The effects of Deepwater Horizon crude oil on ammonia and urea handling in mahi-mahi (Coryphaena hippurus) early life stages.

Many ecologically important fishes, including mahi-mahi (Coryphaena hippurus), and their offspring were directly exposed to crude oil following the Deepwater Horizon (DWH) oil spill. Early life stage fish are especially vulnerable to the toxicity of crude oil-derived polycyclic aromatic hydrocarbons (PAHs). In teleosts, yolk sac proteins are the main energy source during development and are usually catabolized into ammonia or urea among other byproducts. Although excretion of these waste products is sensitive to oil exposure, we know little about the underlying mechanisms of this process. In this study, we examined the effects of crude oil on ammonia and urea handling in the early life stages of mahi. Mahi embryos exposed to 30-32 µg L-1 ∑PAH exhibited increased urea excretion rates and greater accumulation of urea in the tissues before hatch suggesting that ammonia, which is highly toxic, was converted into less-toxic urea. Oil-exposed embryos (6.3-32 µg L-1 ∑PAH) displayed significantly increased tissue ammonia levels at 42 hpf and upregulated mRNA levels of ammonia transporters (Rhag, Rhbg and Rhcg1) from 30 to 54 hpf. However, despite increased accumulation and higher expression of ammonia transporters, the larvae exposed to higher ∑PAH (30 µg L-1 ∑PAH) showed reduced ammonia excretion rates after hatch. Together, the increased production of nitrogenous waste reinforces previous work that increased energy demand in oil-exposed embryos is fueled, at least in part, by protein metabolism and that urea synthesis plays a role in ammonia detoxification in oil-exposed mahi embryos. To our knowledge, this study is the first to combine physiological and molecular approaches to assess the impact of crude-oil on both nitrogenous waste excretion and accumulation in the early life stages of any teleosts.

Ontogeny of urea and ammonia transporters in mahi-mahi (Coryphaena hippurus) early life stages.

The mechanism(s) of ammonia and urea excretion in freshwater fish have received considerable attention; however, parallel investigations of seawater fish, specifically in the early life stages are scarce. The first objective of this study was to evaluate the patterns of ammonia and urea excretion in mahi-mahi (Coryphaena hippurus) up to 102  hours post fertilization (hpf). Similar to other teleosts, mahi embryos are ureotelic before hatch and gradually switch to being ammoniotelic around the time of hatch. The second objective was to characterize mRNA levels of ammonia transporters (Rhag, Rhbg, Rhcg1 and Rhcg2), as well as urea transporter (UT) and sodium hydrogen exchangers (NHE3 and NHE2) during mahi development. As predicted, the mRNA levels of the Rhesus glycoprotein (Rh) genes, especially Rhag, Rhbg and the UT gene were highly consistent with the ontogeny of ammonia and urea excretion rates. Further, the localization of each transporter was examined in larvae collected at 60 and 102 hpf using in situ hybridization. Rhag was expressed in the gills, yolk sac, and operculum. Rhbg was expressed in the gills and upper mouth. Rhcg1 and NHE3 were co-localized in the sub-operculum, and Rhcg2 was expressed in the skin. Together, these results indicate that urea excretion is critical for ammonia detoxification during embryonic development and that Rh proteins are involved in ammonia excretion via gills and yolk sac, possibly facilitated by NHE3.

Combined magnetron sputtering and pulsed laser deposition of TiO 2 and BFCO thin films.

We report the successful demonstration of a hybrid system that combines pulsed laser deposition (PLD) and magnetron sputtering (MS) to deposit high quality thin films. The PLD and MS simultaneously use the same target, leading to an enhanced deposition rate. The performance of this technique is demonstrated through the deposition of titanium dioxide and bismuth-based perovskite oxide Bi2FeCrO6 (BFCO) thin films on Si(100) and LaAlO3 (LAO) (100). These specific oxides were chosen due to their functionalities, such as multiferroic and photovoltaic properties (BFCO) and photocatalysis (TiO2). We compare films deposited by conventional PLD, MS and PLD combined with MS, and show that under all conditions the latter technique offers an increased deposition rate (+50%) and produces films denser (+20%) than those produced by MS or PLD alone, and without the large clusters found in the PLD-deposited films. Under optimized conditions, the hybrid technique produces films that are two times smoother than either technique alone.

A preliminary in vitro and in vivo study of the effects of new anthraquinones on neutrophils and bone remodeling.

