Chiara De Luca

Blood antioxidant status and urinary levels of catecholamine metabolites in β-thalassemia

It has been reported that iron overload in beta-thalassemia leads to an enhanced generation of reactive oxygen species and to oxidative stress. We have studied the oxidant/antioxidant imbalance in the blood of 48 transfusion-dependent beta-thalassemic patients (TLP) (17 males, 31 females, 11-22 year), under chelation therapy, and in 40 sex and age matched healthy controls (CTR). Plasma and lymphocyte levels of vitamin E (Vit E), ubiquinol (CoQ10H2), ubiquinone (CoQ10), plasma concentrations of vitamin A (Vit A), beta-carotene, lycopene, vitamin C (Vit C), total thiols, fatty acid patterns of phospholipids (PL-FA), and plasma and urinary markers of lipoperoxidation (TBA-RM, conjugated dienes, and azelaic acid (AZA), as well as the urinary levels of catecholamine and serotonin metabolites, were evaluated by gas chromatography-mass spectrometry (GC-MS), HPLC and spectrophotometry. Routine laboratory blood analyses were performed on the same samples; 39/48 TLP were HCV positive. Blood samples were collected just before transfusion, the 24 h urine samples the day before. Our results clearly showed that a severe oxidative stress occurs in the plasma of TLP in comparison with CTR. In fact, the levels of lipophilic antioxidants and ascorbate were severely depleted: CoQ10H2 (-62.5%), total CoQ10 (-35.1%), Vit E (-43.8%), beta-carotene (-31.1%), lycopene (-63.7%), Vit A (-35.9%), Vit C (-23.1%). The impairment of the antioxidant status was associated with elevated plasma levels of by-products of lipoperoxidation and urinary concentrations of catecholamine metabolites and of AZA, indicating a high degree of both neurological stress and lipoperoxidation. A significant positive correlation was found between vitamin E and non-transferrin-bound iron (NTBI) (r = -0.81; p < 0.001), while no correlation was found between antioxidant depletion and ferritin serum levels, average blood consumption, or the presence of clinical complications. The administration of selective antioxidants along with an appropriate diet might represent a promising way of counteracting oxidative damage and its deleterious effects on the progression of the disease.

Paraphenylenediamine, a contact allergen, induces oxidative stress and ICAM-1 expression in human keratinocytes.

In an investigation of the role of keratinocytes in the pre‐immunological phase of contract allergy, we have studied the effect parphenylendiamine (PPD) on cell proliferation, membrane lipid peroxidation and the expression of the intercellular adhesion molecule 1 (ICAM‐1). Because PPD undergoes rapid autoxidation in the culture medium, the effect of PPD‐modified medium on keratinocyte proliferation and ICAM‐l expression was also examined.

Study on Cross-Reactivity to the Para Group

In 80 patients, positive to at least one hapten of the para group (para-phenylenediamine, diaminodiphenylmethane, benzocaine, PPD mix), patch tests were carried out with freshly prepared solutions of para-phenylenediamine (PPD) and of 3 selected aromatic compounds related structurally to PPD (para-aminophenol, ortho-aminophenol, hydroquinone). The number of positive reactions correlated with the rate of decomposition of the substances as evaluated by high-pressure liquid chromatography. PPD, which was almost decomposed after 24 h, gave the highest number of positive reactions, followed by ortho-aminophenol and by para-aminophenol, while hydroquinone, which was oxidized to the extent of 35%, did not give any reactions. To evaluate if a different rate of oxidation can modify the patch test response, in the same patients and in 10 normal volunteers, tests were carried out with PPD solutions containing the oxidizing agent silver oxide (0.1%). By this procedure a significant increase in the number of positive responses was observed. The results suggest that the rate of decomposition and therefore the amount of quinone(s) generated, might be the key to eliciting patch test responses to oxidizable aromatic haptens.

Saturated dicarboxylic acids as products of unsaturated fatty acid oxidation.

