Diego Di Lorenzo

Plasma total antioxidant capacity in hemodialyzed patients and its relationships to other biomarkers of oxidative stress and lipid peroxidation.

Abstract Patients undergoing long-term hemodialysis (HD) exhibit increased levels of oxidative stress, likely contributing to the increased rate of cardiovascular disease. The present study represents a critical evaluation of some of the most widely used oxidative indicators, as applied to the monitoring of hemodialysis-associated oxidative stress. Total plasma antioxidant capacity was determined by two independent procedures, the total antioxidant status (TAS) and the ferric reducing ability of plasma (FRAP) methods. Plasma lipid peroxidation was assessed by determining the peroxidation products malonaldehyde and 4-hydroxynonenal (MDA-4HNE) as well as lipid hydroperoxides (“Fox-2” and “d-ROMs” methods). Total plasma thiols and plasma b-tocopherol were also determined. MDA-4HNE levels were higher in HD patients and decreased following HD, possibly due to passive diffusion across dialysis filters. d-ROMs were also higher in HD patients but exhibited a further increase following the dialysis procedure. Serum α-tocopherol did not show any significant differences. Plasma thiols were lower in HD patients and were restored following HD. Plasma total antioxidant capacity determined with either method was unexpectedly higher in HD patients compared to controls, and decreased following HD. These data indicate that, of the biomarkers studied, d-ROMs level is the one more accurately reflecting the oxidative alterations taking place in HD patients, while determination of MDA-4HNE fails to detect oxidation occurring during the HD sessions. In addition, our findings point out that the determination of total antioxidant capacity in HD patients is severely affected by the concomitant fluctuations in plasma urate levels and therefore needs careful interpretation.

Oxidative Stress and Cardiovascular Disease in Dialyzed Patients

Background/Aim: Oxidative damage has been suggested to play a key role in accelerated atherosclerosis and to be involved in cardiovascular disease (CVD) of dialyzed patients who are at risk of increased oxidative stress. The purpose of the present study was to examine the relationship between the severity of CVD and some markers of oxidative stress and antioxidant activity in our hemodialyzed (HD) and peritoneal dialysis (PD) patients. Methods: Plasma reactive oxygen metabolites, malondialdehyde and 4-hydroxynonenal (MDA-4HNE), thiols, α-tocopherol, and total antioxidant status (TAS) were measured in 55 HD and in 16 PD patients. CVD was considered as the result of variably combined cardiac, cerebral, and vascular pathologies which were scored and grouped in a single CVD index and analyzed with respect to the markers of the oxidative status. 16 normal subjects served as controls. Results: All patients showed evidence of increased oxidative stress which was more severe in HD than in PD patients and which was exacerbated by HD. When cardiac, cerebral, and vascular diseases were analyzed separately, plasma MDA-4HNE and TAS were significantly higher in more severely affected HD patients, but not in PD patients. In HD patients the CVD index was directly correlated with both MDA-4HNE and TAS (r = 0.42, p < 0.01; r = 0.39, p < 0.01) and inversely correlated with α-tocopherol (r = –0.32, p < 0.05). MDA-4HNE and TAS were directly correlated in HD patients and inversely correlated in control subjects. Conclusions: Our data show that, in spite of increased antioxidant defense, there is a relationship between the degree of lipid peroxidation and the severity of CVD in HD patients. Moreover, these data underscore the utility of MDA-4HNE, α-tocopherol, and TAS in the evaluation of cardiovascular disease.

Effect of biological and chemical oxidation on the removal of estrogenic compounds (NP and BPA) from wastewater: An integrated assessment procedure

A major source of the wide presence of EDCs (Endocrine Disrupting Compounds) in water bodies is represented by direct/indirect discharge of sewage. Recent scientific literature reports data about their trace concentration in water, sediments and aquatic organisms, as well as removal efficiencies of different wastewater treatment schemes. Despite the availability of a huge amount of data, some doubts still persist due to the difficulty in evaluating synergistic effects of trace pollutants in complex matrices. In this paper, an integrated assessment procedure was used, based on chemical and biological analyses, in order to compare the performance of two full scale biological wastewater treatment plants (either equipped with conventional settling tanks or with an ultrafiltration membrane unit) and tertiary ozonation (pilot scale). Nonylphenol and bisphenol A were chosen as model EDCs, together with the parent compounds mono- and di-ethoxylated nonylphenol (quantified by means of GC-MS). Water estrogenic activity was evaluated by applying the human breast cancer MCF-7 based reporter gene assay. Process parameters (e.g., sludge age, temperature) and conventional pollutants (e.g., COD, suspended solids) were also measured during monitoring campaigns. Conventional activated sludge achieved satisfactory removal of both analytes and estrogenicity. A further reduction of biological activity was exerted by MBR (Membrane Biological Reactor) as well as ozonation; the latter contributed also to decrease EDC concentrations.

