Effect of testosterone metabolites on ABC half-transporter relative gene expression in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder associated with reduced very long-chain fatty acid beta-oxidation, mainly affecting the nervous system, the adrenal cortex and the testes. The clinical manifestations of hypogonadism, alopecia and the impairment of the enzyme 5alpha-reductase, which converts testosterone into dihydrotestosterone, clearly point to an involvement of androgens in this pathology. The disease is characterized by mutations in the ABCD1 gene, which codes for the peroxisomal ABC half-transporter ALDP, and by a broad range of clinical manifestations. The altered function of ALDP can be compensated by the overexpression of proteins belonging to the same family of ABC half-transporters. A promising therapeutic approach is represented by the activation of these proteins by specific agonists. In this study we evaluated the effect of the testosterone metabolite dihydrotestosterone (DHT) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) on the expression of the ABC half-transporters encoded by the ABCD2 and ABCD3 genes, in fibroblasts drawn from controls and from two affected brothers. The two patients presented the same mutation in exon 9 but had different clinical manifestations, one patient being asymptomatic and the second one severely affected. When the cells were stimulated with testosterone metabolites, only the severely affected patient showed a significant increase in ABCD2 mRNA levels, while the ABCD3 expression remained unchanged in both patients.

New findings on X-linked Adrenoleukodystrophy: 5alpha-reductase isoform 2 relative gene expression is modified in affected fibroblasts.

X-linked Adrenoleukodystrophy (X-ALD) is a neurodegenerative disease with an endocrinological component since, in addition to the nervous system, the adrenal cortex and the testis are mainly affected, with corresponding clinical signs. 5Alpha-reductase, a key enzyme in steroid hormone metabolism, catalyzes the conversion of testosterone into the potent androgen dihydrotestosterone and other metabolic steps in steroidogenesis. It is present in two isoforms, 5alpha-reductase isoform 1 and 2, that are encoded by different genes. The isoforms are differently expressed in the tissues, where they have distinct physiological relevance. Our study shows that the expression of isoform 2, evaluated by Real-Time PCR, is significantly altered in fibroblasts from patients affected by X-ALD with respect to controls, whereas isoform 1 is not affected. This is the first demonstration of an alteration of 5alpha-reductase isoform 2 gene expression in X-ALD, that may be related to the steroidogenesis impairment and to the specific organ malfunction.

Effects of the testosterone metabolite dihydrotestosterone and 5 alpha-androstan-3 alpha,17 beta-diol on very long chain fatty acid metabolism in X-adrenoleukodystrophic fibroblasts.

X-Adrenoleukodystrophy (X-ALD) is a peroxisomal disorder associated with the abnormal accumulation of very long chain fatty acids (VLCFA) in plasma and tissues. We have demonstrated that the androgen dihydrotestosterone (DHT) and 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol) have favorable effect on VLCFA metabolism. We have investigated the effect of androgens on peroxisomal beta-oxidation, the incorporation of labelled lignoceric acid into cholesterol esters and VLCFA elongation, in cultured skin-fibroblasts from control and X-ALD patients. The androgens significantly increased peroxisomal beta-oxidation in X-ALD fibroblasts although VLCFA levels were not normalized. The major effect was on the incorporation of labelled lignoceric acid into cholesterol esters, since the enhanced lignoceric acid incorporation into cholesterol ester fraction, which occurred in X-ALD fibroblasts, was reduced towards normal values. In contrast, the androgens had no effect on the elongation pathway.

Blockade of A(2A) adenosine receptors leads to c-fos inhibition in a rat model of brain ischemia.

Adenosine plays an important role in cerebral ischemia by acting on its own receptors, in particular the A(2A)receptor. Its activation leads to excitatory amino acid release thus contributing to the ischemic damage. Blockade by specific antagonists may protect against cytotoxic injury. Our study was aimed to investigate the effect of the blockade of A(2A)receptors, by Sch 58261, on the expression of the early gene c-fos, in a model of permanent middle cerebral artery occlusion (pMCAo), in rats. In the pMCAo model, ischemia was induced in the right hemisphere whereas the contralateral one was considered the control. In our study, we have compared pMCAo rats, pMCAo rats treated with Sch 58261 and sham operated ones.C-fos was markedly expressed in the ischemic hemispheres, whereas lower levels were detected in the contralateral ones of the ischemic animals. The lowest bands were observed in sham operated rats. After treatment with Sch 58261, a considerable reduction in c-fos expression was observed in the ischemic hemispheres, whereas a limited effect was detected in the others. Our results suggest that inhibition of immediate-early gene expression by the A(2A)receptors antagonist Sch 58261 may contribute to its neuroprotective activity.

