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PubMed2003

Tyrosinase protects human melanocytes from ROS-generating compounds.

The effects of two tetrahydroisoquinolines (TIQs), tetrahydropapaveroline (THP) and salsolinol (SAL), on human primary melanocytes were studied. These compounds are naturally occurring alkaloids deriving from the condensation of dopamine with aldehydes. The effects on the viability were studied by treating the cells with variable concentration of THP or SAL; both TIQs were well tolerated up to roughly 30 micro M. At higher concentrations, THP became overtly toxic while SAL showed no cytotoxic effect up to 100 micro M. TIQs treatment determined an impairment of intracellular activity of antioxidant enzymes, like SOD, DT-diaphorase, and glutathione peroxidase. A decrease of alpha-ketoglutarate dehydrogenase activity was also evidenced following TIQs treatment; a very strong diminution was found in THP-treated cells, whose viability was highly decreased. Both TIQs increased tyrosinase-specific mRNA transcription followed, in the case of SAL, by an activation of tyrosinase. In the presence of tyrosinase inhibitors TIQs treatment resulted in a sharp cytotoxic effect even at concentrations normally well tolerated. Taken together these data suggest that tyrosinase represents an outstanding protective mechanism against ROS-generating compounds, once primary detoxifying mechanisms are impaired or not available.

Antioxidants↗

PubMed1995

Rod outer segment (ROS) renewal as a mechanism for adaptation to a new intensity environment. II. Rhodopsin synthesis and packing density.

Using intraocular injection of a 3H-phe and 3H-leu mixture, we found that the net synthesis of rhodopsin is light-intensity-dependent and can adjust when an animal encounters a new lighting environment. Rhodopsin net synthesis dropped dramatically in day 1, 200-lx immigrants; however, preliminary studies show that the opsin mRNA levels in these animal were the same as that found in the 200-lx-native group. This suggests that the changes in the net synthesis of rhodopsin found when an animal is moved to a higher intensity light may be controlled at some point post-transcriptionally. Microspectrophotometric measurements on single rod cells revealed that the differences in whole-eye rhodopsin levels between the two cyclic intensity groups, 3 lx and 200 lx, were also present at the single cell level. This supports previous studies suggesting that the density of rhodopsin per disc varies according to the intensity of light to which the animal is exposed. Individual rods also had differences in packing density of rhodopsin at the base compared to the tip of the same outer segment when the animal had been switched to a new intensity for one half of a turnover period (5 days). This indicates the amount of rhodopsin per disc is regulated and suggests that renewal is the process responsible for creating a modified outer segment. Animals, when switched to a new intensity, can adjust the synthesis of rhodopsin and the number of molecules added per disk to ensure the appropriate packing density of rhodopsin in the rod outer segment (ROS) disk membranes for that level of light intensity.

Adaptation, Ocular↗

PubMed2003

ROS-dependent caspase-9 activation in hypoxic cell death.

Mitochondria are known to play a fundamental role in apoptosis by releasing apoptogenic molecules such as cytochrome c into the cytoplasm, thereby sequentially activating initiator caspase-9. However, the mechanisms of cytochrome c release or caspase-9 activation in response to hypoxia are unclear. In this report, we show that caspase-9 is activated by reactive oxygen species (ROS) without involvement of cytochrome c release in hypoxic injury. In addition, activated caspase-9 induces permeability transition (PT)-independent cytochrome c release, suggesting that caspase-9 may disrupt mitochondrial diffusion limit of cytochrome c and serve to amplify further release of cytochrome c.

Apoptosis↗

PubMedDate not supplied

Nonhypercalcemic analogs of vitamin D stimulate creatine kinase B activity in osteoblast-like ROS 17/2.8 cells and up-regulate their responsiveness to estrogens.