Osteolysis, that is, progressive periprosthetic bone loss, is responsible for approximately 70% of aseptic loosening and implant failure. Usually, it is due to a granulomatous reaction wear-induced, leading to macrophage and osteoclast-mediated bone resorption. At present, there is no established prophylaxis or treatment for this process. For this purpose, as a preliminary investigation, we aimed to study the effects in two directions, inhibition of proinflammatory signals, and bone remodeling activity, of two newly synthesized anthraquinone molecules [N,N'-Diethylamino-2,6-anthraquinone-disulfonamide (GR375) and N,N'-(p-ethoxyphenyl)-2,6-anthraquinone-disulfon amide (GR377)]. Among the pro-inflammatory signals, the ability of the two anthraquinones to interfere with the production of superoxide anion (O(2) (-)), which was assumed as a marker of reactive oxygen species (ROS), was evaluated in an in vitro cell model by testing phagocytes, such as human neutrophils, challenged by the chemotactic agent N-formylmethionyl-leucyl-phenylalanine (FMLP). Both compounds inhibited O(2) (-) production, in a dose-dependent way, without exerting scavenger effects. An in vivo model was applied to investigate their effect on bone remodeling. Fifty-four female Wistar rats were divided into eight groups of six animals each, and a 4-week treatment was applied in two phases. A 25 mg/kg/os dose in the first phase and 12.5-6.25 mg/kg/os doses in the second one were employed. The tibia trabecular bone at the secondary spongiosa level was analyzed, and trabecular bone volume (%TBV), trabecular thickness (TbTh), and apatite lattice parameters were measured. At the highest doses of GR375 and GR377 the %TBV and the TbTh increased by 33.2, 34.6%, and 3.6 and 9.1%, respectively, whereas crystallographic parameters were not significantly different from the untreated group. Our results suggest a simultaneous antiinflammatory and antiosteoclastic activity of both drugs that encourages to perform further research. If it will be confirmed, they could be proposed in a variety of bone diseases, in particular, when acute inflammation is associated to osteolytic processes and, eventually, in the prevention and treatment of periprosthetic osteolysis.

Effect of continuous high hydrostatic pressure on the morphology and cytoskeleton of normal and osteoarthritic human chondrocytes cultivated in alginate gels.

OBJECTIVE: To investigate the effect of continuous high hydrostatic pressure on the cell ultrastructure and cytoskeleton of cultivated normal and osteoarthritic (OA) human chondrocytes. METHODS: The effects of continuous hydrostatic pressure (24 MPa) for 3 hrs on normal and OA chondrocytes were assessed by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and immunofluorescence microscopy (IF). RESULTS: Structural differences at the nuclear, cytoplasmic and cytoskeletal levels were observed between normal and OA chondrocytes. Continuous high hydrostatic pressure severely altered normal chondrocytes that became similar in structural organization to OA chondrocytes and further reduced the number of cell organelles involved in the synthesis of collagen and proteoglycans. IF showed major changes in the distribution of actin and tubulin after pressurization in normal and OA chondrocytes. CONCLUSIONS: The results confirm the major role of pressure on chondrocyte ultrastructure. Continuous high hydrostatic pressure caused structural alterations in normal chondrocytes, which obtained similar, if not identical, characteristics to those typical of osteoarthritic chondrocytes.

Effects of orally administered bisphosphonates on bone loss in a disuse osteopenia model involving the rat.

OBJECTIVE: This study was carried out to evaluate the effectiveness of two newly synthesized bisphosphonates (BPs) [Alendronate (4-amino-1-hydroxybutylidene-1, 1-bisphosphonic acid (AHBuBP)) and Neridronate (6-amino-1-hydroxyhexylidene-1,1-bisphosphonic acid (AHHexBP)], administered orally, in reducing experimentally induced bone loss. METHODS: Unilateral sciatic nerve section was performed on the Sprague-Dawley rat to induce osteopenia in one of the hind limbs. Histomorphometric measurements of the tibial trabecular bone and femur ash content determinations were effected to assess the degree of osteopenia. For comparison Chlodronate (dichloromethylene-1-bisphosphonic acid (Cl2MBP) was employed as the reference drug. CONCLUSIONS: The results of this investigation show that both BPs were significantly active in reducing the osteopenic process in the involved limb and were more active than Chlodronate.

In vitro assessment of phagocytosis of bovine collagen by human monocytes/macrophages using a spectrophotometric method.