Arachidonate release and c-fos expression in various models of hypoxia and hypoxia-hypoglycemia in retinoic acid differentiated neuroblastoma cells.

Hypoxia-hypoglycemia has played an important role in inducing both phospholipase A2 activation and the expression of the early gene c-fos, in the neuroblastoma cell line SK-N-BE, after it has been differentiated by retinoic acid. Under hypoxic-hypoglycemic conditions, arachidonic acid release has found to be significant after 30 min, whereas c-fos expression has required at least 4 h. This model has been obtained by adding glycolytic inhibitor 2-deoxyglucose to the culture and by placing cells in an atmosphere containing 100% N2 for different time periods. This condition has been compared with two different models: NaCN and nitrogen have been used as hypoxic stimuli, without inhibiting the glycolytic pathway, but the same cell cultures have been used. Cell viability and the fall of cellular ATP levels have been evaluated in all the models, in order to monitor and compare the hypoxic cellular damage. Phospholipase A2 activation has been found to be significant in all conditions, even if to a different extent; but only hypoxia combined with the inhibition of the glycolytic pathway, has induced a significant expression of c-fos. It is very difficult to study hypoxic stimuli in 'in vitro' systems. Our study has compared three different models and the one combining gaseous hypoxia and hypoglycemic conditions seems to be very effective in stimulating early events involved in hypoxic phenomena such as phospholipase activation and the expression of the early gene c-fos.

Testosterone metabolites in patients reduce the levels of very long chain fatty acids accumulated in X-adrenoleukodystrophic fibroblasts.

Testosterone metabolites (dihydrotestosterone, DHT) and 5 alpha-androstan-3 alpha,17 beta-diol (3 alpha-diol), but not testosterone itself, were shown to reduce the levels of very long chain fatty acids which accumulate in cultured skin fibroblasts from X-adrenoleukodystrophic patients (X-ALD). In addition, in X-ALD fibroblasts, testosterone is less actively converted into DHT vs. controls (skin fibroblasts retrieved from normal subjects) whereas the additional conversion of DHT to the final product 3 alpha-diol is enhanced. This is the first report of altered testosterone metabolism in X-ALD fibroblasts and of the effects of androgens in lowering the abnormal accumulation of very long chain fatty acids in this type of cells.

The beta-oxidation of arachidonic acid and the synthesis of docosahexaenoic acid are selectively and consistently altered in skin fibroblasts from three Zellweger patients versus X-adrenoleukodystrophy, Alzheimer and control subjects.

The beta-oxidation of [3H] arachidonic acid (AA; 20:4 n-6) and the conversion of [1-14C]eicosapentaenoic acid (EPA, 20:5 n-3) to docosahexaenoic acid (DHA, 22:6 n-3) have been studied in skin fibroblasts from patients with inherited peroxisomal diseases, such as Zellweger (ZW) and X-linked adrenoleukodystrophy (X-ALD), from patients with Alzheimer's disease (AD), a non-inherited neuropathology, and from controls. EPA is not converted to DHA, while there is enhanced formation of the intermediate product 22:5 n-3 in ZW, when compared to X-ALD, AD and controls. We also confirmed that AA is not beta-oxidized to 4,7,10-hexadecatrienoic acid (16:3), a metabolite produced by peroxisomes, while being more effectively converted to the elongation product 22:4, in ZW, in comparison to X-ALD, AD and controls. The data demonstrate a defect in DHA synthesis and in AA beta-oxidation, and the occurrence of associated adaptative modifications in the metabolism of these long chain PUFA, in three Italian ZW patients.

Inhibition of leukocyte leukotriene B4 production by an olive oil-derived phenol identified by mass-spectrometry.