A glucocorticoid receptor-independent mechanism for neurosteroid inhibition of tumor necrosis factor production

We investigated the effect of two neurosteroids, pregnenolone and dehydroepiandrosterone sulfate on lipopolysaccharide-induced tumor necrosis factor (TNF) production in vivo and in vitro. Dehydroepiandrosterone sulfate (0.3-30 mg/kg, i.p.) inhibited serum TNF induced by lipopolysaccharide (2.5 micrograms/mouse, i.p.), without affecting the induction of serum corticosterone. Intracerebroventricular (i.c.v.) administration of dehydroepiandrosterone sulfate (0.2-5 micrograms/mouse) also inhibited brain TNF induced by i.c.v. lipopolysaccharide (2.5 micrograms/mouse). Dehydroepiandrosterone sulfate and pregnenolone (10(-6)-10(-4) M) inhibited TNF production in vitro by lipopolysaccharide-stimulated human peripheral blood mononuclear cells or by the human THP-1 cell line, suggesting that this action might also be relevant in humans. We obtained two lines of evidence that neurosteroids do not inhibit TNF via the glucocorticoid receptor. (1) Dehydroepiandrosterone sulfate and pregnenolone did not activate the alpha 1-acid glycoprotein promoter, a typical effect of glucocorticoids mediated by the glucocorticoid receptor, while strong activation of this promoter was observed with dexamethasone. (2) The inhibitory effect of dehydroepiandrosterone sulfate and pregnenolone on TNF production was not reversed by the glucocorticoid receptor antagonist, mifepristone (RU38486). On the contrary the inhibitory effect of dexamethasone, a classical glucocorticoid and inhibitor of TNF synthesis, was completely reversed by RU38486.

Genistein is an efficient estrogen in the whole body throughout mouse development

The widespread use of diets containing estrogenic compounds raises questions on how relevant the presence of phytoestrogens may be, in order to allow a correct development of the reproductive ability and sexual maturity in humans and animals. The isoflavone genistein is the most estrogenically active molecule present in soy. Here we show that genistein, through an estrogen receptor (ER)-mediated action, modulates gene expression in the whole body of male mice in a dose- and time-dependent manner, at all ages. By luciferase bioassays, we show that genistein-induced ER activation is present in reproductive and nonreproductive organs of the transgenic mice Estrogen Responsive Element (ERE)-tK-LUC, although to an extent that is lower than what observed with the administration of estradiol. Peak activity was registered at genistein doses of 500-5000 microg/kg, at 12 h from the administration by gavage. In the liver, ER-alpha and ER-beta messenger RNAs and two target genes, CYP17 and the progesterone receptor, were modulated by genistein. CYP17 and PR time-dependent induction was similar to that of luciferase. ER-alpha protein level followed an opposite regulation by genistein and estradiol. Genistein passed from the lactating mother to the suckling offspring at levels sufficient to activate gene expression in reproductive and nonreproductive tissues of the pups, with maximal upregulation at 16-24 h. We also followed responsiveness to genistein in the testis, from early development to adult age. Testis are well responsive to genistein as well as to estradiol already at day 14.5 of fetal development, as determined by exposing organotypic cultures from mouse fetus testis. Ovaries were not responsive under the same conditions. Activation of luciferase correlates with an activation of cell proliferation in testis, but not in the ovaries. Prolonged exposure (15 days) to genistein also decreases prostate weight like estradiol. In conclusion, our results show that genistein affects reproductive and nonreproductive organs of male mice in a dose- and time-dependent manner, at all developmental ages.

Iron and Neurodegeneration: Is Ferritinophagy the Link?

Mounting evidence indicates that the lysosome-autophagy pathway plays a critical role in iron release from ferritin, the main iron storage cellular protein, hence in the distribution of iron to the cells. The recent identification of nuclear receptor co-activator 4 as the receptor for ferritin delivery to selective autophagy sheds further light on the understanding of the mechanisms underlying this pathway. The emerging view is that iron release from ferritin through the lysosomes is a general mechanism in normal and tumour cells of different tissue origins, but it has not yet been investigated in brain cells. Defects in the lysosome-autophagy pathway are often involved in the pathogenesis of neurodegenerative disorders, and brain iron homeostasis disruption is a hallmark of many of these diseases. However, in most cases, it has not been established whether iron dysregulation is directly involved in the pathogenesis of the diseases or if it is a secondary effect derived from other pathogenic mechanisms. The recent evidence of the crucial involvement of autophagy in cellular iron handling offers new perspectives about the role of iron in neurodegeneration, suggesting that autophagy dysregulation could cause iron dyshomeostasis. In this review, we recapitulate our current knowledge on the routes through which iron is released from ferritin, focusing on the most recent advances. We summarise the current evidence concerning lysosome-autophagy pathway dysfunctions and those of iron metabolism and discuss their potential interconnections in several neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and Huntington’s diseases; amyotrophic lateral sclerosis; and frontotemporal lobar dementia.