The use of a wound dressing with covering and haemostatic properties significantly improves wound healing. In this study, a lyophilized bovine collagen sponge used for the treatment of wounds and ulcerae has been tested in a cell culture system. Phagocytosis of collagen fragments by human blood monocytes/macrophages has been investigated. For the assessment of collagen ingestion by mononuclear phagocytes, a picrosirius dye specific for collagen molecules has been used. By adapting this histochemical technique to microplate cell culture system, replicate monocyte cultures are assayed. Collagen content is determined by evaluating spectrophotometrically at 540 nm the absorbance of a sirius red/picric acid solution. Using this simple and sensitive method, the phagocytosis of bovine collagen by LPS-stimulated monocytes/macrophages has been ascertained.

[Pulsating tumor of the sternum: enlarged exeresis and reconstruction with marlex-methylmethacrylate prosthesis]. / Tumore pulsante dello sterno: exeresi allargata e ricostruzione con protesi di marlex-methyl-methacrylate.

The authors report on a rare case of pulsating tumor of the sternum. En bloc resection was carried out with removal of the sternal manubrium and body, tracts of the I-II-III rib with the intercostal muscles and part of the left pectoralis major. Reconstruction of the thoracic wall was performed with a marlex MMA sandwich prosthesis. From a histological viewpoint, the carcinoma was well differentiated and compatible with thyroid primitivity. Non complementary antiblastic therapies were provided as all the examinations carried out were negative and the intervention was radical. The patient had been followed-up with serial controls and three complete stagings at one, three and five years after intervention (CT, echography and function tests of the thyroid, bone scintigraphy). At follow-up of more than six years the patient is doing well and is event free. The authors believe, even with the help of reports from the literature that, in sternal metastases from thyroid carcinomas, especially when single and large, surgical therapy is required.

Alpha B crystallin is constitutively expressed in cultures of bovine articular chondrocytes.

alpha B crystallin is a small heat shock protein constitutively expressed in the mammalian lens and in a variety of extraocular tissues. We report here the presence of alpha B crystallin also in bovine articular chondrocytes by means of an immunoblot and immunofluorescence analysis carried out with anti-alpha B crystallin polyclonal antibodies. The expression level of alpha B crystallin can be further induced by a short heat shock treatment of chondrocytes as well as cell treatment with cadmium bromide or calcium ionophore A 23187. The level of alpha B crystallin expression is not modified by treating chondrocytes with interleukin-1 and phorbol 12-myristate 13-acetate. In some preparations the antibodies recognise two bands of alpha B crystallin, probably corresponding to different degrees of protein phosphorylation, but in cells treated with phorbol ester a single band is constantly observed, indicating a complete phosphorylation of alpha B crystallin.

[Surgical stabilization of post-traumatic flail chest. Our experience with 116 cases treated]. / La stabilizzazione chirurgica del lembo costale mobile post-traumatico. Nostra esperienza relativa a 116 casi trattati.

The therapeutic approach in a patient with traumatic flail chest is varied and controversial, both as regards the type of treatment and as regards the surgical techniques to be employed. The authors have examined 116 cases of flail chest treated surgically; these represented 17.7% of the 655 chest traumas operated in the period from July 1975 to March 1993. Increasing experience has reduced indications for surgery: at present it is not performed in cases of serious craniocerebral lesions, of severe pulmonary contusions, or, however, when mechanical ventilation is required. As for surgical techniques, external traction was applied in the first 15 cases treated but then discontinued because of the poor outcome. Osteosynthesis (77 cases), associated with thoracotomy, was carried out with Kirschner wires, Vecsei metal plates, Judet agraffe, alone or variously combined. Recently, a less aggressive personal technique has been employed prevalently: Kirschner wires placed vertically within the chest, bridging between one rib (generally the V or VI, well fixed with metal plates), and the clavicle and/or sternum. The overall mortality rate, prevalently due to associated lesions, was 20.6%. Excluding emergencies, these patients should be operated in deferred emergency within 24-48 hours of trauma. Osteosynthesis, when indicated is the procedure offering the greatest assurance of success. The authors believe that, in selected cases, surgical stabilization is necessary.

Effects of bisphosphonate derivatives on macrophage function.

The effects of three bisphosphonates (BPs), designated 4-amino-1-hydroxybutylidene-1, 1-bisphosphonate (AHBuBP), 6-amino-1-hydroxylidene-1, 1-bisphosphonate (AHHexBP) and chloromethylenebisphosphonate (Cl2MBP), were evaluated on the basis of their effect on the phagocytic activity, lysosomal enzyme release and superoxide anion production in the rat peritoneal macrophage (M phi). AHBuBP was found to inhibit in a concentration-dependent manner the phagocytosis of sheep red blood cells (SRBC). The same activity was seen with the phagocytosis of latex beads, although this effect was independent of calcium concentration. Conversely, AHHexBP and Cl2MBP were weak inhibitors of phagocytosis of both SRBC and latex beads. Inhibition studies on the phorbol myristate acetate-stimulated production of superoxide anion have shown all three BPs to be active. When compared with Cl2MBP, AHBuBP and AHHexBP were shown to be substantially active in inhibiting the release of beta-glucuronidase from ionophore A23187-stimulated rat peritoneal M phi.

