Investigation of the Effect of Rhubarb stalks extracts on Mice Exposed to Oxidative Stress.

Normal blood lipid levels have a crucial role in lowering cardiovascular mortality. This study was designed to investigate the effect of aqueous rhubarb extract on serum glucose, cholesterol, total lipids, peroxynitrite, malondialdehyde, glutathione, and ceruloplasmin levels, as well as glutathione and malondialdehyde levels in the liver, kidney, and heart tissue in mice exposed to oxidative stress. 40 Balb/c mice were randomly allocated into 8 groups (n=5). Group 1: The control group was left eating feed and water without treatment for (15) days. Group 2: A group exposed to oxidative stress by giving hydrogen peroxide at a rate of (0.5%) with drinking water for 15 days. Group 3: A group exposed to oxidative stress induced by hydrogen peroxide at a rate of (0.5%) for 15 days with injecting on the seventh day, daily for a week, with insulin subcutaneously (15) units/kg. Group (4-8): the Groups were exposed to the oxidative stress induced by hydrogen peroxide (0.5%) for 15 days with injecting on the seventh day into the peritoneal cavity with both the cold aqueous and nonprotein extract, the extract of flavonoids at a dose of 400, 400, 0.4, 8.8, 1.96 mg/kg body weight, respectively. All animals were anesthetized on the last day of the experiment, blood samples were obtained for biochemical testing, and tissue samples from the livers were collected for research. The results revealed that the cold crude aqueous, non-proteinous extracts, flavonoids, proteinous precipitate, and proteinous compound caused a significant decrease (P<0.05) in serum glucose, cholesterol, total lipids, peroxynitrite, malondialdehyde levels in kidney, liver, and heart. The recorded data showed a significant increase (P<0.05) in serum glutathione and ceruloplasmin in serum and glutathione levels in liver, kidney, and heart tissues in male mice exposed to oxidative stress. The results showed that all Rhubarb extracts have an antioxidant effect in mice exposed to oxidative stress.

[Reparative regeneration of connective tissue structures of mammals under antioxidant therapy conditions].

The influence of administration of the antioxidant complexes consisting of nonenzymatic antioxidants (alpha-tocopherol acetate preparation) and enzymatic antioxidants (ceruloplasmin) has been studied in rabbits with experimental arthritis. The introduction of alpha-tocopherol acetate (at a daily dose of 4 mg) improved metabolic processes in the organism (decreased in the rate of erythrocyte precipitation, total leukocytes and their stub and segmental forms; increased in erythrocyte count; reduced the glycosaminoglycan content as determined from uronic acid and hexose level; decreased ceruloplasmin activity and malonic dialdehyde level ion blood serum, all at p < 0.05), thus favoring reduction in the total activity of the inflammatory process as judged from hematological and biochemical data. Intra-articular introduction of ceruloplasmin (1.5 mg/kg, once per week) positively influenced the state of joint structures in damaged knee joints of the animals: decreased the activity of ceruloplasmin (from 5.28 ± 0.06 to 3.94 ± 0.01 AU), and malonic dialdehyde level (0.18 ± 0.02 to 0.08 ± 0.01 µM) in the articular fluid (all at p < 0.05). These effects are probably related to the elimination of inefficiency of the antioxidant system in the synovial medium, thus preventing inflammatory destruction of articular tissues, hindering the development of pannus, and assisting the activation of reparative regeneration of connective tissue structures.

Anti-inflammatory activity of lycopene isolated from Chlorella marina on type II collagen induced arthritis in Sprague Dawley rats.

