Carbonic anhydrase integrated into a multimarker approach for the detection of the stress status induced by pollution exposure in Mytilus galloprovincialis: A field case study.

The work was addressed to study the sensitivity of the enzyme carbonic anhydrase (CA) to chemical pollution in the hepatopancreas of the bioindicator organism Mytilus galloprovincialis in the context of a multimarker approach in view of ecotoxicological biomonitoring and assessment application. The study was carried out by means of a transplanting experiment in the field, using caged organisms from an initial population exposed in the field in two areas of interest: Augusta-Melilli-Priolo, an heavy polluted industrial site (eastern Sicily, Italy), and Brucoli (eastern Sicily, Italy) an area not affected by any contamination and selected as a reference site. Mussels in Augusta presented a significant increase in the digestive gland CA activity and gene expression compared to the animals caged in the control site of Brucoli. The CA response in animals from the polluted site was paralleled by proliferation/increase in the size of lysosomes, as assessed by Lysosensor green charged cells, induction of metallothionein, up-regulation of hif-α (hypoxia-inducible factor), metabolic changes associated with protein metabolism, and changes in the condition factor. Biological responses data were integrated with information about sediment chemical analysis and metal residue concentration in animal soft tissues. In conclusion, obtained results highlighted the induction of CAs in the hepatopancreas of Mytilus galloprovincialis following to pollution exposure, and demonstrated its suitability to be integrated into a multimarker approach for the detection and characterization of the stress status induced by pollution exposure in this bioindicator organism.

Expression of metallothionein mRNAs by in situ hybridization in the gills of Mytilus galloprovincialis, from natural polluted environments.

Metallothioneins (MTs), metal-inducible proteins, are crucial proteins for the regulation of essential metals, and are transcriptionally induced in all organisms by certain heavy metals, oxidative stress and inflammation. The gills represent an organ of uptake and loss of metals in which different mechanisms are present controlling the functions directly involved in the maintenance of homeostasis. In this study, the morphological and histomorphological aspects of branchial epithelium in Mytilus galloprovincialis from polluted environment (Faro swamp, Messina, Italy) have been investigated. The reverse transcriptase-polymerase chain reaction (PCR) has been used to isolate complementary DNA of both MT isoforms present from RNA extracted from mussel gills. The respective mRNAs on histological sections have been visualized by in situ hybridization. These methods showed that MT-10 mRNA is expressed at the basal level. In contrast, the MT-20 expression level was very low under basal conditions, while its mRNA increased dramatically in individuals collected in Faro. The presence of acid mucocytes and MTs in the gills may be considered a further defensive mechanism also related to the significantly higher concentration of Cd, Pb and Cr found in gills of M. galloprovincialis from Faro than specimens from the reference site (Goro). The results obtained show that, in stressed mussels, the defensive processes increase to maintain the normal functions of the organs more exposed to the action of polluted substances.

Antidepressant activity of 5-aryl-2,3,5,6-tetrahydroimidazo[2,1-a]isoquinolin-5-ols.

A series of 5-aryl-2,3,5,6-tetrahydroimidazo[2,1-a]isoquinolin-5-ols was prepared and evaluated for potential antidepressant activity in the reserpine-induced hypothermia model and selected central nervous system and autonomic activity tests. Several members of the series, notably the 4-chloro- and 4-fluorophenyl analogues, demonstrated pharmacological activity in the range of imipramine. Both compounds provided a marked potentiation of the 5-hydroxytryptophan-facilitated monosynaptic spike in the spinal cat preparation.

Structural modification of 5-aryl-2,3-dihydroimidazo[2,1-a]isoquinoline platelet activating factor receptor antagonists.

In an effort to determine the effect of modification of the imidazo[2,1-a]isoquinoline portion of the PAF-receptor antagonist SDZ 64-412 (1), several new analogs were prepared and evaluated in vitro and in vivo. One of these, 5-[4-[2-(3,4,5-trimethoxyphenyl)ethyl]phenyl]-2,3-dihydroimidazo [1,2-a]thieno[2,3-c]pyridine (6) was 4-5 times more potent than 1 in inhibiting PAF-induced bronchoconstriction and hemoconcentration when administered po to the guinea pig.

Halogenated mazindol analogs as potential inhibitors of the cocaine binding site at the dopamine transporter.

A series of halogenated (F, Cl, Br, I), pyrimido and diazepino homologs of mazindol were prepared and evaluated for their ability to displace [3H]WIN 35,428 binding and to inhibit uptake of [3H]dopamine (DA) in rat striatal tissue. All of the compounds except for the 2'-chloro (6) and 2'-bromo (16) analogs of mazindol displaced [3H]WIN 35,428 binding and inhibited [3H]DA uptake more effectively than (R)-cocaine. Structure-activity studies indicated that best inhibition of [3H]WIN 35,428 binding occurred in the imidazo series with compounds containing one or two Cl or Br atoms in the 3'- or 4'-position of the free phenyl group. Replacement of the imidazo ring by a pyrimido or diazepino ring enhanced binding inhibition. The most potent inhibitors of [3H]WIN 35,428 binding and [3H]DA uptake were 6-(3'-chlorophenyl)-2,3,4,6-tetrahydropyrimido[2,1-alpha]isoind ol-6-ol (23; IC50 1.0 nM; 8 x mazindol) and 7-(3',4'-dichlorophenyl)-2,3,4,5-tetrahydro-7H-diazepino[2,1-alpha ]isoindol-7-ol (28; IC50 0.26 nM; 32 x mazindol), respectively. No significant differences was found between binding and uptake inhibition. Mazindol and the pyrimido and diazepino homologs 24 and 27 showed a selectivity for the DA uptake over the serotonin (5-HT) uptake site of 5-, 250-, and 465-fold, respectively, and displayed weak or no affinity for a variety of neurotransmitter receptor sites.

