Biomedical subjects

R Santus

Publications and source records attributed to R Santus.

At least 73 records · Page 4Linked to original sources

PubMed1994

Ultraviolet A decreases epidermal growth factor (EGF) processing in cultured human fibroblasts and keratinocytes: inhibition of EGF-induced diacylglycerol formation.

The binding, uptake, and degradation of epidermal growth factor (EGF) has been studied in MRC5 human fibroblasts and NCTC 2544 human keratinocytes following ultraviolet A (UVA) irradiation at doses up to 18.9 J/cm2, which are not lethal to cells under our experimental conditions. A dose-dependent reduction in EGF binding was observed, with an approximately 75% decrease at the maximal studied UVA dose. At lower doses (6 to 12 J/cm2), EGF binding was more affected by ultraviolet A in fibroblasts than in keratinocytes. In both cell types, this effect of UVA appeared to be related to a reduction of the affinity of the EGF receptor for EGF. Kinetic studies by pulse-chase experiments indicated that EGF is more rapidly internalized by keratinocytes than by fibroblasts, and that UVA exposure resulted in a slower decay of EGF intracellular content. A 24-h pretreatment of cells with 5 x 10(-5) M vitamin E strongly reduced the appearance of light-induced lipid peroxidation products, measured via assay of thiobarbituric acid reactive substances formation, but only partially prevented the UVA-induced alterations of EGF processing by cells. Finally, UVA exposure almost completely abolished the EGF-induced increase in diacylglycerol production from 14C-arachidonic acid-labeled lipids in both cell types. These results demonstrate that UVA radiation induces important changes in EGF processing and could participate in the light-induced degenerative processes of the skin.

Antioxidants↗

PubMed1994

Early alterations of actin in cultured human keratinocytes and fibroblasts exposed to long-wavelength radiations. Possible involvement in the UVA-induced perturbations of endocytotic processes.

Exposure of cultured MRC5 human fibroblasts or NCTC 2544 human keratinocytes to mild doses of ultraviolet A (UVA: 320-400 nm) radiations markedly decreased the actin reactivity with fluorescein-labeled phalloidin. This indicates a change in the degree of polymerization of actin and thus in the organization of actin filaments. Such a phenomenon might be involved in the previously reported UVA-induced inhibition of specific and nonspecific endocytotic processes.

Actins↗

PubMed1994

[Potentiation of the photocytotoxic effect of photofrin II: synergistic action of verapamil and lovastatin].

Photofrin II (P2) is at the present time the most used drug in the photochemotherapy of tumors. As previous studies from our group have demonstrated that P2 is taken up by cells mainly via the low density lipoprotein (LDL) receptor pathway, we tried to increase the amount of drug delivered to cells by enhancing the LDL receptor expression. For this purpose, we used hydroxy methyl glutaryl Coenzyme A (HMG-CoA) reductase inhibitors such as lovastatin. In the present work, we show that the calcium antagonist verapamil, which is currently used in human chemotherapy to overcome multidrug resistance, enhances in a synergistic manner the potentiating action of lovastatin on the photocytotoxic effect of P2.

Colonic Neoplasms↗

PubMed1993

An in situ study of beta-glucosidase activity in normal and Gaucher fibroblasts with fluorogenic probes.

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE). Post-cleavage increase of fluorescence with fluoresceinyl (bis) beta-glucopyranoside was appreciably above the rather large pre-cleavage emission. In cells incubated overnight with nonylumbelliferyl-beta-glucoside (UG9) in the presence of bovine serum albumin and in the absence of ApoE, the probe was dealt with as a cytotoxic agent, accumulating in a paranuclear cap, most likely comprising elements of the endoplasmic reticulum (ER) and Golgi apparatus. Targeting of UG9 to lysosomes occurred within 1 to 3 h of preincubation in the presence of ApoE. There was some evidence of specificity, as Gaucher fibroblasts exhibited weaker cleavage of UG9 (by 50 per cent or more) compared to normal fibroblasts, but in the Gaucher cells there was some residual beta-glucosidase activity. Cleavage of UG9 was nearly totally suppressed in Gaucher cells treated with the beta-glucosidase inhibitor, conduritol B epoxide, for 24 h to 7 days. Suppression in the control fibroblasts was evident but to a lesser degree. The in situ method of fluorogenic assay established for beta-glucosidase deficiency, is in principle applicable to enzyme deficiencies in other lysosomal storage diseases, or to evaluate enhanced enzyme activity following gene therapy.

Cells, Cultured↗

PubMed1993

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.