We have reported that pretreatment with 1 alpha, 25(OH)2D3(1, 25) up-regulates responsiveness and sensitivity to 17 beta estradiol (E2) in osteoblast-like cells, as measured by parallel stimulation of [3H]thymidine incorporation into DNA and the specific activity of creatine kinase BB (CK). Increased responsiveness was correlated with increased E2 receptor concentration. In this study, we have extended these observations to new nonhypercalcemic analogs of 1,25. We compared the analogs hexafluoro vitamin D3 (FL), and the side chain modified derivatives: EB 1089 (EB), CB 1093 (CB) and MC 1288 (MC) with 1,25 and 25 (OH)D3(25 D3). Treatment with 30 nM E2 for 4 h stimulated CK activity in ROS 17/2-8 cells by 40%; there was no further increase after 3 daily additions of E2. Treatment by 3 daily additions, at 1 nM, of all analogs except 25 D3 caused a 2-3-fold increase in CK specific activity. This schedule of treatment also upregulated the response to 4 h exposure to 30 nM E2 by 30-70% above the response to vitamin D analogs alone, and by up to 2 fold compared to E2 without pretreatment. At 1 pM, the analogs doubled CK activity, and, except for 1,25, upregulated the response to E2 to levels characteristic of each analog. Pretreatment with vitamin D analogs also increased the sensitivity to E2 by lowering the dose for a comparable response to E2 by one or two orders of magnitude. Stimulation of specific activity of CK by the analogs was paralleled by increases in the steady state level of mRNA for CKB, but not in its half life. Whereas pretreatment by vehicle followed by E2 for 2 h was unable to increase CKB mRNA, pretreatment with the analogs made possible detection of mRNA responsiveness to E2. These results add to the evidence for the interaction of estrogens and antiestrogens with vitamin D metabolites in regulation of bone growth in vitro. They also strengthen the potential for treatment of bone loss, as occurs in postmenopausal osteoporosis, by a combination of nonhypercalcemic vitamin D analogs and estrogens.

Animals↗

PubMed2002

The effect of aging on p38 signaling pathway activity in the mouse liver and in response to ROS generated by 3-nitropropionic acid.

Since mitochondrial dysfunction is a major source of oxidative stress in aged tissues, we asked whether the basal activities of stress response signaling pathway(s) are indicative of oxidative stress in aged tissues. To address this issue we asked whether: (a). aging affects the basal activity of the p38 MAPK stress signaling pathway; (b). the p38 MAPK pathway is activated by 3-nitropropionic acid (3-NPA), an inhibitor of complex II (succinic dehydrogenase) and generator of reactive oxygen species (ROS); (c). aging affects the response of the p38 alpha signaling pathway to 3-NPA. Our studies have shown that the basal kinase activities of p38 alpha, its upstream activator, MKK3, and its downstream substrate, ATF-2, are elevated in livers of aged C57BL/6 male mice and that these kinase activities, which are induced by 3-NPA in young livers, do not occur in aged livers. Furthermore, although aging does not affect their protein pool levels there are specific increases in phosphorylation of threonine residues in the p38 alpha and ATF-2 catalytic sites that might account for the increased basal level kinase activities in the aged livers. Our studies suggest that failure to respond to 3-NPA may be a factor in the susceptibility of aged tissue to oxidative damage, and support our hypothesis that aged tissues (especially liver) develop a state of chronic stress even in the absence of a challenge.

Aging↗

PubMed1998

Reactive oxygen species (ROS) generated by xanthine oxidase in the corneal epithelium and their potential participation in the damage of the corneal epithelium after prolonged use of contact lenses in rabbits.

Prolonged use of contact lenses (for 14 days) evoked an imbalance between the activity of xanthine oxidase (an enzyme belonging to reactive oxygen species-generating oxidases) and catalase (an enzyme belonging to reactive oxygen species-scavenging oxidases) in the corneal epithelium of rabbits. The activity of catalase decreased, while xanthine oxidase activity was very high. Of other enzymes studied in the corneal epithelium, the activities of xanthine oxidoreductase, glucoso-6-phosphate dehydrogenase and succinate dehydrogenase were decreased. In contrast, the activities of lactate dehydrogenase and lysosomal hydrolases (acid beta-galactosidase, dipeptidyl peptidase II) were increased and appeared in animals sacrificed immediately after contact lens removal. In rabbits sacrificed later (after 1 h), an additional increase of lactate dehydrogenase and lysosomal hydrolase activities developed in the superficial layers of the corneal epithelium. Catalase supplementation during use of contact lenses prevented both the significant decrease of catalase activity in the corneal epithelium and the development of additional epithelial damage. In contrast, topical treatment with 3-aminotriazole (an inhibitor of catalase) resulted in the nearly complete loss of catalase activity in the corneal epithelium and the appearance of more serious epithelial damage. We conclude that ROS generated by xanthine oxidase induce additional damage of the corneal epithelium related to the use of contact lenses.