Upon chemical, radiation-induced or enzymatic oxidation, cis-polyunsaturated fatty acids, i.e., C18:2(n-6), C18:3(n-3), C20:2(n-6), C20:3(n-6), C20:3(n-3), C20:4(n-6), C20:5(n-3), C22:2(n-3), C22:4(n-6), C22:6(n-3), were found to generate saturated short and medium-chain length dicarboxylic acids, which can be regarded as a distinctive feature of the particular double bonds positions in the polyunsaturated fatty acid molecule. Two different dicarboxylic acids, which were unambiguously quantified by GC-MS, were produced from a single fatty acid: one deriving from the oxidative splitting at the level of the first double bond in the molecule, the other being two-carbon-atoms lower homologous. Formation of dicarboxylic acids occurred also from triacylglycerols and phospholipids containing cis-polyunsaturated fatty acids. In this case, following oxidation, the diacids remained covalently bound to the starting molecule and transesterification was necessary for identification. Being extremely stable and easily detectable compounds, dicarboxylic acids may be considered potential markers of oxidative attack to both free and esterified unsaturated fatty acids.

Nickel-keratinocyte interaction: a possible role in sensitization

Normal human keratinocytes and the keratinocyte‐derived cell lines NCTC 2544 and A 431, were exposed for different periods (i–5 days) to various concentrations (0·023–46.6 μg/ml) of nickel (Ni2+). A dose‐ and time‐dependent inhibition of cell growth and viability was observed. Cultures exposed to 2·3 μg Ni2+/ml showed approximately 50% cell survival at 5 days. An increase in release of interleukin I by keratinocytes was detected following culture for 24 h with a Ni2+ concentration of 2·3–11·5 μg/ml. Short periods of incubation (30 min) with these concentrations induced an activation of lipoxygenase in leucocytes from healthy subjects, without modifying cell viability. The results suggest that the percutaneous penetration of small amounts of Ni2+ can result in damage to keratinocytes and can initiate sensitization.

The oxyradical-scavenging activity of azelaic acid in biological systems.

We have previously shown that azelaic acid, a C9 dicarboxylic acid, as disodium salt (C(9)2Na) is capable of inhibiting significantly the hydroxylation of aromatic compounds and the peroxidation of arachidonic acid due to reactive hydroxyl radicals (HO.). In this paper we have investigated the ability of C(9)2Na to inhibit the oxyradical induced toxicity towards two tumoral cell lines (Raji and IRE1) and normal human fibroblasts (HF). Oxyradicals were generated either by the addition of polyphenols to the medium, or by direct irradiation of phosphate buffered-saline in which cells were incubated from 15 min prior to incubation in normal medium. The effects of C(9)2Na were compared with those obtained by mannitol (MAN), superoxide dismutase (SOD) and catalase (CAT). C(9)2Na, MAN, SOD and CAT significantly decreased the polyphenol toxicity towards cell lines cultured up to 24 h. After 48 h of incubation the above compounds lost the capability of protecting cells from polyphenol toxicity. This suggests that the toxic role of oxyradicals (O2-., H2O2, HO.) persists for about 24 h and, subsequently other toxic mechanisms must be involved, which are not affected by oxyradical scavengers. SOD and CAT did not show any protective effect on UV induced cytotoxicity, while both C(9)2Na and MAN were capable of reducing significantly the UV damage towards cell lines, even after 48 h incubation. This can be explained by the fact that UV cytotoxicity depends mainly on the generation of HO., that can be scavenged by C(9)2Na or MAN, but not by SOD or CAT. C(9)2Na and MAN were not significantly degraded in the period during which they afford protection against HO..

Lipoperoxidase activity of Pityrosporum: characterisation of by‐products and possible rôle in pityriasis versicolor