Apolipoprotein A-I↗

PubMed1993

Lysosomes, a key target of hydrophobic photosensitizers proposed for photochemotherapeutic applications.

Despite their important biological activity, lysosomes have been generally neglected as important primary targets of photosensitizers, because they are not easily accessible for experiments. This paper reviews factors favoring the localization of photosensitizers in lysosomes and the various experimental approaches which have been used so far for the characterization of the lysosomal staining by various photosensitizing dyes, including porphyrins, chlorins and phenoxazines. The experimental difficulties observed in combining several in vitro techniques for the unambiguous demonstration of lysosomal targeting are examined. New data on tetraphenylporphine derivatives and a pyropheophorbide, as well as previous data on photofrin II, are presented to illustrate the advantages and possibilities of microspectrofluorometry in the study of photosensitizer localization in single living cells. Both spectral and topographic information available from areas smaller than 1 microns2 make it possible to characterize fairly specific sites of localization through the use of specific and vital fluorescent probes of lysosomes, such as Lucifer Yellow. It is also shown by microspectrofluorometry on single living cells that the chronology of the photosensitized reactions induced by specific or unspecific lysosomal photosensitizers can be easily followed. The photosensitized lipofuscin formation observed at the plasma membrane level with the lysosomotropic tetraphenylporphine supports the contention that it is very rare to find a truly specific lysosomal photosensitizer.

Cells, Cultured↗

PubMed1993

Ultraviolet A-induced lipid peroxidation and antioxidant defense systems in cultured human skin fibroblasts.

Cultured human skin fibroblasts from healthy donors were irradiated with 180 kJ.m-2 ultraviolet (UV) A (320-400 nm) and assayed for thiobarbituric acid-reactive substances (TBARS), taken as an indicator of lipid peroxidation. Antioxidant defenses, including total glutathione (GSH) levels, superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase (Cat) activities were simultaneously assayed before and after irradiation. For the various donors, with different activities of these antioxidant systems before irradiation, TBARS correlated positively with SOD activity and negatively with Cat activity, whereas no correlation with GSH level or GSHPx activity was found. These data support the view that O2- is generated by UVA irradiation. They also suggest that H2O2, arising from O2- dismutation by SOD is not completely removed by Cat. Thus, the sensitivity of human fibroblasts to UVA-induced lipid peroxidation depends on a balance between SOD and Cat activities. After UVA irradiation, Cat activity was strongly inhibited, whereas GSH level was slightly decreased. By contrast, GSHPx and SOD activity remained unchanged after UVA irradiation.

Catalase↗

PubMed1993

Exposure to long wavelength ultraviolet radiation decreases processing of low density lipoprotein by cultured human fibroblasts.

Exposure of MRC5 human fibroblasts to UVA radiation (365 nm) resulted in a dose-dependent decrease in low density lipoprotein (LDL) uptake and degradation by cells. Following a 25 J/cm2 irradiation dose, about 45% and 70% reduction in 125I-LDL uptake and degradation were observed, respectively. Under the same conditions, the 14C-sucrose uptake was also decreased to about the same extent as LDL uptake. Cell pretreatment with the antioxidants vitamin E and vitamin C did not prevent the UVA-induced fall in LDL degradation. These results point to the possible effects of UVA radiation on receptor-mediated and nonspecific uptake of exogenous molecules. With special regard to the alterations in receptor-mediated processing of exogenous ligands, such a phenomenon could be of importance in UVA-induced skin degenerative processes.

Cell Line↗

PubMed1993

Lovastatin potentiates the photocytotoxic effect of photofrin II delivered to HT29 human colonic adenocarcinoma cells by low density lipoprotein.

A 24 h preculture of HT29-18 human colonic adenocarcinoma cells with the sterol synthesis inhibitor lovastatin at concentrations of 0.1-0.5 microM markedly increased the photocytotoxic effect of photofrin II delivered to cells by low density lipoproteins. Under the same conditions, LDL binding and photofrin II (PII) uptake by HT29 cells increased about 1.8-fold and 1.5-fold, respectively. These results suggest that hydroxymethylglutaryl-coenzyme A reductase inhibitors could be useful for potentiating the photodynamic therapy of tumors by PII.

Adenocarcinoma↗

PubMed1993

Modulation of the renal effects of contrast media by endothelium-derived nitric oxide in the rat.