We have evaluated the effects of hydroxytyrosol (HT), a potent antioxidant present in olive oil, on the formation of arachidonic acid 5-lipoxygenase metabolites by leukocytes in vitro. HT, a simple phenolic compound, extracted from first-pressure oil, was isolated by HPLC and characterized by gas chromatography/mass spectrometry. HT inhibited in a dose-related manner the production of leukotriene B4 (LTB4) by calcium ionophore-stimulated leukocytes. As expected, similar inhibition was observed for omega-oxidized metabolites of LTB4, namely 20-hydroxy and 20-carboxy-LTB4. The results disclose a new biological activity of olive oil-derived phenols on leukocyte eicosanoid production.

Arachidonic acid synthesis and lipid metabolism in retinoic acid-differentiated neuroblastoma cells.

SK-N-BE cells were differentiated (D) to neurons with retinoic acid. Total n-6 polyunsaturated fatty acids (PUFA), especially arachidonic acid (AA), were increased in D versus ND cells. AA synthesis from linoleic acid (LA) and phospholipid (PL) synthesis from glycerol were initially elevated, whereas both processes were reduced approaching differentiation. At this stage, the incorporation of glycerol in triglycerides was enhanced. Formation of long chain PUFA and synthesis of acceptors (PL) for esterification, during RA-induced SK-N-BE differentiation, appear to be associated.

Changes in arachidonic acid levels and formation and in lipid synthesis in the human neuroblastoma SK-N-BE during retinoic acid-induced differentiation.

We observed that retinoic acid, which differentiates the human neuroblastoma SK-N-BE into mature neurons, induced an elevation in levels of polyunsaturated fatty acids, especially arachidonic acid (20:4 n-6). This effect was not induced by phorbol myristate acetate, another differentiating agent. We then explored the effects of retinoic acid on the formation of arachidonic acid and of docosahexaenoic acid from precursors and on the de novo lipid synthesis from acetate at various stages of differentiation, which was assessed by morphological (cell number and neurite outgrowth) and biochemical (protein content and thymidine incorporation) criteria. At 3 days of incubation with retinoic acid, in the n-6 series, total conversion of linoleic acid, especially to 20:3 n-6, was elevated, in association with preferential incorporation of acetate into phospholipids; in contrast, at 8 days, synthesis of 20-carbon polyunsaturated fatty acids declined, in association with enhanced incorporation in triglycerides. In the n-3 series, eicosapentaenoic acid was converted to docosahexaenoic acid in SK-N-BE, but the conversion was not affected by retinoic acid. During the early stage of neuronal differentiation, therefore, enhanced production of 20-carbon polyunsaturated fatty acids from their precursors occurred, and newly formed fatty acids were preferentially incorporated in phospholipids, possibly in association with membrane deposition. When differentiation was completed, arachidonic acid formation and incorporation of acetate in phospholipids and cholesterol declined with enhanced labeling of storage lipids.

Inhibition of platelet aggregation and eicosanoid production by phenolic components of olive oil.

This study was designed to investigate the in vitro effects of phenolic compounds extracted from olive oil and from olive derived fractions. More specifically, we investigated the effects on platelets of 2-(3,4-di-hydroxyphenyl)-ethanol (DHPE), a phenol component of extra-virgin olive oil with potent antioxidant properties. The following variables were studied: aggregation of platelet rich plasma (PRP) induced by ADP or collagen, and thromboxane B2 production by collagen or thrombin-stimulated PRP. In addition, thromboxane B2 and 12-hydroxyeicosatetraenoic acid (12-HETE) produced during blood clotting were measured in serum. Preincubation of PRP with DHPE for at least 10 min resulted in maximal inhibition of the various measured variables. The IC50s (concentration resulting in 50% inhibition) of DHPE for ADP or collagen-induced PRP aggregations were 23 and 67 microM, respectively. At 400 microM DHPE, a concentration which completely inhibited collagen-induced PRP aggregation, TxB2 production by collagen- or thrombin-stimulated PRP was inhibited by over 80 percent. At the same DHPE concentration, the accumulation of TxB2 and 12-HETE in serum was reduced by over 90 and 50 percent, respectively. We also tested the effects of PRP aggregation of oleuropein, another typical olive oil phenol, and of selected flavnoids (luteolin, apigenin, quercetin) and found them to be much less active. On the other hand a partially characterized phenol-enriched extract obtained from aqueous waste from olive oil showed rather potent activities. Our results are the first evidence that components of the phenolic fraction of olive oil can inhibit platelet function and eicosanoid formation in vitro, and that other, partially characterized, olive derivatives share these biological activities.

