pt compound.

Fibrin gels loaded with cisplatin and cisplatin-hyaluronate complexes tested in a subcutaneous human melanoma model.

Fibrin gels are attractive biomaterials for local delivery of a variety of agents, from drugs to proteins. Similarly, polymer-anticancer-drug conjugates and nanoparticles are emerging as potential candidates for cancer treatment. Combining these different approaches, we have studied the efficacy of fibrin gels loaded with cisplatin (DDP) and a complex of DDP with hyaluronate (DDP-HA) for tumor growth inhibition in a melanoma model. Loaded gels prepared at relatively high fibrinogen concentration (22 mg/ml) showed good in vitro antiproliferative activities, prolonged release of the anticancer drug, and a long persistence (10-15 days) in vivo when implanted subcutaneously (sc) in immunodeficient mice. Gels loaded with DDP or DDP-HA containing 1/3 or even 1/6 of their systemic dose (6 mg/kg) and positioned under the tumor mass in mice bearing a sc human SK-Mel-28 tumor showed an antitumor activity better than that of the original parent compound given intraperitoneally (ip). Moreover, in an additional experiment in vivo, fibrin gels loaded with N-trimethyl chitosan-based nanoparticles containing a DDP-HA complex were assayed, resulting in a further 8 % improvement of anticancer activity, with lesser adverse systemic toxic effects. Taken together, these results suggest that the combination of fibrin gels and drugs complexed with suitable macromolecules holds great promise for loco-regional anticancer therapy of melanoma and other surgically removable cancer types.

Renal fibrogenesis and platinum compounds in a rat model: a novel Pt (II) complex vs. cisplatin.

BACKGROUND/AIM: A new platinum compound, (Pt(O,O'-acac)(γ-acac)(DMS)) (PtAcacDMS), has been shown to possess higher cytotoxic activity than cisplatin on several cancer and chemoresistant cell lines. The aim of the present study was to compare the nephrotoxic effects - particularly renal fibrogenesis- of PtAcacDMS and cisplatin in rats after the subcutaneous administration of a single dose (5 mg/Kg b.w., s.c.) of either compound to ten-day-old rats. MATERIALS AND METHODS: Control and treated rats were killed 1 day (PD11), 7 days (PD17), 21 days (PD31) and 40 days (PD50) after the injection. Kidneys were processed for light and electron microscopy, and platinum determination. Antibodies against E-cadherin (E-cad), vimentin (VIM) and α-smooth muscle actin (αSMA) were used to identify epithelial and mesenchymal cells. RESULTS AND CONCLUSION: Cisplatin produced progressive cortical fibrotic lesions displaying a variable number of VIM-positive tubules and interstitial αSMA-positive cells around. By contrast, PtAcacDMS induced a minimal number of histopathological changes, which declined in the adult samples, while the renal platinum content was generally higher after PtAcacDMS than after cisplatin. The present experimental model was proven suitable to investigate the occurrence of epithelial-mesenchymal transition (EMT) in renal fibrogenesis induced by the platinum-based compounds.

Evaluation of the anti-proliferative activity of three new pyrazole compounds in sensitive and resistant tumor cell lines.