The role of commercially available lycopene (all-trans) from tomato in controlling arthritis has been reported. Even though many reports are available that the cis form of lycopene is more biologically active, no report seems to be available on lycopene (cis and trans) isolated from an easily available and culturable sources. In the present study, the anti-arthritic effect of lycopene (cis and trans) from the algae Chlorella marina (AL) has been compared with lycopene (all-trans) from tomato (TL) and indomethacin (Indo). Arthritis (CIA) was developed in male Sprague dawley rats by collagen and the following parameters were studied. The activities of inflammatory marker enzymes like cyclooxygenase (COX), lipoxygenase (LOX) and myeloperoxidase (MPO) were found to be decreased on treatment with AL when compared to TL and Indo. Changes in Erythrocyte sedimentation rate (ESR), white blood cell (WBC) count, red blood cells (RBC) count, hemoglobin (Hb), C-reactive protein (CRP), rheumatoid factor (RF), and ceruloplasmin levels observed in the blood of arthritic animals were brought back to normal by AL when compared to TL and Indo. Histopathology of paw and joint tissues showed marked reduction in edema on supplementation of AL. Thus these results indicate the potential beneficiary effect of algal lycopene on collagen induced arthritis in rats when compared to TL and even to the commonly used anti-inflammatory drug indomethacin. Therefore lycopene from C. marina would be recommended as a better natural source with increased activity and without side effects in the treatment of anti-inflammatory diseases.

Sex and ceruloplasmin modulate the response to copper exposure in healthy individuals.

Previous studies indicated that sex might influence the response to copper exposure. Ceruloplasmin (Cp) is an indicator of Cu status, but it is not clear whether and how it reflects changes of Cu status among healthy individuals. In this study, 82 apparently healthy women and men were chosen from 800 individuals because their Cp values belonged to the higher and lower 10% of the group Cp distribution curve. Before and after receiving a supplement of 10mg Cu/day (upper limit of daily intake) for 2 months, we performed blood and urinary biochemical measurement of potential Cu markers. We used principal component analysis and linear discriminant analysis to identify blood and/or urinary Cu indicators that showed a differential response to copper. Results showed that Cp values in serum represent a reliable indicator to differentiate subgroups within the normal population in their response to Cu exposure. The response depends on Cp values and on sex, such that women with higher and men with lower Cp values exhibit the greatest response.

Cellular copper content modulates differentiation and self-renewal in cultures of cord blood-derived CD34+ cells.

Several clinical observations have suggested that copper (Cu) plays a role in regulating haematopoietic progenitor cell (HPC) development. To further study this role we used an ex vivo system. Cord blood-derived CD34+ cells were cultured in liquid medium supplemented with Kit- ligand, FLt3, interleukin 6 (IL-6), thrombopoietin and IL-3. Under these conditions, Cu content, measured by atomic absorption, was 7 ng/10(7) cells. Modulation of intracellular Cu was achieved by supplementing the cultures with the Cu chelator tetraethylenepentamine, which reduced cellular Cu (4 ng/10(7) cells), or ceruloplasmin or Cu sulphate that elevated cellular Cu (18 and 14 ng/10(7) cells respectively). The results indicated that low Cu content delayed differentiation, as measured by the surface antigens CD34, CD14 and CD15, colony-forming unit (CFU) frequency and cell morphology, while high Cu accelerated differentiation compared with Cu unmanipulated cultures. As a result, expansion of total cells, CFU and CD34+ cells in low Cu was extended (12-16 weeks), and in high Cu was shortened (2-4 weeks), compared with control cultures (6-8 weeks). These effects required modulation of intracellular Cu only during the first 1-3 weeks of the culture; the long-term effects persisted thereafter, suggesting that the decision process for either self-renewal or differentiation is taken early during the culture. This novel method of controlling cell proliferation and differentiation by copper and copper chelators might be utilized for ex vivo manipulation of HPC for various clinical applications.

[Experimental research on the ultrastructural changes in the cells of the spiral organ]. / Eksperimental'nye issledovaniia ul'trastrukturnykh izmenenii kletok spiral'nogo organa.

Adverse effects of vibration and kanamycin on the spiral organ (SO), SO response to physiotherapeutic electrostimulation and ceruloplasmin were studied in guinea pigs. Vibration and kanamycin provoked the same reaction of the SO receptor cells which can be characterized as metabolic stress. In this case mitochondria suffer most of all. Electrostimulation and administration of protein-antioxidant ceruloplasmin gave rise to protective changes suggesting activation of biosynthetic and energetic processes in the receptor cells.

Ceruloplasmin releases pH-induced inhibition of cell proliferation stimulated by growth factors.

