Comparative effects of nacre water-soluble matrix and dexamethasone on the alkaline phosphatase activity of MRC-5 fibroblasts.

In this study we demonstrate, for the first time, that dexamethasone and BMP-2 stimulated alkaline phosphatase (ALP) activity in MRC-5 fibroblasts, a cell line derived from human fetal lung. Previously we reported that the water-soluble matrix (WSM) of nacre obtained from the inner shell layer of the oyster Pinctada maxima, promoted an increase in ALP activity that was dose-dependent. In this work, we show that the effect of WSM is also time-dependent. As a comparison, the effect of WSM was also tested in bone marrow stromal cells because marrow and other bone surface-derived osteoblast stem cells have the inherent direct potential for osteogenesis. WSM promotes cell proliferation and ALP activity when tested with bone marrow cells in concentrations between 135 and 540 microg protein/mL. The effect of WSM on ALP activity of bone marrow stromal cells is similar to that obtained by dexamethasone. These results imply that MRC-5 fibroblasts respond to differentiating factors that promote osteoblastic phenotype in bone-derived cell cultures.

UV photolysis of catalase revisited: a spectral study of photolytic intermediates.

The 365-nm irradiation of 4.6 microM (approximately equal to 1.1 mg/ml) catalase solutions in pH 7.4 phosphate buffer induces spectral modifications. Difference spectra show maxima at 434, 555, 584 nm at the beginning of the irradiation, then a final spectrum with a maximum at 568 nm and a shoulder at 530 nm is observed. These results suggest the formation of compound III (oxyferrous catalase) and compound II, respectively. In deaerated 0.1 M, pH 8.7 borate buffer, the ferrous catalase is characterized by maxima at 563 and 594 nm. Hydrogen donors such as ethyl alcohol, formate and p-cresol inhibit, but citrate ions enhance the formation of these intermediates. A mechanism involving Fe(III) reduction according to an internal electron transfer is proposed.

Contrasting effects of excess ferritin expression on the iron-mediated oxidative stress induced by tert-butyl hydroperoxide or ultraviolet-A in human fibroblasts and keratinocytes.

Iron and/or ferritin accumulation are known to occur under pathological conditions in many inflammatory skin diseases or in human skin chronically exposed to UV light. Under such conditions, ferritin is believed to play an effective protective role in accommodating and 'deactivating' excess 'free' iron produced by the inflammatory process or the UV illumination. The present study compares the relationship between ferritin over-expression and effects of an oxidative stress induced chemically by tert-butyl hydroperoxide or photochemically by UV-A radiation. As shown by immunoassay, cultured MRC 5 and HS 68 fibroblasts treated for at least one day with transferrin or overnight with non-toxic concentrations of the ferric nitrilotriacetate complex express up to 10 times more ferritin than untreated cells, whereas a five-fold increase is obtained with NCTC 2544 keratinocytes. In all cases a parallel increase in soluble cellular iron is measured by inductive plasma emission spectroscopy. The superoxide dismutase and catalase activities and total glutathione levels are not modified by the iron treatment, whereas a transient increase in the Se-dependent glutathione peroxidase activity of keratinocytes is observed after a short incubation with the iron complex. In keratinocytes and fibroblasts, ferritin over-expression after iron treatment markedly inhibits lipid peroxidation but, paradoxically, not the mortality induced by tert-butyl hydroperoxide. In contrast, this excess ferritin does not protect cells from both the peroxidation and mortality induced by moderate doses (30 J/cm2) of UV-A radiation. As a consequence, protection against oxidative damage by excess ferritin synthesis clearly depends on the nature of the oxidative stress on cell targets and it seems to be of lesser importance in the case of photochemically induced oxidation.

Effect of water-soluble matrix fraction extracted from the nacre of Pinctada maxima on the alkaline phosphatase activity of cultured fibroblasts.