BACKGROUND: In previous papers we demonstrated that the activity of short heteroretinoids as anti-proliferative and pro-apoptotic compounds was deeply linked to their heterocyclic moiety and that ionone-derived 1,5-pyrazoles had the highest anti-proliferative activity in our preliminary experiments. We then demonstrated the high and pharmacologically significant anti-proliferative and apoptotic activities of the pyrazole compounds 2-(1-(4-chlorophenyl)-1H-pyrazol-5-yl)-5-methoxyphenol (EN12-4), 5-methoxy-2-(1-(pyridin-2-yl)-1H-pyrazol-5-yl)phenol (EN12-2A) and 2-(5-(4-methoxyphenyl)-1H-pyrazol-1-yl)pyridine (EN7-2) establishing, especially for EN12-2A, a possible mechanism of action involving the cell microtubular system. METHODS: Here, the anti-proliferative activity of these pyrazole compounds was analyzed in vitro by the MTT assay in six drug-resistant cell lines, five of which were selected after exposure to increasing concentrations of cisplatin (L1210/DDP), doxorubicin (A2780/DX3), 5-fluorouracil (HCT-8/5FU), taxol (A549/T24) and etoposide (MCF-7/VP), and one was obtained by transfection of the ABCG2 membrane transporter (HEK-293/R2). RESULTS: Our data show that these compounds have a similar anti-proliferative activity in nearly all resistant and sensitive cell lines, demonstrating their ability to overcome the most common mechanisms of drug resistance with two exceptions regarding the MCF-7/VP cell line over-expressing the ABCC1 (MRP1) transporter, and the MDR1 over-expressing A2780/DX3 cells, with a calculated RI = 3.2 for EN12-2A, relative to their sensitive cellular counterpart. On the other hand, the taxol-resistant A549/T24 cell line showed a significantly increased sensitivity to our compounds. CONCLUSIONS: Our data suggest that our pyrazole compounds are able to overcome in vitro the most common drug-resistance mechanisms demonstrating a significant anti-proliferative activity and confirming a mechanism of action involving the depolymerization of microtubules.

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.

Design, synthesis, and in vitro evaluation of new naphthylnitrobutadienes with potential antiproliferative activity: toward a structure/activity correlation.

On the grounds of previous encouraging results on the antitumor activity of (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1), we have designed and synthesized two new molecules [(1E,3E)-1,4-bis(4-carboxy-1-naphthyl)-2,3-dinitro-1,3-butadiene (2) and methyl (2Z,4E)-2-methylsulfanyl-5-(1-naphthyl)-4-nitro-2,4-pentadienoate (3)] characterized by a common naphthylnitrobutadiene array but with different structural properties, with the aim of approaching to some structure-activity correlation. When 2 and 3 were analyzed in vitro for their inhibition of cell proliferation and pro-apoptotic properties, the carboxyderivative 2 did not furnish appreciable results. In contrast, 3 (which contains only one of the two naphthylnitroethenyl moieties of the original compound 1) showed remarkable activities in the range of micromolar concentrations (in six over eight cell lines its IC(50)s are in the 1-3 microM range), with a significant improvement compared to 1. In particular, 3 proved able to bind to DNA, to upregulate p53, to block cells in the G2/M phase of their cycle, and to induce apoptosis. Thus, very interestingly, the performance of 3 with respect to 1 shows that a single 1-(1-naphthyl)-2-nitroethene moiety is able to ensure better (on four out of eight of the cell lines tested) or comparable levels of activity. This result suggests that the 'molecular-simplification strategy' could furnish a useful instrument for future design in our antitumor research.

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.

Synthesis, in vitro activity and in vivo toxicity of the new 2,3-dinitrobutadiene derivative (1E,3E)-1,4-bis(2-naphthyl)-2,3-dinitro-1,3-butadiene.

Our interesting results on the antiproliferative (in vitro) and antitumour (in vivo) activities of (1E,3E)-1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene (1-Naph-DNB) have more recently induced us to design and synthesize some new 1,4-diaryl-2,3-dinitro-1,3-butadienes characterized by a common arylnitrobutadiene array but with different geometric and/or functional properties. This task was undertaken with the aim to obtain new compounds with an enhanced antiproliferative activity and, possibly, a different specificity with respect to the original (lead) compound. (1E,3E)-1,4-Bis(2-naphthyl)-2,3-dinitro-1,3-butadiene (2-Naph-DNB) is one of the molecules so obtained, a structural isomer of 1-Naph-DNB provided with a different spatial arrangement. When analyzed in vitro for its inhibition of cell proliferation 2-Naph-DNB showed a remarkable activity in the range of micromolar concentrations, with significant differences, with respect to 1-Naph-DNB, against some cell lines. Furthermore, it was able to significantly trigger apoptosis, to up-regulate p53, to block cells in the G2/M phase of the cell cycle and, finally, to slightly bind to DNA forming interstrand cross-links (ISCL). 2-Naph-DNB was then analyzed for its toxic activity in vivo in CD1 mice. This allowed the determination of toxicity parameters such as the lethal doses (LD) and the maximal tolerated dose (MTD) together with the definition of the spectrum of tissue alterations due to its administration i.v. Altogether our data suggest that the idea of modifying the geometry of the lead compound 1-Naph-DNB deserves further investigation aimed at synthesizing new molecules with similar chemical functionalities but with different spatial requirements, hopefully characterized by still enhanced activities in terms of inhibition of cell proliferation and apoptosis.