Swiss 3T3 fibroblasts can be weakly stimulated to grow by bombesin, epidermal growth factor or ceruloplasmin when cells are maintained in Dulbecco's Modified Essential Medium (DMEM), the pH of which is 7.75. Addition of insulin synergizes with the other mitogens. However, only ceruloplasmin promotes DNA synthesis in Minimum Essential Medium (MEM). The pH in this medium is 7.0. All the other growth factors synergize with the ceruloplasmin effects, but such synergism is not evident with insulin. If the pH in MEM is increased to 7.25 or 7.75 by supplementation with HEPES or NaHCO3, respectively, the results are similar to those found in DMEM. Since the oxidation of iron is increased at alkaline pH, the reoxidation of iron at the cell surface may facilitate growth at alkaline pH. We propose that iron reoxidation is limiting for cell growth and that part of the ceruloplasmin effect is mediated by its action as a terminal oxidase for ferrous iron on the cell surface. Observations consistent with this explanation include: 1) combinations of insulin with bombesin or epidermal growth factors do not promote cell proliferation at pH 7.0; 2) fetal calf serum, which has ferroxidase activity, and ceruloplasmin plus or minus other growth factors stimulate cell proliferation at pH 7.0; and 3) alkaline pH also restores the mitogenic effect of growth factors.

Expression and loading of recombinant heavy and light chain homopolymers of rat liver ferritin.

The full-length genes for the heavy (H) and light (L) chains of ferritin isolated from a rat liver cDNA library were amplified using polymerase chain reaction. Each was inserted at the unique BglII site downstream of the p10 promoter of the baculovirus transfer vector pAcUW21. The genes were transferred separately to infectious Autographa californica nuclear polyhedrosis virus (AcNPV) expression vectors after in vivo homologous recombination. Ferritin homopolymers of either H or L chain were expressed up to approximately 1.5 mg per 100 ml of infected cultures (2.0 x 10(6) cells/ml) of Spodoptera frugiperda, Sf-21, 4 days postinfection. Both recombinant H chain ferritin (rH-Ft) and recombinant L chain ferritin (rL-Ft) assembled as multi-subunit complexes with predicted electrophoretic mobility. Neither rH-Ft nor rL-Ft homopolymers had ferroxidase activity in 50 mM NaCl, as we have reported previously for native ferritin [D. DeSilva, D. M. Miller, D.W. Reif, and S.D. Aust (1992) Arch. Biochem. Biophys. 293,409-415]. When ceruloplasmin, a copper-containing protein, was used as a ferroxidase, rH-Ft loaded iron at rates comparable those obtained with native rat liver apoferritin, but rL-Ft failed to load any iron. The initial rate of Fe(II) oxidation catalyzed by ceruloplasmin was increased in the presence of rH-Ft or rat liver ferritin but not in the presence of rL-Ft. A maximum of about 2500 atoms of iron were incorporated into both rH-Ft and rat liver ferritin. These results demonstrate that both rat liver rH-Ft and rL-Ft homopolymer can be properly produced by the baculovirus expression system and ceruloplasmin can only load iron into H chain ferritin. The physiological significance of these results is discussed.

Embryotoxicity of silver ions is diminished by ceruloplasmin--further evidence for its role in the transport of copper.

The effect of alimentary administration of silver salts upon embryogenesis in rats was studied. Feeding of female rats throughout the term on a regular diet supplemented with AgCl did not cause alterations of their physiological functions, despite the fact that enzymatically active copper-containing ceruloplasmin (CP) was eliminated from the blood plasma. However, developmental abnormalities of embryos, their prenatal death or the 100% mortality of the newborns in the first 24 h of life was seen. Copper content in placenta and fetal tissues was strongly diminished. Cu, Zn-superoxide dismutase (SOD) activity decreased in cytoplasm of embryonic cells along with a drop, though less pronounced, in the tissues of the pregnant females. Embryotoxicity of AgCl was seriously diminished by repetitive injections of native CP to the pregnant rats. Such treatment resulted in an increase of SOD activity in placenta and embryonic tissues. The mortality of the newborns also became less. It is suggested that the embryotoxic effect of AgCl is caused by its ability to interfere with copper metabolism, in particular by altering the copper-transporting function of CP.