Antitumor activity of 5-aryl-2,3-dihydroimidazo[2,1-a]isoquinolines.

A series of 5-aryl-2,3-dihydroimidazo[2,1-a]isoquinolines previously reported to be platelet activating factor (PAF) receptor antagonists were evaluated for potential antitumor activity. Several compounds, such as the 5-(4'-tert-butylphenyl) (65), 5-[4'-(trimethylsilyl)phenyl] (69), and 5-(4'-cyclohexylphenyl) (71) analogs showed very good cytotoxicity against several tumor cell lines. 5-[4'-(Piperidinomethyl)phenyl]-2,3-dihydroimidazo[2,1- a]isoquinoline (SDZ 62-434, 53) was more effective on a milligram per kilogram basis than the clinical cytostatic agent edelfosine (1) in increasing survivors and decreasing tumor volume in the oral mouse Meth A fibrosarcoma assay. It was selected for further development and is currently in phase I clinical trials in cancer patients.

Chemolithoheterotrophy in a metazoan tissue: sulfide supports cellular work in ciliated mussel gills

Hydrogen sulfide, a common constituent of marine intertidal sediments, is both a potent toxin of aerobic cellular respiration and an electron-rich molecule used by some prokaryotic organisms as a source of energy. In ciliated gills from Geukensia demissa, a marine mussel from sulfide-rich sediments, sulfide oxidation supports cellular work. Evidence for this comes from measurements of ciliary beat frequency (fCB) as a measure of ATP turnover rate, the rate of gill oxygen consumption ( m_dot O2) as a measure of ATP production rate, and mitochondrial cytochrome redox state as an indicator of the path of electron flow. Results from experiments performed in the presence and absence of the mitochondrial complex III inhibitor antimycin A to limit endogenous carbon substrate oxidation showed that exposure to sulfide stimulated oxygen consumption and ciliary beating, with cytochrome c being the dominant reduced species. These results, along with the resultant fCB/ m_dot O2 ratio, are qualitatively and quantitatively consistent with the hypothesis that electrons from sulfide oxidation support mitochondrial ATP production. We propose that Geukensia demissa gills use sulfide as a respiratory substrate when given the choice and thus function metabolically as facultative chemolithoheterotrophs. Similar conclusions could not be drawn for the ciliated gills from Mytilus edulis, a marine mussel from aerated habitats, or for the ciliated lungs from the phylogenetically distinct leopard frog Rana pipiens.

ATP production from the oxidation of sulfide in gill mitochondria of the ribbed mussel Geukensia demissa.

The ribbed mussel Geukensia demissa inhabits intertidal Spartina grass marshes characterized by sulfide-rich sediments. Sulfide poisons aerobic respiration, and G. demissa may cope in this seemingly inhospitable environment by oxidizing sulfide in gill mitochondria. Well-coupled mitochondria isolated from G. demissa gills were used to investigate sulfide oxidation and ATP synthesis. State 3 respiration, maximally stimulated by 5 micromol l(-)(1) sulfide with a P/O ratio of 0.89 and a respiratory control ratio (RCR) of 1.40, remained refractory to sulfide at higher concentrations except in the presence of salicylhydroxamic acid (SHAM), an inhibitor of alternative oxidases. Sulfide-stimulated ATP production was 3-5 times greater than that stimulated by malate and succinate, respectively, giving an ATP/sulfide ratio of 0.63. The inhibition of sulfide-stimulated respiration and ATP production by the complex III inhibitors myxothiazol and antimycin A, respectively, suggests that electrons enter the electron transport chain before complex III. Combined with in vivo evidence for electron entry at cytochrome c, these data suggest that more than one type of sulfide-oxidizing enzyme may function in G. demissa gills. The SHAM-sensitive pathway of electron flux may be a critical component of a physiological strategy to tolerate sulfide. We conclude that G. demissa exploits the energy available from its reduced environment by using sulfide as a respiratory substrate for cellular ATP production.

Cyclic oxygen analogues of alkyl-lysophospholipids. Synthesis and neoplastic cell growth inhibitory properties.

Ether phospholipids have demonstrated both in vitro and in vivo activity against a wide variety of tumor cell lines. The known cyclic ether phospholipid, SRI 62-834, was used as the model to prepare eight novel phospholipids containing a cyclic ether. All of the compounds were as effective as ET-18-OCH3 in their ability to activate macrophage-induced cytotoxicity against the Abelson-8.1 tumor cell line but varied in their direct cytotoxic effects. One of the new compounds, SDZ 62-406, was selected for in vivo studies and showed oral and i.v. activity in the mouse MethA fibrosarcoma model in the same range as ET-18-OCH3. No correlation was found between the direct or macrophage-activated cytotoxicity and the ability of the compounds to inhibit or promote platelet-activating factor (PAF)-induced aggregation of human platelets.