RATIONALE AND OBJECTIVES: A possible involvement of endothelium derived relaxing nitric oxide (NO) in the pathogenesis of iodinated contrast media (CM)-induced nephrotoxicity was investigated in the rat. METHODS: Male rats (6 to 12 per group) were uninephrectomized. Six days later, the aorta was clamped above the renal artery and a low-osmolar contrast medium (CM), ioxaglate, was injected (1 mL/min; 3 minutes) via an aortic puncture in the single remaining kidney. Contrast medium was injected with or without the NO-synthase inhibitor L-NAME (100 mg/kg intravenously [i.v.] 5 minutes before CM). One group received L-Arginine, the physiological precursor of NO (100 mg/kg i.v.), 5 minutes before L-NAME. Phenylephrine (300 micrograms/kg; 30 min) was used as a vasoconstrictive NO-independent control. The effects of iohexol, another low-osmolar CM, on creatinine clearance (CrCl) were also studied with and without pretreatment with L-NAME. A control group was subjected to a 3-minute renal ischemia only. Creatinine clearance and urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion were determined before, and 24 and 48 hours after CM administration. Blinded histologic analysis was carried out after completion of the study. RESULTS: When administered alone, neither L-NAME nor L-arginine modified CrCl. Ioxaglate mildly but significantly decreased CrCl at 24 hours (-26.5% of preinjection value). This was similar to the effect observed in the control group subjected to ischemia only. When associated with L-NAME, ioxaglate markedly decreased CrCl (-58 + 11% at 24 hours, P < .05 vs. ioxaglate alone). A similar interaction was noted in the case of iohexol. L-NAME also markedly increased ioxaglate-induced urinary NAG excretion. Phenylephrine had a similar impact on renal function. L-arginine pretreatment reduced the increase in serum creatinine induced by L-NAME+ioxaglate (68 + 17 mumol/L vs. 175 + 59 mumol/L for L-NAME+ioxaglate; P < .05) and urinary NAG excretion. Ioxaglate alone induced only tubular epithelial vacuolization. When associated with L-NAME, this CM induced tubular and vascular lesions, as well as necrosis in the outer medulla. Such histologic effects were clearly inhibited by L-arginine. CONCLUSION: These data indicate that L-NAME, a specific inhibitor of NO-synthase, and phenylephrine, accentuate the nephrotoxicity of CM in the rat. This is consistent with results from the literature showing that CM-toxicity is enhanced by renal ischemia.

Acetylglucosaminidase↗

PubMed1992

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.

Apolipoprotein A-I↗

PubMed1992

Malondialdehyde modification and copper-induced autooxidation of high-density lipoprotein decrease cholesterol efflux from human cultured fibroblasts.

Malondialdehyde modification and copper ion-induced autooxidation of the apo E-free HDL3 fraction of high-density lipoproteins were studied with respect to physico-chemical characteristics and physiological properties of the lipoprotein. Cu(2+)-oxidized HDL was much less modified than MDA-treated HDL, in terms of electrophoretic mobility, lipid peroxidation product content, Lys and Trp amino acid residue level and polymerization of apo A-I. With [3H]cholesteryl linoleate-labeled LDL, an inhibition of cholesterol efflux was observed in the presence of modified HDL, with a more marked effect with MDA-modified HDL. Competition studies with iodinated native HDL demonstrated a decreased binding of modified HDL to cell surface receptors. The decrease in cholesterol intracellular content, determined either by the isotopic equilibrium method or by the enzymatic cholesterol oxidase technic, was less marked in the presence of modified HDL than in the presence of native HDL. MDA-modified HDL was the less effective in decreasing cellular cholesterol content. It is thus suggested that malondialdehyde-induced alteration of HDL, or HDL peroxidation, if occurring in vivo, could contribute to the progress of atherogenesis by decreasing cholesterol efflux from peripheral tissues.

Biological Transport↗

PubMed1992

Photosensitization by Photofrin II delivered to WI26VA4 SV40-transformed human fibroblasts by low density lipoproteins: inhibition of lipid synthesis and fatty acid uptake.

Irradiation with 365 nm light of Wi26VA4 SV40-transformed human fibroblasts cultured for 24 h in the presence of low density lipoproteins loaded with the anticancer porphyrin mixture Photofrin II resulted in a near complete inhibition of [14C]oleic acid incorporation into triacylglycerols, cholesteryl esters and phospholipids. More than 80% reduction of the fatty acid incorporation in all lipid classes was observed following an irradiation dose of 1 J/cm2. The activities of the respective acyltransferases, measured in vitro on cell homogenates, were also markedly diminished, but to a lesser extent than lipid synthesis from oleic acid. Moreover, oleic acid uptake by cells was strongly and rapidly reduced. It is suggested that the rapid inhibition of membrane phospholipid synthesis upon cell photosensitization, due to both a direct inactivation of acyltransferases and to a reduction of fatty acid utilization, could play an important role in the photocytotoxic effect of Photofrin II.