Animals↗

PubMed1998

Plasma copolymer surfaces of acrylic acid/1,7 octadiene: surface characterisation and the attachment of ROS 17/2.8 osteoblast-like cells.

The purpose of this study was: (a) to examine the effect of plasma-gas composition on plasma polymer oxygen/carbon (O/C) ratio, functional group composition and stability in water, and then (b) to examine cell attachment to surfaces containing different concentrations of O/C and functional groups. Oxygen-functionalised surfaces were deposited by means of the plasma copolymerisation of acrylic acid/1,7-octadiene. The use of a diluent hydrocarbon allowed the deposition of surfaces with a range of O/C concentrations. Plasma copolymer surfaces were characterised by X-ray photoelectron spectroscopy (XPS). Changes in functional group composition with % acrylic acid monomer and the non-dispersive and dispersive parts of the surface energy of these plasma copolymers were measured. The solubility of the plasma copolymers was assessed by means of XPS. The degree of attachment of ROS 17/2.8 osteoblast-like cells to plasma copolymer surfaces deemed to be 'stable' in aqueous medium was measured. Tissue culture polystyrene (TCPS) was included as a control. Attachment was found to be greatest to the plasma copolymer surface with an O/C of 0.11. This surface had a carboxylic acid concentration of ca. 3%. Attachment did not correlate with increased surface wettability (i.e. the non-dispersive component of the surface energy).

Acrylates↗

PubMed2003

Cell permeable ROS scavengers, Tiron and Tempol, rescue PC12 cell death caused by pyrogallol or hypoxia/reoxygenation.

The role of superoxide anion (O(2)*-) in neuronal cell injury induced by reactive oxygen species (ROS) was examined in PC12 cells using pyrogallol (1,2,3-benzenetrior), a donor to release O(2)*-. Pyrogallol induced PC12 cell death at concentrations, which evidently increased intracellular O(2)*-, as assessed by O(2)(*-)-sensitive fluorescent precursor hydroethidine (HEt). Caspase inhibitors, Z-VAD-FMK and Z-Asp-CH(2)-DCB, failed to protect cells from injury caused by elevation of intracellular O(2)*-, although these inhibitors had effects on hypoxia- or hydrogen peroxide (H(2)O(2))-induced PC12 cell death. Two known O(2)*- scavengers, Tiron (4,5-dihydroxy-1,3-benzenedisulfonic acid) and Tempol (4-hydroxy-2,2,6,6-tetramethylpiperydine-1-oxyl) rescued PC12 cells from pyrogallol-induced cell death. Hypoxia/reoxygenation injury of PC12 cells was also blocked by Tiron and Tempol. Further understanding of the underlying mechanism of the protective effects of these radical scavengers reducing intracellular O(2)*- on neuronal cell death may lead to development of new therapeutic treatments for hypoxic/ischemic brain injury.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed2002

1,25-Dihydroxyvitamin D3 selectively translocates PKCalpha to nuclei in ROS 17/2.8 cells.

We have investigated protein kinase C (PKC) regulation by 1,25-(OH)2D3 in the rat osteosarcoma cell line ROS 17/2.8 since previous reports have implicated PKC in the 1,25-(OH)2D3-mediated regulation of osteocalcin gene expression (J. Biol. Chem. 267 (1992) 12562; Endocrinology 136 (1995) 5685). Here we report that 1,25-(OH)2D3 increased PKCalpha, but not PKCbetaI, epsilon or zeta, levels in the nuclear fraction in a time-dependent manner. Unlike PMA, 1,25-(OH)2D3 did not alter the association of any of the expressed PKC isoenzymes with the plasma membrane. Treatment with 20 nM 1,25-(OH)2D3 for 15 min, 30 min, 1 h and 24 h increased PKCalpha levels in the nuclear fraction by 2.3- to 2.6-fold. Nuclear PKCalpha expression was also increased with doses of 1,25-(OH)2D3 as low as a 0.05 nM. 1,25-(OH)2D3-mediated stabilization of osteocalcin mRNA (Arch. Biochem. Biophys. 332 (1996) 142) was inhibited with bisindolylmaleimide treatment, suggesting that PKCalpha may be involved in the 1,25-(OH)2D3-mediated regulation of osteocalcin gene expression.