A new approach to the isolation of the water-soluble factors from nacre without any demineralization is described and examined their effect on fibroblast cells in culture. The soluble matrix in pure water from the nacre of Pinctada maxima was analysed by size-exclusion HPLC. Four fractions (SE1-SE4) of the water-soluble matrix (WSM) were further analysed by anion-exchange HPLC. The amino acid composition of the WSM showed that it is mainly composed of glycine and alanine. SE1 and SE4 had different amino acid compositions from the whole WSM. The WSM and SE4 tested on a culture of human foetus lung tissue fibroblasts increased the alkaline phosphatase (ALP) activity. SE1 caused a decrease in ALP activity. Our results support the hypothesis that WSM promotes the differentiation of cells in vitro.

Alteration of lipoproteins of suction blister fluid by UV radiation.

Suction blister fluid is a good representative of the interstitial fluid feeding the epidermal cells. Lipoproteins contained in the suction blister fluid of healthy volunteers are readily photo-oxidized by UV radiation in the wavelength range 290-385 nm. Absorbed light doses equivalent to one minimal erythemal dose absorbed by skin exposed to UVB (e.g. 290-320 nm) are sufficient to induce lipid peroxidation and modification of apolipoproteins A-I, A-II and B. Albumin, which is known to protect serum fully from oxidative stresses, is not so effective against photo-oxidation. Although tryptophan (Trp) residues of proteins contained in the suction blister fluid are photo-oxidized, apolipoprotein A-II, which does not contain any chromophoric Trp residue, is also altered by the UV radiation. With regard to results obtained with a model reconstituted fluid, it is suggested that the radical chain reaction of the lipid peroxidation can transfer the initial photodamage at sites not directly susceptible to photo-oxidation. The biological implications of these results are discussed.

Iron mobilization from ultraviolet-irradiated, iron-saturated, transferrin.

Transferrin is the major iron transport protein of mammalian plasma. The ultraviolet-B irradiation of 1.4 mg/ml iron saturated transferrin solutions (~32 µM Fe(3+)) induces a Fe(3+) loss accompanied by Fe(2+) formation. The initial quantum yield of Fe(3+) loss is wavelength dependent (φ(313 nm)~1.3×10(-3)) and oxygen independent suggesting an intramolecular electron transfer from one of the Fe(3+) ligands. A photolysis of tryptophan residues parallels this photoreduction.

Peroxidation of model lipoprotein solutions sensitized by photoreduction of ferritin by 365 nm radiation.

A mechanistic study involving the 365 nm irradiation of aerated, phosphate-buffered solutions of human high-density lipoproteins (HDL3 fraction) and ferritin was undertaken. The 365 nm irradiation of phosphate-buffered horse spleen ferritin solutions induces the release of Fe2+ in the medium. The initial quantum yield of Fe2+ release on irradiation is 0.002. This quantum yield is oxygen independent. The 365 nm irradiation of mixtures of HDL and ferritin leads to alterations in apolipoproteins as revealed by tryptophan (Trp) oxidation and electrophoretic pattern modification. In parallel with protein damage, lipid peroxidation is induced as shown by hydroperoxide and thiobarbituric acid reactive substances (TBARS) formation. These peroxidations are strongly reduced in 0.1 M formate solution, which suggests chain initiation by .OH radicals or subsequent radicals produced by .OH. They are completely inhibited by desferrioxamine, consistent with propagation by Fe2+ ion. By contrast incubation of HDL in the presence of ferritin and FeSO4 induces only poor auto-oxidation. The biological relevance of this study is discussed.

Copper- and malondialdehyde-induced modification of high density lipoprotein and parallel loss of lecithin cholesterol acyltransferase activation.

Incubation of high density lipoproteins (HDL) with 0.1-10 microM copper ions resulted in a decrease in tryptophan residues and a moderate diminution of lysine residues. Polymerization of apolipoprotein AI (apo A-I) was only observed for the highest concentration of Cu2+. A dose-dependent loss in lecithin cholesterol acyl-transferase (LCAT) activity was noted. Following incubation with 10 mM malondialdehyde, the physicochemical properties of HDL were more pronouncedly affected, in terms of lipid peroxidation products, relative electrophoretic mobility and percentages of intact tryptophan and lysine residues. Polymerization of apo A-I occurred after 40 min incubation, and a time-dependent loss of LCAT activation was noted. Since the deficiency in LCAT activation was observed in relatively mild conditions, when no perturbation of the physico-chemical properties of the particle could be shown, the determination of LCAT activity appears to be a sensitive test for HDL discrete modification.