Inhibition by n-3 fatty acids of arachidonic acid metabolism in a primary culture of astroglial cells.

Docosahexaenoic acid (22:6 n-3) was present in low concentrations in a primary culture of rat brain astroglial cells, when compared to brain cortex. We have thus supplemented these cells with this fatty acid and investigated the effects of its incorporation in cell phospholipids on the conversion of arachidonic acid, 20:4 n-6, through the cyclo and lipoxygenase pathways, after cell stimulation. Docosahexaenoic acid-enriched cells produced less thromboxane B2 and 6-keto-Prostaglandin F1 alpha and markedly less 12-hydroxyeicosatetraenoic acid than unsupplemented cells, after stimulation with the Ca(2+)-ionophore A23187. The production of 15-hydroxyeicosatetraenoic acid from arachidonic acid was slightly increased in docosahexaenoic acid-supplemented cells. We have also supplemented these cells with eicosapentaenoic acid (20:5 n-3) and, in addition to accumulation of this fatty acid in cell phospholipids, we found elevation of 22:5 n-3 and some increment of 22:6, confirming that glial cells are able to convert eicosapentaenoic acid to the long chain, more unsaturated derivatives. In conclusion, n-3 fatty acids, when supplemented to glial cells, appear to modulate the arachidonic acid cascade and to be converted through the elongation and desaturation pathways.

Eicosanoid and inositol phosphate response to platelet-activating factor (PAF) and to a PAF antagonist in rat astroglial cells.

The effects of different concentrations of exogenous platelet-activating factor (PAF) on the formation of arachidonic acid-cyclooxygenase metabolites and on the production of inositol phosphates have been investigated in a primary culture of rat astroglial cells. The cells were used at confluence and the purity was checked by immunostaining of the culture with specific antibodies against glial fibrillary acidic protein. Incubation of the cells with PAF (range 10(-9) to 10(-6) M) resulted in maximal accumulation of total inositol phosphate (620 +/- 60% increment over basal values, P < 0.001) at the concentration of 10(-8) M, after 1 min of stimulation. Smaller inositol phosphate accumulation occurred at higher concentrations of the agonist and at longer stimulation time. After 1 min of stimulation with PAF, the accumulation of the cyclooxygenase metabolites, thromboxane B2 (630 +/- 58 vs 20 +/- 2 pg/mg protein in non-stimulated samples) and 6-keto-prostaglandin F1 alpha (132 +/- 15 vs 55 +/- 7 pg/mg protein in non-stimulated samples) was also maximal at 10(-8) M concentration of the agonist. When the cultures were stimulated with PAF or Ca(2+)-ionophore after preincubation with equimolar concentration of the PAF inhibitor BN 52021, a significant inhibition in the synthesis of both inositol phosphates and cyclooxygenase metabolites occurred only in the PAF-stimulated cells.

Arachidonic acid cycloxygenase and lipoxygenase pathways are differently activated by platelet activating factor and the calcium-ionophore A23187 in a primary culture of astroglial cells.

The aim of our study has been to investigate the metabolism of endogenous arachidonic acid or that of radiolabeled arachidonate in astroglial cells, stimulated with platelet activating factor (PAF) and with the calcium-ionphore A23187. Primary cultures of astroglial cells were obtained from brain cortex of one-day-old rats and were characterized by immunofluorescent staining vs glial fibrillary acidic protein. In labeled cells, diacylglycerol was formed after stimulation with platelet activating factor, whereas mainly the release of labeled arachidonic acid from phospholipids was observed after stimulation with calcium-ionophore. Both PAF and the calcium-ionophore A23187 actively stimulated the formation of the cycloxygenase products PGD2, TXB2 and 6-keto-PGF1 alpha, measured by radio- or enzyme-immunoassay. Differences were observed, instead, in the formation of the lipoxygenase metabolites, the hydroxyeicosateraenoic acids, which were measured by high pressure liquid chromatography (HPLC) with on line radiodetection for the labeled products, and Leukotriene C4, measured by radioimmunoassay. The formation of hydroxyacids by stimulated cells was confirmed by gas chromatography-mass spectrometry (GC-MS). In labeled cells, both agonists induced the formation of 12- and 15-hydroxyeicosatetraenoic acids, whereas stimulation of unlabeled cells with calcium ionophore resulted in formation of 12-hydroxyeicosatetraenoic acid and Leukotriene C4. Our results suggest that in astroglial cells, PAF, a compound which is produced in several tissues including brain, mobilizes a selected arachidonic acid pool, possibly associated with diacylglycerol production, from phospholipids, thus activating the conversion of the released fatty acid via the cyclo and the 12-lipoxygenase pathways.