Mutagenicity of hexahydrophenanthrenes and their oxirane derivatives.

The mutagenicity of some hexahydrophenanthrenes and their corresponding arene oxides was assayed in histidine-dependent mutants of Salmonella typhimurium TA98 and TA100. All the arene epoxides examined were devoid of mutagenic activity, although some of them could alkylate nicotinamide. By contrast, the 1,2,3,9,10, 10a-hexahydrophenanthrene, trans-1,2,3,4,4a,10a-hexahydrophenanthrene, 9-methyl-, 6-methoxy-trans-1,2,3,4,4a,10a-hexahydrophenanthrene, 7-bromo-trans-1,2,3,4,4a,10a-hexahydrophenanthrene and 9-methyl-trans-1,2,3,4,4a,10a-hexahydrophenanthrene were active as mutagens in the presence of S9 mix. A negative result was obtained with octahydrophenanthrene, suggesting that the benzylic double bond is a prerequisite for the mutagenic activities of hexahydrophenanthrenes. Thus, probably a very reactive intermediate (aryloxirane) formed by a secondary metabolism following the primary oxidation of the benzylic double bond by S9 mix could be responsible for the mutagenicity of the hexahydrophenanthrenes.

Potential role of heterologous collagen in promoting cutaneous wound repair in rats.

The effect of native bovine tendon type-I collagen sponges (CONDRESS) on wound repair was evaluated by employing an experimental animal (rat) model which utilized subcutaneously implanted collagen and polyurethane sponges. Lesions were also created in the control groups with the exception that implants were omitted. The fusion of the hypodermal layer was selected as the index of wound repair expressed as % healing. In order to assess the extent of the healing process, parameters of clinical evaluation such as exudate volume, number of polymorphonuclears (PMNs) and macrophages (MOs) were also determined. All studies were effected at time intervals of 24, 48 and 72 h post wounding and implantation. The collagen-treated groups showed a greater healing capacity as compared to the polyurethane sponge-treated and control groups. Likewise, the exudate volume, number of leukocytes and mononuclear-type cells were all significantly higher for the collagen-treated animals than those of the polyurethane sponge-treated and control ones. Furthermore, the healthier appearance of the artificially produced wounds in the collagen sponge-treated groups (when compared to the others after 24 h) further confirmed collagen's validity in the treatment of wounds.

Superoxide anion production and toxicity in V79 cells of six hydroxy-anthraquinones.

This study investigates the cytotoxic and genotoxic effects of various carboxy AQ, 1,4-dihydroxy 6-carboxy AQ, 1,8-dihydroxy 3-carboxy AQ, 1,4-dihydroxy AQ, 1,5-dihydroxy AQ, 1,8-dihydroxy AQ and 2,6-dihydroxy AQ in V79 Chinese hamster cells. The V79 cells were used since, as they contain flavoproteins but not cytochrome P-450, they can bioactive xenobiotics only through the reductive pathway excluding the oxidative one. In addition, the abilities of AQs to stimulate O2-production using both purified flavoproteins (NADH-dehydrogenase, NADPH-cytochrome P-450 reductase) and V79 subcellular fractions (homogenate and microsomes) were assayed. The NADH and NADPH consumption stimulated by AQs in V79 microsomes was also determined. The results showed that the carboxylic-containing drugs and the 1,4-dihydroxy AQ were weak sister chromatid exchange inducers and the most toxic among the six anthraquinones examined. Dicumarol, a potent inhibitor of DT-diaphorase, reduced, rather than potentiated, both the cytotoxicity and genotoxicity caused by these AQs. Thus, the higher superoxide formation rates stimulated by the carboxylic-containing AQs compared to those of the other quinones with all the in vitro systems used, suggested, except for the 1,4-dihydroxy AQ, a possible relationship between cytotoxicity and O2-production. For the 1,4-dihydroxy AQ toxicity, a specific bioactivation route was hypothesized.

Effects of diacerein on the quantity and phagocytic activity of thioglycollate-elicited mouse peritoneal macrophages.