Blotting, Northern↗

PubMed2003

Hypothermia attenuates delayed cortical cell death and ROS generation following CO inhalation.

Carbon monoxide (CO) is the most popular cause of poisoning. The bilateral basal ganglia lesion characterizes the delayed neuronal cell death (DCD). We demonstrated there were both apoptosis and necrosis in the cortex, basal ganglia and hippocampus in a case of human CO accident. To elucidate the mechanism of DCD after CO inhalation, histological studies on the rat brain were conducted. Rats were ventilated with nitrous oxide (sham group), 10% O(2) (hypoxia group) or 1005 ppm CO (CO group) for 90 min, while the pericranial temperature was controlled at either 32, 37, or 39 degrees C during CO inhalation. After reoxygenation for 30 min, the rats were allowed to recover for 48 h. The ratio of eosinophilic and HNE-positive neurons in the cortex were higher in the CO group than in the hypoxia group at 37 degrees C, while the PaO(2) was much lower in the hypoxia than in the CO group. The damage was alleviated in the hypothermia (32 degrees C) as compared with normothermia, while the hyperthermia (39 degrees C) did not significantly increased it. CO inhalation injures neuron by reactive oxygen species (ROS), independent of hypoxia, as can be concluded from the histological comparison of DCD with HNE immunoreactivity.

Aldehydes↗

PubMed1997

Cadmium perturbs calcium homeostasis in rat osteosarcoma (ROS 17/2.8) cells; a possible role for protein kinase C.

The mechanism of the toxic effects of Cd2+ on bone cell function is not completely understood at this time. This study was designed to characterize the effect of Cd2+ on Ca2+ metabolism in ROS 17/2.8 cells. Cells were labeled with (45)Ca (1.87 mM Ca) for 20 h in the presence of 0.01, 0.1, or 1.0 microM Cd2+ and kinetic parameters were determined from (45)Ca efflux curves. Three kinetic compartments described the intracellular metabolism of (45)Ca. Cd2+ (0.01 microM) caused an approximate 9 x increase in Ca2+ flux across the plasma membrane and a decrease in the most rapidly exchanging intracellular Ca2+ compartment (S1). However, there was no change in total cell Ca2+, indicating an increased cycling of Ca2+ across the plasma membrane. Flux between S1 and the intermediate Ca2+ compartment (S2) was also increased and S2 increased significantly. All Cd2+ induced changes in Ca2+ homeostasis were obliterated by concurrent treatment with 0.1 microM calphostin C (CC), a potent protein kinase C (PKC) inhibitor. This data suggests that Cd2+ perturbs Ca2+ metabolism via a PKC dependent process.

Animals↗

PubMed2003

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis.

Exposure to particulate silica (most crystalline polymorphs) causes a persistent inflammation sustained by the release of oxidants in the alveolar space. Reactive oxygen species (ROS), which include hydroxyl radical, superoxide anion, hydrogen peroxide, and singlet oxygen, are generated not only at the particle surface, but also by phagocytic cells attempting to digest the silica particle. Two distinct kinds of surface centers-silica-based surface radicals and poorly coordinated iron ions-generate O(2)(*)(-) and HO(*) in aqueous solution via different mechanisms. Crystalline silica is also a potent stimulant of the respiratory burst in phagocytic cells with increased oxygen consumption and production of O(*)(-), H(2)O(2), and NO leading to acute inflammation and HO(*) generation in the lung. Oxidative stress elicited by crystalline silica is also evidenced by increased expression of antioxidant enzymes such as manganese superoxide dismutase (Mn-SOD) and glutathione peroxidase, and the enzyme inducible nitric oxide synthase (iNOS). Generation of oxidants by crystalline silica particles and by silica-activated cells results in cell and lung injury, activation of cell signaling pathways to include MAPK/ERK kinase (MEK), and extracellular signal-regulated kinase (ERK) phosphorylation, increased expression of inflammatory cytokines (e.g., tumor necrosis factoralpha [TNFalpha], interleukin-1 [IL-1]), and activation of specific transcription factors (e.g., NFkappaB, AP-1). Silica can also initiate apoptosis in response to oxygen- and nitrogen-based free radicals, leading to mitochondrial dysfunction, increased gene expression of death receptors, and/or their ligands (TNFalpha, Fas ligand [FasL]).

