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.

Sensitization of skin fibroblasts to UVA by excess iron.

Human skin chronically exposed to UV light is known to accumulate iron and to have an increased ferritin content as compared to unexposed areas. Iron accumulation is also found in many inflammatory skin diseases. Cultured human fibroblasts loaded with iron by incubation with non-toxic concentrations of the ferric nitrilotriacetate complex have been irradiated with low (up to 15 J/cm2) and moderate (up to 45 J/cm2) UVA doses. At low irradiation doses, lipid peroxidation doubles without affecting the viability of iron-loaded cells. At higher irradiation doses (30 J/cm2) the photocytotoxicity of UVA towards iron-loaded cells increases in a concentration-dependent manner with the iron load. Thus, after exposure to 30 J/cm2 of UVA, the cytotoxicity is about 3-fold greater for cells incubated for 75 min with 100 microM of the ferric complex as compared to those not treated with the ferric complex. Incubation with desferrioxamine, an extremely efficient chelator of ferric ion or vitamin E, a radical scavenger which blocks the lipid peroxidation radical chain, leads to marked inhibition of the sensitizing effects of iron on lipid peroxidation but is less effective for the survival of cells exposed to UVA. A similar concentration-dependent protective effect of desferrioxamine was observed with cultured fibroblasts not treated with the ferric complex. It is suggested that the photoreduction of ferritin and/or other iron-containing proteins plays a significant role in the UVA-induced photocytotoxicity of skin fibroblasts.

Photoinactivation of cellular catalase by ultraviolet radiation.

Photoinactivation of catalase is found to be similar in solution and in human normal skin fibroblasts exposed to ultraviolet B, ultraviolet A and near visible light, and the kinetics of such photoinactivation obey first order processes. The action spectrum, measured for the first time in cells, suggests that catalase photoinactivation in solution and in cells proceeds via similar routes. In both systems, no protective effect was observed with diethyldithiocarbamate, a superoxide dismutase inhibitor, with desferrioxamine, an iron chelator which impedes the production of hydroxyl radical via the Fenton reaction, and with vitamin E which scavenges peroxyl radical to protect against membrane peroxidative process. While the absence of protection by these inhibitors may be anticipated for the photoinactivation of catalase in solution, the lack of effect in cells suggests that reactive oxygen species produced by endogenous photosensitization are not responsible for the enzyme inactivation. Moreover, the already established protective effect of ethanol in solution is also observed in cells, supporting the view that photoinactivation in solution and in cells is due to the same primary events.

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.

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.

Ferrous ion release from ferritin by ultraviolet-A radiations.

Ferritin is the principal protein of iron storage (in the Fe(III) state). The UV-A irradiation of 0.25 microM ferritin solutions (from horse spleen) loaded with 530 microM Fe(III) induces Fe2+ release in the medium. The initial quantum yield is wavelength dependent (phi(365 nm) approximately 2 x 10(-3) but pH and oxygen independent. The Fe2+ release reaches a plateau which strongly depends on pH and oxygen. The amino acid composition of the apoprotein is not altered by the UV irradiation. Addition of formate ions enhances the Fe2+ production, suggesting that the ferritin photoreduction involves an electron transfer from an OH- ligand. The possible importance of this phenomenon in skin photobiology is discussed.