Cell Line, Transformed↗

PubMed1992

[Ultraviolet A radiation and the skin. Implications of activated forms of oxygen. Current trends and newest results].

Involvement of activated oxygen species in the responses of skin to solar ultraviolet radiations, especially ultraviolet A radiations, is being addressed by an increasing number of studies. The aim of this review is to outline the concept of "photooxidative stress". The various activated oxygen species, mechanisms involved in their formation, potential cellular targets, and cell defense mechanisms are discussed. Recently published findings are briefly described to illustrate this fast developing line of research.

DNA↗

PubMed1992

[Antitumor photochemotherapy: biochemical bases, therapeutic uses and perspectives].

The photodynamic therapy of tumors (PDT) is a recent and promising technique for the treatment of tumors which can be reached by the light (directly or by endoscopic illumination). Excellent results are now obtained with hematoporphyrin derivatives such as Photofrin II, provided the concerned tumors are small and well delimited. Porphyrins are transported in blood mainly by lipoproteins, and the low density lipoprotein (LDL) receptor-mediated pathway is probably one of the important factors involved in the selective accumulation of porphyrins by tumor tissues, as cancer cells generally express much more LDL receptors than normal cells. In the present paper, after a brief presentation of the biochemical basis of the light-dependent cytotoxicity of porphyrins, we shall examine the role of lipoproteins, especially LDL, in the transport and the cellular uptake of these compounds. We shall also present recent approaches for the improvement of the PDT efficiency.

Humans↗

PubMed1992

Protection against light-activated photofrin II killing of tumor cells by nitroimidazoles.

Nitroimidazoles are good quenchers of triplet state porphyrins in chemical systems, thereby inhibiting singlet oxygen formation and type II photodynamic reactions. Photobiological studies were performed with EMT-6 tumor cells in vitro utilizing Photofrin II (PII) in combination with etanidazole (ETAN), misonidazole (MISO), and trifluoromisonidazole (TF-MISO). After short-term (1 h) exposure of cells to PII, 5 mM ETAN and MISO had no effect on photoinactivation while 5 mM TF-MISO had a small but significant protective effect. When the intracellular oxygen level was equilibrated with 0.3% oxygen in the gas phase, all three nitroimidazoles produced significant photoprotection at concentrations as low as 0.3 microM. After long-term (24 h) exposure of cells to PII, all three nitroimidazoles demonstrated large photoprotective effects under both aerobic and 0.3% oxygen conditions. At equal concentrations of nitroimidazole, photoprotection was greatest for the most lipophilic compound (TF-MISO) and least effective for the most hydrophilic compound (ETAN). These studies suggest that nitroimidazoles can quench triplet state porphyrins (within cells) to reduce intracellular concentrations of singlet oxygen, the putative toxin in PII photoinactivation. In addition, after long-term exposures to PII when porphyrins have partitioned into cellular membranes and lipid environments, the lipophilicity of this class of photoprotector correlates with effectiveness in these mammalian cells.

Animals↗

PubMed1991

A mechanistic study of the interaction of UVB radiations with human serum lipoproteins.

The tryptophan residues of HDL are important chromophores of the interstitial fluid feeding the epidermal cells. The UVB light readily photoionizes tryptophan residues as demonstrated by the increase in their photolysis yield in presence of N2O. Saturation of the HDL solution with a N2O/O2 mixture (80%:20%, v/v) decreases the peroxidation of HDL lipids, thereby implying that lipid peroxidation is at least partly induced by tryptophan photolysis. Addition of EDTA and desferrioxamine to HDL solutions loaded with either Cu2+ or Fe2+ ions suggests that Fe2+ could be the 'contaminating' trace metal ions that are required to explain the occurrence of lipid photoperoxidation induced by photons absorbed by tryptophan residues.

Chelating Agents↗

PubMed1991

UVA-induced lipid peroxidation in cultured human fibroblasts.

The UVA irradiation of cultured human fibroblasts leads to the formation and to the release of thiobarbituric acid-reactive substances in the supernatant. The major thiobarbituric acid-reactive substance is identified by fluorescence spectroscopy and HPLC, as malondialdehyde or malondialdehyde-forming substances under the thiobarbituric acid assay conditions. Malondialdehyde formation strongly suggests a UVA-induced lipid peroxidation. Lipid peroxidation is also supported by the inhibitory effect of D,L-alpha-tocopherol, the well-known chain breaking antioxidant, by the additional malondialdehyde formation in the dark after the photooxidative stress and by membrane damage revealed by lactate dehydrogenase leakage.

Cell Membrane↗