Animals↗

PubMed1996

Identification of proteolytic activities in ROS 17/2.8 cell lysates which cleave peptide substrates for protein kinase C-mediated phosphorylation.

We have observed two proteolytic activities in cell lysates from the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation, and other peptides containing similar sequences. Both activities are inhibited by Pefabloc, a serine protease inhibitor, while one of the activities is inhibited by either EDTA or aprotinin. The protease inhibitors pepstatin, bestatin, E-64, leupeptin and phosphoramidon do not block either of these proteolytic activities.

Amino Acid Sequence↗

PubMed2002

E2F1 and c-Myc potentiate apoptosis through inhibition of NF-kappaB activity that facilitates MnSOD-mediated ROS elimination.

Overexpression of c-Myc or E2F1 sensitizes host cells to various types of apoptosis. Here, we found that overexpressed c-Myc or E2F1 induces accumulation of reactive oxygen species (ROS) and thereby enhances serum-deprived apoptosis in NIH3T3 and Saos-2. During serum deprivation, MnSOD mRNA was induced by NF-kappaB in mock-transfected NIH3T3, while this induction was inhibited in NIH3T3 overexpressing c-Myc or E2F1. In these clones, E2F1 inhibited NF-kappaB activity by binding to its subunit p65 in competition with a heterodimeric partner p50. In addition to overexpressed E2F1, endogenous E2F1 released from Rb was also found to inhibit NF-kappaB activity in a cell cycle-dependent manner by using E2F1(+/+) and E2F1(-/-) murine embryonic fibroblasts. These results indicate that E2F1 promotes apoptosis by inhibiting NF-kappaB activity.

3T3 Cells↗

PubMed2000

Endogenous parathyroid hormone-related peptide enhances proliferation and inhibits differentiation in the osteoblast-like cell line ROS 17/2.8.

To investigate potential effects of endogenous parathyroid hormone-related peptide (PTHrP) on osteoblast function, ROS 17/2.8 cells were transfected with full-length PTHrP cDNA in a sense or antisense orientation to alter PTHrP production. Compared with vector-transfected control cells, PTHrP-overproducing (sense-transfected) cells showed increased DNA synthesis ([(3)H]-thymidine incorporation) and increased growth (cell number). The extent of apoptosis was compared for the different clones using the terminal deoxynucleotide-mediated dUTP nick-end-labeling assay (TUNEL) and Hoechst staining. No differences in percentages of apoptotic cells were found under basal culture conditions or after 3 days of serum deprivation, which, itself, markedly increased numbers of apoptotic cells. The effect of PTHrP on osteoblast differentiation was assessed by examining two protein markers of differentiation, alkaline phosphatase, and bone morphogenetic protein (BMP)-2. Alkaline phosphatase activity was decreased in sense-transfected cells and increased in antisense-transfected cells, compared with cells transfected with empty vector. PTHrP-overproducing cells also showed decreased numbers of BMP-2-positive cells, whereas antisense-transfected cells showed no difference compared with vector control. The results indicate that: (a) endogenously produced PTHrP can increase growth of these osteoblastic cells by stimulating proliferation while not affecting apoptosis; and (b) the increased cell proliferation produced by PTHrP was accompanied by a reduction in activity or amount of two proteins normally expressed by differentiated osteoblasts.

Alkaline Phosphatase↗

PubMed2003

Fluid flow-induced prostaglandin E2 response of osteoblastic ROS 17/2.8 cells is gap junction-mediated and independent of cytosolic calcium.