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

On 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.

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 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.

Morphological and histochemical evidence of the protective effect of procainamide hydrochloride on tissue damage induced by repeated administration of low doses of cisplatin.

BACKGROUND: The class 1 antiarrhythmic drug procainamide hydrochloride might protect against acute cisplatin-induced nephrotoxicity and hepatotoxicity in mice and rats. In this report, the protective activity of procainamide hydrochloride against renal and hepatic tissue damage induced by repeated administration of low doses of cisplatin was analyzed morphologically and histochemically. MATERIALS AND METHODS: Light microscopy observations were performed on liver, renal and heart samples obtained from female Wistar rats treated twice a week for 10 weeks with 1 mg/kg cisplatin (cumulative dose: 20 mg/kg), with or without 100 mg/kg procainamide hydrochloride (cumulative dose: 2 g). Samples were then submitted to histochemical stainings [i.e. H & E, periodic acid Schiff (PAS) and Sudan Black]. RESULTS: Light microscopy analysis revealed that the coadministration of cisplatin and procainamide hydrochloride significantly reduced tissue alterations both in the kidneys and liver, while in the heart, neither cisplatin nor the combination of cisplatin and procainamide hydrochloride caused any evident tissue damage. CONCLUSION: The morphological and histochemical data confirm that procainamide hydrochloride is able to protect not only from acute cisplatin-induced toxicities, but also from tissue alterations induced in the liver and kidneys by the administration of repeated low doses of cisplatin.

Inhibition of cell proliferation, cytotoxicity and induction of apoptosis of 1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene in gastrointestinal tumour cell lines and preliminary evaluation of its toxicity in vivo.

Our preliminary data suggested that 1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene [Viale M, Ottone M, Chiavarina B, Mariggiò MA, Prevosto C, Dell'Erba C, et al. Preliminary evaluation in vitro of the inhibition of cell proliferation, cytotoxicity and induction of apoptosis by 1,4-bis(1-naphthyl)-2,3-dinitro-1,3-butadiene. Invest New Drug 2004;22:359-67] (Naph-DNB), possesses good characteristics in terms of inhibition of cell proliferation in two cell lines derived from colon and gastric cancers. On this basis and to confirm the specificity of our compound towards gastrointestinal malignancies, we have analyzed the inhibition of cell proliferation, the cytotoxicity and the induction of apoptosis by Naph-DNB in seven cell lines derived from human colon (DLD-1, Lovo, HCT-8 and Colo 741), stomach (HGC-27) and pancreas (Panc-1 and Hup-T4) tumours. For the sake of comparison, cells have also been exposed to four anticancer drugs utilized for the treatment of gastrointestinal malignancies (oxaliplatin, irinotecan, gemcitabine and 5-fluorouracil). Moreover, toxicological data have been obtained in order to define the lethal dose (LD) and maximal tolerated dose (MTD) values and the spectrum of tissue alterations caused by the intraperitoneal (i.p.) and intravenous (i.v.) administration of Naph-DNB. IC50 data obtained by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay suggest that Naph-DNB is generally more active than two or more of the anticancer drugs above in most cell lines: it displayed the lowest activity only in HGC-27 cells, although data concerning the IC75 parameter enlighten a significantly better activity than irinotecan and 5-fluorouracil. Using the equitoxic concentrations IC50 and IC75, we have also evaluated the ability of Naph-DNB and of the other anticancer drugs to kill cells and to induce apoptosis. Our data show that at these concentrations Naph-DNB has a cytotoxic activity comparable or even better than that of some anticancer drugs. Similarly, Naph-DNB induces apoptosis better than the other anticancer drugs in HCT-8 and HGC-27 cells, while in Lovo and Panc-1 cells the induction is comparable. On the basis of toxicological data we defined the LD10, LD50, LD90 (i.p., 17.6, 36.1 and 54.1 mg kg(-1), respectively; i.v., 6.1, 14.1 and 22.0 mg kg(-1), respectively) and the MTD (i.p., 15 mg kg(-1); i.v., 5 mg kg(-1)) parameters. Histochemical analysis has shown that, in general, the administration of even toxic doses of Naph-DNB does not cause great structural injuries, although it can have some effects on the metabolism of glicogen and iron in organs as liver and spleen. In conclusion, our preclinical studies in vitro suggest that Naph-DNB may represent a good anticancer compound for the treatment of generally unresponsive tumours such as those of pancreas, stomach and colon. Moreover, the analysis of its toxic effects has allowed the definition of LD and MTD parameters, which will be used in further experiments in vivo for the definition of its antitumour activity.