Effects of hypoxia and recovery on brain eicosanoids and carbohydrate metabolites in rat brain cortex.

The effects of hypoxia (respiration of 5% O2 for 30 min) and recovery (respiration of air up to 30 min) on brain levels of carbohydrate metabolites, of free arachidonate (FAA) and of several eicosanoids (E) were studied in the rat. Animals were sacrificed before or after 30 min of hypoxia, and during recovery, by microwave radiation (MW). At the end of the hypoxic period, arterial pO2 was reduced to 28 mm Hg and glycemia was elevated. Brain lactate and glucose levels were also elevated, whereas glycogen was unchanged. Levels of free FAA and of E were practically unchanged. During recovery, arterial pO2 values were raised above prehypoxic levels at 5 min and returned to normal within 30 min. The elevated serum glucose declined at 5 min and values returned to normal at 30. Brain glucose was still elevated at 5 min and returned to normal, together with lactate, at 30 min. Brain FAA did not change during recovery, but levels of prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGF2) and thromboxane B2 (TxB2) were raised, at 5 min, and those of 6-keto-PGF1 alpha were reduced with respect to pre-hypoxia. At the end of recovery, all E were lower than during hypoxia. The results indicate that changes of brain E occur only during recovery from hypoxia. At 5 min of recovery, at high arterial pO2 values, concentrations of all E were elevated, with the exception of 6-keto-PGF1 alpha which was reduced. At 30 min a marked reduction of E levels was observed, possibly resulting from increased clearance following elevation of cerebral blood flow.

Differential effects of various vasoactive drugs on basal and stimulated levels of TXB2 and 6-keto-PGF1 alpha in rat brain.

Thromboxane B2 and 6-keto-PGF1 alpha (6KPGF1 alpha), the major stable metabolites of thromboxane and prostacyclin, are present in the CNS, where they appear to be mainly produced within and/or acting upon the vascular district. Their concentrations are of few pg/mg protein in rat brain cortex of animals sacrificed by microwave (MW) radiation, procedure which inactivates tissue enzymes and allows the determination of endogenous "basal" levels of eicosanoids. Levels of 6KPGF1 alpha and especially those of TxB2 increase several fold over the basal values in brain cortex of animals sacrificed by decapitation followed by a few minute interval before analysis (post-decapitation ischemia, PDI). Pretreatment of animals with the vasoactive drug papaverine, resulted in elevation of brain basal levels of 6KPGF1 alpha and with the carbochromene derivative AD6 in reduction of basal levels of TxB2, whereas the calcium antagonist nifedipine and dipyridamole did not modify basal levels of the two eicosanoids. Treatments with papaverine and AD6 reduced the accumulation of TxB2 and enhanced that of 6KPGF1 alpha occurring after PDI, to different extents, both resulting, however, in reduction of the TxB2/6KPGF1 alpha ratio. Nifedipine instead, decreased the release of both eicosanoids and resulted in elevation of the TxB2/6KPGF1 alpha ratio, whereas dipyridamole had no effect. In conclusion, the evaluation of the overall effects of drug treatments on the TxB2/6KPGF1 alpha ratio in cerebral tissue, provided useful informations on the pharmacological modulation of vascular eicosanoids in this district.

Differential effects of probenecid on the levels of endogenous PGF2 alpha and TXB2 in brain cortex.

Probenecid in single or repeated doses does not modify levels of PGF2 alpha and TXB2 in rat brain cortex. After administration of subconvulsant dose of pentamethylene tetrazole (PMT) PGF2 alpha increases sharply and rapidly declines subsequently, whereas the elevation of TXB2 is smaller but of longer duration. After probenecid pretreatment PGF2 alpha levels do not decline up to 30 minutes after the initial peak and are still elevated after 60 minutes. Levels of TXB2 tend to be reduced after pretreatment. Differences in transport process or in biosynthetic compartments for these arachidonic acid (AA) metabolites may account for the observed data.