Regulation of Cu,Zn superoxide dismutase with copper. Caeruloplasmin maintains levels of functional enzyme activity during differentiation of K562 cells.

K562 cells, a human erythroleukaemic cell line blocked for differentiation, commit towards erythrocytes when exposed to haemin (20 microM). The cells synthesize fetal haemoglobins and show site-specific binding of caeruloplasmin, a plasma copper protein. These events are set into motion by haemin. On the assumption that the binding of caeruloplasmin could reflect a greater need for copper, we sought to determine whether the transfer of 67Cu from caeruloplasmin was accelerated in haemin-induced compared with non-induced K562 cells. Cu,Zn superoxide dismutase (CuZnSOD) was the recipient. Haemin induction caused the K562 cells to lose CuZnSOD activity. By 96 h, the level of SOD activity was less than 60% of that of non-induced cells. The loss was confined entirely to the CuZn form, MnSOD activity staying essentially unchanged. Although CuZnSOD activity declined with the haemin induction, the incorporation of [4,5-3H]lysine into immunoprecipitable CuZnSOD protein was unaffected. There was also no change in CuZnSOD mRNA concentration in haemin-induced cells. Thus a loss of enzyme did not correlate with a decline in the synthesis de novo of CuZnSOD protein. When 48 h-induced cells were transferred to a medium supplemented with 0.2 microM-caeruloplasmin, CuZnSOD activity was restored to control levels in 24 h. Caeruloplasmin also stimulated the incorporation of [3H]lysine into immunoprecipitable CuZnSOD protein. Caeruloplasmin addition may have affected a post-translational regulatory site for CuZnSOD biosynthesis, possibly by providing copper for the newly synthesized enzyme.

Studies on the roles of apotransferrin and caeruloplasmin (EC 1.16.3.1) on iron absorption in copper-deficient rats using an isolated vascularly- and luminally-perfused intestinal preparation.

1. Studies have been made on the effects of dietary copper on the iron and Cu distribution in rats and on the metabolic activity and absorptive capacity of intestines perfused both vascularly and luminally. 2. Rats maintained for 4-5 weeks on a Cu-deficient diet (0.4 microgram Cu/kg) had significantly lower plasma, liver and intestinal Cu concentrations and significantly reduced plasma caeruloplasmin and liver cytochrome c oxidase (EC 1.9.3.1) activity compared with controls receiving a Cu-supplemented diet (5 micrograms Cu/kg). Disturbances in Fe metabolism in Cu-deficient rats were evident as shown by a mild anaemia, significantly elevated hepatic Fe concentrations and hypoferraemia. 3. Intestinal glucose uptake from both the luminal perfusion medium (LPM) and vascular perfusion medium (VPM) was unaffected by Cu deficiency despite a significant (25-30%) reduction in oxygen consumption. This was associated with a 40% decline in mucosal cytochrome c oxidase activity. 4. In studies of Fe absorption, Fe uptake from the LPM was unaffected by Cu deficiency while transfer of Fe to VPM was significantly reduced (50%) compared with control preparations. Addition of apotransferrin (1 g/l) to the VPM was without effect in preparations from control rats but significantly increased the transfer of Fe to the VPM in preparations from Cu-deficient rats without affecting Fe uptake from the LPM. 5. The addition of either human or porcine caeruloplasmin (together with apotransferrin) to the VPM, such that the resultant ferroxidase (EC 1.16.3.1) activity of the VPM supernatant fraction was four to five times that of normal rat plasma, was without effect on either Fe uptake, tissue retention or Fe transfer to the VPM by preparations from either Cu-deficient or control rats. 6. These findings offer no evidence in support of the proposed role for caeruloplasmin with its associated ferroxidase activity in Fe absorption in the rat.

Regulation of aortic CuZn-superoxide dismutase with copper. Caeruloplasmin and albumin re-activate and transfer copper to the enzyme in culture.