Effects of environmental pollution on the liver parenchymal cells and Kupffer-melanomacrophagic cells of the frog Rana esculenta.

In vertebrates, the biotransformation processes of xenobiotics are performed mainly by the liver which involves both hepatocytes and Kupffer-melanomacrophagic cells through enzymatic and nonenzymatic mechanisms. In this study, we investigated the liver of Rana esculenta adult frogs collected at two sample rice fields, one heavily polluted and one relatively unpolluted. Water pollution was determined by chemical analysis on tadpoles. The specific activities of some enzymes (glucose-6-phosphate dehydrogenase (G6PDH), acid and alkaline phosphatases (AcPase and AlkPase), succinic dehydrogenase (SDH), and catalase) were studied in the liver of adult frogs to identify the possible changes induced by contamination in the metabolic processes which depend on the function of the liver. The production of reactive oxygen species (ROS) were also evaluated through histochemical techniques. In the polluted samples, hepatocytes showed variations in the activity of G6PDH, AlkPase, and SDH and a moderate to intense ROS expression. Prominent changes were observed in Kupffer cells (KCs) and melanomacrophages (MMPs), both showing intense reactivity for AcPase and catalase and variations in melanin content and distribution. Results thus indicate a general adaptive response of liver parenchyma to environmental pollution. The possible role of both KCs and MMPs as scavengers of foreign substances is discussed.

Expression of natriuretic peptides, nitric oxide synthase, and guanylate cyclase activity in frog mesonephros during the annual cycle.

Natriuretic peptides (NPs), a family of structurally related hormones and nitric oxide (NO), generated by nitric oxide synthase (NOS), are believed to be involved in the regulation of fluid balance and sodium homeostasis. Differential expression and regulation of these factors depend on both physiological and pathological conditions. Both NPs and NO act in target organs through the activation of guanylate cyclase (GC) and the generation of guanosine 3',5'-cyclic monophosphate (cGMP), which is considered a common messenger for the action of these factors. The present study was designed to investigate--by histochemical methods--the expression of some NPs (proANP and ANP) and isoforms of NOS (neuronal NOS, nNOS, and inducible NOS, iNOS) in the mesonephros of Rana esculenta in different periods of the year including hibernation, to evaluate possible seasonal changes in their expression. We also studied the enzyme activity of NOS-related nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) and of GC. The experiments were performed on pieces of kidney of R. esculenta collected in their natural environment during active and hibernating life. The study was carried out using immunohistochemical techniques to demonstrate proANP, ANP, and some NOS isoforms. Antigen capture by enzyme linked immunosorbent assay (ELISA) was also performed to determine the presence of NPs in the frog kidney extract. Enzyme histochemistry was used to demonstrate the NOS-related NADPHd activity at light microscopy; GC activity was visualized at the electron microscope, using cerium as capture agent. The application of the immunohistochemical techniques demonstrated that frog mesonephros tubules express different patterns of distribution and/or expression of ANP and NOS during the annual cycle. Comparing the results obtained on active and hibernating frogs has provided interesting data; the NOS/NADPHd and GC activities showed some variations as well. Furthermore, the presence of NPs in the frog kidney extract was evidenced by dose-dependent response in the ELISA. The data suggest that both ANP and NO are intra-renal paracrine and/or autocrine factors which may modulate the adaptations of frog renal functions to seasonal changes through the action of the cGMP generated from GC activity.

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 -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.