Abstract Modification of pigmentation and damage of melanocytes are characteristic features of skin colonisation by Pityrosporum orbiculare hyphae in pityriasis versicolor (PV). The yeast is lipophylic and lipid‐dependent, capable of oxidising unsaturated lipid components of skin surface, i.e. unsaturated fatty acids, cholesterol and squalene (SQ). The oxidation of unsaturated fatty acids gives rise to dicarboxylic acids (DA) which behave, in vitro, as competitive inhibitors of tyrosinase. In this work, we further investigate the oxidase activity of Pityrosporum in vitro, by evaluating (a) the generation of lipoperoxides in cultures supplemented with fatty acids at various degrees of unsaturation; (b) the mechanism of SQ oxidation; (c) the chemical characteristics of some by‐products of lipoperoxidation; (d) the formation of peroxisomes in fungal cells. In cultures supplemented with the saturated palmitic acid (C16:0) and monounsaturated oleic acid (C18:1 n‐9), low amounts of lipoperoxides were detected by a spectrophotometric test, whereas in cultures supplemented with di‐unsaturated linoleic acid (C 18:2 n‐6), significant concentrations were found. Gas chromatographymass spectrometry analyses showed the generation of linoleic acid hydroperoxides both in Pityrosporum cultures and following incubation of acetone powder of the fungus with the unsaturated fatty acid, indicating the presence of a lipoxygenase activity in the fungus. In cultures supplemented with linoleic acid plus SQ, and increase of lipoperoxide generation was observed and trans‐trans farnesal and squalene epoxides have been identified. Electron microscopic examinations have evidenced peroxisomes in cells grown in the presence of linoleic acid, whereas they were not delected in cultures supplemented with oleic acid and palmitic acid. The metabolic activities of peroxisomes, through the formation of hydrogen peroxide and the subsequent generation of hydroxyl radicals, may account for the peroxidation of SQ, which is not a substrate of lipoxygenase. Following these results, we propose a mechanism for DA generation by Pityrosporum metabolism and hypothesize that the lipoperoxidation process induced by lipoxygenase activity of the fungus may be the key to understanding the clinical appearance of skin manifestation of PV.

Scavenging Activity of Azelaic Acid on Hydroxyl Radicals in Vitro

Azelaic acid is an aliphatic dicarboxylic acid (HOOC-(CH2)7-COOH) which has recently been shown to have some practical therapeutic applications in skin diseases of different etiologies. It possesses diverse biological activities and its mechanisms of action are still under investigation. Azelaic acid, as disodium salt (C(9)2Na), at concentrations from 0.05 mM to 1.0 mM is capable of inhibiting significantly the hydroxylation of 1-tyrosine to 1-DOPA due to hydroxylradicals (HO.) produced by Fenton reaction. Similarly C(9)2Na significantly inhibits the heterogeneous photocatalytic oxidation of toluene to cresols, and the peroxidation of arachidonic acid (C20:4,n6), due to HO. formed by dissolved oxygen in the presence of UV-irradiated semiconductor TiO2 (photo-Fenton type reaction). C(9)2Na decomposition and its by-products formation are quantifiable only at high HO. concentrations. On the contrary, C(9)2Na is not a scavenger of O2-. generated by xanthine-xanthine oxidase system. Under the same experimental conditions, mannitol behaves like C(9)2Na. These data indicate that HO. scavenging capacity of C(9)2Na in vitro, and represent a useful tool for further investigations on the mechanisms of action of azelaic acid in biological systems.

Aflatoxin congener biosynthesis induced by lipoperoxidation

Abstract Lipoperoxidation plays a key role in inducing the production of aflatoxins and their congeners (norsolorinic acid, averufin, averantin, versicolorin A, sterigmatocystin, and O -methyl sterigmatocystin) by different strains of Aspergillus parasiticus and Aspergillus flavus . The stimulating effect was obtained by adding epoxides, hydroperoxides, and carbon tetrachloride to culture media. The presence in the media of a free radical scavenger (cysteamine) was capable of inhibiting the output of aflatoxins and their precursors induced by epoxides, hydroperoxides, and carbon tetrachloride. The stimulating effect of lipoperoxidation would appear to take place before the biosynthesis of norsolorinic acid.

Surface Lipids of Seeds Support Both Aspergillus Parasiticus Growth and Aflatoxin Production

AbstractSeed surface lipids (SSL) play a key role in supporting aflatoxin (AFT) output in oily and starchy seeds following infection with Aspergillus parasiticus. On oily seeds, fungal growth and AFT production occur when the levels of SSL are higher than 0.15% of total oil content of seeds, with a ratio between triglycerides and free fatty acids (TG/FFA) of SSL > 1. If FFA prevail over TG, growth can be inhibited because of the toxicity towards Aspergillus of unsaturated FFA of SSL. Defatted meals of sunflower seeds support a reduced AFT biosynthesis. On starchy seeds, A. parasiticus proliferates in the germ region, the area richest in lipids.