Diacerein (DAR: 1,8-diacetoxy-9,10-dioxo-dihydroanthracene-3-carboxylic acid) is an anthraquinone drug which displays anti-inflammatory effects in experimental animals and antirheumatic activity in humans. The drug was administered for orally for four consecutive days to mice injected intraperitoneally with thioglycollate. The following dose levels of DAR were used: 2.5, 5 and 10 mg/kg/day. At the end of the experimental period the macrophage content of peritoneal exudate was dose-dependent and significantly lower in DAR-treated mice compared with animals that were given saline orally. The macrophages isolated from the peritoneal exudate of mice that received DAR displayed a dose-dependent reduced phagocytosis. The effects of DAR were found to be similar to those of indometacin and dexamethasone, which were used as reference drugs. The ability of DAR to interfere with macrophage functioning may contribute to its overall therapeutic activity.

Rhein reduces proteoglycan loss during the autolytic breakdown of cultured cartilage.

Rhein (R: 1,8-dihydroxy-3-carboxyanthraquinone) is the active metabolite of the drug diacerhein (DAR), an anthraquinone molecule which has recently been proposed for the long-term treatment of osteoarthrosis. In the present study we have examined the effects of rhein, as compared to indomethacin or hydrocortisone, on an in-vitro model of cartilage degradation, represented by the autolytic breakdown of the articular cartilage excised from rabbit knee and cultured for seven days. During this period there is a spontaneous loss of proteoglycans. At the end of the period we measured the amount of proteoglycans which remained bound to the cartilage. The samples treated with R revealed dose-dependent modifications in the amounts of cartilage-bound proteoglycans, with a 40% increase as compared with non-treated samples at the dose of 7 x 10(-5) M. We conclude that R shows a protective effect on the articular cartilage, and that at least a part of the beneficial effect that DAR has shown in the course of clinical trials in osteoarthrosis may be due to direct effects of its active metabolite (R) on cartilaginous tissue.

Rhein: an anthraquinone that modulates superoxide anion production from human neutrophils.

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), the active metabolite of diacetylrhein, which has been reported as an effective antirheumatic drug in man, inhibited superoxide anion production from human neutrophils challenged with N-formylmethionyl-leucyl-phenylalanine (FMLP: IC50, 2 x 10(-5) M) and A23186 (IC50, 10(-5) M), but not with phorbol myristate acetate. In the same concentration range (10(-6)-10(-3) M), the drug did not affect oxy-radical production by a cell-free hypoxanthine-xanthine oxidase system and exerted weak inhibitory effects on FMLP-evoked lysosomal enzyme release. Rhein inhibitory effects on neutrophil functioning may contribute to the overall therapeutic activity of the parent drug, diacetylrhein.

Experimental studies on diacerhein: effects on the phagocytosis of neutrophil cells from subcutaneous carrageenan-induced exudate.

Diacerhein (DAR), a new drug which is particularly suitable for the treatment of osteoarthritis, was studied for its interference with the phagocytic capacity of cells coming from exudates of subcutaneous carrageenan oedema and from the peripheral blood of Sprague-Dawley rats. DAR was found to inhibit phagocytosis in both types of cells examined. This finding indicates that DAR may exert its action by means of a direct effect on the cells involved in the inflammatory process.

Studies in vitro on the effects of rhein on the chemotaxis of human leukocytes.

Rhein (R: 1,8-dihydroxy-3-carboxyanthraquinone) is the active metabolite of the drug diacetylrhein (DAR), an anthraquinone molecule which has recently been proposed for the long-term treatment of osteoarthrosis. Its action mechanism in rheumatic pathology has not been fully explained. It is known that DAR, while not inhibiting the formation of prostaglandins, inhibits certain proteolytic enzymes, and acts on phlogistic cells by lysosomal enzymic and superoxide-anion modifications. Moreover DAR modifies phagocytic functions and the motility of cells. This paper is a contribution to the clarification of the last point, namely the effect of rhein on cell motility. It reports that in vitro no effect of R on random migration was found, but instead a double inhibiting effect on chemotaxis (i.e. a low-dosage and a high-dosage effect). Furthermore, R did not modify the inhibition or induce modification of chemotaxis by vinblastine. Finally R cancelled the stimulating effect of ionic potassium. The results thus indicate that R acts on the chemotaxis of the leukocytes with a complex action at different doses. The action mechanism is probably due to a membrane effect, since rhein (R) did not modify the chemotaxis-inhibiting activity of vinblastine but did interfere with the stimulating effect of K+.