Caeruloplasmin and albumin were compared as potential donors of copper to Cu2Zn2-superoxide dismutase (CuZn-SOD) in culture. Aortas from 15-day copper-deficient chicks were suspended in oxygenated, serum-free, Waymouth medium (752/1) for 24 h. SOD activity was restored when the medium was supplemented with CuCl2, a copper-albumin complex or caeruloplasmin, all present at a level equivalent to 5 microM-copper. Activation did not occur at 4 degrees C or with Cu-EDTA as the supplement. Mn2+ and Zn2+, alone or in combination, did not activate nor enhance the activation achieved by CuCl2. The activation with CuCl2 was not inhibited by cycloheximide or cordycepin. [67Cu]Caeruloplasmin and albumin when added to the medium transferred radioactive copper to at least three cytosolic protein fractions, one of which was determined by immunoprecipitation to be CuZn-SOD. The transfer of 67Cu from caeruloplasmin was inhibited by increasing amounts of unlabelled caeruloplasmin; disodium EDTA (1.0 mM) had no effect on the transfer of copper from caeruloplasmin. These data show that aortic SOD activity, suppressed in copper deficiency, can be restored by incubating the aortas in culture medium supplemented with copper salts. In this system, caeruloplasmin and Cu-albumin appear equally capable of activating aortic CuZn-SOD. Moreover, the transfer of copper into the enzyme structure appears to be the primary event restoring catalytic activity to the enzyme.

Effects of ceruloplasmin and the haptoglobin-hemoglobin complex on the oxygen-dependent reduction in growth of human diploid fibroblasts in serum-free, albumin containing medium.

We examined that growth-promoting activity of two different human albumin (HSA) preparations for human diploid fibroblasts in serum-free RITC 80-7 medium. The activity of one preparation (sample A) was affected markedly by environmental oxygen, whereas the other (sample B) was little affected. Sample B contained ceruloplasmin (Cp) and haptoglobin (Hp) as impurities. To detect the generation of superoxide anion in the media the amount of reduction of cytochrome c that is inhibited by superoxide dismutase (SOD) was determined. In an aerobic environment it was relatively large in comparison with reduction inhibited in a hypoxic environment. Reduction in the sample A with HSA-supplemented medium was relatively large in comparison with that in sample B with HSA-supplemented medium. The reduction of cytochrome c also was inhibited by Cp (25 mg/l) and catalase (4000 units/ml). Moreover, SOD, Cp, catalase and Hp.Hb (but not Hp) partially prevented oxygen-dependent reduction in growth in an aerobic environment when added to sample A HSA-supplemented medium. These results suggest that Cp and Hp.Hb act as an antioxidants in culture.

Ceruloplasmin, a copper-transport protein, can act as a growth promoter for some cell lines in serum-free medium.

Ceruloplasmin (Cp) is a copper-transport protein with ferric oxidase activity found in high concentrations in the plasma of all vertebrates. Five cell lines (TR-1, TR-M, TR-ST, TM3, and TM4) derived from the testis can be grown in hormone-supplemented serum-free medium. Cp stimulates the growth of four of these five cell lines in serum-free medium. The growth stimulation by Cp is not affected by the addition or deletion of free copper, nor does copper itself elicit any significant growth response. Cp can stimulate growth also in the absence of TF suggesting that it is not acting solely to promote Fe(III)-TF binding. A strong interaction is seen between Cp and high density lipoprotein (HDL), with the presence of either decreasing the growth-promoting activity of the other. It is suggested that these cell lines may provide an ideal system for studying the action of Cp at the cellular level.

Immunopotentiation of tumor-bearing and toxohormone treated mice by human ceruloplasmin.

Human ceruloplasmin was shown to have a restorative effect on the decrease of delayed hypersensitivity in sarcoma-180-bearing mice and of helper T-cell activity in cell-free. Ehrlich cancerous ascitic fluid-treated mice. Furthermore, it was also shown that ceruloplasmin augments the in vivo generation of alloreactive cytotoxic cell which consist of cytotoxic T-lymphocytes, cytotoxic macrophages and K cells.