Composition, biophysical properties, and morphometry of plasma membranes in pulmonary interstitial edema.

We evaluated the changes in plasma membrane composition, biophysical properties, and morphology of pulmonary endothelial cells in anesthetized rabbits receiving 0.5 ml. kg(-1). min(-1) saline infusion for 180 min, causing mild interstitial edema. Plasma membrane fractions were obtained from lung homogenates with gradient centrifugation, allowing a sixfold enrichment in caveolin-1. In edematous lungs, cholesterol content and phospholipidic phosphorus increased by 15 and 40%, respectively. These data correlated with morphometric analysis of lungs fixed in situ by vascular perfusion with 2.5% glutaraldehyde, suggesting a relative increase in surface of luminal to interstitial front of the capillary endothelial cells, due to a convoluted luminal profile. In edematous lungs, the fraction of double-bound fatty acids increased in membrane lipids; moreover, the phosphatidylcholine/phosphatidylethanolamine and the cholesterol/phospholipid ratios decreased. These changes were consistent with the increase in fluorescence anisotropy of plasma membrane, indicating an increase in its fluidity. Data suggest that mechanical stimuli elicited by a modest (approximately 4%) increase in extravascular water cause marked changes in plasma membranes that may be of relevance in signal transduction and endothelial cell activation.

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.

Morpho-functional analysis of lung tissue in mild interstitial edema.

Mild pulmonary interstitial edema was shown to cause fragmentation of interstitial matrix proteoglycans. We therefore studied compartmental fluid accumulation by light and electron microscopy on lungs of anesthetized rabbits fixed in situ by vascular perfusion after 0.5 ml.kg(-1).min(-1) iv saline infusion for 180 min causing approximately 6% increase in lung weight. Morphometry showed that a relevant portion (44%) of extravascular fluid is detected early in the alveolar septa, 85% of this fluid accumulating in the thick portion of the air-blood barrier. The arithmetic mean thickness of the barrier increased in interstitial edema from 1.06 +/- 0.05 (SE) to 1.33 +/- 0.06 microm. The harmonic mean thickness increased from 0.6 +/- 0.03 to 0.86 +/- 0.07 microm, mostly due to thickening of the thin portion causing an increase in gas diffusion resistance. Despite some structural damage, the air-blood barrier displays a relatively high structural resistance providing a safety factor against the development of severe edema. It is suggested that the increase in extra-alveolar perivascular space occurs as a consequence of fluid accumulation in the air-blood barrier.

In situ detection of reactive oxygen species and nitric oxide production in normal and pathological tissues: improvement by differential interference contrast.

Reactive oxygen species (ROS), among which nitric oxide (NO) is currently included, play a plethora of (patho)physiological roles. Harman's free radical theory of aging put forth over 40 years ago received full support since then. A nitric oxide hypothesis of aging recently proposed by McCann, is very likely to be the object of widespread investigation in the near future. Therefore, the possibility of localizing at the (sub)cellular level under the light microscope the sites of ROS and NO production with simple and reliable methods appears as a powerful tool for analytic cytology and pathology. Various histochemical methods were developed for visualizing ROS production; a recently improved version to localize superoxide (and possibly also singlet oxygen), based on a DAB-Mn2+ -Co2+ reaction, appears very promising. Since the direct detection of NO is still very difficult, the action sites of NO are currently localized by the identification of NO synthase (NOS). The most widespread method to reveal the catalytic activity of NOS is that of demonstrating the fixation-resistant NADPH diaphorase activity with the tetrazolium salt method. We have improved this method by using a tetrazolium salt whose formazan particles are very thin and lipid insoluble (tetranitroblue tetrazolium, TNBT) and by including a tissue protectant, polyvinyl alcohol, in the incubation medium. Here significant examples of application of the DAB-Mn2+ -Co2+ technique for ROS and the TNBT-PVA method for NOS to normal liver and brain and to solid tumors are presented. We further document the usefulness of Nomarkis's differential interference contrast (DIC) to analyze wide tissue areas where ROS production or NOS activity is low or even nil. The improved version for NOS allowed for the first time to demonstrate NOS activity in liver fat-storing cells and in astrocyte-like cells in the brain.