Comparison of three distinct methods for the detection of circulating tumor cells in colorectal cancer patients

The detection of circulating tumor cells (CTCs) has considerable utility in the clinical management of patients with solid cancers. However, the phenotypic heterogeneity of CTCs and their low numbers in the bloodstream of patients means that no standardized detection method currently exists for these cells. This, together with differences in pre-analytical sample processing, has led to the collection and accumulation of inconsistent data among independent studies. Here, we compare the ability of three methods to detect CTCs in the blood of colorectal cancer patients. Specifically, different aliquots of the same blood sample were screened for the presence of CTCs by a multimarker RT-PCR assay, the standardized CellSearch assay and dHPLC-based gene mutation analysis. In the population tested, none of the blood samples analysed appeared to be positive by all three methods. Of the samples, 75% were positive for the presence of CTCs by the RT-PCR method. Only 20% were positive by the CellSearch assay, while 14.3% of samples displayed gene mutations consistent with the presence of CTCs when the dHPLC method was applied. The samples which were positive for CTCs by the CellSearch assay did not overlap with those that were positive by dHPLC. Interestingly, however, all of these samples were positive when assessed by RT-PCR. Conversely, of the samples that resulted negative by RT-PCR analysis, none appeared to be positive by either of the other methods. These data, therefore, indicate that of the three methods tested, the multimarker RT-PCR assay provides maximal probability of CTC detection. Here, we present the preliminary results of an ongoing clinical study. Future follow-up involving detection of CTCs in the blood of colorectal cancer patients using these three distinct methods will allow us to verify whether either a single method, or a combination of different assays, is necessary to uncover further prognostic significance of circulating tumor cells.

Molecular Imaging, an Innovative Methodology for Whole-Body Profiling of Endocrine Disrupter Action

Endocrine disrupters (EDs) are environment and food contaminants known to alter metabolic functions of mammals by interfering with specific endocrine pathways. Many EDs act on steroid hormone target cells by interacting with intracellular receptors (IRs) like estrogen receptors, androgen receptors, and thyroid hormone receptors; other receptors may be engaged. IRs are ligand-operated transcription factors acting in concert with general or cell-specific coregulators. The newly acquired awareness on the panoply of IR functions has increased the concern on potential, unsought, harmful effects of EDs on human health and has questioned the capability of currently available methodologies to identify and study EDs in the environment and in the food chain. Indeed, current in vivo and in vitro methodologies restrict the analysis to very specific organs or cell systems, with obvious limitations in predicting the systemic metabolic consequences of ED exposure. The emphasis recently laid by Regulatory Authorities, including European Center for the Validation of Alternative Methods, on the generation of in vitro model systems for toxicological analyses discouraged the development of models suitable to envision the whole spectrum of ED body actions required when studying compounds acting through IRs. Molecular imaging now provides the opportunity to quantify ED effects in living organisms enabling, for the first time, to acquire a full comprehension of the systemic effects of acute and prolonged exposure to EDs, solving the issue of the potential harm due to repeated low-dose exposure. The systems here reviewed are of unquestionable toxicological relevance and need to be taken into consideration to improve the methodology currently available and in use.

Seizures can precede cognitive symptoms in late-onset Alzheimer's disease.

This study describes late-onset Alzheimers disease (LOAD) in the mild cognitive impairment (MCI) stage, debuting with seizures in a 72 year-old woman. Prodromal AD was consistently diagnosed with four among amyloidosis and neurodegeneration biomarkers about 1 year after onset of seizures. Genetic assessment demonstrated apolipoprotein E ε2/ε3 genotype and three intronic single nucleotide substitutions, two in presenilin 1 and one in amyloid-β protein precursor genes. This case of seizures at onset of LOAD with severe signs of brain amyloidosis and neurodegeneration but with just MCI leads to a re-appraisal of the intriguing relationship between AD pathology and neuron excitability in humans.

Fibroblasts maintained in 3 dimensions show a better differentiation state and higher sensitivity to estrogens

Cell differentiation and response to hormonal signals were studied in a 3D environment on an in-house generated mouse fibroblast cell line expressing a reporter gene under the control of estrogen responsive sequences (EREs). 3D cell culture conditions were obtained in a Rotary Cell Culture System; (RCCS™), a microgravity based bioreactor that promotes the aggregation of cells into multicellular spheroids (MCS). In this bioreactor the cells maintained a better differentiated phenotype and more closely resembled in vivo tissue. The RCCS™ cultured fibroblasts showed higher expression of genes regulating cell assembly, differentiation and hormonal functions. Microarray analysis showed that genes related to cell cycle, proliferation, cytoskeleton, migration, adhesion and motility were all down-regulated in 3D as compared to 2D conditions, as well as oncogene expression and inflammatory cytokines. Controlled remodeling of ECM, which is an essential aspect of cell organization, homeostasis and tissue was affected by the culture method as assessed by immunolocalization of β-tubulin. Markers of cell organization, homeostasis and tissue repair, metalloproteinase 2 (MMP2) and its physiological inhibitor (TIMP4) changed expression in association with the relative formation of cell aggregates. The fibroblasts cultured in the RCCS™ maintain a better responsiveness to estrogens, measured as expression of ERα and regulation of an ERE-dependent reporter and of the endogenous target genes CBP, Rarb, MMP1 and Dbp. Our data highlight the interest of this 3D culture model for its potential application in the field of cell response to hormonal signals and the pharmaco-toxicological analyses of chemicals and natural molecules endowed of estrogenic potential.