It has been well demonstrated that bone adapts to mechanical loading. To accomplish this at the cellular level, bone cells must be responsive to mechanical loading (mechanoresponsive). This can occur via such mechanisms as direct cell deformation or signal transduction via complex pathways involving chemotransport, hormone response, and/or gene expression, to name a few. Mechanotransduction is the process by which a bone cell senses a biophysical signal and elicits a response. While it has been demonstrated that bone cells can respond to a wide variety of biophysical signals including fluid flow, stretch, and magnetic fields, the exact pathways and mechanisms involved are not clearly understood. We postulated that gap junctions may play an important role in bone cell responsiveness. Gap junctions (GJ) are membrane-spanning channels that physically link cells and support the transport of small molecules and ions in the process of gap junctional intercellular communication (GJIC). In this study we examined the role of GJ and GJIC in mechanically stimulated osteoblastic cells. Following fluid flow stimulation, we quantified prostaglandin E(2) (PGE(2)) (oscillatory flow) and cytosolic calcium (Ca(2+)) (oscillatory and steady flow) responses in ROS 17/2.8 cells and a derivative of these cells expressing antisense cDNA for the gap junction protein connexin 43 (RCx16) possessing significantly different levels of GJIC. We found that the ROS17/2.8 cells possessing increased GJIC also exhibited increased PGE(2) release to the supernatant following oscillatory fluid flow stimulation in comparison to coupling-decreased RCx16 cells. Interestingly, we found that neither osteoblastic cell line responded to oscillatory or steady fluid flow stimulation with an increase in Ca(2+). Thus, our results suggest that GJ and GJIC may be important in the mechanotransduction mechanisms by which PGE(2) is mechanically induced in osteoblastic cells independent of Ca(2+).

Animals↗

PubMed1990

Preparation and characterization of [N alpha-(4-azido-2-nitrophenyl)Ala1,Tyr36]-parathyroid hormone related peptide (1-36)amide: a high-affinity, partial agonist having high cross-linking efficiency with its receptor on ROS 17/2.8 cells.

The synthesis, purification, and structural analysis of the major compounds resulting from photoderivatization of [Tyr36]-parathyroid hormone related peptide (1-36)amide [[Tyr36]PTHrP(1-36)amide] are described. The reaction of the synthetic peptide with 4-fluoro-3-nitrophenyl azide under nonaqueous conditions yields three major products (peaks D-1, D-2, and G), which were purified to homogeneity by reverse-phase high-performance liquid chromatography. Subsequent amino acid analysis showed that the peptides of peaks D-1 and G each lack one lysine residue, while the peptide in peak D-2 lacks one alanine residue, suggesting that these residues are chemically modified by photoderivatization. Sequence analysis of the photoderivatized peptides revealed that compounds D-1 and G were derivatized on Lys13 and Lys11, respectively. Compound D-2 was N-blocked, indicating that this compound is derivatized on the alpha-amino function of Ala1. Both Lys residues of D-2 were quantitatively recovered upon sequencing after digestion with endoproteinase Glu-C. Compounds D-2 and G had apparent KdS of 1 X 10(-9) M and 0.6 X 10(-9) M, respectively, for their receptors on ROS 17/2.8 cells, which are identical with or similar to that of the underivatized [Tyr36]PTHrP(1-36)amide. Compound G had the same adenylate cyclase stimulating potency as the underivatized, synthetic [Tyr36]PTHrP(1-36)amide, whereas compound D-2 was only a partial agonist, having about 25% of the maximal cAMP production. Compound D-1, which is modified on Lys13, retained only 2-4% of its receptor binding affinity and biological activity relative to that of its parent compound.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗

PubMed2001

A new dehydrogeranylgeraniol antioxidant from Saururus cernuus that inhibits intracellular reactive oxygen species (ROS)-catalyzed oxidation within HL-60 cells.

A new triene, 12,13-dehydrogeranylgeraniol (1), was isolated from the aquatic plant Saururus cernuus and its structure determined spectroscopically. Compound 1 inhibits PMA-induced peroxide-catalyzed oxidation of 2',7'-dichlorodihydrofluorescein dye (DCFH) by reactive oxygen species (ROS) within human promyelocyctic HL-60 cells.

Antineoplastic Agents, Phytogenic↗