Modified apolipoprotein pattern after irradiation of human high-density lipoproteins by ultraviolet B.

The ultraviolet B-induced destruction of tryptophan residues and lipid peroxidation of high-density lipoproteins is accompanied by the immediate and marked structural modification of the apolipoproteins, as revealed by SDS-polyacrylamide gel electrophoresis and immunoblot with specific monoclonal antibodies. Formation of several polymers of apolipoprotein A-I, apolipoprotein A-II or both apolipoproteins occurred, although apolipoprotein A-II did not contain any Trp residue. These results suggest that initial photochemical damage can be transferred via intramacromolecular processes to other sites within the same apolipoprotein and by intermacromolecular reactions from apolipoprotein A-I to other apolipoproteins. In both cases, lipid peroxidation enhances the propagation of the initial photochemical damage. The physiological significance of this work is discussed with respect to the low-light doses required for the alterations of the high-density lipoproteins.

Daflon as a cellular antioxidant and a membrane-stabilizing agent in human fibroblasts irradiated by ultraviolet A radiation.

Daflon is a strong inhibitor of Cu(2+)-induced arachidonic acid peroxidation, as revealed by the inhibition of thiobarbituric acid-reactive substance formation in mixed liposomes of phosphatidylcholine and arachidonic acid. Diosmin, the major Daflon constituent, is a good complexant of Cu2+ ions but not of Fe2+ ions. The Cu2+ complex formation may thus explain part of the antioxidant effect. However, Daflon is also a good quencher of the singlet oxygen-induced arachidonic acid peroxidation that does not involve metal ions. When cultured human fibroblasts are incubated with the diosmin-hesperidin mixture constituting Daflon, the latter is as efficient as vitamin E in inhibiting the ultraviolet A-induced lipid peroxidation and plasma membrane damage measured by lactate dehydrogenase release.

Photoreactivity of 5-geranoxypsoralen and lack of photoreaction with DNA.

5-Geranoxypsoralen, commonly called bergamottin, a major furocoumarin contained in bergamot oil, is reported in vitro as a highly photoreactive psoralen. In ethanol, it exhibits quite a high triplet state quantum yield (approximately 0.37). The triplet state is involved in subsequent photochemistry which depends on the initial concentration and on the presence of oxygen. In contrast to most psoralens, absorption and fluorescence data suggest that 5-geranoxypsoralen does not interact with DNA in the dark. No UVA-induced interstrand cross-links in DNA were shown.

Sensitivity of tryptophan and related compounds to oxidation induced by lipid autoperoxidation. Application to human serum low- and high-density lipoproteins.

Tryptamine, serotonin and tryptophan are readily oxidized during the Cu2+-catalyzed peroxidation of arachidonic acid (AA) at neutral pH and under certain experimental conditions which determine their relative susceptibility to oxidation. Thus, in AA micelles, fluorescence spectroscopy demonstrates that positively-charged indoles interact with negatively-charged micelles while Trp remains in the aqueous phase. As a result, serotonin and tryptamine are preferentially oxidized. In egg phosphatidylcholine liposomes loaded with AA, the three substrates interact with vesicles and undergo lipid-induced oxidation. EDTA inhibits the formation of thiobarbituric-reactive substances (TBARS) and prevents the indoles from oxidation. Owing to the intricate contact between the lipidic core and the apolipoproteins, the Trp residues of human serum LDL and HDL3 are very rapidly oxidized, i.e., at least one order of magnitude faster than Tyr HDL and Lys LDL, which are believed to be involved in the binding of these lipoproteins to their cell receptors. Cupric ions are rather specific for the lipid-induced autoxidation of Trp residues of lipoproteins whereas in micelles and liposomes, Mn2+ and Fe2+ can lead to TBARS production and to oxidation of indoles. This specificity is surprising considering the known ability of Fe2+ to catalyze LDL modification (measured by TBARS production) during their incubation with various cells. Biological consequences of the easy lipid-induced oxidation of biologically important indoles are discussed.